Isolation, Structural Determination and Biological Studies on Some Relatives of the Genus Launaea and Carissa from Pakistan

Isolation, Structural Determination and Biological Studies on some Relatives of the Genus Launaea and Carissa from Pakistan

A Dissertation Submitted for

THE PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF DEGREE OF DOCTOR OF PHILOSOPHY IN CHEMISTRY

Shehla Parveen (M.SC., M.PHIL.) Department of Chemistry The Islamia University of Bahawalpur 63100-Bahawalpur, Pakistan September 2012

Contents

Acknowledgements i Summary iv 1 Human Health and the Role of Natural Products 1 1.1 Human Health and Role of Natural Products 2 1.2 FDA Approved Natural Products since 1998-2010 4 1.3 Natural Products under Clinical Trials 7 1.4 Emergence of the Natural Product Research 8 1.5 Decline of Natural Product Research in the Pharmaceutical Industry 9 1.6 Revitalization in Natural Product Research 10 1.7 Hypothesis 12 1.8 Aims and Objectives of the Study 13 2 The Genus Launaea and its Importance 15 2.1 Introduction of the Genus Launaea 16 2.2 Pharmacological Importance of the species of Genus Launaea 16 2.3 Previous Phytochemical Investigation of the species of the Genus Launaea 18 3 Phytochemical Investigations on Launaea nudicaulis 28 3.1 Introduction of Launaea nudicaulis 29 3.2 Botanical Description of Launaea nudicaulis 29 3.3 Scientific Classification of Launaea nudicaulis 30 3.4 Pharmacological Importance of Launaea nudicaulis 30 3.5 Results and Discussion 31 3.5.1 Characterization of nudicholoid ( 116 ) 32 3.5.2 Characterization of nudicualin A ( 112 ) 37 3.5.3 Characterization of nudicualin B ( 113 ) 39 3.5.4 Characterization of nudicualin D (114 ) 42 3.5.5 Characterization of nudicualin C ( 115 ) 44 3.5.6 Characterization of trideca-12-en-4,6-diyne-2,8,9,10,11- pentaol (119 ) 46 3.5.7 Characterization of cholistaquinate ( 121 ) 51 3.5.8 Characterization of cholistaflaside ( 123 ) 55 3.5.9 Characterization of 1-hexatriacontanol ( 109 ) 58 3.5.10 Characterization of elaidic acid ( 110 ) 59 3.5.11 Characterization of oleanolic acid ( 111 ) 60 3.5.12 Characterization of ergosta-7,22-diene-3,5,6-triol ( 117 ) 61 3.5.13 Characterization of benzyl glucopyranoside (118 ) 62 3.5.14 Characterization of 3,7,12-trihydroxycholan-24-oic acid ( 120 ) 63 3.5.15 Characterization of β-sitosterol 3-O-β-D-glucopyranoside ( 67 ) 64 3.5.16 Characterization of 20-hydroxyecdesone (122 ) 65 3.6 Biological studies 67 3.6.1 Antioxidant Assay (DPPH Radical Scavenging Method) 67 3.6.2 Enzyme Inhibition Activities 68 3.6.2.1 Acetylcholinesterase Enzyme Inhibition Activity 68 3.6.2.2 Butyrylcholinesterase Enzyme Inhibition Activity 69

3.6.2.3 Lipoxygenase Enzyme Inhibition Activity 70 3.7 Experimental 72 3.7.1 General Experimental Notes 72 3.7.2 Plant material 73 3.7.3 Extraction and Isolation 74 3.7.4 Experimental data of Isolated Compounds 78 3.7.4.1 Nudicholoid ( 116 ) 78 3.7.4.2 Nudicaulin A ( 112 ) 79 3.7.4.3 Nudicualin B ( 113 ) 80 3.7.4.4 Nudicualin D ( 114 ) 82 3.7.4.5 Nudicaulin C ( 115 ) 83 3.7.4.6 Trideca-12-en-4,6-diyne-2,8,9,10,11-pentanol ( 119 ) 85 3.7.4.7 Cholistaquinate (121 ) 86 3.7.4.8 Cholistaflaside ( 123 ) 88 3.7.4.9 1-Hexatriacontanol ( 109 ) 87 3.7.4.10 Elaidic acid: ( 110 ) 88 3.7.4.11 Oleanolic acid ( 111 ) 88 3.7.4.12 Ergosta-7,22-diene-3,5,6-triol ( 117 ) 89 3.7.4.13 Benzyl glucopyranoside: ( 118 ) 90 3.7.4.14 3,7,12-Trihydroxycholan-24-oic acid ( 120 ) 90 3.7.4.15 β-Sitosterol 3-O-β-D-glucopyranoside ( 67 ) 91 3.7.4.16 20-Hydroxyecdesone ( 122 ) 92 3.7.5 Methanolysis 93 3.7.6 Oxidative Cleavage of the Double bond 93 3.8 Bioassays 94 3.8.1 DPPH Radical Scavenging Activity 94 3.8.2 Acetylcholinesterase Assay 95 3.8.3 Butyrylcholinesterase Assay 95 3.8.4 Lipoxygenase Assay 96 4 Phytochemical investigations on Launaea intybacea 98 4.1 Introduction of Launaea intybacea 99 4.2 Scientific Classification of Launaea intybacea 99 4.3 Pharmacological Importance of Launaea intybacea 99 4.4 Results and discussion 100 4.4.1 Characterization of 6,6'-oxybis(4-allyl-2-methoxyphenol)(126 ) 100 4.4.2 Characterization of lupeol (53 ) 102 4.4.3 Characterization of β-sitosterol (30 ) 103 4.4.4 Characterization of octadecyl ( E)-p-coumarate ( 124 ) 105 4.4.5 Characterization of 3-methoxy-4-hydroxy benzaldehyde ( 125 ) 106 4.4.6 Characterization of 4-hydroxybenzoic acid (127 ) 106 4.4.7 Characterization of 4-hydroxy-3-methoxybenzoic acid (128 ) 107 4.4.8 Characterization of 4-hydroxy-trans-cinnamic acid ( 129 ) 108 4.4.9 Characterization of methyl gallate ( 130 ) 109 4.4.10 Characterization of 3,4-dihydroxybenzoic acid (131 ) 109 4.4.11 Characterization of 4',5,7-trihydroxyflavone (132 ) 110 4.4.12 Characterization of 3',4',5,7-tetrahydroxyflavone (133 ) 111 4.4.13 Characterization of 3,3',5,7-tetrahydroxy-4'-methoxyflavone (134 ) 112 4.4.14 Characterization of apigenin 7 -O-(4'' -O-p-E-coumaroyl -β-D-glucopyranoside ( 135 ) 113

4.5 Biological Studies 114 4.5.1 Antioxidant Assay (DPPH Radical Scavenging Method) 114 4.5.2 Enzyme Inhibition Activities 115 4.5.2.1 Acetylcholinesterase Enzyme Inhibition Activity 115 4.5.2.2 Butyrylcholinesterase Enzyme Inhibition Activity 116 4.5.2.3 Lipoxygenase Enzyme Inhibition Activity 117 4.6 Experimental 119 4.6.1 Plant Material 119 4.6.2 Extraction and Isolation 119 4.6.3 Experimental Data 122 4.6.3.1 6,6'-Oxybis(4-allyl-2-methoxyphenol) (126 ) 122 4.6.3.2 Lupeol ( 53 ) 122 4.7.3 β-Sitosterol ( 30 ) 123 4.6.3.4 Octadecyl ( E)-p-coumarate ( 124 ) 124 4.6.3.5 4-Hydroxy-3-methoxybenzaldehyde ( 125 ) 124 4.6.3.6 4-Hydroxybenzoic acid ( 127 ) 125 4.6.3.7 4-hydroxy-3-methoxybenzoic acid ( 128 ) 125 4.6.3.8 4-Hydroxy-trans-cinnamic acid (129 ) 126 4.6.3.9 Methyl gallate ( 130 ) 126 4.6.3.10 3,4-Dihydroxybenzoic acid ( 131 ) 127 4.6.3.11 4',5,7-Trihydroxyflavone ( 132 ) 127 4.6.3.12 3',4',5,7-Tetrahydroxyflavone ( 133 ) 128 4.6.3.13 3,3',5,7-Tetrahydroxy-4'-methoxyflavone (134 ) 128 4.6.3.14 Apigenin 7-O-(4''-O-p-E-coumaroyl-β-D-glucopyranoside ( 135 ) 129 5 The Genus Carissa and Its Importance 131 5.1 The Introduction of the Genus Carissa 132 5.2 Botanical Description 133 5.3 Pharmacological Importance of Some Species of Genus Carissa 133 5.4 Previous Phytochemical Investigations on Genus Carissa 137 6 Phytochemical Investigations on Carissa opaca 146 6.1 Introduction of Carissa opaca 147 6.2 Botanical Description of Carissa opaca 148 6.3 Scientific Classification of Carissa opaca 148 6.4 Pharmacological Importance of Carissa opaca 149 6.5 Results and Discussion 151 6.5.1 Characterization of cyclic trimer of ethylene terephthalate ( 191 ) 151 6.5.2 Characterization of 2,3-dihydoxy 30-nor-2α,3 β–dihydroxy-urs -12-ene ( 195 ) 153 6.5.3 Characterization of 30-nor -2α,3 α,23-trihydroxyurs-12-ene (196 ) 156 6.5.4 Characterization of (2 S,3 S,4 R,15 E)-2-{[(2 R)-2-hydroxydocosanoyl]amino}eicos-15-ene-1,3,4-triol ( 198 ) 158 6.5.5 Characterization of 3 β,27-dihydroxylup-12-ene ( 189 ) 161 6.5.6 Characterization of lupeol-β-hydroxyoctadecanoate ( 190 ) 162 6.5.7 Characterization of pinoresinol ( 192 ) 163 6.5.8 Characterization of (-) carinol ( 193 ) 164 6.5.9 Characterization of (-) carissanol ( 194 ) 166 6.5.10 Characterization of arjunolic acid (197 ) 167

6.6 Biological Studies 168 6.6.1 Antioxidant Assay (DPPH Radical Scavenging Method) 168 6.6.2 Enzyme Inhibition Activities 169 6.6.2.1 Acetylcholinesterase Enzyme Inhibition Activity 169 6.6.2.2 Butyrylcholinesterase Enzyme Inhibition Activity 170 6.6.2.3 Lipoxygenase Enzyme Inhibition Activity 171 6.7 Experimental 173 6.7.1 General Experimental Procedures 173 6.7.2 Plant Material 173 6.7.3 Extraction and Isolation 174 6.7.4 Experimental Data 177 6.7.4 .1 Cyclic trimer of ethylene terephthalate ( 191 ) 177 6.7.4.2 2,3-Dihydoxy-30-nor-2α,3β–dihydroxy-urs-12-ene ( 195 ) 177 6.7.4.3 30-Nor-2α,3 α,23-trihydroxyurs-12-ene (196 ) 178 6.7.4.4 (2S,3S,4R,15E)-2-{[(2R)-2-Hydroxydocosanoyl]amino} eicos-15-ene-1,3,4-triol (198 ) 179 6.7.4.5 3β, 27-Dihydroxylup-12-ene ( 189 ) 181 6.7.4.6 Lupeol β-hydroxy octadecanoate ( 190 ) 182 6.7.4.7 Pinoresinol ( 192 ) 183 6.7.4.8 (-) Carinol ( 193 ) 183 6.7.4.9 (-) Carissanol ( 194 ) 184 6.7.4.10Arjunolic acid ( 197 ) 185 6.7.5 Methanolysis 186 6.7.6 Oxidative Cleavage of the Double bond 186 Bibliography 187 Annexure 1 206 Annexure 2 216 Annexure 3 223 Annexure 4 228 Annexure 5 233

Summary

The work embodied in this dissertation is mainly concerned with the isolation and characterization of natural chemical constituents having some biological importance. The isolated compounds were either new or previously been reported in the literature and they were characterized by various sophisticated spectroscopic techniques.

The dissertation deals with purification and biological screening of secondary metabolites isolated from three indigenous medicinal plants of

Pakistan namely Launaea nudicaulis , Launaea intybacea and Carissa opaca . The whole dissertation is divided into six chapters. The chapter 1 entitled as “ Human Health and Role of Natural products ” describes significance of terrestrial plants in Human life for the cure of various diseases, some FDA approved natural product drugs, natural products under clinical trials for FDA approval and new challenges in the field of natural products research.

The second chapter entitled as “ The Genus Launaea and its

Importance ” describes the genus Launaea, its economical, pharmacological and phytochemical properties. In this chapter the pharmacology of various isolates from the genus Launaea and biological activities of various species

iv or their extract has been discussed. Almost all secondary metabolites isolated up to date from the different species of Launaea have been described year wise.

The third chapter entitled as “Phytochemical Investigations on

Launaea nudicaulis ” deals with the phytochemical studies on Launaea nudicaulis collected from Cholistan desert covers the isolation, structure elucidation and experimental detail of eight new and same number of known secondary metabolites. The methanolic extract of this plant was divided into n-hexane and ethyl acetate fractions . The n-hexane soluble part yielded four new sphingolipids: nudicualin A (112 ), nudicualin B

(113 ), nudicualin D (114 ), nudicualin C (115 ) together with known compounds, 1-hexatriacontanol (109 ), elaidic acid (110), oleanolic acid

(111 ) and β-sitosterol glucoside (67). The column chromatography of ethyl acetate part yielded four new compounds: a sesquiterpene lactone, nudicholoid ( 116 ), a diacetylene derivative, trideca-12-ene-4,6-diyne-

2,8,9,10,11-pentaol (119 ), quinic acid derivative, cholistaquinate (121 ) and a flavonoid di-C-glycoside, cholistaflaside ( 123 ) along with four known compounds, ergosta-7,22-diene-3,5,6-triol ( 117 ), benzyl glucopyranoside

(118 ), 3,7,12-trihydroxycholan-24-oic acid ( 120 ) and 20-hydroxyecdysone

(122 ). Their structures were assigned by ID and 2D-NMR in combination with EIMS, HR-MS and FAB-MS techniques. Structures of the known compounds were determined by spectroscopic analyses and in comparison with the literature.

The structure elucidation of the compounds isolated from hexane part is published in Journal of Asian Natural Products [1], which is a peer- reviewed journal. The publication has been attached in the end as

Annexure-1.

[1] Naheed Riaz, Shehla Parveen , Muhammad Saleem, Muhammad Shaiq Ali, Abdul Malik, Muhammad Ashraf, Iftikhar Afzal, Abdul Jabbar; Lipoxygenase inhibitory sphingolipids from Launaea nudicaulis, Journal of Asian Natural Products Research, 14 (6), 545–554, 2012.

The manuscript on structure elucidation of all compounds isolated from ethyl acetate part has been published in Phytochemistry Letters which is peer reviewed journal [2] . The publication has been attached in the end as

Annexure-2.

[2]Muhammad Saleem, Shehla Parveen , Naheed Riaz, Muhammad Nawaz Tahir, Muhammad Ashraf, Iftikhar Afzal, Muhammad Shaiq Ali, Abdul Malik and Abdul Jabbar; New Bioactive natural products from Launaea nudicaulis; Phytochemistry Letters, 5, 793-799, 2012.

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Among the compounds isolated from L. nudicaulis, 109 -110, 112 -

116 , 117 , 119-121 , and 123 were subjected to various biological activities.

In antioxidant essay, only the new compound 121 was active with IC 50 value of 60.3. In acetylcholinesterase assay, the two new compounds 121 and 123 exhibited minor activity while others were inactive against this enzyme. In butyrylcholinesterase enzyme inhibition essay the new compound nudicholoid 116 showed significant activity with an IC 50 value of

88.34 and rest of the compounds showed minor activities. The compounds

112 , 113 , 114 and 115 were active against lipoxygenase enzyme inhibition activity with IC 50 value of 193.0, 105.5, 103.5 and 163.8 respectively. All the known compounds isolated except 67 are reported for the first time from this source.

The fourth chapter entitled as “ Phytochemical investigations on

Launaea intybacea ” deals with the structure elucidation and experimental detail of isolated secondary metabolites from this species. The methanolic extract of this species on chromatography yielded one new compound, 6,6'- oxybis(4-allyl-2-methoxyphenol) (126 ) and fourteen known compounds, lupeol ( 53 ), β-sitosterol ( 30 ), octadecyl ( E)-p-coumarate ( 124 ), 4-hydroxy-3- methoxybenzaldehyde ( 125 ), 4-hydroxybenzoic acid ( 127 ), 4-hydroxy-3- methoxybenzoic acid ( 128 ), 4-hydroxy-trans-cinnamic acid (129 ), methyl gallate ( 130 ), 3,4-dihydroxybenzoic acid ( 131 ), 4',5,7-trihydroxyflavone

(132 ), 3',4',5,7-tetrahydroxyflavone ( 133 ), 3,3',5,7-tetrahydroxy-4'- methoxyflavone ( 134 ) and apigenin 7-O-(4''-O-p-E-coumaroyl-β-D-

viii glucopyranoside ( 135 ). All known compounds isolated are reported for the first time from this source as no phytochemical studies have been carried out on this species earlier.

These compounds were screened for various biological activities except 30 , 53 and 111 due to their less amounts. In antioxidant assay, compound 130 showed maximum DPPH free radical scavenging activity with an IC 50 value of 76.2, while 131 and 133 were moderately active. In acetylcholinesterase enzyme inhibition activity, the 124 and 134 showed considerable activity with an IC 50 value of 106.31 and 143.9 respectively.

While 125 , 127 , 129 , 131 -133 and 135 showed minor activity. Significant butyrylcholinesterase enzyme inhibition activities were observed for 124 ,

132 , 134 and 135 with an IC 50 value of 53.9, 93.3, 34.1, and 96.5 respectively. Compounds 125 , 127 , 130 , 131 and 133 were moderately active and 126 , 128 , 129 showed minor activity. Compounds 125 , 126 ,

132 and 135 were significantly active against lipoxygenase enzyme with

IC 50 values of 99.1, 56.37, 83.7, and 48.2 while 124 , 127 -131 and 133 did not show any activity against this enzyme. The experimental procedures and full spectroscopic data have also been included in the same chapter.

The manuscript for the structure elucidation of new as well as known compounds has been published in Journal of Chemical Society of Pakistan which is a peer reviewed journal [3,4]. The published manuscript has been attached in the end as Annexure-3 and Annexure 4 .

[3] Muhammad Saleem, Shehla Parveen , Naheed Riaz, Muhammad Ashraf, Syeda Abida Ejaz, Hafiz Muhammad Waris, Muhammad Shaiq Ali, Abdul Malik And Abdul Jabbar; Launeugenol, a New Eugenol Derived Dimer from Launaea intybacea; Journal of Chemical Society of Pakistan.35(3), 911- 915, 2013. [4] Shehla Parveen , Naheed Riaz, Muhammad Saleem, Jallat Khan, Shabir Ahmad, Muhammad Ashraf, Syeda Abida Ejaz, Rasool Bakhsh Tareen, Abdul Jabbar; Bioactive Phenolics from Launaea intybacea; Journal of Chemical Society of Pakistan,34(6), 1513-1519, 2012.

The structure elucidation of new compound from L. intybacea is under the process of publication in a peer reviewed journal.

The fifth chapter entitled “The genus Carissa and its Importance ” describes economical, pharmacological and phytochemical properties of the genus Carissa . In this chapter medicinal importance and pharmacology of some important species of the genus Carissa has been discussed. The same chapter also describes the review of literature on genus Carissa up to the date and described year wise.

The sixth chapter entitled “ Phytochemical Investigations on

Carissa opaca ” describes the isolation and characterization of four new compounds which include a macrocyclic compound, Cyclic trimer of ethylene terephthalate ( 191 ), two nor-triterpenes: 2,3-dihydoxy-30-nor-

2α,3 β–dihydroxy-urs-12-ene (195 ) and 30-nor-2α,3 α,23-trihydroxyurs-12- ene (196 ) and a new sphingolipid: (2S,3S,4R,15E)-2-{[(2R)-2- hydroxydocosanoyl]amino}eicos-15-ene-1,3,4-triol (198 ) along with eight known secondary metabolites namely 3β, 27-dihydroxylup-12-ene ( 189 ), β- sitosterol glucoside ( 67 ), lupeol β-hydroxy octadecanoate ( 190 ), pinoresinol

(192 ), (-) carinol ( 193 ), (-) carissanol ( 194 ) and arjunolic acid ( 197 ). All the known compounds have never been reported from this source.

All the compounds isolated from C. opaca 189 -191 and 193 -198 were subjected to biological screening essays except 67 and 192 due to their less amounts. In DPPH free radical scavenging essay the two lignins

193 and 194 showed significant activity with an IC 50 value of 84.91 and

83.41 respectively, while compounds 189 -191 and 195 -198 showed minor activities with IC 50 below 500.

In acetylcholinesterase enzyme inhibition essay, none of the compounds was found to be considerably active. Significant butyrylcholinesterase enzyme inhibition activities were observed for carissanol (194 ), with an IC 50 value of 101.81 and new compound 198 against Butyrylcholinesterase enzyme (IC 50 336.21). Among all the tested compounds for lipoxygenase enzyme inhibition activity, 190 , 196 , 197 ,

198 showed activities with IC 50 value below 300 and 189 , 190 , 191 , 193 and 194 didn’t show this activity.

The experimental procedures and complete spectroscopic data of the secondary metabolites isolated from Carissa opaca have been included in this chapter. The structure elucidation of all the compounds, new as well as known is under the process of publication in Journal of Asian Natural

Products which is a peer reviewed journal and attached as Annexure 5 .

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[5] Muhammad Saleem, Shehla Parveen , Naheed Riaz, Muhammad Ashraf, Syeda Abida Ejaz,

Muhammad Farrukh Nisar and Abdul Jabbar, Isolation and characterization of secondary metabolites from Carissa opaca, Journal of Asian Natural Products, Submitted.

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Acknowledgments

First of all, Many Thanks to Almighty Allah Azzawajal for giving me courage to explore His bestowed world as a research student and His prophet (Peace Be Upon Him) for enlightening our concoius with essence of faith in Allah

Writing PhD dissertation is not an easy task and was not possible without supervision of a thoughtful, cooperative and inspirational personality. Words are inadequate to express my heartiest obligation and gratitude to my research supervisor Dr. Abdul Jabbar , Professor, Chemistry Department, The Islamia University of Bahawalpur, Without his inspirational guidance, his enthusiasm, his encouragements, his unselfish help, I could never finish my doctoral work in Islamia University Bahawalpur. I am heartly thankful to him for being so friendly and for being pardon-able for my faults during the research work. I am equally thankful to Prof. Dr. Muhammad Shaiq Ali , H.E.J Research Institute of Chemistry, University of Karachi, for enabling me to utilize excellent facilities of HEJRIC on collaborative basis under his supervision. I wish to express my gratitude to him for solving my research problems and for care.

My research work and dissertation writing would be hard to complete without precious ideas and throughout guideline of Dr. Muhammmad Saleem , Associate Professor, Chemistry Department, The Islamia University of Bahawalpur. I am heartily thankful to him for being so much co-operative, helping and problem solving. Equal gratitudes to Dr. Naheed Riaz , Assistant Professor, Chemistry Department, The Islamia University of Bahawalpur, for checking my PhD dissertation and helping me in paper writing, whose comments and suggestions, co-operative and hard working attitude helped me a lot throughout. My research expertise would never be polished without their guidance and assistance.

I am grateful to the Chairman, Chemistry Department, and Dean Faculty of Science, The Islamia University of Bahawalpur, for their help throughout the course of this study.

I am also thankful to Prof. Dr. Abdul Malik, HEJRIC, University of Karachi, for his help and guidance during my stay at Karachi University. I duly acknowledge Mr. Mehmmood Alam, Director, Bentham's publications, Karachi for his co-operation during my stay in Karachi.

I am grateful to the Dr. Muhammad Arshad (late), Ex-plant Taxonomist, Cholistan Institute for Desert Studies (CIDS), The Islamia University of Bahawalpur for guiding us in plant collection and identifying the specimens. Thanks due to Dr. Muhammad Ashraf, Professor, Biochemistry & Biotechnology, The Islamia University of Bahawalpur for providing the facilities of the biological assay.

I am also thankful to Dr. Muhammad Iqbal Choudary, Director HEJRIC, University of Karachi and Dr Khalid Khan for providing me spectroscopic facilities to complete this work. I am also obliged to Dr. Muhammad Nawaz Tahir, Department of Physics, University of Sargodha, for providing X-ray facilities. I am also thankful to Professor Muhammad Moazzam for solving occasional problems.

A great deal of gratitude is for the Higher Education Commission (HEC) of Pakistan for the award of Full PhD Scholarship and award of International Support Initiative Programme (IRSIP) for South Korea which provided me a chance to broaden my vision and helped in completing my dissertation in time.

My heartiest gratitudes and thanks to my friends Naseem Akhtar, Rizwana Mustafa and Mamona Nazir for being so nice, caring and sharing all moments of stress and happiness with me. Their moral support and useful discussions are appreciable during research and dissertation writing.

My deep gratitudes to my laboratory fellows; Asia Tabassum, Sara Musaddiq, Bushra Jabeen, Nusrat Shafiq, Basharat Ali, Muhammad Akram Naveed, Imran Tousif, Jallat Khan, Abdul Ghaffar and my juniors for their moral support and providing me the valuable assistance and enjoyable moments during research.

I have to remember all those people whose collaboration was essential, all technical and non technical staff (Analytical lab, Stores, Accounts, Library and General Services), Chemistry Department, The Islamia University of Bahawalpur and HEJRIC, University of Karachi.

The more I say in the honour of my parents, the lesser it would be. I just say that I am proud of them. I am proud that I could make their dreams come true. I pray to Allah that He gives me power to make them happy always.

I wish to express my deepest gratitude to my Brothers and sisters for their moral support especially Malik Shahid and Shaista Malik for being adaptive, caring and cooperative.

Shehla Parveen

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1.1 Human Health and Role of Natural Products

The interesting chemicals designated as Natural Products are formed inside the living organisms as a result of their interaction with each other and with the environment. These chemicals of diverse range and complexity are very important to the organisms for their survival and better adaptation to the environment.

The use of natural products as a cure for human illnesses is dated back with the human civilization. Ancient records show the use of natural resources as medicines in various civilizations as written evidences

(Phillipson, 2001). For example, Mandrake roots are reported to possess pain relieving properties, turmeric for blood clotting properties and garlic used to cure circulatory malfunctioning. Successive development in natural product chemistry revealed the presence of countless chemical entities of pivotal importance in natural resources, like muscarine ( 1), penicillin ( 2) nicotine ( 3), cocaine ( 4), quinine ( 5) morphine ( 6), paclitaxel ( 7) and many others.

Natural product based medicines have been originated from various natural resources ranging from terrestrial plants and microorganisms to marine life, vertebrates and invertebrates (Newman et al., 2000). The medicinal importance of the natural products can be evaluated in three different ways:

• Firstly, the rate of introduction of new natural product drugs

with structural diversity and availability as new templates, as

semi synthetic and total synthetic drugs.

• Secondly, the number of successful prevention or cure of

disease by the natural product drugs.

• Thirdly, how commonly natural products drugs are prescribed

for cure of diseases.

An analysis shows that almost 87% of all the categories of human diseases are being cured by natural products and natural product derived chemical entities (Newman et al., 2003). These substances have been very successful due to their structural diversity and mostly complex carbon skeletons as immunosuppressants, antiparasitic, anticoagulant, antibacterial, anticancer and others. For instance, among 90 antibacterial

3 agents approved worldwide from 1982 to 2002, 79% of them were of the natural product origin (Chin et al., 2006).

On the other hand, an analysis of the drugs developed in the years

1982-2002 indicates that 28% of the approved drugs consist of natural product or natural product derived entities, and 24% were based on synthetic natural products or natural product mimics (Newman et al.,

2000). In short, more than 50% of FDA approved drugs are related to the natural products. These facts reveal that how the natural products are important for the development of the new drug candidates and provide lead compounds for further improvement in drug discovery processes.

1.2 FDA Approved Natural Products since 1998-2010.

Most of the FDA approved drugs in the recent years originated from plants, microbes or their synthetic and semi synthetic natural products.

They are related to the treatment of a variety of human diseases as cardiovascular, bacterial, inflammatory, neurological, immunological oncology and many others (Mishra and Tiwari, 2011). Some of the FDA approved natural products, their biological source, approval year and class are being described in the Table 1.1.

Table 1.1: Some of the FDA Approved Natural Products (1998-2010)

Lead Trade Name FDA Disease area Biological References Compound (Drug) Appro- Source val (Year) Mixture of Veregen TM 2006 genital warts Green tea (Phung et al., Catechins (Polyphenon ® 2010) E ointment) Fumagillin (8) (Flisint®) 2005 Oncology Aspergillus (McCowen et fumigates al., 1951), (Gilbert and Granath, 2003) Trabectedin (9) Yondelis® 2007 Oncology Ecteinascidia (Pommier et al., turbinate 1996), (Carter and Keam, 2010 ) Artemisinin (10 ) (Artemotil®) 2000 vivex malaria Artemisia annua (Wright et al., 2010 ) Capsaicin (11 ) (Qutenza®) 2009 neuropathic Genus Capsicum (Knotkova et al., pain 2008; Thresh, 1876) Tigecycline (12 ) (Tygacil®) 2005 Antibiotic Acinetobacter (Kasbekar, baumannii 2006 ) Valrubicin (13 ) (Valstar ®) 2009 anticancer Streptomycetes (Behal, 2000) chemotherapy Everolimus (14 ) (Luveniq TM ) 2010 Immunosuppre Streptomyces (Formica et al., -ssive hygroscopicus 2004) Zotarolimus( 15 ) (Endeavor TM ) 2008 Cardiovascular Streptomyces (Chen et al., hydroscopicus 2007a; Chen et al., 2007b)

These FDA approved natural product drugs are classified into three categories. 1: the compounds having biological activities and isolated from the natural sources (plants, animals and microorganism) are under the group natural products. 2: the compounds derived as a result of semi synthesis from natural product template are categorized as semi synthetic natural products and 3: synthetic compounds obtained after inspiration of a natural product template, are grouped as natural product derived compounds. According to a report, a total of 40 natural products and natural products based drugs have been launched in the market during

1998-2010. Among them 10 are natural products, 20 are semi synthetic natural products and 10 are natural product derived compounds. Majority of these natural drugs are approved for infectious, oncology and cardiovascular diseases (Mishra and Tiwari, 2011).

1.3 Natural Products under Clinical Trials

Although the importance of natural products and success as drugs is in progress, but the natural products drug discovery declined in the past few decades due to some complications like accessibility, complexity in natural products chemistry, long time span etc. Even then more than 100 natural products are under clinical trials in drug discovery and almost the same number is in the preclinical studies (Table 1.2) for drug development processes (Butler, 2008). Majority of these are developed from plants and microbial sources (Harvey, 2008).

Table 1.2: Some of the Natural Products under Clinical Trials

Natural Product Class Clinical Disease Area Biological References trial Source Phase Rostafuroxin ( 16 ) Digitoxygenin II chronic Digitalis (Schoner and derivative arterial lanata Scheiner, hypertension 2007) Trodusquemine (17 ) Cholesterol I Type 2 Squalus (Ahima et al., derivative & Diabetes acanthias 2002; Rao et II al., 2000) (-)-Gossypol ( 18 ) Phenolic II (b) Brain and Genus (Kim et al., aldehyde lung cancer Gossypium 2009; Polsky derivative et al., 1989)

Pyridoxamine ( 19 ) VitaminB 6 II (b) Type 2 (Roje, 2007) analogue Diabetes Tetrameprocol ( 20 ) Lignin I & II leukemia, Larrea (Khanna et al., solid tumors tridentate 2007) and glioma Silybin ( 21 ) Flavonolignin II Cancer Silybum (Gazak et al., chemotherapy marianum 2007; Lee and Liu, 2003) Combretastatin A-4 Stilben III anaplastic Combretum (Escalona- phosphate (22 ) derivative thyroid cancer caffrum Benz et al., (ATC). 2005)

Most of the projects for the natural products are being evaluated as infectious, anticancer and many therapeutic areas. Data shows that most of the lead compounds which are being analyzed in clinical trials are mostly from plant and bacterial sources (Butler, 2005).

1.4 Emergence of the Natural Product Research

Pharmaceutical research in the field of natural products reached at its peak during the years 1970-1980, as a result Western pharmaceutical

8 investment was greatly influenced by natural product research. By 1990, almost 80% of the drugs used worldwide were natural products or natural product analogues (Sneader, 1996).

An analysis shows that the natural products and natural product analogues constitute 52% of the total new chemical entities developed as drugs during the years 1981-2002 and natural product derived drugs are responsible to cure almost 87% of the human diseases all over the world

(Newman et al., 2003).

1.5 Decline of Natural Product Research in the Pharmaceutical

Industry

In spite of the successful story of natural product drugs, there is a slow decline observed during the past two decades in the natural product pharmaceutical research. With the advances in the alternative drug discovery methods such as rational drug design and combinatorial chemistry, pharmaceutical industries decreased or almost cut down the use of natural products in their drug development program (Rishton, 2008);

(Lam, 2007). There are some commercial and scientific reasons that why pharmaceutical industries emphasize on alternative drug discovery methods.

