[Effect of salvia miltiorrhiza combined with roxadustat on wound healing of full-thickness skin defects in diabetic rats and its mechanism].

Objective: To explore the effect of salvia miltiorrhiza combined with roxadustat on wound healing of full-thickness skin defects in diabetic rats and its mechanism. Methods: This study was an experimental study. Twenty male 8-week-old Sprague-Dawley rats were used to successfully establish diabetic model, then full-thickness skin defect wounds on their backs were made. The rats were divided into normal saline group, roxadustat alone group, salvia miltiorrhiza alone group, and roxadustat+salvia miltiorrhiza group according to the random number table, with 5 rats in each group. Immediately after injury, the rats in normal saline group were given 5 mL normal saline by gavage, the rats in roxadustat alone group were given 1.5 mg/mL roxadustat suspension by gavage at 25 mg/kg, the rats in salvia miltiorrhiza alone group were given 18 mg/mL salvia miltiorrhiza suspension by gavage at 300 mg/kg, and the rats in roxadustat+salvia miltiorrhiza group were given 19.5 mg/mL roxadustat and salvia miltiorrhiza suspension at roxadustat 25 mg/kg and salvia miltiorrhiza 300 mg/kg. All were administered once a day for 2 weeks. The wounds at 0 (immediately), 4, 8, and 12 d after injury were observed, and the wound healing rates at 4, 8, and 12 d after injury were calculated (n=5). At 14 d after injury, abdominal aortic blood was collected, and hemoglobin, red cell count, and white blood cell count were detected (n=5). The wound tissue was collected for hematoxylin-eosin staining to observe inflammatory infiltration, skin tissue structure, and neovascularization, for Masson staining to observe the proportion of collagen fiber (n=3), for Western blotting to detect the protein expression levels of vascular endothelial growth factor (VEGF), CD31, interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and IL-1ß (n=3), and for immunohistochemical staining to determine the protein expression levels of epidermal growth factor receptor (EGFR), hypoxia-inducible factor 1α (HIF-1α), and proliferating cell nuclear antigen (PCNA), with sample number of 3. Results: From 0 to 12 d after injury, the wound areas of rats in 4 groups were gradually decreased. At 4 d after injury, the wound healing rates of rats in salvia miltiorrhiza alone group and roxadustat+salvia miltiorrhiza group were significantly higher than those in normal saline group and roxadustat alone group (P<0.05). At 8 d after injury, the wound healing rates of rats in roxadustat alone group and salvia miltiorrhiza alone group were significantly higher than the rate in normal saline group (P<0.05), and the wound healing rate of rats in roxadustat+salvia miltiorrhiza group was significantly higher than the rates in the other 3 groups (with P values all <0.05). At 12 d after injury, the wound healing rates of rats in roxadustat alone group, salvia miltiorrhiza alone group, and roxadustat+salvia miltiorrhiza group were significantly higher than the rate in normal saline group (P<0.05). At 14 d after injury, there were no statistically significant differences in the hemoglobin or red blood cell count of rats in 4 groups (P<0.05). The white blood cell count of rats in roxadustat alone group, salvia miltiorrhiza alone group, and roxadustat+salvia miltiorrhiza group were respectively (24.3±1.2)×109/L, (26.3±2.4)×109/L, and (15.0±0.7)×109/L, which were significantly lower than (33.8±2.7)×109/L in normal saline group (P<0.05); the white blood cell count of rats in roxadustat+salvia miltiorrhiza group was significantly lower than that in roxadustat alone group and salvia miltiorrhiza alone group (with P values both <0.05). At 14 d after injury, a large number of inflammatory cell infiltration, disordered skin tissue structure, and few new blood vessels were observed in the wounds of rats in normal saline group; while a small amount of inflammatory cell infiltration, tight skin tissue structure, and rich neovascularization were observed in the wounds of rats in the other 3 groups. There were no statistically significant differences in the proportion of collagen fiber of wounds in rats among the 4 groups (P>0.05). At 14 d after injury, the protein expression levels of VEGF and CD31 in the wound tissue of rats in roxadustat alone group, salvia miltiorrhiza alone group, and roxadustat+salvia miltiorrhiza group were significantly higher than those in normal saline group (P<0.05), the protein expression level of CD31 in the wound tissue of rats in roxadustat+salvia miltiorrhiza group was significantly higher than the levels in roxadustat alone group and salvia miltiorrhiza alone group (with P values both <0.05). At 14 d after injury, the protein expression levels of IL-6, TNF-α, and IL-1ß in the wound tissue of rats in roxadustat alone group, salvia miltiorrhiza alone group, and roxadustat+salvia miltiorrhiza group were significantly lower than those in normal saline group (P<0.05); the protein expression levels of IL-6 and IL-1ß in the wound tissue of rats in roxadustat+salvia miltiorrhiza group were significantly lower than those in roxadustat alone group and salvia miltiorrhiza alone group (P<0.05); the protein expression level of TNF-α in the wound tissue of rats in roxadustat+salvia miltiorrhiza group was significantly lower than that in salvia miltiorrhiza alone group (P<0.05). At 14 d after injury, the protein expression level of EGFR in the wound tissue of rats in roxadustat+salvia miltiorrhiza group was significantly higher than the levels in the other 3 groups (with P values all <0.05); the protein expression levels of HIF-1α in the wound tissue of rats in roxadustat alone group and roxadustat+salvia miltiorrhiza group were significantly higher than the level in normal saline group (P<0.05), and the protein expression level of HIF-1α in the wound tissue of rats in roxadustat+salvia miltiorrhiza group was significantly higher than that in salvia miltiorrhiza alone group (P<0.05); there were no statistically significant differences in the protein expression level of PCNA in the wound tissue of rats in 4 groups (P>0.05). Conclusions: Roxadustat combined with salvia miltiorrhiza can promote the wound healing of full-thickness skin defects in diabetic rats by promoting blood vessel regeneration and reducing inflammatory response.

