PHYTOCHEMICAL INVESTIGATION OF RHAMNUS TRIQUETRA AND ZIZIPHUS OXYPHYLLA
BY
FARHANA MAZHAR
ROLL NO. 76-GCU-Ph.D-CHEM-2009
SESSION: 2009-2013 DEPARTMENT OF CHEMISTRY GC UNIVERSITY, LAHORE
PHYTOCHEMICAL INVESTIGATION OF RHAMNUS TRIQUETRA AND ZIZIPHUS OXYPHYLLA
Submitted to the GC University Lahore in
partial fulfillment of the requirements
for the award of the degree of
DOCTOR OF PHILOSOPHY IN CHEMISTRY
BY
FARHANA MAZHAR
ROLL NO. 76-GCU-Ph.D-CHEM-2009
SESSION: 2009-2013
DEPARTMENT OF CHEMISTRY GC UNIVERSITY, LAHORE
In the name of Allah, The Most Compassionate,
The Most Merciful
RESEARCH COMPLETION CERTIFICATE
Certified that the research work contained in the thesis entitled “Phytochemical Investigation of Rhamnus triquetra and Ziziphus oxyphylla” has been carried out and completed by Ms. Farhana Mazhar, Roll No. 76-GCU-PhD-CHEM-2009, under my supervision during her PhD (Chemistry) studies in the laboratories of the Department of Chemistry. The quantum and the quality of the work contained in this thesis is adequate for the award of degree of Doctor of Philosophy.
Dated: ------
______Dr. Muhammad Jahangir Supervisor
Submitted through
______Prof. Dr. Ahmad Adnan Controller of Examination Chairman. GC University, Lahore Department of Chemistry, GC University, Lahore.
DECLARATION
I Ms.Farhana Mazhar, Roll No. 76-GCU-PhD-CHEM-2009, student of Ph.D in the subject of Chemistry, session 2009-2013, hereby declare that the matter printed in the thesis titled “Phytochemical Investigation of Rhamnus triquetra and Ziziphus oxyphylla” is my own work and has not been printed, published and submitted as research work thesis or publication in any form in any University, Research Institute etc. in Pakistan or abroad.
Dated: ------______
Signature of Deponent
DEDICATION
Affectionately Dedicated to my Beloved Parents,
For their endless love, support and encouragement
Especially my Brother Syed Kashif Mazhar for his help and patience, for every period I was away
and Family
due to whom Prayers and Cooperation
I became able to reach this status.
ACKNOWLEDGEMENT
In the name of Allah Almighty, who is the most beneficent the merciful. All praise to Almighty Allah who blessed me with knowledge, courage and strength to work and to his Last and Beloved Prophet Hazrat Muhammad (May Peace be upon Him) for whom this wide and huge universe was assembled and who is the pioneer of education and research. Being his spiritual follower and believer, I tried to get educated and explore the artifacts and secrets of the universe.
My deepest gratitude and thanks are extended to my supervisor Dr. Muhammad
Jahangir, Assistant Professor, Department of Chemistry, GC University, Lahore,for his keen interest, proficient suggestions, sympathetic guidance and persistent encouragement, whom intelligence and experiences enabled me to learn a lot. I really acknowledge his tolerance for students diversified attitudes, understanding and experience in different research fields. His support, prayers and guidance helped me to complete this task.
I feel great honour and pleasure in expressing thanks to Prof. Dr. Ahmad Adnan,
Chairman, Department of Chemistry for his guidance and kind behaviour during the course of my studies. He always remains in touch with the new research activities in the world. I would also express my profound thanks to Prof. Dr. Islam Ullah Khan, Ex. Chairman and Dean of Arts and Sciences, for his proficient suggestions, sympathetic guidance and persistent encouragement.
I wish to express my deep, heartfelt and sincere regards to Dr. Athar Abbasi, Associate professor, Department of Chemistry, GC University, Lahore, for his learned guidance, skilled advice, sympathetic and inspiring attitude.
I am very thankful and obliged to Dr. Khalid M. Khan (HEJ Research Institute of
Chemistry, University of Karachi) and Dr. Nisar Ullah (KSA) for providing all out help in spectroscopic analysis.
I would particularly like to express my profound thanks to Dr. Muhammad Shahid,
University of Agriculture, Faisalabad, Mrs. Saiqa Ishtiaq, University College of
Pharmacy, PUNJAB University, Lahore for providing help for pharmacological activities and Mr. Muhammad Ajaib, Department of Botany GC University, Lahore, for providing me the plant material.
I am highly acknowledged Higher Education Commission of Pakistan for financial support for the completion of this worthy task.
The continued encouragement and prays of my loving parents,Abu and Ammi are an ever remaining treasure for me, who always appreciated and guided me to take next step in life and provided me facilities and best working environment. Whatever I am that is because of the countless prayers of my parents, who instilled in me a charm for education and value of being organized. I am greatly indebted to my sister Noreen Mazhar, Ambreen Mazhar and my elder brother Syed Atif Mazhar and my bhabi Sammina Kashif for their divine love, prayers, constant care, encouragement and continuous support throughout my studies.
My sincere appreciations are also extended to my friends and colleagues Dr. Tayyaba
Shahzadi, Raisa Khanum, Saima Idrees, Farah Khalid, Afza Malik, Ume Farwa,
Ammar Yasir and Hafiz M. Faizan Haider and all others for providing me valuable assistance, moral supports and smiling moments during my stay in GCU.
I acknowledge all technical and non technical staff of Chemistry Department for their co-operation and nice behaviour.
Farhana Mazhar
CONTENTS
ACKNOWLEDGEMENT
SUMMARY
CONTENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF SCHEMES
Chapter- 1 INTRODUCTION
1.1 Natural Products 01
1.1.1 Importance of Natural Product as Medicines 01
1.2 Phytochemicals 02
1.2.1 Bioactive Phytochemicals present in Medicinal Plants 02
1.2.2 Classification of Phytochemicals 03-06
1.3 Family Rhamnaceae 06
1.3.1 Genus Rhamnus 07
1.3.2 Rhamnus triquetra 07
1.3.3 Genus Ziziphus 09
1.3.4 Ziziphus oxyphylla 10
1.4 Antioxidant Activity 11- 13
1.5 Antimicrobial Activity 13- 14
1.6 Enzyme Inhibition/ Inhibitory Activities 14- 15
1.7 Hemolytic Activity 16
1.8 Anti- inflammatory and Analgesic Activity 16
1.9 Hepatoprotective Activity 17- 19 Chapter- 2 LITERATURE REVIEW
2.1 Literature review of Rhamnus triquetra 20-19
2.2 Literature review of Ziziphus oxyphylla 28- 40
Chapter- 3 EXPERIMENTAL WORK
3.1 General Experimental Conditions 41
3.1.2 Chromatographic Techniques 41
3.1.3 Detection of Compounds 41
3.2 Rhamnus triquetra
3.2.1 Plant Material 42
3.2.2 Extraction and Fractionation of plant material 42
3.2.3 Isolation and Purification of Compounds of Rhamnus triquetra 44
3.2.4 Characterization of Chemical Constituents of Rhamnus triquetra 46
3.2.4.1 Characterization of Physcion 46
3.2.4.2 Characterization of Madagascin 46
3.3 Ziziphus oxyphylla
3.3.1 Collection and Identification of plant material 47
3.3.2 Extraction and Fractionation
3.3.3 Isolation and Purification of Compounds of Ziziphus oxyphylla 49
3.3.4 Characterization of Chemical Constituents of Ziziphus oxyphylla 51
3.3.4.1 Characterization of ß- Sitosterol 51
3.3.4.2 Characterization of Betulinic Acid 51
3.3.5 Extraction of Essential Oil through Water Distillation 52
3.3.5.1 GC-MS Analysis 52
3.4 Fluorescence Analysis 52
3.5 Physicochemical Studies 53
3.5.1 Loss on drying 53
3.5.2 Total Ash 53
3.5.3 Water soluble Ash 53
3.5.4 Acid Insoluble Ash 54
3.5.5 Determination of n- hexane soluble Extractive value 54
3.5.6 Determination of chloroform soluble Extractive value 54
3.5.7 Determination of ethanol soluble Extractive value 54
3.6 Phytochemical Screening 55
3.6.1 Test for Alkaloids 55
3.6.2 Test for Flavonoids 55
3.6.3 Test for Tannins 55
3.6.4 Test for reducing sugar 56
3.6.5 Test for Terpenoids 56
3.6.6 Test for Saponins 56
3.6.7 Test for Phenolics 56
3.6.8 Test for Cardiac Glycosides 56
3.7 Determination of Antioxidant Activities 57
3.7.1 Chemical and Standards of Antioxidant Activities 57
3.7.2 DPPH Radical Scavenging Activity 57
3.7.3 Total Antioxidant Activity by Phosphomolybdenum method 58
3.7.4 Ferric Reducing Antioxidant Power Assay 58
3.7.5 Total Phenolic Contents 59
3.7.6 Ferric Thiocyanate Assay 60
3.8 Enzyme Inhibition Study 61
3.8.1 In- vitro Acetylcholine esterase Inhibition Assay 61
3.8.2 Protease Inhibition Assay 62
3.9 Antibacterial Activity 63
3.9.1 Strains Used for Antibacterial Activity 63
3.9.2 Agar Well Diffusion method 63
3.10 Hemolytic Activity 63
3.11 Pharmacological Activities 64
3.11.1 Animals 64
3.11.2 Anti-inflammatory Activity 64
3.11.2.1 Carregeenan Induced Rat Paw Edema 65
3.11.2.2 Formalin Mice Paw Edema 65
3.11.3 Analgesic Activity 66
3.11.3.1 Hot Plate method 66
3.11.3.2 Tail Flick method 66
3.11.3.3 Acetic acid Induced writhing method 67
3.11.4 Hepatoprotective Activity 67
3.11.4.1 Chemical and Standards 68
3.11.4.2 Experimental Protocol 68
3.11.4.3 Interval of Dose 68
3.11.4.4 Blood Sampling 68
a) Estimation of Aspartate aminotransferase activity 69
b) Estimation of Alanine aminotransferase activity 69
c) Estimation of Alkaline phosphatase activity 71
d) Estimation of Bilirubin activity 72
3.11.4.5 Histopathology of Liver 73
3.11.5 Statistical Analysis 73 Chapter- 4 RESULTS AND DISCUSSION
4.1 Structure Elucidation of Chemical Constituents of Rhamnus triquetra
4.1.1 New Source Compounds of Rhamnus triquetra 74
4.1.1.1 Structure Elucidation of Physcion 74
4.1.1.2 Structure Elucidation of Madagascin 75
4.1.2 GC- MS Analysis of Chloroform fraction of Rhamnus triquetra 77
4.2 Structure Elucidation of Chemical Constituents of Ziziphus oxyphylla
4.2.1 New Source Compounds from Ziziphus oxyphylla 78
4.2.1.1 Structure Elucidation of ß- Sitosterol 78
4.2.1.2 Structure Elucidation of Betulinic Acid 80
4.2.2 GC- MS Analysis of Chloroform fraction of Ziziphus oxyphylla 82
4.2.3 GC- MS Analysis of Essential Oil of Ziziphus oxyphylla 83
4.3 Fluorescence Analysis 84
4.4 Determination of Physicochemical Parameters 85
4.5 Phytochemical Screening 86
4.6 Antioxidant Activities 88- 98
4.7 Enzyme Inhibition 99
4.7.1 Acetylthiocholine esterase 99-102
4.7.2 Protease Inhibition 102-104
4.8 Antibacterial Activity 104
4.9 Hemolytic Activity 105-107
4.10 Anti- inflammatory Activity 108
4.10.1 Carrageenan Induced Rat Paw Edema Method 108- 110
4.10.2 Formalin Mice Paw Edema 111- 114
4.11 Analgesic Activity 114- 123
4.12 Hepatoprotective Activity 124
4.12.1 Liver Markers/ Liver Function Test 124- 129
4.12.2 Histopathological Study 130- 137
REFERENCES 138- 153
LIST OF TABLES
No. Description Page #
Table 1.1 Isolated Drugs from Natural products and their Uses 2
Table 1.2 Biological functions/ Importance of Bioactive Phytochemicals 3
Table 1.3 Description of Some Antioxidant Plants 13
Table 1.4 Plants having Anti-inflammatory and Analgesic Activity 17
Table 2.1 Previously Isolated Phytochemical Compounds from Genus Rhamnus 20
Table 2.2 Previously Isolated Compounds from Genus Ziziphus 28
Table 3.1 KIT ASSAY Procedure for Assessing ASAT Level 69
Table 3.2 KIT ASSAY Procedure for Assessing ALAT Level 70
Table 3.3 KIT ASSAY Procedure for Assessing ALP Level 71
Table 3.4 KIT ASSAY Procedure for Assessing Total Bilirubin Level 72
Table 4.1 13C and 1H- NMR (300 MHz) Spectral Data of Physcion 75
Table 4.2 13C and 1H- NMR (300 MHz) Spectral Data of Madagascin 76
Table 4.3 GC- MS Analysis of Fr. 1 of CHCl3 fraction of Rhamnus triquetra 77
Table 4.4 GC- MS Analysis of Fr. 4 of CHCl3 fraction of Rhamnus triquetra 78
Table 4.5 13C and 1H- NMR (300MHz) Spectral Data of ß- Sitosterol 79
Table 4.6 13C and 1H- NMR (300MHz) Spectral Data of Betulinic Acid 81
Table 4.7 GC- MS Analysis of Fr. 1 of CHCl3 fraction of Ziziphus oxyphylla 83
Table 4.8 GC- MS analysis of Essential Oil of Ziziphus oxyphylla 83
Table 4.9 Fluorescence Analysis of Rhamnus triquetra 84
Table 4.10 Fluorescence Analysis of Ziziphus oxyphylla 84
Table 4.11 Proximate/ Physicochemical Parameters of Rhamnus triquetra and Ziziphus oxyphylla 85
Table 4.12 Phytochemical Constituents of various extracts of Rhamnus triquetra 86
Table 4.13 Phytochemical Constituents of various extracts of Ziziphus oxyphylla 87
Table 4.14 Percentage Inhibition of DPPH and IC50 values of Rhamnus triquetra 88
Table 4.15 Percentage Inhibition of DPPH and IC50 values of Ziziphus oxyphylla 88
Table 4.16 Total Phenolics, FRAP values, Lipid Peroxidation and
Total Antioxidant Activity of Rhamnus triquetra 89
Table 4.17 FRAP values, Total Antioxidant Activity and Total Phenolic Contents
of Ziziphus oxyphylla 90
Table 4.18 Acetylthiocholine Esterase of Rhamnus triquetra 99
Table 4.19 Acetylthiocholine Esterase of Ziziphus oxyphylla 100
Table 4.20 Trypsin Inhibition of various fractions of Rhamnus triquetra and Ziziphus oxyphylla 102
Table 4.21 Antibacterial Activity of Rhamnus triquetra 104
Table 4.22 Antibacterial Activity of Ziziphus oxyphylla 105
Table 4.23 Hemolytic Activity of Rhamnus triquetra and Ziziphus oxyphylla 106
Table 4.24 Anti-inflammatory Activity of Rhamnus triquetra and Ziziphus oxyphylla by Carregeenan Induced Rat Paw Edema 108
Table 4.25 Anti-inflammatory Activity of Rhamnus triquetra and Ziziphus oxyphylla by Formalin Induced Paw Edema 112
Table 4.26 Analgesic activity of Rhamnus triquetra and Ziziphus oxyphylla
on Hot plate Method 115
Table 4.27 Analgesic activity of Rhamnus triquetra and Ziziphus oxyphylla on Water Bath Method 115
Table 4.28 Analgesic activity of Rhamnus triquetra and Ziziphus oxyphylla by Acetic Acid Writhing Method 116
Table 4.29 Liver Function Test for ASAT Level 124
Table 4.30 Liver Function Test for ALAT Level 124
Table 4.31 Liver Function Test for ALP Level 125
Table 4.32 Liver Function Test for Total Bilirubin 125
Table 4.33 Comparison of Liver Function Tests among Different Animal groups After Treatment 126
Table 4.34 Histopathological Examination of Toxic, Standard and
Methanolic Extracts of Both Plants 137
LIST OF FIGURES
No. Description Page #
Figure 1.1 Diseases due to Free Radical Oxidative Stress 12
Figure 1.2 Side Effects of Synthetic drugs used in treatment of
Alzheimer’s disease (AD) 15
Figure.1.3 Some Natural Antioxidants 19
Figure 4.1 Free Radical Scavenging Activity of Rhamnus triquetra 91
Figure 4.2 IC50 Values of Various Fractions of Rhamnus triquetra 92
Figure 4.3 Free Radical Scavenging Activity of Ziziphus oxyphylla 93
Figure 4.4 IC50 Values of Various Fractions of Ziziphus oxyphylla 93
Figure 4.5 Total Antioxidant Activity of Rhamnus triquetra 94
Figure 4.6 Total Antioxidant Activity of Ziziphus oxyphylla 95
Figure 4.7 FRAP Values of Different Fractions of Rhamnus triquetra 96
Figure 4.8 FRAP Values of Different Fractions of Ziziphus oxyphylla 96
Figure 4.9 Total Phenolic Contents of Different Fractions of Rhamnus triquetra 97
Figure 4.10 Total Phenolic Contents of Different Fractions of Ziziphus oxyphylla 98
Figure 4.11 Inhibition of Lipid Peroxidation of Rhamnus triquetra 99
Figure 4.12 Acetylthiocholine Esterase of Rhamnus triquetra 101
Figure 4.13 Acetylthiocholine Esterase of Ziziphus oxyphylla 102
Figure 4.14 Trypsin Inhibition of Various Fractions of Rhamnus triquetra 103
Figure 4.15 Trypsin Inhibition of Various Fractions of Ziziphus oxyphylla 104
Figure 4.16 Hemolytic Activity of Various Fractions of Rhamnus triquetra 107
Figure 4.17 Hemolytic Activity of Various Fractions of Ziziphus oxyphylla 107
Figure 4.18 Anti-inflammatory Activity of Rhamnus triquetra by Carregeenan Induce Rat Paw Edema 110
Figure 4.19 Anti-inflammatory Activity of Ziziphus oxyphylla by Carregeenan Induced Rat Paw Edema 111
Figure 4.20 Anti-inflammatory Activity of R. triquetra by Formalin Induced Paw Edema 113
Figure 4.21 Anti-inflammatory Activity of Z. oxyphylla by Formalin Induced Paw Edema 114
Figure 4.22 Analgesic activity of Rhamnus triquetra on Hot plate Method 117
Figure 4.23 Analgesic activity of Ziziphus oxyphylla on Hot plate Method 118
Figure 4.24 Analgesic activity of Rhamnus triquetra by Tail Flick Method 120
Figure 4.25 Analgesic activity of Ziziphus oxyphylla by Tail Flick Method 121
Figure 4.26 Analgesic activity of Rhamnus triquetra by Acetic Acid Writhing Method 122
Figure 4.27 Analgesic activity of Ziziphus oxyphylla by Acetic Acid Writhing Method 123
Figure 4.28 Liver Function Test for ASAT Level 128
Figure 4.29 Liver Function Test for ALAT Level 129
Figure 4.30 Liver Function Test for ALP Level 129
Figure 4.31 Liver Function Test for Total Bilirubin 130
Figure 4.32 Histopathological Examination of Normal Group 130
Figure 4.33 Histopathological Examination of Normal Group 131
Figure 4.34 Histopathological Examination of Toxic Group 131
Figure 4.35 Histopathological Examination of Toxic Group 132
Figure 4.36 Histopathological Examination of Toxic Group 132
Figure 4.37 Histopathological Examination of Standard Group 133
Figure 4.38 Histopathological Examination of Standard Group 133
Figure 4.39 Histopathological Examination of Experimental Group (IV) 134
Figure 4.40 Histopathological Examination of Experimental Group (IV ) 134
Figure 4.41 Histopathological Examination of Experimental Group (V ) 135
Figure 4.42 Histopathological Examination of Experimental Group (V) 135
LIST OF SCHEME
No. Description Page #
Scheme 3.1 Extraction Scheme of Rhamnus triquetra 43
Scheme 3.2 Isolation of Chloroform Fraction of Rhamnus triquetra 45
Scheme 3.3 Extraction Scheme of Ziziphus oxyphylla 48
Scheme 3.4 Isolation of Chloroform Fraction of Ziziphus oxyphylla 50
SUMMARY
The work embodied in this dissertation is mainly concerned with the isolation and characterization of chemical constituents from two medicinal plants such as Rhamnus triquetra and Ziziphus oxyphylla and their possible activities like antioxidant, enzyme inhibition, antibacterial, hemolytic, anti-inflammatory, analgesic and hepatoprotective activities of crude extracts of both plants. The isolated compounds from the present work were characterized by using various modern spectroscopic techniques.
Phytochemical studies of R. triquetra had resulted in the isolation of two new source compounds, which had never been isolated so far from this investigated source. The compounds isolated for the first time from R. triquetra were Physcion (Compound no. 3)
and Madagascin (Compound no. 4). The structures of these compounds were determined by IR, 1H and 13C NMR spectroscopy and by comparison with the published data of the closely related compounds.
The phytochemical investigation of Z. oxyphylla led to the isolation of ß- Sitosterol (Compound no. 5) and Betulinic acid (Compound no. 6) for the first time from this plant source. The structures of these compounds were determined by IR, 1H and 13C NMR spectroscopy and by comparison with the published data of the closely related compounds.
Essential oil of Z. oxyphylla was extracted through steam distillation and volatile components were evaluated by GC- MS analysis. About 15 components were present on chromatogram and these were identified by NIST dictionary.
Both plants were subjected to proximate analysis and results obtained were: loss on drying (11.54 and 12.09%), ash values (7.98 and 7.94%) and polar extractive values i.e chloroform (4.96 and 3.68%) were more than non- polar extractive values i.e., n- Hexane (0.79 and 0.62%).
The methanolic extract of both plants were dissolved in distilled water separately and partitioned with n-hexane, chloroform, ethyl acetate and n-butanol successively. These organic fractions and the remaining aqueous fraction were screened for their possible antioxidant activities by different methods: 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH) scavenging activity, total antioxidant activity, ferric reducing antioxidant power (FRAP) assay, total phenolic contents (TPC) and ferric thiocyanate assay. The results revealed that among these fractions ethyl acetate fraction of Rhamnus triquetra showed good % inhibition of DPPH radical (92.01± 0.21) at 60µg/mL concentration, having IC50 value of 7.59± 0.65 with total antioxidant activity (1.840± 0.08), FRAP value (2137.2± 0.58), TPC (121.5± 1.10) and inhibition of lipid peroxidation (61.94± 1.17) while in Ziziphus oxyphylla, chloroform fraction showed maximum % inhibition of DPPH radical
(95.01± 0.37), having IC50 value (13.20±0.27) at 120 µg/mL concentration and also showed highest antioxidant activity (1.723±0.34), FRAP value (339.5±0.57) and TPC (55.06±1.45). The results obtained in the present study indicated that R. triquetra and Z. oxyphylla as the potential sources of natural antioxidants.
In enzyme inhibition, two assays were performed i.e., acetylthiocholine esterase and protease inhibition. Rhamnus triquetra showed maximum % acetylthiocholine esterase (AChE) inhibition in ethyl acetate fraction i.e., 95.51±1.30 and protease inhibition in n- butanol fraction (93.03± 0.65). n- Butanol fraction of Ziziphus oxyphylla showed good % AChE inhibition (86.0±0.02) and protease inhibition (91.05±0.94).
The antibacterial activity of both plants were checked against two Gram- positive (B. subtilis and S. aureus) and four Gram- negative bacteria (E. coli, P. aeruginosa, S. flexenari and S. typhi) by using agar well diffusion method. The results of antibacterial activity of Rhamnus triquetra showed that only ethyl acetate fraction showed significant inhibition against E. coli and B. subtilis i.e., 18 and 14mm and zone of inhibition of ethyl acetate fraction of Ziziphus oxyphylla was observed 18mm each against B. subtilis and S. aureus. Hemolytic or cytotoxic activity of both plant extracts revealed that ethyl acetate fraction of Rhamnus triquetra and n-butanol fraction of Ziziphus oxyphylla have highest toxicity value while n-hexane fraction of both plants have lowest toxicity value.
