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.

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.

Synthesis and inhibition of human acrosin and trypsin and acute toxicity of aryl 4-guanidinobenzoates.

The aryl 4-guanidinobenzoate, 4'-nitrophenyl 4-guanidinobenzoate (NPGB), is a potent inhibitor of sperm acrosin, an enzyme with an essential function in the fertilization process. NPGB prevents fertilization in a number of animal species and is a good lead compound for the development of contraceptive agents. In order to assess the efficacy of other aryl 4-guanidinobenzoates as acrosin inhibitors, 24 of these compounds were synthesized. Their inhibitory activity toward human acrosin was determined and compared with their activity toward human pancreatic trypsin in order to assess whether inhibitor sensitivity differed between these similar enzymes. Nine of the inhibitors were synthesized from phenols approved by the FDA for therapeutic use. The acute toxicity of these inhibitors in mice was determined and compared to that of nonoxynol-9, the most commonly used active ingredient in today's vaginal contraceptive preparations. All of the compounds proved to be potent inhibitors of human acrosin although 3 orders of magnitude difference were observed between the most and least effective inhibitors. Little specificity was present in regard to their inhibition of acrosin and trypsin. All the aryl 4-guanidinobenzoates synthesized from FDA-approved phenols were less toxic than nonoxynol-9, and it is concluded that these 4-guanidinobenzoates are of interest for further development and testing as nonhormonal contraceptive agents.

Specific inhibition of the testicular mitochondrial respiratory chain in vitro by gossypol.

Optically inactive gossypol is an effective male antifertility agent in several mammalian species, while optically active (+)-gossypol has no antifertility effect in the rat and hamster. Recently, it was suggested that the mitochondria of spermatogenic cells may be a subcellular target of gossypol. We are reporting the effects of optically inactive gossypol and (+)-gossypol on the respiratory chain of mitochondria isolated from the testes and liver of rats and hamsters. The mitochondria were incubated with the test compounds and difference spectra were recorded. Complete inhibition of the testicular mitochondrial respiratory chain was observed at a concentration of approximately 75 microM. In contrast, no inhibition of the liver mitochondrial respiratory chain was observed with the test compounds at concentrations as high as 300 microM. These results demonstrate selective inhibition of the testicular mitochondrial respiratory chain by gossypol isomers.

Inhibition of LDH-X by gossypol optical isomers.

Racemic gossypol is an effective male antifertility agent in several mammalian species. However, (+)-gossypol is not an effective male antifertility agent in the rat or the hamster. Previous studies have demonstrated the ability of racemic gossypol to inhibit the testis-specific LDH-X enzyme derived from various mammalian species and have suggested LDH-X as the potential site of gossypol antifertility action. In the present study, the effects of racemic gossypol and the (+) and (-) optical isomers of gossypol on LDH-X derived from rat and hamster testicular cytosol are compared to determine if there is any correlation between the in vitro inhibition of the LDH-X enzyme and in vivo antifertility effects. Both optical isomers of gossypol as well as racemic gossypol inhibit rat and hamster testicular cytosolic LDH-X activity. Inhibition of hamster testicular cytosolic LDH-X activity by (-)-gossypol was less than by either racemic gossypol or (+)-gossypol. Based on the previous reports of racemic gossypol inhibition of LDH-X, therefore, it cannot be simply concluded that LDH-X is the specific site of antifertility action of gossypol since, in the present study, (+)-gossypol, which is not an effective male antifertility agent, also inhibited rat and hamster testicular cytosolic LDH-X.

Properties of a new, long-lasting vaginal delivery system (LASRS) for contraceptive and antimicrobial agents.

