Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis.

As a result of bioassay-guided fractionation, betulinic acid, a pentacyclic triterpene, was identified as a melanoma-specific cytotoxic agent. In follow-up studies conducted with athymic mice carrying human melanomas, tumour growth was completely inhibited without toxicity. As judged by a variety of cellular responses, antitumour activity was mediated by the induction of apoptosis. Betulinic acid is inexpensive and available in abundant supply from common natural sources, notably the bark of white birch trees. The compound is currently undergoing preclinical development for the treatment or prevention of malignant melanoma.

Cancer chemopreventive activity of resveratrol, a natural product derived from grapes.

Resveratrol, a phytoalexin found in grapes and other food products, was purified and shown to have cancer chemopreventive activity in assays representing three major stages of carcinogenesis. Resveratrol was found to act as an antioxidant and antimutagen and to induce phase II drug-metabolizing enzymes (anti-initiation activity); it mediated anti-inflammatory effects and inhibited cyclooxygenase and hydroperoxidase functions (antipromotion activity); and it induced human promyelocytic leukemia cell differentiation (antiprogression activity). In addition, it inhibited the development of preneoplastic lesions in carcinogen-treated mouse mammary glands in culture and inhibited tumorigenesis in a mouse skin cancer model. These data suggest that resveratrol, a common constituent of the human diet, merits investigation as a potential cancer chemopreventive agent in humans.

Activation of the mu-opiate receptor by Vitex agnus-castus methanol extracts: implication for its use in PMS.

The dried ripe fruit of Vitex agnus-castus L. (VAC) is widely used for the treatment of premenstrual syndrome (PMS). A previous study reported that extracts of VAC showed affinity to opiate receptors; however, functional activity was not determined. We tested two different VAC extracts in receptor binding and functional assays. Our objectives were: (1) to confirm the opiate affinity; (2) to rule out interference by free fatty acids (FFA); (3) to determine the mode of action of VAC at the mu-opiate receptor. Methanol extracts of VAC were prepared either before (VAC-M1) or after (VAC-M2) extraction with petroleum ether to remove fatty acids. Both extracts showed significant affinities to the mu-opiate receptor, as indicated by the concentration-dependent displacement of [3H]DAMGO binding in Chinese hamster ovary (CHO)-human mu-opiate receptor (hMOR) cells. The IC50 values were estimated to be 159.8 microg/ml (VAC-M1) and 69.5 microg/ml (VAC-M2). Since the defatted extract not only retained, but exhibited a higher affinity (p<0.001), it argued against significant interference by fatty acids. In an assay to determine receptor activation, VAC-M1 and VAC-M2 stimulated [35S]GTPgammaS binding by 41 and 61% (p<0.001), respectively. These results suggested for the first time that VAC acted as an agonist at the mu-opiate receptor, supporting its beneficial action in PMS.

Chemical and biological characterization and clinical evaluation of botanical dietary supplements: a phase I red clover extract as a model.

Botanical dietary supplements, as compared with nutritional supplements or single-component pharmaceutical drugs, are typically less-refined preparations derived from bulk plant material and, as such, require a modified approach to their development, production, and evaluation. An integrated, multidisciplinary team of scientific and clinical investigators is required in order to develop high quality phytomedicines and rigorously evaluate their safety and efficacy. Research on botanicals involves unique challenges as plant source materials frequently vary in chemical content and may contain unwanted pesticides, heavy metals, contaminant plant species, or other adulterants. Ideally, a botanical formulation should be standardized, both chemically and biologically, by a combination of analytical techniques and bioassays. This combination approach provides multiple measures by which reproducible quality and efficacy of botanical supplements may be achieved, and is particularly useful for botanical products for which the active compound(s) have not yet been identified. Safety and toxicity should be evaluated during the supplement development process in both in vitro and in vivo systems. A number of liquid chromatography-mass spectrometry methods can aid in the assessment of purity, bioavailability, toxicity, metabolism, and molecular target profiling of botanical extracts. Clinical investigators must appreciate the complexity of multi-component phytomedicines and adjust trial protocols accordingly. This review highlights practical considerations of value to basic science and clinical investigators engaged in the study of botanical supplements. Lessons and examples are drawn from the authors' experience in designing and developing a red clover (Trifolium pratense L.) standardized extract for evaluation in Phase I and Phase II clinical trials.

Plant antitumor agents. 31. The calycopterones, a new class of biflavonoids with novel cytotoxicity in a diverse panel of human tumor cell lines.

Three new biflavonoids to which we have accorded the trivial names calycopterone (1), isocalycopterone (2), and 4-demethylcalycopterone (3) and the known flavone 4',5-dihydroxy-3,3',6,7-tetramethoxyflavone (4) were isolated as cytotoxic constituents from the flowers of Calycopteris floribunda Lamk. (Combretaceae). Compounds 1-3 showed a wide range of activity against a panel of solid tumor cell lines. Among the biflavonoids, calycopterone (1) is the major constituent.

