Assessment of the importance of medicinal plants among communities around Khiat Ngong of southern Laos.

A field survey was launched to identify medicinal plants growing in the Khiat Ngong wetlands and surrounding forested areas of Pathoumphone District, Champasak Province in southern Laos. In this area, 418 plants representing approximately 250 species, belonging to at least 200 genera in 93 families of vascular plants, are used by traditional healers to treat more than 95 symptoms. A large number of species are used for treating fever. At least 14 plant species have not been previously reported for having medicinal properties. At least 10 have previously been investigated and have shown interesting biological activity by other researchers, signaling promising candidates for income-generating activities.

Folk Epidemiology Recorded in Palm Leaf Manuscripts of Laos.

In an effort to preserve traditional medicine knowledge and to uncover information about disease patterns and treatment in the Lao People's Democratic Republic (PDR), linguistic experts have scanned centuries-old medical palm leaf manuscripts for disease entries. A list of more than 7000 diseases has resulted, shedding valuable light onto the medical history and traditional medicine heritage of the people of Laos, as well as providing an index for faster research into specific diseases and their traditional treatments.

Accurate mass-time tag library for LC/MS-based metabolite profiling of medicinal plants.

We report the development and testing of an accurate mass-time (AMT) tag approach for the LC/MS-based identification of plant natural products (PNPs) in complex extracts. An AMT tag library was developed for approximately 500 PNPs with diverse chemical structures, detected in electrospray and atmospheric pressure chemical ionization modes (both positive and negative polarities). In addition, to enable peak annotations with high confidence, MS/MS spectra were acquired with three different fragmentation energies. The LC/MS and MS/MS data sets were integrated into online spectral search tools and repositories (Spektraris and MassBank), thus allowing users to interrogate their own data sets for the potential presence of PNPs. The utility of the AMT tag library approach is demonstrated by the detection and annotation of active principles in 27 different medicinal plant species with diverse chemical constituents.

Development of an Antiviral Screening Protocol: One-Stone-Two-Birds.

As prophylactic therapies and vaccines against viral infections continue to improve, drug resistant strains are continuing to arise; therefore it is imperative to develop new therapeutics against these diseases. For highly pathogenic viruses, such as Ebola and H5N1 influenza virus, the need for antivirals is even more urgent due to limited therapeutics against these viruses. Furthermore, the high pathogenicity of such viruses often makes it difficult to work with such agents. In this report, we describe a protocol called "One-stone-two-birds" which provides a safe and efficient screening system to identify anti-flu (entry) and anti-HIV (replication) activities. Using plant extracts as an example, we demonstrate the utility of this protocol in antiviral screening.

Biological evaluation of plants of Laos used in the treatment of tuberculosis in Lao traditional medicine.

Tuberculosis has existed in Southeast Asia for thousands of years. Many traditional treatments involve herbal remedies. Over time, these traditional treatments have had the chance to become refined based on efficacy and safety. It was therefore hypothesized that plants that were used in the past and are still used today to treat symptoms associated with tuberculosis are more likely to contain anti-tubercular compounds than plants that have not been used continuously. To try to deduce which plants were used in Laos in the past, a collection of palm leaf manuscripts was studied and a list of plants used to treat symptoms associated with tuberculosis was compiled. Interviews were then conducted with contemporary healers to see if the same plants are still being used today. Plants that were found in the manuscripts and/or are presently used by healers were collected, extracted and were evaluated in an anti-tubercular assay. This paper presents the methods used to identify and collect plants used to treat symptoms indicative of tuberculosis, and the results of anti-TB assays to test for activity.

Study of antimalarial activity of chemical constituents from Diospyros quaesita.

Bioassay-directed fractionation led to the isolation of seven compounds from a sample of the dried leaves, twigs, and branches of Diospyros quaesita Thw. (Ebenaceae). One of the isolates, betulinic acid 3-caffeate (1), showed in vitro antimalarial activity against Plasmodium falciparum clones D(6) (chloroquine-sensitive) and W(2) (chloroquine-resistant) with IC(50) values of 1.40 and 0.98 microM, respectively. Evaluation of compound 1 in the human oral epidermoid (KB) cancer cell line revealed cytotoxicity at ED(50) of 4.0 microM. In an attempt to reduce the cytotoxicity of 1, the acetylated derivative 1a and betulinic acid (1b) were prepared. Of the seven isolates, diospyrosin (2) was determined to be a new neolignan. In addition to 1, other known compounds isolated in this study were pinoresinol, lariciresinol, N-benzoyl-L-phenylalaninol, scopoletin, and poriferast-5-en-3beta,7alpha-diol. The structure of 2 was elucidated based on spectroscopic data analysis including 1D- and 2D-NMR, and HR-ESI-MS.

