CHAPTER 1

INTRODUCTION

1.1 Background of Study

Malaysia is one of the countries that is rich with tropical retreats; hiding
within it is some of the greatest centres of biodiversity with a very high potential
value for the herbal industry. More than 35,000 plant species have been reported to
be utilised in various human cultures around the world for medical purposes [1]. As
ancient as human history, plants have been extensively used in medical practices and
have proved to be the major remedy in traditional system of medicine due to their
biomedical benefits as well as place in cultural beliefs in many parts of world. Over
the time and with the beginning of societies, human learned to recognize and
categorize plant materials suited for use in meeting the necessities of life. As the
Malaysia herbal medicine market experiences an extraordinary growth, the research
approaches taken have recently included activities to discover and develop the
natural products utilized from the tropical rainforest in this land.

The immense use of herbal medicine due to the great interest shown by public
and the attentiveness of researchers has placed a substantial emphasis and drive in
medicinal plant research. Plainly, such research has been roused by scientific
curiosity in the pharmacological and biological activities of the substances and
mechanisms involved in the natural products. In the broadest sense, natural product
can also be prepared by total synthesis. As the Malaysian herbal medicine has an
extraordinary utilization, researchers have ventured out to develop its rich plant
genetic resources to their full potential as pharmaceutical agents. This included
plants of the genus Zingiber (Family Zingiberaceae) which is widely cultivated in

1
sub-tropical regions around the world such as Zingiber zerumbet known as bitter
ginger and Curcuma Longa (turmeric) [2].

1.2 Zingiberaceae Family

Zingiberaceae is the ginger gamily of angiosperm made up of approximately

52 genera with species added up to 1600 kind [3]. These aromatic perennial herbs
grow in moist areas by its creeping horizontal or tuberous rhizomes of the tropics and
subtropics, including Malaysia reainforest. According to Larsen [4] there are 18
genera of gingers recorded in Malaysia such as Alpinia, Amomum, Boesenbergia,
Burbidgea, Curcuma, Elettaria, Elettariopsis, Etlingera, Geocharis, Geostachys,
Globba, Haplochorema, Hedychium, Hornstedtia, Plagiostachys, Scaphochlamys,
Tamijia, and Zingiber. Gingers often growing in shady area but some of the native
species are able to tolerate the full exposure of sun.

Several species of this family whose bioactive compounds confer its

characteristics flavour, aroma and biological activities properties such as Zingiber,
has made the plant to not only be used as spices, but also as traditional remedies due
to its medicinal properties. Zingiber Zerumbet, traditionally known as bitter ginger,
has a wide spectrum of uses. The leaves and leaf stalks, which are also aromatic,
were served as seasoning to enhance the flavour of foods. Traditionally, the aromatic
underground rhizomes were sliced, dried, and pounded to a powder to treat toothache
while in the form of tea, it is served to treat stomach ache [5].

1.3 Genus Zingiber

The genus Zingiber, belonging to the family Zingiberaceae, is a perennial

plant comprises about 85 species of herbs found abundant mainly in Asia [6]. The
name “Zingiber” is derived from a Sanskrit word which referring to bull's horn.

2
Although different members of this genus are found similar in terms of its
morphology, they differ widely in their pharmacological and therapeutic activities [7].
This genus is believed to be native to India and the Malaysian Peninsula, and it has
been cultivated for so long in so many places throughout Southeast Asia, the Pacific,
and Oceania.

Among the large number of Zingiber species found in Malaysia, the eminent
spice comes from the rhizomes of the plant Zingiber zerumbet with numerous
therapeutic effects have been widely reported in literature and will be widely
cultivated in the topic. Numbers of Zingiber species with its biological activities
distributed within them are listed in Table 1.1

Table 1.1 Traditional medicinal uses of some Zingiber species

Species Functions
Zingiber 1. Boiled and used as an aromatic herbal bath for ladies in
montanum confinement [8].
Zingiber 1. To relief flatulence/stomachache/colic [8].
officinale 2. Treatment for aching joints (e.g., knee joints) and sprains
[8].
Zingiber 1. Eaten raw for treatment for hypertension [8].
zerumbet 2. Used for diseases and conditions such as those of aches,
pains and sprains [8].

