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.

Biodiversity as a source of anticancer drugs.

Natural Products have been the most significant source of drugs and drug leads in history. Their dominant role in cancer chemotherapeutics is clear with about 74% of anticancer compounds being either natural products, or natural product-derived. The biodiversity of the world provides a resource of unlimited structural diversity for bioprospecting by international drug discovery programs such as the ICBGs and NCDDGs, the latter focusing exclusively on anticancer compounds. However, many sources of natural products remain largely untapped. Technology is gradually overcoming the traditional difficulties encountered in natural products research by improving access to biodiverse resources, and ensuring the compatibility of samples with high throughput procedures. However, the acquisition of predictive biodiversity remains challenging. Plant and organism species may be selected on the basis of potentially useful phytochemical composition by consulting ethnopharmacological, chemosystematic, and ecological information. On the conservation/political front, the Convention on Biological Diversity (CBD) is allaying the anxiety surrounding the notion of biopiracy, which has defeated many attempts to discover and develop new natural products for human benefit. As it becomes increasingly evident and important, the CBD fosters cooperation and adaptation to new regulations and collaborative research agreements with source countries. Even as the past inadequacies of combinatorial chemistry are being analyzed, the intrinsic value of natural products as a source of drug leads is being increasingly appreciated. Their rich structural and stereochemical characteristics make them valuable as templates for exploring novel molecular diversity with the aim of synthesizing lead generation libraries with greater biological relevance. This will ensure an ample supply of starting materials for screening against the multitude of potentially "druggable" targets uncovered by genomics technologies. Far from being mutually exclusive, biodiversity and genomics should be the driving force of drug discovery in the 21st century.

Medicinal plants: an important asset to health care in a region of Central Laos.

A field survey of commonly used medicinal plants in the district of Paksan, Bolikhamsai Province in the Lao People's Democratic Republic (Lao P.D.R.) indicates that 55 species of plants, belonging to 49 genera in 31 families of vascular plants, are used in day-to-day medical therapy. Lao names along with uses and preparations for remedies are given. Seven species have medicinal uses that overlap with uses reported in the literature. No medicinal uses have been previously reported for 31 of the species collected based on ethnobotanical field interviews, signifying that their uses may be unique to Laos. Nine of the 31 previously unreported species are mentioned as medicinals multiple times, indicating that further research may be warranted.

Ethnobotany/ethnopharmacology and mass bioprospecting: issues on intellectual property and benefit-sharing.

Ethnobotany/ethnopharmacology has contributed to the discovery of many important plant-derived drugs. Field explorations to seek and document indigenous/traditional medical knowledge (IMK/TMK), and/or the biodiversity with which the IMK/TMK is attached, and its conversion into a commercialized product is known as bioprospecting or biodiversity prospecting. When performed in a large-scale operation, the effort is referred to as mass bioprospecting. Experiences from the mass bioprospecting efforts undertaken by the United States National Cancer Institute, the National Cooperative Drug Discovery Groups (NCDDG) and the International Cooperative Biodiversity Groups (ICBG) programs demonstrate that mass bioprospecting is a complex process, involving expertise from diverse areas of human endeavors, but central to it is the Memorandum of Agreement (MOA) that recognizes issues on genetic access, prior informed consent, intellectual property and the sharing of benefits that may arise as a result of the effort. Future mass bioprospecting endeavors must take heed of the lessons learned from past and present experiences in the planning for a successful mass bioprospecting venture.

An intensely sweet dihydroflavonol derivative based on a natural product lead compound.

The dihydroflavonol dihydroquercetin 3-acetate (1) was isolated as a sweet constituent of the young shoots of Tessaria dodoneifolia (Hook. & Arn.) Cabrera (Compositae). Compound 1 and dihydroquercetin 3-acetate 4'-(methyl ether) (2), a novel synthetic analogue of this natural product lead compound, were rated by a taste panel as being 80 and 400 times sweeter than a 2% w/v sucrose solution, respectively. Synthetic dihydroquercetin 4'-(methyl ether) (3) showed a reduced sweetness intensity when compared to 2, while (+)-dihydroquercetin (4) was devoid of sweetness. Dihydroflavonol derivatives 1-3 represent a new class of potentially noncaloric and noncariogenic intense sweeteners.

Cytotoxic clerodane diterpenes from Polyalthia barnesii.

Three cytotoxic clerodane diterpenes were purified from an ethyl acetate-soluble extract of the stem bark of Polyalthia barnesii, namely, 16 alpha-hydroxycleroda-3,13(14)Z-dien-15,16-olide, a known compound, and two novel compounds, 3 beta, 16 alpha-dihydroxycleroda-4(18),13(14)Z-dien-15,16-olide and 4 beta, 16 alpha-dihydroxyclerod-13(14)Z-en-15,16-olide. These compounds were found to exhibit broad cytotoxicity against a panel of human cancer cell lines.