Anti- inflammatory and analgesic activities of methanolic extracts of both plants were evaluated against Aspirin (standard drug). Carregeenan induced rat paw edema and formalin mice paw edema methods were used for anti- inflammatory activity methods were used. 300mg/Kg dose of aspirin and methanolic extracts of both plants were administered orally. The results revealed that in carrageenan induced rat paw edema, maximum inhibition observed at the end of 3h. 39.12% inhibition showed in aspirin while methanolic extracts of R. triquetra and Z. oxyphylla produced 34.9% and 42.3% inhibition respectively while in formalin mice paw edema, R. triquetra and Z. oxyphylla showed maximum % response i.e., 53.2% and 56.86% response when compared with standard drug (aspirin) 28.10%.
Hot plate, water bath and acetic acid induced methods were used in analgesic activity. In hot plate and water bath methods, the temperature and duration were 51±1ͦ C and 0,1,2,3 and 4 hours respectively. 300mg/Kg methanolic extracts and standard drug (Aspirin) was used in mice and administered via gastric incubation. Statistically, significant activity was recorded during 60- 180 min in both methods but in acetic acid induced writhing
method, methanolic extracts of both plants showed significant reduction in the number of writhings in % response i.e., 34.8% and 41.8% when compared with aspirin (55.1%).
Hepatoprotective activity of methanolic extracts of both plants was evaluated against rifampicin and isonaized induced hepatic damage in rats. Silymarin was used as reference drug. Silymarin at dose of 200mg/Kg and 300mg/Kg of methanolic extracts of both plants were administered orally. The substantially elevated enzyme levels were restored towards normalization significantly by the extracts. The biochemical observations were supplemented with histopathological examination of rat liver sections. The results of this study strongly indicated that R. triquetra and Z. oxyphylla had more potent hepatoprotective action than silymarin, a reference drug, against RIF + INH induced hepatic damage in rats.
INTRODUCTION
Human beings have been using the medicinal plants and herbs as medicine/ drug for a number of diseases throughout the world since time immemorial [1] and also a good source of income [2]. More than 0.259 million species of plants have been distributed all over the world [3] and about 53,000 species are used as medicine [4]. 80% of world population is using the medicinal plants for basic health care because these plants have fewer side effects as compared to other pharmaceuticals. These medicinal plants contain various bioactive molecules/ natural products such as terpenes, flavonoids, phenols, alkaloids, coumarins, tannins etc. that can improve the resistance of body to cellular stress and prevent the cytotoxicity of different agents.
1.1 Natural product: Natural products are the chemical compounds/ constituents which are derived from the natural sources i.e. animals, insects, plant, microorganisms and marine sources [5]. Natural products are divided into two categories Primary Metabolites Secondary Metabolites Former are responsible for the formation of basic building block of plants for their existence [6] while the later have been concerned due to their biological properties and defensive mechanism of plants [7]. Many natural products act used as medicines. Most of the natural products are as protective agents, antioxidants, pheromones and antifeedants attractants etc.
1.1.1 Importance of Natural Products as Medicines: A large numberof natural products/ secondary metabolites such as alkaloids, flavonoids and terpenes [8] derived from plants are used as medicine for different ailments such as anti- tumor, anti-cancer, cardiovascular diseases, antimalarial, antihypertensive, antiplatelets and antiviral [9, 10]. Some of them are given below in Table 1.1.
Table 1.1 Isolated Drugs from Natural products and their Uses [11]:
Sr. Name of Drug/ Plant Family Indication/ Use No Medicine 1 Filipendula ulmaria Rosaceae Aspirin Analgesic, Inflammation 2 Atropa belladorra Styracaceae Benzoin Ophthalmology 3 Cumanmomum Lauraceae Camphor Rheumatic pain camphora 4 Erythroxylm coca Erythroxylaceae Cocaine Analgesic, antitussaive 5 Rauvolfia canescns Apocynaceae Deserpidine Hypertension 6 Ephedrasinica Ephedraceae Epherdrine Respiratory ailments 7 Papaver somniferum Papaveraceae Morphine Analgesic 8 Papaver somniferum Papaveraceae Papaverine Antisplasmodic 9 Chinchona pubescens Rubiaceae Quinine Malaria prophylaxis 10 Rauvolfia serpentine Apocynaceae Resperine Hypertension
1.2 Phytochemicals: Phytochemicals are non- nutritive chemical compounds that are present in plants and have disease preventive properties. These phytochemicals protect the plant cells from pollution, UV exposure, drought, stress and pathogenic attacks [12]. More than 4,000 phytochemicals are present [13] and are classified on the basis of protective function, physical and chemical characteristics [14]. These phytochemicals are found in vegetables, fruits, nuts, legumes, seeds, whole grains, herbs, fungi and plant species [15]. For example, flavonoids are present in fruits and help in slowing the aging process, isoflavones in soya and lycopene is present in tomatoes [16]. Phytochemicals are non- essential nutrients but have important role in preventing human body from heart diseases, high blood pressure, diabetes and cancer [17].
1.2.1 Bioactive Phytochemicals present in Medicinal Plants [18]: Different phytochemicals are present in plants which are bioactive and disease preventing, shown in Table 1.2.
Table 1.2 Biological functions/ Importance of Bioactive Phytochemicals: Sr. Classification Phytochemicals Biological function/ No Importance 1 Antioxidants Flavonoids, polyphenolics, Oxygen free radical, inhibition tocopherols, carotenoids, of lipid peroxidation, quenching ascorbic acid 2 Antimicrobial Alkaloids, terpenoids, Inhibitors of microorganisms, phenolics reduce the fungal infection 3 Anticancer Polyphenols, curcumine, Tumor inhibitors, inhibition of flavonoids, carotenoids lung cancer 4 Detoxifying Tocopherols, phenols, agents indoles, coumarins, reductive Tumourogenesis inhibitors, acids, aromatic inhibitors 0f procarcinogen isothiocyanates, carotenoids 5 Non-starch Delay in nutrient absorption, Lignins, cellulose, polysaccharides water holding capacity, bile hemicelluloses, pectins, gums (NSA) acids and binding toxins 6 Others Terpenoids, alkaloids, Anti- oxidants, biogenic amines, volatile neuropharmacological agents, flavor compounds cancer chemoprevention
1.2.2 Classification of Phytochemicals: In Chemistry, the classification is classified on the basis of: [19] i) Structural skeleton (alkaloids, flavonoids, terpenoids and steroids) ii) Functional groups (alkanes, acids and ketones) iii) Physiochemical properties (volatile oils and organic acids).
The major chemical substances or phytochemicals which are present in plants are shown below:
Anthraquinones Terpenoids
Tannins Alkaloids
Saponins Flavonoids
Phenolics Phytochemicals Coumarins
a) Phenolics: Phenolics [20] are hydroxyl group containing compounds found in plants and are a large and complex group. These are present as polyphenols in fruits of some plants. These phenolics show a number of properties that are beneficial for humans. Antioxidant properties of phenolic compounds act as protecting agents against free radical mediated disease processes [21]. b) Flavonoids: Flavonoids [22] are a polyphenolic compounds that are present in fruits, vegetables, nuts, grains and beverages (e.g. coffee, tea and fruit drinks) act as antioxidants [23]. About 4000 flavonoids are still known and pigments are also included, in higher plants [24]. These included kampferol, quercetin, anthocyanin, hesperidin, myricetin and apigenin. Most of the flavonoids are used as antioxidant, enzyme inhibition, antimicrobial, anti- inflammatory, antiviral, anti-tumor, anti- allergy, anticancer and cytotoxic activities [25]. c) Alkaloids: Alkaloids [26] are a nitrogen containing compounds present in plants, animals, fungi and bacteria. These are the secondary metabolites and most important class of the
phytochemicals. More than 10,000 different alkaloids are present in about 300 plant families. Alkaloids have many pharmacological properties and show different effects such as antimalarial, antihypertensive, anticancer and antiarrhythmic effects [27]. Some alkaloids are showed stimulant property as nicotine and caffeine while morphine is used as analgesic; quinine is as antimalarial drug etc [28]. d) Terpenoids: Terpenoids [29, 30] are a compound whose structures are based on isoprene units (methylbuta-1,3- diene). Plants terpenoids are used extensively for their aromatic qualities. Terpenoids can have medicinal properties such as antimalarial, antimicrobial, anticarcinogenic, antiulcer and hepaticidal and also act as antifeedants [31]. e) Anthraquinones: Anthraquinones are extracted from different plants and have been used since ancient times because of their cathartic and laxative properties [32]. Mostly anthraquinones are present in root, bark and leaves of few species of plant. Anthraquinones have shown potential antipyretic, anti- inflammatory, analgesic, wound healing, antiviral, antifungal and anti- tumor effects [33, 34]. These are also used as food colourants, bugs repellents and textile dyes [35]. f) Tannins Tannins [36], astringent, bitter polyphenols, containing sufficient hydroxyl and other suitable groups such as carboxyl group that either bind and precipitate or shrink proteins. Tannins, a large polyphenolic compounds. Tannins are found in fruits (blueberry, grapes, and persimmon), chocolate, tea and in grasses (sorghum corn) [37]. Tannins containing plant extracts are used as diuretics, against stomach, as astringents, against diarrhea and as antiparasitic, antibacterial, anti-inflammatory, antiviral, antiseptic, antioxidant and haemostatic pharmaceuticals [38, 39]. Tannins are also used in food industry, dyestuff industry and textile industry [40]. In the past few years, tannins have also been studied against cancer through different mechanisms [41].
g) Saponins Saponins [42] are glycosides, present in plants with soap- like foaming properties. The foaming property is produced by shaking the aqueous solution. Saponins have bitter taste and some of them are toxic. Chemically, saponins are glycosylated steroids, steroid alkaloids and triterpenoids [43]. Saponins possess antispasmodic, anti- tumor, anti- inflammatory, anti- proliferative and antidiabetic properties [44]. h) Coumarins Coumarin [45] is an oxygen heterocycle, first isolated in 1820. Coumarin belongs to a group of compounds known as benzopyrones which consist of a benzene ring joined to a pyrone [46]. It is famous for its vanilla- like or freshly- mowed hay fragrance and also shows significant antibacterial and antifungal effects [47]. Coumarins are mostly present in Leguminoseae, Umbelliferae and Rutaceae family and also present in bacteria and fungi [48].
1.3 Family Rhamnaceae: Rhamnaceae belongs to the Buckthorn family having flowering plants, especially trees, shrubs and also vines. 50-60 genera and 870- 900 species are present in family Rhamnaceae. In Pakistan, only 6 genera and 21 species are present [49]. The family is distributed in all over the world but commonly present in tropical and subtropical regions [50].
Importance of Family Rhamnaceae: Economically, Rhamnaceae is essentially used as an ornamental plant and Rhamnus, Paliurus and Hovenia species are cultivated as an ornamentals [51]. Drugs and the material of various brilliant green and yellow dyes have been obtained from various species of Rhamnus [52]. Several species of Rhamnaceae (R. catharica and R. frangula) have been used as laxative. It is a good source of timber. Timber of Alphitonia, Colubrina, Hovenia and Zizyphus species are used for making furniture, carving, musical instrument, construction, lathwork [51]. The importance of this family also includes edible fruits, medicinal soap and varnish plants [53, 54].
Rhamnaceae is one of the plant families that contain anthraquinones. About four genera are certainly known to contain anthraquinones.
1.3.1 Genus Rhamnus: The genus Rhamnus widely spread in east Asia and North America and are found mostly in temperate region, subtropical Northern and Southern hemisphere in parts of Africa and South America [55]. This comprises of 150 species. These are shrubs and small trees, range 1- 10m tall and these are also called buckthorns. The fruits of many species contain a yellow dye and seeds are rich in proteins and the oils of these seeds are used for making printing ink, lubricant oil and soap [55]. The wood of Rhamnus was best for making charcoal and gunpowder in 15th to 19th centuries [56].
Medicinal Importance of Genus Rhamnus: Some species of Rhamnus have been used in common medicines such as gastric, asthma, tumor and hepatic complications for a long time are also used as inflammation, antiviral [57] purgative (laxative) [58] and antihypertensive [59]. Rhamnus also show pharmacological properties due to the presence of secondary metabolites specially flavonoids, anthraquinones, tannins and coumarins [60, 61]. The dried ripe berries of Rhamnus catharticus and bark of Rhamnus frangula are used for constipation [62]. The bark of Rhamnus procumbens is used for habitual constipation, pancreatic stimulant, and laxative and this specie contains emodin which exhibit cardiac and intestinal stimulant, analgesic and central nervous system depressant properties and due to emodin, Rhamnus procumbens possesses anti- inflammatory, anti- cancerous and anticonvulsant properties [62]. The bark of Rhamnus wightti is astringent and deobstruent whereas fruit of Rhamnus virgatus is purgative and emetic [62].
1.3.2 Rhamnus triquetra: Rhamnus triquetra (Wallich) belongs to family Rhamnaceae and are commonly distributed in Himalayas from Azad Jammu and Kashmir to Nepal [62]. It is an evergreen shrub and it grows upto 7m high. Locally, it is also known as Clader.
Taxonomical Position of Rhamnus triquetra:
Vernacular Names of Rhamnus triquetra [63]:
Gardhan India (Punjab) Gaunt India (Dehradun) Gaunta India (Almora) Gaunth India (Garhwal) Katheraa India (Jaunsar) Clader Pakistan (Azad Jammu & Kashmir)
Medicinal Importance of Rhamnus triquetra: The bark extract of Rhamnus triquetra is used in curing of dysentery and diarrhea and is also used as tonic, deobstruent and astringent [64]. Leaf juice is used for malarial fever and to kill intestinal worms [65]. The plant exhibited significant anti-inflammatory and nonspecific antiplasmodic activities [66]. Leaves and fruits extracts are used for hemorrhagic septicemia in cattle [67]. Wood is used for turnery, agricultural and is chopped for fodder [68].
1.3.3 Genus Zizyphus: This genus is represented by more than 80 species through the world but only 6 species are present in Pakistan [69], 17 species are in India [70] and 8 are present in Nepal [71]. This genus is distributed widely in Pakistan, India, indo Malaysia, Australia, Africa and tropic America and few species of this genus are spread in Pacific Islands.
Medicinal Importance of Genus Zizyphus: It has been paid attention to genus Zizyphus because it has remarkable medicinal uses such as anti-inflammatory, antioxidant, antimicrobial, antitumor, hypotensive, hypoglycemia and liver protection [72, 73]. Most of the Zizyphus species are used as folklore medicine of different diseases such as weakness, obesity, digestive disorder, diabetes, diarrhea, fever, insomnia, anemia, loss of appetite, bronchitis, urinary troubles and skin infections [74]. The roots of some species are used in fever, cure wounds and ulcer [75]. Many species of Zizyphus are popular for edible fruit among Zizyphus jujuba and Zizyphus mauritiana are cultivated on commercial scale [51]. Fruits of some species
are used as a medicine in astringency, stomachache, laxative, indigestion, nausea, blood purification, throat troubles and vomiting [76]. The bark of Zizyphus mauritiana is used in ulcers, diarrhea, astringency and wound’s dressing [77]. The wood of Zizyphus rugosa is used for fuel, fruits are consumed by people and leaves are used as fodder by animals [78]. When mixed the bark powder with ghee is used for swelling of cheek and mouth’s ulcer as medicine [79].
1.3.4 Zizyphus Oxyphylla: Zizyphus oxyphylla Edgew is a medium size tree, widely distributed in Pakistan (Swat, Malakand and Gari Habibullah), Azad Jammu and Kashmir, Himalayas and India. The local name of this plant is ‘Mamyanu’ [68].
Taxonomical Position of Zizyphus oxyphylla:
Medicinal Importance of Zizyphus oxyphylla: Zizyphus oxyphylla is used as medicine for some common diseases such as fever, pain, allergy and diabetes [80]. Its roots are boiled in water and filtered. The filtered water is used for jaundice [81]. The plant has been shown to possess antipyretic and antinociceptive in animal models [82]. Fruits of this plant are edible and used in gas trouble. Wood is used as fuel and it serves as a honey bee species [83].
1.4 Antioxidant Activity: Antioxidants are such type of compounds which inhibit or delay oxidation of substrates even these compounds are present in very low concentration as compared to oxidized substrate[84]. Scavenging of reactive oxygen species (ROS) is one of the possible mechanisms of action of antioxidants. ROS formation can be prevented by enzyme inhibition or metal binding. Most of the human diseases as shown in Figure 1.1 are due to the free radical oxidative stress [85] and these diseases can be prevented by antioxidants.
Heart (Hypertension, Cardiac, Fibrosis, Eyes (Retinal Ischemia) Skin (Skin Ageing, degeneration, Sunburn, Cateracts Dermatitis, Melanoma)
Multiorgan (Diabetes, Ageing, Kidney (Chronic Free Radical kidney disease, Chronic, Fatigue) Oxidative Stress Renal, Graft)
Blood Vessels Lungs (Asthama, (Hypertension, Allergies, Cancer Restenosis Brain (Alzheimer, Parkinson, Stroke, Migraine, Trauma, Caner)
Figure 1.1 Diseases due to Free Radical Oxidative Stress
Hydroxyl radicals (OH), hydrogen peroxide (H2O2) and superoxide radicals are the ROS, observed in some cancer cells [86] while H2O2 is one of the main ROS that causes lipid peroxidation and DNA oxidative damage in cells [87]. Many synthetic antioxidants such as BHT, BHA, PG, DG etc. are used but these antioxidants show toxic effect on human health and also mutagenic effect [88] while the natural antioxidants like phenolic compounds, vitamins, essential oils, and plant extracts are used to enhance health and food preservation by rancidity, retarding discolouration or deterioration due to auto- oxidation and such type of natural antioxidants exhibit significant biological activities like antimicrobial, antiviral, antiallergic, antithrombotic, anti-inflammatory, and vasodilatory activities [89]. Phenolic compounds, α- tocopherols and vitamin C are used to inhibit lipid peroxidation and lipoxygenases [90]. Some antioxidants are complex group of enzymes like protease, lipase, DNA repair enzymes, reductase, transferase etc. these enzymes are used for repair of damaged proteins and DNA, oxidized lipids and per oxidized and also stop the chain of propagation of peroxyl lipid radical [91]. These
enzymes are also used to repair the damage biomolecules and reconstitute the damage cell membrane. Some of the antioxidant plants are given below with their chemical constituents and biological activities as shown in Table 1.3.
Table 1.3 Description of Some Antioxidant Plants [92, 93, 94]: Sr.No Botanical Common Chemical Biological Activities Name Name Constituents 1 In cough, for malarial ß-pinene, curcumin, fever, for cleaning Curcuma Turmeric camphene, blood, antifingal, in domestica ß-sitosterol, eugenol gastro- intestinal, insecticide, antifungal 2 Used in chest troubles, Carotenoids, carotenes, Daucus in bronchitis, jaundice, Carrot flavonoids, sugars, caarota tumours, urinary glycosides complaints 3 Aromatic, diuretic, Limonene, purgative, useful in Foeniculum Saunf anisaldehyde, volatile chest, spleen and kidney oil, estragole troubles 4 Polyphenols, ß- Dysentery, leucorrhea, Mangifera sitosterol, vitamin A Mango bronchitis, urinary indica and C, quercetin, gallic discharges acid and ellagic acid 5 Terpenoids, thymol, Expectorant, stomachic, Ocimum Tulsi eugenol, estragole, gastric disorders of sanctum volatile oil children 6 Antipyretic, smallpox, Santalum Safed- ß-sitosterol, ß-santalol, useful in disease of album chandan volatile oil heart, aphrodisiac, bronchitis 7 Chronic fever, Swertia Arginine, xanthones, Chirayita antimalarial, laxative, chirayita swertinin, chiratin febrifuge
1.5 Antimicrobial Activity: Infectious diseases are the world’s leading cause of premature deaths, especially diarrhea which is the main problem with children in many developing countries. Escherichia coli, Pseudomonas spp, Vibrio cholera, Shigella spp, and Staphylococcus aureus etc., are the
common infectious bacterial strains [95]. In recent years, drug resistance to human pathogenic bacteria has been commonly reported from all over the world [96]. Microorganisms have become resistant with the continuous use of antibiotics. So the adverse side effects of antibiotics on host including allergic reaction, hypersensitivity and, depletion of beneficial gut. Research on herbs and medicinal plants has enlarged and their antimicrobial activity has been screened in number of studies [97]. Antimicrobial substances are more effective as compared to antibiotics in the treatment of microbial infection. So much attention has been paid to isolate such active compounds from plants that used in herbal medicine because these medicinal plants have already been used as medicine in various diseases such as gastrointestinal disorders [98], urinary tract infections and skin infections [99]. Thus this has lead to investigate the antimicrobial activity of medicinal plants because they are a rich source of bioactive compounds [100].
1.6 Enzyme Inhibition/ Inhibitory Activities: Plants are a rich source of flavonoids and phenolic contents and showed significant antioxidant activities. Due to such types of secondary metabolites like flavonoids, phenols etc. in plants, they possess effective inhibitors of various enzymes (Cyclo- oxygenase, Xanthine, Lipoxygenase and Cytochrome P450) to prevent some diseases [101]. Acetylcholinesterase (AChE), is used for the breakdown of acetylcholine and the inhibitors of AChE are generally used for the treatment of Alzheimer’s disease (AD), glaucoma and myasthenia gravis but the most important function is to improve the cholinergic function in brain during AD treatment [102]. Different synthetic inhibitors like galanthamine, donepzil, tacrine and rivastigmine, are used for the AD treatment [103] but these inhibitors show a little unpleasant side effects [104] and side effects of these drugs are [105] shown in Figure 1.2.
Anorexia Sleep disturbance Fatigue Side Effects of Synthetic Drugs used in AD Muscle Nausea cramps
Diarrhea
Figure 1.2 Side Effects of Synthetic drugs used in treatment of Alzheimer’s Disease (AD)
In recent studies, it is indicated that the medicinal plants are a rich source of biologically active compounds and are used as a new source of AChE enzyme because anticholinesterase activity has been reported in few plants in the world [106].
Protease inhibitors, small protein molecules have the ability to reduce/ inhibit the action of target proteolytic enzymes [107] and these are present in plants, animals, microorganisms but mainly in plants where they are widely distributed in legumes seeds (tepary bean, soybean, navy bean, cowpea, and pigeon pea) potatoes, cereals. The quantity of inhibitors depends on the multiplicity and physiological status of the plants and on the level of insect damage or infestations [108]. Plant products are a rich source of protein, influence the quality of human diet and of the feed to a great degree, mostly in developing countries. These rich sources of protein have a significant quantity of condensed tannins which are powerful trypsin inhibitors and reduce the digestibility of proteins by preventing their complete hydrolysis in the gut [109]. So these inhibitors have been referred to as an antinutritional factor. Trypsin inhibition mostly occurs by polyphenols [110] and other fodder plants [111]. Although human ingest small amount of polyphenols as compared to animals, in vitro studies on the trypsin inhibition is very important.
1.7 Hemolytic Activity: In vitro, hemolytic activities are now-a- days becoming a latest research area in the drug discoveries [112]. Hemolytic activity is determined by the action of plant extracts on human blood because this is the sign of bioactivity and cytotoxicity [113]. In drug designing, toxicity of the active molecule is a main factor and in this regard, hemolytic activity represents a useful initial point and provides the key information on the relationship between molecules and biological entities at cellular point. Hemolytic activity of some compounds is a marker of cytotoxicity towards normal healthy cells [114]. Generally, saponins, a phytochemical constituent, if present in plants, showed significant hemolytic activity by creating changes in the cell membrane [115]. This activity was performed by spectroscopic method because it is an easy and effective method for the quantitative measurement of hemolysis and also the estimation of various concentrations of biomolecules on the human erythrocytes.
1.8 Anti- inflammatory and Analgesic Activity: WHO (World Health Organization) has estimated that about 80% of population depends on medicinal plants and herbs for their health care and for their livestock [116]. Drugs which are used for the administration of pain (analgesic) and inflammatory conditions like steroidal (corticosteroids) or non- steroidal (aspirin), possess more or less toxic and have side effects such as hearing loss, allergic reaction, renal failure or they may also increase the risk of haemorrhage by affecting platelet function [117]. On the contrary, many medicines which are originated from plants have been used without any unpleasant or side effects. So in recent studies it is indicated that the plants possess anti- inflammatory and analgesic properties because large number of bioactive compounds are present in plants. Purified natural compounds are now isolated from plants to serve as template for synthesis of new anti-inflammatory and analgesic drug with low side effect and toxicity and higher therapeutic value [118]. Plants having anti-inflammatory and analgesic activity are explained in Table 1.4.