Zingiberaceae are important as an ornamental plants, spices, or medicinal

plants. The plants of Zingiber have been widely reported in literature that it is
consumed worldwide as a spice and flavouring agent and has been extensively used
with remarkable therapeutic effects which attributed as traditional folk medicine for
treatment of diarrhoea, allergies, inflammation, fever and bacterial infections [9].
The examples of gingers that used as ornamental plant are the Shell gingers (Alpinia),
Siam or Summer tulip (Curcuma alismatifolia), Ginger lily (Hedychium),
Kaempferia and Ginger (Zingiber). The spices ginger are Ginger (Zingiber),
Galangal or Thai ginger (Alpinia galanga), Myoga (Zingiber mioga), Turmeric

3
(Curcuma). Zingiber officinale or locally known as Halia has been used for many
years as spices and in traditional forms of medicine to treat diarrhoea [4].

Complex mixture of aromatic compounds with a predominance of flavonoid

present in Zingiber species have been reported as having anti-inflammatory [10],
anti-tumoral [11], analgesic [12], anti-oxidant and anti-microbial properties [13]. The
presence of essential oil in some genera of gingers is used in the perfume industry for
examples Alpinia and Hedychium [14].

1.4 Problem Statement

From the former research, Zingiber zerumbet has been reported to contain
wide variety of secondary metabolites such as tannins terpernoids, alkaloids,
flavonoids; that dictates the therapeutic potency of the plants. However, numerous
studies had shown that different parts of plants give different chemical constituents
with different pharmacological and biological activity. Therefore, in this study, the
knowledge on active chemicals constituents of plants and the scientific investigation
to confirm their medicinal values are focused on. The optimization of extraction of
chemical constituents from Z. zerumbet rhizome using cold maceration by using
solvents of varying polarity was evaluated. The yield obtained can be used for
development and usage of terpernoid in Z. zerumbet rhizome in the future by
providing a scientific quantification using HPLC and test of its depigmentation
activities

4
1.5 Significance of Study

Depigmentation potential of plants has received a great deal of thoughts due

to ubiquitous melanin production which has been identified as a major causative
factor in the arisen of several life threatening ailments. Overproduction of melanin
and accumulation thereof are suggested to cause various dermatological
complications, including freckles, spots, and other hyperpigmentation-related
disorders Since the tyrosinase inhibition properties of a species could be stem from
phenolic compounds through inhibition of tyrosinase and DOPA oxidation and
suggested to be related with the downregulation of microphthalmia-associated
transcription factor, tyrosinase, tyrosinase-related protein-1, and tyrosinase-related
protein-2, verified with mRNA and protein expression levels, studies of major
chemical constituents isolated from naturally occurring phytochemicals of dried
rhizome of Z. zerumbet will be looked into. Anti-melanogenic derived from natural
resource received a great deal of attention due to their general safety image,
multifunctional properties and can act as reducing agents, free radical terminators,
metal chelators and singlet oxygen quenchers

1.6 Objectives of Study

Owing to the great interest on secondary metabolites contained in the rhizome

of Zingiber zerumbet, the overall purposes of the study are:
1. To extract the Zingiber zerumbet rhizome by using solvent extraction
method with number of solvents with incresing polarity (cold maceration).
2. To fractionate the crude plant extracted by using vacuum liquid
chromatography.
3. To purify and elucidate the chemical constituents of Zingiber zerumbet
rhizome by TLC and column chromatography
4. To characterize the purified compounds using NMR spectrometry and
identify the qualitative chemical shifts markers.

5
 5. To quantify the chemical constituents present in Zingiber zerumbet by
HPLC.
6. To evaluate the tyrosinase inhibition activity of the crude extract and pure
compounds of Zingiber zerumbet.

