Antinociceptive, genotoxic and histopathological study of Heliopsis longipes S.F. Blake in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: H. longipes S.F. Blake (Asteraceae) is a Mexican plant, whose roots are traditionally used as a condiment, as a mouth anesthetic, and as an antiparasitic. Affinin is the alkamide present in higher amounts in the roots of H. longipes. AIM OF THE STUDY: To date, there are no published studies regarding the relation between the analgesic properties, in vivo cytotoxicity, and DNA-damaging potential of H. longipes ethanol extract (HLEE). MATERIALS AND METHODS: The HLEE was chromatographically fingerprinted to validate its affinin contents. Biological evaluation was conducted in sets of 6-8 CD1(+) mice. Antinociceptive effect was evaluated using the writhing and hot-plate tests, and mutagenic and cytotoxic effects were evaluated with micronucleous test in CD1(+) mice. For histopathological studies, biological samples from liver, heart, kidneys, spleen, lung, and brain were collected and stained. RESULTS: Oral administration of HLEE (3-100 mg/kg) produced a dose-dependent antinociceptive effect in both assays. In micronucleus assay, the variability in the number of micronucleated polychromatic erythrocytes (MNPE) induced, and PE/NE index, the ratio of polychromatic erythrocytes with respect to the number of normochromatic erythrocytes induced by HLEE in the evaluated schedule, were small and nonsignificant. After histopathological results, HLEE showed polioencephalomalacia with 1000 mg/kg dose. CONCLUSIONS: This work provides evidence that HLEE exerts analgesic effects, with no genotoxic effects in vivo. These findings would be an important contribution to explain the use of H. longipes root as an effective analgesic in traditional medicine, and to establish for the first time the absence of genotoxic and cytotoxic effects of the root in bioactive doses in vivo.

Effect of xanthorrhizol, xanthorrhizol glycoside and trachylobanoic acid isolated from Cachani complex plants upon the contractile activity of uterine smooth muscle.

Xanthorrhizol, xanthorrhizol glycoside, and trachylobanoic acid, compounds isolated from medicinal plants that are grouped in the complex known as Cachani have been shown to inhibit the tonic contraction of rat uterus induced by: (a) depolarizing K+ solution (60 mM), (b) CaCl2 (1 mM), and (c) BAY K 8644 (0.3 microM) in a concentration-dependent manner (1-30 micrograms/mL). The inhibitory potency was displayed as follows: xanthorrhizol > xanthorrhizol glycoside > trachylobanoic acid. These results suggest that the assayed compounds might block voltage operated calcium influx in myometrial cells as they displayed a calcium-antagonistic activity. This effect is not due to peripheral receptor activation (beta 2-adrenergic, H2-histaminergic) as neither propranolol nor cimetidine modified the inhibitory effect of the compounds assayed. This is the first report showing that plants belonging to the Cachani complex may contain uterine smooth muscle bioactive substances.

Smooth muscle relaxing compounds from Dodonaea viscosa.

Bioassay-directed fractionation of the chloroform-methanol (1:1) extract of Dodonaea viscosa (L.) Jacq. (Sapindaceae) resulted in the isolation of four active spasmolytic principles: sakuranetin (1), 6-hydroxykaempferyl 3,7-dimethyl ether (2) hautrivaic acid (3), and ent-15, 16-epoxy-9 alpha H-labda-13(16)14-diene-3 beta, 8 alpha-diol (4). All the isolated compounds elicited a concentration-dependent inhibition of the spontaneous and electrically-induced contractions of guinea-pig ileum. Sakuranetin and the ent-labdane inhibited ileum contractions evoked by acetylcholine (Ach), histamine, and barium chloride. In addition, both substances were capable of relaxing contractions of rat uterus induced by Ca2+ in K(+)-depolarizing solution, displacing to the right the concentration-response curves to Ca2+. These results suggest that sakuranetin and ent-15,16-epoxy-9 alpha H-labda-13(16)14-diene-3 beta, 8 alpha-diol produce an interference with calcium metabolism in smooth muscle cells. The spasmolytic activity exhibited by the active principles from D. viscosa, provides the pharmacological basis for the traditional use of the plant as an antispasmodic agent.

