J Acupunct Meridian Stud 2011;4(4):248e256
In Vitro Antiproliferative and Antioxidant Activities and Total Phenolic Contents of the Extracts of Melastoma malabathricum Leaves Z.A. Zakaria 1,2,*, M.S. Rofiee 3, A.M. Mohamed 3, L.K. Teh 2, M.Z. Salleh 2 1
Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 2 Pharmacogenomics Center, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia 3 Faculty of Biotechnology and Life Sciences, Universiti Industri Selangor, Jalan Zirkon A7/A, Seksyen 7, 40000 Shah Alam, Selangor, Malaysia Available online Oct 19, 2011 Received: May 31, 2011 Revised: Jul 9, 2011 Accepted: Jul 14, 2011 KEYWORDS antioxidant activity; antiproliferative activity; Melastoma malabathricum; phenolic compounds
Abstract The present study aims to determine the in vitro antiproliferative and antioxidant activities of various extracts from the leaves of Melastoma malabathricum using various established in vitro assays. The aqueous extract inhibited the proliferation of Caov-3 and HL-60 cell lines, while the chloroform extract exhibited antiproliferative activity against the Caov3, HL-60, and CEM-SS cell lines. The methanol extract demonstrated antiproliferative activity against more cell lines, including the MCF-7, HeLa, Caov-3, HL-60, CEM-SS, and MDA-MB-231 cancer cell lines. Interestingly, all extracts did not inhibit the proliferation of 3T3 cells, thus indicating their noncytotoxic properties. Unlike the chloroform extracts, the aqueous and methanol extracts of M malabathricum (20, 100, and 500 mg/ml) produced high antioxidant activity for the superoxide scavenging assay with only the 500 mg/ml aqueous and methanol extracts exhibited high activity for the 2,2-diphenyl -1picrylhydrazyl radical scavenging assay. The total phenolic content recorded for the aqueous, methanol, and chloroform extracts were 3344.2 19.1, 3055.1 8.7, and 92.5 7.3 mg/100 g of gallic acid, respectively. The M malabathricum leaves possessed potential antiproliferative and antioxidant activities that could be attributed to its high content of phenolic compounds.
1. Introduction Cancer is a major health problem with 1 in 4 deaths occurring in the United States . Despite the advancement
in the therapy and diagnosis for cancer, patients and their families are faced with psychological distress due to cancer’s chronic clinical signs and symptoms. Pain is one of the most frequent and disturbing symptoms experienced by
* Corresponding author. Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. E-mail: [email protected]
Copyright ª 2011, Korean Pharmacopuncture Institute doi:10.1016/j.jams.2011.09.016
Antiproliferative of Melastoma malabathricum patients in all stages of cancer . Despite the widespread dissemination of the World Health Organization’s threelevel ladder, and the demonstration that its appropriate use can relieve pain in more than 90% of cases, pain management remains poor [3e5]. The use of herbal remedies as a complimentary and alternative medicine (CAM) for the treatment of patients with cancer or other chronic diseases have been greatly acknowledged [6,7]. Although less prevalent, CAM approaches have been associated with some success and symptom relief in several diseases, particularly in cancer . Interestingly, the use of CAM was more prevalent among women, and among adults who had one or more existing health conditions or with a higher education, as well as with adults who made frequent medical visits in the previous years or who engaged in leisure-time physical activity . One of the plants that was believed to possess potential anticancer activity and is currently under investigation in our laboratory is Melastoma malabathricum (family: Melastomataceae). The shrub is commonly found locally and known to the Malays as “senduduk” . The plant has been used by practitioners of traditional medicine in Malays to treat various types of ailments such as inflamed wounds, bleeding , pox scars, diarrhea , and hemorrhoids . Scientifically, the ethanol, chloroform, and aqueous