MUEL Ph.D Thesis by N

BIOASSAY-GUIDED ISOLATION AND STRUCTURE ELUCIDATION OF STEROIDAL ALKALOIDS FROM SARCOCOCCA SALIGNA (D.DON) MUEL

Ph.D Thesis

NAEEM ULLAH JAN

CENTER FOR BIOTECHNOLOGY AND MICROBIOLOGY UNIVERSITY OF PESHAWAR SESSION 2010-2011

BIOASSAY-GUIDED ISOLATION AND STRUCTURE ELUCIDATION OF STEROIDAL ALKALOIDS FROM SARCOCOCCA SALIGNA (D.DON) MUEL

NAEEM ULLAH JAN

A thesis submitted to the University of Peshawar in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology and Microbiology

CENTER FOR BIOTECHNOLOGY AND MICROBIOLOGY UNIVERSITY OF PESHAWAR SESSION 2010-2011

In the name of Allah, The Most Gracious, The Most Merciful

"And say: My Lord increase me in knowledge."

(Qur'an, Ta-Ha 20:114)

Author’s Declaration

I, Naeem ullah Jan, solemnly declare that the thesis titled “Bioassay-Guided

Isolation and Structure Elucidation of Steroidal Alkaloids from Sarcococca saligna (D.Don) Muel”” and the work presented in it is my own work and has been generated by me as the result of my own original research.

I confirm that this work was done wholly or mainly while in candidature for a research degree at University of Peshawar and any part of this thesis has not been previously submitted for a degree or any other qualification at this University or any other institution in the country/world. Further, where I have consulted the published work of others, this is always clearly attributed. More over, the views expressed in the thesis, belongs to the author.

At any time if my statement is found to be incorrect the university has the right to withdraw my Ph. D degree.

______Naeem Ullah Jan

Dated: ______

Plagiarism Undertaking

I solemnly declare that the research work presented in the thesis titled “Bioassay-

Guided Isolation and Structure Elucidation of Steroidal Alkaloids from

Sarcococca saligna (D.Don) Muel” is solely my research work with no significant contribution from any other person. Moreover, the complete thesis has been written by me and minor contribution/help (if any) wherever taken has been duly acknowledged and.

I understand the zero tolerance policy of Higher Education Commission (HEC) and

University of Peshawar 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 cited.

I undertake that if I am found guilty of any formal plagiarism in the above titled thesis even after award of Ph. D degree, the University reserves the right to withdraw/revoke my Ph. D degree and that HEC and the University has the right to publish my name on the HEC/University website in the list of students of plagiarized thesis.

______Naeem Ullah Jan

Dated: ______

BIOASSAY-GUIDED ISOLATION AND STRUCTURE ELUCIDATION OF STEROIDAL ALKALOIDS FROM SARCOCOCCA SALIGNA (D.DON) MUEL

This dissertation is submitted by Naeem Ullah Jan as partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biotechnology and Microbiology

Approved By:

1. ______Dr. Bashir Ahmad Meritorious Professor/Dean Research Supervisor

2. ______External Examiner

3. ______Dr. Sumera Afzal Director Centre for Biotechnology and Microbiology

CENTER FOR BIOTECHNOLOGY AND MICROBIOLOGY UNIVERSITY OF PESHAWAR

CERTIFICATE OF APPROVAL This thesis titled “Bioassay-Guided Isolation and Structure Elucidation of

Steroidal Alkaloids from Sarcococca saligna (D.Don) Muel” submitted by Naeem

Ullah Jan is hereby approved and recommended as partial fulfillment for the award

of degree of Doctor of Philosophy in Biotechnology and Microbiology.

1. Supervisor ______

2. External Examiner ______

3. Director ______Centre for Biotechnology and Microbiology University of Peshawar

CENTRE OF BIOTECHNOLOGY AND MICROBIOLOGY UNVERSITY OF PESHAWAR

DEDICATED TO MY PARENTS

ACKNOWLEDGEMENTS

Acknowledgments

All praises to Almighty ALLAH who blessed me with the patience, dynamism and understanding to effectively complete my PhD project successfully, a milestone in my life. My research would not have been possible without the support and guidance of many people whom I highly oblige.

I would like to extend my utmost gratitude and sincerity to my research supervisor Dr.

Bashir Ahmad, Meritorious Professor, Centre of Biotechnology & Microbiology for the continuous support of my Ph.D study and related research, his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. Moreover, his friendly attitude and continuous grooming not only helped me as a researcher but also as an individual. I could not have imagined having a better advisor and mentor for my Ph.D study.

I extend sincere gratitude to Dr.Achyut Adhikari, HEJ, ICCBS, and University of

Karachi, whose support was undivided throughout my PhD project. I am sincerely grateful to him for providing constructive reviews on my project.

I would also like to thank Dr.Sumera Afzal, Director, Centre of Biotechnology and

Microbiology, University of Peshawar for her continuous support and encouragement throughout my research. I am also thankful to my friends Dr.Hamid Tanoli, Dr.Ibrar,

Dr.Sadiq, Dr.Inamullah, Dr.Inayat-ur-Rahman and Dr.Naveed Malik for their co- operation and guidance. I would also like to appreciate the co-operation of all the teaching and non-teaching staff of the department.

I am indebted to the Centre of Biotechnology and Microbiology, University of

Peshawar for providing me the facilities and opportunity for this research. ACKNOWLEDGEMENTS

I am grateful to all my fellow researchers’ and friends especially Mr. Saifullah

Dr.Javed, Dr. Mehboob Alam and Kashif Bashir for their stimulating and fruitful discussions and support.

Finally, I would like to pay my heartiest tribute to my beloved parents and entire family for their belief who supported me at all stages of my career.

Naeem ullah Jan

TABLE OF CONTENTS

LIST OF TABLES …………………………………………………………………… i LIST OF FIGURE …………………………………………………………………….ii PICUTRE …………………………………………………………………………….vi SUMMARY …………………………………………………………………………vii 1 Introduction and Literature Review: ...... 1

1.1 Plant Sarcocococca saligna ...... 5 1.1.1 Introduction of Buxaceae Family ...... 5 1.1.1.1 Genus Sarcococca ...... 5 1.2 Sarccococca saligna ...... 23 1.3 Alkaloid ...... 24 1.3.1 Introduction ...... 24 1.3.2 Steroidal Alkaloids ...... 26 1.3.3 Classification ...... 26 1.3.3.1 Plants steroidal alkaloids ...... 26 1.3.3.2 Marines Steroidal Alkaloids ...... 38 1.3.3.3 Amphibians Steroidal Alkaloids ...... 40 1.4 Spectral characteristics of sarcococca alkaloids...... 42 1.5 Biosynthesis of Steroid Alkaloids from Sarcococca ...... 49 1.5.1 Cholesterol Biosynthesis ...... 50 1.5.2 Biosynthsis of Squalene ...... 51 1.5.3 Biosynthesis of Lanosterol ...... 54 1.5.4 Biosynthesis of Cholesterol ...... 55 1.5.5 Biosynthesis of Pregnenolone from cholesterol ...... 57 1.6 Biosynthesis of Steroid alkaloids from Sarcococca ...... 57 1.7 Aims and Objectives ...... 59 1.7.1 Phytochemical Investigation ...... 59 1.7.2 Bio-Assay of purified isolated compounds ...... 59 1.7.3 Molecular docking of isolated compounds ...... 59 2 Experimental ...... 60

2.1 General Methods of Experiements ...... 60 2.2 Sarcococca saligna ...... 61 2.2.1 Plant material...... 61 2.2.2 Extraction and isolation ...... 61

TABLE OF CONTENTS

2.2.3 Extraction and Fraction of Sarcococca saligna ...... 64 2.3 Characterisation of Isolated Steroidal Alkaloid ...... 66 2.4 Biological Activities of Isolated Steroidal Alkaloids ...... 70 2.4.1 In-vivo biological assay ...... 70 2.4.1.1 Animal Study ...... 70 2.4.1.2 Hepatoprotective Assay ...... 70 2.4.1.3 Antihyperglycemic Activity ...... 72 2.4.2 In-vitro Biological activities...... 74 2.4.2.1 Immunosuppressant Activity ...... 74 2.4.2.2 Antibacterial Activity ...... 79 2.4.2.3 Antifungal Activity ...... 80 2.4.2.4 Antileishmanial Activity...... 80 2.4.2.5 Phytotoxic Activity ...... 81 2.4.2.6 Insecticidal Activity ...... 83 2.4.2.7 Antioxidant Activity ...... 83 2.4.2.8 Cytotoxicity Assay (Anticancer Activity) ...... 84 3 RESULT and DISCUSSION ...... 85

3.1 Structure Elucidation of Isolated Compounds ...... 85 3.1.1 Alkaloid-C (1) ...... 85 3.1.2 Dictyophlebine (2)...... 86 3.1.3 Sarcovagine-D (3) ...... 87 3.1.4 Saracodine (4) ...... 89 3.1.5 Holaphylline (5) ...... 90 3.2 In-vivo Biological Assays ...... 91 3.2.1 Hepatoprotective Assay...... 91

3.2.1.1 Effect of Biomarker Components of S. saligna on CCl4 as an oxidative inducer 91 3.2.1.2 Hepatoprotective potential of S. saligna pure compounds: A Histological Study 94 3.2.1.3 Hepatic Kupffer cells Immunohistochemistry ...... 96 3.2.1.4 Discussion ...... 101 3.2.2 Antihyperglycemic Activity ...... 102 3.2.2.1 Effect of purified compounds on fasting plasma glucose levels ...... 102 3.2.2.2 Fructosamine levels in Blood...... 105 3.2.2.3 Effect of steroidal alkaloids on Systolic blood pressure (SBP) of diabetic rats 105

TABLE OF CONTENTS

3.2.2.4 Oral Glucose Tolerance Test ...... 107 3.2.2.5 Effect on blood lipids ...... 107 3.2.2.6 Effect on Body Weight ...... 107 3.2.2.7 Discussion ...... 111 3.3 In Vitro Biological activity ...... 113 3.3.1 Immunosuppressant Activity ...... 113 3.3.1.1 Effect of steroidal alkaloid on T-cell multiplication ...... 113 3.3.1.2 Effect of steroidal alkaloid on generation of Interleukin-2 ...... 116 3.3.1.3 Cytotoxicity Assay ...... 116 3.3.1.4 Discussion ...... 119 3.3.2 Antibacterial activity ...... 120 3.3.2.1 Anti bacterial activity of compound halophylline (5) ...... 120 3.3.2.2 Antibacterial activity of compound Sarcovagine-D (3) ...... 122 3.3.2.3 Antibacterial activity of compound dictyophlebine (2) ...... 124 3.3.3 Antifungal Activity ...... 131 3.3.4 Antileishmanial Activity ...... 135 3.3.4.1 Anti-promastigote Activity of Compound 4 (Saracodine) ...... 135 3.3.4.2 Anti-promastigote Activity of Compound 5 (Holaphylline) ...... 139 3.3.5 Phytotoxic activity...... 143 3.3.6 Insecticidal Activity: ...... 146 3.3.7 Antioxidant Activity ...... 149 3.3.8 Cytotoxic Activity ...... 152 4 Molecualr docking of isolated steroidal alkaloids against aromatase enzyme in breast cancer ...... 155

4.1 Materials and methods ...... 158 4.1.1 Aromatase Activity ...... 158 4.1.2 Molecular docking simulations ...... 159 4.2 Results and discussion ...... 160 5 Conclusion ...... 165

6 References ...... 166

LIST OF TABLES

Table 1.1 Nature Derived Drug Product Used for Various Treatment ...... 4 Table: 1.2 Steroidal Alkaloids Isoltaed from Sarcococca Genus ...... 7 Table: 2.1 Composition of E-Medium ...... 82 Table 3.1 Effect of Compounds on FPG Level on Diabetic Rats ...... 103 Table 3.2: Effect of Steroidal Alkaloids on Fructosamine of Diabetic Rats ...... 106 Table 3.3 Effect of Steroidal Alkaloids on SBP (mmHg) of Diabetic rats ...... 106 Table: 3.4 The Effect of Steroid Alkaloids from S. saligna on T-cells Multiplication, IL-2 Generation and Cytotoxicity ...... 118 Table 3.5 Zone of inhibition (mm) of Antibacterial Holaphylline (5) compound ..... 121 Table. 3.6 Zone of inhibition (mm) of Antibacterial Sarcovagine-D (3) compound ...... 123 Table 3.7 Zone of inhibition (mm) of Antibacterial Dictyophlebine (2) compound . 125 Table 3.8 Antifungal activity of steroids alkaloid from S.saligna ...... 132 Table 3.9: Anti-leishmanial activity of Saracodine against promastigotes of L. tropica ...... 136 Table 3.10 Anti-leishmanial activity of compound 5 (holaphylline) against promastigotes of L. tropica ...... 140 Table 3.11 Phytotoxic Effect of Steroidal alkaloids ...... 144 Table 3.12 Insecticidal activity of isolated steroidal alkaloids ...... 147 Table 3.13 Antioxidant activity of Steroid alkaloidal from S.saligna ...... 150 Table: 3.14.Anticancer activity of isolated steroidal alkaloids against HeLa cells ... 153

LIST OF FIGURES

Figure 1-1 Examples of True Alkaloids...... 25 Figure 1-2 Examples of Protoalkaloids...... 25 Figure 1-3 Examples of Pusedo-alkaloids ...... 26 Figure 1-4 Example of New Prototype of Buxus Alkaloid...... 27 Figure 1-5 Examples of Buxus Alkaloids having Nitrogen at C-3 and C-20 ...... 28 Figure 1-6 Example of Buxus Alkaloid having C-4 and C-14 subsitution ...... 28 Figure 1-7 Example of Buxus alkaloid with Formation of Cycloartane Ring B ...... 29 Figure 1-8 Examples of Buxus Alkaloid having Presences of Oxyen at Ring B ...... 29 Figure 1-9 Examples of Pregnane –Type Steroidal Alkaloids from Sarcococca and Pachysandra genus ...... 30 Figure 1-10 Examples of Conanine –Type Alkaloids...... 31 Figure 1-11 Examples of Preganane –type alkaloid ...... 31 Figure 1-12 Examples of Isosteroidal and Steroidal Alkaloids ...... 32 Figure 1-13 Example of Cevanine Type Isosteroidal Alkaloid ...... 33 Figure 1-14 Example of Veratramine Alkaloid ...... 33 Figure 1-15 Example of Jervine Isosteroidal Alkaloid ...... 34 Figure 1-16 Example of solanidine Steroidal Alkaloid ...... 34 Figure 1-17 Example of verazine Steroidal Alkaloid ...... 35 Figure 1-18 Example of Solanidine Type Alkaloid from Solanaceae Family ...... 36 Figure 1-19 Example of Spirosolanes Steroidal Alkaloid ...... 36 Figure 1-20 Examples of Solacongestidine Steroidal Alkaloid s ...... 37 Figure 1-21 Example of Solanocapsine Steroidal Alkaloid ...... 37 Figure 1-22 Example of Jurubidine Steroidal Alkaloid ...... 38 Figure 1-23 Example of Thai marine Steroidal Alkaloid ...... 39 Figure 1-24 Example of Marine Steroidal Alkaloid from Corticium niger ...... 39 Figure 1-25 Example of Marine Steroidal Alkaloid from Ritterella Species ...... 40 Figure 1-26 Example of Marine Steroidal Alkaloid from Plakina and Corticium simplex ...... 40 Figure 1-27 Examples of Amphibian Steroidal Alkaloids from Salamandra and Bufo ...... 41 Figure 1-28 Examples of Amphibian Steroidal Alkaloids from Phyllobates Genus ... 41 Figure 1-29 Example of Steroidal Alkaloid Showed UV Absorption at 254nm ...... 42 Figure 1-30 Example of Steroidal Alkaloid for IR Absorption ...... 43

LIST OF FIGURES

Figure 1-31Example of Steroidal Alkaloid Containing Nitrogen Between C-17 and C- 20 form Base Peak at 58 and 72 m/z through Mass Spectroscopy ...... 44 Figure 1-32 Example of Steroidal Alkaloid having Base Peak at M+ -15 due to loss of Secondary Methyl at C-21 Position and Showed Peak at 55,83 and 98 m/z ...... 45 Figure 1-33 Steriodal Alkaloid Showed Peak at 105 m/z in Mass Spectra having Benzoyl Group at C-3 ...... 45 Figure 1-34 Example of Steroidal Alkaloid Showed Fragment ions at 84 and 110 m/z through ring A breaking ...... 46 Figure 1-35 NMR Spectroscopy Techniques Used for Structure Determination of Steroidal Alkaloids which Resonate at Specific Ranges ...... 49 Figure 1-36 Schemic Diagram for Biosynthetic Pathwayof Mavalonic acid ...... 51 Figure 1-37 Schematic Diagram for Biosynthetic Pathway of Squalene ...... 54 Figure 1-38 Schematic diagram for biosynthetic pathway of lanosterol ...... 55 Figure 1-39 Schematic diagram for biosynthetic pathway of Cholesterol ...... 56 Figure 1-40 Schematic diagram for biosynthetic pathway of Pregnenolone from Cholesterol ...... 57 Figure 1-41 Schematic diagram of steroid alkaloids biosynthesis from pregnenolone ...... 58 Figure 2-1 Shemetic Diagram of Different Extract Fractions from Whole Plant Sarcococca saligna by Using Different Solvent System ...... 63 Figure 2-2 Schematic diagram for Isolation of purified compounds from Chlroform fractions of Sarcococca saligna ...... 65 Figure 3-1 Stucture of Isolated Compound Alkaloid –C ...... 86 Figure 3-2 Structure of Isolated Compound Dictyophlebine ...... 87 Figure 3-3 Structure of Isolated Compound Sarcovagine-D ...... 88 Figure 3-4 Structure of Isolated Compound Saracodine...... 89 Figure 3-5 Structure of Isolated Compound Holaphylline ...... 91 Figure 3-6 (A) Effects of S. saligna Steroidal Alkaloids (Sarcovagine-D, Alkaloid-C, Holaphylline), on Hepatic Biochemical parameters MDA (A), GSH (B), and SOD

(C), in CCl4-intoxicated rats ...... 93 Figure 3-7 The Effect of Test Compound on Liver Inflammation and its Histopathological profile ...... 95 Figure 3-8: Effects of steroidal alkaloids on hepatic macrophages (Kupffer cells). ... 98

iii

LIST OF FIGURES

Figure 3-9: (A) Biochemical Tests of The Effects of S.saligna Compounds on CCl4- Induced Liver injury ...... 100 Figure 3-10: Graphical Representation of the Compound Effect on FPG Level on Diabetic Rats ...... 104 Figure 3-11: Compound Effects on OGTT Test ...... 108 Figure 3-12: The Effect of Compounds on Changes in Blood Lipid in Different Groups ...... 109 Figure 3-13: Compounds Effect on Changes in Body Weight in Different Groups 110 Figure 3-14: Effect of Steroidal alkaloid Sarcovagine-D, Alkaloid-C and Holaphylline on T-cells proliferation...... 115 Figure 3-15: Effect of Puriﬁed Test Compounds on the Generation of IL-2 Production from T- Lymphocytes at Different Concentration...... 115 Figure 3-16: Cytotoxic Effect of Steroidal Alkaloids on 3T3 Fbroblast Cell Line ... 117 Figure 3-17 : Antibacterial effect of compounds Holaphylline, Sarcovagine-D and Dictyophlebine against E.coli ...... 126 Figure: 3-18: Antibacterial effect of compounds against Citrobacter ...... 126 Figure 3-19: Antibacterial activity of compounds against S.aureus ...... 127 Figure 3-20: Antibaterial activity of compounds against S.typhi ...... 127 Figure 3-21: Antibacterial activity of compounds against B.subtilus ...... 128 Figure 3-22: Antibacterial activity of compounds against S.boydii ...... 128 Figure 3-23: Antibacerial activity of compounds against M.luteus ...... 129 Figure 3-24: Antibacterial activity of compounds against E.faecalis ...... 129 Figure 3-25: Antibacerial activity of compounds against P.mirablis ...... 130 Figure 3-26: Antibacerial activity of compounds against P.areuginosa ...... 130 Figure 3-27: Antifungal activity of Holaphylline against various fungi ...... 133 Figure 3-28: Antifungal activity of Alkaloid-C against different fungi ...... 133 Figure 3-29: Antifungal activity of Sarcovagine-D against various fungi ...... 134 Figure 3-30: Graphical representation of Saracodine against L.tropica with different cocentration ...... 138 Figure 3-31: Graphical representation of Holaphylline against L.tropica ...... 142 Figure 3-32: Graphical representation of Phytotoxicity activity with various compounds ...... 145

LIST OF FIGURES

Figure 3-33: Graphical representation of insecticidal activity with various compounds ...... 148 Figure 3-34: Graphical representation of antioxidant activity with various steroidal alkaloids ...... 151 Figure 3-35: Graphical representation of anticancer activity with various compounds ...... 154 Figure 4-1: Biosynthetic pathway of estrogen through aromatase enzyme reaction . 157 Figure 4-2: Binding mode of the compound 5 (Holaphylline) inside the catalytic site of aromatase enzyme...... 162 Figure 4-3: A closer view of the molecular interactions between compound 5 and aromatase enzyme ...... 162 Figure 4-4: Electrostatic and steric interactions between compound 5 and aromatase enzyme ...... 163 Figure 4-5: Binding mode of the compound 1 inside the catalytic site of aromatase enzyme ...... 163 Figure 4-6: A closer view of the molecular interactions between compound 1 and aromatase enzyme ...... 164 Figure 4-7: Electrostatic and steric interactions between compound 1 and aromatase enzyme ...... 164

PICTURE

Picture 1: Sarcococca saligna

Summary

The main focus of this PhD research project is the isolation, structure elucidation and bioassay of steroidal alkaloids of medicinally important plant Sarcococca saligna, belongs to Buxaceae family.

