An efficient combination of supercritical fluid extraction and high-speed counter-current chromatography to extract and purify homoisoflavonoids from Ophiopogon japonicus (Thunb.) Ker-Gawler

Supercritical fluid extraction (SFE) was used to extract homoisoflavonoids from Ophiopogon japonicus (Thunb.) Ker-Gawler. The optimization of parameters was carried out using an orthogonal test L-9 (3)(4) including pressure, temperature, dynamic extraction time and the amount of modifier. The process was then scaled up by 100 times with a preparative SFE system under the optimized conditions of 25 MPa, 55 degrees C, 4.0 h and 25% methanol as a modifier. Then crude extracts were separated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/ACN/water (1.8:1.0:1.0:1.2:1.0 v/v). There three homoisoflavonoidal compounds including methylophiopogonanone A 6-aldehydo-isoophiopogonone A, and 6-formyl-isoophiopogonanone A, were successfully isolated and purified in one step. The collected fractions were analyzed by HPLC. In each operation, 140 mg crude extracts was separated and yielded 15.3 mg of methylophiopogonanone A (96.9% purity), 4.1 mg of 6-aldehydo-isoophiopogonone A (98.3% purity) and 13.5 mg of 6-formyl-isoophiopogonanone A (97.3% purity) respectively. The chemical structure of the three homoisoflavonoids are identified by means of ESI-MS and NMR analysis.

The antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo

To investigate the antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo, six bromophenol derivatives 6-(2,3-dibromo-4,5-dihydroxybenzyl)-2,3-dibromo-4,5-dihydroxy benzyl methyl ether (1), (+)-3-(2,3-dibromo-4,5-dihydroxyphenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroisobenzofuran (2), 3-bromo-4-(2,3-dibromo-4,5-dihydroxybenzyl)-5-methoxymethyl-pyrocatechol (3), 2,2',3,3'-tetrabromo-4,4',5,5'-tetrahydroxy-diphenylmethane (4), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (5), 2,2',3-tribromo-3',4,4',5-tetrahydroxy-6'-ethyloxymethyldiphenylmethane (6) were isolated from brown alga Leathesia nana, and their cytotoxicity were tested by MTT assays in human cancer cell lines A549, BGC-823, MCF-7, B16-BL6, HT-1080, A2780, Bel7402 and HCT-8. Their inhibitory activity against protein tyrosine kinase (PTK) with over-expression of c-kit was analyzed also by ELISA. The antitumor activity of ethanolic extraction of Leathesia nana (EELN) was evaluated on S-180-bearing mice. All compounds showed very potent cytotoxicity against all of the eight cancer cell lines with IC50 below 10 mu g/mL. In PTK inhibition study, all bromophenol derivatives showed moderate inhibitory activity and compounds 2, 5 and 6 showed significant bioactivity with the inhibition ratio of 77.5%, 80.1% and 71.4%, respectively. Pharmacological studies reveal that EELN could inhibit the growth of Sarcoma 180 tumor and increase the indices of thymus and spleen to improve the immune system remarkably in vivo. Results indicated that the bromophenol derivatives and EELN can be used as potent antitumor agents for PTK over-expression of c-kit and considered in a new therapeutic strategy for treatment of cancer.

Effect of ethanol extract of alga Laurencia supplementation on DNA oxidation and alkylation damage in mice

Laurencia terpenoid extract (LET) had been extracted from the red alga Laurencia tristicha. The study is to investigate the effects of LET supplementation on DNA oxidation and alkylation damages in mice. Forty healthy kunming mice weighing between 18g and 25g were randomly assigned into 4 groups, each consisting of ten animals. The mice were orally intubated respectively for 60 days with the designed concentrations of LET (25, 50, 100 mg/kg b.w.) for three exposed groups and salad oil (0.2 ml) for the blank group. Food and water were free for the animals. Mice in the blank and exposed groups were sacrificed after the last treatment and the blood of each animal was quickly taken for further experiments. The spontaneous and oxidized DNA damages of peripheral lymphocytes induced by H2O2 were analysed by SCGE. O-6-Methy-guanine (O-6-MeG) was measured by high performance capillary zone electrophoresis. There was no significantly difference in DNA spontaneous damage on peripheral lymphocytes of all the mice. The oxidative DNA damage in the 50 mg/Kg body weight supplement group are 286AU with the oxidation of 10 mu mol/L H2O2, significantly lower than the blank group 332AU (p<0.05). The contents of O-6-MeG in plasma in the 50mg/kg b.w. and 100mg/kg b.w. supplement group were 1.50 mu mol/L andl.88 mu mol/L, significantly lower than that of the blank group, which was 2.89 mu mol/L(p<0.05). The results from the present study indicated that the LET were rich in terpenoids and safety to be taken orally and it could improve antioxidative and decrease DNA damage effectively.