1.7 Scope of Study

The overall purpose of the study is to extract various chemical constituents

with different polarity of solvents for rhizome of Zingiber zerumbet using cold
maceration extraction method. Prior to extraction, silica gel column chromatography
and vacuum liquid chromatography have been used for separation of bioactive
molecules with some analytical tools. Meanwhile, fractions of compounds isolated
by column chromatography were analysed and monitored by thin-layer
chromatography. Additional tests involve the viewing the plate under the UV light
for confirmation of purity and identity of isolated compounds. NMR spectroscopy is
used for characterization of isolated compounds by giving a clear picture of what the
positions of these nuclei are in the molecule and demonstrate which atoms are
present in neighbouring groups. Besides that, Fourier Transform Infrared
Spectroscopy (FTIR), a high-resolution analytical tool is used to identify the
chemical constituents and elucidate the structural compounds. Of known
concentration, High Performance Liquid Chromatography (HPLC) is used to
quantify the individual components that present in the sample. After the active
fraction from plant material is obtained, phytochemical screening can be performed
with the appropriate tests and anti-melanogenic potential of the major compound
extracted evaluated by tyrosinase inhibition assays.

6
 CHAPTER 2

LITERATURE REVIEW

2.1 Description of Zingiber genus

Although Genus of Zingiber plants prefer humid and tropical settings, the
plants are widely cultivated in many countries including some regions that are
seasonably dry. The aromatic, perennial herbs have the characteristics of fleshy
rhizomes either fibrous or growing horizontal which may nurture to 6 metres (20 feet)
in height. Previous phylogenetic analysis has supported that the tribe Zingibereae is
monophyletic [15], this genus is easily recognised among Zingiberaceae either by the
flower character which has the horn-shaped anther crest embracing the upper part of
the style or by the vegetative structure widely known as a pulvinus which has the
existence of a swollen part of the petiole.

Among this genus, many phytochemists were interested in Z. zerumbet as it

has high medical values. Zingiber that is confined to the tropics of the Pacific Island,
Malaysia and Asia is represented by more than 100 species with main distribution is
in Asia [16]. Zingiber zerumbet (L.)Smith is a perennial herb locally known as
“Lempoyang” to the Malay [17] and by various other names, for example, “Yaiimu”
(India) [18], “Hong qiu jiang” (China) [19] and “Jangli adha” (Bangladesh) [20]. The
rhizome is horizontal, hard, biennial, white or yellowish to brown, with a strong
aromatic ginger-like taste, but with some bitterness. Stem is leafy, thick, to 60 cm
high. Leaves are pointed, narrowly or linear-lanceolate, approximately 20 cm to
30cm long and clasping the stem by long sheaths [21]. Commonly known as
shampoo ginger in English, it is named after the aromatic and slimy clear liquid that
present in the matured flowers enclosed within the cone-shaped flower heads with
overlapping scales [22].

7
 Z. zerumbet has cultivated for thousands of years as a spice to enhance the
savour, colour and odour of food. In addition to boosting flavour, bitter ginger is also
known for its remarkable therapeutic effects to treat various ailments by the local
communities. The species is considered as medicinal plants as it has numerous
remedial properties in treating different maladies. Traditionally, the aromatic
underground rhizomes were sliced, dried, and pounded to a powder and it is served
in the form of tea to treat stomach ache [23]. While the cooked and softened rhizome
is used to treat caries, that is an infectiousness disease of microbial origin by pressing
it and left for as how long needed into the hollow [24]. In folk medicine and Chinese
traditional medicine, the leaves is used as a therapy to alleviate joint pain by
increasing blood circulation through the heat and healing properties brought to the
affected area. In addition, the creamy fluid presence in the matured inflorescence can
be squeezed out and used as shampoo and conditioner as it is rich in surfactants [25].

