Isolation, Characterization and Biological Evaluation of Secondary Metabolites from Some Related Varieties of Genus Aerva and Halothamnus
A Dissertation Submitted to
Fulfill the Requirement for the Award of Degree of Doctor of Philosophy in Chemistry
By
Sara Musaddiq (M.Sc., M.Phil.)
Department of Chemistry The Islamia University of Bahawalpur 63100-Bahawalpur, Pakistan July 2014
DECLARATION
Sara Musaddiq “Isolation,
Characterization and Biological Evaluation of Secondary Metabolites from Some
Related Varieties of Genus Aerva and Halothamnus ”
Sara Musaddiq
July 2014
CERTIFICATE
Sara Musaddiq
Aerva Halothamnus
Dr. Abdul Jabbar Chairman
ACKNOWLEDGEMENTS
All praises for Almighty ALLAH, the most merciful and the most beneficent who showered upon me His blessings throughout my efforts for the completion of this research work. He guides us in the darkness and helps us in difficulties. All respects and praises for the Holy Prophet MUHAMMAD (PBUH) who enabled us to recognize our creator. He also showed us the right path and gave a complete code of life.
It is with a profound sense of gratitude, I would like to express my sincere thanks to my supervisor and esteemed guide Professor Professor Dr. Abdul JabbarJabbar, The
Islamia University of Bahawalpur, for his personal care, understanding, stimulating discussion with humble and inspiring guidance throughout the entire studies.
I would like to express my sincere thanks to my teachers, Dr. Muhammad
Saleem and DDDr.Dr. Naheed Riaz for their suggestions, deep involvement, constant and valuable assistance throughout my research work. I am also very grateful to Dr.
Muhammad Saleem for his scientific advice and knowledge and many insightful discussions and suggestions. He is my primary resource for getting my science questions answered and was instrumental in helping me crank out this thesis. Infact he is one of the smartest people I know.
I am grateful and feel pleasure in expressing my thanks to Prof. Dr. Ross
McgearyMcgeary, School of Chemistry and Molecular Biosciences, University of Queensland, Australia for his valuable suggestions and to provide various research facilities at his institute.
I must pay thanks to the Chairman, Chemistry and Dean Sciences of IUB for providing necessary facilities to complete my research work. I also acknowledge the
Director ICCBS, University of Karachi for providing spectroscopic analysis facilities.
I am very thankful to Prof. Dr. Faiz5ul5Hassan Nasim and Prof. Dr.
Muhammad Ashraf, Department of Biochemistry, Biotechnology for performing various bioassays of our pure compounds.
I take this opportunity, to express my heartfelt thanks to Nusrat, Bushra,
Shehla, Naseem, Rizwana, Jallat, Imran, Akram, Basharat sb, Abdul Ghaffar, Iftikhar and Shabir sb for their kind suggestions and generous help rendered throughout my Ph. D studies. Also I can not ignore my little fellows Momina and Mehwish, as we had real fun together. If I have forgotten anyone, I apologize.
I also acknowledge the HEC for financial support. I also wish to thank the technical staff, non5teaching and clerical staff, for their constant support and timely help.
The best outcome from previous years is my best friend, soul5mate, Sajjad
Ahmad, my husband and our kids Asim and Fatima as their smile of love and tolerance of partial depart provided me an additional energy for this completion.
These past several years have not been an easy ride, both academically and personally. I truly thank Sajjad for sticking by my side, even when I was irritable and depressed. I feel that what we both learned a lot about life and strengthened our commitment and determination to each other and to live life to the fullest.
I offer my sincere thanks to my mother5in5law, brother, brothers5in5law and their wives, sisters, sisters5in5law and their husbands, with all of their children for their zeal and support, as their love and possible support are the sources of my inspiration. I would like to thank all my friends and every single soul that has made a contribution in my life.
How I can say thanks to my hard5working parents who have sacrificed their lives for us and provided unconditional love and care. I love them so much, and I would not have made it this far without them.
I will forever be thankful to my friends, my nephew Wasim and Naeem and my niece Saima, Gul Jabeen and Gulshan who really contributed a lot in my life, my real friends, without their help I could never complete my task. I wish these younger fellows the best of luck in the future life.
Sara Musaddiq
Dedicated to
`ç uxÄÉäxw [âáutÇw ft}}tw T{Åtw 9 `ç fãxxà ^|wá Tá|Å ft}}tw? Ytà|Åt ft}}tw
CONTENTS
Summary Chapter 1 Error! Bookmark not defined. Introduction to Natural Products and Their Role in Human Life Error! Bookmark not defined. 1.1. Introduction Error! Bookmark not defined. 1.2. Main Classes of Secondary Metabolites Error! Bookmark not defined. 1.2.1. Terpenes Error! Bookmark not defined. 1.2.2. Alkaloids Error! Bookmark not defined. 1.2.3. Phenolics Error! Bookmark not defined. 1.3. Drugs Based on Natural Origin Error! Bookmark not defined. 1.3.1. Role of Plants in Human Health Error! Bookmark not defined. 1.3.2. Natural Products Isolated from Plants Error! Bookmark not defined. 1.3.3. Natural Products Isolated from Microorganisms Error! Bookmark not defined. 1.3.4. Natural Products Isolated from Marine Sources Error! Bookmark not defined. 1.3.5. Natural Products Isolated from Animal Sources Error! Bookmark not defined. Chapter 2 Error! Bookmark not defined. Phytochemical and Pharmacological Studies on the Genus Aerva Error! Bookmark not defined. 2.1. Family Amaranthaceae Error! Bookmark not defined. 2.1.1. Medicinal Importance of the Family Amaranthaceae Error! Bookmark not defined. 2.2. The Genus Aerva Error! Bookmark not defined.
i
2.2.1. Medicinal Importance of the Genus Aerva Error! Bookmark not defined. 2.2.2. Pharmacological Studies of the Aerva Plants Error! Bookmark not defined. 2.2.2.1. Antimicrobial Activity of the Aerva Plants Error! Bookmark not defined. 2.2.2.2. Diuretic Activity of the Aerva Plants Error! Bookmark not defined. 2.2.2.3 . Immunomodulatory and Anti tumour Activity of the Aerva Plants Error! Bookmark not defined. 2.2.2.4 . Hepatoprotective Activity of the Aerva Plants Error! Bookmark not defined. 2.2.2.5 . Antidiabetic Activity of the Aerva Plants Error! Bookmark not defined. 2.2.2.6. Antioxidant Activity of the Aerva Plants Error! Bookmark not defined. 2.2.2.7. Anti lithiatic Activity of the Aerva Plants Error! Bookmark not defined. 2.2.2.8. Antifungal Activity of the Aerva Plants Error! Bookmark not defined. 2.2.2.9 . Nephroprotective Activity of the Aerva Plants Error! Bookmark not defined. 2.2.2.10. Antiulcer Activity of th Aerva Plant Error! Bookmark not defined. 2.2.2.11. Miscellaneous Activities of the Aerva Plants Error! Bookmark not defined. 2.3. Phytoconstituents Isolated from the Genus Aerva Error! Bookmark not defined. 2.4 Aerva Javanica var. bui Error! Bookmark not defined.
ii
2.4.1. Scientific Classification of Aerva Javanica Error! Bookmark not defined. 2.4.2. Botanical Description of Aerva Javanica Error! Bookmark not defined. 2.4.3. Medicinal Importance of Aerva Javanica Error! Bookmark not defined. 2.4.4. Nutritional Value and Elemental Analysis of Aerva Javanica Error! Bookmark not defined. Chapter 3 Error! Bookmark not defined. Biosynthesis of Flavonoids and Steroids Error! Bookmark not defined. 3.1. Biosynthesis of Natural Products Error! Bookmark not defined. 3.2. What are Flavonoids? Error! Bookmark not defined. 3.2.1. Biosynthesis of Flavonoids Error! Bookmark not defined. 3.2.2. Glycosylation of Flavonoids Error! Bookmark not defined. 3.3. Biosynthesis of Steroids Error! Bookmark not defined. 3.3.1. What are Ecdysteroids? Error! Bookmark not defined. 3.3.2. Biosynthesis of Ecdysteroids Error! Bookmark not defined. Chapter 4 Error! Bookmark not defined. Results and Discussions of Metabolites Isolated from Aerva Javanica Error! Bookmark not defined. 4.1. Results and Discussion Error! Bookmark not defined. 4.2. Structure Elucidation of the Isolated Compound Error! Bookmark not defined. 4.2.1. Structure Elucidation of Aervecdysone A ( 222 )Error! Bookmark not defined. 4.2.2. Structure Elucidation of Aervecdysone B ( 223 )Error! Bookmark not defined. 4.2.3. Structure Elucidation of Aervecdysone C ( 224 )Error! Bookmark not defined. 4.2.4. Structure Elucidation of Aervecdysone D (225 )Error! Bookmark not defined.
iii
4.2.5. Structure Elucidation of 24-Epi-makisterone A (226 )Error! Bookmark not defined. 4.2.6. Structure Elucidation of 5-β-2-deoxyintegristerone A (227 ) Error! Bookmark not defined. 4.2.7. Structure Elucidation of β-ecdysone (215 )Error! Bookmark not defined. 4.2.8. Structure Elucidation of Kaempferol-3-O-β-D-[4 ′′′-p-coumaroyl-α-L- rhamnosyl(1→6)]galactoside ( 228 ) Error! Bookmark not defined. 4.2.9. Structure Elucidation of Kaempferol-3-O-β-D-[4 ′′′-p-coumaroyl-α-L- rhamnosyl(1→6)]3 ′′-p-coumaroylgalactoside ( 229 )Error! Bookmark not defined. 4.2.10. Structure Elucidation of Kaempferol-3-O-β-D-[4 ′′′-p-coumaroyl-α-L- rhamnosyl(1→6)]4 ′′-p-coumaroylgalactoside ( 230 )Error! Bookmark not defined. 4.2.11. Structure Elucidation of kaempferol-3-O-β-D (6-E-p-coumaroyl) glucoside ( 231 ) Error! Bookmark not defined. 4.2.12. Structure Elucidation of Aervfuranoside (232 )Error! Bookmark not defined. 4.2.13. Structure Elucidation of Allantoin ( 233 ) Error! Bookmark not defined. 4.2.14. Structure Elucidation of Mannitol ( 234 )Error! Bookmark not defined. 4.2.15. Structure Elucidation of 1-O-β-D-glucopyranosyl-(2 S,3 S,4 R,8 Z)-2- [(2 R)-2-hydroxyPentacosanoylamino]-8-octadecene-1,3,4-triol (235 )
Error! Bookmark not defined. 4.2.16. Structure Elucidation of (14 E)-2-[(2 R)-2-hydroxyoctadecanoyl] aminotetraeicos-14-ene-1,3,4-triol-1-O-β-D-glucopyranoside ( 236 ) Error! Bookmark not defined.
iv
4.2.17. Structure Elucidation of β-sitosterol ( 106 )Error! Bookmark not defined. 4.2.18. Structure Elucidation of β-sitosterol 3-O-β-D-Glucopyranoside ( 237 )
Error! Bookmark not defined. 4.2.19. Structure Elucidation of Oleanolic Acid ( 238 )Error! Bookmark not defined. 4.2.20. Structure Elucidation of Lupeol ( 113 ) Error! Bookmark not defined. 4.2.21. Structure Elucidation of Hexadecanoic Acid ( 239 )Error! Bookmark not defined. 4.2.22. Structure Elucidation of Gallic Acid ( 240 )Error! Bookmark not defined. 4.2.23. Structure Elucidation of Caffeic Acid ( 241 )Error! Bookmark not defined. 4.2.24. Structure Elucidation of p- Coumaric Acid ( 242 )Error! Bookmark not defined. 4.2.25. Structure Elucidation of Hexadecyl Ferulate ( 243 )Error! Bookmark not defined. 4.2.26. Structure Elucidation of Hexacosyl Ferulate ( 244 )Error! Bookmark not defined. 4.2.27. Structure Elucidation of Eicosanyl trans-p-coumarate ( 245 ) Error! Bookmark not defined. 4.2.28. Structure Elucidation of 1H-Indole-3-carboxylic Acid ( 246 ) Error! Bookmark not defined. 4.2.29. Structure Elucidation of Tricontanol ( 247 )Error! Bookmark not defined. 4.3. Biological Studies of the Compounds Isolated from Aerva javanica Error! Bookmark not defined. Chapter 5 Error! Bookmark not defined.
v
Experimental Error! Bookmark not defined. 5.1. General Experimental Procedures Error! Bookmark not defined. 5.2. Collection and Identification of the Plant Material Error! Bookmark not defined. 5.3. Extraction of the Plant Material and Isolation Error! Bookmark not defined. 5.4. Spectroscopic Data of the Isolated CompoundsError! Bookmark not defined. 5.4.1. Spectroscopic Data of Aervecdysone A ( 222 )Error! Bookmark not defined. 5.4.2. Spectroscopic Data of Aervecdysone B ( 223 )Error! Bookmark not defined. 5.4.3. Spectroscopic Data of Aervecdysone C ( 224 )Error! Bookmark not defined. 5.4.4. Spectroscopic Data of Aervecdysone D ( 225 )Error! Bookmark not defined. 5.4.5. Spectroscopic Data of 24-Epi-makisterone A ( 226 )Error! Bookmark not defined. 5.4.6. Spectroscopic Data of 5-β-2-Deoxyintegristerone A ( 227 ) Error! Bookmark not defined. 5.4.7. Spectroscopic Data of β-ecdysone ( 215 ) Error! Bookmark not defined. 5.4.8. Spectroscopic Data of Kaempferol-3-O-β-D-[4 ′′′-p-coumaroyl-α-L rhamnosyl(1→6)]galactoside ( 228 ) Error! Bookmark not defined.
5.4.9. Spectroscopic Data of Kaempferol-3-O-β-D-[4 ′′′-p-coumaroyl-α-L- rhamnosyl(1→6)]3 ′′-p-coumaroylgalactoside ( 229 ) 135 5.4.10. Spectroscopic Data of Kaempferol-3-O-β-D-[4 ′′′-p-coumaroyl-α-L- rhamnosyl(1→6)]4 ′′-p-coumaroyl galactoside ( 230 )Error! Bookmark not defined.
vi
5.4.11. Spectroscopic Data of Kaempferol 3-O-β-D (6-E-p-coumaroyl) glucoside ( 231 ) Error! Bookmark not defined. 5.4.12 Spectroscopic Data of Aervfuranoside ( 232 )Error! Bookmark not defined. 5.4.13. Spectroscopic Data of Allantoin ( 233 ) Error! Bookmark not defined. 5.4.14. Spectroscopic Data of Mannitol ( 234 ) Error! Bookmark not defined. 5.4.15. Spectroscopic Data of 1-O-β-D-Glucopyranosyl-(2 S,3 S,4 R,8 Z)-2-[(2 R)- 2-hydroxy Pentacosanoyl amino]-8-octadecene-1,3,4-triol ( 235 ) Error! Bookmark not defined. 5.4.16. Spectroscopic Data of(14 E)-2-[(2 R)-2hydroxyoctadecanoyl] amino tetraeicos-14-ene-1,3,4-triol-1-O-β- D-glucopyranoside (236 ) Error! Bookmark not defined. 5.4.17. Spectroscopic Data of β-Sitosterol ( 106 ) Error! Bookmark not defined. 5.4.18.Spectroscopic Data of β-Sitosterol 3-O-β-D-Glucopyranoside ( 237 ) Error! Bookmark not defined. 5.4.19. Spectroscopic Data of Oleanolic Acid ( 238 )Error! Bookmark not defined. 5.4.20. Spectroscopic Data of Lupeol ( 113 ) Error! Bookmark not defined. 5.4.21. Spectroscopic Data of n-Hexadecanoic Acid ( 239 )Error! Bookmark not defined. 5.4.22. Spectroscopic Data of Gallic Acid (240 ) Error! Bookmark not defined. 5.4.23. Spectroscopic Data of Caffeic Acid ( 241 ) Error! Bookmark not defined. 5.5.24. Spectroscopic Data of p-Coumaric Acid ( 242 )Error! Bookmark not defined. 5.4.25. Spectroscopic Data of Hexadecyl Ferulate ( 243 )Error! Bookmark not defined. 5.4.26. Spectroscopic Data of Hexacosyl Ferulate ( 244 )Error! Bookmark not defined.
vii
5.4.27. Spectroscopic Data of Eicosanyl trans-p-coumarate ( 245 ) Error! Bookmark not defined. 5.4.28. Spectroscopic Data of 1H-Indole-3-carboxylic Acid ( 246 ) Error! Bookmark not defined. 5.4.29. Spectroscopic Data of Tricontanol ( 247 ) Error! Bookmark not defined. 5.5. DPPH Free Radical Scavenging Assay Error! Bookmark not defined. 5.6. Enzyme Inhibitory Assay Error! Bookmark not defined. 5.6.1. Acetylcholinesterase Assay Error! Bookmark not defined. 5.6.2. Butyrylcholinesterase Assay Error! Bookmark not defined. 5.6.3. Lipoxygenase Assay Error! Bookmark not defined. Chapter 6 Error! Bookmark not defined. Phytochemistry of Halothamnus Auriculus Error! Bookmark not defined. 6.1. The Genus Halothamnus Error! Bookmark not defined. 6.1.1. Distribution of the Genus Halothamnus Error! Bookmark not defined. 6.1.2. Importance of the Genus Halothamnus Error! Bookmark not defined. 6.2. Halothamnus Auriculus Error! Bookmark not defined. 6.2.1. Botanical Description of Halothamnus Auriculus Error! Bookmark not defined. 6.2.2. Classification of Halothamnus Auriculus Error! Bookmark not defined. 6.2.3. Previous Phytochemical studies of Halothamnus Auriculus Error! Bookmark not defined. 6.3. Results and Discussions of the Isolated Compounds Error! Bookmark not defined. 6.3.1. Structure Elucidation of Allantoic Acid (248 )Error! Bookmark not defined. 6.3.2. Structure Elucidation of Quercetin 3-glucoside ( 249 )Error! Bookmark not defined.
viii
6.3.3. Structure Elucidation of 8-C-glucopyranosylapigenin ( 250 ) Error! Bookmark not defined. 6.3.4. Structure Elucidation of 5, 6-Dihydroxy-3′, 4′,7-Trimethoxy Flavone ( 251 ) Error! Bookmark not defined. 6.3.5. Structure Elucidation of 4′, 5, 7-Trihydroxy-3′, 6-dimethoxy Flavone (252 ) Error! Bookmark not defined. 6.3.6. Structure Elucidation of Quercetin 3′,4′-dimethyl ether ( 253 ) Error! Bookmark not defined. 6.4. General Experimental Error! Bookmark not defined. 6.4.1. Collection and Identification of Plant Error! Bookmark not defined. 6.4.2. Extraction and Isolation Error! Bookmark not defined. 6.5. Spectroscopic Data of the Isolated Compounds Error! Bookmark not defined. 6.5.1. Spectroscopic Data of Allantoic Acid ( 248 )Error! Bookmark not defined. 6.5.3. Spectroscopic Data of Quercetin-3-glucoside ( 249 )Error! Bookmark not defined. 6.5.4. Spectroscopic Data of 8-C-glucopyranosylapigenin ( 250 ) Error! Bookmark not defined. 6.5.5. Spectroscopic Data of 5, 6-dihydroxy-3′, 4′,7-trimethoxy Flavone (251 ) Error! Bookmark not defined. 6.5.6. Spectroscopic Data of 4′, 5, 7-trihydroxy-3′, 6-dimethoxy Flavone ( 252 )
Error! Bookmark not defined. 6.5.7. Spectroscopic Data of Quercetin 3′,4′-dimethyl Ether ( 253 ) Error! Bookmark not defined. 6.5.8. Spectroscopic Data of β-Sitosterol 3-O-β-D-glucopyranoside ( 237 ) Error! Bookmark not defined.
ix
6.5.9. Spectroscopic Data of Oleanolic Acid ( 238 )Error! Bookmark not defined. 6.5.10. Spectroscopic Data of β-Sitosterol ( 106 ) Error! Bookmark not defined. 6.5.11. Spectroscopic Data of Lupeol ( 113 ) Error! Bookmark not defined. References Error! Bookmark not defined.
LIST OF TABLES
Table 4.1: 1H- and 13 C-NMR data of 222 and 223 (CDCl 3, 400 and 100 MHz) 74 Table 4.2: 1H- and 13 C-NMR data of 224 and 225 (DMSO-d6 , 400 and 100 MHz) 79
Table 4.3: 1H- and 13 C-NMR data of 226 (C 5D5N, 500 MHz) 227 and 215 (DMSO, 400 MHz) 83
Table 4.4: 1H- and 13 C-NMR data of 228 (CD 3OD; 500, 125 MHz) 88
1 13 Table 4.5: H- and C-NMR data of 229 and 230 (CD 3OD; 500, 125 MHz) 93
Table 4.6: 1H- and 13 C-NMR data of 231 (CD 3OD; 500, 125 MHz) 95
Table 4.7: 1D and 2D spectral data of 232 (DMSO-d6, 400 and 100 MHz) 99 Table 4.8: Enzyme inhibitory activities of isolated compounds ( 215 , 222-232 ) from Aerva javanica 117 Table 4.9: DPPH free radical scavenging potential of the isolated compounds (215 , 222-233 ) from Aerva javanica 118
Table 6.1: 1H and 13 C NMR data of 249 (CD 3OD , 400 and 100 MHz) and 250 (DMSO, 400 and 100 MHz) 165
1 13 Table 6.2: H- and C-NMR data of 251, 252 and 253 (CD 3OD, 400 MHz) 170
LIST OF FIGURES
Figure 3.1: Flavonoids with 1, 3-diphenyl propane skeleton 44 Figure 3.2: Basic skeleton of chalconoids 45 Figure 3.3: Basic skeleton of auronoids 46 Figure 3.4: Basic skeletons of flavonoids with 1, 3-diphenyl propane 46 Figure 3.5: Flavonoids with 1, 2-diphenyl propane skeleton 47
x
Figure 3.6: Flavonoid with 1,1-diphenyl propane nucleus 47 Figure 3.7: Basic Skeletons of Homo Isoflavonoids 48 Figure 3.8: Enzyme catalaysed glycosylation of flavonoids 54 Figure 4.1: A] Mass fragmentation and B] important HMBC and COSY correlations observed in the spectra of 222 70 Figure 4.2: NOESY correlation observed in the spectrum of 222 71 Figure 4.3: A] Mass fragmentation and B] important HMBC and COSY correlations observed in the spectra of ( 223) 73 Figure 4.4: A] Mass Fragmentation and B] important HMBC and COSY correlations observed in the spectra of 224 76 Figure 4.5: A] Mass fragmentation and B] important HMBC and COSY correlations observed in the spectra of 225 78 Figure 4.6: Important HMBC correlations observed in the spectra of 227 81 Figure 4.7: Important HMBC and COSY correlations observed in the spectra of 228 86 Figure 4.8: Important HMBC and COSY Correlations observed in the spectra of 229 90 Figure 4.9: Important HMBC and COSY correlations observed in the spectra of 230 92 Figure 4.10: Important HMBC and COSY correlations observed in the spectra of 231 95 Figure 4.11: Important HMBC and COSY correlations observed in the spectra 98 Figure 4.12: Important HMBC correlations observed in the spectrum of 233 100 Figure 4.13: Important HMBC and COSY correlations observed in the spectra of 234 101 Figure 4.14: Important HMBC and COSY correlations observed in the spectra of 235 103 Figure 5.1: Purification Protocol of Compounds from Aerva javanica 125 Figure 6.1: Leaf and Fruit of Halothamnus auriculus 159 Figure 6.2: HMBC correlations observed in the spectrum of 248 160 Figure 6.3: HMBC correlations observed in the spectrum of 249 162 Figure 6.4: Important HMBC and COSY correlations observed in the spectra of 250 164 Figure 6.5: HMBC correlations observed in the spectrum of 251 167 Figure 6.6: HMBC correlations observed in the spectrum of 25 2 168 Figure 6.7: HMBC correlations observed in the spectrum of 253 169 Figure 6.8: Isolation Scheme of secondary metabolites from Halothamnus auriculus 173
xi
LIST OF SCHEMES
Scheme 3.1: Synthetic route of shikimate 49 Scheme 3.2: Conversion of shikimate into 4-hydroxycoumaric acid 50 Scheme 3.3: Biosynthetic route for the synthesis of chalcone 51 Scheme 3.4: Enzymatic conversion of chalcone into flavanone (naringenin) 52 Scheme 3.5: Conversion of flavanone into flavone (apigenin) 52 Scheme 3.6: Enzymatic conversion of flavanone into flavonol (kaempferol) 53 Scheme 3.7: Mevalonate pathway; formation of IPP and DMAPP 56 Scheme 3.8: Mevalonate pathway; formation of farnesyl pyrophosphate 57 Scheme 3.9: Conversion of presqualene into squalene 57 Scheme 3.10: Formation of lanosterol and cycloartenol 58 Scheme 3.11: Conversion of lanosterol into zymosterol 59 Scheme 3.12: Conversion of zymosterol into cholesterol 60 Scheme 3.13: Different routes for the formation of ecdysone 63 Scheme 3.14: Mechanism for introduction of 6 ketonic group in ecdysones 64
xii
Summary
Natural product chemistry has experienced explosive and diversified growth, making natural products the subject of much interest and promise in the present day research directed towards drug design and discovery. It is noteworthy that natural products are a source of new compounds with diversified structural arrangements possessing interesting biological activities. Natural products, thus, have played and continue to play an invaluable role in the drug discovery process.
The work embodied in this dissertation deals with isolation of chemical entities from natural sources. In the present work, we selected two medicinal plants of family Amaranthaceae: Aerva javanica and Halothamnus auriculus to explore for their secondary metabolites. The whole dissertation has six chapters.
In Chapter 1 some basic information about natural products and classification of secondary metabolites is discussed. It also deals with important bioactive secondary metabolites and drugs derived from different natural sources.
Chapter 2 incorporates traditional medicinal uses of some species belonging to family Amaranthaceae and genus Aerva . Pharmacological studies and
i
phytochemical survey on various species of the genus Aerva are included in the same chapter. In Literature survey it was found that most of the phyto constituents isolated from genus Aerva belongs to flavonoids and steroids so biosynthesis of these two classes of compounds is discussed in chapter 3 .
Chapter 4 covers detailed structure elucidation of isolated compounds from
Aerva javanica , while in chapter 5 experimental procedures are discussed. The methanolic extract of Aerva javanica showed antibacterial, antioxidant and enzyme inhibitory properties, therefore, the extract was partitioned and ethylacetate fraction was subjected to chromatographic purification. As a result eight new and twenty one known compounds were purified (106 , 113,
215 and 222-247 ). The structures of the new compounds were established due to 1D, 2D NMR and HREIMS and HRFABMS techniques. The structures of known compounds were determined by spectroscopic analyses and in comparison with the literature. Among the isolated compounds
Aervecdysone A-D ( 222-225 ) are ecdysteroids which are well studied as plant and insect growth factors, and derived their name “ecdy ” from the process of molting in insects, called ecdysis. Along with four new ones three known ecdysteroids: 24-epi -makisterone A (226 ), 5-β-2-deoxyintegristerone A (227 ) and β-ecdysone (215 ) are also isolated for the first time from Aerva javanica .
ii
Characterization of these compounds has been published in a peer-reviewed journal [1].
OH HO HO HO
OH O O OH HO HO H OH H OH H OH HO HO HO H H H O O 222 O 223 224
OH HO OH HO HO OH OH OH OH OH OH HO H OH H OH H OH HO H HO HO O H H O O 227 225 226
OH HO
OH
HO H OH HO H O 215
1. Muhammad Saleem, Sara Musaddiq , NaheedRiaz, Muhammad Ashraf, RumanaNasar, and Abdul Jabbar “Ecdysteroids from the flowers of Aerva Javanica , Steroids 78 (2013) 1098–1102 .
The publication has been attached in the end as Annexure-1.
From the same source three new acylated flavonoid glycosides i.e.,
Kaempferol-3-O-β-D-[4 ′′′-p-coumaroyl-α-L rhamnosyl(1→6)]galactoside (228 ),
iii
Kaempferol-3-O-β-D-[4 ′′′-p-coumaroyl-α-L-rhamnosyl(1→6)]3 ′′-p-coumaroyl galactoside (229 ) and Kaempferol-3-O-β-D-[4 ′′′-p-coumaroyl-α-L- rhamnosyl(1→6)]4 ′′-p-coumaroyl galactoside (230 ) are isolated which have been published in another peer reviewd journal [2] along with six known but first time isolated phenolics from this source i.e., gallic Acid ( 240 ), caffeic acid ( 241 ), p-coumaric acid ( 242 ), hexadecyl ferulate ( 243 ), hexacosyl ferulate ( 244 ) and eicosanyl trans-p-coumarate ( 245 ).
OH OH OH HO O HO O HO O O OH O O O OH OH O OH O HO OH OH O HO HO O O HO O HO HO OH O O O OH O O O O O O O O O O HO O O HO 228 229 230
O OH OH O OH O OH OH OH OH OH HO OH HO HO OH OH 240 241 242 O O O O O 16' O
HO 243 HO 244 HO 245 OMe
Sara Mussadiq , NaheedRiaz, Muhammad Saleem, Muhammad Ashraf, Tayaba Ismail and Abdul Jabbar, “ New Acylated Flavonoid Glycosides From Flowers of Aerva javanica”Journal of Asian Natural Product Research, 15 (7), 708–716, 2013 .
The publication is attached in the end as Annexure-2.
iv
Compound 232 is a dibenzofuran glycoside and it is also a new secondary metabolite which is in the process of publication along with some other known secondary metabolites from Aerva javanica .
OH
HO O OH O HO OH O O OH O O H H 231 HO OH HO 106 113
OH O OH HO OH O HO OH OH OH HN HO O O O OH NH HO N O O H OH OH HO O Cl H2N OMe OMe 234 232 233
O OH O OH HN OH 11 HN 18 OH OH O OH HO O O O HO 7 7 HO OH HO 7 OH OH OH 236 235
11
239 OH O OH OH O O HO HO HO O 27 N OH 237 238 H 247 246
Among known compounds Kaempferol 3-O-β-D (6-E-p-coumaroyl) glucoside
(231), allantoin (233), 1-O-β-D-Glucopyranosyl-(2S,3S,4R,8Z)-2-[(2R)-2-hydroxy
v
Pentacosanoyl amino]-8-octadecene-1,3,4-triol (235), (14E)-2-[(2R)-2 hydroxyoctadecanoyl] aminotetraeicos-14-ene-1,3,4-triol-1-O-β-D-glucopyranoside
(236 ), hexadecanoic acid ( 239 ), 1H-Indole-3-carboxylic acid (246) and tricontanol
(247) are reported for the first time from this source.
Chapter 6 deals with the botanical description, occurrence, common usage and medicinal properties of genus Halothamnus and Halothamnus auriculus .
This specie is explored for the first time for its chemical constituents. The experimental procedures and structure elucidation of isolated compounds are discussed in the same chapter. As a result of partitioning of the methanolic extract and chromatography of ethyl acetate soluble fraction resulted in the isolation of eleven metabolites including β-sitosterol (106) ,
Lupeol (113) , allantoin (233), β-sitosterol 3-O-β-D-glucopyranoside (237) , Oleanolic
Acid (238), allantoic acid (248), quercetin 3-glucoside (249), 8-C- glucopyranosylapigenin (250), 5, 6-dihydroxy-7, 3′, 4′-trimethoxy flavone (251) , 4′,
5, 7-trihydroxy-3′, 6-dimethoxy flavones (252) and Quercetin 3′,4′-dimethyl ether
(253) . Their structures were established due to detailed 1D and 2D spectroscopic analyses and comparing with the reported literature. Due to limited lab facilities only new isolates were studied for antioxidant and enzyme inhibitory studies, unfortunately none of the tested compounds was found potent.
vi
OH OH HO O O OH HO HO O OH O O OH OH HO O O H2N N N NH2 H H OH O OH 248 249 O 250 OH O OH HO OH
OMe OMe OH HO O HO O MeO O OMe OMe OMe OH HO MeO OH O OH O OH O 251 252 253
The whole work is in process of publication.
vii
CHAPTER 111
Introduction to Natural Products and Their Role in Human Life
Page 1
1.1. Introduction
Natural products are the compounds synthesized by living organisms.
These compounds may be novel chemo types themselves or sources of other medicinal agents. Impressive number of modern drugs is originated from natural sources which inspired researchers to explore nature and discover precious components hidden in nature for benefit of humanity. Origin of natural product lies in plants, animals and microbes either terrestrial or marine (Nakanishi, 1999). Natural metabolites may be primary or secondary which are not easily distinguishable on the basis of chemical structures or biochemistry e.g., kaurenoic acid ( 1) is an intermediate formed in gibberellins synthesis which is plant growth hormone and abietic acid ( 2) is a resin component which is mostly found in members of the family Fabaceae and
Pinaceae. Both these compounds belong to diterpene series. Former is classified as primary while later as secondary metabolite.
Another example is of amino acid proline ( 3) which is primary metabolite, whereas pipecolic acid ( 4) is classified as secondary metabolite.
OH OH N N H COOH COOH O H O 1 2 3 4
Page 2
As there is no valid distinction between metabolites, they can be classified according to their function. Primary metabolites are products of fundamental metabolic pathways. Functions of primary metabolites are essential for growth and development and therefore occur in all organisms.
Examples include carbohydrates, amino acids, peptides, proteins, nucleic acids, lipids etc. Secondary metabolites are not directly involved in growth and development or other primary function and are limited in distribution.
Living beings produce thousands of these chemicals belonging to different classes. Different organisms produce specific types of secondary metabolites as they desire.
1.2. Main Classes of Secondary Metabolites
1.2.1. Terpenes
Building blocks of terpenoids are five carbon branched skeleton called isoprene unit. Compounds made of a single unit are called hemiterpenes e.g., isoprenol (5), isovaleramide ( 6). C-10 terpenoids contain two isoprene units and are called monoterpenes. Usually monoterpenoids are a complex mixture called essential oils or volatile oils, which are used as flavors and fragrances and in aromatherapy.
Menthol ( 7) usually isolated from mint oils and camphor ( 8) found in camphor laurel , are examples of monoterpenoids. Some Sesquiterpenes (C-15
Page 3
skeleton) act as antifeedents and phytoalexins e.g., capsidiol (9) is a phytoalexin which plants produce against pathogens (Hammerschmidt,
1999). Gossypol ( 10 ) acts as dehydrogenase enzyme inhibitor and also possess antimalarial properties (Polsky et al., 1989 ). Artemisinin ( 11 ) is another anti malarial agent (White, 1997) isolated from plant Artemisia annua .
Example of diterpene (C-20 skeleton) include forskolin ( 12 ) which is a remedy for glaucoma (Wagh et al., 2012), whereas, 4-acetoxydictylactone ( 13 ) possesses antitumor activity (Faulkner, 1988; Ishitsuka et al., 1988). Another diterpene; phorbol ( 14 ) is a tumor promoter (Blumberg, 1988).
O
OH NH2 OH 5 6 O 8 7
H OH O O OH O OH O HO OH H H HO O OH HO O 11 O 9 10 OH O OH OH OAc O H OH OH H O O HO H O O OH OH O 12 13 14
Page 4
The sesterpenes (C-25 skeleton) show antimicrobial compounds fasciospongines A ( 15 ) (Yao and Chang, 2007) and 19-oxofascio-spongine A
(16 ) (Yao et al., 2009). The triterpenes (C-30 skeleton) include anti-HIV agent betulinic acid ( 17 ) (Sun et al., 1998) , Ambrein ( 18 ), a triterpene alchohol, is an analgesic compound (Taha, 1992) while retinol ( 19 ) and β- carotene ( 20 ) are examples of Tetraterpenes (C-40 skeleton) (Arnum, 1998).
HO3SO O SO 3 H O N O N O H COOH
N N HO 15 NH 16 NH 17
OH HO 19
18
20
1.2.2. Alkaloids
Alkaloids are nitrogen containing bioactive compounds produced by almost every class of organisms e.g., plants, animals, microbes etc. Generally these compounds are chemical defenses of organisms e.g., toxic alkaloids are produced by secretory glands or skin of amphibians (Toledo and Jared, 1995).
Some feeding deterrent alkaloids are nicotine ( 21 ), present in tobacco.
Page 5
(Henningfield and Zeller, 2006). Rauwolfia serpentine is a source of an anti arrhythmic alkaloid ajmaline ( 22 ) (Siddiqui and Siddiqui, 1931). Atropine ( 23 ) is isolated from Hyoscyamus niger, it acts as anticholinergic (Mein, 1831).
Caffeine ( 24 ) stimulate CNS and is produced by a number of plants e.g.,
Coffea arabica (Nehlig et al., 1992) . Pilocarpine ( 25 ), isolated from Pilocarpus jaborandi, is used for treatment of glaucoma (Rosin, 1991). Catharanthus roseus synthesizes vinblastine ( 26 ) which is an antineoplastic (Goppi et al., 2003).
OH O H Me CH2OH N H N O Me N OH N N 23 21 H Me 22 HO C2H5 N O Me N Me N N N O O N H H MeO2C O N N C2H5 N C2H5 Me H H Me MeO N HO CO2Me 24 25 26 Me
1.2.3. Phenolics
Phenolics contain hydroxyl groups (one or more) attached to aromatic ring. Phenolics may be classified as phenolic acids, coumarins, lignins, flavonoids, tannins and lignans. Simple phenolic acids include hypogallic acid ( 27 ) that is a very useful drug for chelation of iron (Graziano et al., 1974).
Page 6
Psoralen ( 28 ) is furocoumarin and it is used for the treatment of skin problems, e.g., psoriasis and eczema (Nettelblad et al., 1996). Apigenin ( 29 ) is a flavonoid that is reported to possess anti cancer properties (Chiang et al.,
2006). Sesamin ( 30 ) is an example of lignans (Kamal-Eldin et al., 2011) while tannins include tannic acid ( 31 ) which is used for staining of wood (Halkens,
2001).
OH O OH O OO OH HO O HO
28 29 27 OH O
OH O HO O O O OH O HO OH HO O O O O O O OH O O O 30 HO O OH OH 31 OH OH
1.3. Drugs Based on Natural Origin
According to an analysis (United States), from 1959-1980, more than
25% medications were originated from nature (Bhuwan et al., 2011). A more recent analysis showed that during the year 1998-2004, 21 drugs of natural origin were launched in Japan, Europe and US including an antifungal
Page 7
lipopeptide; caspofungin (32 ), an anti microbial agent fumagillin (33 ) that was isolated from Aspergillus fumigates .
HO OH O H OH N N N H O O HO O NH NH O OH HN O O H2N NH OH HO N
HO 32 NH2
O H MeO
O O N
O H
O COOH HO 33 34
Galantamine (34 ) is used for the treatment of Alzheimer's disease, is isolated from various natural sources e.g., Galanthus caucasicus (Tsakadze et al., 2005) Tigecycline (35 ), an antibiotic that is intravenously used to cure abdominal organs and skin infections (Bhuwan et al., 2011). Micafungin (36 ) is an anti fungal drug which belongs to echiocandins (Lesley, 2012).
Page 8
OH HO O OH O OH H S N O O H2N NH OH N N H H O OH N O O OH O H O O N N HO N CONH2 H OH NH OH O HN OH O HO OH O 35 HO O N 36 O NH
O
In 2011, FDA approved dificid ( 37 ), an antibiotic of actinomycetes origin
(Butler et al., 2013). Vascepa ( 38 ) is a derivative of omega 3 fatty acid. It was approved in 2012 for the treatment of hypertriglyceridemia (Christie et al.,
2013). Further in 2012, albumin binded paclitaxil (39 ), abraxane was approved as an anti cancer agent (Kazuhiko, 2013).
Cl HO OH
O O Cl O O OH O O OH O O O OH O O O O NH O HO O OH 37 O OH O OH O OH O O 39 O O
O 38
Page 9
1.3.1. Role of Plants in Human Health
Throughout the history, plants have been the main source of food, shelter, clothing and ultimately for health care products. In all civilizations plants and herbs have been used as cure e.g., Emblica officinalis (amla) contain anti microbial properties (Saeed and Tariq, 2007) while fruit of amla is used against diabetes (Tiwari et al., 2011). Saraca Asoca (Ashok) is used to cure menstrual pain and uterine disorder (Pradhan et al., 2009). Solanum nigrum
(Makoi) is an anti dysenteric and diuretic (Jain et al., 2011). Santalum Album
(Sandal Wood) is used for skin problems and urinary disorders (Basant et al.,
2008). Ocimum sanctum (Tulsi) is used for fever, vomiting and bronchitis
(Gupta et al., 2002). Lawsennia iermis (Mehndi), an excellent hair conditioner, also contain anti fungal properties (Bosoglu et al., 1998). catharanthusRoseus
(Sada Bahar) crude extract contain antibacterial and anti diabetic properties
(Ibrahim et al., 2011).
