Background
At present, naturally derived products play an important role as source of medicine. Many pharmaceutical agents have been discovered by screening natural products from plants, based on ethnopharmacological data which provides a substantially increased chance of finding active plants relative to a random approach.
Leea indica (Vitaceae), commonly known as ‘Huo Tong Shu’ in Malaysia, have been traditionally used as natural remedy in folk medicine by the locals. It is a perennial shrub which can be found in tropical and subtropical countries, such as Thailand, Malaysia, India and China. The leaves and roots of
L. indica are traditionally used for the treatment of cancer, diabetes, diarrhea, dysentery, spasm and skin diseases [
1,
2]. The leaves are generally consumed by locals either raw or taken as a concoction brewed from fresh leaves. The whole plant is also used as remedy for the relief of headache, body pains and skin complains [
3].
There are limited phytochemical studies reported on
L. indica leaves [
4‐
6] and essential oil of flowers [
7]. To our knowledge, although the leaves of
L. indica is reported to be used in a large number of Malaysian traditional medicine preparations, there is not much recorded data on biological studies of
L. indica leaves. An investigation by Saha et al. [
8] reported that the crude methanol extract from the whole plant of
L. indica showed high antioxidant and nitric oxide inhibitory activities, by employing FTC (ferric thiocyanate), TBA (thiobarbituric acid), DPPH free radical scavenging methods and Griess assay. A later report by Temkitthawaon et al. [
9] indicated that the crude ethanol extract of
L. indica roots showed potent phosphodiesterase inhibitory activity. However, a report by Nurhanan et al. [
10] stated that the crude methanol extracts of leaf, stem and bark of
L. indica did not showed any anti-proliferative activities against the breast cancer cell lines. Additionally, the essential oil of
L. indica flowers showed only moderate antibacterial activity against the tested bacteria [
7]. More recent investigations by Hsiung et al. [
11] and Wong et al. [
12] reported that the ethyl acetate fraction of
L. indica leaves and the mollic acid arabinose isolated from it induced growth-inhibitory effect and apoptosis in Ca Ski human cervical cancer cells.
The current study aimed to investigate the total phenolic content, antioxidant effect and cytotoxic activity of L. indica leaves. The antioxidant potency of L. indica leaves have been investigated, employing three different established in vitro testing systems, such as scavenging activity on DPPH radicals, reducing power assay and superoxide dismutase (SOD) activity assay. The total phenolic content of the leaf extracts was also accessed by Folin-Ciocalteau’s method. To our knowledge, there is no antioxidant study reported for L. indica leaves. Thus, the antioxidant activity of L. indica leaves was evaluated as it has not been determined previously.
In view of the traditional usage of
L. indica in cancer-related diseases and the investigation by Hsiung et al. [
11] and Wong et al. [
4,
12] which indicated the potential use of
L. indica in the treatment of Ca Ski human cervical cancer cells, it was thus necessary to further expand this area of research to other cancer cell lines. According to Malaysian Cancer Statistics [
13], colorectal cancer is one of the leading cancers in Malaysia and a total of 2,866 cancer cases were diagnosed among Malaysians in Peninsular Malaysia in the year 2006. In this study, we evaluated the cytotoxic activity of the extracts against three colon cancer cell lines with varying molecular characteristics, HT-29 (APC, type II truncation and COX-2 constitutive expression), HCT-15 (COX-2 deficient) and HCT 116 (APC, wild-type and COX-2 inducible) [
14]. The resulting information will certainly provide scientific support upon the traditional usage of
L. indica.
Methods
Chemicals and reagents
Gallic acid, BHA (butylated hydroxyanisole), ascorbic acid, DPPH (1,1-diphenyl-2-picrylhydrazyl), potassium ferricyanide, Folin-Ciocalteu’s phenol reagent, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], RPMI 1640 medium and McCoy’s 5A medium were obtained from Sigma-Aldrich Company. Trichloroacetic acid, ferric chloride, ethanol, hexane and ethyl acetate were purchased from Merck Company. Foetal bovine serum, penicillin, streptomycin and fungizone were from PAA Lab (Austria). SOD (superoxide dismutase) kit was purchased from Sigma-Aldrich Company.
