prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/9092/1/Adil-Thesis- Corrected.pdfPlagiarism Undertaking I solemnly declare that research work presented in the thesis titled

PHYTOCHEMICAL INVESTIGATION ON THE CHEMICAL

CONSTITUENTS OF CARALLUMA FLAVA

Thesis Submitted

for

The Partial Fulfillment of the Degree of

DOCTOR OF PHILOSOPHY IN CHEMISTRY

By

MUHAMMAD ADIL RAEES

DEPARTMENT OF CHEMISTRY

Federal Urdu University of Arts, Science & Technology

Gulshan-e-Iqbal Campus, Karachi-75300, Pakistan

(2017)

PHYTOCHEMICAL INVESTIGATION ON THE CHEMICAL

CONSTITUENTS OF CARALLUMA FLAVA

By

MUHAMMAD ADIL RAEES




(2017)

Plagiarism Undertaking

I solemnly declare that research work presented in the thesis titled

“PHYTOCHEMICAL INVESTIGATION ON THE CHEMICAL CONSTITUENTS

OF CARALLUMA FLAVA” is solely my research work with no significant

contribution from any other person. Small contribution/help wherever taken has been

duly acknowledged and that complete thesis has been written by me.

I understand the zero tolerance policy of the HEC and Federal Urdu University of Arts,

Science & Technology towards plagiarism. Therefore I as an Author of the above titled

thesis declare that no portion of my thesis has been plagiarized and any material used

as reference is properly referred/cited.

I undertake that if I am found guilty of any formal plagiarism in the above titled thesis

even after award of PhD degree, the University reserves the rights to withdraw/revoke

my PhD degree and that HEC and the University has the right to publish my name on

the HEC/University Website on which names of students are placed who submitted

plagiarized thesis.

Name of Student: Muhammad Adil Raees

Signature:

Author’s Declaration

I, MUHAMMAD ADIL RAEES hereby state that my PhD thesis titled

“PHYTOCHEMICAL INVESTIGATION ON THE CHEMICAL CONSTITUENTS

OF CARALLUMA FLAVA” is my own work and has not been submitted previously

by me for taking any degree from Federal Urdu University of Arts, Science &

Technology Or anywhere else in the country/world.

At any time if my statement is found to be incorrect even after my Graduate the

university has the right to withdraw my PhD degree.

Name of Student: Muhammad Adil Raees

Signature:

Certificate of Thesis Evaluation

It is certified that the thesis entitled “PHYTOCHEMICAL INVESTIGATION ON

THE CHEMICAL CONSTITUENTS OF CARALLUMA FLAVA” has been

submitted by MUHAMMAD ADIL RAEES to the Graduate Research Management

Committee (GRMC) for reward of the degree of DOCTOR OF PHILOSOPHY (Ph.D)

in CHEMISTRY and has been written under my supervision and is thereby approved.

__________________________ ____________________________

DR. TALAT MAHMOOD PROF. AHMED AL-HARRASI

(Research Supervisor), Chairperson Co-Supervisor

Department of Chemistry University of Nizwa, Oman

FUUAST, Karachi Campus

__________________________

DR. HIDAYAT HUSSAIN

Co-Supervisor

University of Nizwa, Oman




(2017)

Thesis Approved

By

____________________________

DR. TALAT MAHMOOD

Supervisor and Chairperson

Department of Chemistry, FUUAST

___________________________

PROF. DR. RUBINA MUSHTAQ

Dean, Faculty of Science & Technology, FUUAST

__________________________

EXTERNAL EXAMINER

DEDICATED TO MY LOVING

PARENTS

Raees Ahmed

Shahida Raees

&

YOUNGER ROTHERS

Muhammad Faisal Raees

Muhammad Bilal Raees

Without their commitment and support

this work could not be completed on time.

CARALLUMA FLAVA Syn. Crenulluma flava (N.E.Br) Plowes

Syn. Desmidorchis flava Meve & Liede

i

CONTENTS

ACKNOWLEDGEMENT ……………………………………… iv

ABSTRACT ……………………………………………………... vi

KHULASA ………………………………………………………. vii

1. INTRODUCTION …..………………………………………. 1

1.1 Caralluma Flava …………………………………………………….. 6

1.2 Pregnane Glycosides ………………………………………………... 7

1.3 Literature Survey ……………………………………………………. 9

2. PRESENT WORK …………………………………………. 20

3. EXPERIMENTAL ……………………….…………………. 27

3.1 General Note ………………………………………………………… 28

3.2 Extraction and Isolation …………………………………………….. 29

3.2.1 Characterization of Desmiflavaside A (1) …………………… 36

3.2.2 Characterization of Desmiflavaside B (2) …………………… 39

3.2.3 Characterization of Desmiflavaside C (3) …………………… 42

3.2.4 Characterization of Desmiflavaside D (4) …………………… 45

3.2.5 Characterization of Nizwaside (5) …………………………… 48

3.2.6 Characterization of Desflavaside A (6) ……………………… 51

3.2.7 Characterization of Desflavaside B (7) ………………………. 55

3.2.8 Characterization of Desflavaside C (8) ………………………. 59

3.2.9 Characterization of Desflavaside D (9) ……………………... 63

ii

3.3 Biological Activities ………………………………………………… 66

3.3.1 Anticancer Activity …………………………………………. 66

3.3.2 Enzyme Inhibition Activity …………………………………. 73

3.3.3 Antioxidant Activity ………………………………………… 76

3.4 Molecular Docking Studies …………………………………………. 79

4. RESULTS AND DISCUSSION................................................ 87

4.1 New Compounds from Caralluma flava ……………………………. 88

4.1.1 Desmiflavaside A (1) ………………………………………... 88

4.1.2 Desmiflavaside B (2) ………………………………………… 92

4.1.3 Desmiflavaside C (3) ………………………………………… 94

4.1.4 Desmiflavaside D (4) ………………………………………... 97

4.1.5 Nizwaside (5) ………………………………………………... 99

4.1.6 Desflavaside A (6) …………………………………………… 102

4.1.7 Desflavaside B (7) …………………………………………… 106

4.1.8 Desflavaside C (8) …………………………………………… 108

4.1.9 Desflavaside D (9) …………………………………………... 111

4.2 Biological Activities ………………………………………………… 113

4.2.1 Anticancer Activity ………………………………………….. 113

4.2.3 Enzyme Inhibition Activity ………………………………….. 114

4.2.4 Antioxidant Activity ………………………………………… 115

4.3 Molecular Docking Studies ………………………………………….. 116

5. CONCLUSIONS ……………………………………………… 118

6. REFERENCES ………………………………………..……… 120

iii

7. GLOSSARY …………………………………………………... 126

8. APPENDIX ……………………………………………………. 129

9. PUBLICATIONS ……………………………………………... 152

iv

ACKNOWLEDGMENT

First of all, I bow my head to the Omnipresent, Omnipotent and Omniscient Al-Mighty

ALLAH, whose clemency resulted into my success. I would like to pay homage to the

most respectful and sacred personality of the world, Holy Prophet, Hazrat Muhammad

(S.A.W.W). The research work present in this thesis is the result of the many people’s

contributions.

First and foremost, I express my sincere gratitude to the most co-operative supervisor

and chairperson Dr. Talat Mahmood for her constructive supervision, valuable

suggestions, great encouragement, indefinite motivation and scientific support

throughout the research. Her encouraging attitude always raised my morale. I believe

that she has not only guided me in my academics but also in some other matters of life.

I wish to extend my thanks to Dr. Aneela Wahab, Dr. Iffat Mahmood and Dr. Atya

Hassan for their precious time and outstanding suggestions for this thesis writing. They

also contributed greatly in the NMR spectral interpretation and characterization.

My sincere and boundless thanks to Prof. Dr. M. Iqbal Choudhary and Head of NMR

Section, Dr. Atia-tul-Wahab for my training in NMR spectroscopy and operation of

NMR spectrometers at University of Karachi.

I was fortunate enough to perform a part of my research work at University of Nizwa,

the Sultanate of Oman, while doing my job there as NMR Spectroscopist in the NMR

lab, for which I am grateful to Prof. Dr. Ahmed Sulaiman Al-Harrasi for permitting me

to pursue this work in his laboratories along with my job and gave me the opportunity

to become a part of an enthusiastic and ambitious internationally diverse group with

multidisciplinary competences. I believe that I had a profit there from the expertise of

the team as a whole, as well as from each team member individually, which includes

Dr. Hidayat Hussain, Dr. Javid Hussain, Dr. Liaqat Ali, Dr. Najeeb-Ur-Rehman, Dr.

Thomas Dzeha, Dr. Ali Elyassi, Dr. Faruck Lukman-Ul-Hakkim, Dr. Ghulam Abbas,

Mohammad Abdullah Al-Broumi and Ahmed Al-Ghafri. Some very special and joyful

thanks are extended to the Director of Technical Staff, Dr. Obaid Yusuf Khan for the

effective management of NMR and Mass spectroscopy sections as well as his utmost

help and moral support.

v

I cannot forget the cooperation and help that I received from the local Omanis for the

plant collection. The guidance of the local Omani vegetable sellers helped a lot to search

the plant on the high mountains of Oman and I am highly grateful to all of them. I

acknowledge the wonderful and timely jobs carried out by my collaborators Dr. Gilani

at University of Nizwa in Oman for helping me in the plant identification; Dr. Hussain

Yar Khan for carrying out the bioassays for my sample very efficiently.

My gratitude extends to all those who have contributed to my education. I owe very

much to my lab fellows Syed Waseem Ahmed, Syed Wali Shah and Syed Sajjad Haider,

who has always helped me during my studies and proven to be outstanding companion

and good friends. Thank you all for your friendship especially Shah Rizwan Ashrafi for

being there for me whenever I need your help.

At the end, I express my deepest and heartiest gratitude to all my family members,

specially my parents, who are always so loving and caring. They taught me to be dutiful

and serve the nation. They are so wonderful and cooperative that no words can describe.

I could never been able to start and continue this work to the end without their support

and trust. I am proud to have such a loving and enlightened family.

vi

ABSTRACT

The research work embodied in this thesis deals with the isolation of chemical

constituents from the Omani plant named Caralluma flava (N.E.Br) Meve & Liede

along with the spectral characterization, biological activities and computational studies.

Crude crystals (CR) obtained from the squeezed sap of succulent C. flava showed

significant anticancer activity against MDA-MB-231 breast cancer cells and provided

nine new pregnane glycosides namely Desmiflavaside A (1), Desmiflavaside B (2),

Desmiflavaside C (3), Desmiflavaside D (4), Nizwaside (5), Desflavaside A (6),

Desflavaside B (7), Desflavaside C (8) and Desflavaside D (9). The structures of these

isolates were elucidated through spectral analysis using UV, IR, HRSEIMS, NMR and

by comparing with literature reports.

Treatment of MDA-MB-231 breast cancer and SKOV-3 ovarian cancer cells with

compounds 3-5 demonstrated a prominent reduction in the viability of both types of

cancer cells. Furthermore, 3-5 were also tested for their effect on normal breast

epithelial cells (MCF-10-2A) which displayed no significant cytotoxicity on normal

cells. The molecular docking studies of 3-5 revealed that these molecules have a good

potential to bind with the target protein tyrosine phosphatase.

The methanolic extract (ME) of squeezed residual plant and its different fractions also

showed good anticancer activity against MDA-MB-231 cells. Different fractions of

ME, CR and compounds (1-5) were additionally evaluated for enzyme inhibition

(urease, acetylcholinesterase and α-glucosidase) and DPPH antioxidant activities which

displayed no remarkable results.

The research work embodied in this dissertation has resulted four publications as

mentioned in the end of the thesis.

vii

1

1. INTRODUCTION

2

The Sultanate of Oman, despite its arid nature, is endowed with a variety of medicinal

plants (Hussain et al., 2014). It has almost 1204 terrestrial plants and many of them are

used by herbalists in traditional medicine (Alhakmani et al., 2014; Ghazanfar, 1992).

In common with other Gulf countries, several traditional systems of medicinal

treatment are used in Oman. In addition to the use of plants as medicine (Al Tadawee

bil A’ashiab), cupping (Al Hajamah), bone setting (Al Tajbeer) and cauterization

(Wasm, Qai) are also practised. In any of these, specific medicinal plants are used as a

part of the treatment (Ghazanfar, 1994).

Until recently, the only type of cure available to the large section of people in Oman

was traditional medicine. In the past two decades, with the establishment of clinics and

hospitals, traditional medicine (which comprises healing by using plants) has become

less popular but is still used. Many minor ailments such as headaches, stomach upsets,

coughs, colds and fevers are often treated at home with herbal remedies (Ghazanfar and

Al-Sabahi, 1993).

In Sultanate, the traditional knowledge concerning plants and their uses is largely

untapped. In northern and central Oman, there are no educational or formal training

institutes for teaching traditional medicine and the method of cure. Neither is

knowledge documented. Instead, these traditional medicinal knowledge skills are

passed orally from one generation to another or by elders and ancestors through

apprenticeship and once forgotten may not be practised again. Literate healers consult

the available classical work on herbal and traditional medicines for their practice and

still the oral way is highly preferred for transferring and sharing traditional knowledge

(Lupton et al., 2012; Ghazanfar, 1994; Ghazanfar and Al-Al-Sabahi, 1993).

The Sultanate of Oman is a home to large number of succulent and xerophytic

Caralluma species. In Sultanate, the genus is represented by approximately thirteen

species viz., C. flava N.E.Br., C. aucheriana (Decne.) N.E. Br., C. arabica N.E. Br., C.

penicillata (Deflers) N.E.Br., C. quadrangula (Forssk.) N.E.Br., C. hexagona

Lavranos, C. meintjesiana Lavranos, C. dodsoniana Lavranos, C. luntii N. E. Br., C.

adenensis (Deflers) A. Berger, C. edulis (Edgew.) Benth. ex Hook.f., C. tuberculate

N.E.Br., C. awdeliana (Deflers) Berger (Walter and Gillett, 1998; Albers and Meve,

2002; Ghazanfar, 1994; Bruyns et al., 2010; Patzelt, 2015). The scientific studies of the

genus has been carried out in Pakistan, India, Saudi Arabia, Spain, Italy and Nigeria

3

(Fig. 1) but almost no evidences of scientific examination of Caralluma and validation

of its traditional therapeutic uses in the Sultanate of Oman has been found. Out of

Sultanate, numerous in-vitro and very few in-vivo biological investigations of

Caralluma have been conducted on crude extracts and its isolates. Mostly, antidiabetic

studies of Caralluma species are reported but very few species are examined for their

anticancer potential (Fig. 2) (Adnan et al., 2014).

Cancer is reported to be the major public health issue globally and its incidences are

rising across the globe (Burney et al., 2014; Al-Lawati et al., 1999). Among Omani

females, breast cancer was the leading malignancy of the total cancer cases between

1998 to 2007 and continued to be the leading by May, 2014 (Fig. 3 and 4) (Al-Madouj

et al., 2011; WHO, 2014). One out of five Omani women is diagnosed with breast

cancer in her lifetime and a preventive strategy for cancer has not been developed yet

(Renganathan et al., 2014; WHO, 2010).

Taking in to account the facts stated above, the work was undertaken on Omani

medicinal plant for the present doctoral dissertation entitled “Phytochemical

investigation on the chemical constituents of Caralluma flava”. The introduction

provides a brief description of C. flava, structural features of pregnane glycosides, a

review of isolated pregnane glycosides from the genus and their pharmacological

significance. This is followed by a brief discussion of present work containing the

structures of nine new pregnane glycosides isolated for the first time from C. flava.

Further, the breast cancer activity of these pure constituents along with molecular

docking studies are also included.

4

Figure 1: Scientific studies of Caralluma species in different countries (Adnan et al.,

2014).

Figure 2: Pharmacological activities of Caralluma species (Adnan et al., 2014).

5

Figure 3: Cancer incidences of Omani females (WHO, 2014)

Figure 4: Cancer mortality profile of Omani females (WHO, 2014)

Breast

18%

Lymphomas,

multiple

myeloma

10%Leukaemia

10%Colorectum

8%

Stomach

6%

Other

48%

Females Cancer Mortality

400 Deaths

0

50

100

150

200

Breast Thyroid Colorectum Leukaemia

195

53 51 42

Nu

mb

er o

f ca

ses

Cancer

6

1.1 Caralluma Flava

Caralluma flava, commonly known as نبتة الضجع in Arabic language, belongs to the

family Apocynaceae and has various synonyms viz., Desmidorchis flava, Desmidorchis

flavus and Crenulluma flava. It is a regional endemic plant, most often grows on the

Omani-Yemeni border (Mahra-Dhofar) where it extends into the UAE. It grows much

rare in central and northern Oman. C. flava can be found in rocky limestone areas, dry

riverbeds and coastal hills along rocky watercourses. Its succulent Stems are gray in

colour, bluntly square in shape and usually grow straight in clusters. (Patzelt, 2015;

Grulich V, 2015; Formisano, 2009; Mosti, 2004; Albers and Meve, 2002).

