Essential Oils Profile and Biological Activities of Zingiber spp

Sabrina Bong Mei Mei (38558)

A thesis submitted in partial fulfillment of the

Final Year Project 2 (STF 3015) Course

Supervisor: Prof. Dr. Fasihuddin Badruddin Ahmad

Resource Chemistry

Department of Chemistry

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

2015

II

ACKNOWLEDGEMENT

First and foremost, I would like to express my deepest gratitude to my supervisor Prof Dr

Fasihuddin B Badruddin Ahmad for the continuous support of my final year project study,

for his patience, caring, enthusiasm, and immense knowledge. His guidance helped me in

completing my research and also writing of this thesis. My sincere thanks also goes to my

co-supervisor Prof Dr Zaini Assim for giving me guidance whenever I am in need.

Many thanks to Gloria, a master student in the laboratory, she is a helpful and kind hearted

person who always willing to give her guidance in helping me to complete my task.

I would also like to thank my fellow lab matesfor all the fun discussion we have had

throughout the years.

Lastly, I would like to thank my parents for providing me with the financial support in

collecting my samples as well asgiving me endless support throughout my life.

III

DECLARATION

I hereby declare that no portion of the work referred to in this dissertation has been

submitted in support of an application for another degree or qualification to this university

or any other institution of higher learning.

____________________________________

Sabrina Bong Mei Mei

Resource Chemistry

Department of Chemistry

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

IV

TABLE OF CONTENTS

Title and Front Cover I

Acknowledgement II

Declaration III

Table of Contents IV

List of Abbreviations VI

List of Tables VII

List of Figures VIII

Abstract 1

Introduction 3

Literature Review

2.1 Zingiberaceae

2.2 Zingiber species

2.3 Chemical constituents of Zingiber spp

2.4 Biological activities

2.4.1 Antimicrobial

2.4.2 Antioxidant

2.4.3 Antitumor

2.4.4 Cytotoxicity Test

2.4.5 Anti-inflammatory

2.4.6 Insecticidal Test

Materials and Methods

3.1 Plant Material

3.2 Isolation of Essential Oil

5

5

5

8

13

13

16

17

18

18

19

21

21

22

V

3.2.1 Percentage of Essential Oil

3.3 Analysis of Essential Oil

3.3.1 GC-FID.

3.3.2 GCMS

3.3.3 Quantitative Analysis (Kovat Indices)

3.4 Brine Shrimp Lethality Bioassay

3.4.1 Lethality Concentration determination

3.5 Antitermite Test

Result and Discussion

4.1 Determination the Yield of Essential Oil

4.2 Determination the Chemical Composition of Essential Oil

4.3 Brine Shrimp Lethality Bioassay

4.4 Antitermite Test

Conclusion

References

Appendix

23

24

24

24

25

26

27

28

29

29

33

45

49

52

54

63

VI

LIST OF ABBREVIATIONS

Gas Chromatogrphy-Flame Ionization Detector GC-FID

Gas Chromatography-Mass Spectrometry GC-MS

Helium gas He

Microlitre µL

Miligram mg

Mililitre mL

Celcius ºC

Lethal Concentration 50 LC50

Part per million ppm

VII

LIST OF TABLES

Table 4.1 Percentage and physical properties of essential oils. 29

Table 4.2 Percentage chemical composition of ginger oils of imported and

local Zingiber officinale Roscoe.

33

Table 4.3 Percentage chemical composition of ginger oils (Zingiber sp 1). 37

Table 4.4 Percentage chemical composition of rhizome ginger oils (Zingiber

officinale var. rubra).

39

Table 4.5 Percentage chemical composition of rhizome ginger oils (Zingiber

zerumbet).

41

Table 4.6 Brine shrimp lethality results for the essential oils of rhizome 46

Table 4.7 Antitermite results for the essential oils of rhizome 50

VIII

LIST OF FIGURES

Figure 3.1 Zingiber sp. 1 (wild ginger). 21

Figure 3.2 Set up apparatus for hydrodistillation. 22

Figure 3.3 Set up apparatus for cultivation of brine shrimp. 27

Figure 4.1 Essential oil of local Zingiber officinale (rhizome). 30

Figure 4.2 Essential oil of imported Zingiber officinale (rhizome). 30

Figure 4.3 Chemical structure of major compounds presence in the ginger

essential oil.

