Anatomical study of secondary tuberized roots of Harpagophytum procumbens DC and quantification of harpagoside by high-performance liquid chromatography method.

AIM AND BACKGROUND: A botanical study is conducted to provide a standard diagnostic tool. In order to improve the quality assurance of the secondary tuberized roots of Harpagophytum procumbens, derived extract and phytomedicine, a simple, rapid, and accurate high-performance liquid chromatography (HPLC) method was developed to assess the harpagoside. MATERIAL AND MEHODS: This HPLC assay was performed on a reversedphase C18 column with methanol and water (50/50-V/V) as the mobile phase with a flow rate of 1.5 mL/min and using a monitoring wavelength at 278 nm. RESULTS AND CONCLUSION: This method was successfully applied to quantify these bioactive iridoid in an aqueous extract of H. procumbens and in its related phytomedicine "harpagophyton". The result demonstrated that the quantification of harpagoside, indicating that the quality control of the bioactive ingredient in H. procumbens, derived extract and phytomedicine, is critical to ensure its clinical benefits.

The kinetics of exsheathment of infective nematode larvae is disturbed in the presence of a tannin-rich plant extract (sainfoin) both in vitro and in vivo.

The mode of action of bioactive plants on gastrointestinal nematodes remains obscure. Previous in vitro studies showed that exsheathment was significantly disturbed after contact with tannin-rich extracts. However, the role of important factors (extract concentration, parasite species) has not been assessed and no information is available on the occurrence in vivo. These questions represent the objectives of this study. The model incorporated the parasites Haemonchus contortus and Trichostrongylus colubriformis with sainfoin as the bioactive plant. A set of in vitro assays was performed, measuring the changes observed, after 3 h of contact with increasing concentrations of sainfoin, on the rate of artificial exsheathment. The results indicated that sainfoin extracts interfered with exsheathment in a dose-dependent manner and the process overall was similar for both nematodes. The restoration of control values observed after adding PEG to extracts confirms a major role for tannins. A second study was performed in vivo on rumen-cannulated sheep fed with different proportions of sainfoin in the diet to verify these in vitro results. The consumption of a higher proportion of sainfoin was indeed associated with significant delays in Haemonchus exsheathment. Overall, the results confirmed that interference with the early step of nematode infection might be one of the modes of action that contributes to the anthelmintic properties of tanniniferous plants.

Molluscicidal activity against Bulinus truncatus of Croton campestris.

The bark of Croton campestris provided three furano-clerodane named, respectively, velamone, velamolone acetate and velamolone. Fractions containing these diterpenoids were investigated for their molluscicidal activity against Bulinus truncatus, one of the aquatic snail vectors of schistosomiasis. A dichloromethanic extract of root barks, rich in furano-clerodane and molluscicidal at 20ppm was promising as natural molluscicide. Enriched fraction with velamone gave 80% mortality from 25ppm, while its LD(100) was at 50ppm. The LD(80) was ca. at 45ppm, while LD(100) was at 60ppm for the enriched fraction containing velamolone acetate. The velamolone-enriched fraction was less efficient and gave a mortality of 90% at 90ppm. The pure compounds, velamone and velamolone acetate were active at 100% at 3 and 6ppm, respectively. Velamolone showed a molluscicidal activity at 20ppm.

Effects of four tanniferous plant extracts on the in vitro exsheathment of third-stage larvae of parasitic nematodes.

The anthelmintic properties of tanniferous plants and of their secondary metabolites represent one possible alternative to chemotherapy that is currently being explored as a means of achieving sustainable control of gastrointestinal nematodes in ruminants. Previous in vivo and in vitro results suggest that tanniferous plants can have direct anti-parasitic effect against different stages of nematodes. However, the mode of action of the bioactive plant compounds remains obscure. The objectives of the current study were (1) to examine the hypothesis that extracts of tanniferous plants might interfere with the exsheathment of third-stage infective larvae (L3); (2) to assess the role of tannins in the process by examining the consequence of adding an inhibitor of tannins (polyethylene glycol: PEG) to extracts. The effects of 4 tanniferous plant extracts on exsheathment have been examined on L3 of Haemonchus contortus and Trichostrongylus colubriformis. Artificial exsheathment was induced in vitro by adding hypochloride solution to larval suspension. The evolution of exsheathment with time was measured by repeated observations at 10-min interval for 60 min. The selected plants were: genista (Sarothamnus scoparius), heather (Erica erigena), pine tree (Pinus sylvestris), and chestnut tree (Castanea sativa), with tannin contents ranging from 1.5 to 24.7% of DM. Extracts of a non-tanniferous plant (rye grass, tannin content: 0.3% of DM) were included in the assay as negative controls. The extracts were tested at the concentration of 600 microg/ml and the effects were compared to the rate of exsheathment of control larvae in PBS. No statistical differences in the pattern of exsheathment was observed after addition of rye grass or genista extracts for both nematode species and with heather extracts for T. colubriformis. In contrast, pine tree extracts on larvae of both species and heather extracts with H. contortus induced a significant delay in exsheathment. Last, contact with chest nut extracts led to a total inhibition of the process for both nematodes. These results suggest that extracts of tanniferous plants might affect a key process in the very early stages of larval invasion of the host. In most cases, the addition of PEG led to a total or partial restoration towards control values. This suggests that tannins are largely involved in the inhibitory process. However, other secondary metabolites may also interfere with the process that would help to explain some of the differences in response observed between the two nematode species.

