Pharmacologia 3 (10): $3: ISSN 2044-4648 / DOL: 10.5: © 2012 Science Reuters, UK pharmacologia.2012.535.538 ‘The Evaluation of Some Biological Activity of Mentha longifolia (L.) Huds Growing Wild in Iran 'Seyed Mehdi Razavi, “Gholamreza Zarrini and ‘Ghader Molavi "Department of Biology, Faculty of Science, University of Mohaghegh Ardabil, Ardabil, Iran “Department of Animal Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran Abstract: Mentha longifolia (L.) Huds. i a prernial rhizomatous herb dstibuted from Southern Africa, Europe, the Mediterranea region and eastwards into Asia. Mentha species are also generally known under name of Pune in Iran where they have been used for centuries as tones, earminatve, digestive, stomachic, antispasmodic and antiinflammatory agent in folk medicine. In the present work, we sty antimicrobial and ytotoxie activity of the plant extracts, The antibacterial and antifungal activity of the plant extracts were evaluated using disk diffusion method. We evaluated eytotoxcity of 1. longifolia with MTT aasay, as well as, Present finding revealed that the methanol extract ofthe plant leaves were active aginst all tested bacterin and fimgi. The highest inhibitory effect was observed against Erwinia carotovora, a common plant pathogen boteria, with MIC value of 128 jg ml.~* and iaubition zone of 41 mum. The extract also exhibited high anttctrial activity with MIC value ang of 192-512 yg mi.~ and inhibition zone of 3440 mm agains. aureus, Enterococeus faecalis, Streptococcus agalactiae and Escherichia cols. The methanol extract of the plant disployed modest. to strong antifungal activity aguinst Candida Kefir, Candida albicans, Sclerotinia sclerotiorum an Aspergilis niger with MIC value range of 576-800 jg mi." and inhibition zone (of 25-33 mm, Our finding showed tha.M, longifolia methanol extract has eytotoxe ativity. The extuet reduced the viability of MoCoy cells with RC, value of 1.92 mg mL". Itwas be concluded that. fongifola extract cab be used at an antiseptic agent and may be also @ good candidate to construction of a new plant biopeticde Key words: Mentha longifolia, antiseptic, biopesticide INTRODUCTION (AlBayati, 2009) and hepatoprotective ‘The Labiatac family comprised of 220 gencra of — (Nimica-Dukic ef al, 1999), posses antioxidant (Nickavar ef al., 2008), antimicrobial activity aromatic plants which are widely used for various purposes wiorld wide, The gemus Mentha is one of the important members of the family represented by species in the Flora of Tran (Mozaffarian, 1996). Mentha species are generally known under name of Puneh in Iran where they have been used for centuries as tonics, canninative, digestive, stomachic, antispasmodic and anti-inflammatory agent in folk medicine (Amin, 2005), Mentha longifolia (L.) ius, is the most widespread species of the genus in Iran, It is a perennial rhizomatous herb with erects to straggling stems reach 120 em in height. The plant is an extremely variable species with a widespread distribution in Southem Africa, Europe, the Mediterranean region and eastward into Asia (Rechinger, 1982), ‘There is many report on bioactivity of A longifolia in the literature. It was previously shown that the plant Phytochemical studies revealed the presence of different flavonoides (Ghoulami ef al, 2001), monoterpene ketones (Heganauer, 1953), tannins and saponins (Do Nascimento et af, 2009) in the plants of the genus Mentha, Those chemicals aro responsible for different pharmacological and biological activity of these plants. In the recent work, we focus on eytotexie activity and antimicrobial propertios of M. longifolia extracts on some human and plant pathogen microorganisms, MATERIALS AND METHODS. Plant materials: The leaves of M. longifolia were collected ftom around of Ardabil, Iran during June 2008. A voucher specimen was deposited at the herbarium of faculty of seiences, University of Mohaghegh Ardabili, Ardabil, Iran (No. 1389-3), Corresponding Author: Gholareza Zanini, Department of Animal Biology, Faeuity of Natural Sciences, University of Tabriz, ‘Tabriz, ran Tel: +98 0411 3392Pharmacologia 3 (10): 335-538, 2012 