institute of chemical sciences university of …

STUDIES ON THE CHEMICAL CONSTITUENTS OF HEDERA

NEPALENSIS K. KOCH AND CORNUS MACROPHYLLA WALL. EX

ROXB

Ph.D. Thesis

By

ASHFAQ AHMAD KHAN

INSTITUTE OF CHEMICAL SCIENCES

UNIVERSITY OF PESHAWAR,

PESHAWAR, PAKISTAN

FEBRUARY, 2014

STUDIES ON THE CHEMICAL CONSTITUENTS OF HEDERA

NEPALENSIS K. KOCH AND CORNUS MACROPHYLLA WALL. EX

ROXB

By

ASHFAQ AHMAD KHAN

A dissertation

Submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Chemistry

INSTITUTE OF CHEMICAL SCIENCES

UNIVERSITY OF PESHAWAR,

PESHAWAR, PAKISTAN

FEBRUARY, 2014

“Start from the name of ALLAH, the most beneficent, the mostmerciful”

DEDICATED TO

MY

PARENTS AND FAMILY MEMBERS

CONTENTS

Acknowledgments……………………………………………………………………………i

Abstract………………………………………………………………………………............iii

List of the Abbreviations……………………………………………………………………..v

List of the Tables………………………………………………………………………..........vii

List of the Figures…………………………………………………………………………….ix

List of the Schemes……………………………………………………………………...........x

Chapter 1 Part A

1.0. General Introduction……………………………………………………………...1

Chapter 2 Plant Introduction…………………….

2.1. Plant Introduction …………………………………………………………………..........7

2.1.1. Genus Hedera…………………………………………………………….............7

2.1.2. Hedera nepalensis K. Koch………………………………………………………7

2.1.3. Chemical constituents of the genus Hedera…………………………………………...8

2.1.4. Structures of selected compounds reported from the genus Hedera.....................12

2.1.5. Medicinal and pharmacological properties of the genus Hedera…………….........20

Chapter 3 Results and Discussion …………………

3.1. Present work..................................................................................................................22

3.2. New compound isolated from H. nepalensis................................................................22

3.3. Hitherto unreported compounds from H. nepalensis.....................................................23

3.2.1 Hepalensiside A (1) .........................................................................................25

3.3.1. Colchiside A (2)………………………………………………………………27

3.3.2. Pastuchoside A (3)……………........………………………………………….28

3.3.3. Helixoside A (4)……………………………………………………………….29

3.3.4. Kizuta saponin K12 (5)……………………………………………….……….30

3.4. Biological studies……………………………………………………………………..30

3.4.1. Anti-bacterial assay………………………………………………………….30

3.4.2. Anti-fungal assay……………………………………………………………31

3.4.3. Phytotoxicity assay………………………………………………………….31

3.4.4. Insecticidal assay……………………………………………………………31

3.4.5. Brine shrimp (Artemia salina) lethality bioassay…………………………..32

Chapter 4 Experimental………………………….

4.1. General experimental……………………………………………………………….33

4.2. Plant Material……………………………………………………………………….33

4.3. Extraction and isolation…………………………………………………………….34

4.4. Characterization of chemical constituents…………………………………………36

Chapter 5 Plant Introduction…………… Part B

5.1. Plant Introduction ………………………….............................................................61

5.1.1. Genus Cornus…………………………....................................................................61

5.1.2. Cornus macrophylla Wall…………….....................................................................61

5.1.3.Chemical constituents of the genus Cornus ………………………….........................62

5.1.4. Structures of some selected compounds reported from the genus Cornus...............67

5.1.5. Medicinal and pharmacological properties of the genus Cornus…………………..74

Chapter 6 Results and Discussion ………………………….

6.1. Present work……………………………………………………………………..….77

6.2. New compound isolated from Cornus macrophylla………………………………..77

6.2.1 Macrophyllanin A (6)……………………………………………………...82

6.2.2 Macrophyllanin B (7)……………………………………………………....84

6.2.3 Macrophyllanin C (8)………………………………………………………86

6.2.4 Macrophyllanin D (9)……………………………………………………....88

6.3. Hitherto unreported compounds from Cornus macrophylla…………………….....79

6.3.1. Kaempferol (10) ……………………………….............................................90

6.3.2. Taraxasterol (11) ………………………………............................................90

6.3.3. 3β-Hydroxy-18α-olean-28-19β-olide (12)…………………………………91

6.4. Reported compounds from Cornus macrophylla…………………………………...80

6.4.1. Betulinic acid (13) ………………………………..........................................92

6.4.2. Betulin (14) ………………………………....................................................93

6.4.3. Stigmasterol (15) ………………………………............................................94

6.4.4. Lupeol (16) ……………………………….....................................................95

6.4.5. Oleanolic acid (17) ……………………………….........................................95

6.5. Biological studies ………………………………........................................................96

6.5.1 Anti-bacterial assay ………………………………............................................96

6.5.2 Anti-fungal assay.………………………………................................................96

6.5.3 Phytotoxicity assay .………………………………............................................97

6.5.4 Insecticidal assay………………………….......................................................97

6.5.5. Brine shrimp (Artemia salina) lethality bioassay.…………………………...97

Chapter 7 Experimental………………………….

7.1. General experimental…………………………………………………………….....101

7.2. Plant Material…………………………………………………………………….....101

7.3. Extraction and isolation ……………………………………………………………101

7.3.1. Fraction of ethyl acetate phase ……………………………………………....101

7.4. Characterization of chemical constituents………………………………………......105

References ………………………………………………………………………………….132

List of Publication …………………………………………………………………………140

i

Acknowledgments

At first I want to express my unfeigned thanks and praise to Almighty Allah, Who in his great

mercy and benevolences has enabled me to carry out and complete this Ph.D. research work.

I would like to express my special appreciation and thanks to my advisor Professor Dr. Ghias

Uddin, you have been a tremendous mentor for me. I would like to thank you for encouraging

my research and for allowing me to grow as a research scientist. Your advice on both research

as well as on my career have been priceless.

My special gratitude and thanks to my co-supervisor Prof. Dr. Bina Shaheen Siddiqui (HEJ

Research institute of Chemistry, University of Karachi) for her guidance.

I would like to give special thanks to my supervisors Dr. Valerie. A. Ferro and Prof Dr.

Alexander Irvine Gray (SIPBS, University of Strathclyde, Glasgow UK.) for their unflagging

support, guidance and provided me a unique opportunity to work in their research center.

I am particularly indebted to Prof. Mingkui Wang (Chengdu Institute of Biology, Chinese

Academy of Sciences.) who guided me during the course of my Ph.D. His everlasting

encouragements, supervision and generous help facilitate my research work.

I feel great pleasure in expressing my special thanks to Prof. Dr. Yousaf Iqbal (Director,

Institute of Chemical Sciences, University of Peshawar) and all the faculty members of ICS

for their support and co-operation.

My special gratitude and thanks to Prof. Dr. Abdur Rashid (Ex-chairman Botany Department,

University of Peshawar) and Abdul Majid (Lecturer Department of Botany, Hazara

University, Mansehra) for the identification of my plants species on such a short notice.

ii

I want to sincerely acknowledge all my friends and colleagues, Dr. Waliullah, Dr. Alauddin,

Dr. Abdul Latif, Dr. Hamid Hussain, Dr. Muhammad Alamzeb, Anwar Sadat, Muhammad

Alam, Saqib Ali, Mamoon-Ur-Rashid and Abdur Rauf for their encouragement, support and

help all the time.

In the last but not least, I am truly indebted to my parents and family members whose prayers

and support have enabled me to complete the Ph.D. research work. I would like to thank my

parents for allowing me to realize my own potential. All the support they have provided me

over the years was the greatest gift.

I am also greatful to the Higher Education Commission of Pakistan for research funds.

Ashfaq Ahmad Khan

iii

Abstract

The research work carried out for the doctoral dissertation is mainly focused on

bioassayguided isolation, characterization and structure determination of pure chemical

constituents of H. nepalensis K. Koch (Part-A) and C. macrophylla Wall (Part-B).

The structures of the pure chemical constituents were elucidated by advanced spectroscopic

methods.

Part A

Chemical explorations of H. nepalensis were described in part A; resulted in the isolation of

one new triterpenoid saponin; hepalensiside A (1) and four hitherto unreported triterpenoid

saponins; colchiside A (2), pastuchoside A (3), helixoside A (4) and kizuta saponin K12 (5)

from the leaves of H. nepalensis. Various fractions and extract of H. nepalensis were also

evaluated for different bioassays. As a result semi-purified fractions exhibited significant anti-

bacterial activities against P. mirabilis at conc. of 12 μg/ml, against B. cereus 13 and E. coli

11 μg/ml, while the methanolic extract of the H. nepalensis was found to be cytotoxic.

iv

Part B

Part B described the phytochemical studies of C. macrophylla, affording four new

chemical constituents; macrophyllanin A (6), macrophyllanin B (7), macrophyllanin C

(8) and acrophyllanin D (9), together with three hitherto unreported compounds;

kaempferol (10), taraxasterol (11) 3β- hydroxy-18α-olean-28-19β-olide (12) and five

known compounds; betulinic acid (13), betulin (14), stigmasterol (15), lupeol (16) and

oleanolic acid (17). Semi-purified fractions were also evaluated for biological assays, as

a result some fractions were found to be active against B. cereus and Lemna minor (plant)

and were also showed cytotoxic effect.

6 7

.

8 9

v

List of Abbreviations

13C- NMR Carbon-13 Nuclear Magnetic Resonance Spectroscopy

COSY Correlation Spectroscopy

DEPT Distortion less Enhancement by Polarization Transfer

1D NMR One Dimensional Nuclear Magnetic Resonance Spectroscopy

2D NMR Two Dimensional Nuclear Magnetic Resonance Spectroscopy

ESI-MS Electrospray Ionization Mass Spectroscopy

EIMS Electron Mass Impact Spectroscopy

ESI Electrospray Ionization

FAB-MS Fast Atomic Bombardment Mass Spectrometry

Hz Hertz

HMBC Heteronuclear Multiple Bond Correlation

HMQC Heteronuclear Multiple Quantum Coherence

1H-NMR Proton Nuclear Magnetic Resonance Spectroscopy

HR-ESI-MS High Resolution Electrospray Ionization Mass Spectroscopy

HSQC Heteronuclear Single Quantum Coherence

IR Infrared spectroscopy

J Coupling Constant

MS Mass Spectroscopy

MIC Minimum Inhibitory Concentration

m/z Mass to Charge ratio

mp Melting Point

NOESY Nuclear Overhauser Effect Spectroscopy

vi

NMR Nuclear Magnetic Resonance

ppm Parts Per Million

st Stretching

UV-Vis Ultraviolet-Visible Spectroscopy

ῡ Wave number

vii

List of Tables

Table. 2.1. Chemical constituents of the genus Hedera…………………………... 9

Table. 4.1. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 1 in C5D5N…… 37


Table. 4.3. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 3 in C5D5N……... 43

Table. 4.4. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 3 for sugar

moieties at position C-28 ……………………………………………… 45



moieties at position C-28 ……………………………………………... 50



moieties of 5 at position C-28 ……………………………………….. 54

Table. 4.9. Anti-bacterial activity of H. nepalensis………………………………….. 55

Table. 4.10. Anti-fungal profile of H. nepalensis……………………………………... 56

Table. 4.11 Phytotoxicity assay of H. nepalensis……………………………………... 57

Table. 4.12. Insecticidal assay H. nepalensis………………………………….... 59

Table.4.13. Brine Shrimp lethality bioassay of H. nepalensis…………………… 60

Table. 5.1. Chemical constituents of the Genus Cornus……………………………… 64

Table. 7.1. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 6 in CDCl3…….. 105

viii

Table. 7.2. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 7 in CDCl3………. 107

Table. 7.3. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 8 in CDCl3…… 109

Table. 7.4. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 9 in CDCl3……. 111

Table. 7.5. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 10 in CDCl3……... 113



Table. 7.8. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 13 in CDCl3……. 119





Table. 7.13. Anti-bacterial activity of C. macrophylla……………………………….. 128

Table. 7.14. Anti-fungal assay activity of C. macrophylla………………………... 128

Table. 7.15. Phytotoxicity assay of C. macrophylla ………………………………… 129

Table. 7.16. Insecticidal activity of C. macrophylla………………………………….. 130

Table. 7.17. Brine Shrimp activity of C. macrophylla……………………………….. 131

ix

List of Figures

Figure. 3.1. Important HMBC (H→C) correlations of compound 1………….. 27

Figure. 6.1. Key HMBC correlations of 6…………………………………….. 83




x

List of Schemes

Scheme 4.1. Extraction and isolation from H. nepalensis K. Koch……………. 37

Scheme 7.1. Extraction and isolation from C. macrophylla stem……………… 105

Chapter 1

General Introduction

Chapter 1 General introduction

1

1.0. General introduction

Human being is associated with the therapeutic plants from the beginning of the

human civilization. Plants are used as a source of drugs in all most every part of the world.

Long life, everlasting beauty and perfect health has quest for human being. Man enabled to

distinguish facts from imaginary tales through such ingenious expeditions based on his

dreams and on his practical experiences. It moved to the new group as folk medicines and as

the time passed developed into a field of modern medical science. The hard work of human

being describes the history of medication from the primal endeavors of ancient human being

to the modern advanced age of medical sciences and further be divided into the subsequent

periods. First period expanded over Chinese, Indian, Egyptian, Assyrian, Sumerian and

Babylonian civilizations followed by Arabic, Persian, Greek, Roman and at the end the

modern era. The Greeks originated the modern medicines. The knowledge of medicines

from the Greece was gradually shifted to Romans and then to Arabs. After the advancement

in the field of medical science with the Indian and Chinese medicines, it was transformed to

the current Europe. It was commenced by the Muslim rulers in India and combing together

with the indigenous Ayurvedic medicine, which is called as Eastern or Unani medicine.1, 2

The earliest ideas about the use of medicinal plants were recorded by Rigveda (4500-1600

BC) and Ayurveda (2500-600 BC). Numerous groups of herbs in several sets was searched

and explained by Chopra and his research groups. The use of medicinal plants during

Buddhist period was enhanced and substantial attention was given in a scientific mode for

cultivation of such plants.3


2

Among Chinese Shen Nung accumulated a Pharmacopoeia around 2735 BC on

medicinal plants called as Pen Tsao which consisted of forty volumes and many

therapeutic preparations and literature record of medicinal values of some drugs like Ma

Huang and Chang Sheng extracted from Ephedra sinica and Dichroa febrifuga plants.

Another great work compiled by Li Shizhen in the form of Ben Cao Gang Mu is serving as

a reference book for practicing, teaching and supervision in Chinese therapeutic research.

However majority of the literature concerning Chinese medicine originated from Nei

Ching.4, 5

Numerous medicinal plants have been introduced by Egyptian for the treatment of

human diseases in Ebers Papyrus about 1550 BC. The Assyrian and Babylonian fields of

medicine developed at about 650 BC. In 460 BC Hippocrates, the developer of medicines

put forward the foundation of pharmacy. They illustrated and named about 400 samples

having medicinal valve.

Among Romans, Galen the developer of Roman pharmacology wrote about thirty

books on pharmacology and investigated preparations known as galenicals from many

medicinal plants.

