Study on Biological Activities and Essential Oil of Cinnamomum Tamala

STUDY ON BIOLOGICAL ACTIVITIES AND ESSENTIAL OIL OF CINNAMOMUM TAMALA

A DISSERTATION SUBMITTED FOR THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE MASTERS DEGREE OF SCIENCE IN CHEMISTRY

BY Dhan Bahadur G.C. Exam Symbol No.: Chem.426/072 T.U. Regd. No.: 5-2-37-111-2011

CENTRAL DEPARTMENT OF CHEMISTRY INSTITUTE OF SCIENCE AND TECHNOLOGY TRIBHUVAN UNIVERSITY, KIRTIPUR KIRTIPUR,KATHMANDU NEPAL May, 2019 BOARD OF EXAMINER AND CERTIFICATE OF APPROVAL

This dissertation entitled “STUDY ON BIOLOGICAL ACTIVITIES AND ESSENTIAL OIL OF Cinnamomum tamala” by Dhan Bahadur G.C., under the supervision of Prof. Ram Chandra Basnyat, PhD, Central Department of Chemistry, Tribhuvan University, Nepal, is hereby submitted for the partial fulfillment of the Master of Science (M.Sc.) Degree in Chemistry. This dissertation has been accepted for the award of a degree.

______Supervisor Prof. Ram Chandra Basnyat, PhD Central Department of Chemistry Tribhuvan University Kirtipur,Kathmandu, Nepal

Internal Examiner External Examiner Prof. Niranjan Parajuli, PhD Prof. Daman Raj Gautam, PhD Central Department of Chemistry Amrit Science College Tribhuvan University Tribhuvan University Kirtipur,Kathmandu, Nepal Lainchaur, Kathmandu

Head of the Department Prof. Ram Chandra Basnyat, PhD Central Department of Chemistry

Tribhuvan University Kritipur, Kathmandu, Nepal

RECOMMENDATION LETTER

This is to certify that the dissertation work entitled “STUDY ON BIOLOGICAL ACTIVITIES AND ESSENTIAL OIL OF Cinnamomum tamala” has been carried out by Dhan Bahadur G.C. as partial fulfillment for the requirement of Master Degree in Chemistry under my Supervision. To the best of my knowledge, this work has not been submitted to any other degree in this institute.

______Supervisor Prof. Ram Chandra Basnyat, PhD Central Department of Chemistry Tribhuvan, University Kirtipur, Kathmandu, Nepal

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DECLARATION

I, Dhan Bahadur G.C., hereby declare that the work presented herein is genuine work done originally by me and has not been published or submitted elsewhere for the requirement of a degree program. Any literature, data or works done by others, presented in this dissertation are cited, has been given due acknowledgment and listed in the reference section.

______Dhan Bahadur G.C. Central Department of Chemistry Tribhuvan, University Kirtipur, Kathmandu, Nepal

ACKNOWLEDGEMENTS

The successful completion of this study was possible with the support, assistance, motivation and advice of various people. I would like to express my wholehearted gratitude and appreciation to the following:

I would like to express my deepest gratitude and sincere appreciation to my supervisor and Head of the Central Department of Chemistry Prof. Ram Chandra Basnyat, Ph.D., Central Department of Chemistry, for his constant support, inspirable guidance, invaluable suggestions, and regular feedback at all stages of my dissertation work.

My deepest appreciation goes to all my respected teachers of CDC. I would also like to convey my thanks to all the administration staff as well as laboratory staffs of the department for their continuous assistance throughout the dissertation work.

I would especially like to thank National Herbarium and Plant Laboratories (KATH), for the identification of plants. My immense thanks go to Department of Plant Resources, Thapathali, Kathmandu, for providing GC/MS analysis. I am thankful to the authors of books and journals which I have used as a reference in this work.

My special thanks go to all my colleagues whom I have been surrounded and enjoyed, for their co-operation, support and encouragement during the work. I would also like to convey my thanks to my seniors and my juniors for their help and suggestion during this work.

Last but not least, my sincere obligation goes to my dearest family. Thank you so much your love, encouragement, inspiration and cooperation that has made this dissertation possible.

Thank you all. Dhan Bahadur G.C. Email: [email protected]

LIST OF TABLES

Table 1 : Structures of some major compounds found in Cinnamomum tamala. 19

Table 2 : Composition of artificial sea water. 25

Table 3 : Sample collection and identification. 35

Table 4 : Table showing % yield of methanol and hexane extract. 35

Table 5 : Micro-chemical analysis of phytochemicals of leaves. 36

Table 6 : Micro-chemical analysis of phytochemicals of Bark. 36

Table 7 : Total phenolic content of Cinnamomum tamala extracts. 42

Table 8 : Total flavonoid content of Cinnamomum tamala extracts. 44

Table 9 : Calculation of LC50 value of methanol extract of leaves and bark of Cinnamomum tamala. 45

Table 10 : Antibacterial activity of methanol extract of C. tamala leaves and bark. 47

Table 11 : Antifungal activity of methanol extract of C. tamala bark and leaves. 47

Table 12 : Organoleptic properties of essential oil from C. tamala leaves. 48

Table 13 : Chemical constituents of essential oil from Cinnamomum tamala leaves of Bhadgaun, Gulmi. 50

Table 14 : Structures of some major compounds of the Essential Oil from C. tamala leaves. 52

Table 15 : Antioxidant activity of Ascorbic acid. 61

Table 16 : Antioxidant activity of Methanol extract of Bark. 61

Table 17 : Antioxidant activity of Methanol extract (cold 61 percolation) of Leaf. vi

Table 18 : Antioxidant activity of Methanol extract of Leaf. 62 Table 19 : Antioxidant activity of Hexane extract of Bark. 62 Table 20 : Antioxidant activity of Hexane extract of Leaf. 62 Table 21 : Total phenolic content in methanol extract of Bark. 63

Table 22 : Total phenolic content in methanol extract of Leaf 63

Table 23 : Total phenolic the content in hexane extract of Bark. 63 Table 24 : Total phenolic content in hexane extract of Leaf. 64

Table 25 : Total flavonoid content in methanol extract of Bark. 64

Table 26 : Total flavonoid content in hexane extract of Leaf. 64

Table 27 : Total flavonoid content in methanol extract of Leaf. 65

Table 28 : Total flavonoid content in hexane extract of Bark. 65

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LIST OF FIGURES Figure 1 : Industrial uses of medicinal plants. 3 Figure 2 : Cinnamomum tamala leaves. 4 Figure 3 : Cinnamomum tamala bark. 4 Figure 4 : Mechanism of DPPH free radical scavenging by 8 ascorbic acid. Figure 5 : General chemical representation of a polyphenol. 10 Figure 6 : Generic structure of flavonoid. 10 Figure 7 : Flow chart for the extraction, fractionation and analysis of Cinnamomum tamala leaf and bark using Soxhlet apparatus. 22 Figure 8 : Antioxidant activity of standard ascorbic acid. 38 Figure 9 : Comparision of % radical scavenging between ascorbic acid and methanol extract of C. tamala bark. 38 Figure 10 : Comparision of % radical scavenging between ascorbic acid and hexane extract of C. tamala bark. 38 Figure 11 : Comparision of % radical scavenging between ascorbic acid and methanol extract (cold percolation) of C. tamala leaves. 39 Figure 12 : Comparision of % radical scavenging between ascorbic acid and methanol extract of C. tamala leaves. 39 Figure 13 : Comparision of % radical scavenging between ascorbic acid and hexane extract of C. tamala leaves. 40

Figure 14 : IC50 values of different extract of the plant along with ascorbic acid. 40 Figure 15: Variation of absorbance with concentration for standard Gallic acid. 41 Figure 16: Total phenolic content in different Cinnamomum tamala plant extract. 42

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Figure 17: Variation of absorbance with concentration for Quercetin. 43 Figure 18: Total flavonoid content in different C. tamala plant extracts. 44

LIST OF SPECTRA

Spectra 1: GC Chromatogram of the essential oil from C.tamala leaves of Bhadgaun, Gulmi. 49

LIST OF ACRONYMS AND ABBREVIATIONS

C. tamala : Cinnamomum tamala

DMSO : Dimethyl Sulphoxide

DPPH : 1,1-diphenyl-2-picryl hydrazyl

FCR : Folin-Ciocalteau Reagent

GAE : Gallic Acid Equivalent

GC/MS : Gas Chromatography/Mass Spectrometry

IC50 : Inhibitory Concentration for 50% Inhibition

IR : Infra-Red

LC50 : Lethal Concentration for 50% Mortality

MHA : Mueller Hinton Agar

MHB : Mueller Hinton Broth ppm : Part per million

QE : Quercetin Equivalent

ROS : Reactive Oxygen Species

RT : Retention Time

TAC : Total Antioxidant Capacity

TFC : Total Flavonoid Content

TPC : Total Phenolic Content

UV : Ultraviolet

ZOI : Zone of Inhibition

ABSTRACT

The present work is focused on the study of antioxidant, antimicrobial and toxicity of the leaves and bark extract of the Cinnamomum tamala and quality assessments of essential oil of Cinnamomum tamala leaves collected from Gulmi district. The phytochemical screening showed that the plant extracts possess volatile oil, quinones, glycosides, flavonoids, terpenoids, etc. Antioxidant activity studied by DPPH radical scavenging assay showed the

inhibitory concentration (IC50) value of 90.35 μg/mL and 204.31 μg/mL for methanolic and hexane bark extracts respectively as compared to standard

ascorbic acid having IC50 value 55.40 μg/mL. The total phenolic content calculated showed more value for methanolic bark extract 196.5 mg GAE/gm than the methanolic leaf extract 153.41 mg GAE/gm. Similarly, the highest flavonoid content was found in methanol extract of bark 167.82 mg QE/gm. Methanolic extract of bark and leaf demonstrated significant toxicity to A.

salina with LC50 value of 275.42 μg/mL and 251.18 μg/mL respectively. The antimicrobial activity was carried out by well diffusion method which showed the highest ZOI value 15 mm for Staphylococcus aureus by methanolic extract of bark followed by methanolic extract of the leaf (9 mm) as compared to standard neomycin (19 mm). The GC/MS study of essential oil from Cinnamomum tamala of Gulmi reported 13 components. Cinnamaldehyde was found to be the major constituent with 62.49% area in gas chromatogram.

Keywords: Cinnamomum tamala, medicinal plant, antioxidant, antimicrobial activity

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TABLE OF CONTENTS

Page No. BOARD OF EXAMINER II RECOMMENDATION LETTER III DECLARATION IV ACKNOWLEDGEMENTS V LIST OF TABLES VI LIST OF FIGURES VIII LIST OF SPECTRA X LIST OF ACRONYMS AND ABBREVIATIONS XI ABSTRACT XII TABLE OF CONTENTS XIII CHAPTER – I: INTRODUCTION 1-12 1.1 General Background 1 1.2 Introduction to the plant Cinnamomum tamala (Nees Eberm) 3 1.2.1 Identity and classification of the plant 5 1.2.2 Importance 5 1.3 Antioxidants 6 1.3.1 Mechanism of DPPH with antioxidant 7 1.4 Antimicrobial 8 1.5 Polyphenols and Flavonoids 9 1.6 Brine Shrimp Assay 11 1.7 Objectives of the study 11 1.7.1 General Objective 11 1.7.2 Specific Objectives 12 CHAPTER – II LITERATURE REVIEW 13-19 CHAPTER – III MATERIALS AND METHODS 20-34 3.1 Experimental for plant extract 20 3.1.1 Chemicals 20 3.1.2 Equiments 20

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3.1.3 Collection and identification of plant materials 20 3.1.4 Sample preparation 20 3.1.5 Extraction process 21 3.1.6 Phytochemical analysis 22 3.1.7 Biological activities 22 3.1.7.1 Antioxidant Activity 23 3.1.7.1.1 Preparation of the 0.2 mM DPPH solution 23 3.1.7.1.2 Preparation of ascorbic acid solution (Standard) 23 3.1.7.1.3 Preparation of sample solutions 24 3.1.7.1.4 Measurement of DPPH radical scavenging activity 24 3.1.7.2 Brine Shrimp Bioassay 24 3.1.7.2.1 Required Materials 25 3.1.7.2.2 General Procedure of Brine Shrimp Bioassay 25 3.1.7.2.2.1 Sterilization of the apparatus 25 3.1.7.2.2.2 Preparation of the artificial sea water 25 3.1.7.2.2.3 Hatching of the brine shrimp eggs 25 3.1.7.2.2.4 Preparation of samples 26 3.1.7.2.2.5 Procedure for bioassay 26 3.1.7.2.2.6 Data analysis 26 3.1.7.3 Total Phenolic Content 27 3.1.7.3.1 Preparation of the standard gallic acid solution 27 3.1.7.3.2 Construction of the calibration curve 27 3.1.7.3.3 Preparation of the sample solution 27 3.1.7.3.4 Calculation of the total phenolic content (TPC) 27 3.1.7.4 Total Flavonoid Content (TFC) 28 3.1.7.4.1 Preparation of the standard quercetin stock solution 28

