Studies on the Standardization of Rose Water and Assessment of Its Uses/ Role in Herbal Medicine

STUDIES ON THE STANDARDIZATION OF ROSE WATER AND ASSESSMENT OF ITS USES/ ROLE IN HERBAL MEDICINE

By SAFIA ABIDI B.Pharm., M.Pharm.

DEPARTMENT OF PHARMACOGNOSY FACULTY OF PHARMACY AND PHARMACEUTICAL SCIENCES UNIVERSITY OF KARACHI KARACHI-75270, PAKISTAN 2018 STUDIES ON THE STANDARDIZATION OF ROSE WATER AND ASSESSMENT OF ITS USES/ ROLE IN HERBAL MEDICINE

By SAFIA ABIDI B.Pharm., M.Pharm.

Thesis submitted for the partial fulfillment of the degree of

Doctor of Philosophy

DEPARTMENT OF PHARMACOGNOSY FACULTY OF PHARMACY AND PHARMACEUTICAL SCIENCES UNIVERSITY OF KARACHI KARACHI-75270, PAKISTAN 2018

CERTIFICATE

I have gone through the thesis titled “STUDIES ON THE STANDARDIZATION OF

ROSE WATER AND ASSESSMENT OF ITS USES / ROLE IN HERBAL

MEDICINE” submitted by Miss Safia Abidi for the award of Ph.D. degree and certify that to the best of my knowledge it contains no plagiarized material.

Signature & Seal of Supervisor

Prof. Dr. Iqbal Azhar Department of Pharmacognosy Faculty of pharmacy and pharmaceutical Sciences University of Karachi Date :

iii

BOARD OF ADVANCED STUDIES & RESEARCH UNIVERSITY OF KARACHI DECLARATION

I, SAFIA ABIDI d/o SYED TAJAMMUL HUSAIN hereby declare that no part of the work presented by me has been plagiarized from anywhere. Proper references are cited wherever necessary.

I understand that the university reserves the right to cancel the degree if any of the above declaration is proved false before or even after the award of degree.

Signature of Candidate:……………………………..

Name of the Candidate: SAFIA ABIDI

Title of the thesis: “Studies on the Standardization of Rose Water and Assessment of Its Uses / Role in Herbal Medicine” Degree: Ph.D. Department: Pharmacognosy Date:…………………………….

IN THE NAME OF ALLAH THE MOST BENEFICENT THE MOST MERCIFUL

DEDICATION

Dedicated to my parents for their continous support To my husband for be my strength and To my priceless loving sons Aarib and Muhammad

TABLE OF CONTENTS Pg. No.

List of Tables xi List of Figures xv List of Abbreviations xviii Acknowledgements xx Abstract xxii Urdu Translation xxv PART-Ia 1. INTRODUCTION 2 1.1 Introduction PART-Ib LITERATURE REVIEW 7 1.2 Introduction 8 1.3 Morphology 8 1.4 History 9 1.5 Varieties of Rosa damascena 10 1.6 Phytoconstituents of Rosa damascena 10 1.7 Products obtained from Rosa damascena Petals 12 1.8 Traditional Uses of Rosa damascena 12 1.9 Current Uses of Rosa damascena 13 1.10 Toxicity 14 PART-II 2. MATERIAL AND METHODS 15 2.1 List of Chemical and Reagents Used 16 2.2 List of Apparatus/ Equipment Used 18 2.3 List of Microorganisms 20 2.4 List of Animals 21 2.5 List of Solution Used 21

vii

TABLE OF CONTENTS Pg. No.

2.6. Collection and Identification of Rose Species (Flower) 22 2.7. Preparation of Rose water 23 2.8. Evaluation of Physicochemical Parameters 24 2.8.1 Color 24 2.8.2 pH 24 2.8.3 Detection of Saponin 24 2.8.3.1 Foam Test 24 2.8.4 Detection of Tannins 24 2.8.4.1 Lead Acetate Test 24 2.8.4.2 Nitric Acid Test 25 2.8.5 Detection of Triterpenoids 25 2.8.5.1 Liebermann Burchard Test 25 2.8.6 Detection of Fixed Oil 25 2.8.6.1 Spot Test 25 2.8.7 Detection of Flavanoids 25 2.8.7.1 Lead Acetate Test 25 2.8.7.2 Sulphuric Acid Test 25 2.9 Determination of Volatile Constituents by HS-GC-MS 26 2.10 Detection of Functional Groups by FT-IR 26 2.11 Acute toxicity Studies 26 2.11.1 Experimental Animals 26 2.11.2 Experimental Design 27 2.12 Brine Shrimp(Artemia Salina) Lethality Bioassay 27 2.12.1 Shrimp’s Eggs Viability Test 27 2.12.2 Brine Shrimp Lethality Assay 27 2.12.3 Data Analysis 28 2.13 In-Vitro Anti-Oxidant Activity 28 2.13.1 Preparation of Stock Solution 28

viii

TABLE OF CONTENTS Pg. No.

2.13.2 Determination of Reducing Power 28 2.13.3 Statistical Analysis 29 2.14 In-Vitro Anti-Inflammatory Activity 29 2.14.1 Preparation of Stock Solution 29 2.14.2 Inhibition of Albumin Denaturation Method 30

2.14.2.1 Control Solution (50ml) 30 2.14.2.2 Standard solution (50ml) 30 2.14.2.3 Test solution (50ml) 30 2.15 Determination of In-vitro Sun Protecting Factor (SPF) 31 2.16 Anti-Microbial Activity 32 2.16.1 Collection of Isolates 32 2.16.2 Standard Anti-Microbial Agents 33 2.16.3 Anti-Microbial Assay 34 2.17 Microbial Contamination 34 2.17.1 Sample preparation 34 2.17.2 Bacterial contamination 34 2.18 Clinical Studies 35 2.18.1 Clinical Protocol 35 2.19 Glow Measurement 35 2.19.1 Study Protocol 35 2.19.2 Instrumentation 36 2.19.3 Method 36 2.19.4 Statistical Analysis 36 2.20 Determination of Skin Hydration and Oil Content 36 2.20.1 Study Design 36 2.20.2 Statistical analysis 37 2.21 Cream Formulation 37 2.21.1 Placebo Cream and Its Preparation 37

TABLE OF CONTENTS Pg. No.

2.21.2 Cream Formulation and Its Preparation 38 2.22. In-vitro Anti-Inflammatory Activity of Rose Water Based Cream 41 Formulation 2.22.1 Preparation of Stock Solution 41 2.22.2 Inhibition of albumin denaturation method 41 2.23 In -vitro Anti-oxidant Activity of Rose Water Based Cream Formulation 42 2.23.1 Preparation of stock solution 42 2.23.2 Determination of reducing power 42 2.24 Antitussive Activity of Rose Water Based Cough Syrup Formulation 43 2.24.1 Preparation of cough syrup formulations 43 2.25 Screening for Antitussive activity of Cough Syrup 45 2.25.1 Animals Used 46 2.25.2 Method 46 PART-III 3. RESULTS 48 3.1 Evaluation of Physicochemical Parameters 48 3.2 Determination of Volatile Constituents by HS-GC-MS 48 3.3 Detection of Functional Groups by FT-IR 49 3.4 Acute Toxicity Study 49 3.5 Brine Shrimp(Artemia Salina) Lethality Bioassay 49 3.6 In-vitro Anti-Oxidant Assay by Ferric Reducing Power 49 3.7 In-vitro Anti-Inflammatory Activity 50 3.8 Determination of In-vitro Sun Protecting Factor (SPF) 50 3.9 Anti-Microbial Activity 51 3.10 Microbial Contamination 51 3.11. Clinical Studies 52 3.12 Glow Measurement of Skin 52 3.13 Measurement of Skin Hydration and Oil Content 52

TABLE OF CONTENTS Pg. No.

3.14 In-vitro Anti-Inflammatory activity of Rose Water Based Cream 53 Formulation 3.15 In-vitro Anti-Oxidant Activity of Rose Water Based Cream 53 Formulation 3.16 Antitussive Activity of Rose Water Based Cough Syrup Formulation 54 PART-IV 4. DISCUSSION 56 4.1 Evaluation of Physicochemical Parameters 56 4.2 Determination of Volatile Constituents by HS-GC-MS 57 4.3 Detection of Functional Groups by FT-IR 58 4.4 Acute Toxicity Study 58 4.5 Brine Shrimp(Artemia Salina) Lethality Bioassay 49 4.6 In-vitro Anti-Oxidant Assay by Ferric Reducing Power 60 4.7 In-vitro Anti-Inflammatory Activity 62 4.7.1 Chronic Inflammation 62 4.7.2 Recent advancement on inflammation 63 4.8 Determination of In-vitro Sun Protecting Factor (SPF) 65 4.9 Anti-Microbial Activity 67 4.10 Microbial Contamination 67 4.11 Clinical Studies 68 4.12 Glow Measurement of Skin 68 4.13 Measurement of Skin Hydration and Oil Content 69 4.14 In-vitro Anti-Oxidant Activity of Rose Water Based Cream 70 Formulation 4.15 In-vitro Anti-Inflammatory Activity of Rose Water Based Cream 71 Formulation 4.16 Antitussive Activity of Rose Water Based Cough Syrup Formulation 72

TABLE OF CONTENTS Pg. No.

PART-V CONCLUSION AND SUGGESTIONS 75 REFERENCES 181 PUBLICATION 200

xii

LIST OF TABLES

Table No. Title of Tables Pg. No. Part I (a) Table 1.1 General testing parameters for the standardization of herbal 5 medicine Part II (b) Table 1.2.1 Chemical composition of rose essential oil obtained from 10 different parts of the world Table 1.2.2 Traditional uses of different parts of rose flower 12 Table 1.2.3 Pharmacological effects of different types of extracts of Rose 13 flower Part-II Table 2.1 Normalized product function used in the calculation of SPF 32 Table 2.2 Composition of placebo cream 39 Table 2.3 Composition of cream formulation 40 Table 2.4 Composition of cough syrups formulation C1 and C2 44 Table 2.5 Composition of cough syrups formulation C3 and C4 45 Table-3 pH Trend of Rose Water Samples 77 Table-4 Phytochemical Screening of Rose Water Samples 78 Table-5 Volatile Components Present in Rose Water Samples 79 Table-6 FT-IR Absorption Band Assignments of Rose Water Samples 80 Table-7 Acute Toxicity Study of Rose Water at 15, 20,25ml Adult Dose 81 Table-8 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #1 82 Table-9 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #2 82 Table-10 Brine Shrimp (Artemia Salina) Lethality Bioassay of Sample #3 83 Table-11 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #4 83 Table-12 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #5 84 Table-13 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #6 84 Table-14 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #7 85 Table-15 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #8 85

xiii

Table No. Title of Tables Pg. No. Table-16 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #9 86 Table-17 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #10 86 Table-18 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #11 87 Table-19 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #12 87 Table-20 Determination of Absorbance using 05 ml (5.21 g) samples 88 Table-21 Percentage of Ferric Reducing Power Capacity of Rose Water 89 samples (05 ml) Equivalent to 50, 100, 200 µg/ml of Ascorbic Acid as Standard Table-22 Determination of Absorbance Using Ascorbic Acid as Standard 90 Drug Table-23 Determination of Absorbance Using 10 ml (10.35 g) Sample 91 Table-24 Percentage of Ferric Reducing Power Capacity of Rose Water 92 Samples(10 ml) Equivalent to50, 100, 200µg/ml of Ascorbic Acid as Standard Table-25 Determination of Absorbance Using Ascorbic Acid as Standard 93 Drug Table-26 In-Vitro Anti Inflammatory Activity of Rose Water Samples 94 Table-27 In-Vitro Anti Inflammatory Activity of Diclofenac Sodium 95 Table-28 Viscosity Value of Rose Water Samples Used to Calculate Anti- 96 inflammatory activity Table-29 In- Vitro Sun Protecting Factor (SPF) of Rose Water Samples 97 Table-30 In-Vitro Anti-Microbial Analysis of Rose Water Samples#1 98 Table-31 In-Vitro Anti-Microbial Analysis of Rose Water Samples#2 99 Table-32 In-Vitro Anti-Microbial Analysis of Rose Water Samples#3 100 Table-33 In-Vitro Anti-Microbial Analysis of Rose Water Samples#4 101 Table-34 In-Vitro Anti-Microbial Analysis of Rose Water Samples#5 102 Table-35 In-Vitro Anti-Microbial Analysis of Rose Water Samples#6 103 Table-36 In-Vitro Anti-Microbial Analysis of Rose Water Samples#7 104 Table-37 In-Vitro Anti-Microbial Analysis of Rose Water Samples#8 105 Table-38 In-Vitro Anti-Microbial Analysis of Rose Water Samples#9 106

xiv

Table No. Title of Tables Pg. No. Table-39 In-Vitro Anti-Microbial Analysis of Rose Water Samples#10 107 Table-40 In-Vitro Anti-Microbial Analysis of Rose Water Samples#11 108 Table-41 In-Vitro Anti-Microbial Analysis of Rose Water Samples#12 109 Table-42 Bacterial Contamination in Rose Water Samples 110 Table-43 Glow Measurement 111 Table-44 Measurement of Hydration 112 Table-45 Oil Measurement 113 Table-46 Anti-inflammatory Activity of Cream Formulation F1 114 Containing 50g RoseWater Table-46a Overall In-vitro Anti-inflammatory Activity by Protein (Egg 115 albumin) Denaturation of F1 Table-47 Anti-inflammatory Activity of Cream Formulation F2 116 Containing 30g Rose Water Table-47a Overall In-vitro Anti-inflammatory Activity by Protein (Egg 117 Albumin) Denaturation of F2 Table-48 Anti-inflammatory Activity of Placebo Cream F3 Formulation 118 Table-48a Overall In-vitro Anti-inflammatory Activity by Protein (Egg 119 Albumin) Denaturation of F3 Table-49 Anti-inflammatory Activity of Standard Diclofenac Sodium 120 Table-49a Overall In- vitro Anti-inflammatory Activity by Protein (Egg 121 Albumin) Denaturation Table-50 Antioxidant Activity of Cream Formulation F1 Containing 50g 122 Rosewater Table-51 Antioxidant Activity of Cream Formulation F2 Containing 30g 123 Rose Water Table-52 Antioxidant Activity of Placebo Cream Formulation F3 124 Table-53 Antitussive Activity of Cough syrup C1 Formulation at 5 ml 125 Adult Dose Table-54 Antitussive Activity of Cough syrup C1 Formulation at 10 ml 126 adult Dose

Table No. Title of Tables Pg. No. Table-55 Antitussive Activity of Cough Syrup C1 Formulation at 15 ml 127 Adult Dose Table-56 Antitussive Activity of Cough Syrup C2 Formulation at 5 ml 128 Adult Dose Table-57 Antitussive Activity of Cough Syrup C2 Formulation at 10 ml 129 Adult Dose Table-58 Antitussive Activity of Cough Syrup C2 Formulation at 15 ml 130 Adult Dose Table-59 Antitussive Activity of Cough Syrup C3 Formulation at 5 ml 131 Adult Dose Table-60 Antitussive Activity of Cough Syrup C3 Formulation at 10 ml 132 Adult dose Table-61 Antitussive Activity of Cough Syrup C3 Formulation at 15 ml 133 Adult Dose Table-62 Antitussive Activity of Cough Syrup C4 Formulation at 5 ml 134 Adult Dose Table-63 Antitussive Activity of Cough Syrup C4 Formulation at 10 ml 135 Adult Dose Table-64 Antitussive Activity of Cough Syrup C4 Formulation at 15 ml 136 Adult Dose Table-65 Antitussive Activity of Standard Drug (IVY Leaves Dry Extract) 137 Cough at 5 ml Adult Dose Table-66 Antitussive Activity of Standard Drug (Diphenhydramine HCL 138 and Dextromethorphan HBr) Cough Syrup at 10 ml Adult Dose Table-67 Overall Antitussive Activity of Cough Syrup Formulation C1, 139 C2, C3, C4 and Standard Drugs.

xvi

LIST OF FIGURES

Fig. No. TITLES Pg. No.

Fig. 1 GC-MS Analysis of Sample No 9 141

Fig. 2 GC-MS Analysis of Sample No 8 142

Fig. 3 GC-MS Analysis of Sample No 12 143

Fig. 4 FT-IR Analysis of Sample#1 144

Fig.5 FT-IR Analysis of Sample#2 144

Fig. 6 FT-IR Analysis of Sample#3 145

Fig. 7 FT-IR Analysis of Sample#4 145

Fig. 8 FT-IR Analysis of Sample#5 146

Fig. 9 FT-IR Analysis of Sample#6 146

Fig. 10 FT-IR Analysis of Sample#7 147

Fig. 11 FT-IR Analysis of Sample#8 147

Fig. 12 FT-IR Analysis of Sample#9 148

Fig. 13 FT-IR Analysis of Sample#10 148

Fig.14 FT-IR Analysis of Sample#11 149

Fig. 15 FT-IR Analysis of Sample#12 149

Fig. 16 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample#1 150

Fig. 17 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample#2 150

Fig. 18 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample#3 151

Fig. 19 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample#4 151

Fig. 20 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample#5 152

Fig. 21 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample#6 152

Fig. 22 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample#7 153

xvii

Fig. No. TITLES Pg. No.

Fig. 23 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample#8 153

Fig. 24 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample#9 154

Fig. 25 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample#10 154

Fig. 26 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample#11 155

Fig. 27 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample#12 155

Fig. 28 Percentage of Ferric Reducing Power Capacity of Rose Water 156 Samples (05 and 10 ml) Equivalent to 50, 100, 200 µg/ml of Ascorbic Acid as Standard

Fig. 29 In-Vitro Anti-Inflammatory Activity of Rose Water Samples 157

Fig. 30 In-Vitro Anti-Inflammatory Activity of 10 ml Rose Water Samples 157 Compared with 100µg/ml of Standard Drug (Diclofenac Sodium)

Fig. 31 In-Vitro Sun Protecting Factor of Rose Water Samples 158

Fig. 32 Anti-bacterial activity of rose water sample # 1 160

Fig. 33 Anti-bacterial activity of rose water sample # 2 161

Fig. 34 Anti-bacterial activity of rose water sample # 3 162

Fig. 35 Anti-bacterial activity of rose water sample # 4 163

Fig. 36 Anti-bacterial activity of rose water sample # 5 164

Fig. 37 Anti-bacterial activity of rose water sample # 6 165

Fig. 38 Anti-bacterial activity of rose water sample # 7 166

Fig. 39 Anti-bacterial activity of rose water sample # 8 167

Fig. 40 Anti-bacterial activity of rose water sample # 9 168

Fig. 41 Anti-bacterial activity of rose water sample # 10 169

Fig. 42 Anti-bacterial activity of rose water sample # 11 170

xviii

Fig. No. TITLES Pg. No.

Fig. 43 Anti-bacterial activity of rose water sample # 12 171

Fig. 44 Bacterial Contamination :Gram-Positive and Gram-Negative 172 Bacteria Present in Rose Water Samples

Fig. 45 Percentage of Change in Skin Glow After Application of Rose 173 Water Samples

Fig. 46 Comparison of Rose Water Samples Enhancing Glow Effect 173

Fig. 47 Percentage of Change in Skin Hydration After Application of 174 Rose Water Samples

Fig. 48 Comparison of Rose Water Samples Enhancing Hydration Effect 174

Fig. 49 Percentage of Change in Skin Oil Content After Application of 175 Rose Water Samples

Fig. 50 Comparison of Rose Water Samples Enhancing Oil Content Effect 175

Fig. 51 In-Vitro Anti-Oxidant Activity by Ferric Reducing Power of 176 Cream Formulation F1 ,F2 and F3

Fig. 52 Percentage Inhibition of Diclofenac Sodium and Formulated 176 Cream Against Denaturation of Protein

Fig. 53 Relationship Between Ferric Reducing Power Anti-Oxidant 177 Activity and Percentage Inhibition of Protein Denaturation of Cream Formulation F1

Fig. 54 Relationship between Ferric Reducing Power Anti-Oxidant 177 Activity and Percentage Inhibition of Protein Denaturation of Cream Formulation F2

Fig. 55 Antitussive Activity of Rose Water Based Formulation C1 178 Contaning Sample#12 As Active Ingredient

Fig. 56 Antitussive Activity of Rose Water Based Formulation C2 178

xix

Fig. No. TITLES Pg. No. Contaning Sample#9 As Active Ingredient

Fig. 57 Antitussive Activity of Rose Water Based Formulation C3 179 Contaning 50% Sample#12 As Active Ingredient

Fig. 58 Antitussive Activity of Rose Water Based Formulation C4 179 Contaning 50%Sample#9 As Active Ingredient

LIST OF ABBREVIATIONS

TLC Thin Layer Chromatography

HPLC High Performance Liquid Chromatography

GC Gas Chromatogarphy ml Milliliter

Kg Kilogram

Hrs Hours

RPM Rotation per minute

HS-GC-MS Head Space-Gas Chromatography-Mass spectrometry

FT-IR FourierTtransform Infra-Red Spectroscopy

OECD Organization for Economics Co-operation and Development

LC50 Leathal Concentration at which 50% population killed

µg Microgram

KFe3(CN)6 Potassium ferricyanide

TCA Trichloro acetic acid

W/V Weight by volume nm Nanometer

As Absorbance of sample

Ac Absorbance of standard

Mg Milligram

xxi

UV Ultravoilet

SPF Sun Protection Factor

CF Correction Factor

EE Erythemal Effect Spectrum

I Solar Intensity Spectrum

Abs Absorbance

TSA Tryptic Soya Agar

SDA Sabrose dextrose agar

µl Microliter

MHA Muller Hinton Agar

V/V Volume by Volume

BST Brine Shrimp Test

ROS Reactive Oxygen Species

RNS Reactive Nitrogen Species

xxii

ACKNOWLEDGMENTS

First of all thanks and prayers to Almighty Allah without whose blessing and munificence, it would not have been possible to complete this special work praise to his Prophet Hazrat Muhammad (Peace Be Upon Him)who is externally a beam of knowledge and guidance to human resource.

I want to express respectful thanks and sincere gratitude to my research supervisor Prof. Dr. Iqbal Azhar, Dean, Faculty of Pharmacy and Pharmaceutical Science, University of Karachi. Moreover, my Co-supervisor Dr. Zafar Alam Mahmood, Member Board of Studies, Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi. This thesis could not have been written without his guidance, dedication, cooperation, valuable suggestion and passionate support.

I am sincerely obliged to Dr. M. Mohtasheem ul Hasan, Chairman, Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Science, University of Karachi for his cooperation during the work. I am also grateful to Ms. Farah Mazhar, Assistant Professor and Mr. Salman Ahmed, Lecturer Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Science, University of Karachi for their support and help.

I am particularly thankful to Prof. Dr. M. Harris Sohaib, Dr. Rabia Ismail Yousuf, Depertment of Pharmaceutics. Dr.Asia Naz Awan, Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Pharmaceutical Science, University of Karachi. Dr. Tanveer Abbas, Depermtent of Microbiology, Dr. Salman P.C.S.I.R, Mr. Aamir Ali Zaidi, Sanofi Aventis Ptv Limited, Pakistan for providing facility for performing part of my research work in their labs.I also thankful to Getz Pharma (Private) Limited and Abbott Laboratories (Pakistan) Limited for providing standard drugs .

I would like to avail this opportunity to pay my regards to non-teaching staff, specially Mr.Rehan, Mr.Khawaja Azhar, Mr.Abdul Qadir, Mr.Noman Ahmed, Mr.Sahab, Mr.Kashif, Mr.Tauseef, Mr. Mansoor, Mr. Amir of Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Science, University of Karachi, for their technical assistance and help during the research work.

xxiii

This research would never be attainable without the encouragement of my dearest parents, my husband who were always cooperative, helpful and encourage me at every step of higher learning. My sons who sacrificed a lot during research work.

I would like to thanks my friends Dr. Najma Shaheen, Dr. Shela Imam, Dr. Farhana Tasleem, Dr. Nausheen Hameed, Mr. Umer, Ms.Rafia and Ms.Rifat for their constant care, love, and support throughout my research work.

In the end, I want to express my honest gratitude to everyone who helped me throughout the achievement of this destination.

Safia Abidi

xxiv

ABSTRACT

The plant-based medicament isan essential therapeutic weapon to fight against the various human and animal diseases. The present study is based on the rose water obtained from the petals of Rosa damascena Mill available in Pakistan and to correlate its usage and application in personal care preparations. In this study twelve samples of rose water were examined in which eleven samples were collected from the local market / provided by the manufacturer on request, and one hydro distilled sample was prepared on lab scale. The thesis has alienated into five parts. The first part has two portions.

Part I (a) consists of the brief introduction related to the rose water botanical source, the region of the world where rose water distilled, chemical constituents, historical, traditional and medicinal importance. In the firstpart, the value of standardization also highlighted through the introduction of standardization, in the end of the first part, the objectives of the present study is discussed.

Part I (b) deals with the literature review of Rosa damascena. Rose plant widely disseminated in the plant kingdom and currently consumed bya significant amount as a therapeutic agent. Literature data suggested that rose flowers associated with various biological and pharmacological activities, including anti-cancer, cardiovascular, analgesic, anti-inflammatory, anti-oxidant, anti-microbial and helpful in skin care.

Part II describes the experimental work of the present study. Part III shows the results of studies on rose water samples and their physicochemical study, estimation of their volatile components through HS-GC-MS, determination of functional groups through FT- IR, toxicity study, in-vitro antioxidant activity study against standard ascorbic acid, in- vitro anti-inflammatory activities against standard diclofenac sodium, in-vitro sun protection factor determination, anti-microbial activity, bacterial contamination study, cosmaceutical effect on topical application on skin to observe the hydration glow and oil content. Two formulations prepared one is rose water-based cream, study its anti- inflammatory and anti-oxidant effects against standard drugs, and the other is rose water based cough syrup and performed its antitussive effect on rats.

xxv

In the present study, the pH of rose water is within the range from 4-6 which is an acceptable and non-skin irritating pH value and suitable for topical application. The phytochemical study indicates the presence of flavonoids, tannins, saponins, triterpenoids and fixed oil, whereas the volatile components which have detected through HS-GC-MS are phenyl ethyl alcohol, citronellol, pentadecane, heptadecanol, octadecanol, tetracosane, decane and nonane. FT-IR estimation indicates the presence of carbohydrates, an amine group, a hydroxyl group, alkane, alcohol, ester, ether and carboxylic anhydrides. Toxicity study of rose water indicated that it is non-toxic and LC 50 is more than 3500µg/ml. In- vitro antioxidant activity of three samples 12, 9 and 8 shown marked % reduction. The reducing power of sample# 12 recorded as highest 841.66, followed by sample #9 (533.33) and then sample #8 (458.33) against 50µg/ml standard ascorbic acid. The in- vitro anti-inflammatory activity of rose water studied on four different concentration that is 3,5 7 and 10 ml, and it observed that 10 ml of rose water shown protein inhibition activity equivalent to 100µg/ml of standard diclofenac sodium.

Rose water protects from the UV radiation. The sun protecting factor of rose water samples ranges from 3.956 to 0.218. Results of anti-microbial activity indicated that all the test organisms had not shown dose-related sensitivity and thus no zone of inhibition was observed by well diffusion method. The bacterial contamination indicates that rose water samples contain both gram positive and gram negative bacilli. The skin hydration, as well as the glowing effect, significantly increased after the application of rose water.

Two formulations are designed based on rose water one is rose water-based cream, and other is rose water based cough syrup.

Results of in-vitro anti-inflammatory activity by protein (egg albumin) denaturation of cream formulation throughout the concentration range of 50-1000 µg/ml. For F1 cream formulation was 52.2-80.6 % F2 was 41.4-65.2%, and F3(Placebo) was 0.375-43.67% and for standard drug diclofenac sodium observed within the range of 35.33-86.32%. The

IC 50 value of F1 and F2 are 257.39µg/ml and 375.41µg/ml respectively.

The reducing power of formulated rose water-basedcream increased with the increase of concentration (25-1000 µg/ml). The reducing power of cream noted as F1 ranges from

xxvi

11.80 % to 81.55 %, F2 formulation antioxidant activity ranges from 8.05 % to 72.81 % and F3 formulation which is placebo cream 7.38% to 9.12% ascorbic acid equivalents (AES)µg/g.

Four formulations of cough syrup are designed based on rose water and checked their antitussive activity at three different doses (5 ml, 10 ml and 15ml) against standard ivy extract 5 ml/70kg and Dextromethorphan 10 ml/70 kg and it observed that C1 has the highest inhibition of a cough that is mean±S.E.M (9.00±2.08, 4.33±1.76 and 3.00±0.57) at 30min, 60 min and 90 minutes respectively.

Part IV accentuates the overall discussion based on the results of rose water study. Rose water exhibit an anti-oxidant, anti-inflammatory, prevent premature aging, provide nourishment and glow to the skin prevent UV penetration. Rose water cream shows good anti-oxidant and anti-inflammatory activity and rose water based cough syrup has an excellent antitussive effect.

Part V has the general conclusion and suggestions for future studies.

xxvii

xxviii

xxix

Part I (a)

1. Introduction

1.1 INTRODUCTION

For centuries, Rose and its preparations based on rose water and rose oil have been declared as well-established, preferred and demanding commodities with immense consumption in personal care, confectionery, dietary supplements and house hold products along with prominent medicinal role and application. Rosa damascena Mill, also known as Damask rose, is best the known flower, cultivated across the world and available in different colors, most commonly as red and pink. According to the world standards during May and June high quality rose oils are obtained but now a day‟s, cut flower growing condition in Pakistan face a unique challenges because of the high temperature during summer, the good quality flower only grow in the month from October to March but by adopting the advanced agricultural techniques and by the support of government and agencies farmer developed the green house for the better quality production of roses throughout the season. Therefore a huge quantity of roses are available in domestic market, generating substantial revinue for the cultivators and retailers. Although the climatic condition of Pakistan is favouable for the growth of roses and Pakistan produces huge quantity of rose water but the middle eastern counteries such as Iran, Turkey, Saudi-arabia and Oman are also the largest producers of the rose water because of the availability of damask (American society for horticultural science.12 Dec 2011). The major cities of Pakistan where rose flowers grow include, Multan, Rawalpindi, Lahore, Chunnian, Pattoki, Karachi, Hyderabad, Mansehra, and Quetta. (Usman et al., 2014).The genus Rosa, belonging to the family Rosaceae, includes 200 species and more than 20,000 cultivars (Tariq et al., 2016). Globally, roses are grown in a number of countries, such as Iran, Bulgaria, Spain, Italy, and France.

The main products of rose are rose hydrosol, perfume, mouthwash, cream, and jam. It is also used as a flavoring mediator in the preparation of different food items (Nikbakht et al., 2008). The marketed analysis of rose water in Pakistan indicates that the different laboratories are involved in the large production of rose water. The commercially available rose water using in different products are available in spray form, pet bottles of 100, 300 and 750 ml etc and mostly prepared by steam distillation.