The widespread familiarity with the traditional use of medicinal plants

donates to the spread of its knowledge and serves as a foundation for scientific
research of such pharmacological and biological activities. Extensive studies have
presented that regular consumption of the rhizomes also displays anti-hyperlipidemic
effect which tone down intestinal cholesterol absorption, making it useful for treating
heart diseases [26]. Its effectiveness in a broad range of biological activities includes
anti-inflammatory [27], anti-pyretic, antioxidant [28] and other medicinal properties.

Z. zerumbet has aroused the interest of researchers due to its various

applications. Populations around the world use medicinal plants to treat numerous
diseases and ethno pharmacological information serves as a starting point for
developing new drugs. Due to its numerous health benefits, this ginger deserves
special attention and greater diffusion of its culture. Studies of Z. zerumbet have
shown the immense potential of this medicinal plant in the treatment of various
diseases. The analysis of the literature presented in this study has great relevance
because several recent studies have demonstrated the pharmacological activities of Z.
zerumbet. The outcome of these studies provides a view of the current state of
research on bioactive compounds and biological activities of Z. zerumbet and arouses

8
the interest to conduct further studies on tyrosinase enzyme and to target the benefits
of its use as a medicinal plant and to determine whether it can be a source of
molecules that can serve as a model for the discovery new drugs.

2.2 Phytochemicals of Zingiber Species

Zingiber zerumbet or locally known as ‘Lempoyang’ that belongs to

Zingiberaceae family has caught increasing attention from researchers and widely
cultivated throughout the tropical and subtropical areas due to its potential active
ingredient that can be beneficial to human healthcare. Z. zerumbet plant is reported to
contain sesquiterpenoids, flavonoids, aromatic compounds, vanillin, kaempferol
derivatives and other polyphenolic compounds. Polyphenolic compounds are
reported to have multiple biological effects including antioxidant activity. These are
aromatic benzene rings with substituted hydroxyl groups and are secondary
metabolites, ubiquitous to the plant kingdom [29]. Phytochemical investigations on
this plant revealed the isolation of several sesquiterpenes, flavonoids and aromatic
compounds while Zerumbone has been identified as the most active ingredient as it
accounts for the greatest percentage of total substance in Z. zerumbet extract [30]. Z.
zerumbet is known to have medicinal applications, it was used as medicine for
sprains, indigestion and other ailments. In traditional use, the root was ground with a
stone mortar and pestle, and the pulp was placed in a cloth and loosely bound around
the injured area [31]. In Hawaii and Polynesia, it is used against tooth and stomach
ache. To ease the stomach ache, the ground and strained root material were mixed
with water and used as a drink. For a toothache or a cavity, the cooked and softened
root was pressed into the hollow and left for as long as needed [32].

The genus Zingiber is widely used in the world for its medicinal and
biological properties, these properties have been attributed to the chemical
components of Z. zerumbet, mainly consisting in monoterpene and sesquiterpene

9
hydrocarbons. Z. zerumbet plant is reported to contain sesquiterpenoids, flavonoids,
aromatic compounds, vanillin, kaempferol derivatives and other polyphenolic
compounds. Polyphenolic compounds are reported to have multiple biological effects
including antioxidant activity. These are aromatic benzene rings with substituted
hydroxyl groups and are secondary metabolites, ubiquitous to the plant kingdom [33].
The extracts and isolated metabolites of Z. zerumbet have exhibited the following
properties: anti-inflammatory, antioxidant, antidiabetic, anticancer, antimicrobial,
analgesic and antiviral. The previous phytochemical investigations work done on this
plant revealed the isolation of several sesquiterpenes, flavonoids and aromatic
compounds with the sesquiterpenes were the major components, followed by
monoterpenes [34,35].

2.2.1 Monoterpenoids Isolated from Zingiber Species

Monoterpenoids are C10 compounds have been known for several centuries as
components of the fragrant oils obtained from leaves, flowers and fruits produced
from geranyl diphosphate exist in the form of acyclic, monocyclic, or bicyclic
structures depending on the terpene synthase as components [36]. This compound
contributes to the savour and aroma of herbal from which they are isolated.
Structurally, monoterpene may be linear or cyclic that consists of two isoprene units
and has the molecular formula of C10H16. Monoterpenoids is a modified monoterpene
containing oxygen functionality or missing a methyl group.