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3.1.7.4.2 Preparation of the sample solutions 29 3.1.7.4.3 Calculation of the total flavonoid content 29 3.1.7.4.4 Statistical analysis 29 3.1.7.5 Antimicrobial activity 30 3.1.7.5.1 Collection of Test Organisms 30 3.1.7.5.2 Preparation of Working Solution 30 3.1.7.5.3 Preparation of Standard Culture Inoculums 30 3.1.7.5.4 Preparation of Media 30 3.1.7.5.5 Nutrient Agar 31 3.1.7.5.6 Mueller Hinton Agar (MHA) 31 3.1.7.5.7 Screening and Evaluation of Antimicrobial Activity 31 3.2 Experimental for Essential Oil 32 3.2.1 Extraction of Essential Oil 32 3.2.2 Analytical condition for GC/MS 32 3.2.3 Determination of Physical Parameters 32 3.2.3.1 Specific Gravity Determination 32 3.2.4 Determination of Chemical Parameters 33 3.2.4.1 Saponification Value Determination 33 3.2.4.2 Acid Value Determination 33 3.2.4.3 Iodine Value Determination 34 CHAPTER - IV RESULTS AND DISCUSSION 35-52 4.1 Plant Extracts 35 4.1.1 Identification of Selected Plant 35 4.1.2 Percentage Yield 35 4.1.3 Qualitative Analysis of Phytochemicals 35 4.1.4 Antioxidant Activity 37 4.1.5 Determination of total phenolic content 41 4.1.6 Determination of total flavonoid content 43 4.1.7 Brine Shrimp Bioassay 45 4.1.8 Antimicrobial Activity 46 xv

4.2 Essential oil 47 4.2.1 Extraction and Quantification of Essential Oil 47 4.2.2 Organoleptic Properties of Essential oil 48 4.2.3 Chemical analysis of constituents of Essential Oil 48 4.2.4 Determination of Physical Parameters 51 4.2.4.1 Specific Gravity 51 4.1.5 Determination of Chemical Parameters 51 4.1.5.1 Saponification Value 51 4.1.5.2 Acid Value 51 4.1.5.3 Iodine Value 51 CHAPTER – V CONCLUSIONS AND RECOMMENDATIONS 53-54 5.1 Conclusions 53 5.2 Suggestion for further work 54 REFERENCES 55-57 APPENDICES 58-65

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CHAPTER I INTRODUCTION

1.1 General Background Nepal, the country of Mt. Everest, the highest peak of the world and the birth place of Lord Buddha, Lumbini is situated in South Asia. It is a land locked country which occupies 0.03 % and 0.3% land area of the World and Asia respectively. It is situated between the latitude of 26°22' North to 30°27' North and longitude of 80°04' East to 88°12' East. It has a diverse topography and climate. It stretches from east to west with an average length of 885 kilometers and widens from north to south with an average breadth of 193 kilometers which makes the total 1,47,181 sq. km.1 Gulmi district, a part of province no. 5, one of the seventy-seven district of Nepal, is situated between 27o55’N to 28o27’N latitude and longitude between 83o 10’E to 83o 35’ E longitude covering an area of 1149 sq km area, which is 0.78 % of the total area of Nepal. The altitude ranges from 465 m at Ridi to 2690 m at Madaneko lekh and Thapleko lekh.2

Geographically, Nepal has three east-to-west elongated ecological belts. The northern mountain belt (3,000-8,848m) is naturally decorated by an unbroken range of the Himalayas, which contains eight peaks higher than 8,000 meters, including the world's highest peak Mt. Everest (8848 meters). Middle hilly belt (600-3,000m) is enriched by gorgeous hills, valleys and lakes. Terai belt (up to 600m above the sea level) is the plain area situated in the southern part of Nepal, which is usually known as the grain house of the country since most of the crops produced in Nepal are farmed in this region.1

Nepal has a great diversity of flora and fauna due to a unique geographical location with a representative of deciduous and coniferous forests of subtropical and temperate regions to the sub-alpine and alpine, pastures and snow-capped Himalayan peaks with their cold streams, glaciers and lakes.3 A total of 118 different ecosystems have been identified in Nepal, including 112 forest ecosystems, four cultivation ecosystems, one water body ecosystem, and one glacier/snow/rock ecosystem. Nepal is ranked 25th and 11th positions in biodiversity richness in the world and Asia, respectively. Nepal occupies about 1

0.1% of the global area but harbors 3.2% and 1.1% of the world’s known flora and fauna, respectively. About 5.2% of the world’s known mammals, 9.5% birds, 5.1% gymnosperms and 8.2% bryophytes are reported in Nepal. A total of 284 species of flowering plants, 160 animal species and 14 species of herpetofauna are reportedly endemic to Nepal. The diverse climatic and topographic conditions have also favored maximum diversity of agricultural crops, their wild relatives and animal species.4

Biodiversity is closely linked to the livelihoods and economic well-being of most Nepalese people. Biodiversity relates to almost every aspect of Nepalese life, including agricultural productivity, food security, building materials, human health and nutrition, indigenous knowledge, gender equality, culture, climate, water resources and aesthetic value for society. The economy of Nepal is very much dependent on the use of natural resources. The country’s biodiversity is also an important source of revenue.4

Human societies have been in close contact with their environments since the beginning of their formation and used the ingredients of the environment to obtain food and medicine. Awareness and application of plants to prepare food and medicine have been realized through trial and error, and gradually human became able to meet his needs from his surrounding.5 Medicinal plants have been used as a perennial source of traditional remedies for thousands of years and various modem drugs which are used in the treatment of various diseases and disorders have been isolated from natural sources. In fact, plants produce different bioactive molecules, which act as different types of medicines and play an important role in the maintenance of human health.6

The medicinal value of these plants is due to the presence of certain chemical substances scientifically known as phytochemicals that generate definite physiological action on the human body. Phytochemicals are natural bioactive compounds occurring in plants that work with nutrients and fibers to act or protect against diseases. These natural products are the secondary metabolites that are taxonomically extremely diverse in nature forming the source of new drugs based on their modes of pharmacological action.7

The demand in the world market for plant-derived chemicals- pharmaceuticals, fragrances, flavors and color ingredients, alone exceeds several billion dollars per year, figure-1 gives an out-look into the industrial uses of medicinal plants.5

Phyto-pharmaceuticals

Auxiliary Product Drug discovery metabolism

Industrial Medicinal Plants Herbal teas pharmaceuticals

Health Products Traditional

New drugs Medicines

Figure 1: Industrial uses of medicinal plants Essential oil is a concentrated hydrophobic liquid containing volatile (easily evaporated at normal temperatures) chemical compounds from plants. Essential oils are also known as volatile oils, ethereal oils, aetherolea, or simply as the oil of the plant from which they were extracted, such as oil of clove. Essential oil is "essential" in the sense that it contains the "essence of" the plant's fragrance the characteristic fragrance of the plant from which it is derived.8

An essential oil typically obtained from distillation. They are natural aromatic compounds found in seeds, bark, root, flowers and other parts of the plant. Therapeutically, essential exert a wide spectrum of activities like an antiseptic, stimulant, carminative, diuretic, antihelminthic, analgesic, anti-rheumatic, aromatic, counter-irritant and many other activities. Apart from food and pharmaceutical uses, they are also used as insect repellents, insecticides, pesticides and deodorants.9

1.2 Introduction to the plant Cinnamomum tamala Cinnamomum tamala also is known as a Malabar leaf, Indian bay leaf is a moderate sized evergreen tree attaining a height of 8 m and a girth of 50 cm. The genus Cinnamomum has about 250 tropical trees and shrub species.10,11 It 3

is native to South-east Asia, some Pacific Islands and Australia, growing mainly in tropical rain forests at varying altitudes. Natural habitat is in the tropical and subtropical Himalayas at altitudes of 900-2500 m. Natural stands of C. tamala are mostly found in shady moist habitats.12 In Nepal, it is distributed from west to east at an elevation of 450-2000m commercially harvested from 33 districts.13

The etymology is derived from the Greek word ‘kinnamomon’ (meaning spice). The Greek borrowed the word from the Phoenicians, indicating that they traded with the East from early times. The specific epithet ‘tamala’ is after a local name of the plant in India.10

Figure 2: a) Cinnamomum tamala leaves b) Cinnamomum tamala bark

Historically, it is one of the oldest known and used spices having a clove-like the taste and a faint pepper-like odor.12,14,15 Bark is dark brown or blackish, slightly rough, blaze 13 cm. pinkish or reddish –brown with whitish streaks towards the exterior. Leaves lanceolate, glabrous; alternately placed, opposite and short-stalked, 3-nerved from the base. Leaves are bright pink when young in spring, aromatic when crushed. Flowers are pale yellowish in axillary, arising in the terminal and axillary branched clusters about as long as the leaves.10

Leaves are aromatic and traded as a spice and also as a source of various Ayurvedic formulations.16 It flowers from March to May and fruits are ellipsoidal drupe and require approximately one-year attaining maturity.

Hence, the flowers and fruits can be seen at the same time during April - May. Ripe fruits are dark purple in color and contain single seed.12

This species mostly found in the north-west facing slopes of chure and mid hills regions. This species preferred moist and shady places with sandy loam soil rich in organic matters. Well-drained, deep sandy loam, black soil with pH 4-5.5 and rich humus is suitable for the crop. Humid tropical evergreen rain forest conditions favor the best growth of Cinnamon.13

1.2.1 Identity and classification of the plant Kingdom - Plantae Division - Tracheophyta Class - Magnoliopsida Order - Laurales Family - Lauraceae Genus - Cinnamomum

Vernacular name(s) - Dalchini, Tejpat, Sinkouli (Nepali),Tamalpatra (Sanskrit), Tejpatta (Bhojpuri), Tejpat (Danuwar), Lep (Gurung), Sangsornyo (Lepcha), Sorong tetala (Limbu), Tejpat (Newari), Belakhan (Rai), Sijakaulisapha (Sunwar), Dalchini, Lepte (Tamang).13,17

1.2.2 Importance Cinnamon is being used as an anti-inflammatory, antitermitic, nematicidal, mosquito larvicidal, insecticidal, antimycotic and anticancer agent from a long time. It is traditionally used as tooth powder and to treat toothaches, dental problems, oral microbial and bad breath. The essential oil of Cinnamomum tamala has great antibacterial, antioxidant, antidiabetic, antimicrobial and many more properties.18

The Ayurvedic Pharmacopoeia of India indicates the use of dried mature leaves of Cinnamomum tamala in sinusitis, diarrhea, anorexia, coryza and dryness of the mouth. It is popular as a flavoring agent and is inevitable in the preparation of traditional cuisines, especially in the North-eastern region. Although C. tamala leaves are mainly used as spices, the plant has many medicinal properties. It is reported to be hypoglycaemic, stimulant,

carminative and also used in cough, diarrhoea, gonorrhea, rheumatism, irritations, boils, conjunctivitis and itching. Studies have also been conducted for its hypolipidemic effects, anti-diabetic and antioxidant, anti-ulcer, anti- inflammatory, anti-diarrhoeal, immunosuppressive activities.11,18

The essential oil from Cinnamomum tamala exhibits antidermatophytic, antibacterial, antifungal, antihyperglycaemic and antihypercholesterolanemic effects. It is also used in soaps, detergents, cosmetics and perfumes, toothpaste as a fragrance.19

1.3 Antioxidants Antioxidants were broadly defined as “any substance that, when present at low concentrations compared to that of an oxidizable substrate, significantly delays or inhibits oxidation of that substrate” but by most recent definition antioxidants are natural or synthetic substances that may prevent or delay oxidative cell damage caused by physiological oxidants having distinctly positive reduction potentials, covering reactive oxygen species, reactive nitrogen species and free radicals.20,21

In terms of classification of AOA/TAC assays, the antioxidant assays are classified into in-vitro and in-vivo according to their applications but general classification of antioxidant activity/capacity assays are: (1) Hydrogen atom transfer based assays (HAT-based assays): (2) Single-electron transfer based assays (SET-based assays): (3) Mixed mode (SET/HAT) assays: (4) In vitro antioxidant assays and (5) Miscellaneous methods.20

Antioxidants in human beings at the cellular level are related to oxidative stress, characterized by the inability of endogenous antioxidant to counteract the oxidative damage on tissue and organism owing to over production of cellular ROS/RNS and cause oxidative modification of biological macromolecules (lipid, protein, DNA), tissue injury and accelerated cellular death. Free radicals have been blamed at least partially for the development of various diseases such as atherosclerosis, diabetes, mellitus, chronic inflammation, neurodegenerative disorders, Alzheimer's disease, Parkinson's disease cancer and many more.20,21

At present various synthetic antioxidants such as ascorbyl palmitate, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate (PG), tertiary butylated hydroxyquinone (TBHQ) and many more are available. However recent concern over their adverse effects and toxicity has created a need and prompt research for safer and bioactive natural antioxidant present in plant species.22

1.3.1 Mechanism of DPPH with antioxidant The proposed mechanism involves the transfer of a hydrogen atom from an antioxidant to the DPPH molecule to form DPPH-H molecule which is stable with the loss of violet color and does not adsorb at 517 nm. DPPH solutions show a strong absorbance band at 517 nm due to its odd electron appearing a deep violet color; the adsorption vanishes as electron pairs off.23

Antioxidants

RH Rº

DPPH free radical (Violet) DPPH (Yellow)

Ascorbic acid

Ascorbic acid was used as a standard in the DPPH radical scavenging method.