Table 1.4 Plants having Anti-inflammatory and Analgesic Activity [119, 120, 121]: Sr. Botanical Name Common Chemical Constituents Activity No Name Eucalyptus Lemon Flavonoids, terpenes, Anti- 1 citriodora eucalyptus tannins, alkaloids, inflammatory, eucalyptol analgesic Kaempferia Aromatic Methylcinnamate, ethyl- Anti- 2 galangal ginger p- methoxycinnamate, inflammatory, carvone analgesic Manilkara Chickoo Flavonoids, phenolic Analgesic 3 zapota compounds, alkaloids, steroids Murraya Orange Alkaloids, coumarins, α- Analgesic 4 paniculata Jessamine glycol, paniculol Mangifera indica Mango Polyphenols, ß- Anti- sitosterol, triterpenes, inflammatory, 5 vitamin A and C, Analgesic quercetin, gallic acid and ellagic acid Pimpinella Saunf 2-terpineol, p- Narcotic analgesic 6 anisum anisaldehyde, linalool Pletranthus Maxican mint Limonene, terpenes, Anti-inflammatory 7 amboinicus eugenol, myrcene Sinapis arvensis Field mustard Glucosinolates, essential Narcotic analgesic 8 oil Sterculia foetida Jangli badam Fats, cycloprenoid fatty Anti- 9 acids inflammatory, Analgesic Xanthium studtii Banokra Polyphenols, glucosides, Analgesic 10 α and γ- tocopherols
1.9 Hepatoprotective Activity: Liver is an important organ in a body and plays a significant role in metabolism of carbohydrates, proteins and fats and storage of vitamins, glycogens and various metals. Liver also plays an important role in the synthesis of fibrinogen, albumin, and clotting factors [122]. Liver damage is always related with cellular necrosis, increase in tissue lipid peroxidation and depletion in the tissue glutathione levels. In addition, serum levels of many biochemical markers like;
Serum glutamate pyruvate transaminase (SGPT/ ALT) Serum glutamate oxaloacetate transaminase (SGOT/ AST) triglycerides Alkaline phosphatase (ALP) Total bilirubin and Cholesterol are elevated [123]. Following are some liver diseases that are commonly observed. Liver disorder due to impaired metabolic function. Generally disorders associated with bilirubin (jaundice) and fat (liposis) metabolisms are very commonly seen. Hepatic failure (Acute/ chronic) Necrosis, Cirrhosis Hepatitis (viral, toxic, or deficiency type). Toxic liver injury is produced by drugs and chemicals may virtually mimic any form of naturally occurring liver disease. Hepatoprotective effect was studied against
chemicals and drugs induced hepatotoxicity in rats like CCl4, Paracetamol, Alcohol, Galactosamine, Isoniazid and rifampicin, Antibiotics, Aflatoxin, Peroxidised oil etc. Severity of hepatotoxicity is greatly increased if drug is continued after symptoms develop. Rifampicin and isoniazid (RIF+ INH), being the first line drugs used as anti tuberculosis chemotherapy, are known to be associated with hepatotoxicity [124] with an inflammatory response [125The rate of hepatic damage has been increased about 8- 30% in developing countries because by using the combination of these two drugs, oxidative stress is produced which cause hepatic injury [126]. These drugs (INH + RIF) are reported to induce hepatotoxicity and elevated by serum ALAT, ASAL, ALP and total bilirubin levels, presence of focal hepatitis necrosis and portal triaditis [127]. Natural antioxidants as shown in Figure 1.2, are being increasingly suggested as important dietary factors that reduce the oxidative stress [128].
Phenolic Flavonoids diterpenes
Phenolic Acids Catechins
Vitamin E Anthocyanins
Natural Vitamin C Antioxidants Procyanidins
Figure.1.3 Some Natural Antioxidants
In recent years, usage of herbal drugs for the treatment of liver diseases has increased all over the world because these herbal drugs are harmless, free from adverse reactions and are obtained from nature and easily available [129]. So natural remedies from medicinal plants are considered to be effective and safer alternative treatment for hepatotoxicity.
LITERATURE REVIEW
2.1 Literature Review of Rhamnus triquetra: A number of compounds of different classes have been investigated from this genus Rhamnus. Some important compounds with their structures of this genus are given in Table 2.1.
Table 2.1: Previously Isolated Phytochemical Compounds from Genus Rhamnus:
Name of Molecular Ref. No Compounds Formula with Structure Molecular Mass Emodin C15H10O5 130 270.24
Physcion C16H12O5 130 284.26
Aloe- Emodin C16H12O6 130 300.26
Chrysophanol C15H10O4 130 254.24
Rhein C16H10O7 130 314.25
Frangulin A C29H39O9 131 531.61
Nakahalene (2- C14H14O4 132 acetyl-3-methyl- 246.26 6-methoxy naphthalene-1,8- diol) 2-acetyl-3- C18H18O6 132 methyl-6- 330.33 methoxynaphthal C17H14O4 ene-1,8-diacetate 282.29
Physcion-8-O-ß- 131 glucoside
Isotorachrysone C14H14O4 133 246.26
Isotorachrysone C18H18O6 133 peracetate 330.33
6- C15H16O5 133 mehoxysorigenin 276.28
Quercetin-3-O- C16H12O7 133 methylether 316.26
Quercetin C15H10O7 133 302.24
Quercetin-3-O- C24H20O11 133 methyl ether 484.41 peracetate
Madagascin C20H18O5 134 338.35
3- C25H26O5 134 geranyloxyemod 406.47 in
ß- Sorgenin C12H8O4 135 216.19
α- Sorinin C13H9O4 136 229.21
Geshoidin C18H18O9 135 378.33
Musizin C13H12O3 135, 137 216.23
10- C25H24O11 135 Oxoprinoidin 500.45
Rhamnocitrin C16H12O6 135 300.26
Rhamnazin C17H14O7 135 330.29
5- hydroxy-7- C9H8O4 137 methoxyphtalide 180.16
3,5- dihydroxy- C12H13O5 137 7- methoxy- 2- 237.23 methylchromone
Eugenine C12H13O4 137 221.23
Frangulin B C26H32O9 131 488.53
Denee et al, 1981 [138] extracted anthracene derivatives through water- ethanol (H2O: EtOH) mixture of Rhamnus purshiana by XAD-2 column chromatography and also extracted glycosides and aglycones. Abegaz et al, 1999 [139] discovered that Rhamnus prinoides are one of beneficial marketed plants. Marketed plants are important and vulnerable groups of plants whose conservation and investigation is considered with priority, so, they are used as marketed plants as different groups (functional) or important groups are isolated from them are discussed and play revolutionary role in biology and chemistry. The important groups include isoflavonoids, nor-lignans, anthacene and naphthalene derivatives and polyprenylated isoflavonoids are isolated from this plant. Rhamnus prionides are discussed in a research which shows that Rhamnus species is an important plant. Jain et al, 1999 [140] found that there are certain species of plant (trees) which have fuel wood properties; these are such properties which include moisture, silica ash, carbon, nitrogen, and volatile matter. According to research thirty tree species indigenously grown in their natural habitat in sub tropical forest of central India were collected and have fuel wood properties. Among them Rhamnus triquetra was also included. Other tree species were Aceroblongum, Betula alonoides, Grevillea robusta, Limonia acidissina, Lyonia ovalifolia, Madhuca indica, Melia azedarch, Morinda tinctoria, Myrica sapida,
Prunus cornuta, Pyrus pashia, Quercus langinosa and Rhamnus triquetra. This reveals that Rhamnus triquetra are an important plant having fuel wood properties. Ferreira et al, 2004 [141] analyzed different compounds of flavonoid dyes by complementary techniques of LC-ion trap MS and PDA-HPLC. These techniques liquid chromatography, electron spray ionization (LS-ESI) ion trap and mass spectrometry and photodiode array HPLC provides information about analysis and characterization of each dye component of Rhamnus cathartica L. The results of such techniques showed the absorbance of seven components in UV region of spectrum, but the components included quercetin, kampferol, isorhamnetic emodin and other components were not identified from authentic studies.
Venture et al, 2004 [142] purposed a simple method for the purpose of ‘B1, B2, G1 and
G2’ aflatoxins in Rhamnus purshiana by LC joined to mass spectrometry. Aflatoxins were isolated by a combination of MeOH: H2O and then it was purified by solid phase clean up using a polymeric sorbent, not described before, for the purpose of these toxins.
The eluted extract was injected onto a reversed phase C18 short column by way of an isocratic mobile phase ratio of MeOH: H2O (30:70). A particular quadruple mass spectrometry with an electro spray ionization spray working in the positive ion mode was utilized to identify aflatoxins. Recoveries of the full analytical processes were 110% for
B1, 89% for B2, 8% for G1 and 77% for G2. Luigia et al, 2005 [143] extracted anthocyanin constituents from berries of Rhamnus alaternus. The pigments could isolate with 0.1% HCl in methanol and purified by means of a C-18 solid-phase cartridge. HPLC diode array detection-mass spectrometry analysis showed that delphinidin 3- Orutinoside present up to 62.4% of the whole pigments while new anthocyanins are petunidin (15.8%), peonidin with malvidin (8.7%), pelargonidin (4.7%), and 3- O-rutinoside derivatives of cyanidin (8.4%). The simultaneous presence of the six most frequent anthocyanidins recommended that R. alaternus berries, also a good pigment resource, might also be a helpful means for anthocyanin detection. Manojlovic et al, 2005 [144] determined the consequences of antifungal test of the methanol extracts and the main anthraquinone aglycones, alizarin and emodin, of Rubia tinctorum and Rhamnus frangula in contrast with the antifungal activity of the anthraquinone-containing lichen Caloplaca cerina.
Rebai et al, 2007 [145] used the methanolic, petroleum ether, chloroform, ethyl acetate,
Total Oligomers Flavonoids (TOF) enriched extracts, water extract and its fractions A1,
A2 and A3 achieved from aerial parts of Rhamnus alaternus, were examined for the phenolic contents, cytotoxic activity against the K562 human chronic myelogenous leukaemia cell line and L1210 leukaemia murine cells and for antibacterial activity adjacent to Gram positive and Gram negative bacterial reference strains. The antimicrobial and cytotoxic activities in R. alaternus depended on the chemical composition of the extracts. A cytotoxic effect on both the cell lines was shown in the
TOF, ethyl acetate, methanolic, aqueous extracts and A2 fraction, with respectively IC50 values 75, 232, 298, 606 and 571 μg/ml on K562 cells and 198, 176, 767, 560 and 614 μg/ml on L1210 cell line while these bacterial reference strains:, Salmonella typhimurium, Enterococcus faecalis, Salmonella enteritidis, Staphylococcus aureus and Escherichia coli was shown considerable activity with ethyl acetate, TOF extracts and A2 fraction showed. Baya et al, 2008 [146] extracted the volatile compounds from essential oil of R. alaternus L. by using hydro- distillation method. About 94 constituents were identified but the phytol, linalool and ß- damascenone were present in major amount i.e. 16.10%, 15.33% and 5.28% and these were first time reported in this plant. Ben Amma et al, 2008 [147] revealed a distinct antiproliferative effect on human leukemia K 562 cells with flavonoids- enriched extracts from (Tunisian) Rhamnus alaternus roots and leaves. Antioxidative effects via xanthine oxidase assay with IC50 values of 83.33 and 103.96 g/mL and high DPPH radical scavenging action with IC50 values of 7.21 and 18.84 g/mL respectively were detected in the existence of the two tested extracts. Kandwal et al, 2008 [148] established that Rhamnus triquetra (wallich) have its place in family Rhamnaceae (Buckthorns). Rhamnus triquetra is extensively scattered in moderate zone of the world. Kaempferol -7-O-methyl ether was extracted from ethanolic (EtOH) extract of R. triquetra. Research exposed that kaempferol -7-O-methyl ether, pharmacologically show significant non specific anti-plasmodic and anti-inflammatory activities. R. triquetra have shown another significant activity which is revealed that
leave extract contain “Methyl-ether” and “emodin” that reduce CNS depressant activity, this is indications of its biological significance. Locatelli et al, 2009 [149] found a number of species belonging to the genus Rhamnus (Rhamnaceae), including ones which are originate from nearly all the typical plants of the Italian flora, are known to have biologically active anthraquinone derived metabolites. Even though a number of Rhamnus species were so far analyzed, no sequence is existing regarding the content and relative abundances of anthraquinones in R. saxatilis. In this reading we used an easy, consistent, and precise analytical process to find out the anthraquinones in bark of R. saxatilis. This permitted us also to sketch a relative study on the effectiveness of different extraction solvents in ultrasonication time dependent method. Division and quantification of anthraquinones were able to use a C18 column with the mobile phase of H2O: methanol (40:60, v/v, 1% formic acid) at a flow rate of 0.7 mL/min and a wavelength of finding at 254 nm, although a qualitative examination was also accomplished at a wavelength of 435 nm. Finally, the described HPLC technique was used to find a definite chemical fingerprint for this species in contrast with other species from the same family.
2.2 Literature Review of Zizyphus oxyphylla: A number of constituents of different classes have been investigated from this genus Zizyphus. Some important compounds with their structures are given in Table 2.2.
Table 2.2: Previously Isolated Compounds from Genus Zizyphus:
Molecular Name of Formula with Structure Ref. No Compound Molecular Mass
Abyssenine B C25H38N4O4 150 458.6
Abyssenine C C24H36N4O4 150 444.57
Mucronine G C25H38N4O5 150 474.6
Zizyphine D C25H38N4O5 151 474.6
Zizyphine E C24H36N4O5 152 460.57
Adouetine X C28H44N4O4 153 500.6
Lotusanine A C31H42N4O4 154 534.70
Oxyphylline A C42H45N5O6 155 715.85
Amphibine A C33H43N5O4 156 573.73
Amphibine B C39H47N5O5 156 665.83
Amphibine C C36H49N5O5 156 631.81
Amphibine D C36H49N5O5 156 631.81
Amphibine E C38H50N6O5 157 670.9
Amphibine F C29H36N4O4 158 504.63
Mauritine J C37H48N6O5 159 656.82
Nummularine C32H41N5O6 160 B 591.71
Nummularine C32H41N5O7 161 T 619.72
Daechuine S3 C34H53N5O6 162 627.82
Daechuine S7 C28H42N4O5 162 514.66
Sanjoinenine C29H35N3O4 163 489.61
Franganine C28H44N4O4 164
Frangufoline C31H42N4O4 165 534.70
Sanjoinine B C30H40N4O4 163 520.7
Sanjoinine F C31H42N4O5 163 550.7
Frangulanine C28H44N4O4 166 500.7
Hysodricanine C35H45N5O5 167 A 615.77
Hysodricanine C30H43N5O6 168 B 569.7
Nummularine C30H40N4O4 169 D 520.7
Integerrenine C31H42N4O4 170 534.7
Nummularine C39H47N5O6 170 H 681.8
Nummularine C42H45N5O6 171 O 715.9
Xylopyrine B C34H38N4O5 172 582.7
Lotusine A C30H38N4O4 173 518.66
Ramosine C C30H38N4O5 174 534.65
Mauritine A C32H41N5O5 175 575.71
Mauritine B C35H47N5O5 175 617.79
Mauritine E C32H41N5O6 175 591.71
Mauritine F C31H39N5O5 175 561.7
Daechuine S5 C27H42N4O4 163 486.65
O- C36H49N4O6 176 Demehylmucro 647.813 nine D
Mucronine J C27H40N4O4 177 484.64
Nummularine C31H40N4O5 152 C 548.68
Rugosanine A C30H43N5O7 178 585.70
Sativanine C C29H43N5O6 179 557.69
Sativanine M C30H43N5O7 180 585.70
Acharya et al, 1994 [181] performed antinociceptive, anticonvulsant and anti- inflammatory activities on the ethanolic extract of the bark of Zizyphus jujube and these activities were due to the presence CNS depressant activity in this plant. Mathur et al, 1995 [182] studied nitrogen metabolism due to the reason that enzymatic changes occurred because of five (VA) mycorrhizae were studied in glass house circumstances in In Zizyphus nummalaria fruit plant. Gs, NR and GDH functions were increased because of five mycorrhizal plants instead of increasing protein argumentation in Z. nummularia. Such results are very useful for budding extremely proteinaceoues leafy straw of Z. nummularia. Particulars of symbiotic relationships have also been reviewed. Ghannadi et al, 2004 [183] isolated the compounds from the oil of Zizyphus spina – Christi (L) which is extracted from hydro-distillation and exhibiting 92.2% of oil. The main components in the oil were following: 10% Me-hexadecanoate, 9.9% Farnesyl acetone, 9.9% Me- actadecanoate, 9.7% hexadecanol, 8% Et- actadecanoate and 14.0% general acetone. Dahiru et al, 2005 [184] studied the screening effect of ethanolic (EtOH) extract of leaves of Zizyphus mauritiana on carbon tetrachloride resulted in liver malfunction. Z. mauritiana leaf protected rats (200-300 mg/Kg body mass) against carbon tetrachloride injured liver by greatly decreasing alkaline phosphate (ALP), lipid peroxide, aspartate aminotransaminase (ALT) and total bilirubin (TB) and rise advanced to handle. The abstract of two doses also greatly construct up reducing level of Vitamin E and gluthatione and approached to handle. Leaf extract of Z.mauritiana also showed different compounds i.e. saponins, tannins, phenolic contents and flavonoids. Herrera et al, 2005 [185] used Zizyphus sonorensis fruits and studied the physico- chemical and nutritional properties. This study was made by Uniform size, low moisture and colour components were three major traits of this fruit constituent of tannins and nickel of the digestible portions competent concepts of its use. Through Z. sonorensis fruit had some main and significant properties and thought to be most excellent food source. The outstanding qualities and characteristics of digest able part were grate content of total (35g / 100g) and soluble (0.6g/100g) dietary fiber, high level of Cu (0.53mg / 100g) and zinc (4.2 mg / 100g). Seed was distinguished by this greater part of net dietary
fiber (82.1g / 100g) on foundation of fresh weight moisture value was described where as residual values were decided on base of dry weight. Harami et al, 2006 [186] extracted methanolic extracts of such species: Cassia accidentalis Linn, Detarium microcarpum, Ziziphus abyssincia, Z. mauritana, Z. mucronata and Z. spina – Christ wild. Antifungal activity was shown by these species by using agar diffusion method against dermatophytes such as Aspergillus fumigates, Microsporum canis and TrichoPhyton rubrum and T. mentagaphytes. Ataa et al, 2010 [187] extracted the volatile constituents from Z. Jujube (leaves and flowers) and Z. spina- Christi (fruits) and these constituents were analyzed through GC and GC- MS. Phytol (29.1%), hexahydrofarnesyl acetone (9.1%) and borneol (10.1%) were the major constituents present in Z. jujube while dodecanoic acid (22.4%) was the major component of Z. spina- Christi. Njidda et al, 2010 [188] designed this experiment to analyze the chemical constituents, in vitro dry matter digestibility and in vitro fermentation of four dominant species (Leuceana lecocephala, Moringa oleafera, Acacia tortillas and Ziziphus mucronata) in area of testing. Raw protein ranges from 13.96 DM for A. tortillas to 19.42% DM, for L. leucocephala but M. oleafera had the highest value. Lignocelluloses had a range of 21.16g/ 100g DM to 31.39g/ 100g DM for A. tortillas. The TCT (Total condensed tannins) had different range for browsers were 0.25mg/g DM for M obleafera and 2.96mg/g DM for L. leucocephala. Z. mucronata had the greatest value for calcium, magnesium, sodium and potassium rather than phosphorous. In vitro gas and formulation of methane was greatest in Z. mucronata/ VDMD had range of 70.66-72.00 CP. TCT exposed a positive relationship with /VDMD. Nisar et al, 2010 [189] performed antibacterial, antifungal, phytotoxic, cytotoxic and insecticidal activities on crude methanolic and various fractions of Z. oxyphylla Edgew leaves. E. coli, B. subtilis, S. flexenari, P. aeruginosa, S. typhii and S. aureus were used for antibacterial activity and T. longifusus, A. flavus, F. solani, Microsporum canis, C. albicans and C. globerata were used for antifungal activity. Ethyl acetate (EtOAc) fraction showed significant antibacterial activity against B. subtilus while antifungal strains and cytotoxicity showed non significant results. The insecticidal activity present
moderately (40%) in n- hexane and ethyl acetate (EtOAc) fractions while the methanolic extract and all other fractions also showed good phytotoxicity (60- 90%). Ataa et al, 2011 [190] isolated three phenolic compounds i.e., p- hydroxybenzoic acid, rutin and kampferol, first time from Zizyphus spina- christi and these were confirmed by UV, GC- MS and 1HNMR Priyanka et al, 2012 [191] studied the medicinal importance of Z. mauritiana. The leaves were used for extraction and the extraction was done by ether, chloroform
(CHCl3), methanol (CH3OH), 95% ethanol (EtOH) and distilled water for 48h and found various secondary metabolites such as saponins, lignins, tannins, phenols and glycosides. Anwar et al, 2012 [192] isolated the phenolic compounds (p- coumaric acid, rutin, quercitin, apigenin, syringic acid and chlorogenic acid) from methanolic extract of stem of Z. spina- Christi by using the reversed phased HPLC (RPHPLC) and rutin (325.0mg/ 100g) and apigenin (122.90mg/ 100g) were in high concentration in fruits while p- hydroxybenzoic acid, chlorogenic acid and ferulic acid were isolated from the stem of the plant and rutin (15.88mg/ 100g) was again in high concentration. It showed that the phenolic compounds were more present in stem as compared to fruits of Z. spin- Christi. Waqar et al, 2012 [193] obtained oil from n- hexane fraction of stem (WO-1 and WO- 2) and leaves (WO- 5) of Z. oxyphylla Edgew by using solid phase extraction. `Antibacterial, antifungal, phytotoxic, cytotoxic and insecticidal activities were performed on these oil fractions. Six bacteria (E. coli, B. subtilis, S. flexenari, P. aeruginosa, S. typhii and S. aureus) and fungal (T. longifusus, A. flavus, F. solani, M. canis, C. albicans and C. globerata) strains were used. These fractions showed low antifungal activity against M. canis and A. flavus while these fractions were non significant against bacterial strains. WO- 2 and WO-5 showed significant phytotoxicity while WO-1 and WO-5 showed good insecticidal activity against Tribolium castaneum and Rhyzopertha dominica respectively. Cytotoxicity and brine shrimp lethality assay were non significant against these oil fractions. Elyacout et al, 2013 [194] extracted the alkaloids from Z. mauritiana by using gas chromatography- mass spectrometry (GC- MS) and these alkaloids were first time isolated from this plant species.
EXPERIMENTAL WORK
3.1 GENERAL EXPERIMENTAL CONDITIONS Ultraviolet (UV) spectra were taken in methanol and used the instrument i.e., Cecil 7200 Model of 7000 series spectrophotometer. IR (Infrared) spectra were analyzed by KBr disk on Thermo Nicolet, [Model: M2000 Infrared Spectrophotometer. The 1H NMR spectra were measured at 500, 400 and 300 MHz. 13C-NMR spectra were measured at 150, 125 and 100 MHz respectively on Bruker Advance spectrometer. (The 1H-NMR, and 13C-NMR were recorded at HEJ Research Institute of Chemistry, University of Karachi, Pakistan).
3.1.2 CHROMATOGRAPHIC TECHNIQUES Silica gel (Si, 0.063- 0.200mm, 70-230 mesh, E. Merck) was used to perform Column chromatography (CC). Silica gel (GF254, 20×20 cm, 0.5 mm thick, Merck) thin layer chromatography (TLC) plates were used to check the purity of the isolated compounds by using different solvent systems such as n- Hexane: Chloroform, n- Hexane: Ethyl acetate and n- Hexane: Methanol.
3.1.3 DETECTION OF COMPOUNDS The isolated compounds through chromatography were viewed under ultraviolet (UV) light by using 254nm and 366nm wavelengths and Iodine.
SPRAY REAGENT: Ceric sulphate reagent and Dragendorff’s reagent.
CERIC SULPHATE REAGENT: Ceric sulphate (0.1 g) and trichloroacetic acid (1g) were dissolved in 4 ml distilled water.
The solution was boiled and conc. H2SO4 was added drop wise until the disappearance of turbidity. This reagent was used for the detection of compounds.