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.

Quinoline alkaloids from Acronychia laurifolia.

Bioassay-directed fractionation of a root extract of Acronychia laurifolia (Rutaceae) using the KB-V1+ human tumor cell line led to the isolation of six quinoline alkaloids. One of these alkaloids is novel, namely, 2,3-methylenedioxy-4,7-dimethoxyquinoline and the other five were identified as the known compounds, evolitrine, gamma-fagarine, skimmianine, kokusaginine and maculosidine. Two known bis-tetrahydrofuran lignans, sesamolin and yangambin, were also identified. The structure of the new alkaloid was determined by spectroscopic methods. All of the isolates were evaluated against a panel of human cancer cell lines; four of the alkaloids showed weak cytotoxic activity.

A pregnane steroid from Aglaia lawii and structure confirmation of cabraleadiol monoacetate by X-ray crystallography.

The pregnane steroid, (E)-aglawone, along with four known triterpenes, and a known sterol mixture were isolated from the bark of Aglaia lawii (Wight) Saldanha ex Ramamoorty (Meliaceae). The structural determination/identification was accomplished by a combination of 1D- and 2D-NMR spectroscopic techniques. The relative stereochemistry of the known triterpene, 20S,24S-epoxydammarane-3alpha,25-diol acetate, was also unequivocally determined for the first time by X-ray crystallography. The isolates were not active against various human cancer cell lines.

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.

Antifertility effects of gossypol and its impurities on male hamsters.

Recent papers reporting on the male antifertility effects of gossypol have been criticized due to the possible influence of impurities on efficacy and toxicity. Our laboratory has prepared purified gossypol and a fraction containing the total impurities. The effects of these two substances and impure gossypol on the fertility of male hamsters was evaluated. Only pure gossypol produced a decrease in fertility after six weeks of dosing. No effects on male fertility were observed in the other treatment groups. A full recovery of fertility in the pure gossypol group was observed four weeks after cessation of treatment. These data support the claim that gossypol is indeed an active antifertility agent for males. It is also clear that the impurities may have the ability to block or decrease the efficacy of gossypol.

PIP: Recent papers reporting on the male infertility effects of gossypol have been criticized due to the possible influence of impurities on efficacy and toxicity. Our laboratory has prepared purified gossypol and a fraction containing the total impurities. The effects of these 2 substances and impure gossypol on the fertility of male hamsters was evaluated. Only pure gossypol produced a decrease of fertility after 6 weeks of dosing. No effects on male fertility were observed in the other treatment groups. A full recovery of fertility in the pure gossypol group was observed 4 weeks after cessation of treatment. These data support the claim that gossypol is indeed an active antifertility agent for males. It is also clear that the impurities may have the ability to block or decrease the efficacy of gossypol.

Biodiversity prospecting and benefit-sharing: perspectives from the field.

Searching for new biologically active compounds from natural sources starts, obviously, in the field. Plant, microbial or animal materials to be sought and investigated may be selected through a number of approaches. No matter what selection criterion(a) is (are) used, the first step in obtaining the organism concerned is to undertake field collecting work to search for and to collect the organism. Good knowledge on the ecogeographic distribution and precision in the taxonomic identification of the organism(s) sought are crucial if the field work involves the search for a pre-determined organism or set of organisms. Such knowledge and precision during field work are of secondary importance, however, if the search and collection are based on biodiversity or ethnomedical uses, since accurate taxonomic identification may be made at a later date, in a Museum or Herbarium environment. When an individual or institution from a biotechnologically developed country wishes to obtain indigenous raw biological material from a biotechnologically less developed country, an agreement for the procurement of such raw material may be negotiated. Since the effort to search and develop a biologically active compound(s) from natural sources is a long-term process that involves teamwork between field and laboratory scientists, the success of the endeavor will depend in large part on the continued flow of raw material from the field. Goodwill to maintain such a flow may be achieved through appropriate scientific and monetary compensations, both in real-time and in long-term sharing of the benefits of discovery. Only with the prospect of financial return to the supplying country will there be an incentive for the protection of the natural resources towards sustainable use and development, as well as to allow time for continuing explorations and discoveries.

Medicinal plants of Seberida (Riau Province, Sumatra, Indonesia).