1.3.2. Natural Products Isolated from Plants
Plants cannot move and they lack an immune system so alternative defense tools are evolved in plants, involving a variety of secondary metabolites to adapt to environment, to overcome stress and to survive. The role of secondary metabolites for plants can constitute a rationale for use of
Page 10
these chemicals as drugs and food supplements. A few examples of plant derived medicines include capsaicin ( 40 ) which is isolated from chili peppers
(genus Capsicum ). Colchicine ( 41 ) is isolated from Colchicum autumnale and is used in the treatment of gout, pericarditis, and as an anticancer drug
(Graham and Roberts, 1953). Ingenol 3-Oangelate ( 42 ) is an anti tumor and chemotherapeutic compound against skin cancer. It is isolated from Euphorbia peplus (Kedei et al., 2004) . An anti HIV agent is, calanolide A ( 43 ), isolated from Calonphyllum lanigerum , another compound with anti HIV property is prostratin ( 44 ) which is isolated from Homalanthus nutans (Kashman et al.,
1992). Arteether ( 45 ) is an approved anti malarial drug isolated from
OMe MeO OMe MeO HO O H N O MeO O HN O O OH OH 40 OH O 41 42
O H O O O O HO H H O H H O O O H O
OH OCH2CH3 O OH OH 43 44 45
Artemisia annua (Bhuwan et al., 2011).
Page 11
1.3.3. Natural Products Isolated from Microorganisms
Microbial specie produce organic compounds with diverse structural features and having range of biological activities. In the case of shortage of any key nutrient, microbes start producing secondary metabolites e.g.,
Penicillium chrysogenum synthesize penicillin when glucose is exhausted from the medium and lactose is consumed by the fungus (Barrios-Gonzalez et al.,
2005).
H2N HO O OH O OH O O O OH O O Cl OH OH N HO Cl HO O O O HO H H OH O O N N N N H O OMe O H O O O O O HN NH NH OH NH O HOOC 2 47
OH OH 46 HO O OH O HO
HO O OH HO
HO 48 49
Vancomycin ( 46 ), a broad spectrum antibiotic is isolated from
Amycolatopsis orientalis (Butler, 2004). Erythromycin ( 47 ) is an antibacterial
Page 12
drug isolated from Saccharopolyspora erythrae (Dewick, 2002) . Inonotus hispidus produce hispidin ( 48 ) and hispolion ( 49 ). These compounds showed anti viral activity (Ali et al., 2003). Kojic acid (50 ), a skin whitening agent , is isolated from various fungal specie especially Aspergillus oryzae (Yabuta, 1924) . It contain antibacterial properties and also used for the treatment of skin diseases Codinaeopsin ( 51 ) is isolated from Vochysia guatemalensis, and inhibits the growth of Plasmodium falciparum (Kotinik et al., 2008).
Augmentin, a safer antibiotic, is a combination of clavulanic acid ( 52 )
(isolated from Streptomyces clavuligerus ) and amoxicillin ( 53 ) (Butler, 2004).
O H OH O OH
HO N O O CO2H 50 52 H H O NH HO 2 H H N O N S H O N HO N O H OH 51 53 O
1.3.4. Natural Products Isolated from Marine Sources
Marine flora and fauna produce a variety of chemicals for protection and survival in different and harsh habitat. It is believed that sessile organisms have chemical defense for their protection (Jimeno et al., 2004).
Page 13
Many bioactive compounds have been isolated from different marine organisms. Some examples of secondary metabolites isolated from marine sources include Bryostatin 1 ( 54 ), an anticancer compound, isolated from
Bugula neritina (Pettit et al., 1982). Dolostatin 10 ( 55 ) is a cytotoxic peptide, isolated from an ocean mollusk Dolabella auricularia (Vaishampayan et al.,
2000). Squalamine ( 56 ), an antibiotic, is isolated from dogfish sharks (Moore et al., 1993). Halichondrin B ( 57 ), active against breast cancer, is isolated from sponges (Bai et al., 1991).
COOMe HO O O O O H OH N O H H N O O O H O O N H OH N O HO O S O O O COOMe O N
54 55
H H H H H OSO3H O O H O H O H O O O O O O HO H H H H H H H O OH OH H H O O H O O H N OH H NH N 2 O H H 56 57
Page 14
1.3.5. Natural Products Isolated from Animal Sources
Certain animals and animal products are being used as medicine e.g., leeches have been used to treat venous blocking at surgical wound sites
(Whitaker et al., 2004). Anticoagulants are secreted by leech during feeding.
Hirudin is an example from a number of anticoagulants isolated from leech
(Eldor et al., 1996). Whole scorpions, extracts or venoms from the chinese scorpion have been used to treat epilepsy, meningitis, rheumatic diseases and stroke (Rajendra et al., 2004). Chinese scorpion venom contains numerous small neuroactive peptides, phospholipase and mucopolysaccharides that have anti-inflammatory properties (Goudet et al., 2002). Toxins from
Scorpion venoms may act as bronchodilators and may be helpful in curing chronic coughing and chronic bronchitis (Rogers, 1996).
Lead compounds are also extracted from animals e.g., epibatidine (58) , a potent analgesic, was isolated from the skin of the ecuadorian poison frog
(Daly et al., 1994). Carminic acid ( 59) is the oldest known insect natural product as active color ingredient of cochineal, a natural dye produced by an insect dactylopius coccus (Sugimoto et al., 2010). Some examples of chemicals isolated from beetles are PAML908 ( 60 ) and chilocorine B ( 61 ). Myrmicarin
663 ( 62 ) is isolated from ants, scolopin 1 and 2, that are antibacterial are
Page 15
isolated from centipede (Peng et al., 2010). Pederin ( 63 ) is a defensive polyketide of coleoptera (Pavan, 1953).
OH O HO OH O O O Cl N H OH N N H O OH
OH O O 58 HO OH HN HN OH 59 O O H N H O O N 60
H C 3 O N N O O OMe N O H H N OMe MeO H OH H OMe N 61 62 63 OH
O
Page 16
CHAPTER 2
Phytochemical and Pharmacological Studies on the Genus Aerva
Page 17
Medicinal plants and their extracts have been used in unani, sidha and ayurvedic system of medicine for centuries. Plant derived natural products have received considerable attention due to their pharmacological potential.
Plants have played a noteworthy role in improving the value of life.
Innumerable types of plants are spread on the earth and whole plant kingdom is alienated into families on the basis of similar characters in order to make the study easy. Further these families are separated into smaller sections, genera and species.
2.1. Family Amaranthaceae
Amaranthaceae is a wide ranging family consisting of 180 genera and
2,500 species (Müller and Borsch, 2005). Most of the plants of Amaranthaceae are annual and perennial herbs or sub shrubs. Only a few members are trees or climbers. Many species are halophytes growing in salty soils.
Generally the leaves are alternate and/or opposite without stipules and with toothed or entire margins. Mostly bisexual flowers are present which may be solitary or clustered as spikes or panicles. Some exceptions to this general character are also found where unisexual flowers are present.
Regular flowers are present with 1-5 herbaceous perianth. Ovary is superior with one and in rare cases two basal ovules. There are 1-5 stamens inserting from a hypogynous plate. The anthers with 2-4 pollen sacs, spherical pollen
Page 18
grains, 1-3 fused carpels are some common features of the plants of this family (Zaveri et al., 2012).
2.1.1. Medicinal Importance of the Family Amaranthaceae
Plants of this family are used in ayurvedic medicine and modern pharmaceutics. Achyranthes aspera commonly called as Ubat kandri is a potential plant with, pungent, expectorant, purgative and laxative Properties.
Paste of the roots is believed to be useful for pregnant women to stimulate labor pain (Nadkarni, 1979). Leaf juice is useful for eyes disorders and is helpful in improving the vision. Besides these potentials, variety of other actions are associated with different parts of this specie (Qureshi and Bhatti,
2009). Amaranthus virdis is locally known as mariro and has been found to contain wide range of applications e.g., it is useful in burning sensation and urinary tract problems etc. It resolves digestion issues and help to overcome the deficiency of vitamin A and calcium (Qureshi and Bhatti, 2009). Luluris is common name of Digera muricata which is used as laxative and in urinary tract problems (Srivastava, 1989).
Powdered leaves and seeds of Celosia argentea are used for diarrhea and the flowers are used for dysentery, muscular troubles, and haemophythysis while grinded flowers mixture with curd and sugar is a remedy of menorrhagia (Burkill, 1985). Leaf paste of Amaranthus hybridus
Page 19
diminishes poisonous effects of snake bite and insects (Shah et al., 2006).
Amaranthus trilocular is useful against stomach pain in, diarrhea and bladder stone (Shah et al., 2006). These are few examples of medicinal plants of this family, and there is still a big list of herbs and shrubs present which play very important role in serving human. Genus Aerva is one among the important groups of family Amaranthaceae, and few relatives of this genus are growing in Pakistan, which are locally being used for medicinal purposes.
2.2. The Genus Aerva
Aerva is one of the most potential genera of the family Amaranthaceae.
A number of phytochemical investigations have been carried out on different species of Aerva . Generally Aerva plants are perennial herbs with salient morphological characters. The species of this genus are distributed in Africa and temperate regions of Asia (Chawla et al., 2012).
More than 28 species of the genus Aerva are reported in literature, some of them are listed here: A. ambigua, A. brachiata, A. congesta, A. desertorum, A. hainanensis, A. japonica, A. lanata, A. madagassica, A. monsonia, A. revoluta, A. sanguinolenta, A. sansibarica, A. scandens, A. timorensi, A. wighti, A. artemisioides, A. cochinchinensis, A. coriacea, A. glabrata, A. incana, A. javanica, A. leucura, A. microphylla, A. persica, A. sanguinolenta, A. sericea and A. triangularifolia.
Page 20
2.2.1. Medicinal Importance of the Genus Aerva
A. tomentosa is used as demulcent and diuretic and also purgative properties are associated with this plant (Sethi and Sharma, 2011). Seeds and flowers of A. tomentosa are used against rheumatism swelling and headache
(Bakshi et al., 1999; Kirtikar and Basu, 2001).
A. lanata possesses wide spectrum of applications in traditional systems e.g., the whole plant is used in litiasis and diabetes and also as an anthelmintic agent and an expectorant (Rajesh et al., 2011). It is also used in treating kidney stones (Mukerjee et al., 1984), in burn healings and skin problems, clearing uterus after delivery, nasal bleedings, fractures, scorpion stings, bronchitis and as an anti-inflammatory agent (Rajesh et al., 2011).
2.2.2. Pharmacological Studies of the Aerva Plants
Species of genus aerva are widely studied for their pharmacological potential. Literature survey revealed that various activities are associated with different extracts of species. A summary of biological studies mentioned in literature is given as under;
2.2.2.1 . Antimicrobial Activity of the Aerva Plants
Methanolic extract of A. Lanata exhibited antimicrobial activity against gram positive and gram negative bacteria. This study was carried out on
Staphylococcus, Bacillus subtilis , Bacillus cereus (gram positive) while Klebsiella
Page 21
species, Shigella flexneriae, Shigella dysenteriae, Shigella boydii, Shigella shiga,
Shigella sonnnei and Escherichia coli (Gram negative) (Chowdhury et al., 2002) .
In 1998 Gehlot and Bohra estimated antibacterial activity of A. persica and found that aqueous and alcoholic extracts of different parts of plant were active against Salmonella typhi and Staphylococcus aureus (Gehlot and Bohra,
1998). Activity against Staphylococcus aureus and E. coli was observed by perianth lobes of A. tomentosa in 2003 (Jaswant et al., 2003). Significant activities were observed against gram negative bacteria by flavonoids isolated from extracts of A. javanica (Radwan et al., 1999). In 2011 a group of researchers reported significant antimicrobial activity of crude extracts of A. javanica along with six isolates named as kaempferol-3-O-β-D- glucopyranosyl-(1→2)-α-L-rhamnopyranoside-7-O-α-L-rhamnopyranoside
(85 ), apigenin 7-O-glucuronide (86 ), Isoquercetrin (87 ). methylmellein (137 ), hydroxyethyl)-2-(2″-hydroxyethyl)-3,4-dihydrobenzopyran ( 138 ) and 2- 1׳)7- hydroxy-3-O-β –primeveroside naphthalene-1, 4-dione (139 ), The activity was tested against Bacillus subtilis, Staphylococcus aureus, Salmonella typhi,
Trichophyton longifusus, Shigella flexneri, Escherichia coli, Candida albicans,
Pseudomonas aeruginosa, Candida glabrata. Microsporum canis and Fusarium solani, (Sharif et al., 2011). A recent investigation carried out on extracts of different parts of A. javanica i.e., flower and leaves, roots and stem against
Page 22
range of bacteria ( Escherichia coli, Enterobacter aerogenes, Klebsiella penumoniae,
Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonas putida, Salmonella typhimurium, Bacillus cereus, Bacillus subtilis, Staphylococcus aureus,
Staphylococcus epidermidis ) showed that methanolic extracts of flower and leaves had more antibacterial activity (Srinivas and Reddy, 2012).
2.2.2.2 . Diuretic Activity of the Aerva Plants
Significant diuretic activity of extracts (root, stem and leaves) of A. lanata was reported in 1999 showing stem extract better than the other two extracts (Majmudar et al., 1999). Diuretic activity of A. lanata was also reported in 2000 (Vetrichelvan et al., 2000). In another study flowers of A. lanata were found active at the dose of 50 g/L (Goonaratna et al., 1993).
2.2.2.3 . Immunomodulatory and Anti tumour Activity of the Aerva Plants
The whole plant ethanolic extract of A. lanata was evaluated for
Immunomodulatory and anti tumour effects and found that it enhanced total white blood cells count and α-esterase positive cells. The extract was 100% cytotoxic to EAC (Ehrlich ascites carcinoma) and DLA (Dalton lymphoma ascites) cells (Siveen and Kuttan, 2011). Significant cytotoxicity of petroleum ether fraction of extract of A. lanata was shown against daltons lymphoma ascites, ehrlich ascites and B16F10 cell lines in vitro (Nevin and Vijayammal,
2003).
Page 23
2.2.2.4 . Hepatoprotective Activity of the Aerva Plants
Significant hepatoprotective activity was shown by alcoholic extracts of leaf and roots of A. lanata . This study was carried out on albino mice
(Majmudar et al., 1999). A. lanata extracts were also studied against CCl 4 induced liver damage (Nevin and Vijayammal, 2005) and liver damage induced by paracetamol in rats (Manoharan et al., 2008). Activity of Perianth lobes of A. tomentosa is also reported (Jaswant et al., 2003).
2.2.2.5 . Antidiabetic Activity of the Aerva Plants
Alcoholoic extract of A. lanata was found useful in preventing the increased blood sugar level in rats (Vetrichelvan and Jegadeesan, 2002). Leaf extracts of A. lanata were also found to contain anti hyperglycaemic activity.
Leaf extracts were studied on serum glucose and oral glucose tolerance test
(OGTT) where 400mg/kg dose showed significant antihyperglycaemic activity for OGTT (Deshmukh et al., 2008). Leaves of A. Javanica also showed antidiabetic activity in alloxan induced diabetic mice (Srinivas and Reddy,
2009). A more recent investigation performed in 2012 showed that methanolic and aqueous ethanolic extracts of ariel parts of A. lanata significantly reduced blood glucose level in streptozotocin induced diabetic rats, decrease in lipid content was also observed in the same experiment showing antihyperlipidimic activity for A. lanata extracts (Rajesh et al., 2012).
Page 24
2.2.2.6. Antioxidant Activity of the Aerva Plants
In 2006 Ahmad et al. isolated flavonoids from ethylacetate fraction of whole plant of A. persica and evaluated their antioxidant potential. Results showed that all compounds had profound antioxidant activity (Ahmed et al.,
2006). In one study A. tomentosa was subjected to evaluate its methanolic, ethylacetate, DCM and aqueous extracts for their antioxidant potential and total phenolic components. As a result it was found that methanolic extract was rich in total phenolics and was more potent antioxidant than other extracts thus showing structure activity relationship of phenolics and antioxidant activity (Sethi and Sharma, 2011).
2.2.2.7. Anti lithiatic Activity of the Aerva Plants
Leaf extract of A. lanata has been reported to help in excretion of uric acid. A dose of 3.0 mg/kg of body weight of the leaf extract for 28 days increased excretion of uric acid and calcium oxalate in hyperoxaluric rats
(Selvam et al., 2001). In another report, the reduction of oxalate synthesizing enzymes such as glycolic acid oxidase (GAO) in liver and lactate dehydrogenase (LDH) in liver and kidney in calcium oxalate urolithiatic rats was observed for the aqueous extract of A. lanata at a dose of 2g/kg of body weight for 28 days (Soundararajan et al., 2006). The aqueous extract of A. lanata has also been reported to normalize the levels of lipid, cholesterol and
Page 25
triglycerides in ethylene glycol induced calcium oxalate urolithiatic rats
(Soundararajan et al., 2007). The Anti-lithiatic importance of A. lanata is clear after A. lanata became part of Russian patent product PHYTOSORB introduced to remove calculus from gallbladder (Korovaev, 1997).
2.2.2.8. Antifungal Activity of the Aerva Plants
Antifungal activity of methanolic and ethylacetate extract of A. lanata was evaluated in 2002 by a group of researchers. Fungi used in this study included Aspergillus fumigates, Candida albicans, Rhizopus oligosporum,
Aspergillus niger and Hensinela californica (Chowdhury et al., 2002).
2.2.2.9 . Nephroprotective Activity of the Aerva Plants
75, 150 and 300 mg/kg doses of alcoholic extract of whole plant of A. lanata were applied to albino rats having acute renal failure induced by cisplatin and gentamicin and marked reduction in serum creatinine and blood urea was observed (Shirwaikar et al., 2004).
2.2.2.10. Antiulcer Activity of th Aerva Plant
Ethylacetate fraction of methanolic extract of A. javanica has been reported to show moderate anti-ulcer activity with 50% inhibition at a concentration of 0.2 mg/mL (Khan et al., 2012). The ethanolic extract of the roots of A. persica was also found to exhibit antiulcer activity in albino wistar rats. For alcohol induced ulcer model, the extract showed 70.43% protection
Page 26
from ulcers at a dose of 200 mg/kg. The mentioned activity was close to the activity of standard drug, ranitidine. In another assay for pylorus ligation induced ulcer model, the extract showed 36.88% protective effect at a dose of
200 mg/kg while ranitidine showed 62.95% protective effects against ulcers at a dose of 50 mg/kg when compared with the control groups (Vasudeva et al., 2012).
2.2.2.11. Miscellaneous Activities of the Aerva Plants
Extracts of A. javanica kills Plasmodium falciparum ; where leaf, mature fruit and stem extracts showed IC 50 values of 100, 76 and 308 µmol/mL, respectively (Simonsen et al., 2001). A crude extract of A. javanica exhibited weaker acetylcholinestrase inhibitory activity with IC 50 value of 275.2 µg/mL
(Murtaza et al., 2013). 1 % aqueous extract of A. lanata showed good inhibitory activity to prevent the growth of gout causing crystals of monosodium urate monohydrate (MSUM) (Parekh et al., 2009). A. lanata has also showed anti inflammatory activity in its alcoholic and benzene extracts at dose of 800 mg/kg in carageenan induced rat paw edema model
(Vetrichelvan et al., 2000) whereas, the same plant has been used along with other herbs for preparation of a medicated liquor, GRAAL. This product is helpful in treating cardiovascular diseases such as hypertension,
Page 27
atherosclerosis, and coronary heart disease, blood diseases such as anemia, headache, ulcer, and allergic disease (Shengeliya and Mardaleishvili, 2000).
2.3. Phytoconstituents Isolated from the Genus Aerva Name of Sr. Compound Structures Source Ref. No M. Formula 64 Chrysin -7-O- OMe A. persica (Garg et al., OH galactoside 1979) O O OM C23 H24 O12 O OH M.W. 492.429 HO OH O HO OH
65 Aervanone HO OH A. persica (Garg et al., O C21 H22 O10 HO 1980) OH OH M.W. 434.393 O HO O
O
66 Kaempferol-3- OH A. javanica (Saleh et al., galactoside. HO O 1990 )
C21 H20 O11 O M.W. 448.376 OH O OH O
HO OH HO 67 Kaempferol-3- OH A. javanica (Saleh et al., rhamnogalactoside HO O 1990 ) C27 H30 O15 O OH O M.W. 594.518 OH HO O HO O OH O HO HO
Page 28
68 Quercetin-3- OH A. javanica (Saleh et al., OH galactoside 1990 ) HO O C21 H20 O12 M.W. 464.376 O OH O OH O OH HO HO 69 Isorhamnetin-3- OMe A. javanica (Saleh et al., OH galactoside. 1990 ) HO O C22 H22 O12
M.W. 478.403 O OH O OH O OH HO HO 70 Isorhamnetin-3- OH A. javanica (Saleh et al., HO O rhamnosyl-(1→6)- OMe 1990 ) galactoside O (Narcissin) OH O OH C28 H32 O16 HO O M.W. 624.544 HO O OH O HO HO 71 Isorhamnetin -3- (P- OH A. javanica (Saleh et al., HO O coumaroyl)- OMe 1990 ) rhamnogalactoside O C37 H38 O18 OH O OH HO M.W.770.686 O HO O OH O O O HO
OH
Page 29
72 Isorhamnetin-3-O- OH A.lanata (Zadorozhnii OMe β-D-glucoside et al., 1986) HO O C22 H22 O12
M.W. 478.403 O OH O O OH HO
OH OH 73 Chrysoeriol OMe A. javanica (Radwan et OH C16 H12 O6. al., 1999) M.W. 300.263 HO O
OH O
74 Persinol OH A. persica (Ahmed et al., OH C16 H14 O7 2006) MeO O M.W. 318.278 OH
OH O OH 75 Persinosides A HO A. persica (Ahmed et al., O C22 H24 O12 O OH 2006) MeO O M.W. 480.419 OH O HO
OH O
OH 76 Persinosides B OH OH A. persica (Ahmed et al., O C21 H22 O12 HO O 2006) HO O OH M.W. 466.392 OH OH O
77 4',5-Dihydroxy - OH A. persica (Ahmad et al., 3,6,7-trimeth- MeO O 2008) oxy flavone MeO OMe C18 H16 O7 OH O M.W. 344.315 78 5-Hydroxy-3,6, 7 OMe A. persica (Ahmad et al., ,4'-Tetramethoxy MeO O 2008) flavone MeO OMe C19 H18 O7 OH O M.W. 358.342
Page 30
79 Apigenin 7-O- β-D- OH A. persica (Ahmad et al., glucopyrano O O 2008) side O OH HO C21 H20 O10 OH O M.W. 432.377 OH OH
80 2',3,5',6,7- MeO A. persica (Ahmad et al., Pentamethoxy MeO O 2008) OMe flavone MeO OMe C20 H20 O7 O M.W. 372.369 81 3,3' ,5- Trihydroxy- OMe A. persica (Ahmad et al., O O OH 4' -methoxyflavone HO O 2008) HO OH 7-O- β-D- OH OH O glucopyranoside OH
C22 H22 O12 M.W. 478.403 82 5-Hydroxy,7-8- OMe A. persica (Imran et al., dimethoxy MeO O 2009b) flavone C17 H14 O5 OH O M.W. 298.290 83 4´,5,7-Trihydroxy OH A. persica (Imran et al., flavone HO O 2009b) C15 H10 O5 M.W. 270.237 OH O OMe 84 5-Hydroxy, OMe A. persica (Imran et al., 3´,4´,6,7,8 MeO O 2009b) OMe Pentamethoxy MeO flavone OH O C20 H20 O8 M.W. 388.368 HO OH 85 Kaempferol -3-O-β- HO A. javanica (Sharif et al., D-glucopyranosyl- O OH 2011) (1→2)-α-L- O O OH
rhamnopyranoside- O OH O 7-O-α-L- O rhamnopyranoside OH O HO O O OH C33 H40 O20 HO HO M.W. 756.659
Page 31
86 HO OH A. javanica Apigenin 7-O HO (Sharif et al., HOOC glucuronide O O 2011)
C21 H18 O11 O O M.W. 446.361
OH O
87 Isoquercetrin OH A. javanica (Sharif et al., C21 H20 O12 HO O OH 2011)
M.W. 464.376 HO O O OH O OH OH OH
88 Feruloyltyramine A.lanata (Zadorozhnii HO C18 H19 NO 4. H et al., 1986) N M.W. 313.348 MeO O OH
89 HO A.lanata Feruloylhomovanill H (Zadorozhnii NOM yl amine MeO et al., 1986) O C19 H21 NO 5 OH M.W. 343.374 90 Syringic acid OMe A.lanata (Zadorozhnii HO C9H10 O5 OH et al., 1986) M.W. 198.173 O OMe 91 Vanillic acid O OH A.lanata (Zadorozhnii C8H8O4 et al., 1986) M.W.168.147 OMe OH 92 Ferulic acid HO A.lanata (Zadorozhnii C10 H10 O4 OH et al., 1986) MeO M.W.194.184 O 93 Methyl gravillate OH O A. persica (Ahmad et al., C10 H10 O4. OMe 2008) M.W.194.184 OH 94 4-Hydroxy O A. persica (Imran et al., benzaldehyde OH 2009b) H C7H6O2 M.W. 122.121
Page 32
95 4-Hydroxybenzoic O A. persica (Imran et al., acid OH 2009b) HO C7H6O3 M.W. 138.121 96 3-Hydroxybenzoic OH A. persica (Imran et al., acid O 2009b) C7H6O3 HO M.W. 138.121 97 Gallic acid HO A. persica (Imran et al., O C7H6O5 HO 2009b) M.W. 170.120 OH HO 98 3, 4 ʹDihydroxy- OMe A. persica (Imran et al., O 3ʹ,5 ʹ-dimethoxy OH 2009b) propiophenone C11 H14 O5 HO OMe M.W. 226.226 99 Shikimic acid O OH A. lanata . (El-Seedi et al., C7H 10 O5. 1999) M.W. 174.151 HO OH OH 100 4-Ethoxy benzoic OEt A. persica (Ahmad et al., acid 2008) C9H10 O3 M.W.166.174 HO O 101 3-Hydroxy-4 O H A. javanica (Khan et al., methoxy 2012) benzaldehyde C8H8O3 OH M.W.152.147 OMe 102 Ascorbic Acid OH A. persica (Harsh and HO C6H8O6 O Kapoor, 2004) M.W. 176.124 O HO OH 103 Bakuchiol A. sanguin (Gottumukkal C18 H24 O olenta a et al., 2012) M.W. 256.182 HO
Page 33
104 Campesterol (Patterson et C28 H48 O A. persica al., 1991) M.W. 400.680
HH HO
105 7-Ergostenol A. persica (Patterson et C28 H48 O al., 1991) M.W. 400.680
HH HO
106 β- Sitosterol A. persica (Patterson et C29 H50 O al., 1991) M.W. 414.707
HH HO
107 7-Stigmastenol A. persica (Patterson et C29 H50 O al., 1991) M.W. 414.707
HH HO
108 Campestanol A. persica (Patterson et C28 H50 O. al., 1991) M.W. 402.696
HH HO
109 22-Stigmastenol A. persica (Patterson et C29 H50 O al., 1991) M.W. 414.707
HH HO
Page 34
110 α-Amyrin A. javanica (Radwan et
C30 H50 O H al., 1999) M.W. 426.717
HO
111 β-Amyrin A. javanica (Radwan et C30 H50 O. H al., 1999) M.W. 426.717
HO
112 β-Sitosteryl acetate A. persica (Ahmad et al., C31 H52 O2 2008) M.W. 456.743 O
O
113 Lupeol A. persica (Ahmad et al., C30 H50 O 2008) M.W. 426.717
HO
114 Lupeol acetate A. persica (Ahmad et al., C32 H52 O2 2008) M.W. 468.754
O
O
115 Ursolic acid A. javanica (Khan et al., C30 H48 O3. H 2012) M.W. 456.70 OH O H HO
116 Squalene A. javanica (Samejo et al., C30 H50 2012) M.W. 410.71
Page 35
117 Canthin-6-one A. lanata (Zapesochnay N C14 H8N2O N a et al., 1991) M.W. 220.226 O 118 Aervin HO A. lanata (Zapesochnay N C14 H8N2O2 N a et al., 1991) M.W. 236.225 O 119 Methylaervin MeO A. lanata (Zapesochnay N C15 H10 N2O2 N a et al., 1991) M.W. 250.295 O 120 Aervoside OH A. lanata (Zapesochnay O HO C20 H18 N2O7 HO O a et al., 1991) OH M.W. 398.366 N N
O
121 β-Carboline -1- A. lanata (Zapesochnay propionicacid N a et al., 1991) N C14 H12 N2O2 H M.W.240.257 COOH 122 Aervolanin MeO A. lanata (Zapesochnay C15 H14 N2O3 N a et al., 1991) N M.W. 270.283 H
COOH 123 Glycinebetaine O A. japonica (Cai et al., N C5H11 NO 2 O 2001) M.W. 117.079 124 Trigonelline Me A. japonica (Cai et al., C7H7NO 2 N 2001)
M.W. 137.047 O O 125 Amaranthine OH A. (Yang et al., O HO C30 H34 O19 N2 HO O sanguinole 1998) HOOC M.W. 726.593 O O C nta HO N HO HO OH
HOOC N H
Page 36
126 Isoamaranthine OH A. (Yang et al., O HO C30 H34 O19 N2 HO O sanguinole 1998) HOOC M.W. 726.593 O O C nta HO N HO HO OR
HOOC N
OH 127 Celosianin I O A. (Yang et al., HO HO O sanguinole C39 H40 N2O21 . HOOC 1998) O O COO HO N M.W. 872.735 HO HO nta O p coumaroyl
HOOC N COOH H
OH 128 Celosianin II O A. (Yang et al., HO HO O sanguinole C40 H42 N2O22 . HOOC 1998) O O COO HO N M.W. 902.761 HO HO nta O feruloyl
HOOC N COOH H
129 Aervin A OMe A. persica (Imran et al., O O C17 H10 O6 O 2009a) M.W. 310.258 O O 130 Aervin B O O O A. persica (Imran et al.,
C17 H10 O6 O 2009a) M.W. 310.258 OMe O 131 Aervin C OMe A. persica (Imran et al., MeO O O C18 H14 O6 2009a) M.W. 326.30 MeO O O 132 Aervin D OMe A. persica (Imran et al., 11 C32 H41 O8 O O O 2009a) M.W. 553.280 OH O O 133 5,7-Dimethoxy OMe A. persica (Imran et al., coumarin 2009b) C11 H10 O4 OO OMe M.W. 206.195
Page 37
134 5,8-Dihydroxy OH A. persica (Imran et al., coumarin 2009b) C9H6O4. O O M.W. 178.142 OH 135 5,6,7 Trimethoxy MeO OO A. persica (Imran et al., coumarin 2009b) MeO C12 H12 O5 OMe M.W. 236.221 136 2H-1-Benzopyran- A. persica (Imran et al., 2-one 2009b) OO C9H6O2 M.W. 146.143 137 5-Methylmellein CH3 A. javanica (Sharif et al., CH C11 H12 O3 3 2011) M.W. 192.211 O OH O 138 7-(1 A. javanica (Sharif et al., Hydroxyethyl)-2- 2011) HO O (2″-hydroxyethyl)- OH 3,4- dihydrobenzopyra n C13 H18 O3 M.W. 222.280 139 2-Hydroxy -3-O-β - O A. javanica (Sharif et al., OH primeveroside 2011) naphthalene-1,4- O HO dione O OH C21 H24 O13 O OH M.W. 484.407 O O
HO OH HO 140 β-Damascenone O A. javanica (Samejo et al., C13 H18 O 2012) M.W. 190.281
141 Trans-α-Ionone O A. javanica (Samejo et al., C13 H20 O 2012) M.W. 192.2973
Page 38
142 Megastigmatrienon O A. javanica (Samejo et al., e 2012) C13 H18 O
M.W. 190.1358 143 Pentadecanoic acid O OH A. javanica (Radwan et C15 H30 O2 al., 1999) M.W. 242.398
144 6,10,14-Trimethyl- A. javanica (Samejo et al., 2-Pentadecanone O 2012) C18 H36 O
M.W. 268.4778 145 n- Octadecane A. javanica (Samejo et al., C18 H38 14 2012) M.W. 254.49 146 n- Nonadecane A. javanica (Samejo et al., C19 H40 15 2012) M.W. 268.52 147 n- Eicos ane A. javanica (Samejo et al., C20 H42 16 2012) M.W. 282.54 148 n- Heneicosane A. javanica (Samejo et al., C21 H44 17 2012) M.W. 296.58 149 n- Docosane A. javanica (Samejo et al., C22 H46 18 2012) M.W. 310.60 150 n- Tricosane A. javanica (Samejo et al., C23 H48 19 2012) M.W. 324.62 151 n- Tetracosane A. javanica (Samejo et al., C24 H50 20 2012) M.W. 338.65 152 n- Pentacosane A. javanica (Samejo et al., C25H50 21 2012) M.W. 3 52 .40 153 n- Hexacosane A. javanica (Samejo et al., C26H50 22 2012) M.W. 3 66 .42
Page 39
Aerva javanica is an important relative of this genus, which is growing in cholistan desert of Pakistan, and is being used in local medicinal system.
For my Ph.D project, I selected this plant to investigate for its bioactive secondary metabolites.
2.4 Aerva Javanica var. bui
Aerva javanica is a perennial herb growing in Western Himalaya,
Punjab, and Kashmir. Locally it is called “bui” and in English it is known as
“kapok bush” (Burndud, 1990). Some other common names are Bur (Gujrati),
Dodda (Kannad), Gidda (Hindi), Perumpoolai (Tamil), Magavira (Telagu) and Boi kalan. (Punjabi) etc.
2.4.1. Scientific Classification of Aerva Javanica
Kingdom Plantae Subkingdom Tracheobionta Division Magnoliophyta Class Magnoliopsida Subclass Caryophyllidae Order Caryophyllales Family Amaranthaceae Genus Aerva Species javanica Complete name Aerva javanica
Page 40
2.4.2. Botanical Description of Aerva Javanica
Aerva javanica is a perennial woody herb growing as erect clumps 0.3-
1.5 m high. Usually simple stem branches originate from the base but sometimes entangled branches with a complex pattern originate from the base. The variable sized leaves are sessile or may contain a short petiole and are arranged in an alternate fashion. The dioecious flowers occur in the form of clusters on sessile and cylindrical spikes. More slender spikes are present in male plants. Male and female flowers are different e.g., outer petals range from 1.5-2.25 mm in male flowers while in female flowers size of two outer petals ranges 2-3 mm and three inner petals are slightly shorter. In male flowers filaments are delicate, and anthers are almost equal to the perianth, while female flowers lack anthers and reduced filaments are present. Female plant bears a small ovary with short a style. Stigma is rudimentary and compressed and round capsule with size 1-1.5 mm are present. Black and brown colored round or slightly compressed seeds are present which range in size from 0.9-1.25 mm.
2.4.3. Medicinal Importance of Aerva Javanica
Aerva javanica is commonly used in folklore for diuretic and demulcent properties and decoction is used to cure swellings and urinary disorders. The leaves, seeds and roots are used to treat kidney stones. The roots of this plant
Page 41
are chewed for teeth cleaning, the seeds are said to relieve headache and the paste of roots is applied to remove acne from the face. The seeds are used to ease head ache and rheumatism (Imran et al., 2009b; Perry and Metzger,
1980).
2.4.4. Nutritional Value and Elemental Analysis of Aerva Javanica
Nutritional evaluation of A. Javanica revealed that it contains 7.3 % moisture, 70 % carbohydrate, 7.1 % protein, 1.1 % fats, 319.5 kcal/100g of energy and 29.1 % fibre content. Results of elemental analysis of A. Javanica tell the presence of elements (in ppm): Cu (2.135), Mn (0.64), Pb (0.38), Cd
(0.06), Fe (2.77), Cr (3.65), Mg (29.93), Na (28.46) (Hussain et al., 2011).
Page 42
CHAPTER 3
Biosynthesis of Flavonoids and Steroids
Page 43
3.1. Biosynthesis of Natural Products
Literature survey and experimental part revealed that most of the phyto constituents isolated from genus Aerva comprise of flavonoids and steroids, therefore, biosynthesis of these two classes of natural products is discussed in the following section.
3.2. What are Flavonoids?
The word flavonoid has been derived from a Greek word “flavus” which means yellow. The flavonoids are classified among natural phenolics and they constitute an important group of natural compounds (Harbone, 1988). A variety of structural forms of flavonoids exist but they share some common features which include fifteen carbon skeleton in their basic nuclei and two phenyl rings linked by three carbon chain. The basic structures are 1, 3-diphenyl propane
(154) ( Fig. 3.1) 1,2-diphenyl propane ( 172 ) and 1,1-diphenyl propane ( 177 )
(Grotewold, 2006).
B B A C A O B 161 155 A
B B 154 O O A A C C
171 O 166
Figure 3.1: Flavonoids with 1, 3-diphenyl propane skeleton
Page 44
Following various routes or enzymatic actions, 1, 3-diphenylpropane
(154) give rise to different classes of flavonoids. The class which pertains 1, 3- diphenyl propane nucleus is called chalconoid ( 155 ). Chalconoids are further divided into subclasses which include β-chalcanol ( 156 ), β-chalcanone ( 157 ),
α-chalcanone ( 158 ), chalcone ( 159 ) and chalcan 1, 3-dione ( 160 ) (Fig. 3.2)
(Bohm, 1988).
5
5' 1 β 3
1' 3' α β' OH O 155 156 157
O
O O O 158 159 160
Figure 3.2: Basic skeleton of chalconoids
Sometimes three carbon chain condenses to give a third ring originating other classes of compounds, if five member ring is formed then the compounds are called auronoids ( 161 ). Further subclasses of auronoids are aurone ( 162 ), aurononol ( 163 ), auronol ( 164 ) and isoaurone ( 165 ) (Fig. 3.3)
(Bohm, 1988).
Page 45
8 1 9 O 7 O 2 3 2' O 1' O OH O 6 3' 10 5 4 O 4' H 6' O H 5' O O
162 163 164 165
Figure 3.3: Basic skeleton of auronoids
If 3-membered carbon chain gives a six membered ring containing a hetero atom then flavonoids ( 166 ) are originated. Flavonoids may be flavanone ( 167 ), flavanonol ( 168 ), flavones ( 169 ), flavonol ( 170 ), flavan ( 171 ) and anthocyanidine (172 ) (Fig. 3.4) (Hollman and Arts, 2000).
3' 2' 4' 8 1' 9 O 5' O O 7 1 2 6' 4 3 6 10 OH 5 O O O 167 168 169
O O O
OH O 170 171 172
Figure 3.4: Basic skeletons of flavonoids with 1, 3-diphenyl propane
Page 46
1, 2 diphenyl propane ( 173 ), generates basic skeletons of isoflavanone ( 174 ), isoflavonoid ( 175 ) and 3-phenyl coumarin ( 176 ) due to cyclization of three carbon chain (Fig. 3.5) (Dewick, 1988).
159
O O O O
160 161 162
Figure 3.5: Flavonoids with 1, 2-diphenyl propane skeleton
1, 1 diphenyl propane ( 177 ) gives rise to neo flavonoids ( 178 ) (Fig. 3.6)
(Donnelly and Sheridan, 1975).
O O
177 178
Figure 3.6: Flavonoid with 1,1-diphenyl propane nucleus
Page 47
Sometimes flavonoids deviate from fifteen carbon skeleton and an additional carbon atom is added in the basic skeleton which is numbered as C- 11. This class of flavonoids is called homo flavonoid. Different subclasses of homoflavonoids are homoflavone ( 179 ), homoisoflavone ( 180 ), homoisoflavanone ( 181 ), homoisoflavan ( 182 ), rotenoids ( 183 ) etc. (Fig. 3.7)
(Geiger, 1988).
8 11 2' 9 O 1' O O 7 3' 1 2 6 4 3 4' 10 6' 5 5' O O O 179 180 181
O O O
O 182 183
Figure 3.7: Basic Skeletons of Homo Isoflavonoids
3.2.1. Biosynthesis of Flavonoids
Acetyl CoA ( 184 ) is very first starting material for biosynthesis. It is derived from carbohydrates i.e., glucose is converted into glucose-6 phosphate, which after series of steps is converted into phospho enolpyruvate ( 184b ). Pyruvate is formed of phosphoenol pyruvate after elimination of phosphorus and then ultimately pyruvate becomes the
Page 48
precursor of acetyl CoA ( 184 ). Synthesis of coumaroyl CoA is a complicated process and involves shikimate pathway. Glucose 6-phosphate gives D- erythrose-4-phosphate ( 184a ) and phosphoenyl pyruvate ( 184b ). These reagents give 3-dehydro quinate ( 184c ) in the presence of 3-dehydroquinate synthase. Then 3-dehydro quinate dehydratase excite removal of water from the molecule thus synthesizing 3-dehydroshikimate (184d ). Finally shikimate
(99 ′) is formed where shikimate dehydrogenase is the enzyme which catalyzes this formation (Scheme 3.1).
PO H
HO O OH 184a Photosynthesis Glucose Glucose -6P O
- O HO COO - - P O COO O H2O 3-dehydroquinate PO synthase enzyme complex H H O OH OH 184b NAD+ HO -Pi OH OH 184c DAHP
- COO- COO
shikimate O OH dehydrogenase HO OH OH NADPH OH 184d 99' Scheme 3.1: Synthetic route of shikimate
Shikimate ( 99 ′) is converted into 3-phosphoshikimate ( 99a ) which is converted into chorismate ( 99c ) via EPSP intermediate ( 99b ). The enzymes involved in these reactions are shikimate kinase, EPSP synthase and
Page 49
chorismate synthase respectively. Chorismate ( 99c ) give rise to 4 - hydroxy phenyl pyruvate ( 99d ) which after a series of reactions synthesizes 4-hydroxy coumaric acid ( 99e ) (Kim et al., 2013) (Scheme 3.2).
