Antimicrobial Evaluation of Extracts of Psidium Guajva and Syzgium Cumini Against Oral Pathogens

Antimicrobial evaluation of extracts of Psidium guajva and Syzgium cumini against oral pathogens

Lubna Tahir

Department of Microbiology

Faculty of Biological Sciences

Quaid-i-Azam University

Islamabad

2015

Antimicrobial evaluation of extracts of Psidium guajva and Syzgium cumini against oral pathogens

A thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Microbiology

Lubna Tahir

Department of Microbiology

Faculty of Biological Sciences

Quaid-i-Azam University

Islamabad. Pakistan

2015

In the name of Allah, Most gracious, most merciful

"...... let them devote themselves to studies in religion and admonish their comrades when they return to them so that they may guard themselves against evil."

(Sura 9, Verse 121)

CERTIFICATE

This thesis submitted by Lubna Tahir is accepted in the present form by Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, as satisfying the dissertation requirements for the degree of Doctor of Philosophy in Microbiology.

External Examiner I ______

External Examiner II ______

Supervisor: ______

(Prof. Dr. Safia Ahmed)

Chairperson: ______

(Dr. Fariha Hasan)

Dated:------

III

LIST OF EXTERNAL EXAMINERS

1. Dr. John McCall

Professor Robert Gordon University Riverside East Garthdee Road Aberdeen, AB107GJ United Kingdom Tel: +441224262473 Email: [email protected]

2. Dr. Ananda M. Chakrabarty

Professor College of Medicine Department of microbiology and Immunology (MC 790) E-704 Medical Sciences Building 835 South Wolcott Avenue Chicago, Illinois 60612-7344 Email: [email protected]

3. Dr. Albert La Spada

Professor Institute of Genomic Medicine 2880 Torrey Pines science Drive Sanford Consortium for regenerative Medicine MC 0642, Room 3804 University of California, San Diego California 92037-0642 Email: [email protected]

DEDICATED

To my loving parents &

FAMILY

DECLARATION

The experimental work described in this thesis was carried out in the Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan; Department of Applied Chemistry, Pakistan Council of Scientific and Industrial Research, Lahore, Pakistan and Department of Biochemistry, University of Maryland, College Park, USA. I have not presented any part of this work for any other degree. All the assistance and help received during the course of research have been duly acknowledged.

Lubna Tahir

List of Tables

TABLE OF CONTENTS

List of Contents

DEDICATED ...... V

DECLARATION ...... VI

TABLE OF CONTENTS ...... VII

LIST OF TABLES ...... XII

LIST OF FIGURES...... XIV

ABBREVIATIONS...... XVII

ACKNOWLEDGMENTS...... XIX

ABSTRACT ...... XX

1. INTRODUCTION ...... 1

1.1. Aims and Objective ...... 6

2. REVIEW OF LITERATURE ...... 7

2.1. Medicinal Plants and Their Importance ...... 7

2.2. Oral Health and Global Concerns ...... 9

2.3. Oral Health of People of Pakistan ...... 10

2.4. Dental Caries ...... 11

2.5. From Synthetic to Herbal Products ...... 14

VII

List of Tables

2.6. The Ethnobotanical Importance, Bioassay and Phytochemical Review of Selected Plants of the Present Study ...... 17

2.6.1. Syzygium cumuni ethanobotanical importance ...... 17

2.6.2. Psidium guajava ethanobotanical importance...... 18

2.6.3. Morus nigra ethanobotanical importance...... 19

2.6.4. Phoenix dactylifera ethanobotanical importance ...... 21

2.6.5. Mangifera indica ethanobotanical importance ...... 22

2.6.6. Vitis vinifera ethanobotanical importance ...... 23

2.6.7. Dispyros blancoi ethanobotanical importance ...... 24

2.6.8. Litchi chinensis ethanobotanical importance ...... 25

3. MATERIALS AND METHODS...... 27

3.1. Collection of Plant Material ...... 27

3.2. Drying and Extraction ...... 27

3.3. Fractionation...... 27

3.4. Phytochemical Analysis ...... 33

3.4.1. Test for Alkaloids...... 33

3.4.2. Test for Saponins ...... 33

3.4.3. Test for Tannins ...... 33

3.4.4. Test for Flavonoids ...... 34 VIII

List of Tables

3.5. Antibacterial Sensitivity of Crude Extracts against Dental Caries Causing Pathogens (in vitro)...... 34

3.5.1. Test Microorganisms...... 34

3.5.2. Antibacterial Assay of crude extract ...... 34

3.5.3. Minimum Inhibitory Concentration Determination ...... 35

3.6. Formulation of Guava & Jaman Tablets ...... 35

3.7. In-vitro Antibacterial Sensitivity of Syzigium cumunii:Psidinm guajava Tablet against Bacterial Strains ...... 36

3.8. Evaluation of Physical Properties of Syzygium cumuni:Psidinm guajava Tablet 36

3.9. Determination of Bactericidal Activity of Syzygium cumuni: Psidium guajava Chewable Tablet...... 37

3.10. In-vivo Study of Syzygium cumuni : Psidinm guajava Chewable Tablet ...... 37

3.11. Isolation of Bioactive Fractions ...... 37

3.11.1. Flash chromatography ...... 38

3.11.2. Reverse Phase High Performance Liquid Chromatography (RP-HPLC) 45

Sample Preparation ...... 45

3.11.3. Flash Chromatography (FC)...... 45

3.11.4. Preparative Thin Layer Chromatography...... 46

3.12. Bio-assay of Collected Fractions ...... 46

List of Tables

3.13. Detection of Biofilm Formation in Streptococci and Biofilm Inhibition Assay 47

3.14. Scanning Electronic Microscopy (SEM) of the Bacterial Cells after Treatment with Bioactive Components of the Extracts ...... 48

3.15. Analysis of Bioactive Fractions ...... 48

3.15.1. Mass Spectroscopy ...... 49

3.15.2. FT-IR analysis ...... 49

3.15.3. NMR analysis ...... 49

4. RESULTS ...... 50

4.1. Screening for Antibacterial activity ...... 50

4.2. Preliminary Phytochemical Analysis ...... 57

4.3. Tablet Formation for Treating Dental Caries ...... 63

4.3.1. Tablet formulation...... 65

4.3.2. Antibacterial activity of dental caries tablet ...... 65

4.3.3. Bactericidal activity of dental caries tablet by time kills assay method ..... 67

4.3.4. In-Vivo study of dental caries tablet ...... 67

4.4. Isolation of Bioactive Fractions ...... 68

4.4.1. Isolation of Bioactive fraction from ethyl acetate fraction of Psidium guajava 68

List of Tables

4.4.2. Bioassay of collected fractions ...... 69

4.4.3. Fraction MG-15...... 71

4.4.4. Fraction MG-25...... 78

4.4.5. Fraction MG-24...... 83

4.5. Purification of Bioactive compound From Syzygium cumini (JAMUN) ...... 87

4.5.1. Bioassay of collected fractions ...... 87

4.5.2. Fraction MJ-6 ...... 89

4.5.3. Fraction MJ-26 ...... 96

4.6. Biofilm Formation of Syzygium cumini and Psidium guajava Fractions...... 102

4.6.1. Determination of biofilm formation in Streptococci ...... 102

4.6.2. Anti-biofilm formation (using 1% dextrose) by Syzygium cumini and Psidium guajava fractions ...... 102

4.7. Scanning Electron Microscopy (SEM) ...... 104

5. DISCUSSION ...... 108

6. Conclusions ...... 115

Future Prospects ...... 116

References ...... 117

Paper Presentations / Publications ...... 154

List of Tables

LIST OF TABLES

Table 1.1: Plants of medicinal importance used in the present study ...... 5

Table 3.1: Composition of Guava and Jaman Tablets ...... 36

Table 3.2: Percentage composition of solvents used to elute the fractions in given time ...... 40

Table 3.3: Solvent system, their ratios, fractions and Rf values after TLC of ...... 41

Table 3.4: Solvent system, their ratios, fractions and Rf values after TLC of Psidium guajava ...... 43

Table 3.5: Gradient elution systems used for HPLC separations ...... 45

Table 4.1: Total yield obtained after fractionation of crude extract ...... 52

Table 4.2: Phytochemical analysis of Psidium guajava ...... 59

Table 4.3: Phytochemical analysis of Syzygium cuminis...... 59

Table 4.4: Phytochemical analysis of Mangifera indica ...... 60

Table 4.5: Phytochemical analysis of Litchi chinesis...... 60

Table 4.6: Phytochemical analysis of Vitis vinifera ...... 61

Table 4.7: Phytochemical analysis of Phoenix dactylifera...... 61

Table 4.8: Phytochemical analysis of Diospyros blancoi...... 62

Table 4.9: Phytochemical analysis of Moris nigra ...... 62

Table 4.10: Antibacterial activity of dry formulation of P. guajava and S. cumini against dental caries bacteria ...... 64

Table 4.11: Antibacterial activity of different combinations of dry extracts of S. cumini and P. guajava in DMSO ...... 64 XII

List of Tables

Table 4.12: Minimum Inhibitory Concentration (MIC) of chewable tablet ...... 66

Table 4.13: Antibacterial activity of MG-15 sub fractions from Psidium guajava ...... 73

Table 4.14: Antibacterial activity of MG-15 sub- sub fractions from Psidium guajava ...... 74

Table 4.15: FT-IR spectrum of Semi-purified bioactive fraction from MG-15.3.3 isolated from chloroform fraction...... 74

Table 4.16: Antibacterial activity of MG-25 fractions against oral bacteria ...... 79

Table 4.17: FT-IR spectrum of MG-25.1 isolated from MG-25 ...... 79

Table 4.18: Antibacterial activity of MG-24 fractions against oral bacteria ...... 85

Table 4.19: Antibacterial activity of MG-24.2 fractions against oral bacteria ...... 85

Table 4.20: Antibacterial activity of MJ-6 fractions against oral bacteria...... 91

Table 4.21: TLC of bioactive MJ-6 fractions...... 91

Table 4.22: Antibacterial activity of MJ-6.12 and MJ-6.13 fractions against oral bacteria ...... 92

Table 4.23: FT-IR spectrum of MJ-6.1.2 ...... 92

Table 4.24: Antibacterial activity of MJ-26 fractions against oral bacteria...... 97

Table 4.25: FT-IR spectrum of MJ-26.3 isolated from Syzygium cumini ...... 97

Table 4.26: Anti-adherence effect of partially purified fractions of Syzygium cumini and Psidium guajava (with 1% dextrose)...... 103

XIII

List of Figures

LIST OF FIGURES sFigure 3.1: Extraction and fractionation from the leaves of Syzigium cumunii ...... 29

Figure 3.2: Extraction and fractionation from the leaves of Psidium gujava...... 29

Figure 3.3: Extraction and fractionation from the leaves of Morus nigra ...... 30

Figure 3.4: Extraction and fractionation from the leaves of Phonex dactylifera...... 30

Figure 3.5: Extraction and fractionation from the leaves of Mangifera indica ...... 31

Figure 3.6: Extraction and fractionation from the leaves of Vitis vinifera ...... 31

Figure 3.7: Extraction and fractionation from the leaves of Diospyros Blancoi ...... 32

Figure 3.8: Extraction and fractionation from the leaves of Litchi chinensis ...... 32

Figure 3.9: Isolation of bioactive fractions from leaves of Syzygium cumini ...... 42

Figure 3.10: Isolation of bioactive fractions from leaves of Psidium guajava ...... 44

Figure 4.1: Antibacterial activity of crude fractions of leaves of Syzygium cumini ...... 53

Figure 4.2: Antibacterial activity of crude fractions of leaves of Psidium guajava ...... 53

Figure 4.3: Antibacterial activities of crude fractions of leaves of Mangifera indica ...... 54

Figure 4.4: Antibacterial activities of crude fractions of leaves of Litchi chinesis...... 54

Figure 4.5: Antibacterial activities of crude fractions of leaves of Vitis vinifera ...... 55

Figure 4.6: Antibacterial activities of crude fractions of leaves of Phoenix dactylifera...... 55

Figure 4.7: Antibacterial activities of crude fractions of leaves of Diospyros blancoi...... 56

Figure 4.8: Antibacterial activity of crude fractions of leaves of Morus nigra ...... 56

Figure 4.9: Antibacterial activity of dental caries tablet with respect to standard antibiotic against oral bacterial strains...... 66 XIV

List of Figures

Figure 4.10: Effect of variable time durations (0, 5, 10 and 15min) of chewable tablet on S. mutans ...... 67

Figure 4.11: In-vivo studies of dental caries tablets...... 68

Figure 4.12: Antibacterial activity of Psidium guajava fraction ...... 70

Figure 4.13: RP-HPLC chromatogram of fraction MG-15 from Psidium guajava ...... 73

Figure 4.14: FT-IR spectrum of isolated semi-purified fraction from MG-15.3.3 where absorption bands quoted in wave number (cm -1) represents functional groups ...... 75

Figure 4.15: 1H NMR of MG-15.3.3 from Psidium guajava ...... 76

Figure 4.16: Mass spectra of MG-15.3.3 from Psidium guajava...... 77

Figure 4.17: RP-HPLC chromatogram of fraction MG-25...... 78

Figure 4.18: FT-IR spectra of MG-25.1 ...... 80

Figure 4.19: H1 NMR of fraction MG-25.1 of Psidium guajava ...... 81

Figure 4.20: Mass spectra of fraction MG-25.1 of Psidium guajava...... 82

Figure 4.21: FC chromatogram of MG-24 ...... 86

Figure 4.22: FC chromatogram of MG-24.2 ...... 86

Figure 4.23: Antibacterial activity of Syzygium cumini fractions...... 88

Figure 4.24: RP-HPLC chromatogram of MJ-6 showing 11 peaks belonging to 11 fractions ...... 90

