High Performance Liquid Chromatography Determination of flavonoids in semipinnata
Author: Gong Xian-ling, Zhi-Hong Chen, Nian-Ci
【Abstract】 Objective To determine semipinnata of luteolin and apigenin content. Methods ODS C18 analytical column (25
mm × 4.6 mm, 5 μm); mobile phase was methanol -0.4%
phosphoric acid solution (1:1); detection wavelength of 345 nm, flow rate 1 ml * min-1, column temperature at 25 ?.
Results of luteolin linear range 0.02 ~ 0.4 μg, r = 0.999 9,
the average recovery was 96.39% (RSD = 1.95%); apigenin linear range of 0.02 ~ 0.4 μg, r = 0.999 6, the average recovery was 98.10 % (RSD = 0.96%). Conclusion This method is simple, accurate, high sensitivity, good reproducibility can be used
as half of the flag of the determination of two flavonoids.
Key words semipinnata; flavonoids; high-performance liquid
Analysis and Determination of Flavonoids in Pteris semipinnata L. by HPLC
Abstract: ObjectiveTo analyze and determine the content of luteolin and apigenin in Pteris semipinnata L. by HPLC. MethodsThe HPLC system consisted of ODS-C18 column (25 mm ×
4.6 mm, 5 μm), methanol: 0.4% sodium mixture as a mobile phase , detection wavelength at 345nm, 1 ml * min-1 of flow
rate and column temperature at 25 ?. ResultsThe linear range
of luteolin and apigenin were 0.02 ~ 0.4 μg, r = 0.999 9 and
0.02 ~ 0.4 μg, r = 0.999 6 respectively , the recovery were
96.39% (RSD = 1.95%, n = 5) and 98.10% (RSD = 0.96%, n = 5) respectively.ConclusionThe method is simple, accurate, sensitive and reproducible. This method can be used for the quantitative determination of flavonoids in Pteris semipinnata L.
Key words: Pteris semipinnata L.; Flavonoids; HPLC
Pteris Pteris semipinnata L. flag for Pteridaceae Pteris species, widely distributed in Sichuan and the Yangtze River south of the provinces and autonomous regions, bitter, Xin, sexual cool, with evacuation cold, dampness swelling, heat and so the effectiveness of detoxification [ 1]. Semipinnata
entire grass-containing flavonoids [1,2], Fu Yuping et al  examined semipinnata content of total flavonoids, flavonoid compounds with anti-tumor, anti-inflammatory, antibacterial,
antiviral and other effects . In order to further clarify
the semipinnata in flavonoids, this paper analyzed using high performance liquid chromatography determination of semipinnata of luteolin and apigenin content.
An instrument and reagent
1.1 Instrument the U.S. Hewlett-Packard HP 1100 high-
performance liquid chromatography; Mettler's AE 240
electronic analytical balance.
1.2 Standard test drug flavonoids: luteolin (luteolin; 5,7,3 ', 4' TETRA hydroxyflavone), apigenin (apigenin; 5,7,4 ' trihydroxyflavone), provided by Tianjin peak
(content greater than 98%), methanol as a pure
chromatographic, and other reagents were of analytical grade; semipinnata 2002 05 samples collected from the suburbs of Guangzhou, Guangdong Ocean University, Associate Professor Yang Yanjun identification.
2 Methods and Results
2.1 The chromatographic conditions were ODS C18 column for the analytical column (25 mm × 4.6 mm, 5 μm); mobile
phase was methanol -0.4% of the phosphoric acid solution
(1:1); DAD detector, detection wavelength 345nm, flow rate 1
ml * min-1, the column temperature was 25 ?.
2.2 Preparation of solution for the test materials semipinnata crushed herbs, over 40 mesh sieve, saying that the powder obtained is about 2.0 g, plus 20 ml methanol, 80 ?
water bath heated to return the next 2 times, 2 h / times. Filtration, combined filtrate, moved into 25 ml flask, the constant volume, take a small amount of the solution using 0.45 μm microporous membrane filter, as a solution for the test materials, spare.
