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CHEMICAL AND SPECTROSCOPIC STUDIES ON CORDIA ROTHII AND RELATED MEDICINAL PLANTS
Thesis submitted for the fulfilment of the degree of
DOCTOR OF PHILOSOPHY
by
Kehkashan Khan
DEPARTMENT OF CHEMISTRY, UNIVERSITY OF KARACHI, KARACHI-75270, PAKISTAN. April, 2014
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Dedicated to My
Parents,
Family Members,
and Teachers
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Acknowledgements
Countless thanks to Graceful Almighty Allah Subhana-u-Taala who has given me an excellent opportunity for exploring a minor fraction of the bounty of nature and has blessed us with His last Holy Prophet
(Peace be upon Him), whose guidance and teachings lead us on a path towards Almighty Allah.
It is impossible to express my indebtedness to those, who remain involved directly or indirectly in giving this dissertation a final shape.
I am deeply indebted to my research supervisor Prof. Dr. Sadiqa
Firdous, Department of Chemistry, University of Karachi, for her invaluable guidance, constructive criticism, careful advice, constant encouragement and moral support throughout the course of this investigation. She always put her best efforts to facilitate my research work.
I acknowledge with deep gratitude to my co-supervisor
Dr. Munawwer Rasheed, Asst. Prof., Department of Chemistry, and now Assoc. Prof., Center of Excellence in Marine Biology, University of Karachi, for his expert opinion, dedicated and committed efforts, generous support, and keen interest that enabled me in accomplishing
this gigantic task in time.
My humble thanks go to Prof. Dr. Azhar Ali, Chairman, Department
of Chemistry, University of Karachi, for providing necessary facilities.
I am also thankful to Prof. Dr. P. J. A. Siddiqui, Director, CEMB for providing lab space to finalize my thesis write up.
I wish to acknowledge Prof. Dr. M. Iqbal Choudhary H.I., S.I., T.I., Director, H.E.J. Research Institute of Chemistry, International Center for
Chemical and Biological Sciences, University of Karachi, for providing v the facilities of library, spectral analyses and for bioactivity assays under HEC scheme.
I am deeply grateful to Prof. Dr. Viqar Uddin Ahmad H.I., S.I., Khawarizmi Laureate, Distinguished National Professor, H.E.J. Research Institute of
Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, for his kind advice, useful insight, and generous support during this endeavor.
My grateful acknowledgements are to Prof. Dr. Shaheen Faizi S.I. , H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, for her expert advice, valuable comments, and suggestions received over a period of years.
I take this opportunity to express my sincere thanks and appreciation to Dr. Zulfiqar Ali, National Center for Natural Product Research,
School of Pharmacy, University of Mississippi, MS-38677, USA for his helpful comments, valuable criticism and generous help extended in solving many research problems.
I would like to thank Prof. Dr. Aqeel Ahmad for providing help in conducting antibacterial and antifungal assays.
Heartly thanks are due to my colleagues Dr. Sadia Zikr-Ur-Rehman,
Dr. Anila Naz, Dr. Samina Bano, Nida Hassan Ansari, Muhammad
Nadir, Muneeba Khan, Muhammad Ibrahim Jaffery, Rajkumar
Dewani, Farah Safdar, Javeria Khalid, Umme Hani, Rahma Khan, and Sana for providing me the friendly and supportive environment during my research days.
Thanks are also reserved for the technical and non-technical staff of the Department of Chemistry, and ICCBS, University of Karachi.
The monetary assistance of HEC for Instrumental access and for conducting bioactivity assays is gratefully acknowledged that
vi immensely facilitated this research endeavor.
Words cannot express the extensive support extended to me by my parents and family members. Their love, patience, endless care, co-
operation, and prayers encouraged and strengthened me to complete
this dissertation task during a very difficult stage of my life. I thank
them all with the core of my heart.
Kehkashan Khan
April 2014
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PAGE SECTION CONTENT No.
ABSTRACT 1
INTRODUCTION
1.1 General Introduction 5
1.2 Family Boraginaceae 7
1.3 Genus Cordia 8
1.4 Cordia rothii Roem. & Schult 8
1.5 Identification of Metabolites from Genus Cordia Exploiting GC/GC-MS 76
BIOSYNTHESES
2.1 Biosyntheses 77
2.2 Biosynthesis of Saturated Fatty Acids 78
2.3 Biosynthesis of Unsaturated Fatty Acids 80
2.4 Biosynthesis of Cerebrosides 82
2.5 Biosynthesis of Terpenes 83
2.6 Glycosylation Reactions 91
RESULTS AND DISCUSSION
3.1 Characterization of Isolated and Purified Compounds 97
3.1.1 Structure Elucidation of 2′′ -Butoxyethyl 3-[3 ′,5 ′-di( tert -butyl)-4′- 99 hydroxyphenyl]-propanoate, Mairajinol (30 ) ― A New Compound
3.1.2 Characterization of Stigmast-5-en-3β-ol (β-Sitosterol) ( 26) 106
3.1.3 Characterization of (24 S)-Stigmast-5, 22-dien-3β-ol (Stigmasterol) ( 27) 107
3.1.4 Characterization of Octacosan-1-ol ( 74 ) 108
3.1.5 Characterization of Stigmast-5-en-3-O-β-D-glucoside ( β-Sitosterol 109 glucoside) ( 79 )
3.1.6 Characterization of (2 S) Methyl 2-hydroxy-3-(4 ′-hydroxyphenyl)- 110 propanoate (Latifolicinin C) ( 62 )
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3.1.7 Characterization of 1-O-β-D-Glucopyranosyl-(2 S,3 S,4 R,8 Z)-2-[(2 ′R)-2′- 112 hydroxytetracosanoyl amino]-1,3,4-octadecanetriol-8-ene ( 80 )
3.1.8 Characterization of (2 R) 2-Hydroxy-3-(4 ′-hydroxyphenyl) propanoic acid 114 [(2 R) ( p-hydroxyphenyl) lactic acid] ( 81 )
3.1.9 Characterization of Syringaresinol mono-β-D-glucoside ( 82 ) 115
3.1.10 Characterization of 6-Hydroxy-3-oxo-α-ionol 9-O-β-D-glucopyranoside 116 (Roseoside) ( 83 )
3.1.11 Characterization of 3,5-Dihydroxy-megastigma-6,7-dien-9-one-3-O-β-D- 118 glucopyranoside (Staphylionoside D) (84 )
3.1.12 Characterization of (2 E)3-(3 ′,5 ′-Dimethoxy-4′-O-β-D-glucopyranosyl- 120 phenyl)-prop-2-en-1-ol (Syringin) ( 85 )
3.2 Studies on Non-polar to Moderately Polar Fractions of Root, Stem and 122 Leaves Exploiting Gas Chromatography - Flame Ionization Detection (GC-FID) and Gas Chromatography - Mass Spectrometry (GC-MS)
3.2.1 Methodology 122
3.2.2 Results 123
3.2.3 Discussion 135
3.2.4 Structure Elucidation Using GC-EI-MS 158
3.3 Bioactivities 167
3.3.1 Sample Preparation for Bioactivity 168
3.3.2 Bioassays 168
3.3.2.1 Antimicrobial Activity 168
3.3.2.2 Toxicity Studies 170
3.3.2.3 Antioxidant and Immunomodulating Activity 171
3.3.2.4 Insecticidal Activity 174
3.3.2.5 Antiglycation Studies 174
EXPERIMENTAL
4.1 General Experimental 175
4.2 Extraction, Fractionation, Isolation, and Identification Schemes 177
4.2.1 Schemes of work for Root of C. rothii 177
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4.2.2 Schemes of work for Stem of C. rothii 183
4.2.3 Schemes of work for leaves of C. rothii 187
4.3 Characterization of Isolated Compounds 193
4.3.1 Characterization of 2′′ -Butoxyethyl 3-[3 ′,5 ′-di( tert -butyl)-4′- 193 hydroxyphenyl]-propanoate, Mairajinol (30 ) ― A New Compound
4.3.2 Characterization of Stigmast-5-en-3β-ol ( β-sitosterol) ( 26) 194
4.3.3 Characterization of (24 S)-Stigmast-5, 22-dien-3β-ol (Stigmasterol) ( 27) 195
4.3.4 Characterization of (2 S-) Methyl 2-hydroxy-3-(4 ′-hydroxyphenyl)- 196 propanoate (Latifolicinin C) ( 62 )
4.3.5 Characterization of Octacosan-1-ol ( 74 ) 197
4.3.6 Characterization of Stigmast-5-en-3-O-β-D-glucoside ( β-Sitosterol 198 glucoside) (79 )
4.3.7 Characterization of 1-O-β-D-Glucopyranosyl-(2 S,3 S,4 R,8 Z)-2-[(2 ′R)-2′- 199 hydroxytetracosanoyl amino]-1,3,4-octadecanetriol-8-ene ( 80 )
4.3.8 Characterization of (2 R) 2-Hydroxy-3-(4 ′-hydroxyphenyl)-propanoic acid 200 [(2 R) ( p-hydroxyphenyl) lactic acid] (81 )
4.3.9 Characterization of Syringaresinol mono-β-D-glucoside ( 82 ) 201
4.3.10 Characterization of 6-Hydroxy-3-oxo-α-ionol 9-O-β-D-glucopyranoside 202 (Roseoside) (83 )
4.3.11 Characterization of Staphylionoside D (84 ) 203
4.3.12 Characterization of (2 E) 3-(3 ′,5 ′-Dimethoxy-4′-O-β-D-glucopyranosyl- 204 phenyl)-prop-2-en-1-ol (Syringin) ( 85 )
4.4 Identification of Natural Compounds using GC-MS Analyses 204
4.4.1 Gas Chromatographic Data 204
4.4.1.1 Gas Chromatographic Data of C. rothii Roots 204
4.4.1.2 Gas Chromatographic Data of C. rothii Stem 216
4.4.1.3 Gas Chromatographic Data of C. rothii Leaves 226
4.4.2 Gas Chromatographic Electron Impact Mass Spectral (GC-EIMS) Data 232
4.4.2.1 GC-EIMS Data of Identified Constituents 232
4.4.2.2 GC-EIMS Data of Un-identified Constituents 238
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4.5 Bioactivities 243
4.5.1 Sample Preparation of Extracts from Root, Stem, and Leaves for 243 Bioactivities
4.5.2 Bioassays 243
4.5.2.1 Antimicrobial activity 243
4.5.2.2 Toxicity studies 251
4.5.2.3 Antioxidant & Immunomodulatory activities 254
4.5.2.4 Insecticidal activity 260
4.5.2.5 In Vitro Glycation 261
REFERENCES 262
APPENDICES
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Abstract
Chapter 1 of the dissertation discusses the medicinal and pharmacological importance and phytochemistry of Cordia rothii Roem. & Schult and thus the plant was selected for further studies. Chapter 2 summarizes the biosynthesis of selected phytochemicals of this plant.
Chapter 3 and 4 covers the current studies on root, stem and leaves of C. rothii . Altogether 88 phytochemicals are being reported here. 12 were isolated, purified and characterized while 79 were identified using GC-MS studies. 3 metabolites ( 26 , 27 and 62 ) were isolated as well as were also identified in the GC-MS study. Isolated compounds included stigmast-5-en-3β-ol (26 ), (24 S) stigmasta-5,22-dien-3β-ol ( 27 ) 2′′ -butoxyethyl 3-[3 ′, 5 ′-di( tert -butyl)-4′-hydroxy- phenyl]-propanoate (30 ), (2 S) methyl 2-hydroxy-3-(4 ′-hydroxyphenyl)-propanoate (62 ),
octacosan-1-ol ( 74 ), stigmast-5-en-3-O-β-D-glucoside (79 ), (2 S,1 ′S,2 ′S,3′R,7 ′Z)-N-1′-(O-β-D- glucopyranosyl)methyl-2′,3 ′-dihydroxy-heptadec-7′-enyl-2-hydroxytetracosaneamide ( 80 ),
(2 R) 2-hydroxy-3-(4′-hydroxyphenyl)-propanoic acid (81 ), syringaresinol mono-β-D-
glucoside ( 82 ), 6-hydroxy-3-oxo-α-ionol 9-O-β-D-glucopyranoside (83 ), 3,5-dihydroxy-
megastigma-6,7-dien-9-one-3-O-β-D-glucopyranoside (84 ), and 3-(3 ′,5 ′-dimethoxy-4′-O-β-D- glucopyranosyl-phenyl)-prop-2E-en-1-ol (85 ).
30 , trivially named as Mairajinol, is the new phenolic compound purified and characterized from the chloroform extract of roots. Its structure was characterized and found to possess a unique combination of ester and ether functionalities in an alkylated monohydroxy phenyl propanoid system. The possible biogenetic pathway for its formation has also been discussed.
26, 27, 62 , 74 , 79 , and 80 to 85 are known compounds, of these, nine compounds (62 , 74 , 79 , 80 to 85 ) are reported for the first time from this plant. Compound 80 , 82 , 83 , and 84 bears the cerebroside, lignan, norterpenoid, and megastigmane skeleton respectively. The thesis is the first isilation report on aforementioned classes of compounds from the source. All structural elucidations were done using recent sophisticated spectral techniques; EIMS, ESIMS, FABMS, CIMS, HREIMS, UV, IR, 1H and 13 C NMR (both 1D and 2D). Further confirmation of known compounds was done by comparing their data with the previously reported one in literature.
GC-MS analyses revealed the presence of 45, 17, and 17 phytochemicals from non-polar to less polar fractions of root, stem and leaves respectively. These belong to hydrocarbons, fatty acids and their derivatives, aromatics, phenols, sesquiterpenoids, diterpenoids, triterpenoids, phytosteroids and other interesting metabolites. Few metabolites were identified in more than one part or fractions. In total, 79 metabolites were identified. To the best of our knowledge, 60 constituents are identified for the first time from C. rothii . These included:
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1, 2, 4, 5, 7, 11 , 12 , 14 -17 , 19 , 21 -25, 28, 29, 32-40 , 43 -48, 50 , 51 , 53 -59 , 61-73 , and 75 -78 . The IUPAC names of the isolated and identified natural constituents are classified below:
Hydrocarbons
H3C CH3 n 10 n=9, n-tridecane (1) n=10, n-tetradecane (2) n=11, n-pentadecane (3) iso -hexadecane ( 4) n=13, n-heptadecane (6) n=14, n-octadecane (8) n=15, n-nonadecane (10 ) n=12, n-hexadecane (20 ) n=16, n-eicosane (49 ) n=17, n-heneicosane (52 ) n=20, n-tetracosane (86 ) n=22, n-hexacosane ( 87 ) n=24, n-octacosane ( 88 ) Free Fatty Acids HO O R R n 2 5 O HO R R 3 4
n=14, n-hexadecanoic acid ( 13 ) R2=R 3=∆, R 4=R 5= ∆ , octadec-9Z,12 Z-dienoic acid (31 ) n=16, n-octadecanoic acid ( 18 ) R2=R 3=∆, R 4=R 5= H, octadec-9Z-enoic acid (42 ) n=10, n-dodecanoic acid ( 39 ) R2=R 3=∆, R 4=R 5= H, octadec-9E-enoic acid (69 ) n=12, n-tetradecanoic acid ( 41 ) n=15, n-heptadecanoic acid ( 51 ) HO n=18, n-eicosanoic acid (54 ) 11 n=13, n-pentadecanoic acid (78 ) O iso -hexadecanoic acid ( 68 ) Derivatives of Fatty Acids O R n O R=Et, n=10, n-dodecanoic acid ethyl ester ( 5) R=Me, n=20, n-docosanoic acid methyl ester ( 56 ) R=Et, n=12, n-tetradecanoic acid ethyl ester ( 7) R=Me, n=21, n-tricosanoic acid methyl ester ( 57 ) R=Me, n=14, n-hexadecanoic acid methyl ester ( 11 ) R=Me, n=22, n-tetracosanoic acid methyl ester ( 58 ) R=Et, n=14, n-hexadecanoic acid ethyl ester ( 12 ) R=Me, n=24, n-hexacosanoic acid methyl ester ( 59 ) R=Me, n=16, n-octadecanoic acid methyl ester ( 23 ) R=Me, n=6, n-octanoic acid methyl ester ( 66 ) R=Me, n=15, n-heptadecanoic acid methyl ester ( 50 ) R=Me, n=12, n-tetradecanoic acid methyl ester ( 70 ) R=Me, n=18, n-eicosanoic acid methyl ester ( 53 ) R=Me, n=13, n-pentadecanoic acid methyl ester (71 ) R=Me, n=19, n-heneicosanoic acid methyl ester ( 55 ) R=Me, n=10, n-dodecanoic acid methyl ester (77 ) O O 5 O O
hexadec-9Z-enoic acid methyl ester ( 72 ) octadec-9Z, 12 Z, 15 Z-trienoic acid methyl ester ( 73 )
O R2 R5 R 1 O R3 R4
R1=Me, R 2=R 3=∆, R 4=R 5=H, octadec-9Z-enoic acid R1=Et, R 2=R 3=∆, R 4=R 5= H, octadec-9E-enoic acid methyl ester (14 ) ethyl ester ( 16 ) R1=Et, R 2=R 3=∆, R 4=R 5= ∆ , octadec-9Z,12 Z-dienoic R1=Et, R 2=R 3=∆, R 4=R 5= H, octadec-9Z-enoic acid acid ethyl ester ( 15 ) ethyl ester ( 17 ) R1=Me, R 2=R 3=∆, R 4=R 5= ∆ , octadec-9Z, 12 Z-dienoic acid methyl ester (22 ) O R O HO O O O HO hexadecanoic acid 2-hydroxy-1-(hydroxymethyl)- R= OH, nonanedioic acid monomethyl ester ( 47 ) ethyl ester ( 43 ) R= H, 9-oxo-nonanoic acid methyl ester ( 67 )
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Aromatics Benzoic Acid Derivatives O O O O O OH OH
HO HO phenylethene (34 ) acetic acid phenylmethyl ester 4-hydroxy-benzoic acid 4-hydroxy-3-methoxy- (35 ) (45 ) benzoic acid (46 ) Phenylpropanoids O O OH OH HO HO O HO O OH O
(1 E)-4-(3-hydroxy-1-propenyl)- 2′′ -butoxyethyl 3-(3 ′,5 ′-di( tert -butyl)-4′- 3,4-dihydroxy- 2-methoxyphenol ( 28 ) hydroxyphenyl)-propanoate ( 30 ) benzenepropanoic acid ( 40 )
HO O O HO HO OCH3 O O HOOH HO O OH HO OH HO O (2 S) methyl 2-hydroxy-3-(4 ′- (2 R) 2-hydroxy-3-(4 ′-hydroxy- 3-(3 ′,5 ′-dimethoxy-4′-O- β -D-gluco- hydroxyphenyl)-propanoate (62 ) phenyl)-propanoic acid (81 ) pyranosyl-phenyl)-prop-2E-en-1-ol ( 85 ) Sesquiterpenoids & nor-sesquiterpenoids (Megastigmane) O OH OH H H HO OH
O H H H OH O OH H H 5,6,7,8-tetrahydro-7- 2-methyl-2-(4- 5,6,7,8-tetrahydro-7- 5,6,7,8,8a,9,10,10a- isopropenyl-6-methyl-6- methylpent-3-enyl)-2H- isopropenyl-6-methyl-6- octahydro-5,5-dimethyl vinyl naphthalene-1,4- chromen-6-ol ( 21 ) vinyl naphthalene-1,4- anthracene-1,4,8a-triol dione (9) diol (24 ) (29 ) OH H O O HO H HO OH O H OH O HO HO H HO O OH O OH 1,2,3,4,4a,5,6,8a-octahydro-7- 6-hydroxy-3-oxo-α-ionol 9-O-β-D- 3,5-dihydroxy-megastigma-6,7- methyl-4-methylene-1-(1- glucopyranoside ( 83 ) dien-9-one-3-O-β-D- methylethyl)-naphtahlaene (76 ) glucopyranoside (84 ) Diterpenoid & nor-diterpenoid O HO
6,10,14-trimethyl-pentadecan-2-one ( 48 ) 3,7,11,15-tetramethyl-2-hexadecen-1-ol ( 75 ) Triterpenoids
H H H H H
HO HO HO H H H
9,19-cyclolanost-24-en-3β-ol ( 32 ) 24-methylene-9,19-cyclolanostan- 4α,14 α-dimethyl-9,19-cycloergost- 3β-ol ( 33 ) 24(28)-en-3β-ol ( 44 )
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H H H
H HO HO H H olean-12-en-3β-ol ( 60 ) 25-methylene-9,19-cyclolanostan-3β-ol ( 63 ) Phytosteroids
H H H H H H H HO HO H H stigmasta-3,5-diene ( 19 ) stigmasta-4,22-diene-3β-ol ( 25 ) stigmast-5-en-3β-ol (26 )
OH
HO O HO OH HO O
O stigmasta-5,22-dien-3β-ol ( 27 ) stigmasta-3,5-dien-7-one ( 61 ) stigmast-5-en-3-O-β-D-glucoside (79 ) Miscellaneous O O O O OH O NH HO OH O N O H 2,4-dihydroxy-5- 2,3-dihydro-3,5-dihydroxy-6- 5-(hydroxymethyl)-2- (E,E) hepta-2,4-dienal methylpyrimidine ( 36 ) methyl-4H-pyran-4-one ( 37 ) furan-carboxaldehyde ( 38 ) (64 )
HO 25 octacosan-1-ol ( 74 ) 2,7-dimethyl-1,6-octadiene ( 65 ) OH OH O O O HO O HO OH 19 H NH OH OH O O O O HO O H HO O H OH 3 OH OH O 8 (2 S,1 ′S,2 ′S,3 ′R,7 ′Z)-N-1′-(O-β-D-glucopyranosyl)-methyl-2′,3 ′- Syringaresinol mono-β-D-glucoside ( 82 ) dihydroxy-heptadec-7′-enyl-2-hydroxytetracosaneamide ( 80 )
Root extract showed significant antibacterial and antioxidant potential while leaves extract showed significant antifungal, insecticidal, antiglycation and immunomodulatory activity. Leaves extract exhibited significant inhibitory activity against ROS and very strong suppressive effect on PHA stimulated T-cell proliferation. Sub-fractions from leaves also affected the adaptive immune system and showed potent inhibitory activity on the NO. 82, isolated from leaves, exhibited significant inhibitory activity on zymosan activated ROS generation.
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1.1 General Introduction:
Nature not only has created human being but also decorated this universe with the essentials of life and ordered mankind to spread on this earth and discover its enormous creations. Accordingly, since inception, human has been engaged in exploring the secrets of this universe that not only improves the standard of living on earth but also brings human closer to its Creator.
Among all the necessities of life, health related issues have been taken on priority and people remain engaged in solving health problems before the advent of science. They used to prepare extracts, tinctures and other preparations from various plants and animals, possessing diverse bioactive properties ranging from poisonous to healing. In the early ages, due to the lack of scientific knowledge, all these achievements were accidental and based on luck or trial and error (Agosta WC, 1997). It is obvious that the goal of most of the efforts was to identify natural resources possessing medicinal properties and to convert them into medicines. These naturally occurring resources provide the natural products (Baker DD, 2007). Historical evidences confirm that different civilizations have been converting plants into medicines. Even today, the traditional medicine is functional in most parts of the world (Agosta WC, 1997).
Egyptian medicine left the first record near about 2900 BC, preceded by Ebers Papyrus. Mesopotamia first record was dated around 2600 BC and best known Egyptian pharmaceutical record was from 1500 BC. The first document on Chinese Materia Medica was found in 1100 BC and that of Indian Ayurvedic system was in 1000 BC. The Greek philosopher and natural scientist of the ancient Western world, Theophrastus (~ 300 BC) discussed herb’s medicinal properties and pointed out their potentials of changing characteristics by cultivation. Another Greek Physician Dioscorides recorded data of storage, use, and collection of medicinal herbs around 100 AD.
A Roman scientist, Galen (130-200 AD), was famous for practicing complex prescriptions and formulas having several ingredients in his medicines. Later on, Arabs preserved the Western knowledge acquired from 5 th to 12 th centuries during the dark and middle ages and flavoured it with their own preparations. However, Chinese and Indian herbal data, was unfamiliar to Greeco-Roman world (Cragg G and Newman, 2001).
The store-house of medicinal knowledge unveiled by Arab scientists during eighth and ninth centuries remained hidden till 15 th century from the West. Tibb-i-Unani flourished due to the efforts of one of the eminent physicist, Abu Bakar Muhammad Ibn Zakariya, familiar as Al-Razi (865-925) during early middle ages. Another Persian physician, Ibn-e-Sina
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(Avicenna, 980-1037), a follower of Al-Razi, improved the Aristotelian Platonic based medicinal heritage. One of his contributions “Al-Qanun fi al-Tibb” has been taught as text book to the medical students in Europe till 17 th century. Since then, plant kingdom remains the target of scientists all around the world in search of potent therapeutic agents (Najma RM, 2009; Mbwambo ZH et al ., 1996).
Today, the challenging demand of new medicines for the treatment and cure of deadly diseases, by natural products has attracted the attention of not only academia but pharmacopoeia as well. According to a careful analysis, 25-50% currently marketed drugs and two-third anticancer and anti-infective medicines are derived from natural products. No doubts, microbial species’ derived drugs constitute a larger portion of medicines used clinically however, the importance of naturally occurring plant-derived drugs can’t be ignored. In other words, without morphine, vinblastine, vincristine, quinine, artemisinin, etoposide, teniposide and paclitaxel, plant-derived drugs, we would be barehanded (Kingston DGI, 2011).
Since oceans constitute 90% of various life forms on earth. Currently scientists are paying more attention towards marine natural products. The reason for this attraction is the biological diversity of marine ecosystem. Various natural products of marine origin either have been screened or are under clinical trials for the cure of cancer, analgesia, allergy and cognitive diseases. These are also used in other disciplines of life sciences, such as in agrochemical research, nutrition and supplements (Füllbeck M et al ., 2006).
Majority of the underdeveloped world’s population still depend on crude extract of plants for the treatment and cure of various diseases, whereas modern Western scientific medicine favours the use of a single component or pure compound. The use of crude extracts for the treatment of several deadly diseases like cancer and HIV infection in orthodox medicine in the near future cannot be ignored. Today, several drugs comprising plant extracts are being used and available pharmaceutically. For instance, cascaroside A from Rhamnus purshiana is used as laxative; eugenol enriched extracts from Syzygium aromaticum as topical antiseptic and analgesic; whereas emetine from Cephaëlis pecacuanha as emetic and expectorant (Houghton PJ, 2001).
With the technological advancement in chromatographic and analytical techniques, such as TLC, GC, IR, 1D- and 2D-NMR, and MS isolation and identification of a single compound within a complex mixture become easier and the quantity of organic compound required for the identification, has decreased from gram to microgram (Phillipson JD, 2007, Meinwald J, 2011).
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Isolated compounds obtained from a natural source cannot always be utilized pharmaceutically due to its insufficient quantity; however, it helps the researchers in tracing the pharmacophore (part of the molecule attributing the activity). It also helps in identifying the portion of molecule responsible for unwanted side effects. Compounds from natural origin serve as a template in the synthesis of pharmacophore of relevant molecule required for the SAR (structure-activity relationship) studies. By making use of such templates, many important synthetic drugs have been introduced in the market for a common man and hence have reduced the suffering of mankind. For instance aspirin (acetylsalicylic acid) from willow (Salix spp.) and meadowsweet ( Filipendula ulmaria ) are used traditionally for the cure of rheumatism and normal aches and pains (Houghton PJ, 2001).
The adverse side effects of the synthetic drugs observed has led to the revival of the naturally derived medicine. It is believed that amongst 250,000 higher plant species, only 5-15% have been screened for potent therapeutic agents. The increasing number of naturally occurring potent compounds also reveal the chemical and biological diversity of nature (Houghton PJ, 2001).
“The woods are lovely, dark and deep,
But I have promises to keep,
And miles to go before I sleep,
And miles to go before I sleep.”
(Robert Frost) (Tiempo EK et al ., 1999)
1.2 Family Boraginaceae:
Family Boraginaceae in Pakistan is represented by 32 genera and 135 species. It comprises of annual, biennial or perennial herbs and rarely trees or shrublets (Ali SI and Nasir YJ, 1989). Many plants belonging to family Boraginaceae possess medicinal properties and have been used for the treatment of different diseases. A wide range of biological activities such as prostaglandin inhibitory, wound healing, antiplatelet, contraceptive, cardiotonic, anti- inflammatory, antiviral, antitumor and antimicrobial activities are reported. These plants have shown the presence of diverse range of secondary metabolites i.e., phenols, terpenoids, triterpenoids, pyrrolizidine alkaloids, naphthoquinones and flavonoids (Sharma RA et al ., 2009).
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1.3 Genus Cordia :
Plants belonging to genus Cordia of family Boraginaceae are renowned in folk medicine. They have been used as antiandrogenic, anti-inflammatory, antifungal, larvicidal (Dettrakul S et al ., 2009), antiulcerogenic, analgesic (Diniz JC et al ., 2009 ), antimicrobial (Hernandez T et al ., 2007 ), antileishmanial (Mori K et al ., 2008 ), trypanocidal (Vieira NC et al ., 2008 ), expectorant, astringent, and diuretic (de Menezes JESA et al ., 2005a). These are also employed for the treatment of pneumonia, cough, dyspepsia, rheumatism (Diniz JC et al ., 2009 ), as well as a wound-healing promoter (de Menezes JESA et al ., 2005a).
Literature review reveals the presence of broad range of significant phytochemicals. These were either isolated from plant extracts or identified from essential oils of different Cordia species. These metabolites belong to various classes of natural products including hydrocarbons, acids, fatty acids and their derivatives, ceramides, cerebrosides, amino acids, chromenes, phenols, lignans, neo-lignans, flavonoids, flavanol glycosides, terpenoid quinones, plastoquinones, merosesquiterpenoid quinones, terpenoid hydroquinones, monoterpenes, sesquiterpenes, diterpenes, triterpenes, triterpene glycosides (including saponins), steroids, alkaloids, pyrrolizidine alkaloids, carbohydrates, megastigmanes, etc., in its various species. These are summarized in table-1.1.
1.4 Cordia rothii Roem. & Schult:
Cordia rothii Roem. & Schult (Syn. Cordia gharaf (Forssk.) Ehren. ex Asch. (Ali SI and Nasir YJ, 1989), commonly known as Gondani (Mhaskar KS et al ., 2000) is distributed in Pakistan, Sri Lanka, India, Arabia and North Africa. The flowering period is from April to June.
Cordia rothii is a shrub or tree and may attain the height of 9m. The shape of leaves may vary from elliptic oblong or oblanceolate on which nerves are impressed above. The length of Calyx is 3.5 mm and it is tubular-campanulate, shallowly lobed which are obtuse. Corolla is 5mm in length, white in colour and the lobes are ± ligulate-obtuse, glabrous filaments having the length of 1.8mm. The length of anthers is 1mm. Fruit (drupe) is reddish-brown and 11mm in length (Ali SI and Nasir YJ, 1989).
Cordia rothii has been explored to a lesser extent as compared to other species of the genus Cordia and therefore has been selected for further investigations. Simplest natural compounds reported from C. rothii included C-15 to C-39 n-hydrocarbons (Mukat B & Chhaya G, 1980). Literature review of the chemical constituents from C. rothii is summarized in table-1.2 that provides a brief survey of different classes of natural products isolated or identified from it.
8
Cordia rothii Roem. & Schult.
9
The decoction from the bark of the plant is astringent and is used as gargle (Watt G. 1972). The bark is also used for treating stomach disorders and curing chest pains. Recently some phenolic constituents isolated from C. rothii have been evaluated for their antioxidant and antiglycation properties and exhibited marked antioxidant and significant antiglycation properties (Al-Musayeib N et al ., 2011).
The use of bark for heart diseases is also reported in Ayurveda and is found to be better than other cardiotonic drugs. In ddition to that antidote property is also exhibited by the bark. The fruit of the plant also possesses medicinal properties. It is antiseptic, antidiarrhoeal, astringent and provides relief in urinary tract burning sensation. The root of the plant acts as abortifacient and also possesses anti-inflammatory properties. Antidiabetic and antileprotic activities are shown by the whole plant. The plant is also used for fever and dyspepsia as a substitute for the bark of Cordia dichotoma Forst. in Ayurveda (Chauhan MG and Chavan SS, 2009). Fruits are also used in piles and toothache. One of the most important uses of the ethanolic extract (50%) of the aerial parts of C. rothii is the treatment of CNS depression (Asolkar LV et al ., 1965-1981).
The bark of C. rothii provides fibre, which is used for making ropes and caulking boats (Ambasta SSP et al ., 1994). Its fruit is edible and used in pickle. The wood of the plant is also utilized as fuel. Other uses of its wood are for building and agricultural purposes (Watt G, 1972).
10
Table -1.1 : Phytochemicals Isolated and Identified from Genus Cordia :
Structures, Molecular Formulae Source References & Name of Compounds
HYDROCARBONS
C. gilletii Bonesi M et al ., 2011 10 C. sebestina Adeosun CO & Samuel C18 H38 SO, 2012 n-Octadecane
C. gilletii Bonesi M et al ., 2011 11
C19 H40 n-Nonadecane
C. gilletii Bonesi M et al ., 2011 12
C20 H42 n-Eicosane
C. sebestina Adeosun CO & Samuel 13 SO, 2012 C21 H44
n-Heneicosane C. gilletii Bonesi M et al ., 2011 15 Adeosun CO & Samuel C23 H48 C. sebestina n-Tricosane SO, 2012
C. gilletii Bonesi M et al ., 2011 17
C25 H52 n-Pentacosane
C. sebestina Adeosun CO & Samuel 18 SO, 2012 C26 H54 n-Hexacosane
C. gilletii Bonesi M et al ., 2011 19
C27 H56 n-Heptacosane
C. gilletii Bonesi M et al ., 2011 21 C. sebestina Adeosun CO & Samuel C29 H60 SO, 2012 n-Nonacosane
11
C. obliqua Agnihotri VK et al ., 1987 23 C. sebestina Adeosun CO & Samuel C31 H64 SO, 2012 n-Hentriacontane
C. ecalyculata Vell. Lucia SM et al ., 1985 25
C33 H68 n-Tritriacontane
C. sebestina Adeosun CO & Samuel 6 SO, 2012 C14 H28
3-Tetradecene
C. gilletii Bonesi M et al ., 2011 9
C17 H34 1-Heptadecene
C. gilletii Bonesi M et al ., 2011 10
C18 H36 1-Octadecene
C. gilletii Bonesi M et al ., 2011 12
C20 H40 1-Eicosene
C. sebestina Adeosun CO & Samuel 13 13 SO, 2012 C35 H70
17-Pentatriacontene
C. sebestina Adeosun CO & Samuel 9 9 SO, 2012
C35 H70
13-Methyl-Z-14-nonacosene
ACIDS AND THEIR DERIVATIVES
O C. nitida Vahl Pino JA et al ., 2002 OH
C2H4O2 Acetic acid
12
O C. nitida Vahl Pino JA et al ., 2002 O
C6H12 O2 Ethyl butyrate
O C. nitida Vahl Pino JA et al ., 2002 O
C4H8O2 Ethyl acetate
O O C. nitida Vahl Pino JA et al ., 2002
C3H6O2 Ethyl formate
O C. sebestina Adeosun CO & Samuel O SO, 2012 O O
C19 H32 O4
Fumaric acid isobutyl undec-2-en-1-yl ester
FATTY ACIDS AND THEIR DERIVATIVES
OH C. nitida Vahl Pino JA et al ., 2002 O
C5H10 O2 Isovaleric acid
OH C. nitida Vahl Pino JA et al ., 2002 O
C6H12 O2 Hexanoic acid
OH C. curassavica da Camara CAG et al ., O 2007 C9H18 O2 Nonanoic acid
OH C. nitida Vahl Pino JA et al ., 2002 6 O C. gilletii Bonesi M et al ., 2011
C14 H28 O2 C. sebestina Adeosun CO & Samuel Myristic acid SO, 2012
13
OH C. gilletii Bonesi M et al ., 2011 7 O
C15 H30 O2 Pentadecanoic acid
OH C. nitida Vahl Pino JA et al ., 2002 8 O C. sebestina Adeosun CO & Samuel SO, 2012 C16 H32 O2
Hexadecanoic acid
OH C. myxa Tiwari RD et al ., 1967 8 O C. dichotoma Theagarajan KS et al ., 1977 C16 H32 O2 C. myxa & Palmitic acid C. sebestina Miralles J et al ., 1989 C. gilletii Bonesi M et al. , 2011
OH C. myxa Tiwari RD et al ., 1967 10 O C. dichotoma Theagarajan KS et al ., 1977
C18 H36 O2 C. dichotoma Rameshwar D et al ., 2006 Stearic acid C. sebestina Adeosun CO & Samuel SO, 2012
OH C. obliqua Kleiman R et al ., 1964 6 O C. myxa Tiwari RD et al ., 1967
C18 H30 O2 C. myxa & Miralles J et al ., 1989 α-Linolenic acid C. sebestina
OH C. myxa Tiwari RD et al ., 1967 6 4 O C. dichotoma Theagarajan KS et al ., 1977
C18 H34 O2 C. myxa & Miralles J et al ., 1989 Oleic acid C. sebestina C. boissieri A. D.C. Alanis-Guzman MG et al ., 1998 C. boissieri Alanis-Guzman MG et al ., 1995 C. dichotoma Rameshwar D et al ., 2006
OH C. oblique Kleiman R et al ., 1964 4 3 O C18 H30 O2 γ-Linolenic acid
14
OH C. dichotoma Theagarajan KS et al ., 1977 4 6 O C. myxa & Miralles J et al ., 1989 C H O 18 32 2 C. sebestina Linoleic acid C. boissieri A. D.C. Alanis-Guzman MG et al ., 1998 C. boissieri Alanis-Guzman MG et al ., 1995 C. dichotoma Rameshwar D et al., 2006
OH C. dichotoma Theagarajan KS et al ., 1977 12 O C20 H40 O2 Arachidic acid
OH C. dichotoma Theagarajan KS et al ., 1977 14 O
C22 H44 O2 Behenic acid
CERAMIDES
O C. platythyrsa Tapondjou LA HN 17 et al ., 2005 HO OH HO 5 OH
C42 H83 O5N
(2 S,3 S,4 R,8 E)-2N-[(2 ′R)-2′-hydroxy-tetracosanoyl]-octadec-8-ene-1,3,4-triol
CEREBROSIDES
O C. platythyrsa Tapondjou LA
HN 17 et al ., 2005 HO HO OH O HO O HO OH 5 OH
C48 H93 O10 N
(2 S,3 S,4 R,8 E)-1-O-β-D-Glucopyranosyl-2N-[(2 ′R)-2′-hydroxy-tetracosanoyl]-octadec-8-ene-1,3,4-triol
AMINO ACIDS
NH2 O C. dichotoma Theagarajan KS HO S S OH et al ., 1977 O NH2 C. dichotoma Saxena VK & C6H12 N2O4S2 Jain S, 1983 Cystine
15
O C. dichotoma Theagarajan KS et al ., S OH 1977 NH2
C5H11 NO 2S Methionine
O C. dichotoma Saxena VK & Jain S, 1983 H N 2 OH
C2H5NO 2 Glycine
O C. dichotoma Saxena VK & Jain S, 1983 OH C. latifolia Dahot MU & NH2 Roxb Noomrio MH, 1999 C6H13 NO 2 Leucine
O O C. dichotoma Saxena VK & Jain S, 1983 HO OH C. sebestina Dhore MM et al ., 2001 NH2
C5H9NO 4 Glutamic acid
O C. dichotoma Saxena VK & Jain S, 1983 OH C. latifolia Dahot MU & NH2 Roxb Noomrio MH, 1999 C3H7NO 2 Alanine
OH O C. dichotoma Saxena VK & Jain S, 1983 OH NH2
C4H9NO 3 Threonine
O C. dichotoma Saxena VK & Jain S, 1983 HO OH O NH2
C4H7NO 4 Aspartic acid
COOH C. dichotoma Saxena VK & Jain S, N 1983 H
C5H9NO 2 Proline
16
H2N N N C. latifolia Dahot MU & Noomrio HN NH N H Roxb MH, 1999 COOH O N O COOH
C19 H19 N7O6 Folic acid
O C. latifolia Dahot MU & Noomrio H N 2 OH Roxb MH, 1999 NH2
C6H14 N2O2 Lysine
AROMATIC COMPOUNDS
OH C. nitida Vahl Pino JA et al ., 2002 O
C7H6O2 Benzoic acid
H HO C. boisieri DOminguez XA et al ., O 1973a C7H6O2 p-Hydroxybenzaldehyde
OH HO C. macleodii El-Sayed NH et al., 1998 O
C7H6O3 p-Hydroxybenzoic acid
O C. nitida Vahl Pino JA et al ., 2002 O OH
C8H8O3 Methyl salicylate
COOH C. rufescens do Vale AE et al ., 2012 HO
H CO 3
C8H8O4 4-Methyl-protocatechuic acid
O C. nitida Vahl Pino JA et al ., 2002 O OH
C9H10 O3 Ethyl salicylate
17
C. macleodii El-Sayed NH HO O et al ., 1998 HO OH
C9H10 O4 p-Hydroxyphenyl lactic acid
HO O C. macleodii El-Sayed NH et al ., 1998 H3CO OH
C10 H10 O4 Ferulic acid
H3CO C. rufescens do Vale AE et al ., H HO 2012 O H3CO
C9H10 O4 Syringaldehyde
H3COCH3 O C. macleodii El-Sayed NH HO HO NH2 et al ., 1998
C11 H15 NO 4 2-Hydroxy-3-(4-hydroxy-3-methoxyphenyl)- 2-methylpropanamide
OCH3 C. alliodora Jean-Robert I
H3CO OCH3 et al ., 2000a
H3CO O
C13 H18 O5 Methyl 3-(2 ′,4 ′,5 ′-trimethoxyphenyl) propanoic acid
C. sebestina Adeosun CO & Samuel SO, 2012 OH
C14 H22 O 2,4-Bis(1,1-dimethylethyl)-phenol
OH C. sebestina Adeosun CO & Samuel SO, 2012
C15 H24 O Butylated hydroxytoluene
18
OH C. alliodora Jean-Robert I et al ., HO 2000a OH
C16 H22 O3 (2 Z) 2-(3-Hydroxy-3,7-dimethylocta-1,6- dienyl)-1,4-benzenediol
O O C. trichotoma de Menezes JESA
O H et al ., 2004 O O
C12 H16 O5 3-(2 ′,4 ′,5 ′-Trimethoxyphenyl)propanoic acid
OH C. latifolia Siddiqui BS et al ., HO O 2006 O C. latifolia Begum S et al ., 2011 C13 H18 O4 Latifolicinin A
OH C. latifolia Siddiqui BS et al ., HO O 2006 O C. latifolia Begum S et al ., 2011 C11 H14 O4 Latifolicinin B
OH C. latifolia Siddiqui BS et al ., HO O 2006 O C. latifolia Begum S et al ., 2011 C10 H12 O4 Latifolicinin C
OH C. latifolia Siddiqui BS et al ., O O H 2006 O C. latifolia Begum S et al ., 2011 C10 H12 O4 Latifolicinin D
C. latifolia Siddiqui BS et al ., O 2010 H3CO
C14 H14 O2 Latifolidin
19
OCH3 C. latifolia Begum S et al ., 2011 O H3CO C. latifolia Siddiqui BS et al ., O 2010 HO
C11 H14 O5 Cordicinol
HO O Cordia species Rita F et al ., 1995 H C. macleodii El-Sayed NH HO O et al ., 1998 C9H8O4 Caffeic acid
HO O C. rufescens do Vale AE et al ., OCH3 2012 HO O
C10 H10 O4 Methyl caffeate
HO COOH Cordia species Rita F et al ., 1995 O C. macleodii El-Sayed NH HO O OH OH et al ., 1998 OH
C16 H18 O9 Chlorogenic acid
OH C. dentata Ferrari F et al ., 1997a OOH OH O C. dichotoma Yan W et al ., 1996 O C. verbenacea Ticli FK et al , 2005 HO OH C. latifolia Begum S et al ., 2011
C18 H16 O8
Rosmarinic acid
OH C. rufescens do Vale AE et al ., O OCH3 OH O 2012 O
HO OH
C19 H18 O8 Methyl rosmarinate
20
OH C. spinescens Aura LY et al ., 1997 OH
2+ 1/2Ca OH OO OH
O O
Ca(C18 H15 O8)2 Calcium rosmarinate
OH C. spinescens Aura LY et al ., 1997 OH
2+ 1/2Mg OH OO OH
O O
Mg(C 18 H15 O8)2 Magnesium rosmarinate
OH C. spinescens Aura LY et al ., 1997 OH OH O
COO Mg2+ OH O O OH
O O
Mg(C 27 H20 O12 ) Magnesium lithospermate
HO OH C. goetzei Marston A et al ., 1988 O Guerke HO OH C. goetzei Hostettmann K, 1990 O O OH HO Guerke
C30 H24 O9 Cordigone
HO OH OH C. goetzei Marston A et al ., 1988 H O Guerke O H C. goetzei Hostettmann K, 1990 O HO OH Guerke
OH
C30 H24 O9 Cordigol
21
OH C. goetzei Marston A et al ., 1988 Guerke O OH HO O OH
OH O HO
C30 H20O9 Calodenin B
OH C. goetzei Marston A et al ., 1988 Guerke O
HO O OH
OH O HO OH
C30 H22 O9 Afzelone C
LIGNAN
O C. rufescens Souza da Silva SA O A. DC. et al ., 2004 HO O O
OH OH
C18 H8O7 Rufescidride
CO2Me C. rufescens do Vale AE et al ., HO 2012 OH HO
O O
C19 H18 O7 Rufescenolide
NEO-LIGNAN
OH H O C. platythyrsa Tapondjou LA OH et al ., 2005 O O H O
C20 H20 O6 Balanophonin
22
FLAVONOIDS OH O C. boissieri Dominguez XA O
HO O et al ., 1973 O
C17 H14 O6 3,4´-Di-O-methyl kaempferol OH O C. verbenaceae Sertie JAA, 1988 H3CO OCH3
OCH3 DC H3CO O
OCH3 C. verbenaceae Sertié JAA et al .,
C20 H20 O8 DC 1990 Artemetin O Cordia species Rita F et al ., 1995 OH
OH HO O
OH OH
C15 H12 O7 Dihydrorobinetin OH O C. macleodii El-Sayed NH OH OH et al ., 1998 HO O OH C. dichotoma Kuppast IJ et al ., C15 H10 O7 Forst. F. 2006 Quercetin OH O C. macleodii El-Sayed NH et al ., OH 1998 HO O OH C. dichotoma Kuppast IJ
C15 H10 O6 Forst. F. et al ., 2006 Kaempferol OH O C. dichotoma Kuppast IJ OH
OCH Forst. F. et al ., 2006 HO O 3 OH
C16 H12 O7 Isorhamnetin C. globosa Souza da Silva SA H3CO O et al ., 2004
O
C16 H12 O3 7-Methoxyflavone
23
OCH3 C. Jose OF et al ., 2007 H3CO O cylindrostachya OCH3 OH O
C18 H16 O6 5-Hydroxy-3,7,4 ′-trimethoxyflavone OH C. globosa Souza da Silva SA HO O OH et al ., 2004 OCH3 OH O
C16 H12 O7 5,7,3 ′,4 ′-Tetrahydroxy-3-methoxyflavone
FLAVANOL GLYCOSIDES
H O OCH3 C. obliqua Chauhan JS & HO
HO O Linn. Srivastava SK, 1977 O OCH3 OH O HO OH
C23 H26 O11 5,7-Dimethoxytaxifolin-3-O-α-L-rhamnopyranoside OH O C. obliqua Chauhan JS et al .,1978 C. obliqua Agnihotri VK O O
OH O O et al ., 1987 HO OH OH C. obliqua Tiwari KP & C22 H24 O10 Srivastava SK, 1979 Hesperetin 7-O-α-L-rhamnopyranoside OH C. obliqua Srivastava SK, 1979 OH
HO O
O OH O O OH OH OH
C21 H22 O11 Taxifolin-3-rhamnoside OH C. obliqua Srivastava SK & OH HO O Srivastava SD, 1979a
O C. obliqua Agnihotri VK et al ., O O O OH 1987 O OH HO OH HO OH
C27 H32 O15 Taxifolin 3,5-dirhamnoside
24
OH C. obliqua Srivastava SK, 1980 OH H HO O
O O OH OH OH O HO
C20 H20 O11
3',4',5,7-Tetrahydroxyflavanone-3-O-D-xylopyranoside
OH HO Cordia species Rita F et al ., 1995 HO O
O OH OH O O O OH HO O O
HO O OH HO OH
C33 H40 O19 Robinin
OH HO Cordia species Rita F et al ., 1995 HO O C. dentata Ferrari F et al ., 1997a O OH O O O OH C. macleodii El-Sayed NH OH HO OH HO O et al ., 1998 OH
C27 H30 O16 Rutin
O HO Cordia species Rita F et al ., 1995
O H HH O HO O OH
O O O OH O
C38 H54 O12 Datiscoside
OH HO Cordia species Rita F et al ., 1995 HO O
O OCH3 O HO OO OH HO OH
OH O
C28 H34 O15 Hesperidin
25
OH HO C. dentata Ferrari F et al ., HO O 1997 O OH OH O O O OH HO OH HO O OH
C27 H30 O16 Quercetin 3-O-rhamnosyl-(1 →6)-galactoside
HO C. macleodii El-Sayed NH O OH OH et al ., 1998 OH O HO O O OH OH O OH HO O O OH
C29 H34 O17
Quercetin 3-O-β-D-glucopyranosyl-(1 →2)-β-D-glucuronate ethyl ester
OH HO C. macleodii El-Sayed NH HO O et al ., 1998 O OH O O OH O OH HO OH HO HO O HO O O OH O O O O OH OH HO HO O OH
C54 H58 O30 3,3"-Hinokiflavanol dirutinoside
OH C. macleodii El-Sayed NH HOO OH et al ., 1998
O OHO O OH OH OH
C21 H20 O11
Quercetin 3-O-α-L-rhamnoside
OH C. macleodii El-Sayed NH HO O OH et al ., 1998 O O OH OH OHO H OH
C21 H20 O11
Kaempferol-3-O-β-D-glucopyranoside
26
OH C. El-Sayed NH HO O macleodii et al ., 1998 O OH O O OH OH OH
C21 H20 O10
Kaempferol 3-O-α-L-rhamnoside
OH OH C. El-Sayed NH HO O HO O O O macleodii et al ., 1998 OH O HO OH O HO OH
C27 H30 O15 Kaempferol 7-O-neohesperidoside
TERPENOID QUINONES O C. millenii Moir M et al ., 1972 C. millenii Moir M & Thomson RH, H O 1973 C16 H18 O2 C. fragrantissima Mori K et al ., 2008 Cordiachrome A O C. millenii Moir M et al ., 1972 C. millenii Moir M & Thomson RH, H O 1973 C16 H18 O2 C. fragrantissima Mori K et al ., 2008 Cordiachrome B O C. millenii Moir M et al ., 1972 C. millenii Moir M & Thomson RH, 1973 H O C. trichotoma Menezes JESA et al ., 2001 C H O 16 18 2 C. fragrantissima Mori K et al ., 2008 Cordiachrome C O C. millenii Moir M et al ., 1972
O C. millenii Moir M & Thomson RH, H O 1973 C17 H20 O3 Cordiachrome D
O H C. millenii Moir M et al ., 1972
O C. millenii Moir M & Thomson RH, O 1973 C17 H20 O3 Cordiachrome E
27
O C. millenii Moir M et al ., 1972
O C. millenii Moir M & Thomson H O RH, 1973 C17 H20 O3 Cordiachrome F
O CH2OH C. trichotoma Menezes JESA et al ., 2001 O O OH
C17 H18 O5 Oncocalyxone A
PLASTOQUINONES
HO C. sebestina Adeosun CO & H O Samuel SO, 2012 3
C29 H50 O2 α-Tocopherol
TERPENOID HYDROQUINONES
OH C. alliodora Stevens KL et al ., 1973 H O C. alliodora Stevens KL & Jurd L, 1976 OH C. elaeagnoides Manners GD, 1983 C H O 16 20 3 C. fragrantissima Mori K et al ., 2008 Alliodorin
HO C. alliodora Manners GD & Jurd L, O 1977 C H O 16 20 2 Cordiachromene A
OH C. alliodora Manners GD & Jurd L, 1977 H OH C. fragrantissima Mori K et al ., 2008 C16 H20 O2 Cordiaquinol C
OH C. fragrantissima Mori K et al ., 2008
O OH O
C16 H16 O4 Cordiaquinol I (racemate)
28
OH C. fragrantissima Mori K et al ., 2008
H OH OH
C16 H22 O3 Cordiaquinol J
OH CHO C. fragrantissima Mori K et al ., 2008
HO
OH
C16 H20 O4 Cordiaquinol K
OH C. alliodora Manners GD & Jurd L, 1977 OH HO
C16 H20 O3 Allioquinol C
OH C. alliodora Manners GD & OH Jurd L, 1977 OH C. elaeagnoides Manners GD, 1983
C16 H22 O3 Alliodorol
HO OH C. alliodora Manners GD & OH Jurd L, 1977 C. elaeagnoides Manners GD, 1983 OH
C16 H22 O4 Cordallinol
OH H H OH C. alliodora Manners GD & Jurd L, 1977 H OH H H
C16 H22 O3 Cordiol A
OH C. elaeagnoides Manners GD, 1983
OH
C16 H22 O2 Geranyl hydroquinone
29
OH C. elaeagnoides Manners GD, 1983 O
O OH
C17 H22 O4 Methyl alliodorate
HO OH C. elaeagnoides Manners GD, 1983 H
O OH
C16 H20 O4 Cordallinal
C. elaeagnoides Manners GD, 1983 O OH OH
OH
C16 H22 O4 Cyclocordallinol
H C. elaeagnoides Manners GD, 1983 O O HO
C16 H18 O3 Elaeagin
H C. elaeagnoides Manners GD, 1983 O O HO
C16 H16 O3 Dehydroelaeagin
MONOTERPENES
C. nitida Vahl Pino JA et al ., 2002
C. curassavica da Camara CAG et al ., 2007 C10 H14 C.leucomalloides Santos RP et al ., 2006 p-Cymene & C. curassavica C. multispicata Zoghbi B et al ., 2010 Cham.
30
C. gilletii Bonesi M et al ., 2011
C10 H16 α-Terpinene C. curassavica Gomez NE et al ., 1999
C10 H16 β-Terpinene C. curassavica Santos RP et al ., 2006
C. globosa (Jacq.) de Menezes JESA C10 H16 γ-Terpinene H.B.K. et al ., 2006
C. globosa (Jacq.) de Menezes JESA HO H.B.K. et al ., 2006 C10 H18 O Terpinen-4-ol C. verbenacea D.C. Meccia G et al ., 2009
C10 H16 α-Terpinolene C. multispicata Cham. Zoghbi B et al ., 2010
OH
C10 H18 O trans -Piperitol
C. multispicata Cham. Zoghbi B et al ., 2010
O O
C12 H20 O2 Isodihydrocarvyl acetate
O C. curassavica da Camara CAG et al ., 2007
C10 H12 O Cuminaldehyde
C. verbenacea Meccia G et al ., 2009 OO
C12 H20 O2 Fenchyl acetate
31
C. globosa da Camara CAG et al ., 2007
O
O
C14 H24 O2 Lavandulyl isobutyrate
O C. globosa da Camara CAG et al ., 2007 O
C14 H24 O2 Linalyl butyrate
C. multispicata Cham. Zoghbi B et al ., 2010
C14 H22 β-Longipinene
C. curassavica Gomez NE et al ., 1999 C. verbenacea D.C. de Carvalho PM et al ., 2004
C. curassavica Santos RP et al ., 2006 C10 H16 C. curassavica da Camara CAG et al ., 2007 α-Pinene C. leucocephala Moric Viana FA et al ., 2008 C.leucomalloides & Santos RP et al ., 2006 C. curassavica C. gilletii Bonesi M et al ., 2011 C. multispicata Cham. Zoghbi B et al ., 2010 C. verbenacea D.C. Meccia G et al ., 2009 C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006
C. curassavica Santos RP et al ., 2006 C. globosa & da Camara CAG et al ., 2007
C. curassavica C10 H16 C. leucocephala Moric Viana FA et al ., 2008 β-Pinene C.leucomalloides & Santos RP et al ., 2006 C. curassavica C. multispicata Cham. Zoghbi B et al ., 2010 C. verbenacea D.C. Meccia G et al ., 2009 C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006
32
C.leucomalloides & Santos RP et al ., 2006 C. curassavica O
C10 H16 O Camphor
C. curassavica da Camara CAG et al ., H HO 2007
C10 H16 O trans -Pinocarveol
OH C. curassavica da Camara CAG et al ., 2007 C10 H18 O cis -p-Menth-2-en-1-ol
HO C. globosa da Camara CAG et al .,
2007 C10 H18 O C. leucocephala Moric Viana FA et al ., 2008 α-Terpineol C. multispicata Cham. Zoghbi B et al ., 2010 C. globosa (Jacq.) de Menezes JESA et al ., H.B.K. 2006
C. curassavica da Camara CAG et al ., 2007 C H 10 16 C. curassavica Santos RP et al ., 2006 α-Phellandrene C. multispicata Cham. Zoghbi B et al ., 2010
C. verbenacea D.C. de Carvalho PM et al ., 2004 C H 10 16 C. curassavica da Camara CAG et al ., β-Phellandrene 2007 C. multispicata Cham. Zoghbi B et al ., 2010
C. verbenacea D.C. de Carvalho PM et al ., 2004 O O
C12 H22 O2 Citronellyl acetate
33
C. curassavica Gomez NE et al ., 1999 C.leucomalloides & Santos RP et al ., 2006
C. curassavica
C10 H16 C. multispicata Cham. Zoghbi B et al ., 2010 Camphene C. verbenacea D.C. Meccia G et al ., 2009
C. verbenacea D.C. Meccia G et al ., 2009
C10 H16 Tricyclene
C. nitida Vahl Pino JA et al ., 2002
HO
C10 H18 O Geraniol
C. curassavica Gomez NE et al ., 1999 C. nitida Vahl Pino JA et al ., 2002 C H 10 16 C. globosa da Camara CAG et al ., 2007 Myrcene C. leucocephala Moric Viana FA et al ., 2008 C. curassavica Santos RP et al ., 2006 C. multispicata Cham. Zoghbi B et al ., 2010 C. verbenacea D.C. Meccia G et al ., 2009 C. globosa (Jacq.) de Menezes JESA et al ., H.B.K. 2006
HO C. globosa da Camara CAG et al ., 2007 C H O 10 18 C. curassavica Santos RP et al ., 2006 Linalool C. multispicata Cham. Zoghbi B et al ., 2010 C. globosa (Jacq.) de Menezes JESA et al ., H.B.K. 2006
O C. sebestina Adeosun CO & Samuel SO,
2012 C13 H26 O 2-Tridecanone
34
O C. globosa da Camara CAG et al ., 2007
C10 H14 O (E)-Ocimenone
C. curassavica Gomez NE et al ., 1999 O C. leucocephala Moric Viana FA et al ., 2008 C10 H18 O C. verbenacea D.C. Meccia G et al ., 2009 1,8-Cineole
C. curassavica Gomez NE et al ., 1999 C. curassavica da Camara CAG et al ., 2007 C H 10 16 C. leucocephala Moric Viana FA et al ., 2008 Sabinene C. leucomalloides & Santos RP et al ., 2006 C. curassavica C. multispicata Cham. Zoghbi B et al ., 2010 C. verbenacea D.C. Meccia G et al ., 2009 C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006
C. curassavica Gomez NE et al ., 1999
C. nitida Vahl Pino JA et al ., 2002 C H 10 16 C. curassavica Santos RP et al ., 2006 Limonene C. verbenacea D.C. Meccia G et al ., 2009 C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006
C. curassavica da Camara CAG et al ., 2007
OH
C10 H14 O Carvacrol
C. nitida Vahl Pino JA et al ., 2002
OH
C10 H16 O trans -Carveol
C. curassavica Gomez NE et al ., 1999 C. leucocephala Moric Viana FA et al ., 2008
C. multispicata Cham. Zoghbi B et al ., 2010 C10 H16 trans -β-Ocimene C. verbenacea D.C. Meccia G et al ., 2009 C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006
35
C. globosa & da Camara CAG et al ., 2007
OH C. curassavica H C. leucomalloides & Santos RP et al ., 2006 C10 H18 O C. curassavica Borneol C. curassavica Gomez NE et al ., 1999 C. globosa & da Camara CAG et al ., 2007 C. curassavica O O
C12 H20 O2 C. verbenacea D.C. Meccia G et al ., 2009
Bornyl acetate
SESQUITERPENES
C. globosa da Camara CAG et al ., HO 2007
C15 H26 O (Z)-Nerolidol OH C. globosa da Camara CAG et al .,
2007 C15 H26 O C. multispicata Cham. Zoghbi B et al ., 2010 (E)-Nerolidol C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006
C. multispicata Cham. Zoghbi B et al ., 2010
C15 H24 (E,E)- α-Farnesene C. gilletii Bonesi M et al ., 2011 OH
C15 H26 O Farnesol O C. sebestina Adeosun CO & Samuel
4 SO, 2012
C17 H34 O 2-Heptadecanone
O C. gilletii Bonesi M et al ., 2011
C18 H36 O 6,10,14-Trimethyl-pentadecan-2-one
36
O C. sebestina Adeosun CO & Samuel
4 SO, 2012
C19 H38 O 2-Nonadecanone
C. curassavica da Camara CAG et al ., 2007
C15 H22 trans -Calamenene
C. curassavica Hernandez T et al ., 2007
C. globosa da Camara CAG et al ., 2007 C15 H22 cis -Calamenene
C. globosa (Jacq.) H.B.K. de Menezes JESA et al., 2006
C15 H22 trans -Cadina-1(2), 4-diene
C. trichotoma de Menezes JESA et al ., Vell 2005 O
C15 H20 O Calamenene-1,11-epoxide
O C. curassavica Hernandez T et al ., 2007
C15 H24 O Hexahydro-2,5,5-trimethyl-2H-2,4a- ethanonaphthalen-8(5 H)-one
C. globosa da Camara CAG et al ., 2007
C15 H24 cis -Thujopsene
37
C. curassavica da Camara CAG et al ., HO 2007
C15 H26 O Thujopsan-2α-ol C. globosa & da Camara CAG et al ., C. curassavica 2007
C15 H24 Valencene
H C. leucomalloides Santos RP et al ., 2006
H
C15 H24 α-Amorphene C. trichotoma de Menezes JESA Vell et al ., 2005 C. curassavica da Camara CAG et al ., C H 15 20 2007 α-Calacorene C. multispicata Cham. Zoghbi B et al ., 2010
C15 H24 Cyclosativene C. multispicata Cham. Zoghbi B et al ., 2010
C15 H24 α-Ylangene C. multispicata Cham. Zoghbi B et al ., 2010
C15 H24 α-Selinene C. curassavica Hernandez T et al ., 2007 C. curassavica da Camara CAG et al ., 2007 H C. multispicata Cham. Zoghbi B et al ., 2010 C15 H24 β-Selinene C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006
38
C. curassavica Gomez NE et al ., 1999
H
C15 H24 γ-Selinene C. curassavica Gomez NE et al ., 1999
C15 H24 δ-Selinene H C. globosa da Camara CAG et al .,
OH 2007 H H
C15 H24 O 1-Endo-bourbonanol H C. leucocephala Moric Viana FA et al ., 2008 C. leucomalloides & Santos RP et al ., 2006 HH C. curassavica
C. multispicata Cham. Zoghbi B et al ., 2010 C15 H24 β-Bourbonene C. verbenacea D.C. Meccia G et al ., 2009 C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006 C. globosa & da Camara CAG et al ., 2007 C. curassavica
OH H C. curassavica da Camara CAG et al ., H 2007 C. leucomalloides Santos RP et al ., 2006
C15 H26 O Cubebol C. curassavica da Camara CAG et al ., 2007
OH
C15 H26 O epi -Cubebol C. curassavica Hernandez T et al ., 2007 C. globosa & da Camara CAG et al ., 2007 H H C. curassavica
C15 H24 C. multispicata Cham. Zoghbi B et al ., 2010 α-Cubebene C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006
39
C. multispicata Cham. Zoghbi B et al ., 2010 C. verbenacea D.C. Meccia G et al ., 2009 C. curassavica da Camara CAG et al .,
C15 H24 2007 β-Cubebene
C. multispicata Cham. Zoghbi B et al ., 2010
OH
C15 H26 O allo-Hedycaryol
C. trichotoma Margaret K et al ., 1964
H OH
C15 H26 O α-Eudesmol
C. trichotoma Margaret K et al ., 1964 C. curassavica Hernandez T et al ., 2007 H OH
C15 H26 O β-Eudesmol
C. trichotoma Margaret K et al ., 1964
H OH
C15 H26 O γ-Eudesmol
H C. trichotoma Margaret K et al ., 1964
H H OH
C15 H26 O Guaiol
H C. trichotoma Vell de Menezes JESA et al ., 2005 C.leucomalloides Santos RP et al ., 2006 H C. curassavica da Camara CAG et al ., 2007 OH C. multispicata Cham. Zoghbi B et al ., 2010 C15 H26 O C. globosa (Jacq.) de Menezes JESA et al ., 2006 Globulol H.B.K.
