Determination of total flavonoids in three Sedum crude drugs by UV–Vis spectrophotometry

A simple, rapid UV–Vis spectrophotometry method for the determination of total flavonoids in Sedum sarmentosum Bunge., S. lineare Thunb., and S. erythrostictum Migo. was developed, with a good linearity, precision, and stability. The detection wavelength was set at 500 nm, and an extraction solvent was optimized. Through the comparative study of multiple samples of the three plant drugs, their collected seasons and the habitats can be preliminarily ascertained, which may help to control the quality of the medicines to some extent.


INTRODUCTION
Sedum sarmentosum Bunge., S. lineare Thunb., and S. erythrostictum Migo. are three Sedum plant medicines widely used against hepatitis, dysentery, herpes zoster, and swelling in China. Since S. sarmentosum Bunge. is famous for its validity of acute or chronic hepatitis, it has been recorded by Chinese Pharmacopoeia (2010), and various preparations have been developed from it. According to the present reports, the active components of S. sarmentosum Bunge. due to liver protection and alanine aminotransferase (ALT) decrease mainly exist in the water portion and n-butanol portion which principally include the glycosides and flavonoids, suggesting that the medicinal properties of S. sarmentosum Bunge. might result from pharmacologically active bioflavonoids. [1][2][3][4][5] Thus, the total flavonoid determination could reflect the quality of the drug to some extent.
Traditional extraction techniques, such as maceration, heating reflux, and soxhlet extraction, are often effective but time consuming or labor intensive. In contrast, ultrasonic extraction can extract analytes from various matrices in a shorter time. Besides, the quantification of total flavonoids was usually completed by UV-Vis absorption at 500 nm and calculated against a stand reference in routine analyses. [6][7][8][9] Although a number of papers have reported on the quantification of total flavonoids in traditional herbal medicines, few described the determination combined with comparison among several kinds of herbal drugs from different habitats as well as various months of collection together.
This article aimed to develop a rapid simple approach to estimate the quality of the three crude medicines and try to preliminarily explore the relationships among the harvest period, habitat, and drug quality.

Apparatus
KQ-500E ultrasonic apparatus (Kunshan Ultrasonic Instrument Co., Ltd., Beijing, People's Republic of China) was applied here and the outpower was 500 W, with a frequency of 40 kHz. The determination was performed on a 757 CRT UV-Vis spectrophotometer (Lengguang Instrument Co., Ltd., Shanghai, People's Republic of China).

Reagents and materials
The standard rutin was purchased from National Institute for the Control of Pharmaceutical and Biological Products (batch number: 100080-200707); the ethanol and petroleum ether employed were of the analytical reagent grade. Double distilled water was used in this work. and were respectively identified as S. sarmentosum Bunge., S. lineare Thunb., and S. erythrostictum Migo. by Professor Wan Dingrong from College of Pharmacy, South-Central University for Nationalities [ Table 1].

Sample preparation
Each dried plant drug was crushed and passed through a 20-mesh sieve. Then 1.0 g of each sample was accurately taken, mixed with 50 ml of petroleum ether, and extracted ultrasonically twice for 40 min. Poured out the petroleum ether solution. After the sample powder was dried completely, 100 ml of 70% ethanol was added, and subjected to another ultrasonic extraction for 90 min (3 × 30 min) at 55°C, and then filtered into a flask to make a total volume of 100 ml with 70% ethanol.

Standard preparation
A total of 200.0 mg/l of the rutin standard solution was prepared by dissolving rutin reference material in 70% ethanol.

Procedures for the determination of total flavonoids
A total of 4 ml of each sample extraction was pipetted into a 10-ml volumetric flask. The solution was treated with 0.40 ml of the 5% NaNO 2 solution for 6 min and evenly mixed, into which 0.4 ml of the 10% Al(NO 3 ) 3 solution was added and shaked up; then 6 min later, 4ml of the 4% NaOH solution was added to it. The mixture was diluted to the volume with double distilled water, and allowed to stand for 15 min before analyzing against the blank solution.