Hypothalamic monoamine oxidase activity in ovariectomized rats after sexual behavior restoration.

The effect of estradiol benzoate, progesterone and a sequential treatment with both on the activity of the enzyme monoamine-oxidase (MAO) was assessed in mitochondria from hypothalami of ovariectomized rats. A differential effect on the subtypes A and B MAO was found according to the type of treatment. Estradiol benzoate administration decreased MAO activity, mainly that of MAO-A. Progesterone alone had no effect, and sequential treatment with estradiol benzoate plus progesterone restored sexual behavior and produced a significant increase of MAO-A activity, without changes in total MAO activity. Since MAO-A is an isoform of MAO that preferentially uses norepinephrine and serotonin as substrates and MAO-B acts on phenylethylamine and benzylamine as substrates, our findings suggest that the restoration of sexual behavior after the treatment with estradiol benzoate followed by progesterone may be associated with the differential effect exerted by the hormones on MAO subtypes, rather than to the simple decrease in hypothalamic monoamine concentrations as reported in the literature.

Antiestrogen U23,469 induced alterations of catecholamine levels on plasma and central nervous system.

The effect of antiestrogen U23,469 administration in vivo on the concentration of dopamine, norepinephrine and epinephrine in the plasma, cerebral cortex and hypothalamus in ovariectomized rats was investigated. Rats were treated with estradiol benzoate, progesterone and U23,469 in different doses, s.c., daily for 6 days. Control group was injected with sesame oil. Catecholamines were estimated by radioenzymatic assay. Six days of U23,469, estradiol benzoate, progesterone or its combination altered the catecholamine levels compared to the control. Dopamine decreased in plasma with progesterone and U23,469. In the cerebral cortex, progesterone and U23,469 increased significantly and in the hypothalamus all the treatments produced a decrease of catecholamines. The levels of NE were reduced with estradiol benzoate, progesterone and U23,469; there was no significant difference in the norepinephrine levels after different treatments in the cerebral cortex, but the NE levels were significantly decreased in the hypothalamus. Epinephrine showed differences related to the treatment, as in plasma, as in cerebral cortex and hypothalamus. These results suggest that antiestrogen treatment compared with the estradiol benzoate or progesterone may affect the catecholamine levels of the central nervous system and plasma and support the idea that AE could have an indirect effect on the catecholaminergic system.

The zoapatle. XV. Activity of 16 alpha-hydroxy-ent-kauran-19-oic acid isolated from Montanoa hibiscifolia, and its methyl ester on rat and guinea pig uterus.

16 alpha-Hydroxy-ent-kauran-19-oic acid was isolated from Montanoa hibiscifolia. The effects of this acid and its methyl ester on the contractile activity of rat and guinea pig uterine horns were studied. Both inhibited spontaneous, oxytocin-induced and potassium-induced contractile activities. The inhibitory effect produced by the methyl ester was greater than that observed with the acid. The methyl ester was 2-5 times more potent than the acid upon spontaneous and potassium-induced contractions and 11-15 times more potent than the acid upon the contractile activity of uterine smooth muscle induced by oxytocin. Such effects were observed using bath concentrations of 6, 15, 30 and 60 microM of each compound.

Zoapatle. XII. In vitro effect of kaurenoic acid isolated from Montanoa frutescens and two derivatives upon human spermatozoa.

Zoapatle aqueous crude extract (ZACE) from Montanoa tomentosa has been used as an oral contraceptive in traditional Mexican medicine for centuries. Kaurenoic acid, as isolated from Montanoa frutescens, and the methyl esters of 15-hydroxy-dihydro-kaurenoic acid and 15-keto-dihydro-kaurenoic acid were tested in vitro on human sperm motility and viability. The estimated ED50 concentrations immobilization were 374, 126 and 58 micrograms/ml, respectively using 15 X 10(6) sperms/500 microliter. Kaurenoic acid and both of its derivatives displayed only weak to negligible capacity for killing human sperms.

