Total monitoring of the constituents of Danshen tablet using micellar electrokinetic chromatography fingerprinting for antioxidant activity profiling.

A micellar electrokinetic chromatography fingerprint coupled with quantification was developed for assessing the quality consistency of Danshen tablets, a traditional Chinese medicine, with pharmacological activities of activating blood to remove blood stasis and regulating the flow of vital energy to alleviate pain. Square optimization method was proposed to select the suitable background electrolyte. Subsequently, the index of chromatographic fingerprints F was simultaneously employed as an objective function to evaluate capillary electrophoresis conditions. After that, the method was proved to meet the fingerprint analysis criteria by evaluating stability, linearity, limit of detection, limit of quantification, precision, accuracy, and repeatability. Moreover, systematic quantified fingerprint method was used to evaluate chromatographic fingerprints of 30 batches of samples in terms of both quality and quantitation. The result indicated that the chemical compositions of samples were basically similar while their contents showed marked variation. In addition, the fingerprint-efficacy relationship was established using partial least squares model, which can serve as anti-oxidant activity evaluation of Danshen tablets as well as a reference for the selection of active constituents. The proposed method can be applied for the holistic quality control of Danshen tablets.

[Herbal textural and original plants research on medicines from Salvia in China].

On the basis of review and study on literatures of Chinese medicine, combining the history of medicinal plants of Salvia in China, and investigating the morphological description, geography distribution and therapeutic effects, herbal textural research were carried on the medicinal plants from Salvia. The results showed that the original plant of Danshen is S. miltiorrhiza and related species, the original plant of Dian Danshen is S. yunnanensis, the original plant of Li Zhi Cao is S. plebeia, the original plant of Shi Jian Chuan is Bidens pilosa. or S.chinensis, the original plant of Shu Wei Cao is S. japanica, the original plant of Ye Xia Hong is S. kiangsiensis. And the article aimed to point out the relationship of medicinal plants from Salvia and provided new insight and proof to explore the new natural medicine from medicinal plants of Salvia.

Diversity evaluation of Salvia miltiorrhiza using ISSR markers.

Salvia miltiorrhiza is a well-known medicinal plant and functional food in Asia. The range of quality among its germplasms is irregular, which inevitably affects its health and economic benefits. We established a system comprising phenotypic characteristics, ISSR markers, and active constituents to facilitate the comprehensive evaluation of this species. Concerning the phenotypic traits, 55 germplasms were characterized and divided into eight groups. ISSR molecular markers were employed to detect polymorphisms and distinguish germplasms by integrating with phenotypic characteristics. In terms of economic benefits, we were able to screen out the tall-stem and purple-stem types, both with good yields (root biomass) and high contents of active constituents, as members of a preferred-quality group in S. miltiorrhiza. Our study is the first to provide an overall assessment of different S. miltiorrhiza germplasms. The integrated investigation of the phenotypic, genetic, and phytochemical diversity of S. miltiorrhiza germplasms demonstrates the great potential of diverse genotypes to be exploited by plant breeders.

[Micromorphological comparative identification between several Chinese herbal medicines and their counterfeits].

OBJECTIVE: To identify comparatively several commercial Chinese herbal medicines and their counterfeits. METHOD: The micromorphological characters were identified. The shape, surface, section and other characters of the medicinal materials were identified by using anatomical lens and scanning apparatus. Pictures were taken and saved. RESULT: Main micromorphological differences between several Chinese herbal medicine including Lonicera macranthoides, L. similis, Cuminum cyminum, Plantago asiatica, Cuscuta chinensis, Sinapis alba, Salvia miltiorrhiza and their counterfeits were identified. CONCLUSION: The reference for the authenticity identification of Chinese herbal medicine and helpful experiences for the research of the same subject were provided.

RNAi-mediated suppression of the phenylalanine ammonia-lyase gene in Salvia miltiorrhiza causes abnormal phenotypes and a reduction in rosmarinic acid biosynthesis.

Medicinal Salvia miltiorrhiza contains two main groups of active pharmaceutical ingredients: lipid-soluble tanshinones and water-soluble phenolic acids, including rosmarinic acid and salvianolic acid B. Phenylalanine ammonia-lyase (PAL) catalyzes the first step in the phenylpropanoid pathway and is assumed to be closely related to the accumulation of rosmarinic acid and its derivatives. We selected a 217-bp fragment, located at the 3' end of the coding region of PAL1, to establish an RNA interference construct that was introduced into S. miltiorrhiza via Agrobacterium tumefaciens-mediated transformation. PAL-suppressed plants exhibited several unusual phenotypes such as stunted growth, delayed root formation, altered leaves, and reduced lignin deposition. The total phenolic content was decreased by 20-70% in PAL-suppressed lines, and was accompanied by lower PAL activity. Down-regulation of PAL also affected the expression of C4H, 4CL2, and TAT, which are related genes in the rosmarinic acid pathway. Moreover, rosmarinic acid and salvianolic acid B were markedly reduced in PAL-suppressed lines, as detected by HPLC analysis. Our results indicate that PAL is very important for the synthesis of major water-soluble pharmaceutical ingredients within S. miltiorrhiza.