Recently, trends for the drug development program are towards rapid screening, hit identification and then hit to lead development. While natural 9 product drug discovery program needs extract library screening, bioassay guided isolation, structure elucidation and finally large scale production which of course needs greater time span. However instead of the promising success of these alternative drug discovery methods there is still a shortage of lead compounds in the clinical trials especially anticancer, immunosuppressive and metabolic diseases where natural products have been very successful and provided many lead compounds (Koehn and

Carter, 2005). Promising biological sources are there for drug development from natural sources but there is a pressure of short time framework for drug discovery.

1.6 Revitalization in Natural Product Research

Rapid resistance development in pathogens against synthetic drugs, and their un-necessary side effects, once again motivated the researchers to focus on natural products for future drug discovery. Therefore, now there are emerging trends and renewed interest in natural product research for chemical diversity and discovery of new leads. An analysis indicates that among recently introduced drugs, sorafenib is the only drug reaching market from combinatorial chemistry origin (Fig. 1.1) (Newman and Cragg,

2007).

Fig. 1.1: Sorafenib, only FDA approved drug from combinatorial

chemistry

Now it is being realized that natural products are better candidate towards new drugs and drug leads as they possess greater chemical diversity, greater number of chiral centers, increased steric complexity than collection of synthetic compounds (Galm and Shen, 2007). Natural products considered as privileged structures as they are more effective in various types of therapeutic conditions. As they are produced as a result of evolution pressure to interact greater number and variety of proteins and biological targets therefore likely to possess more biological friendliness or drug likeness as compared to synthetic compounds (Evans et al., 1988),

(Martin et al., 2001).

Although a large number of drugs have been introduced to cure almost every type of diseases but still there is need to search new drug candidates for many reasons.

• There is a need to find cheaper and improved medicines for existing

diseases.

• Some medicines currently in use, possess more side effects or much

costly to produce or not easy to store.

• Some diseases are there for which still some good remedies are not

discovered yet.

• Viruses and bacteria responsible for diseases can mutate, evolve and

adapt to the new environmental conditions therefore, they become

resistant to the drugs already in use.

Due to all these facts there is continuous need to introduce new drug candidates to improve health well being and economy. This renewed interest in the natural product drug discovery will be more beneficial to the patients and community and drug discovery process will be more holistic, personalised when equipped with ancient and modern research methodologies in a complementary manner as still more than 80% of the world population depends upon the natural products because of their time tested safety and effectiveness. These facts and needs motivated us to investigate indigenous medicinal plants for their bioactive secondary metabolites.

1.7 Hypothesis

The natural biological resources terrestrial as well as marine produce secondary metabolites for their chemical defense which possess unique chemical structures and play pivotal role in human health. These

12 secondary metabolites designated as natural products are extracted from natural resources and practiced to cure a wide range of human diseases.

1.8 Aims and Objectives of the Study

The natural resources have provided most of the remarkable compounds playing important role in human health ranging from oncology to heart diseases. Although a vast array of the natural resources like plants, microbes and marine organisms have provided incredible remedies to various diseases but still a major part of the natural resources is needed to be explored to solve a wide array of biomedical problems.

Pakistan is endowed with the wealth of medicinal plants which are the natural botanical source of medicines being manufactured in local pharmaceutical industries in Pakistan. These are also the primary source of modern pharmaceutics. Although today, the pharmaceutical industry has become a world of synthetics but with the medicines at higher prices which are not affordable by a common man. Therefore there is a need to develop and organize our medicinal plant research in provision of common indigenous natural drugs prepared from plants used by the local people to cure a number of diseases. Pakistan is rich in plants which are grown wild or cultivated and possess great medicinal potential as evident from their use in Pakistan and India in Unani system of medicine for centuries (Haq,

2008). More than 6000 plant species reported in the country of which

13 about 3200 species are being utilized in various Unani, allopathic and homeopathic medicines (Ahmad et al., 2008) and about 350 plants are being used specifically in herbal medicinal preparations in various

Dawakhana’s in Pakistan (Haq, 2008), but still a very less number of the plants have been subjected to systemic phytochemical studies.

The medicinal and economic importance of the members of family

Compositaea and Apocynacaea is well explored but still some species are un explored as long as their medicinal potential is regarded (Rashid et al.,

2000a); (Sulaiman et al., 2008b); (Gite et al., 2010b). These facts motivated us to work on species of genus Launaea and Carissa. Two species of genus

Launaea namely L. nudicualis , L. intybacea and one of genus Carissa : C. opaca were selected for phytochemical investigations due to their medicinal potential. L. nudicualis , L. intybacea were collected from Cholistan desert while C. opaca from Quaid-e-Azam University Islamabad. Systematic phytochemical studies on above mentioned species provided with interesting results.

2.1 Introduction of the Genus Launaea

The Genus Launaea of the family Asteraceae, consists of 40 species which are mostly glabrous herbs and found in tropical and subtropical regions and mostly adapted to dry, saline and sandy habitat (Ozenda,

2004). Members of this genus usually possess branched stems with alternate leaves which are lobed, hairless and lined with teeth. These are little flattened, prismatic with yellow ligules and afford membranous scaly edges. About 20 species of the genus are found in Pakistan and most of them are used in folk medicine and possess galactogogue, aperient, soporific, and diuretic properties. Some species are used for the treatment of wounds and fever in children (Nasir and Ali, 1972), (Baquar, 1989) and others reported to possess cytotoxic, antitumor and insecticidal activities

(Rashid et al., 2000c).

2.2 Pharmacological Importance of the Species of Genus Launaea

Among medicinally important species of the genus Launaea , L. arborecens is a shrub commonly used for the treatment of diarrhoea and abdominal spasms (Bitam et al., 2008). Methanolic extract of aerial parts of

L. arborescens possess significant antifungal activities against Candida albicans , and Sacharomyce cereviveae and the antibacterial activity against

E. coli , S. aureus , P. aeriginisa and Klebsiella entrecocus (Belboukhari and

Cheriti, 2006). The methanolic extract of the plant reported to possess 16 insecticidal, antifungal and antibacterial properties (Belboukhari and

Cheriti, 2006); (Jbilou et al., 2008).

L. residifolia is a perennial herb usually found in Pakistan, Libya,

Algeria and India. It is a medicinal plant effective for the treatment of hepatic pains. Extracts of L. residifolia showed anti-inflammatory activities and found effective in rat paw oedema induced by carrageenan and inhibitory effects against locomotory activities of mice (Razag et al., 2007).

L. procumbens is a perennial herb found in tropical and subtropical regions generally 30-60 cm tall and it has been reported to show antibacterial activities and to be effective in renal disorders (Ahmad et al.,

2006a), rheumatism (Parikh and Chanda, 2006), and painful urination

(Qureshi and Bhatti, 2008). Studies showed that L. procumbens possess antioxidant and free radical scavenging activities against CCl 4 induced oxidative damage in mice (Khan et al., 2010). L. pinnatifida leaves have been shown to be effective in treatment of diarrhea (Tetali et al., 2009).

Petroleum ether, chloroform, ethanol and water extracts of L. pinnatifida leaves have been examined to show promising antioxidant activities

(Nagalapur and Paramjyothi, 2010).

2.3 Previous Phytochemical Investigations of the Species of Genus

Launaea

Phytochemical investigations on genus Launaea were started with the isolation of two compounds taraxasterol ( 23 ) and taraxeryl acetate ( 24 ) from the roots and leaves of L. pinnatifida (Prabhu and Venkateswarlu,

1969). Palmitic acid ( 25 ), stearic acid ( 26 ), oleic acid ( 27 ) and linoleic acid

(28 ) have been isolated from the roots of L. nudicaulis (Bahadur and

Sharma, 1974). A glycoside of D-(+)-xylose and the aglycon ( 29 ) were isolated from dried powdered leaves of L. nudicaulis (Sushma et al., 1980).

Characterization of 10 sterols from the petroleum ether extract of L. nudicaulis and the unique distribution of ∆7-sterols along with ∆5-sterols namely β-sitosterol ( 30 ), campesterol ( 31 ), stigmasterol ( 32 ), cholesterol

(33 ), 28-isofucosterol ( 34 ), stigma-7-enol (35 ), stigma-7,24(28)dienol ( 36 ),

24-methylcholest-7-enol ( 37 ), cholest-7-enol ( 38 ) and brassicasterol ( 39 ) was carried out (Behari and Gupta, 1980).

The roots of L. mucronata afforded lactucin ( 40 ), lactucin-8-O-acetate

(41 ), 11-β-13-dihydrolactucin ( 42 ), and 11-β,13-dihydrolactucin-8-O- acetate ( 43 ) (Sarg et al., 1982). Chemical investigation of L. nudicaulis

19 yielded β-sitosterol ( 30 ), β-sitosterylacetate (44 ), and taraxasterol ( 23 )

(Majumder and Laha, 1982). Apigenin-7-O-glucoside (45 ), and apigenin-7-

O-gentiobioside ( 46 ), luteolin 7-O-glucoside ( 47 ), luteolin 7-O-gentiobioside

(48 ), luteolin-7-O-rutinoside ( 49 ), luteolin-7,3'-O-diglucoside ( 50 ), luteolin-

7,4'-O-diglucoside ( 51 ), and luteolin-7-O-gentiobioside-4'-O-glucoside ( 52 ), were detected in L. capitata , L. cassiniana , L. resedifolia , L. spinosa , and L. tenuiloba (Mansour et al., 1983).

The petroleum extract of L. nudicaulis contains hydrocarbons, higher fatty acid esters of primary alcohols, triterpene alcohols, triterpene acetates, primary alcohols, triterpene alcohols and sterols namely β- sitosterol ( 30 ), campesterol ( 31 ), stigmasterol (32 ), cholesterol ( 33 ), 28- isofucosterol ( 34 ), stigma-7-enol (35 ), stigma-7,24(28)dienol ( 36 ), 24-

20 methylcholest-7-enol ( 37 ), cholest-7-enol ( 38 ) and brassicasterol ( 39 ) that revealed the plant is a good source of triterpenoids (Behari et al., 1984).

L. asplenifolia contain lupeol ( 53 ), delphinidin ( 54 ), apigenin ( 55 ), luteolin ( 56 ), vitexin ( 57 ), and luteolin-7-O-glucoside ( 47 ) (Gupta et al.,

1985). Chemical investigations of L. asplenifolia led to the identification of octacosanoic acid ( 58 ), lupeol ( 53 ), 7-hydroxy-3',4'-dimethoxyflavone ( 59 ), apigenin ( 55 ), luteolin ( 56 ), apigenin-7-O-β-D-glucoside ( 45 ), vitexin ( 57 ), luteolin-7-O-glucoside ( 47 ), and delphinidin ( 54 ) (Gupta et al., 1985).

Asplenetin ( 60 ), and its glycoside, asplenetin 5-O-neohesperidoside

(61 ), reported from L. asplenifolia (Gupta and Ahmed, 1985). Phytochemical investigations of L. tenuiloba yielded 8-deoxylactucin ( 62 ), cichoriin ( 63 ), esculetin ( 64 ), luteolin 7-O-glucoside ( 47 ), apigenin 7-O-glucoside ( 45 ), and

21 apigenin 7-O-diglucoside ( 65 ) (Salam et al., 1986). Three phenolic compounds were isolated from L. nudicualis namely, cichoriin ( 63 ), aesculetin ( 64 ) and luteolin 7-O-diglucoside ( 47 ) (Sarg et al., 1984).

Phytochemical studies of L. spinosa led to the isolation of 3,4- dihydroscopoletin ( 66 ), cichoriin ( 63 ), esculetin ( 64 ), luteolin 7-O-glucoside

(47 ), stigmasterol ( 32 ), β-sitosterol (30 ), lupeol ( 53 ), β-sitosterol-3-O- glucoside ( 67 ) and friedelin ( 68 ) (Sarg et al., 1987). Apigenin ( 55 ), dihydroxycoumarin ( 69 ), luteolin-7-O-glucoside ( 47 ) and apigenin-5-O- diglucoside ( 70 ) were isolated from L. resedifolia (Saleh et al., 1988).

Phytochemical studies on L. asplenifolia yielded taraxasteryl acetate

(71 ), taraxasterone ( 72 ) taraxasterol ( 23 ), stigmasterol ( 32 ), ethylpalmitate

(73 ), ethylstearate ( 74 ), hexacosanol ( 75 ), octacosanol ( 76 ) and octacosanoic acid ( 58 ) (Gupta et al., 1989). α-Amyrin ( 77 ), α-amyrin acetate

(78 ), moretenol ( 79 ) moretenol acetate ( 80 ) lupeol acetate ( 81 ), ∆ 7- stigmastenol ( 82 ) and ∆ 7-stigmastenol-3-O-glucoside ( 83 ) have been isolated from L. resedifolia (El-Fattah et al., 1990).

Luteolin-7-O-glucoside (47 ), luteolin-7-O-rhamnoside, ( 84 ) apigenin-

7-O-β-D-glucoside (45 ), cichoriin ( 63 ), aesculetin (64 ), ferulic acid ( 85 ) and methylcaffeoate ( 86 ) were reported from L. arborescens. (Giner et al., 1992)

H H

HO AcO 79 80 AcO 81

OH O HO HO O 82 HO OH 83 OH OH O O

RO O OH OCH3

HO HO OCH3 OH OH O 84 R =Rhamnose 85 86

The presence of moreten-3β,19 α-diol ( 87 ), moreten-3β,28-diol ( 88 ),

Ψ-taraxasterol-30-aldehyde ( 89 ), olean-3Ψ,19-diol,( 90 ) stigmasterol ( 32 ) and stigmasterol-β-D-glucoside ( 91 ) and crepidiaside A ( 92 ) was reported in

L. spinosa (Sokkar et al., 1993). Two triterpenes, nudicauline A ( 93 ) and nudicauline B ( 94 ) have been isolated from L. nudicaulis (Zaheer et al.,

2006). Four coumarin compounds namely, cichoriin ( 63 ), esculetin ( 64 ), scopoletin ( 95 ) and isoscopoletin ( 96 ) were isolated from the aerial parts of

L. residifolia showing high antibacterial activity (Gherraf et al., 2006).

Chemical investigation of L. arborescens resulted in the isolation of five new terpenes, 3 β-hydroxy-11 α-ethoxy-olean-12-ene ( 97 ), 9-hydroxy-

11 β,13-dihydro-3-epi-zaluzanin C ( 98 ), 3 β,14-dihydroxycostunolide-3-O-β- glycopyranosyl-14-O-p-hydroxyphenyl-acetate ( 99 ) 3 β,14- dihydroxycostunolide-3-O-β-glucopyranoside ( 100 ), 9 α-hydroxy 4 α,15- 25 dihydrozaluzanin C ( 101 ), 3β-11 α-dihydroxy-olean-12-ene ( 102 ), 3 β- hydroxy-11 α-methoxyolean-12-ene ( 103 ) and 8-deoxy-15-(30-hydroxy-20- methylpropanoyl)-lactucin-30-sulfate ( 104 ) (Bitam et al., 2008).

A triterpenoid saponin 3 β-O-[α-L-rhamnopyranosyl-(1→3)-O-α-L- arabinopyranosyl-(1 →3)-O-β-D-galactopyransyl]-spergulatriol ( 105 ), having antifungal activity, along with glutenol ( 106 ) and hopenol-b ( 107 ) have been isolated from defatted seeds of plant L. pinnatifida (Yadava and

Chakravarti, 2009). Investigation on aerial parts of L. residifolia afforded four flavonoids apigenin ( 55 ), luteolin ( 56 ), apigenin 7-O-β-D-glucoside ( 45 ) and apigenin 7-O-β-D-glucuronide ( 108 ), last two having antimicrobial activity (Moussaoui et al., 2010a).

3.1 Introduction of Launaea nudicaulis

L. nudicaulis is an important member of the genus widely growing in

Cholistan Desert in District Bahawalpur. Locally it is known as jangli booti, bathal, or tariza. It is also found in northern Sahara. The plant is collectively rising with thermophilous plants and distributed in

Mediterranean woodlands, shrublands and moderate to extreme deserts. Its flowering period is April to May. The young leaves can be cooked and eaten and is a good feed for camels. The name Launaea is derived after the name of a French lawyer Jean Claude Mien Mordant de Launay (c.1750-1816) and nudicaulis is from nudus (naked, bare or unclothed) caulis (stem of a plant) bare stem.

3.2 Botanical Description of Launaea nudicaulis

L. nudicaulis is a small perennial herb with taproot and shoot often possess lateral roots. Stem branched, 15-16 cm in length, divaricately branched, leafless or with distant few small leaves at the lower branching, leaves are 2-12 cm, alternate, rosette, dissected, pinnate, dentate or serrate with acute apex and segment and narrow base. There is synflorescence system of flowering with 16-30 flowers on a single capitulum or head with multiple flowering branches, with central branch prolonging, or other overtopping the central. Flowers are with yellow ligules 6-9 mm long, style

29 branched with yellow sweeping hairs. Achenes are 2.6-5.5 mm, pale to grayish, dark brown or black (Kilian, 1997)

3.3 Scientific Classification of Launaea nudicaulis

Kingdom: Plantae

Subkingdom: Angiosperms

Order: Asterales

Family: Asteraceae

Tribe: Cichorieae

Genus: Launaea

Specie: nudicaulis

3.4 Pharmacological Importance of Launaea nudicaulis

L. nudicaulis is used by the local community to cure many diseases.

Its latex is effective for the treatment of constipation. The leaves are used to cure ulcers, cuts, eczema eruptions, rheumatism, swellings, skin itches well as for the treatment of fever in children. The roots are used to treat toothache (Rashid et al., 2000b). The leaves are also used as antipyretic in children (Bhandari, 1988). The aqueous extract of L. nudicaulis showed very interesting antibacterial, antifungal, insecticidal and cytotoxic 30 activities. Its methanolic extract showed very high activity against E. coli which was shown to be concentration dependant (Rashid et al., 2000b). The juice of leaves is reported to show anti-inflammatory activities in rats against acute and chronic inflammation (Nivsarkar et al., 2002).

The plant also possesses hyperglycemic properties against diseased rats (Shabana et al., 1990). It is used in traditional medicine in Oman because of its antimicrobial properties for wound healing and for treatment of abscesses (Salam et al., 1990). Phytotoxic studies of various methanolic and ethyl acetate fraction of L. nudicaulis against radish growth showed that it possess significant allelopathic activity and suggested that the activity may be due to the presence of allelochemicals in the species (Khan et al., 2012).

3.5 Results and Discussion

The phytochemical investigations on L. nudicaulis were carried out which resulted in the isolation of a total of sixteen compounds. Four new secondary metabolites (112 -115 ) were isolated from n-hexane fraction and the same number (116 , 119 , 121 and 123 ) from ethyl acetate fraction. All of these isolated compounds were further subjected to their pharmacological activities. The structure elucidation of eight new secondary metabolites isolated is discussed before the known compounds.

3.5.1 Characterization of nudicholoid (116)

The molecular formula of the compound 116 was confirmed by the

HR-EIMS which displayed the exact mass at 4' H C14 CH O 3 3 OH m/z 364.1581 corresponding to the 10 9 O 1' 2' 2 1 3' 3 8 5 H 7 O 4 H molecular formula C 19 H24 O7 with eight double 6 H 15 H 11 HO 12 O CH3 13 bond equivalences while EIMS showed O 116 molecular ion at m/z 364. The IR spectrum of

116 exhibited stretching absorption bands at 3445 cm -1 due to primary hydroxyl groups, and absorption at 1765 cm -1 indicated the presence of γ- lactone, 1685 cm -1 for carbonyl and absorptions at 1615 and 1605 cm -1 indicated the presence of olefinic system.

This olefinic proton displayed a doublet methine in the 1H NMR spectrum at δ 6.41 (1H, t, J = 1.0 Hz, H-3) due to a conjugated system, which was crossed linked in COSY spectrum with a methylene resonating at δ 4.82 and 4.40. The shift of H-3 and amount of coupling constant (1.0

Hz) revealed that an oxymethylene was present at allelic position to H-3.

The 13 C NMR spectrum showed four most downfield signals at δ 197.0 (C-

2), 179.1 (C-12), 176.4 (C-4) and 175.7 (C-1′). The signal for C-4 was assigned to the olefinic system and its downfield shift was attributed to the

β-effect of a conjugated pentenone ring in 116 . The HMBC correlation of H-

3 ( δ 6.41) with carbons at δ 197.0 (C-2), 176.4 (C-4), 49.7 (C-5) and 134.2

(C-1) confirmed the cyclopentenone moiety. A methine proton resonating at

δ 3.76 (d, J = 10.0 Hz, H-5) showed COSY correlation with an oxymethine at

δ 3.81 (t, J = 10.0 Hz, H-6). The H-6 was further correlated in the same spectrum with another methine at δ 2.51 (H-7) and in HMBC spectrum it was found to interact with the carbon signals at δ 179.1 (C-12) and 41.9 (C-

11) as depicted in Fig. 3.1. This information revealed that a butanolide moiety was also present in 116 .

Fig. 3.1: Important HMBC (→) and COSY (▬) correlations in 116

Further careful analysis of 1H and 13 C NMR data indicate that 116 is a sesquiterpene lactone possessing the same skeleton as have been reported for sesquiterpene lactones from Helianthus species (Gao et al.,

1987) and Reichardia gaditana (Zidorn et al., 2007). The 2-methyl-3- hydroxy propanoate moiety at C-8 could be fixed due to HMBC correlation of H-8 ( δ 4.18) with that of C-1′ (δ 175.7). The remaining assignments were accomplished due to COSY and HMBC spectral data.

The mass spectrum of the compound in addition to the molecular ion peak at m/z 364 showed fragments at m/z 278, 260, 231, 214, 187 and

159, obtained due to the fragmentation pattern as shown in Fig (3.2).

Fig. 3.2: Mass fragmentation pattern in 116

The relative stereochemistry at various centers could be established due to careful analysis of coupling constants, NOESY spectrum (Fig. 3.3) and finally the structure was confirmed through single X-ray crystallographic analysis (Fig. 3.4) to get the structure of 116 as sesquiterpene lactone, which is named as nudicholoid and is a new natural product.

O H3C CH3 O OH H H H O H H HO O CH3 O

Fig. 3.3: Important NOESY correlations in 116

[(3 R,3a S,4 R,9a R,9b S)-9-(hydroxymethyl)-3,6-dimethyl -2,7-dioxo-

2,3,3a,4,5,7,9a,9b-octahydroazuleno[4,5 -b]furan-4-yl(2 R)-3-hydroxy-2- methylpropanoate].

Fig. 3.4: A Computer generated drawing of 116

The skeleton of 116 consists of a seven -membered A

(C5/C6/C7/C9/C14/C15/C16) ring and two fused five -membered rings B

(C9/C10/C11/C12/C14) and C (C15/C16/C17/C19/O7). At C5 and C7, the 3-hydroxy-2-methylpropanoic acid and methyl groups respectively, are attached. At rings B and C there are methanolic and methyl groups present at C12 and C17, respectively. There exists carbonyl at C10 and C19. In the seven-membered ring A, four C-atoms, D (C6/C7/C15/C16) are nearly planar with r. m. s. deviation of 0.0580 Å. This ring seems in chair form with one leg, C5 and two heads C9 and C14. The C5, C9 and C14 are at a distance of -0.7680 (25), 0.3996 (32) and 1.0660 (27) Å, respectively.

The ring B is roughly planar with r. m. s. deviation of 0.0277 Å. The dihedral angle between B/D is 29.66 (6)°. The ring C is not planar as the deviation from mean square plane is 0.1636 Å and from this plane the maximum deviation is of C16 which is 0.2272 (12) Å. The propan-1-ol moiety E (O1/C1/C2/C3) is planar with r. m. s deviation of 0.0048 Å and is oriented at a dihedral angle of 82.70 (16)° with the carboxlate group F

(O2/C4/O3). The O-atom of methanolic group is disordered over two sites with refined occupancy ratio 0.712(5): 0.288(5). The molecules are interlinked due to C-H…O and O-H…O types of H-bondings.

3.5.2 Characterization of nudicualin A (112)

The molecular formula C 42 H84 NO 4 of the compound 112 was deduced by HR-FABMS O 18' HN 1' CH showing molecular 2' 3 OH 14 + HO ion peak [M+H] at 2 3 4 1 15

OH H3C 23 m/z 666.6405 24 112 having two degrees of unsaturation. The IR spectrum showed the presence of O-H (3640 cm -1), secondary amide (1650 cm -1) and olefinic (1625 cm -1) functions. The 1H NMR spectrum of 112 displayed signal for a secondary amide N-H at δ 6.75 (1H, d, J = 8.0 Hz), an oxygenated methylene at δ 3.94

(1H, dd, J = 3.5, 11.5 Hz), 3.57 (1H, dd, 5.5, 11.5 Hz), two oxymethines at δ

3.47 (1H, dd, J = 4.1, 5.3 Hz), 3.43 (1H, dd, J = 4.1, 4.5 Hz). A signal at δ

3.97 (1H, m) was ascribed for the methine proton vicinal to the nitrogen atom of the amide group. The same spectrum also displayed signals for a double bond and two primary methyls.

The 13 C NMR spectrum of 112 fully supported the 1H NMR data and declared it to be a sphingolipid (Kwon et al., 2003),(Ahmad et al., 2006b). A methylene triplet at δ 2.10 ( J = 8.0 Hz) indicated the absence of usual hydroxyl group at C-2. The entire sequence of the skeleton and the substitutions were fixed by 1H-1H COSY and long range HMBC correlations

(Fig. 3.5).

Fig. 3.5: Important HMBC & COSY correlations in 112

The length of the fatty acid chain was determined by characteristic fragments at m/z 268, 282 and the amine chain containing a double bond by the fragments at m/z 441 and 384 (Fig. 3.6).

Fig. 3.6: Mass fragmentation pattern of 112

Methanolysis (Ahmed et al., 2007) of 112 with methanolic HCl provided the base and the methyl ester of fatty acid, which after acetylation

(Muralidhar et al., 2005 ) were analyzed by GC-MS and were identified as methyl octadecanoate ( m/z 298) and 2-acetamino-1,3,4-

38 triacetoxytetraeicosene ( m/z 567). The position of double bond was fixed between C-14,15 by permanganate /periodate oxidative cleavage (Ahmed et al., 2007) of 2-acetamino 1,3,4-triacetoxy-tetraecosene, yielded a binary mixture of carboxylic acids which on methylation and GC-MS analysis provided peaks for 2-acetamino-1,3,4-triacetoxytetradecanoic acid ( m/z

473) and decanoic acid ( m/z 186). The stereochemistry at the three stereogenic centers was determined by 1H NMR spectrum (through chemical shifts and coupling constants) and optical rotations of 112 ([ α]D =

+ 16.9) and its methanolized base ([ α]D = + 11.2) which were found similar with those having (2 S,3 S,4 R)-configurations [(Natori et al., 1994); (Riaz et al., 2007); (Mukhtar et al., 2002).

Based on these evidences, 112 could be assigned the structure

(2S,3S,4R,14E )-2-{[octadecanoyl]amino}tetraeicos-14-ene-1,3,4-triol and named as nudicaulin A which is a new addition to the natural products.

3.5.3 Characterization of nudicualin B (113)

Compound 113 was obtained as a white amorphous powder. The IR O OH spectrum of 113 was 18' 1' CH HN 2' 3 similar to that of 112 . OH 14

HO 2 3 4 The HR-FABMS 1 15 OH H3C 23 24 spectrum showed the 113 molecular ion peak at m/z 682.6355 corresponding to the molecular 39 formula C 42 H84 NO 5 with two degrees of unsaturation indicated that 113 could be an oxidative derivative of 112 . The 1H and 13 C NMR spectra of 113 were almost super imposable to that of 112 with a minor difference of an additional signal for oxymethine at δ 3.94 (1H, dd, J = 3.6, 6.4 Hz) and δ

71.9. The absence of 2H triplet at δ 2.2-2.5 in 1H NMR spectrum revealed that position-2 of the fatty acid is hydroxylated (Kwon et al., 2003). The positions of all the substituents were assigned with the help of 1H-1H COSY and HMBC spectra (Fig. 3.7).

Fig. 3.7: Important HMBC connectivities and COSY correlations in 113

The length of fatty acid and the base chain containing a double bond were tentatively fixed by mass fragmentation (Fig. 3.8). Compound 113 was methanolized and the obtained fragments after acetylation were analyzed by GC-MS, to identify the two fragments: methyl 2-acetoxyoctadecanoate

(m/z 356) and 2-acetamino-1,3,4-triacetoxytetraecosene ( m/z 567).

Fig. 3.8: Mass fragmentation pattern in 113

The double bond was confirmed between C-14,15 of the base chain due to permanganate/periodate oxidative cleavage and GC-MS analysis, which gave peaks at m/z 473 and 186 as were observed for 112 . The configuration at the stereogenic centers was deduced by 1H NMR spectrum

(coupling constants) and optical rotations of 113 ([ α]D = + 39.7) and the subsequent methanolysis products ([ α]D = − 7.1 and + 19.1) which were comparable with those of sphingolipids with a 2 S,3 S,4 R,2 ′R configurations

(Muralidhar et al., 2003); (Natori et al., 1994); (Riaz et al., 2004). Based on these evidences, nudicaulin B (113 ) could be assigned the structure

(2S,3S,4R,14E )-2-{[(2 R)-2-hydroxyoctadecanoyl]amino}tetraeicos-14-ene-

1,3,4-triol which has never been reported so far from any source.

3.5.4 Characterization of nudicualin D (114)

The sphingolipid 114 was obtained as colorless shiny powder with molecular formula C 38 H76 NO 6. The 1H NMR spectrum of 114 was similar to

O OH that of 113 13' OH 20' 1' 3' 12' CH3 with an HN 2' OH 18 HO CH additional 1 2 3 4 3 oxymethine OH 114 signal at δ 3.82 (1H, d, J = 4.4 Hz) corresponding to the carbon at δ 72.8.

This information indicated that 114 could be an oxidative derivative of 113 with slight variations in lengths of both the chains.

The 13 C NMR spectrum of 114 disclosed characteristic signals of sphingolipid as were observed in the spectra of 112 & 113 . The four oxymethines resonated at δ 75.2, 74.3, 72.8, and 72.0 that indicated 114 may be oxidative derivative of 113 with slight variations. Mass fragmentation pattern and 2D NMR data helped to tentatively fix the position of various hydroxyl groups and double bond (Fig. 3.9).

Fig. 3.9: Important HMBC & COSY Interactions in 114

The length of fatty acid and base chain, and position of hydroxyl groups on each chain were confirmed by EIMS spectrum (Fig. 3.10) and methanolysis of 114 , which after acetylation and subsequent GC-MS analysis provided methyl 2,3-diacetoxyunieicosenoate ( m/z 440) and 2- acetamino-1,3,4-triacetoxyoctadecane ( m/z 485).

Fig. 3.10: Mass fragmentation pattern in 114

The position of double bond was fixed between C-12′,13′ by permanganate/periodate oxidative cleavage of methyl 2,3- diacetoxyunieicosenoate ( m/z 440) that on methylation and GC-MS 43 analysis gave methyl esters of 2,3-diacetoxydodecanoic acid ( m/z 474) and octanoic acid ( m/z 158), respectively. The optical rotations of 114 ([ α]D = +

29.9) and the subsequent methanolysis products ([ α]D = − 9.6 and + 16.9) were comparable with the sphingolipids having same configurations (Kwon et al., 2003); (Ahmad et al., 2006b) . Based on these evidences, nudicaulin

D (114 ) was assigned the structure ( 2S,3S,4R, )-2-{[(2 R,3S,12E )-2,3- dihydroxyeicos-12-enoyl]amino}octadecane-1,3,4-triol and is a new addition to the natural product.

3.5.5 Characterization of nudicualin C (115)

Compound 115 with a molecular formula C 49 H94 NO 10 isolated as a colorless gummy solid, was found to be a glycosphingolipid based on its

NMR data O OH 17' CH when 1' 3 HN 2' OH 6'' OH 4'' compared O 14 HO 5'' 2'' O HO 1 2 3 4 3'' OH1'' 15 to that of 26 OH H3C 115 112 and

113 . In addition to the usual signals for a sphingolipid skeleton, the 1H

NMR spectrum of 115 displayed signals for sugar moiety at δ 4.15 (1H, d, J

= 7.6 Hz, H-1''), 3.40 (1H, m, H-2''), 3.28 (1H, t, J = 7.4 Hz, H-3''), 3.23 (1H, t, J = 7.4 Hz, H-4''), 3.16 (1H, m, H-5''), 3.79 (1H, dd, J = 4.9, 10.8 Hz, H-

6''), 3.61 (1H, dd, J = 2.9, 10.8 Hz, H-6''). The 13 C NMR spectrum of 115

44 also supported the above data for a glycosphingolipid. A downfield shift ( δ

68.5) of C-1 was due to the sugar unit attached to it, which was further confirmed by HMBC correlation of anomeric methine at δ 4.15 with C-1 ( δ

68.5) (Fig. 3.11).

Fig. 3.11 : Important HMBC and COSY interactions in 115

The lengths of fatty acid and base chain (containing double bond) were determined by mass fragmentation pattern (Fig. 3.12). Methanolysis of

115 provided methyl ester, a sphingosine base and methylated sugar moiety. Both methylated fatty acid and base on acetylation and GC-MS analysis; were identified as methyl 2-acetoxyheptadecanoate ( m/z 342) and

2-(acetamino)-1,3,4-triacetoxyhexaeicosene ( m/z 595).

Sugar moiety obtained from aqueous layer was determined to be D- glucose by comparing the sign of its optical rotation and spectral data with those of authentic sample (Jin et al., 1994). The GC-MS and chemical analysis helped to fix the position of double bond between C-14,15 in the

45 base chain. The stereochemistry at various stereogenic centers was found similar to those for 113.

