| Salvia miltiorrhiza, named ―Danshen‖ in China, is an important traditional Chinese medicine in the Salvia genus of the Lamiaceae family. Two groups of effective components were found in Danshen: the phenolic acids and tanshinones. Salvia is a large genus with 84 species distributed in China, the active compounds existed in many Salvia are proven common to Danshen, such as Salvia przewalskii, Salvia castanea Diels f. tomentosa Stib., Salvia yunnanensis etc., which are used as ethnomedicinal plants and substitution of Danshen material for the therapy of disease. The usage of Salvia closely related species in some areas influences the circulation and application of S. miltiorrhiza, which is the unique plant of Danshen approved by Chinese pharmacopeia(version 2010). This study aimed to the differences of effective components composition, the influence factors of active compounds accumulation in S. miltiorrhiza and several closely related Salvia species from different habitat regions, to evaluate the relationship between the components and genetic diversity of diverse Salvia species, and to supply the theory evidence of development and exploitation of these closely related species of S. miltiorrhiza. The main results we got were listed as follows:1. Six germplasm of S. miltiorrhiza and several different closely related Salvia species were collected from China, the active compounds from different parts of these Salvia species were evaluated in the whole growth period. Amounts of tanshinones were found in the roots of S. castanea Diels f. tomentosa Stib. and S. przewalskii, the tanshinone IIA content is close to Danshen, which showed gradual increase followed by the growth of plants. The leaves of different Salvia are rich in phenolic acids; rosmarinic acid contents were higher in leaves of S. castanea Diels f. tomentosa Stib., S. przewalskii and Salvia officinalis. While, salvianolic acid B were trace amount in different Salvia species apart from Danshen. The root to shoot ratio of S. miltiorrhiza was gradually enhanced by the plant growth, and lots of fresh roots were obtained at the harvest time. Meanwhile, the activities of two key enzymes in phenolic acids metabolic pathway: phenylalanine ammonia lyase and tyrosine aminotransferase were evaluated. These two enzyme activities were significantly higher in different Danshen than in other Salvia, which was correlated to the high amounts of rosmarinic acid and salvianolic acid B found in Danshen. The two enzyme activities were higher in S. castanea Diels f. tomentosa Stib. and S. przewalskii than in other Salvia species.2. Using high performance liquid chromatography(HPLC) method to analyze the effective components of S. przewalskii from four habitat regions: Mangkang county, Tibet; Litang county, Sichuan; Shangluo and Yangling, Shaanxi. The soil element levels from selected regions were analyzed, and evaluated the relationship between the active compound contents and soil element levels by correlation analysis. The results showed that ferulic acid content of S. przewalskii roots from Mangkang and Litang(wild type) is lower than that of Shangluo and Yangling samples(cultivated); 4.25 mg g-1 DW of ferulic acid at most was found in Yangling sample. However, the caffeic acid level was markedly higher in wild type of S. przewalskii leaves than that of the cultivated ones, which was of 0.64 mg g-1 DW at most found in leaves of Litang sample. The rosmarinic acid contents were different from roots and leaves of selected S. przewalskii. For the tanshinone IIA comparison, the highest level of 24.67 mg g-1 DW was found in S. przewalskii root from Mangkang than that of the cultivated ones. Apart from dihydrotanshinone, almost no other tanshinone was tested in S. przewalskii leaves. The correlation analysis showed that phenolic acids from roots of S. przewalskii were positively related to the potassium and calcium levels in the soil(P <0.05). However, tanshinones from S. przewalskii roots showed negative correlation with manganese content in the soil(P <0.05). The results confirmed significant differences of effective components accumulation in S. przewalskii collected from diverse regions of China, which was mainly affected by the composition of the soil elements.3. Start codon targeted polymorphism(SCo T) and DNA barcoding(psb K-psb I intergenic spacer) methods were performed to analyze the genetic diversity of different Salvia species, the polymorphism of SCo T and sequences of psb K-psb I intergenic spacer were analyzed by unweighted pair group method analysis(UPGMA) and neighbor-joining(NJ) algorithms, respectively. The phylogenetic trees formed by these two methods showed similar results. Different Danshen germplasm formed one group, which are rich in tanshinones and salvianolic acid B, showed similar genetic background. S. przewalskii from different regions and S. castanea Diels f. tomentosa Stib., which are of high tanshinones and rosmarinic acid, formed another group; Salvia roborowskii, Salvia deserta and S. officinalis formed one group, which only contained rosmarinic acid. The present study displayed affluent genetic diversity of different Salvia species.4. The total phenol, total flavone contents, and in vitro ferric reducing ability of different Salvia species extracted by methanol were evaluated. When the sample concentration is 5 mg ml-1, S. miltiorrhiza from different regions showed high total phenols, total flavones contents, and remarkable antioxidant ability. S. przewalskii, S. castanea Diels