Table of Contents
Phytochemical Investigation and Pharmacological Assessment of
Taverniera nummularia D.C and Ifloga spicata (Forssk.) Sch. Bip.
Ph. D. Thesis
By
Yar Muhammad Khan
Department of Chemistry University of Science and Technology Bannu Khyber Pakhtunkhwa, Pakistan
2019
Table of Contents
Table of Contents
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ...... i LIST OF ABBREVIATION ...... ii LIST OF FIGURES ...... iii LIST OF TABLES ...... v ABSTRACT ...... vi Chapter No. 1 INTRODUCTION ...... 1 1.1 Medicinal Plants ...... 1 1.2 Taverniera nummularia D.C ...... 3 1.3 Ifloga spicata (Forssk) Sch.Bip...... 3 1.4 Phytochemicals...... 3 1.4.1 Alkaloids ...... 4 1.4.2 Flavonoids ...... 4 1.4.3 Phenolics ...... 5 1.4.4 Terpenes...... 5 1.4.5 Glycosides ...... 6 1.4.6 Saponins ...... 7 1.4.7 Steroids ...... 7 1.5 Anti-microbial Screening ...... 8 1.6 Antioxidant Activity ...... 9 1.7 DPPH Assay ...... 9 1.8 Hydrogen Peroxide Assay ...... 10 1.9 Cytotoxicity ...... 10 1.10 Anti-Diabetic Activity of Medicinal Plants ...... 11 1.11 Diabetes Mellitus...... 11 1.12 Aims and Objectives ...... 12 Chapter No. 2 LITERATURE REVIEW ...... 13 2.1 Review of Literature...... 13 Chapter No. 3 MATERIALS & METHODS ...... 18 3.0 Materials and Methods ...... 18
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3.1 Collection and Extraction ...... 18 3.2 Fractionation...... 18 3.3 Phytochemicals Screening of the Crude Methanolic Extracts ...... 18 3.3.1 Alkaloids ...... 19 3.3.2 Flavonoids ...... 19 3.3.3 Terpenoids...... 19 3.3.4 Tannins ...... 19 3.3.5 Saponins ...... 20 3.3.6 Sterols ...... 20 3.3.7 Cardiac glycosides ...... 20 3.3.8 Anthraquinones ...... 20 3.4 Antibacterial Assay ...... 20 3.5 Antifungal Assay ...... 20 3.6 DPPH Free Radical Scavenging Activity...... 22 3.7 Hydrogen Peroxide Scavenging Effect ...... 22 3.8 Cytotoxicity ...... 23 3.8.1 Lymphocyte Isolation ...... 23 3.8.2 Trypan Blue Staining ...... 23 3.8.3 Cell Culturing and Treatments ...... 24 3.9 Biochemical Analysis ...... 24 3.10 Superoxide Dismutase Activity (SOD) ...... 24 3.11 Catalase Activity ...... 24 3.12 Peroxidase Activity ...... 25 3.13 TBARS Estimation ...... 25 3.14 Determination of Reactive Oxygen Species (ROS) ...... 25 3.15 In-vivo Pharmacological Assessment of Various Extracts ...... 25 3.15.1 Experimental Animals ...... 26 3.15.2 Induction of Hyperglycemia ...... 26 3.15.3 Experimental Designing...... 26 3.15.4 Acute Toxicity Testing ...... 27 3.16 Investigation of Blood Chemistry ...... 27 3.16.1 Analysis of Serum Chemistry ...... 27 3.16.2 Total Protein Estimation ...... 27
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3.16.3 Urea Determination ...... 28 3.16.4 Creatinine Determination ...... 28 3.16.5 Determination of Bilirubin (Serum)...... 29 3.16.6 Determination of Cholesterol (Serum) ...... 30 3.16.7 High Density Lipoproteins (HDL) ...... 30 3.16.8 Low Density Lipoprotein (LDL)-Cholesterol Determination...... 31 3.16.9 Determination of Triglycerides ...... 31 3.16.10 Determination of Alanine Aminotransferase ...... 31 3.16.11 Determination Aspertate Aminotransferase ...... 32 3.17 Statistical Analyses ...... 32 Chapter No. 4 RESULTS ...... 33 4.0 RESULTS ...... 33 4.1 Phytochemical Analysis ...... 33 4.2 Qualitative Analysis of Taverniera nummularia and Ifloga spicata ...... 33 4.3 In-vitro Pharmacological Evaluation of Various Extracts ...... 34 4.4 Antibacterial Activity of T. nummularia and I. spicata ...... 34 4.5 Antifungal Activity of Taverniera nummularia and Ifloga spicata ...... 36 4.6 DPPH free Radical Scavenging Activity of T. nummularia and I. spicata ...... 39 4.7 Hydrogen Peroxide Scavenging Activity of T. nummularia and I. spicata ...... 40 4.8 Effect of T. nummularia on ROS Content of Lymphocytes ...... 42 4.9 Effect of T. nummularia on Antioxidant Enzymes Activity of Lymphocytes ...... 43 4.10 Effect of T. nummularia on TBARS of Lymphocytes ...... 46 4.11 Effect of Ifloga spicata on ROS content of lymphocytes ...... 47 4.12 Effect of Ifloga spicata on Antioxidant Enzymes Activity of Lymphocytes ...... 48 4.13 Effect of Ifloga spicata on TBARS of Lymphocytes ...... 51 4.14 In-vivo Investigation ...... 52 4.15 Body Weight ...... 52 4.16 Blood Glucose Level ...... 53 4.17 Serum ALP, ALT and Total Bilirubin ...... 55 4.18 Renal Function Tests ...... 57 4.19 Serum Lipid Profile ...... 59 Chapter No. 5 DISCUSSION ...... 62 5.0 Discussion ...... 62
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6.0 Conclusions ...... 68 7.0 Recommendations ...... 68 8.0 References ...... 70
Acknowledgment
ACKNOWLEDGEMENTS
All praises to Almighty Allah, the omnipotent, the most compassionate and His Prophet Muhammad (P.B.U.H), the most perfect among all human being even born on the face of the earth, who is forever a source and knowledge of humanity as a whole. I fell highly privileged to express my gratitude to my worthy supervisor, Dr. Saleem Jan Assistant Professor, Department of Chemistry, University of Science and Technology, Bannu, for his devotion, creativity and keen interest in my work. Special gratitude goes to my Co-Supervisor Dr. Rahmat Ali Khan, Assistant Professor, Department of Biotechnology, University of Science and Technology, Bannu, for his keen interest and constructive suggestions during the research work. He has contributed a lot to complete this manuscript. Any word of thank is insufficient to express my gratitude to Dr. Faizan Ullah Khan, Chairman Department of Botany UST Bannu, for his guidance and encouragement. I am highly obliged to Prof. Dr. Sultan Mehmood, Dean of Sciences, University of Science and Technology, Bannu, for his moral support and co-operation. I would like to express my special thanks to Dr M. Iqbal Zaman, Associate Professor/ Director ASRB for his help time to time. I am also thankful to Dr. Hidayat Ullah Khan Chairman Department of Chemistry, University of Science and Technology, Bannu for his help, encouragement and motivation. I pay my special thanks to Dr. Hizbullah Khan Wazir, Department of Animal Sciences Quaid-i-Azam University Islamabad for his co-operation in my research work. I appreciate the moral support and encouragement of my entire Lab fellows in particular Mr. Adnan Khan (Ph.D. Scholar) Department of Botany UST Bannu, and all technical and non- technical stuff, Department of Chemistry University of Science and Technology Bannu. I wish to record my deep sense of gratitude and sincere thanks to Mr. Aamir Khan Awan, M. Phil Scholar, for his co-operation in my research work. Words are inadequate to express my thanks to all my colleagues and friends.
Yar Muhammmad Khan
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List of Abbreviations
LIST OF ABBREVIATION
POD Peroxidase Activity
CAT Catalase Activity
SOD Superoxide Dismutase Activity
H2O2 Hydrogen Peroxide
Is-I5 Ifloga spicata
Tn-T-1 Taverniera nummularia
ROS Reactive Oxygen Species
F Standard Concentration Factor
HDL High Density Lipoproteins
LDL Low Density Lipoprotein
ALT Serum Alanine Aminotransferase
ALT Serum Alanine Aminotransferase
LSD Least Significant DifferencesTest
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List of Figures
LIST OF FIGURES
Fig. Page Title No. No.
1a Antibacterial Activity of the solvent fractions of Taverniera. nummularia 26
1b Antibacterial activity of the solvent fractions of Ifloga Spicata 27
2a Antifungal activity of the various fractions of Taverniera nummularia 28
2b Antifungal activity of the solvent fractions of Ifloga Spicata 28
3a Antioxidant activity (DPPH method) of Taverniera nummularia 29
3b Antioxidant activity (DPPH method) of Ifloga spicata 30
4a Antioxidant activity (H2O2 scavenging method) of Taverniera nummularia 31
4b Antioxidant activity (H2O2 scavenging method) of Ifloga Spicata 31
5 Effect of T.nummularia methanolic extract on body weight of experimental 42 mice under hyperglycemic conditions induced by alloxan
6 Effect of I. spicata methanolic extract on body weight of experimental mice 43 under hyperglycemic conditions induced by alloxan
7 Effect of T.nummularia crude methanolic extract on blood glucose level of 44 experimental mice under hyperglycemic conditions induced by alloxan
8 Effect of Ifloga spicata crude methanolic extract on blood glucose of 45 experimental mice under hyperglycemic conditions induced by alloxan
9a Effect of Taverniera nummularia and Iflago spicata extract on ALT test. 46
9b Effect of Taverniera nummularia and Iflago spicata extract on ALP test. 47
9c Effect of Taverniera nummularia and Iflago spicata extract on Bilirubin test. 47
10a Effect of Taverniera nummularia and Iflago spicata extract on Urea test. 48
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List of Figures
10b Effect of Taverniera nummularia and Iflago spicata extract on Craetinine test. 49
10c Effect of Taverniera nummularia and Iflago spicata extract on Total 49 cholesterol.
10d Effect of Taverniera nummularia and Iflago spicata extract on Total protein. 50
11a Effect of Taverniera nummularia and Iflago spicata extract on Total 51 Cholesterol test
11b Effect of Taverniera nummularia and Iflago spicata extract on Triglyceride 51 test.
11c Effect of Taverniera nummularia and Iflago spicata extract on HDL test. 52
11d Effect of Taverniera nummularia and Iflago spicata extract on LDL test. 52
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List of Tables
LIST OF TABLES
1.1 Phytochemical Composition of leaf extracts of T. nummularia and I. 25 spicata
2 Effect of T. nummularia on ROS Content of Lymphocytes 32
3.1 Effect of T. nummularia on Antioxidant Enzymes Activity of 33 Lymphocytes
3.2 Effect of Taverniera nummularia on CAT activity (milli Units / 106 cells) 34 of lymphocytes under H2O2 induced oxidative stress
3.3 Effect of Taverniera nummularia on POD activity (nmol / 106cells) of 35 lymphocytes under H2O2 induced oxidative stress
4 Effect of T. nummularia on TBARS of Lymphocytes 36
5 Effect of Ifloga spicata on ROS content of lymphocytes 37
5.1 Effect of Ifloga spicata on Antioxidant Enzymes Activity of Lymphocytes 38
5.2 Effect of Ifloga spicata on antioxidant enzymes (POD) activity of 39 lymphocytes
5.3 Effect of Ifloga spicata on antioxidant enzymes (SOD) activity of 40 lymphocytes
6 Effect of Ifloga spicata on TBARS of Lymphocytes 41
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Abstract
ABSTRACT
The present studies were performed to investigate the phytochemical composition and pharmacological potential of different solvent fractions (methanolic, n-hexane, dichloromethane, ethyl acetate and water) of Taverniera nummularia and Ifloga spicata. The different solvent fractions were tested for various types of phytochemicals, antimicrobial assay, hydrogen peroxide scavenging assay, DPPH (1, 1-diphenyl-2-picrylhydrazyl) free radical scavenging assay and detoxification effects of reactive oxygen species (ROS) on blood lymphocytes of humans under induced stress of H2O2. Methanolic extract of
Taverniera nummularia and Ifloga spicata revealed significantly (p<0.05) higher antibacterial potential against S. aureus. The ethyl acetate, n-hexane and chloroform significantly showed higher antibacterial activity against P. aeruginosa. The different fractions for antibacterial activity used against E. coli were methanolic ethyl acetate, aqueous, n-hexane and chloroform. Maximum activity against fungi was exhibited by chloroform fraction against Aspergillus niger (64%), Aspergillus flavus (62.02%) and Aspergillus fumigates (59.25%). The n-hexane, aqueous, methanolic and ethyl acetate showed higher antioxidant activity were higher for n-hexane, aqueous, methanolic and ethyl acetate as a compare to other fractions. The superoxide dismutase (SOD), catalase (CAT) and peroxidase
(POD) activities of the human blood lymphocytes were significantly (p<0.05) decreased in response to oxidative stress induced by H2O2. The antioxidant enzymes activities were significantly improved by the various leaf fractions of Taverniera nummularia and Ifloga spicata at 50µg/ml under H2O2 stress methanol and aqueous fractions being the most effective ones. The increase in antioxidant enzymes activities showed significant decrease in the reactive oxygen species (ROS) level and thiobarbituric acid (TBARS) content.The in vivo antidiabetic activity of methanolic extract of Taverniera nummularia (T. nummularia) and
Iflago spicata (I. spicata) was investigated in mice. Treatment with alloxan significantly.
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Abstract
The alloxan treated mice exhibited a significant increase in blood glucose level, total bilirubin, ALT, ALP, urea, creatinine, total proteins, triglycerides and LDL cholesterol content as compared with control group (p<0.05). Treatment with methanolic leaf extract of
Taverniera nummularia and Ifloga spicata significantly decreased blood glucose level and resulted in a better body weight under hyperglycemic conditions. Moreover, increases in the content of bilirubin, ALT, ALP, urea, Craetinine, total proteins, triglycerides and LDL cholesterol due to hyperglycemia was normalized by treatment with methanolic extract of both the plant species.
Further column chromatography to find the basic ingredients responsible for this potential will be very useful and interesting. This can result in diversity in natural product and pharmaceutical chemistry.
Keywords: Taverniera nummularia, Ifloga spicata, Lymphocytes, Antimicrobial activity,
Oxidative stress, Oxidative enzymes, Superoxide dismutase, Catalase, Peroxidase.
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Chapter 1 Introduction
Chapter No. 1
INTRODUCTION
1.1 Medicinal Plants
Mankind has been using medicinal plants since the beginning of human civilization. A wide variety of different chemical composites are derived from different groups of plants. These biologically active ingredients protect plants from the attack of herbivores, pathogenic microbes and insect pests [1]. Almost 12,000 such compounds of plant origin have been identified [2]. Most common pharmaceuticals obtain from plants include aspirin, digitoxin, quinine and opium [3]. Herbal drugs are as effective as the synthetic ones with little side effects on human body [1, 2]. Based on the ethno medicinal information, scientists are struggling to identify and isolate biologically more active compounds from various medicinal plants. In 2001 scientists obtained about 122 medicinal compounds from plants, which are now used as medicines [4].
In developed countries many people still use plant based medicines for their health care. The demand and need of herbal medicines is growing with the passage of time. Approximately
5000 therapeutic plant species have been reported in ancient literature and more than 800 plants have been used in medicines [5]. Researchers are struggling to isolate therapeutically more effective compounds from plants. The purpose is that pathogenic bacteria are gaining resistance to synthetic antibiotics all over the world. Recently it has been observed that extracts obtained from various parts of medicinal plants can be used to control variety of disease causing agents [6- 8]. A number of herbal products are used for protection of food and as appetizers. Most of them are also used as drugs in modern times [9]. The chemical compounds isolated from medicinal plants provided basis for the formation of new and effective drugs [10]. Plant products reduce side effects of other chemical products when used
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Chapter 1 Introduction in combinations. Due to these properties they have prodigious therapeutic value [11]. In developing countries, 80% people utilize traditional medicines having plants based compounds [12, 13].
In old-fashion system of medicine, crude extracts of plants were used by humans for treatment of pathogenic diseases [14, 15]. Flowers, leaves, fruits and roots are the rich sources of phytochemicals having antimicrobial tendency [16-18]. The efficiency and mechanism of action of these herbal extracts is still required [18-20] Infections triggered by various pathogenic bacteria are common all over the world. Livestock and other economically important animal species are also affected by these pathogenic bacteria [14,
21]. Bacterial species like Staphylococcus aureus, Escherichia coli, Micrococcus leuteas and
Pseudomonas aeruginosa are causative agent of several types of human diseases [22, 23].
Recently it has been listed most pathogenic bacterial species showing resistance to common existing synthetic antibiotics [24]. Therefore scientists are searching for potent antimicrobial agents of plant origin [25- 27].
Human body may face some negative challenges such as reactive oxygen species (ROS) which are responsible for DNA damages. This negative effect is caused due to agents that can be produced in environment, oxidative agents, ultra violet rays and viruses [28]. During oxidative process H2O2 is formed in the cell which is changed into water through peroxidases, catalases, glutathione and peroxiredoxins [29]. But when these reactions do not occur in a proper way then it will yield hydroxyl free radicals. These radicals may react with the pentose sugar of DNA which led to breakage of DNA molecule [30]. Plants are rich source of antioxidants, which can scavenge these free radicals and protect biomolecules of cells from adverse effects of these free radicals [29, 30].
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Chapter 1 Introduction
1.2 Taverniera nummularia D.C
Taverniera nummularia (Fabaceae) is flowering plant. Height of T. nummularia is from 60 to100 cm. It has trifoliate leaves and is present mostly in Pakistan and India. Traditionally T. nummularia is used as anti-inflammatory, anticancer and for digestive problems. Roots are sweet like licorice and used as an expectorant in cough. T. nummularia leaves are used as poultice for sloughing wounds, applied superficially on swelling, abscess and ulcers. 2 teaspoon filled decoction of roots is given orally two times a day for 3 day in throat problems.
Seeds are fried and used in hoarseness of voice [31, 32].
1.3 Ifloga spicata (Forssk) Sch.Bip.
Ifloga spicata is one of the specific annual plants of family Asteraceae. It has about 5-10 cm height and branches along with stem form globular and cylindrical shape either at lower or upward portion. The stems and branches of the plant make dense globular to cylindrical inflorescence either at base or in the upper portion. It is present in the desserts of Pakistan,
India, Afghanistan, Canary Island and southern Spain to North Africa. Period of flowering is started from February to September. It is traditionally used to treat diabetic, stomach disorders, tooth ache and respiratory tract problems.
1.4 Phytochemicals
Medicinal Plants have secondary metabolites with therapeutic potential. Phytochemicals comprise a large group of compounds found in plants. These phytochemicals provide protection to plants against infection; give different colors, aroma and flavors to the plants.
They also protect the plants from adverse environmental changes, such as pollution, drought, ultraviolet radiations and pathogenic attack [32, 33]. Over 4000 phytochemicals have been reported from different plant species [33, 34]. Phytochemicals are present comparatively in
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Chapter 1 Introduction larger amounts in the outer portions of different plant parts. The amount of phytochemicals in different plants depends upon the type of plant, methods of treating and cooking as well as the conditions in which the plants are grown [35]. These bioactive compounds are pharmacologically active against many diseases including, jaundice, cough, bronchitis, diarrhea, asthma, heart diseases, brain abnormalities, breast/prostate cancers and diabetic mellitus [36]. There are various classes of phytochemicals, includes alkaloids, polyphenol, steroids, saponins, terpenoids, glycosides, flavonoids etc.
1.4.1 Alkaloids
Alkaloids are nitrogen containing heterocyclic compounds with basic characters. [37]. It concerns to the molecular structure of the specific alkaloid as well as the existence and position of the functional group [38]. Crystalline salts are formed without producing water when reacted with acids [39]. Generally alkaloids are found in solid condition but some may be present in liquid state. Alkaloids are dissolved in medium polar solvent like alcohol, and less soluble in highly polar solvents like water. Due to unpleasant taste, alkaloids protect the plants from herbivorous animals and other pathogenic organisms. They are also used as stimulants (e.g. caffeine), narcotics (e.g. morphine) and poisons (e.g. nicotine). Alkaloids are found in nearly each and every part of the plant but the utmost concentrations are found in seeds and roots [40, 41]. Alkaloids have various medicinal properties. For example, lots of indole alkaloids are used as antihypertensive drugs. Quinidine and spareien show antiarrhythmic effects. Quinine is used as antimalarial drug and dimeric indoles, such as vincristine and vinblastine are used as anticancer drugs [42].