It is commonly known as vitamin C (molecular formula C6H8O6 and molecular weight 176.12). It is a natural antioxidant.

+ DPPH free + DPPH radical

7 Ascorbic acid Free radical of ascorbic acid

+ H˙

Hydrogen Keto form of Free radical of free radical ascorbic acid ascorbic acid

Figure 1: Mechanism of DPPH free radical scavenging by ascorbic acid.

Two molecules of DPPH are reduced by one molecule of ascorbic acid. Similarly, gallic acid and quercetin can donate a pair of hydrogen atoms to DPPH molecule and thus get oxidized acting as an antioxidant.

1.4 Antimicrobial Antimicrobial use is known to have been common practice for at least 2000 years. Ancient Egyptians and ancient Greeks used specific molds and plant extracts to treat the infection. In the 19th century, microbiologists such as Louis Pasteur and Jules Francois Joubert observed antagonism between some bacteria and discussed the merits of controlling these interactions in medicine. In 1928, Alexander Fleming became the first to discover a natural antimicrobial fungus known as Penicillium rubens and named the extracted substance penicillin which in 1942 was successfully used to treat a Streptococcus infection.24

An antimicrobial is an agent that kills microorganisms or stops their growth. Antimicrobial medicines can be grouped according to the microorganisms they act primarily against. For example, antibiotics are used against bacteria and antifungals are used against fungi. They can also be classified according to their function. Agents that kill microbes are called microbicidal, while those that merely inhibit their growth are called biostatic. The use of antimicrobial medicines to treat infection is known as antimicrobial chemotherapy, while the use of antimicrobial medicines to prevent infection is known as antimicrobial prophylaxis.7,25

The main classes of antimicrobial agents are disinfectants, which kill a wide range of microbes on non-living surfaces to prevent the spread of illness, antiseptics, and antibiotics. The term "antibiotic" originally described only those formulations derived from living microorganisms but is now also applied to synthetic antimicrobials, such as the sulphonamides, or fluoroquinolones.25

The increase in the occurrence of multiple drug resistance has considerably slowed down the research and development of new synthetic antimicrobial drugs and has demanded the search for innovative antimicrobials from a natural plant source. Such factors necessitated new research focusing on screening of natural products found in medicinally important plants to develop new and efficient drugs against microbial diseases and infections. The antimicrobials should be selectively toxic to the pathogenic microbes but not toxic to the host tissues.7

1.5 Polyphenols and Flavonoids Phenolic compounds are plant substances which possess in common an aromatic ring bearing one or more hydroxyl groups. There are about 8000 naturally occurring plant phenolics and about half of this number are flavonoids. Phenolics possess a wide spectrum of biochemical activities such as antioxidant, antimutagenic, anticarcinogenic as well as the ability to modify the gene expression. Phenolics are the largest group of phytochemicals that account for most of the antioxidant activity in plants or plant products.7,26

Polyphenolic compounds have strong antioxidant activity whereas monophenols are a weak antioxidant. Polyphenolic nature enables them to scavenge injurious free radicals such as superoxide and hydroxyl radicals. There is a positive correlation between phenolic content and free-radical scavenging activity. Phenolic compounds contribute to the quality and nutritional value in terms of modifying color, taste, aroma and favour and also in providing healthbeneficial effects.26

Figure 5: General chemical representation of a polyphenol

Flavonoids are low molecular weight polyphenolic substances based on the flavan nucleus.27 The flavonoids are a class of widely distributed phytochemicals with antioxidant and biological activity. They have structures consisting of two aromatic rings linked by three carbons in an oxygenated heterocycle. The difference in the structures of the heterocycle, or C-ring, classify them as flavonols, flavones, flavanols (catechins), flavanones, anthocyanidins or isoflavonoids.

Figure 6: Generic structure of flavonoid

Flavonols are characterized by a 2,3- double bond, a 4-keto group and a 3- hydroxy group in the C-ring. Flavones lack the 3-hydroxy moiety and flavanones have a saturated C-ring. The 2,3-double bond and 4-keto group are absent from flavanols or catechins. The B-ring of isoflavones is linked to C-3 ring, instead of C-2, as it is for the other flavonoid subclass. Flavonoids, as constituents of plant foods, have been implicated in the reduction of cancer risk. In the Zuphen Elderly Study, flavonoid intake from fruits and vegetables was inversely associated with all-cause cancer risk and cancer of the

alimentary and respiratory tract. Lung cancer risk has been inversely associated with flavonoid and quercetin intake.28

1.6 Brine Shrimp Assay Brine Shrimp bioassay was first proposed by Michael et al. in 1956. Subsequently, it was further developed by others. This lethality assay has been successively employed as a bioassay guide for active cytotoxic and antitumor agents in 1982. Cytotoxicity studies are a useful initial step in determining the potential toxicity of a test substance, including plant extracts or biologically active compounds isolated from plants. Minimal to no toxicity is essential for the successful development of pharmaceutical or cosmetic preparation and in this regard, cellular toxicity studies play a crucial role. For the bioactive compound of either natural or synthetic origin, this lethality test is a rapid and comprehensive test.11 It easily utilizes a large number of organisms for statistical validation and requires no special equipment and a relatively small amount of sample (2-20 mg or less) is necessary. The larvae (nauplii, singular nauplius), about 22 mm long, are large enough to observe without high magnification and small enough for hatching in enormous amount without extensive workspace in a laboratory. Cytotoxicity screening tests provide important information to help to select plant extract with potential antineoplastic properties for future work.29

1.7 Objectives of the study Plants have been used from ancient age for the medicinal benefits. Plants synthesize a large variety of compounds of biological significance. More studies on the phytochemical and biological activity of plant extract may provide the scientific support for their use but still, no much work has been done in this area. This plant has been chosen for this study with the following general and specific objectives.

1.7.1 General Objective The general objective of the study is to determine the phytochemical and biological activities of leaves and bark of Cinnamomum tamala and quality assessments of the essential oil from Cinnamomum tamala leaves with GC/MS.

1.7.2 Specific Objectives The specific objectives are as follows:  To perform phytochemical screening of leaves and bark extracts of Cinnamomum tamala.  To evaluate the antioxidant and toxicity of leaf and bark extracts of Cinnamomum tamala.  To determine the total phenolic and total flavonoid content of leaf and bark extracts of Cinnamomum tamala.  GC/MS analysis of the essential oil of Cinnamomum tamala leaves.

CHAPTER II

LITERATURE REVIEW

The literature survey on Cinnamomum tamala is done by collecting different papers published in different journals via Scholar Google and some are depicted below.

A literature review showed that the plant leaves are widely used in pharmaceutical preparation because of therapeutic efficacy against various diseases and disorders due to the presence of different phytochemicals. The leaves showed the presence of ash, moisture, crude fat, crude fiber, crude carbohydrate, crude protein and different phytochemical content.

An essential oil obtained by hydrodistillation of fresh leaves of Cinnamomum tamala cultivated in North Cacher Hills of North East India was examined by gas chromatography. Fourteen oil components were identified which constituted 93.44% of the oil. Linalool was the main component (60.73%), whereas eugenol and cinnamic aldehyde were detected in trace amounts (<1%). Other components of significant occurrence (3%) in the oil were α- pinene (10.54%), β-pinene (10.42%), Limonene (3.21%) and camphene (3.06%).30

Dev S.L., Kannappan S. & Anuradha C.V., (2007) evaluated the in-vitro antioxidant activity of Indian bay leaf, Cinnamomum tamala T.Nees & Eberm using rat brain synaptosomes as a model system. A methanolic extract of bay leaf was tested for the polyphenolic content and significant increase in the level of lipid and lipid peroxidation product and a decline in antioxidant potential were observed in diabetic rat brain synaptosomes.31

GC/MS analysis of essential oil and oleoresin of C. tamala leaf revealed eugenol as a major component of essential oil and oleoresin and shows antimicrobials potentials against various food born fungi and bacteria. Both have effective antioxidant and antimicrobial activities, however, the essential oil is better than oleoresins. The major constitute of the leaf essential oil contains furanosesquiterpenoids, furanogermenone (59.5%), β-caryophyllene (6.6%), sabinene (4.8%) and curcumenol (2.3%).12 13

Among the leaf of five species of Cinnamomum, namely C. burmanni, C. cassia, C. pauciflorum, C. tamala, and C. zeylanica, C. zeylanica showed the highest DPPH radical scavenging activity, total antioxidant activity and reducing power, while C. tamala exhibited the highest superoxide anion scavenging activity. The study exhibited C. zeylanica had the highest total phenolic content (2708.7 μg GAE/g) while C. tamala had the lowest value (20.62 μg GAE/g), using the standard curve of gallic acid.32

Kumar S. and et al. (2012) studied the antidiabetic, antioxidant and hypolipidemic potential of Cinnamomum tamala, (Buch.-Ham.) Nees & Eberm (Tejpat) oil in streptozotocin (STZ) induced diabetes in rats along with an evaluation of chemical constituents. The GC-MS analysis of essential oil led to the identification and quantification of 31 components which accounted for 99.99% of the total oil. The main volatile components were found as cinnamaldehyde (44.898%), Trans cinnamyl acetate (25.327%), Ascabin (15.249%), Hydro cinnamyl acetate (3.384%), Beta-caryophyllene (2.669%) which comprised of 91.527% of the oil.33

The essential oil obtained by hydrodistillation of C. tamala leaf (2012), characterized by a large amount of oxygenated monoterpenes (92.10%) with cinnamaldehyde (37.85%) and cis-linalool oxide (29.99%) being the major constituents found. The susceptibility of essential oil of C. tamala was tested on five bacterial strains (Staphylococcus aureus, S. aureus, E. faecalis, Escherichia coli, and Pseudomonas aeruginosa) and four fungal strains (Candida albicans, Candida parapsilosis, Aspergillus fumigates and Aspergillus niger. The strongest activity was shown against all the four fungal pathogens, whereas oil was found comparatively less effective against bacterial pathogens.34

Lohani H. and Andola C.H. evaluated the variation of Cinnamomum tamala leaf essential oil in respect with months and tree size class. Study of the chemical constituents revealed that a total of fourteen components identified in 30 cm tree class and seventeen components identified in 60 cm tree class in the month of October. Cinnamaldehyde was principle component and its content was greater in the smaller class tree as compared to higher size class.35

Rahman M. and et al. (2013), evaluated preliminary phytochemical and some pharmacological properties including cytotoxicity, antibacterial and antifungal sensitivity, total phenolic content of the leaves of C. tamala. The extract contained alkaloids, steroids, tannins and reducing sugars. Total phenolic content was found to be 276 gallic acids equivalent/100 g of dried plant material. In brine shrimp lethality assay, it showed moderate toxicity (LC50 =

40 μg/ml and LC90 = 160 μg/ml). The extract inhibited the growth of tests bacteria and fungi with the highest activity observed against the bacteria Salmonella typhi and the fungus Aspergillus nige.29

C. tamala leaf extract showed the presence of many phytochemical moieties such as phenolics, flavonoids, tannins, terpenoids, alkaloids, and saponins. The fungicidal effect was most pronounced against C. albicans and Penicillium spp. The antioxidative activities of extracts were compared with the activities of standard antioxidant compounds BHA and ascorbic acid. Petroleum ether, ethanol, acetone, and chloroform extracts exhibited about 30- 67% antioxidant activity in β-carotene bleaching assay. Aqueous and ethanol extracts exhibited better reducing power which increased gradually with an increasing amount of the extract concentration showing dose-dependent response.36

Choudhary D. and et al. carried out an analysis for quality oriented value chain development of essential oil from bay leaves in India and Nepal based on their altitude and age. A total of fifteen compounds were isolated from oil samples of Nepal. Linalool and 1,8 cineole were major constituents in which linalool content decreased from low altitude to high altitude which varied with altitude but was not significant to the leaves age. 1,8 cineole was found more in high altitude. Essential oil content was higher for new leaves from lower altitudes.37

The antioxidant potential, DPPH radical scavenging assay was carried out by Sudan R. and et al. (2013) and it was observed that methanol extracts showed

high radical scavenging activity with IC50 175 ± 0.32 mg/ml. BHT is a

standard antioxidant which shows an IC50 value of 50 ± 0.62 mM. The quantification of phenolic content in different extracts exhibited that

methanolic extracts contain high phenolic content i.e. 161 ± 0.58 mg/g GAE in comparison to other extracts.21

Dandapat et al. (2013) reported phytochemicals such as tannin, saponins, alkaloids, phenols, and flavonoids possess direct or indirect correlation with therapeutic efficacy against various diseases. Among the phytochemicals, polyphenols were highest (16.7 ± 0.7 g/100g) and flavonoids were lowest (1.0 ± 1.01 g/100g). Antipathogenic efficacy of C. tamala has been tested against, S. typhi, P. aeruginosa, S. aureus, P. mirabilis and B. subtilis by agar diffusion method and broth dilution method and all the strains were affected by methanolic and aqueous leaf extracts of C.tamala. The leaf extracts did not show cytotoxic at 0.2mg.mL - 1mg/mL concentration of aqueous leaf extract but showed haemolysis at 1mg/mL concentration of methanolic leaf extract of C. tamala.6