The Muslims during the early and late middle ages made great contribution to the

field of medical science. During the excellent era of Muslims superpower, Arabs took great

interest in the field of herbal medicines and they translated the work to his own language

from almost all the famous civilizations. Abu Bakr Mohammad Bin Zakaria Al-Razi

(Rhazes) the renowned Arab scholar wrote about 250 phenomenal books. Most part of his

work on medicines was illustrated in Alhavi Kabeer (Continens of Rhazes). Kitab-al-

Mansoori is one of his evident books in which he briefly discussed


3

the Greek-Arab system of medication. For the first time in the history Rhazes described

opium as anesthetic agent. Ali Ibn-e-Rabban Tabari wrote a book ‘Firdous Al-Hikmat’

which consists of seven volumes during the period 833-870 AD. The most famous

philosopher, physician, mathematician and astronomer of the Muslims era Bu-Ali Ibn-e-Sina

(908-1037 AD) known as Avicenna in Europe has written 760 herbal drugs in his book

entitled ‘Qanun fi Al-Tibb’ (The Cannon of Medicine) which was an authentic book of

medicines up to 17th century AD. Al-Idrisi another well-known scientist of the Muslim era

(1099-1166 AD) has done a lot of work in the field of medicinal plants therapy and has

written Kitab Al-Jami-ul-sifat Ashat Al-Nabatat.2 F.W. Serturner (1783-1810) in the first

decade of 19th century, isolated morphine from the dried leaf of Papaver sominiferum L

(opium) that opened a new authentic way for the search of valuable herbal drugs from the

natural resources. Pelletier and Cavantou later on isolated cocaine, nicotine, papaverine,

quinine, strychnine from the plants. In 1870 Hoffmann for the first time identified the

structure of coniine and after that it was synthesized by Ladenburg in 1886. Due to their

physiological importance and complicated chemistry these compounds are considered to be

the first of the real pure natural chemical constituents. Several important findings in the field

of medicine can be attributed to the isolation of potent compounds from the natural

sources.6 Different biologically active compounds are present in plants like, glycosides

(saponin glycoside, cardioactive glycoside, anthraquinone glycosides), volatile oils

(peppermint, clove, cardamom oil), alkaloids (morphine, cocaine, atropine), resins, gums

and mucilages. Several important findings in the field of medicine can be accredited to the

isolation of potent compounds from natural sources.7 Atropine, morphine, digoxin, reserpine

and quinine, are some of the examples of plants drugs still used in the modern day


4

therapeutics. Although majority of the plants have been studied for their medicinal

properties but still the numbers of plants that have not been investigated for their

biologically active components is very long.8 It has been reported that the herbal drugs are

less toxic and free from the more side effects than the synthetic drugs so for the industrial

development there is a great potential for the traditional herbal remedies plants extracts for

the treatment and prevention of disease.9 Even though plants have played significant role

from the early ages of many human diseases as basis for the treatment, one wonders that

only two percent plant species have been subjected to pharmaco-chemical investigations. In

one of the reports of WHO, it is recorded that due the convenient availability and socio-

cultural environment of the conventional medicines, more than half of the world population

is still dependent on these types of medicines.10-17 Synthetic drugs such as salvarasan by

Chakravarthi 18 and the use of the chemotherapy in the early decades of 20th century greatly

decreased the value of herbal drugs and increased the concern of people towards synthetic

drugs. Isolation of antibiotics from the plants and the importance of some of the constituents

of medicinal plants for the treatment against cardiovascular diseases and many types of

cancer, which has been an extensive recovery of attention in the field of natural products.

Although all herbal medicines are not useful as claimed, even though they have provided us

massive field for development and investigation. It is the medicinal agents isolated from the

plants which are the starting materials of many active synthetic and semi synthetic drugs.

The task which needs to be accepted is to validate the clinical values of prescribed herbal

medicines. This challenge requires great determination, persistence and scientific

knowledge. In all over the world, the interest of people developed in the chemical and


5

pharmacological valuation of medicinal plants is growing particularly in tropical and sub-

tropical regions.

The work carried out for doctoral dissertation in view of the facts discussed consists

of isolation, structural determination of chemical constituents and studies on the biological

activities of H. nepalensis K. Koch (Part-A) and C. macrophylla Wall (Part-B).

Chapter 2

Plant Introduction

Chapter 2 Plant introduction (Part-A)

6

Hedera nepalensis K. Koch


7

2.1. Plant Introduction

Plant description

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Apiales

Family: Araliaceae

Genus: Hedera

Specie: H. nepalensis

Binomial name: Hedera nepalensis K. Koch

2.1.1. Genus Hedera

Genus Hedera belongs to the family Araliaceae consists of 70 genera and 700 species

mainly habitating in Europe, North Africa and Asia. Two species H. helix and H. nepalensis

are present in Pakistan. The H. nepalensis is locally known as Arbambal.19

2.1.2. Hedera nepalensis K. Koch

H. nepalensis is a species of the genus Hedera at altitude of about 1000-3000 m.

Plants grow up to 30 m in height, with simple leaves ranging from 2-15 cm long and yellow

flowers. Stem is creeping or climbing to a height of 30 m with adventitious roots. Flower

stalks (length 7-12 mm) and flowering hairy. Petals are yellow crown, stamens are 5 in

number, anthers are 1-2 mm long and pillar is short neck. Fruit is a drupe, flattened, 5-7 mm


8

long, 5-10 mm wide, with orange to red. The plant blooms from October to April. It occurs

mostly in moist soil in shade, at the height of 1000-3000 m and extensively climbs on walls,

rocks, tree trunks by its aerial roots used as a ground cover or decorative climber in gardens

and parks.20

2.1.3. Chemical constituents of the genus Hedera

In 1974 Mahran and his research group reported that the genus Hedera mainly consists

of α-hederin, hederasaponin B and hederasaponin C.21 In 1975 the same group isolated an

alkaloid named emetine from the methanolic extract of H. helix in Egypt.22 Mineo Shimizu

and his research group in 1978 isolated four saponins saponin K3, saponin K6, saponin K10

and saponin K12 from H. rhombea.23 Kizu, H. and his co-workers in 1985 isolated and

characterized four triterpene glycosides named Kizuta saponin K4, K5, K7 and K7C from the

stem bark of the Hedera rhombea.24 In 1985, Kizu, H. and his research group isolated

twelve saponins from the stem bark of H. nepalensis.25 Chandel, R. and Rostogi, R. in 1989,

isolated two saponins from H. nepalensis.26 In the same year Rank Gafner et.al., isolated a

diacetylene, 11 dehydrofaicarinol while Elias, R. and his co-workers in 1991 isolated four

triterpenoids saponins from the leaves of H. helix.27,28 In 1991 Lars, P. C. et.al., isolated

polyacetylenicepoxide from the fruits of H. helix.29 Kwon, B. and his research group in

1997 isolated a rhombenone; dammarane analogue containing 25-oxo from the leaves of H.

rhombea.30 In 2000 Bedir, E. and his research group isolated six triterpene saponins from the

fruits of H. helix.31 Mshvildadze, V. and his research group in 2001 isolated ten triterpenoid

saponins from the berries of H. colchica.32 Subsequently Mshvildadze, V. et.al., in 2005

reported arjunolic acid derivative glycoside from the stems of H. colchica.33 Yamazoe, S. in


9

2007 isolated two polyacetylenes from the extract of Japanese ivy (H. rhombea) flower

buds.34 Sieben, A. and his research group in 2009 isolated three saponins from the leaves

extract of ivy (H. helix).35 The literature survey of the genus Hedera regarding

phytochemistry is summarized in the Table: 2.1.

Table 2.1: Chemical constituents of the genus Hedera

S.

No.

Compounds Species References

1 α-Hederin H. helix 21

2 Hederasaponin B H. helix 21

3 Hederasaponin C H. helix 21

4 Emetine H. helix 22

5 Saponin K3 H. rhombea 23







12 Saponin K7c H. rhombea 24

13 Saponin A H. nepalensis 25

14 Saponin B H. nepalensis 25

15 Saponin D1 H. nepalensis 25


10

16 Saponin D2 H. nepalensis 25

17 Saponin E H. nepalensis 25

18 Saponin F H. nepalensis 25

19 Saponin H H. nepalensis 25

20 Saponin I H. nepalensis 25

21 Saponin K H. nepalensis 25

22 Saponin M H. nepalensis 25

23 Saponin N H. nepalensis 25

24 Saponin P H. nepalensis 25

25 Nepalin-1 H. nepalensis 26

26 Nepalin-2 H. nepalensis 26

27 Diacetylene, 11 dehydrofaicarinol H. helix 27

28 Hederasaponin E H. helix 28

29 Hederasaponin F H. helix 28

30 Hederasaponin H H. helix 28

31 Hederasaponin I H. helix 28

32 Polyacetylenicepoxide H. helix 29

33 Rhombenone H. rhombea 30

34 3-O-β-D-Glu-copyranosyl hederagenin H. helix 31

35 3-O-β-D-Glucopyranosyl-(1→2)-β-D-glucopyranosyl

oleanolic acid

H. helix 31

36 3-O-β-D-Glucopyranosyl-(1→2)-β-D-glucopyranosyl

hederagenin

H. helix 31


11

37 3-O-β-D-Glucopyranosyl hederagenin-28-O-β-D

-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester

H. helix 31

38 Helixosides A H. helix 31

39 Helixosides B H. helix 31

40 Saponin 1 H. colchica 32

41 Colchisides A H. colchica 32



44 Heteroside E2 H. colchica 32

45 Staunoside A H. colchica 32

46 Heteroside I H. colchica 32

47 Scheffleraside II H. colchica 32

48 Hederasaponin D H. colchica 32

49 Colchisides B H. colchica 32

50 Arjunolic acid derivative glycoside H. colchica 33

51 Polyacetylenes H. rhombea 34

52 (Z)-9,10-Epoxy-1-heptadecene-diyn-3-one H. helix 35

53 Falcarinone H. helix 35

54 Falcarinol H. helix 35


12

2.1.4 Structures of selected compounds reported from the genus

Hedera

α-Hederin

Hedersaponin B


13

Hedersaponin C

Hedersaponin D

Emetine


14

Saponin K3

OO

OH

O

CH2OH

COOH

OH

O

HO OH

O OH

Saponin K6


15

OO

OR

OR

OH

CH2OH

CO

O

OOO

CH2OR

OR

RO

OR

OR

OOOR

OR OR

Me

Saponin K10

Saponin K4


16

Saponin K5

Saponin K7

Saponin K7c


17

3-O-β-D-Glu-copyranosyl hederagenin

3-O-β-D-Glucopyranosyl-(1→2)-β-D-glucopyranosyl oleanolic acid


18

3-O-β-D-Glucopyranosyl-(1 → 2)-β-D-glucopyranosyl hederagenin

3-O-β-D-Glucopyranosyl hederagenin-28-O-β-D-glucopyranosyl-(1 → 6)-β-D-

glucopyranosyl ester

Helixosides A


19

Helixosides B

Colchiside A

Polyacetylene


20

2.1.5 Medicinal and pharmacological properties of the genus Hedera

The genus Hedera is known for its economical importance. Acute and chronic anti-

inflammatory profile of H. helix, in rats was reported by Suleyman and his group in 2003.36

The aqueous and methanolic extracts of H. helix were found to reduce blood glucose level in

rabbits to a significant level.37 The plant is traditionally used in folk medicine. It has been

reported that the leaves and berries of H. nepalensis are stimulating, diaphoretic, cathartic

and also used to treat indolent ulcers and abscesses.38 Qureshi and his research group also

reported that the decoction of the leaves is very effective against lice.39 Timen, D. et.al.,

reported the antifungal activity of H. helix extract.40 Agarwal and Rostogi investigated

antimitotic activity of glycosides isolated from the genus Hedera.41 Antileishmanial activity

of the saponins isolated from the H. helix were reported by Majester Savornin and his co-

workers.42 Cioaca et.al., reported the antibacterial activity of the saponins of the H. helix

which are more active against Gram positive bacteria as compared to Gram negative

bacteria.43 Quetin and his researcher investigated cytotoxicity both in vitro and in vivo on

Ehrlich tumor cells of the crude extract of H. helix.44 H. nepalensis leaves are traditionally

used for diabetes treatment.45 Hamayun et.al., in 2006 documented that the leaves of H.

nepalensis are used for the cancer treatment.46 Shah et.al., in 2006 reported that the plants

has hypoglycemic properties and is very effective against rheumatism, fever and pulmonary

infections.47 Inayatullah and his co-workers in 2007 screened the methanolic crude extract

of aerial part of the plant and tested for different bioassays such as cytotoxic, phytotoxic

activity, potato disc antitumor activity and brine shrimp.48 It has been considered one of the

most useful plant containing saponins for the treatment of cough and human ailments.49 The

literature survey showed that saponins from the leaves of H. helix have spasmolytic,


21

antifungal, anthelmintic, molluscicidal, antileishmanial and antimutagenic activities.50 The

fresh leaves and fruits of the H. helix are toxic and cause gastrointestinal irritation,

dermatitis and bloody diarrhea.51

Chapter 3

Results & Discussion

Chapter 3 Results & discussions, (Part A)

22

3.1. Present work

Due to medicinal and biological importance assigned to genus Hedera, chemical

explorations of H. nepalensis were carried out in current studies which resulted in the

isolation and structure determination of one new and four hitherto unreported compounds

from H. nepalensis. Structure of the new compound was identified by advanced

spectroscopic methods including UV, IR, MS, 1D (1H- and 13C-NMR; BB and DEPT) and

2D NMR (J- resolved, COSY-45o, NOESY, HSQC, HMBC) experiments. The known

compounds were determined by comparison their spectral data (1D and 2D NMR) with the

reported compounds in the literature.

3.2. New compound isolated from H. nepalensis

Hepalensiside A (1)

CH2OH

O

O

O

O

O

COOH

HO

HO

HOH

H3CHO

HOHO OH

O


23

3.3. Hitherto unreported compounds from H. nepalensis

Colchiside A (2)

Pastuchoside A (3)


24

Helixoside A (4)

Kizuta saponin K12 (5)

OCH2OH

OOH

HOO

O

HOHO

OH

O

O

O

O

OHO

OHHO

O

OHHO

OH

H3C

O

OHHO

OH

H3C


25

3.2.1. Hepalensiside A (1)

Compound 1 was obtained as a white needle crystal with melting point 235-236

°C. Its molecular formula was determined to be C46H72O16 from the pseudo molecular ion

peak at m/z 903.3012 (calcd. for C46H72O16 880.2313) in HRESI-MS together with the 1H-

and 13C-NMR data.