DRAGENDROFF’S REAGENT: Bismith nitrate (8.0g) was dissolved in distilled water (40mL) and glacial acetic acid (10mL) and then this solution was mixed with 40% potassium iodide (20mL).
3.2 Rhamnus triquetra 3.2.1 Plant Material The plant Rhamnus triquetra was collected from Azad Jammu and Kashmir in March 2010, and identified by Mr. Muhammad Ajaib (Taxonomist), Department of Botany, GC University, Lahore. A voucher number of specimen ‘‘G.C. Herb. Bot. 854’’ has been deposited in the herbarium of the same university.
3.2.2 Extraction and Fractionation: The shade-dried ground whole plant (6 kg) was extracted with methanol (10L × 5) at ambient temperature. The methanolic extract was evaporated on rotary evaporator (Laborta 4000-efficent Heidolph) to obtain the residue (575 g). The residue was dissolved in the distilled water (1L) and then partitioned with n-hexane (1.5L × 3), chloroform (1.5L × 3), ethyl acetate (1.5L × 4) and n-butanol (2L × 4), separately. These organic fractions and remaining aqueous fraction were concentrated separately on rotary evaporator under vacuum. The detailed extraction scheme is shown in scheme 3.1.
Scheme 3.1 Extraction Scheme of Rhamnus triquetra
3.2.3 Isolation and Purification of Compounds of Rhamnus triquetra through Column Chromatography: Silica gel 60 (70-230 mesh) was used for column chromatography to isolate and purify the compounds. Slurry was made by dissolving the silica gel in n-Hexane and then packed the column in order to minimize the air bubbles interruption. Sample about 115g of CHCl3 fraction was loaded carefully so that all the air bubble was taken out. Sample was loaded onto the column after packing of column and start eluting with n- Hexane. Raised the polarity with help of Chloroform in increasing order: 5% at one time and eluted one to two liter for one combination. The polarity further increased using methanol
(CH3OH) up to 100 % in similar way. TLC was carried out to collect or combine the fractions whose Rf values were same. About ten fractions (Fr. 1-10) were finally collected. The Fr-1 was non- polar (oily) in nature and it was subjected to GC- MS analysis and two compounds were identified. The fraction 4 was loaded again on a smaller column, eluted with n- Hexane and CHCl3 (40: 60) and Compound no. 3 was purified. The initial non-polar fractions from column subjected to GC MS analysis and six non- polar compounds were identified. Fraction 7 was subjected to smaller column, eluted with n-Hexane and Chloroform (15: 85) to purify Compound no. 4. The details of identified compounds are shown in scheme 3.2.
3.2.4 Chemical Compounds Characterization of Rhamnus triquetra: 3.2.4.1 Physcion Characterization (Compound no.3):
Physical State: Orange red powder (30mg) Mol. Wt: 284.12
Mol. Formula: C16H12O5 Melting Point: 202 ͦ C -1 IR ῡ cm (KBr): 3440 (-OH), 1675 (unchelated or free carbonyl), 1630 (chelated carbonyl), and 1575 (C=C aromatic)
1HNMR and 13CNMR: Table 4.1
3.2.4.2 Characterization of Madagascin (Compound no.4):
Physical State: Orange red powder (40mg) Mol. Wt: 338.35
Mol. Formula: C20H18O5 Melting Point:
-1 IR ῡ cm (KBr): 3410 (- OH), 1630 (conjugated carbonyl), 1608 (aromatic ring)
1HNMR and 13CNMR: Table 4.2
3.3 Zizyphus oxyphylla: 3.3.1 Plant Material The plant Zizyphus oxyphylla was collected from Azad Kashmir in March 2010 and identified by Mr. Muhammad Ajaib (Taxonomist), Botany Department, GC University, Lahore. Voucher specimen (GC- Herb- Bot- 851) has been submitted in the herbarium of the Botany department of the same University.
3.3.2 Extraction and Fractionation: The whole dried plant (5 kg) was exhaustively extracted with methanol (2.5L × 5) at room temperature. The extract was evaporated under vacuum on rotary evaporator at 40 oC to yield the residue (748 g), which was then dissolved in distilled water (1 L) and then partitioned with n-Hexane (1L ×3), chloroform (2 L × 4), ethyl acetate (2.5 L × 4) and n- butanol (2.5 L × 4) respectively as shown in scheme 3.3. These four organic fraction and remaining water fraction were concentrated separately on rotary evaporator and the residues thus obtained.
Scheme 3.3: Extraction Scheme of Zizyphus oxyphylla
3.3.3 Isolation and Purification of Chemical Constituents of Zizyphus oxyphylla through Column Chromatography:
Chloroform fraction (235g) was subjected to column chromatography to isolate and purify compounds. The column was eluted with n- Hexane (non- polar) solvent with gradient of chloroform and methanol. The fraction 1 was non- polar in nature so it was subjected to GC- MS analysis and three compounds were analyzed. The fraction 3 was subjected with small column and eluted with n-Hexane and Chloroform (80: 20), three major spots were identified and then further purification was done by preparative TLC and Compound no.5 was purified. Fraction 5 was loaded on silica gel and subjected to small column and then eluted with n-Hexane and Chloroform (40: 60) to purify the Compound no. 6. The detailed isolation scheme is shown in scheme 3.4.
3.3.4 Characterization of Chemical Constituents of Zizyphus oxyphylla: 3.3.4.1 Characterization of ß- Sitosterol (Compound no. 5):
ß- Sitosterol Physical State: White crystal (45.3mg) Mol. Wt: 415.4
Mol. Formula: C29H50O Melting Point: 136- 137o C
-1 IR ῡ cm (KBr): 3420 (-OH), 3050, 1653 and 800 (C ═C), 2934 (CH2), 2865 (CH)
1HNMR and 13CNMR: Table 4.5
3.3.4.2 Characterization of Betulinic Acid (Compound no.6):
Physical State: White powder (35.5mg) Mol. Wt: 456.36
Mol. Formula: C30H48O3 Melting Point: 282.3 o C
-1 IR ῡ cm (KBr): 3610 (-OH), 3400- 2615 (-COOH), 1705 (carbonyl region) and 1610 (double bond) 1HNMR and 13CNMR: Table 4.6
3.3.5 Extraction of Essential Oil through Water Distillation: Powder of Zizyphus oxyphylla (500gm) is immersed in water in 1L round bottom flask and then fitted with water condenser to condense water vapours (steam) and collecting flask was fitted at other end of condenser. Then mixture was heated to its boiling temperature and the mixture liquid and oil was collected in collecting (receiving) flask. The essential oil was separated from mixture liquid by using solvent extraction. n- Hexane was used for extraction of essential oil. The n- Hexane layer (organic layer) was evaporated by rotary evaporator and essential oil was left behind and was analyzed for GC- MS.
3.3.5.1 GC- MS Analysis: The constituent analysis of different fractions obtained by column chromatography and essential oil was analyzed by GC- MS. Analysis was performed in three times and blank was run after every sample. The results were analyzed and identified by dictionary NIST 147.LIB as shown in Table 4.3, 4.4, 4.7 and 4.8.
3.4 Fluorescence Analysis [195]: Fluorescence analysis of powder of R. triquetra and Z. oxyphylla was done by using different reagents. Ordinary light and ultra violet light of short (254nm) and long (365nm) wavelengths were used for this analysis. The results were given in Table 4.9 and 4.10.
3.5 Physicochemical Studies [196, 197, 198]: The following physicochemical parameters were studied in dried powder of Rhamnus triquetra and Zizyphus oxyphylla. Loss on drying Total ash Water soluble ash Acid insoluble ash Hexane soluble extractive Chloroform soluble extractive Ethanol soluble extractive The results of these parameters of both plants are tabulated in Table 4.11.
3.5.1 Loss on drying: 4g of powder of both plants (Rhamnus triquetra and Zizyphus oxyphylla) were taken in an evaporating dish separately and then dried at 105˚C in an oven for 2hr till constant weight was obtained. It was cooled in room temperature and then noted the weight after drying and % of loss on drying was calculated by using this formula:
% Loss on drying= [Loss in weight of sample/ weight of sample] × 100
3.5.2 Total Ash: 4g of dried powder of above plants were taken in silica crucible and ignited at 300˚C for 3h until they were white, indicating the free of carbon. Cooled the ash in desiccators and then weighed. Percentage of total ash was calculated by using the following formula:
% Total ash value= [weight of total ash/ weight of sample] × 100
3.5.3 Water soluble Ash: 25ml water was added in the crucible containing total ash and boiled for 10 minutes. The water soluble ash was filtered on an ashless filter paper. Washed the filter paper with hot
water and ignited in a crucible for 30 minutes. The residue was allowed to cool in desiccators and weighed. The % of water soluble ash value was calculated by using the following formula:
% Water soluble ash value= {(Wt. of total ash – Wt. of water insoluble ash)/ Wt. of sample} x 100
3.5.4 Acid Insoluble Ash: 25ml HCl was added in crucible containing total ash and boiled gently for 10 minutes. Filtered and collected the insoluble matter on an ashless filter paper. This filter paper was washed with hot water until the filtrate become neutral. Dried the residue present on ashless filter paper and then weighed. Percentage of acid insoluble ash was calculated from this formula:
% Acid insoluble ash value= [Wt. of acid insoluble ash/ Wt. of sample] x 100
3.5.5 Determination of n-Hexane soluble extractive value: 4g of dried powder of both plants were taken in 100ml of n- Hexane in a conical flask, plugged with cork/ cotton wool and then kept on a rotary shaker for 24 h at 120 rpm. After 24h, filtered the sample and evaporated the filtrate (hexane) to dryness at 105˚C till constant weight was obtained. The % was calculated with reference to the sample taken initially.
3.5.6 Determination of Chloroform soluble extractive value: 4g of dried powder of both plants were taken in 100ml of chloroform in a conical flask, plugged with cork/ cotton wool and then kept on a rotary shaker for 24 h at 120 rpm. After 24h, filtered the sample and evaporated the filtrate to dryness at 105˚C till constant weight was obtained. The % was calculated with reference to the sample taken initially.
3.5.7 Determination of Ethanol soluble extractive value:
4g of dried powder of both plants were taken in 100ml of ethanol in a conical flask, plugged with cork/ cotton wool and then kept on a rotary shaker for 24 h at 120 rpm. After 24h, filtered the sample and evaporated the filtrate to dryness at 105˚C till constant weight was obtained. The % was calculated with reference to the sample taken initially.
3.6 Phytochemical Screening of Rhamnus triquetra and Zizyphus oxyphylla: The Phytochemical screening of crude methanolic fraction, organic fractions and aqueous fraction was performed by using standard methods [199, 200,201] and the results of both plants are reported in Table 4.12 and 4.13.
3.6.1 Test for Alkaloids: 1) Dragendorff’s Reagent: For alkaloid test, the Draggendorff’s reagent was sprayed on TLC card having spots of the samples. The appearance of orange colour indicates the presence of alkaloids. 2) Hager’s Reagent: Hager’s reagent was added in sample solution and yellow white precipitate indicates the presence of alkaloids.
3.6.2 Test for Flavonoids: Two tests were performed for the presence of flavonoids. 1) Lead Acetate test: 5ml lead acetate solution was added in sample solution and yellow colour indicates the presence of flavonoids. 2) Alkaline Reagent test: A few drops of dilute NaOH were added to sample solution, intense yellow colour present and this colour disappeared when added a few drops of dilute HCl. This indicates the presence of flavonoids.
3.6.3 Test for Tannins: 1) Gelatin test: Samples/ extracts were treated with 1% gelatin solution containing NaCl (5ml) and white precipitate indicates the presence of tannins.
2) Neutral FeCl3: Boiled the 0.5g of each extract in 10mL distilled water and then added few drops of 0.1% FeCl3. Appearance of bluish- black or brownish- green colour indicates the presence of tannins.
3.6.4 Test for Reducing Sugars:
1) Fehling’s Reagent: Fehling’s solution (A and B) was added to 0.5g sample solutions in a test tube and then heated. Red precipitate formation indicates the presence of sugar. 2) Benedict’s Reagent: Benedict’s solution was added to 0.5g sample solutions in a test tube and orange red precipitate formation indicates the presence of sugar.
3.6.5 Test for Terpenoids: Two methods were performed for the presence of terpenoids. 1) Salkowski test: 2 ml of chloroform was added to 0.5 g of each extract/ sample and then 3 ml of concentrated H2SO4 was added carefully to form a layer. A reddish brown layer at the interface indicates the presence of terpenoids. 2) Ceric sulphate Reagent: TLC card having sample spots were sprayed by Ammonium cerium (IV) sulphate. Heated the TLC card on TLC heater. The appearance of brown colour indicates the presence of terpenoids.
3.6.6 Test for Saponins (Frothing test) 0.5 g of extract/ sample was added to 5 ml of distilled water in a test tube. The solution was shaken vigorously and observed a stable persistent froth. 3 drops of olive oil was mixed with the frothing and shaken vigorously again after that it was observed for the formation of an emulsion.
3.6.7 Test for Phenolics (Ferric Chloride test): Neutral ferric chloride solution was added to each extract/ fraction. The appearance of bluish green colour indicates the presence of phenolics.
3.6.8 Test for Cardiac Glycosides: 1) Legal’s Reagent test: The sample solution was treated with sodium nitroprusside in pyridine and methanolic alkali solution. A pink/ blood red colour indicates the presence of cardiac glycosides.
2) Kellar Killani test: 5ml distilled water was added in 0.5g of each sample and then added 2ml glacial acetic acid and one drop of ferric chloride and this was underplayed with 1ml conc. H2SO4. A violet or brown colour ring formation shows the presence of cardiac glycosides.
3.7 ANTIOXIDANT ACTIVITY:
3.7.1 Chemicals and Standards of Antioxidant Activity: 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), Butylated hydroxytoluene (BHT), Trolox, 2,4,6-Tripyridyl-s-triazine (TPTZ), Gallic acid and Follin Ciocalteu’s reagent were obtained from Sigma Chemical Company Ltd. (USA) and ferric chloride, ferrous chloride, sodium phosphate, ammonium molybdate, sulphuric acid and organic solvents (n-Hexane, chloroform, ethyl acetate, n-butanol), from Merck (Pvt.) Ltd. (Germany).
3.7.2 DPPH Radical Scavenging Activity Different fractions of plants (Rhamnus triquetra and Zizyphus oxyphylla) were used for the determination of DPPH radical scavenging activity by using the reported method [202]. Step I: Preparation of DPPH: 0.1mM DPPH solution was prepared by dissolving 0.04g of DPPH in 1L of methanol. Step II: Preparation of Various Concentration of Sample: Stock solution of extracts and samples (1000µg/ mL) was prepared by dissolving 0.02g of samples and extracts in 20mL of methanol. Then following solutions were made from stock solution i.e. 500 µg/ mL, 250 µg/ mL, 100 µg/ mL, 60 µg/ mL, 30 µg/ mL and 15 µg/ mL. Step III: Assay: Above stock solution and dilutions were mixed with 3 ml of DPPH and shaken the mixture vigorously. At room temperature, the mixture was allowed to stand for one an hour (60 minutes). After 60 minutes, the absorbance was measured in the spectrophotometer at 517 nm. Methanol was taken as blank. High free radical scavenging activity was observed by the lower absorbance of spectrophotometer.
Step IV: Calculations: The scavenging of free radical of the samples was calculated by using this formula:
% Inhibition of DPPH (Antiradical activity) = {(Acontrol - Asample)/ Acontrol} ×100 The samples were assayed in triplicate and mean values were calculated. The results are reported in Table 4.14 and 4.15
3.7.3 Total Antioxidant Activity by Phosphomolybdenum Method Phosphomolybdenum complex formation method was used to evaluate the total antioxidant activities of various fractions/ samples [203]. Step I: Preparation of Phosphomolybdenum Reagent: Yellow colour of Phosphomolybdenum reagent was prepared by dissolving 5.32g of 28mM sodium phosphate, 2.47g of 4mM ammonium molybdate and 16.7mL of 0.6M
H2SO4 in 500mL of distilled water. Step II: Sample Preparation: 500 µg/mL sample solution was taken from above stock solution. Step III: Assay: Each sample (500 µg/mL) was mixed with 4 mL of phosphomolybdenum reagent solution in capped test tubes and incubated these test tubes in water bath at 95oC for 90 minutes.. The blank solution contained only 4 mL of reagent solution. The samples had been cooled at room temperature and the absorbance was measured at 695 nm against blank. The antioxidant activity was expressed relative to that of butylated hydroxytoluene (BHT). All determinations were assayed in triplicate and mean values were calculated. The results are reported in Table 4.16 and 4.17.
3.7.4 Ferric Reducing Antioxidant Power (FRAP) Assay The FRAP assay was done by using Benzie and Strain method with some modifications [204]. Step I: Preparation of FRAP Reagents: First prepared the different reagents for the preparation of FRAP solution. i) 10mM TPTZ was prepared by dissolving 0.312g in 100mL of water. ii) 20mM FeCl3. 6H2O and 40mM HCl were mixed in 100mL of water.
iii) 300mM acetate buffer was prepared by dissolving 3.1g sodium acetate (CH3COONa.
3H2O) in 16mL acetic acid (CH3COOH) of pH= 3.6 iv) The fresh FRAP solution was prepared by mixing 25mL acetate buffer (pH= 3.6), o 2.5mL TPTZ, and 2.5mL FeCl3. 6 H2O and then warmed at 37 C for 5 minutes. Step II: Sample Preparation: 50 µL of sample solution was taken from above stock solution. Step III: Assay: 100 µL (0.1mL) of each of sample solution and 2990 µL (2.9mL) of FRAP solution were taken in test tubes to make a total volume upto 3 mL. The sample solutions were allowed to react with FRAP solution in the dark to form ferrous tripyridyltriazine complex for 30 minutes. Absorbance of the coloured product [ferrous tripyridyltriazine complex] was then taken at 593nm. The FRAP values were compared with trolox. Results were expressed in TE µM/mL. Step IV: Calculations: The FRAP value was determined by following equation: Y= 0.002x + 0.069 The results are reported in Table 4.16 and 4.17
3.7.5 Total Phenolic Contents Total phenolic contents of various fractions of plants were determined by the reported method [205]. Step I: Preparation of Reagents: i) 10% Na2CO3 was prepared by dissolving 1g Na2CO3 in 10mL of distilled water. ii) 2N Folin-Ciocalteu (FC) reagent Step II: Sample Preparation: 10µL sample solution was taken from stock solution. Step III: Assay:
The 10µL of sample was mixed with 2.8 mL of Na2CO3 (10%) and 0.1 mL of 2N Folin- Ciocalteu (FC) reagent and allowed to stand for 40 minutes. After 40 minutes, the absorbance was measured at 725nm by UV-visible spectrophotometer. Total phenolics were compared with standard (Gallic acid).All the results were expressed in GAE µg/mL.
Step IV: Calculations: The total phenolic contents (TPC) value was determined by using this equation: Y= 0.006x + 0.139 The results of both plants are reported in Table 4.16 and 4.17.
3.7.6 Ferric Thiocyanate (FTC) Assay Inhibition of linoleic acid peroxidation was assayed by thiocyanate method [206]. Step I: Preparation of Reagents: i) 0.02M Disodium hydrogen phosphate solution was prepared by dissolving 0.284g in 100mL of water. ii) 20mM ferrous chloride was prepared by dissolving 0.199g/ 100mL. iii) 3.5% HCl was prepared by dissolving 4.73mL/ 50mL. iv) Linoleic emulsion was prepared by dissolving 0.28 g of linoleic acid, 0.28 g of Tween-20 (as emulsifier) and 50.0 mL of 0.02M phosphate buffer (pH = 7) Step II: Sample Preparation: 500µL of sample solution was taken from above stock solution. Step III: Assay: 500µL of each sample solution was mixed with 2.5 mL of linoleic acid emulsion (pH 7.0) and 2.0 mL of phosphate buffer (pH 7.0). The reaction mixture was incubated at 40oC for 5 days. The mixture without sample was used as control. The 0.1 mL of incubated mixture was taken and mixed with 75% ethanol (5.0 mL), 30% ammonium thiocyanate (0.1 mL) and 0.1 mL ferrous chloride in 3.5% HCl and reaction mixture was allowed to stand at room temperature. Precisely 3 min. after addition of ferrous chloride to the reaction mixture, absorbance was recorded at 500 nm. Step IV: Calculations: The antioxidant activity was expressed as percentage inhibition of peroxidation (IP %) IP% = {1-(abs. of sample) / (abs. of control)} × 100]. The antioxidant activity of BHT was assayed for comparison as reference standard. The results are reported in Table 4.16.
3.8 ENZYME INHIBITION STUDY 3.8.1 In-vitro AChE inhibition assay The Acetylcholine esterase inhibitory (AChE) activity was measured by reported method with minor modification [207, 208]. Step I: Preparation of Reagents i) Prepared phosphate buffer of pH 7 and 8. ii) DTNB was prepared by dissolving 39.6mg of DTNB in 10ml of phosphate buffer of pH 7. iii) Acetylthiocholine iodide was prepared by dissolving 108.35mg of acetylthiocholine iodide in5mL distilled water. iv) 20µL enzyme (acetylthiocholine esterase) was taken from human blood and then added 18mL phosphate buffer of pH 8 for dilution. Step II: Samples Preparation Stock solution was made by adding 20mg of sample in 20mL of methanol. Then further dilutions such as 150µg/ mL, 100 µg/ mL, 50 µg/ mL and 30 µg/ mL were made by this stock solution. Step III: Assay 2.8 ml of phosphate buffer (0.1M, pH 7.8), 100 μl of erythrocytes (acetylcholine esterase), various amount of each extract/ sample solution of different concentrations and 100μl of DTNB were added in test tubes and incubated at 37°C for 15 min . After 15 minutes, 200μl acetylthiocholine (substrate) was added for initiation the reaction. After 30 minutes of incubation, the hydrolysis of acetylthiocholine was monitored at 412 nm. Galanthamine was used as positive control. All the reactions were carried out in triplicate. Step IV: Calculations The percentage inhibition was calculated as follows: % inhibition = [(E – S)/ E] × 100
Where; E is the absorbance of the enzyme without sample and S is the absorbance of enzyme with test sample. The results are recorded in Table 4.18 and 4.19
3.8.2 Protease Inhibition Assay/ Trypsin Inhibitory Activity Assay: Protease inhibition assay of all extracts and purified compounds was carried out by using the method of Jendinak et al with some modification [209, 210] using Nα- benzoyl- DL- arginine- para nitroanilide hydrochloride (BApNA) as a substrate. Step I: Reagent Preparation i) 2mg of trypsin was dissolved in 10mL of 1.0mM HCl. ii) 0.6mM BApNA was prepared by dissolving 20mg of BApNA in 1mL of DMSO. iii) Tris buffer of pH 7.5 was prepared by dissolving 12.1g of tris buffer in 100mL distilled water. iv) 30% acetic acid was prepared by dissolving 30mL glacial acetic acid in 70mL distilled water. Step II: Sample Preparation 100µL sample solution was prepared by dissolving 2mg in 1ml of methanol. Step III: Assay 0.3mL trypsin and 1mL inhibitor (0.1ml sample) was incubated for 15 minutes at room temperature. 50µl BApNA was added after 15 minutes and then added Tris buffer of pH 7.5 to make the final volume 2.5mL. The reaction mixture was incubated for 30 minutes (37˚C). The reaction was terminated/ quenched by adding 30% acetic acid (200µL) and absorbance was measured on UV/VIS spectrophotometer at 410nm due to the formation of para nitroaniline. Phenylmethanesulfonylfluoride (PMSF) was used as positive inhibitor. Step IV: Calculations Trypsin inhibitory potential was calculated by using the following formula: % Inhibition = {(Abs. B – Abs. T)/ Abs. B} × 100 Where ‘B’ is absorbance of blank while ‘T’ is the absorbance of test sample. The results of both plants are reported in Table 4.20
3.9 ANTIBACTERIA ACTIVITY: 3.9.1 STRAINS USED FOR ANTIBACTERIAL STUDY: Gram Positive Bacillus subtilis Staphylococcus aureus Gram Negative Escherichia coli Pseudomonas aeruginosa Shigella flexenari Salmonella typhi
3.9.2 Ager Well Diffusion Method: The antibacterial activity of different fractions of both plants was determined by ager well diffusion method [211, 212, 213]. The crude extract and various fraction solutions (3mg/ml of DMSO) of plants were added into their respective wells of size 6mm in diameter and other wells were supplemented with Imepinem (standard drug). DMSO was used as negative control. Plates were incubated at 37 ºC for 24 hrs for bacteria strains. The antibacterial activity was evaluated by measuring the diameter of the growth inhibition zones (mm) for the organisms and comparing to the standard drug. The results of both plants are tabulated in Table 4.21 and 4.22.