The in vitro antimicrobial activity assay was carried out by agar well diffusion method and in-vitro antioxidant activity was quantified by DPPH radical scavenging assay. Brine shrimp lethality assay was used to evaluate the cytotoxicity of the ethanol extract of the leaves. Ethanolic extract of C. tamala leaf exhibited brime lethality in a dose dependent manner and LD50= 4.86 ± 0.08 μg/ml. The extract also exhibited the scavenging of DPPH free radicals and recorded an IC50 of 26.02 ± 0.61 μg/ml and significant antimicrobial activity against S. epidermis followed by S. aureus.11

Chaudhary P. and Singh P. (2014) evaluated the antibacterial potential of Cinnamomum tamala extracts against two foodborne and spoilage bacteria (isolated from spice mixes), E.coli and Bacillus sp. by Kirby-Bauer disc diffusion method. The extracts showed good antibacterial activity against both and the best diameter of the inhibition zone was obtained against Bacillus sp. (12.6 mm) with the methanolic extract followed by the ethanolic extract. The aqueous extract was found effective only against E.coli with zone size of 8.00mm.38

In vitro antioxidant and cytotoxic activity of ethanolic extract of Cinnamomum tamala leaf carried out by Akter S. et.al (2015) showed the high potential of it. Antioxidant activity of the extract was evaluated by using DPPH free radical 16

scavenging assay and ascorbic acid used as a standard, the IC50 value of leaves

was 13.55 μg/ml while the IC50 value of ascorbic acid was 5.35 μg/ml. Cytotoxic activity evaluated by using brine shrimp lethality bioassay and

vincristine sulfate as a standard, the LC50 value of the ethanolic extract of

Cinnamomum tamala leaves was 17.82 μg/ml whereas LC50 value of vincristine sulfate was 5.24 μg/ml.39

The essential oil extracted from C. tamala varies in phytochemical constituents and concentration of its components with changing geographical and climatic conditions. Sankaran V., et al., (2015) evaluated the chemical analysis of leaf essential oil of Cinnamomum tamala from Arunachal Pradesh, India. The GC-MS analysis of leaf essential oil revealed eugenol (60.2%), α- Phellandrene (11.7%) and β-Phellandrene (7.2%), α-Pinene (2.8%), Elixene (1.8%), cis-Caryophyllene (1.6%), Myrcene (1.5%) and Limonene (1.4%) as its major constituents. The identified compositions owing in a total of 37 components that amounts to 98.6 % of the tested sample. On the contrary Cinnamaldehyde (44.9%) and Trans-cinnamyl acetate (25.33%) composition was found to be higher in essential oil from Southern part of India which had Eugenol composition of only 0.078%.40

Qualitative phytochemical screening of the methanolic extract of Cinnamomum tamala leaves by Hassan W., (2016) is rich in phytonutrients like flavonoids, alkaloids, terpenoids, and tannins. Saponins and Steroids were absent in the tested extracts. Antimicrobial potential of the crude extract and its fractions i.e. aqueous, n-hexane, dichloromethane, and isobutanol showed a variable degree of inhibition zones against all tested microbes except dichloromethane, aqueous fraction and crude extract which were completely inactive against Salmonella typhi (a gram-negative strain). All the extracts showed their best inhibitory activity against B. atropheous amongst which the aqueous extract recorded the highest zone of inhibition measuring 38 mm. Crude, aqueous and DCM extracts were found completely inactive against S. Typhi. The crude extract showed its best activity against B. atropheous (31 mm) while found mildly active against E. coli (10 mm). In the case of fungal activity, the DCM extract showed the highest zone of inhibition (18 mm)

against C. albican while the least value (12 mm) was observed for both aqueous and crude extracts.7

A reviewed on aroma profile of Cinnamomum species revealed that it mainly contains essential oils and important compounds like cinnamaldehyde, eugenol, cinnamic acid, and cinnamate. The oil shows antioxidant, anti- inflammatory, antidiabetic, antimicrobial, anticancer activity and reported to have been used for lipid-lowering and cardiovascular-disease.18

Table 1: Structures of some major compounds found in Cinnamomum tamala S.N. Compounds Structures

1 Cinnamaldehyde

2 Eucatyptol

3 Linalool

4 Geraniol

5 Eugenol

CHAPTER III

MATERIALS AND METHODS

3.1 Experimental for plant extract 3.1.1 Chemicals Methanol (Fisher Scientific), hexane (Merck), acetone (Fischer Scientific), dimethyl sulphoxide used was of analytical grade. Distilled water was purchased from the local market. Chemicals and reagents like gallic acid

ascorbic acid, 1, 1-diphenyl-2-picrylhydrazyl (DPPH), NaNO2, AlCl3, and KOH were available in the laboratory. Reagents and solvents used during the phytochemical analysis such as Meyer’s reagent, Dragendorff’s reagent, Molisch’s reagent, etc were prepared in the laboratory with the chemicals provided in the laboratory of laboratory reagent grade.

3.1.2 Equiments Electric grinder, digital weighing balance, hot air oven, cuvettes, burettes, pipettes, micropipettes, thermometer, condensers, beakers, conical flasks, test tubes, reagents bottles, petri-disc, stands, vial tubes, round bottom flasks, Soxhlet extractor were used during this work. Rotatory evaporator with water bath was used for the evaporation of solvents. Absorbance for DPPH assay and absorbance for total phenolic content was measured by using a spectrophotometer.GC/MS analysis was performed on a gas chromatography mass spectrometer GCMS-QP2010 available in the Department of Plant Resources, Thapathali, Kathmandu.

3.1.3 Collection and identification of plant materials The plant materials, leaves and bark of Cinnamomum tamala were collected from Resunga Municipality-03, Bhadgaun, Gulmi. The plant was identified by Ganga Datta Bhatt, National Herbarium and Plant Resources (KATH), Lalitpur.

3.1.4 Sample preparation The leaves and bark of the plant were collected locally and processed. The collected plant’s parts were washed in tap water to remove the contaminations. Then the leaves and bark were shade dried for a few days. The shade dried

plant parts were grinded into powder form in an electric grinder and stored in a clean plastic bag at a low temperature until the further use.

3.1.5 Extraction process The extraction process was based on the principle- ‘Like dissolves Like’. Both extraction processes were performed; in case of methodology, soxhlet extraction was performed along with a cold percolation of C. tamala leaves extract with methanol.

Soxhlet extraction process: Powdered plant parts (50gm) was wrapped in a filter paper and kept in clean and dry soxhlet. The soxhlet was fitted to the condenser from upside and to the downside round bottom flask with solvent (250ml). The instrumentation was set up and the heat was given to the RB flask with the help of heating mantle maintaining the temperature at 50ºC. The condenser was fitted to the regular water supply through the tap. After sometime solvent evaporates and the sample absorbs the solvent and then the solvent runs down to the RB flask. This process was continued until the solvent running down to the RB flask is almost colorless. The extract obtained was filtered, concentrated and dried in rotatory evaporator maintained at 45ºC. The dried extract was stored in air tight containers at 4ºC for further study.

Cold percolation method: For cold percolation extraction, powdered Cinnamomum tamala leaf (150 gm) was kept in a clean and dry conical flask. Methanol (400 ml) was added to the flask and kept for 48 hours with frequent shaking and filtered. The residue obtained was again soaked with methanol. The process was repeated until the methanol in the sample becomes colorless. Thus obtained filtrate was concentrated with the help of rotator evaporator under reduced pressure by maintaining temperature lower than the boiling point of methanol. Then the concentrated filtrate was kept in a beaker wrapping with aluminium foil containing small pores to facilitate the evaporation of the solvent. After complete evaporation of the solvent, solid methanolic extract was obtained. This leaf extract was stored at 4ºC until doing biological activities.

Dry plant materials Hexane

Hexane extract Residue Methanol Phytochemical screening Total phenolic content Methanol extract Residue Total flavonoid content Antioxidant assay Phytochemical screening Antimicrobial activity Antioxidant activity Brine shrimp bioassay Total phenolic content Total flavonoid content Figure.7: Flow chart for the extraction, fractionation and analysis of Cinnamomum tamala leaf and bark using Soxhlet apparatus

The percentage yields of the extracts were calculated using the following formula,

Wt. of the extract (gm) % Yield of Extract = × 100% …..(1) Wt. of the powdered plant parts (gm)

3.1.6 Phytochemical analysis The method used for phytochemical screening was based on protocol put forward by Ciulei I.41 Basically phytochemical screening helps to identify the bioactive compounds present in plants. The analysis of the presence of main groups of natural constituents present in the different plant extracts was done by the color reaction using different specific reagents. The procedure is given in detail in Appendix A.

3.1.7 Biological activities The different chemical constituents present in the plants are responsible for their biological activity. The biological activity involves the study of the effect of the crude plant extracts/fractions and isolated fraction at arbitrarily fixed

dose levels in a species of organism and prediction of its effect over the entire dosage range.

The present study based on the analysis of methanol and hexane extracts of leaf and bark of Cinnamomum tamala for their antioxidant, antifungal, antibacterial, and brine- shrimp toxicity activity.

3.1.7.1 Antioxidant Activity The method used for antioxidant activity was based on protocol put forward by Blois M.S. (1958).42 The various methods used to measure antioxidant activity of plants can give varying results depending on the specific free radical being used as a reactant. A rapid, simple and inexpensive method to measure antioxidant capacity involves the use of the free radical, 1,1-diphenyl- 2-picrylhydrazyl (DPPH). The percentage of the DPPH free radical scavenging activity was calculated by using the following equation:

Radical scavenging (%) = [(Ao-As)/Ao] × 100 …………… (2)

Where A0 = Absorbance of the control (DPPH solution + methanol)

As = Absorbance of the test sample

The IC50 (50% inhibitory concentration) value is indicated as the effective concentration of the sample that is required to scavenge 50% of the DPPH free

radicals. IC50 values were calculated using the inhibition curve by plotting extract concentration versus the corresponding scavenging effect.

3.1.7.1.1 Preparation of the 0.2 mM DPPH solution DPPH has a molecular weight of 394.32 gm/mol. Thus 100 mL of 0.2 mM solution of DPPH was prepared by weighing the 0.007886 g of the DDPH carefully and dissolving it on methanol and finally maintain the volume to 100 mL and was kept in dark place until the used.

3.1.7.1.2 Preparation of ascorbic acid solution (Standard) 10 mg ascorbic acid was weighed out and dissolved in 10 mL methanol to make the stock solution of 1000 µg/mL (ppm). Then by serial dilution, ascorbic acid solutions having concentration 20 ppm, 40 ppm, 60 ppm, 80 ppm and 100 ppm were prepared.

3.1.7.1.3 Preparation of sample solutions Firstly 15 mg extracts (methanol and hexane) were weighed out and dissolved in 15 mL methanol to make the stock solution of 1000 µg/mL (ppm). Then by serial dilution, extract solutions having concentration 20 ppm, 40 ppm, 60 ppm, 80 ppm, and 100 ppm were prepared.

3.1.7.1.4 Measurement of DPPH radical scavenging activity 2 mL ascorbic acid solution from each concentration was pipetted out and mixed with 2 mL 0.2 mM DPPH solution in triplicate and kept in dark for 30 minutes. 2 mL methanol was mixed with 2 mL 0.2 mM DPPH solution and kept in dark also. Then their absorbance value was measured at 517 nm by using spectrophotometer against methanol and DPPH as a blank.

Similarly, absorbance value for extract and DPPH solution was measured following the same procedure as ascorbic acid. Finally, the calibration curve was drawn taking sample concentration as X-axis and % radical scavenging

activity as Y-axis for both ascorbic acid and sample solution and IC50 values were calculated.

3.1.7.2 Brine Shrimp Bioassay The procedure followed for the brine-shrimp bioassay was carried out according to the procedure by Mayer et al. as being simple, rapid and inexpensive.43

Brine shrimp, Artemia salina is a tiny crustacean. The eggs of brine shrimp are readily available at low cost and they remain viable for years in the dry state. Upon being placed in a brine solution, the eggs hatch within 48 hours providing a large number of larvae (nauplii). These larvae are used in this assay for biological screening. This method is the rapid, inexpensive, simple and in-house approach for screening and monitoring physiologically active plant extracts.

It determines the LC50 values (µg/mL) for the crude extracts. Compounds having LC50 values less than 1000 ppm (µg/mL) are considered as potentially pharmacologically active.

3.1.7.2.1 Required Materials i. Eggs of brine shrimp ii. Artificial sea water iii. Beakers for hatching iv. Table Lamp v. Disposable pipette vi. Micropipette vii. Test tubes and test tube stand

3.1.7.2.2 General Procedure of Brine Shrimp Bioassay 3.1.7.2.2.1 Sterilization of the apparatus All the apparatus used in the experiment were sterilized before their use.