The chemical compositions of species that is native to Thailand, Zingiber

cassumunar (Roxb.) shown that major compounds were sabinene (1), γ-terpinene (2),
and terpinen-4-ol (3). Based on the study done Boonyanugomol [37], Zingiber
montanum was found to have such compounds as found in Z. cassumunar with
differences by absence of compound (2). Compounds (1), (2) and (3) were also
reported by [38] to present in Coral ginger (Zingiber corallinum), a herbal remedy in
traditional Chinese medicine.

10
 (1) (2) (3)

According to Sulaiman [39], the widely distributed monoterpene that were

identified within the rhizome of Z. zerumbet are classified into camphene (4)
followed by camphor (5) and 1,8-cineole (6), confirming the aforementioned study
by Bhuiyan [20]. Camphene is a categorised under the pinane-type monoterpenoids
which is a bicyclic monoterpene that found to be rich in other Zingiber species
including Z. officinale [39], Z. rubens and Z. moran [40]. In a study, it was shown to
reduce triglycerides and plasma cholesterol in hyperlipidemia rats independent of the
reductase activity of HMGA-CoA. However, among all the chemical constituents
discovered, only a few monoterpenoids are widely distributed within the Zingiber
genus.

(4) (5) (6)

2.2.2 Sesquiterpenoids Isolated from Zingiber Species

Plants of the genus Zingiber (Family Zingiberaceae) mainly consisting of

sesquiterpene hydrocarbons such as α-zingiberene (7), ar-curcumene (8), β-

11
bisabolene (9) and β-sesquiphellandrene (10). In addition, gingerols (11) have been
identified as the major active components in the fresh rhizome, whereas shogaols
(12), dehydrated gingerol derivatives, are the predominant pungent constituents in
dried rhizome. The most abundant compounds is (7) which responsible for the
distinctive flavour and aroma of Zingiber species, geranial, (8), (9), (10) and neral
with (11) and (12) as pungent constituents found in lower quantity.

(7) (8)

(9) (10)

(11) (12)

Zingiber nimmonii is an endemic species with the major components of the

rhizome were different from the rhizome obtained from other species. The major
constituents were said to be β-caryophyllene (13), α-humulene (14) and α-cadinol
(15) [35]. These results are also in accordance with Sabulal [6] who showed that Z.
nimmonii is a unique caryophyllene-rich natural source.

12
 The rhizomes of zingiber contain several sesquiterpenes of which the
sesquiterpene hydrocarbon, humulene (14) and the mono cyclic sesquiterpene ketone
zerumbone (16) are the major constituents [39]. The compound (16) isolated from
the roots of Z. zerumbet had shown excellent melanin inhibition property. Although
leaves, roots and stems of bitter ginger species have been cultivated for food
flavouring and widely used in traditional medicine, little or no research has been
done on tyrosinase inhibition properties which gave depigmentation effects by the
active compounds [40].
.

(13) (14)

(15) (16)

13
 CHAPTER 3

EXPERIMENTAL

3.1 Instruments and Chemicals

The extraction method of dried powder rhizome employed the method of cold
maceration with n-hexane, dichloromethane (CH2Cl2), and methanol (MeOH) as the
solvents. Fractions and isolated compounds will be subjected to Thin layer
chromatography (TLC) analysis by using 0.20 mm pre-coated silica gel aluminium
plate (Merck Kieselgel 60 GF254). The mobile phase will be used in the TLC analysis
are Hexane and ethyl acetate and the mixture in several of proportions. The TLC
plate will be visualized under UV light at 254 nm and 365 nm incorporated vanillin
sulphuric acid spraying reagent.

