Effect the accumulation of bioactive constituents of a medicinal plant (Salvia Miltiorrhiza Bge.) by arbuscular mycorrhizal fungi community.

BACKGROUND: Arbuscular mycorrhizal fungi (AMF) form symbiotic relationships with various terrestrial plants and have attracted considerable interest as biofertilizers for improving the quality and yield of medicinal plants. Despite the widespread distribution of AMFs in Salvia miltiorrhiza Bunge's roots, research on the impact of multiple AMFs on biomass and active ingredient accumulations has not been conducted. In this study, the effects of five native AMFs (Glomus formosanum, Septoglomus constrictum, Rhizophagus manihotis, Acaulospora laevis, and Ambispora gerdemannii) and twenty-six communities on the root biomass and active ingredient concentrations of S. miltiorrhiza were assessed using the total factor design method. RESULTS: Thirty-one treatment groups formed symbiotic relationships with S. miltiorrhiza based on the pot culture results, and the colonization rate ranged from 54.83% to 89.97%. AMF communities had higher colonization rates and total phenolic acid concentration than single AMF, and communities also appeared to have higher root fresh weight, dry weight, and total phenolic acid concentration than single inoculations. As AMF richness increased, there was a rising trend in root biomass and total tanshinone accumulations (ATTS), while total phenolic acid accumulations (ATP) showed a decreasing trend. This suggests that plant productivity was influenced by the AMF richness, with higher inoculation benefits observed when the communities contained three or four AMFs. Additionally, the affinities of AMF members were also connected to plant productivity. The inoculation effect of closely related AMFs within the same family, such as G. formosanum, S. constrictum, and R. manihotis, consistently yielded lower than that of mono-inoculation when any combinations were applied. The co-inoculation of S. miltiorrhiza with nearby or distant AMFs from two families, such as G. formosanum, R. manihotis, and Ac. laevis or Am. gerdemannii resulted in an increase of ATP and ATTS by more than 50%. AMF communities appear to be more beneficial to the yield of bioactive constituents than the single AMF, but overall community inoculation effects are related to the composition of AMFs and the relationship between members. CONCLUSION: This study reveals that the AMF community has great potential to improve the productivity and the accumulation of bioactive constituents in S. miltiorrhiza, indicating that it is an effective way to achieve sustainable agricultural development through using the AMF community.

Integrated analysis of lncRNAs, mRNAs, and TFs to identify network modules underlying diterpenoid biosynthesis in Salvia miltiorrhiza.

Long non-coding RNAs (lncRNAs) are transcripts of more than 200 nucleotides (nt) in length, with minimal or no protein-coding capacity. Increasing evidence indicates that lncRNAs play important roles in the regulation of gene expression including in the biosynthesis of secondary metabolites. Salvia miltiorrhiza Bunge is an important medicinal plant in China. Diterpenoid tanshinones are one of the main active components of S. miltiorrhiza. To better understand the role of lncRNAs in regulating diterpenoid biosynthesis in S. miltiorrhiza, we integrated analysis of lncRNAs, mRNAs, and transcription factors (TFs) to identify network modules underlying diterpenoid biosynthesis based on transcriptomic data. In transcriptomic data, we obtained 6,651 candidate lncRNAs, 46 diterpenoid biosynthetic pathway genes, and 11 TFs involved in diterpenoid biosynthesis. Combining the co-expression and genomic location analysis, we obtained 23 candidate lncRNA-mRNA/TF pairs that were both co-expressed and co-located. To further observe the expression patterns of these 23 candidate gene pairs, we analyzed the time-series expression of S. miltiorrhiza induced by methyl jasmonate (MeJA). The results showed that 19 genes were differentially expressed at least a time-point, and four lncRNAs, two mRNAs, and two TFs formed three lncRNA-mRNA and/or TF network modules. This study revealed the relationship among lncRNAs, mRNAs, and TFs and provided new insight into the regulation of the biosynthetic pathway of S. miltiorrhiza diterpenoids.

