Colon Cancer

Colon cancer is a type of cancer that occurs at the colon and rectum. It is also one of the common malignancies in the world. Parkin (1998) stated that colon cancer is the most common type of cancer that occurs in many societies. According to Chong (2012), colon cancer is now the second most common cancer in Malaysia with high mortality in which 90% of the cases occurred in people over the age of 40. Fernandez et al. (2000) stated that the major role that contributes to the cancer initiation, progression and protection is the dietary factors. The common treatment of colon cancer is chemotherapy which employs the usage of many anticancer drugs and cause bad side effects to the patients (American Cancer Society, 2014). Previous research stated that the incidence of colon cancer could be lowered by consuming food containing fruits, vegetables and spices (Bhanot et al., 2011).
Malaysia possesses a vast number of herbs and spices that have medicinal value. One of the most popular is Kaempferia galanga Linn. from the Zingiberecaeae family. It is a monocotyledonous perennial herbs from which can usually be found in Malaysia, Indochina, Thailand, China, Taiwan and India (Kanjanapothi et al., 2004; Umar et al., 2011; Jagadish et al., 2010). The rhizome of K. galanga Linn. which is rich in oil had been used as traditional medicine in the form of decoction or powder to treat indigestion, cold, chest and abdominal pain, headache, expectorant, diuretic and carminative, skin disorders, rheumatism and diabetes mellitus (Kanjanapothi et al., 2004; Jagadish et al., 2010).
Furthermore, it was used in the treatment of dyspepsia and inflammatory tumours (Liu et al., 2010). It was also popular medicine as an aromatic stomachic and incense by Chinese (Kanjanapothi et al., 2004). In China, K. galanga Linn. is famous as spice while in Malaysia, K. galanga Linn. has been used extensively in food which is mostly eaten raw as 'ulam' or made as local health tonic called 'jamu' (Chan et al., 2008). K. galanga Linn. has captured the attention of many for its valuable biological properties such as anti-inflammatory and analgesic (Umar et al., 2012), larvicidal, antimicrobial and antifungal (Helen et al., 2011; Kochutressia et al. 2012), antitumor (Kirana et al., 2003), and antiproliferative (Liu et al., 2010) can be found in the essential oil of K. galanga Linn.. Sukariet al. (2008), Bhuiyan et al. (2008) and Umar et al. (2012) found that ethyl p-methoxycinnamate, the contributor for the bioactivity as the major constituents in the essential oil of K. galanga Linn..
Hence, this study is carried out to screen whether the essential oil extracted from K. galanga Linn. that can be found abundantly in Malaysia has the anticancer properties against HCT116 colon cancer cell line since there is still no anticancer studies done on this cancer cell line. In this study, the essential oil will be extracted from the rhizome by employing the steam distillation method and will be further analyzed to identify the constituents by using Gas Chromatography-Mass Spectrometry technique. The cytotoxicity assay used for screening the anticancer properties will be MTT assay.

1.2 OBJECTIVES
The objectives of this research are to:
1. Extract the essential oil from the rhizome of K. galangal Linn.;
2. Separate and analyze the constituents of the essential oils by using Gas Chromatography-Mass Spectrometry (GC-MS) technique and;
3. Investigate the cytotoxicity effects of the essential oil on HCT116 colon cancer cell line by comparing the essential oil to pure ethyl p-methoxycinnamate compound

1.3 RESEARCH QUESTIONS
What is the potential of essential oil extracted from rhizome K. galanga Linn. in suppressing the proliferation activity of HCT 116 colon cancer cell line?

1.4 LITERATURE REVIEW

1.4.1 The Epidemiology of Colon Cancer
One of the most common causes of cancer related death is colon cancer. According to Center et al. (2009), the rate of this type of cancer from 1982 to 11987 through 1998 until 2002 had increased for both females and males. The highest incidence rate for both men and women are in Eastern European countries, New Zealand, Australia, Germanya and United States (Jemal et al., 2010). The increment was due to several factors such as obesity, smoking habit, heavy alcohol consumption, high intake of diet in red processed meat and low intake of fruits and vegetables (Center et al., 2009). There are 3 stages of colon cancer - Dukes' Stage A in which the malignancy is limited to intestine wall, Dukes' Stage B in which the malignancy has spread to nearest tissue but not to nodes and Dukes' Stage C in which the cancer cells have metastasized in regional lymph nodes.
In a study done by Chin (2001), colon cancer in Malaysia is one of 10 leading cancers in the population whereby the rise of the incidence is believed due to the rise in aging. One of common treatments for colon cancer is conventional chemotherapy which is given in general and private hospitals (Chin, 2001). Chemotherapy is normally used in treating patients with Dukes' B and C colon cancer (Lim et al., 1997). Anticancer drugs that are used in colon cancer chemotherapy are 5-fluorouracil, oxaliplatin, irinotecan and capecitabine (American Cancer Society, 2014). However, this chemical based treatment causes severe side effects as the chemotherapy drugs affect blood forming cells of bone marrow causing lower blood cell count and resulting in increased chance of infection, easily bruising and bleeding as well as fatigue due to lack of oxygen (American Cancer Society, 2014).