3.10 Hemolytic activity: Sharma and Powell et al method [214, 215] was used to determine the hemolytic activity of various fractions of plant. 3mL freshly obtained heparinized human blood was collected from volunteers after consent and counseling and bovine from the Department of Clinical Medicine and Surgery, University of Agriculture. Centrifuged the blood for 5 min at 1000xg, plasma was discarded and the cells were washed three times with 5 mL of chilled (4oC) sterile isotonic PBS (Phosphate-buffered saline) of pH 7.4. Erythrocytes were maintained 108 cells per mL for each assay. Each compound/ extract (100μL) was mixed with erythrocytes (108cells/mL) separately. Samples were incubated at 37oC for 35 minutes and agitated after 10 min. Immediately after incubation the samples were placed
on ice for 5 min then centrifuged for 5 min at 1000xg. Supernatant (100μL) were taken from each tube and diluted 10 time with chilled (4oC) PBS. Triton X-100 (0.1% v/v) was taken as positive control and phosphate buffer saline (PBS) was taken as negative control and pass through the same process. The absorbance was observed at 576 nm using μ Quant (Biotech, USA). The % RBCs lysis for each sample was calculated.
% Hemolysis = {(Sample (Abs) – Blank (Abs)) / Positive control (Abs)} x 100
Each sample was assayed in triplicate and mean values were calculated. The results are reported in Table 4.23.
3.11 Pharmacological Activities: Following pharmacological activities were performed. These activities were: Analgesic activity Anti-inflammatory activity Hepatoprotective activity
3.11.1 Animals: Swiss albino mice (30-35g) and rats (250-300g) of both sexes were used for study of anti- inflammatory, analgesic and hepatoprotective activities. These animals were obtained from College of Pharmacy, University of Punjab, Lahore Pakistan, were used for the study. The animals were housed in standard environmental conditions with natural light/ dark cycle at temperature 28 ±3 ˚C. All the experiments were performed in morning according to current guide lines for the care of the laboratory animals and the ethical guidelines for the investigation of experimental pain in conscious animals.
3.11.2 Anti-inflammatory Activity: Screening of anti-inflammatory activity of methanolic extracts of both plants was done in mice and rats and evaluating their response in time (sec) and volume of paw.
3.11.2.1 Carrageenan Induced Rat Paw Edema: Carrageenan induced paw edema in rats was used to examine the inflammation by using reported method [216]. Rats were divided into three groups of 5 rats each. Group 1 = Negative Control Group 2 = Crude methanolic extract Group 3 = Aspirin (Standard drug) Normal saline (10mL/Kg of body weight) was used to Group 1 (negative control). Group 2 and 3 were administered orally by 300mg/Kg of body weight methanolic extract and aspirin respectively. After 40 minutes of administration of above drugs to each group, 0.1 ml of carrageenan (1% w/v) was injected and observed the inflammatory edema in the right hind paw of sub-plantar region of each rat. The left hind paw served as reference (non- inflammation paw) for comparison. The left and right hind paw volume of control and test compound treated rats were measured by analogue vernier caliper for four hours after carrageenan administered. The results of the anti- inflammatory effects of the test compounds and aspirin are presented in Table 4.24. Percentage inhibition of edema is calculated by the following equation:
% Inhibition = {(Vc-Vt)/Vc} x100 Where ‘Vt’ and ‘Vc are the mean relative changes in the paw volume of the test and control respectively.
3.11.2.2 Formalin Mice Paw Edema: Formalin induced paw edema in mice was done by using reported method with some modification [217]. Mice were divided into three groups of 5 animals each. Group 1= Negative Control Group 2 = Crude Methanolic Extract Group 3 = Aspirin (Standard drug) The negative control group (Group 1) received 10mL/Kg body weight of normal saline whereas mice in Group 2 and 3 received methanolic extract and standard drug (Aspirin) respectively. After 30 minutes of administration, 20µL of 2% formalin was injected subcutaneously to a hind paw of each mouse. The time spent in licking and biting to a
hind paw in early phase (0- 10 min) and later phase (10- 30 min) were recorded. This time spent in licking and biting hind paw showed anti- inflammatory activity which was expressed as % inhibition and the results are reported in Table 4.25. The percentage inhibition was determined by using this formula: % Inhibition= {(A– B)/ A} × 100
Where ‘A’ represents control group while ‘B’ represents experimental group.
3.11.3 Analgesic Activity: Screening of analgesic activity of methanolic extracts of both medicinal plants was done in mice and evaluating their response in time (sec).
3.11.3.1 Hot Plate Test (Thermal Stimulus): Analgesic activity of mice was observed by placing the mice on a hot plate at 51± 1˚ C by using the reported method [218]. Mice were divided into three groups based on administered drugs having 5 mice in each group i.e., Group 1= Treated as negative control (fed with saline water) Group 2= feed with Crude Methanolic extract Group 3 = Treated as positive control, used standard drug (Aspirin) Group 1 received normal saline (10mL/Kg body weight). Group 2 and 3 were administered the 300mg/ Kg each dose of methanolic extract and Aspirin orally respectively. After 30min of treatment with extract and positive control, mice were placed on a hot plate at 51± 1˚ C then reaction time was recorded when the animals licked the hind paw or jumping at 30, 60, 90, 120 and 150 min and the results are shown in Table 4.26.
3.11.3.2 Tail Flick/ Immersion Test (Thermal Stimulus): This test was performed by using the reported method with some modification [219]. Mice were divided into three groups with each group containing 5 mice. Group I = Negative Control Group II = Crude methanolic extract
Group III = Aspirin (Standard drug) Control animals received 10mL/Kg body weight of normal saline orally while 300mg/Kg dose of methanolic extract and aspirin were also administered orally into Group 2 and 3 respectively. An area of tail, 2- 3cm in length, was marked and immersed in water bath at 51± 1˚ C. The withdrawal time of the tail from hot water (in seconds) was noted as the reaction time. The initial readings were taken immediately before administration of sample and standard drug and then taken readings at 30, 60, 90, 120 and 150 min. results are reported in Table 4.27.
3.11.3.3 Acetic Acid Induced Writhing (Chemical Stimulus): In this test, mice were used to check the analgesic activity by antinociceptive test [220]. 5 mice each were distributed into three groups i.e., Group 1 = Control Group 2 = Crude Methanolic extract Group 3 = Standard drug (Aspirin) Mice of Group 1 were treated orally with normal saline while Group 2 and 3 were administered by 300mg/kg methanolic extract and aspirin respectively. The animals were treated orally with sample drug and standard drug 30 minutes prior to the administration of CH3COOH (10mL/Kg of body weight). After administration of acetic acid, animals were placed in individual cages and number of abdominal constrictions was counted for 30 minutes. The abdominal constrictions is defined as a posture with the abdomen flattened, the back depressed and the hind- limbs extended [221].Reduction in the abdominal constrictions as shown in table 4.28 was evaluated for analgesic activity which was expressed as percentage inhibition. % Inhibition = % Inhibition= {(A– B)/ A} × 100
Where ‘A’ represents control group while ‘B’ represents experimental group.
3.11.4 Hepatoprotective Activity: Rats were used for hepatoprotective activity by using methanolic extracts of both plants and saw the role of these medicinal plants in liver diseases against Rifampicin and Isoniazid.
3.11.4.1 Chemicals: Silymarin, standard kits of serum glutamate oxaloacetate (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphate (ALP)and Total Bilirubin were purchased from Sigma- Aldrich Chemicals Co. (Gill Ingham, Dorset, UK) and other reagents and solvents (analytical grade) were purchased from Merck.
3.11.4.2 Experimental Protocol:[222, 223] The rats were divided into five groups of 5 animals each. Group 1= Normal group (control) that received only 1mL/Kg sterile saline orally. Group 2= Hepatotoxicity model (toxic control) that administered 50mg/Kg each isoniazid and rifampicin orally. Group 3= Silymarin (standard) that received 200mg/Kg silymarin suspension via oral route. The silymarin suspension was prepared by mixing the silymarin powder in distill water along with simultaneous does of 50mg/Kg isoniazid and rifampicin each orally.
Group 4 and 5= Experimental groups that received 300mg crude methanolic extract of R. triquetra and Z. oxyphylla along with isoniazid (50mg/Kg) and rifampicin (50mg/Kg) orally. Crude methanolic extract was prepared by dissolving the methanolic extract in distilled water [222].
3.11.4.3 Interval of Dose: Standard drug and extracts were injected orally after every 24 hour for 21 days according to the above protocol.
3.11.4.4 Blood Sampling: [224]
Blood samples were taken from the tail vein of each animal to evaluate liver functions before and after treatment and centrifuged for 10 minutes at 3000rpm to separate the serum that was used for different biochemical tests such Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), alkaline phosphatase (ALAP) and total bilirubin (TB) by using standard kits as shown in Table 4.29, 4.30, 4.31 and 4.32 [225, 226, 227]. a) Estimation of Aspartate aminotransferase (ASAT) activity [228]: Principle:
In this method AST catalyses the transfer of -NH2 group from L- Aspartate to 2- oxoglutarate to form oxaloacetate and L- glutamate. The oxaloacetate is converted into malate when NADH is reduced to NAD and the oxidation of NADH is measured at 340nm when the absorbance of NADH is decreased and proportional to AST activity.
ASAT L- Aspartate + α Ketoglutarate Oxaloacetate + L- Glutamate
MAD Oxaloacetate + NADH +H+ Malate + NAD
Procedure:
In this method, first the working reagent was prepared by pour the contents of bottle L 2
(starter reagent) into bottle L1 (enzyme reagent) and stored at 2- 8˚C for at least three weeks. The absorbance was checked at 340nm at room temperature 37˚C.
Table 3.1 KIT ASSAY Procedure for Assessing ASAT Levels: Sr. No Addition of Reagent T (37˚C)
1 Enzyme Reagent L1 0.8mL
Starter Reagent L2 0.2mL Incubate at the assay temperature for 1min and then add 2 Sample 0.1mL
‘T’ represents the test. Mixed well and measured the initial absorbance (A) after 1 minute and repeated the absorbance reading after every 1, 2 and 3 minutes. Then mean absorbance change per minute (∆A/min) was calculated by using the following formula:
ASAT (SGOT) activity in U/L (37˚C) = ∆A/min × 1746 b) Estimation of Alanine aminotransferase (ALAT) activity [229]: Principle:
ALAT/ SGPT catalyses the transfer of -NH2 group from L- Alanine to 2- oxoglutarate and form pyruvate and L- glutamate. In this reaction the pyruvate also produces L- lactate when react with NADH in the presence of lactate dehydrogenase and NADH also reduces to NAD. The oxidation of NADH is measured by decrease of absorbance at 340nm and is proportional to ALAT activity.
ALAT L- Alanine + α- Ketoglutarate Pyruvate + L- Glutamate
LDH Pyruvate + NADH + H Lactate + NAD
Procedure:
The working reagent was prepared by mixing the starter reagent bottle (L2) with enzyme reagent bottle (L1). This reagent is stable for 3 weeks and stored at 2- 8˚C. The absorbance was measured on UV/VIS spectrophotometer at 340nm at 37˚C.
Table 3.2: KIT ASSAY Procedure for Assessing ALAT Levels: Sr. No Addition of Reagent T (37˚C)
1 Enzyme Reagent L1 0.8mL
Starter Reagent L2 0.2mL Incubate at the assay temperature for 1min and then add 2 Sample 0.1mL
The mean absorbance change per minute (∆A/min) was calculated by this formula:
ASAT (SGOT) activity in U/L (37˚C) = ∆A/min × 1746 c) Estimation of Alkaline phosphatase (ALP) activity [230]: Principle: The p- nitrophenyl phosphate is hydrolysed into p- nitrophenol and phosphate by serum alkaline phosphatase in the presence of Mg2+ as oxidizing agent.
Alkaline Phosphatase p- Nitrophenylphosphate p- Nitrophenol + Phosphate
Procedure:
Working reagent was prepared by dissolving the substrate tablet (T1) in 15mL buffer reagent. The substrate is light and temperature sensitive so take adequate care especially after reconstitution. This reagent was stored for at least 15 days at 2- 8˚C. The absorbance was measured at 405nm at 37˚C.
Table 3.3: KIT ASSAY Procedure for Assessing ALP Levels: Sr. No Addition of Reagent T (37˚C)
1 Working reagent (T1+ buffer reagent) 0.5mL Incubate at the assay temperature for 1min and then add 2 Sample 0.01mL
Mean absorbance change per minute was calculated by using the formula:
Alkaline phosphatase (ALP) activity in U/L (37˚C) = ∆A/min × 2754 d) Estimation of Total Bilirubin Activity [231]: Principle: Bilirubin reacts with diazotized sulphanilic acid to form a coloured compound (pink) in acidic medium and the intensity of coloured compound is directly proportional to the amount of Bilirubin present in sample.
Bilirubin + Diazotized sulphanilic acid Azobilirubin compound (coloured compound) Procedure:
In this method, Total Bilirubin reagent (L1) and Total Nitrite reagent (L2) was used for the estimation of Bilirubin test. The absorbance was measured at 546nm on spectrophotometer at room temperature.
Table 3.4: KIT ASSAY Procedure for Assessing Total Bilirubin Levels: Sr. No Addition of Reagent B (37˚C) T (37˚C)
1 Total Bilirubin Reagent (L1) 1.0 mL 1.0 mL
2 Total Nitrite Reagent (L2) 0.1 mL 0.1 mL 3 Sample 0.05 mL
The sample and working reagent was mixed well and incubated for 10 minutes at room temperature. The absorbance of the test sample (Abs.T) was measured against their respective blank by using the following formula:
Total Bilirubin in mg/dl = Abs.T × 13
3.11.4.5 Histopathology of Liver: [232] After blood sampling and an hour after last dose administration, the animals were sacrificed and removed the liver under mild chloroform anesthesia and washed with distilled water and preserved in 10% formalin at room temperature. Then made a slide and observed the changes in histopathological characteristics and photographs were taken as shown in Fig. 4.32 to 4.42.
3.11.5 Statistical Analysis: [233] Statistics was applied by using student t- test. All the data was presented as mean ± SEM for five mice/ rats in each group. P >0.05 was considered statistically significant and these groups were analyzed by one way ANOVA.
RESULTS AND DISCUSSIONS
4.1 Structure Elucidation of Chemical Constituents of Rhamnus triquetra: 4.1.1 New Source Compounds from Rhamnus triquetra: Two known compounds were isolated first time from the Chloroform fraction of Rhamnus triquetra. 4.1.1.1 Structure Elucidation of Physcion (Compound no. 3):
Physcion was isolated as an orange red powder. Its molecular mass is 284.12 corresponding to the molecular formula C16H12O5 which indicates eleven degrees of unsaturation. The absorption band at 1630 cm-1 and 1675 cm-1in IR spectrum revealed the presence of chelated and non- chelated carbonyl groups, chelated hydroxyl group observed at 3440 cm-1 and aromatic carbon double bond indicated at 1575 cm-1. 1H- NMR spectrum of Compound no. 3 showed aromatic methyl proton at δ= 2.43 (s) and aromatic methoxy protons at δ= 3.92 (s) while the two protons in aromatic rings appeared at δ= 6.67 (d, J= 2.5) and δ= 7.36 (d, J= 2.5). Coupling constant of these aromatic protons showed that both are meta- orientated while the other aromatic signals at δ= 7.62 and at δ= 7.07 displayed on broad singlets. C- 1 and C- 8 showed chelated hydroxyl protons and chemical shift of these two protons appeared were at δ= 12.11 and δ= 12.31.
13 C- NMR of Compound no. 3 indicated 16 signals, one methyl (-CH3) was observed at
δ= 22.7, one methoxy (-OCH3) at δ= 56.7, four methines (-CH) were indicated at δ= 107.1, 120.1, 124.5 and 121.5 and ten quaternary carbons (-C) were observed at δ = 166.2, 162.0, 148.3, 164.0, 190.6, 182.6, 134.8, 114.1, 110.4 and 132.3 by DEPT experiments. Quinone carbonyl groups were indicated at δ= 190.6 and 182.6 while quaternary carbons of chelated hydroxyl groups were observed at δ= 166.2 and 164.0.
Compared the 1H- NMR and 13C- NMR of Compound on.3 with literature, it was concluded that the Compound no. 3 was Physcion.
Table 4.1: 13C and 1H- NMR (300 MHz) Spectral Data of Physcion: C. No Multiplicity (DEPT) 13C- NMR (δ) 1H- NMR (δ) J value (Hz) 1 C 166.2 12.11 (-OH) s 2 CH 107.1 6.67 d, J=2.5 3 C 162.0 - - 4 CH 120.1 7.36 d, J= 2.5 5 CH 124.5 7.62 br. s 6 C 148.3 - - 7 CH 121.5 7.07 br. s 8 C 164.0 12.31 (-OH) s 9 C 190.6 - - 10 C 182.6 - - 4a C 134.8 - - 8a C 114.1 - - 9a C 110.4 - - 10a C 132.3 - -
O- CH3 56.7 3.92 s
CH3 22.7 2.43 s
4.1.1.2 Structure Elucidation of Madagascin (Compound no. 4):
Compound no. 4, orange red powder was isolated from chloroform fraction through column chromatography, eluted by Hex: CHCl3 (40: 60). Its molecular mass is 338.35
corresponding to the molecular formula C20H18O5.IR spectrum of this compound showed that chelated hydroxyl group observed at 3410 cm-1, conjugated carbonyl group at 1630 cm -1 and aromatic ring observed at 1608 cm-1. 1H- NMR spectrum revealed that the chelated hydroxyl group appeared as two sharp singlet signals at δ= 12.11 and 12.31. C- 2 and C- 4 showed doublet at δ= 6.67 and 7.36 with coupling constant J= 2.5 and coupling constant of these protons showed that both protons are meta to each other while C- 5 and C- 7 showed broad singlet at δ= 7.07 and 7.09. Further more, protons of prenyl side chain observed at δ= 4.21 (d, J= 6.6, 1H), δ= 5.48 (d, J= 6.6, 1H), δ= 1.84 (br. s) and δ= 1.77 (br. s). Chemical shift of methylene protons of side chain showed at low field because side chain was attached with oxygen at C-3. 13 C- NMR of Compound no. 4 showed 20 signals, three methyl (-CH3), one methylene (-
CH2), five methines (-CH) and eleven quaternary carbons (-C) were observed. Signals of 13C- NMR indicated that the methyl groups of prenyl side chain were observed that δ=
25.8 and 18.3 and –CH2 at 68.14. On the basis of above evidences and also comparison the 1H- NMR and 13C- NMR signals of this compound with literature, it was concluded that the Compound no.4 was Madagascin.
Table 4.2: 13C and 1H- NMR (300 MHz) Spectral Data of Madagascin: C. No Multiplicity (DEPT) 13C- NMR (δ) 1H- NMR (δ) J value (Hz) 1 C 166.2 12.11 (-OH) s 2 CH 1071 6.67 d, J=2.5 3 C 162.0 4 CH 7.36 d, J= 2.5 5 CH 120.5 7.07 br. s 6 C 148.3 7 CH 121.5 7.09 br. s 8 C 164.0 12.31 (-OH) s 9 C 190.6 10 C 182.6
4a C 134.8 8a C 114.1 9a C 110.4 10a C 132.3
1’ CH2 68.14 4.21 d, j= 6.6 2’ CH 118 5.48 d, j= 6.6 3’ C 142.6
4’ CH3 25.8 1.84 br. s
5’ CH3 18.3 1.77 br. s
CH3 21.7 2.42 s
4.1.2 GC- MS Analysis of Chloroform Fraction of Rhamnus triquetra:
Fraction 1 was taken from column chromatography of CHCl3 fraction which was n- Hexane fraction, was subjected to GC- MS analysis because volatile constituents were present. Two components were identified by GC- MS and these two components were confirmed by matching with NIST dictionary. Fr. 4 was also subjected to column chromatography and eluted with Hex: CHCl3 (40:60) and oily fractions were eluted in start of the column and collected these vials on the bases of Rf values and also analyze by GC- MS analysis and about six components were identify by NIST dictionary. They are listed below in Table 4.3 and 4.4.
Table 4.3: GC- MS Analysis of Fr. 1 of Chloroform fraction of Rhamnus triquetra:
Sr. Compound Molecular Molecular Retention Area Similarity No Name Formula Weight Time % Index 1 Butanoic C6H12O2 116 3.399 56.89 94 Acid ß- Myrcene C10H16 136 6.492 12.18 93 2
Table 4.4: GC- MS Analysis of Fr. 4 of Chloroform fraction of Rhamnus triquetra:
Sr. Compound Name Molecular Molecular Retention Area Similarity No Formula Weight Time % Index Butanoic acid C6H12O2 116 3.393 0.38 92 1 N- Vinylimidazole C5H6N2 94 4.079 2.61 94 2 ß- Myrcene C10H16 136 6.493 0.70 94 3 2- phenylethyl ester C10H11ClO2 198 13.037 15.72 90 4 9- Octadecenoic acid C19H36O2 296 16.774 16.76 88 5 1,2- C24H38O4 390 19.403 14.37 96 6 Benzenedicarboxylic acid
4.2 Structure Elucidation of Chemical Constituents of Zizyphus oxyphylla: 4.2.1 New Source Compounds from Zizyphus oxyphylla: 4.2.1.1 Structure Elucidation of ß- Sitosterol (Compound no. 5):
ß- Sitosterol The Compound no. 5 as white crystals was isolated from chloroform fraction, subjecting to column chromatography eluting with Hex: CHCl3 (80:20). The molecular mass of ß-
Sitosterol is 415.4 deduced the formula C29H51O. In IR spectrum, hydroxyl group (-OH)
was observed at 3420 cm-1, double bond was observed at 3050 cm-1,1653 cm-1 and 800 -1 -1 -1 cm and –CH2 and –CH was observed at 2865 cm and 3050 cm . The 1H- NMR spectrum showed that six methyl protons were present i.e., one primary at δ= 0.88, three secondary at δ= 0.93, 0.84 and 0.75 and two were tertiary, observed at δ= 0.69 and 1.08 while signal at δ= 5.23 for the olefinic proton and oxymethine proton at C- 3 resonated at δ= 3.18. 13C- NMR spectral data showed that 29 carbon atoms are present i.e., six -CH3 groups, eleven - CH2 (methylene), nine – CH (methine) and three –C (quaternary carbon atoms). The olefinic carbons showed signals at δ= 141.5 and 120.9 while oxymethine carbon was observed at 75.1 and the methyl carbons resonated at δ= 19.7, 19.7, 19.3, 18.3, 11.4 and 11.4. On the basis of above evidences and also comparison with literature, it was concluded that the Compound no. 5 was ß- Sitosterol. Table 4.5: 13C and 1H- NMR (300MHz) Spectral Data of ß- Sitosterol: C. No Multiplicity (DEPT) 13C- NMR (δ) 1H- NMR (δ) J value (Hz)
1 CH2 37.2 - -
2 CH2 - - - 3 CH 75.1 3.18 m
4 CH2 42.4 - - 5 C 141.5 - - 6 CH 120.9 5.23 m
7 CH2 31.1 - - 8 CH 31.0 - - 9 CH - - - 10 C 35.6 - -
11 CH2 21.1 - -
12 CH2 - - - 13 C 42.5 - - 14 CH - - -
15 CH2 24.3 - -
16 CH2 27.4 - -
17 CH 55.7 - -
18 CH3 11.4 0.69 s
19 CH3 19.7 1.08 s 20 CH - - -
21 CH3 19.3 0.88 d, J= 6.3
22 CH2 36.0 - -
23 CH2 30.3 - - 24 CH 46.8 - - 25 CH 29.3 - -
26 CH3 19.7 0.93 m
27 CH3 18.3 0.75 m
28 CH2 23.2 - -
29 CH3 11.4 0.84 m
4.2.1.2 Structure Elucidation of Betulinic acid (Compound no. 6):
The Compound no. 6 as white powder was isolated from chloroform fraction as Hex:
CHCl3 (40:60) was used as mobile phase. Molecular mass of Betulinic acid is 456.36
deduced the molecular formula C30H48O3. In IR spectrum, hydroxyl group (-OH) was observed at 3610 cm-1, double bond was observed at 1610 cm-1 , carbonyl group was indicated at 1705 cm-1. The 1H- NMR spectrum showed that three proton integration having six sharp singlets were indicated at δ= 1.23, 0.82, 0.98, 0.96, 0.91 and 1.67. Olefinic methylene was indicated at δ= 4.71 and 4.59 and hydroxyl group at δ= 3.66 as multiplet. 13C- NMR spectral data disclosed the presence of 30 carbon signals i.e., six methyl
(-CH3), eleven methylene (- CH2), six methine (- CH) and six quaternary carbon atoms (-C) and one carboxylic group. 13C- NMR of six sharp signals of methyl groups were at δ= 28.46, 14.4, 16.5, 16.1, 14.67 and 19.36 while the olefinic methylene at C- 29 was indicated at δ= 109.8, hydroxyl group (-OH) at C- 3 was observed at δ= 77.4 and quaternary carbon signal at C- 28 was observed at downfield value δ= 179.3, indicated the presence of carboxylic acid. So with the help of 1H- NMR and 13C- NMR spectral data and also comparison with literature, it was concluded that the Compound no. 6 was betulinic acid (3ß- hydroxylup- 20(29)-en-28-oic acid).
