Phytochemical Screening, Invitro-Anti-Oxidant and Anti Bacterial Activities of the Stem Extracts of Jasminum Abyssinicum (Tembelel) Plant

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Chemistry Thesis and Dissertations

2021-07-27 PHYTOCHEMICAL SCREENING, INVITRO-ANTI-OXIDANT AND ANTI BACTERIAL ACTIVITIES OF THE STEM EXTRACTS OF JASMINUM ABYSSINICUM (TEMBELEL) PLANT

GASHA, HANA http://ir.bdu.edu.et/handle/123456789/12269 Downloaded from DSpace Repository, DSpace Institution's institutional repository

BAHIR DAR UNIVERSITIY

COLLEGE OF SCIENCE

DEPARTMENT OF CHEMISTRY

POSTGRADUATE PROGRAM

PHYTOCHEMICAL SCREENING, INVITRO-ANTI-OXIDANT AND ANTI- BACTERIAL ACTIVITIES OF THE STEM EXTRACTS OF JASMINUM ABYSSINICUM (TEMBELEL) PLANT

BY: HANA GASHA

ADVISOR: BELETE BEDEMO (PHD)

Bahir Dar, Ethiopia

July /2021

CERTIFICATION

The undersigned certify that they have read and hereby recommend for acceptance by the Department of Chemistry, a thesis entitled “PHYTOCHEMICAL INVESTIGATION, ANTIOXIDANT AND ANTIBACTERIAL ACTIVITIES OF THE STEM OF JASSIMINUM ABYSSINICUM” as part of the work recommended in fulfillment of the requirements for a master of science in Chemistry (Organic) at Bahir Dar University.

Board of Examiners

Name Signature Date

BELETE BEDEMO (PhD) ______

(Advisor)

(External examiner) ______

(Internal examiner)1 ______

(Internal examiner)2 ______

Acknowledgment

First, I would like to express my heartfelt glory and praises to my God for his extra ordinary gifts, uninterrupted support, divine guidance providing all aspects regarding my study and being with me in all aspects throughout my life.

I would like to express my deepest gratitude to my adviser Dr. Belete Bedemo for his unreserved cooperation, incredible suggestion, supervision and remarks, appreciable and fatherly hood consultancy. I am greatly thankful to Science College, Bahir Dar University, for providing a scholarship for MSc study. Also, I would like to thank Biology department for supplying necessary materials for antibacterial test and spectrophotometer, and material science department for supplying a Uv-Vis spectrophotometer.

I would like to thank to Mamaru Bitew Alem, a staff members of Chemistry department, Debre Markos University for his unreserved supervision. Also, I would like to thank to Dr. Esubalew Meku, a staff members of Chemistry department, Debre Markos University for supplying a chemical (DPPH) for this study. Finally, my special thanks go to my mother and families for giving me their everlasting love, support, and encouragement throughout my work and I am also grateful to my friends. I am also thanking full to all of those persons who rendered their cooperation when their helps were necessary.

iii

Contents List of Tables ...... viii

List of Acronyms ...... ix

Abstract ...... x

CHAPTER ONE ...... 1

1. INTRODUCTION ...... 1

1.1 Background of the study ...... 1

1.2. Statement of the problem ...... 2

1.3. Objective of the study ...... 3

1.3.1. General objective of the study ...... 3

1.3.2. Specific objective of the study ...... 3

1.4. Significance of the study ...... 3

1.5 Limitation of the study ...... 3

CHAPTER TWO ...... 4

2 LITERATURE REVIEW ...... 4

2.1 The family of J. abyssinicum ...... 4

2.1.1 Genus of Jasminum ...... 5

2.1.2 Previously isolated compounds from family and genus of J. abyssnicum ...... 5

2.2 Phytochemical in medicinal plants ...... 7

2.2.1 Biological activities of phytochemical ...... 8

2.2.2 Classification of Phytochemicals ...... 9

2.3 Antioxidants ...... 9

2.3.1 Classification of Antioxidants ...... 10

2.3.2 Natural Antioxidants ...... 10

2.3.3 Synthetic Antioxidants ...... 10 iv

2.4 Reactive Oxygen species and reactive Nitrogen species ...... 10

2.5 Antimicrobial resistance ...... 11

CHAPTER THREE ...... 13

3. MATERIALS AND METHODS ...... 13

3.1 Study area...... 13

3.2. Chemicals and Materials ...... 13

3.3. Instrumentation ...... 13

3.4. Sample preparation ...... 13

3.4.1. Extraction ...... 13

3.3. Qualitative Phytochemical analysis ...... 14

3.3.1. Test for terpenoids (Salkowski’s Test)...... 14

3.3.2. Test for flavonoids ...... 14

3.3.3. Test for alkaloids (Wagner’s reagent test) ...... 14

3.3.4. Test for steroids (Salkowski’s test) ...... 14

3.3.5. Test for saponins ...... 14

3.3.6. For the test of glycosides (Salkowski`s test) ...... 14

3.3.7. Test for tannins (ferric chloride test) ...... 14

3.3.8. Test for phenolics (ferric chloride test) ...... 15

3.4. Quantitative phytochemical determination ...... 15

3.4.1. Preparation of stock solution ...... 15

3.4.2 Preparation of standard solution ...... 15

3.4.3. Determination of Total Phenolic Content (TPC) ...... 15

3.4.4. Determination of Total Flavonoid Content (TFC) ...... 16

3.4.5. Antioxidant capacity assay ...... 16

3.4.5.1. DPPH radical scavenging assay ...... 16 v

3.4.6. Antimicrobial activity test ...... 16

3.4.6.1. Preparation of test solutions ...... 16

3.4.6.2. Agar diffusion method ...... 17

3.5. Method of data analysis ...... 17

CHAPTER FOUR ...... 18

4 RESULTS AND DISCUSSION ...... 18

4.1 Yield of the extracts of J. abyssinicum ...... 18

4.2. Phytochemical analysis ...... 19

4.2.1. Qualitative preliminary phytochemical analysis ...... 19

4.3. Quantitative analysis for total phenolic and total flavonoid contents ...... 20

4.3.1. Total Phenolic Content ...... 20

4.3.2. Total Flavonoid Content...... 22

4.3.3 Antioxidant capacity assay ...... 24

4.3.3.1 DPPH Radical Scavenging Activity ...... 24

4.3.4 Antibacterial activity ...... 26

4.3.4.1 Antibacterial activities of crude extracts of stems of J. abyssinicum ...... 26

CHAPTER FIVE ...... 29

5. CONCLUSIONS AND RECOMMENDATION ...... 29

5.1. Conclusion ...... 29

5.2. Recommendation ...... 29

6. Refrence ...... 30

APPENDIX ...... 34

List of Figures

Figure 1 : Previously isolated compound from the family and genus of Jasminum ...... 7 Figure 2 : J. abyssinicum ...... 8 Figure 3 : Standard Gallic acid calibration curve...... 21 Figure 4 : Standard Catichen calibration curve ...... 23 Figure 5: Absorbance vs concentration of standards and extracts ...... 25 Figure 6 : % of scavenging capacity vs concentration of standard and extracts...... 26 Figure 7: Antibacterial activities of stem extracts of J. abyssinicum ...... 28

