the fulfillment of degree of doctor of philosophy

i

Isolation of Secondary Metabolites of

Berberis calliobotrys, Caragana ambigua and

Vincetoxicum stocksii and their In Vitro

Screening in Various Bioassays

Thesis Submitted

For

The Fulfillment of Degree of

Doctor of Philosophy

By

Saima Khan

(M.Sc., M.phil.)

Department of Chemistry

The Islamia University of Bahawalpur

(2014-2019)

DECLARTION

iii

DECLARATION

I, Saima Khan, Ph.D. Scholar in Chemistry Department, The Islamia University

of Bahawalpur, hereby declare that the dissertation entitled “Isolation of Secondary

Metabolites of Berberis calliobotrys, Caragana ambigua and Vincetoxicum stocksii

and their In Vitro Screening in Various Bioassays” is done by me under the

supervision of Prof. Dr. Muhammad Saleem, Department of Chemistry, The Islamia

University of Bahawalpur and it has not formed the basis for the award of any other

Degree or other similar title to any candidate of any University. This thesis contains no

material that has been submitted previously, in whole or in part, for the award of any

other academic degree or diploma. I also corroborate that nothing has been integrated in

this research without acknowledgement.

Saima Khan

CERTIFICATE

iv

CERTIFICATE

It is certified that research work presented in this dissertation, entitled “Isolation

of Secondary Metabolites of Berberis calliobotrys, Caragana ambigua and

Vincetoxicum stocksii and their In Vitro Screening in Various Bioassays”, has been

carried out by Ms. Saima Khan under my supervision for the fulfillment of the

requirements of Ph.D. degree in Chemistry during the session 2014-2019. It is hereby

recommended for submission and further process for the award of Ph.D. degree.

Prof. Dr. Muhammad Saleem

Research Supervisor

Department of Chemistry

The Islamia University of Bahawalpur,

Pakistan

The Chairman

Department of Chemistry,

The Islamia University of Bahawalpur

Pakistan

Acknowledgement

v

Acknowledgements

In the name of Allah, the Omnipotent, the most Merciful, the Compassionate,

Who blessed us with the perfect code of life and Whoseelegance resulted into my

success. All respects for the most perfect personality of the world Hazrat Muhammad

(Peace Be Upon Him) who enlighten our minds to recognize our creator.

The writing of acknowledgements after completion of research and writing thesis

is a pleasant task to pay thanks to those who provided their guidance and co-operated. It

is very difficult to write thesis without co-operation, guidance and help of a teacher, for

this reason, I am very thankful to my research supervisor Prof. Dr. Muhammad Saleem

whose suggestions, care and hard work helped me to complete my research work and

thesis. I am obliged to my respected teachers Prof. Dr. Abdul Jabbar, Prof. Dr. Naheed

Riaz and Prof. Dr. Shazia Anjum for their guidance and motivation.I am thankful to my

great mentor Prof. Dr Abdul Rauf who always inspired me and whose precious prayers

helped me in the journey of achieving this task. To facilitate documentation time to time

during the whole study, I am thankful to the Chairman, Department of Chemistry,

I am especially indebted toProf. Dr.Muhammad Ashraf, Sir Hammad Saleem,

Prof. Dr.Gokhan Zengin, Prof. Dr. Ishtiaq Ahmad and Prof. Dr. Fawzi Mohamad

Mohomodally for their cooperation in providing several research supports.

Above all, I wish to express my deepest love and gratitude to my parents,

brothers, sisters, bhabi, uncle, anti and other family members for the encouragement and

support they have provided me and their countless prayers for my success.I am happy to

acknowledge to my sweet nieces Areeba, Aneeka, Laiba, Rameeza and Bareera for

their prayers.

I am also thankful to my sweet friend Mahreen Mukhtar for everything she did

which cannot be expressed in words. May Allah Almighty bless her.

Acknowledgement

vi

Next I am thankful to my dear friend Rabbia Ahmed for her guidance and

assistance as well as moral support during research work.

I am grateful to all of my laboratory fellows Liaqat Ali, Natasha Shazmeen,

Humna Tahir, Romaisa Maqsood, Ghzala Fazal, Saima Muzzafar, Bushra Bashir,

Dur-e-Shahwar Jamil, Maria Aslam, Bushra Siddique, Iqra Siddique, Shabnum

Mustafa, Saima Naz, Umber, Fatima for always being helpful. I thereby express my

thanks to all technical and non-technical staff of the department for their co-operation.

Saima Khan

ABSTRACT

vii

ABSTRACT

Throughout the journey of mankind, reliance of man on plant to alleviate

sufferingsis still considered with more awareness for their therapeutic application.

Petition of tradition medicinal system is because of its less cost, safer and easily

accessibility. Therefore,in the present era, interest in the elucidation of biological

potential and chemical constituent of plant is ever budding among the naturopathic.

In under discussion studies, three medicinal plants: Berberis calliobotrys,

Caragana ambigua and Vincetoxicum stocksii were selected to be investigated for their

secondary metabolites. The whole work done during these studies has been embodied in

this dissertation as five chapters. Chapter 1containsthe introduction ofthe natural

products, their sources and some recent drugs approved by FDA available in market with

natural product framework and offers background of the present study. Chapter

2describesthemedicinal importance, worldwide distribution and reported secondary

metabolites with biological potential from the genera Berberis, Caragana and

Vincetoxicum.It also comprises the detailed literature survey on Berberis calliobotrys,

Caragana ambigua,and Vincetoxicum stocksii.

Chapter 3 discusses the biological screeningin various bioassays(DPPH, ABTS,

FRAP, CUPRAC, phosphomolybdenum, metal chelation, acetylcholinesterase (AChE),

butyrylcholinesterase (BChE), α-glucosidase, α-amylase and tyrosinase) and

phytochemical analyses (total phenolic contents and total flavonoid contents)ofcrude

extracts of three plants with their qualitative UHPLC-MS dataobtained for fractions Bc-

M, Bc-E, BC-W, Ca-M, Ca-E. It also describes theresult and discussion of isolated

compounds from the Berberis calliobotrys, Caragana ambigua, and Vincetoxicum

stocksii with detailed structure elucidation and isolation scheme. The biological activities

of crude extract, with experimental method applied in our investigation are also included.

Biological screening of crude extract in above said assays for Berberis calliobotry sand

Caragana ambigua are also reported in journal of industrial crops and product (Khan, S.,

Nazir, M., Saleem, H., Riaz, N., Saleem, M., Anjum, S. M. M., Zengin, G.,M.,

ABSTRACT

viii

Mukhtar,Tousif, M. I., Mahomoodally, F. M.,Ahemad, N.(2019) Valorization of the

antioxidant, enzyme inhibition and phytochemical propensities of Berberis calliobotrys

Bien. ex Koehne: A multifunctional approach to probe for bioactive natural

products.Industrial Crops and Products,141, 111693; Khan, S., Nazir, M., Raiz, N.,

Saleem, M., Zengin, G., Fazal, G., Saleem, M., Mukhtar, M., Tousif, M. I., Tareen,

R. B., Abdallah, H. H. and Mahomoodally, F. M.(2019). Phytochemical profiling, in

vitro biological properties and in silico studies on Caragana ambigua stocks (Fabaceae):

A comprehensive approach. Industrial Crops and Product, 131, 117-124). The natural

products isolated from Vincetoxicumstocksii are reportedin journal of chemical society of

pakistan (Khan, S., M., Tousif, Raiz, N., M., Raiz, Mukhtar, M., Ahmad, I., Tareen,

R. B., Jabbar, A. and Saleem, M. (2019) Rarely occurring natural products isolated

from Vincetoxicum stocksii,The Journal Of Chemical Society Of Pakistan, 41 (4), 695-

700.

Chapter 4 describes general experimental methods and techniques employed for

biological screening in various bioassays and phytochemical analyses of crude extracts of

three plants with their qualitative UHPLC-MS data obtained. It also contains

spectroscopic characterization of isolated compounds.

Chapter 5elaborates the assays or protocol applied for Berberis calliobotrys,

Caragana ambigua, and Vincetoxicum stocksii extract.

Table of Contents

ix

Table of Contents

DECLARATION ................................................................................................................................... III

CERTIFICATE...................................................................................................................................... IV

ACKNOWLEDGEMENTS ..................................................................................................................... V

ABSTRACT .......................................................................................................................................... VII

CHAPTER 1 ALLURING NATURAL PRODUCTS AND THEIR APPLICATIONS IN DRUG

DEVELOPMENT ................................................................................................................................................... 1

1.1. INTRODUCTION OF NATURAL PRODUCTS ................................................................................... 2 1.2. APPEALING POOLS OF BIOACTIVE NATURAL PRODUCTS ...................................................................... 2

1.2.1. Plants as appealing pool of bioactive natural products....................................................... 3 1.2.2. Microbes as appealing pool of bioactive natural products ........................................................ 7 1.2.3. Marine source as appealing pool of bioactive natural products ................................................ 9

1.3 A QUICK GLANCE ON SOME NATURAL PRODUCT-BASED MEDICINES IN NEAR PAST ........................ 11

CHAPTER 2 ........................................................................................................................................... 22

PREVIOUS PHYTOCHEMICAL INVESTIGATION ON THE

BERBERISCALLIBOTRAYS,CARAGANA AMBIGUA AND VINCETOXICUM STOCKSII ................................ 22

PART A ................................................................................................................................................... 23

2.1. PREVIOUS PHYTOCHEMICAL INVESTIGATION OF THE GENUS BERBERIS................................................. 23 2.2. PREVIOUS PHYTOCHEMICAL INVESTIGATION OF BERBERIS CALLIOBOTRYS ............................................. 28 2.3. CLASSIFICATION OF BERBERIS CALLIOBOTRYS ..................................................................................... 28

PART B ................................................................................................................................................... 30

2.4. PREVIOUS PHYTOCHEMICAL INVESTIGATION OF THE GENUS CARAGANA ............................................... 30 2.5. PREVIOUS PHYTOCHEMICAL INVESTIGATION OF CARAGANA AMBIGUA .................................................. 35 2.6. CLASSIFICATION OF CARAGANA AMBIGUA ........................................................................................... 36

PART C ................................................................................................................................................... 37

2.7. PREVIOUS PHYTOCHEMICAL INVESTIGATION OF THE GENUS VINCETOXICUM ........................................ 37 2.8. PREVIOUS PHYTOCHEMICAL INVESTIGATION OF VINCETOXICUM STOCKSII............................................. 39 2.9. CLASSIFICATION OF VINCETOXICUM STOCKSII ..................................................................................... 39 2.10. RESEARCH HYPOTHESIS ................................................................................................................ 40

2.11. RESEARCH PROBLEM AND OBJECTIVES OF THE STUDY ..................................................................... 40

CHAPTER 3 ........................................................................................................................................... 42

RESULTS AND DISCUSSION: BIOLOGIAL SCREENING AND PHYTOCHEMICAL ANALYSES

OF BERBERIS CALLIOBOTRYS, CARAGANA AMBIGUA AND VINCETOXICUM STOCKSII;AND

ISOLATION OF SECONDARY METABOLITES ............................................................................................... 42

PART A ................................................................................................................................................... 43

3.1. BIOLOGICAL SCREENING OF CRUDE EXTRACTS OF BERBERIS CALLIOBOTRYS ......................................... 43 3.2 PHYTOCHEMICAL ANALYSIS OF SECONDARY METABOLITES FROM BERBERIS CALLIOBOTRYS ................... 45

3.2.1 Total phenolic and flavonoid contents estimation ..................................................................... 45

3.2.2.1 UHPLC-MS Analysis for Identification of Secondary Metabolites of B. ................................. 46 3.2.2.2 Secondary metabolite identification of Bc-M through UHPLC-MS analysis ........................... 46 3.2.2.3 Secondary metabolite estimation of Bc-E through UHPLC-MS analysis ................................ 49 3.2.2. Secondary metabolite determination of Bc-W through UHPLC-MS analysis ............................ 52

3.3. SPECTROSCOPIC CHARACTERIZATION OF SECONDARY METABOLITES ISOLATED FROM B.CALLIOBOTRYS 55 3.3.1. Structure Elucidation of 4-Hydroxybenzoic acid (214) ............................................................ 55 3.3.2. Structure Elucidation of Methyl p-Coumarate (235)................................................................ 56 3.3.3. Structure Elucidation of Octadecyl-p-cumarate (236) ............................................................. 56

3.3.4. Structure Elucidation of Corydaldine (121) ............................................................................ 58 3.3.5. Structure Elucidation of N-methyl Corydaldine (122).............................................................. 58 3.3.6. Structure Elucidation of Armepavine (207) ............................................................................. 59

Table of Contents

x

3.3.7. Structure Elucidation of Berberine (83) .................................................................................. 60 3.3.8. Structure Elucidation of Columbamine (237) .......................................................................... 61 3.3.9. Structure Elucidation of Syringaresinol (238) ......................................................................... 62

PART B ................................................................................................................................................... 66

3.4. BIOLOGICAL SCREENING OF CRUDE EXTRACTS OF CARAGANA AMBIGUA ............................................... 66

3.5. PHYTOCHEMICAL ANALYSIS OF SECONDARY METABOLITES FROM CARAGANA AMBIGUA ........................ 67 3.5.1 Total phenolic and flavonoid contents estimation ..................................................................... 67 3.5.2. UHPLC-MS Analysis to Identify Secondary Metabolites of Caragana ambigua ....................... 68 3.5.2.1. Secondary metabolite estimation of Ca-M through UHPLC-MS analysis .............................. 68 3.5.2.2 Secondary metabolite estimation of Ca-E through UHPLC-MS analysis ................................ 71

3.6. CHARACTERIZATION OF SECONDARY METABOLITES ISOLATED FROM THE FRACTION CA-E ................... 73 3.6.1. Structure Elucidation of Taraxerol (262) ................................................................................ 74 3.6.2. Structure Elucidation of Teraxerol acetate (263) .................................................................... 75

3.6.3. Structure Elucidation of 2′-(4-Hydroxyphenyl)-Ethyl Stearate (264) ........................................ 76 3.6.4. Structure Elucidation of Apigenin (265) ................................................................................. 77 3.6.5. Structure Elucidation of Naringinin (249) .............................................................................. 78 3.6.6 Structure Elucidation of Kaempheride (222) ............................................................................ 79 3.6.7. Structure Elucidation of Quercetin (125) ................................................................................ 80 3.6.8. Structure Elucidation of Quercetin 3-O-β-D-glucopyranoside (198) ........................................ 80 3.4.9. Structure Elucidation of β-Sitosterol 3-O-D-glucopyranoside (172)......................................... 81

PART C ................................................................................................................................................... 83

3.7. BIOLOGICAL SCREENING OF CRUDE EXTRACT OF VINCETOXICUM STOCKSII .......................................... 83 3.8. CHARACTERIZATION OF SECONDARY METABOLITES ISOLATED FROM V. STOCKSII................................. 84

3.8.1. Structure Elucidation of stocksiloate (266) ............................................................................. 84 3.8.2. Structure Elucidation of 4-(4-(methoxycarbonyl) benzyl) phenyl] carbamic acid (267) ............ 86 3.8.3. Structure Elucidation of Bis[di-p-phenyl methane]ethyl Carbamate (268) ............................... 87 3.8.4. Structure Elucidation of 4-Hydroxy-3-Methoxyphenyl 7, 8, 9 Propanetriol (194) ..................... 88 3.8.5. Structure Elucidation of Feruloyl-6-O- β-D-glucopyranoside (197) ......................................... 89 3.8.6. Structure Elucidation of Apocynin (196) ................................................................................. 90

3.8.7. Structure Elucidation of vincetomine (192) ............................................................................. 91

CHAPTER 4 GENERAL EXPERIMENTAL METHODS AND TECHNIQUES ................................. 93

4.1. GENERAL EXPERIMENTAL PROCEDURES ........................................................................................... 94 4.1.1 Chromatographic techniques ............................................................................................ 94 4.1.2 Spectroscopic techniques.................................................................................................. 94

4.2. INSTRUMENTATION AND WORK METHODOLOGY OF UHPLC-MS ....................................................... 94 4.3. ASSESSMENT OF BIOLOGICAL POTENTIAL OF CRUDE EXTRACTS OF BERBERIS CALLIOBOTRYS AND

CARAGANA AMBIGUA ............................................................................................................................................. 95 4.4. COLLECTION, EXTRCATION AND ISOLATION OF METABOLITES OF BERBERIS CALLIOBOTRYS .................... 95

4.4.1. Collection of Berberis calliobotrys ......................................................................................... 95 4.4.2. Extraction and Fractionation ................................................................................................. 95 4.4.3 Isolation and purification of secondary metabolites........................................................... 96

4.5. SPECTROSCOPIC DATA OF THE ISOLATED COMPOUNDS ........................................................................ 98 4.5.1. Spectroscopic data of 4-Hydroxybenzoic acid (214):............................................................... 98 4.5.2. Spectroscopic data of Methyl p-coumarate (235): ................................................................... 98 4.5.3. Spectroscopic data of Octadecyl-p-cumarate (236): ................................................................ 98 4.5.4. Spectroscopic data of Corydaldine (121): ............................................................................... 98

4.5.5. Spectroscopic data of N-methyl Corydaldine (122): ................................................................ 98 4.5.6. Spectroscopic data of Armepavine (207): ............................................................................... 98 4.5.7. Spectroscopic data of Berberine (83): .................................................................................... 99 4.5.8. Spectroscopic data of Columbamine (237): ............................................................................ 99 4.5.9. Spectroscopic data of Syringaresinol (238): ........................................................................... 99 4.5.10. Spectroscopic data of Acanthoside D (208): ......................................................................... 99

4.6. COLLECTION, EXTRCATION AND ISOLATION OF METABOLITES OF CARAGANA AMBIGUA ......................... 99 4.6.1. Collection of Caragana ambiguia .......................................................................................... 99

4.6.2. Extraction and Fractionation ................................................................................................. 99 4.7. SPECTROSCOPIC DATA OF ISOLATED COMPOUNDS ........................................................................... 102

4.7.1. Spectroscopic data of Teraxerol (262): ................................................................................. 102

Table of Contents

xi

4.7.2. Spectroscopic data of teraxerol Acetate (263):...................................................................... 102 4.7.3. Spectroscopic data of 2′-(4-Hydroxyphenyl)-Ethyl Stearate (264): ........................................ 102 4.7.4. Spectroscopic data of Apigenin (265): .................................................................................. 102 4.7.6. Spectroscopic data of Naringinin (249): ............................................................................... 102 4.7.5. Spectroscopic data of kaempheride (222) ............................................................................. 103

4.7.7. Spectroscopic data of Quercetin (125): ................................................................................ 103 4.7.8. Spectroscopic data of Quercetin 3-O-β-D-glucopyranoside (198): ........................................ 103 4.7.9. Spectroscopic data of β-Sitosterol 3-O-D-glucopyranoside (172): ......................................... 103

4.8. COLLECTION, EXTRACTION AND ISOLATION OF METABOLITES OF V. STOCKSII .................................... 103 4.8.1. Collection of V. stocksii ....................................................................................................... 103 4.8.2. Extraction and Fractionation ............................................................................................... 104

4.9. SPECTROSCOPIC DATA OF ISOLATED COMPOUNDS ........................................................................... 106 4.9.1. Spectroscopic data of Stocksiloate (266): ............................................................................. 106

4.9.2. Spectroscopic data of 4-(4-(methoxycarbonyl)benzyl) phenyl]carbamic acid (267): ............... 106 4.9.3. Spectroscopic data of bis [di-p-phenylmethane] ethyl carbamate (268): ................................ 106 4.9.4. Spectroscopic data of 4-Hydroxy-3-Methoxyphenyl 7, 8, 9 Propanetriol (194):...................... 106 4.9.5. Spectroscopic data of Feruloyl-6-O-Dglucopyranoside (197):............................................... 106 4.9.6. Spectroscopic data of Apocynin (196): ................................................................................. 107 4.9.7. Spectroscopic data of Vincetomine (192): ............................................................................. 107

CHAPTER 5 ......................................................................................................................................... 108

BIOASSAYS ......................................................................................................................................... 108

5.1. BIOASSAYS OR PROTOCOL ............................................................................................................. 109 5.2. BIOASSAYS .................................................................................................................................. 109

5.2.3. Total antioxidant capacity evaluation (Phosphomolybdenum method) ................................... 109 5.2.4. DPPH free radical scavenging assay.................................................................................... 109 5.2.5. ABTS free radical scavenging assay ..................................................................................... 110 5.2.6. Metal chelating assay .......................................................................................................... 110 5.2.7. Cupric ion reducing assay ................................................................................................... 110 5.2.8. Ferric reducing antioxidant assay ........................................................................................ 110

5.2.9. Cholinesterase inhibition assay ............................................................................................ 111 5.2.10. α- glucosidase inhibition assay ........................................................................................... 111 5.2.11. α-Amylase inhibition assay................................................................................................. 111 5.2.12. Tyrosinase inhibition assay ................................................................................................ 111 5.3.13. Anti-Urease assay .............................................................................................................. 112

LIST OF ABBREVIATIONS ............................................................................................................... 112

REFERENCES ..................................................................................................................................... 113

PUBLICATIONS: ................................................................................................................................. 130

List of Tables

xv

List of Tables

Table 1.1 Some FDA approved drugs in recent years 20

Table 3. 1Antioxidant properties of B. calliobotrys extracts* 43

Table 3. 2 Enzyme inhibition activities of B. calliobotrys extracts* 44

Table 3. 3Total phenolic and flavonoid contents of B. calliobotrys extracts* 45

Table 3. 4UHPLC-MS analysis of Bc-M fraction 47

Table 3. 5UHPLC-MS analysis of Bc-E fraction 50

Table 3. 6 UHPLC-MS analysis of Bc-W fraction 53

Table 3. 7 1H- and 13C-NMR data of compound 214 (CDCl3, 400 and 125 MHz) 56

Table 3. 8 1H-NMR and 13C-NMR data of compound 235 and 236 (CDCl3, 400 and 100 MHz) 57

Table 3. 9 1H-NMR and 13C-NMR data of compounds 121, 122 and 207 (CDCl3, 400 and 100 MHz) 60


Table 3. 11 1H- and 13C-NMR data of compound 238 and 208 (CDCl3, 400 and 125 MHz) 65

Table 3. 12 Antioxidant properties of C. ambigua extracts* 66

Table 3. 13 Enzyme inhibition activities of C. ambigua extracts* 67

Table 3. 14 Total phenolic and flavonoid content of C. ambigua extracts* 68

Table 3. 15 UHPLC-MS secondary metabolites profile of Fraction Ca-M 69

Table 3. 16 UHPLC-MS secondary metabolites profile of fraction Ca-E 72


Table 3. 18 1H-NMR and 13C-NMR data of compound 264 (CDCl3, 500 and 125 MHz) 77

Table 3. 19 1H- and 13C-NMR data of compound 265 and 249 (DMSO-d6 , 500 and 125 MHz) and 222

(CD3OD, 400 and 100 MHz) 79

Table 3. 20 1H- and 13C-NMR data of compound 125 and 198 (DMSO-d6, 400 and 100 MHz) 81

Table 3. 21 1H-NMR and 13C-NMR data of compound 172 (CDCl3+CD3OD, 400 and 100 MHz) 82

Table 3. 22 Anti-oxidant and anti-urease activities of V. Stocksii (Vs) extracts* 83

Table 3. 23 1H-NMR and 13C-NMR data of compound 266 (CDCl3, 400 and 100 MHz) 85

List of Tables

xvi

Table 3. 24 1H and 13C NMR data of 267 (DMSO-d6, 600 and 150 MHz, respectively), 268 (CD3OD, 600

and 150 MHz, respectively) 88

Table 3. 25 1H and 13C NMR data of 194 and 197 (CD3OD, 600 and 150 MHz) 90

Table 3. 26 1H and 13C NMR data of 196 (CD3OD, 500 and 125 MHz) 91

Table 3. 27 1H and 13C NMR data of 192 (CD3OD, 500 and 125 MHz) 92

List of Figures

xvii

List of Figures

Figure 3. 1 Total ion chromatograms (TICs) of Bc-M fraction 46

Figure 3. 2 Total ion chromatograms (TICs) of Bc-E fraction 49

Figure 3. 3 Total ion chromatograms (TICs) of Bc-W 52

Figure 3. 4 Important HMBC (H→C) csorrelations of 207 60

Figure 3. 5 Important HMBC (H→C) correlations of 238 63


Figure 3. 7 Total ion chromatograms of methanol extractof C. ambigua 68

Figure 3. 8 Total ion chromatograms of ethyl acetate extract (Ca-E) of C. ambigua 71


List of Scheme

xviii

List of Scheme

Scheme 4. 1 Extraction and isolation of secondary metabilities from Berberis calliobotrys

97

Scheme 4. 2 Extraction and isolaltion of secodndary metabolities from Caragana

ambigua 101

Scheme 4. 3 Extraction and islationfo secondary metabolites from vincetoxcicum. Stocksii

105

1

CHAPTER 1

Alluring Natural Products and Their

Applications in Drug Development

Chapter no. 01 Introduction

2

1.1. Introduction of Natural Products

Plants, microbes, marine and terrestrial animals are the leading factories for the

production of fascinating chemicals called as natural products (Ghori et al., 2016). As a

result of overall metabolism, these organisms produce primary and secondary

metabolites. The later have been the focus of researchers to discover and develop modern

medicine; these metabolite are termed as natural products and are the basis of

pyhtomedicne, phytochemistry and natural product chemistry.

