Sedum sarmentosum Bunge., S.lineare Thunb. and S. erythrostictum Migo., whose dried herbs are traditional herbal medicines, all belong to Genus Sedum. As the three plants are closely related with each other, they usually possess similar chemical components. Despite certain differences that existed among their efficacy, all could treat hepatitis, dysentery, swelling poison and so on.1-4 It has be proved that sarmentosin, a water-soluble component contained in S. sarmentosum Bunge., is one of the active components to protect liver and reduce serum alanine aminotransferase level. Many reports on the methods to determine sarmentosin have appeared, including to the IR, HPLC and GC approaches.5-7 The studies also find that the flavonoids included in S. sarmentosum Bunge.8 show a better efficacy in liver protection and the enzyme decrease compared with the water-soluble total glycoside,9 suggesting that flavonoids should also be main active composition to treat hepatitis. Through the qualitative analysis and TLC assay, we have verified that both S. lineare Thunb. and S. erythrostictum Migo. also contained flavonoids. And the preliminary HPLC analysis has shown that the three plant drugs all contain the flavonoid glycosides whose aglycones include quercetin. As HPLC has been a routine methodology to determine the active composition in natural plants both at home and abroad,10-13 in this study, we have developed a HPLC means to determine the quercetin content of the samples collected in various seasons, trying to find out the variation trends of the some flavonoids content vs. the growing periods, obtain the appropriate harvest seasons and preliminarily control the quality of the 3 medicines finally.
Reagents and materials
The quercetin reference substance was purchased from Chinese pharmaceutical and biological product verification station (batch number: 10008-200406); methanol and phosphoric acid employed here were HPLC grade reagents. Deionized (DI)water was used in the experiment.The three plant medicines collected from Jianshi, Huangmei, and Sheshan (Wuhan), Hubei province of China, and were respectively identified as the dried herbs of S. sarmentosum Bunge., S. lineare Thunb. and S. erythrostictum Migo. by Professor Wan Ding-rong from college of pharmacy, South-central University for Nationalities.(Table 1)
High performance liquid chromatography (HPLC) analysis was performed using an Agilent 1200 HPLC system from Agilent (Karlsruhe, Germany), equipped with a quaternary pump, an autosampler, and a VWD UV detector. Quercetin was separated from the sample solutions using a C18 column (4.6×250 mm I.D., particle size 5 µm, Agilent Eclips XDB- C18), with a mobile phase consisting of methanol and 0.40% phosphoric acid(49:51, V/V), at 25°C. The flow rate was 1.0 ml/min , and injections were 20 µl in volume.
Standard solution preparation
10.3 mg of the quercetin standard substance was accurately weighed into a 100 ml volumetric flask and made up to the volume with methanol. After evenly mixed, 2 ml was accurately transferred to a 10 ml flask and was diluted with methenol to the volume. Thus, quercetin standard solution (20.6 µg/ml) was abtained.
About 2.0 g of dried (100°C, to a constant weight) of Sedum sarmentosum, S. lineare and S. erythrostictum. samples powder was taken and weighed accurately and respectively, then 20 ml of methanol was added and refluxed twice( each time for 30 min ).The mixture was filtered into a 50ml volumetric flask. The residue was washed with methanol (2×10 ml), and transferred to the same flask, consecutively, and then made up to the volume with methanol, and shaken until evenly mixed. After that, 20 ml was taken, mixed with 5ml of 25% hydrochloric acid solution, refluxed for 30 min, then cooled immediately and transferred to a 50 ml volumetric flask, and made up to the volume with methanol, finally shaken, filtered through a 0.45 µm millipore filter prior to HPLC analysis.
Determination of the content of quercetin by HPLC
A calibration curve was established for quercetin by injecting 2, 4, 8, 12, 16, 20 µl of the standard solutions twice respectively. Linearity was tested by analyzing the average peak area of quercetin of different injection volume. And then the regression equation(y=74.431x－0.9177) was obtained，and the correlation coefficient （r） was 0.9999. Thus, a good linearity was shown when the quercetin concentration ranged from 41.2 to 412.0 µg/ml. [Figure 1].
The precision was assessed by injecting 20 µl of quercetin standard solution five times respectively. The quercetin content was calculated based on the calibration curve. The content variation (RSD, %) was found to be 1.04%, demonstrating that the instrument used had a high precision.
The 20 µl of the same S. sarmentosum Bunge. sample (Jianshi, April) solutions were injected into the apparatus in a certain period of time (0,2,4,6,8,12 h) respectively, and quercetin content was calculated based on the calibration curve. As a result, the content variation (RSD, %) was 1.65%, revealing that the sample solution was stable within at least 12 h.
Five same dried samples of the three plant medicines harvested in April were taken respectively to prepare the sample solutions according to the method mentioned above prior to the HPLC determination. The consequences showed that the content variation RSD (%) of each sample was less than 2.8%, indicating a good repeatability of this method. [Figure 2, 3, 4]
In the linear range, 1.0 mg of quercetin which was nearly equal to that contained in 2.004 g of dried S. lineare Thunb. Sample collected in June in Huangmei was accurately taken to prepare the solution for recovery test according to the sample preparation method mentioned above. The quercetin content was determined according to the above chromatographic conditions. The recovery test was repeated six times. By comparing obtained quercetin content with that actually injected each time, the average recovery was100.52%, and RSD (%) was 2.2%, showing that developed determination method had a nice accuracy.
Average retain time of quercetin peak was 8.835 min, and the quercetin standard substance HPLC peak was as follows [Figure 3].
A total of 9 samples of three plant medicines collected in different periods were determined by the developed analytical method. The results of the quercetin content were given in the following table (Table 2). And the relations of the content change and the collected months were shown as follows [Figure 2].
All Sedum samples harvested in different seasons were weighed accurately, prepared according to the previously described method and analyzed by injecting thrice into the HPLC. The amounts of quercetin in these Sedum samples are listed in Table 1, and the variation with respect to the harvest season of the three Sedum medicinal plants is shown in Table 2. The results showed that the amounts of quercetin in Sedum sarmentosum, S. lineare and S. erythrostictum, and the content of quercetine in this three drugs can achieve the quality standard required by Chinese Pharmacopoeia.( No less than 0.01%). In conclusion, the contents of quercetin varied in all Sedum medicinal samples harvested in different seasons. The content of quercetin was higher in Sedum sarmen tosum harvested at the beginning of april, but lower in June, and grown a little bit at September，the highest content of quercetin reached 0.0512 percent at april，and then The content of quercetin in S. lineare and S. erythrostictum increased in the later harvest months, and the highest content of quercetin reached 0.0659 and 0.0167 percent at September respectively.
By the HPLC analysis, we found that Sedum sarmentosum, S. lineare. and S. erythrostictum all contained flavonoid glycosides, whose hydrolysate included quercetin. The results suggested that the common flavonoids existed in the 3 plant medicines should be associated with anti-hepatitis activity.14,15
The results also revealed preliminarily a relation between collected season and medicine quality. In detail, the quercetin content of S. lineare Thunb. went up with growing months and reached the climax in October, while both S. erythrostictum Migo. and S. sarmentosum Bunge. had the relatively high amount in April (flowering period), and were the lowest in June (during this period, the growing state of S. sarmentosum Bunge. was poor, and the leaves mostly withered), then increased a little in September, showing preliminarily that the last two would own the best quality if collected in flowering period.
In a word, The HPLC assay of the quercetin in the hydrolysate of the 3 herbal drugs could be used to control the drug quality to a certain extent from the aspect of the related flavonoids content.