extracts of M malabathricum leaves have been shown to possess antinociceptive activity at both the peripheral and central levels [9,12,13]. The antinociceptive activity of the ethanol extract was found to cause activation of opioid receptors. Earlier phytochemistry studies revealed that the plant contained b-sitosterol, a-amyrin, sitosterol-3-O-b-Dglucopyranoside, quercetin, quercitrin, and rutin [14,15]. Several attempts have been made to study the pharmacologic properties of some of these compounds. For example, Susanti et al.  have isolated naringin, kaempferia, and kaempferol-3-O-D-glucoside from the ethyl acetate extract and kaempferol-3-O-(200 ,600 -di-O-p-trans-coumaroyl)-b-glucopyranoside and kaempferol-3-O-D-glucoside from the methanol extract of M malabathricum. Together with their respective crude extracts, these compounds were found to exhibit strong antioxidant activity when assessed using the 2,2-diphenyl -1-picrylhydrazyl (DPPH) radical-scavenging electron spin resonance (ESR) spectroscopic method. Later, Susanti et al.  also reported on the antioxidant potential of extracts of the leaves of white petals of M malabathricum (extracts using n-hexane, ethyl acetate and methanol) as well as the isolated compounds (e.g., a-amyrin, patriscabatrine and auranamide, hyperin, quercetin, quercitrin, kaempferia, kaempferol-3-O-b-D-glucose, and kaempferol-3-O-(200 ,600 -di-O-p-trans-coumaroyl) glucoside. This was assessed using the ferric thiocyanate and DPPH (ultraviolet and ESR spectroscopic) methods. Susanti et al.  also reported on the cytotoxic activity of the M malabathricum flowers (extracted using ethyl acetate, but not methanol) and several compounds isolated from the crude extract of ethyl acetate e.g. naringin, kaempferia, kaempferol-3-O-D-glucoside, kaempferol-3-O-(200 ,600 -di-Op-trans-coumaroyl)-b-glucopyranoside, and kaempferol-3O-D-glucoside. The cytotoxic activity was assessed against a MCF-7 cell line using a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. The anticancer activity of those compounds was demonstrated to involve
249 cell proliferation and changes in the cell morphology. On the other hand, Mazura et al.  reported in vitro anti-inflammatory potential of several compounds (e.g. a.-amyrin, betulinic acid, quercetin and quercitrin) isolated from M malabathricum as indicated by their ability to inhibit the platelet-activating factor (PAF) binding to rabbit platelets using 3H-PAF as a ligand. Quercetin, quercitrin, a.-amyrin, and betulinic acid showed inhibition of the PAF receptor binding to rabbit platelets with IC50 values of 33.0, 45.4, 20.0, and 22.2 mM, respectively. The results were comparable with a known PAF receptor antagonist, cedrol (13.1 mM). Later, Susanti et al.  reported on the antiinflammatory activity of topically applied kaempferol-3O-(200 ,600 -di-O-p-trans-coumaroyl) glucoside and a-amyrin obtained from n-hexane, ethyl acetate, and methanol extracts of M malabathricum using a 12-O-tetradecanoylphorbol-13-acetate-induced mouse ear edema assay. As M malabathricum grows wildly in Malaysia and only a limited number of studies has been carried out to explore its pharmacologic benefits, we have taken the opportunity to evaluate and establish some of its basic pharmacologic properties. The aim of the present study is to determine the in vitro antiproliferative and antioxidant activities of the aqueous, chloroform, and methanol extracts of M malabathricum leaves using the MTT, the DPPH radical scavenging, and superoxide scavenging assays respectively.
2. Materials and methods 2.1. Plant materials The plant M malabathricum has been previously identified by Shamsul Khamis, a botanist from the Institute of Bioscience (IBS), Universiti Putra Malaysia (UPM), Serdang, Selangor, Malaysia, and the voucher specimen, SK 507/03, was deposited at the Herbarium of IBS, UPM. For the present study, the leaves of M malabathricum were collected from their natural habitat in Shah Alam, Selangor, Malaysia in August 2007 to September 2007.
