Enzyme-extracted raspberry pectin exhibits a high-branched structure and enhanced anti-inflammatory properties than hot acid-extracted pectin.

To characterize the structure of purified raspberry pectin and discuss the impact of different extraction methods on the pectin structure, raspberry pectin was extracted by hot-acid and enzyme method and purified by stepwise ethanol precipitation and ion-exchange chromatography isolation. Enzyme-extracted raspberry pectin (RPE50%-3) presented relatively intact structure with molecular weight of 5 × 104 g/mol and the degree of methylation was 39%. The 1D/2D NMR analysis demonstrated RPE50%-3 was a high-branched pectin mainly containing 50% homogalacturonan, 16% branched α-1,5-arabinan and α-1,3-arabinan, 18% ß-1,4-galactan and ß-1,6-galactan. Acid-extracted raspberry pectin (RPA50%-3) contained less arabinan than RPE50%-3. Moreover, RPE50%-3 inhibited the nitric oxide (NO), TNF-α, IL-6 production of lipopolysaccharide-induced macrophages by 67%, 22% and 46% at the dosage of 200 ug/mL, while the inhibitory rate of RPA50%-3 were 33%, 9%, and 1%, respectively. These results suggested that enzyme-extracted raspberry pectin contained more arabinan sidechains and exhibited better immunomodulatory effect.

Structure-activity relationship of Citrus segment membrane RG-I pectin against Galectin-3: The galactan is not the only important factor.

Rhamnogalacturonan I (RG-I) pectin are regarded as strong galectin-3 (Gal-3) antagonist because of galactan sidechains. The present study focused on discussing the effects of more structural regions in pectin on the anti-Gal-3 activity. The water-soluble pectin (WSP) recovered from citrus canning processing water was categorized as RG-I pectin. The controlled enzymatic hydrolysis was employed to sequentially remove the α-1,5-arabinan, homogalaturonan and ß-1,4-galactan in WSP. The Gal-3-binding affinity KD (kd/ka) of WSP and debranched pectins were calculated to be 0.32 µM, 0.48 µM, 0.56 µM and 1.93 µM. Moreover, based on the more sensitive cell line (MCF-7) model, the IC30 value of WSP was lower than these of modified pectins, indicating decreased anti-Gal-3 activity. Our results suggested that the total amount of neutral sugar sidechains, the length of arabinan and cooperation between HG and RG-I played important roles in the anti-Gal-3 activity of WSP, not the Gal/Ara ratio or RG-I/HG ratio. These results provided a new insight into structure-activity relationship of citrus segment membrane RG-I as a galectin-3 antagonist and a new functional food.

LBP-4a improves insulin resistance via translocation and activation of GLUT4 in OLETF rats.

Lycium barbarum polysaccharide (LBP) has been shown to ameliorate insulin resistance, but the identification of compounds from LBP and the mechanisms have not been clarified. In this study, LBP-4a was purified from Lycium barbarum by DEAE cellulose and Sephadex G-100 column chromatography, and the effects of LBP-4a on insulin resistance were investigated. The results indicated that LBP-4a caused translocation of the glucose transporter isoform 4 (GLUT4) to the cell surface, which in turn stimulated glucose uptake, and the effect was sensitive to wortmannin, an inhibitor of phosphoinositol 3-kinase (PI3-K), and SB203580, an inhibitor of p38 mitogen activated protein kinase (p38 MAPK (α, ß)). Furthermore, the effects of LBP-4a on p38 MAPK activities were abrogated by pretreatment of rat adipocytes using SB203580. In summary, LBP-4a improved insulin resistance via translocation and activation of GLUT4 in OLETF rats, and the activation of PI3-K and p38 MAPK contributed to these effects.

Establishment of a pancreatic ß cell proliferation model in vitro and a platform for diabetes drug screening.