Preparation of the extracts: Air-cried plant Ieaves were Soxhlet extracted with n-hexane, dichloromethane and methanol, respectively. The extracts were dried in vactaum (Razavi et at, 2009), Antibacterial assay: The antibacterial activities of the plant extracts were determined against £. coli (PICC 1047), S, aureus (PTCC 1112), B fasts (PTCC 1394), S, agalactiae (PTOC 1321), Eninia carotovora (PIC. 1675) apd 5. aureus (E,) by the dise diffusion method (azavi and Nejad-Bbrahims, 2009). Mulle-Hinton Agar (MEA) (oxoid) was used for preparation of the media for bacteria, The filter paper discs (6 mm in diameter) were individually impregnated with 15 pl of stock solution of| the plant extracts (100 mg mL) and then placed ento the agar plates which had previously been inoculate withthe tested mieroorganisms. The plates were inceulated at 37°C fox 24h, The diameters of inhibition zones were measured jn millimeters, ll the tests were performed in duplicate Gentamicin (10 ig) and Brythromyein (15 pg) served at potitve control, The MICs of the extracts agains the test rnioroorganisms were determined by the Agar dilution method (Razavi and Zari, 2010), Antifungal assay: The aifungel ectivites of the plant extracts were determined against C. kefpr (ATCC 38296), C. albicans (ATCC 14053), 4. niger (PLM 1140), Penicillium sp. and S. sclerotiorum by the dise diffusion method. Sabourand Dextrose Agar (SDA) was ised for preparation of the media forthe Fungal stains, The filter paper dises (6 mm in diameter) wore individually ‘impregnated with 15 pl of stock solution of the extracts (100° mg_ mL) and then placed onto the agar plates Which had previously been inoculated with the tested microorganisms. Amphotericin B 10 yg. dise was applied as positive control. The plates were Inceulated with the fang incubated at 30°C for 4b, The Giameters of inhibition zones were measured in millimeters. All che tests were performed in duplicate The MICS of the extracts against the test microorganisms were determined by the Agar dilution method (Razavi ane Zain, 2010) Cytotoxic assay: MacCoy cell lines (Pastour, ©) were ‘grown in RPMI 1640 (Gibco, No. 51800-019) medium, Each S00 mL. of the medium consisted of 52 g RPMI powder, 1 g of sodium bicarbonate, 19% wh’ of penicillivstreptomycin and supplemented with 10% heat-inactivated Fetal Calf Serum (FCS) in demonized water (Razavi ef al., 2010a), Completed medium was sterilized by filtering through 0.22 jum microbiological filters (Art No. 1107-25), Coll ine was maintained in @ humidified atmosphere of 58% CO, at 37°C in incubator. The stock solutions of methanol extracts of Maiva sylvestris flowers and leaves were prepared by dissolving the compound in DMSO (100 uL,), The final concentration of the extract was 0.70, 0.50, 0.30,0.3, 010 and 0.05 mg mL™. Cells were plated in the appropriate media on 24-well microplates na 500 iL total volume at a density of 6-10'cell mL“. Triplicate wells were treated with media containing different concentration of the extrac, The plates were incubated at 37°C in 5% CO? for time course of 16 h. For evaluating of eall viability, each well was supplemented with 50 il. of a 5:mg ml~') solution of MTT in uncompleted media and treated for 3hat 37°C in 5% CO, The media was carefully removed from each well and 1 ml. of DMSO and placed in room temperature for 20 min. The plates were gently agitated until the eolor reaction was uniform and the OD,» was determined using a spectrophotometer. The amount of MIT converted to formazan is a sign of the mumber of Viable cells, Medis-only treated cells served as the indicator of 100% cell viability. The 5% inhibitory concentration (ICs) was defined as the concentration that retced the absorbance of the untreated wells by 50% of the control in the MTT assay. Viability percentage was evaluated a8 OD eaa/D agi Razavi etal, 20106), RESULTS AND DISCUSSION Table 1 presents the results of antibacterial and antifungal assays, This finding revealed — that _M. longifolia extracts indicated considerable antimicrobial activity. The methanolic extract of the plant leaves were active against all tested bactoria and fungi. The highest inhibitory effect was observed against E. carotovara, common plant pathegen bacterium, with MIC value of 128 jig mL~" and inhibition zone of 41 mm. The extract also exhibited high antibacterial activity with MIC Value rang of 192-512 yg ml.