Role and molecular mechanism of Salvia miltiorrhiza associated with chemical compounds in the treatment of diabetes mellitus and its complications: A review.

Diabetes mellitus (DM) is one of the most prevalent diseases worldwide, greatly impacting patients' quality of life. This article reviews the progress in Salvia miltiorrhiza, an ancient Chinese plant, for the treatment of DM and its associated complications. Extensive studies have been conducted on the chemical composition and pharmacological effects of S miltiorrhiza, including its anti-inflammatory and antioxidant activities. It has demonstrated potential in preventing and treating diabetes and its consequences by improving peripheral nerve function and increasing retinal thickness in diabetic individuals. Moreover, S miltiorrhiza has shown effectiveness when used in conjunction with angiotensin-converting enzyme inhibitors, angiotensin receptor blockers (ARBs), and statins. The safety and tolerability of S miltiorrhiza have also been thoroughly investigated. Despite the established benefits of managing DM and its complications, further research is needed to determine appropriate usage, dosage, long-term health benefits, and safety.

[Effects of organic fertilizer from traditional Chinese medicine residues on growth and soil microbial community of Salvia miltiorrhiza by metagenomic technique].

Soil microbiome is a key evaluation index of soil health. Previous studies have shown that organic fertilizer from traditional Chinese medicine(TCM)residues can improve the yield and quality of cultivated traditional Chinese medicinal materials. However, there are few reports on the effects of organic fertilizer from TCM residues on soil microbiome. Therefore, on the basis of evaluating the effects of organic fertilizer from TCM residues on the yield and quality of cultivated Salvia miltiorrhiza, the metagenomic sequencing technique was used to study the effects of organic fertilizer from TCM residues on rhizosphere microbiome community and function of cultivated S. miltiorrhiza. The results showed that:(1) the application of organic fertilizer from TCM residues promoted the growth of S. miltiorrhiza and the accumulation of active components, and the above-ground and underground dry weight and fresh weight of S. miltiorrhiza increased by 371.4%, 288.3%, 313.4%, and 151.9%. The increases of rosmarinic acid and salvianolic acid B were 887.0% and 183.0%.(2)The application of organic fertilizer from TCM residues significantly changed the rhizosphere bacterial and fungal community structures, and the microbial community composition was significantly different.(3)The relative abundance of soil-beneficial bacteria, such as Nitrosospira multiformis, Bacillus subtilis, Lysobacter enzymogenes, and Trichoderma was significantly increased by the application of organic fertilizer from TCM residues.(4)KEGG function prediction analysis showed that metabolism-related microorganisms were more easily enriched in the soil environment after organic fertilizer application. The abundance of functional genes related to nitrification and denitrification could also be increased after the application of organic fertilizer from TCM residues. The results of this study provide guidance for the future application of organic fertilizer from TCM residues in the cultivation of traditio-nal Chinese medicinal materials and enrich the content of green cultivation technology of traditional Chinese medicinal materials.

