Abstract
Twenty-two methanol and chloroform crude extracts from Guttiferae (Hypericum hookerianum. Wight & Arn; Garcinia speciosa. Wall; Garcinia xanthochymus. Hook. F. ex T. Anderson; Cratoxylum formosum. subsp. pruniflorum. (Kurz) Gogel; and Calophyllum polyanthum. Wall ex Choisy) and Schisandraceae families (Schisandra verruculosa. Gagnap) collected from the northern region of Thailand were tested for antiproliferative activity on B16F10 (melanoma), HeLa (cervical carcinoma), and KB (epidermoid carcinoma) human cancer cell lines using the sulforhodamine-B (SRB) binding assay. All crude extracts showed an interesting antiproliferative activity with a dose-response relationship. The chloroform extract from leaves of G. speciosa. was the most potent in inhibiting cancer cell growth with the GI50 of 4, 6.6, and 3.7 µg/mL in HeLa, KB, and B16F10 cell lines, which were 13-, 20- and 142-fold less potent than doxurubicin, respectively. The information from this study can explain the use of these plants in Thai traditional medicine and suggests the potential for further development of these extracts to new pharmaceuticals.
Introduction
Plants are important sources of lead compounds for research and development of new drugs. Several substances from plants can be used as alternatives for the treatment of several life-threatening diseases, especially cancer and HIV. For example, paclitaxel (Taxol®) is used for the first-line treatment of non–small cell lung cancer, and calanolides (coumarin derivatives from Calophyllum lanigerum.) are known to possess anti-HIV activity (Kashman et al., Citation1992). These successes have spurred efforts for continued discovery of novel natural products with a higher level of activity or reduced toxicity (Grzybek et al., Citation1997). Only a few plants have been investigated so far as sources of potential drugs.
Plants from various parts of Southeast Asia have been screened and found to exhibit significant pharmacologic activities. The Guttiferae plants were found to be rich in secondary metabolites such as xanthonoids, biflavonoids, and triterpenoids (Xu et al., Citation1998). Some of these plants have been used in traditional medicines, and novel bioactive compounds with cytotoxic activity have been previously reported (Cao et al., Citation1998; Xu et al., Citation1998; Kosela et al., Citation1999). For plants in the Schisandraceae family, more than 19 species are widely used in Chinese traditional medicine. These plants have been proved to be rich in lignans and triterpenoids with various biological activities (Li et al., Citation2004). Some triterpenoids showed anti-HIV, antitumor, antihepatitis, and antioxidant activity (Hancke et al., Citation1999; Li et al., Citation2003). For the in vitro. primary screening for anticancer drugs, an assay using the protein-binding dye sulforhodamine B (SRB) has been used by the National Cancer Institute (NCI, USA). The SRB binds to the basic amino acids of cellular macromolecules, and the solubilized stain is measured spectrometrically to determine relative cell growth in treated and untreated cells (Monks et al., Citation1991). Coumarins, isolated from bark of the Myanmar plant Kayea assamica. (Clusiaceae), have been evaluated for their cytotoxicity on human cancer cell lines using the SRB assay. They exhibited strong cytotoxic activity against Col2 (colon), KB (epidermoid), and LNCaP (lung) human cancer cell lines with IC50 values in the range 3.5–13.1 µM (Lee et al., Citation2003).
This work reports the anti proliferative activity of 22 methanol and chloroform extracts from wood and leaves of six Thai plant species on three human cancer cell lines, HeLa (cervical carcinoma), KB (epidermoid carcinoma), and B16F10 (melanoma), which plants have not-been previously been investigated using the SRB assay.
Materials and Methods
Plant samples
Six plants were collected from Chiang Mai Province, Thailand, in November and December 2002. Voucher specimens were authenticated and deposited at the herbarium of the Biology Department, Faculty of Science and Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand.
Preparation of the extracts
Wood and leaves from the plants were separately reduced to small pieces, dried at 40°C in a hot air oven, and comminuted to powder. The dried powder samples (100–300 g) were macerated in methanol for 48 h. The extracts were evaporated under reduced pressure in a rotary evaporator. The residues were re-extracted with chloroform and concentrated by partial evaporation under reduced pressure.
Chemicals and reagents
Ethanol and chloroform were obtained from Merck Ltd (Darmstadt, Germany) and Labscan Asia Co., Ltd. (Thailand), respectively. Fetal calf serum (FCS) was obtained from Gibco BRL (Ontario, Canada). DMEM cell culture medium, dimethylsulfoxide (DMSO), gentamicin, and sulforhodamine B (SRB) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Doxorubicin hydrochloride was purchased from Dabur Pharma Ltd (Hamshire, UK). The National Cancer Institute, Bangkok, Thailand, kindly provided the cancer cell lines.
Samples preparation
Stock solutions of extracts were prepared in DMSO and stored at − 20°C. The frozen samples were diluted with cell culture medium prior to the assay. The concentration ranges of the extracts were 250 to 3 µg/mL.
