Occlusion of vas deferens after a single intraluminal injection of carrageenan and other chemical agents in rabbits.

A direct single injection of a mixture of sclerosing chemical agents into the lumen of the rabbit vas deferens was effective as early as 4 weeks post treatment in 13 of 16 animals. No effect was observed in carrageenan-treated animals.

Hypoglycaemia associated to vaginal insulin placement in rats.

The effect of vaginal placement of insulin was ascertained in rats by measuring plasma glucose levels. A rapid and sustained decrease of plasma glucose levels at 30, 60, 90 and 120 minutes after vaginal insulin administration was observed, returning to basal levels by 240 minutes. Thus, rapid insulin absorption was not curtailed by vaginal mucose. The effect was independent of the rat oestrous cycle phase.

The zoapatle XIV--the effect of oral administration of zoapatle upon progesterone plasma levels in pregnant rabbits.

Oral administration of zoapatle aqueous crude extract (ZACE) made from Montanoa tomentosa or frutescens at doses of 5 mg/day from days 3 through 17 of pregnancy in rabbit, did not modify the normal plasma levels of progesterone. Pregnancy and delivery were similar in controls and zoapatle-treated animals.

The effect of acetylsalicylic acid on menstrual blood loss in women with IUDs.

Fifty-three volunteer women using Copper T 220C IUDs, complaining of increased menstrual bleeding, received per os 1 g, three times a day, of acetylsalicylic acid, for 5 days, during their menstrual periods. Menstrual bleeding for each patient was measured at least once before treatment. Bleeding estimates were also performed from the second to the fifth treatment cycle. From the 53 women admitted to the study, only 13 subjects (24.7%) had pre-treatment menstrual bleeding of more than 80 ml; 40 subjects had less than 80 ml. The group with hypermenorrhea had slightly decreased (not significant) the amount of menstrual blood loss with acetylsalicylic acid intake. On the other hand, 67.1% of women with bleeding less than 80 ml observed a significant increase in menstrual blood loss.

Estimates of post-partum bleeding.

Post-partum bleeding was estimated during the first 72 hours in 106 women with normal delivery at term, all had live child with a body weight between 2.5 and 3.5 Kg. Three groups of women were studied: Group 1 comprised 41 volunteers with normal deliveries to whom no medication had been administered; Group 2 comprised 39 volunteers receiving 20 IU of oxytocin in 250 ml of a 5% glucose solution, immediately after delivery; and in Group 3, 26 volunteers received 0.2 mg of ergonovine maleate, orally three times daily for three days. The average post-partum blood loss in Group 1 during the first 72 hours after delivery was 151.5 +/- 12.5 ml; for Group 2, 155.9 +/- 13.9 ml; and for Group 3, 135.5 +/- 15.9 ml. There were no significant differences among the groups and, most of the blood loss occurred during the first 24 hours after delivery.

The effect of post-partum IUD insertion on post-partum bleeding.

Post-partum bleeding was measured for 72 hours in 66 volunteer healthy women after normal pregnancy and delivery at term. After complete placenta expulsion, a 220C Copper T was placed by the attending physician in 33 volunteer subjects before leaving the delivery room. Similar number of volunteer subjects served as a control. Post-partum bleeding during the 72 hours of study was 134.7 +/- 16.3 ml for the IUD group and 155.9 +/- 16.0 ml for the control group. No differences were detected among the two groups. Hemoglobin levels were the same for both groups, being at 72 hours after delivery, 11.9 +/- 0.28 gm/ml for the IUD group and 12.2 +/- 0.36 for controls. None of the women with immediate post-partum IUD insertion had any changes in the normal characteristics of their post-partum period.

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. 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. VI. Revisited.

A review of new Zoapatle publications was made, including five chemical compounds characterized recently. Relative potency of Kaurenoic acid, Kauradienoic acid (its mixture), Zoapatanol and Montanol was estimated in an in vitro guinea pig uterine assay. Kaurene compounds were several times more potent than Montanol and Zoapatanol as uteroactive substances. Zoapatle aqueous crude extract made from Montanoa frutescens had a strong in vitro uterine potency and unique in vivo characteristics in several biological systems, described in detail in the following five publications.

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. VIII. Ultrastructural changes in endometrium of rats.

Intrauterine administration of Zoapatle aqueous crude extract (ZACE) prepared from Montanoa tomentosa s.s.p. tomentosa, on day 4 of pregnancy, did not alter the normal uterine morphological changes by days 5 and 8 of pregnancy in the rat. On the other hand, ZACE made from Montanoa frutescens caused profound alterations on the uterine structures, such as: focal loss of epithelial lining, thickened blood vessels and alterations in stroma cells in rat endometrium. Morphological alterations correlate well with the antiimplantation effect associated with the intrauterine administration of these plant extracts. Scanning electron micrographs of leaves from Montanoa tomentosa and Montanoa frutescens revealed striking differences in glandular secretion and trichomes.

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 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.