vii

List of Tables

Table :1 Percent occurrence and role of major classes of phytochemicals [27]...... 9 Table 2 : Yields of the extract ...... 18 Table 3 : Preliminary phytochemical screening of stem extracts of J. abyssinicum ...... 19 Table 4 : Concentration and absorbance of standard Gallic acid...... 20 Table 5 :Absorbance and total phenolic content of each extract...... 21 Table 6 : Concentration and absorbance of Catichen standard ...... 22 Table 7 : Absorbance and total flavonoid content of the extract ...... 23 Table 8 : Absorbance and concentration of the standard (ascorbic acid) and extracts...... 24 Table 9: % of scavenging capacity and concentration of standard (AA) and extracts ...... 25 Table 12 : Zones of inhibition and concentration of extracts concentration...... 28

viii

List of Acronyms

WHO World Health Organization

ROS Reactive oxygen spicies

DPPH 2, 2- DiPhenyl-1-picrylhyorazyl

TLC Tin layer chromatograpy

TPC Total Phenolic Content

TFC Total Flavonoid Content

DMSO Dimethylsulfoxide

DCM Dichloromethane

STD Standard deviation

Abstract

Traditional medicinal plants are rich in bioactive compounds or secondary metabolites. Jasminum is one of the ethno-medicinal plants that have a potential of therapeutic functions. Traditional medicine has not only gained popularity and approval, but it is sometimes the only system available in many rural areas. In this study, successive extraction of stem of Jasminum abyssinicum (J.abssynicum) was carried out by using different solvent system and followed by qualitative phytochemical analysis, total flavonoid and phenolic content determination, antioxidant and antibacterial activity tests of the extracts. Qualitative phytochemical screening of the crude extracts obtained from the stems of the plant indicated the presence of alkaloids, flavonoids, phenols, glycoside, steroids, terpenoids, saponins and tannins. The highest total phenolic and total flavonoid content was obtained in ethanol extract, followed by dichloromethane and petroleum ether extract. All stem extracts of J. abyssinicum exhibited antioxidant activity as revealed by DPPH assays. All stem extracts showed antibacterial activity against gram positive (Staphylococcus aureus, Streptococcus pyogenes) and gram negative (Escherichia coli, K. pneumonia) bacteria. The result showed that the extracts of the plant exerted more bactericidal effect on gram positive bacteria than gram negative bacteria.

Key words: antioxidant activity, antibacterial activity, Jasminum abyssinicum, phytochemical, total phenol and total flavonoid content.

CHAPTER ONE

1. INTRODUCTION

1.1 Background of the study

Identification and characterization of natural products for their therapeutic activities such as antioxidant, antimicrobial, antidiabetic, anticancer, and anti-proliferative, activity has received much interest over the past few years. Nowadays, plants are recognized as a rich source of biological vital phytochemical, of which antioxidant compounds (e.g simple phenolics, anthocyanins, stilbenes, flavonoids), are used primarily in food industry [1]. It is considered that consumption of plant-based antioxidants could be connected with lowered risk of occurrence of human chronic diseases that are related to the oxidative stress, including cancer [2].

Natural products being enriched with variety of anticancer, antioxidant, and neuroprotective compounds have a great potential for drug discovery [3]. Currently, drug discovery from plants is a multidimensional research approach including botanical, phytochemical, molecular, and biological techniques providing important and new leads against pharmacological targets in various pathological conditions [4]. According to the World Health Organization (WHO) about 70–95% of the world's population in developing countries relies mainly on plants for their primary health care [5]. Traditional medicine has not only gained popularity and approval, but it is sometimes the only system available in many rural areas. Furthermore, the use of medicinal plants to treat skin infections is very common in many rural areas [6].

Scientists have demonstrated a variety of chemical compounds from plants, notably the secondary phytochemicals known to exhibit antimicrobial activity and which are effective against multidrug resistance microorganisms [4] .Groups of phytochemical compounds commonly implicated for antimicrobial activity in medicinal plants are flavonoids, alkaloids, tannins, and triterpenoids and different essential oils [7].

Jasminum abysinicum (J. abyssinicum) belongs to the plant family Oleaceae [6]. J. abyssinicum is a climbing shrub, with compounded leaves with 5 leaflets and white flower. J. abyssinicum, locally known as “Tembelel” is used in traditional medicine(TM) as wound dressing; for treating vitiligo, eye disease, rheumatic pain and arthritis [8]. Mainstream medicine is increasingly

receptive to the use of antimicrobial and other drugs derived from plants, as traditional antibiotics (product of microorganisms or their synthetic derivatives) are becoming ineffective due to multidrug resistance and hitherto unknown causes of infections[9]. For this reason, plants have become the focus of intense study in recent years to determine whether their traditional uses are supported by actual pharmacological effects or are merely based on folklore [10]. However, to the best of the researchers knowledge antibacterial and antioxidant activity of J. abyssinicum stem extract have never been explored. In this work, we aimed to investigate the phytochemical content, study the antimicrobial and antioxidant activity of stem extract of J. abyssinicum.

