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The word cancer is derived from "crab" in Latin due to the way a cancer adheres to any cells or tissues that it seizes upon like a crab. It is a common term to describe malignancy but there is another medical term for cancer that is neoplasia which from the Greek, means new formation. (Hart, I.R. Metastasis, 2002). Cancer is defined as the rapid creation of abnormal cells that grow beyond their usual boundaries, and which can then invade adjoining parts of the body and spread to other organs. Cancer cells have two main characteristics, which are, uncontrolled growth of cells in the human body and the ability of these cells to migrate from the original site and spread to distant sites which is referred as metastasis. Metastases are the major cause of death from cancer.
Cancer cell growth is different from normal cell growth. According to American Cancer Society, cancer begins when cells in a part of the body start to proliferate out of control. Cancer occurs due to damage of DNA. In a normal cell, when DNA gets damaged, the cell either repairs the damage or undergo apoptosis (programmed cell death) (Sato et al., 2008). But, in cancer cells, the damaged DNA is not repaired. The cell continue to grow, lump together and form a mass of tissue which known as malignant tumor. These new cells will have the same damaged DNA as the first cell does. This happened as a result of defects in the balance between the activity of proto-oncogene, which promote cell proliferation and tumor suppressor, which control the cell cycle (Shehata, 2005).
1.1.2 Cancer health statistics
According to World Health Organization, cancer is a worldwide disease which is labeled as one of the top ten leading causes of death which can attack anyone of us. There are more than 100 types of cancers and it can affect any part of the body. Since the occurrence of cancer increases as individuals' age, most of the cases are seen in adults, middle-aged or older.
According to National Cancer Registry, Malaysia (2006), there were a total of 21,773 cancer cases diagnosed among Malaysians in Peninsular Malaysia in the year 2006. The Age standardized Incidence Rate (ASR) for all cancers in the year 2006 regardless of sex was 131.3 per 100,000. In year 2005, 22,000 Malaysians are killed by cancers and 14,000 of them are below 70 years old. Cancer deaths account for 17.5% of the total deaths among Malaysian.(WHO, 2008)
Figure 1.1 National Cancer Registry Malaysia in 2006 showed the ten most frequent cancers occur in Peninsular Malaysia. (Adopted from: National Cancer Registry Malaysia, 2006)
In the order of frequency, the top five most common types of cancer that kill men are lung, stomach, liver, colorectal and oesophagus cancer while women are breast, lung, stomach, colorectal and cervical cancer in the order of frequency (American Cancer Society, 2008).
Ten most frequent cancers in females,
Peninsular Malaysia 2006
Ten most frequent cancers in males,
Peninsular Malaysia 2006
Figure 1.2: National Cancer Registry Malaysia showed the 10 most frequent cancers occur in both males and females in Peninsular Malaysia in year 2006. (Adopted from: National Cancer Registry Malaysia, 2006)
In Malaysia, breast cancer was claimed as the most common cancer among female and also the most important cancer among population regardless of sex in Peninsular Malaysia. While the cervical cancer was the third most common cancer among women (Figure 1.2). Besides, the National Cancer Registry, Malaysia (2006) showed that among the major races, Chinese women had the highest incidence for both breast and cervical cancer followed by Indian and Malay.
On the other hands, prostate cancer was stated as the forth most common cancer among males. It was highest among the Indians followed by Chinese and Malay.
1.1.3 Treatments and prevention of cancer
Treatment and prevention of cancers continue to be the focus of a great deal of research. There are several ways of treatment to cure the cancer including surgery, radiotherapy, chemotherapy, hormone therapy, bone marrow and stem cell transplants, biological therapies and so on.
Cancer therapy is an interplay among the induction of apoptosis or cell-cycle arrest, inhibition of angiogenesis, overcoming multidrug resistance (MDR) and boosting the immune system (Harendra S Parekh et al., 2009).
More than 30% of cancers could be prevented, mainly by not using tobacco, having a healthy diet, being physically active and preventing infections that may cause cancer (World Health Organization, 2008).
Other than that, cancer also and be prevented by having regular screening examinations. Detection and removal of precancerous growths, as well as the diagnosis of cancers at an early stage increase the probability of treatment and decrease the mortality rate. Cancers that can be diagnosed early through screening include cancers of the breast, colon, rectum, cervix, prostate, oral cavity, and skin.
1.2.1 What is herbal medicine?
Herbal medicine which has been used as the basis of health care throughout the world since the earliest days of mankind is still widely used. Recognition of their clinical, pharmaceutical and economic value is still growing, although this varies widely among countries (Jayasuriya DC, 1990).
Herbal medicine has two special characteristics which distinguish them from chemical drugs. In herbal medicine, crude herb is used and there is need for prolonged usage. There are experiences and evidences that show that there are real benefits in the long-term use of whole medicinal plants and their extracts, since the constituents in them work in conjunction with each other. Medicinal plants are important in pharmacological research and drug development. This is because not only the whole plant constituents are used directly as therapeutic agents, but they also serve as starting materials for the synthesis of drugs or as models for pharmacologically active compounds (Jayasuriya DC, 1990).