Diabetes, a disease resulting from loss of functional ß cells, is globally an increasingly important condition. Based on the islet-differentiation ability of ductal epithelial cells and stimulating ß cell proliferation ability of the Reg Iα gene, we aimed to establish an in vitro pancreatic ß cell proliferation model for screening therapeutic drugs of diabetes in the future. Pancreatic ductal epithelial cells were isolated from male Wistar rats, and induced to differentiate into pancreatic ß cells. Immunofluorescence staining assay, western blot, RT-PCR analysis, and dithizone staining were used to characterize the cells. Rat Reg Iα protein was transiently expressed in vitro by transfection of HEK 293 cells with the PCMV6-entry-REG Ia plasmid, and expression was verified by RT-PCR analysis, proliferation assay, and apoptosis assay. The pancreatic ß cell proliferation model was further validated by a proliferation assay using differentiated pancreatic ß cells treated with transfection supernatant. Finally, we have successfully established an in vitro pancreatic ß cells proliferation model using transiently expressed rat Reg Iα protein and differentiated pancreatic ß cells from pancreatic ductal epithelial cells. This model could be used as a platform to screen new drugs for islet neogenesis to cure diabetes, especially Chinese herbal drugs in the future.

Chlorogenic acid alters the biological characteristics of basophil granulocytes by affecting the fluidity of the cell membrane and triggering pseudoallergic reactions.

It is not clear whether pseudoallergic reactions are caused by similar mechanisms as type I allergic reactions. 3­Caffeoylquinic acid (chlorogenic acid) is an active ingredient in traditional Chinese medicines used for antibacterial, anti-inflammatory and cholagogic purposes. It is assumed to be the reason for the high allergic reaction rates associated with certain traditional Chinese medicine injection solutions. The aim of the present study was to investigate the possible mechanisms through which chlorogenic acid triggers pseudoallergic reactions. The fluidity of the cell membrane was investigated using fluorescence recovery after photobleaching. Western blot analysis was used to measure the phosphorylation levels of the Spleen tyrosine kinase (Syk) protein and Fluo­3/AM fluorescent probes were used to investigate the influx of calcium ions. In addition, fluorescence microscopy and phalloidin were used to determine F­actin depolymerization levels. The secretion rate of ß­hexosaminidase by RBL­2H3 cells clearly increased following treatment with chlorogenic acid and the levels of cytoskeletal disintegration were also markedly increased. Furthermore, we detected an increase in the intracellular calcium ion concentration along with distinct changes in Syk protein phosphorylation and cellular F­actin. These changes indicated that chlorogenic acid affected the restructuring of the cytoskeleton and played a role in cell degranulation. In conclusion, chlorogenic acid may lead to the aggregation of lipid rafts on the cell membrane surface by altering RBL­2H3 cell membrane fluidity, thus triggering Syk­related signal transduction and inducing a truncated type I like allergic reaction.

Antitumor activity of Portulaca oleracea L. polysaccharides against cervical carcinoma in vitro and in vivo.

Portulaca oleracea L. has been used as folk medicine in different countries to treat different ailments in humans. P. oleracea L. polysaccharide (POL-P), extracted from P. oleracea L., is found to have bioactivities such as hypoglycemic and hypolipidemic activities, antioxidant and antitumor activities. In our study, a water-soluble polysaccharide (POL-P3b) was successfully purified from Galium verum L. by DEAE cellulose and Sephadex G-200 column chromatography. To evaluate the anticancer efficacy and associated mechanisms of POL-P3b on cervical cancer in vitro and in vivo, we showed that treatment of HeLa cell with POL-P3b inhibited cell proliferation. In addition, POL-P3b significantly inhibited tumor growth in U14-bearing mice. Further analysis indicated that POL-P3b possesses the activity of inhibiting cervical cancer cell growth in vitro and in vivo at a concentration- and time-dependent manner, and the mechanisms were associated with Sub-G1 phase cell cycle arrest, triggering DNA damage and inducing apoptosis.

[Study of effects and mechanism of phytosterols on chronic abacterial prostatitis].