1.4.2 Plant as Source of Anticancer Agents
Plants has been used in treating cancer for centuries as the nature has interesting sources with tremendous chemical variants found in the plants. The interest in plant-based anticancer agents began in 1950s with the finding and development of vinca alkaloid found in Madagascar periwinkle, Catharanthus roseus G. Don. which were vinblastine and vincristine (Cragg and Newman, 2005). Over 60% of currently used anticancer remedy came from natural sources (Bhanot et al., 2011). In fact, 4 drugs had been approved as anticancer agents which are doxorubicin and estradiol in 2002 as well as chlorophyll and 1-aspartic acid in 2004.
Previous studies suggested that consumption of food rich in fruits, vegetables and spices could lead to lower incidence of colon cancer (Bhanot et al., 2011). They had various biologically active compounds that could inhibit the inflammatory process which later could lead to transformation, excessive proliferation and initiation of carcinogenesis such as inositol, isoflavones, catechins, lycopene, curcumin and genistein. These compounds can be found in the plant extract such as essential oil.
In ancient times, the main role of plant essential oil was as food preservative as well as alternative choice for medicine (Sayyad and Chaudhari, 2010). Edris (2007) stated that essential oil which is normally found in aromatic plants, are complex and multicomponent systems which consists of mainly terpenes with some non-terpene components. Many studies have been done to discover the potential of essential oil as anticancer agents such as the inhibitory action on human hepatoma Hep G2 cells shown by garlic oil. The anticancer properties of the essential oil was observed when garlic essential oil showed potential in inhibiting human hepatoma Hep G2 cells. ??-Bisabolol found in Matricaria chamomilla essential oil induced apoptosis in highly malignant glioma cells.

1.4.3 The Zingiberaceae Family
1.4.3.1 General Morphology of Zingiberaceae Family
The Zingiberaceae is a monocotyledonous plant (Wohlmouth, 2008). According to Sirirugsa (1999), they are perennial aromatic rhizomatous herbs with simple and distichous leaves. The inflorescence is found at the end of the leafy shoot or on lateral shoot. Besides that, the flower of the Zingiberaceous plants is a short-lived, subtle, and highly modified structure and the fruit presents as a capsule (Sirirugsa, 1999). The rhizomes are thick with and rich with essential oil produced by the secretory cells (Wohlmuth, 2008).

1.4.3.2 Habitats and Distribution of Zingiberaceae family
The Zingiberaceae family is recognized based on their similar morphology and habitat. In a study done by Sirirugsa (1999), they exist as ground plants which grow in damp and shady area and normally can be found in tropical forests. However, some of the species can be fully exposed to the sun and found to live in the high altitude as well. They are also seldomly found in secondary forest.
They can be found abundantly in tropica and subtropical areas. South East Asia is well known as the centre distribution for this family. This is because the Malesian region is the region that has the highest distribution of genera and species of this Zingiberaceae family especially in Malaysia, Indonesia, Singapore, Brunei, Philippines and Papua New Guinea (Sirirugsa, 1999).

1.4.3.3 Classification of Zingiberaceae family
According to Sirirugsa (1999), one of the biggest families in the plant kingdom is the Zingiberaceae family with four tribes which are Hedychieae, Zingibereae, Alpineae and Globbeae. Figure 2.2 shows the summary of the taxonomy for Zingiberaceae family. 52 genera with total about 1100 species are identified under these tribes (Wohlmuth, 2008). Meanwhile, Sukariet al. (2008) said that there are about 50 genera and over 1000 species in Zingiberaceae family. In Peninsular Malaysia, it is estimated that there are 150 species of ginger belong to to 23 genera (Sukari et al., 2008).

Table 1.1
Sub family Tribe Genus
Zingiberoideae Hedychieae Boesenbergia
Curcuma
Hedychium
K.
Scaphochlamys
Zingibereae Zingiber
Alpinieae Aframomum
Alpinia
Amomum
Elettaria
Etlingera
Hornstedtia
Globbeae Globba
Costoideae Costus
Tapeinochilus
The sub families, tribes and genera of Zingiberaceae family (Sirirugsa, 1999)

1.4.3.4 Medicinal values of Zingiberaceae family
Zingiberaceae has been used in alternative medicine since the ancient times. The pharmaceutical properties of the Zingiberaceae family are attributed to the essential oil found in the rhizome (Edris, 2007). Zingiber spp. that belong to Zingiberaceae family has been used in treating thrombosis, sea sickness, migraine and rheumatism (Sirirugsa, 1999). In addition, the Curcuma spp. is also used in relieving cough, tuberculosis and diarrhea while the Hedychium has been applied in curing tonsillitis and as a remedy to treat intestinal worm (Sirirugsa, 1999).
There were many previous researches that studied on the biological activities of Zingiberaceae family. Prakash et al. (2013) stated that Z. officinale inhibited the endothelial cell tube formation in skin tumor. In addition, Z. cassumunar and K. parviflora possessed anti allergic activity when tested against antigen-induced ??-hexosaminidase. C. longa and A. galanga showed antioxidant properties as they were capable to act as free radical scavengers (Zaeoung et al., 2005).

1.4.4 The Kaempferia genus
Among 50 species belong to the genus of K. , only four species are popular which are K. galanga Linn., K. rotunda Linn., K. pulchra Ridl. and K. elegans Wall. (Sirirugsa, 1999). They are commonly distributed in Malaysia, Indonesia, India and Indochina (Sirirugsa, 1999).