Fig. 3.12: Mass fragmentation pattern in 115

Based on these evidences, nudicaulin C (115 ) could be assigned the structure ( 2S,3S,4R,14E )-2-{[(2 R)-2-hydroxyoctadecanoyl]amino}tetraeicos-

14-ene-1,3,4-triol-1-O-β-D-glucopyranoside. This compound has been isolated for the first time from any natural source and is a new natural product.

3.5.6 Characterization of trideca-12-en-4,6-diyne-2,8,9,10,11-pentaol

(119)

Compound 119 was obtained as white amorphous solid. The EIMS exhibited molecular ion peak at m/z 254 and HR-EIMS displayed the molecular ion at m/z 254.1128 corresponding to the molecular formula

C13 H18 O5 with five double bond equivalence. The IR spectrum of 119 showed absorption bands at 3390 (O-H), 3080, 2950 (C-H), 2240, 2150

(C≡C) and 1645-1490 cm -1 (C=C).

The 1H NMR spectrum of 119 afforded the signals for three olefinic protons at δ 6.02 (1H, ddd, J = 5.6, OH OH H3C 1 13 OH 7 4 H2C 10.4, 16.8 Hz), 5.34 (1H, dt, J = 1.6, 3 6 5 OH OH 17.2 Hz) and 5.19 (1H, dt, J = 1.6, 10.4 119

Hz) were attributed to the carbons in 13 C NMR spectrum at δ 140.2 (CH) and 116.1 (CH 2). This data indicated that the molecule contains a terminal double bond. The two fragments A and B in 119 could easily be identified through COSY spectrum in which olefinic methine ( δ 6.02) correlated with an oxymethine ( δ 4.12, t, J = 7.6 Hz, H-11), which in turn showed cross peak in the same spectrum with another oxymethine at δ 3.63 (1H, dd, J =

1.6, 7.6 Hz, H-10). The H-10 showed COSY correlation with the oxymethine resonating at δ 3.79 (1H, dd, J = 1.6, 8.0 Hz, H-9), which in turn showed

COSY correlation with another signal at δ 4.42 (1H, d, J = 8.0 Hz, H-8).

Similarly, the oxymethine resonating at δ 3.87 (m, H-2) exhibited cross peaks in COSY spectrum with a methylene found at δ 2.41 (m, H2-3) and a doublet methyl at δ 1.22 ( J = 6.0 Hz, H-1) (Fig. 3.13).

Fig. 3.13: Identification of fragment A and B in 119 based on the

COSY correlations

The 13 C NMR spectrum of 119 displayed altogether 13 signals, which were identified as one methyl ( δ 22.5), two methylene ( δ 116.1, 30.0), six methine ( δ 140.2, 73.9, 73.5, 73.2, 67.2 and 64.6) and four quaternary carbons ( δ 78.2, 77.5, 70.6 and 67.0). The shift of quaternary carbons indicated that they could be oxygenated aliphatic C-atoms. The molecular formula afforded only five oxygen atoms, whereas, the NMR data displayed signals for five oxymethines.

This information with the support of the IR data revealed that 119 possess two triple bonds and the four quaternary carbons were attributed to them. The positions of the triple bonds could be fixed due to HMBC information in which H-9 ( δ 3.79) showed long-range correlation with C-7 ( δ

77.5), whereas, H-8 ( δ 4.42) interacted C-7 ( δ 77.5) and C-6 ( δ 70.6). The H-

3 ( δ 2.41) correlated with C-4 ( δ 78.2) and C-5 ( δ 67.0), whereas, H-2 ( δ

3.87) was found to couple with C-4 ( δ 78.2) (Fig. 3.14).

Fig. 3.14: Important HMBC interactions observed in spectra of 119

This is how the two fragments A and B could be connected through two triple bonds. The above data declared 119 as trideca-12-en-4,6-diyne-

2,8,9,10,11-pentaol, which is a new natural product.

Although we could not determine the stereochemistry at various chiral centers but we tried to make a comparison of the optical rotations and stereocenters with the related synthetic analogues (Prasad and Swain,

2011). In the referred paper, the three synthetic compounds in first row (left to right) have β-OH next to olefinic function and have positive sign of optical rotation, whereas, those analogues in the second row with α-OH showed negative sign of optical rotation. The structural difference of 119 with the acetylenes reported in the (Fig. 3.15) is that the natural acetylene

119 has polyhydroxyl functions adjacent to terminal double bond, whereas, the synthetic compounds have polyhydroxyl functions on the other side of acetylenic functions. As 119 showed positive sign of optical rotation, the stereochemistry of hydroxyl group next to olefinic bond in 119 could be β, and others may have the trans-orientation as shown in Fig. 3.15.

Fig. 3.15: Referred synthetic acetylenes

The EIMS of the compound showed the abundance of ions at m/z

254, 180, 163, 120, 91 and 57 due to the fragments as shown in the Fig.

3.16.

Fig. 3.16: Mass fragmentation pattern in 119

3.5.7 Characterization of cholistaquinate (121)

Compound 121 was obtained as white amorphous solid, its EIMS exhibited molecular ion at m/z 530. The HR-EIMS showed molecular ion at m/z 530.1451 corresponding to the O HO 7 OCH3 molecular formula C 26 H26 O12 with 14 OH O 1 5' 3 5 HO 6' 4' 1' O OH double bond equivalence. The IR 2' O O 8' 1'' spectrum was the evident of hydroxyl, 2'' aromatic and carbonyl functions in 4'' OH 5'' 6'' 8'' 121 . The aromatic region of 1H NMR 121 OH spectrum of 121 showed the signals for two trans-double bonds at δ 7.61

(1H, d, J = 16.0 Hz), 7.51 (1H, d, J = 15.5 Hz), 6.30 (1H, d, J = 16.0 Hz) and

6.18 (1H, d, J = 15.5 Hz), in addition, three o-coupled protons were observed at δ 7.02 (2H, d, J = 8.5 Hz), 6.93 (2H, t, J = 8.5 Hz) and 6.75 (2H, d, J = 8.5 Hz) indicating the presence of two 1,2,3,-trisubstituted benzene rings. This data justified two cinnamoyl derived nuclei in 121 , which accommodated 12 double bond equivalences.

Further analysis of the 1H NMR spectrum of 121 showed the presence of a quartet methine at δ 5.53 (1H, J = 5.0 Hz) which showed

COSY correlation with a methylene signal at δ 2.24 (2H, m) and an oxymethine at δ 5.11 (1H, dd, J = 3.0, 8.5 Hz). The oxymethine ( δ 5.11) was found to couple with another oxymethine resonating at δ 4.33 (1H, ddd, J =

3.0, 6.0, 8.5 Hz), which in turn showed COSY correlation with methylene resonating at δ 2.33 (1H, dd, J = 3.0, 14.0 Hz) and 2.07 (1H, dd, J = 6.0,

14.0 Hz) (Fig. 3.17).

Fig. 3.17: Important HMBC and COSY correlation observed in spectra of

121

The downfield shift of the above two oxymethines ( δ 5.53 and 5.11) revealed that two cinnamoyl moieties must be connected at these two centers. This hypothesis was further confirmed through HMBC correlations of these oxymethines with carbonyl carbons at δ 168.7 and 167.8 of the cinnamoyl moieties. A methoxy group resonating at δ 3.71 exhibited HMBC correlation with the most downfield carbon signal at δ 175.6 indicating a methyl carboxylate function in 121 (Fig. 3.17).

The 13 C NMR data was in agreement with the 1H NMR data as it displayed signals for two cinnamoyl moieties ( δ 168.4, 167.8, 149.7, 149.6,

147.7, 147.6, 146.8, 127.6, 127.5, 123.1, 116.5, 115.1, 114.7, 114.5), three oxymethines ( δ 74.8, 69.0 68.5), two methylenes ( δ 38.5, 38.3) and methyl carboxylate function at δ 175.6 and 53.1. In addition, it displayed an oxygenated quaternary carbon at δ 75.5. This data was comparable with the reported data for quinic acid derivatives (Chuda et al., 1996). Therefore, compound 121 must be methyl (3,4-dianthenobiloyl)quinate.

The relative stereochemistry of quinic acid unit was established by

NOESY experiment (Fig. 3.18).

Fig. 3.18: Important NOESY interactions observed in spectrum of

121

In NOESY spectrum, H-2ax showed NOESY correlation with H-6ax .

The big coupling constant (12.5 Hz) between H-4ax and H-3 indicated that

H-3 must be axial and acyl moiety must be equatorial. The cis -confirmation between H-5 and H-4 was established due to smaller coupling constant (3.0

Hz), which could be justified through dihedral angle observed in molecular model. The stereochemistry at C-1 was found to be similar to that of reported analogues of quinic acid with equatorial carboxylate function

(Chuda et al., 1996)

EIMS of the compound showed fragments at m/z 530.14[M] +, 336,

180 and 163. The abundant ions at m/z 180 and 163 supported the presence of two cinnamoyl derived moieties in the molecule (Fig. 3.19).

Fig. 3.19: Mass fragmentation pattern in 121

Based on these informations, compound 121 was finally characterized as methyl (3 β,4 α-dianthenobiloyl)quinate and is named as cholistaquinate which is a new natural product and has never been reported from any natural source.

3.5.8 Characterization of cholistaflaside (123)

Compound 123 was isolated as yellow amorphous solid, whose

− FABMS (-ve mode) showed pseudo -molecular ion at m/z 579 [M-H] . The molecular formula was established OH as C 26 H27 O15 by HR-FABMS with 2''' 1''' 5''' OH O OH thirteen double bond equivalences. 6' 4' OH HO 8 O 1' 6'' 9 1 2' OH The UV data ( λmax 261, 348 nm) was HO 10 O 4 3 OH HO 5'' 2'' 6 5 suggestive of a flavonoid nucleus OH 1'' OH O (Moussaoui et al., 2010b). The 1H 123

NMR spectrum of 123 showed only four signals in aromatic region at δ 7.99

(1H, d, J = 8.5 Hz), 6.93 (1H, d, J = 8.5 Hz), 6.90 (1H, t, J = 8.5 Hz) and

6.62 (1H, s).

The ABC splitting pattern of the first three signals indicated that the ring C is 1,2,3-trisubstituted, while the fourth signal, correlated in HSQC spectrum with a carbon at δ 103.7, was attributed to H-3. This data suggested that ring A in 123 is fully substituted. The 1H NMR spectrum was also evident of two sugar moieties due to two oxymethines resonating at δ 5.03 (1H, d, J = 10.0 Hz) and 4.84 (1H, d, J = 10.0 Hz), correlated to the carbons appeared at δ 75.1 and 76.5, respectively in 13 C NMR spectrum.

Relatively up-field resonance of these two carbon signals revealed that two sugar units could have C-C linkages with aglycon (Feng et al.,

2007). This idea was further supported due to the up field shifts of C-6 ( δ

108.5) and C-8 ( δ 103.8). Other sugar protons displayed their positions between δ 4.80-3.43. The analysis of coupling constants of H-1′′ and H-1′′′ suggested that both the sugar units must be β-anomers (Feng et al., 2007).

Further analysis of various sugar protons and their respective carbons identified both the sugars to be glucose and xylose (Carnat et al., 1998).

All the assignments in sugar units could be made with the help of 1H-

1H COSY, HSQC and HMBC information. The HMBC correlation of H-1′′ (δ

5.03) with C-6 ( δ 108.5) and that of H-1′′′ with C-8 ( δ 103.8) confirmed the attachment of glucose and xylose units at C-6 and C-8, respectively. Other important HMBC interactions observed in the spectrum of 123 have been shown in Fig. 3.20.

Fig. 3.20: Important HMBC and COSY interactions in 123

The FABMS of the compound showed the typical fragmentation pattern of the flavones di-C-glucosides. The pseudo molecular ion peak in the negative mode observed at m/z 579 [M-H] +. The fragments observed at m/z 561, 519, 489 and 459 were observed due to [M-H-18], [M-H-60], [M-

H-90], [M-H-120] respectively, which confirms the presence of C-hexosyl unit in the molecule. While other fragments observed at m/z 535, 405, 475 were due to the [M-H-44], [M-H-74], [M-H-104] which is characteristic of the de-oxyhexosyl unit in the molecule. Other two characteristic fragments

[aglycone + 83] and [aglycone + 113] confirmed that compound is 6,8-di-C- substitued 2',3',5,7-Tetra-hydroxyflavone (Artur et al., 2008).

Fig. 3.21 : Mass fragmentation pattern observed in 123

Above presented evidences were found to be sufficient to establish the structure of 123 as 5,7,2',3'-tetrahydroxy-6-C-β-D-glucopyranosyl-8-C-β-

D-xylopyranosyl flavonoside and is named as cholistaflaside. This compound is also a new addition into the list of natural products. 57

3.5.9 Characterization of 1-hexatriacontanol (109)

Compound 109 was isolated as white amorphous solid. The molecular formula C 36 H74 O was determined through HR-EIMS having a molecular ion peak [M] + at m/z 522.5739, suggested the presence of saturated hydrocarbon without 36 CH3 double bond equivalence. The 2 OH 3 1 IR spectrum showed the 109 absorptions at 2500-3000 cm -1 (O-H), along with the intense bands at 1460

-1 -1 -1 cm (CH 2, CH 3), 1250 cm (O-CH 2), and 730 cm (C-C). The EIMS of 109 showed peaks differing by 14 mass units, characteristic of the aliphatic compounds.

The 1H NMR spectrum of 109 showed a triplet for the terminal methyl at δ 0.89 (3H, J = 6.6 Hz, H-36), a broad singlet centered at δ 1.23

(64H, br s, CH 2-4-32), and an oxymethylene triplet at δ 3.65 (2H, J = 6.7

Hz, H-1), together with the signals for two saturated methylenes at δ 2.31,

1.57 (2H each, m), respectively. The 13 C NMR spectrum of 109 showed the presence of an oxymethylene at δ 63.3, terminal methyl at δ 14.2 and aliphatic methylenes in the range of δ 25.3-39.2.

The length of the aliphatic chain was fixed to be of 32 carbons on the basis of EIMS having a molecular ion peak at M/z 522. Based on these evidences and comparison with the literature values (Zhang et al., 2011 ) 58

109 was confirmed as 1-hexatriacontanol and is isolated for the first time from this plant.

3.5.10 Characterization of elaidic acid (110)

Compound 110 was obtained as colorless amorphous solid. The

EIMS of the compound showed peak at m/z 282 while its exact mass was calculated from HR-EIMS as O 11 3 9 1 OH 18 10 2 282.2664, corresponding to the 110 molecular formula C 18 H34 O2 with two double bond equivalence. In IR spectrum of 110 absorption peaks for carboxylic group (3350-2240, 1705 cm -1) and olefinic bond (1624 cm -1) were observed. The 1H NMR spectrum of

110 displayed the signals at δ 5.30 (2H, dt, J = 5.2, 16.5 Hz, H-9, 10), 1.85-

1.92 (4H, m, H-8,11), 2.30 (2H, t, J = 5.1 Hz, H-2), 0.85 (3H, t, J = 6.6 Hz,

H-18).

The analysis of 13 C NMR spectrum of 110 revealed resonances for one methyl, fourteen methylenes, two methine and one quaternary carbon atoms. This data suggested that 110 is a long chain carboxylic acid with E- double bond. The length of the chain and position of the double bond was fixed by the EIMS based on fragments at m/z 282, 223, 167, 114 and 60.

The above discussion and comparison with the literature values, 110 was found to be elaidic acid (Swern et al., 1952) and reported first time from this source. 59

3.5.11 Characterization of oleanolic acid (111)

Compound 111 was isolated as colorless crystalline solid. The EIMS of 111 showed the molecular ion peak in HR- 29 30

19 2 1 EIMS spectrum at m/z 456.3640 12

25 26 13 17 COOH 28 corresponding to the molecular formula 1 9 15

3 5 7 27 C30 H48 O3 with seven degrees of unsaturation. HO

2 3 24 The IR spectrum of 111 showed peaks for 111

COOH (3410-2650 cm -1), C=O (1710 cm -1) and C=C (1660 cm -1). The EIMS spectrum showed characteristic fragments at m/z 456 248, 208, 203, and

133 indicated the presence of ∆ 12 -amyrin skeleton (Budzikiewicz et al.,

1963).

The 1H NMR spectrum of 111 showed the signals for a trisubstituted double bond at δ 5.49 (1H, t, J = 3.5 Hz, H-12), oxymethine δ 3.44 (1H, dd,

J = 4.2, 9.8 Hz, H-3) and seven methyl singlets at δ 1.30 (3H, s, H-27), 1.24

(3H, s, H-23), 1.04 (3H, s, H-26), 1.02 (6H, s, H-24,30), 0.97 (3H, s, H-29) and 0.93 (3H, s, H-25). The 13 C NMR spectrum of 111 showed the signals for seven methyl, ten methylene, five methine and seven quaternary carbons. The downfield signals at δ 180.0, 144.8, 122.6 and 78.2 assigned to carboxylic acid, double bond and an oxymethine respectively. The above data completely matched with those reported for the oleanolic acid (Ageta and Ageta, 1984).

3.5.12 Characterization of ergosta-7,22-diene-3,5,6-triol (117)

The compound 117 was isolated as colorless crystalline solid and showed positive test for steroid (Galbraith 28

2 1 26 20 24 25 and Horn, 1969). The molecular formula of 1 9 17 27 18 11 13

15 117 was deduced as C 28 H46 O3 from HR- 1 9

3 5 FABMS showing molecular ion peak at m/z HO 6 OH OH 430.3428 have six degree of unsaturation. 117

The 1H NMR spectrum of 117 showed signals for the olefinic protons at δ

5.23 (1H, br s, H-7) and 5.10 (2H, m, H-22,23) and a multiplet for oxymethines at δ 3.90 (1H, H-3) and 3.49 (1H, br s, H-6). It also showed signals for six methyls at δ 0.97 (3H, s, H-19), 0.94 (3H, d, J = 6.4 Hz, H-

21), 0.83 (3H, d, J = 6.4 Hz, H-28), 0.76 (3H, d, J = 6.4 Hz, H-26), 0.74 (3H, d, J = 6.4 Hz, H-27) and 0.51 (3H, s, H-18).

The 13 C NMR spectra (BB and DEPT) of 117 showed a total of 28 signals for six methyl, seven methylene, eleven methine and four quaternary carbon atoms. This data suggested that 117 have ∆ 7-3,5,6-triol skeleton (Arnold et al., 1974). EIMS of 117 showed two prominent peaks at m/z 412 [M-H2O] + and 376 (M-3H 2O). Comparison of the above data with the literature helped to suggest 117 as ergosta-7,22-diene-3,5,6-triol

(Iorizzi et al., 1988) and isolated first time from L. nudicaulis.

3.5.13 Characterization of benzyl glucopyranoside (118 )

Compound 118 was obtained as colorless amorphous powder whose

HR-EIMS exhibited molecular ion peak at m/z OH 4' 6' O 270.1143 and the molecular formula deduced as OH 5' 2' 1 OH 3' 1' O OH 2 C13 H18 O6 with five double bond equivalence. It

4 6 showed absorption maxima in its UV spectrum at 118

214, 254 and 264 nm. The IR spectrum of 118 showed the absorption bands for aromatic, C=C and oxymethylene.

The 1H NMR spectrum of 118 displayed three signals in the aromatic region at δ 7.41 (2H, d, J = 7.5 Hz, H-3,7), 7.33 (2H, t, J = 7.5 Hz, H-4,6) and 7.25 (1H, dd, J = 7.5 Hz, H-5) suggesting the presence of a mono- substituted benzene ring. While the two doublets were observed at 4.93-

4.67 (1H each, d, J = 12.0 Hz, H-1) for an oxymethylene. Moreover, the signals for sugar moiety were also observed at δ 4.35 (1H, d, J = 7.5 Hz, H-

1'), 3.86-3.64 (2H, m, H-6'), 3.65 (1H, m, H-5'), 3.35 (1H, m, H-4'), 3.24 (1H, m, H-3'), 3.21 (1H, m, H-2').

The 13 C NMR (BB and DEPT) spectrum suggested the presence of mono-substituted benzene ring with resonances at δ 139.0, 129.2 129.3 and 128.6, and a sugar moiety at δ 103.2, 78.1, 78.0, 75.1, 71.7, 71.6, and

62.8. From the above discussion and comparison with the literature data

(Iqbal et al., 2004), 118 was confirmed as benzyl glucopyranoside and has been isolated first time from L. nudicaulis .

3.5.14 Characterization of 3,7,12-trihydroxycholan-24-oic acid (120)

Compound 120 was isolated as O 21 20 colorless crystalline solid and showed OH19 24 OH 17

18 11 13 positive test for steroid. The molecular mass H 15 1 9 of 120 was determined from HR-EIMS 3 5 HO 6 OH H which showed molecular ion peak [M] + at 120 m/z 408.2935 correspond to the molecular formula C 24 H40 O5 with five degree of unsaturation. The IR spectrum showed the absorption bands for

O-H (3450 cm -1) and COOH (3330-2250 and 1705 cm -1).

The 1H NMR spectrum of 120 showed signals for oxymethines at δ

3.94 (1H, t, J = 2.5 Hz, H-12), 3.79 (2H, d, J = 3.0 Hz, H-7), 3.38 (1H, m, H-

3), while three methyl groups were observed at δ 1.01 (1H, d, J = 6.5 Hz, H-

21), 0.91 (1H, s, H-19), 0.70 (3H, s, H-18) suggesting the presence of cholestane skeleton (Omkar et al., 2005).

The 13 C NMR (BB and DEPT) spectrum of 120 showed the presence of three methyl, nine methylene, nine methines and three quaternary carbon atoms. The carboxylic carbon and the three oxmethine were observed at δ 178.2 (C, C-24) and at δ 74.0 (CH, C-12), 72.9 (CH, C-3), 69.0

(CH, C-7) respectively and three methyl carbons showed resonances at 21.1

(C-21), 17.6 (C-19) and 12.9 (C-18). This data when matched with the literature (Danielsson et al., 1962) revealed 120 as 3,7,12- trihydroxycholan-24-oic acid which has never been reported from this source.

3.5.15 Characterization of β-sitosterol 3-O-β-D-glucopyranoside (67)

Compound 67 was isolated as white amorphous solid. Its molecular mass was calculated from HR-FABMS showing peak at m/z 576.4686 consistent with the molecular formula C 36 H62 O5 with six double bond equivalence.

The IR spectrum of 67 showed characteristic bands of the hydroxyl group (3454 cm -1), and tri-substituted double bond (3040, 1644, 815 cm -1).

The EIMS of 67 showed the peak at m/z 414 [M-160] + due to the loss of one

29 28 sugar moiety from the 21 22 20 25 26 19 23 molecule. The fragments at m/z 27 1 18 11 13 7 1 OH 1 399, 396, 381, 329 and 275 5 4' 6' 8 5' O 3 5 H 7 H OH 1' 5 2' O indicated the presence of ∆ OH 3' OH 67 sterol skeleton (Wyllie et al., 1977b). The fragments observed at m/z 273 and 255 are as a result of the loss of (M +-side chain) and (M +-side chain,-

H2O), respectively from molecular ion.

The 1H NMR spectrum showed total six methyl signals splitted as a triplet at δ 0.85 (3H, J = 7.0 Hz, H-29), three doublets at δ 0.92 (3H, J = 6.2

Hz, H-21), 0.84 (3H, J = 6.4 Hz, H-26) and 0.82 (3H, J = 6.4 Hz, H-27), and two singlets at 1.01 (3H, H-19), 0.68 (3H, H-18). The carbinylic and olefinic proton were observed at δ 3.84 (1H, m, H-3) and δ 5.12 (1H, br d, J = 5.4 Hz,

H-6). In addition signals for the sugar moiety were observed at δ 5.34 (1H, d, J = 7.2 Hz, H-1') and 3.82-3.43 (4H, m).

The 13 C NMR (BB and DEPT) spectrum of 67 showed the presence 35 carbons atoms for six methyl, eleven methylene, nine methine and three quaternary carbon atoms and in addition five carbons for the sugar moiety which appeared at δ 103.2 (C-1'), 77.7 (C-3'), 77.8 (C-5'), 75.3 (C-2'), 71.3

(C-4'), 62.0 (C-6'). The above data was in close agreement with the literature values for β-sitosterol-3-O-β-D-glucopyranoside (Iribarren and Pomilio,

1983).

3.5.16 Characterization of 20-hydroxyecdesone (122)

Compound 122 was obtained as colorless crystalline solid and showed positive test for steroid. The molecular formula C 27 H44 O7 was deduced from HR-FABMS showing molecular ion peak at m/z 480.3125.

The UV spectrum showed absorption at 240 nm suggesting the presence of enone system while the IR spectrum showed strong absorption band at

3300 cm -1 and 1650 cm -1 indicating the presence of ecdysone (Levina et al.,

2003).

The 1H NMR spectrum of 122 showed the downfield signal at δ 5.79

OH α 26 (1H, br s, H-7) assigned to the proton 21 OH 25 22 19 23 OH

27 to the enone carbonyl. The signals for 17 18 11 13

15 HO 1 9 the methines geminal to the hydroxyl OH 3 5 6 HO δ group were observed at 3.93 (2H, br s, O 122

H-2), 3.83 (1H, d, J = 11.5 Hz, H-3) and 3.33 (1H, d, J = 6.0 Hz, H-22). The five singlet methyls were observed at δ 1.19 (3H, H-21), 1.18 (3H, H-26),

1.17 (3H, H-27), 0.95 (3H, H-19) and 0.88 (3H, H-18).

The 13 C NMR (BB and DEPT) spectra of 122 showed presence of five methyls, eight methylenes, seven methine and seven quaternary carbon atoms. The carbons of the enone system were observed at δ 206 (C-6),

167.9 (C-8), 122.1 (C-7) whereas the methyls were observed at δ 29.7 (C-

26), 28.9 (C-27), 24.4 (C-19), 21.0 (C-21), 18.0 (C-18).

The EIMS of 122 showed the fragments at m/z 462 (M-H2O), 444 (M-

2H 2O), 426 (M-3H 2O). Facile removal of three water molecules suggests the presence of three tertiary hydroxyl groups in the molecule. The relatively abundant ions at m/z 363 indicated the presence of vicinal dihydroxyl group at C-20 and C-22 due to the cleavage at this position. The fragments

66 at m/z 345, 327, 99 and 81 are characteristic of the ecdysone having 20,22 dihydroxy groups (Prakash and Ghosal, 1979), (Ikekawa et al., 1980). From above discussion and comparison with the literature values revealed 122 as

20-hydroxyecdesone (Singh and Thakur, 1982) and never been reported from this source.

3.6 Biological studies

Among the compounds isolated from L. nudicualis, 109 -110, 112 -

117, 119-121 , and 123 were subjected to various biological activities namely DPPH free radical scavenging, AChE, BChE, and LOX enzyme inhibition activities and 67, 111 , 118 and 122 were not checked due to their less amounts. All the compounds were tested at a concentration of

0.5 mMol well -1.

3.6.1 Antioxidant Assay (DPPH Radical Scavenging Method)

Among the compounds 109 , 110 , 112 -117, 119 -121 and 123 checked for DPPH free radical scavenging activity, only a new compound cholistaquinate ( 121 ), displayed significant activity with an IC 50 value of

60.7, whereas all other compounds were inactive (Table 3.1). Quercetin was used as a positive control at concentration 0.5 mMwell -1 for control as well as test compounds.

Table 3.1: Antioxidant Assay (DPPH Radical Scavenging Activity)

Compound (%) at (IC 50 ) 0.5 mM µM 109 23.9±0.2 NIL 110 12.6±0.5 NIL 112 4.30±0.65 NIL 113 2.76±0.43 NIL 114 5.41±0.79 NIL 115 1.78±0.87 NIL 116 13.6±0.6 NIL 117 2.68±0.42 NIL 119 21.9±0.1 NIL 120 2.16±0.78 NIL 121 92.48±2.54 60.73 123 30.87±0.2 NIL Quercetin 93.21±0.97 16.96±0.14

3.6.2 Enzyme Inhibition Activities

3.6.2.1 Acetylcholinesterase Enzyme Inhibition Activity

In acetylcholinesterase enzyme inhibition assay, all the compounds

109 , 110 , 112 -117, 119 -121 and 123 were tested. The two new compounds cholistaquinate (121) and cholistaflaside ( 123) exhibited minor activity while others were inactive against this enzyme (Table 3.2). Eserine

(0.5 mM well -1) was used as positive control.

Table 3.2: Acetylcholinesterase enzyme inhibition activity

Compound (%) at (IC 50 ) 0.5 mM µM 109 38.52±0.52 NIL 110 10.5±0.3 NIL 112 6.88±0.74 NIL 113 7.56±0.96 NIL 114 9.32±0.69 NIL 115 23.13±0.72 NIL 116 30.9±0.2 NIL 117 5.38±0.98 NIL 119 23.5±0.2 NIL 120 13.88±0.72 NIL 121 59.68±1.21 >200 123 69.33±1.31 >300 Eserine 91.29±1.17 0.04±0.0001

3.6.2.2 Butyrylcholinesterase Enzyme Inhibition Activity

Compounds 109 , 110 , 112 -117, 119 -121 and 123 were subjected to butyrylcholinesterase enzyme inhibition activity. Among all, nudicholoid

(116 ) displayed significant activity with 94.1% inhibition with corresponding IC 50 value of 88.3. The compounds 119 and 121 also showed minor activities while 109 , 112-115 , 117 , 120 and 123 were inactive (Table 3.3).

Table 3.3: Butyrylcholinesterase enzyme inhibition activity

Compound (%) at (IC 50 ) 0.5 mM µM 109 32.11±0.52 NIL 110 12.9±0.4 NIL 112 18.73±0.95 NIL 113 20.01±0.52 NIL 114 13.38±0.45 NIL 115 17.57±0.39 NIL 116 94.10±2.13 88.34±0.11 117 10.34±0.88 NIL 119 58.61±0.18 <400 120 9.26±0.69 NIL 121 48.57±1.78 >200 123 15.68±0.5 NIL Eserine 82.82±1.09 0.85±0.0001

3.6.2.3 Lipoxygenase Enzyme Inhibition Activity

In lipoxygenase enzyme inhibition essay, the four new sphingolipids nudicualin A ( 112) , nudicualin B ( 113) , nudicualid D ( 114) and nudicualin

C ( 115) showed significant activities, while other compounds 109 , 110 ,

116 , 117 , 119 -121 , and 123 were inactive against this enzyme. Baicalein

(0.5 mM well -1) was used as a positive control (Table 3.4).

Table 4.4: Lipoxygenase enzyme inhibition activity

Compound (%) at (IC 50 ) 0.5 mM µM 109 5.4±0.1 NIL 110 12.8±0.5 NIL 112 54.13±1.49 193±1.11 113 73.72±1.23 105±1.34 114 60.10±1.74 163±1.25 115 75.14±1.56 103±1.74 116 10.18±0.6 NIL 117 2.40±0.89 NIL 119 17.40±0.8 NIL 120 32.91±1.05 NIL 121 8.97±0.5 NIL 123 6.0±0.4 NIL Baicalein 93.79±1.2 22.4±1.3

3.7 Experimental

3.7.1 General Experimental Notes

Commercially available solvents i.e. n-hexane, chloroform, ethyl acetate and methanol, were used after distillation for column and thin layer chromatography.

Chromatography

Flash silica gel (230-400 mesh, E-Merk) was used for column chromatography. For preparative thin layer chromatography, precoated silica gel preparative plates (20×20 cm, 0.5 mm thick) were used. Sample purity was checked on the same plates. Ceric sulphate and iodine were used as locating reagents.

Physical Measurements

Buchi 535 melting point apparatus was used for the determination of melting points of the samples. Optical rotations were measured on JASCO

DIP-360 (Japan Spectroscopic Co. Ltd., Tokyo, Japan) digital polarimeter.

Spectroscopy

UV, IR and NMR techniques were used for the identification and characterization of organic compounds. UV spectra were recorded on

Hitachi UV-3200 spectrophotometer. IR spectra were recorded on JASCO A- 72

302 infrared spectrophotometer. Proton nuclear magnetic resonance ( 1H

NMR) spectra were scanned on a bruker AM 300 FT NMR, AM 400 FT NMR,

AM 500 FT NMR while TMS was used as internal standard. The 13 C NMR spectra were recorded at 75, 100 and 125 MHz on the same instruments.

The 2D NMR (HMQC, HMBC, COSY and NOESY) spectra were scanned at 300, 400 and 500 MHz on the same instruments. The chemical shift values ( δ) are reported in ppm and the coupling constant ( J) are in

Hertz. The mass spectra were recorded on variant-MAT 112S and

Finningan MAT-112 and 312A double focusing mass spectrometers connected to DEC PDP 11/34 and IBM AT compatible PC based system respectively. Electron impact, peak matching, field desorption and fast atom bombardment experiments were performed on a MAT-312A or a Jeol-

JMS HX-110 mass spectrometers. High resolution electron impact mass measurements and fast atomic bombardment (FAB) measurements were carried out on Jeol-JMS HX-110 mass spectrometers using glycerol and thioglycerol as the matrix and cesium iodide (CsI) as internal standard for accurate mass measurements.

3.7.2 Plant Material

The whole plant material of L. nudicaulis Hook was collected from

Cholistan Desert, District Bahawalpur (Punjab), Pakistan in April 2008. It was identified by Dr. Muhammad Arshad (Late), Plant Taxonomist, 73

Cholistan Institute for Desert Studies (CIDS), The Islamia University of

Bahawalpur, where a voucher specimen is deposited (0022-LN/CIDS/08).