1.2. Statement of the problem

Plants are widely used in traditional medicine for the treatment of variety of ailments and well known for their haemostatic and wound healing properties. Traditional medicine refers to the sum total of all the knowledge, beliefs and practices that are used in diagnosis, prevention and elimination of physical, mental illness or social imbalance. Today, public health problems are the major issues worldwide. To protect this different disease, peoples use different traditional medicine and modern pharmaceuticals. Medicinal plants are an inexhaustible source of molecules with very different biological and pharmacological activities. Different researchers investigated phytochemical screening, antibacterial activity, antioxidant activity and isolation of molecular compound from many medicinal plants. J. abyssinicum root extract exhibited analgesic and anti- inflammatory activity due to its secondary metabolites, possibly flavonoids, saponins, terpenoids, triterpenes and glycosides [11]. According to previous study crude extracts of leave part of the plant have shown antimicrobial and antifungal activities against selected organisms (E.coli , P. gallinarumthose, M. hemolyticum, S. typhimurium S. agalactae) infectious to human. Aqueous extract of leaves of J. abyssinicum exhibited antibacterial activity against most organisms tested. J. abyssinicum contains polyphenols and triterpens, oligomeric secoiridoid glucosides [7].

However, investigation of phytochemical screening, antioxidant activity and antimicrobial activity of stem of J. abyssinicum has not further studied in Ethiopia. Although J. abyssinicum relatively abundant and widely used in traditional medicine in Ethiopia specially Amhara region around Bahirdar to treat skin disease (scabies), scalp wound etc. This research focuses on

investigation of phytochemicals, antimicrobial activity, and antioxidant activity of stem extract of J. abyssinicum.

1.3. Objective of the study

1.3.1. General objective of the study

The main objective of this study is to investigate phytochemicals content, and study the antioxidant and anti-microbial activity of the stem extracts of J. abyssynicum plant.

1.3.2. Specific objective of the study

The specific objectives of designed work are:

 To extract the stem part of J. abyssinicum using different solvent system successively.  To screen the major phytochemical constituents in each stem extract of J. abyssinicum  To determine the total flavonoids and phenolic contents of each extract.  To analyze the antibacterial and antioxidant activities of each extract.  To compare the antibacterial and antioxidant activities of the extracts with the standards.

1.4. Significance of the study

Recently, there has been much research emphasis on the phytochemical screening, TPC, TFC, antioxidant and antibacterial activity of medicinal plants. Medicinal Plants with these attributes are good resources for general health maintenance and use for treatment of different diseases. In this work, the phytochemical content of the stem of J. abyssinicum for petroleum ether ,dichloro methane and ethanol solvent extracts, its anti-microbial and anti-oxidant activity have been reported. Besides, we have reported the total phenolic and total flavonoid content of the plant under study. Hence, the output of these research will benefit pharmacologists, food scientists and more specifically chemists of the same research area.

1.5 Limitation of the study

 There was no enough chemical inside the store (reagent, solvent etc).  Instrument problem (MS, NMR, IR spectroscopy).

CHAPTER TWO

2 LITERATURE REVIEW

2.1 The family of J. abyssinicum

Traditionally Oleaceae family has significant economic, horticultural and medicinal importance [12]. Many species in the genus Syringa are grown for beautification purposes and their flowers are extracted for essence. In the Liubań district of Belarus, the buds of S. vulgaris L. are processed as medicinal wine for the treatment of joint pain and dried flowers as recreational tea [13]. In the Peninsula Sorrentina (Southern Italy), the bark of F. ornus L. is used for treating diarrhea and lowering cholesterol [14] . The leaves of Olea europaea L. are used in Greece for lowering blood pressure [15].

This family includes 25 genera with approximately 688 species [12] . The species of Syringa oblata var. diatata, Jasminum nudiflorum Lindl., Osmanthus fragrans (Thunb.) Lour., J. sambac (L.) Ait. and Forsythia suspensa are famous ornamental plants and also O. fragrans (Thunb.) Lour. and J. sambac (L.) Ait. also serve as sources of aromatic oil or food [16]. Fraxinus mandshurica Rupr. is an excellent wood, which can be made into furniture and the samara of F. suspensa and the fruits of Ligustrum purpurascens Y. C. Yang are available for medicinal purposes [17]. Phytochemical investigations have revealed that the main chemical constituents from this family are flavonoids, monoterpenoids, iridoids, secoiridoids and phenylethanoid glycosides [18].

J. humile is in Family of Oleaceae a small erect much-branched shrub, growing to 1 m or more tall, commonly found in the Himalayan region and it has green, angular branches. Leaves are hpinnate with 3-7 ovate to lance like leathery leaflets. The last leaflet is some what larger [4]. Inflorescences are lax clusters of yellow tubular flowers at the end of branches. Yellow Jasmine contains indole alkaloids (including gelsemine and gelsedine), iridoids, coumarins, and tannins [4]. Fraxinus, a member in the Oleaceae family, commonly known as ash tree is found in various regions of world such as in western and eastern France, North American, north East Asia, China, north Pakistan, India, Afghanistan, Morocco, and Algeria [19] . In northern areas of Pakistan, root bark and leaves of Fraxinus plant have been traditionally used for the cure of malaria and pneumonia . A range of chemical constituents including secoiridoids, phenyl echinoids’, lignums, flavonoids, and coumarins has been isolated from Fraxinus plant [20]. Metabolites and extracts 4

from this plant have been found to possess variety of biological activities such as anticancer, anti- inflammatory, antioxidative, antimicrobial, hepato protective, antiallergic, skin regenerating, and diuretic [20] .

2.1.1 Genus of Jasminum

Genus Jasminum (Oleaceae) includes beyond 200 species, some of which are used in folk medicine or cultivated to obtain essential oil from the fragrant flowers [21] . The term Jasminum was first mentioned in the ‘‘Materia Medica’’ of Dioscoride [22]. The phytochemical studies of the aerial parts of some species, J. sambac [Soland.], J. mesnyi Hance J. urophyllum Hemsl. and J. nudiflorum Lindl. resulted in the isolation of some secoiridoid glucosides, in particular of oligomeric consisting of oleoside units linked to a cyclopentanoid monoterpene named iridane[22]. Plants of the genus Jasminum are widely distributed in the temperate and semitropical zone of Asia and Africa. The flowers of many Jasminum plants, such as Jasminum sambac, Jasminum polyanthum, Jasminum nudiflorum and Jasminum lang, are used as or folk remedy in China for the treatment of arthritis, hepatitis, conjunctivitis, gastritis and diarrhea [23].

