Studies On The Identification Of Botanical Origins And Cultivation Techniques Of Pulsatilla Koreana

Pulsatilla koreana, a perennial plant belonging to the family of Ranuculaceae, is one of traditonal medicine in China. In recent years, with the ceaseless researches on the chemical constituents and the pharmacological action of Pulsatilla koreana Bercht, the demand of market further increased, and the wild resources cannot meet fundamentally to the needs for that, artificial planting is imperative. The Pulsatilla medicinal plants were distinguished by means of morphological, microscopic and chemical characteristics. And the extraction method of effective components, growth characteristics and the cultivation techniques of Pulsatilla koreana were studied, also established preliminarily the HPLC fingerprints for Pulsatilla koreana so as to provide basis for its quality control. The main results were as follows:Morphological characteristics of Pulsatilla koreana, Pulsatilla chinensis, Pulsatilla dahurica and Pulsatilla turczaninovii were observed. The results showed that4species had significant differences in leaf shape, lobes, sepals and other morphological characteristics. There were small variation within specie and significant differences between species in corolla length, sepal length and achene length, which can be used as indicators of morphological identification.The root of4species consists of periderm, phloem, vascular cambium, and xylem. The phloem Was more developed, accounted for about1/2of transverse radius.The differences of root cross-section between4species were mainly in the xylem. The root xylem of P. koreana was slightly3bunch of shape, which radially arranged in the center, each beamed to outside and had bifurcates, and some conduits arranged irregularly. Xylem conduits of P.chinensis were also in the center, which has3beam tube.P.dahurica and P. turczaninovii were not triarch, P.dahurica had2xylem conduits that radiated out, while xylem conduits of P.turczaninovii had about10to14bunches spreading outward from the center, some bifurcated2times.The differences in powder microscopic characteristics between4species were mainly in the conduit type, presence or absence of stone cells, the number of phloem fibers, the shape of cork cell, and so on. The characteristics can be used to identify4species microscopically. View from the presence or absence of stone cells, P. koreana and P.chinensis had no stone cells, while P. dahurica and P. turczaninovii had a few stone cells. P. chinensis, P. dahurica, and P. turczaninovii had more phloem fibers, while less in P. koreana. The cork cells of P. koreana, P.chinensis and P. turczaninovii were rectangular or near rectangular. However, P. dahurica had several different cork cells, some are rectangular or polygonal or oblong. Furthermore, the vessels of P. dahurica were thicker than other3species, but mainly bordered pits.The extraction conditions of total saponins from P. koreana were studied. The optimum conditons of ultrasound-assisted extraction was in the combination of80%of alcohol concentration,40min of ultrasonic time,1:20of solid to liquid ratio,80W of ultrasonic power and one time of extraction. Among them, alcohol had most significant effect on the extraction of total saponins.The HPLC was used to establish the fingerprint of Pulsatilla medicinal plants, and to evaluate and distinguish the different species of Pulsatilla based on the chromatographic data. The different species of Pulsatilla showed different HPLC fingerprints, but common with10peaks. Q cluster analysis of14 accessions indicated that they were divided into four groups:all accessions from P. koreana were classified into Group Ⅰ, P. ambigua in Group Ⅱ, P. dahurica and P. turczaninovii in Group Ⅲ, and P. chinensis in Group Ⅳ, respectively.The HPLC methods were employed to analyz the fingerprint of different sources of P. koreana. The precision, stability and repeatability of the method was investigated and reached the technical requirements of HPLC fingerprint. The characteristic mode of HPLC fingerprint of P. koreana was established, and23common peaks were identified. The similarity of15lots tested was good, which illustrated the chemical composition of different sources had good consistency. The HPLC fingerprint of P. koreana with high characteristics and specificity can be used for quality control of P. koreana.The growth characteristics and total saponins accumulation in the root of P.koreana were studied. The ontogeny of P. koreana can be divided into five growth stages:budding stage, blooming stage, maturity stage, growth stage and wilting stage. The distribution rates and growth rates of its organs changed greatly during its growth period, the dry matter accumulative curve could be described as "S" shape. The total saponins content of the biennial, triennial, and perennial P.koreana reached the highest in mid-August, total saponin yield per plant reached the highest during the mid-August to the mid-September, then began to decline. Combining the results from the quality and quantity of medicinal material, the best harvest time of P. koreana is from the late August to the mid-September.Using the seeds of P. koreana as materials, basic characters, water absorption characteristics, germination rates and seedling emergence rates of seeds treated by GA3, PEG6000and ultrasonicwaves, were studied in the laboratory. The results showed that seeds had the biological characteristic of fast water absorption, and the results also showed that the three pretreatments with GA3concentration of100mg·L-1for12hours,10%～30%PEG6000for12hours, ultrasonicwaves conditions with40℃,80W,20min had optimum effects on seed germination and seedling emergence, under which seeding emergence rates are above48.0%. Compound fertilizers had obvious effects on the seedlings growth. Fresh weight of leaves in all treatments increased by163.71%on average compared to the CK. Fresh weight of seedling root increased by83.10%on average. The appropriate level for NPK fertilizer was the400～600kg·hm-2. The spraying concentration of trace-element fertilizer was0.10%, spraying every10～15d for3to5times.The results of field experiment conducted based on the D-saturation optimal design with three factors of nitrogen, phosphorus and potassium showed that the effects on the growth, yield and total saponin contents in P. koreana of different proportional of nitrogen, phosphorus and potassium were quite different. The effect of nitrogen, phosphorus and potassium application on the total dry matter accumulation and root yield of P. koreana was as following:nitrogen> phosphorus> potassium, the effect on the content of total saponins was as following:potassium>phosphorus>nitrogen. To reach the goals of high yield and good quality, the optimum proportion was N109.44～123.12kg·hm-2, P2O5113.94～128.34kg·hm-2, K2O143.21～160.99kg·hm-2.