Screening of medicinal plants for induction of somatic segregation activity in Aspergillus nidulans

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Journal of Ethnopharmacology52 (1996) 123-127

Screening of medicinal plants for induction of somatic segregation activity in Aspergillus nidulans A. Ramos Ruiz *a, R.A. De la Torre b, N. Alonso b, A. Villaescusa a, J. Betancourt b, A. Vizoso a aCentro de lnvestigacirn y Desarrollo de Medicamentos, Ave. 26 No. 1605, lndustria Mrdico-Farmacrutica, Ciudadde La Habana, CP 10600, Cuba bLaboratorio Central de Farmacologia, Facultad de Medicina "Dr. Salvador Allende", Ciudadde La Habana, Cuba

Received 2 May 1995; revised 6 February 1996;accepted 12 February 1996

Abstract Knowledge about mutagenic properties of plants commonly used in traditional medicine is limited. A screening for genotoxic activity was carried out in aqueous or alcoholic extracts prepared from 13 medicinal plants widely used as folk medicine in Cuba: Lepidium virginicum L. (Brassicaceae); Plantago major L. and Plantago lanceolata L. (Plantaginaceae); Ortosiphon aristatus Blume, Mentha x piperita L., Melissa officinalis L. and Plectranthus amboinicus (Lour.) Spreng. (Lamiaceae); Cymbopogon citratus (DC.) Stapf (Poaceae); Passiflora incarnata L. (Passifloraceae); Zingiber officinale Roscoe (Zingiberaceae); Piper auritum HBK. (Piperaceae); Schinus terebinthifolius Raddi (Anacardeaceae) and Momordica charantia L. (Cucurbitaceae). A plate incorporation assay with Aspergillus nidulans was employed, allowing detection of somatic segregation as a result of mitotic crossing-over, chromosome malsegregation or clastogenic effects. Aspergillus nidulans D-30, a well-marked strain carrying four recessive mutations for conidial color in heterozygosity, which permitted the direct visual detection of segregants, was used throughout this study. As a result, only in the aqueous extract of one of the plants screened (Momordica charantia) a statistical significant increase in the frequency of segregant sectors per colony was observed, and consequently, a genotoxic effect is postulated. Keywords: Plant mutagenicity; Aspergillus nidulans; Somatic segregation

1. Introduction Toxicological research on plants used in traditional medicine involves, as in drugs of any other origin, short-term tests for genotoxicity that alert on potential risks (e.g., cancer, genetic disorders) * Corresponding author.

for individuals under treatment. However, information on this subject is rather dispersed and scarce for medicinal herbs, when available. Point mutation is generally the primary choice in mutagenicity testing. Assays scoring reversion from auxotrophic markers in bacteria (e.g., Ames test) are usually performed in most protocols. In any case, supplementary tests in eukaryotic

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organisms are needed to record the changes in chromosome structure and number. In this respect, the somatic segregation assay in suitable strains of Aspergillus nidulans allows detection of chromosomal damage caused by aneuploidy and some clastogenic effects, as well as mitotic crossover (Scott and K/ifer, 1982). This test has been applied in more than 150 chemicals including pesticides, fungicides, drugs, solvents, etc. Its validation as part of a battery of genotoxicity assays to detect aneuploidy has been promoted at the Genomic Mutation Program of the European Economic Community by two work teams (Kappas, 1989; Crebelli et al., 1991). In this paper, we made a first report on a rather heterogeneous group of 13 extracts from medicinal plants used in Cuba for the population, which were evaluated for genotoxic activity using the Aspergillus nidulans somatic segregation assay. Therapeutic properties attributed by people to these plants range from diuretic (Lepidium virginicum L., Brassicaceae; Ortosiphon aristatus Blume, Lamiaceae; Piper auritum HBK., Piperaceae), antiinflammatory and antiseptic (Plantago major L. and Plantago lanceolata L., Plantaginaceae), antihypertensive and febrifuge ( Cymbopogon citratus (DC.) Stapf, Poaceae; Mentha x piperita L., Lamiaceae), sedative (Melissa officinalis L., Lamiaceae; Passiflora incarnata L., Passifloraceae), anticonvulsive, antiepileptic and bronchodilator (Plectranthus amboinicus (Lour.) Spreng., Lamiaeeae), antispamodic (Zingiber officinale L., Zingiberaceae), hypoglycemic, vermifuge and antianemic (Momordica charantia L., Cucurbitaceae) to antiulcer effects (Schinus terebinthifolius Raddi, Anacardiaceae) (Roig, 1988). 2. Materials and methods

2.1. Plant material The list of plants studied is given in Table 1. The plant material was collected in Pinar del Rio and La Habana provinces from 1991 to 1992. Properly classified specimens were kept at the herbarium of the Medicinal Plant Experimental Station 'Dr Juan Tom~s Roig' in Giiira de Melena, La Habana. Voucher specimens are cited in parenthesis behind family.