COO- COO- PEP COO- PO COO- H ATP - HO OH Shikimate kinase PO OH EPSP PO O COO OH OH synthase OH Pi 99b 99' 99a Chorismate synthase Pi O - COO - - - COO COO O + OOC NADH NAD O O
O COO- O OH OH H 99c decarboxylation
O O COOH COO- COOH O NH3 NH2
NH3 NH2
OH OH OH OH 99d 99e
Scheme 3.2: Conversion of shikimate into 4-hydroxycoumaric acid
Malonyl CoA ( 184e ), and 4-hydroxy coumaroyl CoA ( 99f ) are precursors for the synthesis of chalcone intermediate. Malonyl CoA (184e) is itself formed by acetyl CoA ( 184 ) (Scheme 3.3). C-15 intermediate “chalcone” plays central role in the biosynthesis of all types of flavonoids (Monitto, 1965). Chalcone
Page 50
synthase is the enzyme involved in synthesis of chalcone ( 184f ) (Weisshaar and Jenkins, 1998).
O OH HO SCoA O SCoA O SCoA H3C SCoA O O O O -CO O 184 184e 2 O O O O OH OH Chalcone synthase CoASH
HO SCoA OH
O HO OH H2O O 99e 99f
OH O 184f
Scheme 3.3: Biosynthetic route for the synthesis of chalcone
Once chalcone ( 184f ) is formed from malonyl CoA ( 184e ) and 4-hydroxy coumaroyl CoA ( 99f), it becomes the precursor for almost all flavonoids.
Formation of flavanone, naringenin ( 184g ) involves isomerization of chalcones (Scheme 3.4). An equilibrium is established between flavanone and respective chalcone and chalcone isomerase catalyse this interconversion
(Monitto, 1981). This enzyme shows stereospecific character and maintains S chirality at C-2 in flavanone. All natural flavanone retain the same stereochemistry at C-2 and are levorotatary (Miyahisa et al., 2006).
Page 51
B OH OH OH H HO O HO O HO O BH OH H OH O OH O OH O H H OH HA A 184f 184g
Scheme 3.4: Enzymatic conversion of chalcone into flavanone (naringenin)
The biosynthesis of flavones is carried out from (2S)-flavanones where the reaction is catalyzed by a membrane-bound cytochrome P450 monooxygenase and flavone synthase II (FSII) (Leonard et al., 2005). Parsly enzyme is also reported to be involved in this transformation (Stotz and
Forkmann, 1981). For the conversion of naringenin (184g ) to flavone apigenin
(29 ), the proposed mechanism suggests that in the first step 2– hydroxyflavanone ( 184h ) is formed. Then dehydratase enzyme catalyzes water elimination transforming the intermediate into flavone, apigenin ( 29 )
(Scheme 3.5) (Stotz and Forkmann, 1981).
OH OH OH OH HO O HO O HO O [O] H2O H H OH O OH O OH O 184g 184h 29
Scheme 3.5: Conversion of flavanone into flavone (apigenin)
Page 52
Again for the synthesis of flavonols, flavonones act as intermediates.
Flavone synthase is the enzyme which brings about the conversion of naringenin ( 184g ) into kempferol ( 184k). Most probable mechanism involves formation of dihydrokaempferol ( 184i) and 2-hydroxy dihydrokaempferol
(184j). Kaempferol ( 184k) is formed by elimination of water molecule from 2- hydroxy dihydrokaempferol ( 184j) (Lau, 2008) (Scheme 3.6).
OH OH HO O HO O [O] H OH OH O OH O 184g 184i
OH OH OH HO O HO O H2O
OH Flavonol synthase OH OH O OH O 184k 184j
Scheme 3.6: Enzymatic conversion of flavanone into flavonol (kaempferol)
3.2.2. Glycosylation of Flavonoids
Glycosylation is common in flavonoids, where one or more hydroxyl groups of flavonoids are bound with sugar molecules. Sugar moiety may get itself attached with flavone nucleus either by O- linkage or by C- linkage and are called O-glycoside and C- glycoside respectively (Fig. 3.8). Different types of enzymes catalyze different types of glycosylations and a number of these
Page 53
enzymes have been isolated e.g., flavonol O-glycoside transferases i.e., 3-O- glucosyl transferase, 3-O-glucosiderhamnosyl transferase, 3-O-glucoside xylosyltransferase were isolated from tulip anthers (Kleinehollenhorst et al.,
1982).
OH
HO O
Flavonol 3-O glucosyltransferase O OH O OH O
HO OH OH Kaempferol-3-glucoside OH
OH HO O
HO O O OH O OH Flavonol 3-O galactoside rhamnosyltransferase OH HO OH O O HO O OH O HO HO OH Kaempferol-3-rutinoside HO O
O OH O OH O
Flavonol 3-O galactosyltransferase HO OH HO
Kaempferol-3-galactoside
Figure 3.8: Enzyme catalaysed glycosylation of flavonoids
Page 54
3.3. Biosynthesis of Steroids
Class of compounds with 1,2-cyclopentanoperhydrophenantherene nucleus is steroid. Based on the origin, steroids are further classified e.g., if steroids are isolated from plants they are called phytosterols, if originated from animals, they are called zoosterols, mycosterols are isolated from fungi and marine sterols are isolated from marine organisms.
Naturally occurring sterols may contain cholestane, stigmastane or ergostane nuclei. It is elaborated that all steroids are derived from two triterpenes i.e., lanosterol ( 185 ) and cycloartenol ( 186 ). Furthermore, 185 is precursor of animal sterols while ( 186 ) is modified by series of transformation into phytosterols. These two parent triterpenes are synthesized by cyclization of squalene ( 116 ). Steps for the formation of squalene involve mevalonate pathway starting from acetyl coA ( 184 ) which is first converted into acetoacetyl CoA (187) where acetoacetyl CoA thiolase brings out this interconversion, and then into 3-hydroxy-3-methylglutaryl-CoA ( 188 ) which is further reduced to mevaldic acid (189 ) and then to mevalonic acid ( 190 ).
HMG-CoA synthase and HMG-CoA reductase are the enzymes which are involved in synthesis of 188 and reduction of 188 to 190.
Mevalonate kinase catalyzes phosphorylation of mevalonic acid to give mevalonic acid-5-diphosphate ( 191 ). Mevalonate–PP decarboxylase, on
Page 55
decarboxylation and dehydration of (191 ) gives isopentenylpyrophosphate
(IPP) ( 192 ). IPP isomerizes to give dimethylallylpyrophosphate (DMAPP)
(193 ) (Scheme 3.7).
H O OO OH H3CC SCoA H3C SCoA - CoA-SH 187 + CoA-SH HO2C O + CH3CO-SCOA O O SCoA 188 H2C C SCoA H2C C SCoA NADP 184
NADPH
ADP ATP NADPH3 NADPH OH OH OH
CH OPP HO2C CH2OH HO C CHO H OO 2 2 190 191 189
- CO2
- H2O
CH3 CH3 isomerase CH2OPP CH2OPP 193 192
Scheme 3.7: Mevalonate pathway; formation of IPP and DMAPP
IPP ( 192 ) and DMAPP ( 193 ) combine to yield geranyl pyrophosphate
(GPP), ( 194 ). In the next step farnesyl pyrophosphate (FPP), (195 ) is formed due to the condensation of GPP ( 194 ) and DMAPP ( 193 ) in the presence of enzyme FPP-synthase (Qureshi and Porter, 1981) (Scheme 3.8).
Page 56
CH3 OPP OPP -HOPP
H OPP
PPO trance 194 cis 194 H
-HOPP 193 PPO
OPP PPO trance 195 cis 195
Scheme 3.8: Mevalonate pathway; formation of farnesyl pyrophosphate
Presqualen ( 196 ) is then formed due to the condensation of two molecules of FPP ( 195 ). 196 on rearrangement gives squalene ( 116 ) and all these steps are catalyzed by squalene synthase (Goldstein and Brown, 1990).
116 on different oxidative and non-oxidative cyclization gives steroids and triterpenes (Abe et al., 1993) (Scheme 3.9).
OPP H H PPO PPO
Enzyme
OPP X-Enz 1, 2 shift 196
H NADP+ 195 NADPH
116
Scheme 3.9: Conversion of presqualene into squalene
Page 57
In case of oxidative pathway a 2, 3 epoxy squalene intermediate ( 197 ) is formed which is the intermediate for the synthesis of lanosterol ( 185 ) (Finar,
2002). 2,3-Epoxysqualene ( 197 ) is first converted into an ionoic intermediate
(198 ) which after a series of 1,2 migrations and removal of protons, is converted into ( 185 ) and ( 186 ). Lanosterol synthase and cycloartenol synthase catalyze these reactions (Scheme 3.10).
H + H H H - + Enz-X 9 8 H - HO - HO O H Rearrangement H
197 198 185 - Rearrangement H H
H H H H H
X-Enz - Enz-X HO + HO H - H H 186
Scheme 3.10: Formation of lanosterol and cycloartenol
Lanosterol ( 185 ) is converted into zymosterol ( 199 ) by demethylation.
In this conversion three methyls are lost. Sequence of conversion of lanosterol to cholesterol has been suggested as first 14-α methyl is oxidized to aldehyde and removed as formic acid to give compound 200, then reduction of 14-α position and 15-β position yield 201 . Then 4-α methyl is oxidized to
Page 58
carboxylic acid, 3-hydroxyl group is converted into ketone then removal of carbon dioxide yield 202 and ketone is reduced back to hydroxyl group, methyl is then oxidized and removed as carbon dioxide to give compound
199 (Scheme 3.11).
R R R
H
HO HO HO 200 185 R
OX
HO O -CO HO HO H H 2 H COOH COOH 202 201
H HO
Scheme 3.11: Conversion of lanosterol into zymosterol
Two alternative pathways are reported for the synthesis of cholesterol
(209 ) from zymosterol ( 199 ) which differs in the step when double bond at C-
24 is reduced i.e., reduction may occur in early steps or in the end of cholesterol biosynthesis. If route I (Scheme 3. 12) is followed then 24-ene is reduced in the final step while in the first step ∆8 ∆7 isomerase converts 199 into cholesta-7,24-dien- 3β-ol ( 203 ) followed by ∆ 5 desaturase which converts
Page 59
203 into 7-dehydrodesmosterol ( 204 ) which is further converted into desmosterol ( 205 ) by ∆ 7 reductase, and finally ∆ 24 reductase transforms desmosterol into cholesterol ( 209 ). In case of route II (Scheme 3.12), ∆ 24 reductase reduces 24-ene in the first step transforming 199 into cholest-8(9)- en- 3β-ol ( 206 ). Then ∆ 8 ∆7 isomerase acts to synthesize lathosterol ( 207 ). ∆ 5 desaturase converts lathosterol into 7-dehydrocholesterol ( 208 ) and in the end ∆ 7 reductase reduces double bond at C-7 finally producing cholesterol
(209 ) (Liscum, 2002) (Scheme 3.12).
Route I Route II
H OH 199
H 206 H H 203 OH OH
204 H H 207 H H OH OH
205 208 H H H H OH OH
H H OH 209
Scheme 3.12: Conversion of zymosterol into cholesterol
Page 60
3.3.1. What are Ecdysteroids?
Ecdysteroids are a class of compounds comprises of polyhydroxylated ketosteroids, with various tails that are structurally similar to androgens.
They are well studied as plant and insect growth factors, and derived their name “ ecdy ” from the process of molting in insects, called ecdysis (Dinan et al., 2001). Ecdysteroids are generally characterized as a basic skeleton containing 27-29 carbon atoms with a long sterol alkyl side chain on C-17 and a 7-en-6-one chromophore group in ring B. Other characteristic feature is the presence of hydroxyl groups at 3 β- and 14 α- positions with further hydroxylation may be observed at C-1, 2, 5, 11, 20, 22, 25, 26 or 27. These hydroxyl functions are then responsible for further derivatization like etherification, esterification and glycosylation etc (Bathori et al., 2008). In most of the insects, the main and most significant ecdysteroid is 20- hydroxyecdysone which was first isolated from crayfish ( Jasus lalandii )
(Hampshire and Horn, 1966).
3.3.2. Biosynthesis of Ecdysteroids
In vivo studies suggested that 7-dehydro cholesterol ( 208 ) is the intermediate for the synthesis of ecdysteroids (Horn et al., 1974; Milner et al.,
1986). Later, several studies (Grieneisen et al., 1991; Warren et al., 1988 ),
(Rudolph and Spaziani, 1992) confirmed that 7-dehydrocholesterol ( 208 ) is
Page 61
converted into trideoxy ecdysone ( 211 ) due to a series of modifications
(Scheme 3.13) in sterol basic nucleus and side chain while maintaining A/B cis ring junction. The mechanism of this conversion is not yet clear but it is postulated that a 3-oxo- ∆4 steroid intermediate ( 210 ) is formed which may be involved in A/B cis ring junction (Davies et al., 1981; Rees and Isaac, 1985).
Compound ( 210 ) serves to produce 2,22,25-trideoxyecdysone ( 211 ), which in turn is transformed into ecdysone ( 215 ) (Kappler et al., 1989; Rees, 1989).
Sequences of terminal hydroxylations to form several derived ecdysones has been studied by using radioactive substrates (Kappler et al., 1989). It was found that 3-dehydro ecdysone ( 220 ) was also produced in addition to α- ecdysone ( 214 ) by lepidoptarin prothoracic gland. As discussed earlier that a
3-oxo- ∆4 steroid intermediate ( 210 ) is formed which either retain oxo function throughout the biosynthesis yielding 3-dehydroecdysone ( 220 ) or hydroxyl function is introduced at the end to give α-ecdysone ( 214 ) Various possibilities are summarized in scheme 3.13. 14-α-hydroxyl group is believed to be introduced before or simultaneously with introduction of 6-oxo-7-ene functionality in the nucleus (Kappler et al., 1989; Rees, 1989). Other hydroxyl groups i.e., 2, 22 and 25 follow the sequence that 25-hydroxylation occurs first yielding ( 212 ), followed by 22 ( 213 ) and 2 hydroxyl ( 214 ) forming in the
Page 62
end (Rees and Isaac, 1985). Finally α-ecdysone ( 214 ) is transformed into 20- hydroxyecdysone ( 215 ), after hydroxylation at C-20. (Scheme 3.13)
H H H H OH OH 209 208
H H O 210
207 216 H H H H HO O
211 217 H OH H OH HO O O O OH OH 212 218 H OH OH H OH HO OH O O OH O OH 213 219 H OH OH H OH HO OH O O OH O OH 214 HO HO H OH 220 H OH HO OH OH O O O OH
HO 215 H OH HO O Scheme 3.13: Different routes for the formation of ecdysone
Page 63
A simpler route was purposed by Ohyama K et al. in 1999 who studied the biosynthesis of 20-hydroxy ecdysone ( 215 ) (Ohyama et al., 1999) and found that ( 207 ) and ( 209 ) are first converted into 7-dehydrocholesterol ( 208 ), where a 5-α, 6-α epoxide is formed transforming ( 208 ) into an intermediate 7- dehydrocholesterol 5-α, 6-α epoxide ( 221 ). In next step 6-β proton is shifted to position 5 and ketone is formed at position 6 giving basic ecdysteroid skeleton ( 211 ) A/B cis ring junction. This intermediate is ultimately converted into 20-hydroxyecdysone ( 215 ) after a series of hydroxylations
(Ohyama et al., 1999) (Scheme 3.14).
H H H H H H HO HO 209 207 HO 208
OH OH
OH
HO H H H OH H OH HO HO HO O H O O 221 211 215
Scheme 3.14: Mechanism for introduction of 6 ketonic group in ecdysones
As far as the intermediacy of 7-dehydrocholesterol is concerned, some groups of researchers claim that it is not a compulsory intermediate rather
Page 64
introduction of double bond at seven position is also possible at later stages of ecdysone biosynthesis. This study suggests the presence of an alternative route for biosynthesis (Hyodo and Fujimoto, 2000).
Species which cannot synthesize cholesterol directly utilize other sterols like sitosterol, campasterol, stigmasterol etc. These species can modify different sterols into cholesterol by dealkylations of side chain. Some omnivorous and phtophagus insect species are reported to synthsize makisterone A, a C-28 ecdysone, by using campasterol. Occurrence of C-29 ecdysone is also reported (Feldlaufer et al., 1991).
Page 65
CHAPTER 4
Results and Discussions of Metabolites Isolated from
Aerva Javanica
Page 66
4.1. Results and Discussion
Methanolic extract of Aerva javanica was divided into n-hexane, chloroform, ethyl acetate and butanol fractions, which were evaluated for antioxidant, antibacterial and enzyme inhibitory potential. The ethyl acetate fraction showed considerable antioxidant activity at concentration of
50µg/ml, and exhibited weak antibacterial activity. Therefore, this fraction was subjected to purification to get bioactive metabolites, as a result, twenty nine pure compounds, mostly steroids, were identified with the help of spectroscopic analysis.
4.2. Structure Elucidation of the Isolated Compound
4.2.1. Structure Elucidation of Aervecdysone A (222)
Compound 222 was isolated as white amorphous powder, which absorbed in the IR region at 3470, 1651 and 1630 cm-1 due to hydroxyl, carbonyl and olefinic functions HO 28 22 24 21 27 20 25 respectively. The UV absorption maxima at 18 O 17 26 19 11 13 9 C D 243 nm indicated the possibility of an α, β- HO 1 10 15 A OH 3 5 B 7 unsaturated carbonyl system. The EIMS of HO O 222 exhibited the molecular ion peak at m/z 222
476 with other fragments at m/z 458, 440, 422, 301 (100) and 283 (Fig. 4.1 A).
The HREIMS of this compound showed molecular ion peak at m/z 476.3132
Page 67
[M] + corresponding to the molecular formula C 28 H44 O6 with seven double bond equivalences (DBE). The 1H-NMR spectrum of 222 (Table 4.1) displayed signals for five tertiary methyls at δ 1.16 (Me-27), 1.12 (Me-21), 1.04 (Me-26),
0.86 (Me-19) and 0.75 (Me-18), and one secondary methyl at δ 0.91 (d, J = 6.4
Hz, Me-28). An olefinic methine resonated at δ 5.75, which showed allylic
COSY correlation with two methines resonating at δ 2.95 (m, H-9) and 2.29
(dd, J = 4.0, 11.3 Hz, H-5). The signal for H-5 ( δ 2.29) was further correlated in
COSY spectrum with a methylene at δ 1.65 and 1.58 (m, H-4) (Fig. 4.1 B). Two vicinal oxymethines were observed in the 1H-NMR spectrum at δ 3.84 (dd, J =
3.8, 11.3 Hz, H-3) and 3.73 (d, J = 3.1 Hz, H-2). Another oxymethine appeared at δ 3.48, which showed COSY interaction with a methylene resonating at δ
2.23 and 2.12, attested for H-22 and H-23 respectively. This data revealed a steroidal skeleton of 222 , which was further substantiated through 13 C-NMR spectrum (Table 4.1). The 13 C-NMR spectrum displayed altogether 28 signals, which were attested for six methyls, seven methylenes, eight methines and seven quaternary carbons due to DEPT experiment. Although the 1H-NMR spectrum displayed mostly tertiary methyl signals as usually observed in case of triterpenoids, but 13 C-NMR data confirmed a steroidal nature of 222 .
The most downfield carbon signals resonating at δ 204.5 (C-6), 121.2 (C-7) and
166.0 (C-8) were attested for an enone system. Other than three oxymethine
Page 68
carbons [ δ 67.4 (C-2), 67.0 (C-3) and 77.0 (C-22)], the spectrum displayed three oxygenated quaternary carbons at δ 84.2 (C-14), 74.7 (C-20) and 73.2 (C-25).
These data, when compared with the literature values, closely resembled with the data of ecdysteroid-type skeleton (Zhou et al., 2005). The various substitutions in 222 were fixed through COSY correlations and HMBC experiment (Fig. 4.1 B), in which the HMBC correlations of olefinic proton ( δ
5.75) was observed with carbons at δ 49.0 (C-5), 204.5 (C-6) and 33.4 (C-9) that helped to fix carbonyl function at C-6. Further, the HMBC correlation of this olefin and of Me-18 ( δ 0.75) with an oxygenated quaternary carbon at δ 84.2
(C-14) confirmed a hydroxyl group at C-14. Me-18 was further correlated with carbons at δ 30.9 (C-12), 47.0 (C-13) and 48.5 (C-17). Another singlet methyl ( δ 1.12) was found to exhibit HMBC correlation with C-17 and hence was designated as Me-21, which showed further HMBC interactions with a quaternary carbon at δ 74.7 (C-20) and an oxymethine at δ 77.0 (C-22). The
HMBC correlations of a doublet methyl ( δ 0.91) with a methylene at δ 34.0 (C-
23), a methine at δ 42.1 (C-24) and an oxygenated quaternary carbon at δ 73.2
(C-25) helped to fix it as Me-28. Two further tertiary methyls ( δ 1.16 and 1.04) were found to interact in HMBC spectrum with C-24 (δ 42.1) and C-25 ( δ 73.2) and therefore were attributed to an isopropyl moiety.
Page 69
m/z406
HO m/z 362 HO m/z 346 m/z319 O 70m/z 114m/z O 130m/z HO 157m/z H OH HO HO H OH H O HO m/z 476 [M]+ O
+ + + HMBC ())and COSY ( 458 [M-H2O ] 440 [M-2H2O ] 422 [M-3H2O ] A B
Figure 4.1: A] Mass fragmentation and B] important HMBC and COSY correlations observed in the spectra of 222
The above whole data was comparable with the data reported for 24-
Epi -makisterone A and other similar compounds (Wu et al., 2010). The main difference was observed in molecular formula which showed an additional
DBE when compared to that of reference compound. The formula of 222 showed seven DBE, whereas, the above discussed data accommodated six
DBE, therefore, the remaining one DBE could be attributed to a pyran ring between C-20 and C-25. The relative stereochemistry at various chiral centers was established due to NOESY correlations (Fig. 4.2), coupling constants and deriding model (Fig. 4.2). A strong NOESY correlation between Me-18 ( δ
0.75) and Me-21 ( δ 1.12) revealed β-orientation of Me-21. This correlation further confirmed equatorial β-attachment of pyran ring at C-17. A weak
Page 70
NOESY correlation between Me-21 and Me-28 ( δ 0.91) revealed β- and axial orientation of Me-28.
OH CH H 3 H H3C O H H CH3 H CH3 H CH3 CH H 3 H H H O H H H H H OH H H HO H HO H H H
Figure 4.2: NOESY correlation observed in the spectrum of 222
Moreover, the absence of NOESY correlation between H-22 (3.48, brd, J = 4.0
Hz) and Me-28 indicated that H-22 must be α-equatorial and 22-OH must be
β-axial . A strong NOESY correlation between Me-19 and H-5 clearly
indicated a cis -junction of ring A and B with β-axial orientation of H-5.
Coupling constant of H-2 at δ 3.73 (1H, d, J = 3.1) and the absence of NOESY
correlation between H-2 and Me-19 ( δ 0.86) revealed that H-2 must be α-
axial , and therefore, 2-OH was established as β- equatorial . This deduction
was further supported by a NOESY correlation between H-2 and H-9 ( δ
2.95). Absence of NOESY correlation between H-3 and H-5 revealed that H-3
must be equatorial and α, therefore, 3-OH must be β-axial . These NOESY-
based inferences were further substantiated due to molecular model. The
stereochemistry of hydroxyl group at C-14 was defined as α- on biogenetic
Page 71
ground. The above discussion led to the structure of 222 as 20,25-epoxy-
2β,3 β,14 α,22 β-tetrahydroxy-5β-ecdysteroid and is named as aervecdysteroid
A, which has recently been published as a new compound (Saleem et al.,
2013).
4.2.2. Structure Elucidation of Aervecdysone B (223)
Compound 223 was also obtained as white amorphous powder. The
EIMS showed molecular ion peak at 474 with HO the same fragmentation pattern as observed for O
222 (Fig. 4.3 A), while the molecular formula HO H OH HO C28 H42 O6 with eight DBE was confirmed due to H O 223 HREIMS. In IR spectrum, absorptions were observed for hydroxyl (3475 cm -1), carbonyl (1635 cm -1) and olefinic (1650 cm -
1) functional groups, whereas, the absorption maxima at 242 nm in UV spectrum indicated the possibility of α, β-unsaturated carbonyl system. This data was superimposable to the data observed for 222 , whereas, the 1H-NMR spectrum of 223 (Table 4.1) was also identical to the spectrum of 222 , with the main difference of the absence of a secondary methyl in 223 , and instead the resonance of an olefinic methylene at δ 5.02 (s) and 4.81 (s) was observed, which was correlated in HSQC spectrum with a methylene at δ 110.7.
Further, the 13 C-NMR spectrum was also missing a methine carbon at δ 42.1,
Page 72
when compared to that of 222 , and additionally the spectrum displayed an olefinic quaternary carbon at δ 153.7. This data confirmed that Me-28 of 222 must have been oxidized to an olefinic system in 223 . The remaining tertiary methyls resonated at δ 1.32 (Me-26), 1.23 (Me-27), 1.14 (Me-21), 0.87 (Me-19) and 0.76 (Me-18). The above deduction was substantiated due to an additional DBE when compared to that of 222 . The HMBC correlation of H-28
(δ 5.02 and 4.81) with C-23 (33.6), C-24 (153.5) and C-25 (72.3) (Fig. 4.3 B) finally confirmed the oxidation of Me-28 into an exo-cyclic double bond.
m/z406 H m/z 362 HO m/z 346 HO m/z319 O 68m/z 112m/z O 128m/z HO 155m/z HO H OH H OH HO H HO O O m/z 474
+ + + HMBC ())and COSY ( 456 [M-H2O ] 438 [M-2H2O ] 420 [M-3H2O ]
A B
Figure 4.3: A] Mass fragmentation and B] important HMBC and COSY correlations observed in the spectra of ( 223)
Based on the above information, and comparison of the data with that of 223 , the compound 223 was characterized as 24,28-dehydro-20,25-epoxy-2β,3 β,14 α,22 β-
Page 73
tetrahydroxy-5β-ecdysteroid and is named as aervecdysteroid B, which has also been published as a new natural product (Saleem et al., 2013).
Table 4.1: 1H- and 13 C-NMR data of 222 and 223 (CDCl 3, 400 and 100 MHz)
Position δH (J = Hz) δC δH (J = Hz) δC 1 1.70, m 36.3 1.72, m 36.2 1.29, m 1.28, m 2 3.73, d (3.1) 67.4 3.72, d (3.2) 67.2 3 3.84, dd (3.8, 11.3) 67.0 3.87, dd (3.2, 11.1) 67.0 4 1.65, m 31.5 1.63, m 31.4 1.58, m 1.57, m 5 2.29, dd (4.0, 11.3) 49.0 2.30, dd (3.5, 11.1) 49.5 6 _ 204.5 _ 205.1 7 5.75, s 121.2 5.76, s 121.4 8 _ 166.0 _ 166.2 9 2.95, m 33.4 2.97, m 33.5 10 _ 38.0 _ 38.1 11 1.66, m 20.3 1.65, m 20.5 1.53, m 1.52, m 12 2.03, m 30.9 2.00, m 30.8 1.80, m 1.81, m 13 _ 47.0 _ 48.7 14 _ 84.2 _ 84.1 15 1.90, m 31.0 1.91, m 31.1 1.47, m 1.45, m 16 1.92, m 20.5 1.92, m 20.4 1.73, m 1.74, m 17 2.28, t (9.2) 48.5 2.27, t (9.5) 48.6 18 0.75, s 17.2 0.76, s 17.2 19 0.86, s 23.7 0.87, s 23.7 20 _ 74.7 _ 76.2 21 1.12, s 20.5 1.14, s 20.0 22 3.48, brd (4.0) 77.0 3.47, brd (3.8) 77.2 23 2.23, m 34.0 2.25, m 33.6 2.12, m 2.10, m 24 1.60, m 42.1 _ 153.5 25 _ 73.2 _ 72.3 26 1.04, s 24.6 1.32, s 29.1 27 1.16, s 29.6 1.23, s 29.8 28 0.91 (d, 6.4) 16.1 5.02, s 110.7 4.81, s
Page 74
4.2.3. Structure Elucidation of Aervecdysone C (224)
Compound 224 was also isolated as white amorphous solid, which showed similar physicochemical properties to that of 222 and 223 , indicating it to be an ecdysteroid also. The OH HO molecular ion peak in EIMS was OH OH observed at m/z 494 with major H OH HO fragments at m/z 476, 458, 442, 363, 345 H O 224 (100), 327, 329 (Fig. 4.4 A). The molecular formula of 224 with six DBE could be determined as C 28 H46 O7 through
HREIMS ( m/z 494.3244). The 1H-NMR spectrum (Table 4.2) displayed similar signals as were observed in the spectrum of 222 with a the difference in substitution pattern at ring A. Instead of two vicinal oxymethines, as were characterized for 222 , the spectrum of 224 also displayed two oxymethines at
δ 3.59 (dd, J = 4.5, 10.4 Hz, H-1) and 3.75 (m, H-3), which do not show mutual correlation in COSY spectrum, rather both were correlated with a methylene resonating at δ 1.57 and 1.24 (Fig. 4.4 B). It was therefore, assumed that the two hydroxyl groups at ring A of 224 must be present at C-1 and C-3, instead
C-2 and C-3 as were found in 222 and 223 . The position of these two hydroxyl groups was substantiated through HMBC spectral analysis, in which Me-19
(δ 0.82, s) exhibited correlation with an oxygenated carbon at δ 66.7 (C-1) (Fig.
Page 75
4.4 B). The other difference between 222 and 224 was observed through the molecular formula of 224 which showed six DBE that indicated the absence of pyran ring as was found in 222 and 223 .
The stereochemistry of hydroxyl group at C-1 was determined as β and axial due to NOESY correlation of H-1 and H-9 and was also substantiated by molecular model. Based on the above data and comparison with the data of
222 -223 , compound 224 was characterized as 1β,3 β,14 α,20 β,22 β,25- hexahydroxy-5β-ecdysteroid and is named as aervecdysteroid C, which we recently have published as another new natural product (Saleem et al., 2013).
m/z 407 m/z 363 OH HO OH
m/z OH 319 OH 87 m/z OH OH 131 m/z OH 175 m/z H OH OH HO H HO O m/z 494 [M]+ O
+ + + ())( 476 [M-H2O ] 458 [M-2H2O ] 442 [M-3H2O ] HMBC and COSY
A B
Figure 4.4: A] Mass Fragmentation and B] important HMBC and COSY correlations observed in the spectra of 224
Page 76
4.2.4. Structure Elucidation of Aervecdysone D (225)
The NMR (Table 4.2) and other spectroscopic data revealed that compound 225 must have the same structure as that of 224 , however, a similar difference was observed between OH HO
224 and 225 , as was observed between OH OH 222 and 223 ; that is the oxidation of Me- H OH HO 28 into an olefinic system. The 1H-NMR H O 225 spectrum of 225 displayed the signals for an olefinic methylene at δ 5.01 (1H, s) and 4.82 (1H, s), whereas, the 13 C-NMR spectrum (Table 4.2) showed the olefinic carbons at δ 154.4 (C-24) and 108.1
(C-28). Another difference was noted in the molecular formula (C 28 H44 O7), which afforded an additional DBE when compared to that of 224 , which was attributed to the olefinic system. The HMBC correlations (Fig. 4.5 B) of H-28 with C-23, C-24 and C-25 confirmed oxidation of methyl-28 in 224 into an olefin in 225 . Based on the above discussion, the structure of 225 was established as 24,28-dehydro-1β,3 β,14 α,20 β,22 β,25-hexahydroxy-5β- ecdysteroid, which is named as aervecdysteroid D and has been published in the year 2013 (Saleem et al., 2013).
Page 77
m/z 363 129 m/z OH m/z 173 85 m/z HO OH OH
OH OH OH
OH OH m/z H OH 319 HO HO O H O m/z 492 [M]+ HMBC ())and COSY ( A B
Figure 4.5: A] Mass fragmentation and B] important HMBC and COSY correlations observed in the spectra of 225
Page 78
Table 4.2: 1H- and 13 C-NMR data of 224 and 225 (DMSO-d6 , 400 and 100 MHz) Position δH (J = Hz) δC δH (J = Hz) δC 1 3.59, dd (4.5, 10.4) 66.7 3.60, dd (4.3, 10.2) 66.9 2 1.57, m 36.6 1.56, m 37.0 1.24, m 1.23, m 3 3.75, m 66.5 3.77, brs 66.3 4 1.54, m 31.5 1.56, m 32.0 1.47, m 1.45, m 5 2.17, m 50.1 2.19, m 49.9 6 - 202.7 _ 203.0 7 5.61, s 121.3 5.63, s 121.4 8 - 165.3 _ 165.2 9 2.99, m 33.2 2.97, m 33.1 10 - 37.6 _ 37.5 11 1.63, m 20.0 1.65, m 20.3 1.51, m 1.52, m 12 1.98, m 31.0 1.97, m 30.8 1.71, m 1.70, m 13 - 46.9 - 46.8 14 - 82.9 - 83.1 15 1.76, m 30.3 1.75, m 31.0 1.48, m 1.48, m 16 1.88, m 20.3 1.90, m 20.4 1.62, m 1.63, m 17 2.27, t (9.2) 48.5 2.29, t (9.4) 48.6 18 0.75, s 17.2 0.76, s 17.1 19 0.82, s 23.9 0.82, s 24.0 20 - 75.8 _ 75.6 21 1.08, s 21.0 1.03, s 20.5 22 3.30, m 75.6 3.43, m 76.1 23 2.22, m 33.2 2.25, m 33.9 1.93, m 1.90, m 24 1.47, m 42.7 _ 154.4 25 - 71.4 _ 71.3 26 1.00, s 29.7 1.22, s 29.8 27 1.03, s 29.4 1.19, s 29.5 28 0.91, d (6.8) 16.6 5.01, s 108.1 4.82, s 1-OH 4.45, br s _ 4.47, brs _ 3-OH 4.34, brs _ 4.36, brs _ 14-OH 4.65, s _ 4.67, s _ 20 -OH 3.62, s _ 3.63 _ 22-OH 4.31, d (4.5) _ 4.57, d (4.7) _ 25-OH 4.14, s _ 4.75, s _
Page 79
4.2.5. Structure Elucidation of 24-Epi-makisterone A (226)
Compound 226 was also found to be an ecdysteroid, as it displayed nearly super imposable NMR (Table 4.3) HO OH and other spectroscopic data to that of 222 OH with the only difference in molecular HO OH formula. The molecular formula afforded HO O 226 one oxygen atom more when compared to that of 222 , and one DBE less than that of 222 . This difference revealed that
226 must be missing pyran ring, rather it has hydroxyl functions at C-20 and
C-25. Further, the whole spectroscopic data was identical to the data reported for 24-Epi-makisterone A (Zhu et al., 2001), hence, compound 226 was found to be the same, which has been isolated for the first time from Aerva javanica.
4.2.6. Structure Elucidation of 5-β-2-deoxyintegristerone A (227)
The UV and IR data of 227 was similar to that observed for other ecdysteroids 222-226 indicating it to be HO OH also an ecdysteroid. The EIMS showed OH OH molecular ion peak at m/z 480 and OH molecular formula C 27 H44 O7 was HO O 227 established through HREIMS. The NMR data (Table 4.3) of 227 was similar to that of 224 , with the difference that 227
Page 80
afforded one carbon atom less than that of 224 . Methyl-28 which resonated in the NMR spectrum of 224 at δ 0.91 (3H, d, J = 6.8) was missing the spectrum of 227, besides the signal for methine-24 was also absent. Rather a methylene resonated at δ 1.30 (1H, m) and 1.61 (1H, m) in the 1H-NMR spectrum of 227, which was correlated with the carbon at δ 42.3 (C-24) in HSQC spectrum. The absence of Me-28 and presence of methylene-24 in 227 , was further substatiated due to HMBC spectrum (Fig. 4.6), in which the methyls of isopropyl moiety at δ 1.05 (H-26) and 1.02 (H-27) exhibited HMBC correlation with methylene at δ 42.3 (C-24). The structure was finally confirmed as 5-β-2- deoxyintegristerone A, which is a known ecdysteroid (Bathori et al., 2002).
This compound has also been isolated for the first time from our investigated source.
OH OH
OH OH
OH HO O
HMBC ())and COSY (
Figure 4.6: Important HMBC correlations observed in the spectra of 227
Page 81
4.2.7. Structure Elucidation of β-ecdysone (215)
Compound 215 was found to be the de-methyl analogue of 226 , as it displayed nearly the same NMR data (Table 4.3), with the difference of the absence of Me-28, which was further OH OH confirmed due to the molecular OH formula C27 H44 O7 with one carbon HO OH atom less than that of 226 . Instead of HO O 215 methine-24 as was observed in the
NMR spectra of 226 , a methylene resonated at δ 1.60 and 1.25 in the 1H-NMR spectra of 215 , which confirmed the above deduction. Further, the HMBC correlation of Me-26 (1.06, s) and Me-27 (1.03, s) with methylene-24 supported the final structure of 215 . Therefore, the comparison of the observed data with the reported one confirmed the compound to be 20- hydroxy ecdysone ( β-ecdysone) which is a well known and most abundant phytoecdysteroid (Zhu et al., 2001). In fact it was the major ecdysteroid in the flower extract of Aerva javanica, which is a known secondary metabolite, and has been reported for the first time from Aerva javanica .
Page 82
Table 4.3: 1H- and 13 C-NMR data of 226 (C 5D5N, 500 MHz) 227 and 215 (DMSO, 400 MHz) Compound 226 compound 227 compound 215
Position δH (J = Hz) δC δH (J = Hz) δC δH (J = Hz) δC 1 2.13, m 38.0 3.64, m 66.9 1.52, m 36.6 1.92, m 1.28, m 2 4.2, br s 68.0 1.50, m 37.0 3.57, br s 66.7 1.31, m 3 4.18, d (11.5) 68.1 3.68, m 66.0 3.75, br s 66.5 4 2.00, m 32.8 1.58, m 32.0 1.57, m 31.5 1.77, m 1.49, m 1.51, m 5 3.02, dd (4.0, 13.5) 51.4 2.17, dd (3.6, 13.2) 49.1 2.19, dd (3.6, 13.2) 50.1 6 - 203.4 203.6 - 202.6 7 6.28, d (2) 121.6 5.63, s 121.7 5.61, br s 120.4 8 - 166.1 - 166.0 - 165.2 9 3.58, t (9.5) 34.6 2.96, t (9.2) 34.9 2.99, t (9.5) 33.1 10 - 38.6 - 38.0 - 37.6 11 1.85, m 21.1 1.66, m 22.1 1.68, m 20.0 1.75, m 1.49, m 1.50, m 12 2.63, m 32.0 2.03, m 31.2 2.00, m 30.8 2.03, m 1.70, m 1.69, m 13 - 48.1 - 47.0 - 46.8 14 - 84.1 - 83.6 82.9 15 2.23, m 31.8 1.84, m 30.8 1.79, m 30.3 1.95, m 1.56, m 1.49, m 16 2.51, q (10, 21) 21.3 1.90, m 20.5 1.87, m 20.2 2.12, m 1.55, m 1.53, m 17 2.97, t (9.5) 49.9 2.25, t (9.2) 49.5 2.24, t (9.2) 48.6 18 1.22, s 17.9 0.74, s 18.5 0.75, s 17.1 19 1.06, s 24.4 0.86, s 23.2 0.82, s 23.8 20 - 76.9 - 76.4 - 75.6
21 1.59, s 21.8 1.05, s 21.0 1.04, s 20.9 22 3.99, dd (4.0, 11.0) 74.6 3.12, t (8.2) 77.0 3.10, t (7.6) 76.1 23 2.16, m 34.4 1.44, m 26.3 1.46, m 26.0 1.58, m 1.00, m 1.08, m 24 2.29, m 41.8 1.61, m 42.3 1.60, m 41.4 1.30, m 1.25, m 25 - 72.0 - 69.0 - 68.6 26 1.31, s * 28.2* 1.05, s* 30.2* 1.06, s* 30.0* 27 1.29, s* 26.5* 1.02, s* 28.9* 1.03, s* 28.9* 28 1.07 , d (6.4) 15.3 - - - - 2-OH 6.15,d (3.4) - 4.38,d (2.4) - 4.38,d (2.4) - 3-OH 6.00, brs - 4.41, d (6.0) - 4.39, d (6.0) - 14 -OH 4.83, - 4.68, s - 4.70, s - 20 -OH 6.37, s - 3.62, s - 3.58, s - 22 -OH 5.58, s - 4.36,d (4.8) - 4.35,d (4.8) - 25 -OH 6.10, d (4.5) - 4.10, s - 4.12, s - *values interchangeable
Page 83
4.2.8. Structure Elucidation of Kaempferol-3-O-β-D-[4 ′′′′′′′′-p-coumaroyl-α-
L-rhamnosyl(1→6)]galactoside (228)
Compound 228 was isolated as yellow amorphous powder whose
HRFABMS in positive mode depicted the molecular formula as C36 H37 O17 with 19 double bond equivalences 5' OH
8 (DBE). The IR spectrum displayed 3' HO O 1' 8a 1 6 4a absorption bands for hydroxyl (3384 3 O OH O 1'' OH -1 -1 HO cm ), conjugated ester (1712 cm ), O 5'' HO 3''' 1''' 3'' O -1 OH conjugated ketone (1685 cm ) and O O O 5''' HO phenyl (1600, 1550, 1520 cm -1) groups, whereas, UV spectrum showed 228 absorption maxima at 257, 267, 314 OH and 357 nm attested for an acylated kaempferol or quercetin glycoside
(Mabry et al., 1970).