1.4.4.1 Kaempferia galanga Linn.
K. galanga Linn. is commonly known as cekur in Malaysia, proh hom in Thailand (Kanjanapothi et al., 2004) and shan-nai in Chinese (Kosuge et al., 1985). The species can be recognized based on the inflorescence that is enclosed in the two horizontal, sessile and flat on the ground leaves with white flowers (Sirirugsa, 1999).. According to Helen et al. (2011), there are 4 to 5 petals for each flower and the sepal is 2 to 3 cm long. In addition, the rhizome is in brown with pungent, sharp, bitter and aromatic characteristic and rich with essential oil. It is popular as spices and flavoring in cooking (Liu et al., 2010).

Figure 1.1
K. galanga Linn. and its rhizomes
(Indrayan et al., 2009)

1.4.4.2 Phytochemical Constituents of K. galanga Linn.
The essential oil extracted from K. galanga Linn. is a complex mixture of various chemical compounds. A few analyses of essential oils of rhizomes of K. galanga Linn. had been done previously by using gas chromatography-mass spectrometry methodology. According to Jagadish et al. (2010), the essential oil contained several terpenes which were pinene, camphene, carvone, benzene, eucalyptol, borneol, methyl cinnamate, pentadecane and ethyl p-methoxycinnamate. However, Sukari et al. (2008) identified 25 chemical components consisted of mainly non-terpenoids components which were ethyl p-methoxycinnamate (58.5%), isobutyl ??-2- furylacrylate (30.9%) and hexyl formate (4.8%). Umar et al. (2012) claimed that ethyl p- methoxycinnamate existed abundantly in 80.05% followed by ??-sitosterol in 9.88%, propionic acid in 4.71%, pentadecane (2.08%), tridecanoic acid (1.81%) and 1,21- docosadiene.
In another study, Helen et al. (2011) revealed 14 components out of 20 peaks resulting from gas chromatography analysis in which 3-carene (16.22%), cyclohexane (13.03%), borneol (10.41%), and camphene (9.78%) were the major components. Other components such as decane (3.34%), pentene (2.68%), 3-pinene (2.20%), santolinatriene (2.15%), isobornyl acetate (1.94%), 2-butyne (1.37%), cyclododecane (1.31%), 2-octyne (1.03%), cyclopentano (0.79%) and 2-nonyne (0.43%) were found as minor components in the essential oil from the rhizome. Meanwhile, Zaeoung et al. (2005) reported that only 7 components could be identified even though 12 compounds were present in the essential oil which the main constituent of the essential oil was ethyl cinnamate (61.8%) followed by ethyl p-methoxycinnamate (18.3%), 1-borneol (7.6%), pentadecane (2.6%), terpinene-4-ol (2.0%), ??-fenchyl alcohol (1.2%), and p-cymen-8-ol (1.1%).
The chemical compounds in the leaf and rhizome are varies. Bhuiyan et al. (2008) analyzed the oils from the leaf and rhizomes which had 108 and 81 compounds respectively. The main component detected in the leaf was linoleoyl chloride with 21.42%. A few other compounds that present abundantly were caryophyllene oxide (11.75%), cubenol (9.66%), caryophyllene (5.60%) and ethyl p-methoxycinnamate (5.56%). Meanwhile, in the rhizome, ethyl p-methoxycinnamate was the main compound with 63.36% followed by ethyl cinnamate (6.31%), 4-cyclooctene-1- methanol (4.61%), caryophyllene oxide (4.37%) and limonene (3.22%). Hence, the oil from the rhizome is more biologically active since it contains higher ethyl p-methoxycinnamate which contributes to the various medicinal properties of K. galanga Linn.