In ancient times, plants were the primary source for human to take care of their needs

including health. Secondary metabolites in plants do not have much decisive role in

development, growth and reproduction, however, they have defensive role against harm

from natural environment and in interspecies competition, revealing their need dependent

production is. Due to such properties, of the secondary metabolites, they have magic role

as drugs and other industrial applications (Justin et al., 2014). Secondary metabolites with

their humongous chemical and structural variety have kept naturopaths inspired for the

most excellent sources of drugs (Newman et al., 2012). With the dawn of mankind

through the ages, folk medicine system gains the fame in India, China and throughout the

world. In these systems, herbal remedy was found more charming tool for treatment of

various ailments which is still fascinating for modern world (Takahashi et al., 2006).

1.2. Appealing Pools of Bioactive Natural Products

Natural products are always considered as active ingredients for drug

development. More than 80% of drugs were purely natural products or derivative of

natural compounds. Various organisms have been identified as major sources of natural

products (Bishayee and Sethi, 2016); they mostly include plants, bacteria, fungi and

several marine sources, however, animals and symbioses have also been included in this

group.


3

1.2.1. Plants as appealing pool of bioactive natural products

Plants are always considered as vital source of life sustainer since the time of

human birth. In many countries, plants and herbal extracts are the imperative sources for

impediment and management of human diseases for therapeutic purposes (Ashtiania et

al., 2018). The well-known antique alkaloid “morphine” (1) was isolated from Papaverm

somniferum. It works on mesenteric plexus of CNS and reduces feeling of pain. It also

reduces shortness of breath due to pulmonary edema.Solanine (2) from Solanum

tuberosum possesses anti-fungal, anti-carcinogenic, anti-convulsant and anti-

inflammatory potential (Kabera et al., 2014). Nicotine (3) purified from solanaceae

species exhibits anti-inflammatory properties. Paclitaxel (4) initially was identified in the

bark of Taxus brevifolia showed anticancer potential. Vincristine (5), vinblastine (6),

vindoline (7) and catharanthine (8) isolated from Catharanthus roseusare also known

anticancer natural products (Safonova and Luca, 2018). The Mitragyna speciosa leaves

were used as folk medicine for remedy of diabetes, diarrhea and for better blood

circulation. Mitragynine (9) with its analogous speciogynine (10), paynantheine (11), and

speciociliatine (12) were identified as major alkaloid in M. speciosa, exhibited anti-

depressant property (Hamid et al., 2017).


4

Isosteroidal alkaloids are abundantly found in the genera Fritillaria and

Veratrum. Peiminine (13), imperialine (14), verticine (15), peimisine (16), and

cyclopamine (17) are their important constituents with anti-inflammatory, anti-

hypertensive, anti-tumor and antitussive properties (Shang et al., 2018). Artemisinin (18)

was isolated from Artemisia annua; its howed antimalarial potential (Shen, 2015). A

pentacyclic triterpene ursolic acid (19) which is potent antioxidant also manifested

hepatoprotective, anti-inflammatory, antimicrobial, anti-tumor and cardioprotective

effects (López-Hortas et al., 2018). Paracaseolin A (20) and D (21) are triterpenoids

which were isolated from Sonneratia paracaseolaris and showed cytotoxic potential

against A549 tumor cell line and anti-H1N1 virus activity respectively (Gong et al.,

2017).


5

A chalcone, isoliquiritigenin (22) was isolated from Glycyrrhiza uralensis, and

Glycyrrhiza inflate possess anti-cancer and cardio protective effects. Echinatin (23),

which have cytotoxic effect on neuraminidases was isolated from Glycyrrhyza echinata

(Zsuzsanna and Perjési, 2016). Isobavachalcone (24) isolated from the twigs of Dorstenia

barteri has displayedanti-inflammatory property (Dzoyem et al., 2017). Two antioxidant

compounds tricin (25) and luteolin 6, 8-di-C-glucoside (26) were isolated from

Stipagrostis plumosa (L) (Hussein et al., 2018).


6

Two commonly found important phytosterols, β-sitosterol (27) and stigmasterol

(28) were isolated by investigating root part of Indigofera heterantha, exhibited potent

anti-diabetic and anti-inflammatory activity respectively (Zeb et al., 2017).

Phyllanthus emblica which is commonly known as amla, has active agents;

emblicanin A (29) and emblicanin B (30) which reveal anti-microbial, anti-viral,

hepatoprotective and anti-cancer potential (Lei et al., 2015).


7

Ainsliaeasin C (31) is acoumarin derivative, which is known as anticoagulant

agent identified from Ainsliaea fragrans (Lei et al., 2015). Sanandajin (32), and ethyl

galbanate (33) are well known sesquiterpenoid coumarin with antibacterial potential; they

were separated from the roots of Ferulap seudalliacea (Dastana et al., 2016).

1.2.2. Microbes as appealing pool of bioactive natural products

Microorganisms possess structurally enormous and significant variety of

therapeutically active secondary metabolites. Penicillin (34) was the first splendid gift by

Alexander Fleming in 1929 from the fungus Penicillium notatum, which established the

basis of new era “the Golden Age of Antibiotics” (Milshteyn, et al., 2014).

Similarly a cyclic peptide, cyclosporin A (35) and a macrolid rapamycin (36)

were isolated from Tolypocladium inflatum and Streptomyces hygroscopicus,

respectively, showe dimmunomodulatory potential (Katz et al., 2016).


8

Bleomycin (37) is an anticancer compound, isolated from Streptomyces verticillus

(Katz et al., 2016), while polycyclic ethanones, alternethanoxin A (38) and B (39) were

identified from Alternaria sonchias cytotoxic metabolites against various cancer cells

(Evidente et al., 2014). Aspergillus aculeatus, a fungusfrom terrestrial source, produces

anti-diabetic compound rubrofusarin (40) (Dewi et al., 2016).


9

1.2.3. Marine source as appealing pool of bioactive natural products

Marine natural products are getting attention among the natural products due to

their versatile pharmacological properties. Terpenoids like dixiamycin A (41) and

dixiamycin B (42), were isolated from marine Actinomycete and Streptomyces sp. SCSIO

02999, possessing significant anti-bacterial activity. Alkaloids, rubrumazine B (43),

echinulin (44) and variecolorin H (45) were identified from Eurotium cristatum EN-220.

Rubrumazine B (43) possesses activity against pathogenic fungus Magnaporthe grisea

while echinulin (44) and variecolorin H (45) exhibited potential againsthuman pathogen

S. aureus (Choudhary et al., 2017). The chemical investigation of Streptomyces strain

CNH189 leads to the isolation of ansalactams B (46), ansalactams C (47) and

ansalactams D (48) which were found to be modest antibacterial agent (Blunt et al.,

2017).


10

Marine sources have also contributed to the bank of anti-cancer agents.

Marinomycin (49) from streptomyces sp. and chinikomycin A (50) from marinispora sp.

were found as active anti-tumor agents. Marinomycin (49) was also found active in

antibacterial assay. A secondary metabolite mechercharmycin A (51) was purified from

Thermo actinomyces sp. YM3-251 showed potent antitumor activity. Arenicolides A

(52), a 26-membered, type 1 polyketide showed modest cytotoxic effect towards HCT-

116 of human colon adenocarcinoma cell line, was isolated from Salinispora arenicola

CNR-005 (Manivasagan et al., 2014).


11

1.3 A Quick Glance on Some Natural Product-Based Medicines in

Near Past

Reliance of pharmaceutical drugs on natural product cannot be denied even in 21st

century. These are always endorsing source for the evolution of innovative drugs. About

one-third FDA-approved drugs during1981 to 2014 have natural product background

(Newman, 2018).They include small molecule based structures, either unaltered natural

metabolites or their derivatives and synthetic natural mimics (Li and Lou, 2018).


12

Following section describe the role of some natural and derived natural products recently

approved as drugs by FDA (Table 1.1).

Oritavancin (53) trade name Orbactiv (Newman et al., 2016), is semisynthetic

compound of natural precursor, chloroeremomycin (54), which was isolated from

Amycolatopsis orientalis (Domenech et al., 2009; Katz and Baltz, 2016). It is

antibacterial, oral drug, promote bacterial cell death by following three ways, interrupting

the cell wall formation through transglycosylation inhibition, cell membrane distortion by

interaction with hydrophobic 4'-chlorobiphenyl methyl (55) group and by offering more

secondary binding positions to resist bacterial strains (Markham, 2014).

Vorapaxar (56) with trade nameZontivity is derivative of himbacine (57) isolated

from plants Galbulimima baccata and G. belgraveana (Pinhey et al., 1961; Butler et al.,

2014). It is used in preventive measurement of cardiological and hematological disorders.

It involves reduction of thrombotic cardiovascular in patient with myocardial infarction

(MI) or peripheral arterial disease (PAD) history. It inhibits cardiovascular events by

inhibiting platelet aggregation related to thrombin (Poole and Elkinson 2014).


13

Ecteinascidin or trabectedin (58) is being marketed under brand name Yondelis as

anti-cancer, intravenous, alkaloid drug (Rinehart et al., 1990; Palanisamy et al., 2017). It

was isolated from Ecteinascidia turbinate.In its action, it binds to the guanine (59) part of

DNA at minor grooves, where it opens the double helix of DNA which eventually lead

the death of soft cancer cell (Petek et al., 2015).Xifaxan is trade name for rifaximin (60)

whichis a semisynthetic compound, derivative of rifamycin B (61) or rifamycin SV (62),

being produced by Amycolatopsis rifamycinica. It is taken orally for treatment for

digestive disorders (Bimer et al., 1972; Saxena et al., 2014).It stops the synthesis of RNA

by interrupting the steps of transcription by binding to the beta subunit of bacterial DNA-

dependent RNA polymerase. As a result controls command for protein synthesis and

inhibits cell growth (Kane and Ford 2016).

https://www.centerwatch.com/drug-information/fda-approved-drugs/drug/100079/xifaxan-rifaximin%20which


14

Obeticholic acid (63) with trade name of Ocaliva, is derived from

chenodeoxycholic acid (64), was first isolated from the bile of the domestic goose. It is

taken orally and therapeutically acts against cirrohsis (Russell, 2003). It activates

farnesoid X-activated receptor in intestine and liver, which stops the production of more

bile acids (65) from cholesterol and prevents accumulation of bile acids near hepatocytes

avoiding liver damage (Markham and Keam, 2016).


15

Midostaurin/Rydapt (66) is an alkaloidal drug derived from staurosporine (67),

which was isolated from Streptomyces staurosporeus (Omura et al., 1977; Zhou et al.,

2019).It is a prominent anticancer oral drug for the patient suffering with FLT3-positive

acute myeloid leukemia (AML) (Beatriz et al., 2018). It enriches bloodstream with white

blood cells killing cancer roots in bone marrow (US FDA 2017). It shows multikinase

inhibitor effect that works by obstruction in cell growth by controlling respective enzyme

(Kim, 2017).

Naldemedine (68), sold under the name of symproic, is derived from naltrexone

(69) (Hussar and Hussar 2018), which is semi-synthetized from oxymorphone (70)

(Sudakin, 2016) formally originatedfrom morphine (1)(Huang, Patent 2008).It was first

isolated from natural source Papaver somniferum (Sharopov et al., 2018). It is

administered orally and acts by hindering peripheral µ-opioid receptors in gastrointestinal

tract tissue which results into lesson the opioids effect on constipation (Markham, 2017).


16

Inotuzumab ozogamicin (71), an anticancer intravenous drug, is marketed as

Besponsa, in which inotuzumab is identified in ovary cells of Chinese hamster (Datta-

Mannan et al., 2018), which is attached to derivative cytotoxic group of “calicheamicins”

(72) isolated from Micromonospora echinospora (Song et al., 2015). Ozogamicin (73) is

cytotoxic part of Besponsal, enters into the cell by the process of endocytosis where

ozogamicin gets separated from antibody part and reached to the nucleus, starts

disintegration of DNA, consequences in cell death (Lamb, 2017).


17

Plazomicin (74) is an intravenous infusion, antibacterial drug traded as zemdr

(FDA Approved Drug Products 2019) It is semisynthetic aminoglycoside derivative of

sisomicin (75) (Gupta, 2017), isolated from Micromonospora inyoensis (Talukdar et al.,

2016). It impede protein production in bacterial cell by developing bonding with its

ribosomal 30S subunit, varies with drug concentration (Shaeer, 2018).

Cannabidiol (76) known as Epidiolexis a natural product isolated from Cannabis

sativa (Fairbaim and Liebmann, 1973; Dyer, 2018). It is taken orally for the treatment of

epilepsy because it has anti-seizures property which makes it valuable for treatment of

Lennox-Gastaut syndrome or Dravet syndrome in patient with age not under 2 (Elliott

and Chan 2018). Omadacycline (77) is known as Nuzyra in market (Markham and Keam

2018). It is derivative of tetracycline (78), isolated from Streptomycessp. (Petković et al.,

2017). It has antibacterial effect, administrated orally and by intravenous infusion. By

binding with protein synthesizing unit of ribosome (70S) with greater affinity it ensures

the excellent antibacterial activity (Rahman and Koh, 2018).


18

Eravacycline (79) trade marked as Xerava (FDA Approved Drug Products 2018)

is anti-biotic, derived from tetracycline (78) isolated from Actinomycete sp. (Butler et al.,

2014). It is taken orally and in injection form to treat infections. It hinders the

prolongation of amino acid chain by binding to responsible ribosomal (30S) unit of

bacteria to inhibit the protein synthesis (Zhanel et al., 2016).

Krintafel is the trade name for tafenoquine (80) (Elliott and Chan 2018), is a

derivative of quinoline (81) “a natural compound” was isolated initially from bark of

Cinchona sp. possess anti-malarial potential (Fernandez-Alvaro et al., 2016). Oral dosage

works to eliminate the roots of dormant parasite in the liver for the patient of age 16 or

older along with the appropriate anti-malarial therapy (Frampton, 2018). Sarecycline (82)

is generic name for seysara, an anti-inflammatory, orally taken drug, derivative of

tetracycline (77) isolated from actinomycetes (Butler et al., 2014); it is applied for skin

infection (Deeks, 2019).


19


20

Table 1.1 Some FDA approved drugs in recent years

Drug

Generic/trade

name

Lead compound/source Therapeutic

effect

Route of

administra

tion

Mode of action Manufacturer/App

roved status

Reference

Oritavancin(53)/

Orbactiv

Chloroeremomycin

(54)/Amycolatopsis orientalis

Dermatology

infaction

oral Interruption in

cell wall

synthesis

The medicine

company (2014)

(Newman et al., 2016),

Domenech et al., 2009),

(Katz, and Baltz, 2016)

(Markham, 2014)

Vorapaxar(56)/

Zontivity

Himbacine(57)/ Galbulimima

baccata and G. belgraveana

Cardic and

heamatology

oral Reduce

thrombotic

Event

Merck

(2014)

(Butler et al., 2014)

(Poole and Elkinson 2014)

Trabectedin(58)/

Yondelis

Trabectedin (58)/

Ecteinascidia turbinate

Oncology intravenous Binding with

DNA

Janssen

(2015)

Palanisamy et al., 2017).

(Petek et al., 2015)

Rifaximin

(60) /Xifaxan

Rifamycin B or Rifamycin

SV (61,62) / Amycolatopsis

rifamycinica

Gastroenterol

ogy

oral Inhibit protein

synthesis

Salix

Pharmaceuticals

(2015)

(Saxena et al., 2014).

(Kane and Ford 2016)

Obeticholic

acid(63)/ Ocaliva

chenodeoxycholic acid (64)/

domestic goose

Hepatology oral FXR activation

and control bile

acid level

Intercept

Pharmaceuticals

(2016)

(Russell, 2003) (Markham

and Keam, 2016)

Midostaurin(66) /

Rydapt

Staurosporin(67)/

Streptomyces staurosporeus

Hematology

and

Oncology

oral multikinase

inhibitor

Novartis

(2017)

Zhou et al., 2019, (Beatriz et

al., 2018). (Kim, 2017)

Naldemedine (68)/

Symproic

Morphine (1)/ Papaver

somniferous

Gastroenterol

ogy

oral Block opioid

receptor

Shionogi

(2017)

(Hussar and Hussar 2018,

(Sudakin, 2016) (Sharopov

et al., 2018). (Markham,

2017)

Inotuzumab

ozogamicin(71)/

Besponsa

Monoclonical

antibody/chinease hamster,

calicheamicins (72)/

Micromonospora

echinospora.

hematology intervenous DNA breakage

and apoptosis of

cell

Pfizer (2017) (Datta-Mannan et al., 2018)

(Lamb, 2017). (Song et al.,

2015)

Plazomicin Sisomicin (75)/ Infaction intervenous Inhibit protein Achaogen (Gupta, 2017), (Talukdar et

https://www.centerwatch.com/drug-information/fda-approved-drugs/therapeutic-area/5/gastroenterology


https://www.centerwatch.com/drug-information/fda-approved-drugs/drugs/company/Salix+Pharmaceuticals

https://www.centerwatch.com/drug-information/fda-approved-drugs/drugs/company/Salix+Pharmaceuticals

https://www.centerwatch.com/drug-information/fda-approved-drugs/drugs/company/Intercept+Pharmaceuticals

https://www.centerwatch.com/drug-information/fda-approved-drugs/drugs/company/Intercept+Pharmaceuticals

https://www.centerwatch.com/drug-information/fda-approved-drugs/drugs/company/Novartis



https://www.centerwatch.com/drug-information/fda-approved-drugs/drugs/company/Shionogi

https://www.centerwatch.com/drug-information/fda-approved-drugs/drugs/company/Pfizer

https://www.centerwatch.com/drug-information/fda-approved-drugs/drugs/company/Achaogen


21

(74)/Zemdr Micromonospora inyoensis disesas synthesis (2018) al., 2016) (Shaeer, 2018)

Cannabidiol(76)/

Epidiolex

Cannabidiol(76)/Cannabis

sativa

Neurology oral anticonvulsant GW

Pharmaceuticals

(2018)

(Elliott and Chan 2018)

(Dyer, 2018)

Omadacycline

(77)/ NUZYRA

Tetracycline(78)/Streptomyce

ssp

antibacterial Oral and

intervenous

Inhibit protein

synthesis

Paratek

Pharmaceuticals

(2018)

(Markham and Keam 2018),

(Petković et al., 2017).

Rahman and Koh, 2018)

Eravacycline (79)

/ Xerava

Tetracycline(78)/Actinomycet

e sp

Gastroenterol

ogy

Intervenous Inhibit protein

synthesis

Tetraphase

Pharmaceuticals (2018)

(Butler et al., 2014) (Zhanel

et al., 2016)

Tafenoquine (80)

/Krintafel

Quinolin(81)/ Cinchona sp Infactious

disoreded

oral prevents the

progress of the

erythrocytic

forms of the

parasite

GlaxoSmithKline

(2018)

(Elliott and Chan 2018)

(Fernandez-Alvaro et al.,

2016) (Frampton, 2018)

Sarecycline (82) /

Seysara

Tetracycline (78) /

actinomycetes.

Dermatology oral Inhibit protein

synthesis

Paratek

Pharmaceuticals

(2018)

(Butler et al., 2014)

(Deeks, 2019)

https://www.centerwatch.com/drug-information/fda-approved-drugs/drugs/company/GW+Pharmaceuticals

https://www.centerwatch.com/drug-information/fda-approved-drugs/drugs/company/GW+Pharmaceuticals

https://www.centerwatch.com/drug-information/fda-approved-drugs/drugs/company/Paratek+Pharmaceuticals




https://www.centerwatch.com/drug-information/fda-approved-drugs/drugs/company/GlaxoSmithKline



22

CHAPTER 2

Previous Phytochemical Investigation on The

Berberis calliobotrys, Caragana ambigua And

Vincetoxicum stocksii

Chapter no. 02 Phytochemical Investigation of genus Berberis

23

Part A

2.1. Previous Phytochemical Investigation of the Genus Berberis

Main genus of family Berberidaceae is Berberis which is distributed worldwide

with its folk medicinal uses documented in Pakistan, India, Japan, China, Asia, Europe,

Africa and America. It grows as evergreen small tree or shrub with yellow flowers and

wood. Thisgenus is classified into three groups on the basis of habitat, Rocky Mountain,

Asiatic and European group.

Many species of the genus Berberis were used as source of indigenous medicines

and as modern available drugs (Khan et al., 2016). B.vulgarisis an important traditional

medicinal plant widely distributed in Iran.It has yellow wood, flowers, and red fruits.

Phytochemical investigation revealed the presence of alkaloids, phenolics, sterols, tannins

and triterpenoids compounds in B.vulgaris. It has been reported to cure inflammation,

vomiting, nausea, itching, heart diseases, diabetes, jaundice, malaria, sore throat and

block the action of acetylcholine (Zadeh et al., 2107).It is a source of isoquinoline

alkaloids including berberine (83) palmatine (84) berberamine (85), 5-

methoxyhydnocarpin (86) etc. Quantitative HPLC analysis showed that methanolic

extract of the whole plant contains 3.9% berberamine (85) and about 1.24% berberine

(83). In China berberine (83) has been used as anti-hyperglycemic agent and inhibits the

increase of blood and liver cancer (Imanshahidi and Hosseinzadeh, 2008).


24

In addition, tejedine (87), baluchistanamine (88), oxyacanthine (89),

isotetrandrine (90), obaberine (91), obamegine (92), aromoline (93), and thaligrisine (94)

were also isolated from B. vulgaris.


25

Some non-basic alkaloids like oxyberberine (95), thalifoline (96), chilenine (97)

and quaternary alkaloid jatrorrhizine (98) were also reported from B. vulgaris (Suau et

al., 1998).

B. aristata locally known as Rasaut, possesses several medicinal properties like in

skin diseases, ear, eye and urinary tract infections and menorrhagia, antibacterial,

antiviral, antioxidant, antifungal, antidiabetic, anti-ulcer, anti-malaria and gastric disorder

etc (Khan et al., 2016). Berberine (83) oxyberberine (95), berbamine (85), aromoline

(93), karachine (99), palmatine (84), oxyacanthine (89), taxilamine (100), epiberberine

(101), jatrorrhizine (98), 1-O-methylpakistanine (102), pakistanine (103), chitraline (104)

and kalashine (105) have been isolated from this plant (Semwal et al., 2018).