Effect of bioactive compounds from Sainfoin ( Onobrychis viciifolia Scop.) on the in vitro larval migration of Haemonchus contortus: role of tannins and flavonol glycosides.

Anthelmintic bioactivity against gastrointestinal nematodes has been associated with leguminous forages supporting the hypothesis of a role of condensed tannins. However, the possibility that other compounds might also been involved has received less consideration. Using bio-guided fractionation, the current study aimed at characterizing the biochemical nature of the active compounds present in sainfoin (Onobrychis viciifolia ), previously identified as an anthelmintic leguminous forage. The effects of sainfoin extracts were evaluated on 3rd-stage larvae (L3) of Haemonchus contortus by using a larval migration inhibition (LMI) assay. Comparison of extracts obtained with several solvent systems showed that the bioactivity was associated with the 70ratio30 acetone/water extract. Further fractionation of the later allowed the separation of phenolic compounds. By use of a dialysis method, compounds were separated with a molecular weight cut-off of 2000 Da. The in vitro anthelmintic effect of the fraction with condensed tannins was confirmed. In the fraction containing molecules of MW <2000 Da, 3 flavonol glycosides were identified as rutin, nicotiflorin and narcissin. At 1200 mug/ml, each inhibited significantly the migration of larvae. Addition of polyvinyl pyrrolidone (PVPP) to both fractions before incubation restored larval migration. These results confirmed the role of both tannins and flavonol glycosides in the anthelmintic properties of sainfoin.

In vitro effects of four tropical plants on the activity and development of the parasitic nematode, Trichostrongylus colubriformis.

The in vitro effects of extracts of four tropical plants (Zanthoxylum zanthoxyloides, Newbouldia laevis, Morinda lucida and Carica papaya) on the egg, infective larvae and adult worms of Trichostrongylus colubriformis were screened for potential anthelmintic properties. Significant effects were observed with the four plants on T. colubriformis but they differed depending on the stage of the parasite. Extracts of each plant induced a dose-dependent inhibition of egg hatching. Using a larval inhibition migration test, the effects on the infective larvae were also detected with the four plant extracts. In contrast, for adult worms, the effects were statistically significant only for N. laevis and C. papaya. No significant activity was shown for M. lucida and Z. zanthoxyloides. These in vitro results suggest the presence of some anthelmintic properties associated with these four plants, which are traditionally used by small farmers in western Africa. These effects need to be studied under in vivo conditions.

In vitro effects of four tropical plants on three life-cycle stages of the parasitic nematode, Haemonchus contortus.

Alcoholic extracts of four tropical plants (Zanthoxylum zanthoxyloides, Newbouldia laevis, Morinda lucida and Carica papaya) were screened in vitro for potential anti-parasitic effects against eggs, infective larvae and adult Haemonchus contortus. Significant effects were obtained with all four plants but differences were observed depending on the parasitic stage. The effects of the four plant extracts were similar on egg hatching and were dose dependent. In contrast, no dose-response relationship was found for infective larvae and adult worms, although more potent effects were usually observed with the highest concentrations. Using a larval inhibition migration test, extracts of fagara (Z. zanthoxyloides) were found to be less active against Haemonchus infective larvae than were the other plants. N. laevis was found to be highly and rapidly effective against adult worms. Overall, these in vitro results suggest that these four plants, traditionally used by small farmers in Western Africa, do possess anti-parasitic properties. These effects remain to be confirmed through in vivo studies.

In vitro effects of three woody plant and sainfoin extracts on 3rd-stage larvae and adult worms of three gastrointestinal nematodes.