PIP: The effects on human sperm motility and viability of 3 compounds isolated from an aqueous extract of Montanoa frutescens Cerv (Compositae, Heliantheae) were tested. The plant is related to the zoapatle, traditionally used as a contraceptive, and known to have uterotonic and luteolytic activity. Kaurenoic acid, its 15 hydroxy- and its methyl ester were prepared from the polar fraction, and dissolved in 95% ethanol at a concentration of 10 mg/ml. Human sperm, suspended in Bigger's Whitten and Wittingham medium at a final concentration of 15 million sperm per 500 mcl were incubated at 37 degrees C. for 60 seconds. Judging sperm motility under phase contrast microscopy, kaurenoic acid was 0.89 times less effective, and the hydroxy kaurenoic acid 2.6 times more effective than verapamil, while the keto derivative was 5.7 times more potent than verapamil at the same concentration. Sperm viability assessed by vital staining was hampered only about 10% by the keto-kaurenoic acid, 21% by kaurenoic acid, and 33% by hydroxy-kaurenoic acid. Doses required for immobilization of 50% of human sperms ranged from 58 to 374 mcg/ml. This is 225 times lower than that reported for gossypol in vitro, and much lower than the millimolar range needed to inactivate sperm with nonoxynol. Thus these drugs may prompt a search for analogs useful in vaginal contraceptives.

The zoapatle. VII. Antiimplantation effect in the rat of zoapatle aqueous crude extract (ZACE) from Montanoa tomentosa and Montanoa frutescens.

Intrauterine administration of zoapatle aqueous crude extract (ZACE) from Montanoa frutescens on the fourth day of rat pregnancy, at concentrations equivalent to 50 mg and 5 mg of dry leaves, was associated with total inhibition of implantation sites. On the other hand, ZACE from Montanoa tomentosa equivalent to 50 and/or 100 mg of dry leaves, prepared and administered in the same fashion, did not inhibit the number of implants by day 11 of pregnancy. However, most implants were found abnormal, of blue color, poor orientation or spacing; these morphological changes could be considered as reabsorption sites.

The zoapatle. IX. In vitro effect of Montanoa tomentosa and Montanoa frutescens upon human sperm and red cells.

The effect of zoapatle aqueous crude extract (ZACE) was further studied and partially characterized upon human and rabbit spermatozoa. ZACE prepared from Montanoa tomentosa s.s.p tomentosa did not influence sperm motility or viability in a wide range of ZACE concentrations tested; on the other hand, ZACE prepared from Montanoa frutescens had immediate and constant inhibitory effect upon motility and decreased cell viability. Red cell lysis was readily observed with ZACE-frutescens, but not with ZACE-tomentosa. The effect of time on ZACE-frutescens potency for inducing red cell lysis was observed.

The zoapatle. X. The in vitro effect of zoapatle aqueous crude extract (ZACE) and histamine upon rat and guinea pig uterine strips.

In vitro histamine response in rat and guinea pig uterine strips was similar to the one observed with zoapatle aqueous crude extract (ZACE). The relaxing effect elicited by ZACE/tomentosa on the in vitro rat uterine strips was not mediated by activation of H-2 histamine receptors; moreover, the increase on uterine contractility elicited by ZACE/tomentosa on the in vitro guinea pig uterine strip was not mediated through the activation of H-1 histamine receptors.

The zoapatle. XI. Effects elicited by Montanoa tomentosa and Montanoa frutescens on rat uterine strips.

Zoapatle aqueous crude extract (ZACE) obtained from Montanoa tomentosa (M.t.) inhibits the in vitro spontaneous contractility pattern of rat uterine tissue. The opposite effect was observed with ZACE from Montanoa frutescens (M.f.) in the same preparation. Both plant extracts, M.t. and M.f., increased the in vitro spontaneous contractility pattern in the uterine guinea pig assay. In depolarized uterine tissue, propranolol (beta-blocker) inhibited the relaxing effect induced by M.t.. Atropine (cholinergic antagonist) abolished the increase in uterus contractility produced by the presence of M.f.

The zoapatle II--botanical and ecological determinants.

A collection of Montanoa (Cerv) specimens was conducted throughout Mexico. Twenty-one specimens were classified, some of them grown in the greenhouse and transplanted in an agricultural experimental field station in the Valley of Mexico. In vitro uterotonic potency was assayed and the results expressed as equivalents of oxytocine, by using estrogenized guinea pig uterine strips. A great variation of uterotonic potency was observed among the wild plants. A clear decrease and uniformity of uterotonic potency was found in plants grown in the experimental field. The study points out the importance of ecological variations in expression of the plant's biological activity.