2.2. Preparation of the aqueous, chloroform, and methanol extracts of M malabathricum leaves The matured leaves of M malabathricum were air dried for 1e2 weeks at room temperature (27 2 C) based on previous studies [9,13]. It is believed that this process did not cause destruction of the bioactive compounds of the leaves of the plants as proven by the presence of antinociceptive, anti-inflammatory, antioxidant, and antibacterial activities as described earlier. Furthermore, the leaves of M malabathricum were air dried to remove the water content of the leaves in order to get the exact amount of samples prior to the soaking process. The dried leaves were then ground into small particles, weighed (40 g), and then sequentially soaked at room temperature for 72 hours with distilled water (dH2O), chloroform, and methanol in the ratio of 1:20 (w/v). Each of the mixture solutions were collected and filtered using Whatman No. 1 filter paper (Whatman, UK) to obtain the aqueous, chloroform, and methanol supernatants. The aqueous extracts of M malabathricum was kept at e80 C for at least 48 hours and then
250 subjected to the freeze-drying process, leading to a yield of 1.98 g (4.95%) of crude extracts. The chloroform and methanol extracts of the plants were evaporated (40 C) under reduced pressure to dryness, which resulted in a yield of 2.14 g (5.35%) and 0.80 g (2%), respectively. All of the crude dried extracts obtained were kept at 4 C and, prior to use, the aqueous extracts were dissolved in dH2O while the chloroform and methanol extracts were dissolved in dimethyl sulfoxide (DMSO) to make up the concentration of 100 mg/ml.
2.3. MTT assay All cell lines cultures of the American Type Culture Collection, namely 3T3 (normal mouse fibroblast), MCF-7 (estrogen-dependent human breast adenocarcinoma), HeLa (human cervical adenocarcinoma), Caov-3 (human ovarian adenocarcinoma), HL-60 (acute promyelocytic leukemia), CEM-SS (T-lymphoblastic leukemia) and MDA-MB-231 (human breast carcinoma) were purchased from the Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. The proliferative activity of extract at diluted ranges of 12.5, 25, 50, and 100 mg/ml was determined using the MTT assay (3- [4, 5 - dimethylthiazol - 2-yl]2,5-diphenyl tetrazolium bromide) .
2.4. Antioxidant assays DPPH radical scavenging activity. The assay for the DPPH free radical scavenging potential is based on the scavenging activity of stable DPPH free radicals . Reaction mixtures containing test samples dissolved in methanol and 200 mM DPPH (Sigma-Aldrich, USA) in ethanolic solution were incubated at 37 C for 30 minutes in a 96-well microtiter plate. After the reaction, absorbance was measured at 520 nm, and percent inhibition was calculated. Superoxide anion radical scavenging activity. The superoxide anion radical scavenging activity was performed using the method adopted by Zakaria and others . This assay is based on the removal rate of xanthine/xanthine oxidasegenerated superoxide by measuring the reduction of nitro blue tetrazolium (NBT). The sample solution (0.1 mg.ml1) in 5% DMSO was added to 1 mL of a mixture of 0.1 mM xanthine and 0.2 mM NBT (Sigma, USA) in a 50-mM potassium phosphate buffer (pH 7.5) containing 0.05 mM ethylenediaminetetraacetic acid. Xanthine oxidase (0.1 mL) (Sigma, USA; 0.8 unit.mL1) diluted in 50 mM phosphate buffer (pH 7.5) was added, and the resulting mixture was incubated at 37 C for 20 minutes. The addition of 2 mL of 2.5 N HCl to the mixtures terminated the reaction, followed by increase of coloration of NBT, which was measured at 540 mM. The percent of removal rate by sample was calculated relative to the control.
2.5. Phytochemical screening of the aqueous, chloroform, and methanol extracts of the M malabathricum leaves The phytochemical screening was carried out on the aqueous, chloroform, and methanol extracts of M
Z.A. Zakaria et al. malabathricum leaves according to the standard screening tests and conventional protocols adopted by Zakaria and others .
2.6. Determination of the total phenolic contents of aqueous, chloroform and methanol extracts of M malabathricum The total phenolic content of extracts obtained from M malabathricum was determined using the FolineCiocalteu method as adopted by Zakaria and others . Briefly, 1.0 mL of each extract was added to 10.0 mL distilled water and mixed with 2.0 mL of FolineCiocalteu phenol reagent (Merck-Schuchardt, Hohenbrun, Germany). The mixture was left at room temperature for 5 minutes and subsequently 2.0 mL of sodium carbonate was added. The blue complex formed was then measured at 680 nm and catechin was used as the standard for the calibration curve. The content of the phenolic compound was derived from the calibration curve. The content of phenolic compound was expressed as mg catechin equivalent/g dry weight (mg.g1). The dry weight indicated was the dry weight of the leaves of M malabathricum.