1.4.2 Flavonoids
Flavonoids are the main metabolites of plants which are derivative of heterocyclic ketones called flavones. In plants about 4000 kinds of flavonoids have been introduced. Mostly
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Chapter 1 Introduction flavonoids are present in fruits and seeds of vegetables in the form of glucosides, methylated derivatives and aglycones [43, 44]. In nature mostly flavonoids are present as aglycon with one or more sugar parts. So, they may be classified as monoglycosides, diglycosides and depending upon the number of sugar molecules present. The glycosidic bond is formed at carbon number 3 or 7 and the sugar may be D-glucose, L-rhamnose, arabinose, galactose or glucorhamnose. The flavonoids without sugar molecules are called aglycones [45].
Flavonoids have various significant biological actions. They have anti-inflammatory, antimicrobial, anti-allergic, antioxidant, vascular activity and cytotoxic antitumor activity
[46]. They are helpful in the protection of cells against the harmful effects of oxidative processes taking place in different types of macromolecules, like carbohydrates, lipids, proteins and DNA [47, 48].
1.4.3 Phenolics
Phenolic compounds are phytochemicals derived from phenols. Most of them are derivative of amino acid phenylalanine. In nature they are mostly present in the form of pigments which give color to the fruits of plants. The phenylalanine lyase enzyme converts phenylalanine into phenolic compounds. It may protect them from pathogenic infection [49].
The maximum and frequently occurring phenolic compound in plants is caffeic acid.
Chlorogenic acid is another phenolic compound which is considered to cause allergic skin diseases in humans [43]. Phenolics give protection against oxidative damage as they are strong antioxidents. Therefore, they are utilized in the prevention of diseases of various types such as those of heart, inflammation as well as cancer [50].
1.4.4 Terpenes
Plants consist of unsaturated hydrocarbons called Terpenes. They occur in liquid form and are highly combustible. Most of them are found in resin, essential oils and oleoresins [39].
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Chapter 1 Introduction
They consist of a large group of naturally occurring lipids which are the derivative of isoprene units. They are polycyclic and are different from each other due to their basic carbon and functional groups skeleton [51]. The general molecular formula of terpenes is (C5 H8) n.
Terpenoids are categorized into mono-terpenes, di-terpenes, tri-terpenes, sesqui-terpenes, etc.
This type of arrangement is based on the number of isoprene units forming particular terpenoids [52]. They are secondary metabolites of plant origin. They keep the plants safe by making the plants inedible to herbivores and other enemies or by making phytoalexins [53].
Most of the terpenes are volatile. Terpenes have the ability to attract the insects for plants pollination and may create bad smell to protect pants herbivores [54]. Terpenoids have medicinal importance as they act as anticancer, antimalarial, anti-ulcer, antimicrobial and diuretic. For example, artemisinin act as an antimalarial drug and taxol act as anticancer drug
[52, 55].
1.4.5 Glycosides
Glycosides are colorless, crystalline, and water soluble phytochemicals. It is consist of carbon, hydrogen and oxygen, and small amounts of nitrogen and sulphur. As they are water soluble, so they are found in the vacuolar sap of plant cells. They are conjugate molecules.
They are consisting of Glucose, (Carbohydrate) part and non-carbohydrate part. The former is called glycone and the latter is called aglycone [39, 43]. Aglycones are signified by alcohol, glycerol or phenol. Glycosides are not involved in any kind of chemical reactions. They are easily split into their components with the help of specific enzymes or inorganic acids.
Glycosides have been categorized into various classes. This classification is based on the type of their carbohydrate component, non-carbohydrate component and their pharmacological effects. It has more medicinal properties. They increase appetite which helps in digestion.
Glycoside has bitter taste and may increase saliva secretion and gastric juice. Extract of some
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Chapter 1 Introduction plants possess cyanogenic groups which are used in preparation of pharmaceutical products.
Similarly Amygdalin is used for cancer prevention and as a suspension of cough. Use of high amount of cyanogenic has harmful effect. So those foods which consist of cyanogenic should be used in proper ways. If not they can cause stomach problems, such as severe gastric irritations and tissue damage [38].
1.4.6 Saponins
Saponins are also secondary metabolites and are the derivatives of Saponaria vaccaria
(Quilaja saponaria). Saponins are used as soap. It is soluble in water and insoluble in ether
[43]. Upon hydrolysis it gives aglycones, and form glycosylated teroids, triterpenoids, and steroid alkaloids [56]. The carbohydrate part of saponin contains glucose, galatose, xylose, arabinose, rhamnose which is bonded to saponogen (non-carbohydrate). In some saponins, the sugar part is replaced by some other molecules such as glucoronic acid. Saponins having a single sugar molecule are called monodesmoside saponins, while those having two sugar molecules are called bidesmosides [57]. In plants the role of saponins is not yet well understood. They, however, keep safe the plants from insects attack and are said to be phytoprotectants. In animals, they possess antibacterial and antifungal effects [58].
1.4.7 Steroids
Steroids are 3- carbon organic compounds present in plants. Steroids also possess medicinal values. They are effective in the prevention of various types of heart diseases and are called cardio glycosides. Mostly they control heart muscles [39]. In case of osteoporosis of mankind and animals anabolic steroids are used [59, 60]. Natural products obtained from plants and many other resources like animals, fungi, bacteria as well as minerals are utilized for the recovery of human beings, plants and animals from different diseases.
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Chapter 1 Introduction
.
1.5 Anti-microbial Screening
Current reports on the resistance of various bacterial and fungal pathogens to synthetic antibiotics, the investigations on the plant based antimicrobials have been increased.
Recently reported by World Health Organization many of the plants species are good source of medicinal compounds. About 80% people in developing countries use traditional herbal drugs and their derivatives in the treatment of different ailments. In developing countries where microbial diseases are endemic, people rely on medicinal plants for the treatment of diseases. However a very little data are available about the authentication of antimicrobial properties of herbal medicine [61].Plants extracts may be used in prevention of various chronic disorders [62]. According to Monthana and Lindequista, (2005) and Bajpai et al.,
(2009), certain foods and molecules of no or little nutritional value isolated from aromatic and medicinal plants possess anti-microbial activities. Besides the prevention of pathogens, food spoilage is also a very serious problem, and needs the safety against microbes [63], thus more protection to our food is needed by adding anti-microbial ingredients isolated from various plants [64]. The medicinal plants are used against food wastage and pathogens like bacteria and fungi etc. and have anti-microbial properties [65]. Fungi are main demolishers of foods products and scraps during storage, rendering them harmful for human ingestion by generating mycotoxins and often by slow down their nutritive value [66, 68]. Due to mycotoxins contamination of the agricultural products throughout the world many food items become unhealthy for human consumption, especially those produced by species of
Aspergillus [68, 69]. Recently reported that more than 300 fungal metabolites are poisonous for human and other animals. Therefore, building of food products with phytochemicals can be healthy approach to protect food products from spoilage by bacteria and fungi of
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Chapter 1 Introduction medicinal plants is due to phytochemicals such as flavonoids, terpenoids, alkaloids, steroids, tannins, quinones, saponins, fatty acids, and gums which are responsible to produce physical action on the body [69].
1.6 Antioxidant Activity
Plants contain bioactive ingredients with high therapeutic potential. Recently various studies conducted by different researchers are showing that various crude extracts obtained from medicinal plants have effective antioxidant activities. Cells form free radicals upon exposure to environmental toxins, allergens, insecticides, pesticides and cigarette smoke. Free radicals are mostly Reactive Oxygen Species (ROS), including singlet oxygen, hydrogen peroxide, superoxide radical, hydroxyl radical, hypochlorite radical, nitric oxide radical and several lipid peroxidases. They can have the capability to bind with proteins of membrane, lipids, nucleic acids, enzymes and other compounds, and cause cell lysis. Antioxidants are free radicals scavengers. Antioxidants may be substances as carotenoids, vit-E, vitamin-C and other molecules like lipoid acid and glutathione. Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) play a vital role in the controle of free radicals produced as result of metabolism or other stress conditions.
1.7 DPPH Assay
The α-Diphenyl-β-picrylhydrazyl method was first presented by Blois (1958) to estimate the radical scavenging potential of chemical entity of a compound, plants extracts or other biological sources. In this procedure antioxidant donate a H+ atom to their corresponding hydrazine and thus incomplete electron of N-atom gets reduced. This method is consider to be the simplest, where the compound or extract sample under study is intermixed with the solution of DPPH and the absorbance taken a pre-defined time interval. Moreover the instrumental techniques are advanced in this era, but the basic approach is given the same.
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Chapter 1 Introduction
1.8 Hydrogen Peroxide Assay
Hydrogen peroxide is the highly reactive oxygen species which are produce inside living cells as a result of metabolism. During normal metabolic process H2O2 is converted to water by peroxiredoxins, glutathione, peroxidases and catalases. However, in conditions when these types of mechanisms are not functional properly, transition metals react with H2O2 and through the Fenton reaction produce hydroxyl radicals. This hydroxyl radical attacks the sugar component of the DNA resulting into single strand breaks. Hydrogen peroxide (H2O2) is produced in the cells during the process of oxidative metabolism.
1.9 Cytotoxicity
Cytotoxicity is the condition in which the immune cell becomes affected by some toxic chemical. Presently, in the clinical trials a large number of techniques one used to treat cancer such chemotherapy, surgery and radiation. The synthetic drugs used in the treatment of cancer possess many side effects to overcome these effects. Therefore there is a dire need for the development and generation herbal medicines. The Kingdom Plantae has blessed us with drugs like those of antitumor and anticancer with special mechanisms of action and structure.
Recently it is found that Diperene resins produced from various species of cotton are very effective in cancer treatment. Similarly Taxol is isolated from Taxus revifolia bark and is effective in treating ovarian, pulmonary and breast cancer. Currently it was observed that
Fagonia cretica possess strong anticancer potential. Blood lymphocytes are immune cells and are important in studying toxicological effects of compounds and chemicals and are a good tool to study immune response of the body to external chemicals.
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Chapter 1 Introduction
1.10 Anti-Diabetic Activity of Medicinal Plants
Medicinal plants the green gold play a crucial role in reducing diabetes by increasing hormonal level, convert glucose to glycogen. Medicinal plants also play a key role in recovering β-cells of islets of Langerhans. Recently various studies have been published by researchers showing that extracts obtained in crude forms from medicinal plants have effective antidiabetic activities [82]. Most of the herbal drug are effective to treat Diabetes mellitus [70].
1.11 Diabetes Mellitus
Diabetes mellitus is a metabolic illness in which there is a high blood sugar level caused by deficiency of insulin to convert glucose into glycogen. [71]. A survey report showed that 171 million people were suffering from diabetes in 2000.In 2012 nearly 1.5 million people died of diabetes by 2030 this number will reach to 366 million [72]. By diabetes 1.5 million deaths were stated to be straight instigated in 2012, [73]. Diabetes mellitus are of three main types.
Type1DM or IDDM is the insulin-dependent diabetes mellitus. It is also called juvenile diabetes results from the pancreas's failure to produce sufficient insulin. The cause of Type 1
DM is unidentified. Adult-onset diabetes (Type 2 DM) is a disorder in which cells fail to response to insulin properly. This form was before declared as "non-insulin-dependent diabetes mellitus" (NIDDM). The most shared cause is extreme body weight and inadequate exercise. Type 3 (Gestational diabetes) which occurs during pregnancy [72]. Diabetic mellitus in long term creates problems such as potential loss of vision (retinopathy), foot ulcers (outlying neuropathy), renal failure (nephropathy), genitourinary, autonomic neuropathy, cardiovascular symptoms as well as sexual dysfunction [72]. Normal range of plasma glucose levels during fasting condition is 3.5-6.7 mmol/L (63-120.6 mg/dl). By using carbohydrate rich meal/food the blood glucose level increases to about 8 mmol/L.
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Chapter 1 Introduction
1.12 Aims and Objectives
Plants are rich source of anti-microbial and natural anti-oxidants. Due to current reports on the resistance of various bacterial and fungal pathogens to synthetic antibiotics, the investigations on the plant based antimicrobials have been increased. The current study was aimed to achieve objectives mentioned below:
1) Determination of the presence of various phytochemical constituents of T.nummularia and
I.spicata.
2) Determination of anti-microbial and anti-oxidant activity of various solvent fractions of
Taverniera nummularia and Ifloga spicata.
3) Determination of cytotoxicity of T.nummularia and I. spicata by using human blood
lymphocytes.
4) Evolution of T. nummularia and I. spicata for anti-diabetic potential.
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Chapter 2 Literature Review
Chapter No. 2
LITERATURE REVIEW
2.1 Review of Literature
Shad et al., (2016) have reported the phytochemical composition and antimicrobial activities of Aerva javinica and Linum ustitssimum only found that A. javinica exhibited the occurrence of saponins, flavonoids and tannin while L. ustitssimum plant had phenolic compounds but do not contain tannin and saponins. They also studied that the extract of A. javinica showd anti- fungal activity [73].
Palombo et al., (2001) reported the bactericidal effect of various parts of 39 plants species against four different species of Graham-positive bacteria (Bacillus cereus, E. faecalis, S. aureus and S. pyogenes) and four species of G-negative bacteria, (Salmonella typhimurium,
E.coli, K.. pneumoniae and P. aeruginosa ). All extracts of plants species inhibited the growth of these bacteria, among the 5 extracts of 5 plant species showed significant antibacterial potential against G-positive bacteria [74].
Quiroga et al., (2001) studied methanolic crude extract of Larrea divaricata, Larrea cuneifolia and Zuccagnia punctata for their antifungal activities against various groups of fungi. Antifungal assay include disk, well diffusion and radial growth inhibition by broth dilution tests. Extracts obtained from Zuccagnia punctata, Larrea cuneifolia and Larrea divaricata, exhibited remarkable activity against most of the fungi tested [75].
Soliman et al., (2002) studied the extracted essential oils from twelve medicinal plants and checked their antifungal activities against four different fungal species (Fusarium moniliforme, Aspergillus flavus, Aspergillus ochraceus, and Aspergillus parasiticu). All these extracts showed significant antimicrobial potential against tested fungal species. Caraway caused inhibition at 2000 ppm against A. parasiticus and A. flavus, while at 3000 ppm
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Chapter 2 Literature Review against F. moniliforme and A. ochraceaus. Anise at⩽500 ppm showed complete inhibition of
F. moniliforme A. ochraceus, A. parasiticus and A. flavus. These results illustrated that the tested fungi showed sensitivity to these oils especially to thyme and cinnamon. It also becomes clear from the results that essential oils of thyme, cinnamon, anise and spearmint were extra effective to stop the fungal growth [76].
Gupta et al., (2004) studied the therapeutic potential of natural dyes obtained from plants.
Eleven natural dyes were studied which showed antimicrobial properties against three species of G-negative bacteria. In the said study it was determined that minimum concentration of three selected dyes, which was effective for inhibition of microbial growth in the case of E. coli and P. vulgaris. Therefore, selected dyes would be beneficial for the dyeing of sheets and gowns for hospital applications, and on objects which cannot be washed easily such as mattresses and furniture [77].
Cai et al., (2004) studied 112 plants species belonging to 50 different families, found that all these extracts have flavonoids, tannins, phenolic acids, coumarins, lignans, curcuminoids quinones, and stilbenes. All these plants showed high antioxidant activity [78].
Morgen et al., (2006) have reported that methanolic extract of onion (Allium cepa) have antioxidant effect. This is due to flavonoids which are polyphenolic substances and thereby used for the treatment of cancer as well cardiovascular diseases. [79].
Sahreen et al., (2011) have reported that methanolic extract of Carissa opaca (MCL)leaves showed significant effect to prevent hepatotoxicity in induced rats with CCl4, elevates level of AST, ALT, ALP, LDH. Different extracts obtained from MCL and silymarin recovered all the changes observed with CCl4-treated rats. MCL phytochemical investigation showed the presence of alkaloids, anthraquinones, cardiac glycosides, tannins, flavonoids, coumarins, terpenoids and phlobatannins [80].
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Chapter 2 Literature Review
Bhatt et al., (2015) studied that Sonchus asper play a vital role to recover wounds, bronchitis, gastrointestinal infections, cough, inflammation, diabetic and cardiac disease. It has been also testified that S. asper also used for the treatment of kidney and liver injuries, inflammations and in jaundice. The methanolic extract of Sonchus asper showed significant effect in an alloxan treated diabetic rats [81].
Sharma et al., (2010) investigated Ficus glomerata plant for antidiabetic activity. It was found that leaves extract of F. Glomerata showed a potent antihyperglycemic effect in an alloxan induce rats [82].
Khan et al., (2016) have reported that the leaves extracts of Acacia modesta plant significantly reduced the elevated level of blood glucose, ALT, ALP, Craetinine, HDL, LDL in an alloxan induced diabetic rats [83].
Ghori et al., (2015) investigated that Glycosmis pentaphylla plant posess various secondry metabolites such as alkaloids, saponins, tannins, flavonoids, glycosides, carbohydrates and proteins. It was also submitted that Glycosmis pentaphylla extract have potent antidiabetic activity in an alloxan induced diabetic rats [84].
Bhatt et al., (2015) studied anti-diabetic activity (in-vitro& in vivo) of Roylea cinerea. The methanolic extract of aerial part of Roylea cinerea (In vitro as well as In vivo) was found to exhibit potent antidiabetic activity. The methanolic extract showed maximum reduction in blood glucose levels [85].
Farahnaz et al., (2016) studied antifungal and antibacterial P.granatum, Losonia inermis,
Murraya koenigii, Sysgium and Capsicum annum. They found that phytochemicals (steroids, tannin, terpeniods, anthocyanin, alkaloids and flavonoids) of leaves of respective plants control and prevent various diseases in vegetables and plants [86].
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Chapter 2 Literature Review
Gislene et al., (2000) has given the phytochemicals and antibacterial potential of different plants and found that extracts of plants Syzygyum joabolanum and Caryophyllus arometicus showed highest antimicrobial(In-vitro) and antidiabetic potential in alloxan treated rats [87].
Njangiru et al., (2017) has explored the effect of extract (aqueous) of Psiduim quajava in alloxan induced mice. Various doses of plant extract 50, 100, 200 and 300 mg/kg body weight were used. The plant extracts significantly recover the hyperglycemic effect in alloxan treated mice [88].
Khan et al., (2017) reported the effect of aqueous extract of Psiduim quajava in alloxan induced mice. Various doses of plant extract 50, 100, 200 and 300 mg/kg body weight were used. The plant extracts significantly recover the hyperglycemic effect in alloxan treated mice
[88].
Khan et al., (2017) investigated the antidiabetic effect of the aerial parts of Galium tricornutum in alloxan treated rats. Various concentration of methanolic extract 200 and 400 mg/kg body weight were used to check acute toxicity and later with glucose tolerance. All the extracts significantly recovered glucose levels in an alloxan induced diabetic rats [89].
Qumar et al., (2010) reported the antidiabetic potential of methanolic and aqueous extracts of
Erythrina indica in induced diabetic rats. In this experiment they injected alloxan through inter peritoneal. They orally administrated the aqueous and methanolic extracts for 10 days and found that the glucose level of rats was reduced towards normal level. From this investigation it was shown that Erythrina indica plant extract may be used against hyperglycemic effect [90].
Dholi et at., (2011) studied hypoglycemic effect of Azadirachta indica plant in diabetic rats.
During study they gave a dose (single) of Azadirachta indica 150 mg/kg body weight for 15
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Chapter 2 Literature Review days. Glucose level in rats was significantly decreased towards normal level. They also reported that the renal and kidneys function were recovered with the oral administration of
Azadirachta indica. From his study they concluded that Azadirachta indica may be used as a vital alternate source in the controlling of diabetes mellitus [91].
Medicinal plants possess various bioactive compound which can be used to treat a disease and a good source for developing valuable and potent novel drug (Hamamouchi; 2002).