44

Figure 4.4 Brine shrimp (Artenia salina) toxicity test of essential oil from

rhizome of local and imported Zingiber officinale Roscoe.

45

Figure 4.5 Brine shrimp (Artenia salina) toxicity test of essential oil from

rhizome of Zingiber officinale var. rubra.

45

Figure 4.6 Brine shrimp (Artenia salina) toxicity test of essential oil from

rhizome of Zingiber zerumbet.

46

Figure 4.7 Antitermite activity of essential oil from rhizome of local and

imported Zingiber officinale Roscoe.

49

Figure 4.8 Antitermite activity of essential oil from rhizome of Zingiber

officinale var. rubra.

49

Figure 4.9 Antitermite activity of essential oil from rhizome of Zingiber

zerumbet

50

1

Essential Oils Profile and Biological Activities of Zingiber spp.

Sabrina Bong Mei Mei

Department of Chemistry

Faculty of Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

The objective of this study was to evaluate and compare the chemical composition of the

essential oil from several Zingiber spp. by hydrodistillation method and later identify the

chemical composition of the essential oil which was separated into three parts namely the

stems, rhizomes and the leaves. Besides, the second objective of this study is to determine

the biological activity of essential oils namely the cytotoxicity and antitermite activity of

the essential oil. For the identification of chemical composition of the Zingiber, gas

chromatography-flame ionization detector (GC-FID) and gas chromatography mass-

spectrometry (GC-MS) were carried out. On the other hand, brine shrimp lethality bioassay

and antitermite test were also carried out in order to determine the biological activities

respectively. Generally, the rhizome part of the oil contain the highest percentage of

essential oil followed by the leaves and the stem. The result obtained from the five

Zingiber species revealed that sesquiterpene and monoterpene compound was the

predominant compound present in the essential oils in general. The cytotoxicity properties

of Zingiber zerumbet (3.00 µL/mL) is the highest followed by Zingiber officinale var.

rubra (LC50=4.92 µL/mL), local Zingiber officinale (LC50=5.54 µL/mL) and imported

Zingiber officinale (LC50=8.03 µL/mL). The antitermite property was found to have

similar trend as the lethality bioassay with Zingiber zerumbet having the lowest LC50 value

of 0.30%, followed by Zingiber officinale var. rubra (LC50=0.50%), local Zingiber

officinale (LC50=0.55%) and imported Zingiber officinale (LC50=0.80%).

Keywords: Zingiber, hydrodistillation, biological activity, GC-FID, GC-MS, essential oil

2

ABSTRAK

Objektif kajian ini adalah untuk menilai dan membandingkan komposisi kimia minyak pati

dari beberapa Zingiber spp. yang diperoleh melalui kaedah pengulingan hidro dan

mengenal pasti komposisi kimia minyak pati yang akan dibahagikan kepada tiga bahagian

iaitu batang, rizom dan daun. Selain itu, objektif kedua kajian ini adalah untuk

menentukan aktiviti biologi minyak pati iaitu ketoksikan dan anti anai-anai ujian minyak

pati. Untuk mengenal pasti komposisi kimia Zingiber, gas kromatografi-api pengesan

pengionan (GC-FID) dan kromatografi gas-spektroskopi jisim (GC-MS) telah dijalankan.

Tambahan lagi, anak udang marin bioesei dan anti anai-anai juga dijalankan untuk

menentukan aktiviti biologi masing-masing. Komposisi kimia minyak pati daripada lima

spesies Zingiber telah dikenal pasti dan aktiviti biologi minyak yang penting telah dinilai.

Secara umumnya , bahagian rizom minyak telah direkod mengandungi peratus yang

tertinggi minyak diikuti oleh daun dan batang. Hasil yang diperolehi daripada lima

spesies Zingiber menunjukkan bahawa sesquiterpene dan monoterpene kompaun adalah

kompaun utama yang terkandung di dalam minyak pati secara keseluruhan. Sifat

sitotoksiti daripada Zingiber zerumbet (3.00 μL / mL ) adalah yang tertinggi diikuti oleh

Zingiber officinale var. rubra (LC50= 4.92 μL / mL ), Zingiber officinale tempatan (LC50 =

5.54 μL / mL ) dan Zingiber officinale import (LC50 = 8.03 μL / mL ). Anti anai-anai

didapati mempunyai trend yang sama seperti marin bioesei dengan Zingiber zerumbet

mempunyai nilai LC50 terendah 0.30 %, diikuti oleh Zingiber officinale var. rubra (LC50 =

0.50 %), Zingiber officinale tempatan (LC50 = 0.55 %) dan yang diimport Zingiber

officinale (LC50= 0.80 %).