According to American Cancer Society, herbal medicine is a major aspect of traditional Chinese medicine. Traditional Chinese medicine focuses on restoring a balance of energy, body and spirit to maintain health rather than treating a particular disease or medical condition. Chinese medicine employs mixtures of naturally occurring herbs or herbs extracts (Yuan & Lin, 2000), and such mixtures are considered integral to the treatment. The foundation of various clinical efficacies observed in patients is believed to rely on numerous interacting combinations of natural products or herbs (Keith et al. , 2005). However, some bioactivities found in many of the natural product extracts disappear when the extracts were fractionated into individual chemical components (Foungbe et al. , 1991; Turner 1996; Schuster 2001). On the other hand, there may be some individual herbs or extracts that play a role in the prevention and treatment of cancer and other diseases when used in combination with conventional treatment.
1.2.2 Selected medicinal herbs in the study
In this study, a preparation containing a combination of 13 herbs is tested for their in vitro cytotoxic activities against human cancer cell lines based on the prior information on the folk medicinal use from Mr. Lim Kok Hong, a local medicinal practitioner and the supplier of the herbs use in this cytotoxic study. He claims that this combination of herbs has demonstrated positive effects on cancer patients. However, the cytotoxic effect and the effective compound in the herbs are not well understood as the formulation has only been used in traditional treatment. Therefore, this study is done to explore the cytotoxicity effect of combination on several cancer cell lines.
The combination of herbs in the preparation might have important synergistic effects which explained the history of successful cancer treatment among the volunteer cancer patients. All of the individual herbs in the formulations have been used traditionally as medicinal herbs and the following table shown some of the traditional uses of the herbs.
Stop bleeding (Moinon Jacy et al., 2009)
Anti-ulcer (Sim KS et al., 2010)
Antidote for snakebite (C. Yoosook et al.,1999)
Relieve herpes symptoms
Allergic and rashes
Burns and eczema (Visut Baimai et al.,1998)
Heat clearing (anti-viral and antibacterial)
(Quality of Prunella Vulgaris (Self heal). Available from: URL: http://www.planetherbs.com/)
(Suherman et al., 2004; Fauziah et al., 2005; Asmah et al., 2006b; Mohd Fadzelly et al., 2006a)
Anti-AIDS (Kusumoto et al., 1992)
(Norfarizan-Hanoon et al., 2009; Mohd Fadzelly et al., 2006b)
(Houttuynia cordata. Available from: URL: http://www.pfaf.org/user/Plant.aspx?LatinName=Houttuynia%20cordata)
Diuretic (promotes the excretion of urine)
Jaundice (Permana D, et al.,1999)
Diuretic (promotes the excretion of urine)
Detoxification (Subhuti Dharmananda, 2004)
Lobelia chinensis lour
Depurative (purifying the blood)
Diuretic (promotes the excretion of urine)
Febrifuge (removes fever)
(Chinese Lobelia. . Available from: URL: http://www.e2121.com/fherb.html)
Glycyrrhiza uralensis fisch
Detoxification (Fachun Wan et al., 2009)
Agrimonia pilosa ledeb
Anti-tumor (Kenichi et al., 1987; Pei et al., 1989a, 1989b)
Elephantopus scaber linn
Febrifuge (removes fever)
Diuretic (promotes the excretion of urine)
Antidote for snakebite
Anti-emetic (Wan Yong Ho et al.,2009)
Nan Fei Shu
Diabetes (Chan, 2006)
Table 1.1: Some of the traditional uses of each individual herbs in the combination of 13 herbs.
1.3 In vitro cytotoxic study
1.3.1 Principle of MTT assay Â
3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay is performed to evaluate the cytotoxicity of the herbs formulation on three types of cancer cell lines (MCF-7, CaSki and PC-3) and normal liver cells (Chang). The MTT assay was performed according to the method of Mossmann who first described the assay in 1983. It is a rapid and quantitative colorimetric method for determination of cell viability and measures the effectiveness of anticancer drugs on killing cancer cells. The MTT assay is well established for investigations of cellular viability in single cell cultures (Hayon T. et al. , 2003).
According to the principle of MTT assay, NADH dependent reduction activity in mitochondria of cells is able to reduce the tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], a pale yellow water-soluble quaternary ammonium dye into formazan, which is dark purple water-insoluble formazan salt crystal (Liu Y, 1999). Thus, the amount or concentration of formazan production is proportional to the number and viability of the cells. After this, DMSO is used to dissolve the formazan salts and the reading of optical density can be used to quantify the amount of formazan salts formed. Formazan dye is quantified by ELISA plate reader as optical density at the wavelength of 554nm (reference wavelength: 690nm). The absorbance is directly proportional to the viable cell number (Tan G.K. , 2005)
1.3.2 Optimization of MTT assay Â
The resulting absorbance of MTT assay is directly proportional to the viable cells number. However, the amount of formazan produced by a given number of cells varies between different cell lines (Tan et al., 2005). This is due to different types of cells have different size and proliferation rate. So, preliminary experiment is required to find out the optimal number of cells to be cultured in the 96-well flat-bottomed microplate for each individual cell line (Kawada et al., 2002). The optimization adjusts the number of culture cells to a density that allows the control cells to grow exponentianally (Kawada et al., 2002). This ensures the measurement of formazan produced at the end of the assay will not exceed the linear range of the assay.