Sigaroodi et al., (2012) studied 12 plant species for the determination of cytotoxicity and antioxidant potential. In this experiment they performed cytotoxicity against brine shrimp and were found that the extracts of Taverniera spartea and Tephrosia persica showed more cytotoxicity. The plant Gleditschia caspica and Taverniera spartea extracts showed significant antioxidant activity [92].
Mesia et al., (2008) reported the cytotoxic potential of various extracts obtain from medicinal plants through MRC-5 cell line. The use of various doses shows significant effect on MRC-5 cell line [93].
Kilani et al., (2008) studied the effect of various medicinal plants extracts on L1210 cells.
Kiani evaluated the effect of extracts and tuber infusion of Cyperus rotundus (L) for cytotoxic activity, and revealed that cytotoxic compound present in it inhibit proliferation and growth of L1210 cells [94].
Hussain et al., (2007) studied various extracts of plant Fagonia cretica possess highly antitumour and cytotoxic potentials [95].
Similarly Dawidar et al., (1989) investigated chemically various extract of I. spicata and reported various phytochemicals [96].
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Chapter 3 Materials and Methods
Chapter No. 3
MATERIALS & METHODS
3.0 Materials and Methods
3.1 Collection and Extraction
The plant material i.e. Taverniera nummularia D.C and Ifloga spicata (Forssk) Sch.Bip) were collected from District Bannu Khyber Pakhtonkhwa, Pakistan. The plant specimens were confirmed by Dr. Fizan Ullah Assistant Professor in the Department of Botany UST, Bannu, and assigned a voucher numbers Tn-T-1(Taverniera nummularia) and Is-I5 (Ifloga spicata) respectively. Samples of both the plant species were kept for future studies in the herbarium of Department of Botany, University of Science and Technology, Bannu.
After collection both the plant species were first washed with running tap water followed by washing with distilled water. After drying under shade the dried material of T. nummularia (5
Kg) and Ifloga spicata (3Kg) were converted to fine powder and dipped in 80% aqueous methanol for seven days, and was subjected to filtration by a Whatman filter paper No.1.
After processing of the extract via rotary (Buchi Rota vapor R-200) at room temperature under vacuum, methanol was evaporated.
3.2 Fractionation
The dried material was kept in 80% methanol (aqueous) and portioned by using n-hexane to defat the material. Remaining filtrate was dissolved in water and fractionation was carried out with a variety of organic solvents like ethyl acetate and chloroform in an order of increasing polarity. All the fractions (five) viz. aqueous, methanolic, hexane, chloroform and ethyl acetate were reduced to dryness by processing them by using a rotary evaporator. For further analysis storage of all the fractions was made at 4C°.
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3.3 Phytochemicals Screening of the Crude Methanolic Extracts To detect the occurrence of different kinds of bioactive constituents in the extracts of plant species selected for study various chemical tests were performed through standard and established protocols.
3.3.1 Alkaloids
The 0.4 g extract of different fractions was mixed with 8 ml 1% HCl. Both the fractions warmed and then filtered. A part of filtrate 2 ml was titrated by Dragendroff’s reagent and
Mayer’s reagent. The turbidity of the precipitate ensures the occurrence of alkaloids [96]
3.3.2 Flavonoids
Ethyl acetate in a quantity of 10 ml was taken and mixed with minute quantity of different extract and boiled for 3 minutes and filtered. A part of filtrate (3ml) was mixed with 1ml of
Ammonium hydroxide. Yellow color appeared indicates the flavonoids presence [96].
3.3.3 Terpenoids
Simultaneously chloroform (2ml) and 3ml of Concentrated H2SO4 was added to 0.5g of each extract. Redish-Browm colouration of the interface indicated terpeniods presence [96].
3.3.4 Tannins
The plant extract (500ug) were put in 10ml of boiling water and then filtered. 0.1%FeCl3 was added which produced brownish green or blue-black coloration which indicated tannins presence [97].
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3.3.5 Saponins
The 0.5g of crude extract was dissolved in water, boiled and allowed to cool with continuous shaking. Persistent forth appeared which indicated saponins presence [96].
3.3.6 Sterols
The extracts 1ml was put into a test tube and adds 1000 ul of Concentrated H2SO4.Red color appearance indicates that Sterols is present [98].
3.3.7 Cardiac glycosides
The extract under study (0.5g) was diluted by (5ml) of D/H2O ,Glacial acetic acid (2ml) was added along with one drop of FeCl3 solution. The brown ring at the interface designates that cardaic glycosides is present [98].
3.3.8 Anthraquinones
Crude extract 0.5g was boiled for few minutes by using water bath, filtered and allowed to cool. Chloroform with equal volume was added to filtrate along with few drops of aqueous
NH3 (10%). The entire mixture was again heated. Appearance of pink rose color indicates
Anthraquinones presence [98].
3.4 Antibacterial Assay
The activity of extract against selected bacterial strains was carried out by agar well diffusion method. For preparation of samples, 15mg/ml dimethyl sulfoxide (DMSO) solutions were constructed for each fraction of the plant. Similarly solutions of Roxithromycin and
Cefixime-USP (1 mg/ml each) were used as control (positive). Pure DMSO was incorporated as a -Ve control. Three bacterial strains viz. Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were cultured on solidified nutrient agar medium in petri
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Chapter 3 Materials and Methods plates separately. Wells were prepared in solidified agar nutrient medium in petridishes.
Using a 0.1 ml pipette, 100 µl of the test sample of each fraction was poured into the individual wells through a micropipette. The petriplates were incubated at 37oC in an incubator for a period of 24 h. After completion of incubation period, zone of inhibition (mm) of all the extracts was measured [99].
3.5 Antifungal Assay
The antifungal activity was determined by using well known agar tube dilution method as given by Duraipandiyan and Ignacimuthu (2009). The solutions of 12mg/ml of methanolic, chloroform, n-hexane, ethyl acetate and water of the plant were prepared in DMSO.
Terbinafine (Positive control/ antifungal agent) at 1 mg/ml was also prepared in the DMSO.
Negative control has only pure DMSO. Fungi were cultured on solidified Sabouraud dextrose agar in test tubes. The sample extracts of various fractions (67µl) with a final concentration of 0.2 mg/ml was put in the test tubes individually. The test tubes were placed in an incubator at 28oC for seven days. The linear growth of all the fungal strains in test tubes was measured in mm. The % inhibition in linear growth of fungi was determined by the formula given below:
% inhibition growth = (dc-dt/dc) x100
Negative control group is represented by c whereas sample growth is given by t.
Source of the Tested Microbial Strains
The tested microbial strain were obtained from Department of Biotechnology Lab having
ATCC codes (American type culture collection-Manassas Virgina USA).
Bacterial Strains
Pseudomonas aeruginosa ATCC 27853,
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Chapter 3 Materials and Methods
Escherichia coli ATCC 25922,
Staphylococcus aureus ATCC 29213
Fungal Strains
Aspergillus flavus ATCC 9643
Aspergillus fumigatus ATCC 204305
Aspergillus niger ATCC 16404
3.6 DPPH Free Radical Scavenging Activity
For antioxidant activity (DPPH free radical scavenging assay, the method of Bibi et al.,
(2011) was used. Pure DPPH (5 mg) was put in CH3-OH (100 ml) in a flask. The O.D of the solutions was taken to 0.9 at 517 nm. A 100 μl of the various solvent fractions of the plant
(250, 500, 1000 μg/ml in individual solvent) was mixed with 900 μl of the DPPH solution.
Incubation of test tubes was done at normal room temperature for 25 minutes. At 517 nm the absorbance of all the tested samples was determined.
(%) age scavenging potential = absorbance (Control) - absorbance (sample) × 100 [(Control absorbance)] During assay Ascorbic acid was used as a positive control.
3.7 Hydrogen Peroxide Scavenging Effect
First prepared 2 mM Hydrogen peroxide solution in in phosphate buffer (50 mM; pH 7.4).0.1 ml of various fractions was taken in to the test tubes and 50 mM phosphate buffer (pH 7.4) was further put to make volume up to 400ul. Stock solutions of the various solvent fractions were made in phosphate buffer to achieve the concentration of 50 µg/ml, 100 µg/ml, 250
µg/ml and 500 µg/ml. 0.6 ml of hydrogen peroxide solution was added to each solvent fraction in the tubes, tubes were subjected to spectrophotometer absorbance. The absorbance
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Chapter 3 Materials and Methods of all fractions was made at 230 nm after an interval of 10 minutes as given by Ruchet al.
(1989). Following equation was used for estimation of hydrogen peroxide scavenging activity.
3.8 Cytotoxicity
3.8.1 Lymphocyte Isolation
The blood samples were collected from volunteers (age 24-26 years old) for the isolation of blood lymphocytes. The blood samples (15 ml) were taken in sterile test tubes and diluted with an equal quantity of phosphate saline buffer having a pH of 7.4. The samples were centrifuged and the precipitate obtained was discarded. A 3 ml solution of ficoll-hypaque was added to 10 ml of blood by using a sterile pipette. After an hour of centrifugation (200 x g) lymphocytes collection was made above the ficoll-hypaque layer and added with a phosphate saline solution.
3.8.2 Trypan Blue Staining
Solution having lymphocytes was diluted by using cell culture medium (RPMI-1640, thermoscientific). The sample of lymphocytes (10 µl) was added to a 0.2% trypan blue stain
(10 µl) and was loaded to a haemocytometer and seen using a light microscope. The dead
(stained) and non-stained cells were counted for determination of the % of viable cells. It was found that 85% of cells per sample were alive.
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3.8.3 Cell Culturing and Treatments
The samples having viable cells and diluted with cell culture medium were subjected to haemocytometer for count. The solutions having viable cells were further diluted to a concentration of 1×108 /ml and incubated either with pure cell culture medium or medium having added plant extracts at concentrations of 1, 10, 100 µg/ml respectively for 120 minutes at a temperature of 37Co. The cultures of cells were subjected to centrifugation
(200×g) for 10 minutes. The pellet having isolated cells was collected whereas supernatant was discarded. The pellet of cells was dispersed in a PBS (1×106) and preserved at -20Co for further experimentation.
3.9 Biochemical Analysis
The cells were taken and lysed by using ultra-sonication. The obtained lysate was utilized for antioxidant enzymes activity and level of the reactive oxygen species determination.
3.10 Superoxide Dismutase Activity (SOD)
For determination of activity of SOD, the procedure of Marklund & Marklund (1974) was followed by taking optical density of the supernatant at 470 nm by using a spectrophotometer.
The activity of SOD was taken as the enzyme amount which is necessary to dismutase in one minute 50% of the superoxide radicals. The activity of SOD unit was demonstrated as mU/106 cells.
3.11 Catalase Activity
A reagent of dichromate/acetic acid having potassium dichromate solution in acetic acid (5%) was prepared (1:3). A 0.2 M H2O2 was mixed with phosphate buffer (0.01 M). The method of
Sinha (1972) was followed for the determination of activity of catalase. Our reaction mixture
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Chapter 3 Materials and Methods had 1000ul of buffer (phosphate buffer 0.01 M), 0.4ml of hydrogen peroxide (2M) and tissue homogenate (0.1 ml). After completion of incubation period with the addition of dichromate- acetic acid reagent (2 ml) the reaction was stopped.Absorbance measured at 530 nm. The
Catalase activity was taken into account as μM of H2O2 consumed/min/mg protein.
3.12 Peroxidase Activity
Peroxidase was determined through the procedure of Carlberg & Mannervik (1975). A homogenate of 0.1 ml was prepared having 0.1 ml guaiacol, 0.3 ml H2O2 (40 mM) and 2.g ml phosphate buffer (50 mM). After a minute of incubation the change in color of mixture was calculated at 470 nm by using spectrophotometer. POD unit was measured as a change in the absorbance of 0.01unit/minute.
3.13 TBARS Estimation
Li et al. (2010) method was used for the estimation of TBARS. The content of TBARS was estimated with absorption (535 nm) at 156 mM/cm. Units of TBARS content was nano-moles per 106 cells.
3.14 Determination of Reactive Oxygen Species (ROS)
Using Hayashi et al., (2007) method reactive oxygen species was determined. A cell suspension (5 µl) or H2O2 (used as standard) was mixed with buffer solution of sodium acetate (pH 4.8) in a well plate. The mixture was subjected to incubation at a 37 Co for 5 minutes. A solution (100 µl) composed of DEPPD and FeSO4 (mixed in ratio of 1:12) was put into the wells and incubated for 60 seconds at a 37 Co. Absorbance of the samples in wells was determined at 505 nm for three minutes at a time period of 5 seconds using a micro plate reader. The ROS level was taken into account as Unit/106 cells.
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Chapter 3 Materials and Methods
3.15 In-vivo Pharmacological Assessment of Various Extracts
3.15.1 Experimental Animals
In present study White Albino mice (age 3-4 weeks), 20-22 g weighed with a mean of 21 g weight were used. White Albino mice kept under control condition i.e temperature 25-27ºC with 12 hrs darkness photoperiod with rodent pellets as feed along with water.
3.15.2 Induction of Hyperglycemia
First the experimental animals were fasted for 8-12 hrs openly allowed to water until the end of this experiment.
Hyperglycemia was brought experimentally by a single intra peritoneal administration of 150 mg/kg body weight of a freshly prepared alloxan10% monohydrate obtained from Sigma
(Steinhein, Switzerland). Blood glucose level was checked using glucometer after 48- Hours after alloxan administration. Mice with 200mg/dL blood glucose level were found diabetic and used for further biochemical investigation.
3.15.3 Experimental Designing
In present study experimental models i.e mice divided randomly into seven groups, each group with five animals (mice). Group I consisted of normal mice orally directed with 0.1 ml physiological saline; Group II consisted of alloxan induced diabetic mice (150mg/Kg) orally administered with 0.1 ml physiological saline; Group III consisted of alloxan treated mice orally managed with 10 mg glibenclamide .Group 1V and Group V consisted of extract of
150mg/kg and 300mg/kg (Taverniera nummularia). Group VI and Group VII consisted of extract of 150mg/kg and 300mg/kg (Ifloga spicata).
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3.15.4 Acute Toxicity Testing
The experiment was undertaken to check the acute toxic behavior of the various extracts of T. nummularia and I. spicata in albino mice. For determination of acute toxicity (Gupta et al.,
2004) [119] method was used. All the mice were divided into groups (n=5). Group I consisted of diabetic control mice orally directed with 0.1 ml physiological saline, Group II consisted of aqueous extracts of T. nummularia at a dose of 150 mg/kg and 300 mg/kg and the extract of Ifloga spicata at dose of 150 mg/kg and 300 mg/kg I. spicata wase orally administrated to each mice. All the mice were examined for 0, 30, 60 min, and followed by 2,
5, 24 and 48 hours for any toxic effect and physiological behavioral changes. Since the extracts of T. nummularia plant were safe at dose of 150mg/kg and 300 mg/kg and I. spicata wase safe at dose of 150mg/kg 300 mg/kg body weight.
3.16 Investigation of Blood Chemistry
In the study we used various assay to check the pharmacologically important activities of extract in mice subjected to alloxan induced diabetes.
3.16.1 Analysis of Serum Chemistry
Different kits (AMP Diagnostics Company) used to estimate Glucose, Creatinine Total protein, Urea, in serum of alloxan induced diabetic mice.
3.16.2 Total Protein Estimation
Principle In order to determine the Total protein (serum), colored complex is estimated in alkaline solution in the presence of salt of copper.
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Method Using kit (AMP Diagnostics).
10 µl sample/ standard (albumin) was taken and mixed well with 1 ml reagent provided in kit.
Absorbance was noted at 550 nm, incubation time period was kept 10 mintus.
For blank 10 µl distilled water Absorbance was again noted at 550nm, incubation time period was kept 10mintus .Using the following formula total protein calculated as,
Sample Absorbance × n
Standard Absorbance
“n” is standard concentration.
3.16.3 Determination of Urea
Method
10 µl blood was mixed with 1ml reagent. Variation and standard optical density (OD) was recorded at 340 nm), time interval was 30 sec and 90 sec .Urea concentration was calculated using formula;
Urea concentration = Sample Optical density (OD) × n
Standard Optical density (OD) n stands for (concentration of standard).
3.16.4 Determination of Creatinine
Principle
Using the kit of AMP Diagnostics Company Ltd. the Craetinine level was estimated from rate of appearance of colorful complex among alkaline pirates.
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Procedure of Assay: 100 µl sample in a test tube was taken and mixed well with 1 ml
Reagent (R) possessing picric acid. After 25 sec time interval, absorbance was recorded at
500nm. 2nd reading (A2) was noted precisely 2 minutes after recording the 1st reading (A1),
The Craetinine concentration was calculated using following formula.
Second absorbance (A2) – (A1) first absorbance sample
A2-A1 standard x n
3.16.5 Determination of Bilirubin (Serum)
Principle
Sodium nitrate reacts with Sulfanilic acid, Diazotized sulfanilic acids were formed when sulfanilic acid reacts with sodium nitrate. Similarly azobilirubin formation took place DMSO presence when bilirubin as a whole reacts with sulfanilic acid (diazotized).
Method: Reagent (R1)1.5 ml was taken along with 0.5ml Reagent (R2) in a test tube, then mixed both the reagents with 100 µl sample. Temperature of the test kept at 37C0 Incubation period was five minutes and Optical density (OD) was measured at 555nm.
1.5 ml working reagent (R1) along with 500ul reagent (R2) mixed with 0.1 ml of sample.
Optical density for sample blank is without Reagent R2.
Using the following formula to calculate the Total bilirubin in serum
Sample Optical density (OD) - sample blank optical density (OD) × F
Standard concentration factor = F
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3.16.6 Determination of Cholesterol (Serum)
Serum Cholesterol was determined using kit AMP Diagnostics
Method:
10 µl Standard /sample was taken along with I ml AMP kit reagent. The sample was compared with blanks and noted optical density (OD) after 60 minutes. Temperature was kept
37Co and wavelength was taken 505.
Through the following formula Cholesterol level was measured.
Optical density (Sample) × n
Optical density Standard
Standard concentration = n
3.16.7 High Density Lipoproteins (HDL)
Two types of reagents R1 and R2 were used in the determination of HDL.
Reagent (R1) possess Antihuman β-lipoprotein antibody while reagent (R2) consist of CHE and cholesterol oxidase.R1 reagent was mixed with sample posse lipoprotein, then after addition of Reagent (R2) color changed to blue with addition of H2O2 produced by reactions of various enzymes indicates the presence of HDL. High density Lipoproteins (HDL) was noted at 593 nm.
Method: Sample (2.5 µl) along with the 2.5 µl calibrator was mixed, than after optical density (OD) was measured. 60 µl reagents R2 was added and mixed well again checked the absorbance after 5mintues incubation. Temperature was kept 37C0 at wavelength 600nm with
1 cm optical path against the blank reagent. HDL concentration was calculated using following formula.
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HDL concentration= (OD2-OD1) sample × n
(OD2-OD1) Calibrator
3.16.8 Low Density Lipoprotein (LDL)-Cholesterol Determination
For determination of LDL cholesterol was calculated from measured value of triglycerides and HDL-cholesterol using the following formula;
Low density lipoprotein (LDL) = TG/5+ HDL– cholesterol
3.16.9 Determination of Triglycerides
Procedure of Assay 10 µl distilled water was mixed with 1 ml of the reagent (R) containing
(Glycerol kinase, Potassium ferrocynate, Mg+2 and ATP), in the said procedure sample and standard used as blank. The temperature was kept 370C for short time (10 mintus) optical density (OD) measured at 500 nm. Triglyceride concentration was determine using the following formula,
Triglyceride concentration = Sample optical density (OD) × n
Standard optical density (OD)
3.16.10 Determination of Alanine Aminotransferase
Serum Alanine aminotransferase (ALT) was determined by using kit (AMP Diagnostics).
Working Reagent was prepared by mixing 400µl of R1 with 400µl of R2.
Procedure: The instructions provided with the kits were followed. 50µl serum mixed well with 800µl of working reagent, then optical density (OD) was measured.
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Calculation: Activity (U/L) = ΔOD/min.x1746
3.16.11 Determination Aspertate Aminotransferase
Serum alanine aminotransferase (ALT) was determined using pre-packed kits made by AMP
Diagnostics (AMP Medizintechnik GmbH, Graze, Austria).