Identification, classification, and expression profile analysis of heat shock transcription factor gene family in Salvia miltiorrhiza.

In response to abiotic stresses, transcription factors are essential. Heat shock transcription factors (HSFs), which control gene expression, serve as essential regulators of plant growth, development, and stress response. As a model medicinal plant, Salvia miltiorrhiza is a crucial component in the treatment of cardiovascular illnesses. But throughout its growth cycle, S.miltiorrhiza is exposed to a series of abiotic challenges, including heat and drought. In this study, 35 HSF genes were identified based on genome sequencing of Salvia miltiorrhiza utilizing bioinformatics techniques. Additionally, 35 genes were classified into three groups by phylogeny and gene structural analysis, comprising 22 HSFA, 11 HSFB, and two HSFC. The distribution and sequence analysis of motif showed that SmHSFs were relatively conservative. In SmHSF genes, analysis of the promoter region revealed the presence of many cis-acting elements linked to stress, hormones, and growth and development, suggesting that these factors have regulatory roles. The majority of SmHSFs were expressed in response to heat and drought stress, according to combined transcriptome and real-time quantitative PCR (qRT-PCR) analyses. In conclusion, this study looked at the SmHSF gene family using genome-wide identification, evolutionary analysis, sequence characterization, and expression analysis. This research serves as a foundation for further investigations into the role of HSF genes and their molecular mechanisms in plant stress responses.

A Novel Based-Network Strategy to Identify Phytochemicals from Radix Salviae Miltiorrhizae (Danshen) for Treating Alzheimer's Disease.

Alzheimer's disease (AD) is a common age-related neurodegenerative disease that strikes millions worldwide. Herein, we demonstrate a new approach based on network target to identify anti-AD compounds from Danshen. Network pharmacology and molecular docking were employed to establish the DS-AD network, which mainly involved apoptosis of neuron cells. Then network scoring was confirmed via Connectivity Map analysis. M308 (Danshenxinkun D) was an anti-AD candidate with a high score (p < 0.01). Furthermore, we conducted ex vivo experiments with H2O2-treated PC12 cells to verify the neuroprotective effect of Salvia miltiorrhiza-containing plasma (SMP), and UPLC-Q-TOF/MS and RT-qPCR were performed to demonstrate the anti-AD activity of M308 from SMP. Results revealed that SMP could enhance cell viability and level of acetylcholine. AO/EB staining and Mitochondrial membrane potential (MMP) analysis showed that SMP significantly suppressed apoptosis, which may be due to anti-oxidative stress activity. Moreover, the effects of M308 and SMP on expressions of PSEN1, DRD2, and APP mRNA were consistent, and M308 can significantly reverse the expression of PSEN1 and DRD2 mRNA in H2O2-treated PC12 cells. The strategy based on the network could be employed to identify anti-AD compounds from Chinese herbs. Notably, M308 stands out as a promising anti-AD candidate for development.

Prediction of active ingredients in Salvia miltiorrhiza Bunge. based on soil elements and artificial neural network.

The roots of Salvia miltiorrhiza Bunge. are commonly used in the treatment of cardiovascular diseases, and tanshinones and salvianolic acids are its main active ingredients. However, the composition and content of active ingredients of S. miltiorrhiza planted in different regions of the soil environment are also quite different, which adds new difficulties to the large-scale and standardization of artificial cultivation. Therefore, in this study, we measured the active ingredients in the roots of S. miltiorrhiza and the contents of rhizosphere soil elements from 25 production areas in eight provinces in China, and used the data to develop a prediction model based on BP (back propagation) neural network. The results showed that the active ingredients had different degrees of correlation with soil macronutrients and trace elements, the prediction model had the best performance (MSE = 0.0203, 0.0164; R2 = 0.93, 0.94). The artificial neural network model was shown to be a method that can be used to screen the suitable cultivation sites and proper fertilization. It can also be used to optimize the fertilizer application at specific sites. It also suggested that soil testing formula fertilization should be carried out for medicinal plants like S. miltiorrhiza, which is grown in multiple origins, rather than promoting the use of "special fertilizer" on a large scale. Therefore, the model is helpful for efficient, rational, and scientific guidance of fertilization management in the cultivation of S. miltiorrhiza.