Inhibition of bromophenols against PTP1B and anti-hyperglycemic effect of Rhodomela confervoides extract in diabetic rats

Protein tyrosine phosphatase 1B (PTP1B) plays an important role as a negative regulator in insulin signaling pathways. PTP1B is an effective target for the treatment of type 2 diabetes mellitus. Four bromophenol derivatives from red algae Rhodomela confervoides, 2,2',3,3'-tetrabromo-4,4',5,5'-tetra-hydroxydiphenyl methane (1), 3-bormo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl) pyrocatechol (2), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (3) and 2,2',3-tribromo-3',4,4',5-tetrahydroxy-6'-ethyloxy-methyldiphenylmethane (4) showed significant inhibitory activity against PTP1B (IC50 were 2.4, 1.7, 1.5 and 0.84 mu mol/L, respectively) as potential therapeutical agents for the treatment of type 2 diabetes mellitus. The anti-hyperglycemic effects of the ethanol extracts from R. confervoides on streptozotocin-induced diabetes (STZ-diabetes) in male Wistar rats fed with high fat diet were investigated. The STZ-diabetic rats treated with medium-dose and high-dose alga extracts showed remarkable reductions in fasting blood glucose (FBG) as compared with the STZ-diabetic control. The results indicate that the in vivo anti-hyperglycemic activity of the R. confervoides extracts can be partially attributed to the inhibitory actions against PTP1B of the bromophenol derivatives and that may be of clinical importance in improving the management of type 2 diabetes mellitus.

Mass spectrometric identification of multihydroxy phenolic compounds in Tibetan herbal medicines

A highly selective and accurate method based on derivatization with dansyl chloride coupled with liquid chromatography-mass spectrometry has been developed for identification of natural pharmacologically active phenolic compounds in extracts of Lomatogonium rotatum plants (Tibetan herbal medicine) obtained by solid-phase extraction. The number of hydroxyl groups on the dansylated phenols was estimated by LC-MS-MS analysis in positive-ion mode. Dansyl derivatization of the compounds introduced basic secondary nitrogen into the phenolic core structures and this was readily ionized when acidic HPLC mobile phases were used. MS fragmentation of the derivatives generated intense protonated molecular ions of m/z [MH](+) (phenol aglycones were transformed into the corresponding free phenols by cleavage of an aglycone bond). Collision-induced dissociation of the protonated molecule generated characteristic product ions of m/z 234 and 171 corresponding to the protonated 5-(dimethylamino)naphthalene sulfoxide and 5 -(dimethylamino) naphthalene moieties, respectively. Selected reaction monitoring based on the m/z [MH](+) to 234 and 171 transitions was highly specific for these phenolic compounds. Characteristic ions with m/z values of [MH - 234](+), [MH 2 x 234](+), and [MH - 3 x 234](+) were of great importance for estimation of the presence of multihydroxyl groups on the phenolic backbone.

Capillary electrophoretic analysis of pharmacologically active xanthone compounds from Swertia przewalskii pissjauk extract

Pharmacologically active xanthone compounds isolated from Swertia przewalskii pissjauk were well separated by capillary electrophoresis (CE) within 5 min, using a running buffer of 25 mM disodium tetraborate at pH 9.0. Quantitative determination was shown to be possible because regression equations revealed a linear relationship between the peak area of each constituent and its concentration, with correlation coefficients of 0.9972-0.9994. The relative standard deviations were between 0.44%-0.73% for migration times and 2.52%-4.28% for peak areas. The dissociation constant of 1,7-O-beta-D-glucopyranosyl-8-hydroxy-3,7-dimethoxyxanthone, 1,8-dihydroxy-3, 7-dimethoxy-xanthone and 1,7-dihydroxy-3,8-dimethoxyxanthone were also measured by the CE method, giving a value of 9.04, 8.94, and 8.59, respectively.