B. lyceum, which is commonly known as Ishkeen, found abundantly in Himalayan

region of Asia. In Pakistan it is found in Gilgit, Swat, and Kashmir. Its powdered root

extract was used as local medicine for diabetes in Himalaya region (Ali et al., 2015). In

addition, it has been used for treatment of fever, eye diseases, liver and kidney disorders,


26

throat pain, internal wounds etc. Its roots are rich in palmatine (84) and berberine (83)

along with minor amount of jhelumine (106), baluchistanamine (88), sindamine (107),

punjabine (108), and Karakoramine (109) (Shabbir, et al., 2012). β-sitosterol (27), butyl-

3-hydroxypropyl phthalate (110), 4-methyl-7-hydroxy-coumarin (111), 4,4-

dimethylhexadeca-3-ol (112) and 3-(4’-(6-methyl-butyl)-phenyl)propan-1-ol (113) were

also obtained from this plant (Ali, et al., 2015).

B. orthobotrys, growing in Gilgit-Baltistan, is used for joint pain therapy.

Phytochemical investigation has revealed the presence of aporphine-benzylisoquinoline

alkaloids specifically berberine (83), oxyacanthine (89), berbamine (85), pakistanine

(103), chitraline (104), kalashine (105) and pakistanamine (114) in B. orthobotrys. Its

aqueous-methanolic extract along with its aqueous, n-butanol and ethyl acetate extract

were found active in various in vitro and vivo anti-arthritic protocols (Alamgeer, et al.,

2017).


27

B. baluchistanicais is well known specie grown in Baluchistan province of

Pakistan and also distributed in other parts of the world. Its root extract is used to cure

cough and internal wound of humans and domestic animals. Literature studies explicate

the isolation of pakistanine (103), pakistanamine (114), (+)-baluchistine (115) and

baluchistanamine (88) from root part of B. baluchistanicais (Khan et al., 2016).B. chitria

is another famous species of genus Berberis found in Pakistan with its local use against

ulcers, eye problems, liver disorder, bleeding piles and enlarged spleen. Chlorogenic acid

(116) was identified as main constituent of B. chitria flower, magnoflorine (117) as main

metabolite in the stem portion along with berberine (83), palmatine (84), jatrorrhizine

(98), isocorydine (118), tetrahydropalmatine (119) and tetrahydroberberine (120)(Singh

et al., 2015).


28

2.2. Previous Phytochemical Investigation of Berberis calliobotrys

Diversity and medicinal importance of the genus Berberis, we selected an under-

explored species B. calliobotrys to investigate for its bioactive metabolites.B. calliobotrys

is a tall shrub with brownish-yellow stem, abundantly distributedin Pakistan and

Afghanistan. In indigenous medicine system it was used for the treatment of backache,

hepatic and stomach disorder, fever pharyngitis and inflammation in Chitral valley of

Pakistan. Modern research elaborates its potentialagainst anti-inflammatory, analgesic,

and antipyretic activity of its crude methanolic extract (Alamgeer et al., 2016). Further,

its extract exhibits antipyretic, anti-convulsant and antimicrobial effects (Rasool et al.,

2015).

2.3. Classification of Berberis calliobotrys

Phylum Tracheophyta

Class Magnoliopsida

Order Magnoliopsida

Family Berberidaceae

Genus Berberis

Species Berberis calliobotrys

Previously berberine (83), aromoline (93), oxyberberine (95), karachine (99), 1-

O-methylpakistanine (102), pakistanine (103), chitraline (104) and kalashine (105),

pakistanamine (114), corydaldine (121), methylcorydaldine (122), N-methyl-6, 7-

dimethoxy-isoquinoline (123) and waziristanine (124) have been isolated from roots of B.

calliobotrys (Srivastava et al., 2015). HPLC analysisof ethyl acetate extract resulted in

the identification of quercetin (125), gallic acid (126), trans ferulic acid (127), caffeic

acid (128), chlorogenic acid (116), vanillic acid (129) and p-coumeric acid (130) (Rasool

et al., 2015).

http://www.catalogueoflife.org/col/browse/tree/id/73328ba4e1446e1ee3602956b66eb127


29

Chapter no. 02 Phytochemical Investigation of genus Caragana

30

Part B

2.4. Previous Phytochemical Investigation of the Genus Caragana

The sub-family Papilionoideae contributes more than 100 species to family

Fabaceae. Its relativesare abundantly distributed in the grass land, desert climate and

rarely in forest climate. Caragana, one of the important genus of this family, is rich

source of various bioactive components including terpenoids, lectin, flavonoids,

stilbenoids, and phenylpropanoids (Meng et al., 2009). Literature of traditional medicine

of Mongolian, Chinese, and Tibetan system explains the therapeutic application of

various species of genus Caragana against numerous disorders including rheumatism,

headache arthritis, hypertension, neuralgia, atherosclerosis, inflammation, cancer and

arthritis (Meng et al., 2009). Literature search further revealed that several species of

Caragana have been studied for their phytochemicals.

Caragana tangutica found inNorth-West of China is used traditionally for remedy

for trauma, hypertension, rheumatic pains injury, cardiovascular and irregular

menstruation (Qiuxia et al., 2009). Varity of components have been isolated from this

plant, they include maackiain (131), medicarpin (132), formononetin (133), cajanin

(134), melilotocarpan A (135), 2-(2', 4'-dihydroxyphenyl)- 3-methyl-6-methoxy

benzofuran (136), bolusanthinIII (137), 7,3'-dihydroxy-5-methoxy isoflavone (138) and

p-ethoxy benzoic acid (139) (Niu et al., 2013).


31

Yang et alhave further reported the isolation of maackiain 3-O-β-D-

glucopyranoside (140), maackiain 3-O-6′-O-methyl malonyl-β-D-glucopyranoside (141),

(-)-maackiain 3-O-6′-O-acetyl-β-D-glucopyranoside (142) and 3-O-6′-O-acrylyl-β-D-

galactopyranoside (143) from ethyl acetate fractionof C. tangutica (Yang et al., 2017).

C. changduensis found in the region of China, traditionally used to remove lymph

in the blood by promoting blood circulation, treat hypertension and prevent elevation of

red blood cells in the blood. It has been reported to produce 6,7,2'-trihydroxy-4'-

methoxyisoflavone(144), (8S)-2,4-dihydroxy-8-hydroxymethyl-4'-methoxydeoxybenzoin

(145), 7,3'-dimethoxy-5'-hydroxy-isoflavone (146), 2,4-dihydroxybenzoate (147), ethyl


32

4-hydroxybenzoate (148), methyl 4-hydroxylbenzoate (149), cararosin A (150),

piceatannol (151) and isoliquiritigenin (152) (Guo et al., 2017).

C. sinica occupies large area of China, Korea, and Japan. Dried roots of C. sinica

are used for curing heumatism, rheumatism, neuralgia, vascular hypertension,

leukorrhagia, migrane, syndrome, arthritis, and wounds. Oligostilbenes are main

constituents of C. sinica showed anti-inflammatory, anti-acetylcholinesterase, and anti-

oxidative activities. Betulinic acid (153), medicarpin (154), pallidol (155), caragasinin C

(156) (Jeong et al., 2017), (+)-α-viniferin (157) miyabenol C (158) and kobophenol (159)

have been reported from C. sinica (Menga et al., 2009).


33

A perennial shrub, C. sukiensis grows insub arid and arid habitat of Eurasia.Its

ethanolic extract shows antimicrobial activity against the fungus Cryptococcus

neoformans (Mandal et al., 2015). Chromatographic separation of ethyl acetate extract of

aerial parts led to the identification of various compounds including sukienside A (160),

cyclosiversigenin (161), sukienside B (162), 3-O-β-D-xylocyclosiversigenin (163), β-

amyrin (164) and 3-β-hydroxy-2,5- melilotigenin C (165).


34

C. jubataiscultivated in Himalayan state, where local people use its aerial parts to

cure cardiovascular diseases, hyper-lipidemia, blood circulation disorders and

hypertension in folkmedicine system. Its mehanolic extract was found to be active in

antioxidant assay (Mandal et al., 2016). Chomatographic analysis of stem, root and

flower parts led to the isolation of different bioactive constituents including lyoniresinol

(166), isoampelopsin F (167), caraphenol A (168), caraphenol B (169), caraphenol C

(170) (Menga et al., 2009).


35

2.5. Previous Phytochemical Investigation of Caragana ambigua

Another part of my PhD project, I selected C. ambigua to be studied for its

bioactive compounds. C. ambigua is an important specie of the genus Caragana found in

Ziarat, Gilgit and Kashmir Valleys of Pakistan at about 7,000 to 12,000 feet above from

sea level. Study conducted on dried roots of C. ambigua reported the presence of

alkaloids, tannins and saponins (Kayani et al., 2007). C.ambigua, also known as Makhi,

is used as fuel wood, fodder and to increase soil fertility (Sarangzai et al., 2013).

Kingdom Plantae

Order Magnoliopsida

Class Fabales

Family Fabaceae

Genus Cagagana

Specie Caragana ambigua


36

2.6. Classification of Caragana ambigua

Methanolic extract of C. ambigua contains ambiguanol (171), E-cinnamic acid

(130), β-sitosterol 3-O-β-D-glucopyranoside (172), 5-hydroxy-3', 4', 6, 7-

tetramethoxyflavone (173), 3, 3', 4', 5, 7-pentahydroxyflavane (174) and calycosin (175)

(Majida et al., 2011).

Chapter no. 02 Phytochemical Investigation of genus Vincetoxicum

37

Part C

2.7. Previous Phytochemical Investigation of the Genus Vincetoxicum

The genus Vincetoxicum of the family Asclepiadaceae, accounting for around 100

species is growing in Europe, Asia and Japan (Guzel et al., 2017). In Pakistan six species

are found namely V. hirundinaria, V. cardiostephanum, V. stocksii, V. sakesarense, V.

arnottianum and V. canescens (Ali et al., 1989). Literature search revealed the presence

of phenanthroindolizidine alkaloids, triterpenoids, flavonoids, steroidal glycosides,

volatile compounds, saponin, phenolics, acetophenone, steroids and alkanols in

Vincetoxicum species which make them responsible for antibacterial and antifungal,

antispazmodic, antimalarial antileishmanial, antidiarrheal, and cytotoxic antifeedant

properties (Guzel et al., 2017).

Phytochemical analysis of the root extract of V hirundinaria (syn: V. officinale)

(Nowaka, et al.,2000) disclosed the presence of many phenanthroindolizidine, flavonoids,

steroids and alkaloids (Lavault et al., 1999), which indicated the reason for it being used

in traditional medicinal system to cure tumor and laxative (Nowaka et al., 2000), cough,

promotes diuresis and vomiting. Compound isolated from roots, included paeonol (176),

4-hydroxyacetophenone (177), syringic aldehyde (178), myristicine (179), (Lavault et al.,

1999), β- sitosterol (27), stigmasterol (28), α-amyrin (180), hancokinol (181) (Nowaka et

al., 2000), cynatratoside E (182), cynatratoside C (183), hirundigoside B (184)

hirundigoside C (185), hirundigoside D (186) (Lavault et al.,1999).

https://en.wikipedia.org/wiki/Diuresis


38

V. pumilum is a perennial herb commonly found in Central Asia. Methanolic

extract root and aerial part evaluation confirmed the presence of (-)-13a-α-antofine (187)

(-)-10 β, 13a-α-antofine N-oxide (188) and (-)-14 β -hydroxy-10 β, 13a-α-antofine N-

oxide (189) (Staerk et al., 2005).

V. nigrum is found in Europe mainly in France, Italy, Spain and Portugal. In

Chinese medicine system it was often used to treat rupture, scabies, scrofula and internal

fevers. (-)-13a-α-Antofine (187) which is phenanthroindolizidine alkaloids has been

found active against antibiotic activities (Nourian et al., 2016).


39

2.8. Previous Phytochemical Investigation of Vincetoxicum stocksii

V. stocksii is a perennial climbing leafy, poisonous, and medicinal important vine

found in Baluchistan, Pakistan. It is used for the treatment of cancer, healing of wounds

and injuries Crude extract of V. stocksii shows antidiarrheal, antispasmodic, antibacterial

and antifungal activities (Mudassir et al., 2005). Phytochemical analysis of the plant

extract exposed the existence of glycosides and tannins while other bioactive agentswere

not found. Antidiarrheal and calcium channel blocking potential are in correspondence

with existence of tannins (Shah et al., 2011). It showed highcytotoxicity against brine

shrimps (Mudassir et al., 2012).

2.9. Classification of Vincetoxicum stocksii

Family Asclepiadaceae

Subfamily Periplocoideae

Class Asclepiadeae

Genus Vincetoxicum

Specie Vincetoxicum stocksii

Literature review revealed the presence of vincetoside (190), vincetolate (191),

vincetomine (192) vincetetrol (193), 4-hydroxy-3-methoxyphenyl 7, 8, 9-propanetriol

(194) 4-hydroxy-3,5-dimethoxybenzoic acid (195) apocynin (196) feruloyl-6-O- β-D-

glucopyranoside (197), quercetin-3-O- β-D-glucopyranoside (198) in the ethyl acetate

soluble fraction of V. stocksii (Tousif et al., 2016).

Chapter no. 02 Research Hypothesis

40

2.10. Research Hypothesis

The selected three under-explored Pakistani plants Berberis calliobotrys,

Caragana ambigua and Vincetoxicum stocksii are being used as folk medicine, and

related specis are rich in variety of bioactive secondary metabolites therefore, it is

hypothesized that these plants might also be producing certain bioactive secondary

metabolites, which contributes to their medicinal use; therefore, if they are isolated they

may provide leads for new drug development.

2.11. Research Problem and Objectives of the Study

Enriched Pakistan land and marvelous importance of folk medicine inspired us to

search out for the compounds which are making these plants important. Despite of the

Chapter no. 02 Research Hypothesis

41

presence of countless medicinal floras in Pakistan, field of natural product chemistry and

drug development is still ignored in the country. Therefore, there is crucial need to study

Pakistani indigenous plantsto getdeeper information on active principals of these natural

machineries. To address a part of this problem, we selected three Pakistani plants;

Berberis calliobotrys, Caragana ambigua and Vincetoxicum stocksii to investigate them

for their novel bioactive secondary metabolites.

The Objective Includes:

1) Collection and extraction of plant materials

2) Preparation of different extracts of all plant materials

3) Biological and chemical screening of various extracts

4) Chromatographic purification of the extracts to get pure secondary metabolites

5) Establishing structures of isolated compounds based on spectroscopy

6) Evaluation of biological potential of extract and isolated compounds

42

CHAPTER 3

Results and Discussion: Biological Screening

and Phytochemical Analyses of Berberis

Calliobotrys, Caragana Ambigua and

Vincetoxicum Stocksii;and Isolation of

Secondary Metabolites

Chapter no.03 Result and Discussion of Berberis calliobotrys

43

Part A

3.1. BiologicalScreening of Crude Extracts of Berberis calliobotrys

Different polarity solvent extracts i.e.Berberis calliobotry smethanol extractBc-

M,Berberis calliobotrys ethyl acetate extract Bc-E, Berberis calliobotrys butanol extracts

Bc-B and Berberis calliobotryswater extract Bc-W were tested for antioxidant potentials

(2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-

sulphonic acid (ABTS), ferric reducing antioxidant power (FRAP), cupric reducing

antioxidant capacity (CUPRAC), phosphomolybdenum and metal chelation assays(Table

3.1) selective enzyme inhibitory assays (acetylcholinesterase (AChE),

butyrylcholinesterase (BChE), α-glucosidase, α-amylase and tyrosinase) (Table 3.2).

Inclusive assessment of antioxidant potential escorted that Bc-W offered the most

convincingresults in the ABTS (451.90 mg TE/g extract), CUPRAC (778.98 mgTE/g

extract), FRAP (718.48 mgTE/g extract) and phosphomolybdenum (2.04 mmolTE/g

extract) antioxidant assays which are in accord to the highest phenolic content of Bc-W

compared to other fractions. Whereas Bc-Moffered more promising results for DPPH

(204.73 mgTE/g extract) and metal chelation (60.32 mgEDTAE/g extract) activities. It

might be because of antagonistic or synergetic and non-phenolic chelator (Marini et al.,

2018).

Table 3. 1Antioxidant properties of B. calliobotrys extracts*

Plant extracts

Radical scavenging assays Reducing power assays Total

antioxidant activity

Ferrous ion chelation

DPPH (mgTE/g extract)

ABTS (mgTE/g extract)

CUPRAC

(mgTE/g extract)

FRAP

(mgTE/g extract)

Phospho molybdenum (mmolTE/g)

Metal chelaing (mgEDTAE/g)

Bc-M 204.73±3.103a 342.25±17.34b 744.72±18.86a 566.20±3.69b 1.88±0.06b 60.32±1.53a

Bc-E 71.54±0.91d 125.24±4.45d 308.47±13.53c 273.96±4.39d 1.93±0.05b 17.03±0.61d

Bc-B 95.93±0.41c 177.76±4.46c 523.30±18.67b 410.57±8.20c 2.01±0.13a 27.36±1.78c

Bc-W 165.73±9.75b 451.90±26.90a 778.98±8.38a 718.48±9.47a 2.04±0.08a 36.44±0.57b

* Values expressed are means S.D. of three parallel measurements. TE: Trolox equivalent;

EDTAE: EDTA equivalent. Different letters indicate differences in the extracts (p< 0.05)


44

All extracts of B. calliobotrys were tested for their inhibitory potential against

AChE, BChE, α-glucosidase, α-amylase, and tyrosinase (Table 3.2). In AChE, following

order was observed Bc-E>Bc-M>Bc-W>Bc-B (5.25>5.16>5.09>4.65

mgGALAE/gextract respectively), while in BChE assay, the Bc-W attributed excellent

(5.66mgGALAE/g extract) inhibition and Bc-E exhibited least anti-BChE potential

(4.11mgGALAE/g extract). Our results were in consistence with literature review which

supports the unusual response of Bc-E in both neurodegenerative assays (Kolar et al.,

2010).

Bc-E, Bc-M and Bc-B extracts contributed comparable potential for α-glucosidase

inhibitory activity while the Bc-W was found inactive. In α-amylase inhibition assay, Bc-

E showed highest inhibition power 0.76 mmolACAE/g extract, followed by Bc-B0.71

mmolACAE/g extract, Bc-M 0.53 mmolACAE/g extract and 0.29 mmolACAE/g

extract.Bc-E exhibited highest inhibitory potential in both anti-diabetic assays (Table

3.2). SimilarlyBc-E extract presented highest inhibition with value 169.92 mgKAE/g

extract, furthermore Bc-M (158.81 mgKAE/g extract) and Bc-B (153.107 mgKAE/g

extract) analysis also exposed their equally effective potential in tyrosinase inhibition.

While the Bc-W was found least active (37.06mgKAE/g extract) in the above assay.

Table 3. 2Enzyme inhibition activities of B. calliobotrys extracts*

Plant extracts

AChE

(mgGALAE/g extract)

BChE (mgGALAE/g

extract

α-Glucosidase (mmolACAE/g

extract)

α-Amylase (mmolACAE/g

extract)

Tyrosinase

(mgKAE/g extract)

Bc-M 5.16±0.11a 5.41±0.06a 1.68±0.02a 0.53±0.02b 158.81±0.59b

Bc-E 5.25±0.03a 4.11±0.29b 1.68±0.01a 0.76±0.02a 169.92±1.18a

Bc-B 4.65±0.02b 5.62±0.12a 1.63±0.03a 0.71±0.08a 153.107±0.73c

Bc-W 5.09±0.11a 5.66±0.06a Na 0.29±0.03c 37.06±0.92d

* Values expressed are means S.D. of three parallel measurements.

GALAE: Galatamine equivalent; KAE: Kojic acid equivalent; ACAE: Acarbose equivalent; na:

not active. Different letters indicate differences in the extracts (p< 0.05)


45

3.2 Phytochemical Analysis of Secondary Metabolites from Berberis

calliobotrys

Different polarity solvent extracts Bc-E, Bc-M, Bc-W and Bc-B of Berberis

calliobotryswereinvestigated for their bioactive contents and UHPLCMS analyses.

3.2.1 Total phenolic and flavonoid contents estimation

The phenolic and flavonoid content investigation revealed the highest phenolic

content in Bc-W with value 94.34 mgGAE/g extracts. In flavonoid estimation assay Bc-E

exhibited 11.11 mgRE/g extract of flavonoid content, while for other extracts the contents

ranges between 1.06-1.45 mgRE/g extract (Table 3.3).

Table 3. 3Total phenolic and flavonoid contents of B. calliobotrys extracts*

Solvents Abbreviation Total phenolic content

(mgGAE/g extract)

Total flavonoid

content (mgRE/g

extract)

Methanol Bc-M 77.70±1.39b 1.06±0.34c

Ethyl acetate Bc-E 50.24±1.05d 11.11±0.37a

Butanol Bc-B 67.69±1.19c 1.08±0.18c

Water Bc-W 94.34±1.11a 1.45±0.20b

* Values expressed are means S.D. of three parallel measurements. GAE: Gallic acid equivalent;

RE: Rutin equivalent. Different letters indicate differences in the extracts (p< 0.05)

Following methods were employed for the determination of total phenolics.

Total phenolic contents were measured through Folin-Ciocalteu method, where a

mixture of 0.25 ml of extract and 1.0 ml of diluted Folin-Ciocalteu reagent (1:9 dilution

ratio) was incubated at room temperature for three min, followed by the addition of 0.75

mL of 1% Na2CO3 solution. The reaction mixture was then incubated for two hours and

the absorbance was recorded at 760 nm using gallic acid was used as standard (Aktumsek

et al., 2013).

For the determination of total flavonoids contents, aluminium trichloride method

was used for evaluation of all four crude extract (Zengin et al 2014). In this method 1.0

mL of 2% methanolic AlCl3 solution was mixed with an equal volume of extract,


46

incubated for 10 min and absorbance was recorded at 415nm. In this assay, rutin was

used as standard compound (Mocan et al., 2017).

3.2.2.1 UHPLC-MS Analysis for Identification of Secondary Metabolites of B.

calliobotrys

Ultra high performance liquid chromatography mass spectrometry (UHPLC-MS)

was used for identification of secondary metabolites Bc-M and the most biological active

fractions i.e.Bc-E and Bc-W.

3.2.2.2 Secondary metabolite identification of Bc-M through UHPLC-MS analysis

UHPLC-MS negative ionization mode analysis of Bc-M conceded the presence of

16 secondary metabolites including alkaloids as major constituents (Figure 3.1) identified

compounds included 9 alkaloids, two fatty acids, two lignins, and one compound from

coumarin and terpene classes (Table 3.4). Indication of alkaloids as major constituents

was in accordance with the literature (Srivastava et al., 2015).

Figure 3. 1Total ion chromatograms (TICs) of Bc-M fraction


47

Table 3. 4UHPLC-MS analysis of Bc-M fraction

S.

No RT(min)

Base

peak(m/z) Peak height AUC Proposed compounds

Compoun

d class Mol. formula Mol. mass

1. 0.62 179.05 292350 1650440 Theobromine(199) Alkaloid C7H8N4O2 180.06

2. 0.624 215.03 681898 2124628 Isobergaptene(200) Coumarin C12H8O4 216.04

3. 0.631 165.04 301308 964565 1-Methylxanthine (201) Alkaloid C6H6N4O2 166.0

4. 0.632 195.05 167445 469761 1,9-Dimethyluric acid(202) Alkaloid C7H8N4O3 196.05

5. 0.64 683.22 368932 2819487 Citbismine C (203) Alkaloid C37H36N2O11 684.23

6. 0.90 191.02 580742 2694188 Citric acid (204) Fatty acid C6H8O7 192.02

7. 7.82 593.26 70421 600931 Aromoline (93) Alkaloid C36H38N2O6 594.27

8. 7.86 372.18 61503 280744 (S)-Autumnaline (205) Alkaloid C21H27NO5 373.18

9. 8.21 595.28 189464 1483642 Berbamunine(206) Alkaloid C36H40N2O6 596.28

10. 8.26 312.16 150069 926471 Armepavine(207) Alkaloid C19H23NO3 313.16

11. 8.27 741.26 61654 708860 Acanthoside D(208) Lignin C34H46O18 742.27


13. 9.12 579.20 322569 2143860 (+)-Syringaresinol O-β-D-

glucoside(209) Lignin

C28H36O13 580.21

14. 9.48 338.14 312848 2398858 Papaverine (210) Alkaloid C20H21NO4 339.14

15. 11.45 329.23 129225 1018956 5,8,12-trihydroxy-9-

octadecenoic acid(211) Fatty acid

C18H34O5 330.24

16. 13.73 221.11 76148 585072 (6S)-dehydrovomifoliol (212) Terpene C13H18O3 222.12

RT: retention time; AUC: area under curve

Chapter no.03 Result and Discussion of Berberis

calliobotrys

48


calliobotrys

49

3.2.2.3Secondary metabolite estimation of Bc-E through UHPLC-MS analysis

Bc-E analysis revealed the existence of 17 secondary metabolites (Table 3.5,

Figure 3.2). It included seven flavonoids and four phenolics as major constituents;

whereas three terpenes, one saponin, propiophenone and one fatty ester were

identified as minor contributor.

