Quantitative Determination and Variation Tendencies of Flavonoids in Five Selaginella Plant Drugs

Introduction: Selaginella species have been used widely for the treatment of inflammatory diseases, diabetes and cancer. In this study, the contents of the total flavonoids and amentoflavone in five Selaginella plant drugs [Selaginella moellendorffii Hieron. (SM), Selaginella delicatula (Desv.) Alston (SD), Selaginella uncinata (Desv.) Spring (SU), Selaginella heterostachys Baker (SH) and Selaginella remotifolia Spring (SR)] from different harvest time were determined by UV-Vis spectrophotometry and HPLC, respectively. The research aimed to preliminarily confirm the appropriate harvest time of Selaginella plant drugs. Methods: The contents of the total flavonoids and amentoflavone in five Selaginella plant drugs from different harvest time will be determined by UV-Vis spectrophotometry and HPLC, respectively. Results: The proposed methods were rapid and reliable for the quantitative determination of the total flavonoids and amentoflavone in the five plant drugs. The total flavonoids and amentoflavone contents were significantly different in the five plant drugs, and SM showed the highest content in the both contents. The variation tendencies of the total flavonoids and amentoflavone contents from July to October indicated the diversity and complexity of the accumulation of the active ingredients in plants from the same genus. Conclusion: In conclusion, the results provided valuable references for evaluating the quality and preliminarily confirming the appropriate harvest time of Selaginella plant drugs.


INTRODUCTION
The genus Selaginella belongs to the family Selaginellaceae, mostly distributed in the subtropics and the tropics. 1 There are more than 60 species in China, and a number of them are used as Chinese traditional or folk medicines.The surveys indicated that 15 Selaginella species were found in Hubei province, among which 14 species were used as medicines for the treatment of various inflammatory diseases, traumatic hemorrhage and some kinds of cancer. 2,3][6][7][8][9][10][11][12] In this study, we applied UV-Vis spectrophotometry and HPLC to determine the total flavonoids and amentoflavone contents of the samples collected in different harvest time, trying to evaluate the quality and preliminarily confirm the appropriate harvest time of the five Selaginella plant drugs.

Materials and reagents
The amentoflavone reference substance was purchased from the National Institutes for Food and Drugs Control of China (batch number: 111902-201101).Acetonitrile of HPLC grade was purchased from Tedia Company, Inc., Fairfield, Ohio, USA; the methanol, ethanol, phosphoric acid were of analytical grade.The plant materials were collected from Yichang in Hubei province, China, in July and October, 2012.They were respectively identified as Selaginella moellendorffii Hieron., Selaginella delicatula (Desv.)Alston, Selaginella uncinata (Desv.)Spring, Selaginella heterostachys Baker, and Selaginella remotifolia Spring by Professor Dingrong Wan (College of Pharmacy, South-Central University for Nationalities, China).All samples were air-dried, pulverized and passed through a 50 mesh sieve.

Apparatus
The HPLC analysis was performed on a Dionex Ultimate 3000 HPLC System, which was composed of a quaternary pump, an autosampler, a column thermostat, temperature-controlled sample trays, an online degasser and an ultraviolet detector.The total flavonoids content was determined by an UV-Visible spectrophotometer (UV-1800PC, Mapada Co., Shanghai, China).

Determination of total flavonoids
Preparation of sample solutions 1.0 g of each sample was accurately weighed, and then 60 mL methanol (90%, v/v) was added and refluxed for 1.5 h.After being cooled to the room temperature, the mixture was weighed again.The loss in weight was replenished with the same solvent, and then, the solution was shaken and filtered.1.00 mL of the successive filtrate was pipetted into a measuring flask of 50 mL and diluted with 90% methanol for the further use.

Preparation of standard solution
An amount of 4.51 mg of amentoflavone reference substance was accurately weighed, dissolved in methanol and brought to the volume in a measuring flask of 50 mL.The concentration of the standard solution was 90.2 μg mL -1 .