Total analytical method for Radix astragali extract using two-binary multi-segment gradient elution liquid chromatography

There is an urgent need for thorough analysis of Radix astragali, a widely used Chinese herb, for quality control purposes. This paper describes the development of a total analytical method for Radix astragali extract, a multi-component complex mixture. Twenty-four components were separated step by step from the extract using a series of isocratic isopropanol-methanol elutions, and then 42 components were separated similarly using methanol-water elutions. Based on the log k(w) and -S of the 66 components obtained from the above procedure and the optimization software developed in our laboratory, an optimum elution program consisting of seven methanol-water segments and four isopropanol-methanol segments was developed to finish the task of analyzing the total components in a single run. Under optimized gradient conditions, the sample of Radix astragali extract was analyzed. As expected, most of the components were well separated and the experimental chromatogram was in a good agreement with the predicted one.

New method for analysis of Chinese herbal complex prescription and its application

Traditional Chinese medicine (TCM) is a great treasure of China, the analysis of which is an arduous task. The viewpoint that all chemical constituents of Chinese herbal complex prescription should be analyzed as a black box is elucidated for the first time. Intelligent multi-mode multi-column chromatographic system (IMMCC) with its hybrids is the basic method and HPLC Unified Method is the breakthrough for the black box analysis. Dang-Gui-Bu-Xue-Tang was selected as a typical TCM and a systematic separation method from non-aqueous mobile phase to pure water mobile phase was put forward in order to convert unknown sample to known sample. The a, c values and UV spectra of 66 components of Astragalus, 78 components of Angelica and 71 components of Dang-Gui-Bu-Xue-Tang were obtained. Intelligent optimization and peak identification method and software for complex samples were developed and the optimum multi-step multi-binary gradient curve of mobile phase for Astragalus was ascertained. The maximum error and minimum error of predicted retention time for all components of Astragalus are 8.62% and 0.05% respectively. All components of Astragalus were compared with those of Angelica and it is found that many components of Astragalus are the same as those of Angelica, while the contents of these components are different. Many components of Dang-Gui-Bu-Xue-Tang are also the same as those of Astragalus and Angelica with different contents.

Discriminating the xanthones in an extract of Swertia franchetiana by retention parameters

The quantitative structure-retention relationship is one of the most actively studied topics in the field of chromatography. In this paper, retention parameters of components were used to discriminate the xanthones in a methanol extract of Swertia franchetiana. The extract was analysed by HPLC under two different multistage linear gradient conditions and the retention parameters calculated from these retention data. It was found that the retention parameters of xanthones are in a specific region in the plot of log k(w) vs. S and the xanthones in the extract could be distinguished from other components by this feature. Furthermore, xanthone aglycones and xanthone glucosides could also be discriminated by retention parameters. Copyright (C) 2005 John Wiley Sons, Ltd.

Herbal supplements: Recent findings in exercise, health and weight loss

Natural herbs have been in use for weight loss purposes since history began. However, the current global obesity epidemic and the rise in obesity-related chronic diseases, including type-II diabetes and cancer, have highlighted the need for novel and effective approaches for herbal remedies. Whilst the popularity of several prescribed and non-prescribed slimming aids and herbal plant supplements have been marketed for their weight loss efficacy, single and multi-ingredient herbal supplements are still being investigated for their single or combined weight loss benefits. Limited research have highlighted an interesting efficacy for several popular herbal plant supplements including caffeine and capsaicin, Ayurvedic preparations and herbal teas, resulting in various degrees of effectiveness including thermogenic, appetite control and psychological benefits such as mood state. Recent research has suggested acute augmented weight-loss effects of combining herbal ingestion with exercise. For example, ingesting green tea, yerba mate and/or caffeine have been shown to increase metabolic rate, and augmented fatty acid metabolism and to increase energy expenditure from fatty acid sources during exercise with various intensities, particularly at low and moderate intensities. Other promising weight-loss effects have also been also reported for combining exercise with multi-ingredient herbal supplements, particularly those that are rich in phytochemicals and caffeoyl derivatives. Combining herbal ingestions with exercise still require further research in order to establish the supplementation most effective protocols in terms of dosage and timing, and to determine the long-term benefits, particularly those related to exercise protocols, and the long term adherence to sustain the weight loss outcomes.

Herbal Dietary Supplements: Safety, Efficacy, and Use by Breast Cancer Survivors

Gemstone Team IMAC (Integrative Medicine and Cancer)