Screening active components from Rubus amabilis for pancreatic ß-cells protection.

CONTEXT: Rubus species (Rosaceae) have been used in folk medicine to treat diabetes due to their hypoglycaemic activity. OBJECTIVE: To screen the active components that act as hypoglycaemic agents in Rubus amabilis Focke and the underlying mechanisms. MATERIALS AND METHODS: Aqueous stem extract of R. amabilis was incubated with MIN6 ß-cells, PBS was used as the blank control. Then the cells were washed, cell membrane-bound components were dissociated and identified by UPLC/MS. Total procyanidins (PCs) in R. amabilis was enriched and the cytotoxicity and anti-proliferation on ß-cell were evaluated by MTT assay. PCs at 25, 50, and 75 µg/mL was applied for 24 h to determine its effects on palmitate (PA)-induced apoptosis and GSIS. Western blotting was employed to detect the protein expression of PI3K/Akt/FoxO1 signalling. The antioxidant indices were also measured. RESULTS: ß-Cell membrane-bound components were identified as three procyanidin B dimers and a C trimer. PCs showed no significant cytotoxicity up to a concentrations of 100 µg/mL. PCs treatment reversed the elevated apoptosis rate and impaired GSIS induced by PA. PCs markedly decreased the intracellular ROS and MDA production and increased the SOD activity. Moreover, PCs promoted the phosphorylation of Akt and FoxO1, and regulated Pdx-1 and Bax expression in MIN6 cells. Discussion and conclusion: The active components that act as hypoglycaemic agents in R. amabilis are procyanidins, which protected MIN6 cells against PA-induced apoptosis by activating PI3K/Akt/FoxO1 signalling. These results indicate that ß-cell extraction, combined with UPLC/MS, is a valid method for screening antidiabetic components from herbal medicines.

Isolation, partial characterisation and immunomodulatory activities of polysaccharide from Morchella esculenta.

BACKGROUND: Polysaccharides extracted from fungi or algae have shown a variety of medical activities, and the exploitation of polysaccharides for application in pharmacy has been very promising. In this study, the immunomodulatory properties of polysaccharides from Morchella esculenta were investigated to identify immunostimulatory activity and potentially contribute to the therapeutic potential of Morchella esculenta. RESULTS: A water-soluble polysaccharide, MEP, was obtained from the fermentation broth of Morchella esculenta. Two fractions of this polysaccharide (MEP-I and MEP-II) were extracted and purified. High-performance gel permeation chromatography analysis showed the average molecular weights of two polysaccharides. Structural properties and compositions of these two fractions were examined by Fourier transform infrared and a high-performance liquid chromatography with an evaporative light scattering detector. Active experiments suggested that the MEP had typical immunostimulatory activity. CONCLUSION: These bioactivity tests showed that the polysaccharides from Morchella esculenta presented significant immune modulating activity, and this finding may offer the basis for the popular use of polysaccharides in functional foods or medicine.

[Optimization of culture conditions for production of extracellular polysaccharides by Morchella esculenta].

OBJECTIVE: To optimize the conditions for exopolysaccharides (EPS) production by Morchella esculenta GIM 5.69. METHODS: A Plackett-Burman design was used to evaluate the effects of variables factors. The optimal combined conditions for maximum polysaccharide production were further optimized by response surface methodology. RESULTS: The optimal fermentation conditions were determined as follows: glucose 30%, cultivation time 5.98 d, rotary speed 217.44 r/min. The average yield of polysaccharide validation experiments was 53.04%, the relative error was 1.64%. CONCLUSION: This research has the vital significance regarding the Morchella esculenta polysaccharide function of the product development and the application.

Separation mechanism of oleanane and ursane pentacyclic triterpenoid isomers by coordination chromatography.

This paper focuses on the study of the separation mechanism of oleanane and ursane pentacyclic triterpenoid isomers by a coordination chromatographic method. Based on the calculation analysis, beta-cyclodextrin (beta-CD) and its derivatives were selected as the suitable agents. The experimental results showed that the resolution of madecassoside isomers with the addition of glucosyl-beta-cyclodextrin (Glu-beta-CD) to the mobile phase (11.95) was higher than that of the addition of beta-CD (9.61) or dimethyl-beta-cyclodextrin (DM-beta-CD) (9.89). The formation of 1:1 inclusion complexes was assumed. The apparent formation constants (K(F)) of pentacyclic triterpenes with beta-CD were determined by HPLC method. For asiaticoside-B, the K(F) value with the addition of Glu-beta-CD (2 534 L/mol) was larger than that with the addition of beta-CD (1 467 L/mol) or DM-beta-CD (1 373 L/mol). According to the infrared characteristic absorbing peaks and simulation, the methyl part of asiaticoside-B might enter into beta-CD cavity while the carbonyl group of asiaticoside-B might not enter into the cavity of beta-CD, and the large glycoside parts were kept outside, forming hydrogen-bond interaction with the exterior of beta-CD cavity. The results of the separation of oleanane and ursane pentacyclic triterpenoid isomers by coordination chromatography indicated that, the separation mechanism might be attributed to the steric differences (the place of methyl group in E cyclic), which lead to different chromatographic behaviors.