2.2. Extract preparation Tinctures and fluid extracts were prepared by the usual alcoholic extraction in a percolator as described by Soler et al. (1992). To obtain the decoctions of P. auritum and M. charantia, 300 to 400 g of fresh leaves were boiled in distilled water for 10 min. The resulting extract was filtered and the decoction was used immediately (M. charantia) or lyophilized (P. auritum). For P. amboinicus, 500 g of dried leaves were refluxed in distilled water during 15 min, the extract was filtered and lyophilized later. In all cases, leaves were used to prepare the extracts, except for Zingiber officinale, where the rhizome was employed.

2. 3. Strain and culture media The diploid strain Aspergillus nidulans D-30 (K/ifer, 1986), carrying four recessive mutations for conidial color, was used. This strain was synthesized via parasexual cycle using haploids FGSC A593(a) and FGSC A594(b) from the Fungal Genetics Stock Center, Atlanta, USA. Mutations were as follows (in parenthesis, the linkage group of the corresponding haploid): yA2 (Ia) = yellow; wA2 (lib) = white; fwA2 (Villa) = fawn; chaA1 (VIIIb) = chartreuse. Somatic segregation occurrence (because of either mitotic crossover, chromosome malsegregation or some kind of clastogenic damage) can be assessed through visual inspection for homozygous colored sectors arising against a background of yellow to green (wild type) conidia. The complete medium (CM) proposed by Scott and K/ifer (1982) was employed.

2.4. Experimental procedure Tests for toxicity and genotoxicity were performed by plate incorporation as previously reported (De la Torre et al., 1989a). Plant extracts were added to melted CM (45°C). Groups of 14 plates for each dose were prepared and four plates were inoculated at the central point with conidia of strain D-30 in order to evaluate the quantitative toxicity after 72 h incubation at 37°C. Colony diameters were measured at this time and a Toxicity Index (TI) expressed as percentage reduction with respect to negative control. The remaining plates, inoculated at five equidistant points, were allowed to grow for six to ten days and then visually inspected for counting color

A.R. Ruiz et al. / Journal of Ethnopharmacology 52 (1996) 123-127

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3. Results and discussion

segregant sectors. The average frequency of colored sectors per colony (FCSC) was determined as an indicator of genotoxic events leading to somatic segregation. At least three concentrations were tested for every extract and experiments were repeated twice. The top concentration in the assay was determined in a previous dose finding experiment. It was chosen as the highest concentration causing no morphological changes in conidiation (e.g. 'fluffy' appearance) that might prevent the adequate scoring of segregant sectors.

The results obtained in this study are summarised in Table 1. As no genotoxic effects were observed in 12 out of the 13 plants screened, only the results corresponding to the maximal concentration assayed are shown in such cases. Toxic effects o n growth of colonies were not observed in 10 of these plants: M. officinalis, Men-

tha x piperita, P. amboinicus, O. aristatus, P. major, P. auritum, S. terebinthifolius, L.

Table 1 Somatic segregation induction in Aspergillus nidulans by some cuban medicinal plants Plant species (Family, voucher specimen number)

Melissa officinalis L. (Lamiaceae,

Extract Type a

% solids

T20

3.72

ROIG 4586)

Mentha × piperita L. (Lamiaceae,

T20

2.55

ROIG 4590)

Ortosiphon aristatus Blume (Lamiaceae, ROIG 4597) Piper auritum HBK. (Piperaceae, ROIG 4519) Schinus terebinthifolius Raddi (Anacardiaceae, ROIG 4513) Plantago major L. (Plantaginaceae, ROIG 5489)

FE

7.40

AEL

2.02

FE

12.64

FE

28.30

FE

7.88

Passiflora incarnata L.