In view of the need for improved vaginal formulations that are contraceptive, that may prevent transmission of sexually transmitted infections, or both, a new delivery system (base formulation; called Long Acting, Sustained Release of Spermicide, or LASRS) was developed that contains bioadhesive and other ingredients with a long history of safety, and was designed to provide long-lasting vaginal retention of the formulation and to minimize possible vaginal irritation caused by incorporated active ingredients. Nonoxynol-9 (N-9) was added as an active ingredient to study the vaginal irritating properties of the formulation and to assess its long-term effectiveness by postcoital spermicidal tests. In the first series of experiments, in vitro studies showed that the formulation spreads rapidly over a cellulose membrane, forming a bioadhesive layer that remained for at least 12 hours. The second series of experiments addressed the safety of the LASRS suppository in rabbits and primates. Even with a very high concentration of N-9 (20.5%), LASRS caused only mild/moderate but acceptable irritation in the rabbit. No vaginal irritation occurred in the primate at an even higher concentration (22.5%). During the third series of experiments, the long-lasting vaginal retention properties were evaluated by postcoital spermicidal tests in the primate. LASRS with N-9 was highly spermicidal even when mating was delayed for 12 hours after placement of the formulation. Spermicidal activity was also observed when 1) mating was delayed for 24 hours after insertion of the formulation, and 2) if the females were mated 2 or even 3 times without reinsertion of the suppository before collection of the vaginal contents. In the final series of tests, the postcoital spermicidal properties of menfegol, another cytotoxic spermicide, were evaluated as were several modifications in the base formulation. Menfegol produced essentially the same results as N-9. Altering the base formulation proved to be nonbeneficial because a decrease in the long-term spermicidal effectiveness was obtained. These results suggest that the LASRS suppository has good vehicle properties for the delivery of active ingredients to the vagina.

Characterization of cysteamine as a potential contraceptive anti-HIV agent.

Cysteamine (beta-mercaptoethylamine, or MEA) is a thiol-reducing agent and has anti-HIV activity. Because of these properties, cysteamine was evaluated as a vaginal contraceptive and tested for its effects on sperm function and on other sexually transmitted microbes. Cysteamine was contraceptive in the rabbit. Conception was inhibited completely when sperm were pretreated with 500 microg/ml cysteamine and was inhibited by more than 60% when 7.5 mg cysteamine was applied vaginally as a suspension in 50% K-Y Jelly. Cysteamine had multiple effects on spermatozoa. Both acrosin (EC 3.4.21.10) and hyaluronidase (EC 3.2.1.35) were reversibly inhibited by cysteamine. Calculated IC50 values were 370 microg/ml and 150 microg/ml for acrosin and hyaluronidase, respectively. Cysteamine behaved as a poor spermicide when activity was measured by the 30-second Sander-Cramer test. However, sperm motility was inhibited completely when cysteamine was preincubated for 10 minutes prior to motility evaluation, at concentrations as low as 50 microg/ml. The calcium ionophore A23187-induced human acrosome reaction was inhibited by cysteamine (IC50 = 0.5 microg/ml). Neither herpes simplex virus nor Neisseria gonorrhoeae was affected by cysteamine at concentrations as high as 500 microg/ml and 100 microg/ml, respectively. Cysteamine appears to have no effect on normal vaginal flora (i.e., lactobacillus). These results, together with published data, strongly support the further development of cysteamine as a topical contraceptive anti-HIV agent.

Evaluation of poly(styrene-4-sulfonate) as a preventive agent for conception and sexually transmitted diseases.

A commercial preparation of a sodium polystyrene sulfonate (designated as N-PSS; its molecular weight is 500000 daltons) was tested as an inhibitor of sperm function and as a preventive agent for conception and the transmission of sexually transmitted diseases. The polymer is an irreversible inhibitor of hyaluronidase and acrosin; its IC50 values are 5.7 microg/mL and 0.5 microg/mL, for hyaluronidase and acrosin, respectively. N-PSS is also a stimulus of human sperm acrosomal loss. It produces maximal acrosomal loss at 2.5 microg/mL. Contraception in rabbits is nearly complete when rabbit spermatozoa are pretreated with 0.5 mg/mL of N-PSS before artificial insemination; however, N-PSS does not immobilize spermatozoa at concentrations as high as 50 mg/mL. N-PSS has broad spectrum antiviral and antibacterial activities. Infection by human immunodeficiency virus and herpes simplex virus are inhibited by N-PSS; 3-log reductions are produced by 7 microg/mL and 3 microg/mL, respectively. N-PSS is active against Chlamydia trachomatis and Neisseria gonorrhoeae. At 1 mg/mL, N-PSS inhibits chlamydial infectivity by more than 90%. N-PSS produces a 3-log reduction in gonococcal growth at 15 microg/mL. In contrast, N-PSS (5 mg/mL) does not affect the growth of Lactobacillus (normal component of the vaginal flora). N-PSS can be classified as a noncytotoxic contraceptive antimicrobial agent. These properties justify bringing a polystyrene sulfonate into clinical trials for its evaluation as a preventive agent for conception and several sexually transmitted diseases.