Diterpenoid tanshinones inhibit gastric cancer angiogenesis through the PI3K/Akt/mTOR signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza Bunge is a kind of Chinese herbal medicine known for activating blood circulation and removing blood stasis, with the effect of cooling blood and eliminating carbuncles, and has been proven to have the effect of treating tumors. However, the inhibitory effect of Salvia miltiorrhiza Bunge extracts (Diterpenoid tanshinones) on tumors by inhibiting angiogenesis has not been studied in detail. AIM OF THE STUDY: This study aimed to investigate the anti-gastric cancer effect of diterpenoid tanshinones (DT) on angiogenesis, including the therapeutic effects and pathways. MATERIALS AND METHODS: This experiment utilized network pharmacology was used to identify relevant targets and pathways of Salvia miltiorrhiza Bunge-related components in the treatment of gastric cancer. The effects of DT on the proliferation and migration of human gastric cancer cell line SGC-7901 and human umbilical vein endothelial cell line HUVECs were evaluated, and changes in the expression of angiogenesis-related factors were measured. In vivo, experiments were conducted on nude mice to determine tumor activity, size, immunohistochemistry, and related proteins. RESULTS: The findings showed that DT could inhibit the development of gastric cancer by suppressing the proliferation of gastric cancer cells, inducing apoptosis, and inhibiting invasion and metastasis. In addition, the content of angiogenesis-related factors and proteins was significantly altered in DT-affected cells and animals. CONCLUSIONS: Results suggest that DT has potential as a therapeutic agent for the treatment of gastric cancer, as it can inhibit tumor growth and angiogenesis. It was also found that DT may affect the expression of the angiogenic factor VEGF through the PI3K/Akt/mTOR pathway, leading to the regulation of tumor angiogenesis. This study provides a new approach to the development of anti-tumor agents and has significant theoretical and clinical implications for the treatment of gastric cancer.

Salvianolic acid extract prevents Tripterygium wilfordii polyglycosides-induced acute liver injury by modulating bile acid metabolism.

ETHNOPHARMACOLOGICAL RELEVANCE: Tripterygium wilfordii polyglycosides (TWP) tablet is the most widely used traditional Chinese medicine preparation for the treatment of rheumatoid arthritis (RA), but the hepatotoxicity often limits its widespread application. In traditional use, Salvia miltiorrhiza has cardioprotective and hepatoprotective effects. Salvianolic acid extract (SA) is a hydrophilic component of Salvia miltiorrhiza and has significant antioxidant and hepatoprotective effects. AIM OF THE STUDY: To investigate the protective effects of SA on the TWP-induced acute liver injury in rats and to explore the related mechanisms by integration of metabolomics and transcriptomics. MATERIALS AND METHODS: SA and TWP extracts were identified by UPLC-Q/TOF-MS. SA (200 mg/kg) was administered for consecutive 7 days. On day 7, TWP (360 mg/kg) was administered by gavage to induce the acute liver injury in rats. Serum biochemical assay and H&E staining were used to evaluate liver damage. Liver metabolomics and transcriptomics were used to explore the potential mechanisms, and further molecular biological experiments such as qPCR and IHC were utilized to validate the relevant signaling pathways. RESULTS: SA can prevent liver injury symptoms caused by TWP, such as elevated liver index, elevated ALT and AST, and pathological changes in liver tissue. Liver metabolomics studies showed that TWP can significantly alter the content of individual bile acid in the liver and SA had the most significant impact on the biosynthetic pathway of bile acids. The transcriptomics results of the liver indicated that the genes changed in the SA + TWP group were mainly involved in sterol metabolism, lipid regulation and bile acid homeostasis pathways. The gene expression of Nr1h4, which encodes farnesoid X receptor (FXR), an important regulator of bile acid homeostasis, was significantly changed. Further studies confirmed that SA can prevent the downregulation of FXR and its downstream signaling induced by TWP, thereby regulating bile acid metabolism, ultimately preventing acute liver injury caused by TWP. CONCLUSION: Our results demonstrated that SA could protect the liver from TWP-induced hepatic injury by modulation of the bile acid metabolic pathway. SA may provide a new strategy for the protection against TWP-induced acute liver injury.

Elucidating the distinctive regulatory effects and mechanisms of active compounds in Salvia miltiorrhiza Bunge via network pharmacology: Unveiling their roles in the modulation of platelet activation and thrombus formation.

Salvia miltiorrhiza Bunge. (DS), as an important traditional Chinese medicine (TCM), has a long history of usage for promoting blood circulation and removing blood stasis. Modern studies have shown that the chemical components of DS have many biological activities such as cardiovascular protection, anti-arrhythmia, anti-atherosclerosis, improvement of microcirculation, protection of myocardium, inhibition and removal of platelet aggregation. Nevertheless, the action mechanism of DS as well its active compounds on platelet activation has not been fully uncovered. This study aimed to find out the potential targets and mechanisms of DS in the modulation of platelet activation and thrombosis, using network pharmacology and biological experimental. These compounds with anti-thrombotic activity in DS, cryptotanshinone (CPT), isoeugenol (ISO) and tanshinone IIA (TSA), together with the corresponding targets being Src, Akt and RhoA are screened by network pharmacology. We confirmed that ISO, CPT and TSA dose-dependently inhibited platelet activation in vitro, mainly by inhibiting agonist-induced clot retraction, aggregation and P-selectin and ATP release. The western blot findings indicated that ISO, CPT, and TSA led to reduced levels of p-Akt and p-ERK in activated platelets. Additionally, ISO and TSA were observed to decrease p-cSrc expression while increasing RhoA expression. ISO, CPT, and TSA demonstrated a potential to restrict the advancement of carotid arterial thrombosis in vivo. We confirm that ISO, CPT and TSA are the key anti-thrombotic active compounds in DS. These active compounds exhibit unique inhibitory effects on platelet activation and thrombus formation by modulating the Akt/ERK and cSrc/RhoA signaling pathways.