40
C. verbenacea D.C. Meccia G et al ., 2009
C15 H24 Italicene
C. trichotoma de Menezes JESA et al ., Vell 2005
O
C15 H24 O Italicene epoxide
C. curassavica da Camara CAG et al ., 2007
C15 H24 α-Bulnesene
C. chacoensis Ramaldo J & Rasendo Y, 1968 H C. verbenaceae D.C. de Carvalho et al ., 2004 C. leucomalloids Santos RP et al ., 2006 H & C.curassavica
C15 H24 C. globosa (Jacq.) de Menezes JESA et al ., 2006 Caryophyllene H.B.K. C. verbenacea Fernandes ES et al ., 2007 C. verbenacea Durate MCT et al ., 2007 C. verbenacea Medeiros R et al ., 2007 C. globosa & de Menezes JESA et al ., 2005 C. curassavica C. globosa & da Camara CAG et al ., 2007 C. curassavica C.leucocephala Moric Viana FA et al ., 2008 C.leucomalloides & Santos RP et al ., 2006 C. curassavica C. gilletii Bonesi M et al ., 2011 C. multispicata Cham. Zoghbi B et al ., 2010 C. verbenacea D.C. Meccia G et al ., 2009
41
C. multispicata Cham. Zoghbi B et al ., 2010 H C. globosa (Jacq.) de Menezes JESA et al ., H.B.K. 2006 H
C15 H24 9-epi -β-Caryophyllene C. multispicata Cham. Zoghbi B et al ., 2010
OH
C15 H24 Caryophylla-4(14),8(15)-dien-5β-ol C. globosa da Camara CAG et al ., 2007
C15 H24 α-Santalene C. curassavica Hernandez T et al .,
HO 2007
C15 H24 O α-Santalol
C. curassavica da Camara CAG et al ., 2007
C15 H24 α-Alaskene C. multispicata Cham. Zoghbi B et al ., 2010
C15 H24 cis -β-Guaiene C. curassavica da Camara CAG et al ., 2007
C15 H24 trans -β-Guaiene
42
C. globosa & da Camara CAG et al ., C. curassavica 2007
C15 H22 ar -Curcumene C. chacoensis Ramaldo J & Rosendo Y, 1968 C. verbenaceae D.C. de Carvalho et al ., 2004 C. curassavica Fernandes ES et al ., 2007 C15 H24 C. globosa da Camara CAG et al ., 2007 α-Humulene C. leucocephala Moric Viana FA et al ., 2008 C. leucomalloides & Santos RP et al ., 2006 C. curassavica C. multispicata Cham. Zoghbi B et al ., 2010 C. verbenacea D.C. Meccia G et al ., 2009 C. globosa (Jacq.) H.B.K. de Menezes et al ., 2006 C. verbenacea D.C. Meccia G et al ., 2009
C15 H24 γ-Curcumene C. globosa & da Camara CAG et al ., C. curassavica 2007
C15 H18 Cadalene
C. curassavica Hernandez T et al .,
OH 2007
C15 H26 O 4-Methyl, 4-ethenyl-3-(1-methyl ethenyl)-1-(1-methyl methanol)-cyclohexane
H OH C. trichotoma de Menezes JESA et al ., 2001 H OH
C15 H26 O2
Trichotomol
43
C. curassavica Gomez NE et al ., 1999
OH
C15 H26 O Elemol
H OH C. trichotoma Menezes JESA et al ., 2001 C. trichotoma Vell de Menezes JESA et al ., 2005 H C. globosa & da Camara CAG et al ., 2007
C15 H26 O C. curassavica α-Cadinol C. multispicata Cham. Zoghbi B et al ., 2010
H OH C. curassavica Gomez NE et al ., 1999
H
C15 H26 O δ-Cadinol
OH H C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006 H
C15 H26 O τ-Cadinol OH C. trichotoma de Menezes JESA et al ., 2004 HO H OH
C15 H28 O3 (+)-1β,4 β,6 α-Trihydroxyeudesmane OH C. trichotoma de Menezes JESA et al ., 2004 HO H OH
C15 H28 O3 (−)-1β,4 β,7 α-Trihydroxyeudesmane
OH C. trichotoma de Menezes JESA et al ., 2004 HO H OH
C15 H28 O3 (+)-1β,4 β,11 α-Trihydroxyoppositane
44
C. globosa & da Camara CAG et al ., C. curassavica 2007 H C. multispicata Cham. Zoghbi B et al ., 2010
C15 H24 C. globosa (Jacq.) de Menezes JESA Aromadendrene H.B.K. et al ., 2006 C. curassavica da Camara CAG et al ., 2007
C15H24 Dehydroaromadendrene
H C. verbenacea D.C. de Carvalho PM et al ., 2004
H C. leucocephala Moric Viana FA et al ., 2008 C. multispicata Cham. Zoghbi B et al ., 2010 C15 H24 C. globosa (Jacq.) de Menezes JESA et al ., Alloaromadendrene H.B.K. 2006 C. globosa & da Camara CAG et al .,
H C. curassavica 2007
C15 H24 Seychellene
CH3 C. globosa da Camara CAG et al ., CH3 2007
H3C CH3 O
C15 H22 O Vulgarone B C.leucomalloides & Santos RP et al ., 2006 H C. curassavica H C. curassavica da Camara CAG et al ., OH 2007 C15 H26 O Viridiflorol C. multispicata Cham. Zoghbi B et al ., 2010 C. curassavica da Camara CAG et al ., H 2007 H OH
C15 H26 O Ledol
45
HO H C. leucomalloides Santos RP et al ., 2006
C15 H26 O α-Bisabolol C. verbenacea D.C. Meccia G et al ., 2009
C15 H24 trans -γ-Bisabolene
H OH C. trichotoma de Menezes JESA Vell et al ., 2005 H C. globosa & da Camara CAG et al .,
C15 H26 O C. curassavica 2007 α-Muurolol C. multispicata Cham. Zoghbi B et al ., 2010
OH C. trichotoma de Menezes JESA Vell et al ., 2005 H C. curassavica da Camara CAG et al .,
C15 H26 O 2007 1,10-di-epi -Cubenol
OH C. globosa da Camara CAG et al ., 2007
C. leucocephala Moric Viana FA et al ., 2008
C15 H26 O cis -Sesquisabinene hydrate OH C. globosa & da Camara CAG et al ., C. curassavica 2007
C15 H26 O trans -Sesquisabinene hydrate HO C. trichotoma Vell de Menezes JESA et al ., 2005
C15 H26 O epi -α-Muurolol
46
H C. trichotoma de Menezes JESA Vell et al ., 2005 H C. multipicata Cham. Zoghbi B et al ., 2010
C15 H24 C. verbenacea D.C. Meccia G et al ., 2009 α-Muurolene
H C. trichotoma de Menezes JESA Vell et al ., 2005 H C. multipicata Cham. Zoghbi B et al ., 2010
C15 H24 γ-Muurolene
H C. trichotoma de Menezes JESA Vell et al ., 2005 H C. curassavica da Camara CAG et al ., 2007
C15 H24 C. multispicata Cham Zoghbi B et al ., 2010 α-Cadinene
H C. trichotoma Vell de Menezes JESA et al ., 2005 C. curassavica da Camara CAG et al ., 2007 H C. multispicata Cham. Zoghbi B et al ., 2010 C15 H24 C. verbenacea D.C. Meccia G et al ., 2009 γ-Cadinene C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006 C. verbenaceae D.C. de Carvalho PM et al ., 2004 C. trichotoma Vell de Menezes JESA et al ., 2005 H C. leucomalloides Santos RP et al ., 2006
C15 H24 C. leucocephala Moric Viana FA et al ., 2008 δ-Cadinene C.leucomalloides Santos RP et al ., 2006 C. multispicata Cham. Zoghbi B et al ., 2010 C. globosa (Jacq.) H.B.K de Menezes JESA et al ., 2006 C. trichotoma Vell de Menezes JESA OH O et al ., 2005
C15 H24 O2 Occidenol C. trichotoma de Menezes JESA Vell et al ., 2005
OH
C15 H24 O Guaia-3,10(14)-dien-11-ol
47
HO C. verbenaceae D.C. de Carvalho PM et al ., 2004 C. curassavica Hernandez T et al ., 2007 C. leucocephala Moric Viana FA et al ., 2008 C. leucomalloides & Santos RP et al ., 2006 C15 H24 O C. curassavica Spathulenol C. globosa & da Camara CAG et al ., 2007 C. curassavica C. multispicata Cham. Zoghbi B et al ., 2010 C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006
C. verbenaceae D.C. de Carvalho PM et al ., 2004 C. leucomalloids & Santos RP et al ., 2006
C. curassavica C15 H24 C. globosa de Menezes JESA et al ., 2006 Bicyclogermacrene C. globosa & da Camara CAG et al ., 2007 C. curassavica C. leucocephala Moric Viana FA et al ., 2008 C.leucomalloides & Santos RP et al ., 2006 C. curassavica C. multispicata Cham. Zoghbi B et al ., 2010 C. verbenacea D.C. Meccia G et al ., 2009 C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006
C. curassavica Hernandez T et al ., 2007 H
H
C15 H24 Isocaryophyllene
H H C. verbenaceae D.C. de Carvalho PM et al ., 2004 O C. globosa & da Camara CAG et al ., 2007 H C. curassavica C15 H24 O C. leucocephala Moric Viana FA et al ., 2008 Caryophyllene oxide C. leucomalloides & Santos RP et al ., 2006 C. curassavica C. multispicata Cham. Zoghbi B et al ., 2010 C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006
48
C. globosa da Camara CAG et al ., 2007 C. leucocephala Moric Viana FA et al ., 2008 C. leucomalloides Santos RP et al ., 2006
C. multispicata Cham. Zoghbi B et al ., 2010 C15 H24 C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006 Germacrene B
H C. globosa da Camara CAG et al ., 2007
C15 H24 β-Sesquiphellandrene
C. leucomalloids Santos RP et al ., 2006 C. globosa da Camara CAG et al ., 2007 C. leucocephala Moric Viana FA et al ., 2008
C. leucomalloides & Santos RP et al ., 2006 C15 H24 C. curassavica Germacrene D C. multispicata Cham. Zoghbi B et al ., 2010 C. verbenacea D.C. Meccia G et al ., 2009 C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006
C. curassavica Gomez NE et al ., 1999
OH
C15 H26 O Germacrene-1,6-dien-5-ol
C. leucomalloides Santos RP et al ., 2006
H
C15 H24 Cadina-1,4-diene
H C. curassavica Hernandez T et al ., 2007
H
C15 H24 Cadina-4(15),10(14)-diene
49
C. leucomalloides & Santos RP et al ., 2006 C. curassavica
C15 H24 Longifolene
C. verbenacea D.C. de Carvalho PM et al ., 2004 C. globosa da Camara CAG et al ., 2007
C. leucocephala Moric Viana FA et al ., 2008 C15 H24 C. curassavica Santos RP et al ., 2006 β-Elemene C. multispicata Cham. Zoghbi B et al ., 2010 C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006
C. curassavica Gomez NE et al ., 1999 C. globosa & da Camara CAG et al ., 2007 C. curassavica C H 15 24 C. multispicata Cham. Zoghbi B et al ., 2010 δ-Elemene C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006
C. curassavica Gomez NE et al ., 1999 C. leucomalloides Santos RP et al ., 2006
C. multispicata Cham. Zoghbi B et al ., 2010 C15 H24 γ-Elemene
H C. globosa da Camara CAG et al ., 2007
H
C15 H24 cis-α-Bergamotene
C. curassavica Gomez NE et al ., 1999 C. verbenacea D.C. Meccia G et al ., 2009 C. globosa (Jacq.) H.B.K. de Menezes JESA et al ., 2006
C15 H24 α-Amorphene
C. multispicata Cham. Zoghbi B et al ., 2010 C. curassavica Santos RP et al ., 2006
C15 H24 trans -α-Bergamotene
50
C. curassavica da Camara CAG et al ., 2007 C. multispicata Cham. Zoghbi B et al ., 2010
H
C15 H24 α-Gurjunene
C. verbenacea D.C. de Carvalho PM et al ., 2004
C. multispicata Cham. Zoghbi B et al ., 2010 C15 H24 β-Gurjunene
C. curassavica Gomez NE et al ., 1999 C. globosa & da Camara CAG C. curassavica et al ., 2007
C15 H24 C. leucocephala Moric Viana FA et al ., 2008 α-Copaene C. leucomalloides & Santos RP et al ., 2006 C. curassavica C. multispicata Cham. Zoghbi B et al ., 2010 C. verbenacea D.C. Meccia G et al ., 2009
C. curassavica Gomez NE et al ., 1999 C. verbenacea D.C. Meccia G et al ., 2009
C15 H24 β-Copaene
C. curassavica da Camara CAG et al ., HO 2007
C15 H24O β-Copaen-4α-ol
C. curassavica Gomez NE et al ., 1999
C15 H24 6,9-Guajadiene
51
MEROSESQUITERPENOID QUINONES
O C. corymbosa Bieber LW et al ., 1990 OH C. curassavica Jean-Robert I et al ., 2000
O
C21 H26 O3 Cordiaquinone A
O O C. corymbosa Bieber LW et al ., 1990 C. linnaei Jean-Robert I et al ., 1998 O C. curassavica Jean-Robert I et al ., 2000 C21 H24 O3 Cordiaquinone B O C. corymbosa Bieber LW et al ., 1994 O O C. linnaei Jean-Robert I et al ., 1999
O
C26 H30 O4 Cordiaquinone C
O O C. corymbosa Bieber LW et al ., 1994 O 22
O
C47 H76 O4 Cordiaquinone D
O O C. linnaei Jean-Robert I et al ., 1998
O
C21 H24 O3 Cordiaquinone E O C. linnaei Jean-Robert I et al ., 1998 O O
O
O
C26 H30 O5 Cordiaquinone F O OH C. linnaei Jean-Robert I et al ., 1998
OH O
C21 H26 O4 Cordiaquinone G
52
O C. linnaei Jean-Robert I et al ., 1998 OH
OH O
C21H26O4 Cordiaquinone H
O C. linnaei Jean-Robert I et al ., 1999 O O
O
C26H30 O4 Cordiaquinone H (revised)
O O C. curassavica Jean-Robert I et al ., 2000
O
C21 H24 O3 Cordiaquinone J
O O C. curassavica Jean-Robert I et al ., 2000
O
C21 H22 O3 Cordiaquinone K
O C. leucocephala Diniz JC et al ., 2009 OH
O
C21 H24 O3 Cordiaquinone L
O OH C. leucocephala Diniz JC et al ., 2009
O
C21 H24 O3 Cordiaquinone M
O C. globifera Dettrakul S et al ., 2009
O
C16 H18 O2 Globiferin
53
OH O C. globosa de Menezes JESA et al ., OCH3 O 2005
O H H O
C27 H20 O6 Microphyllaquinone
O H H H C. globosa de Menezes JESA H3CO H H et al ., 2005 H CO 3 H C. globosa Viera NC et al ., 2008 O H H H
C18 H22 O4
(1a S*,1b S*,7a S*,8a S*)-4,5-Dimethoxy-1a,7a-dimethyl-1,1a,1b,2,7,7a,8,8a-octahydrocyclo- propa-[3,4]cyclopenta[1,2 b]naphthalene-3,6-dione
DITERPENES
C. latifolia Siddiqui BS et al ., HOOC 2006 C. latifolia Begum S et al ., 2011 H COOH
C20 H26 O4 Cordioic acid
C. verbenacea D.C. Meccia G et al ., 2009
C20 H32 Pimaradiene
C. latifolia Siddiqui BS et al ., 2006 HOOC C. latifolia Begum S et al ., 2011 H
C20H28 O2 Cordifolic acid
C. leucocephala Viana FA et al ., 2008 OH Moric C20 H40 O C. gilletii Bonesi M et al ., 2011 Phytol
54
TRITERPENES
C. sebestina Adeosun CO,
Samuel SO, 2012
C30 H50 Squalene
C. verbenacea DC Vande VV et al ., O O 1982 H H H C. multispicata Masanori K et al ., HO H H 2003
O H HO H
C30 H46 O5 Cordialin A
C. verbenacea DC Vande VV et al ., OH OH 1982 H H H HO H H
O H HO H
C30 H50 O5 Cordialin B
H H C. latifolia Begum S et al ., 2011 H O H COOH
H H COOH
C30 H44 O5 Cordinoic acid
C17H35 C. latifolia Begum S et al ., 2011 O O OH OCOOH HO O HO
C36 H50 O9 Cordicilin
55
C. alliodora Chen TK et al ., 1983 C. cylindrostachya Jose OF et al ., 2007 H
H H COOH
HO H
C30 H48 O3 3α-Hydroxy-olean-12-en-27-oic acid
C. alliodora Chen TK et al ., 1983
H
H COOH O H
C30 H46 O3 3-Oxo-olean-12-en-27-oic acid
H C. alliodora Chen TK et al ., 1983 O
H
H COOH O H
C30 H44O4 3,29-Dioxo-olean-12-en-27-oic acid
CHO C. alliodora Chen TK et al ., 1983
H
H H COOH HO H
C30 H46 O4 3α-Hydroxy-29-oxo-olean-12-en-27-oic acid
CH2OH C. alliodora Chen TK et al ., 1983
H
H H COOH HO H
C30 H48 O4 3α-29-Dihydroxy-olean-12-en-27-oic acid
56
COOH C. alliodora Chen TK et al ., 1983
H
H H COOH HO H
C30H46 O5 3α-Hydroxy-olean-12-en-27,29-dioic acid C. obliqua Agnihotri VK et al ., 1987 H C. sebestina Adeosun CO & H Samuel SO, 2012 HO H
C30 H50 O α-Amyrin C. obliqua Agnihotri VK et al .,
H 1987 OH H
H OH H
C30 H50 O2 Betulin HO C. spinescens Nakamura N et al ., 1997 O H
H HO H OH
C30 H52 O4 (20 S,24 S)-3α,6 β,25-Trihydroxy-20,24-epoxy-dammarane HO C. spinescens Nakamura N et al ., 1997 O H
O H O H OH
C32 H54 O5 (20 S,24 S)-3α-Acetoxy-6β,25-dihydroxy-20,24-epoxy-dammarane
57
OH C. spinescens Nakamura N et al ., O 1997 H
H HO H H
C30 H52O3 Cabraleadiol
C. multispicata Kuroyamagi M O O et al ., 1999 OH CHO C. multispicata Kuroyamagi M O H HO et al ., 2001 H
C32 H48 O6 Cordiaketal A
C. multispicata Kuroyamagi M OH et al ., 2001 OH
CH2O O O H HO H
C32 H50 O6 Cordiaketal B
C. multispicata Kuroyamagi M OH et al ., 2001 OH
CH2O O H O H OH
C32 H50 O6 Cordianone
C. multispicata Kuroyamagi M O O et al ., 2001 OH CHO H O H
C32 H48 O5 Cordianal A
58
OH C. multispicata Kuroyamagi M
OH et al ., 2001 CHO H O H
C30 H46 O4 Cordianal B
C. multispicata Kuroyamagi M OH et al ., 2001 OH CHO H H HO H
C30 H48 O4 Cordianal C
C. trichotoma Menezes JESA et al ., 2001 H COOH
H HO H
C30 H48 O3 Oleanolic acid
OH OH C. multispicata Kuroyamagi M et al ., 2003 H HO
O H HO H
C30 H48 O5 Cordianol B
OH HO C. multispicata Kuroyamagi M et al ., 2003 H OCH3 HO
O H HO H
C31 H52 O6 Cordianol C
59
HO O C. multispicata Kuroyamagi M
H et al ., 2003 HO
O H HO H
C30 H48 O5 Cordianol D
OH O C. multispicata Kuroyamagi M
H et al ., 2003 HO OHC
H O H
C30 H46 O5 Cordianol E
HO O C. multispicata Kuroyamagi M O H et al ., 2003 O
O H HO H
C32 H50 O6 Cordianol F
OH O C. multispicata Kuroyamagi M
H et al ., 2003 HO
H O H
C30H48 O4 Cordianol G
OH OH C. multispicata Kuroyamagi M et al ., 2003 H HO
H O H
C30 H48 O4 Cordianol H
60
OH OH C. multispicata Kuroyamagi M et al ., 2003 H OH HO H3CO
O H H
C31 H52 O6 Cordianol I
H H C. piauhiensis dos Santos RP et al ., 2005 H COOH
H COOH HO H
C30 H46 O5 Quinovic acid
H C. piauhiensis dos Santos RP H
H COOH et al ., 2005 H COOH HO H
C30 H46 O5 Cincholic acid
TRITERPENE GLYCOSIDES (including SAPONINS)
C. obliqua Agnihotri VK
H et al ., 1987 H C. obliqua Srivastava SK H et al ., 1983 HO O O H HO OH
C36 H60 O5
Lupa-20(29)-ene-3-O-α-L-rhamnoside
C. obliqua Chauhan JS &
H OH Srivastava SK, HO O H HO OH 1978 OHO O H HO OH O H
C42 H70 O11
Lupa-20(29)-ene-3-O-β-D-maltoside
61
H C. piauhiensis dos Santos RP H et al ., 2005 H COOH
O H HO COOH HO O HO H
C36 H56 O9
Cincholic acid 3 β-O-6-deoxy-β-D-glucopyranoside H C. piauhiensis dos Santos RP H et al ., 2005 H COOH HO O H COOH HO HO O OH H
C36 H56 O10 Quinovic acid 3β-O-β-D-glucopyranoside
OH C. piauhiensis O O OH O OH O OHHO H OH HO O Santos RP HO H O HO O H HO et al ., 2003 O
HO O HO OH C54 H88 O22
3β-O-[α-L-Rhamnopyranosyl-(1 →2)-β-D-glucopyranosyl]-ursolic acid 28-O-[β-D-glucopyranosyl-
(1 →6)-β-D-glucopyranosyl] ester
OH OH C. piauhiensis O O OH OH HO O O OH HO H OH Santos RP O HO HO O H HO O et al ., 2005 O H HO O HO OH C54 H88 O23
3β-O-[α-L-Rhamnopyranosyl-(1 →2)-β-D-glucopyranosyl]-pomolic acid 28-O-[β-D-glucopyranosyl-
(1 →6)-β-D-glucopyranosyl] ester
OH H C. piauhiensis O O OH OH HO O O OH HO H OH O O Santos RP HO O H O HO O HO O H et al ., 2005
HO O OH HO OH OH C59 H96 O26
3β-O-[α-L-Rhamnopyranosyl-(1 →2)-β-D-glucopyranosyl]-Oleanolic acid 28-O-[β-D-xylopyranosyl-
(1 →2)-β-D-glucopyranosyl-(1 →6)-β-D-glucopyranosyl] ester
62
OH C. piauhiensis dos Santos RP et al ., 2005 O H HO OH O H HO HO O O H
HO O HO OH
C42 H68 O13
3β-O-α-L-Rhamnopyranosyl-(1 →2)-β-D-glucopyranosyl pomolic acid
STEROIDS
OH C. curassavica Hernandez T et al ., H 2007 HH HO H
C20 H32 O2 1-Methyl-,(3 β,5 α,17 β)-androst-1-ene-3,17-diol
C. obliqua Agnihotri VK et al ., H 1987
OH H C. trichotoma Menezes JESA et al ., O HO H H 2001 HO OH O C. rufescens do Vale AE et al ., 2012
C35 H60 O6 β-Sitosterol-3β-Ο-β-D- glucopyranoside
C. platythyrsa Atchade T et al ., 2012 H C. rufescens do Vale AE et al ., 2012 H
H H HO
C29 H48 O Stigmasterol
C. sebestina Adeosun CO & Samuel SO, 2012 O
HO
C26 H42 O2 26-Nor -5-cholestene-β-ol-25-one
63
C. myxa Tiwari RD et al ., 1967 H C. boisieri Dominguez XA et al ., 1973a
H C. obliqua Tiwari KP & Srivastava SK, 1979 HH C. obliqua Srivastava SK et al ., 1977 HO C. obliqua Agnihotri VK et al ., 1987 C29 H50 O C. myxa & Miralles J et al ., 1989 β-Sitostrerol C. sebestina C. trichotoma Menezes JESA et al .,2001 C. platythyrsa Tapondjou LA et al .,2005 C. rufescens do Vale AE et al ., 2012
C2H5 C. platythyrsa Tapondjou LA et al ., 2005
H
HH O HO H
C29 H48 O2 Procesterol
C. sebestina Adeosun CO & H Samuel SO, 2012
H
HH O
C29 H48 O2 Stigmast-4-en-3-one
ALKALOIDS
OH C. latifolia Roxb Dahot MU & O Noomrio MH, 1999 N
C6H5NO 2 Niacin
PYRROLIZIDINE ALKALOIDS
O C. myxa Wassel G et al ., 1987
O H
OH N
C13 H21 NO 3 Macrophylline
64
CARBOHYDRATES
OH C. myxa Kassem AA et al ., 1969 O HO OH C. myxa Ifzal SM & Qureshi A, 1976 HO OH C. myxa Bhatty MK et al ., 1978 C H O 6 12 6 Cordia species Reicher F et al ., 1978 D-Glucopyranose C. myxa Karawya MS et al ., 1980 C. dichotoma Saxena VK & Jain S, 1983 C. dichotoma Forst Basu NG et al ., 1984 C. dichotoma Forst Basu NG et al ., 1986 C. latifolia Roxb Dahot MU & Noomrio MH, 1999 C. abyssinica Benhura MAN & Chidewe C, 2002
HOH2C C. myxa Kassem AA et al ., 1969 O HO OH HO C. myxa Ifzal SM & Qureshi A,1976 OH C. myxa Karawya MS et al ., 1980 C6H12 O6 C. latifolia Roxb Dahot MU & Noomrio MH, 1999 D-Fructopyranose C. sebestina Dhore MM et al ., 2001
OH O C. myxa Kassem AA et al ., 1969 HO O HO C. myxa Ifzal SM & Qureshi A, 1976 OH OH C. myxa Karawya MS et al ., 1980
C6H10 O7
D-Galacturonic acid
HO O C. myxa Ifzal SM & Qureshi A,1976 HO OH OH C. myxa Bhatty MK et al ., 1978 C5H10 O5 C. myxa Karawya MS et al ., 1980 D-Xylopyranose C. dichotoma Saxena VK & Jain S, 1983 C. abyssinica Benhura MAN & Chidewe C, 2002
OH C. myxa Ifzal SM & Qureshi A,1976 O OH C. dichotoma Saxena VK & Jain S, 1983 HO C. dichotoma Forst Basu NG et al ., 1986 OH C. abyssinica Benhura MAN & Chidewe C, C5H10 O5
D-Arabinopyranose 2002
65
OH C. dichotoma Forst Basu NG et al ., 1984 O HO OH OH
C5H10 O5
L-Arabinopyranose
OH OH C. myxa Bhatty MK et al ., 1978 O HO OH C. myxa Karawya MS et al ., 1980 OH C. abyssinica Benhura MAN & Chidewe C, C6H12 O6 2002 D-Galactopyranose
OH C. myxa Bhatty MK et al ., 1978 OH O C. abyssinica Benhura MAN & Chidewe C, HO OH HO 2002 C6H12 O6
D-Mannopyranose
OH C. myxa Bhatty MK et al ., 1978 O HO OH HO HN
O
C8H15 NO 6 N-Acetylglusamine
HOOC C. myxa Karawya MS et al ., 1980 HO O HO OH C. dichotoma Saxena VK & Jain S, 1983 OH
C6H10 O7 D-Glucuronic acid
O C. dichotoma Saxena VK & Jain S, 1983 HO OH OH C. latifolia Roxb Dahot MU & Noomrio MH, OH et al ., 1999 C5H10 O5 D-Ribopyranose
OH C. dichotoma Saxena VK & Jain S, 1983 HO O C. abyssinica Benhura MAN & Chidewe HO OH C, 2002
C6H12 O5
L-Rhamnopyranose
66
OH OH OH C. dichotoma Saxena VK & Jain S, 1983 O O HO O OH HO OH OH
C12 H22 O11 Lactose
HOH2C C. boissieri A. D.C. Alanis-Guzman MG O HO HO HO et al ., 1998 OH O O C. trichotoma Menezes JESA et al ., CH2OH OH CH2OH 2001
C12 H22 O11 Sucrose
OH OH C. piauhiensis dos Santos RP OH HO et al ., 2005 OH OH
C6H14 O6 D-Mannitol
MEGASTIGMANE
O C. nitida Vahl Pino JA et al ., 2002
C13 H18 O (E)-β-Damascenone
O H C. globosa da Camara CAG et al ., 2007
C13 H20 O (E)-α-Ionone
MISCELLANEOUS
OH C. boissieri Dominguez XA et al ., HO O 1973a HO OH OH
C7H14 O6 D-(+)-Pinitol
67
O C. boissieri Dominguez XA et al ., 1973a NH HN C. obliqua Tiwari KP & Srivastava SD, 1979 O N O C. ecalyculata Vell Lucia SM et al ., 1985 H NH2 C. collococca L. Vargas L & Alberto J, 1982 C4H6N4O3 C. micrantha Swartz Menezes JESA et al ., 2001 Allantoin C. trichotoma & Tapondjou LA et al ., 2005 C. platythyrsa
O C. nitida Vahl Pino JA et al ., 2002
C9H14 O Isophorone
O OH C. latifolia Begum S et al ., 2011 H O
C11 H16 O3 Cordinol
HO H OH C. latifolia Roxb Dahot MU & O O Noomrio MH, 1999
HO OH
C6H8O6 Ascorbic acid
C2H5O C. nitida Vahl Pino JA et al ., 2002 Ethanol
C. nitida Vahl Pino JA et al ., 2002 HO
C5H12 O Isoamyl alcohol
OH C. sebestina Adeosun CO & Samuel SO, 2012 OH
C5H12 O 3,7-Decadiene-5,6-diol
HO C. nitida Vahl Pino JA et al ., 2002
C6H12 O (2E)-Hex-2-en-1-ol
68
C. nitida Vahl Pino JA et al ., 2002 HO
C6H12 O (3Z)- Hex-3-en-1-ol
C. nitida Vahl Pino JA et al ., 2002 HO
C9H16 O (2E,6Z)-Nona-2,6-dien-1-ol
OH C. nitida Vahl Pino JA et al ., 2002
C6H14 O Hexanol
OH C. globosa da Camara CAG et al ., 10 2007 C18 H38 O Octadecanol
OH C. obliqua Agnihotri VK et al ., 1987 20
C28 H58 O Octacosanol
OH C. obliqua Agnihotri VK et al ., 1987 23
C31 H64 O Hentriacontanol
O C. nitida Vahl Pino JA et al ., 2002
C2H4O Ethanal
O C. nitida Vahl Pino JA et al ., 2002
C6H12 O Hexanal
O C. gilletii Bonesi M et al ., 2011
C. multispicata Cham. Zoghbi B et al ., 2010 C9H18 O Nonanal C. verbenacea D.C. Meccia G et al ., 2009
O C. nitida Vahl Pino JA et al ., 2002 6 C. gilletii Bonesi M et al ., 2011 C14 H28 O Tetradecanal
69
O C. nitida Vahl Pino JA et al ., 2002
C. gilletii Bonesi M et al ., 2011 C6H10 O (E)-2-Hexenal C. verbenacea D.C. Meccia G et al ., 2009
OH C. nitida Vahl Pino JA et al ., 2002
O
C4H8O2 Acetoin
C. sebestina Adeosun CO & O O Samuel SO, 2012
C7H12 O2 3,5-Heptanedione
C. sebestina Adeosun CO & Samuel SO, 2012 O O
C8H14 O2 3-n-Propyl-2,4-pentanedione
C. sebestina Adeosun CO & HO N Samuel SO, 2012 C6H13 NO 1-Ethyl-3-pyrrolidinol
C. nitida Vahl Pino JA et al ., 2002 O
C9H14 O 2-Pentylfuran
C. nitida Vahl Pino JA et al ., 2002 O O
C10 H18 O2 γ-Decalactone
C. latifolia Siddiqui BS et al ., 2010 H O O 12 O O
C24 H40 O4 Latifolinal
70
H C. nitida Vahl Pino JA et al ., 2002 O O
C5H4O2 Furfural
C. nitida Vahl Pino JA et al ., 2002
C8H10 Ethyl benzene
C. nitida Vahl Pino JA et al ., 2002
C8H10 p-Xylene
OH C. nitida Vahl Pino JA et al ., 2002
C7H8O Benzyl alcohol
OH C. nitida Vahl Pino JA et al ., 2002
C8H10 O 2-Phenylethanol
O C. curassavica da Camara CAG et al ., 2007 C7H6O Benzaldehyde
O C. multispicata Cham. Zoghbi B et al ., 2010
O O O
C12 H14 O4 Dillapiole
C. sebestina Adeosun CO & Samuel SO, O 2012 C9H14 O Cryptone
O C. sebestina Adeosun CO & Samuel SO, O O 2012
C8H12 O3 1,4-Dioxaspiro[4,5]decan-8-one
71
C. sebestina Adeosun CO & Samuel SO, 2012 O O
C21 H40 O2 4,8,12,16-Tetramethylheptadecan-4-olide
O C. globosa da Camara CAG et al ., 2007
C13 H20 O Myrac aldehyde
C. nitida Vahl Pino JA et al ., 2002 C. curassavica da Camara CAG et al ., O 2007 C8H8O Phenylacetaldehyde
O O C. curassavica da Camara CAG et al ., 2007
C10 H12 O2 Ethyl phenylacetate
C. sebestina Adeosun CO & Samuel SO, 2012 O O O O
C12 H14 O4 Diethyl phthalate
C. gilletii Bonesi M et al ., 2011 OO
HO
C14 H12 O3 2-Hydroxy-benzoic acid phenylmethyl ester
72
Table-1.2: Phytochemicals Isolated and Identified from Cordia rothii :
FATTY ACIDS AND FATTY ALCOHOL
OH OH 6 6 4 O O
C14 H28 O2 C18 H34 O2 Myristic acid Oleic acid (Daulatabad CMJD et al ., 1992) (Miralles J et al ., 1989 & Daulatabad CMJD et al ., 1992) OH 8 O OH 4 6 O C16 H32 O2 Palmitic acid C18 H32 O2 (Miralles J et al ., 1989 & Linoleic acid Daulatabad CMJD et al ., 1992) (Miralles J et al ., 1989 & Daulatabad CMJD et al ., 1992) OH 10 O OH 6 O C18 H36 O2 Stearic acid C18 H30 O2 (Daulatabad CMJD et al ., 1992) α-Linolenic acid (Miralles J et al ., 1989)
O OH HO O
OH C18 H32 O2 Malvalic acid (Daulatabad CMJD et al ., 1992)
C18 H34 O3 O Ricinoleic acid OH (Daulatabad CMJD et al ., 1992) C19 H34 O2
Sterculic acid 19 OH (Daulatabad CMJD et al ., 1992)
C26 H54 O n-Hexacosanol (Mukat B & Chhaya G, 1980)
73
PHENOLICS, PHENYL PROPANOIDS AND FLAVANOL GLYCOSIDES
COOH O
OH HO OH HO OH C7H6O4 Protocatechuic acid C9H8O4 (Al-Musayeib N et al ., 2011) trans -Caffeic acid (Al-Musayeib N et al ., 2011) OH OOH OH O OH O OCH3 OH O O O HO OH HO OH C18 H16 O8 C H O Rosmarinic acid 19 18 8 Methyl rosmarinate (Al-Musayeib N et al ., 2011) (Al-Musayeib N et al ., 2011)
OH OCH HO O OH 3
O HO O O O OH O OH OH OH O OH OH OH O H O OH OH OH O C21 H20 O11 H OH
Kaempferol-3-O-β-D-glucopyranoside C28 H32 O15 (Al-Musayeib N et al ., 2011) Kaempferide-3-O-α-L-rhamnopyranosyl (1 →6)-β-D- glucopyranoside OCH3 (Al-Musayeib N et al ., 2011) HO O OH O O OH OH OH OH O OH H HO O O O OH C H O OH OH 22 22 11 O O OH OH Kaempferide-3-O-β-D-glucopyranoside OH O H OH (Al-Musayeib N et al ., 2011) C27 H30 O15
Kaempferol-3-O-α-L-rhamnopyranosyl (1 →6)-β-D- OH OH glucopyranoside
HO O OH (Al-Musayeib N et al ., 2011)
O O OH OH OH O H OH
C21 H20 O12 Quercetin-3-O-β-D-glucopyranoside (Al-Musayeib N et al ., 2011)
74
TERPENOID QUINONES
O O O
H H H O O O
C16 H18 O2 C16 H18 O2 C16 H18 O2 Cordiachrome A Cordiachrome B Cordiachrome C (Moir M & (Moir M & (Moir M & Thomson RH, 1973) Thomson RH, 1973) Thomson RH, 1973)
TRITERPENES PYRROLIZIDINE ALKALOIDS
N
O O H O O H HO HO OH O H O O C30 H50 O
β-Amyrin C21 H31 NO 9 (Verma YS et al ., 1978) Floridanine (Wassel G et al ., 1987)
STEROIDS
H H H H H H HH HO HO C29 H48 O C29 H50 O Stigmasterol β-Sitostrerol (Desai HK et al ., 1976) (Desai HK et al ., 1976 & 1977, Verma YS et al ., 1978, Mukat B & Chhaya G, 1980, Miralles J et al ., 1989,)
75
1.5 Identification of metabolites from Genus Cordia exploiting GC/GC-MS: Genus Cordia has also been analysed for the identification of metabolites using GC and GC- MS. Such type of work has also been done on Cordia rothii , but the results were found limited to only n-hydrocarbons and few fatty acids or their derivatives. Table-1.3 below summarizes the work done on identification of metabolites using GC and GC-MS.
Table-1.3: Phytochemical Analyses on Genus Cordia using GC-MS technique:
Year Species Reference
2012 Cordia sebestina Adeosun O & Samuel SO
2011 Cordia gilletii Bonesi M et al .,
2010 Cordia multispicata Cham Zoghbi B et al .,
2009 Cordia verbenacea D.C. Meccia G et al .,
2008 Cordia leucocephala Moric Diniz JC et al .,
2007 Cordia globosa (Jacq.) Hmb., Bonpl. et Kunth da Camara CAG et al ., Cordia curassavica (Jacq.) Roem. et Schult.
2007 Cordia curassavica (Jacq) Roemer & Schultes Hernandez T et al .,
2006 Cordia leucomalloides Santos RP et al ., Cordia curassavica
2006 Cordia globosa (Jacq.) H.B.K. de Menezes JESA et al .,
2005 Cordia trichotoma Vell de Menezes JESA et al .,
2004 Cordia verbenacea D.C. de Carvalho PM et al .,
2002 Cordia nitida Vahl Pino JA et al .,
1995 Cordia boissieri (anacahuita) Alanis-Guzman MG et al .,
1992 Cordia rothii Daulatabad CMJD et al .,
1990 Cordia cylindrostachya Roem. and Schult Fun CE & Svendsen AB
1980 Cordia rothii Mukat B & Chhaya G
76
2.1 Biosyntheses:
In order to live, grow and reproduce, there is a continuous series of chemical transformations occurring within the living bodies. These are mostly regulated by sets of enzymes, interlinked with each other, and thereby producing different selected classes of natural products. These pathways not only supply energy to the plants in the form of ATP but also help in generating the building blocks for the chemical constituents present in the plants. Biosynthetic pathways explain the structural diversity and relationships amongst naturally occurring compounds, and suggest how a complex natural product has been assembled from simpler fragments. Most organisms share basic, similar pathways with or without variations for the biosynthesis of carbohydrates, proteins, fats, nucleic acids, etc. These universal, basic biosynthetic pathways constitute primary metabolism, and the type of compounds originating and participating in these pathways are called primary metabolites. On the other hand, the type of metabolism dealing with the compounds (secondary metabolites) having selected distribution in various forms of life is referred to as secondary metabolism. Particular types of secondary metabolites attribute distinguishing characters to various species, organisms or group of organisms. Primary metabolism is the major source of building blocks for secondary metabolites. Figure- 2.1 shows some of the intermediates involved in the biosynthesis of various secondary metabolites, isolated from Cordia rothii during the current investigation. These include acetyl coenzyme A (acetyl CoA), shikimic acid, mevalonic acid and methylerythritol phosphate, utilized in the acetate, shikimate, mevalonate and methylerythritol phosphate pathways, respectively.
Acetyl-CoA is one of the building blocks utilized in the biosynthesis of secondary metabolites. It is the thioester of acetic acid and is produced by the oxidative decarboxylation of pyruvic acid; a product of glycolytic pathway. Another source of acetyl-CoA is the β-oxidation of fatty acids. Phenols, prostaglandins, macrolide antibiotics, fatty acids, and their derivatives synthesized via acetate pathway are the major secondary metabolites acting as a bridge between various primary-secondary metabolisms (Dewick PM, 2009).
In the present investigation several secondary metabolites have been isolated and purified from root, stem and leaves of the plant. The study has also resulted in the identification of various fatty acids and their derivatives along with many other secondary metabolites through gas chromatography – mass spectrometric studies (GC-MS). The biosynthetic pathway with reference to these metabolites is presented here.
77
Photosynthesis Glycolysis OOH OH OH O hv O CO OP 2 HO HO OH HO OH OH OH
D-Glucose Phosphoenolpyruvate Shikimic acid
Lignin Phenylpropanoids
CoAS O Syringaresinol mono- Latifolicinin C(62) β -D-glucoside (82) Acetyl-CoA (2R)(p-Hydroxyphenyl) lactic acid (81)
Syringin (85) Mairajinol (30) O O O HO HO OH HO SCoA The shikimate pathway Malonyl CoA Mevalonic acid
Fatty acids
Terpenes Steroids 1-Octacosanol (74) β-Sitosterol (26) 1-O-β -D-Glucopyranosyl-(2S,3S,4R,8Z) -2-[(2'R)-2'-hydroxytetracosanoyl- Roseoside (83) amino]-1,3,4-octadecanetriol-8-ene (80) β -Sitosterol glucoside (79)
The acetate pathway Staphylionoside D (84)
Stigmasterol (27)
The mevalonate and methylerythritol phosphate pathways
Figure-2.1: Nature's Biosynthetic Diversity Observed in Current Study on Cordia rothii.
2.2 Biosynthesis of Saturated Fatty Acids:
Fatty acid biosynthesis is referred to as primary metabolic pathway due to its existence in all plant cells. It plays a vital role in cell division, growth and development (Rogalski M and
78
Carrer H, 2011). Plastids, chloroplasts and in some cases, mitochondria are the sites of fatty acid biosynthesis in plants (Sánchez-García A et al ., 2010).
Claisen reactions involved in the biosynthesis of fatty acids (figure-2.2) include a series of reactions for the conversion of acetyl-CoA into malonyl-CoA, catalyzed by acetyl-CoA carboxylase (a 3-component enzyme), can be summarized as follows:
- i) Formation of mixed anhydride using ATP, CO 2 (in the form of HCO 3 ) and coenzyme
biotin (as CO 2 carrier).
O O _ P ATP + HCO3 ADP + biotin carboxylase HO O OH OH mixed anhydride ii) Carboxylation of coenzyme by the nucleophilic attack of cyclic urea on mixed anhydride.
O O O P O HN NH N HO O OH H H biotin carboxylase OH HO S Co-Enz mixed anhydride biotin-enzyme N'-carboxybiotin-enzyme
iii) Nucleophilic attack of acetyl-CoA (enolate anion) on the carbonyl of N′-carboxybiotin- enzyme followed by the loss of biotin-enzyme and formation of malonyl-CoA.
O N O O carboxyltransferase CoAS OH CoAS OH O malonyl-CoA + biotin-enzyme
The conversion of acetyl-CoA into malonyl-CoA increases the acidity of α-hydrogen atoms, thereby furnishing a better nucleophile for the Claisen condensation.
In next few steps (figure-2.2) acetyl-CoA and malonyl-CoA are converted into enzyme-bound thioesters utilizing acyl carrier protein (ACP). This is followed by condensation of malonyl- ACP and acyl-enzyme thioester in the presence of ketosynthase to produce β-ketoacyl-ACP. Stereospecific reduction of β-ketoacyl-ACP into β-hydroxy ester in the presence of NADPH and ketoreductase, and dehydration of β-hydroxy ester in the presence of dehydratase,
79
O O Enz-SH SCoA RH C SEnz acetyl-CoA- 2 acetyl-CoA ACP-transacylase acyl-enzyme thioester Claisen OO condensation RH2C SACP Ketosynthase β-ketoacyl-ACP OO OO ACP-SH HO SCoA HO SACP
malonyl-CoA- e
s
malonyl-CoA malonyl-ACP a ACP-transacylase H t
P c
u
D
d
A
e
r
h
N
t o
t
g
o
e
t
n
e K
e
l
l
c
n
i
y
a
c
h
n
c
i
o
d
j
n
y e
t
a x enoyl O
m e O reductase O dehydration H OH E RH C SACP RH2C SACP RH2C SACP dehydratase 2 NADPH R saturated acyl-ACP α,β-unsaturated β-hydroxyacyl-ACP acyl-ACP
O
RH2C SCoA A o C fatty acyl-CoA S H
th i o H e s 2 O te r a O s e reductase RH2C OH RH2C OH fatty acid alcohol
Figure-2.2: Biosynthesis of Saturated Fatty acids. produces ( E)-α,β-unsaturated ester. Hydrogenation of this ( E)-α,β-unsaturated ester then produces saturated acyl-ACP. The saturated acyl-ACP may combine with malonyl-ACP, further undergoes dehydration and reduction processes, resulting in the elongation of chain length by two carbon atoms and the repetition of the process results in the required chain length. On the other hand it may also release fatty acyl-CoA (a precursor of the free fatty acid). The free fatty acid may undergo reduction to produce the corresponding alcohol (Dewick PM, 2009).
2.3 Biosynthesis of Unsaturated Fatty Acids:
Biosynthesis of unsaturated fatty acids can be achieved through various pathways (Dewick PM, 2009). Enzymes, classified as fatty acid desaturases, facilitate the generation of double bond into the hydrocarbon chain of fatty acid (Chi X et al ., 2011). In most cases the
80 corresponding alkanoic acid is desaturated using oxygen-dependent ∆9-desaturase with NADPH or NADH cofactor, resulting in the introduction of cis double bond at ∆9 position of saturated fatty acid, which appears at other positions due to the course of chain-lengthening or
shortening, for instance, stearoyl (C 18 ) coenzyme A thioester in animals and fungi, whereas
stearoyl (C 18 ) ACP thioester in plants, is converted into oleoyl derivative (figure-2.3).
R = ACP in plants R = CoA in animals, fungi H O H SR
stearic 18:0 9 stearoyl-ACP -desaturase O2 stearoyl-CoA 9-desaturase NADPH O 9 10 SR
oleic 18:1(9c)
Figure-2.3: Generation of Double Bond into the Hydrocarbon Chain.
Further introduction of double bond varies from organism to organism. For example, in case of plant metabolism, the introduction of new double bond is favoured between the methyl terminus and the already existing double bond (figure-2.4) whereas animal metabolism favours the introduction of double bond between the carboxyl terminus and the already existing double bond (figure-2.5) (Dewick PM, 2009).
6 favoured desaturation is at methyl terminus CO-SACP CO-SACP 12 -desaturase 12 9 12 oleic linoleic 18:1 (9c) 18:2 (9c, 12c)
favoured desaturation 15 -desaturase is at methyl terminus
CO-SACP
9 12 15 α -linolenic Figure-2.4: Desaturation in Plant Metabolism. 18:3 (9c, 12c, 15c)
81
favoured desaturation 6 is at carboxyl terminus 6 CO-SACP CO-SACP 6 -desaturase 9 12 9 12 linoleic γ-linoleic 18:2 (9c, 12c) 18:3 (6c, 9c, 12c)
C18 elongase + C2 (malonate)
favoured desaturation 8 5 8 CO-SCoA is at carboxyl terminus 5 CO-SCoA 5 -desaturase 11 14 11 14 arachidonic dihomo-γ-linolenic 20:4 (5c, 8c, 11c, 14c) 20:3 (8c, 11c, 14c)
Figure-2.5: Desaturation in Animal Metabolism.
2.4 Biosynthesis of Cerebrosides:
Cerebroside is one of the members of glycosphingolipids. It is a ceramide having one or more oxygen-linked sugar moiety (Murphy RC and Axelsen PH, 2011), and constitutes various forms of tissues and organs in different forms of life. The saccharide may be a hexose while ceramide consists of a sphingoid base. This sphingoid base consists of a long-chain aminoalcohol attached with a long-chain fatty acid by means of amide linkage. The biosynthesis of cerebroside is initiated with the formation of relevant ceramide and later on the saccharide moiety gets attached with it (Tan RX and Chen JH, 2003).
First step in cerebroside biosynthesis is the condensation of palmitoyl-CoA with L-serine under the catalysis of serine palmitoyltransferase to generate 3-ketosphinganine. 3-Ketospinganine is then reduced by 3-ketosphinganine reductase to produce sphinganine. Acylation of amino moiety of sphinganine in the presence of ceramide synthase afford ceramide.
Ceramide may undergo certain modifications by the process of desaturations or/and hydroxylations resulting in the formation of various compounds, for instance, ( E)-sphing-4- enine and 4-hydroxysphinganine (phytosphinganine) are the two possible common products. The two products may be desaturated to furnish ∆8-cis and trans -isomers. Another important modification of ceramide is the glycosylation of its primary hydroxyl group yielding cerebroside utilizing glucosylceramide synthase (figure-2.6) (Sperling P and Heinz E, 2003).
82
O H OOC CH2OH H3C(H2C)10H2C + SCoA NH3 Palmitoyl-CoA L-Serine serine palmitoyltransferase O
OH
3-ketosphinganine NH2
3-ketosphinganine reductase OH
OH
sphinganine NH2
ceramide synthase Fattyacyl-CoA
O NH
OHOH ceramide
O NH
OHOH UDP-glucose: ceramide β-D-glucosyltransferase
O OHOH NH O O HO OH OH Cerebroside
Figure-2.6: Possible Cerebroside Biosynthesis in Plants.
2.5 Biosynthesis of Terpenes:
Majority of the natural products are isoprenes (terpenes) or their derivatives i.e., isoprenoids (terpenoids) (Lohr M et al ., 2012). These are derived from isopentenyl diphosphate (IPP) or its allylic isomer, dimethylallyl diphosphate (DMAPP). IPP and DMAPP consist of a C 5 unit, called isoprene. Different short-chain terpenoid precursors, for instance, geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranyl geranyl diphosphate (GGPP), are obtained by the sequential condensation of IPP and/or DMAPP. Cyclization and modification of these
83 precursors using terpene cyclases and oxidases, respectively, yield monoterpenes (C 10 ), sesquiterpenes (C 15 ), diterpenes (C 20 ), (figure-2.7) (Okada K, 2011).
Mevalonic acid M ethylerythritol phospha te
O PP O PP isopentenyl PP dimethylallyl PP (IP P ) (C 5) (D M A P P ) (C 5)
Monoterpenes (C ) 10 C 10 Iridoids IPP
Sesquiterpenes (C 15) C 15 IPP x 2
Diterpenes (C ) 20 C 20 IPP
Sesterterpenes (C ) 25 C 25
Triterpenoids (C ) x 2 30 C 30
S teroids (C 18-C 30)
Tetraterpenes (C 40) C 40 Carotenoids
Figure-2.7: Flow Chart Showing Basic Constitutional Units of Terpenes.
The IPP and DMAPP can be derived from mevalonic acid (MVA) or methylerythritol phosphate (MEP). MVA and MEP are used as the two intermediates in mevalonic acid and methylerythritol phosphate pathways, respectively.
Mevalonic acid is formed by the combination of three molecules of acetyl-CoA. The carboxylic acid group is then decarboxylated resulting in the leftover five carbon atoms constituting isoprene unit.
SCoA OH 3 x HOOC OH O acetyl-CoA mevalonic acid isoprene unit (C5)
Another precursor of the isoprene unit is methylerythritol phosphate, which originates from the skeletal rearrangement of straight-chain sugar derivative, deoxyxylulose phosphate.
84
OH OH
O P O P OOH OHOH
deoxyxylulosephosphate methylerythritol phosphate isoprene unit (C 5)
The isoprene units are generally connected with one another through a head-to-tail linkage as in the case of geraniol (C 10 ), farnesol (C 15 ) and geranylgeraniol (C 20 ).
OH OH
geraniol (C10) farnesol (C15)
OH
geranylgeraniol (C20)
But this is not the case always; these may also join together in a tail to tail manner. Examples are squalene (C 30 ) and phytoene (C 40 ) (Dewick PM, 2009).
squalene (C30)
phytoene (C40)
The Mevalonate Pathway:
Mevalonate (MVA) pathway begins with the Claisen condensation of two molecules of acetyl-CoA to produce acetoacetyl-CoA (figure-2.8). Third molecule of acetyl-CoA adds stereospecifically producing 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). Acetyl-CoA molecules are bonded with enzyme through a sulfide bridge. The HMG-CoA produced is finally converted into MVA in a two-step reduction; i.e., thioester to aldehyde (through hemithioacetal) and aldehyde to primary alcohol.
85
acetyl-CoA EnzSH HMG-CoA synthase Claisen O EnzSH O condensation O O O + SCoA SCoA acetoacetyl- SEnz acetoacetyl- SCoA + SEnz CoA synthase O CoA synthase acetyl-CoA enzyme-bound acetoacetyl-CoA acetyl-CoA reaction Stereospecific acetyl group synthase HMG-CoA
HO O HO HO O O OH O reduction to aldehyde NADPH O OH OH HO SCoA HMG-CoA reductase CoAS H HMG-CoA reductase mevaldic acid mevaldic acid hemithioacetal 3-hydroxy-3-methylglutaryl-CoA M-o reductase HMG-CoA rdcint alcohol) to (reduction (HMG-CoA) +
NADPH EnzSH
Phosphorylation reactions O OH ATP-CO 2 OPP OPP HO OH mevalonate 5-diphosphate IPP isomerase decarboxylase mevalonic acid (MVA) isopentenyl PP dimethylallyl PP (IPP) (DMAPP)
Figure-2.8: Generation of IPP and DMAPP via MVA Pathway.
A series of phosphorylation reactions convert MVA (C 6) into mevalonic acid diphosphate which upon decarboxylation-dehydration provides IPP (C 5; isoprene unit). This IPP isomerizes into another isoprene unit, DMAPP (Dewick PM, 2009).
Mevalonate Independent Pathway:
Labeling studies have shown that IPP and DMAPP can also be formed through an alternative pathway utilizing MEP, also known as deoxyxylulose phosphate pathway. It is also assumed that nature favours MEP pathway more, as compared to MVA pathway (figure-2.9). The MEP pathway is mostly observed in plants, algae and many bacteria. The specific sites are chloroplasts.