The qualities, worthless secret and reimbursement of rose water exposed through ancient romantic stories that relate as follow, a royal leader in ancient Persian, for his royal wedding ceremony to a princess, once pre-arranged rose water to be filled in his royal fountains. After their wedding ceremony, as the royal pair was taking a walk through the lovely gardens, the tiny droplet was seen on the plane of the rose water fountain. These tiny droplets were rose oil drop that had produced below the temperate streaming daylight. The royal emperor was intrigued to find that the droplets, when touched had an oily feel and an attractive scent emitted from them. Thus the advantage of rose water along with the rose oil was initially concealed in Persia(BinaRani et al.,2013).Rose water is the most abundant and popular product, obtained from the petals of Rosa damascena plant which contains about 10 – 15 % of the rose oil. Traditionally in the religious ceremonies rose water was used to calm and relax people, (Nikbakhtet al., 2008). Rose water is also significant due to its role in aromatherapy; Roman people enjoyed bathing with rose water (Manus et al., 2009). The benefits of rose water are multiple.It acts as an analgesic (Rakhshandah et al., 2008), anti-inflammatory and soothing agent (Garwood et al., 2009), rich source of antioxidants so help in the construction of the skin tissues and provides strength to skin cells (Garwood et al., 2009) and incorporated in perfumes, collyrium and lotions also work as a blood purifying agent used in many formulation to treat pimples, acne and improve skin complexsion (Hameed and Vohra et al., 2001). Rose oil traps the moisture of the skin acts as a moisturizer and not only helps to treat dry skin but also makes the skin smoother (Luxemag, 2009). Excessive exposure to the ultraviolet radiation damages the elastin fiber of the skin resulting in loss of firmnes. Rose water helps in protecting from the UV radiation (Scirrotto et al., 2013). It acts as a mild sedative, antidepressant, mood enhancing agent and relieves the anxiety (BinaRani et al., 2013). Rose water has hypnotic effects (Rakshanda et al., 2006), antitussive (Shafei et al., 2003) and anti-HIV effects (Mahmood et al., 1996) used as a hair tonic, disinfectant, anti-acne, insect repellant, used in eye infections and different types of cosmetic formulation (soaps, lotions, cream cleanser) (BinaRani et al., 2013), rose water is also incorporate in ointment as emollient produce cooling effect (narayanswami and biswas., 1957). Beside rose flower, the other medicinally important flower used in Pakistan include Acacia nilotica (L.) Del. Used as astringent in diarrhoea and dysentery (Meena et

3 al., 2006), Borago officinalis L. used as antipyretic, diuretic and expectorant (Rizvi et al., 2007), Camellia sinensis L.used as anti-allergic (Yoshikawa et al., 2007), Hibiscus rosa- sinensis L. expectorant, antioxidant, anti-inflammatory (Venkatesh et al., 2008), Prunus persica protect against solar radiation (Kim et al., 2002).

The demand of rose water and other herbal products have many times increased due to their utilization as pharmaceuticals, neutraceuticals and cosmaceutical. Such utilizations also increased the health practiocners and consumers concern regarding the quality of rose water and other natural products. The quality, safety and efficacy of herbal products are associated with the standardization methods.There are many issues associated with the plant raw material but two are more important, first is the verification of the plant species and second is the seasonal effects.American Herbal Product association defines standardization as “Standardization refers to the body of information and control necessary to produce material of reasonable consistency” (Waldesch et al., 2003). For the standardization of any plant-based product certain points are always taken into consideration such as quality and identity of the drug sample, organoleptic and pharmacognostic evaluations, quantitative analysis, the study of the chemical constituent present (phytochemicals), antimicrobial, cytotoxic studies and biological activity evaluation. The activity of any formulation depends upon the chemical constituenst and their quality and quantity. Plant-based therapeutic agents are marketed now adays as standardized preparation either in any form solids (pills, powder) liquid (extracts).These powders and extracts are prepared by the different methods of extraction (percolation, maceration or distillation) with the help of different organic solvents or by simple water. The WHO has established some guidelines for the standardization of herbal drugs, to determine their morphology, habitat, macro, microscopic, histological examinations, qualitative and quantitative assessments of biomakers, physicochemical testing, toxicity study, microbial and radioactive contaminations (Shrikumar et al., 2006).

Table-1.1 General testing parameters for the standardization of herbal medicine

Title Testing Parameters Guidelines

General Data Geographical Good

Harvesting time Agricultural Practice (GAP) Harvesting process Processing

Description Macroscopic According to Identity Pharmacopoeias Microscopic

Chemical

TLC fingerprints

Purity Foreign matter According to Pharmacopoeias Ash/Sulfated ash

The content of extractable matter Water content Constituents with known therapeutic Assay activity (biomarker) According to Pharmacopoeias Constituents with unknown therapeutic activity (marker substances) Titrimetric Photometric HPLC/GC/TLC

Pesticides Contaminants Ph. Eur. Recommended Heavy metals limits for herbal drugs Aflatoxins (Oct. 91) Regulation on aflatoxins (Nov. 90) Ph. Microbiological purity Radioactivity Eur. 1997 Suppl. 1999

The objective of the present study is to establish the methods for standardization of rose water and to explore the medicinal uses of rose water available in Pakistan. At present in Pakistan many manufacturer are producing rose water. Some of the major producers are Mohammad Hashim Tajir Surma, Saeed Ghani (Pvt) Ltd., Qarshi Industries (Pvt) Ltd, Hamdard Laboratories (Waqf) Pakistan, and Marhaba Laboratories (Pvt) Ltd. Such manufacturers also exporting their rose water to fulfill the need of rose water for medicine, cosmetic and neutraceutical industries. There is a dire need to established standard procedures not only for the production of rose water but also with reference to its biological, chemical, pharmaceutical and cosmaceutical applications .The present study deals with establishment of some methods for the standardization of rose water upto the international standards. In this connection total twelve samples of rose water were analyzed, eleven marketed sample collected from the local market or provided by the manufacturer on request, one sample of rose water was prepared by making slight changes in the previous established distillation method on lab scale. Therefore the rose water has tested for physicochemical, antioxidant, anti-inflammatory, cosmaceutical activities (effect rose water on skin hydration glow and oil contents), ultraviolet radiation protection (SPF). Further more the possibility of its medicinal uses and application was also assessed by preparing two formulations one was rose water based cream and evaluate its anti-oxidant and anti-inflammatory properties and second was rose water based cough syurp and to check its in-vivo antitussive effect on rats was studied.

Part I (b)

Literature Review

1.2 INTRODUCTION

The genus Rosa, belonging to the family Rosaceae, includes 200 species and more than 18,000 cultivars (Gudin, 2000). Roses used for ornamental purposes, perfumery industries, medicinally, garden plants, cut flowers or indoor plants. Rose essential oil has a wide range of application in many industries used for scenting and flavoring agent, direct utilization or making a variety of food products (Hassanein et al., 2010).

Among 200 species of rose the most important one is the Rosa damascene Mill which is also known as damask rose (Kaul et al., 2000), damask, rose of Castile, Gul-e- Mohammadi (Loghmani-Khouzani et al., 2007) and king of flowers (Mahboubi et al., 2016) (Nikbakht et al., 2004-2005). Rosa damascene is the hybrid species of R.gallica and R.phoenicia, belonging to the family Rosaceae. Damask rose is grown as an ornamental plant both in houses and in gardens and has a good fragrance effect (Dolati et al., 2011). Beside this, it also possesess nutritional and industrial importance (Boskabady et al., 2011, Jabbarzadeh and Khoshkhuiet al. 2005). This plant is called Damask rose because it was firstly brought to Europe from Damascus (Gudin et al., 2000) and mainly cultivated in Turkey, Bulgaria, Iran, India, Morocco, South France, China, South Italy, Libya, South Russia and the Ukraine (Buttner et al., 2001).

1.3 MORPHOLOGY

It is deciduous perennial bushy growing shrub about 1 to 2 meters or 7 to 3 ft in height having curved prickles, stiff bristle with imparipinnate and compound leaflets (Libester, M. Delmar‟s Integrative Herb Guide for Nurses 2002). Delmar Thomson Learning, Albany: p. 360-370). The fragrance of the rose flower is constant and distinct. The method of propagation of rose plant is by cutting while the total life duration is about 50 years. It produces good quality flowers after three years and the total economic period is about 25 years (Nikbakht et al., 2008).

1.4 HISTORY

It believed that many ancient plants belong to the Rosaceae family. The history of rose is about 25-40 million years old, around 5000 years back Mesopotamian made clay tablets of rose which is the oldest record, but many believers say that the Babylonians were the first who used rose water. The bright image of rose also raised in the Cleopatra era. She was also known as a lover of roses and used roses in her famous baths of aphrodisiac recipes.The Greeks taught the Romans about rose, roman used rose water as an antiseptic and antibacterial agent, they used rose water in washing hands and baths.

In the 11 century, the Turkic people made the syrup of rose water. They preserved the rose water in the copper container. In 14 century the Ottomans physicians used rose products in their medication, they used rose in the form of water, paste, oiland sherbet.

In America, the fossils of rose found that are around 30 million years old. Iran is also among the main countries regarding the production of rose products especially the rose water and rose oil.

Rose water also used in many religious and spiritual ceremonies in ancient times; it also became a religious symbol in the Christian community, they symbol red rose as the blood of Jesus (www.gulsha.com.tr/en/rose-damascena/history-of-rose-water.aspx).

1.5 VARIETIES OF ROSA DAMASCENA

There are two varieties of rose (Huxley 1992). The summer damask has a short duration of flowering, and the autumn damask hasa long duration of flowering. They are morphologically similar and not distinguishable (Jerry Haynes, 2010).Traditionally and commercially the summer rose posseses more importance for making rose water, rose oil, and in perfumery (Lavidet al, 2002).

1.6 PHYTOCONSTITUENTS OF ROSA DAMASCENA

The chemical composition of the Rosa damascena reported from different part of the world include phenyl ethyl alcohol, citronellol, gereniol, nerol, nonadecane, tricosane, gerenyl acetate, eugenol and henicosane.The chemical composition of rose essential oil is listed in Table-1.2.1. Rose absolute consists of phenyl ethyl alcohol (78.4%), gereniol (3.7%), nonadecane (4.4%) and citronellol (9.9%).While phenyl ethyl alcohol, citronellol, nerol and gereniol are the main componenets of rose water (Ulusoy et al., 2009).

Table-1.2.1 Chemical Composition of Rose Essential Oil Obtained from Different Parts of the World

Main Components Origin References

Phenyl ethyl alcohol (70.9%), citronellol (3.7%), rhodinol (2.7%), citranellyl acetate (2.5%), eugenol Pakistan Khan et al., 2005 (1.6%), geraniol (1.5%)

Citronellol (23%), nonadecane (16%), geraniol Iran-Kashan Sadraei et al., 2013 (16%), heneicosane (5%)

Citronellol (14.5-47.5%), nonadecane (10.5-40.5%), Iran-Kashan Sadraei et al., 2013 geraniol (5.5-18%), henicosane (7-14%)

Citronellol (48.2%), geraniol (17%), b-phenyl ethyl Iran-Kashan Mahboubi et al., benzoate (5.4%) and phenyl ethyl alcohol (5.1%) 2011

Citrenellol (35.2%), geraniol (22.2%), nonadecane Turkey Ulusoy et al., 2009 (13.8%), nerol (10.3%)

Citronellol (15.9%-35.3%), geraniol (8.3-32.3%), India Verme et al., 2011 nerol (4-9.6%), nonadecane (4.5-16%), heneicosane (2.6-7.9%)

Linalool (3.4%), nerol (3.1%), geraniol (15.5%), 1- Iran-Guilan Yassa et al., 2009 nonadecene (18.6%), n-tricosane (16.7%), n- pentacozane (5.1%), n-hexa triacosane (24.6%)

Citronellol (38.7%), geraniol (17.2%), nerol (8.3%), Turkey Jirovetz et al., nonadecane (7.2%) 2006

Citronellol (24.5-42.9%), nonadecane (6.4-18.9%), Turkey Bayrak et al., 1994 geraniol (2.1-18.1%), ethanol (0-13.4%), heneicosane (2.3-8.9%), nerol (0.75-7.6%) and 1-nonadecene (1.8-5.4%)

Phenyl ethyl alcohol (27.2%), octadecane (10.5%), India Perumal et al., hexadecane (7.8%) 2012

Citronellol (23.4%), geraniol (19.0%), nonadecane Bulgaristan Gochev et al., (11.9%), nerol (7.5%) 2009

Citronellol (23-28%), geraniol (14-20%), nonadecane Saudi Kurkucoglu et al., (11-16%), nerol (6-11%), linalool (8%) and Arabia 2013 heneicosane (7%)

1.7 PRODUCTS OBTAINED FROM ROSA DAMASCENA PETALS

Among the major products obtained from the rose is the rose water which contains 10- 50% of the rose oil. Other products include rose oil which is very expensive and a large amount of rose petal about 3000 parts make only one part of rose oil (Nikbakht et al., 2008) (Moein et al., 2010) and rose hips both in fresh and dried form are also used (Nikbakht et al., 2008). Different types of extract such as hydrosol, aqueous, ethanolic and absolute are used for a research study and economical purpose (Kurkcuoglu et al., 2003).

1.8 TRADITIONAL USES OF ROSA DAMASCENA

Traditional medication of Iran, rose extract is used for the treatment of different diseases such as menstrual bleeding, chest and abdominal pains, laxative and cardiotonic (Shahriari et al., 2006). Rose essential oil and rose water are also used in the healing of different aliments, rose water are used for eye washing work as antseptic (Gochev et al., 2008) mouth disinfection (Akhmadieva et al., 1992). Abdominal pain chest and bronchial congestion, decoction of dired petals was used to treat fever work as diuretic also play their role in breast pains (Foter et al., 1990).

The traditional uses of Rosa damascena are listed in Table-1.2.2.

Table-1.2.2 Traditional Uses of Different Parts of Rose Flower

Rose Products Traditional Uses References Treatment of chest and abdominal pains Decoction of menstrual bleeding digestive ailments Shahriari et al., 2006 flowers (gentle laxative for constipation) cardiotonic agent for strengthing the heart muscles Rose hips Blood purifier Mahboubi et al., 2016 Decoction of Diuretic relief fever, breast pain,and Foster et al., 1990 dried flower menstrual problems Antiseptic agent for eye washing mouth Rosewater disinfectant antispasmodic agent, bronchial Gochev et al., 2008 and chest infection

1.9 CURRENT USES OF ROSA DAMASCENA

The pharmacological effects of different types of extracts of the rose flower are listed in the Table-1.2.3.

Table -1.2.3 Pharmacological Effects of Different types of Extracts of Rose Flower

Types of extract Effect Reference

Extracts (ethanolic, aqueous) Hypnotic Rakhshandah et al., 2004, 2006

Hydroalcoholic and ethanolic Analgesic Rakhshandah et al., 2008 extracts Hajhashemi et al., 2010

Essential oil Anticonvulsant Kheirabadi et al., 2008 Ramezani et al., 2008

Ethanolic and aqueous extract Antitussive Shafei et al., 2003

Ethanolic, aqueous-ethanolic Bronchodilatory Boskabady et al., 2006 extract,and essential oil Rakhshandah et al., 2010

Aqueous-ethanolic extract Potentiation of HR Boskabady et al., 2011 and contractility

Methanolic extract Anti-HIV Mahmood et al., 1996

Essential oil and absolute Antibacterial Mahmood et al., 1996 extract Andogan et al., 2008 Adwan et al., 2008

Methanol extract Anti-diabetic Gholamhoseinian et al., 2008

Aqueous, Antioxidant Ozkan et al., 2004 hydroalcoholic,ethanolic Kumar et al., 2009 extracts, essential oil Shahriari et al., 2007

Boiled extract Laxative Adwan et al., 2008

Hydroalcoholic extract Anti-inflammatory Maleev et al., 1972

1.10 TOXICITY

LD50 of Rosa damascena and rose absolute when given orally to the rats was more than

5g/kg while the dermal LD50 when study on rabbits was more than 2.5g/kg. The essential oil of Rosa damascena caused the sensitization in the sensitive person the major toxicity sign at very high dose was nephrotoxicity and hepatotoxicity was reported(Lis-balchin et al.,2006).

Part II

2. Materials and Methods

2.1 LIST OF CHEMICALS AND REAGENTS USED

Ascorbic acid (Sigma)

Sabouraud dextrose agar (Oxoid)

Cetomacrogol-1000 (Delta Chemsol)

Cetostearyl alcohol, stearic acid, lanolin (Avonchem)

Diclofenac sodium, Ethanol, Ferric chloride (Fecl3), Methanol, Potassium hydroxide, Tryptic soy agar (Merk)

Glycerine (Genmark)

Hydrochloric acid, Sulphuric acid (JT Baker)

Methyl paraben (Zxchem)

Potassium dihydrogen phosphate, Potassium ferricyanide, Trichloro acetic acid, (Daejung Chemicals)

Propyl paraben, Sodium hydroxide solution (Foodchem International Corporation)

Distilled water(Local Preparation)

Petroleum ether (Atom scientific)

Lead acetate solution (Merck)

Nitric acid (Merck)

Chloroform (Merck)

Acetic anhydride (Merck)

Sulphuric acid (J.T.Baker)

Olive oil (Shakeel Enterprises) 16

Sea salt (Al-azeem enterperisis)

Etoposide (PFIZER Laboratories Ltd.)

Methanol (Merck)

Tryptic Soya Agar (Oxide)

Sabouraud Dextrose Agar (Oxide)

Ciprofloxacin (5µg/ml) (Getz Pharma (Private) Limited)

Amoxicillin(30µg/ml) (Abbott Laboratories (Pakistan) Limited)

Nystatin (100µg/ml) (Wyeth Pakistan Limited (Hawkes Bay Plant).

Muller –Hinton agar (Oxide)

Potassium sorbate (Food Chem)

Xanthane gum (Food Chem)

Citric acid (Oxide)

Sorbitol (Local pharma)

Propylene glycol (Reagents Chemicals)

Sucralose (Reagents chemicals)

Honey flavor (Sigma)

IVY leaf extract (Local Pharma)

2.2 LIST OF APPARATUS/ EQUIPMENT USED

Analytical balance (Sartorius, Germany)

Amber glass bottle, (Ghani glass, Pakistan)

Burette (pyrex, Germany)

Burner(China)

Centrifuge machine (HERMLE Labortechnik GmbH, Germany)

Desiccator(Duran, Germany)

Energy saver bulb (Philips, Pakistan)

Filter paper(Whatmann, England)

Hot plate (Germany)

Incubator (Binder Gmbh Germany)

Laboratory glass wares (Pyrex, England)

Lux meter(LU-1010B, Matrix Electronics, MEXTECH, China)

Ostwald viscometer (Pyrex Germany) pH meter (Systrronics Germany)

Refrigerator (Dawlance, Pakistan)

Relative density bottle 25 ml(Pyrex Germany)

Rotary evaporator (Buchi, Switzerland)

Ultrasonicator (Zhangjiagang Shenke Ultrasonic Electronics Co., Ltd)

UV-Vis-1800 Spectrophotometer (Shimadzu Corporation, Australia)

Volumetric flask 100, 50, 25 ml (Pyrex Germany)

Vortex mixer (Benchmark Scientific, Inc., USA)

Water bath (Thermo Scientific, USA)

Distillation assembly

Separating funnel

Headspace coupled with GC-MS (Shimadzu 2010, Singapore 118227)

Fourier Transformed-Infrared (FT-IR) (SHIMADZU)

Vcare Skin Analyzer MODE:SK-8

2.3 LIST OF MICROORGANISMS

 Pseudomonas aeruginosa (ATCC # 27853)

 Escherichia coli (ATCC # 14169)

 Salmonella typhi (ATCC # 9394)

 Salmonella typhipara A(ATCC # 14028)

 Salmonella typhipara B (ATCC # 14023)

 Streptococcus epidermis(ATCC # 12228)

 Streptococcus fecalis(ATCC # 29212)

 Proteus mirabilis (ATCC # 12543)

 Corynebacterium xerosis (ATCC # 373)

 Staphylococcus aureus(ATCC # 6538)

 Klebsiella pneumonia (ATCC # 5046)

 Enterococcus sp.(ATCC # 12970)

 Candida albicans (ATCC # 10231)

 Aspergillus Spp. (ATCC #16404 )

2.4 ANIMALS USED

Albino Wistar rats

2.5 LIST OF SOLUTION USED

80% Methanol

Take 800ml of methanol and mix it with 200ml of water it make 80% methanol

Phosphate Buffer

Weight 3.99g of sodium hydroxide and dissolved it in 1000ml of water. Weight 13.60g of potassium dihydrogen phosphate and dissolved it in 1000ml of water taken 29.1ml of NaOH from 1000ml solution and 50ml of potassium dihydrogen phosphate solution mixed them and make up volume up to 1000ml with the help of distilled water and check pH was measured with the help of ph meter.

1% Solution of Potassium Ferricyanide:

1g of potassium ferricyanide powder was dissolved in 100ml of distilled water

10% Trichloro acetic acid Solution:

Take 10g of trichloro acetic acid powder was dissolved in 100ml of distilled water.

0.1% Ferric Chloride Solution:

0.1g of ferric chloride dissolved in 100ml distill water

2.6. COLLECTION AND IDENTIFICATION OF ROSE SPECIES (FLOWER)

Rose flower sample purchased from the local market of Karachi which was authenticated by Professor Dr. UsmanGhani Khan, Jinnah University for Women Nazimmabad, Karachi, Pakistan as Damask rose(Rosa damascena Mill.), family Rosaceae. Sample and the voucher specimen number(RD-01-12) is available in Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi herbarium.

Rosa damascena Mill.

Scientific Classification Kingdom Plantae Unranked Angiosperms Unranked Eudicots Unranked Rosids Order Rosales Family Rosaceae Genus Rosa Species R. × Damascena

2.7. PREPARATION OF ROSE WATER

A method reported by Verma et al., 2011 was modified and used to prepare rose water in the present study. The rose water prepared as batch production. Initially, 10 kg of fresh rose petals were procured from local market and after cleaning and sorting contaminations were shade dried at room temperature (25oC) till the moisture around 79.30% has removed. Petals were spreader over five nylon sieves every 4×4 feet in dimension containing 2 kg of petals. The duration of drying process lasted for 12 days. The dried petals were kept in amber colored air tight glass jars and used in the preparation of rose water.Dried rose petal (60g) were added into 1.5L of purified water and distilled for 4hrs to get approximately 800ml of rose water.

Modified Distillation Assembly

For the determination of volatile constituents (essential oil) the rose water was further extracted with petroleum ether. The extraction with petroleum ether was carried out in two steps. In the first step 500ml of rose water was mixed with 500ml of petroleum ether and the mixture was heated to 45oC for 150 minutes using a water bath to move the volatile constituents from aqueous to the organic phase. In the second step, the residue of

23 organic phase from the first step removed,and further 500 ml of fresh petroleum ether was added into the system. The content was again heated to 45oC for 150 minutes to complete the extraction procedure. The essential oils extracted from this procedure was further concentrated in a rotary evaporator equipped with a vacuum pump. During this process, 500 ml of petroleum ether extract was heated to 45OC and vacuumed by rotation of a pump motor at 60 rpm to remove the solvent (petroleum ether). The extract thus obtained subjected to study, the volatile constituent (essential oils) present in rose water (Moein et al., 2014)

2.8. EVALUATION OF PHYSICOCHEMICAL PARAMETERS

2.8.1 Color

Recorded using a dark background (Karthika et al., 2013).

2.8.2 pH

Determined using pH meter directly dip into the different samples of rose water included in the study

2.8.3 Detection of Saponins

2.8.3.1 Foam Test

In 2ml of the sample, 2ml of distilled water was added and shaken well and recorded the foam formation. Presence of foam which lasted for about 10 minutes confirmed the presence of saponins (Tiwari et al., 2011).

2.8.4 Detection of Tannins

2.8.4.1 Lead Acetate Test

The 3-4 drops of 10% aqueous lead acetate solution added into 2 ml of sample and the content was observed for the appearance of white precipitates which confirming the presence of tannins (Ukoha et al., 2011).

2.8.4.2 Nitric Acid Test:

In 2ml of the sample, few drops of dilute nitric acid were added and development of red to the yellow color was noted which indicated the presence of tannins (Saklani et al., 2012).

2.8.5 Detection of Triterpenoids

2.8.5.1 Liebermann Burchard Test

In 2ml of the sample, 1ml of chloroform was add,and few ml of acetic anhydride were also added and heated for few minutes. After cooling, few drops of concentrated sulfuric acid was added along with the side of the test tube. Development of blue color indicated the presence of triterpenoids (Saklani et al., 2012).

2.8.6 Detection of Fixed Oil

2.8.6.1 Spot Test

2ml of the sample taken on a white paper was observe after evaporation and match with control (2 drops of olive oil) on a white paper.The appearance of translucent spot confirmed the presence of fixed oil (Ukoha et al.,2011).

2.8.7 Detection of Flavanoids

2.8.7.1 Lead Acetate Test:

3ml of rose water was mixed with five drops of lead acetate and observed the formation of white colored precipitates.

2.8.7.2 Sulphuric Acid Test:

A small amount of sample was treated with the concentrated sulphuric acid formation of orange color was observed which indicated the presence of flavonoids (Sama et al., 2011).

2.9 DETERMINATION OF VOLATILE CONSTITUENTS BY HS-GC-MS

The direct method of analysis i.e., headspace coupled to gas chromatography-mass spectrometry (HS-GC-MS) was used in the present study. For the analysis and presence of the volatile components of the rose water. Headspace coupled with GC-MS (Shimadzu 2010, Singapore 118227) used for sample agitation; incubation time was 20 min at 120 °C. Separation of constituents achieved with a DB-5 MS capillary column (30 m, 0.25 mm i.d., 0.25 @m) coupled directly to the mass detector; whereas MS spectra recorded by electron impact mode (70 eV). The sample injected in the splitless mode at 220 °C. The initial column temperature was 50 °C, maintained for 5.0 min and then programmed to increase at 5 °C to 240 °C. Data acquisition performed in the full-scan mode (m/z range 35-600) after optimization of the MS.

2.10. DETECTION OF FUNCTIONAL GROUPS BY FT-IR

For the determination of functional groups in samples, Fourier Transformed-Infrared (FT-IR) used. FT-IR matched with the library data. For a liquid sample, 1-2 drop about 0.1 ml of samples was placed in between two sodium chloride plates to form a thin film of the solution and observed (Bruttia et al., 2016)

2.11 ACUTE TOXICITY STUDIES

2.11.1 Experimental Animals

Albino Wistar rats, weighing 187-196 g were obtained from the animal house, Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi.Animals acclimatized to standard laboratory conditions that are 12 hours light and 12 hours dark cycle, and fed standard rat die.t Animals were fasted 12 hours before dosing. The institutional animal ethical guidelines were strictly followed.

2.11.2 Experimental Design

The experiment conducted under the guideline of Organization for Economic Co- operation and Development 423. (OECD 423, Paragraph 23). A total of twelve animals divided into four dosage groups with three animals per dose.groupone serves as control take normal saline, group 2 received dose of 15ml/70kg, group 3 received dose of 20ml/70kg and group 4 received dose of 25ml/70kg. Doses calculated according to the body weight of each rat. Dosing was performed by using the ball –tipped intubation needle affixed with 1 ml syringe.

After administration of a single dose, the animal was observed for behavioral change and any other toxicity symptoms. Results were observed strictly for 30 minutes followed by hourly interval up to 48 hours (Amna et al., 2013).

2.12 BRINE SHRIMP (ARTEMIA SALINA) LETHALITY BIOASSAY

2.12.1 Shrimp’s Eggs Viability Test

To confirm the hatch ability of the shrimp‟s eggs viability test was performed. A high volume beaker filled with sea water (33g of sea salt in 1 L of distilled water). Animals were fasted 12 hours before dosing. A teaspoon full brine shrimp egg was added in seawater and placed in a cultivar for about 48 hours by visual examination checks the swimming of Artemia napuliis indicated how much eggs hatched.

2.12.2 Brine Shrimp Lethality Assay

Brine shrimp lethality assays was performed at the International Center for Chemical and Biological Sciences, University of Karachi, by following the method as described by lilybeth, 2013.

Different concentration of rose water samples prepared by obtaining the final concentration of 10, 100, 1000 µg/ml. With the help of the pipette collected the ten napuliis from the lighter side of the hatching chamber and put into vials containing the different concentrations of rose water extract and 4.5 ml of seawater. Etoposide used as a

27 standard drug. The control setup contained the 4.5 ml of seawater ten napuliis and 0.2 % of distilled water. After 24 hours each vial was observed with the help of magnifying glass and counts the number of survivals and data recorded.

2.12.3 Data Analysis

Data analysis is done through Microsoft excel spread sheet 2007 to determined the LC 50values.

2.13. IN-VITRO ANTI-OXIDANT ACTIVITY

The in- vitro antioxidant activity was determined by reducing power assay (Hinneburg et al., 2006).

2.13.1. Preparation of Stock Solution

100 mg of ascorbic acid dissolvedin sufficient amount of distilled water. The volume was adjusted with distlled water in 100 ml volumetric flask and labeled as a stock solution (1000ug/ml), various dilutions were prepared from the stock solution so that concentration range from 50 – 100 ugs/ ml achieved. It was use as a reference standard drug solution.

 5 ml of stock solution was adjusted to 100 ml with distilled water = 50 µg/ml

 10ml of the stock solution was adjusted to 100 ml with distilled water = 100 µg/ml

 20 ml of stock solution was adjusted to 100 ml with distilled water = 200 µg/ml

2.13.2 Determination of Reducing Power

12 samples of the different concentrations of rose water were separately mixed with 1ml of 80% methanol than 5ml of phosphate buffer of pH 6.6 (0.2 M) was added. Further,

5ml of 1%, (w/v) of potassium ferricyanide [KFe3 (CN)6] was added and mixed and incubated at about 50OC for 20 minutes.At the end of incubation period, 5ml of 10% of trichloroacetic acid (TCA) was added,and the mixture was centrifuged at 3000 rpm for 10 minutes. The lower layer was discarde and the upper layer was collected to 5ml of 28 upper layer part, 1ml of 0.1% w/v ferricchloride solution, 5ml of distilled water was added and mixed. The absorbance of the different sea green colored shade was observed in the solution at 700 nm (Siddhuraju et al., 2003). Increased in the absorbance indicated the increased in reducing power. Different dilution of ascorbic acid (50, 100, 200 µg/ml) was used as a standard drug to compare the activity of test solutions.

Percentage of reduction power (%) =1- [1- As/Ac] x 100

Where:

Ac absorbance of the standard at the different concentration tested

As: absorbance of the sample

2.13.3 Statistical Analysis

Experimental data expressed as mean ± SD.

2.14. IN-VITRO ANTI-INFLAMMATORY ACTIVITY

In- vitro anti-inflammatory activity was determined by inhibition of albumin denatured method (Mizushima and Kobayashi 1968).

2.14.1 Preparation of Stock Solution

100 mg of diclofenac sodium was weighed and transferred to 100 ml volumetric flask and dissolved in sufficient amount of distilled water. The volume was raised up to 100 ml with distilled water and labeled as a stock solution (1000ug/ml), Different concentrations, i.e., 20-100 µg/ml were made using appropriate dilutions of the stock solution,used as a reference standard drug solution.

 1ml of the stock solution diluted to 50 ml with distilled water = 20 µg/ml

 2ml of the stock solution diluted to 50 ml with distilled water = 40 µg/ml

 3ml of the stock solution diluted to 50 ml with distilled water = 60 µg/ml

 4ml stock solution diluted to 50 ml with distilled water = 80 µg/ml

 5ml stock solution diluted to 50 ml with distilled water = 100 µg/ml

2.14.2 Inhibition of Albumin Denaturation Method

2.14.2.1 Control Solution (50ml)

Phosphate buffer saline (28 ml) of pH 6.4 was transferred to freshly prepared egg albumin (2ml) and distilled water (20ml) was added to this to prepare control solution.

2.14.2.2 Standard solution (50ml)

Phosphate buffer saline (28 ml) of pH 6.4 transferred to freshly prepared egg albumin (2 ml) and 20 ml of a solution of different concentrations of diclofenac sodium 20-100 µg/ ml was added to this to prepared standard solution.

2.14.2.3 Test solution (50ml)

Phosphate buffer saline (28 ml) of pH 6.4 was transferred to freshly prepared egg albumin (2 ml) and 20 ml of different rose water samples.All the solutions were incubated at 37±2 oC for 15 minutes and it was then heated at 70oC in a water bath for 15 minutes. The solution was allowed to cool at room temperature. The absorbance was then measured using UV-visible spectrophotometer at 660nm using the vehicle as blank. The percentage inhibition of protein denaturation was calculated from the control using below under formula (Ullah et al., 2014).