Vacuum liquid chromatography (VLC) was then carried out for fractionation
of crude extract using Merck silica gel (230-240 mesh) and dry packed under
vacuum in a funnel with a porous frit and eluted with a gradient system
containing hexane: ethyl acetate: methanol, to allow the packing to dry
completely at each change of solvent polarity, yielding several fractions of extracts.
Gravity column chromatography (CC) will be performed following VLC for the
process of purification. Merck silica gel 60 (70-230 mesh) and Sephadex LH-20
(Sigma) will be used in column chromatography with the solvent system which gave
great separation on TLC analysis used as eluting solvents in column to get desired
pure compounds.

The structure of pure compounds was elucidated using 1H Nuclear Magnetic

Resonance (1H NMR), 1
H-1H COSY and Fourier Transform Infrared (FTIR)
1
spectroscopies. The H NMR spectra was recorded using Bruker Advance

14
spectrometer (400 MHz) and the chemical shifts were recorded in δ ppm with
deuterated acetone (CD3COCD3) as the solvent. The IR spectra were recorded using
Perkin-Elmer series 1600.

3.2 Plant materials and Sample Preparation

The ground powdered rhizomes of Z. zerumbet was collected from Pahang.

The air-dried sample was grounded into fine powder by using electric grinder to
increase the surface area for extraction and the mass of the fine powder will be
recorded. The dry and finely powder of rhizomes samples will be stored into airtight
container until further use.

3.3 Extraction of Zingiber zerumbet

The procedure is adopted from Boonyadist [41] to obtain the maximum yields
of flavonoid compounds, which consequently influence the antioxidant activity.
Extraction of rhizomes of Z. zerumbet will be based on the sequential solvent
extraction in which the powdered raw material is first extracted with n-Hexane,
followed with dichloromethane, for isolation of flavonoid aglycones and less polar
material. A subsequent step with an alcohol (Methanol) will extract flavonoid
glycosides and polar constituents. For powdered sample fruits of Z. zerumbet, 300 g
will be soaked with 1.5 L of n-Hexane, Dichloromethane and Methanol at room
temperature for 72 hours sequentially. The solvent will be decanted through filter,
and fresh solvent added to the flask with swirling, and left to macerate again for 3
days with occasional shaking. . The residue sample will be extracted thrice with same
condition to ensure the completion of extraction process. The total filtrates of each
solvent will be evaporated to dryness by using rotary evaporator to obtain crude
extract. The temperature of the water bath will be set depend on the boiling point of

15
the solvents. The percentage yield of the crude extracts will be calculated using the
formula below and recorded in Table 4.1:

𝑇ℎ𝑒 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 𝑦𝑖𝑒𝑙𝑑 (𝑔)

The percentage of yield (%) =𝑇ℎ𝑒 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑔𝑟𝑜𝑢𝑛𝑑 𝑝𝑜𝑤𝑑𝑒𝑟 𝑝𝑙𝑎𝑛𝑡 𝑢𝑠𝑒𝑑 (𝑔) x 100%

The extracts were concentrated to give sticky brown gum yield of n-hexane
(1.80%), and CH2Cl2 (2.10%) crude extracts, however the yield of MeOH is still to
be determined.

3.4 Thin Layer Chromatography (TLC) Analysis of Crude Extracts

The crude extracts of Z. zerumbet rhizome, fractions and isolated compounds

will be subjected to TLC analysis by using silica gel aluminium sheets (Merck
kieselgel 60 GF254, 0.20 mm). A start line will be drawn about 0.5 cm from the
baseline of the TLC plate. Retention factor, Rf for each spot will be calculated using
the equation below:

Distance travelled by the compound (cm)

Rf = 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡 (𝑐𝑚)

The mobile phase will be used in the TLC analysis are Hexane and ethyl
acetate and the mixture in several of proportions. The TLC plate will be visualized
under UV light at 254 nm and 365 nm incorporated vanillin sulphuric acid spraying
reagent and all the spots shown on the TLC plate will be circled by using pencil. The
ratio system for TLC can be shown below in Table 3.1.