Five compounds were isolated from the chloroform fraction of S.saligna through phytochemical investigation i.e. Alkaloid-C (1), Dictyophlebine (2), Sarcovagine-D

(3), Saracodine (4) and Holaphylline (5). The structure of compounds was determined through modern spectroscopic techniuqes. These steroidal alkaloids were then screened for various In-vivo biological assays such as hepatoprotective and antidiabetic, while also tested In-vitro biological activities such as immunosuppresent, antibacterial, antifungal, phtotoxicity, insecticidal, cytotoxicity and leshmanicidal activity. The compounds were also investigated as steroidal aromatase inhibitors through molecular docking studies against breast cancer.

The selected steroidal alkaloids were screened for In-vivo hepatoprotective and antidiabetic activities. The compounds 1, 3 and 5 markedly decreased hepatic injury by CCl4-injury inducer and mixed inﬂammatory penetration. Therefore, we explored and suggest that steroidal alkaloids from S.saligna could be excellent hepatoprotective agents. The isolated steroidal alkaloids were also tested for the antidiabetic potential and the result showed that compounds 3 and 5 reduced the glucose level significantly in blood and also make better others diabetes associated complications.

The isolated steroidal alkaloids were screened for In-vitro biological assays.

Compound 1, 3 and 5 were screened for immunosuppressant activity. Compounds showed inhibitory activities of T-cell proliferation in the range of 78 to 95% and also inhibit IL-2 production which means the tested compounds were excellent immunosuppressive agents. The compounds 2, 3 and 5 were screened for antibacterial

vii

Summary activity. The compound 2 showed significant antibacterial activity against S.aureus

(79%). The compound 3 showed significant antibacterial activity against

P.aeruginosa (79%), while compound 5 showed good antibacterial activity against

B.subtilus (72%) and P.aeruginosa (69%) respectively. The isolated steroidal alkaloids were screened for antifungal activity against various pathogenic fungi and showed low to moderate antifungal actions. Compounds 1 and 3 showed low antifungal activity while compound 5 showed moderate antifungal activity against selected pathogenic fungi. The selected steroidal alkaloids were also screened for antileshmanial activity. Compounds 4 showed moderate antileshmainal activity while compound 5 showed significant inhibition against the promastigotes of L. tropica.

The phytotoxic effect of compound 5 was observed maximum 66%, while 1 and 2 showed low activity at different concentration level. The isolated steroidal alkaloids were tested for insecticidal activity and among them compound 5 showed maximum activity 65% against T.castaneum, while showed low activity against R.dominica and

C.analis (20% and 10%) respectively.

The selected steroidal alkaloids were screened for antioxidant actions in which compounds 3 and 5 showed significant antioxidant actions and radical scavenging activity increased up to 78% and 80%. The isolated compounds were also screened for anticancer activity in which compound 2 found to be more active with IC50 value

6.13±0.345, while compound 1 showed moderate anticancer activity with IC50 values

12.98±0.235 against HeLa cells lines.

The compounds were to explore as a new steroidal aromatase inhibitors through molecular docking studies in which compound 5 and 1 were active against aromatase enzyme in breast cancer could provide new lead compounds.

viii

Summary

Alkaloid-C (1) Dictyophlebine (2)

Sarcovagine-D (3) Saracodine (4)

Holaphylline (5)

Chapter 1 Introduction and Literature Review

1 INTRODUCTION AND LITERATURE REVIEW:

Human being used plants for the betterment of his life since ancient times in different ways. Plant utilized by human as a food, medicines, shelter and clothing. Plant and microorganism serves as a main source for the development of many types of drug which were used therapeutically [1]. Plants have been used in different ways for treatment like Ayurvedic, Grceco-Arab, Homeopathic and Allopathic.

The old Egyptian civilization ( 1500 BC ) have famous pharmaceutical book called “

Ebers papyrus ” which contain hundreds of drugs mostly originated from plants and also have formulae of different pharmaceutical preparation such as infusions , ointments and pills [2] . Mesopotamia ( 2600 BC ) the first documented record, have hundreds of clay tablets derived from thousands plants such as Cedrus species ( cedar

) , Papaver somniferum ( poppy extract ) , Glycyrrhiza glabra oils , Commiphora species ( myrth ) still used for the treatment of inflammation , infections and colds and coughs [2] . Ayurvedic system is widely implemented in South Asia. Ayurveda and Athrvavada (1200 BC), while Charak samhita and Sushrut samhita ( 500-1000

BC ) have full detailed of over seven hundred medicinal plant [3]. There was well documented record Materia Medica in china over the century. Wu Shi Er Bing Fang

(1100 BC) has a record of 52 prescriptions. The works done by Shennong Herbal (~

100 BC) and Tang Herbal (659 AD) contained 365 and 850 drugs, respectively [2].

The Greeks were famous for development and utilization of herbal medicines in western histories. Dioscorides (100 AD) a famous Greek scientist worked on medicinal plant for their collection, storage and uses. A known scientist Galen (130-

200 AD) in Rome wrote books on drug compounding and formulation, taught pharmacy, and medicines [2].

Chapter 1 Introduction and Literature Review

Plants give us very useful synthetic clues for modern medicines [4]. Plants have ability to synthesise structurally complex bioactive compounds. Therefore plants still retain a historical importance as a source of novel compounds. About 80% of people in the world depend on natural medicines for their primary healthcare [5], while in the survey of WHO shows that 90% of people from developing world rely on the use of medicinal plants [6]. The community pharmacies in the world used quarter of prescription have contain plant extracts or active compound of plant origin [7] .There are about 422,000 flowering plants and out of it more than 50,000 is used for medicinal purpose [8] .Herbal drugs are also used in patient with chronic diseases like breast cancer, liver diseases, HIV, asthma and rheumatologic disorders.

Traditional medicines under the name of Unani system widely practiced in Pakistan for primary health care. Pakistan climatic environment is diverse and is rich source of medicinal plants that naturally grow in abundance like Hazara, Malakand, Azad

Kashmir, Swat, Gilgit Bulthistan, Muree Hills and Baluchistan. There were about

1572 genera and 5521 species identified in Pakistan and local people used only 600 plant species as a source of medicine [9].The search for new compounds from plant still continue which can be used as curative agent for a number of diseases like leishminasis, viral infection, diabetes and many other disorder in human and animals and ultimately these new compounds can be developed as new drug or new lead compound for the foreseeable future.

The chemistry of natural products started from morphine isolation by Serturner since

1803 from opium (Papaver somniferum). In 1860 quinine was isolated from Cinchona bark. Alkaloid Reserpine was isolated in 1952 from Rauwolfia serpentine used as antihypertension [2].

Chapter 1 Introduction and Literature Review

Secondary metabolites produced by plant and microorganisms eliciting pharmacological or toxicological effect on human and animals. Different separation and spectroscopic techniques would be used for the purification and structure determination of compounds [10,11] Secondary metabolites synthesised by plant when needed and play important role in protection of plant serves as a defender from any possible environmental harmful effect [12, 13]. A number of active ingredients reported from plants in last few decades have important therapeutic uses such as artemisinin used for malarial treatment, morphine for severe cardiac pain and qunindine used as antiarrhythmic. The active components from plant and microorgamism can use as a base for synthetic and semi-synthetic preparations through the study of structure activity relationship which are then used those drug for therapeutic purposes. There are many drugs which derived from nature and some of them are given as following in Table1.1.

Bioactives are chemically classified in to several major groups such as glycosides,flavonoids , terpenoids and alkaloids which are active compounds and perform different biological actiivties such as antibacterial .antifungal.antileshmanaial and cytotoxic and therefore a large number of these active compounds were isolated from medicinal plants [14,15] .

Chapter 1 Introduction and Literature Review

Table 1.1 Nature Derived Drug Product Used for Various Treatment

Serial No Drug Compound Indication Lead

nature compound

1 Codeine Natural Narcotic analgesic Plant

2 Orlistat Semi-synthetic antiobestiy Lipstatin

3 Amoxicillin Semi-synthetic antibiotic Penicillin

4 Rosuvastatin Semi-synthetic antilipidemic mevastatin

5 Fluvastatin Synthetic antilipidemic Statin

analogue

6 Qunindine Natural antiarrhythmic Plant

7 Valrubicin Semi-synthetic anticancer Doxorubicin

8 Mycophenolate Synthetic Immunosuppressant Mycophenolic

sodium analogue acid

9 Azithromycin Semi-synthetic Antibiotic Erythromycin

10 Artemisinin Natural Antimalarial Plant

Chapter 1 Introduction and Literature Review

1.1 Plant Sarcocococca saligna

1.1.1 Introduction of Buxaceae Family

Sarcoccoca saligna belong to Buxaceae family. Buxaceae are evergreen shrubs, small trees and some are perennial herbs. It consists of Baxus, Sarcococca, Pachysandra and Simondsia genera. There are about one hundred species. These are generally found at tropical and temperate regions in the world [16], but especially distributed in

Himalayan and Hindokush mountains ranging from west Afghanistan, Pakistan to

East Myanmar including the Philippines, but not found in Australia and South-East

America. Steroidal alkaloids are abundantly found in Buxaceae family and a number of new steroidal alkaloids have been reported [17].

1.1.1.1 Genus Sarcococca

Sarcococca genus is an evergreen shrub, mostly grows in moist and well-drained soil in deep or partial shade. This genus have dark green shining leaves almost free from diseses and pest with an attractive odour from flowers when bloom in January –

February following small red or balck berries. It frequently found in South and East

Asia like Afghanistan Pakistan, India, Nepal, Sarilanka, China, Thailand and

Philippines [18]. Sarcococca genus consist of several species [15] which include ,

S.saligna, S. hookeriana, S. wallichii, S. coriaceae , S. ruscifolia, S.humilis, S.vegans,

S. brevifolia and S. zeylanica. The researcher especially Atta-ur-rehman and M. Iqbal

Choudhary research group performed phytochemical and biological studies of these species [15].

Chapter 1 Introduction and Literature Review

1.1.1.1.1 Phytochemistry Investigation of Genus Sarcococca

Phytochemical investigation of genus Sarcococca was first time reported in 1963

[19] and after that a large number of secondary metabolites mainly steroidal alkaloid have been reported (Table 1.2)

Chapter 1 Introduction and Literature Review

Table: 1.2 Steroidal Alkaloids Isoltaed from Sarcococca Genus

Chapter 1 Introduction and Literature Review

1.1.1.1.2 Biological Importance of Steroidal Alkaloids from Sarcococca Genus

The crude extract and isolated steroidal alkaloid from different species of Sarcococca have exert different pharmacological actions and therefore for the last few decades researcher still showed interest in this genus to explore more its biological actions through its phytochemical studies. Some biological activities of steroidal alkaloid from genus Sarcococca are as following.

1. The bioassay studies of different Sarcococca species extracts fractions and isolated steroidal alkaloids show inhibition of acetyl- and butyrylcholinestrase enzymes [41].

2. Some steroidal alkaloids and crude extract of Sarcococca species have shown antibacterial and antifungal activity [35, 44].

3. Some steroidal alkaloids of Sarcocoacca genus have shown antileishmanial activity

[29].

4. Antiplasmodial activity shown by steroidal alkaloids from Sarcococca genus against P. falciparum [45].

5. Steroidal alkaloid and crude extract fraction from S.saligna used has shown spasmolytic, antidiarrheal, antisecretory and calcium antagonist activity [46].

6. Steroidal alkaloids and different fractions of S. saligna have shown cytotoxic effect

[47]. 22

Chapter 1 Introduction and Literature Review

7. Different extract fractions of S.vegans have anticancer, antiulcer, and antigastric property [37].

8 S.saligna extract fractions have been widely used for management of pain, rheumatism, malaria, and skin problems [25].

9. The aqueous methanolic extract of S.saligna exhibit cardio-suppressive, vasodilator and bronchial relaxant effects [48].

10. S.hookeriana root extract has been used for gout treatment in Nepal [49].

1.2 Sarccococca saligna

There are several species in Sarcococca genus and one of them is S.saligna (syn. S. pruniformis, Buxcaceae) (D.Don) Muell locally called Ladan , is an evergreen dicotyledonous shrub with a scaly buds, found in an areas of high altitudes mountains in Pakistan like Swat, Dir, Manshera, Kashmir and other northern regions of

Pakistan.

S.saligna is 2-3 m tall, green bark and profuse long flexible branches. Leaves are simple lance like, sometime nearly curve like sickle , 5- 9 cm long, 1-2 cm broad, petiole 0.5 -1.3 cm long.Flowers are greenish-white colour mostly sessile, unisexual; bracts ovate 2mm length , sepals ovate , 5mm wide, stamens with 6-7mm long filaments and anthers 2-3mm long.

The literature study of S. saligna shows that crude fractions and steroidal alkaloids have potential of different pharmacological properties. In this research work several steroidal alkaloids were isolated from chloroform fraction of whole plant and their different biological activities were studies.

Chapter 1 Introduction and Literature Review

1.3 Alkaloid

1.3.1 Introduction

The term alkaloid first time originated at the discovery of compound called morphine.

Friedrich Serturner a German scientist in 1805, first isolated morphine which has made a significant step in the field of chemistry and pharmacology [50, 51]. Later on in 1817 to 1821 Joseph Pelletier and Joseph Benaime pharmacists isolated a number of alkaloids such as brucine, quinine, caffeine and veratrine [52, 53]. The meaning of alkaloid was first introduced by a W. Meissner in 1819 which means alkali- like compounds [54]. Alkaloid defined as an organic nitrogenous base compound either from plant or animal origin that may have marked physiological actions on man or animals and in which one or more nitrogen atom is an integral part of a heterocyclic ring system or part of a side chain of a molecule [55].

There are about more than 20,000 alkaloids so far identified as secondary metabolites and have more structural variations in it than any other class of compounds. Alkaloids are classified in to three sub-classes on the basis of structural variation.

1) True Alkaloids

Alkaloids usually derived from amino acid and have heterocyclic ring with nitrogen are called true alkaloids. These are pharmacologically active substances even with low doses. They have bitter taste and mostly occur in white crystalline powder form with exception of nicotine which is in a brown liquid form. Amino acids are the primary precursor of true alkaloid such as L-tyrosine, L- lysine, L-trytophan and L- histidine [56, 57]. Examples of true alkaloid are morphine, atropine (Fig 1-1)

Chapter 1 Introduction and Literature Review

2) Proto-alkaloids

Alkaloids having nitrogen is not a part of heterocyclic ring but present in the side chain part and usually biosynthesised from amino acid are said to be Protoalkaloids

[58] .The main amino acid precursors of protoalkaloid are L-tyrosine, L-tryptophan.

Examples of Protoalkaloid are mescaline, and yohimbine (Fig 1-2)

3) Pseudo-alkaloid

Alkaloids which are not biosynthetically originated from amino acid but nitrogen incorporated later in a part of side chain are called pseudo-alkaloids [58]. Examples of pseudo-alkaloids are solanidine, sarcorucinine-B and pinidine (Fig 1-3)

Figure 1-1 Examples of True Alkaloids

Figure 1-2 Examples of Protoalkaloids

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Figure 1-3 Examples of Pusedo-alkaloids 1.3.2 Steroidal Alkaloids

Steroidal alkaloids are include in pseudo-alkaloids and called steroidal amines. They have contained either a normal steroidal skeleton (cyclopentenophenanthrene), or modified steroidal nucleus having nitrogen in the ring or in a side chain [55]. The precursor of steroid alkaloid biosynthesis is mevalonic acid instead of amino acid.

1.3.3 Classification

Steroidal alkaloids are abundantly found in plant especially in certain families of plant kingdom such as Buxaceae, Apocyanaceae, Solanceae, and Liliaceae. They are also found in some marine organisms and amphibians. Therefore steroidal alkaloid can be classified in to three major categories.

1.3.3.1 Plants steroidal alkaloids

Steroidal alkaloids abundantly found in higher plants belonging from the angiosperms family while exists very rarely in gymnosperms family. Therefore steroidal alkaloids can be divided in to four classes on the basis of plant origins. a) Buxaceae family steroidal alkaloids b) Apocynaceae family steroidal alkaloids c) Liliaceae family steroidal alkaloids

Chapter 1 Introduction and Literature Review d) Solanaceae family steroidal alkaloids

1.3.3.1.1 Buxaceae family Steroidal alkaloids

Plants belonging to Buxaceae family are the major source of steroidal alkaloids.

Research studies revealed that steroidal alkaloids were found majorly in genus Buxus,

Sarcococca, and Pachysandra. There were more than 96 steroidal alkaloids isolated from the genus Sarcococca while, more than 250 steroidal alkaloid isolated from

Buxus genus [43].

Cyclobuxine-D a new prototype of steroidal alkaloid, which contains cyclopropane ring and have C-4 and C-14 substitution pattern isolated in 1964 from B. microphylla

(Fig 1-4). It was intermediate product of lanosterol and cholesterol –type steroids

[17].

Figure 1-4 Example of New Prototype of Buxus Alkaloid The nitrogen positions in Buxus alkaloids have majorly at C-3 and C-20 and an example are buxepidine and beleaubxine (Fig 1-5) [59, 60].

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Figure 1-5 Examples of Buxus Alkaloids having Nitrogen at C-3 and C-20 Buxus alkaloids can be classified on the basis of basic skeleton which is as following.

1. 9β, 19-cyclo-4, 4,14α-trimethyl-5α-pregnane derivatives

It contain a cyclopentanophenanthrene ring with C-4 and C-14 substituted position and cyclobuxapaline-C is an example (Fig 1-6) [61].

Figure 1-6 Example of Buxus Alkaloid having C-4 and C-14 subsitution 11. Abeo-9(10-19)-4, 4,14α-trimethyl-5α-pregnane derivatives

This kind of alkaloids, the bonds breaks at C-9/ C-10 and cycloartane skeleton form in ring B. The specific example is Papilamine (Fig 1-7) [62].