PIP: 21 specimens of the zoapatle Montanoa (Cerv) plant were collected from sites throughout Mexico with a variety of climatic, humidity, soil composition, and altitude conditions to assess their uterotonic properties. Some plants were then grown in an agricultural experimental field station of the National Institute of Agricultural Research, where all the variables were known, allowing for a valid comparison between species and plants. In vitro uterotonic potency was assayed and the results expressed as equivalents of oxytocin. The wild plants were found to vary greatly in uterotonic potency, perhaps reflecting differences in ecological conditions. The most active uterotonic zoapatle aqueous crude extract was the Montanoa frutescens, and the least active was M. mollissima, the former having approximately 4 times the uterotonic potency of the latter. The plants grown in the agricultural field station showed a considerable decrease in uterotonic potency, but valid comparisons must await assay in winter to match the stage in the plant life when the wild samples were assayed. Plant age, the part of the plant from which the uterotonic principle is extracted, climate, altitude, and soil constituents are other possible influences on uterotonic potency. A sample of the zoapatle aqueous crude extract with the highest uterotonic potency will be assayed for its luteolytic activity in an animal model.

The zoapatle III--biological and uterotonic properties of aqueous plant extract.

Differences in uterotonic activity were observed between zoapatle Montanoa (Cerv.), plants growing in their natural habitat and plants growing in an experimental agricultural plot. Details of an in vitro analogic model for assaying uterotonic potency in guinea pig strips is described. Important species differences on the uterine response to zoapatle aqueous crude extract were noticed in rats, hamsters, guinea pigs, cats and Rhesus monkeys. The need for proper biological evaluation of chemical substances already isolated from zoapatle specimens, is mentioned, and the advantages of working with zoapatle specimens grown under controlled ecological conditions are pointed out.

PIP: An in vitro analogic method for comparing the uterotonic potency of dialysed zoapatle aqueous crude extract (DZ-ZACE) with oxytocin is described. Uterine strips from estrogenized guinea pigs were selected as the appropriate animal model after trials with estrogenized rats, hamsters, cats, and Rhesus monkeys. Results of the in vitro uterotonic effect produced by 21 different zoapatle Montanoa plants collected from different parts of Mexico and grown in an experimental agricultural field station indicate considerable difference, with less variation in uterotonic oxytocin/equivalents among the plants grown at the field station than in wild specimens. The general reduction in uterotonic potency of plants grown at the field station may be due to differences in the time of collection: wild specimens were collected in winter 1980, while field station specimens were collected just after flowering in early summer 1981. The findings clearly demonstrate the influence of the plant environment on the expression of biological response. Because of its lack of toxicity, abundance, ease of production, and the availability of an efficient analogic in vitro assay for the evaluation of relative uterotonic potency, ZACE may eventually be the source of a new agent to intercept pregnancy.

The zoapatle IV--toxicological and clinical studies.

The zoapatle aqueous crude extract has been used in Mexico for the last 5 centuries for the induction of labor, treatment of post-partum bleeding problems, and as a menses inducer. Today, it is sold in street markets, and its long documented history of use by humans could be taken as indirect evidence of a lack of toxicity. Rigorous pharmacological and clinical studies described here, fully confirm the empirical observations.