MEP is produced from pyruvic acid and glyceraldehydes 3-phosphate; both are intermediates of glycolytic pathway. In this pathway, pyruvic acid is decarboxylated via thiamine diphosphate-mediated decarboxylation process resulting in the formation of acetaldehyde- equivalent enamine bound product. The enamine undergoes nucleophilic addition reaction
with glyceraldehyde 3-phosphate and produces 1-deoxy-D-xylulose-5-phosphate 86
H H 3COH O O H C R 1 DXP synthase 3 OH NS + DXP synthase O P O 2 OH H 3 CR pyruvic acid + TPP/pyruvate-derived D -Glyceraldehyde 3-phosphate Thiamine diphosphate e n a m ine (TPP)
O OH OH H C H C 3 O P 3 OH O P O P I sp C OH O OH O O OH H O P OH
1 -d e o xy -D -x y lu los e O
5-phosphate H
P
C
D p
s
+ A I
N TPP O O OH OH P P CTP O O P O O R Is pD HO OH OH OH OH OH NH 2 4- (C D P )- 2- C - m e th y l-D -erythritol MEP N
R = O N O
P
E
p
T
s
A HOOH I
O O OH P O O OH O P O HO O O -CMP P I sp G O OH P P I sp F H OH O O O R OH OH OH OH OH 2-phospho-4-(CDP)-2- C -methyl-D-erythritol 2 -C -methyl-D-erythritol-2,4-cyclophosphate
O PP isopentenyl PP (IPP) O PP Isp H OH IPP isomerase 4-hydroxy-3-m ethyl- but-2-enyl diphosphate O PP dimethylallyl PP (DMAPP)
Figure-2.9: Generation of IPP and DMAPP via MEP Pathway.
(deoxyxylulose phosphate). This deoxyxylulose phosphate is rearranged involving reverse aldol-aldol reaction followed by reduction to produce MEP (C 5 isoprene unit). MEP reacts with cytidine triphosphate (CTP), generating cytidine diphospho-derivative. This derivative
upon phosphorylation produces 2-phospho-4-(CDP)-2-C-methyl-D-erythritol which releases cytidine phosphate to form cyclic phosphoanhydride. Reductive processes lead to IPP and DMAPP through 4-hydroxy-3-methyl-but-2-enyl diphosphate.
87
Terpenes having (C 5)n carbon skeleton are grouped into; Hemiterpenes (C 5), Monoterpenes
(C 10 ), Sesquiterpenes (C 15 ), Diterpenes (C 20 ), Sesterterpenes (C 25 ), Triterpenes (C 30 ),
Tetraterpenes (C 40 ), etc., (figure 2.7).
In the biosynthesis of hemiterpenes, isopentenyl PP (IPP) and dimethylallyl PP (DMAPP) are reactive intermediates of MVA and MEP pathway which upon coupling produces complex terpenoid structures (figure-2.7). To produce a monoterpene diphosphate, geranyl PP, DMAPP and IPP are coupled together in the presence of enzyme geranyl diphosphate synthase. The mechanism is initiated with the ionization of DMAPP to the allylic cation, which is added to the IPP double bond with the stereospecific loss of proton (figure-2.10).
OPP OPP H Hs Dimethylallyl PP geranyl diphosphate allylic cation R synthase DMAPP IPP
OPP OPP HR Hs geranyl diphosphate synthase geranyl PP (GPP)
E OPP
Figure-2.10: Biosynthesis of Monoterpene.
Simple addition of IPP to the growing chain is not an acceptable idea to prove biosynthesis of triterpenes. Thus squalene, a triterpene, is produced by the tail-to-tail combination of two molecules of farnesyl diphosphate (FPP). Farnesyl diphosphate synthase facilitates the formation of FPP by the addition of IPP to GPP. The C-2 double bond of one of the FPP molecule attacks the farnesyl allylic cation, which in turn was formed from another molecule of FPP. The resulting tertiary cation loses a proton stereospecifically to form a cyclopropane ring (presqualene PP). Squalene is formed by the NADPH reduction of rearranged presqualene PP. Only one enzyme, squalene synthase, catalyzes all these reactions (figure-2.11). 88
OPP Squalene synthase Farnesyl PP (FPP)
PPO 3 2 Allylic cation Farnesyl PP (FPP)
HH OPP
Tertiary cation
H OPP NADPH
sq u alen e H sy n th ase Presqualene PP
H H
S qu alen e
Figure-2.11: Biosynthesis of Triterpene.
Squalene undergoes a series of reactions including, epoxidation, cyclization, migration of hydride and methyl groups through concerted Wagner-Meerwein rearrangements to form protosteryl cation. The initial reaction is catalyzed by squalene epoxidase in the presence of a flavoprotein, O 2, and NADPH as co-factors. In plants, prosteryl cation, in the presence of cycloartenol synthase, produces cycloartenol with the loss of protons at C-10 methyl. Desmethylated forms of triterpenoids having tetracyclic ring system of lanosterol are characterized as steroids. In photosynthetic organisms, cycloartenol serves as steroid precursor while lanosterol is the precursor in non-photosynthetic organisms. Plant origin
major sterols contain a C 1 or C 2 substituents at C-24 as compared to animal origin cholesterol
(C 27 ). The most common plant sterols campesterol and sitosterol are 24-methyl and 24-ethyl derivatives of cholesterol respectively (figure-2.12).
S-adenosylmethionine (SAM) facilitates C-methylation of cycloartenol in the presence of sterol C-24-methyl transferase, providing a tertiary carbocation at C-25. This carbocation undergoes Wagner-Meerwein 1,2-hydride shift, followed by the loss of proton from C-24 yielding 24-methylene side-chain. The 24-methylene undergoes further methylation in the
89
squalene epoxidase H H cyclizations squalene H O2, FAD, NADPH H HO O H
(3S)-2,3-oxidosqualene protosteryl cation (squalene oxide)
cycloartenol synthase
Ad H Me S H R 26 24 25 27 sterol C-24 methyl- HO transferase H cycloartenol Ad Me S H R 24' 24-methylenesterol sterol C-24 methyl- C-methyltransferase H transferase
H
24-methylenesterol NADPH C-methyltransferase
fucosterol sitosterol
2 4 s e t s e a r t o H l c r P u e D d d e u A r c N l t o a r s e e t 4 s 2
Figure-2.12: Biosynthesis of Cycloartenol.
90 presence of SAM, giving rise to another carbocation (figure-2.12). This carbocation, after losing proton, forms 24-ethylidene side-chain, which on reduction produces sitosterol. Alternatively, allylic isomerization followed by reduction also leads to sitosterol (Dewik PM 2009).
2.6 Glycosylation Reactions:
Glycosylation reactions involve attachment of sugar moieties to aglycones or to an already existing sugar moiety in the molecules to produce a di- or polysaccharide. Sugar unit may get attached to aglycone via O-, S-, N- or C-linkages. Nucleoside diphosphosugars, for instance, uridine diphosphoglucose (UDP glucose) facilitates the process of glycosylation. These types of reactions may be referred to as a simple nucleophilic displacement reaction of S N2 type (Dewick PM, 2009).
The Shikimate Pathway:
The biosynthetic approach, dealing with the metabolism of aromatic amino acids and phenylpropanoids is referred to as shikimate pathway. The pathway is observed in plants and microorganisms only because aromatic amino acids are essential amino acids, present in plants and microorganisms but not in animals. One of the important intermediates in this pathway is the Shikimic acid.
L-Phenylalanine and L-tyrosine; shikimate-derived aromatic amino acids, provide basic phenylpropane (C 6C3) units, constituting many natural products, including flavonoids, coumarins, cinnamic acids and lignans. A broad range of alkaloids and simple benzoic acid derivatives are also reported to form through shikimate pathway.
C C O 6 2
OH R NH R 2
C 6C 3 R = H , L -phenylalanine R = O H , L -tyrosine R = CH 2CH 3
C 6C 1
Phosphoenolpyruvate (PEP) and D-erythrose-4-phosphate, metabolites of glycolytic and pentose phosphate cycle, respectively, reacts to form 3-deoxy-D-arabino -heptulosonic acid 7-phosphate (DAHP). Removal of phosphoric acid from DAHP and later on an intramolecular
91 aldol-type reaction produces a carbocyclic intermediate 3-dehydroquinic acid. 3-Dehydroquinic acid undergoes dehydration under the catalysis of 3-dehydroquinase to give 3-dehydroshikimic acid, which on reduction yields Shikimic acid (figure-2.13).
O
OH O OH Glycolytic pathway P O O PEP PO PO H H DAHP HO OH Pentose phosphate HO O synthase cycle OH OH H DAHP
D-erythrose 4-Phosphate
3-dehydroquinate NAD+ synthase
O OH OOH HO COOH dehydration O H 3-dehydro- O OH O OH quinase OH OH HOOH OH 3-dehydroshikimic acid 3-dehydroquinic acid
NADPH shikimate dehydrogenase
OOH
HO OH OH
Shikimic acid
Figure-2.13: Biosynthesis of Shikimic Acid.
ATP-based phosphorylation reaction of shikimic acid produces shikimic acid-3-phosphate. Nucleophilic attack of hydroxyl oxygen present at C-5 of shikimic acid 3-phosphate on PEP gives an addition-elimination product 5-enolpyruvylshikimic acid-3-phosphate (EPSP).
92
1,4-elimination of EPSP produces chorismic acid (figure-2.14). Chorismic acid is a very important branch point compound of Shikimate pathway.
H OOH OOH OH PO ATP O EPSP Shikimate synthase HO OH Kinase PO OH PEP OH OH Shikimicacid Shikimicacid3-phosphate
O OH OOH O OH H -POH HH -POH FMNH2 chorismate OH synthase OH PO O COOH PO O O OH OP OH O OH O EPSP Chorismic acid
Figure-2.14: Biosynthesis of Chorismic Acid
Claisen rearrangement converts chorismic acid into prephenic acid. During the transformation, phosphoenolpyruvate-derived side chain of chorismic acid gets attached
directly to the carbocyclic ring and hence providing the fundamental C 6C3 phenylpropanoid carbon skeleton (figure-2.15).
O OH O OH O O OH HO O OH OH O
Chorismic acid Chorismic acid (pseudoequatorial conformer)
O O O OH HO O Claisen rearrangement O HO O OH chorismate mutase OH OH Chorismic acid (pseudoaxial conf ormer) Prephenic acid
Figure-2.15: Biosynthesis of Prephenic Acid.
93
Biosynthesis of L-phenylalanine and L-tyrosine from prephenic acid differs from organism to organism. Decarboxylative aromatization, transamination, and an additional oxidation reaction (in case of tyrosine), are involved. However, the different orders of these reactions define their routes. Normally prephenic acid undergoes decarboxylative aromatization to
produce phenylpyruvic acid which generates L-phenylalanine via PLP-dependent transamination. However, NAD +-dependent dehydrogenase enzyme catalyzes decarboxylative aromatization of prephenic acid and converts it into 4-hydroxyphenylpyruvic acid.
4-Hydroxyphenylpyruvic acid then undergoes transamination to provide L-tyrosine.
In another route, prephenic acid before undergoing decarboxylative aromatization, is
deoxoaminated to yield L-arogenic acid which in the presence of appropriate stress and enzymes is converted either into L-phenylalanine or L-tyrosine or both (figure-2.16) (Dewick PM, 2009).
OOH
OH O OH O Chorismic acid
Chorismate mutase
OOH O OOH O OH H O + O NAD O O p rephenate preph enate dehydratase dehydrogenase H OH OH 4-Hydroxyphenylpyruvic acid Prephenic acid Phenylpyruvic acid
PLP prephenate PLP phenylpyruvate PLP aminotransferase aminotransferase
OOH O OOH O OH H NH NAD+ NH 2 2 O NH2 arogen ate dehydratase
OH OH
L -Ty rosine L-Arogenic acid L-Phenylalanine
Figure-2.16: Biosynthesis of L-phenylalanine and L-tyrosine.
94
Phenylpropanoids are important class of natural products, responsible for the biotic and abiotic stimuli. Depending upon the environmental and physiological stresses, phenylpropanoids acts as indicators or even mediators for the plant. They support survival of plant habitats in non-native area. Polymeric phenylpropanoids, for example, lignin, etc., provide considerable strength to gymnosperms and angiosperms against mechanical and environmental threats and harms (Vogt T, 2010).
Two of the C 6C3 building blocks, L-phenylalanine and L-tyrosine, constitute a broad range of natural products as precursors. First step in their biosynthetic pathway in plants is the deamination of side-chain to produce corresponding ( E)-cinnamic acid. For instance,
cinnamic acid and 4-coumaric acid are produced from L-phenylalanine and L-tyrosine, respectively. Hydroxylation and methylation produces several other cinnamic acid derivatives following the typical ortho -oxygenation pattern of shikimate pathway metabolites. CoA esters and aldehydes can also undergo aromatic methylation and hydroxylation. Hydroxycinnamyl alcohols; precursors of lignans and lignin, may also be produced from cinnamic acid reduction by means of CoA esters and aldehydes (figure-2.17).
C-alkylation of phenols is also a very common phenomenon facilitated by SAM while DMAPP work as O-alkylating agent (Dewick PM, 2009). Biosynthetic approach of ether formation is supported by the reaction between an alcohol and an ester catalyzed by alkyl- dihydroxyacetone phosphate synthases (ADPSs) (de María PD et al ., 2010).
95
HO O HO O
NH2
phenylalanine ammonia lyase
L-Phenylalanine Cinnamic acid
cinnamate 4-hydroxylase
HO O HO O HO O HO O HO O HO O
NH2
tyrosine p -coum arate HO caffeic acid MeO OH caffeic acid MeO OMe ammonia 3-hydroxylase O-methyl MeO O-methyl OH OH OH OH lyase OH transferase OH transferase L-Tyrosine 4-Coumaric acid Caffeic acid Ferulic acid 5-Hydroxyf erulic Sinapic acid acid
HO HO HO
MeO MeO OMe OH OH OH 4-Hydroxycinnamyl alcohol Coniferyl alcohol Sinapyl alcohol
Polymers x 2 x n
Lignans Lignin Figure-2.17: Biosynthetic Approach for Phenylpropanoids.
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3.1 Characterization of Isolated and Purified Compounds:*
In the present study root, stem, and leaves of C. rothii were extracted separately for the isolation, purification and finally characterization of natural products. Purifications ( vide experimental) were performed mainly utilizing solvent-solvent partitioning and chromatographic techniques. The structural elucidation was done exploiting various techniques of spectroscopy including UV, IR, Mass and 1D and 2D NMR. The spectral studies and discussion resulted in characterization of compounds either new or already reported in literature. A list of compounds and their sources, indexed as going to appear in discussion is given below. Moreover, various extracts, fractions and pure compounds have
also been subjected to various bioassay techniques.
Section Name of Compound Source Fraction Comments no. (trivial name) (number)** (scheme no.)
3.1.1 2′′ -Butoxyethyl 3-[3 ′,5 ′-di( tert -butyl)-4′- Root New natural hydroxyphenyl]-propanoate KC-C (4.1a) constituent (Mairajinol) ( 30 ) – A new compound 3.1.2 Stigmast-5-en-3β-ol Stem Reported from (β-Sitosterol) (26 ) CRSH (4.5) & C. rothii Leaves BCH (4.8) 3.1.3 (24 S)-Stigmasta-5,22-dien-3β-ol Root Reported from (Stigmasterol) (27 ) KAcMe (4.4) C. rothii 3.1.4 Octacosan-1-ol ( 74 ) Leaves First reporting BD (4.8) from C. rothii
3.1.5 Stigmast-5-en-3-O-β-D-glucoside Leaves First reporting (β-Sitosterol glucoside) ( 79 ) BA (4.8) from C. rothii 3.1.6 (2 S) Methyl 2-hydroxy-3-(4 ′- Leaves First reporting hydroxyphenyl)-propanoate C4 (4.9) from C. rothii (Latifolicinin C) (62 )
* A portion of this work has already been published as; “Isolation of Phytochemicals from Cordia Rothii (Boraginaceae) and Evaluation of their Immunomodulatory Properties”, S. Firdous, K. Khan, S. Z. Rehman, Z. Ali, S. Soomro, V. U. Ahmad, M. Rasheed, M. A. Mesaik and S. Faizi, Records of Natural Products , Vol. 8, No. 1, 51-55, (2014). ** Arranged in the order of their isolation in the experimental schemes
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3.1.7 1-O-β-D-Glucopyranosyl-(2 S,3 S,4 R,8 Z)-2- Leaves First reporting [(2 ′R)-2′-hydroxytetracosanoyl amino]- BA3 (4.8) from C. rothii 1,3,4-octadecanetriol-8-ene ( 80 )
3.1.8 2-Hydroxy-3-(4 ′-hydroxyphenyl)- Leaves First reporting propanoic acid C7 (4.9) & from C. rothii ((2 R) ( p-Hydroxyphenyl) lactic acid) ( 81 ) CRMB (4.10)
3.1.9 Syringaresinol mono-β-D-glucoside ( 82 ) Leaves First reporting D5 (4.10) from C. rothii
3.1.10 6-Hydroxy-3-oxo-α-ionol 9-O-β-D- Leaves First reporting glucopyranoside (Roseoside) ( 83 ) D5 (4.10) from C. rothii 3.1.11 3,5-Dihydroxy-megastigma-6,7-dien-9- Leaves First reporting
one-3-O-β-D-glucopyranoside D5 (4.10) from C. rothii (Staphylionoside D) ( 84 )
3.1.12 3-(3 ′,5 ′-Dimethoxy-4′-O-β-D-gluco- Leaves First reporting
pyranosyl-phenyl)-prop-2E-en-1-ol D5 (4.10) from C. rothii (Syringin) (85 )
98
3.1.1 Structure Elucidation of 2 ′′ -butoxyethyl 3-[3 ′,5 ′-di( tert -butyl)-4′-hydroxy- phenyl]-propanoate, Mairajinol (30 ) ― A New Compound:
OO 1'' 2'' 1''' 2''' 1 O 2
3
1' 2' 8' 6' 12' 9' 3' 5' 13' 4' 7' 11'
10' OH 14'
Figure-3.1: 2''-Butoxyethyl 3-[3',5'-di(tert-butyl)-4'-hydroxyphenyl]propanoate, Mairajinol (30).
In the present study, a new natural product, trivially named as Mairajinol ( 30 ) has been isolated and identified. The compound possesses a unique combination of ester and ether functionalities in an alkylated monohydroxy phenylpropanoid system (figure-3.1).
The compound 30 was obtained as a colourless amorphous solid from the root extract of the plant (scheme-4.1a). Its UV spectrum exhibited absorption maxima for ester at 205 and for benzenoid moiety at 225, and 275 nm, while the IR spectrum showed peaks for hydroxyl group at ranging between 3717 to 3572, saturated straight-chain at 2956, 2924, 2855, 1461, and 741, an ester carbonyl function at 1735 and a strong absorption of aliphatic ether at 1125 cm -1. Other intense absorption peaks at 1600 and 1470 cm -1 for aromatic ring were also observed. The EIMS spectrum displayed molecular ion peak at m/z 378 whereas HREIMS spectrum provided exact mass at m/z 378.2750 was consistent with the formula (C 23 H38 O4) showing five degrees of unsaturation in the molecule. 13 C NMR (BB, DEPT) spectrum was in complete agreement with the molecular formula displaying twenty three carbons in the structure. The 13 C NMR spectrum in the BB and DEPT mode exhibited seven quaternary carbons, seven methylenes, two methines, and seven methyls. The 1H NMR spectrum showed
two proton singlet at δH 6.97 for H-2' and H-6', indicating the symmetrical substitution pattern of aromatic system and the presence of gallic acid moiety in the molecule (Kandil FE et al ., 13 1999). However, in gallic acid C-2' and C-6' resonate at δC 110.5 in C NMR spectrum (Lu Y and Foo LY. 1999) whereas in compound 30 these carbons appeared at a downfield value of
δC 124.8, and also showing a one bond correlation with H-2', H-6' in the HSQC plot. These chemical shifts as well as other NMR resonances for 3',4',5' tri-substituted aryl system at
δC 136.0 (C-3', C-5') and 152.2 (C-4') and for tert -butyl groups at δH 1.41 (18H, s, H-8'-10',
99
H-12-14') and δC 34.1 (C-7' and C-11'') are reminiscent for the presence of butylated hydroxytoluyl (BHT) moiety in the structure (Chen C-T et al ., 2005). HMBC spectrum has crucial correlations for long range 2J and 3J couplings between H-2' and H-6' with C-3' and C-
5' ( δC 136.0) and C-4' ( δC 152.2) respectively (figure-3.2).
H H
OO 1'' 2'' 1''' 2''' 1 O H 2 H H H 3
1' H 6' 5' 4' 11' OH
Figure-3.2: HMBC Correlation of 30.
Important fragments at m/z 219, 205, and 91 in the EIMS spectrum further supported the presence of butylated hydroxytoluyl (BHT) segment in the molecule (figure-3.3). The base + peak at m/z 57 assignable to [C 4H9] may be primarily due to the fragments of 3° t-butyl moiety, although 1° n-butyl could also be a contributer. Similarly ester bond cleavage + + furnished fragment ion peak at m/z 278 [C 17 H26 O3] and m/z 102 [C 6H14 O] whereas a + significant cleavage resulted in fragment ion peak at m/z 219 [C 15 H23 O] . It is noteworthy that BHT has been reported as a natural constituent from freshwater phytoplankton and Azadirachta indica A. Juss (Neem) (Babu B and Wu J-T, 2008; Siddiqui BS et al ., 2004), and also from genus Cordia (Adeosun CO and Samuel SO, 2012).
C4H9 = 57 C17H26O3 = 278 C3H7 = 43 O O O
C6H14O = 102 C15H23O = 219
C14H21O = 205 OH
C H O = 91 6 3 C4H9 = 57
Figure-3.3: Mass Fragmentation Pattern of 30.
The 1H NMR spectrum exhibited two mutually coupled double doublets integrating for two protons each. These resonated at δH 2.62 ( J = 7.5, 8.5 Hz, H-2) and 2.86 ( J = 7.5, 8.5 Hz, H-3) ______suggesting the presence of an isolated CH 2 CH 2 system flanked between the two
100 quaternary carbons i.e. C-1′ of the phenyl ring and C-1 of the carbonyl carbon in the
molecule. A down field chemical shift ( δH 2.86) was assigned to the benzylic CH 2 (H-3),
which showed one bond correlation with δC 31.0 (C-3) in the HSQC spectrum. The other
adjacent methylene appearing at δH 2.62 (H-2), exhibited cross peak for δC 36.4 (C-2) in HSQC plot. The 1H-1H shift correlation spectroscopy (COSY-45°) further confirmed the mutual relationship between these methylenes. These data are comparable with those of the methylenes reported for 3-(4 ′-hydroxyphenyl)-propanoic acid (phloretic acid) ( 2a ) in literature (Owen RW et al ., 2003). In the HMBC plot, H-2 and H-3 showed long range 1 connectivities for C-1' ( δC 131.1) and C-1 ( δC 173.2). H NMR spectrum further displayed
signals of two deshielded protons at δH 4.21 (2H, dd, J = 4.5, 5.0 Hz, H-1″) exhibiting one
bond HSQC correlation with methylene carbon at δC 63.7 (C-1″). These correlations suggested that the mentioned methylene is attached with the oxygen of ester functionality. The data was also comparable with the reported data of the structurally related compounds in literature as observed in the case of 4-methylpentyl 3-(4 ′-hydroxyphenyl) propionate (isohexyl dihydro-p-coumarate) ( 3a ) and 4-methylpentyl 3-(3 ′,4 ′-dihydroxyphenyl)- propionate (isohexyl dihydrocaffeate) ( 4a ) (Marco JA et al ., 1994) (figure-3.4).
A unique set of four methylene protons attached with a central etheral oxygen at δH 3.60 (2H, dd, J = 4.5, 5.0 Hz, H-2″) and 3.44 (2H, t, J = 7.0, H-1″′ ) displayed one bond HSQC
interaction with C-2″ ( δC 68.6) and C-1″′ ( δC 71.2) respectively. The presence of this segment 2 3 was also confirmed by long range J correlations of H-1″ and J of H-1″′ with C-2″ ( δC 68.6). 2 3 Other long range J and J connectivities of H-2″′ and H-2″ resonating at δH 1.53 and 3.60 respectively with C-1″′ ( δC 71.2) was in complete agreement with the presence of this moiety in the molecule. It was further confirmed through COSY-45° plot that showed correlations between H-1″ ( δH 4.21) and H-2″ ( δH 3.60) together with another pair of COSY interaction between H-1″′ ( δH 3.44) and H-2″′ ( δH 1.53). The multiplet of 2H at δH 1.53 exhibited one bond HSQC correlation with C-2″′ (δC 31.9). A triplet of three protons resonating at δH 0.86 (3H, t, J = 7.0 Hz, H4 ″′ ) confirmed the presence of terminal methyl group of straight aliphatic chain in the molecule, and showed cross peaks with its carbon at δC 14.1 (3H, C-4′′′ ). Two
methylene protons (H-3''') of the terminal ethyl unit resonating as broad singlet at δH 1.24 also displayed one bond HSQC correlation with methylene carbon C-3″' present at δC 22.7. Based on these findings the structure of the compound 30 was elucidated as 2 ′′ -butoxyethyl 3-[3 ′,5 ′- di( tert -butyl)-4′-hydroxyphenyl]-propanoate and name mairajinol was assigned to it. Natural existence of 30 was also supported by the natural occurrence of BHT in genus Cordia and isolation of many other structurally related compounds from it (Adeosun CO and Samuel SO, 2012, Babu B and Wu J-T 2008 and Siddiqui BS et al ., 2004).
101
There are several examples of natural products bearing ether bonds, for instance terpenoids, lipids, polyketides, carbohydrates, flavonoids and antibiotics (de María PD et al ., 2010).
Reported literature provided examples of the natural occurrence of various C 6-C3 acids and their esters, and ether, for example, 2a (Owen RW et al ., 2003), 3a and 4a (Marco JA et al ., 1994), and 3-(3',4',5'-trimethoxyphenyl)-propanoic acid (3,4,5-trimethoxydihydrocinnamic acid) ( 5a ), (Das B et al ., 1996) as well as the acetyl derivative of ( E)-2-hydroxyethyl 3,4,5- trimethoxy-cinnamate, (2-(acetyloxy)-ethyl ( E)-3-(3 ′,4 ′,5 ′-trimethoxyphenyl)-2-propenoate) (6a ) (DellaGreca M et al ., 2007).
O R1 OR
R2 R3 R1 R2 R3 R
2a HOHHH 3a H OH H (CH2)3CH(CH3)2 4a OH OH H (CH2)3CH(CH3)2 5a OCH3 OCH3 OCH3 H
O
H3CO O O O H3CO 6a OCH3
Figure-3.4: Compounds Structurally Related with Mairajinol ( 30 )
Several fatty alcohols hexanol ( 7a ) (Pino JA et al ., 2002), n-octacosanol ( 8a ), and n-hentriacontanol ( 9a ) (Agnihotri VK et al ., 1987) and n-hexacosanol ( 10a ) (Mukat B and Chhaya G, 1980) have been isolated from genus Cordia . The methyl ester of 5a ; methyl 3-(3 ′,4 ′,5 ′-trimethoxyphenyl)-propanoate) (11a ) has also been found naturally (Anuradha V et al ., 2004). ( E)-3-(4 ′-hydroxy-3′-methoxyphenyl)-2-propenoic acid (ferulic acid) ( 12a ) has already been identified from genus Cordia (El-Sayed NH et al ., 1998) (figure-3.5).
102
HO n 7a n = 2 8a n = 24 9a n = 27 10a n = 22 O O
H3CO H OCH3 OH
H3CO HO
OCH3 OCH3
11a 12a
Figure-3.5: Compounds Structurally Related with Mairajinol (30).
The possible biosynthetic pathway for 30 may be sequenced as follows; L-tyrosine, a precursor of phenylpropanoids, formed through shikimic acid ( vide biosynthesis section: 2.6 and scheme-3.1) pathway from prephenic acid is deaminated by enzyme tyrosine ammonia lyase (TAL) thereby converting it into ( E)-3-(4 ′-hydroxyphenyl)-2-propenoic acid (p-coumaric acid) ( 13a ) (Knaggs AR, 2001). Hydrogenation of 13a converts it into 2a (Owen RW et al ., 2003) (scheme- 3.1).
O H OH
HO H
13a
Figure-3.6: Compound Structurally Related with Mairajinol (30).
As discussed earlier, a number of fatty alcohols are reported from genus Cordia , for example, 7a , 8a , 9a , and 10a (figure-3.5). 2-(Dodecyloxy)-1-ethanol (2-hydroxyethyl lauryl ether) (14a ) (Yingpeng Z et al ., 2009), 2-(decyloxy)-1-ethanol ( 15a ) (Koz FFY et al ., 2009), 2,2 ′- oxydiethanol (diethylene glycol) ( 16a ), and 2-(2 ′-n-butoxyethoxy)-ethanol (diethyleneglycol monobutyl ether) ( 17a ) (Humaira Siddiqi, Ph.D. Dissertation 2012) are naturally occurring mono alkyl ethers of ethylene glycol (figure-3.7a). Condensation of such 2-alkoxyethanols with 2a may lead to the esters analogous to 30.
103
OR
HO n
14a n = 11, R = CH3 15a n = 9, R = CH3 16a n = 2, R = OH 17a n = 2, R = O(CH2)3CH3
Figure-3.7a: Naturally Occurring Monoalkylethers of Ethylene Glycol
The isolation of esters of C 6-C3 acids with glycol, for example, 2-hydroxyethyl ester of p-coumaric acid; 2 ′′ -hydroxyethyl ( E)-3-(4 ′-hydroxyphenyl)-2-propenoate (ariscucurbin-B) (18a ) and 2 ′′ -hydroxyethyl ( Z)-3-(4 ′-hydroxyphenyl)-2-propenoate (ariscucurbin-C) ( 19a ) have been reported earlier (Wu T-S et al ., 1999). Similar esterification is also possible with 2a to yield 2 ′′ -hydroxyethyl 3-(4 ′-hydroxyphenyl)-propanoate ( 20a ). Successive O-alkylation of alcohol and C-alkylation of phenol at activated positions ortho to OH group (Dewick PM, 2009) may occurr generating 30. Alternatively, 18a or 19a (figure-3.7b) may undergo reduction via NADPH yielding 20a , followed by O-alkylation and C-alkylation of phenolic group (Dewick PM, 2009) as discussed earlier to probably furnish the skeleton for 30 (scheme-3.1).
O R1 OR
R2 R3 1 2 3 ______R R R R
18a H OH H CH2CH2OH(E) 19a H OH H CH2CH2OH (Z)
Figure 3.7b: Compounds Structurally Related with Mairajinol ( 30 )
104
O OH O
OH NH HO OH HO 2 OH
Shikimic acid L-Tyrosine
O O OH OH HO OH O
HO HO 13a 18a = E / 19a = Z
[H] NADPH [H] NADPH
O O OH OH HO OH O O-alkylation
HO HO 2a 20a
O O O O O C-alkylation O 3 3 HO HO 30
Scheme-3.1: Proposed Biosynthesis of 30.
105
3.1.2 Characterization of Stigmast-5-en-3β-ol (β-Sitosterol) (26 ):
29 28
21 24 26 20 22 18 23 25 12 17 11 27 19 13 16 1 9 14 2 10 8 15 3 5 7 HO 4 6
The compound 26 was obtained as white shiny crystals from stem and leaves, as appearing in schemes-4.5 and 4.8 respectively. The EIMS showed an intense molecular ion peak, [M] +, at m/z 414. The molecular formula of the compound was deduced from its HREIMS that + afforded exact mass at m/z 414.3855 (calcd. for [C 29 H50 O] , 414.3862). Fragment ions at m/z 399, 396, 381, 329, and 303, diagnostic of ∆5-unsaturation for sterols, were also observed in + + the EIMS spectrum. Major peaks at m/z 255 for [M-C10 H21 -H2O] and 273 for [M-C10 H21 ] were also indicative of the presence of the 26 .
The 1H NMR spectrum showed the presence of six methyl signals, characteristic of a steroidal pattern. The two tertiary methyl singlets integrating for three protons each resonated at
δH 0.66 (H-18) and 0.99 (H-19) whereas the three secondary methyl groups, H-27, H-26, and
H-21 appeared as doublets at δH 0.79 (3H, J = 7.0 Hz), 0.81 (3H, J = 6.5 Hz) and 0.90 (3H, J = 6.5 Hz). A triplet of the primary methyl H-29 was observed at 0.83 (3H, J = 7.0 Hz) was
also indicative of the steroidal skeleton. A downfield one proton multiplet resonating at δH 5.33 (H-6) indicated the presence of an olefinic ∆5- bond in the molecule (Rubinstein I et al ., 1976), which was also supported by the major fragment ions displayed in the EIMS. Signals of 29 carbon atoms in the 13 C NMR (BB and DEPT) spectrum were assignable to the three quarternary carbons, nine methines, eleven methylenes, and six methyls present in the compound. The data discussed above and its comparison with the reported literature, suggested that the purified compound 26 is β-sitosterol (Kolak U et al ., 2005) .
26 is generally a wide spread metabolite in terrestrial plants and has been reported from various species of family Boraginaceae (Hoang QH et al ., 2009), genus Cordia (Menezes JESA et al ., 2001), and C. rothii (Mukat B and Chhaya G, 1980).
106
3.1.3 Characterization of 24( S)-Stigmasta-5,22-dien-3β-ol (Stigmasterol) (27 ):
29 28
21 24 26 20 22 18 23 25 12 17 11 27 19 13 16 1 9 14 2 10 8 15 3 5 7 HO 4 6
Compound 27 was obtained as white amorphous solid from roots (scheme-4.4). Its 1H NMR spectrum displayed six methyl groups. Signals of two methyl appeared as singlets at δH 0.67
(3H, H-18) and 0.99 (3H, H-19); three methyl groups as doublet at δH 0.85 (3H, J = 6.4 Hz,
H-26), δH 0.78 (3H, J = 6.4 Hz, H-27), and δH 1.06 (3H, d, J = 6.6 Hz, H-21) and an other methyl as triplet at δH 0.81 (3H, J = 7.2 Hz, H-29). A pair of downfield double doublets
present at δH 4.99 (1H, J = 8.4, 15.2 Hz, H-23) and 5.13 (1H, J = 8.4, 15.2 Hz, H-22), indicated the presence of two olefinic protons. The large coupling constant of these protons
established their trans -geometry. Another olefinic proton (H-6) was observed at δH 5.33 as broad singlet. Furthermore, a one proton multiplet of H-3 resonating at δH 3.50 suggested the presence of a hydroxymethine proton in the molecule.
On the basis of above findings in 1H NMR as well as its 13 C NMR data ( vide experimental) and its comparision with the reported literature (Azizudin and Choudhary MI, 2008), the isolated compound 27 is identified as stigmasterol.
The compound 27 commonly occur in plant kingdom, and has been reported earlier from C. rothii (Desai HK et al ., 1976), genus Cordia (do Vale AE et al ., 2012,) and family Boraginaceae (Andhiwal CK et al ., 1985).
107
3.1.4 Characterization of Octacosan-1-ol (74 ):
27
28
1 HO 2
Compound 74 was obtained as white amorphous powder from leaves (scheme-4.8) of + C. rothii . The [M-H2O] ion peak was confirmed through HREIMS that provided exact mass + at m/z 392.4413 (calcd. for [C 28 H56 ] , 392.4382), and helped in deducing the molecular formula. The molecular mass was also confirmed by the chemical ionization mass spectrometery (CIMS-negative mode). The presence of a major fragment ( pseudo -molecular + ion peak) in the EIMS spectrum at m/z 392 due to [M-H2O] suggested that the compound is possibly a 1° alcohol. Appearance of various fragment ions 14 mass units apart in the EIMS spectrum indicated the presence of straight-chain hydrocarbon. The base peak present at m/z + 57 was due to [C 4H9] .
The presence of absorption bands in a rather simple IR spectrum at ranging between 3490 to 3350 cm -1 and 1467 cm -1 further supported the respective presence of hydroxyl and methylene 1 groups in the molecule. The H NMR spectrum displayed a triplet for terminal methyl at δH
0.86 (3H, J = 6.0 Hz, H-28). Another downfield triplet resonating at δH 3.62 (2H, J = 6.6 Hz, H-1) was assigned to methylene group adjacent to the hydroxyl function. A broad singlet of methylene integrating for a large number of protons resonated at δH 1.24 (H-3 to H-27) further confirmed the presence of long chain hydrocarbon in the molecule. Another downfield signal
resonating as multiplet at δH 1.54 and integrating for two protons was assigned to methylene 13 group (H-2). The C NMR spectrum showed only one methyl signal resonating at δC 14.1 (C- 28), further supported the presence of a terminal methyl group in the molecule. The hydroxy methyl group appeared at δC 63.10 (C-1). The data discussed above and its comparison with the reported literature values (Safder M et al ., 2009) led to conclude compound 74 as octacosan-1-ol.
Various fatty alcohols have been reported earlier from other species of the genus Cordia . These included; hexanol (Pino JA et al ., 2002), octacosanol and hentriacontanol (Agnihotri VK et al ., 1987) and n-hexacosanol (Mukat B and Chhaya G, 1980). The current study reports C. rothii as the new source of octacosan-1-ol, however, previously it has been reported from other species of the family Boraginaceae (Ul'chenko NT et al ., 1991).
108
3.1.5 Characterization of Stigmast-5-en-3-O-β-D-glucoside ( β-Sitosterol glucoside)
(79):
29 28
21 24 26 20 22 18 23 25 12 17 11 27 19 13 OH 16 6' 1 9 4' 14 5' O 2 10 8 15 HO 1' 3 2' 7 HO 3' 5 OH 4 6
The compound 79 was purified as white amorphous solid from leaves of C. rothii (scheme- 4.8). The 1H NMR spectrum data was found to be similar to β-sitosterol ( vide section 3.1.2), except the typical sugar protons resonating between δH 3.94-4.53 and the corresponding 13 doublet of anomeric proton at δH 5.02 (1H, J = 7.7, H-1′). C NMR further confirmed the
presence of sugar moiety, the anomeric carbon showed resonance at δC 102.5 (CH, C-1′) and
the typical hydroxylated carbons of sugar moiety were observed in the range of δC 62.8-78.6. The HREIMS showed quasimolecular ion at m/z 415.3979, due to aglycone.
13 C NMR (BB and DEPT) further showed the presence of six methyl, twelve methylene, fourteen methine and three quaternary carbons. Discussed observations and their comparison with the previously reported data, particularly 1H NMR and 13 C NMR data (Faizi S et al .,
2001), led to identify the purified compound 79 as stigmast-5-en-3-O-β-D-glucoside.
Literature revealed that this is the first report of the 79 from C. rothii . Although it’s presence from other species of genus Cordia (Menezes JESA et al ., 2001) and the family Boraginaceae (Hoang QH et al ., 2009) has been reported earlier.
109
3.1.6 Characterization of (2 S) Methyl 2-hydroxy-3-(4 ′-hydroxyphenyl)-propanoate
(Latifolicinin C) (62 ):
O OH
1 2 H3CO 3 1' 4' OH
Compound 62 was purified as an amorphous powder from leaves (scheme-4.9). The EIMS of 62 showed a weak molecular ion peak [M] + at m/z 196. The HREIMS afforded exact mass at + m/z 196.0747 (calcd. for [C 10 H12 O4] , 196.0736), corresponding to the five degrees of unsaturation in the molecule. Molecular mass of 196 was further confirmed by the CIMS and FABMS in the +ve ion mode that displayed [M+H] + peak at m/z 197. The appearance of base peak at m/z 107 in the EIMS spectrum indicated the presence of p-hydroxy benzyl entity in the molecule (Siddiqui BS et al ., 2006) which was also evident from the 1H and 13 C NMR spectra of the compound. The IR spectrum showed an intense absorption at 1730 cm -1 for ester carbonyl and a broad absorption ranging between 3434 to 3289 cm -1 typical of hydroxyl group. Other strong absorptions observed at 1444, 1514, and 1608 cm -1 were indicating the presence of unsaturation (aromaticity) in the molecule.
1 The H NMR displayed a pair of doublets integrating for two protons each at δH 6.73 (2H, J =
8.4 Hz, H-3′, H-5′) and δH 7.05 (2H, J = 8.3 Hz, H-2′, H-6′) indicating the presence of 1,4- disubstituted aromatic ring system in the molecule. It was also supported by 13 C NMR in which the corresponding carbons resonated at δC 115.3 (CH, C-3′, C-5′) and 130.6 (CH, C-2′, C-6′) respectively as two pairs of aromatic methines. In 1H NMR spectrum benzylic
methylene appeared as double doublets at δH 2.88 (1H, J = 6.5 Hz, 14.0 Hz, H-3a) and δH 3.04
(1H, J = 4.4 Hz, 14.0 Hz, H-3b) whereas corresponding carbon resonated at δC 39.6 (CH 2, 13 C-3) in C NMR. A one proton broad triplet at δH 4.39 ( J = 5.6 Hz, H-2) with its carbon 1 resonating at δC 71.4 (CH 2, C-2) suggested the presence of hydroxyl moiety at C-2. In H
NMR spectrum a three proton singlet present at δH 3.75 (3H, 1-OCH 3) indicated the presence of methoxy group in the molecule. It was further supported by 13 C NMR spectrum where this 13 methoxy carbon appeared at δC 52.4 (CH 3). The downfield signal in the C NMR spectrum resonated at δC 174.6 (C, C-1) was assigned to the carbonyl carbon confirming the conclusions drawn by 1H NMR that H-2 was attached to the carbon bearing the hydroxyl function. The data discussed above was suggestive of the compound 62 as (2 S) methyl 2-hydroxy-3-(4 ′-hydroxyphenyl)-propanoate trivially named as Latifolicinin C. Comparing
110 experimental values with the reported literature (Siddiqui BS et al ., 2006) confirmed the elucidation.
This is the first report of isolation of compound 62 from C. rothii leaves. It was also identified in the stem through GCMS (section 3.2). However, it has been reported earlier from other species of genus Cordia (Siddiqui BS et al ., 2006) and family Boraginaceae (Ara I et al ., 2012).
111
3.1.7 Characterization of 1-O-β-D-Glucopyranosyl-(2 S,3 S,4 R,8 Z)-2-[(2 ′R)-2′-hydroxy tetracosanoyl amino]-1,3,4-octadecanetriol-8-ene (80):
24' OH
O 2' OH 1' 6'' NH OH 4'' 5'' O 6 8 HO O 3 2'' 1 2 4 9 HO 3'' 1'' 5 7 OH OH 18
The compound was purified as white gelatinous gummy material from the leaves (scheme- 4.8). The ESIMS provided [M+H]+ peak at m/z 844.7004. In the EIMS spectrum + [M-C6H12 O6] peak was observed at m/z 663, suggesting the presence of hexose moiety in the 1 molecule. The appearance of the doublet of anomeric proton in H NMR spectrum at δH 4.94 (1H, J = 8.0 Hz, H-1′′ ), further confirmed the presence of sugar moiety in the molecule. The larger coupling constant of the anomeric doublet attributed it to be the β-anomer. The rest of
the signals of sugar moiety appeared at δH 3.86 (1H, m, H-5′′ ), 4.01 (1H, t, J = 8.0 Hz, H-2′′ ), 4.19 (2H, m, H-3′′ , 4 ′′ ), 4.35 (1H, dd, J = 5.1 Hz, 12.0 Hz, H-6′′ a), and 4.50 (1H, m, H-6′′ b). Conclusive evidence for the presence of sugar moiety were drawn from 13 C NMR spectrum
where anomeric carbon resonated at δC 105.5 (CH, C-1′′ ) and the five other oxygenated
carbons were observed at δC 62.6 (CH 2, C-6′′ ), 71.5 (CH, C-4′′ ), 75.1 (CH, C-2′′ ), 78.4 (CH, C-5′′ ) and 78.5 (CH, C-3′′ ).
The 1H NMR spectrum displayed a broad triplet integrating for six protons and resonating at
δH 0.85 ( J = 6.9 Hz, H-18, 24 ′). It was assignable to the two terminal methyl groups with a methylene group adjacent to it. Supporting signal for the terminal methyl groups, like those 13 present in long hydrocarbon chains, was also observed at δC 14.3 (CH 3, C-18, 24 ′) in the C
NMR spectrum. A broad singlet at δH 1.29 (H-4′-H-21 ′) integrating for various methylene groups indidcated the presence of straight-chain hydrocarbon. Strong signal in the region between δC 29.5-30.0 (CH 2, C-4′-C-21 ′) further confirmed the presence of methylenes of long chain hydrocarbon. A typical one proton doublet resonating in the downfield region of 1H
NMR at δH 8.56 ( J = 9.0 Hz, NH) suggested the presence of amide proton. The corresponding
carbon signals were observed at δC 51.7 (CH, C-2). Such chemical shift values are typical of 13 carbon atom attached to nitrogen of the amide group. Similarly the C NMR signal at δC 175.7 (C, C-1′) indicated the existence of carbonyl carbon of amide group. Important 1H―1H
112
COSY interaction was observed between amide proton (resonating at δH 8.56) and H-2 proton at δH 5.28 (Kang SS et al ., 1999). This H-2 proton also showed cross peak linkages with C-1 1 1 (δC 70.4), C-3 ( δC 75.9) and C-4 ( δC 72.4) (Zhang W-K et al ., 2007). H- H COSY interaction
of H-2 with H-1a ( δH 4.52), H-1b ( δH 4.70) and H-3 also supported this view (Kang SS et al ., 1999).
13 The downfield C NMR signal at δC 130.2 (CH, C-9) and 130.4 (CH, C-8) indicated the presence of two olefinic carbon atoms, equivalent to a double bond in the molecule. The geometry of double bond was assigned to be Z on the basis of the chemical shifts of vicinal
carbon atoms resonating at δC 27.9 (CH 2, C-7) and 27.6 (CH 2, C-10) (Zhang W-K et al ., 2007). The position of double bond at C-8 was confirmed through HMBC spectrum, which showed interactions between H-5a, H-5b resonating at δH 2.05 (2H, m, H-3′, H-5a) and 1.98
(1H, m, H-5b) respectively with C-6 and C-7 appearing at δC 26.8, 27.9 respectively.
Similarly H-8 resonating at δH 5.50 (2H, m, H-8, H-9) showed cross peak with C-7 appearing
at δC 27.9 (Tapondjou LA et al ., 2005). On comparing the observations, discussed above, with the data reported in literature (Zhang W-K et al ., 2007) compound 80 was identified as 1-O-β-
D-glucopyranosyl-(2 S,3 S,4 R,8 Z)-2-[(2 ′R)-2′-hydroxytetracosanoyl amino]-1,3,4-octadecane- triol-8-ene.
To the best of our knowledge, C. rothii is found to be a new source of compound 80 , as well as this is the first report of its occurrence from genus Cordia and family Boraginaceae. However, its geometrical isomer “ E” has already been reported from genus Cordia (Tapondjou LA et al ., 2005).
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3.1.8 Characterization of (2 R) 2-Hydroxy-3-(4 ′-hydroxyphenyl) propanoic acid [(2 R) ( p-Hydroxyphenyl) lactic acid] (81):
HO 3 1' 4' OH 2 1 O OH
Compound 81 was obtained as a crystalline solid from C. rothii leaf extract (scheme-4.9). The EIMS spectrum of the compound showed weak molecular ion peak at m/z 182. The chemical ionization technique in the positive ion mode was utilized to verify the molecular mass of the compound. It displayed [M+H] + peak at m/z 183. The HREIMS afforded exact mass at
m/z 182.0578 (calcd. for C 9H10 O4, 182.0579) from which the molecular composition of the
compound was deduced as C 9H10 O4.
The IR spectrum showed absorption bands 1738 cm -1 for C=O, and a broad band ranging from 3536 to 3391 cm -1 for O-H group. Intense absorption bands at 1613 and 1448 cm -1 indicated the presence of unsaturation in the molecule. Altogether 13 C NMR showed resonances of nine carbon atoms, with only one methylene, five methines and three quaternary carbons.
1 H NMR spectrum showed two doublets of ortho -coupled aromatic protons at δH 6.72 (2H, J
= 8.4 Hz, H-3′, H-5′) and δH 7.09 (2H, J = 8.4 Hz, H-2′, H-6′) indicating the presence of 1,4-disubstituted aromatic ring in the molecule. In the 13 C NMR spectrum, the corresponding two pairs of aromatic methine appeared at δC 115.7 (CH, C-3′, C-5′) and δC 131.4 (CH, C-2′,
C-6′). A pair of double doublets at δH 2.81(1H, J = 7.5 Hz, 14.0 Hz, H-3a) and δH 3.00 (1H, J = 4.3 Hz, 14.0 Hz, H-3b) was assigned to benzylic protons and the corresponding methylene 13 carbon was observed at δC 40.4 in the C NMR spectrum. The presence of p-hydroxy benzyl moiety was in complete agreement with the base peak appearing at m/z 107 in the EIMS and 1 HREIMS spectra. A downfield signal in the H NMR spectrum at δH 4.31 (1H, J = 4.3 Hz, 7.5 Hz, H-2) was indicative of the oxygenated proton (Siddiqui BS et al ., 2006), which was also 13 verified by the chemical shift of its corresponding carbon at δC 72.3 in the C NMR spectrum.
Carbonyl carbon resonating at δC 175.4 (C, C-1), together with the carbonyl absorption in IR at 1738 cm -1 and conclusions drawn from 1H NMR indicated that a carboxylic acid group is present in the molecule. These findings led to the identification, that 81 is (2 R) 2-hydroxy-3- (4 ′-hydroxyphenyl)-propanoic acid. Confirmation was done by comparing the observed data with reported values (Wu T-S et al ., 1998).
To the best of our knowledge, this is the first report of the presence of 81 from C. rothii . It was reported earlier from other species of the genus Cordia (El-Sayed NH et al ., 1998).
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3.1.9 Characterization of Syringaresinol mono-β-D-glucoside (82 ):
Ha OH Hb 6''' O HO 5''' O 5' 4''' 1'''O Hb Ha HO 2''' 4' 5 3''' H 8 OH 1' O H 3' 6 O 2 1 5 O H 1'' 5'' O 4 2 H 4'' Ha Hb 3'' OH O
Compound 82 was purified as amorphous solid from C. rothii leaves (scheme-4.10). The ESIMS spectrum afforded molecular ion peak at m/z 579.1988. The 1H NMR spectrum
displayed two broad singlets for methoxy groups resonating at δH 3.84 (6H, 3 ′′ -OCH 3 and 5 ′′ - 13 OCH 3) and 3.85 (6H, 3 ′-OCH 3 and 5 ′-OCH 3). In the C NMR spectrum the corresponding pairs of methoxy carbons, C-3′′ -OCH 3 and C-5′′ -OCH 3; and C-3′-OCH 3 and C-5′-OCH 3 1 resonated at δC 56.9 and 57.2 respectively and further supported the H NMR findings. Similarly a pair of singlets corresponding to the two pairs of aromatic methines were 1 observed at δH 6.64 (2H, H-2′′ /H-6′′ ) and 6.71 (2H, H-2′/H-6′) in the H NMR. The respective 13 aromatic methines appeared at δC 104.6 (C-2′′ , C-6′′ ) and 104.9 (C-2′, C-6′) in the C NMR. 1 The H NMR displayed another pair of diastereotopic protons at δH 3.91 (2H, dd, J = 2.5 Hz, 9.0 Hz, H-4a, H-8a) and 4.27 (2H, m, H-4b, H-8b). The appearance of several pairs of signals in 1H NMR with their corresponding signals in 13 C NMR, were indicative of some sort of symmetrical substitution or dimerization in the molecule.
A downfield singlet of an anomeric proton appeared at δH 4.83 (1H, H-1′′′ ) along with other resonances of protons bearing hydroxyl group at δH 3.19 (1H, m, H-5′′′ ), 3.40 (2H, m, H-3′′′ , H-4′′′ ), 3.66 (1H, dd, J = 5.0 Hz, 11.5 Hz, H-6′′′ a) and 3.77 (1H, dd, J = 2.0 Hz, 12.0 Hz, H- 6′′′ b) showed the presence of sugar moiety in the molecule. The presence of a sugar moiety was further confirmed by the 13 C NMR spectra, in which the anomeric carbon signal was
observed at δC 105.5 (C-1"') and the other resonances of carbons bearing hydroxyl groups resonated at δC 62.6 (CH 2, C-6′′′ ), 71.4 (CH, C-4′′′ ), 75.7 (CH, C-2′′′ ), 77.8 (CH, C-3′′′ ) and 78.3 (CH, C-5′′′ ). HMBC interactions further correlated the connectivities of aromatic units, sugar moiety, and C-3 units in the molecule. Based on these findings 82 was identified as syringaresinol mono-β-D-glucoside. Previously published literature (Lami N et al ., 1991) further helped in confirming the experimental values. A detailed literature search revealed that this is the first report of compound 82 from the titled plant, genus Cordia , and family Boraginaceae.
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3.1.10 Characterization of 6-Hydroxy-3-oxo-α-ionol 9-O-β-D-glucopyranoside (Roseoside) (83 ):
OH 6' 10 5' 4' O HO H 12 11 2' 1' HO 3' 9 8 7 OH O 1 6 2 HO 5 3 4 13 O
Compound 83 was obtained as viscous syrup from C. rothii leaves (scheme-4.10). The EIMS did not show [M] + peak instead the highest mass peak was observed at m/z 224. The FABMS +ve spectrum of the compound showed [M+H] + peak at m/z 387, which was further confirmed by ESIMS spectrum displaying [M+H] + peak at m/z 387.2063. The highest mass peak in EIMS appeared at m/z 224, possibly due to the loss of sugar moiety from the molecule. This argument was supported by the corresponding resonances in the 1H NMR spectrum. The signals for typical anomeric and other protons of sugar moiety were observed at δH 4.34 (1H, d, J = 7.8 Hz, H-1′), and 3.17 (1H, dd, J = 7.5 Hz, 9.0 Hz, H-2′), 3.22 (1H, m, H-5′), 3.25 (1H, m, H-4′), 3.34 (1H, m, H-3′), 3.63 (1H, dd, J = 6.0 Hz, 12.0 Hz, H-6′a) and 3.85 (1H, dd, J = 1.8 Hz, 12.0 Hz, H-6′b).
The 13 C NMR (BB, DEPT) spectrum provided signals for four methyl, two methylene, nine methine and four quarternary carbon atoms (concluding nineteen carbons in the molecule). 1 The H NMR spectrum showed a downfield olefinic singlet at δH 5.84 (1H, H-4), indicating the presence of a trisubstituted double bond in the molecule. Another set of downfield olefinic
signals resonating at δH 5.85 (1H, d, J = 12.6 Hz, H-7) and 5.86 (1H, dd, J = 8.4 Hz, 12.6 Hz, H-8) showed the presence of another double bond with trans configuration linked to a 13 quaternary carbon at one end. In the C NMR spectrum these olefinic carbons resonated at δC 127.2 (CH, C-4), 131.6 (CH, C-7), 135.3 (CH, C-8) and 167.4 (C, C-5). In the 1H NMR spectrum a methine resonating as a broad singlet at δH 4.56 (1H, H-9) was identified as 13 1 carbinol, with its corresponding carbon resonating at δC 77.3 in the C NMR. H NMR spectrum also gave information of four methyl signals, typical of monoterpenes, which were clearly identified at δH 1.02 (3H, s, H-12), 1.03 (3H, s, H-11), 1.29 (3H, br. d, J = 6.0 Hz, H- 10) and 1.94 (3H, s, H-13). The chemical shift values and multiplicities of methyls resonating at δH 1.29 (H-10) and 1.94 (H-13) were suggestive of carbinol and an olefinic methyl respectively.
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1 In H NMR spectrum, a pair of doublet with Jgem = 16.8 Hz resonating at δH 2.14 (1H, H-2a) and 2.51 (1H, H-2b) was identified as the diastereostopic methylene with its corresponding
carbon resonated at δC 50.7 ppm. Olefinic proton H-7 displayed long range connectivity with C-5 and C-6 indicating the side chain attachment with C-6. Further correlations and comparison of these data with the values reported in literature (Champavier Y et al ., 1999 and
Murai Y et al ., 2001) led to identify 83 as 6-hydroxy-3-oxo-α-ionol 9-O-β-D-glucopyranoside.
A thorough literature search revealed that this is the first report of the isolation of compound 83 from C. rothii , genus Cordia , and family Boraginaceae.
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3.1.11 Characterization of 3,5-Dihydroxy-megastigma-6,7-dien-9-one-3-O-β-D-gluco-
pyranoside (Staphylionoside D) (84 ):
O 10 9 OH 11 12 8 6' H 5' 1 7 HO 4' O 2 6 HO 3' 2' 1' 3 5 4 OH O 13 OH
Compound 84 was purified as amorphous solid from leaves (scheme-4.8). The EIMS did not provide [M] + peak, hence its molecular mass was deduced with the help of FABMS (+ve) giving [M+H] + at m/z 387. The ESIMS further confirmed the molecular mass by providing + exact mass for [M+H] peak at m/z 387.1952, corresponding to molecular formula C 19 H31 O8. 13 C NMR (BB, DEPT) spectrum displayed nineteen carbon atoms that included four methyl, three methylene, seven methine and five quaternary carbons and was in agreement with the
molecular formula C 19 H31 O8.
Characteristic signals for the protons of a sugar moiety were detected in 1H NMR and 13 confirmed by C NMR. These included a downfield anomeric proton resonating at δH 4.46
(1H, d, J = 8.0 Hz,H-1′) and other protons at hydroxylated carbons resonating at δH 3.14 (1H, dd, J = 8.0 Hz, 9.0 Hz, H-2′), 3.28 (1H, m, H-4′), 3.29 (1H, m, H-5′), 3.35 (1H, m, H-3′), 3.70 (1H, m, H-6′a) and 3.88 (1H, m, H-6′b). Corresponding signals in 13 C NMR were also observed at 102.7 (CH, C-1′), 75.1 (CH, C-2′), 71.6 (CH, C-4′), 77.9 (CH, C-5′), 78.1 (CH, C-
3′), and 62.7 (CH 2, C-6′), respectively. These data recommended the presence of a D-glucose moiety in the molecule.
1 The H NMR spectrum displayed a downfield singlet at δH 5.83 (1H, s, H-8) assignable to an 13 olefinic proton. The corresponding carbon resonated at δC 101.2 in C NMR spectrum, characteristic of an allenic moiety. The appearance of two quaternary carbon atoms at δC 120.1 (C-6), and 200.9 (C-7), further supported the presence of allenic moiety in the 13 molecule. Another quaternary carbon resonating at δC 211.5 in C NMR, and a downfield 1 methyl resonating at δH 2.18 in the H NMR spectrum suggested the presence of an acetyl group in the molecule. In the 1H NMR spectrum, diastereotopic protons appeared as doublet of double doublet at δH 1.45 (2H, J = 4.0 Hz, 11.5 Hz, 13.0 Hz, H-2a, H-4a), 2.08 (1H, J = 2.0 Hz, 4.0 Hz, 13.0 Hz, H-4e) and 2.36 (1H, J = 2.0 Hz, 3.5 Hz, 13.0 Hz, H-2e). A set of three proton singlets of methyl groups appeared at δH 1.15 (3H, H-12), 1.37 (3H, H-11), and 1.38
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(3H, H-13) along with a terminal methyl ketone (discussed above) suggested that the aglycone may be a monoterpenoid.
All these findings and comparing these values with reported data from literature (Yu Q et al ., 2005) led to identify the compound 84 as staphylionoside D. This is the first report of the occurrence of 84 from C. rothii , genus Cordia and family Boraginaceae.
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3.1.12 3-(3 ′,5 ′-dimethoxy-4′-O-β-D-glucopyranosyl-phenyl)-prop-2E-en-1-ol (Syringin)
(85 ):
O HO 1 2 2' 3' 6'' 5'' O 4'' HO 3 1' 4' O 1'' OH 6' 5' 2'' OH HO 3'' O
Compound 85 was obtained as colourless non-crystalline solid from the butanol extract of the C. rothii leaves. (scheme-4.10). The FABMS +ve spectrum of the compound afforded [M+H] + peak at m/z 373. Diagnostic sugar moiety signals in 1H NMR were found resonating
at δH 4.86 (1H, d, J = 7.8 Hz, H-1′′ ) for anomeric proton and at δH 3.20 (1H, m, H-5′′ ), 3.41 (2H, m, H-3′′ , H-4′′ ), 3.47 (1H, m, H-2′′ ), 3.65 (1H, dd, J = 5.5 Hz, 12.0 Hz, H-6′′ a), 3.77 (1H, dd, J = 2.5 Hz, 12.0 Hz, H-6′′ b) for other protons attached to the carbon bearing hydroxyl groups.