Table 4.6: 13C and 1H- NMR (300MHz) Spectral Data of Betulinic Acid: C. No Multiplicity (DEPT) 13C- NMR (δ) 1H- NMR (δ) J value (Hz)
1 CH2 37.5 - -
2 CH2 25.0 - - 3 CH 77.4 3.66 m 4 C 37.8 - - 5 CH 53.3 - -
6 CH2 - - -
7 CH2 35.0 - - 8 C 41.1 - - 9 CH - - - 10 C 36.2 - -
11 CH2 23.1 - -
12 CH2 24.6 - - 13 CH 37.5 - - 14 C 40.6 - -
15 CH2 - - -
16 CH2 37.4 - - 17 C - - - 18 CH 47.3 19 CH 47.8 2.34 dd, J= 10.3, 5.2 20 C 28.3 - -
21 CH2 - - -
22 CH2 150.28 - -
23 CH3 28.46 1.23 s
24 CH3 14.4 0.82 s
25 CH3 16.5 0.98 s
26 CH3 16.1 0.96 s
27 CH3 14.67 0.91 s 28 COOH 179.3 - -
29(a,b) CH2 109.8 4.71, 4.59 s, s
30 CH3 19.36 1.67 s
4.2.2 GC- MS Analysis of Fr. 1 of Chloroform fraction of Zizyphus oxyphylla:
Fraction 1 of CHCl3 fraction of Zizyphus oxyphylla which was n- Hexane fraction (100%) was subjected to GC- MS analysis because volatile/ oily constituents were present. Three components were isolated by GC- MS analysis and these three components were identified by using NIST dictionary.
Table 4.7: GC- MS Analysis of Fr. 1 of Chloroform fraction of Zizyphus oxyphylla
Sr. Compound Name Molecular Molecular Retention Area Similarity No Formula Weight Time % Index 1 Butanoic Acid C6H12O2 116 3.4 7.60 93
2 ß- Myrcene C10H16 136 6.497 1.75 93
3 1,2- Benzenedicarboxylic C24H38O4 390 19.414 86.86 96 acid
4.2.3 GC- MS Analysis of Essential oil through Water Distillation: The essential oil was extracted through water distillation by using solvent extraction and subjected to GC- MS analysis for evaluation of its volatile components. The chromatogram showed that about 15 components were present and all were identified by NIST dictionary which are listed below (Table 4.8).
Table 4.8: GC- MS analysis of Essential Oil of Zizyphus oxyphylla:
Sr. Compound Name Molecular Molecular Retention Area Similarity No Formula Weight time % Index
1 Ethane, 1, 1- diethoxy C6H14O2 118 4.325 45.84 97 sec- Butyl Methyl 2 C H O 100 5.041 1.44 94 ketone 6 12
3 Amyl ethyl ether C7H16O 116 5.259 4.96 91 Methyl benzene/ 4 C H 92 5.481 1.02 92 Antisal 7 8
5 tert- butyl Ethyl ketone C7H14O 114 6.529 0.73 97
6 Ethyl isopropyl ether C5H12O 88 7.048 31.42 85 3,3- Dimethyl- 2- 7 C H O 114 7.109 2.93 95 pentanone 7 14
8 3- Methyl- 2- hexanone C7H14O 114 7.947 0.72 93
9 p- Xylene/ p- Xylol C8H10 106 8.624 1.13 96
10 m- Ethyl toluene C9H12 120 11.004 0.86 93
11 p- Cymene C10H14 134 12.289 2.89 93 4, 7- dimethoxy- 5- (2- 12 C H O 222 19.376 1.04 82 propenyl)-benzene 12 14 4
13 Hexatriacontane C36H74 506 21.146 2.85 94
4.3 Fluorescence Analysis: The fluorescence analysis of powder of R. triquetra and Z. oxyphylla is given in Table 4.9 and 4.10. Table 4.9: Fluorescence Analysis of Rhamnus triquetra: Short Wavelength Long Wavelength Sr. No Reagent Ordinary Light (254nm) (366nm)
1 66% H2SO4 Light brown Bright green Light brown 2 50% H2SO4 Light brown Light green Light brown 50% HNO Orange Bright green Dark brown 3 3 Yellow 4 5% FeCl3 Dark brown Light green Dark brown 5 5% NaOH Dark brown Light green Dark grey 6 Conc. KOH Light brown Light green Light grey 7 Aniline Reddish brown Dark green Dark red 8 Water Colourless Light green Light grey 9 Chloroform Colourless Light green Light brown 10 Powder Light Brown Colourless Dark brown
Table 4.10: Fluorescence Analysis of Zizyphus oxyphylla: Short Wavelength Long Wavelength Sr. No Reagent Ordinary Light (254nm) (366nm)
1 66% H2SO4 Light brown Light green Brown 2 50% H2SO4 Light brown Light green Light brown 3 50% HNO3 Dark brown Dark green Dark grey 4 5% FeCl3 Pale yellow Dark green Brown 5 5% NaOH Yellow Light green Light brown 6 Conc. KOH Pale yellow Dull green Dark grey 7 Aniline Dark red Dark green Dark brown 8 Water Colourless Light green Colourless 9 Chloroform Colourless Light green Colourless 10 Powder Light Brown Light green Colourless
The powder of both plants was study under ordinary light and UV light of both short (254nm) and long (366nm) wavelength. The formation of different colour showed the presence of specific compound/ constituent and use as an initial diagnostic tool of the test sample/ drug. After treating the both plants with various reagents such as 66% and 50%
H2SO4, HNO3, FeCl3, NaOH, Conc. KOH, Aniline etc., different shades of brown and green colour were observed under day light and UV- light at short and long wavelength [234].
4.4 Determination of Physicochemical Parameters: The results of physicochemical parameters of both plants are presented in Table 4.11.
Table 4.11: Proximate/ Physicochemical Parameters of Rhamnus triquetra and Zizyphus oxyphylla: Observations Observations of Sr. No Parameters of Rhamnus Zizyphus triquetra oxyphylla 1 Loss on drying 11.54% 12.09% 2 Total ash value 7.98% 7.94% 3 Water soluble value 1.04% 1.46% 4 Acid insoluble value 0.34% 0.67% 5 n- Hexane soluble extractive value 0.79% 0.62% 6 Chloroform soluble extractive value 4.96% 3.68% 7 Ethanol soluble extractive value 3.44% 2.91%
The physical constant evaluation of the drug is an important parameter in the detecting adulteration or improper handling of drugs. The loss on drying (11.54% and 12.09%) of Rhamnus triquetra and Zizyphus oxyphylla is not too much so it could inhibit or discourage the bacteria, fungi or yeast growth. The ash value determination is also important. The total ash value evaluates the presence or absence of foreign inorganic matter such as silica or metallic salts [235]. Total ash value of powder of R. triquetra was 7.98% and Z. oxyphylla was 7.94%, water soluble ash was 1.04% and 1.46% of both plants and acid insoluble ash value was 0.37% and 0.64% respectively. Extractive values are used for evaluating the quality and purity of samples or drugs of which cannot be
readily estimated by other means. Extractive yield of powder of both plants were higher
in CHCl3 (4.96% and 3.68%) followed by ethanol (3.44% and 2.91%) and then n- Hexane (0.79% and 0.62%). It showed that the polar constituents are more present than non- polar constituents.
4.5 Phytochemical Screening of Rhamnus triquetra and Zizyphus oxyphylla: Table 4.12: Phytochemical Constituents of Various Extracts of Rhamnus triquetra: Chlorofo Ethyl Methano n-Hexane n-Butanol Aqueous Chemical tests rm acetate l extract extract extract extract extract extract
Tests for
Alkaloids ++ -- + + ++ -- A. Dragendroff’s ++ _ + + ++ + test
B. Hager’s test
Tests for Flavonoids +++ + ++ +++ ++ + A. Lead acetate test ++ -- ++ +++ ++ + B. Alkaline reagent test Test for Tannins A. Gelatin test +++ _ -- + + -- B. Ferric Chloride +++ -- -- + + -- test Test for Carbohydrates ++ -- -- + + ++ A. Fehling’s test ++ -- -- + -- + B. Benedict’s test Test for Terpenoids ++ + ++ +++ ++ + A. Salkowski test ++ + ++ +++ + -- B. Cerric Sulphate test Test for Saponins + -- -- + + _ A. Frothing test Test for Phenolics ++ -- ++ +++ + + A. Ferric Chloride test Test for Cardiac glycosides + -- -- + -- _ A. Legal’s test + -- -- + -- _ B. Kellar Killani test
Table 4.13: Phytochemical Constituents of Various Extracts of Zizyphus oxyphylla: Chemical tests Methanol n-Hexane Chloroform Ethyl n-Butanol Aqueous extract extract extract acetate extract extract extract
Tests for Alkaloids ++ -- ++ +++ +++ + Dregandroff’’s test ++ _ ++ +++ +++ + A. Hager’s test
Tests for Flavonoids +++ _ + ++ + _ A. Lead acetate test ++ -- + ++ + _ B. Alkaline reagent test
Test for Tannins A. Gelatin test +++ _ -- + + -- B. Ferric Chloride test +++ -- -- + + -- Test for Carbohydrates A. Fehling’s test ++ + -- + + +++ B. Benedict’s test ++ + -- + + +++ Test for Terpenoids A. Salkowski test +++ + ++ +++ +++ + B. Cerric Sulphate test +++ + ++ +++ +++ + Test for Saponins A. Frothing test + -- + ++ + -- Test for Phenolics A. Ferric Chloride test ++ -- ++ ++ +++ -- Test for Cardiac glycosides A. Legal’s test + -- -- + + _ B. Kellar Killani test + -- -- + -- _
The phytochemical screening of Rhamnus triquetra was done on all fractions and it was
showed that the chloroform (CHCl3), ethyl acetate (EtOAc) and n- butanol (BuOH) fractions contain the terpenoids, flavonoids and phenolic contents more as compared to alkaloids, tannins, cardiac glycosides and saponins (Table 4.12). These constituents were also present in methanolic extract while the sugar contents were more present in aqueous
extract and absent in polar fractions such as CHCl3, EtOAc and BuOH. In Zizyphus oxyphylla, flavonoids, alkaloids, terpenoids and phenolics were rich in polar
fractions such as CHCl3, EtOAc and BuOH and these were absent in non polar fraction (n- hexane) and aqueous extract (Table 4.13) while the sugar/ carbohydrates were mostly present in aqueous fraction as compared to other polar fractions.
4.6 Antioxidant Activities: The antioxidant activities/ potential of both plant (Rhamnus triquetra and Zizyphus oxyphylla) extracts were summarized in Table 4.14, 4.15, 4.16 and 4.17.
Table 4.14: Percentage Inhibition of DPPH and IC50 values of Rhamnus triquetra:
Concentration in % Scavenging of DPPH IC50 of DPPH Sr. No Extracts/ Fractions assay (µg/ml) radical Assay (µg/ml) Crude Methanolic 250 89.01±0.89 extract 120 62.89±0.21 1 70.26±0.27 60 52.1±0.86 30 40.47±0.61 n-Hexane fraction 1000 71.43±0.49 500 60.28±0.58 2 182.99±1.48 250 42.36±0.71 120 33.29±0.62 Chloroform fraction 250 87.49±0.31 120 71.24±0.29 3 60.09±0.54 60 54.72±0.10 30 41.09±0.37 EtOAc fraction 60 92.01±0.21 4 30 89.72±0.68 7.59±0.65 15 51.2±0.89 n-Butanol fraction 60 80.43±0.73 5 30 62.69±0.91 37.98±1.35 15 48.71±0.68 Remaining aqueous 500 79.93±0.34 6 extract 250 60.47±0.90 94.73±0.74 120 45.21±0.39 BHT 60 91.49±0.13 7 30 75.54±0.07 12.10±0.29 15 42.62±0.04
Table 4.15: Percentage Inhibition of DPPH and IC50 values of Zizyphus oxyphylla:
Extracts/ Concentration in % Scavenging of IC50 of DPPH Sr.No Fractions assay (µg/ml) DPPH radical Assay(µg/ml) Crude Methanolic 250 81.15±0.98 68.61±0.48 extract 120 62.73±0.17 1 60 55.02±0.68 30 38.07±0.16 n-Hexane fraction 1000 53.87±0.63 282.5±1.89 500 49.14±0.78 2 250 31.85±0.52 120 17.80±0.41
Chloroform 120 95.01±0.37 13.20±0.27 3 fraction 60 76.01±0.21 30 53.72±0.14 Ethyl acetate 60 73.39±0.65 38.98±0.54 4 fraction 30 53.71±0.87 15 39.94±0.12 n-Butanol 60 79.03±0.79 29.79±1.30 5 fraction 30 63.74±0.95 15 36.93±0.86 Remaining 500 67.83±0.43 455.21±0.47 6 aqueous fraction 250 41.17.±0.97 120 21.91±0.31 BHT 60 91.49±0.13 12.10±0.29 7 30 75.54±0.07 15 42.62±0.04
Table 4.16: Total Phenolics, FRAP values, Lipid Peroxidation and Total Antioxidant Activity of Rhamnus triquetra: Total Inhibition of Total Sr. Phenolics FRAP Value Sample lipid antioxidant No (GAE mg/g of TE (µM/ml) peroxidation activity extract) Crude Methanolic 114.17±1.63 2156.7±0.91 57.12±0.73 1.740±0.01 1 extract 2 n-Hexane fraction 9.3±0.51 266.5±0.46 46.07±0.51 0.100±0.026 3 Chloroform fraction 80.74±1.29 1565.2±0.94 53.39±0.56 1.359±0.43 Ethyl acetate 121.5±1.10 2137.2±0.58 61.94±1.17 1.840±0.08 4 fraction 5 n-Butanol fraction 98.17±1.54 1709.3±0.19 49.68±0.92 1.537±0.002 Remaining aqueous 28.83±1.2 357.5±0.57 54.21±0.88 1.183±0.016 6 extract 7 BHT 62.91±0.16 1.2186±0.015 8 Blank 1.15 2.30
Table 4.17: FRAP values, Total Antioxidant Activity and Total Phenolic Contents of Zizyphus oxyphylla:
FRAP Value Fractions/ Total antioxidant Total Phenolics (GAE mg/g Sr. No TE (µM/ml) Extracts activity ±S.E.M a) of extract) ±S.E.M a) ±S.E.M a) Crude 1 Methanolic 150.53±0.94 1.223±0.04 86.67±1.45 extract n-Hexane 2 21.5±0.19 0.187±0.02 14.3±0.31 fraction Chloroform 3 339.5±0.57 1.723±0.34 142.65±1.20 fraction Ethyl acetate 4 296.01±0.85 1.406±0.41 55.06±1.45 fraction n-Butanol 76.19±1.32 5 125±0.49 1.523±0.07 fraction Remaining 6 107.3±0.64 1.138±0.013 25.33±1.29 aqueous fraction 7 BHT 1.2186±0.015 8 Blank 2.30 1.15
The DPPH scavenging activity of the methanolic extract and fractions of plants were studied due to its hydrogen donating ability/ radical scavenging activity at room temperature. When DPPH solution was mixed with sample/ substance, it donate a hydrogen atom and give rise to reduce form of 1,1-diphenyl-2-picryl hydrazine (yellow colour) from 1,1-diphenyl-2-picryl hydrazyl (violet colour). The change of colour from purple to yellow and decrease of absorbance was due to the reaction between the antioxidants and radicals and the absorbance was measured at wavelength 517 nm and the results of both plants were shown in Table 4.14 and 4.15. Radical scavenging activity was increased with increasing the percentage of free radical inhibition [236]. Figure 4.1 indicated that the ethyl acetate fraction of Rhamnus triquetra showed the highest DPPH scavenging activity of 92.01± 0.21 of concentration 60µg/mL when compared with butylated hydroxyl toluene (BHT), it also showed highest percentage free radical inhibition (91.49±0.13) but less that EtOAc fraction while n- Hexane fraction revealed the lowest scavenging value of 71.43± 0.49 at a concentration 1000µg/mL. The present
study showed that the methanolic extract and ethyl acetate (polar fraction) indicated the similar or higher antioxidant activities as compared to BHT (standard) and this is due to the presence of bioactive constituents such as flavonoids, polyphenols including tannins [237].
100
80
60
40
20
% inhibition of DPPH RadicalDPPHof % inhibition 0 0 200 400 600 800 1000 1200 Concentration (µg/ml)
n-Hexane fr. Chloroform fr. Ethyl acetate fr. n- Butanol fr. Aqueous fr. Methanolic fr. BHT
Figure 4.1: Free Radical Scavenging Activity of Rhamnus triquetra
The IC50 value of crude methanolic extract and fractions was calculated from the curves plotted. IC50 is the concentration of fraction causing 50 percent inhibition of absorbance and its lower value reflects the better antioxidant activity of the fraction. The IC50 value of ethyl acetate fraction was lower (7.59 ± 0.65 µg/ml) relative to butylated hydroxytoluene (BHT) having IC50 value of 12.10 ± 0.29 µg/ml. Ethyl acetate fraction showed highest antioxidant activity with low IC50 (7.59± 0.65) as compared to other fractions and the IC50 values of other fractions were decreased in this order i.e., n-butanol fraction (37.98± 1.35) > chloroform fraction (60.09± 0.54) > crude methanolic extract (70.26± 0.27) > aqueous fraction (94.73± 0.74) >n-Hexane fraction (182.99± 1.48), respectively as shown in Figure 4.2.
182.99 200 180 160 140 120 94.73 100 70.26 60.09 80 60 37.98 12.1 of DPPH Assay DPPH of 40 7.59
50 20
IC 0
Fractions
Figure 4.2: IC50 Values of Various Fractions of Rhamnus triquetra
Chloroform fraction of plant Zizyphus oxyphylla exhibited highest % inhibition (95.01± 0.37) of DPPH radical at concentration 120μg/ ml as compared to other fractions while the other fractions were in this order i.e; methanolic extract ˃ n- butanol fraction ˃ ethyl acetate fraction ˃ remaining aqueous fraction ˃ n- Hexane fraction. IC50 value of chloroform fraction showed lowest value i.e.13.07±0.27 µg/mL as compared to a reference standard i.e. BHT (IC50 = 12.01± 0.29μg/ml). The n- butanol fraction also showed good value of IC50 = 29.79± 1.30μg/ml), ethyl acetate showed moderate value of
IC50 = 38.98± 0.54 μg/ml and n- hexane and aqueous fractions indicated non- significant
IC50 value i.e. 282.5± 1.89 and 455.21± 0.47 respectively in Table 4.15 (Figure 4.4).
100 90 80 70 60 50 40 30 20
% Inhibition of DPPH Radical DPPH of % Inhibition 10 0 0 200 400 600 800 1000 1200
Concentration (µg/ml) Hex. Fr. Chloroform fr. EtOAc fr. BuOH fr. Aq. fr. Methanolic fr. BHT
Figure 4.3: Free Radical Scavenging Activity of Zizyphus oxyphylla
600 455.21 500 400 282.5 300 200 68.61 100 13.2 38.98 29.79 12.1
of DPPH Radical DPPH of 0
50 IC
Fractions
Figure 4.4: IC50 Values of Various Fractions of Zizyphus oxyphylla Phosphomolybednum method was used to determine the total antioxidant activity of plants Rhamnus triquetra and Zizyphus oxyphylla. In this method, molybdenum (VI) reduces to molybdenum (V) in various fractions of plant and it was measured by spectrophotometer at wavelength of 695nm due to the formation of a green phosphate Mo (V) complex at acidic pH [238]. BHT was used as a reference standard to compare antioxidant activities of different fractions of plants. In Figure 4.5, Rhamnus triquetra
showed that the EtOAc fraction displayed highest antioxidant activity i.e. 1.840± 0.08 followed by slightly less potent methanolic fraction (1.740± 0.01), BuOH fraction
(1.537± 0.002), CHCl3 fraction (1.359± 0.43) and remaining aqueous fraction (1.183± 0.016) while n- Hexane fraction showed the lowest antioxidant capacity/ activity i.e. 0.100± 0.026 (Table 4.16).
2.5 1.84 1.74 2 1.537 1.359 1.5 1.183 1.2186
1 Absorbance 0.5 0.1 0 Methanolic n-Hexane fr. Chloroform Ethyl n-Butanol fr. Aqueous fr. BHT extract fr. acetate fr. Fractions
Figure 4.5: Total Antioxidant Activity of Rhamnus triquetra The results of Zizyphus oxyphylla in Table 4.17 (Figure 4.6) showed that the chloroform showed highest antioxidant activity (1.723± 0.34) and the other fractions were decreased in the following order: BuOH fraction (1.523± 0.07)> EtOAc fraction (1.406± 0.41)> methanolic fraction (1.223± 0.04)> aqueous fraction (1.138± 0.013)>n- Hexane fraction (0.187± 0.02).
2 1.723 1.523 1.8 1.406 1.6 1.22 1.4 1.138 1.218 1.2 1 0.8 0.6 Absorbance 0.4 0.187 0.2 0 Crude n-Hexane Chloroform Ethyl n-Butanol Remaining BHT Methanolic soluble soluble acetate soluble aqueous extract fraction fraction soluble fraction fraction fraction Fractions Figure 4.6: Total Antioxidant Activity of Zizyphus oxyphylla
The FRAP (ferric reducing antioxidant power) assay involved redox potential of the antioxidants. The FRAP assay is a simple, useful and reproducible method used to measure the reducing ability of the antioxidant in the reaction medium [239]. In this assay, ferric-tripyridyltriazine (Fe-III-TPTZ) complex is reduced to the ferrous (Fe-II) form at low pH and produce an intense blue colour and this change of colour is measured by UV/ VIS spectrophotometer at 593nm [240]. A colour development means that an antioxidant (reductant) is present. The results in Table 4.16 (Figure 4.7) of Rhamnus triquetra showed that ethyl acetate (EtOAc) fraction was found to be significantly higher value (2137.2± 0.58 μM/ml) than other fractions which followed the order of crude methanolic extract (2116.7 ± 0.91 μM/ml) > n-butanol fraction (1709.3 ± 0.19 μM/ml) > chloroform fraction (1565.2 ± 0.94 μM/ml) while the aqueous fraction (357.5± 0.57 μM/ml) and n-Hexane fraction (266.5± 0.46 μM/ml) showed lowest FRAP values respectively.
2500 2116.7 2137.2 1709.3 2000 1565.2
1500
1000 266.5 357.5
500 38.63 FRAP value TE(µM/ml) value FRAP 0 Methanolic n-Hexane Chloroform Ethyl n-Butanol Aqueous fr. Blank extract fr. fr. acetate fr. fr. Fractions
Figure 4.7: FRAP Values of Different Fractions of Rhamnus triquetra
In Zizyphus oxyphylla plant, Table 4.17 revealed that CHCl3 showed highest FRAP value (339.5±0.57 µM/mL), while EtOAc fraction showed good FRAP value (296.01± 0.85 µM/mL) and methanolic extract and BuOH fraction showed the moderate values (150.53±0.94 µM/mL and 125± 0.49 µM/mL) respectively. Aqueous fraction and n- Hexane fraction showed very less values i.e. 107.3±0.64 µM/mL and 21.50±0.19
µM/mL. High FRAP values indicated that the polar fractions such as CHCl3, EtOAc, BuOH and methanol contain the large number of flavonoids and phenolic contents (Figure 4.8).