This cactus like plant is edible. Its bright yellow flowers and seed pods are eaten after

the rain and the sap filled juicy stems are collected by local people for food. Due to its

extreme bitterness, addition of lemon and spices are preferred before eating. Fresh plant

as well as its dried powdered forms are available in the vegetable and local markets

(Patzelt, 2015; Raees et al., 2016). The plant has been used for generations in traditional

Omani society due to its medicinal properties. Referenced to the reasons mentioned

earlier, scanty information regarding its medicinal uses have been documented only in

Arabic text as given below (سلطنۃ عمان دیوان البالط السطالنی(.

للحروق: یؤخذ نبات الضجججع الطرو ویسججحى يتي ینججمر ناعما ویونججر ثو نهن ی •

ه مكان الحرق.

ا یضاف إلمه • لممون وملح.للسكر: یهرس ویؤكل غضًّ

للطحال والكب : نفس الطریهة السا هة. •

لإلمساك: یؤكل غضا قبل الطوام. •

للغازات: یشر عنمر الضجع و الطوام ق ر كو . •

لضغط ال م: یُونر النبات ویُشر ق ر كو شاو مرتم فن الموم ون إضافة شنء. •

Translation:-

• For burns: Crush the fresh stems until it becomes soft, squeeze it with cloth and then

apply on the burns.

• Diabetes: Eat juicy stems with salt and lemon.

• Spleen and liver: The same method as above.

• Constipation: Eat before taking meal.

• Abdominal gas: Drink a cup of its juice after food.

• Blood pressure: Drink its juice twice a day without adding anything.

7

The phytochemical investigation of C. flava has not been performed yet and no

chemical constituent has been reported so far except the only available data on the

chemical composition of its floral scent volatiles (Jürgens et al., 2006). However,

several other Caralluma species have been investigated and numerous potent

compounds have been isolated, characterized and reported. On reviewing the literature

undertaken on the phytochemistry of genus Caralluma, it could be observed that

pregnane glycosides are one of its major constituents.

1.2 Pregnanes Glycosides

The C-21 steroidal saponins having the usual per-hydro-1,2-cyclopentanophenanthrene

ring system with a two carbon chain at C-17 and β-oriented Me groups at C-10 and C-

13 are known as pregnanes. Most frequently, pregnane derivatives bear a hydroxyl

group at C-14 which possess β-configuration. The configuration at C-5 is α except for

molecules containing a C-5 double bond. Pregnanes have a β-oriented C-3 hydroxyl

group (Adnan et al., 2014). In 1989, Deepak et al. reported some characteristic features

of pregnanes which are given below.

1- Presence of Double bond at C-5.

2- Fusion of rings B and C is always trans.

3- Fusion of rings C and D is cis when a hydroxyl group is present at C-14 and trans

when H is present at C-14.

4- Additional hydroxyl groups at 5α, 6β, 7α, 8β, 11α, 12α or 12β, 14β, 15α, 16α, 17α

or 17β, 20 and 21 which may be partially esterified.

5- Presence of carbonyl group at positons C-l, C-12, C-15 and C-20.

OH

HOH

H

H

AB

C D1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

Basic skeleton of pregnane (Adnan et al., 2014; Deepak et al., 1989)

8

In pregnane glycosides, the glycoside is attached to an alcoholic OH group of the

aglycone portion, most frequently at C-3, and is usually found as a linear saccharide

chain rather than a branched. The most common and successful employed method of

preparative isolation of pregnane glycosides is column chromatography (Deepak et al.,

1997; Al-Massarani and El-Shafae, 2011). Several types of sugar units have been

detected in pregnane glycosides. The most common are:

9

1.3 Literature Survey

In 1988, four pregnane glycosides viz., boucerosides A-I (10), B-I (11), A-II (14) and

B-II (15) were reported from CHCl3 soluble fraction of the MeOH extract obtained

from dried aerial parts of C. aucheriana. Compounds 10 and 11 contained molecular

formula C62H88O21 whereas 14 and 15 possessed C62H90O21. Preparative HPLC was

used for the purification of these compounds. Later in 1990, two more pregnane

glycosides viz., boucerosides CNC (12) and CNO (13) having molecular formula

C56H80O16 were reported from the same fraction. Pregnane glycosides 10 and 11 had

the similar pregnane skeleton but differed from each another in the sugar moiety, as this

difference was also observed in 14 and 15. A couple of benzoyl groups at position C-

12 and C-20 of the pregnane were present in all of these six molecules (Hayashi et al.,

1988; Ahmad and Basha, 2007; Tanaka et al., 1990).

Lee-Juian et al. (1994) reported pregnane glycosides viz., carumbelloside I (16) and II

(17) isolated from n-BuOH and EtOAc fractions of EtOH extract of fresh whole C.

umbellate plant, respectively. Silica gel column chromatography was used for the

isolation. The structural characterization of these compounds were achieved using

different NMR techniques. TOCSY experiments were reported to be very useful to

discover the correlations among protons of each sugar units (Lee-Juian et al., 1994)

In 2001 and 2002, Abdel-Sattar and his co-workers published two reports on seven

pregnane glycosides namely penicillosides A-G (18-24) isolated from the EtOH extract

of C. penicillata. The plant was collected from Saudi Arabia. All seven compounds (18-

24) had the benzoyl groups present in them at differrent positions and the glycoside

chain was attached at C-3 of the pregnane part. Only compound 18 was found to be a

disaccharide whereas the remaining (19-24) were trisaccharides containing glucose,

digitalose, cymarose, allomerose and thevetose units (Abdel-Sattar et al., 2002, 2001).

10

11

12

13

Abdel-Sattar et al. (2007) carried out phytochemical investigation on C. russeliana and

reported three pregnane glycosides viz., russeliosides E-G (25-27) isolated from CHCl3

soluble fraction of the EtOH extract, via chromatography over silica gel column and

RP-18 column on HPLC. All of these three isolates had the similar aglycone portion

carrying benzoyl group at C-12 but differed from each other on the basis of sugar

moiety (Abdel-Sattar et al., 2007).

Two pregnanes characterized as 12β-O-benzoyl-3β,11α,14β‚(20R)-pentahydroxy-

pregn-5-ene (28) and 11α-O-benzoyl-3β,12β,14β,(20R)-pentahydroxy-pregn-5-ene

(29) were isolated from the fresh whole C. pauciflora plant. The phytochemical

investigation was carried out by Reddy et al. (2011). No glycoside was found to be

attached in these molecules (Reddy et al., 2011).

Elsebai et al. (2015) reported that the MeOH extract of C. retrospiciens yielded

pregnane glycoside viz., retrospinoside (30), C34H58O13, m.p. 208-210 oC, [α]D -2.3 (c,

2.2, MeOH), after fractionation and repeated chromatographic separation on silica gel

columns. The disaccharide chain attached at C-3 of the pregnane was found to be

comprised of glucose and digitalose units. As the molecule contained many hydroxyl

groups, therefore, in order to obtain better quality of spectra for assignments and

additional information, 30 was acetylated with acetic anhydride-pyridine.

Spectroscopic measurements including 1D and 2D NMR, and HRMS were performed

on the acetylated product (Elsebai and Mohamed, 2015).

Some of the isolated pregnane glycosides from various Caralluma species are listed in

Table 1.

14

15

16

Table 1: Pregnane glycosides of various Caralluma species.

S. No. Specie Name Reference

1. C. tuberculata Caratuberside C-G Abdel-Sattar et al., 2008

2. C. dalzielii Caradalzielosides A-E Oyama et al., 2007

3. C. russeliana. Russeliosides A-D Al-Yahya et al., 2000

4. C. fimbriata. Stalagmoside V Kunert et al., 2008

5. C. umbellata Carumbelloside I and II Lee-Juian et al., 1994

6. C. penicillata Penicillosides A-C Abdel-Sattar et al., 2001

7. C. stalagmifera Stalagmosides I-V Kunert et al., 2006

8. C. indica Indicosides I and II Kunert et al., 2006

9. C. lasiantha Lasianthoside A and B Qiu et al., 1999

10. C. retrospiciens Retrospinoside Elsebai et al., 2015

17

No pharmacological activity has been performed on C. flava except the antioxidant

activity of its aqueous EtOH extract. The investigation showed weaker antioxidant

activity in the DPPH and promising activity in phosphomolybdenum assay (Marwah et

al. 2007). However, extracts of various Caralluma species along with their isolated

pregnane glycosides have been investigated for their pharmacological potential.

Different fractions of the EtOH extract of C. tuberculata were tested on the growth and

viability of various types of cancer cells by Waheed et al. (2011) and his co-workers.

Its EtOAc fraction was reported to be the most anti-proliferative against MDA-MB-468

(46% ± 2.8%) and MCF-7 (94% ± 4.0%) breast cancer cell lines. Pregnane glycoside

viz., 12-O-benzoyl-20-O-acetyl-3β‚12β,14β,20β-tetrahydroxy-pregnan-3-yl-O-β-D-

glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-3-methoxy-β-D-ribopyranoside

(31) isolated from this active fraction showed moderate micromolar cytotoxicity against

both breast cancer cells (IC50: MDA-MB-468 = 25-50 μM; MCF-7 = 6.25-12.5 μM)

(Waheed et al., 2011).

In 2013, Abdallah et al. and his co-workers tested C. quadrangular MeOH extract and

its various fractions for their cytotoxic activity against MCF-7 breast cancer cells. The

phytochemical investigation of the bioactive chloroform fraction yielded four pregnane

glycosides (32-35) having benzoyl groups attached at C-12 and C-20 of the pregnane

skeleton. The cytotoxicity of these isolates were tested against the same cancer cell line

using doxorubicin as positive control. Pregnane glycoside 32 (IC50 = 4.8 μM), 33 (IC50

= 2.0 μM) and 35 (IC50 = 8.0 μM) showed promising cytotoxic activities. Whereas, 34

was reported to be inactive (Abdallah et al., 2013).

Recently (2015), the MeOH extract of six indigenous folk medicinal plants (Arnebia

decumbens, Caralluma sinaica, Fagonia tenuifolia, Lavandula pubescens, Sonchus

oleraceus, Verbesina encelioides) growing wild in Saudi Arabia were tested for their

anticancer activity using three cancer cell lines (breast, lung and central nervous

system). Among these plants extracts tested, the most potent was C. sinaica which

showed strong anticancer activity against MCF-7, SF-268 and NCI-H460 cancer cell

lines with IC50 = 0.60, 2.01 and 0.02 μg/L, respectively (Albalawi et al., 2015).

18

OH

RO

Me

MeOO

O

OMe

Me

Pregnane Glycoside (31): R = Glc (1-4) Glc (1-4) 3-Methoxy-ribopyranose

19

20

2. PRESENT WORK

21

In the present study, organic extract (CH2Cl2:MeOH, 1:1) of squeezed sap from fresh

C. flava stems afforded shiny yellow crude crystals (CR) whereas the squeezed residual

plant was dried and powdered to extract with methanol (ME) which was solvent

partitioned to finally obtain various fractions (n-hexane: HX; dichloromethane: DM;

ethyl-acetate: EA; n-butanol: BU and aqueous: AQ). Crystals (CR), ME and its

fractions were first investigated for their effect on breast cancer cells (MDA-MB-231).

Interestingly, CR appeared to be more effective than ME and its fractions.

Crude crystals (CR) were subjected to column chromatography using gradients of

CH2Cl2:MeOH which provided nine new pregnane glycosides namely desmiflavaside

A-D (1-4), nizwaside (5) and desflavaside A-D (6-9). The structures of these isolates

(1-9) were established through various spectroscopic techniques including 1D (1H-1H

TOCSY, NOE) and 2D NMR (NOESY, 1H-1H COSY, HMBC), UV, IR and mass

spectrometry such as ESIMS and HRESIMS.

The effects of purified compounds 3-5 against MDA-MB-231 breast cancer cells and

SKOV-3 ovarian cancer cells in culture were examined. All tested compounds showed

significant anticancer activities. Compounds 3-5 were also tested for their cytotoxic

effect on normal breast epithelial (MCF-10-2A) cells and results illustrated that 3 was

slightly cytotoxic towards normal cells whereas 4 and 5 showed no major cytotoxic

effects and their cytotoxicity was selective for the cancer cells only.

Molecular docking studies of compounds 3-5 illustrated that all these three ligand

molecules have a good potential to accurately interact with the target protein tyrosine

phosphatase. Compound 4 demonstrated a comparatively promising docking energy as

compared to 3 and 5.

Furthermore, urease, acetylcholinesterase and α-glucosidase enzyme inhibition and

DPPH antioxidant activities of CR, isolated compounds (1-5) and different fractions of

ME were determined. The results showed no remarkable activity.

The structures of nine new pregnane glycosides isolated from CR are shown on the

following pages.

22

Structures of New Pregnane Glycosides

Desmiflavaside A (1)

Desmiflavaside B (2)

23

Desmiflavaside C (3)

Desmiflavaside D (4)

24

Nizwaside (5)

Desflavaside A (6)

25

Desflavaside B (7)

Desflavaside C (8)

26

Desflavaside D (9)

27

3. EXPERIMENTAL

28

3.1 General Note

Fresh whole C. flava plants were collected in December 2013 from the Al-Hajar

(Arabic: جبال الحجر means "Stone Mountains") mountainous region of Sultanate of

Oman including Jabal-Akhdar (Arabic: الجبل األخضر means "Green Mountains") and

Jabal-Shams (Arabic: جبل شمس, means "The Mountain of Sun") and were identified by

Dr. Gilani (plant taxonomist). A voucher specimen (BSBHPR-012/2013) was

submitted in the Herbarium of the University of Nizwa, Sultanate of Oman.

The IR and UV spectra were measured on ATR-Tensor 37 and Shimadzu UV-240

spectrophotometers, respectively. Optical rotations of the molecules were

recorded on KRUSS-P-P3000 polarimeter. The ESIMS and HRESIMS spectrum were

measured on Waters Quattro Premier XE Mass and Agilent 6529B Q-TOF Mass

spectrometers, respectively. The 1H-NMR was recorded on Bruker spectrometer

operating with cryoprobe prodigy at 600 MHz, while 13Carbon NMR was

obtained at 150 MHz. The chemical shifts and coupling constant values are

reported in part per million (ppm) and in hertz (Hz), respectively.

Silica gel PF254 (Merck) was used for column chromatography (CC) and for TLC pre-

coated aluminum sheet. Plates of TLC were visualized in the UV-light at 254 and 366

nm and spray of ceric sulfate reagent was used as locating agent.

All the experimental work along with spectroscopic techniques were performed

at the University of Nizwa (UoN). The anticancer activities were performed by Dr.

Husain Yar Khan at UoN. Enzyme inhibition and antioxidant activities were performed

by Dr. Ghulam Abbas.

29

3.2 Extraction and Isolation

Drops of sap exudate oozed out from fresh broken stems of succulent C. flava (30 kg)

were collected by gentle squeezing the juicy stems into a small beaker until it was filled

with 40 mL of sap (Scheme I). It was then extracted with a mixture of CH2Cl2-MeOH

(1:1) by slowly adding the solvent that turned the sap into white milky precipitates

which were left to settle down. The supernatant liquid which dissolved the major

portion of the sap was successively filtered with the help of whatman filter paper and

the filtrate was left for 24 hours in fume hood for drying. After the evaporation of

solvent, the dried beaker was found to have yellow shiny crude crystals (CR; 1.0 g)

(Fig. 5).

A B C

Figure 5: (A) Sap exudate of C. flava (B) Sap precipitates (C) Crude crystals

The crystals (CR) were subjected to CC (n-hexane-CH2Cl2; CH2Cl2; CH2Cl2-MeOH in

order of increasing polarity) which ultimately furnished 10 fractions (Frcn-1 to Frcn-

10) on combining the eluates on the basis of TLC (Scheme II). Frcn-7 obtained on

elution with CH2Cl2-MeOH (8.5:1.5) showed two major spots on TLC which on

separation through preparative TLC (two times; EtOAc-MeOH, 4.0:6.0) gave

Desmiflavaside A (1; 5.0 mg) and Desmiflavaside B (2; 7.0 mg). Frcn-3 on elution with

CH2Cl2-MeOH (9.5:0.5) yielded Desmiflavaside C (3; 6.7 mg). Frcn-1 on elution with

CH2Cl2-MeOH (9.6:0.4) yielded Desmiflavaside D (4; 6.2 mg). Frcn-2 on elution with

CH2Cl2-MeOH (9.6:0.4) provided Nizwaside (5; 12.0 mg). Frcn-4 obtained on elution

30

with CH2Cl2-MeOH (9.2:0.8) was further subjected to CC (CH2Cl2; CH2Cl2-MeOH in

order of increasing polarity) which furnished 5 fractions (Frcn-4-I to Frcn-4-V) on

combining the eluates on the basis of TLC (Scheme III). Frcn-4-III on elution with

CH2Cl2-MeOH (9.6:0.4) afforded two compounds Desflavaside A (6; 5.0 mg) and

Desflavaside B (7; 5.2 mg) after separation through preparative TLC (two times;

EtOAc-MeOH; 9.0:1.0). Frcn-4-IV obtained on elution with CH2Cl2-MeOH (9.5:0.5)

was further subjected to CC (CH2Cl2; CH2Cl2-MeOH in order of increasing polarity)

which resulted 5 fractions (Frcn-4-IV-1 to Frcn-4-IV-5) on combining the eluates on

the basis of TLC (Scheme III). Frcn-4-IV-4 obtained on elution with CH2Cl2-MeOH

(9.5:0.5) afforded two compounds Desflavaside C (8; 4.0 mg) and Desflavaside D (9;

4.8 mg). The rest of the fractions containing several compounds in the minor quantities

were not pursued further in the present working.