Separation rule of oleanane and ursane pentacyclic triterpenoids isomers from nature plants by coordination chromatography.

Many of oleanolic and ursolic pentacyclic triterpenoid isomers generally coexist. There is a small difference in their structures. Based on coordination chromatography theory, a reversed-phase high-performance thin-layer chromatography (HPLC) method has been investigated for improving the isomers' resolution by adding suitable agents in mobile phase, and the separation rule was summarized. With the calculation analysis, the space sizes of isomers were in the range of 3.77-5.65 Å. The total minimum energy in the inclusion of guest and ß-CD had the biggest reduction, compared with the energy in the simple mixture of guest and ß-CD (such as "asiaticoside-B" and "ß-CD," from 196.4406 to 95.0670 kJ mol(-1)). So, ß-CD (the cavity space size is in the range of 6.00-6.50 Å) and its derivatives were selected as the suitable agents. The experiment results showed that the resolution might be improved by adding the hydrophilic ß-CD derivatives in mobile phase, such as Glu-ß-CD, when the isomer structures carry big hydrophilic groups.

[Investigation on the convertion between ginkgolide B and its derivative].

A derivative decomposed from ginkgolide B (GB) was isolated by semi-preparative chromatography. The results of the high performance liquid chromatography (HPLC) showed that the retention time of the derivative was approximately three times of that of GB with ultraviolet detection (UV), and the derivative can not exist independently without the presence of GB. The UV spectrum showed that the lamdamax of the derivative was 212.1 nm and the epsilonmax was 2.29 x 10(4), which were approximately 100 times higher than those of GB. This means a new conjugation bond has been formed and that means that the conjugation bond's pii-pi* electron jump occured in the derivative. Liquid chromatography-mass spectrometry showed the molecular ion peaks of the derivative in the positive mode and negative mode were m/z 429.1 (M + Na)+ and m/z 405.2 (M-H)-, respectively. The relative molecular mass of the derivative is 406.2. The derivative showed the same mode of fragment ion as GB in the mass spectrum, which might be due to the loss of a H2O from GB. Thermal stability of GB was greater than that of the derivative. They were both easily to be dissolved in dilute alkali. When the pH gradually increases, the ring-opening speed of the derivative is higher than that of GB. The derivative was obviously affected by solvent and higher temperatures. When GB was dissolved in PEG, the peak of the derivative disappeared at 50 degrees C for 15 h or at 120 degrees C for 4 h. Besides the main peak of GB, there appeared a small peak with a retention time of 1.2-3.0 min after heating 4 h at 120 degrees C. The result showed that the derivative was in a high energy state, and GB had a better stabilization. They coexisted and converted to each other. Under some specific conditions, the derivative could all convert to GB.

[Separation and determination of madecassic acid in triterpenic genins of Centella asiatica by high performance liquid chromatography using beta-cyclodextrin as mobile phase additive].

Centella asiatica (L. ) Urban is a tropical medicinal plant with a long history of therapeutic uses. Madecassic acid and terminolic acid, which are a pair of structural isomers, are two constituents of Centella asiatica. A method using reversed-phase high performance liquid chromatography in which beta-cyclodextrin (beta-CD) was the additive in mobile phase has been developed for separation of the structural isomers and determination of madecassic acid. The two compounds can be isolated with high resolution on a C18 reversed-phase column with the addition of beta-CD in the mobile phase. The separation mechanism of the isomers was discussed. It was assumed that the separation of the isomers might have been resulted from different inclusion forces of complexes with beta-CD. The effects of beta-CD concentration and the pH of mobile phase on resolution were investigated. It was found that the resolution of the isomers increased with the increase of beta-CD concentration when the mobile phase consisted of methanol-water (65 : 35, v/v) at pH 4. The correlation coefficient (r2) of the linear calibration curve between peak area and concentration of madecassic acid was 0.998 9 in the range of 0.1 - 5.0 g/L. This method was successfully used to determine the madecassic acid in triterpenic genins of Centella asiatica.