Figure 4.25: FT-IR spectra of fraction MJ-6.11.2 of Syzygium cuminii...... 93

Figure 4.26: H1 NMR spectra of fraction MJ-6.11.2 of Syzygium cuminii...... 94

Figure 4.27: Mass spectra of MJ-6.11.2 fraction of of Syzygium cumini ...... 95

Figure 4.28: RP-HPLC chromatogram of fraction MJ-26 showing three fractions...... 96

Figure 4.29: FT-IR spectra of fraction MJ-26.3 ...... 98 XV

List of Figures

Figure 4.30: 1H NMR spectra of fraction MJ-26.3...... 99

Figure 4.31: 13C NMR spectra of fraction MJ-26.3...... 100

Figure 4.32: Mass spectra of fraction MJ-26.3...... 101

Figure 4.33: Screw cap tubes showing biofilm formation by S. mutans (A) and anti-biofilm (B) effect of selected bioactive fraction (MJ-26) ...... 103

Figure 4.34: Scanning electron microscopy of E.coli, untreated (a), treated with Psidium guajava fractions (b &c) and with Syzygium cumini fractions (d & e) for 5 hrs ...... 105

Figure 4.35: Scanning electron microscopy of S. mutans, untreated (a), treated with Syzygium cumini fractions (b &c) and with Psidium guajava fractions (d & e) for 5 hrs ...... 106

Figure 4.36: Scanning electron microscopy of P. aureginosa, untreated (a), treated with Psidium guajava fractions (b &c) and with Syzygium cumini fractions (d & e) for 5 hrs ...... 107

XVI

Abbreviations

ABBREVIATIONS

% Percentage µ Micron 13H-NMR Carbon NMR 1H-NMR Proton NMR ATCC American Type Culture Collection BAF Bioactive Fraction BHI Brain Heart Infusion CFU Colony Forming Unit

CH3OH Methanol

CHCl3 Chloroform CHD Coronary Heart Diseases CSP-QS Competence- stimulating peptide-quorum sensing DCM Di- chloromethane DMFT Decayed Missing Filled Teeth DMSO Dimethyl sulphoxide EIS Electrochemical Impedance Spectroscopy EtOAc Ethyl acetate FC Flash chromatography FceR1 Fc Epsilon receptor 1 FT-IR Fourier Transform Infra-Red Spectroscopy GA Glutaraldehyde GNP Gross National Production GTFs Glucosyltransferases Hrs Hours MG Maryland Guava (collected from Pakistan) MGG Monogalloyl glucosides MHz Mega herds MIC Minimum Inhibitory Concentration

XVII

Abbreviations

MJ Maryland Jamun(Collected from Pakistan) MS Mass Spectroscopy NHANES National Health and Nutrition Examination Survey NMR Nuclear Magnetic Resonance ºC Degree centigrade OD Optical Density

OsO4 Osmium tetraoxide PA Proanthocyanidin PCSIR Pakistan Council of Scientific and Industrial Research Ph Potential of hydrogen of ion PHC Primary Health care PTLC Preparative thin layer chromatography QS Quorum sensing Rf Retention Factor RP-HPLC Reverse phase-High performance liquid chromatography Rpm Revolution per minute RP-TLC Reverse phase-Thin Layer chromatography SEM Scanning electron microscope TLC Thin Layer Chromatography Umd University of Maryland UV Ultra violet WHO World Health Organization

XVIII

Acknowledgment

ACKNOWLEDGMENTS

First of all thanks to Allah almighty, the sympathetic, the merciful, the supreme being of universe, the head of stream of all knowledge, who blessed me with loving parents, health, talented teachers and cooperative friends and enabled me to accomplish this work successfully. I offer my humble thanks to the compassionate; Holly Prophet Muhammad the most perfect and exalted among and of ever born on the surface of ,(ﺼلﻰ ١هلل عليه وسلم ) earth, who is forever torch of guidance and knowledge for humanity as a whole.

I would like to thank my supervisor, Professor Dr. Safia Ahmed, for her excellent supervision, guidance and the opportunity to undertake research within her group. Her devotion to and knowledge of Microbiology combined with the ability to share these traits with me is greatly appreciated.

I am deeply indebted to, Dr. Salma Rahman, Chief Scientific Officer (CSO), ACRC, PCSIR, Labs. Complex, Lahore and Dr. Herman O Sintim UMD, USA, whose suggestions and encouragement helped me throughout my research work and writing of thesis. I would also like to thanks Dr. Fariha Hasan, Chairperson Department of Microbiology, Quaid-i-Azam University, Islamabad, for her positive motivation.

I would like to offer my heartiest thanks to Dr. Shahzad Alam, Director General PCSIR Labs. Complex, Lahore for giving me every possible opportunity for the accomplishment of this degree.

I am grateful to Higher Education Commission, Pakistan for providing me the opportunity of IRSIP for completing my PhD and my parent organization, PCSIR Laboratories Complex, Lahore for giving me the opportunity to carry out higher studies.

I would like to thank many people who have helped me out in accomplishment of this task. Rabia Nazir, Naeem Khan, Mohammad Naeem, Mohammad Zaheer, Naqi Hussain, Mohammad Rizwan, Afsheen Arshad, Zia-ur-Rehman, Zeeshan Ali, Hifza Akhtar and all the technical staff from pharmaceutical section, ACRC, PCSIR Labs. Lahore. I would like to thanks my Lab fellows from Department of chemistry and biochemistry, University of Maryland, USA especially to Benjamin Roembke and Shizuka for helping me a lot in my experiments there. I would like to thanks my friends and fellows Ayesha Aslam, Irum Perveen, Qaiser Fareed, Shama Adnan from Department of Microbiology, QAU. Islamabad

All the credit goes to my parents who always stood beside me throughout my life. I would also like to acknowledge my sister and brothers, for their love, good wishes and moral support.

Lubna Tahir

XIX

Abstract

ABSTRACT

The search for bioactive compounds from plants has always been of great interest for scientists who are looking for new drugs from natural resources as according to WHO survey 80% of world population still rely on traditional medicines. Keeping this in mind the present research was conducted with a focus on isolation of some bioactive fractions from medicinal plants with strong antibacterial activities against dental caries causing pathogens. Eight plants were selected on the basis of their traditional uses, literature survey and ethnobotanical importance, Gaff (Diospyros blancoi), Grapes (Vitis vinifera), Jamaun (Syzygium cumuni), Gauva (Psidium guajava), Mango (Mangifera indica), Litchi (Litchi chinensis), Dates (Phoenix dactylifera) and Mulberry (Morus nigra). Extracts of all these plants in different solvents i.e. n-hexane, ethyl acetate, chloroform, methanol and water were tested for antibacterial activities against six oral bacteria (Streptococcus mitis, Streptococcus mutans, Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa). Two plants i.e. Jamaun (Syzygium cumuni), Gauva (Psidium guajava)) were selected on the basis of antibacterial activity and preliminary phytochemical analysis. Tablets were formulated form the crude extracts of the selected plants Syzygium cumuni and Psidium guajava and their efficacy was clinically tested. The in-vivo studies revealed the efficacy of dental caries tablets equal to 54%. Bioassay guided purification of the crude extracts of the two plants were carried out using different chromatotographic techniques (flash, column and thin layer chromatography). Out of 60 fractions obtained as a result of different chromatographic techniques, total of 5 fractions having bioactive compounds were further selected from both the plants. The anti-biofilm assay revealed the biofilm inhibition of almost between 90-95%. The FT-IR analysis revealed the presences of nitrile and aromatic groups in the bioactive fractions owing to the anti-caries activity of the extract. Scanning electron microscopic studies of cells after treatment with active fractions of extracts revealed that the extracts affected the integrity of the cell wall to considerable extent. Hence, it can be safely concluded that the inhibition of the dental caries causing bacteria by crude and partially purified extracts of Psidium guajava and Syzygium cumini was due to presences of various active compounds that act in synergy to cause biological effect which might not be recapitulated using single entities. Different combinations of ethyl acetate extracts of these plants will prove helpful in treating dental caries in a more natural and safe way in this world of searching for discovery of safe drug.

Introduction

1. INTRODUCTION

Pakistan has got a very diverse flora which is rich in medicinal plants. Almost 6000 species of flowering plants are there, out of which 400- 600 are of medicinal importance (Nasir et al., 1972; Hamayun, 2005). These plants with therapeutic properties are the main source of traditional medicine system, which is prevailing for thousands of years in nearly all the civilizations from ancient times (Newman et al., 2000). In many developing countries of the world people are still relying on traditional medicines for their health care, even in developed countries people are turning towards herbal remedies due to myriad reasons. Screening of medicinal plants on the basis of their location and their use in folklore medicines indicate their importance in determining the large number of diverse plant derived metabolites of therapeutic importance (Evans, 2002). A plant is considered as a medicinal plant when “it is used to relieve, prevent or cure the diseases or to alter any pathological process or that is used as a source of drug or their precursor” (Arias, 1999). The use of the plant derived drugs in modern medicines for curing and treating different diseases like skin diseases, tuberculosis, diabetes, jaundice, mental illness, cancer and hypertension paved its way from the traditional curing system (Chatterjee et al., 2006).

Caries, wide spread oral mucosal diseases and periodontal diseases are the major oral health problems in developing countries (Saparamadu, 1984) that are prevalent in all age, demographic and socio-economic groups, either male or female irrespective of geographic location in the world. The main factor that can aid in improvement and non- occurrence of these diseases is individual attitude and their knowledge, related to health beliefs. Dental caries is most prevalent in Latin America, South Asia, and the Middle East and least common in China (Petersen, 2008). It is also the most common childhood disease; five-times more common than childhood asthma. It is also the primary pathological cause of tooth loss in children (Hicks et al., 2004a). In dental caries the occurrence increases with age due to denture use and poor hygiene. The presentation of caries varies among people but the risk factors and developmental stages are the same (Guido et al., 2011). According to a survey conducted by the National Health and 1

Introduction

Nutrition Examination Survey (NHANES) in United States (1992-2004), adults between the ages of 20-64, there was a decline in cases to 97% in 1990’s but still the prevalence is high, affecting 92% of class of people and in developing countries it is 96% (Hicks et al., 2004a). Dental caries etiology has four main factors: bacteria, time, susceptible tooth surface and fermentable carbohydrates (Keyes and Jordan, 1964; König, 1971). Along with these factors there are certain behavioral and socio-demographic factors that are likely to increase the risk of caries. These include; poor oral hygiene, age, improper tooth brushing habits, plaque and sugar-containing drinks (Declerck et al., 2008).

Dental caries results from the discrepancy between different risk factors and natural protective factors over time and eventually leads to tooth loss by demineralization or cavitation (Hicks et al., 2004b). The pH of a healthy oral cavity is around 6.2 to 7.0, hence oral problem starts when pH gets lower as it is under acidic environment that demineralization and cavity development starts (Freeth, 1999). The usual recommended procedure in case of severe tooth decay is its extraction for relieving pain (Petersen, 2005), but in other cases the main focus is on use of antibiotics to prevent tooth decay (Joshi and Joshi, 2005; Amadi et al., 2007).There are different antibiotics commercially available to treat oral infections like erythromycin, cephalosporin and penicillin, metronidazole and tetracycline (Philippe et al., 2007), but these chemicals modify the oral flora with undesirable side effects like diarrhea, tooth staining and vomiting (Park et al., 2003) and their use also leads to resistant bacteria (DiazGranados et al., 2008). Chlorhexidine, Cetypridinium chloride, amine fluoride or products containing such agents have higher antibacterial properties but are reported to exhibit toxicity and are even linked to oral cancer (Knoll-Köhler and Stiebel, 2002), which itself is a threatening situation.

Due to the high prevalence of oral disease, increased microbial resistance against antibiotics, the adverse effects of some antibacterial agents used in dentistry and the financial problems in developing countries there is, in fact, a global need to search for alternative prevention and treatment (Tichy and Novak, 1998; Badria and Zidan, 2004). Therefore, the search for alternative products is of paramount importance. Developing 2

Introduction drugs from natural products is gaining intrest because of structural diversity, low production cost and diverse used of different bioavtive compounds to treat different diseases and even in this regards focous is on medicinal plants as they are being used traditionally for generations to treat different infections (Alvin et al., 2014).

