Danshen Germplasm Collection And Evaluation Studies

Salvia miltiorrhiza Bunge is a well-known traditional Chinese herb. Its root contains two major groups of identified biologically active compounds, the hydrophilic caffeic-acid-derived phenolic acids, and various lipophilic tanshinones belonging to the diterpene quinines, and has been formulated and used clinically for the treatment of various conditions such as cardiovascular and cerebrovascular diseases. With continued pharmacologic studies on its secondary metabolites, additional biological activities including antioxidant, anti-thrombosis, anti-hypertension, antivirus, antitumor, and so on, have been reported. With the constantly rising demand and severely destroy of the wild resources of S. miltiorrhiza, the cultivar of S. miltiorrhiza increased year by year. As ordinary outcrossing plant, it brought much difficult to identify, evaluate and utilize of S. miltiorrhiza for its rich variety, complex character and widespread variation. So, there is a great significant for combining the modern techniques to studying, exploting, protecting and evaluating the germplasm resources of S. miltiorrhiza.Taking the cultivar and wild germplasms of S. miltiorrhiza from Shaanxi, Shandong, Sichuan, Henan, and Hebei as materials and referring to the national germplasm description standard, we established the description criterion, data standard and germplasm description table. The studying included ISSR molecular marker, HPLC fingerprint and antioxidant activity test system based on 267 germplasm of S. miltiorrhiza. Identify and distinguish different germplasms from the morphological and molecular level. And evaluate the germplasms from the morphological traits, agronomic traits and the contents of active components. The main results are as follows:(1) Based on formulation of description standard in S. miltiorrhiza, we investigated many factors such as geography factors, entironment, morphological traits and agronomic traits and so on. In addition, we collected 267 germplasms of S. miltiorrhiza in total. Analysed and appraised these germplasms on biological traits, agronomic traits and active components.(2) Observation of morphological and agronomic traits: The high yield No.1, high yield No.2, high stem, purple stem, big leaf, small leaf, light green leaf, crinkle leaf, glabrous leaf, round leaf, narrow leaf and yellow root were selected and took them to 3 germplasm nurseries in Huxian, Shangzhou and Pucheng for close observation. The results showed that different germplasms have some differences in leaf shape, leaf size, shape of leaf edge, leaf colour, plant height, stem colour, first and second grade branch number, flower colour, root diameter, root length, yield of individual plant, and content of active constituents. Different germplasms can be identified and distinguished according to their agriculture and apparent characteristics. There are significant difference in leaf size, plant height, root length, root diameter, yield of individual plant, and content of active constituents. We obtained two fine types named High yield No.1 and Purple stem by comprehensive evaluation. The characteristics are as follows: high yield, good growth trend, strong resistance and content of active constituent higher than other types. In addition, Wrinkle leave type showed higher lipophilic constituents than other types.(3) Compared the content of active component: We established the chemical fingerprint of S. miltiorrhiza by HPLC and determined the contents of rosmarinic acid, salvianolic acid B, dihydrotanshinone, crptotanshinone, and tanshinone II_A. The results showed there are significant differences between different germplasms. The high yield No. 1, purple stem, big leaf and small leaf type were possessed high content of water-soluble constituent. In addition, crinkle type possessed high content of lipo-soluble constituent.(4) DNA fingerprints by ISSR analysis were figured out in this paper. The orthogonal testing was used in this study through adjusting template DNA concentration, Mg²⁺ concentration, primer concentration, dNTP and Taq polymerase contents, and annealing temperature. The PCR amplification conditions were optimized. Meanwhile, 11 primers were selected from 60 ISSR primers. A total of 328 bands were obtained, among which 312 were polymorphic bands. The average percentage of polymorphic bands was 95.1 %. By the genomic distance clustering, the population could be divided into 2 main branches and 8 minor branches. ISSR based phylogeny were majority consistent with the based on morphological characters.(5) The antioxidant activities of acetone and methanol extracts of the roots and leaves of S. miltiorrhiza were evaluated with several antioxidant testing systems (DPPH radical scavenging assay, superoxide radical scavenging assay,β-carotene-linoleic acid assay (β-CLA), ferric reducing/antioxidant power assay, and a plasmid DNA damage protection potential assay). The total phenolics (TP) and flavonoids (TF) were measured. Qualitative and quantitative analyses of the major phenolic acids were also carried out by HPLC. The results showed that the mechanisms underlying the antioxidant effects of these extracts were distinct and diverse. In particular, the methanol extract of leaves (ML) exhibited high scavenging activity against DPPH radicals and strong reducing power, which were accompanied by high TP, TF, and salvianolic acid B contents. ML also showed high inhibitory activity (stronger than BHT and VE) against linoleic acid oxidation. Because of several high antioxidant activity results, S. miltiorrhiza must be considered as a promising natural antioxidant for the medicine and food industry.