The 13C-NMR spectrum of compound 1 displayed 46 carbon signals of which 30 could

be attributed to aglycone moeity. Spectroscopic analysis including proton, 13C-NMR (Table:

4.1 vide experimental) and 2D NMR experiments showed that compound 1 is a sapogenin

triterpenoid. The 1H-NMR spectrum showed six tertiary methyl groups at δH 0.86 (3H, s, H-

30), 0.93 (3H, s, H-29), 0.99 (3H, s, H-25), 1.03 (3H, s, H-27), 1.07 (3H, s, H-26) and δH 1.09

(3H, s, H-24) and the corresponding 13C-NMR signals at δC 24.3 (C-30), 32.3 (C-29), 18.8 (C-

25), 20.1 (C-27), 17.2 (C-26), and δC 13.5 (C-24). Two olefinic protons were observed at δH

6.63 (1H, dd, J =10.5, 4.5 Hz, H-11), 5.73 (1H, d, J =10.5 Hz, H-12) while the olefinic

carbons were present at δC 125.9 (C-11), 127.1 (C-12), 133.2 (C-13), and δC 136.6 (C-18). A

pair of hydroxy methylene protons appeared at δH 3.91 and 4.32 connected to a carbon at δC


26

63.9 (C-23) in HSQC spectrum while a carboxylic carbon appeared at δC 178.9 (C-28) which

is a characteristic peak in hederagenin type of skeleton. The sugar moieties were identified as

L-arabinose, L-rhamnose and D-ribose in a ratio of 1:1:1 by acidic hydrolysis followed by GC

analysis and comparison the corresponding aldononitrile peracetates with the authentic

samples prepared in the manner described in the literature.52 The assignments of the NMR

signals associated with the aglycone moiety were made from HSQC, HMBC, 1H–1H COSY,

and NOESY experiments. The data revealed that the 1 has common hederagenin aglycone

skeleton. The two double bonds located at position 11 and position 13 was confirmed by the

HMBC spectrum (Figure: 3.1). A downfield shift of carbon C-3 was resonating at δC 81.2

indicated that oligosaccharide moiety attached at position C-3. The HMBC spectrum

confirmed the interglycosidic connectivities of 1 through correlations of anomeric proton at

δH 5.05 (H-1') of arabinose and C-3 (δC 81.2) of aglycon, the anomeric proton of rhamnose at

δH 6.32 (H-1'') with C-2' of arabinose (δC 75.3), similarly the anomeric proton of ribose at δH

5.96 (H-1''') showed connectivity with C-3'' (δC 81.2) of rhamnose. In view of the above

evidences the structure of 1 was established as 3β-O-[β-D-ribopyranosyl-(1→3)-α-L-

rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl] hederagenin-11,13-dien-28-oic acid.


27

Figure: 3.1 Important HMBC (H→C) correlations of compound 1

3.3.1. Colchiside A (2)

Compound 2 was obtained as white powder from the butanolic fraction. The HRESI-

MS indicated the molecular ion peak at m/z 604. 1171 a.m.u representing the molecular

formula C35H56O8 (calcd. for C35H56O8 604.1230).The NMR data (Table: 4.2 vide

experimental) showed that one anomeric carbon resonated at δC 106.3 and anomeric proton

resonated at δH 5.12. The carbon C-3 resonated at δC 83.4 and C-28 at δC 182.6 confirmed that

the sugar chain attached to C-3. The structure was elucidated as 3-O-(β -D-xylopyranosyl)-


28

hederagenin. The spectroscopic data (1H- and 13C-NMR) was in accordance with the data

reported for Colchiside A in the literature.53

3.3.2. Pastuchoside A (3)

Pastuchoside 3 was isolated as white powder. The molecular formula C53H86O21 was

determined on the basis HRESI-MS m/z 1528.7190 a.m.u (calcd. for C71H116O35 1528.7186).

The NMR data (Table: 4.3, 4.4 vide experimental) and acid hydrolysis of 3 yielded glucose,

arabinose and rhamnose as sugars and the skeleton was hederagenin. Therefore, the structure

of pastuchoside A (3) was determined as 3β -O-{α -L-rhamnopyranosyl-(1→2)-[α-

Lrhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)]-α-Larabinopyranosyl}-28-O-[α-L-

rhamnopyranosyl-(1→4)-β-Dglucopyranosyl-(1→6)-β-D-glucopyranosyl]-hederagenin. The

spectral data (1H- and 13C-NMR) was identical with the data reported for Pastuchoside A (3)

in the literature.54


29

3.3.3. Helixoside A (4)

Compound 4 was afforded from the butanolic fraction as a colourless amorphous

powder. The HRESI-MS indicated molecular ion peak at m/z 1143.0221 [M+Na]+

corresponding to the molecular formula C54H88O24 (calcd. for C54H88O24 1143.0217). The IR

spectrum for 4 showed an absorption bands for hydroxyl at 3410 cm-1, 1696 cm-1 for

carboxylic group and 1732 cm-1 for ester group. The proton and 13C-NMR data (Table: 4.5,

4.6 vide experimental) were in complete agreement with the data reported for Helixoside A

(4) in the literature and characterized as 3-O-β-D-glu-copyranosyl hederagenin.55


30

3.3.4. Kizuta saponin K12 (5)

OCH2OH

OOH

HOO

O

HOHO

OH

O

O

O

O

OHO

OHHO

O

OHHO

OH

H3C

O

OHHO

OH

H3C

Compound 5 was obtained as white powder from the butanolic fraction. The HRESI-MS

indicated molecular ion peak at m/z 1198.1593 a.m.u. (calcd. for C59H90O25 1198.1589)

showing the molecular formula C59H90O25. The IR spectrum for 5 showed an absorption

bands at 3414 cm-1 for hydroxyl, 1704 cm-1 for carboxylic group and 1739 cm-1 for ester

group. The proton and 13C-NMR data (Table: 4.7, 4.8 vide experimental) were in complete

agreement with the data reported for Kizuta saponin K12 (5) in the literature.56

3.4. Biological studies

3.4.1 Anti-bacterial assay

Various fractions n-hexane (G1), dichloromethane (H1), ethyl acetate (G2) and

methanol (H2) of stem and aerial parts of H. nepalensis were examined for anti-bacterial

assay against the four selected bacterial stains; Escherichia coli, Bacillus cereus, Proteus

mirabilis, Staphylococcus aureus, which showed good activity with zone of inhibition


31

ranging from 9-13 mm at concentration of 28 μg/ml (Table: 4.9 vide experimental). The G1

fractions showed good activity against P. mirabilis at conc. of 12 μg/ml while H2 and H3

exhibited good activity against B. cereus and E. coli at conc. of 13, 11 μg/ml respectively.

The remaining fraction (G-2, G3) was found almost inactive against tested pathogen.

3.4.2. Anti-fungal assay

Various fractions of the stem and aerial parts of H. nepalensis were also tested

against selected fungal strains (Candida albicans, Aspergillus flavus, Microsporum canis,

Fusarium solani and candida glabrata). None of the tested extract/ fractions showed

significant activity. (Table: 4.10 vide experimental).

3.4.3. Phytotoxicity assay

Different fractions of the stem and aerial parts of H. nepalensis were examined for

their in vitro phytotoxic bioassay. Two fractions of the aerial parts of H. nepalensis revealed

significant activities at highest doses against Lemna minor plant. Dichloromethane extract

(H1) of the stem of H. nepalensis revealed good activity, while MeOH and MeOH: H2O

(1:1) extracts revealed moderate activities at highest doses. (Table: 4.11 vide experimental).

3.4.4. Insecticidal assay

Insecticidal assay of the various fractions of the stem and aerial parts of H.

nepalensis were carried out by contact toxicity method.57 Neither of the fractions showed

significant activities (Table: 4.12 vide experimental).


32

3.4.5. Brine shrimp (Artemia salina) lethality bioassay

Various fractions of the stem and aerial parts of H. nepalensis were evaluated

for brine shrimp (Artemia salina) lethality bioassay. The n-hexane and EtOAc fractions

showed non-significant results, while the methanolic fraction was found to be cytotoxic at

highest dose (1000 μg/mL). Similarly dichloromethane fraction of the stem of H. nepalensis

showed no cytotoxicity. (Table: 4.13 vide experimental).

Chapter 4

Experimental

Chapter 4 Experimental (Part-A)

33

4.1. General experimental

Infrared spectra (IR) were obtained in solid state through FT/IR-4200 spectrometer.

Ultraviolet spectra (UV) were recorded on UNICAM UV 300 spectrophotometer. Optical

rotations were recorded on AA-100 polarimeter in chloroform at 20°C. Melting points of the

samples were recorded on Buchi 535 melting point apparatus. Finnigan MAT 112 11/34

computer system was used for recording of mass spectra. High resolution mass spectra

(HRMS) were employed for accurate mass measurement.

1H- and 13C-NMR spectra were recorded on Bruker AVANCE-400, AVANCE-600

MHz for 1H and 100, 125 MHz for 13C nuclei with TMS (tetramethylsilane) as internal

reference using CDCl3 as a solvent, the chemical shifts () values are given in ppm while J

values are given in Hz. Column chromatography was performed on silica gel (Si 60, 70-230

mesh) and precoated aluminum cards (0.2 mm thickness) with silica gel 60PF254 (Merck)

were used. Purity of the samples was checked by TLC and visualized by checking in UV

light and by spraying with I2 vapours and ceric sulphate solution.

4.2. Plant Material

The leaves of Hedera nepalensis were collected from Bara Gali, Khyber

Pakhtunkhwa, Pakistan in July 2009. The plant was identified by Abdul Majid of the

Department of Botany, Hazara University, Khyber Pakhtunkhwa, Pakistan. A voucher

specimen No. BT-576 was deposited in the herbarium of Hazara University.


34

4.3. Extraction and isolation

Leaves of H. nepalensis (400 g) were repeatedly (x3) extracted at room temperature

with methanol. After concentration under vacuum the syrupy liquid was treated with n-

BuOH to get a crude extract of saponins (80 g) which was subjected to column

chromatography on silica gel with solvent system CHCl3-MeOH-H2O ( 26:14:3) to afford

three fractions (1-3). Fraction 3 the most polar triterpene was further subjected to high

performance liquid chromatography (HPLC) eluted with MeOH-H2O (20% to 80% of

MeOH) to yield compound 1 (20 mg), compound 2 (26 mg), compound 3 (38 mg) and

mixture of 4 and 5 (85 mg), which was further subjected to column chromatography

afforded compound 4 (19 mg) and 5 (32 mg) (Scheme: 4.1).


35

Percolation with MeOH(three times) at room temp.

Not pursued

Leaves of H. nepalensis(400 g)

n-BuOH

Crude extract of saponins(80 g)

Column chromatography

Not pursued

HPLC(500 mg offraction 3)

1 (20 mg) 2 (26 mg) 3 (38 mg) mixture(85 mg)


4 (19 mg) 5 (32 mg)

Fraction 2(35 g)

Fraction 3(12 g)

Fraction 1(32 g)

Aqueous layer (140 g)

Scheme: 4.1: Extraction and isolation from H. nepalensis K. Koch


36

4.4. Characterization of chemical constituents

Hepalensiside A (1)

Physical data

Yield: 20 mg

UVλmax ( in MeOH: 242 (4.62) nm

IR max cm-1: 1742, 1754 (ester/ lactone carbonyls), 1630 (C=C)

HR-ESI-MS: 903. 3012 [M+Na]+ (calcd. for C46H72O16 880.2313)

1H- and 13C-NMR : Table- 4.1.


37

Table: 4.1. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 1 in C5D5N

Position δH Multiplicity (J in Hz) δC Multiplicity

1a /1b 1.16/1.89 m 38.6 CH2

2a/2b 2.05/2.30 m 25.8 CH2

3 4.28 dd (11.5, 4.2) 81.2 CH

4 - - 42.8 C

5 1.75 m 47.6 CH

6a /6b 1.43/1.72 m 18.4 CH2

7a/7b 1.25/1.39 m 32.7 CH2

8 - - 42.7 C

9 2.11 m 53.8 CH

10 - - 36.7 C

11 6.63 dd (10.5, 4.5) 125.9 CH

12 5.73 d (10.5) 127.1 CH

13 - - 133.2 C

14 - - 42.2 C

15a /15b 1.02/1.93 m 25.7 CH2

16a / 16b 1.78/2.20 m 33.5 CH2

17 - - 48.9 C

18 - - 136.6 C

19a / 19b 2.11/2.68 m 41.2 CH2

20 - - 32.9 C

21a/21b 1.32/1.68 m 37.8 CH2


38

22a / 22b 1.48/2.58 m 36.4 CH2

23a/23b 3.91/4.32 m 63.9 CH2

24 1.09 s 13.5 CH3

25 0.99 s 18.8 CH3

26 1.07 s 17.2 CH3

27 1.03 s 20.1 CH3

28 - - 178.9 C

29 0.93 s 32.3 CH3

30 0.86 s 24.3 CH3

1' 5.05 d (6.8) 104.8 CH

2' 4.01 dd (10.2, 6.8) 75.3 CH

3' 4.58 m 75.3 CH

4' 4.11 m 69.7 CH

5'a/5'b 3.65/4.23 d (10.8)/ m 47.6 CH2

1'' 6.32 d (6.2) 101.3 CH

2'' 4.92 dd (10.7, 6.2) 72.1 CH

3'' 4.76 m 81.2 CH

4'' 4.35 m 72.4 CH

5'' 4.17 m 70.3 CH

6'' 1.53 d (6.4) 18.3 CH3

1''' 5.96 d (6.6) 104.6 CH

2''' 4.41 dd (10.3, 6.6) 72.7 CH


39

3''' 4.47 m 68.3 CH

4''' 4.10 m 69.7 CH

5a'''/5'''b 4.12/4.32 dd (10.5, 3.2)/m 65.2 CH2

Colchiside A (2)

Physical data

Yield: 26 mg


IR max cm-1: 3401 (hydroxyl), 3056 (C-H stretching

aromatic), 2917 (C-H stretching aliphatic)

HR-ESI-MS: 604.1171 (calcd. for C35H56O8 604.1230)

1H- and 13C-NMR : Table- 4.2.


40



1a /1b 1.16/1.89 m 38.6 CH2

2a/2b 2.05/2.30 m 25.8 CH2

3 4.28 dd (10.8, 4.1) 83.4 CH

4 - - 42.8 C

5 1.75 m 47.6 CH

6a /6b 1.43/1.72 m 18.4 CH2

7a/7b 1.25/1.39 m 32.7 CH2

8 - - 42.7 C

9 2.11 m 53.8 CH

10 - - 36.7 C

11a/11b 1.92 dd (10.5, 3.3) 125.6 CH2

12 5.68 d (10.5) 127.3 CH

13 - - 134.1 C

14 - - 42.2 C

15a /15b 1.02/1.93 m 25.7 CH2

16a / 16b 1.78/2.20 m 33.5 CH2

17 - - 48.9 C

18 1.48 m 45.7 CH

19a / 19b 2.11/2.68 m 41.2 CH2

20 - - 32.9 C

21a/21b 1.32/1.68 m 37.8 CH2


41

22a / 22b 1.48/2.58 m 36.4 CH2

23a/23b 3.90/4.32 m 62.3 CH2

24 1.09 s 13.5 CH3

25 0.95 s 18.4 CH3

26 1.04 s 16.7 CH3

27 0.97 s 19.8 CH3

28 - - 182.6 C

29 0.93 s 30.5 CH3

30 0.86 s 26.7 CH3

1' 5.12 d (6.8) 106.3 CH

2' 4.03 dd (10.7, 6.8) 75.4 CH

3' 4.38 m 78.2 CH

4' 4.21 m 71.7 CH

5'a/5'b 3.45/4.31 dd (10.5, 2.2)/ m 66.8 CH2


42

Pastuchoside A (3)

Physical data

Yield: 38 mg

UVλmax (in MeOH: 259 (4.56) nm



HR-ESI-MS: 1528.7190 a.m.u (calcd. for C71H116O35 1528.7186)

1H- and 13C-NMR : Table- 4.3, 4.4.