.Phytochemicals isolated from plants that are used in traditional medicines are considered safe and effective alternative to synthetic chemicals (Prabu et al., 2006), as the medicinal importance of these plants have been well documented. This situation diverted the efforts towards finding natural products as the potential medicine for treating dental caries that are safe to use. Presence of wide variety of secondary metabolites in medicinal plants having in vitro antimicrobial activities provide a hope for novel drug compounds (Lewis and Ausubel, 2006). For the treatment of different diseases like constipation, asthama, fever, eimeriosis, cancer (Cousins & Huffman 2002; Saganuwan 2010; Dkhil et al., 2014). Because of increase side effects and toxicity of antibiotics, herbal medicines are getting popular (Agarwal et al., 2005 and Bonifacio et al., 2014). Plants are the complex storehouse of undiscovered bioactive compounds with prospective to be used in medicines (Plotkin, 1988). Medicinal plants contain diverse substances that can be used to treat different infections and diseases, like isolation of Cocaine, Reserpine from Rauwolfia serpentine (Patwardhan et al., 2004). Octadecanoic acid methyl ester and hexadecanoic acid methyl ester isolated from root extract of Jatropha curcas Linn. Has strong anti-inflammatory activity (Othman et al., 2015). Verrucarin A, isolated from the Myrothecium roridum inhibits interleukin-8 production from human promyelocytic leukemia cells, Leptosins, produced from different strains of Leptosphaeria sp. originally isolated from alga Sargassum tortile, have in vivo cytotoxic activity(Hasan et al., 2015). Cannabinoid, the active compound from Cannabis sativa has strong anti-cancer activity (Pisanti et al., 2009). Some plants like Acacia nilotica , Psidium guajava , Terminalia chebula Retz have strong bacterial urease, which play an important role in urinary tract infections inhibitory effect (Bai et al., 2014).

In the present investigation, Diospyros blancoi, Vitis vinifera, Syzygium cumuni, Psidium guajava, Mangifera indica, Litchi chinensis, Phoenix dactylifera, Ocimum basilicum, and 3

Introduction

Morus nigra, were tested for their activity against dental carries causing bacterial strains. These plants are common and used as traditional remedies against different diseases. Plants descriptions and their use are listed in Table 1.1.

Introduction

Table 1.1: Plants of medicinal importance used in the present study

Common Botanical Occurrence Family Part References plant name name used Jamun, Jambul Syzygium Found throughout Asia,India Myanmar Myrtaceae Leaves (Kirtikar and Basu, 1975; cumuni and east indes Bailey et al., 1976) Guava Psidium Found in South America, Africa Myrtaceae Leaves (Stone, 1971; Rios et al., guajava European and Asia, native to Mexico, 1977) widely distributed to tropical and subtropical areas of the world. Mulberry Morus nigra Native to south-western Asia, Asia, South Moraceae Leaves (Mabberley, 1997; Özgen America, Africa, Europe et al., 2009) Dates Phoenix Native to North Africa, Persian Gulf. Iraq, Arecaceae Leaves (Lim, 2012) dactylifera UAE, Oman, Libya,Pakistan, Egypt, Saudi Arab, Sudan, Europe and USA are the top producers of dates

Mango Mangifera Large, evergreen native from tropical Anacardiceae Leaves (Calabrese, 1993; Ross, indica Asia, but introduced wherever the climate 2007) is warm, therefore now found in tropics and subtropics Grapes Vitis vinifera Found in Europe, Asia, North America Vitaceae Leaves (Rossetto et al., 2002; under continental–temperate climatic Crespan, 2004; Sefc et conditions, subtropical, Mediterranean al., 2009) Gaff, Mabolo Diospyros Small to large evergreen tree,Found in Ebenaceae Leaves (Ghani, 1998; Stuart, blancoi Bangladesh, Pakistan, India and endemic 2010) to Philippine Leechi, litchi, Litchi Sub-tropical species from South China, Sapindaceae Leaves (Gontier et al., 2000; laichi, lichu, chinensis Vietnam, Indonesia and Philippines Bhat and Al-daihan, lizhi) 2014; Davidson, 2014)

Introduction

1.1. Aims and Objective

The main aim of the study was to identify and develop possible herbal remedy for oral diseases specially for treating dental caries. To fulfill the aim following objectives were laid down

1. Collection of plants from different areas of Pakistan 2. Evaluation of antibacterial activity of crude extracts and fractions of these plants 3. Evaluation of the phytochemical components of crude extracts of selected plants using standard biochemical tests. 4. To develop a crude herbal tablet to treat dental caries, and its efficacy testing. 5. Isolation of bioactive fractions using different chromatographic techniques and their structural characterization using MS, NMR and FT-IR analysis 6. In-vitro assays of antibacterial effect of selected bioactive fractions against different oral pathogens 7. Anti-biofilm ability of the final selected fractions against the S. mutans 8. Morphological studies of different microbial strains after reatment with the selected plant’s fractions.

Review of Literature

2. REVIEW OF LITERATURE 2.1. Medicinal Plants and Their Importance

People have been exploring nature in general and plants in particular since ancient times in search of natural medicines that has resulted in the use of medicinal plants to treat various diseases (Verpoorte, 1999). The therapeutic use of plants dates back to Akkadian and Sumerian civilizations in third millennium BC, Hippocrates (ca. 460-377BC) identified approximately 400 different plant species that can be used for medicinal purpose (Sarker and Nahar, 2007). Eleven percent of 255 drugs that are considered as basic by the World Health Organization (WHO) are plant based and in addition to this many of the synthetic drugs are also derivatives from natural precursors (Wong et al., 2009). The importance of these traditional medicines (that are derived from plant extracts) can also be established from the fact that almost 80% of the world’s population relies on them (Sandhya et al., 2006) and the famous traditional systems of Ayurveda, Homeopathy, Unani and Siddha accounts for 95% of these medicinal herbs (Satyavati et al., 1976). Additionally homeopathetic based “Material Medica”, established by the Romans, Egyptians, Greeks, Chinese, Babylonians and the people of India and Pakistan, and “Greek medicines”, initially accustomed by Romans and Arabs was (Ackerknecht, 1973) further enriched with the Indian and Chinese medicines that latter on was adopted by Europe are all herbal based majorly. Further, Natural products present a great contribution of drugs that are introduced in market in last 25 years (Newman et al., 2003; Newman and Cragg, 2007) resulting in increased availability (i.e. approximately 80%) of products that are of plant origin with sales exceeding US $ 65 billion in 2003 (Patwardhan et al., 2004). Hence, it can be safely concluded that plants with medicinal importance have marked the era of drug discovery and had played important role owing to their small dimensions, unique structural diversity and ability to get absorbed and metabolized (Sarker and Nahar, 2007).

Plants synthesize different useful substances majority of which are secondary metabolites and almost 12,000 of them have been isolated (Lai and Roy, 2004). Natural products 7

Review of Literature owing to their diverse structures, synthesizable analogues (Paterson and Anderson, 2005) and frequent usage (Chin et al., 2006) become an important source of medicine. The importance of natural products is further enhanced by considering their long synthetic routes, high process cost and with low yield overall making their synthesis tiresome and less feasible. (Paterson and Anderson, 2005). Further, natural products are biological friendly than their synthetic counter-parts as they are synthesized in the living system (Koehn and Carter, 2005; Sundrarajan and Gowri, 2011) making them ideal candidates for drug development for treating various diseases (Balunas and Kinghorn, 2005; Drahl et al., 2005; Ganapaty et al., 2006; Schmidt et al., 2007; Monzote et al., 2014; Rosales- Mendoza et al., 2014).

Traditional plants of medicinal values are used to treat different diseases like asthma, esophageal cancer, constipation, hypertension, fever, rabies and eimeriosis (Cousins and Huffman, 2002; Saganuwan, 2010; Dkhil et al., 2014), and for that purpose they are applied as infusions or tinctures, or as component mixture in soups and porridges that can be used orally, topically in the form of lotions, tonics, as poultices etc. Different parts of the plants are used in treating multifarious infections like urinary tract, respiratory system, gastrointestinal and skin (Rios et al., 1977; Gyawali, 2014; Kedia et al., 2014). At present the increase in multiple drug resistant pathogenic bacteria like MDR Klebsiela pneumonia, methicillin resistant Helicobacter pylori, Staphylococcus aureus have further revitalized the interest in medicinal plants that have antimicrobial properties (Voravuthikunchai et al., 2005). Flavonoids, phenolic compounds like protosappanin E, neosappanone A, caesalpin P, 7,3′,4′-trihydroxy-3-benzyl-2H-chromene, lipids and triterpenoids from different parts of C. sappan have found to have strong antibacterial potential and they have also strong tendency to develop into an antibiotic(Protosappanin, 1986; NAMIKOSHI and SAITOH, 1987)

The highly varied methodology that involves the drug discovery includes the observation, report and experimental method of native drugs and evaluation of their biological activities is done under ethanopharmacology (Fabricant and Farnsworth, 2001; Chau et al., 2014). The formation of complementary and alternative medicine research centers in 8

Review of Literature different countries i.e., Germany and USA predicted noble future prospects for these therapies in the world (Khan et al., 2004). Herbal medicines are getting popular due to increase side effects and toxicity of different allopathic medicines. (Agarwal et al., 2005; Bonifácio et al., 2014).

Plants have evolved multiple defense systems for their survival because of plethora of rivals in order to fight the environmental stresses (Ballhorn et al., 2009). Plants are in fact the complex storehouse of undiscovered bioactive compounds with great potential to be used in medicines (Plotkin, 1988). Basically the chemicals produce by plants are divided into two categories, the primary and the secondary metabolites (Agosta, 1996). The primary metabolites are involved in the synthesis of the basic building blocks of the plant, while the secondary metabolites are involved in the defense mechanism of the plant against different microbial infections (Cowan, 1999). From medicinal perspective the important secondary metabolites include flavonoids, alkaloids, terpenes, tannins and phenolic compounds (Kazmi et al., 1994; Omulokoli et al., 1997; Cosentino et al., 1999; Edeoga et al., 2005). Hence, unlimited opportunities for drug discovery have been provided by natural products like plant extracts, whether pure compounds or standardized extracts because of their chemical diversity (Cos et al., 2006) and all the plants that are used in traditional medicines contain diverse substances that can be used to treat different infectious and chronic diseases (Duraipandiyan et al., 2006; Aziz et al., 2014). It was with the isolation of morphine from Papaver somniferum by Serturner followed by isolation of quinine from Chinchona tree (1860) that chemistry of natural product actually began. Later, Koler isolated Cocaine that is used for treating paralysis caused by those nerve endings that are involved in pain transmission. Similarly, Reserpine which was an antihypertensive alkaloid was isolated from Rauwolfia serpentine (Patwardhan et al., 2004).

2.2. Oral Health and Global Concerns

In spite of all the improvement that is being made in oral health, problems still persists on the global scale (Pearson et al., 2001). Oral diseases like tooth loss, orpharyngeal cancers,

Review of Literature dental caries, oral mucosal, periodontal diseases, and HIV/AIDS-related oral diseases are the main public health problems globally (Petersen, 2005; Javed et al., 2014). Out of total 291 diseases and injuries evaluated in global burden of disease, untreated tooth decay has highest rate of prevalence between 70-90% of populations (Marcenes et al., 2013) and it is also one of the most common reason for tooth extraction. In many developed countries the treatment of oral diseases is very expensive. About 5-10% of public health funds are related to oral health and in developing countries this expenditure is very low and that only cover the pain relief and emergency care and these conditions also get worse by poor living conditions, poverty and failure of government in providing the sufficient health care (Pack, 1998). Many epidemiological studies confirmed the poor health conditions of adults in developing countries as evidenced by (Ronderos et al., 2001) in people of Amazon rainforest who were suffering from oral diseases. Similarly, there is evidence of age dependent caries in Chinese (Luan et al., 1989) and Thailand people with high caries prevalence (91%) for ages between 30-39 years (Baelum et al., 2002). High caries experience (72.4%) was also reported with little proof of previous restorative treatment (Shah et al., 2009) and high prevalence (87%) of periodontal problems occurs in Vietnamese villagers (Uetani et al., 2006). In 1984 there was a ratio of one dentist to 1905 people (Miyazaki et al., 1989; Miyazaki et al., 1995). Studies on different ethnic groups residing in UK showed a mean Decayed Missing Filled Teeth (DMFT) of 9.9 and 2.8 in Indians and Bangladeshis (Pearson et al., 2001). Despite all the advancement, caries is still a problematic case in adults (Bernabé et al., 2014). DMFT studies were carried out in line with previous studies (Becker et al., 2007), where the alphabets D, M, F and T stands for untreated caries, teeth missing due to carries, filling and index per tooth, respectively. According to this study only clinical examinations are carried out to assess oral health and no advance techniques like dental radiographs are permissible considering the fact that many nations might not be equipped with these facilities. WHO latter reported that radiographic assessment to be an important contributor in assessing oral health as its absence can likely cause estimation of data.

2.3. Oral Health of People of Pakistan

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In Pakistan, the health care system comprises of both public and private sector which, include clinics, hakeems, herbalists, quacks, traditional healers and homeopaths. Public sector contributes only 23% on health, while 77% comes from private sector and even from that, only 30% of the population utilizes the Primary Health Care (PHC) facility of public sector because of many factors (Shaikh et al., 2010) with 3-4% of GNP being spend on health which was only 1-2 % more than what was there in 2008 (Hameed, 2008). Lack of public policies, cultural restriction, low human development of country, limited resources and health priorities are some of the factors responsible for poor oral health of people (Harchandani, 2012). People of Pakistan are overburdened with periodontal diseases and dental caries and the problem is further enhanced due to inappropriate oral health care systems (Khan et al., 2004). Almost 90% of the oral diseases remain untreated as health care services are insufficient in the country and are available only to 55-85% population resulting in non-availability and inadequate medical facilities to the rural population, women, children and the poor (Bille and Aslam, 2003; Nishtar et al., 2013). The situation gets worsen considering the focused nature of the treatment available which is capable of coping with the widespread nature of diseases prevailing in the country (Bille and Aslam, 2003). To make the conditions more worse, people suffering from various diseases are more prone to dental diseases, like poor oral health is associated with coronary heart diseases (CHD), (Javed et al., 2007; Bokhari et al., 2014). The conditions are worst among the elder people (Khan et al., 2004; Pawelec et al., 2014).