1 In the H NMR spectrum a broad downfield singlet resonating in the aromatic region at δH 6.75 (H-2′/H-6′) and integrating for two equivalent protons were suggestive of the presence of a tetrasubstituted symmetrical benzene ring in the molecule. Resonances of the corresponding 13 aromatic protons in C NMR spectrum were observed at δC 154.4, 135.3, 130.3 and 105.5 that further supported the presence of an aromatic ring in the molecule. Another downfield resonating signal appearing as doublet of triplet at δH 6.32 (1H, J = 5.5 Hz, 16.0 Hz, H-2) and
a broad doublet of doublet at δH 6.54 (1H, J = 1.5 Hz, 16.0 Hz, H-3) indicated the presence of a disubstituted olefinic bond bearing trans stereochemistry. The long range coupling constant (1.5 Hz) displayed by H-3 with the aromatic protons (H-2′ and H-6′) confirmed the attachment of C-3 to the aromatic quaternary carbon. The 13 C NMR spectrum confirmed the 1 presence of this olefinic bond between C-2 ( δC 130.1) and C-3 ( δC 131.3). In the H NMR
spectrum, a six proton singlet resonating at δH 3.85 (6H, s, 3 ′-OCH 3 and 5 ′-OCH 3) was assigned to the two methoxy groups attached to the aromatic ring. The integration and appearance of signals of these methoxy groups indicated that these are substituted symmetrically and possess magnetically equivalent protons. In the 13 C NMR spectrum the 1 two methoxy carbon atoms resonated at δC 57.1 (3 ′-OCH 3 and 5 ′-OCH 3). In the H NMR spectrum, a downfield double doublet appearing at δH 4.22 with 2H integral, and having a J value of 5.5 Hz, was assigned to the methylene (H-1) adjacent to the C-2 olefinic proton. The
120 downfield shift showed that this is an oxymethylene and its corresponding carbon resonating 13 at δC 63.6 ppm in the C NMR spectrum further confirmed its presence in the molecule. On the basis of the data discussed above and comparing it with the reported data in literature (Greca MD et al ., 1998), the isolated compound 85 was characterized as 3-(3 ′,5 ′-dimethoxy-
4′-O-β-D-glucopyranosyl-phenyl)-prop-2E-en-1-ol.
A detailed literature search revealed that this is the first report of the occurrence of 85 from C. rothii , however, it has been reported earlier from other species of genus Cordia (Andrew M et al ., 1990) and family Boraginaceae (Bergaoui A et al ., 2004).
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3.2 Studies on Non-polar to Moderately Polar Fractions of Root, Stem and Leaves
Exploiting Gas Chromatography - Flame Ionization Detection (GC-FID) and
Gas Chromatography - Mass Spectrometry (GC-MS):
3.2.1 Methodology:
Owing to the medicinal importance, the genus Cordia has been explored well for its phytochemicals. The literature presents many previous studies but it is noteworthy that, GC- FID and GC-MS have been exploited only to a lesser extent. Only few citations are available for this genus ( vide table 1.3, section: 1.5). Therefore, C. rothii was subjected to the detailed chemical investigation using GC-MS and GC-FID techniques for its phytochemicals.
Thus various fractions and sub-fractions were obtained from classical solvent extraction and chromatography ( vide experimental). Twelve fractions coded as 2A, 4A, KC-PE, KC-C, 6A, KEA-PE, KEA-C, KA-PE, KA-C, KM-PE, KM-C, and KM-EA, obtained from C. rothii roots (scheme-4.1 to 4.3), were subjected to GC-FID analysis . Since their GC-FID chromatograms were different, these were also analyzed on GC-MS. Similarly nine non-polar to moderately polar fractions, SH, SC, SEA, SME 10%, SME 20%, SME 30% ABCD, SME 30% EFGH, SME 30% IJKL, and SME 30% MNO, obtained from methanol extract of C. rothii stem, using solvent of different polarity (scheme-4.6a and 4.6b) were also analyzed on GC-FID and GC-MS. The study was also extended to five non-polar to moderately polar fractions, HEXCRU, HS-GC-MS, HS28, A(KK), and B(KK), obtained from methanol extract of C. rothii leaves (schemes-4.7 and -4.8).
A protocol as reported in earlier communications (Siddiqui BS et al ., 2004, 2005, 2009, Azmat S et al ., 2010) was used for the GC-FID and GC-MS analyses of these 26 fractions and sub-fractions with little variation. Identifications were carried out mostly with the help of electronic mass spectral survey (NIST, 2005) and other literature. Their order of elution was confirmed and authenticated using retention indices (Kovats E, 1958; Van den dool H and Kratz PD, 1963) published in literature. Constituents were quantified by the area normalization method using the TIC responses on GC-MS
Other metabolites were identified by comparing their EIMS data with those presented in their first isolation reports, for instance, cordiachromene A ( 21 ), cordiachrome C ( 9), cordiaquinol C ( 24 ), and cordiol A ( 29 ) (Manners GD & Jurd L, 1977 and Moir M & Thomson RH, 1973).
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Several constituents remain unidentified, may be these were not part of the mass spectral library used in the present study. Co-elution of the compounds is also a factor that can be compensated using 2D-GC-MS, which unfortunately wasn’t available. Mass spectrum of co- eluted compounds results in additive spectra, which are difficult to hunt in libraries. The concentration variation in co-eluted compounds, even if similar may increase complexity of mass spectrum from case to case (Stein SE, 1999).
Hexadecanoic acid ( 13 ) was found present in most fractions of root (except in the fraction 6A and KEA-C). It was also present in all studied fractions of stem, and leaves. Overall it was found in maximum concentration and is therefore served as a retention time reference. Similarly, hexadecanoic acid methyl ester ( 11 ), octadecanoic acid methyl ester ( 23 ), and octadecanoic acid ( 18 ), found in all fractions of stem also served to calculate the relative retention times. These retention times of these metabolites together with reported retention indices, were helpful in tracing shifts in retention times or scan numbers during GC analyses, if any.
GC-FID and GC-MS were out of order in the Department of Chemistry, University of Karachi. Therefore, analyses were carried out at H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, utilizing HEC Pakistan funded scheme. The author was unable to use the retention time locking features, flow and pressure adjustments, and maintenance of the quality of columns, in different sets of analyses because these samples were submitted in different time intervals, in an outside analytical facility. Hence an expected retention time shift was observed among these different analyses. These variations, after identification of individual mass spectra, were correlated with reported retention indices and calculated relative retention times using 11 , 13 and an unavoidable, usual contaminant di-isooctyl phthalate (a plasticizer).
3.2.2 Results:
Current gas chromatographic investigation revealed the presence of 152 phytoconstituents comprising 95, 29, and 28 constituents in various sub-fractions from root, stem and leaves of C. rothii respectively. In all 79 were identified. 45 constituents were identified in the root extracts, 17 in the extract of stem and 17 in the leaves exrtract. Out of 79 constituents, to the best of our knowledge, 60 have been identified for the first time from C. rothii . These include 32 from root, 14 from stem and 14 from leaves of C. rothii . However, 57 constituents have been reported for the first time from genus Cordia (31, 13 and 13 natural constituents identified from root, stem, and leaves respectively). Out of the total identified constituents, 34 constituents have been reported for the first time from family Boraginaceae including 21, 6 and 7 metabolites from root, stem, and leaves respectively. 123
In the study of sub-fractions 2A, obtained from the root extracts of the plant, was found to give the most promising results with 25 constituents in the GC chromatogram. Out of these 19 phytochemicals were identified comprising 76% by composition. However, among the sub- fractions of the stem, fraction SH was the most productive with 31 constituents. Out of these 22 were identified, constituting ~71% by composition. Similarly in the studies of the sub- fractions from leaves, fraction HS-GC-MS, exhibited maximum success with 31 constituents. 19 of these were identified, constituting 61% approximately.
45 Phytoconstituents were identified from root while 50 can’t be identified; 17 constituents were identified in stem and 12 remained unidentified. In case of leaves, the identified and unidentified constituents were found to be 17 and 11 respectively. Altogether 152 phytochemicals were observed in the GC-MS studies on root, stem, and leaves of C. rothii .
The various fractions and sub-fractions obtained from root, stem, and leaves on average has resulted in the identification of more than approximately 44%, 82%, and 75% constituents, on the basis of their quantification using TIC responses in GC-MS. Out of these 152 botanicals, 79 were identified comprising approximately 52% on TIC response in concentration.
The results of qualitative and quantitative analyses of the aforementioned 26 fractions from root, stem, and leaves, are presented in individual tables of each fraction vide experimental . Structures of the identified constituents are compiled in respective figures-3.9, 3.10, and 3.11. The qualitative and quantitative data of collective results of different individual fractions obtained from root, stem and leaves are summarized in tables -4.13, 4.23, and 4.29 respectively. Complete results are presented in table-3.1.
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Table-3.1: Results of GC-MS Studies on Root, Stem, and Leaves of C. rothii . Constituent (given number)# Source Report RI References n-Tridecane ( 1) R a,b 1300 Kováts E 206, 1958 n-Tetradecane ( 2) R a,b 1400 Kováts E 206, 1958 n-Pentadecane ( 3) R b 1500 Kováts E 206, 1958 iso -Hexadecane ( 4) R a - - n-Dodecanoic acid ethyl ester R a,b,c 1554 Beaulieu JC & Grimm (Lauric acid ethyl ester) ( 5) CC, 2001 n-Heptadecane ( 6) R b 1700 Kováts E 206, 1958 n-Tetradecanoic acid ethyl ester R a,b,c 1798 Isidorov VA et al ., 2001 (Myristic acid ethyl ester) ( 7) n-Octadecane ( 8) R, S - 1800 Kováts E 206, 1958 5,6,7,8-tetrahydro-7-isopropenyl- R c n.a. Moir M & Thomson RH, 6-methyl-6-vinyl naphthalene- 1973 1,4-dione (Cordiachrome C) ( 9) n-Nonadecane ( 10 ) R, S - 1900 Kováts E 206, 1958 n-Hexadecanoic acid methyl ester R, S, L a,b 1938 Ansorena D et al ., 2000 (Palmitic acid methyl ester) ( 11 ) n-Hexadecanoic acid ethyl ester R, L a,b,c 1975 Andrade EHA et al ., 1998 (Palmitic acid ethyl ester) ( 12 ) n-Hexadecanoic acid R, S, L - 1983 Ansorena D et al ., 2000 (Palmitic acid) ( 13 ) Octadec-9Z-enoic acid methyl R, S a,b 2107 Ansorena D et al ., 2000 ester (Oleic acid methyl ester) ( 14 ) Octadec-9Z,12 Z-dienoic acid R a,b,c 2162 Demyttenaere JCR et al ., ethyl ester 2003 (Linoleic acid ethyl ester) ( 15 ) Octadec-9E-enoic acid ethyl ester R a,b,c 2168 Demyttenaere JCR et al ., (Elaidic acid ethyl ester) ( 16 ) 2003 Octadec-9Z-enoic acid ethyl ester R a,b,c 2171 Siddiqui BS, et al ., 2004 (Oleic acid ethyl ester) ( 17 ) n-Octadecanoic acid R, S, L - 2177 Ansorena D et al ., 2000 (Stearic acid) ( 18 )
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Constituent (given number)* Source Report RI References Stigmasta-3,5-diene ( 19 ) R, S, L a,b,c 2525 Estimated value from NIST* n-Hexadecane ( 20 ) R B 1600 Kováts E 206, 1958 2-Methyl-2-(4-methylpent-3- R a,c n.a. Manners GD & Jurd L, enyl)-2H-chromen-6-ol 1977 (Cordiachromene A) ( 21 ) Octadec-9Z,12Z-dienoic acid R, S, L a,b 2098 Palmeira SFJ et al ., 2001 methyl ester (Linoleic acid methyl ester) ( 22 ) Octadecanoic acid methyl ester R, S, L a,b,c 2134 Ansorena D et al ., 2000 (Stearic acid methyl ester) ( 23 ) 5,6,7,8-tetrahydro-7-isopropenyl- R a,c n.a. Manners GD & Jurd L, 6-methyl-6-vinylnaphthalene-1,4- 1977 diol (Cordiaquinol C) ( 24 ) Stigmasta,4-22-diene-3β-ol ( 25 ) R a,b,c 2739 Estimated value from NIST* Stigmast-5-en-3β-ol R, S - 2731 Estimated value from (β-Sitosterol) ( 26 ) NIST* Stigmasta-5,22-diene-3β-ol R - 2739 Estimated value from (Stigmasterol) ( 27 ) NIST* (1 E)-4-(3-Hydroxy-1-propenyl)- R, S a,b,c 1727 Yousuf M et al ., 1998 2-methoxyphenol (Coniferyl alcohol) ( 28 ) 5,6,7,8,8a,9,10,10a-Octahydro- R a,c 2274 Manners GD & Jurd L, 5,5-dimethylanthracene-1,4,8a- 1977 triol (Cordiol A) ( 29 ) Octadec-9Z,12Z-dienoic acid R, S, L - 2157 Ansorena D et al ., 2001 (Linoleic acid) ( 31 ) 9,19-Cyclolanost-24-en-3β-ol R a,b 2816 Estimated value from (Cycloartenol) ( 32 ) NIST* 24-Methylene-9,19- R a,b,c 2834 Estimated value from cyclolanostan-3β-ol NIST* (24-Methylenecycloartanol) ( 33 ) Phenylethene (Styrene) ( 34 ) R a,b,c 895 Kondjoyan N et al ., 1997
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Constituent (given number)* Source Report RI References Acetic acid phenylmethyl ester R a,b,c 1184 Bauchot AD et al ., 1998 (Benzyl acetate) ( 35 ) 2,4-Dihydroxy-5- R a,b,c 1118 Estimated value from methylpyrimidine (Thymine) ( 36 ) NIST* 2,3-Dihydro-3,5-dihydroxy-6- R a,b,c 1167 D' Arcy BR et al ., 1997 methyl-4H-pyran-4-one ( 37 ) 5-(Hydroxymethyl)- 2-furan- R a,b 1256 D' Arcy BR et al ., 1997 carboxaldehyde (Hydroxymethyl furfuraldehyde) ( 38 ) n-Dodecanoic acid R a,b 1566 Lee S-R et al ., 1991 (Lauric acid) ( 39 ) 3,4-Dihydroxy- benzene- R a,b 1790 Estimated value from propanoic acid NIST* (Hydrocaffeic acid) ( 40 ) n-Tetradecanoic acid R, S, L - 1774 Ansorena D et al ., 2000 (Myristic acid) ( 41 ) Octadec-9Z-enoic acid R, S, L - 2161 Priestap HA et al ., 2003 (Oleic acid) ( 42 ) Hexadecanoic acid 2-hydroxy-1- R a,b,c 2498 Estimated value from (hydroxymethyl)-ethyl ester NIST* (2-Monopalmitin) ( 43 ) 4α,14 α-Di-methyl-9,19- R a,b,c 2760 Estimated value from cycloergost-24(28)-en-3β-ol NIST* (Cycloeucalenol) ( 44 ) 4-Hydroxy-benzoic acid R, S a,b 1522 Jerkovic I et al ., 2010 (p-Salicylic acid) ( 45 ) 4-Hydroxy-3-methoxy-benzoic R, S a,b 1608 Alissandrakis E et al ., acid (Vanillic acid) ( 46 ) 2005 Nonanedioic acid monomethyl S a,b,c 1539 Estimated value from ester (Azelaic acid monomethyl NIST* ester) ( 47 ) 6,10,14-Trimethyl-pentadecan-2- S, L A 1845 Aligiannis N et al ., 2001 one (Hexahydrofarnesyl acetone) ( 48 ) n-Eicosane ( 49 ) S - 2000 Kováts E 206, 1958
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Constituent (given number)* Source Report RI References n-Heptadecanoic acid methyl S, L a,b 2028 Rostad CE & Pereira WE, ester 1986 (Margaric acid methyl ester) ( 50 ) n-Heptadecanoic acid S a,b 2077 Alissandrakis E et al ., (Margaric acid) ( 51 ) 2005 n-Heneicosane ( 52 ) S B 2100 Kováts E 206, 1958 n-Eicosanoic acid methyl ester S a,b 2224 Chen PH et al ., 2002 (Arachidic acid methyl ester) ( 53 ) n-Eicosanoic acid S A 2230 Donnelly JR et al ., 1993 (Arachidic acid) ( 54 ) n-Heneicosanoic acid methyl S a,b,c 2318 Rostad CE & Pereira WE, ester 1986 (Cerotic acid methyl ester) ( 55 ) n-Docosanoic acid methyl ester S, L a,b 2531 Rostad CE & Pereira WE, (Behenic acid methyl ester) ( 56 ) 1986 n-Tricosanoic acid methyl ester S, L a,b,c 2632 Rostad CE & Pereira WE, (Tricosylic acid methyl ester) 1986 (57 ) n-Tetracosanoic acid methyl ester S, L a,b 2674 Estimated value from (Lignoceric acid methyl ester) NIST* (58 ) n-Hexacosanoic acid methyl ester S a,b,c 2872 Estimated value from (Hexacosylic acid methyl ester) NIST* (59 ) Olean-12-en-3β-ol S B 3237 Radulovic NS et al ., 2009 (β–Amyrin) ( 60 ) Stigmasta-3,5-dien-7-one S a,b,c 2696 Estimated value from (β-Saccharostenone) ( 61 ) NIST (2 S-) Methyl 2-hydroxy-3- S A 1642 Siddiqui BS et al ., 2006 (4 ′-hydroxyphenyl)-propanoate (Latifolicinin C) (62 ) 25-methylene-9,19- S a,b,c 2834 Estimated value from cyclolanostane-3β-ol NIST* (Cyclolaudenol) (63 )
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Constituent (given number)* Source Report RI References (E,E) 2,4-Heptadienal ( 64 ) L a,b,c 994 Triqui R & Bouchriti N, 2003 2,7-Dimethyl-1,6-octadiene ( 65 ) L a,b,c 968 Estimated value from NIST* n-Octanoic acid methyl ester L a,b,c 1112 Rostad CE &Pereira WE, (Caprylic acid methyl ester) ( 66 ) 1986 9-Oxo-nonanoic acid methyl ester L a,b,c 1439 Elmore JS et al ., 2002 (Methyl azelaaldehydate) ( 67 ) iso -Hexadecanoic acid ( 68 ) L a,c - - Octadec-9E-enoic acid L a,b 2175 Estimated value from (Elaidic acid) ( 69 ) NIST* n-Tetradecanoic acid methyl ester L a,b,c 1695 Rostad CE & Pereira WE, (Myristic acid methyl ester) ( 70 ) 1986 n-Pentadecanoic acid methyl ester L a,b 1784 Rostad CE and Pereira (Methyl pentadecanoate) ( 71 ) WE, 1986 Hexadec-9Z-enoic acid methyl L a,b 1886 Estimated value from ester (Palmitoleic acid methyl NIST* ester) ( 72 ) Octadec-9Z,12 Z,15 Z-trienoic acid L a,b 2099 Senatore F & Bruno M, methyl ester 2003 (Linolenic acid methyl ester) ( 73 ) 3,7,11,15-Tetramethyl-hexadec- L a 2118 Esteban J et al ., 1999 2-en-1-ol (Phytol) ( 75 ) τ-Cadinene ( 76 ) L a,b,c 1484 Oprean R et al ., 1998 n-Dodecanoic acid methyl ester L a,b 1500 Donnelly JR et al ., 1993 (Lauric acid methyl ester) ( 77 ) n-Pentadecanoic acid L a 1851 Berdague J-L et al ., 1991 (Pentadecylic acid) ( 78 ) n-Tetracosane ( 86 ) L b 2400 Kováts E 206, 1958 n-Hexacosane ( 87 ) L - 2600 Kováts E 206, 1958 n-Octacosane ( 88 ) L b 2800 Kováts E 206, 1958
Identified/total: 79/152; % Identified (TIC): ~52 Key: # Numbers given to the constituents are arranged in the order of their appearance in experimental R = root; L = leaves; S = stem; a = New from C. rothii ; b = New from genus Cordia ; c = New from family Boraginaceae; * estimated values mentioned in NIST are on a column different from the column used in the study
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RO H3C CH2 CH3 n n O n n = 9, n-Tridecane ( 1), C 13 H28 n=12, iso - n =10, R = CH 2CH 3, n = 10, n-Tetradecane ( 2), C 14 H30 hexadecane n-Dodecanoic acid ethyl ester n = 11, n-Pentadecane ( 3), C 15 H32 (4), C 16 H34 (lauric acid ethyl ester, 5), C 14 H28 O2 n = 12, n-Hexadecane ( 20 ), C 16 H34 n = 12, R = CH 2CH 3, n = 13, n-Heptadecane ( 6), C 17 H36 n-Tetradecanoic acid ethyl ester n = 14, n-Octadecane ( 8), C 18 H38 (myristic acid ethyl ester, 7), C 16 H32 O2 n = 15, n-Nonadecane ( 10 ), C 19 H40 n =14, R = CH 3, n-Hexadecanoic acid methyl ester RO
(palmitic acid methyl ester, 11 ), C 17 H34 O2 O
R = CH 3, Octadec-9Z-enoic acid methyl ester n = 14, R = CH 2CH 3,
(oleic acid methyl ester, 14 ), C 19 H36 O2 n-Hexadecanoic acid ethyl ester
R = CH 2CH 3, Octadec-9Z-enoic acid ethyl ester (palmitic acid ethyl ester, 12 ), C 18 H36 O2
(oleic acid ethyl ester, 17 ), C 20 H38 O2 n =14, R = H, R = H, Octadec-9Z-enoic acid n-Hexadecanoic acid
(oleic acid, 42 ), C 18 H34 O2 (palmitic acid, 13 ), C 16 H32 O2 n =16, R = H, RO n-Octadecanoic acid O (stearic acid, 18 ), C 18 H36 O2
R = CH 2CH 3, Octadec-9Z,12 Z-dienoic acid n =16, R = CH 3, ethyl ester n-Octadecanoic acid methyl ester
(linoleic acid ethyl ester, 15 ), C 20 H36 O2 (stearic acid methyl ester, 23 ), C 19 H38 O2
R = CH 3, Octadec-9Z,12 Z-dienoic acid n =10, R = H, methyl ester n-Dodecanoic acid (lauric acid, 39 ),
(linoleic acid methyl ester, 22 ), C12 H24 O2
C19 H34 O2 n = 12, R = H, R = H, Octadec-9Z,12 Z-dienoic acid n-Tetradecanoic acid (myristic acid, 41 ),
(linoleic acid, 31 ), C 18 H32 O2 C14 H28 O2
O O HO O O HO Octadec-9E-enoic acid ethyl ester Hexadecanoic acid 2-hydroxy-1-(hydroxymethyl)-
(elaidic acid ethyl ester, 16 ), C 20 H38 O2 ethyl ester (2-monopalmitin, 43 ), C 19 H38 O4
Figure-3.9(a): Hydrocarbon, Fatty Acids and Fatty Acid Derivatives Identified in the Root of C. rothii
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O HO
O H O Cordiachromene A ( 21 ), C 16 H20 O2 Cordiachrome C ( 9), C 16 H18 O2 OH OH H H OH
H H OH OH H H
Cordiaquinol C ( 24 ), C 16 H20 O2 Cordiol A ( 29 ), C 16 H22 O3
Figure-3.9(b): Sesquiterpenoids Identified in the Root of C. rothii
O OH
HO (1 E)-4-(3-Hydroxy-1-propenyl)-2-methoxyphenol Phenylethene (styrene, 34 ), C 8H8 (coniferyl alcohol, 28 ), C 10 H12 O3 O O
O NH N O H Acetic acid phenylmethyl ester 5-Methyl-2,4-pyrimidinediol (benzyl acetate, 35 ), C 9H10 O2 (Thymine, 36 ), C 5H6N2O2 O O O OH HO OH O 5-(Hydroxymethyl)-2-furancarboxaldehyde 2,3-Dihydro-3,5-dihydroxy-6-methyl- (Hydroxymethylfurfuraldehyde, 38 ), C 6H6O3 4H-pyran-4-one ( 37 ), C 6H8O4
O O C OH OH
HO OH R OH 3,4-Dihydroxy- benzenepropanoic acid (hydrocaffeic acid, 40 ), C 9H10 O4 R = H, 4-Hydroxy- benzoic acid
(p-salicyclic acid, 45 ), C 7H6O3
R = OCH 3, 4-Hydroxy-3-methoxy- benzoic acid
(vanillic acid, 46 ), C 8H8O4
Figure-3.9(c): Aromatics, Phenolics and Miscellaneous Constituents Identified in the Root of C. rothii
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H H H H H HO
Stigmasta-3,5-diene ( 19 ), C 29 H48 Stigmasta-4,22-diene-3β-ol ( 25 ), C 29 H48 O
H H H H
H H H H HO HO Stigmast-5-en-3β-ol Stigmasta-5,22-diene-3β-ol
(β-sitosterol, 26 ), C 29H50 O (Stigmasterol 27 ), C 29 H48 O
H H H H
HO HO H H 9,19-Cyclolanost-24-en-3β-ol 24-Methylene-9,19-cyclolanostan-3β-ol
(cycloartenol, 32 ), C 30 H50 O (24-methylenecycloartanol, 33 ), C 31 H52 O
H
HO H
4α,14 α-Dimethyl-9,19-cycloergost-24(28)-en-3β-ol
(cycloeucalenol, 44 ), C 30 H50 O
Figure-3.9(d): Triterpenoids and Phytosterols Identified in the Root of C. rothii
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HO O O
O O Nonanedioic acid monomethyl ester 6,10,14-Trimethyl-pentadecan-2-one (hexahydrofarnesyl acetone, 48 ), C H O (azelaic acid monomethyl ester, 47 ), C 10 H18 O4 18 36
O H C CH CH R n 3 2 3 O n n =15, R = CH 3, n-Heptadecanoic acid methyl ester n=16, n-Eicosane ( 49 ), C 20 H42
(margaric acid methyl ester, 50 ), n=17, n-Heneicosane ( 52 ), C 21 H44
C18 H36 O2 n =15, R = H, n-Heptadecanoic acid
(margaric acid, 51 ), C 17 H34 O2 H n =18, R = CH 3, n-Eicosanoic acid methyl ester (arachidic acid methyl ester, 53 ), H C H O HO 21 42 2 H n =18, R = H, n-Eicosanoic acid Olean-12-en-3β-ol ( β-amyrin, 60 ), C 30 H50 O
(arachidic acid, 54 ), C 20 H40 O2 n =19, R = CH 3, n-Heneicosanoic acid methyl ester (cerotic acid methyl ester, 55 ),
C22 H44 O2 n =20, R = CH 3, n-Docosanoic acid methyl ester (behenic acid methyl ester, 56 ), O C23 H46 O2 Stigmasta-3,5-dien-7-one n =21, R = CH 3, n-Tricosanoic acid methyl ester (β-saccharostenone, 61 ), C H O (tricosylic acid methyl ester, 57 ), 29 46
C24 H48 O2 n =22, R = CH 3, n-Tetracosanoic acid methyl ester H (lignoceric acid methyl ester, 58 ), H
C25 H50 O2 n =24, R = CH , n-Hexacosanoic acid methyl ester HO 3 H (hexacosylic acid methyl ester, 59 ), 25-Methylene-9,19-cyclolanostan-3β-ol
C27 H54 O2 (cyclolaudenol, 63 ) O
O H OH H HO Methyl 2-hydroxy-3-(4 ′-hydroxyphenyl)-propanoate HO (latifolicinin C, 62 ), C 10 H12 O4 H 9,19-Cyclolanost-24-en-3β-ol
(cycloartenol, 32 ), C 30 H50 O Figure-3.10: Phytochemicals Identified in the Stem of C. rothii .
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O
(E,E) 2,4-Heptadienal ( 64 ), C 7H10 O
2,7-Dimethyl-1,6-octadiene ( 65 ), C 10 H18 O n O O O n =6, n-Octanoic acid methyl ester O 9-Oxo-nonanoic acid, methyl ester (methyl caprylate, 66 ), C 9H18 O2 (methyl azelaadehydate, 67 ), C H O n =12, n-Tetradecanoic acid methyl ester 10 18 3
(methyl myristate, 70 ), C 15 H30 O2 HO n =13, n-Pentadecanoic acid methyl ester 11 O (methyl pentadecanoate, 71 ), C 16 H32 O2 iso -Hexadecanoic acid ( 68 ), C H O n =10, n-Dodecanoic acid methyl ester 16 32 2 HO (methyl laurate, 77 ), C 13 H26 O2 O Octadec-9E-enoic acid (elaidic acid, 69 ),
C18 H34 O2 O O
O O
Hexadec-9Z-enoic acid methyl ester Octadec-9Z,12 Z, 15 Z-trienoic acid methyl ester
(palmitoleic acid methyl ester, 72 ), C 17 H32 O2 (linolenic acid methyl ester, 73 ), C 19 H32 O2
HO
3,7,11,15-Tetramethyl-2-hexadecen-1-ol
(phytol, 75 ) C 20 H40 O 1,2,3,4,4a,5,6,8a-octahydro-7-methyl-4- H3C CH3 n methylene-1-(1-methylethyl)-naphtahlaene
n=20, n-tetracosane (86 ), C 24 H50 (τ- Cadinene) ( 76 ), C 15 H24
n=22, n-hexacosane ( 87 ), C 26 H54 HO n n=24, n-octacosane ( 88 ), C 28 H58 O n =13, n-Pentadecanoic acid
(pentadecylic acid, 78 ), C 15 H30 O2
Figure-3.11: Phytochemicals Identified in the Leaves of C. rothii
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3.2.3 Discussion:
The present study can be considered as an attempt for utilizing rather a simple 1D-GC-MS technique for the metabolomic studies on the chemical constituents present in the non-polar to moderately-polar fractions of the plant. For instance, a fraction from the extract of the stem coded as SME 30% ( vide section: 4.2.2) was subjected to column chromatography. Using isocratic elution with solvent mixture EtOAc:MeOH (70:30) fifteen eluates (SME-A to SME-O) were collected. Collections were done on the basis of colour intensity of eluates. The TLC profiles of the major constituents of these fractions were quite similar. It was expected that fractions SME-A to SME-O might also have different minor metabolites, masked in the major constituents. Later, these were pooled into four major fractions on the basis of their TLC profiles and coded as SME 30% ABCD, SME 30% EFGH, SME 30% IJKL and SME 30% MNO respectively, assuming that these fractions might contain different metabolites. Each of these pooled sub-fractions was then sequentially subjected to detail GC-FID/GC-MS studies. This strategy resulted in interesting findings. n-hexadecanoic acid methyl ester ( 11 ) n-hexadecanoic acid ( 13 ), octadec-9Z-enoic acid methyl ester ( 14 ), n-octadecanoic acid methyl ester ( 23 ), octadec-9Z-enoic acid ( 42 ), n- octadecanoic acid ( 18 ), n-eicosanoic acid methyl ester ( 53 ), n-docosanoic acid methyl ester ( 56 ), n-tricosanoic acid methyl ester ( 57 ), and n-tetracosanoic acid methyl ester ( 58 ) (arranged in the order of elution and appearance) were observed in all the afore mentioned pooled fractions. Compound 31 was eluted from the column in initial fractions only and was not observed at all in the chromatogram of the later fractions i.e., SME 30% EFGH, SME 30% IJKL and SME 30% MNO. However, constituent 22 did not appear in the chromatogram of the late eluting fractions and was only observed in the initial three pooled fractions. Constituents 19 , 26 and 60 were common in fractions SME 30% ABCD and SME 30% EFGH only whereas metabolite 54 and 55 were only observed in the middle fraction i.e., SME 30% EFGH. A minor amount of metabolite 55 tailed in fraction SME 30% IJKL also. The overall metabolomic profile of the study is given in figure-3.8.
For the ease of understanding, discussion has been written as per chemical classes of the metabolites, which in turn are arranged in the order of elution as per experimental. Short but supporting information on their natural existence and biological importance has also been given.
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Figure-3.8: Metabolomic Profile of Phytoconstituents Identified in GC-MS Studies.
The present study has resulted in the identification of various hydrocarbons as shown below:
H3C CH2 CH3 n
n =9, n-Tridecane ( 1), C 13 H28 n =10, n-Tetradecane ( 2), C 14 H30
n =11, n-Pentadecane ( 3), C 15 H32 n =12, n-Hexadecane ( 20 ), C 16 H34
n =13, n-Heptadecane ( 6), C 17 H36 n =14, n-Octadecane ( 8), C 18 H38
n =15, n-Nonadecane ( 10 ), C 19 H40 n =16, n-Eicosane ( 49 ), C 20 H42
n =17, n-Heneicosane ( 52 ), C 21 H44 n =20, n-Tetracosane ( 86 ), C 24 H50
n =22, n-Hexacosane ( 87 ), C 26 H54 n =24, n-Octacosane ( 88 ), C 28 H58
1, 2, 3, 6, 8, 10 , and 20 were identified from the roots, 49 and 52 from the stem, while 86 , 87 , and 88 were identified from the leaves of C. rothii . These findings show that root being underground probably has retained the volatiles, while leaves, do not. This is the first report of 1 and 2 from C. rothii and genus Cordia . Rest of the hydrocarbons identified in the current study, have been reported earlier from the C. rothii and family Boraginaceae (Maggi F et al ., 2009; Santana O et al ., 2012; Papageorgiou VP and Assimopoulou AN, 2003). Beside these, iso -hexadecane ( 4) has also been identified, which has not been reported earlier from this plant.
12 iso -hexadecane ( 4), C 16 H34
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Several hydrocarbons are reported as natural constituents from different plants. 1, 2, 3, 4, 6, 8, 10 , 20 , 49 , 52 , 86 , 87 , and 88 all have been reported as the natural constituents from various sources e.g., benzene extract of Trichodesma amplexicaule Roth (Singh B and Singh S, 2003); heartwood extract of Manilkara bidentata (Cocker W and Shaw SJ, 1963); plant extract of Acrostichum aureum (Uddin SJ et al ., 2012); leaves extract of Moschosma polystachyum Linn (Rajkumar S and Jebanesan A, 2004); seed oil of Cassia glauca (Kumar D et al ., 2013); Valencia orange oil (Hunter GLK and Brogden JrWB, 1966); essential oil of Sesuvium portulacastrum (Magwa ML et al ., 2006); essential oil of Carduus pycnocephalus L. (Al-Shammari LA et al ., 2012).
Crinum ornatum (Ait) Bury is a medicinal plant, used traditionally against various illnesses. Its essential oil possesses cytotoxicity due to the presence of higher amount of hydrocarbons 49 and 52 (Oloyede GK et al ., 2010). Compound 6, reported as major constituents (4.33%) of Angelica purpuraefolia Chung essential oil, has been found to exhibit immunotoxic activity against Aedes aegypti L. (Park YJ et al ., 2010). Similarly 52 is used to control Aedes aegypti mosquito population, responsible for dangue hemorrhagic fever (Bhutia YD et al ., 2010). Several hydrocarbons have been used in the cosmetics composition of skin (Park SH et al ., 2010)
Major portion of studied extracts, particularly those extracted with non-polar solvents, consists of free fatty acids and their methyl esters. Fatty acids are commonly occuring important phytochemicals. Certain fatty acids are used for the prevention of cardiovascular diseases. Tumor cells are killed by the carboxylic acid group of free fatty acids. It has also been observed that these fatty acids show selective behaviour of inhibition for tumor cell growth (Zhu YP et al ., 1989). These are also found to possess antioxidant, anticancer, and hypotensive properties (Zhao X et al ., 2013). Saturated free fatty acids identified in the present study are listed below:
HO n O
n =10, n-Dodecanoic acid (lauric acid, 39 ), C 12 H24 O2
n = 12, n-Tetradecanoic acid (myristic acid, 41 ), C 14 H28 O2
n =13, n-Pentadecanoic acid (pentadecylic acid, 78 ), C 15 H30 O2
n =14, n-Hexadecanoic acid (palmitic acid, 13 ), C 16 H32 O2
n =15, n-Heptadecanoic acid (margaric acid, 51 ), C 17 H34 O2
n =16, n-Octadecanoic acid (stearic acid, 18 ), C 18 H36 O2
n =18, n-Eicosanoic acid (arachidic acid, 54 ), C 20 H40 O2
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HO 11
O iso -hexadecanoic acid ( 68 ), C 16 H32 O2
Compounds 13 , 18 , 39 and 41 were identified from roots; 51 , and 54 from stem, and 78 from leaves. 13 , 18 , and 41 have been reported earlier from seed oil of C. rothii (Daulatabad CMJD et al ., 1992) and family Boraginaceae (Velasco L and Gofman FD, 1999; Özcan T, 2008). 13 , 18 , 41 , 54 and 78 have been previously reported from genus Cordia (Pino JA et al ., 2002; Bonesi M et al ., 2011; Miralles J et al ., 1989; Rameshwar D et al ., 2006; Theagarajan KS et al ., 1977) whereas 39 and 51 are reported for the first time from the plant under study and genus Cordia . However, these constituents have been reported previously from other species of the family Boraginaceae (Velasco L and Gofman FD, 1999; Papageorgiou VP and Assimopoulou AN, 2003; Özcan T, 2008). This is also the first report of iso -hexadecanoic acid ( 68 ) from C. rothii and family Boraginaceae.
Compound 13 was the most abundant saturated fatty acid distributed in nature. Among the commercial sources, it was most plentiful in palm oil (lipid library, 2013). 13 , 39 , and 41 have also been identified in essential oil of Carduus pycnocephalus L. (Al-Shammari LA et al ., 2012); roots of Solanum erianthum (Chen Y-C et al ., 2013); and fruits of Mandragora autumnalis (Hanuš LO et al ., 2006).
13 , 18 , 39 , 41 , 51 , 54 , and 78 have been reported in the fatty acid composition of the leaves of Excoecaria agallocha , of these 13 , 18 , 39 , and 41 were found to possess antimicrobial properties (Agoramoorthy G et al ., 2007). 13 and 78 attribute antimicrobial property to Andrographis paniculata (Wei LS et al ., 2011). Role of fatty acid mixture consisting of 13 (~30%) and 18 (~17%) along with some other unsaturated acids has been evaluated on the cell proliferation (immunomodulatory property), and found to show inhibitory effect (Renner L et al ., 2013). It was observed that 13 exhibited significant antithrombic activity (Yang N-Y et al ., 2011). 13 , 18 , 51 , and 54 have been identified in desmodium elegans . These compounds possess no cytotoxicity but are reported to be moderately antibacterial and insecticidal against various microorganisms (Khan A and Usman R, 2012). Compound 39 exhibited significant inhibitory activity against Gram-positive and -negative bacteria bacteria (Kabara J et al ., 1972; Fischer CL et al ., 2012) and has been found to exhibit inhibitory effect on proliferation of prostate cancer cells (Liu J et al ., 2009). 39 and 41 have been reported as 5α-reductase inhibitor and antimycobacterial (Abe M et al ., 2009; Saravanakumar DEM et al ., 2008). Strong antifungal activity of 39 has also been reported (Altieri C et al ., 2009). 18 can comprise 80% of the total fatty acid in gangliosides. There is a report that combination of
138
13 , 18 , and 51 that significantly inhibited in vitro prostate cancer in humans (Kim E-K et al ., 2010).
Derivatives of fatty acids produce signals in plants. These play important role in plant development, defense and communication between plants and with different organisms as well (Hans W, 2002). Following fatty acid methyl esters were identified in the current study.
O n O
n =6, n-Octanoic acid methyl ester (caprylic acid methyl ester, 66 ), C 9H18 O2
n =10, n-Dodecanoic acid methyl ester (lauric acid methyl ester, 77 ), C 13 H26 O2
n =12, n-Tetradecanoic acid methyl ester (myristic acid methyl ester, 70 ), C 15 H30 O2
n =13, n-Pentadecanoic acid methyl ester (pentadecylic acid methyl ester, 71 ), C 16 H32 O2
n =14, n-Hexadecanoic acid methyl ester (palmitic acid methyl ester, 11 ), C 17 H34 O2
n =15, n-Heptadecanoic acid methyl ester (margaric acid methyl ester, 50 ), C 18 H36 O2
n =16, n-Octadecanoic acid methyl ester (stearic acid methyl ester, 23 ), C 19 H38 O2
n =18, n-Eicosanoic acid methyl ester (arachidic acid methyl ester, 53 ), C 21 H42 O2
n =19, n-Heneicosanoic acid methyl ester (cerotic acid methyl ester, 55 ), C22 H44 O2
n =20, n-Docosanoic acid methyl ester (behenic acid methyl ester, 56 ), C 23 H46 O2
n =21, n-Tricosanoic acid methyl ester (tricosylic acid methyl ester, 57 ), C 24 H48 O2
n =22, n-Tetracosanoic acid methyl ester (lignoceric acid methyl ester, 58 ), C 25 H50O2
n =24, n-Hexacosanoic acid methyl ester (hexacosylic acid methyl ester, 59 ), C 27 H54 O2
Saturated fatty acids 39 , 41 , 78 , 13 , 51 , 18 and 54 were also identified as their methyl esters, with the respective numbers 77 , 70 , 71 , 11 , 50 , 23 , and 53. 55 -59 , and 66 were also identified in current investigation. 11 and 23 were identified in the root, 50 , 53 , and 55 -59 in stem, and 66 , 70 , 71 , and 77 , from leaves of the plant. The methyl esters; 23 , 55 , 57 , 59 , 66 , and 70 are reported for the first time from C. rothii, genus Cordia , and family Boraginaceae. This is also the first report of the presence of 11 , 50 , 53 , 56 , 58 , 71 , and 77 from C. rothii and genus Cordia but these metabolites have been reported earlier from other species of family Boraginaceae (Kawata J et al ., 2008; Santana O et al ., 2012; Papageorgiou VP and Assimopoulou AN, 2003).
Fatty acid methyl esters are usual constituents of plants. These are particularly present in seeds and seed oils. For example 11 , 23 , 50 , 53 , 55 , 56 , 57 , 58 , 59 , 70 , 71, and 77 have been reported as natural constituents in the extracts and essential oils of Plumbago zeylanica Linn. (Ajayi GO et al ., 2011); Satureja thymbra and Satureja cuneifolia (Goren CA et al ., 2003); Rorippa indica L. (Ananthi P and Kumari BDR, 2013b); Carduus pycnocephalus L. (Al-
139
Shammari LA et al ., 2012); Plastrum Testudinis (a Chinese traditional medicine) (Wang T-T et al ., 2012) Nelumbo nucifera (Jeon S et al ., 2009) Cephalaria species (Kırmızıgül S et al ., 2012), and Phoenix dactylifera L. (Azmat S et al ., 2010).
Compound 77 is found to reduce the oral bacterial arginine deiminase system activity, responsible for dental plaque (Barboza-Silva E et al ., 2009). 23 exhibited inhibitory activity against Staphylococcus aureus , Escherichia coli and Candida albicans (Mubarak AD et al ., 2012). Protective effect of 11 was investigated against silica-induced pulmonary fibrosis in rats (Sharawy MH et al ., 2013). 11 was also used as an antiinflammatory agent and as fragrance (Ananthi P and Kumari BDR, 2013a) and is reported to regulate amount of melanin in human melanocytes during melanogenesis (Jeon S et al ., 2009). 58 has been found to possess anti-diabetic property (Shilpa K et al ., 2009). 11 , 23 , and 70 has been evaluated for growth inhibitory and insecticidal activity (Farag M et al ., 2011, McFarlane JE and Henneberry GO, 1965). Use of 70 as food additive has also been reported (Anon, 1961).
In the present investigation, ethyl esters of relatively short chained and even numbered saturated fatty acids 39 , 41 , and 13 were also identified from the root. Thus n-dodecanoic acid ethyl ester ( 5), n-tetradecanoic acid ethyl ester ( 7), and n-hexadecanoic acid ethyl ester ( 12 ) are reported for the first time from the C. rothii , genus Cordia and family Boraginaceae
O n O
n =10, n-Dodecanoic acid ethyl ester (lauric acid ethyl ester, 5), C 14 H28 O2
n = 12, n-Tetradecanoic acid ethyl ester (myristic acid ethyl ester, 7), C 16 H32 O2
n = 14, n-Hexadecanoic acid ethyl ester (palmitic acid ethyl ester, 12 ), C 18 H36 O2
Compound 5 has been isolated from the pine needles of Cedrus deodara (Zhang JM, 2010); 7 from the seeds and leaves of Phoenix dactylifera L. (Azmat S et al ., 2010); and 12 in the essential oil of Carduus pycnocephalus L (Al-Shammari LA et al ., 2012) and Rorippa indica L. (Ananthi P and Kumari BDR, 2013b).
12 showed hepatoprotective activity (Lu L et al ., 2012) and was also found to exhibit nematicidal, pesticidal, antiandrogenic, and antioxidant properties, besides its use as a flavouring agent in food (Ananthi P and Kumari BDR, 2013b). Risk of experimental tuberculosis is found to be reducing in monkeys by the subcutaneous dose of 5 and 7, and more specifically of 12 (Negre L et al ., 1938). Antimutagenic activities of 5, 7, and 12 have been evaluated against Salmonella typhimurium (Kimuras S and Okazat H, 1986). 7 and 12 have also been evaluated for their biofouling activity (Refaat J et al ., 2009).
140
Unsaturated fatty acids play a vital role in the human body too. For example these are responsible for the normal growth of cells, nerves, and blood vessels inside a body. These are reported to regulate energy requirement and oxygen transportation in the body and decrease the level of cholesterol in blood (Igwe OU and Okwu DE, 2013; Liu J et al ., 2009).
The unsaturated fatty acids identified in the study included octadec-9Z,12 Z-dienoic acid ( 31 ), octadec-9Z-enoic acid ( 42 ), and octadec-9E-enoic acid ( 69 ). Free linolenic acid was not identified in the present study whereas its methyl ester ( 73 ) was identified ( vide infra ).
HO
O
Octadec-9Z,12 Z-dienoic acid (linoleic acid, 31 ), C 18 H32 O2 HO
O
Octadec-9Z-enoic acid (oleic acid, 42 ), C 18 H34 O2
Octadec-9E-enoic acid (elaidic acid, 69 ), C 18 H34 O2
Monounsaturated fatty acid 42 and 69 are geometric isomers while 31 is a polyunsaturated fatty acid. 31 and 42 were identified from root and stem, and 69 from leaves. 31 and 42 were already reported from the C. rothii seed oil (Daulatabad CMJD et al ., 1992) and from other species of genus Cordia (Alanis-Guzman MG et al ., 1995; Alanis-Guzman MG et al ., 1998). This is the first report of occurrence of 69 from C. rothii and genus Cordia , however, 31 , 42 , and 69 has been reported earlier from the other species of family Boraginaceae (Yunusova SG et al ., 2012; Velasco L and Gofman FD, 1999; Özcan T, 2008).
Compounds 31 , 42 , and 69 have been identified in various fatty acid compositions obtained from Excoecaria agallocha (Agoramoorthy G et al ., 2007); Crinum x powellii bulbs (Kissling J et al ., 2005); Melia azedarach L. (Farag M et al ., 2011); and Pinus tabulaeformis (Chen J et al ., 2011).
Constituent 31 has inhibited acetylcholinesterase (Kissling J et al ., 2005) and also reported to reduce blood pressure during hypertension (Al-Bishri WM, 2013). It has also been found to exhibit cytotoxic effect against human breast cancer cells and hepatocarcinoma (Kwak H-Y et al ., 2008). 31 is an important contributor in the process of fertilization and reproduction (Jungheim ES et al ., 2013). It is an active nematocidal agent (Stadler M et al ., 1994). 42 (~46%) along with some other saturated fatty acids, exhibits inhibitory effect on cell proliferation (Renner L et al ., 2013). It has been found to inhibit human telomerase activity significantly (Oda M et al ., 2002) and also exhibit significant antithrombic (Yang N-Y et al .,
141
2011) and antimicrobial property (Wei LS et al ., 2011). Proliferative effect of 69 on human umbilical vein smooth muscle cells has been observed (Li X-P et al ., 2013). Derivatives of 69 are reported for their anti-inflammatory and antitumor potentials (Myhren F et al ., 2012). Together 31 and 42 have been found to possess antimicrobial properties and also inhibit growth of transplanted tumors in mice (Agoramoorthy G et al ., 2007). These have also been found to exhibit strong anti α-glucosidase activity against enzymes of type 2 diabetes (Su C-H et al ., 2013). 31 and 69 have been found to exhibit significant growth regulating effect in seedlings of Pinus tabulaeformis (Chen J et al ., 2011).
It is noteworthy that all the saturated and unsaturated fatty acids; 13 , 18 , 31 , 39 , 41 , 42 , 51 , 54 , 69 , and 78 , identified in this study have also been identified in the ginseng fine roots. Such combination of acids is reported to be useful in improving blood circulation (Hwang IC, 2013).
The esters of unsaturated fatty acids identified in the present study included following:
RO n O
n=3, R= CH 3, Octadec-9Z-enoic acid methyl ester (oleic acid methyl ester, 14 ), C 19 H36 O2
n=3, R=CH 2CH 3, Octadec-9E-enoic acid ethyl ester (elaidic acid ethyl ester, 16 ), C 20 H38 O2
n=3, R=CH 2CH 3, Octadec-9Z-enoic acid ethyl ester (oleic acid ethyl ester, 17 ), C 20 H38 O2
n=1, R= CH 3, Hexadec-9Z-enoic acid methyl ester (palmitoleic acid methyl ester, 72 ), C 17 H32 O2
RO
O
R=CH 2CH 3, Octadec-9Z,12 Z-dienoic acid ethyl ester (linoleic acid ethyl ester, 15 ), C 20 H36 O2
R=CH 3, Octadec-9Z,12 Z-dienoic acid methyl ester (linoleic acid methyl ester, 22 ), C 19 H34 O2 O
O
Octadec-9Z,12 Z, 15 Z-trienoic acid methyl ester (linolenic acid methyl ester, 73 ), C 19 H32 O2
Out of these, esters 14 , 15 , 16 , 17 , and 22 were identified in roots whereas 72 and 73 were identified from leaves. These ethyl esters of unsaturated fatty acids ( 15 , 16 , and 17 ) and methyl esters of unsaturated fatty acids ( 14 , 22 , 72 , and 73 ) are reported for the first time from C. rothii and genus Cordia . The presence of these methyl esters of unsaturated fatty acids from family Boraginaceae, has already been reported previously (Papageorgiou VP and Assimopoulou AN, 2003; Maggi F et al ., 2009).
Compounds 14 , 15 , 16 , 22 , 72 , and 73 are also among the common metabolites and Nigella glandulifera Freyn (Nguyen DH et al ., 2011); Carduus pycnocephalus L. (Al-Shammari LA
142 et al ., 2012); Satureja thymbra and Satureja cuneifolia (Goren CA et al ., 2003); Azadirichta indica A. Juss. (Siddiqui BS et al ., 2004); Brachystegia eurycoma (Igwe OU and Okwu DE, 2013); Crinum x powellii (Kissling J et al ., 2005); Melia azedarah L. (Farag M et al ., 2011) and Cassia glauca (Kumar D et al ., 2013) has been reported as their source.
Compound 14 has been characterized as the skin depigmenting agent (Nguyen DH et al ., 2011). 14 and 22 possessed insecticidal and growth inhibitory properties (Farag M et al ., 2011). Both of these esters are also reported to prolong the life of mice having transplanted tumors (Zhu YP et al ., 1989). 17 is also reported to exhibit strong antimicrobial activity against some oral microorganisms and hence can be used to cure oral infections (Huang CB et al ., 2010). 14 , 22 , and 73 have been identified as strong antimicrobial agents (Mubarak AD et al ., 2012).
Fatty acid ester 15, being the major constituent, in the seed oil of Brachystegia eurycoma , has been found responsible for its antibacterial activity (Igwe OU and Okwu DE, 2013). It has also been identified as acetylcholinesterase inhibitor (Kissling J et al ., 2005) and has been used to cure spermatogenic tissue degeneration induced by hydrogenated peanut oil in rats (Aaes-Jorgensen E et al ., 1957). 72 is also reported as a strong antimicrobial agent against oral pathogens (Huang CB et al ., 2010). 73 has been identified (~5%) from lotus flower essential oil and plays a key role in gray hair treatment and as a tanning agent (Jeon S et al ., 2009). The antimicrobial and antioxidant property of Andrographis paniculata was reported due to the presence of 73 in its leaves extract (Wei LS et al ., 2011). It has also been evaluated for its cytotoxic behaviour against human breast cancer cells and hepatocarcinoma (Kwak H-Y et al ., 2008).
It is interesting to note that stearic acid ( 18 ) and its various analogues or derivatives, formed either by desaturation and/or esterification were found to be present in one or other extracts of different parts of the plant species studied. These included: oleic acid ( 42 ), elaidic acid ( 69 ) and linoleic acid ( 31 ) as unsaturated C-18 acids; 23 , 14 , 22, and 73 , as methyl esters of 18 , 42 , 31 and linolenic acid respectively; and 17 , 16 and 15 , ethyl esters of 42 , 69 and 31 respectively (scheme-3.2a).
Nonanedioic acid monomethyl ester (azelaic acid monomethyl ester, 47 ) was identified in the fractions of stem of the plant; and was the only dicarboxylic acid identified in this study. It was found methylated at one terminus. In GC-MS chromatograms of the stem, its peak appeared shouldering with peak of 4-hydroxy-3-methoxy-benzoic acid ( 46 ). 47 has been reported for the first time from C. rothii , genus Cordia , and family Boraginaceae.
143
O 16 O 23 methylation
O ethylation HO desaturation HO 16 5 7 5 7 O O O 16 69 18 (9E) desaturation
HO 6 7 O HO desaturation HO desaturation 42 6 6 4 (9Z ) O O Linolenic acid 31 (9Z,12Z,15Z) (9Z,12Z) esterification
methylation esterification RO 6 7 O 14: R = CH O O 3 R 17: R = CH CH 6 6 4 2 3 O O 73 15: R = CH2CH3 22: R = CH3 Scheme-3.2a: Biogenetic Correlation Amongst Various Phytochemicals Identified During GC-MS Studies.
144
Earlier 47 has been identified in the flowers of Azadirachta indica A. Juss. (Siddiqui BS et al ., 2009) and reported to exhibit tyrosinase inhibition property (Schallreuter KU and Wood JW, 1990). It has been used in the synthesis of anti-HIV and anti-cancer compounds (Gung BW and Dickson H, 2002).
HO O
O O
Nonanedioic acid monomethyl ester (azelaic acid monomethyl ester, 47 ), C 10 H18 O4
It is interesting to note that 9-oxo-nonanoic acid methyl ester ( 67 ) identified in current study from leaves, is the reduced form of 47 and such biosynthetic relations are well established (section: 2.2). This is the first report of 67 from C. rothii, genus Cordia , and family Boraginaceae.
O O O
9-Oxo-nonanoic acid methyl ester (Methyl azelaadehydate, 67 ), C 10 H18 O3
An interesting biosynthetic relation can be inferred between metabolites 73 , 67 and 47 . 73 can undergo oxidative cleavage to 67 , which in turn can serve as a precursor of 47 or vice versa. 47 was identified from extract of stem while 67 and 73 from the extract of leaves of plant under current investigation. Thus these metabolites support and confirm the identification of constituents in one or other way (scheme-3.2b).
O
O 73 [O]
O O
O 67
[O]
O OH
O 47 O
Scheme-3.2b: Biogenetic relationship between different metabolites
145 A Glyceride, hexadecanoic acid 2-hydroxy-1-(hydroxymethyl)-ethyl ester ( 43 ) commonly known as 2-monopalmitin has also been identified for the first time in the roots of C. rothii. it is also reported for the first time in genus Cordia and family Boraginaceae.
It has been identified earlier in the Gloiopeltis tenax extract. The extracts containing 43 exhibited antioxidant and antimicrobial properties (Zheng J et al ., 2012). It is reported to strongly inhibit growth of bifidobacteria in infants (Powell HJ and May JT, 1981) and helps in palmitic acid absorption in human infant (Filer FJJr et al., 1969). It is also a precursor for triglyceride biosynthesis in intestine (Brown JL and Johnston JM, 1964) and has been recognized as a potent aromatase inhibitory phytoconstituent (Li Y-H et al ., 2009).
O HO O HO
Hexadecanoic acid 2-hydroxy-1-(hydroxymethyl)-ethyl ester (2-monopalmitin, 43 ), C 19 H38 O4
Current study revealed that C. rothii has plenty of fatty acids and their derivatives. This may be a reason for its trivial use in ayurvedic system for cure and as supplement.
GC-MS studies on C. rothii has also led to the identification of various aromatics, which are discussed as benzoic acid derivatives, phenyl propanoids, hydroquinones, phenols, etc. ( vide infra ). Phenylethene ( 34 ) and acetic acid phenyl methyl ester ( 35 ) were rather simpler aromatics identified from C. rothii root.
O
O
Phenylethene Acetic acid phenylmethyl ester
(vinyl benzene, 34 ) C 8H8 (benzyl acetate, 35 ). C 9H10 O2
Natural occurrence of 34 in minor amounts has been reported in some fruits and vegetables. However, it affects human health badly (Mooney A et al ., 2006). Long exposure of 34 has been found to develop neuropsychiatric symptoms (Edling C et al ., 1993). Ester 35 was identified as one of the constituents of bud oil of Eugenia caryophyllata Thunberg (clove). The oil is reported to possess insecticidal activity (Kim E-H et al ., 2003). Literature search revealed that it is the first report of occurrence of 34 and 35 from C. rothii , genus Cordia, and family Boraginaceae. 35 is also used as a fragrance constituent, although it imposes numerous toxicological effects on human health, for instance, skin, and eye irritation, reproductive toxicity, genotoxicity, and carcinogenicity (McGinty D et al ., 2012).
146 Along with other biological activities, plant phenolics are more famous for possessing free radical scavenging properties. Hence antioxidant property of plant extracts reflects its phenolic content (Kumar D et al ., 2013). Research showed that food containing larger proportions of phenolic compounds may help to reduce health related issues, owing to their antiinflammatory, antimutagenic, antioxidant and antibacterial characteristics (Alves MJ et al ., 2013). Phenolics, identified in the present study are discussed as benzoic acid derivatives, phenyl propanoids and sesquiterpene hydroquinones. Phenyl propanoids, itself, encompass a wide range of structural classes possessing diversified biological functions (Dixon RA and Paiva NL, 1995).
4-Hydroxy-benzoic acid ( 45 ) and 4-hydroxy-3-methoxy-benzoic acid ( 46 ) are the two benzoic acid derivatives identified from the root extract of C. rothii . This is the first report of identification of 45 and 46 from C. rothii and genus Cordia . Both constituents are reported earlier from other species of Boraginaceae family (Ahmad I et al ., 2003; Naz S et al ., 2006).
O O C OH C OH
O OH OH 4-Hydroxy-benzoic acid 4-Hydroxy-3-methoxy-benzoic acid
(p-salicyclic acid, 45 ), C 7H6O3 (vanillic acid, 46 ), C 8H8O3
Compound 45 is an antioxidant and beside other plants it has also been isolated from rice hull (Cho J-Y et al ., 1998). It commonly serves as the starting material for the preparation of its esters (parabens). These esters are used as preservatives in ophthalmic solutions, beverages, foodstuffs, pharmaceuticals and cosmetics (Dey G et al ., 2005, Liao C et al ., 2013). Parabens in foodstuffs may affect level of thyroid hormones in humans (Koeppe ES et al ., 2013). Thus cytotoxicity of 45 has also been evaluated (Wang C et al ., 2013). Significant nematicidal activity (Aoudia H et al ., 2012), antimicrobial activity (Cho J-Y et al ., 1998), has also been observed for 45 . It induces alkaline phosphatase activities on rat osteoblastic UMR106 cells (Yang X et al ., 2005).