According to Tan et al. (2004), the incubation time of three days after drugs treatment is sufficient for drug-induced cell death to occur.
In this study, the MTT assay was carried out according to the optimization for screened cell lines that have been done by Tan et al. (2004). The three cancer cell lines and normal liver cell screened in this study has been optimized by Tan et al. (2004), as shown in the table below.
Number of cells per well
wavelength used / reference wavelength (nm)
Table 1.2: Optimization of cell lines screened. Adopted from Tan et al. (2004).
After addition of prepared plants extracts, the cells are incubated for three days. The optimal incubation period after addition of MTT dye is three hours. After the solubilization of the formazan, the optical density values are recommended to read within one hours after addition of DMSO (Chan, 2006)
1.3.2 Advantages and limitations of MTT assay Â
There are several advantages as well as disadvantages of MTT assay. The main advantages of MTT assay is the speed of the assay which able to process numbers of sample at the same time. It is a simple and inexpensive quantitative assay which is capable of handling large number of samples due to it can be performed by using 96-well microplates by using multiwall spectrophotometer (Tan et al., 2005; Mosmann, 1983). Besides, the optical density after dissolving the formazan crystal with DMSO can be read after 10 minutes and it is stable within one hour (Tan at el., 2005). Furthermore, MTT assay is highly sensitive as it can detect as few as 3 viable cells per well (Tan at el., 2005).
However, it cannot be denied that there are several disadvantages of MT assay. Extra precautions are required when handling the assay as both of the MTT dye and DMSO which are the main chemical reagents in this assay are hazardous and carcinogenic (Tan at el., 2005). Other than that, MTT assay depends on the number of cells present and the motichondial activity per cell (Denizot and Lang, 1986). This shows that the results might be affected by the metabolic state of the tested cells. Furthermore, this assay cannot distinguish between cytostatic and cytocidal effects. So, it is unable to differentiate dead cell with no enzymatic activity frm static cells with very low enzymatic activity (Staton et al., 2004)
1.4 Research objectives
The objective of this study is to evaluate the cytotoxicity of extracts of a preparation containing a combination of 13 herbs in various types of human cancer cell lines including MCF-7, CaSki, PC-3 and a normal liver cell line (Chang cell) in vitro by using MTT assay.
At the same time, this study also proposes to determine the type of solvent that can most effectively extract the anticancer compounds according to the extract polarity that are responsible for the cytotoxic activity in the herbs formulation.
CHAPTER 2.0: MATERIALS AND METHODS
2.1.1 plant materials
A preparation containing a combination of 13 herbs used in this study was obtained from Mr. Lim Kok Hong, a medicinal practitioner at Tangkak, Johor. They were then extracted and processed at department of Physiology, Faculty of Medicine, University of Malaya. The following table shows the herbs which contained in the combination.
Lobelia chinensis lour
Glycyrrhiza uralensis fisch
Agrimonia pilosa ledeb
Elephantopus scaber linn
Nan Fei Shu
2.1.2 Cell lines
The secondary cell lines used for the screening purpose were obtained from Cell Culture Laboratory, Department of Physiology, Faculty of Medicine, University of Malaya.
The cell lines used in this study are stated as below:
Normal hepatic cell (control)
Human breast adenocarcinoma
Human cervix adenocarcinoma
Human prostate adenocarcinoma
Note: Photo of each cell line taken from the inverted microscope is shown in Appendices A.