16
 Table 3.1 Ratio of n-hexane and EtOAc used for TLC analysis
n-hexane EtOAc
5 0
4 1
3 2
1 1
2 3
1 4
0 5
Total volume used = 5 mL

3.5 Vacuum Liquid Chromatography (VLC) of Z. zerumbet Crude Extracts

Vacuum liquid chromatography involves step gradient elution, and the

column is allowed to run dry after each fraction is collected. The extracts will be
fractionated with silica gel 60 (230-400 mesh). A part of silica need to be
homogenized with the extract and shall be placed on the top of silica (residual part)
and dry packed under vacuum in a funnel with a porous frit and eluted with a
gradient system containing hexane: ethyl acetate: methanol, to allow the packing to
dry completely at each change of solvent polarity, yielding several fractions of
extracts. These fractions will be analysed with TLC and which gives the same profile
could be combined.

3.6 Gravity Column Chromatography (CC)

Solvent system with suitable and great separation on TLC analysis will be
used as eluting solvents in column chromatography to get desired pure compounds.

17
Merck silica gel 60 (70-230 mesh) and Sephadex LH-20 (Sigma) will be used in
column chromatography. The slurry method will be used for preparing the column.
First, the column will be rinsed with the solvent system to give the stationary phase
an even base and prevent concentration and streaking of the bands as they come off
the column and are collected. In a separate flask or beaker, solvent with
approximately one and a half times the volume of silica to be added is measured. The
silica gel will be mixed with solvent to make it become slurry. Then, the column will
be compacted with cotton wool and 0.5 cm of soil will be place on top of the cotton
wool. The slurry silica gel will be packed into the column until it filled up ¾ of the
column. The column is tapped gently during the addition of slurry silica gel to
encourage bubbles to rise and the silica to settle. The inside of the column will be
rinsed by pipetting solvent down the inside edge once all the slurry silica gel is added.
The solvent is drained until the solvent level is just even with the surface of the
stationary phase. Next, the crude portioned need to be placed into the column and the
column will be eluate with the same solvent system followed by mixture of different
solvent systems in various ratios.

3.7 Structural Determination of Secondary Metabolites

The structural compounds will be determined by using spectroscopic methods.

The structure of secondary metabolite compounds will be determined by using Gas
Chromatography-Mass Spectrometry (GC-MS), Nuclear Magnetic Resonance (NMR)
and Fourier Transform Infrared Spectroscopy (FTIR). GC-MS will be used to give
information on the molecular weight of fragment ions. 1H-NMR and 13
C-NMR will
be used to determine the number of carbon and hydrogen respectively and to
determine their environment. FTIR will be used to obtain the functional groups in the
compound.

18
3.8 Determination of Anti-tyrosinase Activity of Pure Compounds

Tyrosinase inhibition was determined using the modified dopachrome

method with L-3,4-dihydroxyphenylalanine (L-DOPA) as substrate [42]. Assays
were carried out in a 96-well microtitre plate and a plate reader was used to measure
absorbance at 475 nm with 700 nm as reference. Samples to be tested were dissolved
in 50% dimethylsulphoxide (DMSO). Each well contained 40 μL of sample with
80μL of phosphate buffer (0.1 M, pH 6.8), 40 μL of tyrosinase (31 unit/mL) and 40
μL of L-DOPA (2.5 mM). Each sample was accompanied by a blank that had all the
components except L-DOPA. Results were compared with a control consisting of 50%
DMSO in place of sample. The percentage tyrosinase inhibition was calculated using
the formula below:

Absorbance control − Absorbance sample

% Inhibition = × 100%
Absorbance control

3.9 Quantification by HPLC

HPLC analysis is carried out using an High Performance Liquid

Chromatography apparatus equipped with a Agilent DAAD detector (Agilent, USA)
and an Agilent Eclipse plus C18 column (100 mm×4.6 mm, 3.5 μm) (Agilent, USA).
While the mobile phase that shall be used are (A) 0.1% phosphoric acid and (B)
acetonitrile which will be used for washing and re-equilibration of the column.
The standard curve is calibrated by using standard kaempferol (Sigma-
Aldrich, Co. USA) with DAAD detection at 254 nm for the quantification. The
quantification is achieved by comparing the retention times of UV-Vis spectra and of
pure standards.