Plant sample collection and identification
The fresh leaves of L. indica were collected from Seremban, Negeri Sembilan, Malaysia in February 2011. The plants were identified by Dr Yong Kien Thai of Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia and a voucher specimen (herbarium no: KLU47724) was deposited at the herbarium of the Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.
The extracts were prepared as previously described [
15]. Briefly, the leaves of
L. indica (2.70kg) were washed, dried (38°C) and ground to fine powder (1.60kg, 59.26%). The dried, ground leaves (300.30g) were extracted with ethanol (3x 1.5L) at room temperature yielding a dark green crude ethanol extract (27.80g, 9.26%). The ethanol extract (24.80g) was further extracted with hexane to give a hexane-soluble extract (5.80g, 23.39%) and a hexane insoluble residue. The hexane-insoluble residue was further partitioned between ethyl acetate–water (1:1, 100ml: 100ml) to give an ethyl acetate-soluble extract (3.60g, 14.52%). The water layer was freeze-dried to give a brown coloured fractionated water extract (3.60g, 14.52%). All the extracts (ethanol, hexane, ethyl acetate and water) were kept in the dark at 4°C for not more than one week prior to evaluation of total phenolic content, antioxidant effect and cytotoxicity.
Determination of total phenolic content
The concentrations of phenolic compounds in the extracts of
L. indica leaves were measured according to the Folin-Ciocalteu method as previously described [
16]. Briefly, extract solution (0.02ml) at different concentrations (concentrations ranging from 0 to 20mg/ml) was mixed with 1.58ml of distilled water. Folin-Ciocalteu’s phenol reagent (0.1ml) was then added to each test tube. After 3min, 0.3ml of saturated sodium carbonate solution was added to the mixture. The reaction mixtures were incubated in dark at 40°C for 30min. The absorbance was measured at 765nm with a spectrophotometer. All extracts were assayed in triplicate. Gallic acid solutions with concentrations ranging from 25 to 1000mg/l were used for calibration. A dose response linear regression was generated by using the gallic acid standard absorbance and the levels in the samples were expressed as gallic acid equivalents (mg of GAEs/g of extract).
Scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals
The scavenging activity of the extracts of
L. indica on DPPH radicals was measured according to the method as previously described [
16]. Briefly, extract solution with different concentrations (concentrations ranging from 0 to 5mg/ml) was mixed with 0.8% of DPPH solution. The reaction mixtures were incubated at room temperature and allowed to react for 30minutes in the dark. All measurements were done in dim light. The absorbance was measured at 520nm with a spectrophotometer. All assays were conducted in triplicate. The scavenging activity (%) on DPPH radical was calculated according to the following equation:
Scavenging activity (%)=[(Acontrol-Asample)/Acontrol] x 100%; where Acontrol is the absorbance of the control and Asample is the absorbance of the tested extract.
The scavenging ability of the extracts was expressed as EC50 value, which is the effective concentration at which 50% of DPPH radicals were scavenged. The EC50 value was obtained from the graph of scavenging activity (%) versus concentration of samples. Ascorbic acid was used as positive reference standard.
Reducing power assay
The reducing power of the prepared extracts was determined according to method as previously described [
16]. Briefly, extract solution at different concentrations (concentrations ranging from 0 to 0.8mg/ml) was added with 2.5ml of 0.2M phosphate buffer (pH 6.6) and 2.5ml of 1% (w/v) solution of potassium ferricyanide. The mixture was incubated in a water bath at 50°C for 20min. Following this, 2.5ml of 10% (w/v) trichloroacetic acid solution was added and the mixture was then centrifuged at 1000rpm for 10min. A 2.5ml aliquot of the upper layer was combined with 2.5ml of distilled water and 0.5ml of a 0.1% (w/v) solution of ferric chloride. The absorbance was measured at 700nm with a spectrophotometer. All assays were conducted in triplicate. Ascorbic acid was used as positive reference standard.