Figure 1.2
Structure of ethyl p-methoxycinnamate

1.4.4.3 Medicinal properties of K. galanga Linn.
K. galanga Linn. posseses many medicinal values and has been used as traditional medicines for centuries especially in Asia. Previously, K. galanga Linn. was used traditionally in treating hypertension, pectoral and abdominal pains, headache, toothache, coughs, dyslexia and rheumatism (Kanjanapothi et al., 2003; Liu et al., 2010; Sirisangtragul & Sripanidkulchai, 2011). Besides that, they are also used as the ingredient in tonic, stomachic, cough and mixture for women after childbirth (Sukari et al., 2008).
K. galanga Linn. is well-known as analgesic. Leaves and the rhizomes were claimed to have anti-inflammatory effects and had been used to in treating swelling, stomach ache, toothache, rheumatism and headache (Mitraet al., 2007). However, Tewtrakul and Subhadhirasakul (2007) clarified that the volatile oils of K. galanga Linn. had weak anti-inflammatory activity as the extracts were seemingly inactive against the activity of ??-hexosaminidase.
In another research done by Umar et al. (2012), the ability of chloroform fraction of the rhizomes extract to show highest inhibitory effect on carrageenan- induced edema and non-selectively exhibit the activities of cyclooxygenase 1 and 2 had clarified the ability of K. galanga Linn. to fight against inflammation due to the presence of ethyl p-methoxycinnamate. Besides that, it was found that ethyl p-methoxycinnamate exhibited significant potential as anti-inflammatory by inhibiting pro-inflammatory cytokines, and as anti-angiogenic as well as this compound decreased the level of vascular endothelial growth factor by inhibiting the differentiation and migration of endothelial cells (Umar et al., 2012).
Essential oil extracted from K. galanga Linn. had strong anti microbial effect on the human pathogenic bacteria, S. typhi and Enterobacteraerogens (Helen et al., (2011). Similar result was observed for both bacteria when ethanol, methanol, petroleum ether, chloroform and aqueous extracts were tested in a research done by Kochutressia et al. (2012). According to Helen et al. (2011), there waas no inhibition of activities observed on B. megaterium, B. amyloliquefaciens, Mycobacterium spp., X. campestris, S. thermophilus. Besides that, it was found that essential oil from the rhizomes had no inhibitory effect on S. aureus, E. coli and P. aerugenosa (Helen et al., 2011). However, the bacteria showed the opposite effect when tested with ethanol, methanol, petroleum ether, chloroform and aqueous extracts (Kochutressia et al., 2012).
K. galanga Linn. is proven to have antifungal properties as well. C. albicans were one of the fungus that showed significant antifungal properties when tested with both volatile oils and methanol, petroleum ether, chloroform and aqueous extracts of K. galanga Linn. (Helen et al., 2011 and Kochutressia et al., 2012). The various types of extracts showed positive antimicrobial activity on A. flavus, A. sfumigaus as well (Kochutressia et al., 2011). However, the antifungal activity seems to be depending on the types of extract as well. The methanol, petroleum ether, chloroform and aqueous extracts of K. galanga Linn. was observed to have strong antifungal properties on A. niger in the study done by Kochutressia et al., (2012). In contrast, the volatile oil from the rhizomes of K. galanga Linn. had weak activity against A. niger (Helen et al. (2011). The similar weak effect was observed on K. maxianus.
Several studies were done on the anticancer properties of K. galanga Linn.. The earliest was done by Kosuge et al. (1985) on cervical cancer HeLa cells. It was found that the methanol extracts of the rhizomes were highly cytotoxic to cervical cancer HeLa cells. Zaeoung et al. (2005) said that water extract of K. galanga Linn. showed a slight cytotoxic effect on human colon adenocarcinoma LS174T and human breast adenocarcinoma MCF-7 while volatile oil extracted from K. galanga Linn. had the ability to prevent the proliferation of two cell lines. Kirana et al. 2003 proved that K. galanga Linn. had the cytotoxicity activity on MCF-7 and on HT-29 colon cancer as well even though the higher concentration was required.
Besides that, the extract of K. galanga Linn. also showed the inhibition of SW 620 human colon adenocarcinoma cells, PA-1 human ovarian teratocarcinoma cells and DU-145 human epithelial prostate cancer cells (Jagadish et al., 2010). In addition, Liu et al. (2010) had proven that ethyl p-methoxycinnamate purified from the essential oil of the rhizome of K. galanga Linn. was able to induce apoptosis in HepG2 hepatocellular liver carcinoma cells in a dose-dependent manner. The rhizome of K. galanga Linn. was proven to have anti-tumor potential due to its inhibitory action on 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced Epstein-Barr virus activation in Raji cells (Vimala et al., 1999).

1.4.5 Colon Cancer Cell Lines
Various cell lines had been derived from cancer cells for research purposes. In previous research, several colon cancer cell lines had been used in screening the anticancer property of K. galanga Linn. The cell lines used were LS174T, HT-29 and SW620. These cells have different genetic background. However, they were derived from patients with colon adenocarcinoma and shared same morphology which is epithelial. According to American Type Culture Collection, ATCC (2012), LS 174T colon cancer cell line was derived from a female patient at Dukes' Stage B and there was no other chromosomal abnormality except one X chromosome was found to be missing; making the number of chromosomes to be only 45 .
According to ATCC (2012), HT-29 colon cancer cell line is other types of colon cancer cell lines derived from patient at Dukes' Stage A in which 45% of 22 different autosomal chromosomes were rearranged. Unknown material was added to the short arms of chromosomes 3, 7 and 13 and to the long arm of chromosome 4. There was deletion of unknown material detected from the short arms of chromosomes 10 and 17. A translocation involving chromosomes 6 and 14 together with duplication of chromosomal DNA in chromosomes 9 were observed. In addition, there was an isochromosome 13 found and insertion of unknown material on the short arm of chromosome 3 in HT-29 cell lines (ATCC, 2012).
Colon SW620 cell line was derived from a patient at Dukes' Stage C (ATCC, 2012). Cytogenetic analysis showed that there were rearrangements to the chromosomes - an addition of unknown material to the short arms of chromosome 2 and 8 as well as to the long arms of chromosomes 3, 6, 7, 10, and 16; deletion of unknown material from the short arm of chromosomes 3 and from the long arm of chromosomes 4, 5, and 18 as well as translocation of short arm of chromosome 8 and long arm of chromosome 13 (ATCC, 2012).
Meanwhile, HCT116 colon cancer cell line is another colon cancer cell line which was obtained from a patient with Dukes' Stage A. The structural abnormalities was due to rearrangements whereby there was addition of unknown material to the short arms of chromosomes 16 and 18 as well as to the long arm of chromosome 10 (ATCC, 2012).