3.7.3 Extraction and Isolation

The whole plant of L. nudicaulis (26 kg) was shade dried ground and extracted with methanol. The methanolic extract was evaporated under vacuum to a dark greenish mass (1.2 kg) which was suspended in water and extracted with n-hexane and ethyl acetate. The n-hexane soluble fraction (150 g) was subjected to column chromatography over silica gel eluting with n-hexane, n-hexane-dichloromethane, dichloromethane, dichloromethane-methanol and methanol in increasing order of polarity to give seven fractions (1-7).

The fraction 1 obtained from n-hexane-DCM (8.5:1.5) was a binary mixture which on purification using n-hexane-DCM (8.4:1.6) as eluent yielded 1-hexatriacontanol ( 109 ) from the head and elaidic acid ( 110 ) from the tail fraction. The fraction 2 obtained by n- hexane-DCM (6.8:3.2), showed a single spots on TLC, and purified using n- hexane-DCM (6.4:3.6) to get oleanolic acid ( 111 ). The fractions 3 obtained from pure DCM were subjected to column chromatography eluted with DCM-MeOH (9.8:0.2) provided nudicaulin A (112 ). The fractions 4 collected at DCM-MeOH

(9.7:0.3) further subjected to silica gel column chromatography using DCM-

MeOH (9.5:0.5) as eluent to get β-sitosterol 3-O-β-D-glucopyranoside ( 67 ). 74

The fraction 5 was collected at DCM-MeOH (9.4:0.6) subjected to column chromatography using same solvent system yielded nudicaulin B ( 113 ), whereas the fractions 6 obtained from DCM-MeOH (9.3:0.7) was chromatographed on silica gel using solvent system dichloromethane- methanol (9.2:0.8) provided nudicaulin D (114 ). The fractions 7 obtained from DCM-MeOH (9.1:0.9) were further purified using solvent system DCM-

MeOH (9:1) yielded nudicaulin C (115 ).

The ethyl acetate fraction (250 g) was subjected to silica gel column chromatography eluting with n-hexane, n-hexane-dichloromethane, dichloromethane, dichloromethane-methanol and methanol in increasing order of polarity. As a result six fractions (1-6) were obtained. Fraction 1 obtained at 100% DCM showed single spot on TLC and subjected to column chromatography using n- hexane-DCM (0.2:9.8) and provided nudicholoid

(116 ). Fraction 2 obtained at DCM-MeOH (9.7:0.3) when further subjected to column chromatography using solvent DCM-MeOH (9.6:0.4) yielded

Ergosta-7,22-diene-3,5,6-triol ( 117 ) from head fraction and benzyl glucopyranoside ( 118 ) from the tail fractions.

Fraction 3 obtained at DCM-MeOH (9.5:0.5) on further column chromatography using DCM-MeOH (9.4:0.6) provided trideca-12-en-4,6- diyne-2,8,9,10,11-pentaol ( 119 ). Fraction 4 collected at DCM-MeOH

(9.3:0.7) showed two spots on TLC which on further purification by column

75 chromatography using DCM-MeOH (9.2:0.8) yielded 3,7,12- trihydroxycholan-24-oic acid ( 120 ) and cholistaquinate ( 121 ) one after the other. Fraction 5 obtained at DCM-MeOH (9.1:0.9) showed single spot on

TLC and on further purification at DCM-MeOH (9.0:1.0) provided 20- hydroxyecdysone ( 122 ). Fraction 6 obtained at DCM-MeOH (8.5:1.5) was further subjected to repeated column chromatography, using the same solvent system as eluent yielded cholistaflaside ( 123 ).

Launaea nudicualis (26 kg)

Finally ground, extracted with methanol and concentrated to dry mass Methanolic extract (1.2 kg)

n-Hexane Water extract extract

subjected to silicagel column chromatography using n-hex,DCM and methanol Aqueous Ethyl acetate fraction soluble

Fr. 1 Fr. 2 Fr. 3 Fr. 4 Fr. 5 Fr. 6 Fr. 7 n-hex/DCM n-hex/DCM DCM DCM/MeOH DCM/MeOH DCM/MeOH DCM/MeOH (8.5:1.5) (6.8:3.2) (100%) (9.7:0.3) (9.4:0.6) (9.3:0.7) (9.1:0.9)

n-hex/DCM n-hex/DCM n-hex/DCM n-hex/DCM n-hex/DCM n-hex/DCM n-hex/DCM (8.4:1.6) (6.4:3.6) (9.8:0.2) (9.5:0.5) (9.4:0.6) (9.2:0.8) (9:1)

1-Hexatriacontanol (109) Nudicaulin A Nudicualin B Nudicaulin C & Elaidic acid (110) (112) (113) (115)

sitosterol 3-O- -D- Nudicualin D Oleanolic acid glucopyranoside (67) (114) (111)

subjected to silicagel column chromatography using n-hex,DCM and methanol

Fr. 1 Fr. 2 Fr. 3 Fr. 4 Fr. 5 Fr. 6 DCM DCM/MeOH DCM/MeOH DCM/MeOH DCM/MeOH DCM/MeOH (100%) (9.7:0.3) (9.5:0.5) (9.3:0.7) (9.1:0.9) (8.5:1.5)

DCM/MeOH DCM/MeOH DCM/MeOH DCM/MeOH DCM/MeOH (0.2:9.8) DCM/MeOH (9.6:0.4) (9.4:0.6) (9.2:0.8) (9.0:1.0) (8.5:1.5)

nudicholoid Trideca-12-en-4,6-diyne- 20-hydroxyecdesone (116) 2,8,9,10,11-pentaol (119) (122)

Ergosta-7,22-diene- 3,7,12-Trihydroxycholan- Cholistaflaside 3,5,6-triol (117) 24-oic acid (120)& (123) Benzyl glucopyranoside Cholistaquinate (121) (118)

Scheme 3.1: Protocol for the isolation of compounds 67 and 109-123 from methanolic extract of L. nudicaulis. 77

3.7.4 Experimental Data of Isolated Compounds

3.7.4.1 Nudicholoid (116)

ααα 25 4' Crystals from ethylacetate (25 mg); [ ]D : H C14 CH O 3 3 OH 10 9 O 1' 2' -1 2 1 3' +28.7 (c = 0.21, MeOH) ; IR (KBr) ν max cm : 3 8 5 H 7 O 4 H 6 H 15 H 11 3445, 3110, 2940, 1765, 1685, 1615, 605; HO 12 O CH3 13 1 O H NMR (CD 3OD, 500MHz) δ: 6.41 (1H, d, J 116 = 1.0 Hz, H-3), 4.85 (1H, m, H-8), 4.82 (1H, dd, J = 1.0, 18.0 Hz, Ha-15), 4.40 (1H, d, J = 18.0 Hz, Hb-15), 3.81 (1H, t, J

= 10.0 Hz, H-6), 3.76 (1H, d, J = 10.0 Hz, H-5), 3.70 (1H, dd, J = 7.5, 11.0

Hz, Ha-3'), 3.63 (1H, dd, J = 5.6, 11.0 Hz, Hb-3'), 2.84 (1H, d, J = 10.5, 13.0

Hz, Ha-9), 2.66 (1H, m, H-11), 2.65 (1H, m, H-2'), 2.51 (1H, q, J = 10.0 Hz,

H-7), 2.42 (3H, s, H-14), 2.39 (1H, dd, J = 1.5, 13.0 Hz, Hb-9), 1.45 (3H, d,

13 J = 7.0 Hz, H-13), 1.16 (3H, d, J = 7.0 Hz, H-4'); C NMR (CD 3OD,

125MHz) δ: 197.0 (C-2), 179.1 (C-12), 176.4 (C-4), 175.7 (C-1'), 148.4 (C-

10), 134.2 (C-1), 133.3 (C-3), 82.1 (C-6), 72.0 (C-8), 64.9 (C-3'), 63.1 (C-15),

59.2 (C-7), 49.7 (C-5), 45.3 (C-9), 43.9 (C-2'), 41.8 (C-11), 21.4 (C-14), 15.4

(C-13), 13.9 (C-4'); HR-EIMS : m/z 364.1522 [M] + (calcd. for C 19 H24 O7,

364.1520); EIMS : m/z 364 [M] +.

3.7.4.2 Nudicaulin A (112)

O White amorphous solid

18' HN 1' CH 2' 3 (39 mg); M. P.: 106- OH 14 HO 108 0C; [ααα]D24 : +16.9 ( c 2 3 4 1 15 OH H3C 23 = 0. 2, MeOH); IR (KBr) 112 24

νmax cm -1: 3640, 2910, 1650, 1625; 1H NMR (CDCl 3, 400MHz) δ: 6.75 (1H, d,

J = 8.0 Hz, N-H), 5.31 ( 2H, dt, J = 5.2, 16.5 Hz, H-14,15), 3.97 (1H, m, H-

2), 3.94 (1H, dd, J = 3.5, 11.5 Hz, Ha-1), 3.57 (1H, dd, J = 5.5, 11.5 Hz, Hb-

1), 3.47 (1H, dt, J = 5.5, 11.5 Hz, H-4), 3.43 (1H, dd, J = 4.1, 4.5 Hz, H-3),

2.10 (2H, t, J = 8.0 Hz, H-2'), 1.93-1.86 (4H, m, H-13,16), 1.56, 1.32 (each

H, m, H2-5), 1.51 (2H, m, H-3'), 1.30-1.15 (56H, brs, H- 6-12, 17-23, 4'-

17'), 0.79 (6H, t, J = 7.0 Hz, H-18',24); 13 C NMR (CDCl 3, 100MHz) δ: 174.4

(C1'), 130.6 (C-14 or 15), 129.6 (C-14 or 15), 75.7 (C-3), 72.4 (C-4), 60.9 (C-

1), 51.9 (C-2), 36.4 (C-2'), 32.6 (C-5), 32.4 (C-13,16), 31.5-29.2 (C- 6-12,

17-23, 4'-17'), 25.7 (C-3'), 13.9 (C-18',24); HR-EIMS: m/z 665.6330 (calcd. for C 42 H83 O4, 665.6322); HR-FABMS [M+H] +: m/z 666.6405; EIMS: m/z

665, 552, 498, 441, 386, 384, 356, 339, 282, 268.

Methyl ester derived from 112

1 H NMR (CDCl 3, 400MHz) δ: 3.51 (3H, s, OCH 3), 2.32 (2H, t, J = 6.5 Hz, H-

2'), 1.51 (2H, m, H-3'), 1.19–1.28 (28H, br s, CH 2-4'-17'), 0.84 (3H, t, J = 6.8

Hz, CH 3-18'); GC-MS: m/z 298. 79

Acetylsphingamine derived from 112

[ααα]D24 : +11.2 ( c = 0. 01, MeOH); 1H NMR (CDCl 3, 400MHz) δ: 8.06 (1H, d, J

= 7.6 Hz, NH), 5.31 (2H, dt, J = 5.2, 15.1 Hz, H-14,15), 4.56 (1H, dd, J =

4.3, 5.1 Hz, H-4), 4.50 (1H, m, H-2), 4.42 (1H, dd, J = 5.6, 10.3, Hz, Hb-1),

4.33 (1H, dd, J = 3.3, 10.3 Hz, Hb-1), 4.17 (1H, dd, J = 3.2, 5.1 Hz, H-3),

2.00 (6H, 2 x CH 3CO), 1.98 (6H, 2 x CH 3CO), 1.13–1.21 (26H, brs, CH 2-7-

12, 17-23), 0.89 (3H, t, J = 6.5 Hz, CH 3-24); GC-MS : m/z 567.

3.7.4.3 Nudicualin B (113)

O White amorphous OH 18' 1' CH HN 2' 3 solid (35 mg); M. P.: OH 14 HO 0 24 2 3 4 114-116 C; [ααα]D : + 1 15

OH H3C 23 24 39.7 ( c =1.5 MeOH); 113

-1 1 IR (KBr) νmax cm : 3334, 3215, 2919, 1651, 1622, 1602; H NMR (CDCl 3,

400MHz) δ: 7.42 (1H, d, J = 8.8 Hz, NH), 5.29 (2H, dt, J = 6.8, 15.3 Hz, H-

14,15), 3.99 (1H, m, H-2), 3.94 (1H, dd, J = 3.6, 6.4 Hz, H-2'), 3.71 (1H, dd,

J = 4.0, 11.6 Hz, Ha-1), 3.63 (1H, dd, J = 4.0, 11.6 Hz, Hb-1), 3.45 (1H, dd,

J = 3.9, 4.2 Hz, H-3), 3.41 (1H, dt, J = 4.2, 6.3 Hz, H-4), 1.90 (4H, m, H-

13,16), 1.59 (2H, m, H-5), 1.52 (2H, m, H-3'), 1.29-1.15 (36H, brs, H-6-12,

17-23,14'-17'), 0.77 (6H, t, J = 6.4 Hz, H-18',24); 13 C NMR (CDCl 3,

125MHz) δ: 175.7 (C-1'), 130.6 (C-14 or 15), 129.7 (C-14 or 15), 75.5 (C-3),

72.1 (C-4), 71.9 (C-2'), 61.0 (C-1), 51.5 (C-2), 32.8 (C-5), 32.5 (C-13,16), 80

32.3 (C-3'), 31.3-28.5 (6-12, 17-23, 4'-17'), 13.9 (C-18',24); HR-EIMS: m/z

681.6280 (calcd. for C 42 H83 O5, 681.6271); HR-FABMS [M+H] +: m/z

682.6355; EIMS: m/z 663 [M-H2O] +, 456, 398, 383, 339, 283, 279, 167,

113.

Methyl ester derived from 113

24 1 [ααα]D : – 7.1 ( c = 0.01 MeOH); H NMR (CDCl 3, 400MHz) δ: 4.12 (1H, t, J =

6.9 Hz, H-2'), 3.53 (3H, s, MeO), 1.98 (3H, s, MeCO), 1.17–1.27 (28H, brs,

CH 2-4'-17'), 0.83 (3H, t, J = 6.7 Hz, CH 3-18'); GC-MS: m/z 356.

Acetylsphingamine derived from 113

[ααα]D24 : + 19.1 ( c = 0.015, MeOH); 1H NMR (CDCl 3, 400MHz) δ: 7.96 (1H, d, J

= 7.9 Hz, NH), 5.33 (2H, dt, J = 5.5, 15.5 Hz, H-14,15), 4.66 (1H, dd, J =

4.1, 5.0 Hz, H-4), 4.55 (1H, m, H-2), 4.44 (1H, dd, J = 5.4, 10.2 Hz, Ha-1),

4.33 (1H, dd, J = 3.1, 10.2 Hz, Hb-1), 4.19 (1H, dd, J = 3.1, 5.0 Hz, H-3),

2.00 (12H, 4 x MeCO), 1.15–1.25 (26H, brs, CH 2-7-12, 17-23), 0.85 (3H, t, J

= 6.6 Hz, CH 3-24); GC-MS: m/z 567.

3.7.4.4 Nudicualin D (114)

Colorless shiny powder O OH 13' OH 20' 1' 3' CH HN 2' 12' 3 (57 mg); M. P.: 139- OH 18 HO CH 0 ααα 24 1 2 3 4 3 141 C; [ ]D : + 29.9 ( c =

OH 114 0.22, MeOH); IR (KBr)

νmax cm -1: 3440, 3296, 2914, 1651, 1616; 1H NMR (CDCl 3, 400MHz) δ: 7.42

(1H, d, J = 8.8 Hz, NH), 5.21 (2H, dt, H-12',13'), 3.94 (1H, m, H-2), 3.82

(1H, d, J = 4.3 Hz, H-2'), 3.62 (1H, dt, J = 4.3, 6.2 Hz, H-3'), 3.60 (1H, dd, J

= 4.8, 11.6 Hz, Ha-1), 3.54 (1H, dd, J = 3.2, 11.6 Hz, Hb-1), 3.35 (1H, dd, J

= 4.1, 4.8 Hz, H-3), 3.31 (1H, dt, J = 4.8, 5.1 Hz, H-4), 1.81 (4H, m, H-11',

14'), 1.58 (2H, m, H-4'), 1.52 (2H, m, H-5), 1.06 (44H, brs, H- 6-17, 5'-10',

15'-19'), 0.68 (6H, t, J = 6.4 Hz, H-18',24); 13 C NMR: (CDCl 3, 100MHz) δ:

174.8 (C1'), 130.5 (C-12' or 13'), 129.6 (C-12' or 13'), 75.2 (C-3), 74.3 (C-2'),

72.8 (C-3'), 72.0 (C-4), 60.8 (C-1), 51.7 (C-2), 32.9 (C-5), 32.3 (C-11',14'),

31.6 (C-4'), 29.4-22.4 (6-17, 5'-10', 15'-19'), 13.7 (C-18',24); HR-EIMS: m/z

641.5682 (calcd. for C 38 H75 NO 6, 641.5594); HR-FABMS: [M+H] +: m/z

642.5680; EIMS: m/z 456, 398, 383, 456, 339, 283, 279, 167, 113.

Methyl ester derived from 114

24 1 [ααα]D : + 9.6 ( c = 0.012 MeOH); H NMR (CDCl 3, 400MHz) δ: 5.28 (2H, dt, J

= 5.6, 16.0 Hz, H-12,13), 4.19 (1H, d, J = 4.3 Hz, H-2'), 4.11 (1H, dt, J =

4.3, 6.1 Hz, H-3'), 3.50 (3H, s, MeO), 2.00 (6H, s, 2 x MeCO), 1.15–1.25 82

(22H, brs, H- 5'-10', 15'-19'), 0.90 (3H, t, J = 6.5 Hz, CH 3-20'); GC-MS: m/z

440.

Acetylsphingamine derived from 114

[ααα]D24 : + 16.9 ( c = 0.012 MeOH); 1H NMR (CDCl 3, 400MHz) δ: 8.16 (1H, d, J

= 7.9 Hz, NH), 4.56 (1H, dd, J = 3.9, 5.2 Hz, H-4), 4.50 (1H, m, H-2), 4.39

(1H, dd, J = 5.1, 11.0 Hz, Ha-1), 4.30 (1H, dd, J = 3.0, 11.0 Hz, Hb-1), 4.15

(1H, dd, J = 3.0, 5.2 Hz, H-3), 2.00 (12H, 4 x MeCO), 1.19–1.30 (24H, brs,

H-6-17), 0.86 (3H, t, J = 6.5 Hz, CH 3-18); GC-MS: m/z 485.

3.7.4.5 Nudicaulin C (115)

Colorless gummy O OH 17' 1' CH3 2' solid (44mg); HN OH 6'' OH 4'' O 14 ααα 24 HO 5'' 2'' O [ ]D : + 32.1 ( c = HO 1 2 3 4 3'' 1'' 15 OH 26 OH H3C 0.12, MeOH); IR 115

(KBr) νmax cm -1: 3346, 3242, 2910, 1650, 1624, 1606; 1H NMR (CDCl 3

400MHz) δ: 7.48 (1H, d, J = 8.8 Hz, NH), 5.26 (2H, dt, J = 5.4, 17.3 Hz, H-

14,15), 4.09 (1H, m, H-2), 3.92 (1H, dd, J = 4.8, 11.3 Hz, Ha-1), 3.71 (1H, dd, J = 3.4, 11.3 Hz, Hb-1), 3.88 (1H, t, J = 6.9 Hz, H-2'), 3.43 (1H, dt, J =

4.8, 5.8 Hz, H-4), 3.12 (1H, dd, J = 3.8, 4.8 Hz, H-3), 1.86 (4H, m, H-13,16),

1.64, (2H, m, H-3'), 1.52 (2H, m, H-5), 1.28-1.12 (58H, br s, H- 6-12, 17-25,

4'-16'), 0.77 (6H, t, J = 6.4 Hz, H-17',26); Glucose moiety δ: 4.15 (1H, d, J =

7.6, H-1''), 3.79 (1H, dd, J = 4.9, 10.8 Hz, Ha-6''), 3.61 (1H, dd, J = 2.9, 10.8

Hz, Hb-6''), 3.40 (1H, m, H-2''), 3.28 (1H, t, J = 7.4 Hz, H-3''), 3.23 (1H, t, J

13 = 7.4 Hz, H-4''), 3.16 (1H, m, H-5''); C NMR (CDCl 3, 100MHz) δ: 175.7 (C-

1'), 130.6 (C-14 or 15), 129.7 (C-14 or 15), 76.1 (C-3), 74.2 (C-4), 71.9 (C-

2'), 68.5 (C-1), 51.5 (C-2), 34.2 (C-3'), 32.1 (C-5), 32.4 (C-13,16), 30.9-28.1

(C- 6-12, 17-25, 4'-16'), 13.9 (C-18', 24); Glucose moiety δ: 102.8 (C1''),

76.5 (C-5''), 76.1 (C-3''), 73.1 (C-2''), 69.7 (C-4''), 61.2 (C-6''); HR-FABMS

[M-H] -: m/z 855.6877 (calcd. for C 49 H94 NO 10 , 856.6877); EIMS: 694, 662,

394, 367, 307, 269, 141.

Methyl ester derived from 115

24 1 [ααα]D : -11.2 ( c = 0.011 MeOH); H NMR (CDCl 3, 400MHz) δ: 4.15 (1H, t, J =

6.6 Hz, H-2'), 3.56 (3H, s, CH 3O), 1.99 (3H, s, CH 3CO), 1.18–1.29 (26H, brs,

H-4'-16'), 0.86 (3H, t, J = 6.9 Hz, H-17'); GC-MS: m/z 342.

Acetylsphingamine derived from 115

[ααα]D24 : + 19.1 ( c = 0.013 MeOH); 1H NMR (CDCl 3, 400MHz) δ: 7.90 (1H, d, J

= 8.2 Hz, NH), 5.29 (2H, dt, J = 5.8, 16.1 Hz, H-14,15), 4.60 (1H, dd, J =

4.0, 5.1 Hz, H-4), 4.52 (1H, m, H-2), 4.40 (1H, dd, J = 5.3, 10.6 Hz, Ha-1),

4.30 (1H, dd, J = 3.0, 10.6 Hz, Hb-1), 4.16 (1H, dd, J = 4.0, 5.1 Hz, H-3),

2.01 (12H, 4 x MeCO), 1.16–1.26 (30H, brs, H-7-12, 17-25), 0.87 (3H, t, J =

6.2 Hz, H-26); GC-MS: m/z 595.

3.7.4.6 Trideca-12-en-4,6-diyne-2,8,9,10,11-pentaol (119)

White amorphous solid (10 mg); [ααα]D25 : OH OH H3C 1 13 OH 7 4 H C +7.2 (c = 0.82, MeOH); IR (KBr) νmax 2 3 6 5 OH OH -1 119 cm : 3390, 3080, 2950, 2240, 2150,

1645, 1490; 1H NMR (CD 3OD, 500MHz) δ: 6.02 (1H, ddd, J = 5.6, 10.4,16.8

Hz, H-12), 5.34 (1H, dt, J = 1.6,17.2 Hz, Ha -13), 5.19 (1H, dt, J = 1.6, 10.4

Hz, Hb-13), 4.42 (1H, d, J = 8.0 Hz, H-8), 4.12 (1H, t, J = 7.6 Hz, H-11),

3.87 (1H, m, H-2), 3.79 (1H, dd, J = 1.6, 8.0 Hz, H-9), 3.63 (1H, dd, J = 1.6 ,

7.6 Hz, H-10), 2.41 (2H, m, H-2), 1.22 (3H, d, J = 6.0 Hz, H-1); 13 C NMR

(CD 3OD, 125MHz) δ: 140.2 (C-12), 116.1 (C-13), 78.2 (C-4), 77.5 (C-7), 73.9

(C-11), 73.5 (C-9), 73.2 (C-10), 70.6 (C-6), 67.2 (C-2), 67.0 (C-5), 64.6 (C-8),

30.0 (C-3), 22.5 (C-1); HR-EIMS: m/z 254.1128 [M] + (calcd. for C 13 H18 O5,

254.1154); EIMS: m/z 254 [M] +, 236, 195, 171, 147, 117, 87.

3.7.4.7 Cholistaquinate (121)

ααα 25 O White amorphous solid (11 mg); [ ]D : HO 7 OCH 3 -257 (c = 0.27, MeOH); IR (KBr) ν OH O 1 max 5' 3 5 HO 6' 4' 1' O OH -1 2' cm : 3430, 1730, 1675, 1600-1550; O O 8' 1'' 1 2'' H NMR (CD 3OD, 500MHz) δ: 7.61

4'' OH (1H, d, J = 16.0 Hz, H-3''), 7.51 (1H, d, 5'' 6'' 8'' 121 OH J = 15.5 Hz, H-3'), 7.02 (2H, d, J = 8.5

Hz, H-9', 9''), 6.93 (2H, t, J = 8.5 Hz, H-8', 8''), 6.75 (2H, d, J = 8.5 Hz, H-7', 85

7''), 6.30 (1H, d, J = 16.0 Hz, H-2''), 6.18 (1H, d, J = 15.5 Hz, H-2'), 5.53

(1H, q, J = 7.5 Hz, H-3), 5.11 (1H, dd, J = 3.0, 8.5 Hz, H-4), 4.33 (1H, ddd, J

= 3.0, 6.0, 8.5 Hz, H-5), 3.71 (3H, s, OCH 3), 2.33 (1H, dd, J = 3.0, 14.0 Hz,

Ha-6), 2.07 (1H, dd, J = 6.0, 14.0 Hz, Hb-6), 2.26 (1H, dd, J = 8.5, 13.5 Hz,

Ha-2), 2.14 (1H, dd, J = 7.0, 8.5 Hz, Hb-2); 13 C NMR: (CD 3OD, 125MHz) δ:

175.1 (C-7), 168.4 (C-1''), 167.8 (C-1'), 149.7 (C-5'), 149.6 (C-5''), 147.7 (C-

3''), 147.6 (C-3'), 146.8 (C-6',6''), 127.6 (C-4''), 127.5 (C-4'), 123.1 (C-9',9''),

116.5 (C-7',7''), 115.1 (C-8',8''), 114.7 (C-2''), 114.5 (C-2'), 75.7 (C-1), 74.8

(C-4), 69.0 (C-3), 68.5 (C-5), 53.1 (OCH 3), 38.5 (CH 2, C-6), 38.3 (CH 2, C-2);

HR-EIMS : m/z 530.1451 [M] + (calcd. for C 26 H26 O12 , 530.1424); EIMS: [M] + m/z 530, 336, 180, 163.

3.7.4.8 Cholistaflaside (123)

Yellow amorphous solid (10 mg); OH

2''' [ααα]D20 : -25.3 (c = 0.46, MeOH); UV 1''' 5''' OH O OH 6' 4' (MeOH) λmax (log ε) nm: 261 (4.16), OH HO 8 O 1 1' HO 6'' 9 2' OH -1 10 348 (4.45); IR (KBr) νmax cm : O 4 3 OH HO 5'' 2'' 6 5

OH 1'' OH O 3445, 3310, 1666, 1602, 1568,

123 1 1506,1456; H NMR (CD 3OD,

500MHz) δ: 7.99 (1H, d, J = 8.5 Hz, H-6′), 6.93 (1H, d, J = 8.5 Hz, H-4′),

6.90 (1H, t, J = 8.5 Hz, H-5′), 6.62 (1H, s, H-3), 5.03 (1H, d, J = 10.0 Hz, H-

1′′), 4.95 (1H, m, H-2′′′), 4.84 (1H, d, J = 10.0 Hz, H-1′′′), 4.01 (2H, m, H-

2′′,4 ′′′), 4.07 (1H, m, H-3′′′), 3.92 (1H, dd, J = 5.3, 12.0 Hz, Ha -6′′), 3.85

(1H, dd, J = 2.0, 12.0, Hz, Hb -6′′), 3.72, 3.62 (each H, m, H2-5′′′), 3.44 (1H, m, H-5′′), 3.43 (2H, m, H-3′′), 3.42 (2H, m, H-4′′); 13 C NMR (CD 3OD,

125MHz) δ: 184.2 (C-4), 167.2 (C-5), 163.1 (C-7), 162.5 (C-2), 151.5 (C-9),

145.4 (C-2′), 144.8 (C-3′), 130.2 (C-6′), 123.4 (C-1′), 117.1 (C-5′), 117.0 (C-

4′), 108.5 (C-6), 105.8 (C-10), 103.8 (C-8), 103.7 (C-3), 83.0 (C-5′′), 82.0 (C-

3′′), 79.1 (C-3′′′), 76.5 (C-1′′′), 75.3 (C-2′′′), 75.1 (C-1′′), 73.1 (C-2′′), 71.1 (C-

˗ 4′′), 70.4 (C-4′′′), 70.0 (C-5′′′), 63.1 (C-6′′); HR-FABMS [M-H] : m/z 579.1360

˗ (calcd. for C 26 H28 O17 , 579.1349); FABMS [M-H] : m/z 579

3.7.4.9 1-Hexatriacontanol (109)

36 White amorphous solid (65 mg); CH3 0 -1 2 M. P.: 89 C; IR (KBr) νmax cm : OH 3 1 109 3000-2500, 2850, 1635, 1460,

1250, 730; 1H NMR (CDCl 3, 400MHz) δ: 3.65 (2H, t, J = 6.7 Hz, H-1), 2.31

(1H, m, H-2), 1.57 (4H, m, H-3), 1.23 (64H, brs, H-4-35), 0.89 (3H, t, J = 6.6

Hz, H-36); 13 C NMR (CDCl 3, 100MHz) δ: 63.3 (C-1), 39.2 (C-2), 32.5-25.3

(C-3-35), 14.2 (C-36); HR-EIMS : m/z : 522.5743 (calcd. for C 36 H74 O,

522.5752).

3.7.4.10 Elaidic acid: (110)

White amorphous solid (30 O 11 3 9 1 OH 0 18 10 2 mg); M. P.: 43-44 C; IR (KBr) 110 νmax cm -1: 3350-2240, 1705,

1624, 1460, 1250; 1H NMR (CDCl 3, 400MHz) δ: 5.30 (2H, dt, J = 5.2, 16.5

Hz, H-9,10), 1.92-1.85 (4H, m, H-8, 11), 2.30 (2H, t, J = 5.1 Hz, H-2), 0.85

(3H, t, J = 6.6 Hz, H-18); 13 C NMR (CDCl 3, 100MHz) δ: 178.2 (C-1),

130.5,129.6 (C-9,10), 32.7 (C-8,11), 32.3 (C-2), 13.9 (C-18); HR-EIMS: m/z :

282.2650 (calcd. for C 18 H34 O2, 282.2664); EIMS: 282, 223, 167, 114, 60.

3.7.4.11 Oleanolic acid (111)

29 30 White crystalline solid (30 mg); M.P.: 304-

19 2 1 306 0C; [ααα]D25 : +78.9 0 (c = 0.15, CHCl 3); IR 12

25 26 13 17 COOH -1 28 (KBr) νmax cm : 3410-2650, 1710, 1660, 1 9 15

3 5 27 7 1 δ HO 820; H NMR (CDCl 3, 400MHz) : 5.49 (1H, t,

2 3 24 111 J = 3.5 Hz, H-12), 3.44 (1H, dd, J = 4.2, 9.8

Hz, H-3), 1.30 (3H, s, H-27), 1.24 (3H, s, H-23), 1.04 (3H, s, H-26), 1.02

(6H, s, H-24,30), 0.97 (3H, s, H-29), 0.93 (3H, s, H-25); 13 C NMR (CDCl 3,

100MHz) δ: 180.0 (C-28), 144.8 (C-13), 122.6 (C-12), 78.2 (C-3), 55.9 (C-5),

48.2 (C-9), 46.7 (C-17), 42.2 (C-19), 41.7 (C-14), 40.7 (C-18), 39.2 (C-8),

38.7 (C-4), 38.5 (C-1), 37.2 (C-10), 33.8 (C-21), 32.9 (C-29), 32.5 (C-7), 32.4

(C-22), 30.6 (C-20), 28.2 (C-23), 27.7 (C-15), 27.8 (C-2), 25.9 (C-27), 23.4 88

(C-30), 23.4 (C-11), 23.2 (C-16), 18.3 (C-6), 17.2 (C-26), 15.5 (C-24), 15.3

(C-25); HR-EIMS: m/z 456.3643 (calcd. for C 30 H48 O3, 456.3640); EIMS: m/z [M] + 456, 248, 208, 203, 133.

3.7.4.12 Ergosta-7,22-diene-3,5,6-triol (117)

25 28 White crystalline solid (15 mg); [ααα]D : -18.5

21 26 20 24 2 5 19 (c 0.20, CHCl 3); UV (MeOH) λmax nm (log ε): 17 27 18 11 13

15 -1 1 9 238 (3.7); IR (KBr) νmax cm : 3460, 1677;

3 5 6 HO 1H NMR (CDCl 3, 400MHz) δ: 5.23 (1H, br s, OH OH 117 H-7), 5.10 (2H, m, H-22,23), 3.90 (1H, m,

H-3), 3.49 (1H, br s, H-6), 0.97 (3H, s, H-19), 0.94 (3H, d, J = 6.4 Hz, H-21),

0.83 (3H, d, J = 6.4 Hz, H-28), 0.76 (3H, d, J = 6.4 Hz, H-26), 0.74 (3H, d, J

= 6.4 Hz, H-27), 0.51 (3H, s, H-18); 13 C NMR: (CDCl 3, 100MHz) δ: 143.3 (C-

8), 135.3 (C-22), 131.9 (C-23), 117.3 (C-7), 75.8 (C-5), 73.0 (C-6), 67.1 (C-

3), 55.8 (C-17), 54.5 (C-14), 43.5 (C-13), 43.0 (C-9 ), 42.6 (C-24), 40.3 (C-

20), 39.1 (C-12), 38.7 (C-4), 36.8 (C-10), 32.9 (C-25), 32.6 (C-1), 30.3 (C-2),

27.8 (C-16), 22.7 (C-15), 21.8 (C-11), 20.9 (C-21), 19.7 (C-26), 19.4 (C-27),

18.2 (C-19), 17.4 (C-28), 12.1 (C-18); HR-EIMS: m/z 430.3428 (Calcd. for

C28 H46 O3, 430.3364); EIMS: m/z [M-OH] + 412, 379, 269, 251, 199, 159, 95,

55.