The c4h, tat, hppr and hppd genes prompted engineering of rosmarinic acid biosynthetic pathway in Salvia miltiorrhiza hairy root cultures.

Rational engineering to produce biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Here we capitalized on our previously described gene-to-metabolite network in order to engineer rosmarinic acid (RA) biosynthesis pathway for the production of beneficial RA and lithospermic acid B (LAB) in Salvia miltiorrhiza hairy root cultures. Results showed their production was greatly elevated by (1) overexpression of single gene, including cinnamic acid 4-hydroxylase (c4h), tyrosine aminotransferase (tat), and 4-hydroxyphenylpyruvate reductase (hppr), (2) overexpression of both tat and hppr, and (3) suppression of 4-hydroxyphenylpyruvate dioxygenase (hppd). Co-expression of tat/hppr produced the most abundant RA (906 mg/liter) and LAB (992 mg/liter), which were 4.3 and 3.2-fold more than in their wild-type (wt) counterparts respectively. And the value of RA concentration was also higher than that reported before, that produced by means of nutrient medium optimization or elicitor treatment. It is the first report of boosting RA and LAB biosynthesis through genetic manipulation, providing an effective approach for their large-scale commercial production by using hairy root culture systems as bioreactors.

[Differences in shapes and properties and microscopic frameworks of wild and cultivated Radix Salviae Miltiorrhizae from different regions].

OBJECTIVE: To compare the differences in shapes and properties and the microscopic frameworks of the wild and cultivated Radix Salviae Miltiorrhizae from different regions. METHOD: The differences in the shapes and properties, the characters of transverse sections, the powder and disintegrated tissue of roots were compared using microscopic measurement and statistics analysis. RESULT: The wild Radix Salviae Miltiorrhizae had several long cylinder roots with rough flaky squama skin and brown red or wine culour, the cultivated had root of many branch with cling skin and brick-red or chestnut culour. The difference of microscopic histological structure was that the xylem vessel of wild Radix Salviae Miltiorrhizae had bunched vessel with the rank form of big diameter alternating with small diameter, and had stone cell on samples from some producing region, the xylem vessel of the cultivated had no bunched vessel and no stone cell with the rank form of tangential radial. Radix Salviae Miltiorrhizae cultivated in Sichuan Province is called original-region medicinal materials and named Chuandanshen. Chuandanshen had the differences with the Salviae Miltiorrhizae Radix cultivated in other region. The root of Chuandanshen had 1.2 cm diameter, and was bulky and fat with solid fabric and the fracture with brownish yellow color and cutin-alikeness, its xylem vessel of transverse section of root was thin with the rank form of tangential radial, and 19-24 vascular bundle and a few wood fiber. CONCLUSION: Salviae Miltiorrhizae Radix of the wild and the cultivated, of the original-region (Chuandanshen) and the other-region, have the differences in the shapes and properties, and the microscopic frameworks. The character can be identified by the differences in the shapes of medicinal materials, and the rank form of vascular bundle of transverse section of root.

Direct purification of tanshinones from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography without presaturation of the two-phase solvent mixture.

Tanshen, the rhizome of Salvia miltiorrhiza Bunge, is a famous Traditional Chinese Medicine for multiple therapeutic remedies. This work presents the isolation and purification of tanshinone I and tanshinone IIA from the extract of the rhizome of S. miltiorrhiza by using high-speed counter-current chromatography (CCC) without presaturation of the two-phase solvent mixture. The CCC method combines the results of CCC solvent system selection and components analyses of solvent mixture by GC, and thus it is possible to add accurately each individual solvent to prepare single saturated solvent phase without presaturation. The optimum CCC solvent system is a system of hexane-ethyl acetate-ethanol-water (8:2:7:3, v/v), which has been determined by usual solvent system selection and CCC runs. As a result, over 98% pure tanshinone IIA and over 94% pure tanshinone I have been obtained by using less solvent volume. Their structures have been identified by ESI-MS, NMR spectra.

[Study on configuration fabric and germinative conditions of Salvia miltiorrhizy seeds].

OBJECTIVE: To provide theoretic warrant and technical reference for Salvia miltiorrhizr standardization planting, by carrying out various systemic studies such as observation of seeds configuration fabric, idiosyncrasy of water absorption and groping germinating conditions. METHOD: In the study of configuration fabric, seeds were observed and taken photos by scanning electronic microscope, and heft method was used for measuring changes of water absorption velocity and dehydration velocity. Seeds germination conditions were probed into under the national test regulations for crop seeds and related prescription from international standards. RESULT: (1) There was a layer of slime about 10-20 microm thickness covering epicarp of Danshen seeds. The slime formed as diamond meshwork (reseau) and the weight of it was 8%-10% of total seeds weight. (2) The speed of water absorption of seeds was extremely rapid. The weight of seeds could increase above 10 times as original while the dehydration velocity was quite low. (3) The optimal temperature for the seeds germination is around 25 degrees C, and the germination rate of the new seeds gained yearly was above 75%, but the rate would decrease sharply as years went by. It was also found that the seeds germination power and exponent of vigor were quite high under the temperature transformation between 23 degrees C, 28 degrees C. Such treatments as pre-cool, PEG treatment and infusing with GA3 could increase the rate of seeds germination capacity obviously.