% inhibition =Abs control- Abs test x 100

Abs control

Whereas,

Abs: absorbance

The viscosity of the solution determined by using Ostwald viscometer.The result of the experiment was expressed in mean ± SD.

2.15. DETERMINATION OF IN-VITRO SUN PROTECTING FACTOR (SPF)

The in-vitro determination of SPF of different rose water formulations were studied by spectrophotometric method. The different samples of rose water solution were filtered through Whatman No1 filter paper and collected in a conical flask.The absorbance value was determined between 290 and 320 every 5 nm increments, using water as a blank. The reading is taken in triplicateand the determination made at each point. The SPF was calculated using the below equation (Mansur et al., 1986).

320 SPF spectrophotometric=CFx ∑ EE (λ) x I (λ) x Abs(λ) 290

Where;

CF: Correction factor (=10)

EE(λ): Erythemal effect spectrum

I(λ): Solar intensity spectrum

Abs (λ): Absorbance of sunscreen product

EE (λ) x I (λ) values are constant (Sayre et al., 1979) the absorbance value obtain between 290 to 320 nm were multiplied by a correction factor (10) to obtain the SPF values. Data were expressed as the mean of triplicate.

Table-2.1 Normalized Product Function Used in the Calculation of SPF

Wavelength λ nm EE (λ) × I (λ) (Normalized)

290 0.0150

295 0.0817

300 0.2874

305 0.3278

310 0.1864

315 0.0839

Total 1

2.16. ANTI-MICROBIAL ACTIVITY

2.16.1 Collection of Isolates

ATCC cultures were collected from the Department of Microbiology, University of Karachi which include the following:

 Pseudomonas aeruginosa (ATCC # 27853)

 Escherichia coli (ATCC # 14169)

 Salmonella typhi (ATCC # 9394)

 Salmonella typhipara A(ATCC # 14028)

 Salmonella typhipara B (ATCC # 14023)

 Streptococcus epidermis(ATCC # 12228)

 Streptococcus fecalis(ATCC # 29212)

 Proteus mirabilis (ATCC # 12543)

 Corynebacterium xerosis (ATCC # 373)

 Staphylococcus aureus(ATCC # 6538)

 Klebsiella pneumonia (ATCC # 5046)

 Enterococcus sp.(ATCC # 12970)

The bacterial culture grown on Tryptic Soya Agar (TSA) plates for 24 hours at 35 oC. Each bacterial culture then washed with 10 ml serial saline the washing is repeated thrice and centrifuged at 3000 rotation per minutes for about 15 minutes and thencollect the sediments (cells). The cells of each bacterial species were re-suspended in 5 ml saline,and the standard loopful of each portion streaked on individual TSA agar plates, incubated at 35 OC for 24 hours and finally, a culture dilution of 1×108 / ml was prepared to test antimicrobial activity.

Similarly, the fungal ATCC culture collected from Sanofi-Aventis Pakistan.Which includes the following:

 Candida albicans (ATCC # 10231)

 Aspergillus Spp. (ATCC #16404 )

Fungal culture grown on Sabouraud Dextrose Agar (SDA) slants for 48 hours at 25oC. Gentally scraped the colonies by using serial wire loops into 10 ml of sterile saline, centrifuge at 3000 rpm for 15 minutes the sediments were collected and re-suspended in 10 ml sterile saline the procedure was repeated thrice and finally loop full of culture were streaked on agar plates which incubated at 25 OC. Finally, the culture dilution of 1×108/ l was ready to performed antifungal activity (Tatke et al., 2015).

2.16.2 Standard Anti-Microbial Agents

Antibiotic Ciprofloxacin (5µg/ml) and Amoxicillin (30µg/ml) were used as a standard for antibacterial activity.Amoxicillin was provide by Abbott Laboratories (Pakistan) Limited; Ciprofloxacin was provided by Getz Pharma (Private) Limited. Nystatin (100µg/ml) was

33 used for antifungal activity as standard provided by Wyeth Pakistan Limited (Hawkes Bay Plant). Standard stored in the refrigerator between 4-8 oC.

2.16.3 Anti-Microbial Assay

Agar well diffusion method reported by (Perez et al., 1990) was adapted to performed antimicrobial activity. TSA and SDA Agar prepared as described by the manufacturer (Oxide) 25 ml of media were then poured into a sterile petri dish and allowed to solidify when the media is adjusted make a well with the help of serial borer and 100µl of the test compound with the help of micropipette pour into the well. Plates were incubated at 37OC for 24-72 hours to observe the zone of inhibition. By measuring the zone of inhibition the result was calculated and compared with the standard (Tatke et al., 2015).

2.17. MICROBIAL CONTAMINATION

2.17.1 Sample preparation

Each rose water sample (1ml) was taken in a test tube separately and adds 9ml of sterile distilled water was added and shake well. Serial dilution prepared by transferring 1ml of first dilution (10-1) from each serial test tube into other 9ml of distilled water to make (1:100) procedure was repeate for making (1:1000) dilution.

2.17.2 Bacterial contamination

The Muller–Hinton agar was taken in plates and marked original, (1:10), (1:100), (1:1000) dilution with the help of sterilized pipette, serially diluted each sample was taken and dropped 1ml of each sample on the surface of MHA plates and the drops spread. The plates were left upright for dry then incubated at 37oC for 24 hours. After 24 hours bacterial growth on plates was observed and results were noted and the following equation calculated the colony count.

CFU= per ml= An average number of colonies for a dilution x 50 x dilution factor (Thoha et al., 2012).

2.18. CLINICAL STUDIES

2.18.1 Clinical Protocol

The experimental protocol was approved by Institutional Bioethics Committee, University of Karachi, Resolution No. IBCPH 22.

2.19. GLOW MEASURMENT

2.19.1 Study Protocol

 Glow measurement study was conducted at the Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi- Pakistan.

 Twenty healthy females subjects 20-25 years old having normal skin type participated in the study.

 All subjects checked for any dermatitis and skin allergy problem at the beginning of the study, including any previous skin treatment with any cosmetic.

 Forearm skin area (6× 3) was selected for the right study hand as control and left hand as a test.

 During the study period the subject has washed their hand normally without applying any soap or cleansing agents on the area marked on the forearm skin.

 The total duration of the study was three weeks during that period subject‟s were not allowed to expose the marked area directly to the sunlight.

 Samples applied in the form of a spray. All subjects applied the sample two puffs twice a day one in the morning and other in the evening

 Reading was taken using Luxmeter on a regular basis till the three weeks application completed.

 All subjects were pre-informed about the nature of the test.

 Written consent was signed by the subject‟s before the start of the study

2.19.2 Instrumentation

To measure the glow lux meter used. Readings in lux indicate a measure that how much photon is reflected back by the marked area of the skin. A 10 volt energy saver bulb was used to throw the light on the marked area.

2.19.3 Method

An area 6×3 cm on the forearm of each participant considered as a test. Rose water samples (2 puffs) was applied on a test area of the forearm of each participant twice aday and observation recorded each day until the end of three weeks the applications stopped. The reading obtained by keeping the probe of lux meter a constant distance away and in the same intensity environment range from 5 to 7 luxes (Saraf et al., 2010).

2.19.4 Statistical Analysis

The result expressed as mean ± SD. Using SPSS version 20.0 software with the help of one-way analysis of variance (ANOVA) shows that there is a difference between control and test groups.

2.20 DETERMINATION OF SKIN HYDRATION AND OIL CONTENT

2.20.1 Study Design

This study performed at Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi-Pakistan.

Exclusion criteria

 Participants involved in the study was not had any previous history of hypersensitivity and also was not having any skin problem like cut, wounds, dermatitis on the forearm area.

 Systemic and topical application of any cosmetics formulation such as moisturizer, sunscreens,etc.02 weeks before and during the study was not allowed

Inclusion criteria

 10 participant selected for each sample

 Females with age group between 20-25 years were selected.

 The participant with normal skin type

 The study performed for 03 weeks.

 During the study period, the participant was not allowed to use any skin formulation on the marked form area

 All the procedures performed according to the guideline and written consent signed by each participant

The skin hydration and oil contents were analyzed by using Vcare Skin Analyzer MODE:SK-8. Two puffs of rose water samples were applied on the marked forearm area twice daily one in the morning and other in the evening,and the readings were taken daily for three weeks. The percent increase in hydration and oil content was plotted against the weeks (Saraf et al., 2010).

2.20.2 Statistical analysis

The result expressed in mean ± SD by using SPSS version 20.0 with the help of one-way analysis of variance (ANOVA) followed by independence t-test.

2.21. CREAM FORMULATION

2.21.1. Placebo Cream and Its Preparation

The oil phase of the placebo cream formulation (F3) that is Cetostearyl alcohol, Cetomacrogol-100, Lanolin, Stearic acid, Glycerin mixed at 75oC by using a hot plate.

Whereas, for the preparation of aqueous phase purified water was heated in the separately in a 2500ml capacity beaker around 80oC ± 2. Upon continuous stirring added the methylparaben, propylparabenand borax and the temperature brought to 75oC ± 2. The oil phase was added to the water phase and stirred continuously for about 1- 2 minutes mixing continued till the required consistency of the cream isachieved.Then reduced the temperature to 45oC by using a cold water bath and stopped the mixing and stored the cream in a well-closed container and kept at room temperature (Imam et al., 2015). List of ingredients are mentioned in Table-2.2.

2.21.2 Cream Formulation and Its Preparation

Two sets of the cream formulations were prepared using ingredients shown in the Table- 2.3 one containing rose water 30g (F1) and the other contains 50 g (F2). The oil phase of the cream formulation that were cetostearyl alcohol, cetomacrogol-100, lanolin, stearic acid, glycerin was mixed at 75oC ± 2 with constant stirring by using a hot plate. While for the preparation of aqueous phase, purified water was heated separately in a 2500 ml capacity beaker at 80oC ± 2. Upon constant stirring methylparaben, propylparaben and borax were added and the temperature brought to 75oC ± 2. The two phases were mixed with continuous stirring for about 1-2 minutes finally rose water was added with constant stirring till cream formed. The temperature was further reduced to 45 oC using a cold water bath.The cream was stored in airtight container at room temperature for further studies (Imam et al., 2015).

Table-2.2 Composition of Placebo Cream

S.No Ingredient Used as Amount (gram)

01 Cetostearyl alcohol Emulsifying agent 12.5

02 Cetomacrogol-100 Emulsifying agent 2.5

03 Lanolin Emollient 20

04 Stearic acid Emollient 10

05 Glycerin Humectants 50

06 Liquid paraffin Barrier 15

07 Methylparaben Preservative 2

08 Propyl paraben Preservative 0.2

09 Borax Emulsifier, Preservative, Buffering 1 agent

10 Distill water Vehicle q.s to make 500 g

11 Total weight 500 gram

Table-2.3 Composition of Cream Formulation

S.No Ingredient Used as Amount (Gram)

01 Cetostearyl alcohol Emulsifying agent 12.5

02 Cetomacrogol-100 Emulsifying agent 2.5

03 Lanolin Emollient 20

04 Stearic acid Emollient 10

05 Glycerin Humectants 50

06 Liquid paraffin Barrier 15

07 Methylparaben Preservative 2

08 Propyl paraben Preservative 0.2

09 Borax Emulsifier, Preservative 1

Buffering agent

10 Active (rosewater) Sunprotecting agent 30g and 50g sample # 9

11 Distill water Vehicle q.s to make 500g

12 Total weight 500 gram

2.22. IN-VITRO ANTI-INFLAMMATORY ACTIVITY OF ROSE

WATER BASED CREAM FORMULATION

In-vitro anti-inflammatory activity determined by inhibition of albumin denatured method (Gautam et al., 2013; Mizushima and Kobayashi 1968).

2.22.1 Preparation of Stock Solution

250 mg of diclofenac sodium was weighed and transferred to 100 ml volumetric flask and dissolved with sufficient amount of distilled water. Make up the volume up to 100 ml with distilled water and labeled the flask as a stock solution (2500 µg/ml). From that stock solution, different concentrations (50-1000 µg/ml) were made,used as a reference drug solution.

 1 ml of stock solution diluted to 50 ml with distilled water to make 50 µg/ml

 3 ml of stock solution diluted to 50 ml with distilled water to make 150 µg/ml

 6 ml of stock solution diluted to 50 ml with distilled water to make 300 µg/ml

 15 ml of stock solution diluted to 50 ml with distilled water to make 750 µg/ml

 20 ml of stock solution diluted to 50 ml with distilled water to make 1000 µg/ml

The similar procedure was adopted to prepare the stock solution and dilution of the cream formulation (F1, F2 and F3) with ethanol (30% V/V).

2.22.2 Inhibition of albumin denaturation method

The method as described by the Mizushima and Kobayashi was followed with slight modifications. 2ml of egg albumin from the fresh hen egg was taken,28ml phosphate buffer pH 6.4 and 20ml (50µg/ml,150µg/ml, 300µg/ml, 750µg/ml and 1000µg/ml) dilutions of F1, F2, F3 and Standard) were taken. Incubated the mixture at 37oC for about 30min and then heated at 70oC for about 15 min cooled the mixture and observed absorbance at 660 nm using the vehicle as blank. The percentage inhibition of protein

41 denaturation was calculated from the control using the following formula (Ullah et al., 2014).

% inhibition =Abs control- Abs test x 100 Abs control

Whereas,

Abs: absorbance

The viscosity of the solution determined by using Ostwald viscometer.The result of the experiment expressed in mean ± SD.

2.23 IN-VITRO ANTI-OXIDANT ACTIVITY OF ROSE WATER

BASED CREAM FORMULATION

The in vitro antioxidant activity was determined by reducing power assay (Calabrone et al., 2015; Hinneburg et al., 2006).

2.23.1 Preparation of stock solution

Ascorbic acid 200mg dissolved in sufficient amount of distilled water. The volume was adjusted with distilled water in 50 ml volumetric flask to make a stock solution of (2000µg/ml) from that stock solution various dilutions (25, 100, 500 and 1000 µg/ml) prepared.

 0.625 ml of stock solution was adjusted to 50 ml with distilled water = 25µg/ml

 2.5 ml of stock solution was adjusted to 50 ml with distilled water = 100 µg/ml

 12.5 ml of stock solution was adjusted to 50 ml with distilled water = 500µg/ml

 25 ml of the stock solution adjusted to 50 ml with distilled water = 1000µg/ml

The similar procedure was adopted to prepare the stock solution and dilutions of cream formulations with ethanol.

2.23.2 Determination of reducing power

2.5 ml of F1 cream formulation (ranges 25- 1000 µg/ml) were separately mixed with 2.5 ml of 0.2 M phosphate buffer pH 6.6 and 2.5 ml of (1%,w/v) potassium ferricyanide. The mixture incubated at 50 oC for 20 minutes. At the end of incubation, 2.5 ml of (10%, W/V) trichloroacetic acid (TCA) was added to the mixture and then centrifuged at 3000 rpm for 10 minutes. Subsequently, 2.5 ml of supernatant mixed with 2.5 ml of distilled water and 0.5 ml of FeCl3 (0.1 %, W/V), the absorbance was recorded at 700 nm using UV-VIS spectrophotometer.Ascorbic acid used as a reference standard. The percentage reduction was calculated by using the following formula (Rohman et al., 2010).

Percentage of reduction power (%) =1- [1- As/Ac] x 100

Where:

Ac: Absorbance of the standard at the different concentration tested

As: Absorbance of the sample

The same procedure was adopted to calculate the reduction power of F2 and F3 formulations.

2.24 ANTITUSSIVE ACTIVITY OF ROSE WATER BASED COUGH

SYRUP FORMULATION

2.24.1 Preparation of cough syrup formulations

Four sets of cough syrupformulation were made and named as C1,C2,C3, and C4; ingredients mentioned in the Table-2.4 and 2.5.The syrup prepared as follows:

 In 10 ml of rose water was added methylparaben, propyl parabens and mixed well until a clear solution was formed.

 In a separate beaker, xanthan gum was soaked in rose water.

 Mixed sorbitol, glycerin and propylene glycol in a separate beaker, then added sucralose and honey flavor, upon complete mixing, IVY leaf extract was added.

 Finally mixed all the above solutions in a separate 2000ml beaker than potassiumsorbate and citric acidwas added.

 The solution was heated to obtain clear solution.

 Make up the volume with rose water (C1, sample#12), C2,sample#9, C3 50% sample # 12 + 50% purified water, C4 50% sample # 9 + 50% purified water.

Table-2.4 Composition of Cough Syrups Formulation C1 and C2

S.No Ingredient Used as Amount (Gram)

01 Ivy leaf extract Active ingredient 7.14

02 Methylparaben Preservative 1.20

03 Propylparaben Preservative 0.20

04 Potassium sorbate Liquid pharmaceutical 1.50 preservative 05 Xanthan gum Thickening agent 2.50

06 Citric acid Preservative 0.70

07 Sorbitol Sweetener 200.0 (Diet sugar) 08 Glycerine Soothing agent 25.0

09 Propylene glycol Non-active enabling agent or 50.0 excipient

10 Sucralose Artificial sweetener 1.50

11 Honey flavor Flavoring agent 2.5

12 Rose water (lab distilled Active ingredient Q.S for 1 L or marketed)

Table-2.5 Composition of Cough Syrups Formulation C3 and C4

S.No Ingredient Used as Amount ( gram)

01 Ivy leaf extract Active ingredient 7.14

02 Methylparaben Preservative 1.20

03 Propylparaben Preservative 0.20

04 Potassium sorbate Liquid pharmaceutical 1.50 preservative

05 Xanthan gum Thickening agent 2.50

06 Citric acid Preservative 0.70

07 Sorbitol Sweetener 200.0 (Diet sugar)

08 Glycerine Soothing agent 25.0

09 Propylene glycol Non-active enabling agent or 50.0 excipient

10 Sucralose Artificial sweetener 1.50

11 Honey flavor Flavoring agent 2.5

12 50% rose water + 50% Active ingredient Q.S for 1 L purified water

2.25 SCREENING FOR ANTITUSSIVE ACTIVITY OF COUGH SYRUP

The antitussive activity described previously by Miyagoshiet al. and also performed by Pulok K Mukherjee was adopted to evaluate the antitussive activity of the cough syrup formulations C1, C2, C3 and C4.

2.25.1 Animals Used

Wister albino rats of either sex weight range from 180-200g, provided by Department of Pharmacology, Faculty of pharmacy and pharmaceutical sciences, maintained on a standard diet, light and dark cycle.Ethical clearance for experimenting obtained from Institutional Bioethics Committee, University of Karachi. The animals were divide in to 07 Groups; Group 1 served as control group received only the normal saline, Group 2 received C1 formulation, Group 3 C2 formulation, Group 4 C3 formulation, Group 5 C4 formulation, Group 6 Standard drug IVY -Special extract EA575 and Group 7 received standard drug of Dextromethorphan.

2.25.2 Method

Sodium hydrogen sulfite solution prepared by dissolving 1 g of sodium hydrogen sulfite in 2 ml of distilled water and 0.7 ml of concentrated sulphuric acid required for the formation of sulfur dioxide, the reaction is as follow:

2NaHSO3 + H2SO3-a 2SO2- + Na2SO4 +2H2O

This protocol was for one rat after the formation of sulfurdioxide gas rat was exposed to the gas for 45 seconds.

The procedure for the cough induction through sulfurdioxide was took place in desecrator. In the base of desecator water was filled on which sodium hydrogen sulfide solution was placed in the small jar porous aluminum sheet has covered the base on which rat is present the sulphuric acid added with the help of pipette. The frequency of coughs were observed for all the animals of all groups at zero minutes, before drug administration and thirty, sixty and ninety minutes after drug administration (Marina et al., 2008).

Part III

3. Results

RESULTS

Current research investigation was under taken to study phytochemical screening along with the determination of biological activities of rose water obtained from the petals of Rosa damascene useful in its standardization as well as to identify and report the possible role and utilization of rose water in different phytopharmaceutical preparations. The most important approach of present groundwork, a part from physicochemical investigation was to evaluate the therapeutic significance of rose water and to develop a correlation between traditional uses with the results obtained through different pharmacological screening test utilizing in-vitro bioassays methadologies and through topical application of formulations

3.1 EVALUATION OF PHYSICOCHEMICAL PARAMETERS

For physico-chemical analysis of eleven different rose water samples were collected from local market or kindly supplied by the manufacturers one rose water sample was prepared in the lab and thus twelve samples were analysed.

Rose water was observed as transparent with a characteristic odor with pH between 4-6 shown in Table -3. The phytochemical analysis has been highlighted in Table-4. The test performed include, detection of saponin, tannins, triterpenoids, fixed oils and sulphuric acid and flavonoids. All these test were observed positive in sample # 8, 9 and 12.

3.2 DETERMINATION OF VOLATILE CONSTITUENTS BY HS-GC-MS

The volatile components of rose water samples were analyzed through HS-GS-MS, targeting detection of eight most important volatile components e.g. Phenylethyl alcohol, citronellol, pentadecane, heptadecanol, octadecanol, tetracosane, decane and nonane. It was observed that test samples behave in different manners with respect to presence of these volatile components. In terms of volatile constituents both qualitatively and quantitatively sample # 9 was found rich in volatile constituents as compare to other samples, followed by sample# 12 and sample#8 Result shown in Table-5.

3.3 DETECTION OF FUNCTIONAL GROUPS BY FT-IR

The result of detection of the functional group through FT-IR of rose water samples have been shown in Fig. 1 to 12. Data presented in Table-6 was used as a refrence for the interpretation of functional groups. All samples indicated presence of respective functional groups, however sample # 8, 9 and 12 were noted to be quite close in terms of spectral similarity.

3.4 ACUTE TOXICITY STUDIES

For acute toxicity determination, five samples were subjected. These include sample# 6, 7, 8, 9 and 12. Results presented in Table-7. All the animals in 15 ml/70kg, 20 ml/70kg and 25ml/70kg adult dose categories did not showed signs of any irregularity during whole 03 days of study duration after oral administration of rose water samples. The only transient clinical sign that was most pronounced at 25 ml/70kg adult dose included mild sedation. The animals noted as dull and inactive immediately after dosing, but the sign was disappeared after 120 minutes. The motor functions were normal with no signs of gait abnormality. The mucous membranes were normal in all animals, and there were no noticeable changes in the color of the eyes. All the animals except the control groups defecated semi-formed wet droppings/pellet that could not fit the description of outright diarrhea. There was no sign of acute toxicity seen during the 48 hours of study. There was no animal death in all sets of animals.

3.5 BRINE SHRIMP (ARTEMIA SALINA) LETHALITY BIOASSAY

The results of brine shrimp lethality assay of rose water samples shown in the Table 8- 19. Results obtained from the study showed that all rose water samples tested had

LC50˃ 3000µg/ml mention in Fig. 13-24, indicating practically non-toxic.

3.6 IN-VITRO ANTI-OXIDANT ASSAY BY FERRIC REDUCING POWER

Results of the in-vitro antioxidant activity of twelve rose water samples based on the calculation of reducing power capacity have presented in Table-20-25. Tables-20 and 23.

49 indicated the results of the absorbance determined at 700nm using 5ml and 10ml of each rose water sample, whereas Table-21 and 24 highlights the percentage of reducing power capacity. Calculated by respective absorbance value using 50µg/ml, 100µg/ml and 200µg/ml of ascorbic acid as standard drug respectively. The reducing power of sample# 12 recorded as highest 841.66, followed by sample # 9 (533.33) and then sample #8 (458.33) against 50µg/ml standard ascorbic acid. However, the significant reduction in reducing power observed as the concentration of standard drug (ascorbic acid) increased. Against 100µg/ml of ascorbic acid, the reducing power of sample # 12, 9 and 8 was recorded as 30.42, 19.27 and 16.56 respectively, while against 200µg/ml of ascorbic acid the reducing power further decreased to 10.27, 6.51 and 5.59 respectively .The overall reducing power of the three samples (sample # 12, 9 and 8) against 50,100 and 200µg/ml concentration of ascorbic acid using 5 and 10 ml sample size has been presented in Fig. 25.

3.7 IN-VITRO ANTI-INFLAMMATORY ACTIVITY

The anti-inflammatory activity of rose water samples at four different dosage level (3, 5, 7 and 10ml) was determined using inhibition of albumin denaturation against standard drug diclofenac sodium at five different concentrations (20, 40 .60, 80,100µg/ml).The results have been shown in the Table-26 and represented in Fig. 26. In the present study concentration-dependent, anti-inflammatory activity observed. Effect of 10 ml sample was noted to be equivalent to 100µg/ml of diclofenac sodium activity as mention in Fig- 27. The anti-inflammatory activity of standard drug diclofenac sodium was mentioned in Table-27 while viscosities of samples were mention in Table-28.

3.8 DETERMINATION OF IN-VITRO SUN PROTECTING FACTOR

The SPF value of the 12 samples of rose water ranged from 3.956 to 0.218. The highest value was observed in sample # 9 which was 3.956 and the lowest value of sample# 10 0.218. The results are shown in the Table-29

3.9 ANTI-MICROBIAL ACTIVITY

The anti-microbial analysis was performed by following the standard agar well diffusion method against the ATCC cultures of following bacteria:

 Pseudomonas aeruginosa (ATCC-27853)  Escherichia coli (ATCC-14169)  Salmonella typhi (ATCC-9394)  Salmonella typhi para A(ATCC-14028)  Salmonella typhi para B(ATCC-14023)  Streptococcus epidermis(ATCC-12228)  Streptococcus fecalis(ATCC-29212)  Proteus mirabilis(ATCC-12453)  Corynebacterium xerosis(ATCC-373)  Staphylococcus aureus (ATCC-6538)  Klebsiella pneumonia (ATCC-5046)  Enterococcus spp.(ATCC-12970)

Moreover, following fungal species:

 Candida albicans (ATCC-10231)  Aspergillus spp. (ATCC # 16404)

The antimicrobial assay of all the twelve rose water samples showed no zones of inhibition against standard agar well diffusion method .Results shown in the Table-30-41 and fig of anti-bacterial activity was shown in fig # 32-43.

3.10 MICROBIAL CONTAMINATION

Table-42 indicates results of bacterial contamination as identified after the microbiological analysis of all the rose water samples. Fig. 44 shows the bacterial contamination. The bacterial contamination was noted in all commercial samples tested in the present study sample # 6 showes 130CFU/ml. Whereas sample # 12 which was

51 freshly extracted in the lab, showed no bacterial contamination. The bacterial species which were identified including both gram-positive and gram-negative bacilli.

3.11 CLINICAL STUDIES

3.12 GLOW MEASURMENT OF SKIN

The statistically significant difference between groups, i.e., control and test after using a different sample of rose water as determined by independence t-test followed by one-way analysis of variance (ANOVA) revealed that the glowing skin effect was statistically significant after application of rose water. Sample # 09 showed the highest glow control reading was 14.73±7.42 and test reading was 20.83±7.54. The lowest glow was showed by the sample # 06 with 24.10±13.09 for control and 24.50±13.62 for the test group. The results expressed in mean±S.D summarized in Table-43. The intensity of glow plotted against the time interval is presented in Fig. 45. The comparison of different samples shown in Fig. 46.

3.13 MEASURMENT OF SKIN HYDRATION AND OIL CONTENT

The experimental results expressed in mean±SD, by using SPSS version 20.0 apply independence t-test followed by one-way analysis of variance (ANOVA). The results shown in the Tables 44-45 indicated that during 03 weeks of study, sample # 09 showed a great increase in hydration that is 27.33±6.78 for control and 32.03±6.21 for test the oil measurement was as follow 18.61±3.99 for control and 20.40±3.73 for the test. While sample # 06 showed the lowest increase in hydration that was 24.91±8.63 for control and test 25.39±9.19 and the oil measurement was as follow 19.23±4.01 for control 22.40±4.10 for the test. The increase in hydration and oil content of skin was shown in Fig. 47, 49. While the comparison study of samples are mentioned in Fig. 48, 50.

3.14 ANTI-INFLAMMATORY ACTIVITY OF ROSE WATER BASED CREAM FORMULATION

The in-vitro anti-inflammatory effect of cream formulation prepared from sample # 9 evaluated against denaturation of egg albumin at two different dose level 50 g and 30 g (formulation # F1 and F2). The percentage inhibition of protein (albumin) denaturation of diclofenac sodium observed within the range of 35.33-86.32% and cream formulation F1 was 52.2-80.6 % F2 was 41.4-65.2%, and F3(Placebo) was 0.375- 43.67% throughout the concentration range of 50-1000 µg/ml.inhibitory concentration

(IC50) value of cream formulation F1 was recorded as 257.39µg/ml, F2 formulation IC50 was 375.41 µg/ml at correlation coefficient value (R) of 0.856 and 0.923 respectively. The inhibition of protein denaturation significantly increased with the increase of concentration as shown in Fig. 52. The elaborated result of in-vitro anti-inflammatory activity of cream formulation and diclofenac sodium are given in Tables-46-49. A strong positive correlation mention in Fig. 53, 54 between the reducing power antioxidant and anti-inflammatory activity was observed for F1 (y=0.023x + 55.92, R2=0.856) and F2 (Y=0.024x + 40.99, R2=0.923) formulation.

3.15 ANTI-OXIDANT ACTIVITY OF ROSE WATER BASED CREAM FORMULATION

The reducing power of cream formulation F1, F2, and F3 at four different concentrations 25, 100, 500 and 1000 µg/ml was found to be, F1 ranges from 11.80 % to 81.55%, F2 formulation antioxidant activity ranges from 8.05 % to 72.81 % and F3 formulation which was placebo cream 7.38% to 9.12%. The results of reducing power was calculated as ascorbic acid equivalents (AES)µg/g shown in Table-50-52. It was seen that by increasing the amount of rose water and also by increasing the concentration of formulations anti-oxidant activity increased shown in Fig. 51.

3.16 ANTITUSSIVE ACTIVITY OF ROSE WATER BASED COUGH SYRUP FORMULATION

The antitussive activity of cough syrup formulation C1, C2, C3 and C4 at three different doses 5ml/ 70kg, 10 ml/70kg and 15ml/ 70kg shown in the Tables-53-66 and Fig. 55-58. The experimental results expressed in mean±SEM by using SPSS version 20.0 apply the one-way analysis of variance (ANOVA) Tukey‟s post hoc test. The results shown in the Table-67 indicates that at 60 min and 90 min all samples showed significant inhibition in cough P <0.05.

Part IV 4. Discussion

DISCUSSION

Plants deliver a large number of precious molecules which can further be explored for the development of new bioactive compounds or phytopharmaceuticals. During the past decades, thousands of plants extracts have been screened and hundreds of compounds of various therapeutic categories were identified. Plants contain valuable secondary metabolites such as, polyphenols, terpenes, saponins, fatty acids, flavonoids and tannins that have been shown to display important biological actions. In view of all these important aspects and role of bio-active compounds available through various parts of plants. The present research was taken into consideration and designed to explore the role of rose water obtained from Rosa damascena and to develop a suitable stable topical cream formulation with the aim to utilize the cream against skin disorder, UV radiation and other adverse environmental conditions affecting skin by judging the sun protecting factor, hydration and glowing effects of rose water. In addition various rose water samples were aalysed with respect to physico-chemical parameters to established its standardization and to explore their biological role by determing anti-tussive, anti-oxidant and anti-inflammatory effects.

4.1 EVALUATION OF PHYSICOCHEMICAL PARAMETERS

For the stability and performance of any pharmaceutical, preparation pH plays a vital role relating to its usage and application. Any chemical change in the product mostly due to variation in pH which gives the idea of the quality of the finished products for its application upon skin (Smaoui et al., 2012).

The pH of human skin ranges from 4.5 to 6. The regular washing of skin with soaps and detergents skin lost its acidities. For that reason, a moisturizer that ranges in the acidic range should be used to normalize the skin. Satisfactory and suitable pH range of moisturizers should be between 5-8 (Saraf et al., 2010). The pH of all rose water samples tested shown in Table-3 were found under acceptable limits and non-skin irritating and thus were observed suitable for topical application.