43



1a /1b 1.06/1.75 m 39.6 CH2

2a/2b 2.04/2.28 m 26.8 CH2

3 4.14 dd (10.6, 4.4) 81.7 CH

4 - - 43.9 C

5 1.73 m 48.7 CH

6a /6b 1.46/1.62 m 18.3 CH2

7a/7b 1.28/1.32 m 33.5 CH2

8 - - 40.7 C

9 2.13 m 49.8 CH

10 - - 36.9 C

11a/11b 1.88 dd (10.3, 2.4) 24.6 CH2

12 5.63 d (10.3) 127.3 CH

13 - - 142.5 C

14 - - 42.7 C

15a /15b 1.04/1.91 m 27.8 CH2

16a / 16b 1.73/2.18 m 24.2 CH2

17 - - 48.4 C

18 1.49 m 42.3 CH

19a / 19b 2.13/2.62 m 47.2 CH2

20 - - 31.7 C

21a/21b 1.33/1.59 m 34.4 CH2


44

22a / 22b 1.42/2.53 m 33.2 CH2

23a/23b 3.43/4.36 m 64.3 CH2

24 1.05 s 13.3 CH3

25 0.93 s 17.4 CH3

26 1.03 s 17.7 CH3

27 0.95 s 20.7 CH3

28 - - 178.2 C

29 0.92 s 32.8 CH3

30 0.88 s 23.3 CH3

1' 4.48 d (6.6) 106.32 CH

2' 3.73 dd (11.3, 6.6) 75.4 CH

3' 3.84 m 78.2 CH

4' 4.02 m 71.7 CH

5'a/5'b 4.15/3.54 dd (10.6, 2.7)/ m 66.8 CH2

1'' 5.36 d (6.4) 102.7 CH

2'' 3.97 dd (10.4, 6.4) 72.6 CH

3'' 3.84 m 72.5 CH

4'' 4.35 dd (6.8, 3.1) 73.9 CH

5'' 3.53 m 70.6 CH

6'' 1.45 d (6.4) 18.2 CH3

1''' 6.22 d (6.7) 105.5 CH

2''' 4.94 dd (10.1, 6.7) 75.3 CH


45

3''' 5.45 m 76.4 CH

4''' 3.94 m 79.4 CH

5''' 3.87 m 76.6 CH

6'''a/6'''b 4.43/4.26 dd (12.3, 2.8)/m 61.8 CH2

1'''' 4.75 d (6.7) 101.8 CH

2'''' 3.75 m 72.9 CH

3'''' 3.85 m 72.3 CH

4'''' 3.41 dd (7.3, 1.4) 73.3 CH

5'''' 4.04 m 70.8 CH

6'''' 1.32 d (6.7) 17.3 CH3

Table: 4.4. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 3 for sugar moieties

at position C-28

1'a/1'b 5.36 dd (6.9, 2.6)/m 95.8 CH2

2' 3.33 m 73.6 CH

3' 3.55 m 78.2 CH

4' 3.46 m 70.8 CH

5' 3.62 m 78.3 CH

6'a/6'b 4.12/3.65 dd (11.5, 2.8)/m 69.4 CH2

1'' 4.43 d (7.3) 104.5 CH

2'' 3.26 dd (10.4, 7.3) 75.6 CH

3'' 3.45 m 76.4 CH

4'' 3.56 m 79.4 CH


46

5'' 3.37 m 76.4 CH

6''a/6''b 3.45/3.75 dd (11.2, 2.7)/m 61.6 CH2

1''' 4.62 d (6.8) 102.4 CH

2''' 3.64 m 72.7 CH

3''' 3.87 dd (7.6, 3.4) 73.3 CH

4''' 3.47 m 73.6 CH

5''' 4.07 m 70.9 CH

6''' 1.28 d (6.4) 18.5 CH3


47

Helixoside A (4)

Physical data

Yield: 19 mg

UVλmax (in MeOH: 272 (4.72) nm



HR-EI-MS: 1143.0221 [M+Na]+ (calcd. for C54H88O24 1120.0132)

1H- and 13C-NMR : Table- 4.5, 4.6.


48



1a /1b 1.04/1.74 m 39.7 CH2

2a/2b 2.02/2.26 m 26.3 CH2

3 3.84 dd (10.2, 4.2) 84.7 CH

4 - - 43.5 C

5 1.71 m 48.8 CH

6a /6b 1.44/1.64 m 18.4 CH2

7a/7b 1.26/1.33 m 33.6 CH2

8 - - 40.5 C

9 2.14 m 49.4 CH

10 - - 36.5 C

11a/11b 1.94 dd (10.4, 2.4) 24.4 CH2

12 5.64 d (10.4) 126.3 CH

13 - - 143.5 C

14 - - 42.5 C

15a /15b 1.02/1.92 m 28.3 CH2

16a / 16b 1.74/2.16 m 24.6 CH2

17 - - 48.5 C

18 1.53 m 42.3 CH

19a / 19b 2.12/2.60 m 47.4 CH2

20 - - 31.3 C

21a/21b 1.32/1.58 m 34.3 CH2


49

22a / 22b 1.40/2.54 m 33.4 CH2

23a/23b 3.46/4.35 m 64.5 CH2

24 1.08 s 13.4 CH3

25 0.97 s 17.4 CH3

26 1.01 s 17.7 CH3

27 0.93 s 20.8 CH3

28 - - 178.3 C

29 0.96 s 32.5 CH3

30 0.81 s 23.4 CH3

1' 4.52 d (7.5) 104.4 CH

2' 3.35 dd (10.4, 7.5) 81.5 CH

3' 3.46 m 77.6 CH

4' 3.57 m 71.3 CH

5' 3.58 m 78.4 CH

6'a/6'b 4.21/3.43 dd (11.2, 2.5)/m 62.4 CH2

1'' 4.69 d (7.4) 104.2 CH

2'' 3.34 dd (10.8, 7.4) 76.1 CH

3'' 3.67 m 77.8 CH

4'' 3.57 m 71.5 CH

5'' 3.35 m 78.5 CH

6''a/6''b 3.44/3.78 dd (11.6, 2.5) 62.9 CH2


50

Table: 4.6. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 4 for sugar moieties at

position C-28

1' 5.36 d (7.9) 95.7 CH

2' 3.26 dd (10.5, 7.9) 73.8 CH

3' 3.74 m 78.0 CH

4' 3.45 m 70.9 CH

5' 3.45 m 77.5 CH

6'a/6'b 4.02/3.34 dd (10.5, 2.4)/ m 69.5 CH2

1'' 4.35 d (7.8) 104.6 CH

2'' 3.32 dd (10.9, 7.8) 75.1 CH

3'' 3.64 m 77.6 CH

4'' 3.55 m 71.6 CH

5'' 3.25 m 78.0 CH

6''a/6''b 3.37/3.64 dd (11.3, 4.8)/m 62.7 CH2


51

Kizuta saponin K12 (5)

OCH2OH

OOH

HOO

O

HOHO

OH

O

O

O

O

OHO

OHHO

O

OHHO

OH

H3C

O

OH

HOOH

H3C

Physical data

Yield: 32 mg


IR max cm-1: 3414 (hydroxyl), 1704 for carboxylic group

1739 cm-1 for ester group 3035 (C-H stretching


HR-ESI-MS: 1198.1593(calcd. for C59H90O25 1198.1589)

1H- and 13C-NMR : Table- 4.7, 4.8.


52



1a /1b 1.03/1.72 m 38.5 CH2

2a/2b 2.01/2.20 m 25.7 CH2

3 3.86 dd (10.8, 4.3) 83.8 CH

4 - - 44.5 C

5 1.73 m 47.3 CH

6a /6b 1.35/1.54 m 18.6 CH2

7a/7b 1.30/1.32 m 33.1 CH2

8 - - 39.4 C

9 2.02 m 48.5 CH

10 - - 37.4 C

11 1.74 dd (10.2, 2.6)/ m 24.2 CH2

12 5.32 d (10.2) 124.4 CH

13 - - 142.7 C

14 - - 42.4 C

15a /15b 1.01/1.94 m 28.4 CH2

16a / 16b 1.72/2.13 m 24.5 CH2

17 - - 48.3 C

18 1.49 m 42.2 CH

19a / 19b 2.31/2.46 m 47.1 CH2

20 - - 31.2 C

21a/21b 1.31/1.53 m 34.2 CH2


53

22a / 22b 1.42/2.52 m 33.5 CH2

23a/23b 3.34/4.32 m 64.2 CH2

24 1.02 s 13.5 CH3

25 0.95 s 17.5 CH3

26 1.05 s 17.1 CH3

27 0.94 s 20.4 CH3

28 - - 178.1 C

29 0.92 s 32.2 CH3

30 0.78 s 23.3 CH3

1' 5.43 d (7.7) 102.5 CH

2' 3.65 m 73.3 CH

3' 3.79 m 71.4 CH

4' 4.53 m 73.8 CH

5' 3.46 m 70.2 CH2

1'' 4.52 d (7.4) 106.2 CH

2'' 3.74 dd (8.8, 7.4) 75.1 CH

3'' 3.87 m 78.1 CH

4'' 4.12 m 71.3 CH

5''a/5''b 4.21/3.45 d (10.4)/ m 66.4 CH

6'' 1.34 d (6.3) 18.5 CH3


54

Table: 4.8. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 5 for sugar moieties at

position C-28

1' 4.57 d (7.3) 102.3 CH

2' 3.51 dd (9.2, 7.3) 72.1 CH

3' 3.73 m 73.3 CH

4' 3.35 m 73.7 CH

5' 4.11 m 69.4 CH

6'a/6'b 1.19 dd (6.3, 2.1) /m 17.4 CH2

1'' 5.21 d (6.7) 96.1 CH

2'' 3.21 dd (10.5, 6.7) 72.9 CH

3'' 3.42 m 77.3 CH

4'' 3.51 m 70.2 CH

5'' 3.57 m 77.4 CH

6''a/6''b 4.09/3.49 dd (11.2, 2.4)/m 69.2 CH2

1''' 4.34 d (6.6) 104.1 CH

2''' 3.16 dd ( 10.3, 6.6) 75.3 CH

3''' 3.23 m 75.6 CH

4''' 3.34 m 79.2 CH

5''' 3.45 m 76.3 CH

6''' 1.26 d (7.2) 18.3 CH3


55

Table: 4.9. Anti-bacterial activity of H. nepalensis

Keywords: X = Non significant, aerial parts = G, stem = H, G1 = n-hexane, G2

EtOAc, G3 = MeOH, H1 = Dichloromethane, H2 = Methanol, H3 = MeOH: H2O

(1:1), P. m = Proteus mirabilis, S. a = Staphylococcus aureus, E. c = Escherichia

coli, B. c = Bacillus cereus

Fractions E. c B. s P. m S. a

G1 x x 12 X

G2 x x X X

G3 x x X X

H1 10 x X 9

H2 x 13 X X

H3 11 x X X

DMSO (–) x x X X

Imipenum 10 µg/Disc (+) 34 32 28 23


56

Table: 4.10. Anti-fungal profile of H. nepalensis

Fractions Name of Fungus % inhibition Std. Drug Mic (µg/mL)

G1

C. a 0 Miconazole (110.8)

A. f 0 Amphotericin B (20.20)

M. c 0 Miconazole (98.4)

F. s 13 Miconazole (73.25)

C. g 18 Miconazole (110.8)

G2






G3






H1






H2 C. a 0 Miconazole (110.8)


57

Keywords: Aerial parts = G, stem = H, G1 = n-hexane, G2 = EtOAc, G3 = MeOH,

H1 = dichloromethane, H2 = Methanol, H3 = MeOH: H2O (1:1), C. a = Candida albicans,

A. f = Aspergillus flavus, M. c =Microsporum canis, F. s = Fusarium solani, C. g= Candida

glabrata

.

Table: 4.11. Phytotoxicity assay of H. nepalensis

Fractions

Compounds

Conc.

(µg/mL)

No. of fronds% Growth

regulation

Std. Drug

Conc.

(µg/mL)Samples Control

G1

1000 02

20

70

0.015100 01 30

10 0 10

G2 1000 09 70





H3







58

100 14 20 20 0.015

10 17 10

G3

1000 07

20

70

0.015100 15 30

10 16 10

H1

1000 09

20

50

0.015100 11 40

10 16 15

H2

1000 10

20

45

0.015100 15 20

10 16 10

H3

1000 13

20

40

0.015100 15 20

10 16 10

. Keywords: Aerial parts = G, stem = H, G1 = n-hexane, G2 = EtOAc,

G3 = MeOH, H1 = dichloromethane, H2 = Methanol, H3 = MeOH: H2O (1:1)


59

Table: 4.12. Insecticidal assay of H. nepalensis

Fractions Name of Insects % Mortality

G1

Tribolium castaneum 0

Rhyzopertha dominica 0

Callosbruchus analis 20

G2




G3




H1




H2




H3





H1 = dichloromethane, H2 = Methanol, H3 = MeOH: H2O (1:1)


60

Table: 4.13. Brine Shrimp lethality bioassay of H. nepalensis

FractionsDoses

(µg/mL)

No. of

shrimps

No. of

survivors

LD50

(µg/mL)STD. Drug

LD50

(µg/mL)

G1

1000 30 22

413892.80 Etoposide 7.4625100 30 25

10 30 26

G2

1000 30 25

4251653 Etoposide 7.4625100 30 27

10 30 28

G3

1000 30 16

1050.50 Etoposide 7.4625100 30 21

10 30 28

H1

1000 29 19

1039111.1 Etoposide 7.4625100 29 22

10 29 23

H2

1000 29 15

981.641 Etoposide 7.4625100 29 22

10 29 20

H3

1000 29 13

607.64 Etoposide 7.4625100 29 22

10 29 28


H1 = dichloromethane, H2 = Methanol, H3 = MeOH: H2O (1:1)

Chapter 5

Plant Introduction

Cornus macrophylla Wall. Ex. Roxb

Chapter 5 Plant introduction (Part-B)

61

5.1. Plant Introduction

Plant description

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Cornales

Family: Cornaceae

Genus: Cornus

Specie: C. macrophylla

Binomial name: Wall

5.1.1. Genus Cornus

Genus Cornus belongs to the family Cornaceae consists of 55 species, distributed in

the temperate region. It is represented in Pakistan by three species namely C. macrophylla,

C. capitata and C. oblonga. Cornus species are represented by shining colorful flowers and

fruits, generally grown as ornamental plants.58

5.1.2. Cornus macrophylla Wall

C. macrophylla is a medium sized tree, 12-15 m tall. Leaves are 7-15 cm long, 3- 9

cm broad, hairs are modified, apex acute 1.5- 4 cm long petioles. Stamens are equal or

slightly longer than petals, anthers are yellow or rarely blue. Cylindrical style slightly

expanded at apex to apparently clavate, 2-4 mm stigma subcapitate, broader than style

slightly lobed. Fruits are purplish black or bluish black. The flowers are hermaphrodite,

pollinated by insects. It has flowering period from April to June.