“ and inhibition zone of 3440 mm against S. aureus, E. faecalis, S. agalactiae and E. coli. ‘The methanol extract of M, Tongifolia was found to have inhibitory effect against meticillin resistant strain of S. aureus (E38), 98 well as. Moreover, the methanolic extract of the plant displayed modest to strong. antifungal activity against C. kefir, C. albicans, S. sclerotiorum and A. niger wit, MIC value range of 376-800 pig mL.~* and inhibition zone of 26-33 mm (Table 2)Pharmacologia 3 (10): 335-538, 2012 ‘Tele: Ansbactal ec of A lola seats Metin exe (Sma m=) Dishorometiane eet (15 mg miL=) Eyton G0) Gentumicn (9p) ‘Trentet microorganism [nition zone MIC (ae yp) Ibi zone MIC nip.) nition re re) tition zane nen ‘Bcheritda col TSI 1160 16 » ‘Supiplecocour ee pity 1160 1" 20 reeroceccs fea sais a 16 ‘Srepococeus ceaactae wore rete “rwiacaraonora 4138 1160 en Sepinvocccus ens 25500 _ ‘Tuble2:Anifinga ets of A nga xcs Mananobe ett (3 msi) Iubion zope MIC uniugL) 3ST sas 2.800 230 36.80 “rent Comte Cond aicans Asperger Pencil sp ‘Steratiawleretonoy ‘The finding of present study has also showed that Gichloromethanie and hexanic extract have no considerable antimicrobial effect against tested rmicroorganians. On the other hand, our finding showed that -M. longifolia methanol extract has modest cytotoxic activity. The extract reduced the viability of MeCoy cells with IC, value of 1.92 mg mL ig. 1) nas previously documented that methanol, ethanol and water extracts of M. longifolia have a good antimicrobial activity Ckrani and Inam-ul-baq, 1980; Jawad ef al, 1988; Akroumn ef a, 2009), However, no activity was reported against B. coli, S. aureus and C. albicans i these studies. Our finding depited that the methanolic extract of the plant very active against the mentioned human pathogen microorganisms, Therefore, st ean be concluded that Iranian sample of M. fongfolia has great antimicrbial potential than other samples and could be garded asa speifie chemotype ofthe species. Phytochemical stuties revealed the presence of flavonoides in great quantity in M. longifolia (Ghoulami ef al, 2001; Alroum ef af, 2009). Many flavonoides like quercetin, Iuolin, apigenin and kaempferol glycosylated derivatives were found in the plant leaves. It was realized that this compound are responsible for high antimicrobial activity of the plant methanol, ethanol and water extract. It ean be abo concluded that high antimicrobial activity of the methanolic extract than dichloromethane or hexane ones should be attributed to prescience of flavonoides as pola compounds. It has also been shown that different falyeosylated Hlavonoides exert a synergism effect in antimicrobial activity, Moreover, nonpolar. volatile compounds like menthol isolated from AM longifolia essential oil vere found to have strong antimicrobial activity (AFBayali 2009) Dicorendane sac (18mg a") Inhbion zope MIC ug) 1166 rie “Amstrics i210 ‘Wb zone) 2 » B 28 a ass ‘Concestarion (gent) 07 Fig. 1: Cytotoxic activity of methanolic extract of M. longifolia on Me-Coy cell line using MTT test, ‘Bars indicate standard error ‘The results herein reported showed that the plant methanolic extract has a great potential to suppress a moetiillin resistant strain of S. aureus (E38), In the last decades, continuous using of antibiotics has caused some pathogen microorganisin species ultimately evolved resistance to some antibiotics. The utilize of medicinal plat extracts or plant derived chemicals with antimicrobial activity could dissolve the problem. ‘The results of present study shovted that M. longifolia methanol exact possess modest cytotoxic and antiproiferative activity. It is assumed that this biological effect of the plant extract i dependent to presence of phenolie compounds like favanoides, A previous paper also demonstrated the M. longifolia extracts indicated high antioxidant potential (Nickavar etal, 