As mentioned earlier, plants metabolites (phytochemicals) are natural compounds protect

56 from different diseases and maintain the human health. These compounds also defend the plant against insects and environmental hazards and also give good appearance and fragrance to plants. Phytochemicals exhibit properties and work as an anti-cancer, antioxidant, anti-inflammatory, and immunity potentiating agents. There are about 150 plants metabolites that have been investigated and classified according to their physical, chemical characteristic and protective functions (Saxena et al., 2013). Different metabolites which are found in plants are alkaloids, triterpenoid, steroids, gums, mucilages, tannins, phenolic compounds, flavanoids, sterols, resins and fatty acids (Swadhini et al., 2011). One of the largest groups of phytochemicals are alkaloids which have anti-microbial, anti-fungal, anti-hypertensive, anti-arrhythmic, anti-malarial and anti-cancer effects. Tannins used against diarrhea anti-inflammatory, antiseptic, as astringent and used as a diuretic. Flavonoids are hydroxyl phenolic compounds showed the activity as anti-microbial, antioxidant, cytotoxic, anti-tumor, anti-inflammatory, oestrogenic activity, anti-allergic enzyme inhibition and vascular activity.Terpenoids have the activity as anticarcinogenic, anti-ulcer, anti-malarial, septicidal and antimicrobial etc (Saxena et al., 2013; Madhukar, 2013; Yadav et al., 2014; Joy and Alam, 2012; Sahoo et al., 2012).

In the present study, all samples of rose water have almost the same physical state- that they were transparent with a characteristic smell and pH ranges in between 4-6. By performing the different chemical tests, it revealed that saponin, triterpenoids, tannins, and flavonoids are presents in most of the samples these active metabolites are responsible for most of the biological activity of the rose water.Result mention in Table-4.

4.2 DETERMINATION OF VOLATILE CONSTITUENT BY HS-GC-MS

The present study describes a direct analysis method headspace coupled to gas chromatography-mass spectrometry (HS–GC–MS) for the determination of volatile compounds present in the rose water. Among different operational conditions of headspace, the highest efficiency achieved at 120 °C and 20 min of incubation time. In comparison with generally used hydro-distillation (HD), HS–GC–MS method is found very simple, needs a low amount of sample, convenient to handle without using any

57 solvent and requires shorter analysis time. It has high trapping capability for volatile and thermally responsive compounds. The main components recognized by this method were phenylethyl alcohol, Citronellol, pentadecane, heptadecanol, octadecanol,tetracosane, decane and nonane. These components are present in all samples but high in sample # 9, 12 and 8. The graph of GC-MS was mentioned in Fig. # 1-3. Results mention in Table-5

4.3 DETECTION OF FUNCTIONAL GROUPS BY FT-IR

The FT-IR analysis is a most commonly used method as a „fingerprint‟ device in phytochemical screening for associating natural oriented samples. The FT-IR spectrum reveals peaks or spectral bands by the vibrations of individual bonds or functional groups. Many functional groups can recognize by their characteristic vibrations and create the spectrum, FT-IR spectroscopy is a non-destructive and most reliable method of assigning a compound to its group (Ganapathy et al., 2014). The results of FT-IR peak values suggested the presence of different functional groups such as alkanes, aldehydes/ketones, alkene, ethers, amines and aromatics in different tested rose water extracts.

The FT-IR spectrums Fig. 4-15 revealed that the peaks arise in the range 3600 cm⁻¹ to 920 cm⁻ ¹. The tested samples showed intense peaks from 3400 cm⁻¹ to 3600 cm⁻¹, due to the characteristic stretching vibration of N-H and O-H from alkaloids, polyphenols amino acids, while the absorption peaks from 2800 cm⁻¹ to 2900 cm⁻¹ appeared from the C - H symmetric stretching of CH3 and CH2 group of lipid region and ester group. While the peak at 2600 cm⁻¹ revealed the strong, broad O-H stretching of carboxylic acids structure, the absorbance peaks from 1700 cm⁻¹ to1850 cm⁻¹ from C = O stretch bending to indicated the presence of conjugated aldehyde and carbonyl group. Peaks from 1450 cm⁻¹ to1470 cm⁻¹ showed the alkane group. The absorbance peaks from1100 cm⁻¹ to 1200 cm⁻¹ appeared in a sample from C - O stretching vibration, indicated the presence of alcohol, ether, carboxylic acid and anhydride.

4.4 ACUTE TOXICITY STUDIES

Acute toxicity is caused by a mediator when administered in one or additional doses more than a phase not greater than 24 hours and involves harmful possessions to the organism

58 throughout a single or small term exposure. Acute toxicity study has also been worn during the range of starting doses for phase-I human and animal studies, and provide information related to acute overdosing in humans and animals. The testing base on the route of substance administered to the animal and therefore it is classified from Class-1 to Class-5 for oral, dermal, gas inhalation, vapor/dust/mist inhalation and injection. Dosing can be repetitive during the administration of test material by a range of routes of exposure, including gavaging which involves stomach intubation or forced feeding, injection, skin, painting, and inhalation. The acute toxic class process is a step-wise method, involves the use of three animals of a single sex per step. Depending on the mortality and moribund status of the animals, on average 2 to 4 steps may be essential to allow a decision on the acute toxicity of the substance. The OECD Guideline 423 (2001) provides a reproducible method that uses few animals as per appendices 2, 3 and 4. Rose water studied for its acute toxicity was done on Wister albino rats. Rose water was administered orally to animals observation recorded up to 48 hours. There were no abnormalities seen in the behavior of animals. Also, no change in the respiratory system, gastrointestinal and execratory system was observed. The slightly sedative effect was noted. The result was shown in Table-7.

4.5 BRINE SHRIMP (ARTEMIA SALINA) LETHALITY BIOASSAY

Toxicity testing of the herbal extract is on the same procedure as the conventional medicine, both in-vitro and in-vivo methods employed for the toxicity testing, in-vivo methods are done on mice and rats whereas in-vitro toxicity testing was performed by using a model such as BST (Brine shrimp lethality test). The main advantage of this toxicity testing is the use of shrimp, there is much homogeneity in the eggs, and the nauplii are highly sensitive to chemicals. Their eggs are easily available and within 16-24 hours hatched and nauplii formed.At 24thhour, the nauplii are exposed to the test chemical to determine the LC50 (concentration causing 50 % lethality) of the test compound. For screening the bioactivity, cytotoxicity, toxicity, pesticidal and gastroprotective activity of medicinal plants BST method used. This method is harmless, consistent, low-cost and suitable bioassay tool (Meyer et al., 1982). The results obtained from the brine shrimp lethality test of rose water samples showed no toxicity up to

1000µg/ml dose. Results shown in Table-8-19 and the graph of percent mortality and calculated LC50 were mentioned in fig-16-27. Over all results of brine shrimp test indicated rose water as a safe product with respect to its usage and application.

4.6 IN-VITRO ANTI-OXIDANT ASSAY BY FERRIC REDUCING POWER

Oxygen free radicals that appear as an outcome of the respirative phase of oxidative phosphorylation may hit biological macromolecules similar to cellular DNA, open- handed rise to single- and double-strand breaks that may finally cause cell aging, cardiovascular diseases, mutagenic changes and cancerous tumor growth. When natural defences of the living being (enzymatic, non-enzymatic or dietary origin) weighed down by an extreme generation of reactive oxygen species (ROS), a condition of „oxidative stress‟ occur, in which cellular and extracellular macromolecules (proteins, lipids, and nucleic acids) can undergo oxidative injury, causing tissue damage. Utilization of foods naturally having antioxidant action is the main competent way of fighting such tissue injuries, undesired transformation and preventing fitness risks. The chemical variety of natural antioxidants (AOX) makes it tricky to detach, distinguish, and calculate individual antioxidants beginning a complex food/biological template. Moreover, the whole antioxidant influence is often more significant to assess health valuable effects because of the supportiveact of individual antioxidant species. Antioxidant capacity assays may be classified as electron transfer (ET) − and hydrogen atom transport (HAT)−based assays, although in some cases, this two mechanism may not differentiate with different limitations. The results obtained are hardly comparable because of the different mechanisms, redox potentials, pH and solvetheno dependencies, etc. of various assays (Apak et al., 2013).

ROS can originate from both endogenous and exogenous source. The exogenous source includes physical anxiety, environmental contamination, radiation, chemicals. While endogenous source includes supplement, metabolism, and aging procedures. An excess amount of free radicals if not removed from the endogenous system may lead to the weak immune system, cell injury, generate a typical protein and change in gene expression,

60 which may cause the initiation and propagation of many diseases and disorders. Many enzymes play their role in the scavenging of free radicals such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). If the number of anti-oxidants inadequate than the rate of cellular injury increases with the much faster rate.To overcome this problem natural anti-oxidants of plant origin utilized. Synthetic antioxidants had negative health issues and also a limitation of the application (Anu et al., 2013, Doss and Pugalenthi, 2012).

A variety of analytical methods were used to verify the efficiency of natural anti-oxidants (as either crude extracts or pure active compounds from plants) such as 2,2-diphenyl-1- picrylhydrazyl (DPPH), total radical-trapping antioxidant potential (TRAP), nitric oxide, ferric reducing antioxidant power (FRAP), oxygen radical absorbing capacity(ORAC) and Trolox equivalent capacity (TEAC) etc. These techniques are extensively suitable to their sensitivity and accurateness and are capable of proposing a total profile of the antioxidant content of foodstuff (Sulaiman et al., 2013). It is well reputable that the reducing capacity of bioactive compound connected to the anti-oxidant activity. Thus, the existence of reductants in the sample or standard solution will characterize the reduction of the Fe/ferricyanide complex to the ferrous form. Earlier research information recommended that the reducing power show antioxidant activity by donating an electron atom to stop the free radical chain reaction.thequantity of Fe+2 complex can be monitored by measuring the formation of Perl's Prussian blue at 700 nm (Chavan et al., 2014)

The ferric reducing power capacity of rose water samples summarized in Table-20-25. This is the comparative study in which reducing power capacity of different volumes of rose water samples that is (10ml, 5ml)was analyzed equivalent to 50, 100 and 200 µg/ml of standard ascorbic acid. The significant reducing power capacity of sample # 12 recorded as highest 841.66 followed by sample # 9 (533.33) and the third highest value was of sample # 08 (458.33) against 50µg/ml of ascorbic acid. The results against 50 µg/ml, 100µg/ml and 200µg/ml of ascorbic acid for both volumes 5ml and 10 ml are mentioned in Table-21 and 24 and Fig. 28. The phytochemical screening of rose water samples confirms the presence of flavonoids, tannin, triterpenoids and saponins. All of

61 these compounds have shown antioxidant activity which is also previously reported in various studies (Sandhar et al., 2011, Zhang and lin, 2008, Grassman, 2005, gulcinet al. 2004). Moreover by this research rose water can proposed as a good source of natural antioxidant for cosmetic and topical pharmaceutical formulations. Due to increase demand for natural ingredients consumption, the cosmetologists have shown great interest to incorporate natural ingredients in their formulations. However proper isolation and identification of active constituents can helped in the management of many topical and systematic diseases.

4.7 IN-VITRO ANTI-INFLAMMATORY ACTIVITY

It is a typical biological procedure in response to tissue damage, microbial pathogen contamination and chemical irritation. This biological process furthermore involves, innate the adaptive immune systems (Pan et al., 2010). At an injured location, inflammation initiated by movement of immune cells from blood vessels and discharge of mediators followed by recruitment of inflammatory cells and liberation of reactive oxygen species (ROS), reactive nitrogen species (RNS) and pro-inflammatory cytokines to remove foreign pathogens, resolving infection and repairing injured tissues (Libby, 2007; Medzhitov, 2008). Thus, the chief purpose of inflammation is valuable for a host's protection. In general, usual inflammation is fast and self-limiting, but an abnormal declaration and extended inflammation causeassorted chronic disorders.

4.7.1 Chronic Inflammation

It can cause injury to a host tissue than bacterial infection. Various ROS and RNS such as

•O2− (superoxide anion), •OH (hydroxyl radical), H2O2 (hydrogen peroxide), nitric oxide

(NO), and O2 (singlet oxygen) generate by inflammatory cells, damage cellular biomolecules including nucleic acids, proteins and lipids, causing cellular and tissue damage, which in turn augment the condition of inflammation (Pan et al., 2009). Inflammatory chemicals formed by inflamed and immune cells also hit normal tissues surrounding the infected tissue, causing oxidative injury and extensive tissue inflammation (Libby, 2007; Pan et al., 2009).

4.7.2 Recent advancement on inflammation

It is the well-recognized reality that chronic inflammation is related to a broad range of progressive diseases such as cancer, neurological disease, metabolic disorder and cardiovascular disease (Pan et al., 2009). Numerous studies propose the removal of chronic inflammation as a most important method to avoid various chronic diseases. Epidemiological studies provide persuasive proof that natural dietary compounds that humans consume as food have many biological activities. Among these natural bioactive compounds, flavonoids extensively documented for their biological and pharmacological effects, together with antiviral, anti-carcinogenic, antioxidant, antimicrobial, anti- inflammatory, anti-angiogenic and anti-thrombogenic properties (Garcia-Lafuente et al., 2009; Libby, 2007; Pan et al., 2010). Epidemiologic studies point out that the occurrence of chronic disease and cancer is inversely connected with the consumption of fruits and vegetables rich in phenolics (Garcia-Lafuente et al., 2009), and this attributed to their possible anti-inflammatory activities.

Inflammation is a complicated process, driven by pre-existing conditions such as infection or injury or genetic changes. These conditions have resulted in triggering signaling cascades, activation of transcription factors, gene expression, increased of levels of inflammatory enzymes, and release of various oxidants and pro-inflammatory molecules (cytokines &chemokines) in immune or inflammatory cells (Medzhitov, 2008). In these conditions, excessive oxidants and inflammatory mediators have a harmful effect on normal tissue, including toxicity, loss of barrier function, abnormal cell proliferation, inhibit the normal function of tissues and organs, and at last leading to systemic disorders (Libby, 2007; Medzhitov, 2008). Over the past two decades, many studies disclosed that chronic inflammation is a serious element in many human diseases and situation, together with obesity, cardiovascular diseases (atherosclerosis, coronary diseases, cerebrovascular disorder, heart failure and cardiomyopathy), neurodegenerative diseases (Alzheimer & Parkinson), diabetes, aging, metabolic disorder and cancers

There are two iso forms of cyclo-oxygenase (COX) enzymes, i.e., COX-1 and COX-2 participate means to function in our body. The COX-1 enzyme is articulated in the majority tissues and is accountable for maintenance roles for usual physiological

63 functions. COX-2 induced by pro-inflammatory cytokines, growth factors, oncogenes, carcinogens and tumor promoters and not visible in regular physiological conditions. COX-2 take part in a serious function in both inflammation and control of cell growth (Dannhardt and Kiefer, 2001). Thus, botanical COX-2 that inhibits the action or appearance of COX-2 might be an essential object for cancer chemoprevention or anti- inflammation (Ko et al., 2011). Nitric oxide synthase (NOS) is another key enzyme, which produces nitric oxide (NO) via oxidation of the terminal guanidine nitrogen atom of L-arginine. Nitric oxide has also been proposed to be avital mediator of tumor growth. Inducible isoforms of cyclooxygenase (COX-2) and nitric oxide synthase (iNOS) are primarily accountable for the manufacture of large amounts of assorted mediators. These mediators such as cytokines, chemokines, prostaglandins (PGs) and nitric oxide (NO) are implicated in a variety of processes including inflammation and carcinogenesis (Ko et al., 2011; Yang et al., 2007). For that reason, selective inhibitors of COX-2 or NOS enzyme action may be an option to hold back of these genes and sluggish down the inflammation or carcinogenic process.

Protein denaturation is the famous reason for unusual inflammation-based diseases, due to protein denaturation its actual structure diminished and it will not perform its actual enzymatic and biological activity properly (Ratnasurya et al., 2015). The viscosity of protein solution further supports the inhibition of protein denaturation.When protein is denatured, its viscosity increased. The viscosity of protein solution will be affected by the external factor (pH, temperature, % of solvent and ionic strength) and the physicochemical nature of the protein (Szymanska et al., 2008; Alizadehfard 1995). Previously reported that on increasing the concentration of solution protein denaturation is also increasing (Gautum et al., 2013; Vennila and Anitha 2015; Prased et al., 2013; Chandra et al., 2012). There are many factors which influence the viscosity of protein solution shape, concentration, size, molecular weight, intermolecular attraction, flexibility, charge and degree of hydration. Due to the large molecular size protein solution had higher viscosities.

In past several years, research on phytochemicals having anti-inflammatory property improved. Most of the secondary plant metabolites such as polyphenols had great

64 medicinal importance, In the present study, the in-vitro anti-inflammatory activity of rose water samples is due to the presence of polyphenolic content such as flavonoids, tannins, and saponins. These compounds inhibit protein denaturation.Previously reported that many enzymes (cyclooxygenase-2, tyrosine kinase and neutrophil degranulation) inhibit by flavonoids. These polyphenolic compounds established both anti-oxidant and anti- inflammatory activity (Shallangwa et al., 2015).

The anti-inflammatory activity of rose water is due to the presence of polyphenolic compounds and saponins that bind with surface action and produce an anti-inflammatory response. The result of in-vitro anti-inflammatory activity of rose water samples was shown in Table-26 and mentioned in Fig. 29. The comparision of 100µg/ml standard diclofenac sodium drug with the samples are mentioned in Fig. 30.

For the in vivo study of anti-inflammatory effect the main problem related to the approvals from the ethical committee (Shallangwa et al., 2015). Therefore, researchers move towards the in-vitro technique to study the inhibitory effect of the drug on protein denaturation that‟s why in-vitro experiments are the good approach towards the formation of topical medication. It is a simple and cheap method.

4.8 DETERMINATION OF IN- VITRO SUN PROTECTING FACTOR (SPF)

Sunscreen effectiveness expressed with the help of sun protecting factor (SPF). An in- vitromethod in which at every 5 nm interval absorbance of the sample have calculated. SPF is the ratio of ultraviolet energy requirement for the production of minimal erythemal dose (MED) both protected and unprotected skin (Malsawmtluangi et al., 2013).

WHO (world health organization) classified UV radiation as carcinogenic, containing many side effects such as photodermatosis,the aging effect on skin,mutagenicity, and loss of immunity of skin. By energy and adverse effect, the ultraviolet light divided into three categories UV-A, UV-B, and UV-C. Among these, the UV-C (200-280 nm) is the most dangerous one, which is filtered by the natural filter ozone layer. Due to the filtration of

UV-C the portion of ultraviolet radiation which reaches to the earth is UV-A and UV-B among them UV-B is more in energy 30-40 times to UV-A (320-400nm) (Malsawmtluangi et al., 2013, M Mishra et al., 2011, More et al., 2003, Kale et al. 2011, Mansur et al., 1986).

The natural and synthetic ingredients are now a days and also in the history have been used as a sunscreen (Chemicals protect against ultraviolet radiation) (Nair and Parida, 2001). Sunscreen which made from the plant extract is good as an antioxidant and Play an impressive role in many sunscreen preparations (Shekar et al., 2012). There are many studies which show the good effect of a natural chemical constituent as they delay, prevent and reverse the effect of ultraviolet radiation (Stratton et al., 2000, Afaqet al. 2005). The group of naturally obtain photoprotective compounds include polyphenolic compounds and phenolic acids (Robbins et al., 2003).

Rose water is a good as sunscreen alone if we compare our results of samples with the sunprotection factor rating the result is ranging between 50-75 %. The highest protection against ultraviolet radiation showed by sample # 9 which is around 75 % followed by sample # 8 which is 70% protection against ultraviolet radiation. The results listed in the Table-29 and Fig-31. The phytochemical screening of rose water shows the presence of polyphenolic compounds tannin, saponin, flavonoids and triterpenoid. These phytoconstituents protect from Free radical. Which are formed by the ultraviolet radiation cause sunburn and skin related diseases. Antioxidant donates an electron to neutralized such free radicals and prevents the cells and tissues from damage (Davies et al., 2002).

Rose water sunscreen activity improved by the incorporation of any vehicles for the making of any sunscreen formulation, the behavior of the skin, pH of the formulation (use of esters, emulsifiers) can increase the SPF value of rose water from 10-15 %.

The in-vitro SPF determination method is economical,simple, required less time and gave the rapid result.Photoprotective agents efficiency determined by in-vivo method but it required the ethical permeation (Bambal et al., 2014). Nowadays the uses of sunscreen made from herbal preparation are widely used because of low side effects and multiple healing effects. (Guyer et al., 2003)

4.9 ANTI-MICROBIAL ACTIVITY

In the present study the antimicrobial effect of rose water study against standard ATCC cultures which includes Pseudomonas aeruginosa (ATCC-27853),Escherichia coli (ATCC-14169), Salmonella typhi (ATCC-9394), Salmonella typhipara A(ATCC-14028), Salmonella typhipara B(ATCC-14023),Streptococcus epidermidis(ATCC-12228), Streptococcus fecalis(ATCC# 29212), Proteus mirabilis(ATCC-12453) Corynebacteriu- mxerosis(ATCC # 373), Staphylococcus aureus(ATCC-6538), Klebsiella pneumonia (ATCC-5046)Enterococcus sp.(ATCC-12970), Candida albicans(ATCC-10231), Aspergillus Spp. (ATCC # 16404) the activity was executed using stock, 5%, 10% and 15% test sample respectively. Standard drug provided by the manufacturer upon request Ciprofloxacin 5µg and Amoxicillin 30 µg was employed as a positive control for antibacterial screening and Nystatin was used for anti-fungal screening results as shown in Table-30-41 and it was observed that rose water in the form of distillate was not show antimicrobial effect which is also supported by previous research conducted by (Ulusoy et al., 2009) previous reported positive antimicrobial activity data of Rosa damascena petals extract, it was seen that for performing antimicrobial activity either the researcher concentrate the aqueous extract or dry, or use any organic solvent for the extraction (Shohayeb et al., 2014; Taktke et al., 2015; Halawani et al.,2014; Vasanthakumar et al., 2015). The anti-bacterial work images are mention in Fig. 32-43.

4.10 MICROBIAL CONTAMINATION

The determination of colony forming unit of any solution and suspension was known as Miles and Misra Method. In 1938 Miles and Misra firstly invented this technique. This technique is easy, fast and effortless to process, but it necessitates highly capable microbiologist and also restricted environmental condition. Any bacteria when growing on the agar forming visible colonies. On the first step, the proper absorption of the solution on agar surface and place the plates in an incubator in an inverted position by providing the favorable condition that is temperature and humidity. The bacteria were grown within 18-24 hours. From 1938 this method of calculating CFU is most commonly used (biotechnology forumns: determination of bacterial CFU Using „Miles and Misra

67 technique. The result of bacterial contamination was mention in Table-42.11 out of 12 extracts were found to be microbiologically contaminated while only 1 extract showed no growth on MHA. Growth on all the plates showed three distinct types of colonies. Their microscopic examination revealed Gram-positive Bacilli and Gram-negative Bacilli mention in Fig. # 44.

4.11 CLINICAL STUDIES

4.12 GLOW MEASURMENT OF SKIN

The extent of gloss is performed to assess the effectiveness of skin and hair care, decorative cosmetics and individual care goods. A digital lux meter is a delicate gadget for measuring glow and intensity and for that reason used in a presentstudy.Independent t-test followed by one-way analysis of variance (ANOVA) concealed that glowing skin outcome after the application of the rose flower extracts was significantly improved. The glowing effects on skin were experiential during the study period, i.e. from 1st week till the end of 3rd week between control and test hand. The appreciably improved in skin glowing effect may be due to the application of rose water extract containing the polyphenolic compounds.These photochemical is natural efficient component for skin glowing silken and skin rejuvenating. The natural antioxidant participate a vital position in neutralize the destructive effect of free radicals and adding up a young-looking glow to skin. Flavanoids are definitely valuable for the skin and act as an antioxidant, avoid early aging, nourish the skin, stop skin darkening, support healthier skin by serving obstruct UV penetration, reducing free radical destructive andmaintain the skin hydrated, even, slippery and supple, bestowing a healthy glow. Due to its strong antioxidant effect, it may be suitable for either counteracting the effect of aging or regenerating tired stress-ridden skin and pink cheeks and unblemished skin, fewer lines, minimized pores and strengthen the skin. Given the experimental data result which is mention in Table-43 and Fig. 45. It was suggested that the rose water significantly improves the radiance of the skin. Difference in an increase of enhancement of skin glow by application of different rose water sample is mentioned in Fig. 46.

4.13 DETERMINATION OF SKIN HYDRATION AND OIL CONTENT

The hydration of the stratum corneum and lipids of the skin surface play an important role on the general appearance and function of the skin (Rawlings et al., 2004, Marty et al., 2002). The human skin is the utmost part of the body,and it is indirect contact with the environment (Carvellol et al., 2008). The human skin can divide into four types dry, normal, oily and a combinational.The largest of all organ of human is skin which is about 20 square feet (1-5) human skin consists of different layers the uppermost is the stratum corner which is also known as the epidermis and consists of layers of dead cells. There are many factors which affect the skin (external and endogenous factors) (Thorleifsson et al., 2003, Egawa et al., 2002, Flynn et al., 2001). The topical use of different types of soap, detergent, and irritants (alcohol and hot water) disturbed the skin (Yosipovitch et al., 2004). Due to the effect of all of these things the skin behave abnormally.Different types of problems arise in which the most common one is the dehydration of the skin, which lead to the dryness of the skin such as cracking, scaling, itching and redness, roughness and tightening of the skin (Loden et al., 1995).

Maintenance of hydration of skin layers are very important around 30% of water content must be present in the first upper layer of skin to keep them active alive and give protection against different infection. If the percentage of water content becomes low up to 10% skin is in the condition of dehydration. For the recovery of the skin hydration different types of moisturizers are available which maintain the health of the skin (Loden et al., 2005). Nowadays different preparations of moisturizers are available in the market containing different synthetic ingredients which have a toxic effect on the skin, so these toxic components are replaced with the natural ingredients (Kapoor et al., 2008, Kapoor et al., 2007). Plant-based natural beautifying preparations are available having fewer side effects for the rehydration of different skin. In the present study we had taken different marketed samples of rose water and observed their effects on skin hydration also one sample was extracted in the lab by adopting simple distillation method. For the study of skin hydration a biomechanical and the electrical technique were applied. A small skin hydration meter was used having an LCD screen on which the result showed digitally.

Among different rose water samples, sample#9 showed great increase in hydration and oil content of the skin while sample#6 showed the least increase as mentioned in the Table-44,45 and Fig. 47-50.

4.14 ANTI-OXIDANT ACTIVITY OF ROSE WATER BASED CREAM FORMULATION

The oxidative stress is solitary of the most important mechanism for skin aging and dermatological conditions (Chaudhari et al., 2011). UV radiation from sunlight is the most common exogenous cause injurious to the skin.The constant exposure to environmental factor leads to alterations in the connective tissue due to the formation of lipid peroxides and ROS, as well as enzymes action, which results in numerous skin disorders (Kaur et al., 2006). The free radical construction naturally restricted by various beneficial compounds known as antioxidants. These are radical scavengers protecting the human body by inhibition of various oxidizing chain reactions. Reactive oxygen species generated exogenously act in response to a variety of biomolecules present in the skin and play a vital role in skin disorders (Yamakoshi et al., 2003). In this view, topical application of antioxidants provides a competent strategy to enrich the endogenous cutaneous system, leading to a reduction in the UV-radiation mediate oxidative damage and avoid oxidative stress-mediated diseases (Lin et al., 2003). In the present study in- vitro anti-oxidant activity of rose water-based cream formulation was done by ferric reducing power assay on four different concentrations 25, 100, 500 and 1000 µg/ml and it was noticed that rose water base cream has good anti-oxidant property. Anti-oxidant property of cream formulation increases by increasing the amount of rose water. The F1 formulation (50g rose water) had percentage reduction power ranges from 11.80 % to 81.55% while F2 ranges from 8.05 % to 72.81 %. By comparing the results with the placebo cream (F3 ranges 7.38% to 9.12% ) which do not have rose water show a mark difference in antioxidant activity. The anti-oxidant activity of cream formulation was mentioned in Table-50-52 and Fig. 51.

4.15 ANTI-INFLAMMATORY ACTIVITY OF ROSE WATER BASED CREAM FORMULATION

Inflammation is the essential way in which the body reacts to irritation, infection, or other tissue injuries; the explanation features being a pain, puffiness, reddishness and heat (Stankov et al., 2012). The inflammatory reaction is the body‟s normal protection strategy as a result of injury to body tissues andthe majority of the body defense elements are found in the blood (Patil et al., 2015). Inflammation occurs in response to physical injuries, intense irritating chemicals, intense heat and infection by viruses and bacteria (Ali and Siddiqui, 2013). Five basic signs were used by the ancients to describe inflammation; heat (color), redness (rubor), pain (dolor), swelling (tumor), and loss of function (functionless), based on visual observation (Punchard et al., 2004). Celsus named the first four signs in ancient Rome (30–38 B.C.) while the last sign was named by Galen (A.D 130–200) (Punchard et al., 2004). The heat feeling is typical as a result of the increased blood movement into the environmentally cooled extremities through the dilated blood vessels and this also results in the increased redness; due to the increased erythrocytes transient through the inflamed area.The edema is the result of penetration of cells into the damaged area, increased channel of fluid from dilated and permeable blood vessels into the surrounding tissues, and deposition of connective tissue in prolonged inflammatory responses. The inflammatory reaction is main because it; disposes of pathogens and cell debris, prevent damaging agent from spreading to the nearby tissues, and sets the stage for the repair process. Inflammation wasvaried as it ranges from the acute inflammation which is allied with the infection of the skin by S. aureus (the humble boil) to chronic inflammation that results in remodeling of the artery wall in atherosclerosis and the bronchial wall in chronic bronchitis and asthma. The immune system cells involved in these processes include; basophils, T-cells, neutrophils, mast cells and B-cells (Punchard, 2004). In the present study rose water based cream formulated and checked for anti-inflammtory activity by in-vitro protein denaturation method by incorporating rose water in two different concentration that is 50g in F1 cream formulation and 30g in F2 cream formulation, It was noticed that by increasing the amount of rose water its anti-inflammatory activity was also increased. F1 anti-

71 inflammatory activity ranges from 52.2-80.6 % while for F2 41.4-65.2% results are mention in Table-46-49 and in Fig. 52. If we compare the result with the standard drug diclofenac sodium it was seen that F1 formulation has very close to the standard that is

35.33-86.32%, the IC50 value of F1 was 257.39µg/ml and F2 was 375.41 µg/ml was mentioned in Fig. 53-54.

