CytotoxiC , antioxidant , and antimiCrobial aCtivities of Ampelopsis brevipedunculAtA and pArthenocissus tricuspidAtA ( vitaCeae )

Cyclohexane and methanol extracts of leaves and inflorescences of Ampelopsis brevipedunculata and Parthenocissus tricuspidata are shown to exert significant cytotoxic action on both estrogen-dependent (MDA-MB361) and estrogen-nondependent (MDA-MB-453) breast cancer cell lines. Methanol extracts of P. tricuspidata exhibited higher cytotoxicity for the MDA-MB-453 cell line (inflorescence: IC50 =111.45 ± 2.56 μg/ml; leaves: IC50= 56.76 ± 7.11 μg/ml) than for MDA-MB-361. Cyclohexane extracts of A. brevipedunculata leaves exhibited high cytotoxicity against MDA-MB-453 (IC50 =78.32 ± 0.1 μg/ml) and the estrogen-dependent MDA-MB-361 cell line (IC50 =97.40 ± 2.61 μg/ml). The highest DPPH-scavenging ability was exhibited by methanol extracts of P. tricuspidata inflorescences, with IC50=7.55±0.07 μg/ml. The tested extracts possessed weak antimicrobial activity.


INTRODUCTION
Plants of the family Vitaceae belong to the specific life form of climbing plants.Based on their characteristic adaptive strategy regarding water supply, use of light energy, and some other ecological factors, their structural and functional adaptations define them as a specific ecological type (M e t c a l f e and C h a l k , 1950).
The cytotoxicity, antioxidative properties, and antimicrobial activities of two species of climbing plants, Ampelopsis brevipedunculata (Maxim.)Trautv.and Parthenocissus tricuspidata (Sieb.& Zucc.)Planch.(Vitaceae), were studied using in vitro tests.Both species originate from E and SE Eurasia.Introduced to Serbia as horticultural species, they acclimated successfully and today are in use as garden plants.Ethanol extracts of A. brevi-pedunculata berries have traditionally been used to treat liver disease, and the value of such treatment was confirmed by in vitro and in vivo tests.The proposed mechanism of action was through inhibition of reactive oxygen species generation from primary injured hepatocytes in the presence of Fe(II), as well as through induction of cellular stress gene expression (Ya b e et al., 1997; Ya b e and M a t s u i , 1998; Wu et al., 2004).Different classes of compounds -ionone, phenylpropanoid glycosides, and hydroquinone glucosides, as well oligostilbenes (ampelopsins) and triterpenes could be responsible for the pharmacological activity of A. brevipedunculata (O s h i m a and U e n o , 1993; I n a d a et al., 1991; X u et al., 1995).Also, A. brevipedunculata extracts inhibited the formation of collagen fibers by rat hepatic M cells (Ya b e and M a t s u i , 1997) and showed antimutagenic action (L e e and L i n , 1988).Stilbene derivatives have been well studied in CytotoxiC, antioxidant, and antimiCrobial aCtivities of Ampelopsis brevipedunculAtA and pArthenocissus tricuspidAtA (vitaCeae) the stem, wood, and leaves of P. tricuspidata.Isolated stilbenes possessed strong antioxidant properties, as well antiplasmodial activity in vitro (K i m et al., 2005;S o n et al., 2007;Ta n a k a et al., 1998).There are no data on antimicrobial activity of these two Vitaceae species, except for antiviral activity of A. brevipedunculata (S u n et al., 1986).
In view of the well-known antioxidant, anticancer, and estrogenic activities possessed by stilbene derivatives (M u r i a s et al., 2005) and by flavonoids and caffeic acid derivatives (G a l a t i and O ' B r i e n , 2004; S a l e e m et al., 2004), the aim of present study was to investigate potential antioxidant, cytotoxic, and antimicrobial activities of extracts of leaves and inflorescences of P. tricuspidata and A. brevipedunculata.

Plant material
Leaves and inflorescences of Ampelopsis brevipedunculata and Parthenocissus tricuspidata were collected in June of 2007 in the Botanical Garden in Belgrade (Serbia).Voucher specimens are preserved in the herbarium of the Institute of Botany, Botanical Garden, University of Belgrade (BEOU 16253; BEOU 16252).The plant material was then dried and ground into powder.

Extraction
Dried leaves (100 g), inflorescences (50 g), and fruits (10 g) of P. tricuspidata and A. brevipedunculata were macerated with cyclohexane (1: 10; 2 x two days), followed by methanol (70%, v/v) extraction using the same procedure.The solvents were evaporated under reduced pressure at 40 o C. The composition of all extracts was monitored by TLC and HPLC.Different in vitro tests were used to assay the cytotoxicity and antimicrobial activities of cyclohexane and methanol extracts and the antioxidant activity of methanol extracts.