In Vitro Effects of Tartary Buckwheat-Derived Nanovesicles on Gut Microbiota.

Evidence suggests that plant-derived nanovesicles may play a significant role in human health. Tartary buckwheat has several physiological activities; however, its underlying health-promoting mechanism remains unclear. In this study, first, Tartary buckwheat-derived nanovesicles (TBDNs) were collected, their structures were analyzed, and microRNA sequencing was performed. Next, target prediction and functional verification were conducted. Finally, the effects of TBDNs on gut microbiota and short-chain fatty acid levels were evaluated. The average size of TBDNs was 141.8 nm diameter. Through the sequencing analyses, 129 microRNAs, including 11 novel microRNAs were identified. Target gene prediction showed that some microRNAs could target functional genes in Escherichia coli and Lactobacillus rhamnosus-related physiological processes. TBDNs significantly promoted the growth of E. coli and L. rhamnosus, enhanced the diversity of fecal microorganisms and increased the short-chain fatty acid levels. These findings provided a new nutritional perspective for Tartary buckwheat and were conducive to promote the development and utilization of Tartary buckwheat.

Comparative transcriptome analyses of different Salvia miltiorrhiza varieties during the accumulation of tanshinones.

Salvia miltiorrhiza (Labiatae) is an important medicinal plant in traditional Chinese medicine. Tanshinones are one of the main active components of S. miltiorrhiza. It has been found that the intraspecific variation of S. miltiorrhiza is relatively large and the content of tanshinones in its roots of different varieties is also relatively different. To investigate the molecular mechanisms that responsible for the differences among these varieties, the tanshinones content was determined and comparative transcriptomics analysis was carried out during the tanshinones accumulation stage. A total of 52,216 unigenes were obtained from the transcriptome by RNA sequencing among which 23,369 genes were differentially expressed among different varieties, and 2,016 genes including 18 diterpenoid biosynthesis-related genes were differentially expressed during the tanshinones accumulation stage. Functional categorization of the differentially expressed genes (DEGs) among these varieties revealed that the pathway related to photosynthesis, oxidative phosphorylation, secondary metabolite biosynthesis, diterpenoid biosynthesis, terpenoid backbone biosynthesis, sesquiterpenoid and triterpenoid biosynthesis are the most differentially regulated processes in these varieties. The six tanshinone components in these varieties showed different dynamic changes in tanshinone accumulation stage. In addition, combined with the analysis of the dynamic changes, 277 DEGs (including one dehydrogenase, three CYP450 and 24 transcription factors belonging to 12 transcription factor families) related to the accumulation of tanshinones components were obtained. Furthermore, the KEGG pathway enrichment analysis of these 277 DEGs suggested that there might be an interconnection between the primary metabolic processes, signaling processes and the accumulation of tanshinones components. This study expands the vision of intraspecific variation and gene regulation mechanism of secondary metabolite biosynthesis pathways in medicinal plants from the "omics" perspective.

Diversity and function of culturable actinobacteria in the root-associated of Salvia miltiorrhiza Bunge.