2.1.2 Previously isolated compounds from family and genus of J. abyssnicum

Phytochemical investigations have revealed that the main chemical constituents from this family are flavonoids, monoterpenoids, iridoids, secoiridoids and phenylethanoid glycosides. However only Seoiridoids have been isolated .Seoiridoids are a group of compounds in the cyclopentane monoterpene derivatives formed by the cleavage of the cyclomethene oxime compounds at C-7 and C-8. Secoiridoids have shown a variety of pharmacological effects including anti-diabetic, anti-inflammatory, neuroprotective, anti-cancer and anti-obesity [12].

where R1 = H R2= CH3 R3 = Glc

Figure 1 : Previously isolated compound from the family and genus of Jasminum

2.2 Phytochemical in medicinal plants

Phytochemicals are biologically active, naturally occurring chemical compounds found in plants, which provide health benefits for humans further than those attributed to macronutrients and micronutrients [24]. They protect plants from disease and damage and contribute to the plant’s color, aroma and flavor [25]. In general, the plant chemicals that protect plant cells from environmental hazards such as pollution, stress, drought, UV exposure and pathogenic attack are called as phytochemicals. Recently, it is clearly known that they have roles in the protection of human health, when their dietary intake is significant. Wide -ranging dietary phytochemicals are found in fruits, vegetables, legumes, whole grains, nuts, seeds, fungi, herbs and spices. Broccoli, cabbage, carrots, onions, garlic, whole wheat bread, tomatoes, grapes, cherries, strawberries, raspberries, beans, legumes, and soy foods are common sources.

Phytochemicals accumulate in different parts of the plants, such as in the roots, stems, leaves, flowers, fruits or seeds. Many phytochemicals, particularly the pigment molecules, are often concentrated in the outer layers of the various plant tissues. Phytochemicals are also available in supplementary forms, but evidence is lacking that they provide the same health benefits as dietary phytochemicals[24].

Figure 2 : J. abyssinicum

2.2.1 Biological activities of phytochemical

Phytochemicals are secondary plant metabolites and have biological properties such as antioxidant activity, antimicrobial effect, modulation of detoxification enzymes, stimulation of the immune system, decrease of platelet aggregation and modulation of hormone metabolism and anticancer property. There are more than thousand known and many unknown phytochemicals. It is well- known that plants produce these chemicals to protect themselves, but recent researches demonstrate that many phytochemicals can also protect human against disease. The phytochemicals present in plants are responsible for preventing disease and promoting health have been studied extensively to establish their efficacy and to understand the underlying mechanism of their action. Such studies have included identification and isolation of the chemical components, establishment of their biological potency both by in vitro and in vivo studies in experimental animals and through epidemiological and clinical-case control studies in man. Study findings suggest that phytochemicals may reduce the risk of coronary heart disease by preventing the

oxidation of low density lipoprotein (LDL) cholesterol, reducing the synthesis or absorption of cholesterol [24].

2.2.2 Classification of Phytochemicals

The exact classification of phytochemicals has not been given so far, because of their diverse forms and structures. Classically, the phytochemicals have been classified as primary or secondary metabolites, depending on their role in plant metabolism. Primary metabolites include the common sugars, amino acids, proteins, purines and pyrimidines of nucleic acids, chlorophylls etc. Secondary metabolites are the remaining plant chemicals such as alkaloids, terpenes, flavonoids, lignans, plant steroids, curcumines, saponins, phenolics and glucosides. Literature survey indicates that phenolics are the most common and structurally most diverse plant chemicals [26].

Table :1 Percent occurrence and role of major classes of phytochemicals [27].

Class of Occurrence Role in health care secondary as natural phytochemical product (%)

Phenolics 45 Anti-oxidants, anticanerous anticancerous, cytotoxicants, anti- microbialsand vasodilating

Terpenoids and 27 Anti-microbial, detoxifying agents,strengthners, antirheumatics, Steroids anti-malarial, hepaticidal

Alkaloids 18 Neuropharmaceuticals, anticancerous, sedatives, antimicrobials, insecticidal

Other chemicals 10 Anti-inflammatory, Immunostimulating

2.3 Antioxidants

Antioxidants are the molecules that prevent cellular damage caused by oxidation of other molecules. Oxidation reactions are known to produce free radicals, which are highly reactive

species containing one or more unpaired electrons in their outermost shell. Once they are formed, the chain reaction starts. Antioxidant reacts with these free radicals and terminates this chain reaction by removing free radical intermediates and inhibits other oxidation reactions by oxidizing themselves. Though oxidation reactions are crucial for life, they can also be damaging. Plants and animals have a complex system of multiple types of antioxidants, such as vitamin C and vitamin E, as well as enzymes, such as catalase (CAT), superoxide dismutase (SOD), and various peroxidases. Oxidative stress plays a key role in causing various human diseases, such as cellular necrosis, cardiovascular disease, cancer, neurological disorder, Parkinson’s dementia, Alzheimer’s disease, inflammatory disease, muscular dystrophy, liver disorder, and even aging (Amit and Priyadarsini Besides, there are some antioxidants in the form of micronutrients which cannot be manufactured by the body itself such as vitamin E, β-carotene, and vitamin C, and hence these must be supplemented in the normal diet [28].

2.3.1 Classification of Antioxidants

Antioxidants can be classified into two major types based on their source, i.e., natural and synthetic antioxidants [29].

2.3.2 Natural Antioxidants

Natural antioxidants either are synthesized in human body through metabolic process or are supplemented from other natural sources, and their activity very much depends upon their physical and chemical properties and mechanism of action [29].

2.3.3 Synthetic Antioxidants

Synthetic antioxidants are artificially produced or synthesized using various techniques. Basically they are polyphenolic compounds mainly that capture the free radicals and stop the chain reactions. Polyphenolic derivatives usually contain more than one hydroxyl or methoxy group. Ethoxy quinine is the only heterocyclic, N-containing compound reported to be used as antioxidant in the food [30].

2.4 Reactive Oxygen species and reactive Nitrogen species Oxidative stress is an increase in the steady-state levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), Also, it is a decreased antioxidant defense mechanism, and 10

occurs when the amount of ROS and RNS is more than the defensive system can remove [31]. As a result, the antioxidant competence is challenged with various problems and which in turn makes the body system unable to maintain redox homeostasis. This condition is, at least in part, associated with the occurrence of several chronic diseases [32]. It is suggested to be the main cause for several chronic diseases: adiposity, diabetes and diabetic complications, arthritis, neurodegenerative diseases, cancer, chronic kidney disease, cardiovascular diseases, endothelial cell dysfunction, aging and atherosclerosis [31, 33, 34]. In addition, increased caloric intake, decreased energy expenditure, alcoholism and smoking are suggested to contribute to generation of more ROS than needed [32].

