View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Directory of Open Access Journals ORIGINAL ARTICLE Study on biological activities of Mansao hymenaea (DC.) A. Gentry leaf extracts Rugkeart Chirunthorn1, Tanomjit Supavita2, Niwan Intaraksa3, Sopa Kummee4 , Natemata Junkong5, Benjaporn Chisorn5, and Arunporn Itharat6 Abstract Chirunthorn, R., Supavita, T., Intaraksa, N., Kummee, S., Junkong, N., Chisorn, B., and Itharat, A. Study on biological activities of Mansao hymenaea (DC.) A. Gentry leaf extracts Songklanakarin J. Sci. Technol., 2005, 27(Suppl. 2) : 489-495 Petroleum ether extract (1) and ethanolic extract (2) of garlic vine leaves [Mansoa hymenaea (DC.) A. Gentry] were investigated for biological activities using the following tests : brine shrimp toxicity test, pediculi- cidal activity (in vitro) test, cytotoxicity test against COR L-23 (large cell lung carcinoma) using the SRB assay, antimicrobial activity and antioxidant activity by the DPPH radical scavenging assay. The cytotoxic activity against brine shrimp and headlice as well as the EC50 of antioxidant activity of 1 were higher than those of 2. However cytotoxicity of 1 against lung cancer cell line was less than that of 2 (IC50 = 35.39 and 6.44 µg/ml, respectively). Antifungal activities against three fungi (Tricophyton rubrum, T. mentographytes, Micro- sporum gypseum) of 1 were higher than those of 2 but 2 possesed higher antibacterial activity against Staphy- lococcus spp than 1. Key words : garlic vine, Mansoa hymenaea, pediculicidal, brine shrimp, cytotoxicity, antimicrobial, antioxidant 1B.Sc. in Pharm., and MPA., 2M.Sc. in Pharm. (Pharmaceutical Botany), Assoc. Prof., 3B.Ed., Scientist, 4M.Sc. (Microbiology), Scientist, 5B.Sc. in Pharm. student, 6Ph.D. (Pharmacognosy), Assoc. Prof., Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla Univer- sity, Hat Yai, Songkhla 90112, Thailand Corresponding email: [email protected] Received, 11 November 2004 Accepted, 28 April 2005 Songklanakarin J. Sci. Technol. Biological activities of Mansao hymenaea Vol. 27 (Suppl. 2), 2005 : Thai Herbs 490 Chirunthorn, R., et al. ∫∑§—¥¬àÕ √—°…凰’¬√µ‘ ®‘√—π∏√ ∂πÕ¡®‘µ ÿ¿“«‘µ“ π‘«√√≥ Õ‘π∑√—°…“ ‚ ¿“ §”¡’ ‡πµ√¡“µ“ ®—π§ß ‡∫≠®æ√ ‰™¬»√ ·≈– Õ√ÿ≥æ√ Õ‘∞√—µπå °“√»÷°…“ƒ∑∏‘Ï∑“ß™’«¿“æ¢Õß “√ °—¥®“°„∫°√–‡∑’¬¡‡∂“ [Mansoa hymenaea (DC.) A. Gentry] «. ߢ≈“π§√‘π∑√å «∑∑. 2548 27(©∫—∫摇»… 2) : 489-495 °“√»÷°…“ƒ∑∏‘Ï∑“ß™’«¿“æ¢Õß “√ °—¥„∫°√–‡∑’¬¡‡∂“ [Mansoa hymenaea (DC.) A. Gentry] ∑’Ë °—¥¥â«¬ ªî‚µ√‡≈’¬¡Õ’‡∑Õ√å ·≈–‡Õ∑∏“πÕ≈ ‚¥¬°“√∑¥ Õ∫ ƒ∑∏‘Ï¶à“‰√πÈ” ƒ∑∏‘Ï¶à“‡À“ ƒ∑∏‘χªìπæ‘…µàÕ‡´≈≈å¡–‡√Áߪե (COR L23) „™â«‘∏’ SRB assay ƒ∑∏‘ϵâ“π‡™◊ÈÕ·∫§∑’‡√’¬ ·≈–ƒ∑∏‘ϵâ“πÕπÿ¡Ÿ≈Õ‘ √–¥â«¬«‘∏’ DPPH assay º≈°“√∑¥≈Õßæ∫«à“ “√ °—¥™—Èπªî‚µ√‡≈’¬¡Õ’‡∑Õ√å· ¥ß ƒ∑∏‘Ï¶à“‰√πÈ” ‡À“ ·≈–ƒ∑∏‘ϵâ“πÕπÿ¡Ÿ≈Õ‘ √–‰¥â¥’°«à“™—Èπ‡Õ∑“πÕ≈ º≈¢Õߧ«“¡ ‡ªìπæ‘…µàÕ‡´≈≈åªÕ¥¢Õß “√ °—¥™—Èπªî‚µ√‡≈’¬¡Õ’‡∏Õ√å¶à“‡´≈≈å¡–‡√Áߪե‰¥âπâÕ¬°«à“ “√ °—¥™—Èπ‡Õ∑“πÕ≈ (IC50 = 35.39 ·≈– 6.44 ¡§°./¡≈. µ“¡≈”¥—∫) “√ °—¥™—Èπªî‚µ√‡≈’¬¡Õ’‡∏Õ√å· ¥ßƒ∑∏‘Ï¶à“‡™◊ÈÕ√“ 3 ™π‘¥ (Trichophyton rubrum, T. mentographytes, Microsporum gypseum) ¥’°«à“ “√ °—¥™—Èπ‡Õ∑“πÕ≈ ·µà “√ °—¥™—Èπ‡Õ∑“πÕ≈· ¥ßƒ∑∏‘Ï¶à“‡™◊ÈÕ ·∫§∑’‡√’¬™π‘¥ Staphyllococcus spp. ¥’°«à“ “√ °—¥™—Èπªî‚µ√‡≈’¬¡Õ’‡∑Õ√å ¿“§«‘™“‡¿ —™‡«∑·≈–‡¿ —™æƒ°…»“ µ√å §≥–‡¿ —™»“ µ√å ¡À“«‘∑¬“≈—¬ ߢ≈“π§√‘π∑√å «‘∑¬“‡¢µÀ“¥„À≠à Õ”‡¿ÕÀ“¥„À≠à ®—ßÀ«—¥ ߢ≈“ 90112 Garlic vine (Mansoa hymenaea, synonym Thailand, and in the case of containing bioactive names: Cydista acquinoctalis, Mansoa alliacea, compounds, it may be of commercial use as a Pachyptera hymenaea and Pseudocalymna medicine. alliaceum) is a woody vine. The leaves of this vine, when crushed smell nearly the same as crushed Material and Methods garlic (Allium sativum). Mansoa hymenaea is a native plant of Brazil and Peru (Thetburanatham, 1. Plant material 1987). It is an ornamental plant in Thailand because The leaves of Mansoa hymenaea were of its attractive large pink flowers. In South Africa collected from Hat Yai, Songkhla, Thailand, in July, it is used traditionally to treat rheumatoid arthritis 2002. The herbarium specimen of the plant was and as a muscle relaxant (Luna et al., 1984). Pre- compared with the herbarium in Southern Center vious research on this plant has shown that its wood of Thai Traditional Medicine, Faculty of Pharma- contains a naphoquinone compound (lapachone). ceutical Sciences, Prince of Songkla University. The methanolic extract of Mansoa hymenaea wood had cytotoxic activity against V-79 cells (colon can- 2. Extraction µ o cer cell line) (IC50 = 60 g/ml) (Itokawa et al., The leaves were dried at 50 C , and the dried 1992). The dichloromethane and methanolic ex- leaves (350 g) were macerated for 3-5 days with tracts of Mansoa hymenaea leaves showed anti- petroleum ether (3 × 500 ml) and ethanol (3 × 500 fungal activity against Trichophyton mentagro- ml) The organic solvents were evaporated to dry- phytes and Microsporum gypseum (10 mg/disc) ness under reduced pressure. (Freixa et al., 1998). Surprisingly, little research has been reported on this plant worldwide. Studies 3. Biological activities assay on the leaves of this plant should be further carried 3.1 Brine shrimp toxicity assay out. This plant grows very well in most the parts of The leaf extracts were first dissolved in Songklanakarin J. Sci. Technol. Biological activities of Mansao hymenaea Vol. 27 (Suppl. 2), 2005 : Thai Herbs 491 Chirunthorn, R., et al. DMSO (10 mg/ml). These samples were diluted volume of 5 ml. The cell pellet obtained by cen- with artificial sea water to afford concentrations in trifugation (1000 g , 5 min) was resuspended in 10 the range of 0.1-1.0 mg/ml. Each dilution was tested ml of RPMI medium to make a single cell suspen- in triplicate for brine shrimp toxicity test (Meyer et sion and viable cells were counted by the trypan al., 1982). The LD50 values were obtained by probit blue exclusion method using a haemocytometer. analysis using the SPSS for Windows version 10. The cell suspension was then diluted with medium 3.2 In vitro assay for pediculicidal activity to give a final cell concentration of 1 × 103 cells/ Headlice were collected from school girls well. One hundred l/well of these cell suspensions suffering from this headlice were used in this as- were seeded in 96-well microtiter plates and incu- say. The headlice were stored in a bottle with its lid bated to allow for cell attachment. After 24 h the punched with small holes until required for the as- cells were treated with the leaf extracts. The leaf say. The