Ginkgo biloba by High Performance Liquid Chromatography compound content of total flavonoids in Ginkgo biloba
【Abstract】 Objective To establish a compound of Flavonoids in Ginkgo biloba Ginkgo biloba in the determination. Methods
using high performance liquid chromatography, using Symmetry
C18 column (250 mm × 4.6 mm, 5 μm), methanol 0.4%
phosphoric acid (52:48) as mobile phase, flow rate of 1.0 ml * min -1, detection wavelength 360 nm, column temperature was 35 ?. Results The linear relationship was good, the average recovery was 98.35%, RSD of 1.17% (n = 5). Conclusion The method is simple, separation is good, accurate, specific and can be used as quality control of Ginkgo leaf compound.
Key words compound Ginkgo biloba; flavonoids; quercetin;
kaempferol; isorhamnetin; high-performance liquid
chromatography
Abstract: ObjectiveTo establish the determination method of flavonoids in compound Ginkgo biloba Leaves Tablets. MethodsThe analysis was performed on a Symmetry C18 column
(4.6 mm × 250 mm, 5 μm) with methanol 0.4% phosphoric
acid (52:48) as mobile phase at a flow rate of 1.0 ml * min-1,
and at a column temperature of 35 ?. The detection wavelength
was 360nm.ResultsThe linearity of this method was good.The average recovery of added samples was 98.35% with RSD 1.17%. (n = 5). ConclusionThe method is convient with a good separating degree for the determination of flavonoids and can be used to evaluate the quality of compound Ginkgo biloba Leaves Tablets.
Key words: Compound Ginkgo biloba Leaves Tablets; Flavonoid; Quercetin; Kaempferol; Isorhamnetin; HPLC
Ginkgo leaf is a compound of Ginkgo biloba extract and Guangxi Pueraria Mirifica extract, etc. made of compound preparation, with blood circulation Huayu, reduce blood fat,
removing free radicals and anti-aging effects and the role of
[1]. Ginkgo leaves contain quercetin, kaempferol, isorhamnetin and other flavonoid compounds, one of its main active ingredient. Ginkgo biloba leaves the determination of
flavonoids in many ways, such as derivative spectrophotometry [2], near-infrared spectroscopy [3], spectrophotometry [4], polarography [5], fluorescence spectrometry [6] , coulometric titration [7], capillary electrophoresis [8] and high performance liquid chromatography [9]. And the ginkgo leaves and preparations of Pueraria both formulations Determination of Flavonoids in Ginkgo biloba has not been reported. In this paper, high-performance liquid chromatography in Ginkgo leaf compound quercetin, kaempferol and isorhamnetin content of
compounds such as flavonoids, methods, fast and easy, separation result is good, a strong specificity. Ginkgo biloba can be used for quality control of compound.
An instrument and reagent
1.1 Instrument U.S. PerKinElmer Inc. Series 200 High
Performance Liquid Chromatography (including the Series 200 UV detector, Series 200 binary pump, 600 Series interface and chromatography workstation).
1.2 reagent quercetin, kaempferol and isorhamnetin reference substance (China Pharmaceutical and Biological Products Institution, batch, respectively 10081
200406,110861 200405,110860 200407). Ginkgo leaf
compound-based research group in Guangxi Science and
Technology Agency Fund under the three developed new products.
Chromatography of pure methanol, water, high pure water, phosphoric acid as the AR.
2 Methods and Results
2.1 Chromatographic conditions Symmetry C18 column
(4.6 mm × 250 mm, 5 μm, the United States Waters
Corporation), the mobile phase was methanol: 0.4% phosphoric acid solution (52:48), flow rate 1 ml * min-1, detection of
wavelength of 360 nm, column temperature 35 ?, injection
volume 20 μl.
2.2 Solution Preparation
2.2.1 Preparation of reference substance solution
precision that learn from phosphorus pentoxide drying overnight quercetin, kaempferol and isorhamnetin appropriate reference substance (15.6,17.0,10.0 mg) Purchase 100 ml Liang Ping, add methanol Micro-heat allows the dissolved and diluted to the scale, shake, spare.
2.2.2 Preparation for the test product solution to take this product 10, except for coating, and research fine, fine, said appropriate amount (about containing Ginkgo flavonol glycosides 19.2 mg), cone-shaped bottle home with Cyprus,
precision by adding methanol, 20 ml, Mesa, saying that given the weight, ultrasound treatment 30 min, let cool, and then said that given the weight by using methanol to complement the weight loss, shaking, filtration. Precise amount of filtrate continued to take 10 ml, set 100 ml conical flask, add
methanol 10 ml, 25% hydrochloric acid 5 ml, shaken, heated in a water bath set back 30 min, rapidly cooled to room temperature, transferred to a 50 ml flask in methanol was diluted to scale, shake, with 0.45 μm microporous membrane
filtration, the filtrate continued to take that too.