Discovery of Natural Ah Receptor Antagonists from Salvia miltiorrhiza Bunge and Synthesis of Analogs for Tumor Immunotherapy.

IDO/TDO/Kyn/AhR signaling plays a crucial role in regulating innate and adaptive immunity, and targeting Ah receptor (AhR) inhibition can potentially redirect immune cells toward an antitumoral phenotype. Therefore, AhR is an attractive drug target for novel small molecule cancer immunotherapies. In this study, natural products tanshinolic A-D (1-4), the first adducts composed of ortho-naphthoquinone-type tanshinone and phenolic acid featuring a unique 1,4-benzodioxan hemiacetal structure, were isolated and characterized from the roots of Salvia miltiorrhiza Bunge. Luciferase reporter gene assay revealed that these adducts exhibited significant AhR inhibitory activity. A linear strategy was developed to construct a cis-3,4-disubstituted 1,4-benzodioxan hemiacetal structure. Encouragingly, in both in vitro and in vivo experiments, (±)-13e demonstrated the ability to inhibit tumor cell proliferation, promote INF-γ secretion in CD8+ T cells, and inhibit PD-1/PD-L1 signal transduction, which could exert tumor inhibition properties by inhibiting AhR activity, positioning it as a promising candidate for tumor immunotherapy.

Porous phenolic resin regulated by alcohol-based deep eutectic solvent for selective extraction and separation of tanshinones from Salvia miltiorrhiza.

Deep eutectic solvents (DES), regarded as promising green solvents, have gained attention due to their distinctive properties, particularly in analytical chemistry. While the use of DES in solvent extraction and separation has been extensively studied, its application in the synthesis of adsorbents has just begun. Phenolic resin, with its polyhydroxy structure and stable spherical morphology, could serve as an effective as adsorbents for enrichment of active ingredients in herbal medicine. Designing adsorbents with high selectivity and adsorption capacity presents a critical challenge in the enrichment of active ingredients in herbal medicine. In this study, alcohol-based DESs were employed as regulators of morphology and structure instead of organic solvents, facilitating the creation of polyhydroxy structure, adjustable pores and high specific surface areas. The resulting DES-regulated porous phenolic resin demonstrated enhanced extraction and separation capacity for active ingredients compared to conventional spherical phenolic resin owing to the alcohol-based DES offering more interaction modes with the analytes.

Ultrasonic-assisted activated carbon separation removing bacterial endotoxin from salvia miltiorrhizae injection.

Ultrasonic-assisted activated carbon separation (UACS) was first employed to improve product quality by regulating adsorption rate and removing bacterial endotoxin from salvia miltiorrhizae injection. The adsorption rate was related to three variables: activated carbon dosage, ultrasonic power, and pH. With the increase of activated carbon dosage from 0.05 % to 1.0 %, the adsorption rates of salvianolic acids and bacterial endotoxin increased simultaneously. The adsorption rates at which bacteria endotoxins increased from 52.52 % to 97.16 % were much higher than salvianolic acids. As the ultrasonic power increased from 0 to 700 W, the adsorption rates of salvianolic acids on activated carbon declined to less than 10 %, but bacterial endotoxin increased to more than 87 %. As the pH increased from 2.00 to 8.00, the adsorption rate of salvianolic acid dropped whereas bacterial endotoxin remained relatively stable. On the basis of response surface methodology (RSM), the optimal separation conditions were established to be activated carbon dose of 0.70 %, ultrasonic power of 600 W, and pH of 7.90. The experimental adsorption rates of bacterial endotoxin were 94.15 %, which satisfied the salvia miltiorrhizae injection quality criterion. Meanwhile, salvianolic acids' adsorption rates were 1.92 % for tanshinol, 4.05 % for protocatechualdehyde, 2.21 % for rosmarinic acid, and 3.77 % for salvianolic acid B, all of which were much lower than conventional activated carbon adsorption (CACA). Salvianolic acids' adsorption mechanism on activated carbon is dependent on the component's molecular state. Under ideal separation conditions, the molecular states of the four salvianolic acids fall between 1.13 % and 6.60 %. The quality of salvia miltiorrhizae injection can be improved while maintaining injection safety by reducing the adsorption rates of salvianolic acids to less than 5 % by the use of ultrasound to accelerate the desorption mass transfer rate on the activated carbon surface. When activated carbon adsorption was used in the process of producing salvia miltiorrhizae injection, the pH of the solution was around 5.00, and the proportion of each component's molecular state was tanshinol 7.05 %, protocatechualdehyde 48.93 %, rosmarinic acid 13.79 %, and salvianolic acid B 10.28 %, respectively. The loss of useful components was evident, and the corresponding activated carbon adsorption rate ranged from 20.74 % to 41.05 %. The average variation rate in plasma His and IgE was significant (P < 0.05) following injection of 0.01 % activated carbon, however the average variation rate of salvia miltiorrhizae injection was dramatically decreased with the use of UACS and CACA (P > 0.05). The ultrasonic at a power intensity of 60 W/L and the power density of 1.20 W/cm2 may resolve the separation contradiction between salvianolic acids and bacterial endotoxin, according to experiments conducted with UACS at different power intensities. According to this study, UACS has a lot of potential applications in the pharmaceutical manufacturing industry and may represent a breakthrough in the field of ultrasonic separation.