The root-associated actinobacteria play important roles in plant growth, nutrient use, and disease resistance due to their functional diversity. Salvia miltiorrhiza is a critical medicinal plant in China. The root actinobacterial community structure has been studied; however, the functions of root-associated actinobacteria of S. miltiorrhiza have not been elucidated. This study aimed to decipher the diversity and function of the culturable root-associated actinobacteria in plant growth using culture-dependent technology and culturable microbe metagenomes. We isolated 369 strains from the root-associated actinobacteria, belonging to four genera, among which Streptomyces was dominant. Besides, the functional prediction revealed some pathways related to plant growth, nitrogen and phosphorus metabolism, and antagonistic pathogens. We systematically described the diversity and functions of the culturable root-associated actinobacteria community. Our results demonstrated that the culturable root-associated actinobacteria of S. miltiorrhiza have rich functionalities, explaining the possible contribution of culturable root-associated actinobacteria to S. miltiorrhiza's growth and development. This study provides new insights into understanding the function of the culturable root-associated actinobacteria and can be used as a knowledge base for plant growth promoters and biological control agent development in agriculture.

Effects of Native Arbuscular Mycorrhizae Isolated on Root Biomass and Secondary Metabolites of Salvia miltiorrhiza Bge.

Arbuscular mycorrhiza fungi (AMFs) are a group of soil-dwelling fungi that form symbiotic associations with plants, to mediate the secondary metabolism and production of active ingredients in aromatic and medicinal plants. Currently, there is little research on Salvia miltiorrhiza Bge. inoculation with native AMFs and the concomitant effects on growth and secondary metabolites. In this study, S. miltiorrhiza was treated with eight AMFs, i.e., Glomus formosanum; Gl. tenebrosum; Septoglomus constrictum; Funneliformis geosporum; Rhizophagus manihotis; Ambispora gerdemanii; Acaulospora laevis; Ac. tuberculata, to investigate the influence of AMF inoculation on biomass and secondary production under greenhouse conditions in S. miltiorrhiza roots. The results showed that mycorrhiza formation rates were between 54.83 and 86.10%. Apart from Ac. laevis and Gl. tenebrosum treatment, the roots biomass of the other treatment groups was effectively increased, and the fresh and dry weight of the plant inoculated with Fu. geosporum were increased by 86.76 and 86.95%, respectively. Specifically, AMF treatments also impacted on phenolic acids production; inoculation with both Fu. geosporum or Ac. laevis significantly reduced total phenolic acids, whereas the other treatments effectively increased these levels, of which Gl. formosanum generated significant levels. Most AMF-plant symbiotic experiments facilitated phenolic acid accumulation in the secondary metabolites of S. miltiorrhiza (except Ac. laevis). This study showed that most native AMFs inoculation with S. miltiorrhiza promoted roots growth and increased secondary metabolites production (especially phenolic acids). Going forward, inoculation of native AMF is a promising method to improve the quality and yield of S. miltiorrhiza and should be considered during production.

Cryptotanshinone: A review of its pharmacology activities and molecular mechanisms.

As a natural quinone compound, the medicinal value of cryptotanshinone (CT) has received increasing attentions, but there is no systematic literature review that describes the pharmacological activity of CT. This paper reviewed the pharmacology researches of CT, with a primary focus on its anti-tumor activity. We also discussed the underlying molecular mechanisms, and proposed future outlooks. In addition to anti-tumor activity, CT was found to have anti-inflammatory, neuroprotective, cardioprotective, visceral protective, anti-metabolic disorders and other abilities. Furthermore, the potential molecular mechanisms contributing to the anti-tumor effect of CT likely involve the following aspects: the induction of apoptosis, targeting of ER and AR, reversion of MDR, combined pharmacotherapy, and the inhibition of cell proliferation, migration, and invasion. We also found that different pharmacological effects involved various signaling pathways. Among them, STAT3-related signaling pathways played a vital role in the CT-mediated induction of tumor cell apoptosis and proliferation, while NF-κB signal pathway also was essential for inhibition of inflammation by CT. Furthermore, CT could significantly enhance the activities of several anticancer drugs and reverse their resistances in tumors. Therefore, we proposed suggestions for future studies of CT, including enhancing anti-tumor activity by targeting STAT3-related receptors, targeting NF-κB-related pathways to inhibit inflammatory responses, enhancing anti-tumor efficacy by combining with anti-tumor drugs, and further studying the dose-effect relationship to ensure safer and more effective applications of CT.

