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This study is aimed to establish a high-performance liquid chromatography (HPLC) method for simultaneous determination of skimmin, scopolin and umbelliferone in Saussurea hieracioides. Samples were analyzed on a Wondasil C18-WR column (4.6 mm x 250 mm, 5 microm) with methanol (A) and water containing 0.1% phosphate (B) as mobile phases for gradient elution at a flow rate of 1.0 mL x min(-1). The detection wavelength and column temperature were set at 325 nm and 35 degrees C, respectively, and the sample size was 10 microL. The results showed that skimmin, scopolin and umbelliferone were simultaneously achieved within 40 min under the above conditions. A good linearity was observed in the range of 0.18-5.6 microg (r = 1.000 0), 0.060-1.8 microg (r = 0.999 9), 0.032-0.97 microg (r = 0.999 8) for skimmin, scopolin and umbelliferone, respectively, with the average recoveries of 99.16% (RSD = 0.41%), 100.3% (RSD = 0.79%), 102.2% (RSD = 0.87%). The method is simple, accurate and reproducible and can be used for the quality control of S. hieracioides.
This study established an HPLC fingerprint of Tibetan medicine Shaji Gao from different habitats and lay a foundation for Shaji Gao varieties identification and preparation process. The chromatographic condition was as follow: Agilent zorbax SB-C18 (4.6 mm x 250 mm, 5 μm) eluted with the mobile phases of acetonitrile and 0.4% phosphoric acid water in gradient mode. The flow rate was 1.0 mL x min(-1), and the detection wavelength was set at 360 nm. The fingerprints of 15 batches Shaji Gao were carried out by similarity comparation, 7 chromatographic peaks were extracted as the common peaks of fingerprint, 3 peaks were identified, which were quercetin, kaempferol and isorhamnetin. The similarity degrees of 14 batches of samples were above 0.9 and 1 batch of samples was below 0.9. This is the first established fingerprint of Shaji Gao by using HPLC. This method has good precision, stability and repeatability that it could provide basis for quality control and evaluation of Shaji Gao.
To evaluate the efficacy and safety associated with anti-hypoxia effect and establish the quality standard for Brassicea Radix extract, the investigations of acute toxicity and subacute toxicity were carried out to preliminarily appraise the toxicity, and the models of normal pressure hypoxia, acute cerebral ischemia and sodium nitrite poisoning in mice were used to evaluate the effect of enhancing anoxia endurance. Then according to the methods described in the Appendix of Chinese Pharmacopoeia (2010 edition), the sulfuric acid-phenol method was applied to determine the content of polysaccharide, and the water, ash and insoluble matter in water inspections were carried out and the control medicinal herb was identified with the samples by qualitative TLC. The results indicated that ① the toxic effects (LD₅₀) of mice was 56.73 g•kg⁻¹ by oral administration of Brassicea Radix extract, while Dm and Dn were respective 86.80 g•kg•d⁻¹ and 35.55 g•kg•d⁻¹;②the determined effective dosage of Brassicea Radix extract which could enhance anoxia endurance was 0.388 g•kg⁻¹•d⁻¹; ③ the methods of TLC and the content of polysaccharide were established. The method of quality control has been recorded in Sichuan Province Standard for Tibetan Medicine, which is reliable, accurate and simple, with good reproducibility. Meanwhile, given the prominent effect on anti-hypoxia and good safety, it provided important basis for clinic safe and effective usage and the development of health products.