19
3.10 Flow Chart of Experimental

Ground powder of rhizome

of Z.zerumbet (300 g)

Cold maceration: n-hexane,

CH2Cl2, and MeOH

n-hexane, CH2Cl2, and MeOH

Concentration by rotary evaporator

CH2Cl2 crude MeOH crude

n-hexane crude
extracts
i. TLC
ii VLC
Fraction Fraction Fraction

i. TLC
ii CC
Pure compounds Pure compounds Pure compounds

Quantificatio Structure Anti-tyrosinase

n by HPLC elucidation by activities
NMR and FTIR

Figure 3.1 Flow chart of experimental

20
 CHAPTER 4

EXPECTED RESULT

4.1 Extraction on the Rhizome of Zingiber zerumbet

The rhizome of Z.zerumbet was dried and ground into powder. The powdered
rhizomes (300 g) was extracted by cold maceration technique with n-hexane,
CH2Cl2, and MeOH using 1.25 L of solvent each time. The extracts were filtered
and concentrated using rotary evaporator in vacuo at 50ºC, 40ºC, and 42ºC to obtain
n-hexane, CH2Cl2, and MeOH crude extracts of Z. zerumbet respectively. The
available crude extracts obtained were listed in Table 4.1.

Table 4.1 Yield of crude extracts of Z. zerumbet rhizomes

Dried sample n-hexane Extract CH2Cl2 Extract MeOH Extract
Part
(g) (CAH) % (CAD) % (CAM) %
Rhizome 300 1.80 2.10 TBD

4.2 Thin Layer Chromatography (TLC) Analysis of Crude Extracts

TLC analysis was carried out on the crude extracts of n-hexane, CH2Cl2, and
MeOH with different ratio of n-hexane and EtOAc as the solvent system. The Rf
values of spots that were prominent was calculated and the pictures of TLC plates
under shortwave UV light and after applying vanillin sulphuric acid spraying agent

21
were shown at the Appendix A while the retention factor, Rf for each spot for Z.
zerumbet crude extracts is tabulated in Table 4.2.

Table 4.2 Rf values for TLC profile of Z. zerumbet crude extracts

Solvent System Ratio
Crude Extract Rf Value Colour
(n-hexane:EtOAc)
n-hexane 4:1 0.25 Purple
0.50 Purple
0.63 Purple
3:2 0.30 Purple
0.50 Purple
0.68 Purple
1:1 0.40 Purple
0.55 Purple
0.70 Purple
2.3 0.70 Purple
0.73 Purple
0.80 Purple
1:4 0.78 Purple
0.88 Purple
0:5 0.63 Purple
0.75 Purple
0.88 Purple
CH2Cl2 4:1 0.50 Purple
3:2 0.60 Purple
0.13
1:1 0.20 Purple
0.70 Purple
2:3 0.33 Purple
0.83 Purple
1:4 0.50 Purple
0:5 0.78 Purple

22
MeOH 4:1 0.50 Purple
3:2 0.60 Purple
0.13 Purple
1:1 0.20 Purple
0.70 Purple
2:3 0.33 Purple
0.83 Purple
1:4 0.50 Purple
0:5 0.78 Purple

4.3 Purification of Z. zerumbet Crude Extracts

The compounds identified under the TLC plated are isolated by Vacuum
Liquid Chromatography into simpler mixtures and later purified into a single
compound by Gravity Column chromatography to carry out further study on the
structures and their biological activities

4.4 Biological Activities of Pure Compounds

The tyrosinase inhibition activity of the pure compounds would be

determined after isolation and purification..

4.5 Quantification of Pure Compounds

The quantification of pure compounds using HPLC would be determined

upon completion of bioassay activities.

23
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APPENDIX A TLC results of various ratio of n-hexane: EtOAc

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30