Functional characterization of CYP81C16 involved in the tanshinone biosynthetic pathway in Salvia miltiorrhiza.

Danshen, the dried roots and rhizomes of Salvia miltiorrhiza Bunge (S. miltiorrhiza), is widely used in the treatment of cardiovascular and cerebrovascular diseases. Tanshinones, the bioactive compounds from Danshen, exhibit a wide spectrum of pharmacological properties, suggesting their potential for future therapeutic applications. Tanshinone biosynthesis is a complex process involving at least six P450 enzymes that have been identified and characterized, most of which belong to the CYP76 and CYP71 families. In this study, CYP81C16, a member of the CYP71 clan, was identified in S. miltiorrhiza. An in vitro assay revealed that it could catalyze the hydroxylation of four para-quinone-type tanshinones, namely neocryptotanshinone, deoxyneocryptotanshinone, and danshenxinkuns A and B. SmCYP81C16 emerged as a potential broad-spectrum oxidase targeting the C-18 position of para-quinone-type tanshinones with an impressive relative conversion rate exceeding 90%. Kinetic evaluations andin vivo assays underscored its highest affinity towards neocryptotanshinone among the tested substrates. The overexpression of SmCYP81C16 promoted the accumulation of (iso)tanshinone in hairy root lines. The characterization of SmCYP81C16 in this study accentuates its potential as a pivotal tool in the biotechnological production of tanshinones, either through microbial or plant metabolic engineering.

SmDXS5, acting as a molecular valve, plays a key regulatory role in the primary and secondary metabolism of tanshinones in Salvia miltiorrhiza.

Red sage, the dry root and rhizome of the herbaceous plant Salvia miltiorrhiza Bunge, is widely used for treating various diseases. The low content of tanshinones (terpenoids) has always restricted development of the S. miltiorrhiza industry. Here, we found that SmDXS5, a rate-limiting enzyme-coding gene located at the intersection of primary and secondary metabolism, can effectively change the transcription level and secondary metabolome profile of hairy roots of S. miltiorrhiza, and significantly increase the content of tanshinones. Agrobacterium rhizogenes was used to infuse S. miltiorrhiza explants, and hairy roots of S. miltiorrhiza expressing the SmDXS5 gene were obtained successfully. We identified 39 differentially accumulated metabolites (DAMs) by metabolomics based on ultra-high performance liquid chromatography quadrupole exactive mass spectrometry and multivariate statistics. These DAMs might be key metabolites of SmDXS5 gene regulation. RNA sequencing was used to compare gene expression between the hairy roots of the SmDXS5 overexpressing group and the blank control (BC) group. Compared with the BC group, 18,646 differentially expressed genes were obtained: 8994 were upregulated and 9,652 downregulated. The combined transcriptome and metabolome analyses revealed that the mevalonate and methylerythritol phosphate pathways and synthase gene expression levels in the SmDXS5 overexpressing group were upregulated significantly, and the accumulation of tanshinone components was increased significantly, which promoted the process of glycolysis and promoted the transformation of carbohydrates to secondary metabolism. Moreover, the expression of SmPAL, the first rate-limiting enzyme gene of the phenylpropane pathway, decreased, reducing the accumulation of phenolic acid, another secondary metabolite. Therefore, SmDXS5 can be defined as a 'valve' gene, mainly responsible for regulating the distribution of primary and secondary metabolic flow of tanshinones in S. miltiorrhiza, and for other secondary metabolic pathways. The discovery of SmDXS5 and its molecular valve function in regulating primary and secondary metabolism will provide a basis for the industrial production of tanshinone components, and cultivation of high quality S. miltiorrhiza.

Preparation, structure characterization, and specific gut microbiota properties related to anti-hyperlipidemic action of type 3 resistant starch from Canna edulis.