Working Reagent was prepared by mixing 400µl of R1 with 400µl of R2.
Procedure: All those instructions provided with kits were kept in mind. The procedure for the determination of AST was the same as that for ALT. Again 50µl serum mixed well with
800µl of working reagent, then optical density (OD) was measured.
Calculation: Activity (U/L) = Optical density (ΔOD)
Mint x1746
3.17 Statistical Analyses
The data of cytotoxicity were subjected to analysis of variance (two ways ANOVA) whereas that of antimicrobial was analyzed by one way ANOVA. The mean values were matched by using least significant differences test (LSD).
Ethics Approval
The experimental protocol for the use of human blood lymphocytes as well use of Mice as experimental model was approved by research attics committee, Department of
Biotechnology University of Science and Technology Bannu.
Informed consent was obtained from the participants for blood collection and use of lymphocytes in experimental manipulation.
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Chapter No. 4
RESULTS
4.0 RESULTS
4.1 Phytochemical Analysis
Phytochemical analysis of various extracts was carried out and they were found of vital significance for the treatment of various ailments.
4.2 Qualitative Analysis of Taverniera nummularia and Ifloga spicata
Present investigation revealed that various bioactive constituents are present in leaf extracts of Taverniera nummularia and Ifloga spicata (Table 1:1).
Table1.1 Phytochemical Composition of leaf extracts of T. nummularia and I. spicata
Constituents T.nummularia extract I. spicata extract
Alkaliod + +
Steroids _ +
Terpeniods + +
Tannins + +
Glycosides + +
Saponins + +
Flavonides + +
Anthraquinon _ _
+ (presence) and (absence)
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4.3 In-vitro Pharmacological Evaluation of Various Extracts
In-vitro preliminary evaluation of both plants extracts T. nummularia and I. spicata various bioactive assay were used. The results obtained are as follows.
4.4 Antibacterial Activity of T. nummularia and I. spicata
The study revealed that CH3OH extract of T.nummularia possessed significantly higher antibacterial activity against S. aureus as compared to other fractions (p<0.05). Antibacterial activity of the ethyl acetate and chloroform fractions against S. aureus was statistically similar but significantly higher than aqueous and n-hexane fractions (Fig. 1a).The ethyl acetate, n-hexane and chloroform fractions exhibited higher and statistically similar antibacterial activity against P. aeruginosa. The ranking of various fractions for antibacterial activity against E. coli was as: methanolic = ethyl acetate = aqueous > n-hexane = chloroform.
Similarly methanolic extract of I. spicata exhibited significantly higher antibacterial activity against Pseudomnas aeruginosa as compared to other fractions (p<0.05). Antibacterial activity of the methanolic, ethyl acetate, n-hexane and chloroform fractions against S. aureus was statistically similar but significantly higher than aqueous aqueous fraction (Fig. 1b). The ranking of various fractions for antibacterial activity against E. coli was as: ethyl acetate = methanolic = aqueous > n-hexane = chloroform.
The readings of antibacterial activity were compared with a scale designed by Sirajuddin et al
(2012).
According to the scale the biological compounds having zone of inhibition less than 11-
14mm have non-significant activity. Those having zone of inhibition 15-17mm one are low antibacterial activity. While those having zone of inhibition greater than 20 mm are considered with a significantly higher antibacterial activity.
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In our study the methanolic fraction of T.nummularia showed significantly higher antibacterial activity against E.coli and S. aureus
Similarly the ethyl acetate and aqueous fraction have significantly higher antibacterial activity against E.coli, S. aureus and P. aeruginosa. The ethyl acetate, Hexane, chloroform and aqueous fraction have low antibacterial activity.
Similarly the methanolic fractions of I. spicata have significantly higher bactericidal activity against E.coli and P. aeruginosa. The aqueous and ethyl acetate fractions of I. spicata also slowed significantly higher antibacterial activity against E.coli. Whereas n-Hexane, chloroform and fractions have low antibacterial activity.
45 a 40 a a 35 30 25 b b b b c c 20 b c c 15 d b b 10 e c c 5 f d d
0
Zoneinhibitionon (mm)
Aqueous Aqueous Aqueous
n-hexane n-hexane n-hexane
Methanolic Methanolic Methanolic
Chloroform Chloroform Chloroform
Ethyl aectate Ethyl Ethylaectate Ethylaectate
Positivecontrol Positivecontrol Positivecontrol
Negetivecontrol Negetivecontrol Negetivecontrol Staphylococcus aureus Pseudomonas aeruginosa Escherichia coli Bacterial strains
Figure 1a: Antibacterial activity of the various fractions of Taverniera nummularia against
Staphylococcus aureus (Least Significant difference: 1.449), Pseudomonas aeruginosa (Least
Significant difference: 0.648) and Escherichia coli (Least Significant difference: 1.053).
Means with similar English letters don’t differ significantly.
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45 a 40 a 35 30 25 a b 20 c b b b 15 b b b b c c c c 10 c d 5 d e d
0
Zoneinhibitionon (mm)
Aqueous Aqueous Aqueous
n-hexane n-hexane n-hexane
Methanolic Methanolic Methanolic
Chloroform Chloroform Chloroform
Ethylaectate Ethylaectate Ethylaectate
Positivecontrol Positivecontrol Positivecontrol
Negetivecontrol Negetivecontrol Negetivecontrol Staphylococcus aureus Pseudomonas aeruginosa Escherichia coli Bacterial strains
Figure 1b: Antibacterial activity of the solvent fractions of I. spicata against S. aureus (least significant difference: 4.309), P. aeruginosa (least significant difference: 0.815) and E. coli
(least significant difference: 3.257). Means with similar English letters don’t differ significantly.
4.5 Antifungal Activity of Taverniera nummularia and Ifloga spicata
The various fractions of T.nummularia showed antifungal activity against the tested fungal strains. Maximum antifungal activity was reported against Aspergillus fumigates (59.25%),
Aspergillus flavus (62.02%) and Aspergillus niger (64%) was shown by chloroform fraction.
Next to the chloroform, methanolic fraction was more effective against Aspergillus fumigatus
(56.65%), n-hexane against Aspergillus flavus (48.5%) and ethyl acetate against Aspergillus niger (50.9%) (Fig. 2a).
Similarly various fractions of Ifloga spicata revealed significant antifungal activity against various fungus strains including Aspergillus fumigatus (59.050%), Aspergillus flavus (61%) and Aspergillus niger (64 %) was shown by chloroform fraction. Next to the chloroform, methanolic fraction was more effective against Aspergillus fumigatus (59 %), n-hexane
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Chapter 4 Results against Aspergillus flavus (46.260%) and ethyl acetate against Aspergillus niger (50.58%)
(Fig.2b).
The criteria for antifungal activity was based on percent inhibition in linear growth of fungi
(Sirajudin et al 2012).
According to the scale compounds showing 70% growth of inhibition were consider with a significant antifungal activity. Those compounds sowing 60-70 % inhibition of fungal growth were consider as good,50-60% inhibition activity was moderate, whereas compounds showing less than 50% inhibition of fungal growth were taken as non-significant.
In our studies chloroform fraction of T.nummularia has good antifungal activity against
Aspergillus niger, Aspergillus flavus and Aspergillus fumigatus. Similarly the methanolic fraction has moderate fungicidal potential against Aspergillus niger and Aspergillus fumigatus. Other fractions have low and non-significant antifungal activity against tested fungal strains.
Similarly chloroform fraction of I. spicata has good antifungal activity against all the strains of fungi. While ethyl acetate, aqueous fraction and n-hexane have low and non-significant antifungal activity against tested fungal strains.
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120 a a a 100 80 b b b c d c 60 c c e d d e f d f 40 20 f g g 0
-20
Aqueous Aqueous Aqueous
n-hexane n-hexane n-hexane
Methanolic Methanolic Methanolic
Chloroform Chloroform Chloroform
Ethylaectate Ethylaectate Ethylaectate
Positivecontrol Positivecontrol Positivecontrol
%inhibition in linear growth offungus
Negetivecontrol Negetivecontrol Negetivecontrol Aspergillus flavus Aspergillus fumigatus Aspergillus niger Fungal strains
Figure2a: Antifungal activity of the various fractions of Taverniera nummularia against
Aspergillus flavus (Least Significant difference: 2.749), Aspergillus fumigatus (Least
Significant difference: 1.883) and Aspergillus niger (Least Significant difference: 0.702).
Means with similar English letters don’t differ significantly.
120 a a a 100 80 b b b b c c c 60 c c d d d e e e 40 20 f f f 0
-20
Aqueous Aqueous Aqueous
n-hexane n-hexane n-hexane
Methanolic Methanolic Methanolic
Chloroform Chloroform Chloroform
Ethylaectate Ethylaectate Ethylaectate
Positivecontrol Positivecontrol Positivecontrol
%inhibition in linear growth offungus
Negetivecontrol Negetivecontrol Negetivecontrol Aspergillus flavus Aspergillus fumigatus Aspergillus niger Fungal strains
Figure 2b: Antifungal activity of the solvent fractions of Ifloga spicata against A. flavus
(least significant difference: 4.015), A. fumigatus (least significant difference: 2.17) and A. niger (least significant difference: 1.595). Means with similar English letters don’t differ significantly.
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4.6 DPPH free Radical Scavenging Activity of T. nummularia and I. spicata
Antioxidant activity was measured as % reactive oxygen species scavenging activity of
DPPH, using ascorbic acid as a standard. Results showed that significantly higher antioxidant activity was recorded for methanolic extract followed by n-hexane and aqueous fractions at
1000µg/ml (p<0.05). At lowest concentration (250 µg/ml) the antioxidant activity was higher for methanolic extract (Fig. 3a).
Similarly the various fraction of Ifloga spicata showed significantly efficacy in scavenging of
DPPH free radicals with order of Methanol>Ethyl acetate>Chloroform>n-Hexane as shown in (Fig. 3b).
1000µg/ml 500µg/ml 250µg/ml
100 a 90 b d c 80 e f g f 70 h j i l n k m kl 60 o p 50 40 30
DPPH DPPH inhibition) (% 20 10 0 Methanolic Ethyl acetate n-Hexane Chloroform Aqueous Ascorbic acid Extract
Figure 3a: Antioxidant activity (DPPH method) of Taverniera nummularia (Least
Significant difference: 0.273). Means with similar English letters don’t differ significantly.
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1000µg/ml 500µg/ml 250µg/ml
100 a 90 b 80 c c d d 70 e e f f 60 g i f h h 50 i i i 40
30 DPPH DPPH inhibition) (% 20 10 0 Methanolic Ethyl acetate n-Hexane Chloroform Aqueous Ascorbic acid Extract
Figure 3b: Antioxidant activity (DPPH method) of Ifloga spicata (Least Significant difference: 0.293). Means with similar English letters don’t differ significantly.
4.7 Hydrogen Peroxide Scavenging Activity of T. nummularia and I. spicata
H2O2 scavenging activity was concentration dependent. All the solvent fractions were found to have significant scavenging ability (Fig. 4a). At higher concentration of the extracts
(500 µg/ml) maximum scavenging activity was shown for ethyl acetate fraction (85%).
Whereas lowest for aqueous (40%). H2O2 scavenging activity of the various solvent fractions of Taverniera nummularia were were occurred in basipetal manner of ethyl acetate
>methanolic extract > n-hexane > chloroform and aqueous. Fig. 4b revealed that ethyl acetate fraction (83.33%) of Ifloga spicata showed maximum scavenging activity as compare with aqueous (8.33%) and other fractions with order of ethyl acetate >methanolic extract > n- hexane > chloroform and aqueous.
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500µg/ml 250µg/ml 100µg/ml 50µg/ml
120 a b 100 b d c e 80 f g g 60 h h h h j i %inhibition 40 k k k l l m n 20 no o
0 Methanolic Ethyl acetate n-Hexane Chloroform Aqueous Control Extract
Figure 4a: Antioxidant activity (H2O2 scavenging method) of T. nummularia (Least
Significant difference: 2.850). Means with similar English letters don’t differ significantly.
500µg/ml 250µg/ml 100µg/ml 50µg/ml
120 a a 100 a ab b 80 c d e 60 f g gh hi i ghi
%inhibition 40 j j j k l kl kl m m 20 n 0 Methanolic Ethyl acetate n-Hexane Chloroform Aqueous Ascorbic acid Extract
Figure 4b: Antioxidant activity (H2O2 scavenging method) of I. spicata (least significant difference: 3.779). Means with similar English letters don’t differ significantly.
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4.8 Effect of T. nummularia on ROS Content of Lymphocytes
The H2O2 stress resulted in elevated level of ROS in lymphocytes as compared to control (Table 2a). Under normal condition extract of
T.nummularia has no significant on ROS contents in lymphocytes. However the increase in ROS due to H2O2 treatment was significantly reduced by various fractions of T.nummularia. Most effective concentrations of the various fractions were 5ug/ml and 50ug/ml. The level of ROS (p>0.05) was significantly recovered by methanol and ethyl acetate fraction. 6 Table 2: Effect of Taverniera nummularia on ROS content (Units / 10 cells) of lymphocytes under H2O2 induced oxidative stress Treatment Fractions Mean
Methanol Ethyl acetate n-hexane Chloroform Aqueous
Control 0.134±0.02g-j 0.134±0.02g-j 0.134±0.02g-j 0.134±0.02g-j 0.134±0.02g-j 0.134±0.02de
a a a a a a H2O2 (100µM) 0.284±0.04 0.284±0.04 0.284±0.04 0.284±0.04 0.284±0.04 0.284±0.04
Extract 0.5 µg/ml 0.132±0.01g-j 0.166±0.01e-h 0.153±0.04f-i 0.129±0.01g-j 0.154±f0.02-i 0.147±0.12d
Extract 5 µg/ml 0.130±0.06g-j 0.142±0.07g-j 0.123±0.02h-j 0.127±0.02g-j 0.125±0.04g-j 0.129±0.04de
Extract 50 µg/ml 0.100±0.01j 0.131±0.03g-j 0.125±0.01g-j 0.108±0.06ij 0.122±0.05h-J 0.117±0.03e
ab ab ab ab a-d b H2O2 (100µM)+Extract 0.5 µg/ml 0.264±0.08 0.278±0.01 0.257±0.06 0.253±0.05 0.245±0.01 0.259±0.04
e-g ab a-d d-f b-d c H2O2 (100µM)+Extract 5 µg/ml 0.173±0.01 0.255±0.04 0.236±0.04 0.201±0.02 0.234±0.01 0.220±0.03
e-h ab a-c c-e a-d c H2O2 (100µM)+Extract 50 µg/ml 0.162±0.01 0.269±0.02 0.251±0.01 0.203±0.05 0.239±0.04 0.225±0.03
Mean 0.172±0.02c 0.207±0.01a 0.195±0.03ab 0.179±0.02bc 0.192±0.01ab ± represents value of standard error. Means sharing common English letters are statistically similar. (Treatment: Least significant difference 0.021, Fractions: Least significant difference 0.017, Treatment x Fraction: Least significant difference 0.048).
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4.9 Effect of T. nummularia on Antioxidant Enzymes Activity of Lymphocytes
The SOD, CAT and POD activities of lymphocytes were significantly decreased under H2O2 induced oxidative stress as compared to control (p<0.05). Under unstressed conditions the various leaf fractions of T.nummularia at 5µg/ml and 50µg/ml significantly increased antioxidant
enzymes activity than control. The extracts at all the concentrations minimized adverse effects of H2O2 stress on antioxidant enzymes activity. Among the various fractions, methanol and aqueous fraction was significantly more effective (Table 3.1-3.3).
6 Table 3. 1: Effect of Taverniera nummularia on SOD activity (milli Units / 10 cells) of lymphocytes under H2O2 induced oxidative stress
Treatment Fractions Mean
Methanol Ethyl acetate n-hexane Chloroform Aqueous
Control 9.244 ±1.05de 9.244 ±1.05 de 9.244±1.05de 9.244±1.05de 9.244±1.05de 9.244 ±1.05b
k k k k k e H2O2 (100µM) 4.036 ± 0.31 4.036 ± 0.31 4.036±0.31 4.036 ±0.31 4.036±0.31 4.036 ± 0.31
Extract 0.5 µg/ml 10.648±3.14a-d 10.124±1.98 a-e 9.152±2.10e 9.110± 1.69ef 9.610± 2.13b-e 9.729 ±2.21ab
Extract 5 µg/ml 10.284±1.97a-e 9.552 ±2.18c-e 10.156±3.19a-e 9.870±1.26b-e 11.082 ± 3.16ab 10.189±2.36a
Extract 50 µg/ml 10.749±2.16a-c 10.478±1.79a-e 9.658 ± 2.17b-e 7.668± 1.95fg 11.484 ±2.10a 10.007±2.03a
k i-k jk i-k g-i d H2O2 (100µM)+Extract 0.5 µg/ml 5.297 ±0.98 5.016 ± 0.41 4.492 ± 0.50 4.896 ±0.21 6.302 ± 0.71 5.201 ±0.56
gh hi i-k k ij cd H2O2 (100µM)+Extract 5 µg/ml 7.367 ±0.31 6.094 ± 0.95 5.330 ± 0.24 4.172 ±0.37 5.770 ± 0.29 5.747± 0.43
gh i-k i-k i-k fg c H2O2 (100µM)+Extract 50 µg/ml 7.547 ±0.29 5.262 ±0.38 5.112 ± 0.20 4.966±0.29 7.635 ± 1.00 6.104±0.43
Mean 8.147± 040a 7.476±0.41b 7.148±0.44bc 6.745± 0.12c 8.145 ±0.38a ± represents value of standard error. Means with similar English letters don’t differ significantly. (Treatment: Least significant difference 0.662, Fractions: Least significant difference 0.523, Treatment x Fraction: Least significant difference 1.481).
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6 Table 3. 2: Effect of Taverniera nummularia on CAT activity (milli Units / 10 cells) of lymphocytes under H2O2 induced oxidative stress
Treatment Fractions Mean
Methanol Ethyl acetate n-hexane Chloroform Aqueous
Control 2.810±0.13b-e 2.810± 0.13b-e 2.810± 0.13b-e 2.810±0.13b-e 2.810± 0.13b-e 2.810± 0.13a
n n n n n c H2O2 (100µM) 0.946±0.17 0.946± 0.17 0.946± 0.17 0.946± 0.17 0.946±0.17 0.946± 0.17
Extract 0.5 µg/ml 3.356± 0.21ab 2.750±0.20 b-e 2.356± 0.19e-h 2.690± 0.15b-e 2.707± 0.18b-e 2.772± 0.19a
Extract 5 µg/ml 3.230± 0.24a-c 2.550± 0.18d-f 2.618± 0.13c-e 2.886 ± 0.10b-e 2.876±0.12b-e 2.832± 0.15a
Extract 50 µg/ml 3.698± 0.19a 2.534± 0.31d-f 3.126± 0.54a-d 2.716± 0.40b-e 2.942±0.37b-e 3.003± 0.36a
f-j n n j-n i-m b H2O2 (100µM)+Extract 0.5 µg/ml 1.902± 0.11 0.988± 0.02 0.932± 0.18 1.448± 0.12 1.672± 0.26 1.388± 0.14
d-g mn mn j-n h-l b H2O2 (100µM)+Extract 5 µg/ml 2.460± 0.10 1.009± 0.31 1.052± 0.28 1.290±0.10 1.724± 0.30 1.507± 0.22
e-i l-n k-n j-n g-k b H2O2 (100µM)+Extract 50 µg/ml 2.316 ± 0.14 1.084± 0.29 1.188± 0.28 1.292±0.18 1.842± 0.20 1.544± 0.22
Mean 2.590± 0.40a 1.834± 0.47c 1.879± 0.39c 2.0097±0.58 bc 2.1899± 0.39b
± represents value of standard error. Means with similar English letters don’t differ significantly. (Treatment: Least significant difference 0.299,
Fractions: Least significant difference 0.236, Treatment x Fraction: Least significant difference 0.670).
