GENOTOXICITY TESTING OF THE METHANOL EXTRACT OF THE PLANT COTINUS COGGYGRIA AND GALLIC ACID ON DROSOPHILA MELANOGASTER

The genotoxic activity of methanol extract obtained from the stem of Cotinus coggygria Scop. and synthetic gallic acid were investigated using the Drosophila sex-linked recessive lethal test (or SLRL test). In the tested methanol extract of C. coggygria (1 g), 62.50 mg of pyrocatechol equivalent of phenols was detected. Also, 46.76 mg of flavonoids and 15.75 mg of nonflavonoids were observed in 1 g of dry weight of extract. Methanol extract of C. coggygria in a concentration of 5% and 5% synthetic gallic acid were shown to be clearly genotoxic, inducing sex-linked recessive lethal mutations on the X-chromosome of Drosophila melanogaster males in all three broods.


INTRODUCTION
Cotinus is a small genus of the family Anacardiaceae with two species: Cotinus coggygria Scop.(syn.: Rhus cotinus L.) and Cotinus obovatus Raf., American smoketree.Cotinus coggygria is a deciduous, polygamous shrub or little tree up to 7 m tall.It has a wide distribution from Southern Europe, the Mediterranean, Moldova, and the Caucasus to Central China and the Himalayas (Novaković et al., 2007).Flora of Serbia defines two varieties of Cotinus coggygria: var.laevis with form atropurpurea and var.arenaria (Josifović et al., 1973).
Leaves and young branches are utilized for the production of essential oil with a terpenic scent for use in perfumery (Tsankova et al., 1993).Yellow/ orange color can be obtained from the root and stem of Cotinus cogygria and can be used for fabric coloring.Leaves and bark are a good source of tannins (Grieve, 1971).
In folk medicine, the plant is used for its antiseptic, anti-inflamatory, antimicrobial, antihemorrhagic, and wound-healing effects and against diarrrhoea (Demirci et al., 2003).The dried leaf and twig of C. coggygria are used in Chinese traditional medicine to eliminate «dampness» and «heat», and as an antipyretic (Huang, 1999).and the voucher specimen was deposited (16178, BEOU) at the Department of Botany, Faculty of Biology, University of Belgrade.

Chemicals
Total soluble phenolic compounds in methanol extract of C. coggygria stem were determined with Folin-Ciocalteu reagent (FC) using pyrocatechol as a standard.Methanol extract was soluted to a soluted to a to a concentration of 0.02 g/mL.Of the soluted extract, 0.5 mL was mixed with 2.5 mL of FC reagent (previously diluted 10-fold with distilled water) and 2 mL of NaHCO 3 (7.5%).After 15 min of stirring at 45ºC, the absorbance was measured at 765 nm on a spectrophotometer (ISKRA, MA9523-SPEKOL 211).
The concentration of total phenolic compounds in the C. coggygria stem was determined as mg of pyrocatechol equivalent/g of dry weight of extract using an equation obtained from the standard pyrocatechol graph.All samples were analyzed in three replications.
The flavonoid fraction was precipitated by mixing 10 mL of the extract dissolved in methanol (0.02 g/mL) with 10 mL of HCl (1: 3) and 5 mL of HCHO (8 mg/mL).After 24 h, the mixture was filtered through filter paper (Whatman No. 5).Nonflavonoid components were determined from the filtrate with Folin-Ciocalteu reagent using the same spectrophotometric method as for determining total phenolic concentration; absorbance was measured at 765 nm on a spectrophotometer.Nonflavonoid content was expressed as milligrams of pyrocatechol per gram of dry weight through the calibration curve with pyrocatechol.All samples were analyzed in three replications.
Flavonoid content was determined from the residium of total phenolic and nonflavonoid content.Flavonoid content was expressed as milligrams of pyrocatechol per mg of extract.All samples were analyzed in three replications.

Genotoxicity
The sex-linked recessive lethal test for mutagenicity mutagenicity (SLRL test) was performed with laboratory stocks of was performed with laboratory stocks of Drosophila melanogaster (obtained from the Umea Stock Center, Sweden).Canton-S line flies had a normal phenotype (wild type), while Basc line flies were characterized by individuals homozygous for an Xchromosome balancer carrying three genetic markers: Bar (B), which produces a narrow eye shape in homo-and hemizygous conditions and a kidney-shaped eye when heterozygous in females (the character can be regarded as partially dominant); white-apricot (w a ), which alters the red eye color to light-orange and is expressed only in homozygous females and hemizygous males; and scute (sc), which is a recessive mutation that reduces the number of thoracic bristles [the given mutation is linked with a long inversion on the X-chromosome, necessary for suppression of crossover that could potentially change the existing gene combinations on the treated chromosome (Lee et al., 1983)].
The stocks were maintained and all experiments performed under optimal conditions (t = 25ºC, relative humidity = 60%, 12/12 h light/dark regime) on a standard nutritive medium for Drosophila (corn flour, yeast, agar, sugar, and nipagin to prevent the occurrence of mold and infections).