2.1.3 Apparatus/ Equipments
Autoclave machine (Tomy, Japan)
Blender (Sumeet, Gujarat)
Centrifuge (Hettich, USA)
ELISA microplate reader
Freeze dryer (Heto, USA)
Laminar flow chamber (Holten LaminAir, Denmark)
Liquid nitrogen tank (Cryomed, USA)
Magnetic stirrer and magnetic bar
Multichannel pipettor (10-200Âµl)
Olympus CK-2 inverted microscope (Olympus, Japan)
Pipettor (10-100Âµl, 20-1000Âµl)
Rotary evaporator (Buchi, Switzerland)
Weighing machine (Ohaus, USA)
2.1.3 Chemicals and consumables
15ml and 50ml centrifuge tubes
Conical flasks and beakers (Pyrex, England)
Filter paper (Whatman, England)
Funnel (Pyrex, England)
Laboratory bottles(100ml, 250ml, 1L, 2L, 5L)
Sterile blue and yellow pipette tips (Axygen, USA)
95% ethanol (industrial grade) (R&M Chem, UK)
Hexane (industrial grade) (R&M Chem, UK)
Ethyl Acetate (industrial grade) (R&M Chem, UK)
Petri dishes (Pyrex, England)
Round-bottom flasks and adapter (Schott Duran, Germany)
Separating funnel (Isolab, German)
Cell Culture and in vitro cytotoxicity testing
Culture flasks (25cm3, 75cm3) (TPP, Switzerland)
Cryovials (TPP, Switzerland)
Filtration unit (10L) to sterile culture media (orange Scientific, Belgium)
Dimethyl sulphoxide (DMSO) (Applichem, Denmark)
Phosphate Buffer Saline (PBS) tablet (Oxoid, UK)
Trypsin 2.5% (Flowlab, Australia)
Ethylenediaminetetraacetic acid (EDTA) (Sigma, USA)
Foetal Bovine Serum (FBS) (PAA, Austria)
Dulbecco's Modified Eagles Medium (DMEM) (Flowlab, Australia)
Roswell Park Memorial Institute medium (RPMI) (Flowlab, Australia)
L-glutamine (Applichem, Denmark)
Liquid nitrogen (MOX, Malaysia)
Penicillin/ Streptomycin (PAA, Austria)
MTT dye (Applichem, Denmark)
Sterile syringe filter (0.22Âµm) (TPP, Switzerland)
96-wells flat-bottomed microplate (TPP, Switzerland)
2.2.1 Outline of the whole project
* If there is a significant cell death compared to the control, then quantitative determination of cell death will be done using MTT assay.
4.1 Preparation of herbs extract
The combination of thirteen herbs was extracted by using four solvents including 95% ethanol, hexane, ethyl acetate and water. The four different solvents have different polarities which can extract different fraction of the herbs. The procedures of preparing herb extraction are as follows:
Dry, grind to powder
Soak in ethanol for 3 days at room temperature (repeat for 3 times)
Filtration and evaporation of solvent
Filtration and Liquid-liquid extraction
Evaporation of solvent Water : Ethyl Acetate, ratio 1:1
Filtration, evaporation of solvent and freeze dry
*Hexane extract is air-dried in the fume cupboard for around 3 weeks.
The frozen cells (MCF-7, HT-29, CaSki and PC-3 and Chang) were removed from the nitrogen tank and thawed immediately at room temperature. After that, the cells were expanded in a 25cm cell culture flask using DMEM complete media which consists of 10 % heat inactivated fetal bovine serum (FBS), 1% of L-glutamine and 1% of penicillin/streptomycin. All cell lines were then incubated in humidified atmosphere of 95% air / 5% CO2 at 37oC. The media was changed every 2-3 days when the colour of the media change from red to yellowish in order to continuously supply the cells with nutrient. When the cells are 80-90% confluence, they were sub-cultured into a new flask or cryopreserved into the liquid nitrogen tank (for long term storage) or -80oC freezer (for short term storage). (Han H. Aung et al.,2007)
Procedure for thawing, subculturing, cryopreserving cells and cell counting are described in the following sections: 4.2.1, 4.2.2, 4.2.3, 4.2.4.
4.2.1 Thawing the cells
Procedures for thawing the cryopreserved cells are as follows:
Identify the cells to be removed from the liquid nitrogen tank
Immediately thaw the frozen cells to room temperature (RT) in 37ËšC waterbath
(or put under running tap water)
When the samples are in liquid form, clean the vial cap with 70% alcohol
Immediately transfer to 10 ml of culture medium in centrifuge tube
Centrifuge at 1000 rpm for 5 min
(Whitish pellet will be seen)
Discard supernatant and resuspend with 3 - 5 ml of culture medium
Pour into 25 ml culture flask
Examine under inverted microscope with phase contrast optic
Incubate > 6 hours (37ËšC) and change the medium
4.2.2 Subculturing / Expanding the cells
Procedures for subculturing / expanding the cells are shown as follows:
Examine cell culture under inverted microscope
Select "confluent" cells for subculture purpose
Discard media and rinse with 2 - 3 ml of PBS
Add trypsin + EDTA solution (2 - 3 ml)
Incubate ~ 2 min (37ËšC)
Tap gently and add culture medium
Split some of the medium into another culture flask or discard
Label (type of cell line, date, etc) the culture flask and examine under microscope
Incubate at 37ËšC, 5% CO2, 95% humidity
4.2.3 Cryopreservation of the cells
For satisfying cell recovery, the number of cells should ideally be between 1 Ã- 103 - 1 Ã- 106 per ml of freezing mixture. Procedures for cryopreservation of cells are shown as follows:
Prepare freezing mixture (90% FBS + 10% DMSO)
Discard media, rinse with PBS and add trypsin + EDTA solution,
incubate for ~ 2 min
Tap gently and transfer all the solution into centrifuge tube
Add appropriate amount of culture medium (~ 10 ml)
Centrifuge at 1500 rpm for 5 min and discard the supernatant
Add 1 ml of freezing mixture for each tube and mix well
Transfer to labeled cryovial and put into freezer (-80ËšC)
After 1 - 2 days, transfer the vial to liquid nitrogen tank (for long storage period)
4.2.4 Cell counting
Cell counting was performed using a hemocytometer. The number of cell viability was quantitated by trypan blue exclusion assay for the cell plating. The cells were diluted to a concentration of 1 Ã- 105 cells/ml by using the specific medium for the particular cell line. The process for the cell counting was performed as follows:
Place 80 Âµl of complete medium in a 1 ml eppendorf tube
Add 10 Âµl of the cell suspension
Add 10 Âµl of 0.25% trypan blue
Mix and incubate at 37ËšC (1 - 2 min)
Take a sample of the cell suspension and count in haemocytometer
Cell count calculation:
Cells per ml = M x DF x 1 ml
2 x 10-4
* M = average number of cells in one square, Dilution factor, DF = 10
For plating 3000 cells in 180 Âµl medium per well: consider there are 100 wells per plate, therefore 3 x 105 cells are needed in one 96-well microplate.