The aromatic region of 1H NMR spectrum of 228 (Table 4.4) afforded two pairs of o-coupled doublets [ δ 7.88 (2H, d, J = 8.8 Hz), 6.68 (2H, d, J = 8.8
Hz) and 7.18 (2H, d, J = 8.4 Hz), 6.60 (2H, d, J = 8.4 Hz)] attributed to two p- substituted benzene rings. The first pair of signals could be assigned to the ring B of kaempferol moiety, whereas, the other pair of aromatic protons was identified for a E-p-coumaroyl. The protons of E-olefin moiety of p-
Page 84
coumaroyl resonated at δ 7.40 (1H, d, J = 16.4 Hz) and 6.00 (1H, d, J = 16.4
Hz). Besides, the same spectrum displayed two m-coupled doublets at δ 6.17
(1H, d, J = 1.6 Hz) and 6.04 (1H, d, J = 1.6 Hz) corresponding to the protons
H-6 and H-8 of ring A of kaempferol nucleus. The 1H NMR spectrum also showed signals for two sugar moieties between δ 4.68-3.33 with anomeric protons resonating at δ 4.58 (1H, d, J = 8.0 Hz) and 4.32 (1H, br s). Their corresponding carbons appeared in 13 C NMR spectrum at δ 105.0 and 100.1 respectively. The amount of coupling constants of these two protons revealed that the sugars must be β and α hexoses respectively. The coupling constant ( J
= 2.8 Hz) of an oxymethine of β-sugar at δ 3.55 due to H-4 indicated it to be a galactose unit, whereas the resonance of a doublet methyl at δ 1.12 indicated that the α-sugar could be rhamnose.
The 13 C NMR spectrum of 228 (Table 4.4) was in full agreement with the mass and proton data as it displayed signals for kaempferol nucleus ( δ
181.0, 164.2, 161.0, 158.0, 157.5, 133.7, 104.5, 98.8, 93.7), p-coumarate ( δ 166.8,
159.8, 145.5, 129.6, 125.4, 115.4, 113.4) and two sugar moieties ( δ 105.0, 100.1,
73.8, 73.6, 73.5, 70.5, 70.3, 69.0, 68.3, 66.4, 66.0, 16.7). All the assignments were accomplished through HSQC and COSY data, whereas, the position of various substituents was located through the HMBC analysis (Fig. 4.7). The anomeric proton ( δ 4.58, H-1'') of galactose moiety showed long range
Page 85
correlation with a quaternary carbon at δ 133.7 (C-3), whereas, the HMBC correlation of anomeric hydrogen ( δ 4.32) of rhamnose with the methylene at
δ 66.0 suggested its attachment to C-6 of the galactose. Relatively downfield shift ( δ 66.0) of this methylene carbon substantiated the above deduction.
Further, the downfield shift ( δ 4.68) of H-4 of rhamnose unit and its HMBC correlation with the carbonyl carbon at δ 166.8 confirmed the connectivity of coumaroyl moiety on C-4 of rhamnose (Fig. 4.7).
OH
HO O
O OH O OH HO O HO O OH O O O HO
HMBC ())and COSY ( OH
Figure 4.7: Important HMBC and COSY correlations observed in the spectra of 228
Acid hydrolysis of 228 provided three products which were separated by solvent extraction. The ethyl acetate layer contains kaempferol and coumaric acid. The glycone part was separated from the aqueous layer and was
Page 86
purified by preparative thin layer chromatography using a solvent system of
EtOAc-MeOH-H2O-AcOH (4:2:2:2), and thus were identified as D-galactose and L-rhamnose through their optical rotation values ([α]D23 +7.5˚ (c, 0.2 in
H2O) for L-rhamnose and comparison of the retention time of their trimethylsilyl (TMS) ethers with that of the standards in gas chromatography
(GC). The above discussed data was further compared with the related published compounds and found to be closely resembled to that of isorhamnetin-3-O-β-D-[4 ′′′-p-coumaroyl-α-L-rhamnosyl-(1→6)]galactoside
(Saleh et al., 1990 ) with the only difference that 228 contains kaempferol nucleus instead of isorhamnetin. Based on the observed data and above discussion, compound 228 was finally determined as kaempferol-3-O-β-D-
[4 ′′′-p-E-coumaroyl-α-L-rhamnosyl(1→6)]galactoside, which we have recently published as a new natural product (Mussadiq et al., 2013).
Page 87
Table 4.4: 1H- and 13 C-NMR data of 228 (CD 3OD; 500, 125 MHz) Position δH (J = Hz) δC 2 _ 157.5 3 _ 133.7 4 _ 181.0 4a 104.5 5 _ 161.0 6 6.04, d (1.6 ) 98.8 7 _ 164.2 8 6.17, d (1.6 ) 93.7 8a _ 158.0 1′ _ 121.5 2′,6 ′ 7.88, d (8.8) 130.9 3′,5 ′ 6.68, d (8.8) 114.7 4′ _ 160.4 1′′ 4.58, d (8.0) 105.0 2′′ 3.60, t (7.8) 70.5 3′′ 3.43, dd (2.8, 7.8) 73.5 4′′ 3.55, d (2.8) 68.3 5′′ 3.33, m 73.6 6′′ 3.72, dd (4.7, 10.8) 66.0 3.53, dd (3.0, 10.8) 1′′′ 4.32 br s 100.1 2′′′ 3.41, br d (6.4) 70.3 3′′′ 3.50, dd (6.4, 9.0) 69.0 4′′′ 4.68, t (9.6) 73.8 5′′′ 3.48, m 66.4 6′′′ 1.12, d (6.4) 16.7 1′′′′ _ 125.4 2′′′′,6 ′′′′ 7.18, d (8.4) 129.6 3′′′′,5 ′′′′ 6.60, d (8.4) 115.4 4′′′′ _ 159.8 7′′′′ 7.40, d (16.4) 145.5 8′′′′ 6.00, d (16.4) 113.4 9′′′′ _ 166.8
Page 88
4.2.9. Structure Elucidation of Kaempferol-3-O-β-D-[4 ′′′′′′′′-p-coumaroyl-α-
L-rhamnosyl(1→6)]3 ′′′′′′-p-coumaroylgalactoside (229)
Compound 229 was also isolated as yellow amorphous powder, which exhibited similar UV and IR data to that of 228 A pseudo -molecular ion peak
was observed at m/z 887 in positive 5' OH
8 3' HO O 1' FABMS while the molecular formula 8a 1 6 4a 3 O OH O 1'' OH C45 H42 O19 with 25 DBE was established HO O 5'' HO 3''' 1''' 3'' O O O through positive HRFABMS ( m/z O O O 5''' HO 887.2404). The 1H-NMR spectrum of 229
228 229 (Table 4.5) was similar to that of with OH OH fewer additional protons resonated at δ
[(7.55, d, J = 16.0 Hz), (7.29, d, J = 8.8 Hz), (6.73, d, J = 8.8 Hz) and (6.31, d, J =
16.0 Hz)] were attested for another p-coumaroyl moiety. This data indicated that the compound 229 must be analogue to 228 , with an additional p- coumaroyl moiety. The 13 C NMR spectrum (Table 4.5) of 229 was also in full agreement with the proton and mass data as it displayed signals for two p- coumarates [( δ 166.9, 160.0, 146.0, 130.0, 125.0, 116.1, 114.0) and ( δ 167.2, 160.4,
146.0, 130.1, 125.0, 116.1, 114.0)] along with usual signals for galactose, rhamnose and kaempferol nucleus as were observed in the spectra of 228 .
The attachment of additional coumaroyl unit was observed at C-3 of
Page 89
galactose due to down field shift of H-3 and its HMBC correlation with a carbonyl carbon at δ 167.2 Other HMBC and COSY correlations are shown in
Fig. 4.8.
OH
HO O
O OH O OH HO O HO O O O O O O HO
OH OH
HMBC ())and COSY (
Figure 4.8: Important HMBC and COSY Correlations observed in the spectra of 229
All C-H assignments were completed due to COSY, HSQC and HMBC analysis, and finally in comparison with the data of 228 , compound 229 was characterized as Kaempferol-3-O-β-D-[4 ′′′-p-coumaroyl-α-L-rhamnosyl
(1→6)] 3′′-p-coumaroyl galactoside, which we also recently published as a new natural product (Mussadiq et al., 2013).
Page 90
4.2.10. Structure Elucidation of Kaempferol-3-O-β-D-[4 ′′′-p-coumaroyl-α-L- rhamnosyl(1→6)]4 ′′-p-coumaroylgalactoside (230)
Compound 230 was also isolated as light yellow amorphous solid, which showed a similar positive FABMS
5' OH data as was observed for 229 , and the 8 3' HO O 1' 8a 1 6 4a 3 same formula (C 45 H42 O19 ) indicated that O OH O 1'' OH HO 230 has similar structural features as that O 5'' HO 3''' 1''' 3'' O OH O of 229 . The UV and IR data were also O O 5''' O O found similar to the data of 229. Only a little difference was observed in the 1H- 230 OH OH NMR spectrum (Table 4.5) of 230 , in which H-4 of galactose shifted downfield ( δ 5.29, d, J = 2.8), whereas, H-3 shifted upfield when compared to that of 229 . This information revealed that one coumaroyl moiety has acylated C-4 of galactose instead of C-3 as was found in compound 229 . This inference was substantiated through HMBC spectral analysis in which H-4 ( δ 5.29), of galactose showed long range correlation with a carbonyl carbon at δ 167.0 (Fig. 4.9). Other hydrogen and carbon nuclei of kaempferol, galactose, rhamnose, and two coumaroyl groups resonated at their usual positions (Table 4.5). Further 2D-NMR analysis and acid hydrolysis led to the structure of compound 230 as
Page 91
Kaempferol-3-O-β-D-[4 ′′′-p-coumaroyl-α-L-rhamnosyl(1→6)]4 ′′-p- coumaroylgalactoside which has recently been published as a new secondary metabolite (Mussadiq et al., 2013).
OH
HO O
O OH O OH HO O HO O OH O O O O O
OH OH HMBC ())and COSY (
Figure 4.9: Important HMBC and COSY correlations observed in the spectra of 230
Page 92
Table 4.5: 1H- and 13 C-NMR data of 229 and 230 (CD 3OD; 500, 125 MHz) Position δH (J in Hz) δC δH (J = Hz) δC 2 _ 158.2 _ 158.0 3 _ 134.5 _ 134.1 4 _ 181.6 _ 181.0 4a _ 104.0 _ 104.5 5 _ 159.8 _ 159.9 6 6.20, d (2.0) 99.0 6.23, d (1.6) 99.0 7 _ 162.7 _ 160.0 8 6.31, d (2.0) 94.1 6.36, d (1.6) 94.0 8a _ 159.8 _ 159.2 1′ _ 121.4 _ 121.0 2′,6 ′ 8.04, d ( 8.8 ) 131.6 8.03, d (8.8) 131.3 3′,5 ′ 6.85, d (8.8) 115.0 6.83, d (8.8) 115.8 4′ _ 160.0 _ 160.0 1′′ 4.78, d (7.6) 106.1 4.79, d (8.4) 105.5 2′′ 4.08, t (7.6) 69.0 3.80, t (8.4) 71.5 3′′ 4.80, dd (2.8, 7.6) 75.5 3.74,dd (2.8, 8.4) 72.5 4′′ 3.95, d (2.8) 66.5 5.29, d (2.8) 69.0 5′′ 3.55, m 73.6 3.64, m 73.2 6′′ 3.68, dd (4.9, 11.1) 67.0 3.69, dd (4.6, 10.8) 66.0 3.58 dd (2.9, 11.1) 3.51, dd (3.1, 10.8) 1′′′ 4.44, br s 100.2 4.40 (br s) 100.0 2′′′ 3.53, brd (6.5) 70.1 3.54, m 70.5 3′′′ 3.64, dd (6.5, 7.2) 69.4 3.66, dd (3.2, 13) 69.5 4′′′ 4.83, t (7.2) 74.0 4.77, m 74.1 5′′′ 3.62, m 66.0 3.52, m 67.1 3.15 6′′′ 1.05, d (6.4 ) 17.1 1.00, d (6.4) 17.1 1′′′′ _ 125.0 _ 125.0 2′′′′,6 ′′′′ 7.36, d (8.4) 130.0 7.30, d (8.4) 130.1 3′′′′,5 ′′′′ 6.76, d (8.4) 116.1 6.75 ,d (8.8) 116.0 4′′′′ _ 160.0 _ 159.5 7′′′′ 7.66, d (16.0) 146.0 7.59 , d (16.0) 146.0 8′′′′ 6.32, d (16.0) 114.0 6.20, d (16.0) 114.9 9′′′′ _ 166.9 _ 167.1 1′′′′′ _ 125.0 _ 125.0 2′′′′′,6 ′′′′′ 7.29, d (8.8) 130.1 7.33, d (8.4) 130.1 3′′′′′,5 ′′′′′ 6.73, d (8.8) 116.1 6.75, d (8.4) 116.0 4′′′′′ _ 160.4 _ 159.5 7′′′′′ 7.55, d (16.0) 146.0 7.63, d (15.6) 146.0 8′′′′′ 6.30, d (16.0) 114.0 6.15, d (15.6) 114.9 9′′′′′ _ 167.2 _ 167.0
Page 93
4.2.11. Structure Elucidation of kaempferol-3-O-β-D (6-E-p-coumaroyl) glucoside (231)
The compound 231 was also isolated as yellow amorphous powder. Its positive FABMS showed molecular ion at m/z 595 whereas positive
HRFABMS showed OH
[M+H]+ at 595.1449 HO O OH O HO OH corresponding to O O OH O O OH molecular formula 231
C30 H27 O13. Similarity of UV and IR data to that of compounds 228-230 suggested the compound 231 must also be an acylated flavone glycoside. Its
1HNMR spectrum (Table 4.6) displayed the signals for kaempferol nucleus, one sugar unit and one coumaroyl moiety. The amount of coupling constant of anomeric proton ( δ 4.85, d, J = 7.6) suggested β hexose, which was confirmed as glucose by optical rotation value i.e., [α]D20 +48.5˚(c, 10 in H 2O)
(Mukhtar et al., 2004) and comparison with standard after acid hydrolysis.
The connectivity of the sugar units was determined due to HMBC experiment (Fig 4.10) in which the anomeric proton of glucose moiety ( δ 4.85,
H-1'') showed long range correlation with a quaternary carbon at δ 133.3 (C-
3), whereas, the downfield shift and the HMBC correlation of H-6 resonating at δ [(4.35, dd, J = 4.6, 10.6) and (4.17, dd, J = 3.8, 10.6 Hz)] of glucose to a
Page 94
carbonyl carbon at δ 166.8 confirmed the connectivity of coumaroyl moiety on C-6 of glucose (Fig. 4.10). (Ren et al., 2011). Based on the observed data and comparison with the literature, compound 231 was determined as kaempferol-3-O-β-D (6-E-p-coumaroyl) glucoside which is a known secondary metabolite, but isolated for the first time from discussed source.
OH
HO O OH O HO OH O O OH O O OH HMBC ())and COSY (
Figure 4.10: Important HMBC and COSY correlations observed in the spectra of 231
Table 4.6: 1H- and 13 C-NMR data of 231 (CD 3OD; 500, 125 MHz) Position δH (J in Hz) δC Position δH (J in Hz) δC 2 _ 158.0 1′′ 4.85, d (7.6) 105.2 3 _ 133.3 2′′ 3.78, t (7.6) 72.2 4 _ 179.4 3′′ 3.91, m 74.3 4a _ 105.0 4′′ 3.97, m 70.5 5 _ 160.1 5′′ 3.53, m 75.7 6 6.21, d (2.0) 98.6 6′′ 4.35, dd (4.6, 10.6) 67.0 4.17, dd (3.8, 10.6) 7 _ 163.0 1′′′ _ 125.0 8 6.33, d (2.0) 94.1 2′′′,6 ′′′ 7.39, d (8.4) 130.4 8a _ 158.7 3′′′,5 ′′′ 6.66, d (8.4) 117.2 1′ _ 123.2 4′′′ _ 160.2 2′,6 ′ 8.04, d ( 8.8 ) 131.5 7′′′ 7.58, d (16.0) 145.1 3′,5 ′ 6.85, d (8.8) 114.6 8′′′ 6.28, d (16.0) 114.2 4′ _ 160.0 9′′′ _ 167.0
Page 95
4.2.12. Structure Elucidation of Aervfuranoside (232)
Compound 232 was isolated as pale yellow gummy material. The positive FABMS OH 6'' O OH 5'' 1'' exhibited a pseudo - HO 3' OH HO OH 3'' 9 1 HO 8 9a 9b O O 6' O molecular ion peak 1' 5' 7 5 3 5a 4a HO 6 O 4 Cl at m/z 619 [M+H] + OMe OMe 232 with an M+2 peak at m/z 621. The molecular formula C 26 H31 ClO 15 with 11 double bond equivalence
(DBE) could be established through positive HRFABMS. The IR spectrum exhibited absorption bands for hydroxyl group (3410 cm -1), aromatic system
(1595, 1560 and 1525 cm -1) and for methoxyl group at (1030 cm -1). The UV spectrum of 232 showed absorption maxima at 215, 239, 264, 298, 308 and 337 suggesting the presence of oxygen-substituted aromatic system.
The aromatic region of 1H NMR spectrum of 232 (Table 4.7) showed only two singlets at δ 7.79 and 7.51, whereas, two oxymethyl singlets resonated at δ 4.06 and 4.03. The presence of two sugar moieties were evidenced due to the appearance of two anomeric protons at δ 5.12 (1H, d, J =
7.5 Hz) and 4.48 (1H, br s), whereas, other sugar protons displayed their positions between δ 3.08-3.83. Besides, the same spectrum displayed a doublet methyl at δ 1.01 ( J = 6.0 Hz). The analysis of coupling constants of
Page 96
anomeric protons revealed the sugars to be β- and α-hexoses respectively, in addition the resonance of doublet methyl indicated that the α-hexose could be rhamnose.
The 13 C NMR spectrum (BB and DEPT) of 232 (Table 4.7) showed 26 signals for three methyl, one methylene, twelve methine and ten quaternary carbons.
The total twelve sp 2 carbon atoms ( δ 158.4, 158.3, 151.2, 141.9, 141.7, 140.9,
140.3, 114.4, 112.8, 112.3, 111.9 and 111.7) in 13 C NMR spectrum were attested for two benzene rings that afforded 08 DBE, whereas 02 DBE was attributed to two sugar units. The remaining 01 DBE could only be attested for another ring system, which was supposed to be present in the form of a furan ring between two benzene rings. The above discussion led to the identification of a chloroderivative of dibenzofuran glycoside.
The two sugar moieties were identified as glucose and rhamnose due to acidic hydrolysis of compound 232 followed by comparative TLC of the hydrolyzed sugars with the authentic samples of glucose and rhamnose and their optical rotation values [ α]D20 +50.5˚(c, 10 in H 2O) (Mukhtar et al., 2004) and [ α]D23 +7.5˚ (c, 0.2 in H 2O) (Liocharova et al., 1989). The substitutions on dibenzofuran nucleus were fixed due to HMBC spectral information in which the anomeric proton ( δ 5.12) of glucose showed long range interaction with the quaternary carbon at δ 151.2 (C-2) of benzene ring (Fig. 4.11). H-1 ( δ 7.79)
Page 97
exhibited HMBC correlation with the carbons at δ 151.2 (C-2), 114.4 (C-3)
158.3 (C-4a) and 112.8 (C-9a) whereas, H-6 ( δ 7.51) was correlated with the carbons at δ 112.8 (C-9a), 111.9 (C-9b), 140.3 (C-7) and 158.4 (C-5a). The two methoxyl groups resonating at δ 4.03 and 4.06 were fixed at C-4 and C-6 due to their HMBC correlation with the carbons at 141.9 and 140.3 respectively.
The HMBC interaction of anomeric proton of rhamnose ( δ 4.48) with the methylene carbon ( δ 66.0) of glucose confirmed its attachment at C-6 of glucose (Fig. 4.11). All the assignments were accomplished due to COSY,
HSQC and HMBC experiments and comparison with literature data which finally led to the structure 232 which is a new natural product and is named as aervfuranoside.
OH O OH HO OH OH HO O O HO O
HO O Cl OMe OMe HMBC ())and COSY (
Figure 4.11: Important HMBC and COSY correlations observed in the spectra of 232
Page 98
Table 4.7: 1D and 2D spectral data of 232 (DMSO-d6, 400 and 100 MHz) Position δH (J in Hz) δC Position δH (J in Hz) δC 1 7.79, s 112.3 3′ 3.36, m 73.2 2 _ 151.2 4ʹ 3.08, dt (5, 9.5) 71.9 3 _ 114.4 5ʹ 3.57, m 75.5 4 _ 141.3 6′ 3.83, dd (7, 11.5) 66.0 3.42, br d (10) 4a _ 158.3 1′ʹ 4.48, br s 100.5 5a _ 158.4 2′ʹ 3.59, m 70.1 6 _ 140.9 3′ʹ 3.15, m 69.7 7 _ 140.3 4′ʹ 3.38, m 70.6 8 _ 141.7 5′ʹ 3.30, m 68.1 9 7.51, s 111.7 6′ʹ 1.01, d (6.0) 17.7 9a _ 112.8 2′ -OH 5.21, br s _ 9b _ 111.9 3′ -OH 5.50, br s _ 4-OCH 3 4.06, s 61.6 4ʹ -OH 4.59, d (5) _ 6-OCH 3 4.03, s 60.8 2ʹ′-OH 4.49, d (4) _ 1′ 5.12, d (7.5) 101.5 3′ʹ-OH 5.20, br s _ 2′ 3.34, m 76.3 4′ʹ-OH 4.34, d (6) _
4.2.13. Structure Elucidation of Allantoin (233)
Compound 233 was obtained as needle like crystals whose EIMS showed molecular ion peak at m/z 158.0. The HREIMS O
HN 3 5 showed molecular formula C4H6N4O3 due to peak 1 NH N 7 O O H observed at m/z 158.1148. The IR spectrum showed H2N 233 absorption bands due to carbonyl groups at 1690 and
1705 cm -1 and for N-H stretching at 3430 cm -1. The 1H-NMR of 233 (Sec.
5.4.13) showed resonance for only one methine at δ 5.24 (1H, d, J = 8.0), which was correlated in HSQC spectrum with the carbon at δ 62.4. Other proton signals observed at δ 8.03 (1H, s), 6.87 (1H, d, J = 8.0) and 5.75 (2H, s) were attributed to amine and amide functions. In 13 C-NMR spectrum (Sec. 5.4.13)
Page 99
three downfield resonances were observed at δ 156.7, 157.3, and 173.5, in addition, a methine carbon showed its position at δ 62.4. A strong COSY correlation was found between the methine proton ( δ 5.24) and an amide proton ( δ 6.87), whereas, a weak COSY correlation was observed between the methine proton and another amide hydrogen ( δ 8.03). In the HMBC experiment the methine proton showed correlations with the carbon signals at δ 156.7, 157.3 and 173.5 (Fig. 4.12).
O O HN N NH2 N H O H 233
Figure 4.12: Important HMBC correlations observed in the spectrum of 233
Careful analysis of all above data and comparison with literature values revealed compound 233 as a known natural product; allantoin (Sripathi et al.,
2011), which has been isolated for the first time from our investigated source.
4.2.14. Structure Elucidation of Mannitol (234)
Compound 234 was isolated as white amorphous powder, which exhibited a molecular ion peak in EIMS at m/z 182.0, while OH OH OH HREIMS of 234 depicted the molecular formula as HO OH OH
C6H14 O6. The 1H NMR spectrum of 234 (Sec. 5.4.14) 234 displayed signals due to an oxygenated methylene at δ 3.61 (ddd, J = 3.2, 6.0,
Page 100
10.8 Hz) and 3.37 (dd, J = 6.0, 10.8 Hz) and two oxymethines at δ 3.54 (t, J =
7.2 Hz) and 3.45 (m). The oxymethylene hydrogens were correlated in HSQC spectrum with the carbon at δ 63.8, whereas, the oxymethine protons were correlated with the carbons at δ 69.7 and δ 71.3. The 13 C NMR (BB and DEPT)
(Sec. 5.4.14) displayed total three carbon signals, while the molecualr formula showed six carbons, double the number observed in NMR data. This observation revealed that compound 234 must be a symmetrical dimer. All th eassignments were confirmed with the help of HMBC and COSY experiments (Fig. 4.13). The above data was compared to the reported data for mannitol (Lee et al., 2010), therefore, compound 234 was found to be the same.
OH OH OH HO OH OH
HMBC ())and COSY (
Figure 4.13: Important HMBC and COSY correlations observed in the spectra of 234 4.2.15. Structure Elucidation of 1-O-β-D-glucopyranosyl-(2S,3S,4R,8Z)-2-
[(2R)-2-hydroxyPentacosanoylamino]-8-octadecene-1,3,4-triol (235)
Compound 235 was isolated as a white amorphous powder. FABMS
(positive) showed molecular ion peak at m/z 858. while HRFABMS depicted
Page 101
the molecular formula O OH HN as C 49 H96 NO 10 due to OH 18 OH O the molecular ion HO O HO 5 OH OH peak observed at m/z 235
858.7029 . The IR spectrum displayed prominent absorption bands at 3430
(hydroxyl), 1635 (secondary amide) and 1620 (olefinic group). In 1H-NMR a resonance of a proton was observed at δ 7.32 (1H, d, J = 7.8 Hz), which was attested for an amide function. Another signal at δ 5.25 (2H, dt, J = 5.5, 17.2
Hz) was identified for an isolated double bond. The amount of coupling constant of olefinic hydrogen revealed E-geometry of the double bond.
Further an oxymethylene resonated at δ 3.95 (1H, dd, J = 5.0, 10.8) and 3.67
(1H, dd, J = 4.6, 10.8), which was correlated with a carbon at δ 68.2, showed
COSY correlation with a methine proton at δ 4.22 (m, H-2). This methine was correlated in HSQC spectrum with the carbon at δ 51.5. The oxygenated region of the spectrum further displayed three oxymethines at δ 3.91 (1H, t, J
= 7.5, H-2ʹ), 3.59 (1H, m, H-4) and 3.14 (1H, t, J = 6.8, H-3), whereas, another set of protons resonating in the same region at δ 4.49 (1H, d, J = 7.6 Hz, H-
1ʹʹ ), 3.69 (1H, dd, J = 10.4, 4.8 Hz, H-6ʹʹ ), 3.57 (1H, dd, J = 10. 8, 3.4, H-
6ʹʹ ), 3.45 (1H, m, H-2ʹʹ ), 3.33 (1H, t, J = 7.6, H-3ʹʹ ), 3.23 (1H, t, J = 7.6, H-
4ʹʹ ), 3.20 (1H, m, H-5ʹʹ ) was attributed to a sugar moiety. The 1H-NMR
Page 102
spectrum (Sec. 5.4.15) also displayed broad singlet at δ 1.26 indicating presence of long chain along with signals for chain terminating methyls at δ
0.76 (6H, t, J = 6.4 Hz). The above discussed data revealed a ceramide nature of 235 . The 13 C-NMR spectrum (Sec. 5.4.15) supported the above data for a glycosphingolipid showing specific signals at δ 176.2 for carbonyl, δ 51.5 for azamethine and δ 130.2 and 129.9 for a double bond along with the signals for oxygenated methine and aliphatic chain. Relatively downfield shift (δ
68.2) of C-1 indicated the attachment of sugar unit which was further confirmed by HMBC correlation of anomeric methine with C-1 and vice versa (Fig. 4.14 ).
O OH HN OH OH O HO O HO OH OH
HMBC ())and COSY (
Figure 4.14: Important HMBC and COSY correlations observed in the spectra of 235 The whole discussed data closely resembled to the data reported for 1-O-β-D- glucopyranosyl-(2 S,3 S,4 R,8Z)-2-[(2 R)-2-hydroxyPentacosanoylamino]-8- octadecene-1,3,4-triol, which is a known secondary metabolite isolated for the first time from discussed source (Kang et al., 2001).
Page 103
4.2.16.Structure Elucidation of (14E)-2-[(2R)-2-hydroxyoctadecanoyl] amino tetraeicos-14-ene-1,3,4-triol-1-O-β-D-glucopyranoside (236)
Compound 236 O OH HN was isolated as a OH 11 OH O white HO O HO 7 OH OH amorphous 236 solid, which exhibited exactly similar NMR data (Sec. 5.4.16) as was observed for 235 . The only difference was observed in the molecular formula or the lengths of the aliphatic chains when compared to that of 235 . The FABMS showed the mole- cular ion peak at m/z 844, whereas, the HRFABMS showed [M+H] + at m/z
844.6871 establishing molecular formula C 48 H94 NO 10 . The above discussed data and revealed the compound 236 to be (14 E)-2-[(2 R)-2- hydroxyoctadecanoyl]amino}tetraeicos-14-ene-1,3,4-triol-1-O-β-D-glucopyran
-oside (Riaz et al., 2012) which is a known secondary metabolite isolated for the first time from the discussed source.
4.2.17. Structure Elucidation of β-sitosterol (106)
Compound 106 was isolated as shining needles, which was found to be sitosterol due to EIMS fragmentation pattern, in which the molecular ion peak was observed at m/z 414 with other characteristic ions at m/z 399, 396,
Page 104
381, 329, 275, 273 and 255 for sitosterol nucleus (Pateh et al., 2009). The molecular formula C 29 H50 O with five degrees of H H HO unsaturation was confirmed through 106
HREIMS.
The IR spectrum of compound 106 showed the absorption bands for hydroxyl group at 3450 cm -1 along with 3030, 1645, and 816 cm -1 for C-H and
C=C groups. The 1H-NMR spectrum (Sec. 5.4.17) of compound showed a specific pattern of a steroidal nucleus as it displayed two singlet methyl at δ
0.70 and 1.04, three secondary methyl at δ 0.90 (3H, d, J = 6.5 Hz), 0.85 (3H, d,
J = 6.5 Hz) and 0.83 (3H, d, J = 6.5 Hz) and one primary methyl at δ 0.87 (3H, t,
J = 7.0 Hz). An olefinic proton resonated at δ 5.35 was attested for H-6 of sitosterol nucleus. The oxymethine of sitosterol was observed at δ 3.33 as multiplet and its orientation was determined as α on the basis of its coupling constant thus confirming a 3 β-hydroxy function. The comparative study of its
NMR spectroscopic data with that of reported one confirmed its identity as β- sitosterol (Ahmad et al., 2009), which was further confirmed through comparative TLC with the authentic sample in our lab.
Page 105
4.2.18. Structure Elucidation of β-sitosterol 3-O-β-D-Glucopyranoside (237)
Compound 237 was the major component in the flower extract of Aerva javanica and was isolated as amorphous solid. The IR spectrum of this
compound showed bands 29 28
21 27 at 3450, 3040, 1445 and 816 18 20 23 25
19 17 26 -1 11 13 cm for hydroxyl and 15 HO 1 9 3 5 7 olefinic system. The EIMS O HO O HO OH spectrum showed similar 237 fragmentation pattern as was observed for 106 (Pateh et al., 2009) indicating that compound 237 must also be a sitosterol. However, the FABMS showed a higher mass by 162 amu indicating that 237 must be a glycoside of 106 . The molecular formula C 35 H61 O6 with six DBE could be determined by HRFABMS m/z 577.4461 [M+H] + that also confirmed glycosidic nature of 237 . The 1H-
NMR data (Sec. 5.4.18) were similar to β-sitosterol ( 106 ) except additional signals for a sugar unit at δ 5.33 (1H, d, J = 7.2 Hz) and 3.82-4.42 (5H, m). The
13 C-NMR spectrum of the same compound disclosed the presence of 35 carbon signals which were identified as six methyl, twelve methylene, fourteen methine and three quaternary carbon atoms by DEPT experiment.
On the basis of above evidences and comparison of 13 C-NMR values with the published data (Ahmad et al., 2009), the structure of the compound 237 could
Page 106
be established as β-sitosterol 3-O-β-D-glucoside, which is a common phytochemical.
4.2.19. Structure Elucidation of Oleanolic Acid (238)
Compound 238 was isolated as white solid. Its EIMS displayed [M]+ at m/z 456 while HREIMS showed molecular 28 29 ion peak at m/z 456.3599 corresponding to 19 21
25 11 13 17 OH molecular formula C30 H48 O3. The IR spectrum 15 1 9 O 3 5 7 27 of 238 showed a broad absorption bands at HO 23 24 238 3410-2430 cm -1 for a chelated hydroxyl group as an indication of a carboxylic function, which was substantiated due to the absorption band at 1710 cm -1 due to a carbonyl group. Other absorption bands observed at 1665 and 815 cm -1 were attributed to a trisubstituted double bond. In 1H-NMR spectrum seven singlet methyl were observed at δ
1.11, 1.04, 0.96 , 0.94, 0.90, 0.89 and 0.85. Most downfield signal was observed at δ 5.25 (1H, t, J =3.45 Hz) and was assigned to H-12. The oxymethine resonating at δ 3.47 (1H, dd, J = 4.4, 11.9 Hz ) was assigned to H-3. The amount of coupling constant of H-3 revealed its α-axial orientation and β- equatorial of hydroxyl function could be established. The 13 C-NMR (BB and
DEPT) experiments revealed the presence of seven methyl, ten methylene, five methine and seven quaternary carbon atoms. The comparison of 13 C-
Page 107
NMR values with the published data, confirmed the compound as oleanolic acid (Gohari et al., 2009), which has been isolated for the first time from flowers of Aerva javanica
4.2.20. Structure Elucidation of Lupeol (113)
Compound 113 was isolated as white solid and was also found a
pentacyclic triterpenoid The molecular formula 29
30 20 21 as C 30 H50 O with six DBE could be determined 27 19
25 11 13 17 28 through HREIMS, which exhibited molecular 15 1 9 3 5 7 26 ion at m/z 426.3858 . HO 23 24 113 The IR spectrum of compound 113 showed absorption band for hydroxyl group at 3460 cm -1 and for a terminal double bond at 3070, 1650, and 875 cm -1. The EIMS spectrum exhibited molecular ion peak at m/z 426, along with characteristic fragment ions at m/z
385, 220, 218 and 207 attested for a lupane series (Fotie et al., 2006). The 1H
NMR spectrum of 113 showed seven singlet methyl signals at δ 1.62, 1.05,
0.98, 0.96, 0.90, 0.87 and 0.82 due to a pentacyclic triterpenoid, whereas, the signals at δ 4.71 and 4.69 were attributed to a terminal olefin. The oxymethine resonating at δ 3.22 (1H, dd, J = 4.4, 9.8 Hz) was identified as H-
3. The value of coupling costant of H-3 ( J = 4.4, 9.8 Hz) suggested it to be α- axial and thus 3-OH function as β-equatorial. Above discussed data when
Page 108
compared to literature was found identical to the data reported for lupeol
(Jamal et al., 2008), which is a common phytochemical but has been reported from our investigated for the first time.
4.2.21. Structure Elucidation of Hexadecanoic Acid (239)
Compound 239 was obtained as white amorphous solid whose EIMS showed [M] + at m/z 256. The high resolution O
2 HO 1 3 analysis of the same peak depicted the molecular 11 16 239 formula as C16 H32 O2 with one degree of unsaturation.
The IR spectrum exhibited characteristic absorption due to a carboxylic acid function at 3390 and 1715 cm -1.
The 1H-NMR of 239 showed a triplet at δ 2.16 (2H, t, J = 7.2 Hz) identified as a methylene attached to carbonyl function, which showed COSY correlation with a multiplet at δ 1.43 (4H, m), which in turn was correlated with a broad singlet of several aliphatic methylene at δ 1.23-1.08. In addition to these signals, the resonance of a triplet methyl at δ 0.71 (3H, t, J = 6.4 Hz) indicated the aliphatic chain ending with methyl. This data was fully justified for a fatty acid. The 13 C-NMR spectrum substantiated the idea as it afforded the carbonyl carbon at δ 175.6, with other signals resonated at δ 22.3-33.8 and
13.6. The length of the fatty acid chain could be determined due to mass
Page 109
spectrum and finally the compound was confirmed as n-hexadecanoic acid
(239 ) (Yang et al., 2011).
4.2.22. Structure Elucidation of Gallic Acid (240) Compound 240 was obtained as crystalline solid, which showed absorption bands at 3415, 3377, 1705, 1510, 1455 cm−1 in IR spectrum for an aromatic carboxylic acid, whereas, the formula C9H10 O5 O OH 7 with 5 DBE could be determined through HREIMS. In 1
3 5 UV spectrum absorption bands were observed at 215 and HO OH OH 265 nm. The 1H-NMR spectrum (Sec. 5.4.22) showed only 240 one singlet in the aromatic region at δ 6.98 and one chelated hydroxyl proton at δ 11.62. In 13 C-NMR most down field signal at δ 177.6 was assigned to acid carbonyl, while the signals at δ 144.8 and δ 137.9 were assigned as oxygenated centers at aromatic system. The signals for another aromatic quaternary carbon and two symmetrical methine were observed at δ 122.0 and δ 109.6 respectively. Comparison of spectroscopic data with literature values revealed that the compound was gallic acid (Imran et al., 2009).
4.2.23. Structure Elucidation of Caffeic Acid (241) Compound 241 was isolated in the form of O 6 7 9 yellow powder whose molecular ion peak was 1 OH 4
HO 3 + observed at m/z 180 in EIMS. In HREIMS [M] OH 241
Page 110
observed at m/z 180.0419 corresponded to molecular formula C9H8O4 with six DBE. In UV spectrum absorption bands were observed at 214, 289, 315 nm. The IR spectrum of 241 was comparable with that of 240 with the additional absorption bands at 1620 for an olefinic system, which indicated compound 241 must also be an aromatic carboxylic acid with additional double bond.
The aromatic region of 1H-NMR spectrum (Sec. 5.4.23) showed an ABX pattern at δ 7.02 (1H, d, J = 2.0 Hz), 6.89 (1H, dd, J = 2.0, 8.0 Hz) and 6.73 (1H, d, J = 8.0 Hz). Another pair of signals at δ 7.62 (1H, d, J = 16.0 Hz) and 6.31
(1H, d, J = 16.0 Hz) was identified for a conjugated E-olefin. The 13 C-NMR data (Sec. 5.4.23) displayed altogether nine carbons including five methines
108.2, 115.5, 121.5, 114.8, 144.2 and four quaternary carbons δ 125.5, 144.6,
145.0, 167.0. The whole data was identical to the data reported for caffeic acid
(Chen et al., 2007), which is an important phytochemical. Therefore, our isolated compound 241 was also found to be the same.
4.2.24. Structure Elucidation of p- Coumaric Acid (242)
The UV and IR data of compound 242 was almost similar to that of 241 .
The HREIMS ( m/z 164.156) depicted the molecular O 6 7 5 9 OH formula as C9H8O4 with six DBE. The 1H-NMR 1 8 2 4 HO 3 242
Page 111
spectrum of 242 displayed signals for a p-substituted benzene ring at δ 7.36
(2H, d, J = 8.4 Hz) and 6.56 (2H, d, J = 8.0 Hz) and conjugated E-olefin at δ
7.30 (1H, d, J = 15.6 Hz,) and 6.31 (1H, d, J = 15.6 Hz , H-8). The 13 C-NMR showed seven signals including four methines at δ 129.6, 115.2, 115.0, 143.0 and three quaternary carbons at 167.4 160.1, 125.1. Comparison of the data with that of 241 and with published literature (Ahmad et al., 2009) confirmed the compound 242 to be p- coumaric acid, which is also a known but important phytochemical.
4.2.25. Structure Elucidation of Hexadecyl Ferulate (243)
The EIMS showed molecular ion peak at m/z 418 with prominent fragments at m/z 177 and 194. The HREIMS ( m/z O 6 7 1' 9 O 418.3080) depicted the molecular 1 2' 16' 4 11
HO 3 formula as C26 H42 O4 , while the IR OMe 243 spectrum showed absorption bands for hydroxyl (3415 cm -1), carbonyl
(1720 cm -1), olefinic (1630 cm -1) and aromatic system (1545, 1460 cm -1). In UV spectrum absorption bands appeared at 234, 290 and 325 nm. In the 1H-NMR spectrum the signals at δ 6.97 (1H, dd, J = 2, 7.4 Hz), 6.96 (1H, d, J = 2 Hz) and 6.80 (1H, d, J = 7.4 Hz) were attributed to a 1, 3, 4-tri-substituted benzene ring. Usual signal for a conjugated E-olefin resonated at δ 7.54 (1H, d, J = 16
Hz) and 6.22 (1H, d, J = 16 Hz). In addition, the spectrum displayed methoxyl
Page 112
proton at δ 3.84, (3H, s) and oxymethylene at δ 3.53 (2H, t, J = 6.8 Hz). This oxymethylene was correlated with another methylene at δ 1.68 (2H, m) which in turn was correlated to a broad singlet of several methylenes ( δ 1.17) ending with a triplet methyl at δ 0.80 ( J = 6.8 Hz). The chemical shift of oxymethylene indicated its direct attachment with a carboxylate moiety, which was further confirmed through HMBC correlation of methylene ( δ 3.53) with the carbonyl carbon ( δ 168.0). Other aromatic 13 C signals were found at 146.7, 144.5, 144.0,
125.0, 120.4, 115.8, 114.5, 109.5, along with the signals for an alkoxy chain.