2.2.3 Preparation of negative control solution, in addition to taking a prescription outside of Ginkgo biloba extract the remaining herbs and accessories, according to the
provisions of Technology prepared into tablets, press materials for the test solution made from the negative control solution preparation method.
2.3 The precise amount of the production of standard curve to take the above-mentioned reference substance solution
1.0,2.0,3.0,4.0,5.0 ml, respectively, 10 ml Liangping in the home, using mobile phase was diluted to scale, shake, each sample 20 μl, according to the above color spectrum
measurement conditions in order to reference substance concentration C (μg * ml-1) as the abscissa, the peak area A of the vertical axis, draw the standard curve as follows:
Quercetin: A = 80 074C - 52 255, r = 0.999 4, linear
range of 15.6 ~ 78.0 μg * ml-1
Kaempferol: A = 63 168C 35 558, r = 0.998 2, the linear
range of 17.0 ~ 85.0 μg * ml-1
Isorhamnetin: A = 60 465C 20 840, r = 0.999 6, the linear range of 10.0 ~ 50.0 μg * ml-1
2.4 The precision of experimental precision drawing reference substance solution, 20 μl according to the above
chromatographic conditions, continuous sampling five times, three kinds of reference substances of the peak area RSD were 0.87%, 0.58% and 1.20%, indicating good precision instruments. Reposted elsewhere in the paper for free download http://
2.5 repeated experiment approved to take the same side effect of GBE (20.05101 million), in accordance with 2.2.2, "Preparation for the test product solution," according to the law under the preparation of five copies of the parallel solution of the tested products, according to the above-
mentioned chromatographic conditions were measured by the total flavonol glycosides contents were 9.10,9.18, 9.20, 9.03, 9.27 mg / piece, RSD 1.01%, indicating good repeatability of this Act.
2.6 stability test to take the same group of test items for the solution (20.05101 million), according to the above chromatographic conditions are 0,5,7,21,24 h sample were determined and calculated the average total flavonol glycoside content of 9.16 mg / tablets, RSD of 1.60%. The results showed
that for the test items within the solution at 24 h was basically stable.
2.7 The average recovery experiments of known content to take tablets (20.05101 million) fine powder amount of precision that set, and placing a plug cone bottle, a total of
five copies, respectively, by adding sophisticated quercetin, kaempferol and isorhamnetin Mixed reference substance
solution, 10 ml (concentrations of 0.156,0.170,0.100 mg * ml-
1), according to 2.2.2 "for the solution of test preparation
materials," operating under the law, each sample 20 μl,
record chromatograms, calculation the recovery rate, the results in Table 1.
Table 1 compound in Ginkgo Ginkgo biloba flavonol glycosides recoveries experimental determination results
(omitted)
2.8 Interference experiments were drawing a blank precision reference substance solution for the test product solution and the negative control solution,20 μl, According
to the above chromatographic conditions, sample measurement, the results of quercetin (tR = 17.9 min), Kaempferol (tR = 33.4 min), isorhamnetin (tR = 38.1 min) peaks were well separated from other components, and the negative control in the corresponding position without chromatographic peaks, indicating prescription of other components and accessories on the determination results without interference. Chromatography shown in Figure 1.
Figure 1 Effects of Gingko Biloba HPLC chromatogram of compound (abbreviated)
2.9 Determination of the sample taken four batches of
samples, according to 2.2.2 Preparation for the test materials under the solution, according to the above chromatographic conditions were determined, we calculate the quercetin, kaempferol and isorhamnetin content, press-style translated
into a total flavonol glycoside content [10]. The results in Table 2.
= Total flavonol glycosides (quercetin kaempferol and isorhamnetin content content) × 2.51
Table 2 Sample Ginkgo flavonol glycosides determination results (omitted)
3 Discussion
Take reference substance solution and the negative control solution, respectively by UV scanning at 200 ~ 300 nm both with strong absorption in the 300 ~ 400 nm absorption of a smaller negative control solution, so choose 360 nm as quercetin, mountain Nai Su, isorhamnetin measured wavelength.
Experiment selected methanol water, methanol 0.4%
phosphoric acid aqueous solution as mobile phase, and proportion of mobile phase composition of the selection results show that the "2.1" under the system better, a good separation of all peaks, sharp peak shape, retention time, moderate.
The experimental results show that using the HPLC method, the main active ingredients of compound effect of GBE quercetin, kaempferol, isorhamnetin and other flavonoid
compounds were determined method is simple, accurate, precision, good repeatability, prescription the other components of the Ginkgo biloba flavonoids determination of non-intrusive, method specificity strong, can be used to
compound the quality control of Ginkgo leaf.
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