2.4. Dental Caries

Archaeological studies showed that tooth decay dates back into prehistory (Suddick and Harris, 1989). From ancient times, people had doubted some possible links between dental caries and some form of living organism called as tooth worm in Sumerian text around 500 BC, but it took another 6000 years to reveal the true identity of tooth worms. Miller in 1890, in his book “Microorganisms of the Human Mouth”, suggested a chemo-parasitic theory regarding dental caries (Miller, 1890) which suggests the correlation between tooth decay and consumption of fermentable carbohydrates which 11

Review of Literature probably is converted to acids by the oral microorganisms and ultimately resulting in demineralization of teeth. This theory along with gelatinous microbic plaques, known as dental plaque by Williams and Black (Black, 1898; Williams, 1898) provided the basis of our today’s concept of etiology of dental caries. Smooth surface caries, enamel caries, secondary caries, root caries, pit and fissure caries and child hood caries are some of the caries categories that are usually considered by researchers and clinicians. Oral diseases, a major health issue in the world (Petersen, 2005) is economically affecting people of developed countries as 10% of the health expenditure is related to dental care (Petersen, 2005). Even though there is an improvement in oral health in most of the developed countries of the world but still dentally disadvantaged people exist in these countries, usually people with low socio-economic status (Jamieson et al., 2007). Streptococcus mutans and Streptococcus mitis are the most important agents of human dental caries. First bacteria to colonize human mouth after birth are Viridans streptococci and among them, the principal colonizing strains are Streptococcus oralis, Streptococcus mitis and Streptococcus mitis which is also an important pathogen in bacterial endocarditis (Linder et al., 1983; Duval and Leport, 2008).

It was in 1924 when Clarke did the microbiological studies on human dental plaque and first time observed Streptococci (Clarke, 1924) and in 1960 Keyes first time discovered the infectious transmittable nature of dental caries while working on gnotobiotic rodents and also discovered that certain Streptococci bacteria (later identified as Streptococci mutans) are the main causative organism of dental caries (Fitzgerald, 1960). This and many other studies confirmed the infectious nature of dental caries (Beck and Drake, 1975). The oral cavity of human is considered as a complex ecosystem which has both acid producing and acid tolerant bacteria. Almost 700 different bacterial species have been known for human oral cavity (Paster et al., 2001; Aas et al., 2005; Paster et al., 2006), and nearly 200-300 species have been identified for dental plaque (Islam et al., 2007) using different culture dependent and independent techniques. Dental caries the other name of tooth decay, is the most prevailing disease in human population, five times greater than asthma (Van Houte, 1994) and is considered as irreversible bacterial infection of teeth (Shivakumar et al., 2009). There is irreversible proteolytic destruction 12

Review of Literature of collagen matrix by bacterial population, which causes the demineralization of dentine. The interaction between bacteria and its surrounding epithelium are acute elements in bacterial infections (Finlay and Cossart, 1997; Finlay and Falkow, 1997; Meyer et al., 1997) and if left untreated will result in pain, infection and tooth loss depending on the severity.

Dental plaque, a sticky substance that sticks to the surface of the teeth, is considered as complex biofilm that is also the main cause of development of dental caries (Benson et al., 2004). In fact the development of dental plaque depends on the result of interaction between the plaque adhesion to the tooth surface and the physical shear forces involved in dislodging and removal of plaque (Roberts, 2005) and if it is not removed properly and routinely, tooth decay will flourish (Hardie, 1992). The mature dental plaque is embedded in a matrix of bacteria and host polymer that includes proteins, DNA secreted by cells and polysaccharides (Baelum et al., 2002; Fejerskov et al., 2003; Featherstone, 2004, 2008) and this provide the bacteria with protection against host defenses and predators, from desiccation and enhanced resistance against antimicrobial compounds (Scheie and Petersen, 2004). Steptococcus mutans, S. mitis, S. constellatus, S. sanguis, S. mitis, S. salivarius, S. anginosus, S. gordonii, S. intermedius and S. oralis are some of the primary acid tolerant bacteria that are associated with dental plaque (Dye et al., 2007). Accumulation of plaque in gingival and sub-gingival regions led to shifting of the micro- flora from gram positive to gram negative which can cause the periodontal diseases (Iwaki et al., 2006). Dental caries and specifically the persistent dental caries is linked with the high blood pressure, diabetes, heart diseases and sometimes multiple sclerosis along with continuous pain that gets aggravated by cold, heat, sugar and drinks (Wright and Hart, 2002; Taylor et al., 2004). There are different bacteria that have been involved in different systemic diseases like aspiration pneumonia (Scannapieco, 1999), osteomyelitis in children (Dodman et al., 2000), cardiovascular disease (Beck et al., 1996; Wu et al., 2000), bacterial endocarditis (Berbari et al., 1997) and preterm low birth weight (Offenbacher et al., 1998; Buduneli et al., 2005), but surprisingly no efficient strategies have been developed to fight oral diseases. Some of the reasons that oral pathogens are not been eradicated are linked to difficulty of studying microbial 13

Review of Literature inhabitants of oral cavity. The complexity of oral ecosystem makes the pathogenical species difficult to target (Socransky et al., 1998). According to Koch’s postulates not a single ethiological organism can be identified owing to the formation of red complex with the periodontal pathogen that is involved in the illness (Darveau, 2010), and large number of oral bacteria cannot be cultured (Paster et al., 2006).

Usually, in a healthy mouth the pH is around 6.2 to 7 and when it is disturbed and reaches around 5 and 5.5 (Freeth, 1999; Barad et al., 2014) the problem with mouth arises as in this acidic environment the tooth’s demineralization starts resulting in mineral loses which eventually leads to breakdown of tooth (cavity).

Streptococcus mutans is considered as main etiologic organism of human dental caries and certain factors like ability to form biofilms, tolerate frequent and rapid environmental fluctuations and metabolizing carbohydrates are considered as responsible for the virulence of this bacterium (Banas and Vickerman, 2003; Lemos and Burne, 2008). In addition to that, the mutans is also associated with bacterial endocarditis, the inflammation of heart valves. There is a natural way to repair dental caries by remineralization, usually minerals from saliva that diffuse back in caries lesion (Featherstone, 2004). Synthesis of the extracellular polysaccharides by S. mutans from sucrose through glucosyltransferases (GTFs) is considered another important virulence factor that causes caries in humans (Loesche, 1986) which not only facilitate the adhesion and accumulation of the organism on the tooth surface but also provide protection against host immune defences along with provision of increased resistance against antibiotics and gene expression (Watnick and Kolter, 2000). This combination of virulence properties allow the mutans to colonize the surface of tooth and modify the nonpathogenic to highly cariogenic dental biofilm that ultimately leads to caries formation (Jeon et al., 2011).

2.5. From Synthetic to Herbal Products

Treatment of infectious diseases, the second primary cause of death in the world is problematic today because of severe side effects of different antimicrobials and the

Review of Literature growing resistance against all the lifesaving drugs due to their continuous use (Sydney et al., 1980; Mehrgan et al., 2010). The issue becomes much worst with almost 70% of bacteria that cause common infections in hospitals develop resistance to atleast one of the common antibiotic that is used to treat them (Nascimento et al., 2000; Bisht et al., 2009). Even the antibacterial agents can enhance the development of resistant bacterial strains (Tenover, 2006). Different antibiotics like erythromycin and penicillin are effective against dental caries in humans and animals but because of their adverse effects they are not recommended clinically (Kubo et al., 1992). Chlorohexidine, penicillin, cephalothin, ampicillin, methicillin and digluconate are some of the other antibiotics that are used to treat dental caries (Iwaki et al., 2006) and recently, development of resistant have also been observed in cariogenic bacteria against these antibiotics. The development of resistant strains and the associated side effects of these medicines have resulted in diversion of research towards screening of natural products (plants) for anti-caries activity as some plants have shown potential against dental caries causing pathogens (Tsai et al., 2008; Tellez et al., 2009). Today, oral care products that are combined with medicinal plant extracts are gaining high interests due to their low toxicity (Knoll-Köhler and Stiebel, 2002; Neumegen et al., 2005) as compared to oral care products that contain different antimicrobial agents like cetylpyridinium chloride, amine fluorides, triclosan and chlorhexidine that are not only toxic but also cause staining of teeth.

The commercially available chemicals even alter the oral microbiota along with certain undesirable side effects (Park et al., 2003; Tenover, 2006) and bacterial resistance if not all, to most of the antibiotics commonly available to treat oral infections (Palombo, 2011). An alteration in the microenvironment like wounds, malnutrition, abrasions and different pathological conditions enhances the disease development (Madenspacher et al., 2013). Also, the presence of ethanol that is commonly found in mouth washes have been linked to oral cancer (Knoll-Köhler and Stiebel, 2002; Neumegen et al., 2005; Lachenmeier and Sohnius, 2008; McCullough and Farah, 2008). Therefore, the search for alternative methods and products continues and for that the phytochemicals isolated from plants that are used in traditional medicines are proving to be the good alternative to synthetic chemicals (Prabu et al., 2006). As a result of indiscreet use of allopathic drugs 15

Review of Literature and improper diagnosis of microbial infections not only lead to untargeted therapy but it also gives way to resistant pathogens (Kumarasamy et al., 2010; Ewam, 2014).

The use of natural remedies from medicinal plants are proving to be good as an alternative for the adverse effects of antibiotics like supra infections, hyper sensitivity and teeth staining and because of this and many other drug resistant factors a search for new antibiotics continues persistently. In fact the failure of different chemotherapeutics and increasing resistant against antibiotics also lead to the screening of different medicinal plants for their potential use against these microbial pathogens (Vaghasiya and Chanda, 2009). The significant contribution of medicinal plants to the drug industry all over the world was due to the increasing number of phytochemical and biological studies. In developing countries the herbal medicines are proving to be an important source of products in order to treat different infectious diseases and also to overcome the problems related with the available antimicrobial agents. Herbal remedies are getting popularity as they provide safe alternative for treating various type of cancers (Lampronti et al., 2003; Yadav and Agarwala, 2011; Yarney et al., 2013; Tiwari et al., 2014). These remedied are gaining intrest because of their multi-dimentional health benefits like they are even used in different alternative treatment like acupuncture, massage therapy and various traditional practitioners. It is very well recognized that different secondary metabolites produced by plants like terpenoids, flavonoids, alkaloids, tannins are providing a new source of antimicrobial substances which is helping us in combating the new resistant pathogens. The medicinal uses of the plants range from administration of leaves, stem, barks, seeds and roots to using of decoction from different plants (Ogbulie et al., 2007). There has been an increase in the demand of different herbal products in the last few decades, even in countries like United States herbal remedies are in use in the form of different dietary supplements (Briskin, 2000; Rahal et al., 2014; Tiwari et al., 2014). Many herbal remedies have stood the test of time for treating various bacterial infections because of their efficacy and potency with some having the scientific evidence for their usefulness as well.

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2.6. The Ethnobotanical Importance, Bioassay and Phytochemical Review of Selected Plants of the Present Study

In Pakistan a variety of different plants have been used to treat different infectious diseases and they are also used to treat/prevent different oral diseases. Inhabitants of the areas also have the indigenous knowledge about the medicinal uses of the plants which they inherit from their ancestors (Bhardwaj and Gakhar, 2005). The details of occurance and the parts used of selected plants in present study are given in Table 1.1.

2.6.1. Syzygium cumuni ethanobotanical importance

Kingdom: Plantae Phylum: Angiosperm Class: Magnoliopida Order: Myrtales Family: Myrtaceae Genus: Syzygium Species: S. cumini

Syzygium cumini is a slow growing species which can reach the height of upto 30 m and can live for more then 100 years. The leaves turn to a leathery dark green on maturation with a yellow midrib. The fruit, leaves, bark and seeds of this plant are used traditionally as a remedy for diabetes in many areas of the world (Teixeira et al., 1997; Tripathi and Kohli, 2014). (Shinde et al., 2008) reported the anti-diabetic effect due to inhibition of α- glucosidase. Leaves are also used to make teeth and gum strong, for fever, dremopathy and to treat leucorrhoea (Warrier et al.). It is also used to treat constipation, diabetes and inhibit blood discharge in faeces (Bhandary et al., 1995; Rastogi et al., 2001; De Bona et al., 2014). The leaves have antimicrobial activity (Chandrasekaran and Venkatesalu, 2004; Oliveira et al., 2007). Antioxidant activity is due to phenolic compounds present in the leaves (Ravi et al., 2004; Bajpai et al., 2005; Md et al., 2009; Gowri and Vasantha, 2010; Mohamed et al., 2013). Seed extract is used to lower the blood pressure (Morton, 1987), it is also used to treat skin wounds (Oliveira et al., 2007). Barks and seeds have anti-inflammatory and anti- diarrheal effect (Indira and Mohan, 1993). It is also used to treat fever, dermopathy gastropathy (Warrier et al.). 17

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2.6.1.1. Bioassay and Phytochemical review of Syzygium cumuni

Gallic acid, tannins, vitamin C and anthocyanins that includes malvidinglucoside, petunidin, cyaniding and different other compounds were reported in this plant (Martinez and Del Valle, 1981; Chadha, 1985). Its oil consists of α and β pinene, 1, 3, 6-octatriene, trans-caryophyllene, α caryophyllene, delta -3-carene and alimonene (Mohamed et al., 2013). Leaves are rich source of acylated flavonol glycosides (Mahmoud et al., 2001), myricetin, quercetin, myricetin 3-O-4-acetyl-L-rhamnopyranoside (Timbola et al., 2002), esterase, galloyl carboxylase (Bhatia et al., 1974) and triterpenoids (Gupta and Sharma, 1974). Triterpenes/steroids, glycosides, carbohydrates, alkaloids, flavonoids, saponins, tannins and amino acids has been reported from this plant (Tripathi and Kohli, 2014).