2.5 Antimicrobial resistance

Antimicrobial resistance (AMR) is the ability of a microbe to resist the effects of medication that once could successfully treat the microbe. AMR is driven by widespread antibiotic use, increasing a serious public health threat, and a worldwide problem that can cross international boundaries and spread between continents with ease [35]. The wide availability of effective antibiotics is under threat, endangering modern health care [36]. It is suggested that the extensive use and misuse of antimicrobials during the last decades have been associated with the explosion of antimicrobial resistance [37]. What gives AMR more apprehension worldwide is, medical procedures such as organ transplantation, cancer chemotherapy, diabetes management and major surgery become very high risk without effective antimicrobials for prevention and treatment of infections [38]. On the other hand, the forecasts of the economic costs for AMR are similar to those of a 2°C rise in global average surface temperature, above preindustrial levels [36]. Africa in general and east Africa in particular is vulnerable region to AMR with a high load of infectious diseases [39].

A study reported that the burden of AMR is comparable to the combined effect of influenza, tuberculosis and HIV/AIDS. It is also reported that the impact is highest in infants and the elderly. Disturbingly, the study also reported that 39% of all AMR were caused by bacteria that are resistant to last-line antibiotics, making them very difficult or impossible to treat [40]. As per the report of World Health Organization, new resistance mechanisms are emerging and spreading globally, threatening the ability of scientists in the field to treat common infectious diseases, resulting in prolonged illness, disability, and death [38]. Besides the existing resistance to

antibiotics, the development of new antibiotics is deteriorating at the same time [41]. On the other hand, poor solubility, stability, and side effects are leading to inefficiency to the currently available antibacterial drugs .These combined issues urged the researchers in the field to explore strategies, creation of other means of dealing with bacterial disease to overwhelmed such tough microbes [42].

CHAPTER THREE

3 MATERIALS AND METHODS

3.1 Study area

The stem of J. abyssinicum was collected from Bahir Dar Tiss abay kebele, 565 Km North West of Addis Ababa, Ethiopia. The Experiment was conducted at Bahir Dar University department of chemistry post graduate research Laboratory.

3.2 Chemicals and Materials

Laboratory chemicals and reagents used for this study were DCM, PE, ethanol, ammonia (NH3), hydrochloric acid (HCl), sulfuric acid (H2SO4), sodium hydroxide (NaOH), 10% aqueous FeCl3,

Iodine in potassium iodide, Folin-ciocalteu reagent, sodium carbonate(Na2CO3), aluminum chloride (AlCl3), ascorbic acid, Gallic acid, Catechin, DPPH reagent, gentamycin, Muller Hinton Agar and Distilled water was used throughout the experiment.

3.3 Instrumentation

The necessary apparatus and instruments used for this study were Mortar and Pestle, for grind plant material, electronic beam balance for mass measurement, electrical shaker to mix and extract the mixture well, Rotary Evaporator for the removal of solvent from the mixture, UV-visible spectrophotometer, for the absorbance measurement, Whatman No.1 filtrate paper for filtration purpose.

3.4. Sample preparation

The selected samples were thoroughly washed with tap water to remove all the dust particles. The cleaned stem was air dried in the absence of light and ground into a powder fine texture and stored at room temperature in a plastic bag prior to use.

3.4.1. Extraction

The powdered stem sample was successively extracted with petroleum ether, dichloromethane and ethanol using maceration technique for 48 hours in each solvent. The extract was filtered and the solvent from each extract was removed using Rotary evaporator under reduced pressure. The resulting semidried mass of each extract was stored in fridge until used for further experiments.

3.3. Qualitative Phytochemical analysis

3.3.1. Test for terpenoids (Salkowski’s Test)

Extracts of 3 mL were taken and 2 mL of chloroform and 2 mL of concentrated H2SO4 were added along the sides of the tube. The reddish-brown/grayish color in the interface is considered positive for the presence of terpenoids [43].

3.3.2. Test for flavonoids

2 mL of the extract was treated with 2 mL of dilute NH3 solution and a few drops of concentrated H2SO4. A formation of yellow color indicates the presence of flavonoid [44]

3.3.3. Test for alkaloids (Wagner’s reagent test)

Extracts were dissolved individually in dilute hydrochloric acid and filtered. Then the filtrates were treated with Wagner’s reagent (iodine in potassium iodide). Formation of brown/ reddish precipitate indicates the presence of alkaloids [45].

3.3.4. Test for steroids (Salkowski’s test)

Two mL of each crude extract was mixed with 2 mL of chloroform and concentrated sulfuric acid was added sidewise. A red color was produced in the lower layer indicated the presence of steroid [46].

3.3.5. Test for saponins

Two mL of crude extract was mixed with 5mL of distilled water in a test tube and it was shaken vigorously. The formation of dark green was taken as an indication for the presence of saponins [44]

3.3.6. For the test of glycosides (Salkowski`s test)

Two mL of each crude extract was mixed with 2mL of chloroform. Then 2 mL of concentrated sulfuric acid was added carefully and shaken gently. A reddish brown color was observed which indicates the presence of steroidal ring [44]

3.3.7. Test for tannins (ferric chloride test)

Two mL of the extract was added to 2 mL of water, and 2 drops of diluted ferric chloride 14

solution was added. A dark green or blue green coloration indicates the presence of tannins [47, 48].

3.3.8. Test for phenolics (ferric chloride test)

To 3 mL of extract, 2 mL of distilled water followed by drops of 10% aqueous FeCl3 solution was added. Formation of blue or green indicates the presence of phenols [49, 50].

3.4. Quantitative phytochemical determination

3.4.1. Preparation of stock solution

A stock solution of 100 ppm of Gallic acid and Catechine were prepared by dissolving 0.1g of Gallic acid and catechine by distilled water in 1000 mL volumetric flask for total phenolic and flavonoid content determination respectively. Ascorbic acid solution (400 ppm) was prepared by dissolving 0.04 grams of ascorbic acid using ethanol in a 100 mL volumetric flask for DPPH assay.

3.4.2 Preparation of standard solution

Serious of solutions of 10, 20, 30, 40 and 50 ppm and 20, 40, 60 and 80 ppm from 100 ppm of stock solution of gallic acid and catechine were prepared for constructing the calibration curve of Gallic acid and catechine using serial dilution law, respectively., 25, 50, 100, 200 and 400 ppm of ascorbic acid was prepared in ethanol for DPPH assay.