OBJECTIVE: To investigate the inhibitory effects of phytosterols on abacterial prostatitis and discuss the possible mechanism. METHOD: Xiaozhiling-induced chronic prostatitis model were used to observe the inhibitory effect of phytosterols on abacterial prostatitis. The changes of serum IL-2, IL-1beta and TNF-alpha were evaluated by enzyme-linked immunosorbent assay (ELISA). The expression of COX-2 and 5-LOX were evaluated by Western blot and immunohistochemistry. RESULT: Treated by phytosterols (150 mg x kg(-1)), the number of white blood cells in xiaozhiling-induced chronic abacterial prostatitis rats was obviously decreased, the density of lecithin corpuscle in prostatic secretion increased and closed to control group. The edema, inflammatory infiltration of prostate were partly recovered compared with model group. The proliferation of chronic prostatitis were obviously decreased in phytosterols groups compared with model group in histological sections. Phytosterols could obviously reduce the serum IL-1beta, TNF-alpha, prostate COX-2 and 5-LOX expression and improve IL-2 level. CONCLUSION: These results demonstrated that phytosterols had good therapeutic effects on chronic abacterial prostatitis. Participation of immune regulation and inhibiting COX-2 and 5-LOX expression may be the mechanisms of action.

[Anaphylactoid reaction induced by Qingkailing injection via basophils cells degranulation].

OBJECTIVE: To observe the effects of Qingkailing injection on RBL-2H3 cell degranulation and histamine release, and discuss the possible mechanism of anaphylactoid reaction induced by Qingkailing injection. METHOD: RBL-2H3 cells were incubated with Qingkailing injection for 30 min. Then the morphological changes of cells were observed by transmission electron microscopy. Cell degranulation rate was detected by Alcian blue dye assay, Annexin V binding assay and beta-hexosaminidase assay, and cell histamine release rate was detected by ELISA. RESULT: Different concentration of Qingkailing injection can induce the typical morphological changes in RBL-2H3 cell with degranulation. The rates of degranulation and histamine release in Qingkailing injection treated cells were significantly increased and dose-dependent. CONCLUSION: RBL-2H3 cell degranulation and histamine release can be induced by single administration of Qingkailing injection, and then induced anaphylactoid reaction, which may be one of the possible mechanisms of serious adverse induced by Qingkailing injection for the first administration in clinic.

Astilbic acid induced COLO 205 cell apoptosis by regulating Bcl-2 and Bax expression and activating caspase-3.

AIM: To investigate the effect of astilbic acid (3beta, 6beta-dihydroxyolean-12-en-27-oic acid, AA) on human colorectal carcinoma COLO 205 cell proliferation and apoptosis. METHODS: Proliferation of COLO 205 cells was measured by MTT assay. Content of DNA in COLO 205 cell was measured by modified diphenylamine assay. AA-induced morphological changes was observed with fluorescence microscope and transmission electron microscope. DNA fragmentation was visualized by agarose gel electrophoresis. Apoptosis rate and cell cycle distribution were determined by flow cytometric analysis. Expressions of Bcl-2 and Bax proteins were visioned by immunohistochemical analysis. The change of relative mitochondrial transmembrane potential (MTP) in COLO 205 cell was analyzed with FCM after rhodamine 123 staining. RESULTS: The IC50 (96 h) of AA for inhibiting COLO 205 cell proliferation was 61.56+/-0.34 micromol/L. AA induced a marked concentration- and time-dependent inhibition of COLO 205 cell proliferation and reduced the DNA content in COLO 205 cell. Cells treated with AA 64 micromol/L showed typical morphological changes of apoptosis and DNA ladder pattern. The cell cycle was arrested in G0/G1 phase, and the apoptosis rate was 28.25 % for COLO 205 cells treated with AA 64 micromol/L for 48 h. Meanwhile the expression of Bcl-2 protein was decreased while that of Bax was increased and relative MTP was decreased as well. DEVD-CHO 1 micromol/L could increase the viability of COLO 205 cells treated with AA for 48 h. CONCLUSION: AA showed potent inhibitory activity on COLO 205 cells proliferation, and could induce COLO 205 cells apoptosis through disturbing DNA replication, down-regulating Bcl-2 expression, and up-regulating Bax expression, lowering relative MTP, and activating caspase-3 pathway.