4.16 ANTITUSSIVE ACTIVITY OF ROSE WATER BASED COUGH SYRUP

Cough is one of the most widespread symptoms that lead patients to look for medical care from doctors. Under normal circumstances, the principal job of coughing is to defend the respiratory tract from inhaling irritants, and the stimulation of vagal afferent nerves activates the cough reflex in the extra pulmonary airways and mediates by brainstem (Young & Smith, 2011). Under disease situation, coughing becomes extreme and patients often self-medicate for an acute cough relatedwith temporary upper respiratory tract infections (“colds”). However, some serious respiratory diseases, such as asthma, chronic obstructive pulmonary disease (COPD), and lung cancer, have a commonindication of a chronic cough (Mackenzie et al., 2004). So, the chronic cough is being one more serious medical problem than the acute or a subacute cough. Existing antitussive drugs generally divided into two categories, i.e., central or peripheral ones, based on their site of action. The antitussives suppressing coughby the central pathway comprise the majority of currently used cough suppressants (Reynolds et al., 2004). Although opioids including codeine phosphate are the most effective antitussive, their use is limited by undesirable side effects such as addiction (Kamei, 2002). Peripheral acting antitussives such as levodropropizine and guaifenesin generally inhibit the responsiveness of airway nerve subtypes that evoke a cough, but these drugs are not used widely (Dicpinigaitis, 2006). As a result, effective and well-tolerated cough medicines remain to be searched for the clinical requirement.in the present study the effect of rose water based cough syrup on sulphur di-oxide induced cough in experimental animals at three different dose level that is 5ml/70kg , 10ml/70kg and 15ml/70kg and by using four different formulation C1 (sample # 12 as active), C2 (sample # 9 as active), C3 (50% sample #12+ 50% distill water) and C4 (50% sample#9 + 50% distill water)against two

72 different standard that is ivy extract 5ml/70kg and Dextromethorphan 10ml/70kg was observed that at 30 minute after introduction of sulphur-di-oxide and cough syrup administration the number of cough bouht in C1 formulation at three different dose level is as follow mean±S.E.M (9.00±2.08 , 4.33±1.76 and 3.00±0.57) which is minimum in all the formulation followed by C2 formulation which is as follow (10.33±1.20, 9.66±1.85 and 6.00±2.08) the reaming result is mention in Table-53-66.by comparing the result with the standard it was seen that standard ivy extract 5ml/70kg dose at 30 minute show no cough bouts but Dextromethorphan 10ml/70kg show 8.00±5.13 cough bouts which is more than C1 formulation by using SPSS version 20.0 apply the one-way analysis of variance (ANOVA) Tukey‟s post hoc test the results shown in the Table-67 indicates that at 60 min and 90 min all samples show significant inhibition in cough P <0.05. The reduction in cough was mention in Fig. 55-58.

Part V

5. Conclusion and Suggestions

5. CONCLUSION AND SUGGESTIONS FOR FURTHER STUDIES

Studies on phytopharmaceuticals provide the vast prospect for the researcher to illustrate the primary principle to choose natural products for additional in detail to establish their pharmacological and toxicological studies and to explore their possible utilization as a therapeutic, neutraceutical and cosmaceutical agents. Application of rose water in phytopharmaceuticals is increasing worldwide due to its impressive bioactive properties. The present study provided a sound, safe and standardized beneficial effects of rose water obtained from the petals of Rosa damascena Mill available in Pakistan. The overall results of the present studies supported the application of rose water as anti-oxidant, anti- inflammatory, anti-aging, skin lightening, skin moisturizing and sun protecting agent. However, as an extension program, we suggest that the evaluating the rose water containing formulations should be designed and explore its new therapeutic and cosmetic dimension to observe and establish its properties as well for better application.

Results of the present study can conclude that;

- Rose water has shown excellent safety profile and could be used in phytopharmaceuticals, neutraceuticals and cosmeceuticals.

- The present research demonstrated effective antioxidant, anti-inflammatory, sun- protective properties showing its utilization in personal care formulations.

- The present study also supports its utilization as skin moisturizing agents

- The method used in this research study is quick, simple and economic and also easy to handle for the evaluation of rose water-based cream formulation and rose water based cough syrup.

- Present work also extended an opportunity in understanding the interactions between topically applied substances and the epidermal biochemistry.

- Study on rose water represents one of the most essential attractive and great future applications. Therefore further research studies are necessary to explore the mechanisms of action against various skin issues to designeda safe and effective product.

TABLES

Table-3 pH Trend of Rose Water Samples

sample● 01 02 03 04 05 06 07 08 09 10 11 12

pH 6.77 6.06 3.19 4.84 3.43 4.45 4.97 6.02 6.22 5.85 4.46 5.72

●: Source & manufacturer‟s names are available upon request

Table-4 Phytochemical Screening of Rose Water Samples

Detection of Detection of Detection of Fixed Detection of Detection of SAMPLE● Tannins Triterpenoids Oils Saponins Flavanoids

01 + + + + -

02 + + + + -

03 + - - + +

04 + + + ++ ++

05 + + + ++ +

06 + ++ + ++ ++

07 + + + ++ +

08 + ++ + ++ +

09 + ++ ++ +++ ++

10 + + + + ++

11 + ++ ++ ++ -

12 + ++ + ++ ++

●: source & manufacturer‟s name are available upon request (+)Low concentration, (++) Moderate concentration, (+++) High concentration, (-) Absent / not present

Table-5 Volatile Components Present in Rose Water samples

SAMPLES

Constituents 01 02 03 04 05 06 07 08 09 10 11 12

PERCENTAGE OF VOLATILE CONSTITUENTS

Phenylethyl Alcohol 4.7 1.2 3.2 0.2 1.7 0.7 2.01 2.76 5.08 3.2 3.1 5.02

Citronellol 0.2 0.4 0.4 0.1 1.2 0.4 1.7 2.9 3.06 2.1 2.2 3.01

Pentadecane 1.02 - 0.3 4.6 0.3 T 3.4 7.6 8.82 1.2 1.7 6.9

Heptadecanol 0.2 0.2 0.7 1.2 0.4 T 0.2 0.54 0.69 0.2 1.8 1.2

Octadecanol 0.6 0.7 0.1 0.1 0.9 T 0.9 1.93 2.62 0.2 t 2.4

Tetracosane 0.6 - t 0.2 t 1.2 1.2 3.1 3.37 0.1 t 3.32

Decane - 0.1 t 0.4 t 0.4 0.1 0.3 0.34 0.4 0.2 0.2

Nonane - - t 0.3 t 0.3 0.1 0.26 0.2 t 0.2 0.2

(-) component is absent (t) component is less than 0.1%

Table-6 FT-IR Absorption Band Assignments of Rose Water Samples

Peak's wavelength Possible functional Intensity/ Assignment (cm-1) groups

3400-3600 O-H and N−H O-H stretching vibration of hydroxyl groups (mainly lipids and proteins) and N-H stretching vibration mainly carbohydrates proteins.

2800-2900 C−H C-H lipid region, esters groups.

2600 O−H Strong, broad O-H stretching carboxylic acid.

1750-1850 C=O C=O stretching conjugated aldehyde and strong stretching anhydride and a carbonyl group

1450-1470 C=C Weak medium stretching of alkane group, aromatic ring.

1100-1200 C-O C-O stretching of alcohol, ether, ester and carboxylic acid anhydride.

Note: The cumulative FT-IR chart of all twelve samples of rose water, the ranges of wavelength cm-1 indicate the presecnce of specific group in the sample.

Table-7 Acute Toxicity Study of Rose Water at 15, 20, 25ml Adult Dose

Observed Parameters Before Drug After 30 After 1 After 2 After 4 After 24 After 48 Minutes Hour Hour Hour Hour Hour

Change in skin/fur NC NC NC NC NC NC NC

Sense/ alertness NC NC NC NC NC NC NC

Hyperactivity NC NC NC NC NC NC NC

Change in respiration NC NC NC NC NC NC NC

Urination NC NC NC NC NC NC NC

Piloerection NC NC NC NC NC NC NC

Salivation NC NC NC NC NC NC NC

Convulsion/coma NC NC NC NC NC NC NC

Paralytic effect NC NC NC NC NC NC NC

Sedation NC NC MS MS MS NC NC

Death NC NC NC NC NC NC NC

Note: in all twelve samples of rose water same effect was observed at specific dose NC: No Change in Parameters MS: Mild Sedation Only Seen at Higher Dose (25ML)

BRINE SHRIMP (Artemia salina) LETHALITY BIOASSAY Table-8 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #1

Dose µg/ml No. of No. of % Standard % Shrimps Survivors Mortality Drug Mortality

10 30 25 16.66

100 30 22 26.66

1000 30 21 30 Etoposide 46.66%

Mean ±S.D 24.44±6.94  Values are recorded as Mean ± S.D

Table-9 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #2

No. of No. of % Standard % Dose µg/ml Shrimps Survivors Mortality Drug Mortality

10 30 23 23.33

100 30 23 23.33

1000 30 25 16.66 Etoposide 46.66%

Mean ±S.D

21.10±3.85  Values are recorded as Mean ± S.D

Table-10 Brine Shrimp (Artemia Salina) Lethality Bioassay of Sample #3

Dose µg/ml No. of No. of % Standard % Shrimps Survivors Mortality Drug Mortality

10 30 21 30

100 30 23 23.33

1000 30 22 26.66 Etoposide 46.66%

Mean ±S.D

26.66±3.33  Values are recorded as Mean ± S.D

Table-11 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #4

Dose µg/ml No. of No. of % Standard % Shrimps Survivors Mortality Drug Mortality

10 30 24 20

100 30 25 16.66

1000 30 22 26.66 Etoposide 46.66%

Mean ±S.D

21.10±5.09  Values are recorded as Mean ± S.D

Table-12 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #5

No. of No. of % Standard % Dose µg/ml Shrimps Survivors Mortality Drug Mortality

10 30 21 30

100 30 22 26.66

1000 30 25 16.66 Etoposide 46.66%

Mean ±S.D

24.44±6.94

 Values are recorded as Mean ± S.D

Table-13 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #6

No. of No. of % Standard % Dose µg/ml Shrimps Survivors Mortality Drug Mortality

10 30 25 16.66

100 30 22 26.66

1000 30 21 30 Etoposide 46.66%

Mean ±S.D

24.44±6.94

 Values are recorded as Mean ± S.D

Table-14 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #7

No. of No. of % Standard % Dose µg/ml Shrimps Survivors Mortality Drug Mortality

10 30 23 23.33

100 30 22 26.66

1000 30 21 30 Etoposide 46.66%

Mean ±S.D

26.66±3.33

 Values are recorded as Mean ± S.D

Table-15 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #8

No. of No. of % Standard % Dose µg/ml Shrimps Survivors Mortality Drug Mortality

10 30 26 13.33

100 30 24 20

1000 30 22 26.66 Etoposide 46.66%

Mean ±S.D

19.99±6.66

 Values are recorded as Mean ± S.D

Table-16 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #9

No. of No. of % Standard % Dose µg/ml Shrimps Survivors Mortality Drug Mortality

10 30 25 16.66

100 30 23 23.33

1000 30 18 40 Etoposide 46.66%

Mean ±S.D

26.66±12.02

 Values are recorded as Mean ± S.D

Table-17 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #10

No. of No. of % Standard % Dose µg/ml Shrimps Survivors Mortality Drug Mortality

10 30 23 23.33

100 30 18 40

1000 30 25 16.66 Etoposide 46.66%

Mean ±S.D

26.66±12.02

 Values are recorded as Mean ± S.D

Table-18 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #11

No. of No. of % Standard % Dose µg/ml Shrimps Survivors Mortality Drug Mortality

10 30 26 13.33

100 30 24 20

1000 30 23 23.33 Etoposide 46.66%

Mean ±S.D

18.88±5.09

 Values are recorded as Mean ± S.D

Table 19 Brine Shrimp (Artemia salina) Lethality Bioassay of Sample #12

No. of No. of % Standard % Dose µg/ml Shrimps Survivors Mortality Drug Mortality

10 30 23 23.33

100 30 25 16.66

1000 30 24 20 Etoposide 46.66%

Mean ±S.D

19.99±3.33

 Values are recorded as Mean ± S.D

REDUCING POWER CAPACITY OF ROSE WATER SAMPLES

Table-20 Determination of Absorbance using 05 ml (5.21 g) samples

Quantity Absorbance at 700 nm Sample● (ml) Mean ± S.D

01 05 0.023 ± 0.005

02 05 0.034 ± 0.003

03 05 0.022 ± 0.002

04 05 0.03 ± 0.01

05 05 0.023 ± 0.002

06 05 0.005 ± 0.001

07 05 0.008 ± 0.001

08 05 0.055 ± 0.004

09 05 0.064 ± 0.199

10 05 0.004 ± 0.001

11 05 0.006 ± 0.001

12 05 0.101 ± 0.001

Data are mean of absorbance at700nm (n=3)±S.D, (p<0.001) ●: Source & manufacturer‟s name are available upon request

Table-21 Percentage of Ferric Reducing Power Capacity of Rose Water Samples (05 ml) Equivalent to 50, 100, 200 µg/ml of Ascorbic Acid as Standard

%age of reduction power capacity

Mean of Sample● Equivalent to Equivalent to absorbance Equivalent to 100µg 50µg of ascorbic 200µg of of ascorbic acid acid ascorbic acid

01 0.023 191.66 6.927 2.33

02 0.034 283.33 10.24 3.45

03 0.022 183.33 6.62 2.23

04 0.03 250 9.03 3.05

05 0.023 191.66 6.92 2.33

06 0.005 41.66 1.50 0.50

07 0.008 66.66 2.40 0.81

08 0.055 458.33 16.56 5.59

09 0.064 533.33 19.27 6.51

10 0.004 33.33 1.20 0.40

11 0.006 50 1.80 0.61

12 0.101 841.66 30.42 10.27

●: Source & manufacturer‟s name are available upon request

Table-22 Determination of Absorbance Using Ascorbic Acid as Standard Drug

Standard (Ascorbic Acid) Absorbance at 700 nm Absorbance at 700 nm µg/ml Mean ± S.D

50 0.014 0.009 0.014 0.012±0.002

100 0.332 0.33 0.335 0.332±0.002

200 0.985 0.986 0.98 0.983±0.003

 Values are recorded as Mean ± S.D

REDUCING POWER CAPACITY OF ROSE WATER SAMPLES

Table-23 Determination of Absorbance Using 10 ml (10.35 g) Sample

Sample● Quantity Absorbance at 700 nm

(ml) Mean ± S.D

01 10 0.081 ± 0.002

02 10 0.043 ± 0.003

03 10 0.072 ± 0.002

04 10 0.071 ± 0.029

05 10 0.042 ± 0.004

06 10 0.008 ± 0.001

07 10 0.008 ± 0.001

08 10 0.18 ± 0.005

09 10 0.037 ± 0.002

10 10 0.02 ± 0.006

11 10 0.006 ± 0.001

12 10 0.37 ± 0.006

Data are mean of absorbance at700nm (n=3)±S.D

●: Source & manufacturer‟s name are available upon request

Table-24 Percentage of Ferric Reducing Power Capacity of Rose Water Samples (10

ml) Equivalent to50, 100, 200µg/ml of Ascorbic Acid as Standard

Sample● %age of reduction power capacity

Mean of Equivalent to50µg Equivalent to Equivalent to absorbance of Ascorbic Acid 100µg of Ascorbic 200µg of Acid Ascorbic Acid

01 0.081 52.59 17.60 13.68

02 0.043 27.92 9.34 7.26

03 0.072 46.75 15.65 12.16

04 0.071 46.10 15.43 11.99

05 0.042 27.27 9.13 7.09

06 0.008 5.19 1.73 1.35

07 0.008 5.19 1.73 1.35

08 0.18 52.59 17.60 13.68

09 0.037 116.88 39.13 30.40

10 0.02 12.98 4.34 3.37

11 0.006 3.89 1.30 1.01

12 0.37 240.25 80.43 62.5

●: Source & manufacturer‟s name are available upon request

Table-25 Determination of Absorbance Using Ascorbic Acid as Standard Drug

Standard (Ascorbic acid) Absorbance at 700 nm Absorbance at 700 nm

µg/ml Mean ± S.D

50 0.154 0.155 0.153 0.154±0.001

100 0.459 0.462 0.46 0.460±0.001

200 0.592 0.593 0.59 0.592±0.001

 Values are recorded as Mean ± S.D

INVITRO ANTI-INFLAMMATORY ACTIVITY BY PROTEIN(ALBUMIN) DENATURATION METHOD

Table-26 In-Vitro Anti Inflammatory Activity of Rose Water Samples

Sample● 01 02 03 04 05 06 07 08 09 10 11 12

Volume % Inhibition of Protein Denaturation Mean ± S.D

3ML(3.09g) 93.2± 0.01 92.06±0.05 95.1±0.01 80.7±0.15 21.4±0.34 50.5±1.43 38.2±0.27 28.8±0.06 52.0±0.06 23.7±2.60 6.73±2.81 71.6±0.24

5ML(5.21g 10.44±0.04 -6.5±0.49 62.70±0.28 40.54±0.88 29.33±0.31 57.5±0.10 14.8±0.88 91.0±0.14 95.2±0.01 84.4±1.98 50.6±1.16 40.0±0.08

7ML7.32g 77.09±0.05 77.1±0.01 93.9±0.05 66.2±0.05 83.42±0.05 81.4±0.01 64.4±0.02 88.1±0.06 60.7±0.14 71.6±0.06 64.6±0.11 51.1±1.15

10ML10.35g 99.22±0.32 69.65±0.08 81.22±0.03 57.82±0.01 95.16±0.34 85.6±1.66 82.8±0.06 96.8±0.08 98.6±0.06 99.5±0.04 99.0±0.08 68.1 ±0.08

●: Source & manufacturer‟s name are available upon request ●: Values are recorded as Mean ± S.D

Table-27 In-Vitro Anti Inflammatory Activity of Diclofenac Sodium

Standard(diclofanec sodium) % inhibition of protein denaturation

µg/ml Mean ± S.D

20 1.841 ± 0.184

40 50.47 ± 0.739

60 90.33 ± 0.05

80 94.36 ± 0.01

100 96.71 ± 0.09

 Values are recorded as Mean ± S.D

Table-28 Viscosity Value of Rose Water Samples Used to Calculate Anti-inflammatory Activity

Sample● 01 02 03 04 05 06 07 08 09 10 11 12

Volume Viscosity of Rose Water Samples

3ML(3.09g) 0.181 0.406 0.407 0.4033 0.5680 0.4532 0.205 0.274 0.275 0.186 0.1800 0.473

5ML(5.21g 0.256 0.540 0.548 0.568 0.587 0.476 0.331 0.304 0.361 0.234 0.2128 0.541

7ML7.32g 0.366 0.555 0.558 0.578 0.560 0.566 0.519 0.448 0.573 0.517 0.2510 0.585

10ML10.35g 0.541 0.575 0.579 0.607 0.541 0.567 0.540 0.537 0.530 0.602 0.2599 0.627

Table-29 In- Vitro Sun Protecting Factor (SPF) of Rose Water Samples

Samples ● SPF

01 0.637

02 0.386

03 1.373

04 0.800

05 0.242

06 0.439

07 0.704

08 3.612

09 3.956

10 0.218

11 0.460

12 1.854

●: Source & manufacturer‟s name are available upon request

Table-30 In-Vitro Anti-Microbial Analysis of Rose Water Samples #1

Microorganism ATCC # Mean Values Standard (bacteria species) Stock 5% 10% 15% Ciprofloxacin Amoxicillin 5 µg 30 µg Pseudomonas 27853 0 0 0 0 0 0 Aeruginosa Escherichia coli 14169 0 0 0 0 0 19mm Salmonella typhi 9394 0 0 0 0 35mm 18mm Salmonella typhipara A 14028 0 0 0 0 35mm 21mm Salmonella typhipara B 14023 0 0 0 0 27mm 30mm Enterococcus spp. 12970 0 0 0 0 22mm 24mm Proteus mirabilis 12543 0 0 0 0 0 0 Streptococcus fecalis 29212 0 0 0 0 18mm 0 Staphylococcus aureus 6538 0 0 0 0 0 0 Klebsiela pneumonia 5046 0 0 0 0 0 11mm Corynebacterium 373 0 0 0 0 36mm 41mm Xerosis Streptococcus epidermidis 12228 0 0 0 0 34mm 30mm Anti-Fungal Activity

Microorganism ATCC # SAMPLE Standard Fungul species Stock 5% 10% 15% Nystatin Candida albicans 10231 0 0 0 0 11mm Aspergillus Spp. 16404 0 0 0 0 10mm

Table-31 In-Vitro Anti-Microbial Analysis of Rose Water Samples # 2 Microorganism ATCC # Mean Values Standard (bacteria species) Stock 5% 10% 15% Ciprofloxacin Amoxicillin 5 µg 30 µg Pseudomonas 27853 0 0 0 0 0 0 Aeruginosa Escherichia coli 14169 0 0 0 0 0 19mm Salmonella typhi 9394 0 0 0 0 35mm 18mm Salmonella typhiparaA 14028 0 0 0 0 35mm 21mm Salmonella typhiparaB 14023 0 0 0 0 27mm 30mm Enterococcus spp. 12970 0 0 0 0 22mm 24mm Proteus mirabilis 12543 0 0 0 0 0 0 Streptococcus fecalis 29212 0 0 0 0 18mm 0 Staphylococcus aureus 6538 0 0 0 0 0 0 Klebsiela pneumonia 5046 0 0 0 0 0 11mm Corynebacterium 373 0 0 0 0 36mm 41mm Xerosis Streptococcus 12228 0 0 0 0 34mm 30mm epidermidis Anti-Fungal Activity Microorganism ATCC # Sample Standard Fungul species Stock 5% 10% 15% Nystatin Candida albicans 10231 0 0 0 0 11mm Aspergillus Spp. 16404 0 0 0 0 10mm

Table-32 In-Vitro Anti-Microbial Analysis of Rose Water Samples #3 Microorganism ATCC # Mean Values Standard (bacteria species) Stock 5% 10% 15% Ciprofloxacin Amoxicillin 5 µg 30 µg Pseudomonas 27853 0 0 0 0 0 0 Aeruginosa Escherichia coli 14169 0 0 0 0 0 19mm Salmonella typhi 9394 0 0 0 0 35mm 18mm Salmonella typhipara A 14028 0 0 0 0 35mm 21mm Salmonella typhipara B 14023 0 0 0 0 27mm 30mm Enterococcus spp. 12970 0 0 0 0 22mm 24mm Proteus mirabilis 12543 0 0 0 0 0 0 Streptococcus fecalis 29212 0 0 0 0 18mm 0 Staphylococcus aureus 6538 0 0 0 0 0 0 Klebsiela pneumonia 5046 0 0 0 0 0 11mm Corynebacterium 373 0 0 0 0 36mm 41mm Xerosis Streptococcus epidermidis 12228 0 0 0 0 34mm 30mm

Anti-Fungal Activity Microorganism ATCC # Sample Standard Fungal species Stock 5% 10% 15% Nystatin Candida albicans 10231 0 0 0 0 11mm Aspergillus Spp. 16404 0 0 0 0 10mm

100

Table-33 In-Vitro Anti-Microbial Analysis of Rose Water Samples #4 Microorganism ATCC # Mean Values Standard (bacteria species) Stock 5% 10% 15% Ciprofloxcin Amoxicilline 5 µg 30 µg Pseudomonas 27853 0 0 0 0 0 0 Aeruginosa Escherichia coli 14169 0 0 0 0 0 19mm Salmonella typhi 9394 0 0 0 0 35mm 18mm Salmonella typhipara A 14028 0 0 0 0 35mm 21mm Salmonella typhipara B 14023 0 0 0 0 27mm 30mm Enterococcus spp. 12970 0 0 0 0 22mm 24mm Proteus mirabilis 12543 0 0 0 0 0 0 Streptococcus fecalis 29212 0 0 0 0 18mm 0 Staphylococcus aureus 6538 0 0 0 0 0 0 Klebsiela pneumonia 5046 0 0 0 0 0 11mm Corynebacterium 373 0 0 0 0 36mm 41mm Xerosis Streptococcus 12228 0 0 0 0 34mm 30mm epidermidis Anti-Fungal Activity Microorganism ATCC # Sample Standard Fungul species Stock 5% 10% 15% Nystatin Candida albicans 10231 0 0 0 0 11mm Aspergillus Spp. 16404 0 0 0 0 10mm

101

Table-34 In-Vitro Anti-Microbial Analysis of Rose Water Samples #5 Microorganism ATCC # Mean Values Standard (bacteria species) 5% 10% 15% Ciprofloxacin Amoxicillin 5 µg 30 µg Pseudomonas 27853 0 0 0 0 0 0 Aeruginosa Escherichia coli 14169 0 0 0 0 0 19mm Salmonella typhi 9394 0 0 0 0 35mm 18mm Salmonella typhipara A 14028 0 0 0 0 35mm 21mm Salmonella typhipara B 14023 0 0 0 0 27mm 30mm Enterococcus spp. 12970 0 0 0 0 22mm 24mm Proteus mirabilis 12543 0 0 0 0 0 0 Streptococcus fecalis 29212 0 0 0 0 18mm 0 Staphylococcus aureus 6538 0 0 0 0 0 0 Klebsiela pneumonia 5046 0 0 0 0 0 11mm Corynebacterium 373 0 0 0 0 36mm 41mm Xerosis Streptococcus 12228 0 0 0 0 34mm 30mm epidermidis Anti-Fungal Activity

Microorganism Sample Standard ATCC # Fungul species Stock 5% 10% 15% Nystatin

Candida albicans 10231 0 0 0 0 11mm Aspergillus Spp. 16404 0 0 0 0 10mm

102

Table-35 In-Vitro Anti-Microbial Analysis of Rose Water Samples #6 Microorganism ATCC # Mean Values Standard (bacteria species) Stock 5% 10% 15% Ciprofloxacin Amoxicillin 5 µg 30 µg Pseudomonas 27853 0 0 0 0 0 0 Aeruginosa Escherichia coli 14169 0 0 0 0 0 19mm Salmonella typhi 9394 0 0 0 0 35mm 18mm Salmonella typhipara A 14028 0 0 0 0 35mm 21mm Salmonella typhipara B 14023 0 0 0 0 27mm 30mm Enterococcus spp. 12970 0 0 0 0 22mm 24mm Proteus mirabilis 12543 0 0 0 0 0 0 Streptococcus fecalis 29212 0 0 0 0 18mm 0 Staphylococcus aureus 6538 0 0 0 0 0 0 Klebsiela pneumonia 5046 0 0 0 0 0 11mm Corynebacterium 373 0 0 0 0 36mm 41mm Xerosis Streptococcus 12228 0 0 0 0 34mm 30mm epidermidis Anti-Fungal Activity Microorganism ATCC # Sample Standard Fungul species Stock 5% 10% 15% Nystatin Candida albicans 10231 0 0 0 0 11mm Aspergillus Spp. 16404 0 0 0 0 10mm

103

Table-36 In-Vitro Anti-Microbial Analysis of Rose Water Samples #7 Mean Values Standard Microorganism ATCC # Ciprofloxacin Amoxicillin (bacteria species) Stock 5% 10% 15% 5 µg 30 µg Pseudomonas 27853 0 0 0 0 0 0 Aeruginosa Escherichia coli 14169 0 0 0 0 0 19mm Salmonella typhi 9394 0 0 0 0 35mm 18mm Salmonella typhipara A 14028 0 0 0 0 35mm 21mm Salmonella typhipara B 14023 0 0 0 0 27mm 30mm Enterococcus spp. 12970 0 0 0 0 22mm 24mm Proteus mirabilis 12543 0 0 0 0 0 0 Streptococcus fecalis 29212 0 0 0 0 18mm 0 Staphylococcus aureus 6538 0 0 0 0 0 0 Klebsiela pneumonia 5046 0 0 0 0 0 11mm Corynebacterium 373 0 0 0 0 36mm 41mm Xerosis Streptococcus epidermidis 12228 0 0 0 0 34mm 30mm Anti-Fungal Activity Microorganism ATCC # Sample Standard Fungul species Stock 5% 10% 15% Nystatin Candida albicans 10231 0 0 0 0 11mm Aspergillus Spp. 16404 0 0 0 0 10mm

104

Table-37 In-Vitro Anti-Microbial Analysis of Rose Water Samples #8 Microorganism ATCC # Mean Values Standard (bacteria species) Stock 5% 10% 15% Ciprofloxacin Amoxicillin 5 µg 30 µg Pseudomonas 27853 0 0 0 0 0 0 Aeruginosa Escherichia coli 14169 0 0 0 0 0 19mm Salmonella typhi 9394 0 0 0 0 35mm 18mm Salmonella typhipara A 14028 0 0 0 0 35mm 21mm Salmonella typhipara B 14023 0 0 0 0 27mm 30mm Enterococcus spp. 12970 0 0 0 0 22mm 24mm Proteus mirabilis 12543 0 0 0 0 0 0 Streptococcus fecalis 29212 0 0 0 0 18mm 0 Staphylococcus aureus 6538 0 0 0 0 0 0 Klebsiela pneumonia 5046 0 0 0 0 0 11mm Corynebacterium 373 0 0 0 0 36mm 41mm Xerosis Streptococcus epidermidis 12228 0 0 0 0 34mm 30mm

Anti-Fungal Activity Microorganism ATCC # SAMPLE Standard Fungul species Stock 5% 10% 15% Nystatin Candida albicans 10231 0 0 0 0 11mm Aspergillus Spp. 16404 0 0 0 0 10mm

105

Table-38 In-Vitro Anti-Microbial Analysis of Rose Water Samples #9 ATCC # Mean Values Standard Microorganism (bacteria species) Stock 5% 10% 15% Ciprofloxacin Amoxicillin 5 µg 30 µg Pseudomonas 27853 0 0 0 0 0 0 Aeruginosa Escherichia coli 14169 0 0 0 0 0 19mm Salmonella typhi 9394 0 0 0 0 35mm 18mm Salmonella typhipara A 14028 0 0 0 0 35mm 21mm Salmonella typhipara B 14023 0 0 0 0 27mm 30mm Enterococcus spp. 12970 0 0 0 0 22mm 24mm Proteus mirabilis 12543 0 0 0 0 0 0 Streptococcus fecalis 29212 0 0 0 0 18mm 0 Staphylococcus aureus 6538 0 0 0 0 0 0 Klebsiela pneumonia 5046 0 0 0 0 0 11mm Corynebacterium 373 0 0 0 0 36mm 41mm Xerosis Streptococcus 12228 0 0 0 0 34mm 30mm epidermidis Anti-Fungal Activity Microorganism ATCC # Sample Standard Fungul species Stock 5% 10% 15% Nystatin Candida albicans 10231 0 0 0 0 11mm Aspergillus Spp. 16404 0 0 0 0 10mm

106

Table-39 In-Vitro Anti-Microbial Analysis of Rose Water Samples #10 Mean Values Standard Microorganism ATCC # Ciprofloxacin Amoxicillin (bacteria Species) Stock 5% 10% 15% 5 µg 30 µg Pseudomonas 27853 0 0 0 0 0 0 Aeruginosa Escherichia coli 14169 0 0 0 0 0 19mm Salmonella typhi 9394 0 0 0 0 35mm 18mm Salmonella typhipara A 14028 0 0 0 0 35mm 21mm Salmonella typhipara B 14023 0 0 0 0 27mm 30mm Enterococcus spp. 12970 0 0 0 0 22mm 24mm Proteus mirabilis 12543 0 0 0 0 0 0 Streptococcus fecalis 29212 0 0 0 0 18mm 0 Staphylococcus aureus 6538 0 0 0 0 0 0 Klebsiela pneumonia 5046 0 0 0 0 0 11mm Corynebacterium 373 0 0 0 0 36mm 41mm Xerosis Streptococcus epidermidis 12228 0 0 0 0 34mm 30mm

Anti-Fungal Activity Microorganism Sample Standard ATCC # Fungul species Stock 5% 10% 15% Nystatin Candida albicans 10231 0 0 0 0 11mm Aspergillus Spp. 16404 0 0 0 0 10mm

107

Table-40 In-Vitro Anti-Microbial Analysis of Rose Water Samples #11 Microorganism ATCC # Mean Values Standard (bacteria species) Stock 5% 10% 15% Ciprofloxcin Amoxicilline 5 µg 30 µg Pseudomonas 27853 0 0 0 0 0 0 Aeruginosa Escherichia coli 14169 0 0 0 0 0 19mm Salmonella typhi 9394 0 0 0 0 35mm 18mm Salmonella typhipara A 14028 0 0 0 0 35mm 21mm Salmonella typhipara B 14023 0 0 0 0 27mm 30mm Enterococcus spp. 12970 0 0 0 0 22mm 24mm Proteus mirabilis 12543 0 0 0 0 0 0 Streptococcus fecalis 29212 0 0 0 0 18mm 0 Staphylococcus aureus 6538 0 0 0 0 0 0 Klebsiela pneumonia 5046 0 0 0 0 0 11mm Corynebacterium 373 0 0 0 0 36mm 41mm Xerosis Streptococcus 12228 0 0 0 0 34mm 30mm epidermidis Anti-Fungal Activity Microorganism SAMPLE Standard ATCC # Fungul species Stock 5% 10% 15% Nystatin Candida albicans 10231 0 0 0 0 11mm Aspergillus Spp. 16404 0 0 0 0 10mm