2.6.2. Psidium guajava ethanobotanical importance

Kingdom: Palntae Phylum: Angiosperm Class: Magnoliopida Order: Myrtales Family: Myrtaceae Genus: Psidium Species: P. guajava

Psidium guajava is an evergreen shrub native to tropical America and occurs up to 1600 m above sea level. The leaves are thick and leathery. Psidium guajava is commonly used to treat the respiratory and gastrointestinal diorders and also act as anti-inflammatory medicine (Gutiérrez et al., 2008). The polyphenols in ethyl acetate extract have antioxidant property (Hsieh et al., 2005; Hsieh et al., 2007a; Rai et al., 2010) and they also have anticancer, anti-allergic and anti-inflammatory activity (Ojewole, 2006; Hsieh et al., 2007b). The leaves are used for different purposes in different parts of the world. In US they are used as antibiotic and in diarrhea (Smith et al., 1992; Lutterodt, 1994), In Mexico for diarrhea, rheumatic pain, tooth ache and ulcers (Heinrich et al., 1998), for cough (Heinrich et al., 1998; Leonti et al., 2001), for caries, fever, wound, vaginal

Review of Literature haemorrhage (Linares et al., 1988; Arqueta-Villamar et al., 1994; Heinrich et al., 1998), in diabetes mellitus (Mejia and Reng, 1995; Teixeira et al., 2003; Oh et al., 2005; Mukhtar et al., 2006; Huang et al., 2011). Leaf extract also showed anti-allergic activity (Seo et al., 2005), leaf oil has highest anti-proliferative activity on P388 cell lines, as anticancer agent (Lee and Park, 2010; Ahn et al., 2014), as antidiabetic and as antibacterial (Sato et al., 2000; Begum et al., 2004; Mukhtar et al., 2006; Nair and Chanda, 2007). It is also used as hepato-protective agent (Moura et al., 2012), anti- plaque. The leaves have anti-adherence effect on the early colonizers of oral bacteria (Razak and Rahim, 2003; Razak et al., 2006).

2.6.2.1. Bioassay and Phytochemical review of Psidium guajava

Phytochemistry of leaves reveal the presences of essential oil that contains α and β pinene, menthol, isopropyl alcohol, caryophyllene oxide, terpenyl acetate and cardinene and curcumene (Li et al., 1999; bin Zakaria and Mohd, 2010). Saponins and flavonoids with oleanolic acids have been isolated from leaves (Arima and Danno, 2002). P. guajava contain broad spectrum phytochemicals which include enzymes, proteins and minerals (Deo and Satri, 2003), alkaloids, steroids, tannins, glycosides, flavonoids and saponins (Narayana et al., 2001; Perez-Perez et al., 2014), also lutein, lycopene and zeaxanthine (Tee et al., 1997; Hobert and Tietze, 2001), triterpenoid and sesquiterpenoid, and one new pentacyclic triterpenoid psidiumoic acid was also reported in Psidium guajava (Begum et al., 2004; Begum et al., 2007). It also contains polyphenols and carotenoids (Seshadri and Vasishta, 1965). The flavonoids present in leaf and bark of the plant like quercetin, morin glycosides and quercetin glycosides have antimicrobial activity (Arima and Danno, 2002; Qa'dan et al., 2005; Chah et al., 2006). Shu (Shu et al., 2010) reported two new compounds i.e., ellagicacid-4-O-beta-D- glucopyranoside and quercetin-3-O (6"-galloyl) beta-D-galactopyranoside. α- humulene, valerenol and germacrene D was reported in stem oil (Khadhri et al., 2014), veridiflorol and trans-caryophylene was present in leaf oil.

2.6.3. Morus nigra ethanobotanical importance

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Kingdom: Plantae Phylum: Angiosperm Class: Rosids Order: Rosales Family: Moraceae Tribe: Moreae Genus: Morus Species: M. nigra

Morus nigra is deciduous tree that grows to 39 ft tall and 49 ft broad. The leaves are usually long on vigorous shoots with upper rough surface and stiff hairs. Leaves are used to increase the milk yield in dairy animals (Srivastava et al., 2003). Fruit is used to prepare natural dye, medicine (Imran et al., 2010). It has antibacterial, antifungal, antiviral, antinematodal, antioxidant, anti-cancer, cytotoxic, anti-inflammatory, and immune-regulating activities (Kim et al., 1999; Chang et al., 2002; Hansawasdi and Kawabata, 2006; Arabshahi-Delouee and Urooj, 2007; Song et al., 2007; Zhang et al., 2009; Qadir et al., 2014). This plant also has anti-parasitic and anthelmintic activity (Badar et al., 2011; Babar et al., 2012), anti-diarrheal properties are reported by Jung (Jung et al., 2011). The leaves are used to reduce cholesterol and blood pressure (Zhang et al., 2009). It is useful in treating diabetes, arthritis and rheumatis (Wang et al., 2009). It also possess hepatoprotective ability (Mallhi et al., 2014).

2.6.3.1. Bioassay and Phytochemical review of Morus nigra

Coumarins, flavonoids, phenols are reported in leaves of Morus nigra (Zhang et al., 2009). It also consist of sterols, isoprenoid-substituted phenolic compounds, prenylated flavonoids, stillbene derivatives and arylbenzofurans (Lin and Tang, 2007; Ercisli and Orhan, 2008; Pawlowska et al., 2008; Imran et al., 2010). Broad range of anthocyanins was also reported in this species (Veberic et al., 2015 ). Betulinic acid, β-sitosterol and germanicol are also reported that are responsible for anti-inflammatory activity of the plant (Padilha et al., 2010). Flavonoids have hepato-protective activity (Ali et al., 2013). Alkaloids, phenols and flavonoids are also reported in this plant (Özgen et al., 2009; Malik et al., 2012).The polyphenolics act as radical scavengers and reducing agents 20

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(Natić et al., 2015). Chromatographic root extract separation confirms the presences of 2- arylbenzofurans moracin C, mulberrofuran H, mulberrofuran Y and the prenylated flavonoids and the Diels–Alder adducts with cytotoxic activity (Zelová et al., 2014).

2.6.4. Phoenix dactylifera ethanobotanical importance

Kingdom: Plantae Phylum: Angiosperm Class: Commelinids Order: Arecales Family: Arecaceae Genus: Phoenix Species: P. dactylifera

Phoenix dactylifera usually grows up to 70-75 ft, growing in clumps or singly. The leaves have spines on petiole and pinnate. Research confirms the presences of antidiarheal (Al- Taher, 2008; Baliga et al., 2011), anti inflammatory and anti proliferative (Elberry et al., 2011) activity in this plant. Antibacterial (Bawazir and Saddiq, 2010; Perveen et al., 2012; Sooad and Ramesa, 2012), antimicrobial, anti-mutagenic and antioxidant activity is also reported (Vayalil, 2002; Mansouri et al., 2005; Mohamed and AL-okbi, 2005; Bawazir and Saddiq, 2010; Jassim and Naji, 2010; Bokhari and Perveen, 2012). It is beneficial to manage brain ischemia (Kalantaripour et al., 2012). Antifungal (Abd-El- Khair and Haggag, 2007; Joseph et al., 2008; Yasmin et al., 2008; Mdee et al., 2009; Abou-Bakr, 2011; Boulenouar et al., 2011; Yanar et al., 2011; Bokhari and Perveen, 2012), antiviral (Jassim and Naji, 2010), anti–cancer and antioxidant (Bawazir and Saddiq, 2010) Zineb et al., 2012) activities are also reported in this plant. It is used to treat liver injury (Al-Qarawi et al., 2003). Phoenix dactylifera pollens are used to treat male infertility problems and to increase testosterone and estrogen level (Miller et al., 2003; Bahmanpour et al., 2006; Bastway Ahmed et al., 2010; Bawazir and Saddiq, 2010). It also possesses antidiabetic activity (Mard et al., 2010; Zangiabadi et al., 2011; Fatima et al., 2012; Patel et al., 2012). It also possesses anti-inflammatory activity (Eddine, 2013; Zhang et al., 2013).

2.6.4.1. Bioassay and Phytochemical review of Phoenix dactylifera

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Cinnamic acids, flavonoid glycosides, flavanols are reported phenolic compounds (Vembu et al., 2012) in this plant. Steroids like, α-sitosterol, campesterol cholesterol, stigmasterol, Anthocyanins (Vembu et al., 2012). Fatty acids like oleic acid, linolenic acid, palmitolieic and linoleic acid is also reported in dates (Abdu, 2011). (El-Gazzar and

El-Far, 2009) reported CCl4 induced hepatotoxicity by Phoenix dactylifera. Column chromatography revelead β- Sitosterol acetate, clionasterol acetate, β-Sitosterol caproate, cerotic acid, lignoceric acid, behenic acid, in n-hexane fraction and, luteolin-7-O- glucoside, Isorhamnetin-3-O-glucoside, naringin, apigenin and rutin in ethyl acetate fractions of date (Abbas and Ateya, 2011).

2.6.5. Mangifera indica ethanobotanical importance

Kingdom: Plantae Phylum: Angiosperm Class: Magnoliopsida Order: Sapindales Family: Anacardiaceae Genus: Mangifera Species: M. indica

Mangifera indica is a tall tree that grows up to 115-131ft with some species giving fruit even after 300 years. The leaves are evergreen, alternate and simple. They are orange pink when young which change to dark red and green as they mature. Used to treat scalds, burns, throat infection (Chadha, 1985), antidiabetic (Muruganandan et al., 2005), antioxidant, antibacterial (Srinivasan et al., 1982; Stoilova et al., 2005; Awa-Imaga, 2011; Badmus et al., 2011; Mustapha et al., 2014), antimicrobial (Enikuomehin, 1998), antiviral (Zhu et al., 1993). It is also used as anti -tumor (Yoshimi et al., 2001), as anti- allergic (Rivera et al., 2006), antidiabetic (Núñez-Sellés, 2005) dental caries (Muanza et al., 1994) as anti-inflammatory and neuroprotective agent (Lemus-Molina et al., 2009).

Review of Literature

Amrita (Bhowmik et al., 2009) reported a reduced glucose absorption in ethanol extracts of stem and bark. It also possesses anti-trypanosomal potentials (Ibrahim et al., 2014).

2.6.5.1. Bioassay and Phytochemical review of Mangifera indica

Chemical constituents of the different parts of M. indica L. are reviewed by Ross (Ross, 2007) and Scartezzini and Speroni (Scartezzini and Speroni, 2000). Mangiferin, protocatechic acid, γ-aminobutyric acid, shikimic acid, catechin, alanine, glycine, kinic acid and the tetracyclic triterpenoids cycloart-24-en-3β,26-diol, 3-ketodammar-24 (E)-en- 20S,26-diol, C-24 epimers of cycloart-25 en 3β,24, 27-triol and cycloartan-3β,24,27-triol was reported in bark of the mango (Scartezzini and Speroni, 2000). It possess monogalloyl glucosides (MGG) which are identified as ester-linked glucose (Krenek et al., 2014).

2.6.6. Vitis vinifera ethanobotanical importance

Kingdom: Plantae Phylum: Angiospermae Class: Magnoliopsida Order: Rhamnales Family: Vitaceae Genus: Vitis Species: V.vinifera

Fruit is an important source of nutrients (vitamins: A, C, B1, B2, B6) (Amanatidou et al., 1999) Jayaprakasha et al., 2003), Vitamin E (Anastasiadi et al., 2008). Grape seed extract is rich in (Proanthocyanidin) PAs used as antioxidant, increase the collagen cross-links in the tissues (Bedran‐Russo et al., 2007; Anastasiadi et al., 2008), oligomeric PACs is also reported (Macheix and Fleuriet, 1990; Smullen et al., 2007).Seed extract is used to manufacture new antibacterial agents without modifying the oral equilibrium (Furiga et al., 2009; Wu, 2009). The leaf extract have antibacterial activity (Nirmala and

Review of Literature

Narendhirakannan, 2011; Ahmad et al., 2014) and antifungal activity (Askari et al., 2012). Rutin and β-sitosterol glycoside were isolated from ethyl acetate fraction. Grape skin also has antimicrobial activity (Nayak et al., 2010). The chloroform fraction is effective against Herpes simplex virus type-1 and Parainfluenza viruses (Orhan et al., 2009). The ethyl acetate extract has strong anti-oxidant activity as well.