3.7.4.13 Benzyl glucopyranoside: (118)

White needles (16 mg); M.P.: 124-125 0C; [ααα]D25 : - OH 4' 6' O OH 5' 2' 58.5 (c 0.68, MeOH); UV (MeOH) λmax nm: 214 1 OH 1' O 3' OH 2 -1 (3.76), 254 (3.88), 264 (2.70); IR (KBr) νmax cm :

4 6 1 δ 118 3384, 1637, 1250; H NMR (CD 3OD, 500MHz) :

7.41 (2H, d, J = 7.5 Hz, H-3,7), 7.33 (2H, t, J = 7.5 Hz, H-4,6), 7.25 (1H, dd,

J = 7.5 Hz, H-5), 4.93-4.67 (each H, d, J = 12.0 Hz, H-1), 4.35 (1H, d, J =

7.5 Hz, H-1'), 3.86-3.64 (2H, m, H-6'), 3.65 (1H, m, H-5'), 3.35 (1H, m, H-

4'), 3.24 (1H, m, H-3'), 3.21 (1H, m, H-2'); 13 C NMR : (CD 3OD, 125MHz) δ:

139.0 (C-2), 129.2 (C-3,7), 129.3 (C-4,6), 128.6 (C-5), 103.2 (C-1'), 78.1 (C-

5'), 78.0 (C-3'), 75.1 (C-2'), 71.7 (C-1), 71.6 (C-4'), 62.8 (C-6'); HR-EIMS: m/z 270.1191 (calcd. for C 13 H18 O6, 270.1143).

3.7.4.14 3,7,12-Trihydroxycholan-24-oic acid (120)

White powder (18 mg); [ααα] 25 : +27.8 (c = O D 21 20 24 OH19 OH 0.45, MeOH); M.P.: 251-252 0C; IR (KBr) 17

18 11 13 H -1 1 15 νmax cm : 3450, 3330-2250, 1705, 1250; H 1 9

3 5 6 δ HO OH NMR (CD 3OD, 500MHz) : 3.94 (1H, t, J = H 120 2.5 Hz, H-12), 3.79 (2H, d, J = 3.0 Hz, H-7),

3.38 (1H, m, H-3), 1.01 (1H, d, J = 6.5 Hz, H-21), 0.91 (1H, s, H-19), 0.70

(3H, s, H-18); 13 C NMR (CD 3OD, 125MHz) δ: 178.2 (C-24), 74.0 (C-12), 72.9

(C-3), 69.0 (C-7), 48.0 (C-17), 47.5 (C-13), 43.2 (C-14), 43.0 (C-5), 41.0 (C-

8), 40.4 (C-6), 36.7 (C-20 ), 36.5 (C-4), 35.9 (C-9), 35.8 (C-1), 32.3 (C-23),

32.0 (C-22), 31.2 (C-11), 29.6 (C-2), 28.6 (C-16), 27.9 (C-9), 24.2 (C-15),

21.1 (C-21), 17.6 (C-19), 12.9 (C-18); HR-EIMS: m/z 408.29350 (calcd. for

C24 H40 O5, 408.2923); EIMS: m/z [M-2H2O] + 372, 354, 300, 271, 253, 226,

145, 81, 55.

3.7.4.15 β-Sitosterol 3-O-β-D-glucopyranoside (67)

29 White crystalline solid (30 mg); 28

21 22 20 25 ααα 25 26 [ ]D : -14.7 (c 0.40, MeOH); 19 23

27 11 13 17 -1 18 IR (KBr) νmax cm : 3454, 3040, 15 OH 1 4' 6' 8 5' 1 O 3 5 H 7 H 1644, 1616, 1590-1545; H OH 2' 1' O OH 3' OH 67 NMR (CDCl 3, 400MHz) δ: 5.12

(1H, brd, J = 5.4 Hz, H-6), 3.84 (1H, m, H-3), 1.01 (3H, s, H-19), 0.92 (3H, d, J = 6.2 Hz, H-21), 0.85 (3H, t, J = 7.0 Hz, H-29), 0.84 (3H, d, J = 6.4 Hz,

H-26), 0.82 (3H, d, J = 6.4 Hz, H-27), 0.68 (3H, s, H-18), sugar moiety: 5.34

(1H, d, J = 7.2 Hz, H-1'), 3.82-3.43 (4H, m, H-2'-6'); 13 C NMR: (MeOH

125MHz) δ: 142.1 (C-5), 122.3 (C-6), 80.9 (C-3), 56.7 (C-14), 56.2 (C-17 ),

50.6 (C-24), 49.8 (C-9), 43.6 (C-4), 43.0 (C-13), 40.6 (C-12), 39.4 (C-22),

38.6 (C-1), 37.1 (C-20), 37.0 (C-10), 33.1 (C-7), 32.8 (C-8), 29.8 (C-2), 29.6

(C-16), 29.3 (C-23), 25.6 (C-25), 25.0 (C-15), 23.1 (C-28), 21.4 (C-11), 19.6

(C-27), 19.3 (C-19), 19.0 (C-21), 18.1 (C-26), 12.2 (C-18), 11.6 (C-29), sugar

91 moiety: 103.2 (C-1'), 77.7 (C-3'), 77.8 (C-5'), 75.3 (C-2'), 71.3 (C-4'), 62.0 (C-

6'); HR-FABMS: m/z 576.4686 (calcd. for C 35 H60 O6, 576.4689); EIMS: m/z

[M] + 414, 399, 396, 381, 329, 275, 273, 255.

3.7.4.16 20-Hydroxyecdesone (122)

ααα 25 OH White crystalline solid (17 mg); [ ]D : 26 21 OH 25 22 19 - 23 OH +41.95 (c = 0.25, Pyr); IR (KBr) νmax cm 27 17 18 11 13 1 1 15 : 3300, 1650, 1435, 1380, 1060; H HO 1 9 OH 3 5 6 HO NMR (CD 3OD, 400MHz) δ: 5.79 (1H, brs, O 122 H-7), 3.93 (2H, brs, H-2), 3.83 (1H, dist,d, J = 11.5 Hz, H-3), 3.33 (1H, d, J = 6.0 Hz, H-22), 1.19 (3H, s, H-21),

1.18 (3H, s, H-26), 1.17 (3H, s, H-27), 0.95 (3H, s, H-19), 0.88 (3H, s, H-

18); 13 C NMR (CD 3OD, 100MHz) δ: 206.0 (C-6), 167.9 (C-8), 122.1 (C-7),

85.2 (C-14), 78.4 (C-22), 77.9 (C-20), 71.2 (C-25), 68.7 (C-2), 68.5 (C-3),

51.7 (C-5), 50.5 (C-17), 48.1 (C-13), 42.4 (C-24), 39.2 (C-10), 37.3 (C-1),

35.1 (C-9), 32.8 (C-4), 32.5 (C-12), 31.7 (C-15), 29.7 (C-26), 28.9 (C-27),

27.3 (C-23), 24.4 (C-19), 21.5 (C-11,16), 21.0 (C-21), 18.0 (C-18); HR-EIMS: m/z 480.3125 (calcd. for C 27 H44 O7, 480.3150); EIMS: m/z [M-H2O] + 462,

444, 426, 411, 363, 345, 327, 320, 309, 301, 300, 285, 269, 250, 173, 161,

117, 99.

3.7.5 Methanolysis

Compounds 112-115 (12 mg each) were refluxed separately with 6 ml of 1N HCl and 25 ml of MeOH for 15 h. The reaction mixture was then extracted with n-hexane to obtain the corresponding fatty acid methyl esters, which were analyzed by GC-MS after acetylation with Ac 2O-Py. The aq. layer from 112-114 was evaporated, and the residue was acetylated.

Purification over Sephadex LH-20 and elution with CH 2Cl 2/MeOH 1:1 gave the corresponding acetylated sphingosines, which was analyzed by GC-MS.

The aq. layer from 115 was evaporated to dryness, and the residue was separated by silica gel column chromatography as sphingosine base and methylated sugar. The base was acetylated and analyzed by GC-MS. The sugar was identified as methyl α-D-glucopyranoside by comparing optical rotation and spectral data with those in the literature. [α]D + 76.2 ( c =

0.0024, MeOH). Rf 0.45 (EtOAc/MeOH/H 2O; 5:2:0.5).

3.7.6 Oxidative Cleavage of the Double bond

To the solution of acetylsphingamines of compound 112-114 and the methyl ester derived from 115 (4 mg each) in acetone, added 1mL of 0.04M solution of K 2CO 3, 6 mL of an aqueous solution 0.025M KMnO 4 and 0.09M

NaIO 4 in 100 ml round bottom flask. The reaction was allowed to proceed at

37 0C for 18h. After acidification with 5N H 2SO 4, the solution was decolorized with a 1M solution of oxalic acid and extracted with Et 2O (3 93 to10 mL). The combined organic extracts were dried over Na 2SO 4, filtered, and concentrated. The resulting carboxylic acids were methylated with ethereal solution of diazomethane and analyzed by GC-MS.

3.8 Bioassays

3.8.1 DPPH Radical Scavenging Activity

The DPPH free radical scavenging activities of pure compounds isolated from L. nudicualis were examined by comparison with that of known antioxidant, quercetin by using the method of Lee and Shibamoto

(Lee and Shibamoto, 2001). Briefly, various amounts of the compounds

(500 µg/mL, 250 µg/mL, 125 µg/mL, 60 µg/mL, 30 µg/mL, 15 µg/mL) were mixed with 3 ml of methanolic solution of DPPH (0.1mM). The mixture was shaken vigorously and allowed to stand at room temperature for one hour. Then absorbance was measured at 517 nm against methanol as a blank in the spectrophotometer. Lower absorbance of spectrophotometer indicated higher free radical scavenging activity.

The percent of DPPH decoloration of the samples was calculated according to the formula:

Antiradical activity = Acontrol – Asample × 100 Acontrol

Each sample was assayed in triplicate and mean values were calculated.

3.8.2 Acetylcholinesterase Assay

The Acetylcholinesterase (AChE) inhibition activity was performed according to the method used by Ellman (Ellman et al., 1961) with slight modifications. Total volume of the reaction mixture was 100 µL. It contained 60 µL Na 2HPO 4 buffer with concentration of 50 mM and pH 7.7.

Ten µL test compound (0.5 mM well -1) was added, followed by the addition of 10 µL (0.005 unit well -1) 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 30 min of incubation at 37ºC, absorbance was measured at

405 nm. Synergy HT (BioTek, USA) 96-well plate reader was used in all experiments. 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 by the help of following equation.

Inhibition (%) = Control – Test × 100

Control

3.8.3 Butyrylcholinesterase Assay

The Butyrylcholinesterase (BChE) inhibition activity was performed according to the reported method (Ellman et al., 1961) with slight modifications. Total volume of the reaction mixture was 100 µL containing

60 µL, Na 2H PO 4 buffer, 50 mM and pH 7.7. Ten µL test compound 0.5 mM well -1, followed by the addition of 10 µL (0.5 unit well -1) BChE. The contents were mixed and pre-read at 405 nm and then pre-incubated for 10 mins at

37ºC. The reaction was initiated by the addition of 10 µL of 0.5 mM well -1 substrate (butyrylthiocholine bromide) followed by the addition of 10 µL

DTNB, 0.5 mM well -1. After 30 min of incubation at 37ºC, absorbance was measured at 405 nm. Synergy HT (BioTek, USA) 96-well plate reader was used in all experiments. 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 with the help of following equation.

Inhibition (%) = Control – Test × 100

Control

IC 50 values (concentration at which there is 50% enzyme inhibition) of compounds were calculated using EZ–Fit Enzyme kinetics software (Perella

Scientific Inc. Amherst, USA).

3.8.4 Lipoxygenase Assay

Lipoxygenase (LOX) activity was assayed according to the reported method (Tappel, 1953) but with slight modifications. A total volume of 200

µL assay mixture contained 140 µL sodium phosphate buffer (100 mM, pH

8.0), 20 µL test compound and 15µL (600U) purified lipoxygenase enzyme

(Sigma, USA). The contents were mixed and pre-read at 234 nm and pre incubated for 10 minutes at 25°C. The reaction was initiated by addition of

25 µL substrate solution. The change in absorbance was observed after 6 min at 234 nm. Synergy HT (BioTek, USA) 96-well plate reader was used in all experiments. All reactions were performed in triplicates .The positive and negative controls were included in the assay. Baicalein (0.5 mM well -1) was used as a positive control. The percentage inhibition was calculated by formula given below.

Inhibition (%) = Control – Test × 100

Control

4.1 Introduction of Launaea intybacea

L. intybacea is a biennial herb, also called as wild lettuce. It is cosmopolitan in nature and has adapted to dry conditions and commonly found in coastal areas (Kirtikar and Basu, 1999). The species is commonly found in north and Central America, West Indies and tropical parts of Asia

(Pokharkar et al., 2007). Flowering period is January to march.

4.2 Scientific Classification of Launaea intybacea

Kingdom: Plantae

Subkingdom: Angiosperms

Order: Asterales

Family: Asteraceae

Tribe: Cichorieae

Genus: Launaea

Specie: intybacea

4.3 Pharmacological Importance of Launaea intybacea

Root and the aerial parts of the plant have been used by the local people as a folk remedy to treat jaundice, as blood purifier, glactogogue, hepatomegaly, dyspepsia, skin diseases, dry cough and galactoriya (Kirtikar

99 and Basu, 1999);(Handa et al., 1986). L. intybacea has also been used in folk medicine for the treatment of liver disorders (Gite et al., 2010a).

Various solvent extracts of the aerial parts of the L. intybacea have been evaluated against CCl 4-induced hepatoprotective injury in albino rats and the results showed significant hepatoprotective activity by reducing the serum total bilirubin, direct bilirubin, and SGPT and SGOT levels (Gite et al., 2010a). Despite of its local use as medicine and hepatoprotective activities, no phytochemical investigations have been carried out on this plant.

4.4 Results and Discussion

The phytochemical studies of the methanolic extract of L. intybacea resulted in the isolation of a new natural product, 6,6'-oxybis(4-allyl-2- methoxyphenol) (126 ) along with fourteen known secondary metabolites

(30 , 53 , 111 , 124 , 125 , and 127 -135 ). As no chemical constituents have been reported previously from this species thus all known compounds were new source. The structures of the isolated compounds were determined by various spectroscopic techniques.

4.4.1 Characterization of 6,6'-oxybis(4-allyl-2-methoxyphenol) (126)

Compound 126 was isolated as colorless viscous oil. The HR-EIMS displayed molecular ion peak at m/z 342.1459 and the molecular formula deduced as C 20 H22 O5 with 10 double bond equivalence. The IR spectrum of 100

126 displayed absorption bands due to O-H (3600 cm -1) and C=C (1645-

1500 cm -1). The UV spectrum showed the absorption maxima at 206 and

272. The 1H NMR spectrum of 126 displayed two signals in the aromatic region at δ 6.80 (d, J = 2.0 Hz) and δ 6.60 (d, J = 2.0 Hz) coupled with carbons at δ 116.5, 110.7 in the HMQC spectrum.

The Coupling constant and their COSY correlations with each other suggested that they are meta to each. Moreover 9' 9 8 7' both these protons exhibited allelic COSY 5 4' 6' 1 3 correlations with a methylene observed at δ 3.40 H3CO 2' O OCH3 OH OH (d, J = 7.0 Hz) which was further correlated with 126 a signal at δ 5.94 (tdd, J = 6.5, 10.0, 17.0, Hz) which further showed COSY correlation with two olefinic protons at δ 5.08 (tdd, J = 1.0, 2.0, 17.0 Hz) and δ 5.02 (tdd, J = 1.0, 2.0, 10.0 Hz) (Fig. 4.1)

Fig. 4.1: Important HMBC and COSY correlations in 126

The above data suggested the presence of an allyl group on the aromatic ring between the two aromatic meta protons. This concept was 101 further supported when methylene at δ 3.40 showed HMBC correlations with aromatic carbons at δ 116.5 and 110.7. The HMBC correlations of aromatic protons with carbons at δ 141.5, 138.6 and 150.2 suggested that the ring is oxygenated at the three positions. A methoxy δ 3.80 observed in

HSQC at δ 56.8 exhibited HMBC correlations with the aromatic carbon δ

150.2.

Careful analysis of all the 1D and 2D NMR data revealed that the compound is 3-allyl-5-methoxybenzene-1, 2-diol. But the molecular mass of the compound as m/z 342 is exactly double to this moiety so it was deduced that the compound must be a dimer. Moreover the spectral data was compared with its monomer obovatol (Kwak et al., 2008). Based on these evidence the compound was found to be 6,6'-oxybis(4-allyl-2- methoxyphenol) which is a new natural product.

4.4.2 Characterization of lupeol (53)

Compound 53 was isolated as white crystalline solid. Its molecular mass was calculated from HR-EIMS which 30 22 29

1 9 displayed molecular ion peak at m/z 426.3830 21 1 2 18 25 11 2 6 28 consistent with the molecular formula C 30 H50 O. 1 1 5 H 8 3 5 7 27 It IR spectrum displayed characteristic bands for HO

23 24 hydroxyl group at 3450 cm -1, and terminal 53 double bond at 3070, 1645 and 880 cm -1. The EIMS of 53 showed 102 fragments at m/z 385 [M ˗41] +, 220 [M ˗C15 H26 ]+, 218 [M ˗C14 H20 O] +, and 207

[M ˗ C16 H27 ]+ which are characteristic for lupane skeleton (Perveen et al.,

2009).

The 1H NMR spectrum of 53 displayed the signals for seven tertiary methyl groups at δ 1.64, 1.08, 0.97, 0.94, 0.85, 0.79 and 0.77 (3H each, s).

The carbinylic proton resonance was observed as double doublet at δ 3.21

(J = 9.9, 4.5 Hz). The chemical shift of H-3 and coupling constants revealed that hydroxyl group at C-3 possess β and equatorial configuration.

The signals due to olefinic proton were observed at δ 4.65 and 4.75

(1H each, br s), while a sextet of one proton at δ 2.37 (J = 5.4, 10.5, 10.5 Hz) might be assigned to 19 β-H. The physical and spectral data of 53 completely matched with the literature values for lupeol (Perveen et al.,

2009) which was furthered confirmed through co-TLC with the authentic sample.

4.4.3 Characterization of β-sitosterol (30)

Compound 30 was purified as colorless needles. The molecular mass was calculated from HR-EIMS showing molecular ion peak at m/z

414.3850, consistent with the molecular formula C 29 H50 O which corresponds to five degree of unsaturation. The IR spectrum showed

103 absorption bands for hydroxyl group (3450 cm -1) and tri-substituted double bond (3050, 1650 and 815 cm -1).

The EIMS spectrum of 30 displayed fragments at m/z 399, 396, 381,

29 329 and 303. The fragments at m/z 329 28 21 22 20 25 26 19 and 303 were characteristics for the sterols 23 27 12 17 18 11 5 15 which possess ∆ -unsaturation (Wyllie et 1 8

3 5H 7 H al., 1977a). Other important fragments were HO 30 observed at m/z 273 and 255, indicating the loss of [M ˗ side chain] + and [M

˗ side chain ˗H2O] +, respectively.

The 1H NMR spectrum of 30 showed the carbinylic and olefinic protons at δ

3.51 (1H, m, H-3), and δ 5.32 (1H, m, H-6). Moreover it showed six methyl resonances of which two were tertiary δ 1.22 (3H, H-19), 0.67 (3H, H-18), three secondary δ 0.98 (3H, J = 6.2 Hz, H-21), 0.84 (3H, J = 6.5 Hz, H-26),

0.82 (3H, J = 6.5 Hz, H-27) and a primary at δ 0.85 (3H, J = 7.0 Hz, H-29).

The 13 C NMR (BB and DEPT) spectrum of 30 showed the presence of a total 29 carbon signals for six methyl, eleven methylene, nine methine and three quaternary carbons. From above data and as well as comparison with the literature 30 was confirmed as β-sitosterol (Rubinstein et al.,

1976); (Holland et al., 1978).

104

4.4.4 Characterization of octadecyl ( E)-p-coumarate (124)

The compound 124 was isolated as colorless amorphous solid. The

HR-EIMS displayed molecular ion peak at m/z 416.3288 consistent with the molecular formula C27 H44 O3. The IR spectrum showed absorption bands for hydroxyl (3540 cm -1) and carbonyl (1715 cm -1) functionalities while its

UV absorbance at 227 and 312 nm showed the presence of substituted benzene ring.

The 1H NMR spectrum showed signals for trans olefinic protons at δ

7.60 (1H, d, J = 18.0 Hz), 6.25 (2H, d, J = O 7 1' 1 2 2 3 9 18.0 Hz), an aromatic ring splitted as A B , 8 O (CH2)16CH3

HO 5 δ 7.43 (2H, d, J = 7.8 Hz), 6.81 (2H, d, J = 124

7.8 Hz), an oxymethylene at δ 4.21 (2H, t, J = 6.8 Hz) and an aliphatic chain at δ 1.70 (2H, m), 1.25 (30H, br s), 0.88 (3H, t, J = 6.0 Hz) indicated that 124 could be (E)-p-coumarate.

The length of the aliphatic chain was confirmed through the molecular ion peak at m/z 416. Moreover analysis of 13 C NMR data and comparison with the literature values confirmed the compound 124 as octadecyl ( E)-p-coumarate (Bohlmann et al., 1979).

105

4.4.5 Characterization of 3-methoxy-4-hydroxy benzaldehyde (125)

Compound 125 has been isolated as white crystal. Its molecular formula C 8H8O3 was deduced from HR-EIMS which showed molecular ion peak at m/z 152.0473. The UV spectrum showed absorption maxima at

234, 281 and 310 nm indicated the presence of phenolic chromophore. The

IR spectrum showed the peaks at 1746 cm -1 and 1642 cm -1 indicated the presence of aryl aldehyde group.

The 1H NMR spectrum showed the presence of aldehyde at δ 9.74

7 (1H, s), three aromatic protons at δ 7.42 (1H, dd, J = 1.6, 8.4 CHO 1

Hz), 7.29 (1H, d, J = 1.6 Hz), 7.09 (1H, d, J = 8.4 Hz) with 5 3 OCH3 ABX splitting pattern and a methoxy signal at δ 3.94 (3H, s). OH 125 The 13 C NMR spectrum showed eight carbon signals including one methyl, three methine and four quaternary carbons. Thus 125 was 3,4- disubstituted aromatic aldehyde. From above data and comparison with the literature the compound 125 was found to be 3-methoxy-4- hydroxybenzaldehyde (Mukonyi and Ndiege, 2001).

4.4.6 Characterization of 4-hydroxybenzoic acid (127)

Compound 127 was isolated as white crystalline solid with molecular formula C 7H6O3 deduced from HR-EIMS with the molecular ion peak at m/z

138.0321. The UV absorption maxima at 222 and 310 nm indicating the

106 presence of phenolic system while IR spectrum showed absorption bands at

3515 cm -1 for hydroxyl group as well as a broad band at 3335-2730 cm -1 along with intense band at 1710 cm -1 indicated the presence carboxylic group.

The 1H NMR spectrum of 127 showed the presence of aromatic ring

7 with A 2B2 splitting pattern with two doublets at δ 7.86 (2H, d, J COOH 1

13 = 8.8 Hz) and 6.80 (2H, d, J = 8.8 Hz) where as its C NMR 5 3 showed five carbon signals for four methine and the three OH 127 quaternary carbons at δ 169.5, 163.7, 132.9, 123.0 and 115.9. The above data was completely matched to the data reported for 4-hydroxybenzoic acid (Kazmi et al., 1994).

4.4.7 Characterization of 4-hydroxy-3-methoxybenzoic acid (128)

Compound 128 was isolated as white crystals. The HR-EIMS showed the molecular ion peak at m/z 168.0419 corresponding to the molecular

7 formula C 8H8O4 with five double bond equivalence. Analysis COOH 1

3 of UV spectrum of 128 indicated the presence of phenolic 5 OCH3 chromophore while IR spectrum with absorption bands at OH 128 3510, 3335-2730, 1705 cm -1 showed the presence of hydroxyl and carbonyl functionalities.

The 1H NMR spectrum of 128 was almost super imposable to that of

125 in the aromatic region which showed signals at δ 7.55 (1H, d, J = 2.0

107

Hz, H-2), 7.53 (1H, dd, J = 2.0, 8.5 Hz, H-6), and 6.82 (1H, d, J = 8.5 Hz, H-

5). The 13 C NMR spectrum showed eight carbon signals for one methyl, three methine and four quaternary carbons at δ 169.8, 152.5, 148.6, 127.2,

125.2, 115.7, 113.8 and 56.3.

The above data was sufficient enough to conclude 128 as 4-hydroxy-

3-methoxybenzoic acid (Sang et al., 2002).

4.4.8 Characterization of 4-hydroxy-trans-cinnamic acid (129)

Compound 129 was isolated as yellow amorphous solid. The molecular formula found to be C 9H8O3 from a molecular ion peak in HR-

EIMS at 164.04754 which indicated six double bond equivalences.

The 1H NMR spectrum of 129 was similar to that of 124 displaying

δ signals at 7.44 (2H, d, J = 8.6 Hz), 7.22 (1H, d, J = O 7 3 1 9 15.8 Hz), 6.80 (2H, d, J = 8.6 Hz), 6.23 (1H, d, J = 8 OH HO 15.8 Hz) typical for coumaric acid. This concept was 5 129 further confirmed by the analysis of 13 C NMR spectrum having total seven carbon signals at δ 170.6, 158.7, 138.2, 129.4, 126.8, 115.5 and 114.6.

From this discussion and all other related information from UV, IR and comparison with the literature, the compound 129 is identified as 4- hydroxy-trans-cinnamic acid (Cho et al., 1998).

108

4.4.9 Characterization of methyl gallate (130)

The compound 130 was obtained as white crystalline solid. The HR-

EIMS of 130 showed molecular ion peak at m/z 7 COOCH3 1 184.0345 correspond to the molecular formula C 8H8O5

5 3

(calcd. for C 8H8O5, 184.0371). The UV spectrum showed HO OH OH absorptions at 278 and 283 nm whereas IR spectrum 130 showed peaks at 3350, 1690 at 1619 cm -1 indicating the presence of aromatic carboxylic system.

The 1H NMR spectrum showed only two signals, one in the aromatic region at δ 7.03 (2H, s, H-2,5) and a signal for the methoxy group at δ 3.80

(3H, s, OCH 3), whereas six signals at δ 169.0, 146.5, 139.7, 121.5, 110.0 and 56.3 were observed in its 13 C NMR spectrum. From above discussion and comparison with the literature values, 130 was confirmed as methyl gallate (Banday et al., 2012).

4.4.10 Characterization of 3,4-dihydroxybenzoic acid (131)

Compound 131 was obtained as white crystalline solid. Its molecular mass was calculated from HR-EIMS as 154.0254 and the molecular formula deduced as C 7H6O4 with five degree of unsaturation. The UV spectrum showed absorption at 289, 258 and 215 nm while IR spectrum displayed absorption bands at 3505, 3235-2857, 1710 cm -1.

109

The 1H NMR spectrum was similar to that of compound 128 showing signal in the aromatic region at δ 7.42 (1H, dd, J = 1.5, 8.5 Hz), 7 COOH 1 7.41 (1H, d, J = 1.5 Hz), 6.78 (1H, d, J = 8.5 Hz). Thus 128 3 5 could be 3,4-dihydroxy derivative of compound 128 whereas OH OH 131 the 13 C NMR spectrum showed seven carbon signals at δ

169.4, 151.1, 145.2, 124.2, 123.8, 117.7 and 115.7. From all above discussion 131 was found to be 3,4-dihydroxybenzoic acid (Ayinde et al.,

2007).

4.4.11 Characterization of 4',5,7-trihydroxyflavone (132)

Compound 132 was isolated as yellow needles. The HR-EIMS showed the molecular ion peak at m/z 270.0512 having the molecular formula

C15 H10 O6 with eleven double bond equivalence. The UV spectrum showed the bands at 340 and 269 nm typical for flavonoid nucleus (Dordevic et al.,

2000) while the IR spectrum showed absorption bands at 3454, 1650 and

1590 cm -1.

The 1H NMR spectrum of showed the downfield signal in the aromatic 3' OH region at δ 7.85 (2H, d, J = 8.5 Hz), 6.93 (2H, d, J 1 1' HO 7 9 O 5' 2 2 = 8.5 Hz) suggested the A B splitting pattern in 3 10 4 5 ring B and the upfield aromatic signals were OH O 132 observed at δ 6.45 (1H, d, J = 2.0 Hz), 6.20 (1H, d, J = 2.0 Hz) for ring A while the singlet for H-3 was observed at δ 6.58. The 13 C NMR spectrum

110 showed altogether thirteen carbon signals at δ 183.9, 166.3, 166.1, 163.2,

162.1, 159.4, 129.4, 123.2, 117.0, 105.3, 103.8, 100.1 and 95.0. All this data suggested 132 as 4',5,7-trihydroxyflavone (Moussaoui et al., 2010b).

4.4.12 Characterization of 3',4',5,7-tetrahydroxyflavone (133)

Compound 133 was isolated as yellow needles. The HR-EIMS showed molecular ion peak at m/z 286.0435 corresponds to the molecular formula

C15 H10 O6 with eleven double bond equivalence. The UV, IR data was similar as for 132 .

The 1H NMR showed a double doublet, a singlet and a doublet at δ

7.39 (1H, dd, J = 2.5, 7.5 Hz, H-6'), 7.37 (1H, br s, H-2'), 6.90 (1H, d, J =

8.5 Hz, H-5') respectively instead of two doublets OH 3' OH as in 132 indicated the presence of additional 1 1' HO 7 9 O 5'

3 hydroxyl group in ring C with ABX splitting 10 4 5 OH O pattern which was supported by its 13 C NMR 133 spectrum due to signal at δ 147.0 (C-3') while the rest of the signals in the

1H NMR were appeared at δ 6.53 (1H, s, H-3), 6.43 (1H, d, J = 2.5 Hz, H-8),

6.20 (1H, d, J = 2.0 Hz, H-6) suggesting that 133 is C-3' oxidative derivative of 132 . Further analysis of 13 C NMR spectrum and comparison with the literature revealed that compound 133 is 3',4',5,7- tetrahydroxyflavone (Sashida et al., 1983); (Wagner et al., 1976).

111

4.4.13 Characterization of 3,3',5,7-tetrahydroxy-4'-methoxyflavone

(134)

Compound 134 was isolated as yellow prism. The UV spectrum showed the absorptions at 259 and 375 OH 3' OCH3

1 suggesting the presence of flavonoid nucleus 1' HO 7 9 O 5'

10 3 (Dordevic et al., 2000). The IR spectrum 4 5 OH OH O showed absorption bands at 3450, 1655, and 134

1595 cm -1. The molecular mass was calculated from HR-EIMS due to molecular ion peak at m/z 316.0583 which correspond to the molecular formula C 16 H12 O7.

The 1H NMR spectrum of 134 was almost similar to that of compound 133 and displayed signals at δ 7.99 (1H, d, J = 1.6 Hz, H-2'),

7.63 (1H, dd, J = 1.6, 8.4 Hz, H-6'), 6.91 (1H, d, J = 8.4 Hz, H-5'), 6.40 (1H, br s, H-8), 6.16 (1H, br s, H-6). But the signal for H-3 was missing which mean C-3 is oxygenated which was further supported due to presence of oxygenated olefinic carbon at δ 133.6 (C-3) and an additional signal for the methoxy group was observed at δ 3.93 (3H, s, OCH 3). The 13 C NMR spectrum displayed 16 signals for one methyl, five methane and ten quarternary carbons. From above discussion and as well as comparison with the literature revealed 134 as 3,3',5,7-tetrahydroxy-4'-methoxyflavone

(Mullen et al., 2002); (Yuan et al., 2007).

112

4.4.14 Characterization of apigenin 7 -O-(4'' -O-p-E-coumaroyl-β-D- glucopyranoside (135)

O OH 3' OH 9''' 3''' 4'' 6'' O 1''' 1 1' O O O 5' HO 7 7''' 5''' 3'' OH 1'' 9 HO 10 4 3 5 OH O 135

Compound 135 was isolated as yellow amorphous powder. The UV spectrum was similar to that for the above discussed flavonoids. The HR-

FABMS showed the molecular ion peak at m/z 579.1499 [M] + and the molecular formula was calculated to be C 30 H27 O12 with sixteen double bond equivalence.