Advanced applications of counter-current chromatography in the isolation of anti-tuberculosis constituents from Dracaena angustifolia.

Through several steps of in vitro based bioassay-guided fractionation, three highly potent anti-mycobacterial constituents (1-2 microg/mL minimum inhibitory concentrations) were isolated from Dracaena angustifolia Roxb. (Dracaenaceae). Isolation was expedited due to the application of new techniques in counter-current chromatography (CCC). The first of these applications, gradient-array CCC, enables the expansion of the high-resolution window (sweet spot) by applying successive CCC separations in different solvent systems to a crude extract. Further fractionation was also performed using the recently designed 1L fast-centrifugal partition chromatography (FCPC) rotor with the solvent system hexane:methyl-tert butylether:acetonitrile (10:1:10). Results indicated that gradient-array CCC and high-capacity FCPC can facilitate drug discovery efforts from complex natural products, increase reproducibility of separation schemes, and provide more rigid dereplication of previously isolated bioactive compounds.

Rourinoside and rouremin, antimalarial constituents from Rourea minor.

Bioassay-directed fractionation of the antimalarial active CHCl(3) extract of the dried stems of Rourea minor (Gaertn.) Aubl. (Connaraceae) liana led to isolation of two glycosides, rourinoside (1) and rouremin (2), as well as five known compounds, 1-(26-hydroxyhexacosanoyl)-glycerol (3), 1-O-beta-D-glucopyranosyl-(2S,3R,4E-8Z)-2-N-(2'-hydroxypalmitoyl)-octadecasphinga-4,8-dienine, 9S,12S,13S-trihydroxy-10E-octadecenoic acid, dihydrovomifoliol-9-beta-D-glucopyranoside, and beta-sitosterol glucoside. Compounds 1-3 showed weak in vitro activities against Plasmodium falciparum. Their structures and stereochemistry were elucidated by spectroscopic methods and selected enzyme hydrolysis.

Antimalarial compounds from Grewia bilamellata.

Bioassay-directed fractionation led to the isolation of 12 compounds from a sample of the dried leaves, twigs, and stems of Grewia bilamellata. Five of the isolates, 3alpha,20-lupandiol (1), grewin (2), nitidanin (4), 2alpha,3beta-dihydroxy-olean-12-en-28-oic acid (5), and 2,6-dimethoxy-1-acetonylquinol (6), showed varying degrees of in vitro antimalarial activity against Plasmodium falciparum, but were devoid of significant cytotoxicity to the human oral epidermoid KB cancer cell line. Of the 12 isolates, compounds 1, 2, and 3 (bilagrewin) were determined to be a new triterpene, a new coumarinolignan, and a new neolignan, respectively. Other known compounds isolated in this study were 8-O-4' neolignan guaiacylglycerol-beta-coniferyl ether isomers (threo and erythro), cleomiscosin D, icariol A(2), ciwujiatone, and daucosterol. The structures of 1-3 were elucidated and identified on the basis of spectroscopic data including 1D and 2D NMR analysis.

Ethnopharmacological evaluation of the informant consensus model on anti-tuberculosis claims among the Manus.

Ethnobotanists often utilize predictive models to analyze the potential of indigenously used medicinal plants. The most common of these prognostic models is the informant consensus model. This study evaluates use of this model through the analytical ethnopharmacology of Manus Province, Papua New Guinea (PNG). The informant consensus model enables researchers to prioritize plants for pharmacognostic evaluation, based on the relative frequency of plants cited in anthropological interviews. Fieldwork on Manus Island, PNG, led to the identification of 43 species of plants used in traditional medicine for persistent respiratory symptoms. Plants were collected, dried, micro-extracted using a new technique generated in our laboratory, and evaluated in vitro against Mycobacterium tuberculosis. The results, in the form of IC(50) values and modified selectivity indices (SI), were compared to the results of the anthropological models of informant consensus, and statistically compared through linear regression and t-tests. Results were not statistically significant (alpha=0.1), leading to the conclusions that the informant consensus assumptions were inaccurate in predicting anti-mycobacterial activity among the Manus for anti-TB claims.

