In vivo antitumoural activity and acute toxicity study ofLarrea divaricata Cav. extract

PHYTOTHERAPY RESEARCH, VOL. 11, 521–523 (1997)

SHORT COMMUNICATION

In Vivo Antitumoural Activity and Acute Toxicity Study of Larrea divaricata Cav. Extract

Claudia Anesini,1 Jose Boccio,2 Graciela Cremaschi,3 Ana Genaro,3 Marcela Zubillaga,2 Leonor Sterin Borda3 and Enri S. Borda2 w 1

Department of Pharmacology, School of Dentistry, Buenos Aires University Radioisotope Laboratory, School of Pharmacy and Biochemistry, Buenos Aires University 3 CEFYBO-CONICET, Buenos Aires, Argentina 2

In the present work, we analysed the effect of the extract of Larrea divaricata Cav. on a mammary carcinoma chemically induced with N-nitroso-N-methylurea in female rats. The extract was administered in a dose of 25 mg/ kg, subcutaneously, beginning 20 days after tumour appearance. Of a total of 75 tumours treated with extract, a regression of 10 tumours (13%) was observed, a stabilization of 60 tumours (80%) and an increment of 5 tumours (6%). None of the 80 control tumours decreased (0%) and only 12 tumours remained static (15%). The survival time of the animals treated with the extract was significantly higher (p < 0.05) than the survival time of the controls. The LD50 in females was lower than in male mice. Data indicate that the aqueous extract of Larrea divaricata Cav. could be considered tumouristatic as the extract blocked progress of the tumour. © 1997 John Wiley & Sons, Ltd. Phytother. Res. 11, 521–523, 1997 No. of Figures: 2. No. of Tables: 1.

No. of References: 9

Keywords: Larrea divaricata Cav.; antitumoural activity; acute toxicity.

INTRODUCTION Antimicrobial, antitumoural and antiinflammatory activities of Larrea divaricata Cav. have been documented (Smart et al., 1968; Ratera and Ratera, 1980). In a previous work (Anesini et al., 1996), we have found that an aqueous extract from the leaves of this plant exerted an antiproliferative activity in vitro on a T lymphoma cell line (BW 5147); but on normal lymphocytes it had no antiproliferative activity triggering only a stimulatory effect. Also, the antiproliferative effect was observed on concanavalin A (Con A) stimulated lymphocytes without changes in cell viability, suggesting a suppressive but not a cytotoxic effect against hyperproliferative lymphocytes. Moreover, the antiproliferative effect of the extract was higher in the T lymphoma cell line than in the Con A stimulated lymphocytes. On the other hand, the mechanism of action of the extract on BW 5147 cells appeared to be related to arachidonic acid metabolism, via leukotriene synthesis (Anesini et al., 1996). Also, the increase in cAMP level has been associated with its antiproliferative activity but would not be mediated by the activation of b adrenergic and histaminergic receptors (Anesini et al., 1996). A complete regression of a spontaneous mammary carcinoma of a pregnant rat was found by in vivo administration of the extract (Anesini et al., 1996). Here, we analyse the in vivo effect of the extract on a w

Correspondence to: Enri S. Borda, CEFYBO-CONICET, Serrano 669, 1414, Buenos Aires, Argentina Contract grant sponsor: Buenos Aires University Contract grant number: OD 014 CCC 0951–418X/97/070521–03 $17.50

© 1997 John Wiley & Sons, Ltd.

mammary carcinoma chemically induced with N- nitrosoN-methylurea in female rats. The acute toxicity of the extract was examined and the LD50 of the extract in female and male mice is also reported. MATERIALS AND METHODS Preparation of the extract. The extract from air dried leaves

of Larrea divaricata Cav. (Anesini and P´erez, 1994), was prepared as previously reported (Anesini et al., 1996). An aqueous suspension of the extract was prepared by redissolving the residue in distilled water. A concentration of 1540 mg/mL was obtained and different dilutions were made to obtain the experimental concentrations used in this study. The vehicle alone was used as control. Induction of mammary carcinomas. Mammary tumours in female Sprague-Dawley rats (200 g), were induced by three i.p. injections of the carcinogen N-nitroso-N-methylurea (NMU) at a dose of 50 mg/kg, when the animals were 50, 80 and 110 days old (Gullino et al., 1975). Tumour sizes were measured at weekly intervals with a caliper and their growth parameters and histopathological properties were tested. Five animals that did not receive any extract were randomly chosen for histological examination. To do this, these animals were necropsied when the tumours were at least 2 cm in diameter, the tumours were classified according to their histological appearance (Rivera et al., 1994). Determination of the in vivo antitumoural activity of the extract. Rats were divided into two groups: 20 animals Accepted (revised) 7 April 1997

522

C. ANESINI ET AL.

Figure 1. Comparison of the progression of mammary tumours in a control untreated representative rat (A) and an extract treated representative rat (B). The untreated rat did not receive any extract, while the treated rat was administered with 25 mg/kg of Larrea divaricata Cav. extract subcutaneously, thrice a week, beginning on day 20 of tumour evolution. The size of tumours was expressed in cm and the progression was registered with the time. The different symbols represent different tumours in the rat. Each rat had 3–6 tumours.

treated with the extract (25 mg/kg) and 25 animals that received only vehicle (control group). Both were administered subcutaneously thrice a week beginning 20 days after the tumour appearance. Acute toxicity studies. To determine the DL50 of the extract,

female and male Albino Swiss (20–25 g) mice were divided into 8 groups of 10 animals each. Each group received increasing doses of the aqueous solution (25–25 000 mg/kg, i.p.). The DL50 for male and female mice were determined as described (Litchfield and Wilcoxon, 1949).