Kata kunci: Zingiber, pengulingan hidro, aktiviti biologi, GC-FID, GC-MS, minyak pati

3

Chapter 1

INTRODUCTION

Zingiberaceae is one of the largest families of the order Zingiberales which consist

of 47 genus and almost 1500 species distributed throughout the tropics mainly in Southeast

Asia (Holttum, 1950). Based on the cultivation history of ginger, ginger was originated

from China and later spread to India, Southeast Asia, West Africa and finally to the

Caribbean (Weiss, 1997; McGee, 2004). Zingiber (ginger), a member of the Zingiberaceae

family is one of the most consumed spices in many Asian countries in their daily diet

(Demin and Yingying, 2010).

Today, ginger widely used in the food, beverage, and confectionary industries to

produce pickles, ginger beer, chutney, ginger and wine (Wang et al., 2011). Ginger is also

used in the production of ginger candy or known as Injimurappa in Tamil in South India

(Souza et al. 2005). In Malaysia, Zingiber cassumunar is used to treat ashtma and

rheumatism disease (Amatayakul et al., 1979). According to Vendruscolo et al. (2006),

Zingiber officinale Roscoe has been used extensively in folk medicine to treat pain,

inflammation, arthritis, urinary infections, and gastrointestinal disorders.

The essential oil of ginger is volatile at room temperature. This makes them

sometimes to be known as volatile oils or ethereal oils. There are many different ways that

has been used to extract the essential oil of the plant namely the water and steam

distillation (Sellar, 2011). Besides, ginger has been studied on its chemical composition

using both GC-MS and GC-FID applications (Sultan et al., 2005; Singh et al., 2008). The

essential oil of ginger mainly composed of monoterpenes and sesquiterpenes. Aldehydes

and alcohols are also present. The essential oils' constituents such as (-)-linalool was found

to be responsible to antagonize different pain responses stimulated by chemical stimulus

4

such as acetic-induced, by a thermal stimulus or by a tissue injury produced by formalin

injection (Vendruscolo et al., 2006).

Moreover, ginger was found to be rich in bioactive compounds and many

researches have been done in order to study the beneficial properties of ginger and its

extracts (Grzannaet al., 2005). Essential oils of ginger are of interest because of its richness

in various functional compounds mostly terpene, monoterpene and sesquiterpenes, which

give the oil its biological activity (Daferera et al., 2002). There are many studies has been

done on the biological activity of ginger such as the antioxidant test using free radical

scavenging properties and also study on antimicrobial activities of the ginger compounds

(Singh et al., 2008; Sasidharan et al., 2010; Takahashi et al., 2011; Bellik et al., 2013).

However, comparative studies of essential oil of ginger from different species have

not been performed in detail during the past. Plus, there is also very limited of a more

specific study on biological activity of essential oil of Zingiber species especially the

toxicity test using brine shrimp method and also study of antioxidant activity using 2,2’-

diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method.

Therefore, the aim of this study is to determine the chemical components of

essential oil of ginger from five selected Zingiber species using GC-FID and GC-MS and

to evaluate the biological activities of its essential oil in order to apply for future

application. The more specific objectives are:

a. To extract the essential oil of several Zingiber spp. by hydrodistillation method and

identify the chemical composition of the essential oil.

b. To determine the biological activities of Zingiber spp. by performing brine shrimp

lethality bioassay and antitermite test.

5

Chapter 2

LITERATURE REVIEW

2.1 Zingiberaceae

Zingiberaceae family consist of almost 47 genus that are mainly distributed around

Southeast Asia (Holttum, 1950). There are two subfamilies in the Zingiberarceae family

which are Costideae and Zingiberoideae (Ravindran and Nirmala, 2004). Besides,

Zingiberaceae members are mostly of terrestrial and aromatic herb. The members of

Zingiberaceae are often annual or perennial herbaceous plant as they are made up of

fleshly or fibrous root (Ravindran et al., 2005). Moreover, they often have a creeping

rhizome (Lock, 1985). They are able to survive in all sorts of habitats such as limestone

rocks and lowlands. However, majority of gingers are terrestrial where they tend to grow

in humid, damp and shady area with sufficient sunlight (Larsen et al., 1999).