If M = 50 , cell per ml = 50 x 10 x 1 ml
2 x 10-4
= 2.5 x 106
Hence, volume of cell suspension needed = 3 x 105 x 1 ml
2.5 x 106
= 0.12 ml
Therefore, 0.12 ml of cell suspension will be pipette out and make up to final volume of 18 ml with complete culture medium.
In vitro cytotoxicity screening of plant extracts
The preliminary in vitro screening of herbs extracts against various cancer cells and normal liver cells will be evaluated by modified colorimetric MTT assay (Tan et al., 2005).
All of the crude extracts were first screened at the highest concentration(100 ug/ml). If the crude extracts show the cytotoxic effect in the cancer cells, then serial dilution of the plant extracts was prepared and quantitative determination of cell death is done by using MTT assay. Each test was set up in six wells where three as test wells and other three as control wells ( repeat 3 times intra-assay). The test wells contained culture medium, cultured cells and plant extracts while the control wells only contained extracts and culture medium.
The plant extracts were prepared by dissolving 5 mg of plant crude extract (ethanol, hexane, and ethyl acetate extracts) in 1 ml of DMSO (except water extract was dissolved in culture medium), followed by 10 times dilution. The stock solution obtained had a concentration of 5 mg/ml. The stock solution is then diluted in culture medium to a concentration range of between 25-100 Âµg/ml (Figure 2.1) .
The preparation of plant extract was shown as below:
50 mg of plant crude extracts were dissolved in 1 ml of DMSO (50 mg/ml)
The sample was filtered with 0.22 Âµl disposable syringe filter
The sample was diluted 10x by in culture medium(5 mg/ml)
1 ml of test extract with a series of concentration was prepared as follows:
Stock solution (Âµl)
Culture medium with 10% DMSO (Âµl)
Culture medium (Âµl)
Concentration in 1000 Âµl (Âµg/ml)
Final concentration of test extract in the 96-well microplate (Âµg/ml)
Figure 2.1 Preparation of plant extracts for in vitro cytotoxicity testing. The volume prepared here was enough for 50 wells or 8 tests. Each test was performed in at least triplicate and repeated for at least 2 times.
The in vitro cytotoxicity screening of human cancer cell lines using MTT assay involed a five days process for adherent cells.
On day 1, the cancer cell lines and normal liver cells were trypsinised and counted using a haemocytometer. The cells were then plated into a 96-well flat-bottomed microplate at 3000 cells (MCF-7 and PC-3) and 4000 cells (Chang and CaSki cells) per well in 180ul of culture medium and incubated overnight at about 37 oC. The incubation process enabled the cells to adhere to the plate.
On the next day, 20ul of plant extracts were loaded into each well. Then the plate was incubated for 72 hours in 37 oC.
On day 5, 50ul of MTT solution with concentration of 2 mg/ml were added to each well and further incubated for 3 hours at the same temperature. The formed formazan salt was then dissolved with DMSO. The optical density (concentration of formazan) was determined by using ELISA microplate reader within 1 hour, at wavelength 554nm. (reference wavelength: 690nm) The assay was performed in triplicate and the average absorbance values were calculated.
Determination of IC50 value
The IC50 value is the dose of extract that inhibits cell growth to 50% control levels.
The percentage of inhibition for each plant extract was calculated using the formula stated below:
Percentage of Inhibition = 1- Mean OD of extract - OD of its background___ Ã-100%
Mean OD of solvent control- OD of its background
The IC50 value was determined from the curve where percentage of inhibition (%) versus concentration of plant extract( Âµg/ml). Since the highest concentration tested was 100 Âµg/ml, the IC50 value is > 100 Âµg/ml if the percentage of inhibition does not exert 50% even at this highest concentration test.