3.1.7.2.2.2 Preparation of the artificial sea water Sea water was prepared by dissolving following chemicals in 1 Liter of distilled water. Table 2: Composition of artificial sea water S.N. Composition Amount (g/L) 1 NaCl 23.5

2 Na2SO4 4 3 KCl 0.68

4 H3BO3 0.027

5 MgCl2.2H2O 10.68

6 CaCl2.2H2O 1.78

7 NaHCO3 0.197 8 NaEDTA 0.0003

3.1.7.2.2.3 Hatching of the brine shrimp eggs About 50 mg eggs of brine shrimp were sprinkled on the artificial sea water taken in the beaker and the beaker was covered with aluminum foil. Several small pores were made to facilitate the passage of heat and light. Then the beaker was kept for 48 hours illuminating with the bulb (60 Watt) at room temperature.

3.1.7.2.2.4 Preparation of samples 20 mg extract was weighed out and dissolved in 2 mL methanol to make a stock solution of concentration 10,000 ppm (µg/mL). From that stock solution, solutions of concentration 1000 µg/mL, 100 µg/mL, and 10 µg/mL were prepared by serial dilution method. 2 mL solutions from each solution (1000 ppm, 100 ppm, and 10 ppm) were transferred to nine different test tubes, three for each concentration. Similarly, 2 mL methanol was taken in three test tubes (as a blank). After labeling these test tubes, they were kept for 24 hours to evaporate the solvent (methanol).

3.1.7.2.2.5 Procedure for bioassay After the complete evaporation of the solvent, 5 mL artificial sea water was added and the solution was mixed thoroughly to suspend the residue. Then ten mature brine shrimp nauplii were transferred into all twelve test tubes. After 24 hours, the numbers of the survivors were counted with the help of the disposable pipettes.

3.1.7.2.2.6 Data analysis

LC50 value is the lethal concentration dose required to kill 50 % of the organisms used in bioassay. It can be determined as follows, If ‘n’ is the number of replicates (here three), ‘x’ is the log of the concentration of the solution in µg/mL (log10, log100 and log1000 in this experiment) and ‘y’ is the Probit for average survivors for all replicates, Then we have, [ Σy− βΣx ] α = ……………….(3) n Σx Σy Σxy − β = n ……………….(4) (Σx) Σ x − n Now, From Probit regression, Y= α + βX…………………..…(5) (Y − α) X= ………………….…(6) β

Where Y is a constant having value 5 for calculating LC50 value. Hence,

LC =Antilog (X) …………...…(7)

3.1.7.3 Total Phenolic Content The total phenolic content in plant extract was analyzed by Folin-Ciocalteu colorimetric method based on oxidation-reduction reaction as described by Singleton V.B. and Ross J.A. (1965).44 Gallic acid is used as a standard.

3.1.7.3.1 Preparation of the standard gallic acid solution Firstly, 1000 µg/mL stock solution of gallic acid was prepared by dissolving 10 mg gallic acid in 10 mL methanol. Various concentrations of gallic acid such as 20, 40, 60, 80 and 100 µg/mL were prepared by serial dilution of the stock solution.

3.1.7.3.2 Construction of the calibration curve Gallic acid solution (1 mL) from each concentration was poured into test tubes. Then, 5 mL of 10 % Folin-Ciocalteu reagent (FCR) and 4 mL of 7 %

sodium carbonate solution (Na2CO3) were added to these test tubes to get a total volume of 10 mL. The blue colored mixture was shaken well and incubated for 30 minutes at 40 ºC in a water bath. Finally, the absorbance of the solution was measured at 760 nm wavelength using spectrophotometer against a blank solution containing all reagents except gallic acid. All the experiments were carried out in triplicate. The average absorbance values obtained at different concentrations of gallic acid were used to plot the calibration curve.

3.1.7.3.3 Preparation of the sample solution Stock solution (10000µg/mL) of the extract was prepared by dissolving 50 mg extract in 5 mL methanol. Then triplicate of concentrations of the extract 1000 µg/mL was prepared by serial dilution and their absorbance values were measured following the same procedure as explained above for gallic acid.

3.1.7.3.4 Calculation of the total phenolic content (TPC) The total phenolic content was calculated using equation 8, cV C= ……………(8) m

Where, C = Total content of the phenolic compounds (mg/g) in gallic acid equivalent c = Concentration of gallic acid established from the calibration curve (mg/mL) V = Volume of extract (mL)

m = Weight of the plant extract (mg) 3.1.7.3.5 Statistical analysis Data were recorded as a mean of three determinations of absorbance for each concentration, from which the linear correlation coefficient (R2) value was calculated. The regression equation is given as,

y=mx +c …………(9) Where, y = Absorbancey=mx +c……………(9) of the extract

m = Slope from the calibration curve

x = Concentration of the extract

c = Intercept

Using this regression equation, the concentration of the extract was calculated. Thus with the calculated value of the concentration of the extract, the total phenolic content was calculated from the equation (9).

3.1.7.4 Total Flavonoid Content (TFC) The total flavonoid content of the plant extract was determined by the aluminium chloride colorimetric Assay.45 Quercetin is used as a standard.

3.1.7.4.1 Preparation of the standard quercetin stock solution Quercetin stock solution of concentration 1000 µg/mL (ppm) was prepared by dissolving 20 mg of quercetin in 20 mL of methanol. Then various concentrations of quercetin such as 20, 40, 60, 80 and 100 µg/mL were prepared by serial dilution of the stock solution. An aliquot of 1 mL quercetin of each concentration in methanol was poured into 20 mL test tube containing

4 mL distilled water. Then, at the zero time, 0.3 mL 5% NaNO2 was added to

the test tube. After 5 minutes, 0.3 mL of 10% AlCl3 and after 6 minutes 2 mL of 1 M NaOH were added to the mixture. Immediately the total volume of the mixture was made up to 10 mL by the addition of 2.4 mL distilled water and mixed thoroughly. Finally, the absorbance of the pink color mixture was measured at 510 nm wavelength using spectrophotometer against a blank solution containing all the reagents except quercetin. The average absorbance 28

values obtained for different concentrations of quercetin were used to plot the calibration curve.

3.1.7.4.2 Preparation of the sample solutions Stock solution (10000µg/mL) of the extract was prepared by dissolving 50 mg extract in 5 mL methanol. Then triplicate of concentrations of the extract 1000 µg/mL was prepared by serial dilution and their absorbance values were measured following the same procedure as explained above for quercetin.

3.1.7.4.3 Calculation of the total flavonoid content The following formula was used to calculate the total flavonoid content of the extract, cV C= ……………(10) m

Where, C = Total Flavonoid Content (in mg/g)

in Quercetin Equivalent (QE) c = Concentration of quercetin established from calibration curve in mg/mL V = Volume of the extract (in mL) m = Weight of the plant extract (in g)

3.1.7.4.4 Statistical analysis Data were recorded as a mean of three determinations of absorbance for each concentration, from which the Linear Correlation Coefficient (R2) value was calculated. The regression equation is given as, y=mx +c……………(11) Where, y = Absorbance of the extract m = Slope from the calibration curve x = Concentration of the extract, c = Intercept Using this regression equation, the concentration of the extract was calculated. Thus with the calculated value of the concentration of extract, the flavonoid content was calculated by equation (11).

3.1.7.5 Antimicrobial activity In biological screening, the effect of the crude plant extract or fraction at fixed dose level in species of the organism was studied. In this work, the antibacterial and antifungal assay was performed. Agar well diffusion method was used in the study of screening and the evaluation of the antibacterial and antifungal activity of the crude plant extracts. Inhibition of the bacterial and fungal growth was tested by agar well diffusion method and measured in the form of the zone of inhibition (ZOI). The antimicrobial assay was performed at NIST college and Central Department of Microbiology, Kathmandu.

3.1.7.5.1 Collection of Test Organisms The microbial strains were identified strains that were obtained from NIST College, Khusibu, Kathmandu. The four studied strains included two different types of bacteria and a fungus. Gram-positive bacteria: Staphylococcus aureus Gram-negative bacteria: Escherichia coli, Salmonella typhimurium Fungus: Candida albicans

3.1.7.5.2 Preparation of Working Solution 50 mg/mL of working solution was made by transferring 0.005 g of each crude extract to sterile vial aseptically containing 1 ml of DMSO solvent. The extract was dissolved in DMSO. After making up a stock solution, the test tubes were capped, sealed and stored in the refrigerator (2-8 ºC) until use.

3.1.7.5.3 Preparation of Standard Culture Inoculums The test organisms to be tested were aseptically touched with the help of the inoculating loop from primary culture plate. Then it was transferred into a test tube 10 ml of sterile liquid media of nutrient broth and incubated overnight at 37 ºC in the incubator.

3.1.7.5.4 Preparation of Media The media used in the study were prepared according to the manufacturer’s recommendation. The detailed procedure is given below:

3.1.7.5.5 Nutrient Agar It was prepared by adding the distilled water to 28 g of nutrient agar in the appropriate size of the conical flask and made final volume 1000 ml (28 g/liter). Then it was boiled with continuous shaking and autoclaved at 121 ºC for 15 minutes. The sterilized media was allowed to cool about 50 ºC. They were distributed in the sterilized petri-plates of 90 mm diameter in the ratio of 25 ml per plate aseptically and labeled properly. Plates were then left as such for solidification.

3.1.7.5.6 Mueller Hinton Agar (MHA) The Muller Hilton Agar medium was prepared according to the manufacturer's recommendations. For this, 9.5 grams of media was suspended into 250 ml distilled water, boiled to dissolve and sterilized by autoclaving at 15 lb pressure and 121°C for 15 minutes. It was then allowed to cool about 50°C and poured to petri-plates in 20 ml/plate quantities. The plates were left as such for solidification.

3.1.7.5.7 Screening and Evaluation of Antimicrobial Activity Already prepared Sterile Mueller-Hinton Agar (MHA) plates were dried to remove the excess of moisture from the surface of the media. The sterile cotton swab was dipped into the standard inoculums and the excess of the inoculums was removed by pressing and rotating against the upper inside wall of the tube above the liquid level and then swabbed carefully all over the plates. The plate was rotated through an angle of 60° after each swabbing. Finally, the swab was passed round the edges of the agar surface. The inoculated plates were left to dry for a day in laminar air flow. The wells were made in the incubated media plates with the help of sterile cork borer of the diameter of 6mm and labeled properly. Then, 20 μl of the working solution of the plant extracts was loaded into the respective wells with the help of micropipette. DMSO was used as negative control and Neomycin was used as a positive control in the separate well for the antibacterial activity while itraconazole was used as a positive control for antifungal activity. The plates were then left for half an hour with the lid closed so that extracts diffuse into the media. The plates were incubated overnight at 37°C.

The plates were then observed for the zone of inhibition (ZOI) produced by the antibacterial and antifungal activity of plant extracts and the inhibition zones were measured by the use of a scale.

3.2 Experimental for Essential Oil 3.2.1 Extraction of Essential Oil The essential oil from the leaves of Cinnamomum tamala was extracted by hydrodistillation using Clevenger apparatus. 100 grams of fresh leaves of Cinnamomum tamala were cut into small pieces and kept into round bottom flask along with some distilled water. The content of the flask was heated to boiling. Heating was continued for 3 hours and allowed to stand for some time and the stopper of the Clevenger apparatus was opened. The water was drawn out slowly until the surface of the oil layer corresponded to the preparation line and allowed to stand for some time. Finally, the surface of the layer was lowered to zero lines and the volume of the oil was measured at the same condition. The process was repeated several times. Finally, the oil was

collected in a glass vial over dry Na2SO4 and stored in cool at 4ºC until use.

3.2.2 Analytical condition for GC/MS GC/MS analysis was performed on a gas chromatography mass spectrometer GCMS-QP 2010 under the following condition: 1 μL with split ratio 1:90; Helium as a carrier gas with an RTX-5MS column of dimension 30m × 0.25mm × 0.25 μm, temperature programmed at 50, 150 and 250ºC with a hold time of 0.0, 0.0 and 5.0 min identification was accomplished by comparison of MS with those reported in NIST 17 and FFNSC 1.3 libraries.