Selection of the detection wavelength
According to the absorption spectra of the standard solution and the sample solution, both amentoflavone and the sample had strong absorptions at 270 nm.Therefore, 270 nm was chosen as the detection wavelength.
Optimization of the sample extraction SD (October 2012) was employed here to explore the optimum extraction process with different extraction solvents, extraction time and material/solvent ratios.The sample solutions prepared under different conditions were determined by measuring the absorbance at 270 nm with an UV-Vis spectrophotometer.The optimum conditions were noted by calculating the extraction yield of total flavonoids on the basis of the calibration curve.

Determination of amentoflavone
Preparation of sample solutions 1.0 g of each sample was accurately weighed, and then 60 mL methanol (90%, v/v) was added and refluxed for 1.5 h.After being cooled to the room temperature, the mixture was weighed again.The loss in weight was replenished with the same solvent, and then, the solution was shaken and filtered through a 0.45 μm membrane into an amber glass HPLC vial before analysis.
Preparation of stock standard solution 5.05 mg of amentoflavone reference substance was accurately weighed and moved into a volumetric flask, dissolved and made up to 10 mL with methanol.The concentration of the stock standard solution was 505.0 μg mL -1 .

Chromatographic conditions
Based on the results of the full wave scanning of the sample solution and the absorbing wavelength of the standard solution of amentoflavone, 270 nm was chosen as the detection wavelength.Reverse phase separation was performed at 30 o C using a BDS Hypersil C-18 column (4.6×250 mm) with a particle size of 5 μm.The mobile phase composed of 65% water (containing 0.1% H 3 PO 4 ) and 35% acetonitrile.The flow rate was kept at 1 mL•min -1 and the injection volume was 10 μL.The chromatographic conditions above were optimized to separate the primary marker peaks of each sample with good resolution (R＞1.5) and theoretical plate numbers (amentoflavone: n＞8000).The compounds were identified by comparing the retention times of sample solution with that of the reference standard amentoflavone (Figure 1).The concentration of the amentoflavone was calculated according to the equation of the calibration curve.Three parallel operations were performed for each sample.

Determination of total flavonoids by UV-Vis spectrophotometry
Calibration curve Standard solutions of amentoflavone with different volumes (0.50, 1.00, 2.00, 3.00, 4.00, 5.00, 6.00, 7.00 mL) were spiked into measuring flasks and diluted with methanol to 50 mL, followed by shaking.The absorbances of the solutions were measured at 270 nm using a spectrophotometer against an appropriate blank solution.Using the absorbability as ordinate and the concentration as abscissa, the specification curve was obtained.The calibration curve presented a linear absorbance response within the concentration range of 0.902-12.628μg•mL -1 .The regression equation was Y=0.0853X + 0.0011 with r=0.9999, showing a good linearity.
Validation SD collected in October 2012 was used for validation.To measure precision, the absorbance of the sample solution was measured by an UV-Vis spectrophotometer for six times.The average absorption was 0.390, and the relative standard deviation (RSD) was 0.13%, demonstrating that the instrument used had a high precision.Repeatability test was carried out with SD powder for six times.The average content of the total flavonoids was 13.73 mg•g -1 with RSD of 0.77%, indicating a good repeatability of this method.As for the stability, the absorbance of the sample solution was measured at 0, 2, 4, 6, 8, 10, 12, 24 h after sample preparation.The results showed that the absorption of the solution was stable (RSD=0.38%)if the measurement was carried out within 24 h.The accuracy was determined by calculating the recovery.Nine samples solution were prepared according to the previously described method and evenly divided into three groups, then added with 1.00, 2.00, 3.00 mL of amentoflavone (concentration: 90.2 μg•mL -1 ) at each group respectively before analyzing by an UV-Vis spectrophotometer.Recoveries of the total flavonoids obtained are shown in Table 1.The mean recovery was 98.50% and RSD was 1.11%, confirming the accuracy of this method.