Powdered (2.0 kg) of air dried squeezed residual plant material (= remaining herbage

after squeezing out sap) was repeatedly (thrice) extracted with methanol at normal room

temperature. The syrupy concentrate (ME; 170 g), obtained on solvent removal from

the combined extracts under reduced pressure, was partitioned between EtOAc and H2O

(AQ; 35 g) (Scheme IV). The EtOAc phase was treated with Na2CO3 which separated

the acidic form from the neutral fraction. The EtOAc layer carrying the neutral fraction

was washed with water (Na2SO4) and treated with activated charcoal. It was then

filtered and its filtrate was successfully solvent freed under reduced pressure giving the

neutral fraction (N; 100 g). The charcoal bed was repeatedly eluted with MeOH-C6H6

(1:1) which on usual work-up gave another part of neutral fraction (N'; 20 g). Both N

and N' were mixed together after comparison of their TLC (silica gel PF254; CH2Cl2-

MeOH; 9.5:0.5). The total neutral fraction (TNF; 120 g) thus obtained was partitioned

into hexane soluble (HX; 30 g) and hexane insoluble portions (Scheme V). The hexane

insoluble fraction was again partitioned into DCM soluble (DM; 40 g) and DCM

insoluble portions. The DCM insoluble fraction was again partitioned into EtOAc

soluble (EA; 30 g) and EtOAc insoluble portions. The EtOAc insoluble fraction was

further partitioned into n-BuOH soluble (BU; 18 g) and BuOH insoluble portions. The

darkish BuOH insoluble fraction was very minor in quantity with several spots on TLC,

therefore neglected.

31

SCHEME IV

Extraction and Isolation

Fresh Caralluma flava plant

Stems squeezing by hands

Squeezed sap Squeezed residual plant material

+ CH2Cl2-MeOH

(1:1)

Drying and grindingPrecipitation

Powdered materialFiltration

Residue Filtrate

Neglected Crude crystals(CR)*

Drying at room temperature

Whatman filter paper

SCHEME I

* Denotes the sample exhibiting anticancer activity.

SCHEME II

32

SCHEME II

* Denotes the compounds exhibiting anticancer activity.

CR

CC

(n-hexane-CH2Cl2; CH2Cl2; CH2Cl2-MeOH

in order of increasing polarity)

Frcn-5

Preparative TLCEtOAc-MeOH(4.0:6.0)

Desmiflavaside A (1) Desmiflavaside B (2)*

Frcn-7Frcn-6Frcn-4Frcn-3Frcn-2Frcn-1 Frcn-8 Frcn-9 Frcn-10

Desmiflavaside D (4)*

Nizwaside (5)*

Desmiflavaside C (3)*

CC

CH2Cl2-MeOH

(9.6:0.4)

CC

CH2Cl2-MeOH

(9.6:0.4)

CC

CH2Cl2-MeOH

(9.5:0.5)

SCHEME III

33

SCHEME III

Desflavaside C (8) Desflavaside D (9)

CC

CH2Cl2:MeOH

(9.5:0.5)

Frcn-4

CC

(CH2Cl2; CH2Cl2-MeOH


Frcn-4-I Frcn-4-II Frcn-4-VFrcn-4-IVFrcn-4-III

Preparative TLCEtOAc-MeOH(9.0:1.0)

Desflavaside A (6) Desflavaside B (7)

CC

(CH2Cl2; CH2Cl2-MeOH


Frcn-4-IV-1 Frcn-4-IV-4Frcn-4-IV-3Frcn-4-IV-2 Frcn-4-IV-5

34

* Denotes the fractions exhibiting anticancer activity.

+ MeOH (3 times repeatedly;room temprature)

Powdered Caralluma flava

Methanolic Extract

Solvent removed under vaccum

Crude Extract (ME)*

+EtOAc

+ H2O

EtOAc Phase Aqueous Phase (AQ)*

+Aqueous Na2CO3 (4%)

EtOAc Phase

1) General work up2) Charcoal

EtOAc eluate Charcoal bed

solvent removed underreduced pressure

MeOH-C6H6

(1:1)

MeOH-C6H6 eluate

solvent removed under vaccum

combined

Total neutral fraction(TNF)

Aqueous Na2CO3 phase

(Not worked up)

Neutral fraction(N')

Neutral fraction(N)

SCHEME IV

SCHEME V

35

SCHEME V

+ n-Hexane

n-Hexanesoluble fraction

(HX)*

n-Hexane insoluble fraction

+ Dichloromethane (DCM)

DCM soluble fraction

(DM)*

DCM insoluble fraction

+EtOAc

EtOAc insoluble fraction

EtOAcsoluble fraction

(EA)*

+ n-BuOH

n- BuOHinsoluble fraction

(neglected)

n- BuOHsoluble fraction

(BU)*

Total neutral fraction(TNF)

* Denotes the fractions exhibiting anticancer activity.

36

3.2.1 Characterization of Desmiflavaside A (1)

It was obtained as white solid which was readily soluble in MeOH. Its molecular

formula was established as C47H72O19.

Percentage purity: 90%

[α]D25: –4.5 (CH3OH, c 0.04)

UV (CH2Cl2) λmax (log ε): 241 (3.66), 283 (3.30) nm

IR (KBr): 3400, 1710, 1660, 1610, 1450, 1060 cm-1

ESI-MS (m/z): 963.1 [M+Na]+ (89) (C47H72NaO19).

HR-ESIMS: 963.4553 (calculated for C47H72NaO19, 963.4560).

1H and 13C NMR (600 and 150 MHz respectively, CD3OD): Table 2

37

Table-2: 1H and 13C NMR data of Desmiflavaside A (1)x,y

No. Multiplicity δH (J, Hz) δC

1. C-H2 1.76 (m), 1.02 (m) 38.3

2. C-H2 1.85 (m), 1.49 (m) 30.4

3. C-H 3.58 m 79.4

4. C-H2 1.63 (m), 1.29 (m) 35.2

5. C-H 1.00 (m) 45.6

6. C-H2 1.56 (m), 1.30 (m) 21.9

7. C-H2 1.32 (m), 1.20 (m) 30.0

8. C-H 1.72 (m) 41.4

9. C-H 1.06 (m) 50.5

10. C - 37.1

11. C-H2 2.09 (m), 1.03 (m) 28.4

12. C-H2 1.46 (m), 1.41 (m) 40.1

13. C - 48.0

14. C - 82.4

15. C-H 5.60 (m) 77.3

16. C-H2 2.50 (m), 1.75 (m) 27.3

17. C-H 1.62 (m) 54.8

18. C-H3 1.11 (s) 15.6

19. C-H3 0.84 (s) 12.8

20. C-H 4.03 (m) 66.0

21. C-H3 1.07 (d, 6.6) 21.9

Bz(15)

C=O C - 167.8

1ʹ C - 131.7

2ʹ, 6ʹ C-H, C-H 8.09 (dd, 2.0, 7.0) 130.7

3ʹ, 5ʹ C-H, C-H 7.47 (t, 7.0) 129.5

4ʹ C-H 7.60 (t, 7.0) 134.2

38

Table 2: Continued ………


Dig

1″ C-H 4.30 (d, 7.8) 102.8

2″ C-H 3.55 (m) 71.3

3″ C-H 3.18 (m) 85.7

4″ C-H 4.15 (d, 2.4) 74.8

5″ C-H 3.60 (m) 71.6

6″ C-H3 1.26 (d, 6.0) 17.5

OMe OCH3 3. 49 (s) 58.5

Glc-I

1‴ C-H 4.57 (d, 7.8) 104.1

2‴ C-H 3.19 (m) 75.8

3‴ C-H 3.33 (m) 77.8

4‴ C-H 3.26 (m) 71.8

5‴ C-H 3.43 (ddd, 2.0, 6.0, 8.3) 77.4

6‴ C-H2 4.12 (dd, 2.0, 12.0) 70.3

3.76 (dd, 6.0, 12.0)

Glc-II

1‴′ C-H 4.38 (d, 7.8) 105.0

2ʹ‴ C-H 3.16 (m) 75.1

3ʹ‴ C-H 3.32 (m) 78.0

4ʹ‴ C-H 3.25 (m) 71.6

5ʹ‴ C-H 3.24 (m) 78.0

6ʹ‴ C-H2 3.85 (dd, 2.4, 12.0) 62.0

3.64 (dd, 5.4, 12.0)

x Values were assigned using NOESY, 1H-1H COSY, HMBC and HSQC 2D-NMR

spectra, as well as by comparing with literature reports for other pregnane glycosides.

y DEPT experiments assessed for multiplicity determination and J values are

mentioned in parentheses.

39

3.2.2 Characterization of Desmiflavaside B (2)




[α]D25: –4.1 (CH3OH, c 0.05)


IR (KBr): 3350, 1710, 1665, 1625, 1450, 1060 cm-1




40

Table 3: 1H and 13C NMR data of Desmiflavaside B (2)x,y


1. C-H2 1.66 (m), 1.50 (m) 39.5

2. C-H2 1.84 (m), 1.48 (m) 30.3

3. C-H 3.59 (m) 79.4

4. C-H2 1.63 (m), 1.28 (m) 35.2

5. C-H 0.99 m 45.6

6. C-H2 1.58 (m), 1.33 (m) 21.7

7. C-H2 1.19 (m) 29.9

8. C-H 1.68 (m) 41.5

9. C-H 1.03 (m) 50.3

10. C - 37.1

11. C-H2 2.11 (m), 0.99 (m) 28.3

12. C-H2 1.66 (m), 1.50 (m) 39.5

13. C - 49.0

14. C - 83.7

15. C-H 5.63 (m) 77.3

16. C-H2 2.69 (m), 1.87 (m) 32.9

17. C-H 2.91 (m) 60.8

18. C-H3 1.03 (s) 16.4

19. C-H3 0.83 (s) 12.8

20. C - 218.2

21. C-H3 2.26 (s) 32.6

Bz (15)

C=O C 167.6

1ʹ C 131.5

2ʹ, 6ʹ CH, CH 8.08 (m) 129.6

3ʹ, 5ʹ CH, CH 7.48 (m) 130.7

4 ʹ CH 7.60 (m) 134.4

41



Dig

1″ C-H 4.29 (d, 7.8) 102.8

2″ C-H 3.55 (m) 71.6

3″ C-H 3.18 (m) 85.7

4″ C-H 4.16 (d, 2.4) 74.8

5″ C-H 3.59 (m) 71.3

6″ C-H3 1.25 (d, 6.6) 17.5

OMe OCH3 3.47 (s) 58.6

Glc-I

1‴ C-H 4.57 (d, 7.8) 104.1

2‴ C-H 3.19 (m) 75.8

3‴ C-H 3.33 (m) 77.7

4‴ C-H 3.25 (m) 71.8

5‴ C-H 3.43 (m) 77.4

6‴ C-H2 4.12 (dd, 1.8, 12.0) 70.3

3.75 (dd, 6.6, 12.0)

Glc-II

1ʹ‴ C-H 4.38 (d, 7.8) 105.0

2ʹ‴ C-H 3.17 (m) 75.1

3ʹ‴ C-H 3.32 (m) 78.0

4ʹ‴ C-H 3.26 (m) 71.6

5ʹ‴ C-H 3.23 (m) 78.0

6ʹ‴ C-H2 3.85 (dd, 1.8, 12.0) 62.7

3.64 (dd, 6.6, 12.0)





42

3.2.3 Characterization of Desmiflavaside C (3)




[α]D25: –4.5 (CH3OH, c 0.05)


IR (KBr): 3410, 1710, 1615, 1450, 1060 cm-1




43

Table 4: 1H and 13C NMR data of Desmiflavaside C (3)x,y


1. C-H2 1.66 (m), 1.04 (m) 38.0

2. C-H2 1.82 (m), 1.28 (m) 30.3

3. C-H 3.63 (m) 78.5

4. C-H2 1.63 (m), 1.20 (m) 35.5

5. C-H 1.13 (m) 45.6

6. C-H2 1.37 (m), 1.27 (m) 29.8

7. C-H2 2.03 (m), 1.13 (m) 28.6

8. C-H 1.69 (m) 41.2

9. C-H 1.08 (m) 47.4

10. C - 36.9

11. C-H2 1.72 (m), 1.46 (m) 27.5

12. C-H 4.95 (dd, 4.5, 12.0) 79.5

13. C - 53.7

14. C - 87.2

15. C-H2 1.89 (m), 1.67 (m) 32.3

16. C-H2 2.03 (m), 1.66 (m) 26.0

17. C-H 2.25 (m) 50.9

18. C-H3 1.09 (s) 10.0

19. C-H3 0.82 (s) 12.4

20. C-H 5.24 (dd, 6.0, 10.0) 75.6

21. C-H3 1.20 (d, 6.0) 19.7

Bz(12)

C=O C - 168.0

1ʹ C - 132.0

2ʹ, 6ʹ C-H, C-H 8.03 (d, 7.2) 130.7

3ʹ, 5ʹ C-H, C-H 7.50 (t, 7.2) 129.8

4 ʹ C-H 7.68 (t, 7.2) 134.3

44



Bz(20)

C=O C - 167.4

1ʹ C - 132.0

2ʹ, 6ʹ C-H, C-H 7.73 (d, 7.2) 130.3

3ʹ, 5ʹ C-H, C-H 7.22 (t, 7.2) 129.4

4ʹ C-H 7.51 (t, 7.2) 133.8

CymI

1″ C-H 4.85 (dd, 1.8, 9.6) 97.1

2″ C-H2 2.04 (m), 1.51 (m) 36.6

3″ C-H 3.83 (m) 78.5

4″ C-H 3.25 (m) 83.8

5″ C-H 3.79 (dq, 6.0, 10.0) 69.9

6″ C-H3 1.19 (d, 6.0) 18.5

OMe O-CH3 3.41 (s) 58.4

CymII

1‴ C-H 4.57 (dd, 1.8, 9.6) 102.7

2‴ C-H2 2.31 (m), 1.34 (m) 37.3

3‴ C-H 2.96 (t, 9.0) 76.9

4‴ C-H 3.18 (m) 81.6

5‴ C-H 3.25 (dd, 6.0, 10.0) 73.2

6‴ C-H3 1.26 (d, 6.0) 18.3

OMe O-CH3 3.41 (s) 57.3

x Values were assigned using NOESY, 1H-1H COSY, HMBC and HSQC 2D NMR




45

3.2.4 Characterization of Desmiflavaside D (4)




[α]D25: –4.1 (CH3OH, c 0.05)


IR (KBr): 3410, 1715, 1610, 1455, 1070 cm-1




46

Table 5: 1H and 13C NMR data of Desmiflavaside D (4)x


1. C-H2 1.67 (m), 1.04 (m) 38.0

2. C-H2 1.83 (m), 1.28 (m) 30.3

3. C-H 3.62 (m) 78.5

4. C-H2 1.63 (m), 1.20 (m) 35.5

5. C-H 1.13 (m) 45.6

6. C-H2 1.37 (m), 1.28 (m) 29.8

7. C-H2 2.02 (m), 1.13 (m) 28.6

8. C-H 1.69 (m) 41.2

9. C-H 1.09 (m) 47.4

10. C - 36.9

11. C-H2 1.71 (m), 1.46 (m) 27.5

12. C-H 4.95 (dd, 4.5, 12.0) 79.5

13. C - 53.7

14. C - 87.2

15. C-H2 1.90 (m), 1.67 (m) 32.3

16. C-H2 2.03 (m), 1.66 (m) 26.0

17. C-H 2.26 (m) 50.9

18. C-H3 1.09 (s) 10.0

19. C-H3 0.82 (s) 12.4

20. C-H 5.24 (dd, 6.0, 10.0) 75.6

21. C-H3 1.19 (d, 6.0) 19.7

Bz (12)

C=O C - 168.0

1ʹ C - 132.0

2ʹ, 6ʹ C-H, C-H 8.03 (d, 8.5) 130.7

3ʹ, 5ʹ C-H, C-H 7.50 (t, 7.8) 129.8

4 ʹ C-H 7.68 (t, 7.8) 134.3

47



Bz (20)

C=O C - 167.4

1ʹ C - 132.0

2ʹ, 6ʹ C-H, C-H 7.73 (d,7.8) 130.3

3ʹ, 5ʹ C-H, C-H 7.22 (t, 7.8) 129.4

4ʹ C-H 7.51 (t, 7.8) 133.8

CymI

1″ C-H 4.86 (dd, 2.0, 12.0) 97.1

2″ C-H2 2.04 (m), 1.51(m) 36.6

3″ C-H 3.82 (m) 78.5

4″ C-H 3.25 (m) 83.8

5″ C-H 3.80 (m) 69.8

6″ C-H3 1.19 (d, 6.0) 18.5

OMe O-CH3 3.41 (s) 58.4

CymII

1‴ C-H 4.59 (dd, 2.0, 12.0) 102.5

2‴ C-H2 2.32 (m), 1.43 (m) 37.9

3‴ C-H 3.40 (m) 80.1

4‴ C-H 3.26 (m) 83.5

5‴ C-H 3.39 (m) 72.7

6‴ C-H3 1.36 (d, 6.0) 18.7

OMe O-CH3 3.46 (s) 58.2

Glc

1ʹ‴ C-H 4.43 (d, 7.8) 104.1

2ʹ‴ C-H 3.15 (dd, 7.8, 9.0) 75.5

3ʹ‴ C-H 3.32 (t, 8.7) 78.0

4ʹ‴ C-H 3.22 (m) 71.8

5ʹ‴ C-H 3.22 (m) 78.3

6ʹ‴ C-H2 3.85 (dd, 2.0, 12.0) 63.0

3.62 (dd, 6.0, 12.0)

x Values were assigned using 1D and 2D NMR spectra.