Compound 46 is a derivative of vanillin. Its ortho -analogue is used as a flavouring agent (Kim S-J et al ., 2010). It is noteworthy that the dried fruits of C. rothii are used in traditional Indo-Pak foods. 46 has also been identified in various mushroom species (Alves MJ et al ., 2013). 46 is reported to possess antimicrobial (Alves MJ et al ., 2013), antioxidant, and antihypertensive potential (Kumar S et al ., 2012). It is one of the constituents of Chinese medicine used for the treatment of certain bone disorders (Huang W et al ., 2010) and its
147 glycoside derivatives are used in curing ailments of systemic autoimmune disorders (Yin Y and Dong J, 2013). It treats obesity by inhibiting pancreatic lipase (Jo YH et al ., 2013) and also inhibits tyrosinase activity (Mu Y et al ., 2013).
Three phenolics in the presented GC-MS study possessed the phenyl propanoid (C 6-C3) skeletons and were identified as (1 E)-4-(3-Hydroxy-1-propenyl)-2-methoxyphenol ( 28 ), 3,4-dihydroxy-benzenepropanoic acid ( 40 ) and methyl 2-hydroxy-3-(4-hydroxyphenyl)- propanoate ( 62 ). 28 and 40 were identified from roots whereas 62 was identified from stem.
To our knowledge, this is the first report of; 28 from C. rothii , genus Cordia , and family Boraginaceae. The identification of 28 is further supported by the isolation and characterization of (2 E) 3-(3 ′,5 ′-dimethoxy-4′-O-β-D-glucopyranosyl-phenyl)-prop-2-en-1-ol (85 ), trivially known as syringin, from leaves of plant specie under study (scheme-4.10). On comparing 28 with 85, it can be observed that later has an additional hydroxymethyl group ortho - to the phenolic hydroxyl function. The hydroxyl group, in turn is
O-Glycosylated with β-D-Glucopyranose.
O O OH OH HO HO (2E)-4-(3-Hydroxy-1-propenyl)-2- OH methoxyphenol 3,4-Dihydroxy-benzenepropanoic acid (coniferyl alcohol, 28 ), C H O 10 12 3 (hydrocaffeic acid, 40 ), C 9H10 O4 O O OH O GlcO OH HO O 3-(3 ′,5 ′-Dimethoxy-4′-O-β-D- (2 S) Methyl 2-hydroxy-3-(4 ′- glucopyranosylphenyl)-prop-2E-en-1-ol hydroxyphenyl)-propanoate
(syringin, 85 ), C 17 H25 O9 (latifolicinin C, 62 ), C 10 H12 O4
Compound 40 is also being reported for the first time from C. rothii and genus Cordia . However, it has been reported earlier from other species of family Boraginaceae (Pedersen JA, 2002). It is interesting to note that 62 has also been isolated and characterized in the current study from the leaves. The thesis is simultaneously reporting identification of 62 from the stem using GC-MS and its isolation from the leaves of C. rothii . However, it has been reported earlier from Cordia latifolia (Siddiqui BS et al ., 2006) and other species of family Boraginaceae (Ara I et al ., 2012).
148 Earlier alcohol 28 has been identified from essential oil of flowers of Acalypha hispida and it serves as a pheromone for honey bee and tephritid fruit fly (Onocha PA et al ., 2011; Keeling CI et al ., 2003; and Hee AKW and Tan KH, 2005). Thus in plant it may be serving to attract insects. 28 has also been reported as the proliferation stimulating agent (Yang X et al ., 2005). Polymers of 28, its salts and other derivatives are reported to be used as anticancer agents (Matsukawa T et al ., 2012), analgesics, circulation enhancing botanicals, antiobese and immunostimulatory agents (Yazawa S et al ., 2007).
Radical scavenging effect of 40 has been reported (Siquet C et al ., 2006). 40 has also exhibited in vivo and in vitro anti-inflammatory effects (Larrosa M et al ., 2009). Curative effectiveness of 40 against UV-B damages were tested in vitro (human conjunctival cells) and in vivo (rabbit) and has been reported (Larrosa M et al ., 2008). Oxidized polymerization products of 40 exhibited virucidal effect against influenza virus A (Mentel R et al ., 1983). It also increases peristalsis movement of intestine (Czok G et al ., 1972). Compound 62 has also been evaluated for nematicidal activity (Begum S et al ., 2011).
The sesquiterpenoid quinone Cordiachrome C ( 9), identified through GC-MS studies in the C. rothii roots was also found present in several other species of the genus Cordia (Moir M et al ., 1972; Moir M and Thomson RH, 1973). It has also been reported earlier from Cordia rothii (Moir M and Thomson RH, 1973). Its leishmanicidal activity has been evaluated (Mori K et al ., 2008). Beside that significant antimycobacterial activity of 9 has also been reported. Further antimalarial and antifungal activity, and cytotoxicity has also been evaluated (Dettrakul S, 2009).
The reduced form of 9, Cordiaquinol C ( 24 ), was also identified in the current study from the root. Identification of 9 and 24 mutually confirmed their presence. 9 may form when cordiaquinol C undergoes oxidation preventing the system from oxidative stresses.
O Red OH
Ox.
H H O OH
Cordiachrome C ( 9), C 16 H18 O2 Cordiaquinol C ( 24 ), C 16 H20 O2
Cordiachromene A ( 21 ) is a rearranged sesquiterpenoid hydroquinone identified in the present study from the roots. Another terpenoid hydroquinone Cordiol A ( 29 ) was also identified from root. 29 can also be a contributor for antioxidation potential of C. rothii . The presence of these hydroquinones 24 and 29 along with other phenolics 21 , 40 , 46 , 62 , 28 and specially 45 led us to evaluate potential of current species as antioxidant/immunomodulating agent
149 (section: 3.3.2.3). Cordiachrome C ( 9), cordiachromene A ( 21 ), cordiaquinol C ( 24 ), and cordiol A ( 29 ), all are related biogenetically (Manners GD and Jurd L, 1977; Mori K et al ., 2008).
OH H H OH
HO
H O OH H H
Cordiachromene A ( 21 ), C 16 H20 O2 Cordiol A ( 29 ), C 16 H22 O3
This is the first report of occurrences of 24 , and 29 from C. rothii , although these compounds have been reported earlier from genus Cordia (Manners GD and Jurd L, 1977). This is the first report of compound 21 from C. rothii . However, it has been reported previously from other species of genus Cordia (Manners GD and Jurd L, 1977). Compound 21 , 24 , and 29 were isolated from Cordia alliodora (Manners GD and Jurd L, 1977). 24 has exhibited good leishmanicidal activity with IC 50 value of 4.5 µg/mL in leishmania major assay (Mori K et al ., 2008) while 21 was found to possess antiinflammatory property (Benslimane AF, et al ., 1995). Cytotoxic and antibacterial properties of 21 isolated from ascidian Aplidium antillense have also been evaluated (Benslimane AF et al ., 1988).
Current study has also led to the identification of a bicyclic sesquiterpene (1a,4a β,8a α) 1,2,3,4,4a,5,6,8a-octahydro-7-methyl-4-methylene-1-(1-methylethyl)-naphthalene, trivially known as τ-cadinene ( 76 ), from the C. rothii roots. This is the first report of 76 from the plant under study, genus Cordia , and family Boraginaceae. It has been identified earlier in the various essential oils, e.g., essential oil from leaves of Callicarpa integerrima (Chai L et al ., 2010). Anticancer potential of Asimina triloba essential oil against human breast and lung carcinoma has been evaluated. This typical oil comprises ~83% sesquiterpenes, of these, approximately 70% have been identified as derivatives of 76 (Farag MA, 2010).
τ- Cadinene ( 76 ), C 15 H24
3,7,11,15-Tetramethyl-hexadec-2-en-1-ol or simply phytol ( 75 ) is a diterpene identified in the current study from the leaves. This is also the first report of 75 from the species under investigation. However, it has been reported earlier from other species of genus Cordia (Viana FA et al ., 2008) and family Boraginaceae (Theeraphan M et al ., 2007).
150 HO
3,7,11,15-Tetramethyl-hexadec-2-en-1-ol (phytol, 75 ) C 20 H40 O
Phytol has also been reported earlier in the leaves extract of Andrographis paniculata (Wei LS et al ., 2011) and in mulberry in a concentration as high as ~25% (Liu F et al ., 2013). It is studied well for its biological activities. For instance, antidiabetic (Elmazar MM et al ., 2013), antitrypanosomal (Bero J et al ., 2013), antiplasmodial (Grace MH et al ., 2012), antiarthritis (Zhu W et al ., 2012), anticonvulsant (due to its modulating effect on neurotransmitter system) (Costa JP et al ., 2012a) and antituberculosis potentials of 75 have been reported (Singh R et al ., 2012). The extracts containing 75 are also reported to possess antibacterial (Liu F et al ., 2013), antioxidant, and anticancer (Wei LS et al ., 2011) activities. It has been observed as a selective inhibitor of acetylcholinesterase and butrylcholinesterase (Fang Z et al ., 2012) and HepG2 cells (antiproliferative agent) (Wang X et al ., 2013) and plays an important role in tryptophan-niacin metabolism (Matsuda H et al ., 2013), glucolipid metabolism, and signaling pathways (Lin X et al ., 2012). Acute toxicity and cytotoxicity of 75 has also been studied in detail (Satyal P et al ., 2012, Costa JP et al ., 2012b, Liska J et al ., 2012).
6,10,14-Trimethyl-pentadecan-2-one ( 48 ), a dinorditerpenoid is also identified for the first time from stem and leaves of the plant species undertaken for current investigation. It has been reported previously from other species of genus Cordia (Bonesi M et al ., 2011) and family Boraginaceae (Theeraphan M et al ., 2007).
O
6,10,14-Trimethyl-pentadecan-2-one (hexahydrofarnesyl acetone, 48 ), C 18 H36 O
Compound 48 , trivially known as hexahydrofarnesyl acetone possess a long lasting fresh jasmine, celery odor, and is used in jasmine compositions (Bedoukian Research Inc., 2013). It is also present in the essential oil of Carduus pycnocephalus L (Al Shammari et al ., 2012), seed oil of Cassia glauca (Kumar D et al ., 2013) and has also been identified as the major constituent (~62%) in the essential oil of Sagittaria trifolia . This oil exhibits antimicrobial properties and is effective for skin related disorders and in childbirth (Zheng X et al ., 2006).
Triterpenes are rarely identified through GC-MS. These larger molecules are usually non- volatiles and elute late from columns at elevated temperature. Therefore, if any, usually undergo thermal degradation. Some triterpenes/triterpenoids can survive thermal degradation
151 and thus the current study has resulted in the identification of five triterpenoids, thermally stable under the analytical conditions. All of these have alcoholic function in common. These included 9,19-cyclolanost-24-en-3β-ol (cycloartenol, 32 ), 24-methylene-9,19-cyclolanostan- 3β-ol, (24-methylene-cycloartanol, 33 ), 4 α,14 α-dimethyl-,9,19-cycloergost-24(28)-en-3β-ol, (cycloeucalenol, 44 ), olean-12-en-3β-ol ( β–amyrin, 60 ), and 25-methylene-9,19- cyclolanostan-3β-ol (cyclolaudenol, 63 ).
H H H H
HO HO H H
9,19-Cyclolanost-24-en-3β-ol 24-Methylene-9,19-cyclolanostan-3β-ol
(cycloartenol, 32 ), C 30 H50 O (24-methylenecycloartanol, 33 ), C 31 H52 O
H H
HO H H HO H
4α,14 α-Dimethyl-9,19-cycloergost-24(28)- Olean-12-en-3β-ol
en-3β-ol (cycloeucalenol, 44 ), C 30 H50 O (β-amyrin, 60 ), C 30 H50 O
H H
HO H
25-Methylene-9,19-cyclolanostan-3β-ol (cyclolaudenol 63 ), C 31 H52O
Triterpenoids 32 , 33 , and 44 are identified from the roots while 60 and 63 are identified in stem of the species undertaken in the study. α or β-isomers of amyrin exhibit almost identical mass spectrum. Isolation of β-amyrin in earlier studies from C. rothii (Verma SY et al ., 1978) was found helpful in confirming the mass spectrum of identified constituent as β-amyrin. This is the first report of 32 from C. rothii and genus Cordia , however, literature survey has
152 confirmed its presence earlier from other species of family Boraginaceae (Wretensjö I and Karlberg B, 2002). On the other hand, 33 , 44 , and 63 have been reported for the first time from C. rothii , genus Cordia , and family Boraginaceae.
Compound 32 has been isolated from the leaves of Garcinia subelliptica (Ito T et al ., 2013a) and also in large amounts from Crataegus monogyna . It possesses anti-inflammatory (Ahumada C et al ., 1997), anti-cancer and analgesic (Bigoniya P, 2013), anti-alzheimer (acetylcholinesterase inhibition) (Jung HA et al ., 2010), anti-cancer (Sultana S et al ., 2003) and antituberculative (Saludes JP et al ., 2002) properties. It has also been found to exhibit inhibitiory effect against tumor necrosis factor (TNF-α production) (Yang X et al ., 2008). 32 and derivatives of 32 have also been reported to reduce blood glucose level; during postprandial effect (Fukuoka D et al ., 2010). 32 and its derivatives have also been recognized as anti-wrinkle and skin treatment agents (Thorel JN, 2005).
Triterpenoid 33 has been isolated from the roots of Salvia blepharochlaena (Kolak U et al ., 2005) and Aloe vera gel (Tanaka M et al ., 2006). It showed antihyperglycemic (Tanaka M et al ., 2006) and analgesic properties (Floriani AEO et al ., 1998). Active cytotoxic role of 33 has also been evaluated against lymphocytic leukemia (Oeksuz S et al ., 1994). 32 and 33 have been found to exhibit promising anti-inflammatory property (Akihisa T et al ., 1996) whereas their ferulates have been confirmed as the potent inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (Akihisa T et al ., 2001).
Compound 44 has been reported from the stem of Tinospora crispa and has been evaluated for its cardiac contractility (Kongkathip N et al ., 2002). It inhibits carcinogenesis (Akihisa T et al ., 2006) and aromatase activity (Li Y-H et al ., 2009). It has also been identified as a constituent of thread used in Ayurvedic medicine for the treatment of fistula (Gewali MB et al ., 1990). In another study anti-inflammatory potential of ferulates of 32 , 33 , and 44 has also been evaluated (Akihisa T et al ., 2000).
Leaves of Garcinia subelliptica furnished 60 (Ito T et al ., 2013a), which is also identified in the current study. Moreover, 60 has also been isolated from Haloxylon salicornicum and tested for its antituberculative activity (Bibi N et al ., 2010). Further reported biological activities of 60 included; anti-inflammatory (Akihisa T et al ., 1996), anti-herptic (Sritularak B et al ., 2013), antibacterial (Choi JW et al ., 2012), insect antifeedant (Kannan S et al ., 2013), and anti-inflammatory and analgesic properties (Chicca A et al ., 2012). It has also been found to reduce oxalate toxicity (Geetha K et al ., 2010). It is recognized having high potential as
153 NO inhibitor for curing arthritis inflammation (Shih M-F et al ., 2010) and collagen-induced platelet aggregation inhibitor (Ching J et al ., 2010).
The biosynthesis of the triterpenoids identified in current studies are related to each other. 2,3-Oxidosqualene; an intermediate formed from squalene (Dewick PM, 2009), undergoes cyclization via two different routes, giving rise to either β-amyrin ( 60 ) or cycloartenol ( 33 ) (figure-2.12, section: 2.5) (Henry M, 2005). SAM facilitates alkylation at C-24 of cycloartenol ( 32 ) to generate 24-methylenecycloartanol ( 33 ). 33 may lose α-methyl from C-4 to give cycloeucalenol ( 44 ) (schem-3.2c) (Dewick PM, 2009). Cyclolaudenol ( 63 ) is the positional isomer of cycloartenol. The triterpenes are precursors of phytosterols.
Phytosteroids are an important class of natural products. All these metabolites are quiet known and common in higher plants. Important biological functions of the phytosterols included LDL-cholesterol reducing potential and anti-colon cancer activity (Zhao X. et.al. , 2013) Phytosteroids identified in this study included; stigmasta-3,5-diene ( 19 ), stigmasta- 4,22-diene-3β-ol ( 25 ), stigmast-5-en-3β-ol ( 26 ), and stigmasterol ( 27 ) from roots while stigmasta-3,5-dien-7-one ( 61 ) from the stem. 19 , 25 , and 61 have been reported for the first time from C. rothii , genus Cordia , and family Boraginaceae. Phytosterol 27 has been reported earlier from C. rothii (Desai HK et al ., 1976), genus Cordia (do Vale AE et al ., 2012) and family Boraginaceae (Andhiwal CK et al ., 1985). Compound 26 is wide spread in plants of family Boraginaceae (Hoang QH et al ., 2009) and has been reported in various species of genus Cordia (Menezes JESA et al ., 2001) including C. rothii (Mukat B and Chhaya G, 1980). Beside identifications in GC-MS studies, stigmasterol ( 27 ) has also been isolated from root (scheme-4.4) while β-sitosterol ( 26) was also characterized from stem and leaves (scheme-4.8) respectively, in the current study.
Phytosteroid 19 has been isolated from the stem bark of Bombax ceiba (Faizi S et al ., 2011). It is the dehydrated form of 26 , which has also been identified in the present study. It has been reported in the vegetable oils containing 26 , cooked or treated at high temperature (Frankel EN, 2010). It has also been experienced that some time 26 may undergo dehydration in gas chromatographic analysis, which is usually performed under high temperature/pressure conditions to produce 19 . 25 was also identified in Ginkgo biloba L. leaves (Wang C-Z et al ., 2008).
154 O (3S)-2,3-oxidosqualene (squalene oxide)
H H H H HO H HO β-Amyrin (60) H Cycloartenol (32)
Alkylation at C-24
H H a-CH loss from C-4 H 3 H H
H H HO HO HO H H
β-Sitosterol (26) 24-Methylene cycloartanol (33) Cycloeucalenol (44) Scheme-3.2c: Biogenetic Relation between Identified Triterpenoids
155 H H H H HO
Stigmasta-3,5-diene ( 19 ), C 29 H48 Stigmasta-4,22-diene-3β-ol ( 25 ), C 29 H48 O
H H H H
H H H H HO HO
Stigmast-5-en-3β-ol (24 S)-Stigmasta-5,22-dien-3β-ol
(β-sitosterol, 26 ), C 29 H50 O (stigmasterol, 27 ), C 29 H48 O
O
Stigmasta-3,5-dien-7-one ( β-saccharostenone, 61 ), C 29 H46 O
β-Sitosterol ( 26 ), also identified in the n-hexane extract of seeds of Phoenix dactylifera L. (Azmat S et al ., 2010), is known to possess anti-inflammatory and anti-pyretic (Patel B and Rajput A, 2012), and antioxidant and antidiabetic potentials (Radika MK et al ., 2013). It has also been found to exhibit tyrosinase inhibitory (Munoz E et al ., 2013) and radical scavenging properties (He R et al ., 2013). It is a proven anticancer against certain cancers (Baliga MS et al ., 2013), human cancer cell lines (HL-60) (Nahata A et al ., 2013) and human natural killer (NK) cells (SW-1990 cell line) (Cheng J et al ., 2012). It is also reported to contribute antioxidant and anticancer property to Andrographis paniculata leaves extract (Wei LS et al ., 2011). In vitro Th1/Th2 immunomodulatory potential of 26 has also been evaluated in which it showed significant Th2-inclination with strong antiinflammatory potential (Ku C-M and Lin J-Y, 2013).
Stigmasterol ( 27 ) has been isolated from the aerial parts of Tannacetum polycephalum (Azizudin and Choudhary MI, 2008) and Haloxylon salicornicum and has exhibited antituberculative (Bibi N et al ., 2010), antibacterial (Gazi HR et al ., 2012) and antioxidant potential (Hassanein RA et al ., 2012). It is also reported to possess strong cytotoxic effect
156 against human cancer cell line, Raji (lymphoma) and thus can be used as an anticancer drug (Teh SS et al ., 2012). 27 together with some other phytoconstituents, isolated from Vitex parviflora leaves is recognized to be responsible for its antimutagenic property (Ragasa CY et al ., 2003). 26 and 27 have also been evaluated for their apoptosis-inducing property against human liver cancer cells (SMMC-7721) (Li Q-Y et al ., 2012).
Phytosteroid 61 is also one of the constituents of Poa huecu (Rofi RD and Pomilio AB, 1982). 61 was identified as the fungal resistance biomarker, and has also been isolated from the leaves of Vitis vinifera (Batovska DI et al ., 2008). It is also reported from the seeds of Phoenix dactylifera L. (Azmat S et al ., 2010).
Steroidal derivatives are responsible for several physiological functions in human body. For instance, fertility, birth, and sexual growth are controlled by steroidal hormones. Structure- activity relationship (SAR) study showed that side chains of phytosterols are responsible for their antibacterial activity (Tanaka A et al ., 2013).Therapeutical importance includes cure against allergies, arthritis, and cancer (Igwe OU and Okwu DE, 2013). Therefore, compounds identified from the plant species undertaken for the current study can be used to address such illnesses.
2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one ( 37 ), and 5-(hydroxy-methyl)-2- furancarboxaldehyde ( 38 ) have also been identified in the extracts of C. rothii roots. 37 and 38 are the metabolites from hexoses (Shaw PE et al ., 1971; Fagerson IS, 1969). 37 is reported for the first time from C. rothii , genus Cordia and family Boraginaceae. This is also the first report of 38 from C. rothii and genus Cordia , however 38 has been reported previously from other species of family Boraginaceae.
O O O OH HO OH O 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran- 5-(Hydroxymethyl)-2-furancarboxaldehyde
4-one ( 37 ) C 6H8O4 (hydroxymethyl furfuraldehyde, 38 ) C 6H6O3
Compound 37 is a food component which furnishes flavour enhancing caramel taste, especially in baked cookies (Nishibori S and Kawakishi S, 1990). It is a constituent of food composition particularly used in therapies. It controls energy metabolism and autonomic nerve activity (Beppu Y et al ., 2008). Mango peels contain higher amounts of 37 due to which it exhibits strong antioxidant and cell proliferation inhibition activity (Ali MR et al ., 2012). Antioxidant and ABTS radical-scavenging potential of this compound have also been evaluated (Hwang IG et al ., 2013). Mutagenic and DNA strand cleavage (Hiramoto K et al .,
157
1997) and tyrosinase and melanin formation inhibitory property of 37 has also been evaluated (Yamashita M, 2006).
Ethanol extract of leaves and flowers of Madhuca longifolia has shown significant antimicrobial properties due to the presence of 37 and 38 , along with a few other similar compounds in it (Kalaivani M and Jegadeesan M, 2013). 38 is also considered as an active uterotonic agent (Sewram V et al ., 2001). Curative effect of 38 against cardiovascular abnormalities and diabetes mellitus (Cao G et al ., 2013) and its significant HSP70 mRNA expression-enhancing activity in HL-60 cells has also been documented (Ito T et al ., 2013b).
(E,E) 2,4-Heptadienal ( 64 ) was identified for the first time from the leaves extract of the investigated plant specie. This is also the first report of 64 from genus Cordia and family Boraginaceae. 64 has been identified from the Kiwi fruit (Jordán MJ et al ., 2002). The characteristic stale odour of Chinese dark fermented tea is due to the contribution of 64 (Xu X et al ., 2007).
O
(E,E) 2,4-Heptadienal ( 64 ), C 7H10 O
Thymine ( 36 ) is also identified in the root of C. rothii . It is a nitrogenous base and a basic component of DNA. Antimicrobial and anticancer potential of 36 can be increased by conjugating it with chitosan. It inhibits proliferation of cancer cells without showing toxicity for normal cells (Kumar S et al ., 2012a). 36 has been reported to use in the synthesis of anti- HIV compounds (Sun J-B et al ., 2013). Various derivatives of 36 have been used for the treatment of damaged skin (Vince R, 2013).
O
NH
N O H
5-Methyl-2,4-pyrimidinedione (Thymine, 36 ), C 5H6N2O2
3.2.4 Structure Elucidation Using GC-EI-MS:
The intensity of [M] + ion peak was observed to be weaker in case of relatively long-chain hydrocarbons. The fragmentation product of straight chain hydrocarbons are primary carbocations which are unstable, therefore, fragmentation is least favoured, resulting in the reasonable intensity of [M] + ion peak as observed in the case of short-chain hydrocarbons identified in the study. The characteristic fragmentation pattern for saturated hydrocarbons is a series of cluster ion peaks separated from each other with 14 mass units (CH 2, m/z 14), or its 158 integral multiple (Silverstein RM and Webster FX, 1996; Pavia DL et al ., 2001). For + example, mass spectrum of 1 showed strong molecular ion peak [M, C 13 H28 ] at m/z 184.
Cluster of peaks were observed at 14 mass (CH 2) units apart. For instance, cleavage of bond between C1 and C2 resulted in the loss of methyl radical with the formation of dodecyl + carbocation [C 12 H25 ] at m/z 169. Cleavage of bond between C3 and C4 resulted in the + + formation of propyl radical [C 3H7] and decyl carbocation [C 10 H22 ] , at m/z 141 and so on. + The base peak in the identified hydrocarbons appeared mostly at m/z 57 due to [C 4H9] . On the basis of rather characteristic fragments and comparison of the above discussed mass spectrum with electronic mass spectral library (NIST, 2005) hydrocarbons 2, 3, 6, 8, 10 , 20 , 49 , 52 , and 86-88 were identified in various fractions and sub-fractions of different extracts.
Straight-chain monocarboxylic acids have shown weaker [M] +. α-cleavage in short-chain acids provided peaks at [M-17] + and [M-45] + due to the loss of –OH and –COOH, respectively. The mass spectrum of long-chain fatty acids contained series of fragment ions, separated by 14 mass units from each other. Another characteristic fragmentation pattern of aliphatic acids having γ-hydrogen is the McLafferty rearrangement which gave a stronger + peak [C 2H4O2] at m/z 60 (Silverstein RM and Webster FX, 1996; Pavia DL et al ., 2001).
R H + O R Radical Induced Cleavage RH H O O C C R R H H O O + C Charge Induced Cleavage Figure-3.12: Mclafferty Rearrangement Mechanism
+ In the present study n-hexadecanoic acid ( 13 ) showed a medium intensity [M, C 16 H32 O2] at + + m/z 256. α-cleavage resulted in the loss of OH [M-17] and the acylium ion [CH 3(CH 2)14 CO] + was observed at m/z 239. β-cleavage provided a diagnostic peak [H 2C=C(OH)OH] for acids at m/z 60, formed as a result of McLafferty rearrangement. Fragment ion series, 14 mass units apart, was also evident. Similar findings and comparison of the observed data with electronic mass spectral library (NIST, 2005) resulted in identification of various saturated fatty acids (18 , 39 , 41 , 51 , 54 , and 78 ).
Weak [M] + ion peak can be observed in certain cases of unbranched fatty acid methyl esters. This [M] + is still weaker in case of ethyl esters. Acylium ion [ R C O]+ resulting from the
α-cleavage, gives the most important recognizable peak of esters. It results from the loss of corresponding methoxy or ethoxy group from an ester. Another important characteristic peak results from the β-cleavage reaction i.e. , McLafferty rearrangement. This gives the diagnostic + + base peak [C 3H6O2] at m/z 74 in straight-chain methyl esters and [C 4H8O2] at m/z 88 in the
corresponding ethyl esters (Silverstein RM and Webster FX, 1996; Pavia DL et al ., 2001). 159
H O + Radical Induced Cleavage H H O O C C H H O O + C Charge Induced Cleavage
Figure-3.13: Mclafferty Rearrangement Mechanism for Methyl Esters
H O + Radical Induced Cleavage H H O O C C H H + O O C Charge Induced Cleavage
Figure-3.14: Mclafferty Rearrangement Mechanism For Ethyl Esters
In the case of n-tetradecanoic acid ethyl ester ( 7), identified in the current study, mass of m/z + 256 was assigned to molecular ion peak [M, C 16 H32 O2] . α-cleavage resulted in diagnostic + + loss of ethoxy radical [M-45] or the corresponding acylium fragment ion [H 3C(CH 2)12 CO] , + detected at m/z 211. McLafferty fragment ion at m/z 88 [H 2C=C(OH)OCH 2CH 3] , a characteristic of ethyl esters was also observed in the mass spectrum. The base peak at m/z 57 + was for butyl carbocation [C 4H9] . Alkyl part of the ester furnished homologous series of + fragment ions having general formula [(CH 2)nCOOCH 2CH 3] , which were 14 mass uints apart from each other. This characteristic fragmentation pattern when compared with electronic mass spectral library (NIST, 2005) confirmed n-tetradecanoic acid ethyl ester ( 7). In the similar fashion 5 and 12 were also identified. n-Octadecanoic acid methyl ester ( 23 ) can be discussed as an example for fatty acid methyl + ester (FAMEs). The spectrum showed molecular ion peak [M, C 19 H38 O2] of medium intensity at m/z 298. α-cleavage resulted in a diagnostic methoxy radical loss [M-31] + + generating the acylium fragment ion [H 3C(CH 2)16 CO] at m/z 267. McLafferty rearrangement + ion [H 2C=C(OH)OCH 3] appeared as base peak at m/z 74. The series of ions of general + formula [(CH 2)nCOOCH 3] separated by 14 mass units were also identified. This fragmentation pattern when compared with electronic mass spectral library (NIST, 2005) confirmed the presence of n-octadecanoic acid methyl ester ( 23 ). Various other FAMEs, 11 , 50 , 53 , 55 , 56 - 59 , 66 , 70 , 71 , 76 , and 77 were also identified on similar basis.
+ Medium intensity molecular ion peak [M, C 18 H32 O2] was observed for octadec-9Z,12 Z- dienoic acid (31 ) at m/z 280. β-cleavage resulted in a McLafferty rearranged fragment ion + [H 2C=C(OH)OH] at m/z 60 with comparatively weak intensity. Base peak was observed at
160 m/z 81. Rest of the identification was based on comparing this fragmentation pattern with electronic mass spectral library (NIST, 2005) which indicated this spectrum to be of octadec- 9Z,12 Z-dienoic acid (31 ).
+ Weak intensity molecular ion peak [M, C 18 H34 O2] was identified at m/z 282 for both isomers of monoenoic fatty acids; octadec-9Z-enoic acid (42 ) and octadec-9E-enoic acid ( 69 ). Loss of water resulted in the fragment ion peak [M-18] + at m/z 264. Low intensity McLafferty + rearranged fragment ion [H 2C=C(OH)OH] , due to β-cleavage was detected at m/z 60. Base peak appeared at m/z 55. Confirmation of identification was based on comparison of this mass fragmentation pattern with electronic mass spectral data base (NIST, 2005), which showed these spectra to be of 42 and 69 (E/Z isomers of each other).
+ Low intensity molecular ion peak [M, C 19 H36 O2] was identified at m/z 296 in the EIMS of octadec-9Z-enoic acid methyl ester (14 ). A high intensity peak [M-32] + at m/z 264, indicated + the loss of methanol. Fragment ion [M-CH 2COOCH 3-H] furnished another characteristic peak at m/z 222 (Hallgren B et al ., 1959). Hydrocarbon fragment ion series having the general + formula of [C nH2n-1] at m/z 180, 166, and 152 was also identified. Base peak at m/z 55 was also characteristic of unsaturated fatty acid methyl ester (Lipid library, 2013). This particular mass fragmentation pattern when compared with electronic mass spectral library (NIST, 2005) confirmed the spectrum to be of octadec-9Z-enoic acid methyl ester ( 14 ).
High intensity molecular ion peak was detected at m/z 308 in the EIMS of octadec-9Z, 12 Z- dienoic acid ethyl ester (15 ). Loss of ethoxy gave fragment ion peak [M-45] + at m/z 263. Relative abundance of fragment ion [M-45] + was higher in contrast to [M-46] +. Homologous + series of the ions of general formula [C nH2n-3] were observed at m/z 67, 81, 95, 109,123, etc (Lipid library, 2013). Rest of the confirmation was achieved by comparing this particular mass fragmentation pattern with electronic mass spectral database (NIST, 2005). Hence the spectrum was characterized as of octadec-9Z, 12 Z-dienoic acid ethyl ester ( 15 ). Using the similar strategy other unsaturated fatty acid esters including 16 , 17 , 22 , 72 , and 73 were identified.
Nonanedioic acid monomethyl ester (azelaic acid monomethyl ester, 47 ) was observed as the only dicarboxylic acid methylated only at one terminal. Molecular ion peak [M] + was not observed in GC-MS. Loss of methoxy radical gave medium intensity peak [M-31] + at m/z + 171. Fragment ion [M-OCH 3-OH] resulted in the base peak at m/z 152 while loss of acetate + ion [M-CH 3OCO] was detected at m/z 143. McLafferty fragment ion peak was also identified at m/z 74. Comparison with the electronic mass spectral library (NIST, 2005) confirmed this spectrum to be of nonanedioic acid monomethyl ester ( 47 ).
161
In case of aromatics, the intensity of molecular ion peak is usually very high as the aromatic ring stabilizes it. Alkyl-substituted benzene ring gives a prominent peak at m/z 91, due to the rearranged product of benzyl cation; a tropylium ion. Another characteristic peak of aromatic hydrocarbons originates when the alkyl group attached to benzene ring is propyl or larger and may undergo McLafferty rearrangement (Silverstein RM and Webster FX, 1996; Pavia DL et al ., 2001).
+ Styrene ( 34 ) showed molecular ion [M, C 8H8] as base peak at m/z 104. This is due to the formation of highly resonance stabilized cationic radical. Loss of ethylene molecule gave rise to medium intensity peak [M-28] + at m/z 78. This mass spectrum was rather simple and was also available in the electronic mass spectral library (NIST, 2005).
+ Acetic acid phenylmethyl ester (35 ) displayed strong molecular ion peak [M, C 9H10 O2] at m/z 150. Tropylium ion peak typical of alkyl-substituted benzene was also observed at m/z 91. Base peak originated due to characteristic loss of acidic moiety of ester molecule resulting in the formation of benzyl alcohol at m/z 108. This mass fragmention pattern was also simple and upon comparison with electronic mass spectral library (NIST, 2005), 35 was identified as acetic acid phenyl methyl ester.
One of the benzoic acid derivatives was 4-hydroxy-benzoic acid ( 45 ). Molecular ion peak [M] + was observed at m/z 138. Loss of hydroxyl function due to the stabilized acylium + + resulted in the base peak [M-17] at m/z 121. Fragment ion [M-45, M-CO 2H] provided medium intensity peak at m/z 93. The mass spectrum of 45 after confirming from the electronic mass spectral database (NIST, 2005) was identified as of 4-hydroxy-benzoic acid.
Molecular ion peak [M] + for methoxy analogue of 45 ; 4-hydroxy-3-methoxy-benzoic acid (46 ) appeared at m/z 168. Other major fragment ion peaks observed were due to the loss of carboxylic acid function [M-45] + at m/z 123, loss of methyl [M-15] + at m/z 153, loss of methoxy [M-31]+ at m/z 137, and loss of hydroxyl [M-17] + at m/z 151. This fragmentation pattern on comparison with electronic mass spectral data base (NIST, 2005) confirmed the spectrum of 46 to be of 4-hydroxy-3-methoxy-benzoic acid.
4-(3-Hydroxy-1-propenyl)-2-methoxyphenol ( 28 ), 3,4-dihydroxy- benzenepropanoic acid (40 ) and methyl 2-hydroxy-3-(4-hydroxyphenyl)-propanoate (62 ) were phenolics possessing basic skeleton of phenyl propanoid.
In the mass spectrum of 28, [M] + was observed at m/z 180, due to the resonance stabilized benzene ring. Cleavage of bond, β to ring, provided the base peak at m/z 137. Presence of alkyl-substituted benzene ring was ascertained by the characteristic tropylium ion peak at m/z
162
91. The electronic mass spectral data base (NIST, 2005), also verified the mass spectrum to be of 4-(3-hydroxy-1-propenyl)-2-methoxyphenol ( 28 ). The mass spectrum of (2 E) 3-(3 ′,5 ′- dimethoxy-4′-O-β-D-glucopyranosyl-phenyl)-prop-2-en-1-ol ( 85 ) an analogue to 28 isolated and purified in the current study (scheme-4.10) also supported these conclusions.
3,4-Dihydroxy-benzenepropanoic acid ( 40 ) in its EIMS displayed relatively medium intensity molecular ion peak at m/z 182. Fragment ion at m/z 91 was due to the tropylium ion. Another fragment ion at m/z 123, resulting from the cleavage of bond β to the ring displayed as the base peak in the mass spectrum. Comparison of the data with electronic mass spectral library (NIST, 2005) characterized the compound as 3,4-dihydroxy-benzenepropanoic acid ( 40 ).
Methyl 2-hydroxy-3-(4-hydroxyphenyl)-propanoate (62 ) was another phenyl propanoid showing a less intense [M] + at m/z 196. Fragment ion [M-18] + appearing at m/z 178 was due the loss of water molecule and it established the presence of sec -hydroxy moiety. Cleavage of bond β to ring provided base peak at m/z 107. Tropylium ion peak was also observed. On comparison with electronic mass spectral database (NIST, 2005) the spectrum was identified as methyl 2-hydroxy-3-(4-hydroxyphenyl)-propanoate (62 ). Compound 62 was also isolated and purified in the current study and the mass spectrum of isolated and identified constituent were identical.
+ A prominent peak at m/z 161 arised due to the fragment ion [C 10 H9O2] formed by the loss of side chain from cordiachromene A ( 21 ). Loss of methyl group from the side chain generated fragment ion [M-15] + at m/z 229. A relatively weak molecular ion peak was observed at m/z 244. All these observations were in accordance with the previously published mass spectral data of cordiachromene A (Manners GD and Jurd L, 1977).
The peak observed at m/z 244 in the EIMS of cordiaquinol C (24 ) was identified as its molecular ion as well as base peak. The pattern in the mass spectrum indicated the presence of naphthalene derivative. Loss of methyl radical provided the fragment ion [M-15] + at m/z 229. Conclusively many other fragment ions almost of the same intensity (as discussed in the experimental section: 4.4.2.1) were identified. Confirmation by comparison with the already published data (Manners GD and Jurd L, 1977), verified the mass spectrum under study to be of Cordiaquinol C.
In case of 29 , an intense molecular ion peak was observed at m/z 262. A peak corresponding to the loss of water [M-18] + was observed at m/z 244. Base peak was detected at m/z = 136. Similarly, many other fragment ions of approximately same intensity (as discussed in the experimental section: 4.4.2.1) were identified and confirmed by comparison with the already
163 reported literature (Manners GD and Jurd L, 1977). These findings led to conclude and identify the mass spectral data of 29 to be of cordiol A.
Cordiachrome C ( 9) mass fragmentation pattern was deduced with the help of the fragmentation pattern of Cordiachrome B discussed by Moir and Thomson in their article (Moir M and Thomson RH, 1973). According to them 9 may fragment at the alicylic ring junction giving rise to the fragment ions at m/z 174 and 108 as shown below.
O
H O O m/z = 242
O m/z = 174 m/z = 108
Further peak representing the loss of methyl was observed as the base peak at m/z 227. In contrast to Cordiachrome B, the peak appearing at m/z 108 was found to be weaker. Further confirmation was done by comparing intensities of different fragment ion peaks (vide section 4.4.2.1) in previously reported data (Moir M and Thomson RH, 1973).
6,10,14-Trimethyl-pentadecan-2-one ( 48 ), is a linear dinor-diterpenoid while 3,7,11,15- tetramethyl-hexadec-2-en-1-ol ( 75 ) is a diterpene. In case of 48 , [M] + was not observed. + Fragment ion peak [M-H2O] at m/z 250 was due to the loss of water molecule from the + compound. Fragment ion peak [M-C3H6O] at m/z 210 corresponded to the loss of terminal + methyl ketone while fragment ion [C 3H6O] itself was detected as base peak at m/z 58 (Rao CB and Pullaiah KC, 1982). This fragmentation pattern upon comparison with electronic mass spectral database (NIST, 2005) was suggestive of the mass spectrum 48 to be of 6,10,14-trimethyl-pentadecan-2-one.
+ Molecular ion peak was also not observed in the case of 75 . Loss of water molecule [M-H2O] + was displayed at m/z 278. The fragment ion [M-H2O-CH 3] provided an intense peak at m/z + 263. Loss of propyl radical [M-C3H7] gave rise to peak at m/z 253. Another fragment ion [M- + H2O-C2H5] at m/z 249 was indicative of the loss of ethyl radical with water. Peaks at m/z + + + 196, 126, and 111 were suggestive of the [M-C6H12 O] , [M-C8H14 O] and [M-C7H11 O] fragment ions respectively. This typical fragmentation pattern when compared with electronic mass spectral library (NIST, 2005) and a reference data in literature (de Souza NJ and
164
William RN, 1969) characterized the mass spectrum 75 to be of 3,7,11,15-tetramethyl- hexadec-2-en-1-ol.
Mass spectra alone cannot distinguish the closely related isomers. The mass spectrum obtained from the non-polar fractions for amyrin ( 60 ) was unable to confirm its identity as α or β-isomer, however, literature survey revealed the presence of β-amyrin in an earlier report on C. rothii (Verma YS et al ., 1978). Therefore, 60 was confirmed as β-amyrin.
In the mass spectrum of compound 32 , weak intensity molecular ion [M] + was observed at m/z 426. Loss of methyl resulted in a medium intensity peak [M-15] + at m/z 411. Loss of side chain [M-111] + also furnished a typical fragment ion peak at m/z 315. Another important peak [M-140] + showing the loss of ring A was observed at m/z 286. This typical fragmention led to conclude the spectrum to be of cycloartenol ( 32 ) (Aplin RT and Hornby GM, 1966). Similarly other triterpeoids; 24-methylenecycloartanol ( 33 ), cycloeucalenol ( 44 ), and cyclolaudenol (63 ) were confirmed in various fractions ( vide experimental) by mass spectral library search (NIST 2005).
Stigmasta-3,5-diene ( 19 ), stigmasta-4,22-diene-3β-ol ( 25 ), β-sitosterol ( 26 ), stigmasterol ( 27 ) and stigmasta-3,5-dien-7-one ( 61 ) were identified from different non-polar fractions. The originally reported mass spectra of these compounds from electronic mass spectral library (NIST, 2005) were found to be matching.
In case of 61 , strong intensity molecular ion peak [M] + was observed at m/z 410. Loss of side chain from C-17 gave characteristic fragment ion peak [M-141] + at m/z 269. Two other very typical fragment ions of cholesta-3,5-dien-7-one moiety were identified at m/z 187 and 174. Both of these peaks arise due to the cleavage of ring C (Baldé AM et al ., 2000). These findings upon comparison with the electronic mass spectral library (NIST, 2005), were suggestive of the spectrum to be of stigmasta-3,5-dien-7-one.
2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one ( 37 ) and 5-(hydroxymethyl)-2-furan- carboxaldehyde ( 38 ) are known metabolites of Glucose. Compound 37 showed moderate + + intensity molecular ion peak [M] at m/z 144. Fragment ion [CH 3CO] appearing at m/z 101 indicated the loss of acylium ion from the molecule. Another major fragment ion peak + supporting the base peak and observed at m/z 43 was formed due to the loss of [C 4H5O3] + from the molecule. Loss of carbon monoxide from fragment [C 4H5O3] furnished another + + major fragment ion peak [C 3H5O2] at m/z 73. Loss of water from [C 3H5O2] further yielded + fragment ion peak [C 3H3O] at m/z 55, confirming the molecule as 2,3-dihydro-3,5- dihydroxy-6-methyl-4H-pyran-4-one (Kim M-O and Baltes W, 1996).
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O -OC C OH -CO OH O C HO OH C C O O O m/z = 144 m/z = 101 m/z = 73
The molecular ion peak of 38 was strong. Fragment ion peak [M-OH] + of lower intensity was observed due to the loss of hydroxyl group appeared at m/z 109. Loss of carbon monoxide [M-CO] + from molecule appeared as base peak at m/z 97. Loss of hydroxyl function followed by loss of methyl furnished peaks at m/z 81 and 69 respectively (Shen Y-M and Mu Q-Z, 1990).
O O O -OH -CO O OH O
m/z = 126 m/z = 109 m/z = 81
-CO
O O O -OH -CH OH 3
m/z = 97 m/z = 81 m/z = 69
-CO
H2C CH2 CH C CH H H2C m/z = 53 m/z = 53
In case of 36 , strong intensity molecular ion peak was observed at m/z 126. Fragment ion [M- + + HNCO] was observed due to formation of acylium ion [C 4H5NO] at m/z 83. Loss of carbon + + monoxide from fragment ion [C 4H5NO] furnished an intense peak for fragment ion [C 3H5N] at m/z 55 (Jerry MR et al ., 1965). Further confirmation of the mass spectrum was performed by comparing the data with the electronic mass spectral library (NIST, 2005). These observations identified the spectrum of compound 36 to be of thymine.
O O
NH -HNCO -CO HN N O HN H m/z = 126 m/z = 83 m/z = 55
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3.3 Bioactivities:*
*COLLABORATORS: Root’s bioactivities were evaluated under the supervision and collaboration with Prof. Dr. Aqeel Ahmad (Department of Microbiology, University of Karachi, Karachi-75270). Stem and leaves’ bioactivities were performed with Prof. Dr. M. Iqbal Choudhary and Dr. Muhammad Ahmed Mesaik at the International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi. A portion of this work has already been published as;
“Isolation of Phytochemicals from Cordia Rothii (Boraginaceae) and Evaluation of their Immunomodulatory Properties”, S. Firdous, K. Khan, S. Z. Rehman, Z. Ali, S. Soomro, V. U. Ahmad, M. Rasheed, M. A. Mesaik and S. Faizi, Records of Natural Products , Vol. 8, No. 1, 51-55, (2014).
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3.3.1 Sample Preparation for Bioactivity:
In general extracts (as mentioned in respective schemes) were provided to the collaborators for the evaluation of bioactivity, along with the information of solvents showing maximum solubility. Bioactivities of the pure compounds were also evaluated after complete structure elucidation.
3.3.2 Bioassays:
3.3.2.1 Antimicrobial Activity:
Antibacterial Activity:
In vitro antibacterial activity * of various fractions of root extract of C. rothii (schemes-4.1- 4.3, section: 4.2.1) was performed against 13 Gram-negative and 15 Gram-positive bacteria table-4.30 and 4.31). Fractions KA-PE, KA-C, KM-C and KM-EA exhibiting good activity were further evaluated for their minimum inhibitory concentrations (MIC) (table-4.32, section: 4.5.2.1).
In vitro antibacterial activity ** of the mother extract of the stem of the plant was performed against Gram-negative and Gram-positive bacteria. At the sample concentration of 03 mg/ml in DMSO, 10 µg/disk standard drug (Imipenum) in a well of 6mm (diameter) Std., the extract showed no promising activity (table-4.33, section: 4.5.2.1).
In vitro antibacterial activity ** of the mother extract and hexane, ethyl acetate, and butanol soluble fractions of the leaves of the plant was performed against Gram-negative and Gram- positive bacteria. At the sample concentration of 03 mg/ml in DMSO, 10 µg/disk standard drug (Imipenum) in a well of 6mm (diameter) Std., the mother extract of the leaves showed no antibacterial activity. Hexane fraction showed non-significant activity against Bacillus subtilis , ethyl acetate fraction exhibited non-significant activity against Pseudomonas aeruginosa , whereas butanol fraction exhibited no antibacterial activity at all (table-4.33, section: 4.5.2.1).
*Antibacterial Assay using Disk Diffusion method was performed in collaboration and under Supervision of Prof. Dr. Aqeel Ahmad, Department of Microbiology, University of Karachi.
** Antibacterial Assay using agar well diffusion method was performed in collaboration with International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi.
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Antifungal Activity:
In vitro antifungal activity * of various fractions obtained from root of the C. rothii was evaluated against different fungi (section 4.5.2.1). Non-significant antifungal activity was observed.
In vitro antifungal activity ** of the mother extract of the stem of the plant was performed against different fungi. At the sample concentration of 400 µg/ml in DMSO, 27 °C with the incubation period of 7 days, the mother extract of the stem showed non-significant activity (table-4.34, section: 4.5.2.1).
In vitro antifungal activity ** of the mother extract and the hexane, ethyl acetate, and butanol soluble fractions of the leaves of the plant was performed against different fungi. At the sample concentration of 400 µg/ml of DMSO, 27 °C with the incubation period of 7 days, the mother extract of the leaves showed non-significant antifungal activity, hexane soluble fraction showed no-activity while ethyl acetate fraction exhibited significant activity against Microsporum canis and non-significant activity against Fusarium solani . Butanol soluble fraction exhibited good activity against Aspergillus flavus and Microsporum canis and non- significant activity against Fusarium solani (table-4.34, section: 4.5.2.1).
Antileishmanial Activity:***
Antileishmanial activity of the fractions obtained from the leaves of C. rothii using solvents of varying polarity, i.e., hexane, ethyl acetate, and butanol was also evaluated. No leishmanicidal activity was observed for any of the fractions at the sample concentration of >100 µg/mL, and incubation temperature 22 °C after 72 h against standard drug Amphotericin B (0.50 ± 0.02 µg/mL) (table-4.35, section: 4.5.2.1).
*Antifungal Assay using Disc Diffusion method was performed in collaboration and under Supervision of Prof. Dr. Aqeel Ahmad, Department of Microbiology, University of Karachi.
** Antifungal Assay using agar tube dilution method and *** Antileishmanial Assay using 96-well serial dilution protocol were performed in collaboration with International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi.
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3.3.2.2 Toxicity Studies:
Phytotoxicity:*
In vitro phytotoxicity of the mother extract of the stem of C. rothii was determined against fronds. Paraquat was used as the standard drug. The mother extract exhibited moderate activity at higher level of dose only (table-4.36, section: 4.5.2.2).
In vitro phytotoxicity of mother extract of the leaves of the plant and its fractions in hexane, ethyl acetate, and butanol was determined against fronds using Paraquat as the standard drug. The mother extract of the leaves and its hexane and ethyl acetate soluble fractions showed non-significant activity whereas butanol soluble fraction exhibited no phytotoxicity at all (table-4.36, section: 4.5.2.2).
Brine Shrimp ( Artemia Salina ) Lethality Activity of Leaves:**
Hexane, ethyl acetate and butanol soluble fraction of the leaves extract of the plant were evaluated for their Brine shrimp ( Artemia salina ) lethality bioassay. No cytotoxicity was observed in any of the fractions tested (table-4.37, section: 4.5.2.2).
Cytotoxicity Studies:***
Hexane soluble fraction was also evaluated for cytotoxicity using 3T3 (fibroblast cells). The sample showed 41.68 ± 0.05 IC 50 as compared to control cycloheximide's IC 50 (0.51 ± 0.05) exhibiting no cytotoxic effect on the 3T3 (fibroblast cells) (table-4.38, section: 4.5.2.2).
*Phytotoxicity Assay using modified protocol of Prof. Mc Laughlin et al., (1991), **Brine Shrimp ( Artemia Salina ) Lethality Bioassay, and *** Cytotoxicity Test (3T3-NIH) were performed in collaboration with International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi.
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3.3.2.3 Antioxidant and Immunomodulating Activity:
Antioxidant Activity: *
Antioxidant activity of various fractions of roots of plant (schemes-4.1-4.3, section: 4.2.1) was evaluated using literature protocol (Lee SK et al., 1998) . Only a few fractions exhibited antioxidant activity (table-4.39, section: 4.5.2.3).
Immunomodulatory Studies:**
Immunomodulatory properties of crude extracts, fractions and pure compounds isolated from HEXCRU, CRME, and CRMB were evaluated using oxidative burst, PHA stimulated T-cell proliferation, and nitric oxide (NO), and cytotoxicity assays using 3T3-NIH mouse fibroblast cell line. In preliminary chemiluminescence assay screening, fraction CRM showed no inhibitory activity against reactive oxygen species (ROS) production at >100 µg/mL. Fraction CRME showed significant inhibitory activity whereas fraction HEXCRU and CRMB were found to be moderate at the initial screening doses, 25, 100, and 200 µg/mL (table-4.40, figure-4.1, section: 4.5.2.3).
Fraction HEXCRU exhibited very strong suppressive effect on phytohaemagglutinin (PHA) activated T-cell proliferation stimulated by thymidine incorporation method (Neilson M and
Gerwien J, 1998). The IC 50 value was found to be < 3.12 µg/mL (figure-4.2, section: 4.5.2.3).The effect of sub-fractions and pure compounds on ROS production was observed. Almost all the sub-fractions exerted low to moderate inhibitory effect on ROS production with <50% inhibition. Moreover, pure compound 82 exerted significant inhibitions of ROS production on whole blood phagocytes and neutrophils ROS production with IC 50 of 18.0 and 11.3 µg/mL respectively (table-4.41 and 4.42, section: 4.5.2.3) (Firdous S et al. , 2014).
______
*Antioxidant Bioassay (DPPH method) was performed in collaboration and under Supervision of Prof. Dr. Aqeel Ahmad, Department of Microbiology, University of Karachi.
**Immunomodulating Bioassay was performed in collaboration with International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi. A portion of this work has already been published as;
“Isolation of Phytochemicals from Cordia Rothii (Boraginaceae) and Evaluation of their Immunomodulatory Properties”, S. Firdous, K. Khan, S. Z. Rehman, Z. Ali, S. Soomro, V. U. Ahmad, M. Rasheed, M. A. Mesaik and S. Faizi, Records of Natural Products , Vol. 8, No. 1, 51-55, (2014).
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Among 18 different sub-fractions, fractions BD, BCH, A(KK), B(KK), BA2, C2, C6, and D3 showed significant inhibition on NO production (table-4.41, section: 4.5.2.3). The inhibitory effect ranges between 60 to 80%, with sub-fraction B(KK) scoring maximum inhibitory effect. However, the pure compounds did not show any significant activity on NO production, except in case of 62 where moderate inhibition (43.09%) was observed (table-4.42, section: 4.5.2.3) (Firdous S et al. , 2014).
Almost all sub-fractions exhibited significant immunomodulatory effect on T-cell proliferation with inhibition greater than 85% at concentration of 200 µg/mL. Interestingly sub-fraction D-4, obtained from fraction CRMB showed variation (table-4.43, section: 4.5.2.3). Pure compounds did not show any anti-proliferative effect in similar experiments,
however, compound 82 moderately inhibited the cell proliferation with IC50 of 14.6 µg/mL (table-4.44, section: 4.5.2.4) (Firdous S et al. , 2014).
Cytotoxicity of compounds 62 and 82, which were found to be active as immunomodulating agents, was studied at three different concentrations levels (0.5, 5 and 25 µg/mL) on 3T3-NIH mouse fibroblast cell line. The IC 50 was found to be > 25 µg/mL (Firdous et al. , 2014).
It is worth mentioning that compound 62 was reported as a very important regulator of normal and malignant cell growth (Markaverich BM et al ., 1988), and aglycone of compound 82 significantly reduced NO production in LPS-stimulated BV-2 microglia cells (Kim KH et al ., 2010). Moreover, cytotoxicity of compounds 26 (Gerard L, 2008), 79 (Jayaprakasha GK et al ., 2010), 80 (Zhu Y et al ., 2013), 84 (Ninomiya K et al ., 2007), and 85 (Fang Z et al ., 2010) is also reported in literature (Firdous S et al. , 2014).
Immunomodulatory activity of the sub-fractions and pure compounds obtained from the plant species was determined by chemiluminiscence technique for oxidative burst ROS, assay for nitric oxide and T-cell proliferation assay. Luminol based detection system was applied. Some of the sub-fractions and pure compounds were studied for their response on innate immune including oxidative burst of phagocytes, which is the very first step in host defense mechanism. Stimulants like zymosan, phorbol 12-myristate 13-acetate (PMA), or N-formyl- methionyl-leucyl-phenylalanine (fMLP) initiate oxidative burst and generate, superoxide, hydroxyl radical, hydrogen peroxide and hypochlorous acid. Exposure of these radicals leads to their oxidation and generate excited aminophthalate, which emits photons (light) indicating levels of generated ROS (El Ashry et al ., 2013). Results indicated that some of the sub- fractions exhibited moderate inhibitory activity against oxidative burst where as pure compound 82 exerts significant inhibitory activity on zymosan activated ROS generation thus could have potential to inhibit some of the enzymatic pathways in ROS generation. Alternatively, it might possess scavenging property for free radicals generated during this 172 process. In addition to ROS production, effect of these substrates on NO generation in the stimulated macrophages was also monitored. LPS treated J774.2 was used to induce the production of NO by activation of inducible nitric oxide synthase (iNOS). Out of 18 different sub-fractions, only 8 fractions showed potent activity for NO inhibition. One of the pure compounds, 62 is found to have a moderate inhibitory activity against NO production in macrophages. Compounds 62 and 82 showed moderate antiproliferative effect on human lymphocytes. These results are intriguing and suggest that compounds from C. rothii could act as potential inhibitor for autoimmune disorders and transplant rejection and can also be used as an anti-inflamatory agent for the treatment of chronic inflammatory diseases.
Statistical Analysis:
Results are expressed as mean % inhibition of three determinations at level of significance P ≤ 0.05* P ≤ 0.005** and are calculated using ANOVA.
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3.3.2.4 Insecticidal Activity: *
Mother extract of the stem of C. rothii was evaluated for its insecticidal activity by contact toxicity method. Permethrin (235.9 µg/cm 2) was taken as standard drug. At the concentration level of 1019.10 µg/cm 2, sample showed non-significant activity (table-4.45, section: 4.5.2.4).
The mother extract of the leaves of the plant and its fractions of varying polarity i.e., hexane, ethyl acetate, and butanol were evaluated for their insecticidal activity by contact toxicity method. Taking standard drug Permethrin (235.9 µg/cm 2) and sample (1019.10 µg/cm 2), it was observed that the mother extract showed non-significant activity, hexane fraction exhibited moderate activity against Callosbruchus analis , however, ethyl acetate fraction exhibited good activity against Callosbruchus analis and significant activity against Sitophilus oryzae . Butanol fraction showed good activity against Callosbruchus analis (table- 4.45, section: 4.5.2.4).
3.3.2.5 Antiglycation Studies: **
Hexane, ethyl acetate and butanol fractions of the leaves extract of C. rothii were evaluated for their antiglycation properties. Hexane fraction exhibited excellent % inhibition (77.41 %) and IC 50 value (771.8 ± 0.899 µg). Ethyl acetate fraction showed less inhibition (51.3 %) as
compared to standard's (85.9 %) and IC 50 of 67.3 ± 0.01 µg. Butanol fraction exhibited even
lesser % inhibition (23.4 %) with no IC 50 value as compared to standard Rutin (85.9 %
inhibition and IC 50 41.9 ± 2.3 µg) (table-4.46, section: 4.5.2.5). The concentration of sample taken was 1 mg.
*Insecticidal Activity by Contact Toxicity Method and ** In Vitro Glycation were performed in collaboration with International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi.
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4.1 General Experimental: 4.1.1 Chemicals:
Organic solvents; n-hexane, dichloromethane, chloroform, ethyl acetate, acetone, methanol, and butanol used, were purchased from Merck. For spectroscopy solvents of spectral grade were used.
4.1.2 Spectroscopy:
UV data was taken on Thermo Scientific (Model: Evolution 300) UV-Visible Spectrophotometer and Thermo Electron, UV-Visible Spectrophotometer with visionpro software. IR was recorded on Bruker Vector 22.
EIMS was performed on JEOL, The MS route JMS-600H, HREIMS on Thermo Finnigan MAT 95 XP and FABMS (+ve/-ve) on JEOL JMS-HX110 Mass Spectrometer. ESI-MS was taken on Applied Biosystem Q-STAR XL, in the positive ion mode.
1 The H NMR experiments were recorded in CDCl 3, C 5D5N, C 3D6O, and CD 3OD on Bruker Avance 300, 400, 500, and 600 spectrometers working at 300, 400, 500, and 600 MHz respectively. 13 C NMR (BB & DEPT) were measured at 75, 100, 125, and 150 MHz. Chemical shifts ( δ) were calculated in ppm and coupling constant ( J) in Hertz (Hz).
4.1.3 Chromatography:
Silica gel 60 (70-230 mesh, E. Merck, Damstadt, Germany) was used for column chromatography (CC) and flash column chromatography (FCC). Sephadex LH-20 (25-100 µm; Sigma-Aldrich), and C-18 (25-40 µm, 10 nm; Macherey-Nagel) were used to perform reverse phase column chromatography. Vacuum liquid chromatography (VLC) was
conducted using silica gel 60 HF 254 (Merck).
