A new sesquiterpene synthase catalyzing the formation of (R)-ß-bisabolene from medicinal plant Colquhounia coccinea var. mollis and its anti-adipogenic and antibacterial activities.

The sesquiterpene ß-bisabolene possessing R and S configurations is commonly found in plant essential oils with antimicrobial and antioxidant activities. Here, we report the cloning and functional characterization of a (R)-ß-bisabolene synthase gene (CcTPS2) from a Lamiaceae medicinal plant Colquhounia coccinea var. mollis. The biochemical function of CcTPS2 catalyzing the cyclization of farnesyl diphosphate to form a single product (R)-ß-bisabolene was characterized through an engineered Escherichia coli producing diverse polyprenyl diphosphate precursors and in vitro enzyme assay, indicating that CcTPS2 was a high-fidelity (R)-ß-bisabolene synthase. The production of (R)-ß-bisabolene in an engineered E. coli strain harboring the exogenous mevalonate pathway, farnesyl diphosphate synthase and CcTPS1 genes was 17 mg/L under shaking flask conditions. Ultimately, 120 mg of purified (R)-ß-bisabolene was obtained from the engineered E. coli, and its structure was elucidated by detailed spectroscopic analyses (including 1D and 2D NMR, and specific rotation). Four chimeric enzymes were constructed through domain swapping, which altered the product outcome, indicating the region important for substrate and product specificity. In addition, (R)-ß-bisabolene exhibited anti-adipogenic activity in the model organism Caenorhabditis elegans and antibacterial activity selectively against Gram-positive bacteria.

[Protective effect and mechanism of total lignans from Tibetan medicinal Herpetospermum seeds on carbon tetrachloride-induced liver fibrosis in rats].

This experiment was aimed to discuss the protective effect and mechanism of total lignans from Tibetan medicinal Herpetospermum seeds on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Forty-eight male Sprague-Dawley rats were randomly divided into the blank control group (G1), model group (G2), total lignans high, middle and low dose groups (400,200, and 100 mg•kg⁻¹â€¢d⁻¹)(G3/4/5) and the glycyrrhizin positive control group (25 mg•kg⁻¹â€¢d⁻¹)(G6), n=8 in each group. The rats in blank group received normal feeding; the rats in model group, total lignans low, middle and high dose groups and glycyrrhizic group were subcutaneously in jected with 3 mL•kg⁻¹ olive oilsolution containing 40%CCl4 every two or three days for Eight weeks. During the course, the rats inblank group and model group were orally administered with 2 mL normal saline, and the rats in total lignans groups and the glycyrrhizin positive control group received corresponding doses of drugs by intragastric administration. Eight weeks later, after the the last time modeling, the rats were sacrificed. Then the biochemical analysis was used to determine alanine aminotransferase(ALT), aspartate aminotransferase(AST), and alkaline phosphatase (ALP) levels in serum while enzyme-linked immuno sorbent assay(ELISA) was applied for detecting transforming growth factor ß1(TGF-ß1), hyaluronic acid(HA), hydroxy-proline(HYP) and superoxide dismutase(SOD) levels in serum. HE and Masson's trichrome stainings were conducted in liver tissues to observe the pathological variations and grades of hepatic fibrosis. The results showed that as compared with the model group, the levels of ALT, AST, ALP, TGF-ß1, HA, HYP and SOD in serum of total lignans groups were significantly decreased (P<0.05, P<0.01); levels of SOD in the liver tissue homogenate were increased(P<0.05, P<0.01); the pathological damage of the liver tissues were relieved (P<0.05, P<0.01), and liver fibrosis scores were decreased(P<0.05, P<0.01). The above experimentsindicated that total lignans from Tibetan medicinal Herpetospermum seeds can effectively reduce carbon tetrachloride-induced hepatic injury of rat liver fibrosis, reduce the degree of liver fibrosis, and its mechanism maybe associated with down-regulating TGF-ß1 expression.

Protective Effects of MDG-1, a Polysaccharide from Ophiopogon japonicus on Diabetic Nephropathy in Diabetic KKAy Mice.

