AROMATASE INHIBITORY AND CYTOTOXIC ACTIVITIES OF CHEMICAL CONSTITUENTS FROM THE VIETNAMESE MEDICINAL PLANT BANCHI-LIEN ( SCUTELLARIA BARBATA D . DON )

Aromatase inhibitory and cytotoxic activities were determined for apigenin, luteolin and the new diterpene named scutebarbalactone VN, which were obtained by bioassay-guided fractionation and isolation from the methanol extract of the Vietnamese medicinal plant Banchi-lien (Scutellaria barbata D. Don). In the aromatase inhibition assay, an IC50 value of 3.36 M was found for scutebarbalactone VN, while IC50 values of 7.2 M and 7.95 M were found for the positive controls aminoglutethimide and -estradiol, respectively. In the cytotoxicity assays using a panel of human cancer cell lines, scutebarbalactone VN showed promising anticancer activity with IC50 ranging from 2.15 to 8.3 M compared with those of the positive control ellipticine ranging from 1.0 to 2.1 M. Apigenin and luteolin were found to be inactive in both assays.


INTRODUCTION
There is an increasing need in Vietnam for anti-cancer and cancer preventive medicines, but only about 10 % of the Vietnamese cancer patients have access to modern therapy and medicaments [1].The major part still relies on traditional herbal medicines for their cure.Therefore it is our desire to investigate those plants scientifically, so that they could be used more effectively.
Our biological screening of Vietnamese medicinal plants with a traditional use to treat tumor using the cytotoxicity assay revealed that the plant Ban-chi-lien (Scutellaria barbata D. Don, also known under the synonym Scutellaria rivularis Wall., family Lamiaceae) possesses remarkable anti-cancer properties.Bioassay-guided fractionation of the methanol extract of the whole plant then resulted in the isolation of a new neoclerodane diterpenelactone, named as scutebarbalactone VN, together with the flavones apigenin and luteolin.Their isolation and structure elucidation were described in a separate report [2].In this article we report about the aromatase inhibitory and cytotoxic activities of these chemical constituents, as well as about the bioassay-guided fractionation and isolation, which led to them.

Plant material
The whole plants of Scutellaria barbata were collected in Hoa Binh province in November 2003.A voucher specimen (accession code BCL1) is deposited at the Department of Animal Cell Technology, Institute of Biotechnology, VAST, Hanoi, Vietnam.After the collection, the whole plants were air-dried, stored in an air-conditioned storage room, and powdered mechanically just before extraction.

Extraction and Isolation:
The aqueous extract was made according to traditional preparation method by gentle boiling of 100 g of the air-dried and powdered plant sample with water in about 10 hours.Water was then removed from the extract by evaporation in vacuo to yield a water extract residue.The methanol extract was prepared by extraction with methanol at room temperature until exhaustion.The combined methanol extracts were concentrated in vacuo at 40 o C to yield the methanol extract residue.
For the bioassay-guided fractionation, the methanol extract residue was partitioned successively between n-hexane, dichloromethane and water.The dichloromethane extract was chromatographed on a Sephadex LH-20 column and eluted with methanol to yield 4 combined fractions R4 -R7.

Assay for Inhibition of Aromatase activity:
Aromatase inhibitory activity was determined by measuring the fluorescent intensity of the product, which is formed from the dibenzylfluorescein (DBF) dealkylation under the catalysis of aromatase [3].The assay was conducted in 96-well microtiter plates (Cat # 3915, Corning Costar).The substrate DBF and aromatase enzyme (baculovirus / insect cell-expressed) were obtained from Gentest Corporation.A cofactor/serial dilution (C/SD) buffer was prepared in 50 mM potassium phosphate, pH 7.4.This buffer contains 2.6 mM NADP + , 6.6 mM glucose-6phosphate, 6.6 mM MgCl 2 , 4 unit/ml of glucose-6-phosphate dehydrogenase, and 1 mg/ml albumin.100 l of test substance or extract and C/SD buffer in the ratio of 1:9 were added.The plate was then pre-incubated at 37 o C for 10 minutes, and the reaction was initiated by addition of 100 l of a pre-warmed enzyme/substrate (E/S) mixture.The E/S mixture contained potassium phosphate buffer (pH 7.4), DBF (0.2 M final), enzyme (2 pmol/ml final) and 4 mg/ml albumin.The plate was continued to be incubated at 37 o C for 30 minutes.Reactions were terminated by addition of 75 l 2 N NaOH.To develop adequate signal to background ratio, the plate (with lid) was then incubated at 37 o C for 2 hours.Fluorescence was measured at 485 nm excitation and 538 nm emission wavelength with a fluorescence plate reader.Data were exported and analyzed using an Excel spreadsheet and the IC 50 values were determined by the TableCurve software.
Samples were tested in triplicate and the mean values were used to prepare doseresponse curves.Aminoglutethimide, Naringenin, -estradiol (Sigma, St. Louis, MO) were used as positive controls.