Detection of superoxide dismutase (SOD) activity
SOD activity was measured using water-soluble tetrazolium salt (WST) according to the method described by [
17]. This method utilizes Dojindo’s WST-1, which can produce a water soluble formazan dye upon reduction with superoxide anion. After addition of all the working solution and extract solution with different concentrations (concentrations ranging from 0 to 20mg/ml) in each well as described in the SOD kit manual, the ninety-six-well microplate was agitated and incubated at 37°C for 20min. Absorbance was taken using microplate reader (Oasys UVM340) at 450nm. Percentage inhibition of each sample was calculated by using following equation: {[(B1 – B3) - (S – B2)]/(B1 – B3)} x 100 where B1, B2, B3 and S were the absorbance at 450nm for Blank 1, Blank 2, Blank 3 and sample, respectively. BHA was used as positive reference standard.
Cell lines and culture medium
The colon cancer cell lines HT-29, HCT-15 and HCT-116 were purchased from American Type Culture Collection (ATCC, USA). The HCT-15 cells were maintained in RPMI 1640 medium; HCT 116 and HT-29 cells in McCoy’s 5A medium, supplemented with 10% foetal bovine serum, 2% penicillin or streptomycin and 1% of fungizone. The cells were cultured in a 5% CO2 incubator (Shel Lab water-jacketed) kept at 37°C in a humidified atmosphere.
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay
The cytotoxic activities of samples were evaluated using MTT assay according to the method described by Mosmann [
18]. Cytotoxicity of each extract was expressed as IC
50 value, which is the concentration of extract that reduced the viability of the cells by 50% compared to the control, which were treated with 0.5% DMSO. Three replicate plates were performed for each sample.
Cis-platin was used as positive reference standard.
Statistical analysis
The antioxidant data in the present study were subjected to one-way analysis of variance (ANOVA) and the significance of the difference between the means was determined by the Duncan’s multiple range tests at 95% least significant difference (p<0.05). The Pearson correlation analysis was performed to determine the correlation between total phenolic content and antioxidant activity of the extracts. Statistical significance was set at p<0.05. The IC50 values for cytotoxic activity were obtained by non-linear regression using GraphPad Prism statistical software.
Conclusions
This study was designed to investigate the phenolic content, antioxidant effect and cytotoxic activity of
L. indica leaf extracts. The antioxidant activity of the extracts correlated well with the total phenolic contents and indicated that phenolic compounds are dominant contributors to the antioxidant activity of the extracts. This finding is supported by published manuscript [
32] which indicates that phenolic compounds have the abilities to quench lipid peroxidation, prevent DNA oxidative damage and scavenge the reactive oxygen species. Overall, the fractionated water extract of
L. indica leaves which contained the highest amount of phenolic compounds, exhibited outstanding reducing power, strong DPPH radical scavenging activity and pronounced inhibition rate in SOD assay. All the four extracts exert no damage to the selected colon cancer cells (HT-29, HCT-15 and HCT-116) in the MTT assay.
The data obtained in these testing systems clearly establish the antioxidant potency of the fractionated water extract of L. indica leaves. Future studies should be carried out to identify the active compounds in the fractionated water extract, in order to provide more convincing evidence. An investigation into this phenomenon is now underway.
Acknowledgements
This work was supported by research funding from University of Malaya (UMRG RG047/11BIO). We are also grateful to Dr Yong KT from Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia for plant identification.
Author details
1Institutional address: Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. 2Institutional address: Biology Division, Centre for Foundation Studies In Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
RNS prepared the extracts and carried out the total phenolic content as well as the antioxidant studies. NS worked on the cytotoxicity screening. SSK co-worked on antioxidant assays and analyzed the data for antioxidant assays. WNA evaluated the data and edited the manuscript. SKS designed the current project, supervised the work and wrote the manuscript. All authors have read and approved the final manuscript.