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6 Table 3. 3: Effect of Taverniera nummularia on POD activity (nmol / 10 cells) of lymphocytes under H2O2 induced oxidative stress
Treatment Fractions Mean
Methanol Ethyl acetate n-hexane Chloroform Aqueous
Control 4.892±0.73ab 4.892±0.73ab 4.892±0.73ab 4.892±0.73ab 4.892±0.73ab 4.892±0.73ab
e e e e e d H2O2 (100µM) 2.272±0.18 2.272±0.18 2.272±0.18 2.272±0.18 2.272±0.18 2.272±0.18
Extract 0.5 µg/ml 4.622±0.27A-C 3.258± 0.14de 4.973±0.18ab 5.671±0.35a 5.217±0.28a 4.748±0.24b
Extract 5 µg/ml 4.650 ±0.16a-c 5.049±0.29ab 4.689±0.13a-c 5.160±0.20a 5.538±0.34a 5.017± 0.22ab
Extract 50 µg/ml 2.272±0.27e 5.312±0.27a 5.136±0.19a 5.250±0.42a 5.814±0.29a 5.434±0.29a
de e e e e cd H2O2 (100µM)+Extract 0.5 µg/ml 2.733±0.10 2.399±0.17 2.197±0.10 2.370±0.11 2.222±0.18 2.384± 0.26
de de de de c-e c H2O2 (100µM)+Extract 5 µg/ml 2.624±0.09 3.226±0.22 2.568±0.16 2.939±0.13 3.364±0.11 2.944± 0.14
de de de e b-d c H2O2 (100µM)+Extract 50 µg/ml 2.804±0.11 2.694±0.18 2.699±0.21 2.411±0.20 3.782±0.19 2.878± 0.18
Mean 3.782±0.38ab 3.638±0.25b 3.678±0.17ab 3.871±0.32ab 4.137±0.58a
± represents value of standard error. Means with similar English letters don’t differ significantly. (Treatment: Least significant difference 0.592,
Fractions: Least significant difference 0.468, Treatment x Fraction: Least significant difference 1.325).
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4.10 Effect of T. nummularia on TBARS of Lymphocytes
The lymphocytes treated with H2O2 exhibited higher TBARS activity (nano-moles per 106 cells) over control group. Under unstressed conditions various fractions of T.nummularia significantly reduced TBARS content in comparison to control (Table 4a). The raise in TBARS activity due to H2O2 treatment was significantly reduced by various fractions. The most effective concentrations were ug/ml and 50ug/ml. Among the various fractions, ethyl acetate, n-hexane and chloroform fractions were significantly more effective in reducing TBARS content under oxidative stress (p<0.05). 6 Table 4: Effect of Taverniera nummularia on TBARS (nano-moles/ 10 cells) of lymphocytes under H2O2 induced oxidative stress
Treatment Fractions Mean Methanol Ethyl acetate n-hexane Chloroform Aqueous Control 0.696±0.09f-j 0.696±0.09f-j 0.696±0.09f-j 0.696±0.09f-j 0.696±0.09f-j 0.696±0.01 d a a a a a H2O2 (100µM) 1.014±0.17 1.014±0.17 1.014±0.17 1.014±0.17 1.014±0.17 1.014 ±0.03a Extract 0.5 µg/ml 0.535±0.06k-m 0.631±0.07h-k 0.652±0.04 h-k 0.624 ±0.10h-l 0.652±0.07h-k 0.619 ±0.07e Extract 5 µg/ml 0.484±0.08lm 0.609±0.04i-l 0.683±0.07g-j 0.651±0.09h-k 0.589±0.02i-m 0.603±0.06e Extract 50 µg/ml 0.465±0.09m 0.587±0.04i-m 0.568±0.03 j-m 0.641±0.09h-k 0.558±0.02J-M 0.564±0.05e a-c a-c ab b-d d-h b H2O2 (100µM)+Extract 0.5 µg/ml 0.903±0.03 0.907±0.09 0.994± 0.05 0.874±0.08 0.756±0.08 0.887±0.12 c-f a-c a-d c-e d-h bc H2O2 (100µM)+Extract 5 µg/ml 0.831±0.05 0.910±0.04 0.883± 0.07 0.849±0.10 0.754±0.01 0.845±0.05 e-i c-e c-g a-c e-i c H2O2 (100µM)+Extract 50 µg/ml 0.72 ±0.09 0.844±0.05 0.813± 0.09 0.950±0.08 0.718±0.05 0.810 ±0.07 Mean 0.707±0.06b 0.7749±0.03a 0.788±0.02a 0.787±0.06a 0.717±0.046b ± represents value of standard error. Means with similar English letters don’t differ significantly. (Treatment: Least significant difference 0.062, Fractions: Least significant difference 0.049, Treatment x Fraction: Least significant difference 0.139).
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4.11 Effect of Ifloga spicata on ROS content of lymphocytes
The ROS level was higher in lymphocytes treated with H2O2 as compare to control group (Table 5).One group of lymphocytes which was not
treated with H2O2 and supplemented with various solvent fractions of Ifloga spicata has no alteration in ROS level. One increase in ROS level by
H2O2 stress was significantly recovered by Ifloga spicata extracts at 5 µg /ml and 50µg/ml methanolic and chloroform fractions being the most
effective ones (p>0.05).
Table 5: Effect of Ifloga spicata on ROS content of lymphocytes
Treatment Fractions Mean Methanol Ethyl acetate n-hexane Chloroform Aqueous E1 E2 E3 E4 E5 Control 0.1340 ±0.01g-j 0.1340±0.03g-j 0.1340±0.03g-j 0.1340 ±0.02g-j 0.1340 ±0.02g-j 0.1340 ±0.01de
H2O2 (100µM) 0.2840 ±0.5a 0.2840 ±0.02a 0.2840 ±0.016a 0.2840 ±0.05a 0.2840±0.01a 0.2840 ±0.016a Extract 0.5 µg/ml 0.1322±0.2 g-j 0.1664±0.05e-h 0.1532±0.05f-i 0.1298±0.01g-j 0.1540±0.05f-i 0.1471 ±0.02d Extract 5 µg/ml 0.1300±0.16 g-j 0.1418 ±0.03e-j 0.1226 ±0.04h-j 0.1268±0.2g-j 0.1250±0.01g-j 0.1292±0.05de Extract 50 µg/ml 0.1000±0.1 J 0.1312±0.01g-j 0.1246 ±0.02g-j 0.1078±0.04i-j 0.1216±0.04h-i 0.1170±0.03 e
H2O2 (100µM)+Extract 0.5 µg/ml 0.2642 ± ab 0.2778 ±0.013ab 0.2568±0.06 ab 0.2528±0.01ab 0.2448±0.03a-d 0.2593 ±0.01b
H2O2 (100µM)+Extract 5 µg/ml 0.1726±.0.1 e-g 0.2546±0.02ab 0.2364±0.01a-d 0.2013±0.011d-e 0.2338 ±0.01b-d 0.2197 ±0.001c
H2O2 (100µM)+Extract 50 µg/ml 0.1622 ±e-h 0.2694 ±0.01ab 0.2510 ±0.02a-c 0.2030 ±0.3c-e 0.2394 ±0.4a-d 0.2250 ±0.02c Mean 0.1724±0.01 c 0.2074 ±0.02a 0.1953 ±0.04ab 0.1799 ±0.07b-c 0.1921±0.03ab ± represents value of standard error. Means with similar English letters don’t differ significantly. (Treatment: Least significant difference 0.0218, Fractions: Least significant difference 0.0173, Treatment x Fraction: Least significant difference 0.0489).
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4.12 Effect of Ifloga spicata on Antioxidant Enzymes Activity of Lymphocytes
In compression to control group the SOD, CAT and POD activities of lymphocytes were significantly decreased under H2O2 induced oxidative
stress (p<0.05). Under normal conditions the various leaf fractions of Ifloga spicata at 50 µg/ml significantly increased antioxidant enzymes
activity than control. The extracts at all the concentrations minimized adverse effects of H2O2 stress on antioxidant enzymes activity. Among the
various fractions, methanol and aqueous fraction was significantly more effective (Table 5.1-5.3).
Table 5.1: Effect of Ifloga spicata on antioxidant enzymes (CAT) activity of lymphocytes
Treatment Fractions Mean Methanol Ethyl acetate n-hexane Chloroform Aqueous E1 E2 E3 E4 E5 Control 2.8100±0.03b-e 2.8100± 0.06b-e 2.8100±0.03b-e v 2.8100±0.03b-e 2.8100 ±0.03b-e 2.8100 ±0.01a
H2O2 (100µM) 0.9460±0.1n 0.9460 ±0.02n 0.9460±0.002n 0.9460±0.04n 0.9460 ±0.01n 0.9460 ±0.003c Extract 0.5 µg/ml 3.3560±0.305ab 2.7500±0.05b-e 2.3560 ±0.03e-h 2.6900 ±0.06b-e 2.7072±0.03b-e 2.7718 ±0.05a Extract 5 µg/ml 3.2300±0.01abc 2.5500±0.14d-f 2.6180±0.05c-e 2.8858±0.02b-e 2.8760 ±0.04b-e 2.8320 ±0.05a Extract 50 µg/ml 3.6980±0.02a 2.5340±0.01d-e 3.1260±0.04a-d 2.7158 ±0.3b-e 2.9420±0.03b-e 3.0032 ±0.03a
H2O2 (100µM)+Extract 0.5 µg/ml 1.9020 ±0.05f-j 0.9880 ±0.001n 0.9320 ±0.03n 1.4480±0.01j-m 1.6720±0.02i-m 1.3884 ±0.02b
H2O2 (100µM)+Extract 5 µg/ml 2.4600 ±0.03d-g 1.0088±0.09mn 1.0520±0.04nm 1.2900±0.04j-m 1.7240 ±0.06h-l 1.5070 ±0.04b
H2O2 (100µM)+Extract 50 µg/ml 2.3160 ±0.01e-i 1.0840 ±0.001mn 1.1880 ±0.01k-n 1.2920±0.02j-m 1.8422 ±0.03j-k 1.5444 ±0.01b Mean 2.5898 ±0.02a 1.8339 ±0.06c 1.8785±0.01c 2.0097 ±0.01bc 2.1899 ±0.001b ± represents value of standard error. Means with similar English letters don’t differ significantly. (Treatment: Least significant difference 0.2996, Fractions: Least significant difference 0.2369, Treatment x Fraction: Least significant difference 0.6700).
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Table 5.2: Effect of Ifloga spicata on antioxidant enzymes (POD) activity of lymphocytes
Treatment Fractions Mean
Methanol Ethyl acetate n-hexane Chloroform Aqueous E2 E1 E3 E4 E5
Control 4.8920 ±0.012ab 4.8920±0.21ab 4.8920± 0.03ab 4.8920±0.23ab 4.8920 ±0.04ab 4.8920 ±0.32b
H2O2 (100µM) 2.8280 ±0.03de 2.8280±0.01de 2.8280 ±0.12de 2.8280±0.11de 2.8280 ±0.21de 2.8280 ±0.02cd
Extract 0.5 µg/ml 5.0260 ±0.023ab 3.6460±0.3cd 5.8888± 0.00a 5.8888±0.04a 5.2174 ±0.04ab 5.1334 ±0.05ab
Extract 5 µg/ml 4.9400 ±0.033ab 5.1744 ±0.20ab 5.0534± 0.09ab 5.1600 ±0.21ab 5.5380 ±0.23a 5.1732±0.40ab
Extract 50 µg/ml 6.0382 ±0.01a 5.5900 ±033a 5.3374±0.01a 5.2900±0.11ab 5.8140 ±0.05a 5.6139±0.014a
H2O2 (100µM)+Extract 0.5 µg/ml 2.7326 ±0.02de 2.3994±0.11e 2.3888± 0.04e 2.3702±0.02e 2.2180 ±0.12e 2.4218±0.01d
H2O2 (100µM)+Extract 5 µg/ml 2.6240±0.05de 3.2262 ±0.03c-e 2.5680± 0.03de 2.9398 ±0.02c-e 3.3640 ±0.23c-e 2.9444 ±0.11cd
H2O2 (100µM)+Extract 50 µg/ml 3.2520 ±0.12c-e 2.4460 ±0.02de 2.6990± 0.04de 2.4926±0.01de 4.0960±0.32bc 2.9971 ±0.02c
Mean 4.0416 ±0.06ab 3.7752±0.001b 3.9569 ±0.02ab 3.9827±0.23ab 4.2459 ±0.22a
± represents value of standard error. Means with similar English letters don’t differ significantly. (Treatment: Least significant difference 0.4350,
Fractions: Least significant difference 0.4350, Treatment x Fraction: Least significant difference 1.2305)
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Table 5.3: Effect of Ifloga spicata on antioxidant enzymes (SOD) activity of lymphocytes
Treatment Fractions Mean
Methanol Ethyl acetate n-hexane Chloroform Aqueous E2 E1 E3 E4 E5
Control 9.244 ±0.23de 9.244±0.22de 9.244 ±0.01de 9.244 ±0.21de 9.244 ±0.36de 9.244 ±0.12b
H2O2 (100µM) 4.036 ±0.32k 4.036 ±032k 4.036±0.03k 4.036 ±0.04k 4.036 4.036±033e ±011k±0.01k
Extract 0.5 µg/ml 10.648±0.32a-d 10.124±00a-e 9.152±0.06e 9.110 ±0.23ef 9.610 ±0.21b-e 9.729 ±0.02ab
Extract 5 µg/ml 10.284±0.33a-e 9.552±0.4cde 10.156 ±0.55a-e 9.870±0.03b-e 11.082±0.01ab 10.189 ±0.24a
Extract 50 µg/ml 10.749±0.43abc 10.478 ±0.36a-e 9.658±0.6b-e 7.668 ±0.33fg 11.484 ±0.09a 10.007 ±056a
H2O2 (100µM)+Extract 0.5 µg/ml 5.297±0.22ijk 5.016±0.22ijk 4.492±0.23jk 4.896±0.6i-k 6.302 ±0.66g-i 5.201 ±0.42d
H2O2 (100µM)+Extract 5 µg/ml 7.367 ±0.22gh 6.094 ±0.01hi 5.330±0.22i-k 4.172±0.31k 5.770±0.33ij 5.747 ±0.06cd
H2O2 (100µM)+Extract 50 µg/ml 7.547 ±0.33gh 5.262 ±0.21ijk 5.112 ±0.01i-k 4.966 ±0.22i-k 7.635±0.55fg 6.104 ±0.1c
Mean 8.1466±0.11a 7.4757±0.22b 7.1475 ±0.33bc 6.7453±0.15c 8.1454±0.22a
± represents value of standard error. Means with similar English letters don’t differ significantly. (Treatment: Least significant difference 0.5239,
Fractions: Least significant difference 0.6627, Treatment x Fraction: Least significant difference 1.4818).
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4.13 Effect of Ifloga spicata on TBARS of Lymphocytes
In the present study the lymphocytes treated with H2O2 exhibited higher TBARS activity (nano-moles per 106 cells) over control group. Under unstressed conditions various fractions of I. spicata did not significantly affect TBARS in comparison to control (Table 6). Various fractions at
all the concentrations tested recovered significantly the elevated TBARS activity due to H2O2 treatment was significantly reduced by various fractions at all the concentrations tested. The most effective concentrations were 5ug/ml and 50ug/ml. Among the various fractions, methanol and aqueous fraction was significantly more effective in reducing TBARS content under oxidative stress (p<0.05). Table 6: Effect of Ifloga spicata on TBARS activity of lymphocytes Treatment Fractions Mean Methanol Ethyl acetate n-hexane Chloroform Aqueous E1 E2 E3 E4 E5 Control 0.6960±0.001f-j 0.6960 ±0.002f-j 0.6960 ±0.11f-j 0.6960±0.00f-j 0.6960 ±0.02f-j 0.6960 ±0.1a
H2O2 (100µM) 1.0144±0.00a 1.0144±0.09a 1.0144 ±0.00a 1.0144 ±0.001aA 1.0144±0.003a a 1.0144 ±0.00a Extract 0.5 µg/ml 0.5352 ±0.02k-m 0.6310±0.21h-k 0.6522 ±0.22h-k 0.6236±0.22h-l 0.6518 ±0.0021h 0.6188 ±0.001e Extract 5 µg/ml 0.4840±0.002lm 0.6088 ±0.31i- 0.6834±0.01g-j 0.6506 ±0.07h-k 0.5888±0.01i-m 0.6031 ±0.03e Extract 50 µg/ml 0.4652 ±0.00m 0.5874 I±0.01i-m 0.5680 ±0.20j-m 0.6410±0.10h-k 0.5580 ±0.001j-m 0.5639 ±0.03e
H2O2 (100µM)+Extract 0.5 µg/ml 0.9034 ±0.03abc 0.9074±0.56abc 0.9938±0.00ab 0.8742±0.003bcd 0.7564±0.01d-h 0.8870 ±0.11b
H2O2 (100µM)+Extract 5 µg/ml 0.8306±0.001c-f 0.9102 ±0.001abc 0.8832±0.51abcd 0.8492±0.005cde 0.7540 ±0.002d-h 0.8454 ±0.05bc
H2O2 (100µM)+Extract 50 µg/ml 0.7260 ±0.006e-i 0.8444 ±0.006cde 0.8130 ±0.00c-g 0.9500±0.002abc 0.7178±0.22e-i 0.8102 ±0.12c Mean 0.7068±0.03b 0.7749 ±0.004a 0.7880±0.00a 0.7874 ±0.00a 0.7171±0.00b ± represents value of standard error. Means with similar English letters don’t differ significantly. (Treatment: Least significant difference 0.0625, Fractions: Least significant difference 0.0494, Treatment x Fraction: Least significant difference 0.0625).
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4.14 In-vivo Investigation
In order to check the effect of Taverniera nummularia and Ifloga spicata extract on alloxan induced diabetic mice various parameters were studied.
4.15 Body Weight
In present studies it was checked that alloxan considerably decreased body weight of mice than control group measured at an interval of 5 days for 15 days. Co-administration of different doses of both plants Taverniera nummularia and Ifloga spicata extract rescue significantly in the body weight. Both plant species minimize adverse effects on body weight when supplement at 300 mg/kg body weight (Figure 5&6).
Day 0 Day 5th Day 10th Day 15th
200 a a a b 195 b b c c c c c cd c 190 ef f fg g 185 h h 180 i
Body weight weight Body (g) 175
170
165 Normal Control Diabetic Control Glibenclamide 150 (mg/Kg B.W) 300 (mg/Kg B.W) Treatments
Figure 5: Effect of T.nummularia methanolic extract on body weight of experimental mice under hyperglycemic conditions induced by alloxan (LSD: 1.725). Means with similar English letters are statistically similar.
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Day 0 Day 5th Day 10th Day 15th
200 a a a 195 cd c cd cd cd cd de 190 fg de jk h hi ij 185 jk h 180 l 175 hi
170 Body weight weight Body (g) 165 160 155 Normal Control Diabetic Control Glibenclamide 150 (mg/Kg B.W) 300 (mg/Kg B.W) Treatments
Figure 6: Effect of I. spicata methanolic extract on body weight of experimental mice under hyperglycemic conditions induced by alloxan (LSD: 1.533). Means with similar English letters are statistically similar.
4.16 Blood Glucose Level
Glucose level of blood significant and continuous increase in alloxan treated groups after 15 days of the treatment (Figure 7&8).Groups of mice treated with Tarveneria nummularia, Ifloga spicata and glibenclamide exhibited recovery significant in the level of blood glucose than diabetic control measured at 0th day-15st days of the experiment. The most effective dose of
Taverniera nummularia and Ifloga spicata for reducing blood glucose level was 300 mg/kg body weight after 10 and 15 days of alloxan treatment.
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Day 0 Day 5th Day 10th Day 15th
350 a a 300 c b d e 250 f g g 200 i i j i j h l k 150 n m n
Glucose level 100 50 0 Normal Control Diabetic Control Glibenclamide 150 (mg/Kg B.W) 300 (mg/Kg B.W) Treatment
Figure 7: Effect of T.nummularia crude methanolic extract on blood glucose level of experimental mice under hyperglycemic conditions induced by alloxan (LSD): Means with similar English letters are statistically similar.