Purity of compounds was checked on silica gel GF 254 precoated cards (0.2 mm thickness, E.
Merck, Darmstadt, Germany). RP-18 F 254s aluminum sheets (20 x 20 cm, Merck, Darmstadt, Germany) were used for reverse phase TLC. Spots were visualized either by placing TLC plates in the iodine vapour tanks or under UV rays of two different frequencies; 254 and 366 nm. A spraying reagent containing the solution of 0.1 g ceric sulphate and 1g trichloroacetic acid in 4 ml distilled water and enough concentrated sulphuric acid for dissolution was also used.
Gas Chromatography-Flameionization Detection (GC-FID) analyses were performed on a SPB-5® capillary column (30 m length, 0.25 mm ID and 0.25 µm df), installed in a Shimadzu GC-17A. Helium was used as carrier gas at a flow rate of 1 ml/min. Initial temperature of the
175 oven was kept at 70 °C and programmed to 260 °C at a rate of 5 °C/min. Injector was set at 280 oC.
For Gas Chromatography-Mass Spectrometric Detection (GC-MS) experiments a Hewlett- Packard 5890 gas chromatograph was combined with a Jeol, JMS-600 mass spectrometer operating in EI mode with ion source at 250 oC and electron energy at 70 eV. Injector was set at 260 oC with splitting ratio 1:30. Analyses were performed on the aforementioned program used in GC-FID except the temperature rate was kept at (i) 10 oC/min for fractions coded 4A, 2A, 6A, and KEA-C, (ii) 8 oC/min for fractions coded KA-C, KM-PE, KM-C, KM-EA, KEA- PE, KC-PE, and KC-C, (iii) 7 oC/min for fractions coded KA-PE, SH, SEA, SC, SME 10%, SME 20%, SME 30% ABCD, SME 30% EFGH, SME 30% IJKL, and SME 30% MNO, and (iv) 5 oC/min for fractions coded HEXCRU, HS-GC-MS, HS28, A(KK), and B(KK). ZB- 5MS ®capillary column (30 m × 0.25 mm and 0.25 µm film thickness) was used. Mass spectral survey was performed using MS-libraries (NIST, 2005). It is to mention that the GC- FID and GC-MS analyses were performed from an outsource laboratory.
Recycling Preparative HPLC (Model LC-908W/G10/G30/C60) was used to purify compounds from the highly polar fraction. Purity of compounds was checked on silica gel 60
GF 254 precoated cards (0.2 mm thickness) and RP-18 F 254S aluminum sheets (20 x 20 cm, Merck, Darmstadt, Germany).
4.1.4 Plant Material:
Aerial parts of the plant (leaves and stem) were collected in December 2007, and roots in July 2010 from the local nursery of Karachi and University of Karachi Campus, respectively. Prof. Dr. Surraya Khatoon, Taxonomist, Department of Botany, University of Karachi, identified the plant material which is deposited in the herbarium of the same department (voucher specimen number 85853).
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4.2 Extraction, Fractionation, Isolation, and Identification Schemes:
4.2.1 Schemes of work for Root of C. rothii :
Fresh, powdered (4.5 Kg) roots of C. rothii was soaked in hexane for defatting and the marc left was percolated sequentially in chloroform, ethyl aceate, acetone, methanol, and acetone:methanol (50:50)* (2 x 10 L) at room temperature. All the extracts were filtered and evaporated separately in vacuo giving residues of hexane (2A, 11.95 g), chloroform (4A, 12.05 g), ethyl acetate (6A, 7.89 g), acetone (KA, 3.52 g), methanol (KM, 3.02 g), and acetone:methanol (KAcMe, 1.45 g) extracts.
4A and 6A were then treated with hexane and chloroform to fractionate these into hexane soluble KC-PE (2.08 g) and KEA-PE (2.87 g), and hexane insoluble KC-C (7.25 g) and KEA-C (1.97 g) matter. KC-C was dissolved in chloroform and subjected to flash column chromatography (FCC) affording 78 fractions using the eluents hexane, ethyl acetate, methanol, and water in the increasing order of polarity. Fractions number 1-7 (1.52 g), eluted with hexane:ethyl acetate (9.5:0.5), were pooled together and re-chromatographed on FCC following the same procedure mentioned above, provided 56 fractions. Fractions number 32-36 eluted with hexane:ethyl acetate (9.5:0.5) afforded 5.0 mg of pure amorphous powder of mairajinol ( 30 ) (scheme-4.1).
KA and KM were then partitioned into hexane soluble (KA-PE, 0.97 g) and (KM-PE, 1.02 g), chloroform soluble (KA-C, 0.54 g) and (KM-C, 0.45 g), ethyl acetate soluble (KA-EA, 0.28 g) and (KM-EA, 0.24 g), acetone soluble (KA-A, 0.26 g) and (KM-A, 0.35 g), and methanol soluble (KA-M, 0.22 g), and (KM-M, 0.43 g), respectively (schemes-4.2 and 4.3).
KAcMe was subjected to column chromatography (CC), and seven major fractions (1-7) were collected using the solvents hexane, ethyl acetate, and methanol through gradient elution. Fractions number 3-5 (0.24 g) were re-chromatographed with hexane:ethyl acetate (7.5:2.5) in isocratic elution over silica gel affording six fractions (1-6). Fractions number 2 and 3 were combined, and subjected to preparative thin layer chromatography (PTLC) yielding purified compound, identified as stigmasterol ( 27 , 1.79 mg) (scheme-4.4).
Twelve sub-fractions of ground roots; i.e., 2A, 4A, KC-PE, KC-C, 6A, KEA-PE, KEA-C, KA-PE, KA-C, KM-PE, KM-C, and KM-EA (schemes-4.1-4.3, tables-4.1-4.13) were subjected to GC and GC-MS analyses.
______*All the ratios of solvent combination, mentioned in parentheses, are in v/v
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Powdered Roots (4.5 Kg) 1)Hexane x 2* 2) F iltered
Filtrate Marc Evap. in vacuo 1) Chloroform x 2 2) F iltered Residue 2A GC/GC-MS Analysis Filtrate M arc 1-19 ( ) Evap. in vacuo (Scheme-4.1b) T able-4.1 Residue 4A GC/GC-MS Analysis (8,11, 13, 18 re-identified and 20) Table-4.2 1) H exane 2) Filtered
Filtrate S olid M ass E vap. in vacuo 1) Chloroform 2) Filtered Residue Filtrate KC-PE Evap. in vacuo GC/GC-MS Analysis (8, 9, 11, 13-16, 18 Residue re-identified, and 21-27) KC-C Table-4.3 GC/GC-MS Analysis (9, 13, 18, 19, 21, 22, 25, 26 re-identified, and 28, 29) Table-4.4
(FCC) (Hexane, Ethyl acetate, Methanol, and Water)
1 --- 7 ------78
(FCC, Hexane:Ethyl acetate, 9.5:0.5)
1 --- 31 32 ------3637 56
2''-Butoxyethyl 3-[3',5'-di(tert-butyl-4'-hydroxyphenyl]-propanoate (Mairajinol, 30), 5.01mg Isolated New Compound
Scheme-4.1a: Extraction, Isolation, and Identification of constituents from C. rothii Roots.
*Integral with sign of multiplication shows the repetition of step, e.g., x 2 shows the particular step is repeated twice and so on. 2A, 4A, KC-PE, and KC-C were submitted for antibacterial, antifungal, and antioxidant activity.
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Marc (from scheme-4.1a) 1) Ethyl acetate x 2 2) Filtered
Filtrate Marc Evap. in vacuo 1)Acetone x 2 2) Filtered Residue 6A Filtrate Marc GC/GC-MS Analysis (6, 7, 11, 20 re-identified) Evap. in vacuo 1)Methanol x 2 Table-4.5 2) Filtered Residue 1) Hexane KA** 2) Filtered (Scheme-4.2) Filtrate Marc Evap. in vacuo 1) Acetone:Methanol (1:1)x 2 2) Filtered Hexane Soluble Hexane Insoluble Residue Evap. in vacuo Chloroform KM*** (Scheme-4.3) Filtrate Marc Filtrate Residue Evap. in vacuo KEA-PE Evap. in vacuo GC/GC-MS Analysis Residue (9, 11, 13, 14, 18, 21, Residue KAcMe**** 25, 26, 29 re-identified, KEA-C (Scheme-4.4) and 31-33) GC/GC-MS Analysis Table-4.6 (34 and 35) Table-4.7
Scheme-4.1b: Extraction and Identification of constituents from C. rothii Roots.
** ,*** ,****continued in next schemes. 6A, KEA-PE, KEA-C, KA, and KM were submitted for antibacterial, antifungal, and antioxidant activity.
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Residue KA** 1) Hexane 2) Filtered
Hexane Soluble Hexane Insoluble Evap. in vacuo 1) Chloroform 2) Filtered Residue KA-PE GC/GC-MS Analysis Chloroform Soluble Chloroform Insoluble (11, 13, 14, 18, 19 Evap. in vacuo 1) Ethyl acetate 21, 26 re-identified) 2) Filtered Table-4.8 Residue KA-C Ethyl acetate Insoluble GC/GC-MS Analysis Ethyl acetate Soluble (9, 13, 21, 25, 26, Evap. in vacuo 1) Acetone 29 re-identified) 2) Filtered Table-4.9 Residue KA-EA Acetone Soluble Acetone Insoluble Evap. in vacuo 1) Methanol 2) Filtered Residue 3) Evap. in vacuo KA-A Residue KA-M
Scheme-4.2: Extraction and Identification of constituents from C. rothii Roots .
**KA from Scheme-4.1b. KA-PE, KA-C, KA-EA, KA-A, and KA-M were submitted for antibacterial, antifungal, and antioxidant activity.
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Residue KM*** 1) Hexane 2) Filtered
Hexane Soluble Hexane Insoluble Evap. in vacuo
Residue KM-PE GC/GC-MS Analysis (11, 13, 18, 21, 25, 26, Chloroform soluble Chloroform Insoluble 32, 33 re-identified, and 36-44) Evap. in vacuo Table-4.10 1) Ethyl acetate 2) Filtered Residue KM-C GC/GC-MS Analysis (11, 13, 18, 21, 25, Ethyl acetate soluble Ethyl acetate Insoluble 26, 33, 42, 43 re-identified) 1) Acetone Table-4.11 Evap. in vacuo 2) Filtered Residue KM-EA GC/GC-MS Analysis Acetone Soluble Acetone Insoluble (11, 13, 21, 24, 25, 26, 28, 1) Methanol 40, 41 re-identified, and 45, 46) Evap. in vacuo Table-4.12 2) Filtered 3) Evap. in vacuo Residue KM-A Residue KM-M
Scheme-4.3: Extraction and Identification of constituents from C. rothii Roots.
***KA from Scheme-4.1b. KM-PE, KM-C, KM-EA, KM-A, and KM-M were submitted for antibacterial, antifungal, and antioxidant activity.
181
Residue KAcMe**** (CC) (Hexane, Ethyl acetate, and Methanol)
1 2 3 4 5 6 7
(CC) (Hexane:Ethyl acetate, 7.5:2.5)
1 2 3 4 5 6
PTLC (Hexane:Ethyl acetate, 5.5:4.5)
Stigmasterol (27), 1.79 mg Isolated Known Compound
Scheme-4.4: Isolation and Identification of constituents from C. rothii Roots.
****KAcMe from Scheme-4.1b.
182
4.2.2 Schemes of work for Stem of C. rothii :
5 Kg dried, chopped pieces of stem of the plant was soaked in methanol (3 x 20 L) at room temperature for 15 days each. The extracts obtained were combined, filtered and evaporated in vacuo to provide 20 g residue CRS. The CRS was subjected to liquid-liquid partitioning and extracted sequentially with hexane, ethyl acetate, and butanol. The hexane fraction CRSH (6.02 g) after usual workup was subjected to column chromatography (CC) yielding 120 fractions. Fractions number 71-90 were pooled together and re-chromatographed using silica gel. It provided white shiny crystalline compound identified as β-sitosterol ( 26 ) (scheme-4.5).
Due to the interesting results in GC-MS studies from roots, another extraction protocol was followed to get maximum volatiles from the stems. Thus carefully cut dried stem of C. rothii (5kg) was soaked in methanol and filtered off. Filtrate was evaporated under reduced pressure and the residue obtained was extracted with hexane, resulting in hexane soluble (SH, 13.34 g) and hexane insoluble part. The hexane insoluble part was extracted with chloroform, resulting in chloroform soluble (SC, 2.97 g) and chloroform insoluble portions. The chloroform insoluble part was further treated with ethyl acetate that divided it into ethyl acetate soluble (SEA, 2.75 g) and ethyl acetate insoluble portion. The marc left was extracted with same procedure successively, using the mixtures of ethyl acetate:methanol in the ratios of 90:10 (SME 10%, 1.34 g), 80:20 (SME 20%, 0.98 g), and 70:30 (SME 30%, 4.45 g). The last combination used was giving yellowish shade showing further extractable in this fraction. Therefore, extraction with ethyl acetate:methanol (70:30) was repeated several times, yielding extracts, coded ‘A’ to ‘O’. These fractions on the basis of TLC were pooled as, SME 30% ABCD (1.02g), SME 30% EFGH (0.59 g), SME 30% IJKL (0.75 g), and SME 30% MNO (0.64 g) respectively. All these aforementioned 9 extracts coded SH, SC, SEA, SME 10%, SME 20%, SME 30% ABCD, SME 30% EFGH, SME 30% IJKL, and SME 30% MNO were subjected to GC-FID and GC-MS analyses. Their chromatograms showed the presence of 31, 16, 17, 14, 26, 19, 23, 16, and 15 compounds respectively. These metabolites were primarily identified via electronic mass spectral library (NIST, 2005) and identification of these was confirmed by their Retention Indices. Scheme-4.6a and 4.6b illustrates the complete picture of the extraction protocol used for the isolation and identification of chemical constituents from C. rothii stem while table-4.14-4.23 expressed the results and findings.
______*All the ratios of solvent combination, mentioned in parentheses, are in v/v
183
Stem(5 Kg) 1) MeOH x 3 2) Filtered
Marc Filtrate Evap. in vacuo Residue CRS 1) Distilled water 2) Hexane ( Solvent-solvent separation)
Aqueous Layer 1) E.A. 2) Solvent-solvent separation Hexanelayer
1) Dried with anhydrous Na2SO4 2) Filtered E.A. layer
1) Dried with anhydrous Na2SO4 2) Filtered
Na2SO4 Filtrate 1) Charcoaled 2) Filtered Na2SO4 Filtrate Evap. in vacuo
Residue Charcoal bed Filtrate CRSE Evap. in vacuo
Residue CRSH
(CC) (Hexane, Ethyl acetate, Methanol, and Water)
1 --- 30 --- 71 --- 90 ------120 Aqueous layer 1) Butanol 2) Solvent-solvent separation β-Sitosterol (26) Isolated Known Compound Aqueous layer Butanol layer
Evap. in vacuo
Residue CRSB
Scheme-4.5: Extraction, Isolation, and Identification of constituents from C. rothii Stem.
CRS was submitted for antibacterial, antifungal, phytotoxic, and insecticidal activity.
184
Stem (5 Kg) 1) MeOH x 2 2) Filtered
Filtrate Marc Evap. in vacuo
Residue CRS Hexane
Hexane Soluble Hexane Insoluble SH Chloroform GC/GC-MS Analysis (8, 10, 11, 13, 18, 23, 41 re-identified, Chloroform Soluble Chloroform Insoluble and 47-61) SC Table-4.14 Ethyl acetate GC/GC-MS Analysis (11, 13, 18, 23, 28, 46, 53, 56, 58, 60 Ethyl acetate Soluble Ethyl acetate re-identified, and, SEA Insoluble 62 and 63) GC/GC-MS Analysis Table-4.15 (11, 13, 18, 23, 45, 46, 53, 57, 58, 60, 63 re- identified) Ethyl acetate:Methanol Table-4.16 (90:10)
Ethyl acetate:Methanol (90:10) Soluble Ethyl acetate:Methanol (90:10) Insoluble SME 10% GC/GC-MS Analysis (11, 13, 14, 18, 23, 42, 46, Ethyl 49, 53, 56 re-identified) acetate:Methanol Table-4.17 (80:20)
Ethyl acetate:Methanol (80:20) Soluble Ethyl acetate:Methanol (80:20) SME 20% Insoluble GC/GC-MS Analysis (11, 13, 14, 18, 22, 23, 26, Continued.... 31, 41, 42, 45, 46, 53-58, 60, 63 re-identified) Table-4.18
Scheme-4.6a: Extraction, Isolation, and Identification of constituents from C. rothii Stem.
185
Ethyl acetate:Methanol (80:20) Insoluble
Ethyl acetate:Methanol (70:30)
SME 30% Soluble SME 30% Insoluble
ABCDEFGHIJKLMNO
Ethyl acetate:Methanol (70:30) Ethyl acetate:Methanol (70:30) Soluble SME 30% ABCD Soluble SME 30% IJKL GC/GC-MS Analysis GC/GC-MS Analysis (11, 13, 14, 18, 19, 22, (11, 13, 14, 18, 22, 23, 26, 31, 42, 46, 23, 42, 46, 53, 55, Ethyl acetate:Methanol (70:30) 53, 56, 57, 58, 60, 63 56, 57, 58, 60 Soluble SME 30% MNO re-identified) re-identified) Table-4.19 Table-4.21 GC/GC-MS Analysis (11, 13, 14, 18, 23, 42, 46, 53, 56, 57, 58 re-identified) Ethyl acetate:Methanol (70:30) Soluble SME 30% EFGH Table-4.22 GC/GC-MS Analysis (11, 13, 14, 18, 19, 22, 23, 26, 42, 46, 53-58, 60, 63 re-identified) Table-4.20 Scheme-4.6b: Extraction, Isolation, and Identification of constituents from C. rothii Stem.
186
4.2.3 Schemes of work for leaves of C. rothii :
Uncrushed dried leaves (5 Kg) of C. rothii were soaked in methanol (3 x 20 L), at room temperature, each time for 15 days. The extract obtained was filtered and evaporated under reduced pressure to give residue CRM (85.23 g). CRM was treated with water and extracted sequentially with n-hexane (HEXCRU, 57.72 g), ethyl acetate (CRME, 11.14 g), and butanol (CRMB, 12.24 g) to yield corresponding phases. Fraction HEXCRU was partitioned into aqueous methanol and hexane soluble layer which upon evaporation under reduced pressure provided methanol soluble (CRMHM, 14.78 g) and hexane soluble residues (HS-GC-MS, 28.73 g) respectively (scheme-4.7). HEXCRU and HS-GC-MS fractions were submitted for GC-FID and GC-MS and also subjected to further fractionation and sub-fractionation. GC- FID and GC-MS analyses resulted in identification of 11 metabolites from HEXCRU and 19 from HS-GC-MS, which were 11 , 13 , 18 , 42 , 64-69 , and 73 ; and 11 , 13 , 19 , 23 , 41 , 48 , 50 , 53 , 57 , 58 , and 64 -67, 70-73 , and 75 respectively.
The HS-GC-MS provided different fractions when subjected to vacuum liquid chromatography on elution with solvents of different polarities. The sub-fractions were collected from n-hexane (HS28, 6.90 g), carbon tetrachloride (BCT, 3.16 g), dichloromethane (BD, 4.25 g), chloroform (BCH, 3.41 g), ethyl acetate (BE, 4.53 g), acetone (BA, 3.57 g) and methanol (HHM, 2.51 g) (scheme-4.8). HS28 on GC analyses revealed presence of 14 metabolites ( 11 , 13 , 22 , 23 , 42 , 50 , 56 , 57 , 68, 70 , 71 , 73 , 76 , and 77 ). From BD, an insoluble solid settled down, which on washing yielded purified compound octacosan-1-ol ( 74 , 1.58 mg). Fraction BCH provided crystalline solid, through re-crystallization in chloroform it afforded a compound identified as β-sitosterol ( 26, 1.38 mg). BE was subjected to column chromatography on silica gel using isocratic mobile phase chloroform:methanol (6.75:3.25). It provided 79 fractions. Of these, fractions number 1-15 and 16-53 were pooled on the basis of TLC, yielding two sub-fractions A(KK) (1.99 g)and B(KK) (0.98 g) respectively. Faractions A(KK) and B(KK) were analysed on GC-MS and resulted in identification of 04 (13 , 41 , 42 , 48 ) and 09 ( 13 , 18 , 41 , 42 , 48, 78 , 86-88 ) constituents respectively.
Fraction BA, which was obtained from VLC, upon column chromatography on silica gel with isocratic mobile phase, chloroform:methanol (9:1) yielded 31 fractions. On standing at room temperature, insoluble solid separated out from the first three fractions of this column, which on the basis of TLC were combined. Upon filtration and washing with cold chloroform:methanol (9:1), it furnished a single compound identified as stigmast-5-en-3-O-β-
D-glucoside ( 79 , 1.67 mg). The filtrate upon vacuum evaporation yielded a residue (HHA ______
*All the ratios of solvent combination, mentioned in parentheses, are in v/v
187
Leaves (5 Kg) 1) MeOH (20 L x 3) 2) Filtered 15 days each
Marc Filtrate Evap. in vacuo
Residue CRM (85.23g) 1) Distilled water 2) Hexane (Solvent-solvent separation)
Hexane layer Aqueous Layer
1) Dried with anhydrous Na2SO4 1) Ethyl acetate 2) Filtered 2) Solvent-solvent separation
Aqueous layer Na2SO4 Filtrate Ethyl acetate layer 1) Charcoaled 1) Dried with anhydrous Na2SO4 1) Butanol 2) Filtered 2) Filtered 2) Solvent-solvent separation
Aqueous layer Butanol layer Charcoal bed Filtrate Na SO Filtrate 2 4 Evap. in vacuo Evap. in vacuo Evap. in vacuo
Residue Residue Residue CRME** CRMB*** HEXCRU (57.72 g) (11.14 g) (12.24g) GC/GC-MS Analysis (11, 13, 18, 42 re-identified, and 64-69, and 73) Table 4.24 1) Hexane 2) MeOH (Solvent-solvent separation)
Methanol soluble Hexane soluble Evap. in vacuo Evap. in vacuo Residue Residue CRMHM (14.78 g) HS-GC-MS* (28.73 g) GC/GC-MS Analysis (11, 13, 19, 23, 41, 48, 50, 53, 57, 58, 64-67, 73 re-identified, 70-72, and 75) Table 4.25
Scheme-4.7: Extraction and Identification of constituents from C. rothii leaves. *,** ,*** Continued in next schemes ______CRM, HEXCRU, CRME, and CRMB were submitted for antibacterial, antifungal, phytotoxic, chemiluminescence, and insecticidal activity whereas HEXCRU, CRME, and CRMB were evaluated for Brine Shrimp lethality, antileishmanial, and antiglycation activity. CRMHM and HS-GC-MS were tested for Inhibition oxidative burst on whole blood phagocytes (ROS) and Nitric oxide % Inhibition. Effect of HEXCRU, CRMHM, and HS-GC-MS on PHA induced T-cell proliferation was also checked.
188
HS-GC-MS* (28.73 g) VLC
(CH ) CO CH OH Hexane CCl4 CH2Cl2 CHCl3 EtOAc 3 2 3 HS28 BCT BD BCH BE BA HHM (6.90 g) (3.16 g) (4.25 g) (3.41 g) (4.53 g) (3.57 g) (2.51 g) Insoluble matter settled down, Re- washed crystallization CC on Silica gel β CHCl3:MeOH -Sitosterol (26) (6.75:3.25) Octacosan-1-ol Filtrate (1.38 mg) (74) (1.58 mg) Re-isolated Isolated Known Compound First Report from Species 1 --- 15 16 --- 53 54 --- 79
GC/GC-MS Analysis A(KK) B(KK) BE3 (11, 13, 22, 23, 42, 50, 56, 57, 68, 70, CC on 71, 73 re-identified, GC/GC-MS GC/GC-MS Silica gel and 76 and 77) Analysis Analysis CHCl3:MeOH Table-4.26 (13, 41, 42, 48 (13, 18, 41, 42, 48 (9.0:1.0) re-identified) re-identified, Table-4.27 and 78, 86-88) Table-4.28 1 2 3 --- 10 --- 20 --- 31
1) Insoluble matter settled down 2) Filtered
β Filtrate Stigmast-5-en-3-O- -D-glucoside (79) (1.67 mg) Evap. in vacuo Isolated Known Compound Residue First Report from Species HHA-1-3 (0.58 g)
CC on Silica gel (CHCl3:MeOH, 9.25:0.75)
1 --- 4 5 6 --- 8 9 --- 12 --- 32 (BA1) (BA2) (BA3)
1-O-β-D-Glucopyranosyl-(2S,3S,4R,8Z)-2-[(2'R)-2'- hydroxytetracosanoylamino]-1,3,4-octadecanetriol-8-ene (80) (51.2 mg) Isolated Known Compound First Report from Species & Genus
Scheme-4.8: Extraction, Isolation, and Identification of constituents from C. rothii leaves ______HS28, BCT, BD, BCH, A(KK), B(KK), and BA2 were scanned for Inhibition oxidative burst on whole blood phagocytes (ROS) and Nitric oxide % Inhibition. All these sub-fractions were also evaluated for their effect on PHA induced T-cell proliferation. Isolated, purified compounds, 74 , 26, 79 , and 80 were tested for ROS (in whole blood and neutrophils), and NO production, and their effect on T-cell proliferation were also evaluated.
189
1-3, 0.58 g). This was subjected to column chromatography on silica gel using isocratic solvent system, chloroform:methanol (9.25:0.75) and provided 32 fractions. On the basis of TLC, fractions 1-4, 6-8, and 9-12 were combined to afford sub-fractions; BA1, BA2, and BA3 respectively. Major fraction BA3 was further purified and provided a chromatographically pure white gelatinous solid identified as 1-O-β-D-glucopyranosyl- (2 S,3 S,4 R,8 Z)-2-[(2 ′R)-2′-hydroxytetracosanoyl amino]-1,3,4-octadecanetriol-8-ene (80 , 5.12 mg).
The residue CRME ( vide scheme-4.7) was partitioned into neutral fraction CRMEE (5.46 g) and acidic fraction CRMEA (0.99 g) by treating it with 4% aq. Na 2CO 3 and ethyl acetate followed by acidification with 5% aq. HCl and ethyl acetate solution, respectively. The
CRMEE was loaded on silica gel column (scheme-4.9). Using gradient elution (chloroform, ethyl acetate, and methanol), it afforded 118 fractions. Out of these, fractions numbers 16-24, 27-29, and 37-48 were pooled and coded as C2, C4, and C7 while fraction C7 was chromatographically pure. Fraction C2 was screened for immunomodulatory properties. Fraction C4 upon repeated column chromatography on silica gel using chloroform:methanol (9.8:0.2 v/v) as eluent yielded (2 S) methyl 2-hydroxy-3-(4 ′-hydroxyphenyl)-propanoate ( 62 , 3.25 mg). Fraction C7 provided another crystalline compound (2 R) 2-hydroxy-3-(4 ′- hydroxyphenyl)-propanoic acid (81 , 5.89 mg) via recrystallization.
The residue CRMB (12.24 g) was subjected to column chromatography on silica gel, with gradient elution using chloroform, methanol, and water sequentially. 79 fractions (scheme- 4.10) were obtained. Fraction number 29 (D1), 30 (D2), 31 (D3), and 32 (D4) were screened for immunomodulatory properties while other fractions were combined on the basis of TLC. Combined fractions containing fractions number 33-40 (D5, 2.85 g) was fractionated on column chromatography using silica gel with gradient elution. Sequential elution with
chloroform and methanol provided 68 fractions. Among these, combined fractions number 36-51 upon repeated column chromatography on silica gel using dichloromethane:methanol
(9:1) furnished pure compound syringaresinol mono-β-D-glucoside ( 82 , 4.56 mg). In another attempt combined fractions number 52-59 from fraction D5 on silica gel column chromatography using dichloromethane:methanol (9:1) yielded 91 fractions. Three different compounds namely: 6-hydroxy-3-oxo-α-ionol 9-O-β-D-glucopyranoside ( 83 , 3.15 mg); staphylionoside D ( 84 , 4.25 mg); and 3-(3 ′,5 ′-dimethoxy-4′-O-β-D-glucopyranosyl-phenyl)- prop-2E-en-1-ol ( 85 , 2.35 mg) were obtained and purified from the pooled sub-fractions number 13-29 of this column using preparative RP-18 column on recycling HPLC using methanol:water (7:3) as mobile phase.
190
Residue CRME** (11.14 g) 1) Ethyl acetate 2) 4% Na2CO3
Ethyl acetate layer Aqueous layer Evap. in vacuo 1) Acidify with 5% HCl 2) Ethyl acetate (Solvent-solvent separation) Residue CRMEE (5.46 g) Aqueouslayer Ethylacetate-acidiclayer
1) Dried with anhydrous Na2SO4 2) Filtered
Filtrate Na2SO4 Evap. in vacuo
Residue CRMEA (0.99 g) CC on Silica gel (Chloroform, Ethyl acetate, and Methanol)
1 --- 16 --- 24 ---27 --- 29 --- 36 37 --- 48 --- 118
C2 C4 C6 C7 CC on Silica gel (Chlorof orm:Methanol, Crystallization 9.8:0.2)
(2R) 2-Hydroxy-3-(4'- 1 --- 5 --- 9 --- 21 hydroxyphenyl)- propanoic acid (81) (5.89 mg) Isolated Known Compound First Report from Specie (2S-) Methyl 2-hydroxy-3- (4'-hydroxyphenyl) propanoate (62) (3.25 mg) Isolated Known Compound Also identified in GC-MS studies from SC First Report from Species
Scheme-4.9: Extraction, Isolation, and Identification of constituents from C. rothii leaves.
Fractions CRMEE, C2, C6, and CRMEA were tested for Inhibition oxidative burst on whole blood phagocytes (ROS) and Nitric oxide % Inhibition. The effect of these sub-fractions on PHA induced T-cell proliferation was also checked. Moreover, purified compounds 62 and 81 were screened for ROS (in whole blood and neutrophils), and NO production, and their effect on T-cell proliferation were also evaluated.
191
CRMB*** (12.24 g) CC on Silica gel (Chloroform, Methanol, and Water)
1 --- 2930 31 32 33 --- 40 41 --- 46 47 --- 53 54 --- 70 --- 79
D1 D2 D3 D4 D5 CRMB C CRMBD CRMBE (2.85 g) (1.93 g) (1.97 g) (2.25 g)
CC on Silica gel CC on Sephadex (LH-20) (Chloroform, Methanol) (Water, Methanol)
1 --- 23 --- 36 --- 51 52 --- 59 --- 68 1 2 3 --- 10 --- 48
CC on RPSilica CC on Silica gel (Water, Methanol) (Dichloromethane: Methanol, 9:1)
1 --- 6 --- 8 --- 19 1 --- 20 --- 51 --- 54 Crystallization
Syringaresinol mono-β -D-glucoside (82) 2-Hydroxy-3-(4'-hydroxyphenyl)propanoic acid (81) (4.56 mg) (3.91 mg) Isolated Known Compound Re-isolated FirstReportfromSpecies&Genus CConSilicagel (Dicholoromethane:Methanol, 9:1)
1 --- 13 --- 29 --- 40 --- 91
Recycling HPLC RP-18 Column (Methanol:Water,7:3)
C-8 C-9 C-10
6-Hydroxy-3-oxo-α-ionol Staphylionoside D (84) 3-(3',5'-dimethoxy-4'-O-β -D-glucopyranosyl 9-O-β -D-glucopyranoside (83) (4.25 mg) -phenyl)-prop-2E-en-1-ol (85) (2.35 mg) (3.15 mg) Isolated Known Compound Isolated Known Compound Isolated Known Compound First Report from Species & First Report from Species First Report from Species & Genus Genus
Scheme-4.10: Isolation of Pure Compounds from C. rothii Leaves.
______D1-D4 were evaluated for Inhibition oxidative burst on whole blood phagocytes (ROS) and Nitric oxide % Inhibition and their effect on PHA induced T-cell proliferation was also determined. Beside these, pure compounds 82 -85 were tested for ROS (in whole blood and neutrophils), and NO production. The effect of these compounds on T-cell proliferation was also evaluated.
192
4.3 Characterization of Isolated Compounds.
4.3.1 Characterization of 2 ′′ -Butoxyethyl 3-[3 ′,5 ′-di( tert -butyl)-4′-hydroxyphenyl]- propanoate (Mairajinol, 30) ― A New Compound:
O O 1'' 2'' 1''' 2''' 1 O 2 3
1' 2' 8' 6' 12' 9' 3' 5' 13' 4' 7' 11'
10' OH 14'
Colourless amorphous solid isolated from roots (scheme-4.1a)
-1 IR νmax (KBr) cm : 3717 to 3572, 2956, 2924, 2855, 1735, 1600, 1470, 1461, 1374, 1125, 741.
UV/Vis λmax (CHCl 3) nm: 205, 225, 275.
+ + + EIMS m/z (rel. intensity, %): 378 (40) [M, C 23 H38 O4] ; 278 (4) [C 17 H26 O3] or [M-C6H14 O] ; + + + + 219 (30) [C 15 H23 O] or [M-C8H15 O3] ; 102 (5) [C 6H14 O] or [M-C17 H26 O3] ; 91 (14) + + + + [C 6H3O] ; 57 (100) [C 4H9] or [M-C19 H29 O4] ; 43 (97) [C 3H7] .
+ HREIMS m/z (rel. intensity, %): 378.275 (M , calcd. for C 23 H38 O4, 378.2770, ∆ = -0.002).
Error (ppm) = 1.0 x 10 6 (-0.002)/378.275 = -5.7.
FABMS (+ve) m/z : 379 [M+1] +.
FABMS (-ve) m/z : 377 [M-1] -.
1 H NMR (500 MHz, CDCl 3) δH: 0.86 (3H, t, J = 7.0 Hz, H-4″′ ), 1.24 (2H, br. s, H-3″′ ), 1.41 (18H, s, H-8′-H-10 ′, H-12 ′-H-14 ′), 1.53 (2H, m, H-2″′ ), 2.62 (2H, dd, J = 7.5 Hz, 8.5 Hz, H- 2), 2.86 (2H, dd, J = 7.5 Hz, 8.5 Hz, H-3), 3.44 (2H, t, J = 7.0 Hz, H-1″′ ), 3.60 (2H, br. dd, J = 4.5 Hz, 5.0 Hz, H-2″), 4.21 (2H, dd, J = 4.5 Hz, 5.0 Hz, H-1″), 6.97 (2H, s, H-2′, 6 ′).
13 C NMR (100 MHz, CDCl 3) δC: 14.1 (CH 3, C-4″′ ), 22.7 (CH 2, C-3″′ ), 30.3 (CH 3, C-8′-C10 ′,
C-12 ′-C-14 ′), 31.0 (CH 2, C-3), 31.9 (CH 2, C-2″′ ), 34.1 (C, C-7′, C-11 ′), 36.4 (CH 2, C-2), 63.7
(CH 2, C-1″), 68.6 (CH 2, C-2″), 71.2 (CH 2, C-1″′ ), 124.8 (CH, C-2′, C-6′), 131.1 (C, C-1′), 136.0 (C, C-3′, C-5′), 152.2 (C, C-4′), 173.2 (C, C-1).
193
4.3.2 Characterization of Stigmast-5-en-3β-ol (β-sitosterol) (26 ):
29 28
21 24 20 26 22 18 23 25 12 17 11 27 19 13 16 1 9 14 2 10 8 15 3 5 7 HO 4 6
White shiny crystals isolated from stem (scheme-4.5) and leaves (scheme-4.8).
+ + EIMS m/z (rel. int., %): 414 (13) [C 29 H50 O] or [M] ; 399 (4), 396 (6), 381 (5), 329 (7), 303 + + + + (5), 273 (7) [C 19 H29 O] or [M-C10 H21 ] ; 255 (9) [C 19 H27 ] or [M-C10 H21 -H2O] ; 105 (24), 95 + (26), 85 (15), 79 (26), 71 (22), 57 (77), 55 (100) [C 4H7] .
+ HREIMS : m/z 414.3855 [M] (calcd. for C 29 H50 O, 414.3862, ∆ = -0.0007).
Error (ppm) = 1.0 x 10 6 (-0.0007)/414.3855 = -1.6.
1 H NMR (500 MHz, CDCl 3) δH: 0.66 (3H, s, H-18), 0.79 (3H, d, J = 7.0 Hz, H-27), 0.81 (3H, d, J = 6.5 Hz, H-26), 0.83 (3H, t, J = 7.0 Hz, H-29), 0.90 (3H, d, J = 6.5 Hz, H-21), 0.99 (3H, s, H-19), 3.50 (1H, m, H-3), 5.33 (1H, m, H-6).
13 C NMR (125 MHz, CDCl 3) δC: 11.9 (CH 3, C-29), 12.1 (CH 3, C-18), 18.8 (CH 3, C-21), 19.1
(CH 3, C-27), 19.4 (CH 3, C-19), 19.8 (CH 3, C-26), 21.1 (CH 2, C-11), 23.1 (CH 2, C-28), 24.3
(CH 2, C-15), 26.2 (CH 2, C-23), 28.3 (CH 2, C-16), 29.2 (CH, C-25), 31.7 (CH 2, C-2), 31.9
(CH 2, C-7), 31.9 (CH, C-8), 34.0 (CH 2, C-22), 36.2 (CH, C-20), 36.5 (C, C-10), 37.3 (CH 2, C-
1), 39.8 (CH 2, C-12), 42.2 (C, C-13), 42.3 (CH 2, C-4), 45.9 (CH, C-24), 51.3 (CH, C-9), 56.1 (CH, C-17), 56.8 (CH, C-14), 71.8 (CH, C-3), 121.7 (CH, C-6), 140.8 (C, C-5).
194
4.3.3 Characterization of (24 S)-Stigmast-5,22-dien-3β-ol (Stigmasterol) (27 ):
29 28
21 24 26 20 22 18 23 25 12 17 11 27 19 13 16 1 9 2 14 10 8 15 3 5 7 HO 4 6
White amorphous solid isolated form roots (scheme-4.4)
1 H NMR (400 MHz, CDCl 3) δH: 0.67 (3H, s, H-18), 0.78 (3H, d, J = 6.4 Hz, H-27), 0.81 (3H, t, J = 7.2 Hz, H-29), 0.85 (3H, d, J = 6.4 Hz, H-26), 0.99 (3H, s, H-19), 1.06 (3H, d, J = 6.6 Hz, H-21), 3.50 (1H, m, H-3), 4.99 (1H, dd, J = 8.4 Hz, 15.2 Hz, H-23), 5.13 (1H, dd, J = 8.4 Hz, 15.2Hz, H-22), 5.33 (1H, br.s, H-6).
13 C NMR (100 MHz, CDCl 3) δC: 11.8 (CH 3, C-18), 11.9 (CH 3, C-29), 19.0 (CH 3,
C-26), 21.2 (CH 3, C-21), 21.2 (CH 3, C-19), 21.1 (CH 2, C-11), 21.2 (CH 3, C-27), 24.3 (CH 2,
C-15), 26.1 (CH 2, C-28), 28.2 (CH 2, C-16), 31.7 (CH 2, C-2), 31.9 (CH 2, C-7), 31.9 (CH, C-8),
31.9 (CH, C-25), 36.2 (C, C-10), 37.3 (CH 2, C-1), 39.8 (CH 2, C-12), 39.8 (CH,
C-20), 42.3 (CH 2, C-13), 42.3 (CH 2, C-4), 50.2 (CH, C-9), 51.3 (CH, C-24), 56.1 (CH, C-17), 56.8 (CH, C-14), 71.8 (CH, C-3), 121.7 (CH, C-6), 129.2 (CH, C-23), 138.3 (CH, C- 22), 140.8 (C, C-5).
195
4.3.4 Characterization of (2 S-) Methyl 2-hydroxy-3-(4 ′-hydroxyphenyl)-propanoate (Latifolicinin C) (62 ):
O OH
1 2 H3CO 3 1' 4' OH
Amorphous powder isolated form leaves (scheme-4.9).
+ + + EIMS m/z (rel. intensity, %): 196 (2) [M, C 10 H12 O4] ; 178 (2) [C 10 H10 O3] or [M-H2O] ; 147 + + + + + (2) [C 9H7O2] or [M-CH 5O2] ; 137 (12) [C 8H9O2] or [M-C2H3O2] ; 119 (2) [C 8H7O] or + + [M-C2H5O3] ; 108 (9), 107 (100) [C 7H7O] , 91 (8), 83 (5), 77 (11), 65 (2).
+ HREIMS m/z : 196.0747 [M] (calcd. for C 10 H12 O4, 196.0736, ∆ = 0.0011).
Error (ppm) = 1.0 x 10 6 (0.0011)/196.0747 = +5.6.
+ FABMS (+) m/z : 197 [M+H] , C 10 H13 O4.
CI (+) m/z : 197 [M+H] +.
-1 IR νmax (CDCl 3) cm : 3434 to 3289, 1730, 1608, 1514, 1444.
1 H NMR (300 MHz, CDCl 3) δH: 2.88 (1H, dd, J = 6.5 Hz, 14.0 Hz, H-3a), 3.04 (1H, dd, J =
4.4 Hz, 14.0 Hz, H-3b), 3.75 (3H, s, 1-OCH 3), 4.39 (1H, br.t, J = 5.6 Hz, H-2), 6.73 (2H, d, J = 8.4 Hz, H-3′, H-5′), 7.05 (2H, d, J = 8.3 Hz, H-2′, H-6′).
13 C NMR (75 MHz, CDCl 3) δC: 39.6 (CH 2, C-3), 52.4 (CH 3, -OCH 3), 71.4 (CH, C-2), 115.3 (CH, C-3′/C-5′), 128.2 (C, C-1′), 130.6 (CH, C-2′/C-6′), 154.6 (C, C-4′), 174.6 (C, C-1).
196
4.3.5 Characterization of Octacosan-1-ol (74 ):
27
28
1 HO 2
White amorphous powder isolated from leaves (scheme-4.8).
-1 IR νmax (KBr) cm : 3490 to 3350, 2919, 2850, 1467, 723.
+ + + EIMS m/z (rel. intensity, %): 392 (12) [C 28 H56 ] or [M-H2O] ), 111 (23), 97 (51), 85 [C 6H13 ] + + (25), 83 (71), 71 (46) [C 5H11 ] , 69 (72), 57 (100) [C 4H9] .
+ HREIMS : m/z 392.4413 [M-H2O] (calcd. for C 28 H56 , 392.4382, ∆ = 0.0031).
Error (ppm) = 1.0 x 10 6 (0.0031)/392.4413 = +7.8
CI (+):m/z 409 [M-H] +.
1 H NMR (300 MHz, CDCl 3) δH: 0.86 (3H, t, J = 6.0 Hz, H-28), 1.24 (50H, br.s, H-3 to H-27), 1.54 (2H, m, H-2), 3.62 (2H, t, J = 6.6 Hz, H-1).
13 C NMR (75 MHz, CDCl 3) δC: 14.1 (CH 3, C-28), 22.7 (CH 2, C-27), 25.7 (CH 2, C-3), 29.7
(CH 2, C-4 to C-25), 31.9 (CH 2, C-26), 32.8 (CH 2, C-2), 63.10 (CH 2, C-1).
197
4.3.6 Characterization of Stigmast-5-en-3-O-β-D-glucoside ( β-Sitosterol glucoside) (79 ):
29 28 21 24 20 26 22 18 23 25 12 17 27 19 11 13 16 OH 1 6' 9 4' 2 14 15 HO 5' O 10 8 1' 3 HO 7 3' 2' O 5 OH 4 6
White amorphous solid isolated form leaves (scheme-4.8).
+ + + EIMS m/z (rel. intensity, %): 415 (5) [C 29 H51O] or [M-C6H11 O5] ; 396 (19) [C 29 H49 ] or + + + + [M-C6H12 O6] ; 255 (4) [C 25 H38 O5] or [M-C10 H21 -H2O] ; 135 (5), 85 (71) [C 6H13 ] ; 83 + [C 6H11 ] (100), 69 (16).
+ + + HREIMS : m/z 415.3979 [M-C6H11 O5] or [C 29 H51 O] (calcd. for [C 29 H51 O] , 415.3940, ∆ = 0.0039). Error (ppm) = 1.0 x 10 6 (0.0039)/415.3979 = +9.4
1 H NMR (400 MHz, C5D5N) δH: 0.65 (3H, s, H-18), 0.84 (1H, m, H-9), 0.85 (6H, d, J = 7.2 Hz, H-26, H-27), 0.89 (3H, t, J = 7.4 Hz, H-29), 0.92 (3H, s, H-19), 0.95 (1H, m, H-14), 0.98 (3H, d. J = 6.5 Hz, H-21), 0.99 (1H, m, H-1a), 1.01 (1H, m, H-24), 1.03 (1H, m, H-15a), 1.10 (1H, m, H-12a), 1.09 (1H, m, H-17), 1.10 (1H, m, H-22b), 1.26 (1H, m, H-16a), 1.26 (2H, m, H-23a,b), 1.30 (2H, m, H-28a,b), 1.37 (1-H, m, H-8), 1.38 (1H, m, H-20), 1.41 (3H, m, H- 11a,b, H-22a), 1.55 (2H, m, H-7a, H-15b), 1.69 (1H, m, H-25), 1.73 (1H, m, H-1b), 1.74 (1H, m, H-2a), 1.85 (1H, m, H-16b), 1.96 (2H, m, H-7b, H-12b), 2.10 (1H, m, H-2b), 2.46 (1H, t, J = 10.2 Hz, H-4b), 2.70 (1H, m, H-4a), 3.94 (1H, m, H-3, H-5′), 4.03 (1H, t, J = 8.0 Hz, H-2′), 4.27 (2H, t, J = 8.0 Hz, H-3′, H-4′), 4.38 (1H, dd, J = 5.2 Hz, 11.8 Hz, , H-6′b), 4.53 (1H, dd, J = 2.2 Hz, 11.8 Hz, H-6′a), 5.02 (1H, d, J = 7.7, H-1′), 5.33 (1H, br.d, J = 2.2 Hz, H-6).
13 C NMR (75 MHz, C 5D5N) δC: 12.0 (CH 3, C-18), 12.2 (CH 3, C-29), 19.0 (CH 3, C-21), 19.2
(CH 3, C-26), 19.4 (CH 3, C-19), 20.0 (CH 3, C-27), 21.3 (CH 2, C-11), 23.4 (CH 2, C-28), 24.5
(CH 2, C-15), 26.4 (CH 2, C-23), 28.5 (CH 2, C-16), 29.5 (CH, C-25), 30.2 (CH 2, C-2), 32.0
(CH, C-8), 32.2 (CH 2, C-7), 34.2 (CH 2, C-22), 36.4 (C, C-10), 36.4 (CH, C-20), 37.5 (CH 2, C-
1), 39.3 (CH 2, C-4), 39.9 (CH 2, C-12), 42.5 (C, C-13), 46.0 (CH, C-24), 50.3 (CH, C-9), 56.2
(CH, C-17), 56.8 (CH, C-14), 62.8 (CH 2, C-6′), 71.7 (CH, C-4′), 75.3 (CH, C-2′), 78.1 (CH, C-5′), 78.4 (CH, C-3), 78.6 (CH, C-3′), 102.5 (CH, C-1′), 121.9 (CH, C-6), 140.9 (C, C-5).
198
4.3.7 Characterization of 1-O-β-D-Glucopyranosyl-(2 S,3 S,4 R,8 Z)-2-[(2 ′R)-2′-hydroxy- tetracosanoyl amino]-1,3,4-octadecanetriol-8-ene (80 ):
24' OH
O 2' OH 1' 6'' NH OH 4'' 5'' O 6 8 HO O 3 2'' 1 2 4 9 HO 3'' 1'' 5 7 OH 18 OH
White gelatinous gummy solid isolated from leaves (scheme-4.8).
+ + + EIMS m/z (rel. intensity, %): 663 (84) [C 42 H81 NO 4] or [M-C6H12 O6] ; 632 (17) [C 41 H78 NO 3] + + or [M-C6H12 O6-CH 3OH] ; 463 (14), 439 (21), 422 (16), 408 (53), 368 (19) [C 24 H48 O2] ; 355 (43), 248 (61), 54 (100).
ESIMS m/z: 844.7004 [M+H] +.
HRESIMS : 844.6877 (calcd. for C 48 H94 NO 10 , 844.6946, ∆ = - 0.0069)
Error (ppm) = 1.0 x 10 6 (-0.0069)/844.6877 = +8.0
1 H NMR :(300 MHz, C 5D5N) δH: 0.85 (6H, br.t, J = 6.9 Hz, H-18, 24 ′), 1.29 (36H, br. s, H-4′- H-21 ′), 1.72 (6H, m, H-6, H-22 ′, H-23 ′), 1.98 (1H, m, H-5b), 2.05 (3H, m, H-3′, H-5a), 2.18 (4H, m, H-7,10), 3.86 (1H, m, H-5″), 4.01 (1H, t, J = 8.0 Hz, H-2″), 4.19 (3H, m, H-4, H-3′′ , H-4′′ ), 4.30 (1H, m, H-3), 4.35 (1H, dd, J = 5.1 Hz, 12.0 Hz, H-6″a), 4.50 (1H, m, H-6″b), 4.52 (1H, m, H-1a), 4.57 (1H, m, H-2′), 4.70 (1H, dd, J = 6.6 Hz, 10.8 Hz, H-1b), 4.94 (1H, d, J = 8.0 Hz, H-1′′ ) 5.28 (1H, m, H-2), 5.50 (2H, m, H-8, H-9), 8.56 (1H, d, J = 9.0 Hz, NH).
13 C NMR (75 MHz, C 5D5N) δC: 14.3 (CH 3, C-18, 24 ′), 22.9 (CH 2, C-23 ′), 26.8 (CH 2, C-6),
27.6 (CH 2, C-10), 27.9 (CH 2, C-7), 29.5-30.0 (CH 2, C-4′-C-21 ′), 32.1 (CH 2, C-22 ′), 34.0
(CH 2, C-5), 35.5 (CH 2, C-3′), 51.7 (CH, C-2), 62.6 (CH 2, C-6″), 70.4 (CH 2, C-1), 71.5 (CH, C-4″), 72.4 (CH, C-4, C-2′), 75.1 (CH, C-2″), 75.9 (CH, C-3), 78.4 (CH, C-5″), 78.5 (CH, C- 3″), 105.5 (CH, C-1″), 130.2 (CH, C-9), 130.4 (CH, C-8), 175.7 (C, C-1′).
199
4.3.8 Characterization of (2 R) 2-Hydroxy-3-(4 ′-hydroxyphenyl)-propanoic acid [(2 R) ( p-hydroxyphenyl) lactic acid] (81 ):
HO 3 1' 4' OH 2 1 O OH
Crystalline solid isolated from leaves (Scheme-4.9 and 4.10).
+ + + EIMS m/z (rel. intensity, %): 182 (2) [M, C 9H10 O4] ; 147 (1) [C 9H7O2] , 137 (2) [C 8H9O2] , + + + 119 (2) [C 8H7O] , 107 (100) [C 7H7O] , 91 (15), 79 (11), 78 (16), 77 (48) [C 6H5] , 65 (14), 53 (14).
+ HREIMS m/z : 182.0578 [M] (calcd. for C 9H10 O4, 182.0579, ∆ = -0.0001).
Error (ppm) = 1.0 x 10 6 (-0.0001)/182.0578 = -0.5.
CIMS (+) m/z : 183 [M+H] +.
-1 IR νmax (KBr) cm : 3536 to 3391, 3235, 1738, 1613, 1448.
1 H NMR (300 MHz, CD 3CD 2OD) δH: 2.81 (1H, dd, J = 7.5 Hz, 14.0 Hz, H-3a), 3.00 (1H, dd, J = 4.3 Hz, 14.0 Hz, H-3b), 4.31 (1H, dd, J = 4.3 Hz, 7.5 Hz, H-2), 6.72 (2H, d, J = 8.4 Hz, H-3′/H-5′), 7.09 (2H, d, J = 8.4 Hz, H-2′/H-6′).
13 C NMR (75 MHz, CD 3CD 2OD) δC: 40.4 (CH 2, C-3), 72.3 (CH, C-2), 115.7 (CH, C-3′/C-5′), 129.2 (C, C-1′), 131.4 (CH, C-2′/C-6′), 156.9 (C, C-4′), 175.4 (C, C-1).
200
4.3.9 Characterization of Syringaresinol mono-β-D-glucoside (82 ): Ha OH Hb 6''' O 5''' 4''' O Hb Ha HO 1''' O 5' 5 HO 3''' 2''' 4' H 8 O OH 1' H 3' 1 6 5 O O 2 1'' 5'' H 4 4'' O H 3'' 2 OH Ha Hb O
Amorphous solid isolated from leaves (scheme-4.10).
ESIMS m/z : 579.1988 [M+H] +.
HRESIMS : 579.2077 (calcd. for C 28 H35 NO 13 , 579.2098, ∆ = -0.0021)
Error (ppm) = 1.0 x 10 6 (-0.0021)/579.2077 = +3.5.
1 H NMR (500 MHz, CD 3OD) δH: 3.13 (2H, m, H-1, H-5), 3.19 (1H, m, H-5″′ ), 3.40 (2H, m, H-3″′ , H-4″′ ), 3.66 (1H, dd, J = 5.0 Hz, 11.5 Hz, H-6″′ a), 3.77 (1H, dd, J = 2.0 Hz, 12.0 Hz,
H-6″′ b), 3.84 (6H, br.s, 3 ″, 5 ″-OCH 3), 3.85 (6H, br.s, 3 ′, 5 ′- OCH 3), 3.91 (2H, dd, J = 2.5 Hz, 9.0 Hz, H-4a, H-8a), 4.27 (2H, m, H-4b, H-8b), 4.71 (1H, d, J = 4.5 Hz, H-6), 4.76 (1H, d, J = 4.0 Hz, H-2), 4.83 (1H, br.s, H-1″′ ), 6.64 (2H, s, H-2″/H-6″), 6.71 (2H, s, H-2′/H-6′).
13 C NMR (125 MHz, CD 3OD) δC: 55.5 (CH, C-5), 55.7 (CH, C-1), 56.9 (CH 3, C-3″, C-5″-
OCH 3), 57.2 (CH 3, C-3′, C-5′-OCH 3), 62.6 (CH 2, C-6′″ ), 71.4 (CH, C-4′″ ), 72.9 (CH 2, C-4),
73.0 (CH 2, C-8), 75.7 (CH, C-2′″ ), 77.8 (CH, C-3′″ ), 78.3 (CH, C-5′″ ), 87.2 (CH, C-2), 87.6 (CH, C-6), 104.6 (CH, C-2″/C-6″), 104.9 (CH, C-2′/C-6′), 105.5 (CH, C-1″′ ), 132.0 (C, C-1″), 133.0 (C, C-4″) 133.1 (C, C-4′), 149.4 (C, C-3″/C-5″), 154.4 (C, C-3′/C-5′).
201
4.3.10 Characterization of 6-Hydroxy-3-oxo-α-ionol 9-O-β-D-glucopyranoside (Roseoside) (83 ):
OH 6' 10 5' 4' O HO H 12 11 2' 1' HO 3' 9 8 7 OH O 1 6 2 HO 5 3 4 13 O
Viscous syrup isolated from leaves (scheme-4.10).
+ + EIMS m/z (rel. int., %): 224 (8), 207 (14) [C 13 H19 O2] or [M+H-C6H12 O6] ; 206 (33) + + + [C 13 H18 O2] or [M- C6H12 O6] ; 166 (16), 149 (100) [C 10H13 O] , 135 (25), 124 (79), 123 (43), 106 (25).
+ FABMS (+) m/z : 387 [M+H] ,
ESIMS m/z : 387.2063 [M+H] +.
HRESIMS : 387.2018 (calcd. for C 19 H31 O8, 387.2049, ∆ = -0.0031).
Error (ppm) = 1.0 x 10 6 (-0.0031)/387.2018 = +7.7
1 H NMR (600 MHz, CD 3OD) δH: 1.02 (3H, s, H-12), 1.03 (3H, s, H-11), 1.29 (3H, d, J = 6.0 Hz, H-10), 1.94 (3H, s, H-13), 2.14 (1H, d, J = 16.8 Hz, H-2a), 2.51 (1H, d, J = 16.8 Hz, H- 2b), 3.17 (1H, dd, J = 7.5 Hz, 9.0 Hz, H-2′), 3.22 (1H, m, H-5′), 3.25 (1H, m, H-4′), 3.34 (1H, m, H-3′), 3.63 (1H, dd, J = 6.0 Hz, 12.0 Hz, H-6′a), 3.85 (1H, dd, J = 1.8 Hz, 12.0 Hz, H- 6′b), 4.34 (1H, d, J = 7.8 Hz, H-1′), 4.56 (1H, br.s, H-9), 5.84 (1H, s, H-4), 5.85 (1H, d, J = 12.6 Hz, H-7), 5.86 (1H, dd, J = 8.4 Hz, 12.6 Hz, H-8).
13 C NMR (150 MHz, CD 3OD) δC: 19.6 (CH 3, C-13), 21.2 (CH 3, C-10), 23.5 (CH 3, C-11),
24.7 (CH 3, C-12), 42.5 (C, C-1), 50.7 (CH 2, C-2), 62.8 (CH 2, C-6′), 71.7 (CH, C-4′), 75.3 (CH, C-2′), 77.3 (CH, C-9), 78.0 (CH, C-5′), 78.1 (CH, C-3′), 80.0 (C, C-6), 102.8 (CH, C-1′), 127.2 (CH, C-4), 131.6 (CH, C-7), 135.3 (CH, C-8), 167.4 (C, C-5), 201.4 (C, C-3).
202
4.3.11 Characterization of Staphylionoside D (84 ):
O 10 9 OH 11 12 8 6' H 5' 1 7 HO 4' O 2 6 HO 3' 2' 1' 3 5 4 OH O 13 OH
Amorphous solid isolated from leaves (scheme-4.10).
EIMS m/z (rel. int., %): 118 (3), 87 (12), 85 (83), 84 (4), 83 (100), 82 (5), 78 (17), 63 (27).
+ FABMS (+) m/z : 387 [M+H] .
ESIMS (+) m/z : 387.1952 [M+H] +.
HRESIMS : 387.2018 (calcd. for C 19 H31 O8, 387.1996, ∆ = 0.0022).
Error (ppm) = 1.0 x 10 6 (0.0022)/387.2018 = -5.9
1 H NMR (500 MHz, CD 3OD) δH: 1.15 (3H, s, H-12), 1.37 (3H, s, H-11), 1.38 (3H, s, H-13), 1.45 (2H, ddd, J = 4.0 Hz, 11.5 Hz, 13.0 Hz, H-2a, H-4a), 2.08 (1H, ddd, J = 2.0 Hz, 4.0 Hz, 13.0 Hz, H-4e), 2.18 (3H, s, H-10), 2.36 (1H, ddd, J = 2.0 Hz, 3.5 Hz, 13.0 Hz, H-2e), 3.14 (1H, dd, J = 8.0 Hz, 9.0 Hz, H-2′), 3.28 (1H, m, H-4′), 3.29 (1H, m, H-5′), 3.35 (1H, m, H-3′), 3.70 (1H, m, H-6′a), 3.88 (1H, m, H-6′b), 4.35 (1H, tt, J = 4.0 Hz, 11.5 Hz, H-3), 4.46 (1H, d, J = 8.0 Hz, H-1′), 5.83 (1H, s, H-8).
13 C NMR (125 MHz, CD 3OD) δC: 26.6 (CH 3, C-10), 29.4 (CH 3, C-11), 30.8 (CH 3,
C-13), 32.3 (CH 3, C-12), 37.0 (C, C-1), 46.6 (CH 2, C-2), 48.1 (CH 2, C-4), 62.7 (CH 2, C-6′), 71.6 (CH, C-4′), 72.4 (C, C-5), 72.6 (CH, C-3), 75.1 (CH, C-2′), 77.9 (CH, C-5′), 78.1 (CH, C-3′), 101.2 (CH, C-8), 102.7 (CH, C-1′), 120.1 (C, C-6), 200.9 (C, C-7), 211.5 (C, C-9).