Chapter 1 Introduction and Literature Review

Figure 1-7 Example of Buxus alkaloid with Formation of Cycloartane Ring B Some buxane alkaloids skeleton have observed modification due to the presences of different oxygen groups, absences of one or both methyl at C-4 or C-14 and the locations of double bonds. Ο6-buxafurnamine and O10- buxafurnamine (Fig-8) were good examples of these type alkaloids [63].

Figure 1-8 Examples of Buxus Alkaloid having Presences of Oxyen at Ring B Pachysandra and Sarcococca genus alkaloids have usually simple pregnane- type steroidal alkaloids structure with nitrogen atoms at carbon position number 3 and 20.

Examples of this kinds of alkaloid are epipachysandrine A [15], isolated from

P.procumbens and Salonine-A from S. saligna (Fig 1-9) [26].

Chapter 1 Introduction and Literature Review

Figure 1-9 Examples of Pregnane –Type Steroidal Alkaloids from Sarcococca and Pachysandra genus 1.3.3.1.2 Apocynaceae Family Steroidal Alkaloids

Steroidal alkaloids abundantly found in Apocynaceae family. There are more than 150 steroidal alkaloids isolated from its different genra such as Funtumia, Holarrhena,

Kibatalia, Malovetia and Paravallaris [64]. Goutrael and co-workers in 1960s and early 1970s first time performed phytochemical investigation of its different genera in

France [65]. Steroidal alkaloids belong from Apocynaceae can be divided structurally in to two types.

1. Conanine- type Alkaloids

Conanine-type alkaloids have five- membered heterocyclic ring, attached to ring D of the basic steroidal nucleus. The ring may be pyrrolidine or pyrroline heterocyclic ring and oxygen or amine group is present at C-3 of ring A. A classic example is conessine was isolated from plants of genera Holarrhena, Funtumia, and Malovetia. These types of alkaloids are important for scientist because important hormones can synthesise from it through very simple chemical reactions [66]. The examples are holonamine

[67], and Conessine [68] which were in (Fig 1-10).

Chapter 1 Introduction and Literature Review

Figure 1-10 Examples of Conanine –Type Alkaloids 2) Pregnane-type Alkaloids

Pregnanae-type alkaloids have pregnane nucleus with amino groups on C-3 or C-20 or at both positions. An example of this type is funtuphyllamines A and C in (Fig

1.11) isolated from Funtumia africana [69].

Figure 1-11 Examples of Preganane –type alkaloid

1.3.3.1.3 Liliaceae family Steroidal Alkaloids

Liliaceae family include genera Fritillaria, Veratrum, Petilium, Korolkowia,

Rhinopetalum, Northoliron and Zygadenus which produced more than 300

steroidal alkaloids, exert pharmacological action and used as well for therapeutic

purpose [17, 70].Liliaceae family posses’ steroidal alkaloids have common

structural characteristics, is a C27 cholestane nucleus with 5 or 6 carbocyclic or

Chapter 1 Introduction and Literature Review

heterocyclic rings and because of its carbon skeleton, it can be divided in to two

groups.

e) Isosteroidal Alkaloids f) Steroidal Alkaloids

The structure of both alkaloids shown below in (Fig 1-12)

Figure 1-12 Examples of Isosteroidal and Steroidal Alkaloids The distinguish characteristics of Isosteroidal alkaloids are C-nor-D-homo-[14(13-

12)-abeo] ring system in their basic structure as in above figure. On the basis of linkages between E and F rings can further divided in to sub-types.

A. Cevanine Alkaloids

B.Veratramine Alkaloids

C.Jervine Alkaloids

1. Cevanine Alkaloids

The common characteristics of Cevanine alkaloids is a hexacyclic benzo [7, 8],

[fluoreno [2, 1-b] quinolizine skeleton. Different alkaloids isolated from Veratrum and Fritillaria species. Impericine is an example of these type alkaloids (Fig 1-13)

[71].

Chapter 1 Introduction and Literature Review

Figure 1-13 Example of Cevanine Type Isosteroidal Alkaloid 2. Veratramine Alkaloid

The common feature of Veratramine alkaloids is the presences of an aromatic ring.

However many derivatives of this group contain unaromatized ring D. These types of alkaloids have been isolated from Veratrum and Fritillaria species. An example of this type of alkaloids is 20-Isoveratramine in (Fig 1-14) [72].

Figure 1-14 Example of Veratramine Alkaloid 3. Jervine Alkaloids

The common feature of these alkaloids has C-nor-D homosteriodal alkaloid with the presences of hexacyclic and furan ring E, bound to a piperidine ring, forming an ether bridge between C-17 and C-23. An example of this type alkaloid was peimisine in

(Fig 1-15) [73].

Chapter 1 Introduction and Literature Review

Figure 1-15 Example of Jervine Isosteroidal Alkaloid b) Steroidal Alkaloids

The basic structure of steroidal alkaloids as shown above figure contains six membered rings C and five membered rings D. It can be further divided in to sub- classes.

1) Solanidine Alkaloids

These are steroidal alkaloids have amino group incorporated into indolizine ring to make hexacyclic structure. Naturally it arise from epiminocholestanes , Veratrum and

Fritillaria are main source of this type alkaloids. An example was solanidine as shown in (Fig 1-16) [74].

Figure 1-16 Example of solanidine Steroidal Alkaloid

Chapter 1 Introduction and Literature Review

2) Verazine Alkaloids

The common feature of these types of alkaloids is 22, 26- imino-cholestan heterocyclic structure. Alkaloids from this group have been isolated from Veratrum and Fritillaria species. An example was verazine in (Fig 1-17) [73].

Figure 1-17 Example of verazine Steroidal Alkaloid

4) Solanaceae Family of Steroidal Alkaloids

Steroidal alkaloids are abundantly found in different genera of Solanaceae family and approximately more than 200 alkaloids were isolated from various species of

Solanum and Lycopersicon. Their basic structure possesses C27 cholestane skeleton which can be further divided into following types [17]. a) Solanidine

These alkaloids found mainly in Solanaceae and Liliaceae family. An example of this type alkaloid was 3-O-β –Lycotriaoside in (Fig 1-18) which was isolated from S. lyratrum plant [75].

Chapter 1 Introduction and Literature Review

Figure 1-18 Example of Solanidine Type Alkaloid from Solanaceae Family g) Spirosolanes

The common characteristics of these alkaloids have methyl piperidine ring F, formed oxazaspirane unit by joining C-22 at α position to the steroidal skeleton. An example of this type alkaloid was veramine as ahown in (Fig 1-19) [76].

Figure 1-19 Example of Spirosolanes Steroidal Alkaloid h) Solacongestidine

The common structure of this kind of alkaloids have 5-methylpiperidine ring which is attached at C-20 through 2-position of the heterocycle ring in the main steroidal skeleton. An example of these kind alkaloids were Veralkamine from Vertanum album and etionile from Solanum and Veratrum species as shown in (Fig 1-20) [77,

17].

Chapter 1 Introduction and Literature Review

Figure 1-20 Examples of Solacongestidine Steroidal Alkaloid s d) Solanocapsine

These alkaloids found very rarely. These alkaloids contains epiminocyclohemiketal group in their basic structure and found mostly in Solanum species. An example of this kind of alkaloids was Solacapsine in (Fig 1-21) which isolated from

S.capsicastrum [78].

Figure 1-21 Example of Solanocapsine Steroidal Alkaloid e) Jurubidine

These alkaloids contain amino group at C-3 position and spirostane skeleton in their structure. An example of this kind of alkaloids was jurubidine as in (Fig 1-22) [79].

Chapter 1 Introduction and Literature Review

Figure 1-22 Example of Jurubidine Steroidal Alkaloid 1.3.3.2 Marines Steroidal Alkaloids

The marines animals especially invertebrates produced a number of steroid alkaloids.

Corticium a Thai marine’s sponge produced 4-acetoxy–plakaminamineB in (Fig 1-

23), a stigmastane kind of steroid alkaloid [80]. There are number steroidal alkaloids which have cytotoxic effect were isolated from Corticium niger a Philippine sponge and the example was Plakaminamine I in (Fig 1-24) [81]. There is another series of secondary metabolites called dimeric alkaloids which have also cytotoxic effect were isolated from different species of Ritterella and Cephalodiscus. The steroidal alkaloids isolated from these species have similar structural characteristics in which two highly oxygenated C27 steroidal unit are together through a pyrazine ring at C-2 and C-3 and usually form either 5/5 or 5/6 spiroketals. An example of this series of group was Ritterazine-A as in (Fig 1-25) [82]. Plakina a marine sponge genus produced steroidal alkaloids which have antimicrobial activities and an example was

Plakinamine A in (Fig 1-26) [83]. Cortistatins A-D is a series of 9 (9-10) - abeo- andorstane kind of steroidal alkaloids which have been recently isolated from marine sponge Corticium simplex and an example of this series was Cortistatin A(Fig 1-26) in [84].

Chapter 1 Introduction and Literature Review

Figure 1-23 Example of Thai marine Steroidal Alkaloid

Figure 1-24 Example of Marine Steroidal Alkaloid from Corticium niger

Chapter 1 Introduction and Literature Review

Figure 1-25 Example of Marine Steroidal Alkaloid from Ritterella Species

Figure 1-26 Example of Marine Steroidal Alkaloid from Plakina and Corticium simplex 1.3.3.3 Amphibians Steroidal Alkaloids

There were more than 30 steroidal alkaloids isolated from the skin of various species of amphibians such as Salamandra, Phyllobates, and Bufo which protect the skin from fungal and bacterial infections [17]. The skin gland of these species secretes alkaloids which have basic skeleton as shown in below figure and have some common characteristics in their structure which include cis fusion between A and B ring and also ring A expanded to form isoxazoline system. Examples of these types of alkaloids were samandarin, bufotalin A, and bufotalin B as in (Fig 1-27) [85, 76, 86].

Chapter 1 Introduction and Literature Review

Figure 1-27 Examples of Amphibian Steroidal Alkaloids from Salamandra and Bufo Batrachotoxins (-), batrachotoxinin A and homobatrachotoxin in (Fig 1-28), a highly poisonous steroidal alkaloids were found in different species of poison –dart frog of genus Phyllobates [87].

Figure 1-28 Examples of Amphibian Steroidal Alkaloids from Phyllobates Genus

Chapter 1 Introduction and Literature Review

1.4 Spectral characteristics of sarcococca alkaloids

Steroidal alkaloids of Sarcococca species can be different from other types of steroidal alkaloids by their special spectral features. UV, IR, MS spectroscopy, H1 and

2D NMR spectroscopy technique would be used for structure determination.

1. UV Spectrophotometry

Steroidal alkaloids of Sarcococca species have shown terminal UV absorption; while mostly functional groups show UV absorption. An example was sarcovagine-C in

(Fig- 1-29) which shows 254nm UV absorption of α, β –unsaturated carbonyl group

[36].

Figure 1-29 Example of Steroidal Alkaloid Showed UV Absorption at 254nm B) IR Spectrophotometry

IR technique is used for the detection of functional groups in steroidal alkaloids at different points such as NH (3300-3500 cm-1), OH (3200-3600 cm-1), C=O (1700-

1750 cm-1), C=C (1600- 1680 cm-1 ). Salignarine-C was an example (Fig 1-30), which showed IR absorption at 3345 (NH), 3330(OH), C=O (1645) cm-1 [22].

Chapter 1 Introduction and Literature Review

Figure 1-30 Example of Steroidal Alkaloid for IR Absorption C) Mass Spectromtery

Mass spectrometry technique is used for determination of molecular mass and fragmentation of steroidal alkaloid in structure elucidation. Most of the Sarcococca alkaloids have specific mass spectra features which are as follows.

1. The alkaloids having nitrogen groups, fragmentation occur between α and β carbon bond to the nitrogen atom. The resulting fragment ions form by the breakdown of nitrogen containing side chain on the ring D mostly form the base peak at m/z 58 and

72 , which showed N-methyl-N-ethyliminium and N, N-dimethyl-ethyliminium , breakdown from the place of C-17 as in ( Fig 1-31 ).

Chapter 1 Introduction and Literature Review

Figure 1-31Example of Steroidal Alkaloid Containing Nitrogen Between C-17 and C-20 form Base Peak at 58 and 72 m/z through Mass Spectroscopy 2) Some Sarcococca alkaloids have double bond between C-16 and C-17 as in

Sarovagenine-C, which showed peak base of M+ -15 due to elimination of secondary methyl at C-21 position in ( Fig 1-32 ) [34].

3) Some Sarcococca alkaloids like sarcovaginine-C shown below ( Fig 1-32 ) have tigolyl or senecoyl group at C-3 position which showed characteristics peaks at m/z

55, 83, and 89 [34] . The senecoyl group of some these alkaloids give similar peaks at the same position but can be easily distinguished from 1H-NMR spectra [36].

4) Compounds having peak at m/z 105 in the mass spectra show benzoyl group at C-3 position and an example is Axillarine-C shown in (Fig 1-33) [15].

Some other possible fragmentation occurs in structure sarconidine which shows fragments ions at m/z 84 and 110 through result of breaking ring A [17]. The structure of compound shown in (Fig 1-34)

Chapter 1 Introduction and Literature Review

Figure 1-32 Example of Steroidal Alkaloid having Base Peak at M+ -15 due to loss of Secondary Methyl at C-21 Position and Showed Peak at 55,83 and 98 m/z

Figure 1-33 Steriodal Alkaloid Showed Peak at 105 m/z in Mass Spectra having Benzoyl Group at C-3

Chapter 1 Introduction and Literature Review

Figure 1-34 Example of Steroidal Alkaloid Showed Fragment ions at 84 and 110 m/z through ring A breaking D: 1H and 2D- NMR Spectroscopy

Steroidal alkaloids from Sarcococca species have common cyclopentenophenanthrene skeleton which possess different groups such as hydroxyl, carbonyl, acetoxy, methoxy, and olefins. Sarcococca alkaloids spectras have some common characteristics which are as following.

1 1. H-NMR is used to detect the CH3, CH2 and CH protons of the basic skeleton,

resonate mostly in the range of δ 0.6 to 2.6, as overlapping signals. However 2D-

NMR techniques and its data interpretation are also useful for structure

determination.

2. Most of the known steroidal alkaloids from Sarcococca species posess two

tertiary CH3 moieties at C-18 and C-19, resonating in the range of δ 0.6 to 1.2.

Generally CH3 protons vibrated up-field at C-18 than CH3 protons at C-19.

3. Most of Sarcococca alkaloids contain a secondary methyl at C-21, which shows

as a doublet in the range of δ 0.9 to 1.4.

Chapter 1 Introduction and Literature Review

4. Alkaloids from Sarcococca species possess 3H or 6H downfield singlets in the

range of δ 2.0 to 2.4, because of Na or Nb mono-or dimethyl moieties at C-3 or C-

20.

5. These alkaloids also contain usually N-senecioyl or N-tigloyl group at C-3, C-4΄

and 5΄ methyl protons of C-3 senecioyl functional group resonate as singlets in the

range of δ 1.7 to 2.1, whereas methine proton at C-2΄ shows a singlets at a range

of δ 5.4 as observe in ( Fig 1- 35 ) salignarine-D [22]. C-4´ and C-5´ methyl of

tigloyl functions resonate as doublets in the range of δ 1.7 to 1.9 and as a singlet in

the range of δ 1.7 to 2.0. The methine proton at C-3´ shows as quartet in the range

of δ 6.2 to 6.5 as in example of sarcovaginine B ( Fig 1-35 ) [20].

6. There are protons which resonate downfield multiplets in the range of δ 2.8 –

5.2 are the feature of methine protons, geminal to the hydroxyl, acyloxy and

amidic groups. The presence of an acetoxy function has also been observed in the

downfield shift of geminal methine by 1ppm as compared to a hydroxyl function.

The C-2 and C-4 are the common position for oxygenation in the skeleton while

generally C-3 position has amidic function as observed in sarcovagenine-B (Fig 1-

35).

7. Vinylic hydrogens at C-6 and C-16 resonate in some cases in the range of δ 5.2-5.8.

Chapter 1 Introduction and Literature Review

Figure 1-35 NMR Spectroscopy Techniques Used for Structure Determination of Steroidal Alkaloids which Resonate at Specific Ranges 1.5 Biosynthesis of Steroid Alkaloids from Sarcococca

Metabolic reactions occur enzymatically in living things. Over the last few decades scientist try to understand biosynthetic pathway of secondary metabolites and to study the main precursor product relationship. Natural products can be divided in to primary and secondary metabolites. Primary metabolites are essential for normal activity of living organisms which includes amino acid, sugars and nucleotide bases. Secondary metabolites are produced when needed and biosynthesized from primary metabolites

Examples are alkaloids, terpenes, flavonoids, phenols etc.

Steroid alkaloids from Sarcococca species have pregnane-type skeleton in their basic structure with C-20 graded side chain and it follows the same biosynthetic pathway as other pregnane-type steroidal alkaloids. The biosynthetic pathway sequence is as following.

Acetate------Cholestrol------Pregnenolone------Steroidal alkaloids

Chapter 1 Introduction and Literature Review

1.5.1 Cholesterol Biosynthesis

Cholesterol biosynthesis starts by condensation of acetyl-CoA molecule which is then continuous followed by the enzymatic cyclization and rearrangement reactions and it biosynthesis can be divided in to four steps [87].

1. Mevalonic acid synthesis

2. Mevalonic acid conversion into squalene

3. Formation of lanosterol

4. Lanosterol conversion into cholesrerol.

1. Biosynthesis of Mevalonic acid

Mevalonic acid biosynthetic pathway involves initially the reduction reaction of carbon dioxide through photosynthetic pathway to form glucose as shown in (Fig 1-

36). The glucose then converted in to phosphoenol pyruvate (PEP) via glycolysis reaction. The PEP goes isomerisation and decarboxylation react with Co-enzyme A form acetyl co-enzyme A (acetyl-CoA). The acetoacetyl-CoA then form through condensation of two acetyl-CoA molecules in the presence of thiokinase enzymes with elimination of CoASH as shown in ( Fig 1-36 ) .The acetoacetyl-CoA was then combine with another acetyl-CoA in the presence of 3-Hydroxy-3-methyl glutaryl-

CoA synthetase enzyme to synthesized 3-Hydroxy-3-methyl glutaryl-CoA ( HMG-

CoA) as shown in (Fig 1-36). Mevalonic acid (MVA) form via HMG-CoA molecule reduced in the presence of NADPH and HMG-CoA reductase enzymes as shown in

Fig 1-36.

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Figure 1-36 Schemic Diagram for Biosynthetic Pathwayof Mavalonic acid 1.5.2 Biosynthsis of Squalene

Biosynthesis of squalene occurs with the following scheme. The compound mevalonic acid phosphorylated with ATP in the presence of mevalonic kinase to produce 5-phosphomevalonic acid ( phospho-MVA) as in below figure and then 5-

Phospho-MVA phosphorylated again with presences of ATP and phosphomevalonate kinase enzyme to yield 5-pyrophosphomevaloic acid ( pyrophospho MVA) as shown in Fig 1-37. The compound pyrophospho MVA converted in to Iso pentenyl pyrophosphate (IPP) through decarboxylation and dehydration with the presence of pyrophosphomevalonate decarboxylase and ATP as shown in Fig 1-37. The enzyme

IPP isomerase act on IPP which yield its isomer 3,3-dimethylallyl pyrophosphate (

Chapter 1 Introduction and Literature Review

DMAPP) as shown in Fig 1-37 by trans elimination of phosphate [88].The IPP and

DMAPP condensed asymmetrically in the presence of geranyl transferase enzyme produced geranyl pyrophosphate (GPP) as shown in fig below. The GPP and IPP also condensed in a similar manner in the presence of isoprenyl transferase enzyme, which give a product farnesyl pyrophosphate (FPP) as shown in Fig 1-37 [89]. The condensation of two molecules FPP in a tail- to-tail manner through catalysed of microsomal enzyme squalene synthetase to produce an intermediate product presqualene pyrophosphate (PSPP) as shown in scheme.