f. tomentosa Stib. and S. officinalis displayed higher activities than other Salvia, which approached the activities of Danshen materials. After correlation analysis, the total phenols and total flavones contents from different parts of Salvia species were identically and significantly correlated with the antioxidant ability, which showed diverse and powerful biological activity.5. The essential oils from fresh leaves and flowers of S. miltiorrhiza, S. przewalskii, S. officinalis and S. deserta were performed by simultaneous distillation extraction method; the compounds and relative peak area were identified by GC-MS method. The results showed that different leaves and flowers of Salvia are rich in essential oils; and the oil yield was influenced by plant species and tissue specificity. The main compounds in S. miltiorrhiza leaf and flower oils were aromadendrene oxide-(1) and β-caryophyllene; leaf and flower oils of S. przewalskii are of different levels of limonene. The leaf and flower oils of S. officinalis are rich in β-thujone and α-thujone, respectively. The primary compounds in S. deserta oils are ledol and β-phellandrene, respectively. A principal component analysis(PCA) was performed with 11 common components in all Salvia essential oils, PC1 and PC2 could distinguish different Salvia species with diverse compounds. Meanwhile, cluster analysis with all the compounds identified in Salvia oils showed that S. miltiorrhiza and S. przewalskii formed one group, S. officinalis and S. deserta formed another group, which showed similarities respectively. The composition of essential oils from different Salvia species is mainly determined by the genetic diversity and tissue specificity of Salvia plants.6. The hairy root cultures of S. castanea Diels f. tomentosa Stib. was inoculated with Agrobacterium rhizogenes ATCC 15834 for the first time. The transformation rate was 3.92 to 8 %. The fast-growing and high-tanshinone-content hairy root line was selected for enlargement culture, and chose 6,7-V liquid medium as the appropriate culture substrate. Tanshinone IIA content of the hairy root cultured in 6,7-V medium for 30 days was of 0.91 mg g-1 DW, which was significantly higher than that of S. miltiorrhiza hairy root, and this is in accordance with the situation in their parent plants. The result showed that hairy root culture of S. castanea Diels f. tomentosa Stib. is an effective approach for tanshinones achievement in vitro. The hairy root growth curve was evaluated based on the fresh and dry weights, and an S-curve was present in the whole growth period. Thus, 18 day-old hairy root cultured in 6,7-V medium at the intermediate growth stage was favorable for elicitation.7. Three typical elicitors: Ag+, methyl jasmonate(Me JA) and yeast extract elicitor(YE) were applied in the hairy root cultures of S. castanea Diels f. tomentosa Stib. for the growth and effective components evaluation. Ag+(15 μM) promoted the fresh weight of hairy root, and a 1.8-fold increase of tanshinone IIA content was obtained compared with that of the control. The dry weight of hairy root was increased by 200 μM Me JA addition, cryptotanshinone and tanshinone IIA levels were increased to 1.5-fold and 1.99-fold than that of the control, respectively. The dry weight of the hairy root was polished up to 1.73-fold higher than control by 200 mg l-1 YE treatment. An activation of dihydrotanshinone with 1.95 ±0.09 mg g-1 DW was present after YE addition. Meanwhile, the cryptotanshinone level in the hairy root was promoted to 8.37-fold(2.84±0.33 mg g-1 DW) than that of the control. A 2.77-fold increase of tanshinone IIA(2.52±0.67 mg g-1 DW) was also noticed by the YE treatment. The result showed that yeast extract is the most effective elicitor for tanshinone accumulation in the hairy root cultures of S. castanea Diels f. tomentosa Stib. After the time course assay, the selected elicitors showed multiple influences on effective components at 0 d, 0.5 d, 1 d, 2 d, 4 d and 7 d, and most striking increments of tanshinones were observed at the end of the treatment period.8. The fragments of four key genes located in the tanshinone metabolic pathway were homology-based cloned from S. castanea Diels f. tomentosa Stib. hairy root, which are the 3-hydroxy-3-methyl glutaryl coenzyme A reductase(HMGR), 1-deoxy-D-xylulose 5- phosphate reductoisomerase(DXR), isopentenyl diphosphate isomerase(IPPI) and geranylgeranyl diphosphate synthase(GGPPS). The relative expression levels of these four genes were evaluated by treatment of diverse optimum concentration of elicitors. The results showed that HMGR, DXR and GGPPS transcripts were apparently promoted within 12 h treated by 15 μM Ag+, the maximums of different gene expression levels were found at 24 h with 3-fold, 2-fold and 4.6-fold increase compared with that of the control, respectively. Me JA(200 μM) showed multiple effects on gene expression levels in this hairy root culture. After 200 mg l-1 of YE addition, the relative expression level of IPPI was severely up-regulated within 12 h, which was 13.9-fold higher than that of the control, HMGR and GGPPS transcripts approached their maximums of 5.6-fold and 16.7-fold relative to the control at 24 h, respectively. The expression level of DXR was increased to 5.9-fold than control after YE treatment for 4 d. The up-regulations of tanshinone metabolic related genes were markedly influenced by YE treatment, which was better than Ag+ and Me JA. |
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