Figure 3. 2Total ion chromatograms (TICs) of Bc-E fraction


50

Table 3. 5UHPLC-MS analysis of Bc-E fraction

S.No RT(min) Base

peak(m/z)

Peak

height AUC Proposed compounds

Compound

class

Mol.

formula Mol.mass

1. 1.478 153.0192 22487 90608 3,4-DihydroxybenzoicAcid

(213) Phenol C7H6O4 154.0264

2. 3.085 137.0245 34358 292148 p-Salicylic acid (214) Phenol C7H6O3 138.0318

3. 8.095 193.0505 53525 194273 Kakuol (215) Propiophenone C10H10O4 194.0577

4. 8.201 193.0505 109241 438072 Ferulic acid (127) Phenol C10H10O4 194.0579

5. 8.41 197.0455 48343 197418 2-Hydroxy-3,4-

dimethoxybenzoic Acid (216) Phenol C9H10O5 198.0526

6. 9.103 361.1659 62848 241533 Hydroxyisonobilin (217) Terpene C20H26O6 362.1729

7. 9.388 431.1676 44075 187186 Agecorynin C (218) Flavonoid C22H24O9 432.1427

8. 10.052 385.0937 26167 106111 Melisimplexin (219) Flavonoid C20H18O8 386.1013

9. 10.205 449.1824 29095 122594 Neoglabrescin A (220) Terpene C23H30O9 450.1892

10. 10.313 355.0447 27799 126517 Demethyl-torosaflavone D

(221) Flavonoid C18H12O8 356.052

11. 10.626 299.0565 37211 164743 Kaempferide(222) Flavonoid C16H12O6 300.0638

12. 11.833 343.0831 182566 829314 Wightin (223) Flavonoid C18H16O7 344.0904

13. 11.834 457.076 26137 127558 Epigallocatechin 7-O-gallate

(224) Flavonoid C22H18O11 458.0833

14. 13.749 355.1196 61220 258942 Phellodensin D (225) Flavonoid C20H20O6 356.127

15. 16.714 455.3528 47344 336630 Betulinic Acid (153) Terpene C30H48O3 456.3601

16. 18.202 585.4862 54451 397558 Erythrinasinate A (226) Fatty ester C38H66O4 586.4933

17. 19.502 466.2828 36583 239994 Browniine (227) Akaloid C25H41NO7 467.2901

18. 19.503 517.2814 869799 5182485 Corchoroside B(228) Saponin C29H42O8 518.2885

RT: retention time; AUC: area under cure


calliobotrys

51


calliobotrys

52

3.2.2. Secondary metabolite determination of Bc-W through UHPLC-MS

analysis

UHPLC-MS analysis of Bc-Wfractionshowed the presence of 17 different

secondary metabolites (Table 3.6, Figure 3.3), comprising alkaloids as major

contributor along with phenolic glycosides, lignins, coumarin, polyphenol,

azoglycoside and epoxide glycoside.

Figure 3. 3Total ion chromatograms (TICs) of Bc-W


53

Table 3. 6UHPLC-MS analysis of Bc-W fraction

s

Sr no

RT(min) Base

peak(m/z)

Peak


Class Mol.

formula

Mol.

mass

1. 3.1066 215.0344 1206736 6457920 Isobergaptene (200) Coumarine C12H8O4 216.0414

2. 3.1081 195.0527 604637 2393090 1,9-Dimethyluric acid (202) Alkaloid C7H8N4O3 196.06

3. 3.1091 179.0578 836570 4195362 Theobromine (199) Alkaloid C7H8N4O2 180.0651

4. 3.1093 165.0422 898017 3313658 1-Methylxanthine (201) Alkaloid C6H6N4O2 166.0495

5. 2.821 383.1305 771964 2901209 Macrozamin(229) Azoglycosid

e

C13H24N2O11 384.1376

6. 3.584 290.0889 971805 16903584 Sarmentosin epoxide (230) Epoxide

Glycoside

C11H17NO8 291.0962

7. 3.919 191.02 1541306 14019079 Citric acid (204) Alkaloid C6H8O7 192.0272

8. 9.825 329.0886 193706 1260922 3'-Glucosyl-2',4',6'-

trihydroxyacetophenone (231)

Phenolic

glycoside

C14H18O9 330.096

9. 10.725 711.2169 2493221 29663968 1-O-Feruloyl- β-D-

glucoside(232)

Phenolic

glycoside

C16H20O9 356.1135


11. 10.891 771.2376 675048 9684476 4-O-β-D-Glucosyl-sinapate

(233)

Phenolic

glycoside

C17H22O10 386.1233

12. 11.19 595.2825 294169 1702692 Berbamunine (206) Alkaloid C36H40N2O6 596.2895

13. 11.263 777.2394 124972 1568819 Acanthoside D (208) Lignin C34H46O18 742.27

14. 11.84 312.1613 473597 2215616 Armepavine (207) Alkaloid C19H23NO3 313.1685

15. 13.775 579.209 161877 1069149 (+)-Syringaresinol O-β-D-

glucoside (209)

Lignin C28H36O13 580.2162

16. 12.937 338.1407 945834 6727954 Papaverine (210) Alkaloid C20H21NO4 339.1479

17. 17.616 293.177 269353 1933409 Gingerol (234) Polyphenol C17H26O4 294.1842

Chapter no.03 Results and Discussionof Berberies calliobotrys

54


55

3.3. Spectroscopic Characterization of Secondary Metabolites

Isolated From B. calliobotrys

The Bc-Mwas divided into ethyl acetate (Bc-E), butanol (Bc-B) and water

(Bc-W) fractions. Since the Bc-E and Bc-W exhibited good potential in

aforementioned bioassays, they were separately subjected to chromatography to

getpuresecondary metabolites. Silica gel column chromatography of Bc-E fraction

yielded six compounds: 4-hydroxybenzoic acid/p-salicylic acid (214), methyl p-

coumarate (235), octadecyl-p-coumarate (236) corydaldine (121) methylcorydaldine

(122) and armepavine (207).Bc-W on chromatographic purificationled to the isolation

of four compounds berberine (83), columbamine (237), syringaresinol (238) and

acanthoside D (208).

3.3.1. Structure Elucidation of 4-Hydroxybenzoic acid (214)

The IR spectrum of white crystalline solid of compound

214, exhibited the absorption bands for carboxylic acid at 3590-

2425 and 1730 cm-1, while characteristic absorptions at 1620-

1550 cm-1 were recorded for aromatic system. The EIMS

spectrum showed the molecular ion peak at m/z 138. The HREIMS analysis of the

same ion displayed molecular ion at m/z138.0320, attested the molecular formula as

C7H6O3 with 5 DBE.

The 1H NMR spectrum of 214 (Table 3.7) offered three signals, two

resonances at δH7.86 (2H, d, J = 8.7 Hz, H-2,6) and 6.76 (2H, d, J = 8.7 Hz, H-3,5)

were attributed to a p-substituted aromatic system, while the most downfield singletat

δH 10.9 was attributed to carboxylic hydroxyl proton.

The 13C NMR spectrum of 214 (Table 3.7) exhibited five carbon resonances

which were differentiated as three quaternary and two methine carbons with the help

of DEPT spectra. Signals at δC 171.5 and 162.7 were assigned to acid carbonyl and

oxygenated quaternary carbon atoms respectively. Other signals at δC 131.8(C-2, 6),

124.0 (C-4) and 116.8 (C-3, 5) were assigned to aromatic carbons. The above

recorded data was superimpose-able to the literature reported data of 4-


56

hydroxybenzoic acid (Yoshioka et al., 2004). Thus compound 214 was found the

same, which is a well-known phytochemical.

Table 3. 71H- and 13C-NMR data of compound 214 (CDCl3, 400 and 125 MHz)

Position δH (J in Hz) δC

1 - 124.0

2,6 7.86 (2H, d, 8.7) 131.8

3,5 6.76 (2H, d, 8.7) 116.8

4 - 162.7

7 - 171.5

3.3.2. Structure Elucidation of Methyl p-Coumarate (235)

Compound 235was obtained as white solid, which showed absorption bands

for hydroxyl, carbonyl, and double bond at 3450, 1730 and 1650 cm-1 respectively in

theIR spectrum. The EIMS spectrum displayed

molecular ion peak at m/z 178, while the HR-EIMS

(m/z178.0641) supported the molecular formulaas

C10H10O3 with 6 DBE. The 1H-NMR spectrum of

235(Table 3.8) displayed the presence of two pairs of

doublets resonating at δH7.60 (2H, d, J = 9.0 Hz) and δH 6.90 (2H, d, J = 9.0 Hz)

contributing for the presence of p-substituted aromatic system. Furthermore two more

doublets of one proton each were seenat δH 7.59 (J = 16.0 Hz)andδH 6.45 (J = 16.0

Hz), which were attested for two hydrogens in trans relationship of an olefinic

system. A sharp singlet was observed for oxygenated methyl at δH3.54.

13C-NMR data of compound 235 (Table 3.8), displayed 10 signals which were

distinguished as three quaternary carbons (δC168.2 (C-9), 159.0 (C-4) 125.5(C-1)), six

methines (δC146.4 (C-7), 131.2 (C-2, 6), 117.0 (C-3, 5) 116.0 (C-8)) and a methyl

carbon (δC50.9, C-1'). Comparison with the reported values, compound 235 was

identified as methyl ester of p-coumarate (Daayf et al., 1997).

3.3.3.Structure Elucidation of Octadecyl-p-cumarate (236)


57

Compound 236 was purified as white

amorphous solid. IR spectrum depicted the

distinctive absorption peaks at 3410 cm-1 for

OH, 3150 cm-1 for sp2 methines,1758cm-1

for carbonyl, 1610 cm-1 for conjugated C=C and 1585-1514 cm-1 for aromatic

system.EIMS analysis disclosed the molecular ion at m/z 430, whereas the molecular

formula C28H46O3 with 6 DBE could be determined due to the HR-EIMS

(m/z430.3445).

Aromatic region of the 1H-NMR spectrum of 236 (Table 3.8) was nearly

identical to that of 235, sinceit also afforded signals for p-substituted benzene ring and

E-olefinic system. The spectrum of 236 was missing signal for methoxyl proton,

instead it displayed signal for an oxygenated methylene at δH 4.20 (2H, J=6.6 Hz),

which in1H-1H-COSY spectrum was correlated with another methylene at δH1.69,

which in turn showed COSYinteractionwith a broad signal at δH 1.34-1.27 for various

methylenes, and ending with a triplet methyl atδH 0.85 (J=7.3 Hz). These signals

witnessed an alkyl-p-coumarate skeleton for compound 236.

13C-data of 236 (Table 3.8) was also similar to the data of 235, but with

additional signals for oxygenated methylene at δC 60.3, aliphatic methylenes at δC

29.7-23.4 and methyl signal resonance at δC 15.0. Presented data of alkyl-p-coumarate

along with its molecular mass assisted to assign it as octadecyl-p-coumarate (236),

which is a known compound as supported by literature (Nidiry et al., 2011).

Table 3. 81H-NMR and 13C-NMR data of compound 235 and 236 (CDCl3, 400 and 100 MHz)

Position 235 236

δH(J in Hz) δC δH(J in Hz) δC

1 - 125.5 - 130.2

2,6 7.60 (2H, d, 9.0) 131.2 7.50 (2H, d,8.7 ) 132.3

3,5 6.90 (2H, d, 9.0) 117.0 6.70 (2H, d,8.7 ) 116.4 4 - 159.0 - 156.4

7 7.59 (1H, d, 16.0) 146.4 7.48 (1H, d,15.9 ) 145.3

8 6.45 (1H, d, 16.0) 116.0 6.32 (1H, d,15.9 ) 114.6

9 - 168.2 - 168.0

1′ 3.54 (1H, s) 50.9 4.20 (2H, t, 6.6) 60.3

2′ - - 1.69 (2H, m) 27.0

3′ - - 1.34 (2H, m) 25.7

4′ - - 1.29 (2H, m) 29.7

5′ - - 1.28 (2H, m) 29.4

6′-17′ - - 1.27 (2H, m) 29.4


58

18′ - - 1.35 (2H, m) 23.4

19′ - - 0.85 (3H, t, 7.3) 15.0

3.3.4.Structure Elucidation of Corydaldine (121)

Compound 121 was also obtained as white

amorphous powder, whose UV spectrum in methanol

showed absorption bands at 301 and 287 nm which are

characteristics of isoquinoline alkaloid. Its IR spectrum

disclosedabsorption bands at 3390, 1660 and 1640-1450 cm-

1 for N-H, amide and aromatic groups respectively. The HR-EIMS of 121 showed

molecular ion at m/z 207.0979, which was calculated formolecular formula as

C11H13NO3 with 6 DBE. The 1H-NMR spectrum (Table 3.9) of 121 displayed two

singlets in the aromatic region (δH 7.31, 1H, s, 6.70, 1H, s) and two singlets for

methoxylprotons (δH3.89 3H, s, and 3.87, 3H, s,).Another singlet of one hydrogen at

δH 6.22 could be attributed toamino group as lactum.Besides, the spectrum showed

two triplets atδH 3.52 (2H, J=7.5 Hz) and 2.90 (2H, J=7.5 Hz), which showed COSY

correlation with eachother.

The 13C-NMR spectrum (Table 3.9) of 121 offered signals for 11 carbon

atoms which were differentiated as two methyls (δC 50.2 and 49.5), two methylenes

(δC 40.1 and 28.0), two methines (δC 115.2 and 110.2,) and five quaternary (δC 167.1,

152.9, 148.5, 128.5, 118.2) carbons. In HMBC spectrum amine proton at δH 6.22, and

methylene proton of H-3 at 3.52 (t, 7.5 Hz) showed correlation with carbonyl carbon

(C-1, δC 167.1) and which was in consistence with UV-studies, supporting the

presence of isoquinoline nucleus. Comparison of under discussion data with the

literature led to the identification of compound 121 as corydaldine (Shamma and

Podczasy, 1971, Atan et al., 2011), which is a known phytochemical.

3.3.5. Structure Elucidation of N-methyl Corydaldine (122)

The amorphous powder of compound 122 disclosed similar UV spectrum as

that of 121, while its IR spectrum missed the band due to

amine group, which suggested an N-alkylated derivative of

121. The EIMS spectrum displayed molecular ion peak at


59

m/z 221, while HREIMS established the molecular formula C12H15NO3 with 6 DBE

(221.1043 calculated 221.1052). The 1H-NMR spectrum (Table 3.9) of 122 was also

nearly super-imposable to that of 121 with only one difference; as the spectrum

missed signal for N-H and instead it showed an additional methyl group atδH 3.21

(s).This information substantiated the above deduction of N-alkylated derivative of

121. Furthermore the 13C-NMR spectrum (Table 3.9) of 122 also exhibited an

additional signal for N-methyl at δH 47.5 presenting HMBC correlations with

carbonyl carbon at δC165.2, thatconfirmed122 as N-methyl derivative of 121,whichis

named as is N-methylcorydaldine, another known phytochemical (Shamma and

Podczasy, 1971).

3.3.6. Structure Elucidation of Armepavine (207)

Compound 207 was isolated as white crystalline solid.The IR spectrum of this

compound showed absorption bands at 3420 cm-1 (O-H), 3120 cm-1 (=C-H) and 1649-

1466 cm-1 (Ar-C=C). EIMS spectrum afforded molecular ion peak at m/z 313, while

HREIMS analysis of the same ion (m/z313.1672) depicted the molecular formula as

C19H23NO3 with 9 DBE.Aromatic region of 1H NMR spectrum of 207 (Table 3.9)

displayed two resonances at δH 7.21 (2H, d, J = 8.8 Hz, H3', 5') and δH 6.81 (2H, d, J

= 8.8 Hz, H2', 6'), while two methoxyl proton signals and one N-methyl resonance

were seen at δH 3.88 (3H, s), 3.87, (3H, s) and 3.22 (3H, s) respectively. Two singlet

protons appeared at 6.72 (1H, s) and 7.31(1H, s) for C-5 and C-8. One downfield

triplet appeared at 4.24 (1H, t, 7.0) for proton near to nitrogen along with a doublet at

3.01 (2H, d, 6.8) for C-7. Two more triplets for methylene resonated at 3.51 (2H, t,

7.2) and 2.82 (2H, t, 7.2). This data indicated compound 207 could be also an

isoquinoline derivative having partial similarity with structure of compound

122expect an extra benzene ring. Furthermore the 13C-NMR spectrum of compound

207 also exhibited additional five signals as compared to 122 for seven carbons

(Table 3.9), which were predicted as two quaternary carbons δC 154.3 (C-1') and δC

135.4 (C-4') and two methines δC 117.3 (C-2', 6') and δC131.0 (C-3', 5') and one

methylene resonating at δC 41.0 (C-7'). The HMBC interaction (Fig 3.4) of methine

proton at δH 4.24 with δC C-8 (117.2), C-9 (119.8), -3', 5' (131.0) and with N-methyl

(δC 45.5) confirmed attachment of benzyl group at C-1 of isoquinoline part.


60

Comparison with reported data confirmed that 207 is a known phytochemical

namedas armepavine (207) (Marek et al., 1997).

Figure 3. 4 Important HMBC (H→C) csorrelations of 207

Table 3. 91H-NMR and 13C-NMR data of compounds 121, 122 and 207 (CDCl3, 400 and 100 MHz)

Position 121 122 207

δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz) δC

1 - 167.1 - 165.2 4.24 (1H, t, 7.0) 72.9

2 - - - - - -

3 3.52 (2H, t, 7.5) 40.1 3.49 (2H, t, 7.0) 39.7 3.51 (2H, t, 7.2) 40.1

4 2.90 (2H, t, 7.5) 28.0 2.80 (2H, t, 7.0) 29.0 2.82 (2H, t, 7.2) 30.0

5 6.70 (1H, s) 110.2 6.72 (1H, s) 111.3 6.72 (1H, s) 113.7

6 - 152.9 - 151.9 - 150.9

7 - 148.5 - 149.1 - 149.6

8 7.31(1H, s) 115.2 7.31(1H, s) 116.2 7.31(1H, s) 117.2

9 - 118.2 - 119.7 - 119.8

10 - 128.5 - 127.5 - 127.9

1' - - - - - 154.3

2', 6' - - - - 6.81 (2H, d, 8.8) 117.3

3', 5' - - - - 7.21 (2H, d, 8.8) 131.0

4' - - - - - 135.4

7' - - - - 3.01 (2H, d, 6.8). 41.0

1'-OH - - - - 4.5(1H, s) -

2-NH 6.22(1H, s) - - - - -

2-NMe - - 3.21(3H, s) 47.5 3.22 (3H, s) 45.5

6-OMe 3.89(3H, s) 50.2 3.87(3H, s) 53.8 3.88 (3H, s) 51.4

7-OMe 3.87(3H, s) 49.5 3.86(3H, s) 51.2 3.87 (3H, s) 49.2

3.3.7. Structure Elucidation of Berberine (83)

Compound 83 was isolated as yellow crystalline solid, which displayed

absorption bands in the IR spectrum at 1612-1490 cm-1 for aromatic system and at


61

1041 cm-1 for methylenedioxy group. Molecular ion peak at m/z 336 was observed in

EIMS spectrum, while HREIMS analysis helped to establishing molecular formula as

C20H18NO4 with 13 DBE (336.1218 calculated 336.1230). The 1H-NMR spectrum

(Table 3.10) of 83 exhibited six signals in aromatic region presenting one AB-system

at δH 8.11 (1H, d, J = 9.2 Hz) and 7.99 (1H, d, J = 9.2

Hz), whereas, four singlets at δH 9.75(1H, s),8.70

(1H, s), 7.65 (1H, s), and 6.97 (1H, s), offered an

indication for three tetra-substituted benzene ring

systems. The same spectrum also revealed presence

of two methoxylgroups due to signals at δH 4.21 (3H,

s) and 4.10 (3H, s) and three signals for methylene

protons at δH 6.11 (2H, s), 4.93 (2H, t, J=6.4) and 3.75 (2H, t, J=6.4) suggested the

presence of three methylene protons. Downfield shift of methylene group at δH 6.11

(2H, s) revealed the presence of methylendioxy group. All aboveproton data was

inconsistent with the presence of berberine nucleus. The 13C-NMR of 83 (Table 3.10)

disclosed resonances of 20 carbon atoms, which were distinguished as nine quaternary

(δC152.1, 150.8, 150.0, 143.2, 139.4, 167.3, 163.6, 124.7, 122.0), six methines (δC

145.2, 164.4, 124.7, 121.7, 109.4, 106.4) three methylenes (δC103.6, 57.1, 28.3), and

two methoxy carbons (δC 63.8, 59.0). The down field signal for methylene (δC 103.6)

substantiated methylenedioxy group, while two other methylenes (δC 57.1, 28.3) were

connected to nitrogen atom and aromatic system respectively.

All spectroscopic data was in full agreement with the reported data of

forberberine (83) (Hsieh et al., 2004; Yu et al., 2014), which is also a known

phytochemical.

3.3.8. Structure Elucidation of Columbamine (237)

Yellow amorphous solid of compound 237

exhibited IR spectrum abosprtion bands for O-H

(3350 cm-1) and aromatic system (1620-1540 cm-

1). High resolution analysis of the molecular ion

(m/z338.1377) observed in EIMS revealed the


62

molecular formula as C20H20NO4 with 13 DBE.The 1H-NMR spectrum (Table 3.10) of

237 was found barely identical to that of 83 with only one difference that the

spectrum missed signal for methylendioxy, however, an additional methoxyl signal

(δH 3.83, s) was ascertained, which indicated the hydrolyzing of methylendioxy group

of 83. The13C-NMR spectrum (Table 3.10) of 237 afforded 21 resonances with no

signal for methylenedioxy and additional signal for methoxyl group when compared

to the spectrum of 83. Thus compound 237 was found to becolumbamine 237 (Hsieh

et al., 2004), which is also a known natural product.


Position 83 237

δH (J in Hz) δC δH(J in Hz) δC

1 7.65 (1H, s) 106.4 7.63 (1H, s) 103.8

1a - 122.0 - 123.0 2 - 150.0 - 148.0

3 - 152.1 - 150.1

4 6.97 (1H, s) 109.4 6.97(1H, s) 104.4

4a - 163.6 - 164.6

5 3.75 (2H, t,6.4) 28.3 3.73 (2H, t,6.6) 39.3

6 4.93 (2H, t, 6.4) 57.1 4.83 (2H, t, 6.6) 56.1

7 - - - -

8 9.75 (1H, s) 145.2 9.68 (1H,s) 144.2

8a - 125.4 - 123.4

9 - 143.2 - 142.2

10 - 150.8 - 152.8 11 7.99 (1H, d, 9.2) 164.4 7.87 (1H, d, 8.7) 130.4

12 8.11 (1H, d, 9.2) 124.7 8.10 (1H, d, 8.7) 125.7

12a - 167.3 - 167.3

13 8.70 (1H, s) 121.7 8.68 (1H, s) 121.3

13a - 139.4 - 138.4

9-OMe 4.21 (3H, s) 63.8 4.12 (3H, s) 60.4

10-OMe 4.10 (3H, s) 59.0 4.12 (3H, s) 57.0

14 (-OCH2O-) 6.11 (s) 103.6 - -

2-OH - - 5.49(s) -

3-OMe - - 3.83(s) 56.2

3.3.9.Structure Elucidation of Syringaresinol (238)

Compound 238 was separated as

white amorphous solid. The IR spectrum

showed characteristics absorption bands for

OH (3340 cm-1), aromatic system (1620-

1430 cm-1) and C-O-C linkage (1380, 1650

cm-1). The EIMS exhibited molecular ion

peak at m/z 418, whereas HR-EIMS spectrum deduced the molecular formula as


63

C22H26O8 with 10 DBE (418.1612 calculated 418.1628). The 1H-NMR spectrum

(Table 3.11) of 238 presented the signal for aromatic protons at δH 6.60 for methine.