62

Suitable for light (sandy), medium (loamy) heavy (clay) soils and can be grown in

heavy clay soils. It can grow in semi-shade or no shade. It prefers moist soils. It is

distributed in Afghanistan, Japan, China, Himalayas Pakistan and India at 1500 m to 2700 m

elevation. It is a comparatively common in Pakistan.59

5.1.3. Chemical constituents of the genus Cornus

The earliest evidence regarding phytochemical studies on the genus Cornus trace

back to 1960 when Rudolf and Nair carried out chemical examination of C. Stolonifera, and

reported hyperin from its stem bark.60 In 1973, Du and Francis identified and characterized

anthocyanins, cyanidin 3-rhamnosylgalactoside, pelargonidin 3-galactoside, cyanidin 3-

galactoside, pelargonidin 3-rhamnosylgalactoside and delphinidin 3-galactoside by

chromatographic spectrophotometric and chemical analysis.61 In the same year Jensen and

his research group isolated cornin and dihydrocornin from the leaves of C. nuttallii.62

Chikaon Ishino and his co-workers in 1988 isolated a cytotoxic compound halleridone from

the fruits of C. controvers.63 Hatano, et.al., in 1989 isolated cornusiin A, cornusiin B,

cornusiin C, tellimagranin I, tellimagranin II, isoterchebin, gemin D, 1,2,3-tri-O-galloyl-β-

D-glucose, 1,2,6,-tri-O-galloyl-β-D-glucose, 2,3,-di-O-galloyl-D-glucose, 1,2,3,6-tetra-O-

galloyl-β-D-glucose and camptothin B from the fruits of C. officinalis, and characterized

them by chemical and spectroscopic analysis.64 In 1990 Takuo, et.al., also isolated

digalloylglucose from the fruits of C. officinalis.65 Hatano, et.al., in 1990 isolated further

compounds cornoside and cornusiin G and 7-O-galloylsecologanol from the fruits of C.

officinalis.66 In 1998, Slimestad and Andersen isolated four anthocyanins from the fruits of

C. suecica, and characterized their structure as cyanidin 3-O- chromatographic β-(2'-

glucopyranosyl chemical-O-β-galactopyranoside, cyanidin 3-O-β-(2'-glucopyranosyl-O-β-


63

gluco-pyranoside), cyanidin 3-glucoside and cyanidin 3-galactoside.67 Subsequently Kim

and Kwak isolated dimethyltetrahydrofuran cis-2, 5-dicarboxylate, a furan derivative from

the fruits of C. officinalis.68 In 1998, Stermitz and Krull studied iridoid glycosides;

scandoside methyl ester, Scandoside geniposide, monotropein, hastatoside and galioside

from the fruit of C. Canadensi.69 Lee, D. and his research group in 2000 reported phenolic

compounds; (-)-2,3-digalloyl-4-(E)-caffeoyl-L-threonic acid, (-)-2-galloyl-4-(E)-caffeoyl-L-

threonic acid, (-)-4-(E)-cafeoyl-L-threonic acid and kaempferol 3-O-α-L-rhamnoside from

the leaves of C. Controversa.70 Tanaka, et.al., in 2001 isolated iridoid glucosides; 6α-

dihydrocornic acid, 6β-dihydrocornic acid and 3, 3ꞌ-di-O-methylellagic acid 4-(5ꞌꞌ-acetyl)-α-

l-arabinofuranoside along with three similar compounds; stenophyllin H1, dihydrocornin

and cornin from the C. capitata adventitious roots culture.71 Tanaka and his research group

in 2003 isolated 1, 2, 3, 4, 6-penta-O-galloyl-β-D-glucose from the root of C. capitata.72

Vareed, et al., in 2006 isolated delphinidin 3-O-rutinoside, cyanidin 3-O- glucoside and

delphinidin 3-O-glucoside from the C. alternifolia.73 In 2007, Lee, D. and his research group

isolated flavonoids from the fruits of C. kousa as kaempferol, astragalin and isoquercitin.74

In 2008 Tural and Koca characterized major anthocyanins, cyanidin 3-O-rutinoside and

pelargonidin 3-O-glucoside from the fruits C. mas.75 In 2008 Lee, D. and his group isolated

and characterized a lignan glycoside, (7'S,8'R)-dihydrodehydroconiferyl alcohol-4'-O-β-D-

xylopyranoside along with its aglycone, (7'S,8'R)-dihydrodehydroconiferyl alcohol from C.

kausa.76 Pawlowska et.al., phytochemically studied the methanolic fruits extract of C. mas

and reported flavonoids, quercetin 3-O-xyloside, quercetin 3-O-rhamnoside, quercetin 3-O-

rutinoside, quercetin 3-O-galactoside, quercetin 3-O-glucoside, quercetin 3-O-glucuronide,

a kaempferol 3-O-galactoside and aromadendrin 7-O-glucoside.77 Subsequently Lee, D.


64

et.al., isolated cytotoxic triterpenoids from the fruits of C. Kausa and characterized as

arjunolic acid, asiatic acid, betulinic acid, betulinic aldehyde, lupeol, taraxasterol,

tormentic acid, ursolic acid, ursolic aldehyde, and 19-hydroxyasiatic acid.78 Recently Wali

Ullah isolated 3ꞌ-O-methyl-3,4-methylenedioxy ellagic acid, betulin, betulinic acid and

cornallegic acid from the stem bark of C. macrophylla.79 The compounds reported in the

literature from the genus Cornus are summarized in Table: 5.1.

Table: 5.1. Chemical constituents of the Genus Cornus

S. No. Compounds Species References

1 Hyperin C. stolonifera 61

2 Delphinidin 3-galactoside C. mas 62

3 Cyanidin 3-galactoside C. mas 62

4 Cyanidin 3-rhamnosylgalactoside C. mas 62

5 Pelargonidin 3-galactoside C. mas 62

6 Pelargonidin 3-rhamnosyl–galactoside C. mas 62

7 Dihydrocornin C. nuttallii 63

8 Cornin C. nattullii 63

9 Halleridone C. controvers 64

10 Cornusiin A C. officinalis 65

11 Cornusiin B C. officinalis 65

12 Cornusiin C C. officinalis 65

13 Tellimagranin I C. officinalis 65

14 Tellimagranin II C. officinalis 65

15 Isoterchebin C. officinalis 65

16 Gemin D C. officinalis 65

17 Camptothin B C. officinalis 65

18 1,2,3-Tri-O-galloyl-β-D-glucose C. officinalis 65


65

19 1,2,6-Tri-O-galloyl-β-D-glucose C. officinalis 65

20 1,2,3,6 –Tetra-O-galloyl-β-D-glucose C. officinalis 65

21 2,3,-di-O-galloyl-D-glucose C. officinalis 65

22 Digalloylglucose C. officinalis 66

23 Cornoside C. officinalis 67

24 Cornusiin G C. officinalis 67

25 7-O-galloylsecologanol C. officinalis 67

26 Cyanidin 3-O- chromatographic β-(2ꞌ-

glucopyranosyl, chemical –O-β-

galactopyranoside

C. suecica 68

27 Cyanidin 3-O-β-(2ꞌ -glucopyranosyl-O-β-

gluco-pyranoside)

C. suecica 68

28 Cyanidin 3-glucoside C. suecica, 68

29 Cyanidin 3-galactoside C. suecica 68

30 Dimethyltetrahydrofurancis-2,5dicarboxy-

late

C. officinalis 69

31 Monotropein C. canadensis 70

32 Hastatoside C. canadensis 70

33 Scandoside geniposide C. Canadensis 70

34 Galioside C. Canadensis 70

35 Scandoside methyl ester C. Canadensis 70

37 (-)-2,3-Digalloyl-4-(E)-caffeoyl-L-threonic

acid

C. controversa 71

38 (-)-2-Galloyl-4-(E)-caffeoyl-L-threonic acid C. controversa 71

39 (-)-4-(E)-Cafeoyl-L-threonic acid C. controversa 71

40 kaempferol 3-O-α-L-rhamnoside C. controversa 71

41 3,3'-Di-O-methylellagic acid 4-(5''-acetyl)-

α- L-arabinofuranoside

C. capitata 72

42 6α-dihydrocornic acid C. capitata 72


66

43 6β-dihydrocornic acid C. capitata 72

44 Stenophyllin H1 C. capitata 72

48 1,2,3,4,6-Penta-O-galloyl-β-D-glucose C. capitata 73

49 delphinidin 3-O-rutinoside C. alternifolia 74

50 delphinidin 3-O-glucoside C. alternifolia 74

51 cyanidin 3-O-glucoside. C. alternifolia 74

52 Kaempferol C. kousa 75

53 Astragalin C. kousa 75

54 Isoquercitin C. kousa 75

55 cyanidin 3-O-rutinoside C. mas 76

56 pelargonidin 3-O-glucoside C. mas 76

57 7'S,8'R-dihydrodehydroconiferyl alcohol-4'-

O-β-D-xylopyranoside

C. kousa 77

58 7'S,8'R-dihydrodehydroconiferyl alcohol C. kousa 77

59 Quercetin 3-O-xyloside C. mas 78

60 Quercetin 3-O-rhamnoside C. mas 78

61 Quercetin 3-O-rutinoside C. mas 78

62 Quercetin 3-O-galactoside C. mas 78

63 Quercetin 3-O-glucoside C. mas 78

64 Quercetin 3-O-glucuronide C. mas 78

65 Kaempferol 3-O-galactoside C. mas 78

66 Aromadendrin 7-O-glucoside C. mas 78

67 Lupeol C. kousa 79

68 Taraxasterol C. kousa 79

69 Betulinic acid C. kousa 79

70 Betulinic aldehyde C. kousa 79

71 Ursolic aldehyde C. kousa 79

72 Arjunolic acid C. kousa 79

73 Tormentic acid C. kousa 79

74 Asiatic acid C. kousa 79


67

75 19-Hydroxyasiatic acid C. kousa 79

76 3'-O-Methyl-3,4-methylenedioxy ellagic

acid

C. macrophylla 80

77 Betulin C. macrophylla 80

78 Cornallegic acid C. macrophylla 80

5.1.4. Structures of selected compounds reported from the genus

Cornus

Hyperin

Delphinidin 3-O-galactoside


68

O

OGlc

OCH3OOH

Cornin

1, 2, 3-Tri-O-galloyl-β-D-glucose

OH

O

O

H

OHOHH

H

O O

HO

HOOH

HO

O

HO

HOHO

HOHO

HO

1, 2, 6-Tri-O-galloyl-β-D-glucose


69

OH

HO

O

H

OOH

H

O O

O

HO

HOOH

OH OH

OH

O

HOOH

OH

HO

HO

HOHO

1, 2, 3, 6-Tetra-O-galloyl-β-D-glucose

HO

O

OH

Halleridone Cornoside

O

OGlc

OCH3O

OHOH O

OGlc

OCH3OOOH

Galioside Hastatoside


70

O

OGlc

OHO

OHOH O

OGlc

OCH3OOH

OH

Monotropein Scandoside methyl ester

O

OOH

O

O

O

OHHO O

O

HOOH

OH

OHHO

HO

(-)-2,13-Digalloyl-4-(E)-caffeoyl-L-threonic acid


71

(-)-4-(E)-Caffeoyl-L-threonic acid (-)-2-Galloyl-4-(E)-caffeoyl-L-threonic acid

OH

O

O

H

OOH

H

O O

O

HO

HOOH

OH OH

OH

O

HOOH

OH

HO

O

HO

HOHO

HOHO

HO

1, 2, 3, 4, 6-Penta-O-galloyl-β-D-glucose


72

3, 3'-Di-O-methylellagic acid 4-(5'-acetyl)-α-L-arabinofuranoside

OH

OHHO OH

O

HO

HHO

H

HOH

H

HO

HO

6-β-Dihydrocornic acid Stenophyllin H1


73

Kaempferol Astraglin

Isoquercitin Arjunolicacid

Taraxasterol


74

Asiatic acid Tormentic acid

O

O

OO

OCH3

O

O

O

CH3

O

H3CO

O

OO

OCH3

HO

O

O

Cornallegic acid 3'-O-Methyl-3, 4-methylenedioxy ellagic acid

5.1.5. Medicinal and pharmacological properties of the genus Cornus

Several reports about the importance of the genus Cornus in preservation of food and

in traditional medicine have been published.80 One of a mostly grown Cornus specie, C.

officinalis was used in Chinese herbal medicine such as a tonic, analgesic and diuretic

agents.81 The fruits of Cornus species were used to improve the functions of liver and

kidney.82 Anti-bacterial, anti-allergic, anti-microbial, anti-malarial and anti-histamine

activities of many Cornus species are also documented in the literature.83 Many fruits of

Cornus species were studied for their anthocyanin contents. Anthocyanins are a class of


75

phenolic compounds that give excellent colours to several vegetables, fruits and having

anticancer, anti-inflammatory, anti-diabetic and antioxidant activities.84-87 In China and

Korea C. controversa was used as a tonic and an astringent.88 It is further reported that the

ethanolic extract of C. mas possess useful activities against Pseudomonas aeruginosa,

Micrococcus luteus and Proteus vulgaris.89 The stones of the fruits have antioxidant

properties90 while C. kousa fruits are edible, attractive and are fermented to wine in some

areas of China where this plant is grownn.91 For immuno-regulatory activity the extract of C.

kousa fruits have been reported in the literature.92,93 The genus Cornus is well known for

tannins particularly hydrolysable tannins and iridoid compounds.94-96 Tannins and related

compounds were documented to have anti-oxidative, anti-microbial,97 anti-cancer,98 and

anti-HIV activities.99

Chapter 6

Results & Discussions

Chapter 6 Results & discussion (Part-B)

77

6.1. Present work

Medicinal and biological uses attributed to the genus Cornus, chemical exploration

of C. macrophylla was carried out in the current study resulted in the isolation and

determination of four new, five reported and three hitherto unreported compounds from C.

macrophylla. Structures of the new compounds were identified by advanced spectroscopic

methods including UV, IR, MS, 1D (1H- and 13C-NMR; BB and DEPT) and 2D NMR (J-

resolved, COSY-45o, NOESY, HSQC, HMBC) experiments. The known compounds were

determined by comparison their spectral data with the reported compounds in the literature.

6.2. New compounds isolated from C. macrophylla

Macrophyllanin A (6)


78

Macrophyllanin B (7)

Macrophyllanin C (8)

H3CCOO

O

O

1

4 610

11 18

27

282513

2119


79

Macrophyllanin D (9)

6.3. Hitherto unreported compounds from C. macrophylla

Kaempferol (10)

Taraxasterol (11)


80

3β- Hydroxy-18α-olean-28-19β-olide (12)

6.4. Reported compounds from C. macrophylla

Betulinic acid (13)

Betulin (14)


81

Stigmasterol (15)

Lupeol (16)

Oleanolic acid (17)


82

6.2.1. Macrophyllanin A (6)

Compound 6 was obtained as an amorphous white powder. Its molecular

formula was established as C30H46O2 according to the molecular ion peak at m/z

438.3234 a.m.u (calcd. for C30H46O2 438.3230) in the HRESIMS together with the 1H-

and 13C-NMR data.

The IR spectrum for 6 showed an absorption band for lactonic carbonyl group at

1754 cm-1. The 1H-NMR spectrum (Table: 7.1. vide experimental) exhibited seven methyl

signals as singlet in the range of δH 0.85-1.01 ppm and one olefinic proton at δH 5.36 (H-12).

The 13C-NMR spectrum showed resonances for thirty carbon atoms including seven methyl,

ten methylene, five methine and eight quaternary carbons. These characteristic NMR data

suggested that 6 is a pentacyclic triterpene. The 13C-NMR signals at δC 122.4 (C-12) and δC

141.6 (C-13) indicated the presence of a double bond which together with H-18 doublet of

doublet at δH 3.28 (1H, dd, J = 14.3, 4.6 Hz, H-18) led to place the double bond at position

C-12. The downfield chemical shift at δC 86.2 and δC 179.8 showed an oxygen-connected

methine carbon and a carbonyl carbon atom respectively. The 13C-NMR data (Table: 7.1.

vide experimental) of 6 was similar to those of oleanolic acid except the chemical shift of

the oxygen-linked carbon.100 The structure of 6 was further revealed from 2D-NMR spectral

data (Figure: 6.1). The most downfield proton at δH 4.14 (H-21) showed its connectivity


83

with the carbon at δC 86.2 (C-21) in the HSQC spectrum. This oxygen-linked carbon was

assigned as C-21 from the HMBC correlations between the proton at δH 4.14 (H-21) and C-

19, C-29, C-30. The HMBC correlation between the proton at δH 4.14 (H-21) and C-28

further confirmed a lactone group between C-21 and C-28. The other part of 6 was

determined to be the same as that of oleanolic acid except the absence of the hydroxyl group

at position C-3. On the basis of spectral evidences compound 6 was established and

characterized as olea-12-en-28, 21 β- olide.

