POTENTIAL ANTITUMOR ACTIVITY OF TWO POLYGONUM SPECIES

Two Bangladeshi Polygonum species, P. barbatum (L.) Hara var. barbata (common name ‘bekhanjabaj’) and P. stagninum Buch.-Ham. ex Meissn. (common name ‘ratooti sag’ or ‘bara bishkatali’), are perennial herbs of the family Polygonaceae. The genus Polygonum L. is well-known for its use in oriental traditional medicine systems for the treatment of various ailments including fever, pain, inflammation, infections, cancer and tumors. The extracts of P. barbatum var. barbata and P. stagninum were assessed for potential antitumor properties using the potato disc assay. All extracts showed a considerable level of potential antitumor activity. The petroleum ether extract of P. barbatum var. barbata and the n-hexane and ethyl acetate extracts of P. stagninum, having IC50 values of 290, 200 and 180 μg/disc, respectively, were the most active among the extracts. The methanol extracts of both plants were the least active and had an IC50 value of >400 μg/disc. Overall, the extracts of P. stagninum showed better antitumor activity potential than the extracts of P. barbatum var. barbata.


INTRODUCTION
Polygonum barbatum (L.) Hara var.barbata, common name 'bekhanjabaj' , and P. stagninum Buch.-Ham.ex Meissn., common name 'ratooti sag' or 'bara bishkatali' , are perennial herbs of the family Polygonaceae (Balza et al., 1989;GRIN database, 2011;Kirtikar and Basu, 1999).Both species grow widely in marshy and aquatic places, by the sides of the rivers, seasonally flooded roadsides ditches and small ponds throughout Bangladesh, India and Thailand, and also in many other countries in south-east Asia.The genus Polygonum L. is well-known for producing bioactive compounds, and also for its use in oriental traditional medicine systems for the treatment of various ailments including fever, pain, infections, inflammation, cancer and tumors (Dr Duke's Phytochemical and Ethnobotanical Database, 2011).While previous phytochemical studies on P. stagninum revealed the presence of cinnamic acid derivatives, flavonoids and proanthocyanidin polymers (Balza et al., 1989;Datta et al., 2002), P. barbatum var.barbata has recently been reported to yield acetophenone, sitosterone, and a sesquit-erpenes viscozulenic acid (Mazid et al., 2011).The analgesic, anti-inflammatory and diuretic properties of the extracts of P. barbatum var.barbata have also been documented recently (Mazid et al., 2009).However, to the best of our knowledge, there is no report on any bioactivity studies of P. stagninum available to date.As part of our continuing bioactivity and phytochemical studies of the Polygonum species (Datta et al., 2000;2001a, b;2002, 2004a,b;2007;Mazid et al., 2009Mazid et al., , 2011)), we report on the potential antitumor activity of the extracts of P. barbatum var.barbata and P. stagninum using the potato disc assay.

Plant materials
The aerial parts of Polygonum barbatum (L.) Hara var.barbata and Polygonum stagninum Buch.-Ham.ex Meissn.were collected from Kajla, Rajshahi, Bangladesh, authenticated by Professor Naderuzzaman (Department of Botany, University of Rajshahi, Bangladesh), and the voucher specimens, BKD2004-1 and BKD2004-2, respectively, representing this collection have been retained in the Herbarium of the Department of Botany, University of Dhaka, Dhaka, Bangladesh.

Extraction of plant material
The sun-dried and ground aerial parts of P. barbatum var.barbata (650 g) were extracted with methanol (MeOH, 4 L) using maceration for 5 days, and the sun-dried and ground aerial parts of P. stagninum (800 g) were extracted, successively, with n-hexane, ethyl acetate (EtOAc) and MeOH, 4 L each.The duration of each of these extractions was 5 days at room temperature.The extracts were concentrated by evaporation under reduced pressure at 40° C.

Fractionation: solvent partitioning
The MeOH extract of P. barbatum var.barbata was made to 90% aq.MeOH extract and subjected to solvent portioning with petroleum ether (PE).The resulting aq.MeOH extract was further partitioned with chloroform (CHCl 3 ) and finally with EtOAc.All solvent fractions were concentrated by evaporation under reduced pressure at 40° C.

