Toxicity studies and phytochemical screening of four medicinal plants in the caribean

Revista de Fitoterapia 2005; 5 (S1) COMUNICACIONES BREVES · SHORT COMMUNICATIONS V Coloquio Europeo de Etnofarmacología 244 Revista de Fitoterapia 2005; 5 (S1) V Coloquio Europeo de Etnofarmacología SHORT COMMUNICATIONS · COMUNICACIONES BREVES 245 Toxicity studies and phytochemical screening of four medicinal plants in the caribean SAENZ, M.T.; GARCIA, M.D. Laboratorio de Farmacognosia, Facultad de Farmacia, Universidad de Sevilla, ESPAÑA Key words: Anredera leptostachys, Bixa orellana, Eryngium foetidum, Haematoxylon campechianum, toxicity studies, phytochemical screening. Introduction A large number of single and compound herbal drugs are used in the traditional Caribbean system of medicine for the treatment of diverse ailments (Robineau, 1989, Germosen-Robineau, 1995). Four such medicinal plants which are used against diseases where long term treatment is required were selected for the present study. The parts used in this study and various medicinal properties attributed to these plants are given in TABLE 1.There are limited reports on the phytochemical constituents and on some of the pharmacological activities of these plants. However, data are available on their side or toxic effects in the literature. In this study the acute and chronic toxicity of these plants in mice was evaluated and a phytochemical screening was preformed. Materials and methods Plant material The flowery plants were identified at the Jardin Botanico Nacional Dr. Rafael M. Moscoso (Santo Domingo) by Dr. F. Jimenez. The samples studied were: Tuber of Anredera leptostachys (Moq.) Steenis (Basellaceae) (Jimenez, 1445), seed of Bixa orellana L. (Bixaceae) (Jimenez, 1477), leaves of Eyngium foetidum L. (Apiaceae) (Jimenez, 1768) and leaves and woods of Haematoxylon campechianum L. (Caesalpinaceae) (Jimenez, 1617 and 1478 respectively). Toxicity The respective part of the plant was infused in boiling water and then macerated for 24 hours. The macerate was filtered and the solvent evaporated at low pressure. The water-free residue was suspended in normal saline and used in the experiments. Swis mice of both sexes, aged 7-8 weeks, weighing 25-35 g, and fed purina chow diet and water ad libitum were used. Acute toxicity A total of 120 swiss mice were randomly alloted to different control and treatment groups. The extracts in each case was injected intraperitoneal by in doses between 100 and 2000 mg/Kg. The general symtoms of toxicity and mortality were observed for 48 h following method described by Gallego (Gallego, 1986). Chronic toxicity A total of 144 swiss mice were randomly alloted to different treated and control groups. The extracts were injected intraperitoneally. The dose selected was 100 mg/kg body weigth per day, which is 1/5 of de pharmacologically active dose for a period of 2 months, following method described by Galllego (Gallego, 1986).The different parameters were subjected to statistical analysis by Student’s test. Phytochemical screening The different plants parts were pulverized and successively extracted with petroleum ether, methanol and water.The extracts were concentrated under reduced pressure and the chemicals constituents of the residue were determined. Results and discussion The results of the acute toxicity studies (LD50) are summarized in TABLE 2. The leaf of H. campechianum exhibited the most highest toxicity (215,12 ± 10,70 mg d.r./Kg). However, E. foetidum showed no signs of toxicity in the dose range of 200 mg d.r./Kg to 1400 mg d.r./Kg. The results of the chronic toxicity studies are summarized in TABLES 3, 4 AND 5. During chronic treatment loss of body weight or other abnormalities of the vital organ were observed as compared to the control. The results of phytochemical studies are presented in TABLE 6. Animals administered with A. leptostachys extract showed no significant changes during the acute treatment. However, on chronic