Day 0 Day 5th Day 10th Day 15th
350 a a 300 c b d e 250 g f i h i 200 k j l m n o 150 q p q
Glucose level 100 50 0 Normal Control Diabetic Control Glibenclamide 150 (mg/Kg B.W) 300 (mg/Kg B.W) Treatment
Figure 8: Effect of Ifloga spicata crude methanolic extract on blood glucose of experimental mice under hyperglycemic conditions induced by alloxan (LSD): Means with similar English letters are statistically similar.
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Chapter 4 Results
4.17 Serum ALP, ALT and Total Bilirubin
In alloxan induced mice irrespective of pancreas other body parts such as liver and kidney also affected by diabetes mellitus. The alloxan treated mice exhibited a significant increase in total bilirubin, ALT, and ALP as compared with control group. Second treatment with extracts of
Taverniera nummularia and Ifloga spicata significantly decreased total bilirubin, ALT, and ALP level caused by alloxan (figure No 9a,b,c). First results showed the elevated serum level of total bilirubin (1.81±0.7), ALP (203.7±9.7) and ALT (49±4.1), in a comparison of normal control group having total bilirubin (0.72±0.6), ALP (118.2±9.4) and ALT (27±4.2).
60 a a 50 b b c c 40 c d d d 30
ALT ALT (u/l) 20 10
0
B.W B.W B.W B.W
Glibenclamide Glibenclamide
Normal control Normal control
Diabeticcontrol Diabeticcontrol
Extractmg/Kg 150 Extract mg/Kg 150 Extract mg/Kg 300 Extract mg/Kg 300 Taverniera nummularia Ifloga spicata Treatment
Figure 9a: Effect of Taverniera nummularia and Ifloga spicata extract on ALT test. Means with similar English letters are statistically similar
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250 a a 200 b b c d d c 150 e e
100 ALP ALP (µ/l)
50
0
Glibenclamide Glibenclamide
Normal control Normal control
Diabeticcontrol Diabeticcontrol
Extractmg/Kg 150 B.W Extract mg/Kg 300 B.W Extract mg/Kg 150 B.W Extract mg/Kg 300 B.W Taverniera nummularia Ifloga spicata Treatment
Figure 9b: Effect of Taverniera nummularia and Iflago spicata extract on ALP test. Means with similar English letters are statistically similar.
a a
) 1.6 1.4 1.2 b ab 1 bc bc b 0.8 c b b 0.6 0.4 0.2
0
Bilirubin (mg/dl
B.W B.W B.W B.W
Glibenclamide Glibenclamide
Normal control Normal control
Diabeticcontrol Diabeticcontrol
Extract mg/Kg 150 Extract mg/Kg 300 Extract mg/Kg 150 Extract mg/Kg 300 Taverniera nummularia Ifloga spicata Treatment
Figure 9c: Effect of Taverniera nummularia and Ifloga spicata extract on Bilirubin test. Means with similar English letters are statistically similar.
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4.18 Renal Function Tests
The effect of extracts of Taverniera nummularia and Ifloga spicata on urea, creatinine and total proteins are shown in the Figure No10a,b,c The diabetic groups of mice when compared to control group (p<0.05) elevate the serum level of urea, creatinine, and total proteins. The most effective dose of both the plant species in protecting kidneys from adverse effects on hyperglycemia was reported at 300 mg/kg body weight.
140 a a 120 b b d c 100 c d 80 60 e e 40 Urea(mg/l) 20
0
B.W B.W B.W B.W
Glibenclamide Glibenclamide
Normal control Normal control
Diabeticcontrol Diabeticcontrol
Extract mg/Kg 150 Extract mg/Kg 300 Extract mg/Kg 150 Extract mg/Kg 300 Taverniera nummularia Ifloga spicata Treatment
Figure 10a: Effect of Taverniera nummularia and Iflago spicata extract on Urea test. Means with similar English letters are statistically similar.
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2.5 a a 2 1.5 b b b c 1 c d d d 0.5
0
Creatinine(mg/dl)
B.W B.W B.W B.W
Glibenclamide Glibenclamide
Normal control Normal Normal control
Diabeticcontrol Diabeticcontrol
Extract mg/Kg 150 Extract mg/Kg 300 Extract mg/Kg 150 Extract mg/Kg 300 Taverniera nummularia Ifloga spicata Treatment
Figure 10b: Effect of Taverniera nummularia and Iflago spicata extract on Craetinine test.
Means with similar English letters are statistically similar.
250 a a 200 b c b b d c 150 e d 100 50
0
TotalCH(mg/dl
Glibenclamide Glibenclamide
Normal control Normal control
Diabeticcontrol Diabeticcontrol
Extract mg/Kg 150 B.W Extract mg/Kg 300 B.W Extract mg/Kg 150 B.W Extract mg/Kg 300 B.W Taverniera nummularia Ifloga spicata Treatment
Figure 10c: Effect of Taverniera nummularia and Ifloga spicata extract on Total cholesterol Means with similar English letters are statistically similar.
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10 a a 9 b b 8 bc bc 7 c c cd d 6 5 4 3 2 1
0
Totalproteins (mg/dl)
B.W B.W B.W B.W
Glibenclamide Glibenclamide
Normal control Normal control
Diabeticcontrol Diabeticcontrol
Extract mg/Kg 150 Extract mg/Kg 300 Extract mg/Kg 150 Extract mg/Kg 300 Taverniera nummularia Ifloga spicata Treatment
Figure 10d: Effect of Taverniera nummularia and Ifloga spicata extract on Total protein. Means with similar English letters are statistically similar.
4.19 Serum Lipid Profile
Results presented in Figure 11a,b,c,d showed that alloxan treated group exhibited significantly higher triglycerides and LDL cholesterol level as compared to untreated control group (p<0.05).
On the other hand alloxan treatment significantly decreased the level of HDL cholesterol than untreated control group. The application of Taverniera nummularia and Ifloga spicata extracts significantly ameliorated adverse effects of alloxan on serum lipid profile. Extracts of both the plant species significantly decreased level of triglycerides and LDL cholesterol under hyperglycemic conditions. On the other hands extracts of both the plant species increased the level of HDL cholesterol. Effectiveness of both the extracts was higher when applied at 300 mg/kg body weight.
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250 a a 200 b c b b d c 150 e d 100 50
0
TotalCH(mg/dl
Glibenclamide Glibenclamide
Normal control Normal control
Diabeticcontrol Diabeticcontrol
Extract mg/Kg 150 B.W Extract mg/Kg 300 B.W Extract mg/Kg 150 B.W Extract mg/Kg 300 B.W Taverniera nummularia Ifloga spicata Treatment
Figure 11a: Effect of Taverniera nummularia and Ifloga spicata extract on Total Cholesterol test. Means with similar English letters are statistically similar.
250 a b a b 200 c d c e d e 150 100
TG TG (mg/dl) 50
0
B.W B.W B.W B.W
Glibenclamide Glibenclamide
Normal control Normal control
Diabeticcontrol Diabeticcontrol
Extract mg/Kg 150 Extract mg/Kg 300 Extract mg/Kg 150 Extract mg/Kg 300 Taverniera nummularia Ifloga spicata Treatment
Figure 11b: Effect of Taverniera nummularia and Ifloga spicata extract on Triglyceride test. Means with similar English letters are statistically similar.
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50 a b a b 45 c 40 d 35 d c 30 e e 25 20 15 HDL(mg/dl) 10 5
0
B.W B.W B.W B.W
Glibenclamide Glibenclamide
Normal control Normal control
Diabeticcontrol Diabeticcontrol
Extract mg/Kg 150 Extract mg/Kg 300 Extract mg/Kg 150 Extract mg/Kg 300 Taverniera nummularia Ifloga spicata Treatment
Figure 11c: Effect of Taverniera nummularia and Ifloga spicata extract on HDL test. Means with similar English letters are statistically similar.
160 a a 140 b b 120 d c d c 100 e e 80 60 40 LDL LDL (mg/dl) 20
0
B.W B.W B.W B.W
Glibenclamide Glibenclamide
Normal control Normal control
Diabeticcontrol Diabeticcontrol
Extractmg/Kg 300 Extract mg/Kg 150 Extract mg/Kg 300 Extract mg/Kg 150 Taverniera nummularia Ifloga spicata Treatment
Figure 11d: Effect of Taverniera nummularia and Ifloga spicata extract on LDL test. Means with similar English letters are statistically similar.
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Chapter 5 Discussions
Chapter No. 5
DISCUSSION
5.0 Discussion
Using of herbal medicines in the treatment of pathogenic diseases has been increased in recent years due to their effectiveness, sustainability and local availability [101]. The antimicrobial potential of plants is due to the occurrence of a variety of phytochemicals [102, 103].The disadvantages of the presently available synthetic drugs having propelled the discovery of novel pharmacological therapeutic agents from plants [104]. During present investigation two medicinal plants viz. T. nummularia and Ifloga spicata were evaluated for the composition of phytochemicals, antimicrobial, antioxidant and antidiabetic potential. The leaf crude methanolic extract of both the plant species exhibited the presence of alkaloids, tannins, glycosides, saponins and flavonoids. The other phytochemicals such as steroids, terpenoids and saponins were only found in leaf extract of I. spicata. It is reported in the previous studies that extracts of plant possesses various phytochemicals as steroids, terpenoids, flavonoids, saponins and alkaloids
[100]. Leaf extract of lawsonia inermis showed the presence of phytochemical constituents’
Terpenoids, tannins, saponins and alkaloids. Capsicum annum leaves extract detected phytochemical constituents which are steroids, flavonoids, tannins and alkaloids. Syzygium cumini leaves extract showed the presence of phytochemical constituent’s terpenoids, flavonoids, tannins and alkaloids. Whereas steroids, saponins and anthocyanins were absent [105]. These phytochemicals play crucial roles not only in the control of pathogenic diseases but also function as natural antioxidants [106]. The antimicrobial activities of natural compounds are helpful in the discovery of new and novel antibiotic compounds which can be used in the control of pathogenic
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Chapter 5 Discussions diseases of both animals and plants [107]. During current studies methanolic extract of
T.nummularia showed higher antibacterial activity E.coli and S. aureus. The ethyl acetate, n- hexane and chloroform fractions were highly effective against P. aeruginosa. On the other hand methanolic extract of Ifloga spicata was more effective against Pseudomnas aeruginosa. The ethyl acetate and chloroform fractions of Ifloga spicata inhibited the growth of S. aureus. Ethyl acetate fraction was more effective against E.coli. These results are in agreement with previous findings of [108], that methanolic extract of different parts of 39 plant species showed antibacterial activity against four species of G-positive bacteria (Bacillus cereus, E.faecalis, S.aureus and S.pyogenes) and four species of G-negative bacteria, namely
E.coli, K.pneumoniae, P.aeruginosa and Salmonella typhimurium). Various parts of plants such as leaves, stem, root, flowers, fruits and seeds are rich sources of phytochemicals with antimicrobial potential [109-111]. Therefore, the antibacterial activity of the T. nummularia and
I. spicata could be correlated with the presence of different types of phytochemicals present in them. The leaf extracts of the T.nummularia and I. spicata showed that maximum antifungal potential against A. fumigates, A. flavus and A. niger was shown by chloroform and methanolic fractions. Fungi are major causative agents of many human and plant diseases [112]. Use of plant extracts in the inhibition of fungal growth has been reported previously [101]. Studies of various extracts obtained from Zuccagnia punctata, Larrea cuneifolia and Larrea divaricata, showed substantial antifungal activity against most of the tested fungi. Similarly, Prosopanche americana also prevented the growth of yeast [102] Larrea divaricata has shown that methanolic and chloroform extracts of plants were highly effective in the inhibition of mycelia growth in
Aspergillus species. Hydrogen peroxide (H2O2) is produced in the cells during the process of oxidative metabolism. During normal metabolic process H2O2 is converted to water by
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Chapter 5 Discussions peroxiredoxins, glutathione, peroxidases and catalases [113]. However, when such mechanisms do not function properly, H2O2 reacts with transition metals and through the Fenton reaction produce hydroxyl radicals. This hydroxyl radical attacks the sugar component of the DNA resulting into single strand breaks of the DNA molecule [114]. During current studies the various solvent fractions of T. nummularia and Ifloga spicata exhibited antioxidant activity measured as DPPH and H2O2 scavenging method. The antioxidant activity of methanolic, n- hexane and aqueous fractions was higher. Previous studies have shown that oxidative stress is associated with pathogenesis and various chronic diseases and therefore antioxidant behavior is one of the most commonly determined biological activities for biologically active compounds
[115]. The T.nummularia and I. spicata exhibited antioxidant activity and therefore must be tested for cytotoxic and anticancer activities because compounds having a good antioxidant activity are potentially anticancer agents [116]. Similarly previously 112 plants species belonging to 50 different families, all these plants showed high antioxidant activity [105]. The methanolic fraction of T.nummularia and Ifloga spicata exhibited significantly higher antioxidant activity .It was also reported by Morgen in 2006 that methanolic extract of onion
(Allium cepa) have antioxidant effect [80].
Blood lymphocytes are immune cells and are important in studying toxicological effects of compounds and chemicals and are a good tool to study immune response of the body to external chemicals. In current studies various solvent fractions of T. nummularia and I. spicata were investigated in the amelioration of oxidative stress imposed by treating human blood lymphocytes with H2O2. It was observed that H2O2 induced stress was related with a higher level of reactive oxygen species and TBARS content. Similarly, the activity of antioxidant enzymes
SOD, POD and Catalase was decreased. Treatment of lymphocytes with the methanolic and
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Chapter 5 Discussions
aqueous fractions of T. nummularia and I. spicata under H2O2 induced stress effectively increased activity of antioxidant enzymes and resulted in a lower ROS content. The removal of reactive oxygen species by natural compounds is correlated with their antioxidant activity [117].
Water and methanol are polar solvents and are used to extract polar compounds. Both of these solvents have low toxicity and can be used with success in animal models. Similar results were found by Yen et al. (2001) when blood lymphocytes were treated with aqueous extract of P. oleracea under H2O2 induced oxidative stress. Oxidative stress results in damages to DNA and cause mutation. This study established that phytochemicals present in methanol and aqueous extracts are rich sources of antioxidants with a potential to detoxify ROS generated as a result of oxidative stress. Previous studies of (Al-Terehi, 2012) have shown that Glycyrrihza glabra root extract was highly effective to reduce oxidative stress in experimental animals. Plant extracts contain phenolics and flavonoids which have antioxidant activity because of their potential of donor proton to free radicals. Moreover, they have protective effects on antioxidant enzymes because they prevent oxidation of proteins by free radicals [118]. These reactive oxygen species cause damages to DNA resulting into mutation which leads to cancer [119]. It was established that T. nummularia and Ifloga spicata minimized adverse effects of H2O2 induced oxidative stress on human blood lymphocytes. Diabetes mellitus is a metabolic disorder in which there is a raise in blood sugar level that either results from acquired or inherited scarcity in the insulin production by the pancreatic islet cells of Langerhans or as results of improper response to insulin by the peripheral tissues [1-2]. In 2000 estimated that 171 million people were suffering from diabetes, and this number could total 366 million by 2030 [3-5]. 2012, 1.5 million deaths were reported to be directly caused by diabetes [6]. Therefore, in current studies in vivo antidiabetic activity of methanolic extract of Taverniera nummularia and Iflago spicata was
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Chapter 5 Discussions investigated in mice. Treatment with methanolic leaf extract of Taverniera nummularia and
Ifloga spicata significantly decreased blood glucose level and resulted in a better body weight under hyperglycemic conditions. The methanolic extract of T. nummularia and I. spicata possesses phytochemicals such as terpenoids, which play an important role in lowering blood pressure (diastolic) and reduce the sugar level in blood. Similar findings were obtained from other studies from other that blood glucose lowering potential may be attributed to the occurrence of various bioactive compounds such as flavonoids, phenols, Terpenoids ,alkaloids, tannins and sterols [35,40]. It was found that increase in the content of bilirubin, ALT, ALP, urea, craetinine, total proteins, triglycerides and LDL Cholesterol due to hyperglycemia was normalized by treatment with methanolic extract of both the plant species. The reinstatement of
ALT levels after treatment also indicates a recovery of insulin secretion. In the diabetic rats the raise in ALP level has been reported [15]. ALP was significantly inverted by the extract of
Taverniera nummularia and Ifloga spicata. Present results are reliable with the study conducted by Parmar et al 2010 who reported that aqueous methanol plant extract of Phyllanthus niruri,
Aloe vera and Aegle marmelos decreased the elevated ALT, ALP and bilirubin levels in paracetamol induced liver injury in mice. Similar results were reported by Ali et al., (2003) in which leaf extract of Hibiscus sabdariffa significantly ameliorated paracetamol induced toxicity on liver function. These beneficial effects of methanolic extracts of both the studied plant species may be attributed to their phytochemical composition. The current study is similar to that of
Piera et al., (2015) who found that a plant species C. pareira contained terpenoids which were heart-friendly and were very effective among diabetic and high blood pressure patients. The functional kidney status can be checked by blood analysis. [16].The level of serum urea, craetinine remains normal unless there is pathogenesis. The raise level of urea and creatinine
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Chapter 5 Discussions indicate kidney injuries in alloxan treated diabetic rats [17]. Present results of diabetic groups showed higher blood urea and creatinine. The data from current study showed that plant extract of Taverniera nummularia and Ifloga spicata restored raise level of urea and creatinine in blood.
The extract treatment showed more significant effects in lowering urea concentration both in diabetic and non-diabetic rats. There is no consistent to our study because no such study is conducted on any species of T.nummularia and I. spicata up till now. In diabetic rats raise in glucose level was related with a raise in concentration of triglycerides and cholesterol present in the normal diabetic conditions [18-20]. The extract of T.nummularia and I.spicata at 200mg/kg and 400mg/kg dose level, recovered the diabetes induced hyperlipidemia as compared the diabetic control group. Both extract at (200mg/kg b/wt) treated diabetic group restore the raise levels of high density lipoproteins, low density lipoproteins and total cholesterol were found significant and triglyceride was found to be less significant. In extract (400mg/kg b. wt.) treated rats reduction in the levels of serum lipids were found significant. In non-diabetic extract treated group the levels of serum lipids were found more significant as compared with the normal control group showing the hypolipidemic effect of T. nummularia and I. spicata plant. Similar findings were obtained from other studies [21-23]. In alloxan treated rats the raise in serum lipid is significantly restored after treatment with extract of T. nummularia and I. spicata. These results suggested that this plant extract can be used for treatment of diabetes and its complications. It can also be used to control abnormal and enhance levels of renal biochemical marker and liver enzymes both in diabetic and non-diabetic individuals.
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6.0 Conclusions
The crude methanolic extract of both the T. nummularia and Ifloga spicata exhibited the presence of alkaloids, tannins, glycosides, saponins and flavonoids. The various solvent fractions of T. nummularia and Ifloga spicata exhibited antibacterial (S. aureus, P. aeruginosa, E. coli) and antifungal (Aspergillus fumigatus, Aspergillus flavous, Aspergillus niger) activity.
The DPPH free radical scavenging activity was higher at 500 µg/ ml for methanolic extract of both the plant species. The H2O2 free radical scavenging activity was higher at 500 µg/ ml for ethyl acetate extract of both the plant species. The decrease in TBARS and ROS content of lymphocytes under oxidative stress by various fractions of the T. nummularia effectively protected membrane lipid peroxidation of the lymphocytes. The methanolic and aqueous fractions were significantly more effective on antioxidant enzymes of lymphocytes under oxidative stress.
In vivo both plants extract showed significant effect against the elevated level of serum Bilirubin
ALP, ALT,serum lipid profile and renal tests.
The most effective dose was 300 mg/ kg body was found in present experiment.
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Recommendations
7.0 Recommendations
1. The various solvent fractions of T. nummularia and Ifloga spicata may be tested for anti- cancer
activity using cancer cell lines (Overian, Mamary and Pulmonary).
2. Beneficial effects of the two studied plant species on DNA of lymphocytes under oxidative stress
should be evaluated.
3. Isolation and characterization of bioactive compounds should be made by using advanced
chromatographic techniques and their effect as agents against different ailments may be
determined.