Figure: 6.1: Key HMBC correlations of 6


84

6.2.2. Macrophyllanin B (7)

Compound 7 was obtained as a white solid with melting point 244-246 °C. Its

molecular formula was determined as C32H50O4 from the molecular ion peak at m/z

498.7432 a.m.u. (calcd. for C32H50O4 498.7428) in HRESIMS together with the 1H- and

13C-NMR data.

The IR spectrum for 7 exhibited an absorption band for lactonic carbonyl group at

1755 cm-1. The 1H-NMR spectrum (Table: 7.2. vide experimental) exhibited seven methyl

singlets in the range of δH 0.84-1.01 showing the nature of 7 as pentacyclic triterpenoid. The

13C-NMR spectrum showed resonances for thirty carbon atoms including eight methyl, ten

methylene, six methine and eight quaternary carbons. The 1H- and 13C-NMR data (Table:

7.2. vide experimental) of 7 were characteristic of an oleanane skeleton and were in

complete agreement with the proposed structure.101 In addition 1H-NMR showed a singlet at

δH 2.05 (3H) assigned to the methyl of the acetyl group while a doublet of doublet at δH 4.82

(1H, dd, J = 11.8, 4.5 Hz, H-3) was attributed to H-3 proton geminal to the acetyl group. The

13C-NMR spectrum showed the presence of three methine carbon signals at δC 80.4 (C-3),

45.7 (C-18) and δC 85.3 (C-19). The two quaternary carbons resonating at δC 169.6 and δC

178.7 were attributed to the carbonyl of the acetate and lactone group respectively.

H3CCOO

O

O

1

4 610

11 18

27

282513

2119


85

The key HMBC correlations of 7 showed cross links between the down field signal

at δH 4.82 (H-3) with the carbonyl of acetyl group and with δC 54.7 (C-5), 25.8 (C-23) and δC

16.4 (C-24). The proton at δH 3.95 (H-19) showed connectivity with the carbon at δC 85.3

(C-19) in the HSQC spectrum. The proton signal at δH 1.76 (H-18) showed correlation with

δC 36.8 (C-14) and δC 30.5 (C-16). The methyl protons resonating at δH 0.74 (3H, H-23)

showed correlations with δC 80.4 (C-3), 37.6 (C-4) and δC 54.7 (C-5). The methyl protons

resonating at δH 0.94 (3H, H-25) showed correlations with δC 38.9 (C-1), 54.7 (C-5) and δC

50.2 (C-9) while the protons resonating at δH 1.01 (3H, H- 26) showed correlations with δC

35.5 (C-7), 41.5 (C-8), 50.2 (C-9) and δC 36.8 (C-14). The methyl protons resonating at δH

0.84 (3H, H-30) showed correlation with δC 85.3 (C-19), 46.2 (C-20), 31.5 (C-21) and δC

22.7 (C-29). Hence the structure of 7 (macrophyllanin B) was elucidated as 3β-

acetoxyolean-28,19β-olide.

Figure: 6.2. Key HMBC correlations of 7


86

6.2.3. Macrophyllanin C (8)

Compound 8 was obtained as colorless needles with melting point 272-277 °C. Its

molecular formula was established as C30H46O2 from the molecular ion peak at m/z

438.3435 a.m.u (calcd. for C30H46O2 438.3431) in the HRESIMS together with the 1H-

and 13C-NMR data.

The IR spectrum for 8 showed absorption bands for lactonic carbonyl at 1757cm-1

and C=C bond at 1630 cm-1. The 1H-NMR spectrum (Table: 7.3. vide experimental)

exhibited seven methyl protons signals in the range of δH 0.83-1.01. The 13C-NMR spectrum

showed thirty carbon atoms including seven methyl, ten methylene, five methine and eight

quaternary carbons. These characteristic NMR data suggested that 8 is a pentacyclic

triterpene.

The 1H-NMR spectrum showed five methyl groups at δH 0.85 ( 3H, s, H-25), 1.03

(3H, s, H-26), 0.83 (3H, s, H-27), 0.94 (3H, s, H-29), 0.96 (3H, s, H-30) and two methyl as

doublet at δH 0.96 (3H, d, J = 6.8 Hz, H-23), δH 0.90 (1H, d, J = 6.2 Hz, H-24). The 13C-

NMR signals (Table: 7.3. vide experimental) for two quaternary carbons at δC 136.4, 139.6


87

indicated the presence of a double bond located at C-3 (C-5) and signals at δC 86.1 (C-19),

δC 179.9 (C-28) showed an oxygenated carbon and a carbonyl carbon respectively.

The structure of the 8 was further revealed by 2D-NMR spectra (Figure: 6.3). The

proton resonating at δH 3.98 (H-19) showed connectivity with the carbon at δC 86.1 (C-19)

in the HSQC spectrum. The oxygenated carbon was assigned as C-19 due to the HMBC

correlations between the proton at δH 3.98 (H-19) and δC 32.3 (C-21), 23.9 (C-29) and δC

28.7 (C-30). The HMBC correlation between the proton at δH 3.98 (H-19) and the carbon at

δC 179.9 (C-28) supported a lactone group between C-19 and C-28. The methyl protons

resonated at δH 0.96 (3H, H-23) showed correlations with δC 136.4 (C-3), 26.3 (C-4) and δC

139.6 (C-5), while the protons at δH 0.90 (3H, H-24) showed correlations with δC 136.4 (C-

3), 26.3 (C-4), 139.6 (C-5) and δC 21.3 (C-23). The methyl protons at δH 0.84 (3H, H- 25)

showed correlations with δC 42.2 (C-1), 139.6 (C-5) and δC 50.3 (C-9). The methyl proton at

δH 0.94 (3H, H-29) showed correlation with δC 86.1 (C-19), 46.1 (C-20), 32.3 (C-21) and δC

28.7 (C-30). In view of the spectral data 8 (macrophyllanin C) was characterized as A-neo-

18α-olean-3(5)-en-2819β-olide.

O

HHH

H

H

H

O



88

6.2.4. Macrophyllanin D (9)

Compound 9 was assigned the molecular formula C27H36O7 on the basis of molecular

ion peak at m/z 495.2368 [M + Na]+ (calcd. for C27H36O7 472.2253) in the HRESIMS. The IR

spectrum showed absorption bands for OH at 3416 cm−1 and C=O at 1713 cm−1.

O H

OH

OO

1

2 45

7

9

10

12

1415

16

1718

19

20OH

HHH

O O1'2' 2''3'

5'

4' 1''

The 1D-NMR (Table: 7.4. vide experimental) and HSQC spectroscopic data of

compound 9 displayed signals for α, β-unsaturated carbonyl system at δH 7.55 (1H, s, H-1),

159.7 (C-1), 136.4 (C-2) and δC 209.7 (C-3). A trisubstituted double bond was evidenced

from a carbon at δC 142.2 (C-6) and a proton at δH 5.53 (1H, d, J= 5.4 Hz, H-7) and δC 126.4

(C-7). A pair of oxymethylene protons were present at δH 4.03 (2H, s, H-20) and the

corresponding carbon at δC 67.4 (C-20). Four methyl signals were present at δH 1.22 (3H,

s，H-16), δC 16.8 (C-16), δH 1.20 (3H, s，H-17), 23.7 (C-17), δH 0.91 (3H, d, J = 6.4 Hz，H-

18), 14.4 (C-18) and δH 1.73 (3H, s，H-19), δC 10.2 (C-19). Another methylene protons

showed up at δH 2.84 (1H, dd, J = 10.5, 2.5 Hz, H-5a) and 2.15 (1H, dd, J = 10.5, 5.7 Hz, H-

5b). In addition five methines were identified at δH 2.48 (1H, m，H-4) δC 44.2 (C-4)，δH 2.38

(1H, s，H-8) δC 42.1 (C-8)，δH 3.24 (1H, m, H-10) δC 54.1 (C-10)，δH 1.60 (1H, m, H-11)

δC 42.6 (C-11) and δH 1.08 (1H, d, J = 5.3 Hz, H-14), δC 35.6 (C-14).


89

The 1H–1H COSY correlations indicated the spin system H-1–H-10, H-7–H-8–H-14

and H-12–H-11–H-18 moieties. NOESY correlations were observed between H-8 and H-4.

These data suggested that 9 possess the tigliane (phorbol) backbone similar to those of the

known compound sapintoxin A.101 Other characteristic resonances included signals

for a tigloyl group and an acetyl moiety. In the HMBC spectrum (Figure: 6.4), H-12 proton

showed a 3J correlation with the carbonyl carbon of the tigloyl group (δC 167.6), confirming

the location of the tigloyl group at δC 76.2 (C-12). Thus, the structure of 9 was determined as

12-O-tiglylphorbol-4-deoxy-4β- phorbol-13- acetate.



90

6.3.1. Kaempferol (10)

The mass spectrum displayed the molecular ion peak at m/z 286.0423 a.m.u.

representing the molecular formula C15H10O6 (calcd. for C15H10O6 286.0419). The 1H-NMR

(Table: 7.5. vide experimental) showed AAˈBBˈ system ortho related proton at δH 7.02 (2H,

d, J = 9 Hz, H-3ˈ, H-5ˈ) and δH 8.13 (2H, d, J = 9 Hz, H-2ˈ, H-6ˈ). The meta related two sets

of doublets was present at δH 6.24 (1H, J = 1.80 Hz, H-6) and δH 6.48 (1H, J = 2.1 Hz, H-8).

The spectral data were similar to the reported compounds in the literature.102

10

6.3.2. Taraxasterol (11)

Compound 11 was isolated as a white powder. HRESIMS showed the [M]+ peak at

m/z 425.3124 a.m.u corresponding to the molecular formula C30H50O (calcd. for C30H50O

425.3120). The 1H- and 13C-NMR (Table: 7.6 vide experimental) of 11 showed seven

methyl signals and thirty carbon signals. The down field region in the 13C-NMR spectrum

showed a quaternary carbon signals at δC 154.8 and germinal methylene carbon signals at δC

107.5. The 1H- and 13C-NMR valves were identical to the taraxasterol reported in the

literature.103


91

11

6.3.3. 3β- Hydroxy-18α-olean-28-19β-olide (12)

Compound 12 displayed the [M]+ peak at m/z 456.378 a.m.u corresponding to the

molecular formula C30H48O3 (calcd. for C30H48O3 456.3779). The IR spectrum revealed

absorption bands of a hydroxyl group (3421cm-1) and δ- lactone function at (1739cm-1). The

1H-NMR (Table: 7.7. vide experimental) for 12 represents signals for seven methyls in the

region δH 0.75 to 1.01 showing the nature of triterpenoid. The 1H-NMR showed signal at δH

3.25 (1H, dd, J = 11.5, 5 Hz, H-3) which supported a β-oriented hydroxyl group. In addition,

the13CNMR spectrum showed the presence of one methine carbon signal at δC 77.93 (C-3)

and one carbonyl carbon signal at δC 179.4 (C-28). 1H- and 13C- NMR signals were similar

to the data in the literature.104


92

12

6.4.1. Betulinic acid (13)

Compound 13 was isolated as white amorphous powder from the ethyl acatate

fraction of C. macrophylla. Its mass spectrum displayed the [M]+ peak at m/z 456 a.m.u

showing the molecular formula C30H48O3. Its IR spectrum exhibited absorption bands for

OH, C=O, C=C at 3510, 1716 and 1615 cm-1 respectively. The 1H- and 13C-NMR data

(Table: 7.8. vide experimental) were found identical to the reported betulinic acid.105, 106

13


93

6.4.2. Betulin (14)

Compound 14 was isolated as a colourless powder. EIMS spectrum showed molecular

ion peak at m/z 442 a.m.u. corresponding to the molecular formula C30H50O2. The IR

spectrum indicated distinctive absorption bands for hydroxyl group at 3448 cm-1 and olefinic

carbons at 1630 cm-1. The 1H- and 13C-NMR spectral data (Table: 7.9. vide experimental)

showed thirty carbons including six methyl, twelve methylene, six methine and six

quaternary carbon atoms. Carbon signals appearing at δC 150.8 (C-20) and δC 109.5 (C-29)

are the prominent peaks for betulin kind of skeleton. A single proton broad doublet at δH

3.19 (br d, J = 3.6 Hz, H-3) and a multiplet at δH 2.32 were assigned to H-3 and H-19

respectively. The spectral data was in correspondence with the reported betulin in the

literature.107

14


94

6.4.3. Stigmasterol (15)

Compound 15 was isolated as white needle like crystal. Its mass spectrum showed

the parent molecular ion peak at m/z 412.0123 a.m.u corresponding to the molecular formula

C29H48O (calcd. for C29H48O 412.0119). The 1H-NMR (Table: 7.10. vide experimental) for

compound 15 showed methyl signals from δH 0.85 to 1.06. The 13C-NMR for 15 showed

twenty nine carbon atoms consist of six methyl, nine methylene, eleven methine and three

quaternary carbon signals. The down field region in the 13C-NMR showed a quaternary

carbon signal at δC 141.1 (C-5) and methane carbon signals at δC 121.9 (C-6), 138.4 (C-22)

and δC 129.3 (C-23). The spectral data were identical to the stigmasterol reported in the

literature.108

15


95

6.4.4. Lupeol (16)

Lupeol was isolated as white crystals with melting point 209-2110 C. The mass

spectrum exhibited the molecular ion peak at m/z 426.2344 showing to C30H 50O (calcd. for

C30H50O 426.2340). The IR spectrum showed absorption band for hydroxyl group at 3400

cm-1. The 1H- and 13C-NMR spectral data (Table: 7.11. vide experimental) of 16 showed a

pentacyclic triterpinoid of lupane type and after comparison the spectral data with the

reported values confirmed the structure of compound 16 as lupeol.103

16

6.4.5. Oleanolic acid (17)

Compound 17 was isolated as a white amorphous powder. The mass spectrum

showed molecular ion peak at m/z 456.3452 a.m.u corresponding to the molecular formula,

C30H48O3 (calcd. for C30H48O3 456.3448). The 1H-NMR (Table: 7.12. vide experimental)

spectrum of 17 showed seven methyl groups at δH 0.77, 0.79, 0.91, 0.93, 1.21, 0.83 and δH

0.98. A doublet-doublet of one proton at δH 2.83 and a triplet of one vinyl proton at δH 5.24

were attributed to H-18 and H-12 respectively. One methine proton at δH 3.20 (1H, dd, J =

11.2, 4.4 Hz, H-3) represents that 17 has hydroxyl group at position C-3. The spectral data


96

(Table: 7.12. vide experimental) were similar to the reported oleanolic acid in the

literature.101

17

6.5. Biological studies

6.5.1. Anti-bacterial assay

The anti-bacterial activity of crude extract and their various fractions along with

their pure isolated compounds were evaluated against four selected bacterial strains;

Proteus mirabilis, Staphylococcus aureus, Escherichia coli and Bacillus cereus (Table:

7.13. vide experimental). The ethyl acetate and methanolic fractions were found to be

active against B. cereus with the inhibitory zone of 13, 14 mm at concentration of (32

μg/mL).

