DARU Volume 10, No. 4, 2002 162

CYTOTOXIC ACTIVITY OF MEDICINAL PLANTS USED INIRANIAN TRADITIONAL MEDICINE ON TWO STRAINS OF

SACCHAROMYCES CEREVISIAE

GHOLAM HOSEIN SHAHIDI', MAHMOOD REZA MOEIN", ALl REZAFOROUMADI"', FAROKH ROKHBAKHSH-ZAMIN""

'Department of Plant Protection, School of Agriculture, Kerman University, Kerman. "Departmentof Pharmacognosy, School of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran.

'''Department of Medicinal Chemistry, School of Pharmacy, Kerman University of Medical Sciences,

Kerman.""Department of Microbiology,IslamicAzadUniversity,Kerman,Iran.

ABSTRACT

The effects of methanolic crude extracts of some medicinal plants, which are used in Iraniantraditional medicine on RS322N (rad52) of the yeast strians of Saccaromyces cerevisiae wereinvestigated, using agar diffusion method and the concentration which produced 12 mm inhibitionzone (IC12)was determined. The RS322N IC12for Dorema ammoniacum (oleogum resin), Buniumpersicum (fruit) and Illicium verum (fruit) were 3.14, 5.58 and 5.27 mg/ml respectively. Based onthese findings,oleogumresin of Doremaammoniacumis a potentialcytotoxicagent and a goodcandidate for further studies.

Keywords: Cytotoxic, Screening, Medicinal Plants, Saccharomyces cerevisiae, Topoisomerase I.

INTRODUCTIONScreening of medicinal plants to find new cyto-toxic agents is a fruitful way for the discovery ofanticancer drugs.Camptothecin (cpt), an anticancer natural pro-duct which was isolated from Camptothecaacuminata (1) showed strong antitumor andanticancer activity in several studies, but itsmechanism of action had not been identifieduntil 1985 (2). It was found that cpt inhibitsDNA topoisomerase I (top I) by trapping areversible topI-drug-DNA ternary complex. TopI makes a transient single-strand break in DNAsupercoil (3) and its inhibition is mechnism fordrugs which show anticancer activity. Recently,the lower eukaryote, Saccharomyces cerevisiae,has been used for screening of top I inhibitors(4,5,6,7). This assay depends on the fact thatyeast strains lacking the gene for the rad 52DNA repair pathway are sensitive to agentswhich acts like camptothecin. The assay iscarried out by measuring the growth inhibitionof repair-deficient yeast in comparison to a wildtype yeast strain which has the same permeabi-lity mutation (4,6,7). In the present investigationcytotoxic activity of three medicinal plants usedin Iranian traditional medicine has been investi-gated by this method.

MATERIAL AND METHODSFresh oleo-gum-resin of Dorema ammoniacumD.Don was collected from Larestan , Fars pro-vince (south of Iran). Fruits of Illicium verumMill. and Bunium persicum (Boiss.) B.Fedtschwere purchased from medicinal plants store andsamples were identified by Department ofPharmacognosy, School of Pharmacy, TehranUniversity of Medical Sciences. RS I88N(RAD+) and RS322N (rad52) were from Labo-ratory of Biotechnology, Department of PlantProtection, School of Agriculture, Kerman Uni-versity, Kerman. The fresh material was dried inair under shade and then ground to a finepowder. The powder plant materials (5gm) wereextracted with 80% methanol (3x50 ml). Thealcohol extract was filtered and the solvent wasremoved under reduced pressure at 40°Cand theresulting extract was stored in refrigerator forbiological studies. For performing bioaasay (6)YPD agar medium was prepared by followingconstituents per liter; yeast extract 109, peptone20g, dextrose 20g and agar 15 g. RS I88N(RAD+) and RS322N (rad52) were plated onYPDA in 9x9 cm plates. Wells of 6 mm apartwere made in the plates (9 wells in each plate)with sterilized cork borer. Methanolic extractwas dissolved in DMSO- methanol (1:1 VN)

Correspondence: G.H. Shahidi, Department of Plant Protection, School of Agriculture, Kerman University,Kerman, Iran. e-mail:shahidi_hosein@yahoo.com. '

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Cytotoxicity of two Iranian medicinal plants 163

Table-!. Ethnobotanical information and cytotoxic activity of three medicinal plants used in Iraniantraditional medicine.