Effect of (+)-gossypol on fertility in male hamsters.

(+)-Gossypol was isolated from the bark of Thespesia populnea and tested for its ability to inhibit the fertility of male hamsters. Male hamsters of proven fertility were treated orally for 54 days with 40 mg/kg of (+)-gossypol, 40 mg/kg of racemic gossypol, or 5% gum acacia (vehicle control) and were mated with estrous female hamsters during the fourth and seventh weeks of treatment. Both the control and the (+)-gossypol-treated animals exhibited normal fertility throughout the experiment. The racemic gossypol-treated animals were infertile when evaluated during both the fourth and seventh weeks of treatment. Morphologic examination of the testicular tissue could not explain the loss of fertility. These data demonstrate the inability of (+)gossypol to decrease male fertility and suggest that the activity of racemic gossypol may be due primarily to the presence of the (-) optical isomer.

Theobromine toxicity on Sertoli cells and comparison with cocoa extract in male rats.

The target cell(s) of theobromine toxicity on rat testes and reproductive toxicity induced by pure theobromine and cocoa extract are evaluated in the present studies. Theobromine (500 mg/kg x 7 days) inhibited body weight gain in treated rats. Decreased cauda epididymal sperm reserve (38%), seminiferous tubule fluid (STF) volume (33%), lactate concentration in STF (22%), inhibition of binding activity of androgen binding protein (ABP, 21%) and reduced ABP content in STF were also observed in theobromine-treated animals. Cocoa extract containing an equivalent amount of theobromine did not produce significant toxicity in treated rats. Theobromine concentrations in serum and testes from pure theobromine-treated rats were 1.8- and 1.6-fold higher, respectively, than that in rats treated with cocoa extract. The results support Sertoli cells as the primary target cells of theobromine toxicity. The lower theobromine concentrations in serum and testes of cocoa extract-treated rats could account for the lower toxicity in these animals.

Analysis of gossypol and gossypol-acetic acid by high-performance liquid chromatography.

Gossypol, a bis-sesquiterpenoid cotton pigment, is of current interest as a male fertility-regulating agent. For the purposes of analyzing material to be studied biologically, a method is described for the analysis of gossypol by high-performance liquid chromatography. This has been used for examining the purity of gossypol-acetic acid using a UV-absorbance ratio technique.

PIP: Gossypol, a bis-sesquiterpenoid cotton pigment, is of current interest as male fertility-regulating agent. To analyze material fo biological study, a method is described for the analysis of gossypol by high performance chromatography. This has been used for examining the purity of gossypol-acetic acid using a ultraviolet-absorbance ratio technique.

Identification of senecionine and senecionine N-oxide as antifertility constituents in Senecio vulgaris.

The MeOH extract of Senecio vulgaris L., administered po to rats on Days 1-10 postcoitum, significantly decreased the number of normal fetuses per pregnant rat found at autopsy on Day 16. Additional experiments showed a similar activity for its hepatotoxic constituents senecionine and senecionine N-oxide, suggesting that the latter two compounds were probably responsible for the effect seen with the extract. No antifertility effects were seen in MeOH extract-treated hamsters.

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.

The lack of mutagenicity of aryl 4-guanidinobenzoates in the transplacental micronucleus assay in mice.

Two acrosin inhibitors, 4'-methylumbelliferyl 4-guanidinobenzoate and 2'-carbomethoxyphenyl 4-guanidinobenzoate, were tested for mutagenicity in the transplacental micronucleus assay and the mouse bone marrow micronucleus assay. The compounds were administered intraperitoneally at doses of 125 mg/kg and 250 mg/kg to pregnant mice. Fetal peripheral blood and maternal bone marrow cells were examined at 36 h for the frequency of micronucleated polychromatic erythrocytes. Neither compound induced micronuclei in maternal or fetal tissues. The ratio of polychromatic erythrocytes to normochromatic erythrocytes was not affected by the drug treatments indicating that the compounds had no effect on the cell cycle or mitosis in these tissues and that they were not cytotoxic. Both compounds, which show promise as vaginal contraceptives, were not mutagenic in this study.