The potato disc assay
The potato disc assay (Ferigni et al., 1982;McLaughlin, 1991;McLaughlin and Rogers, 1998;Coker et al., 2003) was used to assess the antitumor activity of the extracts of the two Polygonum species.Agrobacterium tumefaciens Cambia SR009-EHA-105, obtained from the Department of Biochemistry, University of Dhaka, Dhaka-1000, Bangladesh and cultured in King's B culture medium, was used to initiate tumor growth in fresh, disease-free and redskinned potato discs.Dimethyl sulfoxide (DMSO) was used to prepare test extracts, and appropriate volumes of test solutions were dispensed to achieve 50, 100, 200 and 400 µg/disc.More than 20% tumor inhibition was considered significant (Ferigni et al., 1982).Data were statistically analyzed by ANOVA.

RESULTS AND DISCUSSION
The PE, CHCl 3 , EtOAc and MeOH extracts of P. barbatum var.barbata, and the n-hexane, EtOAc and MeOH extracts of P. stagninum were assessed for antitumor activity using the potato disc assay (Ferigni et al., 1982;McLaughlin, 1991;McLaughlin and Rogers, 1998;Coker et al., 2003), which is a well-accepted assay for the primary screening of plant extracts, fractions or purified compounds for potential anticancer and antitumor activity.The extracts dose-dependently inhibited the growth of gall tumor caused by Agrobacterium tumefaciens.The IC 50 values as well as % inhibition at various concentrations of the extracts are presented in Table 1.The results were compared with that of the positive control, the well-known anticancer drug vincristine sulphate (3.125 µg/disc), which completely inhibited the growth of gall tumor on potato discs.All extracts exhibited a considerable level of antitumor activity.The PE extract of P. barbatum var.barbata and the n-hexane and EtOAc extracts of P. stagni-num, having IC 50 values of 290, 200 and 180 µg/ disc, respectively, were the most active of all the extracts.The MeOH extracts of both plants were the least active and had an IC 50 value of >400 µg/disc.Overall, the extracts of P. stagninum showed better antitumor activity profiles than the extracts of P. barbatum var.barbata.
The inhibition of A. tumefaciens-induced tumors (or Crown Gall) in potato disc tissue is an assay based on antimitotic activity, and can detect a broad range of known and novel antitumor effects (McLaughlin and Rogers, 1998).It has been shown that the inhibition of Crown Gall tumor initiation on potato discs and subsequent growth showed good correlation with the compounds and extracts active in the 3PS leukemic mouse assay (Cocker et al., 2003).Ferrigni et al. (1982) demonstrated that the potato disc tumor assay was statistically more predictive of 3PS activity than either the 9KB or the 9PS cytotoxicity assays.This assay is an animalsparing, fairly rapid, inexpensive and reliable bioassay that provides useful indication of anticancer and antitumor activity of test samples by the inhibition of the development of crown gall tumor on the disc of potato tubers.This assay is based on the hypothesis that antitumor agents might inhibit the initiation and growth of tumors in both plant and animal systems, because certain tumorogenic mechanisms are similar in plants and animals (Cocker et al., 2003).Crown gall tumor is a neoplastic disease in plants caused by a specific strain of the Gramnegative bacterium, A. tumefaciens.The bacterium possesses large Ti (tumor inducing) plasmids that carry genetic information (T-DNA) which upon infection transforms normal or wounded plant cells into autonomous tumor cells (Binns and Thomashow, 1988).The Ti-plasmid causes the plant's cells to multiply rapidly without going through apoptosis, resulting in tumor formation similar in nucleic acid content and histology to human and animal cancers (Coker et al., 2003).
The antitumor potential demonstrated by the extracts of two Polygonum species in the present study is in line with the traditional uses of various Polygonum species in the treatment of tumors.These plants could be used as a source of potent antitumor agents for antitumor drug development.Balza, F., Abramowski, Z., Towers, G. h. N. and P. Wiriychitra (1989).Identification of proanthocyanidin polymers as the piscicidal constituents of mammea siamensis, Polygonum stagninum and Diospyros diepenhorstii.Phytochemistry 28, 1827-30.A 100% inhibition was observed with the positive control, vincristine sulphate at a concentration of 3.125 µg/disc.There was no inhibition caused by the negative control, DMSO.

Table 1 .
Antitumor activity of the extracts of Polygonum barbatum var.barbata and Polygonum stagninum