[Microecology and geoherbalism of traditional Chinese medicine].

Microecology was directly or indirectly involved in the growth and development, metabolism process, and component accumulation of traditional Chinese medicine(TCM) in various ways, which affected the formation and changes of the geoherbalism of TCM. It was one of the main tasks of traditional Chinese medical microecology(TCMM) to reveal the relationship among microecological structure and its change rule and the quality effect of TCM. The heterogeneity of soil environment caused by geographical and climatic factors, as well as the discreteness limitation caused by isolation factors such as distance and host selection, were the main causes of the differentiation of microecological geography of TCM. The microecology of TCM had important influences and contributions on the distinctive origin and quality of Dao-di herbs, which was mainly reflected in the formation of excellent germplasm(including disease and insect resistance, drought resistance, salt resistance, cold resistance, etc.), the increase of yield, the formation of medicinal parts, the metabolism and accumulation of effective components, the time limit of harvesting, and the toxicity, increasing efficiency or reducing toxicity of TCM in the processing, the changes of product efficiency after introduction, and the authenticity of fungus medicine. With the vigorous development of metabonomics and modern information technology, the following aspects would become the future research trends, including the microecologically mediated biogenic pathway of chemical components, the metabolic synthesis reactor of TCM based on the microecological quantitative effect relationship, the cultivation of genuine Chinese medicine based on reconstruction of microecological structure, the origin identification barcode traceability technology, and the toxicity reduction and efficiency enhancement technology of TCM based on the microecological.

[Research and prospect of Traditional Chinese Medical Microecology].

In the long-term evolution, microbes and hosts coexist widely, forming a symbiotic microecosystem and resulting the complex interactions of the metabolism. With the application of microecological theory in Chinese materia medica science, two main points have been accepted gradually. On the one hand, the prevention and treatment of human diseases by traditional Chinese medicines can be achieved through the correction and adjustment of the imbalance of the human microecosystem. On the other hand, the microecosystem can regulate the quality of traditional Chinese medicines in real time, and further affect their curative effect. Thus, a new discipline, Traditional Chinese Medical Microecology, has been gradually established. In this review, the background, theoretical structure, research directions, key problems and the relationship with human microecology of Traditional Chinese Medical Microecology were systematically summarized and prospected for promoting its development. Moreover, this review provides a reference protocol for further discoursing the microecological mechanism involving the efficacy of traditional Chinese medicines.

[DNA marker-assisted selection of medicinal plants (â ) .Breeding research of disease-resistant cultivars of Panax notoginseng].

The study is aimed to estimate the diversity of arbuscular mycorrhizal fungi (AMF) in the main producing areas of Salvia miltiorrhiza.Diversity of AMF was surveyed directly on spores isolated from the field soil, collected from 20 sites of 8 provinces. Identification of the AMF was made by observation of spore morphology. At least 27 recognized AMF species were identified in samples from field soil, belonging to seven genera of AMF-Acaulospora, Glomus, Funneliformis, Ambispora, Rhizophagus, Pacispora, and Claroideoglomus. Acaulospora and Glomus were the dominant genera, respectively including nine and eight species. A. laevis (90%), R.manihotis (80%), A. brieticulata (75%), A. tuberculata (70%) were the dominant species.Colonization rate was determined,colonization was easily found, but the colonization intensities were low, the colonization rate remained at 10.92%-25.93%. The similarity between provinces is generally low, and the similarity coefficients were from 0.20 to 0.57.

[Correlation analysis between effectiveness of element in rhizonsphere soil and quality of Danshen (Salvia miltiorrhiza)].

