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.

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.

Catalytic strategy of citrate synthase: effects of amino acid changes in the acetyl-CoA binding site on transition-state analog inhibitor complexes.

Acetyl-CoA enol has been proposed as an intermediate in the citrate synthase (CS) reaction with Asp375 acting as a base, removing a proton from the methyl carbon of acetyl-CoA, and His274 acting as an acid, donating a proton to the carbonyl [Karpusas, M., Branchaud, B., & Remington, S.J. (1990) Biochemistry 29, 2213]. CS-oxaloacetate (OAA) complexes with the transition-state analog inhibitor, carboxymethyl-CoA (CMCoA), mimic those with acetyl-CoA enol. Asp375 and His274 interact intimately with the carboxyl of the bound inhibitor. While enzymes in which these residues have been changed to other amino acids have very low catalytic activity, we find that they retain their ability to form complexes with substrates and the transition-state analog inhibitor. In comparison with the value of the chemical shift of the protonated CMCoA carboxyl in acidic aqueous solutions or its value in the wild-type ternary complex, the values in the Asp375 mutants are unusually low. Model studies suggest that these low values result from complete absence of one hydrogen bond partner for the Gly mutant and distortions in the active site hydrogen bond systems for the Glu mutant. The high affinity of Asp375Gly-OAA for CMCoA suggests that the unfavorable proton uptake required to stabilize the CMCoA-OAA ternary complex of the wild-type enzyme [Kurz, L.C., Shah, S., Crane, B.R., Donald, L.J., Duckworth, H.W., & Drysdale, G.R. (1992) Biochemistry (preceding paper in this issue)] is not required by this mutant; the needed proton is most likely provided by His274. This supports the proposed role of His274 as a general acid.(ABSTRACT TRUNCATED AT 250 WORDS)

The rate of formation of transition-state analogues in the active site of adenosine deaminase is encounter-controlled: implications for the mechanism.

We have previously shown that purine riboside, when bound to adenosine deaminase, forms a complex in which C-6 of the purine is tetrahedral [Kurz, L. C., & Frieden, C. (1987) Biochemistry 26, 8450]. We now report the rates of formation of enzyme-inhibitor complexes of two types, those which do and those which do not form such tetrahedral intermediates. In both cases, the rates are encounter-controlled since the progress curves for formation of the complexes are well-described by a simple second-order approach to equilibrium and the rate constants show an inverse solvent viscosity dependence. Assuming that the formation of the intermediate-analogue complex is preceded by an initial ground-state analogue complex, the lifetime of that ground-state complex must be less than approximately 20 microseconds. All of the enzyme-inhibitor complexes studied share three characteristics: (1) the complexes generate large UV-difference spectra; (2) a substantial solvent isotope effect is found on the enzyme's affinity for the inhibitors; and (3) a new signal appears in the CD spectra of the complexes. Two of the nucleosides studied, 1-deazapurine riboside and 1-deaza-adenosine, form complexes which appear to mimic a ground-state rather than a reactive intermediate when bound to adenosine deaminase. We find that the values for the association rate constants for those inhibitors which form intermediate analogues are very similar to that for adenosine. The presence of a significant solvent isotope effect on the affinity of all inhibitors is attributable in part to a large transfer isotope effect on the free ligand and in part to an effect on the bound ligand. This complicates use of the solvent isotope effect in applications of the multiple isotope method for estimating intrinsic isotope effects and commitment factors.

Bovine fibroblast growth factor: comparison of brain and pituitary preparations.

Bovine brain and pituitary fibroblast growth factors (FGF) have been compared with regard to their chemical and biological properties. Pituitary and one preparation of brain FGF (Prep A) contain a basic mitogenic activity, which migrates to the same position on electrophoresis in acid pH gels as detected by incorporation of [methyl-3H]-thymidine into BALB/c 3T3 cells. In contrast, another preparation of brain FGF (Prep B) contains two mitogens, one (20-30%) indistinguishable from the basic components in pituitary and brain (Prep A) FGF preparations and an acidic activity (70-80%), pl 5-6, that migrates more slowly on acid gels, corresponding to the acidic component of brain FGF described previously (Thomas, K. A., M. C. Riley, S. K. Lemmon, N. C. Baglan, and R. A. Bradshaw. 1980. J. Biol. Chem. 255:5517-5520.) In agreement with that report, none of the mitogens comigrates with fragments of myelin basic protein. Pituitary FGF was virtually inactive, brain (Prep A) FGF had a small amount of activity, and brain (Prep B) FGF was highly potent (50% maximal stimulation at 15-30 ng/ml) in stimulating the growth of human umbilical vein endothelial (HUVE) cells. The acidic component of brain FGF, which is much more unstable at pH 8.5 than the basic one, can be protected by reducing agents, whereas the basic constituent of brain FGF as well as pituitary FGF is unaffected by reducing conditions. Thus, brain FGF preparations may contain two distinct mitogenic activities, one that is acidic and contains HUVE cell activity, and a basic mitogen that is similar to and may be identical with pituitary FGF.

Brain fibroblast growth factor: nonidentity with myelin basic protein fragments.

Fibroblast growth factor (FGF) from bovine brain has been reported to be a family of three polypeptide fragments derived by limited proteolysis from myelin basic protein (MBP) (Westall, F. C., Lennon, V. A., and Gosopodarowicz, D. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 4675-4678). However, fragments of sequence similar to the proposed active ones, generated from bovine MBP by acid proteases, are inactive in stimulating [3H]thymidine incorporation in BALB/c 3T3 cells. Further, the principal active component of the brain FGF preparation (Gospodarowicz, D., Bialecki, H., and Greenburg, G. (1978) J. Biol. Chem. 253, 3736-3743) which can be recovered in high yield from isoelectric focusing in sucrose has a pI between 4.8 and 5.8 in contradistinction to the MBP fragments (pI approximately 10) and is not retained on a column of chicken anti-bovine MBP-Sepharose. Therefore, although the reported preparation of brain FGF gives an increase in activity units/mg of protein of about 1000-fold over the crude brain extract, the main protein components, the MBP fragments, do not possess the mitogenic activity. Additional purification of as much as 50- to 100-fold may be required to obtain a homogeneous preparation of the real brain FGF.