3. Results DMSO alone was found to be ineffective in exhibiting antiproliferative activity as indicated by its failure to produce IC50 against all of the cell lines used in this study (data not shown). The antiproliferative activities of the aqueous, chloroform, and methanol extracts of M malabathricum leaves against the normal cell line (3T3) are illustrated in Fig. 1. Interestingly, not all extracts produced antiproliferative or cytotoxic effects on the cells as indicated by the percentage of viability of the 3T3 cells that were generally above 70%. Fig. 2 shows the antiproliferative profiles of the aqueous, chloroform, and methanol extracts of M malabathricum leaves against the MCF-7 cancer cell lines. Only the methanol extract exhibited antiproliferative activity against the MCF-7 and the estimated IC50 value recorded was 87 mg/ml. The effects of the aqueous, chloroform, and methanol extracts of M malabathricum leaves on the proliferative potentials of the HeLa cancer cell lines is illustrated in Fig. 3. The methanol and chloroform extracts of the M malabathricum leaves were found to produce IC50 values of approximately 88 and 96 mg/ml, respectively. The abilities of the aqueous, chloroform, and methanol extracts of the M malabathricum leaves to inhibit the proliferation of Caov-3 cancer cell lines are demonstrated in Fig. 4. All extracts exhibited antiproliferative activity with the chloroform, followed by the methanol and aqueous extracts of M malabathricum leaves producing IC50 values of 34, 41, and 58 mg/ml, respectively. Fig. 5 shows the antiproliferative profiles of the aqueous, chloroform, and methanol extracts of M malabathricum leaves against the HL-60 cancer cell lines. All extracts inhibited the proliferation of HL-60 cells with the aqueous, methanol, and chloroform extracts producing IC50 values of approximately 11, 13, and 30 mg/ml, respectively.
Antiproliferative of Melastoma malabathricum
Figure 1 In vitro antiproliferative activity of the aqueous, chloroform, and methanol extracts of M malabathricum leaves against 3T3 normal cell lines.
The antiproliferative profiles of the aqueous, chloroform, and methanol extracts of M malabathricum leaves against the CEM-SS cancer cell line are shown in Fig. 6. Only the chloroform and methanol extracts of M malabathricum leaves exhibited antiproliferative activity against CEM-SS with the approximate IC50 values of 22 and 30 mg/ml, respectively. The antiproliferative profiles of the aqueous, chloroform, and methanol extracts of M malabathricum leaves against the MDA-MB 231 cancer cell lines are shown in Fig. 7. Only the methanol extract of M malabathricum leaves exhibited antiproliferative activity against MDA-MB 231 with the approximate IC50 value of 59 mg/ml, respectively. Comparison of IC50 was also performed between the extracts of M malabathricum leaves and tamoxifen, a standard antitumor drug (Table 1). Overall, tamoxifen was a more potent antiproliferative agent compared with the three extracts of the plant as indicated by its lower IC50 values.
Table 2 shows the antioxidant activities of the aqueous, chloroform, and methanol extracts of M malabathricum assessed using the DPPH radical scavenging and superoxide scavenging assays. The 20, 100, and 500 mg/ml methanol extract produced approximately 13%e98% of DPPH radical scavenging activities, while the aqueous followed by the chloroform extract produced 7%e70% and 0%e18% radical scavenging activities, respectively. For the superoxide scavenging activity, the 20, 100, and 500 mg/ml methanol extract produced approximately 91%e99% effects, while the aqueous and chloroform extracts caused approximately 80%e96% and 28%e53% scavenging activities, respectively. In addition, the evaluation of the total phenolic content of the three extracts (6.25 mg/ml) indicates that the aqueous and methanol extracts contain the highest phenolic content, which are approximately 3344 and 3055 mg/100 g gallic acid, respectively. The total phenolic content in chloroform extract was approximately 92 mg/100 g of gallic acid (Table 3).
Figure 2 In vitro antiproliferative activity of the aqueous, chloroform, and methanol extracts of M malabathricum leaves against MCF-7 cancer cell lines.
Z.A. Zakaria et al.
Figure 3 In vitro antiproliferative activity of the aqueous, chloroform, and methanol extracts of M malabathricum leaves against HeLa cancer cell lines.