3.2.3 Determination of Physical Parameters 3.2.3.1 Specific Gravity Determination The specific gravity of the oil was determined using a simple weight basis relationship with distilled water.46 An ignition tube, previously cleaned and dried, was weighed and its weight was determined to be W. The tube was

filled with the oil and weighed as W1. The same procedure was performed using the same tube containing water and its weight was noted as W2. Then, the specific gravity (D) was calculated using the formula 12:

W1 – W …………. (12) D = W2 - W

3.2.4 Determination of Chemical Parameters 3.2.4.1 Saponification Value Determination Saponification value of the oil was determined by refluxing the sample oil and titrating against acid using phenolphthalein as an indicator.46 Oil (0.5gm) was accurately weighed in a conical flask. The oil was dissolved in 10 ml of absolute alcohol and then 10 ml of 2.5N KOH solution was added. This procedure was performed in duplicate and blank experiment omitting the oil. The flask was refluxed on a sand bath for about two hours. It was cooled and then a few drops of phenolphthalein indicator was added. The unreacted KOH was titrated with standard N/2 oxalic acid until the pink color just disappeared. Then, saponification value was determined using the following equation,

Saponification Value (S.V.) = 56 × (V1 – V2)1000/ (2×1000×W)…. (13)

Where, W = weight of the oil taken

V1 = volume of the N/2 oxalic acid for blank

V2 = volume of the N/2 oxalic acid for sample

3.2.4.2 Acid Value Determination The acid content in the oil was determined by following acid-base titration using phenolphthalein as an indicator.47 Oil (0.5 gm) was accurately weighed into a 250 ml conical flask. To this, 15 ml of neutral 95% alcohol and 2-3 drops of 1% phenolphthalein solutions were added. The free acid was then titrated with a standard 0.1N aqueous potassium hydroxide solution adding the alkali dropwise at a uniform rate of about 30 drops per minute. The content of the flask was continuously agitated. The first appearance of the red coloration for 10 seconds was considered as the endpoint. Then, the acid value (A.V.) was calculated using the following formula: A.V. = 5.61 (number of ml of 0.1N KOH/weight of sample in gram) …… (14)

3.2.4.3 Iodine Value Determination Oil (0.25 gm) was accurately weighed and introduced into a conical flask of 250 ml capacity and it was dissolved in 10 ml chloroform. 25 ml of iodobromide solution was added to it accurately measured from burette and was allowed to stand for 30 minutes protected from sunlight.46

Then 30 ml of 1N potassium iodide and 100 ml of distilled water was added and the liberated iodine was titrated with N/10 solution of sodium thiosulphate shaking thoroughly after each addition of thiosulphate. When iodine color became quite pale, 1 ml of 1% starch solution was added and the titration was continued until the blue color was discharged.

A blank test was carried out at the same time with the same quantities of chloroform and iodobromide solution and titrating as above.

Iodine number = 1.269 (V1 – V2)/W …………. (15) Where, W = weight of sample taken

V1= number of ml of thiosulphate consumed by the blank test

V2= number of ml of thiosulphate consumed by the actual test

CHAPTER IV

RESULTS AND DISCUSSION

4.1 Plant Extracts 4.1.1 Identification of Selected Plant The plant was collected from the western region, Resunga Municipality-03, Gulmi, Nepal and identified by Ganga Datta Bhatt, National Herbarium and Plant Resources (KATH), Lalitpur, Nepal.

Table 3: Sample collection and identification Identified plant Common Name Family Place of Collection Cinnamomum tamala Dalchini, Tejpat or Lauraceae Gulmi District Sinkouli Province No-05, Nepal

4.1.2 Percentage Yield Table 4: Table showing % yield of methanol and hexane extract Specific Part of plant Methanol Extract (%) Hexane Extract (%)

Leaves 26.21 2.04 Bark 31.37 3.52

The percentage yield of methanol and hexane extract of Cinnamomum tamala leaves and bark were found to be 26.21 %, 2.04%, 31.37 %, and 3.52% respectively.

4.1.3 Qualitative Analysis of Phytochemicals The micro-chemical analysis of a crude extract of Cinnamomum tamala leaves and bark in methanol and hexane extract depicted the presence of a class of phytochemicals as shown in table 4 and 5. The presence of phytochemicals was confirmed by the appearance of specific colors as visualized by microscope.

Table 5: Micro-chemical analysis of phytochemicals of leaves S.N. Phytochemicals Colour Hexane Methanol 1 Volatile oil Yellow + + 2 Alkaloids Reddish gray - - 3 Saponin Light maroon - + 4 Carbohydrates Violet - + 5 Terpenoids Reddish gray + + 6 Coumarins Yellow - + 7 Flavonoids Orange + + 8 Steroids Yellowish - - 9 Quinones Deep red + + 10 Phenolic compounds Greenish blue + + 11 Glycosides Violet - + Where,‘+’ means present and ‘ –‘ means absent

The result shows the presence of most of the phytochemicals in the polar extract, methanol. Volatile oil, quinones and terpenoids were present in both solvents, hexane and methanol. The absence of alkaloids may be due to the decomposition of alkaloids because of heat as soxhlet extractor was used to prepare the extracts as well as concentrating the extracts by rota-evaporator.

Table 6: Micro-chemical analysis of phytochemicals of Bark S.N. Phytochemicals Colour Hexane Methanol 1 Volatile oil Yellow + + 2 Alkaloids Reddish gray - - 3 Saponin Light maroon - + 4 Carbohydrates Violet - + 5 Terpenoids Reddish gray + + 6 Coumarins Yellow - + 7 Flavonoids Orange + + 8 Steroids Yellowish - - 9 Quinones Deep red + + 10 Phenolic compounds Greenish blue + + 11 Glycosides Violet - + Where,‘+’ means present and ‘ –‘ means absent

The result shows the presence of most of the phytochemicals in the polar extract, methanol. Volatile oil, quinones and terpenoids were present in both solvents, hexane and methanol. The phytochemicals present in the bark extracts are similar to that of leaf extracts.

The results presented in the table above are slightly different than the data present in the literature of this plant. Because the result of phytochemical screening for the same sample may vary from the screening of same phytochemical constituent due to variation in altitude of plants, different environmental conditions, method and time of sample collection, extraction procedure and also due to lab setup and chemical grades.

4.1.4 Antioxidant Activity Antioxidant activity of each plant extract was measured by using DPPH free radical given by Blois.42 DPPH is scavenged by antioxidants through the donation of proton forming the reduced DPPH. DPPH solutions show a strong absorbance band at 517 nm appearing as deep violet color. The color changes from purple to yellow after reduction, which can be quantified by its decrease of absorbance at wavelength 517 nm. The degree of decolorization indicates the free radical scavenging potentials i.e. antioxidant potentials of the sample.

DPPH assay was conducted for different extracts of Cinnamomum tamala bark and leaves, using ascorbic acid as standard. In this assay, different concentrations of different extract solutions and ascorbic acid solution were incubated at room temperature and their absorbance was recorded at 517 nm by using a spectrophotometer. Antioxidant results on a decrease of absorbance proportional to the concentration and antioxidant activity of the compound itself.

The antioxidant potential is in an inverse relation with IC50 value, which can be calculated from the linear regression of the % inhibition versus antioxidant

activity. The lower value of IC50 indicates high antioxidant potential. The control used involved DPPH and methanol omitting the sample extracts. In the present study, the percentage scavenging of the DPPH radical was concomitantly increased with the increase in the concentration of the extract from 20 – 100 μg/ml. 37

The comparison of the percentage of radical scavenging between various plant extracts and ascorbic acid as standard are shown in the figure below. 100 90 80 70 60 50 % scv of ascorbic acid 40 % scv of methanol bark 30 20 % of % of free radical Scavenging 10 0 0 20 40 60 80 100 120 Concentration (μg/ml) Figure 9: Comparision of % radical scavenging between ascorbic acid and methanol extract of C. tamala bark.

The IC50 value of methanolic extract of C. tamala bark was 90.35 μg/ml as compared to that of standard IC50 value of ascorbic acid 55.40 μg/ml.

100 90 80 70 % scv of ascorbic acid 60 % scv of hexane bark 50 40 30 20 % of % of free radical scavenging 10 0 0 20406080100120 Concentration (μg/ml)

Figure 10: Comparision of % radical scavenging between ascorbic acid and hexane extract of C. tamala bark

The IC50 value of hexane extract of C. tamala bark was 204.31 μg/ml as

compared to that of standard IC50 value of ascorbic acid 55.40 μg/ml.

100 90 80 70 % scv of ascorbic acid 60 50 % scv of methanol leaves (cold 40 percolation) 30 20

% of % of free radical scavenging 10 0 0 20 40 60 80 100 120 Concentration (μg/ml)

Figure 11: Comparision of % radical scavenging between ascorbic acid and methanol extract (cold percolation) of C. tamala leaves

The IC50 value of methanolic extract (cold percolation) of C. tamala leaf was

125.5 μg/ml as compared to that of standard IC50 value of ascorbic acid 55.40 μg/ml.

100 90 80 70 % scv of ascorbic acid 60 50 Methanol leaves 40 30 20

% of % of free radical scavenging 10 0 0 20 40 60 80 100 120 Concentration (μg/ml) Figure 12: Comparision of % radical scavenging between ascorbic acid and methanol extract of C. tamala leaves

The IC50 value of methanolic extract of C. tamala leaf was 127.63 μg/ml as

compared to that of standard IC50 value of ascorbic acid 55.40 μg/ml.

100 90 80 % scv of ascorbic acid 70 % scv of hexane leaves 60 50 40 30 20 % of % of free radical scavenging 10 0 0 20 40 60 80 100 120 concentration (μg/ml)

Figure 13: Comparision of % radical scavenging between ascorbic acid and hexane extract of C. tamala leaves

The IC50 value of hexane extract of C. tamala leaf was 211.45 μg/ml as compared to that of standard IC50 value of ascorbic acid 55.40 μg/ml.

The IC50 values of plant extracts along with the standard ascorbic acid is shown in the graph below.

250

211.45 204.31 200

150 125.5 127.63 g/mL) μ

100 90.35 value (

50 55.4 IC 50

0 Ascorbic acid MeOH bark MeOH leaves MeOH leaves Hexane bark Hexane (percolation) leaves

Figure 14: IC50 values of different extract of the plant along with ascorbic acid

The concentration of the plant extract required to cause 50 % inhibition of

DPPH free radicals, also referred to as the IC50, is determined from the inhibition of DPPH radicals caused by the different concentrations of the 40

extract. Antioxidant activity is inversely proportional to the IC50 values i.e.

extract or fraction or compounds having small IC50 values are more potent

antioxidants than those having larger IC50 values.

Comparatively, the IC50 values of the methanol extract of leaves and bark are lower than their corresponding hexane extracts which imply methanol extracts have high antioxidant properties as compared with ascorbic acid. Moreover,

methanol extracts of bark showed high radical scavenging activity with IC50

90.93μg /ml as compared to the methanol extract of the leaf having IC50 127.63μg/ml. Thus, they can act as potential natural antioxidants.

4.1.5 Determination of total phenolic content The total soluble phenols present in the methanolic and hexane extract of the plant were evaluated by using Folin-cocalteu reagent (FCR) according to the standard procedure given by Singleton V.B., Ross J.A (1965) involving gallic acid as standard.44 Polyphenols in the plant extracts react with specific redox reagent (FCR) to form a blue complex exhibits a broad light absorption with a maximum at 760 nm that can be quantified by the UV-visible spectrometry. The intensity of light absorption at that wavelength is proportional to the concentration of phenols. The observation for absorbance for different concentration of standard gallic acid was illustrated through graphical representation, X-axis being plotted for concentration and Y-axis for absorbance. The numerical data is shown in Appendix C. The absorbance curve for standard gallic acid is shown in figure 15.

0.25

0.2

0.15

0.1 Absorbance 0.05 Absorbance Linear (Absorbance) 0 0 20 40 60 80 100 120 Concentration (μg/ml)

Figure 15: Variation of absorbance with concentration for standard Gallic acid. 41

Using calibration curve and absorbance values of methanol and hexane extract of bark and leaves of Cinnamomum tamala (1000 μg/mL), total phenolic content was obtained as 196.50, 153.41, 27.62 and 21.50 mg per gram gallic acid equivalent (mg GAE/gm) respectively as represented in table 7.

Table 7: Total phenolic content of Cinnamomum tamala extracts S.N. TPC Methanol extract Hexane extract (mg GAE/gm) (mg GAE/gm) 1 Leaves 153.41 21.5

2 Bark 196.5 27.62

More conveniently total phenolic content in plant extracts is represented in bar diagram in figure 17.

250

196.5 200 153.41 150

100

50 27.62 21.5 0 Methanol bark Hexane bark Methanol leaf Hexane leaf

Figure 16: Total phenolic content in different Cinnamomum tamala plant extract

From the above result, it is vivid that methanol extracts of C. tamala bark and leaves contain high phenolic compounds 196.50 mg GAE/gm and 153.41 mg GAE/gm in comparison to their respective hexane extracts 27.62 mg GAE/gm and 21.5 mg GAE/gm respectively. Among them, the methanol extracts of C. tamala bark has the highest phenolic content among the four extracts.

Phenolic compounds have been known to possess high antioxidant properties due to their free radical scavenging properties. It has been reported that extract containing a large amount of polyphenol content possesses a great antioxidant activity. Although the quantitative determination of phenolic compounds in plant extracts are hampered by their structural complexity, diversity, nature of

analytical assay method, selection of standard and presence of interfering substances.

4.1.6 Determination of total flavonoid content The aluminum chloride colorimetric assay was used for the estimation of total flavonoid present in the methanolic and hexane extracts of the plant according to the standard procedure given involving quercetin as standard. The flavonoids of the plant extracts in the presence of aluminum chloride forms an acid liable complexes, has an intense yellow fluorescence which was observed under UV spectrophotometer at 510 nm. The intensity of light absorption at that wavelength is proportional to the concentration of flavonoids. The numerical data of absorbance of different concentration of standard quercetin is shown in Appendix C.

The calibration curve for standard quercetin is shown in the figure below.