The length of the alkyl chain could be fixed due to molecualr formula as C 16 and finally the data was compared with the reported data of hexadecyl ferulate (Bernards and Lewis, 1992), which is a know phytochemical and has been isolated for the first time from Aerva javanica .
4.2.26. Structure Elucidation of Hexacosyl Ferulate (244)
Compound 244 was isolated as white amorphous powder. The
HREIMS showed molecular ion peak at O 6 7 1' m/z 558.4634 corresponding to the 9 O 1 2' 26' 4 21
HO 3 molecular formula C36 H62 O4. The UV OMe 244 and IR data of the compound 244 resembled to that of 243 indicating its similar nature. Aromatic region of the
1H-NMR spectrum afforded the signals for a conjugated E-olefin at δ 7.55
Page 113
(1H, d, J = 15.6 Hz) and 6.23 (1H, d, J = 15.6 Hz), and for aromatic ring at δ
6.99 (1H, dd, J = 1.2, 8.0 Hz), 6.97 (1H, d, J = 1.2 Hz) and 6.77 (1H, d, J = 8.0
Hz). This information revealed that compound 244 is also an alkyl ferulate with a longer chain length as compared to that of 243 . The 13 C-NMR data of
244 also supported above deduction and finally the whole data was identical to the reported data of hexacosyl ferulate, which is also a known natural product (Wandji et al., 1990).
4.2.27. Structure Elucidation of Eicosanyl trans-p-coumarate (245)
Compound 245 was obtained as white amorphous powder. Its EIMS showed molecular ion peak [M] + at O m/z 444. By HREIMS, the molecular 7 1' 5 9 O 1 2' 20' 15 formula C29 H48 O3 was deduced due to HO 3 245 [M] + at m/z 444.3600. The UV, IR and most of NMR data of compound 245 were super imposable to the data of 244 .
Only difference was observed in 1H-NMR where signals observed at δ 7.40
(2H, d, J = 8.4 Hz, H-2, 6), 6.80 (2H, d, J = 2 Hz H-3, 5), 7.58 (1H, d, J = 16.0
Hz, H-7) and 6.29 (1H, d, J = 16.0 Hz, H-8) showed presence of trans p- coumaroylate moeity in 245 instead of ferulate group in 244. All data supported the structure for long chain ester of p- coumaric acid and
Page 114
confirmed as eicosanyl trans-p-coumarate after comparison with literature
(Mahmood et al., 2003).
4.2.28. Structure Elucidation of 1H-Indole-3-carboxylic Acid (246)
Compound 246 was isolated as white solid, whose EIMS showed molecular ion peak at m/z 161, whereas, the HREIMS O 1' OH 5 showed molecular ion peak at m/z 161.0472 6 4 3 2 7 9 N1 corresponding to molecular formula C9H7NO 2. The IR 8 H 246 spectrum dispalyed absorption bands for carboxylic acid moiety at 3410-2410 cm -1 (hydroxyl) and 1715 cm -1 (carbonyl). Other absorption bands were observed at 1625 and 1535 attested for an aromatic system. The UV spectrum showed absorption maxima at 271, 279, 286 nm for indole nucleus.
In the 1H NMR spectrum four signals were observed in the aromatic region at δ 8.20 (1H, dd, J = 2, 7.8 Hz), 7.39 (1H, dd, J = 1.6, 8.0 Hz), 7.30 (1H, t, J = 7.5 Hz) and 7.28 (1H, t, J = 7.5 Hz). Coupling constants and splitting pattern of these signals suggested a 1, 2 substituted aromatic system. Another sharp singlet was observed at δ 7.80 (1H, s, H-2) correlated in HSQC spectrum with the carbon at δ 136.2. Downfield shift of this carbon revealed the presence of a hetero atom in its vicinity. Further in broad band (BB) 13 C
Page 115
NMR spectrum, total nine carbon signals appeared, which were separated due to DEPT 135 as five methine and four quaternary carbon. Careful analysis of HMBC and COSY spectra helped to establish the structure of 246 as 1H-Indole-3-carboxylic acid, which was further confirmed through the comparison of observed NMR data with the reported data of same compound (Aldrich Library). This metabolite has also been purified for the first time from flowers of Aerva javanica .
4.2.29. Structure Elucidation of Tricontanol (247)
Compound 247 was isolated as white amorphous powder. The EIMS showed [M] + at m/z 438 whereas HREIMS showed
1 HO 30 molecular ion peak at m/z 438.4793 corresponding to 27 247 molecular formula C 30 H62 O. Characteristics fragment ion at m/z 420 [M-H2O] + along with small fragments with loss of two methylene units was observed indicating the compound to be a long chain hydrocarbon.
The IR displayed signals for hydroxyl group (3430 cm -1), saturated hydrocarbon (2925 cm -1) and C-O (1050 cm -1).
In 1H-NMR spectrum oxygenated methylenes at δ 3.63 (2H, t, J = 6.0
Hz) and 1.54 (2H, m, H-2), a broad singlet at δ 1.29-1.23 for long chain hydrocarbon and at δ 0.87 (3H, t, J = 6.0 Hz) for terminal methyl were observed. The 13 C-NMR spectrum of compound dispalyed the signal for
Page 116
oxygenated methylene at δ 63.1, aliphatic chain at δ 29.3-32.8 and terminal methyl at δ 14.1. The structure was further confirmed by HSQC, HMBC and
COSY experiments. On the basis of above discussed data, the compound 247 was established as tricontanol which was further confirmed by comparison with literature (Nogueira et al., 1996).
4.3. Biological Studies of the Compounds Isolated from Aerva javanica
The compounds 215 , 222 -232 were evaluated for DPPH free radical scavenging potential (Table 4.8) and enzyme inhibitory activity against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and lipoxygenase
(LOX) (Table 4.9). Only the test compound 231 was found a potential inhibitor of the enzyme lipoxygenase with an IC 50 value of 53.5 µM, whereas, other compounds showed neither the antioxidant nor the enzyme inhibitory potential.
Table 4.8: Enzyme inhibitory activities of isolated compounds ( 215 , 222-232 ) from Aerva javanica No. AChE AChE (IC 50 ) BChE BChE LOX LOX (%) µµµM (%) (IC 50 ) µµµM (%) (IC 50 ) µµµM
215 52.51±0.12 <400 38.24±0.34 <700 40.12±0.44 <700 222 64.63±0.19 215.31±0.07 27.07±0.14 <800 45.21±0.14 <700 223 65.03±0.15 212.24±0.10 30.12±0.19 <700 48.23±0.16 <700 224 54.02±0.15 218.30±0.10 41.17±0.18 <700 46.22±0.17 <700 225 61.23±0.15 215.19±0.12 36.15±0.20 <700 46.23±0.14 <700 226 66.53±0.18 222.41±0.08 29.05±0.14 <700 44.21±0.17 <700 227 55.61±0.14 <400 41.14±0.28 <700 42.11±0.42 <600 228 65.1±0.25 205.1±0.05 62.1±0.21 304.1±0.17 64.4±0.11 212.3±0.15 229 66.92±0.14 198.01±0.21 39.61±0.14 <700 30.75±0.21 <700 230 67.86±0.10 197.03±0.25 40.01±0.14 <700 31.67±0.25 <700 231 76.42±0.17 165.91±0.17 41.96±0.52 <700 63.61±0.04 53.51±0.05
Page 117
232 68.56±0.32 198.6±0.11 53.13±0.18 <400 62.88±0.18 235.61±0.52 Eserine 91.29±1.17 0.04±0.0001 82.82±1.09 0.85±0.001 - - Baicalein - - - - 93.79±1.2 22.4±1.3
Table 4.9: DPPH free radical scavenging potential of the isolated compounds (215 , 222-233 ) from Aerva javanica No. DPPH DPPH No DPPH DPPH (%) (IC 50 ) µµµM (%) (IC 50 ) µµµM 215 18.32±0.34 <1000 228 20.73±0.18 <900 222 9.19±0.22 <900 229 18.64±0.18 <900 223 11.02±0.25 <900 230 19.44±0.23 <900 224 9.90±0.30 <900 231 15.22±0.18 <1000 225 8.06±0.15 <900 232 18.36±0.25 <900 226 12.06±0.27 <1000 233 3.8±0.95 Nil 227 16.25±0.16 <1000 Quercetin 93.21±0.97 16.96±0.14
All the measurements were done in triplicate and statistical analysis was performed by Microsoft Excel 2003. Results are presented as mean ± sem. The compounds were prepared in methanol with a concentration of 0.5 mM.
Page 118
CHAPTER 5
Experimental Data of Isolates from Aerva Javanica
Page 119
5.1. General Experimental Procedures
Materials and Methods
Spectroscopic methods
The UV spectra were recorded on U-3200 HITACHI or Schimadzu UV-
240 spectrophotometer (Duisburg, Germany), whereas, the IR spectra were
recorded as KBr pellets on JASCO 320-A infrared spectrometer/Shimadzu
460 spectrometer (Duisburg, Germany). The optical rotations were measured
on a JASCO DIP-360 polarimeter (Tokyo, Japan). The EIMS, HREIMS,
FABMS and HRFABMS were recorded on Finnigan (Varian MAT,
Waldbronn, Germany) JMS H×110 with a data system and JMSA 500 mass
spectrometers, respectively. The 1H-NMR spectra were recorded on Bruker
AM-400 and 500 MHz in duterated solvents, while the 13C-NMR spectra
were recorded at 100 MHz or 125 MHz, respectively, on the same
instruments. The 2D-NMR spectra were also measured on the same
instruments operating at 400 and 500 MHz. The chemical shift values ( δ) are
reported in ppm and the coupling constant ( J) are in Hz.
Materials
Commercially available solvents were used after distillation at their
respective boiling points for extraction of plant material and
chromatographic techniques. Column chromatography was performed
Page 120
using silica gel (Keiselgel-70-230 mesh, E-Merck) and silica gel (Keiselgel-
230-400 mesh, Darm stadt, Germany) as stationary phase packed in glas s
columns, eluted by using gradient of organic solvents. Chromatographic
separations were monitored using aluminium sheets precoated with silica
gel 60 F 254 (20×20 cm, 0.2 mm thick; E-Merck; Darmstadt, Germany). UV
light (254 and366) was used to see fluorescence of chromatograms, and ceric
sulphate solution was sprayed on chromatograms followed by heating to
locate UV inactive spots.
5.2. Collection and Identification of the Plant Material
The flowers of Aerva javanica Burm were collected in September 2010 from the Cholistan Desert of Bahawalpur, near IUB and were identified by
Dr. Muhammad Arshad (late), Plant Taxonomist, Cholistan Institute for
Desert Studies (CIDS), The Islamia University of Bahawalpur, where a voucher specimen is deposited (AJ/CIDS-10-102).
5.3. Extraction of the Plant Material and Isolation
The shade dried flowers (4 kg) of A. javanica were extracted thrice with
MeOH (15 L). The combined CH 3OH extract was evaporated, under reduced pressure, to dryness and the residue (250 g) was divided into n-hexane (50 g), chloroform (50 g), ethyl acetate (75 g), n-butanol (25 g) and water-soluble (50 g) fractions. The ethyl acetate fraction (75 g) was subjected to column
Page 121
chromatography over silica gel eluting with n-hexane, n-hexane-CHCl 3,
CHCl 3 and CHCl 3-CH 3OH in increasing order of polarity to get ten fractions
(A-J). Fraction J (4.8 g) from the main column at CHCl 3-CH 3OH (8:2) was again subjected to silica gel column chromatography using CHCl 3-CH 3OH
(9.5:0.5) as mobile phase that gave three sub fractions J 1-J3. Fraction J 3 (1.5 g) was subjected to column chromatography over silica gel eluting with a mobile phase of CHCl 3-CH 3OH (9.3:0.7) to get 24-Epi -makisterone A (226 , 25 mg) with minor impurities, which were removed by passing it through sephadex LH-20. The sub-fraction J 2 (1.8 g) on further column chromatography using mobile phase of CHCl 3-CH 3OH in increasing order of polarity yielded further three (J 2a -J2c ) semi-pure fractions. The semi-pure fraction J 2b , obtained with CHCl 3-CH 3OH (9.8:0.2), on further column chromatography furnished β-ecdysone (215 , 35 mg) and 5-β-2- deoxyintegristerone A (227 , 12 mg).
Fraction I (5.5 g) eluted with CHCl 3-CH 3OH (9.0:1.0) on further silica gel chromatography eluting with CHCl 3 yielded three sub fractions (I 1-I3).
Sub-fraction I 3 (80 mg) was further purified by repeated silica gel column chromatography eluting with CHCl 3-CH 3OH (9.7:0.3) to get a mixture of two compounds, which was separated into aervecdysone C (224 , 19 mg) and aervecdysone D (225 , 22 mg) on RP-8 TLC plates using
Page 122
water:methanol:acetonitrile (60:35:5)., Sub fraction I 1 (1.5 g) on chromatography over silica gel column eluting with CHCl 3:CH 3OH (98:2) followed by preparative reversed phase thin layer chromatography [(RP-8
TLC, water:methanol:acetonitrile: (60:35:5)] furnished compounds aervecdysone A (222) (21 mg) and aervecdysone B (223 , 17 mg). Fraction H
(15 g) from the main column obtained with CHCl 3:CH 3OH (9.5:0.5) was rechromatographed over silica gel eluting with gradient of n-hexane:EtOAc to get six sub fractions (H 1-H6). The sub fraction H 6, after repeated silica gel column chromatography using an isocratic of CHCl 3:CH 3OH (9.8:0.2) yielded allantoin (233, 60 mg) and mannitol (234, 55 mg) . Compound aervfuranoside
(232, 25 mg) was isolated from H 6 on silica gel column chromatography eluting with isocratic of 5% CH 3OH in EtOAc, and on passing through sephadex LH-20. The sub fraction H 5 (3.5 g) obtained with n-hexane:EtOAc
(4:6) was further purified over silica gel column eluted with a mobile phase of n-hexane:EtOAc in increasing polarity order. Compound kaempferol-3-O-
β-D-[4 ′′′-p-coumaroyl-α-L rhamnosyl(1→6)]galactoside (228 , 35 mg) and kaempferol-3-O-β-D-[4 ′′′-p-coumaroyl-α-L-rhamnosyl(1→6)]3 ′′-p-coumaroyl galactoside (229 , 28.5 mg) were obtained with n-hexane:EtOAc (2.5:7.5) from the column, whereas, kaempferol-3-O-β-D-[4 ′′′-p-coumaroyl-α-L- rhamnosyl(1→6)]4 ′′-p-coumaroyl galactoside (230 , 25 mg) and kaempferol 3-
Page 123
O-β-D (6-E-p-coumaroyl) glucoside (231 , 40 mg) were obtained eluted with n- hexane:EtOAc (2.5:7.5). The main fraction G (4.0 g) obtained with pure CHCl 3 on further silica gel column chromatography gave two sub fractions G 1 and
G2. The sub-fraction G 1, on further silica gel column chromatography eluted with CHCl 3:CH 3OH (9.9:0.1) yielded ceramides 235 (12 mg) and 236 (15 mg) with , while the sub-fraction G 2 yielded β-sitosterol ( 106, 110 mg) under the same conditions. Fraction D (3.5 g) obtained from the main column with n- hexane:CHCl 3 (3:7) on repeated silica gel column chromatography yielded three compounds; lupeol ( 113, 22 mg) was eluted with n-hexane:CHCl 3 (5:5), oleanolic acid (238, 90 mg) at n-hexane:CHCl 3 (4:6) and β-sitosterol 3-O-β-D- glucopyranoside ( 237, 70 mg) at CHCl 3: CH 3OH (9.8:0.2). The main fraction B
(12.0 g), which was eluted with n-hexane:CHCl 3 (1:1), was further chromatographed on silica gel column to get further four sub fractions (B 1-
B4).The sub fraction B 4 (2.5 g) obtained from n-hexane:CHCl 3 (4:6) was subjected to repeated silica gel chromatography eluting with n-hexane:CHCl 3
(3.5:7.5) to get gallic acid (240, 15.5 mg). Another sub-fraction B 3 (2.0 g) was also purified on silica gel column eluted with isocratice of n-hexane:CHCl 3
(6:4) to afford p-coumaric acid ( 242, 32 mg) and caffeic acid (241, 22 mg).
Coloumn chromatography of B1 (0.5 g) yielded hexadecanoic acid ( 239 , 40 mg) at n-hexane:CHCl 3 (7:3).
Page 124
The fraction A (4.5 g) obtained from main column with n- hexane:CHCl 3 (8:2) was again subjected to silica gel column chromatography using gradient of n-hexane:CHCl 3 to get three sub-fractions (A 1-A3). The sub- fraction A 3 (2.5 g) was further purified on silica gel column eluting with n- hexane:CHCl 3 (6.5:3.5) to get eicosanyl trans-p-coumarate ( 245, 22 mg) and hexadecyl ferulate (243, 31.5 mg) whereas, hexacosyl ferulate (244, 24.5 mg) was obtained from the same column with n-hexane:CHCl 3 (6.7:3.3). The sub fraction A 2 on further silica gel column chromatography, yielded 1H-Indole-
3-carboxylic acid (246, 17 mg) with an eluent of n-hexane:CHCl 3 (8.0:2.0) and tricontanol (247, 35 mg) at n-hexane:CHCl 3 (9.5:0.5). A summarized schematic diagram of isolation procedure has been given in (Fig 5.1).
Methanolic extract of Aerva javanica
Water
EtOAc Hexane
Hexane part Water part CHCl3 part EtOAc part Butanol part
CC over flash silica using varying polarity of solvents
Fract. A Fract. B Fract. D Fract. G Fract. H Fract. I Fract. J
243 - 247 239-240 106, 113, 238 235-237 228-234 222-225 215, 226-227
Figure 5.1: Purification protocol of compounds from Aerva javanica
Page 125
5.4. Spectroscopic Data of the Isolated Compounds
5.4.1. Spectroscopic Data of Aervecdysone A (222): White amorphous
powder (21mg); [ α]D26 : + 22.5˚ ( c 0.17, HO 28 22 24 21 20 25 27 18 MeOH); UV (MeOH) λ max (ε) nm: 243 (4.1); O 17 26 19 11 13 9 -1 1 HO 1 IR (KBr) νmax cm : 3470, 1651, 1630; H- 10 15 H OH 3 5 7 HO H NMR (CDCl 3, 400 MHz): δ 5.75 (1H, s, H- O 222 7), 3.84 (1H, dd, J = 3.8, 11.3 Hz, H-3), 3.73
(1H, d, J = 3.1 Hz, H-2), 3.48 (1H, brd, J = 4.0 Hz, H-22), 2.95 (1H, m, H-9), 2.29
(1H, dd, J = 4.0, 11.3 Hz, H-5), 2.28 (1H, t, J = 9.2 Hz, H-17), 2.23 (1H, m, H-
23a), 2.12 (1H, m, H-23b), 2.03 (1H, m, H-12a), 1.92 (1H, m, H-16a), 1.90 (1H, m, H-15a), 1.80 (1H, m, H-12b), 1.73 (1H, m, H-16b), 1.70 (1H, m, H-1a), 1.66
(1H, m, H-11a), 1.65 (1H, m, H-4a), 1.60 (1H, m, H-24), 1.58 (1H, m, H-4b),
1.53 (1H, m, H-11b), 1.47 (1H, m, H-15b), 1.29 (1H, m, H-1b), 1.16 (3H, s, H-
27), 1.12 (3H, s, H-21), 1.04 (3H, s, H-26), 0.91 (3H, d, J = 6.4 Hz, H-28), 0.86
(3H, s, H-19) and 0.75 (3H, s, H-18); 13 C-NMR (CDCl 3, 100 MHz): δ 204.5 (C-
6), 166.0 (C-8), 121.2 (C-7), 84.2 (C-14), 77.0 (C-22), 74.7 (C-20), 73.2 (C-25), 67.4
(C-2), 67.0 (C-3), 49.0 (C-5), 48.5 (C-17), 47.0 (C-13), 42.1 (C-24), 38.0 (C-10),
36.3 (C-1), 34.0 (C-23), 33.4 (C-9), 31.5 (C-4), 31.0 (C-15), 30.9 (C-12), 29.6 (C-
27), 24.6 (C-26), 23.7 (C-19), 20.6 (C-16), 20.5 (C-21), 20.3 (C-11), 17.2 (C-18),
16.1 (C-28); EIMS: m/z 476 [M] +, 458 [M-H2O] + (5), 440 [M-2H 2O] + (4), 422 [M-
Page 126
3H 2O] + (2), 362 (30), 346 (62), 328 (65), 283 (5), 157 (4), 114 (4), 96 (7); HREIMS: m/z 476.3132 [M] + (calcd. 476.3138 for C 28 H44 O6).
5.4.2. Spectroscopic Data of Aervecdysone B (223): White amorphous
powder (17 mg); [ α]D26 : + 20.5˚ ( c 0.11, HO
O MeOH); UV (MeOH) λ max (ε) nm: 242 (4.07); IR
HO (KBr) νmax cm -1: 3475, 1650, 1635; 1H-NMR H OH HO H (CDCl 3, 400 MHz): δ 5.76 (1H, s, H-7), 5.02 O 223 (1H, s, H-28a), 4.81 (1H, s, H-28b), 3.87 (1H, dd, J = 3.2, 11.1 Hz, H-3), 3.72 (1H, d, J = 3.2 Hz, H-2), 3.47 (1H, brd, J = 3.8
Hz, H-22), 2.97 (1H, m, H-9), 2.30 (1H, dd, J = 3.5, 11.1 Hz, H-5), 2.27 (1H, t, J
= 9.5 Hz, H-17), 2.25 (1H, m, H-23a), 2.10 (1H, m, H-23b), 2.00 (1H, m, H-12a),
1.92 (1H, m, H-16a), 1.91 (1H, m, H-15a), 1.81 (1H, m, H-12b), 1.74 (1H, m, H-
16b), 1.72 (1H, m, H-1a), 1.65 (1H, m, H-11a), 1.63 (1H, m, H-4a), 1.57 (1H, m,
H-4b), 1.52 (1H, m, H-11b), 1.45 (1H, m, H-15b), 1.32 (3H, s, H-26), 1.28 (1H, m, H-1b), 1.23 (3H, s, H-27), 1.14 (3H, s, H-21), 0.87 (3H, s, H-19) and 0.76 (3H, s, H-18); 13 C-NMR (CDCl 3, 100 MHz): δ 205.1 (C-6), 166.2 (C-8), 153.5 (C-24),
121.4 (C-7), 110.7 (C-28), 84.1 (C-14), 77.2 (C-22), 76.2 (C-20), 72.3 (C-25), 67.2
(C-2), 67.0 (C-3), 49.5 (C-5), 48.7 (C-13), 48.6 (C-17), 38.1 (C-10), 36.2 (C-1), 33.6
(C-23), 33.5 (C-9), 31.4 (C-4), 31.1 (C-15), 30.8 (C-12), 29.8 (C-27), 29.1 (C-26),
23.7 (C-19), 20.5 (C-11), 20.4 (C-16), 20.0 (C-21), 17.2 (C-18); EIMS: m/z 474 (3)
Page 127
[M] +, 456 [M-H2O] + (5), 438 [M-2H 2O] + (4), 420 [M-3H 2O] + (2), 362 (25), 346
(63), 328 (65), 319 (62), 301 (100), 283 (7), 155 (12), 128 (5), 112 (9), 94 (11);
HREIMS: m/z 474.2977 [M] + (calcd. 474.2981 for C 28 H42 O6).
5.4.3. Spectroscopic Data of Aervecdysone C (224): White amorphous
26 OH powder (19 mg); [ α]D : + 11.0˚ ( c 0.0187, HO MeOH); UV (MeOH) λ max (ε) nm: 242 OH OH (4.06); IR (KBr) νmax cm -1: 3465, 1650, 1630; H OH HO H 1H-NMR (DMSO-d6, 400 MHz): δ 5.61 (1H, O 224 s, H-7), 4.65 (1H, s, OH-14), 4.45 (1H, brs,
OH-1), 4.34 (1H, brs, OH-3), 4.31 (1H, d, J = 4.5 Hz, OH-22), 4.14 (1H, s, OH-
25), 3.75 (1H, m, H-3), 3.62 (1H, s, OH-20), 3.59 (1H, dd, J = 4.5, 10.4 Hz, H-1),
3.30 (1H, m, H-22a), 2.99 (1H, m, H-9), 2.27 (1H, t, J = 9.2 Hz, H-17), 2.22 (1H, m, H-23a), 2.17 (1H, m, H-5), 1.98 (1H, m, H-12a), 1.93 (1H, m, H-23b), 1.88
(1H, m, H-16a), 1.76 (1H, m, H-15a), 1.71 (1H, m, H-12b), 1.63 (1H, m, H-11a),
1.62 (1H, m, H-16b), 1.57 (1H, m, H-2a), 1.54 (1H, m, H-4a), 1.51 (1H, m, H-
11b), 1.48 (1H, m, H-15b), 1.47 (2H, m, H-4b, 24), 1.24 (1H, m, H-2b), 1.08 (3H, s, H-21), 1.03 (3H, s, H-27), 1.00 (3H, s, H-26), 0.91 (3H, d, J = 6.8 Hz, H-28),
0.82 (3H, s, H-19) and 0.75 (3H, s, H-18); 13 C-NMR (DMSO-d6, 100 MHz): δ
202.7 (C-6), 165.3 (C-8), 121.3 (C-7), 82.9 (C-14), 75.8 (C-20), 75.6 (C-22), 71.4
(C-25), 66.7 (C-1), 66.5 (C-3), 50.1 (C-5), 48.5 (C-17), 46.9 (C-13), 42.7 (C-24),
Page 128
37.6 (C-10), 36.6 (C-2), 33.24 (C-9), 33.20 (C-23), 31.5 (C-4), 31.0 (C-12), 30.3 (C-
15), 29.7 (C-26), 29.4 (C-27), 23.9 (C-19), 21.0 (C-21), 20.3 (C-16), 20.0 (C-11),
17.2 (C-18), 16.6 (C-28); EIMS: m/z 494 (3) [M] +, 476 [M-H2O] + (5), 458 [M-
2H 2O] + (4), 442 [M-3H 2O] + (2), 363 (76), 345 (100), 327 (65), 301 (8), 283 (5), 175
(4), 131 (3), 113 (7), 95 (12); HREIMS: m/z 494.3239 [M] + (calcd. 494.3244 for
C28 H46 O7).
5.4.4. Spectroscopic Data of Aervecdysone D (225): White amorphous
26 OH powder (22 mg); [ α]D +13.0˚ ( c 0.0187, HO
OH MeOH); UV (MeOH) λ max (ε) nm: 244 OH
(4.07); IR (KBr) νmax cm -1: 3460, 1645, 1620; H OH HO H 1H-NMR (DMSO-d6; 400 MHz): δ 5.63 O 225 (1H, s, H-7), 5.01 (1H, s, H-28), 4.82 (1H, s,
H-28), 4.75 (1H, s, OH-25), 4.67 (1H, s, OH-14), 4.57 (1H, d, J = 4.7 Hz, OH-22),
4.47 (1H, brs, OH-1), 4.36 (1H, brs, OH-3), 3.77 (1H, br s, H-3), 3.63 (1H, s,
OH-20), 3.60 (1H, dd, J = 4.3, 10.2 Hz, H-1), 3.43 (1H, m, H-22), 2.97 (1H, m,
H-9), 2.29 (1H, t, J = 9.4 Hz, H-17), 2.25 (1H, m, H-23a), 2.19 (1H, m, H-5), 1.97
(1H, m, H-12a), 1.90 (2H, m, H-16a, 23b), 1.75 (1H, m, H-15a), 1.70 (1H, m, H-
12b), 1.65 (1H, m, H-11a), 1.63 (1H, m, H-16b), 1.56 (2H, m, H-2a, 4a), 1.52
(1H, m, H-11b), 1.48 (1H, m, H-15b), 1.45 (1H, m, H-4b), 1.23 (1H, m, H-2b),
1.22 (3H, s, H-26), 1.19 (3H, s, H-27), 1.03 (1H, s, H-21), 0.82 (3H, s, H-19) and
Page 129
0.76 (3H, s, H-18); 13 C-NMR (DMSO-d6, 100 MHz): δ 203.0 (C-6), 165.2 (C-8),
154.4 (C-24), 121.4 (C-7), 108.1 (C-28), 83.1 (C-14), 76.1 (C-22), 75.6 (C-20), 71.3
(C-25), 66.9 (C-1), 66.3 (C-3), 49.9 (C-5), 46.8 (C-13), 48.6 (C-17), 37.5 (C-10),
37.0 (C-2), 33.1 (C-9), 33.9 (C-23), 32.0 (C-4), 31.0 (C-15), 30.8 (C-12), 29.8 (C-
26), 29.5 (C-27), 24.0 (C-19), 20.5 (C-21), 20.4 (C-16), 20.3 (C-11), 17.1 (C-18);
EIMS: m/z 492 (3) [M] +, 474 [M-H2O] + (4), 363 (75), 345 (99), 327(63), 319 (65),
301 (9), 283 (5), 173 (12), 129 (11), 111 (15), 93 (13), 68 (22); HREIMS: m/z
492.3082 [M] + (calcd. 492.3087 for C 28 H46 O7).
5.4.5. Spectroscopic Data of 24-Epi-makisterone A (226): White crystals
(25 mg); [ α]D25 : +17.0˚ ( c 0.02, MeOH) ; UV HO OH (MeOH) λ max (ε) nm: 242 (4.06); IR (KBr) OH
νmax cm -1: 3460, 1645, 1620; 1H-NMR HO OH HO (C5D5N, 500 MHz): δ 6.37 (1H, s, OH-20), O 226 6.28 (1H, d, J = 2.0 Hz, H-7), 6.15 (1H, d, J
= 3.4 Hz, OH-2), 6.10 (1H, s, OH-25), 6.00 (1H, brs, OH-3), 4.83 (1H, s, OH-
14), 5.58 (1H, s, OH-22), 4.20 (1H, br s, H-2), 4.18 (1H, d, J = 11.5 Hz, H-3),
3.99 (1H, dd, J = 4.0, 11.0 Hz, H-22), 3.58 (1H, t, J = 9.5 Hz, H-9), 3.02 (1H, dd,
J = 4.0, 13.5 Hz, H-5), 2.97 (1H, t, J = 9.5 Hz, H-17), 2.63 (1H, m, H-12a), 2.51
(1H, q, J = 10.5, 21.0 Hz, H-16a), 2.29 (1H, m, H-24), 2.23 (1H, m, H-15a), 2.16
(1H, m, H-23a), 2.13 (1H, m, H-1a), 2.12 (1H, m, H-16b), 2.03 (1H, m, H-12b),
Page 130
2.00 (1H, m, H-4a), 1.95 (1H, m, H-15b), 1.92 (1H, m, H-1b), 1.85 (1H, m, H-
11a), 1.75 (1H, m, H-11b), 1.59 (3H, m, H-21), 1.58 (1H, m, H-23b), 1.31 (3H, s,
H-26), 1.29 (3H, s, H-27), 1.22 (3H, s, H-18), 1.07 (3H, d, J = 6.4 Hz, H-28) and
1.06 (3H, s, H-19); 13 C-NMR (C5D5N, 125 MHz): δ 203.4 (C-6), 166.1 (C-8),
121.6 (C-7), 84.1 (C-14), 76.9 (C-20), 74.6 (C-22), 72.0 (C-25), 68.1 (C-3), 68.0 (C-
2), 51.4 (C-5), 49.9 (C-17), 48.1 (C-13), 41.8 (C-24), 38.6 (C-10), 38.0 (C-1), 34.6
(C-9), 34.4 (C-23), 32.8 (C-4), 32.0 (C-12), 31.8 (C-15), 28.2 (C-26), 26.5 (C-27),
24.4 (C-19), 21.8 (C-21), 21.3 (C-16), 21.1 (C-11), 17.9 (C-18), 15.3 (C-28); EIMS: m/z 494 [M] +, 442 [M-3H 2O] + (2), 423 (1), 363 (76), 345 (100), 327(65), 301 (8),
283 (5), 175 (4), 157 (4), 131 (3), 113 (7), 95 (12); HREIMS: m/z 494.3241 [M] +
(calcd. for C 28 H46 O7, 494.3244).
5.4.6. Spectroscopic Data of 5-β-2-Deoxyintegristerone A (227): White
needles (12 mg); [ α]D26 : +20.1˚ ( c 0.02, HO OH ε OH CHCl 3 ); UV (MeOH) λ max ( ) nm: 242 OH (4.06); IR (KBr) νmax cm -1: 3460, 1645, OH HO 1620; 1H-NMR (DMSO-d6, 500 MHz): δ O 227 5.63 (1H, br s, H-7), 4.68 (1H, s, OH-14),
4.41 (1H, d, J = 6.0 Hz, OH-3), 4.38 (1H, d, J = 2.4 Hz, OH-2), 4.36 (1H, d, J =
4.8 Hz, OH-22), 4.10 (1H, s, OH-25), 3.68 (1H, m, H-3), 3.64 (1H, m, H-1), 3.62
(1H, s, OH-20), 3.12 (1H, t, J = 8.2 Hz H-22), 2.96 (1H, t, J = 9.2 Hz, H-9), 2.25
Page 131
(1H, t, J = 9.2 Hz, H-17), 2.17 (1H, dd, J = 3.6, 13.2 Hz, H-5), 2.03 (1H, m, H-
12a), 1.90 (1H, m, H-16a), 1.84 (1H, m, H-15a), 1.70 (1H, m, H-12b), 1.66 (1H, m, H-11a), 1.61 (1H, m, H-24a), 1.58 (1H, m, H-4a), 1.56 (1H, m, H-15b), 1.55
(1H, m, H-16b), 1.50 (1H, m, H-2a), 1.49 (2H, m, H-4b, 11b), 1.44 (1H, m, H-
23a), 1.31 (1H, m, H-2b), 1.30 (1H, m, H-24b), 1.05 (6H, s, H-21, 26), 1.02 (3H, s, H-27), 1.00 (1H, m, H-23b), 0.86 (3H, s, H-19), 0.74 (3H, s, H-18); 13 C-NMR
(DMSO-d6, 125 MHz): δ 203.6 (C-6), 166.0 (C-8), 121.7 (C-7), 83.6 (C-14), 77.0
(C-22), 76.4 (C-20), 66.9 (C-1), 69.0 (C-25), 66.0 (C-3), 49.5 (C-17), 49.1 (C-5),
47.0 (C-13), 42.3 (C-24), 38.0 (C-10), 37.0 (C-2), 34.9 (C-9), 32.0 (C-4), 31.2 (C-
12), 30.8 (C-15), 30.2 (C-26), 28.9 (C-27), 26.3 (C-23), 23.2 (C-19), 22.1 (C-11),
21.0 (C-21), 20.5 (C-16), 18.5 (C-18); EIMS: m/z 480 [M] +, 438 [M-3H 2O] + (2),
363 (75), 345 (95), 319 (70), 301 (11), 283 (6), 161 (5), 117 (4), 99 (6), 81 (12);
HREIMS: m/z 480.3082 [M] + (calcd. for C 27 H44 O7, 480.3087).
5.4.7. Spectroscopic Data of β-ecdysone (215): White needles (35 mg); [α]D26 :
+19˚ ( c 0.02, CHCl 3 ); UV (MeOH) λ max OH HO (ε) nm: 242 (4.06); IR (KBr) νmax cm -1: OH
HO 3450, 1644, 1625; 1H-NMR (DMSO-d6, OH HO 500 MHz): δ 5.61 (1H, br s, H-7), 4.70 O 215 (1H, s, OH-14), 4.39 (1H, d, J = 6.0 Hz,
OH-3), 4.38 (1H, d, J = 2.4 Hz, OH-2), 4.35 (1H, d, J = 4.8 Hz, OH-22), 4.12
Page 132
(1H, s, OH-25), 3.75 (1H, br s, H-3), 3.58 (1H, s, OH-20), 3.57 (1H, br s, H-2),
3.10 (1H, t, J = 7.6 Hz, H-22), 2.99 (1H, t, J = 9.5 Hz H-9), 2.24 (1H, t, J = 9.2 Hz,
H-17), 2.19 (1H, dd, J = 3.6, 13.2 Hz, H-5), 2.00 (1H, m, H-12a), 1.87 (1H, m, H-
16a), 1.79 (1H, m, H-15a), 1.69 (1H, m, H-12b), 1.68 (1H, m, H-11a), 1.60 (1H, m, H-24a), 1.57 (1H, m, H-4a), 1.53 (1H, m, H-16b), 1.52 (1H, m, H-1a), 1.51
(1H, m, H-4b), 1.49 (1H, m, H-15b), 1.46 (1H, m, H-23a), 1.28 (1H, m, H-1b),
1.25 (1H, m, H-24b), 1.08 (1H, m, H-23b), 1.04 (3H, s, H-21), 1.03 (3H, s, H-
27), 0.82 (3H, s, H-19) and 0.75 (3H, s, H-18); 13 C-NMR (DMSO-d6, 125 MHz):
δ 202.6 (C-6), 165.2 (C-8), 120.4 (C-7), 82.9 (C-14), 76.1 (C-22), 75.6 (C-20), 68.6
(C-25), 66.7 (C-2), 66.5 (C-3), 50.1 (C-5), 48.6 (C-17), 46.8 (C-13), 41.4 (C-24),
37.6 (C-10), 36.6 (C-1), 33.1 (C-9), 31.5 (C-4), 30.8 (C-12), 30.3 (C-15), 30.0 (C-
26), 28.9 (C-27), 26.0 (C-23), 23.8 (C-19), 20.9 (C-21), 20.2 (C-16), 20.0 (C-11),
17.1 (C-18). EIMS: m/z 480 [M] +, 438 [M-3H 2O] + (2), 363 (74), 345 (90), 319 (65),
301 (10), 283 (8), 161 (7), 117 (9), 99 (6), 81 (11); HREIMS: m/z 480.3082 [M] +
(calcd. for C 27 H44O7, 480.3087).
Page 133
5.4.8. Spectroscopic Data of Kaempferol-3-O-β-D-[4 ′′′′′′′′-p-coumaroyl-α-L rhamnosyl(1→6)]galactoside (228): Yellow amorphous solid (35 mg); [α] 26 D:
-45.5 (0.11, MeOH); UV λmax : 257, 267, 5' OH
8 -1 3' 314, 357 nm ; IR (KBr) νmax cm : 3384, HO O 1' 8a 1
6 4a 3 1712, 1685, 1600, 1550, 1520; 1H-NMR O OH O 1'' OH HO O (CD 3OD, 500 MHz): δ 7.88 (2H, d, J = 8.8 5'' HO 3''' 1''' 3'' O OH O Hz, H-2´,6´), 7.40 (1H, d, J = 16.4 Hz, H- O O 5''' HO 7´´´´) 7.18 (2H, d, J = 8.4 Hz, H-
2´´´´,6´´´´), 6.68 (2H, d, J = 8.8 Hz, H- 228
OH 3´,5´), 6.60 (1H, d, J = 8.4 Hz, H-
3´´´´,5´´´´), 6.17 (1H, d, J = 1.6 Hz, H-8), 6.04 (1H, d, J = 1.6 Hz, H-6), 6.00 (1H, d, J = 16.4 Hz, H-8´´´´), 4.68 (1H, t, J = 9.6 Hz, H-4´´´), 4.58 (1H, d, J = 8.0 Hz,
H-1´´), 4.32 (1H, br s, H-1´´´), 3.72 (1H, dd, J = 4.7, 10.8 Hz, H-6a´´), 3.60 (1H, t,
J = 7.8 Hz, H-2´´), 3.55 (1H, d, J = 2.8 Hz, H-4´´), 3.53 (1H, dd, J = 3.0, 10.8 Hz,
H-6b´´), 3.50 (1H, dd, J = 6.4, 9.0, Hz, H-3´´´), 3.48 (1H, m, H-5´´´), 3.43 (1H, dd, J = 2.8, 7.8 Hz, H-3´´), 3.41 (1H, br d, J = 6.4 Hz, H-2´´´), 3.33 (1H, m, H-
5´´), 1.12 (3H, d, J = 6.4 Hz, H-6´´´); 13 C-NMR (CD 3OD, 125 MHz): δ 181.0 (C-
4), 166.8 (C-9´´´´), 164.2 (C-7), 161.0 (C-5), 160.4 (C-4´), 159.8 (C-4´´´´), 158.0 (C-
8a), 157.5 (C-2), 145.5 (C-7´´´´) 133.7 (C-3), 130.9 (C-2´,6´), 129.6 (C-2´´´´,6´´´),
125.4 (C-1´´´´), 121.5 (C-1´), 115.4 (C-3´´´´,5´´´´), 114.7 (C-3´,5´), 113.4 (C-8´´´´),
Page 134
105.0 (C-1´´), 104.5 (C-4a), 100.1 (C-1´´´), 98.8 (C-6), 93.7 (C-8), 73.8 (C-4´´´),
73.6 (C-5´´), 73.5 (C-3´´), 70.5 (C-2´´), 70.3 (C-2´´´), 69.0 (C-3´´´), 68.3 (C-4´´),
66.0 (C-6´´), 66.4 (C-5´´´), 16.7 (C-6´´´); HRFABMS: m/z 741.2045 [M+H] +
(calcd. 741.2030 for C 36 H37O17 ).