108

Table-41 In-Vitro Anti-Microbial Analysis of Rose Water Samples#12 Mean Values Standard Microorganism ATCC # Ciprofloxacin Amoxicillin (bacteria species) Stock 5% 10% 15% 5 µg 30 µg Pseudomonas 27853 0 0 0 0 0 0 Aeruginosa Escherichia coli 14169 0 0 0 0 0 19mm Salmonella typhi 9394 0 0 0 0 35mm 18mm Salmonella typhipara A 14028 0 0 0 0 35mm 21mm Salmonella typhipara B 14023 0 0 0 0 27mm 30mm Enterococcus spp. 12970 0 0 0 0 22mm 24mm Proteus mirabilis 12543 0 0 0 0 0 0 Streptococcus fecalis 29212 0 0 0 0 18mm 0 Staphylococcus aureus 6538 0 0 0 0 0 0 Klebsiela pneumonia 5046 0 0 0 0 0 11mm Corynebacterium 373 0 0 0 0 36mm 41mm Xerosis Streptococcus 12228 0 0 0 0 34mm 30mm epidermidis Anti-Fungal Activity Microorganism ATCC # Sample Standard Fungul species Stock 5% 10% 15% Nystatin Candida albicans 10231 0 0 0 0 11mm Aspergillus Spp. 16404 0 0 0 0 10mm

109

Table-42 Bacterial Contamination in Rose Water Samples Sample● Original 1:10 (10-1) 1:100 (10-2) 1:1000(10-3) 01 TNTC TNTC TNTC TNTC 02 TNTC TNTC TNTC TNTC 03 TNTC TNTC TNTC TNTC 04 TNTC TNTC 230 200 05 TNTC 150 100 67 06 TNTC 260 250 230 07 TNTC 120 105 100 08 TNTC 180 150 130 09 TNTC 200 185 120 10 TNTC TNTC TNTC TNTC 11 TNTC TNTC TNTC TNTC 12 - - - - ●: source & manufacturer‟s name are available upon request Note: TNTC= Too numerous to count (-)= no colony found Formula: CFU/ml = average number of colonies for a dilution × 50 ×dilution factor Sample#05: 150 × 50× 10-1 = 75 CFU/ml Sample#06: 260 × 50× 10-1= 130 CFU/ml Sample#07: 120 × 50× 10-1= 60 CFU/ml Sample#08: 180 × 50× 10-1= 90 CFU/ml Sample#09: 200 × 50× 10-1= 100 CFU/m

110

Table-43 Glow Measurment Samples ● Mean Values ± S.D Significance Control Test 01 16.40±8.44 19 .17±9.66 02 14.93± 8.45 20.37±13.17 03 22.03±12.43 22.47±12.59 04 26.03±15.65 28.93±16.87 05 23.73±13.66 26.20±14.25 p<0.05 06 24.10±13.09 24.50±13.62 07 21.33±4.91 24.70±4.61 08 18.33±8.81 21.23±9.14 09 14.73±7.42 20.83±7.54 10 19.47±4.97 23.17±3.78 11 24.40±14.72 25.63±14.62 12 23.43±13.76 26.03±15.06 ●: Source & manufacturer‟s name are available upon request  Values are recorded as Mean ± S.D  Mean difference is significant at the 0.05 level as analyzed by independence t-test followed by one-way analysis of varience (Anova)

111

Table-44 Measurement of Hydration Samples● Mean Values ± S.D Significance Control Test 01 25.230±5.69 27.42±5.89 02 30.74±10.17 31.48±8.88 03 23.82±9.01 27.09±9.77 04 28.62±7.37 31.38±6.67 05 25.84±10.82 27.84±10.62 p<0.05 06 24.91±8.63 25.39±9.19 07 32.77±12.39 37.47±11.91 08 36.55±11.36 40.32±8.81 09 27.33±6.78 32.03±6.21 10 36.42±6.92 38.34±7.45 11 27.22±6.86 29.68±6.22 12 30.55±8.50 35.10±9.76 ●: Source & manufacturer‟s name are available upon request

 Values are recorded as Mean ± S.D  Mean difference is significant at the 0.05 level as analyzed by independence t-test followed by one-way analysis of varience (Anova)

112

Table-45 Oil Measurement Samples● Mean Values ± S.D Significance Control Test 01 19.08±4.53 22.40± 3.98 02 20.77±4.47 22.86±4.26 03 19.83±2.92 22.75±2.94 04 26.20±6.82 27.74±6.20 05 25.78±12.51 26.66±12.33 p<0.05 06 19.23±4.01 22.40±4.10 07 21.71±7.70 24.08±6.09 08 21.58±3.82 24.73±3.95 09 18.61± 3.99 20.40±3.73 10 19.95± 4.02 22.76± 3.66 11 26.96±13.79 28.91±12.63 12 24.80±9.85 27.27±9.71 ●: Source & manufacturer‟s name are available upon request

 Values are recorded as Mean ± S.D  Mean difference is significant at the 0.05 level as analyzed by independence t-test followed by one-way analysis of varience(ANOVA)

113

ANTI-INFLAMMATORY ACTIVITY OF CREAM FORMULATIONS

Table-46 Anti-Inflammatory Activity of Cream Formulation F1 Containing 50g RoseWater Concentration Wavelength (AC-AS)A A/AC A/AC*100 µg/ml 660 nm

50 1.007 1.113 0.525 52.2 150 0.764 1.356 0.639 63.9 300 0.734 1.386 0.653 65.3 750 0.629 1.491 0.703 70.3 1000 0.399 1.721 0.806 80.6 Mean±S.D

66.46±10.31 AC=Absorbance of control; AS=Absorbance of sample ; A= (AC-AS)

114

Table-46 (a) Overall in- vitro Anti-inflammatory Activity by Protein (Egg albumin) Denaturation of F1

Cream Formulation F1

Concentration µg/ml % Inhibition Viscosity (CPS)

50 52.2 0.69

150 63.9 0.73

300 65.3 0.82

750 70.3 0.91

1000 80.6 0.95

Mean±S.D 66.46±10.31 0.82±0.11

115

Table-47 Anti-inflammatory Activity of Cream Formulation F2 Containing 30g RoseWater

Concentration Wavelength (AC-AS)A A/AC A/AC*100 µg/ml 660 nm

50 1.242 0.878 0.414 41.4 150 1.229 0.891 0.42 42 300 0.996 1.124 0.53 53.01 750 0.895 1.225 0.577 57.7 1000 0.736 1.384 0.652 65.2 Mean±S.D

51.86±10.24 AC=Absorbance of control; AS=Absorbance of sample; A= (AC-AS)

116

Table-47 (a) Overall Anti-inflammatory Activity by Protein (Egg Albumin) Denaturation of F2

Cream Formulation F2

Concentration µg/ml % inhibition Viscocity (cps)

50 41.4 0.63

150 42 0.69

300 53.01 0.70

750 57.7 0.78

1000 65.2 0.80

Mean±S.D 51.86±10.24 0.72±0.06

117

Table-48 Anti-inflammatory Activity of Placebo Cream F3 Formulation

Concentration Wavelength (AC-AS)A A/AC A/AC*100 µg/ml 660 nm 50 0.802 -0.003 -0.003 -0.375 150 0.662 0.137 0.171 17.14 300 0.54 0.259 0.324 32.41 750 0.683 0.116 0.145 14.51 1000 0.45 0.349 0.436 43.67 Mean±S.D

21.47±17.00 AC=Absorbance of control; AS=Absorbance of sample ; A= (AC-AS)

118

Table-48 (a) Overall In vitro Anti-inflammatory Activity by Protein (Egg Albumin) Denaturation of F3

Placebo Cream F3

Concentration µg/ml % inhibition Viscocity (cps)

50 -0.375 0.23

150 17.14 0.09

300 32.41 0.34

750 14.51 0.30

1000 43.67 0.42

Mean±S.D 21.47±17.00 0.27±0.12

119

Table-49 Anti-inflammatory Activity of Standard Diclofenac Sodium

Concentration wavelength (AC-AS)A A/AC A/AC*100 µg/ml 660 nm

50 1.371 0.749 0.353 35.33

150 1.303 0.817 0.385 38.53

300 0.757 1.363 0.642 64.29

750 0.456 1.664 0.784 78.49

1000 0.29 1.83 0.863 86.32

Mean±S.D

60.59±23.02

AC=Absorbance of control; AS=Absorbance of sample ; A= (AC-AS)

120

Table-49 (a): Overall in-vitro Anti-inflammatory Activity by Protein (Egg Albumin) Denaturation

Standard Drug Diclofenac Sodium

Concentration µg/ml % inhibition Viscosity (cps)

50 35.33 0.98

150 38.53 1.03

300 64.29 1.04

750 78.49 1.08

1000 86.32 1.10

Mean±S.D 60.59±23.02 1.04±0.04

121

ANTI-OXIDANT ACTIVITY OF CREAM FORMULATION

Table-50 Antioxidant Activity of Cream Formulation F1 Containing 50g Rosewater

Concentration Standard Formulation as/ac 1-as/ac 1-(1-as/ac) Percentage of Reduction Absorbance (as) µg/ml Absorbance Power Capacity (ac) 700 nm 700 nm Equivalent to Ascorbic Acid

25 0.149 0.017 0.114 0.88 0.114 11.40

100 0.46 0.19 0.413 0.586 0.413 41.30

500 0.78 0.58 0.743 0.256 0.743 74.35

1000 1.03 0.84 0.815 0.184 0.815 81.55

As=absorbance of sample; ac=absorbance of standard

122

Table-51 Antioxidant Activity of Cream Formulation F2 Containing 30g RoseWater

Concentration Standard Formulation as/ac 1-as/ac 1-(1-as/ac) Percentage of Reduction Absorbance (as) µg/ml Absorbance (ac) Power Capacity Equivalent 700 nm 700 nm to Ascorbic Acid

25 0.149 0.012 0.0805 0.919 0.080 8.05

100 0.46 0.132 0.2867 0.713 0.286 28.69

500 0.78 0.51 0.653 0.346 0.653 65.38

1000 1.03 0.75 0.728 0.271 0.728 72.81

As=absorbance of sample; ac=absorbance of standard

123

Table-52 Antioxidant Activity of Placebo Cream Formulation F3

Concentration Standard Formulation as/ac 1-as/ac 1-(1-as/ac) Percentage of Reduction Absorbance (as) 700 µg/ml Absorbance (ac) Power Capacity Equivalent 700 nm nm to Ascorbic Acid

25 0.149 0.011 0.0738 0.926 0.073 7.38

100 0.46 0.067 0.145 0.854 0.145 14.56

500 0.78 0.085 0.108 0.891 0.108 10.89

1000 1.03 0.094 0.091 0.908 0.091 9.12

As=absorbance of sample; ac=absorbance of standard

124

ANTITUSSIVE ACTIVITY OF COUGH SYRUP FORMULATION C1,C2, C3, C4 AND STANDARD DRUGS

Table-53 Antitussive Activity of Cough Syrup C1 Formulation at a 5ml Adult Dose

Animal wt (g) Dose (ml) O min 30 min 60 min 90 min

180 0.18 12 05 0 01

176 0.176 23 10 01 0

182 0.182 97 12 01 0

Mean ± S.D

179.33±3.05 0.179±0.003 44±46.22 9±3.60 0.66±0.577 0.33±0.577

0 min is after cough induction 30, 60and 90 min after drug given  Values are recorded as Mean ± S.D

125

Table-54 Antitussive activity of Cough Syrup C1 formulation at a 10ml adult dose

Animal wt (g) Dose (ml) O min 30 min 60 min 90 min

176 0.35 130 01 0 02

170 0.34 123 05 02 0

156 0.312 122 07 0 0

Mean ± S.D

167.33±10.26 0.334±0.019 125±4.35 5±3.05 0.66±1.15 0.66±1.15

0 min is after cough induction 30, 60and 90 min after drug given  Values are recorded as Mean ± S.D

126

Table-55 The Antitussive Activity of Cough Syrup C1 Formulation at a 15ml Adult Dose

Animal wt (g) Dose (ml) O min 30 min 60 min 90 min

180 0.54 81 02 0 0

173 0.519 80 03 0 0

169 0.507 89 04 0 0

Mean ± S.D

174±5.56 0.522±0.016 83.33±4.93 3±1 0 0

0 min is after cough induction

30, 60 and 90 min after drug given

 Values are recorded as Mean ± S.D

127

Table-56 Antitussive Activity of Cough Syrup C2 Formulation at a 5ml Adult Dose

Animal wt (g) Dose (ml) O min 30 min 60 min 90 min

182 0.18 33 08 0 04

198 0.198 56 12 02 0

187 0.187 78 11 03 0

Mean ± S.D

189±8.18 0.188±0.009 55.66±22.50 10.33±2.08 1.66±1.52 1.33±2.30

0 min is after cough induction

30, 60 and 90 min after drug given

 Values are recorded as Mean ± S.D

128

Table-57 Antitussive Activity of Cough Syrup C2 Formulation at a 10ml Adult Dose

Animal wt (g) Dose (ml) O min 30 min 60 min 90 min

179 0.35 107 06 0 0

170 0.34 98 12 0 0

172 0.34 122 11 0 0

Mean ± S.D

173.66±4.72 0.34±0.005 109±12.12 9.66±3.21 0 0

0 min is after cough induction 30, 60 and 90 min after drug given  Values are recorded as MEAN ± S.D

129

Table-58 Antitussive Activity of Cough Syrup C2 Formulation at a 15ml Adult Dose

Animal wt (g) Dose (ml) O min 30 min 60 min 90 min

183 0.54 23 03 0 0

180 0.54 54 10 0 0

182 0.546 32 05 0 0

Mean ± S.D

181.66±1.52 0.542±0.003 36.33±15.94 6±3.60 0 0

0 min is after cough induction

30, 60 and 90 min after drug given

 Values are recorded as MEAN ± S.D

130

Table-59 Antitussive Activity of Cough Syrup C3 Formulation at a 5ml Adult Dose

Animal wt (g) Dose (ml) O min 30 min 60 min 90 min

176 0.176 113 03 0 0

170 0.170 109 05 0 0

173 0.173 122 05 0 0

Mean ± S.D

173±3 0.173±0.003 114.66±6.65 4.33±1.15 0 0

0 min is after cough induction 30, 60 and 90 min after drug given  Values are recorded as Mean ± S.D

131

Table-60 Antitussive Activity of Cough Syrup C3 Formulation at a 10ml Adult dose

Animal wt (g) Dose (ml) O min 30 min 60 min 90 min

182 0.364 103 Death

180 0.36 97 10 05 0

179 0.358 123 12 0 0

Mean ± S.D

180.33±1.52 0.36±0.003 107.66±13.61 11±1.41 2.5±3.53 0

0 min is after cough induction 30, 60 and 90 min after drug given  Values are recorded as Mean ± S.D

132

Table-61 The Antitussive Activity of Cough Syrup C3 Formulation at a 15ml Adult Dose

Animal wt (g) Dose (ml) O min 30 min 60 min 90 min

178 0.53 93 02 0 0

170 0.51 67 22 07 02

190 0.57 98 34 0 0

Mean ± S.D

179.33±10.06 0.536±0.030 86±16.64 19.33±16.16 2.33±4.04 0.66±1.15

0 min is after cough induction 30, 60 and 90 min after drug given  Values are recorded as Mean ± S.D

133

Table-62 Antitussive Activity of Cough Syrup C4 Formulation at a 5ml Adult Dose

Animal wt (g) Dose (ml) O min 30 min 60 min 90 min

185 0.18 33 09 0 01

180 0.18 28 10 0 02

178 0.17 70 12 0 0

Mean ± S.D

181±3.60 0.176±0.005 43.66±22.94 10.33±1.52 0 1±1

0 min is after cough induction 30, 60 and 90 min after drug given  Values are recorded as Mean ± S.D

134

Table-63 The Antitussive activity of Cough Syrup C4 Formulation at a 10ml Adult Dose

Animal wt (g) Dose (ml) O min 30 min 60 min 90 min

178 0.35 93 05 0 0

179 0.358 98 09 0 0

170 0.34 105 11 0 0

Mean ± S.D

175.66±4.93 0.349±0.009 98.66±6.02 8.33±3.05 0 0

0 min is after cough induction 30, 60 and 90 min after drug given  Values are recorded as Mean ± S.D

135

Table-64 Antitussive Activity of Cough Syrup C4 Formulation at a 15ml Adult Dose

Animal wt (g) Dose (ml) O min 30 min 60 min 90 min

175 0.528 50 06 0 0

156 0.468 87 07 01 0

189 0.567 43 05 0 0

Mean ± S.D

173.33±16.56 0.521±0.04 60±23.64 6±1 0.33±0.577 0

0 min is after cough induction 30, 60 and 90 min after drug given  Values are recorded as Mean ± S.D

136

Table-65 Antitussive Activity of Standard Drug (IVY Leaves Dry Extract) Cough Syrup at 5ml Adult Dose

Animal wt (g) Dose (ml) O min 30 min 60 min 90 min

172 0.172 99 0 0 0

175 0.175 90 0 0 0

171 0.171 88 0 0 0

Mean ± S.D

172.66±2.08 0.172±0.002 92.33±5.85 0 0 0

0 min is after cough induction 30, 60and 90 min after drug given  Values are recorded as Mean ± S.D

137

Table-66 Antitussive Activity of Standard Drug (Diphenhydramine HCL and Dextromethorphan HBr) Cough Syrup at 10ml Adult Dose

Animal wt (g) Dose (ml) O min 30 min 60 min 90 min

156 0.156 45 18 5 9

158 0.158 56 5 8 4

156 0.156 139 1 1 0

Mean ± S.D

156.66±1.15 0.156±0.001 80±51.3 8±8.88 4.66±3.51 4.33±4.50

0 min is after cough induction 30, 60 and 90 min after drug given  Values are recorded as MEAN ± S.D

138

Table-67 Overall Antitussive Activity of Cough Syrup Formulation C1, C2, C3, C4 and Standard Drugs.

Frequency of cough (mean±SEM) Treatment Dose 0 min 30min 60 min 90min Control 119.66±29.33 6.66±0.33 16.66±3.33 20.00±5.00 Standard ivy extract 5ml 92.33±3.38 0.00±0.00 0.00±0.00* 0.00±0.00* Standard dextro ** 10ml 80.00±29.67 8.00±5.13 4.66±2.02* 4.33±2.60* 5ml 44.00±26.68 9.00±2.08 0.66±0.33* 0.33±0.33* C1 10ml 125.00±2.51 4.33±1.76 0.66±0.66* 0.66±0.66* 15ml 83.33±2.84 3.00±0.57 0.00±0.00* 0.00±0.00* 5ml 55.66±12.99 10.33±1.20 1.66±0.88* 1.33±1.33* C2 10ml 109.00±7.00 9.66±1.85 0.00±0.00* 0.00±0.00* 15ml 86.00±9.60 6.00±2.08 0.00±0.00* 0.00±0.00* 5ml 114.66±3.84 4.33±0.66 0.00±0.00* 0.00±0.00* C3 10ml 107.66±7.85 8.33±2.18 1.66±1.66* 0.00±0.00* 15ml 86.00±9.60 19.33±9.33 2.33±2.33* 0.66±0.66* 5ml 43.66±13.24 10.33±0.88 0.00±0.00* 1.00±0.57* C4 10ml 98.66±3.48 8.33±1.76 0.00±0.00* 0.00±0.00* 15ml 60.00±13.65 6.00±0.57 0.33±0.33* 0.00±0.00* *P <0.05 significant as compared to control ** Diphenhydramine HCL and Dextromethorphan HBr  Values are recorded as Mean ± SEM  Mean difference is significant at the 0.05 level (P<0.05,Tukey‟s test)as analyzed by one –way analysis of varience (Anova)

139

FIGURES

140

Fig. 1 GC-MS Analysis of Sample No 9

S.No Compound Retension Time (RT)

1 Phenylethylalcohol 11.621

2 Citronellol 14.562

3 Pentadecane 18.751

4 Heptadecanol 20.275

5 Octadecanol 20.41

6 Tetracosane 23.089

7 Decane 11.232 8 Nonane 7.600

141

Fig. 2 GC-MS Analysis of Sample No 8

S.No Compound Retension Time (RT)

1 Phenylethylalcohol 12.594

2 Citronellol 15.867

3 Pentadecane 13.367

4 Heptadecanol 23.44

5 Octadecanol 24.635

6 Tetracosane 22.93

7 Decane 11.20 8 Nonane 7.5

142

Fig. 3 GC-MS Analysis of Sample No 12

S.No Compound Retension Time (RT)

1 Phenylethylalcohol 11.43

2 Citronellol 14.62

3 Pentadecane 13.03

4 Heptadecanol 20.21

5 Octadecanol 20.12

6 Tetracosane 23.09

7 Decane 11.20 8 Nonane 7.31

143

Fig. 4 FT-IR Analysis

Fig. 5 FT-IR Analysis

144

Fig. 6 FT-IR Analysis

Fig. 7 FT-IR Analysis

145

Fig. 8 FT-IR Analysis

Fig. 9 FT-IR Analysis

146

Fig. 10 FT-IR Analysis

Fig. 11 FT-IR Analysis

147

Fig. 12 FT-IR Analysis

Fig. 13 FT-IR Analysis

148

Fig.14 FT-IR Analysis

Fig. 15 FT-IR Analysis

149

Sample No 1

35 y = 0.0095x + 20.919 R² = 0.5633 30 % mortality 25 Linear (% 20 mortality)

15 LC50= 3232.22 10

0 0 200 400 600 800 1000 1200

% Mortality

Fig. 16 Brine Shrimp (Artemia salina) Lethality Bioassay

Sample No 2 25 y = -0.007x + 23.701 R² = 0.9932 20 % mortality Linear (% mortality) 15

LC50= 3757.14

0 0 500 1000 1500

% Mortality

Fig. 17 Brine Shrimp (Artemia salina) Lethality Bioassay

150

Sample No 3 35 y = 0.0055x + 24.624 R² = 0.8182

30 % mortality

25 LC50= 5076 20

0 0 200 400 600 800 1000 1200 % Mortality

Fig. 18 Brine Shrimp (Artemia salina) Lethality Bioassay

Sample No 4 30 y = 0.0085x + 17.96 R² = 0.8363 25 % mortality

Linear (% mortality) 20

LC50= 4005 15

0 0 200 400 600 800 1000 1200 % Mortality

Fig. 19 Brine Shrimp (Artemia salina) Lethality Bioassay 151

Sample No 5 35 y = -0.0125x + 29.071 R² = 0.9744 30 % mortality 25 Linear (% mortality) 20

15 LC50= 1744.16

0 0 200 400 600 800 1000 1200 % Mortality

Fig. 20 Brine Shrimp (Artemia salina) Lethality Bioassay

Sample No 6 35 y = 0.0095x + 20.919 R² = 0.5633

30 % mortality

25 Linear (% mortality)

LC50=3232.22 20

0 0 200 400 600 800 1000 1200 % Mortality

Fig. 21 Brine Shrimp (Artemia salina) Lethality Bioassay 152

Sample No 7 35 y = 0.0055x + 24.624 R² = 0.8182 30 % mortality 25 Linear (% mortality)

20 LC50= 5076 15

0 0 200 400 600 800 1000 1200 % Mortality

Fig. 22 Brine Shrimp (Artemia salina) Lethality Bioassay

Sample No 8

30 y = 0.011x + 15.924 R² = 0.8172 25 % mortality 20 Linear (% mortality)

15 LC50= 3098.18

0 0 200 400 600 800 1000 1200

% Mortality

Fig. 23 Brine Shrimp (Artemia salina) Lethality Bioassay

153

Sample No 9 45 y = 0.0215x + 18.698 R² = 0.961 40 % mortality 35 Linear (% mortality) 30 LC = 1490.95 25 50 20 15 10 5 0 0 200 400 600 800 1000 1200 % Mortality

Fig. 24 Brine Shrimp (Artemia salina) Lethality Bioassay

Sample No 10 45 y = -0.0145x + 32.036 40 R² = 0.4372 35 % mortality 30 Linear (% mortality) 25 LC50= 1283.57 20 15 10 5 0 0 200 400 600 800 1000 1200 % Mortality

Fig. 25 Brine Shrimp (Artemia salina) Lethality Bioassay

154

Sample No 11 25 y = 0.0075x + 16.109 R² = 0.6512

20 % mortality Linear (% mortality) 15 LC50= 4842.85

0 0 200 400 600 800 1000 1200

% Mortality

Fig. 26 Brine Shrimp (Artemia salina) Lethality Bioassay

Sample No 12 25 y = 0.0018x + 19.742 R² = 0.0825 % mortality 20 Linear (% mortality)

LC50= 30260 15

0 0 200 400 600 800 1000 1200 % Mortality

Fig. 27 Brine Shrimp (Artemia salina) Lethality Bioassay

155

900

800 700 600 500 50 400 100 300 200

200 % reduction power capacitypower reduction% 100 0 sample 12 sample 9 sample 8 sample 12 sample 9 sample 8 5 ml 10ml Samples

Fig. 28 Percentage of Ferric Reducing Power Capacity of Rose Water Samples (05 and 10 ml) Equivalent to 50, 100, 200 µg/ml of Ascorbic Acid as Standard

156

100%

80%

VOLUME OF SAMPLE 60% (10ML) VOLUME OF SAMPLE (7ML) 40% VOLUME OF SAMPLE (5ML) VOLUME OF SAMPLE 20% (3ML)

% % Inhibition ofProtien Denaturation 1 2 3 4 5 6 -20% 7 8 9 10 11 12

Sample No

Fig. 29 In-Vitro Anti-Inflammatory Activity of Rose Water Samples

120

100

20 % % inhibition of proteindenautration

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Standard 100µg/ml Sample 10ml

Fig. 30 In-Vitro Anti-Inflammatory Activity of 10ml Rose Water Samples Compared with 100µg/ml of Standard Drug (Diclofenac Sodium)

157

SPF 4.5 4

3.5 3 2.5 2 SPF 1.5

Sun protected Factor protected Sun 1 0.5 0 1 2 3 4 5 6 7 8 9 10 11 12

Samples no Fig. 31 In-Vitro Sun Protecting Factor of Rose Water Samples

158

ANTI-BACTERIAL ANALYSIS OF ROSE WATER SAMPLES

159

Fig. 32 Anti-bacterial activity of rose water sample#1

Cip-ciprofloxcin, Amc-amoxicilline, s.fec- Streptococcus fecalis, s.epi- Streptococcus epidermis, k.p- Klebsiella pneumonia, proteus- Proteus mirabilis, S.aureus- Staphylococcus aureus,p.auro- Pseudomonas aeruginosa, c.xerosis- Corynebacterium xerosis, entero- Enterococcus sp., s.typhi- Salmonella typhi, Para B- Salmonella typhipara B, Para A- Salmonella typhipara A, E.coli- Escherichia coli

160

Fig 33 Anti-bacterial activity of sample#2 s.epi- Streptococcus epidermis, PA- Pseudomonas aeruginosa,SA- Staphylococcus aureus, CX-Corynebacterium xerosis, S. Para B Salmonella typhipara B-, S.Fec- Streptococcus fecalis, EC- Escherichia coli, Ent- Enterococcus sp., Kp- Klebsiella pneumonia, Proteus Proteus mirabilis, Para A- Salmonella typhipara A, S.typhi Salmonella typhi 161

Fig. 34 Anti-bacterial activity of sample#3

Ent- Enterococcus sp., para B- Salmonella typhipara B, SA- Staphylococcus aureus, SF- Streptococcus fecalis, KP- Klebsiella pneumonia, PA Pseudomonas aeruginosa-, Proteus Proteus mirabilis, Para B- Salmonella typhipara B, ST- Salmonella typhi, Cx- Coryneba- cterium xerosis, SE- Streptococcus epidermis, EC- Escherichia coli

162

Fig. 35 Anti-bacterial activity of sample#4 Kp- Klebsiella pneumonia, EC- Escherichia coli, SA- Staphylococcus aureus, S Para A- Salmonella typhipara A, S Para B- Salmonella typhipara B, ENT- Enterococcus sp., SE- Streptococcus epidermis, PM- Proteus mirabilis, PA- Pseudomonas aeruginosa, CX- Corynebacterium xerosis, ST- Salmonella typhi, SF- Streptococcus fecalis

163

Fig. 36 Anti-bacterial activity of sample#5 PA- Pseudomonas aeruginosa, EC- Escherichia coli, KP- Klebsiella pneumonia,SA- Staphylococcus aureus, ENT- Enterococcus sp., PM Proteus mirabilis, S Para A- Salmonella typhipara A, ST- Salmonella typhi, CX- Corynebacterium xerosis, S Para B- Salmonella typhipara B, EC- Escherichia coli, SA- Staphylococcus aureus

164

Fig. 37 Anti-bacterial activity of sample#6 S Para A- Salmonella typhipara A, S Para B- Salmonella typhipara B, EC- Escherichia coli, SA- Staphylococcus aureus, PA- Pseudomonas aeruginosa, ENT- Enterococcus sp., CX-Corynebacterium xerosis, PM- Proteus mirabilis, K.P- Klebsiella pneumonia, SE- Streptococcus epidermis, ST- Salmonella typhi, SF- Streptococcus fecalis

165

Fig. 38 Anti-bacterial activity of sample#7 EC-Escherichia coli, ENT Enterococcus sp., SF- Streptococcus fecalis, S.a- Staphylococcus aureus, SP A- Salmonella typhipara A, PM- Proteus mirabilis, CX- Corynebacterium xerosis, S.E- Streptococcus epidermis, ST- Salmonella typhi, para B- Salmonella typhipara B, KP- Klebsiella pneumonia, PA- Pseudomonas aeruginosa 166

Fig. 39 Anti-bacterial activity of sample#8 EC Escherichia coli, SP B- Salmonella typhipara B,S.a Staphylococcus aureus, Pa- Pseudomonas aeruginosa, P.M- Proteus mirabilis, SP A Salmonella typhipara A-, KP- Klebsiella pneumonia, CX- Corynebacterium xerosis, SF Streptococcus fecalis, SE- Streptococcus epidermis, ST- Salmonella typhi, ENT- Enterococcus sp

167

Fig. 40 Anti-bacterial activity of sample # 9 S.a- Staphylo coccus aureus, P.a- Pseudomonas aeruginosa, SF- Streptococcus fecalis, P.m- Proteus mirabilis, CX- Corynebacterium xerosis, ST- Salmonella typhi,EC- Escherichia coli, ENT- Enterococcus sp., SP B- Salmonella typhipara B, PM- Proteus mirabilis, S.E- Streptococcus epidermis, SP A Salmonella typhipara A 168

Fig. 41 Anti-bacterial activity of sample # 10 SF- Streptococcus fecalis, SP A- Salmonella typhipara A, SP B- Salmonella typhipara B, KP- Klebsiella pneumonia, CX- Corynebacterium xerosis, SA- Staphylococcus aureus, ENT- Enterococcus sp., P.a Pseudomonas aeruginosa, ST- Salmonella typhi, SE- Streptococcus epidermis, PM- Proteus mirabilis, EC- Escherichia coli

169

Fig. 42 Anti-bacterial activity of sample # 11 SP B- Salmonella typhipara B, ST- Salmonella typhi, P.a- Pseudomonas aeruginosa, Pm Proteus mirabilis, EC- Escherichia coli, SF- Streptococcus fecalis, CX- Corynebacterium xerosis, SA- Staphylococcus aureus, SP A- Salmonella typhipara A, S.E- Streptococcus epidermis, K.P- Klebsiella pneumonia, ENT- Enterococcus sp.