2.6.6.1. Bioassay and Phytochemical review of Vitis vinifera

Extracts of Vitis vinifera consisits of different phytochemicals of medicinal importance. Fatty acids (Stafford et al., 1974) triterpenes (Zhang et al., 2004), flavonoids (Liggins et al., 2000; Anastasiadi et al., 2008), amino acids (Bolin and Petrucci, 1985), hydroxycinnamic acids (Liggins et al., 2000), 5-hydroxymethyl-2-furaldehyde (Palma and Taylor, 2001). Linoleic acid, polyphenol (Anastasiadi et al., 2008) and anthocyanins (Wu et al., 2014) were reported in this plant. Riverero-Cruz (Rivero-Cruz et al., 2008) reported oleanolic acid, oleanolic aldehyde, linoleic acid, linolenic acid, betulin, betulinic acid, 5-(hydroxymethyl)-2-furfural, rutin, β-sitosterol and β-sitosterol glucoside. Lower quantities of triterpenic acid were reported in dry extracts of Vitis vinifera (Caligiani et al., 2013). Gallic acid, epicatechin, p-OH-benzoic acid and vanillic acid are present in grape pomace as reported by Oliveria (Oliveira et al., 2013).

2.6.7. Dispyros blancoi ethanobotanical importance

Kingdom: Plantae Phylum: Angiospermae Class: Asterids Order: Ericales Family: Ebenaceae Genus: Diospyros Species: D. blancoi

Dispyros blancoi is a tropical tree that grows to 2,400 ft from the sea level.It requires a good amount of rainfall to grow and it almost take 6-7 years to give out the fruit but those trees that grow through propagation give fruit in 3-4 years. Traditionally this plant is used as antifungal, for insomnia and hiccough, internal hemorrhage, dysponea, vermifuge and

Review of Literature vermicide, antifebrile, astringent and bactericidal (Tezuka et al., 1973; Ganapaty et al., 2006). The plant extract of this family also possess anticonvulsant activity as well (Adzu et al., 2002). It possesses the antidiarrhoeal activity (Rahmani et al., 2012). Juice of leave and bark is used to treat diarrhea, snakebite and dysentery (Ghani, 1998). It also possesses different biological activities like antibacterial, antiprotozoal, cytotoxicity, antidiarrhoeal, antifungal and molluscicidal activity (Maridass et al., 2008). Anti- inflammatory and analgesic properties are also reported in this plant extract (Maridass et al., 2008). The fruit is used in a formulation to treat allergic and inflammatory diseases (Ahn et al., 2014). The volatile compounds from Dispyros blancoi are reported to have anti-cancer potential as well (Amar Dev, 2014).

2.6.7.1. Bioassay and Phytochemical review of Diospyros blancoi

The triterpenoids belonging to lupine, ursane and oleanane series possess anti- inflammatory activity (Chopra and Chopra, 1969). The phytochemical analysis revealed the presences of tannin (Howlader et al., 2012), alkaloids (Shoba and Thomas, 2001), flavonoids (Galvez et al., 1993), sterols, saponins, terpenes and reducing sugars (Otshudi et al., 2005). The leaf extract contain isoarborinol methyl ether and α- and β-amyrin fatty esters (Ragasa et al., 2009). Ghias Uddin (Uddin and Rauf, 2012) reported the presence of terpenoids, anthraquinones and steroids in the root extracts, steroids, terpenoids and tannins in leave extract.

2.6.8. Litchi chinensis ethanobotanical importance

Kingdom: Plantae Phylum: Angiospermae Class: Rosids Order: Sapindales Family: Sapindaceae Genus: Litchi Species: L.chinensis

Litchi chinensis is an ever green tree that reaches the height of 33-92ft. The leaves are longer with leaflets in 2-4 pairs and because of adaptation to repel water during

Review of Literature developing they are called lauroid leaves (Menzel and Waite, 2005). It is used in coughing, to cure stomach ulcers, to treat diabetes and obesity and in analgesic actions (Morton, 1987; Obrosova et al., 2010). It is further used to kill intestinal worms. It has anti-cancer activity as well (Xiao et al., 2004). The seeds have antiviral, antiplatelet, antihyperglycemic, antihyperlipidemic (Chen et al., 2007). It has anti-tumor, anti- inflammatory, anti-viral and anti-bacterial activity (Mahato and Sen, 1997; Liu et al., 2013; Bhat and Al-daihan, 2014; Wen et al., 2014). Hepatoprotective activity against paracetamol induced liver damage in rats was reported. The alchoholic extract of leaves possess anti-analgesic and anti-inflammatory activity (Barad et al., 2014).

2.6.8.1. Bioassay and Phytochemical review of Litchi chinensis

Previous researchers reported several phytochemcial in this plant. Flavonoids in fruits are effective against breast cancer (Xu et al., 2011). Shukla (Shukla et al., 2014) reported antioxidant activity due to higher phenolic contents in the leaf extracts (Liu et al., 2009; Jiang et al., 2013). It has cardio-protective activity (Yang et al., 2010). Terpenoids are responsible for cytotoxic activity (Xu et al., 2011). Antimicrobial activity is due to phenolic compounds (Cowan, 1999). Procyanidin (PA2), (PB2) and epicatechin was also isolated from leaves (Castellain et al., 2014). 2-(2-hydroxyl-5-(methoxycarbonyl) phenoxy) benzoic acid with stigmasterol, kaempferol, butylated hydroxytoluene, isolariciresinol, 3,4-dihydroxyl benzoate, ethyl shikimate and methyl shikimate were isolated from in the pericarp (Jiang et al., 2013).

Materials and Methods

3. MATERIALS AND METHODS

Nine different medicinal plants (Diospyros blancoi, Vitis vinifera, Syzygium cumuni, Psidiun guajava, Morus nigra, Litchi chinensis, Phoenix dactylifera and Mangifera indicad) depending on their ethanobotanical importance were initially selected for said studies and finally two plants Syzygium cumini and Psidium guajava were selected for further studies.

3.1. Collection of Plant Material

Plant materials were collected from different areas of Rawalpindi, Islamabad and Lahore, Pakistan from March-May, 2010 respectively and were then identified by Dr. Abdul Nasir Khalid from Department of Plant Sciences, University of the Punjab, Lahore, Pakistan and sample number was assigned to the final two selected plants Syzygium cumini (LAH 0578) and Psidium guajava (0579) and their samples were deposited at the herbarium of University of the Punjab, Lahore, Pakistan.

3.2. Drying and Extraction

The shade-dried and powdered aerial parts of the selected plants material (1.0 Kg each) were subjected for extraction by cold maceration method successively with methanol (2.0 L) at room temperature with occasional shaking for a period of one week in order to extract and maximize the yield of the compounds. The extract was filtered using Whattman 40 filter paper and same process was repeated twice using same volume of solvent for each time. These extracts were concentrated using Rotavapor-R20 at 40 º C to obtain crude semi solid mass.

3.3. Fractionation

Defined amount of crude extracts were then subjected to fractionation process after storing weighed amount of the crude extract in airtight jar for biological screening of all the leaves. The crude extract of Syzygium cumuni leaves was then suspended in distilled

Materials and Methods water (1L) followed by addition of n-hexane (1.5 L) along with stirring and hexane layer was collected and concentrated under vacuum at 40º C and it was coded as n-hexane fraction (12.0 g). Next, chloroform fraction (10.0 g) and ethyl acetate fraction (16.0 g) was collected using same methodology what was left behind was labelled as the aqueous fraction (7.0 g) as shown in Figure 3.1.

Similarly, the crude methanolic extract of leaves of Psidium guajava (66.0 g) was further fractionated as mentioned earlier to obtain n-hexane (14.0 g), chloroform (10.0 g), ethyl acetate (20.0 g) and aqueous (10.0 g) fractions as depicted in Figure 3.2. Crude methanolic extract (40 g) was obtained from leaves of Morus nigra, 5.0 g of the extract was kept for biological activity and after fractionation 10.0 g of n-hexane, 8.0 g of chloroform, 14.0 g of ethyl acetate and 3.0 g of water soluble fraction was obtained (Figure 3.3). The crude methanolic extract (30.0 g) obtained from leaves of Phoenix dactylifera was further fractionated in order to obtain n-hexane (6.0 g), chloroform (8.0 g), ethyl acetate (8.0 g) and aqueous (4.0 g) fractions as presented in Figure 3.4. Likewise, the crude methanolic extract and fractionation results of Mangifera indica, Vitis vinifera, Diospyros Blancoi, Litchi chinensis and Ocimum basilicum are shown in Figure 3.5, Figure 3.6, Figure 3.7 & Figure 3.8.

Materials and Methods

Figure 3.1: Extraction and fractionation from the leaves of Syzigium cumunii

Figure 3.2: Extraction and fractionation from the leaves of Psidium gujava 29

Materials and Methods

Figure 3.3: Extraction and fractionation from the leaves of Morus nigra

Figure 3.4: Extraction and fractionation from the leaves of Phonex dactylifera

Materials and Methods

Figure 3.5: Extraction and fractionation from the leaves of Mangifera indica

Figure 3.6: Extraction and fractionation from the leaves of Vitis vinifera

Materials and Methods

Figure 3.7: Extraction and fractionation from the leaves of Diospyros Blancoi

Figure 3.8: Extraction and fractionation from the leaves of Litchi chinensis

Materials and Methods

3.4. Phytochemical Analysis

The crude methanolic extract of the plants were screened for the presence of different groups of secondary metabolites like saponins, alkaloids, tannins and flavonoids (Evans, 2002; Parekh and Chanda, 2008).

3.4.1. Test for Alkaloids

Mayer’s reagent: 5 g of potassium iodide was dissolved in 20 mL of distelled water and 0.355g of mercuric chloride was dissolved in 60 mL of water. The two solutions were mixed and volume was made upto 1000 mL with distilled water.

Dragendorff’s reagent:

Solution A: 20 g of tartaric acid and 1.7 g of basic Bismith nitrate was dissolved in 80 mL of distilled water

Solution B: 16 g of Potassium iodide was dissolved in 40 mL of distilled water

Both these solutions were then mixed in 1:1 ratio. Plant extract (0.5 g) was mixed with 5.0 mL of 1% aqueous solution of HCl on water bath and was filtered and divided in 2 parts and both the parts were treated with Mayers reagent and Dragendorff reagent individually. Precipitation or turbidity was observed was an indication of alkaloids

3.4.2. Test for Saponins

Plant extract (0.5g) was dissolved in water and shaken vigorously. Appearance of froth indicated the presence of saponins.

3.4.3. Test for Tannins

Plant extract (0.5 g) was dissolved and boiled in 20 mL of distilled water in the test tube and then filtered. After filtration 0.1% FeCl3 was added to the filtrate. Appearance of blue-black, green or blue-green precipitates showed presence of tannins.

Materials and Methods

3.4.4. Test for Flavonoids

Prepared extract (0.5 g) was shaken in peteroleum ether to remove all the fatty material. The defatted extract was then dissolved in 20 mL of 80% ethanol, warmed and then it was filtered. The filterate was further divided in two parts, to part A, 4 mL of 1% KOH was added, apperence of dark yellow colour indicated the presences of flavonoids. To part B, Magnesium turnings (3 pieces) and few drops of conc. HCl was added. Appearance of pink, red or orange colour indicated the presence of flavonoids.

3.5. Antibacterial Sensitivity of Crude Extracts against Dental Caries Causing Pathogens (in vitro). 3.5.1. Test Microorganisms

Six bacterial strains Streptococcus mutans (ATCC 25175) Streptococcus mitis, Staphylococcus aureus (ATCC 12600), Pseudomonas aeruginosa (ATCC 29999), Bacillus subtilis, and Escherichia coli (procured from Microbiologics Pakistan) were used as test organisms for antibacterial activity. The strains obtained from Department of Chemistry and Biochemistry, University of Maryland, USA were also used for Bioassay; Streptococcus mutans, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa. These were then sub-cultured on Nutrient agar, Luria-Bertani and Brain Heart plates and incubated aerobically at 37 ºC for 24 hrs.

3.5.2. Antibacterial Assay of crude extract

For antibacterial activity the agar well diffusion method and disc diffusion method was used (Parekh and Chanda, 2008; Aneja et al., 2010). Overnight bacterial cultures in nutrient broth was vortexed and turbidity was adjusted by adding sterile saline, while using 0.5 Mc Farland turbidity standard as reference until 106 colony forming unit (CFU/mL) was obtained and used as inocula. Nutrient agar and Brain heart infusion agar medium was used for antibacterial activity. Media was sterilized by autoclaving with pH 7 followed by seeding with 10.0 mL of bacterial inoculum after it was cooled down to 45 ºC. Each extract (100 µL) was propelled in the wells of already inoculated specific media

Materials and Methods agar plates for each organism. The plates were allowed to stand for 10-15 minutes for proper diffusion of the extract and were incubated at 37ºC for 24 hrs (Khokra et al., 2008; Aneja and Joshi, 2009). The diameter of zone of inhibition for each concentration was measured and compared with standard antibiotic (ciprofloxacin) and negative control DMSO. Assay was done in duplicate.

3.5.3. Minimum Inhibitory Concentration Determination

Minimum Inhibitory Concentration (MIC) for the methanolic leaf extracts was determined by modified agar well diffusion method (Cappuccino and Sherman, 2008). Twofold serial dilutions of extracts were prepared. Leaves extract were first reconstituted in 20% DMSO. Dilution with sterile water was done to get the decreasing concentration range of 19 mg/mL. Inoculated growth medium without plant extract was used as control, and un-inoculated medium was used as blank. Each dilution (100 μL) was than introduced into wells in the specific media agar plates with inoculum (106 CFU/mL) of the test microbial strain. All plates were incubated aerobically at 37˚C for 24 hrs and MICs were recorded after 24 hrs of incubation.