48

3.2.5 Characterization of Nizwaside (5)




[α]D25: –4.0 (CH3OH, c 0.04)


IR (KBr): 1710, 3400, 1620, 1060, 1450 cm-1




49

Table 6: 1H and 13C NMR data of Nizwaside (5)x


1. C-H2 0.94 (m), 1.65 (m) 38.0

2. C-H2 2.05 (m), 2.13 (m) 28.7

3. C-H 3.65 (m) 78.5

4. C-H2 1.22 (m), 1.68 (m) 35.5

5. C-H 1.27 (m) 45.6

6. C-H2 1.44 (m), 1.71 (m) 27.5

7. C-H2 1.53 (m), 2.01 (m) 36.6

8. C-H 1.70 (m) 41.2

9. C-H 1.10 (m) 47.4

10. C - 36.9

11. C-H2 1.66 (m), 2.02 (m) 26.0

12. C-H 4.95 (dd, 4.5, 12.0) 79.4

13. C - 53.7

14. C - 87.2

15. C-H2 1.38 (m), 1.83 (m) 29.8

16. C-H2 1.66 (m), 1.90 (m) 32.3

17. C-H 2.25 (m) 50.9

18. C-H3 1.09 (s) 10.0

19. C-H3 0.82 (s) 12.4

20. C-H 5.24 (dd, 6.0, 10.0) 75.7

21. C-H3 1.35 (d, 6.0) 18.9

Bz (12)

C=O C 168.0

1ʹ C 132.0

2ʹ/6ʹ C-H, C-H 8.03 (dd, 1.2, 8.4) 130.7

3ʹ/5ʹ C-H, C-H 7.50 (t, 8.4) 129.8

4ʹ C-H 7.67 (t, 8.4) 134.3

50



Bz (20)

C=O C 167.4

1ʹ C 132.0

2ʹ/6ʹ C-H, C-H 7.73 (dd, 1.2, 8.4) 130.3

3ʹ/5ʹ C-H, CH 7.22 (t, 8.4) 129.5

4ʹ C-H 7.52 (t, 8.4) 133.9

Allom

1″ C-H 4.71 (d, 8.4) 102.6

2″ C-H 3.36 (m) 72.5

3″ C-H 3.61 (m) 83.7

4″ C-H 3.30 (m) 73.6

5″ C-H 3.33 (m) 72.5

6″ C-H3 1.19 (d, 6.0) 18.9

OMe O-CH3 3.59 (s) 62.5

Cym-I

1‴ C-H 4.57 (dd, 2.0, 9.0) 102.2

2‴ C-H2 1.22 (m), 1.67 (m) 35.5

3‴ C-H 3.83 (m) 78.5

4‴ C-H 3.67 (m) 83.8

5‴ C-H 3.64 (m) 71.2

6‴ C-H3 1.22 (d, 6.0) 18.2

OMe O-CH3 3.40 (s) 57.4

Cym-II

1ʹ‴ C-H 4.84 (dd, 2.0, 9.0) 97.1

2ʹ‴ C-H2 1.55 (m), 2.02 (m) 36.6

3ʹ‴ C-H 3.83 (m) 78.7

4ʹ‴ C-H 3.24 (m) 83.9

5ʹ‴ C-H 3.80 (m) 69.9

6ʹ‴ C-H3 1.20 (d, 6.0) 18.5

OMe O-CH3 3.41 (s) 58.4

x Values were assigned using 1D and 2D NMR spectra.

51

3.2.6 Characterization of Desflavaside A (6)




[α]D25: –5.4 (CH3OH, c 0.06)


IR (KBr): 3410, 1710, 1610, 1455, 1060 cm-1




52

Table 7: 1H and 13C NMR data of Desflavaside A (6)x,y


1. C-H2 1.66 (m), 1.06 (m) 38.0

2. C-H2 1.82 (m), 1.28 (m) 30.3

3. C-H 3.62 (m) 78.5

4. C-H2 1.63 (m), 1.20 (m) 35.5

5. C-H 1.14 (m) 45.6

6. C-H2 1.38 (m), 1.28 (m) 29.8

7. C-H2 2.03 (m), 1.13 (m) 28.7

8. C-H 1.70 (m) 41.2

9. C-H 1.09 (m) 47.4

10. C 36.9

11. C-H2 1.72 (m), 1.47 (m) 27.5

12. C-H 4.94 (dd, 4.2, 12.0) 79.5

13. C 53.7

14. C 87.2

15. C-H2 1.89 (m), 1.67 (m) 32.3

16. C-H2 2.03 (m), 1.66 (m) 26.0

17. C-H 2.25 (m) 50.8

18. C-H3 1.09 (s) 10.0

19. C-H3 0.82 (s) 12.4

20. C-H 5.24 (m) 75.6

21. C-H3 1.20 (d, 6.6) 19.7

Bz (12)

C=O C 168.0

1ʹ C 132.0

2ʹ, 6ʹ C-H, C-H 8.03 (dd, 1.0, 6.0) 130.7

3ʹ, 5ʹ C-H, C-H 7.50 (t, 6.0) 129.8

4 ʹ C-H 7.67 (t, 6.0) 134.3

53



Bz (20)

C=O C 167.4

1ʹ C 132.0

2ʹ, 6ʹ C-H, C-H 7.73 (dd, 1.0, 6.0) 130.3

3ʹ, 5ʹ C-H, C-H 7.22 (t, 6.0) 129.4

4ʹ C-H 7.51 (t, 6.0) 133.8

Cym-I C-H 4.85 (dd, 2.0, 6.0) 97.1

1″ C-H2 2.03 (m), 1.51 (m) 36.6

2″ C-H 3.36 (m) 80.4

3″ C-H 3.24 (m) 83.9

4″ C-H 3.81 (m) 69.9

5″ C-H3 1.18 (d,6.0) 18.5

6″ O-CH3 3.40 (s) 57.5

OMe

Glc

1‴ C-H 4.33 ( d, 7.2) 106.2

2‴ C-H 3.17 (m) 75.5

3‴ C-H 3.33 (m) 77.9

4‴ C-H 3.24 (m) 78.5

5‴ C-H 3.28 (m) 78.0

6‴ C-H2 3.90 (dd, 2.0, 11.7) 63.0

3.64 (dd, 6.0, 11.7)

Cym-II

1ʹ‴ C-H 4.56 (dd, 2.0, 10.0) 102.5

2ʹ‴ C-H2 2.30 (m), 1.40 (m) 37.5

3ʹ‴ C-H 3.82 (m) 78.5

4ʹ‴ C-H 3.17(m) 83.8

5ʹ‴ C-H 3.35 (m) 72.5

6ʹ‴ C-H3 1.29 (d, 6.0) 18.1

OMe O-CH3 3.41 (s) 58.4

54



Allom

1″‴ C-H 4.70 (d, 8.0) 102.1

2″‴ C-H 3.31 (m) 72.9

3″‴ C-H 3.94 (t, 3.5) 83.2

4″‴ C-H 3.32 (m) 83.9

5″‴ C-H 3.81 (m) 70.1

6″‴ C-H3 1.35 (d, 6.0) 18.9

OMe O-CH3 3.58 (s) 61.9





55

3.2.7 Characterization of Desflavaside B (7)




[α]D25: –4.7 (CH3OH, c 0.05)


IR (KBr): 3415, 1710, 1610, 1455, 1070 cm-1




56

Table 8: 1H and 13C NMR data of Desflavaside B (7)x,y


1. C-H2 1.66 (m), 1.04 (m) 38.0

2. C-H2 1.82 (m), 1.27 (m) 30.3

3. C-H 3.62 (m) 78.5

4. C-H2 1.62 (m), 1.19 (m) 35.5

5. C-H 1.14 (m) 45.6

6. C-H2 1.38 (m), 1.28 (m) 29.8

7. C-H2 2.03 (m), 1.13 (m) 28.6

8. C-H 1.69 (m) 41.2

9. C-H 1.09 (m) 47.4

10. C 36.9

11. C-H2 1.71 (m), 1.46 (m) 27.5

12. C-H 4.95 (dd, 4.5, 12.0) 79.5

13. C 53.7

14. C 87.2

15. C-H2 1.89 (m), 1.67 (m) 32.3

16. C-H2 2.03 (m), 1.66 (m) 26.0

17. C-H 2.25 (m) 50.9

18. C-H3 1.09 (s) 10.0

19. C-H3 0.82 (s) 12.4

20. C-H 5.23 (m) 75.6

21. C-H3 1.19 (d, 6.0) 19.7

Bz(12)

C=O C 168.0

1ʹ C 132.0

2ʹ, 6ʹ C-H, C-H 8.03 (dd, 1.2, 8.0) 130.7

3ʹ, 5ʹ C-H, C-H 7.50 (t, 8.0) 129.8

4 ʹ C-H 7.68 (t, 8.0) 134.3

57



Bz(20)

C=O C 167.4

1ʹ C 132.0

2ʹ, 6ʹ C-H, C-H 7.73 (dd, 1.0, 7.8) 130.3

3ʹ, 5ʹ C-H, C-H 7.22 (t, 7.8) 129.4

4ʹ C-H 7.51 (t, 7.8) 133.8

Cym-I

1″ C-H 4.85 (dd, 2.0, 9.6) 97.1

2″ C-H2 2.03 (m), 1.51 (m) 36.6

3″ C-H 3.82 (m) 78.5

4″ C-H 3.24 (m) 83.8

5″ C-H 3.80 (m) 69.9

6″ C-H3 1.19 (d,6.0) 18.8

OMe O-CH3 3.41 (s) 58.4

Glc

1‴ C-H 4.41( d, 7.8) 104.3

2‴ C-H 3.15 (m) 75.6

3‴ C-H 3.33 (m) 78.0

4‴ C-H 3.24 (m) 78.5

5‴ C-H 3.22 (m) 78.3

6‴ C-H2 3.85 (m), 3.62 (m) 63.1

Cym-II

1ʹ‴ C-H 4.57 (dd, 2.0, 9.6) 102.5

2ʹ‴ C-H2 2.30 (m), 1.40 (m) 37.6

3ʹ‴ C-H 3.36 (m) 80.2

4ʹ‴ C-H 3.19 (m) 84.3

5ʹ‴ C-H 3.37 (m) 72.5

6ʹ‴ C-H3 1.36 (d, 6.0) 18.4

OMe O-CH3 3.40 (s) 57.6

58



Thev

1″‴ C-H 4.43 (d, 7.8) 104.2

2″‴ C-H 3.22 (m) 75.2

3″‴ C-H 3.16 (m) 86.3

4″‴ C-H 3.34 (m) 82.9

5″‴ C-H 3.42 (m) 72.6

6″‴ C-H3 1.36 (d,6.0) 18.5

OMe O-CH3 3.61 (s) 61.2

x Values were assigned using NOESY, 1H-1H COSY, HMBC and HSQC 2D NMR




59

3.2.8 Characterization of Desflavaside C (8)




[α]D25: –6.1 (CH3OH, c 0.06)


IR (KBr): 3415, 1715, 1615, 1455, 1070 cm-1




60

Table 9: 1H and 13C NMR data of Desflavaside C (8)x,y


1. C-H2 1.66 (m), 1.04 (m) 38.0

2. C-H2 1.82 (m), 1.27 (m) 30.3

3. C-H 3.64 (m) 78.5

4. C-H2 1.63 (m), 1.19 (m) 35.5

5. C-H 1.14 (m) 45.6

6. C-H2 1.38 (m), 1.28 (m) 29.8

7. C-H2 2.03 (m), 1.12 (m) 28.7

8. C-H 1.69 (m) 41.2

9. C-H 1.09 (m) 47.4

10. C 36.9

11. C-H2 1.71 (m), 1.47 (m) 27.5

12. C-H 4.95 (dd, 4.8, 12.0) 79.5

13. C 53.7

14. C 87.2

15. C-H2 1.89 (m), 1.67 (m) 32.3

16. C-H2 2.03 (m), 1.66 (m) 26.0

17. C-H 2.25 (m) 50.9

18. C-H3 1.09 (s) 10.0

19. C-H3 0.82 (s) 12.4

20. C-H 5.24 (m) 75.7

21. C-H3 1.19 (d, 6.6) 19.7

Bz (12)

C=O C 168.0

1ʹ C 131.9

2ʹ, 6ʹ C-H, C-H 8.03 (dd, 1.2, 7.8) 130.7

3ʹ, 5ʹ C-H, C-H 7.49 (t, 7.8) 129.8

4 ʹ C-H 7.68 (t, 7.8) 134.3

61



Bz (20)

C=O C 167.4

1ʹ C 131.9

2ʹ, 6ʹ C-H, C-H 7.73 (dd, 1.2, 7.8) 130.3

3ʹ, 5ʹ C-H, C-H 7.22 (t, 7.8) 129.5

4ʹ C-H 7.51 (t, 7.8) 133.9

Cym-I

1″ C-H 4.86 (dd, 2.0, 6.0) 97.1

2″ C-H2 2.03 (m), 1.51 (m) 36.6

3″ C-H 3.82 (m) 78.5

4″ C-H 3.25 (m) 83.7

5″ C-H 3.80 (m) 69.9

6″ C-H3 1.19 (d, 6.0) 18.5

OMe O-CH3 3.41 (s) 58.4

Glc-I

1‴ C-H 4.47 ( d, 7.8) 104.0

2‴ C-H 3.22 (m) 74.9

3‴ C-H 3.38 (m) 76.7

4‴ C-H 3.50 (m) 80.9

5‴ C-H 3.50 (m) 76.4

6‴ C-H2 3.91 (dd, 12.0, 2.0) 62.1

3.80 (dd, 12.0, 6.0)

Glc-II

1ʹ‴ C-H 4.38 (d, 7.8) 104.6

2ʹ‴ C-H 3.22 (m) 75.3

3ʹ‴ C-H 3.33 (m) 78.1

4ʹ‴ C-H 3.28 (m) 79.5

5ʹ‴ C-H 3.33 (m) 77.8

6ʹ‴ C-H2 3.64 (dd, 6.0, 12.0) 62.4

3.86 (dd, 11.8, 2.1)

62



Cym-II

1‴″ C-H 4.59 (dd, 1.7, 9.6) 102.5

2‴″ C-H2 3.32 (m), 1.43 (m) 37.7

3‴″ C-H 3.40 (m) 80.1

4‴″ C-H 3.25 (m) 83.8

5‴″ C-H 3.39 (m) 72.6

6‴″ C-H3 1.37 (d, 6.0) 18.7

OMe O-CH3 3.44 (s) 57.9





63

3.2.9 Characterization of Desflavaside D (9)




[α]D25: –3.9 (CH3OH, c 0.04)

IR (KBr): 3410, 1715, 1610, 1450, 1075 cm-1




64

Table 10: 1H and 13C NMR data of Desflavaside D (9)x,y


1. C-H2 1.74 (m), 0.99 (m) 38.3

2. C-H2 1.85 (m), 1.51 (m) 30.4

3. C-H 3.64 (m) 79.5

4. C-H2 1.69 (m), 1.31 (m) 35.3

5. C-H 1.07 (m) 45.7

6. C-H2 1.45 (m), 1.27 (m) 22.2

7. C-H2 1.32 (m), 1.26 (m) 30.0

8. C-H 1.60 (m) 41.4

9. C-H 0.91 (m) 50.9

10. C 36.9

11. C-H2 2.02 (m), 1.09 (m) 28.8

12. C-H2 1.37 (m), 1.26 (m) 42.0

13. C 54.7

14. C 85.7

15. C-H2 1.95 (m), 1.53 (m) 33.2

16. C-H2 1.90 (m), 1.81 (m) 20.4

17. C-H 1.63 (m) 57.7

18. C-H3 1.07 (s) 15.5

19. C-H3 0.83 (s) 12.6

20. C-H 3.98 (m) 79.0

21. C-H3 1.27 (d, 6.0) 21.3

Glc-I

1″ C-H 4.58 (d, 7.7) 104.1

2″ C-H 2.19 (m) 75.8

3″ C-H 3.34 (m) 77.9

4″ C-H 3.26 (m) 71.9

5″ C-H 3.44 (m) 77.4

6″ C-H2 4.13 (dd, 2.0, 11.6) 70.3

3.75 (dd, 2.0, 11.6)