treatment, the average weigth increase in this group was significative. This effect might be due to carbohydrates present in this extract. The observations of the vital organs were normal. In acute treatment test B. orellana extract did not exhibit toxicity. At the higher dose, a decrease locomotor activity was observed. During the chronic treatment 5% of male mice and 2% of female mices developed hepatic toxicity but the others abnormalities were no observed. This seed contains greases, carotenes, sterols and triterpenes, leucoanthocyanins, catecols, tannis and carbohydrates. The importance of this seed in asthenia may be due to greases and carbohydrates content. This effect colouring may be attributed to carotenes. When it ingested over a period of several weeks, the seed produces carotenemia, a yellowed of the skin often firstly observed in the palms of the hands or on the soles of the feed (Tyler, 1988). Fuente: www.fitoterapia.net Revista de Fitoterapia 2005; 5 (S1) COMUNICACIONES BREVES · SHORT COMMUNICATIONS V Coloquio Europeo de Etnofarmacología 246 Revista de Fitoterapia 2005; 5 (S1) V Coloquio Europeo de Etnofarmacología SHORT COMMUNICATIONS · COMUNICACIONES BREVES 247 The acute toxicity studies E. foetidum extract did not present signs of toxicity. During chronic treatment no loss of body weight nor other abnormalities in the vital organs were observed. The leaves of this plant contain flavonoids, catechol tannins, carbohydrates and saponins. The leaves and the wood of H. campechianum produced stimulation, pilo-erection and rapid irregular respiration preceding death during acute treatment. During the chronic treatment 37.5% and 24% of the animals died with the leaves and the wood extracts. There was significant changes in average body weight. This leaves and the wood showed too toxic lesions in kidney and liver (TABLE 5), something less strong with the wood. The leaves of this plant contain mainly the lactones and alkaloids and the wood contains especially the alkaloids and saponins. These compounds may be responsible for the observed toxicity and the undiserable effects. The acute and chronic toxicity as a result of the present investigation may provide important evidence concerning the therapeutic safety of these drugs and assist in assessment of possible meausures to be introduced before these plants can be safely used in the health care system of the Caribbean. Acknowledgements: The autors express their sincere thanks to Dr. Germosen-Robineau (Enda Caribe) for providing the facilities and to Dr. F. Jimenez for identification of the plant material. References Aguilar, J.: Relaciones de unos aspectos de la Flora utile de Guatemala, Ed. Universidad de Guatemala, 1966. Adjanohoun, E.: Contribution aux études ethnobotaniques et floristiques à la Dominique. Ed.Acct, Paris, 1985. Beuzer, R.: La santé par les plantes des Antillés Francaises. Ed. Emile Désormaux. Pointe-à-Pitre, Guadalupe, 1973. Gallego, M.T.: Toxicidad subcrónica de Viscum cruciatum Sieber. Tesis Doctoral, Facultad de Farmacia, Sevilla, 1986. Germosen-Robineau, L.(ed.): Hacia una Farmacopea caribeña(edición Tramil 7). Ed. Enda-Caribe, UAG &Universidad de Antioquia, Santo Domingo, 1995. Martinez, M.: Las plantas medicinales de Mexico. Ed. Botas, Mexico,, 1959. Nuñez, E.: Plantas medicinales de Costa Rica y su folklore. Ed. Ciudad Universitaria Rodrigo Facio, San José de Costa Rica, 1975. Robineau, L. (ed.): Towards a caribbean pharmacopoeia(edición Tramil 4). Ed. Enda Caribe & ANAH, Honduras, 1989. Stehle, H., Sthehle, M.: Flora médicinale illustrée. Ed. Imprimerie officielle, Basse Terre. Guadalupe, 1962. Tyler, V.E., Brady, L.R., Robbers, J.E.: Pharmacognosy, Ed. Lea & Febiger, Philadelphia, 1988. Weniger, B.: La Medècine populaire dans le plateau Central d’Haiti. Thèse Univ., Metz, 1985. Wong, W.: Some folk medicinal plants from Trinidad. Econom. Bot. 30:103-142, 1976. Plant Part used Uses or disease treated References Anredera leptostachys (Moq.) Steenis (Basellaceae) Tuber Leaf Bone fracture Anti-inflammatory Analgesic Vulneraly Emollient Germosen-Robineau 1995 Adjanohun, 1985 Adjanohun, 1985 Nuñez, 1975 Beuzer, 1973 Bixa orellana L. (Bixaceae) Seed Root Leaf Shock, Asthemia Emollient Diabetes Emmenagogue, Diuretic, Antidiarrheal Germosen-Robineau 1995 Martinez, 1959 Wong, 1976 Aguilar, 1966 Eryngium foetidum L. (Apiaceae) Leaf Whole plant Gastralgia, Diarrhea, Hypertension Vulneraly Febrifuge, Antirheumatic Emmenagogue Robineau, 1989 Weniger, 1985 Stehle, 1962 Wong, 1976 Haematoxylon campechianum L. (Caesalpinaceae) Leaf Wood Amenorrhea,Anemia Shock Anemia Amenorrhea Diarrhea Germosen-Robineau 1995 Wong,1976 Germosen-Rob.1995 Martinez, 1959 TABLE 1. Medicinal uses of the plants under investigation for the presents studies Fuente: www.fitoterapia.net Revista de Fitoterapia 2005; 5 (S1) COMUNICACIONES BREVES · SHORT COMMUNICATIONS V Coloquio Europeo de Etnofarmacología 246 Revista de Fitoterapia 2005; 5 (S1) V Coloquio Europeo de Etnofarmacología SHORT COMMUNICATIONS · COMUNICACIONES BREVES 247 mg d.r./Kg ± SE g plant/Kg ± SE Anredera leptostachys (Moq.) Steenis Tuber 1043.38 ± 137.14 61.07 ± 7.93 Bixa orellana L. Seed 1092.25 ± 202.64 19.50 ± 3.62 Eryngium foetidum L. Leaves 1470.03 ± 124.35 11.12 ± 0.94 Haematoxylom campechianum L. Leaves Woods 215.12 ± 10.70 498.14 ± 66.38 0.76 ± 0.04 24.05 ± 3.20 TABLE 2. LD50 obtained with different plants nº of mice treated Mortality (days) 15 30 45 60 Total of dead animals Lethality (%) Control 24 0 0 1 1 2 8.3 A. leptostachys Tuber 24 0 1 1 1 3 12.5 Bixa orellana Seeds 24 0 1 1 1 3 12.5 E. foetidum Leaves 24 0 0 2 2 4 16.6 H. campechianum Leaves Wood 24 24 1 2 2 4 0 1 2 3 9 6 37.5 ** 24.0 * * p≤ 0.05 ** p≤ 0.001 TABLE 3. Quantitative data on the mortality induced in mice on chronic treatment with different plant extracts. Male mince Pre-treatment average body weight Female mince Pre-treatment average body weight Male mince Post-treatment average body weight Female mince Post-treatment average body weight Control 33,60 ± 0,30 27,02 ± 0,32 36,84 ± 0,39 * 29,01 ± 0,38 * Tuber of A. leptostachys 32,32 ± 0,27 29,17 ± 0,25 34,80 ± 0,32 * 30,90 ± 0,42 * Seeds of B. orellana 34,40 ± 0,32 28,43 ± 0,27 33,90 ± 0,42 28,06 ± 0,50 Leaves of E. foetidum 34,82 ± 0,31 28,94 ± 0,38 35,20 ± 0,49 27,86 ± 0,49 Leaves of H. campechianum 32,70 ± 0,28 29,43 ± 0,20 30,23 ± 0,41 *27,10 ± 0,57 * Wood of H. campechianum 34,90 ± 0,25 32,98 ± 0,24 32,20 ± 0,49 * 30,89 ± 0,49 * * p0,05 ** p0,001 TABLA 4. Quantitative data on the average body weight in mice on chronic treatment with different plants extracts. Fuente: www.fitoterapia.net Revista de Fitoterapia 2005; 5 (S1) COMUNICACIONES BREVES · SHORT COMMUNICATIONS V Coloquio Europeo de Etnofarmacología 248 Revista de Fitoterapia 2005; 5 (S1) V Coloquio Europeo de Etnofarmacología SHORT COMMUNICATIONS · COMUNICACIONES BREVES 249 Kidney (Glomerular inflammation) Degeneration on the renal tubule Liver (Hepatic toxicity) (Kupffer’s cel.) Spinal cord males females males females males females males females males females Control 0 0 0 0 0 0 normal Tuber of A. leptostachys 0 0 0 0 0 0 normal Seeds of B. orellana 0 0 0 0 5% 2% normal Leaves of E. foetidum 0 0 0 0 0 0 normal Leaves of H. campechianum 10% 20% 30% 50% 20% 40% normal Wood of H. campechianum 5% 15% 20% 30% l0% 25% Normal TABLE 5. Disease observed on the vital organs. A. leptostachys (Tuber) B. orellana (Seed) E. foetidum (Leaf) H.campechianum (Leaf) H.campechianum (Wood) greases + ++ - - - carotenes - +++ + ++ + sterols and triterpenes + +++ + ++ - quinones - - - + - lactones - + - +++ + leucoanthocyannins - ++ - ++ +++ flavonoids - + ++ ++ + catechol tannins - ++ +++ - - pyrogallol tannins - - - +++ +++ alkaloids - - - +++ ++ carbo-hydrates +++ +++ +++ +++ +++ saponins ++ + ++ - +++ + (weakly positive), ++ (positive), +++ (very positive), - (negative) TABLE 6. The compounds identified in the phytochemical study. Fuente: www.fitoterapia.net