Miliusanes, a class of cytotoxic agents from Miliusa sinensis.

Bioassay-directed fractionation of the leaves, twigs, and flowers of Miliusa sinensis Finet and Gagnep. (Annonaceae) led to the isolation of a new class of potential anticancer lead molecules. They are a cluster of compounds composed of a C(18) carbon skeleton, a known but heretofore unnamed type, which we have designated as miliusane. Two known (1 and 2) as well as 20 new miliusanes (3-22) have been isolated and identified. They belong to two substructural classes of miliusanes. One subclass (1-19) was determined to be composed of a gamma-lactone spiro-ring system, the opening of which led to the second group of compounds (21 and 22) containing a tetrahydrofuran ring system. Compounds 1-3, 5, 8, 9, 18, 20, and 21 demonstrated significant cytotoxic activity in our cancer cell line panel comprising KB, Col-2, LNCaP, Lu-1, MCF-7, and HUVEC. The structures were determined by spectroscopic and chemical methods. The structure of miliusate was further confirmed by X-ray crystallographic analysis. The absolute stereochemistry of miliusanes was established by the Mosher ester method. Forty-two modified miliusane derivatives were also prepared and evaluated for their cytotoxic activities.

Pyrrole carboxamidine tryptase inhibitors from Leptonychia pubescens.

The root and stem bark extracts of a Nigerian sample of Leptonychia pubescens Keay (Sterculiaceae) were found to inhibit the serine protease tryptase, a potential therapeutic target for the treatment of asthma and chronic obstructive pulmonary disease (COPD). Bioassay-guided isolation led to the identification of 1-beta-ribofuranosylbrunfelsamidine as the active component with a tryptase IC (50) of 3 microM. Brunfelsamidine was also isolated, but was only weakly active.

Sesquiterpenes and butenolides, natural anti-HIV constituents from Litsea verticillata.

In a continuing investigation, six sesquiterpenes, as well as five butenolides were isolated from the leaves and twigs of Litsea verticillata Hance (Lauraceae). Among the isolates, three were determined to be new sesquiterpenes: isolitseanes A (1), B (2) and C (3); three known sesquiterpenes were identified as oxyphyllenodiol B (4), 1,2,3,4-tetrahydro-2,5-dimethyl-8-(1-methylethyl)-1,2-naphthalenediol (5), and chromolaevanedione (6). The butenolides include the novel litseabutenolide (9) and four known compounds: 3- epi-litsenolide D2 (7), cis-listenolide D1 (8), 4-hydroxy-2-methylbut-2-enolide (10), and hydroxydihydrobovolide (11). Isolates 2, 4, 5, 7, 9, 10 and 11 were found to inhibit HIV-1 replication in a green fluorescent protein (GFP)-based reporter cell line (HOG.R5) with IC50 values of 38.1, 54.6, 91.0, 9.9, 40.3, 129.8, and 122.7 microM, respectively. Structure elucidation and identification were based on spectroscopic means including 1D and 2D NMR analyses.

Constituents of the leaves and twigs of Calyptranthes pallens collected from an experimental plot in Southern Florida.

Fractionation of the chloroform-soluble extract of the leaves and twigs of Calyptranthes pallens, collected from an experimental plot in a hardwood forest of southern Florida, using a hormone-dependent human prostate carcinoma (LNCaP) tumor cell line, led to the isolation of a phloroglucinol derivative with a novel carbon skeleton, pallenic acid (1), and a new triterpenoid, 3beta-hydroxy-18alpha,19alpha-urs-20-en-28-oic acid (2). The known compound methylene-bis-aspidinol (3) was selectively active against the human oral epidermoid carcinoma (KB) cell line. Several known compounds of the ellagic acid, lignan, phloroglucinol, sterol, and triterpene types were also obtained in the present investigation.

New 3-O-acyl betulinic acids from Strychnos vanprukii Craib.