Besides, the members of Zingiberaceae family are usually perennial herb plant with

stout rhizomes (Dev, 2006). According to Wu (2000), Zingiberaceae are herbs mostly

acquire tuberous or non-tuberous rhizomes as most of them bear tuber-bearing roots. Their

stems are usually short whereby as their pseudostems are made of leaf sheaths which often

replaced the stems. Their leaves are mostly distichous and simple. Moreover, leaves that

grow towards the base of plant are often bladeless. Ligule is usually present in the plant

member of Zingiberaceae while petiole is present in some but not all. Lastly, the leaf

blade is either suborbicular or lanceolate to narrowly strap-shaped (Wu, 2000).

2.2 Zingiber species

The genus Zingiber of the family Zingiberaceae consists of around 85 species

which are mainly distributed throughout the tropics mainly in East Asia and tropical

Australia (Sabulal et al., 2006). The rhizomes are usually branched, tuberous and aromatic.

6

Leaves are distichous whereby it often held in plane or parallel to the rhizome. The

pseudostems are often erect and leafy and petiole is swollen and cushion like (Wu, 2000).

According to Larsen et al. (1999), the bracts of Zingiber will change from green into red

during fruiting stage. Moreover, the flowers are often ephemeral where they only live for a

few hours (Larsen et al., 1999).

The most well-known Zingiber species, Zingiber officinale is commonly used as

spice and also medicinal purposes in the past thousand years (Ali et al., 2008). It was found

that the yield of essential oil of the rhizome of Zingiber officinale was around 2-3%

which includes both sesquiterpenes and monoterpenes. There were also other

constituents found such as lipophilic pungent constituents, gingerol, starch (50%), fats,

mineral (5%), sugar, vitamins, proteins and amino acids (Blaschek et al., 1998). It was

proven that1,8-cineole is a major compound found from Zingiber officinale that exhibit

antimycotic activity (Sharma et al., 2013). Besides, other studies reported that Zingiber

officinale is a good source of antioxidant which it has the potential to be made into an

antioxidant supplement (Shirin Adel and Jamuna, 2010). In the present study, reports

shown that the phenolic compounds in the essential oil of Zingiber officinale contain

antimicrobial and antioxidant properties (Bellik, 2014). Rhizome of Zingiber officinale var.

rubra which is known as halia bara is used in folk medicine (jamu) to treat various illness

such as postpartum medicine, relieving rheumatic pain, stomach discomfort, and even

tumours (Ibrahim et al., 2008).

Zingiber cassumunar Roxb. which is commonly known as ‘plai’ is widely used in

Asian countries such as Thailand and Indonesia to treat muscular pain, asthma, respiratory

and also act as mosquito repellant (Wanauppathamkul, 2003; Pithayanukul et al., 2007).

Terpinen-4-ol and sabienene were the major component found in the ‘plai’ oil that is

responsible for the antimicrobial properties (Giwanon et al., 2000). Moreover, (E)-1(3,4-

7

dimethoxyphenyl) butadiene which is the constituent isolated from the hexane extract of

the Zingiber cassumunar showed anti-inflammatory properties (Jeenapongsa et al., 2003).

Zingiber spectabile consist of Terpinen-4-ol, α-terpineol, and β-pineneas the major

constituents (Hasnah et al., 2001).The flavonoids and curcuminoids found in the Zingiber

spectabile was reported to possessed antioxidant properties which make them a potential

source to act as preservatives in the food industry (Sivasothy et al., 2011). Moreover,

Zingiber spectabile is normally grown as an ornamental plant (Wolff et al., 1999).

Zingiber zerumbet is a wild ginger that is widely cultivated for medicinal purposes

in the village. It is also known as ‘lempoyang’ in Malaysia (Saadiah and Halijah, 1995).

Besides, Zingiber zerumbet is traditionally used to treat constipation, fever and also

function as pain reliever in Southeast Asia (Perry, 1980). In India, the essential oil of

Zingiber zerumbet is widely used as perfume in soaps and toiletries (Wolff et al., 1999).

According to Somchit et al. (2005), it was prove that the ethanol extract of rhizomes of

Zingiber zerumbet exhibits both analgesic and anti-pyretic properties. The rhizome of

Zingiber zerumbet also showed anti-inflammatory activity with zerumbone as the

dominant chemical constituent of the rhizome oil (Chien et al., 2008).