203
4.3.12 Characterization of (2 E) 3-(3 ′,5 ′-Dimethoxy-4′-O-β-D-glucopyranosyl-phenyl)- prop-2-en-1-ol (Syringin) (85 ):
O HO 1 2 2' 3' 6'' 5'' O 4'' HO 3 1' 4' O 1'' OH 5' 2'' HO 3'' OH O
Colourless non crystalline solid isolated from leaves (scheme-4.10)
EIMS m/z (rel. int., %): 145 (12), 127 (11), 115 (21), 101 (22), 99 (20), 87 (19), 85 (29), 73 (100).
FABMS (+) m/z : 373 [M+H] +.
1 H NMR (500 MHz, CD 3OD) δH: 3.20 (1H, m, H-5′′ ), 3.41 (2H, m, H-3′′ , H-4′′ ), 3.47 (1H, m, H-2′′ ), 3.65 (1H, dd, J = 5.5 Hz, 12.0 Hz, H-6′′ a), 3.77 (1H, dd, J = 2.5 Hz, 12.0 Hz, H-6′′ b),
3.85 (6H, s, 3 ′-OCH 3 and 5 ′-OCH 3), 4.22 (2H, dd, J = 1.5 Hz, 5.5 Hz, H-1), 4.86 (1H, d, J = 7.8 Hz, H-1′′ ), 6.32 (1H, dt, J = 5.5 Hz, 16.0 Hz, H-2), 6.54 (1H, br.d, J = 16.0 Hz, H-3), 6.75 (2H, s, H-2′/H-6′).
13 C NMR (125 MHz, CD 3OD) δC: 57.1 (CH 3, 3 ′, 5 ′-OCH 3), 62.6 (CH 2, C-6′′ ), 63.6 (CH 2, C- 1), 71.4 (CH, C-4′′ ), 75.7 (CH, C-2′′ ), 77.9 (CH, C-5′′ ), 78.4 (CH, C-3′′ ), 105.3 (CH, C-1′′ ), 105.5 (CH, C-2′ and C-6′), 130.1 (CH, C-2), 131.3 (CH, C-2), 135.3 (C, C-4), 154.4 (C, C-3′ and C-5′).
4.4 Identification of Natural Compounds using GC-MS Analyses
4.4.1 Gas Chromatographic Data:
4.4.1.1 Gas Chromatographic Data of C. rothii Roots:
Continuing from next page
204
Table-4.1: Compounds identified through GC-MS in 2A (scheme-4.1a). S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time (RT) %
1. n-Tridecane ( 1) C13 H28 184 193 11.02 1.29
2. n-Tetradecane ( 2) C14 H30 198 255 13.60 1.51
3. n-Pentadecane ( 3) C15 H32 212 315 16.08 1.45
4. iso -Hexadecane ( 4) C16 H34 226 372 18.51 1.86
5. n-Dodecanoic acid ethyl C14 H28 O2 228 - - - ester ( 5) (co-eluted with 4)
6. n-Heptadecane ( 6) C17 H36 240 427 20.75 1.33
7. n-Tetradecanoic acid ethyl C16 H32 O2 256 479 22.92 1.80 ester ( 7)
8. n-Octadecane ( 8) C18 H38 254 480 22.97 2.40 9. UI-isomer of Cordiachrome - - 503 23.96 6.26 C
10. Cordiachrome C ( 9) C16 H18 O2 242 508 24.13 8.51
11. n-Nonadecane ( 10 ) C19 H40 268 529 25.00 2.26
12. n-Hexadecanoic acid methyl C17 H34 O2 270 545 25.67 3.34 ester ( 11 )
13. UI-1 - - 553 26.00 2.43
14. n-Hexadecanoic acid ethyl C18 H36 O2 284 577 27.00 7.35 ester ( 12 )
15. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 586 27.38 7.99 16. UI-2 - - 623 28.92 3.19
17. Octadec-9Z-enoic acid C19 H36 O2 296 625 29.07 3.47 methyl ester ( 14 )
18. Octadec-9Z,12 Z-dienoic acid C20 H36 O2 308 652 30.13 3.21 ethyl ester ( 15 )
19. Octadec-9E-enoic acid ethyl C20 H38 O2 310 655 30.25 5.24 ester ( 16 )
20. Octadec-9Z-enoic acid ethyl C20 H38 O2 310 666 30.72 9.88 ester ( 17 )
21. n-Octadecanoic acid ( 18 ) C18 H36 O2 284 671 30.92 3.63
22. UI-3 - - 682 31.52 5.43 23. UI-4 - - 728 33.40 4.50
24. Plasticizer - - 809 36.67 6.25
25. Stigmasta-3,5-diene (19 ) C29 H48 396 1009 45.00 5.39 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
205
Table-4.2: Compounds identified through GC-MS in 4A (scheme-4.1a).
S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time (RT) %
1. n-Hexadecane ( 20 ) C16 H34 226 372 18.50 1.1
2. n-Octadecane ( 8) C18 H36 254 479 22.43 1.9
3. n-Hexadecanoic acid C17 H34 O2 270 544 25.63 8.84 methyl ester ( 11 )
4. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 582 27.22 28.68
5. n-Octadecanoic acid ( 18 ) C18 H36 O2 284 667 30.75 15.35
6. UI-3 - - 679 31.38 16.47
7. UI-4 - - 725 33.30 10.52
8. Plasticizer - - 807 36.58 17.13 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
Table-4.3: Compounds identified through GC-MS in KC-PE (scheme-4.1a).
S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time (RT) %
1. n-Octadecane ( 8) C18 H38 254 507 22.47 1.17
2. Cordiachrome C ( 9) C16 H18 O2 242 517 22.85 14.75
3. UI-5 - - 521 23.00 6.22 4. UI-6 - - 560 24.50 0.50
5. UI-7 - - 567 24.77 5.67
6. n-Hexadecanoic acid C17 H34 O2 270 573 25.00 5.80 methyl ester ( 11 )
7. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 597 25.95 5.55
8. Cordiachromene A ( 21 ) C16 H20 O2 244 607 26.33 5.48
9. UI-8 - - 613 26.58 0.58
10. UI-9 - - 622 26.93 0.50
11. UI-10 - - 634 27.35 1.00
12. UI-11 - - 652 28.03 0.37
13. Octadec-9Z,12 Z-dienoic C19 H34 O2 294 656 28.20 1.59 acid methyl ester ( 22 )
14. Octadec-9Z-enoic acid C19 H36 O2 296 659 28.33 1.95 methyl ester (14 ) 15. UI-12 - - 670 28.73 1.07
206
Table-4.3:continued S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time(RT) %
16. n-Octadecanoic acid methyl C19 H38 O2 298 672 28.82 1.18 ester ( 23 )
17. UI-13 - - 677 29.02 5.48
18. Octadec-9Z,12 Z-dienoic acid C20 H36 O2 308 688 29.43 1.93 ethyl ester ( 15 )
19. Octadec-9E-enoic acid ethyl C20 H38 O2 310 691 29.53 4.62 ester ( 16 )
20. n-Octadecanoic acid ( 18 ) C18 H36 O2 284 692 29.58 2.83
21. UI-isomer of UI-10 - - 699 29.85 1.41
22. UI-14 - - 725 30.85 5.00
23. UI-15 - - 729 31.00 1.44
24. UI-16 - - 732 31.12 1.46
25. UI-17 - - 736 31.27 1.14
26. UI-18 - - 740 31.45 0.94
27. Cordiaquinol C ( 24 ) C16 H20 O2 244 759 32.15 2.24
28. UI-19 - - 773 32.70 1.42
29. UI-20 - - 782 33.03 1.33
30. UI-21 - - 791 33.38 2.03
31. UI-22 - - 804 33.88 1.33
32. UI-23 - - 814 34.27 1.00
33. Plasticizer - - 854 35.82 6.80
34. Stigmasta-4,22-diene-3β-ol C29 H48 O 412 1228 50.20 1.21 (25 )
35. Stigmast-5-en-3β-ol ( 26 ) C29 H50 O 414 1287 52.47 2.17
36. Stigmasterol ( 27 ) C29 H48 O 412 1433 58.27 0.46
37. UI-24 - - 1463 59.23 0.46 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
207
Table-4.4: Compounds identified through GC-MS in KC-C (scheme-4.1a).
S. no. Name of Compound Molecular Molecular Scan Retention *Rel. formula weight no. time (RT) %
1. (1 E)-4-(3-Hydroxy-1- C10 H12 O3 180 472 21.12 5.03 propenyl)-2-methoxyphenol (28 )
2. Cordiachrome C ( 9) C16 H18 O2 242 516 22.82 0.75
3. UI-5 - - 520 22.97 2.48 4. UI-7 - - 566 24.73 2.36
5. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 594 25.82 5.49
6. Cordiachromene A ( 21 ) C16 H20 O2 244 607 26.32 13.00
7. UI-10 - - 634 27.35 1.70
8. Octadec-9Z,12Z-dienoic C19 H34 O2 294 655 28.25 0.99 acid methyl ester ( 22 )
9. UI-12 - - 670 28.73 2.69
10. UI-13 - - 677 29.00 10.53
11. n-Octadecanoic acid ( 18 ) C18 H36 O2 284 690 29.50 3.35
12. UI-isomer of UI-10 - - 699 29.85 2.59
13. UI-25 - - 717 30.53 3.02
14. UI-14 - - 725 30.85 7.49 15. UI-18 - - 740 31.43 3.71 16. UI-19 - - 773 32.70 2.98
17. UI-21 - - 790 33.35 3.22
18. Cordiol A ( 29 ) C16 H22 O3 262 797 33.62 3.41
19. UI-22 - - 804 33.88 4.60
20. UI-26 - - 842 35.35 3.02
21. Plasticizer - - 853 35.77 4.53 22. UI-27 - - 955 39.70 3.45
23. UI-28 - - 1025 42.38 2.20
24. Stigmasta-3,5-diene ( 19 ) C29 H48 396 1096 45.12 1.25
25. Stigmasta-4,22-diene-3β-ol C29 H48 O 412 1227 50.15 2.24 (25 )
26. Stigmast-5-en-3β-ol ( 26 ) C29 H50 O 414 1284 52.35 3.91 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
208
Table-4.5: Compounds identified through GC-MS in 6A (scheme-4.1b). S. no. Name of Compound Molecular Molecular Scan Retention *Rel. formula weight no. time % (RT)
1. n-Hexadecane ( 20 ) C16 H34 226 372 18.25 0.82
2. n-Heptadecane ( 6) C17 H36 240 427 20.50 0.20
3. n-Tetradecanoic acid C16 H32 O2 256 479 22.75 0.35 ethyl ester ( 7) 4. UI-isomer of - - 500 23.66 0.68 Cordiachrome C
5. n-Hexadecanoic acid C17 H34 O2 270 545 25.25 3.65 methyl ester ( 11 )
6. UI-29 - - 576 26.95 17.96
7. UI-3 - - 680 31.43 28.18
8. UI-4 - - 727 33.39 19.49
9. Plasticizer - - 807 36.58 28.66
UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
209
Table-4.6: Compounds identified through GC-MS in KEA-PE (scheme-4.1b). S. no. Name of Compound Molecular Molecular Scan Retention *Rel. formula weight no. time (RT) %
1. Cordiachrome C ( 9) C16 H18 O2 242 517 22.85 32.61
2. UI-5 - - 521 23.00 6.56 3. UI-7 - - 566 24.73 10.59
4. n-Hexadecanoic acid C17 H34 O2 270 573 25.00 1.44 methyl ester ( 11 )
5. UI-30 - - 575 25.10 1.87
6. UI-31 - - 579 25.23 0.97 7. Plasticizer a - - 590 25.65 1.61
8. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 594 25.82 3.75
9. UI-32 - - 603 26.15 2.63
10. Cordiachromene A ( 21 ) C16 H20 O2 244 607 26.33 3.13 11. UI-10 - - 634 27.35 0.74
12. Octadec-9Z-enoic acid C19 H36 O2 296 658 28.30 1.21 methyl ester ( 14 )
13. UI-12 - - 669 28.70 1.23 14. UI-13 - - 676 28.97 1.53
15. Octadec-9Z,12 Z-dienoic C18 H32 O2 280 679 29.08 3.13 acid ( 31 )
16. n-Octadecanoic acid ( 18 ) C18 H36 O2 284 690 29.50 2.05 17. UI-33 - - 767 32.47 1.40
18. UI-19 - - 772 32.65 1.51
19. UI-20 - - 781 33.00 1.45
20. UI-21 - - 790 33.35 1.58
21. Cordiol A ( 29 ) C16 H22 O3 262 796 33.58 1.20
22. UI-22 - - 803 33.85 1.52
23. UI-23 - - 813 34.23 1.08
24. Plasticizer - - 853 35.77 7.81
25. Stigmasta-4,22-diene-3β- C29 H48 O 412 1227 50.15 1.75 ol ( 25 )
26. Stigmast-5-en-3β-ol (26 ) C29 H50 O 414 1285 52.38 4.13
27. Cycloartenol ( 32 ) C30 H50 O 426 1360 55.27 0.66
28. 24-Methylene C31 H52 O 440 1436 58.20 0.85 cycloartanol ( 33 ) UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
210
Table-4.7: Compounds identified through GC-MS in KEA-C (scheme-4.1b).
S. no. Name of Compound Molecular Molecular Scan Retention *Rel. formula weight no. time (RT) %
1. Phenyl ethene ( 34 ) C8H8 104 65 2.77 18.81
2. UI-34 - - 105 4.47 10.13
3. UI-35 - - 124 5.28 3.79 4. UI-36 - - 150 6.39 15.15
5. UI-37 - - 168 7.16 6.71
6. Acetic acid C9H10 O2 150 193 8.22 4.39 phenylmethyl ester (35 )
7. UI-38 - - 306 13.03 1.38
8. UI-39 - - 558 23.75 8.10
9. UI-isomer of UI-39 - - 561 23.88 3.84
10. UI-isomer of C16 H20 O2 244 641 27.28 25.41 Cordiachromene A
11. UI-40 - - 821 34.94 2.30 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
Table-4.8: Compounds identified through GC-MS in KA-PE (scheme-4.2).
S. no. Name of Compound Molecular Molecular Scan Retention *Rel. formula weight no. time % (RT)
1. n-Hexadecanoic acid C17 H34 O2 270 828 20.93 7.93 methyl ester ( 11 )
2. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 852 21.47 15.03
3. Cordiachromene A ( 21 ) C16 H20 O2 244 875 21.97 11.31
4. Octadec-9Z-enoic acid C19 H36 O2 296 925 23.05 9.08 methyl ester ( 14 )
5. Octadecanoic acid ( 18 ) C18 H36 O2 284 960 23.64 2.8
6. UI-41 C19 H40 268 1073 26.25 12.96
7. Plasticizer - - 1148 27.88 24.19
8. Stigmasta-3,5-diene ( 19 ) C29 H48 396 1672 39.30 11.80
9. Stigmast-5-en-3β-ol ( 26 ) C29 H50 O 414 2125 49.21 4.90 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
211
Table-4.9: Compounds identified through GC-MS in KA-C (scheme-4.2).
S. no. Name of Compound Molecular Molecular Scan Retention *Rel. formula weight no. time % (RT)
1. Cordiachrome C ( 9) C16 H18 O2 242 515 22.77 13.00
2. UI-5 - - 520 22.97 2.17
3. UI-7 - - 565 24.70 2.14
4. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 591 25.70 4.17
5. Cordiachromene A ( 21 ) C16 H20 O2 244 606 26.27 8.67
6. UI-13 - - 676 28.97 14.14
7. UI-isomer of UI-3 - - 679 29.08 7.62
8. UI-14 - - 723 30.77 7.85
9. Cordiol A ( 29 ) C16 H22 O3 262 795 33.53 2.84
10. UI-22 - - 803 33.85 2.38
11. Plasticizer - - 852 35.81 4.2
12. UI-42 - - 912 38.03 12.46
13. UI-43 - - 960 39.88 2.74
14. Stigmast-5-en-3β-ol ( 26 ) C29 H50 O 414 1278 23.87 3.32
UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
212
Table-4.10: Compounds identified through GC-MS in KM-PE (scheme-4.3).
S. no. Name of Compound Molecular Molecular Scan Retention *Rel. formula weight no. time (RT) %
1. Thymine ( 36 ) C5H6N2O2 126 63 5.38 0.48
2. 2,3-Dihydro-3,5- C6H8O4 144. 100 6.82 0.60 dihydroxy-6-methyl-4H- pyran-4-one ( 37 )
3. 5-(Hydroxymethyl)-2- C6H6O3 126 155 8.93 0.53 furancarboxaldehyde ( 38 ) 4. UI-44 - - 301 14.53 0.71
5. n-Dodecanoic acid ( 39 ) C12 H24 O2 200 373 17.32 0.72
6. 3,4-Dihydroxy- C9H10 O4 182 464 20.83 0.62 benzenepropanoic acid (40 )
7. n-Tetradecanoic acid ( 41 ) C14 H28 O2 228 487 21.70 0.99
8. n-Hexadecanoic acid C17 H34 O2 270 572 24.98 2.17 methyl ester ( 11 )
9. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 595 25.85 6.67
10. Cordiachromene A ( 21 ) C16 H20 O2 244 607 26.32 7.70
11. UI-13 - - 677 29.00 6.04
12. n-Octadec-9Z-enoic acid C18 H34 O2 282 681 29.15 8.98 (42 )
13. n-Octadecanoic acid ( 18 ) C18 H36 O2 284 690 29.50 4.38 14. UI-14 - - 724 30.80 2.36
15. 2-Monopalmitin ( 43 ) C19 H38 O4 330 838 35.20 4.73
16. Plasticizer - - 854 35.80 30.14 17. UI-isomer of C 18:2 FFA- - - 908 37.94 3.68 I
18. Stigmasta-4,22-diene-3β- C29 H48 O 412 1227 50.15 3.58 ol ( 25 )
19. Stigmast-5-en-3β-ol ( 26 ) C29 H50 O 414 1288 52.50 9.59
20. Cycloeucalenol ( 44 ) C30 H50 O 426 1318 53.65 1.34
21. Cycloartenol ( 32 ) C30 H50 O 426 1361 55.30 2.00
22. 24-Methylene cycloartanol C31 H52 O 440 1435 58.25 2.00 (33 ) UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
213
Table-4.11: Compounds identified through GC-MS in KM-C (scheme-4.3).
S. no. Name of Compound Molecular Molecular Scan Retention *Rel. formula weight no. time (RT) %
1. n-Hexadecanoic acid C17 H34 O2 270 572 25.00 1.02 methyl ester ( 11 )
2. UI-45 - - 575 25.08 0.68
3. n-Hexadecanoic acid C16 H32 O2 256 593 25.77 3.05 (13 )
4. Cordiachromene A ( 21 ) C16 H20 O2 244 607 26.32 4.20
5. UI-13 - - 676 28.97 6.88
6. Octadec-9Z-enoic acid C18 H34 O2 282 678 29.03 6.54 (42 )
7. UI-isomer of UI-3 - - 680 29.12 5.60
8. n-Octadecanoic acid C18 H36 O2 284 690 29.50 2.75 (18 )
9. UI-14 - - 724 30.82 3.21
10. UI-46 - - 741 31.47 1.99
11. UI-33 - - 767 32.47 3.15
12. UI-21 - - 790 33.35 2.43
13. 2-Monopalmitin ( 43 ) C19 H38 O4 330 837 35.15 3.72
14. Plasticizer - - 853 35.77 18.66
15. UI-42 - - 915 38.42 19.99
16. Stigmasta-4,22-diene-3β- C29 H48 O 412 1224 50.08 4.00 ol ( 25 )
17. Stigmast-5-en-3β-ol ( 26 ) C29 H50 O 414 1283 52.30 9.01
18. 24-Methylene C31 H52 O 440 1430 57.92 3.1 cycloartanol ( 33 ) UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
214
Table-4.12: Compounds identified through GC-MS in KM-EA (scheme-4.3). S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time (RT) %
1. UI-44 - - 300 14.50 8.21
2. 4-Hydroxy-benzoic acid C7H6O3 138 349 16.38 0.79 (45 )
3. 4-Hydroxy-3-methoxy- C8H8O4 168 372 17.33 0.94 benzoic acid ( 46 )
4. 3,4-Diydroxy- C9H10 O4 182 464 20.82 1.74 benzenepropanoic acid (40 )
5. (1 E) 4-(3-Hydroxy-1- C10 H12 O3 180 471 21.08 4.29 propenyl)-2- methoxyphenol (28 )
6. n-Tetradecanoic acid C14 H28 O2 228 486 21.65 0.87 (41 )
7. n-Hexadecanoic acid C17 H34 O2 270 573 25.00 0.92 methyl ester ( 11 )
8. n-Hexadecanoic acid C16 H32 O2 256 593 25.77 4.19 (13 )
9. Cordiachromene A ( 21 ) C16 H20 O2 244 606 26.27 6.78
10. UI-12 - - 669 28.70 2.01
11. UI-13 - - 677 29.00 9.32
12. UI-isomer of UI-3 - - 680 29.12 5.11
13. UI-14 - - 724 30.82 5.32 14. UI-18 - - 740 31.43 3.68
15. Cordiaquinol C ( 24 ) C16 H20 O2 244 758 32.12 4.37
16. UI-21 - - 790 33.35 4.06 17. Plasticizer - - 853 35.77 16.55
18. UI-47 - - 881 36.85 2.22
19. UI-48 - - 893 37.32 3.12
20. UI-49 - - 901 37.62 2.61 21. UI-28 - - 1024 42.35 2.37
22. UI-50 - - 1195 48.92 1.51
23. Stigmasta-4,22-diene- C29 H48 O 412 1226 50.12 2.84 3β-ol ( 25 )
24. Stigmast-5-en-3β-ol ( 26 ) C29 H50 O 414 1284 52.35 6.19 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
215
Table-4.13: Identification of Chemical Constituents from Root - Summary
Sample Total constituents Total constituents % age composition of identified code resulting in MS identified constituents (TIC response)
2A 25 19 71.90 4A 8 5 55.87 KC-PE 37 15 52.93 KC-C 26 10 39.42 6A 9 4 5.02 KEA-PE 28 12 55.91 KEA-C 11 2 23.20 KA-PE 9 7 62.85 KA-C 14 5 32.20 KM-PE 22 17 57.08 KM-C 18 9 37.39 KM-EA 24 11 33.92
Overall 95 45 ~44.00 results
4.4.1.2 Gas Chromatographic Data of C. rothii Stem:
Continuing from next page
216
Table-4.14: Compounds identified through GC-MS in SH (Scheme-4.6a). S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time(RT) %
1. Nonanedioic acid C10 H18 O4 202 628 16.60 1.21 monomethyl ester ( 47 )
2. n-Tetradecanoic acid ( 41 ) C14 H28 O2 228 735 18.92 1.03
3. n-Octadecane ( 8) C18 H38 254 753 19.32 0.62
4. 6,10,14-Trimethyl- C18 H36 O 268 782 19.95 0.63 pentadecan-2-one ( 48 )
5. n-Nonadecane ( 10 ) C19 H40 268 813 20.62 1.58
6. n-Hexadecanoic acid C17 H34 O2 270 830 20.98 19.78 methyl ester ( 11 )
7. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 864 21.72 15.62
8. n-Eicosane ( 49 ) C20 H42 282 870 21.85 3.56
9. n-Heptadecanoic acid C18 H36 O2 284 885 22.18 1.85 methyl ester ( 50 )
10. n-Heptadecanoic acid ( 51 ) C17 H34 O2 270 909 22.70 1.67
11. n-Heneicosane ( 52 ) C21 H44 296 924 23.02 2.29
12. n-Octadecanoic acid C19 H38 O2 298 939 23.35 7.91 methyl ester ( 23 )
13. n-Octadecanoic acid ( 18 ) C18 H36 O2 284 963 23.87 4.24 14. UI-51 - - 976 24.15 2.07 15. UI-52 - - 1030 25.32 3.75
16. n-Eicosanoic acid methyl C21 H42 O2 326 1040 25.53 3.65 ester ( 53 ) 17. UI-53 - - 1055 25.87 5.45
18. n-Eicosanoic acid ( 54 ) C20 H40 O2 312 1062 26.02 1.80
19. n-Heneicosanoic acid C22 H44 O2 340 1087 26.55 1.85 methyl ester ( 55 )
20. n-Docosanoic acid methyl C23 H46 O2 354 1136 27.62 3.28 ester ( 56 ) 21. Plasticizer - - 1150 27.92 6.54
22. n-Tricosanoic acid methyl C24 H48 O2 368 1194 28.88 1.40 ester ( 57 )
23. n-Tetracosanoic acid C25 H50 O2 382 1262 30.35 1.92 methyl ester ( 58 )
24. n-Hexacosanoic acid C27 H54 O2 410 1453 34.50 0.54 methyl ester ( 59 ) 25. UI-54 - - 1564 36.90 0.63 26. UI-55 - - 1646 38.68 1.48
217
Table-4.14: Continue S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time % (RT) 27. UI-56 - - 2149 49.68 1.16
28. Olean-12-en-3β-ol ( 60 ) C30 H50 O 426 2321 53.45 0.78
29. Stigmasta-3,5-dien-7-one C29 H46 O 410 2381 54.71 0.44 (61 ) 30. UI-57 - - 2454 56.27 0.32 31. UI-58 - - 2513 57.62 0.91 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
Table-4.15: Compounds identified through GC-MS in SC (scheme-4.6a). S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time (RT) %
1. 4-Hydroxy-3-methoxy- C8H8O4 168 625 16.53 4.66 benzoic acid ( 46 ) 2. UI-59 - - 675 17.62 2.81
3. Methyl 2-hydroxy-3-(4 ’- C10 H12 O4 196 696 18.08 2.00 hydroxyphenyl)propanoate (62 )
4. (1 E)-4-(3-Hydroxy-1- C10 H12 O3 180 726 18.73 2.43 propenyl)-2-methoxyphenol (28 )
5. n-Hexadecanoic acid C17 H34 O2 270 828 20.93 17.82 methyl ester ( 11 )
6. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 857 21.57 30.30
7. n-Octadecanoic acid methyl C19 H38 O2 298 938 23.33 8.66 ester ( 23 )
8. n-Octadecanoic acid ( 18 ) C18 H36 O2 284 960 23.80 6.53
9. n-Eicosanoic acid methyl C21 H42 O2 326 1039 25.52 4.27 ester ( 53 )
10. n-Docosanoic acid methyl C23 H46 O2 354 1135 27.6 3.90 ester ( 56 ) 11. Plasticizer - - 1148 27.88 9.63
12. n-Tetracosanoic acid methyl C25 H50 O2 382 1260 30.50 0.80 ester ( 58 ) 13. UI-55 - - 1640 38.61 1.91 14. UI-56 - - 2142 49.54 2.42
15. Olean-12-en-3β-ol ( 60 ) C30 H50 O 426 2316 53.27 1.28
16. Cyclolaudenol ( 63 ) C31 H52 O 440 2501 57.31 1.35 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
218
Table-4.16: Compounds identified through GC-MS in SEA (scheme-4.6a).
S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time % (RT)
1. 4-Hydroxy-benzoic acid C7H6O3 138 607 16.15 1.16 (45 )
2. 4-Hydroxy-3-methoxy- C8H8O4 168 627 16.58 1.55 benzoic acid ( 46 )
3. n-Hexadecanoic acid C17 H34 O2 270 829 20.97 11.22 methyl ester ( 11 )
4. Plasticizer a - - 853 21.48 7.95
5. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 859 21.62 19.11
6. n-Octadecanoic acid C19 H38 O2 298 939 23.35 5.35 methyl ester ( 23 )
7. n-Octadecanoic acid ( 18 ) C18 H36 O2 284 962 23.85 4.69
8. n-Eicosanoic acid methyl C21 H42 O2 326 1040 25.53 2.89 ester ( 53 )
9. UI-53 - - 1054 25.83 4.26
10. Plasticizer - - 1151 27.95 31.68
11. n-Tricosanoic acid methyl C24 H48 O2 368 1193 28.85 1.45 ester ( 57 )
12. n-Tetracosanoic acid C25 H50 O2 382 1262 30.35 1.47 methyl ester ( 58 )
13. UI-60 - - 1326 31.73 2.73
14. UI-55 - - 1647 38.70 1.58
15. UI-56 - - 2146 49.66 1.23
16. Olean-12-en-3β-ol ( 60 ) C30 H50 O 426 2321 53.39 0.78
17. Cyclolaudenol ( 63 ) C31 H52 O 440 2510 57.51 0.87 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
219
Table-4.17: Compounds identified through GC-MS in SME 10% (scheme-4.6a).
S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time % (RT)
1. 4-Hydroxy-3-methoxy- C8H8O4 168 624 16.52 2.30 benzoic acid ( 46 )
2. n-Hexadecanoic acid C17 H34 O2 270 827 20.92 20.95 methyl ester ( 11 )
3. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 855 21.53 35.55
4. n-Eicosane ( 49 ) C20 H42 282 868 21.80 3.52
5. Octadec-9Z-enoic acid C19 H36 O2 296 921 23.04 0.80 methyl ester ( 14 )
6. n-Octadecanoic acid C19 H38 O2 298 937 23.30 7.92 methyl ester ( 23 )
7. Octadec-9Z-enoic acid ( 42 ) C18 H34 O2 282 948 23.67 1.20
8. n-Octadecanoic acid ( 18 ) C18 H36 O2 284 959 23.78 6.64
9. UI-52 - - 1024 25.05 2.00
10. n-Eicosanoic acid methyl C21 H42 O2 326 1038 25.50 4.51 ester ( 53 )
11. n-Docosanoic acid methyl C23 H46 O2 354 1135 27.60 3.50 ester ( 56 )
12. Plasticizer - - 1147 27.87 6.94
13. UI-55 - - 1638 38.57 2.07
14. UI-56 - - 2124 49.27 2.07 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
220
Table-4.18: Compounds identified through GC-MS in SME 20% (scheme-4.6a). S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time % (RT) 1. UI-44 - - 541 14.76 0.56
2. 4-Hydroxy-benzoic acid C7H6O3 138 604 16.13 0.52 (45 )
3. 4-Hydroxy-3-methoxy- C8H8O4 168 623 16.53 1.17 benzoic acid ( 46 ) 4. UI-59 - - 673 17.60 0.62
5. n-Tetradecanoic acid ( 41 ) C14 H28 O2 228 734 18.90 1.13
6. n-Hexadecanoic acid methyl C17 H34 O2 270 828 20.93 23.76 ester ( 11 )
7. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 860 21.63 20.94
8. Octadec-9Z,12 Z-dienoic C19 H34 O2 294 921 22.95 3.19 acid methyl ester ( 22 )
9. Octadec-9Z-enoic acid C19 H36 O2 296 925 23.05 5.76 methyl ester ( 14 )
10. n-Octadecanoic acid methyl C19 H38 O2 298 938 23.33 5.08 ester ( 23 )
11. Octadec-9Z,12 Z-dienoic C18 H32 O2 280 947 23.52 3.19 acid ( 31 )
12. Octadec-9Z-enoic acid ( 42 ) C18 H34 O2 282 950 23.58 5.80
13. n-Octadecanoic acid ( 18 ) C18 H36 O2 284 961 23.83 5.48
14. n-Eicosanoic acid methyl C21 H42 O2 326 1037 25.50 2.04 ester ( 53 )
15. n-Eicosanoic acid ( 54 ) C20 H40 O2 312 1059 25.95 1.95
16. n-Heneicosanoic acid C22 H44 O2 340 1086 26.53 1.78 methyl ester ( 55 ) 17. UI-61 - - 1095 26.73 1.77
18. n-Docosanoic acid methyl C23 H46 O2 354 1135 27.60 2.39 ester ( 56 ) 19. Plasticizer - - 1148 27.88 6.25
20. n-Tricosanoic acid methyl C24 H48 O2 368 1192 28.83 1.12 ester ( 57 )
21. n-Tetracosanoic acid methyl C25 H50 O2 382 1260 30.32 1.16 ester ( 58 ) 22. UI-54 - - 1559 36.82 0.47 23. UI-55 - - 1637 38.56 0.79
24. Stigmast-5-en-3β-ol ( 26 ) C29 H50 O 414 2131 49.35 1.39
25. Olean-12-en-3β-ol ( 60 ) C30 H50 O 426 2308 53.19 0.79
26. Cyclolaudenol ( 63 ) C31 H52 O 440 2499 57.27 0.91 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
221
Table-4.19: Compounds identified through GC-MS in SME 30% ABCD (scheme-4.6b).
S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time (RT) %
1. 4-Hydroxy-3-methoxy- C8H8O4 168 620 16.40 0.83 benzoic acid ( 46 )
2. n-Hexadecanoic acid methyl C17 H34 O2 270 827 20.92 27.89 ester ( 11 )
3. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 856 21.53 18.27
4. Octadec-9Z,12 Z-dienoic acid C19 H34 O2 294 920 22.93 4.70 methyl ester ( 22 )
5. Octadec-9Z-enoic acid C19 H36 O2 296 924 23.02 8.65 methyl ester ( 14 )
6. n-Octadecanoic acid methyl C19 H38 O2 298 937 23.30 4.10 ester ( 23 )
7. Octadec-9Z,12 Z-dienoic acid C18 H32 O2 280 946 23.50 3.71 (31 )
8. Octadec-9Z-enoic acid ( 42 ) C18 H34 O2 282 949 23.55 6.19
9. n-Octadecanoic acid ( 18 ) C18 H36 O2 284 959 23.77 4.54
10. n-Eicosanoic acid methyl C21 H42 O2 326 1037 25.47 2.19 ester ( 53 )
11. n-Docosanoic acid methyl C23 H46 O2 354 1133 27.56 1.93 ester ( 56 )
12. Plasticizer - - 1146 27.83 4.35
13. n-Tricosanoic acid methyl C24 H48 O2 368 1189 28.78 0.91 ester ( 57 )
14. n-Tetracosanoic acid methyl C25 H50 O2 382 1258 30.25 1.11 ester ( 58 )
15. UI-55 - - 1633 38.44 0.66
16. Stigmasta-3,5-diene ( 19 ) C29 H48 396 1664 39.12 0.59
17. Stigmast-5-en-3β-ol ( 26 ) C29 H50 O 414 2117 49.08 1.23
18. Olean-12-en-3β-ol ( 60 ) C30 H50 O 426 2297 52.92 0.72
19. Cyclolaudenol ( 63 ) C31 H52 O 440 2486 56.98 0.79 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
222
Table-4.20: Compounds identified through GC-MS in SME 30% EFGH (scheme-4.6b). S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time (RT) %
1. 4-Hydroxy-3-methoxy- C8H8O4 168 620 16.56 0.63 benzoic acid ( 46 )
2. n-Hexadecanoic acid methyl C17 H34 O2 270 824 20.85 21.92 ester ( 11 )
3. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 854 21.50 21.28
4. Octadec-9Z,12 Z-dienoic acid C19 H34 O2 294 917 22.90 3.80 methyl ester ( 22 )
5. Octadec-9Z-enoic acid C19 H36 O2 296 921 22.95 4.94 methyl ester ( 14 )
6. n-Octadecanoic acid methyl C19 H38 O2 298 934 23.23 7.23 ester ( 23 )
7. Octadec-9Z-enoic acid ( 42 ) C18 H34 O2 282 945 23.48 4.45
8. n-Octadecanoic acid ( 18 ) C18 H36 O2 284 957 23.73 4.97 9. UI-52 - - 1024 25.20 3.06
10. n-Eicosanoic acid methyl C21 H42 O2 326 1035 25.43 3.18 ester ( 53 )
11. n-Eicosanoic acid ( 54 ) C20 H40 O2 312 1055 25.87 1.83
12. n-Heneicosanoic acid methyl C22 H44 O2 340 1082 26.46 2.14 ester ( 55 ) 13. UI-61 - - 1090 26.65 1.73
14. n-Docosanoic acid methyl C23 H46 O2 354 1130 27.48 2.26 ester ( 56 ) 15. Plasticizer - - 1143 27.77 6.58
16. n-Tricosanoic acid methyl C2 4H48 O2 368 1186 28.71 1.05 ester ( 57 )
17. n-Tetracosanoic acid methyl C25 H50 O2 382 1253 30.18 1.14 ester ( 58 ) 18. UI-55 - - 1624 38.27 1.15
19. Stigmasta-3,5-diene ( 19 ) C29 H48 396 1656 38.94 1.74
20. Stigmast-5-en-3β-ol ( 26 ) C29 H50 O 414 2108 48.70 1.45 21. UI-56 - - 2119 48.93 1.63
22. Olean-12-en-3β-ol ( 60 ) C30 H50 O 426 2282 52.59 0.80
23. Cyclolaudenol ( 63 ) C31 H52 O 440 2466 56.57 0.84 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
223
Table-4.21: Compounds identified through GC-MS in SME 30% IJKL (scheme-4.6b).
S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time (RT) %
1. 4-Hydroxy-3-methoxy- C8H8O4 168 620 16.42 0.50 benzoic acid ( 46 )
2. n-Hexadecanoic acid methyl C17 H34 O2 270 828 20.93 29.49 ester ( 11 )
3. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 854 21.50 23.76
4. Octadec-9Z,12 Z-dienoic C19 H34 O2 294 921 22.95 4.26 acid methyl ester ( 22 )
5. Octadec-9Z-enoic acid C19 H36 O2 296 924 23.02 8.76 methyl ester ( 14 )
6. n-Octadecanoic acid methyl C19 H38 O2 298 937 23.30 6.74 ester ( 23 )
7. Octadec-9Z-enoic acid ( 42 ) C18 H34 O2 282 948 23.55 5.01
8. n-Octadecanoic acid ( 18 ) C18 H36 O2 284 959 23.78 4.26
9. n-Eicosanoic acid methyl C21H42 O2 326 1038 25.50 3.39 ester ( 53 )
10. n-Heneicosanoic acid C22 H44 O2 340 1086 26.53 2.46 methyl ester ( 55 )
11. n-Docosanoic acid methyl C23 H46 O2 354 1134 27.60 2.70 ester ( 56 )
12. Plasticizer - - 1148 27.88 3.45
13. n-Tricosanoic acid methyl C24 H48 O2 368 1190 28.81 1.40 ester ( 57 )
14. n-Tetracosanoic acid methyl C25 H50 O2 382 1258 30.30 1.61 ester ( 58 )
15. UI-55 - - 1639 38.56 1.38
16. Olean-12-en-3β-ol ( 60 ) C30 H50 O 426 2310 53.10 0.70 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
224
Table-4.22: Compounds identified through GC-MS in SME 30% MNO (scheme-4.6b).
S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time (RT) %
1. UI-62 - - 310 9.70 9.49
2. 4-Hydroxy-3-methoxy- C8H8O4 168 620 16.40 0.30 benzoic acid ( 46 )
3. n-Hexadecanoic acid methyl C17 H34 O2 270 825 20.87 16.28 ester ( 11 )
4. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 853 21.48 26.45
5. Octadec-9Z-enoic acid C19 H36 O2 296 922 22.97 6.58 methyl ester ( 14 )
6. n-Octadecanoic acid methyl C19 H38 O2 298 935 23.25 5.54 ester ( 23 )
7. Octadec-9Z-enoic acid ( 42 ) C18 H34 O2 282 945 23.47 4.85
8. n-Octadecanoic acid ( 18 ) C18 H36 O2 284 957 23.73 6.59
9. UI-52 - - 1026 25.23 4.19
10. n-Eicosanoic acid methyl C21 H42 O2 326 1036 25.45 3.77 ester ( 53 )
11. n-Docosanoic acid methyl C23 H46 O2 354 1131 27.53 2.75 ester ( 56 )
12. Plasticizer - - 1145 27.82 7.92
13. n-Tricosanoic acid methyl C24 H48 O2 368 1187 28.74 1.79 ester ( 57 )
14. n-Tetracosanoic acid methyl C25 H50 O2 382 1256 30.22 1.84 ester ( 58 )
15. UI-55 - - 1631 38.39 1.64 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
225
Table-4.23: Identification of the Chemical Constituents from Stem - Summary
Sample Total constituents Total constituents % age composition of identified code resulting in MS identified constituents (TIC response)
SH 31 22 77.65 SC 16 12 84.00 SEA 17 11 50.54 SME 10% 14 10 86.89 SME 20% 26 20 89.55 SME 30%- 19 17 88.35 ABCD SME 30%- 23 18 85.65 EFGH SME 30%- 16 14 95.04 IJKL SME 30%- 15 11 76.74 MNO
Overall 29 17 ~81.60 results
4.4.1.3 Gas Chromatographic Data of C. rothii Leaves:
Continuing from next page
226
Table-4.24: Compounds identified through GC-MS in HEXCRU (scheme-4.7).
S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time (RT) %
1. (E,E) Hepta-2,4-dienal C7H10 O 110 152 6.88 0.48 (64 )
2. 2,7-Dimethyl-1,6- C10 H18 138 223 7.65 0.52 octadiene ( 65 )
3. n-Octanoic acid methyl C9H18 O2 158 318 9.25 0.40 ester ( 66 )
4. 9-Oxo-nonanoic acid C10 H18 O3 186 705 18.62 1.60 methyl ester (67 )
5. n-Hexadecanoic acid C17 H34 O2 270 1214 29.20 7.17 methyl ester ( 11 )
6. iso -Hexadecanoic acid C16 H32 O2 256 1252 30.02 15.28 (68 )
7. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 1267 30.35 4.98 8. UI-63 - - 1349 32.12 2.44
9. Octadec-9Z,12 Z,15 Z- C19 H32 O2 292 1366. 32.57 0.28 trienoic acid methyl ester (73 )
10. Octadec-9E-enoic acid C18 H34 O2 282 1417 33.58 20.46 (69 )
11. Octadec-9Z-enoic acid C18 H34 O2 282 1422 33.70 17.14 (42 )
12. n-Octadecanoic acid ( 18 ) C18 H36 O2 284 1435 33.98 14.29 13. UI-64 - - 1449 34.28 1.43
14. Isomer of octadec- - - 1478 34.90 2.18 9Z,12 Z-dienoic acid-II
15. UI-65 - - 1583 37.23 0.64
16. Plasticizer - - 1711 39.93 13.99 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
227
Table-4.25: Compounds identified through GC-MS in HS-GC-MS (scheme-4.7).
S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time (RT) %
1. (E,E) Hepta-2,4-dienal ( 64 ) C7H10 O 110 152 6.27 0.17
2. 2,7-Dimethyl-1,6-octadiene C10 H18 138 223 7.80 0.27 (65 )
3. n-Octanoic acid methyl C9H18 O2 158 318 9.85 0.26 ester ( 66 ) 4. UI-66 - - 562 15.12 0.25
5. 9-Oxo-nonanoic acid C10 H18 O3 186 705 18.22 1.10 methyl ester (67 ) 6. UI-67 - - 806 20.40 0.49
7. n-Tetradecanoic acid C15 H30 O2 242 1019 25.00 1.30 methyl ester ( 70 )
8. n-Tetradecanoic acid ( 41 ) C14 H28 O2 228 1067 26.03 0.51 9. UI-68 - - 1101 26.77 0.42
10. n-Pentadecanoic acid C16 H32 O2 256 1117 27.12 0.62 methyl ester ( 71 )
11. 6,10,14-Trimethyl- C18 H36 O 268 1135 27.50 2.93 pentadecan-2-one ( 48 ) 12. UI-69 - - 1153 27.88 0.59 13. UI-phytol isomer-I - - 1169 28.23 0.65
14. Hexadec-9Z-enoic acid C17 H32 O2 268 1189 28.67 0.73 methyl ester ( 72 )
15. n-Hexadecanoic acid C17 H34O2 270 1216 29.25 11.12 methyl ester ( 11 )
16. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 1273 30.48 4.24
17. n-Heptadecanoic acid C18 H36 O2 284 1302 31.10 1.31 methyl ester ( 50 )
18. Octadec-9Z,12 Z,15 Z- C19 H32 O2 292 1371 32.60 13.06 trienoic acid methyl ester (73 )
19. 3,7,11,15-Tetramethyl- C20 H40 O 296 1383 32.85 11.70 hexadec-2-en-1-ol ( 75 )
20. n-Octadecanoic acid methyl C19 H38 O2 298 1390 33.00 5.72 ester ( 23 ) 21. UI-70 - - 1401 33.25 1.68
228
Table-4.25: Continue
S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time (RT) %
22. UI-71 - - 1409 33.42 3.56 23. UI-72 - - 1414 33.53 2.86 24. UI-Phytol isomer-II - - 1462 34.57 1.86
25. Eicosanoic acid methyl C21 H42 O2 326 1546 36.38 1.91 ester ( 53 )
26. Isomer of octadecenoic acid - - 1553 36.53 3.56 methyl ester 27. Plasticizer - - 1719 40.12 16.16 28. UI-73 - - 1736 40.48 3.71
29. n-Tricosanoic acid methyl C24 H48 O2 368 1767 41.15 1.90 ester ( 57 )
30. n-Tetracosanoic acid C25 H50 O2 382 1835 42.62 2.27 methyl ester ( 58 )
31. Stigmasta-3,5-diene (19 ) C29 H48 396 2194 50.38 3.05 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
229
Table-4.26: Compounds identified through GC-MS in HS28 (scheme-4.8).
S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time (RT) %
1. UI-74 - - 145 6.12 0.01
2. τ- Cadinene ( 76 ) C15 H24 204 792 20.08 0.05
3. n-Dodecanoic acid methyl C13 H26 O2 214 806 20.38 1.81 ester ( 77 )
4. n-Tetradecanoic acid C15 H30 O2 242 1017 24.95 3.05 methyl ester ( 70 )
5. n-Pentadecanoic acid C16 H32 O2 256 1115 27.07 0.70 methyl ester ( 71 )
6. n-Hexadecanoic acid C17 H34 O2 270 1222 29.37 26.63 methyl ester ( 11 )
7. iso -Hexadecanoic acid ( 68 ) C16 H32 O2 256 1260 30.24 0.66
8. n-Hexadecanoic acid ( 13 ) C18 H36 O2 284 1271 30.43 0.81
9. n-Heptadecanoic acid C18 H36 O2 284 1299 31.03 2.07 methyl ester ( 50 )
10. Octadec-9Z,12 Z-dienoic C19 H34 O2 294 1361 32.38 7.63 acid methyl ester ( 22 )
11. Octadec-9Z,12 Z,15 Z- C19 H32 O2 292 1376 32.70 25.64 trienoic acid methyl ester (73 )
12. n-Octadecanoic acid methyl C19 H38 O2 298 1390 33.00 10.17 ester ( 23 )
13. Octadec-9Z-enoic acid ( 42 ) C18 H34 O2 282 1414 33.52 3.33 14. UI-phytol isomer-II - - 1461 34.53 2.83 15. Isomer of octadecenoic acid - - 1552 36.50 4.25 methyl ester
16. n-Docosanoic acid methyl C23 H46 O2 354 1695 39.58 2.52 ester ( 56 ) 17. Plasticizer - - 1730 39.75 3.20
18. n-Tricosanoic acid methyl C24 H48 O2 368 1764 41.08 1.98 ester ( 57 ) UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
230
Table-4.27: Compounds identified through GC-MS in A(KK) (scheme-4.8). S. Name of Compound Molecular Molecular Scan Retention *Rel. no. formula weight no. time (RT) %
1. n-Tetradecanoic acid ( 41 ) C14 H28 O2 228 1088 26.60 9.81 2. UI-75 - - 1130 27.52 4.93
3. 6,10,14-Trimethyl- C18 H36 O 268 1137 27.67 14.13 pentadecan-2-one ( 48 ) 4. UI-76 - - 1207 29.18 10.74
5. n-Hexadecanoic acid ( 13 ) C16 H32 O2 256 1284 30.85 30.75 6. UI-77 - - 1352 32.33 6.60 7. UI-78 - - 1367 32.65 7.84
8. Octadec-9Z-enoic acid C18 H34O2 282 1439 33.98 0.53 (42 ) 9. UI-79 - - 1508 35.72 7.60 10. Plasticizer - - 1719 40.30 7.06 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
Table-4.28: Compounds identified through GC-MS in B(KK) (scheme-4.8). S. Name of Compound Molecular Molecula Scan Retention *Rel. % no. formula r weight no. time (RT)
1. n-Tetradecanoic acid C14 H28 O2 228 1093 26.72 7.00 (41 )
2. 6,10,14-Trimethyl- C18 H36 O 268 1139 27.70 7.54 pentadecan-2-one ( 48 )
3. n-Pentadecanoic acid C15 H30 O2 242 1181 28.62 3.92 (78 ) 4. UI-76 - - 1209 29.23 6.44
5. n-Hexadecanoic acid C16 H32 O2 256 1294 31.07 25.26 (13 ) 6. UI-78 - - 1370 32.72 4.39
7. Octadec-9Z-enoic acid C18 H34 O2 282 1439 34.22 20.64 (42 )
8. n-Octadecanoic acid C18 H36 O2 284 1453 34.52 7.93 (18 )
9. n-Tetracosane ( 86 ) C24 H50 338 1609 37.92 4.64 10. Plasticizer - - 1725 40.43 5.08
11. n-Hexacosane ( 87 ) C26 H54 366 1762 41.32 4.29
12. n-Octacosane ( 88 ) C28 H58 394 1930 45.09 2.86 UI = Unidentified; *Calculated using peak area response on TIC (response factor not calculated)
231
Table-4.29: Identification from Leaves - Summary
Sample Total constituents Total constituents % age composition of identified code resulting in MS identified constituents (TIC response)
HEXCRU 16 11 82.60 HSGCMS 31 19 64.17 HS28 18 14 87.05 AKK 10 04 55.22 BKK 12 09 84.08
Overall 28 17 ~74.60 results
4.4.2 Gas Chromatographic Electron Impact Mass Spectral (GC-EIMS) Data:
Constituents were identified in various fractions of the root, stem and leaves of the plant under study as mentioned in various tables (section-4.4.1). Other spectral data remained unidentified. To avoid repetition, the GC-MS m/z data with their % relative abundances in parenthesis is given in two separate headings. First heading contain data of identified constituents and second heading contain data of constituents which remained unidentified. Relative retention times calculated using palmitic acid ( 13 ) are mentioned as RRT.
4.4.2.1 GC-EIMS Data of Identified Constituents:
+ n-Tridecane (1): RRT, 0.40; 184 (33) [M , C 13 H28 ], 169 (15), 141 (15), 127 (16), 113 (15), 99 (16), 85 (58 ), 71 (71), 57 (100).
+ n-Tetradecane (2): RRT, 0.51; 198 (38) [M , C 14 H30 ], 169 (14), 141 (16), 127 (15), 113 (19), 99 (20), 85 (57 ), 71 (84), 57 (100).
+ n-Pentadecane (3): RRT, 0.59; 212 (24), [M , C 15 H32 ], 155 (20), 141 (16), 127 (10), 113 (21), 99 (30), 85 (68 ), 71 (76), 57 (100).
+ iso -Hexadecane (4): RRT, 0.68; 226 (28) [M , C 16 H34 ], 196 (3), 183 (31), 169 (20), 141 (10), 99 (26), 85 (64 ), 71 (77), 57 (100).
+ n-Dodecanoic acid ethyl ester (5): RRT, 0.68; 228 (20) [M , C 14 H28 O2], 199 (12), 183 (31), 157 (22), 143 (14), 129 (18), 115 (17), 101 (46), 88 (78), 73 (19).
+ n-Heptadecane (6): RRT, 0.76; 240 (24) [M ; C 17 H36 ], 183 (17), 155 (18), 127 (21), 113 (22), 85 (64 ), 71 (97), 57 (100).
232
+ n-Tetradecanoic acid ethyl ester (7): RRT, 0.84; 256 (34) [M , C 16 H32 O2], 211 (16), 157 (19), 140 (14), 129 (24), 101 (23), 88 (54).
+ n-Octadecane (8): RRT, 0.87; 254 (3) [M , C 18 H38 ], 183 (4), 155 (6), 141 (7), 127 (8), 113 (10), 99 (14), 85 (54), 71 (78), 57 (100).
+ Cordiachrome C (9): RRT, 0.88; 242 (36) [M , C 16 H18 O2], 227 (100), 213 (22), 200 (23), 187 (28), 174 (23), 160 (12), 129 (12).
+ n-Nonadecane (10 ): RRT, 0.91; 268 (4) [M , C 19 H40 ]; 211 (4), 169 (8), 127 (12), 99 (21), 85 (56), 71 (75), 57 (100).
+ n-Hexadecanoic acid methyl ester (11 ): RRT, 0.97; 270 (22) [M , C 17 H34 O2], 239 (11), 227 (20), 185 (7), 143 (25), 129 (10), 87 (76), 74 (100), 55 (15).
+ n-Hexadecanoic acid ethyl ester (12 ): RRT, 0.99; 284 (60) [M , C 18 H36 O2], 239 (26), 199 (16), 157 (28), 115 (10), 101 (61), 88 (100), 57 (25).
+ n-Hexadecanoic acid (13 ): RRT, 1.00; 256 (65) [M ;C 16 H32 O2], 227 (11), 213 (45), 157 (21), 129 (57), 85 (31), 73 (100), 69 (60).
+ Octadec-9Z-enoic acid methyl ester (14 ): RRT, 1.07; 296 (16) [M ; C 19 H36 O2], 264 (98), 222 (50), 111 (52), 97 (82), 74 (77), 55 (100).
+ Octadec-9Z, 12 Z-dienoic acid ethyl ester (15 ): RRT, 1.10; 308 (5) [M ; C 20 H36 O2], 263 (11), 178 (4), 175 (13), 150 (8), 123 (16) 109 (32), 95 (62), 81 (96), 67 (100).
+ Octadec-9E-enoic acid ethyl ester (16 ): RRT, 1.10; 310 (44) [M ; C 20 H38 O2], 264 (100), 222 (46), 193 (10), 180 (25), 166 (23), 97 (63), 69 (36).
+ Octadec-9Z-enoic acid ethyl ester (17 ): RRT, 1.12; 310 (8) [M ; C 20 H38 O2], 264 (86), 222 (21), 180 (17), 110 (37), 69 (84), 55 (100).
+ n-Octadecanoic acid (18 ): RRT, 1.13; 284 (73) [M ; C 18 H36 O2], 241 (39), 185 (36), 129 (60), 97 (38), 85 (37), 73 (100), 57 (74).
Stigmasta-3,5-diene (19 ): RRT, 1.82; 396 (100), 381 (33), 288 (18), 275 (20), 213 (21), 147 (45), 105 (28), 81 (30).
+ n-Hexadecane (20 ): RRT, 0.70; 226 (28) [M ; C 16 H34 ], 196 (3), 183 (31), 169 (20), 141 (10), 99 (26), 85 (64 ), 71 (77), 57 (100).
Cordiachromene A (21 ): RRT, 1.02; 244 (4), 229 (3), 161 (100), 88 (6).
233
+ Octadec-9Z, 12 Z-dienoic acid methyl ester (22 ): RRT, 1.06; 294 (26) [M ; C 19 H34 O2], 263 (17), 179 (10), 136 (18), 109 (47), 95 (76), 81 (100), 67 (96), 55 (58).
+ n-Octadecanoic acid methyl ester (23 ): RRT, 1.08; 298 (45) [M ; C 19 H38 O2], 255 (36), 199 (19), 143 (33), 129 (14), 87 (94), 74 (100), 55 (19).
Cordiaquinol C (24 ): RRT, 1.24; 244 (100), 229 (27), 215 (9), 189 (10), 175 (39), 162 (9), 161 (19), 107 (11).
+ Stigmasta-4,22-diene-3β-ol (25 ): RRT, 1.94; 412 (28) [M ; C 29 H48 O], 351 (18), 300 (16), 271 (27), 255 (44), 173 (19), 83 (66).
Stigmast-5-en-3β-ol (26 ): RRT, 2.03; 414 (100), 396 (71), 381 (82), 329 (48), 303 (63), 273 (30), 213 (54), 161 (53), 145 (67), 107 (64), 81 (62).
+ Stigmasterol (27 ): RRT, 2.25; 412 (10) [M ; C 29 H48 O], 229 (19), 147 (22), 124 (35), 95 (60), 81 (53), 69 (67), 55 (100).
(1 E)-4-(3-Hydroxy-1-propenyl)-2-methoxyphenol (28 ): RRT, 0.82; 180 (84) + [M ; C 10 H12 O3], 152 (12), 147 (12), 137 (100), 124 (44), 91 (28), 77 (12).
+ Cordiol A (29 ): RRT, 1.30; 262 (54) [M ; C 16 H22 O3], 244 (44), 229 (64), 215 (16), 175 (67), 136 (100), 131 (20), 71 (42).
+ Octadec-9Z,12 Z-dienoic acid (31 ): RRT, 1.09; 280 (28) [M ; C 18 H32 O2], 264 (7), 202 (9), 179 (10), 137 (15), 123 (18), 109 (36), 95 (65), 81 (89), 67 (100), 55 (65).
+ Cycloartenol (32 ): RRT, 2.14; 426 (1) [M ; C 30 H50 O], 393 (17), 365 (12), 339 (12), 286 (14), 203 (26), 135 (30), 81 (56).
+ 24-Methylene cycloartanol (33 ): RRT, 2.25; 440 (5) [M ; C 31 H52 O], 407 (28), 353 (6), 300 (8), 203 (17), 175 (31), 135 (36), 95 (72).
+ Phenyl ethene (34 ): RRT, *; 104 (100) [M ; C 8H8], 103 (39), 89 (1), 83 (6), 78 (26), 77 (14), 51 (6). *13 was not identified in the fraction containing 34 .
+ Acetic acid phenyl methyl ester (35 ): RRT, *; 150 (53) [M ; C 9H10 O2], 109 (9), 108 (100), 107 (15), 91 (27), 79 (8), 65 (2). * 13 was not identified in the fraction containing 35 .
+ Thymine (36 ): RRT, 0.21; 126 (100) [M ; C 5H6N2O2], 95 (5), 83 (8), 55 (52), 43 (100).
2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (37 ): RRT, 0.26; 144 (41) [M +;
C12 H24 O2], 126 (3), 115 (5), 101 (32), 85 (1), 72 (27), 55 (24), 44 (74), 43 (100).
234
+ 5-(Hydroxymethyl)-2-furancarboxaldehyde (38 ): RRT, 0.34; 126 (60) [M ; C 6H6O3], 109 (15), 97 (100), 81 (12), 69 (37), 53 (28).
+ n-Dodecanoic acid (39 ): RRT, 0.67; 200 (6) [M ; C 12 H24 O2], 171 (6), 157 (28), 129 (28), 97 (32), 85 (22), 73 (100), 60 (92).
+ 3,4-Dihydroxy-benzenepropanoic acid (40 ): RRT, 0.81; 182 (17) [M ; C 9H10 O4], 123 (88), 105 (63), 91 (63), 77 (52), 55 (50).
+ n-Tetradecanoic acid (41 ): RRT, 0.86; 228 (10) [M ; C 14 H28 O2], 199 (4), 185 (12), 171 (6), 143 (5), 129 (9), 87 (59), 73 (100).
+ Octadec-9Z-enoic acid (42 ): RRT, 1.09; 282 (6) [M ; C 18 H34 O2], 264 (36), 220 (13), 165 (12), 123 (23), 111 (38), 97 (75), 69 (90), 55 (100).
+ 2-Monopalmitin (43 ): RRT, 1.36; 330 [M was not observed; C 19 H38 O4], 299 (6), 239 (57), 213 (7), 171 (8), 134 (24), 112 (23), 98 (73), 57 (80).
Cycloeucalenol (44 ): RRT, 2.08; 426 (5), 408 (21), 300 (8), 245 (10), 189 (21), 173 (35), 121 (42), 107 (53), 95 (92), 55 (100).
+ 4-Hydroxy-benzoic acid (45 ): RRT, 0.68; 138 (66) [M ; C 7H6O3], 121 (100), 110 (3), 93 (26), 81 (3), 74 (1), 65 (24), 55 (10).
+ 4-Hydroxy-3-methoxy-benzoic acid (46 ): RRT, 0.76; 168 (100) [M ; C 8H8O4], 153 (75), 136 (16), 125 (31), 108(10), 97 (40), 79 (15), 63 (12).
Nonanedioic acid monomethyl ester (47 ): RRT, 0.76; 171 (45), 152 (100), 143(12), 124 (28), 111 (41), 83 (54), 74 (97), 55 (60).