2008). Antioxidants may act as fee radicle seavengers which suppress the free radiele damages in biological systems and then may be associated with aPharmacologia 3 (10): 335-538, 2012 reduced risk of cancer, Therefore, M. longifolia can be regarded as chemopreventive agent against cance. On the hand, Present findings. showed that the Ms longifolia extract exhibited a gocd inhibitory effect against plant pathogen microorganisms, E, carotovora and S. sclerotiorum. The former microorganism is a pathogen bacterium causes soft rot in a wide range of finite and vegetable species and is considered as a serious problem in horticulture (Stange, 2003). The lates crganism couses stem rot in many plants and it is one of the most prevalent plant pathogen fungi. Therefore, the extract can be used ata biopesticide. Reoently, the use of synthetic pesticides is claimed to negatively affect the environment and actually it dose not represent an appropriate tool for the control of plant pathogens developing resistance. So, natural derived materials can bee exploited at an alternative for synthetic pesticides (Razavi and Nejad-Hbrahimi, 2009) CONCLUSION Tt was concluded that Mf longifolia can posses antiseptic activity. The utilizing ofthis plant in treatment of sore throat andl throat irvitation inthe Tranian folk medicine is validated scientifically by the results obtained in the present study. REFERENCES: Akroum, S.,D. Bendjeddou, D. Satta and K. Lalaoui, 2009, Antibacterial activity and soute toxicity effect of flavonoides extracted trem Mentha longifolia, Aan. Burasia J, Sei Rea,, 4: 93-96, Al-Bayati, F.A, 2009. Isolation and identification of ‘antimicrobial compound from Mentha longifolia L. leaves grow wild in Iraq Am. Clin, Microbiol. Antimicrab,, 8: 20-26, Amin, G., 2005. Popular Medicinal Plants of Iran. View chancellorship of Research, Tebran University of Medical Seiences, Tebran, Iran, pp: 70. Do Nascimento, EMM, FG, Rodriques, AR, Campos and A. Costa, 2009. Phytochemical prospection, toxicity and antimicrobial activity of Mfentha arvensis (Labiatae) fiom northeast of—Brazil Pharmacognosy, 1: 210-212, Ghoulami, SA., LL, Idrissi and S. Fkih-Tetouani, 2001 Phytochemical study of Mentha longifolia of Moroevo, Fitoterapia, 72: 596-598. Heganaver, R, 1953, The context of essential oil and ccarvone in various species of mint. Bev, Schweitz Bot. Ges, 63: 90-102. Irani, Mand Inam-ul-hag, 1980. Screening of medicinal plants for antimicrobial activity. Fitoterapia Part 1, 51: 231-235 Jawad, AM, HJ. Jaffer, A. ALNaib, H. Saber and NAW. Razzak, 1988. Antimicrobial activity of some Iraqi plants, Fitoterapia, 59: 130-133, Mozaifarian, V.A., 1996, Dictionary of Iranian Plant Names. Farhang Moaser Publisher, Tehran, Pages: 407 ‘Nickavar, B., A. Alinaghi and M. Kamalinejad, 2008, Evaluation of the antioxidant properties of five ‘Mentha species. Iranian J, Pharm, Res., 7: 203-208. ‘Nimica-Dukic, N.,M. Popavie, V. Iakovlijevic, A. Szabo and 0. Gasie, 1999, Pharmacological studies of Mentha longifolia phenolic extracts, IL Hepatoprotective activity. Pharm. Biol, 37: 221-224 Razavi, SM. and S. Nejad-Ebrahimi, 2009, Chemical composition, allelopatic and cytotoxic effects of essential oils of flowering tops and leaves of Crambe orientalis from Iran, Nat Prod, Res.,23: 1492-1498, Razavi, SM, A. Ghasemiyan, 8. Salehi and F. Zab, 2009, Screening of biological activity of Zosima absinthifolia fouits exteacts, Eur. Asia J. Biosei., 4: 25-28. Razavi, SM, and G. Zarrini, 2010, Bioactivity of aviprin and —aviprin-3-O-glucoside, two linear fuanocoumarins from Apiaceae. Russ. J. Bicorg. Chem, 36: 359-362 Razavi, SM, G. Zanini, 8. Zalui and S. Mohammadi 2010a, Biological activity of Prangos uloptera DC. roots, a medicinal plants from Tran, Nat. Prod Res,, 24: 797-803. Razavi, SM, S, Zahri,Z, Motamed and G. Ghasemi, 2010, Biosereening of axypeucedanin, a known, farmocoumarin, Iran J. Basic Med, Sei, 13: 133-138, Rechinger, KH, 1982. Floralranica. Vol. 150, Akademishe Druck University, Velagsanstalt, Graz, Pages: 462. Strange, RLN,, 2003. Introduction to Plant Pathology. John Wiley and Sons Ltd, UK., ISBN: 9780470849736, Pages: 464,