Type 3 resistant starch (RS3) was developed from Canna edulis (Ce) native starch (NS) through dual enzymatic hydrolysis and recrystallization. Thereafter, the processed Ce-RS3 was subjected to systematic characterizations for its structural properties, anti-hyperlipidemic effect, and in vivo gut microbiota modulatory function. The Ce-RS3 content was increased to 49.11% after processing under optimal conditions. Compared with NS, Ce-RS3 maintained its B-type crystallization without introducing new chemical groups. Meanwhile, it displayed coarse surfaces, higher crystallinity, more ordered structures, and a higher proportion of chains with degree of polymerization (DP) 37-100. Ce-RS3 intervention significantly alleviated dyslipidemia in hyperlipidemic mice, which was associated with increased gut microbial diversity and unique microbial enrichment, potentially mediated by its fine structure. These observations are valuable for developing RS3 from C. edulis for prebiotics applications and support the potential strategy that utilizes well-designed RS to modulate specific bacterial populations to improve health.

Primary and secondary metabolites produced in Salvia miltiorrhiza hairy roots by an endophytic fungal elicitor from Mucor fragilis.

Salvia miltiorrhiza is one of the most commonly used medicinal materials in China. In recent years, the quality of S. miltiorrhiza has attracted much attention. Biotic and abiotic elicitors are widely used in cultivation to improve the quality of medicinal plants. We isolated an endophytic fungus, Mucor fragilis, from S. miltiorrhiza. We compared the effects of endophytic fungal elicitors with those of yeast extract together with silver ion, widely used together as effective elicitors, on S. miltiorrhiza hairy roots. Seventeen primary metabolites (amino acids and fatty acids) and five secondary metabolites (diterpenoids and phenolic acids) were analyzed after elicitor treatment. The mycelium extract promoted the accumulation of salvianolic acid B, rosmarinic acid, stearic acid, and oleic acid in S. miltiorrhiza hairy roots. Additionally, qPCR revealed that elicitors affect the accumulation of primary and secondary metabolites by regulating the expression of key genes (SmAACT, SmGGPPS, and SmPAL). This is the first detection of both the primary and secondary metabolites of S. miltiorrhiza hairy roots, and the results of this work should help guide the quality control of S. miltiorrhiza. In addition, the findings confirm that Mucor fragilis functions as an effective endophytic fungal elicitor with excellent application prospect for cultivation of medicinal plants.

A specific gut microbiota and metabolomic profiles shifts related to antidiabetic action: The similar and complementary antidiabetic properties of type 3 resistant starch from Canna edulis and metformin.

The relationship between gut microbiota and type 2 diabetes mellitus (T2DM) has drawn increasing attention, and the benefits of various treatment strategies, including nutrition, medication and physical exercise, maybe microbially-mediated. Metformin is a widely used hypoglycemic agent, while resistant starch (RS) is a novel dietary fiber that emerges as a nutritional strategy for metabolic disease. However, it remains unclear as to the potential degree and interactions among gut microbial communities, metabolic landscape, and the anti-diabetic effects of metformin and RS, especially for a novel type 3 resistant starch from Canna edulis (Ce-RS3). In the present study, T2DM rats were administered metformin or Ce-RS3, and the changes in gut microbiota and serum metabolic profiles were characterized using 16S-rRNA gene sequencing and metabolomics, respectively. After 11 weeks of treatment, Ce-RS3 exhibited similar anti-diabetic effects to those of metformin, including dramatically reducing blood glucose, ameliorating the response to insulin resistance and glucose tolerance test, and relieving the pathological damage in T2DM rats. Interestingly, the microbial and systemic metabolic dysbiosis in T2DM rats was effectively modulated by both Ce-RS3 and, to a lesser extent, metformin. The two treatments increased the gut bacterial diversity, and supported the restoration of SCFA-producing bacteria, thereby significantly increasing SCFAs levels. Both treatments simultaneously corrected 16 abnormal metabolites in the metabolism of lipids and amino acids, many of which are microbiome-related. PICRUSt analysis and correlation of SCFAs levels with metabolomics data revealed a strong association between gut microbial and host metabolic changes. Strikingly, Ce-RS3 exhibited better efficacy in increasing gut microbiota diversity with a peculiar enrichment of Prevotella genera. The gut microbial properties of Ce-RS3 were tightly associated with the T2DM-related indexes, showing the potential to alleviate diabetic phenotype dysbioses, and possibly explaining the greater efficiency in improving metabolic control. The beneficial effects of Ce-RS3 and metformin might derive from changes in gut microbiota through altering host-microbiota interactions with impact on the host metabolome. Given the complementarity of Ce-RS3 and metformin in regulation of gut microbiota and metabolites, this study also prompted us to suggest possible "Drug-Dietary fiber" combinations for managing T2DM.