Antitumor activity assay
The effects of extracts on the growth of human cancer cell lines were evaluated according to the procedure of the NCI for the in vitro. anticancer drug screening using the protein-binding dye SRB to assess cell growth (Skehan et al., Citation1990). Three human cancer cell lines, HeLa, KB, and B16F10, were used. Cells were routinely maintained as adherent cell cultures in DMEM medium supplemented with 10% heat-inactivated FCS and 50 µ/mL of gentamicin at 37°C in a humidified air incubator containing 5% CO2. Each cell line was plated at a density of 1.0 × 105 cells/mL for KB and B16F10 and 2.0 × 105 cells/mL for HeLa in 96-well plates and allowed to attach overnight. Cells were then exposed to five serial concentrations of extracts for 48 h. After incubation, the adherent cells were fixed in situ., washed, and dyed with SRB. The bound dye was solubilized, and the absorbance was measured at 492 nm in a microplate reader. The dose-response curves were generated, and the GI50, corresponding with the concentration of compounds that inhibit 50% of the cell growth, was determined (Monks et al., Citation1991). Doxorubicin hydrochloride was used as positive control.
Results and Discussion
The GI50 values of extracts on HeLa, KB, and B16F10 cell lines are shown in . Final concentration of DMSO (≤ 0.25%) did not interfere with the biological activities tested (data not shown). Extracts exhibited a dose-dependent growth inhibitory effect on all the cancer cell lines. Most of the GI50 values of any particular extract were not significantly different in three cell lines. The chloroform extracts from wood and leaves of all plants also showed stronger inhibition than the methanol extracts.
Table 1.. Effect of methanol and chloroform extracts from wood and leaves of the selected Thai plants in the families family Guttiferiae and Schisandraceae on the growth of human cancer cell lines
The chloroform leaves extract of G. speciosa. showed the most potent inhibitory effect with GI50 values of 4.0, 6.6, and 3.7 µg/mL on HeLa, KB, and B16F10 cell lines, respectively. These values were 13, 20, and 142-fold less potent than doxorubicin, the positive control, which gave GI50 values of 300, 330, and 26 nM on HeLa, KB, and B16F10 cell lines, respectively. The strong growth inhibitory effects were also detected in the chloroform leaf extract of Calophyllum polyanthum. and the chloroform wood extracts of H. hookerianum., G. speciosa., and G. xanthochymus. with GI50 values of less than 20 µg/mL. The stronger inhibitory effects of most of the chloroform extracts compared with the methanol extracts might be due to the presence of more active compounds, nonpolar compounds, which are more soluble in chloroform.
Moderate inhibitory effects were found in the methanol leaf extract of G. speciosa., the chloroform leaf extract of G. xanthochymus., the chloroform wood extract of Cratoxylum formosum. subsp. pruniflorum., and the methanol wood extract of H. hookerianum.. The methanol leaf extracts of Cratoxylum formosum. subsp. pruniflorum., Calophyllum polyanthum., and S. verruculosa. and the methanol wood extract of Calophyllum polyanthum. showed no inhibitory activity at any concentration.
G. speciosa. also has been studied for antiviral activity. Protostane triterpenes isolated from bark and stem showed significant inhibitory activity againt HIV-1 RT (Rukachaisirikul et al., Citation2003). Xanthones and cinnamate esters from H. hookerianum. have been extracted and have shown cytotoxic effect on human cancer cell lines MCF7, NCI-H460, and SF-268 (Wilairat et al., Citation2005). Xanthones isolated from wood of Calophyllum teysmannii. var. inuphylloide. have been tested for immunomodulator activity, and some were shown to significantly suppress the mitogenic response of lymphocyte to phytohemagglutinin (Gonzalez et al., Citation1999).
Flavones, vitexin, friedelin, and berulin from leaves and xanthones from wood of G. xanthochymus. that had Nerve Growth Factor(NGF)-potentiating activity have been reported (Chanmahasathien et al., Citation2003). Wood of Cratoxylum formosum. subsp. pruniflorum. has been described to contain quercetin, hyperoside, xanthones, mangiferin, and isomangifin (Nguyen & Harrison, Citation1999). In the Guttifeare family, coumarins isolated from bark of the Myanmar plant Kayea assamica. (Clusiaceae) have been evaluated for their cytotoxicity in human cancer cell lines using the SRB assay. They exhibited strong cytotoxic activity against Col2 (colon), KB (epidermoid), and LNCaP (lung) human cancer cell lines with IC50 values in the range 3.5–13.1µM (Lee et al., Citation2003).
The results of this study suggest a potential for the plants with significant growth inhibitory activity for possible further study and development of the pure compounds in the crude extracts to new pharmaceuticals.
Acknowledgments
This work was partly supported by the Thailand Research Fund (TRF) under the RGJ-PhD program.
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