The aqueous extract of M malabathricum was found to contain only flavonoids, the chloroform extract was found to contain flavonoids and steroids, and the methanol extract was found to contain saponins, flavonoids, condensed tannin, and steroids (Table 4).
4. Discussion Cancer cells exist under a state of oxidative stress because this condition increases their potential to survive by activating redox signaling that may lead to the activation of prosurvival factors such as NFkB and AP-1  and the inactivation of tumor suppressor genes such as p53  or mutations . In addition, mild levels of reactive oxygen species have been shown to induce the proliferation of cancer cells . Epidemiologic studies suggest that the
Figure 5 In vitro antiproliferative activity of the aqueous, chloroform, and methanol extracts of M malabathricum leaves against HL-60 cancer cell lines.
consumption of a phytochemical-rich diet (i.e., vegetables and fruits) could be associated with a reduced risk of cancer . Plants contain high levels of a diverse range of phytochemicals, which include polyphenols like flavonoids, hydrolysable, and condensed tannins . In particular, polyphenols have been shown to exert anticancer effects via mechanisms that include antioxidant, antiproliferative, and anti- inflammatory activities as well as their effects on subcellular signalling pathways, apoptosis, and induction of cell-cycle arrest . Therefore, foods rich in antioxidant phytochemicals are important for the prevention of diseases related to oxidant stress such as heart disease and cancer.
Figure 4 In vitro antiproliferative activity of the aqueous, chloroform and methanol extracts of M malabathricum leaves against CaoV3 cancer cell lines.
Antiproliferative of Melastoma malabathricum
Figure 6 In vitro antiproliferative activity of the aqueous, chloroform, and methanol extracts of M malabathricum leaves against CEM-SS cancer cell lines.
There are considerable scientific and public interests in the important role that antioxidants may play in health care, such as cancer chemopreventive and antiinflammatory agents and by reducing the risk of cardiovascular mortality . The present study has revealed the potential anticancer and antioxidant properties of the various extracts of M malabathricum. Together with the previously reported anti-inflammatory and antinociceptive activities, we have confirmed the findings made by various other researchers . The free radical scavenging property, together with the anti-inflammatory activity, are believed to be part of the mechanisms by which this plant is effective in the antiproliferative activity as described earlier . From the data obtained, the methanol extract was considered to be the most effective extract because it inhibited the proliferation of all cancer cells. The phytochemical screening of the methanol extract demonstrated the presence of flavonoids, condensed tannins, saponins, and steroids. The aqueous extract was only effective
against the Caov-3 and HL-60 cells, and this activity could be associated with the sole presence of flavonoids in the extract. On the other hand, the chloroform extract exhibited antiproliferative activity against the Caov-3, HL60, and CEM-SS cells, and its phytochemical screening indicates the presence of flavonoids and steroids. Interestingly, not all extracts exerted an inhibitory effect on the proliferation of the 3T3 (normal), which indicates their noncytotoxic effect. In terms of the phytochemical constituents’ effects, flavonoids, which are present in the aqueous, chloroform, and methanol extracts of M malabathricum, inhibited the proliferation of MCF-7 , HeLa , HT-29 , HL-60 , and K-562  cells. Tannins, which are found in the methanol extract, are also reported to inhibit MCF-7 , HeLa , HT-29 , HL-60, and K-562  cells. Saponins, which are detected in the methanol extract of M malabathricum, have been reported to inhibit the proliferation of HL-60 and K-562 . It is believed that the synergistic actions of these compounds within the methanol extract contribute to the observed
Figure 7 In vitro antiproliferative activity of the aqueous, chloroform, and methanol extracts of M malabathricum leaves against MDA-MB231 cancer cell lines.
Z.A. Zakaria et al.
Comparison of the IC50 values between tamoxifen and the various extracts of M malabathricum leaves.
IC50 values (mmg/ml)
Tamoxifen M malabathricum extracts
Aqueous Chloroform Methanol
ND ND ND ND
8 ND ND 87
4 ND 96 88
ND 58 34 41
ND 11 30 13
3 ND 22 30
ND ND ND 59
ND [ Not detected because the concentration of extracts/drugs required to exhibit IC50 value were above 100 mg/ml (the highest concentration used).