30.. RS188N

25

- - - Linear ( RS322N Dorema ammoniacum )

,.. Linear (RS322N Bunium persicum ) - ~-EE 20-

- -Linear (RS322Nlliciumverum)L

- -~...

Q)c

2 15c0..:c 10:Ec

- - -- -

-

5

0 .

0.2 0.4 0.6

log of concentration (mg/ml)

..- --

- ..- -(?

-/10. -v--

---- [~J

" L"

0.8 1 1.2 1.4

Figure 1: Linear regression analysis of log concentration vs zone size in two strains ofSaccharomycess cerevisiae

and a 100 ~I of2.5, 5, 10 and 20 mglml sampleswere placed .in each well. Cpt was used aspositive control (3.125 ~glml). Plates were incu-bated at 30'C for 72 h and the diameter of inhi-bition zone was measured. Activity was basedon size differences of inhibition zones betweenthe two strains. IC12(dose that produces a l2mmzone) was determined by linear regressionanalysis of log concentration versus zone size(Figure 1).

RESULTS AND DISCUSSIONYeast rad 52 mutant (RS322N) is hypersensitiveto cpt (IC]2of 4 ~glml) while RSl88N strain isnot (IC12of 800 ~glml). It has been shown thatthe presence of DNA top I is necessary forgrowth inhibition of cpt (9). Cpt is thought toincrease the life time of top I-DNA covalentintermediate and apparently this intermediate isrecognized as DNA damage by a DNA repairsystem. In Saccharomyces cerevisiae the Rad52

Family Umbelliferae Umbelliferae Magnoliaceae

Botanical name Dorema ammoniacum D. Bunium persicum (Boiss) Illicium verumDon B. Fedtsch Mill

Common name VashaJ Gum Amoniacum Zireh Kuhi/ Wild Caraway Badeyan-e Katai/PersianlEnglish Star AnisePlantpart Latex Fruit Fruit

Traditional uses Gasteric Problems Anti-septic Anti-septicantihelminitic Carminative Carminative

IC12RS332 3.14 5.58 5.97

IC12RS188 >20 >20 >20

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Shahidi et al

DNA repair system must be the predominatepathway for the repairment the cpt-stabilizedenzyme-DNA covalent complex. Rad 52 mut-ants ale much more sensitive to cpt than otherrepair-proficient strains because they are unableto complete the DNA repair process (4,9). Inthis study these strains were used for screeningof natural products with cytotoxic potenciessimilar to cpt (6,7). It has been reported that anextract is active if it shows selective activityagainst one or more repair-deficient yeasts (ICl2less than one-third that of the wild type yeast)with ICl2 less than 2000 Ilglml (6). In anotherreport it has been indicated that 12-hydroxy-chiloscyphone, a sesquiterpenes from Chilos-cyphus rivu/aris (Schard.) Hazlinsky (Hepati-caceae, Lophocoleaceae), showed selective bio-activity in yeast-based DNA-damaging assay(ICl2 RS322=88 Ilglml , ICl2 RSI88>1000)

164

(11). In the present study Dorema ammonicumD.Don showed the most selective cytotoxicity(ICl2RS322N: 3.14mglml and ICl2 RS188N>20mglmJ) (Table 1). An unpublished work hasshown that Feru/a assa-foetida L. and F.gumosa Boiss. (two Iranian endemic plantsbelonging to Umbelliferae) have cytotoxic com-ponents responsible for topI inhibition. Thecompounds responsible for the cytotoxic activityremain to be investigated.

ACKNOWLEDGMENT

We are grateful of Research Council of KermanUniversity of Medical Sciences for financialsupport (grant No. 310) for this investigationand Mr. ASaberi for his kind collaboration.

This paper is dedicated to Mr. A.R.Afzalipourthe founder of Universities at Kerman.

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