The PSPP is then changed in to another intermediate cyclobutane cation as shown in

Fig 1-37 through elimination of pyrphospahte from PSPP give carbonium ion on cyclopropane which then goes on rearrangement to form cyclobutane cation intermediate as shown in (Fig 1-37). The squalene molecule form as shown in Fig 1-

37 through conversion of cyclobutane cation intermediate in the presences of

NADPH. The NADPH gives hydride ion to central carbon atom of intermediate cyclobutane cation where it opens and neutralises carbonium ion.

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Figure 1-37 Schematic Diagram for Biosynthetic Pathway of Squalene 1.5.3 Biosynthesis of Lanosterol

An enzymatic cyclization of squalene is important step in the biosynthesis of steroids and triterpenoids as shown in Fig 1-38. The epoxidation reaction start from squalene in the presence of squalene epoxidase enzyme with the help of molecular oxygen and

NADPH to form 2,3-epoxysqualene as shown in scheme below . The next and important reaction is the cyclization 2, 3-epoxysqualene which converted in to prosterol in the presences of squalene oxide cyclase enzyme as shown in figure below.the mechanism of cyclization reaction start by an attack of proton on oxide which then followed by electron shifting form ring closure with the carbonium ion formation at C-20 position of prosterol molecule. The prosterol converted in to lanosterol molecule through series reaction of 1, 2-trans shifting of hydrogen and methyl functions with the loss of proton at C-9 as shown in figure [90]. The same reaction also showed parallel by chair-boat-chair-boat form with the trans-syn-trans- anti-trans- anti configuration with the stereochemistry at the stereogenic centres as shown in Fig 1-38 .

Chapter 1 Introduction and Literature Review

Figure 1-38 Schematic diagram for biosynthetic pathway of lanosterol 1.5.4 Biosynthesis of Cholesterol

The conversion of lanosterol into cholesterol is an important step in the biosynthesis of steroids as in below scheme. It involve three step reaction, first the removal of three methyl group at C-4 and C-14 position through oxidative reaction to form zymosterol compound as shown in below scheme, secondly shifting of double bond in ring B to form desmosterol, and thirdly the double bond in the side chain of desmesterol reduce in the presence of microsomal reductase enzyme to form cholesterol as shown in Fig 1-39 below.

Chapter 1 Introduction and Literature Review

Figure 1-39 Schematic diagram for biosynthetic pathway of Cholesterol

Chapter 1 Introduction and Literature Review

1.5.5 Biosynthesis of Pregnenolone from cholesterol

Pregnenolone biosynthesis pathway initiate from cholesterol through stepwise hydroxylation at C-20 and C-22 to form 20-Hydroxycholesterol which further goes to oxidative cleavage between hydroxyls, through peroxide result in the formation of pregnenolone as shown in ( Fig 1-40). These reactions catalysed in the presence of mixed function oxidase enzymes with the help of NADPH and molecular oxygen.

Figure 1-40 Schematic diagram for biosynthetic pathway of Pregnenolone from Cholesterol 1.6 Biosynthesis of Steroid alkaloids from Sarcococca

The investigation of steroidal alkaloids formation from Sarcococca showed that nitrogen atom is inserted at the later stage of steroid skeleton as shown in Fig 1-41 .It is suggested that the biosynthesis of pregnane-type steroidal alkaloids quite easily through reductive amination of steroidal ketone i.e.pregnenolone as shown in figure

1-41. These reactions proceeded through enzymatic oxidation and reduction, form an oxidised product progesterone as shown below and reduced compound as shown in

Chapter 1 Introduction and Literature Review

Fig 1-41, respectively. The compound then goes under reductive amination to yield the compounds as shown Fig 1-41 below.

Figure 1-41 Schematic diagram of steroid alkaloids biosynthesis from pregnenolone

Chapter 1 Introduction and Literature Review

1.7 Aims and Objectives

The literature study of steroid alkaloids from S.saligna has shown the medicinal importance and therefore the researcher still interested to explore more its biological actions through its phytochemical studies. The aims and objectives of current work are as following.

1.7.1 Phytochemical Investigation a) Isolation and purification of compounds

Column chromatography will be used for isolation and purification of compounds from the choloroform fractions of S.saligna plant by using various solvents systems. b) Structure Elucidation of purified compounds

UV, IR, 1H, 13C- NMR, 2D-NMR and mass spectroscopy will be used for structure determination of purified compounds.

1.7.2 Bio-Assay of purified isolated compounds

Compounds will be screend for various biological activities such as immunosuppressent, hepatoprotective, antidiabetic, antibacterial, antifungal, phytotoxicity, insecticidal, cytotoxicity and leshmanicidal activity.

1.7.3 Molecular docking of isolated compounds

The compounds will be investigated as steroidal aromatase inhibitors through molecular docking studies against breast cancer

Chapter 2 Experimental

2 Experimental

2.1 General Methods of Experiements

1. Physical Constant

Melting points instrument Yanaco-MP-S3 was used for compounds melting point.

JASCO Digital polarimeter (Model DIP-3600) was used to measure optical rotation in chloroform and methanol.

2. Instrumentation

A. Infrared Instrument: JASCO- A302 IR spectrophotometer was used for

recording of IR spectra in chloroform solution with compounds.

B. Ultra-violet Instrument: UV spectra for absorption of compounds were recorded

by using Hitachi UV-3200 spectrophotometer in methanol.

C. Mass Spectrometry: Varian MAT 312 mass spectrometry were used for mass

spectra measured by double focusing and Jeol JMS 600 and HX 110 mass

spectrometry were used to measured FAB and HREI-MS spectra .

D. Nuclear Magnetic Resonance : Bruker AC-300 , AM-400 and AMX-400 MHz

1 spectroscopy were used to measured H-NMR spectra , while 75, 100, 125 and

150 MHz were used for 13C-NMR spectra. DEPT 90⁰ and 135⁰ were used for

measuring carbon signals multiplicities. COSY 45⁰ were used to determined

homonuclear 1H-1H connectivity. HMQC were used to determine one- bond 1H-

13C connectivity. HMBC were used to determine two-and three-bond 1H-13C

connectivity. 1H-NMR chemical shift are measure in δ (ppm) while coupling

constant (J) in Hz.

E. Chromatography: Column chromatography technique were used on Merck

salica gel 60 (100-310 mesh sizes), Merck alumina (100-250 mesh size) and LH-

Chapter 2 Experimental

20 Sephadex for isolation of steroidal alkaloids. Compounds purification were

checked through pre coated salica gel TLC plates ( E.Merck , F254) under UV

light at 254nm and 266nm for fluorescence spots. Dragendroff,s reagent spary

was used for detection of alkaloids on TLC plates.

F. Dragendroff,s Reagent Composition :

A Solution: Mix 0.85g bismuth nitrate in 10mL acetic acid and in 40L of water.

B Solution: Mix 8g of potassium iodide in 20mL of water. Then mix equal volume of A and B solution and stored in a dark vessel as a stock solution. So before use mixed one mL stock solution with 2mL acetic acid and 10mL water.

2.2 Sarcococca saligna

2.2.1 Plant material

Sarcococca saligna (D.Don) Muel whole plant (40 Kg) was collected in June 2014 from Miandam, District Swat, Khyber Pukhtoonkhwa, Pakistan. The plant was identified by Assistant Professor Dr. Jilani, a botanist at Botany department,

University of Peshawar, Pakistan and specimen voucher (But.20098 (pup) was submitted in herbarium section.

2.2.2 Extraction and isolation

The whole plant (40Kg) of S. saligna was dried in a shade and crushed in to powder.

The powder was soaked in to MeOH/H2O mixture ratio of 8:2 in 35L for 20 days. The methanolic extract was filtered under vacuum and become concentrated (2Kg). The concentrated methanolic extract was then dissolved in distilled water (2L). The mixture was then defatted with 25L n-hexane; give n-hexane extract (254g). The fatty material removed from aqueous extract and then extracted with chloroform at pH 6 to give extract of chloroform (200g). The rest of aqueous fractions extracted with ethyl 61

Chapter 2 Experimental acetate (150g) and butanol 25L, give extract of butanol (100g). Dragendroff,s spray was used for detection of alkaloids from chloroform fraction through TLC.The chloroform fraction was then subjected to column chromatography for further isolation and purification of alkaloids as in Fig 2-1.

Chapter 2 Experimental

Sarcococca saligna (Whole Plant) 40 Kg

Extracted with MeOH-H2O (8:2) 35 L for 20 days, under vaccum

Crude methanolic extract 2kg

Dissolved in Distilled water (2 L)

Soluble Insoluble Extraction with n-hexane (25 L)

Aqueous extract

n-hexane extract (254g) Extraction with Chloroform at pH-6 (25 L)

Chloroform extract at Aqueous extract pH-6 (200gm)

Extraction with Ethyl acetate (15 L)

Ethylacetate extract Aqueous extract (150gm)

Extraction with Butanol (25 L)

Aqueous extract Butanol extract (100gm)

Figure 2-1 Shemetic Diagram of Different Extract Fractions from Whole Plant Sarcococca saligna by Using Different Solvent System

Chapter 2 Experimental

2.2.3 Extraction and Fraction of Sarcococca saligna

The chloroform extract was subjected for further fractionation to column chromatography on silica gel as shown in Fig 2-2. The solvent used for extraction was n-hexane: ethyl acetate: and a few drops of diethylamine for increased the polarity. A mixture of semi purified compounds isolated from chloroform extract. Different solvents ratio of n-hexane: ethyl acetate and few drops of diethylamine used for isolation and purification of compounds. TLC results show that chloroform fraction contains alkaloids. Several subfractions were obtained which then subjected to repeated column chromatography on alumina gel for isolation of purified compounds, eluted with solvents.

Fractions F1 to F3 were obtained from chloroform extract with different solvent- solvents polarities. F1 (5.2 g) fraction afford from chloroform extract with ratio of 90:

10 of n-hexane: ethyl acetate with a few drops of diethylamine solvent through salica gel column chromatography. The fraction NF-23 was obtained from F1, which shows alkaloids on TLC , can go further through process of purification on alumina column with n-hexane, ethylacetate and few drops of diethylamine solvents affords two alkaloidal compound NF-23-4 (1) and NBEA2 (2) as shown in below figure.

Similarly with increase of solvents n-hexane: ethyl acetate polarities of fraction F2 and F3 through alumina column chromatography, purified isolated compounds obtained which were NA-8 (3), NS-55 (4) and NF-73-31(5) as shown in Fig 2-2.

The purified compounds were detected on TLC by showing single spots with the help of dragendroffs spray. The purified isolated compound NA-8 (3) get from F2 while

NS-55 (4) and NF73-31 (5) from F3 fractions.

Chapter 2 Experimental

Chloroform extract

(200 gm)

Column Chromatography (CC) (Si-gel), eluted with n-hexane: EtOAc few drops of diethylamine

n-hex: EtOAc n-hex: EtOAc n-hex: EtOAc 80:20 with few 75:25 with few 90:10 with a few drops of DEA drops of DEA Drops of DEA

F2 (3.8g) F3 (2.5g) F 1 (5.2g)

Alumina Alumina CC Alumina CC CC

Semi pure Compound NA (230mg) Semi pure Semi pure Compound NS Compound NF-23 (235mg) (215mg)

Alumina Alumina CC Semi pure Compound NF CC Semi pure Compound 73 (185mg) NBEA (85mg) Compound NA-8 (3) (180mg) Alumina CC Alumina CC Compound NF-23-4 (1) ( 15 5mg) Alumina CC Compound NF 73- 31(5) (135mg)

Compound NBEA-2 (2) (55mg)

Compound NS 55(4) (125 mg)

Figure 2-2 Schematic diagram for Isolation of purified compounds from Chlroform fractions of Sarcococca saligna

Chapter 2 Experimental

2.3 Characterisation of Isolated Steroidal Alkaloid

1. Compound 1

The fraction F1 (5.2g) obtained from chloroform extract through salica column chromatography by using of n-hexane: ethyl acetate (90:10) solvents with a few drops of diethylamine. This fraction F1 was further subjected on neutral alumina column chromatography with a same solvent elution gives impure subfraction NF 23 and

NBEA which then further eluted on alumina CC to obtained pure compound 1.

2. Compound 2

The purified compound 2 (55mg) was obtained from fraction F1 on neutral alumina column chromatography with elution by specific solvent system as shown in Fig 2-2.

Chapter 2 Experimental

The solvent ratio used for this elution was n-hexane: ethylacetate (9: 1) with few drops of diethylamine.

3. Compound 3

The Fraction F2 (3.8 g) was subjected on neutral alumina chromatography which eluted with n-hexane/ethylacetate (8:2) with a few drops of diethylamine solvents, yielded a compound (3).

Physical Appearance: white powder

Yield: 180 mg

Rf: 0.45 (n-hexane/EtOAc/Et2NH2 in 8:2:0.5)

25 [α]D : + 30⁰ (с = 0.04, CHCl3)

UV ( MeOH ) nm( log Ɛ ) : λmax 212 ( 2.5 ).

EI MS m/z ( rel . int. % ) : 442 ( 4), 424 ( 11), 99(4),

FAB +ve MS : m/z 440.

HREI MS m/z : 440.3395 ( calcd for C28H44N2O3, 440.3389 ) .

Chapter 2 Experimental

4. Compound 4

The fraction F3 (2.5 g) obtained from chloroform extract was further subjected on neutral alumina column chromatography eluted with n-hexane/ EtOAc (75:25: few drops of Et2NH) of solvents to afford compound (4) as shown in Fig 2-2.

Chapter 2 Experimental

5. Compound 5

The compound 5 was obtained from the fraction F3 by subjecting on alumina column chromatography, eluted with n-hexane: ethyl acetate (75:25) with a few drops of diethylamine solvents as shown in Figure 2-2.

Chapter 2 Experimental

2.4 Biological Activities of Isolated Steroidal Alkaloids

2.4.1 In-vivo biological assay

The isolated compounds from S.saligna were tested for different In-vivo biological assay.

2.4.1.1 Animal Study

A healthy male albino rats aged 2–3 months were fed standard rodent diet. The experimental study was approved by the animal ethical committee of the Centre of

Biotechnology and Microbiology, University of Peshawar. All animals received humane care and all protocols involving the animals were in compliance with the guidelines approved by the Institutional Ethics Committee of Centre of Microbiology and Biotechnology, University of Peshawar adhering to the guidelines of the

Institutional Animal Care and Use Committee (IACUC) for animal studies [91].

2.4.1.2 Hepatoprotective Assay

2.4.1.2.1 In-vivo study design

Male albino rats of the normal control group received vehicle while CCl4 treated group were inject 0.5ml/Kg CCl4 dissolve in olive oil intraperitoneally two times a day for two days. The positive control group was pretreated with silymarin at a dose of 200mg/Kg [48] and the other groups were treated with compounds (3), (5), and

(1) at a dose of 20mg/Kg, for three days prior of CCl4 injection [93] and during the

CCl4 injections for 2 days.

2.4.1.2.2 Quantitative Evaluation of Liver Histopathology Assessment

After 24 h of last CCl4 injection, all animal groups were dissected and the liver was cut into pieces were put in isotonic saline solution. The liver tissues were rapidly excised and ﬁxed in neutral buﬀered formalin dehydrated through a graded series of 70

Chapter 2 Experimental isopropyl alcohol, suspeneded in paraffin, and cut into 5 µm thick sections and stained with hematoxylin-eosin (H&E). The liver tissues were then studied and examined under bright field microscope at different magnification using Nikon 90i microscope.

Following under diﬀerent condition histopathological analysis was carried out. The necrotic area was measured by in 20 diﬀerent liver sections of each group using the

NIS-elements software from Nikon, Japan. The damaged/injured area of the liver around the central vein was expressed in percentage compared to the whole area of the section.

2.4.1.2.3 Immunohistochemistry

For immunohistochemistry, 4 µm thin liver slides were used. The slides were deparaﬃnized in xylene and dehydrated in graded alcohol. The liver sections were incubated for 1 h with primary antibodies for liver macrophages, clone ED1 (clone

ED1,abcam) (diluted 1:50).After thoroughly washing with PBS, the sections were then incubated with the secondary antibody, Texas Red-conjugated goat anti-mouse

IgG (1:50) for 45 min. Then the slides were counterstained with DAPI, and mounted, while the expression proﬁle and cellular localization of liver macrophages were analyzed by ﬂuorescence microscopy (Nikon 90i, Japan).

2.4.1.2.4 Biochemistry of blood

To study the function and damage of liver, blood was collected from heart for serum and chemistry analyzer (Roche) were used to measure the level of ALT, AST and

ALP enzymes.

2.4.1.2.5 MDA, SDA and Glutathione Determination

To measure the liver antioxidant action, the animals were dissected and quickly excised the liver which was then frozen at -80⁰C for storage. The samples of hepatic

Chapter 2 Experimental tissue were melt and blended in equal volumes of cold phosphoric buffer saline at concentration of 50mM (pH 7.4), 20 min centrifuged and kept at 4⁰C. The kits pack available commercially used for MDA, SOD, GSH and protein (Sigma-Aldrich,

USA).

2.4.1.2.6 Statistical Analysis

SPSS software was used for data analysis. Significant differences between the samples were checked by one-way ANOVA. Values in the text are mean ± SD, standard deviation.Differences at P<0.05, or P<0.01were considered as significant.

2.4.1.3 Antihyperglycemic Activity

Compounds (3), (4) and (5) were screened for antidiabetic assay

2.4.1.3.1 In-vivo study design

A whole night the rats were in fasted condition and then made diabetic by injecting streptozotocin intraperitonely, prepared freshly in (3mM) citrate buffer of pH 4.5 at dose of (40mg/Kg) [95]. After a week rats having stabilized diabetes with a fasting plasma glucose (FPG) level was of >220mg/dl and the animals were diabetic considered. After a week of STZ injection the treatment were started and the 8th day was considered as the 1st day of treatment.

Group 1 received normal saline twice daily (1ml/Kg s.c) and served as diabetic control group. Group (II) was treated with active compound (3) with a 5mg/ Kg dose subcutaneously twice a day. Group (III) was treated with active compound (4) subcutaneously with a dose of 5mg/Kg, twice a day. Similarly Group 1V was treated with active compound (5) twice a day subcutaneously with a dose 5mg/Kg. Group V was treated with a standard drug glibenclamide at a dose of 1mg/Kg/day. All the compounds and standard drug were dissolved in 10ml normal saline.

Chapter 2 Experimental

To determine blood glucose level, blood was collected in a heparinized glass tube by pricking the capillary vessels in the tail tip and centrifuged at 4⁰C for 10 minutes. The plasma was separated and keeps at -20⁰C until used for determination of fasting plasma glucose (FPG). To determined fructosamine and lipid level in serum, half of blood was collected in an ordinary vial which is then left for clot in order to separate the serum. Blood glucose was determined by using glucose oxidase method. The basic principle of this method is to transform glucose into gluconic acid by glucose

sample.

1. GOD enzyme

2. Glucose standard (400mg/dl)

3. Working solution

procedure was as following

Chapter 2 Experimental

Mixed the solution vigoursly and then stored for 10 minutes at 37⁰C. A cuvette used for sample and standard for absorbance against blank at 520 nm wave length of spectrophotometer. The formula for glucose calculations are as following

Conc of glucose (mg/dl) = A Sample / A Std ˣ Conc of Std solution, While A is absorbance, Conc is Concentration and Std is Standard.