400 339.5 296.01 350 300 250 150.53 200 125 150 107.3 100 50 21.5 3.63
FRAP Value TE(µM/ml) ValueFRAP 0 Crude n-Hexane Chloroform Ethyl n-Butanol Remaining Blank Methanolic soluble soluble acetate soluble aqueous extract fraction fraction soluble fraction fraction fraction
Fractions Figure 4.8: FRAP Values of Different Fractions of Zizyphus oxyphylla
Flavonoids and Phenolic contents/compounds show antioxidant action/activity on human health and fitness which is due to their redox properties, which make them to act as hydrogen donators, reducing agents, and singlet oxygen quenchers [241]. These antioxidants also act as metal-chelating process. Similarly, these phenolic compounds show different biological activities as anti inflammatory, antibacterial, anti-viral, anticarcinogenic, anti-allergic, immune- stimulating agents and estrogenic [242]. FC (Folin- Ciocalteau) reagent was used to determine the total polyphenols in samples. This reagent oxidizes the phenolate ions resulted in the production of complex molybdenum- tungsten (blue) which can be detected at 725nm by using spectrophotometer [242]. In present results as shown in Figure 4.9 (Rhamnus triquetra), ethyl acetate fraction possessed the highest quantity of total phenolic contents (121.5 ± 1.10 mg/g), followed by methanolic extract (114.17 ± 1.63 mg/g), n- Butanol fraction (98.17 ± 1.54 mg/g), ethyl acetate fraction (80.74 ± 1.29 mg/g), aqueous fraction (28.83 ± 1.2 mg/g) while n- Hexane fraction exhibited the lowest total phenolic contents (9.3± 0.51 mg/g) (Table 4.16).
160 140 114.17 121.5 120 98.17 100 80.74 80 60 40 28.83 19.3 16.49
20 Total Phenolics (GAE mg/g) (GAE Phenolics Total 0 Methanolic n-Hexane fr. Chloroform Ethyl n-Butanol fr. Aqueous fr. Blank extract fr. acetate fr. Fractions
Figure 4.9: Total Phenolic Contents of Different Fractions of Rhamnus triquetra
The results of Zizyphus oxyphylla in Table 4.17 revealed that the CHCl3 fraction showed the highest amount of phenolic contents (142.65± 1.20 mg/g), while the other fractions were in this order i.e. methanolic extract (86.67± 1.45 mg/g) ˃ BuOH fraction (76.19±
1.32 mg/g) ˃ EtOAc fraction (55.06± 1.45 mg/g) ˃ aqueous fraction (25.33± 1.29 mg/g) and the n- hexane fraction showed the lowest value (14.30± 0.31 mg/g) (Figure 4.10).
180 160 142.65 140 120 100 86.67 76.19 80 55.06 60 40 25.33 14.3 16.49
Total Phenolics (GAE mg/g) (GAE Phenolics Total 20 0 Crude n-Hexane Chloroform Ethyl acetate n-Butanol Remaining Blank Methanolic soluble soluble soluble soluble aqueous extract fraction fraction fraction fraction fraction Fractions
Figure 4.10: Total Phenolic Contents of Different Fractions of Zizyphus oxyphylla
The ferric thiocyanate (FTC) method was used to measure the amount of peroxidation at the early stage of linoleic acid emulsion during incubation. In this method ferric chloride was formed when peroxide reacts with ferrous chloride and this ferric chloride then reacts with ammonium thiocyanate to produce ferric thiocyanate (a reddish pigment). Absorbance of low values calculated through the FTC method indicated the high antioxidant activity [243]. The results revealed that ethyl acetate (EtOAc) fraction rendered the maximum inhibition of lipid peroxidation (61.94 ± 1.17) when compared with standard (BHT) (62.91 ± 0.16) and n-Hexane fraction showed minimum value (46.07 ± 0.51), so the other fractions of this plant (Rhamnus triquetra) were in this order: crude methanolic fraction > aqueous fraction > chloroform fraction > n- butanol fraction (Table 4.16 and Figure 4.11).
70 61.94 62.91 57.12 60 53.39 54.21 49.68 50 46.07
40
30
20
10
0 Inhibiition peroxidation :ipid of Inhibiition Methanolic n-Hexane fr. Chloroform Ethyl n-Butanol fr. Aqueous fr. BHT extract fr. acetate fr. Fractions
Figure 4.11: Inhibition of Lipid Peroxidation of Different Fractions of Rhamnus triquetra 4.7 Enzyme Inhibition: Enzymes play a vital role in the control of many physiological functions of the cells. Several diseases are induced within the living system by over activity of different enzymes. By keeping in view, the enzyme inhibition of extracts were evaluated against acetylthiocholine esterase and protease inhibition. 4.7.1 Acetylthiocholine Esterase of Rhamnus triquetra and Zizyphus oxyphylla: Acetylthiocholine esterase of both plants was summarized in Table 4.18 and 4.19.
Table 4.18: Acetylthiocholine Esterase of Rhamnus triquetra: Sr. AChE inhibition Sample Concentration AChE IC50 (mg/mL) No (%) Methanolic extract 150 67.8±1.07 100 61.51± 1.29 74.85 ±0.73 1 50 47.93±0.92 30 31.6± 0.76 n-Hexane fraction 150 35.7±0.09 100 30.5± 0.17 229.45± 0.48 2 50 21.07±1.00 30 15.76±1.23 Chloroform 150 82.74±0.41 fraction 100 75.3± 0.83 46.28±1.07 3 50 54.4± 0.07 30 39.1± 0.19
Ethyl acetate 150 95.5±1.30 fraction 100 81.43± 1.48 12.62±0.12 4 50 67.9±0.85 30 51.17±1.31 n-Butanol fraction 150 85.1±0.23 100 73.4± 0.71 35.76±1.08 5 50 59.14±0.46 30 43.37±0.11 Aqueous fraction 150 54.3±1.24 100 42.63± 0.93 129.61±1.17 6 50 35.7± 1.35 30 23.1±1.01 Galanthamine 150 92.47±0.41 100 84.21± 0.09 13.26±0.73 7 50 67.04±0.10 30 49.72± 0.23
Table 4.19: Acetylthiocholine Esterase of Zizyphus oxyphylla: Sr. AChE inhibition Sample Concentration AChE IC50 (mg/mL) No (%) Methanolic extract 150 68.2±1.24 100 51.7± 1.09 92.68±0.38 1 50 39.53± 1.37 30 27.94±1.41 n-Hexane fraction 150 44.6±1.29 100 37.24± 1.07 165.15±0.94 2 50 23.03±1.00 30 15.61±0.36 Chloroform 150 70.5 ±1.01 fraction 100 61.71± 1.63 70.59±0.41 3 50 46.62± 0.17 30 34.9± 0.09 Ethyl acetate 150 81.13±0.30 fraction 100 72.91± 1.83 26.48±0.92 4 50 59.63±0.80 30 47.0±0.31 n-Butanol fraction 150 86±0.02 100 74.23± 0.09 9.58±0.08 5 50 63.7±1.26 30 52.26±1.17 Aqueous fraction 150 58.8± 0.71 100 51.03±0.26 98.70±0.21 6 50 42.21±0.07 30 36.83±0.31 Galanthamine 150 92.47±0.41 100 84.21± 0.09 13.26±0.73 7 50 67.04±0.10 30 49.72± 0.23
Acetylcholine esterase enzyme is used for the breakdown of acetylcholine and this enzyme is used for the treatment of Alzheimer’s disease (AD) and the important advantage of AChE is to enhance the cholinergic function in the brain in the treatment of AD [244]. Different synthetic inhibitors (donepzil, rivastigmine, tacrine and galanthamine) for the AD treatment [245] show some unpleasant side effects [246] such as diarrhea, anorexia, fatigue, nausea, muscle cramps and sleep disturbance [247] so medicinal plants are used as new source of AChE enzyme because this activity of few plants has already been reported in the world [248]. AChE inhibition was determined by UV/ VIS spectrophotometer method and absorbance was measured at 412nm. In this assay hydrolysis of DTNB occur and produce yellow anion of thionitrobenzoic acid. Results in Table 4.18 revealed that the ethyl acetate fraction of R. triquetra showed significant % of AChE inhibition (95.5± 1.30) with IC50 (12.62 ± 0.12) at 150 µg/mL concentration when compared with standard reference, Galanthamine (92.47±0.41) having IC50 value (13.26±0.73) at same concentration (150 µg/mL) while the IC50 values of other fractions were in this order: n- Butanol (35.76± 1.08) > CHCl3 (46.28± 1.07) > methanolic extract (74.85± 0.73) > aqueous (129.61± 1.17) > n- Hexane (229.45± 0.48) (Figure 4.12).
120 67.8 91.5 92.47 100 82.74 85.1 80 35.7 54.3 60 40 20 %ageInhibition 0
Fractions Figure 4.12: Acetylthiocholine Esterase of Rhamnus triquetra
In Z. oxyphylla, n- butanol fraction exhibited good AChE inhibition (86.0±0.02) with lowest IC50 value (9.58±0.08 mg/ mL) in Table 4.19, indicating that it contained the best inhibition of the enzyme relative to standard reference and decreasing order of other fractions were: EtOAc > CHCl3 > MeOH > Aqueous > n- Hexane in Figure 4.13.
120
100 92.47 86 81.13 80 68.2 70.5 58.5 60 37.24
40 %age Inhibition
20
0 Crude n-hexane Chloroform Ethylacetate n-butanol Aqueous Galanthamine methanolic Fraction Fraction Fraction Fraction Fraction extract
Figure 4.13: Acetylthiocholine Esterase of Zizyphus oxyphylla
4.7.2 Protease Inhibition/ Trypsin Inhibition Assay: Protease inhibition of both plants was tabulated in Table 4.20. Table 4.20: Trypsin Inhibition of Various Fractions of Rhamnus triquetra and Zizyphus oxyphylla: % Protease Inhibition % Protease Inhibition Sr. No Sample ±S.E.M (R. triquetra) ±S.E.M (Z. oxyphylla) 1 Methanolic extract 57.69±0.93 69.55±0.31 2 n- Hexane fraction 21.42±0.47 22.76±0.51 3 Chloroform fraction 69.47±1.04 80.26±0.73 4 EtOAc fraction 81.69±1.31 91.05±0.94 5 n- Butanol fraction 93.03±0.65 78.94±0.87 6 Aqueous fraction 31.31±0.29 47.34±0.92 7 PMSF 85.4±1.23 85.4±1.23
Protease inhibitors are mainly found in plants which have the ability to inhibit the action of target proteolytic enzymes. Proteases play an important role in the normal physiological functions of the cells. A number of diseases such as cancer, emphysema, pulmonary and arthritis are reported to be induced by over activity of protease. Serine protease inhibitors types are mostly used to inhibit the trypsin, chymotrypsin or elastase [249]. The flavonoids are used as inhibitor against trypsin and it has been reported in the literature. Therefore, this study was carried out to find out the trypsin inhibitory potential of extracts and purified compounds [250]. The results of R. triquetra showed in Table 4.20 that the n- Butanol fraction showed highest inhibition i.e., 93.03 ± 0.65 while the decreasing order of other fractions were: EtOAc fraction (81.69±1.31) > CHCl3 fraction (69.47±1.04) > methanolic extract (57.69±0.93) > aqueous fraction (31.31±0.29)> n- Hexane fraction (21.42±0.47). These results were compared with PMSF (standard reference) having trypsin inhibition 85.4 ± 1.23 as shown in Figure 4.14.
93.03 100 81.69 85.4 90 69.47 80 57.69 70 60 50 31.31 40 21.42 % % Inhibition 30 20 10 0 Methanolic n- Hexane Chloroform EtOAc n- Butanol Aqueous PMSF extract fraction fraction fraction fraction fraction Fractions
Figure 4.14: Trypsin Inhibition of Various Fractions of Rhamnus triquetra The other plant Z. oxyphylla in Table 4.20 showed that the EtOAc fraction exhibited significant trypsin inhibition 91.05±0.94 and n- Hexane showed lowest inhibition (22.76±0.51) when compared with PMFS (85.4 ± 1.24) and the other fractions were in this order: CHCl3 (80.26±0.73)> n- Butanol (78.94±0.87)> MeOH (69.55±0.31)> Aqueous fraction (47.34±0.92) as shown in Figure 4.15 .
120 80.26 91.05 85.4 100 69.55 78.94 80 47.34 60 40 22.76
% % Inhibition 20 0 Methanolic n- Hexane Chloroform EtOAc n- Butanol Aqueous PMSF extract fraction fraction fraction fraction fraction
Fractions
Figure 4.15: Trypsin Inhibition of Various Fractions of Zizyphus oxyphylla
4.8 Antibacterial Activity: The results of antibacterial activity of both plants were given in Table 4.21 and 4.22. Table 4.21: Antibacterial Activity of Rhamnus triquetra: Zone of Inhibition (mm)
Zone of Crude Sr. Bacterial Inhibition Ethyl Methanolic n- Hexane Chloroform n- Butanol Aqueous No Species of std. drug acetate Extract (Imipenum) Escherichia 25 10 _ 10 18 _ _ 1 coli Bacillus 50 _ _ _ 14 _ _ 2 subtilis Shigella 28 10 _ 10 12 10 _ 3 flexenari Staphylococc 48 8 _ _ _ _ _ 4 us aureus Pseudomonas 23 _ _ 10 8 10 _ 5 aeruginosa Salmonella 28 ______6 typhi
Table 4.22: Antibacterial Activity of Zizyphus oxyphylla: Zone of Inhibition (mm)
Sr. Bacterial Zone of Crude n- Hexane Chloroform Ethyl n- Butanol No Species Inhibition Methanolic acetate Aqueous of std. drug Extract (Imipenum) 1 Escherichia 25 _ _ 8 10 _ _ coli 2 Bacillus 50 _ _ _ 18 _ _ subtilis 3 Shigella 28 ______flexenari 4 Staphylococcus 48 _ _ _ 18 _ _ aureus 5 Pseudomonas 23 _ _ 10 _ _ _ aeruginosa 6 Salmonella 28 ______typhi
Antibacterial activity of R. triquetra in Table 4.21 showed not much significant inhibition but the ethyl acetate fraction showed moderate inhibition as compared to other fractions and methanolic extract. Ethyl acetate fraction showed significant inhibition against E. coli and B. subtilis as compared to other bacterial strains. The zone of inhibition 18mm was observed against E. coli and 14mm against B. subtilis and an average zone (12 - 8mm) was observed against S. flexenari and P. aeruginosa. Antibacterial activity of Z. oxyphylla in Table 4.22 showed non- significant activity/ inhibition. In this plant only ethyl acetate fraction showed low significant activity against B. subtilis and S. aureus when compared to standard drug (Imipenum) while crude methanolic extract and other fractions showed negative results against these bacterial strains. Zone of inhibition of ethyl acetate fraction was observed at 18mm diameter each against B. subtilis and S. aureus.
4.9 Hemolytic Activity: Hemolytic activity of different fractions of both plants was observed in human serum and the results were given in Table 4.23.
Table 4.23: Hemolytic Activity of Rhamnus triquetra and Zizyphus oxyphylla:
Hemolytic activity of Hemolytic activity of S. No Samples R. triquetra Z. oxyphylla (Mean % ± S.D) (Mean % ± S.D) 1 Methanolic extract 52.7±0.63 63.6±0.47 2 n- Hexane fraction 12.9±0.34 11.2±0.29 3 Chloroform fraction 41±0.41 47.1±0.27 Ethyl Acetate 4 fraction 75.3±0.19 78.2±0.21 5 n- Butanol fraction 63.1±0.52 81.3±0.41 6 Aqueous extract 35.1±0.18 31.2±2.51 7 PBS 0.00±0.00 0.00±0.00 8 Triton ×100 98.0±1.54 98.0±1.54
In vitro hemolytic activities are now -a- days becoming new area of study [251] because this activity is the indicator of cytotoxicity and bioactivity. Hemolysis is a process that maintains the blood in fluid state and confines it into the circulatory system. This is a highly evolved and complicated process which is achieved by the interaction of number of biological systems including platelets, vessel wall, fibrinolysis, coagulation, kininogen and complement pathway [252]. Any change in these biological systems such as platelets, vessel wall, fibrinolysis, coagulation, kininogen and complement pathway causes different diseases related to kidney, heart, liver and malignancy [253]. During any change in these biological systems the Hemolysis maintain the blood fluid state and confuses it into the circulatory system. Different experiments proved that this activity exists in the plant and these plants can be used as drug in such conditions. The results obtained from this activity and serum electrolytes are indicating the diuretic action of these extracts. Hemolytic activity is one of the biological activities, determined due to saponins. Because saponins have the ability to hemolysis human erythrocytes by form pores in cell membrane based on affinity of aglycon moiety. [254]. Many researchers have been used this activity for the evaluation of toxicity of different plants [255]. The hemolytic activity was determined by spectroscopic method. All fractions of R. triquetra showed hemolytic activity such as methanolic extract
(52.7±0.63), CHCl3 fraction (41±0.41), n- Butanol fraction (63.1±0.52), n- Hexane fraction (12.9±0.34) and aqueous fraction (35.1±0.18) while EtOAc fraction revealed
maximum toxicity i.e., 75.3±0.19 when compared with positive control i.e., Triton X100 (98.0± 1.54) in Figure 4.16.
120 98 100 75.3 80 63.1 52.7 60 41 35.1
% Hemolysis Hemolysis % 40 12.9 20
0 Methanolic n- Hexane Chloroform Ethyl n- Butanol Aqueous TRITON X extract fraction fraction Acetate fraction extract 100 fraction
Fractions Figure 4.16: Hemolytic Activity of Various Fractions of Rhamnus triquetra The results of Z. oxyphylla in Figure 4.17 revealed that the n- Butanol fraction showed high toxicity (81.3±0.41) when compared with positive control (Triton X 100) while ethyl acetate fraction showed moderate toxicity value i.e. 78.2±0.21 while the other fractions were in this order i.e. methanolic extract (63.6±0.47) > CHCl3 (47.1±0.27) > aqueous fraction (31.2±2.51) > n- Hexane (11.2±0.29).
120 98
100 78.2 81.3 80 63.6 47.1 60 31.2 40 % % Hemolysis 11.2 20 0 Methanolic n- Hexane Chloroform Ethyl n- Butanol Aqueous TRITON × extract fraction fraction Acetate fraction extract 100 fraction Fractions
Figure 4.17: Hemolytic Activity of Various Fractions of Zizyphus oxyphylla
4.10 Anti-inflammatory Activity: The anti-inflammatory activity of methanolic extract of Rhamnus triquetra and Zizyphus oxyphylla were determined by carrageenan induced rat paw edema and formalin mice paw edema. 4.10.1 Carrageenan Induced Rat Paw Edema Method: Carrageenan induced rat paw edema of both plants were summarized in Table 4.24.
Table 4.24: Anti-inflammatory Activity of Rhamnus triquetra and Zizyphus oxyphylla by Carregeenan Induced Rat Paw Edema:
Time (hours) 1hr 2hr 3hr 4hr Control 0 0 0 0 Methanolic extract 34.86% of R. triquetra 18.23% 20.83% 27.24%
(300mg/kg) Methanolic extract of Z. oxyphylla 14.75% 20.72% 35.52% 42.29 % (300mg/kg) Aspirin 12.67% 15.06% 22.29% 39.12% (300mg/kg)
Inflammation is such physiological responses which occur by variety of injurious agents like bacterial infection, physical pain, chemicals and other phenomenon [256]. Inflammation takes place due to autoimmune diseases and is a basic contributor of different infectious and non infectious diseases such as diabetes, cancer, cardiovascular diseases, Alzheimer’s and arteriosclerosis [257, 258]. Inflammatory process is used to shelter our body from different diseases by releasing cells and prevent infections [259]. So novel anti-inflammatory agents could be discovered from medicinal plants because a number of phytoconstituents are present which are used for different diseases.
Carrageenan induced rat paw edema method is most widely used for anti-inflammatory evaluation of medicinal plants. It is used as harmful agent to induce experimental inflammation/ swelling for screening of compounds/ constituents that possess anti- inflammatory activity. When Carrageenan is injected into rat paw, a severe swelling/
inflammation produced which was visible within 30 minutes. [260, 261]. The lower the volume of paw, better the anti-inflammatory activity. This method is suitable in vivo model to calculate the value of anti-inflammatory agents, which take action by inhibiting the mediators of severe inflammation [262]. Therefore, the results as shown in Table 4.24 (Figure. 4.18 and 4.19) showed that in this method, Aspirin (standard drug) and test drug (methanolic extract of R. triquetra and Z. oxyphylla) exhibited a significant reduction in the volume of paw edema in rats. Sample drugs and aspirin showed maximum inhibition at the end of three hours. Aspirin showed maximum inhibition at 39.12% while methanolic extract of both plants produced 34.9% and 42.3% inhibition respectively.
34.86
27.24 Methanolic extract300mg/kg 20.826
18.2
39.12
22.3 Aspirin 300mg/kg
15 Dose 300mg/ Kg 300mg/ Dose 13
0
0 Control 0
0
Percentage Response 0 10 20 30 40 50 4hr 3hr 2hr 1hr
Figure 4.18: Anti-inflammatory Activity of Rhamnus triquetra by Carregeenan Induce Rat Paw Edema
42.29
35.52 Methanolic extract 300mg/kg 20.72
14.75
39.116
22.3 Aspirin 300mg/kg 15
Dose 300mg/ Kg 300mg/ Dose 13
0
0 Control 0
0
Percentage Response 0 10 20 30 40 50 4hr 3hr 2hr 1hr Figure 4.19: Anti-inflammatory Activity of Zizyphus oxyphylla by Carregeenan Induced Rat Paw Edema 4.10.2 Formalin Induced Mice Paw Edema: In formalin mice paw edema, 2% solution of formalin was injected into hind limb paw of mice and methanolic extract of Rhamnus triquetra and Zizyphus oxyphylla showed significant activity when compared with control and standard drug (Aspirin) as shown in Table 4.25.
Table 4.25: Anti-inflammatory Activity of Rhamnus triquetra and Zizyphus oxyphylla by Formalin Induced Paw Edema: % Response of % Response of 0-15 mins (1st Phase) 15- 30 mins (2nd Phase) R. triquetra Z. oxyphylla
Time Methanolic Methanolic (min) Methanolic Methanolic extract of extract of 1st 2nd 1st 2nd extract of extract of Z. R.triquetra Z. oxyphylla Phase Phase Phase Phase R.triquetra oxyphylla (300mg/kg) (300mg/kg) 66.0± 3.16 71.6 ± 0.978 12.4 ± 0.74 6.6 ± 0.93 53.2% 96.33 56.86 93.1% % % Aspirin 110 ± 3.14 35 ± 1.41 28.10% 80.54% (300mg/ kg)
After 30 minutes of administered the does orally, formalin is injected into the fore limb paw of mice subcutaneously, intense pain reaction produces. So this effect is seen in two phases i.e., initial phase (0- 15mins) is supposed to be mediated through modulation of neuropeptides [263] while the second phase (15- 30mins) is supposed to be mediated through release of inflammatory mediators like prostaglandin, etc. Methanolic extracts of both plants significantly decrease the paw licking and biting episode at both phases when compare with standard drug (aspirin). R. triquetra and Z. oxyphylla showed maximum % response i.e., 53.2% and 56.86% response when compared with standard drug (aspirin) 28.10% in 1st phase and 96.3% and 93.3% response in 2nd phase with reference to aspirin i.e., 80.54% as shown in Table 4.25.
96.33
Methanolic extract 53.2
80.54 Aspirin
Dose mg/kg Dose 28.1
0 Control 0
%age Responce 0 20 40 60 80 100 120
2nd Phase 1st Phase
Figure 4.20: Anti-inflammatory Activity of R. triquetra by Formalin Induced Paw Edema
93.1 Methanolic extract 56.86
80.54 Aspirin
Dose mg/kg Dose 28.1
0 Control 0
%age Responce 0 20 40 60 80 100 120 2nd Phase 1st Phase
Figure 4.21: Anti-inflammatory Activity of Z. oxyphylla by Formalin Induced Paw Edema
4.11 Analgesic Activity: Analgesic activity was determined by hot plate, water bath and acetic acid methods and all methods were summarized in Table 4.26, 4.27 and 4.28.