CHAPTER TWO
MATERIALS AND METHODS

2.1 Materials and Glasswares
The materials used in this study were 96- well plate (Falcon ??), 6-well plate (Falcon ??), eppendorf tubes, Falcon tubes, 1000 ??l pipette tips, 100 ??l pipette tips, 10 ??l pipette tips, 9 cm2 culture dish (Nunclon ' Surface), 5 ml serological pipette tips, and 10 ml serological pipette tips. The glasswares used are universal bottles, 50ml beakers, 100 ml measuring cylinders, 100 ml round bottom flasks, 250 ml conical flasks and 500 ml separating funnel.

2.2 Chemicals
The chemicals used in this study were dichloromethane (QR??C'), DMEM (Sigma-Aldrich??) DMSO Biotechnology Standard (Sigma-Aldrich??), DMSO Industrial Standard (R&M Chemicals), Ethyl p-methoxycinnamate (Tokyo Chemical Industry), PBS (Gibco??), FBS (Gibco??), TrypLE' Express Enzyme (Gibco??) and penicillin-streptomycin antibiotic (Gibco ??).

2.3 Reagents
The reagents used in the assay was MTT (Duchefa Biochemier).

2.4 Instruments
The instruments used in this research were steam distillation extractor, rotary evaporator (B??CHI Rotavapor B-421), gas chromatography-mass spectrometer (Perkin Elmer Clarus?? 680), inverted light microscope (Nikon Eclipse T100), incubator (HERA Cell 150), multi detection microplate reader (Infinite M200 Nanoquant/Tecan), micropipettes, autoclave and biosafety cabinet.
2.5 METHODS
2.5.1 Extraction of Essential Oil
2.5.1.1 Sample preparation
Rhizomes of K. galanga Linn. were purchased from market at Chow Kit Road, Kuala Lumpur. The samples were washed and air dried. The samples then were chopped into smaller pieces.

2.5.1.2 Extraction of essential oil
Approximately 3 kg of samples were transferred into 5-L round bottom flask and placed on another 5-L round bottom flask containing tap water. The tap water was heated at 100??C and the steam was allowed to go through the samples for 8 hour. The essential oil was collected by using liquid liquid extraction. 150 ml of dichlorometane was mixed with 150 ml of distillate in the 500 ml separating funnel. The mixture was shaken vigorously and the mixture was allowed for 3 minutes to separate. Two layers of solution were formed and the bottom layer was collected. Then, the collected solution was filtered by using sodium sulphate. The solution underwent in vacuo concentration process using rotary vaporator to get the essential oil. The essential oil was stored at -4??C for further analyses.

2.5.2 Analysis of Essential Oil
2.5.2.1 Gas Chromatography-Mass Spectrometry
The essential oil was analyzed using GC-MS. The essential oil was dissolved in dichloromethane at 1% concentration. A volume of 1 ??l of sample was injected in the column. Helium was used as the carrier gas with a flow rate of 1 ml/min. The oven was initially kept at 40 ??C and programmed at 4 ??C per minute until it reached 250 ??C and held for 10 minutes. The sample was run for 60 minutes. The compounds were identified by comparing the mass spectrum obtained with the mass spectra of the National Institute of Standard and Technology (NIST) MS library.

2.5.3 Subculturing of HCT 116 Colon Cancer Cells
The confluency of the cells was checked under the linverted light microscope. The DMEM medium from HCT116 colon cancer cells was first removed and the cells were washed with PBS. A volume of 1 ml of TrypLE' Express Enzyme was added to the cells and the cells were incubated for approximately 3 minutes to detach the cells from the culture dish. A volume of 2 ml of complete DMEM medium - added with 10% of FBS and 1% of penicillin-streptomycin antibiotic; was added to the trypsinized cells to stop the enzymatic reaction making the total volume of the cell suspension was 3 ml. The HCT 116 colon cancer cells were subcultured at the ratio of 1:10 in 9mm 2 culture dish and the media was replaced every 2 to 3 days.

2.5.4 Cytotoxicity Test
2.5.4.1 Seeding of cells
The DMEM medium from HCT116 colon cancer cells was first removed and the cells were washed with PBS. A volume of 1 ml of TrypLE' Express Enzyme was added to the cells and the cells were incubated for approximately 3 minutes to detach the cells from the culture dish. A volume of 9 ml of DMEM medium was added to the trypsinized cells to stop the enzymatic reaction making the total volume of the cell suspension was 10 ml. Then, 100 ??l of the cell suspension was taken out and then was placed in each of 96 - well plate except for three wells that were reserved for MTT. The 96 ' well plate was incubated for 24 hours at 37 ??C with 5% of CO2.