Identification and characterization of a novel tyrosine aminotransferase gene (SmTAT3-2) promotes the biosynthesis of phenolic acids in Salvia miltiorrhiza Bunge.

Rosmarinic acid (RA) and salvianolic acid B (SAB) are main phenolic acids in Salvia miltiorrhiza Bunge have been widely used in the treatment of cardiovascular and cerebrovascular diseases due to their excellent pharmacological activity. RA is a precursor of SAB, and tyrosine transaminase (TAT, EC 2.6.1.5) is a crucial rate-limiting enzyme in their metabolism pathway. This study identified a novel TAT gene, SmTAT3-2, and found that it is a new transcript derived from unconventional splicing of SmTAT3. We used different substrates for enzymatic reaction with SmTAT1, SmTAT3 and SmTAT3-2. Subcellular localization of SmTAT1 and SmTAT3-2 was completed based on submicroscopic techniques. In addition, they were overexpressed and CRISPR/Cas9 gene edited in hairy roots of S. miltiorrhiza. Revealed SmTAT3-2 and SmTAT1 showed a stronger affinity for L-tyrosine than SmTAT3, localized in the cytoplasm, and promoted the synthesis of phenolic acid. In overexpressed SmTAT3-2 hairy roots, the content of RA and SAB was significantly increased by 2.53 and 3.38 fold, respectively, which was significantly higher than that of overexpressed SmTAT1 strain compared with EV strain. These findings provide a valuable key enzyme gene for the phenolic acids metabolism pathway and offer a theoretical basis for the clinical application.

Metagenomic sequencing revealed the regulative effect of Danshen and Honghua herb pair on the gut microbiota in rats with myocardial ischemia injury.

In recent years, more and more evidence has shown that the disorder of gut microbiota (GM) is closely correlated with myocardial ischemia (MI). Even though the Danshen and Honghua herb pair (DHHP) is widely used in treating cardiovascular disease in China and exhibits obvious clinical efficacy on MI, the anti-MI mechanism of DHHP remains and needs to be explored in depth. Thus, in this study, we investigated whether the amelioration effect and molecular mechanism of DHHP on MI were related to regulating GM through pharmacodynamics evaluation and metagenomic sequencing. Histopathological testing results showed that DHHP treatment could alleviate the pathological changes of myocardial tissue in the acute MI (AMI) rats induced by isoproterenol (ISO), especially structural disorder, irregular distribution, and enlargement of the myocardial space. These pathological changes were all alleviated to some extent by DHHP treatment. Biochemical analysis results suggested that compared with the control group, the serum levels of AST, CTn-I, CK-MB, and TNF-α in model group rats were notably decreased, and the CAT and SOD levels in serum were markedly increased. These abnormal trends were significantly reversed by DHHP treatment. Furthermore, metagenomic sequencing analysis results indicated that DHHP could improve disorders in the composition and function of GM in AMI rats, mainly reflected in increasing diversity and richness, and obviously enhancing the abundance of Bacteroides fluxus, B. uniformis, B. stercoris, Roseburia hominis, Schaedlerella arabinosiphila, and R. intestinalis, and reducing the abundance of Enterococcus avium and E. canintestini, which were associated with purine metabolism, tyrosine metabolism, cyanoamino acid metabolism, and glutathione metabolism. In conclusion, DHHP may attenuate ISO-induced MI by regulating the structure, composition, and function of GM, thus contributing to further our understanding of the anti-MI mechanisms of DHHP and providing new therapeutic ideas and diagnostic targets for the clinical studies of MI.

A Nondestructive Methodology for Determining Chemical Composition of Salvia miltiorrhiza via Hyperspectral Imaging Analysis and Squeeze-and-Excitation Residual Networks.