170

Fig. 43 Anti-bacterial activity of sample # 12 c.xerosis- Corynebacterium xerosis, ENTERO- Enterococcus sp., S.Typhi- Salmonella typhi, PARA B- Salmonella typhipara B, PARA A- Salmonella typhipara A, E-Coli- Escherichia coli, S.fec- Streptococcus fecalis, s.epi- Streptococcus epidermis, k.p- Klebsiella pneumonia, Proteus- Proteus mirabilis, S.aureus- Staphylococcus aureus, p. aero Pseudomonas aeruginosa 171

Gram-positive Bacilli Gram-negative Bacilli Gram-negative Bacilli Fig. 44 Bacterial Contamination Gram-Positive and Gram-Negative Bacteria Present in Rose Water Samples

172

35 CONTROL TEST

10 % of Glow Measurment Glowof%

0 1 2 3 4 5 6 7 8 9 10 11 12 SAMPLE NO

Fig. 45 Percentage of Change in Skin Glow after Application of Rose Water Samples

Control : On which rose water not applied

Test: on which rose water applied

during 03 week of glow study increase of glow of skin by

use of different rose water samples

Difference of Test and Control Reading Control and Test of Difference 0 1 2 3 4 5 6 7 8 9 10 11 12 13 sample no.

Fig. 46Comparison of Rose Water Samples Enhancing Glow Effect

173

45 CONTROL 40 TEST 35 30 25 20 15 10

% of Hydration Measurment Hydrationof% 5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Sample No.

Fig. 47 Percentage of Change in Skin Hydration after Application of Rose Water Samples

Control : On Which Rose Water not applied

Test: on Which Rose Water Applied

Difference Test of and ControlGroups

1 2 3 4 5 6 7 8 9 10 11 12 Sample No.

Fig. 48 Comparison of Rose Water Samples Enhancing Hydration Effect

174

35 CONTROL TEST 30

% % ofOil Measurment 10

0 1 2 3 4 5 6 7 8 9 10 11 12 Sample No.

Fig. 49 Percentage of Change in Skin Oil Content after Application of Rose Water Samples

Control : On which rose water not applied

Test: on which rose water applied

During the 03 week of oil measurment study the increase in oil contant among different samples of rose water Series1 3.32 3.17 3.15 2.92 2.81 2.37 2.47 2.09 1.95 1.79 1.54

0.88 Difference of Test and Control Groups Control and Test of Difference 1 2 3 4 5 6 7 8 9 10 11 12 sample no.

Fig. 50 Comparison of Rose Water Samples Enhancing Oil Content Effect

175

50 F1 40 F2

30 F3 % % Of ReductionPower 20

0 25 100 500 1000

Concentration µg/ml Fig. 51 In-Vitro Anti-Oxidant Activity by Ferric Reducing Power of Cream Formulation F1, F2 And F3

100

F1 40 F2 F3 F4 20

0 % Inhibition of Protein Denaturation Proteinof Inhibition% 50 150 300 750 1000

-20 Concentration µg/ml

Fig. 52 Percentage Inhibition of Diclofenac Sodium and Formulated Cream against Denaturation of Protein 176

90 y = 0.023x + 55.92 80 R² = 0.856 IC50=257.39 70 % age of

60 reduction power

50 % Inhibition of protein denatured

40 Percentage 30

0 0 200 400 600 800 1000 1200 Concentration Fig. 53 Relationship between Ferric Reducing Power Anti-Oxidant Activity and Percentage Inhibition of Protein Denaturation of Cream Formulation F1

80 y = 0.024x + 40.99 70 R² = 0.923 Ic50=375.41 60

40 % age of

Percentage reduction 30 power

20 % inhibition of protein 10 denaturation

0 0 200 400 600 800 1000 1200 Concentration µg/ml

Fig. 54 Relationship between Ferric Reducing Power Anti-Oxidant Activity and Percentage Inhibition of Protein Denaturation of Cream Formulation F2

177

ANTITUSSIVE ACTIVITY OF ROSE WATER BASED COUGH SYRUP FORMULATIONS

140

120

100

80 5 ml dose

60 10 ml dose No coughof 15 ml dose 40

0 0 min 30 min 60 min 90 min Time

Fig. 55 Antitussive Activity of Rose Water Based Formulation c1 Contaning Sample # 12 as Active Ingredient

120

100

60 5 ml dose

10 ml dose No of cough of No 40 15 ml dose

0 0 min 30 min 60 min 90 min Time

Fig. 56 Antitussive Activity of Rose Water Based Formulation C2 Contaning Sample # 9 as Active Ingredient

178

120

100

60 5 ml dose

10 ml dose No of cough of No 40 15 ml dose

0 0 min 30 min 60 min 90 min Time

Fig. 57 Antitussive Activity of Rose Water Based Formulation C3 Contaning 50% Sample # 12 as Active Ingredient

120

100

60 5 ml dose

10 ml dose No of cough of No 40 15 ml dose

0 0 min 30 min 60 min 90 min Time

Fig. 58 Antitussive Activity of Rose Water Based Formulation C4 Contaning 50% Sample # 9 As Active Ingredient

179

References

180

REFERENCES

A.K. Mishra, A., Mishra, P. Chattopadhyay (2011). Evaluation of sun protection factor of some marketed formulations of sunscreens by ultraviolet spectroscopic method. J. Current Pharma. Res. 5(1),32-35.

Adwan, G., Mhanna, M., (2008). Synergistic effects of plant extracts and antibiotics on Staphylococcus aureus strains isolated from clinical specimens. Middle East J. sci. res. 3:134–139.

Afaq, F., Malik, A., Syed, D., Maes, D., Matsui, M. S., Mukhtar, H. (2005).Pomegranate fruit extract modulates UVB-mediated phosphorylation of mitogen-activated protein kinases and activation of nuclear factor kappa B in normal human epidermal keratinocytes, Photochem Photobiol, 81: 38–45.

Akhmadieva, A., Zaichkina, S.I., Ruzieva, R.K., Ganassi, E.E. (1992). The protective action of a natural preparation of anthcyan(pelargonidin-3, 5-diglucoside) Radio biologiia,33:433-435.

Ali, R. and Siddiqui, N. (2013). Biological Aspects of Emerging Benzothiazole: A Short Review. Journal of Chemistry,

Alizadehfard, M., R, and Wiley. D. E. (1995).Viscosity of whey protein solutions. Irranian J Polymer Sci. Tech., 4(2); 126-133.

American Society for Horicultural Science (2011). A rosy future for Pakistan’s Cut flower industry, Science Daily.

Andoğan, B.C., Baydar, H., Kaya, S., Demirci, M., Özbaşar, D., Mumcu, E. (2008). Antimicrobial activity and chemical composition of some essential oils. Arch Pharm Res; 25: 860–864.

178

Anu, S., Deepashree, G. H. M., Shruthi, S. Singh, V., Bole, S., Balu, S. and Vedamurthy, A. B. (2013). Phytochemical screening, antioxidant and antibacterial activity of Piper longum. International Journal of Current Research,5(2),148-152.

Apak, R., Güçlü, K., Demirata, B., Özyürek, M., Çelik, S.E., Bektaşoğlu, B., Berker, K.I.andÖzyurt, D. 2007. Comparative evaluation of various total antioxidant capacity assays applied to phenolic compounds with the CUPRAC assay. Molecules 12: 1496-1547.

B.H. More., S. N. Sakharwade., S.V.Tembhurne., D.M.Sakarkar.(2013).Evaluation of sunscreen activity pf cream containing leaves extract of Buteamonosperma for topical application. International Journal of research in cosmetic Science. 3(1):1- 6.

Bambal, V., and Mishra, M. (2014). Evaluation of in vitro sunscreen activity of herbal cream containing extract of Curcuma longa and Buteamonosperma. World J Pharmaceut Res., 3(2); 3026-3035.

Bayra, A., Akgul, A. (1994). Volatile oil composition of Turkish rose(Rosa damascena). J Sci Food Agric: 64: 441-448.

Boskabady, M.H., Kiani, S., Rakhshandah, H,.(2006). Relaxant effects of Rosa damascena on guinea pig tracheal chains and its possible mechanism(s) J Ethnopharmacol: 106: 377–382.

Boskabady, M.H., Shafei, M. N., Saberi, Z., and Amini, S. (2011).Pharmacological effects of Rosa damascene, Iranian Journal of Basic Medical Sciences, 14(4), 295-307.

Bruttia, R., Magua, M.M., Agorkua, E.S., and Govendera, P, P.(2016). Alternative method for qualitative analysis of specific non-volatile organic compounds present in South African water systems.S. Afr. J. Chem: 69:244-253.

179

C. Malsawmtluangi, D. K., Nath, I., Jamatia, L. Ralte, E. Zarzoliana, L. Pachuau (2013). Determination of sun protection factor (SPF) number of some aqueous herbal extracts. J. App. Pharm. Sci.; 3(09): 150-151.

Calabrone, L., Larocca, M., Marzocco, S., Martelli, G., Rossano, R.,(2015). Total phenols and flavonoids content, antioxidant capacity and lipase inhibition of root and leaf horseradish (Armoraciarusticana) extracts. Food and Nutr Sci.,6:64-74.

Chandra, S., Chatterjee, P., Dey, P., Bhattacharya, S., (2012). Evaluation of i-vitro anti- inflammatory activity of coffee againt the denaturation of protein.Asian Pacific J Trop Biomed, 2(1); S178-S180.

Chavan, R. W., Saboo, S. S., Tapadiya, G.G., Khadabadi, S.S. (2014). Evaluation of phytochemical and anticancer potential of Aphanamixispolystachya(Wall.) parker stem bark using in-vitro models. In-vitro models.Int. J.Pharm. Sci Rev Rev., 28(1);138-142.

Choudhary, S.P., Kanwar, M., Bhardwaj, R., Gupta, B.D., Gupta, R.K,. (2011). Epibrassinolide ameliorates Cr (VI) stress via influencing the levels of indole-3- acetic acid, abscisic acid, polyamines and antioxidant system of radish seedlings. Chemosphere: 84 592–600

Cravellol, B., Ferri, A. (2008). Relationships between skin properties and environmental parameters.Skin Res Technol.14:180-6.

Dannhardt, G.and Kiefer, W,. (2001). Cyclooxygenase inhibitors: Current status and future prospects, European Journal of Medicinal Chemistry, 36: 109–126

Davies, H., Bignell, G. R., Cox C.(2002).Mutations of the BRAF gene in human cancer, Nature 417(6892)949-954.

Dicpinigaitis, P.V. (2006). Current and future peripherallyacting antitussives. Respir Physiol Neurobiol: 152:356–62.

180

Dolati, K., Rakhshandeh, H., Shafei, M.N. (2011). Antidepressent like effect of aqueous extract from Rosa damascena in mice. Avicenna, J. Phytomed. 1:91-7.

Doss, A., and Pugalenthi, M.(2012). Evaluation of antioxidant activity and phytochemical screening of Malusdomesticaborkh (apple) and Phaseolus vulgaris L.(green beans). Journal of Pharmaceutical and Scientific Innovation, 1(3), 1-4.

Dr. N. Gunavathy., Dr. S. Padmavathy., Dr. S. C. Murugavel (2014). Phytochemical evaluation of musaacuminata bract using screening, FTIR and uv-vis spectroscopic analysis. Journal of international academic research for multidisciplinary: 2:1.

Eberhardt, T.L., Li, X., Shupe, T.F., and Hse, C.Y. (2007). Chinese Tallow Tree (SapiumSebiferum) utilization: Characterization of extractives and cell-wall chemistry. Wood Fiber Sci. 39: 319-324.

Egawa, M., Oguri, M., Kuwahara, T. (2002). Effect of exposure of human skin to a dry environment, Skin Res Technol:8:212-8.

Flynn, T.C., Petros, J., Clark, R.E. (2001). Dry skin and moisturizer. Clinc. Dermatol: 19:387-92.

Foster, S., Duke, J.A. (1990). Rosa rugosa Thunb. In Medicinal Plants. New York , NY.

Garcia-Lafuente, A., Guillamon, E., Villares, A., Rostagno, M.A., Martinez, J.A. (2009). Flavonoids as anti-inflammatory agents: implications in cancer and cardiovascular disease. Inflamm Res 58:537552

Garwood, J.A., (2009). Rose Garden. Elle Canada. http: / /www.ellecanada.com / beauty / face / rose-garden /a/ 24145

Gautam, R.K., Sharma, S., Sharma, k.(2013).Comparative evaluation of anti-arthritic activity of Pongamiapinnata (Linn.) Pierre and Punicagranatum Linn.An in-vitro study.Int J Pharm Pharmaceut Sci., 5(4); 721-724.

181

Gholamhoseinian, A., Fallah, H., sharifi-far, F., Mirtajaddini, M. (2008). The inhibitory effect of some Iranian plantstracts on the alpha glucosidase. Iran J Basic Med Sci.:11:1–9.

Gochev, V., Jirovetz, I., Wlcek, K. (2009). Chemical composition and antimicrobial activity of historical rose oil from Bulgaria. J. Essent. Oil Bear Plants: 12:1-6.

Gochev, V., Wlcek, K., Buchbauer, G.,(2008). Comparative evaluation of antimicrobial activity and composition of rose oil from various geographic origins, in particular Bulgarian rose oil, Nat Prod. Commun.:3:1063-1068.

Gudin, S., Rose (2000). Genetics and breeding, Plant Breed. Rev: 17,159-189.

Guyer, S.F., Afaq, F., Mukhtar, H. (2003). Photochemoprotection of skin cancer by botanical agents. Photodermatol Photoimmunol Photomed :2:26-28.

H. M. Arif Ullah, Sayera, Zaman, Fatematuj Juhara, Lucky, Akter, S. M. Tareq, Emranul, Haque M, Asum and Rajib, Bhattacharajee, (2014). Evaluation of antinociceptive, in-vivo & in-vitro anti-inflammatory activity of ethanolic extract of Curcuma Zedoaria rhizome, BMC Complementary and Alternative medicine: 14:346

Hajhashemi, V., Ghannadi, A., Hajiloo, M. (2010). Analgesic and anti-inflammatory effects of Rosa damascenahydroalcoholic extract and its essential oil in animal models. Iran J Pharm Res: 9:163.

Halawani, E. M., (2014). Antimicrobial activity of Rosa damascena petals extracts and chemical composition by gas chromatography-mass spectrometry (GC/MS) analysis. Afr. J. Microbiol. Res. 8(24):2359-2367.

Hameed, A., S. B. Vohora (2001). Indian system of Medicine Skin Diseases. CBS Publishers and Distributors, India Pp. 1-174.

Hassanein, A.M.A., (2010). Improved quality and quantity of winter flowering in rose (Rosa spp.) by controlling the time and type of pruning applied in autumn. World J. Agric. Sci., 6:260-267.

182

Hazra, K. M., Roy, R. N., Sen, S. K., and Laska, S. (2007). Isolation of antibacterial pentahydroxy flavones from the seeds of Mimusopselengi Linn. Afr. J. Biotechnol. 6 (12): 1446-1449.

Hinneburg, I., Dorman, D.H.J., and Hiltunen, R. (2006). Antioxidant activities of extracts from selected culinary herbs and spices. Food Chem., 97:122-129.

Hinneburg, I., Dorman, H. J. D. and Hiltunen, R. (2006), Antioxidant activities of extracts from selected culinary herbs and spices. Food Chemistry,97, 122-129.

Huxley, A, ed. (1992). New RHS Dictionary of Gardening. Macmillan

Imam, S., Azhar, I., Mahmood, Z. A., (2015). In-vitro evaluation of sun protection factor of a cream formulation prepared from extracts of Musa accuminata (L.), Pisidiumgujava (L.) and Pyruscommunis (L.).Asian J Pharm Clin Res: 8: 3: 234- 237.

J. L. (1982). Brine shrimp: a convenient general bioassay for active plant extracts; Planta

Jabbarzadeh, Z., Khosh-khui, M. (2005). Factors affecting tissue culture of damask rose (Rosa damasecna Mill). Sci. Hortic 105:475-82.

Jerry Haynes (2010), History of Roses: Damask Roses

Jirovetz, I., Eller, G., Buchbauer, G., (2006). Chemical composition, antimicrobial activities and ador description of some essential oils with characteristic floral-rosy scent and of their principal aroma compounds. Recent Res Dev. Agron. Hortic, 2:2:1-12.

Joy, A. and Alam, Md. A.(2012). Quantitative structure activity relationship (QSAR) of cardiac glycoside: the development of predictive in vitro cytotoxic activity model. Der Pharmacia Lettre, 4(4):1246-1269.

183

Kale, S., Gaikwad, M., and Bhandare, S. (2011). Determination and comparison of in vitro SPF of topical formulation containing Lutein ester from Tageteserecta L. flowers, Moringaoleifera Lam seed oil and Moringaoleifera lam seed oil containing Lutein ester. Int. J. Res Pharmaceut and Biomed Sci., 2: 1220-1224.

Kamei, J. (2002). Delta-opioid receptor antagonists as a new concept for central acting antitussive drugs. Pulm Pharmacol Ther 15:235–40

Kapoor, S., Saraf, S. (2017) Regulatory bodies and truth about cosmetic ingredients. Farmavita.net, 6 Nov.

Kapoor, S., Saraf, S., (2008). Risk analysis tools for toxicological profile of cosmetics. Inter J Toxicol: 5(2):1-20.

Karthika, P., Jayshree, N., (2013). Formulation and evaluation of sunscreen cream containing flower extract of Delonix Regia. Int J Pharm Integ Life Sci., 1(6); 111- 129.

Kaul, V. K., Singh, V., Singh, B. (2000) Damask rose and marigold: prospective industrial crops. J Med Aromat Plant Sci: 22: 313-318.

Kaur, G., Tirkey, N., Bharrhan, S., Chanana, V., Rishi, P., and Chopra K.(2006). Inhibition of oxidative stress and cytokine activity by curcumin in amelioration of endotoxin-induced experimental hepatotoxicity in rodents. Clin. Exp. Immunol: 145: 313–321.

Khan, M.A., Rehman, S. (2005). Extraction and analysis of essential oil of Rosa species. Int. J. Agric. Biol.: 7: 973-974.

Kheirabadi, M., Moghimi, A., Rakhshande, H., Rassouli, M.B. (2008). Evaluation of the anticonvulsant activities of Rosa damascena on the PTZ induced seizures in wistar rats. J. Biol. Sci. 8:426–430.

184

Kim, Y. H., H. E Yang., B. K Park, M. Y. Heo, B. K, Jo, H. P. Kim (2002). The extract of the flower of Prunuspersica, a new cosmetic ingredient, protects against solar ultraviolet-induced skin damage in vivo. J. Cosmet. Sci. 53(1):27-34.

Ko, H., Hofer, H.B., Pichler, B., Buchananan, K.A., Sjostrom, P.J., Mrsic-Flogel TD. (2011). Functional specificity of local synaptic connections in neocortical networks, Nature 5; 473 (7345):87-91.

Kumar, N., Bhandari, P., Shamsher, S., Bari, B.(2009) Antioxidant activity and ultra- performance LC-electrospray ionization-quadrupole time-of-flight mass spectrometry for phenolics-based fingerprinting of Rose species: Rosa damascena, Rosa bourboniana and Rosa brunonii. Food ChemToxicol: 47:361–367.

Kurkcuoglu, M., Abdel-Mageed, A, Baser, K.H.C.,(2013). The composition of TaifRose oil, J Essent Oil Res: 25:364-367.

Kurkcuoglu, M., Baser, K.H.C. (2003). Studies on Turkish Rose Concrete, Absolute and Hydrosol. Chemistry of Natural Compounds: 39: 375-379.

Lavid, N., J. Wang, M. Shalit, I. Guterman, E. Bar, T. Beuerle, N. Menda, S. Shafir, D. Samir, Z.Adam, A. Vainstein, D. Weiss, E. Pichersky and E. lewinsohn,(2002). O-Methyltransferases involved in the biosynthesis of volatile phenolic derivatives in rose petals. Plant Physiology, 129:1899-1907.

Libby, P. (2007). Inflammatory mechanisms: the molecular basis of inflammation and disease Nutr Rev 65:S140-S146

Libester, M. (2002). Delmar’s Integrative Herb Guide For Nurses. Delmar Thomson Learning, Albany: p360-370.

Lilybeth, F. Olowa and Olga M. Nuñeza, (2013). Brine Shrimp Lethality Assay of the Ethanolic Extracts of Three Selected Species of Medicinal Plants from Iligan City, Philippines. International Research Journal of Biological Sciences :2(11), 74-77.

185

Lin, J. Y., Selim, M.A., , C.R. (2003). UV photoprotection by combination topical antioxidants vitamin C and vitamin E. J Am Acad Dermatol : 48: 866–74.

Lis-Balchin, M., (2006). Aromatherapy science: a guide for healthcare professionals. London, England: Pharmaceutical Press.

Loden, M.(1995). Biophysical methods of providing objective documentation of the effects of moisturizing creams.Skin Res Technol.1:101-8.

Loden, M. (2005).The clinical benefits of moisturizers. JEADV; 19:672-88.

Loghmani-Khouzani, H., Sabzi-Fini, O., Safari.(2007) J. Essential oil composition of Rose damascena Mill cultivated in central Iran. Scientia Iranica : 14: 316-319.

LuXemag, (2009). Rose Water. Luxury Magazine. http: / /www. Luxemag.org/skin- body/rose-water.html.

M. Usman, M.Ashfaq, S. Taj, and M. Abid. (2014). An economic analysis of cut-rose flower in Punjab, Pakistan, J. Anim., Plant Sci. 24(2).

Mackenzie, A.J., Spina, D., Page, C.P. (2004). Models used in the development of antitussive drugs. Drug Discov. Today 1:297–302.

Madhukar, C.(2013). Phytochemical screening of cumin seeds extract. Report and Opinion,5(1):57-58.

Maheshwari, Raaz., Bina, Rani., (2013). Intrinsic benefits and therapeutic application of rose water, UJP 2013, 02 (02): page 4-7.

Mahmood, N., Piacente, S., Pizza, C., Burke, A., Khan, Al., Hay, A.J. (1996). The anti- HIV activity and mechanisms of action of pure com-pounds isolated from Rosa damascena. Biochemical and Biophysical Research Communication: 229(1):73- 79.

Mahmoodreza, Moien., Mohammad, M., Zarshenas and Shiva, Dilnawaz, (2014). Chemical composition analysis of rose water from Iran: 52:10.

186

Maleev, A., Neshtev, G., Stoianov, S., Sheikov, N. (1972). The ulcer protective and antiinflamatory effect of Bulgarian rose oil. Eksp Med Morfol: 11:55–60.

Mansur, J. S., Breder, M. N. R., Mansur, M. C. A., Azulay, R. D. (1986). Determinação do fator de proteção solar porespectrofotometria. An. Bras.Dermatol., Rio de Janeiro, v. 61, p. 121-124.

Manus ,E.,(2005). Rose water-An Age-Old flavoring for dinner and dessert. Plants and gardens News. http: / /www.bbg.org/ gar2/ topics / Kitchen/ 2005 su_rosewater. html.

Marina, G. D., Kekuda, P., T.R.I, Sudarshan, S.J.I. (2008). Antitussive activity of ethanolic extract of Curcuma aromatic rhizomes on sulfur dioxide induced cough in mice, ASL, XVII(3): 36-40.

Marty, J.P., (2002), NMF and cosmetology of cutaneous hydration.Ann Dermatol Venereol; 129:131-6.

Medzhitov, R. (2008). Origin and physiological roles of inflammation. Nature 454:428- 435. Mencarelli

Meena, P.D., Kaushik, P, Shukla, S, Soni., A.K, Kumar, M., Kumar, A.(2006). Anticancer and antimutagenic properties of Acacia nilotica (L.)on 7,12- dimethylbenz (a) anthracene-induced skin papillomagenesis in swiss albino mice.Asian Pac J Cancer Prev. 7(4):627-632.

Meyer, B. N., Ferrigni, N. R., Putman, J. E., Jacobson, L. B., Nichols, D. E. and McLaughlin

Mizushima, Y., Kobayashi, M.,(1968). Interaction of anti ‐inflammatory drugs with serum proteins, especially with some biologicallyactive proteins. Journal of Pharma Pharmacology, 20:169-173.

Moein, M., Karami, F., Tavallali, H., Ghasemi,Y.(2010). Composition of the essential oil of rosadamascena Mill from south of Iran. Iran J. Pharmaceut Sci: 6: 59-62.

187

Mohaddese, Mahboubi. (2016). Rosa damascena as holy ancient herb with novel application. Journal of Traditional and Complementary Medicine :6 : 10-16.

Nair, C.K., Parida, D.K., Nomura, T., (2001). Radioprotector in radiotherapy. J Radiant Res. 3; 42(1):21-37.

Narayanswami, V. andBiswas, K. (1957). Rose growing centers and Rose industry in India. CSIR, New Delhi, India.pp.1-113.

Nikbakht, A., Kafi, M., Mirmasoudi, M., Babalar. (2004-2005). M. Micropropagation of Damask rose (Rosa damasecna Mill.) cvsAzaran and Ghamsar. International J of Agriculture and Biology. 7(4):535-538.

Nikbakht, A., Kafi, M. (2008). Astudy on the relationships between Iranian people and Damask rose (Rosa damascena) and its therapeutic and healing properties. Acta Hort (ISHS) 790:251-4.

O.F. Amna, Noorain Hashim, Noorhaim Abdullaha (2013).Acute and oral subacute toxicity study of ethanolic sample of cosmos caudatus leaf in Sprague Dawley Rats.

OECD Guideline for Testing of Chemicals.(2001). Acute Oral Toxicity – Acute Toxic Class Method, 423.

OECD Guidelines for the testing of chemicals: 423; 2001. Acute oral toxicity- Acute Toxic Class Method.

Özkan, G. Sagdiç, O. Baydar, (2004). H. Antioxidant and antibacterial activities of Rosa Damascena flower extracts. Int. J. Food Sci. Techno,10:277–281.

Pan, M.H., Lai, C.S., Dushenkov, S. Ho. CT. (2009).Modulation of inflammatory genes by natural dietary bioactive compounds. J. Agric Food Chem. 57:4467-4477

Pan, M.H., Lai, C.S., Ho. CT. (2010). Anti-inflammatory activity of natural dietary flavonoids Food Func.1:15-31.

188

Patil, V., Ghag-Sawant, M., Narayakkadan, A., Asrondkar, A., Bobade, A. and Ramaiya, M. (2015).Biological Evaluation of Benzothiazole-Thiourea Derivatives, Plant Extracts and their Combinations.International Journal of Scientific and Research Publications, 5(12); 227-283.

Perez, C., Paul, M., Bazerque, P. (1990).An Antibiotic assay by the agar well diffusion method. Acta. Bio. Med. Exp. 15: 113-115

Perumal, K., Moorthy, T.A. Sambanda, Savitha, J.S. (2012). Characterization of essential oil from offered temple flower Rosa damascena Mill. Asian J. Exp. Biol. Sci.3:330-334.

Prasad, S.B., Yashwant and Aeri, V. (2013). In vitro anti-inflammatory activity of Raupya (silver) Bhasma. J. Chem. Pharmceut. Res.,5:194-197.

Punchard, N.A., Whelan, C.J, and Adcock, L. (2004). Journal of Inflammation, 1:1

Rakhshandah, H., Boskabady, M.H., Mossavi, Z., Gholami, M., Saberi, Z. (2010).The Differences in the relaxant effects of different fractions of Rosa damascena on guinea pig tracheal smooth muscle.Iran J Basic Med Sci.13:126–132.

Rakhshandah, H., Hosseini, M., Dolati, K. (2004). Hypnotic effect of Rosa damascena in Mice. Iran J. Pharmac. Res.3:181–185.

Rakhshandah, H., Hosseini, M. (2006). Potentiation of pentobarbital hypnosis by Rosa damascena in mice.Indian J. Exp. Biol. 44:910–912.

Rakshanda, H., Hosseini, M., (2006).Potentiation of Phenobarbital hypnosis by Rosa damascena in mice. Indian Journal of Experimental Biology, 44(11):910-912.

Rakshandeh, H., Vahdati-Mahhadian, N., Dolati, K.,Hosseini, M. (2008). Antinoceptive effect of Rosa damascena in mice. Journal of Biological Sciences, 8(1):176-180.

189

Ramezani, R., Moghimi, A., Rakhshandeh, H., Ejtehadi, H., Kheirabadi, M. (2008).The effect of Rosa damascena essential oil on the amygdala electrical kindling seizures in rat. Pak. J. Biol. Sci.11:746–751.

Ratnasooriya, W.D., Jayakody, J.R.A.C., Pathirana, R.(2015). In vitro anti-rheumatoid arthritic activity of Sri Lankan orthodox black tea (Camellia sinensis L.).Int J Pharm Practice Pharm Sci., 2(3);93-98.

Rawlings, A.V., Harding, C.R.(2004). Moisturization and skin barrier function. Dermatol Ther, 17:43-8.

Reynolds, S.M., Mackenzie, A.J., Spina, D. (2004). The pharmacology of cough.Trends PharmacolSci 25:569–76.

Rizvi, M. A. A., Saeed, H.N. Zubairy. (2007). Medicinal Plants-History, Cultivation and Uses. Hamdard Institute of Advanced studies & Research. Hamdard University, Pakistan, pp.1-216.

Robbins, R. J. (2003). Phenolic acids in foods: An overview of analytical methodology. Journal of Agricultural and Food Chemistry, 51:2866-2887.

Rohman, A., Riyanto, S., Yuniarti, N., Saputra, W. R., Utami, R and Mulatsih, W. .(2010). Antioxidant activity, total phenolic, and total flavanoid of extracts and fractions of red fruit (Pandanusconoideus Lam). Int. Food Res, J., 17:97-106.

Sahoo, S.S., Shukla, S., Nandy, S and Sahoo, H. B. (2012). Synthesis of novel coumarin derivatives and its biological evaluations.European Journal of Experimental Biology, 2(4):899-908.

Saklani, S., Kothiyal. C. S., Badoni. P.P. Dogra.S. (2012). Antimicrobial activity, nutritional profile and phytochemical screening of wild edible fruit of Rubusellipticus Int. J.Med.Arom.Plants.2(2).269-274.

Sama, K., Raja. A.X.V., (2011). Preliminary phytochemical screening of root bark of Delonix regia.IRJP:2(10):42-43.

190

Saraf, S. and C.D. Kaur. (2010). Phytoconstituents as photoprotective novel cosmetic formulations. Pharmacogn. Rev., 4: 1-11.

Saraf, S., S. Sahu, C.D. Kaur and S. Saraf (2010). Comparative measurement of hydration effects of herbal moisturizers. Pharmacogn. Res., 2: 146-151

Saxena, M., Saxena, J., Nema, R., Singh, D. and Gupta, A. (2013). Phytochemistry of medicinal plants. Journal of Pharmacognosy and Phytochemistry, 1(6):168-182.

Sayre, R. M., Again, P. P., Levee, G. J., Marlowe, E. (1979). Comparison of in vivo and in vitro testing of sunscreening formulas. Photochem. Photobiol., Oxford, v. 29, p. 559-566.

Scirrotto, J. (2009). Soothing Solution for Sensitive Skin. WebMD for Marie Claire Magazine. http://www.webMD.com/ Skin-beauty/ features/ soothing-solution- sensitive-skin.

Shafei, N.M., Rakhshandah, H., Boskabady, H.M. (2003). Antitussive effect of Rosa damascena in Guinea pig. Iranian Journal of Pharmaceutical Research: (2):231- 234.

Shahriari, S., Yasa, N., Mohammadirad, A., Khorasani, R., Abdollahi, M. (2006). In vivo antioxidant potentials of Rosa damascena petals extract from Guilan. Iran, comparable to a-tocopherol.Int J Pharmacol: 3: 187-190.

Shahriari, S., Yasa, N., Mohammadirad, A., Khorasani, R., Abdollahi, M. (2007). In vitro antioxidant potential of Rosa damascene extract from guilan, Iran comparable to - tocopherol. Int. J. Pharmacol: 3:187–190.