DPPH test
The DPPH radical scavenging activity of methanol extracts was measured according to the modified method described previously by K u n d a k o v i ć et al. (2006).Dry plant material was dissolved in methanol in a concentration 1 mg/ml.Doseresponse curves were constructed and IC 50 values were calculated.All measurements were performed in triplicate.

TBA test
The inhibitory effect of MeOH extracts on lipid peroxidation (LP) in liposomes was determined using the modified spectrophotometric method described by K u k i ć et al. (2006).Different quantities (10-250 μl) of 1% extract solution were used, and LP was induced with FeSO 4 and ascorbic acid.The absorbances of supernatants were measured at 533 nm.

Cytotoxicity in vitro
Cytotoxicity was tested on the MDA-MB-361 (estrogen-dependent) and MDA-MB-453 (estrogen-nondependent) breast cancer cell lines and on healthy peripheral blood mononuclear cells (PBMC).Ellagic acid and cis-DDR were used as standard substances.

Treatment of cell lines
Stock solutions (50 mg/ml) of extracts made in dimethylsulfoxide (DMSO) were dissolved in media corresponding to the required working concentrations.Neoplastic MDA-MB-361 cells (7000 cells per well) and neoplastic MDA-MB-453 cells (3000 cells per well) were seeded into 96-well microtiter plates, and five different doubly diluted concentrations of investigated extracts or compounds were added to the wells 24 h later (after cell adherence).The nutrient medium was RPMI 1640 medium, supplemented with L-glutamine (3mM), streptomycin (100 lg/ml), and penicillin (100 IU/ml), 10% heat inactivated (56˚C) fetal bovine serum (FBS), and 25 mM Hepes.The reaction of the medium was adjusted to pH 7.2 with bicarbonate solution.

Determination of cell survival
Cell survival was determined indirectly by measuring total cellular protein by the Kenacid Blue R (KBR) dye-binding method (C l o t h i e r , 1995).Briefly, after 72 h of continuous extract action, the medium was discarded and target cells were washed twice with warm (37˚C) phosphate-buffered saline (PBS).Target cells were then fixed for 20 min with 150 μl of a mixture of methanol and acetic acid (3:1), stained for 2-3 h with 0.04% Coomassie Brilliant Blue R-250 in 25% ethanol and 12% glacial acetic acid, and washed, after which bound dye was dissolved in desorbing solution (1 M potassium acetate, 70% ethanol).Absorbance (A) at 570 nm was measured 2 h later.To get cell survival (%), absorbance of samples with cells grown in the presence of various concentrations of the investigated agent was divided by the control optical density (the A of control cells grown only in nutrient medium) and multiplied by 100.The value of A of the blank was always subtracted from A of the corresponding sample with target cells.The IC 50 concentration was defined as the concentration of an agent inhibiting cell survival by 50% compared to a vehicle-treated control.All experiments were done in triplicate.

Antimicrobial activity
Two different methods were used to determine antimicrobial activities: the agar-diffusion method and broth microdilution assay The complete procedure for the agar-diffusion method was described by A c a r and G o l d s t e i n (1996).The investigated extracts (100 mg/ml) were dissolved in methanol (50%, v/v) or DMSO and then poured into agar or diluted to the highest concentration.The results of agar diffusion assays were evaluated by measuring the inhibition zone (in mm) after incubation.Each assay in this experiment was repeated twice.Ampicillin (10 μg/tbl), amikacin (10 μg/tbl), and nystatin (100 U/tbl) served as positive controls.
Broth microdilution assay was used to determine the minimal inhibitory concentration and minimal bactericidal or fungicidal concentration (MBC) according to C a n d a n et al. (2003).
The MIC is defined as the lowest concentration of the extract at which the microorganism does not demonstrate visible growth.The MBC is defined as the lowest concentration of the extract at which inoculated microorganisms were completely killed.All determinations were performed in duplicate, and two positive growth controls were included.