2.5.4.2 MTT Assay
The essential oil in 60% concentration diluted in Biotechnology Standard DMSO was taken for 2??l and mixed with 1 ml of DMEM medium to make a stock solution. The stock solution was diluted by using DMEM medium using two-fold serial dilution to get the concentration of 0.120 %, 0.060 %, 0.030 % and 0.015 %. Cisplatin and Ethyl p-methoxycinnamate were diluted using the same procedure to get 0.05000 mg/ml, 0.02500 mg/ml, 0.01250 mg/ml, 0.00625 mg/ml and 1.00 mg/ml, 0.500 mg/ml, 0.250 mg/ml and 0.1205 mg/ml respectively. The present medium in 96 ' well plate was replaced with the medium containing the diluted samples by adding 100 ??l to each well in triplicate for each concentration except the control wells that were filled with 0.2 % of Biotechnology Standard DMSO and 5 mg/mLMTT. The 96 ' well plate was incubated at 37 ??C for 24 hours with 5% CO2. After 24 hours, the media was discarded and 30 ??l of 5 mg/mL MTT solution was added to each wells and incubated for 3 to 4 hours at 37 ??C. After that, 150 ??L of DMSO (industrial grade) was added into each wells to stop the reaction. Wells with purple colour indicate that the cancer wells were alive while wells with yellow colour showed that the cells were no longer viable. Then, after 1 hour, the 96 ' well plates was read using a 96 'well micro plate reader at 570 nm with 630 nm as reference.

CHAPTER THREE
RESULTS AND DISCUSSION

3.1 EXTRACTION YIELD OF ESSENTIAL OIL
Essential oil is rich with many bioactive compounds that have high values especially in medical field. However, the compounds are normally present in lower concentration. Hence, suitable extraction method should be chosen so that the bioactive compounds can be extracted from its source since many factors can influence the yield and purity such as volatility and polarity of the compounds.
In this study, steam distillation was used in extracting essential oil from the rhizomes of K. galanga Linn. Steam distillation is one of the most commonly used conventional methods in extraction. This method does not require any usage of solvent so solvent wastage can be avoided. Besides that, this extraction method is also environmental-friendly since only steam is used to extract the essential oil from the source.
The yield of the essential oil extracted from the rhizomes of K. galanga Linn. by using steam distillation method is presented in Table 3.1.

Table 3.1
Extraction yield d the essential oil from the rhizomes of K. galanga Linn.
Extraction Properties of the essential oil Weight of fresh sample (g) Weight of essential oil extracted (g)
Total yield (%)
Steam distillation Yellow, slightly viscous
3085.40 16.95 0.55
Total yield = (Weight of essential oil/ weight of fresh sample) ?? 100 %

From Table 3.1, the essential oil obtained from 3085.40 g of rhizomes of K. galanga Linn. was 0.55% (w/w) in yellow and slightly viscous after 8 hours of extraction. According to Helen et al. (2011), the extraction of rhizomes for 6 hours produced 0.15% (v/w) of essential oil from 195.68 g of fresh rhizomes. However, in a study done by Zaeoung et al. (2005), higher yield of essential oil could be obtained which was 1.3% (w/w) from 1000 g of fresh rhizomes in the same extraction period. Bhuiyan et al. (2008) stated that 1.05% of essential oil was obtained from 200 g of fresh rhizomes as well. Hence, the yield of essential oil is not influenced by the time taken for the extraction process and the weight of fresh rhizomes used in the extraction.
Meanwhile, in a research that used different varieties of K. galanga Linn. for extraction in equivalent extraction period, the extraction gave different amount and color of essential oil for both varieties. The Kasthuri variety yielded 1.88% of slightly viscous, light yellow essential oil while Rajani variety produced 1.76% of slightly viscous, pale yellow essential oil (Indrayan et al., 2007). Thus, the amount of essential oil that can be extracted is depending on the varieties of K. galanga Linn. used in the extraction. This means the variety of K. galanga Linn. used in this study does not contain much essential oil in the rhizome. Apart from that, essential oil with low viscosity is produced when steam distillation extraction method is employed.

3.2 GAS CHROMATOGRAPHY- MASS SPECTROMETRY (GC-MS) ANALYSIS OF ESSENTIAL OIL
Various types of compounds with different concentration are present in the essential oil. One of the well-known methodologies that can be used to determine the constituents of the essential oil is Gas Chromatography-Mass Spectrometry (GC-MS). The compounds present in the essential oil were determined based on comparison of their mass fragmentation patterns to the NIST library.
The GC-MS analysis result of the essential oil could be seen in the GC chromatogram in Figure 3.1. There were 5 major peaks observed representing 5 major compounds existed in the essential oil were detected.

Figure 3.1
GC chromatogram of essential oil

The 5 major peaks in the essential oil from rhizomes of K. galanga Linn. were then compared to NSIT library and the compounds were identified. The compounds are summarized in Table 3.2 with percentage relative and retention time.
Table 3.2
Major compounds present in the essential oils from rhizome of K. galanga Linn.
Peak No. Compound Retention Time,RT (min)
1 endo-Borneol 19.12
2 (E)-methyl cinnamate 26.41
3 Ethyl cinnamate 29.22
4 Ethyl p-methoxycinnamate 37.62
5 Bis (2-ethylhexyl) phthalate 54.85
Mass spectra and structure for the compounds as in Appendix A

In GC-MS, the compounds will be separated according to their mass. The retention time is the time taken for the compound to be isolated from the essential oil which is inversely proportional to the mass of the compound. Based on Table 3.2, it can be said that endo-Borneol has the smallest molecular mass since the retention time, RT was the earliest, 19.12 min whereas the compound with the largest mass detected was Bis (2-ethylhexyl) phthalate since the RT was the latest which was 54.85 min.
The percentage relative of each compound was calculated by using the area under each peak. The result is shown in Figure 3.2.