One doubletresonating at δH 4.75 (dd, J = 11.2, 7.24 Hz) was attributedfor oxygenated

methineand doublet of doublets at δH 4.31 (dd, J = 11.2, 7.24Hz) and 3.94 (2H, dd,

11.2, 7.24) were deduced for oxygenated methylene. Another singlet at δH 5.54 (1H, s,

4', 4'') was assigned for phenolic proton. One singlet appeared at δH 3.93 with

integration of four methoxyl groups attached with aromatic system. This1H-NMR

spectral data for compound 238 revealed the bis-tetrahydrofuran nucleus which was

confirmed by the13C-NMR spectra that displayed the resonances for eight carbon

atoms, being attributed to three quaternary (δC 147.2, 166.4, 164.3), one methylene (δC

86.2), three methine (δC 103.8, 71.8, 54.2) and one methoxy (δC 56.4). Further dimer

skeleton was confirmed by HMBC correlation (Figure 3.5) of signal at δH 4.75 (1H,

dd, J=3.40) with δC 71.8(C-4,8) and with its own carbon at δC 86.2.Also the double

carbon atoms in molecular formula substantiated dimeric nature of 238. The whole

obtained data was similar with the reported data for compound syringaresinol (238),

which is a known lignin (Monthong et al., 2011).

Figure 3. 5Important HMBC (H→C) correlations of 238

3.3.10. Structure Elucidation of Acanthoside D (208)

Compound 208 was

found to be glucoside of

238, since most of its 1H-

and 13C-NMR spectra

(Table 3.11) displayed

similar signals to that of


64

238. The only difference seen was the resonances due to hexose moiety. The aromatic

proton displayed its position at δH 5.31 (1H, d, J = 7.0 Hz) along with multiplet in the

range of δH 3.70-3.30, offered a symmetric glucoside lignin, whereas, the 13C-NMR

spectrum (Table 3.11) fully agreed with proton data because of carbon resonances

atδC 103.8, 77.3, 76.3, 70.8, 77.1 and 62.2.The sugar connectivity with aromatic

system could be determined due to HMBC (Figure 3.6) interactionof anomeric

hydrogen at δH 5.31with C-4 (δC 72.8) of aromatic system. These deductions were

substantiated due to molecular formula as C34H46O18 with 10 DBE based on HREIMS

(m/z 742.2678) analysis.Further the whole data was similar with the reported data for

compound acanthoside D (208), which is also a known lignin (Lami et al., 1991).

Figure 3. 6Important HMBC (H→C) correlations of 208


65

Table 3. 111H- and 13C-NMR data of compound 238 and208 (CDCl3, 400 and 125 MHz)

Position

238 208

δH (J in Hz) δC δH (J in Hz) δC

1,5 3.12 (1H, m) 54.2 3.11 (1H, m) 53.2 2,6 4.75(1H, d, 3.40) 86.2 4.77(1H,d, 12) 87.2

4a,8a 4.31 (2H, dd, 11.2, 7.24) 71.8 4.33 (2H, dd, 12.0, 8.16) 72.8

4b,8b 3.94 (2H, dd, 11.2, 7.24) 4.09 (2H, dd, 12.0, 8.16)

1',1'' - 164.3 - 165.0

2',2'' 6.60 (1H, s) 103.8 6.63 (1H, s) 103.4

3',3'' - 147.2 - 148.2

4',4'' - 166.4 - 166.9

5',5'' - 147.2 - 148.2

6',6'' 6.60 (1H, s) 103.8 6.63 (1H, s) 103.4

3'-OCH3 3.93 (3H, s) 56.4 3.93 (3H, s) 57.4

3''-OCH3 3.93 (3H, s) 56.4 3.93 (3H, s) 57.4

5'-OCH3 3.93 (3H, s) 56.4 3.93 (3H, s) 57.4 5''-OCH3 3.93 (3H, s) 56.4 3.93 (3H, s) 57.4

4'-OH, 5.54 (1H, s) - - -

4''-OH 5.54 (1H, s) - - -

1''a, 1'b - - 5.31 (1H, d, 7.0) 103.8

2''a, 2'b - - 3.45 (1H, t, 8.4) 77.3

3''a, 3'b - - 3.52 (1H, m) 76.3

4''a, 4'b - - 3.34 (1H, t, 7.2) 70.8

5''a, 5'b - - 3.31 (1H, m) 77.3

6''a, 6'b

- - 3.70 (1H, dd, 12.2,3.9)

3.61 (1H, dd, 12.2, 3.9)

62.2

Chapter no.03 Results and Discussionof Caragana ambigua

66

Part B

3.4. Biological Screening of Crude Extracts of Caragana ambigua

Different polarity solvent extracts C. ambigua methanol extract (Ca-M), C.

ambigua hexane extract (Ca-H), C. ambigua ethyl acetateextract (Ca-E) and C.

ambigua water extract (Ca-W) were tested for antioxidant potentials (DPPH, ABTS,

FRAP, CUPRAC, phosphomolybdenum and metal chelation assays (Table 3.12) and

inhibitory assays (acetylcholinesterase (AChE), butyrylcholinesterase (BChE), α-

glucosidase, α-amylase and tyrosinase (Table 3.13)).

Comprehensive assessment of antioxidant potential revealed that Ca-E shared

most convincing result in the DPPH (83.32 mgTE/g extract), ABTS (421.94 mg TE/g

extract), CUPRAC (405.26 mgTE/g extract), FRAP (617.89 mgTE/g extract) and

phosphomolybdenum (2.70 mmolTE/g extract) antioxidant assays which are in

concurrence to its highest phenolic content while Ca-H was found least active in all

above assays except phosphomolybdenum assay(1.96 mmolTE/g extract) where it

was found next to the Ca-E. Moreover Ca-H was more potent in metal chelation

assays (74.27 mgEDTAE/g extract). It might be because of antagonistic or synergetic

and non-phenolic chelator (Marini et al., 2018; Khan at al., 2019).

Table 3. 12Antioxidant properties of C. ambigua extracts*

Plant extracts

Radical scavenging assays Reducing power assays Total antioxidant

activity

Ferrous ion

chelation

DPPH (mgTE/g extract)

ABTS (mgTE/g extract)

FRAP (mgTE/g extract)

CUPRAC (mgTE/g extract)

Phosphomolybdenum

(mmolTE/g)

Metal chelating

(mgEDTAE/g)

Ca-M 55.05±2.00b 103.12±6.10b 163.108±1.99b 204.66±9.17b 1.30±0.10a 23.82±1.24b

Ca-E 83.32±6.22d 421.94±13.93d 405.26±11.15d 617.89±7.00d 2.70±0.06a 53.96±3.15c

Ca-H 13.106±0.19a 60.10±3.75a 85.36±8.82a 161.88±8.71a 1.96±0.12a 74.27±3.71d

Ca-A 60.16±3.01c 115.52±1.22c 227.96±3.13c 236.98±2.11c 1.47±0.06a 10.32±0.21a

* Values expressed are means S.D. of three parallel measurements; means with different superscript letters in the same column are significantly (p <0.05) different TE: Trolox equivalent;

EDTAE: EDTA equivalent

All extracts of C.ambigu were tested for their inhibitory potential against

AChE, BChE, α-glucosidase, α-amylase, and tyrosinase (Table 3.13). In AChE and BChE

assays, Ca-H attributed high potential (4.81 mgGALAE/g extract and 4.95mgGALAE/g


67

extract respectively). In α-amylase assay Ca-H (0.81 mmolACAE/g extract) showed

highest potential followed by Ca-E (0.75 mmolACAE/g extract), Ca-M (0.54

mmolACAE/g extract) and Ca-W (0.81 mmolACAE/g extract). Furthermore Ca-H

(1.68 mmolACAE/g extract) and Ca-E (1.67mmolACAE/g extract) exhibited

comparable potential for α-glucosidase followed by Ca-A (0.78 mmolACAE/g

extract) while Ca-M was found to be inactive.

Similarly in tyrosinase assays extract presented the following sequences Ca-E

>Ca-H> Ca-M > Ca-A (185.80> 176.01> 172.08> 38.57 mgKAE/g extract)

respectively.

Table 3. 13Enzyme inhibition activities of C. ambigua extracts*

Plant

extracts

AChE

(mgGALAE/g

extract)

BChE

(mgGALAE/g

extract

α-Amylase

(mmolACAE/g

extract)

α-Glucosidase

(mmolACAE/g

extract)

Tyrosinase

(mgKAE/g

extract)

Ca-M 4. 55±0.08b 2.91±0.41a 0.54±0.02b Na 172.08±1.29b

Ca-E 3.18±0.24a 3.57±0.12a 0.75±0.01b 1.67±0.01c 185.80±1.45d

Ca-H 4.81±0.32b 4.95±0.30b 0.81±0.02b 1.68±0.01b 176.01±1.10c

Ca-A Na Na 0.08±0.01a 0.78±0.10a 38.57±1.83a

* Values expressed are means S.D. of three parallel measurements; means with different superscript letters in the same column are significantly (p <0.05) different; GALAE: Galatamine

equivalent; KAE: Kojic acid equivalent; ACAE: Acarbose equivalent; na: not active.

3.5. Phytochemical Analysis of Secondary Metabolites from

Caragana ambigua

Different polarity solvent extracts Ca-M, Ca-H, Ca-E, and Ca-W were

investigated for their bioactive contents and UHPLCMS analyses.

3.5.1 Total phenolic and flavonoid contents estimation

Different polarity solvent extracts Ca-M, Ca-H, Ca-E, and Ca-W were tested

for total phenolic and flavonoid contents (Table 3.14), Analysis of results revealed the

highest phenolic and flavonoid contents in Ca-E with value 85.87mgGAE/g and 66.45

mgGAE/g extract respectively (Table 3.14).


68

Table 3. 14Total phenolic and flavonoid content of C. ambigua extracts*

Extracts Abbreviation Total phenolic content

(mgGAE/g extract)

Total flavonoid content

(mgRE/g extract)

Methanol Ca-M 37.37±0.21b 17.36±0.27b

Ethyl acetate Ca-E 85.87±2.96d 66.45±0.37d

n-Hexane Ca-H 33.79±2.15a 15.06±0.16a

Aqueous Ca-A 44.60±0.89c 58.69±0.15c

* Values expressed are means S.D. of three parallel measurements; means with different

superscript letters in the same column are significantly (p <0.05) different; GAE: Gallic acid

equivalents; RE: Rutinequivalents

3.5.2. UHPLC-MS Analysis to IdentifySecondary Metabolites of Caragana

ambigua

UHPLC-MSwas used for identification of secondary metabolites in Ca-M and Ca-E.

3.5.2.1. Secondary metabolite estimation of Ca-M through UHPLC-MS analysis

UHPLC-MS in negative ionization mode analysis of fraction Ca-M

revealedthe presence of 16 secondary metabolites (Table 3.15,figure 3.7) including

nine-flavonoid, one compound from each class of alkaloid, saponin, terpenoids,

coumarin, glycoside, fatty acid and lignin.

Figure 3. 7Total ion chromatograms of methanol extractof C. ambigua


69

Table 3. 15UHPLC-MS secondary metabolites profile of Fraction Ca-M

S. No RT

(min)

Base

peak(m/z)

Peak


Compound

class

Molecular

formula

Molecula

r mass

1 0.626 215.03 1091409 3957998 Isobergaptene (200) Coumarin C12H8O4 216.03

2 0.668 683.22 118383 750095 Citbismine C (203) Alkaloid C37H36N2O11 684.23

3 7.864 885.26 213028 1807731 Kaempferol-3-isorhamninoside-7-

rhamnoside (239) Flavonoid C39H50O23 886.27

4 7.921 739.20 71637 579281 Robinin (240) Flavonoid C33H40O19 740.21

5 0.868 290.08 120873 391537 Sarmentosin epoxide (230) Glycoside C11H17NO8 291.09

6 8.719 609.14 163999 1095266 Robinetin -3-rutinoside (241) Flavonoid C27H30O16 610.15

7 9.107 579.20 99994 696046 (+)-Syringaresinol O-β-D-glucoside

(209) Lignin C28H36O13 580.21

8 9.118 623.16 300142 2545922 Tricetin 7-methyl ether 3'-

glucoside-5'-rhamnoside (242) Flavonoid C28H32O16 624.17

9 9.992 1165.53 123692 1068560 Calendasaponin C (243) Terpene C54H86O25 1166.54

10 11.442 299.05 92724 709453 Kaempferide (223) Flavonoid C16H12O6 300.06

11 11.451 329.23 139562 1367030 5,8,12-trihydroxy-9-octadecenoic

acid (211) Fatty acid C18H34O5 330.24

12 11.463 517.17 90511 631280 Phellamurin (244) Flavonoid C26H30O11 518.17

13 12.166 941.51 291245 4112074 Jujubasaponin IV (245) Saponin C48H78O18 943.91

14 13.114 355.11 250807 1863271 Phellodensin D (226) Flavonoid C20H20O6 356.12

15 13.179 353.10 139571 1054198 2,3-Dehydrokievitone (246) Flavonoid C20H18O6 354.11

16 14.099 337.10 119429 901058 (-)-Glyceollin I (247) Flavonoid C20H18O5 338.11



70


71

3.5.2.2Secondary metabolite estimation of Ca-E through UHPLC-MS analysis

UHPLC-MS analysisof Ca-E disclosed the presence of 26 secondary

metabolites including twenty flavonoids as a larger group, two fatty acids, two

phenolics and one coumarin class of compounds (Table 3.16,Figure 3.8). The

presence of aforementioned metabolites is in accord with the reported literature of

genus Caragana (Mandal et al., 2016).

Figure 3. 8Total ion chromatograms of ethyl acetate extract (Ca-E) of C.

ambigua


72

Table 3. 16UHPLC-MS secondary metabolites profile of fraction Ca-E

S.

No RT(min)

Base

peak(m/z)

Peak


Compound

class

Molecular

formula

Molecular

mass 1 9.014 342.13 84673 578292 Sphalleroside A (248) Phenolic C16 H22O8 341.12

2 9.7 272.06 399343 5624494 (±)-Naringenin (249) Flavonoid C15H12O5 271.06

3 9.815 288.06 89978 746052 2,6,3',4'-Tetrahydroxy-2-benzylcoumaranone (250) Flavonoid C15H12O6 287.05

4 9.936 534.17 150660 1191160 Phellatin (251) Flavonoid C26H30O12 533.16

5 10.005 300.06 413659 3059898 Kaempferide (222) Flavonoid C16H12O6 299.05

6 10.268 270.05 159020 1635646 Demethyltexasin (252) Flavonoid C15H10O5 269.04

7 10.573 284.06 501543 4588296 Texasin (253) Flavonoid C16H12O5 283.06 8 11.225 298.04 86311 592383 8-Methoxycoumestrol (254) Flavonoid C16H10O6 297.04

9 11.268 370.10 699817 6989022 Neouralenol (255) Flavonoid C20H18O7 369.09

10 11.313 516.16 202694 1939688 Vitexin 2''-O-(2'''-methylbutyryl) (256) Flavonoid C26H28O11 515.15

11 11.335 386.10 290376 2984308 Melisimplexin (219) Flavonoid C20H18O8 385.09

12 11.336 270.05 697173 7127388 Demethyltexasin(252) Flavonoid C15H10O5 269.04

13 11.372 284.06 126016 804765 Texasin(253) Flavonoid C16H12O5 283.06 14 11.445 300.06 1136224 1.2E+07 Kaempferide(222) Flavonoid C16H12O6 299.05

15 11.476 518.18 649168 6253294 Phellamurin (240) Flavonoid C26H30O11 517.17

16 11.628 370.10 170849 3223872 Neouralenol(255) Flavonoid C20H18O7 369.09

17 11.978 386.13 84864 632581 Samidin (257) Coumarin C21H22O7 385.12

18 12.014 330.24 104995 1162332 5,8,12-trihydroxy-9-octadecenoic acid (211) Fatty acid C18H34O5 329.23

19 12.113 370.10 202388 2028171 Neouralenol(255) Flavonoid C20H18O7 369.09 20 12.117 352.09 210748 1953994 Psoralidin oxide (258) Flavonoid C20 H16O6 351.08

21 12.177 372.12 171632 1949428 7,8,3',4',5'-Pentamethoxyflavone (259) Flavonoid C20H20O7 371.11

22 12.486 370.10 192907 2199607 Neouralenol(255) Flavonoid C20H18O7 369.09

23 13.118 356.12 3375776 3.6E+07 Phellodensin D (226) Flavonoid C20H20O6 355.11

24 13.164 294.18 99663 1247070 Gingerol (234) Polyphenol C17H26O4 293.17

25 13.985 314.24 101844 1191607 9,10-Epoxy-18-hydroxystearate (260) Fatty acid C18H34O4 313.23

26 14.268 368.12 524153 6224450 Aurmillone (261) Flavonoid C21H20O6 367.120



73

3.6. Characterization of Secondary Metabolites Isolated from the

Fraction Ca-E

Sincethe ethyl acetate soluble part (Ca-E) exhibited good potential in

biochemical assays therefore was selected for purification of itssecondary metabolites.

Silica gel column chromatographic separation yielded nine known compounds;

taraxerol (262), taraxerol acetate (263), 2-(4′-hydroxyphenyl)-ethyl stearate (264),

apigenin (265), kaempferide (222), naringinin (249), quercetin (125), quercetin 3-O-

β-D-glucopyranosideside (198) and β-sitosterol 3-O-D-glucopyranoside (172).


74

3.6.1. Structure Elucidation of Taraxerol (262)

Crystalline solid of compound 262 exhibited absorption bands in the IR

spectrum for hydroxyl, olefinic hydrogen and

double bond at 3483, 2945 and 1655 cm-1

respectively. The EIMS spectrum showed molecular

ion peak at m/z 426, whereas the HREIMS assisted

to establish molecular formula as C30H50O with 6

DBE (m/z 426.3840). The 1H-NMR spectrum of 262

(Table 3.17) displayed eight tertiary methyl signals

atδH1.03, 0.97, 0.96, 0.88, 0.87, 0.85, 0.82, 0.81. The proton signal resonating at δH

3.70 (1H, dd, J = 9.0, 4.2 Hz) was assigned to H-3, whereas the most downfield signal

appeared at δH 5.49 (H-15, dd, J = 8.0, 3.4 Hz) was attributed todouble bond in 262.

Furthermore, various methylene resonances displayed their positionsas multiplet

between δH 2.02-1.34, which suggested compound 262 could be a triterpenoid

skeleton, which was substantiated by 13C-NMR data (Table 3.17). The 13C-NMR

broad-band spectrum showed 30 carbons signals, which were differentiated by DEPT

experiment as eight methyls, five methines, ten methylenes and seven quaternary

carbons. The olefinic carbons showed their positionsat δC 157.7 (C) and 115.9 (CH),

whereas, oxymethine appeared at δC 70.9. Comparatively downfield shift of the

olefinic quaternary carbon (δC 157.7) indicated taraxe-14-ene-3-β-ol type of

triterpenoid (Deng et al., 2004).

HMBC correlations of the olefinic proton (δH 5.49) showed HMBC correlations

(Figure 3.9) with carbons at δC 157.7 (C-14), 37.0 (C-16), 35.9 (C-13), and 31.2 (C-8)

which further confirmed the C-14 position for double bond. Furthermore HMBC

interaction of tertiary methyls of H-27 (δH 0.88) and H-18 (δH 0.81) with 157.7 (C-14)

supported the proposed structure. This data was matched with the reported data in

literature for taraxerol (Deng et al., 2004), thuscompound 262 was found to be the

same, which is a common phytochemical but isolated for the first time from our

investigated source.


75

Figure 3. 9 Important HMBC (H→C) correlations of 262

3.6.2. Structure Elucidation of Teraxerol acetate (263)

Compound 263was also isolated as white amorphous powder. Along with

similar IR absorption bands, its spectrum afforded

strong characteristic absorption for ester at 1730 cm-1.

The EIMS spectrum showed molecular ion peak at

m/z 468, while HR-EIMS analysis (m/z 468.3962) of

the same peak depicted the molecular formula

C32H52O2 with 7 DBE. The 1H- and 13C-NMR dataof

263 (Table 3.17) was nearly identical to that of 262 with the only difference was that

it offered additional signalsfor an acetyl moiety (δH 2.02, δC 22.0 &

172.1).Aforementioned data supported the presence of acetyl group and its position

was confirmed by downfield shift of H-3 at δH 4.43, which supported the replacement

of (3-OH) with acetyl group in 263. All spectral values were foundin full agreement

with known phytochemical taraxerol acetate, already reported in literature (Deng et

al., 2004).


76


Position 262 263

δH(J in Hz) δC δH(J in Hz) δC

1 1.88 (2H, dd, 13.9, 3.9) 19.1 1.90 (2H, dd, 14.3, 2.8) 36.8

2 1.55 (2H, m) 24.2 1.49 (2H, m) 23.2

3 3.70 (1H, dd, 9.0, 4.2) 70.9 4.43 (1H, dd, 9.0, 4.4) 79.9

4 - 38.1 - 38.1

5 0.82 (1H, m) 56.1 0.87 (1H, m) 55.6 6 1.49 (2H, m) 17.5 1.55 (2H, m) 18.1

7 1.32 (2H, m) 33.3 1.30 (2H, m) 33.2

8 - 31.2 - 30.2

9 1.41 (1H, t, 5.8) 49.1 1.43 (1H, t, 5.6) 49.2

10 - 37.2 - 37.0

11 1.57 (2H, m) 18.9 1.66 (2H, m) 17.1

12 1.89 (2H, m) 31.0 1.30 (2H, m) 35.2

13 - 35.9 - 37.1

14 - 157.7 - 158.2

15 5.49(1H, dd, 8.0, 3.4) 115.9 5.53(1H, dd, 8.2, 3.7) 116.6

16 3.36 (2H, t, 6.8) 37.0 3.38 (2H, t, 5.72) 36.1

17 - 35.5 - 37.9

18 0.81 (1H, m) 47.3 0.83 (1H, m) 48.2

19 1.99 (2H, m) 41.8 1.81 (2H, m) 41.7

20 - 37.7 - 38.1

21 1.41(2H, t, 5.1) 33.7 1.36 (2H, t, 5.1) 35.3

22 1.52(2H, t, 2.8) 26.9 1.65 (2H, t, 2.8) 29.0

23 0.82 (3H, s) 21.3 0.83 (3H, s) 21.3

24 0.81 (3H, s) 21.4 0.81 (3H, s) 28.6

25 1.03 (3H, s) 26.0 1.03 (3H, s) 28.9

26 0.85 (3H, s) 29.4 0.85 (3H, s) 29.0

27 0.88 (3H, s) 29.5 0.88 (3H, s) 30.3 28 0.87 (3H, s) 29.1 0.87 (3H, s) 29.3

29 0.96 (3H, s) 27.8 0.96 (3H, s) 28.1

30 0.97 (3H, s) 18.9 0.97 (3H, s) 18.8

1′ - - - 172.1

2′ - - 2.02 (3H, s) 22.0

3.6.3. Structure Elucidation of 2′-(4-Hydroxyphenyl)-Ethyl Stearate (264)

TheIR spectrum of compound 264 displayed distinguished absorption bands at

3440 (O-H), 1729 (C=O), 1520-

1469 (Ar-C=C) and 1210 cm-1(C-

O), whereas, The HREI-MS

analysis afforded molecular ion at

m/z 404.3286 attested for the molecular formula C26H44O3 with five DBE.The 1H-

NMR spectrum of compound 264 (Table 3.18) offered two doublets at δH 7.15 (2H, d,

J = 8.8 Hz) and 6.75 (2H, d, J = 8.8 Hz); the A2B2 splitting pattern indicated the


77

presence of para-substituted benzene ring.The same spectrum showeda resonance at

δH 4.24 (2H, t, J = 6.6 Hz), which was correlated in COSY spectrum with another

triplet methylene resonance at δH 3.70 (2H, t, J = 6.6 Hz). The chemical shifts of these

two methylenes revealed their attachment with carboxylate function and phenyl ring,

respectively.Other distinct triplet methylene signal at δH 2.75 (2H, t, J = 6.7 Hz) and a

methyl signal at 0.88δH (3H, t, J = 6.8 Hz) showed their COSY relation with a broad

signal of various methylenes between δH 1.63-1.11.This data indicated that compound

264 could be a fatty ester of p-hydroxyphenylethyl.