The effect of ketoconazole on fertility of male rats.

The effect of ketoconazole on the fertility of male rats was evaluated. Three days of oral dosing with ketoconazole at 200 mg/kg reduced fertility compared to controls. A complete loss of fertility was observed after doses of 400 mg/kg. There was no change in the testicular weight, epididymal sperm concentration or epididymal weight between the control and treatment groups. Motility was reduced in the high-dose group and forward progression was reduced in both dosing groups compared to control. These data support previous observations in the dog and primate that orally administered ketoconazole alters sperm viability. Although ketoconazole is too toxic for contraceptive application, its derivatives may be useful for this purpose.

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.

In vitjro spermicidal activity of gossypol.

The in vitro spermicidal effect of gossypol, gossypol acetic acid and gossypol-polyvinylpyrrol idone was investigated. Gossypol-polyvinylmulations. Thus, gossypol in an appropriate chemical form may be useful as a spermicide.

Comparison of the spermicidal activity and acute toxicity of nonoxynol-9 and agent 741 [alkylphenoxy polyethoxy ethanol(10)].

A spermicidal agent called "741" is used in the People's Republic of China as a substitute for nonoxynol-9 in vaginal contraceptive preparations. The relative spermicidal activity of these two agents was evaluated using dog, rabbit, monkey and human semen. Acute LD50 determinations in mice were also performed. The spermicidal activity and acute toxicity of both agents were either identical or very similar in all species. Since agent 741 is less expensive than nonoxynol-9 to manufacture and equally as potent in vitro, it is an alternative spermicide for vaginal contraceptive formulations.

Inhibition of lactate dehydrogenase-X by imino-derivatives of gossypol:structure activity relationship.

Six imino-derivatives (II, III, IV, V, VI, VII) of gossypol (I) have been synthesized, and their effects were evaluated on the purified mouse lactate dehydrogenase-X. Three of these derivatives (V, VI, VII) with aldehyde groups substituted with hydrophobic functionalities showed equivalent or more inhibitory effects on lactate dehydrogenase-X than gossypol, whereas three other derivatives (II, III, IV) with aldehyde groups substituted with hydrophilic functional groups lost the ability to inhibit lactate dehydrogenase-X. It is suggested that two aldehyde groups of gossypol are not essential to inhibit lactate dehydrogenase-X. Furthermore, the hydrophobic property of the gossypol molecule seems to play a more important role in inhibiting lactate dehydrogenase-X. Therefore, lactate dehydrogenase-X inhibition by gossypol may not be associated with its antifertility mechanism, because the aldehyde group of gossypol is known to be required for its antifertility effect.

Vaginal contraceptive activity of aryl 4-guanidinobenzoates (acrosin inhibitors) in rabbits.

Nine aryl 4-guanidinobenzoates were synthesized as inhibitors of the sperm enzyme acrosin. These esters were prepared from 4-guanidinobenzoic acid and a number of phenols which had been approved by the FDA for clinical use. The vaginal contraceptive activity of the inhibitors was evaluated in the rabbit at nonspermicidal concentrations (0.1 mg/ml). All the inhibitors except the 2'-carboxamidophenyl and the 2'-isopropyl-5'-methylphenyl 4-guanidinobenzoates caused significant reductions in fertilization compared to the controls. Several of the aryl 4-guanidinobenzoates appeared to be particularly effective. Nonoxynol-9, under the same conditions but at 10- and 100-fold higher concentrations, also showed an antifertility effect. However, even at these increased dose levels, the contraceptive efficacy of nonoxynol-9 was no higher than that of most of the inhibitors and was less consistent than that of the most active aryl 4-guanidinobenzoates. The relatively high in vivo antifertility activity exhibited by several of the aryl 4-guanidinobenzoates encourages their further evaluation as vaginal contraceptive agents.