Figure 3.2
Graph represents the percentage relative of compounds in essential oil

Based on the Figure 3.2, the highest percent of chemical compound available in the essential oil was ethyl p-methoxycinnamate with 55.68%. Results of previous study showed that ethyl p-methoxycinnamate was identified as the highest compound present in the essential oil (Bhuiyan et al., 2008; Sukari et al., 2008; Umar et al., 2012). However, the percentage of yield is different. Bhuiyan et al. (2008) revealed that 63.36% of ethyl p-methoxycinnamate was present in the essential oil extracted from the rhizomes of K. galanga Linn. Meanwhile, Umar et al. (2012) identified higher percent of ethyl p-methoxycinnamate in the essential oil which were 80.05%. However, there was only 58.50% of ethyl p-methoxycinnamate in the essential oil in study done by Sukari et al. (2008).
The different percentage of ethyl p-methoxycinnamate present in the essential oil is due to the extraction method used in extracting the essential oil. Umar et al. (2012) utilized soxhlet extraction method whilst the other researchers used steam distillation extraction method in extracting the essential oil. This means the utilization of steam distillation extraction method seems to have affected the efficiency of ethyl p-methoxycinnamate extraction from the rhizomes.
Many studies that were done previously showed that other major compounds present in the essential oil were different as well. In contrast to the compounds listed in Table 4.2, Bhuiyan et al. (2008) listed 4-cyclooctane -1 methanol, caryophyllene, limonene and ethyl cinnamate as major compounds in essential oil. Sukari et al. (2008) claimed that E-citral, zerumbone, camphor, and isobutyl ?? -2- furylacrylate as main constituents of essential oil from rhizomes of K. galanga Linn. Propionic acid, ??- sitosterol, pentadecane and tridecanoic acid were identified as the main chemical compounds found in essential oil in a study done by Umar et al. (2012). To conclude, the constituents of essential oil are influenced by the type and the procedure of extraction used in extracting the essential oil.

3.3 CYTOTOXICITY ASSAY ON HCT 116 COLON CANCER CELL LINE
The essential oil obtained from the rhizome of K. galanga Linn. was tested on HCT 116 cancer cells with different concentrations in order to observe the cytotoxicity effect of the essential oil on the cells. The cells were observed under the inverted light microscope after 24 hours of treatment. The cell viability was observed as seen in Figure 3.3.


Figure 3.3
HCT 116 colon cancer cells when (A) untreated, (B) treated with DMSO, (C) treated with 0.015% of essential oil, (D) treated with 0.030 % of essential oil, (E) treated with 0.060 % of essential oil and (F) treated with 0.120 % of essential oil.

Based on figure 3.3, it could be seen that the cell viability was reduced after 24 hours of treatment with essential oil compared to the untreated cells. Besides that, the cell viability decreased as the concentration of essential oil increased since the least viable cells were observed when treated with highest concentration of essential oil (0.120%).
Meanwhile, it could be seen that the cell viability in the untreated HCT 116 colon cancer cells had no obvious difference with the DMSO treated HCT 116 cancer cells. According to Augustijns (2007), DMSO with concentration more than 0.2 % could compromise the cell integrity and suppress the enzyme activity. Hence, the usage of DMSO that acted as the solvent in this study for the essential oil had no cytotoxicity effect on the HCT 116 cancer cell line.
The essential oil treated cells were then treated with MTT to measure the cell viability colorimetrically. The result of MTT assay is shown in Figure 3.4.

Figure 3.4 MTT assay of treated HCT 116 colon cancer cells with (A) 0.120 %, (B) 0.06 %, (C) 0.03% and (D) 0.015 % of essential oil

MTT assay is an enzyme based assay in which the yellow solution of MTT can only be converted to water insoluble blue formazan in the presence of mitochondrial dehydrogenases. Since there is mitochondrial functionality loss in nonviable cells, only viable cells have the ability to carry out the MTT conversion. Besides that, the formazan production is proportional to the number of viable cells present. Therefore, it could be observed in Figure 3.2 that a large amount of formazan was produced in the wells containing cells treated with the lowest concentration of essential oil (0.015%), indicating that the cell viability was the highest. In contrast, least amount of formazan was formed in the wells containing cells treated with highest concentration of essential oil, implicating that the cell viability was the lowest.
Next, the amount of formazan formed was quantified by using microplate reader. The percentage of cell viability for each treatment was calculated and presented in Figure 3.5.

Figure 3.5
Cell viability for HCT 116 colon cancer cell line when treated with each treatment in different concentration (??SD, n=3)
In Figure 3.5, it is shown that the lowest concentration of essential oil, 0.015 % showed the highest percentage of cell viability which was 98.92%. Meanwhile, the highest concentration of essential oil (0.120%) showed only 79.77% of viable HCT 116 cancer cells. Therefore, it could be deduced that as the concentration of essential oil increased, more HCT 116 colon cancer cells could be inhibited. Hence, essential oil from K. galanga Linn. exhibited the properties as antiproliferative agent in a dose-dependent manner.
In comparison of cytotoxicity effects exhibited by essential oil to ethyl p-methoxycinnamate, it was observed that essential oil had the ability to inhibit the growth of HTC 116 colon cancer cells similarly with ethyl p-methoxycinnamate. Since ethyl p-methoxycinnamate was detected as the most abundant compound found in essential oil from the rhizome of K. galanga Linn., it could be stated that the presence of ethyl p-methoxycinnamate in the essential oil that contributed to the antiproliferation properties of the essential oil.
IC50 value is the concentration of inhibitor required to inhibit half of the cell proliferation activity. The IC50 value of the essential oil was determined by constructing a graph of concentration of essential oil against percentage of cell viability as in Figure 3.6.