The 13C-NMR spectrum (Table 3.18) of compound 264 showed signals for

ester carbonyl (δC 178.5), p-substituted aromatic system (δC 153.1, 164.0, 163.0,

117.0) saturated methylenes (δC 67.3, 35.1) and fatty acid chain (δC 35.0, 26.3-21.1,

14.6). The fatty acid chain length could only be fixed due to mass spectrometric data.

Thus compound 264 was finally identified as 2′-(4-hydroxyphenyl)-ethyl stearate,

which was further supported by the reported literature (Ruberto and Tringali, 2004,

Acevedo et al., 2000).

Table 3. 181H-NMR and 13C-NMR data of compound 264 (CDCl3, 500 and 125 MHz)

Position δH (J in Hz) δC

1 - 164.0

2,6 7.15 (2H, d, 8.8) 163.0

3,5 6.75 (2H, d, 8.8) 117.0

4 - 153.1

7 3.70 (2H, d, 6.6) 34.9

8 4.24 (2H, d, 6.6) 67.3

1′ - 178.5

2′ 2.75 (2H, t, 6.7) 35.0

3′ 1.63 (2H, m) 26.3

4′-16′ 1.11-1.38 (26H, br s) 24.0-27.7

17′ 1.22 (2H, m) 21.1

18′ 0.88 (3H, t, 6.8) 14.6

3.6.4. Structure Elucidation of Apigenin (265)

Yellow amorphous powder of compound 265 showed a yellow spot on TLC,

which remained yellow on heating with ceric

sulphate, which indicated a flavonoid

compound. The IR spectrum exhibited

absorption bands for O-H (3420 cm-1), C=O

(1665 cm-1), and Ar-C=C (1610-1520 cm-1).


78

The EIMS spectrumshowedmolecular ion peak atm/z 270, while the HREIMS

disclosed the molecular formula as C15H10O5since it afforded molecular ion peak

atm/z270.0534.The1H-NMR spectrum of compound 265 (Table 3.19) offered five

signals in aromatic region at δH 7.68 (2H, d, J = 9.2 Hz) and 6.72 (2H, d, J = 9.2 Hz),

6.30 (1H, d, J = 2.1 Hz),6.13 (1H, d, J = 2.1 Hz) and 6.74 (1H, s). The first two

signals splitted at A2B2 spin system were assigned to a p-substituted benzene ring,

which next two meta-coupled doublets were attributed to a tetra-substituted benzene

ring. The last singlet could be attested for H-3, which indicated compound 265

belongs to flavone class of compounds.

The 13C-NMR of compound 265 (Table 3.19) showed thirteen resonances for

fifteen carbon atoms which were distinguished as six oxygenated quaternary carbons

due to the shiftsat δC 165.8,162.9, 160.0, 158.5 and151.6, whereas,the most downfield

resonance at δC 179.6 was assigned carbonylic carbon. With the help of 2D techniques

and comparison with the reported data, compound 265 was found to be apigenin,

which is a known phytochemical (Liu et al., 2013).

3.6.5. Structure Elucidation of Naringinin (249)

TheHREI-MS of 249 displayed molecular ion peak at m/z272.0580, which was

calculated for the molecular formula as C15H12O5

with 10 DBE, whereas most of its NMR data (Table

3.19) was superimposable to that of265. The

difference observed wasthat NMR signals for

olefenic CH-3 (in 265) were absent in the spectrum

of 249, and instead an oxymethine displayed its

positions as: [δH 5.32(dd, J=5.3, 11, H-2), δC 77.4 (C-2)] and methylene (δH 3.10 (d,

J=12, H-3a) and 3.03 (d, J=12, H-3b), δC 44.4 (C-3)].This information led to establish

that the double at C-2 is reduced, which substantiated the information obtained from

the molecular formula and DBE. The NMR of compound 249 has been provided in

table 3.19, which was found identical to the reported data for naringinin, which is a

known phytochemical (Olsen et al., 2008).


79

3.6.6Structure Elucidationof Kaempheride (222)

Compound 222was isolated as a yellow crystalline solid, and due to its

behavior on TLC was also found to be a flavonoid.Its

IR spectrum afforded absorption bands at 3410 (O-H),

1700 (C=O), 1620- 1520cm-1 (Ar-C=C),

whilethemolecular formula as C16H12O6could be

determined due to HREI-MS analysis (m/z

300.0634).The 1H NMR spectrum of 222 revealed the

presence of a p-substituted benzene ring C and two meta-coupled doublets were

attested for benzene ring B. When compared with the spectrum of 265, it missed

signal for H-3, which indicated compound 222 must be a flavonol-derivative. The

same spectrum also displayed an additional resonance due to methoxyl proton at δH

3.61.

The 13C-NMR spectrumof 222(Table 3.19) supported the mass and 1H- NMR

data. The position of methoxy group at C-4was found through HMBC correlations.

Finally the comparison of the spectroscopic data with literature values, compound

222was elucidated as kaempheride which is also a known phytochemical (Anđelković

et al.,2017).

Table 3. 191H- and 13C-NMR data of compound 265 and 249 (DMSO-d6 , 500 and 125 MHz) and 222

(CD3OD, 400 and 100 MHz)

Position

265 249 222

δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz) δC

1 - - - - - -

2 - 151.6 5.32(2H, dd,5.3, 11), 77.4 - 146.9

3 6.74 (1H, s) 104.5 3.10 (d,12, H-3a)

3.03 (d,12, H-3b) 44.4 - 137.7

4 - 179.6 - 174.6 - 176.9

4a - 104.7 - 103.7 - 104.0

5 - 162.9 - 162.0 - 162.7

6 6.13 (1H, d, 2.1) 99.3 6.12 (1H, d, 2.1) 99.8 6.30 (1H, d, 2.0) 99.1

7 - 165.8 - 166.2 - 164.4

8 6.30 (1H, d, 2.1) 93.8 6.33 (1H, d, 2.2) 94.5 6.35 (1H, d, 2.0) 92.8

8a - 160.0 - 161.0 - 160.1

1' - 123.1 - 124.1 - 123.1 2' 7.68 (2H, d, 9.2) 128.8 7.17 (2H, d, 9.0) 129.1 7.65 (2H, d, 9.1) 127.8

3' 6.72 (2H, d, 9.2) 117.8 6.70 (2H, d, 9.0) 118.1 6.73 (2H, d, 9.1) 118.0

4' - 158.5 - 159.1 - 157.5

5 6.72 (2H, d, 9.2) 117.8 6.70 (2H, d, 9.0) 117.1 6.73 (2H, d, 9.1) 118.0

6' 7.68 (2H, d, 9.2) 128.8 7.17 (2H, d, 9.0) 128.7 7.65 (2H, d, 9.1) 127.8

4'-OMe - - - - 3.61(3H, s) 59.5


80

3.6.7. Structure Elucidation of Quercetin (125)

Compound 125 was obtained as pale yellow crystalline solid that exhibited

characteristic absorption bands forO-H (3428-3369 cm-1), C=O (1708 cm-1) and C=C

(1665-1475cm-1) in the IR spectrum. HREIMS

spectrum disclosed molecular ion at m/z 302.0430,

which depicted the molecular formula as C15H10O7

with 11 DBE.The 1H NMR spectrum (Table 3.20) of

125 displayed five signals in the aromatic region at

δH7.72 (1H, d, J = 2.0 Hz), 7.59 (1H, dd, J = 8.2, 2.0

Hz), 6.75 (1H, d, J = 8.4 Hz),6.33 (1H, d, J = 2.0 Hz)

and 6.29 (1H, d, J = 2.0 Hz). The first three splitted at ABX pattern revealed a1,3,4-

trisubstituted benzene ring Bwhile the other two m-coupled resonances were

attributed to ring A of flavonoid. The 13C NMR spectra (Table 3.20) of 125 displayed

altogether fifteen carbon signals which were identified as ten quaternary carbons

(δC176.9, 165.4, 163.0, 158.5, 147.9, 147.6, 145.9, 137.1, 105.0) and five methines

(δC123.2, 121.0, 116.0, 98.9, 93.8). The data was fully fit to the NMR values reported

for quercetin (Xiuyun et al., 2006), which is a potent antioxidant natural product.

3.6.8. Structure Elucidation of Quercetin 3-O-β-D-glucopyranoside (198)

The compound 198 was also purified as yellow amorphous powder, which

absorbed in the IR spectrum at 3340-3200, 1610-1459 cm-1due tohydroxyl group,

conjugated ketone and phenyl group, respectively. The EIMS spectrum exhibited the

heaviest ion at m/z 454, which was analyzed in

HREIMS as 464.0950 for the molecular formula

as C21H20O12 with 12 DBE. The aromatic region

of the1H NMR spectrum of 198 (Table 3.20) was

identical to that of 125 indicating the same

flavonoidal skeleton. However, the same

spectrum displayed resonance of an anomeric

proton at δH 5.33 (1H, d, J = 7.0 Hz) along withother oxymethine signals at δH 3.70-

3.30 (Table 3.20). This data indicated quercetin glycoside nature of 198, which was

substantiated due to 21resonances in the 13C NMR spectrumof 198 (Table 3.20).The


81

carbon signals due to aglycone part were similar to that of 125, while sugar signals

displayed their positions at δC 102.8, 78.3, 76.1, 70.9, 77.2 and 62.2.The attachment of

sugar moiety was established at C-3 due to HMBC correlation of anomeric proton (δH

5.33) with quaternary carbon at δC 136.4 (C-3). This data showed complete

resemblancewith the already published data for quercetin 3-O-β-D-glucopyranoside

(198) (Sukito and Tachibana, 2014).

Table 3. 201H- and 13C-NMR data of compound 125 and 198 (DMSO-d6, 400 and 100 MHz)

Position 125 198

δH(J in Hz) δC δH (J in Hz) δC

1 - - - -

2 - 147.9 - 153.8

3 - 137.1 136.4

4 - 176.9 - 180.9

4a - 105.0 - 105.0

5 - 163.0 - 163.1

6 6.29 (1H, d, 2.0) 98.8 6.17 (1H, d, 1.9) 101.2

7 - 165.4 - 165.9

8 6.33 (1H, d, 2.0) 93.8 6.34 (1H, d, 1.9) 94.8

8a - 158.5 - 157.6

1' - 123.2 - 123.7

2' 7.72 (1H, d, 2.0) 116.0 7.69 (1H, d, 2.1) 118.8

3' - 145.9 - 147.2

4' - 147.6 - 151.1

5' 6.75 (1H, d, 8.4) 116.1 6.90 (1H, d, 8.2) 117.2

6' 7.59 (1H, d, 8.2, 2.0) 121.0 7.60 (1H, dd, 8.2, 2.1) 124.4

1'' - - 5.33 (1H, d, 7.0) 102.8

2'' - - 3.45 (1H, t, 8.4) 78.3

3'' - - 3.52 (1H, m) 76.1

4'' - - 3.34 (1H, t, 7.2) 70.9

5'' - - 3.31 (1H, m) 77.2

6'' - - 3.70 (1H, dd, 12.2,3.9)

3.61 (1H, dd, 12.2, 3.9) 62.2

3.6.9. Structure Elucidation of β-Sitosterol 3-O-D-glucopyranoside (172)

Compound 172 was purified as a white amorphous powder.The IR spectrum offered

distinctive peaks for O-H (3451 cm-1) =C-

H (2922-2871 cm-1) and1655 cm-1 (C=C)

functionalities. EI-MS spectrum

displayed molecular ion peak at m/z576

with base peak at m/z 414.It aided finding

the molecular formula as C35H61O6 with

six DBE. The 1H-NMR spectrum of 172


82

(Table 3.21) showed the presence of two singlets methyls at δH 0.91and 0.88

attributed to Me-19 and Me-18, three doublet methyls atδH 0.96 (3H, d, J = 6.6 Hz),

0.90 (d, J = 6.3 Hz), 0.82 (J = 7.2 Hz) and primary methyl resonated at δH0.89 (3H, t,

J = 7.2 Hz) assigned to Me-21, Me-26, Me-27 and Me-29, respectively. A multiplet at

δH 3.27 and a doublet of doublet at δH 5.26 were observed with characteristic coupling

constant value (J = 9.4, 4.9 Hz) typical for H-3 and H-6 of a compound having

steroidal skeleton.In additional, resonances for the sugar moiety at δH 4.22 (1H, d, J =

7.7 Hz, 1), 2.90 (1H, t, m, 2), 3.28 (1H, t, m, 3), 3.03 (1H, m, 4), 3.10 (1H, m, H-

5), 4.13 (1H,dd, J=11.5,3.10,6a) and 4.39 (1H,dd,J=11.7, 2.9, 6b).

The 13C-NMR spectrum of 172 (Table 3.21) depicted 35 carbon signals, of

which twenty nine resonances wereattributed toβ-sitosterol skeleton while remaining

signals for sugar moiety were seen in the spectrumat δC 101.0 (C-1), 72.0 (C-2), 76.0

(C-3), 70.9 (C-4), 75.5 (C-5) and 60.5 (C-6). The position of sugar moiety was

established through HMBC correlations in which the anomeric proton H-1′ (δH 4.22)

showed correlation with C-3 (δC 81.1) of sterol skeleton.Further comparative TLC and

data correlation with the reported data confirmed the compound 172 as β-sitosterol 3-

O-β-D-glucopyranoside (Akhtar et al., 2010).

Table 3. 211H-NMR and 13C-NMR data of compound 172 (CDCl3+CD3OD, 400 and 100 MHz)

Position δH (J in Hz) δC Position δH (J in Hz) δC

1 3.88 (2H, m) 38.3 19 0.91 (3H, m) 20.7

2 1.87 (2H, m) 30.2 20 1.97 (1H, m) 38.5

3 3.29 (1H, m) 81.1 21 0.96 (3H, d, 6.6) 19.3

4 3.81 (2H, m) 43.3 22 1.18 (2H, m) 41.2

5 - 141.1 23 1.90 (2H, m) 31.5

6 5.26 (1H, dd, 9.4, 4.9) 121.3 24 1.42 (1H, m) 52.0

7 2.17 (2H, m) 34.3 25 1.88 (2H, m) 27.0

8 1.22 (1H, m) 34.2 26 1.82 (3H, d, 6.3) 21.3

9 1.52 (1H, m) 49.3 27 0.82 (3H, d, 7.2) 36.0

10 - 38.3 28 1.30 (2H, m) 24.6 11 1.57 (2H, m) 23.2 29 0.89 (3H, t, 7.2) 19.3

12 1.87 (2H, m) 43.7 1' 4.22 (1H, d, 7.7) 101.0

13 - 45.3 2' 2.90 (1H, m) 72.0

14 1.26 (1H, m) 58.6 3' 3.28 (1H, m) 76.0

15 1.91 (2H, m) 23.10 4' 3.03 (1H, m) 70.9

16 1.46 (1H, m) 33.7 5' 3.10(1H, m) 75.5

17 1.51 (1H, m) 56.9

6' 4.13(1H, dd,11.5,3.10)

4.39 (1H, dd,11.7,2.9)

60.5

18 0.88(3H, s) 12.9

Chapter no.03 Results and Discussionof Vincetoxicum Stocksii

83

Part C

3.7. Biological Screening of Crude Extract of Vincetoxicum Stocksii

The crude V.stocksiihexane soluble (Vs-H) and ethyl acetate soluble (Vs-E)

extracts were evaluated for anti-oxidant, anti-urease, anti-- glucosidase and AChE

inhibition activity (Table 3.22). Vs-H showed IC50 value of 29.01±0.01 μg/mL in

antioxidant assays, while Vs-Eexhibite IC50 value of 42.00±0.03 μg/ mL, which is

more potent in antioxidant activity. Similar behavior was seen in anti-urease assay, in

which Vs-E was found more active in comparison toVs-H (IC50= 29.01±0.01,

22.00±0.03 μg/ mLrespectively).Comparable response was recorded for both the

extracts in anti-- glucosidase activity, while in AChE inhibition activity Vs-E

showed IC50 value of 16.54±0.02μg/ mL, while Vs-E exhibited 30.23±0.02μg/ mLof

IC50. Thus overall response of crude extracts towards the key enzyme revealed good

potential of Vs-E that invites further investigation.

Table 3. 22Anti-oxidant and anti-urease activities of V. Stocksii (Vs) extracts*

Plant Material/Standard

Anti-oxidant Activity (DPPH assay)

Anti-urease Activity Anti--glucosidase

Activity

AChE inhibition activity

Inhibition % at 0.5 mg/ml

IC50μg/

mL


IC50μg/

mL


IC50μg/

mL


IC50μg/

mL

Vs-H 82.83±0.01 29.01±0.01 92.83±0.01 29.01±0.0

1

91.52±0.14

41.51±0.11

70.47±0.08

30.23±0.02

Vs-E 70.87±0.03 42.00±0.03 80.87±0.01 22.00±0.0

3

85.23±0.16

40.38±0.12

91.47±0.08

16.54±0.02

DPPH

(Std) 92.02±0.67

37.48±0.0

1 - -

- - - -

Thiourea (Std)

- - 96.60±0.01 21.31±0.1

4 - - - -

Ascorbase (Std)

- - - - 92.23±0.1

5 37.38±0.1

2 - -

Eserine

(Std) - - - -

- - 91.27±1.1

7

0.04±0.01

* Values expressed are means S.D. of three parallel measurements


84

3.8. Characterization of Secondary Metabolites Isolated from V.

stocksii

SinceVs-E discloses superior prospective in biochemical assays, therefore was

preferred to be investigatedfor its secondary metabolites.It was subjected to silica gel

column chromatography to get one new: methyl 2-hydroxy-3-(2-hydroxy-5-(3-

methylbut-2-enyl)phenyl)-2-(4-hydroxyphenyl) propanoate/ stocksiloate (266), and

two rarely occuring natural products: (4-(4-(methoxycarbonyl) benzyl) phenyl)

carbamic acid (267) and bis[di-p-phenylmethane]ethyl carbamate (268), in addition to

abovefour known compounds, 4-hydroxy-3-methoxyphenyl-7,8,9 propanetriol(194),

feruloyl-6-O-β-D-glucopyranoside (197), apocynin (196) and vincetomine (192).

3.8.1. Structure Elucidationof stocksiloate (266)

Compound 266 was purified as white amorphous solid, which showed IR

absorption bands at 3505, 1737 and 1545 cm-1attestingthe presence of hydroxyl

group, ester function and aromatic ring

respectively. In the1H NMR spectrum of 266

(Table 3.23) signals splitting in aromatic region

manifested the presence of A2B2 and ABX

systems at H 7.60 (2H, d, J = 9.2 Hz), 6.85 (2H,

d, J = 8.8 Hz) and at H 6.54 (1H, dd, J = 8.4,

2.0 Hz), 6.48 (1H, d, J = 8.4 Hz), 6.42 (1H, d, J = 2.0 Hz) respectively. A methylene

resonated at H 3.07 (d, J = 7.2 Hz), whilean olefinic methine signal was seen at H

5.07 (m). The two allelic methyl protons displayed their positionsat H1.65 (d, J = 0.8

Hz) and 1.56 (brs) to disclosea prenyl moiety in the structure. In addition the spectrum

afforded signals for a methoxyl groupat H 3.75 (s), and another methylene at 3.39

(1H, d, J = 12.4 Hz) and 3.42 (1H, d, J =12.4 Hz) respectively.The 13C NMR

spectrum for 266 (Table 3.23) depicted 19 carbon resonances attributed to 21 carbon

atoms. COSY and HSQC correlations identified resonances at C 17.7 (CH3), 25.9

(CH3), 28.7 (CH2), 123.6 (CH) and 128.4 (C) for the prenyl moiety.Carboxylate

system was indicated by downfield signal at C 172.1 of quaternary carbon. Moreover


85

the 13C NMR spectrum also exhibited resonances at C 39.6 (-CH2), 53.6 (-OMe), and

86.7 (C) along with two benzene ring signals as shown in Table 3.23.

The HMBC data indicated correlation of methylene proton at C 3.07 with

aromatic carbons at C132.9 (C-9) and 128.4 (C-8) asserting the linkage of prenyl

group to tri-substituted benzene ring through C-8. Similarly further correlations in

HMBC shows linking of H-9 (H 6.42) with C-10 (C 28.7), H-3 (H3.39 and 3.42)

with C-2 (C 86.7) and C-4 (C 125.3) and methoxyl proton with C-1 (C 172.1).

These all allocations were substantiatedcombining information fromHSQC, HMBC

and COSY spectral data. After comparing data with the theoretically calculated values

given by ACD lab software, compound 266 was found to have given structure and

was given the name stocksiloate (266) (Khan et al., 2019).

Table 3. 231H-NMR and 13C-NMR data of compound 266 (CDCl3, 400 and 100 MHz)

Position δH(J in Hz) δC HMBC

1 - 172.1 2 - 86.7

3 3.42 (1H, d,13.8)

3.39 (1H, d,13.8)

39.6 86.7, 125.3

4 - 125.3

5 - 155.0

6 6.48 (1H, d, 8.4) 115.0 125.3, 128.4

7 6.54 (1H, dd,8.4,2.0) 129.7 128.4, 155.0

8 - 128.4

9 6.42 (1H, d, 2.0) 132.9

10 3.07 (2H, d, 7.2) 28.7 128.4, 123.6

11 5.07 (1H,m) 123.6 12 - 132.9

13 1.56 (3H, brs) 17.7

14 1.65 (3H, d,0.8) 25.9 123.6, 164.9,

17.7

1′ - 128.4 130.0

2′, 6′ 7.60 (2H, d, 9.2) 130.0 158.7

3′, 5′ 6.85 (2H, d, 8.8) 116.4 158.7

4′ - 158.7

1′′ 3.75 (3H,s) 53.6 172.1


86

3.8.2. Structure Elucidation of 4-(4-(methoxycarbonyl) benzyl) phenyl] carbamic

acid (267)

White amorphous powder of 267displayed IR absorption bandsat 3385-2470,

1730, 1705, 1605, 1545 and 1490 cm-1that

depicted the presence of carboxylic group,

amide group and aromatic system. The

heaviest ion peak at m/z 282 [M-H2O] was

seen in the EIMS spectrum of

267,however, the molecular formula C16H16N2O4, with 10 DBE could be determined

due to HR-FABMS (m/z301.1189) analysis in positive mode, thus indicating the

molecular mass of 267as 300 amu. The 1H NMR spectrum of 267(Table 3.24)

afforded four signals in the aromatic region at H 7.33 (2H, d, J = 8.4 Hz), 7.32 (2H,

d, J = 8.4 Hz), 7.10 (2H, d, J = 8.4 Hz) and 7.08 (2H, d, J = 8.4 Hz), attested for two

p-substituted benzene rings in the molecule. The most downfield signals at H 9.50

(1H, s) and 8.49 (1H, s) in the spectrum were designated to two secondary amines. A

singlet resonance at H 3.78 (2H, -CH2) was attributed to methylene protons and their

presence between two aromatic systems was confirmed with the help of HSQC and

HMBC correlations.The 13C NMR spectrum (Table 3.24) displayed12 carbon

resonances at C 153.9 (C), 152.5 (C), 137.6 (C), 137.0 (C), 135.5 (C), 134.8 (C),

128.8 (2CH), 118.2 (2CH), 51.4 (CH3) and 39.9 (CH2). The IR data and quaternary

carbon resonances at C 153.9 and 152.5 predicted carbamic acid moieties in 267

(Chang et al., 2011).