The 1H NMR spectrum was similar to 132 with the signals at δ 7.89

(2H, d, J = 8.5 Hz, H-2',6'), 6.94 (2H, d, J = 9.0 Hz, H-3',5'), 6.84 (1H, d, J =

2.0 Hz, H-8), 6.66 (1H, s, H-3), and 6.53 (1H, d, J = 2.0 Hz, H-6), which suggested the presence of apigenin moiety in the molecule (Moussaoui et al., 2010b). In addition, it showed the signals for p-E-coumaroyl at δ 7.71

(1H, d, J = 16.0 Hz), 7.49 (2H, d, J = 8.5 Hz), 6.82 (2H, d, J = 9.0 Hz), 6.41

(1H, d, J = 15.5 Hz) (Cho et al., 1998) and glucose moiety at δ 5.16 (1H, d, J

= 8.0 Hz), 4.96 (1H, t, J = 9.5 Hz), 3.81 (1H, m), 3.78 (1H, m), 3.68 (1H, m),

3.61 (1H, dd, J = 9.5, 8.0 Hz).

113

The attachment of the sugar moiety was found to be C-7 due to the downfield shifts of C-7 ( δ 164.7) and that of coumeroyl moiety to the C-4 of the sugar due to the downfield shift of C-4'' ( δ 72.1). All presented data and above discussion was suggested that 135 as apigenin 7 -O-(4'' -O-p-E- coumaroyl-β-D-glucopyranoside (Singh et al., 1986).

4.5 Biological Studies

The compounds isolated from L. intybacea, 124 -135 were subjected to various biological activities i.e. DPPH free radical scavenging, AChE,

BChE, and LOX enzyme inhibition activities. 30 , 53 and 111 were not tested due to their less amounts. All the compounds were tested at a concentration of 0.5mMol well -1.

4.5.1 Antioxidant Assay (DPPH Radical Scavenging Method)

The antioxidant activities were carried out by using DPPH free radical scavenging method. Significant antioxidant activity was observed for methyl gallate ( 130 ) with an IC 50 value of 76.2 and 3,4-dihydroxybenzoic acid (131 ) and 3',4',5,7-tetrahydroxyflavone (133) showed moderate activity with an

IC 50 value 211.5 and 146.3 respectively while 124 -129 , 133 , 134 and 135 did not show significant antioxidant activity (Table 4.1). Quercetin was used as a positive control at concentration 0.5 mMwell -1 for control as well as test compounds.

114

Table 4.1: Antioxidant Assay (DPPH radical scavenging activity)

Compound (%) at (IC 50 ) 0.5 mM µM 124 23.9±0.2 NIL 125 22.6±0.6 NIL 126 45.27±0.24 NIL 127 24.7±0.8 NIL 128 18.5±0.3 NIL 129 14.3±0.4 NIL 130 85.6±0.6 76.2±0.4 131 69.0±0.1 211.5±0.1 132 21.9±0.1 NIL 133 78.4±0.2 146.3±0.04 134 28.9±0.8 NIL 135 24.1±0.2 NIL Quercetin 93.21±0.97 16.96±0.14

4.5.2 Enzyme Inhibition Activities

The enzyme inhibition activities were checked by following assays.

4.5.2.1 Acetylcholinesterase Enzyme Inhibition Activity

The octadecyl ( E)-p-coumarate ( 124) and 3,3',5,7-tetrahydroxy-4'- methoxyflavone ( 134 ) showed considerable acetylcholinesterase enzyme inhibition activity with an IC 50 value of 106.31 and 143.9 respectively.

While 3-Methoxy-4-hydroxy benzaldehyde ( 125 ), 4-hydroxybenzoic acid

115

(127 ), 4-hydroxy-trans-cinnamic acid ( 129 ), 3,4-dihydroxybenzoic acid

(131 ), 4',5,7-trihydroxyflavone (132 ), 3',4',5,7-tetrahydroxyflavone (133 ), and apigenin 7 -O-(4'' -O-p-E-coumaroyl-β-D-glucopyranoside (135 ) showed minor activity and the compounds 126 , 128 and 130 were inactive (table

4.2). Eserine (0.5 mM well -1) was used as positive control.

Table 4.2: Acetylcholinesterase enzyme inhibition activity

Compound (%) at (IC 50 ) 0.5 mM µM 124 82.2±0.1 106.31±0.31 125 52.5±0.3 <400 126 45.28±0.14 NIL 127 49.5±0.8 <400 128 36.6±0.2 NIL 129 56.9±0.1 <400 130 31.9±0.3 NIL 131 56.9±0.5 <400 132 50.5±0.2 <400 133 58.7±0.6 <400 134 75.1±0.4 143.9±0.22 135 53.5±0.2 <400 Eserine 91.29±1.17 0.04±0.0001

4.5.2.2 Butyrylcholinesterase Enzyme Inhibition Activity

Significant butyrylcholinesterase enzyme inhibition activities were observed for octadecyl ( E)-p-coumarate ( 124) , 4',5,7-trihydroxyflavone

116

(132 ), 3,3',5,7-tetrahydroxy-4'-methoxyflavone ( 134 ) and apigenin 7 -O-(4'' -

O-p-E-coumaroyl-β-D-glucopyranoside (135 ) with an IC 50 value of 53.9,

93.3, 34.1 and 96.5 respectively. Compounds 125 , 127 , 130 , 131 and 133 were moderately active with IC 50 of 241.1, 230.6, 120.2, 152.3, and 106.8 respectively. 126, 128 and 129 showed minor activity (Table 4.3).

Table 4.3: Butyrylcholinesterase enzyme inhibition activity

Compound (%) at (IC 50 ) 0.5 mM µM 124 91.2±0.2 53.9±0.05 125 59.9±0.4 241.1±0.3 126 53.11±0.25 <400 127 59.6±0.1 230.6±0.7 128 50.3±0.2 <400 129 53.4±0.4 <300 130 64.8±0.5 120.2±0.3 131 62.7±0.6 152.3±0.2 132 83.8±0.3 93.3±0.3 133 80.1±0.3 106.8±0.2 134 93.2±0.4 34.1±0.2 135 78.6±0.3 96.5±0.2 Eserine 82.82±1.09 0.85±0.0001

4.5.2.3 Lipoxygenase Enzyme Inhibition Activity

3-Methoxy-4-hydroxy benzaldehyde ( 125 ), 6,6'-oxybis(4-allyl-2- methoxyphenol) (126 ), 4',5,7-trihydroxyflavone (132 ) and apigenin 7 -O-(4'' -

117

O-p-E-coumaroyl-β-D-glucopyranoside (135 ) were significantly active against lipoxygenase enzyme with IC 50 values of 99.1, 56.37, 83.7, 48.2 while compounds 127 , 128 , were not significantly active with an IC 50 value less than 400. The compounds 124 , 129 -131 , 133 and 134 were inactive against lipoxygenase enzyme. Baicalein (0.5 mM well-1) was used as a positive control (Table 4.4).

Table 4.4 Lipoxygenase enzyme inhibition activity

Compound (%) at (IC 50 ) 0.5 mM µM 124 45.6±0.1 NIL 125 82.6±0.3 99.1±0.2 126 91.74±0.23 56.37±0.07 127 57.5±0.8 <400 128 53.7±0.2 <400 129 5.4±0.1 NIL 130 12.8±0.5 NIL 131 27.3±0.7 NIL 132 83.6±0.2 83.7±0.1 133 42.0±0.6 NIL 134 17.3±0.4 NIL 135 93.8±0.2 48.2±0.3 Baicalein 93.79±1.2 22.4±1.3

118

4.6 Experimental

4.6.1 Plant Material

The whole plant L. intybacea was collected from Cholistan Desert

(District Bahawalpur, Punjab), Pakistan in April 2008 and was identified by

Dr. Muhammad Arshad (Late), Plant Taxonomist, Cholistan Institute for

Desert Studies (CIDS), The Islamia University of Bahawalpur, Pakistan where a voucher specimen (0023-LI/CIDS/08) is deposited.

4.6.2 Extraction and Isolation

The whole plant of L. intybacea was shade dried (18 kg), ground and extracted with methanol. The methanolic extract was evaporated under vacuum to a dark greenish mass (700 g) which was subjected to column chromatography over silica gel using the solvent dichloromethane, dichloromethane-methanol and methanol with increasing order of polarity to give seven fractions (1-7).

The fraction 1 obtained from 100% DCM was chromatographed to get three sub fractions. The sub Fr. 1 showed two spots on TLC and on final purification using the solvent system n-hexane-DCM (2.0:8.0) β-sitosterol

(30 ) and at n-hex-DCM (3.0:7.0) yielded lupeol ( 53 ). The sub Fr. 2 collected at n-hex-DCM (1.0:9.0) when subjected to column chromatography yielded oleanolic acid ( 111 ) while the sub Fr. 3 at 100% DCM yielded octadecyl ( E)-

119 p-coumarate (124 ). The fraction 2 obtained at DCM-MeOH (9.9:0.1) was further subjected to silica gel column chromatography and on final purification yielded 4-hydroxy-3-methoxybenzaldehyde ( 125 ) from the head fraction and 6,6'-oxybis(4-allyl-2-methoxyphenol) (126 ) from the tail fraction under the same solvent system. The fractions 3 collected at DCM-

MeOH (9.8:0.2) showed two spots on TLC which on further column chromatography at solvent system DCM-MeOH (9.7:0.3) afforded 4- hydroxybenzoic acid ( 127 ) and 4-hydroxy-3-methoxybenzoic acid ( 128 ) from head and tail fractions. The fraction 4 obtained from DCM-MeOH

(9.6:0.4) further subjected to silica gel column chromatography using DCM-

MeOH (9.5:0.5) afforded coumaric acid ( 129 ). The fraction 5 eluted at DCM-

MeOH (9.4:0.6) further yielded two sub fractions. The sub Fr.1 subjected to column chromatography at DCM-MeOH (9.4:0.6) as eluent yielded methyl gallate ( 130 ) and 3,4-dihydroxybenzoic acid ( 131 ) one after the other. Sub

Fr. 2 on final purification at DCM-MeOH (9.3:0.7) yielded 4',5,7- trihydroxyflavone ( 132 ). The fraction 6 obtained at DCM-MeOH (9.2:0.8) gave two sub fractions. The sub Fr. 1 on final purification using silica gel column chromatography at DCM-MeOH (9.2:0.8) yielded 3',4',5,7- tetrahydroxyflavone ( 133 ) and sub Fr. 2 on purification at DCM-MeOH

(9.0:1.0) yielded 3,3',5,7-tetrahydroxy-4'-methoxyflavone ( 134 ) while the fraction 7 eluted at DCM-MeOH (8.8:1.2) was further purified to give apigenin 7 -O-(4'' -O-p-E-coumaroyl)-β-D-glucopyranoside ( 135 ).

120

Scheme 4.1: Protocol for the isolation of compounds 30 , 53 , 111 , 124 -

135 from Launaea intybacea

121

4.6.3 Experimental Data

4.6.3.1 6,6'-Oxybis(4-allyl-2-methoxyphenol) (126)

Colorless viscous oil (13 mg); UV (MeOH) λmax 9' 9 8 ν 7' (log Ɛ) nm: 206 (4.58), 272 (4.46); IR (KBr) max 5 4' 6' -1 1 1 3 cm : 3600, 1610-1500, 1645; H NMR H CO O OCH 3 2' 3 δ OH OH (CD 3OD, 500MHz) : 6.80 (2H, d, J = 2.0 Hz, 126 H-6, 6'), 6.60 (2H, d, J = 2.0 Hz, H-4,4'), 5.94

(2H, tdd, J = 6.5, 10.0, 17.0 Hz, H-8,8'), 5.08 (2H, tdd, J = 1.0, 2.0, 17.0 Hz,

Ha-9,9'), 5.04 (2H, tdd, J = 1.0, 2.0, 10.0, Hb-9, 9'), 3.80 (6H, s, OCH 3),

3.40 (4H, d, J = 7.0 Hz, H-7, 7'); 13 C NMR (CD 3OD, 125MHz) δ: 150.2 (C-3,

3'), 141.5 (C-1,1'), 138.8 (C-8,8'), 138.6 (C-2,2'), 132.0 (C-5,5'), 116.5 (C-

6,6'), 115.8 (C-9,9'), 110.7 (C-4,4'), 40.7 (C-7,7'); HR-EIMS: m/z 342.1459

(calcd. for C20 H22 O5, 342.1467).

4.6.3.2 Lupeol (53)

30 Colorless needles (5 mg); M.P.: 214-215°C;

22 29 ααα 25 - 1 9 [ ]D : +27 ( c = 0.10, CHCl 3); IR νmax (CHCl 3) cm 21 1 2 18 25 11 2 6 1 1 28 : 3455, 3075, 1645, 880; H NMR (CDCl 3, 1 1 5 H 8 3 5 7 27 400MHz) δ: 3.21 (1H, dd, J = 4.5, 9.9 Hz, H-3), HO

23 24 53 1.07, 0.96, 0.93, 0.83, 0.78, 0.76 (3H, s, each

Me), 4.75 and 4.62 (2H, br s, 1H each, H-29), 1.64 (3H, br s, H-30); 13 C

122

NMR (CDCl 3, 100MHz) δ: 150.7 (C-20), 109.3 (C-29), 40.2 (C-17), 78.8 (C-

3), 55.4 (C-5), 50.5 (C-9), 48.3 (C-18), 47.8 (C-19), 42.9 (C-14), 40.9 (C-8),

39.9 (C-22), 38.8 (C-4), 38.7 (C-1), 38.2 (C-13), 37.2 (C-10), 35.6 (C-16),

34.3 (C-7), 29.9 (C-21), 28.2 (C-23), 27.6 (C-2), 27.4 (C-15), 25.3 (C-12),

20.9 (C-11), 19.3 (C-30), 18.4 (C-6), 18.3 (C-28), 16.3 (C-25), 15.9 (C-26),

15.6 (C-24), 14.7 (C-27); HR-EIMS: m/z 426.3830 (calcd. for C 30 H50 O,

426.3861); ElMS: m/z 426 [M]+, 411, 408, 393, 385, 220, 218, 207, 189,

139.

4.7.3 β-Sitosterol (30)

0 29 Colorless needles (5 mg) ; M.P.: 135 C; 28

21 22 25 20 26 ααα 25 19 [ ]D : +35.6 ( c = 0.22, CHCl 3); IR νmax 23

12 27 17 18 11 (CHCl 3)cm -1: 3450, 3055, 1650, 815; 1H 15 1 8 H H 3 5 7 NMR (CDCl 3, 500MHz) δ: 5.32 (1H, m, H- HO 30 6), 3.51 (1H, m, H-3), 1.22 (3H, s, H-19),

0.98 (3H, d, J = 6.2 Hz, H-21), 0.85 (3H, t, J = 7.0 Hz, H-29), 0.84 (3H, d, J

= 6.5 Hz, H-26), 0.82 (3H, d, J = 6.5 Hz, H-27), 0.67 (3H, s, H-18); 13 C NMR

(CDCl 3, 125MHz) δ: 140.6 (C-5), 121.9 (C-6), 71.6 (C-3), 56.7 (C-14), 56.3

(C-17), 50.8 (C-9), 50.5 (C-24), 42.5 (C-4), 42.4 (C-13), 40.5 (C-12), 37.5 (C-

1), 36.6 (C-10), 36.5 (C-20), 34.2 (C-22), 32.3 (C-7), 32.2 (C-8), 32.0 (C-2),

29.3 (C-23), 28.4 (C-16), 26.4 (C-25), 24.5 (C-15), 23.1 (C-28), 21.1 (C-11),

19.8 (C-27), 19.4 (C-19), 19.1 (C-21), 18.8 (C-26), 12.1 (C-29), 11.8 (C-18);

123

HR-EIMS: m/z 414.3850 (calcd. for C 29 H50 O, 414.3861); EIMS: m/z 414,

399, 396, 381, 329, 275, 273, 255.

4.6.3.4 Octadecyl ( E)-p-coumarate (124)

O White amorphous powder (15mg); M.P.: 7 1' 3 1 9 0 8 O (CH2)16CH3 98-100 C; UV (MeOH) λmax (log Ɛ) nm: 204

HO 5 124 (3.98), 227 (3.86), 312 (4.11), 425 (2.67);

1H NMR (CD 3OD, 300MHz) δ: 7.60 (1H, d, J = 18.0 Hz, H-7), 7.43 (2H, d, J

= 7.8 Hz, H-2, 6), 6.81 (2H, d, J = 7.8 Hz, H-3, 5), 6.25 (1H, d, J = 18.0 Hz,

H-8), 4.21 (2H, t, J = 6.8 Hz, H-1'), 1.70 (2H, m, H-2'), 1.25 (30H, brs, H-3'-

17'), 0.88 (3H, t, J = 6.0 Hz, H-18'); 13 C NMR (CD 3OD, 75MHz) δ: 167.5 (C-

9), 157.5 (C-4), 144.2 (C-7), 129.9 (C-2,6), 115.9 (C-3,5), 115.7 (C-3), 64.6

(C-1'), 22.6-31.9 (C-2'-17'), 14.1 (C-18'); HR-EIMS: m/z 416.3288 (calcd. for

C27 H44 O3, 416.3290).

4.6.3.5 4-hydroxy-3-methoxybenzaldehyde (125)

0 7 White needles (25 mg); M.P.: 81-82 C; UV (MeOH) λmax (log CHO 1 Ɛ) nm: 234 (4.19), 281 (4.02), 310 (3.01); IR (KBr) νmax cm -1:

5 3 OCH3 3286, 1657, 1608, 1513, 1245, 1150, 1082, 1042, 831; 1H OH 125 NMR (CD 3OD, 400MHz) δ: 9.74 (1H, s, H-7), 7.42 (1H, dd, J

= 1.6, 8.4 Hz, H-6), 7.29 (1H, d, J = 1.6 Hz, H-2), 7.09 (1H, d, J = 8.4 Hz, H-

5), 3.94 (3H, s, OCH 3); 13 C NMR (CD 3OD, 100MHz) δ: 193.1 (C-7), 155.0 (C-

124

4), 148.4 (C-3), 131.8 (C-1), 126.0 (C-6), 114.8 (C-5), 112.1 (C-2), 56.5

(OCH 3); HR-EIMS: m/z 152.0471 (calcd. for C 8H8O3, 152.0473).

4.6.3.6 4-Hydroxybenzoic acid (127)

0 7 Crystalline solid (10 mg); M.P.: 213-214 C; UV (MeOH) λmax (log COOH 1 Ɛ) nm: 222 (3.80); 310 (3.89); IR (KBr) νmax cm -1: 3515, 3335-

5 3 2730, 1710; 1H NMR (CD 3OD, 400MHz) δ: 7.86 (2H, d, J = 8.8 OH 127 Hz, H-2,6), 6.80 (2H, d, J = 8.8 Hz, H-3,5); 13 C NMR (CD 3OD,

100 MHz) δ: 169.5 (C-7), 163.7 (C-4), 132.9 (C-2,6), 123.0 (C-1), 115.9 (C-

3,5); HR-EIMS: m/z 138.0321 (calcd. for C 7H6O3, 138.0316).

4.6.3.7 4-Hydroxy-3-methoxybenzoic acid (128)

Crystalline solid (22 mg); M.P.: 210 0C; UV (MeOH) λmax (log Ɛ) 7 COOH 1 nm: 289 (4.5), 258 (4.7), 215 (4.9); IR (KBr) νmax cm -1: 3510,

3 5 OCH3 3335-2730, 1705; 1H NMR (CD 3OD, 500MHz) δ: 7.55 (1H, d, OH 128 J = 2.0 Hz, H-2), 7.53 (1H, dd, J = 2.0, 8.5 Hz, H-6), 6.82

(1H, d, J = 8.5 Hz, H-5), 3.88 (3H, s, OCH 3); 13 C NMR (CD 3OD, 125MHz) δ:

169.8 (C-7), 152.5 (C-4), 148.6 (C-3), 127.2 (C-1), 125.2 (C-6), 115.7 (C-5),

113.8 (C-2), 56.3 (OCH 3); HR-EIMS : m/z 168.0419 (calcd. for C 8H8O4,

168.0422).

125

4.6.3.8 4-Hydroxy-trans-cinnamic acid (129 )

0 O Yellow powder (15 mg); M.P.: 210-212 C; UV 7 3 1 9 8 OH (MeOH) λmax (log Ɛ) nm: 292 (4.69), 308 (4.50); IR HO 5 (KBr) ν cm -1: 3400, 3300-2200, 1680, 1620, 129 max

1420, 1380; 1H NMR (CD 3OD, 400MHz) δ: 7.44 (2H, d, J = 8.6 Hz, H-2,6),

7.22 (1H, d, J = 15.8 Hz, H-7), 6.80 (2H, d, J = 8.6 Hz, H-3,5), 6.23 (1H, d, J

= 15.8 Hz, H-8); 13 C NMR (CD 3OD, 100MHz) δ: 170.6 (C-9), 158.7 (C-4),

138.2 (C-8), 129.4 (C-2,6), 126.8 (C-1), 115.5 (C-3,5), 114.6 (C-7); HR-

EIMS : m/z 164.0475 (calcd. for C 9H8O3, 164.0473).

4.6.3.9 Methyl gallate (130 )

0 7 Crystalline solid (20 mg); M.P.: 157-158 C; UV COOCH3 1 (MeOH) λmax (log Ɛ) nm: 278 (3.78), 283 (4.10); IR (KBr) νmax

5 3 HO OH cm -1: 3375, 1698, 1619; 1H NMR (MeOH, 500MHz) δ: 7.03 OH 130 (2H, s, H-2,5), 3.80 (3H, s, OCH 3); 13 C NMR (MeOH, 125

MHz) δ: 169.0 (C-7), 146.5 (C-3,5), 139.7 (C-4), 121.5 (C-1), 110.0 (C-2,6),

56.3 (OCH 3); HR-EIMS : m/z 184.0345 (calcd. for C 8H8O5, 184.0371)

126

4.6.3.10 3,4-Dihydroxybenzoic acid (131)

0 7 Crystalline solid (15 mg); M.P.: 198-200 C; UV (MeOH) λmax COOH 1 (log Ɛ) nm: 289 (4.5), 258 (4.7), 215 (4.9); IR (KBr) νmax cm -1: 3 5 OH 3505, 3235-2857, 1710; 1H NMR (CD 3OD, 500MHz) δ: 7.42 OH 131 (1H, dd, J = 1.5, 8.5 Hz, H-6), 7.41 (1H, d, J = 1.5 Hz, H-2),

13 6.78 (1H, d, J = 8.5 Hz, H-5); C NMR (CD 3OD, 125MHz) δ: 169.4 (C-7),

151.1 (C-4), 145.2 (C-3), 124.2 (C-1), 123.8 (C-6), 117.7 (C-2), 115.7 (C-5);

HR-EIMS : m/z 154.0254 (calcd. for C 7H6O4, 154.0266).

4.6.3.11 4',5,7-Trihydroxyflavone (132)

0 3' OH Yellow needles (35 mg); M.P.: 352 C; UV (MeOH)

1 1' HO 7 9 O 5' λmax (log Ɛ) nm: 269 (4.2), 340 (4.32); IR (KBr)

3 10 4 5 νmax cm -1: 3454, 1650, 1590; 1H NMR (CD 3OD, OH O 132 500MHz) δ: 7.85 (2H, d, J = 8.5 Hz, H-3',5'), 6.93

(2H, d, J = 8.5 Hz, H-2', 6'), 6.58 (1H, s, H-3), 6.45 (1H, d, J = 2.0 Hz, H-8),

6.20 (1H, d, J = 2.0 Hz, H-6); 13 C NMR (MeOH, 125MHz) δ: 183.9 (C-4),

166.3 (C-2), 166.1 (C-7), 163.2 (C-9), 162.1 (C-4'), 159.4 (C-5), 129.4 (C-

3',5'), 123.2 (C-1'), 117.0 (C-2',6'), 105.3 (C-10), 103.8 (C-3), 100.1 (C-6),

95.0 (C-8); HR-EIMS: m/z 270.0512 (calcd. for C 15 H10 O6, 270.0528).

127

4.6.3.12 3',4',5,7-Tetrahydroxyflavone (133)

0 OH Yellow needles (18 mg); M.P.: 325 C; UV (MeOH) 3' OH λmax (log Ɛ) nm: 253 (4.5), 265 (4.6), 347 (3.7); IR 1 1' HO 7 9 O 5' -1 1 3 ν 10 4 (KBr) max cm : 3455, 1650, 1590; H NMR 5 OH O 133 (CD 3OD, 500MHz) δ: 7.39 (1H, dd, J = 2.5, 7.5,

Hz, H-6'), 7.37 (1H, br s, H-2'), 6.90 (1H, d, J = 8.5 Hz, H-5'), 6.53 (1H, s, H-

3), 6.43 (1H, d, J = 2.5 Hz, H-8), 6.20 (1H, d, J = 2.0 Hz, H-6); 13 C NMR

(CD 3OD, 125MHz) δ: 183.8 (C-4), 166.3 (C-2), 166.1 (C-7), 163.2 (C-9),

159.4 (C-5), 151.0 (C-4'), 147.0 (C-3'), 123.7 (C-1'), 120.3 (C-6'), 116.8 (C-

5'), 114.2 (C-2'), 105.3 (C-10), 103.9 (C-3), 100.1 (C-6), 95.0 (C-8); HR-

EIMS m/z: 286.0435 (calcd. for C 15 H10 O6, 286.0477).

4.6.3.13 3,3',5,7-Tetrahydroxy-4'-methoxyflavone (134)

0 OH Yellow prism (22mg); M.P.: 259 C; UV (MeOH) 3' OCH3 λmax (log Ɛ) nm: 259 (4.5), 375 (3.63); IR (KBr) 1 1' HO 7 9 O 5' ν -1 1 10 3 max cm : 3450, 1655, 1595; H NMR 4 5 OH

OH O (CD 3OD, 400MHz) δ: 7.99 (1H, d, J = 1.6 Hz, 134 H-2'), 7.63 (1H, dd, J = 1.6, 8.4 Hz, H-6'), 6.91 (1H, d, J = 8.4 Hz, H-5'),

6.40 (1H, brs, H-8), 6.16 (1H, brs, H-6), 3.93 (3H, s, OCH 3); 13 C NMR

(CD 3OD, 100MHz) δ: 178.7 (C-4), 166.5 (C-7), 162.8 (C-5), 159.5 (C-2),

158.6 (C-9), 151.5 (C-3'), 148.7 (C-4'), 133.6 (C-3), 123.9 (C-6'), 122.4 (C-

128

1'), 116.2 (C-5'), 114.3 (C-2'), 105.6 (C-10), 100.5 (C-6), 94.9 (C-8), 56.8

(OCH 3); HR-EIMS: m/z 316.0583 (calcd. for C 16 H12 O7, 316.0583).

4.6.3.14 Apigenin 7-O-(4''-O-p-E-coumaroyl-β-D-glucopyranoside (135)

O OH 3' OH 9''' 3''' 4'' 6'' O 1''' 1 1' O O O 5' HO 7 7''' 5''' 3'' OH 1'' 9 HO 10 4 3 5 OH O 135

White amorphous solid (15 mg); [ααα]D25 : -98.0 ( c = 0.08, MeOH); UV (MeOH)

λmax (log Ɛ) nm: 268 (4.5), 320 (4.6); IR (KBr) νmax cm -1: 3286, 1657, 1608,

1513, 1245, 1150, 1082, 1042, 831; 1H NMR (CD 3OD, 500MHz) δ: 7.89

(2H, d, J = 8.5 Hz, H-2', 6'), 7.71 (1H, d, J = 16.0 Hz, H-3'''), 7.49 (2H, d, J =

8.5 Hz, H-5''',9'''), 6.94 (2H, d, J = 9.0 Hz, H-3', 5'), 6.84 (1H, d, J = 2.0 Hz,

H-8), 6.82 (2H, d, J = 9.0 Hz, H-6''', 8'''), 6.66 (1H, s, H-3), 6.53 (1H, d, J =

2.0 Hz, H-6), 6.41 (1H, d, J = 15.5 Hz, H-2'''), 5.16 ( 1H, d, J = 8.0 Hz, H-

1''), 4.96 (1H, t, J = 9.5 Hz, H-2''), 3.81 (1H, m, H-5''), 3.78 (1H, m, H-3''),

3.68-3.61 (2H, m, H-6'') 13 C NMR (CD 3OD, 125MHz) δ: 184.1 (C-4), 168.5

(C-1'''), 166.8 (C-2), 164.7 (C-7), 163.0 (C-4'), 163.0 (C-5), 161.5 (C-7'''),

159.0 (C-9), 147.4 (C-3'''), 131.3 (C-5''',9'''), 129.7 (C-2',6'), 127.1 (C-4'''),

123.1 (C-1'), 117.1 (C-3',5'), 116.9 (C-6''',8'''), 114.7 (C-2'''), 104.2 (C-3),

101.5 (C-6), 101.2 (C-1''), 96.1 (C-8), 76.5 (C-5''), 75.6 (C-3''), 74.9 (C-2''),

129

72.1 (C-4''), 62.2 (C-6''); HR-EIMS: m/z 579.1499 [M+H] + (calcd. for

C30 H27 O12 , 579.1505).

130

131

5.1 The Introduction of the Genus Carissa

Carissa is a small genus of about 30 species and belongs to family

Apocynaceae (Dogbane family)(Hooker, 1882; Trease and Evans, 2002). The members of this genus are distributed mostly in tropical and subtropical regions of Asia, Australia and Africa (Li et al., 2012). The name Carissa is derived from the Indian name which contains a bitter and poisonous glucoside called “carissin” in their bark. Most of the plants of this genus are herbs, shrubs and small trees which possess attractive glossy foliage, sweet scented white flowers, decorative and edible red colored fruit. Some of the members are decorative and ornamental and make beautiful hedges.

Two species of the genus Carissa are found in Pakistan namely C. carandas and C. opaca . C. carandas is extensively grown as a hedge plant and found in dry cultivated areas. Its fruits are edible and purple black when ripe and its young branches and leaves are very glabourous, whereas

C. opaca is wild plant extensively found in drier parts of Punjab, Himalayas and up to 6000 feet in Murree.

The fruit is a berry which is edible and rich source of magnesium, phosphorus, calcium and vitamin C. Beside fruit, plants are poisonous as they possess sharp thorns, therefore they are used as safety hedges

(Nazimuddin and Qaiser, 1983b).

132

5.2 Botanical Description

The plants which belong to the genus Carissa are shrubs with thick branches ranging from 2 to 10 m in height. They possess spines straight or secondary curved, leaves opposite and curvaceous, 3-8 cm long, thick and soft. Inflorescence is a terminal auxiliary, peduncled, trichotomous cyme, flowering is observed throughout the year. The flowers are 1-5 cm in length with five lobed pink corollas, and possess fragrance therefore important as garden plants. Fruit are in form of juicy berry which is somewhat elliptical and 1.5-6 cm in length dark purple in color and possess mostly a flavor of strawberry and apple.

5.3 Pharmacological Importance of some Species of Genus Carissa

Some species of the genus Carissa are medicinally very important which are being utilized in folk remedies for various diseases as C. carandas , C. edulis , C. spinarum , C. opaca , C. lanceolata , and C. congesta .

Carissa edulis: is a thorny shrub and is frequently found in Africa

(Bentley et al., 1984). Its fruit is edible and roots with the pungent taste are used in folk medicine against sickle cell anemia and hernia and also used in the conditions of oedema, toothache, cough, ulcers (Abate, 1989); (Addis et al., 2001), chest complaints (Bently et al., 1984), rheumatism (Giday,

2001), headache, rabies and as diuretic (Addis et al., 2001). The antidiabetic activity of the C. edulis has been studied and observed that

133 oral administration of the ethanolic extract significantly lowered the blood glucose level in streptozotocin (STZ) diabetic and as well as normal rats (El-

Fiky et al., 1996). Root extract showed significant diuretic activity in rats

(Nedi et al., 2004). Aqueous extract of the root possesses significant anti-

HSV activity against both wild type and resistant strain of HSV (Tolo et al.,

2006). The aqueous extracts of root bark, stem bark, leaves, fruit and seeds showed analgesic activity where the fruit with highest activity (Ibrahim et al., 2007). Root bark extract of the plant showed considerable anticonvulsant activity and showed dose dependant inhibition of the convulsion which support ethno medicinal use of the plant to treat epilepsy

(Ya'u et al., 2008).

Carissa carandas : a shrub commonly found in south Asia and Thailand.

Roots of the plant have been used as folk medicine for diarrhea, stomachache, anthelmintic symptoms (Taylor et al., 1996) and extract of the roots used to lower the blood pressure (Chatterjee and Roy, 1965). Fruit possess analgesic, anti-inflammatory (Sharma et al., 2007) and lipase activity (Mala and Dahot, 1995). The ethanolic extract of the roots of C. carandas possesses histamine releasing activity (Joglekar and Gaitonde,

1970).

Significant analgesic and anti-inflammatory activities have been shown by ethanolic extract of C. carandas fruit in experimental animals

134

(Sharma et al., 2007). Anticancer activities of the extract of leaves, fruit ripe and unripe of C. carandas were carried out in various organic solvents and showed that chloroform extract of the leaves and n-hexane extract of the unripe fruit possess significant activity against Caov-3 with EC50 value of

7.72 and 2.94µg/ml (Sulaiman et al., 2008c). This group also studied antioxidant activities of fresh ripe and unripe fruit of C. carandas which revealed that although all fraction possesses good antioxidant activities but chloroform fraction of the unripe fruit possess greatest activity (Sulaiman et al., 2008a).

Ethanolic extract of the root of C. carandas displayed

Hepatoprotective activities against CCl 4 and paracetamol induced hepatotoxicity, data indicate that oral administration of the ethanolic extract reduced activities of serum marker enzymes, bilirubin and lipid peroxidation in a dose dependant manner (Hegde and Joshi, 2009).