5.4.9. Spectroscopic Data of Kaempferol-3-O-β-D-[4 ′′′′′′′′-p-coumaroyl-α-L- rhamnosyl(1→6)]3 ′′′′′′-p-coumaroyl galactoside (229): Yellow amorphous
solid (28.5 mg); [α] 26 D: -55.5 (0.15, OH
HO O MeOH); UV λmax nm: 208, 228, 268,
O 356; IR (KBr) νmax cm -1: 3385, 1715, OH O OH HO O 1680, 1605, 1550, 1520; 1H-NMR HO O O O O (CD 3OD, 500 MHz): δ 8.04 (2H, d, J O O HO
= 8.8 Hz, H-2´,6´), 7.66 (1H, d, J =
229 16.0 Hz, H-7´´´´), 7.55 (1H, d, J = OH OH 16.0 Hz, H-7´´´´´), 7.36 (2H, d, J = 8.4
Hz, H-2´´´´,6´´´´), 7.29 (1H, d, J = 8.8 Hz, H-2´´´´´,6´´´´´), 6.85 (2H, d, J = 8.8 Hz,
H-3´,5´), 6.76 (1H, d, J = 8.4 Hz, H-3´´´´,5´´´´), 6.73 (1H, d, J = 8.8 Hz, H-
3´´´´´,5´´´´´), 6.32 (1H, d, J = 16.0 Hz, H-8´´´´), 6.31 (1H, d, J = 2.0 Hz, H-8), 6.30
(1H, d, J = 16.0 Hz, H-8´´´´´), 6.20 (1H, d, J = 2.0 Hz, H-6), 4.83 (1H, t, J = 7.2
Hz, H-4´´´), 4.80 (1H, dd, J = 2.8, 7.6 Hz, H-3´´), 4.78 (1H, d, J = 7.6 Hz, H-1´´),
4.44 (1H, br s, H-1´´´), 4.08 (1H, t, J = 7.6 Hz, H-2´´), 3.95 (1H, d, J = 2.8 Hz, H-
Page 135
4´´), 3.68 (1H, dd, J = 4.9, 11.1 Hz, H-6a´´), 3.64 (1H, dd, J = 6.5, 7.2 Hz, H-
3´´´), 3.62 (1H, m, H-5´´´), 3.58 (1H, dd, J = 2.9, 11.1 Hz, H-6b´´), 3.55 (1H, m,
H-5´´), 3.53 (1H, br d, J = 6.5 Hz, H-2´´´), 1.05 (3H, d, J = 6.4 Hz, H-6´´´); 13 C-
NMR (CD 3OD, 125 MHz): δ 181.6 (C-4), 167.2 (C-9´´´´´), 166.9 (C-9´´´´), 162.7
(C-7), 160.4 (C-4´´´´´), 160.0 (C-4´´´´), 160.0 (C-4´), 159.83 (C-5), 159.81 (C-8a),
158.2 (C-2), 146.0 (C-7´´´´,7´´´´´), 134.5 (C-3), 131.6 (C-2´,6´), 130.1 (C-
2´´´´´,6´´´´), 130.0 (C-2´´´´,6´´´), 125.0 (C-1´´´´,1´´´´´), 121.4 (C-1´), 116.2 (C-
3´´´´,5´´´´), 116.1 (C-3´´´´´,5´´´´´), 115.0 (C-3´,5´), 114.08 (C-8´´´´), 114.01 (C-
8´´´´´), 106.1 (C-1´´), 104.0 (C-4a), 100.2 (C-1´´´), 99.0 (C-6), 94.1 (C-8), 75.5 (C-
3´´), 74.0 (C-4´´´), 73.6 (C-5´´), 70.1 (C-2´´´), 69.0 (C-2´´), 69.4 (C-3´´´), 67.0 (C-
6´´), 66.5 (C-4´´), 66.0 (C-5´´´), 17.1 (C-6´´´); HRFABMS : m/z 887.2404 [M+H] +
(calcd. 887.2399 for C 45 H43O19 ).
Page 136
5.4.10. Spectroscopic Data of Kaempferol-3-O-β-D-[4 ′′′′′′′′-p- coumaroyl-α-L-rhamnosyl(1→6)]4 ′′′′′′-p-coumaroyl galactoside (230):
Yellow amorphous solid (25 mg); OH
HO O [α] 26 D: -52.5 (0.11, MeOH); UV λmax
O nm: 207, 226, 268, 355 ; IR (KBr) νmax OH O 1'' OH HO O cm -1: 3382, 1715, 1683, 1604, 1550, 1521; HO O OH O 1H-NMR (CD 3OD, 500 MHz): δ 8.03 O O O O (2H, d, J = 8.8 Hz, H-2´,6´), 7.63 (1H, d,
J = 15.6 Hz, H-7´´´´´), 7.59 (1H, d, J = 230
OH OH 16.0 Hz, H-7´´´´), 7.33 (2H, d, J = 8.4
Hz, H-2´´´´´,6´´´´´), 7.30 (2H, d, J = 8.4 Hz, H-2´´´´,6´´´´), 6.83 (2H, d, J = 8.8 Hz,
H-3´,5´), 6.75 (1H, d, J = 8.4 Hz, H-3´´´´,5´´´´), 6.74 (1H, d, J = 8.4 Hz, H-
3´´´´´,5´´´´´), 6.36 (1H, d, J = 1.6 Hz, H-8), 6.23 (1H, d, J = 1.6 Hz, H-6), 6.20
(1H, d, J = 16.0 Hz, H-8´´´´), 6.15 (1H, d, J = 16.0 Hz, H-8´´´´´), 5.29 (1H, d, J =
2.8 Hz, H-4´´), 4.79 (1H, d, J = 8.4 Hz, H-1´´), 4.77 (1H, m, H-4´´´), 4.40 (1H, br s, H-1´´´), 3.80 (1H, t, J = 8.4 Hz, H-2´´), 3.74 (1H, dd, J = 2.8, 8.4Hz, H-3´´),
3.69 (1H, dd, J = 4.6, 10.8Hz, H-6a´´), 3.66 (1H, dd, J = 6.2, 7.2Hz, H-3´´´), 3.64
(1H, m, H-5´´), 3.54 (1H, br d, J = 6.3 Hz, H-2´´´), 3.52 (1H, m, H-5´´´), 3.51
(1H, dd, J = 3.1, 10.8Hz, H-6b´´), 1.00 (3H, d, J = 6.4 Hz, H-6´´´); 13 C-NMR
(CD 3OD, 125 MHz): δ 181.0 (C-4), 167.1 (C-9´´´´), 167.0 (C-9´´´´´), 160.0 (C-4´,
Page 137
7), 159.9 (C-5), 159.5 (C-4´´´´,C-4´´´´´), 159.2 (C-8a), 158.0 (C-2), 146.0 (C-7´´´´,
7´´´´´), 134.1 (C-3), 131.3 (C-2´,6´), 130.18 (C-2´´´´,6´´´), 130.13 (C-2´´´´´,6´´´´),
125.0 (C-1´´´´, C-1´´´´´), 121.0 (C-1´), 116.08 (C-3´´´´,5´´´´), 116.01 (C-
3´´´´´,5´´´´´), 115.8 (C-3´,5´), 114.94 (C-8´´´´), 114.91 (C-8´´´´´), 105.5 (C-1´´),
104.5 (C-4a), 100.0 (C-1´´´), 99.0 (C-6), 94.0 (C-8), 74.1 (C-4´´´), 73.2 (C-5´´), 72.5
(C-3´´), 71.5 (C-2´´), 70.5 (C-2´´´), 69.5 (C-3´´´), 69.0 (C-4´´), 67.1 (C-5´´´), 66.0
(C-6´´), 17.1 (C-6´´´); HRFABMS: m/z 887.2410 [M+H] + (calcd. 887.2399 for
C45 H43O19 ).
5.4.11. Spectroscopic Data of Kaempferol 3-O-β-D (6-E-p-coumaroyl) glucoside (231): Yellow amorphous solid (40 mg) ; UV λmax nm: 208, 270, 358;
IR (KBr) νmax cm -1: 3374, 1725, 1681, 1565, 1515 ; OH
HO O 1H-NMR (CD 3OD, 400 MHz): δ 8.04 (2H, d, J =
O 8.8 Hz, H-2´,6´), 7.58 (1H, d, J = 16.0 Hz, H- OH O O OH O O 7´´´), 7.39 (2H, d, J = 8.4 Hz, H-2´´´,6´´´), 6.85
HO OH (2H, d, J = 8.8 Hz, H-3´,5´), 6.66 (1H, d, J = 8.4
Hz, H-3´´´,5´´´), 6.33 (1H, d, J = 2.0 Hz, H-8), 231 OH 6.28 (1H, d, J = 16.0 Hz, H-8´´´), 6.21 (1H, d, J =
2.0 Hz, H-6), 4.85 (1H, d, J = 7.6 Hz, H-1´´), 4.35 (1H, dd, J = 4.6, 10.6 Hz, H-
6´´), 4.17 (1H, dd, J = 3.8, 10.6 Hz, H-6´´), 3.91 (1H, m, H-3´´), 3.78 (1H, t, J =
7.6 Hz, H-2´´), 3.97 (1H, m, H-4´´), 3.53 (1H, m, H-5´´); 13 C-NMR (CD 3OD, 100
Page 138
MHz): δ 179.4 (C-4), 167.0 (C-9´´´), 163.0 (C-7), 160.1 (C-5), 160.2 (C-4´´´), 160.0
(C-4´), 158.7 (C-8a), 158.0 (C-2), 145.1 (C-7´´´), 133.3 (C-3), 131.5 (C-2´,6´),
130.4 (C-2´´´,6´´), 125.0 (C-1´´´), 123.2 (C-1´), 114.6 (C-3´,5´), 117.2 (C-3´´´,5´´´),
114.2 (C-8´´´), 105.2 (C-1´´), 105.0 (C-4a), 98.6 (C-6), 94.1 (C-8), 70.5 (C-4´´), 75.7
(C-5´´), 74.3 (C-3´´), 72.2 (C-2´´), 67 (C-6´´); FABMS m/z 595 [M+H] +;
HRFABMS: m/z 595.1449 [M+H] + (calcd.595.1452 for C 30 H27 O13 ).
5.4.12 Spectroscopic Data of Aervfuranoside (232): Pale yellow gum (25
mg); [α]D25 −24.7 OH 6'' O OH 5'' HO 1'' OH (c 0.1, MeOH); 3' HO OH 3'' 9 1 HO 8 9a 9b O O 6' O 1' 5' UV (MeOH) λmax 7 5 3 5a 4a HO 6 O 4 Cl OMe OMe nm: 215, 239, 264, 232 298, 308 and 337;
IR (KBr): 3410, 1595, 1560, 1525, 1030 cm -1; 1H-NMR (DMSO-d6, 400 MHz): δ
7.79 (1H, s, H-1), 7.51 (1H, s, H-9), 5.50 (1H, brs, OH-3´), 5.21 (1H, brs, OH-2´),
5.20 (1H, brs, OH-3´´), 5.12 (1H, d, J = 7.5 Hz, H-1´), 4.59 (1H, d, J = 5 Hz, OH-
4´), 4.49 (1H, d, J = 4 Hz, OH-2´´), 4.48 (1H, br s, H-1´´), 4.34 (1H, d, J = 6 Hz,
OH-3´´), 4.06 (3H, s, OMe), 4.03 (1H, s, OMe), 3.83 (1H, dd, J = 7.0, 11.5 Hz, H-
6a´), 3.59 (1H, m, H-2´´), 3.57 (1H, m, H-5´), 3.42 (1H, brd, J = 10.0 Hz, H-
6b´), 3.36 (1H, m, H-3´), 3.38 (1H, m, H-4´´), 3.34 (1H, m, H-2´), 3.30 (1H, m,
H-5´´), 3.15 (1H, m, H-3´´), 3.08 (1H, dt, J = 5.0, 9.5 Hz, H-4´), 1.01 (1H, d, J
Page 139
= 6 Hz, H-6´´); 13 C-NMR (DMSO-d6, 100 MHz): δ 158.4 (C-5a), 158.3 (C-4a),
151.2 (C-2), 141.7 (C-8), 141.3 (C-4), 140.9 (C-6), 140.3 (C-7), 114.4 (C-3), 111.7
(C-9), 111.2 (C-1), 112.8 (C-9a), 111.9 (C-9b), 101.5 (C-1´), 100.5 (C-1´´), 76.3 (C-
2´), 75.5 (C-5´), 73.2 (C-3´), 71.9 (C-4´), 70.6 (C-4´´), 70.1 (C-2´´), 69.7 (C-3´´),
68.1 (C-5´´), 66.0 (C-6´), 61.6 (4-OCH 3), 60.8 (6-OCH 3), 17.7 (C-6´´); FABMS m/z 619 [M+H] +; HRFABMS: m/z 619.1426 [M+H] + (Calcd. 619.1430 for
C26 H32 ClO 15 ).
5.4.13. Spectroscopic Data of Allantoin (233): Needle like crystals (60 mg);
-1 1 O IR (KBr) νmax cm : 3430, 1690, 1705; H-NMR (DMSO- HN 3 5 1 NH d6, 400 MHz): δ 10.51 (1H, s, NH-1), 8.03 (1H, s, NH-3), N 7 O O H H2N 6.87 (1H, d, 8.0 Hz, NH-6), 5.75 (2H, s, NH 2-8), 5.24 (1H, 233 d, J = 8.0 Hz, H-5); 13 C-NMR (DMSO-d6, 100 MHz): δ
157.3 (C-2), 173.5 (C-4), 62.4 (C-5), 156.7 (C-7); EIMS: m/z 158.0; HREIMS:
158.1148 (calcd. 158.1150 for C4H6N4O3).
5.4.14. Spectroscopic Data of Mannitol (234): White amorphous powder
(55 mg); [α]D23 : +4.60 (c 0.1, MeOH); IR (KBr) νmax : OH OH
2 4 6 OH HO 1 3 5 3430, 1775 cm -1; 1H-NMR (DMSO-d6, 400 MHz): δ 4.39 OH OH 234 (2H, d, J = 5.6 Hz, OH-2, 5), 4.31 (2H, t, J = 6.0 Hz,
OH-1, 6 ), 4.12 (2H, d, J = 7.2 Hz, OH-3, 4), 3.61 (2H, ddd, J = 3.2, 6.0, 10.8 Hz,
H-1a, 6a), 3.54 (2H, t, J = 7.2 Hz, H-3, 4), 3.45 (2H, m, H-2, 5), 3.37 (2H, dd, J =
Page 140
6.0, 10.8 Hz, H-1b, 6b); 13 C-NMR (DMSO-d6, 100 MHz): δ 63.8 (C-1, 6), 69.7 (C-
3, 4), 71.3 (C-2, 5); EIMS: m/z 182.0 [M] +, 147 (12), 133 (20), 121 (14), 115 (14),
109 (15), 103 (61), 91 (16), 84 (18), 73 (100), 61 (77), 56 (22), 43 (40); HREIMS: m/z 182.1712 (calcd. 182.1718 for C6H14 O6).
5.4.15. Spectroscopic Data of 1-O-β-D-Glucopyranosyl-(2S,3S,4R,8Z)-2-
[(2R)-2-hydroxy Pentacosanoyl amino]-8-octadecene-1,3,4-triol (235):
White amorphous powder (12 mg); IR (KBr) νmax : 3430, 2920, 1635, 1620 cm -1;
1H-NMR (CDCl 3, 400 O OH HN MHz): δ 7.32 (1H, d, J = OH 18 OH O 7.8 Hz, -NH), 5.25 (2H, HO O HO 5 OH OH dt, J = 5.5, 17.2 Hz, H- 235 8, 9), 4.49 (1H, d, J = 7.6
Hz, H-1ʹʹ ), 4.22 (1H, m, H-2), 3.95 (1H, dd, J = 5.0, 10.8 Hz H-1a), 3.91 (1H,
t, J = 7.5 Hz, H-2ʹ), 3.69 (1H, dd, J = 4.8, 10.4 Hz, H-6a ʹʹ ), 3.67 (1H, dd, J =
4.6, 10.8 Hz, H-1b), 3.59 (1H, m, H-4), 3.57 (1H, dd, J = 3.4, 10.8 Hz, H-6b ʹʹ ),
3.45 (1H, m, H-2ʹʹ ), 3.33 (1H, t, J = 7.6 Hz, H-3ʹʹ ), 3.23 (1H, t, J = 7.6 Hz,
H-4ʹʹ ), 3.20 (1H, m, H-5ʹʹ ), 3.14 (1H, t, J = 6.8 Hz, H-3), 1.66 (2H, m, H-
3ʹ), 1.58 (2H, m, H-5), 1.18–1.26 (br s), 0.76 (6H, t, J = 6.4 Hz, H-25, 18 ʹ); 13 C-
NMR: (CDCl 3, 100 MHz): δ 176.2 (C-1ʹ), 130.2 (C-8), 129.9 (C-9), 101.5 (C-
1ʹʹ ), 76.7 (C-5ʹʹ ), 75.8 (C-3ʹʹ ), 74.8 (C-3), 74.2 (C-4), 73.9 (C-2ʹ), 73.2 (C-
Page 141
2ʹʹ ), 70.4 (C-4ʹʹ ), 68.2 (C-1), 62.0 (C-6ʹʹ ), 51.5 (C-2), 33.6 (C-3ʹ), 32.6 (C-
7), 32.4 (C-9),30.4 (C-5), 28.9 (C-6), 28.6-31.2 (10 ʹ-17 ʹ, 4-24), 14.8 (C-25,
18 ʹ); HRFABMS: m/z 858.7029 [M] + (calcd 858.7032 for C 49 H96 NO 10 ).
5.4.16. Spectroscopic Data of (14E)-2-[(2R)-2 hydroxyoctadecanoyl] amino}tetraeicos-14-ene-1,3,4-triol-1-O-β- Dglucopyranoside (236): White
Gummy Solid O OH HN OH 11 (15 mg); IR (KBr) OH O O HO ν HO 7 max : 3540, 2910, OH OH 236 1650, 1625, 1455,
1070, 720 cm -1; 1H-NMR (CDCl 3, 400 MHz): δ 7.38 (1H, d, J = 8.8 Hz, -NH),
5.22 (2H, dt, J = 5.5, 17.2 Hz, H-14, 15), 4.25 (1H, d, J = 7.5 Hz, H-1ʹʹ ),
4.12 (1H, m, H-2), 3.91 (1H, dd, J = 4.6, 11.4 Hz, H-1a), 3.85 (1H, t, J = 6.8
Hz, H-2ʹ), 3.76 (1H, dd, J = 5.0, 10.6 Hz, H-6a ʹʹ ), 3.75 (1H, dd, J = 4.0,
11.4 Hz, H-1b), 3.56 (1H, dd, J = 3.0, 10.5 Hz, H-6b ʹʹ ), 3.45 (1H, m, H-4),
3.41 (1H, m, H-2ʹʹ ), 3.30 (1H, t, J = 7.5 Hz, H-3ʹʹ ), 3.21 (1H, t, J = 7.5 Hz,
H-4ʹʹ ), 3.14 (1H, m, H-5ʹʹ), 3.10 (1H, dd, J = 3.8, 4.8 Hz, H-3), 1.65 (2H, m, H-3ʹ), 1.50 (2H, m, H-5), 1.12–1.26 (br s), 0.78 (6H, t, J = 6.5 Hz, H-24,
18 ʹ); 13 C-NMR (CDCl 3, 100 MHz): δ 175.8 (C-1ʹ), 130.7 (C-14), 129.8 (C-
15), 103.5 (C-1ʹʹ ), 76.8 (C-5ʹʹ ), 75.7 (C-3), 75.5 (C-3ʹʹ ), 74.0 (C-4), 73.0
Page 142
(C-2ʹʹ ), 72.7 (C-2ʹ), 70.1 (C-4ʹʹ ), 68.7 (C-1), 62.1 (C-6ʹʹ ), 51.8 (C-2),
34.6 (C-3ʹ), 32.3 (C-5), 32.5 (C-13,16), 28.1-31.1 (C-6-12, 17-23,4 ʹ-17 ʹ),
15.1 (C-24, 18 ʹ); FABMS: m/z 844.0 [M+H] +; HRFABMS m/z 844.6871
:(calcd. 844.6878 for C 48 H94 NO 10 ).
5.4.17. Spectroscopic Data of β-Sitosterol (106): White needles (110 mg);
[α]D25 : +35.5˚ ( c 0.22, CHCl 3); IR (KBr)
29 28 -1 1 21 27 νmax cm : 3450, 3030, 1645 and 816; H-
18 20 23 25 3 19 11 13 17 26 NMR (CDCl , 300 MHz): δ 5.35 (1H, m, 15 1 9
3 5 7 H-6), 3.33 (1H, m ,H-3), 1.04 (3H, s, Me- HO 106 19), 0.90 (3H, d, J = 6.5 Hz, Me-21), 0.87
(3H, t, J = 7.0 Hz, Me- 29), 0.85 (3H, d, J = 6.5 Hz, Me-26), 0.83 (3H, d, J = 6.5
Hz, Me-27), and 0.70 (3H, s, Me-18); 13 C-NMR (CDCl 3, 75MHz): δ 141.8 (C-5),
120.8 (C-6), 73.0 (C-3), 56.5 (C-14), 55.8 (C-17), 49.5 (C-9), 48.8 (C-24), 43.0 (C-
13), 41.7 (C-4), 40.1 (C-12), 37.2 (C-20), 37.0 (C-10), 36.3 (C-1), 34.0 (C-22), 33.1
(C-7), 32.5 (C-8), 31.8 (C-2), 29.0 (C-23), 28.2 (C-16), 25.3 (C-15), 23.1 (C-28),
22.2 (C-25), 21.1 (C-11), 20.1 (C19), 19.8 (C-27), 19.2 (C-21), 18.9 (C-26), 12.0 (C-
18), 11.9 (C-29); EIMS: m/z 414 (15) [M] +, 399 (10), 396 (12), 381 (75), 329 (28),
275 (15), 273 (17), 255 (35); HREIMS: m/z 414.3857 [M] + (calcd. 414.3861 for
C29 H50 O).
Page 143
5.4.18.Spectroscopic Data of β-Sitosterol 3-O-β-D-Glucopyranoside (237):
White crystalline solid (70 29 28 27 21 mg); [ α]D25 : +15˚ ( c 0.5, 18 20 23 25
19 26 11 13 17 CHCl 3 ); IR (KBr) V max cm -1: OH 15 1 9 O 5 7 HO 3 3450, 3040, 1445, 816; 1H- HO O OH 237 NMR (CD 3OD, 400 MHz): δ
5.33 (1H, d, J = 7.2 Hz, H-1ʹ), 5.15 (1H, br d, J = 5.5 Hz, H-6), 3.81 (1H, m, H-
3), 3.82-4.42 (6H, m, Gluc-H), 1.01 (3H, s, C-19), 0.90 (3H, d, J = 6.5 Hz, C-21),
0.82 (3H, t, J = 7.0 Hz, C-29), 0.80 (3H, d, J = 6.5 Hz, C-26), 0.78 (3H, d, J = 6.5
Hz, C-27), 0.62 (3H, s, C-18); 13 C-NMR (CD 3OD, 100 MHz): δ 140.0 (C-5), 120.1
(C-6), 102.4 (C-1ʹ), 81.0 (C-3), 76.5 (C-3ʹ), 76.3 (C-5ʹ), 73.4 (C-2ʹ), 71.0 (C-
4ʹ), 62.0 (C-6ʹ), 57.0 (C-17), 55.9 (C-14), 51.4 (C-24), 48.7 (C-9), 42.9 (C-13),
42.5 (C-4), 41.5 (C-12), 40.2 (C-22), 39.0 (C-1), 37.8 (C-20), 37.0 (C-10), 34.6 (C-
7), 33.0 (C-8), 29.8 (C-2), 29.2 (C-16), 29.0 (C-23), 25.4 (C-25), 25.0 (C-15), 23.8
(C-28), 21.9 (C-11), 19.0 (C-27), 18.5 (C-19), 17.1 (C-21), 16.2 (C-26), 12.0 (C-18) and 11.5 (C-29); EIMS: m/z 414 (12) [M] +, 399 (9), 396 (8), 381 (75), 329 (25), 275
(11), 273 (15), 255 (50); FAMBS: m/z 577 [M+H] +; HRFABMS: m/z 577.4461
[M+H] + (calcd. for C 35 H61 O6, 577.4468).
Page 144
5.4.19. Spectroscopic Data of Oleanolic Acid (238): White crystalline solid
(90 mg); [ α]D25 : +80˚ ( c 0.07, CHCl 3 ); IR (KBr) 28 29
19 21 νmax cm -1: 3400, 1710, 1665, and 815; 1H-NMR
25 11 13 17 OH 15 (CDCl 3, 400 MHz): δ 5.25 (1H, t, J = 3.4 Hz, H- 1 9 O 3 5 7 27 HO 12), 3.47 (1H, dd, J = 4.4, 11.9 Hz ,H-3), 2.81 23 24 238 (1H, dd, J = 3.6, 13.2 Hz, H-18), 1.11, 1.04, 0.96
, 0.94, 0.90, 0.89 and 0.85 (3H, each s, Me); 13 C-NMR (CDCl 3, 100Hz): δ 181.3
(C-28), 144.1 (C-13), 122.1 (C-12), 78.0 (C-3), 55.6 (C-5), 47.9 (C-9), 47.5 (C-17),
46.9 (C19), 42.1 (C-14), 41.2 (C-18), 39.7 (C-4), 39.5 (C-8), 38.2 (C-1), 37.0 (C-
10), 32.9 (C-21), 32.7 (C-22), 32.6 (C-29), 31.6 (C-7), 31.3 (C-20), 28.3 (C-23), 27.0
(C-2), 26.5 (C-15), 25.3 (C-27), 23.0 (C-11), 22.9 (C-30), 22.6 (C-16), 18.4 (C-6),
17.3 (C-26), 15.9 (C-24), 15.3 (C-25); EIMS: m/z 456 (4) [M] +, 248 (98), 208 (12),
203 (60) and 133 (53); HREIMS: m/z 456.3599 [M] + (calcd. 456.3603 for
C30 H48 O3).
5.4.20. Spectroscopic Data of Lupeol (113): White solid (22 mg) ; [α]D25 : +37˚
29 (c 0.2, CHCl 3 ); IR (KBr) νmax : 3460, 3070, 1650,
30 20 21 -1 1 27 19 and 875 cm ; H-NMR (CDCl 3, 400 MHz): δ
25 11 13 17 28 15 4.74, 4.66 (2H, br s, 1 H each, H-29), 3.22 (1H, 1 9
3 5 7 26 HO dd, J = 4.4, 9.8 Hz, H-3), 1.62, 1.05, 0.98 , 0.96, 23 24 113
Page 145
0.90, 0.87 and 0.82 (3H, each s, Me); 13 C-NMR (CDCl 3, 100 MHz): δ 151.5 (C-
20), 110.0 (C-29), 78.6 (C-3), 55.6 (C-5), 50.0 (C-9), 48.2 (C-18), 47.6 (C19), 42.8
(C-14), 40.5 (C-17), 39.7 (C-4), 38.8 (C-1), 38.7 (C-22), 38.3 (C-13), 38.3 (C-8),
37.1 (C-10), 34.6 (C-16), 31.6 (C-7), 29.8 (C-21), 27.5 (C-2), 26.8 (C-15), 25.4 (C-
12), 28.3 (C-23), 21.0 (C-11), 20.1 (C-30), 18.4 (C-6), 18.3 (C-28), 17.3 (C-26), 16.8
(C-27), 15.9 (C-24), 15.3 (C-25); EIMS: m/z 426 (13) [M] +, 411 (22), 408 (28), 391
(40) , 385 (17) , 220 (70), 207 (20), 189 (100) and 139 (65); HREIMS: m/z
426.3858 [M] + (calcd. 426.3862 for C 30 H50 O).
5.4.21. Spectroscopic Data of n-Hexadecanoic Acid (239): White
amorphous solid (40 mg); IR (CHCl 3) νmax cm -1: 3390 O
2 HO 1 3 1 11 16 and 1715; H-NMR (CDCl 3, 400 MHz ): δ 2.16 (2H, t, J 239 = 7.2 Hz, H-2), 1.43 (2H, m, H-3), 1.08 (22H, br s, H-4 to H-14), 1.23 (2H, m, H-15) and 0.71 (3H, t, J = 6.4 Hz, H-16); 13 C-NMR
(CDCl 3, 100 MHz): δ 175.6 (C-1), 33.8 (C-2), 24.6 (C-3), 28.6-31.2 (C-4 to C-14),
22.3 (C-15) and 13.6 (C-16); EIMS: m/z 256 (14) [M] +, 211 (23), 197 (15), 185
(16), 157 (24), 129 (13), 101 (17), 73 (30); HREIMS: m/z 256.4237 [M] + (calcd.
256.4241 for C 16 H32 O2).
Page 146
5.4.22. Spectroscopic Data of Gallic Acid (240): Crystalline solid (15 mg);
UV (CD 3OD) νmax : 215, 265 nm; IR (KBr) νmax cm -1: 3415, O OH 7 1 3377, 1705, 1510, 1455 cm −1 ; 1 H-NMR (CD 3OD, 400 MHz ): δ 5 3 HO 4 OH 11.62 (s, carboxylic OH) and 6.98 (2H, s, H-2, 6); 13 C-NMR OH 240 (CD 3OD, 100 MHz): δ 177.6 (C-7), 144.8 (C-3,5), 137.9 (C-4),
122.0 (C-1) and 109.6 (C-2,6); EIMS: m/z 170.0 [M] +; HREIMS: m/z 170.0211
[M] + (calcd. 170.0215 for C 9H10 O5).
5.4.23. Spectroscopic Data of Caffeic Acid (241): Yellow powder (22 mg);
ν -1 O UV λmax (EtOAc): 214, 289, 315; IR (KBr) max cm : 6 7 5 9 OH 1 8 3400, 1740, 1620, 1514, 1445, 1265 ; 1 H-NMR 2 HO 4 3 241 OH (CDCl 3+CD 3OD, 400 MHz): δ 7.62 (1H, d, J = 16.0 Hz,
H-7), 7.02 (1H, d, J = 2.0 Hz, H-2), 6.89 (1H, dd, J = 2.0, 8.0 Hz, H-6), 6.73 (1H, d, J = 8.0 Hz , H-5), 6.31 (1H, d, J = 16.0 Hz, H-8); 13 C-NMR (CDCl 3+CD 3OD,
100 MHz): δ 145.0 (C-4), 144.6 (C-3), 144.2 (C-7), 125.5 (C-1), 121.5 (C-6), 115.5
(C-5), 114.8 (C-8), 108.2 (C-2), 167.0 (C-9); EIMS: m/z 180.0 [M] +; HREIMS: m/z
181.0456 [M] + (calcd. 180.0423 for C9H8O4).
Page 147
5.5.24. Spectroscopic Data of p-Coumaric Acid (242): Yellow powder (20
ν O mg); UV λmax (MeOH): 311, 285, 224; IR (KBr) max
OH cm -1: 3395, 3300-2710, 1705; 1H-NMR (CDCl 3, 400 HO 242 MHz): δ 7.36 (2H, d, J = 8.4 Hz, H-2, 6), 7.30 (1H, d, J
= 15.6 Hz, H-7), 6.56 (2H, d, J = 8.0 Hz, H-3, 5), 6.31 (1H, d, J = 15.6 Hz , H-8);
13 C-NMR (CDCl 3, 100 MHz): δ 125.1 (C-1), 129.6 (C-2,6), 115.0 (C-3,5), 160.1
(C-4), 143.0 (C-7), 115.2 (C-8), 167.4 (C-9); EIMS: m/z 164.0 [M] +; HREIMS: m/z
164.0466 [M] + (calcd. for C9H8O4 164.0473).
5.4.25. Spectroscopic Data of Hexadecyl Ferulate (243): White powder (22
mg); UV λmax (EtOAc) nm: 234, 290, O 6 7 1' 325; IR (KBr) νmax cm -1: 3415, 2900, 9 O 1 2' 16' 4 11 HO 3 1720, 1630, 1545, 1460, 1265, 850; 1H- OMe 243
NMR (CDCl 3+CD 3OD, 400 MHz): δ
7.54 (1H, d, J = 16.0 Hz, H-7), 6.97 (1H, dd, J = 2.0, 7.4 Hz, H-6), 6.96 (1H, d, J
= 2 Hz, H-2), 6.80 (1H, d, J = 7.4 Hz , H-5), 6.22 (1H, d, J = 16.0 Hz, H-8), 3.84,
(3H, s, OMe), 3.53 (2H, t, J = 6.8 Hz, H-1´), 1.68 (2H, m, H-2´), 1.17 (26H, H-3´-
H-15´), 0.80 (3H, t, J = 6.8 Hz, H-16´); 13 C-NMR (CDCl 3+CD 3OD, 100 MHz): δ
125.0 (C-1), 109.5 (C-2), 144.5 (C-3), 146.7 (C-4), 115.8 (C-5), 120.4 (C-6), 144.0
(C-7), 114.5 (C-8), 168.0 (C-9), 63.0 (C-1´), 30.0 (C-2´), 30.5-29.5 (C-3´- C-15´)
Page 148
and 13.6 (C-16´); EIMS: m/z 418.4 [M]+ (20), 194 (100), 177 (55), 150 (40);
HREIMS: m/z 418.3080 (418.3083 calcd. for C 26 H42 O4).
5.4.26. Spectroscopic Data of Hexacosyl Ferulate (244): White powder (17
mg); UV λmax (EtOAc): 233, 327 nm; IR O 6 7 1' 9 O -1 1 2' 26' (KBr) νmax cm : 3420, 1700, 1650, 1460, 4 21
HO 3 OMe 244 1265, 720; 1H-NMR (CDCl 3+CD 3OD,
400 MHz): δ 7.55 (1H, d, J = 15.6 Hz, H-7), 6.99 (1H, dd, J = 1.2, 8.0 Hz, H-6),
6.97(1H, d, J = 1.2 Hz H-2), 6.77 (1H, d, J = 8.0 Hz , H-5), 6.23 (1H, d, J = 15.6
Hz, H-8), 3.88, (3H,s, OMe), 3.54 (2H, t, J = 6.7 Hz, H-1´), 2.01 (1H, m, H-2´),
1.18 (46H, H-3´- H-25´), 0.82 (3H, t, J = 6.8 Hz, H-26´); 13 C-NMR
(CDCl 3+CD 3OD, 100 MHz): δ 168.2 (C-9), 145.5 (C-4), 144.8 (C-3), 143.0 (C-7),
125.1 (C-1), 123.0 (C-6), 115.5 (C-5), 115.0 (C-8), 108.5 (C-2), 63.2 (C-1´), 31.1
(C-2´), 30.5-29.5 (C-3´- C-25´) and 14.8 (C-26´); EIMS: m/z 558.4 [M] +, 194
(100), 177 (57), 150 (43); HREIMS: m/z 558.4634 (calcd. 558.4648 for C 36 H62 O4).
5.4.27. Spectroscopic Data of Eicosanyl trans-p-coumarate (245): White
amorphous powder (22 mg); UV λmax O 7 1' 5 9 O 1 2' 20' (EtOAc): 232, 324 nm; IR (KBr) νmax 15
HO 3 -1 245 cm : 3400, 1695, 1615, 1470, 1165, 910,
730; 1H-NMR (CDCl 3+CD 3OD, 400
MHz): δ 7.58 (1H, d, J = 16.0 Hz, H-7), 7.40 (2H, d, J = 8.4 Hz, H-2, 6), 6.80 (2H,
Page 149
d, J = 2.0 Hz, H-3,5), 6.29 (1H, d, J = 16.0 Hz, H-8), 4.24 (2H, t, J = 6.7 Hz, H-
1´), 1.20 (36H, H-2´- H-19´), 0.85 (3H, t, J = 6.8 Hz, H-20´); 13 C-NMR (CDCl 3+
CD 3OD, 100 MHz): δ 159.5 (C-4), 143.4 (C-7), 131.0 (C-2,6), 124.8 (C-1), 115.2
(C-3, 5), 114.8 (C-8), 168.0 (C-9), 64.2 (C-1´), 30.5-29.5 (C-2´- C-19´) and 14.5 (C-
20´); EIMS: m/z 444 [M ] + (3), 167 (20), 166 (35), 164 (100), 147 (68), 125 (9), 107
(11), 83 (17) 69 (28), 57 (52); HREIMS: m/z 444.3600 (calcd. 444.3603 for
C29 H48 O3).
5.4.28. Spectroscopic Data of 1H-Indole-3-carboxylic Acid (246): White
solid (20 mg); UV λmax (EtOH): 271, 279, 286 nm; IR O 1' OH 5 (CHCl 3) νmax cm -1: 3410, 1715, 1625, 1535; 1H-NMR 6 4 3 2 7 9 N1 8 H (CDCl 3+CD 3OD, 400 MHz ): δ 8.20 (1H, dd, J = 2.0, 7.8 Hz, 246 H-5), 7.80 (1H, s, H-2), 7.39 (1H, dd, J = 1.6, 8.0 Hz, H-8),
7.30 (1H, t, J = 7.5 Hz, H-7), 7.28 (1H, t, J = 7.5 Hz, H-6); 13 C-NMR
(CDCl 3+CD 3OD, 100 MHz): δ 136.2 (C-2), 119.0 (C-3), 124.0 (C-4), 121.5 (C-5),
124.3 (C-6), 123.0 (C-7), 111.4 (C-8), 137.0 (C-9) and 166.7 (C-1´); EIMS: m/z
161.1 [M] +; HREIMS: m/z 161.0472 [M ] + (calcd. 161.0477 for C 9H7NO 2).
5.4.29. Spectroscopic Data of Tricontanol (247): White amorphous solid (25
mg) ; IR (CHCl 3) νmax cm -1: 3430, 1460 and 720-735; 1H-NMR 1 HO 30 27 (CDCl 3, 400 MHz ): δ 3.63 (2H, t, J = 6.0 Hz, H-1), 1.54 (2H, 247
Page 150
m, H-2), 1.46 (2H, m, H-3), 1.29-1.23 (48H, br s , H-4 to H-27), 1.38 (2H, m, H-
28), 1.30 (2H, m, H-29) and 0.87 (3H, t, J = 6.0 Hz, H-30); 13 C-NMR (CDCl 3, 100
MHz): δ 63.1 (C-1), 32.8 (C-2), 25.7 (C-3), 29.7-29.3 (C-4 to C-27), 31.9 (C-28),
22.7 (C-29) and 14.1 (C-30); EIMS: m/z 438 (15) [M] +, 420 (3), 392 (1), 336 (2),
308 (3), 265 (2), 223 (5), 195 (4), 180 (6), 167 (12), 139 (26), 125 (50), 111 (70), 97
(91), 83 (41), 69 (100), 55 (81); HREIMS: m/z 438.4793 [M] + (calcd. 438.4801 for
C30 H62 O).
5.5. DPPH Free Radical Scavenging Assay
The stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) was used for the determination of antioxidant activity (Koleva et al., 2002). Different concentrations of compounds in DMSO were added at an equal volume (5
µL) to 90 µL of 100 µM methanolic DPPH in a total volume of 100 µL in 96- well plates. The contents were mixed and incubated at 37 oC for 30 minutes.
The absorbance was measured at 517 nm, whereas, quercetin was used as standard antioxidant. The experiments were carried out in triplicates. The activity was determined by the following formula:
%age scavenging activity = [100 - (Abs of test compound/Abs of control) X 100
5.6. Enzyme Inhibitory Assay
5.6.1. Acetylcholinesterase Assay
Page 151
The AChE inhibition activity was performed according to the reported method (Ellman et al., 1961) with slight modifications. 100 µL of the reaction mixture contained 60 µL Na 2HPO 4 buffer with concentration of 50 mM and pH 7.7, 10 µL test compound (0.5 mM well-1) was added, followed by the addition of 10 µL (0.005 unit well -1) the enzyme. The contents were mixed and pre-read at 405 nm. Then contents were pre-incubated for 10 min at 37ºC.
The reaction was initiated by the addition of 10 µL of 0.5 mM well -1 substrate
(acetylthiocholine iodide), followed by the addition of 10 µL DTNB (0.5 mM well-1). After 15 min of incubation at 37ºC, absorbance was measured at 405 nm using 96-well plate reader. All experiments were carried out with their respective controls in triplicate. Eserine (0.5 mM well -1) was used as a positive control. The percent inhibition was calculated through the following equation:
Inhibition (%) = Control – Test × 100 Control
5.6.2. Butyrylcholinesterase Assay
The BChE inhibition activity was performed according to the method
(Ellman et al., 1961) with slightmodifications. Total volume of the reaction mixture was 100 µL containing 60 µL, Na 2HPO 4 buffer, 50 mM and pH 7.7.