65



Glc-II

1‴ C-H 4.38 ( d, 7.8) 104.3

2‴ C-H 3.17 (m) 75.6

3‴ C-H 3.32 (m) 78.0

4‴ C-H 3.26 (m) 78.5

5‴ C-H 3.34 (m) 78.3

6‴ C-H2 3.86 (dd, 6.0, 11.6) 63.1

3.75 (dd, 12.0, 6.0)

Glc-III

1ʹ‴ C-H 4.36 (d, 7.9) 104.4

2ʹ‴ C-H 3.12 (m) 75.2

3ʹ‴ C-H 3.25 (m) 78.0

4ʹ‴ C-H 3.27 (m) 79.0

5ʹ‴ C-H 3.34 (m) 77.8

6ʹ‴ C-H2 3.84 (dd, 1.6, 11.6) 62.7

3.64 (dd, 6.0, 11.6)

Dig

1‴″ C-H 4.34 (d, 7.8) 102.7

2‴″ C-H 3.63 (m) 71.3

3‴″ C-H 3.22 (m) 85.8

4‴″ C-H 4.18 (d, 2.6) 74.8

5‴″ C-H 3.58 (m) 71.3

6‴″ C-H3 1.27 (d, 6.0) 17.6

OMe O-CH3 3.61 (s) 58.6





66

3.3 Biological Activities

3.3.1 Anticancer Activity

Breast (MDA-MB-231) and ovarian (SKOV-3) cancer cells were maintained in

dulbecco's modified eagle medium (DMEM) and the media was supplemented with 1%

antimycotic antibiotic and 10% fetal bovine serum (FBS). Cancer cells were cultured

in 5% CO2 humidified atmosphere at 37 oC. Normal breast epithelial (MCF-10-2A) cell

line was propagated in DMEM/F-12 supplemented with 5% horse serum, 500 ng/ml

hydrocortisone, 20 ng/ml EGF, 10 mg/ml insulin, 0.1 mg/ml cholera toxin, 100 units/ml

penicillin, and 100 mg/ml streptomycin in a 5% CO2 atmosphere at 37 oC. A 5 mg/ml

stock solution of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT) was prepared in phosphate buffer saline (PBS).

Cells were seeded in 96-well (1 x 104/well) culture plates. After 24 hours of incubation,

normal growth medium was replaced with either fresh medium or various

concentrations of test samples in medium, diluted from a 2 mg/ml (for Doxorubicin,

CR, ME and its fractions) or 2 mM stock (for compounds 3-5). After 24 hours of

incubation, MTT solution was added to each well (5 mg/ml in PBS for Doxorubicin,

CR, ME and its fractions whereas 0.1 mg/ml in DMEM for compounds (3-5) and

incubated further for 4 hours at 37 oC. Upon termination, the supernatant was aspirated

and the MTT formazan, formed by metabolically viable cells, was dissolved in a

solubilization solution containing DMSO (100 μl) by mixing for 5 minutes on a

gyratory shaker. The absorbance was measured at 540 nm on an Ultra Multifunctional

Microplate Reader. Absorbance of control was considered as 100% cell survival.

Doxorubicin was used as positive control. Values are presented as Mean ± SD of four

duplicates.

67

Figure 6: Anticancer activity of crude crystals, methanolic extract and its fractions against breast cancer cells (MDA-MB-231).

0

20

40

60

80

100

120

CR ME HX DM EA BU AQ

Cel

l S

urv

ival

(%)

Control 25 ug/ml 50 ug/ml 75 ug/ml 100 ug/ml DOX 100 µg/ml

68

Table 11: IC50 values of crude crystals, methanolic extract and its fractions for MDA-

MB-231 cancer cells.

Samples IC50 µg/ml

CR 73.6

ME 89.9

HX 78.7

DM 84.0

EA > 100

BU > 100

AQ > 100

69

Compound 3 Compound 4 Compound 5

Figure 7 (A): Anticancer activity of compounds 3-5 against MDA-MB-231 breast

cancer cells.

70


Figure 7 (B): Anticancer activity of compounds 3-5 against SKOV-3 ovarian cancer

cells.

71


Figure 7 (C): Anticancer activity of compounds 3-5 against MCF-10-2A normal

breast epithelial cells.

72

Table 12: IC50 values of compounds 3-5 for ovarian (SKOV-3) and breast (MDA-MB-

231) cancer cell lines.

Compounds IC50 (μM)

SKOV-3 MDA-MB-231

3 64.50 47.01

4 38.32 19.97

5 37.97 25.84

73

3.3.2 Enzyme Inhibition Activity

Urease Enzyme Inhibition: A solution comprising 25 μL of Jack bean Urease, 55 μL

of phosphate buffer and 100 mM urea was incubated with 5 μL (0.5 mg/mL) of the test

samples (CR, HX, DM, EA, BU, AQ, compounds 1-5) at 30 °C for 15 minutes in 96-

well plate. The production of NH3 was measured by indophenol method and used to

find the urease inhibitory activity. The phenol reagent (45 μL, 1% w/v C6H5OH and

0.005% w/v C5FeN6Na2O) and alkali reagent (70 μL, 0.5% w/v NaOH and 0.1%

NaOCl) were added to each well and the absorbance was measured at 630 nm after fifty

minutes, using a microplate reader (Molecular Device, USA). The assays were

performed at pH 8.2 (0.01 M K2HPO4.3H2O, 1.0 mM EDTA and 0.01 M LiCl2). The

experiment was replicated three times.

α-Glucosidase enzyme Inhibition: In this assay, 0.1 mg of α-glucosidase (type-1, from

Saccharomyces cerevisiae) was dissolved in 10 mL of phosphate buffer (pH 6.8). In

96-well plate, 20 μL of sample (CR, HX, DM, EA, BU, AQ, compounds 1-5) of

concentration 0.5 mg/mL was premixed with 120 μL of 50 mM phosphate buffer (pH

6.8) and 20 μL of 5 mM p-nitrophenyl α-D-glucopyranoside. The reaction mixture was

pre-incubated at 37 ◦C for 5 minutes. After that 20 μL α-glucosidase was added in

reaction wells and incubated at 37 ◦C for 15 minutes. The reaction was terminated by

the addition of 100 μL Na2CO3 (200 mM). Inhibition activity was determined

spectrophotometrically at 400 nm on spectrophotometer (SpectroMax Molecular

Devices, USA).

Acetylcholinesterase Enzyme Inhibition: In this assay, 96-well plate was used and

each reaction well contained 25 μL of 15 mM acetylcholinesterase (type VI-S from

electric eel) dissolved in water, 125 μL of 3 mM DTNB in Buffer (50 mM Tris–HCl,

pH 8.0), and 25 μL of test sample (CR, HX, DM, EA, BU, AQ, compounds 1-5) of

concentration 0.5 mg/mL dissolved in DMSO. In blank sample 25 μL inhibitor sample

was replaced by 25 μL of Tris-HCl buffer. In control sample also 25 μL of Tris-HCl

buffer was used in place of 25 μL of sample. 96-well plate was then incubated at 25 oC

for 15 minutes. Thereafter, 25 μL acetylcholinesterase (0.2 U/mL) was added to the

wells and the absorbance was measured five times consecutively after every 45 seconds.

The reaction was monitored for 5 minutes at 405 nm.

The percentage inhibition for above enzymes assays were calculated using the equation:

Inhibition % = 100 – (ODtest well

/ODcontrol

) × 100.

74

Table 13: Enzyme inhibition activities of crude crystals and fractions of methanolic

extract.

Samples

(0.5 mg/ml)

Urease enzyme

inhibition (%)

α-Glucosidase

enzyme Inhibition

(%)

Acetylcholinesterase

Inhibition (%)

CR 15 NA NA

HX 10 NA NA

DM NA 20 10

EA 12 15 10

BU NA NA 15

AQ NA 10 12

Thiourea 90 - -

Acrabose - 72 -

Galantamine - - 90

NA = Not Active

The values are mean of three replicates.

75

Table 14: Enzyme inhibition activities of compounds 1-5

Compound

(0.5 mg/ml)

Urease enzyme

inhibition (%)

α-Glucosidase enzyme

inhibition (%)

Acetylcholinesterase

enzyme inhibition (%)

1 12 NA NA

2 15 NA NA

3 NA NA NA

4 NA NA NA

5 NA NA NA

NA = Not Active


76

3.3.3 DPPH radical scavenging activity

Free radical scavenging capacity of CR, fractions (HX, DM, EA, BU, AQ) and

compounds 1-5 was determined by measuring the change in absorbance of DPPH (l,l-

Diphenyl-2-picrylhydrazyl radical) at 515 nm using spectrophotometer. In this assay,

reaction mixture was comprised of 95 µL (0 .3mM) of methanolic solution of DPPHֹ

and 5 µL of the test samples (0.5 mg/mL) dissolved in DMSO. Ascorbic acid was used

as standard. Following equation was used for measuring the scavenging activity.

77

Table 15: DPPH radical scavenging activity of crude crystals and fractions of

methanolic extract.

Fraction (0.5 mg/mL) DPPH radical scavenging (%)

CR 12

HX 28

DM 25

EA 15

BU 30

AQ NA

Ascorbic Acid 95

NA = Not Active


78

Table 16: DPPH radical scavenging activity of compounds 1-5

Compounds (0.5 mg/mL) DPPH radical scavenging (%)

1 10

2 15

3 NA

4 NA

5 15

NA = Not Active


79

3.4 Molecular Docking Studies

Compounds 3-5 were used as ligand molecules and their structures were obtained by

using ChemBioDraw software. The three dimensional structure of the protein tyrosine

phosphatases (PTPs) was obtained from the protein Data Bank (PDBID: 4RH5). The

MOL format was first converted to SMILE format using an OpenBabel tool and then

transferred onto the Molecular Operating Enviroment (MOE) software. The coordinates

of the ligand compounds and target protein were optimized by MOE software which

represented the most stable conformation and minimum energy. The drugable

properties (toxicity, molecular weight and partition coefficient) of these pregnane

glycosides were evaluated using Molinspiration server

[http://www.molinspiration.com].

Docking studies were carried out to investigate any binding interactions between the

PTPs target sites with compounds 3-5 by employing MOE software. Default parameters

were used and the energy of binding interaction at every step of the simulation was

measured using atomic affinity potentials. Docking studies were performed by Dr. Syed

Aun Muhammad, Bahauddin Zakariya University, Multan, Pakistan.

80

Figure 8: (A) Protein tyrosine phosphatase binding sites.

81

Figure 8: (B) Analysis of protein tyrosine phosphatase binding sites by MOE software.

(C) View of tyrosine cavity demonstrated the interaction of ligands to protein tyrosine phosphatase.

82

Figure 9 (A): Molecular docking views of ligand (3).

83

Figure 9 (B): Molecular docking views of ligand (4).

84

Figure 9 (C): Molecular docking views of ligand (5).

85

Figure 10: Heat map showing the toxicity analysis and pharmacokinetics of compounds 3-5.

86

Table 17: Binding energies of compounds 3-5 as ligands with target protein tyrosine phosphatase.

Compound mseq S E_conf E_place E_score1

1 -19.4042 1.707 -108.7528 -19.4042

1 -22.4449 0.5044 40.1191 -22.4449

1 -20.0319 0.5974 -106.7344 -20.0319

87

4. RESULTS

AND

DISCUSSION

88

4.1 New Compounds from Caralluma flava

4.1.1 Desmiflavaside A (1)

The IR spectrum of compound 1 showed bands at 1610, 1710 and 3400 cm-1 which

indicated the presence of aryl ring, ester and hydroxyl functionalities in the molecule.

The ESIMS data analysis showed pseudo-molecular ion peak at m/z 963.1 [M+Na] in

positive ion mode and HRESIMS demonstrated quasi-molecular ion peak at m/z

963.4553 [M+Na]+ (calculated for C47H72NaO19, 963.4560). Based on HRESIMS and

13C NMR data, the molecular formula of the compound was suggested to be C47H72O19.

The 13C NMR (Broad band decoupled and DEPT) displayed signals of 47 carbons

which include 28 carbons of aglycone moiety and 19 carbons of glycoside part.

The structure of the aglycone portion was interpreted from NMR data (Table 2) which

demonstrated one doublet of secondary methyl at δH 1.07 (Me-21); δC 21.9 and two

singlets of tertiary methyls at δH 0.84; δC 12.8 and δH 1.11; δC 15.6 assigned to Me-19

and Me-18, respectively. It further showed signals of seven methines including two

oxymethines at δH 3.58 (m, 1H, H-3); δC 79.4 and δH 5.60 (t, J = 8.4 Hz, 1H, H-15); δC

77.3 and two quaternary carbons (C-10 and C-13) appeared at δC 37.1 and 48.0,

respectively (Abdallah et al., 2013; Abdel-Sattar et al., 2001, 2002, 2007; Hayashi et

al., 1988). Moreover, the signal of oxygenated quaternary carbon at δ 82.4 is

characteristic for pregnanes having an OH group at C-14 (Elsebai et al., 2015; Tanaka

et al., 1990). The significant HMBC interactions (Fig. 11) of Me-19 with C-1, C-5, C-

9 and C-10; Me-18 with C-12, C-13, C-14, and C-17; H-20 with C-13, C-16, C-17 and

C-21; H-15 with C-13, C-14, C-16 and C-17 justified the 3,14,20-trioxygenated

pregnane skeleton (Abdallah et al., 2013; Abdel-Sattar et al., 2001, 2002, 2007; Tanaka

et al., 1990).

The presence of one benzoyl group was indicated by its 1H NMR signals at δH 8.09 (dd,

J = 2.0, 7.0 Hz, H-2′/H-6′), δH 7.47 (t, J = 7.0 Hz, H-3′/H-5′), δH 7.60 (t, J = 7.0 Hz, H-

4′) and from an ester carbonyl resonance at δC 167.8 in the 13C NMR spectrum

(Abdallah et al., 2013; Abdel-Sattar et al., 2002; Tanaka et al., 1990; Hayashi et al.,

1988). The attachment of this benzoyl group to C-15 was confirmed by the HMBC

interaction of H-15 (δH 5.60) with benzoyl ester C=O (δC 167.8). The structure of

aglycone part was confirmed by its important connectivities in 1H-1H COSY and 1D

TOCSY spectra (Fig. 12).

89

The presence of three sugar anomeric proton signals at δH 4.57 (d, H-1ʹʹ); δH 4.38 (d,

H-1ʹʹʹ) and δH 4.30 (d, H-1ʹʹʹʹ); one Me group at δH 1.26 (d, H-6''); and one sugar OMe

group at δH 3.49 (s) in the NMR spectra suggesting it to be a triglycoside (Abdallah et

al., 2013; Abdel-Sattar et al., 2007; De Leo et al., 2005). The 13C NMR signals at δC

102.8 (C-1ʹʹ), 71.3 (C-2ʹʹ), 85.7 (C-3ʹʹ), 74.8 (C-4ʹʹ), 71.6 (C-5ʹʹ), 17.5 (C-6ʹʹ), 58.5

(OMe); δC 104.1 (C-1ʹʹʹ), 75.8 (C-2ʹʹʹ), 77.8 (C-3ʹʹʹ), 71.8 (C-4ʹʹʹ), 77.4 (C-5ʹʹʹ), 70.3 (C-

6ʹʹʹ) and δC 105.0 (C-1ʹʹʹʹ), 75.1 (C-2ʹʹʹʹ), 78.0 (C-3ʹʹʹʹ), 71.6 (C-4ʹʹʹʹ), 78.0 (C-5ʹʹʹʹ), 62.0

(C-6ʹʹʹʹ) resulted in establishing the presence of one D-digitalose and two glucoside

sugar units (Abdel-Sattar et al., 2001; Lee-Juian et al., 1994; Al-Massarani et al., 2012).

The β-linkages of the three sugar units were evident from the large coupling constants

(J = 7.8) of the anomeric protons (H-1ʹʹ, H-1ʹʹʹ, H-1ʹʹʹʹ) (Abdel-Sattar et al., 2007; De

Leo et al., 2005).