Most studies on the effects of tanniferous plants on nematodes have examined forages but have neglected the woody plants. Therefore, in vitro effects of extracts from 3 woody plants (Rubus fructicosus, Quercus robur, Corylus avellana) have been tested on trichostrongyles and compared to sainfoin, a legume forage. Because some in vivo results indicated that the effects of tannins differed depending on the parasitic species and/or stages, the effects were measured on 3rd-stage larvae (L3) and adult worms of Teladorsagia circumcincta, Haemonchlus contortus and Trichostrongylus colubriformis. The effects of plant extracts varied according to the plant sources, the parasite species and stages. For the woody plants, significant inhibitory effects were obtained on both stages of abomasal species. Results for T. colubriformis were more variable. Effects of sainfoin extracts were significant on T. colubriformis and H. contortus L3, and on abomasal adult worms. In order to assess the implications of tannins, polyethylene glycol (PEG), an inhibitor of tannins, was added to hazel tree, oak and sainfoin extracts. Without PEG, significant inhibitory effects on L3 and adult worms were confirmed. After addition of PEG, the larval migration and motility of adult worms were restored in most cases. These results confirm variations in effects depending on factors related to plants or parasites and suggest that tannins are partly responsible for the effects.

Identification of the alkaloids of Galipea officinalis by gas chromatography-mass spectrometry.

Galipea officinalis Hancock, a Venezuelan shrubby tree which is acclaimed in folk medicine for its many healing properties, is the only species of the genus to contain tetrahydroquinoline alkaloids. A GC-MS method has been developed in order to analyse the essential oil, hexane and chloroform extracts of the trunk bark of this plant, without prior derivatisation of the alkaloidal components. A study of the MS fragmentation patterns of the components permitted the identification of five new minor quinoline alkaloids together with the known alkaloids. In addition, the method could also be used for the characterisation of alkaloids within the genus Galipea.

Three clerodane diterpenoids from Croton eluteria Bennett.

Three furanoid clerodanes have been isolated from the stem bark of Croton eluteria Bennett. Their structures have been established by spectroscopic methods. The compounds were named cascarillin B (7alpha-acetoxy-3,4,15,16-diepoxy-12-oxo-cleroda-13(16),14-dien-20-al), cascarillin C (7alpha-acetoxy-15,16,12,20-diepoxy-20-hydroxy-cleroda-3,4,13(16),14-triene) and cascarillin D (7alpha-acetoxy-3,4,15,16-diepoxy-cleroda-13(16),14-dien-20-al).

[Study on the structure and activity of new phenolic acid compounds from Erigeron breviscapus].

AIM: To isolate and identify the active constituents of Erigeron brevisapus (Vant.) Hand.-Mazz. METHODS: The chemical constituents were isolated by silica gel column chromatography and two new compounds were obtained. Their structures were elucidated by IR, MS, 1HNMR, 13CNMR, DEPT and 2DNMR. The injury of BCMEC (bovine cerebral microvascular endothelial cell) was determined by lactic dehydrogenase (LDH), the ability of the drugs anti-oxidation and scavenging oxidation of free radical was measured by colorimetric method. RESULTS: Two new compounds have been identified as 1-O-methyl-3, 5-O-dicaffeoyl quinic acid methyl ester (III) and 5-O-caffeoyl quinic acid butyl ester (IV). CONCLUSION: Compounds III and IV are new compounds. Compound III can protect BCMEC injury by LPC.

New lanostanoids from Ganoderma lucidum that induce NAD(P)H:quinone oxidoreductase in cultured hepalcic7 murine hepatoma cells.

Two new lanostanoids were isolated from the basidiocarp of Ganoderma lucidum and were identified as 26,27-dihydroxy-5 alpha-lanosta-7,9(11),24-triene-3,22-dione (1) and 26-hydroxy-5 alpha-lanosta-7,9(11),24-triene-3,22-dione (2) by their respective spectral data. Crude extracts and the isolated compounds were tested for their potential to induce NAD(P)H:quinone oxidoreductase (QR), a phase 2 drug-metabolizing enzyme, as an approach to detect potential cancer chemopreventive activity. Compound 2 doubled the specific activity of QR at a concentration of 3.0 micrograms/ml, whereas compound 1 was significantly less active (1.7-fold induction at 20 micrograms/ml). In addition, both compounds weakly inhibited sheep vesicle cyclooxygenase 1 activity at a test concentration of 40 micrograms/ml.

Iridoids from Caryopteris x clandonensis.

In continuation of our phytochemical studies on Caryopteris x clandonensis (Lamiaceae), three further iridoids were isolated from the methanolic extract of the stems. Their structures were established by 1D and 2D NMR and MS analysis as a C-6 epimer of 8-O-acetylharpagide (6-epi-8-O-acetylharpagide), a derivative of harpagide which contained the unusual feature of a 3',4' seco-glycopyranosyl moiety (clandonoside II) and a methyl cetal of 8-O-acetylharpagide aglucone hydrate named clandonensine.