The mass fragmentation pattern in EIMS spectrum was observed to m/z

208, 223, 240, 256, 267 and 282) in which ion m/z 282 (100%) showed isocyanato

nature. This along with supporting the presence of carbamic acid function also gives

the reason for less stability of this function than isocyanato ion. The generation of this

ion is possibly due to dehydration of molecular ion of carbamic acid group. This leads

to the conclusion that all the fragments in this spectrum were due to dehydrated

isocyanato ion derived from 267.

Among 10 DBE, 8 were attributedto two aromatic systems, while remaining

two DBE supported the presence of two carbamic acid moieties, which was


87

substantiated by the HMBC correlation of most downfield carbon atoms at C 153.9

and 153.9 with two amine protons at H 9.50 and 8.49 respectively. Similarly HMBC

correlation of methoxyl proton at H 3.62 with carbon at C 153.9 indicated

methylcarbamate moiety. N-aryl linkage between two carbamic acid groups was

found by HMBC correlations of two amine protons with the carbon resonances at C

137.6, 137.0 and 118.2 respectively. The presence of methylene group between two

aromatic rings was predicted by the HMBC interactions of these aromatic ring

carbons (C 167.5, 166.8 and 128.8) with methylene proton (H 3.78). After

interpreting the data from HSQC, COSY and HMBC correlations the compound 267

depicted to have the given structure ([4-(4-(methoxycarbonyl) benzyl) phenyl]

carbamic acid) (Khan et al., 2019).

3.8.3. Structure Elucidation of Bis[di-p-phenylmethane]ethyl Carbamate (268)

Compound 268 was found to be derivative of 267, which missed IR absorption for

carboxylic acid functional group at 3385-2470 while other absorption bands were

nearly in same region as were recorded

for compound 267. The molecularion

at m/z 342 was clearly observed in

EIMS spectrum,while, the molecular

formula C19H22N2O4, with 10 DBE

could be determined due to HR-EIMS,

thus indicating the molecular mass of 268 as 342.1579 (342.1580 calcd. for

C19H22N2O4). The 1H NMR spectrum (Table 3.24) of compound 268 showed two

doublets in the aromatic region at H7.30 (4H, J = 8.4 Hz) and 7.08 (4H, J = 8.4 Hz)

presenting the possibility of twop-substituted benzene rings in the form of

symmetrical dimer. There was an imine proton signal at H8.6. A singlet of methylene

proton at H3.84 showed correlation with 13C resonance at H 41.4 in HSQC spectrum.

The proton signals at H 4.16 (4H, q, J = 7.2 Hz) and 1.29 (6H, t, J = 7.2 Hz) were

assignedto ethoxy group. The downfield shift of methylene proton was attributed to

itsattached to carboxylate function.The 1H NMR data of compound 268 was fully

supported by 13C NMR spectrum (Table 3.24) exhibiting eight signals for carbon

resonances which were identified as three quaternary carbons (C 156.2, 138.1 and


88

137.5), two methine (C 130.1 and 120.1), two methylene (C 41.1 and 61.8) and

one methyl (C 14.9). In accordance to the molecular formula there should be 19

carbon atoms, resulting in prediction of two symmetrical p-substituted aromatic rings

and dimeric nature of compound 265. After analyzing the data from HMBC

correlation 268 was identified to be bis[di-p-phenylmethane]ethyl carbamate, a

naturally occurring carbamate derivative (Williamand et al., 1981).

Table 3. 241H and 13C NMR data of 267 (DMSO-d6, 600 and 150 MHz, respectively), 268 (CD3OD,

600 and 150 MHz, respectively)

Position 267 268

H (J in Hz) C HMBC H (J in Hz) C HMBC

1 - 137.6 - 138.1

2 7.33 (1H, d, 8.4) 118.2 7.30 (1H, d, 8.4) 120.1

3 7.08 (1H, d, 8.4) 128.8 7.08 (1H, d, 8.4) 130.1

4 - 135.5 - 137.5

5 7.08 (1H, d, 8.4) 128.8 7.08 (1H, d, 8.4) 130.1

6 7.33 (1H, d, 8.4) 118.2 7.30 (1H, d, 8.4) 120.1

7 3.78 (2H, s) 39.9 128.8, 166.8 3.84 (2H, s) 41.1

8 - 134.8 - 137.5

9 7.10 (1H, d,8.4) 128.8 7.08 (1H, d, 8.4) 130.1

10 7.32(1H, d,8.4) 118.2 7.30 (1H, d, 8.4) 120.1

11 - 137.0 - 138.1

12 7.32 (1H, d,8.4) 118.2 7.30 (1H, d, 8.4) 130.1

13 7.10 (1H, d,8.4) 128.8 7.08 (1H, d,8.4) 120.1

1 - 153.9 - 156.2

2 - 152.5 - -

1 3.62 (3H, s) 51.4 153.9 4.16 (2H, q, 7.2) 61.8 14.9, 156.2

2 - - 1.29 (3H, t,7.2) 14.9 61.8

NH 9.50 (s) and 8.49 (s) - 137.6,118.2,1

37.0,118.2,

153.9

8.6(s) - 156.2, 130.1,

120.1

3.8.4.Structure Elucidation of 4-Hydroxy-3-Methoxyphenyl 7, 8, 9 Propanetriol

(194)

Compound 194 was scanned for IR data that disclosed hydroxyl group and

aromatic ring, whereas, the EIMS spectrum

displayed molecular ion peak at m/z 214. The

HREIMS analysis of the same ion (m/z 214.0850)

depicted the molecular formula C10H14O5 with four

DBE. The 1H-NMR spectrum (Table 3.25) of

compound 194 exhibited three signals in aromatic

region at δH7.70 (1H, d, J = 8.4 Hz), 6.77 (1H, d, J = 8.4, 2.0 Hz) and 6.95 (1H, d, J=


89

1.9 Hz). The ABX splitting pattern revealed a 1, 3, 4-trisubstituted benzene

ring.Among other signals in the same spectrum at δH 4.47 (1H, d, J = 6.6 Hz) and δH

3.68 (1H, q, J = 5.4) were attributed to the two oxymethine. The later methine signal

showed correlation in COSY with the first methine and in addition with an

oxymethylene resonating at δH 3.54 (1H, dd, J = 11.2, 7.0) and 3.43 (1H, dd, J =11.4,

4.2). This indicated a phenylpropanoid moiety in 194. A methoxyl proton resonance

was also seen in the same spectrum at δH3.85. The 13C-NMR spectrum (Table 3.25)

substantiated the above deduction. It displayed ten signals altogether, which were

identified as three quaternary (δC 148.4, 146.7 and 166.4),five methine (δC 120.6,

115.4, 111.4, 77.2 and 74.6), one methylene (δC 64.1) and a methoxyl carbon

resonance at δC 56.2.This data led to the structure of 194 as be 4-hydroxy-3-

methoxyphenyl-7,8,9-propanetriol/1-(4-hydroxy-3-methoxyphenyl) 1,2,3,

propanetriol (Warashina et al., 2005).

3.8.5. Structure Elucidation of Feruloyl-6-O- β-D-glucopyranoside (197)

White amorphous solid of 197displayed IR absorption bands for hydroxyl

group, carbonyl group, double bond and

aromatic moiety at 3442, 1710, 1655, 1608-

1540 cm-1respectively. The molecular ion

peak at m/z 356 was foundby EIMS

spectrum, while the HR-EIMS analysis

showed heaviest ion peak at m/z 356.1110

calculated for the molecular formula C16H20O9 with seven DBE. In addition to the

signals due to a 1,3,4-trisubstituted benzene ring, aromatic region ofthe 1H-NMR

spectrum (Table 3.25) of 197 also showed two doublets at δH 7.59 (1H, d, J = 15.4

Hz) and δH 6.37 (1H, d, J = 15.4 Hz) due to a conjugated trans double bond. This

leads to the indication of caffeoyl derived nature of 197. Further, along with methoxyl

signal (δH 3.88, s) as was found in the spectrum of 194, the spectrum displayed

resonance of an anomeric proton at δH 5.06 (1H, J = 7.2 Hz) indicatinga β-hexose

sugar in 197. The other resonances due to sugar moiety displayed their positionsat δH

3.91 (1H, d, J =6.3), 3.70 (1H, dd, J = 12.0, 5.3 Hz), 3.62 (1H, dd, J = 12.0, 2.8 Hz),

3.52 (1H, t, J = 7.2 Hz), 3.42 (1H, m), 3.12 (1H, m) the resonance at δH 3.88 was

depicted to methoxyl proton.The 13C-NMR spectrum (Table 3.25) exhibited sixteen


90

signals, ten out of which were foundto be methine at δC 146.5, 124.1, 116.5, 115.2,

111.3, 97.8, 77.9, 77.2, 75.1 and 71.6, one for methylene δC 64.2 and four were

attestedfor quaternary carbons at δC168.7, 150.2, 146.9 and 127.3 on the basis of

DEPT analysis. The position of sugar moiety and site of connectivity was confirmed

due to the HMBC correlations of methylene of sugar moiety (δH3.70 and 3.62) with

the carbonyl carbon at δC 165.1 (C-9), confirming the linkage of aglycone and glycon

at C-6 sugar part instead of at C1.Based onthe above discussion and comparing data

with the already reported data, compound 197 was characterized as feruloyl-6-O-

Dglucopyranoside (Dallacqua and Innocenti 2004)

Table 3. 251H and 13C NMR data of 194 and 197 (CD3OD, 600 and 150 MHz)

Position

194 197

δH (J in Hz) δC δH (J in Hz) δC

1 - 166.4 - 127.3

2 6.95 (1H, d, 1.9) 111.4 7.13 (1H, d,2.0) 111.3

3 - 148.4 - 146.9

4 - 146.7 - 150.2 5 6.70 (1H, d, 8.4) 115.4 6.81 (1H, d, 8.0) 116.5

6 6.77 (1H, dd, 8.4, 2.0) 120.6 7.02 (1H, d,8.0, 2.0) 124.1

7 4.47 (1H, d,6.6) 74.6 7.59 (1H, d, 15.4) 146.5

8 3.68 (1H, q,5.4) 77.2 6.37 (1H, d, 15.4) 115.2

9 3.54 (1H, dd, 11.2, 7.0) 64.1 - 168.7

3.43 (1H, dd,11.4, 4.2)

3-OMe 3.85 (3H, s) 56.2 3.88 (3H, s, OMe) 56.4

1 - - 5.06 (1H, d,7.2) 97.8

2 - - 3.52 (1H, t,7.2) 75.1

3 - - 3.12 (1H, m) 71.6

4 - - 3.91 (1H, d, 6.3) 77.2

5 - - 3.42 (1H, m) 77.9

6 - - 3.70 (1H, dd, 12.0, 5.3) 64.2

- - 3.62 (1H, dd, 12.0, 2.8)

3.8.6. Structure Elucidation of Apocynin (196)

1H-NMR spectrum of compound 196also showed the presence of a 1,3,4-

trisubstituted benzene ring like in previous two compounds,

as it displayed three resonances at δH 7.20 (1H, d, J = 1.8

Hz), 7.10 (1H, dd, J = 8.5, 2.0 Hz) and 6.86 (1H, d, J = 8.5

Hz). Similarly, methoxyl proton also resonated at δH 3.89.

However, the spectrum of 196 showed an addition methyl

singlet at δH 2.52, indicated the presence of an acetyl

function in 196. The acetyl function was substantiated due to characteristic absorption


91

and at 1715 cm-1 and by 13C-NMR spectrum, which displayed the most downfield

resonance at δC 194.6 (Table 3.26). Other carbon signals were found at their usual

positions, however, acetyl methyl showed its position at δC25.8. This data led to a

substituted acetophenone nucleus, which was further supported due to the molecular

formula as C9H10O3 based on HREIMS analysis (m/z 166.0650). The position of

methoxyl group at C-3 could be fixed due to HMBC interaction of methoxyl proton

(δH 3.89) with aromatic carbon at δC 144.6. After interpreting the spectral data and

correlating it with the reported data compound 196 was predicted to be 1-(4-hydroxy-

3-methoxyphenyl) ethanone, which is also known as apocynin (Kim et al.,2011).

Table 3. 261H and 13C NMR data of 196 (CD3OD, 500 and 125 MHz)

Position δH(J in Hz)) δC

1 - 128.6

2 7.20(1H, d, 1.8) 109.2

3 - 144.6

4 - 148.6 5 6.86 (1H, dd, 8.5) 113.3

6 7.10 (1H, dd,8.5, 2.0) 123.6

7 - 194.6

3-OMe 3.89 (3H, s) 55.6

7-Me 3.52 (3H, s) 25.8

3.8.7. Structure Elucidation of vincetomine (192)

Compound 192 was also obtained as white amorphous solid. The IR spectrum

of 192 displayed absorption bands for primary amine and

aromatic system at 3260, 1605 and 1570 cm-1 respectively.

The molecular ion peak at m/z 213 was seenin theEIMS

spectrum of 192, whereas,the HREIMS spectrum (m/z

213.1140 calcd. 213.1154) depicted the molecular formula as

C14H15NO with 08DBE.The 1H-NMR spectrum(Table 3.27)

for 192 offered the proton resonances in the aromatic region

at δH 7.35 (2H, J = 8.4 Hz), 7.28 (2H, d, J = 8.2), 7.06 (2H, d,

J = 8.4 Hz) and 7.04 (2H, d, J = 8.3 Hz), all splitted at A2B2. They were attributed to

two p-substituted benzene rings. Signals for a methylene proton and methoxy protons

were seen at δH 3.74 (s) and 3.58 (s) respectively.The most downfield signal at δH

8.45 (2H, s) was assigned to anamino group. In 13C-NMR spectrum (Table 3.27) of

192,nine carbon resonances due to14 carbons atoms were observed δC134.7,


92

136.1,153.5, 137.2, 128.4, 117.8, 116.0,51.0, 39.5. Based on the DEPT experiment,

these were identified as three methine (δC 128.4, 117.8 and 116.0), one methylene (δC

39.5), one methyl (δC 51.0) and four quaternary carbon atoms (δC 153.5, 137.2, 136.1,

134.7). Relatively downfield shift of methylene proton (δH 3.74) was attributed to its

presence between two benzene rings, which was substantiateddue to the HMBC

correlations of this proton signal with aromatic carbons at δC 136.1 (C1), 134.7 (C1'),

128.4 (C-2, 6, 3', 5'). The position of methoxyl group was confirmed by HMBC

correlation with carbon resonance at δC 153.5. Similarly the HMBC correlation of

amino proton with carbon resonance at δC 137.2 confirmed its attachment.

Combination of the above and comparison with the literature values, the compound

192was identified as vincetomine which is a known metabolite (Tousif et al., 2016).

Table 3. 271H and 13C NMR data of 192 (CD3OD, 500 and 125 MHz)

Position δH(J in Hz) δC HMBC

1 - 136.1 -

2 7.35 (1H, d,8.4) 128.4 116.0, 153.5

3 7.06 (1H, d,8.4) 116.0 -

4 - 153.5 -

5 7.06 (1H, d,8.4) 116.0 -

6 7.35 (1H, d, 8.4) 128.4 116.0, 153.5

7 3.74 (2H,s) 39.5 136.1, 128.4, 134.7, 117.8

1 - 134.7 -

2 7.04 (1H, d,8.3) 117.8 128.4, 137.2

3 7.28 (1H, d,8.3) 128.4 -

4 - 137.2 -

5 7.28 (1H, d,8.3) 128.4 128.4, 137.2

6 7.04 (1H, d,8.3) 117.8 -

6-OMe 3.58 (3H, s, OMe) 51.0 137.2

1-NH2 8.45 (1H, s) - -

93

CHAPTER 4

General Experimental Methods and

Techniques

Chapter no. 04 General Experiment Methods and Techniques

94

4.1. General Experimental Procedures

Phytochemical investigation of Berberis calliobotrys, Caragana ambigua and

Vincetoxicum stocksii involved various techniques including chromatography,

spectroscopy, mass spectrometry and UHPLC-MS which are described as follow;

4.1.1 Chromatographic techniques

For column chromatography, silica gel (230-400 mesh, Merck) was used as

stationary phase, while commercial grade distilled solvents were employed as mobile

phase. Gradient elution column chromatographic method was employed using n-

hexane, dichloromethane, ethyl acetate and methanol as mobile phase solvents in

single or various compositions. Fractions obtained from the column were analyzed on

pre-coated silica gel GF254 (Merck) on aluminum sheet of having 0.25mm thickness.

The separated spots of colored compounds were identified through naked eye.

Colorless UV active compounds on developed TLC plates were visualized under UV

lamp at 254 and 366 nm. UV inactive metabolites on chromatograms were visualized

with the aid of Iodine and or ceric sulphate (ceric sulphate in 10% H2SO4) solution.

4.1.2 Spectroscopic techniques

In order to get UV spectrum, UV spectrophotometer, Schimadzu UV-240 and

U-3200 Hitachi, was employed.Jasco-320-A infrared spectrophotometer was used for

obtaining IR spectra by KBr disc method to gather information about the presence of

different functional groups. 1H-NMR spectra at 400 and 500 MHz and 13C-NMR

spectra at 100 and 125 MHz were measured on Bruker AM instruments in deuterated

solvents. TMS was used as internal reference. Mass spectrometers of Finnigan MAT-

112 and MAT-113 were used in order to record HR-EI-MS, HR-FAB-MS, FAB-MS,

EI-MS spectra.

4.2. Instrumentation and Work Methodology of UHPLC-MS

The crude extract(s) was divided on solubility basis into various fractions.

Secondary metabolicpictures of the obtained fractions werescanned by RP-UHPLC-


95

MS. UHPLC of Agilent 1290 Infinity LC system coupled to Agilent 6520 Accurate-

Mass Q-TOF mass spectrometer with dual ESI source was used. The UHPLC was

equipped with Agilent Zorbax Eclipse XDB-C18 column of 2.1 x 150 mm, 3.5 micron

(P/N: 930990-902), whereas, the temperature of auto-sampler and column was

maintained at 4°C and 25°C, respectively. Mobile phase A comprises 0.1% formic

acid solution in water, whereas, 0.1% formic acid solution in acetonitrile was used as

mobile phase B, and the flow rate was kept as 0.5 mL/min. 1.0 μL of methanol extract

was injected for the time of 25 min, and 5 min were used for post-run time. Nitrogen

gas with flow rate of 25 and 600 L/hour was used as a source of nebulizing and drying

gas respectively and temperature was maintained at 350°C. The fragmentation voltage

was optimized to 125 V. Analysis was performed with a capillary 1 voltage of 3500 V

(Saleem et al, 2018, khan et., 2019)

4.3. Assessment of Biological Potential of Crude Extracts of Berberis

calliobotrys and Caragana ambigua

All prepared crude extracts and their fractions were tested for total phenolic,

flavonoid content, DPPH, ABTS, FRAP, CUPRAC,phosphomolybdenum, metal

chelation, AChE, BChE, α-glucosidase, α-amylase and tyrosinase according to

protocols discussed below in Chapter No5.

4.4. Collection, Extraction and Isolation of Metabolites of Berberis

calliobotrys

4.4.1. Collection of Berberis calliobotrys

B.calliobotrys stem part was collected from Ziarat Valley Baluchistan, in

October 2013, and was authenticated by Dr. R. B. Tareen, botanist in the Botany

Department of University of Baluchistan, Quetta, Pakistan.

4.4.2. Extraction and Fractionation

Plant material (15 Kg) was shade dried (for 20 days), chopped and extracted

with methanol (20 L thrice) at room temperature. The extract was concentrated on

rotary evaporator to get dark brown mass of 105 g, which was extracted with ethyl


96

acetate and butanol to get 25g and 20 g fractions leaving behind 40 g of water

fraction. All crude extracts (methanol, ethyl acetate, butanol, water) were tested for

antioxidant activity (DPPH, ABTS, FRAP, CUPRAC, phosphomolybdenum a metal

chelation assays) and enzyme inhibition activity (AChE, BChE, α-glucosidase, α-

amylase and tyrosinase).

4.4.3 Isolation and purification of secondary metabolites

The ethyl acetate soluble fraction (25g) was subjected to silica gel column

chromatography eluting with n-hexane, n-hexane-dichloromethane, dichloromethane,

dichloromethane-methanol and methanol in order of increasing polarity resulted in

further seven fractions (B1-B7). The fractions B1and B2 were not processed as they

contained low polar oily compounds. B3 fraction which was obtained from main

column with n-hexane-dichloromethane at (6:4) ratio was processedfor further

purification by repeated silica gel column chromatography, finally to get compounds

214 (16 mg) and 235 (10 mg) with n-hexane-dichloromethane at polarity (5:5) and

(3:7) respectively. Fraction B4 on repeated CC yielded compound 236 (10 mg) at n-

hexane-dichloromethane (5:5). Three sub fraction B5a-B5c were obtained from B5

fraction, out of these, B5c yielded compound 121 (12 mg) at of n-hexane:

dichloromethane (1:9).Compound 122 (14 mg) was obtained from B6, eluting

withpure dichloromethane, while compound 207 (8 mg) was isolated from sub-

fraction of B6 named as B6a; with dichloromethane and methanol (9.5:0.5) (Scheme

4.1).

Water fraction (40 g) was subjected to vacuum liquid chromatography (VIC),

using reverse phase (RP) silica gel as stationary phase, which resulted in five

fractions, BB1-BB5. Fraction BB1 obtained with methanol: water (1:9), was again

subjected to reverse phase column chromatography (RP-CC) and yielded compound

83 (7 mg) with methanol: water (1.5:8.5). Fraction BB3 obtained with methanol and

water (3:7), on further RP-CC, yielded two compounds 237 (8 mg) and 238 (10 mg)

with methanol: water (3.5:6.5) and (4:6) respectively. Compound 208(10 mg) was

purified from BB4 with methanol: water (5:5) also using RP-CC (Scheme 4.1).


97

Scheme 4. 1Extraction and isolation of secondary metabolites from

Berberis calliobotrys


98

4.5. Spectroscopic data of the isolated compounds

4.5.1. Spectroscopic data of 4-Hydroxybenzoic acid (214):

White Crystalline solid (16mg); IR (KBr) cm-1:3590-2425(OH), 1730 (C=O),

1620-1550 (Ar-C=C); 1H-NMR and 13C-NMR See Table3.7; EIMS: m/z 138 [M+];

HREIMS: m/z138.0320 (138.0317calcd. for C7H6O3).

4.5.2. Spectroscopic data of Methyl p-coumarate (235):

White Crystalline solid (10mg); IR (KBr) cm-1: 3450 (O-H), 1730 (C=O),

1650 (C=C); 1H-NMRand 13C-NMR See Table 3.8; EIMS: m/z 178 [M+]; HREIMS:

m/z 178.0641 (178.0630 calcd. for C30H50O).

4.5.3. Spectroscopic data of Octadecyl-p-cumarate (236):

Crystalline solid (10mg);IR (KBr) cm-1: 3410 (O-H), 3150 (H-C=C), 1610

(C=C), 1585-1514 (aromatic); 1H-NMRand13C-NMR See Table 3.8; EIMS:m/z 430

[M+]; HREIMS: m/z 430.3445 (430.3447 calcd. for C28H46O3).

4.5.4. Spectroscopic data of Corydaldine (121):

White amorphous solid (12 mg); IR (KBr) cm-1: 1660 (amide), 1640-1450

(aromatic); 1H-NMR and 13C-NMR See Table 3.9; EIMS: m/z 207 [M+]; HREIMS:

m/z 207.0979 (207.0985 calcd. for C11H13NO3).