Cytotoxicity assay
Cytotoxicity assays were performed according to a method developed by Monks, which is being used at the National Institute of Health (USA) as a standard method for the evaluation of the cytotoxic potential of compounds or extracts using a panel of human cancer cell lines [4].Cell lines were grown in 96-well microtiter plates with each well containing 190 l medium.After 24 hrs, 10 l of the test samples dissolved in 10% DMSO were added to the wells.The cells were then cultured for additional 48 hrs, fixed with trichloroacetic acid, and stained with sulforhodamine B, followed by the determination of the optical densities at 515 nm using a Microplate Reader (BioRad).The cytotoxicities were calculated and expressed as IC 50 values.Extracts and fractions having an IC 50 < 20 g/ml and pure compounds having an IC 50 < 10 M were considered as cytotoxicity active.The cancer cell lines KB (human epidermoid carcinoma), LNCaP (hormone dependent prostate cancer), LU (human lung cancer), MCF7 (human breast cancer) were used for the assays.Ellipticine was used as a positive control.

Bioassay-guided fractionation and isolation of chemical constituents
Primary biological screenings of the extracts (see Tables 1 and 2) showed that S. barbata was active in both aromatase inhibitory and cytotoxic assays, and that the activities were retained in the methanol extract, while the water extract showed no activity.The fractionation of the active methanol extract was shown in Figure 1 and performed as described in the experimental section.
In order to guide chemical isolation, cytotoxicity assays of the fractions were performed.The results were shown on Table 3.

Aromatase Inhibitory and Cytotoxic Activities of the Chemical Constituents
The aromatase inhibitory and cytotoxic activities of the isolated compounds 1 -3 were determined and presented in Table 4 and 5.In the aromatase inhibition assay, an IC 50 value of 3.36 M was found for scutebarbalactone VN (3), while IC 50 values of 7.2 M and 7.95 M were found for the positive controls aminoglutethimide and -estradiol, respectively.In the cytotoxicity assays scutebarbalactone VN showed promising anticancer activity with IC 50 ranging from 2.15 to 8.3 M compared with those of the positive control ellipticine ranging from 1.0 to 2.1 M.Apigenin (1) and luteolin (2) were found to be inactive in both assays.

CONCLUSIONS AND DISCUSSION
In conclusion, bioassay-guided fractionation of a methanol extract from the whole plant of Scutellaria barbata D. Don led to the isolation of one new diterpene compound scutebarbalactone VN, and two known flavones apigenin and luteolin.The new compound scutebarbalactone VN was shown to possess strong aromatase inhibitory activity with an IC 50 value of 3.36 µM and an evidently selective cytotoxic activity against the breast cancer cell line MCF7 ( IC 50 = 2.15 µM).
Despite its popularity in traditional medicine, the Vietnamese S. barbata has not been scientifically studied before, while plants of other origins, especially the Chinese plant, have been the subject of many scientific investigations.Recent findings have been published, showing its anticancer properties in various human cancers [5], especially in breast [6], ovarian [7], lung [8], colon [9] and cervix cancers [10].Characteristic compounds were found to be flavonoids and neoclerodane diterpenelactones.The observed activities were mostly assigned to the flavonoid constituents like apigenin, luteolin and others [11], while the biological activities of the neoclerodane diterpenelactones have not yet been studied.
The results of this study on the Vietnamese plant S. barbata demonstrated that: a) The anticancer activities of the plant and its chemical constituents are remarkable and therefore the plant deserves further investigations.b) Scutebarbalactone VN is worth to serve as a lead for future drug development against estrogen-dependent cancers; c) The Vietnamese plant contains similar (apigenin and luteolin) but also different (scutebarbalactone VN) chemical constituents than the Chinese plant; d) Any conclusions obtained with the Chinese plant should be applied with cautions when transferring to the Vietnamese plant.

Table 1 :
Aromatase Inhibitory Activity of the Primary Extracts.

Table 4 :
Aromatase Inhibitory Activity of the Chemical Constituents.

Table 5 :
Cytotoxicity of the Chemical Constituents.