3.4.3. Determination of Total Phenolic Content (TPC)

The total phenolic content of the crude extracts were determined by using Folin-Ciocalteu method. A series of 10, 20, 30, 40 and 50 ppm of standard Gallic acid was prepared from Gallic acid stock solution. 4 mL of each of the standard and plant extract were mixed with 1 mL of Folin-Ciocalteu reagent separately and allowed to stand for 6 minutes. Then 1 mL of 10% sodium carbonate solution was added to the reaction mixture. Then absorbance was recorded after 30 minute at 765 nm on UV-Vis spectrophotometer. Total phenolic content of the extracts of dichloromethane and ethanol was calculated as Gallic acid equivalents (mgGAE/g).

3.4.4. Determination of Total Flavonoid Content (TFC)

Aluminum chloride complex forming assay was used to determine the total flavonoid content of the extracts. Catichen was used as a standard to make the calibration curve and flavonoid content was determined as Catichen equivalent [51] [52]. Standard Catichen solution (20, 40, 60 and 80 ppm) was prepared. 1 mL of each of the catichen solution and the plant extract were mixed with 5 mL of distilled water and then with 0.3 mL of 5% Sodium nitrite separately and allowed to stand for 5 minutes for mixing. Then 0.3 mL of 10% Aluminum chloride solution was added and allowed to stand for 6 minutes at room temperature, then 2 mL solution of 1M Sodium hydroxide was added sequentially and distilled water was added until the final volume reaches 10 mL. Finally the absorbance of the reaction mixtures was recorded at 510 nm on UV-Vis spectrophotometer. Total flavonoid content of each extract was calculated as Catichene equivalents (mgCE/g).

3.4.5. Antioxidant capacity assay

3.4.5.1. DPPH radical scavenging assay

Free radical scavenging activities of the extracts and isolated compounds were determined by using 2, 2- Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method [51]. A fresh 0.004% solution of DPPH was prepared in ethanol. Standard solutions of 25, 50, 100, 200 and 400 ppm of ascorbic acid used as reference and extracts were prepared. 4 mL of ascorbic acid and extracts of each concentration were mixed with 1 mL solution of DPPH separately and allowed to stand in darkness for 20 minutes. Control was prepared by taking 2 mL of DPPH and 3 mL of ethanol. Finally, the uv-vis absorbance was measured and the percentage inhibition of DPPH by extracts was calculated by using the following formula: 퐴−퐵 푋100 %Inhibition = 퐴

Where A is the absorbance of pure DPPH in oxidized form while B is the absorbance of sample taken after 20 minutes of reaction with DPPH.

3.4.6. Antimicrobial activity test

3.4.6.1. Preparation of test solutions

Test solutions (4000 ppm) were prepared by dissolving 4 mg of each of crude extract in 16

1 mL of dimethyl sulfoxide. 2000, 1000, 500 and 250 ppm of crude extract were prepared by serial dilution method from 4000 ppm solution.

3.4.6.2. Agar diffusion method

The disk diffusion method with Muller Hinton agar was used to evaluate antimicrobial activity. Using a sterile cotton swab, the fresh culture of bacteria strains were swabbed on the surface of sterile agar plates. Each plant extract of the test solution was poured on the surface of agar plates inoculated including with culture bacteria in Petri dishes by using 5 mm diameter sterile discs (Whatman No 3 paper). Standard disc of the antibiotic disc (gentamicine, 30µg/disc) was serving as the positive antibacterial control and for negative control the DMSO solution was used. Then the plates were incubated for 24 hours at 37˚C. After 24 hours, zone of inhibition was observed and recorded in millimeter. The tests were performed in duplicates for each microorganism evaluated and the final results were presented as arithmetic average (Mean ±Std). Antibacterial activity was recorded if the zone of inhibition was greater than 6 mm [53].

3.5. Method of data analysis

The total phenolic and Flavonoid content of the stem extracts of Jasminum abyssinicum by petroleum ether, dichloromethane and ethanol were calculated as Gallic acid equivalents V (mgGAE/g) and as Catichene equivalents (mgCE/g) respectively. T  C Where T is the total M phenolic and flavonoid content in mg/g of the extracts as GAE and CE for total phenolic and flavonoid content respectively, C is the concentration of Gallic acid and Catichen established from the calibration curve in mg/mL, V is the volume of the extract solution used in mL and M is the weight of the extract used in g.

The percentage inhibition of DPPH by extracts was calculated by using following formula

퐴−퐵 % Inhibition = 푋100 퐴

Where A is the absorbance of pure DPPH in oxidized form while B is the absorbance of sample taken after 20 minutes of reaction with DPPH.

CHAPTER FOUR

4 RESULTS AND DISCUSSION

The powder stem of J. abyssinicum (300 g) was subjected to successive extraction petroleum ether, dichloromethane and ethanol. The extracts were analyzed for phytochemical screening, total phenolic, total flavonoid, antioxidant (antioxidant activities by DPPH) and antibacterial activities. The dichloromethane extract was also subjected to isolation, and antioxidant (antioxidant activities by DPPH) and antibacterial activities were done by isolated compounds.

4.1 Yield of the extracts of J. abyssinicum

Successive extraction from 300 g powder stem of J. abyssinicum by PE, DCM and ethanol solvent gave the following result. Table 2 : Yields of the extract

The yield of the extract should depend on the polarity of the solvent used during extraction. Solubility of the natural products by the solvent could also determine the yield. In this work, the highest yield was obtained in ethanol extract (11.2 g) followed by DCM (8.5g) and PE (2.9 g) (Table 2).

4.2. Phytochemical analysis

4.2.1. Qualitative preliminary phytochemical analysis

The result of phytochemical screening of stem extracts of J. abyssinicum using PE, DMC and ethanol successively have been tabulated (Table 3). In the present study, phytochemical screening test of PE, DCM and ethanol extracts of stem of J. abyssinicum revealed the presence of saponins, galycosides, alkaloids, tannins, phenols, flavonoids, steroids and terpenenoids. The result showed that DCM and ethanol solvent system extracts higher bioactive compounds compared to PE solvent. Previous study reported that polyphenols, oligomeric secoiridoid glucosides, triterpens steroids, flavonoids, and saponins were presented on ethanol and water extract of leave of J. abyssincium [7].