Ten µL test compound 0.5 mM well -1 was added followed by the addition of
Page 152
10 µL (0.5 unit well -1) BChE (Sigma Inc.). The contents were mixed and pre- read at 405 nm and thenpre-incubated for 10 min at 37ºC. The reaction was initiated by the addition of 10 µL of 0.5 mM well -1 substrate
(butyrylthiocholine chloride). Followed by the addition of 10 µL DTNB, 0.5 mM well -1. After 15 min of incubation at 37ºC, absorbance was measured at
405 nm using 96-well plate reader Synergy HT, Biotek, USA. All experiments were carried out with their respective controls in triplicate. Eserine (0.5 mM well-1) was used as positive control. The percent inhibition was calculated by the help of following equation.
Inhibition (%) = Control – Test × 100 Control
5.6.3. Lipoxygenase Assay
The lipoxygenase activity was assayed according to the method of
Tappel et al. (Tappel, 1953) with slight modifications. A total volume of 200 ml assay mixture contained 160 ml sodium phosphate buffer (100 mM, pH
8.0), 10 ml of test compound, and 20 ml purified lipoxygenase (Sigma
Chemicals, Seelze, Germany). The contents were pre-incubated for 10 min at
258 °C. The reaction was initiated by the addition of 10 ml linoleic acid
(substrate solution). The change in absorbance was observed after 6 min at
234 nm. All reactions were performed in triplicates in 96-well microplate
Page 153
reader (Synergy HT, Biotek, Winooski, Vermont, USA). Baicalein (0.5 mM well -1) was used as a positive control. The percentage inhibition was calculated by the formula given below.
Inhibition (%) = Control – Test × 100 Control
Where Control = Total enzyme activity without inhibitor
Test = Activity in the presence of test compound
Note: In all enzyme inhibitory assays, the IC 50 values were calculated using
EZ–Fit Enzyme Kinetics software (Perrella Scientific Inc. Amherst, USA). All the measurements were done in triplicate and statistical analysis was performed by Microsoft Excel 2003. Results are presented as mean ± sem.
Page 154
CHAPTER 6
Phytochemistry of Halothamnus auriculus
Page 155
6.1. The Genus Halothamnus
The genus Halothamnus of the plant family Amaranthaceae was formerly placed in the family Chenopodiaceae. The word Halothamnus comes from the Greek word, Halo means “salt” and thamnus means “bush”, which explains the habitat to be salty and also the accumulation of salt in the plants.
Mostly Halothamnus species are small shrubs and subshrubs. In young stages the branches are green or olive-green. Sometimes light yellow collenchymatous lines are also present in the branches. The sessile, alternate leaves are simple, half-terete or flat and slightly fleshy. Often short curled hairs grow in the axils of the leaves. The branches terminate into inflorescences which is long, lose and panicle like. The bisexual flowers are solitary and sessile and arise from the axils of green bracts and two bracteoles. Bracts contain two distinct parts; lower part is leaf like and upper part is smaller and scale-like and as long as bracteoles. Bracteoles are scale- like and contain membranous margins. The green tepals are erect and slightly connate at base. All the tepals are almost equal in length and their margins are membranous. Five epitepalous stamens are present with band shaped filaments, which arise from a fleshy hypogynous disc. Anthers are short with flat appendages. Pistil contain ovoid ovary whose upper part is conical style like. The pollen grains differentiate the species as pollens in
Page 156
different specie differ in number and diameter (Kothe-Heinrich, 1993).
Nearly 21 specie of the genus are known so far, which are listed below: H. afghanicus , H. auriculus , H.beckettii , H.bamianicus , H. bottae , H. cinerascens , H. ferganensis , H. glaucus , H. hierochunticus , H. iranicus , H. iliensis , H. iraqensis , H. kermanensis , H. lancifolius , H. somalensis , H. schurobi , H. sistanicus , H. seravschanicus , H. subaphyllus , H. turcomanicus and
H. oxianus .
6.1.1. Distribution of the Genus Halothamnus
Halothamnus bottae is distributed in Saudi Arabia, UAE, Yemen and
Oman. Halothamnus glaucus is found in Eastern Turkey, Armenia,
Turkamanistan, Azerbaijan and up to China. Halothamnus subaphyllus grows in the same region and also in Pakistan and Afghanistan (Kothe-Heinrich,
1991).
6.1.2. Importance of the Genus Halothamnus
Like several other plants, the relatives of this genus are also known to have various commercial importance. For example H. subaphyllus and H. glaucus are grown as fodder plant for grazing animals (Kinzikaeva, 1968).
Traditionally, H. subaphyllus has been used for hair strengthening and women's diseases. Also it has been used to treat scabies, anthrax and wound
Page 157
healing in sheep (Larin, 1951; Sachobiddinov, 1948). Further, this species has been used as tissue dying agent and in soap formation (Enden, 1944). This plant is further reported to accumulate boron in the leaves (Amanova and
Kinzikaeva, 1973). Various parts of H. somalensis have been used for the remedy against parasitic worm disease (Dawo and Tibbo, 2005). Some species of this genus are reported to contain alkaloids (Adylov, 1970).
6.2. Halothamnus Auriculus
Halothamnus auriculus is a good fodder plant which is distributed in
Northern Iran, Afghanistan, Turkmenistan, Pakistan (Baluchistan), Tajikistan,
Kirghistan and Usbekistan . In these areas, it is grown for recultivation of grazing land. The plant is reported to accumulate boron in leaves.
6.2.1. Botanical Description of Halothamnus Auriculus
Halothamnus auriculus is a short woody and bushy plant that grows up to a height about 90 cm. The striated branches of the plant are with somewhat bluish green colour, contain fleshy and flat leaves. The shape of the leaves may be oval, triangular, round or kidney shaped. The leaf with membranous margins may be of 6.8 cm in length and up to 4.5 cm in width. The bracteoles are scale like, shorter with membranous margins and grow parallel to the perianth . Wide stigmas are present which are trim at the apex.
Page 158
Figure 6.1: Leaf and Fruit of Halothamnus auriculus
6.2.2. Classification of Halothamnus Auriculus
Kingdom Plantae Unranked Angiosperms Order Caryophyllales Family Amaranthaceae Subfamily: Salsoloideae Genus: Halothamnus Species H. auriculus 6.2.3. Previous Phytochemical studies of Halothamnus Auriculus
Literature search revealed that previously no phytochemical studies were carried out on this species. The importance of this plant in folk medicine system, whereas, lack of scientific data on this plant, prompted us to investigate it for its bioactive secondary metabolites. In this study 11 metabolites have been isolated and characterized for the first time.
Page 159
6.3. Results and Discussions of the Isolated Compounds
6.3.1. Structure Elucidation of Allantoic Acid (248)
Compound 248 was isolated as white needle like crystals. Its EIMS showed molecular ion peak at m/z 176.0 [M] +, whereas, O OH the HREIMS depicted the molecular formula C4H8N4O4 O O
H2N N N NH2 with 3 DBE. The IR spectrum showed absorption H H 248 bands at 1710, 1645 and 3435 cm -1 for carbonyl and amine functions respectively. In the 1H NMR spectrum of 248 , only one signal was observed at δ 5.30, correlated through HSQC experiment with a carbon resonating at δ 65.7. In addition to this carbon, the 13 C NMR spectrum displayed two more signals at δ 161.1 and 178.0. In HMBC spectrum (Fig 6.2), the methine hydrogen at δ 5.30 exhibited long-range correlation with both the quaternary carbons ( δ 161.1 and 178.0). The above discussed data could only be fitted for allantoic acid, which is a known secondary metabolite and is reported for the first time from our investigated source.
O OH
O O
H2N N N NH2 H H
248
Figure 6.2: HMBC correlations observed in the spectrum of 248
Page 160
6.3.2. Structure Elucidation of Quercetin 3-glucoside (249)
The compound 249 was obtained as yellow amorphous powder, which exhibited the pseudo-molecular ion at m/z 465 OH
+ HO O [M+H] in positive FABMS. The high OH resolution analysis ( m/z 465.3839) of the same O OH O OH ion revealed the molecular formula as O HO C21 H21 O12 . The IR spectrum showed prominent OH OH 249 bands at 3240 for hydroxyl, 1665 for conjugated ketone, 1606 and 1520 cm -1 for phenyl groups. The UV spectrum showed absorption maxima at 255, 278, 315 and 332 nm as characteristic feature of quercetin glycoside (Mabry et al., 1970).
The aromatic region of the 1H-NMR spectrum of 249 displayed five signals at
δ 7.70 (1H, d, J = 2.0 Hz), 7.58 (1H, dd, J = 2.0, 8.4), 6.86 (1H, d, J = 8.4 Hz),
6.36 (1H, d, J = 1.6 Hz, H-8) and 6.18 (1H, d, J = 1.6 Hz, H-6). The first three signals splitted at ABX pattern were attributed to ring B of quercetin nucleus, whereas, remaining two were attested for ring A. A sugar moiety was also found in 249 due to the resonance of anomeric proton at δ 5.23 (1H, d, J = 7.2
Hz), and other sugar protons in the range of δ 3.72-3.22. The amount of coupling constant of anomeric proton suggested the sugar to be a β hexose and was identified as glucose due to acidic hydrolysis followed by
Page 161
comparative TLC of the hydrolyzed sugar with the authentic sample of glucose and its optical rotation values [ α]D20 +50.0˚(c, 10 in H 2O ) (Mukhtar et al., 2004). The 13 C NMR spectrum of 249 (Table 6.1) supported proton data, as it displayed signals for 21 carbon atoms including fifteen carbons for flavonoid nucleus and six for sugar unit. The sugar connectivity was found at
C-3 due to the HMBC correlation (Fig 6.3) of anomeric proton ( δ 5.23) with a quaternary carbon at δ 135.6 (C-3). The whole discussed data resembled with the data of quercetin 3-O-β-D-glucoside (Guvenalp and Demirezer, 2005), a known compound, which is isolated for the first time from this source.
OH
HO O OH
O OH O OH O
HO OH OH HMBC ())and COSY (
Figure 6.3: HMBC correlations observed in the spectrum of 249
Page 162
6.3.3. Structure Elucidation of 8-C-glucopyranosylapigenin (250)
The compound 250 was isolated as yellow amorphous powder, whose molecular formula C 21 H21 O10 was OH HO established through positive O HO OH OH HRFABMS ( m/z 433.3849 [M+H] +). HO O The IR and UV data was similar to the OH O data of 249 to support a flavonoid 250 nucleus In 1H-NMR (Table 6.1) two ortho coupled doublets were observed at
δ 8.02 (2H, d, J = 8.8 Hz) and 6.89 (2H, d, J = 8.8 Hz), attested for p-substituted aromatic ring B of flavonoid nucleus. The other two singlets observed at δ
6.76 and 6.26 were attested for H-3 of ring C and H-6 of ring A respectively.
This data revealed that ring A must be penta-substituted. Besides, the same spectrum displayed signals in the range of δ 4.68-3.23 due to a sugar moiety.
The 13 C-NMR spectrum (Table 6.1) of 250 was in accordance with other data suggesting a flavone glycoside. A sugar proton at δ 4.68 (1H, d, J = 7.6 Hz) was correlated through HSQC spectrum with the carbon resonating at δ 73.3.
This hydrogen ( δ 4.68) showed HMBC (Fig. 6.4) correlation in aromatic region with the carbons at δ 162.5 (C-7), 155.9 (C-8a) and 104.5 (C-8), This observation led to the idea of a C-linked sugar in 250 . Further careful analysis of HMBC data established the sugar linkage at C-8. Comparison with
Page 163
literature data confirmed the compound 250 to be 8-C- glucopyranosylapigenin (Wen et al., 2007), which is a known phytochemical but isolated for the first time from Halothamnus auriculus
OH HO O HO OH OH HO O
OH O
HMBC ())and COSY (
Figure 6.4: Important HMBC and COSY correlations observed in the spectra
of 250
Page 164
Table 6.1: 1H and 13 C NMR data of 249 (CD 3OD , 400 and 100 MHz) and 250 (DMSO, 400 and 100 MHz) Compound 249 Compound 250 Position δH (J = Hz) δC δH (J = Hz) δC 2 - 159.0 - 163.9 3 - 135.6 6.76, s 102.4 4 - 179.4 - 182.1 4a - 105.7 - 104.0 5 - 163.0 - 155.9 6 6.18, d (1.6) 99.9 6.26, s 98.1 7 - 165.9 162.5 8 6.36, d (1.6) 94.7 104.5 8a 2.97, m 158.4 2.97, m 160.3 1′ _ 123.0 _ 121.6 2′ 7.70, d (2.0) 117.6 8.02, d (8.8) 128.9 3′ - 145.8 6.89, d (8.8) 115.8 4′ - 149.8 - 161.1 5′ 6.86, d (8.4) 116.0 6.89, d (8.8) 115.8 6′ 7.58, dd (2.0, 8.4) 123.2 8.02, d (8.8) 128.9 1′′ 5.23, d (7.2) 104.4 4.68, d (7.6) 73.3 2′′ 3.48, t (8.4) 78.1 3.23, m 81.8 3′′ 3.52, m 75.7 3.28, t (8.4) 78.6 4′′ 3.34, t (7.2) 71.2 3.37, m 70.5 5′′ 3.22, m 78.3 3.82, m 70.8 6′′ 3.72, dd (2.4, 12.0) 62.6 3.70, dd (6.0, 11.2) 61.2 3.59, dd (5.2, 12.0) 3.58, dd (5.6, 11.0) 5-OH - - 13.1, s - 2′′ -OH - - 4.90, d (5.2) - 3′′-OH - - 4.88, d (4.0) - 6′′ -OH - - 4.59, t (5.6) -
Page 165
6.3.4. Structure Elucidation of 5, 6-Dihydroxy-3′, 4′,7-Trimethoxy
Flavone (251)
Compound 251 was also isolated as yellow amorphous powder, which absorbed in the IR region at 3455, 1670,
OMe 1615, 1505, 1465, 1310 cm -1 for hydroxyl, MeO O OMe carbonyl, phenyl and methoxyl groups HO OH O respectively. The EIMS Spectrum displayed 251 molecular ion at m/z 344 while in HREIMS the molecular ion peak was observed at m/z 344.0891 corresponding to the molecular formula C 18 H16 O7.
The UV spectrum showed absorptions maxima at 278, 280, 345 and 370 nm for an oxygen-substituted flavonoid skeleton (Mabry et al., 1970).
In the 1H-NMR spectrum (Table 6.2), an ABX system of protons resonated at
δ 7.38 (1H, dd, J = 1.6, 8.4Hz), 6.86 (1H, d, J = 8.4 Hz) and 7.23 (1H, d, J = 1.6
Hz), was attested for ring B of the flavonoid. In addition the spectrum displayed two singlets at δ 6.49 and 6.46, which were assigned to H-8 and H-
3 respectively. Three methoxyl function displayed their position at δ 3.88,
3.86 and 3.85. The 13 C-NMR spectrum (Table 6.2) was in full agreement with
1H NMR spectrum and formula as it displaying 18 carbon signals. The three methoxyl functions were fixed at C-7, C-3′ and C-4′ due to HMBC spectral analysis (Fig. 6.5) The whole data of compound 251 was further compared to
Page 166
the literature values and were found identical the data of 5, 6-dihydroxy- 3′,
4′, 7-trimethoxy flavones (Nagao et al., 2002) which is a known compound but isolated for the first time from Halothamnus auriculus .
OMe
MeO O OMe
HO OH O
251
Figure 6.5: HMBC correlations observed in the spectrum of 251
6.3.5. Structure Elucidation of 4′, 5, 7-Trihydroxy-3′, 6-dimethoxy Flavone (252) Compound 252 was also found to be an oxygenated flavonoid, as it
exhibited the IR and UV data similar to
compound 251 (Mabry et al., 1970). The OH
HO O OMe molecular formula C 17 H14 O7 of 252 MeO showed one carbon less than that of 251 , OH O 252 whereas, the aromatic region of the 1H-
NMR spectrum (Table 6.2) was also identical to that of 251 . However, the
same spectrum afforded two methoxyl groups at δ 3.82 and 3.84 instead of
three as were found in 251 . Based on this information, it was concluded that
252 must has an extra hydroxyl group when compared to that of 251 . The
Page 167
position of the two methoxyl groups at C-6 and C-3’ could be identified due
to HMBC interaction of the two methoxyl groups i.e at δ 3.84 and 3.82 with
C-6 ( δ 133.2) and C-3’ ( δ 147.1) respectively (Fig. 6.6). Combination of the
whole data led to the structure of 252 as 4′, 5, 7-trihydroxy-3′, 6-dimethoxy
flavone (Miski et al., 1983) which is a known natural product but has been
isolated for the first time from our investigated source .
OH
HO O OMe
MeO OH O
252
Figure 6.6: HMBC correlations observed in the spectrum of 25 2
6.3.6. Structure Elucidation of Quercetin 3′,4′-dimethyl ether (253)
Compound 253 was found to be dimethyl ether of quercetin as the aromatic region showed five signals at δ 7.61 OMe
(1H, dd, J = 8.4, 2 Hz), 7.30 (1H, d, J = 2 Hz), HO O OMe 6.90 (1H, d, J = 8.4 Hz), 6.38 (1H, d, J = 2 Hz) OH OH O and 6.17 (1H, d, J = 2 Hz). The first three 253 signals splitted at ABX pattern were attributed to ring B, whereas, the rest two were attested for ring A. This data indicated that C-3 position in 253
Page 168
must be substituted, which was substantiated due to the resonance of C-3 at δ
133.6 in 13 C-NMR spectrum (Table 6.2). Two methoxyl units resonated in the
NMR spectra of 253 at δ 3.81 and 3.83, which were fixed at C-3’ and C-4’ due to HMBC spectral analysis. The molecular formula C 17 H14 O7 also supported the above deduction, whereas, the remaining data like IR and UV values were similar to that of 251-252 . Further the comparison of these data with the reported values in literature helped to deduce the structure of 253 as quercetin3′,4′-dimethyl ether, which has been reported for the first time from
Halothamnus auriculus (Pavanasasivam and Sultanbawa, 1975).
OMe
HO O OMe
OH OH O 253
Figure 6.7: HMBC correlations observed in the spectrum of 253
Page 169
Table 6.2: 1H- and 13 C-NMR data of 251, 252 and 253 (CD 3OD, 400 MHz)
Compound 251 compound 252 compound 253
Position δH (J = Hz) δC δH (J = Hz) δC δH (J = Hz) δC 2 - 163.9 - 164.5 - 159.0 3 6.46, s 104.1 6.65, s 105.3 - 133.6 4 - 182.0 - 182.4 - 182.5 4a - 105.2 - 105.5 - 104.5 5 - 152.9 - 153.5 - 163.0 6 - 132.0 - 133.2 6.17, d (2.0) 99.2 7 158.0 - 159.0 - 166.0 8 6.49, s 93.9 6.55, s 90.4 6.38, d (2.0) 94.4 8a - 153.3 - 154.2 - 158.0 1′ - 123.0 - 123.5 - 123.8 2′ 7.23, d (1.6) 108.3 7.36, d (1.6) 110.3 7.30, d (2.0) 108.0 3′ - 146.5 - 147.1 - 148.5 4′ - 149.0 - 149.1 - 149.0 5′ 6.86, d (8.4) 116.0 7.12, d (8.0) 114.6 6.90, d (8.4) 116.0 6′ 7.38, dd (1.6, 121.5 7.45, dd (1.6, 8.0) 128.3 7.61, dd (2, 8.4) 121.7 8.4) 3′-OMe 3.88, s 56.8 3.82, s 55.6 3.83, s 56.0 4′-OMe 0.85, s 56.3 - - 3.81, s 55.5 6-OMe - - 3.84, s 64.6 - - 7-OMe 3.86, s 60.3 - - - -
Note: Result and discussion of compounds 106 , 113 , 233 , 237 and 238 is
already discussed in chapter 4 under Sec. 4.2.17 , 4.2.20 , 4.2.13 , 4.2.18
and 4.2.19 respectively.
6.4. General Experimental
Detailed procedure is already discussed in Sec. 5.1 on page 122.
6.4.1. Collection and Identification of Plant
The whole plant of Halothamnus auriculus (4 kg) was collected in
September 2010 from Ziarat, Baluchistan and was identified by Prof. Dr.
Page 170
Rasool Bakhsh Tareen, Department of Botany, University of Baluchistan,
Quetta, where the voucher specimen has been deposited in the herbarium.
6.4.2. Extraction and Isolation
The shade dried whole plant of H. auriculus (3 kg) was extracted thrice with MeOH (15 L). The methanolic extract was evaporated to dryness under vacuum, and the residue (50 g) was divided into n-hexane (4 g), ethylacetate
(22 g), butanol (12 g) and water (12 g) layer. Column chromatography of the ethylacetate fraction over silica gel eluting with n-hexane, n-hexane-EtOAC ,
EtOAC and EtOAC -CH 3OH in increasing order of polarity yielded eight fractions (HA 1-HA 8).
HA 7 (3 g) was collected from the main column with EtOAc:MeOH
(9.5:0.5). This fraction was rechromatographed on silica gel column and was eluted with isocratic of n-hexane:ethylacetate (3.0:7.0) to get three sub fractions (HA 7a-c). Further purification of HA 7a under the same conditions yielded 251 (22 mg) and 233 (35 mg). HA 7c on further chromatography yielded 250 (20 mg) at n-hexane: ethyl acetate (3:7).
HA 5 (2.5g) was obtained from the main column with n-hexane:ethyl acetate
(2.0:8.0). This fraction was subjected to silica gel column chromatography using a gradient of n-hexane: ethyl acetate to get three sub fractions (HA 5a-c).
The Sub fraction HA 5a on repeated silica gel column chromatography yielded
Page 171
253 (35 mg) when eluted with n-hexane:ethyl acetate (6:4) and 252 (21 mg) with n-hexane:ethyl acetate (5.5:4.5). Other sub-fraction HA 5c afforded 248 and 249 (20 mg) under the same conditions.
The main fraction HA 4 (4 g) collected from the main column with n- hexane:ethyl acetate (4.0:6.0),on further silica gel column chromatography with an isocratic of n-hexane:ethyl acetate (3:7) yielded 237 (150 mg) as major component of halothamnus auriculus .
HA 2 (2 g), collected from the main column with n-hexane:ethyl acetate
(8.0:2.0), was rechromatographed on silica gel column eluting with a gradient of n-hexane:ethyl acetate to get three sub-fractions (HA 2a-c). The sub fraction
HA 2b was purified on silica gel column using n-hexane:ethylacetate (9.0:1.0) as mobile phase to get 106 (55 mg) and 238 (45mg).
HA 1 (3.0 g) obtained from the main column with n-hexane was further
purified on silica gel column when eluted with a gradient of n-hexane and
ethyl acetate yielding three sub-fractions (HA 1a-c). The sub-fraction HA 1c on
further silica gel column chromatography afforded 113 (13 mg) when eluted
with n-hexane:ethyl acetate (7.5:2.5) (scheme 6.8).
Page 172
Methanolic extract of Halthamnus auriculus
Water
Hexane
Water part Hexane part
EtOAc
Water part EtOAc part
Butanol CC over flash silica using Butanol part varying polarity of solvents n-hexane: ethyl acetate
HA HA1 HA2 HA3 HA4 HA5 HA6 HA7 8
238, 106 237 233, 250, 251
113 248, 249, 252, 253
Figure 6.8: Isolation Scheme of secondary metabolites from Halothamnus auriculus
6.5. Spectroscopic Data of the Isolated Compounds
6.5.1. Spectroscopic Data of Allantoic Acid (248): Needle like crystals (12
mg); IR (KBr): 3435, 1710, 1645cm -1; 1H-NMR (D 2O, 500 MHz): δ 5.30 (1H, s);
13 C-NMR (D 2O, 125 MHz): δ 178.0 (C-6, 7), 161.1 (C-2), 65.7 (C-4, 5). EIMS:
m/z 176.0 [M] +; HREIMS: m/z 176.0539 [M] + (176.0546 calcd. for C 4H8N4O4).
Page 173
6.5.2. Spectroscopic Data of Allantoin (233): Needle like crystals (35 mg); IR
(KBr): 3435, 1778 cm -1; 1H-NMR (Pyr, 500 MHz): δ 10.04 O HN NH (1H, s, NH-1), 8.70 (1H, s, NH-3), 8.60 (1H, d, 8.5 Hz, O N O H H N 2 NH-6), 6.88 (2H, s, NH 2-8), 6.38 (1H, dd, J = 8.8, 1.5 Hz, 233 H-5); 13 C-NMR (Pyr, 125 MHz): δ 159.5 (C-2), 174.9 (C-4),
64.5 (C-5), 158.6 (C-7); EIMS: m/z 158.0 [M] +; HREIMS: m/z 158.0436 [M] +
(158.0440 calcd. for C4H6N4O3).
6.5.3. Spectroscopic Data of Quercetin-3-glucoside (249): Yellow
amorphous powder (20 mg); UV λmax : 255, 278, OH
HO O OH 315 and 332 nm; IR (KBr): 3240, 1665, 2925,
O 1606, 1520; 1H-NMR (CD 3OD , 400 MHz): δ 7.70 OH O OH O (1H, d, J = 2.0 Hz, H-2′), 7.58 (1H, dd, J = 2.0, HO OH 8.4 Hz, H-6′), 6.86 (1H, d, J = 8.4 Hz, H-5′), 6.36 OH 249 (1H, d, J = 1.6 Hz, H-8), 6.18 (1H, d, J = 1.6 Hz,
H-6), 5.23 (1H, d, J = 7.2 Hz, H-1″), 3.72 (1H, dd, J =2.4, 12.0 Hz, H-6″), 3.59
(1H, dd, J = 5.2, 12.0 Hz, H-6″), 3.52 (1H, m, H-3″), 3.48 (1H, t, J = 8.4 Hz, H-
2″), 3.34 (1H, t, J =7.2 Hz, H-4″), 3.22 (1H, m, H-5″); 13 C-NMR (CD 3OD , 100
MHz): δ 179.4 (C-4), 165.9 (C-7), 163.0 (C-5), 159.0 (C-2), 158.4 (C-8a), 149.8 (C-
4′), 145.8 (C-3′), 135.6 (C-3), 123.2 (C-6′), 123.0 (C-1′), 117.6 (C-2′), 116.0 (C-5′),
105.7 (C-4a), 104.4 (C-1″), 99.9 (C-6), 94.7 (C-8), 78.3 (C-5″), 78.1 (C-2″), 75.7 (C-
Page 174
3″), 71.2 (C-4″), 62.6 (C-6″); FABMS: m/z 465.0 [M+H] +; HRFABMS: 465.3839
[M+H] + (465.3842 calcd. for C 21 H21 O12 ).
6.5.4. Spectroscopic Data of 8-C-glucopyranosylapigenin (250): Yellow
powder (20 mg); UV λmax : 372, 345, 281, OH HO O 278 nm; IR (KBr): 3450, 1665, 1650, 1505, HO OH OH 1467 cm -1; 1H-NMR (DMSO-d6 , 400 HO O MHz): δ 13.1 (1H, s, OH-5), 8.02 (2H, d, J OH O 250 = 8.8 Hz, H-2′, 6′), 6.89 (2H, d, J = 8.8 Hz,
H-3′, 5′), 6.76 (1H, s, H-3), 6.26 (1H, s, H-6), 4.90 (1H, d, J = 5.2 Hz, OH-2″),
4.88 (1H, d, J = 4.0 Hz, OH-3″), 4.68 (1H, d, J = 7.6 Hz, H-1″), 4.59 (1H, t, J =
5.6 Hz, OH-6″), 3.82 (1H, m, H-5″), 3.70 (1H, dd, J = 6.0, 11.2 Hz, H-6″), 3.58
(1H, dd, J = 5.6, 11.0 Hz, H-6″), 3.37 (1H, m, H-4″), 3.28 (1H, t, J = 8.4 Hz, H-
3″), 3.23 (1H, m, H-2″); 13 C-NMR (DMSO-d6 , 100 MHz): δ 182.1 (C-4), 163.9
(C-2), 162.5 (C-7), 161.1 (C-4), 160.3 (C-8a), 155.9 (C-5), 128.9 (C-2′,6′), 121.6 (C-
1′), 115.8 (C-3′, 5′), 104.5 (C-8), 104.0 (C-4a), 102.4 (C-3), 98.1(C-6), 81.8 (C-2′′),
78.6 (C-3′′), 73.3 (C-1′′), 70.8 (C-5′′), 70.5 (C-4′′), 61.2 (C-6′′); FABMS: m/z 433
[M+H] +; HRFABMS: m/z 433.3849 [M+H] + (433.3854 calcd. for C 21 H21 O10 ).
Page 175
6.5.5. Spectroscopic Data of 5, 6-dihydroxy-3′, 4′,7-trimethoxy Flavone
(251); Yellow powder (22 mg); UV λmax : 278, 280, 345, 370 nm; IR: 3455, 1670,
1655, 1505, 1465, 1310 cm -1; 1H-NMR ( CD 3OD, 400 MHz): δ 7.38 (1H, dd, J =
8.4, 1.6 Hz, H-6′), 7.23 (1H, d, J = 1.6 Hz, H-2′), 6.86 (1H, d, J = 8.4 Hz, H-5′),
6.49 (1H, s, H-8), 6.46 (1H, s, H-3), 3.88 (3H, s, 3′-OMe), 3.86 (3H, s, 7-OMe),
3.85 (3H, s, 4′-OMe); 13 C-NMR ( CD 3OD, 100 MHz): δ 182.0 (C-4), 163.9 (C-2),
158.0 (C-7), 153.3 (C-8a), 152.9 (C-5), 149.0 (C-4′), 146.5 (C-3′), 132.0 (C-6),
123.0 (C-1′), 121.5 (C-6′), 116.0 (C-5′), 108.3 (C-2′), 105.2 (C-4a), 104.1 (C-3), 93.9
(C-8), 60.3 (7-OCH 3), 56.8 (3′-OCH 3), 56.3 (4′-OCH 3); EIMS: m/z 344 [M] +;
HREIMS: m/z 344.0891 (344.0896 Calcd. for C 18 H16 O7).
6.5.6. Spectroscopic Data of 4′, 5, 7-trihydroxy-3′, 6-dimethoxy Flavone
(252): Yellow powder (21 mg); UV λmax : 215,
OH 271, 278, 345, 351 nm; IR (KBr): 3452, 1671,
HO O OMe 1656, 1502, 1462, 1314 cm -1; 1H-NMR
MeO OH O (CD 3OD, 400 MHz): δ 7.45 (1H, dd, J = 1.6, 252 8.0 Hz, H-6′), 7.36 (1H, d, J = 1.6 Hz, H-2′),
7.12 (1H, d, J = 8.0 Hz, H-5′), 6.65 (1H, s, H-3), 6.55 (1H, s, H-8), 3.84 (3H, s, 6-
OMe), 3.82 (3H, s, 3′-OMe); 13 C-NMR (CD 3OD, 100 MHz): δ 182.4 (C-4), 164.5
(C-2), 159.0 (C-7), 154.2 (C-8a), 153.5 (C-5), 149.1 (C-4′), 147.1 (C-3′), 133.2 (C-
6), 128.3 (C-6′), 123.5 (C-1′), 114.6 (C-5′), 110.3 (C-2′), 105.5 (C-4a), 105.3 (C-3),
Page 176
90.4 (C-8), 64.6 (6-OCH 3), 55.6 (3′-OCH 3). EIMS: m/z 330 [M] +; HREIMS: m/z
330.0732 [M] + (330.0740 calcd. for C 17 H14 O7).
6.5.7. Spectroscopic Data of Quercetin 3′,4′-dimethyl Ether: (253): Yellow crystals (35 mg) ; UV λmax . 286, 352, 255 nm; IR (KBr): 3415, 1748, 1655, 1610,
1310 cm -1; 1H-NMR (CD 3OD, 400 MHz): δ 7.61 (1H, dd, J = 8.4, 2 Hz, H-6′),
7.30 (1H, d, J = 2 Hz, H-2′), 6.90 (1H, d, J = 8.4 OMe
HO O Hz, H-5′), 6.38 (1H, d, J = 2 Hz, H-8), 6.17 (1H, OMe OH d, J = 2 Hz, H-6), 3.83 (3H, s, 3′-OMe), 3.81 (3H, OH O
253 s, 4′-OMe); 13 C-NMR (CD 3OD, 100 MHz): δ
182.5 (C-4), 166.0 (C-7), 163.0 (C-5), 159.0 (C-2), 158.0 (C-8a), 148.5 (C-3′), 149.0
(C-4′), 133.6 (C-3), 123.8 (C-1′), 121.7 (C-6′), 116.0 (C-5′), 108.0 (C- 2′), 104.5 (C-
4a), 99.2 (C-6), 94.4 (C-8), 56.0 (3′-OMe), 55.5 (4′-OMe). EIMS: m/z 330 [M] +.
HREIMS: 330.0735 (330.0740 calcd. for C 17 H14 O7).
6.5.8. Spectroscopic Data of β-Sitosterol 3-O-β-D-glucopyranoside (237):
White solid (150 mg); IR
(KBr): 3440, 1640, 1615, 1585-
OH 1540 cm -1: 1H-NMR O HO HO O OH (CDCl 3+CD 3OD, 400MHz): δ 237 5.29 (1H, d, J = 7.8 Hz, H-1′),
5.17 (1H, br d, J = 5.6 Hz, H-6), 3.76 (1H, m, H-3), 3.90-4.57 (6H, m, Glc-H),
Page 177
0.97 (3H, s, Me-19), 0.88 (3H, d, J = 6.4 Hz, Me-21), 0.78 (3H, t, J = 6.8 Hz, Me-
29), 0.76 (3H, d, J = 6.4 Hz, Me-26), 0.74 (3H, d, J = 7.0 Hz, Me-27), 0.67 (3H, s,
Me-18): 13 C-NMR (CDCl 3+CD 3OD, 100MHz): δ 140.3 (C-5), 120.4 (C-6), 105.3
(C-1′), 80.9 (C-3), 77.3 (C-5′), 75.8 (C-3′), 72.1 (C-2′), 71.2 (C-4′), 62.1 (C-6′), 57.2
(C-17), 53.2 (C-14), 49.9 (C-9), 47.3 (C-24), 41.9 (C-13), 41.5 (C-12), 40.7 (C-4),
37.5 (C-20), 37.1 (C-10), 36.6 (C-22), 36.3 (C-1), 33.9 (C-7), 33.2 (C-8), 29.8 (C-
25), 28.0 (C-23), 27.7 (C-2), 25.9 (C-16), 25.7 (C-15), 23.9 (C-28), 22.8 (C-11), 18.7
(C-27), 18.1 (C-19), 17.5 (C-21), 14.7 (C-26), 13.8 (C-18) and 12.5 (C-29):
FABMS: m/z 576.0 [M] +; HRFABMS: m/z 576.4376 [M] + (576.4389 calcd. for
C35 H60 O6).
6.5.9. Spectroscopic Data of Oleanolic Acid (238): White amorphous solid
(45 mg); IR (KBr): 3403, 1716, 1664, and 822
cm -1; 1H-NMR: (CDCl 3, 400 MHz): δ 5.28 (1H,
OH t, J = 3.8 Hz, H-12), 3.49 (1H, dd, J = 4.4, 11.9 O
HO Hz ,H-3), 2.84 (1H, dd , J = 3.6, 13.2 Hz, H-18),
238 1.13, 1.06, 0.98 , 0.95, 0.92, 0.88 and 0.82 (3H, each s, Me); 13 C-NMR: (CDCl 3, 100 MHz); δ 179.8 (C-28), 144.3 (C-13), 122.2
(C-12), 79.0 (C-3), 53.9 (C-5), 48.9 (C-9), 47.1 (C-17), 46.6 (C19), 41.4 (C-14),
41.0 (C-18), 40.4 (C-8), 39.2 (C-4), 37.6 (C-1), 37.3 (C-10), 33.1 (C-7), 33.0 (C-21),
32.8 (C-29), 32.5 (C-22), 31.0 (C-20), 29.6 (C-23), 26.9 (C-2), 26.7 (C-15), 25.7 (C-
Page 178
27), 23.8 (C-11), 22.6 (C-30), 22.3 (C-16), 19.6 (C-26), 18.7 (C-6), 14.8 (C-24),
14.6 (C-25); EIMS: m/z 456.0 [M] +; HREIMS: m/z 456.3603 [M] + (456.3613 calcd. for C 30 H48 O3 ).
6.5.10. Spectroscopic Data of β-Sitosterol (106): White needle like crystals
(55 mg); IR (KBr): 3457, 3052, 1648, and 817
cm -1; 1H-NMR (CDCl 3, 400 MHz): δ 5.23 (1H,
m, H-6), 3.49 (1H, m ,H-3), 1.03 (3H, s, Me- H H HO 106 19), 0.89 (3H, d, J = 6.4 Hz, Me-21), 0.85 (3H, t, J = 6.8 Hz, Me- 29), 0.82 (3H, d, J = 6.5 Hz, Me-27), 0.73 (3H, d, J = 6.5 Hz,
Me-26), and 0.71 (3H, s, Me-18); 13 C-NMR (CDCl 3, 100 MHz): δ 140.9 (C-5),
123.7 (C-6), 76.2 (C-3), 56.7 (C-17), 55.4 (C-14), 51.3 (C-9), 49.3 (C-24), 46.1 (C-
13), 42.5 (C-4), 39.2 (C-12), 39.0 (C-10), 38.6 (C-20), 36.9 (C-1), 36.8 (C-8), 35.2
(C-7), 32.9 (C-22), 30.0 (C-23), 29.1 (C-2), 26.8 (C-15), 25.3 (C-16), 22.2 (C-28),
22.1 (C-25), 20.1 (C-26), 20.0 (C-11), 19.9 (C-27), 19.6 (C19), 17.8 (C-21), 17.6 (C-
18), 12.8 (C-29). EIMS: m/z 414 [M] +; HREIMS: m/z 414.3857 [M] + (414.3861 calcd. for C 29 H50 O).
Page 179
6.5.11. Spectroscopic Data of Lupeol (113): White solid (13 mg); IR (KBr):
3458, 3063, 1645, and 840 cm -1; 1H-NMR (CDCl 3,
400 MHz): δ 4.73 (1H, br s, H-29), 4.59 (1H, br s,
H-29), 3.41 (1H, dd, J = 4.4, 9.8 Hz, H-3), 1.58, 1.03,
HO 0.93 , 0.91, 0.89, 0.84 and 0.79 (3H, each s, Me); 13 C- 113 NMR (CDCl 3, 100 MHz): δ 153.0 (C-20), 108.1 (C-
29), 79.0 (C-3), 53.4 (C-5), 49.4 (C19), 49.3 (C-18), 47.2 (C-9), 44.8 (C-13), 43.7
(C-14), 40.4 (C-17), 40.1 (C-4), 39.0 (C-8), 38.2 (C-22), 37.0 (C-10), 36.0 (C-1),
32.5 (C-7), 30.0 (C-16), 29.7 (C-21), 27.9 (C-2), 27.3 (C-12), 27.0 (C-23), 25.9 (C-
15), 21.7 (C-11), 21.5 (C-30). 19.4 (C-25), 18.9 (C-28), 18.1 (C-6), 17.8 (C-27), 17.6
(C-24), 17.1 (C-26). EIMS: m/z 426.0 [M] +; HREIMS: 426.3858 [M] + (426.3862 calcd. for C 30 H50 O).
Page 180
References
Abe, I., Rohmer, M. and Prestwich, G. D. (1993). Enzymatic cyclization of squalene and oxidosqualene to sterols and triterpenes. Chemical Reviews , 93, 2189-2206.
Adylov, T. A. (1970). Âdovitye i alkaloidonosnye rasteniâ Karakulevodčeskich pastbiŝ Uzbekistana. - Taškent:. Izdat. Fil. Akad. Nauk Uzbeksk. SSR .
Ahmad, E., Malik, A., Ahmad, M. M., Munawar, M. A., Nagra, S. A., Anwar, J., Qazi, M. A., Sharif, A., Afza, N. and Ashraf, M. (2008). Investigation of antimicrobial constituents from Aerva persica . Journal of the Chemical Society of Pakistan , 30, 936-941.
Ahmad, S., Ahmad, I., Saleem, M., Jabbar, A., Rehman, N. U., Hassan, S. S. U., Akhtar, K. S. and Chaudhry, M. I. (2009). Secondary metabolites from Alhaji maurorum. Journal of Chemical Soceity of Pakistan , 31, 960-963.
Ahmed, E., Imran, M., Malik, A. and Ashraf, M. (2006). Antioxidant activity with flavonoidal constituents from Aerva persica . Archives of Pharmacal Research, 29, 343-347.
Ali, N. H. A., Mothana, R. A. A., Lesnau, A., Pilgrim, H. and Lindequist, U. (2003). Antiviral activity of Inonotus hispidus . Fitoterapia , 74, 483-485.
Amanova, N. M. and Kinzikaeva, G. K. (1973). Marevye (Chenopodiaceae Vent.) Ûžnogo Tadžikistana kak nakopiteli bora. - Izv. Akad. Nauk Tadžiksk. SSR. Otd. Biol. Nauk , 3, 18-24.
Page 181
Arnum, S. D. V. (1998). Vitamin A in kirk-othmer encyclopedia of chemical technology. In Vitamin A , pp. 99-107. New York: John Wiley.
Bai, R., Paull, D. K., Herald, C. L., Malspeis, L., Pettit, G. R. and Hamel, E. (1991). Halichondrin B and homohalichondrin B, marine natural products binding in the vinca domain of tubulin. Discovery of tubulin based mechanism of action by analysis of differential cytotoxicity data. The Journal of Biological Chemistry , 266, 15882-15889.