The quality assurance of bulk medicinal materials, crucial for botanical drug production, necessitates advanced analytical methods. Conventional techniques, including high-performance liquid chromatography, require extensive pre-processing and rely on extensive solvent use, presenting both environmental and safety concerns. Accordingly, a non-destructive, expedited approach for assessing both the chemical and physical attributes of these materials is imperative for streamlined manufacturing. We introduce an innovative method, designated as Squeeze-and-Excitation Residual Network Combined Hyperspectral Image Analysis (SE-ReHIA), for the swift and non-invasive assessment of the chemical makeup of bulk medicinal substances. In a demonstrative application, hyperspectral imaging in the 389-1020 nm range was employed in 187 batches of Salvia miltiorrhiza. Notable constituents such as salvianolic acid B, dihydrotanshinone I, cryptotanshinone, tanshinone IIA, and moisture were quantified. The SE-ReHIA model, incorporating convolutional layers, maxpooling layers, squeeze-and-excitation residual blocks, and fully connected layers, exhibited Rc2 values of 0.981, 0.980, 0.975, 0.972, and 0.970 for the aforementioned compounds and moisture. Furthermore, Rp2 values were ascertained to be 0.975, 0.943, 0.962, 0.957, and 0.930, respectively, signifying the model's commendable predictive competence. This study marks the inaugural application of SE-ReHIA for Salvia miltiorrhiza's chemical profiling, offering a method that is rapid, eco-friendly, and non-invasive. Such advancements can fortify consistency across botanical drug batches, underpinning product reliability. The broader applicability of the SE-ReHIA technique in the quality assurance of bulk medicinal entities is anticipated with optimism.

Insights into accumulation of active ingredients and rhizosphere microorganisms between Salvia miltiorrhiza and S. castanea.

Salvia miltiorrhiza is an important traditional herbal medicine, and its extracts could be used for treating cardiovascular disease. Although these medicinal compounds are functionally similar, their wild relative, S. castanea, produces significantly different concentrations of these compounds. The reason for their differences is still unknown. In a series of soil and plant-based analyses, we explored and compared the rhizosphere microbiome of S. miltiorrhiza and S. castanea. To further investigate the geographical distribution of S. castanea, MaxEnt models were used to predict the future suitable habitat areas of S. castanea in China. Results revealed the distributions and structure of the rhizosphere microbial community of S. miltiorrhiza and S. castanea at different times. In addition, differences in altitude and soil moisture resulting from changes in climate and geographical location are also critical environmental factors in the distribution of S. castanea. The findings of this study increase our understanding of plant adaptation to their geographical environment through secondary metabolites. It also highlights the complex interplay between rhizospheric factors and plant metabolism, which provides the theoretical basis for the cultivation of S. miltiorrhiza and the use of S. castanea resources.

Salviamilthone A-O, diterpenoid quinones from Salvia miltiorrhiza.

Fifteen undescribed diterpenoid quinones salviamilthone A-O (1-15), together with three known diterpenoid quinones (16-18), were isolated from the roots of Salvia miltiorrhiza Bunge. Their structures were elucidated using 1D and 2D NMR data, while the relative and absolute configurations were confirmed by NOESY correlations and comparison between experimental and calculated ECD spectra. In the evaluation of bioactivities, salviamilthone J (10), salviamone (18) (10 µM) significantly increased cell viability and decreased the expression of IL-1ß in lipopolysaccharide-induced BEAS-2B cells. These data provide the molecular justification for the usage of Salvia miltiorrhiza in treating acute lung injury.