Shallangwa, G. A., Musa, H. and Nyaga, G. T. (2015). In vitro evaluation of ethanolic extracts of Zingiberofficinale, Sygziumaromaticum and their 1:1 extracts blend on protein denaturation during acute inflammation J. Prog. Res. Chem., 1(1); 1-8.

191

Shekar, M., Shetty, S., Lekha, G. & Mohan, K. (2012). Evaluation of in vitro antioxidant property and radio protective effect of the constituent medicinal plants of a herbal sunscreen formulations. International Journal of Pharmaceutical Frontier Research: 2(2): 90-96.

Shohayeb, M., EI-Sayed, S., Hameed, A., Bazaid, S.A., Maghrabi, I. (2014).Global J. of Pharmacol: 8(1):01-07.

Shrikumar, S., Maheshwari, U., Sughanti, A., Ravi, T.K. (2006). WHO guidelines for standardization of herbal drugs. Pharminfo. net: 2: 78-81.

Siddhuraju, P., Becker, K. (2003). Antioxidant properties of various solvent extracts of total phenolic constituents from three different agroclimatic origins of drumstick tree (Moringaoleifera Lam.) leaves. J. Agric. Food Chem: 51, 2144–2155.

Smaoui, S., Hlima, H.B., Jarraya, R., Kamoun, N.G., Ellouze, R. and Damak, M. (2012). Cosmetic emulsion from virgin olive oil: Formulation and bio-physical evaluation. African J. Biotech., 11(40); 9664-967.

Stankov, S. (2012). Definition of inflammation causes of inflammation and possible anti- inflammatory strategies. The Open Inflammation Journal, 5, 19.

Stratton, S.P., Dorr, R.T., Alberts, D.S. (2000). The state-of-the-art in chemoprevention of skinCancer. European Journal of Cancer 36: 1292-1297.

Sulaiman, M., Tijani, H.I., Abubakar, B.M., Haruna, S., Hindatu, Y., Mohammed, J.N., Idris,Abdulrahman. (2013). An overview of natural plant antioxidant: analysis and evaluation. Advanc. In Biochem, 1(4); 64-72.

Swadhini, S.P., Santhosh, R., Uma, C., Mythili, S. and Sathiavelu, A. (2011). Phytochemical screening and antimicrobial activity of five edicinal plants against Myrothecium sp. International Journal of Pharma and Bio Sciences, 2(1):272- 279.

192

Szymanska, A., Hornowski, T., Kozak, M. and Slosarek, G. (2008). The SAXS and rheological studies of HEWL amyloid formation. Acta Physica Polonica A, 114(2); 447-454.

Tatke, P., Satyapal, U.S., Mahanjan, D.C., Naharwar, V. (2015) .Phytochemical analysis, in-vitro antioxidant and antimicrobial activities of flower petals of Rosa damascena. IJPPR,7(2):246-250.

Tatke, P.A, Patil, P.S, Gabhe, S.Y. (2015). In vitro antioxidant and free radical scavenging activity of extracts of Rosa damascena flower petals. Am J. Phytomed Clin. Ther. 3:589–601.

Thoha, T.B., Christopher, K., Simeon, J.E,. and Sikirat, M.(2012). Antibacterial and Anti-Inflammatory Activities of Anacardiumoccidentale Leaves and Bark Extracts. Crown Journal of. Medicine, 2: 20-24.

Thorleifsson, A., Wulf, H.C. (2003). Emollients and the response of facial skin to a cold environment. Br. J. Dermatol. 148:1149-52.

Tiwari, P., Kuma, B., Kaur, M., K aur, G., and Kaur, H. (2011). Phytochemical screening and extraction .International Pharmaceitica Sciencia, 1(1):98-106.

Ukoha, P.O., Cemaluk, E. A. C., Nnamdi, O. L., and Madus, E. P. (2011). Tannins and other phytochemical of the Samanaeasaman pods and their antimicrobial activities. African Journal of Pure and Applied Chemistry, 5(8):237-244.

Ullah, H.M.A., Zaman, S., Juhara, F., Akter, L., Tareq, S.M., Masum, E. H. and Bhattacharjee, R. (2014). Evaluation of antinociceptive, in-vivo&in-vitro anti- inflammatory activity of ethanolic extract of Curcuma zedoariarhizome. BMC Complement and Alternat Med., 14(346);1-12.

Ulusoy, S., Bosglmez-Tinaz, G., Secilmis-Canbay, H.(2009). Tocopherol, Carotene, Phenolic contents and antibacterial properties of rose essential oil, hydrosol and absolute. CurrMicrobiol: 59:554-558.

193

Usman, Tariq, Atif, Riaz, M. Jafar, Jaskani and Zahir, A., Zahir. (2016).Screening of PGPR isolates for plant growth promotion of Rosa damascena. Int. J. Agric. Biol., Vol. 18, No. 5.

Vasanthakumar, D., Aruna, D., Mujeebunisa, M., Saravanan, S., and Divya, V. (2015). Antibacterial activity of Rosa damascena petal extracts against the fish pathogen Aeromonashydrophila. Euro. J. Exp. Bio., 5(8):56-59.

Venkatesh, S. J. Thilagavathi, D. Shyam Sunder (2008). Anti-diabetic activity of flower of a. Hibiscus rosa-sinensis.Fitoterapia. 79(2):79-81.

Vennila, V., and Anitha, R.(2015). In vitro evaluation of anti-arthritic activity in different solvent extracts from Cuscutareflexa. World J. Pharmacy and Pharmaceut Sci., 4(4); 1340-1350.

Verma,S.R.,Padalia, C.R., Chauhan, A.,(2011). Chemical investigation of the volatile components of shade-dried petals of damask rose (Rosa damascena Mill). Arch Biol Sci: 63:1111-1115.

Waldesch, F.G., Konigswinter, B.S., Remagen, H.B.(2003). Herbal medicinal products- Scientific and regulatory basis for development quality assurance and marketing authorization,published by medpharmstuttagart and CRC press, Washington DC, : 37-52. www.gulsha.com.tr/en/rose-damascena/history-of-rose-water.aspx

Yadav,M., Chatterji, S., Gupta, S.K. and Watal, G.(2014). Preliminary phytochemical screening of six medicinal plants ued in traditional medicine. International Journal of Pharmaceutical Sciences, 6(5):539-542.

Yamakoshi, Y., Umezawa, N., Ryu. A. (2003). Active Oxygen Species Generated from -• 1 Photoexcited Fullerene (C60) as Potential Medicines: O2 versus O2.J. Am. Chem. Soc: 125 (42), pp 12803–12809.

194

Yang, L., Chen, D., Duan, R., Xia, L., Wang, J., Qurashi, A., Jin, P., Chen, D. (2007). Argonaute 1 regulates the fate of germline stem cells in Drosophila. Development 134 (23): 4265--4272.

Yassa, N, Masoomi, F., Rankouhi, S.F., Hadjiakhoondi A.(2009). Chemical composition and antioxidant activity of the extract and essential oil of Rosa damascena from Iran population of Guilan; Dara., 2009;17:175-180.

Yoshikawa, M. S. Nakamura Y. Kato, Kmatsuhira, H. Matsuda. (2007). Medicinal flowers XIV. New acylatedoleanane – type triterpeneoligoglycosides with anti allergic activity from flower buds Chinese tea plants Camellia sinensis. Chem Pharm Bull 55(4):598-605.

Yosipovitch, G. (2004). Dry skin and impairment of barrier function associated with itch- new insights. Int. J. Cosmet. Sci.26:1-7

Young, E.C., Smith, J.A. (2011). Pharmacologic therapy for cough. Curr. Opin. Pharmacol. 11:224–30.

195

Abidi et al., IJPSR, 2018; Vol. 9(12): 1000-08. E-ISSN: 0975-8232; P-ISSN: 2320-5148

IJPSR (2018), Volume 9, Issue 12 (Research Article)

Received on 06 April, 2018; received in revised form, 08 June, 2018; accepted, 14 June, 2018; published 01 December, 2018 PHOTOPROTECTIVE AND ANTIOXIDANT ACTIVITIES ALONG WITH PHYTOCHEMICAL INVESTIGATION OF ROSE WATER Safia Abidi, Najma Shaheen, Iqbal Azher and Zafar Alam Mahmood * Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi, Karachi, Pakistan.

Keywords: ABSTRACT: Ultraviolet radiation (UV) from sun has significant Skin barrier, Spectroscopy, detoriating and degenerative effects on human skin and thus responsible Rose damask, Ultraviolet light for much skin disease. The most authentic and realistic approach to Correspondence to Author: overcome this issue is to use or apply a barrier between sun rays and Dr. Zafar Alam Mahmood exposed skin surface using suitable products called sunscreens or UV Member Board of Studies, protectors. Sunscreens usually include both natural and synthetic Department of Pharmacognosy, molecule and have great application in cosmaceuticals. In the present Faculty of Pharmacy and Pharmaceutical study one such natural product-rose water was evaluated for sun Sciences, University of Karachi, protecting ability of the rose water samples (SPF) using in-vitro Karachi, Pakistan. spectroscopic method. A correlation was also established by evaluating E-mail: [email protected] the antioxidant activity in total 12 samples of rose water were evaluated for SPF and antioxidant activity Among all the samples the SPF value of sample #09 showed high sunscreen activity 3.956 followed by sample #08 with SPF value of 3.612. The rose water sample distilled in the lab indicated SPF value as 0.800. All the samples showed antioxidant activity (free radical scavenging ability). The phytochemical investigation indicates the presence of polyphenolic compounds such as triterpenoids, saponin, flavanoids, tannins and fixed oil. The overall SPF and antioxidant activity of rose water may be responsible for the utilization in cosmaceuticals to achieve reasonable skin protecting effect and to prolong the skin aging. INTRODUCTION: Damask rose botanically Rose is a valuable and important main material for known as Rosa damascena mill., belongs to the fragrance and beautifying purpose. From the rose, family Rosacea, a very popular family containing rose water, rose oil, concrete and absolute are also many ornamental plants. Rose has more than 200 extracted 2. Many naturally occurring materials are species and cultivar are also above 18000 1. good source of antioxidant and used for beautifying Cultivation of rose is very old about 3000 BC it is purpose for their good sunscreen and antioxidant reported in China, Northern Africa and Western activities. The outer most part of the body is skin Asia. which is in direct contact with sun and ultraviolet

QUICK RESPONSE CODE radiation which causes serious damage to the skin DOI: not only potentiate the oxidation but also accelerate 10.13040/IJPSR.0975-8232.9(12).1000-08 some other diseases such as erythema, photoaging, edema, sunburn and cell formation etc 3. Sun is the Article can be accessed online on: main source of ultraviolet radiation and further www.ijpsr.com divided in to UVA (320-400) UVB (280-320) and DOI link: http://dx.doi.org/10.13040/IJPSR.0975-8232.9(12).1000-08 UVC (200-280).

International Journal of Pharmaceutical Sciences and Research 1000 Abidi et al., IJPSR, 2018; Vol. 9(12): 1000-08. E-ISSN: 0975-8232; P-ISSN: 2320-5148

Majority of light that reaches to the earth that is OHG Germany), phosphate buffer (sigma about 90% is UVA having high penetration power Germany) potassium ferricyanide (daejung reagent causes immediate tanning of the skin. About 4 - 5% chemical Korea), trichloroacetic acid (Scharlau of the ultraviolet radiation is made up of UVB Spain), acetic acid and ferric chloride. which is 1000 times more potent than UVA to cause skin burn. Whereas most dangerous one is Instruments: Double beam ultraviolet and visible UVC short term exposure, cause serious damage to spectrophotometer (Shimadzoo), centrifuge the skin 4. Human body has its own force of machine (HERMLE Labortechnik GmbH, defense which neutralized the reactive oxidative Germany), weighing balance (Satorius), pH meter species and helpful in the repairing of tissues as (Systronics) and incubator were utilized in this well as prevention of radical formation. When body study. are suffering from different diseases and chronic Phytochemical Analysis: infections these defense forces are become weaker Qualitative Phytochemical Analysis: Qualitative in that conditions, medicinal plants provide phytochemical analysis of secondary metabolites strength to the body increase the quality of life in was performed as described by the standard patients 5. Objectives of present study is to procedure to check the presence and absent of the determine the photoprotective and antioxidant metabolites in samples of rose water, such as test of activities along with the phytochemical saponins (foam test), triterpenoids (Libermann investigation of 12 rose water samples to correlate burchard test), tannins (lead acetate test, nitric acid through application in various forms as available in 6 7 test) , fixed oil (spot test) and flavonoids (lead local market. 8 acetate test, sulphuric acid test) . FT-IR Analysis: Determination of the functional group in the samples were analyzed through Fourier Transform - Infrared spectro photometer (FT-IR).For liquid samples about 0.1 ml of the solution was spared over sodium chloride plates to form a thin film and observed under FT-IR spectrophotometer the spectrum was recorded between 400 - 4000 cm -1 and matched with the library data for functional group conformation 9. FIG. 1: ROSA DAMASCENA MILL. Sunscreen Activity: In-vitro determination of SPF MATERIALS AND METHODS: (sun protecting factor) was performed as method Sample Collection and Preparation of Rose described by Mansur et al., (1986). The absorbance Water: Total 12 samples of rose water were used of the extracts was determined from 290 nm to 320 in present study, 6 samples were collected from nm at every 5 nm interval using distilled water as market, 4 samples were provided by Mohammad blank. The in-vitro SPF value was calculated by Hashim Tajir Surma laboratories and the last using the following formula sample were prepared in the lab using 320 hydrodistillation method as reported by Verma et SPF spectrophotometric = CF × ∑290 EE (λ) × I (λ) × Abs (λ) al., 2011. All samples were filtered through Whatman filter paper and stored at 6 ºC in a Where CF (correlation factor) is 10, EE (λ) is refrigerator for further analysis and study. Sample erythmogenic effect of radiation with wavelength, and the voucher specimen number (RD-01-12) is (λ), Abs is spectrophotometric absorbance value at available in Department of Pharmacognosy, wavelength (λ). The value of EE (λ) *I (λ) are Faculty of Pharmacy and Pharmaceutical Sciences, constant. The absorbance value is multiplied with University of Karachi herbarium. EE (λ)*I (λ) and then their summation is taken and multiplied with correction factor to obtain the SPF Chemicals: Chemicals were utilized in the study is values 10. of Analytical grade, methanol (Merck schuchardt

International Journal of Pharmaceutical Sciences and Research 1001 Abidi et al., IJPSR, 2018; Vol. 9(12): 1000-08. E-ISSN: 0975-8232; P-ISSN: 2320-5148

Antioxidant Activity: RESULTS AND DISCUSSIONS: Reducing Power Ability (RPA): The reducing Phytochemical Screening: The qualitative power of samples was quantified by the following phytochemical screening of rose water as presented method. Rose water (5 ml and 10 ml) was mixed in Table 1 revealed presence of polyphenolic with 1 ml of 80% methanol, phosphate buffer (5ml, compounds such as triterpenoids, saponins. pH 6.6) and potassium ferricyanide (5 ml, 1.0%) flavonoids, tannins and fixed oil in majority of were added, mixtures were incubated at 50 ºC for samples. 20 min. After incubation 5 ml of trichloroacetic acid (10%) was added and the mixture was These phytochemicals are secondary bioactive centrifuged at 3000 rpm for 10 min. The upper constituent of plant have both medicinal and layer (5 ml) of the solution is mixed with 5 ml nutritional value and are able to provide protection distilled water and ferric chloride (1.0 ml, 0.1%), to the plant and also helpful in maintaining human than absorbance of different colored solution was health, working as anti-cancer agent, anti-oxidant, neuro-pharmacological agents and detoxification measured through spectrophotometer at 700 nm. 12 The same procedure was followed for ascorbic acid agent . which was used as standard drug at concentration of (50, 100 and 200 µg/ml) 11.

TABLE 1: PHYTOCHEMICAL SCREENING OF ROSE WATER SAMPLES Sample● Detection of Detection of Detection of Detection of Detection of tannins triterpenoids fixed oils saponins flavanoids 01 + + + + - 02 + + + + - 03 + - - + + 04 + + ++ ++ 05 + + + ++ + 06 + ++ + ++ ++ 07 + + + ++ + 08 + ++ + ++ + 09 + ++ ++ +++ ++ 10 + + + + ++ 11 + ++ ++ ++ - 12 + ++ + ++ ++ ●: source & manufacturer’s name are available upon request (+) Present, (-) Absent / not present FT-IR Analysis: The FT-IR absorbption spectrum the alkane group. The absorbance peaks from1100 of rose water samples are given in Fig. 2 - 13 cm-1 to 1200 cm-1 were appeared in sample from C - respectively. For the analysis of functional groups O stretching vibration, indicated the presence of fourier transformer infrared spectroscopy is a safe alcohol, ether, carboxylic acid and anhydride. and time saving method. The FT-IR spectrums Results are shown in Table 2. revealed that the peaks arise in the range 3600cm-1 to 920 cm-1. The tested samples showed intense Sunscreen Activity (SPF): The healing property of peaks from 3400 cm-1 to 3600 cm-1 due to the different medicinal plants is depending upon the characteristic stretching vibration of N-H and O-H presence of their secondary metabolites such as alkaloids, glycosides, tannins, saponin and from alkaloids, polyphenols amino acids, while the 13 absorption peaks from 2800 cm-1 to 2900 cm-1 flavonoids . In the present decade compound were appeared from the C - H symmetric stretching obtained from the natural sources gained the considerable attention as a photoprotective agent. of CH3 and CH2 group of lipid region and ester group. While the peak at 2600 cm-1 revealed the There are wide Varity of chemicals which act as strong broad O-H stretching of carboxylic acids sunscreens and show their absorbance in certain structure, the absorbance peaks from 1700 cm-1 to part of the UV spectrum. Extracts of plant had a 1850 cm-1 from C = O stretch bending to indicated wide range of natural compounds which cover the the presence of conjugated aldehyde and carbonyl full range of UV spectrum wavelength. Due to group. Peaks from 1450 cm-1 to1470 cm-1 showed genotoxicity of ultraviolet radiation cause mutation

International Journal of Pharmaceutical Sciences and Research 1002 Abidi et al., IJPSR, 2018; Vol. 9(12): 1000-08. E-ISSN: 0975-8232; P-ISSN: 2320-5148 in the gene was the first step in the development of al., 2011. The SPF value ranges from 0.386 of skin cancer .Ultraviolet radiation penetrate in to the sample # 02 to 3.956 of sample #09. Sun protecting skin cause DNA damage and free radicals activities of samples was represented in Table 3 formation 14. In this research 12 extracts of Rosa and graphical representation was mentioned in Fig. damascena were evaluated for their SPF by in-vitro 14. spectroscopic method as described by Mansur et

FIG. 2: THE FT-IR SPECTRUM OF ROSE WATER FIG. 3: THE FT-IR SPECTRUM OF ROSE WATER SAMPLE NO. 1 SAMPLE NO. 2

FIG. 4: THE FT-IR SPECTRUM OF ROSE WATER FIG. 5: THE FT-IR SPECTRUM OF ROSE WATER SAMPLE NO. 3 SAMPLE NO. 4

FIG. 6: THE FT-IR SPECTRUM OF ROSE WATER FIG. 7: THE FT-IR SPECTRUM OF ROSE WATER SAMPLE NO. 5 SAMPLE NO. 6

FIG. 8: THE FT-IR SPECTRUM OF ROSE WATER FIG. 9: THE FT-IR SPECTRUM OF ROSE WATER SAMPLE NO. 7 SAMPLE NO. 8

International Journal of Pharmaceutical Sciences and Research 1003 Abidi et al., IJPSR, 2018; Vol. 9(12): 1000-08. E-ISSN: 0975-8232; P-ISSN: 2320-5148

FIG. 10: THE FT-IR SPECTRUM OF ROSE WATER FIG. 11: THE FT-IR SPECTRUM OF ROSE WATER SAMPLE NO. 9 SAMPLE NO. 10

FIG. 12: THE FT-IR SPECTRUM OF ROSE WATER FIG. 13: THE FT-IR SPECTRUM OF ROSE WATER SAMPLE NO. 11 SAMPLE NO. 12

TABLE 2: FT-IR ABSORPTION BAND ASSIGNMENTS Peak's wave length Possible functional Intensity/ Assignment (cm-1) groups 3400-3600 O-H and N−H O-H stretching vibration of hydroxyl groups (mainly lipids and proteins) and N-H stretching vibration mainly carbohydrates proteins 2800-2900 C−H C-H lipid region, esters groups 2600 O−H .Strong broad O-H stretching carboxylic acid 1750-1850 C=O C=O stretching conjugated aldehyde and strong stretching anhydride and carbonyl group 1450-1470 C=C Weak medium stretching of alkane group, aromatic ring 1100-1200 C-O C-O stretching of alcohol, ether, ester and carboxylic acid anhydride The FT-IR spectra of the samples conforms the presence of hydrocarbon, alcoholic, esters, aromatic principle and polyphenolic compounds. TABLE 3: IN-VITRO SUN PROTECTING FACTOR different types of diseases but also effect the topical (SPF) OF ROSE WATER SAMPLES part of body (skin), specially that area which is in Samples ● SPF 01 0.637 direct contact of sunlight. When the oxygen and 02 0.386 nitrogen species are imbalance in the body the body 03 1.373 is in the state of oxidative stress which is due to the 04 1.854 05 0.242 presence of free radical’s. 06 0.439 07 0.704 08 3.612 09 3.956 10 0.218 11 0.460 12 0.800 ●: source & manufacturer’s name are available upon request Antioxidant Activity: The phytochemical screening is helpful in the determination of the bioactive metabolite which play their role in the scavenger for free radicals which not only effect FIG. 14: GRAPHICAL REPRESENTATION OF SPF VALUES OF ALL SAMPLES the internal organs of the body by producing

International Journal of Pharmaceutical Sciences and Research 1004 Abidi et al., IJPSR, 2018; Vol. 9(12): 1000-08. E-ISSN: 0975-8232; P-ISSN: 2320-5148

TABLE 4: SPF RATING CHART day having radical scavenging mechanism for the SPF rating % of UV radiation blocked from the skin treatment of many diseases 15, 16. Antioxidants are 2 50 4 75 important in the prevention of UV radiation. The 5 80 major indicator of antioxidant activity is the 10 90 reducing capability of Fe3+/ ferricyanide complex 15 93 25 96 to ferrous form. The free radicals are inhibiting due With the Help of table 4 it was estimated that rose water to the presence of reductones which perform samples had ability to stop 25-75% of the ultraviolet radiation antioxidant activity by donate their electron to free penetration in to the skin. radicals 17. The antioxidant activity of Rosa water Antioxidants provide protection to the living being samples are shown in Table 5 - 6. The higher ferric from the hazardous effect of reactive oxygen reducing power value the greater the antioxidant species. There are many medicine which are now a activity.

TABLE 5: PERCENTAGE OF FERRIC REDUCING POWER CAPACITY OF ROSE WATER SAMPLES (05 ml) WITH RESPECT TO 50, 100, 200 µg/ml OF ASCORBIC ACID AS STANDARD Sample● mean of absorbance % age of reduction power capacity Equivalent to 50 µg of Equivalent to 100 µg of Equivalent to 200 µg of ascorbic acid ascorbic acid ascorbic acid 01 0.023 191.66 6.927 2.33 02 0.034 283.33 10.24 3.45 03 0.055 458.33 16.56 5.59 04 0.064 533.33 19.27 6.51 05 0.023 191.66 6.92 2.33 06 0.005 41.66 1.50 0.50 07 0.008 66.66 2.40 0.81 08 0.022 183.33 6.62 2.23 09 0.03 250 9.03 3.05 10 0.004 33.33 1.20 0.40 11 0.006 50 1.80 0.61 12 0.101 841.66 30.42 10.27 ●: source & manufacturer’s name are available upon request

TABLE 6: PERCENTAGE OF FERRIC REDUCING POWER CAPACITY OF ROSE WATER SAMPLES (10 ml) WITH RESPECT TO 50, 100, 200 µg/ml OF ASCORBIC ACID AS STANDARD Sample● mean of absorbance % age of reduction power capacity Equivalent to 50 µg of Equivalent to 100 µg of Equivalent to 200 µg of ascorbic acid ascorbic acid ascorbic acid 01 0.081 52.59 17.60 13.68 02 0.043 27.92 9.34 7.26 03 0.072 46.75 15.65 12.16 04 0.071 46.10 15.43 11.99 05 0.042 27.27 9.13 7.09 06 0.008 5.19 1.73 1.35 07 0.008 5.19 1.73 1.35 08 0.18 116.88 39.13 30.40 09 0.037 24.02 8.04 6.25 10 0.02 12.98 4.34 3.37 11 0.006 3.89 1.30 1.01 12 0.37 240.25 80.43 62.5 ●: Source & manufacturer’s name are available upon request CONCLUSION: Natural antioxidants are very sunburn, phototoxicity and photosensitivity 18. On effective for reducing the oxidative stress. It was the basis of present study on rose water shows that conformed that consumption of antioxidant due to the presence of polyphenolic compound substance provide prevention for the formation of saponins and flavavoids rose water provide free radicals. Free radicals effect the skin as it is in protection from the ultraviolet radiation and work direct contact with the solar light which is the as sun protecting agent by inhibiting the formation major cause of skin damage and responsible for of free radicals which is the main cause of skin different types of skin related problems such as related problems.

International Journal of Pharmaceutical Sciences and Research 1005 Abidi et al., IJPSR, 2018; Vol. 9(12): 1000-08. E-ISSN: 0975-8232; P-ISSN: 2320-5148

Now a days people avoid to use synthetic 7. Rajesh H, Rao SN, Prathima KS, Megha RN, Rejeesh EP and Lovelyn J: Phytochemical analysis of aqueous extract antioxidants because of their higher toxicity level of Ocimum sanctum Linn. International Journal of so the researchers was focused on the discovery of Universal Pharmacy and BioSciences 2013; 2(2): 462-468. natural compounds having antioxidant effects, 8. Prabhavathi RM, Prasad MP and Jayaramu M: Studies on natural products are safe, low in cost and qualitative and quantitative phytochemical analysis of 19 Cissus quadrangularis. Adv. Appl. Sci. Res 2016; 7(4): effective. In the present study in-vitro SPF and 11-17. antioxidant method was adopted, as in-vitro 9. Bruttia R, Magua MM, Agorkua ES and Govendera PP: Alternative method for qualitative analysis of specific non- methods are safe cost effective and not required volatile organic compounds present in South African water any ethical approval. In-vitro spectrophotometric is systems. S. Afr. J. Chem 2016; 69: 244-253. the approved method for the analysis of SPF and 10. Mesíasa LG, Romero Qwisgaarda AM, Untiverosa GPC, Kobayashia LCP, Shimabukurob LEM and Sugaharac antioxidant activity of the compound. AAK: Comparison of the photoprotective effects of sunscreens using spectrophotometric measurements or the ACKNOWLEDGEMENT: The authors are survivability of yeast cells exposed to UV radiation. Rev thankful to the entire staff of the Department of Soc Qum Perœ 2017; 83(3): 295-307. 11. El Jemli M, Kamal R, Marmouzi L, Zerrouki A, Cherrah Y Pharmacognosy, Faculty of Pharmacy and and Alaoui K: Radical-scavenging activity and ferric Pharmaceutical Sciences. University of Karachi. reducing ability of Juniperus thurifera (L.), J. oxycedrus For their entire support and technical assistance for (L.), J. phoenicea (L.) and Tetraclinis articulata (L.). Advances in Pharmacological Sciences 2016; 1-6. FT-IR analysis of functional group detection in test 12. Shaheen N, Imam S, Abidi S, Sultan RA, Azhar I and samples. Mahmood ZA: Comparative pharmacognostic evaluation and standardization of Capsicum annuum l. (red Chilli). CONFLICT OF INTEREST: There is no conflict IJPSR: 2018; 9(7): 1000-15. 13. Rezvani-Kamran A, Salehi I, Shahidi S, Zarei M, of interest of all authors in this study. Moradkhani S and Komaki A. Effects of the hydroalcoholic extract of Rosa damascena on learning and REFERENCES: memory in male rats consuming a high-fat diet. Pharmaceutical Biology 2017; 55(1): 2065-2073. 1. Halwani EM: Antimicrobial activity of Rosa damascena 14. Ebrahimzadeh MA, Enayatifard R, Khalili M, Ghaffarloo petals extracted and chemical composition by gas- M, Saeedi M and Charati JY: Correlation between sun chromatography-mass spectrometry (GC/MS) analysis. protection factor and antioxidant activity, Phenol and Afr. J. Microbiol. Res. 2014; 11: 2359-2367 Flavonoid contents of some medicinal plants. IJPR 2014; 2. Erbaş S and Baydar H: Variation in scent compounds of 13(3): 1041-1047. oil-bearing Rose (Rosa damascene Mill.) Produced by 15. Chaudhary M and Naithani V: Composition of antioxidant headspace solid Phase micro-extraction, hydrodistillation potential of Ezenus versus Marketed Herbal Products in and solvent extraction. Rec. Nat. Prod. 2016; 10: 5555- India: an In-vitro Study. American J. Phytomed. Clin. 565. Therapeut 2014; 2(1): 045-060. 3. Balakrishnan KP and Aswamy NN: Botanicals as 16. Sulaiman M, Tijani HI, Abubakar BM, Haruna S, Hindatu sunscreens: Their role in the prevention of photoaging and Y, Mohammed JN and Abdulrahman I: An overview of skin cancer. International Journal of Research in Cosmetic natural plant antioxidants: analysis and evaluation. Science 2011; 1: 1-12. Advanc. In Biochem 2013; 1(4): 64-72. 4. Simo A, Kawal N, Paliyath G and Bakovic M: Botanical 17. Ahmed D, Mehboob Khan M and Saeed R: Comparative antioxidants for skin health in the world of cosmeceuticals analysis of phenolics, flavonoids, and antioxidant and .International Journal of Advanced Nutritional and Health antibacterial potential of methanolic, hexanic and aqueous Science 2014; 2(1):67-88 extracts from Adiantum caudatum Leaves. Antioxidants 5. Saravanan M, Mondal DB, Samra K and Sasikala V: In- 2015; 4(4): 394-409. vitro qualitative and quantitative analysis of certain 18. Dunaway S, Odin R, Zhou L, Ji L, Zhang Y and Ana L: nutraceuticals as diuretic and antioxidant for hepatobiliary Kadekaro, Natural antioxidants: Multiple mechanisms to disorders (HBD). International Journal of Pharma Sciences protect skin from solar radiation. Forontiers in pharmaco- and Research (IJPSR) 2014; 5(12): 896-902. logy 2018; 9(392): 1-14. 6. María R, Shirley M, Xavier C, Jaime S, David V, Rosa 19. Imam S, Azhar I, Perveen S, Hussain SG and Mahmood S and Jodie D: Preliminary phytochemical screening, total ZA: Studies on in-vitro antioxidant activity and total phenolic content and antibacterial activity of thirteen flavonoid contents of a cream formulation to correlate its native species from Guayas province Ecuador. Journal of anti-aging effect. World J. of Pharm Research 2015; 4(1): King Saud University–Science xxx(2017)xxx–xxx 1647-1655. How to cite this article: Abidi S, Shaheen N, Azher I and Mahmood ZA: Photoprotective and antioxidant activities along with phytochemical investigation of rose water. Int J Pharm Sci Res 2018; 9(12): 1000-08. doi: 10.13040/IJPSR.0975-8232.9(12).1000-08.

All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. This article can be downloaded to ANDROID OS based mobile. Scan QR Code using Code/Bar Scanner from your mobile. (Scanners are available on Google Playstore)

International Journal of Pharmaceutical Sciences and Research 1006

Abidi et al., IJPSR, 2018; Vol. 9(12): 1000-08. E-ISSN: 0975-8232; P-ISSN: 2320-5148

Reviewer’s recommendations: 1. Specify designation and current full address of corresponding author. 2. Check for spelling, grammar and punctuation error(s). 3. References are out of format, see Instructions to Authors.

International Journal of Pharmaceutical Sciences and Research 1007