0.25

0.2

0.15

0.1

Absorbance Absorbance 0.05 Linear (Absorbance)

0 0 20 40 60 80 100 120 Concentration (μg/ml) Figure 17: Variation of absorbance with concentration for Quercetin

Using calibration curve and absorbance values of methanol and hexane extract of bark and leaves of Cinnamomum tamala (1000 μg/ml), total flavonoid content was obtained as 167.82, 128.24, 25.90 and 22.85 mg per gram quercetin equivalent (mg GAE/gm) respectively as represented in table 8.

Table 8: Total flavonoid content of Cinnamomum tamala extracts S.N. TFC Methanol extract Hexane extract (mg QE/gm) (mg QE/gm) 1 Leaves 128.24 22.85

2 Bark 167.82 25.90

More conveniently total flavonoid content in plant extracts is represented in bar diagram in figure18.

180 167.82 160 140 128.24 120 100 80 60

40 25.9 22.85 20 0 Methanol bark Hexane bark Methanol leaf Hexane leaf

Figure 18: Total flavonoid content in different C. tamala plant extracts

From the above result, it is vivid that methanol extracts of C. tamala bark and leaves contain high flavonoid compounds 167.82 mg QE/gm and 128.24 mg QE/gm in comparison to their respective hexane extract 25.9 mg QE/gm and 22.85 mg QE/gm respectively. Among them, the methanol extract of C. tamala bark has the highest flavonoid content among the four extracts.

Flavonoid compounds are capable of effectively scavenging the free radicals because of their phenolic hydroxyl group and possess antioxidant properties. Their antioxidant properties depend on their structure, particularly hydroxyl position in the molecule. Although the quantitative determination of flavonoid compounds in plant extracts are hampered by their structural complexity, diversity, nature of analytical assay method, selection of standard and presence of interfering substances.

4.1.7 Brine Shrimp Bioassay Brine shrimp assay of methanol extract of leaves and bark of C. tamala were done to assess the toxicity of these extract. The newly hatched brine shrimp nauplii were exposed to different concentrations of extracts (10 µg/ml, 100 µg/ml and 1000 µg/ml) and their toxicity towards nauplii was evaluated by

calculating LC50 (µg/ml) value. LC50 is defined as the concentration that kills

50% of test organisms exposed to it. Extracts having LC50 values less than1000 µg/ml are supposed to be pharmacologically active or toxic. The

calculation of the LC50 value of methanol extract of leaves and bark of C. tamala was summarized in the following table.

Table 9: Calculation of LC50 value of methanol extract of leaves and bark of Cinnamomum tamala 2 Name Conc x = No. of No. of xy x β α X LC50 of in log Z alive Replicate = the µg/mL larvae (n) Anti Extract (Z) (y) log X MeOH 10 1 7 7 1 Extract 100 2 6 3 12 4 -1.5 8.66 2.44 275.42 of C. 1000 3 4 12 9 tamala leaf Σx = 6 Σy= 17 Σxy=31 Σx2 = 14 MeOH 10 1 8 8 1 extract 100 2 7 14 4 -2.5 11.0 2.40 251.18 of C. 1000 3 3 3 9 9 tamala bark Σx = 6 Σy= 18 Σxy=31 Σx2 = 14

The degree of lethality was found to be directly proportional to the concentration of that extracts that is maximum mortalities of the brine shrimp larvae took place at the concentration of 1000 μg/mL and least mortalities

were in 10 μg/mL. Those having LC50 values less than 1000 μg/ml are

supposed to be pharmacologically active. The table showed that the LC50 value of methanol extract of C. tamala leaves was calculated to be

275.42μg/ml. The LC50 value is lower which can be generalized as the methanolic leaf extract was toxic towards Artemia salina larvae.

Similarly, the LC50 value of the methanolic extract of C. tamala bark was found to be 251.18μg/mL. Since this value is also lower than 1000 μg/ml, the extract is also toxic towards Artemia salina larvae.

Brine shrimp lethality assay is a convenient system for monitoring biological activities of various plant species. Although this method does not provide any adequate information regarding the mechanism of toxic action, it is a very useful method for the assessment of the toxic potential of various plant extracts. This method provides preliminary screening data that can be backed up by more specific bioassays once the active compounds have been isolated.

4.1.8 Antimicrobial Activity The diameter of zone of inhibition (ZOI) produced by the plant extracts on particular bacteria or fungus was measured for the estimation of their antimicrobial activity. The potential of the methanol fraction of the Cinnamomum tamala leaf and bark extract to inhibit the growth of bacteria at a fixed concentration (50 mg/ml) was evaluated according to the procedure described in section and the results were expressed in terms of diameter of zone of inhibition including the diameter of well (6 mm). The area around the antimicrobial disk where there is no growth of micro-organisms is called the zone of inhibition. The minimum concentration of the plant extract that hinders the growth of microorganisms is called minimum inhibitory concentration while the minimum concentration that kills the microorganisms completely is called minimum bactericidal concentration.

Results obtained from the antibacterial screening of different extracts are given in table10.

Table 10: Antibacterial activity of methanol extract of C. tamala leaves and bark S.N. Bacterial strains Zone of Inhibition(mm)

Positive Negative C. tamala C. tamala control control leaf extract bark extract Neomycin DMSO 1 Escherichia coli 24 0 - - (ATCC 25922)

2 Staphylococcus 19 0 9 15 aureus (ATCC 25923) 3 Salmonella 21 0 - - typhimurium (ATCC 14028)

The table 10 indicates that the methanol extract of Cinnamomum tamala leaves possessed 0 mm, 9 mm and 0 mm zone of inhibition for Escherichia coli, Staphylococcus aureus and Salmonella typhimurium respectively. The zone of inhibition for methanol extract of Cinnamomum tamala bark resulted in 0 mm, 15 mm and 0mm for Escherichia coli, Staphylococcus aureus and Salmonella typhimurium respectively. There is significant inhibitory activity against the gram-positive bacteria Staphylococcus aureus but not any inhibitory activity against gram-negative bacteria Escherichia coli and Salmonella typhimurium as gram-positive bacteria are more sensitive than gram-negative bacteria.

Table 11: Antifungal activity of methanol extract of C. tamala bark and leaves S.N. Fungal strain Zone of Inhibition(mm) Positive control C. tamala C. tamala Itraconazole leaf extract bark extract

1 Candida albicans 17 - -

The table above summarizes the results obtained from the determination of antifungal activity of the methanol fractions of Cinnamomum tamala bark and leaves at fixed concentration (50 mg/ml) on the tested organisms according to the procedure described in section and the results are expressed in diameter of zone of inhibition (mm) including the diameter of well (6 mm).

The result doesn’t show any inhibitory activity against the growth of the fungi used under study among the two fractions as it multi-drug resistant fungi which were evident from no diameter of the zone of inhibition.

4.2 Essential oil 4.2.1 Extraction and Quantification of Essential Oil The essential oil present in the fresh leaves of Cinnamomum tamala was obtained by hydrodistillation method using Clevenger apparatus. The percentage yield of the essential oil was found to be 1 % (v/w) on a fresh weight basis.

4.2.2 Organoleptic Properties of Essential oil The essential oil obtained from Cinnamomum tamala was found to have the following organoleptic properties.

Table12: Organoleptic properties of essential oil from C. tamala leaves Plant Appearance Colour Aroma Cinnamomum Slightly viscous and Colorless, transparent or Sweet, strongly tamala non- sticky slightly yellowish or fragrant greenish

4.2.3 Chemical analysis of constituents of Essential Oil The chromatogram of Cinnamomum tamala essential oil from Bhadgaun, Gulmi by GC/MS is shown in spectra 1.

(x1,000,000)

TIC (1.00) 9 1.00

0.75 12 0.50

0.25 7 5 4 2 1 10 8 13 11 6 3 10 20 30 40 50 60 70

Spectra1: GC chromatogram of the essential oil from C.tamala leaves of Bhadgaun, Gulmi.

The GC/MS analysis of the Cinnamomum tamala leaf essential oil along with mass library search, NIST 17 and FFNSC 1.3 led to the identification and quantification of 13 components which accounted for 100% of the total oil of sample from Bhadgaun, Gulmi. Most of them were monoterpenes and sesquiterpenes.

The major components present in the sample studied were Cinnamaldehyde (E) (62.49%), Cinnamyl acetate (22.54%), Hydrocinnamaldehyde (2.55%), Neral (2.28%), Linalool (1.76%), Benzaldehyde (1.45%) and Geranyl acetate (1.27%). All the constituents present in the essential oil of Cinnamomum tamala from Bhadgaun, Gulmi is tabulated in table 13.

Table 13: Chemical constituents of essential oil from Cinnamomum tamala leaves of Bhadgaun, Gulmi S.N. / Compounds Retention Area Area % Peaks Time (Rt) 1 Benzaldehyde 12.903 177023 1.45 2 Hept-5-en-2-one <6-methyl-> 14.172 175496 1.44 3 2,3-octanedione 18.284 53234 0.44 4 Linalool 19.589 214398 1.76 5 Hydrocinnamaldehyde 22.651 311339 2.55 6 Cinnamaldehyde <(Z)-> 25.371 61688 0.51 7 Neral 26.338 346491 2.84 8 Borane,[1,2-bis(1- 26.975 182945 1.50 methylethyl)butyl] 9 Cinnamaldehyde <(E)-> 27.863 7623724 62.49 10 Geranyl acetate 32.657 154797 1.27 11 Trans-alpha-Bergamotene 34.299 75008 0.61 12 Cinnamyl acetate <(E)-> 35.348 2749700 22.54 13 Nerolidol 40.963 74887 0.61 12200730 100.00

Upadhaya et.al (1994) reported linalool (54.66 %) as a major component of Nepal bay leaf essential oil. They reported 22 compounds in the oil. A total of 72 compounds were identified in the bay leaf oil from North East India (Rana et.al 2009). Eugenol was found as a major compound in the leaf oil and its concentration varied from 35.1 – 94.3%. Choudhary et.al (2013) reported 17 compounds and eugenol was absent in the oil whereas cinnamaldehyde (39.14%) and linalool (54.66%) was the constituents with a higher percentage from Chamoli, India. Choudhary et.al reported 31 components from the bay leaf essential oil from Udayapur, Nepal and records linalool as the major constituents with 48.59%. The present study reports 13 compounds from the essential oil of Cinnamomum tamala leaves from Bhadgaun, Gulmi and records cinnamaldehyde as the major constituents with a content of 62.49%.

The percentage yield of the chemical constituents from the same species collected from different areas was highly varied in GC chromatogram. For instance, the % yield of linalool is found to be 1.76 % from the sample of Gulmi, where it was 13.89 % from Sanobharyang, Nepal. This shows that the

% yield of chemical constituents in the essential oil of C. tamala leaves tends to depend upon the geographical identity and different eco-climate identity.

Besides of the Climatic conditions and size of trees, the altitude also plays an important role in the presence of chemical constituent from C. tamala leaf oil. For example, Cinnamaldehyde was found as a major product from the C. verum sample collected from Madagascar, India whereas, Eugenol was recorded as a major product from the same species collected from North-east region of Himalayan. It is expected that the essential oil contents depend on organs and geographical identity and different eco-climatic identity. It is also concluded that variation in the essential oil chemicals is due to geographical divergence and abiotic and biotic stress, seasons, sunlight, UV radiation, etc.

4.2.4 Determination of Physical Parameters 4.2.4.1 Specific Gravity The specific gravity of the essential oil of Cinnamomum tamala was found to be 0.961.

4.1.5 Determination of Chemical Parameters 4.1.5.1 Saponification Value Saponification value of a oil is the number of milligrams of potassium hydroxide required to completely saponify one gram of the ester particularly oil. The saponification value of the essential oil of Cinnamomum tamala was found to be 84.1. It means 84.1 mg of KOH is required to saponify 1 gm of the oil sample.

4.1.5.2 Acid Value The acid value of the essential oil of Cinnamomum tamala leaves was found to be 3.34. The acid value measures the presence of free acids present in the sample.

4.1.5.3 Iodine Value The iodine value of the essential oil from Cinnamomum tamala leaves was found to be 102.39 which reflect the degree of unsaturation in the oil sample.

Table 14: Structures of some major compounds of the Essential Oil from C. tamala leaves S.N. Compounds Structures

1 Cinnamaldehyde

2 Benzaldehyde

3 Linalool

4 Cinnamyl acetate

5 Geranyl acetate

6 Neral

CHAPTER – V

CONCLUSIONS

5.1 Conclusions The phytochemical investigations have shown that the plant extracts are rich sources of secondary metabolites. Phytochemical screening of hexane extracts of leaf and bark of C. tamala showed the presence of volatile oil, polyphenols, terpenoids, quinines and flavonoids and that of methanol extract showed the presence of glycosides, saponin, volatile oil, flavonoids, terpenoids, coumarins, quinines and polyphenols. Steroids are absent in both the extract in both solvents.

The determination of DPPH radical scavenging activities and subsequently

IC50 of the plant extracts showed a varying degree of antioxidant property. The

IC50 values of the plant different extracts exhibited that the methanol extract of C. tamala bark was the most potent natural antioxidant among all extracts

which was confirmed by comparing its IC50 value to the standard. The methanol extract of C. tamala leaves also showed considerable antioxidant property. Methanolic percolation extract of the plant leaf showed better antioxidant property than in soxhlet extract of leaves. However, hexane extract of

C. tamala leaves and bark because of very high IC50 values are termed as a poor antioxidant.

Both methanol extract of leaves and bark contained a higher amount of phenolic and flavonoid content as compared to the corresponding hexane extracts. This might be one of the major factors for methanol extracts of C. tamala leaves and bark exhibiting strong antioxidant activity. Among the leaves and bark, TPC and TFC were found to be higher in bark extracts.