Selection of the detection wavelength
The absorption spectra of the three sample extraction and rutin solutions were obtained. The absorption peak of rutin was at 510 nm and S. lineare Thunb. at 500nm, while both S. lineare Thunb. and S. erythrostictum Migo. solutions had strong absorptions at a little less than 500 nm. Comprehensively, 500 nm was chosen as the detection wavelength.

Optimization of the extraction solvent
Since flavonoids contain some hydroxyl groups, they often dissolve easily in alcohol or alcohol-water mixtures. The S. sarmentosum Bunge. sample (Jianshi, June 28, 2006) was employed here, trying to find out the optimum solvent for sample extraction. The relationship between alcohol concentration and the yield of the total flavonoids is shown in Figure 1. In the figure, we could see that the extraction yield kept increasing and reached the top when the ethanol concentration went up to 70%, and then began to fall sharply as the ethanol concentration continued growing. Hence, the optimum extraction solvent turned out to be 70% ethanol.

Method validation
All the sample solutions applied here were made from the S. sarmentosum Bunge. sample collected in Jianshi, June 28, 2006.
The linearity of the method was tested by analyzing different amounts of the standard solution (0, 0.2, 0.5, 1, 2, 3, 4 ml, respectively) coupled with isometric color development reagents according to the procedures for the determination of total flavonids. A good linear response was shown (r > 0.999) over the range of 4-80 μg/ml, with an equation A = 12.155C + 0.005, in which A meant the absorbance value while C (mg/mL) represented the concentration of the rutin solution.  Exactly took 4 ml of the S. sarmentosum Bunge. sample solution, operated according to the colorimetric method mentioned above and determined successively for five times. The average absorption was 0.489, and the RSD attained was 0.09%, demonstrating that the instrument used had a high precision.
Recovery experiment was performed to evaluate the accuracy of the methods. 1.0g of S. sarmentosum Bunge. sample powder were spiked with 0.5ml rutin solution (containing 10 mg of rutin) prior to the extraction. The spiked samples were analyzed in six copies. Recoveries of total flavonoids obtained are shown in Table 2, which informed us that the method possessed a nice accuracy.
After the extraction of S. sarmentosum Bunge., the sample mentioned above was dealt with according to the  procedures for the determination of total flavonoids; absorptions were measured every 20 min ranging from 0 min to 120 min [ Figure 2]. It's clear that the absorption of the solution was relatively stable (RSD = 0.78%) if the measurement was carried out within 120 min. Thus, all the analyses should be performed within 2 h after the color reaction.

Sample analysis
The present method was applied to the analyses of flavonoid contents in three crude drugs from Genus Sedum and the results are displayed in Table 3. It is obvious that all the related plant drugs contain flavonoids, which may be associated with their common pharmacological actions.
A relationship between total flavonoid content and harvest season is also revealed by  Figure 5]. It seemed  samples collected in the west of Hubei Province (Jianshi or Yichang) usually possessed higher flavonoid quantities than those collected in the east. Therefore, it was reasonable to deduce that Western Hubei should be a more suitable habitat to collect the three crude drugs from genus Sedum than the east.

CONCLUSION
As the components contained in a Chinese traditional medicine are various and complicated, the strong polar glycosides, i.e., S. sarmentosum Bunge. glycoside (Sarmentosin), are not the only active composition associated with hepatitis treatment. Flavonoid ingredients are also the key point. Therefore, the flavonoid quantities reflected the quality of the three medicines from one aspect. Studies on total flavonoid determination of the S. sarmentosum Bunge., S. lineare Thunb., and S. erythrostictum Migo. samples collected from different habitats and months clearly manifested that the flavonoid contents are closely related to the collected seasons, which may help to judge the optimum harvest time of the three plant drugs, so that the quality could be preliminarily controlled. The results also suggested that the flavonoid content changed with the habitat. The samples harvested in the west of Hubei usually possessed higher flavonoid contents compared with those collected in the east.
The method established for the flavonoid determination in three crude drugs of genus Sedum was simple, direct, and accurate, providing a valuable reference for quality control.