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Chapter 6 References
8.0 References
1 Tapsell, L.C., Hemphill I. and Cobiac L. 2006. Health benefits of herbs and spices: the
past, the present, the future. Med. J. Aust. 185 (4 Suppl): S4–24
2 Lai, P.K. and Roy, J. 2004. Antimicrobial and chemopreventive properties of herbs and
spices. Curr. Med. Chem. 11 (11): 1451–1460.
3 Fabricant, D.S. and Farnsworth, N.R. 2001. The value of plants used in traditional
medicine for drug discovery. Environ. Health Perspect. 109 (1): 69-75.
4 Tony, S. 1968. Plants in the development of modern medicine. Chines Medicine, 235-
268.
5 Chopra, R.N., Nayar, S.L. and Chopra, I.C. 1956. In Glossory of Indian Medicinal Plants,
Coun. of Sci. and Ind. Research, New Delhi. 1: 197.
6 Sudhakar, Y., Kuotsu, K. and Bandyopadhyay, A.K. 2006. Buccal bioadhesive drug
delivery-a promising option for orally less efficient drugs. J. of Contr. Release, 114 (1):
15-40.
7 Khan, R.A., Bukhari, I.A., Nawaz, S.A. and Choudhary, M.I. 2006. Acetylcholinesterase
and butyrylcholinesterase inhibitorypotential of some Pakistani medicinal plants. J Basic
Appl. Sciences. 2: 7-10.
8 Oyetayo, V.O. and Oyetayo, F.L. 2006. Phytochemical screening and antibacterial
properties of siam weed, Chromoleana odorata, leaf against aerobic isolates of wound. J.
Appl. Environ. Sci. 2 (1): 7-11.
9 Mousavi, S.M., George, W. R., David, S.S. Mirzargar and Omidbaigi, R. 2011.
Antibacterial activities of a new combination of essential oils against marine bacteria.
Aqu.e Internatinal, 19 (1): 205-214.
70 | P a g e
Chapter 6 References
10 Sirinvansan, D., Perumalsamy, L.P., Nathan, S. and Sures, T. 2001. Antimicrobial
activity of certain Indian medicinal plants used in folkloric medicine. J.
Ethnopharmacology, 74 (3): 217-222.
11 Gilani, A. and Atta-ur-Rahman. 2005. Trends in Ethnopharmacology. J.
Ethnopharmacol., 100 (1-2): 43-49.
12 Arunkumar, S. and Muthuselvam, M. 2009. Analysis of phytochemical constituents and
antimicrobial activities of Aloe vera against clinical pathogens. World J. Agr. Sciences,
5: 572-576.
13 Kinghorn, A.D. 2001. Pharmacognosy in the 21st century. J. Pharm. and Pharmacology,
53 (2): 135-148.
14 Alviano, D.S. and Alviano, C.S. 2009. Plant extracts: search for new alternatives to treat
microbial diseases. Curr. Pharm. Biotechnol. 10: 106–121.
15 Ullah, I., Wakeel, A., Shinwari, Z.K., Jan, S.A., Khalil, A.T. and Ali, M. 2017.
Antibacterial and Antifungal Activity of Isatis Tinctoria L. (Brassicaceae) Using the
Micro-Plate Method. Pak. J. Bot., 49 (5): 1949-195
16 Zhang, R., Eggleston, K., Rotimi, V. and Zeckhauser, R.J. 2006. Antibioticresistance as a
global threat: evidence from China, Kuwaitand the United States. Glob. Health, 2: 6.
17 Dorman, H.J. & Deans, S.G. 2000. Antimicrobial agents from plants: antibacterial
activity of plant volatile oils. J. Appl. Microbiology, 88: 308–316.
18 Talib, W.H. and Mahasneh, A.M. 2010. Antimicrobial, cytotoxicity and phytochemical
screening of Jordanian plants used in traditional medicine. Molecules, 15, 1811–1824.
19 Ruberto, G., Baratta, M.T., Deans, S.G., Dorman H.J. 2000. Antioxidant and
antimicrobial activity of Foeniculum vulgare and Crithmum maritimum essential oils.
Planta Med., 66, 687– 693
71 | P a g e
Chapter 6 References
20 Cruz, M.C., Santos, P.O., Barbosa, Alviano, C.S., Antoniolli, A.R. 2007. Antifungal
activity of Brazilian medicinal plants involved in popular treatment of mycoses. J
Ethnopharmacol., 111, 409–412
21 Williamson, D.A., Heffernan, H., Sidjabat, H., Roberts, S.A., Paterson, D.L. and Smith,
M. 2008. Impact of antibiotic resistancein gram-negative bacilli on empirical and
definitive antibiotictherapy. Cli. Infect. Diseases, 1: 14–20.
22 Cheesbrough, M. 1984. Microscopic examination of specimens.In: Medical Laboratory
Manual for Tropical Countries, Oxford, Publishers, United Kingdom. 32–33
23 Peirano, G. 2008. Multi resistant enterobacteriaceae new threat toan old prob. Expert
Review of Anti-infective Therapy, 6: 657–669
24 Eggleston, K., Zhang, R. and Zeckhauser, R.J. 2010. The global challenge of
antimicrobial resistance: insights from economic analysis. Int J Environ Res Public
Health. 7, 3141–3149
25 Bibi, Y., Nisa, S., Chaudhary, F.M. and M. Zia. 2011. Antibacterial activity of some
selected medicinal plants of Pakistan. BMC Complement. Alter. Med, 11: 52.
26 Tariq A., Adnan, M., Amber, R., Pan, K., Mussarat, S. and Shinwari, Z. K. 2016.
Ethnomedicines and anti-parasitic activities of Pakistani medicinal plants
against Plasmodia and Leishmania parasites. Ann Clin Microbiol Antimicrob, 15:52
27 Kalim, M.D., Bhattacharyya, D., Banerjee, A. and Chattopadhyay, S. 2010. Oxidative
DNA damage preventive activity and antioxidant potential of plants used in Unani system
of medicine. BMC Complementary and Alter. Me, 10: 77.
28 Habiba, U., Ahmad, M., Shinwari, S., Sultana, S., Shinwari, Z.K. and Zafar, M. 2016.
Antibacterial and Antifungal Potential of Himalayan Medicinal Plants for Wound
Infections. Pak. J. Bot., 48 (1): 371-375
72 | P a g e
Chapter 6 References
29 Arts, I.C., and Hollman, P.C. 2005. Polyphenols and disease risk in epidemiologic
studies. The Amer. J. of Clin. Nutrition, 81 (1), 317S-325S.
30 Gibson, E.L., Wardal, J. and Watts, C.J. 1998. Fruit and vegetable consumption,
Nutritional knowledge and Beliefs in mother and children. Appetite, 31: 205-228.
31 Shashikant, P., Bhupesh, R.P., Shukla, V. J. 2015. Preliminary phytochemical evaluation
of Taverniera nummularia Baker. (Jethimala) root. Intern. J. Univer.Pharm. and Bio
Sciences, 4(1), 23-28.
32 Meagher, R.B., McKinney, E.C. and Kandasamy, M.K. 1999. Isovariant dynamics
expand and buffer the responses of complex systems: the diverse plant actin gene
family. The Plant Cell, 11 (6), 995-1005.
33 King, A. and Young, G.1999. Characteristics and occurrence of phenolic
phytochemicals. J. of the Amer. Dietetic Association, 99 (2), 213-218.
34 Mahato, S.B. and Sen, S. 1997. Advances in triterpenoids research, 1994. Phytochemistry,
44: 1185-1236.
35 Marles, R.J. and Farnsworth, N.R. 1995. Antidiabetic plants and their active
constituents. Phytomedicine, 2 (2): 137-189.
36 Kim, H.B., Bang, H.S., Lee, H.W., Seuk, Y.S. and Sung, G.B. 1999. Chemical
characteristics of mulberry syncarp. Korean J. Med. Crop. Sci. 47: 3206-3209.
37 Mueller-Harvey, I., McAllan, A.B. 1992. Tanins, their Biochemistry and Nutritional
Properties. In: Advances in Plant Cell Biochemistry and Biotechnology, Vol. 1, Morrison
I.M. ed. JAI Press. Ltd. London (UK): 152-217.
38 Sarker, S.D. and Nahar, L. 2007. Chemistry for Pharmacy Students General, Organic and
Natural Product Chemistry. England: John Wiley and Sons: 283-359.
39 Firn, R. 2010. Nature’s Chemicals. Oxford University Press, Oxford: 74-75.
73 | P a g e
Chapter 6 References
40 Madziga, H.A., Sanni, S. and Sandabe, U.K. 2010. Phytochemical and Elemental
Analysis of Acalypha wilkesiana Leaf. J. Am. Science, 6 (11): 510-514.
41 Wink, M., Schmeller, T., Latz-Briining, B. 1998. Modes of action of allelochemical
alkaloids. Interaction with neuroreceptors, DNA and other molecular targets. J. of Chem.
Ecology, 24: 1888-1937.
42 Rao, R.V.K., Ali, N., Reddy, M.N. 1978.Occurrence of both sapogenins and alkaloid
lycorine in Curculigo orchioides. Indian J. Pharma Science. 40: 104-105.
43 Kar, A, 2007. Pharmacognosy and Pharmacobiotechnology (RevisedExpanded Second
Edition). New Age International Limited Publishers New Delhi: 332-600.
44 Pridham, J.B. 1960. Pergamon Press, New York. 34-35.
45 Haborne, J.B. and Baxter, H. 1999. The handbook of natural flavonoids, Volume 1 and 2.
Chichester, UK. John Wiley and Sons.
46 Pretorius, J.C. 2003. Flavonoids: A Review of its Commercial Application Potential as
Infective Agents. Current Medicinal Chemistry Anti-infective Agents, 2: 335-353.
47 Tapas, A.R., Sakarkar, D.M., Kakde, R.B. 2008. Flavonoids as Nutraceutials: A review.
Tropical J. of Pharm. Research,. 7: 1089-1099.
48 Atmani, D., Nassima, C., Dina, A., Meriem, B., Nadjet, D., Hania, B.2009. Flavonoids in
Human Health: From Structure to Biological Activity. Curr. Nut. and Food Science, 5:
225-237.
49 Puupponen-Pimiä, R., Nohynek, L., Ammann, S., Oksman-Caldentey, K. M. and Buchert,
J. 2008. Enzyme-assisted processing increases antimicrobial and antioxidant activity of
bilberry. J. of Ag. and food chemistry, 56 (3): 681-688.
50 Mandal, S.M., Chakraborty, D. and Dey, S. 2010. Phenolic acids act as signaling
molecules in plant microbe symbiosis. Plant Signal Behav., 5: 359-368.
74 | P a g e
Chapter 6 References
51 Elbein, A.D. and Molyneux, R.J. 1999. Comprehensive Natural Products Chemistry, Vol.
3 Barton, D., Nakanishi, K., ed. Amsterdam.129.
52 Langenheim, J.H. 1994. Higher plants terpenoids. A phyocentric overview of their
ecological roles. J. Chem. Ecology. 20: 1223-1280.
53 McCaskill, D. and Croteau, R.1998. Some caveats for bioengineering terpenoids
metabolism in plants. Trends in Biotechnology, 16: 349-355.
54 Degenhardt, J., Gershenzon, J., Baldwin, I.T. and Kessler, A. 2003. Attracting friends to
feast on foes. Engineering terpenes emission to make crop plants more attractive to
herbivore enemies. Curr. Opinion in Biotechnology, 14: 169-176
55 Dudareva, N., Pichersky, E., Gershenzon, J. 2004. Biochemistry of plant volatiles. Plant
Physiology. 135: 1893-1902.
56 Bolhmann, J., Meyer-Gauen, G. and Croteau, R. 1998. Plant terpenoid synthases:
Molecular Biology and Phylogenetic Analysis. Proc. Natl. Acad. Sci. 95: 4126-4133.
57 Lasztity, R., Hidvegi, M. and Bata, A. 1998. Saponins in food. Food Review
International, 14: 371-390.
58 Lacaille-Dubios, M.A. and Wagner, H. 2000. Bioactive saponins from plants. An update.
In Studies in Natural Products Chemistry, Atta-ur –Rahman ed. Elsevier. Science.
Amsterdam, 21: 633-687
59 Maurya, R., Singh, G., R., Singh, G. and Yadav, P.P. 2008. Antiosteoporotic agents from
natural sources. Studies in Nat. Prod. Chemistry, Vol. 35. Elsevier: 517-545.
60 Madziga, H.A., Sanni, S. and Sandabe, U.K. 2010. Phytochemical and Elemental
Analysis of Acalypha wilkesiana Leaf. J. Amer. Science, 6 (11): 510-514.
61 Jadhav, R. and Puchchakayala, G. 2012. Hypoglycemic and antidiabetic activity of
flavonoids: boswellic acid, Ellagic acid, quercetin, rutin on streptozotocin-nicotinamide
induced type 2 diabetic rats. Int J Pharm Pharm Sci., 42 (2): 51-256
75 | P a g e
Chapter 6 References
62 Saxena, A. and Vikram, N.K. 2004. Role of selected Indian plants in management of type
2 diabetes: a review. J. Alt. Comple. Med., 10, 369-378.
63 Bajpai, V.K., Al-Reza, S.M., Choi, U.K., Lee, J.H. and Kang, S.C., 2009. Chemical
composition, antibacterial and antioxidant activities of leaf essential oil and extracts of
Metasequioa glyptostroboides Miki ex Hu. Food and Chem. Toxicology, 47 (8): 1876-
1883.
64 Cushnie, T.T. and Lamb, A.J., 2005. Antimicrobial activity of flavonoids. International J.
Antim. Agents, 26 (5): 343-356.
65 Parekh, J., Jadeja, D. and Chanda, S. 2006. Efficacy of aqueous and methanol extracts of
some medicinal plants for potential antibacterial activity. Turkish J. Biology, 29 (4): 203-
210.
66 Dellavalle, P.D., Cabrera, A., Alem, D., Larrañaga, P., Ferreira, F. and Dalla Rizza, M.,
2011. Antifungal activity of medicinal plant extracts against phytopathogenic fungus
Alternaria spp. Chilean J. Agr. Research, 71 (2): 231.
67 Essawi, T. and Srour, M., 2000. Screening of some Palestinian medicinal plants for
antibacterial activity. J. of Ehnopharmacology, 70 (3): 343-349.
68 Webster, D., Taschereau, P., Belland, R.J., Sand, C. and Rennie, R.P. 2008. Antifungal
activity of medicinal plant extracts; preliminary screening studies. J.
Ethnopharmacology, 115 (1): 140-146.
69 Rios, J.L. and Recio, M.C. 2005. Medicinal plants and antimicrobial activity. J.
Ethnopharmacology, 100 (1-2): 80-84.
70 Jadhav, R. and Puchchakayala, G. 2012. Hypoglycemic and antidiabetic activity of
flavonoids: boswellic acid, Ellagic acid, quercetin, rutin on streptozotocin-nicotinamide
induced type 2 diabetic rats. Int. J. Pharm. Sci. 42 (2): 51-256
76 | P a g e
Chapter 6 References
71 Yadev, S., and Vats, V. 2002. Medicinal plants of India with antidiabetic potential. J.
Ethnopharmac. 81: 81-100.
72 Mahasnesh, A.M. 1986. Antimicrobial activity of plant extract and pure judicin from
Artemisia judaica. Jordan Med. J., 20: 17-24.
73 Glombitza, K.W. and Pauli, K. 2003. Fucols and phlorethols from the brown alga
Scytothamnus australis hook (chnoosporaceae). Botanica Marina, 46: 315–320.
74 Shad, A.A., Asmat, S., Bakht, J., Jan, S. and Khan, M.A. 2016. Antimicrobial potentials
and phytochemical analysis of desert cotton (A. Javanica) and flax (L.
Ustitatissimum). Pak. J Pharm. Sciences, 29 (3): 861-868.
75 Palombo, E.A. and Semple, S.J. 2001. Antibacterial activity of traditional Australian
medicinal plants. J. Ethnopharmacology, 77 (2): 151-157.
76 Quiroga, E N., Sampietro, A.R., and Vattuone, M.A. 2001. Screening antifungal activities
of selected medicinal plants. J. Ethnopharmacolog, 74 (1): 89-96.
77 Soliman, K. M. and Badeaa, R.I. 2002. Effect of oil extracted from some medicinal plants
on different mycotoxigenic fungi. Food and Chem. Toxicology, 40 (11): 1669-1675.
78 Gupta, D., Khare, S.K. and Laha, A. 2004. Antimicrobial properties of natural dyes
against Gram‐negative bacteria. Coloration Technology, 120: 167-171.
79 Cai, Y., Luo, Q., Sun, M. and Corke, H. 2004. Antioxidant activity and phenolic
compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life
sciences, 74: 2157-2184.
80 Morgen, L. 2006. Quercetin content in yellow onion (Allium cepa L.) Effects of
cultivation methods, curing and storage. Doctoral Dissertation. ISSN 1652-6880, ISBN
91-576-7145-1.
77 | P a g e
Chapter 6 References
81 Sahreen, S., Khan, M.R. and Khan, R.A. 2011. Hepatoprotective effects of methanol
extract of Carissa opaca leaves on CCl 4-induced damage in rat. BMC Complem. and
altern. Medicine, 11: 48.
82 Bhatt, U., Sati, S., Chandra, S., Kumar, S., Anthwal, A., Singh, R., ingh, D., Kumar, N.,
Kumar, A. and Bahuguna, R. 2015. Evaluation of In Vivo and In Vitro Anti-diabetic
Activity of Roylea cinerea. Int. J. Pharm. Sci. Rev. Res, 32, 210-213.
83 Sharma, V.K., Kumar, S., Patel, H.J. and Hugar, S. 2010. Hypoglycemic activity of Ficus
glomerata in alloxan induced diabetic rats. Inter. J. of Pharmac. Sciences Review and
Research, 1: 18-22.
84 Bushra, S., Muhammad I. and Hamid I. 2018. Evaluating the effect of Acacia modesta
leaves extract on blood glucose, serum Lipids, liver and kidney functions in diabetic and
non-diabetic rats. Brazilian J. of Pharmac. Sciences, 54 (2):17467.
85 Ghori, S.S., Khan, M.R. and Kaab-e-Alam, A.H.A. 2015. Evaluation of
antihyperlipidemic activity of ethanolic extact of glycosmis pentaphylla in hyperlipidemic
wistar rats. Intern. J. of Pharma. Sciences and Research, 6 (2): 288-292.
86 Khalighi-Sigaroodi, F., Ahvazi, M., Hadjiakhoondi, A., Taghizadeh, M., Yazdani, D.,
Khalighi-Sigaroodi, S. and Bidel, S. 2012. Cytotoxicity and antioxidant activity of 23
plant species of Leguminosae family. Iranian J. of Pharmac. Research, 11 (1): 295.
87 Nascimento, G.G., Locatelli, J., Freitas, P.C. and Silva, G.L., 2000. Antibacterial activity
of plant extracts and phytochemicals on antibiotic-resistant bacteria. Brazilian J.
Microbiology, 31 (4): 247-256.
88 Nascimento, G.G., Locatelli, J., Freitas, P.C. and Silva, G.L. 2000. Antibacterial activity
of plant extracts and phytochemicals on antibiotic-resistant bacteria. Brazilian J.
Microbiology, 31 (4): 247-256.
89 Khan, S.T., Ahmed, M., Khan, R.A., Mushtaq, N. and Khan, N. 2017. Anti-diabetic
78 | P a g e
Chapter 6 References
potential of aerial parts of Galium tricornutum (Dandy) Rubiaceae. Tropical J. Pharma.
Research, 16 (7): 1573-1578.
90 Kumar, A.Y., Nandakumar, K., Handral, M., Talwar, S. and Dhayabaran, D. 2011.
Hypoglycaemic and anti-diabetic activity of stem bark extracts Erythrina indica in normal
and alloxan-induced diabetic rats. Saudi Pharm. Journal, 19 (1): 35-42.
91 Dholi, S.K., Raparla, R., Mankala, S.K. and Nagappan, K. 2011. In vivo antidiabetic
evaluation of Neem leaf extract in alloxan induced rats. Journal Appl .Pharm. Science, 1
(4), pp.100-105.