In the evaluation of the antibacterial activity of methanol extract of Cinnamomum tamala bark and leaves showed significant inhibitory activity against the growth of positive bacteria Staphylococcus aureus, 15mm and 9 mm respectively in comparison with standard neomycin (19 mm) but did not show any inhibitory activity towards gram-negative bacteria Escherichia Coli and Salmonella typhimurium. This fact can be described by the presence of a

unique outer membrane that excludes the extract from penetrating the cell in gram-negative bacteria which is absent in case of gram-positive type. The antifungal assay showed that there is no inhibitory activity against the growth of the fungi (Candida albicans) used under study among the methanol leaves and bark fractions. From brine shrimp bioassay, it can be inferred that the bark and leaves of the

Cinnamomum tamala have the LC50 values less than 1000, so it’s pharmacologically active.

Cinnamaldehyde was found to be the predominant constituent with 62.49% along with cinnamyl acetate (22.54%) followed by neral, hydrocinnamaldehyde, benzaldehyde, linalool, geranyl acetate from the GC/MS analysis of the essential oil from Cinnamomum tamala leaves from Gulmi.

5.2 Suggestion for further work Phytochemical analysis of the plants showed the presence of many biologically active compounds which can be attributed to the potential biological and pharmacological activities, it is desired to prepare the plant extracts in other major solvents apart from discussed here so that wide varieties of phytochemicals in higher amount can be extracted. Total phenolic and flavonoid content validated the idea behind the use of the traditional medicinal plant to treat different diseases and could be used sources of active compounds in a future study.

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APPENDIX

(A) Phytochemical Screening Protocol 1) Test for Volatile Oils To about 500 mg extract, 0.5 mL methanol was added, shaken vigorously and filtered. Few drops of the filtrate were put on a filter paper by means of a capillary tube. Yellow spot persistent even after evaporation of the solvent indicates the presence of volatile oils.

2) Test for Alkaloids About 500 mg extract was dissolved in 3 mL of 2 % (v/v) HCl. The solution was equally divided into two test tubes and the following tests were performed. i. Meyer’s Test Few drops of Meyer’s reagent were added to the first part. Formation of a pale yellow precipitate indicates the presence of alkaloids.

ii. Dragendorff’s Test Few drops of Dragendorff’s reagent were added to the second part. Formation of an orange-red precipitate indicates the presence of alkaloids.

3) Test for Terpenoids

To about 200 mg extract, 2 mL of chloroform (CHCl3) and then 3 mL

concentrated sulphuric acid (H2SO4) was added carefully. Formation of reddish-brown coloration at the interface indicates the presence of terpenoids.

4) Test for Coumarins To about 1 mL of extract, 1 mL of 10 % sodium hydroxide (NaOH) solution was added. Formation of yellow color indicates the presence of coumarins.

5) Test for Flavonoids/Shinoda’s Test About 200 mg extract was dissolved in 2 mL methanol. To this solution, a small piece of magnesium and 4-5 drops of concentrated hydrochloric acid (HCl) were added. Formation of orange color indicates the presence of flavonoids.

6) Test for Quinones

To about 2 mL extract, 1 mL freshly prepared ferrous sulfate (FeSO4) solution

and few crystals of ammonium thiocyanate (NH4SCN) were added and the

solution was treated with conc. sulphuric acid (H2SO4) drop by drop. The appearance of persistent deep red coloration indicates the presence of quinones.

7) Test for Polyphenols/ FeCl3 Test To about 1 mL extract, 1ml distilled water was added followed by the addition

of a few drops of 10 % (w/v) ferric chloride (FeCl3) solution. The appearance of greenish blue coloration indicates the presence of polyphenols.

8) Test for Glycosides About 500 mg extract was dissolved in 2 mL methanol and divided into two parts and the following tests were performed. i. Molisch’s Test

The first part was treated with 5 mL of Molisch’s reagent and conc. H2SO4 was added drop by drop from the side of the test tube without disturbing the solution. The appearance of a violet ring at the junction of two liquids which on shaking turns the solution into violet color indicates the presence of glycosides. ii. To the second part 2 mL of 25 % (v/v) NH4OH solution was added and shaken vigorously. The appearance of the cherry red color indicates the presence of glycosides.

9) Test for Reducing Sugars To about 1 mL extract, 1mL distilled water was added followed by addition of 1 mL Fehling’s reagent (1,1 mixture of Fehling’s solution A and B). Then the mixture was warmed over a water bath for 30 minutes. The appearance of a brick red precipitate indicates the presence of reducing sugars.

10) Test for Saponins About 500 mg extract was treated with hot water followed by shaking for 30 seconds. Formation of thick forth indicates the presence of saponins.

11) Test for Tannins About 200 mg extract was boiled adding 10 mL distilled water. The mixture

was cooled, filtered and few drops of FeCl3 solution were added to the filtrate. The appearance of blue-black precipitate indicates the presence of tannins.

12) Preparation of Reagents 1. Meyer’s Reagent,

Mercuric chloride, HgCl2 (0.679 g) was weighed in a 50 mL volumetric flask and dissolved in distilled water. To this solution, 2.5 g potassium iodide (KI) was added. The scarlet red precipitate was dissolved by shaking and volume was made up to the mark by adding distilled water.

2. Dragendorff’s Reagent

Bismuth nitrate, Bi(NO3)3 (4.000 g) was dissolved in 5 N nitric acid (10 mL) to make solution A. Next, potassium iodide, KI (13.5 g) was dissolved in distilled water (20 mL) to make solution B. These two solutions were mixed together in a 50 mL volumetric flask.

Picric acid (0.25 g) was dissolved in 50 mL distilled water to make an aqueous picric acid solution. The solution was neutralized with sodium bicarbonate

(NaHCO3). A strip of Whatman no. 1 filter paper was dipped in the prepared solution. The paper was dried completely and protected from external contamination. Thus prepared sodium picrate paper was used for Cyanogenic Glycoside detection.

3. Molisch’s Reagent α-Naphthol (5.000 g) was dissolved in 50 mL methanol to prepare Molisch’s reagent.

4. Neutral Ferric Chloride (FeCl3) Solution Ferric chloride crystals (1.000 g) were dissolved in 100 mL distilled water. To this solution, sodium carbonate crystals were added little by little with stirring until the slight turbidity was persistent. Finally, the mixture was filtered and the colorless filtrate was used as neutral ferric chloride solution.

(B) Antioxidant Activity Table 15: Antioxidant activity of Ascorbic acid S.N. Concentration Absorbance mean % of (ppm) scavenging 1 20 0.337 0.401 0.403 0.400 19.01 2 40 0.312 0.322 0.323 0.319 35.38 3 60 0.209 0.208 0.216 0.211 57.12 4 80 0.136 0.143 0.144 0.141 71.42 5 100 0.054 0.057 0.057 0.056 88.47

IC50 55.40 Blank 0.494

Table 16: Antioxidant activity of Methanol extract of Bark S.N. Concentration Absorbance mean % of (ppm) scavenging 1 20 0.411 0.428 0.418 0.419 15.68 2 40 0.372 0.376 0.380 0.376 24.45 3 60 0.337 0.346 0.343 0.342 31.27 4 80 0.260 0.266 0.260 0.262 47.33 5 100 0.228 0.239 0.223 0.230 53.68

IC50 90.35 Blank 0.498

Table 17: Antioxidant activity of Methanol extract (cold percolation) of Leaf S.N. Concentration Absorbance mean % of (ppm) scavenging 1 20 0.494 0.491 0.506 0.497 16.32 2 40 0.457 0.463 0.457 0.459 22.56 3 60 0.429 0.435 0.438 0.434 26.83 4 80 0.411 0.418 0.416 0.415 29.97 5 100 0.345 0.347 0.349 0.347 41.46

IC50 125.50 Blank 0.594

Table 18: Antioxidant activity of Methanol extract of Leaf S.N. Concentration Absorbance mean % of (ppm) scavenging 1 20 0.399 0.405 0.405 0.403 19.89 2 40 0.384 0.389 0.388 0.387 23.04 3 60 0.358 0.365 0.360 0.361 28.21 4 80 0.341 0.343 0.348 0.344 31.67 5 100 0.301 0.299 0.315 0.305 39.43

IC50 127.63 Blank 0.504

Table 19: Antioxidant activity of Hexane extract of Bark S.N. Concentration Absorbance mean % of (ppm) scavenging 1 20 0.971 0.970 0.978 0.973 8.16 2 40 0.934 0.931 0.952 0.939 11.41 3 60 0.917 0.912 0.910 0.913 13.83 4 80 0.826 0.840 0.836 0.834 21.28 5 100 0.792 0.799 0.794 0.795 24.97

IC50 204.31 Blank 1.06

Table 20: Antioxidant activity of Hexane extract of Leaf S.N. Concentration Absorbance mean % of (ppm) scavenging 1 20 0.457 0.450 0.449 0.452 7.82 2 40 0.428 0.439 0.423 0.430 12.34 3 60 0.416 0.423 0.415 0.418 14.67 4 80 0.385 0.391 0.394 0.390 20.49 5 100 0.367 0.380 0.372 0.373 23.89

IC50 211.45 Blank 0.491

(C) Total Phenolic Content Table 21: Total phenolic content in methanol extract of Bark S.N. Sample Wt. of dry Absorbance GAE GAE TPC as Mean solution extract per conc. conc. GAE × (µg/ml) mL C C = m (g) (µg/ml) (mg/ml) 1 1000 0.001 0.399 195.00 0.195 195.00

2 1000 0.001 0.406 200.50 0.200 200.50 196.50

3 1000 0.001 0.393 193.50 0.193 193.50

Table 22: Total phenolic content in methanol extract of Leaf S.N. Sample Wt. of dry Absorbance GAE GAE TPC as Mean solution extract per conc. conc. GAE × (µg/ml) mL C C = m (g) (µg/ml) (mg/ml) 1 1000 0.001 0.311 153.23 0.153 153.23

2 1000 0.001 0.316 155.50 0.155 155.50 153.41

3 1000 0.001 0.308 151.50 0.151 151.50

Table 23: Total phenolic content in the hexane extract of Bark S.N. Sample Wt. of dry Absorbance GAE GAE TPC as Mean solution extract per conc. conc. GAE × (µg/ml) mL C C = m (g) (µg/ml) (mg/ml) 1 1000 0.001 0.060 27.89 0.027 27.89

2 1000 0.001 0.058 26.98 0.026 26.98 27.62 3 1000 0.001 0.061 28.01 0.028 28.01

Table 24: Total phenolic content in the hexane extract of Leaf S.N. Sample Wt. of dry Absorbance GAE GAE TPC as Mean solution extract per conc. conc. GAE × (µg/ml) mL C C = m (g) (µg/ml) (mg/ml) 1 1000 0.001 0.048 21.50 0.021 21.50

2 1000 0.001 0.052 23.50 0.023 23.50 21.50

3 1000 0.001 0.044 19.50 0.019 19.50

(D) Total Flavonoid Content Table 25: Total flavonoid content in methanol extract of Bark S.N. Sample Wt. of dry Absorbance QE conc. QE TFC as Mean solution extract per C conc. QE × (µg/ml) mL (µg/ml) C = m (g) (mg/ml) 1 1000 0.001 0.346 169.26 0.169 169.26

2 1000 0.001 0.341 166.79 0.166 166.79 167.82

3 1000 0.001 0.342 167.43 0.167 167.43

Table 26: Total flavonoid content in the hexane extract of Leaf S.N. Sample Wt. of dry Absorbance QE conc. QE conc. TFC as QE Mean × solution extract per C C = (µg/ml) mL (µg/ml) (mg/ml) m (g) 1 1000 0.001 0.053 22.67 0.022 22.67

2 1000 0.001 0.056 24.05 0.024 24.05 22.85

3 1000 0.001 0.051 21.83 0.021 21.83

Table 27: Total flavonoid content in methanol extract of Leaf S.N. Sample Wt. of dry Absorbance QE conc. QE TFC as Mean solution extract per C conc. QE × (µg/ml) mL (µg/ml) C = m (g) (mg/ml) 1 1000 0.001 0.267 129.87 0.129 129.87

2 1000 0.001 0.261 126.79 0.126 126.79 128.24

3 1000 0.001 0.264 128.08 0.128 128.08

Table 28: Total flavonoid content in the hexane extract of Bark S.N. Sample Wt. of dry Absorbance QE conc. QE conc. TFC as QE Mean × solution extract per C C = (µg/ml) mL (µg/ml) (mg/ml) m (g) 1 1000 0.001 0.058 25.43 0.025 25.43

2 1000 0.001 0.062 27.21 0.027 27.21 25.90

3 1000 0.001 0.057 25.08 0.025 25.08

Antimicrobial activity

GC/MS Analysis

Soxhlet apparatus

Antimicrobial activity