Test procedure
Three-day-old Canton-S males (test group 1, N = 30) were starved in empty bottles for 5 h prior to the treatment, then transferred and fed in bottles containing filter paper soaked with 5% methanol extract for 24 h.After another 24 h of recovery on a standard medium, each male was mated individually to three Basc females in bottles, which yielded brood I. Two days later, males were transferred to new set of vials containing three virgins of the Basc line (thus creating brood II).After three days, males were transferred again to fresh vials containing three Basc virgins (brood III).These males stayed with females for three days and were removed afterwards.Females were left alone for five days to lay eggs, and then removed.
Another group of individuals of the same age (test group 2, N = 15 males) was treated with 5% synthetic gallic acid, the solvent 1% sucrose (test group 3, N = 30 males) serving as a negative control (Lewis and Bacher, 1968).
After F 1 emerged in all three-test groups, brother-sister mating was allowed for several days, and 10 females from each vial were put individually into new vials.Each vial would give the progeny of one treated X-chromosome.In F 2, the phenotypes were scored according to eye color and shape.The absence of wild type males indicated the presence of a recessive lethal agent induced by the test substance.
The total number of treated X-chromosomes is equal to the sum of lethal and non-lethal cultures, and the frequency of sex-linked recessive lethal cultures was calculated from the ratio between the number of lethal cultures to the total number of treated X-chromosomes.Significance of the percentage difference of lethal cultures was determined through testing for large independent samples by testing the difference between proportions (Petz, 1985).

RESULTS
In methanol extract of C. coggygria (1 g), 62.50 mg of pyrocatechol equivalent of phenols was detected.Also, 46.76 mg of flavonoids and 15.75 mg of nonflavonoids were detected in 1 g of dry weight of extract.Results of determining total phenolic, flavonoid, and phenolic, flavonoid, and , flavonoid, and nonflavonoid content are given in Table 1.
The results of testing the genotoxic effect of methanol extract (test group 1) and synthetic gallic acid (test group 2) are shown in Table 2.In our experiment, a 5% concentration of methanol extract was shown to be clearly genotoxic, inducing significant increases in the frequency of mutants in all three broods (I, II, and III).Also, 5% synthetic gallic acid induced sex-linked recessive lethal mutations on the X-chromosome of Drosophila melanogaster males in all three stages of spermatogenesis.

DISCUSSION
Plant extracts and essential oils, as well as their constituents, are used in the food, cosmetics, and pharmaceutical industries (Stammati et al., 1999).Extracts of many plant species have been examined for a number of biological activities so far, and their antimicrobial, anti-inflammatory, antioxidant, antimutagenic, and cancer-preventive effects have been partially described (Baricevic and Bartol, 2000;Mitić et al., 2001;vujošević and Blagojević, 2004;Faried et al., 2007).
Phytochemical investigation of methanol extract of the plant Cotinus coggygria led to the isolation of several phenolic compounds (Stathopoulou et al., 2007;Zdunić et al., 2007).Our results demonstrate that in methanol extract of C. coggygria (1 g), 62.50 mg of pyrocatechol equivalent of phenols are detected.
Phenols are very important plant constituents because of their scavenging ability due to their hydroxyl groups (Hatano et al., 1989).Phenolic henolic compounds may contribute directly to antioxidative action (Duh et al., 1999).It is suggested that polyphenolic compounds have inhibitory effects on mutagenesis and carcinogenesis in humans when up to 1.0 g is ingested daily from a diet rich in fruits and vegetables (Tanaka et al., 1998;Yoshida et al., 2000;Tsuda et al., 2004).
From alcoholic extract of C. coggygria, gallic acid and its derivatives methyl gallate and pentagalloyl glucose were isolated (Westenburg et al., 2000).Polyphenolic gallic acid and its derivatives are biologically active compounds present in many plants (Kahkonen et al., 1999;Lee et al., 2000).They are widespread in plant foods and beverages such as tea and wine and are present in Cotinus coggygria, both in the free state and as part of the tannin molecule (Trpinac et al., 1983).
Many plants and herbs have potential antioxidant activity.Gallic acid is a strong natural antioxidant (Aruoma et al., 1993;Heinonen et al., 1998;Khan et al., 2000;Zheng and Wang, 2001).It was reported as a free radical scavenger and as an inducer of differentiation and apoptosis in leukemia, lung cancer, and colon adenocarcinoma cell lines, as well as in normal lymphocyte cells (Inoue et al., 1994;Kawada et al., 2001;Salucci et al., 2002;Sohi et al., 2003).Several plant species with anti-cancer activity have already been discovered, one of them being Cotinus coggygria.Gallic acid from this plant has been shown to display selective cytotoxicity against tumor cells and to induce apoptosis in tumor cells (Isuzugawa et al., 2001).
In the present study, we examined the genotoxicity of methanol extract of the plant Cotinus coggygria and synthetic gallic acid using a short test for detection of mutagenicity under in vivo conditions.Our results suggest, as evident from Table 2, that the components of methanol extract of Cotinus coggygria in a concentration of 5% induced sex-linked recessive lethal mutations on the X-chromosome of Drosophila melanogaster (test group 1) in all three broods (I, II, and III).We used synthetic gallic acid for comparative analysis (test group 2).This polyphenolic acid was shown to be clearly genotoxic, inducing significant increases in the frequency of mutants in both post-meiotic (spermatids and spermatozoids) and pre-meiotic (spermatocytes) germ cell lines of the eukaryotic species Drosophila melanogaster.
Employing in vivo experimental methods, the present study showed a significant genotoxic effect genotoxic effect effect of gallic acid on Drosophila melanogaster.Also, methanol extract of the plant Cotinus coggygria induced mutations in male germinative cells of this eukaryotic species, while certain chemical components (except gallic acid) in methanol extract manifested a genotoxic effect.Further studies are needed to prove the genotoxicity of these chemical substances.