Lastly, Zingiber ottensii is a unique ginger which is quite different from the other

species as it has an ink-coloured rhizome (Sirat, 1994). In Thailand, it is traditionally used

for medicinal purposes as the rhizomes are extensively used externally for relieving

bruises and also inflammations. In Malaysia, the essential oil of the rhizomes of Zingiber

ottensii has been determined using GC and GC-MS. There was twenty-six components

identified and zerumbone (25.6%) was the dominant constituent (Sirat and Nordin, 1994).

On the other hand, Thubthimthed et al. (2005) reported that the presence of twenty-eight

components in the essential oil of Zingiber ottensii with zerumbone (40.1%) as the major

8

component. It was also proven that the essential oil exhibits moderate toxicity in the brine

shrimp lethality test (Thubthimthed et al., 2005).

2.3 Chemical constituents of Zingiber spp.

According to Petersen and Amstutz (2008), secondary metabolite is defined as a

group of natural compounds that does not directly involved in the growth, reproduction

and development of an organism. Besides, it is commonly act as a biological warfare agent

of an organism as it plays a role to fight against predator, disease and parasites (Petersen

and Amstutz, 2008).

Generally, the essential oil of plant consists of 30% to 65% of monoterpenoids and

sesquiterpenoids (Dev, 2006). Monoterpenes is a compound that is made up of two

numbers of isoprene units which has the molecular formula of C10H16 as it contains 10

carbons. Sesquiterpenes on the other hand is a compound that is made up of 15 Carbon and

has a high boiling point which is higher than 200 °C (Fasihuddin and Hasmah, 1993).

Based on the study by Sivasothy et al. (2011), the leaf oil of Zingiber officinale var.

Rubrum (halia bara) was rich in both sesquiterpenoids and monoterpenoids. However,

monoterpenoids was more abundant in the rhizome which comprised of mainly camphene

1.

Sharma et al. (2013) had done a study on the phytochemistry of ginger (Zingiber

officinale) oil from the rhizome. Camphene 1 was found to be the main compound of

monoterpenene and zingiberene 2 was the main compound of sesquiterpene. The study

also proved that camphene 1 and zingiberene 2 had shown fungicidal nature (Sharma et al.,

2013). Moreover, the sesquiterpene hydrocarbons namely zingiberene 2 from ginger was

found to possessed antiviral, antifever and antigestation properties (Millar and Notprod,

1998).

9

Kamaliroosta et al. (2013) also studied on the dried rhizomes of Zingiber officinale.

The essential oil consists of a total seventeen compounds. Zingiberene 2 was the most

abundant compound. On the other hand, camphene 1 was present but slightly less in

concentration than β-Phellandrene 3 which both belongs to the monoterpene hydrocarbons.

The findings are in agreement with the work of Sharma et al. (2013) as they both studied

on the same species. However, β-Phellandrene 3 is predominant for monoterpene

hydrocarbons reported by Kamaliroosta et al. (2013) and camphene 1 was dominant

monoterpene hydrocarbons in the report of Sharma et al. (2013). This may be due to the

condition of ginger that has been used whereby Kamaliroosta et al. (2013) used dried

rhizome while Kamaliroosta et al. (2013) used fresh ginger rhizome.

Besides that, Sasidharan and Nirmala Menon (2010) had done a study to compare

the chemical composition of fresh and dry ginger. It was found that zingiberene 2 was the

major chemical compound of sesquitepene identified in the fresh and dry ginger oil.

However, the monoterpene compound was lower in dry ginger oil when compared to the

fresh ginger oil whereby their main monoterpene hydrocarbon was champene 1. Moreover,

oxygenated compound is found to be more abundant in fresh ginger oil (Sasidharan and

Nirmala Menon, 2010).

CH2

CH3CH3

CH3

CH3

CH3

CH3

CH3

CH2

CH3 CH3

1 2 3

10

There are also studies done on the chemical composition of crude extracts of

Zingiber officinale which was extracted using Soxhlet extraction. The chemical

composition might differ from the essential oil of other Zingiber as the method of

extraction differs and the examined product is different. Hasan et al. (2012) studied the

chemical composition of crude extract isolated from Zingiber officinale which identified

zingiberene 2 from sesquiterpene hydrocarbons, α-curcumene 4 from monoterpene

hydrocarbons and gingerol 5 from phenolic hydrocarbons. From the studies, it can be

concluded that phenolic hydrocarbons can be found from the crude extracts but not in the

essential oil of the Zingiber as shown in the previous study (Sivasothy et al., 2011).

