Chemical Composition of Essential Oil and Crude Extract Fractions And

Yarmouk University

Faculty of Science

Department of Biological Sciences

Chemical Composition of Essential Oil and Crude Extract Fractions and

their Antibacterial Activities of Capparis spinosa L. and Capparis

cartilaginea Decne. from Jordan.

Ahmmad Al-Shayeb

Supervisor

Dr. Riyadh Muhaidat © Arabic Digital Library - Yarmouk University

Co – supervisor

Dr. Mahmoud Al-Qudah

Program: Biotechnology

2012

0 © Arabic Digital Library - Yarmouk University Dedication

This thesis is dedicated to my parents

for their love, endless support

and encouragement.

To my grandparents,

sisters and brotherss..

and

I ACKNOWLEDGMENTS

First of all, i thank Almighty God for giving me the courage and the

determination, as well as guidance in conducting this research study, despite all

difficulties.

I would like to take this great opportunity to express my gratitude and

indebtness towards my esteemed guide Dr. Riyadh Muhaidat for his constant

encouragement, continuous support, valuable suggestions and timely advise.

I am extremely thankful to Dr. Mahmoud Al-Qudah for co-advising me

during this work and for his great encouragement during the course of this study by

providing me with all the facilities that lead to successful completion of my

project.

Malkawi and Dr. Hala Al-Jaber for being members of the examining committee.

I would like to express my sincere thanks and heartful gratitude to all my

graduate batch mates for their help and constant support.

There are no words, which can express my gratitude towards my Parents,

sisters, and brothers Mohamad, Abdullah and Moutasem for their unlimited

patience, immense care and faith in me.

II List of Contents Subject Page Dedication…………………………………………………………………. I

Acknowledgments…………………………………………………………. II

List of Contents……………………………………………………………. III

List of Tables………………………………………………………………. V

List of Figures……………………………………………………………… VI

List of Appendices………………………………………………………… VII

List of Abbreviations……………………………………………………… VIII

Abstract…………………………………………………………………… IX

CHAPTER ONE: Introduction……………………………………………….….1

1. Introduction………………………………………………………...... 1

CHAPTER TWO: Literature Review………………………………………..….5

2.1. Capparis spinosa L ……………………………………………………….…...5

2.3. Capparis sepiaria L. …………………………………………………….……8

2.4. Capparis zeylanica L. …………………………………………………..…….9

2.5. Capparis grandiflora Wall. ex Hook.F. & Thomson………………..………...9

2.6.Rationale and objectives………………………………………………………10

CHAPTER THREE: Materials and methods……………………………….....13

3.1. Plant material………………………………………………………………....13

3.2. Extraction of crude extracts...... 13

3.3. Isolation of essential oil………………………………………………………14

III 3.4. GC–FID analysis……………………………………………………………..14 3.5. GC–MS analysis……………………………………………………………...14 3.6. Identification of constituents…………………………………………………15 3.7. Antimicrobial study…………………………………………………………..15 3.7.1. Tested bacterial species…………………………………………..15 3.7.2. Antibacterial activity assay by the agar well diffusion

Method…………………………………………………….…… 16

3.7.3. Determination of Minimal Inhibitory Concentration (MIC)……. 17 3.8. Phytochemical screening…………………………………………..……...... 18

CHAPTER FOUR: Results………………………………………………..…....19

4.1. Analysis of essential oils from C. cartilaginea and C. spinosa…………….…19

4.2. Antibacterial activities of crude extract fractions collected from C. cartilaginea………………………………………………………………………...... 26 4.3. Minimal inhibitory concentrations (MIC) of crude extract fractions collected from C. cartilaginea…………………………………………..….…….30 4.4. Antibacterial activities of crude extract fractions collected from C. spinosa……………………………………………………………………….….....33 4.5. Minimal inhibitory concentrations (MIC) of crude extract fractions collected from C. spinosa…………………….……………………………….….….36 © Arabic Digital Library - Yarmouk University 4.6. Phytochemical screening for secondary metabolites in tested fractions of C. cartilaginea……………………………………………………………..……. 39 4.7. Phytochemical screening for secondary metabolites in tested fractions of C. spinosa………………………………………………………………… 39 CHAPTER FIVE: Discussion………………………………………..………..41 5.1. Essential oils…………………………………………………………….…...41 5.2. Antimicrobial activity………………………………………………………44 5.3. Screening for major phytochemical groups…………………………….…48 Conclusions………………………………………………………………….……50 Recommendations…………………………………………………………….….51 6. References………………………………………………………………….….52

IV List of Tables Table Page

4.1. Essential oil composition (%) of aerial parts collected from C.

Cartilaginea………………………………………………………………. 19

4.2. Essential oil composition (%) of aerial parts collected from C. C. spinosa……………………………………...... 22

4.3. Common chemical components between C. cartilaginea and

C. spinosa…………………………………………………………. 24

4.4. Classification of the components of the essential oils collected from

C. cartilaginea and C. spinosa………………………………………. 25

4.5. Antibacterial activities of crude extract fractions (400 μg/mL) collected

from C. cartilaginea………………………………………………….. 27

4.6. Minimal inhibitory concentrations 0,& JP/-1) of crude extract fractions collected from C. cartilaginea…………………………….. 31

from C. spinosa...... 34

4.8. Minimal inhibitory concentrations 0,& JP/-1) of crude extract Fractions collected from C. spinosa……………………...…...…..…. 37