3.6. Formulation of Guava & Jaman Tablets

Guava & Jaman tablets were formulated (in a batch of 5000 tablets) in local pharmaceutical company, from crushed leaves and extracts of these leaves in following composition (Table 3.1). The average weight of the formulated tablet was 182 mg, with quantity of active ingredient equal to 10 mg / tablet.

Materials and Methods

Table 3.1: Composition of Guava and Jaman Tablets

Ingredients/tablet Quantity (mg) Gauva extracts 5 Jaman extracts 5 Avecil pH 10.2 20 Lactose 41 Starch 70 Mg Stearate 3 Talcum 3 Primojel 35 Total weight 182

3.7. In-vitro Antibacterial Sensitivity of Syzigium cumunii:Psidinm guajava Tablet against Bacterial Strains

Antibacterial sensitivity was carried out by using agar diffusion method as described earlier. Zone of inhibition produced by each tablet was measured and is then compared with standard antibiotic (ciprofloxacin) and the negative control.

3.8. Evaluation of Physical Properties of Syzygium cumuni:Psidinm guajava Tablet

3.8.1 Colour: Colour of tablet was observed with naked eye

3.8.2 Weight per tablet: Weight of the tablet was measured using analytical balance

3.8.3 Friability and disintegration time: It was determined by using friability and disintegration tester of Erweka according to specification of British Pharmacopiaoe (Pharmacopoeia, 2003).

Materials and Methods

3.9. Determination of Bactericidal Activity of Syzygium cumuni: Psidium guajava Chewable Tablet

Bactericidal activity was done by using time kill assay which provided information of the rate at which microorganisms are killed. Cell suspensions of dental caries bacteria were prepared by growing them in respective nutrient and BHI broth and their turbidity was adjusted according to 0.5 McFarland standards. After incubation the samples were withdrawn at 0, 15, 30, 60 and 120 minutes, serial dilutions were made in 0.9% normal saline and 100 µL aliquots were inoculated by spread plate method on the agar plates. The number of viable colonies was counted after incubation at 37˚C overnight. Viable colonies were than calculated to determine the colony-forming unit (CFU/mL).

3.10. In-vivo Study of Syzygium cumuni : Psidinm guajava Chewable Tablet

In-vivo studies of Syzigium cumunii: Psidinm guajava chewable tablets were also conducted. For this purpose 100 subjects were randomly selected from different areas of Rawalpindi, Islamabad and Lahore who were suffering from dental caries from mild to severe stage and who willingly volunteered themselves for this study. They were given the tablets and the questionnaire under the guidance and continuous supervision of Dr. Benzair Chand, Dentist from PCSIR Labs Complex Lahore, Pakistan for a period of 3 months. After 3-4 months the questionnaire was collected back.

3.11. Isolation of Bioactive Fractions

After fractionation, preliminary evaluation and antibacterial assay the ethyl acetate fraction of Psidium guajava and Syzygium cumuni were selected for further isolation studies using flash chromatography, column chromatography (normal and reverse phase) as required and preparative thin layer chromatography.

Materials and Methods

3.11.1. Flash chromatography

The ethyl acetate fraction of Syzygium cumuni was further purified on flash chromatograph (Model: Combi flash Rf 200i, Teledyne ISCO) using 120 g HP silica column with CV 192 mL-85 mL/ min at 225psi.

Approximately 6.0 g of ethyl acetate extract of Syzygium cumuni was mixed with silica and it was loaded in the column as a uniform layer. Mobile phase starting from n-hexane followed by ethyl acetate, chloroform, methanol and water in different set ratios (

Materials and Methods

Table 3.2) were used and fractions were collected. Approximately 240 fractions were collected for Syzygium cumuni which were subjected to TLC using TLC plates (silica gel

60 F254 with fluorescent indicator). Fractions with similar Rf values were combined and total of 26 groups were formed (Table 3.3), Figure 3.9. Same procedure was followed for ethyl acetate fraction of Psidium guajava and total of 239 fractions were collected using n-hexane, ethyl acetate, chloroform and methanol. Fractions with similar Rf values were combined and total of 29 groups were formed. The details of the groups, mobile phases and Rf values of the collected fractions are given in Table 3.4. The sepeartion of bioactive fractions from Psidium guajava was summarized in Figure 3.10.

Materials and Methods

Table 3.2: Percentage composition of solvents used to elute the fractions in given time

Time (min) %age composition of each solvent used 0 100% n- hexane 5 90% n- hexane : 10 % ethyl acetate 25 50% n-hexane: 50% ethyl acetate 50 100% ethyl acetate 55 90% ethyl acetate : 10 % Chloroform 75 50% ethyl acetate: 50% Chloroform 100 100% Chloroform 105 90% Chloroform: 10% Methanol 125 50% Chloroform: 50% Methanol 150 100% Methanol 155 90 % Methanol: 10% Water 175 50% water: 50% Methanol

Materials and Methods

Table 3.3: Solvent system, their ratios, fractions and Rf values after TLC of

Syzygium cumini

Weight of extract Collected Mobile phase Ratio of solvents Fractions (g)

MJ- 1 n-hexane:CHCl3: CH3OH 1:8:1 0.151 MJ-2 -do- 2:7:1 0.182 MJ-3 EtOAC : Hexane 5:5 0.454 MJ-4 -do- -do- 0.53 MJ-5 -do- -do- 0.65 MJ-6 -do- -do- 0.65 MJ-7 -do- -do- 0.77 MJ-8 -do- -do- 0.833 MJ-9 -do- -do- 0.892

MJ-10 CHCl3: CH3OH 7: 3 0.68 MJ-11 -do- 7 : 3 0.67 MJ-12 -do- 5 : 5 074 MJ-13 -do- 5 : 5 0.70

MJ-14 CH3OH: CHCl3 6 : 4 0.72 MJ-15 -do- 6 : 4 0.24

MJ-16 CH3OH:CHCl3: n-Hexane 3: 3: 4 0.33

MJ-17 CHCl3 Drops of methanol 0.48

MJ-18 EtOAC: CHCl3 6 : 4 0.35

MJ-19 CH3OH 100% 0.38

MJ-20 CH3OH: CHCl3 3 : 7 0.91 MJ-21 -do- 3 : 7 0.73 MJ-22 -do- 2.5 : 7.5 0.82 MJ-23 -do- 2.5 : 7.5 0.54 MJ-24 -do- 100% methanol 0.84

MJ-25 CH3OH 100% 0.53

MJ-26 CH3OH : n -Hexane: CHCl3 2: 4: 4 0.43

Materials and Methods

Figure 3.9: Isolation of bioactive fractions from leaves of Syzygium cumini

Materials and Methods

Table 3.4: Solvent system, their ratios, fractions and Rf values after TLC of Psidium guajava

Collected Fractions Mobile phase Ratio of solvents Rf value MG-1 EtOAC :n- hexane 5:5 0.112 MG-2 -do- 5: 5 0.13 MG-3 -do- 5:5 0.12

MG-4 CHCl3: Methanol 7 : 3 0.12

MG-5 EtOAC:n-hexane: CH3OH 5:2 :3 0.12 MG-6 -do- -do- 0.14 MG-7 -do- -do- 0.13 MG-8 -do- -do- 0.13 MG-9 -do- -do- 0.12

MG-10 CHCl3 : CH3OH 7: 3 0.46 MG-11 -do- -do- 0.42

MG-12 EtOAC:n-hexane:CH3OH 5: 2: 3 0.18 MG-13 -do- -do- 0.3

MG-14 EtOAC:CH3OH 7:3 0.56

MG-15 EtOAC: CHCl3:CH3OH 5:3:2 0.75 MG-16 -do- -do- 0.21

MG-17 CHCl3: n-hexane:CH3OH 3: 1: 3 0.88

MG-18 CHCl3: n-hexane:CH3OH 6: 2: 2 0.53

MG-19 EtOAC:n-hexane:CH3OH 5: 1: 4 0.83 MG-20 -do- -do- 0.75

MG-21 CHCl3:CH3OH 4: 6 0.75 MG-22 -do- -do- 0.5 MG-23 -do- -do- 0.3

MG-24 EtOAC:CH3OH 4: 6 0.18

MG-25 CH3OH 100% 0.62

MG-26 n-hexane:CH3OH 4:6 0.18

MG-27 CH3OH 100% 0.33

MG-28 CH3OH 100% 0.47

MG-29 CH3OH 100% 0.25

Materials and Methods

Figure 3.10: Isolation of bioactive fractions from leaves of Psidium guajava

Materials and Methods

3.11.2. Reverse Phase High Performance Liquid Chromatography (RP-HPLC)

Separations of the fractions MJ-6, MJ-26, MJ-16, MG-25 and MG-15 (Figure 3.9) were further achieved by taking the fractions for gradient elution on HPLC (Model Varian, Prostar). The separation was performed on a COSMOSIL C18 column (length 250mm with 2.1mm i.d) with a mixture of acetonitrile and Methanol. Mobile phase A consisted of Acetonitrile (HPLC grade) and Methanol served as mobile phase B using gradient elution system (Table 3.5).

Table 3.5: Gradient elution systems used for HPLC separations

Sr. No. Time Flow Rate Effluent A Effluent B (min) (mL/min) (%) (%) 1 Pre run 0.8 50 50 2 10 0.8 30 70 3 15 0.8 0 100 4 30 0.8 0 100

A= Acetonitrile, B=Methanol

Sample Preparation

For preparation of stock solutions, methanol soluble fraction was dissolved in methanol (HPLC grade) and the sample was vortex (Vortex: super-mixer CAT.No.1290) and also sonicated (Digital Ultrasonic Cleaner H-B 4818 T) for 5 minutes to achieve uniform mixing. Distilled water (1:1) ratio was added resulting in crystallization of some compounds which were filtered using Titan 3, PVDF 0.2 µm filter and the filtrate hence obtained was washed with acetonitrile and that fraction was labeled as Acetonitrile fraction. Same procedure was followed for all the samples for HPLC.

3.11.3. Flash Chromatography (FC)

From the fraction MG-24, 0.25 g of extract was dissolved in 4.0 mL of 10 % methanol in chloroform (Model-Reveleris X2 by GRACE). Mobile phase i.e. n-hexane, EtOAc, 45

Materials and Methods dichloro methane (DCM) and chloroform was used in order of increasing polarity. Two fractions were obtained on combining elutes on the basis of TLC and were named as MG-24.1 and MG-24.2 (Figure 3.10). MG-24.2 was again subjected to FC as a result of bioassay, over silica column (silica 40 µm, 12.0 g, 200 psi and 2.4-12 g sample loading capacity) starting from 10% methanol in DCM till 100% methanol and 12 fractions were obtained. The other fraction containing several compounds was not followed up in present work. The 12 sub-fractions obtained from MG-24.2 were then subjected to bioassay using the agar well diffusion method.

3.11.4. Preparative Thin Layer Chromatography

Preparative TLC was used for isolation of compounds from fraction MJ-12, MJ-3. Samples were dissolved in respective solvents. Silica TLC plates of size 20×20cm were used. Mobile phase was adjusted and selected and 100 mL of the mobile phase was added in the TLC tank and covered with the lid in order to pre-saturate the tank. The dissolved sample was loaded on the entire length of the plate 2-3 times. Developed Plates were air dried and visualized under UV light at 254 nm. Clearly distinct bands were marked and separated. They were washed with the solvents 3-4 times. The resultant sample was filtered, allowed to evaporate at room temperature and subjected to bioassay according to method already described (Aneja et al., 2010).These fractions were not considered in present work due to low activity.

The fractions that have shown maximum activity against the selected bacteria were further purified using the above mentioned procedures and were analyzed by using different spectroscopic techniques.

3.12. Bio-assay of Collected Fractions

The biological activity (antibacterial activity) of all the collected fractions was done using Disc diffusion assay as described earlier. Circular disc from Whatman No. 1 filter paper was made of 6 mm diameter in size. The dry fractions were dissolved in methanol and the discs were impregnated with equal volume of the extracts (50 µL). Nutrient agar, Luria-

Materials and Methods bertani and Brain heart infusion agar media plates were prepared according to given instructions (Aneja et al., 2010). A 24hr old culture was used to prepare the inoculum and turbidity of inoculum was adjusted to 0.5 Mc Farland standards (Schrader and Harries, 2006). The discs were aseptically placed over the plates seeded with each of the test pathogens. The plates were then incubated in an upright position at 37ºC for 24 h and zone of inhibition was then measured. Zone of inhibition less than 12 mm diameter were considered having low, between 12 and 16 as moderate and more than 16 mm were considered as highly active antibacterial activity (Indu et al., 2006). The selected fractions on the basis of antibacterial activity were further taken for purification.