Table 2 Antioxidant activity of various extracts of M malabathricum leaves assessed by DPPH radical scavenging and superoxide scavenging assays. Sample
DPPH radical scavenging (%)
Superoxide scavenging (%)
20 100 500 20 100 500 20 100 500
7.1 1.5 20.5 1.9b 69.8 6.1c 0.0 0.0a 2.1 0.3a 17.9 0.2b 13.6 3.9d 43.0 1.8e 97.3 0.1f
80.0 2.2v 87.9 1.2v 95.6 0.3w 28.1 0.8x 38.3 0.3y 53.2 0.9z 91.3 0.2vw 98.5 0.7w 98.5 0.5w
Data differed significantly (p < 0.05) when compared with each other within the DPPH radical scavenging column. Data differed significantly (p < 0.05) when compared with each other within the superoxide scavenging column.
Total phenolic content of various extracts of M malabathricum leaves.
Total phenolic content (gallic acid mg/100 g)
Aqueous Chloroform Methanol
3344.2 19.09a 92.46 7.25b 3055.1 8.68c
Data differed significantly (p < 0.05) when compared with each other within the total phenolic content column. TPC [ total phenolic content, expressed as mg equivalent of gallic acid per 100 g of dry weight (gallic acid mg/100 g); TPC value > 1000 mg gallic acid/100 g. a,b,c,
antiproliferative activity of the extract against various cancer cells. We have also studied the total phenolic content and antioxidant activity of the extracts. Extracts with high total phenolic content, including the aqueous and methanol extracts, were found to exhibit high antioxidant capacity when assessed by the DPPH radical scavenging and superoxide scavenging assays. The linear co-relation between the high total phenolic content and the antioxidant activity observed was in line with previous reports . It is plausible to suggest that the antioxidant and free radical scavenging properties of those extracts contribute directly or indirectly to the antiproliferative activity of extracts . The presence of those compounds in the respective extract of M malabathricum were in line with our previous phytochemical screening of the leaves M malabathricum, which demonstrated the presence of flavonoids, tannins, saponins, and steroids, but no alkaloids . Flavonoids, saponins, and tannins have all been reported to possess antioxidant and anticancer activities [28,37,38]. The
presences of these compounds are believed to contribute partly to the observed antiproliferative activity through the antioxidant and free radical scavenging effects. Several antiproliferative mechanisms could be plausibly suggested based on the types of phytochemical constituents detected in each of the extracts. Flavonoids, which are detected in the aqueous, chloroform, and methanol extracts, have been previously reported to (1) induce the cyclin-dependent kinase inhibitors or the Ca2þ-dependent apoptotic mechanism, (2) modulate the cell cycle arrest at the G1/S phase, (3) inhibit the cell-survival kinase and the inflammatory transcription factors, or (4) down-regulate the antiapoptotic gene products [39,40]. Other than that, flavonoids can also modulate the expression of proinflammatory genes, such as nitric oxide synthase and cyclooxygenase-2 [41,42], thus indicating its potential in modulating the oxidative processes. Saponins, which are only detected in the methanol extract, were reported to induce an apoptosis response on cancer cells through the permeabilization of the mitochondrial membranes .
Antiproliferative of Melastoma malabathricum
Table 4 The phytochemical constituents of the leaves of M malabathricum aqueous, chloroform, and methanol extracts. Constituents
Flavonoids Triterpenes Tannins Alkaloids Saponins Steroids
Extracts of M malabathricum leaves Aqueous
D D D D
þ [ indicates the presence of respective compound; [ indicate the absence of respective compound.
Saponins were also reported to cause the death of cells  or the disruption of cell cycles . The latter mechanism was achieved via a reduction in the number of cells in G0/ G1 phase followed by the initial increases in S and G2/M or via inhibition of the nuclear factor-kappa B (NF-kB) . In conclusion, the present study demonstrated that the M malabathricum leaves extracts possessed antiproliferative activity against various types of cancer cells, which could be attributed to their antioxidant property and high polyphenolic contents and, thus, will require further extensive studies.
Acknowledgments This study was supported by the research grant from the Ministry of Science, Technology and Innovation, Malaysia (Project Vote Number: 02-01-01-SF0182).
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