6.5.2. Anti-fungal assay

Various fractions of the stem and aerial parts of C. macrophylla were tested against

five selected fungal strains, Candida albicans, Aspergillus flavus, Microsporum canis,

Fusarium solani and candida glabrata. As results none of them were showed any significant

inhibition. (Table: 7.14. vide experimental)


97

6.5.3. Phytotoxicity assay

Various fractions of the stem and aerial parts of C. macrophylla were evaluated for

their in vitro phytotoxic bioassay. All the three fractions of the aerial parts of C.

macrophylla showed significant activities at highest doses against Lemna minor plant.

Acetone extract of the stem of C. macrophylla showed good activity, while MeOH and

MeOH:H2O (1:1) extracts showed moderate activities at highest doses (Table: 7.15. vide

experimental).

6.5.4. Insecticidal assay

The various fractions of the stem and aerial parts of C. macrophylla were evaluated

for the insecticidal assay by using contact toxicity method. All fractions were found to be

inactive at different concentrations (Table: 7.16. vide experimental).

6.5.5 Brine shrimp (Artemia salina) lethality bioassay

Various fractions of the stem and aerial parts of C. macrophylla were evaluated for

brine shrimp lethality bioassay. The n-hexane and EtOAc fractions of the aerial parts of C.

macrophylla showed no cytotoxicity, while methanolic fraction found to be cytotoxic. In the

same way acetone fraction of the stem of C. macrophylla showed no cytoxicity, on the other

hand MeOH and MeOH:H2O (1:1) fractions showed cytotoxicity at highest doses109 (Table:

7.17. vide experimental).

Chapter 7

Experimental

Chapter 7 Experimental (Part-B)

101

7.1. General experimental

The general experimental conditions have already been discussed in chapter 4

(section 4.1.) page no. 35

7.2. Plant Material

The stem bark of C. macrophylla was collected from Bara Gali, Khyber

Pakhtunkhwa, Pakistan in July 2009. The plant was identified by Abdul Majid of the

Department of Botany, Hazara University, Khyber Pakhtunkhwa, Pakistan. A voucher

specimen No. AH-112 was deposited in the herbarium of Hazara University.

7.3 Extraction and isolation

C. macrophylla (6 kg) stem bark was extracted with ethanol at room temperature.

The whole extract was filtered and concentrated under reduced pressure by using rotary

evaporator to obtain a reddish coloured gummy solid (0.5 kg). It was fractionated with

increasing polarity to n-hexane soluble fraction (F1), chloroform soluble fraction (F2) and

ethyl acetate soluble fraction (F3). The two fractions (F1, F2) were not pursued in the

current study.

7.3.1 Fraction of ethyl acetate phase

The ethyl acetate phase was evaporated using rotary evaporator under reduce

pressure and finally the extract dried over anhyd. Na2SO4. The powdery residue (40 g) was

obtained and subjected to column chromatography using silica gel 60 (Merck, 70-230 mm)

eluated with n-hexane and n-hexane_EtOAc solvent in the increasing order of polarity. The


102

eluates were combined on the basis of TLC profile to yield six fractions (AC-1 to AC-6).

Fractions AC-1, AC-3 and AC-4 were not pursued in the present study due to their minute

quantity. Fraction AC-2 was loaded on silica gel column with n-hexane and n-

hexane_EtOAc in increasing order of polarity yielded three fractions ACa, ACb and ACc.

Fraction ACa on prep TLC using n-hexane–EtOAc (7:3) gave three pure compounds, 13

(35 mg), 14 (19 mg), and 16 (21 mg). Fraction ACb following the same procedure and

developing solvent system yielded 2 pure compounds, 17 (28 mg) and 10 (23 mg). Fractions

ACc furnished two pure compounds 11 (31 mg) and 15 (42 mg). Fractions AC-5 was

subjected to column chromatography with n-hexane_EtOAc (4:1) and then chloroform:

methanol (24:1) to afforded two new compounds, 6 (15 mg), 7 (21 mg) and one known

compound, 12 (19 mg) (Scheme: 7.1).

Fraction AC-6 was subjected to silica gel column chromatography, eluted with n-

hexane_EtOAc in the order increasing polarity yielded two fractions AC6a and AC6b.

Fraction AC6a yielded one new compound, 8 (23 mg) on column chromatography and

fraction AC6b yielded another new compound, 9 (17 mg) on silica gel column

chromatography.


103

Scheme: 7.1. Extraction and isolation from C. macrophylla stem.

Percolation with EtOH(three times) at room temp.

n-hexane soluble fractionF1

Aqueous fraction

Solvent removedunder vaccum

Not pursued

Stem bark of Cornus macrophy lla(6 kg)

Ethanolic extract

Solvent removed under vaccum

Solid residue(0.5 kg)

Water suspension

H2O

CHCl3

Aqueous fractionChloroform soluble fractionF2

Not pursued

EtOAc

Aqueous fractionEtOAc soluble fractionF3

Not pursuedPursued

n-hexane


104

Solvent removedunder vaccum

EtOAc soluble fractionF3

Powdered residue(40 g)


Notpursued

6(15 mg)

AC1 AC2 AC3 AC4 AC5 AC6

Notpursued

i.CCii.TLC

i.CCii.TLC

CC

ACaACb

ACc

AC6a AC6b

CCCC7

(21 mg)

12(19 mg)

8(23 mg)

9(17 mg)

13(35 mg)

16(21 mg)

14(19 mg)

11(31 mg)

15(42 mg)

17(28 mg)

10(23 mg)


105

7.4 Characterization of chemical constituents

Macrophyllanin A (6)

Physical data

Yield: 15 mg

UVλmax ( in CHCl3: 236 (4.73) nm

IR max cm-1: 1742, 1754 (ester/ lactone carbonyls), 1634 (C=C)

HR-ESI-MS m/z : 438.3234 (calcd. for C30H46O2 438.3230)

1H- and 13C-NMR: Table - 7.1.

Table: 7.1. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 6 in CDCl3


1a /1b 1.55/1.02 m 39.9 CH2

2a/2b 1.67/1.52 m 18.6 CH2

3a/3b 1.63/1.42 m 42.1 CH2

4 - - 33.6 C

5 0.73 d (7.4) 56.4 CH

6a /6b 1.49/1.30 m 18.2 CH2


106

7a/7b 1.44/1.29 m 33.5 CH2

8 - - 41.5 C

9 1.63 m 50.1 CH

10 - - 38.2 C

11a /11b 1.92/1.91 m 24.5 CH2

12 5.36 t (4.6) 122.4 CH

13 - - 141.6 C

14 - - 42.9 C

15a /15b 2.17/1.19 m 30.2 CH2

16a / 16b 2.11/1.96 m 29.9 CH2

17 - - 44.5 C

18 3.28 dd (14.3, 4.6) 46.6 CH

19a / 19b 1.84/1.33 m 25.4 CH2

20 - - 46.0 C

21 4.14 m 86.2 CH

22a / 22b 2.06/1.83 m 25.5 CH2

23 0.85 s 27.9 CH3

24 0.88 s 15.5 CH3

25 0.90 s 16.5 CH3

26 1.01 s 15.7 CH3

27 0.93 s 13.8 CH3

28 - - 179.8 C

29 0.96 s 23.7 CH3

30 0.89 s 28.7 CH3


107

Macrophyllanin B (7)

Physical data

Yield: 21 mg


IR max cm-1: 1735, 1755 (ester/ lactone carbonyls)

3054 (C-H, aromatic), 2945(C-H, aliphatic)

HR-EI-MS m/z : 498.7432 (calcd. for C32H50O4 498.7428)

1H- and 13C-NMR: Table- 7.2.



1a/2b 1.79 m 38.9 CH2

2a/2b 1.82 m 35.4 CH2

3 4.82 dd (11.8, 4.5) 80.4 CH

4 - - 37.6 C

5 2.55 m 54.7 CH

6a /6b 1.63/1.30 m 19.2 CH2

7a/7b 1.37/1.29 m 35.5 CH2

8 - - 41.5 C


108

9 2.42 t (4.5) 50.2 CH

10 - - 36.2 C

11a /11b 1.85/1.83 m 24.5 CH2

12a/ 12b 1.35/1.14 m 22.6 CH2

13 2.01 m 34.7 CH

14 - - 36.8 C

15a /15b 2.02/1.13 m 25.6 CH2

16a/ 16b 2.14/1.85 m 30.5 CH2

17 - - 32.6 C

18 1.76 m 45.7 CH

19 3.95 m 85.3 CH

20 - - 46.2 C

21a/ 21b 1.46/1.22 m 31.5 CH2

22a/ 22b 2.03/1.82 m 26.8 CH2

23 0.74 s 25.8 CH3

24 0.72 s 16.4 CH3

25 0.94 s 17.4 CH3

26 1.01 s 16.4 CH3

27 0.95 s 12.7 CH3

28 - - 178.7 C

29 0.91 s 22.7 CH2

30 0.84 s 27.8 CH3

31 2.05 s 21.5 CH3

32 - - 175.7 C


109

Macrophyllanin C (8)

Physical data

Yield: 23 mg


IR max cm-1: 1730, 1757 (ester/ lactone carbonyls)

3054 (C-H stretching aromatic), 2945(C-H

stretching aliphatic), 1630 (C=C)

HR-ESI-MS : 438.3435 (calcd. for C30H46O2 438.3431)

1H- and 13C-NMR: Table-7.3.



1a/2b 1.82 m 42.2 CH2

2a/2b 1.72/1.83 m 37.3 CH2

3 - - 136.4 C

4 2.62 m 26.3 CH

5 - - 139.6 C

6a /6b 1.58/1.35 m 19.6 CH2

7a/7b 1.32/1.24 m 32.4 CH2


110

8 - - 40.7 C

9 2.51 m 50.3 CH

10 - - 49.7 C

11a /11b 1.82/1.78 m 23.5 CH2

12a / 12b 1.29/1.09 m 26.5 CH2

13 2.02 m 36.4 CH

14 - - 39.7 C

15a /15b 2.09/ 2.23 m 28.2 CH2

16a / 16b 2.09/1.80 m 31.9 CH2

17 - - - C

18 1.82 s 46.7 CH

19 3.98 s 86.1 CH

20 - - - C

21a / 21b 1.45/1.23 m 32.3 CH2

22a / 22b 2.02/1.83 m 25.2 CH2

23 0.96 d (6.8) 21.3 CH3

24 0.90 d (6.2) 16.4 CH3

25 0.85 s 19.1 CH3

26 1.03 s 14.0 CH3

27 0.83 s 13.5 CH3

28 - - 179.9 C

29 0.94 s 23.9 CH3

30 0.96 s 28.7 CH3


111

Macrophyllanin D (9)

Physical data

Yield: 17 mg


IR max cm-1: 3416 (hydroxyl), 1713 (carbonyl group)

2945(C-H stretching aliphatic), 1630 (double bond)

HR-ESI-MS: 495.2368 [M + Na]+ (calcd. for C27H36O7 472.2253)




1 7.55 s 159.7 CH

2 - - 136.4 C

3 - - 209.7 C

4 2.48 m 44.2 CH

5a/5b 2.84/2.15 dd (10.5, 2.5)/dd (10.5, 5.7) 29.6 CH2

6 - - 142.2 C

7 5.53 d (5.4) 126.4 CH


112

8 2.38 m 42.1 CH

9 - - 77.8 C

10 3.24 m 54.1 CH

11 1.60 m 42.6 CH

12 5.47 d (9.7) 76.2 CH

13 - - 65.4 C

14 1.08 d (5.3) 35.6 CH

15 - - 25.6 C

16 1.22 s 16.8 CH3

17 1.20 s 23.7 CH3

18 0.91 d (6.4) 14.4 CH3

19 1.73 s 10.2 CH3

20a/20b 4.03/3.99 s 67.4 CH2

1′ - - 167.6 C

2′ - - 128.5 C

3′ 6.84 m 137.4 CH

4′ 1.80 d (7.0 ) 14.4 CH3

5′ 1.84 s 12.2 CH3

1" - - 179.4 C

2" 2.10 s 23.4 CH3


113

Kaempferol (10)

Physical data

Yield: 23 mg

UV max (MeOH): 265 (3.62) nm

IR max cm-1: 3400 (hydroxyl), 2944 (C–H), 1458 (C=C)


1H- and 13C-NMR: Table -7.5.



1 - - -

2 - - 147.1 C

3 - - 136.2 C

4 - - 175.8 C

5 - - 161.5 C

6 6.24 d (1.80) 98.6 CH

7 - - 164.3 C

8 6.48 d (2.1) 93.8 CH

9 - - 157.2 C


114

10 - - 103.4 C

1ˈ - - 122.4 C

2ˈ 8.13 d (9.1) 129.8 CH

3ˈ 7.02 d (9.1) 115.6 CH

4ˈ - - 159.5 C

5ˈ 7.02 d (9.2) 115.6 CH

6ˈ 8.13 d (9.2) 129.7 CH

Taraxasterol (11)

Physical data

Yield: 31 mg

UV max (MeOH): 237 (3.55) nm

IR max cm-1: 3445 (hydroxyl), 1615 (C=C)

HR-ESI-MS m/z : 425.3124 [M - H ]+ (calcd. for C30H50O 425.3120)



115



1a /1b 1.70/0.95 m 38.7 CH2

2 1.60 m 37.3 CH2

3 3.15 m 79.1 CH

4 - - 38.6 C

5 0.71 m 55.4 CH

6a /6b 1.53/1.37 m 18.6 CH2

7a/7b 1.38 m 34.2 CH2

8 - - 40.2 C

9 1.31 s 50.4 CH

10 - - 37.2 C

11a /11b 1.53/1.27 m 23.7 CH2

12a / 12b 1.67/1.12 m 26.2 CH2

13 1.58 m 39.1 CH

14 - - 42.1 C

15a /15b 1.67/.96 m 26.6 CH2

16a / 16b 1.22/1.15 m 38.2 CH2

17 - - 34.4 C

18 0.95 m 48.6 CH

19 2.34 m 39.5 CH

20 - - 154.8 C

21a/21b 2.40/2.18 m 25.4 CH2

22 1.36 m 38.7 CH2

23 0.97 s 28.1 CH3

24 0.78 s 15.3 CH3

25 0.90 s 16.2 CH3

26 0.98 s 15.9 CH3

27 0.92 s 14.7 CH3


116

28 0.85 s 19.5 CH3

29 1.01 d (7.1) 25.5 CH3

30a/30b 4.61/4.58 s 107.5 CH2

3β- Hydroxy-18α-olean-28-19β-olide (12)

Physical data

Yield: 19 mg

UV max (MeOH): 247 (4.35) nm

IR max cm-1: 3421 (hydroxyl), 1739 (lactone carbonyls)




117



1a /1b 1.57/1.03 m 38.9 CH2

2a/2b 2.32/1.88 m 27.33 CH2

3 3.25 dd ( 11.5, 5) 77.93 CH

4 - - 38.8 C

5 0.74 d, (11.9) 55.4 CH

6a /6b 1.50/1.31 m 18.1 CH2

7a /7b 1.45/1.30 m 33.6 CH2

8 - - 40.5 C

9 1.64 d (2.6) 51.2 CH

10 - - 37.2 C

11a /11b 1.91/1.91 m 26.5 CH2

12a / 12b 1.66/1.11 m 20.6 CH2

13 1.57 m 35.9 CH

14 - - 39.9 C

15a /15b 2.18/1.20 m 27.8 CH2

16a / 16b 2.12/1.97 m 31.9 CH2

17 - - 33.5 C

18 1.76 m 46.6 CH

19 3.05 m 86.0 CH

20 - - 46.0 C

21a / 21b 1.48/1.24 m 32.2 CH2

22a / 22b 2.06/1.83 m 25.5 CH2

23 0.77 s 27.9 CH3

24 0.75 s 15.3 CH3

25 0.94 s 16.5 CH3

26 1.01 s 15.5 CH3

27 0.96 s 13.6 CH3

28 0.83 - 179.4 C


118

29 0.90 s 23.9 CH3

30 0.86 s 28.7 CH3

Betulinic acid (13)

Physical data

Yield: 35 mg

UV max (MeOH) in nm: 241 (4.22) nm

IR max cm-1: 3510 (hydroxyl), 1716 (carbonyl group), 1615 (C=C).