Three new betulinic acid derivatives, 3beta-O-trans-feruloylbetulinic acid (1), 3beta-O-cis-feruloylbetulinic acid (2), and 3beta-O-cis-coumaroylbetulinic acid (4), along with two known triterpenes, 3beta-O-trans-coumaroylbetulinic acid (3) and ursolic acid (6) were isolated from the leaves and twigs of Strychnos vanprukiiCraib. All isolates showed moderate anti-HIV activity with IC50 values ranging from 3 to 7 microg/mL (5 to 15 microM) in an indicator cell line for HIV infectivity. The structures of the new isolates were elucidated by spectroscopic techniques including 1D and 2D NMR spectroscopy. In addition, the structure of 1 was confirmed by X-ray crystallography.

Bioactive constituents from Asparagus cochinchinensis.

Bioassay-directed fractionation of the dried roots of Asparagus cochinchinensis led to the isolation of a new spirostanol saponin, asparacoside (1), two new C-27 spirosteroids, asparacosins A (2) and B (3), a new acetylenic derivative, 3' '-methoxyasparenydiol (4), and a new polyphenol, 3'-hydroxy-4'-methoxy-4'-dehydroxynyasol (6), as well as five known phenolic compounds, asparenydiol (5), nyasol (7), 3' '-methoxynyasol (8), 1,3-bis-di-p-hydroxyphenyl-4-penten-1-one (9), and trans-coniferyl alcohol (10). Compounds 1, 6, and 8 demonstrated moderate cytotoxicities in a panel comprised of KB, Col-2, LNCaP, Lu-1, and HUVEC cells, with IC(50) values ranging from 4 to 12 microg/mL. The structures were determined by spectroscopic and chemical methods.

Isolation and absolute stereochemistry of coussaric acid, a new bioactive triterpenoid from the stems of Coussarea brevicaulis.

Coussaric acid (1), a triterpenoid based on an ursane skeleton, and an oleanane-type triterpene acid, 3-epi-spathodic acid (2), as well as four known compounds, barbinervic acid, scutellaric acid, stigmasterol and stigmasterol glucoside, have been isolated from an EtOAc-soluble extract of the stems of Coussarea brevicaulis. The structures of compounds 1 and 2 were elucidated on the basis of spectroscopic investigation, and single-crystal X-ray crystallography was used to confirm the structure of 1. The absolute stereochemistry of 1 was established by chemical transformations and by the Mosher ester procedure. The potential of the isolates and chemical transformation products to induce quinone reductase was evaluated in mouse Hepa lclc7 hepatoma cells.

Natural anti-HIV agents. Part IV. Anti-HIV constituents from Vatica cinerea.

In a continuing search for anti-HIV compounds from plants of Vietnam, 19 compounds, including a new triterpene, were isolated from an extract of the leaves and stem of Vatica cinerea. The new triterpene was determined to be a cycloartane triterpenoid with 29 skeletal carbons and was assigned the name vaticinone (1). The known triterpenes included three cycloartanes, a lanostane, two dammaranes, three lupanes, an ursane, and an oleanane. A chlorophyll isolate was identified as pheophorbide a (13). The majority of the triterpenes, the sesquiterpene, 1-hydroxycyclocolorenone, and pheophorbide a showed anti-HIV activity, with the chlorophyll being the most active, demonstrating an IC(50) value of 1.5 microgram/mL (2.5 microM), while being completely devoid of toxicity up to a concentration of 20 microgram/mL (33.8 microM). Vaticinone (1) was found to inhibit the replication of HIV-1, with an IC(50) value of 6.5 microgram/mL (15.3 microM; selective index = 1.4). The structures of these isolates were determined by spectral data including 1D and 2D NMR spectra.

Antimalarial agents from plants. III. Trichothecenes from Ficus fistulosa and Rhaphidophora decursiva.

Bioassay-directed fractionation of an extract prepared from the dried leaves and stem barks of Ficus fistulosa Reinw. ex Blume (Moraceae) led to the isolation of verrucarin L acetate (1), together with 3alpha-hydroxyisohop-22(29)-en-24-oic acid, 3beta-gluco-sitosterol, 3,4-dihydro-6,7-dimethoxyisocarbostyril, 3,4,5-trimethoxybenzyl alcohol, alpha-methyl-3,4,5-trimethoxybenzyl alcohol, indole-3-carboxaldehyde, palmanine, and aurantiamide acetate. Roridin E (2) was identified in a subfraction from the dried leaves and stems of Rhaphidophora decursiva Schott (Araceae). Verrucarin L acetate and roridin E were characterized as macrocyclic trichothecene sesquiterpenoids and found to inhibit the growth of Plasmodium falciparum with IC 50 values below 1 ng/ml.