Ethnobotanical approach versus random approach in the search for new bioactive compounds: support of a hypothesis.

CONTEXT: Whether natural product drug discovery programs should rely on wild plants collected "randomly" from the natural environment, or whether they should also include plants collected on the basis of use in traditional medicine remains an open question. OBJECTIVE: This study analyzes whether plants with ethnomedical uses from Vietnam and Laos have a higher hit rate in bioassay testing than plants collected from a national park in Vietnam with the goal of maximizing taxonomic diversity ("random" collection). MATERIALS AND METHODS: All plants were extracted and subjected to bioassay in the same laboratories. Results of assays of plant collections and plant parts (samples) were scored as active or inactive based on whether any extracts had a positive result in a bioassay. Contingency tables were analyzed using χ(2) statistics. RESULTS: Random collections had a higher hit rate than ethnomedical collections, but for samples, ethnomedical plants were more likely to be active. Ethnomedical collections and samples had higher hit rates for tuberculosis, while samples, but not collections, had a higher hit rate for malaria. Little evidence was found to support an advantage for ethnomedical plants in HIV, chemoprevention and cancer bioassays. Plants whose ethnomedical uses directly correlated to a bioassay did not have a significantly higher hit rate than random plants. DISCUSSION: Plants with ethnomedical uses generally had a higher rate of activity in some drug discovery bioassays, but the assays did not directly confirm specific uses. CONCLUSIONS: Ethnomedical uses may contribute to a higher rate of activity in drug discovery screening.

An ethnobotanical survey of medicinal plants of Laos toward the discovery of bioactive compounds as potential candidates for pharmaceutical development.

CONTEXT: An ethnobotany-based approach in the selection of raw plant materials to study was implemented. OBJECTIVE: To acquire raw plant materials using ethnobotanical field interviews as starting point to discover new bioactive compounds from medicinal plants of the Lao People's Democratic Republic. METHODS: Using semi-structured field interviews with healers in the Lao PDR, plant samples were collected, extracted, and bio-assayed to detect bioactivity against cancer, HIV/AIDS, TB, malaria. Plant species demonstrating activity were recollected and the extracts subjected to a bioassay-guided isolation protocol to isolate and identify the active compounds. RESULTS: Field interviews with 118 healers in 15 of 17 provinces of Lao PDR yielded 753 collections (573 species) with 955 plant samples. Of these 955, 50 extracts demonstrated activity in the anticancer, 10 in the anti-HIV, 30 in the anti-TB, and 52 in the antimalarial assay. Recollection of actives followed by bioassay-guided isolation processes yielded a series of new and known in vitro-active anticancer and antimalarial compounds from 5 species. DISCUSSION: Laos has a rich biodiversity, harboring an estimated 8000-11,000 species of plants. In a country highly dependent on traditional medicine for its primary health care, this rich plant diversity serves as a major source of their medication. CONCLUSIONS: Ethnobotanical survey has demonstrated the richness of plant-based traditional medicine of Lao PDR, taxonomically and therapeutically. Biological assays of extracts of half of the 955 samples followed by in-depth studies of a number of actives have yielded a series of new bioactive compounds against the diseases of cancer and malaria.

Methods in ethnopharmacology.

A variety of pharmacological models are utilized in the evaluation of ethnomedicine. Most investigations are focused on developing new leads for therapeutic agents. However, there should be more efforts focused on the development of ethnomedicines because of their accessibility and acceptability in areas where modern medicine is not readily available. Testing methods to identify the active agents must be carefully selected utilizing information from ethnoanthropology, ethnobotany, phytochemistry, toxicology and pharmacology. New pharmacological models focused on cellular and molecular mechanisms can be used for ethnomedical evaluations but with great caution since they are based on known mechanisms of actions and limited by knowledge of the disease state.

Reproductive toxicity of theobromine and cocoa extract in male rats.

The toxicities of theobromine and cocoa extract on the reproductive tract of male rats were compared in the present study. A cocoa powder extract containing 117 mg theobromine/g extract was prepared using 85% boiling methanol. Sprague-Dawley rats were weighed and dosed daily for 31 days with vehicle, 250 mg/kg theobromine, 2.14 g/kg cocoa extract (117 mg theobromine/g extract), or 0.43 g/kg cocoa extract by oral gavage. The animals were sacrificed on day 32. One testis and epididymis were removed and weighed. The epididymis was saved for the determination of epididymal sperm reserves. The remaining testis was fixed by whole body glutaraldehyde perfusion and processed for morphologic examination. A decrease in body weight gain and epididymal weights were observed in theobromine and high-dose cocoa-extract-treated groups. Theobromine and high-dose cocoa extract caused vacuolation within the Sertoli cell, abnormally shaped spermatids, and failed release of late spermatids in treated animals. Most of the vacuolations were found in the earlier and middle stage seminiferous tubules (stages I to VIII). However, the frequency of some parameters of testis alterations were significantly lower in the high-dose cocoa-extract-treated group compared to the theobromine-treated group. These data demonstrate the ability of a cocoa extract containing theobromine to alter testis structure in a similar pattern but with reduced intensity compared to that observed after oral exposure to pure theobromine.

Furanoid lignans from Larrea tridentata.

From the leaves and stems of Larrea tridentata six new furanoid lignans, compounds 1-6, have been isolated and their structures determined through interpretation of physical and spectroscopic properties. The use of 1D and 2D nOe experiments was of particular importance in assigning the stereochemistry.

Studies on zoapatle. V. Correlation between in vitro uterine and in vivo pregnancy interruption effects in guinea pigs.