Research progress in the relationship between type 2 diabetes mellitus and intestinal flora.

Type 2 diabetes mellitus (T2DM) is a common clinical chronic disease, while its pathogenesis is still inconclusive. Intestinal flora, the largest micro-ecological system in the human body, is involved in, meanwhile has a major impact on the body's material and energy metabolism. Recent studies have shown that in addition to obesity, genetics, and islet dysfunction, the disturbance of intestinal flora may partly give rise to diabetes. In this paper, we summarized the current research on the correlation between T2DM and intestinal flora, and concluded the pathological mechanisms of intestinal flora involved in T2DM. Moreover, the ideas and methods of prevention and treatment of T2DM based on intestinal flora were proposed, providing theoretical basis and literature reference for the treatment of T2DM and its complications based on the regulation of intestinal flora.

[Therapeutic effect of anti-CXCL1 neutralizing antibody on acute ulcerative colitis in mice].

OBJECTIVE: To evaluate the therapeutic effect of CXCL1 monoclonal antibody on dextra sulfate sodium (DSS)-induced acute ulcerative colitis (UC) in mice, and to elucidate its effect on the expressions of TNF-α, IFN-γ, IL-17 and IL-10 as well as neutrophil infiltration.
 Methods: Female BALB/c mice were randomly divided into a normal group (DSS-), a disease group (DSS+saline), an anti-CXCL1 antibody group (DSS+anti-CXCL1 Ab) and a treatment control group (DSS+IgG Ab). The DSS+saline, DSS+anti-CXCL1 Ab and DSS+anti-CXCL1 Ab groups were given 3.5% DSS solution as drinking water to induce acute intestinal inflammation, while the normal control was given distilled water freely. The DSS+anti-CXCL1 Ab mice were intraperitoneal injected with anti-CXCL1 Ab (4 mg/kg) on the 3rd and 6th day. Same amount of rat IgG Ab was given in the DSS+IgG Ab group. The normal group and the disease group were injected with 0.9% sodium chloride solution. The value of disease activity index (DAI) and the injury of colorectal tissue were measured. The levels of TNF-α, IFN-γ, IL-10 and IL-17 in colonic tissues of mice were detected by RT-PCR. Myeloperoxidase (MPO), a specific marker of neutrophils was measured by immunohistochemistry.
 Results: Compared with the normal control group, DAI score and colorectal injury score in the disease group were significantly increased, but the DAI and colorectal in the mice with acute ulcerative colitis tissue damage score were significantly reduced after anti-CXCL1 Ab intervention. Compared with the normal control group, mRNA levels of TNF-α, IFN-γ and IL-17 in the colorectal tissues were significantly elevated (P<0.05) in the disease group while the IL-10 was decreased; these effects were attenuated by anti-CXCL1 Ab intervention (P<0.05). Immunohistochemistry showed that the infiltration of neutrophils (MPO+) in the colon tissue was significantly increased in the disease group, while the anti-CXCL1 Ab treatment could significantly reduce the neutrophil infiltration in colon tissue (P<0.05).
 Conclusion: Anti-CXCL1 Ab relieves the progression of DSS-induced acute ulcerative colitis by suppressing proinflammatory expression and neutrophil infiltration.