The Selective 1D TOCSY NMR experiment was employed on the anomeric proton

signals of the three hexose units at δH 4.30 (d, J =7.8), 4.38 (d, J =7.8) and 4.57 (d, J

=7.8) and their respective resonances H-1 to H-6 were assigned. Further, the NOESY,

COSY, HSQC and HMBC correlations resulted in confirming the presence of one D-

digitalose and two glucoside sugar units (Abdel-Sattar et al., 2001; Lee-Juian et al.,

1994). This was aided by comparison with previous reports for similar systems. The

identification of digitalose unit in desmiflavaside A (1) was assessed from its upfield

shifted OMe group at δH 3.49, δC 58.5 in comparison to the downfield OMe groups of

allomerose and thevetose (δH > 3.55, δC > 61.0). In addition, the characteristic doublet

at δH 4.15, assigned to H-4 of the digitalose moiety, had a distinctly small coupling

constant (d, J =2.4 Hz) that was consistent with the equatorial orientation of this proton

and its weak coupling to the axially oriented H-3 and undetected coupling to H-5.

Furthermore, the presence of a NOESY correlation between H-3/H-4 (δH 3.18/ δH 4.15),

and the absence of a similar correlation between H-2/H-4 (δH 3.55) required an

equatorial orientation of H-4 (Abdel-Sattar et al., 2001; Al-Massarani et al., 2012).

The connectivities of the sugar units to each other and of the digitalose sugar unit to

pregnane skeleton was confirmed via key HMBC correlations (Fig. 11): D-digitalose

anomeric proton H-1ʹʹ and C-3; H-3 and C-1ʹʹ; D-glucose-1 anomeric proton H-1ʹʹʹ and

C-4ʹʹ; H-4ʹʹ and C-1ʹʹʹ; D-glucose-2 anomeric proton H-1ʹʹʹʹ with C-4ʹʹʹ; and H-4ʹʹʹ with

C-1ʹʹʹʹ.

90

The relative stereochemistry at C-3 was established from the comparison of NMR

values with previous reports on related pregnane glycosides as well as from significant

2D NOESY correlation between H-3 with H-5 (De Leo et al., 2005; Abdel-Sattar et al.,

2001, 2007; Tanaka et al., 1990). The relative stereochemistries of C-15, C-17, and C-

21 were obtained from 1D NOE and 2D NOESY correlations. Thus, NOESY

correlations between H-15 with Me-18 and Me-21 confirmed that these groups exist on

one face of the molecule. Further, 1D NOE correlations between H-15 with H-16β; H-

16β with H-17, and H-17 with Me-21 further supported the configurations of C-15, C-

17 and C-21 (Abdel-Sattar et al., 2002). However, the configuration assigned to C-20

was tenuous due to the free rotation around the C-17 to C-20 carbon-carbon bond. From

the above data, the structure of 1 was assigned as 15-O-benzoyl-pregnane-

3β,14β,15α,20-tetraol 3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-

D-digitalopyranoside.

91

OH

OH

OO

O

MeO

OO

HO

OHO

HO

OH1''

3''

1'''3'''

6''

6'''6''''

1''''3''''

1

4

6

9 14

131115

21

19

OH

OHOH

OHO

O

H

Figure 11: Significant HMBC interactions of 1

OH

OH

OO

O

MeO

OO

HO

OHO

HO

OH1''

3''

1'''3'''

6''

6'''6''''

1''''3''''

1

4

6

9 14

131115

21

19

OH

OHOH

OHO

O

H

Figure 12: — Significant 1H-1H COSY and 1D TOCSY interactions of 1

92

4.1.2 Desmiflavaside B (2)

Compound 2 showed pseudo-molecular ion peak at m/z 961.1 [M+Na] in positive ion

mode ESIMS. Based on the molecular ion peak in the HRESIMS spectrum (observed

961.4403 (calculated for C47H70NaO19, 961.4404) its molecular formula was found to

be C47H70O19.

The spectral data of 2 illustrated its close resemblance with desmiflavaside A (1). The

1H and 13C NMR resonances (Table 3) for the aglycone part and three sugar moieties

were almost similar to those of compound 1 (Table 2) except with few differences in

the pregnane skeleton. The most prominent changes appeared in the chemical shifts of

the methyl doublet at δH 1.07 (d, 6.6 Hz, H-21); δC 21.9 and oxymethine multiplet at δH

4.03 (m, H-20); δC 66.0 present in 1 that were absent and replaced by a singlet methyl

signal at δH 2.26 (s, H-21); δC 32.6 and a ketone signal (δC 218.2) in the NMR spectra

of 2 (Abdel-Sattar et al., 2002). This was further confirmed from HRESIMS of 2 which

illustrated two units of mass less than 1 (961.4403 vs 963.4553). Moreover, the

dissimilarities were also evident by comparison of HMBC interactions (Fig. 13); Me-

21 to C-17 and C-20; H-17 to C-20 and C-21. Therefore, the structure of compound 2

was elucidated as 15-O-benzoyl-pregnane-3β,14β,15α-triol 3-O-β-D-glucopyranosyl-

(1→4)-β-D-glucopyranosyl-(1→4)-β-D-digitalopyranoside.

93

O

OH

OO

O

MeO

OO

HO

OHO

HO

OH1''

3''

1'''3'''

6''

6'''6''''

1''''3''''

1

4

6

9 14

131115

21

19

OH

OHOH

OHO

O

H


94

4.1.3 Desmiflavaside C (3)

The IR spectrum of compound 3 exhibited bands at 3410, 1710 and 1615 cm-1

indicating the presence of OH, ester C=O and aryl group in the molecule.

Desmiflavaside C showed a pseudo-molecular ion peak in ESIMS at m/z 871.4 [M+Na]

corresponding to the molecular formula C49H68O12 based on HRESIMS spectrum

(observed 871.4606 (calculated for C49H68NaO12, 871.4608). The 1H NMR spectrum

(Table 4) indicated two singlet resonances for two tertiary methyl protons at δ 1.09 (3H,

s, Me-18) and 0.82 (3H, s, Me-19) and one three protons doublet resonance for a

secondary Me group at δ 1.20 (3H, d, J= 6.0 Hz, Me-21). Its 13C NMR spectrum also

supported the presence of these three Me groups by displaying signals at δ 10.0 (Me-

18), 12.4 (Me-19), and 19.7 (Me-21), respectively. The presence of three oxymethine

groups in molecule were supported by its NMR spectra which displayed signals at δH

3.63 (1H, m, H-3); δC 78.5, δH 4.95 (1H, J= 4.5, 12.0 Hz, dd, H-12); δC 79.5 and δH

5.24 (1H, J= 6.0, 10.0 Hz, dd, H-20); δC 75.6 along with signals of two quaternary

carbons at δ 36.9 (C-10), 53.7 (C-13) and one oxygenated quaternary carbon at δ 87.2

(C-14) (Abdallah et al., 2013; Abdel-Sattar et al., 2007, 2002, 2001; Tanaka et al.,

1990). All the above data confirmed the pregnane skeleton of compound 3. Further

complete structure confirmation was achieved by COSY, 1D 1H-1H TOCSY (Fig. 15)

and HMBC correlations (Fig. 14). The 3,14 dioxygenated pregnane moiety was

confirmed by its key HMBC interactions between Me-21 (δ 1.19) with C-13, C-17, and

C-20; H-20 (δ 5.24) with C-13, C-16, C-17 and C-21; Me-19 (δ 0.82) with C-1, C-5,

C-9 and C-10; Me-18 (δ 1.09) and C-12, C-13, C-14, and C-17 (Abdallah et al., 2013;

Abdel-Sattar et al., 2007, 2002, 2001; Tanaka et al., 1990).

The 1H NMR spectra of desmiflavaside C showed signals for two benzoyl groups Bz-

12: δH 8.03 (J= 7.2 Hz, br d, H-2'/H-6'), 7.68 (J= 7.2 Hz, t, H-4'), 7.50 (J= 7.2 Hz, d, H-

3'/H-5') and Bz-20: δH 7.73 (J= 7.2 Hz, br d, H-2'/H-6'), 7.50 (J= 7.2 Hz, t, H-4'), 7.22

(J= 7.2 Hz, d, H-3'/H-5') (Abdallah et al., 2013; Tanaka et al., 1990; Hayashi et al.,

1988). These two benzoyl moieties were attached to C-12 and C-20 based on significant

HMBC interactions of H-12 and H-20 to the C=O resonances at δc 168.0 and 167.4,

respectively.

The NMR spectra displayed two sugar anomeric signals at δH 4.85 (J = 2.0, 12.0 Hz,

dd, H-1ʹʹ); δC 97.1 (C-1ʹʹ) and 4.57 (J = 1.8, 9.6 Hz, dd, H-1ʹʹ′); δC 102.7 (C-1ʹʹ′). NMR

95

spectra further exhibited two sugar methyl group signals at δH 1.19 (J = 6.0 Hz, d, H-

6′′); δC 18.5 (C-6ʹʹ) and δH 1.26 (J = 6.0 Hz, d, H-6‴); δc 18.3 (C-6‴) and two sugar OMe

group signals at δH 3.41 (s); δC 58.4 and δH 3.41 (s); δC 57.3 which indicated compound

3 to be a diglycoside with a β-linkage (J = 8.4–9.6 Hz; large coupling constant) (De

Leo et al., 2005; Abdel-Sattar et al., 2007).

The selective 1D TOCSY interactions showed the presence of two cymarose sugar units

which were confirmed by significant HSQC and HMBC interactions and were aided by

comparing NMR data with previous reports on cymarose (Abdel-Sattar et al., 2002,

2007). Attachment of the disaccharide chain to C-3 of the pregnane moiety was

confirmed from HMBC correlations between first cymarose anomeric proton (H-1ʹʹ)

and C-3. It was interesting to note that one cymarose unit was glycosylated at C-4 and

was evident from the downfield shift observed for C-4Cym-I [δ 83.8 (C-4Cym-I) vs 81.6

(C-4Cym-II)]. Moreover 3J HMBC cross peaks between H-1ʹʹʹ (H-1Cym-II) and C-4ʹʹ (C-

4Cym-I) indicated the sequence of the disaccharide chain.

The relative stereochemistry at C-3 was established by the significant NOESY

interaction of H-3 with H-5 and was confirmed by comparison of NMR data of C-3 [δH

3.65 (m, H-3); δC 78.5 (C-3)] with previous reports on related pregnane glycosides [Lit:

δH 3.63-3.68 (m, H-3); δC 78.1-78.4 (C-3)] (De Leo et al., 2005). Furthermore, the α-

configuration of H-12 and H-17 were positively identified based on NOE interactions

between H-12 with H-11α and H-17α and was further supported from the lack of

interaction of H-12 with Me-18. On the other hand, the stereochemistry of C-20 was

identified to be S from the NOE interaction of H-20 with H-21 and H-18, and H-l6α

with H-21 and the lack of NOE interactions between H-21 and H-18 (Tanaka et al.,

1990; Qiu et al., 1997; Abdel-Sattar et al., 2001 and 2007; Itokawa et al., 1988).

However, the configuration assigned to C-20 was tenuous due to the free rotation

around the C-17 to C-20 carbon-carbon bond. Based on above mentioned data, the

structure of 3 was elucidated as 12,20-di-O-benzoyl-20S-pregnane-3β,12β,14β,20-

tetraol 3-O-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside.

96

O

OH

OOO

OMe

OHO

OMe

1''3''1'''3'''

6''6'''

1

4

6

9 14

131115

21

19O

OO

H

H


O

OH

OOO

OMe

OHO

OMe

1''3''1'''3'''

6''6'''

1

4

6

9 14

131115

21

19O

OO

H

H


97

4.1.4 Desmiflavaside D (4)

Compound 4 showed molecular ion peak at m/z 1033.5 [M+Na]+ in ESIMS having

molecular formula C55H78O17 obtained through HR-ESIMS (observed 1033.5125

(calculated for C55H78NaO17, 1033.5137).

The IR spectrum of 4 was identical to desmiflavaside C (3) having same functional

groups such as hydroxyl, carbonyl and aromatic ring (3410, 1715 and 1610 cm-1).

The NMR spectral data of 4 (Table 5) was comparable to 3 (Table 4) as it possesses

same pregnane aglycone skeleton and two benzoyl units present at C-12 and C-20.

Further, the three sugar units in 4 were positively suggested from the anomeric signals

at δH 4.86; δC 97.1; δH 4.59; δC 102.5 and δH 4.43; δC 104.1. The two cymarose sugar

units along with their attachments were completely identical to those in 3 but an

additional glucose unit was found in 4 which was present at C-4 of Cymarose-II. This

was positively identified from the significant HMBC interaction (Fig. 16) of H-4''' to

C-1'''' and from the downfield shift in the 13C NMR value of C-4''' (δ 83.5) in 4 compared

to C-4''' (δ 81.6) in 3. Thus, the structure of desmiflavaside D was characterized as

12,20-di-O-benzoyl-20S-pregnane-3β,12β,14β,20-tetraol 3-O-β-D-glucopyranosyl-

(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside.

98

O

OH

OOO

OMe

OO

OMe

1''3''1'''3'''

6''6'''

1

4

6

9 14

131115

21

19O

OO

H

H

OHO

HO

6''''

1''''3''''

OH

OH


99

4.1.5 Nizwaside (5)

Compound 5 had a close resemblance with 4 i.e having pregnane skeleton, two benzoyl

groups and three sugar moieties. The dissimilarity was the presence of 6-deoxy-3-O-

methyl-D-allopyranose in 5 instead of D-glucopyranose.

The IR spectrum of compound 5 showed the presence of hydroxyl (3400cm-1), ester

(1710cm-1) and aromatic ring (1610cm-1) in the molecule. Its molecular formula was

found to be C56C80H16 based on a quasi-molecular ion peak at m/z 1031.5332

(calculated for C56H80NaO16, 1031.5338).

The 1H and 13C NMR spectra of 5 (Table 6) showed resonances for three methyls at δH

1.09 (s, 3H, Me-18); δC 10.0 (C-18); δH 0.82 (s, 3H, Me-19); δC 12.4 (C-19); δH 1.35

(d, J = 6.6 Hz, 3H, Me-21); δC 18.9 (C-21) and three oxygenated methine signals at δH

3.62 (m, 1H, H-3); δC 78.5 (C-3); δH 4.95 (dd, J = 4.8, 12.0 Hz, 1H, H-12); δC 79.4 (C-

12) and δH 5.24 (q, J = 6.6 Hz, 1H, H-20); δC 75.7 (C-20) which were assigned to the

pregnane part (Abdallah et al., 2013; Abdel-Sattar et al., 2001, 2002, 2007; Tanaka et

al., 1990). This was further confirmed by its COSY, selective 1D TOCSY and HMBC

interactions (Fig. 17 and 18). The significant HMBC interactions of Me-19 with C-1,

C-5, C-9, and C-10, Me-18 with C-12, C-13, C-14, and C-17; CH2-11 with C-8, C-9,

C-12, and C-13; H-20 with C-13, C-16, C-17, and C-21; H-12 with C-9, C-11, C-13,

C-14, C-17 and Me-18 confirmed the 3,12,20-trioxygenated pregnane skeleton

(Abdallah et al., 2013; Abdel-Sattar et al., 2001, 2002, 2007; Hayashi et al., 1988). The

pregnane skeleton bearing OH group at C-14 was justified by its characteristic peak

appeared at δH 87.2 in 13C NMR spectrum (Elsebai et al., 2015; Tanaka et al., 1990).

Further, the NMR signals indicated the presence of two benzoyl groups in compound

5. These benzoyl groups were located at C-12 and C-20 which were confirmed by the

prominent HMBC interactions of H-12 (δ 4.95) and H-20 (δ 5.24) with carbonyl

carbons of benzoyl groups (δ 168.0 and 167.4 respectively) (Abdallah et al., 2013;

Tanaka et al., 1990; Hayashi et al., 1988).

NMR spectra (Table 6) showed signals of three sugar units which were positively

identified as one 6-deoxy-3-O-methyl-D-allopyranose and two cymarose (= 2,6-

dideoxy-3-O-methyl-ribohexose) (Abdel-Sattar et al., 2002). The interactions of sugar

units with each other and with aglycone moiety were identified by their significant

100

HMBC correlations (Fig. 17). Thus, the structure of compound 5 was elucidated as

12,20-di-O-benzoyl-pregnane-3β,12β,14β,20-tetraol 3-O-β-D-cymaropyranosyl-

(1→4)-β-D-cymaropyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D-allopyranoside.

101

O

OH

1

4

6

9 14

131115

21

19O

OO

H

H

OO

O

OMe

OO

OMe

OHO

OMeOH

1''3''

1'''3'''

6''6'''6''''

1''''3''''


O

OH

1

4

6

9 14

131115

21

19O

OO

H

H

OO

O

OMe

OO

OMe

OHO

OMeOH

1''3''1'''

3'''

6''6'''6''''

1''''3''''


102

4.1.6 Desflavaside A (6)

The molecular formula of compound 6 was established as C62C90H21 based on quasi-

molecular ion peak in the HR-ESIMS at m/z 1193.5885 (calculated for C62H90NaO21,

1193.5872).