Calibration curve
Different volumes of the stock standard solution (0.10, 0.20, 0.50, 1.00, 2.00, 3.00, 3.50 mL) was accurately pipetted, diluted to a final volume of 10 mL with methanol, and shaken to the uniform to obtain standard solutions with different concentration.Those standard solutions were injected into the HPLC system in duplicate, and the average peak areas were calculated.The regression equation was Y=63.415X + 0.3959 with the correlation coefficient r=0.9999.The results showed that a good correlation exists between the peak areas (Y) and the amount of amentoflavone (X) within the range of 0.0505-1.7675μg.

Validation
In HPLC analysis, the stock solution and powder of SD (collected in October 2012 ) were used for the validation examination.The precision was evaluated by assaying the sample solution under the selected optimal chromatographic conditions for six times.The RSD of the determination results was 0.09%.The repeatability was estimated by preparing and determining the SD sample simultaneously in six replicates.The RSD of the amentoflavone was 0.45%.These results informed us that the precision and repeatability of the method would suffice for the routine quality control requirements of Chinese medicine.In addition, the sample solution was assayed at 2, 4, 6, 8, 10, 12, 24 h after sample preparation to assess the stability of amentoflavone.The results manifested that the analyte was stable in 24 h with RSD of 0.35%.Accuracy was measured by spiking known amount of the analyte at three different concentration levels.The recovery for amentoflavone ranged from 98.75 to102.83%,while the RSD was 1.19%, indicating a good accuracy of the method (Table 2).

Sample analysis
Total flavonoids and amentoflavone (Figure 2 and 3) were determined by the above-mentioned methods.The results are listed in Table 3.

DISCUSSION
The results showed that the amount of the total flavonoids and amentoflavone in SR and SU samples had no obvious changes between Julycollected ones and October-collected ones,.Comparing the amount of amentoflavone and the total flavonoids in SH, it was shown that both of them in October were higher than those in July.The amentoflavone content in SD harvested in autumn was much higher than that in summer, while the total flavonoids content kept changeless.In addition, the varying amount of the total flavonoids in SM was adverse to the content of amentoflavone at different harvest seasons.The results informed us that the contents of the total flavonoids and amentoflavone in the five Selaginella plant drugs presented different variation tendencies from summer to autumn, especially in SD and SM.Therefore, we think it maybe not so perfect to evaluate the quality or confirm the harvest time of plant drugs by using the content of a single active compound or a group of active substance as the sole index.With a small proportion of the amentoflavone in the first four plant drugs, it would be more reasonable to evaluate the quality and confirm the best harvest time primarily based on the total flavonoids content with the assistant of amentoflavone content.Therefore, it was feasible to collect SR and SU in summer or in autumn, whereas SH and SD were suggested to be harvested only in autumn.Because the amentoflavone had a high proportion in the total flavonoids (1/3~1/2) in SM, both of them played important roles in evaluating the quality of SM.As a result, it would be appropriate to collect SM in summer or in autumn.
As we all know, Chinese traditional medicines usually contained many complex bioactive chemical constituents.And more often, its bioactivities is not due to the presence of one component but the presence of a mixture of bioactive components.Our studies demonstrated that the amount of the main active components and a single bioactive component had different change tendencies with the growth of medicinal plants.Thus, we think that if a single component has high proportion in a group of active substance, its content will be significant to use for the assessment of the plant drugs.If not, the content of the group of substance should be paid more attention for quality estimation, unless some single component shows particularly strong pharmacological activities.

CONCLUSION
The methods established in this study had been proved simple, rapid and reliable for quantitative determination of the total flavonoids and amentoflavone contents in the five Selaginella plant drugs.And the experimental data provided valuable references for evaluating the quality and preliminarily confirming the appropriate harvest time of Selaginella plant drugs.