The value of plants used in traditional medicine for drug discovery.

In this review we describe and discuss several approaches to selecting higher plants as candidates for drug development with the greatest possibility of success. We emphasize the role of information derived from various systems of traditional medicine (ethnomedicine) and its utility for drug discovery purposes. We have identified 122 compounds of defined structure, obtained from only 94 species of plants, that are used globally as drugs and demonstrate that 80% of these have had an ethnomedical use identical or related to the current use of the active elements of the plant. We identify and discuss advantages and disadvantages of using plants as starting points for drug development, specifically those used in traditional medicine.

Absolute configuration of novel bioactive flavonoids from Tephrosia purpurea.

[structure: see text] Three novel flavonoids, (+)-tephrorins A (1) and B (2) and (+)-tephrosone (3), were isolated from Tephrosia purpurea. Their structures were elucidated by NMR spectral analysis, and their absolute configurations were determined by Mosher ester methodology. Compounds 1 and 2 are flavanones containing an unusual tetrahydrofuran moiety. Compounds 1-3 were evaluated for their potential cancer chemopreventive properties using a cell-based quinone reductase induction assay.

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.

Constituents of Eugenia sandwicensis with potential cancer chemopreventive activity.

A triterpenoid, 3beta-cis-p-coumaroyloxy-2alpha,23-dihydroxyolean-12-en-28-oic acid (1), and two natural products, 3beta-trans-p-coumaroyloxy-2alpha,23-dihydroxyolean-12-en-28-oic acid (2) and 23-trans-p-coumaroyloxy-2alpha,3beta-dihydroxyolean-12-en-28-oic acid (3), were isolated from a chloroform-soluble extract of the stems of Eugenia sandwicensis, along with 10 known compounds. Of these compounds, 2 showed significant inhibitory activity (79.2% at 4 microg/ml) in a 7,12-dimethylbenz[a]anthracene-induced mouse mammary organ culture assay system of relevance to cancer chemoprevention. Gallic acid was isolated as an antioxidative constituent of an ethyl acetate-soluble extract of E. sandwicensis stems. Isolates 1-3 were characterized on the basis of spectral and chemical evidence.

Cytotoxic prenylated flavanones from Monotes engleri.

From the leaves of Monotes engleri, five prenylated flavanones were isolated as constituents that displayed cytotoxic activity against several human cancer cell lines. There of these substances are novel, namely, 6-(1,1-dimethylallyl)naringenin, 6-(1,1-dimethylallyl)eriodictyol and 3'-O-methyl-6-(1,1-dimethylallyl)-eriodictyol, with the other two active substances being the known flavanones, 6,8-diprenyleriodictyol and hiravanone. Additionally, two novel, but non-cytotoxic, biogenetically related flavanones were isolated, 6-[(2RS)-hydroxy-3-methyl-3-butenyl]-8-prenyleriodictyol and 5,4'-dihydroxy-4",4"-dimethyl-5"-methyl-5"H-dihydrofurano[2",3": 6,7]flavanone. The structures of the new compounds were determined by spectral analysis 1D- and 2D-NMR experiments.

Cytotoxic constituents from the roots of Tovomita brevistaminea.

Two known xanthones, trapezifolixanthone and manglexanthone were isolated as cytotoxic constituents from the CHCl3 extract of the roots of Tovomita brevistaminea by bioassay-guided fractionation using the KB cell line. In addition, a new compound, tovophenone C, and two known compounds, tovophenones A and B which are benzophenones, were found to be inactive constituents in this investigation. The structure of the new isolate was determined by detailed analysis of spectroscopic parameters including its 1D and 2D NMR spectroscopy data.

Cytotoxic biflavonoids from Selaginella willdenowii.

Bioactivity-guided fractionation of the leaves of Selaginella willdenowii afforded three known biflavones, 4',7"-di-O-methylamentoflavone, isocryptomerin and 7"-O-methylrobustaflavone, that were significantly cytotoxic against a panel of human cancer cell lines. Non-cytotoxic isolates were also obtained, namely, amentoflavone, bilobetin, robustaflavone and 2",3"-dihydroisocryptomerin, a new dihydrobiflavone. The structure for the new biflavonoid was unambiguously assigned by a combination of spectroscopic methods.

Xanthones from the twigs of Mammea siamensis.

1,2-Dimethoxy-5-hydroxyxanthone, a new xanthone, was isolated from the twigs of Mammea siamensis, in addition to six known xanthones (5-hydroxy-1-methoxy-, 1,3-dimethoxy-5-hydroxy-, 2,5-dihydroxy-1-methoxy-, 1,7-dihydroxy-, 1,3,7-trihydroxy- and 3,5-dihydroxy-1-methoxyxanthone). Structures for these compounds were deduced from their spectral data.