Table 4.26: Analgesic Activity of Rhamnus triquetra and Zizyphus oxyphylla on Hot Plate Method: Time Control R. triquetra Z. oxyphylla (Sec) Methanolic +ve Control Methanolic +ve Control -ve Extract (Aspirin) Extract (Aspirin) Control (300mg/Kg) (300mg/Kg) (300mg/Kg) (300mg/Kg) 0 0 17.203 ± 3.796 12.424± 2.532 18.02 ± 0.525 13.127± 2.669 30 0 23.74 ± 0.853 25.678± 0.861 24.94 ± 0.651 27.678± 0.778 60 0 18.02 ± 0.993 14.436± 0.501 19.08 ± 0.961 13.688± 0.475 90 0 15.78 ± 0.917 20.326± 0.318 14.88 ± 1.255 20.326± 0.318 120 0 19.24 ± 0.948 22.284± 0.724 18.1 ± 0.915 21.684± 0.904 150 0 16.94 ± 1.102 19.908± 0.763 16.388 ± 1.261 18.708± 0.854 180 0 20.28 ± 0.681 14.704± 0.557 21.3 ± 1.199 16.104± 0.369 210 0 13.92 ± 0.734 20.684± 0.508 10.74 ± 0.941 21.484± 0.654 240 0 18.34± 0.361 22.284± 0.724 17.74 ± 0.941 22.284± 0.724 270 0 14.168 ± 0.885 20.484± 0.489 15.272 ± 0.698 21.084± 0.731
Table 4.27: Analgesic Activity of Rhamnus triquetra and Zizyphus oxyphylla on Water Bath Method: Time Control R. triquetra Z. oxyphylla (Sec) Methanolic +ve Control Methanolic +ve Control -ve Extract (Aspirin) Extract (Aspirin) Control (300mg/kg) (300mg/kg) (300mg/kg) (300mg/kg) 0 0 3.708 ± 0.087 2.197± 0.459 4.612 ± 0.411 2.07± 0.493 30 0 4.812 ± 0.048 4.274± 0.7611 5.544 ± 0.104 4.171± 0.754 60 0 6.048 ± 0.126 3.953± 0.252 7.01 ± 0.166 3.956± 0.251 90 0 6.826 ± 0.259 3.82± 0.196 8.196 ± 0.188 3.746± 0.229 120 0 7.918 ± 0.518 4.976± 0.189 8.908 ± 0.159 4.936± 0.212 150 0 8.11 ± 0.296 3.72± 0.204 10.672 ± 0.501 3.73± 0.201 180 0 9.006 ± 0.389 3.953± 0.252 11.744± 0.278 3.833± 0.299 210 0 7.732 ± 0.308 2.673± 0.153 10.624 ± 0.391 2.66± 0.163 240 0 5.124 ± 0.433 2.761± 0.102 5.114± 0.5365 2.801± 0.18
Table 4.28: Analgesic Activity of Rhamnus triquetra and Zizyphus oxyphylla by Acetic Acid Writhing Method: Time (min) % Response % Response (1st Phase) (2nd Phase) Methanolic extract of R. 34.8 % 16.8% triquetra (300mg/kg) Methanolic extract of Z. oxyphylla (300mg/kg) 41.8% 17.0%
Aspirin (300mg/kg) 55.07% 26.0%
Pain is an unpleasant experience that is the net cause of a complex interaction of ascending and descending nervous systems involving physiological, psychological, biochemical and neocortical processes [264]. Pain affects the all area of person’s life like sleep, emotions, thoughts and daily activities. The pain treatment and management is probably one of the most common and a difficult aspect of medicinal practice. Analgesic treatment is an area by two main classes of analgesic drugs. i.e., non- steroidal anti- inflammatory drugs (NSAIDs) and opioids. These two classes of analgesic drugs produce severe side effects i.e., renal damage, gastrointestinal disturbance (with NSAIDs) etc [265, 266]. Number of steroidal and NSAIDs are available in the market and possess good potential as anti-inflammatory and antipyretic drugs such as aspirin, diclofenac, indomethacin etc but they cause unpleasant, undesired and severe adverse side effects on liver and gastrointestinal way. Therefore, a new and more powerful anti-inflammatory and antipyretic drug are needed to substitute the chemical therapeutics. [267]. In hot plate method, results of Rhamnus triquetra and Zizyphus oxyphylla in Table 4.26 (Figure 4.22 and 4.23) showed that when methanolic extract was compared with aspirin as standard drug at different interval of time then methanolic effect showed significant effect on the latency of nociceptive (analgesic) response in mice than aspirin. Statistically, significant results were recorded during 60- 180min after oral administration of drug.
20.484
22.284
20.684
14.704
19.908 Aspirin 300mg/kg 22.284
20.326
14.436
25.678
12.424
14.168 Dose(mg/ Kg) 18.34
13.92
20.28 Crude methanolic fraction 16.94 300mg/kg 19.24
15.78
18.02
23.74
17.203
Response in sec 0 5 10 15 20 25 30 4.5hr 4.0hr 3.5hr 3hr 2.5hr 2.0hr 1.5hr 1hr 0.5hr 0hr
Figure 4.22: Analgesic Activity of Rhamnus triquetra on Hot plate Method
21.084 22.284 21.484 16.104 18.708 Aspirin 300mg/kg 21.684 20.326 13.688 27.678 13.127
15.272 Dose (mg/ Kg) (mg/ Dose 17.74 10.74 21.3 Crude methanolic fraction 16.388 300mg/kg 18.1 14.88 19.08 24.94 18.02
Respnse in sec 0 10 20 30 40 4.5hr 4.0hr 3.5hr 3hr 2.5hr 2.0hr 1.5hr 1hr 0.5hr 0hr
Figure 4.23: Analgesic Activity of Zizyphus oxyphylla on Hot plate Method
Methanolic extracts of both plants by tail flick method, were observed in Table 4.27 that these two plants showed significant analgesic effect at different intervals of time when compared with Aspirin (Figure 4.24 and 4.25). In this evaluation of tail flick model, the temperature and duration were 51±1ᴏC and 0, 1, 2, 3 and 4 hours respectively. 300mg/Kg test drug and standard drug was used in mice and administered via gastric incubation. The results of sample drugs were compared with negative control and positive control (aspirin) and the results showed that significant activity was observed during 60- 180min after oral administration of drug. The most important significant observation is that methanolic extract of both plants have prolonged as compared to Aspirin.
2.761 2.673 3.953 3.72 Aspirin 300mg/kg 4.976 3.82 3.953 4.274 2.197
5.124 7.732 9.006 8.11 Crude methanolic extract 300mg/kg 7.918 6.82 Dose(mg/ Kg) 6.04 4.81 3.708
0 0 0 0 Control 0 0 0 0 0
Response in sec 0 2 4 6 8 10
4.0hr 3.5hr 3hr 2.5hr 2.0hr 1.5hr 1hr 0.5hr 0hr
Figure 4.24: Analgesic Activity of Rhamnus triquetra by Tail Flick Method
2.801 2.66 3.833 3.73 Aspirin 300mg/kg 4.936 3.746 3.956 4.171 2.07
5.114 10.624 11.744 10.672 Crude methanolic extract 8.908 300mg/kg 8.196
7.01 Dose(mg/ Kg) 5.544 4.612
0 0 0 0 Control 0 0 0 0 0
Response in sec 0 2 4 6 8 10 12 14 4.0hr 3.5hr 3hr 2.5hr 2.0hr 1.5hr 1hr 0.5hr 0hr Figure 4.25: Analgesic Activity of Zizyphus oxyphylla by Tail Flick Method
In Table 4.28, acetic acid induced writhing method of methanolic extracts of both plants and standard drug showed that the Aspirin showed more significant analgesic effect as compared to these two plants but these two plants also showed moderate analgesic effect as shown in Figure. 4.26 and 4.27. R. triquetra and Z. oxyphylla produced 34.8% and 41. 8% protection while Aspirin showed maximum of 55.08% protection of analgesia.
55.08 Aspirin 300mg/kg 26.07
34.8 Crude methanolic extract 300mg/kg
Dose mg/kg Dose 16.8
0 Control 0
Percentage Response 0 20 40 60 80 1st Phase 2nd Phase
Figure 4.26: Analgesic Activity of Rhamnus triquetra by Acetic Acid Writhing Method
55.16
Aspirin 300mg/kg 26
41.8 Crude methanolic extract 300mg/kg
Dose mg/kg Dose 17
0 Control 0
Percentage Response 0 20 40 60 80
1st Phase 2nd Phase
Figure 4.27: Analgesic Activity of Zizyphus oxyphylla by Acetic Acid Writhing Method So the phytochemical study of biologically active methanolic extracts of both plants showed significant anti-inflammatory and analgesic effect due to the presence of phytochemical constituents i.e., terpenoids, flavonoids, alkaloids, phenolic contents etc.
4.12 Hepatoprotective Activity: 4.12.1 Liver markers/ Liver Function Test: These enzyme assays/ tests were used as a helpful quantitative marker for the estimation of liver damage. These serum enzymes involved to estimate and evaluate the hepatocellular injury in Table 4.29, 4.30, 4.31, 4.32 and 4.33
Table 4.29: Liver Function Test for ASAT Level Rat Before Treatment After Treatment No. Group Group Group Group Group Group Group Group Group Group
I II III IV V I II III IV V R- I 2.23 2.74 2.74 2.85 3.06 3.02 22.69 5.86 12.23 9.42 R- II 2.74 2.74 2.73 3.18 2.60 2.92 21.06 7.23 10.33 5.90 R- III 2.74 2.86 2.74 2.42 2.56 2.84 19.55 9.74 7.95 4.90 R- IV 3.49 2.74 3.49 3.21 2.28 2.95 28.05 5.23 9.74 3.81 R- V 2.75 2.69 2.86 2.79 3.21 2.99 27.38 4.74 10.83 5.72 Mean 2.79 ± 2.76 ± 2.92 ± 2.89 ± 2.74 ± 2.95 ± 23.75 6.57 ± 10.17 5.95 ± 0.20 0.03 0.14 0.14 0.17 0.03 ± 1.70 0.89 ± 0.71 0.94 S.D 0.45 0.06 0.33 0.32 0.38 0.07 3.79 2.01 1.59 2.11 T- 5.38 2.96 2.52 3.01 Value P- p> p> p> p>
Value 0.05 0.05 0.05 0.05 p> 0.05= Significant
Table 4.30: Liver Function Test for ALAT Level Rat Before Treatment After Treatment No. Group Group Group Group Group Group Group Group Group Group
I II III IV V I II III IV V R- I 2.74 3.13 2.74 3.06 3.34 3.23 42.12 10.07 15.95 11.42 R- II 3.49 2.67 3.03 2.61 2.76 2.75 39.96 9.49 20.94 8.36 R- III 2.69 2.74 2.74 3.33 2.47 2.98 48.88 9.75 21.89 13.80 R- IV 2.74 3.03 3.23 2.56 2.71 2.75 44.44 11.69 23.80 11.92 R- V 2.74 2.88 2.67 2.69 3.12 3.05 35.94 10.74 17.93 15.29 Mean 2.88 ± 2.89 ± 2.89 ± 2.86 ± 2.88 ± 2.95 ± 42.27 10.35 20.10 12.16 0.15 0.08 0.10 0.15 0.15 0.09 ± 2.16 ± 0.39 ± 1.40 ± 1.17 S.D 0.34 0.19 0.23 0.33 0.35 0.21 4.84 0.88 3.14 2.623 T- 17.42 12.46 6.20 9.01 Value P- p> p> p> p>
Value 0.05 0.05 0.05 0.05 p> 0.05= Significant
Table 4.31: Liver Function Test for ALP Level
Rat Before Treatment After Treatment No. Group Group Group Group Group Group Group Group Group Group
I II III IV V I II III IV V R- I 2.75 2.91 3.14 2.75 3.06 2.89 47.93 9.50 13.55 8.26 R- II 2.77 2.74 2.82 3.29 2.94 2.97 46.81 8.26 17.51 7.75 R- III 3.24 2.87 2.75 2.75 2.36 3.04 53.86 5.35 14.75 9.38 R- IV 2.57 3.04 2.74 3.08 2.67 2.99 49.57 6.97 17.17 10.75 R- V 2.74 2.74 2.91 2.87 2.59 2.99 56.85 8.57 13.39 8.97 Mean 2.82 ± 2.87 2.88 ± 2.95 ± 2.72 2.98 ± 51.01 ± 7.73 ± 15.27 ± 9.02 ± 0.11 ± 0.05 0.07 0.10 ± 0.12 0.03 1.89 0.72 0.88 0.52 S.D 0.25 0.13 0.17 0.23 0.28 0.06 4.23 1.61 1.96 1.15 T- 11.21 7.41 5.77 7.80 Value P- p> 0.05 p> p> 0.05 p> Value 0.05 0.05 p> 0.05= Significant
Table 4.32: Liver Function Test for Total Bilirubin
Rat Before Treatment After Treatment No. Group Group Group Group Grou Group Group Group Group Group I II III IV p V I II III IV V R- I 0.28 0.28 0.34 0.27 0.25 0.30 2.76 0.47 0.95 0.52 R- II 0.33 0.24 0.24 0.26 0.22 0.29 3.89 0.35 0.71 0.38 R- III 0.23 0.24 0.38 0.27 0.29 0.28 1.89 0.43 0.91 0.31 R- IV 0.29 0.27 0.28 0.27 0.31 0.34 3.49 0.69 0.65 0.67 R- V 0.33 0.34 0.27 0.31 0.28 0.28 3.19 0.48 0.62 0.73 0.28 0.29 ± 0.22 ± 0.29 ± 0.28 ± 0.30± 3.04±0. 0.47± 0.76±0. 0.52± Mean ±0.0 0.02 0.02 0.03 0.01 0.01 34 0.06 07 0.08 1 S.D 0.04 0.04 0.06 0.02 0.03 1.53 0.77 0.13 0.15 0.18 T- 3.52 2.85 2.47 2.66 Value P- p> p> p> 0.05 p> 0.05 Value 0.05 0.05 p> 0.05= Significant
Table 4.33: Comparison of Liver Function Tests among Different Animal Groups After Treatment:
Sr. Total Parameters ASAT ALAT ALP No Bilirubin 1 Group I 2. 94± 0.03 2.95± 0.09 2.98 ± 0.02 0.30 ± 0.01 2 Group II 23.75 ± 1.69 42.27 ± 2.17 51.01 ± 1.89 3.04 ± 0.34 3 Group III 6.57 ± 0.89 10.35 ± 0.39 7.73 ± 0.73 0.48 ± 0.06 4 Group IV 10.16 ± 0.71 20.11 ± 1.41 15.27 ± 0.88 0.77 ± 0.07 5 Group V 5.95 ± 0.94 12.16 ± 1.17 9.02 ± 0.52 0.52 ± 0.08 Statistical comparison of 6 _ 20.21* _ 39.32* -48.03* -2.74 Group I with Group II Statistical comparison of 7 17.18a 31.92a 43.28a 2.56 Group II with Group III Statistical comparison of 8 13.59a 22.17a 35.74a 2.27 Group II with Group IV Statistical comparison of 9 17.8a 30.11a 41.98a 2.52 Group II with Group V * Significant p- value represents comparison of Group- I with Group- II a Significant p- value represents comparison of Group- II with Group- III, IV and V.
Liver is the most important organ, used to play essential role in regulating various physiological processes and some essential functions (metabolism, secretion and storage) in our body [268]. The liver is the site of metabolism of nutrients (carbohydrates, proteins and lipids) and excretion of waste metabolites. Liver cell have the antioxidant defense system against oxidative stress by natural antioxidants (Ascorbic acid, Vitamin E etc.) and antioxidant enzymes (catalase, SOD, GPx etc.)[269].Chemical- mediated hepatic injury is the general indication of drug toxicity [270] and more than 50% of acute liver failure cases. Drug- induced liver diseases can be predictable i.e., high incidence and
dose related or may be unpredictable i.e., low incidence and may/ may not be dose related. Most of the predictable hepatotoxins are predictable in animal testing or clinical part of drug development. Silymarin, which is commonly known as ‘milk thistle’, has been used as natural remedies for liver and biliary tract diseases [271]. This drug is mostly used to protect and regenerates the liver diseases such as jaundice, cirrhosis and hepatitis [272] and is good protection in several models of experimental liver disease. Silymarin acts by antilipid peroxidative, antioxidative, antifibrotic and liver regenerating mechanisms [273]. Silymarin/ water thistle has low level of toxicity but it also possess side effects such as allergic skin rashes and gastrointestinal disturbance [274]. Natural products and their active principles are a good source for the treatment of liver disorders [275]. Several plasma proteins are synthesized in the liver, and liver regulates the plasma lipids and lipoprotein. Therefore, the level of total bilirubin can be used as an indicator of liver function while the ALP, AST and ALP tests are considered as good markers of hepatic injury and hepatocellular integrity. Standard kits were used to measure the activities/ tests of these enzymes before the starting treatment and after treatment of rats with drugs. Aspartate amino transferase (ASAT) enzyme mostly present in liver, kidney, heart, muscles and pancreas. This enzyme is evaluated in tissue damage of liver and heart especially. The decreased level of this enzyme is mostly seen in pregnancy and Vitamin- B deficiency. Alanine amino transferase (ALAT) enzyme is mostly found in liver and it is also present in lesser amount in heart and other tissues. It is more useful to diagnose liver function as compared to ASAT [276]. Alkaline phosphatase (ALP) is produced in liver, bones, placenta of a pregnant woman, intestine and kidneys. This enzyme is mostly produce in liver as compared to bones or other organs. Large amount of ALP is released into the blood serum due to the damage liver cells, rapid bone growth or bone disease. Bilirubin is a brownish yellow substance, is present in bile. Bilirubin circulates in direct and indirect form in the bloodstream. This substance is produced when liver breaks down old RBC’s. Bilirubin level is increased in blood serum as liver detoxifies and excrete bilirubin [277]. Rats were used for the evaluation of hepatoprotective activity as experimental animals because these animals show similar genetically determined acetyl transferase activity as in human and are more sensitive to INH- induced hepatotoxicity due to high amidase
activity which results in release large amount of acetyl hydrazine, which induces hepatotoxicity. Various cellular defense mechanisms consisting of enzymatic and non- enzymatic components have been reported in RIF and INH- induced hepatotoxicity [278]. The compiled data after the completion of study was given in Table 4.33. Group I was normal group, isoniazid (INH) and rifampicin (RIF) with 50mg/Kg each was used for Group II, 200mg/Kg silymarin (standard) was used for Group III with INH and RIF of above dose, 300mg/ Kg methanolic extract of R. triquetra was used for Group IV which was experimental group with above dose of INH and RIF and methanolic extract of Z. oxyphylla was used for Group V with INH and RIF. The liver markers were raised as compared to treated and untreated levels in Group II (toxic group) which showed that the INH and RIF drugs injured the liver and indicated the increased oxidative stress when treated with INH and RIF [279]. The levels of these markers were significantly reduced in Group III, IV and V respectively after 21 days and the results are given in Figure 4.28, 4.29, 4.30 and 4.31. The results indicated that the both plants (Rhamnus triquetra and Zizyphus oxyphylla) reduced the toxicity of RIF+ INH as compared to silymarin (standard).
25.0000
20.0000
15.0000
10.0000 ASAT level U/L level ASAT 5.0000
0.0000 GROUP GROUP I GROUPII GROUP III GROUP V(Z.O) IV(R.T) BEFORE 2.7952 2.7596 2.9170 2.8947 2.7455 AFTER 2.9460 23.7464 6.5654 10.1618 5.9523 Groups
Figure 4.28: Liver Function Test for ASAT Level
45.0000 40.0000 35.0000 30.0000 25.0000 20.0000 15.0000
ALAT level U/L level ALAT 10.0000 5.0000 0.0000 GROUP- GROUP-I GROUP-II GROUP-III GROUP-IV(Rh) V(Zizy) BEFORE 2.8846 2.8952 2.8882 2.8560 2.8826 AFTER 2.9538 42.2724 10.3514 20.1057 12.1640 Groups
Figure 4.29: Liver Function Test for ALAT Level
60.0000
50.0000
40.0000
30.0000
20.0000 ALP level U/L level ALP 10.0000
0.0000 GROUP IV GROUP V GROUP I GROUPII GROUP III (R.T) (Z.O) BEFORE 2.8196 2.9260 2.8760 2.9514 2.7261 AFTER 2.9810 51.0084 7.7335 15.2743 9.0235
Groups
Figure 4.30: Liver Function Test for ALP Level
3.5000 3.0000 2.5000 2.0000 1.5000 1.0000
Total Bilirubin mg/dl Bilirubin Total 0.5000 0.0000 GROUP I GROUPII GROUP III GROUP IV(R.T) GROUP V (Z.O) BEFORE 0.2970 0.2727 0.2997 0.2824 0.2756 AFTER 0.3010 3.0412 0.4839 0.5784 0.9948
Groups
Figure 4.31: Liver Function Test for Total Bilirubin 4.12.2 Histopathological Study of Normal, Toxic, Standard and Experimental Groups:
Figure 4.32: Histopathological Examination of Normal Group
Figure 4.33: Histopathological Examination of Normal Group
Figure 4.34: Histopathological Examination of Toxic Group
Figure 4.35: Histopathological Examination of Toxic Group
Figure 4.36: Histopathological Examination of Toxic Group
Figure 4.37: Histopathological Examination of Standard Group
Figure 4.38: Histopathological Examination of Standard Group
Figure 4.39: Histopathological Examination of Experimental Group (IV)
Figure 4.40: Histopathological Examination of Experimental Group (IV)
Figure 4.41: Histopathological Examination of Experimental Group (V)
Figure 4.42: Histopathological Examination of Experimental Group (V)
Histopathological study of Group I (normal) showed a normal architecture of the liver lobules, Group II (toxic) showed the moderate portal area expansion along with duct proliferation and mild cytoplasmic degradation, cell ballooning degradation, steatosis, and confluent necrosis and these were shown in Figure 4.32 to 4.36. There was also no visible fibrosis, vascular injury, sinosidal congestion, sinosidal congestion and acute hepatitis in Group II. Group III i.e., silymarin (200mg/ Kg) along with INH + RIF (50 mg/ Kg) showed mild occasional focal lytic necrosis, steatosis and cholestasis. Mild duct proliferation and portal area expansion were also showed in Figure 4.37 and 4.38 while the cell ballooning, confluent necrosis, fibrosis or interface hepatitis, cytoplasmic degradation and vascular injury were not present. Co- administration of methanolic extract (300mg/ Kg) of R. triquetra along with 50mg/ Kg each INH + RIF in Group IV examined mild portal area expansion, duct proliferation, cholestasis and steatosis with no cell ballooning degradation, cytoplasmic degeneration, no visible vascular injury or inflammation, inosidal congestion, fibrosis or interface hepatitis as shown in Figure 4.39 and 4.40. Co- administration of methanolic extract (300mg/ Kg) of Z. oxyphylla along with INH + RIF (50mg/ Kg each) showed no portal area expansion with normal duct proliferation. Cholestasis, steatosis, and visible vascular inflammation, inosidal congestion or fibrosis were not found but mild cell ballooning degradation and cytoplasmic degradation were found in experimental Group V as shown in Figure 4.41 and 4.42. These results were compiled in Table 4.34 and the results clearly indicated that the methanolic extract of both plants has hepatoprotective effect which is comparable to effect of silymarin (standard).
Table 4.34: Histopathological Examination of Toxic, Standard And Methanolic Extracts of Both Plants: Normal Standard Experimenta Experimenta Sr. Toxic Observations (Group I) (Group l Group IV l Group V No (Group II) III) (R. T) (Z. O) 1 Cytoplasmic - Mild - - Mild degradation 2 Cell - Mild - - - ballooning degradation 3 Steatosis - Mild Mild Mild - 4 Cholestasis - - Mild Mild - 5 Interface - - - - Normal Hepatitis hepatic duct 6 Vascular - - - - - injury/ Inflammation 7 Necrosis - Confluent Occasionall Focal lytic Focal lytic necrosis y Focal necrosis necrosis lytic necrosis 8 Duct - Moderately Mild Mild Portal duct proliferation 9 Fibrosis - - - - - 10 Sinosidal - - - - Mild congestion 11 Portal area - Moderately Mild Mild - expansion
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