The zoapatle plant, Montanoa tomentosa, has an extensive ethnomedical history of use in fertility regulation. Several fractions and isolates of this plant were evaulated in in vitro and in vivo tests to identify and characterize active constituents which may be responsible for its antifertility effects. The guinea pig was the animal model. Two types of in vitro activity were observed, a uterotonic type effect and an inhibition of spontaneous contractions of the uterine muscle. The in vivo effects appear to correlate with the ability of a fraction/isolate to inhibit the spontaneous contractions in in vitro testing. The non-polar fractions/isolates accounted for the inhibition of spontaneous contraction effects and the in vivo activity. The more polar fractions/isolates accounted for the uterine stimulant activity.

Studies on zoapatle, II. Leucanthanolide, a novel sesquiterpene lactone from Montanoa leucantha ssp. leucantha.

PIP: A sesquiterpene lactone given the trivial name leucanthanolide, from the Mexican zoapatle plant (Montanoa leucantha), long used as an herbal remedy, was isolated and was found to have cytotoxicity but no uterine activity. The compound was isolated from the fraction containing zoapatanol, a substance being investigated for its ability to induce menses, abortion and labor. The compound has a molecular formula of C19H2606, a 5-membered lactone ring, an ester group, and a germacradienolide skeleton. It was evaluated for abortifacient activity in pregnant guinea pigs by intraperitoneal injection on day 22 of gestation. 3 of 5 animals had abnormal fetuses, but there was no evidence of early uterine activity. Cytotoxic testing in vitro was done on KB and P-388 test systems in cell culture. The ED50 of leucanthanolide was 0.57 mcg/ml (KB) and 0.93 (P-388). In comparison, the ethyl acetate fraction had an ED50 of 1.35 mcg/ml and 5.2 mcg/ml respectively.^ieng

Studies on Aristolochia III. Isolation and biological evaluation of constituents of Aristolochia indica roots for fertility-regulating activity.

An ethanol extract of Aristolochia indica roots decreased fertility in both rats and hamsters when administered postcoitally (days 1-10 and 1-6, respectively). Petroleum ether (A), CHCl3 (B), and aqueous (C) fractions, tested similarly in rats, were inactive and/or toxic. Partition of fraction B afforded non-acidic (D) and acidic (E) fractions. Savinin (1), isolated from fraction D and not previously reported from the Aristolochiaceae , was inactive when administered postcoitally to rats. Aristolochic acid-I (2), reported previously from A. indica and isolated from fraction E, was inactive when administered postcoitally to rats and toxic when administered postcoitally to hamsters. (12S)-7,12- Secoishwaran -12-ol (3), previously reported from A. indica and isolated from fraction A, did not interrupt pregnancy when administered to mice on day 6 of pregnancy. Four additional compounds, aristolic acid (4) [prepared from aristolochic acid-I (2)], methyl aristolate (5) [prepared by methylating aristolic acid (4)], and cis- and trans-p-coumaric acid (both oblate commercially), were similarly tested in mice and found to be inactive. Aristolic acid (4), and the cis- and trans-p-coumaric acids also were inactive when administered postcoitally (days 1-10) to rats. Seven compounds reported previously from A. indica were also isolated, as were a new naphthoquinone, aristolindiquinone (6) (fraction E), and magnoflorine (fraction C).

Current status of plant products reported to inhibit sperm.

PIP: This report reviews research on plant-derived agents that prevent sperm production if taken orally by the male or that incapacitate or kill sperm on contact if used vaginally by the female. It would be of great value to develop fertility inhibitors that are totally selective for reproductive systems and enzymes, and there is a possibility that a plant-derived drug may have this effect. Plants that have been studied for their fertility inhibiting effects in the male include: Aristolochia indica L. (Aristolochiaceae); Azadirachta indica A. Juss (Meliaceae); Balanites roxburghii Planch. (Zygophyllaceae); Calotropis procera (Ait) R.Br. (Asclepiadaceae); Carica papaya L. (Caricaceae); Catharanthus roseus (L.) G. Don (Apocynaceae); Dieffenbachia seguine (Jacquin) Schott. (Araceae); Ecaballium elaterium A. Richard (Cucurbitaceae); Gossypium species (Malvaceae); Hibiscus rosa-sinensis L. (Malvaceae); Hippophae salicifolia D. Don (Elaeagnaceae); Leucaena glauca (L.) Benth. (Leguminosae); Lonicera ciliosa Poir. (Caprifoliaceae); Lupinus termis Forsk. (Leguminosae); Malvaviscus conzattii Greenm. (Malvaceae); Momordica charantia L. (Curcurbitaceae); Ocimum sanctum L. (Labiatae); Prunus emarginata Walp. (Rosaceae); and Withania somnifera (L.) Dunal (Solanaceae). A large number of plants have been randomly selected and screened for spermicidal activity "in vitro" and several seem promising. Those species found to be active and the nature of the active principle(s), when known, are presented in a table as are plant-derived chemical substances of known or partially known structure reported to be spermicidal "in vitro." Plants warrant systematic study as potential sources of sperm-agglutinating compounds. Of 1600 Indian plants tested, 90 showed positive semen coagulating properties. There seems to be a lack of correlation among experimental results obtained by different groups of investigators, between data obtained "in vitro" and "in vivo," and between experimental results and information found in folklore. Factors complicating the adequate assessment of plants affecting male fertility are inadequate numbers of vehicle-treated controls, poor experimental design, problems related to insolubility of crude plant extracts, variation in routes of administration, diversity in reproductive function and control among various laboratory species, and problems in identifying plant names consistently.^ieng