Table 3 : Preliminary phytochemical screening of stem extracts of J. abyssinicum

Class of compounds Test reagent Type of Solvent

Petroleum ether DCM Ethanol

Alkaloids Wagner + +++ ++

Phenolics ferric chloride test + ++ +++

Flavonoids H2SO4 + ++ +++

Steroids Salkowski’s Test + +++ ++

Terpenoids Salkowski’s Test + +++ +++

Saponins Shaking with water - ++ -

Tannins ferric chloride + +++ ++

Glycosides NaOH test - ++ ++

Key: “+++” present in very high amount “++” present in midium amount “+” present in very small amount “-“not present

4.3. Quantitative analysis for total phenolic and total flavonoid contents

4.3.1. Total Phenolic Content

Phenolic compounds, especially phenolic acids, play an important role in the overall radical scavenging ability. Phenolic compounds were considered as a major group of compounds that contributed to the antioxidant activity [54]. The total phenolic content (TPC) of each extracts of J. abyssinicum stem sample was measured according the Folin-Ciocalteau spectrophotometric method. The total phenolic content of each stem extracts using the Folin-Ciocalteu’s reagent was determined in terms of Gallic acid equivalent. The regression equation of the calibration curve of the Gallic acid standard for the present study was y= 0.0011x - 0.0887, R2 = 0.99891. The total phenolic content of each extracts of stem of J. abyssinicum was determined from this regression equation and the results were (Table 5).

Table 4 : Concentration and absorbance of standard Gallic acid

0.05 Y=0.0011X- 0.0887 R2=0.99891 0.04

0.03

0.02

Absorbance (nm) Absorbance 0.01

0.00

10 20 30 40 50 Concentration (ppm)

Figure 3 : Standard Gallic acid calibration curve.

Table 5 :Absorbance and total phenolic content of each extract.

The total phenol content of crude extracts of stem samples of J. abyssinicum was ranged from 98.91 to 142.88 mg GAE/g of dried weight (Table 5). The highest total phenol content was obtained from ethanol extract (142.8) followed by DCM extract (124.05) and the least value were recorded in PE extract (98.91) mg GAE/g of dried weight. Mostly phenolic compounds were extracted by polar solvents [55]. The present result also showed more phenolic compounds were extracted by polar solvent.

4.3.2. Total Flavonoid Content

Aluminum chloride complex forming assay was used to determine the total flavonoid content of the extracts. Catichen was used as standard and flavonoid content was determined as Catichen equivalent (mgQE/g) [56]. The total flavonoid content (TFC) of stem extracts of J. abyssinicum by PE, DCM and ethanol were determined as Catichen equivalent (mgCE/g). To perform the calculations of total flavonoid content, a standard curve is needed which is obtained from a series of different Catichen concentrations. In this study 20, 40, 60 and 80 ppm concentration of Catichen were used to construct the calibration curve. The calibration curve was constructed by drawing absorbance verses concentration and a straight line with an equation of y = 0.00783x+2.1745 and a linear regression coefficient (R2) of 0.9793 were obtained.

Table 6 : Concentration and absorbance of Catichen standard

2.9 y=0.00783x +2.1745 2 2.8 R =0.9793

2.7

2.6

Absorbance 2.5

2.4

2.3 20 30 40 50 60 70 80 Concentration (ppm)

Figure 4 : Standard Catichen calibration curve

Table 7 : Absorbance and total flavonoid content of the extract

The result showed that the total flavonoid content (TFC) of crude extracts of stems of J. abyssinicum was ranged from 50.846 to 83.141 mg CE/g. The highest total flavonoid content was 23

obtained in ethanol extract (83.141) followed by medium polarity DCM extract (60.586) and the least total flavonoid content was recorded in PE crude extract (50.846) mg CE (Catichene equivalent)/g of dried weight (Table 7). The result revealed that the amount of flavonoid extracts from the plant depends on the polarity of the solvent.

4.3.3 Antioxidant capacity assay

4.3.3.1 DPPH Radical Scavenging Activity

Antioxidants react with DPPH (which is a stable free radical) and is reduced to the DPPHH. As a result, the absorbance’s decreased in which the radical changes from DPPH to the DPPH-H form. The degree of discoloration indicates the scavenging potential of the antioxidant compounds or extracts in terms of hydrogen and electron donating ability [57].

The antioxidant activity of crude extracts of J. abyssinicum and isolated compounds of DCM extract were determined using DPPH (diphenylpicrylhydrazyl) assay. DPPH radical was dissolved by ethanol that forms purple color solution and the maximum absorbance (1.3901) was recorded at 517 nm used as control. The purple color of DPPH is changed when mixed with the crude extracts (the color change depends on antioxidant ability) indicates that scavenging of the radicals by antioxidants and decreases the absorbance. This demonstrates that the antioxidants found in extracts quench the free radicals. In the present study, percentage of inhibition was determined to evaluate the antioxidant activity of the extracts which is able to inhibit free radicals.

Table 8 : Absorbance and concentration of the standard (ascorbic acid) and extracts.

Absorbance at 517 nm Concentration(ppm) AA(standard) EE DCME PEE 25 0.1685 1.0763 1.2044 1.3325 50 0.1455 0.8292 0.9573 1.0996 100 0.115 0.5981 0.7262 0.9093 200 0.0585 0.401 0.5271 0.6891 400 0.0025 0.208 0.353 0.498 Blank solution = 1.3901

AA 1.4 EE DCME 1.2 PEE

1.0

0.8

0.6

Absorbance 0.4

0.2

0.0

0 50 100 150 200 250 300 350 400 450 Concentretion

Figure 5: Absorbance vs concentration of standards and extracts

Table 9: % of scavenging capacity and concentration of standard (AA) and extracts

% of scavenging capacity Concentration(ppm) AA EE DCM PE

25 87.878 22.574 13.359 4.144

50 89.533 40.349 31.134 20.898

100 91.727 56.976 47.759 34.587

200 95.792 71.153 62.082 50.428

400 99.821 85.037 74.606 64.175

100

40 AA EEE DCME 20 PEE

% of scavenging% of capacity 0

0 50 100 150 200 250 300 350 400 450 Concentration(ppm)

Figure 6 : % of scavenging capacity vs concentration of standard and extracts.

The result of antioxidant activity of extracts showed that AA antioxidant activity higher than all. Ethanol extract antioxidant activity higher than DCM extract and the least value antioxidant activity was recorded by EE. Reported the ethanol extract from the Millingtonia hortensis L. leaves exhibited the highest DPPH radical scavenging activit [58].