The IR spectrum of 6 showed the presence of OH, ester C=O and aryl moieties in the

molecule. Further, the NMR spectrum (Table 7) indicated the resonances for two

tertiary and one secondary methyls at δH 1.09 (δC 10.0), δH 0.82 (δC 12.4) and δH 1.20

(δC 19.7). The pregnane skeleton of compound 6 was suggested by the appearance of

three oxymethine group signals at δH 3.63 (δC 78.5), δH 4.94 (δC 79.5) and δH 5.24 (δC

75.6) along with two quaternary carbons at δC 36.9 (C-10), 53.7 (C-13) and a

characteristic oxygenated C-14 at δC 87.2 (Abdallah et al., 2013; Abdel-Sattar et al.,

2001, 2002, 2007; Tanaka et al., 1990). The significant COSY, selective 1D TOCSY

and HMBC interactions (Fig. 19 and 20) also confirmed the pregnane moiety of 6. The

presence of two benzoyl groups illustrated by their resonances in 1H NMR spectrum at

δ 8.03 (H-2'/H-6'), 7.67 (H-4') and 7.50 (H-3'/H-5') were assigned to benzoyl group at

C-12 and signals at δ 7.73 (H-2'/H-6'), 7.51 (H-4') and 7.22 (H-3'/H-5') defined to

benzoyl group at C-20 (Abdallah et al., 2013; Tanaka et al., 1990; Hayashi et al., 1988).

Furthermore, the HMBC interactions of H-12 and H-20 to the C=O carbons at δC 168.0

and 167.0 confirmed their respective locations, respectively.

The tetraglycosidic nature of desflavaside A was positively identified by the resonances

for four anomeric sugar centers at δH 4.85 (J = 2.0, 6.0 Hz, dd, H-1ʹʹ); δC 97.1 (C-1ʹʹ);

δH 4.33 (J = 7.2 Hz, d, H-1ʹʹ′); δC 106.2 (C-1ʹʹ′); δH 4.56 (J = 2.0, 10.0 Hz, dd, H-1ʹʹʹʹ);

δC 102.5 (C-1ʹʹʹʹ); δH 4.70 (J = 6.0 Hz, d, H-1ʹʹʹʹʹ); δC 102.1 (C-1ʹʹʹʹʹ); three deoxy sugar

methyl resonances at δH 1.18 (J = 6.0 Hz, d, H-6′′); δC 18.5 (C-6ʹʹ); δH 1.29 (J = 6.0 Hz,

d, H-6ʹʹʹʹ); δc 18.1 (C-6ʹʹʹʹ); δH 1.35 (J = 6.0 Hz, d, H-6ʹʹʹʹʹ); δc 18.9 (C-6ʹʹʹʹʹ) and three

sugar OMe resonances at δH 3.40 (s); δC 57.5; δH 3.41 (s); δC 58.4; δH 3.58 (s); δC 61.9.

The above mentioned resonances and large coupling constants (8.4–9.6 Hz) indicated

that the group attached at C-3 of the aglycone portion is a tetraglycoside with a β-

linkage (Abdel-Sattar et al., 2002, 2007; De Leo et al., 2005). The 13C NMR, selective

1D TOCSY and 1H-1H COSY spectra (Fig. 20) positively suggested that the

tetrasaccharide is comprised of one glucose, one 6-deoxy-3-O-D-methylallose and two

cymarose (=2,6-dideoxy-3-O-methyl-ribohexose) units which were strongly supported

103

by a comparison of the spectra with previous compounds 4 and 5. Connectivity of the

saccharide chain to C-3 of the pregnane part was confirmed from significant HMBC

interactions between the first cymarose anomeric proton (H-1ʹʹ) and C-3. Moreover, the

sequence of the tetrasaccharide in the chain was confirmed from the H-1ʹʹʹʹʹ to C-4ʹʹʹʹ;

H-4ʹʹʹʹ to C-1ʹʹʹʹʹ; H-1ʹʹʹʹ to C-4ʹʹʹ; H-4ʹʹʹ to C-1ʹʹʹʹ; H-1ʹʹʹ to C-4ʹʹ and H-4ʹʹ to C-1ʹʹʹ 3J

correlations.

The relative stereochemistry at C-3 was established by the prominent NOESY

interaction of H-3 with H-5 and was further confirmed by comparison of NMR values

related to this position [δH 3.62 (m, H-3); δC 78.5 (C-3)] with literature reports of related

pregnane glycosides Lit: δH 3.63-3.68 (m, H-3); δC 78.1-78.4 (C-3) (De Leo et al.,

2005). Further, the α-configurations of H-12 and H-17 were confirmed from NOE

interactions of H-12 with H-11α together with H-17α and positively supported by lack

of NOE correlations with Me-18. On the other hand the stereochemistry of C-20 was

confirmed as S by a similar analysis to that reported for other pregnane glycosides viz.,

an NOE correlation between H-20 with H-21 and H-18 on the one hand and H-l6α with

H-21 together with lack of an NOE correlation between H-21 and H-l8 (Itokawa et al.,

1988; Abdel-Sattar et al., 2001, 2007; Qiu et al., 1997; Tanaka et al., 1990). However,

the configuration assigned to C-20 was tenuous due to the free rotation around the C-

17 to C-20 carbon-carbon bond. Therefore, the structure of 6 was deduced as 12,20-di-

O-benzoyl-20S-pregnane-3β,12β,14β,20-tetraol 3-O-6-deoxy-3-O-methyl-β-D-

allopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-

cymaropyranoside.

104

O

OH

OOO

OMe

OO

1''3''1'''3'''

6''6'''

1

4

6

9 14

131115

21

19O

OO

H

H

OO

6''''

1''''3''''

OH

OHOMe

O

OMe

3'''''

6'''''

OH

HO

1''''' HO


105

O

OH

OOO

OMe

OO

1''3''1'''3'''

6''6'''

1

4

6

9 14

131115

21

19O

OO

H

H

OO

6''''

1''''3''''

OH

OHOMe

O

OMe

3'''''

6'''''

OH

HO

1''''' HO


106

4.1.7 Desflavaside B (7)

The molecular formula of compound 7 was identical to 6 that is C62C90H21 (HRESIMS

observed 1193.5881, calculated for C62H90NaO21, 1193.5872). IR spectrum of 7

showed bands at 3415, 1710, 1610 cm-1 indicated the presence of OH, ester C=O and

aromatic ring in the molecule.

The NMR data of aglycone part of 7 (Table 8) was similar to 6 (Table 7) whereas the

difference was observed in the glycoside chain indicating the existence of β-D-

thevetopyranosyl terminal sugar unit in 7 instead of β-D-allopyranosyl sugar. The

presence of terminal β-D-thevetopyranosyl unit was positively supported by 13C NMR

data of this unit [δC 104.2 (C-1′′′′′), 75.2 (C-2′′′′′), 86.3 (C-3′′′′′), 82.9 (C-4′′′′′), 72.6 (C-

5′′′′′), 18.5 (C-6′′′′′), 61.2 (OMe)] with the literature [Lit: δC 104.2 (C-1), 75.0 (C-2),

86.1 (C-3), 82.6 (C-4), 72.3 (C-5), 18.5 (C-6), 61.0 (OMe)] (De Leo et al., 2005). On

behalf of above differences, the structure of 7 was assigned as 12,20-di-O-benzoyl-20S-

pregnane-3β,12β,14β,20-tetraol 3-O-β-D-thevetopyranosyl-(1→4)-β-D-

cymaropyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-cymaropyranoside.

107

O

OH

OOO

OMe

OO

1''3''1'''3'''

6''6'''

1

4

6

9 14

131115

21

19O

OO

H

H

OO

6''''

1''''3''''

OH

OHOMe

O

MeO 3'''''

6'''''

OH

HO

1''''' HO


108

4.1.8 Desflavaside C (8)

The molecular formula of compound 8 was determined as C61C88H22 based on quasi-

molecular ion peak in the HR-ESIMS at m/z 1195.5673 (calculated for C61H88NaO22,

1195.5665).

The aglycone portion of 8 was characterized as 12,20-di-O-benzoyl-20S-pregnane-

3β,12β,14β,20-tetraol by comparison of its NMR data (Table 9) with that of 7 (Table

8). The appearance of four anomeric signals in the NMR spectrum at δH 4.86 (J = 2.0,

6.0 Hz, dd, H-1ʹʹ); δC 97.1 (C-1ʹʹ); δH 4.47 (J = 7.8 Hz, d, H-1ʹʹ′); δC 104.0 (C-1ʹʹ′); δH

4.38 (J = 7.8 Hz, d, H-1ʹʹʹʹ); δC 104.6 (C-1ʹʹʹʹ); δH 4.59 (J = 1.7, 9.6 Hz, dd, H-1ʹʹʹʹʹ); δC

102.5 (C-1ʹʹʹʹʹ); two deoxy sugar Me group signals at δH 1.19 (J = 6.0 Hz, d, H-6′′); δC

18.5 (C-6ʹʹ); δH 1.37 (J = 6.0 Hz, d, H-6ʹʹʹʹʹ); δc 18.7 (C-6ʹʹʹʹʹ); two sugar OMe groups at

δH 3.44 (s); δC 57.9; δH 3.41 (s); δC 58.4 represented that the tetrasaccharide is

comprised of two glucose and two cymarose (2,6-dideoxy-3-O-methyl-ribohexose)

units (Abdel-Sattar et al., 2002). Attachment of the tetraglycoside to C-3 of the

pregnane moiety was confirmed from HMBC interactions (Fig. 22) between the

cymarose-1 anomeric proton (H-1ʹʹ) and C-3. Moreover, the sequence of the

tetrasaccharide chain was confirmed from the H-1ʹʹʹʹʹ to C-4ʹʹʹʹ; H-4ʹʹʹʹ to C-1ʹʹʹʹʹ; H-1ʹʹʹʹ

to C-4ʹʹʹ; H-4ʹʹʹ to C-1ʹʹʹʹ; H-1ʹʹʹ to C-4ʹʹ and H-4ʹʹ to C-1ʹʹʹ 3J correlations. Therefore, the

structure of desflavaside C was assigned as 12,20-di-O-benzoyl-20S-pregnane-

3β,12β,14β,20-tetraol 3-O-β-D-cymaropyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-

β-D-glucopyranosyl-(1→4)-β-D-cymaropyranoside.

109

O

OH

OOO

OMe

OO

1''3''1'''3'''

6''6'''

1

4

6

9 14

131115

21

19O

OO

H

H

OO

6''''

1''''3''''

OH

OH

O

3'''''

6'''''

OH

HO

1''''' HO

OH

HOOMe OH


110

O

OH

OOO

OMe

OO

1''3''1'''3'''

6''6'''

1

4

6

9 14

131115

21

19O

OO

H

H

OO

6''''

1''''3''''

OH

OH

O

3'''''

6'''''

HO

1''''' HO

OH

HOOMe OH

18


111

4.1.9 Desflavaside D (9)

The HRESIMS analysis of compound 9 showed the quasi-molecular ion peak at m/z

1005.4876 (calculated for C46H78NaO22, 1005.4882) assigned for molecular formula

C46H78O22.

Two major differences in the NMR signals of the aglycone of 9 (Table 10) were

observed as compared to compound 8 (Table 9). Firstly, the NMR peaks for two

benzoyls were completely absent in compound 9. Secondly, the methylene signals for

ring C appeared at δH 1.26 (m) and 1.37 (m) in the 1H NMR spectrum of 9 instead of

an oxymethine signal at δH 4.95 (1H, m, H-12) which was also supported by the

presence of a methylene signal at δC 42.0 in the 13C NMR spectrum instead of a methine

signal at δC 79.5 (C-12). Therefore, the aglycone portion of 9 was characterized as 20S-

pregnane-3β,14β,20-triol.

Further analysis of the NMR spectra of desflavaside D indicated the presence of four

anomeric signals at δH 4.58 (J = 7.7 Hz, d, H-1ʹʹ); δC 104.1 (C-1ʹʹ); δH 4.38 (J = 7.8 Hz,

d, H-1ʹʹ′); δC 104.3 (C-1ʹʹ′); δH 4.36 (J = 7.9 Hz, d, H-1ʹʹʹʹ); δC 104.4 (C-1ʹʹʹʹ) and δH 4.34

(J = 7.8 Hz, d, H-1ʹʹʹʹʹ); δC 102.7 (C-1ʹʹʹʹʹ) suggested compound 9 to be tetrasaccharide

glycoside. Its 1D and 2D NMR spectra displayed the presence of one digitalose (3-O-

methyl-6-deoxy-galactopyranose) and three glucose units.

Attachment of the saccharide chain to C-3 and the sequence of the saccharides in the

chain was confirmed from H-1ʹʹʹʹʹ to C-4ʹʹʹʹ; H-4ʹʹʹʹ to C-1ʹʹʹʹʹ; H-1ʹʹʹʹ to C-4ʹʹʹ; H-4ʹʹʹ to

C-1ʹʹʹʹ; H-1ʹʹʹ to C-4ʹʹ and H-4ʹʹ to C-1ʹʹʹ; H-1ʹʹ to C-3; H-3 to C-1ʹʹ 3J HMBC correlations

(Fig. 24). From the foregoing evidences, compound 9 was characterized as 20S-

pregnane-3β,14β,20-triol 3-O-β-D-digitalopyranosyl-(1→4)-β-D-glucopyranosyl-

(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside.

The NMRs of all the isolated compounds (1-9) indicated that none of the compound

was isolated in a completely pure state and minor impurities were observed in the

NMRs of all the isolates. It was also to be noted that no known compound was isolated

from C. flava in our research.

112

OH

OH

OO

1''3''

6''

1

4

6

9 14

131115

21

19

H

HO

OOO

1'''3'''

6'''

OO

6''''

1''''3''''

OH

OH

O

3'''''

6'''''

OH

HO

1''''' HO

OH

HOOMe OH

HO


OH

OH

OO

1''3''

6''

1

4

6

914

131115

21

19

H

HO

OOO

1'''3'''

6'''

OO

6''''

1''''3''''

OH

OH

O

3'''''

6'''''

OH

HO

1''''' HO

OH

HOOMe OH

HO


113

4.2 Biological Activities

4.2.1 Anticancer Activity

Crude crystals (CR), methanolic extract (ME) and its fractions (HX, DM, EA, BU, and

AQ) were tested for their cytotoxic effect on MDA-MB-231 breast cancer cells.

Treatment of these cells with test samples at various concentrations (25, 50, 75 and 100

µg/ml) demonstrated a significant reduction in the cells viability (Fig. 6). Fractions EA,

BU and AQ showed IC50 (concentration required to inhibit 50% cell growth) > 100

µg/ml whereas ME and its fractions HX and DM showed prominent results having IC50

values 89.9, 78.7 and 84.0 µg/ml, respectively (Table 11). Interestingly, CR (IC50: 73.6

μg/ml) appeared to be more cytotoxic than ME and all of its fractions and induced

50.7% and 59.6 % of cancer cell growth reduction at a concentration of 75 and 100

µg/ml, respectively.

The cytotoxic effect of isolated compounds (3-5) from CR was also assessed using

various cell lines. The treatment of MDA-MB-231 breast cancer cells and SKOV-3

ovarian cancer cells with 3-5 at different concentrations (25, 50, 75 and 100 µM) caused

a prominent decrease in the viability of both types of cancer cells (Fig. 7). Compound

4 (IC50: 19.97 µM) was found to have most cytotoxic effect against MDA-MB-231 cells

whereas compound 5 (IC50: 37.97 µM) showed highest degrees of cell growth inhibition

against SKOV-3 cells (Table 12). Compound 3 showed moderate anti-proliferative

effect against both cancer cell lines with IC50 values 47.01 µM (MDA-MB-231) and

64.50 µM (SKOV-3). Moreover, the IC50 values of compound 4 (MDA-MB-231: 19.97

µM; SKOV-3: 38.32 µM) against both types of cancer cells were closely similar to that

of 5 (MDA-MB-231: 25.84 µM; SKOV-3: 37.97 µM). However, the IC50 values of

compounds 4 and 5 were significantly lower than the IC50 values of compound 3.

Furthermore, when MCF-10-2A normal breast cells were treated with increasing

concentrations (25, 50, 75 and 100 µM) of compounds 3-5 (Fig. 7C), only compound 3

showed slightly toxicity towards normal cells whereas both pregnane glycosides 4 and

5 had no major cytotoxic effect on normal cells and exhibited closely similar results.

114

4.2.2 Enzyme Inhibition Activity

Enzyme inhibition activities of ME fractions (HX, DM, EA, BU, and AQ), CR and

isolated compounds (1-5) were evaluated against urease, α-glucosidase and

acetylcholinesterase enzymes, where it was correlated with known standards viz.,

thiourea (IC50: 88.24 µg/ml), acrabose (IC50: 38.25 µg/ml) and galantamine (IC50: 10.14

µg/ml) (Dalai et al., 2014), respectively (Table 13 and 14). The results showed no

significant enzyme inhibition activity against these enzymes, therefore the IC50 values

of all the tested samples were not calculated. Since the results of inhibition were not

significantly higher or correlative to the known standard, therefore, we didn’t further

evaluated the higher concentration of the fractions and compounds for enzyme

inhibition activity.