An antioxidant sinapic acid ester isolated from Iberis amara.

The isolation of 6-O-sinapoyl sucrose (1) from Iberis amara seeds and an evaluation of its antioxidative properties in comparison with sinapic acid and ascorbic acid are reported.

Novel quinoline alkaloid from trunk bark of Galipea officinalis.

The isolation of N-methyl-4-hydroxy-3-(2',3'-epoxyisobutyl)-2-quinolone (1), a new natural compound, and candicine (2) from Galipea officinalis trunk bark is reported. This is the first report of candicine in the genus Galipea.

Direct characterization of isoquinoline alkaloids in a crude plant extract by ion-pair liquid chromatography-electrospray ionization tandem mass spectrometry: example of Eschscholtzia californica.

An ion-pair HPLC-ESI-MS-MS method has been developed for the direct and rapid characterization of isoquinoline alkaloids in a crudely purified extract of the aerial parts of Eschscholtzia californica (Papaveraceae). This plant was chosen because of its increasing use in pharmaceutical industries and because its well known alkaloid composition allows the optimization of the experimental procedure through an on-line analytical sequence. Thus, 14 isoquinoline alkaloids of different types were detected and characterized. The identities of these compounds were confirmed unambigously by their fragmentation and UV spectra obtained by LC-diode-array detection. Various experiments including tandem mass spectrometry and in-orifice collision induced dissociation were performed and prove that MS-MS is a very efficient technique to identify these compounds. An explanation for each isoquinoline alkaloid type MS-MS fragmentation pattern is proposed and indicates similar neutral and/or radical losses. The order of the fragmentation depended on the type of compound but the lost fragments were similar.

Two new caffeoyl conjugation from Erigeron breviscapus.

Two new constituents with a novel basic skeleton were isolated from Erigeron breviscapus. On the basis of chemical and spectroscopic evidences, the structures of the new compounds were elucidated as 1R,3R-dihydroxy-4S,5R-dicaffeoyloxy cyclohexane carboxylic acid methyl ester (V), 1,4-dihydroxy-3R,5R-dicaffeoyloxy cyclohexane carboxylic acid methyl ester (VI).

Alkaloids from Galipea officinalis.

Galipea officinalis Hancock is a shrubby tree reputed in folk medicine for its many properties which may be related to its alkaloid composition. We present in this paper the structural elucidation of five quinoline alkaloids: cuspareine, cusparine, and galipine, that have been previously described, demethoxycusparine, newly isolated in this species of Galipea and a new quinoline alkaloid named galipinine which is the 3',4'-methylenedioxycuspareine.

In vitro antimalarial activity of penduline, a bisbenzylisoquinoline from Isopyrum thalictroides.

Two bisbenzylisoquinolines, tetrandrine and penduline, were purified from Isopyrum thalictroides. When tested for antimalarial activity in vitro, penduline was efficient at concentrations fivefold lower than those of tetrandrine. In highly synchronized parasite cultures, penduline mostly interfered between the 8th and the 32nd hours of the parasite cycle.

Enrichment in Specific Soluble Sugars of Two Eucalyptus Cell-Suspension Cultures by Various Treatments Enhances Their Frost Tolerance via a Noncolligative Mechanism.

A cell-suspension culture obtained from the hybrid Eucalyptus gunnii/Eucalyptus globulus was hardened by exposure to lower temperatures, whereas in the same conditions cells from a hybrid with a more frost-sensitive genotype, Eucalyptus cypellocarpa/Eucalyptus globulus, were not able to acclimate. During the cold exposure the resistant cells accumulated soluble sugars, in particular fructose and sucrose, with a limited increase in cell osmolality. In contrast, the cell suspension that was unable to acclimate did not accumulate soluble sugars in response to the same cold treatment. To an extent similar to that induced after a cold acclimation, frost-hardiness of the cells increased after a 14-h incubation with specific soluble sugars such as sucrose, raffinose, fructose, and mannitol. Such hardening was also observed for long-term cultures in mannitol-enriched medium. This cryoprotective effect of sugars without exposure to lower temperatures was observed in both the resistant and the sensitive genotypes. Mannitol was one of the most efficient carbohydrates for the cryoprotection of eucalyptus. The best hardiness (a 2.7-fold increase in relative freezing tolerance) was obtained for the resistant cells by the cumulative effect of cold-induced acclimation and mannitol treatment. This positive effect of certain sugars on eucalyptus freezing tolerance was not colligative, since it was independent of osmolality and total sugar content.