Induction of mammary carcinomas To characterize the tumours induced with NMU, histopathological assays were performed. The histopathological studies showed that 98% of the induced tumours were malignant mammary carcinomas and 2% of the tumours were benign adenomatous hyperplasias. Histological classification of tumours showed that the predominant type was adenocarcinoma (48%), followed by cribiformic carcinoma (32%), comedocarcinoma (17%) and papillary carcinoma (1%). Metastases were observed in lungs, spleen, liver and smooth tissues. NMU induced 3–6 tumours per rat.

Statistical analysis. Differences in cumulative mortalities

were evaluated by Student‘s t-test. Determination of the in vivo antitumoural activity of the extract RESULTS AND DISCUSSION

Table 1. Effect of the extract on some growth parameters of tumours and survival time Tumour parametera

Control

Treated

Total tumours Stabilization Regression Progression Survival timeb

80 12 (15%) 2 (0%) 68 (85%) 38 ± 20 days

75 60 (80%) 10 (13%) 5 (6%) 70 ± 14 days

a The results are expressed as percentage of tumours that decreased, increased or remained static in control and extract treated animals. b The results are expressed in days as mean ± SD of 20 treated and 20 control animals. The differences are significant according to Student‘s t-test.

Phytother. Res. 11, 521–523 (1997)

The extract demonstrated an antitumoural activity when administered subcutaneously in a dose of 25 mg/kg thrice a week to female rats that had chemically induced tumours. This dose was selected as in previous studies performed in vitro, it corresponds to the range of the medium effective concentration of the extract. Figure 1 shows the in vivo antitumoural action of the extract along with the evolution of tumours. While untreated tumours (control group, Fig. 1A) exhibited a continuous progression (increase of 50% in surface area) the extract-treated tumours (treated group, Fig. 1B) stopped their progression and remained static and one underwent partial regression (decrease of 50% in surface area) (Table 1). Table 1 also shows a tumouristatic action because 80% of the extract-treated tumours remained static in comparison with the controls. The extract also increased significantly the animal survival time of about 90% (Table 1). Moreover the results are considered of sufficient interest © 1997 John Wiley & Sons, Ltd.

ACUTE TOXICITY OF L. DIVARICATA

523

Figure 2. Acute toxicity study of the Larrea divaricata Cav. extract. Data are expressed as percentage deaths for female (j) and male (r) mice (A), or probit of data following the Litchfield study of LD50 (B). The number of animals that died after a single intraperitoneal administration of the extract in different doses (25–25000 mg/kg) in Albino Swiss mice is shown.

to warrant further studies as this type of carcinoma is very similar to the mammary human carcinomas (McGuire and De La Garza, 1973).

Acute toxicity studies To investigate the acute toxicity of the extract, different doses were administered to female and male mice and the LD50 was calculated. As shown in Fig. 2 (panel A and B), male mice were significantly more resistant to the extract than female mice as the DL50 in males was 10 000 (8196 to

12 200) mg/kg, whereas the LD50 in female mice was 4000 (3030–5276) mg/kg, the effective dose being 150 times less than the LD50 for female mice and 500 times less than the LD50 for male mice. No sign of macroscopic toxicity was observed below 3000 mg/kg of the extract in female and male mice. With higher concentrations signs of apnoea were found in some animals. Work is now in progress to identify the active compounds. Acknowledgement The authors thank Buenos Aires University for the grant (OD 014) that supports this work.

REFERENCES

Anesini, C., Genaro, A., Cremaschi, G., et al. (1996). In vivo and in vitro antitumoral action of Larrea divaricata cav. Acta Physiol. Pharmacol. Ther. Latinoam. 46 (1), 33–40. Anesini, C., Genaro, A., Cremaschi, G., Sterin Borda, L., Cazaux, C., and Borda, E. (1996). Immunomodulatory action of Larrea divaricata Cav. Fitoterapia LXVII, 4, 329–333. Anesini, C., and Perez, ´ C. (1994). Screening of plants used in Argentine folk medicine for antimicrobial activity. J. Ethnopharmacol. 39, 119–128. Gullino, P., Pettigrew, H., and Grantham, H. (1975). N-nitrosomethylurea as mammary gland carcinogen in rats. J. Cancer Inst. 54, 401–404. Litchfield, J., and Wilcoxon, F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp.

© 1997 John Wiley & Sons, Ltd.

Ther. 96, 99–113. McGuire, W., and De La Garza, M. (1973). Similarity of estrogen receptor in human and rat mammary carcinoma. J. Clin Endocrinol. Metab. 36, 548–552. Ratera, E. L., and Ratera, M. O. (1980). Plantas de la Flora Argentina Empleadas en Medicina Popular, pp. 1–105. Hemisferio Sur, Buenos Aires. Rivera, E., Andrade, N., Martin, G., et al. (1994). Induction of mammary tumours in rat by intraperitoneal injection of NMU. Cancer Lett. 86 (2), 223–228. Smart, C. R., Hogle, H. H., Robins, R. K., Broom, A. D., and Bartholomew, D. (1968). An interesting observation on nordihydroguaiaretic acid (NSC 4291; NDGA) and a preliminary report. Cancer Chemother. Rep. (part 1) 53, 147–151.

Phytother. Res. 11, 521–523 (1997)