92 Khalighi-Sigaroodi, F., Ahvazi, M., Hadjiakhoondi, A., Taghizadeh, M., Yazdani, D.,
Khalighi-Sigaroodi, S. and Bidel, S. 2012. Cytotoxicity and antioxidant activity of 23
plant species of Leguminosae family. Iranian J. of Pharm. Research, 11 (1): 295.
93 Mesia, G.K., Tona, G.L., Nanga, T.H., Cimanga, R.K., Apers, S., Cos, P., Maes, L.,
Pieters, L. and Vlietinck, A.J. 2008. Antiprotozoal and cytotoxic screening of 45 plant
extracts from Democratic Republic of Congo. J. of Ethnopharmacology, 115 (3): 409-
415.
94 Kilani, S., Sghaier, M.B., Limem, I., Boubakar, J., Bhouri, W., Skandrani, I., Neffati, A.,
Ammarb, R.B., Dijoux-Franca, M.G., Ghedira, K. and Chekir-Ghedira, L. 2008. In vitro
evaluation of antibacterial, antioxidant, cytotoxic and apoptotic activities of the tubers
infusion and extracts of Cyperus rotundus. Bioresource Tech., 99, 9004-9008.
95 Hussain, A., Zia, M. and Mirza, B., 2007. Cytotoxic and Antitumor Potential of Fagonia
cretica L. Turkish J. Biology, 31 (1): 19-24.
96 Harborne, J. B., Greenham, J., & Williams, C. A. (1973). Phytochemical
analysis. Chapman and Hall company Ltd, London, 1, 5-6.
97 Sofowora, A. Medicinal plants and traditional medicine in Africa. 1993. Ibadan, Nigeria:
Spectrum Books Google Scholar.
79 | P a g e
Chapter 6 References
98 Trease, G. E., & Evans, W. C. (1989). A text book of pharmacognosy Academic
press. London, 198, 22-40.
99 Ahmad, B., Ali, N., Bashir, S., Choudhary, M. I., Azam, S., & Khan, I. (2009).
Parasiticidal, antifungal and antibacterial activities of Onosma griffithii Vatke. African
Journal of Biotechnology, 8(19).
100 Dawidar, A. M., Metwally, M. A., Abou-Elzahab, M., Abdel-Mogib, M. 1989. Chemical
constituents of I. spicata. Pharmazie, 44(3), 235.
101 Haq, I.J., Gray, M.A., Garnett, J.P., Ward, C. and Brodlie, M., 2016. Airway surface
liquid homeostasis in cystic fibrosis: pathophysiology and therapeutic
targets. Thorax, 71(3), pp.284-287.
102 Nascimento, G.G., Locatelli, J., Freitas, P.C. and Silva, G.L., 2000. Antibacterial activity
of plant extracts and phytochemicals on antibiotic-resistant bacteria. Brazilian J.
Microbiology, 31 (4): 247-256.
103 Gibbons, S. 2004. Anti-staphylococcal plant natural products. Nat. Prod. Reports, 21 (2):
263-277.
104 Cordell, G.A. 1993. Pharmacognosy. New roots for an old science. Studies in natural
products chemistry. Vol. 13: Bioactive natural products (Part A). Elsevier.
105 Ayyanar, M. and Subash-Babu, P. 2012. Syzygium cumini (L.) Skeels: A review of its
phytochemical constituents and traditional uses. Asian Paci. J. Trop. Biomedicine, 2 (3):
240-246
106 Umamaheswari, M. and Chatterjee, T.K. 2008. In vitro antioxidant activities of the
fractions of Coccinia grandis L. leaf extract. African J. Trad., Complem. and Alternative
Medicines, 5 (1): 61-73.
107 Sandhu, D.S. and Heinrich, M. 2005. The use of health foods, spices and other botanicals
in the Sikh community in London. Phytotherap. Res. 19: 633-642.
80 | P a g e
Chapter 6 References
108 Palombo, E.A. and Semple, S.J. 2001. Antibacterial activity of traditional Australian
medicinal plants. J. Ethnopharmacology, 77 (2-3): 151-157.
109 Dorman, H.J.D. and Deans, S.G., 2000. Antimicrobial agents from plants: antibacterial
activity of plant volatile oils. J. Appl. Microbiology, 88 (2), pp.308-316.
110 Talib, W.H. and Mahasneh, A.M., 2010. Antimicrobial, cytotoxicity and phytochemical
screening of Jordanian plants used in traditional medicine. Molecules, 15 (3): 1811-1824.
111 Ahmed, S., Ahmad, M., Swami, B.L. and Ikram, S. 2016. A review on plants extract
mediated synthesis of silver nanoparticles for antimicrobial applications: a green
expertise. J. Ad. Research. 7 (1): 17-28.
112 Shad, A.A., Asmat, S., Bakht, J., Jan, S. and Khan, M.A., 2016. Antimicrobial potentials
and phytochemical analysis of desert cotton (A. javanica) and flax (L.
ustitatissimum). Pak. J. Pharmac. Sciences, 29 (3): 861-868.
113 Rhee, S.G., Kang, S.W., Jeong, W., Chang, T.S., Yang, K.S. and Woo, H.A., 2005.
Intracellular messenger function of hydrogen peroxide and its regulation by
peroxiredoxins. Curr. Opinion in Cell Biology, 17 (2): 183-189.
114 Gafrikova, M., Galova, E., Sevcovicova, A., Imreova, P., Mucaji, P. and Miadokova, E.,
2014. Extract from Armoracia rusticana and its flavonoid components protect human
lymphocytes against oxidative damage induced by hydrogen peroxide. Molecules, 19 (3):
3160-3172.
115 Duthie, G.G., Duthie, S.J. and Kyle, J.A. 2000. Plant polyphenols in cancer and heart
disease: implications as nutritional antioxidants. Nutr. Research Reviews, 13 (1):.79-106.
116 Dudonné, S., Vitrac, X., Coutiere, P., Woillez, M. and Mérillon, J.M., 2009. Comparative
study of antioxidant properties and total phenolic content of 30 plant extracts of industrial
interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. J. Agri. and Food
Chemistry, 57 (5): 1768-1774.
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117 Ames, B.N., Shigenaga, M.K. and Hagen, T.M., 1993. Oxidants, antioxidants, and the
degenerative diseases of aging. Proceedings of the National Academy Sciences, 90 (17):
7915-7922.
118 Dimitrios, B. 2006. Sources of natural phenolic antioxidants. Trends in Food Science and
Technology, 17 (9): 505-512.
119 Gafrikova, M., Galova, E., Sevcovicova, A., Imreova, P., Mucaji, P. and Miadokova, E.,
2014. Extract from Armoracia rusticana and its flavonoid components protect human
lymphocytes against oxidative damage induced by hydrogen peroxide. Molecules, 19 (3):
3160-3172.
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Chapter 6 References
9.0 Appendices
Ifloga spicata
Analysis of Variance Table for ROS
Source DF SS MS F P Extract 4 0.02979 0.00745 4.87 0.0010 Treat 7 0.74330 0.10619 69.41 0.0000 Extract*Treat 28 0.04031 0.00144 0.94 0.5556 Error 160 0.24476 0.00153 Total 199 1.05816
Grand Mean 0.1894 CV 20.65
Statistix 8.1 Analysis of Variance Table for POD
Source DF SS MS F P Extract 4 4.595 1.1488 1.18 0.3200 Treat 7 301.141 43.0202 44.33 0.0000 Extract*Treat 28 28.095 1.0034 1.03 0.4273 Error 160 155.275 0.9705 Total 199 489.107
Grand Mean 4.0005 CV 24.63
Statistix 8.1 Analysis of Variance Table for CAT
Source DF SS MS F P Extract 4 15.040 3.7599 13.07 0.0000 Treat 7 120.134 17.1620 59.65 0.0000 Extract*Treat 28 9.356 0.3341 1.16 0.2771 Error 160 46.032 0.2877 Total 199 190.562
Grand Mean 2.1003 CV 25.54
Analysis of Variance Table for SOD
Source DF SS MS F P Extract 4 60.96 15.239 10.83 0.0000 Treat 7 1095.75 156.536 111.22 0.0000 Extract*Treat 28 70.34 2.512 1.78 0.0141 Error 160 225.19 1.407 Total 199 1452.24
Grand Mean 7.5321 CV 15.75
Statistix 8.1 Analysis of Variance Table for TBARS
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Chapter 6 References
Source DF SS MS F P Extract 4 0.25144 0.06286 5.03 0.0008 Treat 7 4.43938 0.63420 50.72 0.0000 Extract*Treat 28 0.39184 0.01399 1.12 0.3226 Error 160 2.00054 0.01250 Total 199 7.08321
Grand Mean 0.7549 CV 14.81
Statistix 8.1 Antibacterial Completely Randomized AOV for EC
Source DF SS MS F P Extract 6 2735.71 455.952 132 0.0000 Error 14 48.43 3.459 Total 20 2784.14
Grand Mean 15.540 CV 11.97
Completely Randomized AOV for PA
Source DF SS MS F P Extract 6 726.391 121.065 559 0.0000 Error 14 3.030 0.216 Total 20 729.420
Grand Mean 9.7414 CV 4.78
Completely Randomized AOV for SA
Source DF SS MS F P Extract 6 2151.12 358.521 59.2 0.0000 Error 14 84.78 6.056 Total 20 2235.90
Grand Mean 14.050 CV 17.51
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Chapter 6 References
Antifungal Statistix 8.1 Completely Randomized AOV for AFMG
Source DF SS MS F P Extract 6 16056.0 2675.99 1731 0.0000 Error 14 21.6 1.55 Total 20 16077.6
Grand Mean 50.230 CV 2.48
Completely Randomized AOV for AN
Source DF SS MS F P Extract 6 15942.6 2657.09 3200 0.0000 Error 14 11.6 0.83 Total 20 15954.2
Grand Mean 50.069 CV 1.82
Completely Randomized AOV for Aflavus
Source DF SS MS F P Extract 6 16099.9 2683.31 510 0.0000 Error 14 73.6 5.26 Total 20 16173.5
Grand Mean 47.278 CV 4.85
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Chapter 6 References
Statistix 8.1 Analysis of Variance Table for H2O2
Source DF SS MS F P Conc 3 8699.5 2899.82 547.13 0.0000 Extract 5 49077.7 9815.54 1851.95 0.0000 Conc*Extract 15 2133.8 142.25 26.84 0.0000 Error 48 254.4 5.30 Total 71 60165.3
Grand Mean 47.290 CV 4.87 Analysis of Variance Table for DPPH
Source DF SS MS F P Extract 5 3476.39 695.278 8.09 0.0000 Error 48 4126.44 85.968 Total 53 7602.83
Grand Mean 60.722 CV 15.27
Statistix 8.1 Analysis of Variance Table for Glucose
Source DF SS MS F P Days 3 135.19 45.065 18.69 0.0000 Treat 4 1184.60 296.150 122.81 0.0000 Days*Treat 12 265.13 22.094 9.16 0.0000 Error 40 96.46 2.412 Total 59 1681.38
Grand Mean 184.03 CV 0.84 Statistix 8.1
Analysis of Variance Table for BLGLUC
Source DF SS MS F P Day 3 3838 1279.3 2703.93 0.0000 Treat 4 187562 46890.4 99110.4 0.0000 Day*Treat 12 6080 506.7 1070.96 0.0000 Error 40 19 0.5 Total 59 197499
Grand Mean 197.19 CV 0.35
Analysis of Variance Table for BW
Source DF SS MS F P Day 3 140.70 46.901 86.25 0.0000 Treat 4 1238.76 309.691 569.53 0.0000 Day*Treat 12 300.88 25.073 46.11 0.0000 Error 40 21.75 0.544 Total 59 1702.10
Grand Mean 185.00 CV 0.40
Completely Randomized AOV for ALP
Source DF SS MS F P Treat 4 11763.1 2940.78 695 0.0000
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Chapter 6 References
Error 10 42.3 4.23 Total 14 11805.4
Grand Mean 163.28 CV 1.26
Completely Randomized AOV for ALT
Source DF SS MS F P Treat 4 873.350 218.337 91.5 0.0000 Error 10 23.850 2.385 Total 14 897.200
Grand Mean 39.379 CV 3.92
Completely Randomized AOV for Bilirubin
Source DF SS MS F P Treat 4 1.30103 0.32526 4.19 0.0302 Error 10 0.77707 0.07771 Total 14 2.07809
Grand Mean 0.8407 CV 33.16 Std Error (Diff of 2 Means) 0.2276
Completely Randomized AOV for Creat
Source DF SS MS F P Treat 4 5.66956 1.41739 329 0.0000 Error 10 0.04306 0.00431 Total 14 5.71262
Grand Mean 1.0679 CV 6.14
Completely Randomized AOV for HDL
Source DF SS MS F P Treat 4 1029.52 257.379 205 0.0000 Error 10 12.58 1.258 Total 14 1042.10
Grand Mean 33.217 CV 3.38
Completely Randomized AOV for LDL
Source DF SS MS F P Treat 4 8774.08 2193.52 2851 0.0000 Error 10 7.69 0.77 Total 14 8781.77
Grand Mean 105.22 CV 0.83 Std Error (Diff of 2 Means) 0.7162
Completely Randomized AOV for TCH
Source DF SS MS F P Treat 4 14336.4 3584.09 567 0.0000 Error 10 63.2 6.32 Total 14 14399.6
Grand Mean 153.43 CV 1.64
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Completely Randomized AOV for TG
Source DF SS MS F P Treat 4 6928.45 1732.11 2843 0.0000 Error 10 6.09 0.61 Total 14 6934.54
Grand Mean 167.97 CV 0.46
Completely Randomized AOV for Urea
Source DF SS MS F P Treat 4 9834.45 2458.61 1759 0.0000 Error 10 13.97 1.40 Total 14 9848.42
Grand Mean 94.963 CV 1.24
Taverniera nummularia
Statistix 8.1
Grand Mean 2.1003 CV 25.54
Grand Mean 3.8212 CV 27.77
Analysis of Variance Table for ROS
Source DF SS MS F P Treat 7 0.74330 0.10619 69.41 0.0000 Extract 4 0.02979 0.00745 4.87 0.0010 Treat*Extract 28 0.04031 0.00144 0.94 0.5556 Error 160 0.24476 0.00153 Total 199 1.05816
Grand Mean 0.1894 CV 20.65
Analysis of Variance Table for TBARS
Source DF SS MS F P Treat 7 4.43938 0.63420 50.72 0.0000 Extract 4 0.25144 0.06286 5.03 0.0008 Treat*Extract 28 0.39184 0.01399 1.12 0.3226 Error 160 2.00054 0.01250 Total 199 7.08321 Grand Mean 0.7549 CV 14.81
Statistix 8.1 4/16/2017, 4:27:28 PM
Analysis of Variance Table for SOD
Source DF SS MS F P Extract 4 60.96 15.239 10.83 0.0000 Treat 7 1095.75 156.536 111.22 0.0000 Extract*Treat 28 70.34 2.512 1.78 0.0141 Error 160 225.19 1.407
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Chapter 6 References
Total 199 1452.24
Grand Mean 7.5321 CV 15.75
Antibacterial Statistix 8.1 5/7/2017, 6:01:48 AM
Completely Randomized AOV for SA
Source DF SS MS F P Extract 7 2372.34 338.905 565 0.0000 Error 16 9.60 0.600 Total 23 2381.93
Grand Mean 14.831 CV 5.22
At least one group variance is near zero, variance-equality tests cannot be computed.
Component of variance for between groups 112.769 Effective cell size 3.0
Completely Randomized AOV for Ecoli
Source DF SS MS F P Extract 7 3600.41 514.344 1626 0.0000 Error 16 5.06 0.316 Total 23 3605.47
Grand Mean 16.512 CV 3.41
At least one group variance is near zero, variance-equality tests cannot be computed.
Component of variance for between groups 171.342 Effective cell size 3.0
Extract Mean Aqueous 15.300 Chloroform 9.9333 Crude 20.300 Ethyl acet 15.033 Methanolic 15.800 n-Hexane 10.433 Negetive c 0.0000 Positive c 45.300 Observations per Mean 3 Standard Error of a Mean 0.3247 Std Error (Diff of 2 Means) 0.4592
Completely Randomized AOV for PA
Source DF SS MS F P Extract 7 1117.46 159.637 695 0.0000 Error 16 3.68 0.230 Total 23 1121.13
Grand Mean 9.7267 CV 4.93
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Chapter 6 References
Antifungal Statistix 8.1 5/7/2017, 6:41:59 AM
Completely Randomized AOV for AF
Source DF SS MS F P extract 7 16124.2 2303.46 1068 0.0000 Error 16 34.5 2.16 Total 23 16158.7
Grand Mean 47.995 CV 3.06
Std Error (Diff of 2 Means) 1.1993
Completely Randomized AOV for AFM
Source DF SS MS F P extract 7 16144.2 2306.32 2017 0.0000 Error 16 18.3 1.14 Total 23 16162.5
Grand Mean 49.771 CV 2.15
Completely Randomized AOV for AN
Source DF SS MS F P extract 7 16066.8 2295.26 8322 0.0000 Error 16 4.4 0.28 Total 23 16071.2
Grand Mean 49.540 CV 1.06
Antidiabetic Statistix 8.1 Analysis of Variance Table for BW
Source DF SS MS F P Day 3 137.64 45.881 41.99 0.0000 Treat 4 1309.68 327.420 299.66 0.0000 Day*Treat 12 491.94 40.995 37.52 0.0000 Error 40 43.71 1.093 Total 59 1982.97
Grand Mean 188.01 CV 0.56
Statistix 8.1 Completely Randomized AOV for Prot
Source DF SS MS F P Treat 4 11.9971 2.99928 14.4 0.0004 Error 10 2.0802 0.20802
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Chapter 6 References
Total 14 14.0773
Grand Mean 6.6867 CV 6.82
Completely Randomized AOV for ALP
Source DF SS MS F P Treat 4 12322.7 3080.67 727 0.0000 Error 10 42.4 4.24 Total 14 12365.1
Grand Mean 157.87 CV 1.30
Completely Randomized AOV for ALT
Source DF SS MS F P Treat 4 1099.73 274.933 98.2 0.0000 Error 10 28.00 2.800 Total 14 1127.73
Grand Mean 35.133 CV 4.76
Completely Randomized AOV for Bilirubin
Source DF SS MS F P Treat 4 1.19863 0.29966 10.2 0.0015 Error 10 0.29373 0.02937 Total 14 1.49236
Grand Mean 0.8660 CV 19.79
Completely Randomized AOV for Creat
Source DF SS MS F P Treat 4 4.97467 1.24367 1582 0.0000 Error 10 0.00786 0.00079 Total 14 4.98253
Grand Mean 1.0639 CV 2.64 Completely Randomized AOV for HDL
Source DF SS MS F P Treat 4 817.175 204.294 140 0.0000 Error 10 14.557 1.456 Total 14 831.731
Grand Mean 35.971 CV 3.35 Completely Randomized AOV for LDL
Source DF SS MS F P Treat 4 8908.41 2227.10 3415 0.0000 Error 10 6.52 0.65 Total 14 8914.93
Grand Mean 105.73 CV 0.76
Completely Randomized AOV for TCH
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Source DF SS MS F P Treat 4 15588.5 3897.12 646 0.0000 Error 10 60.3 6.03 Total 14 15648.8
Grand Mean 149.57 CV 1.64
Completely Randomized AOV for TG
Source DF SS MS F P Treat 4 4083.44 1020.86 1424 0.0000 Error 10 7.17 0.72 Total 14 4090.61
Grand Mean 174.85 CV 0.48
Completely Randomized AOV for Urea
Source DF SS MS F P Treat 4 9284.30 2321.07 1318 0.0000 Error 10 17.61 1.76 Total 14 9301.91
Grand Mean 85.866 CV 1.55
Completely Randomized AOV for Prot
Source DF SS MS F P Treat 4 8.7625 2.19062 7.56 0.0045 Error 10 2.8989 0.28989 Total 14 11.6613Grand Mean 6.8133 CV 7.90
Analysis of Variance Table for Glucose
Source DF SS MS F P Treat 4 191421 47855.3 10136.0 0.0000 Days 3 3629 1209.6 256.20 0.0000 Treat*Days 12 13829 1152.4 244.08 0.0000 Error 40 189 4.7 Total 59 209067
Grand Mean 192.07 CV 1.13
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