The results in the experiments expressed as the mean Â± SD, were analyzed using Student's paired-t-test. A value of p < 0.01 was considered statistically significant.
CHAPTER 3.0: RESULTS
3.1 In vitro cytotoxicity of screened plant extracts
The extracts of the preparation contained combination of 13 herbs obtained using different solvents (ethanol, hexane, water and ethyl acetate) were screened the cytotoxicity activity against three types of human cancer cell lines ( MCF-7, PC-3 and CaSki) and compared to a normal human liver cell line (Chang). According to USA National Cancer Institute (NCI) guidelines, a plant is only considered as a potent cytotoxic agent if the IC50 value is less than 20Âµg/ml.
3.1.1 Cytotoxicity activity of the preparation contained combination of 13 herbs at 100Âµg/ml on screened cell lines.
The four extracts of the combination of 13 herbs including ethanol crude extracts, hexane extracts, water extract and ethyl acetate extract were first screened against three types of human cancer cell lines (CaSki, MCF-7 and PC-3) as well as normal hepatic cell (Chang) at concentration of 100Âµg/ml. All of the extract show inhibitions on the tested cancer cell lines as shown in the Figure 3.1, so serial dilution of the plant extracts were prepared and quantitative determination of cell death is done by using MTT assay.
Figure 3.1: Percentage of inhibition against types of extracts at the concentration of 100Âµg/ml on all of the tested cell lines
3.1.1 Cytotoxicity activity of the preparation contained combination of 13 herbs at serial concentration (0, 25, 50, 75, 100Âµg/ml) on screened cell lines.
After the serial dilution of the plant extracts were prepared and screened on all of the tested cell lines, the data showed that the percentage of inhibition of the ethanol crude extracts, hexane extracts and water extract were less than 50% on all of the tested cancer cell lines, including CaSki, MCF-7 and PC-3 as shown in Figure 3.2, Figure 3.3 and Figure 3.4. Hence, the IC50 value of these extracts against the screened cell lines were assumed to be more than 100Âµg/ml which are consider as weak or no significant cytotoxic effect against the tested cell lines.
However, the ethyl acetate extract showed a significant effect of inhibition more than 50% against all of the tested cancer cell lines which are CaSki (Figure 3.2), MCF-7 (Figure 3.3) and PC-3 (Figure 3.4) but not to the normal hepatic Chang cells as shown in Figure 3.5. Hence, the IC50 values of the ethyl acetate extract against the tested cancer cell lines are considered less than 100Âµg/ml. After the serial concentrations of these extracts were screened on all of the cancer cell lines as well as the normal hepatic cells to determine the IC50 value of the extract against the cell lines, the IC50 value of ethyl acetate against CaSki is 81Âµg/ml, while MCF-7 and PC-3 cell lines are 87.5Âµg/ml.
Figure 3.2: Percentage of inhibition against CaSki versus concentration of extracts
Figure 3.3: Percentage of inhibition against MCF-7 versus concentration of extracts
Figure 3.4: Percentage of inhibition against PC-3 versus concentration of extracts
Figure 3.5: Percentage of inhibition against Chang cells versus concentration of extracts
Figure 3.6: Percentage of inhibition against tested cell lines versus concentration of ethyl acetate extract.
In this study, the ethyl acetate extract show most potent effect compare to other extracts. However, the percentages of inhibition against tested cell lines are slightly different.
According to Figure 3.6, shows that ethyl acetate extract have higher percentage of inhibition against CaSki cell lines with the lower IC50 value of 81Âµg/ml, following by MCF-7 and PC-3 which share the almost same IC50 value of 87.5Âµg/ml. On the other hand, the percentage of inhibition against normal hepatic Chang cells is less than 30%.
Summary of results obtained in this study
The ethanol crude extract, hexane and water extracts of the combination of 13 herbs showed no significant cytotoxic activity on three tested cancer cell lines (CaSki, MCF-7 and PC-3) as well as normal hepatic cell line (Chang) as their IC50 values were estimated higher than 100Âµg/ml.
Meanwhile, the ethyl acetate crude extract of the combination of 13 herbs showed the most potent cytotoxic effect among the four extracts screened on all of the tested cancer cell (CaSki, MCF-7 and PC-3), in which their IC50 values are 81Âµg/ml against CaSki cell lines and 87.5Âµg/ml against both of the MCF-7 and PC-3 cell lines. On the other hand, the ethyl acetate extract showed no cytotoxic effect on the normal hepatic cell line (Chang) as the percentage of inhibition is less than 30% as shown in Figure 3.6.
However, according to USA National Cancer Institute (NCI) guidelines, a plant is only considered as a potent cytotoxic agent if the IC50 value is less than 20Âµg/ml. Thus, we can conclude that the cytotoxicity activity of ethyl acetate extract of the preparation contained a combination of 13 herbs are considered as weak cytotoxicity effect.