EI-MS m/z (rel.int. %): 456 [M]+ (37), 438 (10), 411 (7), 248 (38),

234 (25), 207 (53), and 189 (100).



119



1a /1b 1.51 m 38.5 CH2

1a /1b 1.29 m 27.8 CH2

3 3.30 dd, (10.4, 3.9) 78.6 CH

4 - - 37.7 C

5 1.44 m 55.6 CH

6a/6b 1.56 m 18.6 CH2

7a /7b 1.49 m 34.5 CH2

8 - - 40.8 C

9 1.37 m 50.3 CH

10 - - 37.1 C

11a/11b 1.42 m 20.9 CH2

12a/12b 1.77 m 25.6 CH2

13 1.40 m 37.3 CH

14 - - 42.6 C

15a/15b 1.79 m 30.8 CH2

16a/16b 1.45 m 32.4 CH2

17 - - 56.5 C

18 1.79 m 46.7 CH

19 3.04 br s 49.2 CH

20 - - 150.5 C

21a/21b 1.45 m 29.5 CH2

22a/22b 1.76 m 32.1 CH2

23 0.80 s 27.7 CH3

24 0.83 s 15.6 CH3

25 0.79 s 16.4 CH3

26 0.87 s 16.6 CH3

27 1.15 s 14.7 CH3

28 - - 180.5 C


120

29a/29b 4.56/4.43 m 109.8 CH2

30 1.55 s 19.37 CH3

Betulin (14)

Physical data

Yield: 19 mg

UV max (CHCl3): 244 (4.1) nm

IR max : 3448 (hydroxyl), 1630 (C=C)

EI-MS m/z (rel. int. %): 442 [M]+ (30) 412 (6), 234 (23), 220 (27), 207 (50), 189

(100), 175 (32)




1a/1b 1.41 m 38.9 CH2

2a/2b 1.60 m 20.7 CH2

3 3.19 dd (11.2, 3.6) 78.8 CH

4 - - 38.6 C

5 1.39 m 54.9 CH


121

6a/6b 1.52 m 17.8 CH2

7a/7b 1.47 m 33.9 CH2

8 - - 40.7 C

9 1.36 m 54.8 CH

10 - - 36.9 C

11a/11b 1.46 m 27.7 CH2

12a/12b 1.57 m 24.8 CH2

13 1.39 m 36.9 CH

14 - - 42.4 C

15a/15b 1.59 m 26.9 CH2

16a/15b 1.34 m 28.8 CH2

17 - - 45.9 C

18 1.71 m 49.8 CH

19 2.32 m 48.6 CH

20 - - 150.8 C

21a/21b 1.55 m 29.3 CH2

22a/22b 1.53 m 34.5 CH2

23 0.93 s 27.7 CH3

24 0.79 s 15.8 CH3

25 0.73 s 15.7 CH3

26 0.95 s 14.8 CH3

27 1.01 s 14.5 CH3

28 3.31 m 60.6 CH2

29a/29b 4.64/4.55 s 109.5 CH2

30 1.82 s 19.7 CH3


122

Stigmasterol (15)

HO

H

H

H

H

Physical data

Yield: 42 mg

UV max (MeOH) in nm: 235 (4.53) nm

IR max cm-1: 3475 (hydroxyl), 1615 (C=C)

HR-ESI-MS m/z : 412.0123 (calcd. for C29H48O 412.0119)

1H- and 13C-NMR: Table -7.10.



1a /1b 1.72/0.94 m 37.4 CH2

2a/2b 1.61 m 31.8 CH2

3 3.49 dd, (10.5, 4.2) 72.1 CH

4a/4b - - 42.4 CH2

5 0.72 m 141.1 C

6a /6b 5.12 m 121.9 CH2

7a/7b 1.37 m 31.8 CH2

8 - - 32.1 C


123

9 1.33 m 50.3 CH

10 - - 36.8 C

11a /11b 1.52/1.26 m 21.2 CH2

12a / 12b 1.66/1.11 m 39.8 CH2

13 1.57 m 42.4 C

14 - - 57.1 CH

15a /15b 1.65/.95 m 24.4 CH2

16a / 16b 1.21/1.14 m 28.5 CH2

17 - - 56.3 CH

18 1.06 m 12.2 CH

19 1.25 d 19.5 CH3

20 1.03 m 40.6 CH

21 0.92 m 21.1 CH

22 4.61 m 138.4 CH

23a/23b 4.60 s 129.3 CH2

24 0.77 t (7.1) 51.4 CH3

25 0.90 s 32.1 CH

26 1.06 d ( 6.6) 21.2 CH3

27 1.01 d ( 6.6) 21.1 CH3

28 0.85 s 25.5 CH3

29 0.98 s 12.4 CH3


124

Lupeol (16)

Physical data

Yield: 21 mg

UV max (MeOH): 210 (4.53) nm

IR max cm-1: 3400 (hydroxyl), 2944 (C–H), 1458 (C=C)

HR-ESI-MS m/z : 426.2344 (calcd. for C30H50O 426.2340)




1a /1b 1.63 /0.94 m 38.9 CH2

2a/2b 1.60 m 20.7 CH2

3 3.18 dd (9.6, 6.2) 78.8 CH

4 - - 38.6 C

5 0.69 m 54.9 CH

6a /6b 1.51/1.38 m 17.8 CH2

7a/7b 1.41 m 33.9 CH2

8 - - 40.7 C


125

9 1.33 m 54.8 CH

10 - - 36.9 C

11a /11b 1.35 m 27.7 CH2

12a /12b 1.69 /1.09 m 24.8 CH2

13 1.56 m 36.9 CH

14 - - 42.4 C

15a /15b 1.56/0.95 m 26.9 CH2

16a/16b 1.46 m 28.8 CH2

17 - - 45.9 C

18 1.56 m 49.8 CH

19 2.34 m 48.6 CH

20 - - 150.8 C

21a/21b 1.25 m 29.3 CH2

22a/22b 1.20 m 34.5 CH2

23 0.94 s 27.7 CH3

24 0.76 s 15.8 CH3

25 0.83 s 15.7 CH3

26 0.98 s 14.8 CH3

27 0.94 s 14.5 CH3

28 0.81 s 60.6 CH3

29a/29b 4.64/4.55 s 109.5 CH2

30 1.72 s 19.7 CH3


126

Oleanolic acid (17)

Physical data

Yield: 28 mg

UV max (MeOH): 254 (4.34) nm

IR max cm-1: 3423 (hydroxyl), 1615 (C=C), 1706 (C=O)





1a /1b 1.61/0.93 m 38.9 CH2

2a/2b 1.62 m 26.7 CH2

3 3.20 dd (11.2, 4.4) 78.6 CH

4 - - 38.6 C

5 0.72 m 54.7 CH

6a /6b 1.531.36 m 18.8 CH2

7a/7b 1.43 m 33.1 CH2

8 - - 40.3 C

9 1.32 m 49.8 CH

10 - - 36.6 C


127

11a /11b 1.35 m 23.7 CH2

12 5.24 t (3.5) 122.4 CH

13 1.55 m 143.5 C

14 - - 42.3 C

15a /15b 1.54/.94 m 26.8 CH2

16a/16b 1.45 m 23.5 CH2

17 - - 45.2 C

18 2.83 dd (13.2, 3.6) 41.4 CH

19a/19b 2.35 m 45.6 CH2

20 - - 30.4 C

21a/21b 1.24 m 34.5 CH2

22a/22b 1.21 m 32.4 CH2

23 0.77 s 27.9 CH3

24 0.79 s 15.8 CH3

25 0.91 s 15.4 CH3

26 0.93 s 16.7 CH3

27 1.21 s 26.2 CH3

28 0.83 - 182.8 C

29 0.98 s 33.2 CH3

30 1.01 s 23.4 CH3


128

Table: 7.13. Anti-bacterial activity of C. macrophylla

Sample P. m S. a E .c B. c

F1 12 X x X

F2 x 11 x X

F3 12 X x 13

F4 8 10 x 14

F5 10 11 x 8

Betulin (13) x X x X

Betulinic acid (14) x X x X

DMSO (Negative Control) x X x X

Imipenem 10µg/Disc (Positive

Control)

28 23 34 32

Keywords: F1= n- hexane; F2= CHCl3; F3= EtOAc; F4= MeOH; F5= crude extract.

P. m= Proteus merablus; S. a= Staphylococcus aureus; E. c= Escherichia coli; B.

c= Bacillus cereus

Table: 7.14. Anti-fungal assay activity of C. macrophylla

Fractions/Extracts Name of Fungus % inhibition Std. Drug Mic (µg/mL)

F1

C. a 0 Miconazole (110.8)A. f 0 Amphotericin B (20.20)M. c 20 Miconazole (98.4)F. s 15 Miconazole (73.25)C. g 0 Miconazole (110.8)

F2


F3


A1C. a 0 Miconazole (110.8)A. f 05 Amphotericin B (20.20)


129

Keywords: Aerial parts = F, stem = A, F1 = n-hexane, F2 = EtOAc, F3 = MeOH, A1 =

Acetone, A2 = Methanol, A3 = MeOH:H2O (1:1), C. a = Candida albicans, A. f = Aspergillus

flavus, M. c = Microsporum canis, F. s = Fusarium solani, C. g= Candida glabrata

(Table: 7.15.: Phytotoxicity assay of C. macrophylla

Extract/Fractions

CompoundsConc.

(µg/mL)

No. of fronds% Growthregulation

Std. DrugConc.

(µg/mL)Samples Control

F11000 06

20

70

0.015100 14 3010 18 10

F21000 06

20

700.015100 16 20

10 18 10

F3

1000 06

20

70

0.015100 14 30

10 18 10

A1

1000 10

20

50

0.015100 12 40

10 17 15

A2

1000 11

20

45

0.015100 16 20

10 18 10

M. c 25 Miconazole (98.4)F. s 20 Miconazole (73.25)C. g 0 Miconazole (110.8)

A2


A3



130

A3

1000 12

20

40

0.015100 16 20

10 18 10

Keywords: Aerial parts = F, stem = A, F1 = n-hexane, F2 = EtOAc, F3 = MeOH,

A1 = Acetone, A2 = Methanol, A3 = MeOH: H2O (1:1)

Table: 7.16. Insecticidal activity of C. macrophylla

Fractions Name of Insects % Mortality

F1Tribolium castaneum 0Rhyzopertha dominica 0Callosbruchus analis 20



A1Tribolium castaneum 0Rhyzopertha dominica 20Callosbruchus analis 0



Keywords: Aerial parts = F, stem = A, F1 = n-hexane, F2 = EtOAc, F3 = MeOH, A1 =

Acetone, A2 = Methanol, A3 = MeOH: H2O (1:1)


131

Table: 7.17. Brine Shrimp activity of C. macrophylla

FractionsDoses

(µg/mL)No. of

shrimpsNo. of

survivorsLD50

(µg/mL) STD. DrugLD50

(µg/mL)

F11000 30 22

413892.80 Etoposide 7.4625100 30 2510 30 26

F21000 30 25

4251653 Etoposide 7.4625100 30 2710 30 28

F31000 30 16

1050.50 Etoposide 7.4625100 30 2110 30 28

A11000 29 19

1039111.1 Etoposide 7.4625100 29 2210 29 23

A21000 29 15

981.641 Etoposide 7.4625100 29 2210 29 20

A31000 29 13

607.64 Etoposide 7.4625100 29 2210 29 28

Keywords: Aerial parts = F, stem = A, F1 = n-hexane, F2 = EtOAc, F3 = MeOH,

A1 = Acetone, A2 = Methanol, A3 = MeOH: H2O (1:1)

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List of Publication

140

1. Ghias Uddin, Ashfaq Ahmad Khan, Muhammad Alamzeb, Saqib Ali, Mamoon-Ur-

Rashid, Anwar Sadat, Muhammad Alam, Abdul Rauf and Wali Ullah. Biological

screening of ethyl acetate extract of Hedera nepalensis stem. African Journal of

Pharmacy and Pharmacology Vol. 6(42), pp. 2934-2937, 15 November, 2012 Available

online at http://www.academicjournals.org/AJPP, DOI: 10.5897/AJPP12.828. ISSN

1996-0816 © 2012 Academic Journals.

2. Muhammad Alamzeb, M. Rafiullah Khan, Saqib Ali, Syed Qaiser Shah, Mamoon-Ur-

Rashid, Ashfaq Ahmad Khan. Bioassay guided isolation and characterization of anti-

microbial and anti-trypanosomal agents from Berberis glaucocarpa Stapf. African

Journal of Pharmacy and Pharmacology /23.01.13/3444. OPEN ACCESS JOURNALS.

http://www.academicjournals.org/AJPP.

3. Ghias Uddin, Waliullah, Abdur Rauf, Bina Shaheen Siddique, Ashfaq Ahmed, Chand

Bibi, Muhammad Qaiser and Sadiq Azam. Phytochemical Screening and Antimicrobial

activity of Cornus macrophylla. Middle-East Journal of Scientific Research 9(4); 516-

519,2011 ISSN 1990-9233 IDOSI publication, 2011

4. Ghias Uddin, Waliullah, Bina Shaheen Siddiqui, Muhammad Alam, Anwar Sadat,

Ashfaq Ahmed and AlaUddin "Chemical Constituents and Biological Screening of

Grewia optiva Drummond ex Burret Whole Plant." American-Eurasian J Agric &

Environ Sci 11.4 (2011): 542-546.

5. Muhammad Alam, Ghias Uddin, Anwar Sadat, Naveed muhammd, Ashfaq Ahmad

Khan and Bina S. Siddiqui "Evaluation of Viburnum grandiflorum for its in-vitro

pharmacological screening." African Journal of Pharmacy and Pharmacology6.22

(2012): 1606-1610.

List of Publication

141

6. Ghias Uddin , Ashfaq A Khan , Anwar Sadat , Igoli O John , Dima Semaan , Carol

Clements, Bina S. Siddiqui, Valerie A. Ferro, Alexander I. Gray

Antidiabetic and Antimicrobial potential of pentacyclic terpenoids from Cornus

macrophylla Wall. ex Roxb. (Submitted)

7. Muhammad Alamzeb,*,† Ajmal Khan,‡ Qaiser Jamal,*, ‡ Syed Akram Shah,‡ M. Rafiullah

Khan,§ Saqib Ali,┴ Mamoon-Ur-Rashid,§ Ashfaq Ahmad Khan§

Novel and highly active Anti-Leishmanial agents against Leishmanial clinical field

isolates KHW23 from Berberis glaucocarpa Stapf. (Submitted)

institute of chemical sciences university of …

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