The protective effect of an extract of Salvia miltiorrhiza Bunge (Danshen) on cerebral ischemic injury in animal models: A systematic review and meta-analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Cerebral ischemia is a common disease that seriously threatens the health of human beings. Tanshinone IIA (TSA) is a fat-soluble compound isolated from the traditional Chinese medicine Danshen. Recent studies have shown that TSA plays a significant protective role in the animal models of cerebral ischemic injury. AIM OF THE STUDY: The meta-analysis was to evaluate the protective effect of Danshen (Salvia miltiorrhiza Bunge) extract (TSA) in cerebral ischemic injury, aiming at providing scientific evidence for clinical application of TSA in the treatment of cerebral ischemia in patients. MATERIALS AND METHODS: All relevant studies published in PubMed, Web of Science, Cochrane Library, China National Knowledge Infrastructure (CNKI), Wanfang Database, Chinese Scientific Journals Database (VIP) and Chinese Biomedicine Database (CBM) before Jan 2023 were systematically retrieved. The methodological quality was assessed by SYRCLE's risk of bias tool for the animal studies. Data was analyzed using Rev Man 5.3 software. RESULTS: A total of 13 studies were included. Compared with the control group, TSA significantly reduced the expression of glial fibrillary acidic protein (GFAP) (mean difference [MD], -1.78; 95% CI, [-2.13, -1.44]; P < 0.00001) and high mobility group protein B1 (HMGB1) (MD, -0.69; 95% CI, [-0.87, -0.52]; P < 0.00001). TSA also inhibited the activation of brain nuclear factor κB (NF-κB) (MD, - 0.36; 95% CI, [-0.41, -0.32]; P < 0.00001), malondialdehyde (MDA) (MD, -0.90; 95% CI, [-1.66, -0.13]; P = 0.02), cysteine protease-3 (Caspase-3) (MD, -1.39; 95% CI, [-1.98, -0.81]; P < 0.00001), and reduced cerebral infarction volume(MD, -16.26; 95% CI, [-20.76, -11.77]; P < 0.00001), brain water content (MD, -4.89; 95% CI, [-7.06, -2.71]; P < 0.0001) and neurological deficit scores (MD, -1.19; 95% CI, [-1.48, -0.89]; P < 0.00001). Additionally, TSA increased the brain content of superoxide dismutase (SOD) (MD, 68.31; 95% CI, [10.41, 126.22]; P = 0.02). CONCLUSIONS: The result of this study showed that TSA had a protective effect on cerebral ischemic injury in animal models, and the mechanism is associated with the reduction of inflammation and oxidative stress, and the inhibition of cell apoptosis. However, the quality of included studies may affect the accuracy of positive results. Therefore, more high-quality randomized controlled animal experiments are need for meta-analysis in the future.

[Modification of C20 oxidase in tanshinone biosynthesis pathway].

Tanshinones are one of the main effective components of Salvia miltiorrhiza, which play important roles in the treatment of cardiovascular diseases. Microbial heterogony production of tanshinones can provide a large number of raw materials for the production of traditional Chinese medicine(TCM) preparations containing S. miltiorrhiza, reduce the extraction cost, and relieve the pressure of clinical medication. The biosynthetic pathway of tanshinones contains multiple P450 enzymes, and the catalytic element with high efficiency is the basis of microbial production of tanshinones. In this study, the protein modification of CYP76AK1, a key P450-C20 hydroxylase in tanshinone pathway, was researched. The protein modeling methods SWISS-MODEL, Robetta, and AlphaFold2 were used, and the protein model was analyzed to obtain the reliable protein structure. The semi-rational design of mutant protein was carried out by molecular docking and homologous alignment. The key amino acid sites affecting the oxidation activity of CYP76AK1 were identified by molecular docking. The function of the obtained mutations was studied with yeast expression system, and the CYP76AK1 mutations with continuous oxidation function to 11-hydroxysugiol were obtained. Four key amino acid sites that affected the oxidation acti-vity were analyzed, and the reliability of three protein modeling methods was analyzed according to the mutation results. The effective protein modification sites of CYP76AK1 were reported for the first time in this study, which provides a catalytic element for different oxidation activities at C20 site for the study of the synthetic biology of tanshinones and lays a foundation for the analysis of the conti-nuous oxidation mechanism of P450-C20 modification.

Functional Study and Efficient Catalytic Element Mining of CYP76AHs in Salvia Plants.

Salvia is a large genus with hundreds of species used in traditional Chinese medicine. Tanshinones are a highly representative class of exclusive compounds found in the Salvia genus that exhibit significant biological activity. Tanshinone components have been identified in 16 Salvia species. The CYP76AH subfamily (P450) is crucial for the synthesis of tanshinone due to its catalytic generation of polyhydroxy structures. In this study, a total of 420 CYP76AH genes were obtained, and phylogenetic analysis showed their clear clustering relationships. Fifteen CYP76AH genes from 10 Salvia species were cloned and studied from the perspectives of evolution and catalytic efficiency. Three CYP76AHs with significantly improved catalytic efficiency compared to SmCYP76AH3 were identified, providing efficient catalytic elements for the synthetic biological production of tanshinones. A structure-function relationship study revealed several conserved residues that might be related to the function of CYP76AHs and provided a new mutation direction for the study of the directed evolution of plant P450.

Integrated Multiomics and Synergistic Functional Network Revealed the Mechanism in the Tolerance of Different Ecotypes of Salvia miltiorrhiza Bge. to Doxycycline Pollution.