CHAPTER 4.0: DISCUSSION
4.1 Selection of plant samples
The herbs preparation contained combination of 13 herbs was selected in this study based on its popular traditional use among the volunteer cancer patients. According to Mr. Lim Kok Hong, the local Chinese medicinal herbs practitioner and supplier of the herbs, this hers formulation have been traditionally used for anticancer and health care purpose. Many volunteer cancer patients have demonstrated positive effects after consuming the herbs formulation for a certain period of time. Many of early and critical stage volunteer cancer patients who suffer from cervical, breast, prostate, colan and brain cancers have been cured or became healthier after the use of the formulation. He claimed that each of the herbs in the formulation was believed to have their own specific effects on the body and can be use as a health care product in daily life. He also claimed that most of the herbs contained in the formulation have the anti-cancer and detoxification effects which do not cause harm to our body.
4.2 Preparation of the crude extracts
Traditionally, Chinese herbs are consumed in the form of boiled crude extract or water decoction which normally consists of mainly polar compounds. However, some polar, semi-polar or non-polar compounds can be extracted on prolonged boiling. As the compounds that contribute to anticancer effect maybe of any polarity, so different solvents (ethanol, hexane, water and ethyl acetate) were used in this study to extract different compounds contained in the combination of the 13 herbs according to their polarity. The purpose was to increase the chance of extracting the compound(s) that exert cytotoxic effects towards the human cancer cell lines.
The extraction method used in this study is infusion method which is prepared by leaving the plants material to soak in the solvent at the room temperature for a period of time with intermittent shaking, followed by filtration to separate away the plants debris.
The choices of the solvents should be considered carefully. An ideal solvent for extraction should not cause the extract to complex or dissociate, be easy to remove or evaporated at low heat, preservative in action, be inert, non-toxic and not easily flammable. According to 'like dissolves like' principle, non-polar solvents will extract out non-polar substances and polar compound will be extracted out by polar solvents. Alcoholic solvents can efficiently penetrate the cell membrane and extract high amounts of the endocellular components. Ethanol was chosen as the preliminary extraction solvent due to it meet all above criteria. Ethanol able to dissolve and extract a wide range of compounds from the polar ,semi-polar to non-polar. However, there are some studies showed that the effects of crude preparation of extract are generally slower in onset and less dramatic than those of the purified active compounds. Therefore, the crude ethanolic extract was further fractionated by organic solvent partition to get the hexane, ethyl acetate and water extracts. Hexane with the lowest polarity among the four extraction solvents was used to extract the non-polar compounds. While ethyl acetate was used to extract the semi-polar compounds and water with the highest polarity extracted the polar compounds from the preparation contained a combination of 13 herbs.
After extraction, the solvent was eliminated by rotary evaporation at the appropriate temperature to avoid some of the thermolabile compounds to be degraded by high temperature (Silva et al., 1998). After that, the concentrated extracts were left in the fume cupboard to ensure all of the solvents used to extract the compound were totally eliminated. Then the extracts were send to freeze dry to eliminate the water contained in the extracts which may interfere with the assay. The dried crystal-like extracts were easier to be weighted and stored or reconstituted in a different solvent for further processing.
4.3 In vitro cytotoxicity of plants extracts on human cancer cell lines
According to USA National Cancer Institute (NCI) guidelines, only extracts with IC50 value less than 20Âµg/ml are considered as a potent cytotoxic agent. In this study, each test was performed in triplicate and repeated at least 3 times to obtain the best curve result. Student t-test was performed to determined whether the readings are reliable and significant or not.
Referring to USA National Cancer Institute (NCI) guidelines, none of the tested plants extractsin this study had the potent cytotoxic effect as their IC50 values were more than 20Âµg/ml and some of the extracts did not exhibited percentage of inhibition more than 50% against the tested cancer cell lines. However, there is ethyl acetate extract which exhibited percentage of inhibition more than 50% on all the tested cancer cell lines in which their IC50 values are 81Âµg/ml against CaSki cell lines and 87.5Âµg/ml against both of the MCF-7 and PC-3 cell lines. Besides, all of plants extracts were found to be non-toxic to normal hepatic cell (Chang) as all the plants extracts have exhibited percentage of inhibition less than 30%.
Even though none of the plants extracts exhibited strong cytotoxic effect, the ethyl acetate extract is the most prominent among all the 4 extracts tested against all the testes cancer cell lines. It has the highest percentage of inhibition more than 50%, with IC50 value range between 75-100Âµg/ml on all of the tested cancer cell lines, especially to CaSki cells. This might indicate that the active compound(s) or the compound(s) responsible for the anti-cancer properties of the 13 herbs in the formulation is within this fraction. As the ethyl acetate is a solvent with medium polarity, so the compound(s) been extracted by ethyl acetate Purification of this fraction can be done and test again with the same cancer cell lines.
In the preparation contained combination of 13 herbs are traditionally used for anti-cancer and health care purpose to treat or prevent from getting of cancer. Despite of its history of traditional medical uses in many successful cases, there is no scientific evidence to prove the cytotoxic activities of this combination. However, some of the individual herbs in the combination do have some documented scientific data.