2.3 Preparation of reference substance solution
Weigh luteolin reference substance 2.50 mg, plus constant volume of methanol to 25 ml, as a reserve liquid. Weigh Apigenin reference substance 2.50 mg, plus constant volume of methanol to 25 ml, as a reserve liquid. Take two kinds of
stock solution of 15ml, plus methanol 45 ml, made of luteolin, apigenin concentrations were 0.02 μg * μl -1 mixture of the
reference substance, as a reference substance solution. Reposted elsewhere in the paper for free download http://
2.4 linear relationship between the study
Precision drawing reference substance solution 1,2.5,5,10,20 μl followed by injection, in accordance with the above-mentioned chromatographic conditions were measured peak area, peak area of luteolin, respectively
68.30,168.95,331.70,698.26,1415.26; apigenin peak area, respectively 74.91,161.53,346.67,743.72,1509.54. To peak area (Y) and quality (X) regression. Obtain the regression equation in Table 1. Table 1 Regression equations and linear range
2.5 precision experimental precision drawing luteolin and apigenin were 0.02 μg * μl-1 of the reference substance
solution of 10 μl, repeat injection five times a reference substance luteolin peak area average 699.53, RSD = 0.86% ,
apigenin peak area average of 742.83, RSD = 1.75%.
2.6 stability test to take samples for test solution was measured at 0,2,4,6,8 h, results show that the determination of conditions, in the determination of the results are stable within 8 h, luteolin and apigenin RSD were 2.50%, 2.74%.
2.7 to reproduce the experiment by sample method of
measuring samples of the same batch of Pteris semipinnata measured five times, luteolin and apigenin were average content of 0.240 8,0.211 6 mg * g-1, RSD were 2.20%, 2.46% .
2.8 The average recovery obtained experimental precision, said samples of known concentration of the half flag, and then adding the concentration of precision were 0.02 μg * μl-1
luteolin and apigenin reference substance solution, 11 ml,
according to preparation method for the solution of test product preparation, determined by sample method to calculate recovery. The results shown in Table 2 ~ 3. Table 2 luteolin recovery of experimental results (a little) Table 3 apigenin
recovery of experimental results (a little)
2.9 samples were collected from the above-mentioned
determination of luteolin, apigenin, and reference substance solution of 10 μl, sample solution 10 μl injection of high-
performance liquid chromatography, chromatographic conditions
were determined according to 2.1, luteolin standard chromatogram shown in Figure 1, apigenin standard chromatogram shown in Figure 2, reference substance solution chromatograms shown in Figure 3, the sample solution chromatogram shown in
Figure 4.3 batches of samples luteolin and apigenin content in the results shown in Table 4. Table 43 batches of samples of the determination results (omitted)
3.1 luteolin and apigenin in the 340 ~ 360 nm absorption peak between the larger, but because of luteolin absorption peak at 350 nm at up to the maximum, apigenin absorption peak at 340 nm reached at the most, comprehensive consideration, select 345 nm as detection wavelength.
3.2 Determination of semipinnata Law of luteolin and
apigenin content, simple, accurate, high sensitivity, good reproducibility can be used as half of the flag of the determination of two flavonoids.
 DING Heng-shan. Chinese medicinal spores botany
[M]. Shanghai: Shanghai Science and Technology Press, 1982:104.
 Xu Guojun, Hong-yin, XU Luo-shan, et al. Chinese
medicine school, the next book [M]. Beijing: Chinese Medical Science and Technology Publishing House, 1996:1337.
 Fu Yuping, Gou Zhan-ping, Hu Juying, et al.
Spectrophotometer at different times semipinnata total flavonoids [J]. Shizhen country Sinopharm Medicine, 2001,12 (12): 1080.
 PEI Ling Peng, Hui-Bo-di, Kim Lian, et al.
Physiological activity of flavonoids and Preparation
Technology [J]. Food Science and Technology, 2004,25 (2): 203. Reposted elsewhere in the paper for free download http: / /