FE

16.20

(Passifloraceae, ROIG 4587) Plectranthus amboinicus (Lour.) Spreng. (Lamiaeeae, ROIG 4579)

AEL

Plantago lanceolata L.

FE

23.80

FE

12.25

T50

0.34

AE

0.99

Lepidium virginicum L. (Brassicaceae, ROIG 4596)

(Plantaginaeeae, ROIG 4588) Cymbopogon citratus (DC.) Stapf (Poaceae, ROIG 4593) Zingiber officinale Roscoe (Zingiberaceae, ROIG 4595)

Momordica charantia L. (Cucurbitaceae, ROIG 4520)

0.92

Assayed concentration (mg/ml)b EtOH 0.52% 0.29 EtOH 0.65% 0.25 EtOH 0.40% 1.40 0 18.20 EtOH 0.60% 2.53 EtOH 1.20% 5.66 EtOH 0.40% 1.57 EtOH 0.32% 1.30 0 2 EtOH 0.40% 4.76 EtOH 0.24% 1.23 EtOH 0.46% 0.20 0 0.01 0.10 1.00 Chloral hydrate 6 mM c

TI

0 0 0 1

4 -2 -5 -6 -8 -14 18 34 -1 I -2 63

Colonies analyzed

FCSC

100 100 100 100 97 100 390 190 100 100 100 88 100 100 100 100 150 150 100 100 510 135 97 108 206 211 176 190 100

0.79 0.56 0.47 0~43 0.57 0.53 0.30 0.24 0.75 0.76 0.57 0.53 0.25 0.22 0.67 0.60 0.35 0.37 0.74 0.48 0.79 0.52 0.42 0.68 0.30 0.54* 0.77** 0.55* 2.46**

aAbbreviations for type of extract were as follows: T20, 20% tincture; T50, 50% tincture; FE, fluid extract; AE, aqueous extract; AEL, aqueous extract lyophilized. bFor each plant, the upper row corresponds to the negative control and the lower one to the maximal concentration assayed; negative control was always the solvent used in the extraction in a final concentration equal to that of the maximal dose assayed. The ethanol concentration is given in % by volume. ¢Positive control. Data for genotoxicity was normalized according to x/(x + 0.5/previous to statistical analysis (analysis of variance); *P < 0.05, **P < 0.01 (Dunnett test)

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A.R. Ruiz et al./Journal of Ethnopharmacology 52 (1996) 123-127

virginicum, P. lanceolata and P. incarnata. In some cases, even colony growth was stimulated by extracts, as shown by a negative TI. In those 10 plants, there were no significant differences in FCSC as compared to the corresponding negative control. With respect to this absense of genotoxicity in Aspergillus nidulans, it could be pointed out that, using fluid extracts from L. virginicum and O. aristatus, we did not find micronuclei induction in mouse bone marrow either. Besides, no sperm anomalies were observed in mice exposed to the same extract from P. amboinicus (unpublished results). C. citratus and Z. officinale showed appreciable toxicity. For both plants, antimicrobial activity has been previously demonstrated (Kokate and Varma, 1971; Moleyar y Narasimban, 1988; Mascolo et al., 1989). For C. citratus, the absence of genotoxicity has been reported in different assays: gene mutation in Bacillus subtilis (Unsurungsie, 1982); mitotic segregation and point mutation in A. nidulans and micronucleus induction in mouse bone marrow (De la Torre et al., 1989b). Besides, Kauderer et al. (1989) proved antimutagenic activity of fl-myrcene, a sesquiterpenic component of the essential oil, in mammalian tests in vitro (locus hprt in Chinese Hamster cells, chromosome aberrations and sister chromatid exchange in human lymphocytes). There is scarce information about genotoxicity of Z. officinale, although Qureshi et al. (1989) reported no sperm anomalies in animals treated for three months. Finally, M. charantia caused a significant increase in the FCSC, approximately two-fold with respect to the control at all tested concentrations, although it showed no quantitative toxicity. This suggested that the decoction from M. charantia leaves was genotoxic in this assay. Cytotoxic and antitumoral activities have also been demonstrated for the fruits of this plant (Takemoto et al., 1982; Jilka et al., 1983). Besides, inhibition of DNA and RNA synthesis in mammalian cell cultures was reported for ethanolic extracts from its seeds (Zhu et al., 1990). However, antimutagenic activity of ether extracts from the green fruit has been reported (Guevara et al., 1990).

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