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1、DIFFERENTIATION OF CIRSIUM SPP. BY TLC AND HPLC-MS205Copyright 2005 John Wiley the total flavonoid content varied from 0.81 to 3.67%. In C. setosum only one flavonoid (linarin; 1.36 2.83%) was assignable. The HPLC method was validated for linearity, limit of detection ( 1.7 ng on-column), peak purit
2、y, repeatability ( 2.3%) and accuracy (recovery rates of spiked samples were between 99.2 and 101.6%). Copyright 2005 John Wiley high performance liquid chromatography; mass spectrometry; flavonoids; Cirsium japonicum; Cirsium setosum.* Correspondence to: M. Ganzera, Department of Pharmacognosy, Ins
3、titute of Pharmacy, University of Innsbruck, Innrain 52, 6020 Innsbruck, Austria. Email: Markus.Ganzerauibk.ac.atINTRODUCTIONTwo of Cirsium species (family Compositae) are import- ant components of traditional Chinese medicine, which is becoming increasingly popular in the Western world.Cirsium seto
4、sum Willd. (field thistle; Xiaoji) and C.japonicum DC (Japanese field thistle; Daji) are used internally and externally to treat diverse kinds of bleed- ing (e.g. haematuria, spitting of blood, uterine bleeding)and inflammation (Kim and Kim, 2003; Pharmacopoeia of the Peoples Republic of China, 2000
5、). The anti- haemorrhagic principle in C. japonicum was found to be pectolinarin (2; Ishida et al., 1987); other known constitu-ents of this species include additional flavonoids e.g. hispidulin-7-neohesperidoside (1), linarin (3), luteolin (4), polyacetylenes and phytosterols (Miyaichi et al., 1995
6、; Park et al., 1997; Fei et al., 2003; Jordon-Thaden and Louda, 2003). The recently reported liver-enzyme- activating effects of extracts of C. japonicum appear to be of pharmacological interest, with 1 being the most active (Park et al., 2004). C. setosum is known tocontain several flavonoids (main
7、ly 3, plus syringin and astragalin); pharmacological studies have not been performed with this species (Redyuk et al., 1977; Syrchina et al., 1997). Both species are herbaceous perennials, 0.51.0 m in height, with lance-shaped, spiny-toothed leaves andpurple to pink flowers. The aerial parts, which
8、are usedmedicinally, are difficult to distinguish morphologically,but differentiation of the dried and cut crude drug is even more challenging, and has been described thus far only by microscopic study (Moeck, 1998; Pharmacopoeia of the Peoples Republic of China, 2000). As analytical methods for the
9、 determination of the most important constituents of both species are still not available, this study aimed to achieve twogoals: first, the development of a simple TLC method for the unambiguous differentiation of C. japonicum and C. setosum, and second the elaboration of an HPLC method that would p
10、ermit the quantitative analysis of all compounds of interest. The second methodology was validated and applicable HPLC-MS experiments were conducted in order to ensure peak identity and precision of the obtained results.Structures206M. GANZERA ET AL.Copyright 2005 John Wiley solutions of 2 and 3 ser
11、ved as reference standards. The mobile phase was a mixture of ethyl acetate:formic acid:acetic acid:water (12:1.5:1.5:4, v/v). The plates were developed for 10 cm, dried and sprayed with natural products spray reagent (a 1% solution of 2-aminoethyl diphenylborinate in methanol). After gentle heating
12、, the sheets were inspected under UV light at 366 nm forfluorescent spots: linarin (2; yellow spot at Rf 0.44) and pectolinarin (3; brown spot at Rf 0.38) were clearly separ- ated in the extracts (when present).HPLC analysesSample preparation. The finely powdered plant material(0.500 g) was extracte
13、d five times with 5 mL methanol by sonication (10 min each; room temperature). After centrifugation, the extracts were combined into one25 mL volumetric flask, and the volume completed to the mark with extraction solvent. Prior to injection, allsolutions were filtered through Phenex (Phenomenex, Tor
14、rance, CA, USA) 0.45 m cellulose-acetate mem-brane filters: each sample was analysed by HPLC in triplicate.Calibration. A standard stock solution was prepared by dissolving 2.00 mg of 2, 3 and 4 in 5.00 mL methanol. Five additional calibration levels were prepared by diluting this solution in the ra
15、tio 1:2 with methanol. Within the range of concentrations injected (4001.6 g/mL) the detector response was linear (see Table 1 for calibrationdata; compound 1 was quantified based on the calibra- tion data of the structurally similar 2). The limit of de- tection (with a signal-to-noise ratio of 3) w
16、as determined by further diluting the standard solutions. The solutionswere stable for at least 1 month if stored at 4C (con-firmed by re-assaying).Accuracy. Accuracy was determined by spiking sample CJ 1 (500 mg) with known concentrations of standard compounds 2, 3 and 4, representing 100% of the expected value. Owing to low amounts of standard materials available, recovery was determined at one concentration level only. The spiked sample was ex- tracted and