PIP: For the last 5 centuries in Mexico the zoapatle aqueous crude extract (ZACE) has been used for the induction of labor, treatment of postpartum bleeding problems, and as a menses inducer. Despite widespread and continuous use of ZACE on the part of the Mexican population and the absence of documented side effects or toxicological phenomenon associated with its use, it was decided to undertake acute and subacute toxicological studies prior to clinical studies in volunteer subjects in Mexico and Sweden. Plant specimens for the studies were collected in different batches at several locations near the National University of Mexico. The material was properly identified at the university's botanical herbarium. ZACE was prepared by boiling 100 g of dry leaves in 400 ml of distilled water for 20-30 minutes, filtered through gauze, and the final volume obtained by using a rotor evaporator 50-60 degrees Centigrade with light negative pressure, to 100 ml. The ZACE concentration was 1 g dry leaves/ml. The extract was tested in an "in vitro" guinea pig uterine strip assay. For the 1st series of studies in Mexico, 60 female Sprague-Dawley rats, 8 weeks old, and 6 mongrel female dogs, were used. 25 rats were used as control and 35 tested. All animals were maintained in a controlled laboratory condition with 12/12 hour light/darkness cycle. 2 dogs served as control, and 4 were treated. All experimental animals received, through a rubber cannula, 5 ml/KG of ZACE daily. Control animals received distilled water. Each animal was carefully observed for 30 minutes after fluid intake. At the end of the study, the rats were sacrificed with ether, and the dogs with phenobarbital. Complete anatomapathological examinations were performed. 6 control and 8 treated rats died during the study, and all deaths were associated with accidental placement of the rubber cannula. Behavioral changes were observed in neither rats nor dogs throughout the study. No changes were recorded in body weights. No macro nor microscopic alteration was found in either group of rats. Results of hematological examinations did not differ between the 2 groups. It was concluded that ZACE is devoid of acute and subacute toxicity. In addition to the clinical observations reported by Gallegos in Mexico, where 10 normally menstruating volunteer women received 15 or 30 gm/day of zoapatle dry leaves extract orally for 3-9 consecutive days without clinical evidence of any side effects, clinical investigations were conducted by Landgren et al. in Sweden. 6 women in early pregnancy were given zoapatle; 6 women in early pregnancy served as a control group. A significant dilation of the cervix was found in each of the subjects treated with zoapatle decocts. A menstrual like cramp was reported by all and bleeding occurred in 4 of 6 subjects. No gastrointestinal symptoms were observed. No pain or bleeding nor significant dilatation of the cervix was evident prior to surgery. Immediately prior to vacuum extraction, cervical dilatation was less than 5 mm in all subjects. In sum, ZACE was devoid of toxic complication or side effects.

The zoapatle V--the effect of kauradienoic acid upon uterine contractility.

Kauradienoic acid was obtained from the hexanic extract of M. tomentosa (Cerv) leaves by chromatographic separation. This substance influenced the in vitro contractility of the rat, dog and guinea pig uterine strips. It also induced strong contractions of the guinea pig uterus in vivo when administered intravenously, without changes on arterial blood pressure. The effects produced by the plant infusion, the hexanic extract and pure species were compared. The hexanic of other utero-active compounds in M. tomentosa in addition to those already described is discussed.

PIP: The investigation of uterotonic effects of the hexanic extract (HE) of M. tomentosa leaves, as well as that (-)-kaura-9(11), 16-dien-19-oic acid, henceforth referred to as kauradienoic acid (KA) is described. Pregnant and nonpregnant female Wistar rats, guinea pigs, and dogs were used in this study. Strips obtained from 5 animals had spontaneous rhythmical contractions constant in amplitude and frequency. Muscular tone and frequency of contractions were notably increased by addition of 20 ul/ml of zoapatle aqueous crude extract (ZACE). The preparation returned to the original spontaneous contractility pattern after washing with a modified Ringer solution. Strips obtained from 5 gravid guinea pigs showed scarce spontaneous contractility which was strongly augmented by addition of ZACE. Regular spontaneous contractions were observed in over 30 assays of uterine strips obtained from 5 nongravid rats. The addition of ZACE drastically inhibited the spontaneous contractility. Constant and reproducible results were obtained in gravid rats by addition of ZACE. A gradual decrease in amplitude of contractions was observed. The addition of ZACE depressed the uterine contractility (UC) of 8 uterine strips from 2 animals. The addition of ZACE in gravid dogs in 8 uterine strips was accompanied by an increase of muscular tonus and frequency. A gradual decrease in the amplitude of the rat spontaneous UC was an effect produced by the HE. Addition of water suspension of HE elicited an immediate increase of guinea pig's uterine motility. Results were similar with those observed with ZACE. KA inhibited the "in vitro" rat UC and increased the "in vitro" guinea pig contractions of uterine musculature in a similar fashion as observed with ZACE and HE.