On the other hand, Bua-in and Paisooksantivatana (2009) studied the essential oil

of Zingiber montanum that was collected from various part of Thailand. It was found that

the main constituent in the essential oil of the rhizome of Zingiber montanum were

sabinene 6, terpinen-4-ol 7, and DMPBD ((E)-1(3,4-dimethylphenyl)butadiene) 8. The

results also proved that there were no significant differences in chemical components of

the essential oil between various locations of Thailand (Bua-in and Paisooksantivatana,

2009). Terpinen-4-ol 7 has been found to be exhibits antibacterial and anti-inflammatory

activities (Poonsukcharoen, 2004).

In addition, it was also reported that (E)-1-(3,4-dimethoxyphenyl) butadiene,

DMPBD 8 from Zingiber cassumunar exhibits anti-inflammatory activity by inhibiting the

cyclooxygenase (CO) and lipoxygenase (LO) pathways (Jeenapongsa et al. 2003) . Besides,

another nonpolar substance identified was phenylbutenoids 9 which was found in the

rhizome extracts of Zingiber montanum was reported to possessed anti-inflammatory and

also insecticidal activity (Phonsena et al. 2006).

11

CH3

CH3

CH3

CH3

4

OH

O

CH3

CH3

O

OH

5

CH2

H

CH3

CH3

CH3

OH

CH3

CH3

6 7

12

OCH3

O

CH3

CH2

OCH3

OCH3

OCH3

OCH3

8 9

Meanwhile, Maizura et al. (2010) reported that the total phenolic content of ginger

extract (Zingiber officinale) and proved that the phenolic compounds was the major

contributors to the antioxidant activity observed. Singh et al. (2008) also reported that there

is a significant antimicrobial activity shown by the essential oils of Zingiber officinale

which was caused by phenolic compounds present. The phenolic compounds from the

ginger essential oil and oleoresin such as zingerone 10, eugenol 11 and gingerols 5 had

shown to exhibits both antimicrobial and antioxidant properties (Pawar et al. 2011; Ding et

al. 2012).

Thus, it can be seen that the essential oil of Zingiber is dominated by

sesquiterpenes hydrocarbon, monoterpenes hydrocarbon, phenolic compounds and other

oxygenated sesquiterpenes and monoterpenes.

13

OH

CH3

O

OCH3

O

CH3

OH

CH2

10 11

2.4 Biological activities

2.4.1 Antimicrobial

In Nigeria, a research was done by Malu et al. (2008) on the antibacterial activity of

the crude extract of Zingiber officinale. It was shown that n-hexane, ethyl acetate and

Soxhlet extract of ginger rhizome showed inhibition in bacterial growth whereby Soxhlet

extract was found to be the most active in terms of antibacterial activity among all.

However, there was no inhibition took place for the water extract of the ginger rhizome.

The antibacterial property of the extract was found to be attributed by the presence of

zingiberene as it is the main constituent (Malu et al., 2008). Moreover, the different in

antibacterial potency of the extracts may be due to the nature of solvents used for the

ginger extraction (Sigh et al., 2008).

Based on a study done by Sivasothy et al (2011), the leaf and rhizome of Zingiber

officinale var. rubrum Theilade was found to possessed moderate antibacterial activity

against every bacterial strains tested using micro-dilution assay. The Gram-positive

bacteria are more sensitive toward the essential oil of leaf and rhizome of the Zingiber

14

officinale var. rubrum Theilade. This might be due to the variation of chemical constituents

presence in different part of the ginger where by ß-caryophyllene (31.7%) and camphene

(14.5%) was found to be the most abundant components found in the leaf and rhizome oil

respectively. Report also indicates that Gram-negative bacteria are more resistance to

essential oil (Sivasothy et al., 2011).

Similarly, Hasan et al. (2012) found that crude extracts isolated from Zingiber

officinale using hexane and methanol solvents affects more significant against Gram-

positive bacteria compared to Gram-negative from the agar-well diffusion assay. The

active ingredients found in both extracts was gingerol and shogaol which might be

responsible to the antimicrobial activity tested (Hasan et al., 2012). From the result, the

high resistance of Gram-negative bacteria is due to the highly hydrophilic surfaces whereas

the membrane of Gram-positive bacteria has negative charge that alters their resistance

towards the extracts (Michielin et al., 2009).