petroleum ether extract (1) and ethanol extract (1 and 2) and vincristine sulphate were ini- extract (2) were prepared in a carbopol gel base at tially dissolved in DMSO. Vincristine sulphate six different concentrations (10, 20, 30, 40, 50, (Sigma, Lot No. 34H0447) was used as a positive 60 % w/w in 100 mg gel base). One gram of this control. Both of the leaf extracts were diluted in gel preparation was spread onto glass slides (2.5 × the medium to produce 8 concentrations, and 100 7.5 cm) and 10-15 headlice were put on the gel of ml/well of each concentration was added to the extracts, and the percentage of dead headlice plates (6 replicates) to obtain final concentration calculated at half hour intervals for 3 hours, and of 0.1, 0.5, 1, 5, 10, 25, 50, 100 µg/ml for extract compared with hair gel base used as control. LD50 and 0.05, 0.1, 0.5, 1, 5, 10, 25, 50, 100 nM for values were calculated by the prism program. vinblastine sulphate. The final mixture used for 3.3 In vitro assay for cytotoxic activity treating the cells contained not more than 1% of (1) Human cell lines the solvent, the same as in solvent control wells. Large cell lung carcinoma (COR-L23) The plates were incubated for exposure times of cell lines were used. COR-L23 cells were estab- 72 hours. At the end of each exposure time, the lished and kindly provided by Dr. P. Twentyman medium was removed, the wells were washed and Dr. P. Rabbitts of MRC Clinical Oncology & with medium, and then 200 ml of fresh medium Radiotherapeutics Unit, Cambridge, UK. They were added. The plates were incubated for a recovery cultured in RPMI 1640 medium supplemented period of 6 days and the cell number was analysed with 10% heated inactivated foetal bovine serum, by the SRB assay. 1% of 2 mM L-glutamine, 50 IU/ml penicillin and (2) Sulphorhodamine B (SRB) assay 50 g/ml streptomycin. The cells were maintained The antiproliferative assay, SRB (sulpho- o at 37 C in a 5% CO2 atmosphere with 95% humid- rhodamine B) used to assess growth inhibition, ity (Keawpradub et al., 1997; Keawpradub et al, was performed according to method of Skehan et 1999). According to their growth profiles, the opti- al (1990). This colorimetric assay estimates cell mal plating densities of each cell line were deter- number indirectly by staining total cellular protein mined as 1 × 103 cells/well to ensure exponential with the dye SRB (Skehan et al.,1990). In the as- growth throughout the experimental period and to say, cells were fixed by layering 100 ml of ice-cold ensure a linear relationship between absorbance at 40% trichloroacetic acid (TCA, Aldrich Chemical 492 nm and cell number was analysed by the SRB Co.) on top of the growth medium. Cells were assay. In the assay, cells were washed with magne- incubated at 4oC for 1 h., after the plates were sium - and calcium- free phosphate buffered saline washed five times with cold water, excess water (PBS) (Oxoid Ltd.). PBS was decanted and cells drained off and the plates left to dry in air. SRB were detached with 0.025% of 1ml trypsin-EDTA stain (50 ml; 0.4% in 1% acetic acid) (Sigma Chem (Sigma Chem Co.) and PBS was added to a final Co., St.Louis, U.S.A.) was added to each well and Songklanakarin J.