Ophiopogon japonicus is a traditional Chinese medicine that might be effective for treating type 2 diabetes. Recent research confirmed that MDG-1, a polysaccharide from O. japonicas, activates the PI3K/Akt signaling pathway and improves insulin sensitivity in a diabetic KKAy mouse model, but little is known about its effects on diabetic nephropathy. In this study, KKAy mice were orally administered distilled water (control group), MDG-1, or rosiglitazone for 12 weeks. Blood glucose levels were tested every two weeks for the fed mice. At 6 and 12 weeks, blood samples were collected for biochemical examination. At the end of the experiment, all kidney tissues were collected for histological examination and western blot analysis. Results show that MDG-1 (300 mg/kg) significantly decreased the levels of blood glucose, triglycerides, blood urine nitrogen and albumin, and significantly inhibited the expression of transforming growth factor-beta 1 and connective tissue growth factor. Moreover, MDG-1 could alleviate glomerular mesangial expansion and tubulointerstitial fibrosis in the diabetic mice, as confirmed by histopathological examination. These data indicated that MDG-1 ameliorates renal disease in diabetic mice by reducing hyperglycemia, hyperinsulinemia, and hyperlipidemia, and by inhibiting intracellular signaling pathways.

[Effect of MDG-1, a polysaccharide from Ophiopogon japonicas, on diversity of lactobacillus in diet-induced obese mice].

For understanding the effect of MDG-1, a water-soluble ß-D-fructan polysaccharide from Ophiopogon japonicas, on intestinal microecological balance, especially on the changes of lactobacillus, sixty 8-week-old male C57BL/6J mice were given a high-fat diet for six weeks and were also gavaged with saline once a day simultaneously. Then the mice which is below 30 grams or dropped more than 10% through lavage were eliminated and the rest were randomly divided into four groups: diet-induced obese (DIO) model group (n = 12, gavaged with saline), low-dose MDG-1 group (n = 12, gavaged with MDG-1, 75 mg · kg(-1)) , medial-dose MDG- 1 group (n = 12, gavaged with 150 mg · kg(-1)), and high-dose MDG-1 group (n = 12, gavaged with 300 mg · kg(-1)) according to the weight and blood glucose; the model group and MDG-1 group were placed on a high-fat diet while the normal control group (n = 12, gavaged with saline) were kept on a low-fat diet through the experiment. After 12-weeks of treatment, feces samples were collected and cultured for intestinal microecological balance analysis. Then the intestinal probiotics were cultured through traditional methods combined with modified gradient gel electrophoresis (DGGE) method. The changes of lactobacillus in each treatment group were also detected by a statistical analysis of the total number of the intestinal flora. We have established the phylogenetic tree by 16S rDNA sequencing and use some molecular identification methods such as PCR-DGGE to analyse the changes of the dominant bacteria floras, and also get the pure culture. In conclusion, different concentrations of MDG-1 can increase the number of the intestinal probiotics, especially Taiwan lactobacillus and Lactobacillus murinus, and improve their diversity and promote proliferation in a dose-dependent way.

[Observation on therapeutic effect of blood-letting puncture combined with red-hot needle therapy on knee osteoarthritis].

OBJECTIVE: To observe the therapeutic effect of blood-letting puncture combined with red-hot needle therapy on knee osteoarthritis. METHODS: One hundred and twenty-nine cases were randomly divided into a bloodletting puncture plus red-hot needle therapy group (n=73) and a routine acupuncture group (n=56). The blood letting puncture plus red-hot needle therapy group were treated by blood-letting puncture at Weizhong (BL 40), and red-hot needle pricking Heding (EX-LE 2), Dubi (ST 35), Xiyan (EX-LE 5), Yinlingquan (SP 9), Yanglingquan (GB 34), Xuanzhong (GB 39) and Ashi points, twice each week, 4 times constituting one course. The routine acupuncture group were treated by routine acupuncture at the same points as those for red-hot needle pricking, once each day, two weeks constituting one course. RESULTS: After treatment for 2 courses, the joint pain score and the illness serious index were 2.68+/-0.88 and 4.25+/-1.02, and 4.68+/-1.89 and 7.46+/-2. 13 in the two groups, respectively, with very significant differences before and after treatment in the two groups (P<0.01), the former being better than the later (P<0.05). The clinical cured rate and the total effective rate were 37.0% and 94.5% in the blood-letting puncture plus red-hot needle therapy group and 19.6% and 89.3% in the routine acupuncture group, with a very significant difference between the two groups(P<0. 01). CONCLUSION: Blood-letting puncture combined with red-hot needle therapy has obvious therapeutic effect on knee osteoarthritis.