Ethanolic extract of roots of C. carandas possesses significant activity against tonic seizers (Hegde et al., 2009). Antidiabetic activities of methanolic extract and ethyl acetate fraction of the plant have been studied. The polyphenolic, flavonoid and flavanone components of various fractions of the plant showed considerable antidiabetic activity (Tankar et al., 2011). The berries of C. carandas are rich source of proteins, lipids, sugar and iron. LC-MS/MS analysis of the plant revealed the presence of

135 different polyphenolic compounds and peel extract of the fruit showed high antioxidant activity (Patil et al., 2012).

Aqoueous, ethanol, methanol and ethyl acetate extracts of the leaves of C. carandas were screened and results showed that methanolic extract possess the highest activity more than standard tetracycline (Agarwal et al.,

2012). The lipid lowering activity of the aqueous and methanol (1:1) extract of the leaves of C. carandas in hyperlipidimic rats were studied, and observed that it causes a considerable decrease in body weight, cholesterol, triglyceride, HDL and LDL with the activity similar to Atrovastatin (Sumbul and Ahmed, 2012).

Carissa spinarum : Investigations on the ethanolic extract of the root of the

C. spinarum possess significant antipyretic activity as tested on albino mice which reduced the body temperature in a dose dependant manner

(Hedge and Joshi, 2010c). Ethanolic extract of the plant also possess hepatoprotective and antioxidant activities, oral pretreatment of the extract in mice significantly reduce the hepatotoxicity in a dose dependant manner and possess strong antioxidant activity (Hedge and Joshi, 2010b). Studies on ethanolic extract of the roots of C. spinarum revealed that it possess significant antiarthritic activities (Hedge and Joshi, 2010a).

Methanolic extract of the C. spinarum was investigated for wound healing properties on burn wound model rats (Sanwal and Kumar, 2011).

136

Anticancer activities of n-butanol fraction of the aqueous extract of the C. spinarum stem revealed that apotosis is induced by n-butanol fraction of the plant through mitochondrial dependant pathway in HL-60 cells (Sehar et al., 2011). More over C. spinarum is used for treatment of chronic joint pains (Wambugu et al., 2011), gastrointestinal disorders, parasitic infections (Teklehaymanot and Giday, 2007) and anaplasmosis (Grade et al., 2009b).

Carissa lanceolata: is a poisonous herb commonly found in tropical areas of Western Australia, Queensland and Northern Territory (Webb, 1969),

(Wheeler et al., 1992). All parts of the plants are medicinally important and used by local people for the treatment of various diseases like respiratory infections, toothache, cleaning of sours, cold and flu, chest pains and rheumatism (Reid and Betts, 1979).

5.4 Previous Phytochemical Investigations on Genus Carissa

Phytochemical investigations on genus Carissa started with the isolation of quebrachitol ( 136 ) from twigs of C. edulis (Victor, 1965). Polar glycosides have been isolated from C. carandas which upon enzymatic and mild acid hydrolysis afforded odoroside H ( 137 ), digitoxigenin ( 138 ), 14,15- anhydrodigitoxigenin ( 139 ), glucose ( 140 ) and D-digitalose ( 141 ) (Rastogi et al., 1967). Five cardiac glycosides were isolated from C. spinarum, three of

137 them were identified as odoroside H ( 137 ), evomonoside ( 142 ) and odoroside G ( 143 ) (Rastogi et al., 1969).

O O O O OH HO OCH H 3 OH H O H OH HO OH H CO O H OH OH 3 H OH HO 136 137 H 138 O O O

O OH H OH H HO H CO H 3 H HO OH HO H O H OH H OH O HO 140 139 141 HO OH O O O OH O O HO H HO O OH H H OH O HO O H OH HO HO O H OH H CO O 142 3 OH H 143

A lignin carinol ( 144 ) has been reported from C. congesta (Pal et al.,

1975). Phytochemical investigations on C. edulis yielded 2'- hydroxyacetophenone ( 145 ), vanillin ( 125 ), catalponol ( 146 ), carissone

(147 ), 3,4'-dihydroxypropiophenone ( 148 ), coniferaldehyde ( 149 ), scopoletin ( 95 ), isofraxidin ( 150 ), (-)-nortrachelogenin ( 151 ), carinol ( 144 ), 138 carissanol ( 152 ), (+)-lariciresinol ( 153 ), (-)-secoisolariciresinol ( 154 ) and (-)- olivil ( 155 ) (Hans et al., 1983). 2-hydroxyacetophenone ( 145 ) was isolated as principal volatile compound from root of C. edulis (Bentley et al., 1984).

Phytochemical studies on C. edulis resulted in isolation of carissone

(147 ), cryptomeridiol ( 156 ), β-eudesmol ( 157 ), carissanol ( 152 ), β- carissanol ( 158 ), α-carissanol ( 159 ), germacrenone ( 160 ) (Hans et al.,

1985). Soluble phenolics and insoluble proanthocyanadins are also reported from C. edulis (Reed, 1986).

139

Ursolic acid ( 161 ) and naringin ( 162 ) were isolated from C. spinarum

(Mathurum et al., 1998). Phytochemical investigations of the C. lanceolata carried out to afford carissone ( 147 ), dehydrocarissone ( 163 ) and carindone

(164 ) (Lindsay et al., 2000). Four pentacyclic triterpenoids namely carissin

(165 ), 3-β-hydroxy-27-p-E-coumeroyloxyurs-12-en-28-oic acid ( 166 ), oleanolic acid ( 111 ) and ursolic acid ( 161 ) were isolated from fresh leaves of

C. carandas (Siddiqui et al., 2003).

140

OH OH O HO HO O O COOH O O HO OH OH O HO HO 161 HO OH OH 162 163

OH HO COOH O COOH O O HO OO OH O HO O OCH O 3 OH 166 164 165 OH

A germacrane derivative carenone ( 167 ), 3'-(4''-methoxyphenyl)-3'- oxo-propionyl hexadecanoate ( 168 ), germacrenone ( 160 ), coniferaldehyde

(149 ), pinorsinol ( 169 ), (-)-nortrachelogenin ( 151 ), (-)-carissanol ( 152 ), (-)- secoisolariciresinol ( 154 ), (-)-carinol ( 144 ), and (-)-olivil ( 155 ), are reported from the stem of C. spinarum (Rao et al., 2005). Investigations on essential oils of flower of C. opaca resulted in the isolation of palmitic acid ( 25 ), benzyl salicylate ( 170 ), carissone (147 ), benzyl benzoate ( 171 ), α-farnesene

(172 ) (Rai et al., 2005).

141

Antibacterial compounds have been isolated from the roots of C. lanceolata by Hettiarachchi et al namely 2'-hydroxyacetophenone ( 145 ) , carinol ( 144 ) and carissone ( 147 ) (Hettiarachchi et al., 2009).

C. carandas provided compounds sesquiterpene glucoside, carandoside ( 173 ), (6 S,7 R,8 R)-7a-[( β-glucopyranosyl) oxy]lyoniresinol ( 174 ),

(6 S,7 S,8 S)-7a-[( β-glucopyranosyl)oxy]lyoniresinol ( 175 ), carissanol ( 152 ) and (-)-nortrachelogenin ( 151 ) (Wangteeraaprasert and Likhitwitayawuid,

2009).

C. edulis is screened for its bioactive metabolites and resulted in the isolation of lupeol ( 53 ), oleuropin ( 176 ), carissol ( 177 ) and β-amyrin ( 178 ) and these compounds were evaluated against viral strains of Herpes simples virus types 1, lupeol ( 53 ) was found to possess significant antiviral activity (Tolo et al., 2010).

142

Phytochemical investigations on C. opaca yielded flavonoids, terpenoids, coumerins, anthraquinones and cardiac glycosides as well as isoquercetin ( 179 ), vitexin ( 57 ), myricetin ( 180 ) and kaempferol ( 181 ) from methanolic extract of the plant (Sahreen et al., 2011a). Antibacterial compounds 2'-hydroxyacetophenone ( 145 ), a lignin, carinol ( 144 ) and carissone ( 147 ) have been isolated from C. lanceolata and isolated compounds showed significant antibacterial activity (Hettiarachchi et al.,

2011).

C. macrocarpa yielded pentacyclic triterpenes namely β-amyrin ( 178 ), methyl oleanolate ( 182 ), oleanolic acid ( 111 ), 3-α-hydroxyoleane-11-en-

28,13-β-olide ( 183 ) and ursolic acid ( 161 ) from its leaves (Moodley et al.,

2011).

143

C. spinarum was subjected to phytochemical investigations and evaluated for different activities, twelve compounds isolated namely scopoletin ( 95 ), (-)-nortrachelogenin ( 151 ), carissanol ( 152 ), (-)-carinol

(144 ), (+)-cycloolivil ( 184 ) , (+)-8-hydroxypinoresinol ( 185 ), (-)-olivil ( 155 ), (-

)-secoisolariciresinol ( 154 ), (+)-pinoresinol ( 186 ), carissone ( 147 ), odoroside

H ( 137 ) and evomonoside ( 142 ) (Wangteeraprasert et al., 2012).

144

Analysis of aerial parts of C. carandas showed the presence of various chemical constituents like alkaloids, flavonoids, phenolics, saponins and glycosides (Motwani et al., 2012). Stigmasterol (32 ), ursolic acid ( 161 ), lupeol ( 53 ), campesterol, 17-hydroxy-11-oxo-nor-β-amyrone ( 187 ) and urs-

12-ene-3β,22 β-diol-17-carboxylic acid ( 188 ) have been isolated from petroleum ether extract of the roots of C. spinarum (Hedge et al., 2012).

145

146

6.1 Introduction of Carissa opaca

Carissa opaca is indigenous to the subcontinent and commonly found in various regions of Pakistan, India, Burma and Ceylon. The plant has also been introduced in South East Asia, Indonesia, Malaysia and

Cambodia. It is known by various local names in different regions:

Karandan or Karendang in Indonesia, Kerenda, Bernda in Malaysia,

Caramba, caraunda in Phillipines, Caranda in spainish, Carandeira in

Portuguese, Nam phrom in Thai, Garna in Hindi and Gerna, Karanda or

Kakranda in Pakistan (Lim, 2012). Its fruit is eaten and flowers used in perfumery.

C. opaca is a cold tolerant bush which grows from sea level to 1800 meter in Himalayas. Most commonly it is grown in the semi arid coastal areas of the tropical regions. It can also be found in moist areas as in the base of the hills and tropical rain forests. In Pakistan, it is frequently found in Punjab, Himalayas and up to 6,000 ft in Murree (Nazimuddin and

Qaiser, 1983a). Its fruit is berry with sweet-sour taste which is edible and also used to make pickles while its leaves and branches are used as fodder for cattle. The twigs of plant also used as fire wood. As the plant possesses sharp thorns and form excellent hedges therefore it is also used for safety and ornamental purposes (Abbasi et al., 2012).

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6.2 Botanical Description of Carissa opaca

C. opaca is sprawling native ever green shrub which grows up to 3.5 meter high but usually 1-2 meter tall. Branches are glabourous and possess sharp simple or forked thorns, which arise between the petiole and

2.5-3.5 cm long. Leaves are glabourous, opposite, broadly ovate to oblong, shiny, leathery, dark green above, light green dull below. Inflorescence consists of terminal cymes commonly 3 flowered. Flowers are white or light rose scented, on 1.5-2.5 cm long peduncles; calex is synsepalous, corolla tube cylinder, synpetalous, 8-12 mm long. The fruit is broadly ovoid or ellipsoid 6-8 mm long berry, bluntly pointed, pinkish black or reddish purple when ripe, contains 2-4 small flat seeds embedded in pulp possess milky juice and edible (Lim, 2012); (Nazimuddin and Qaiser, 1983a).

6.3 Scientific Classification of Carissa opaca

Kingdom: Plantae

Order: Gentianales

Family: Apocynaceae

Subfamily: Rauvolfioideae

Tribe: Carisseae

Genus: Carissa

Species: opaca

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6.4 Pharmacological Importance of Carissa opaca

Different parts of the plant are used in folk medicine to treat various diseased conditions. The stem of the plant has been used as healing agent and as stimulant in Thailand (Wutthamawech, 1997). In India, the roots are used as purgative and as remedy for snakebite (Pakrashi et al., 1968).

The bark is effective for chicken pox and skin diseases (Grade et al.,

2009a). The plant also possesses hepatoprotective activities (Abbasi et al.,

2009). The roots and bark together are boiled and the extract is effective for the treatment of respiratory diseases such as asthma (Jabeen et al., 2009).

The decoction of fresh leaves of C. opaca is effective in the treatment of jaundice and hepatitis which is used orally (Abbasi et al., 2009).

The leaves of the plant are also used for tanning (Ravishankar et al.,

1991). As compared to twigs, fruit of the plant is a rich source of many essential nutrients as fat, protein, fiber and as well as significant amount of potassium, magnesium, iron, zinc copper and chromium; which minimizes the threat of many chronic diseases (Sarla et al., 2011). It is also effective in the conditions of cough, fever, eye disorders and diarrhea and as purgative

(Adhikari et al., 2007). The fruit of C. opaca in combination with roots of

Mimosa pudica are effective as aphrodisiac (Acharaya and Rai, 2009). In

Pakistan, the fruit and leaves of the plant have been used as cardiotonic

(Jabeen et al., 2009), (Saghir et al., 2001). The plant has also been known

149 to possess wound healing properties, therefore effective in animal horn injuries, maggot wounds and applied directly on the infected area (Dinesh et al., 1997).

Systematic pharmacological studies on the C. opaca provided scientific basis for the use of plant in folk medicine. Aqueous and chloroform extract of C. opaca leaves were tested for antioxidant activities, the presence of high concentration of flavonoids and phenolics were correlated with strong antioxidant activities of the fruit extract (Sahreen et al., 2010). Methanolic extract of C. opaca leaves showed hepatoprotective activities against CCl 4 induced liver damage in rats thus the methanolic extract possess significant hepatoprotective action against liver injuries.

This property was ascribed due to its membrane stabilizing and antioxidant activities (Sahreen et al., 2011b).

The ethanolic extract of the plant has been screened for its antimicrobial activity against thirteen different strains of gram positive and gram negative bacteria; the extract was significantly effective against food poisoning bacteria i.e. Streptococcus pyrogenes, Streptococcus aureus and

Bacillus cereus and fungus which provide the reason why this plant is used extensively as folk remedy for various ailments (Sarla et al., 2011).

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6.5 Results and Discussion

The systematic phytochemical studies on C. opaca resulted in the isolation of altogether eleven compounds; of them four (191 , 195, 196 and

198 ) were new and seven (67 , 189 , 190 , 192 , 193 , 194 and 197 ) known secondary metabolites which were all new source. All the compounds were characterized spectroscopically.

6.5.1 Characterization of cyclic trimer of ethylene terephthalate (191)

Compound 191 was isolated as colorless crystals. The HR-EIMS showed the molecular ion peak at m/z

576.1267 consistent with the molecular formula C 30 H24 O12 with 19 double bond equivalence. It showed UV absorptions at

228 and 320 nm while the IR spectrum showed absorptions for aromatic CH (3250 cm -1), ester (1760 cm -1) and oxymethylene (1250 cm -1). The 1H NMR spectrum displayed only two singlets at δ 8.08 and 4.66 while the analysis of 13 C NMR (BB and DEPT) spectrum displayed the signals at δ 165.2,

133.8, 129.7 and 62.7.

The HMBC correlations of aromatic proton at δ 8.08 (δc 129.7) with δ

129.7, 165.2 and 133.8 showed the presence of 1, 4-dicarboxylated

151 aromatic system and HMBC correlations of oxymethylene δH 4.66 ( δC 62.7) with the carbons δ 165.2 and 62.7 indicated the presence of dioxyethylene ester of aromatic 1,4-dicarboxylic acid (Fig. 6.1).

Fig. 6.1: Important HMBC correlations observed in spectra of 191

The HR-EIMS showed the molecular mass at m/z 576 which showed that compound 191 could be a cyclic trimer. Furthermore single X-ray crystallographic analysis confirmed the structure to be cyclic trimer of poly

(ethylene terephthalate)(Fig. 6.2). Literature showed that this compound is obtained as by product during the synthesis of Polyethylene terephthalate

(PET) polymer from monoethylene glycol (MEG) and terephthalic acid (TPA) or dimethyl terephthalate (DMT) along with other oligomers as side product

(Kim and Lee, 2012). No report for occurrence in natural sources or biological activities of 191 has been cited. Thus it is isolated for the first time from any natural source.

152

Fig. 6.2: A computer generated drawing of 191

6.5.2 Characterization of 30-Nor-2,3-dihydoxy-2ααα,3 β–dihydroxy-urs-

12-ene (195)

Compound 195 was isolated as white amorphous solid. The HR-EIMS showed the molecular ion peak at m/z 428.3654 consistent with the

29 molecular formula C 29 H48 O2 with six double 19 21 12 22

25 11 26 13 17 bond equivalence. The IR spectrum of 195 28 HO 1 9 15 2 showed absorption bands due to secondary 5 7 27 HO 4

24 hydroxyl groups (3415 cm -1) and olefinic system 23 195 (1630 cm -1). Molecular formula and analysis of 13 C NMR spectrum showed that the skeleton consist of 29 carbons atoms.

The 1H NMR spectrum of 195 showed six tertiary methyl resonances at δ 1.11, 1.01, 0.96, 0.84, and 0.80 corresponding to carbon values δ 24.0, 153

17.2, 17.5, 17.8, 17.6, and 29.3. Multiplicities of the carbons were determined by using DEPT experiments which revealed presence of seven methyl, nine methylene, seven methine and six quaternary carbon atoms in

195 . This information from 1H NMR along with molecular formula and 13 C

NMR suggested that the compound is a triterpene. The resonance for olefinic proton observed at δ 5.23 (1H, dd, J = 3.5, 4.0 Hz, H-12) supported the presence of double bond at C-12 and C-13 with 13 C NMR values at δC

126.7 and 139.7, respectively. The secondary methyl (H-29) observed at δH

0.89 ( J = 6.5) ( δC 21.5) and doublet for the (H-18) at δH 2.21 (1H, d, J = 11.5

Hz) suggested that compound 195 belong to the ursane series of triterpenoids.

The absence of C-30 doublet methyl and COSY correlation of H-19, δH

1.35 (1H, m), (δC 40.4) with a methylene observed at δH 1.62, 1.47 (2H, m,

H-20) indicated that the compound is 30-nor triterpene. This concept was further confirmed with HMBC correlations of H-29 with methylene at δC

30.7 (C-20) and 40.4 (C-19) and 54.3 (C-18) (Fig. 6.3).

Fig. 6.3: Important HMBC and COSY interactions in 195

154

Further analysis of the 1H NMR spectrum revealed that signal for H-3 was observed at δH 2.91 (1H, d, J = 10.0 Hz) which showed COSY correlation with an oxymethine observed at δH 3.61 (1H, ddd, J = 4.0, 4.5,

10.0 Hz, H-2) with carbon δC 69.5 as observed in HSQC this observation suggests that 3-positions in 195 is oxygenated. This fact was supported through HMBC correlations of H-3 with δC 69.5 (C-2), as well as δC 48.5 (C-

1), 40.5 (C-4), 29.3 (C-23), and δC 17.2 (C-24). Thus from all these facts it could be revealed that the compound 195 is 30-nor-3,4-dihydroxyurs-12- ene.

The stereochemistry of hydroxyl groups at C-2 and C-3 could be assigned by careful analysis of NOESY spectrum, molecular modeling, and coupling constants. The larger coupling constant ( J = 10.0 Hz) of H-3 as well as absence of NOESY correlation with H-25 suggested the α and axial configurations to this proton, while the splitting pattren and larger coupling consatant for H-2 (ddd, J = 4.0, 4.5, 10.0 Hz) suggested the β and axial orientation to the H-2. This observation was also confirmed through the

NOESY interaction of H-2 with H-25 (Fig. 6.4).

155

Fig. 6.4: Important NOESY correlations of 195

Moreover the remaining assignments were in accordance with the data reported for α-amyrin (Abd-El-Fattah et al., 1990). Based on these evidence, the compound 195 was found to be 2,3-dihydoxy 30-nor-2α,3 β– dihydroxyurs-12-ene which is a new natural product.

6.5.3 Characterization of 30-nor -2ααα,3 ααα,23-trihydroxyurs-12-ene (196)

Compound 196 was isolated as white amorphous solid. The HR-EIMS of the compound showed the molecular ion peak at m/z 444.3603 which correspond to the molecular formula C 29 H48 O3 with six double bond equivalence. The IR spectrum was similar to that for 195. The 1H NMR

spectrum showed similar pattern as observed in 29

19 21 that of 195 except the missing of a methyl 12 22 25 11 26 13 17 28

HO 1 9 15 singlet and appearance of a double doublet for 2

5 7 27 HO 4 an oxygenated methylene at δ 3.53 (1H, d, J = 23 24 OH 11.0 Hz, Ha-23) and 3.39 (1H, d, J = 11.0 Hz, 196

Hb-23) corresponding to the carbon at δ 71.3 (CH 2) which indicated that

156

CH3-23 has been oxidized which was further confirmed by HMBC correlation of H-3 with C-23 ( δ 71.3). The structure was further confirmed by HMBC and COSY correlations (Fig.6.5).

Fig. 6.5: Important HMBC and COSY correlations of 196

The stereochemistry at C-2 and C-3 positions was confirmed through coupling constant and NOESY correlations. Here H-2 and H-3 both displayed NOESY correlations with H-25 (Fig. 6.6). Thus the compound 196 could be 30-nor -2α,3 α,23-trihydroxyurs-12-ene.

Fig. 6.6: Important NOESY interactions observed in 196

157

6.5.4 Characterization of (2 S,3 S,4 R,15 E)-2-{[(2 R)-2-hydroxydocosanoyl] amino}eicos-15-ene-1,3,4-triol (198)

Compound 198 was isolated as white amorphous solid. The HR-

FABMS showed O OH 1' 22' HN 2' the molecular ion OH 16 20 HO 2 3 4 peak at m/z 1 15 19 OH 198 681.6271 and the molecular formula deduced as C 42 H83 NO 5 which indicated the presence of two double bond equivalences. The IR absorption bands at 3355, 1644 and 1625 cm -1 indicated the presence of hydroxyl groups, amide and olefinic groups.

The 1H NMR spectrum of 198 displayed signals for a secondary amide at δ 7.40 (1H, d, J = 8.5 Hz), an oxygenated methylene at δ 3.71 (1H, dd, J = 4.5, 12.0 Hz) and 3.63 (1H, dd, J = 4.5, 11.5 Hz), three oxymethines at δ 3.94 (1H, dd, J = 3.5, 8.0 Hz), 3.41 (1H, dt, J = 4.2, 6.3 Hz), 3.40 (1H, dd, J = 3.9, 4.5 Hz) and a signal at δ 3.98 (1H, m) was assigned to the methine proton vicinal to the nitrogen atom of the amide group. In addition signal for the olefinic protons and primary methyls were observed at δ 5.30

(2H, dt, J = 5.0, 16.5 Hz) and 0.77 (6H, t, J = 7.0 Hz) respectively.

In the 13 C NMR spectrum the signal for amide carbonyl was observed at δ 175.6, three oxymethine at δ 75.5, 72.1, 71.8, oxymethylene

158 at δ 60.9, and a methine bonded to nitrogen atom at δ 51.4, olefinic carbons at δ 130.6, 129.6, aliphatic chain at δ 22.5-31.7 and methyls at δ

13.9. The signals at δC 32.4 next to the double bond indicated the trans geometry of the double bond (Higuchi et al., 1994; Shibuya et al., 1990). All this data suggested 198 to be a sphingolipid (Ahmad et al., 2006b); (Riaz et al., 2012) and the entire sequence of the skeleton and substitutions were fixed by HSQC, 1H-1H COSY and long range HMBC correlations (Fig. 6.7).

Fig. 6.7: Important HMBC and COSY interaction of 198

The length of the fatty acid chain was fixed by the characteristic fragments at m/z 339 and 280 and the amide chain containing a double bond at m/z 308 (M-2H 2O) and 225 (Fig. 6.8).

Fig. 6.8: Mass fragmentation pattern in 198 159

Methanolysis of the 198 with methanolic HCl provided aliphatic oxygenated amine and methyl ester of the fatty acid (Ahmad et al., 2007).

These fragments were acetylated (Muralidhar et al., 2005) and analysed by

GC-MS and were identified as methyl 2-acetoxydocosanoate m/z 412 and

2-acetamino-1,3,4-triacetoxy-eicosene m/z 512. The position of the double bond was fixed between C-15 and C-16 by permanganate/periodate oxidative cleavage (Ahmad et al., 2007) of 2-acetamino-1,3,4-triacetoxy- eicosene yielding a mixture of carboxylic acids which on methylation and

GC-MS analysis provided peaks for 2-acetamino-1,3,4- triacetoxypentadecanoic acid m/z 473 and pentanoic acid m/z 102. The configuration at the stereogenic centers could be determined by the optical rotation of 198 ([ α]D = + 39.5) and its methanolysis products ([ α]D = ˗ 6.9 and + 19.3) which were comparable to those of the sphingolipids with (2 S,

3S, 4 R, 2' R) configurations (Muralidhar et al., 2003), (Natori et al., 1994),

(Riaz et al., 2004). Based on these evidences and experiments the structure of the 198 could be assigned as (2S,3S,4R,15E)-2-{[(2R)-2- hydroxydocosanoyl]amino}eicos-15-ene-1,3,4-triol.

160

6.5.5 Characterization of 3 β,27-dihydroxylup-12-ene (189)

Compound 189 was isolated as white crystalline solid. The molecular ion peak was observed in HR-EIMS at m/z 442.3813 consistent with the molecular formula C 30 H50 O2 with six degree of unsaturation. The IR spectrum displayed absorption bands for hydroxyl 30 29 22

-1 19 groups (3300-3255 cm ) and geminal methyl 21

25 11 26 13 17 28 -1 groups/isopropyl group (1370, 1344, 1332 cm ). 1 9 15 CH2OH 3 5 7 27 The EIMS of 189 , in addition to the molecular ion HO

23 24 peak at m/z 442 showed characteristic fragment 189 ions at m/z 234 and 207 due to retro Diels alder cleavage and at m/z 191 by the loss of isopropyl group from 234 suggested that 189 is lup-12-ene

(Budzikiewicz et al., 1963). More over the base peak was observed at 203 rather 234 which suggested that CH 3-27 has been oxidized to -CH 2OH

(Budzikiewicz and Thomas, 1980).

The 1H NMR spectrum displayed signal for olefinic, an oxymethylene and carbinylic proton at δ 5.12 (1H, t, J = 3.6 Hz), 3.51 (1H, d, J = 10.9 Hz), 3.18 (1H, d, J = 10.9 Hz) and 3.21 (1H, dd, J = 4.9, 10.8

Hz), respectively. The chemical shift and coupling constant for H-3 suggested the hydroxyl group with β and equatorial orientation in 189

(Pyrek, 1979). Moreover the 1H NMR showed signals for five tertiary methyls at δ 1.10 (3H, H-28), 0.97 (3H, H-23), 0.96 (3H, H-26), 0.92 (3H, H-24),

161

0.77 (3H, H-25) and two secondary methyl at δ 0.85 (3H, J = 5.2 Hz, H-29),

0. 79 (3H, J = 6.5 Hz, H-30).

Analysis of 13 C NMR spectrum (BB & DEPT) revealed the presence of a total of thirty carbons: which were seven methyl, ten methylene, six methine and seven methyl groups. The oxygenated methine and methylene were observed at δ 79.0 (C-3) and 69.9 (C-27) respectively while the olefinic carbons at δ 138.7 (C-13) and 125.0 (C-12). From above discussion and comparison with the literature, compound 189 was confirmed as 3β,27- dihydroxylup-12-ene (Siddiqui et al., 1989) and has been isolated for the first time from this source.

6.5.6 Characterization of lupeol-β-hydroxyoctadecanoate (190)

Compound 190 was isolated as white amorphous solid whose high resolution mass spectrum revealed the 30 29 22

19 molecular ion peak at m/z 736.6214 which was 12 21

25 13 17 26 28 attested for the molecular formula C 50 H88 O3 1 9 15

3 5 7 27 with seven degree of unsaturation. The IR O 1' 23 24

O 2' spectrum showed absorptions for secondary 3'4' 18' 11 HO hydroxyl group (3455 cm -1), trisubstituted 190 double bond (3075, 1444, 720 cm -1) and an ester group (1730 cm -1). The 1H

NMR of 190 was almost superimposable to that of compound 53 with few additional signals for an oxymethine at δ 3.96 (1H, m), a methylene δ 2.50

162

(1H, dd, 2.8, 16.0 Hz), 2.46 (1H, ddd, 3.2, 5.6, 16.0 Hz), a primary methyl at δ 0.83 (3H, t, J = 6.0 Hz) and a broad singlet for long hydrocarbon chain at δ 1.20 (28H, br s). Thus the compound 190 is a lupeol with esterification by a hydroxylated long chain at C-3 (Perveen et al., 2009). 190 was hydrolysed to identify the long chain fatty ester part whose HR-EIMS showed a peak at m/z 282.2550 calculated for the formula C 18 H34 O2.

Moreover comparison of IR and 1H NMR data with the literature values revealed the acid part as 3-hydroxyoctadecanoic acid (Pyrek, 1979). Thus the compound 190 is lupeol-β-hydroxy octadecanoate which has been confirmed from 13 C NMR spectral data and exactly matched with literature values (Wahyuono et al., 1987).

6.5.7 Characterization of pinoresinol (192)

The compound 192 was obtained as white amorphous solid. The exact mass was calculated though HR-EIMS which displayed molecular ion peak at m/z 358.1442 calculated for the molecular formula C 20 H22 O6 with ten double bond equivalents. The eight could be attributed to the two benzene rings as evident from the UV spectrum with the λmax at 224 and

280 nm and 1H NMR signals at δ 6.88 (2H, d, J = 1.6 Hz), 6.87 (2H, d, J =

8.0 Hz), 6.81 (2H, dd, J = 1.6, 8.0 Hz), 4.72 (2H, d, J = 4.0 Hz) with the corresponding carbon values at δ 145.2, 143.7, 132.9, 118.9, 114.2 and

108.5.

163

The IR data ( νmax: 3505, 3475, 1615, 1520 cm -1) suggested the presence of aromatic moieties and hydroxyl function in the molecule.

Moreover absorption bands for aromatic ether (1275, 1210 cm -1) and aliphatic ethers (980, 945 cm -1) were also observed. Further analysis of 1H

NMR spectrum showed signals at δ 4.24-3.86 OCH3 3' 4' OH (4H, dd, J = 6.8, 8.8 Hz) and 3.12 (2H, m) for O 1' 5' 8 6 two fused tetrahydrofuran rings which H 1 5 H 2 4 1' compensated rest of the two DBE (Achenbach 5' O

HO 3' et al., 1983) where as 13 C NMR spectrum OCH3 192 displayed carbons at δ 85.8, 71.6 and 54.1.

The two methoxyl groups were observed at δ 3.89 (6H, s, 2OCH 3) appeared in 13 C NMR spectrum at δ 56.5. Thus all above discussion revealed 192 to be a tetrahydrofuran lignin and this was further confirmed to be pinoresinol when matched with the literature values (Aguiar et al., 2012).

6.5.8 Characterization of (-) carinol (193)

The molecular formula of compound 193 was found to be C 20 H26 O7 with eight double equivalences as calculated from the HR-EIMS with the molecular ion peak at m/z 378.1643. While the EIMS displayed molecular ion peak at m/z 378. The IR spectrum revealed the presence of hydroxyl function, aromatic moieties (3500, 3432, 1610, 1520 cm -1) and aromatic ether (1270, 1210 cm -1) in the molecule. The compound with the UV

164 absorption bands at 228 and 284 nm suggested the presence of two benzene rings with ABX splitting pattern as evident from 1H NMR spectrum which displayed the aromatic signals at δ 6.85 (1H, d, J = 8.0 Hz), 6.82 (1H, d, J = 8.0 Hz), 6.81 (1H, d, J = 2.8 Hz), 6.79 (1H, dd, J = 2.8, 8.0 Hz), 6.70

(1H, d, J = 2.8 Hz), 6.69 (1H, dd, J = 2.8, 8.0 Hz), 6.67 (1H, d, J = 8.0 Hz).

In addition the two oxymethylenes were observed OH H3CO 3' 1' 7' 9' 8' OH δ 8 at 3.68 (2H, ddd, J = 2.2, 2.4, 11.0 Hz) and 3.51 9 OH HO 5' 7 1 H (2H, q, J = 11.6 Hz) while the two methylenes at

5 3 δ 2.96 (1H, dd, J = 2.8, 14.0 Hz), 2.48 (1H, dd, J OCH3 OH 193 = 11.6, 2.8 Hz) and 2.80 (2H, s, H-7'), a methine at δ 2.05 (1H, m) and two methoxy groups at δ 3.86 (3H, s) and 3.84 (3H, s) respectively.

The analysis of 13 C NMR spectrum displayed signals for an oxygenated quaternary carbon at δ 76.6, four methylenes at δ 65.2, 61.0,

40.2, 31.7 and a methine at δ 47.4 in addition to the signals for two aromatic moieties. From above discussion and spectral data, the compound

193 was found to be (-)carinol which was further confirmed when completely overlapped with the literature values (Pal et al., 1975).