HPLC analysis
Phytochemical analysis using the HPLC method showed the presence of flavonoids and phenolic acids in aerial parts of the investigated plants.The identified compounds of A. brevipedunculata were caffeic acid, ellagic acid, quercitrin, and luteolin-7-O-glucoside in methanol extracts of inflorescences; and kempferol, quercetin, rutin, and luteolin-7-Oglucoside in methanol extracts of leaves.The presence of quercetin-3-O-glucoside, caffeic acid, and luteolin-7-O-glucoside was confirmed in methanol extracts of inflorescences of P. tricuspidata, while quercetin-3-O-glucoside as well was detected in methanol extracts of its leaves.The stilbene derivative piceatannol was identified only in leaf extracts of P. tricuspidata.These compounds were previously recorded in both plants.The major compounds in root extracts of A. brevipedunculata were oligostilbenes, while flavonoids and ionone derivatives were isolated from leaves (O s h i m a and U e n o , 1993; I n a d a et al., 1991; X u et al., 1995).Caffeic acid esters with antioxidant activity and stilbene derivatives were isolated from the leaves of P. tricuspidata (K i m et al., 2005; S a l e e m et al., 2004;Ta n a k a et al., 1998).

Antioxidant and radical scavenging activity
Methanol extracts of leaves and inflorescences of P. tricuspidata and A. brevipedunculata possessed dose-dependent DPPH radical scavenging activity (Table 1).The highest effect was exhibited by methanol extracts of P. tricuspidata inflorescences, with IC 50 =7.55±0.07μg/ml.An inhibitory effect on LP was exerted by methanol extracts of inflorescences of A. brevipedunculata (IC 50 =0.033±0.001mg/ml) and leaves of P. tricuspidata (IC 50 =0.96±0.05mg/ml).Leaf extracts of A. brevipedunculata did not attain IC 50 (44.4%).Methanol extracts of fruits of neither species had any inhibitory effects on LP.

Antimicrobial activity
The given plant extracts manifested weak antimicrobial activity against the tested bacteria and fungi, with the exceptions of cyclohexane extracts of inflorescences and methanol extracts of inflorescences and leaves of P. tricuspidata, which showed moderate antimicrobial activity against C. albicans and Micrococcus flavus, with MIC 3.25 mg/ml.Antiviral activity has been reported for A. brevipedunculata (S u n et al., 1986), but there are no data on antibacterial or antifungal activity of the two studied plants.

DISCUSSION
Vitaceous plants are known as a good source of stilbenoids, which are potent chemopreventive agents (J a n g et al., 1997).Anticancer activity is usually  Wu et al. (2004) showed that methanol extracts of stems and roots of A. brevipedunculata possess strong antioxidant activity against linoleic acid peroxidation and plasmid DNA oxidation, as well as hydroxyl-and DPPH-scavenging activity.Stilbene derivatives from stems of P. tricuspidata, especially piceatannol (K i m et al., 2005), and caffeic acid derivatives from its leaves (S a l e e m et al., 2004) were found to be potent in inhibition of LP in rat liver homogenate and in DPPH and superoxide anion scavenging (DPPH: IC 50 =4.56-14.17μg/ml; O 2 -: IC 50 = 0.58-7.39μg/ml).
Our results are in accordance with previously published data because the presence of piceatannol, flavonoids, and phenolic acids was confirmed in methanol extracts of P. tricuspidata, while ellagic acid, phenolic acids, and flavonoids were recorded in methanol extracts of A. brevipedunculata.Those compounds are well-known antioxidants (G e r h ä u s e r et al., 2003), and their presence could explain the strong inhibition of LP and DPPH radical-scavenging activity.Also, since natural polyphenols exert anticancer action by scavenging free reactive oxygen species (M u r i a s et al., 2005), significant cytotoxicity of the tested methanol extracts could be connected with their presence.Piceatannol, a very potent antitumor agent (P o t t e r et al., 2002), was detected only in methanol extracts of P. tricuspi-data, with very high cytotoxicity against the MDA-MB-453 breast cancer cell line.
Malignant cells from both estrogen receptorpositive (ER+) and estrogen receptor-negative (ER-) cell lines were sensitive to hexanol extracts of A. brevipedunculata and P. tricuspidata, and to methanol extracts of P. tricuspidata, indicating that some compounds whose antiproliferative action is ERdependent are present in the mentioned extracts.The absence of antiproliferative action of methanol extracts of A. brevipedunculata on ER receptorpositive malignant MDA-MB 361 cells suggests that estrogen receptor-dependent antiproliferative activity was lost in these extracts.Ellagic acid, present in methanol extracts of A. brevipedunculata inflorescences, exhibited high cytotoxicity for nonestrogen-dependent tumor cells, with IC 50 =74.53± 3.23 μg/ml, and could contribute to the antiproliferative action of those extracts.
The cytotoxicity of cyclohexane extracts, especially those of P. tricuspidata leaves and A. brevipedunculata inflorescences, should be further studied because of its significant strength against the estrogen-dependent MDA-MD-361 cell line.