4.9. Major groups of secondary metabolites detected in crude extract fractions of C. cartilaginea…………………………………………….. 39

4.10. Major groups of secondary metabolites detected in crude extract fractions of C. spinosa……………………………...…….…………………… 39

V List of Figures

Figure Page

4.1. Sensitivity of different bacterial species to A: petroleum ether, B:

hexane, C: methanol, D: butanol and E: water crude extract fractions

(400 μg/mL) from C. cartilaginea.……………………………………….. 28

4.2. 0LQLPDOLQKLELWRU\FRQFHQWUDWLRQV 0,& JP/-1) of A: petroleum

ether, B: hexane, C: methanol, D: butanol and E: water crude extract

fractions from C. cartilaginea against different bacterial species…... 32

4.3. Sensitivity of different bacterial species to A: petroleum ether, B:

hexane, C: methanol, D: butanol and E: water crude extract fractions © Arabic Digital Library - Yarmouk University

(400 μg/mL) from C. spinosa. 35

4.4. 0LQLPDOLQKLELWRU\FRQFHQWUDWLRQV 0,& JP/-1) of A: petroleum

ether, B: hexane, C: methanol, D: butanol and E: water crude extract

fractions from C. spinosa against different bacterial species.…………… 38

5.1. Hydrolysis products of glucosinolates……………….…………………. 43

VI List of Appendices

Appendix Page I The procedures and reagents used for screening of major phytochemical groups………………………………………………….……66

1. Reagents used for screening tests……………………...……………………….66

2. Screening for alkaloids………………………………...……………………….66

3. Screening for Flavonoids…………………………………………………….....67

4. Screening for Tannins…………………………………………………………..68

5. Screening for Anthraquinones………………………………………………….69 © Arabic Digital Library - Yarmouk University

VII List of Abbreviations

ATCC American Type Culture Collection

DMSO Dimethyl sulfoxide

GC-MS Gas Chromatography/Mass Spectrometry

GC-FID Gas Chromatography /Flame Ionization Detector

MIC Minimum Inhibitory Concentration

Mm Millimeter

J Microgram RI Retention Indices

SE Standard Error © Arabic Digital Library - Yarmouk University

VIII Abstract

The chemical composition of essential oils and antibacterial activity of crude

extract fractions and their phytochemical screening from the Aerial parts of C. spinosa L.

and C. cartilaginea Decne. (Capparaceae) were examined. Hydrodistillation of the fresh

flowering aerial parts of C. cartilaginea and C. spinosa yielded 0.1 % and 0.04 % pale

yellowish oils for C. cartilaginea and C. spinosa, respectively. GC/MS analysis of the

essential oils revealed a complex mixture of compounds including nitrogen and sulfur

containing compounds, oxygenated sesquiterpens and monoterpene hydrocarbons. Fifty

nine components were identified in the oil of the C. cartilaginea representing about 95.38

% of the total composition of the oil. The essential oil of C. cartilaginea was dominated

by methyl isothiocyanate (31.81 %), isopropyl isothiocyanate (18.25 %), isobutyl

isothiocyanate (5.40 %), 3-p-menthene (5.18 %), occidol (4.33 %), carissone (3.11 %)

and ethyl isothiocyanate (2.55 %). A total of 40 constituents were identified in the oil of

the C. spinosa amounting to 92.89 % of the total oil content.C. spinosa was dominated © Arabic Digital Library - Yarmouk University

by isopropyl isothiocyanate (28.92 %), methyl isothiocyanate (25.60 %), butyl

isothiocyanate (16.65 %), 3-p-menthene (3.08 %), 2-butenyl isothiocyanate (2.24 %) and

3-methylthio-1-hexanol (2.03 %) as major constituents. The antibacterial activity of

Petroleum ether, water, butanol, methanol and hexane crude extracts obtained from the

aerial parts of C. spinosa and C. cartilaginea was examined by agar well diffusion

method. Different fractions exhibited good to moderate degrees of activity against most

of the bacterial examined. The tested fractions of C. cartilaginea were most active

against Streptococcus faecalis. While, tested fractions of C. spinosa were most active

IX against Staphylococcus epidermidis and Streptococcus faecalis. The Minimal inhibitory

concentrations (MIC) was determined for all active fractions using amended agar method.

The butanol fraction of C. cartilaginea was most active with MIC of JP/ The

crude extracts fractions of C. cartilaginea and C. spinosa were screened for major groups

of secondary metabolites through Phytochemical screening. Water fraction of C.

cartilaginea and C. spinosa contained alkaloids, flavonoids and tannins. However, no

secondary metabolites were detected in Petroleum ether, butanol, methanol and hexane

fractions of C. cartilaginea. Alkaloids, tannins were detected in methanolic fraction of C.

spinosa. While, butanol fraction of C. spinosa contained only tannins. No secondary

metabolites were detected in Petroleum ether and hexane fractions of of C. spinosa.