New bioactive aromatic compounds from Vismia guianensis.

Five benzophenones, vismiaguianones A-E, and two benzocoumarins, vismiaguianins A and B were isolated from the CHCl3 extract of the roots of Vismia guianensis by bioassay-directed fractionation using the DNA strand-scission assay and KB cell line. Of the isolates obtained, vismiaguianone B exhibited DNA strand-scission activity, whereas vismiaguianones D and E and vismiaguianin A were found to be significantly cytotoxic.

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.

Limonoids from Azadirachta excelsa.

Activity-directed fractionation of a stem extract of Azadirachta excelsa using KB (human oral epidermoid carcinoma) cells led to the isolation of four meliacin-type limonoids. Two of these constituents were novel, namely, 2,3-dihydronimbolide and 3-deoxymethylnimbidate, and these were purified along with the known compounds, nimbolide and 28-deoxonimbolide. The structures of the new compounds were determined by spectroscopic methods. Nimbolide and 28-deoxonimbolide were broadly cytotoxic when evaluated against a panel of human cancer cell lines, while the two novel compounds were inactive in this regard. The defection of nimbolide and 28-deoxonimbolide as cytotoxic constituents was facilitated by an electrospray LC/MS dereplication procedure.

Cell-cycle specific cytotoxicity mediated by rearranged ent-kaurene diterpenoids isolated from Parinari curatellifolia.

Two structurally novel cytotoxic ent-kaurene diterpenoids, 13-methoxy-15-oxozoapatlin and 13-hydroxy-15-oxozoapatlin, were isolated from the root bark of Parinari curatellifolia, together with the known compound, 15-oxozoapatlin, on the basis of bioactivity-guided chromatographic fractionation and found to demonstrate broad-spectrum cytotoxic activity against a panel of cultured human cancer cell lines. The structures of these compounds were determined by analysis of their spectroscopic data. The presence of an alpha, beta-unsaturated carbonyl group in 13-methoxy-15-oxozoapatlin suggested that the cytotoxic potential of this compound could be mediated through reaction with cellular nucleophiles by means of a Michael-type addition. The compound 13-methoxy-15-oxozoapatlin reacted with the nucleophiles L-cysteine and beta-mercaptoethanol. The adduct with beta-mercaptoethanol was isolated, structurally characterized and found to be approximately 5-fold less cytotoxic than 13-methoxy-15-oxozoapatlin itself. The compound 13-methoxy-15-oxozoapatlin did not interact with DNA nor guanosine, and it was not mutagenic for Salmonella typhimurium strain TM677. The effects of 13-methoxy-15-oxozoapatlin on the growth of human cancer cells were analyzed utilizing cultured ZR-75-1 breast cancer cells. Biosynthesis of DNA, RNA and protein was reduced in treated cells, and accumulation at the G2/M phase of the cell cycle was observed. The compound 13-methoxy-15-oxozoapatlin did not mediate antimitotic activity with dibutyryl cAMP-treated cultured astrocytoma cells, suggesting that the cell cycle effect is G2 specific. No antitumor activity was observed when athymic mice carrying KB cells were treated with 13-methoxy-15-oxozoapatlin. These data indicate that the cytotoxic activity of 13-methoxy-15-oxozoapatlin is mediated in part by covalent reaction with a cellular component (such as sulfhydryl-containing protein) by means of a Michael-type addition, and this results in the blockage of cell-cycle progression.

Evaluation of the mutagenic potential of endod (Phytolacca dodecandra), a molluscicide of potential value for the control of schistosomiasis.

Extracts of the fruit of Phytolacca dodecandra (endod) demonstrate molluscicidal and other biological activities. Since this plant is indigenous to some countries where schistosomiasis is a common problem, it has been proposed that it may be socioeconomically feasible to employ endod as an aid in the control of this disease through its use to control the snail vector. As an initial step in the safety assessment of this substance, its mutagenic potential was determined utilizing Salmonella typhimurium strain TM677. The seeds and fruit of Phytolacca americana, also molluscicidal, were additionally evaluated for mutagenic potential. Using a variety of conditions, no mutagenic activity could be demonstrated for any of the extracts tested. Thus, subject to the results of future safety assessment, endod remains a viable candidate as a useful molluscicide.

Catharanthus alkaloids XXXII: isolation of alkaloids from Catharanthus trichophyllus roots and structure elucidation of cathaphylline.

Further examination of the cytotoxic alkaloid fractions of Catharanthus trichophyllus roots afforded nine alkaloids. Two of these alkaloids, lochnericine and horhammericine, are responsible for part of the cytotoxic activity. The structure elucidation of cathaphylline, a new beta-anilino acrylate derivative, is described.