In order to investigate the content and distribution of available element in the rhizonsphere soil of the growing areas of Salvia miltiorrhiza Bunge, the contents of available element (N,P,K,B,Cu,Zn,Fe,Mn) in 26 soil samples were tested and evaluated. The results showed that the contents of available P and Fe were very plentiful, available K, Cu and Zn were rich, available N and Mn were deficient, available B was extremely deficient in all growing areas of S. miltiorrhiza of eight provinces in China. The correlation analysis showed that the contents of eight kinds of available elements were varying degree correlation. The stepwise regression analysis between the contents of available elements of rhizonsphere soil and ten kinds of active ingredients of Danshen (Salviae Miltiorrhizae Radix et Rhizoma) were researched. The results showed that the rates of contribution of available N,B,Mn and Fe to quality of Danshen were relatively large and they were the significant factors, and the other factors did not show statistical significance. The recommended fertilizing strategies is that the usage of N,B and Mn fertilizers should be controlled according to different stages of growth of S. miltiorrhiza, and P fertilizer should be reduced in all growing areas of S. miltiorrhiza.

[A novel method for testing sterility of injections based on biothermodynamics].

This study aims at trying to establish a novel method of sterility test for injections based on biothermodynamics, in order to overcome the deficiencies of routine sterility tests such as long detecting cycle, low sensitivity and prone to misjudgments. A biothermodynamics method was adopted to rapidly detect the microorganism contamination of injections by monitoring the heat metabolism during the growth of microbe. The growth rate equal to or greater than zero and the heat power difference of P(i) and P(0) with three folds higher than the noise of baseline were chosen as indexes to study the heat change rule of microbe. In this way, the effectiveness of the new method to detect strains required by conventional sterility test or in injection samples was also investigated. Results showed that the Gram-positive bacteria, Gram-negative bacteria and fungi demanded by sterility testing methodology could be detected by biothermodynamics method within 10 hours, with the sensitivity lower than 100 CFU x mL(-1). Meanwhile, this method was successfully applied to the sterility test of Compound Yinchen injection (FFYC), Shuanghuanglian powder injection (SHL) and Compound Triamcinolone injection (TAND) which were sterilized with different degrees. Therefore, the biothermodynamics method, with advantages of fast detection and high sensitivity, could be a complementary solution for conventional sterility tests.

[Analysis of endophytic fungi community of Ligusticum chuanxiong using PCR-DGGE].

OBJECTIVE: To research the diversity of endophytic fungal communities among Ligusticum chuanxiong growing at 5 areas in Sichuan province, and illuminate the developing mechanism of geoherbs from the microecological perspective. METHOD: The PCR-DGGE and DNA sequencing techniques were used to analyze the endophytic fungi community of L. chuanxiong. RESULT: The community of endophytic fungi present difference among different growing areas. Though minor difference were found among individuals at the same area, similarity among individuals from the same growing areas were higher significantly than those from different growing areas. Compared with the other 4 growing areas, L. chuanxiong from Shiyang town, Dujiangyan city had more abundant endophytic fungi and low similarity to others, and which probably had special types of fungi. CONCLUSION: The abundant and stable endophytic fungal community is an important factor for the development of geoherb L. chuanxiong at Shiyang town, Dujiangyan city.

[Isolation and identification of endophytic fungi from Ligusticum].

OBJECTIVE: To investigate the correlation between entophytic fungal community, habitations and varieties of Ligusticum. METHOD: The solidified potato dextrose agar (PDA) of plates was applied for the isolation of the endophytic fungi, and the identification was completed by spot-planting method. RESULT: Fifty strains of the entophytic fungi were isolated from the rhizome of L. chuanxiong collected from 6 habitations. They were morphologically identified as belonging to 13 genera, 4 families, 3 orders and 1 class. CONCLUSION: There were some differences at quantity, species and distributing of the entophytic fungi in different habitats and varieties of Ligusticum, which was suggested that entophytic fungal community is related with specific habitat.