Bakshi, G. D. N., Sarma, P. S. and Pal, D. C. (1999). In A lexicon of medicinal plants in India , pp. 61-63. Calcutta: Naya Prokash.
Barrios-Gonzalez, J., Fernandez, F. J., Tomasini, A. and Mejia, A. (2005). Secondary metabolites production by solid state fermentation. Malaysian Journal of Microbiology , 1, 1-6.
Basant, B., Chaurasia, O. P., Zakwan, A. and Singh, S. B. (2008). Traditional medicinal plants of cold desert Ladakh used against kidney and urinary disorders. Journal of Ethnopharmacology , 118, 331-339.
Bathori, M., Pongracz, H., Methe, I., Kalman, A. and Argay, G. (2002). 5-α- and 5-β-2-deoxyintegristerone A, a 5-alpha and 5-beta isomer pair of ecdysteroids isolated from the Silene genus. Biomedical Chromatography , 16, 373-378.
Bathori, M., Toth, N., Hunyadi, A., Marki, A. and Zador, E. (2008). Phytoecdysteroids and anabolic androgenic steroids structure and effects on humans. Current Medicinal Chemistry , 15, 75-91.
Page 182
Bernards, M. A. and Lewis, N. G. (1992). Alkyl ferulates in wound healing potato tubers. Phytochemistry, 31, 3409-3412.
Bhuwan, B., Mishra and Tiwari, V. K. (2011). Natural products in drug discovery: clinical evaluations and investigations. Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 1-62.
Blumberg, P. M. (1988). Protein kinase C as the receptor for the phorbol ester tumor promoters: sixth rhoads memorial award lecture Cancer Research , 48, 1-8.
Bohm, B. A. (1988). The minor flavonoids. In The Flavonoids , (ed. J. B. Harborne), pp. 329-388: Chapman and Hall.
Bosoglu, A., Birdane, F. and Solmaz, H. (1998). The effect of Henna ( Folium lawsoniae ) paste in ringworm in calves. Indian Veterinary Journal , 75, 83-84.
Burkill, H. M. (1985). In The useful plants of west tropical Africa , vol. 1.
Burndud. (1990). Wanggalili; Yinjibarndi and Ngarluma Plants. In Juluwarlu Aboriginal Corporation , pp. 17.
Butler, M. S. (2004). The role of natural product in chemistry and in drug discovery. Journal of Natural Product , 67, 2141-2153.
Butler, M. S., Blaskovich, M. A. and Cooper, M. A. (2013). Antibiotics in the clinical pipeline in 2013. Journal of Antibiotics , 66, 571-591.
Cai, Y., Sun, M. and Corke, H. (2001). Identification and distribution of simple and acylated betacyanins in the Amaranthaceae. Journal of Agriculture and Food Chemistry , 49, 1971−1978.
Page 183
Chawla, P., Chawla, A., Vasudeva, N. and Sharma, S. K. (2012). A review of chemistry and biological activities of the genus Aerva , A desert plant,. Acta Poloniae Pharmaceutica - Drug Research , 69, 171-177.
Chen, Y. H., Chang, F. R., Lin, Y. J., Wang, L., Chen, J. F., Wu, Y. C. and Wu, M. J. (2007). Identification of phenolic antioxidants from Sword Brake fern (Pteris ensiformis Burm.). Food Chemistry , 105, 48-56.
Chiang, L. C., Ng, L. T., Lin, I. C., Kuo, P. L. and Lin, C. C. (2006). Anti proliferative effect of apigenin and its apoptotic induction in human Hep G2 cells. Cancer Letters , 237, 207-214.
Chowdhury, D., Sayeed, A., Islam, A., M, S. A. B. and Khan, G. R. A. M. (2002). Antimicrobial activity and cytotoxicity of Aerva lanata . Fitoterapia , 73 , 92-94.
Christie, M. B., Rene, A. B. and Paresh, N. S. (2013). Icosapent ethyl for the treatment of hypertriglyceridemia. Expert Opinion on Pharmacotherapy , 14 , 1409-1416.
Daly, J. W., Gusovsky, F., Myers, C. W., Yotsuyamashita, M. and Yasumoto, T. (1994). 1st Occurrence of tetrodotoxin in a dendrobatid frog ( Colostethus Inguinalis ), with further reports for the bufonid genus atelopus. Toxicon , 32 , 279-285.
Davies, T. G., Dinan, L. N., Lockley, W. J. S., Rees, H. H. and Goodwin, T. W. (1981). Mechanism of formation of the A/B cis ring junction of ecdysteroid in the locust Shistocerca gregaria . Biochemical Journal , 194, 53-62.
Page 184
Dawo, F. and Tibbo, M. (2005). Anthelmintic effect of Halothamus somalensis in arsi-bale goats. Livestock Research for Rural Development , 17, 1-8.
Deshmukh, T. A., Bapuso, V. Y., Sachin, L. B. and Subash, L. B. (2008). Antihyperglycaemic activity of alcoholic extract of Aerva lanata (L.) juss. ex Schults leaves in alloxan induced diabetic mice. Journal of Applied Biomedicine , 6, 81-87.
Dewick, P. M. (1988). In Isoflavonoids. In The Flavonoids , (ed. J. B. Harborne): pp. 125. Chapman and Hall.
Dewick, P. M. (2002). Medicinal natural products: A biosynthentic approach, pp. 520. Great Britain: John Wiley and Son.
Dinan, L., Harmatha, J. and Lafont, R. (2001). Chromatographic procedures for the isolation of plant steroids. Journal of Chromatography A, 935, 105-123.
Donnelly, D. M. X. and Sheridan, M. H. (1975). Neoflavonoids In The Flavonoids , (eds. B. Harborne T. Mabry and H. Mabry), pp. 802. London: Chapman and Hall.
El-Seedi, R, H., Sobaih and Revista, A.-M. S. (1999). Triterpenes and flavonol glycosides from Aerva javanica . Latinoamericana de Quimica , 27, 17- 21.
Eldor, A., Orevi, M. and Rigbi, M. (1996). Role of the leech in medical therapeutics. Blood Reviews , 10, 201-209.
Ellman, G. L., Courtney, K. D., Andres, V. and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology , 7, 88-95.
Page 185
Enden, O. A. (1944). Krasil'nye rasteniâ Turkmenii. - Trudy Turkmensk. Fil. Akad. Nauk SSSR , 5.
Faulkner, D. J. (1988). Marine natural products. Natural Product Report , 20, 269-309.
Feldlaufer, M. F., Weirich, G. F., Lusby, W. R. and Svoboda, J. A. (1991). Makisterone C, a 29 carbon ecdysteroid from developing embryos of cotton stainer bug. Desdercus fasciatus . Archives of Insect Biochemistry and Physiology, 18, 71-79.
Finar, I. L. (2002). Stereochemistry and the chemistry of natural products. In Organic Chemistry , vol. 2: English Language Book Society and Longman Group Limited.
Fotie, J., Bohle, D. S., Leimanis, M. L., Georges, E., Rukunga, G. and Nkengfack, A. E. (2006). Lupeol long chain fatty acid esters with antimalarial activity from Holarrhena floribunda . Journal of Natural Product , 69, 62-67.
Garg, S. P., Bhushan, R. and Kapoor, R. C. (1979). Chrysin-7-Ogalactoside; a new flavonoid from Aerva persica . Indian Journal of Chemistry, 17B, 416-417.
Garg, S. P., Bhushan, R. and Kapoor, R. C. (1980). Aervanone, a new flavonoid from Aerva persica . Phytochemistry , 19, 1265-1267.
Gehlot, D. and Bohra, A. (1998). Antimicrobial activity of various plant extracts of Aerva persica . Advances in Plant Sciences , 11, 109-111.
Geiger, H. (1988). Miscellaneous flavonoids. In The Flavonoids, Advances in Research , (ed. J. B. Harborne), pp. 389-397: Chapman and Hall.
Page 186
Gohari, A. R., Saeidnia, S., Hadjiakhoondi, A., Abdoullahi, M. and Nezafati, M. (2009). Isolation and quantificative analysis of oleanolic acid from Satureja mutica Fisch. & C. A. Mey. Journal of Medicinal Plants , 8, 65- 69.
Goldstein, J. L. and Brown, M. S. (1990). Regulation of the mevalonate pathway. Nature , 343, 425-430.
Goonaratna, C., Thabrew, I. and Wijewardena, K. (1993). Does Aerva lanata have diuretic properties? Indian Journal of Physiology and Pharmacology , 37, 135-137.
Goppi, P. G., Broglia, C., Merli, F., Dell'Olio, M., Stelitano, C., Iannitto, E., Federico, M., Bertè, R., Luisi, D., Molica, S. et al. (2003). Vinblastine, bleomycin, and methotrexate chemotherapy plus irradiation for patients with early-stage, favorable hodgkin lymphoma. Cancer , 98, 2393-2401.
Gottumukkala, V. R., Kandaswamy, K., Gopalakrishnan, M. and Mukhopadhyay, T. (2012). Isolation and characterization of a potent antimicrobial compound from Aerva sanguinolenta Blume.: an alternative source of bakuchiol. Journal of Pharmaceutical Research , 5, 174-176.
Goudet, C., .Chi, C. W. and Tytgat, J. (2002). An overview of toxins and genes from the venom of the Asian scorpion Buthus martensi Karsch. Toxicon , 40, 1239-1258.
Graham, W. and Roberts, J. B. (1953). Intravenous colchicine in the treatment of gouty arthritis. Annals of the Rheumatic Diseases , 12, 16-19.
Page 187
Graziano, J. H., Grady, R. W. and Cerami, A. (1974). The identification of 2,3- dihroxybenzoic acid as a potentially useful iron chelating drug. Journal of Pharmacology and Experimental Therapeutics , 190, 570-575.
Grieneisen, M. L., Warren, J. T., Sakurai, S. and Gilbirt, L. I. (1991). A putative route to ecdysteroids: Metabolism of cholesterol in vitro by mildly disrupted prothoracic glands of Manduca sexta . Insect Biochemistry , 21, 41- 51.
Grotewold, E. (2006). The Stereochemistry of Flavonoids. In The Science of Flavonoids , pp. 1-4. New York: Springer Science Business Media.
Gupta, S. K., Prakash, J. and Srivastava, S. (2002). Validation of traditional claim of Tulsi, Ocimum sanctum Linn. as a medicinal plant. Indian Journal of Experimental Biology , 40, 765-773.
Guvenalp, Z. u. and Demirezer, L. O. U. (2005). Flavonol glycosides from Asperula arvensis L. Turkish Journal of Chemistry , 29, 163 -169.
Halkens, S. B. A. (2001). The use of tannic acid in the local treatment of burn wounds: intriguing old and new perspectives. Wounds , 13, 144-158.
Hammerschmidt, R. (1999). Phytoalexins: What have we learned after 60 years? Annual Reviews of Phytopathology , 37, 285-306.
Hampshire, F. and Horn, D. H. S. (1966). Structure of crustecdysone, a crustacean moulting hormone. Chemical Communication, 2, 37-38.
Harbone, J. B. (1988). The flavonoids, pp. 17. London: Chapman and Hall.
Page 188
Harsh, G. R. and Kapoor, B. B. S. (2004). Ascorbic acid contents of some arid zone medicinal plants of Rajasthan. Oikoassay , 19, 49-50.
Henningfield, J. E. and Zeller, M. (2006). Nicotine psychopharmacology research contributions to United States and global tobacco regulation: a look back and a look forward. Psychopharmacology (Berl.) , 184, 286-291.
Hollman, P. C. H. and Arts, I. C. W. (2000). Flavonols, flavones and flavanols - nature, occurrence and dietary burden. Journal of the Science of Food and Agriculture , 80, 1081-1093.
Horn, D. H. S., Middleton, E. J., Thomson, J. A. and Wilkie, J. S. (1974). Metabolism of some possible ecdysone precursors in calliphora stygia . Journal of Insect Physiology , 20, 2433-2445.
Hussain, J., Khan, F. U., Ullah, R., Muhammad, Z., Rehman, N. U., Shinwari, Z. K., Khan, I. U., Zohaib, M., Imad-ud-din and Hussain, S. M. (2011). Nutrient evaluation and elemental analysis of four selected medicinal plants of Khyber Pakhtoon khwa, Pakistan. Pakistan Journal of Botany , 43, 427-434.
Hyodo, R. and Fujimoto, Y. (2000). Biosynthesis of 20-hydroxyecdysone in ajuga hairy roots: the possibility of 7-ene introduction at a late stage. Phytochemistry , 53, 733-737.
Imran, M., Ibrahim, M., Riaz, N. and Malik., A. (2009a). Structure determination of Aervins A-D, new coumaronochromone analogues from Aerva persica ,by 1D and 2D NMR spectroscopy. Magnetic Resonance in Chemistry , 47, 532-536
Page 189
Imran, M., Riaz, N., Ibrahim, M., Ahmad, E., Rasool, M. A., Malik, A. and Moazzam, M. (2009b). Further phytochemical studies on Aerva persica . Journal of Chemical Society of Pakistan , 31, 126-130.
Ishitsuka, M. O., Kusumi, T. and Kakisawa, H. (1988). Antitumor xenicane and norxenicane lactones from the brown alga Dictyota dichotoma . Journal of Organic Chemistry , 53, 5010-5013.
Jain, R., Sharma, A., Gupta, S., Sarethy, I. P. and Gabrani, R. (2011). Solanum nigrum : Current perspectives on therapeutic properties. Alternative Medicine Review , 16, 78-85.
Jamal, A. K., Yaacob, W. A. and Din, L. B. (2008). A Chemical study on Phyllanthus reticulates . Journal of Physical Science , 19, 45-50.
Jaswant, B., Ragunathan, V. and Sulochana, N. (2003). A rare flavonol glycoside from Aerva tomentosa Forsk. as antimicrobial and hepatoprotective agent. Indian Journal of Chemistry. Section B , 42, 956-958.
Jimeno, J., Faircloth, G., Sousa-Faro, Fernández, J. M., Scheuer, P. and Rinehart, K. (2004). New marine derived anticancer therapeutics. A journey from the sea to clinical trials. Marine Drugs , 2, 14-29.
Kamal-Eldin, A., Moazzami, A. and Washi, S. (2011). Sesame seed lignans: potent physiological modulators and possible ingredients in functional foods & nutraceuticals. Recent Patents on Food, Nutrition and Agriculture , 3, 17- 29.
Page 190
Kang, S. S., Kim, J. S., Son, K. H., Kim, H. P. and Chang, H. W. (2001). Cyclooxegenase-2 inhibitory cerebrocides from phytolaccae Radix. Chemical and Pharmaceutical Bulletin , 49, 321-323.
Kappler, C., Hetro, C., Durst, F. and Hoffmans, J. A. (1989). Enzymes involved in ecdysone biosynthesis. In Ecdysone , (ed. I. Koolman), pp. 161-166. Stuttgart: George thieme Verlag.
Kashman, Y., Gustafson, K. R., Fuller, R. W., Cardellin, J. H., McMahon, J. B., Currens, M. J., Buckheist, R. W., Hughes, S. H., Cragg, G. M. and Boyd, M. R. (1992). The calanolides, a novel HIV-inhibitory class of coumarin derivatives from the tropical rainforest tree, Calophyllum lanigerum . Journal of Medicinal Chemistry , 35, 2735-2743.
Kazuhiko, B. (2013). Pharmaceutical properties of abraxane and highlight of its clinical study. Drug Delivery System , 28, 180-188.
Kedei, N., Lundberg, D. J., Toth, A., Welburn, P., Garfield, S. H. and Blumberg, P. M. (2004). Characterization of the interaction of ingenol 3- angelate with protein kinase C. Cancer Research , 64, 3243-3255.
Khan, A. W., Jan, S., Parveen, S., Khan, R. A., Asma Saeed, Tanveer, A. J. and Shad, A. A. (2012). Phytochemical analysis and enzyme inhibition assay of Aerva javanica for ulcer. Chemistry Central Journal , 6, 76.
Kim, M.-J., Kim, B.-G. and Ahn, J.-H. (2013). Biotechnological products and process engineering, Biosynthesis of bioactive O-methylated flavonoids in Escherichia coli . Applied Microbiology and Biotechnology , 97, 7195-7204.
Page 191
Kinzikaeva, G. K. (1968). Aellenia. In P. N. Občinnikov (Hrsg.) Flora Tadžikskoj SSR , vol. 3, pp. 419-425. Nauka: Leningrad.
Kirtikar, K. R. and Basu, B. D. (2001). Indian medicinal plants, vol. 1, pp. 2066- 2077. Dehradun: Oriental Enterprises.
Kleinehollenhorst, G., Behrens, H., Pegels, G., Srunk, N. and Wiermann, R. (1982). Formation of flavonol 3-O-diglycosides and flavonol 3-O-triglycosides by enzyme extracts from anthers of tulipa cv. Apeldoorn. Z Naturforsch , 37C, 587-599.
Koleva, I. I., Beek, T. A. v., Linssen, J. P. H., Groot, A. d. and Evstatieva, L. N. (2002). Screening of plant extracts for antioxidant activity: a comparative study on three testing methods. Phytochemical Analysis , 13 , 8-17.
Korovaev, V. M. (1997). Method for removing calculus from gallbladder, vol. RU 2097054 C1 19971127 (ed. Russ).
Kothe-Heinrich, G. (1991). The genus Halothamnus (Chenopodiaceae) in Southwest Asia. Flora et Vegetatio Mundi , 9, 43-52.
Kothe-Heinrich, G. (1993). Revision der gattung Halothamnus (Chenopodiaceae). Bibliotheca Botanica , 143, 174-176.
Kotinik, Renee, Clardy and Jon. (2008). Codinaeopsin, an antimalarial fungal polyketide. Organic Letters , 10, 4149-4151.
Larin, I. V. (1951). Kormovye rasteniâ senokosov i pastbuŝ SSSR 2. Moskva: Leningrad.
Page 192
Lau, T. (2008). “A Healthy way to live”: The occurance, bioactivity, biosynthesis, and synthesis of kaempferol. Chemistry, 150.
Lee, E. J., Lee, J. Y., Kim, J. S. and Kang, S. S. (2010). Phytochemical studies on Lonicerae Flos (1) - Isolation of iridoid glycosides and other constituents. Natural Product Sciences , 16, 32-38.
Leonard, E., Yan, Y., Lim, K. H. and Koffas, M. A. G. (2005). Investigation of two distinct flavone synthases for plant specific flavone biosynthesis in Saccharomyces cerevisiae . Applied and Environmental Microbiology , 71, 8241- 8248.
Lesley, J. S. (2012). Micafungin: A review of its use in the prophylaxis and treatment of invasive candida infections. Drugs , 72, 2141-2165.
Liocharova, N. A., Hatano, K., Shaskov, A. S., Knirel, Y. A., Kochetkov, N. K. and Pier, G. B. (1989). The structulre and serologic distribution of an extracellular neutral polysaccharide from pseudomonas aeruginosa immunotype. The Journal of Biological Chemistry , 264, 15569-15573.
Liscum, L. (2002). Cholesterol biosynthesis. In Biochemistry of lipids, proteins and membranes eds. D. E. Vance and J. E. Vance, pp. 409-430.
Mabry, T. J., Markham, K. R. and Thomas, M. B. (1970). The systematic identification of flavonoids, pp. 354. NewYork: Springer-Verlag.
Mahmood, U., Kaula, V. K., Acharya, R. and L.Jirovetz. (2003). p-Coumaric acid esters from Tanacetum longifolium . Phytochemistry , 64, 851-853.
Page 193
Majmudar, F. I., Shah, M. B., Patel, K. N. and Shah, B. K. (1999). Aerva lanata - Its diuretic and hepatoprotective activity. Indian Journal of Natural Product , 15, 9-12.
Manoharan, S., jaswanth, A., Sangottuvelu, S., nandhakumar, J., Duraisamy, R., karthikeyan, D. and Mallegaswari, R. (2008). Hepatoprotective activity of Aerva lanata Linn. against paracetamol induced hepatotoxicity in rats. Research Journal of Pharmacy and Technology , 1, 398- 400.
Mein, H. F. G. (1831). Ueber die darstellung des atropins in weissen kristallen" On the preparation of atropine as white crystals. Annalen der Pharmacie , 6, 67-72.
Milner, N. P., Nali, M., Gibson, J. M. and Rees, H. H. (1986). Early stages of ecdysone biosynthesis: the role of 7-dehydrocholesterol. Insect Biochemistry , 16, 17-23.
Miski, M., Ulubelen, A. and Mabry, T. J. (1983). 6-Hydroxyflavones from Thymbra spicata . Phytochemistry , 22, 2093-2094.
Miyahisa, I., Funa, N., Ohnishi, Y., Martens, S., Moriguchi, T. and Horinouchi, S. (2006). Combinatorial biosynthesis of flavones and flavonols in Escherichia coli . Applied Microbiology and Biotechnology , 71, 53-58.
Monitto, P. (1965). Biosynthesis of natural products, pp. 279. NewYork: Ellis Harwood Limited, John Willey and Sons.
Monitto, P. (1981). Biosynthesis of natural products, pp. 45. NewYork: Ellis Harwood Limited, John Willey and Sons.
Page 194
Moore, K. S., Wehrli, S., Roder, H., Rogers, M., Forrest, J. N., McCrimmon, D. and Zasloff, M. (1993). Squalamine: an aminosterol antibiotic from the shark. Proceedings of the National Academy of Sciences of the United States of America, 90, 1354-1358.
Mukerjee, T., Bhalla, N., Sing, A. G. and Jain, H. C. (1984). Herbal drugs for urinary stones. Indian drugs , 21, 224-228.
Mukhtar, N., Malik, A., Riaz, N., Iqbal, K., Tareen, R. B., Khan, S. N., Nawaz, S. A., Siddiqui, J. and Choudhary, M. I. (2004). Pakistolides A and B, novel enzyme inhibitory and antioxidant dimeric 4-(glucosyloxy) benzoates from Berchemia pakistanica . Helvetica Chimica Acta , 87, 416-424.
Müller, K. and Borsch, T. (2005). Phylogenetics of amaranthaceae using matK/trnK sequence data evidence from parsimony, likelihood and bayesian approaches. Annals of the Missouri Botanical Garden , 92, 66-102.
Murtaza, S., Ghous, T., Ahmed, S., Ullah, R. S. and Abbas, A. (2013). On- line antiacetylcholine esterase activity of extracts of Oxystelma esculentum , Aerva javanica and Zanthoxylum armatum . Journal of Chemical Society of Pakistan , 35, 801-804.
Mussadiq, S., Naheed, R., Saleem, M., Ashraf, M., Ismail, T. and Jabbar, A. (2013). New acylated flavonoid glycosides from flowers Of Aerva Javanica . Journal of Asian Natural Product Research, 708-716.
Nadkarni, K. M. (1979). In Indian Materia Medica , vol. I, pp. 21-22. Bombay: Popular Prakashan Pvt. Ltd.
Page 195
Nagao, T., Abe, F., Kinjo, J. and Okabe, H. (2002). Antiproliferative constituents in plants 10. Flavones from the leaves of Lantana montevidensis Briq. and consideration of structure-activity relationship. Biological and Pharmaceutical Bulletin , 25, 875-879.
Nakanishi, K. (1999). A historical perspective of natural products chemistry. In Comprehensive Natural Products Chemistry , vol. 1 (ed. S. Ushio), pp. 23-40. Amsterdam: Elsevier Science B.V.
Nehlig, A., Daval, J. L. and Debry, G. (1992). Caffeine and the central nervous system: mechanisms of action, biochemical, metabolic and psychostimulant effects. Brain Research Reviews , 17, 139-170.
Nettelblad, H., Vahlqvist, C., Krysander, L. and Sjöberg, F. (1996). Psoralens used for cosmetic sun tanning: an unusual cause of extensive burn injury. Burns , 22, 633-635.
Nevin, K. G. and Vijayammal, P. L. (2003). Effect of Aerva lanata on solid tumor induced by DLA cells in mice. Fitoterapia , 74, 578-582.
Nevin, K. G. and Vijayammal, P. L. (2005). Effects of Aerva lanata against hepatoxicity of carbontetrachloride in rats. Environmental Toxicology and Pharmacology , 20, 471-477.
Nogueira, M. A., oliveira, J. S. D. and Ferraz, S. (1996). Nematicidal hydrocarbons from Mucuna aterrima . Phytochemistry , 42, 997-998.
Ohyama, K., Kushiro, T., Nakamura, K. and Fujimoto, Y. (1999). Biosynthesis of 20-hydroxyecdysone in ajuga hairy roots: fate of 6 alpha- and
Page 196
6 beta-hydrogens of lathosterol. Bioorganic and Medicinal Chemistry , 7, 2925-2930.
Parekh, B. B., Vasant, S. R., Tank, K. P., Raut, A. and Vaidya, A. D. B. (2009). In vitro growth and inhibition studies of monosodium urate monohydrate crystals by different herbal extracts. Journal of Infectious Disease , 5, 225-230.
Pateh, U. U., Haruna, A. K., Garba, M., Iliya, I., Sule, I. M., Abubakar, M. S. and Ambi, A. A. (2009). Isolation of stigmasterol, β-sitosterol and 2- hydroxyhexadecanoic acid methyl ester from the rhizomes of stylochiton lancifolius pyer and kotchy (araceae). Nigerian Journal of Pharmaceutical Sciences, 8, 19 - 25.
Patterson, G. W., Sihua, X. and Salt, T. A. (1991). Sterols of caryophyllales with emphasis on amaranthaceae. Phytochemistry , 30, 523-526.
Pavan, B. (1953). The toxic principle isolated from blister beetles, Paederus fuscipes Curt. Physiologia Comparata Et Oecologia , 3, 307.
Pavanasasivam, G. and Sultanbawa, M. U. S. (1975). Chemical investigation of ceylonese plants. Part XII. (+)-3,4′,5,7-Tetrahydroxy-3′-methoxyflavanone [(+)-dihydroisorhamnetin and 3,5,7-trihydroxy-3′,4′-dimethoxyflavone (dillenetin): two new natural products from Dillenia indica L. Journal of Chemical Society, Perkin Trans , 1, 612-613.
Peng, K., Kong, Y. and Zhai, L. (2010). Two novel antimicrobial peptides from centipede venoms. Toxicon , 55, 274-279.
Page 197
Perry, L. M. and Metzger, J. (1980). Medicinal plants of East and Southeast Asia, pp. 9. London: MIT Press, Cambridge.
Pettit, G. R., Cherry, H. L., Doubek, D. L., Herald, D. L., Arnold, E. and Clardy, J. (1982). Isolation and structure of bryostatin 1. Journal of American Chemical Society , 104, 6846-6848.
Polsky, B., Segal, S., Baron, P., Gold, J., Ueno, H. and Armstrong, D. (1989). Inactivation of human immunodeficiency virus in vitro by gossypol. Contraception 39, 579-587.
Pradhan, P., Joseph, L., Gupta, V., Chulet, R., Arya, H., Verma, R. and Bajpai, A. (2009). Saraca asoca (Ashoka): A Review. Journal of Chemical and Pharmaceutical Research , 1, 62-71.
Qureshi, N. and Porter, J. W. (1981). Biosynthesis of isoprenoid compounds, vol. 1 eds. J. W. Porter and S. L. Spurgeon, pp. 47-94. New York: Wiley.
Qureshi, R. and Bhatti, G. R. (2009). Folklore uses of amaranthaceae family from Nara Desert, Pakistan. Pakistan Journal of Botany , 41, 1565-1572.
Radwan, H. M., Nazif, N. M. and Hamdy, A. A. (1999). The lipid and flovonoidal constituents of Aerva javonica var. bovi webb in hook. F. and their antimicrobial activity. Egyptian Journal of Pharmaceutical Sciences , 40, 167- 178.
Rajendra, W., Armugam, A. and K.Jeyaseelan. (2004). Toxins in anti- nociception and anti-inflammation. Toxicon , 44, 1-17.
Rajesh, R., Chitra, K. and Paarakh, P. M. (2012). Anti hyperglycemic and anti hyperlipidemic activity of aerial parts of Aerva lanata Linn. Juss. in
Page 198
streptozotocin induced diabetic rats. Asian Pacific Journal of Tropical Biomedicine, S924-S929.
Rajesh, R., Chitra, K. and Padmaa, M. P. (2011). Aerva lanata (Linn.) Juss ex Shult- An overview. Indian Journal of Natural Product Resources , 2, 5-9.
Rees, H. H. (1989). Pathways of biosynthesis of ecdysone. In Ecdysone , (ed. J. Koolman), pp. 152-160. Stuttgart: George thieme Verlag.
Rees, H. H. and Isaac, R. E. (1985). Biosynthesis and metabolism of ecdysteroids and methods of isolation and identification of the free and conjugated compounds. Methods in Enzymology , 111, 377-410.
Ren, X., Shen, L.-l., Muraoka, O. and Cheng, M. (2011). Synthesis of quercetin 3-O-[6″- O-(trans-p-Coumaroyl)]-β-D-glucopyranoside. Journal of Carbohydrate Chemistry , 30, 119-131.
Riaz, N., Parveen, S., Saleem, M., Ali, M. S., Malik, A., Ashraf, M., Afzal, I. and Jabbar, A. (2012). Lipoxygenase inhibitory sphingolipids from Launaea nudicaulis . Journal of Asian Natural Products Research , 14, 545-554.
Rogers, D. F. (1996). Scorpion venoms: taking the sting out of lung disease. Thorax , 51, 546-548.
Rosin, A. (1991). Pilocarpine. A miotic of choice in the treatment of glaucoma has passed 110 years of use. Oftalmologia (Romania) , 35, 53-55.
Rudolph, P. H. and Spaziani, E. (1992). Formation of ecdysteroids by Y organs of the crab Menippi merceneria. II incorporation of cholesterol into 7 dehydro cholesterol and secretion products in vitro. General and Comparative Endocrinology , 88, 235-242.
Page 199
Sachobiddinov, S. S. ( 1948). Dikorastuŝie lekarstvennye rasteniâ Srednej Azii. In Gosizdat Uz.SSR . Taškent.
Saeed, S. and Tariq, P. (2007). Antibacterial activities of Emblica officinalis and Coriandrum sativum against Gram negative urinary pathogens. Pakistan Journal of Pharmaceutical Sciences , 20, 32-35.
Saleem, M., Mussadiq, S., Riaz, N., Zubair, M., Ashraf, M., Nasar, R. and Jabbar, A. (2013). Ecdysteroids from the flowers of Aerva javanica . Steroids , 78, 1098-1102.
Saleh, N. A. M., Mansjur, R. M. A. and Markham, K. R. (1990 ). An acylated isorhamnetin glycoside from Aerva javanica . Phytochemistry , 29, 1344- 1345.
Samejo, M. Q., Memon, S., Bhanger, M. I. and Khan, K. M. (2012). Chemical compositions of the essential oil of Aerva javanica leaves and stems. Pakistan Journal of Analytical and Environmental Chemistry , 13, 48-52.
Selvam, R., Kalaiselvi, P., Govindaraj, A., Murugan, V. B. and Kumar, A. S. S. (2001). Effect of Aerva lanata leaf extract and vediuppo chunnam on the urinary risk factors of calcium oxalate urolithiasis during experimental hyperoxaluria. Pharmacological Research , 43, 89-93.
Sethi, A. and Sharma, R. A. (2011). Antioxidant activity with total phenolic constituents from Aerva tomentosa forsk. International Journal of Pharma and Bio Sciences , 2, 596-603.
Shah, S. R. U., Qasim, M., Khan, I. A. and Shah, S. A. U. (2006). Study of medicinal plants among weeds of wheat and maize in Peshawar region. Pakistan Journal of Weed Sciences , 12, 191-197.
Page 200
Sharif, A., Ahmed, E., Malik, A., Hassan, M.-U.-., Munawar, M. A., Farrukh, A., Nagra, S. A., Anwar, J., Ashraf, M. and Mahmood, Z. (2011). Antimicrobial constituents from Aerva javanica . Journal of the Chemical Society of Pakistan , 33, 439-442.
Shengeliya, A. O. and Mardaleishvili, V. A. (2000). A medicated liquor for treating cardiovascular system diseases and blood diseases, vol. WO 2000050052 A1 20000831 (ed. P. I. Appl).
Shirwaikar, A., Issac, D. and Malini, S. (2004). Effect of Aerva lanata on cisplatin and gentamicin models of acute renal failure. Journal of Ethnopharmacology , 90, 81-86.
Siddiqui, S. and Siddiqui, R. H. (1931). Chemical examination of the root of Rauwolfia Serpentina Benth. Journal of Indian Chemical Society , 8, 667-680.
Simonsen, H. T., Nordskjold, J. B., Smitt, U. W., Nyman, U., Palpu, P., Joshi, P. and Varughese, G. (2001). In vitro screening of Indian medicinal plants for antiplasmodial activity. Journal of Ethnopharmacology , 74, 195- 204.
Siveen, K. S. and Kuttan, G. (2011). Immunomodulatory and anti tumour activity of Aerva lanata ethanolic extract, Immunopharmacology and Immunotoxicology , 33, 423-432.
Soundararajan, P., Mahesh, R., Ramesh, T. and Begum, V. H. (2007). Hypolipidimic activity of Aerva lanata on ethylene glycol induced calcium oxalate urolithiasis in rats. Pharmacology Online , 1, 557-563.
Page 201
Soundararajan, P., Mahesh, R., Ramesh, T. and Beguum, V. H. (2006). Effect of Aerva lanata calcium oxalate urolithiases in rats. Indian Journal of Experimental Biology , 44, 981-986.
Srinivas, K. R. and Reddy, V. M. (2009). Antihyperglycaemic activity of ethanol extract of Aerva javanica leaves in alloxan- induced diabetic mice. Journal of Pharmacy Research , 2, 1259-1261.
Srinivas, P. and Reddy, S. R. (2012). Screening for antibacterial principle and activity of Aerva javanica (Burm .f) Juss. ex Schult. Asian Pacific Journal of Tropical Biomedicine, S838-S845.
Sripathi, S. K., Gopal, P. and Lalitha, P. ( 2011). Allantoin from the leaves of Pisonia grandis . International Journal of Pharmacy and Life Sciences, 2, 815- 817.
Srivastava, R. C. (1989). Drug plant resources of central India, pp. 21. New Delhi: Today and Tomorrow’s Printers & Publishers.
Stotz, G. and Forkmann, G. (1981). Conversion of flavanone to flavone, dihydroflavonol and flavonol with an enzyme system from cell cultures of Parsley. Z Natureforcsh , 36 C, 737-741.
Sugimoto, N., Tada, A., Suematsu, T., Arifuku, K., Saito, T., Ihara, T., Yoshida, Y., Kubota, R., Tahara, M., Shimizu, K. et al. (2010). Absolute quantification of carminic acid in cochineal extract by quantitative NMR. Shokuhin Eiseigaku Zasshi , 51, 19-27.
Sun, C., Wang, H.-K., Kashiwada, Y., Shen, J.-K., Cosentino, L. M., Chen, C.-H., Yang, L.-M. and Lee, K.-H. (1998). Anti-AIDS Agents. Synthesis and
Page 202
structure activity relationships of betulin derivatives as anti-HIV agents. Journal of Medicinal Chemistry , 41, 4648-4657.
Taha, S. A. (1992). Studies on the mode of action of ambrein as a new antinociceptive compound. Japanese Journal of Pharmacology , 60, 67-71.
Tappel, A. L. (1953). The mechanism of the oxidation of unsaturated fatty acids catalyzed by hematin compounds. Archieves of Biochemistry and Biophysics , 44, 378-395.
Tiwari, V., Kuhad, A. and Chopra, K. (2011). Emblica officinalis corrects functional, biochemical and molecular deficits in experimental diabetic neuropathy by targeting the oxido-nitrosative Stress mediated inflammatory cascade. Phytotherapy Research , 25, 1527-1536.
Toledo, R. C. and Jared, C. (1995). Cutaneous granular glands and amphibian venoms. Comparative Biochemistry and Physiology , 111, 1-29.
Tsakadze, M. D., Shota, A. S., Richsivoi, Z. and Abdulatip, A. (2005). Alkaloid and phenolic compounds of Galanthus caucasicus , Magnolia obovata , Cocculus laurifolius , and Veratrum lobelianum grown in Georgia. Molecular Diversity , 9, 41-44.
Vaishampayan, U., Glode, M., Du, W., Kraft, A., Hudes, G., Wright, J. and Hussain, M. (2000). Phase II Study of dolastatin-10 in patients with hormone-refractory metastatic prostate adenocarcinoma. Clinical Cancer Research , 6, 4205-4208.
Page 203
Vasudeva, N., Sethi, P., Sharma, S. K., Kumar, S. and Sharma, S. (2012). Antiulcer potential of the ethanolic extract of A. persica Merrill root in rats. Journal of Acupuncture and Meridian Studies , 5, 80-86.
Vetrichelvan, T. and Jegadeesan, M. (2002). Antidiabetic activity of alcoholic extract of Aerva lanata (L.) juss. ex Schults in rats. Journal of Ethnopharmacology , 80, 103-107.
Vetrichelvan, T., Jegadeesan, M., Palaniappan, M. S., Murali, N. P. and Sasikumar, K. (2000). Diuretic and anti-inflammatory activities of Aerva lanata in rats. Indian Journal of Pharmaceutical Sciences , 62, 300-302.
Wagh, V. D., Patil, P. N., Surana, S. J. and Wagh, K. V. (2012). Forskolin: Upcoming antiglaucoma molecule. Journal of Postgraduate Medicine , 58, 199-202.
Wandji, J., Nkengfack, E. A. and Omum, Z. T. (1990). A New prenylated isoflavone and long chain esters from two erythrina Species. Journal of Natural products , 53, 1425-1429.
Warren, J. T., Sakurai, S., Rountree, D. B., Gilbirt, L. I., Lee, S.-S. and Nakanishi, K. (1988 ). Regulation of the ecdysteroid titer of Manduca sexta : Reappraisal of the role of the prothoracic gland. Proceedings of the National Academy of Sciences, USA , 85, 958-962.
Weisshaar, B. and Jenkins, G. I. (1998). Phenylpropanoid biosynthesis and its regulation. Current Opinion in Plant Biology , 1, 251-257.
Page 204
Wen, P., Han, H., Wang, R., Wang, N. and Yao, X. (2007). C-glycosylfavones and aromatic glycosides from Campylotropis hirtella . Asian Journal of Traditional Medicines , 2, 149-153.
Whitaker, I. S., Izadi, D. and Oliver, D. W. (2004). Hirudo medicinalis and the plastic surgeon. British Journal of Plastic Surgery , 57, 348-353.
White, N. J. (1997). Assessment of the pharmacodynamic properties of antimalarial drugs in vivo. Antimicrobial Agents and Chemotherapy , 41, 1413-1422.
Wu, P., Xie, H., Tao, W., Miao, S. and Wei, X. (2010). Phytoecdysteroids from the rhizomes of Brainea insignis . Phytochemistry , 71, 975-981.
Yabuta, T. (1924). The constitution of kojic acid, a gamma-pyrone derivative formed by Aspergillus oryzae from carbohydrates. Journal of the Chemical Society , 125, 575-587.
Yang, G., Janicsak, M.-H., Mathe, G., Carabot-Cuervo, I. and Alfredo. (1998). Betaine distribution in the Amaranthaceae Blunden. Biochemical Systematics and Ecology , 27, 87-92.
Yang, N.-Y., Liu, L., Tao, W.-W., Duan, J.-A., Liu, X.-H. and Huang, S.-P. (2011). Antithrombotic lipids from Semen persicae . Natural Product Research , 25, 1650-1656.
Page 205
Yao, G. and Chang, L. C. (2007). Novel sulfated sesterterpene alkaloids from the marine sponge Fasciospongia sp. Organic Letters , 9, 3037-3040 .
Yao, G., Kondratyuk, T. P., Tan, G. T., Pezzuto, J. M. and Chang, L. C. (2009). Bioactive sulfated sesterterpene alkaloids and sesterterpene sulfates from the marine sponge Fasciospongia sp. Journal of Natural Products , 72, 319-323 .
Zadorozhnii, A. M., Zapesochnaya, G. G., Pervykh, L. N., Shchavlinskii, A. N., Kovtun, L. S. and Svanidze, N. V. (1986). Investigation of the herb Aerva lanata . I-O-acylglycosides of flavonoids. Khimiko-Farmatsevticheskii Zhurnal, 20, 855 -858.
Zapesochnaya, G. G., Pervykh, L. N. and Kurkin, V. A. (1991). A study of the herb Aerva lanata . III. Alkaloids. Chemistry of Natural compounds, 27, 336-340.
Zaveri, M., Movaliya, V. and Setty, M. (2012). Pharmacogostical studies on aerial parts of Aerva Javanica . Advance Research in Pharmaceuticals and Biologicals , 2, 250-253.
Zhou, R., Li, B.-G. and Zhang, G.-L. (2005). Chemical study on Cyathula officinalis Kuan. Journal of Asian Natural Products Research , 7, 245-252.
Zhu, N., Kikuzaki, H., Vastano, B. C., Nakatani, N., Karwe, M. V., Rosen, R. T. and Ho, C. T. (2001). Ecdysteroids of quinoa seeds (Chenopodium quinoa Willd.). Journal of Agriculture and Food Chemistry , 49, 2576-2578.
Page 206