2.4.1.3.2 Data Analysis

The results were in mean ±SD and analyzed by using ANOVA. The Tukey’s test was applied to measure repeated ANVOA. The Values of p ≤0.05 were considered to be statistically significant

2.4.2 In-vitro Biological activities

2.4.2.1 Immunosuppressant Activity

2.4.2.1.1 Material

The material and instrument used for assay were lithium-heparin blood collection tube with an internal vacuum for suck the blood (BD Biosciences) , serum of fetal bovine from Thermo Scientific Hyclone , Rosewell Park Memorial Institute-1640 ( RPMI-

1640) Medium from Mediatech Inc (USA), Phytohemagglutin( PHA) from Sigma

Aldrich (USA), antibiotic penicillin/streptomycin and separation medium for lymphocytes (LSM) obtained from Invitrogen ( USA) , 3H-thymidine obtianed from

Amersham (UK) , filters made of glass fiber obtained from Conncetorate AG (

Switzerland), trypan blue from Amresco (USA) , phorbol-12-myristate-13-acetate (

PMA) from MP Biomedicals (France), Streptavidin-HRP, clear high –binding, ELISA kit and plate sealer, microplates made from polystyrene, reagent pack as a substrate containing H2O2 and TMB ( Tetramethylbenzidine ) from R&D system Inc ( USA)).

Chapter 2 Experimental

Carbon tetrachloride (CCl4), and gelatin, olive oil from Sigma Chemical Co ( USA), dimethyl sulfoxide (DMSO) from Fisher Scientific (Loughborough,UK), for cell viability { 3-( 4,5-dimethylthiazole-2-yl)-2-5-diphenyltetrazolium bromide (MTT) used obtained from Acros organics (USA)

2.4.2.1.2 Lymphocyte proliferation assay based on radioactive 3H-thymidine

For determination of lymphocytes proliferation assay, standard method of Frobel et al was used [92]. A written informed consent was obtained from the human volunteer for use of blood for experimental purposes. Blood was taken by puncturing the vein from a healthy volunteer’s human being for the separation of lymphocytes and poured into Lithium-heparin sterile tube having vacuum which then mixed properly. The

Ethics “Centre of Biotechnology and Microbiology, University of Peshawar” approved vide No/ 9355/VC dated 12/12.2012.The blood mixed with a 2mL- glutamine poured in equal volume of 1640-RPMI in a tube. Diluted blood of 9 ml was poured on 5 mL LSM in 15 mL sterile centrifuge tube for layered. Care should be taken for not displace the two layered and centrifuged for 20 min at 25◦C. Between the blood plasma and LSM phase mononuclear cells present in buﬀy layer was removed carefully into 15 mL sterile centrifuge tube containing insuﬃcient RPMI-

1640. At 4◦C for 10 min the cells were washed at 300 × g by centrifugation. The peripheral blood mononuclear cells (PBMCs) in the form of pellets were re-suspended in RPMI-1640 containing 10% fetal bovine serum (FBS). The numbers of cells were estimated after trypan blue dilution at 1:1(v/v) on light microscope at 10X magniﬁcation .For proliferation assay,3H-thymidine was diluted 1.0 µCi/ml to a 20

Chapter 2 Experimental

µCi/mL concentration with sterile RPMI1640 and stored at −20◦C in 5 ml aliquot.

The dose eﬀect of the test compounds in triplicate were mapped and labeled for assaying in sterile 96-well round bottomed plates. The PBMCs (1.2×105 cells) at the concentration of 50 µL was grown with PHA of 50 µL to reach a concentration of 5

µg/ml, then added FBS RPMI-1640 and the test compounds of 50 µL made to a ﬁnal concentration of 10 µg/mL. The culture was incubated for 72 h in a humidiﬁed atmosphere of 5% CO2 at 37◦C. The 25 µL [methyl-3H] thymidine was added at 0.5

µCi in each well which was further kept for more 18 h. After incubation of mononuclear cells with radioactive 3H-thymidine was found to be incorporated into the DNA of dividing cells in each well determines the multiplication of T-cells. The cells harvester (Connectortae AG, Switzerland) was used for cell harvesting on glass

filters. Vacuum suction was applied for drying the filters. The filter left for drying was then put into the scintillation tubes. Liquid scintillation called CytoScint was used to estimate the radioactivity as count per minute (cpm) by measuring the insertion of radioactive thymidine in the dividing cells and then put the tubes in a counter scintillation obtained from Beckman (USA).

2.4.2.1.3 Determination of IL-2 generation by cell culture

Fresh T-lymphocytes was used to investigate the eﬀect of steroidal alkaloids on the production of IL-2.T-cells proliferation method was used for isolation of PBMCs from fresh venous blood. For this purpose 50 µL of cell suspension [2.5×106 cell/mL,

50 µL of phytohemagglutinin (PHA) final concentration of 20ng/mL], 50µL of phorbolmyristate acetate (PMA, final concentration of 20ng/mL),and 50µL of the samples (final concentration of 0.5, 5.0, or 20 µg/mL were added in flat-bottomed 96- well plates. It was stored at 37◦C for 18h in a 5% CO2 incubator and ELISA was performed for IL-2 estimation from collected supernatants

Chapter 2 Experimental

2.4.2.1.4 Enzyme – linked Immunosorbent Method for Interleukin 2.

IL-2 ELISA Kit (ab174444) was used for determination of Interleukin-2.

Recombinant anti-interleukin-2 was diluted to give a concentration of 4µg/mL which was then used at 100µL/well to stick in polystyrene ﬂat-bottom micro-plates. The

ELISA plate sealers were used for sealing the coated plates and stored at 25◦C for

24h.Buﬀer solution (0.05% Tween 20 in PBS, pH 7.2–7.4) of 300 µL were used three times to wash the plates and solution of antibody aspirated followed by the addition of

100µL blocking buﬀer in each well and were kept at 25◦C for 1h

2.4.2.1.5 Estimation of IL-2 ELISA

The treated cells collected from supernatant were estimated for IL-2. The 100 µL of culture supernatant samples (1.0% bovine serum albumin, 0.05% polysorbate-20) in

TBS were added in each 96-well micro-plates contained conﬁned coated antibody as mentioned and were stored at 25◦C for 2 h. To each well were added a 100 µL of

200ng/mL goat biotinylated anti-human interleukin-2 antibody. Repeatedly washing was done followed by the addition of working solution of streptavidin-HRP of 100 µL in each well which was then incubated in the dark at room temperature for 20 min.

Again the washing step was carried out and in each well a 100 µL of substrate solution was added and left at 25◦C in dark for 20 min followed by the addition of 50

µL of stock solution in each well. The plate photometer was used at 450 nm to measure the optical density of each well.

Chapter 2 Experimental

2.4.2.1.6 3T3 cells and MTT Cytotoxicity assays

The 3T3 NIH mouse embryo fibroblast cells were used to performed In-vitro cytotoxicity by Scudiero et al [94]. DMEM formulated with 10% FBS was used for maintained the 3T3 cells. These cells have adherent property and therefore removed by using trypsin/EDTA from the surface of culture flask. Removed the medium and sterilized phosphate buffer solution was used to clean out dead or damaged cells from the cells in each flask. The 0.25% of solution of trypsin/EDTA was added in each flask containing attached cells which was then stored at 37⁰C for 2-3 min. Gently tapped the flask for observation of detached cells under the microscope from flask surface which was then after added 10% FBS containing media. Centrifuge tubes of

15mL were used for cells collection at 1200 rpm. The precipitation was resuspended in complete media and for observation used powerful microscope, for counting used neubauer chamber.

The 96-well flat- bottomed plate containing 6 × 103 cell/well in 100µL complete media were used MTT assay on the 3T3 cells which was then incubated in a 5% CO2 incubator at 37⁰C for 24 h. when the cells were attached on plates, replaced the media by 200µL of media containing the test samples at different concentration (0.5, 5, and

50µg/mL) which was then placed in a 5% CO2 incubator at 37⁰C for 48 h. The cell viability was checked of each test samples by using 0.5mg/mL of MTT in complete media for 4 h. After that removed the supernatant and then added 100µL of DMSO in each well to dissolve the formazan complex formed by the action of mitochondrial dehydrogenases. After 1 min of gentle shaking the plates were observed at 540nm and determined the optical density. The result were examined and represented in mean± SD by using Graph pad Prism software

Chapter 2 Experimental

2.4.2.2 Antibacterial Activity

The compounds (5), (3) and (2) were screened for antibacterial activity. These compounds were used against various pathogenic bacteria which include Escherichia coli, Citrobacter, Staphylococcus aureus, Salomonella typhi, Bacillus subtilus,

Shigella boydii, Micrococcus luteus, Enterococcus faecalis, Proteus mirablis and

Pseudomonas aeruginosa available at Centre of Biotechnology and Microbiology,

University of Peshawar and Pakistan Council of Scientific and Industrial Research,

Peshawar, Pakistan. The method used for this activity was well diffusion agar method

[96, 97].

The test bacteria were inoculated in 10mL nutrient broth and then incubated at 37⁰C for 24 hrs. After incubation 0.6mL bacterial cultures of broth was poured on a molten agar at 45⁰C, shake it for proper mixing and transferred to sterile petri dish. The agar plates were left an hour for hardness, formed the wells at equal distance in the plates through metallic cork bore. The solutions of isolated compounds were prepared at a concentration of 4mg/mL in dimethyl sulfoxide (DMSO). Transferred 100µl from the stock solution of the compounds samples into the each wells of the agar plates.

Amoxicillin was used as standard while DMSO as a negative control. The plates were incubated at 37⁰C for 24 hrs. The antibacterial activity of compounds was determined by zone of inhibition and compared with a standard drug. The formula for antibacterial activity in % percentage was calculated

Chapter 2 Experimental

2.4.2.3 Antifungal Activity

The antifungal activity of isolated compounds was screened by using agar tube dilution method [98, 99]. Stock solutions of the tested compounds at a concentration of 24mg/ml were prepared in sterile DMSO. Sabouraud Dextrose Agar (SDA) was used for the fresh culture of fungal strains. Slants prepared by dispensing 4ml of SDA medium in to sterilized test tubes. Transferred 66.6µl amount of tested sample from stock solution in each test tube along with 7 days old fungus culture introduced. They were incubated for 7 days at 28⁰C and after 7 days period the linear growth of test fungi was checked. Inhibition in % percentage was determined by comparison with positive control. Standard drug used as positive control was Amphotericin-B and

Fluconazole [100,101].

2.4.2.4 Antileishmanial Activity

2.4.2.4.1 Parasite Culture

then placed in an incubator at 260C. After each third day the medium was changed.

To develop promastigoes into metacyclic stage the cultures were placed for 10-14 days and then suspended the promastigoes into the centrifuge tube which rotated at

2000 RPM for 12 minutes in Sigma centrifuge. The medium was spill off leaving the

Chapter 2 Experimental pellets only. These pellets were the suspended again in 2-4mL growth medium and new cultures were developed.

2.4.2.4.2 Anti-Promastigote Activity of Compounds

The purified compounds (5) and (4) were screened for leishmaniacidal activity. From viable promastigotes bulk culture (3.7×107/mL), 1×105 promastigotes/well in 200µL fresh M-199 medium were seeded in 96-well plate. The compounds were prepared in four different concentrations and control was palced in one row of 12 wells with growth medium only. The 96 wells plates were incubated at 26oC for 48 hours and

Improved Neubauer Haemocytometer was used to count the number of promastigotes in each well (treated and control).

The formula for % inhibition of promastigotes was

Linear regression analysis was used to calculate IC50 by GraphPad Prism 6 Software

[102, 103].

2.4.2.5 Phytotoxic Activity

Phytotoxicity of purified compounds was screened by using Lemna bioassay protocol

[100, 104]. The sterilized E-medium was used for the growth of L.minor as in Table

2-1. The test samples were prepared in methanol at a concentration of 5mg/ml for stock solution. 10, 100 and 1000µg/ml concentration take from stock solution and poured in separate flask for evaporation of solvent at room temperature. 20ml of E- medium and ten healthy L.minor plants with a rosette of three fronds were transferred in each flask. The flasks contain E.medium and L.minor served as negative control and the flask contain standard (Paraquat 0.015µg/ml) served as positive control or

Chapter 2 Experimental

standard growth inhibitor. These flasks were incubated at growth chamber for seven

days (28±1⁰C). The Phytotoxic activity was checked by counting number of dead

fronds in each flask after seven days of incubation. The formula for % inhibition was

given below

Table: 2.1 Composition of E-Medium

Concentration S. No Constituents Formula mg/ml)

1515 1 Potassium nitrate KNO 3 3.62 2 Manganous chloride 5.40 3 Ferric chlorid

4 0.12 Sodium molybdate

5 492 Magnesium sulphate

6 Calcium nitrate 1180

7 Zinc sulphate 0.22

8 Potassium 680 dihydrogenphosphate 9 Ethylene 11.20 diaminetetraacetic acid 10 Boric acid 2.86

11 Copper sulphate 0.22

Chapter 2 Experimental

2.4.2.6 Insecticidal Activity

The purified compounds were screened for insecticidal activity against Callosbruchus analis, Tribolium castaneum and Rhyzopertha dominica. A test sample of 4mg dissolved in 3ml of acetone for prepared stock solution. A filter paper of 90mm was placed in plates and test sample (850.10µg/cm2) loaded on filter paper and left for

24hrs to evaporate the solvent. After a day nine insects were incubated for 24hrs at a temperature of 28⁰C±1⁰C in each plate. Permethrin at a concentration of 235µg/cm2 as standard while acetone as a negative control. Mortality rate was determined in percentage (%) by dividing test sample result with positive control [104, 105].

The formula used for mortality percentage is given below

2.4.2.7 Antioxidant Activity

The purified compounds from S.saligna were screened for antioxidant potential with a reported method of DPPH radical scavenging assay [106]. The test compounds solutions were made by dissolving in DMSO and mixed 5µl of test solutions into 95µl

DPPH ethanolic solution. The solution then transferred into 96-well micro plate and stored at 37⁰C for 30 minutes. The absorbance of test solution was measured at

517nm with multiple reader spectrophotometers (Spectra-Max 3400). Ascorbic acid was used as standard. The percentage (%) radical scavenging potential of test solution was measured by comparing it standard.

The formula is given below.

RSA (%) = 100-(Optical Density test sample/ Optical Density standard sample) × 100

Chapter 2 Experimental

2.4.2.8 Cytotoxicity Assay (Anticancer Activity)

The isolated purified compounds from S.saligna were screened for cytotoxicity assay.

MTT (3-(4, 5-dimethyl-thiazole-2-yl)-2, 5-diphenyltetrazolium bromide) standard colorimetric protocol used with 96-micro well plates (flat –bottom) for determination of cell viability. HeLa cell lines were cultured in a Modified Dulbecco᾽s eagle᾽s medium supplementing with 10% fetal bovine serum at temperature of 37oC in a humidified atmosphere with 5% CO2, Penicillin (100IU/mL), Streptomycin

(200µg/mL) in three flasks. The cell lines were grown by diluting in a specific medium and haemocytometer instrument used for counting the cells. The culture concentration 1˟ 105 cells/mL was prepared and poured in to 96-well micro plate and

100 µL cultures contain in each well. The culture was incubated for whole night and medium was changed with freshly prepared medium (200 µL) for different compounds concentration (0.5-10 µM). Each well 2µg/mL was added after 72 hrs and then further [47].

Chapter 3 Results and Discussion

3 RESULT AND DISCUSSION

The following compounds were isolated from S.saligna through phytochemical investigation

3.1 Structure Elucidation of Isolated Compounds

3.1.1 Alkaloid-C (1)

Compound 1 was isolated as white powder from the chloroform fraction of S.saligna

(Fig 2-2) by subsequent elution of sub-fraction NF23 on neutral alumina column with increasing solvent polarities of EtOAc / n- hexane with a few drops of diethylamine.

25 ⁰ The optical rotation of compound 1 [α] D:-30 (c0.04, CHCl3) indicated the presence of chiral centres, while the UV spectrum displayed absorption at 239nm. The HREI

+ MS of compound displayed the [M ] at m/z 359.3125 (calcd for C24H41NO,

359.3126). The 1H-NMR of compound 1 showed two up-field singlets at δ 0.65 and

0.98, having properties of C-18 and C-19 angular methyls. A C-21 secondary methyl was showed at δ 0.86 doublets (J21, 20 = 5.0 Hz), while signal at δ 2.14 for 6H singlet was ascribed to NMe2 protons. A C-20 methine proton was appeared at δ 2.42 respectively while multiplet at δ 3.04 was assigned to the C-3 methine proton. A methoxy proton was present δ 3.33 of a 3H singlet. At δ 5.34 downfield signal was present due to the C-6 methine proton.

The 13C-NMR spectra of compound 1 demonstrated total of 24 carbons which contained six methyl, eight methylene, seven methine, and three quaternary carbons.

The spectroscopic data of compound 1 similar with a reported compound isolated from Sarcococca pruniformis [27] and Sarcococca saligna [25]. The structure of compound shown in Fig 3-1

Chapter 3 Results and Discussion

Alkaloid-C

Figure 3-1 Stucture of Isolated Compound Alkaloid –C

3.1.2 Dictyophlebine (2)

The IR spectra of compound 2 showed absorption at 3352 (NH), 2925 (CH) cm-1

+ while the HREI MS displayed the [M ] at m/z 360.3029 (C24H44N2, calcd 360.3034).

The 1H-NMR of compound 2 showed signal of two up-field 3H singlets at δ 0.61 and

0.75, for protons of C-18 and C-19 angular methyls. The C-21 secondary methyl protons were appeared at δ 0.83 (3H, d, J 21, 20 = 6.4Hz). The Nb-Me2 protons was at δ

13 2.13, while Na-Me2 protons was resonate at δ 2.39 as 3H singlet. The C-NMR spectra of compound 2 demonstrate resonance of all 24 carbons which also include six methyl, nine methylene, seven methine, and two quaternary carbons.

Chapter 3 Results and Discussion

Dictyophlebine

Figure 3-2 Structure of Isolated Compound Dictyophlebine

3.1.3 Sarcovagine-D (3)

Chapter 3 Results and Discussion

respectively. The 13C-NMR spectra of compound 3 demonstrate resonance of all 28 carbons which include seven methyl, seven methylene, eight methine, and six quaternary carbons. The spectral features of compound 3 showed that it was sarcovagine-D previously reported from Sarcococca vagans [36] as shown in Fig 3-3.

Figure 3-3 Structure of Isolated Compound Sarcovagine-D

Chapter 3 Results and Discussion

3.1.4 Saracodine (4)

+ The HREI MS spectrum showed the [M ] at m/z 402.3579 having formula C26H46N2O

( calcd , 402.36558 ) , while UV absorption of compound 4 appeared at 203nm.

1 The H-NMR of compound 4 showed singlets at δ 0.72 (CH3-18) and 0.80 (CH3-19) for the angular tertiary methyls respectively. The presences of 3H doublet at δ 1.14 (J

20, 21 = 6.5 Hz) was due to C-21 secondary methyl protons. The N, N-dimethyl protons were present at resonance of δ 2.21 (6H singlet). The N-acetyl methyl was present at resonance of δ 2.01/2.07 (3H split singlet), while other 3H split singlet at δ 2.71/2.75 resonance was showed Nb-methyl protons. The methine group resonanated at δ

3.58/4.61 was due to C-20 methine protons. The compound 4 spectral data was similar with previous reported data of compound which was isolated from Sarcococca saligna [28] and was identified as known compound Saracodine shown in Fig 3-4.

Figure 3-4 Structure of Isolated Compound Saracodine

Chapter 3 Results and Discussion

3.1.5 Holaphylline (5)

The compound 5 was isolated as sticky light yellowish powder from the chloroform fractions of S.saligna (Fig 2-2) by subjecting on alumina column chromatography, increasing polarities of repeated elution of n-hexane/ EtOAc and few drops of diethylamine.