Tetracycline pollution in soil irreversibly damages the biosafety of plants by inhibiting the mitochondrial function. Some traditional Chinese medicine (TCM) plants, such as Salvia miltiorrhiza Bunge, have a strong tolerance to mitochondrial damage. We comprehensively compared the doxycycline (DOX) tolerances of two ecotypes of S. miltiorrhiza in the Sichuan and Shandong provinces and found that the Sichuan ecotype had a lower yield reduction, more stable accumulation of medicinal ingredients, higher mitochondrial integrity, and a more robust antioxidant system. The synergetic response networks under DOX pollution of both ecotypes were constructed using RNA sequencing and ultrahigh-performance liquid chromatography-tandem mass spectrometry. The differentiation of the downstream pathways of aromatic amino acids (AAAs) produced variations in the DOX tolerance of S. miltiorrhiza in different regions. The Sichuan ecotype maintained redox homeostasis and xylem development by activating salvianolic acid and indole biosynthesis, while the Shandong ecotype balanced chemical and mechanical defenses by regulating the flavonoid biosynthesis. Rosmarinic acid, a downstream AAA molecule, maintains the mitochondrial homeostasis of plant seedlings under DOX pollution by targeting the ABCG28 transporter. We also highlight the significance of downstream AAA small molecules in guiding the development of bio-based environmental pollution remediation agents.

Integrated proteomics and phosphoproteomics profiling reveals the cardioprotective mechanism of bioactive compounds derived from Salvia miltiorrhiza Burge.

BACKGROUND: Natural products are an important source for discovering novel drugs due to their various pharmacological activities. Salvia miltiorrhiza Burge (Danshen) has been shown to have promising therapeutic potential in the management of heart diseases, making it a candidate for cardiovascular drug discovery. Currently, there is limited quantitative analysis of the phosphorylation levels of Danshen-derived natural products on a proteome-wide, which may bias the study of their mechanisms of action. PURPOSE: This study aimed to evaluate the global signaling perturbation induced by Danshen-derived bioactive compounds and their potential relationship with myocardial ischemia/reperfusion (IR) injury therapy. STUDY DESIGN: We employed quantitative proteome and phosphoproteome analysis to identify dysregulated signaling in IR injury hearts from mice. We compared changes induced by Danshen-derived compounds based on IR-associated phospho-events, using an integrative approach that maps relative abundance of proteins and phosphorylation sites. METHODS: Isobaric chemical tandem mass tags (TMT) labeled multiplexing strategy was used to generate unbiased quantitative proteomics and phosphoproteomics data. Highly accurate and precise TMT quantitation was performed using the Orbitrap Fusion Tribrid Mass Spectrometer with synchronous precursor selection MS3 detection mode. Mass spectrometric raw files were analyzed with MaxQuant (2.0.1.0) and statistical and bioinformatics analysis was conducted with Perseus (1.6.15). RESULTS: We quantified 3661 proteins and over 11,000 phosphosites in impaired heart tissue of the IR mice model, expanding our knowledge of signaling pathways and other biological processes disrupted in IR injury. Next, 1548 and 5545 differently expressed proteins and phosphosites were identified by quantifying the proteome and phosphoproteome of H9c2 cells treated by five Danshen bioactive compounds respectively. Results revealed the vast differences in abilities of five Danshen-derived bioactive compounds to regulate phosphorylation modifications in cardiomyocytes, with dihydrotanshinone I (DHT) showing potential for protecting against IR injury by modulating the AMPK/mTOR signaling pathway. CONCLUSIONS: This study provides a new strategy for analyzing drug/natural product-regulated phosphorylation modification levels on a proteome-wide scale, leading to a better understanding of cell signaling pathways and downstream phenotypic responses.

Effect of ophiopogonin D on the pharmacokinetics and transport of cryptotanshinone during their co-administration and the potential mechanism.

Cryptotanshinone and ophiopogonin D are sourced from herbs with similar indications. It is necessary to evaluate their interaction to provide a reference for their clinical prescriptions. The co-administration of cryptotanshinone (30 and 60 mg/kg) and ophiopogonin D was carried out in Sprague-Dawley rats and the pharmacokinetics of cryptotanshinone were analyzed. The Caco-2 cells were employed to evaluate the transport of cryptotanshinone, and the metabolic stability was studied in the rat liver microsomes. Ophiopogonin D significantly increased the Cmax (from 5.56 ± 0.26 to 8.58 ± 0.71 µg/mL and from 15.99 ± 1.81 to 185.12 ± 1.43 µg/mL), half-life (21.72 ± 10.63 vs. 11.47 ± 3.62 h and 12.58 ± 5.97 vs. 8.75 ± 2.71 h) and decreased the clearance rate (0.697 ± 0.36 vs. 1.71 ± 0.15 L/h/kg) and (60 mg/kg and 0.101 ± 0.02 vs. 0.165 ± 0.05 L/h/kg) of cryptotanshinone. In vitro, ophiopogonin D significantly suppressed the transport of cryptotanshinone with the decreasing efflux rate and enhanced the metabolic stability with the reducing intrinsic clearance. The combination of cryptotanshinone and ophiopogonin D induced prolonged exposure and suppressed the transport of cryptotanshinone, which indicated the decreased bioavailability of cryptotanshinone.