3.13. Detection of Biofilm Formation in Streptococci and Biofilm Inhibition Assay

Brain heart infusion broth was prepared according to manufacturer instructions with 1% D-glucose and after sterilization 3.0 mL of this broth was shifted to sterilized screw cap tubes under aseptic conditions. The broth was then inoculated with overnight culture of S. mutans and S. mitis. Selected bioactive constituents of Psidium guajava and Syzygium cumini (inhibitor of biofilm formation) were also added in different concentrations to give the desired concentration of extract from stock solution (100 mg/mL of DMSO). The controls were also prepared without the extracts. They were incubated at 37 ºC for 18 hrs at an angle of 30 ºC. After incubation, pH was noted and then supernatant was carefully removed without disturbing the cells that adhere with the walls of the tube. Washed the tube of biofilm with 85% normal saline, then 3.0 mL of saline was added in the tube and shake the tube well to separate the cells adhered with wall (Yadav, 2012). Then OD was measured at 550 nm using Varian-fluorescence spectrophotometer (Model: SYS-FL- ECI). The effect of an inhibitor measured as the percentage decrease in reaction rate. Percent inhibition is calculated as:

OD without inhibitor − OD with inhibitor Percent Inhibition = × 100 OD without inhibitor

Materials and Methods

3.14. Scanning Electronic Microscopy (SEM) of the Bacterial Cells after Treatment with Bioactive Components of the Extracts

Scanning electron microscope (SEM) of HITACHI Model S-4700 with operation mode of ultrahigh resolution, having capture resolution of 12800×960, was employed for observing the different morphological changes in the selected bacterial strains caused by the bioactive constituents of Psidium guajava and Syzygium cumini. Bacteria of the mid- exponential growth phase were selected for SEM analysis with cell density equal to 108 to 1010 CFU/mL since a high density of the cell is required for SEM images (Hartmann et al., 2010). Bacterial cultures were incubated with and without the selected bioactive constituents of the above mentioned plants at 37ºC for 5-7 hrs. The cells were harvested by centrifugation at 3,000 rpm for 15 min using Eppendorf AG Moldel 22331 centrifuge. The broth free culture was then washed with 3 changes of phosphate buffer with pH 7, 10 minutes for each wash. The cells were fixed in 2 % glutaraldehyde solution, for 60 minutes at room temperature and further fixation was completed by placing the samples overnight at 4ºC. The sample was collected on nucleopore filter (0.4 µm) held in plastic swinney filter holder. The cells free of excess glutaraldehyde (GA) was washed with 3 changes of phosphate buffer, 10 minutes for each wash. The cells were fixed in 1% osmium tetra oxide (OsO4) in phosphate buffer for 30-90 minutes. The samples were washed free of osmium with 3 changes of double distilled water, 10 minutes for each change. After final wash the filters were removed to multiwall plates for further processing. The cells are hydrated with 75-100% ethanol with 10 minutes for each change, with 3 changes of ethanol 100 % with 10 minutes for each change. The filters were then filterd to containers for critical drying under absolute ethanol and they were mounted on stubs using sticky tab adhesives. Conductive carbon paint was used to ring stub:filter junction and stubs were coated with gold:palladium alloy.

3.15. Analysis of Bioactive Fractions

The isolated bioactive fractions were subjected for spectroscopic analysis.

Materials and Methods

3.15.1. Mass Spectroscopy

The bioactive fractions (MJ-6, MJ-26, MG-15 and MG-25) were further analyzed for their mass on IJEOL ACCU TOF-CS with ESI ionization method

3.15.2. FT-IR analysis

Further analysis was done using IR spectroscopic method. In the current study all the samples were analyzed by Fourier transform infrared (FT-IR) spectroscopy (Thermo Nicolet NEXUS 670 FTIR), having Range: 400-4000 cm-1, Resolution: <0.1cm-1.

3.15.3. NMR analysis

Compounds were dissolved in deutrated solvent. Deutrated methanol was used for compounds isolated from ethyl acetate fractions of plants. Solution was pipetted in clean NMR tubes for analysis purpose. NMR spectra was recorded using Bruker AV-400 MHz high resolution (server am 400.umd.edu) was used for 1H-NMR and Bruker DRX-500 MHz high resolution (server console 500.umd.edu) was used for overnight Carbon NMR, and processing software was used to perform different NMR experiments.

Results

4. RESULTS

The present work represents a study of the importance of natural products particularly those that are derived from higher plants, in relation of drug development. Eight different medicinal plants were selected for the study in order to find some anti-caries substances from natural resources.

All the selected plant materials were collected, dried and extracted with methanol. When further fractionation of crude extract was done in different solvents, maximum yield was obtained in methanol extract (25-66 g), while hexane, chloroform and ethyl acetate fractions were generally 8-20 g and very low concentration was achieved in aqueous fraction (2-7g) except Psidium guajava yielding upto10 g (Table 4.1).

4.1. Screening for Antibacterial activity

The crude fractions of all the selected plants were then subjected for their antibacterial activity against 6 selected oral pathogens. Susceptibility evaluation of all the bacteria, made by disc diffusion method showed that all the fractions of Psidium guajava and Syzygium cumini were effective against the selected bacteria. The antibacterial assay revealed the maximum efficacy of ethyl acetate fraction against all the tested bacteria. The ethyl acetate and methanol fractions were active against all the tested bacteria with maximum activity observed in ethyl acetate fraction of Syzygium cumini against B. subtilis (26 mm), S. aureus (25.5 mm) and S. mutans (20.5 mm) (Figure 4.1) with least activity in chloroform fraction of Syzygium cumini against S. mutans (11 mm). The aqueous and chloroform fractions were inactive against S. mitis. All the extracts of Psidium guajava were found active against all the tested bacterial strains with maximum activity observed against B. subtilis (28 mm). Aqueous extracts showed the maximum mean zone of inhibition of 22 mm against P. aeruginosa and E. coli. The ethyl acetate fraction gave 22 mm zone against P.aeruginosa (Figure 4.2). The distribution of antibacterial activity in low polar extracts indicates the non-polar to slightly polar nature of bioactive compounds.

Results

Results obtained from fractions of Mangifera indica indicated the distributiom of activity in methanol and ethyl acetate fractions (Figure 4.3). Both these fractions were active against 5 out of 6 selected bacteria, with maximum activity observed against S. mutans and E. coli (15 mm). The aqueous, n-hexane and chloroform fractions were not active against S. mutans and S. mitis.

Methanolic extract of Litchi chinesis was found active against S. mutans (20 mm). It was found active against P. aeruginosa and E. coli (13 mm), S. aureus (10 mm) and B. subtilis (14 mm). Rest of the fractions showed no activity (Figure 4.4). Results for the activity of Vitis vinifera extract indicated that the methanolic fractions were active against all the tested strains except S. mitis (Figure 4.5) with maximum activity against S. aureus with 19 mm zone of inhibition. The chloroform fraction also exhibited considerable activity against P. aeruginosa (14 mm), E. coli (10 mm) and B. subtilis (16 mm). Antibacterial activity of Phoenix dactylifera fractions indicates that n-hexane frcation was inactive against all the bacterial strains. The aqueous, methanol and ethyl acetate fraction was active against three bacterial strains, while the chloroform fraction was active against B. subtilis only (Figure 4.6).

Antibacterial activity was found to be moderate in chloroform and ethyl acetate crude extract of Diospyros blancoi against all tested bacteria with maximum inhibition zone of 20 mm against S. aureus and S. mutans in ethyl acetate extract. Aqueous and n-hexane fraction was almost inactive against all the bacteria (Figure 4.7). In case of Morus nigra, the ethylacetate fraction was found active against four bacterial strains, with no activity in n-hexane and aqueous fractions (Figure 4.8).

Results

Table 4.1: Total yield obtained after fractionation of crude extract

Plant Methanol n-Hexane Chloroform Ethyl acetate Aqueous Fraction Fraction Fraction Fraction Fraction (g) (g) (g) (g) (g) Syzygium cumini 66 12 10 16 7 Psidium guajava 66 14 10 20 10 Mangifera indica 33 8 9 8 3 Litchi chinesis 25 4 10 7 2 Vitis vinifera 33 5 10 11 2 Moris nigra 40 10 12 10 3 Phoenix 30 6 8 8 4 dactylifera Diospyros 25 5 8 8 2 blancoi

Results

Aqueous Methanol n-hexane Chloroform Ethyl acetate DMSO Ciprofloxacin

Zone Zone of inhibition (mm) 5

0 Streptococcus Streptococcus Pseudomonas Escherichia Coli Staphlycoccus Bacillus subtilis mutans mitis aeruginosa aureus Bacterial Strain

Figure 4.1: Antibacterial activity of crude fractions of leaves of Syzygium cumini

Aqueous MeOH n-hexane CHCl3 EtoAc DMSO Ciprofloxacin

5 Zone Zone of inhibition (mm) 0 Streptococcus Streptococcus Pseudomonas Escherichia Coli Staphlycoccus Bacillius subtilius -5 mutans mitis aeruginosa aureus Bacterial Strain

Figure 4.2: Antibacterial activity of crude fractions of leaves of Psidium guajava

Results

Aqueous MeOH n-hexane CHCl3 EtOAc DMSO Ciprofloxacin

Zone Zone of inhibition(mm) 5

0 Streptococcus Streptococcus Pseudomonas Escherichia Coli Staphlycoccus Bacillus subtilis mutans mitis aeruginosa aureus Bacterial Strain

Figure 4.3: Antibacterial activities of crude fractions of leaves of Mangifera indica

Aqueous MeOH n-hexane CHCl3 EtoAc DMSO Ciprofloxacin

Zone Zone of inhibition (mm) 5

0 Streptococcus Streptococcus Pseudomonas Escherichia Coli Staphlycoccus Bacillius subtilius mutans mitis aeruginosa aureus Bacterial Strain

Figure 4.4: Antibacterial activities of crude fractions of leaves of Litchi chinesis

Results

Aqueous MeOH n-hexane CHCl3 EtoAc DMSO Ciprofloxacin

Zone Zone of inhibition (mm) 5

0 Streptococcus Streptococcus Pseudomonas Escherichia Coli Staphlycoccus Bacillius subtilius mutans mitis aeruginosa aureus Bacterial Strain

Figure 4.5: Antibacterial activities of crude fractions of leaves of Vitis vinifera

Aqueous MeOH n-hexane CHCl3 EtoAc DMSO Ciprofloxacin

Zone Zone of inhibiton(mm) 5

0 Streptococcus Streptococcus Pseudomonas Escherichia Coli Staphlycoccus Bacillius subtilius mutans mitis aeruginosa aureus Bacterial Strain

Figure 4.6: Antibacterial activities of crude fractions of leaves of Phoenix dactylifera

Results

Aqueous MeOH n-hexane CHCl3 EtoAc DMSO Ciprofloxacin

30 25 20 15 10

5 Zone Zone of inhibition (mm) 0 Streptococcus Streptococcus Pseudomonas Escherichia Coli Staphlycoccus Bacillius subtilius mutans mitis aeruginosa aureus Bacterial Strain

Figure 4.7: Antibacterial activities of crude fractions of leaves of Diospyros blancoi

Aqueous MeOH n-hexane CHCl3 EtoAc DMSO Ciprofloxacin

30 25 20 15 10

5 Zone Zone of inhibition(mm) 0 Streptococcus Streptococcus Pseudomonas Escherichia Coli Staphlycoccus Bacillius subtilius mutans mitis aeruginosa aureus Bacterial Strain

Figure 4.8: Antibacterial activity of crude fractions of leaves of Morus nigra

Results

4.2. Preliminary Phytochemical Analysis

All the crude extracts were subjected to preliminary phytochemical analysis using already reported different biochemical tests.

Phytochemical analysis of Psidium guajava revealed the presences of moderate quantities of alkaloid, in ethyl acetate and methanol extracts. Moderate quantities of flavonoids were also present in hexane fraction. High concentration of tannins and flavonoids were found in aqueous and ethyl acetate fractions. Tannins and saponins were absent in hexane fraction (Table 4.2).

Phytochemical analysis of Syzygium cumini indicated the presence of high concentration of tannins and flavonoids in ethyl acetate fraction. Saponins were found absent in hexane fraction. Moderate quantities of alkaloids were found in ethyl acetate fraction and of tannins in aqueous fraction (Table 4.3).

Alkaloids were present in low quantities in Mangifera indica, while tannins, saponins and flavonoids were also present in these fractions. Moderate quantities of tannins were present in ethyl acetate fraction and flavonoids in hexane fraction. High concentration of flavonoids was found in ethyl acetate fraction (Table 4.4).

Similarly, phytochemical analysis of Litchi chinesis revealed the presences of moderate concentration of tannins, alkaloid (Mayer test) and flavonoids in ethyl acetate fraction. Moderate quantities of tannins were found in hexane fraction. Alkaloids were absent in hexane (Mayer’s test) methanol (Dragendorff test) and aqueous fraction. Flavonoids were absent in hexane fraction (Table 4.5).

High concentration of tannins was present in methanol fraction of Vitis vinifera. Flavonoids and tannins were found absent in hexane fraction. Tannins were absent in aqueous fraction. Saponins were present in all the fractions. Low quantities of alkaloids, saponins, tannins and flavonoids were found in ethyl acetate and chloroform fraction (Table 4.6).

Results

The phytochemical analysis of Phoenix dactylifera revealed the presences of alkaloids and tannins in all the extracts. High concentrations of alkaloids are present in methanol and ethyl acetate fraction. Low quantities of tannins are found in all the extracts. Saponins and flavonoids were absent in hexane fraction (Table 4.7).

Phytochemical analysis of Diospyros blancoi indicated the presences of saponins in high quantities in hexane and ethyl acetate fraction. Moderate quantities of alkaloids, tannins were found in hexane, ethyl acetate and methanol fraction. Flavonoids were absent in hexane fraction. While tannins were also absent in chloroform fraction (Table 4.8).

Analysis of Moris nigra revealed the presences of moderate quantities of saponins in chloroform and methanol fraction. Moderate quantities of tannins were also present in methanol fraction. Tannins and flavonoids were absent in hexane fraction. Saponins were absent in aqueous fraction (Table 4.9).

Results

Table 4.2: Phytochemical analysis of Psidium guajava