Keywords: Capparis, Essential oil, Crude extract fractions, Phytochemicals,

Minimal inhibitory concentrations (MIC), gas chromatography–

mass spectrometry (GC–MS). © Arabic Digital Library - Yarmouk University

X Chapter One

1. Introduction.

Capparaceae (the caper family) is a dicot family in the

order Brassicales. It includes about 45 genera and 800 species of herbs,

climbers, shrubs, and trees distributed in tropical and subtropical regions; a

few of which occur in arid regions (Short and Cowie, 2011). The

Capparaceae, like all other families of the Brassicales, is distinctive in

producing glucosinolates (Hall et al., 2008; Simpson, 2010). These secondary

metabolites are glycosides that serve as feeding repellents and sources of

flavoring agents (i.e., mustard oils) (Levetin and McMahon, 2006). When the

plant is crushed, glucosinolates break down to produce the chemically

reactive compounds isothiocyanates and nitrils, which are responsible for the

pungent taste and odor. For example, the bitterness of cooked cauliflower and © Arabic Digital Library - Yarmouk University

Brussels sprouts was shown to be due to the glucosinolate sinigrin (Van

Doorn et al., 1998). Glucosinolates are currently of considerable interest for

use in natural and commercial pesticides and of great potential for mitigating

cancer. Consumers of high levels of plants-producing glucosinolates are

suggested to benefit from a lower risk of cancer. Preliminary research studies

on isothiocyanates derivatives from Brassica vegetables indicated that these

products act as potent inducers of cytoprotective enzymes and inhibitors of

1 carcinogenesis in vitro (Cornblatt et al., 2007). As an alternative to, or in

combination with, antibiotic-based therapies for treating infectious diseases,

isothiocyanate derivatives have striking inhibitory effect against Gram-

positive and Gram-negative bacterial strains, and thus might be useful in

controlling human pathogens through the diet (Tierens et al., 2001). They can

also act synergistically with less efficient antibiotics (streptomycin) to control

bacterial growth (Saavedra et al., 2010).

In addition to glucosinolates, members of the Capparaceae have the

ability to elaborate a series of flavonoids and occasionally pyrrolidine

alkaloids with unique chemical structures and pharmacological properties.

For example, 5,3´-dihydroxy-3,6,7,8,4´-pentamethoxyflavone isolated from

Polanisia dodecandra was found to inhibit a broad panel of cancerous cells in

different organs such as central nervous system, lung, ovary, and colon. The

leukemia cell lines cultured in vitro (Wiart, 2006).

Capparis commonly known as (the Caper) is the largest genus of the

Capparaceae comprising more than 250 species of shrubs, trees, and woody

climbers distributed over a wide range of habitats in the tropics, subtropics

and temperate regions of the world (Jacobs, 1965; Fici, 1993). The genus is

native to the Mediterranean region with a few species distributed in arid

regions of south-west and central Asia (Zohary, 1966; Romeo et al., 2007).

2 Capparis includes species that are of major interest for human nutrition

and health. Flower buds and unripe fruits of several Capparis species, such as

C. spinosa L. and C. ovata Desf., are traditionally consumed in various

countries as food and sources of flavor (Tlili et al., 2011). Capers are also

used in folk medicine to cure various ailments such as rheumatism, anemia,

arthritis, and gout (Romeo et al., 2007). Phytomedicinal studies have shown

the presence of numerous beneficial compounds, supporting the potential use

of Capparis for therapeutic and culinary purposes (Tlili et al., 2011;

Musallam et al., 2011). These compounds include isothiocyanates,

thiocyanates, sulphides and their oxidative products (Tlili et al., 2011).

Species of caper were found to have a variety of pharmacological properties

such as antimicrobial, antihelmintic, antidiabetic, antiallergic, antisclerosis,

anti-oxidative, anticancer, anti-inflammatory, antihepatotoxic,

Capparis constitutes a conspicuous element of the vegetation in the

Mediterranean and Irano-Turanian regions (Zohary, 1966). Here in Jordan,

members of the genus grow wildly in mountainous, arid, and semi-arid

habitats. Zohary (1966) described four different Capparis species growing

wild in Jordan: C. spinosa L., C. ovata Desf., C. cartilaginea Decne., and C.

decidua (Forssk.) Edgew. A few phytochemical and antimicrobial studies of

Jordanian Capparis species appeared in the literature, and where studies have

3 been conducted, considerable emphasis has been placed on the most abundant

C. spinosa, leaving the remaining species virtually unexplored. C. spinosa is a

well known species for its traditional use in the treatment of back pains in

Jordan. As a comparative research work, we aimed here to examine two

sympatric Capparis species, namely C. spinosa and C. cartilaginea, for

chemical composition of essential oil and screen crude extract fractions from