Field enquiries on the plants used to treat diseases in villages of Seberida Municipality indicated that a large number of plant species (at least 100) are being used in therapy. Many of the uses, however, are magical in nature. Those in which a cause-effect relationship may be established (56) are presented in this paper. A review of the ethnomedical and experimental literature showed that medicinal plant uses in Seberida fall into three categories: those for which uses are corroborated by similar medicinal uses for the same plant or different species of the same genus in other cultures, those for which uses of the plant or species of the same genus are corroborated by evidence of relevant pharmacological activity in the experimental literature and those for which the medicinal uses are not corroborated. A discussion of these categories is presented. Taken as a whole, the medicinal uses of plants in Seberida are characterized by a remarkably high proportion of plants used to treat fevers and malaria and by a high proportion of species of which the leaves are used (externally or internally) for medicinal purposes. Comparison with other studies reported in the literature seems to indicate that a high frequency of the use of leaves in therapy may be a part of a larger cultural phenomenon among the tropical forest tribes of Southeast Asia and the southern Pacific Islands. Possible rationales for this type of use are offered.

Traditional herbal drugs of southern Uganda, I.

One-hundred four plant species used medicinally by herbalists from three southern Ugandan tribes were collected and identified. The collection includes a large portion of the materia medica of the Abayanda of the southwest region, as well as species used by herbalists of the Baganda and Bakiga Tribes. Literature searches were performed in preparation for further collections, and for collaborative laboratory validation of in vitro antimicrobial activity. Literature data provide support for ethnomedical claims for a number of species used in Uganda for disease treatment.

Plant-derived sweetening agents: saccharide and polyol constituents of some sweet-tasting plants.

Samples of the sweet-tasting species Acanthospermum hispidum DC. (Compositae) (aerial parts), Boscia salicifolia Oliv. (Capparidaceae) (stem bark), Hovenia dulcis Thunb. (Rhamnaceae) (peduncles) and Inga spectabilis Willd. (Leguminosae) (arils) were acquired as part of a continuing search for high-intensity natural sweeteners of plant origin. Following their preliminary safety evaluation, the sweetness of these plants was traced to large amounts of sugars and polyols by taste-guided fractionation, which were identified and quantified using gas chromatography/mass spectrometry. The combined yields of sugars and polyols in the A. hispidum, B. salicifolia, H. dulcis, and I. spectabilis samples investigated were 6.9, 10.1, 18.4 and 12.1% w/w, respectively. These yields are much higher than the total saccharide and polyol content (2.4% w/w) of the sweet dried fruits of Thladiantha grosvenorii (Swingle) C. Jeffrey (Cucurbitaceae), a species which has previously been reported to contain more than 1% w/w of the intensely sweet triterpene, mogroside V. The dried leaves of Symplocos tinctoria (L.) L'Hérit. (Symplocaceae), which were not appreciably sweet, were found to contain only 2.0% w/w of sugars. The results of this investigation, therefore, suggest that unless the saccharide and/or polyol content of a plant part is well over 5% w/w, then it is unlikely to exhibit an overtly sweet taste, unless an intense sweetener is present.

Traditional herbal drugs of southern Uganda. Part III: isolation and methods for physical characterization of bioactive alkanols from Rubus apetalus.

The East African plant Rubus apetalus Poir. was collected as a component of an ethnobotanical survey in southern Uganda. No phytochemical investigations of this plant have been found in the literature. Preliminary antimicrobial susceptibility tests performed in Uganda indicated biological activity against several bacterial and one fungal human pathogen. Bulk re-collection of Rubus apetalus was accomplished and crude extraction performed in preparation for further testing. Two chemical fractions of the crude extract were active in the antimicrobial susceptibility assay. Fractionation of one of the active crude fractions led to the isolation and elucidation of a mixture of related compounds that exhibit antimicrobial activity against Staphylococcus aureus (MIC=62 microg/ml), Streptococcus faecalis (16 microg/ml) and Candida albicans (32 microg/ml).

Traditional herbal drugs of Southern Uganda, II: literature analysis and antimicrobial assays.

Continuing field interviews brought the total species used for disease treatment by herbalists of the majority Baganda Tribe of southern Uganda to 168. Literature searches provided support for the ethnomedical claims for a number of these species, and provided criteria for the species classification into four categories of use validation. They also helped guide the selection of species for recollection, for chemical extraction and further testing in laboratories of the Uganda Ministry of Health and the University of Illinois at Chicago. Many species proved active against microorganisms in several susceptibility assays conducted in Uganda and the US.

Sources of the anti-implantation alkaloid yuehchukene in the genus Murraya.

The genus Murraya has been widely used in traditional medicine in east Asia. In view of the recent isolation of the anti-implantation alkaloid yuehchukene from M. paniculata a search has now been made for other natural sources of this alkaloid within the genus. In this paper we report findings for nine taxa of Murraya.