165

6.5.9 Characterization of (-) carissanol (194)

Compound 194 showed molecular ion peak in HR-EIMS at m/z

376.1554 consistent with the molecular formula OH 7' OH H3CO 3' 1' 8' 9' C20 H24 O7 with nine degree of unsaturation. The 8 O 9 HO 5' 7 1 H 194 showed UV absorptions at λmax 226 and 282

3 ν 5 nm. The IR data [ max : 3510, 3430, 1615, 1520, OCH3 OH 1275, 1215, 980, 945 cm -1] indicated the presence 194 of aromatic ring, hydroxyl group aromatic and aliphatic ether functionalities.

The 1H NMR spectrum was almost super imposable to that of 194 but with few differences: an additional oxy methine at δ 5.20 (1H, d, J = 6.4

Hz) and the signals for one oxymethylene were absent. While in the 13 C

NMR spectrum the differences were due to this oxymethine appeared at δ

111.1 and shift in the signals of aliphatic region at δ 101.0, 78.9, 71.0,

43.2, and 32.5. All this discussion and spectral data suggested compound

194 as (-) carissanol which is further confirmed by comparison with the literature values (Achenbach et al., 1983).

166

6.5.10 Characterization of arjunolic acid (197)

The molecular mass of 197 was calculated from the HR-EIMS

29 30 spectrum which showed molecular ion peak at

19 21 m/z 488.7061 corresponds to the molecular 12 22 25 13 17 26 COOH 28 HO 1 9 15 formula C 30 H48 O5 with seven double bond 2 27 3 5 7 HO equivalence. The IR spectrum displayed 23 24 OH absorption bands for carboxylic group (3300- 197

2250, 1680 cm -1) and tri-substituted double bond (3070, 1445, 720 cm -1).

The 1H NMR spectrum displayed six methyl singlets at δ 1.17, 1.02,

0.93, 0.90, 0.81, 0.68 (3H each, s), olefinic multiplet at δ 5.23 (1H, m), two oxymethines at δ 3.68 (1H, m), 3.35 (1H, d, J = 9.5 Hz) and one oxy methylene at δ 3.50 (1H, d, J = 11.0 Hz), 3.26 (1H, d, J = 11.0 Hz). The 13 C

NMR spectrum displayed a total of 30 signals of which six methyls, ten methylene, six methine and eight quaternary carbons were observed. The carboxylic carbon and oxymethines were observed at δ 180.5, 78.2, 69.0, while oxymethylene at δ 66.1. All this data suggested 197 to be an 2,3,-24- trihydroxyolean-12-ene triterpene acid (Ahmad and Rahman, 1994),

(Agrawal, 1992). Thus when matched with literature data, 197 was confirmed as 2α,3 β,24-trihydroxyolean-12-en-28-oic acid (Arjunolic acid);

(Bisoli et al., 2008).

167

6.6 Biological Studies

The compounds 189 -191 and 193 -198 purified from C. opaca were subjected to various biological assays namely DPPH free radical scavenging,

AChE, BChE, and LOX enzyme inhibition activities and the results thus obtained are discussed below. 192 and 67 were not checked for activity due to low amount.

6.6.1 Antioxidant Assay (DPPH radical scavenging method)

In antioxidant assay, the two lignins (-) carinol (193) and carissanol

(194) showed significant activity with an IC 50 value of 84.91 and 83.41 respectively, while compounds 189 -191 and 195 -198 showed minor activities with IC 50 below 500. Quercetin was used as a positive control at concentration 0.5 mMwell -1 for control as well as test compounds (Table.

6.1).

168

Table 6.1: Antioxidant Assay (DPPH radical scavenging activity)

Compound (%) at (IC 50 ) 0.5 mM µM

189 17.98±0.55 <500

190 21.19±0.13 <500

191 22.58±0.22 <500

193 88.24±0.16 84.91±0.07

194 90.11±0.18 83.41±0.17

195 25.14±0.63 <500

196 19.76±0.58 <500

197 15.24±0.86 <500

198 12.41±0.75 <500

Quercetin 93.21±0.97 16.96±0.14

6.6.2 Enzyme Inhibition Activities

The enzyme inhibition activities were checked by following assays.

6.6.2.1 Acetylcholinesterase Enzyme Inhibition Activity

Compounds 189 -198 were subjected to acetylcholinesterase enzyme inhibition essay but none of them was found to be considerably active

169 against this enzyme. The compounds were tested at a concentration of 0.5 mM well -1 (Table 6.2). Eserine was used as positive control.

Table 6.2: Acetylcholinesterase enzyme inhibition activity

Compound (%) at (IC 50 ) 0.5 mM µM

189 38.52±0.52 <700

190 25.31±0.18 <700

191 38.56±0.21 <700

193 32.11±0.16 <700

194 53.61±0.14 <400

195 43.21±0.82 <700

196 42.17±0.66 <700

197 53.81±0.52 <400

198 39.61±0.16 <700

Eserine 91.29±1.17 0.04±0.0001

6.6.2.2 Butyrylcholinesterase Enzyme Inhibition Activity

Significant butyrylcholinesterase enzyme inhibition activities were observed for carissanol (194) , with an IC 50 value of 101.81 and 198 which is new natural product was also active against Butyrylcholinesterase enzyme (IC 50 , 336.21). Compounds 189 -191 , 193 , and 195 -197 didn’t

170 show any significant activity (Table 6.3). Eserine (C= 0.5 mMwell -1) was used as positive control.

Table 6.3: Butyrylcholinesterase enzyme inhibition activity

Compound (%) at (IC 50 )

0.5 mM µM

189 58.61±0.18 <400

190 23.61±0.22 <700

191 53.21±0.17 <400

193 48.91±0.17 <700

194 88.51±0.12 101.81±0.24

195 25.31±0.33 <700

196 51.21±0.61 <400

197 53.21±0.11 <400

198 62.18±0.27 336.21±0.14

Eserine 82.82±1.09 0.85±0.0001

6.6.2.3 Lipoxygenase Enzyme Inhibition Activity

Among all the compounds tested for lipoxygenase enzyme inhibition activity, the compounds 190 , 196 -198 showed activities with IC 50 value below 300 and 189 -191 , 193 and 194 were almost inactive against lipoxygenase enzyme.

171

All compounds and the control were used at a concentration of 0.5 mM well -1 (Table 6.4). Baicalein was used as a positive control.

Table 6.4: Lipoxygenase enzyme inhibition activity

Compound (%) at (IC 50 ) 0.5 mM µM

189 20.11±0.58 <800

190 55.21±0.63 <300

191 49.61±0.82 <500

193 48.21±0.68 <500

194 44.58±0.46 <500

195 47.51±0.18 <500

196 51.21±0.82 <300

197 52.37±0.57 <300

198 55.31±0.17 <300

Baicalein 93.79±1.2 22.4±1.3

172

6.7 Experimental

6.7.1 General experimental Procedures

Column chromatography was carried out using silica gel F 254 (230-

400 mesh) as stationary phase and distilled solvents were used as mobile phase. Thin layer chromatography (TLC) was carried out using aluminium sheets pre-coated with silica gel 60 F 254 (20×20 cm, 0.2 mm thick; E.

Merck). TLC plates were visualized under UV lamp of at 254 and 366 nm wave length and by spraying with ceric sulfate reagent solution (on heating). Optical rotation was measured on JASCO DIP-360 polarimeter. IR spectra were recorded on Shimadzu 460 spectrometer, while UV spectra were scanned on a Hitachi UV-3200 spectrophotometer ( λmax in nm). 1H and 13 C NMR data was measured on Bruker instrument operating at 500,

400 and 125, 100 MHz respectively. 2D experiments (COSY, HMQC and

HMBC) were also performed on the same instrument operating at 500, and

400 MHz frequency. EIMS, HR-EIMS, FABMS and HR-FABMS were measured on JMS H×110 with a data system and JMSA 500 mass spectrometers, respectively.

6.7.2 Plant Material

The whole plant C. opaca was collected from Quaid-e-Azam University

Islamabad, Pakistan in June 2008 and was identified by Mr. Furrakh Nisar,

173

Plant Taxonomist, University of Gujarat, Gujrat, where a voucher specimen is deposited (212-CO/BOT/08).

6.7.3 Extraction and Isolation

The shade dried aerial parts of C . opaca (20 kg) were crushed and extracted with methanol thrice. The methanolic extract was concentrated under reduced pressure to get green gummy mass (600 g) which subjected to silica gel column chromatography eluting with n-hexane, n- hexane:dichloromethane (DCM), DCM, DCM:methanol and methanol in increasing order of polarity. As a result seven fractions (1-7) were obtained.

Fraction 1 on gradient elution using n-hexane-DCM (1:1) on further chromatography yielded 3 β,27-dihydroxylup-12-ene ( 189 ). Fraction 2 as eluted with 100% DCM yielded lupeol β-hydroxy octadecanoate ( 190 ) at n- hexane-DCM (0.1:9.9). The fraction 3 obtained at DCM-MeOH (9.8:0.2) when subjected to column chromatography yielded further two sub fractions. The sub Fr. 1 showed a single spot on TLC and on further purification using column chromatography, cyclic trimer of poly (ethylene terephthalate) ( 191 ) was eluted at DCM-MeOH (9.9:0.1), while the sub Fr. 2 when subjected to column chromatography at DCM-MeOH (9.8:0.2), yielded two compounds pinoresinol ( 192 ) from the head fraction and (-) carinol

(193 ) from the tail fraction.

174

Fraction 4 eluted at DCM-MeOH (9.6:0.4) further yielded two sub fractions. The sub Fr. 1 on further purification by using silica gel column chromatography using the solvent system DCM-MeOH (9.7:0.3) yielded (-)- carissanol ( 194 ). The second sub fr. 2 on repeated silica gel column chromatography yielded 2,3-dihydoxy 30-nor-2α,3 β–dihydroxy-urs-12-ene

(195 ) at DCM-MeOH (9.6:0.4). The fraction 5 collected at DCM-MeOH

(9.4:0.6) when subjected to repeated column chromatography further yielded two subfractions. The sub Fr. 1 on column chromatography resulted in the isolation of 30-nor-2α,3 α,23-trihydroxyurs-12-ene ( 196 ) at

DCM-MeOH (9.5:0.5) and sub Fr. 2 yielded arjunolic acid ( 197 ) and

(2S,3S,4R,15E)-2-{[(2R)-2-hydroxydocosanoyl]amino}eicos-15-ene-1,3,4-triol

(198 ) at DCM-MeOH (9.4:0.6) one after the other. The fraction 6 collected at

DCM-MeOH (9.2:0.8) yielded β-sitosterol glucopyranoside ( 67 ) on further purification. The fraction 7 collected at DCM-MeOH (9.0:1.0) but this fraction shoewed no significant spot on TLC.

175

Scheme 6.1: Protocol for the isolation of compounds 67 and 189 -198 from Carissa opaca.

176

6.7.4 Experimental Data

6.7.4 .1 Cyclic trimer of ethylene terephthalate (191)

O O White crystalline solid (12 mg); M.P.:

O O 5 318 0C; UV (MeOH) λmax (log Ɛ) nm: 228 5 O O (3.80), 320 (3.89); IR (KBr) νmax cm -1: 3250, O 4 O 2 1 1 δ 2 3 1760, 1250; H NMR (CDCl 3, 400MHz) : 1 3 O O 4 8.08 (12H, s, H-2), 4.66 (12H, s, H-4); 13 C O O 191 NMR (CDCl 3, 100MHz) δ: 165.2 (C-3),

133.8 (C-1), 129.7 (C-2), 62.7 (C-4); HR-EIMS : m/z 576.1266 (calcd. for

C30 H24 O12 , 576.1267); EIMS : m/z 576, 533, 445, 162, 148, 104.

6.7.4.2 30-Nor-2,3-dihydoxy-2ααα,3 β–dihydroxy-urs-12-ene (195)

α 24 29 White amorphous solid (12 mg); [αα]D : +75.0

19 21 12 22 (c = 0.56, MeOH); UV (MeOH) λmax (log Ɛ) nm: 25 11 26 13 17 28 HO 1 9 15 -1 2 210 (3.82); IR (KBr) νmax cm : 3415, 2935,

5 7 27 HO 4 1655, 1630, 1375, 1365, 1065, 1035, 830; 1H 23 24 195 NMR (CD 3OD, 500MHz) δ: 5.23 (1H, dd, 3.5,

4.0, H-12), 3.61 (1H, ddd, J = 4.0, 4.5, 10.0 Hz, H-2), 2.03 (1H, ddd, J =

3.5, 4.0, 4.0, Hz, Ha-22), 1.92 (1H, ddd, J = 3.0, 4.0, 4.5 Hz, Hb-22), 2.91

(1H, d, J = 10.0 Hz, H-3), 2.21 (1H, d, J =11.5 Hz, H-18), 1.97, 1.78 (each

H, m, H2-16), 1.93 (2H, m, Ha-11), 1.93 (2H, m, Hb-11), 1.90, 1.62 (each H,

177 m, H2-15), 1.62, 1.47 (each H, m, H2-20), 1.60, 1.40 (each H, m, H2-8),

1.57, 1.42 (each H, m, H2-6), 1.56 (1H, m, H-9), 1.35 (1H, m, H-19), 1.43

(each H, m, H2-21), 1.11 (3H, s, H-27), 1.01 (3H, s, H-24), 1.01 (3H, s, H-

25), 0.96 (3H, s, H-28), 0.89 (3H, d, J = 6.5 Hz, H-29), 0.84 (3H, s, H-26),

0.81 (1H, m, H- 5), 0.80 (3H, s, H-23); 13 C NMR (CD 3OD, 125MHz) δ: 139.7

(C-13), 126.7 (C-12), 84.4 (C-3), 69.5 (C-2), 56.6 (C-5), 54.3 (C-18), 49.8 (C-

9), 48.5 (C-1), 43.3 (C-14), 40.8 (C-8,17), 40.5 (C-4), 40.4 (C-19), 39.2 (C-

10), 38.1 (C-22), 34.2 (C-7), 31.7 (C-21), 30.7 (C-20), 29.3 (C-23), 29.1 (C-

15), 25.3 (C-16), 24.4 (C-11), 24.0 (C-27), 21.5 (C-29), 19.5 (C-6), 17.8 (C-

28), 17.6 (C-26), 17.5 (C-25), 17.2 (C-24); HR-EIMS : m/z 428.6921 [M] +

(calcd. for C 29 H48 O2, 428.6922).

6.7.4.3 30-Nor-2ααα,3 ααα,23-trihydroxyurs-12-ene (196)

α 24 29 White amorphous solid (11mg); [αα]D : +58 (c =

19 21 12 22 0.50, MeOH); UV (MeOH) λmax (log Ɛ) nm: 212; 25 11 26 13 17 28 HO 1 9 15 -1 2 IR (KBr) νmax cm : 3520, 3480, 3365, 2935, 5 7 27 HO 4 23 2935, 1655, 1665, 1375, 1365, 1065, 1035, 24 OH 196 1 830; H NMR (CD 3OD, 500MHz) δ: 5.23 (1H, dd, J = 3.5, 4.0 Hz, H-12), 3.88 (1H, td, J = 2.0, 3.0, 11.5 Hz, H-2), 3.59

(1H, d, J = 2.0 Hz, H-3), 3.53 (1H, d, 11.0 Hz, Ha-23), 3.39 (1H, d, 11.0 Hz,

Hb-23), 2.21 (1H, d, J = 11.0 Hz, H-18), 1.97, 1.79 (each H, m, H2-16), 1.96

(2H, m, H-11), 1.90, 1.61 (each H, m, H2-15), 1.75 (1H, m, H-9), 1.67, 1.39

178

(1H each, m, H-2,7), 1.62, 1.48 (each H, m, H2-20), 1.60, 1.42 (each H, m,

H2-6), 1.59, 1.54 (2H, m, H-21), 1.38 (1H, m, H-19), 1.30 (each H, m, H2-

1), 1.13 (3H, s, H-27), 1.02 (3H, s, H-25), 0.96 (3H, s, H-28), 0.89 (3H, d, J

13 = 6.5 Hz, H-29), 0.84 (3H, s, H-26), 0.77 (3H, s, H-24); C NMR (CD 3OD,

125MHz) δ: 139.8 (C-13), 126.6 (C-12), 78.7 (C-3), 71.3 (C-23), 67.2 (C-2),

54.4 (C-18), 48.4 (C-9), 44.1 (C-5), 43.2 (C-14), 42.5 (C-4), 42.3 (C-1), 40.8

(C-8,17), 40.4 (C-19), 39.1 (C-10), 38.1 (C-22), 33.8 (C-7), 31.8 (C-21), 30.7

(C-20), 29.1 (C-15), 25.3 (C-16), 24.4 (C-11), 24.1 (C-27), 21.5 (C-29), 18.6

(C-6), 17.8 (C-28), 17.7 (C-26), 17.6 (C-25), 17.5 (C-24); HR-EIMS: m/z

444.69 [M] + (calcd. for C 29 H48 O3, 444.69); EIMS : m/z 444.69 [M] +.

6.7.4.4 (2S,3S,4R,15E)-2-{[(2R)-2-Hydroxydocosanoyl]amino}eicos-15- ene-1,3,4-triol (198)

O White amorphous OH 1' 22' HN 2' solid (26 mg); OH 16 20 HO 3 4 2 24 1 15 19 [ααα]D : + 38.5 (c = OH 198 0.0025, MeOH);

IR (KBr) νmax cm -1: 3335, 3215, 2920, 1623, 1602; 1H NMR (CDCl 3,

500MHz) δ: 7.40 (1H, d, J = 8.5 Hz, NH), 5.30 (2H, dt, J = 5.0, 16.5 Hz,

15,16-H), 3.98 (1H, m, H-2), 3.94 (1H, dd, J = 3.5, 8.0 Hz, H-2'), 3.71 (1H, dd, J = 4.5, 12.0 Hz, Ha-1), 3.63 (1H, dd, J = 4.5, 11.5 Hz, Hb-1), 3.41 (1H, dt, J = 4.2, 6.3 Hz, H-4), 3.40 (1H, dd, J = 3.9, 4.5 Hz, H-3), 1.91 (4H, m, H-

179

14,17), 1.69, 1.48 (each H, m, H2-3'), 1.59, 1.30 (each H, m, H2-5), 1.15-

1.31 (56H, brs, H-6-13,18-19,4'-21'), 0.77 (6H, t, J = 7.0 Hz); 13 C NMR

(CDCl 3, 125 MHz) δ: 175.6 (C-1'), 130.6 (C-15 or 16), 129.6 (C-15 or 16),

75.5 (C-3), 72.1 (C-4), 71.8 (C-2'), 60.9 (C-1), 51.4 (C-2), 34.2 (C-3'), 32.4

(C-14,17), 31.7 (C-5), 29.2-31.5 (6-13,18-19,4'-21'), 13.8 (C-20,22'); HR-

- FABMS : [M-H] , m/z 680.6271 (calcd. for C 42 H83 NO 5, 681.6272); EIMS : m/z

648 [M- CH 3OH] +, 453, 439, 408, 398, 383, 370, 339, 308, 280, 265, 225,

125, 111, 97, 83, 71, 57, 44.

Methyl ester derived from 198

1 H NMR (CDCl 3, 300MHz): δ 4.11 (1H, t, J = 6.5, H-2'), 3.54 (3H, s, MeO),

1.97 (3H, s, MeCO), 1.15-1.28 (38H, br s, CH 2-3'-21'), 0.84 (3H, t, J = 6.5,

CH 3-22'); GC-MS: m/z 384.

Acetylsphingamine derived from 198

1H NMR (CDCl 3, 300MHz): δ 7.42 (1H, d, J = 8.0, NH), 5.33 (2H, dt, J = 5.0,

16.5, H-14,15), 4.62 (1H, dd, J = 4.4, 5.5 Hz, H-4), 4.22 (1H, m, H-2), 4.34

(1H, dd, J = 4.5, 11.5 Hz, H-1), 4.20 (1H, dd, J = 3.1, 5.0 Hz, H-3), 2.00

(12H, 4×MeCO), 1.15-1.26 (26H, br s, CH 2-5-14,17-19), 0.85 (3H, t, J = 6.5

Hz, CH 3-20); GC-MS: m/z 511.

180

6.7.4.5 3β, 27-Dihydroxylup-12-ene (189)

White needles; M.P.: 194-196 0C; [ααα]D25 : +67.9 (c 30

29 22 = 0.419, CHCl 3); UV (MeOH) λmax (log Ɛ) nm: 214; 19 21

25 11 26 13 17 ν -1 28 IR (KBr) max cm : 3300-3255, 1605, 1370,

1 9 15 CH2OH 1 3 5 7 27 1344, 1330, 1124, 1000; H NMR (CDCl 3, HO 23 24 400MHz) δ: 5.12 (1H, t, J = 3.6 Hz, H-12), 3.51 189 (1H, d, J = 10.9 Hz, Ha-27), 3.21 (1H, dd, J = 4.9, 10.8 Hz, H-3), 3.18 (1H, d, J = 10.9 Hz, Hb-27), 1.10 (3H, s, H-28), 0.97 (3H, s, H-23), 0.96 (3H, s,

H-26), 0.92 (3H, s, H-24), 0.85 (3H, d, J = 5.2 Hz, H-29), 0.79 (3H, d, J =

6.5 Hz, H-30), 0.77 (3H, s, H-25); 13 C NMR (CDCl 3, 100MHz) δ: 138.7 (C-

13), 125.0 (C-12), 79.0 (C-3), 69.9 (C-27), 55.2 (C-5), 54.0 (C-18), 47.6 (C-

9), 42.0 (C-14), 40.0 (C-8), 39.4 (C-19), 39.3 (C-20), 38.8 (C-1), 38.6 (C-17),

38.0 (C-4), 36.9 (C-10), 35.1 (C-22), 32.8 (C-7), 30.6 (C-21), 28.1 (C-23),

27.2 (C-2), 26.0 (C-16), 23.3 (C-11,15), 23.3 (C-28), 21.3 (C-29), 18.3 (C-6),

17.3 (C-30), 16.7 (C-24), 15.7 (C-26), 15.6 (C-25); HR-EIMS : m/z

442.38134 [M] + (calcd. for C 30 H50 O2, 442.3810); EIMS : m/z 442, 412, 234,

207, 203, 191, 189, 175, 133, 123, 122, 107, 95, 94, 91, 77, 69, 65, 55,

51.

181

6.7.4.6 Lupeol β-hydroxy octadecanoate (190)

ααα 25 30 White amorphous powder (23mg); [ ]D : -25.1

29 22 ν -1 19 (c = 0.28, CHCl 3); IR (KBr) max cm : 3455, 12 21

25 13 17 1 26 3075, 1730, 1460, 1444, 1380, 720; H NMR 28 1 9 15

3 5 7 27 (CDCl , 400MHz) δ: 4.66 (1H, brs, Ha-29), 4.54 O 3 1' 23 24 O 2' (1H, brs, Hb-29), 4.53 (1H, m, H-3), 3.96 (1H,

3'4' 18' 11 HO m, H-3'), 2.50 (1H, dd, 2.8, 16.0 Hz, Ha-2'), 190 2.46 (1H, ddd, 3.2, 5.6, 16.0 Hz, Hb-2'), 1.68 (3H, s, H-30), 1.20 (28H, br s,

H-4'-17'), 1.03 (3H, s, H-26), 0.99 (3H, s, H-23), 0.98 (3H, s, H-27), 0.83

(3H, t, J = 6.0 Hz, H-18'), 0.82 (3H, s, H-25), 0.79 (3H, s, H-28), 0.76 (3H, s,

H-24); 13 C NMR (CDCl 3, 125MHz) δ: 172.8 (C-1'), 150.9 (C-20), 109.3 (C-

29), 81.4 (C-3), 68.2 (C-3'), 55.4 (C-5), 50.3 (C-9), 48.3 (C-18), 48.0 (C-19),

43.0 (C-17), 42.8 (C-14), 41.6 (C-2'), 40.8 (C-8), 40.0 (C-22), 38.3 (C-1),

38.0 (C-13), 37.8 (C-4), 37.1 (C-10), 36.6 (C-16), 35.5 (C-7), 34.2 (C-4'),

(29.9-25.1 (C5'-C17'), 29.8 (C-21), 28.0 (C-23), 27.4 (C-15), 25.1 (C-12),

23.7 (C-2), 20.9 (C-11), 19.3 (C-30), 18.2 (C-6), 18.1 (C-28), 16.6 (C-26),

16.1 (C-24), 15.9 (C-27), 14.5 (C-25), 14.1 (C-18'); HR-EIMS : m/z 736.6214

[M] + (calcd. for C 50 H88 O3, 736.6733); EIMS : m/z [M] +, 708, 681, 663, 468,

409, 408, 393, 298, 218, 203, 189, 147, 135, 121, 109, 95.

182

6.7.4.7 Pinoresinol (192)

White amorphous solid (5 mg); UV (MeOH) OCH3 3' 4' OH λmax (log Ɛ) nm: 224 (4.10), 280 (3.70); IR O 1' 5' 8 6 (KBr) νmax cm -1: 3430, 1615, 1520, 1275, H 1 5 H 2 4 1' 5' O 1210, 980, 945; 1H NMR (CDCl 3, 400MHz) δ:

HO 3' 6.88 (2H, d, J = 1.6 Hz, H-2'), 6.87 (2H, d, J = OCH3 192 8.0 Hz, H-5'), 6.81 (2H, dd, J = 1.6, 8.0 Hz, H-

6'), 4.72 (2H, d, J = 4.0 Hz, H-2,6), 4.24-3.86 (4H, dd, J = 6.8, 8.8 Hz, H-

4,8), 3.89 (6H, s, 2OCH 3), 3.12 (2H, m, H-1,5); 13 C NMR (CDCl 3, 100MHz) δ:

145.2 (C-3'), 143.7 (C-4'), 132.9 (C- 1'), 118.9 (C-6'), 114.2 (C-5'), 108.5 (C-

2'), 85.8 (C-2,6), 71.6 (C-4,8), 56.5 (2OCH 3), 54.1 (CH, C-1,5); HR-EIMS : m/z 358.1442 (calcd. for C 20 H22 O6, 358.1440); EIMS : m/z 378 [M] +.

6.7.4.8 (-)Carinol (193)

White amorphous powder; [ααα]D25 : -24 (c = 0.25, OH H CO 1' 3 3' 7' 9' OH 8' EtOH); UV (MeOH) λmax (log Ɛ) nm: 228 (4.10), 8 9 OH HO 5' 7 H -1 1 284 (3.70); IR (KBr) νmax cm : 3500, 3432, 1610,

5 3 1520, 1270, 1210; 1H NMR (CDCl 3, 400MHz) δ: OCH3 OH 193 6.85 (1H, d, J = 8.0 Hz, H-5'), 6.82 (1H, d, J =

8.0 Hz, H-5), 6.81 (1H, d, J = 2.8 Hz, H-2'), 6.79 (1H, dd, J = 2.8, 8.0 Hz, H-

6'), 6.70 (1H, d, J = 2.8 Hz, H-2), 6.69 (1H, dd, J = 2.8, 8.0 Hz, H-6), 6.67

183

(1H, d, J = 8.0 Hz, H-6), 3.86 ( 3H, s, OCH 3), 3.84 ( 3H, s, OCH 3), 3.68 (2H, ddd, J = 2.2, 2.4, 11.0 Hz, H-9), 3.51 (2H, q, J = 11.6 Hz, H-9'), 2.96 (1H, dd, J = 2.8, 14.0 Hz, Ha-7), 2.80 (2H, s, H-7'), 2.48 (1H, dd, J = 2.8, 11.6

Hz, Hb-7), 2.05 (1H, m, H-8); 13 C NMR (CDCl 3, 100MHz) δ: 146.6 (C-3'),

146.5 (C-3), 144.5 (C-4'), 143.9 (C-4), 132.2 (C- 1), 128.0 (C-1'), 123.6 (C-

6'), 121.6 (C-6), 113.1 (C-2'), 111.4 (C-2), 76.6 (C-8'), 65.2 (C-9'), 61.0 (C-9),

47.4 (C-8), 40.2 (C-7'), 31.7 (C-7); HR-EIMS: m/z 378.164 [M] + (calcd. for

C20 H26 O7, 378.1678); EIMS: m/z 378 [M] +.

6.7.4.9 (-) Carissanol (194)

25 White amorphous powder (16 mg); [ααα]D : -16 (c = OH 7' OH H3CO 3' 1' 8' 9' 0.75, EtOH); UV (MeOH) λmax (log Ɛ) nm: 226 8 O 9 HO 5' 7 ν -1 1 H (4.10), 282 (3.70); IR (KBr) max cm : 3510, 3430,

5 3 1615, 1520, 1275, 1215, 980, 945; 1H NMR OCH3 OH δ 194 (CDCl 3, 400MHz) : 6.82 (1H, d, J = 8.4 Hz, H-5'),

6.81 (1H, d, J = 8.4 Hz, H-5), 6.78 (1H, d, J = 2.8 Hz, H-2'), 6.76 (1H, d, J =

2.8 Hz, H-2), 6.68 (1H, dd, J = 2.8, 8.4 Hz, H-6'), 6.67 (1H, dd, J = 2.8, 8.0

Hz, H-6), 5.20 (1H, d, J = 6.4 Hz, H-9'), 3.82 (6H, s, 2OCH 3), 3.75 (2H, dd, J

= 4.0, 9.6 Hz, H-9), 2.80 (1H, dd, J = 2.8, 7.6 Hz, H-7'), 2.71 (1H, dd, J =

5.2, 13.9 Hz, Ha-7), 2.50 (1H, dd, J = 10.4, 13.9 Hz, Hb-7), 2.30 (1H, m, H-

8); 13 C NMR (CDCl 3, 100MHz) δ: 146.5 (C-3'), 146.4 (C-3), 144.8 (C-4'),

144.7 (C-4), 131.9 (C-1), 128.1 (C-1'), 123.0 (C-6'), 121.1 (C-6), 114.4 (C-5),

184

114.2 (C-5'), 112.9 (C-2'), 111.1 (C-2), 101.0 (C-9'), 78.9 (C-8'), 71.0 (C-9),

56.6 (2OCH 3), 43.2 (C-7'), 32.5 (C-7); HR-EIMS: m/z 376.1554 [M] + (calcd. for C 20 H24 O7, 376.1538); EIMS: m/z 193, 175, 164, 163, 150, 139, 138,

137, 131, 124, 123, 122, 107, 94, 91, 77, 65, 55, 51.

6.7.4.10 Arjunolic acid (197)

White needles (11 mg); M.P.: 337-340 0C; 29 30

19 19 21 [ααα]D : +63.9 (c = 0.52, EtOH); IR (KBr) νmax 12 22

13 25 17 -1 26 COOH cm : 3070, 3300-2250, 1680, 1620, 1445, 28 HO 1 9 15 2 27 3 5 7 720; 1H NMR (CD OD, 500MHz) δ: 5.23 (1H, HO 3 23 24 OH m, H-12), 3.68 (1H, m, H-2) 3.50 (1H, d, J = 197 11.0 Hz, Ha-23), 3.26 (1H, d, J = 11.0 Hz,

Hb-23), 3.35 (1H, d, J = 9.5 Hz, H-3), 1.17 (3H, s, H-27), 1.02 (3H, s, H-26),

0.93 (3H, s, H-30), 0.90 (3H, s, H-29), 0.81 (3H, s, H-25), 0.68 (3H, s, H-

24); 13 C NMR (CD 3OD, 125MHz) δ: 180.5 (C-28), 143.5 (C-13), 122.5 (C-12),

78.2 (C-3), 69.0 (C-2), 66.1 (C-23), 48.2 (C-9), 48.1 (C-1), 48.0 (C-5), 46.8

(C-17), 46.3 (C-19), 44.2 (C-4), 42.4 (C-14), 42.2 (C-18), 39.9 (C-8), 39.5 (C-

10), 34.1 (C-21), 33.5 (C-29), 33.3 (C-22), 32.6 (C-7), 30.9 (C-20), 28.6 (C-

15), 26.0 (C-27), 23.8 (C-16), 23.7 (C-30), 23.5 (C-11), 17.5 (C-26), 17.2 (C-

25), 18.6 (C-6), 13.2 (C-24); HR-EIMS : m/z 488.3543 [M] + (calcd. for

C30 H48 O5, 488.3501).

185

6.7.5 Methanolysis

Compounds 198 (12 mg) were refluxed separately with 6 ml of 1N

HCl and 25 ml of MeOH for 15 h. The reaction mixture was then extracted with n-hexane to obtain the corresponding fatty acid methyl esters, which were analyzed by GC-MS after acetylation with Ac 2O-Py. The aq. layer from

198 was evaporated, and the residue was acetylated. Purification over

Sephadex LH-20 and elution with CH 2Cl 2/MeOH 1:1 gave the corresponding acetylated sphingosines, which was analyzed by GC-MS. The aq. layer from 198 was evaporated to dryness, and the residue was separated by silica gel column chromatography as sphingosine base. The base was acetylated and analyzed by GC-MS.

6.7.6 Oxidative Cleavage of the Double bond in 198

To the solution of methyl ester of compound 198 (4 mg) in acetone, added 1 ml of 0.04 M solution of K 2CO 3, 6 ml of an aqueous solution 0.025

M KMnO 4 and 0.09 M NaIO 4 in 100 ml round bottom flask. The reaction was allowed to proceed at 37 0C for 18 h. After acidification with 5 N H 2SO 4, the solution was decolorized with a 1M solution of oxalic acid and extracted with Et 2O (3 to10 ml). The combined organic extract was dried over Na 2SO 4, filtered, and concentrated. The resulting carboxylic acids were methylated with ethereal solution of diazomethane and analyzed by GC-MS.

186

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