Bellik (2014) recently studied on the antimicrobial activity of the essential oil and

oleoresin of Zingiber officinale in Algeria. It was revealed that Staphylococcus aureus was

the most affected microorganism by the essential oil Escherichia coli was the least affected

strain. However, Escherichia coli exerted the most effect from oleoresin (Bellik, 2014).

Therefore, the finding is in agreement from the previous work done by (Sivasothy et al.,

2011) whereby E. coli is most resistance toward Zingiber essential oil.

Besides, Emmanueal et al. (2013) done antimicrobial test on Zingiber officinale

using agar disc diffusion method. Both the essential oil and extracts of Zingiber officinale

were used to test the antimicrobial activity towards both fungi and bacteria strains. Study

shows that essential oil and ethyl acetate extract inhibited all the strains tested. While,

15

aqueous extract, ethanol extract, hexane extract and methanol extract did not inhibit a

significant antimicrobial activity (Emmanueal et al., 2013).

Sasidharan and Menon (2010) studied on the antimicrobial activity of the rhizome

of Zingiber officinale by comparing both dry and fresh ginger oil. The dry ginger oil

influenced most in the Pseudomonas aeruginosa as they contain more monoterpene

hydrocarbon which may be more active toward Pseudomonas aeruginosa. Meanwhile,

Aspergillus niger was highly affected by the antimicrobial activity of the fresh ginger oil

that contains more oxygenated compound (Sasidharan and Menon, 2010). This study

shows that the wet and dry ginger oil have different inhibition towards certain bacteria.

This is because the chemical variationof the wet and dry Zingiber is different which

resulting them to react differently.

Chairgulprasert et al. (2005) reported the antibacterial activity of the essential oil

and crude extracts from dried rhizomes of Zingiber wrayi var. halabala with petroleum

ether and dichloromethane. This study showed that the concentration of both petroleum

ether and dichloromethane extracts is directly proportional with the antibacterial activity

against Bacillus substilis, Escherichia coli, Staphylococcus aureus and Sarcina sp.

Meanwhile, trans-anethole 17 (96.8%) would be the volatile component that contributed to

the antibacterial activity in this case as the concentration was the highest (Chairgulprasert

et al., 2005).

According to Sharma et al. (2013), the essential oil of Zingiber officinale was

active in antifungal activities towards all tested fungi. From the In vivo experiment, the

ginger oil performed a fungistatic nature but it was found to be fungicidal in higher

concentration. The fungicidal nature of the essential oil was reported to cause by the

monoterpenes namely the camphene and zingiberene (Sharma et al., 2013).

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2.4.2 Antioxidant

El-Ghorab et al. (2010) studied on the antioxidant activity of the ginger using both

fresh and dry ginger. The result showed that antioxidant activity for both fresh and dry

ginger essential oil in DPPH radical scavenging method. The FRAP antioxidant test also

found comparative results. Camphene would be the main contributor to the antioxidant

property of the ginger. Thus, the results suggest ginger to be used as a potential antioxidant

source in the food industry (El-Ghorab et al., 2010).

Jeena et al. (2013) found that ginger oil could inhibit the oxygen radicals in lipid

peroxidation, scavenging of superoxide and hydroxyl radical in vitro. However they

showed moderate scavenging activity against the stable free radicals namely DPPH and

ABTS. This may be due to the mixture of different functional groups of compounds with

various polarity which resulting on different antioxidant activity (Jeena et al., 2013).

Moreover, many reports found that phenolic groups also play an important role in

antioxidant activity (Ruberto and Baratta, 2000).

Shirin Adel and Jamuna Prakash (2010) studied the antioxidant properties of the

ginger (Zingiber officinale) root using free radical scavenging activity by DPPH (2,2-

diphenyl-1-picrylhydrazyl), reducing power and total antioxidant activity by

phosphomolybdenum method. It was found that antioxidant components such as

flavonoids were higher in hot water extracts at 100°C compared to water extracts (30°C).

The study also proved that the antioxidant component of ginger is more effective in

alcoholic media. (Shirin Adel and Jamuna Prakash, 2010). From this study, it is proven

that antioxidant activity of the ginger will be affected directly by the media used and also

the temperature applied to the water extracts.