6,10,14-Trimethyl-pentadecan-2-one (48 ): RRT, 0.90; 268, M + was not observed, 250 (20), 194 (16), 179 (24), 124 (28), 85 (48), 71 (76), 58 (96), 43 (100).
+ n-Eicosane (49 ): RRT, 1.01; 282 (8) [M; C 20 H42 ] , 169 (7), 129 (12), 99 (23), 85 (59), 71 (79), 57 (100).
+ n-Heptadecanoic acid methyl ester (50 ): RRT, 1.02; 284 (24) [M or C 18 H36 O2] , 241 (23), 199 (11) 143 (28), 129 (17), 87 (80), 74 (100).
+ n-Heptadecanoic acid (51 ): RRT, 1.04; 270 (38) [M or C 17 H34 O2] , 227 (30), 185 (19), 171 (21), 129 (55), 73 (86), 57 (100).
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+ n-Heneicosane (52 ): RRT, 1.06; 296 (4) [M or C 21 H44 ] , 225 (6), 169 (5), 127 (13), 99 (22), 85 (63 ), 71 (79), 57 (100).
+ n-Eicosanoic acid methyl ester (53 ): RRT, 1.18; 326 (44) [M or C 21 H42 O2] , 295 (10), 283 (30), 199 (10), 143 (31), 129 (11), 87 (84), 74 (100).
+ n-Eicosanoic acid (54 ): RRT, 1.20; 312 (100) [M or C 20 H40 O2] , 269 (44), 213 (27), 171 (35), 129 (78), 83 (55), 73 (99), 57 (96).
+ n-Heneicosanoic acid methyl ester (55 ): RRT, 1.23; 340 (34) [M or C 22 H44 O2] , 297 (21), 241 (9), 199 (11), 143 (25), 97 (24), 87 (76), 74 (100).
+ n-Docosanoic acid methyl ester (56 ): RRT, 1.28; 354 (38) [M or C 23 H46 O2] , 311 (22), 255 (13), 199 (11), 143 (32), 111 (9), 87 (90), 74 (99).
+ n-Tricosanoic acid methyl ester (57 ): RRT, 1.34; 368 (81) [M or C 24 H48 O2] , 337 (13), 325 (36), 269 (12), 143 (42), 129 (22), 87 (94), 74 (100).
+ n-Tetracosanoic acid methyl ester (58 ): RRT, 1.41; 382 (64) [M or C 25 H50 O2] , 339 (32), 283 (14), 199 (13), 143 (37), 129 (12), 87 (84), 74 (100).
+ n-Hexacosanoic acid methyl ester (59): RRT, 1.60; 410 (76) [M ; C 27 H54 O2], 367 (33), 207 (14), 143 (35), 129 (20), 87 (81), 74 (100), 57 (38).
+ Olean-12-en-3β-ol (60 ): RRT, 2.46; 426 (44) [M or C 30 H50 O] , 411 (24), 391 (6), 353 (5), 315 (13), 257 (16), 218 (100), 189 (87), 147 (46), 135 (75), 121 (59), 81 (57).
+ Stigmasta-3,5-dien-7-one (61 ): RRT, 2.52; 410 (91) [M or C 29 H46 O] , 395 (7), 269 (31), 187 (38), 174 (100), 161(36) 135 (16), 81 (20).
Methyl 2-hydroxy-3-(4-hydroxyphenyl)-propanoate (62 ): RRT, 0.84; 196 (8) [M +;
C10 H12 O4], 178 (9), 137 (6), 107 (100), 91 (5), 77 (4).
+ Cyclolaudenol (63 ): RRT, 2.65; 440 (10) [M ; C31 H52 O], 422 (24), 407 (50), 175 (60), 147 (75), 121 (60), 107 (64), 95 (96), 69 (100).
+ (E,E) Hepta-2,4-dienal (64 ): RRT, 0.21; 110 (20) [M ; C 7H10 O], 95 (10), 81 (100), 79 (8), 77 (7), 68 (8), 67 (8), 53 (14).
+ 2,7-Dimethyl-1,6-octadiene (65 ): RRT, 0.26; 138 (14) [M ; C 10 H18 ], 123 (11), 107 (10), 95 (20), 82 (11), 69 (100), 61 (25), 42 (53 ).
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+ n-Octanoic acid methyl ester (66 ): RRT, 0.32; 158 (5) [M ; C 9H18 O2], 127 (24), 115 (16), 101 (10), 87 (51), 74 (100), 69 (6), 55 (18).
+ 9-Oxo-nonanoic acid methyl ester (67 ): RRT, 0.61; 186 [M was not observed, C 10 H18 O3], 155 (20), 143 (40), 111 (62), 87 (71), 83 (44), 74 (100), 69 (21), 55 (74).
+ iso -Hexadecanoic acid (68 ): RRT, 0.99; 256 (84) [M ; C 16 H32 O2], 213 (49), 185 (21), 157 (28), 129 (62), 85 (33), 73 (100), 60 (75).
+ Octadec-9E-enoic acid (69 ): RRT, 1.11; 282 (5) [M ; C 18 H34 O2], 264 (30), 222 (12), 111 (28), 97 (45), 83 (65), 69 (66), 55 (100).
+ n-Tetradecanoic acid methyl ester (70 ): RRT, 0.82; 242 (4) [M ; C 15 H30 O2], 211 (9), 199 (17), 185 (4), 157 (8), 143 (24), 87 (80), 74 (100).
+ n-Pentadecanoic acid methyl ester (71 ): RRT, 0.89; 256 (6) [M ; C 16 H32 O2], 225 (8), 213 (16), 199 (8), 143 (24), 129 (15), 87 (78), 74 (100).
+ Hexadec-9Z-enoic acid methyl ester (72 ): RRT, 0.94; 268 (10) [M ; C 17 H32 O2], 152 (21), 109 (36), 97 (46), 87 (47), 74 (60), 69 (65), 55 (100).
+ Octadec-9Z,12 Z,15 Z-trienoic acid methyl ester (73 ): RRT, 1.07; 292 (4) [M ; C 19 H32 O2], 236 (10), 149 (19), 135 (20), 121 (26), 108 (51), 95 (84), 79 (100).
3,7,11,15-Tetramethyl-hexadec-2-en-1-ol (75 ): RRT, 1.08; 296 [M + was not observed;
C20 H40 O], 196 (2), 123 (25), 111 (10), 95 (15), 81 (22), 71 (100), 57 (28), 43 (22).
+ τ-Cadinene (76 ): RRT, 0.68; 204 (26) [M ; C 15 H24 ], 161 (100), 148 (6), 133 (12), 119 (20), 105 (23), 93 (16), 81 (14).
+ n-Dodecanoic acid methyl ester (77 ): RRT, 0.67; 214 (10) [M ; C 13 H26 O2], 183 (13), 171 (14), 143 (16), 87 (60), 74 (100), 69 (18), 55 (28).
+ n-Pentadecanoic acid (78 ): RRT, 0.92; 242 (18) [M ; C 15 H30 O2], 199 (20), 143 (28), 129 (16), 73 (62), 60 (28), 55 (62), 43 (100).
+ n-Tetracosane (86 ): RRT, 1.22; 338 (14) [M ; C 24 H50 ], 169 (6), 141 (15), 127 (14), 99 (28), 85 (62), 71 (79), 57 (100).
+ n-Hexacosane (87 ): RRT, 1.33; 366 (4) [M ; C 26 H54 ], 239 (12), 183 (15), 155 (12), 99 (16), 85 (48), 71 (68), 57 (100).
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+ n-Octacosane (88 ): RRT, 1.45; 394 (6) [M ; C 28 H58 ], 141 (12), 127 (13), 113 (18), 99 (21), 85 (53), 71 (78), 57 (100).
Plasticizer : 279 (40), 390 (2), 167 (53), 149 (100), 113 (11).
Plasticizer a: 256 (5), 223 (6), 205 (5), 167 (40), 149 (100), 129 (4).
4.4.2.2 GC-EIMS Data of Unidentified Constituents:
Unidentified-1: 258 (4), 242 (20), 227 (16), 186 (36), 175 (100), 108 (44), 93 (33), 77 (14).
Unidentified-2: 294 (67), 256 (56), 244 (48), 175 (44), 155 (40), 109 (52), 95 (66), 67 (100).
Unidentified-3: 284 (10), 276 (14), 244 (100), 229 (44), 175 (40), 161 (30), 136 (33), 105(36), 91 (20).
Unidentified-4: 274 (6), 244 (100), 229 (26), 203 (22), 175 (34), 136 (20), 105(7), 91 (8).
Unidentified-5: 242 (14), 227 (100), 200 (25), 187 (27), 174 (20), 131 (19), 91 (20), 77 (14).
Unidentified-6: 268 (6), 242 (7), 227 (31), 187 (19), 174 (15), 141 (11), 115 (16), 91 (26), 71 (84).
Unidentified-7: 242 (14), 227 (100), 201 (48), 186 (27), 173 (36), 145 (16), 108 (22), 91 (47), 77 (26).
Unidentified-8: 256 (4), 240 (10), 212 (8), 172 (31), 161 (12), 115 (24), 83 (23), 69 (100).
Unidentified-9: 256 (27), 206 (22), 175 (24), 159 (23), 97 (33), 69 (39), 55 (76), 43 (100).
Unidentified-10: 272 (7), 257 (4), 202 (3), 175 (100), 160 (3), 132 (15), 91 (4), 77 (4).
Unidentified-11: 286 (5), 271 (6), 244 (19) 226 (19), 175 (23), 111 (16), 97 (49), 83 (58), 69 (55), 55 (95).
Unidentified-12: 244 (47), 229 (16), 175 (35), 161 (100), 147 (28), 133 (40), 121 (69), 105 (21), 77 (24).
Unidentified-13: 244 (68), 229 (56), 175 (62), 161 (50), 136 (68), 81 (73), 67 (88), 55 (100).
Unidentified-isomer of UI-10: 272 (4), 257 (4), 175 (100), 161 (6), 132 (8), 109 (8), 77 (7), 55 (9).
Unidentified-14: 244 (100), 229 (27), 203 (34), 175 (56), 161 (37), 136 (31), 115 (17), 91 (20).
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Unidentified-15: 244 (100), 229 (37), 187 (24), 173 (52), 161 (25), 148 (26), 136 (100), 109 (36).
Unidentified-16: 244 (91), 229 (32), 207 (24), 187 (23), 175 (42), 169 (60), 136 (100), 109 (99).
Unidentified-17: 242 (100), 227 (48), 213 (74), 199 (20), 173 (26), 147 (26), 115 (27), 91 (26).
Unidentified-18: 274 (62), 203 (26), 187 (11), 173 (63), 161 (51), 111 (45), 104 (100), 91 (68).
Unidentified-19: 242 (62), 227 (100), 212 (17), 198 (20), 165 (16), 128 (13), 95 (17), 77 (13).
Unidentified-20: 304 (100), 257 (35), 240 (67), 225 (52), 203 (39), 196 (28), 181 (45), 115 (48).
Unidentified-21: 274 (100), 227 (23), 189 (8), 173 (25), 145 (6), 111 (11), 97 (25), 83 (25).
Unidentified-22: 274 (32), 242 (31), 227 (64), 187 (50), 174 (44), 161 (28), 147 (100), 136 (29), 95 (44), 77 (24).
Unidentified-23: 304 (8), 258 (11), 225 (100), 173 (20), 161 (24), 115 (31), 97 (32), 71 (40).
Unidentified-24: 436 (1), 418 (1), 373 (2), 281 (2), 261 (3), 207 (5), 175 (5), 123 (16), 109 (28), 95 (55), 55 (100).
Unidentified-25: 244 (92), 229 (98), 187 (26), 175 (37), 161 (44), 136 (44), 107 (42), 91 (44), 77 (31).
Unidentified-26: 334 (44), 262 (16), 229 (19), 183 (100), 173 (19), 136 (17), 95 (16), 81 (30).
Unidentified-27: 334 (100), 316 (20), 247 (45), 207 (43), 183 (39), 115 (29), 91 (28), 77 (44).
Unidentified-28: 415 (4), 355 (8), 319 (65), 250 (68), 211 (100), 182 (40), 149 (17), 115 (17), 55 (28).
Unidentified-29: 284 (28), 256 (40), 197 (29), 157 (27), 129 (36), 85 (68), 71 (93), 57 (100).
Unidentified-30: 258 (4), 242 (20), 227 (16), 186 (36), 175 (100), 108 (44), 93 (33), 77 (14).
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Unidentified-31: 242 (36), 227 (28), 186 (24), 136 (29), 128 (18), 108 (82), 93 (100), 77 (28).
Unidentified-32: 242 (64), 227 (100), 213 (17), 186 (38), 173 (35), 145 (13), 115 (29), 91 (28), 77 (20).
Unidentified-33: 260 (28), 242 (68), 227 (46), 175 (68), 162 (71), 131 (36), 91 (57), 77 (32), 55 (60).
Unidentified-34: 140 (5), 112 (62), 110 (100), 109 (41), 105 (18), 85 (11), 77 (25), 75 (78).
Unidentified-35: 140 (7), 112 (65), 110 (100), 109 (71), 105 (25), 91 (5), 77 (29), 75 (89).
Unidentified-36: 124 (6), 123 (100), 118 (8), 107 (5), 93 (7), 89 (14), 77 (54), 51 (6).
Unidentified-37: 140 (4), 140 (26), 122 (14), 107 (31), 105 (100), 91 (37), 78 (23), 77 (43).
Unidentified-38: 285 (4), 189 (10), 160 (17), 128 (34), 101 (100), 91 (4), 87 (48), 71 (9).
Unidentified-39: 246 (1), 244 (37), 227 (100), 213 (21), 187 (47), 174 (48), 136 (33), 91 (29), 77 (22).
Unidentified-isomer of UI-39: 246 (1), 244 (43), 227 (100), 213 (21), 187 (36), 174 (35), 136 (22), 91 (14), 77 (8).
Unidentified-Isomer of Cordiachromene A: 244 (8), 229 (2), 161 (100), 162 (9), 131 (2), 115 (3), 91 (3), 69 (5).
Unidentified-40: 282 (4), 274 (74), 242 (70), 227 (100), 187 (61), 136 (58), 95 (58), 77 (14).
Unidentified-41: 358 (11), 300 (8), 285 (10), 113 (28), 99 (29), 85 (76), 71 (93), 57 (100).
Unidentified-Isomer of UI-3: 244 (74), 229 (80), 174 (100), 161 (92), 147 (51), 136 (84), 115 (41), 91 (81), 77 (46).
Unidentified-42: 662 (32), 647 (100), 591 (6), 443 (6), 316 (8), 253 (10), 191 (17), 57 (56).
Unidentified-43: 341 (5), 281 (10), 207 (12), 149 (12), 95 (19), 81 (52), 69 (100).
Unidentified-44: 136 (100), 110 (10), 89 (29), 77 (9), 63 (7), 51 (6), 43 (5).
Unidentified-isomer of C 18:2 FFA-I: 280 (7), 264 (22), 109 (27), 95 (55), 81 (73), 67 (85), 55 (100), 41 (93).
Unidentified-45: 258 (6), 175 (100), 161 (4), 132 (8), 107 (4), 91 (4), 69 (6), 44 (27).
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Unidentified-46: 274 (84), 259 (32), 204 (32), 166 (96), 161 (100), 151 (49), 77 (57), 69 (49).
Unidentified-47: 300 (24), 272 (20), 241 (28), 173 (21), 161 (100), 151 (44), 91 (17), 77 (21).
Unidentified-48: 272 (100), 211 (16), 197 (8), 169 (10), 139 (4), 91 (3), 55 (5).
Unidentified-49: 348 (100), 333 (17), 251 (20), 187 (14), 161 (52), 115 (26), 91 (21), 69 (22).
Unidentified-50: 416 (4), 400 (12), 358 (16), 289 (17), 281 (37), 207 (61), 151 (92), 137 (68), 55 (100).
Unidentified-51: 284 (11), 240 (7), 220 (13), 155 (11), 127 (14), 85 (64), 71 (80), 57 (100).
Unidentified-52: 294 (8), 199 (39), 167 (100), 155 (43), 139 (35), 121 (38), 81 (59), 57 (87).
Unidentified-53: 310 (5), 279 (19), 211 (40), 167 (89), 153 (100), 137 (91), 97 (76), 55 (92).
Unidentified-54: 392 (89), 362 (12), 281 (39), 180 (32), 135 (70), 95 (72) 83 (100), 69 (86).
Unidentified-55: 394 (100), 370 (7), 275 (22), 252 (13), 218 (56), 158 (28), 143 (49), 135 (59), 81 (43).
Unidentified-56: 410 (50), 395 (40), 381 (100), 269 (24), 204 (25), 174 (18), 145 (34), 121 (31), 69 (23).
Unidentified-57: 438 (50) 355 (26), 281 (36), 216 (41), 161 (57), 121 (84), 95 (100), 69 (69).
Unidentified-58: 440 (10), 422 (52), 407 (96), 289 (28), 175 (71), 147 (80), 124 (86), 95 (100), 55 (81).
Unidentified-59: 182 (97), 152 (73), 137 (50), 111 (56), 95 (60), 81 (71), 69 (64), 55 (100).
Unidentified-60: 354 (2), 297 (4), 203 (5), 185 (100), 139 (7), 112 (19), 71 (23), 57 (23).
Unidentified-61: 308 (24), 237 (44), 166 (52), 151 (88), 119 (41), 95 (92), 81 (100), 69 (66).
Unidentified-62: 132 (6), 101 (11), 87 (18), 75 (21), 57 (100), 45 (81), 41 (26).
Unidentified-63: 265 (53), 264 (78), 97 (75), 87 (64), 83 (83), 55 (100), 43 (75).
Unidentified-64: 280 (39), 105 (100), 95 (51), 81 (58), 67 (66), 55 (55), 42 (54).
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Isomer of octadec-9Z,12 Z-dienoic acid-II: 280 (44), 264 (10), 202 (15), 179 (20), 137 (25), 123 (20), 109 (50), 95 (76), 81 (97), 67 (100), 55 (48).
Unidentified-65: 312 (76), 73 (15), 69 (100), 56 (19), 55 (34), 43 (85).
Unidentified-66: 150 (10), 121 (20), 91 (19), 81 (100), 77 (31), 67 (35), 55 (20), 42 (29).
Unidentified-67: 183 (6), 180 (17), 152 (15), 137 (35), 111 (100), 95 (8), 87 (20), 74 (27).
Unidentified-68: 254 (20), 222 (16), 180 (18), 101 (21), 96 (75), 83 (58), 73 (66), 57 (100).
Unidentified-69: 220 (10), 149 (32), 123 (56), 109 (38), 95 (55), 82 (90), 68 (62), 57 (100).
UI-isomer of phytol-I: 278 (16), 185 (24), 123 (75), 111 (50), 95 (60), 81 (83), 69 (87), 57 (100).
Unidentified-70: 286 (4), 209 (8), 123 (26), 108 (22), 95 (60), 84 (87), 71 (100), 55 (47).
Unidentified-71: 386 (2), 222 (10), 123 (66), 109 (35), 95 (87), 83 (81), 69 (100), 55 (82).
Unidentified-72: 304 (2), 264 (11), 222 (18), 108 (44), 95 (87), 79 (100), 67 (93), 55 (95).
UI-isomer of phytol-II: 278 (5), 123 (100), 111 (27), 95 (66), 82 (67), 68 (83), 57 (67), 43 (11).
UI-isomer of octadecenoic acid methyl ester: 282 (2), 264 (24), 222 (13), 111 (37), 97 (27), 83 (40), 74 (100), 69 (34).
Unidentified-73: 263 (44), 245 (27), 111 (35), 97 (52), 83 (55), 74 (100), 69 (57), 55 (63).
Unidentified-74: 120 (60), 105 (100).
Unidentified-75: 280 (14), 177 (18), 151 (32), 137 (39), 109 (30), 95 (100), 82 (75), 68 (71).
Unidentified-76: 308 (4), 280 (10), 125 (39), 107 (42), 97 (16), 84 (83), 71 (100), 57 (73).
Unidentified-77: 282 (10), 251 (8), 235 (8), 185 (11), 129 (14), 97 (22), 85 (100), 69 (31).
Unidentified-78: 295 (7), 247 (7), 152 (9), 137 (8), 111 (11), 97 (12), 85 (100), 71 (20).
Unidentified-79: 410 (15), 347 (11), 281 (16), 151 (23), 111 (66), 95 (43), 83 (95), 71 (100).
Unidentified-isomer of Cordiachrome C: 242 (40), 227 (100), 213 (22), 199 (20), 187 (36), 174 (25), 115 (16), 91 (20).
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4.5 Bioactivities:
4.5.1 Sample Preparation of Extracts from Root, Stem, and Leaves for Bioactivities:
For antiglycation bioassay sample of plant extract was prepared in 1mg/ml of dimethyl sulfoxide (DMSO). For other bioactivities, samples were prepared as given in respective experimental sections. Conclusively crude extracts, fractions, and sub-fractions obtained and pure compounds isolated from these extracts, were also evaluated for various biological activities discussed in section: 4.5.2.
4.5.2 Bioassays:
4.5.2.1 Antimicrobial activity:
Antibacterial Activity by Disk Diffusion Method:*
Roots extracts were screened for in vitro antibacterial bioassay using disk diffusion method (Bauer et al., 1966). Stock solution having concentration of 50 mg/ml was used to prepare sterile disk of extracts/samples to be tested. Briefly 10 µl containing 500 µg sample was loaded onto the disks. Test cultures (2 hours old) grown in Mueller Hinton broth was used to seed Mueller-Hinton agar plates. Sterile cotton swabs were utilized for seeding. Finally prepared disks were then incubated for 24 h at 37 ºC loaded at different places of agar surfaces. Control was prepared by loading 10 µl sterile DMSO on another disk. Zone of inhibition (mm) was measured to assess antibacterial effects of samples (tables-4.30 and 4.31).
*Antibacterial Assay using Disk Diffusion method was performed in collaboration and under Supervision of Prof. Dr. Aqeel Ahmad, Department of Microbiology, University of Karachi.
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Table-4.30: In vitro antibacterial bioassay of different fractions of the roots extract of C. rothii against Gram-positive bacteria.
Zone of Inhibition (mm) Name of Bacteria 2A 4A KC-PE KC-C 6A KEA-PE KEA-C KA KA-PE KA-C KA-EA KM-PE KM-C KM-EA KM-M
Bacillus cereus - - - - 9 10 10 - 11 12 9 - 11 9 -
Bacillus subtilis 11 9 10 10 9 11 10 9 - 11 - - 9 9 9
Bacillus thereugenesis - - - 10 - - - - 11 15 - - 11 11 - Corynebacterium - - - 9 - - 9 - 12 16 9 10 13 10 - diphtheria Corynebacterium - - - - - 13 11 - - 15 10 - 15 - - hofmanii Corynebacterium 9 - 9 - 9 10 - 10 13 18 - 12 16 13 - xerosis Micrococcus leutus 10 - 9 9 10 10 9 9 - 15 9 - 10 13 - Micrococcus leutus 10 9 10 - 10 12 13 - 14 23 10 11 20 14 - ATCC9341 Methicillin resistant - - - - - 10 9 - - 15 9 9 12 10 - Staphylococcus aureus Staphylococcus aureus 11 9 9 - - 9 11 9 9 12 - - - - - Staphylococcus aureus 9 - 9 9 10 13 9 - 12 15 10 - 12 - 11 Ab188 Staphylococcus - 9 11 10 9 - - 10 17 18 - 11 13 15 - epidermidis Streptococcus fecalis 10 - 9 - - 9 - - 15 20 - - 13 14 - Streptococcus 14 9 11 11 12 10 10 11 10 17 10 - 15 9 - saprophyticus Streptococcus pyogenes ------12 16 - 9 10 11 - - = Inactive
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Table-4.31: In vitro antibacterial bioassay of different fractions of the roots extract of C. rothii against Gram-negative bacteria.
Zone of Inhibition (mm) Name of Bacteria 2A 4A KC-PE KC-C 6A KEA-PE KEA-C KA KA-PE KA-C KA-EA KM-PE KM-C KM-EA KM-M
Enterobacter 11 - 10 10 10 12 12 10 12 13 11 10 12 10 - Escherichia coli ATCC ------14 12 9 11 15 10 - 10 11 8739 Esherichia coli ------
E. coli MDR - - - 10 - 13 9 - - - 9 - - - -
Klebsiella pneumonae 12 10 13 12 10 11 10 12 11 15 - 11 11 10 -
Proteus mirabilis ------17 - 11 14 12 - Pseudomonas ------10 15 - 10 12 10 aeroginosa Pseudomonas 13 9 11 11 11 - - 11 10 13 - - 10 10 - aeroginosa ATCC Salmonella typhi 12 - 10 13 13 9 - 10 ------
Salmonella paratyphi A - - - - - 13 12 - - 12 10 - - - -
Salmonella paratyphi B ------11 9 - - 9
Shigella dysentery 14 11 10 11 14 - - 12 ------
Shigella flexenerii 14 - 10 13 14 ------= Inactive, KA-A, KA-M, KM, and KM-A did not exhibit antibacterial activity against any of the gram-positive or -negative bacteria tested.
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Evaluation of Minimum Inhibitory Concentration (MIC) in µg/disk:
The fractionated root samples of the plant exhibiting strong antibacterial activity ≥ 15 mm (zone of inhibition/concentration in µg) in preliminary screening were evaluated for their minimum inhibitory concentration (MIC). Out of 13 Gram-negative bacteria tested, fraction KA-C was found to be active only against Proteus mirabilis , Pseudomonas aeroginosa , E.coli ATCC 8739, and Klebsiella pneumonae with the values of 250, 125, 62.5, and 31.25 respectively. Samples at different concentrations ranging from 31.25- 250 µg/mL, diluted serially twice, were used (tables-4.32).
Table-4.32: Minimal inhibitory concentration ( µg/disk) of different fractions of root extract of the plant against Gram -positive bacteria.
Name of Bacteria KA-C KM-C KM-EA KA-PE
Bacillus thereugenesis 31.25 - - - Corynebacterium diphthirea 250 - - - Corynebacterium hofmanii 125 250 - - Corynebacterium xerosis 250 125 - - Micrococcus leutus 31.25 - - - Micrococcus leutus ATCC9341 250 31.25 - - MRSA 62.5 - - - Staphylococcus aureus AB188 <31.25 - - - Staphylococcus epidermidis 250 - 250 250 Streptococcus saprophyticus 62.5 62.5 - - Streptococcus pyogenes 250 - - - Streptococcus fecalis 125 - - 31.25
Samples tested were found inactive against Gram-positive bacteria; Bacillus cereus , Bacillus subtilis , and Staphylococcus aureus, and Gram-negative bacteria; Enterobacter , Salmonella typhi , Salmonella paratyphi B, Pseudomonas aeroginosa ATCC 9027, Salmonella typhi A, Shigella dysentery , E.coli , E.coli MDR, Shigella flexenerii .
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Antibacterial Activity by Agar Well Diffusion Method:*
Antibacterial activity of the crude methanol extract of the stem (CRS) and leaves (CRM) of the plant with its various fractions weres performed by agar well diffusion method. Microbial strains used in this study are given in table-4.33. A single colony of bacterial culture was inoculated in nutrient broth and incubated at 37 ºC for 24 h. 10 µl of fresh bacterial culture was added into a soft agar tube. After shaking well, it was poured onto the nutrient agar containing plate and allowed to solidify on the lawn. 6mm-diameter wells were prepared and marked with sample code. 100 µl sample was added in respective agar well plate according to bacterial culture. Other wells were supplemented with DMSO and reference antibacterial drug (imipenum). The plates were then incubated at 37 ºC for 24 h (Alves TMA et al., 2000 and Stepanovi ć S et al., 2003). The results were recorded in terms of zone of inhibition in 1mm (table-4.33).
Table-4.33: In vitro antibacterial bioassay of crude methanol extract (CRS) of stem and leaves of C. rothii .
Zone of Inhibition (mm) Name of Bacteria CRS CRM HEXCRU CRME CRMB
Escherichia coli a - - - - - Bacillus subtilis b - - 11 - - Shigella flexenari a - - - - - Staphylococcus aurea b - - - - - Pseudomonas aeruginosa a - - - 9 - Salmonella typhi a - - - - -
a = Gram-negative bacteria, b = Gram-positive bacteria
*Antibacterial Assay using agar well diffusion method was performed in collaboration with Prof. Dr. M. Iqbal Choudhary at International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi
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Antifungal Activity by Disk Diffusion Protocol:*
Disk diffusion protocol (Bauer et al., 1966) was applied to analyze in vitro antifungal activity of crude root extracts. In order to prepare homogeneous suspension, fungal culture was spin rapidly in small tubes fastened with screw caps. This suspension was used to seed Sabouraud's dextrose agar (SDA) plates. Sterile filter disks (500 µg of sample each) were placed at different positions onto the surface. The plates were then incubated for 1 week at room temperature. Sterile DMSO loaded disk was used as control. Antifungal effects of samples were evaluated by measuring zones of inhibition (mm).
Out of 19 samples tested; 2A, 4A, KC-PE, KC-C, 6A, KEA-PE, KEA-C, KA, KA-PE, KA-C, KA-EA, KA-A, KM, KM-PE, KM-EA, KM-A, and KM-M, only fraction KEA-PE showed activity against Fusarium sp ., and Saccharomyces cerevisiae with zone of inhibition of 13 and 9 mm respectively. None of the sample was found to be active against Aspergillus flavus , Aspergillus niger , Penicillium sp. Rhizopus , Candida albican , Candida albican ATCC 0383, Aspergillus flavus , Microsporum gypseum , Trichophyton rubrum , Trichophyton mentagrophytes , Trichophyton tonsurans , Helminthosporum fungi.
*Antifungal Assay using Disc Diffusion method was performed in collaboration and under the supervision of Prof. Dr. Aqeel Ahmad, Department of Microbiology, University of Karachi.
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Antifungal Bioassay by Agar Tube Dilution Protocol:*
Antifungal bioassay of crude methanol extract of the stem (CRS) and leaves (CRM) with its various fractions, were determined by agar tube protocol (Choudhary MI et al., 1995 & Janaki S and Vijayasekaram V, 1998). In this assay, test samples were prepared by dissolving 24 mg of crude extract in 1 ml sterile DMSO. After media preparing and steaming, 4 ml volume was dispensed into screw caps tubes and autoclaved at 121 ºC for 15 minutes. After sample loading tubes were allowed to solidify in slanting position at room temperature. The 6mm 2 fungus growth (culture) was inoculated and the tubes were then incubated at 27-29 ºC for 3-7 days. Percentage inhibitions of fungal growth were calculated as shown in table-4.34.
Table-4.34: In vitro antifungal bioassay of the crude methanol extract of stem and leaves of the plant.
% Inhibition Name of Fungus CRS CRM HEXCRU CRME CRMB
Trichphyton longifusus - - - - - Candida albicans 0 0 0 0 0 Aspergillus flavus 15 20 0 0 60 Microsporum canis 35 35 0 70 60 Fusarium solani 30 35 0 20 20 Candida glabarata 0 0 0 0 0
*Antifungal Assay using agar tube dilution method was performed in collaboration with Prof. Dr. M. Iqbal Choudhary at International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi
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In vitro Antileishmanial Bioassay:
Sterile 25 cm 2 tissue culture flask was used to culture leishmanial promastigotes at 25 ºC supplemented with 25 mM HEPES and 10% HIFBS in tissue culture medium M-199. Minimum volume of PBS was used to dilute parasites, centrifuged at 3000 rpm. Fresh medium was then used to dilute parasites making up the final concentration of 2.0 x 10 6 parasites/ml. 50 µl of absolute MeOH or DMSO was used to dissolve one mg of extract and the volume was then made up till 1.0 ml with the culture medium.10 µl having different concentrations of extract and 90 µl of the parasite culture was added in the culture in a 96 well microtiter plate. Amphotericin B and pentamidine were used as positive control while 10 µl of PBS, as negative control.The control organism multiplied 3 to 6 times in the dark incubated at 25 ºC for 3 to 5 days in the dark. Lastly, improved Neubauer chamber was used to examine culture/count parasites microscopically. ED 50 values were calculated (table-4.35).
Table-4.35: Antileishmanial bioassay of fractions of C. rothii leaves.
Sample IC 50 (µg / mL) ± S.D HEXCRU > 100 CRME > 100 CRMB > 100
*Antileishmanial Assay using 96-well serial dilution protocol was performed in collaboration with Prof. Dr. M. Iqbal Choudhary at International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi
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4.5.2.2 Toxicity studies:
Phytotoxic Bioassay:*
Stock solution was prepared by dissolving 30 mg of crude methanol extract of stem (CRS) and leaves (CRM) along with its various fractions, in 1.5 mL of solvent. Stock solution was pipette out for three 10, 100 and 1000 µg/mL inoculated flasks. The solvent was then evaporated overnight. 20 ml of working E-medium and then Lemna minor plant 20 fronds/flask was added. E-medium was taken as negative and reference (standard drug) plant growth inhibitors and promoters as positive control. The flasks were then placed in growth cabinet for 7 days. The number of fronds were then counted and recorded on the seventh day. Results were then analyzed as growth regulation in percentage and calculated with reference to the negative control (table-4.36) (Atta-ur-Rahman 1991 and Finny DJ, 1971).
Table-4.36: In vitro phytotoxic bioassay of the crude methanol extract (CRS) of stem and leaves of the plant against fronds.
Concentration of Extract % Growth Regulation (µg/mL) CRS CRM HEXCRU CRME CRMB
1000 40 30 21.7 19.0 10 100 10 10 17.3 14.2 0 10 5 0 13.0 4.7 0
*Phytotoxic Assay was performed in collaboration with Prof. Dr. M. Iqbal Choudhary at International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi
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Cytotoxicity Test (Brine Shrimp Lethality Bioassay):*
Samples from different fractions of leaves were prepared by dissolving 20 mg material in 2 ml of solvent. 5, 50 and 500 µl of prepared samples were transferred to vials. After evaporating solvent overnight at room temperature, 2-days hatched and matured napulii were placed 10 larvae / vial. Volume was then made upto 5 ml with seawater and incubated at 25- 27 ºC for 24 h under illumination. Other vials were supplemented with solvent and reference cytotoxic drug (Etoposide). The data was then analyzed with Finney computer program to determine LD 50 values with 95% confidence intervals (table-4.37) (Alves TMA et al., 2000, Kivack B et al., 2001, Carballo LJ et al., 2002, and Mayer BN et al., 1982).
Table-4.37: Brine Shrimp ( Artemia salina) Lethality bioassay of different fractions of C. rothii leaves. LD (µg/mL) Dose ( µg/mL) 50 HEXCRU CRME CRMB 1000
100 7.4625 7.4625 7.4625
10
______
*Brine Shrimp ( Artemia Salina ) Lethality Bioassay was performed in collaboration with Prof. Dr. M. Iqbal Choudhary at International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi
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Cytotoxicity Test (3T3-NIH):*
3T3–NIH mouse embryo fibroblast cell line (American Type Culture Collection “ATCC”, Manassas, VA 20108, USA) was used to assess cytotoxicity of samples. Dulbecco’s Modified Eagle’s Medium (DMEM) treated with 10% fetal bovine serum (FBS), was used to culture 4 3T3-NIH cells. Cells (6 x 10 cells/mL) were incubated in 5% CO 2 environment at 37 °C for 24 h. These cells after media removal, were treated in triplicates with different concentrations
(0.5, 5.0, and 25 µg/mL) of the fractions. Cells were again incubated in CO2 environment at 37 °C for another 48 h. 0.5 mg/mL MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide) was added and incubation was continued for further 4 h. DMSO was added to solubilize formazan after removing supernatant. Followed by one minute shaking, absorbance was taken at 540 nm (Scudiero DA et al., 1988).
Table-4.38: Cytotoxicity of extract of leaves of the plant.
Sample (code) IC 50 ± SD
HEXCRU 41.68 ± 0.05
Control IC 50 ± SD Cycloheximide 0.51 ± 0.05
*Cytotoxicity Test (3T3-NIH) was performed in collaboration under the supervision of Prof. Dr. M. Iqbal Choudhary at International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi
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4.5.2.3 Antioxidant & Immunomodulatory activities.
Antioxidant Bioassay (DPPH method):*
The method adopted for antioxidant bioassay was developed by Lee SK et al., 1998. Ethanol was used to make 300 µM solution of 1,1-diphenyl-2-pierylhydrazyl (DPPH). Dimethylsulfoxide (DMSO) was used to prepare test sample solution. 10 µL test sample and 90 µL DPPH constituting reaction mixture was added in 96-well microtiter plates. The incubation period of plates was 30 minutes at 37 °C. Spectrophotometer was used to measure absorbance at 515 nm. Comparison between the DMSO treated control and test sample
provided the % inhibition exhibited by test sample. Control used was Ascorbic acid. The EC 50 concentration of test sample was calculated in µg/mL. This value determined the concentration of test sample to scavenge 50 % DPPH (table-4.39).
% Inhibition = (Absorbance of the control) – (Absorbance of test sample) × 100 Absorbance of the control
Table-4.39: Antioxidant bioassay of different fractions of C. rothii roots.
Sample Code % Inhibition ± SD EC 50 µg/ml 2A 61.11±0.02 187.5 4A 51.67±0.02 375 KC-PE 43.88±0.01 - KC-C 46.58±0.01 - 6A 58.63±0.03 375 KEA-PE 67.65±0.01 93.75 KEA-C 56.11±0.02 375 KA 59.89±0.01 187.5 KA-PE 23.56±0.03 - KA-C 63.84±0.02 187.5 KA-EA 33.45±0.01 - KA-A 71.76±0.01 46.875 KA-M 53.95±0.05 375 KM 76.79±0.01 46.875 KM-PE 62.94±0.07 93.75 KM-C 58.27±0.02 375 KM-EA 57.0±0.01 375 KM-A 69.24±0.02 46.875 KM-M 72.58±0.02 93.75
*Antioxidant Bioassay (DPPH method) was performed in collaboration and under Supervision of Prof. Dr. Aqeel Ahmad, Department of Microbiology, University of Karachi.
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Immunomodulating Bioassay:
Isolation of Human Polymorphoneutrophils (PMNs):
Heparinized blood was obtained by vein puncture aseptically from healthy volunteers (25-38 years age). The buffy coat containing PMNs were collected by dextran sedimentation and cells were isolated after the lymphocyte separation medium (LSM) (MP Biomedicals, Ohio, USA) density gradient centrifugation. PMNs were collected from bottom of the tube. Cells were washed twice and suspended in Hank’s Balance Salt Solution [Ca and Mg free] (HBSS -- ) pH, 7.4 (Sigma-Aldrich Steinheim, Germany). Neutrophils were purified from RBCs using hypotonic solution. Cells were adjusted to their required concentration using Hank’s Balance Salt Solution Ca +2 and Mg +2 (HBSS ++ ) obtained from Sigma-Aldrich, Steinheim, Germany (Colin DA and Monteil H, 2003).
Chemiluminescence Bioassay:
Luminol-enhanced chemiluminescence assay was performed following Waleed et al., (Koko WS et al., 2008). 25 µL of diluted (1:50) whole blood in HBSS++ was incubated with 25 µL of serially diluted compounds. The concentration ranges between 1 to100 µg/mL. Control wells contain HBSS ++ and the diluted whole blood but no compound. Test was performed in Corning White 96 wells plates (New York, USA). Culture was incubated at 37 °C for 20 minutes in the thermostated chamber of the lab system luminoscan RS (Vienna, Virginia).
25 µL of opsonized zymosan-A ( Saccharomyces cerevisiae origin), followed by 25 µL luminol (7 × 10 -5 M) (Alfa Aesar, Karlsruhe, Germany) along with HBSS ++ were added to each well to obtain a 100 µL volume/well. The luminometer results were recorded in Relative Light Unit (RLU) (table-4.40, figure-4.1).
*Immunomodulating Bioassay was performed in collaboration with Dr. Muhammad Ahmed Mesaik at International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi. A portion of this work has already been published as;
“Isolation of Phytochemicals from Cordia Rothii (Boraginaceae) and Evaluation of their Immunomodulatory Properties”, S. Firdous, K. Khan, S. Z. Rehman, Z. Ali, S. Soomro, V. U. Ahmad, M. Rasheed, M. A. Mesaik and S. Faizi, Records of Natural Products , Vol. 8, No. 1, 51-55, (2014).
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Figure-4.1: Effect of fractions on ROS production by whole blood phagocytes determined by chemiluminescence technique and oxidative burst study.
RLU = relative light unit
Table-4.40: Effect of methanol extract and fractions of the C. rothii leaves on ROS production determined by chemiluminescence technique (Firdous S et al. , 2014).
Sample IC 50 ± SD ( µg/mL) Methanol extract (CRM) >100 n-Hexane fraction (HEXCRU) 62.4 ± 7.0 EtOAc fraction (CRME) 29.4 ± 2.8 BuOH fraction (CRMB) 75.4 ± 11.5 Control (Ibuprofen) 11.8 ± 1.2
Nitrite Concentration in Mouse Macrophage Culture Medium:
The mouse macrophage cell line J774.2 (European Collection of Cell Cultures, UK) was cultured in IWAKI’s 75 cc flask (Asahi Techno Glass, Tokyo, Japan) in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich, Steinheim, Germany) that contain 10% fetal bovine serum (GIBCO, New York, USA) supplemented with 1% streptomycin/penicillin. Flasks 6 were kept at 37 °C in humidified air containing 5% CO 2. Cells (10 cells/mL) were then transferred to a 24-well plate. The Nitric oxide synthase (NOS-2) in macrophages was induced by the addition of 30 µg/mL E. coli lipopolysaccharide (LPS) (DIFCO Laboratories Michigan, USA). The test compounds were added at 25 µg/mL concentration. Soon after LPS stimulation cells were re-incubated at 37 °C in 5% CO 2. Finally the cell culture supernatant was collected after 48 h for analysis. Nitrite accumulation in cell culture supernatant was measured (Jacobs F et al., 1998). In brief 50 µL of 1% sulfhanilamide in 2.5% phosphoric
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Table-4.41: Effect of sub-fractions on oxidative burst on whole blood phagocytes and on NO production by mouse macrophages J774.2. Results are expressed as mean % inhibition of three determinations (Firdous S et al. , 2014).
Sub- Inhibition %Inhibition Sub- Inhibition %Inhibition fractions Oxidative Burst Nitric oxide fractions Oxidative Burst on Nitric on Whole Blood (NO) Whole Blood Oxide (NO) Phagocytes 25 µg/mL Phagocytes (ROS) 25 µg/mL (ROS) 20 µg/mL 20 µg/mL CRMHM 43.7 53.37 BA2 28.6 61.36 HS-GC- 23.9 45.89 CRMEE 35.7 19.13 MS HS28 36.4 42.83 C2 34.6 73.62 BCT 33.9 49.82 C6 30.8 65.14 BD 47.3 61.22 CRMEA 20.9 28.68 BCH 21.9 60.5 D1 49.1 59.15 A(KK) 29.3 73.49 D2 41.8 59.79 B(KK) 15.8 85.02 D3 43.3 63.11 BA1 19.4 52.68 D4 47.1 49.43
Table-4.42: IC 50 of compounds against ROS production in whole blood and neutrophils, and effect of pure compounds at 25 µg/mL on NO production by mouse macrophages J774.2. Results are expressed as mean ± SD of three determinations (Firdous S et al. , 2014).
Compound ROS ROS IC 50 % Compound ROS IC 50 ROS IC 50 % No. IC 50 Neutrophils Inhibition No. Whole Neutrophils Inhibition Whole (µg/mL) NO blood (µg/mL) NO blood (µg/mL) (µg/mL) 74 >100 >50 16.27 83 >100 >50 -15.62 26 >100 >50 -3.06 84 >100 >50 12.31 79 >100 >50 -11.52 85 >100 >50 26.49 80 >100 >50 6.66 Ibuprofen* 11.2 ± 1.8 1.2 ± 0.1 - 62 >100 >50 43.09 NG-Methyl - - 65.65 81 >100 >50 9.29 L-arginine 82 18.0 ± 11.3 ± 13.86 acetate** 1.3 0.0
*Control, **standard Inhibitor of nitric oxide synthase used as control
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acid and 50 µL of 0.1% naphtyl-ethylenediamine dihydrochloride in 2.5% phosphoric acid were added to 50 µL of culture medium. After 10 minutes of incubation at room temperature the absorbance was read at 550 nm. Micro molar concentrations of nitrite were calculated from a standard curve, which was generated using sodium nitrite as reference compound.
T-cell Proliferation Bioassay:
Cell proliferation was evaluated by standard thymidine incorporation assay following a reported method (Neilson M & Gerwein J, 1998). Briefly, cells were obtained from peripheral blood of healthy individuals and then cultured at a concentration of 5 × 10 5/mL in a 96-well round bottom IWAKI’s tissue culture plates (Asahi Techno Glass, Tokyo, Japan). Preliminary experiments were conducted to determine the optimum concentration of PHA on T-cell proliferation. PHA concentration of 5µg/mL was found to be optimum and hence used in experiments. Cells were stimulated with 5 g/mL of PHA-P (Sigma Co. St. Louis, USA). 200 µg/mL concentrations of compounds were added, each in triplicate. The plates were incubated ° for 72 h at 37 C in 5% CO 2 incubator. After 72 h, cultures were pulsed (0.5 µCi/well) with tritiated thymidine (Amersham Pharmacia Biotech, Buckinghamshire, UK) and further incubated for 18 h. Cells were harvested onto a glass fiber filter (Connectorate Dietikon, Switzerland) using cell harvester (Ionotech Dottikon, Switzerland). The tritiated thymidine incorporated into the cells was measured by a liquid scintillation counter (LS 6500, Beckman Coulter, USA). Results were expressed as mean count per minute (CPM). The inhibitory activity of compounds on T lymphocyte proliferation was calculated using the formula:
Inhibitory Activity (%) = {Control group (CPM) – Experimental group (CPM)} x100 ______Control group (CPM)
Table-4.43: Effect of sub-fractions of leaves (200 µg/mL) on PHA induced T-cell proliferation using human peripheral blood T Lymphocytes. Results are expressed as mean % inhibition of three readings (Firdous S et al. , 2014).
Sub- % Sub- % Sub- % fractions Inhibition fractions Inhibition fractions Inhibition 200 µg/mL 200 µg/mL 200 µg/mL CRMHM 99.7 A(KK) 99.6 C6 99.6 HS-GC-MS 99.8 B(KK) 99.6 CRMEA 99.5 HS28 99.7 BA1 99.7 D1 87.5 BCT 97.0 BA2 99.7 D2 88.8 BD 99.6 CRMEE 95.1 D3 93.4 BCH 99.7 C2 99.8 D4 -24.5
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Table-4.44: Effect of the pure compounds (12.5 to 50 µg/mL) isolated from the plant on T-cell proliferation. IC 50 values are expressed as mean of three determinations (Firdous S et al. , 2014).
Compound T-cell proliferation Compound T-cell proliferation IC 50 No. IC 50 (µg/mL) No. (µg/mL) 74 >50 81 >50 26 >50 82 14.6 ± 0.3 79 >50 83 >50 80 >50 84 >50 62 27.5 ± 1.9 85 >50
Figure-4.2: Effect of HEXCRU on Phytohamagglutinin (PHA-P) dependant T-cell Proliferation.
CPM = counts per minute +ve = Cell activated with PHA, -ve = Cell without PHA activation
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4.5.2.4 Insecticidal activity:
Insecticidal Activity by Contact Toxicity Method:*
Crude methanol extract of stem (CRS) and leaves (CRM) with its various fractions were loaded on the filter paper which was put in the plate. After evaporation of solvent for 24 h, 10 insects of each species were put in each plate (test and control). The plates were then incubated at 27 ºC for 24 h with 50% relative humidity in growth chamber. The number of survivals of each species was counted and percentage mortality was calculated (table-4.45) (Collins PJ 1998, Champ BR 1981, Tabassum R 1997 and Atta-ur-Rahman et al., 2001).
Table-4.45: Insecticidal activity of methanol extracts of stem and leaves of the plant against different insects.
% Mortality Name of Insects CRS CRM HEXCRU CRME CRMB Tribolium castaneum 0 0 0 0 0 Sitophilus oryzae - - - 100 0 Rhyzopertha dominica 20 0 0 0 0 Callosbruchus analis 0 20 40 60 60 Trogoderma granarium - - - - -
*Insecticidal Activity by Contact Toxicity Method was performed in collaboration with Prof. Dr. M. Iqbal Choudhary at International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi
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4.5.2.5 In Vitro Glycation:
In 96-well plate assay, each well was filled with 60µl reaction mixture consisting of 20µl BSA (10mg/ml), 20µl of glucose anhydrous (50mg/ml), and 20µl test sample (Nakagawa T et al., 2002). Glycated control contained 20µl BSA, 20µl glucose and 20µl sodium phosphate buffer. On the other hand, blank contained 20µl BSA and 40µl sodium phosphate buffer. The reaction mixture was then incubated at 37 °C for 7 days (Yamaguchi F et al., 2000). After 7 days’ incubation, 6µl 100% TCA was added into each well and centrifuged (15000 rpm) at 4 ºC for 4 minutes (Mastuura N et al., 2002). The pellet was rewashed after centrifugation with 60µl 5% ice cold TCA (Yamaguchi F et al., 2000). The supernatant containing glucose, inhibitor and interfering substances was removed. The pellet containing AGEs-BSA was dissolved in 60µl PBS (Mastuura N et al., 2002). Fluorescence spectrum (ex.370nm) was assessed and change in fluorescence intensity (ex.370nm to em 440nm) based on AGEs were monitored using spectrofluorimeter RF-1500 (Mastuura N et al., 2002).
Table-4.46: Antiglycation bioassay of fractions of C. rothii leaves.
Sample % Inhibition IC 50 ± SEM [Mm] Hex. Ext. 77.41 771.8 ± 0.899 µg EtOAc Ext. 51.3 - BuOH Ext. 23.4 -
______
*In Vitro Glycation was performed in collaboration with Prof. Dr. M. Iqbal Choudhary at International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry and Dr. Panjwani Center for Molecular Medicine and Drug Research, University of Karachi
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APPENDIX-I
List of Publications
1. Khan K , Firdous S, Choudhary MI. ( 2008 ) Bioactivities of the Indigenous Cordia Species. Proc. 11 th Int. Symp. Nat. Prod. Chem. Oct. 29 – Nov. 01, University of Karachi, Pakistan. 2. Khan K , Firdous S, Mesaik MA, Choudhary MI. ( 2010) Immunomodulatory Studies on Cordia Species. Proc. 12th Int. Symp. Nat. Prod. Chem. Nov. 22-25, University of Karachi, Pakistan. 3. Khan K , Rasheed M, Firdous S, Faizi S. ( 2012 ) Exploring Nature’s Phytochemical Diversity. Proc.13 th Int. Symp. Nat. Prod. Chem. Sept. 22-25, University of Karachi Pakistan. 4. Firdous S, Khan K , Zikr-Ur-Rehman S, Ali Z, Soomro S, Ahmad VU, Rasheed M, Mesaik MA and Faizi S. ( 2014) Isolation of Phytochemicals from Cordia rothii (Boraginaceae) and Evaluation of their Immunomodulatory Properties. Records of Natural Products, 8, 51-55.
289
APPENDIX-II List of Tables S. no. Content Page #
1.1 Phytochemicals Isolated and Identified from Genus Cordia 11
1.2 Phytochemicals Isolated and Identified from Cordia rothii 73
1.3 Phytochemical Analyses on Genus Cordia using GC-MS Technique 76
3.1 Results of GC-MS Studies on Root, Stem, and Leaves of C. rothii 125
4.1 Compounds identified through GC-MS in 2A (scheme-4.1a) 205
4.2 Compounds identified through GC-MS in 4A (scheme-4.1a) 206
4.3 Compounds identified through GC-MS in KC-PE (scheme-4.1a) 206
4.4 Compounds identified through GC-MS in KC-C (scheme-4.1a) 208
4.5 Compounds identified through GC-MS in 6A (scheme-4.1b) 209
4.6 Compounds identified through GC-MS in KEA-PE (scheme-4.1b) 210
4.7 Compounds identified through GC-MS in KEA-C (scheme-4.1b) 211
4.8 Compounds identified through GC-MS in KA-PE (scheme-4.2) 211
4.9 Compounds identified through GC-MS in KA-C (scheme-4.2) 212
4.10 Compounds identified through GC-MS in KM-PE (scheme-4.3) 213
4.11 Compounds identified through GC-MS in KM-C (scheme-4.3) 214
4.12 Compounds identified through GC-MS in KM-EA (scheme-4.3) 215
4.13 Identification of Chemical Constituents from Root - Summary 216
4.14 Compounds identified through GC-MS in SH (scheme-4.6a) 217
4.15 Compounds identified through GC-MS in SC (scheme-4.6a) 218
4.16 Compounds identified through GC-MS in SEA (scheme-4.6a) 219
4.17 Compounds identified through GC-MS in SME 10% (scheme-4.6a) 220
4.18 Compounds identified through GC-MS in SME 20% (scheme-4.6a) 221
4.19 Compounds identified through GC-MS in SME 30% ABCD 222 (scheme-4.6b)
290
4.20 Compounds identified through GC-MS in SME 30% EFGH 223 (scheme-4.6b) 4.21 Compounds identified through GC-MS in SME 30% IJKL 224 (scheme-4.6b) 4.22 Compounds identified through GC-MS in SME 30% MNO 225 (scheme-4.6b) 4.23 Identification of the Chemical Constituents from Stem - Summary 226
4.24 Compounds identified through GC-MS in HEXCRU (scheme-4.7) 227
4.25 Compounds identified through GC-MS in HS-GC-MS (scheme- 228
4.7)
4.26 Compounds identified through GC-MS in HS28 (scheme-4.8) 230
4.27 Compounds identified through GC-MS in A(KK) (scheme-4.8) 231
4.28 Compounds identified through GC-MS in B(KK) (scheme-4.8) 231
4.29 Identification of Chemical Constituents from Leaves - Summary 232
4.30 In vitro antibacterial bioassay of different fractions of the roots extract of 244
C. rothii against Gram-positive bacteria
4.31 In vitro antibacterial bioassay of different fractions of the roots extract of 245
C. rothii against Gram-negative bacteria
4.32 Minimal inhibitory concentration ( µg/disk) of different fractions of root 246
extract of the plant against Gram-positive bacteria.
4.33 In vitro antibacterial bioassay of crude methanol extract (CRS) of stem 247
and leaves of C. rothii
4.34 In vitro antifungal bioassay of the crude methanol extract of stem and 249
leaves of the plant
4.35 Antileishmanial bioassay of fractions of C. rothii leaves 250
4.36 In vitro phytotoxic bioassay of the crude methanol extract (CRS) of stem 251
and leaves of the plant against fronds
291
4.37 Brine Shrimp ( Artemia salina) Lethality bioassay of different fractions of 252
C. rothii leaves
4.38 Cytotoxicity of extract of leaves of the plant 253
4.39 Antioxidant bioassay of different fractions of C. rothii roots 254
4.40 Effect of methanol extract and fractions of the C. rothii leaves on ROS 256
production determined by chemiluminescence technique
4.41 Effect of sub-fractions on oxidative burst on whole blood phagocytes and 257
on NO production by mouse macrophages J774.2. Results are expressed
as mean % inhibition of three determinations
4.42 IC 50 of compounds against ROS production in whole blood and 257
neutrophils, and effect of pure compounds at 25µg/mL on NO production
by mouse macrophages J774.2. Results are expressed as mean ± SD of
three determinations
4.43 Effect of sub-fractions of leaves (200 µg/mL) on PHA induced T-cell 258
proliferation using human peripheral blood T Lymphocytes. Results are
expressed as mean % inhibition of three readings
4.44 Effect of the pure compounds (12.5 to 50 µg/mL) isolated from the plant 259
on T-cell proliferation. IC 50 values are expressed as mean of three
determinations
4.45 Insecticidal activity of methanol extracts of stem and leaves of the plant 260
against different insects
4.46 Antiglycation bioassay of fractions of C. rothii leaves 261
292
APPENDIX-III List of Figures
S. no. Content Page #
2.1 Nature’s Biosynthetic Diversity Observed in Current Study on Cordia 78
rothii
2.2 Biosynthesis of Saturated Fatty Acids 80
2.3 Generation of Double Bond into the Hydrocarbon Chain 81
2.4 Desaturation in Plant Metabolism 81
2.5 Desaturation in Animal Metabolism 82
2.6 Possible Cerebroside Biosynthesis in Plants 83
2.7 Flow Chart Showing Basic Constitutional Units of Terpenes 84
2.8 Generation of IPP and DMAPP via MVA Pathway 86
2.9 Generation of IPP and DMAPP via MEP Pathway 87
2.10 Biosynthesis of Monoterpene 88
2.11 Biosynthesis of Triterpene 89
2.12 Biosynthesis of Cycloartenol 90
2.13 Biosynthesis of Shikimic Acid 92
2.14 Biosynthesis of Chorismic Acid 93
2.15 Biosynthesis of Prephenic Acid 93
2.16 Biosynthesis of L-Phenylalanine and L-Tyrosine 94
2.17 Biosynthetic Approach for Phenylpropanoids 96
3.1 2′′ -Butoxyethyl 3-[3 ′,5 ′-di( tert -butyl)-4′-hydroxyphenyl]-propanoate, 99
Mairajinol (30 )
3.2 HMBC Correlation of 30 100
3.3 Mass Fragmentation Pattern of 30 100
3.4 Compounds Structurally Related with Mairajinol ( 30 ) 102
293
3.5 Compounds Structurally Related with Mairajinol ( 30 ) 103
3.6 Compound Structurally Related with Mairajinol (30 ) 103
3.7a Naturally Occurring Monoalkylethers of Ethylene Glycol 104
3.7b Compounds Structurally Related with Mairajinol ( 30 ) 104
3.8 Metabolomic Profile of Phytoconstituents Identified in GC-MS Studies 136
3.9a Hydrocarbon, Fatty Acids and Fatty Acid Derivatives Identified in the 130
Root of C. rothii
3.9b Sesquiterpenoids Identified in the Root of C. rothii 131
3.9c Aromatics, Phenolics and Miscellaneous Constituents Identified in the 131
Root of C. rothii
3.9d Triterpenoids and Phytosterols Identified in the Root of C. rothii 132
3.10 Phytochemicals Identified in the Stem of C. rothii 133
3.11 Phytochemicals Identified in the Leaves of C. rothii 134
3.12 Mclafferty Rearrangement Mechanism for Acids 159
3.13 Mclafferty Rearrangement Mechanism for Methyl Esters 160
3.14 Mclafferty Rearrangement Mechanism for Ethyl Esters 160
4.1 Effect of fractions on ROS production by whole blood phagocytes 256
determined by chemiluminescence technique and oxidative burst
study
4.2 Effect of HEXCRU on Phytohamagglutinin (PHA-P) dependant 259
T-cell Proliferation
294
APPENDIX-IV List of Schemes
S. no. Content Page #
3.1 Proposed Biosynthesis of 30 105
3.2a Biogenetic Correlation Amongst Various Phytochemicals Identified 144
During GC-MS Studies
3.2b Biogenetic Relationship Between Different Metabolites 145
3.2c Biogenetic Relation Between Identified Triterpenoids 155
4.1a Extraction, Isolation and Identification of Constituents from C. rothii 178
Roots
4.1b Extraction, and Identification of Constituents from C. rothii Roots 179
4.2 Extraction and Identification of Constituents from C. rothii Roots 180
4.3 Extraction and Identification of Constituents from C. rothii Roots 181
4.4 Isolation of Constituents from C. rothii Roots 182
4.5 Extraction and Isolation of Pure Compound from C. rothii Stem 184
4.6a Extraction, Fractionation and Identification Scheme of C. rothii Stem 185
4.6b Extraction, Fractionation and Identification Scheme of C. rothii Stem 186
4.7 Extraction and Identification of constituents from C. rothii Leaves 188
4.8 Extraction, Isolation and Identification of Constituents from C. rothii 189
Leaves
4.9 Extraction and Isolation of Pure Compounds from C. rothii Leaves 191
4.10 Isolation of Pure Compounds from C. rothii Leaves 192
295