115

4.2.3 DPPH radical scavenging activity

The fractions (HX, DM, EA, BU, and AQ) of methanolic extract (ME), CR and

pregnane glycosides (1-5) were screened for their antioxidant activity taken ascorbic

acid as standard (IC50: 20.69 µg/ml) (Table 15 and 16). All the tested samples showed

less than 50% inhibition of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, therefore

were not proceed further for IC50 determination. Since the results of inhibition were not

significantly higher or correlative to the known standard, therefore, we didn’t further

evaluated the higher concentration of the fractions and compounds for antioxidant

activity.

116

4.3 Molecular Docking Studies

Molecular docking studies were carried out to ascertain if it was possible to predict any

important binding orientations required of small molecule drug candidates with

suggested protein target molecules for the purposes of being able to predict the affinity

and activity to an acceptable degree by such compounds. The normal role of protein

tyrosine phosphatases (PTPs) enzyme is to regulate cell proliferation and tumor

suppression while its overexpression leads to breast cancer. Therefore, newly

synthesized PTPs inhibitors have a therapeutic role.

Pregnane glycosides 3-5 were used as ligand molecules to inhibit the PTPs

overexpression activity in order to understand the drug receptor interaction. The

isolated binding pocket of PTPs contained amino acid residues: ALA-707, ARG-875,

ASP-872, ASP-879, GLU-704, ILE-705, ILE-868, ILE-873, LEU-861, LEU-866,

LYS-876, MET-703, PRO-706, PRO-867, THR-857 and TYR-869 (Fig. 8). Results of

the present studies illustrated that the targeted enzyme was docked by these ligands and

sixteen tyrosine residues demonstrated interactions with these inhibitor molecules. The

ASP-872, PRO-706, ASP-879 and ARG-875 amino acid residues of protein target were

mainly interacting with the solvent-residues (H-OH), arene-H of aromatic rings and

electro-negative O-hetero atoms of compound (3) (Fig. 9A). The solvent residues (H-

OH) of compound (4) exhibited binding affinity with ALA-707 and PRO-857, while

the ASP-879 amino acid interacted with the hydroxyl group of the C41-42 group of the

ligand molecule (Fig. 9B). Similarly, compound (5) demonstrated that the arene-H of

the aromatic ring possess promising binding affinity with LYS-876; ALA-707, PRO-

706 and PRO-867 with the C1-O7 group and ASP-879 with carbonyl group (Fig. 9C).

Compound (3) underwent the most important interactions with GLU-704 (acidic

contact), PRO-706 (sidechain donor), ASP-872 (acidic receptor contact), LYS-876

(basic contact), ARG-875 (basic receptor contact), ASP-879 (acidic contact), ILE-705

(greasy contact) and LEU-851 (greasy contact). Similarly, compound (4) interacted

with ARG-875 (basic receptor contact), PRO-857 (greasy receptor contact), ASP-879

(acidic receptor contact), ASP-872 (acidic contact), LEU-856 (greasy contact), ALA-

707 (greasy receptor contact) and ARG-875 (basic receptor contact) while compound

(5) showed good interactions with ALA-707, PRO-857, PRO-705, LEU-851 (greasy

117

receptor contact), GLU-704 (acidic receptor contact), ASP-879, ASP-872 (acidic

receptor contact), LYS-876 (basic receptor contact).

In terms of binding interaction with different amino acid residues, compound 4

exhibited better affinity as compared to 3 and 5. In the best docked position, 4 formed

three hydrogen bonds with the receptor, involving residues ASP-879, PRO-857 and

ALA-707. Residue ASP-879 was strongly involved in hydrogen bond formation with

pregnane glycosides 3-5 showing a similar orientation of these molecules, which was

also obvious from a closer analysis of their relative docking positions (Fig. 9).

All ligand molecules demonstrated the best interaction with target protein and their

calculated docked energies were found to be: desmiflavaside C (3), -19.4042 kcal/mol,

desmiflavaside D (4), -22.4449 kcal/mol and nizwaside (5), -20.0314 kcal/mol (Table

17). Compound 4 showed a comparatively promising binding energy as compared to 3

and 5.

118

5. CONCLUSIONS

119

In conclusion, this study found that the plant of C. flava growing in the high mountains

of the Sultanate of Oman possesses promising anti-cancer properties and can be used

to cope with breast cancer in the Sultanate which has been one of the most common

cancer in Omani females and a leading cause of their mortality. Our findings clearly

showed that both, the plant extract and its exudate sap possess significant anticancer

activity towards breast cancer.

Among the five different fractions of the methanolic extract of C. flava, hexane fraction

showed the most promising anticancer effects in-vitro towards breast cancer. The

therapeutical activities of this plant were carried out scientifically and reported for the

first time in this research. Further work on isolation and characterization of the possible

bioactive compounds contained therein is required for further investigation. Moreover,

in-vivo verification of the anti-cancer therapeutic effectiveness of the active

components are also needed.

In comparison with C. flava methanolic extract and all of its fractions, the viscous sap

of the plant was found to be the most antiproliferative against breast cancer and turned

out to be a rich source of pregnane glycosides. On chemical investigation, it first yielded

crude shiny yellow crystals and later their chromatographic separation provided nine

new pregnane glycosides namely desmiflavaside A (1), desmiflavaside B (2),

desmiflavaside C (3), desmiflavaside D (4), nizwaside (5), desflavaside A (6),

desflavaside B (7), desflavaside C (8) and desflavaside D (9).

The anticancer properties of the sap of C. flava attributed to the pregnane glycosides

present therein. The result of anticancer activities illustrated that all the pregnane

glycosides tested viz., desmiflavasides C (3) and D (4) and nizwaside (5) exhibited

significant potential to effectively suppress the growth of ovarian and breast cancer

cells without disturbing the growth of normal breast epithelial cells. These pregnane

glycosides can have anticancer effects on other cancer cell lines as well which need to

be further explored.

The molecular docking study results of three pregnane glycosides namely

desmiflavasides C (3) and D (4) and nizwaside (5) illustrated that all these quite

extended ligand molecules have a good potential to accurately interact with the target

protein tyrosine phosphatase sites reasonably specifically to inhibit its overexpression

activity which leads to breast cancer. This is why, these isolated pregnane glycosides

120

from C. flava can serve as strong candidates for future anti-cancer drug development

and therefore need to be further explored for their anti-cancer action in the cure of breast

cancer, in-vivo models of tumorigenesis and an understanding of the mechanisms

thereof.

The plant of C. flava showed no significant potential to inhibit urease,

acetylcholinesterase and α-glucosidase enzymes and therefore may not be considered

to be very much effective in the treatment of Alzheimer’s disease, diabetes, peptic ulcer

and infection-induced urinary stones. Further, the scientific examination for the

traditional claims for antidiabetic use of this plant may also not be justified. Further

studies are needed to explore the possible claimed actions of this plants. The antioxidant

ability of the plant was also found to be weak. It is important to note that the failure of

a plant extract or isolated compounds to demonstrate in-vitro activity during the general

screening process does not necessarily imply a total absence of inherent medicinal

value. The possible presence of synergistic interactions between the different

constituents in crude preparations may result in activities that are not exhibited by

isolated compounds, and should not be excluded. Furthermore, the plant or extract may

react differently in-vivo.

As modern cultures and scientific advances spread around the world, the breadth of the

knowledge store of traditional healers still remains crucial. The full significance of the

indigenous knowledge forfeited may not be realized. It is thus important that the

knowledge be documented and the traditional use given some credence through modern

scientific studies.

121

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تالمف: سهم مبارک عب ہللا الو مبی(

127

7. GLOSSARY

128

Plant sap

Sugary water solution in a plant is known as plant sap.

COSY

Homonuclear correlation spectroscopy is the first and very popular

2D NMR experiment and used for the identification of spins which are coupled to each

other.

NOESY

A two dimensional NMR technique used to determine NOE correlation between

protons within a molecule and useful for analyzing the spatial proximity of protons to

find the stereochemistry and conformation of molecules.

HMBC

Heteronuclear Multiple-Bond Correlation is a two dimensional NMR experiment used

for the identification of long-range (refers to 2- or 3-bonds) couplings between 1H and

13C.

HSQC

The Heteronuclear Single Quantum Coherence experiment is a highly sensitive and an

inverse 2D-NMR experiment used to describe the direct shift correlations in a 1H-13C

system.

HOHAHA or TOCSY

A two dimensional homonuclear correlation experiment used to determine scalar (J)

coupling system of protons with in a spin network. The spectrum resembles with 2D

COSY spectrum. This technique is useful in analyzing the overlapped crowded spectra

and popular for analyzing the oligosaccharides and peptides where molecules are

usually composed of discrete subunits (spin systems) ie. Saccharide units or amino-

acids.

DEPT

Distortionless Enhancement by Polarisation Transfer (DEPT) is a one dimensional

NMR experiment. Which is used for increasing the sensitivity of 13C observation and

for editing of carbon spectra. The technique allows the distinction between C, CH, CH2

and CH3 carbons. Intensity of carbon signals are enhanced via polarization transfer

from more sensitive to less sensitive nuclei such as proton.

129

Broadband

It is a completely decoupled 13C-NMR technique and shows all four types of carbons

(C, CH, CH2, CH3) in a single spectrum.

IC50

It is the inhibitor concentration at which response is reduced by half.

IR Spectrum

Infra-red (IR) spectroscopy is one of the most important analytical technique and IR

spectrum is obtained by the absorption of infra-red radiations which triggers the

molecular vibrations and provide information about the absence and presence of certain

functional groups in the molecule.

UV spectrum

Transition of valence electrons to higher energy state occurs when UV radiation strikes

on an organic compound and molecule absorb these radiations. Ultraviolet

spectroscopy (UV) provides information about the presence of double and triple bonds

and conjugated π systems in the molecule.

Coupling Constant

In proton NMR spectra, signals are split into multiplet, doublet of doublet or singlet etc.

This splitting occurs due to chemically nonequivalent protons on the same carbon atom

or on adjacent carbon. Coupling constant (J) is independent of the applied magnetic

field strength and is expressed in Hertz.

Stereochemistry

The branch of chemistry refers to the three dimensional spatial deposition of atoms

present in molecule.

Glycosides

Glycosides are naturally occurring molecules found in various plants. Basically, these

are molecules in which a sugar is attached to a non-sugar molecule, usually small

natural molecule. Glycosides play various functions in all living organisms.

130

8. APPENDIX

131

List of Tables

1: Pregnane glycosides of various Caralluma species……………………. 16

2: 1H and 13C NMR data of Desmiflavaside A (1)…………….……….. 37

3: 1H and 13C NMR data of Desmiflavaside B (2)……………………… 40

4: 1H and 13C NMR data of Desmiflavaside C (3)……………………… 43

5: 1H and 13C NMR data of Desmiflavaside D (4)……………………… 46

6: 1H and 13C NMR data of Nizwaside (5)……………………………… 49

7: 1H and 13C NMR data of Desflavaside A (6)………………………… 52

8: 1H and 13C NMR data of Desflavaside B (7)………………………… 56

9: 1H and 13C NMR data of Desflavaside C (8)………………………… 60

10: 1H and 13C NMR data of Desflavaside D (9)………………………… 64

11: IC50 vlaues of crude crystals, methanolic extract and its fractions for

breast cancer cells (MDA-MB-231)…………………………………… 68

12: IC50 values of compounds 3-5 for ovarian (SKOV-3) and breast (MDA-

MB-231) cancer cell lines……………………………………………… 72

13: Enzyme inhibition activities of crude crystals and fractions of

methanolic extract……….…………………………………………….. 74

14: Enzyme inhibition activities of compounds 1-5……………………… 75

15: DPPH radical scavenging activity of crude crystals and fractions of

methanolic extract……………...……………………………………… 77

16: DPPH radical scavenging activity of compounds 1-5………………….. 78

17: Binding energies of compounds 3-5 as ligands with target protein

tyrosine phosphatase…………………………………………………… 86

132

List of Figures

1: Scientific studies of Caralluma species in different countries…………. 4

2: Pharmacological activities of Caralluma species……………………… 4

3: Cancer incidences of Omani females…………………………………... 5

4: Cancer mortality profile of Omani females…………………………….. 5

5: (A) Sap exudate of C. flava (B) Sap precipitates (C) Crude crystals…… 29

6: Anticancer activity of crude crystals, methanolic extract and its

fractions against MDA-MB-231 cancer cells………..………………… 67

7: (A) Anticancer activity of compounds 3-5 against MDA-MB-231 breast

cancer cells…………………………………………………………….. 69

7: (B) Anticancer activity of compounds 3-5 against SKOV-3 ovarian

cancer cell line…………………………………………………………. 70

7: (C) Anticancer activity of compounds 3-5 against MCF-10-2A normal

breast epithelial cell line……………………………………………….. 71

8: (A) Protein tyrosine phosphatase binding sites………………………… 80

8: (B) Analysis of protein tyrosine phosphatase binding sites by MOE

software………………………………………………………………… 81

8: (C) View of tyrosine cavity demonstrated the interaction of ligands to

protein tyrosine phosphatase…………………………………………… 81

9: (A) Molecular docking views of ligand (3)…………………………….. 82

9: (B) Molecular docking views of ligand (4)…………………………….. 83

9: (C) Molecular docking views of ligand (5)……………………………... 84

10: Heat map showing the toxicity analysis and pharmacokinetics of

compounds 3-5……………………………………………………….. 85

133

11: Significant HMBC interactions of 1……………………………………. 91

12: Significant 1H-1H COSY and 1D TOCSY interactions of 1…………… 91














134

List of Abbreviations

Bz Benzoyl

COSY correlated spectroscopy

CHCl3 Chloroform

Hz hertz

NOESY nuclear over-hauser enhancement spectroscopy

MeOH methanol

EtOH ethanol

s singlet

m multiplet

d doublet

ppm parts per million

EtOAc ethyl acetate

BuOH butanol

DCM dichloromethane

Glc glucose

Thev thevetose

Dig digitalose

Allom allomethylose

Cym cymarose

MOE molecular operating environment

PTPs protein tyrosine phosphatases

CC column chromatography

135

1H and 13C NMR Spectra of Desmiflavaside A (1)

136

Selective 1D TOCSY NMR of Desmiflavaside A (1)

137

1H and 13C NMR Spectra of Desmiflavaside B (2)

138

Selective 1D TOCSY NMR of Desmiflavaside B (2)

139

1H and 13C NMR Spectra of Desmiflavaside C (3)

140

Selective 1D TOCSY NMR of Desmiflavaside C (3)

141

1H and 13C NMR Spectra of Desmiflavaside D (4)

142

Selective 1D TOCSY NMR of Desmiflavaside D (4)

143

1H and 13C NMR Spectra of Nizwaside (5)

144

Selective 1D TOCSY NMR of Nizwaside (5)

145

1H and 13C NMR Spectra of Desflavaside A (6)

146

Selective 1D TOCSY NMR of Desflavaside A (6)

147

1H and 13C NMR Spectra of Desflavaside B (7)

148

Selective 1D TOCSY NMR of Desflavaside B (7)

149

1H and 13C NMR Spectra of Desflavaside C (8)

150

Selective 1D TOCSY NMR of Desflavaside C (8)

151

1H and 13C NMR Spectra of Desflavaside D (9)

152

Selective 1D TOCSY NMR of Desflavaside D (9)

153

9. PUBLICATIONS

154

155

156

157

158

CURRICULUM VITAE

I, Mr. Muhammad Adil s/o Raees Ahmed was born on June 7th, 1987 in Karachi,

Pakistan. I had my secondary school education with 1st division from Dhaka Boys

Secondary School, Karachi. I did higher secondary education with 1st division from

Saint Patrick’s Govt. College, Karachi during 2005-2006. I earned the degree of

Bachelors in Science (Analytical Chemistry) from Department of Chemistry, Federal

Urdu University, Karachi during 2007-2010. I joined University of Karachi as Trainee

NMR Spectroscopy in 2011. Along with my job, I continued my education and took

admission in M.S leading to Ph.D program in Department of Chemistry, Federal Urdu

University in 2012. I had an opportunity to join University of Nizwa, Sultanate of Oman

in the position of NMR Spectroscopist in 2013 and worked there with 600 MHz NMR

spectrometer having CryoProbe. I had my training in Advanced NMR Methods offered

by Bruker in Fällanden/Zürich, Switzerland in 2015. I performed most of my

experimental work of M.S and Ph.D in University of Nizwa, Sultanate of Oman. It is

the most attractive opportunity for me to complete my Ph. D. under the kind supervision

of Dr. Talat Mahmood.

Documents

prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/9092/1/Adil-Thesis- Corrected.pdfPlagiarism Undertaking I solemnly declare that research work presented in the thesis titled