HR-ESI-MS showed pseudo molecular ion peaks [M+ H] at m/z 330 corresponding to the molecular formula C22H35NO [Calculated as C22H35NO +H = 330.2797]. The IR spectrum indicated the presence of an amide carbonyl (1730 cm-1).

1 H-NMR (CD3Cl, 400 MHz) showed the signals of 3 protons each at δ 0.90, 1.0, 2.10

13 and 2.62 correspond to three CH3-18, CH3-19 CH3-21 and CH3-N. The C-NMR.

showed that compound 5 was holaphylline previously from Holarrhena floribunda plant as shown in Fig 3-5 [107]. It was first time isolated from Sarcococca species and reported.

Chapter 3 Results and Discussion

Figure 3-5 Structure of Isolated Compound Holaphylline

3.2 In-vivo Biological Assays

3.2.1 Hepatoprotective Assay

3.2.1.1 Effect of Biomarker Components of S. saligna on CCl4 as an oxidative

inducer

To check the eﬀect of pure compounds against CCl4-induced hepatic injury, malondialdehyde generation, glutathione level and superoxide dismutase enzymes levels in the liver were estimated (Fig 3-9). The level of MDA was drastically increased (P < 0.05) by CCl4 intoxication, however treatment with sarcovagine-D (3), holaphylline (5) and alkaloid-C (1) has reduced the elevated level of MDA as shown

(Fig 3-9A). In CCl4 intoxicated rats, the hepatic antioxidant enzyme SOD level dramatically decreased (P < 0.01), while the activities of antioxidant enzymes in the liver markedly increased (P < 0.05) by co-treatment with pure compounds (Fig 3-9B).

The GSH concentrations in the rat liver were signiﬁcantly decreased by

Chapter 3 Results and Discussion

intraperitoneal injection of CCl4 compared to control group (P < 0.01; Fig 3-9C).

However, upon treatment with S. saligna biomarkers, the level of GSH was elevated

(P < 0.05; Fig 3-9 C).

Chapter 3 Results and Discussion

Figure 3-6 (A) Effects of S. saligna Steroidal Alkaloids (Sarcovagine-D, Alkaloid-C, Holaphylline), on Hepatic Biochemical parameters MDA (A), GSH (B), and SOD (C), in CCl4-intoxicated rats

Chapter 3 Results and Discussion

3.2.1.2 Hepatoprotective potential of S. saligna pure compounds: A Histological

Study

The normal control rats liver which was dissect into sections were dye with reagent hematoxylin and eosin showed normal liver histology which is liver cords cells lined with endothelial cells with clearly defined curved area (Fig 3-10A). However on other side the CCl4-treated group liver sections showed declined production and damaged or injured hepatocytes containing hyaline bodies (Fig 3-10B). A lot of different inflamed cell penetrated, was available at the central vein space. In the space of damage place, liver injury was rare extended, especially soon at the side of lesion lined (Fig 3-10B). The shaped stability of the periportal and non-parenchymal cells were attained and decreased the pathological changes of CCl4 by treating standard drug silymarin at 200 mg/Kg as mentioned in Fig 3-10C. Instead of this some inflammatory cells were still found in the injured place around the central vein.

However when the compound sarcovagine-D (3) treated at 20 mg/Kg dose showed protection of liver membrane stability against CCl4 oxidative inducer injury and its appearance was normal as mentioned in Fig 3-10D. Similarly, other compounds holaphylline (5) and alkaloid-C (1) showed protection of liver in contrast to the CCl4 control group (Fig 3-10F). It also decreased the CCl4-induced pathological changes as the vicinity of sinusoidal lined with endothelial cells observed in normal liver (Fig 3-

10E). Therefore, the compounds result showed dramatically decreased the diseases state induced by oxidative stress to the liver in contrast to the positive control group.

Chapter 3 Results and Discussion

Figure 3-7 The Effect of Test Compound on Liver Inflammation and its Histopathological profile

Chapter 3 Results and Discussion

3.2.1.3 Hepatic Kupffer cells Immunohistochemistry

Inflammation of liver is associated with activation and migration of Kupffer cells into the hepatic cords of liver. Upon hepatic injury these macrophages secrete pro- inflammatory cytokines such as TNF-α and IL-6. In the normal control group, CD68+ immune-reactive cells with distinct slender nuclei were present in the sinusoidal spaces as well as a few around the central vein of liver (Fig 3-11A). The slender shaped nuclei of Kupffer cells were identified using DAPI staining as shown in

Figure 3-11A From ﬂuorescence microscopy it is obvious that the resident macrophages having characteristic elongated shape in the sinusoidal spaces (Fig 3-

11A,B). In CCl4 induced liver injury, the Kupffer cells were found to be densely stained and numerous in number (Fig 3-11B) in the injured area around the central vein. From DAPI staining, the nuclei of mixed inflammatory cells infiltrate was identified around the central vein (Fig 3-11B). Huge number of Kupffer cells was present around the injured central vein compared to normal control group (Figure 3-

116B). Silymarin treatment has slightly reduced the number of activated Kupﬀer cells

(Fig 3-11C) around the injured portion of liver compared to the CCl4 model group were further confirmed from the DAPI staining (Fig 3-11C). Interestingly, treatment with sarcovagine-D (3) and alkaloid-C (1) decreased (Fig 3-11D) the activated macrophages around the injured central vein (Fig 3-11E) to level similar like silymarin treatment but DAPI staining revealed inflammatory infiltrate around the central vein compared to normal control group (Figs 3-11D,E). However, treatment with holaphylline (5) limited the activity of hepatic macrophages (Fig 3-11F), despite the CCl4 treatment as shown in the double channeled immunohistochemistry (Fig 3-

11F). The macrophages were present in the sinusoidal spaces with distinct

Chapter 3 Results and Discussion morphological features more in number compared to macrophages distribution in the normal control group as shown Fig 3-11 below.

Chapter 3 Results and Discussion

Figure 3-8: Effects of steroidal alkaloids on hepatic macrophages (Kupffer cells).

Chapter 3 Results and Discussion

Biochemical studies were conducted to estimate the serum level of ALT, AST, and

ALP in experimental groups. The group treated with CCl4 showed membrane injury and damage of hepatocytes (Fig 3-12), also drastically increased the serum level of

ALT, AST and ALP. The level of ALT, AST, and ALP decreased by treated with standard silymarin but not to the optimum levels which showed that some hepatocytes necrosis still remained. However when treated with compounds Sarcovagine-D (3)

Holaphylline (5) and Alkaloid-C (1) showed good hepatoprotective results and reduced the level of enzymes ALP, ALT and AST more than silymarin standard drug as mentioned in Fig 3-12.

Chapter 3 Results and Discussion

Figure 3-9: (A) Biochemical Tests of The Effects of S.saligna Compounds on CCl4-Induced Liver injury

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Chapter 3 Results and Discussion

3.2.1.4 Discussion

The innate immune system activates after necrosis intensify the initial tissue injury during acute hepatitis and not necessarily cause the liver damage by inflammatory response. When the liver injured by CCl4 inducer, the aim of kupffer cell activation and hiring natural killer cells, neutrophils and monocytes in hepatic is to eliminate remove dead hepatocytes and this process is important for the reproduction of missed tissue, an examples are concanavalin-A[114], lipopolysaccharide [115] and acetaminophen hepatotoxicity, initiate the inflammatory response appearing neutrophils after liver injury in an hour and hiring the macrophages and monocytes within 24–48 h [116, 117]. The purified isolated steroidal alkaloids compounds from this plant showed positive protection results by depressing the injury to the hepatocytes without affecting more tissue in this in vivo study. The cytochrome P450 dependent monooxygenases activated metabolically by depositing CCl4 in the hepatocytes -the liver parenchymal cell to synthesize very high active metabolites, such as (CCl3OO−) and (CCl3−) radicals [118],which causing hepatotoxicity like liver cells death, degeneration and fibrosis [119, 120]. The generation of these free radical cause lipid oxidation by cover the cellular antioxidant defense system. In all this process the hepatic macrophages called kupffer cells play an important role in changing the severity of liver inflammation [121,122].It has been proposed that when liver injury occur, different pro inflammatory agents such as TNF-α and MCP are generated by kupffer cells, stimulated the stellate cells of liver to increase expression of extracellular matrix protein in chronic hepatic inflammation which utterly produce hepatic injury [122, 123]. The finding of our study showed that the isolated steroidal alkaloid from S. saligna reduced liver inflammation by firstly reducing the T-cells multiplication and amount of IL-2 which change the entire inflammation reactions and

101

Chapter 3 Results and Discussion as well non cytotoxic, secondly acts as antioxidant and act as a free radicals scavenger which is produced by the hepatocytes . The In vivo study further showe d that these steroidal alkaloids markedly decreased hepatic injury by CCl4-injury inducer and mixed inﬂammatory penetration. Therefore, we explored and suggest that steroidal alkaloids from S. saligna could be excellent immunosuppressive and hepatoprotective agents which have excellent therapeutic potential.

3.2.2 Antihyperglycemic Activity

3.2.2.1 Effect of purified compounds on fasting plasma glucose levels

The alteration of FPG levels in study groups produced was shown in fig 3-13 or table

3-2.There were no significant difference between groups at base line as shown in table

3-2. The FPG levels decreased significantly in treated groups compared to baseline, while the temporal patterns of these reductions were different among groups. In treated group 4 , the decrease was significant even from the 1st week of treatment and the level decrease from 250.45±22 mg/dl to 208.43±11*¤ ( p< 0.05) and finally the value reduced up to 140.43±25 mg/dl ( p<0.05) at week 4 , whereas the treated group

2 and 3 showed significant decreases only from the 2nd week of treatment and the level reduced from 265.24±21 to 179.65±26 ( p < 0.05) in treated group 2 , while the level reduced from 257.45±25mg/dl to 195.65±24*¤ ( p < 0.05 ) in treated group 3 which were further reduced at week 4. These results were comparable to reference group V glibenclamide. At the end all the treated groups showed significantly reduced the FPG levels compared to control group

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Chapter 3 Results and Discussion

Table 3.1 Effect of Compounds on FPG Level on Diabetic Rats

Groups Baseline Week 1 Week 2 Week 3 Week 4

Control Group 1 252.54±19 250.34±23 279.87±25 294.65±26 327.43±22 ( mg/dl)

Treated Group 2 265.25±21 241.37±15 179.65±12*¤ 165.95±15*¤ 142.56±15*¤ ( mg/dl)

Treated

Group 257.45±28 231.78±25 195.65±18*¤ 187.45±32*¤ 182.54±29*¤

3(mg/dl)

Treated Group 4 250.67±12 208.43±11*¤ 165.65±29*¤ 154.15±19*¤ 140.43±19*¤ ( mg/dl)

Reference Group 5 264.65±25 208.65±30*¤ 188.56±21*¤ 162.23±12*¤ 137.56±25*¤ (mg/dl)

* P≤0.05, compared with control group ¤ P≤0.05, compared with baseline

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Chapter 3 Results and Discussion

Baseline Week 1 400 Week 2 Week 3 Week 4 300 ¤ ¤ ¤ ¤ ¤ ¤ * ¤ * * * * ¤ * ¤ ¤ * ¤ ¤ 200 ¤ * ¤ * * * * * *

100

Fasting plasma glucose level

Group 1 control Group 2 Treated Group 3 Treated Group 4 Treated Group 5 Treated

Figure 3-10: Graphical Representation of the Compound Effect on FPG Level on Diabetic Rats

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Chapter 3 Results and Discussion

3.2.2.2 Fructosamine levels in Blood.

At the baseline, the significant differences of fructosamine levels were negligible among the study groups. Following purified steroidal alkaloids treatment compared to baseline, the level in treated group 4 statistically significantly (p <0.05) decreased even from the 2nd week of treatment and the values dropped to (272.8±8.3*) from

(328.2±12.5) and continued up to the last day of study. However, the decreased in treated group 3 and 2 were significant ((p < 0.05) only at the end point compared to baseline. The fructosamine values were shown in Table 3-3 below.

3.2.2.3 Effect of steroidal alkaloids on Systolic blood pressure (SBP) of diabetic

rats

The steroidal alkaloids of S.saligna showed improvement in systolic blood pressure

(SBP) as compared to baseline but these effects were statistically non-significant as shown in Table 3-4.

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Chapter 3 Results and Discussion

Table 3.2: Effect of Steroidal Alkaloids on Fructosamine of Diabetic Rats Fructosamine Baseline Week 2 Week 4 ( µmol/L) Group 1 330.6±15.2 334.3±15.2 338.6±14.8

Group 2 325.7±14.8 298.6±9.5 291.3±10.3*

Group 3 312.8±16.4 295.3±7.2 285.4±13.9*

Group 4 328.2±12.5 272.8±8.3* 261.2±11.7*

Group 5 310.5±11.6 294.9±11.2* 278.4±8.5*

Table 3.3 : Effect of Steroidal Alkaloids on SBP (mmHg) of Diabetic rats Groups Baseline Week 2 Week 4

Group 1 125.3±6.5 129.6±2.4 133.7±1.5

Group 2 132.2±5.2 128.6±4.2 127.9±4.6

Group 3 129.8±4.2 126.4±3.4 126.7±4.2

Group 4 126.7±5.1 124.6±4.6 123.9±4.7

Group 5 128.3±6.3 126.4±3.8 124.5±3.9

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Chapter 3 Results and Discussion

3.2.2.4 Oral Glucose Tolerance Test

The groups receiving Steroidal alkaloids of S.saligna were significantly decreased glucose excursions during the 1st and 2nd week OGTT as was the case with reference drug. These results show continuous hypoglycemic effects of treated group 4, 3 and 2 with respect to the total AUC after the glucose challenge and the result differentiate with diabetic control group as in Fig 3-14.

3.2.2.5 Effect on blood lipids

The effects of steroidal alkaloids on blood lipids are shown in Fig 3-15. Compared to baseline the treatd group 4 , 2 and 3 produced changes in lipid levels by -2.5, -4.4, and -4.6mg/dl in total cholesterol (TC) respectively ( p<0.08 and p<0.01 ), -3.7, -3.9 and,-4.8mg/dl in low density lipoprotein cholesterol ( LDL-c ) ( respectively ( p<

0.03 and p<0.01 ) , +2.5, +3.6, and +4.2 mg/dl in high density lipoprotein cholesterol

( HDL-c) ( p<0.01 and p<0.004 respectively ), and -2.8,-3.2 and -4.5 mg/dl in triglyceride ( Tg) ( p<0.05 and p<0.01 respectively ).

3.2.2.6 Effect on Body Weight

Steroidal alkaloids exerted favourable effects on body weights as in Fig 3-16, however there is no statistically significant improvement was achieved in treated group (p<0.068) compared to baseline.

107

Chapter 3 Results and Discussion

20 Group 5 Group 4 Group 3 Group 2 15 Group 1

OGTT

0 0 50 100 150 Time (min)

Figure 3-11: Compound Effects on OGTT Test

108

Chapter 3 Results and Discussion

6 TC LDL-c HDL-c Tg

-2

changes in blood lipid (mg/dl) -4

-6

Group 1 Group 2 Group 3 Group 4 Group 5

Figure 3-12: The Effect of Compounds on Changes in Blood Lipid in Different Groups

109

Chapter 3 Results and Discussion

Changes in Body Wieght (g) Base line 250 Week 4

200

150

100

Body weight (g)

0 Group 1 Group 2 Group 3 Group 4 Group 5

Figure 3-13: Compounds Effect on Changes in Body Weight in Different Groups

110

Chapter 3 Results and Discussion

3.2.2.7 Discussion

A disorder characterized by increasing urine excretion is said to be diabetes. Diabetes mellitus is one of the common types of diabetes, which is characterized by chronic metabolic alteration of glucose [124]. The diabetic patient have improper carbohydrates, protein and lipid metabolism which could lead to severe complication like ketosis, polyurea, polyphasia, retinopathy and as well cardiovascular problems

[125]. Oral antidiabetic agents and insulin currently used for the treatment of diabetes produces severe adverse effects on human body such as liver complications, lactic acidosis and diarrhea [126]. On the other hand medicinal plants provide a potential source of treating diabetes mellitus which have less side effects or adverse effects.

The pharmacological actions of the active compounds from plants decrease the actions of α-amylase and other parameters of blood could cause hyperglycemic condition [127].

The aim of study was to explore the antidiabetic potential of isolated steroidal alkaloid of S.saligna chloroform fraction in diabetic rats induced by STZ. The result showed that compounds holaphylline (5) and sarcovagine-D (3) reduced the glucose level significantly in blood while saracodine (4) produced moderate changes reduction in blood glucose level. These results are well supported by previous findings which showed that the extract of ethyl acetate and petroleum ether from S.sligna reduced the glucose level significantly in blood of 18h fasted diabetic rats induced by STZ compared to 0h fasted diabetic rats induced by STZ. The low doses used in our study are due to the fact that we isolated pure compounds whereas the previous study used crude extracts which are generally used in higher doses [128].

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Chapter 3 Results and Discussion

The gradual decline in glucose tolerance during OGTT may be the result of the compounds to reach specific target tissues and elicit a response to achieve the normal level of glucose in blood. Although the proper mechanism of actions is still unknown but earlier studies have shown that hypoglycemic action of alkaloids is by preventing the glucose diffusion through the enteric epithelium which ultimately decrease the level of glucose in blood and improve glucose tolerance [129]. Fructosamine is an important tool for detecting short period changes in glucose control and also indicated of increased mortality and morbidity in patients with final stage renal disease undergoing hemodialysis [130]. Administration of holaphylline (5) sarcovagine-D (3) significant decreased serum fructosamine level in diabetic rats in our study

Lipids play a vital role in the hyperglycemic condition. The abnormal lipid level in diabetes produce a condition of hypertriglyceridemia and high cholesterol level in blood called a condition hypercholesterolemia. The levels of serum lipid rise in diabetic rats such as cholesterol and triglycerides and therefore elicit coronary heart diseases [131]. Normally insulin activates an enzyme lipoprotein lipase which metabolized trigylcerides [132]. The function of insulin is to enhance the deposition of fatty acid into adipose tissue and increase the formations of triglycerides.

Furthermore insulin also inhibits lipolysis. Lipolysis is not inhibited in diabetes which ultimately leads to a condition of hyperlipidemia and also the level of free fatty acid in serum increased because of outflow free fatty acid from adipose tissue and esterification –trigylcerides lipolysis cycle is shift in favor of lipolysis [132]. HDL is a lipoprotein which inhibiting atherogenesis and shift cholesterol from the peripheral tissue into the liver which help in protecting the coronary heart diseases. The finding of our study also reveal that by administration of holaphylline (5) and sarcovagine-D

(3) treated groups showed significant improvement in blood lipids and also elevated

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Chapter 3 Results and Discussion the HDL-cholesterol level in diabetic rats. These results also are in agreement with the previous findings [128].

This study concluded that steroidal alkaloids holaphylline (5) and sarcovagine-D (3) isolated from chloroform crude extract of S.saligna possess good hypoglycemic and also ameliorate others diabetes associated complications. Further research is needed using a range of doses to explore the other possible beneficial effects in diabetes mellitus and its molecular mechanism of antidiabetic action.

* P≤0.05, compared with control group

¤ P≤0.05, compared with baseline

3.3 In Vitro Biological Activity

3.3.1 Immunosuppressant Activity

3.3.1.1 Effect of steroidal alkaloid on T-cell multiplication

The immunosuppressant drug (cyclosporine and tacrolimus) administered for protection of organ transplant rejection by depressing the T-cells multiplication through reduced the production of IL-2. The T-cells proliferation inhibition also occurs by stopping the signal passage of IL-2 receptor through IL-2R antibodies.

Therefore first we investigate the effects of tested compounds on T-cells proliferation.