4.3.4 Antibacterial activity

4.3.4.1 Antibacterial activities of crude extracts of stems of J. abyssinicum

Antibacterial activities of DCM and ethanol stem extracts of J. abyssinicum were evaluated by using Agar well diffusion method. Four different bacteria were used in this study to evaluate antibacterial activities of the plant extract. Two gram negative bacteria (Escherichia coli and Klebsiella pneumonia) and the remaining two were gram positive bacteria (Staphylococcus aureus and Streptococcus pyrogens) were used in Agar well diffusion method. Antibacterial activities of DCM and ethanol extracts at 4000, 2000, 1000, 500 and 250 ppm were diffused into incubated plates in which bacteria was cultured and inhibition zone values of each extracts of 1000 ppm was recorded and the result was put in Table 12.

The crude extracts of DCM exhibited better antibacterial activity followed by ethanol extract. The DCM crude extract showed maximum antibacterial activity followed by ethanol crude extracts against gram positive bacteria (Staphylococcus aureus and Streptococcus Pyogenes). The antibacterial activity of crude extracts of stem of J. abyssinicum was observed on the range between 12.643 to 18.410 mm inhibition zones. The highest inhibition zone was displayed by DCM crude extract which was 18.41 mm inhibition zone followed by ethanol extract (13.85 mm inhibition zone) against Staphylococcus aureus. The DCM crude extract also showed the maximum antibacterial activity (14.4 mm inhibition zone) followed by ethanol extract (13.636 mm inhibition zone) against Streptococcus Pyogenes (Table 12). Among those gram positive bacteria Streptococcus Pyogenes was more susceptible towards all crude extracts.

The crude extracts of stem of J. abyssinicum were also showed antibacterial activity against gram negative bacteria (Escherichia coli and Klebsiella pneumonia). Ethanol crude extract exhibited better antibacterial activity which had 18.28 mm inhibition zone followed by DCM extract (15.75 mm inhibition zone) against Escherichia coli.

Maximum activity was exhibited by DCM extract (17.95 mm inhibition zone) followed by ethanol extract which had 12.643 mm zone against Klebsiella pneumonia (table 12). Among those gram negative bacteria Escherichia coli is more susceptible in DCM extracts and Klebsiella pneumonia is more susceptible in ethanol extract.

Thus, the present study showed that the stem extracts of J. abyssinicum by DCM and ethanol were possessed significant antibacterial activity and provides possible rationalization to the traditional use of this plant wound dressing. The result also showed that stem extracts of J. abyssinicum had better activity towards gram positive bacteria compared to gram negative bacteria. Generally Gram-negative bacteria are more resistant to antimicrobial agents compared with Gram-positive bacteria because they are covered with a phospholipid membrane carrying the structural lipopolysaccharide impermeable to antibacterial substance [59]. All crude extracts were showed weaker inhibition zone than that of gentamicine (standard antibiotic). Phytochemical groups extracted in each solvent are responsible for this antibacterial activities (Table 3). Alkaloids, flavonoids, phenolics and other secondary metabolites have antibacterial activity. Plants with alkaloids have been determined to exhibit antibacterial properties 27

and are used in medicines for reducing headache and fever [60]. [60]Antibacterial activity of plant extract are well known and probably caused by the alkaloids [61]. The maximum antibacterial activity observed by DCM in the present study was also probably by alkaloids extracted by DCM exclusively than ethanol solvents (Table 3).

Table 10 : Zones of inhibition and concentration of extracts concentration.

Test bacteria Conc(ppm) Mean zone of inhibition + S.D(mm)

DCM Ethanol Gentamicine

Staphylococcus aureus 1000 18.41 ± 0.3540 13.85 ± 0.2585 30 ± 0

Streptococcus 1000 14.40 ± 0.606 13.643 ± 0.3535 29.5 ± 0.3535 pyogenes Klebsiella pneumonia 1000 17.95 ± 0.540 12.643 ± 0.2573 29.5 ± 0.2549

Escherichia coli 1000 15.75 ± 0.6614 18.28 ± 0.70711 28 ± 0.7071

35 30 25 20 15 10 5 0 S.aurues S.pyogenes E.coil K.pneumonia DCM EE Gentamicine

Figure 7: Antibacterial activities of stem extracts of J. abyssinicum

CHAPTER FIVE

5. CONCLUSIONS AND RECOMMENDATION

5.1. Conclusion

Qualitative phytochemical screening test of PE, DCM and ethanol extracts of Stems of J. abyssinicum revealed the presence of flavonoids, alkaloids, phenols, glycosides, steroids, terpinoids, saponins and tannins. The ethanol crude extract apparently contained most of the flavonoid and phenolic compounds followed by DCM extract. All the crude extracts possessed antioxidant activity as evidenced by DPPH assays. The highest antioxidant activity was exhibited by ethanol extract followed by DCM and PE extract. The highest flavonoid and phenolic content of the extract has significant linear correlation to the antioxidant activity of the extract. Thus, the better antioxidant activity of ethanol extract may be due to its high content of flavonoid and phenolic compound. This may be due to the ability of constituents to donate hydrogen atoms or electrons. Antibacterial effects of stem extracts of J. abyssinicum showed different degrees of inhibition zone against both Gram positive and Gram negative bacteria. The DCM extract showed better antibacterial activity relative to ethanol and extract.

5.2. Recommendation

 Isolation and characterization of bioactive compounds on the crude extracts of the stems, flowers, root, and fruits of J. abyssinicum by using different solvent system should be done.  Anti-microbial studies by using other kinds of bacteria, malaria and fungi on the crude extracts and isolated compounds (pure compounds) should be done.  Determination of the cytotoxity level of the extracts from all parts of the plant also should be done.

6. Refrence

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APPENDIX

Appendix 1: Preliminary qualitative phytochemical test

Alkaloid Terpinoid Phenol Steroide EE,DCM,PE EE,DCM,PE EE,PDCM,PE EE,DCM,PE

Glycosid Flavonoid Saponin Tanin EE,DCM, EE,DCM,PE EE,DCM,PE EE,DCM,PE PE

Appendix 2 : Solutions of extracts for DPPH assay : A(25), B(50), C(100), D(200) and E(400).

Appendix 3 : Zone of inhibition of extracts at different concentration against gram positive and gram negative bacteria