4.5.5. Spectroscopic data of N-methyl Corydaldine (122):

White amorphous solid (14mg); IR (KBr) cm-1: 1665 (amide), 1645-1460

(aromatic); 1H-NMR and 13C-NMR See Table 3.9; EIMS: m/z 221 [M+]; HREIMS:

m/z 221.1043 (221.1052 calcd. for C12H15NO3)

4.5.6. Spectroscopic data of Armepavine (207):

White crystalline solid (10 mg); IR (KBr) cm-1: 3420 (O-H), 3120 (=C-H),

1649-1466 (Ar-C=C); 1H-NMR and 13C-NMR See Table 3.9; EIMS: m/z 313 [M+];

HREIMS: m/z 313.1670 (313.1678 calcd. for C19H23NO3).


99

4.5.7. Spectroscopic data of Berberine (83):

Yellow Crystalline solid (7mg); IR (KBr) cm-1: 1612-1490 (Ph-C=C), 1041 (-

OCH2O-); 1H-NMR and 13C-NMR See Table 3.10; EIMS: m/z 336 [M+]; HREIMS:

m/z 336.1218 (336.1230 calcd. for C20H18NO4).

4.5.8. Spectroscopic data of Columbamine (237):

Yellow powdered (8mg); IR (KBr) cm-1: 3350 (O-H) 1620-1540 (Ph-

C=C);1H-NMR and 13C-NMR See Table 3.10; EIMS: m/z 338 [M+]; HREIMS: m/z

338.1377 (338.1387 calcd. for C20H20NO4).

4.5.9. Spectroscopic data of Syringaresinol (238):

White powdered (10mg); IR (KBr) cm-1: 3340 (O-H) 1620-1530 (C-O-C),

1620-1430 (aromatic), 1H-NMR and 13C-NMR See Table 3.11; EIMS: m/z 418

[M+]; HREIMS: m/z 418.1612 (418.1628 calcd. for C22H26O8).

4.5.10. Spectroscopic data of Acanthoside D (208):

White powder (10mg); IR (KBr) cm-1: 3340 (O-H), 1630-1410 (Ar-C=C),

1390, 1655 (C-O-C); 1H-NMR and 13C-NMR See Table 3.11; EIMS: m/z742 [M+];

HREIMS: m/z742.2678 (742.2684 calcd. for C34H46O18).

4.6. Collection, Extraction and Isolation of Metabolites of Caragana

ambigua

4.6.1. Collection of Caragana ambigua

Caragana ambigua whole plant was collected from Ziarat Valley Baluchistan,

in May 2014, and was authenticated by Dr. R. B. Tareen, Botanist in the Botany

Department of University of Baluchistan, Quetta, Pakistan.

4.6.2. Extraction andFractionation

Plant material (15 Kg dried) was chopped and extracted with methanol (20 L

thrice) at room temperature. Extract was concentrated on rotary evaporator to get dark


100

brown mass (120 g), which was extracted with n-hexane and ethyl acetate to get 39

and 23.9 g fractions respectively. All crude extract (methanol, n-hexane, ethyl acetate,

water) were tested for antioxidant activity (DPPH, ABTS, FRAP, CUPRAC,

phosphomolybdenum a metal chelation assays) and enzyme inhibition activity

(AChE, BChE, α-glucosidase, α-amylase and tyrosinase).

The ethyl acetate fraction (23.9g) was subjected to silica gel column

chromatography and eluted with n-hexane, n-hexane-ethyl acetate, ethyl acetate, ethyl

acetate-methanol and methanol in order of increasing polarity resulting in further

twelve fractions (C1-C12). Fraction C3 was subjected for further purification resulted

in the purification of compound 262 (9mg). C4 was divide into sub-fractions C4a and

C4b on further silica gel column chromatography (CC). Sub fraction C4a on CC, led

to isolation of 263 (12mg) with n-hexane: ethyl acetate (5:5) while compound 264

(15mg) was isolated from C4b with n-hexane: ethyl acetate (4:6).

C5 fraction yielded compound 265 (10mg) with n-hexane: ethyl acetate (5:5).

Compound 222(12mg) was obtained from C8 fraction with n-hexane: ethyl acetate

(3:7). Three sub fractions (C9a-C9c) were obtained from C9 fraction, among which

fraction C9b yielded compound 249 (10mg) with n-hexane: ethyl acetate (2:8). While

fraction C9c yielded compound 125 (10mg) with n-hexane-ethyl acetate (1.5: 8.5).

Fraction C10 on CC gave compound 198 (8mg) eluting with ethyl acetate. While

compound 172 (15mg) was isolated from C11 with ethylacetate: methanol (9.5:0.5).

All this process is summarized in Scheme 4.2.


101

Scheme 4. 2 Extraction and isolation of secondarymetabolites from

Caragana ambigua


102

4.7. Spectroscopic Data of Isolated Compounds

4.7.1. Spectroscopic data of Teraxerol (262):

Crystalline solid (10 mg); IR (KBr) cm-1: 3483 (O-H), 2945 (=C-H), 1655

(C=C); 1H-NMR (400 MHz, CDCl3) and 13C-NMR (CDCl3, 100 MHz): See Table

3.17; EIMS: m/z 426 [M+]; HREIMS: m/z 426.3840 (426.3861 calcd. for C30H50O).

4.7.2. Spectroscopic data of teraxerol Acetate (263):

Crystalline solid (12mg); IR (KBr) cm-1: 1730 (C=O), 1655 (C=C); 1H-NMR

(400 MHz, CDCl3): and 13C-NMR (CDCl3, 100 MHz) See Table 3.17: EIMS: m/z

469 [M+]; HREIMS: m/z 468.3962 (426.3967 calcd. for C32H52O2).

4.7.3. Spectroscopic data of 2′-(4-Hydroxyphenyl)-Ethyl Stearate (264):

White amorphous solid (15mg); IR (KBr) cm-1: 3440 (O-H), 1729 (O-C=O),

1520-1469 (Ar-C=C), 1210 cm-1(C-O); 1H-NMR (500 MHz, CDCl3) and 13C-NMR

(CDCl3, 125 MHz): See Table 3.18 EIMS: m/z 404 [M+]; HREIMS: m/z 404.3286

(404.3290 calcd. for C26H44O3).

4.7.4. Spectroscopic data of Apigenin (265):

Yellow powder solid (12mg); IR (KBr) cm-1: 3302 (O-H), 1520-1610 (Ar-

C=C); 1H-NMR (DMSO-d6, 500 MHz): See Table.3.19; 13C-NMR (DMSO-d6, 125

MHz,): See Table EIMS: m/z 270 [M+]; HREIMS: m/z 270.0534 (270.0528 calcd.

for C15H10O5).

4.7.6. Spectroscopic data of Naringinin (249):

Colorless solid (12mg); IR (KBr) cm-1: 3302-3300) (O-H), 1660 (C=O), Ar-

C=C (1620-1525 cm-1); 1H-NMR (DMSO-d6, 500 MHz): 13C-NMR (DMSO-d6, 125

MHz,): See Table 3.19 EIMS: m/z 272 [M+]; HREIMS: m/z 272.0580 (272.0685

calcd. for C15H12O5).


103

4.7.5. Spectroscopic data of kaempheride (222)

Yellow crystalline solid (12mg); IR (KBr) cm-1: 3410 (O-H), 1700 (C=O),

1620-1520 (Ar-C=C); 1H-NMR (DMSO-d6, 500 MHz): See Table 3.19; 13C-NMR

(DMSO-d6, 125 MHz,): See Table EIMS: m/z 300 [M+]; HREIMS: m/z 300.0634

(300.0620 calcd. for C16H12O6).

4.7.7. Spectroscopic data of Quercetin (125):

Pale yellow crystalline solid (10mg); IR (KBr) cm-1: 3428-3369 (O-H), 1708

(C=O), 1665-1475 (C=C); 1H-NMR (CD3OD, 400 MHz) and13C-NMR (CD3OD 100

MHz) See Table 3.20: EIMS: m/z 302 [M+]; HREIMS: m/z 302.0430 (302.0427


4.7.8. Spectroscopic data of Quercetin 3-O-β-D-glucopyranoside (198):

Pale yellow crystal (8mg); IR (KBr) cm-1: 3340-3200 (O-H), 1720 (C=O),

1617-1459 (Ar-C=C); 1H-NMR (DMSO-d6, 400 MHz): 13C-NMR (DMSO-d6, 100

MHz) See Table 3.20 EIMS: m/z 270 [M+]; HREIMS: m/z 464.0950 (464.0955


4.7.9. Spectroscopic data of β-Sitosterol 3-O-D-glucopyranoside (172):

Pale yellow crystal (15mg); IR (KBr) cm-1: 3451(O-H), 2922-2871 (=C-H),

1655 (C=C); 1H-NMR (CDCl3+CD3OD, 400MHz):.13C-NMR (CDCl3+CD3OD, 100

MHz,): See Table 3.21 EIMS: m/z 576 [M+].

4.8. Collection, Extraction and Isolation of Metabolites of V. stocksii

4.8.1. Collection of V.stocksii

Vincetoxicum stocksii was collected from Ziarat valley in September 2011,

Baluchistan, which and was identified by Prof. Dr. Rasool Bakhsh Tareen,

Department of Botany, University of Baluchistan, Quetta, Pakistan, where a voucher

specimen (RBT-VS-11) has been deposited in the herbarium.


104

4.8.2. Extraction and Fractionation

The plant was dried under shade for 15 days, ground into semi-powder (20 kg)

and was extracted with methanol (18 L) for 5 days (twice). The solvent was

evaporated under vacuum to get a dark brown gummy mass (217 g). The crude

methanolic extract was suspended in water (2 L) and was extracted with n-hexane and

ethyl acetate to get 105 g and 81 g fractions respectively. The water-soluble part was

weighed after drying as 30 g.

The ethyl acetate fraction (81 g) was subjected to column chromatography over

silica gel eluting with n-hexane, n-hexane-ethyl acetate, ethyl acetate, ethyl acetate-

methanol in increasing order of polarity to get ten fractions (V1-V10).The Fraction

V5 (6 g) obtained with n-hexane:ethyl acetate (4:6) was further chromatographed on

silica gel column eluting with n-hexane: ethyl acetate to get 3 sub-fractions VS1-VS3.

The sub-fraction VS2 (3.9 g) which was subjected to repeated silica gel column

chromatography eluting with n-hexane: ethyl acetate (3.9:7.5) to get compound 266

(21 mg). The sub-fraction VS3 (1.5 g), which was also purified on silica gel column

eluting with n-hexane: ethyl acetate (2:8), provided compound 196 (12 mg).

The fraction V3 (2.8 g) obtained from the main column with n-hexane: ethyl

acetate (6:4) was further subjected to silica gel column to give compound 194 (19 mg)

and 192 (17 mg) when eluted with n-hexane:ethyl acetate (5:5). Fraction V6 (1.5 g)

which was eluted with n-hexane: ethyl acetate (3:7) on further purification with silica

gel and isocratic elution with n-hexane:ethyl acetate (3:7) yielded compound 267(6

mg). The main fraction V9 (5.0 g) from the first column eluted with pure ethyl acetate

give two sub-fractions VV1-VV2 on further silica gel column chromatography. The

sub-fraction VV2 when further purified on silica gel column eluting with ethyl

acetate:methanol (9:1) yielded compound 268 (11 mg) and 197 (15 mg) (Scheme

4.3).


105

Scheme 4. 3 Extraction and isolation of secondary metabolites from

Vincetoxicum. Stocksii


106

4.9. Spectroscopic Data of Isolated Compounds

4.9.1.Spectroscopic data of Stocksiloate (266):

White amorphous solid (6 mg); IR (KBr) cm-1: 3505 (O-H), 1737 (C=O),

1545 (Ph-C=C); 1H-NMR (400 MHz, CDCl3) 13C-NMR (CDCl3, 100 MHz) See

Table 3.23.

4.9.2. Spectroscopic data of 4-(4-(methoxycarbonyl)benzyl)phenyl] carbamic acid

(267):

Amorphous solid (21 mg) ;IR (KBr) cm-1: 3385-2470 (COOH), 1730 (O=C-

NH2), 1605-1490 (Ph-C=C); 1H-NMR (600 MHz, DMSO-d6and 13C-NMR ((DMSO-

d6, 150 MHz) See Table 3.24:EIMS: m/z 282 [M-H2O]; HR-FABMS: m/z 301.1189

[M+H]+ (calcd. 301.1188 for C16H17N2O4 corresponding to the formula as

C16H16N2O4).

4.9.3.Spectroscopic data of bis[di-p-phenylmethane]ethyl carbamate (268):

Amorphous solid (19 mg); IR (KBr) cm-1: 1730 (O=C-NH2), 1605-1490 (Ph-

C=C); 1H-NMR (600 MHz, DMSO-d6) and13C-NMR ((DMSO-d6, 150 MHz): See

Table 3.24EIMS: m/z 342; HR-EIMS: m/z 342.1579 (342.1580 calcd. for

C19H22N2O4,).

4.9.4 .Spectroscopic data of 4-Hydroxy-3-Methoxyphenyl 7, 8, 9 Propanetriol

(194):

White amorphous solid (11 mg); IR (KBr) cm-1: 3433 (O-H), 1610-1560 (Ph-

C=C); 1H-NMR (600 MHz, CD3OD,) and 13C-NMR (CD3OD, 125 MHz) See Table

3.25: EIMS: m/z 214; HR-EIMS: m/z 214.0850 (214.0841calcd. for C10H14O5).

4.9.5. Spectroscopic data of Feruloyl-6-O-Dglucopyranoside (197):

White amorphous solid (15 mg); IR (KBr) cm-1: 3442 (O-H), 1710 (C=O),

1655(C=C), 1608-1540 (Ph-C=C); 1H-NMR (600 MHz, CD3OD) and13C-NMR

(CD3OD, 150 MHz) See Table 3.25: EIMS: m/z 356; HR-EIMS: m/z356.1110

(356.1106 calcd. for C16H20O9).


107

4.9.6. Spectroscopic data of Apocynin (196):

White amorphous powder (12 mg); IR (KBr) cm-1: 3450 (O-H),1715 (C=O),

1610-1550 (Ph-C=C); 1H-NMR (500 MHz, CD3OD) and 13C-NMR (CD3OD, 150

MHz)See Table 3.26EIMS: m/z 166; HR-EIMS: m/z166.0650 (166.0630 calcd. for

C9H10O3).

4.9.7. Spectroscopic data of Vincetomine (192):

White amorphous powder (17 mg); IR (KBr) cm-1: 3260 (-NH2), 1606-1570

(Ph-C=C); 1H-NMR (500 MHz, CD3OD,) and13C-NMR (CD3OD, 125 MHz) See

Table 3.27 EIMS: m/z 213; HR-EIMS: m/z213.1140 (213.1154 calcd. for

C14H15NO).

108

CHAPTER 5

Bioassays

Chapter no. 05 Bioassays

109

5.1. Bioassays or Protocol

Different polarity solvent extracts (methanol, hexane, ethyl acetate, and water)

of Berberis calliobotrys and C. ambigua were tested for antioxidant potentials

(DPPH, ABTS, FRAP, CUPRAC, phosphomolybdenum, metal chelation assays)

(Table 3.1, 3.12) and enzyme inhibitory assays (acetyl cholinesterase (AChE),

butyrylcholinesterase (BChE), α-glucosidase, α-amylase and tyrosinase)(Table 3.2,

3.13).

5.2. Bioassays

5.2.3. Total antioxidant capacity evaluation (Phosphomolybdenum method)

Reagent used for evaluation of total antioxidant activity of the extract was

prepared by mixing 0.6 M sulfuric acid, 4 mM ammonium molybedate and 28 mM

sodium phosphate. 3 mL of reagent solution was mixed with 0.3 mL of the sample

and incubated at 95 °C for 90 min. The absorbance were recorded at 695 nm in the

presence of trolox equivalents as standard (Grochowski et al 2017)

5.2.4. DPPH free radical scavenging assay

The stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) was used as the

target species for the measurement of antioxidant activity. 1.0 mL of sample

concentrations was mixed with 4.0 mL of 0.004% methanolic DPPH in 96-well

plates, incubated for 30 minutes at 37 °C and absorbance was recorded at 517nm

(Zengin et al., 2016).

Another method employed for determining anti-oxidant activity is given as

follow;

The stable radical 1, 1-diphenyl-2-picrylhydrazyl l (DPPH) was used for

measurement of antioxidant activity. Different concentrations of sample were

prepared in DMSO. 5 µL of each sample concentrations and 90 µL of 100 µM DPPH

(methanolic) was added in 96-well plates of 100 µL. Mixed and incubated for 30


110

minutes at 37oC. At 517nm the absorbance was recorded. Quercetin (125) was used as

reference compound. Inhibition% and IC50 value was measured (Koleva et al., 2002).

5.2.5. ABTS free radical scavenging assay

First step involved the production of ABTS by mixing ABTS solution (7 mM)

with potassium persulfate (3.85 mM) and allowed to rest at 25 °C in dark for 12-16 h.

ABTS solution was then diluted up to the absorbance of 0.700 ± 0.02 at 734 nm, and

was mixed with sample solution (ABTS: Sample,1:2), and was the mixture was

incubated at 25 °C for 30 min. The absorbance was recorded at 734 nm, while trolox

was used as reference standard (Zengin et al.,2016).

5.2.6. Metal chelating assay

2.0 mL of the sample solution was mixed with 0.05 mL of 2 mM ferrous

chloride solution followed by the addition of 0.2 mL of ferrozine (5 mM) as reaction

initiator. Absorbance was recorded at 562 nm by using disodium edentate (EDTA) as

standard (Zengin et al., 2016).

5.2.7. Cupric ion reducing assay

0.5 mL of the sample solutions were added to reaction mixture of 1.0 mL of

CuCl2 (10 mM), 1.0 mL of neocuproine (7.5 mM), and 1.0 mL of NH4Ac buffer (1.0

M, pH 7.0), incubated at 25 °C for 30 min and the absorbance was recorded at 450

nm, where trolox equivalents were used to express the measurement unit (Marini et

al., 2018).

5.2.8. Ferric reducing antioxidant assay

0.1 mL of the sample solutions were mixed with freshly prepared 2.0 mL

FRAP reagent which was prepared by 300mM of acetate buffer (pH 3.6) with TPTZ

10 mM (2,4,6-tris(2-pyridyl)-S-triazine and 20 mM of ferric chloride with ratio of

10:1:1 (v/v/v). The absorbance was recorded at 593 nm at room temperature after

incubation for 30 min, whereas, trolox was used as standard (Bahadori et al., 2017).


111

5.2.9. Cholinesterase inhibition assay

Ellman’s method is used for determination of Cholinesterase inhibition

(Grochowski et al., 2017).The sample solutions, Ellman’s reagent (DTNB (5,5-dithio-

bis (2-nitrobenzoic) acid, 125 µL) and enzymes (acetylcholinesterase/

butyrylcholinesterase) are mixed in ratio 2:5:1 in the presence of buffer solution of

Tris-HCl with pH 8.0 in 96-wellplates and allowed for 15 min at 25 °C followed by

the addition of 25 µL of acetylthiocholine iodide as the substrates for AChE and

butyrylthiocholine chloride for BChE. Absorbance was measured at 405nm after 10

min in the presence of galatamine as positive control (Lazarova et al., 2015).

5.2.10. α- glucosidase inhibition assay

4-Nitrophenyl β-D-glucopyranoside was used as substrate in α-glucosidase

inhibition assay. 50 µL of the sample solutions were mixed with equal volume of 50

µL of enzyme (in phosphate buffer with pH 6.8) in the presence of 50 µL of PNPG

(10 mM) as substrate in 96-well microplate reader. After 15 min incubation at 37 °C,

The reaction was stopped by adding 50 µL of sodium carbonate. The absorbance was

recorded at 400 nm, using acarbose as positive control (Mocan et al., 2016)

5.2.11. α-Amylase inhibition assay

Caraway-Somogyi iodine/potassium iodide (IKI), commonly available

method, was used for evaluation of anti-α-amylase activity. 25 µL sample solution

was mixed with 50 µL of α-amylase solution (prepared in phosphate buffer, pH 6.9)

in 96-well-microplates and incubated for 10 min at 37 °C followed by the addition

of50 µL of starch solution (0.05%). After 10 min of incubation at 37 °C, 25 µL of 1.0

M HCl solution was used to stop the reaction. The absorbance was measured at 630

nm by adding iodine-potassium iodide with acarbose as positive inhibitor (Lazarova

et al., 2015).

5.2.12. Tyrosinase inhibition assay

In Tyrosinase inhibition measurement, L-DOPA was used as substrate.25 μL

of the extract solutions and 40 μL of tyrosinase (from mushroom, EC 1.14.18.1,

Sigma-Aldrich) solution were mixed in the presence of phosphate buffer (100 μL, pH


112

6.8) in a 96-well microplate. After incubation at room temperature for 15 min the

reaction was initiated by adding 40 μL of L-DOPA. The reaction mixture was further

incubated at room temperature for 10 min; absorbance was measured at 492 nm by

using Kojic acid as reference (Zengin et al., 2016).

5.3.13. Anti-Urease assay

Berthelot assay with slightly modification is used for anti-urease activity. 10

µl of phosphate buffer (pH 7.0), 10 µl of tested sample, 25 µl of enzyme was added in

96-well plates and was incubated for 5 mints at 37ºC.After addition of 40 µl of urea

solution (20mM) incubation for 10 mints was done followed by the addition of 115 µl

of freshly prepared phenol hypochlorite reagent. For color production more

incubation for 10 mints was done. At 625 nm absorbance was recorded. Inhibition%

was calculated. EZ-Fit Enzyme Kinetics Software was used to calculate IC50 values

(Weatherburn, 1967).

List of Abbreviations

TLC Thin layer chromatography

LC liquid chromatography

HR-LC-MS High resolution liquid chromatography mass spectrometry

HSQC Heteronuclear singular quantum coherence

HMBC Heteronuclear multiple bond correlation

MS Mass Spectrometry

MN Molecular Network

DPPH 1, 1-diphenyl-2-picryl hydrazyl

EtOAc Ethyl acetate

MeOH Methanol

rha rhamnopyranoside

glu glucopyranoside

References

113

References

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Publicatoins

130

Publications:

“Rarely occurring natural products isolated from Vincetoxicum stocksii”,Saima

Khan, Mahreen Mukhtar, Muhammad Imran Tousif, Naheed Raiz, Mamona Nazir,

Liaquat Ali, Rasool Bakhsh Tareen and Muhammad Saleem Journal of the Chemical

Society of Pakistan, Vol. No. 41 (4), 695-700 (2019).

Phytochemical profiling, in vitro biological properties and in silico studies

onCaraganaambigua stocks (Fabaceae): A comprehensive approach, Saima

Khan, Mahreen Mukhtar, MamonaNazir, NaheedRaiz, Muhammad Saleem,

Gokhan Zengin, Gazala Fazal, Hammad Saleem, Muhammad Imran Tousiff,

Rasool Baksh Tareen, Hassan H. Abdallahh, Fawzi M. MahomoodallyIndustrial

Crops & Products,131, 117–124, (2019).

“Valorization of the antioxidant, enzyme inhibition and phytochemical

propensities of Berberis calliobotrys Bien. Ex Koehne: A multifunctional

approach to probe for bioactive natural products”. Saima Khan, Mamona Nazir,

Hammad Saleem, NaheedRaiz, Muhammad Saleem, Syed Muhammad

MuneebAnjum, Gokhan Zengin, Mahreen Mukhtar, Muhammad Imran Tousif,

Fawzi M. Mahomoodally, Nafees Ahemad, Industrial Crops & Products, Vol.141,

Article 111693, (2019).

“Isolation and Enzyme Inhibitory Studies of Steroids and Alkaloidal Steroid Saponins

from Solanum surattense Burm.f.”Mahreen Mukhtar, Saima Khan MamonaNazir,

Naheed Riaz, Muhammad Imran Tousif, Muhammad Ashraf, Muhammad Shaiq

Ali, Ishtiaq Ahmad, Abdul Jabbar and Muhammad Saleem. Journal of Chemical

Society of Pakistan ·Vol. 40, No. 03, (2018).

the fulfillment of degree of doctor of philosophy

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