We tested four mitogens to increase the efficiency of T-cells multiplication assay and

PHA was among the good activator at 5µg/mL (Fig 3-6). The inhibitions of pure samples were then checked on PHA and samples showed suppressive activity of T- cell with an IC50 value which is less than 10µg/mL (Fig 3-6). Therefore these compounds can be used as a drug for prevention of graft rejection. The steroid alkaloid Sarcovagine-D (3), Holaphylline (5) and Alkaloid –C (1) showed different result against T-cell proliferation. The pure compounds, Sarcovagine-D showed

113

Chapter 3 Results and Discussion

78±0.2, Holaphylline showed 95±2.5, while Alkaloid-C showed 82 ±4.5 T-cell proliferation inhibitions, when used at less than 10µg/mL concentration (Table 3-1).

The purified compounds showed inhibitory activities of T-cell proliferation in the range of 78 to 95% which are summarized in Fig.3-6 and Table 3-1. Tacrolimus and cyclosporine used as a standard drug for comparison study.

114

Chapter 3 Results and Discussion

Figure 3-14: Effect of Steroidal alkaloid Sarcovagine-D, Alkaloid-C and Holaphylline on T-cells proliferation.

Figure 3-15: Effect of Puriﬁed Test Compounds on the Generation of IL-2 Production from T- Lymphocytes at Different Concentration.

115

Chapter 3 Results and Discussion

3.3.1.2 Effect of steroidal alkaloid on generation of Interleukin-2

The tested samples for inhibition of T-cells multiplications which is activated through the production of the cytokine IL-2 by PHA activated T-cells. The IL-2 is responsible for T-cells proliferation as well other immune cells which play role in cellular and adaptive immune response. All the tested compounds showed excellent suppressive effects on IL-2 production with an IC50 value less than 5.0µg/mL as shown in Fig. 3-7 and Table 3-1.

3.3.1.3 Cytotoxicity Assay

The cytotoxicity activity of pure compounds was study on mice fibroblast cell-lines

(3T3) in order to examine that the immunosuppressant action was not only due to their cellular toxicities. The compounds showed result and found that the IC50 value was around 11.5µg/mL except for Sarcovagine-D (3). One compound holaphylline (5) consider being safe and have no impact up to 50µg/mL (Table 3-1 and Fig 3-8).

Holaphylline (5) was found to be less toxic and therefore was selected for in-vivo testing.

116

Chapter 3 Results and Discussion

Figure 3-16: Cytotoxic Effect of Steroidal Alkaloids on 3T3 Fbroblast Cell Line

117

Chapter 3 Results and Discussion

Table: 3.4 The Effect of Steroid Alkaloids from S. saligna on T-cells Multiplication, IL-2 Generation and Cytotoxicity

Tested Compounds T-cell Inhibition of IL-2 3T3, IC50 in with standard drug multiplication , % generation ,IC in 50 µg/mL inhibition ( mean ± µg/mL SD ) at 10µg/mL

Sarcovagine-D (3) 78±4.5 2.95±0.6 3.5±0.3

Holaphylline (5) 95±1.2 1.35±0.15 25±1.7

Alkaloid-C (1) 82±0.8 0.9±0.5 14±1.8

Cyclosporine (Std 1) 99±0.8 <0.05 -

Cyclohexamide (Std 2) - - 1.2±0.4

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Chapter 3 Results and Discussion

3.3.1.4 Discussion

The immunity play has an important role to protect the body from any foreign particle or xenobiotics such as bacteria and virus which cause disease or potentially dangerous for body. During autoimmune diseases or organ transplanted from donor the body immune system recognize it as an outside tissue and start ﬁght against its [108].

Leukocytes play an important role in the immunity process and kill any foreign tissue called body immune response [109]. White blood cells are different types and T- lymphocytes from it play an important role in all kind of immunity process [110].The purpose of this biological assay was to investigate steroidal alkaloids that could be lead drugs to stop the proliferation of T-cells and having excellent property of hepatoprotective agents without causing cytotoxicty. As we know that damage of hepatocytes in viral hepatitis is because of immunity reaction and itself virus is not involved in it. We also check the impact of steroidal alkaloids on PHA-activated T- cells which activate specifically CD4 and T-cells [111]. The result of compounds showed inhibition against T-cells proliferation which was further study the effect of

T-cells activated through PHA on generation of IL-2, which causes proliferation and as well other immunity cells multiplications. The CD4+ T-cells generated mostly IL-2 cytokines to diﬀerent stimulating agent response through which activation of T-cells via receptor of T-cells and major histocompatibility complexes I and II antigen- presenting cells. The IL-2 cytokine is not detectable in normal healthy subject blood and its level rise drastically when a subject exhibit infection. The IL-2 generation increased rapidly by the PHA+PMA stimulation, reported in optimization protocol

[112].The PHA and TCR responsible for crosslinking by binding sugar glycosidically on T-cells surface protein, which provokes signals 1 and 2 through linkage of co- stimulatory factor. All these activities take place on surface cell and consequently

119

Chapter 3 Results and Discussion involve various signaling pathways. The phorbol 12-myristate 13-acetae (PMA) is structurally similar to a plant isolated compound Phorbol from Croton tiglium. PMA enter into cytoplasm l through cell membrane and activates protein kinase C enzyme as it structure is resemble to natural diacylglycerol PKC activator. When T-cells stimulated, generation of IL-2 start by PKC activation. The evaluation of steroidal alkaloid compounds is important for research as immunosuppressive compounds and showed excellent immunosuppressive properties. We also evaluated compounds for cytotoxicity, which showed inhibition of T-cells proliferation and IL-2 production is not because of cytotoxicity. The steroidal alkaloids isolated from S.saligna have been used as a source of medicines for many diseases and serve as a basis for many pharmaceutical used [113] and therefore these compounds were study for their anti- inﬂammatory eﬀect.

3.3.2 Antibacterial activity

Antibiotic resistance is the major problem in treating the infectious diseases.

Therefore the search of new antibiotic is needed to solve the resistance problem [133].

Some of bacteria like Staphylococcus aureus showed resistance to various antibiotics such as pencillin G, tetracyclines and macrolides [134].

3.3.2.1 Anti bacterial activity of compound halophylline (5)

The antibacterial activity of compound holaphylline (5) was tested against the specific bacteria; E.coli, Citrobacter, S.aureus, S.typhi, B.subtilus, S.boydii, M.luteus,

E.faecalis, P.mirablis and P.aeruginosa. The zone of inhibition was compared with standard drug amoxicillin (10µg/ml) and was calculated % inhibition as well. The summarized results obtained were shown in Table (3-5). The compound holaphylline

(5) showed mild to moderate antibacterial activity against the bacteria in Table 3-5.

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Chapter 3 Results and Discussion

Table 3.5 Zone of inhibition (mm) of Antibacterial Holaphylline (5) compound

S.NO Bacterial Holaphylline (5) ( 100 µg/100ml) Standard Drug

Strains

Zone of Zone of Zone of

inhibition inhibition inhibition

( mm) ( %) ( mm)

1 E.coli 15 65 23

2 Citrobacter ------20

3 S.aureus 12 50 24

4 S.typhi 10 56 18

5 B.subtilus 18 72 25

6 S.boydii ------10

7 M.luteus 10 67 15

8 E.faecalis 08 40 20

9 P.mirablis ------12

10 P.aeruginosa 15 69 22

Standard Drug = Amoxicillin (10µg/discs)

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Chapter 3 Results and Discussion

3.3.2.2 Antibacterial activity of compound Sarcovagine-D (3)

The antibacterial activity of compound Sarcovagine-D (3) was tested against the selected bacteria; E.coli, Citrobacter, S.aureus, S.typhi, B.subtilus, S.boydii, M.luteus,

(3) showed significant activity against P.aeruginosa (79%), while good antibacterial effect against E.coli (70%), S.aureus (67%), S.typhi (67%), M.luteus (67%) respectively. The compound showed moderate activity against E.faecalis (60%) and

Citrobacter (56%) respectively. The compound was inactive against bacteria S.boydii

, B.subtilus and P.mirablis respectively.

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Chapter 3 Results and Discussion

Table. 3.6 Zone of inhibition (mm) of Antibacterial Sarcovagine-D (3) compound

S.NO Bacterial Sarcovagine-D (3) Standar d Strains ( 100 µg/100ml) Drug

Zone of Zone of Zone of inhibition inhibition inhibition ( mm) ( %) ( mm) 1 E.coli 18 70 26

2 Citrobacter 10 56 18

3 S.aureus 16 67 24

4 S.typhi 12 67 18

5 B.subtilus ------22

6 S.boydii ------18

7 M.luteus 12 67 18

8 E.faecalis 12 60 20

9 P.mirablis ------14

10 P.aeruginosa 22 79 28

Standard Drug = Amoxicillin (10µg/discs)

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Chapter 3 Results and Discussion

3.3.2.3 Antibacterial activity of compound dictyophlebine (2)

The antibacterial activity of compound dictyophlebine (2) was tested against the selected bacteria; E.coli, Citrobacter, S.aureus, S.typhi, B.subtilus, S.boydii, M.luteus,

(dictyophlebine) showed significant active against S.aureus (79%), while good antibacterial effect against P.aeruginosa (65%) respectively. The compound showed moderate activity against E.faecalis (50%), E.coli (41%), S.typhi (55%), B.subtilus

(54%), and M.luteus (56%), respectively. The compound was inactive against bacteria S.boydii, Citrobacter and P.mirablis respectively.

The result shows that compounds have significant to moderate antibacterial property and can be used in various infectious diseases. This result also supports the compounds isolated from S.saligna in against various pathogenic bacteria and can be used in to treat different infection such as sinusitis and pharyngitis [42, 44]. The antibacterial activity of compounds also showed in Figures (3-17 to 3-26) against mentioned pathogenic bacteria.

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Chapter 3 Results and Discussion

Table 3.7 Zone of inhibition (mm) of Antibacterial Dictyophlebine (2) compound

S.NO Bacterial Dictyophlebine (2) Standard Drug Strains (100 µg/100ml) Zone of Zone of Zone of inhibition inhibition inhibition ( mm) ( %) ( mm) 1 E.coli 09 41 22

2 Citrobacter ------15

3 S.aureus 22 79 28

4 S.typhi 10 55 18

5 B.subtilus 12 54 22

6 S.boydii ------15

7 M.luteus 10 56 18

8 E.faecalis 10 50 20

9 P.mirablis ------14

10 P.aeruginosa 18 65 28

Standard Drug = Amoxicillin (10µg/discs)

125

Chapter 3 Results and Discussion

Holaphylline Sarcovagine-D Dictyophlebine Standard 100

% Zone% of inhibtion 20

E.coli

Figure 3-17 : Antibacterial effect of compounds Holaphylline, Sarcovagine-D and Dictyophlebine against E.coli

Holaphylline Sarcovagine-D Dictyophlebine Standard 100

% Zone% of inhibtion 20

Citrobacter

Figure: 3-18: Antibacterial effect of compounds against Citrobacter

126

Chapter 3 Results and Discussion

Holaphylline Sarcovagine-D Dictyophlebine Standard 100

% Zone% of inhibtion 20

S.aureus

Figure 3-19: Antibacterial activity of compounds against S.aureus

Holaphylline Sarcovagine-D Dictyophlebine Standard 100

% Zone% of inhibtion 20

S.typhi

Figure 3-20: Antibaterial activity of compounds against S.typhi

127

Chapter 3 Results and Discussion

Holaphylline Sarcovagine-D Dictyophlebine Standard 100

% Zone% of inhibtion 20

B.subtlius

Figure 3-21: Antibacterial activity of compounds against B.subtilus

Holaphylline Sarcovagine-D Dictyophlebine Standard 100

% Zone% of inhibtion 20

0 S.boydi

Figure 3-22: Antibacterial activity of compounds against S.boydii

128

Chapter 3 Results and Discussion

Holaphylline Sarcovagine-D Dictyophlebine

100 Standard

% Zone% of inhibtion 20

M.luteus

Figure 3-23: Antibacerial activity of compounds against M.luteus

Holaphylline Sarcovagine-D Dictyophlebine Stnadard 100

% Zone% of inhibtion 20

E.faecalis

Figure 3-24: Antibacterial activity of compounds against E.faecalis

129

Chapter 3 Results and Discussion

Holaphylline Sarcovagine-D Dictyophlebine Standard 100

% Zone% of inhibtion 20

P.mirablis

Figure 3-25: Antibacerial activity of compounds against P.mirablis

Holaphyllline Sarcovagine-D Dictyophlebine Standard 100

% Zone% of inhibtion 20

P.areuginosa

Figure 3-26: Antibacerial activity of compounds against P.areuginosa

130

Chapter 3 Results and Discussion

3.3.3 Antifungal Activity

The isolated pure compounds were tested as an antifungal agent against different fungal species such as A.niger, A.flavus, P.notatum, C.albicans, and A.treus. The following Table 3.8 and Figure 3.27 to 3-29 were shown and presented the antifungal activty. The compound 5 (holaphylline) showed mild to moderate antifungal activity against A.niger, P.notatum and C.albicans (25%, 28% and 30% respectively) while inactive against A.flavis and A.treus . Similarly the compound 1

(alkaloid-C) showed low activity against pathogenic A.flavus and A.treus (10% and

9% respectively) while inactive against A.niger , P.notatum and C.albicans . The compound 3 (sarcovagine-D) also screened for antifungal activity and the result showed that it has low activity against A.niger and C.albicans (15% and 20% respectively) while inactive against A.flavus, P.notatum and A.treus.

These pathogenic fungus species produce different diseases in human, animal and plant. The aspergillus species can cause localized or systemic aspergillosis diseases

[135] such as A.flavus can spoil cotton seed and also pollute peanuts during harvesting and storage [136]. Similarly C.albicans causes skin, ear and bronchial candidiasis

[137]. P.notatum can cause infection in low immunity people such as pulmonary and cardiac infection [138]. Therefore the researcher is trying to isolate the compounds from medicinal plants which could be used effectively as an antifungal agent to treat various fungal diseases [139]. The tested steroidal alkaloid showed mild to moderate antifungal activity which can be enhanced by co-administration of synthetic antifungal drug and also by investigating structure activity relationship of tested compounds.

131

Chapter 3 Results and Discussion

Table 3.8 Antifungal activity of steroids alkaloid from S.saligna

Fungi Standard Holaphylline (5) Alkaloid-C (1) Sarcovagine-D Name antifungal (% Inhibition ) ( % Inhibtion) (3) (% 24µg/ml Inhibition) ( MIC)

A.niger Fluconazole ( 55 ) 25 - 15

A.flavus Fluconazole ( 70) - 10 -

P.notatum Fluconazole ( 65) 30 - -

C.albicans Amphoteracin-B ( 28 - 20 90)

A.treus Fluconazole (40) - 9 -

132

Chapter 3 Results and Discussion

A.niger A.flavus 100 P.notatum C.albicans 80 A.treus

% Zone% of inhibtion 20

0 Holaphylline

Figure 3-27: Antifungal activity of Holaphylline against various fungi

A.niger A.flavus 100 P.notatum C.albicans

80 A.treus

% Zone% of inhibtion 20

0 Alkaloid C

Figure 3-28: Antifungal activity of Alkaloid-C against different fungi

133

Chapter 3 Results and Discussion

50 A.niger A.flavus 40 P.notatum 30 C.albicans A.treus 20

%inhibtion of Zone 10

0 Sarcovagine-D

Figure 3-29: Antifungal activity of Sarcovagine-D against various fungi

134

Chapter 3 Results and Discussion

3.3.4 Antileishmanial Activity

In the present study, the anti-promastigote activity of compounds 5 (Holaphylline) and 4 (Saracodine) was evaluated against Leishmania tropica. The results were expressed as the percent inhibition in parasite numbers. The % inhibition was calculated by comparison to untreated controls. The average number of promastigotes in control group after 48 hours was 90. The IC50 (the inhibitory concentration of compound that reduced 50% of the Leishmania tropica promastigotes in comparison to control) values was also calculated for each compounds by using GraphPad Prism 6

Software. The results of the percent inhibition of quadruplicate for each concentration of each compound and their respective IC50 values along with 95% confidence intervals are shown in the following tables.

3.3.4.1 Anti-promastigote Activity of Compound 4 (Saracodine)

Table 3-9 indicates the anti-leishmanial activity (mean percent inhibition) of compound 4 (Saracodine) against promastigotes of L. tropica. Promastigotes were exposed to four different concentrations (100µM, 75µM, 50µM, and 25µM) of compound 4 for 48 hours at 26o C.

As clearly shown in Table 3-9 and Fig 3-30, Saracodine ( 4 ) compound eliminated

71.12% of the promastigotes when it was used in 100 µM concentration, in 75 µM concentration eliminated 63.9% promastigotes, in 50 µM concentration eliminated

54.18% promastigotes and the lowest concentration (25 µM) eliminated 38.88% of the promastigotes. Using percent inhibition IC50 value was calculated in GraphPad

Prism Software as 0.6352 µM (95% Confidence intervals = 0.4470 to 0.9025 µM).

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Chapter 3 Results and Discussion

Table 3.9: Anti-leishmanial activity of Saracodine against promastigotes of L. tropica

Tested Different Prosmastigotes % Mean % IC50 compound conc in ×104 inhibition Inhibition ± µM SD 20 77.8

25 72.2

Saracodine 100 31 65.6 71.12 ± (4) 5.20 28 68.9

25 72.2

Saracodine 75 27 70 63.9 ± 8.41 (4) 38 57.8

40 57.8

35 61.1 0.6352 Saracodine 50 34 61.1 (4) 49 45.6 54.18 ± 8.69 47 47.8

59 34.4

Saracodine 25 49 45.6 (4) 52 42.2 38.88 ± 5.98 60 33.3

25 72

Pentamidine 100 28 69 63.5±6.09 * 40 55

38 58

42 53

40 55

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Chapter 3 Results and Discussion

38 58 57.5±7.54

Pentamidine 75 32 64 *

36 60

Pentamidine 42 53 * 50 48 47 52.5±9.65

45 50

48 46

Pentamidine 55 39 33.75±5.81 * 25 70 22

65 28

* Standard Drug

137

Chapter 3 Results and Discussion

Saracodine % inhibition STD % inhibition 100

values 50 40

0 100M 75M 50M 25M

Figure 3-30: Graphical representation of Saracodine against L.tropica with different cocentration

138

Chapter 3 Results and Discussion

3.3.4.2 Anti-promastigote Activity of Compound 5 (Holaphylline)

Table 3-10 indicates the anti-leishmanial activity (mean percent inhibition) of compound 5 (Holaphylline) against promastigotes of L. tropica. Promastigotes were exposed to four different concentrations (100µM, 75µM, 50µM, and 25µM) of holaphylline (5) for 48 hours at 26o C.

As clearly shown in Table 3-10 and Fig 3-11, compound 5 (holaphylline) eliminated

82.5% of the promastigotes when it was used in 100 µM concentration, in 75 µM concentration eliminated 76.68% promastigotes, in 50 µM concentration eliminated

65% promastigotes and the lowest concentration (25 µM) eliminated 47.22% of the promastigotes. Using percent inhibition IC50 value was calculated in GraphPad Prism

6 Software as 0.6675 µM (95% Confidence intervals = 0.4722 to 0.9437 µM).

In the present study, compound 5 (holaphylline) showed significant inhibition against the promastigotes of L. tropica and their potency was excellent in 100 µM and 75 µM concentrations. Based on these results it is concluded that holaphylline (5) might become suitable antileishmanial agent. The previous research and literature of

Sarcococca species also support our result against Leishmania species and isolated compounds of S.coriacea also shown good antileishmanial activity [140].