Recent study have reported that the methanol extract of Pereskia bleo possessed cytotoxic effects against T-47D cells which is human ductal breast epithelial tumor cell line with IC50 value of 2Âµg/ml (Tan et al., 2005). Another investigation by Er et al., YEAR indicated the antiproliferative and mutagenic activities of aqueous and methanol extracts of Pereskia bleo leaves against mouse mammary cancer cells (4T1) or normal mouse fibroblast cells (NIH/3T3).
As reported by Wing-Yan Li et al. (2007), the water extract of Agrimonia pilosa Ledeb had the highest growth inhibitory effect on human lung adenocarcinoma (A549) cell and human breast cancer (MCF-7) cell. Other than that, 'Agrimoniin' from A. pilosa was also reported as a potent anti-tumor tannin with effects possibly due to enhancement of immune response in host animals (Miyamoto K et al., 1987; Murayama T. et al., 1992).
According to Wong et al. (2009), Scutellaria barbata might modulate the apoptosis by upregulating the apoptotic pathway and downregulating the survival pathway in prostate cancer cells, thus suggesting that Scutellaria barbata possesses chemopreventive properties and has potential for cancer treatment. Moreover, an aqueous extract from the herb Scutellaria barbata D. Don (BZL 101), is currently in phase II clinical trial in patients with advanced breast cancer. Preclinical studies suggest that this herb has antitumor activity for breast cancer and preliminary clinical data suggest that it is tolerable in patients with metastatic breast cancer (Fong S. et al., 2008).
The ethanolic crude extract of Rodgersia sambucifolia showed a potent cytotoxic effect against the proliferation of human leukemia cell (K562)with IC50 value of 22.0Î¼g/ml and a lower cytotoxicity against Human promyelocytic leukemia cell (HL60) with IC50 value of 63.0Î¼g/ml (Kim Kah Hwi et al., 2009).
Recently, there are quite a number of studies showed that ethanol extract of Elephantopus scaber Linn have the cytotoxic effect against various types of cancer cell lines. For examples, Liang et al. (2008) and Xu Et al. (2006) reported that ethanol extract of Elephantopus scaber Linn have cytotoxic effect on human hepatocarcinoma (SMMC7721), human colon carcinoma (Caco-2) and human cervical carcinoma (HeLa).
Eun-Hye Jo et al. (2004) reported that ethanol extract of Chinese licorice root, Glycyrrhiza uralensis has an estrogen-like activity and anti-cancer effects against MCF-7 human breast cancer cells. Besides, Hawthorne S et al. (2008) also showed that licorice extract reduced prostate specific antigen release and reduced cultured prostate cancer cell line (LNCaP) proliferation.
According to Su Hyun Hong et al. (2006), they previously reported that water extract of Houttuynia cordata can inhibit cell proliferation and induce apoptosis in human breast carcinoma cells. After that, they investigated the biochemical mechanisms of anti-proliferative effects by the methanol extract of Houttuynia cordata in human lung carcinoma A549 cells. It was also found that the methanol extract could inhibit the cell growth in a dose-dependent manner.
In year 2005, Li et al. reported that the endophytic fungi isolated from Hedyotis diffusae were screened and results showed that the endophytic fungi exhibited anti-tumor activities on human gastric tumor cell line (BGC-823).
On the other hand, Kusumoto et al (1992) have reported that the Strobilanthes crispus have the cytotoxic effect to inhibit the growth of adult T-cell leukemia. In year 2006, Rahmat et al. showed that its methanolic extract have IC50 values at the range between 22-29Âµg/ml against HepG-2, Caco-2 and MDA-MB-231 .
According to Chong S.S. (2009), ethanolic crude extract of Nan Fei Shu showed weak cytotoxic effect on HT-29 and Skov-3 cancer cell lines, in which the IC50 values ranged from 40-90Âµg/ml and the ethyl acetate extract showed stronger cytotoxic activity on human breast adenocarcinoma cell lines (MCF-7), human colonic adenocarcinoma cell lines (HT-29), human ovarian adenocarcinoma cell lines (Skov-3) as well as normal hepatic cells (Chang) . Their IC50 values ranged from 30-60Âµg/ml.
4.4 In vitro cytotoxicity of plants extracts on human normal hepatic cell line (Chang)
In this study, all of the ethanol, hexane, ethyl acetate and water extracts tested against the normal hepatic cell line (Chang) showed no IC50. This showed that the percentage of inhibition of all of the extracts against the control normal hepatic cell lines (Chang) is less than 50%. Therefore, this showed that the preparation contained combination of 13 herbs is totally safe to be consumed, as may not contain cytotoxic compound on liver.
Even though one of the herb contained in the combination which is Nan Fei Shu was reported have strong cytotoxic effect on normal hepatic Chang cells, but the cytotoxic effect disappear or become weak when using in the combination. This may due to