Figure 3.6
IC50 of essential oil
Based on figure 3.6, the lowest percentage of cell viability observed was 79.77% when 0.120% of essential oil was treated. However, the percentage of non viable HCT 116 cancer cells did not exceed 50% of the population even though after treated with the highest concentration of essential oil. Therefore, the IC50 value of the essential oil was more than 0.120%.
Since 55.68% of ethyl p-methoxycinnamate was found in the essential oil (Table 3.2), it could be said that in 0.120% of essential oil, there was approximately 0.067% of ethyl p-methoxycinnamate present in the essential oil. Even though only small percent of ethyl p-methoxycinnamate present in the essential oil in the highest concentration of essential oil, the cytotoxicity effect could be considered as relatively strong when compared to the cytotoxicity effect shown by the highest concentration of ethyl p-methoxycinnamate (1.000 mg/mL) on HCT 116 cancer cells. Thus, the essential oil worked best in inhibiting the cell proliferation compared to ethyl p-methoxycinnamate.

CHAPTER FOUR
CONCLUSION AND FUTURE WORK

Recently, there is a rise in the number of colon cancer cases worldwide. However, not many researches focus on essential oil from herbs and spices as one of potent remedy for this disease is done. The capability of K. galanga Linn. as anticancer treatment had been studied previously. However, there were few studies done which used different cell lines. Hence, different hence the effects observed were different as well. Thus, this study was initiated to evaluate the anticancer properties of the essential oil extracted from therhizome of K. galanga Linn. on HCT 116 colon cancer cell line.
The first stage in this study was the extraction of essential oil from the rhizome of K. galanga Linn. by using steam distillation method. When compared to other studies (Helen et al., 2011; Zaeoung et al., 2005; Bhuiyan et al., 2008; Indrayan et al., 2007), the yield of essential oil depended on varieties of K. galanga Linn. used in the extraction. Besides that, the time taken for the steam distillation process to run and the amount of fresh samples used showed no influence on the yield of essential oil obtained.
The second stage was the analysis of the essential oil by using GC-MS. The major compounds identified were different with previous studies (Bhuiyan et al., 2008; Sukari et al., 2008; Umar et al., 2012) indicating that different extraction method and procedure used affected the presence of various chemical constituents in the essential oil. Ethyl p-methoxycinnamate was found as the most compound present in the essential oil which was similar to the findings by Bhuiyan et al., (2008), Sukari et al., (2008) and Umar et al., (2012). However, the difference in percentage of yield showed that steam distillation was not an efficient extraction method to extract ethyl p-methoxycinnamate from the rhizome of K. galanga Linn..
The final stage of this study aimed to screen for anticancer properties of the essential oil using the simple MTT assay. The IC50 value for essential oil was more than 0.120%. It was found that the essential oil exhibited greater inhibitory action on the proliferation activity of HCT 116 cancer cells even though only small amount was used when compared to the inhibitory effect exerted by pure ethyl p-methoxycinnamate compound.
To conclude, the objectives of this study were successfully fulfilled. The essential oil obtained from the rhizome of K. galanga Linn. has the potential to be the remedy in treating colon cancer. The anticancer property of the essential oil was proved to be contributed by the presence of ethyl p-methoxycinnamate.
It is suggested for future research to identify whether the essential oil from the rhizome of K. galanga Linn. has the ability to induce apoptosis or necrosis in the cancer cells in order to develop the essential oil as one of novel anticancer drugs. The reasons behind this is that it is crucial to ensure that apoptosis is induced instead of necrosis to prevent inflammation and toxicity to other healthy living cells. It is recommended to utilize the flowcytometry analysis in evaluating the anticancer properties so that the exact potency of the essential oil for colon cancer treatment can be confirmed.


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APPENDIX A
GAS CHROMATOGRAPHY ' MASS SPECTROMETRY ANALYSIS
Appendix B1 Peak (1) Mass spectrum at Rt = 19.12'

Appendix B2 Compound endo-Borneol identified at Rt = 19.12
Appendix B3 'Peak (2) Mass spectrum at Rt = 26.41

Appendix B4 Compound (E)-methyl cinnamate identified at Rt = 26.41
Appendix B5 Peak (3) Mass spectrum at Rt = 29.22


Appendix B6 Compound ethyl cinnamate identified at Rt = 29.22

Appendix B7 Peak (4) Mass spectrum at Rt = 37.62

Appendix B8 Compound ethyl p-methoxycinnamate identified at Rt = 37.62

Appendix B9 Peak (5) Mass spectrum at Rt = 54.85
Appendix B10 Compound Bis (2-ethylhexyl) phthalate identified at Rt = 54.85s



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