139

Chapter 3 Results and Discussion

Table 3.10 Anti-leishmanial activity of compound 5 (holaphylline) against promastigotes of L. tropica

Tested Different Prosmastigotes % Mean % IC50 compound conc in µM ×104 inhibition Inhibition ± SD

10 88.9

Holaphylline 18 80 (5) 100 15 83.3 82.5 ± 4.83

20 77.8

12 86.7

Holaphylline 75 20 77.8 76.68 ± (5) 7.87 0.6675 23 74.4

29 67.8

Holaphylline 50 30 66.7 65.0 ± 2.96 (5) 32 66.4

35 61.1

45 34.4

Holaphylline 25 50 45.6 47.22 ± (5) 2.65 49 42.2

46 33.3

25 72

Pentamidine* 100 28 69 63.5±6.09

40 55

38 58

42 53

75 40 55

Pentamidine* 38 58 57.5±7.54

140

Chapter 3 Results and Discussion

32 64

36 60

Pentamidine* 42 53

50 48 47 52.5±9.65

45 50

25 48 46

Pentamidine* 55 39 33.75±5.81

70 22

65 28

* Standard Drug

141

Chapter 3 Results and Discussion

Holaphylline % inhibition

100 STD % inhibition

values 50 40

0 100M 75M 50M 25M

Figure 3-31: Graphical representation of Holaphylline against L.tropica

142

Chapter 3 Results and Discussion

3.3.5 Phytotoxic activity

The isolated compounds were tested for their phytotoxic effect at 10,100 and 1000

µg/ml concentration. The result showed that phytotoxic effect was concentration dependent. The compound 5 (holaphylline) showed maximum growth inhibition

(66%) at a concentration of 1000µg/ml while showed low activity (17 and 33 %) at 10 and 100 µg/ml concentration. The phytotoxicity of alkaloid-C (1) showed very low effect (13, 16 and 33 %) at a concentration of 10, 100 and 1000µg/ml respectively.

The compound 2 (dictyophlebine) also showed low phytotoxic activity i.e. 13%, 26% and 36 % at 10, 100 and 1000 µg/ml concentration which is shown in Table 3-11 and presented in figure 3-32. The screening of weedicides action of compounds isolated from plant is very valuable and useful for agriculture purpose [141]. The previous study of S.saligna also supported our result and showed that compound have low phytotoxic effect [44].

143

Chapter 3 Results and Discussion

Table 3.11 Phytotoxic Effect of Steroidal alkaloids

Compound No of fronds Sample No of No of Growth Sample (3 Conc ( fronds fronds died regulation fronds/Plant) µg/ml ) survived ( % )

30 10 25 5 17 Holaphylline 30 100 20 10 33 (5) 30 1000 10 20 66

Alkaloid-C (1) 30 10 26 4 13 30 100 22 8 26 30 1000 20 10 33

Dictyophlebine 30 10 26 4 13 (2) 30 100 22 8 26 30 1000 19 11 36

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Chapter 3 Results and Discussion

Holaphylline Alkaloid-C Dictyophlebine STD 100 90 80 70 60 50 40 30

% growth% regulation 20 10 0 10g/ml 100g/ml 1000g/ml

STD = Paraquat

Figure 3-32: Graphical representation of Phytotoxicity activity with various compounds

145

Chapter 3 Results and Discussion

3.3.6 Insecticidal Activity:

The isolated compounds from S.saligna were tested for insecticidal effect against the insects; T.castaneum, R.dominica and C.analis. The results were shown in table 3-12 and Fig 3-33 which showed that compound 5 (holaphylline) has maximum insecticidal activity (65%) against T.castaneum , while showed low activity against

R.dominica and C.analis (20% and 10%). Compound 3 (sarcovagine-D) showed mild actions against T.castaneum (45%), while not active against R.dominica and

C.analis. Compound 1 (Alkaloid-C) observed low activity against R.dominica (20%), while inactive against T.castaneum and C.analis insect. The result obtained showed that some compounds were active against specific insects while others compounds were inactive against insects and the study was supported by previous literature and research [44].

146

Chapter 3 Results and Discussion

Table 3.12 Insecticidal activity of isolated steroidal alkaloids

% Mortality of % Mortality of % Mortality % % Name of Compound 5 Compound 3 ( of Compound Mortality Mortality Insects ( Holaphylline) Sarcovagine-D) 1 ( Alkaloid- of +ve of –ve C ) Control control

T.castaneum 65 45 ---- 100 ----

R.dominica 20 -- 20 100 ----

C.analis 10 ------100 -----

+Ve control: Permethrin (239.50µg/cm2)

147

Chapter 3 Results and Discussion

Holaphylline Sarcovagine-D Alkaloid-C STD 100

% Mortality%

0 T.castaneum R.dominica C.analis

Figure 3-33: Graphical representation of insecticidal activity with various compounds

148

Chapter 3 Results and Discussion

3.3.7 Antioxidant Activity

The isolated steroidal alkaloids from S.saligna were tested for antioxidant activity and the results are shown in Table 3-13 and demonstrated in Fig 3-34. The compounds 3

(sarcovagine-D) and 5 (holaphylline) showed significant antioxidant activity by increasing concentration from 100-400 µg/ml. The radical scavenging activity increased up to 78% and 80% of both compound sarcovgine-D (3) and holaphylline

(5) at 400µg/mL. The compound 1 (alkaloid-C) and 2 (dictyophlebine) showed low antioxidant activity (20%, 10%) at a concentration of 400µg/ml. The result data of antioxidant activity of isolated compounds was also supported by previous research and literature of S.saligna [142].

149

Chapter 3 Results and Discussion

Table 3.13 Antioxidant activity of Steroid alkaloidal from S.saligna

S.No Tested Compound Con .(µg/ml) % Radical Scavenging Activity 100 15 1 200 35 Sarcovagine-D (3) 300 55

400 78

100 20 2 Holaphylline (5) 200 35

300 60

400 80

100 -- 3 Alkaloid-C (1) 200 --

300 10

400 20

100 -- 4 Dictyophlebine (2) 200 ---

300 ----

400 10

100 45 5 Ascorbic acid ( Standard) 200 65

300 80

400 95

150

Chapter 3 Results and Discussion

Sarcovagine-D Holaphylline Alkaloid-C Dictyophlebine Standard 100

% RSA% 40

g/mll g/ml g/ml g/ml 100 200 300 400

Figure 3-34: Graphical representation of antioxidant activity with various steroidal alkaloids

151

Chapter 3 Results and Discussion

3.3.8 Cytotoxic Activity

Some xenobiotics have cytotoxic potential which kill the cancer cells instead of normal healthy cells. The human body contains various cells which paly important role in immune system. These cells include natural killers; cytotoxic cells and lymphokine activated cells are responsible to kill abnormal cells [143]. Those substances which have cytotoxic property can be used in different diseases such as inflammation, AIDS and cancer [143].

The isolated steroidal alkaloids were tested for anticancer activity as shown in Table

3-12. Compound 1 (alkaloid-C) showed moderate anticancer activity against HeLa cancer cells with IC50 values 12.98±0.235. Compound 2 (dictyophlebine) showed good cytotoxic activity against HeLa cells with IC50 value 6.13±0.345 while 5

(holaphylline) showed less effect on cancer cells with IC50 value 23.88±0.243. The effect of these compounds also presented in Fig 3-35.

The result showed that steroidal alkaloids isolated from this plant can be used for the management of cancer. This study was supported by previous activity of steroidal alkaloids isolated from S.saligna which used as a cytotoxic for cancer cells [47].

Sarsaligates A and B, sarcorucinine and sarcovagine isolated from S.saligna were exhibited anticancer property [47].

152

Chapter 3 Results and Discussion

Table: 3.14.Anticancer activity of isolated steroidal alkaloids against HeLa cells

S.NO COMPOUNDS IC50±SD ( µg/ml )

1 Alkaloid-C (1) 12.98±0.235

2 Dictyophlebine (2) 6.13±0.345

3 Holaphylline (5) 23.88±0.243

3 Doxorubicin (Std) 2.10±0.14

153

Chapter 3 Results and Discussion

Alkaloid-C

Dictyophlebine Holaphylline Standard 30

value

IC 10

Figure 3-35: Graphical representation of anticancer activity with various compounds

154

Chapter 4 Molecular Docking

4 MOLECUALR DOCKING OF ISOLATED STEROIDAL ALKALOIDS

AGAINST AROMATASE ENZYME IN BREAST CANCER

Women breast cancer is one of the leading causes of death in many parts of the world

[144]. The treatment of breast cancer is surgery with radiotherapy. In addition, hormone therapy is also used with or without chemotherapy, depending on tumors condition. The potent endogenous estrogen is estradiol, which is produced from androgens by the catalytic action of aromatase enzyme. The aromatase is a cytochrome P450 enzyme complex in nature, which is found in high concentration in different organs of both males and females [145]. Aromatase is a well-established drug target for management of most breast cancers, because, the pathological modulation of estrogens are involved in majority of the breast cancer. Aromatase enzyme inhibitors are an effective modality of treatment for estrogen related breast tumors. It is an enzyme complex, which is based on two protein components and is bound to the endoplasmic reticulum [145, 146]. One of its component is cytochrome

P450 aroma , a hemoprotein which catalyze the conversion of androgens (C19) into phenolic estrogens (C18) by removing methyl group [145, 147]. NADPH-cytochrome

P450 reductase is the second protein, which mobilize reducing equivalents to cytochrome P450arom. The reaction requires three subsequent oxidation steps to convert one mole of androgen into one mole of estrogen with the help of aromatase enzyme [Fig 4- 1]. Therefore aromatase inhibitors (AIs) are critical drugs to inhibit aromatase enzyme for better treatment and management of estrogen dependent breast cancer in post-menopausal women [144]. Clinically available aromatase inhibitors

(AIs) include both steroidal and non-steroidal AIs. Steroidal AIs are slightly superior to others, because of their less reactivity towards heme moiety of aromatase enzyme, and due to their irreversible enzyme inhibition, which is called “suicide inhibition”.

155

Chapter 4 Molecular Docking

In this context, a study was designed, to probe new yet effective steroidal AIs isolated from a medicinal plant Sarcococca saligna, as potential lead compounds for management of breast cancers. The aim was to explore isolated steroidal alkaloids against aromatase enzyme in assistance by molecular docking simulations to understand molecular interaction between the enzyme and ligands.

The first aromatase inhibitor was aminoglutethimide (AG) clinically used in late

1970s [148] but due to its high toxicity, more disadvantages and lack of selectivity at last removed from market [ 149, 150 ]. Therefore the scientist much focus on the development of more effective, selective and safe aromatase inhibitors which eventually synthesized second generation Formestane drug [151] and third generation

Exemestane , Anastrozole and Letrozole aromatase inhibitors which are much more effective , safe and selective [ 152].

Aromatase inhibitors (AIs) can be divided in to two groups. One is Non-steroidal and other is steroidal inhibitors [152, 153]. The non-steroidal are the derivatives of

AG and also include mostly imidazole or triazole compounds such as anastrazole and letrozole while steroidal aromatase inhibitors are the derivatives of natural substrate androstenedione for the aromatase enzyme such as exemestane and formestane [154].

156

Chapter 4 Molecular Docking

Figure 4-1: Biosynthetic pathway of estrogen through aromatase enzyme reaction

157

Chapter 4 Molecular Docking

There are many reasons to develop steroidal instead of non-steroidal aromatase inhibitors. They are different in their mechanism of actions such as steroidal aromatase inhibitors bind to the natural substrate androstenedione site on the aromatase enzyme while non-steroidal aromatase inhibitors bind to the heme group of the aromatase enzyme [155]. Secondly steroidal aromatase inhibitors irreversibly deactivate aromatase enzyme and therefore called it “suicide” inhibition [156].

Instead of effectiveness and selectivity third generations aromatase inhibitors have still major side effects such as resistance produced in the lengthy treatment of breast cancer and increase bone erosion [157]. Therefore the search of new aromatase inhibitors is important which are effective and safe in treatment of breast cancer.

Molecular docking of compounds was important tool for the search of novel aromatase inhibitors based on 3D structure of human placental aromatase enzyme

(pdp code 3EQM) [158]. The molecular docking of non-steroidal aromatase inhibitors and its 3D-QSAR studies is to search effective, safe and specific aromatase inhibitors

[159]. After a while numbers of novel steroidal aromatase inhibitors synthesized by changing the A-and D- rings of aromatase substrate androstenedione and were evaluated for inhibitory activity [158, 160, 161].According to Cepa et al that for the enzyme –drug interaction three important structural characteristics , one is ring-A planarity, second is 5α-sterochemistry and the stability of cyclopentanone D-ring

[158].

4.1 Materials and methods

4.1.1 Aromatase Activity

Stressor et al., method was used with slight changes for the inhibition of aromatase enzyme activity. Gentest kit was used for conducting the experiment with gene

158

Chapter 4 Molecular Docking aromatse cytochrome P450 (CYP19) and a substrate O-benzyl fluorescein benzyl ester (DBF) was used. The alkyl group was eliminated from DBF during biochemical reaction which further hydrolyzed to release a fluorescein product. During experiment, 100 µL of cofactor, comprising 78.4 µL of 50 mM phosphate buffer (pH

7.4); 20 µL of 20x NADPH-generating system (26 mM NADP+, 66 mM glucose-6- phosphate, and 66 mM MgCl2); and 1.6 µL of 100 U/mL glucose-6-phosphate dehydrogenase, were added to a 96-well plate and mixture was preincubated in waterbath at 37 °C for 12 min. Biochemical reaction was started by pipetting 100 µL of enzyme/substrate (E/S) mixture comprising 77.3 µL of 50 mM phosphate buffer

(pH 7.4); 12.5 µL of 16 pmol/mL CYP19; 0.2 µL of 0.2 mM DBF, and 10 µL of the test sample or 10% DMSO as a negative control or Exemestane as a positive control/standard. Fluorimetric analysis was done at the excitation wavelength of 490 nm and emission wavelength of 530 nm with the cutoff limit at 515 nm. IC50 values were calculated form percent inhibition using Graphpad software.

4.1.2 Molecular docking simulations

Study molecular docking of compounds OMEGA pre-generated multi-conformer library FRED 2.1 [162] was used as mentioned above. All compounds were drawn and optimized via MMFF as geometry optimization and energy minimization tool for all ligand. All water molecules were removed from crystal structure of the protein molecule (PDB ID: 3EQM) followed by assigning of appropriate charges to all atoms of the protein keeping the protonation states in view to get most accurate results.

FRED 2.1 strategy is to exhaustively dock/score all possible positions of each ligand in the binding site. The exhaustive search is based on rigid rotations and translations of each conformer within the binding site defined by a box. FRED filtered the poses ensemble by rejecting the ones that clash with the protein (aromatse) or do not have

159

Chapter 4 Molecular Docking enough contacts with the protein. The final poses can then be scored or re-scored using one or more scoring functions. In this study, the smooth shape-based Gaussian scoring function (shapegauss) was selected to evaluate the shape complementarily between each ligand and the binding pocket. The default FRED protocol was used except for the size of the box defining the binding sites. In an attempt to optimize the docking-scoring performance we performed exhaustive docking with shapegauss applying the "Optimization" mode. The "Optimization" mode involves a systematic solid body optimization of the top ranked poses from the exhaustive docking. 3 different boxes were explored for LOX (PDB ID: 3EQM). Three different simulations were carried out with an added value of 8 Å around the reference ligand.

4.2 Results and discussion

The aim of present study is to explore the isolated pure steroidal alkaloids from

Sarcococca saligna, as new steroidal aromatase inhibitors through molecular docking studies. Therapeutically effective AIs are clinically proven chemotherapeutic agents for the treatment of breast cancer in women. Interesting findings were observed after experimental and computational investigations.

All the test compounds were inactive accept compound 5 (holaphylline) and 1

(alkaloid-C), due to their bulky structures in comparison to the active site of

Aromatase enzyme. The IC50 values of compound 5 and 1 were 12.91±0.01µL and

138.27±0.01µL, respectively. The standard drug Exemestane showed potent activity in comparison to the test compounds, having IC50 values of 0.052±0.01µL. Molecular insights based on molecular docking revealed the reason behind lower activity of compound 5 and 1 in as in Fig 4-2 and 4-5.

Compound 5 showed important interactions with the critical amino acid residues.

Carbonyl moiety of the compound was found to be favourably interacting through

160

Chapter 4 Molecular Docking hydrogen bonding with Met374 at the distance of 3.016 Å. Other end, tertiary nitrogen of compound 1 (Alkaloid-C) was detected to be contacting with Ala306 Å via hydrogen bonding. Apart from these two favourable contacts no other reasonable features was observed. Planarity of tetracyclic core of steroidal nucleus with respect to the co-factor was missing, which could be a factor responsible for lower enzyme binding affinity. The major reason was the overall length of the molecular, which led to slight steric hindrance and fitting inside the binding pocket, hence resulted in lower activity. In case of compound 1(Alkaloid-C), methoxy group plated a vital role in binding with aromatase enzyme. Methoxy group was simultaneously bound to

Met374 and Arg115 via hydrogen bonding at the favourable distances of 2.710 Å and

2.712 Å, respectively. Compound 1 looks to be longer than compound 5, which resulted in its strained conformation inside active site of aromatase. The terminal tertiary nitrogen of compound 1 was attached with Ser478 through hydrogen bonding in an unfavourable bent conformation, which led to far lesser activity than compound

5. Both compounds showed favorable electrostatic interactions with the active site of enzyme but the shape and steric bulk of the compounds were the limiting factor in their inhibitory effects. Rests of the compounds were found inactive because their molecular size created a barrier towards their binding inside the active site of enzyme.

This study highlighted the potential of steroidal alkaloids as possible anticancer agents by targeting aromatase enzyme provided that new lead compounds should be generated after extensive modifications guided by computational and experimental tools.

161

Chapter 4 Molecular Docking

Figure 4-2: Binding mode of the compound 5 (Holaphylline) inside the catalytic site of aromatase enzyme

Figure 4-3: A closer view of the molecular interactions between compound 5 and aromatase enzyme

162

Chapter 4 Molecular Docking

Figure 4-4: Electrostatic and steric interactions between compound 5 and aromatase enzyme

Figure 4-5: Binding mode of the compound 1 inside the catalytic site of aromatase enzyme

163

Chapter 4 Molecular Docking

Figure 4-6: A closer view of the molecular interactions between compound 1 and aromatase enzyme

Figure 4-7: Electrostatic and steric interactions between compound 1 and aromatase enzyme

164

Chapter 5 Conclusion

5 CONCLUSION

The steroidal alkaloids isolated from Sarcococca species were pharmacologically active and have shown various biological actiivties.The fruit extract of Sarcococca saligna showed antibacterial, antifungal, antioxidant and insecticidal activity.

Therefore bio-assay guided isolation and structure elucidation of steroidal alkaloids from S.saligna were performed for their ethnobotanical importance.

Five compounds were isolated from chloroform fractions of S.saligna and the compounds were alkaloid-C (1), dictyophlebine (2), sarcovagine-D (3), saracodine

(4) and holaphylline (5). These compounds were then screened for different in-vivo and in-vitro biological activitiest. The compound 5, 3 and 2 showed significint antibacterial activity against certain bacteria while it showed low to moderate antifungal activity against various pathogenic fungi. The compounds 5 posses significant while 4 showed moderate antileshmainal activity against L.tropica. The compounds also showed mild to moderate phytotoxic and insecticidal activity. The compounds 3 and 5 showed significant antioxidant activity while compound 2 and 1 have anticancer activity against HeLa cells lines.

The compound 1, 3 and 5 were screened for immunosuppressant and hepatoprotective activity which showed excellent immunosuppressant and hepatoprotective actions.

The isolated steroidal alkaloids were explore for the antidiabetic potential and the result showed that compounds 5 and 3 reduced the glucose level significantly in blood and also make better others diabetes associated complications . The compounds were to explore as a new steroidal aromatase inhibitors through molecular docking studies in which compound 5 and 1 were active against aromatase enzyme in breast cancer could provide new lead compounds.

165

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