High Performance Liquid Chromatography Shouwu Oral Solution in the content of stilbene glucoside
【Abstract】 Objective To establish Shouwu Oral Solution in the 2,3,5,4 '4-Hydroxy-stilbene 2 O β D
glucoside content in high-performance liquid chromatography (HPLC) determination method. Method column was Waters Corporation Symmetry C18 column (3.9 mm × 250 mm, 5 μm),
mobile phase of acetonitrile - water (eighty-three past five
p.m.), flow rate 1.0 ml / min, detection wavelength was 320
nm. The results of 2,3,5,4 ' 4-hydroxy-stilbene 2 O
β D Glucosidase in the range of 0.45 ~ 1.8 μg good
linear relationship (r = 0.999 987), 8 h of RSD 0.7% . Conclusion This method of measuring the active ingredient Shouwu Oral Solution stilbene glucoside content, high sensitivity, quantitative and accurate method is simple, reproducible, and can effectively control the Shouwu fine quality oral liquid preparations.
【Key Words】 Polygonum multiflorum; stilbene glycoside; high-performance liquid chromatography; determination
Abstract: ObjectiveAn HPLC method was established for the determination of 2,3,5,4 ' tetrahydroxystilbene 2 O
β D glucopyranoside in Shouwujing Oral Solution. MethodsA Symmetry-C18 column (3.9 mm × 250 mm, 5 μm) with a
mobile phase composed of acetonitrile water (17:83) was
used. The detection wavelength was 320 nm and the flow rate was 1.0 ml / min. Results The calibration curves were linear between 0.45 ~ 1.8 μg (r = 0.999 9), the 8 hours' precision
RSD was 0.7%. ConclusionThe method is simple, rapid and accurate and is suitable for the determination of 2,3,5,4 '
tetrahydroxystilbene 2 O β D glucopyranoside
in Shouwujing Oral Solution.
Key words: Polygonum multiflorum Thunb.; Stilbenes; HPLC;
Shouwu Oral Solution alcohol extracted from the fleece-
flower root and other accessories adding flavor to a certain concentration to be diluted, with Ukraine refund, Yuet color, Yangjing blood, anti-aging power of its active ingredients
stilbene glycosides have significant pharmacological activity [ 1]. Modern pharmacological studies have shown that: Shouwu and contains stilbene glycoside compounds have anti-aging,
improve immune function, such as the role of prevention and
treatment of atherosclerosis and liver protection . Clinical studies have shown that: two stilbene glucoside in the prevention and treatment of age-related diseases such as
hyperlipidemia, hypertension, chronic bronchitis, diabetes mellitus; mental neurological diseases such as neurasthenia, epilepsy, and many other aspects have more significant effects . To control the intrinsic quality of products, using HPLC on the fleece-flower root of the active ingredient for
determination of stilbene glycoside study, are reported below.
1 Equipment and reagents
Waters HPLC, Waters 1525 Binary pump, AUTOSAMPLER 717 injector, Waters 2487 dual wavelength UV detector; M32 chromatography workstation; Shimadzu AUW120D one hundred
thousandth of electronic analytical balance; stilbene glucoside reference substance from Chengdu Biology Technology Co., Ltd. China Cisco Systems to provide, and the second styrene glycoside content is more than 98%. Chromatography pure acetonitrile mobile phase, water for ultra-pure water,
oral liquid water for the distilled water, ethanol, etc. AR.
2 Methods and Results
2.1 Shouwu Oral Solution prepared fleece-flower root,
Sichuan real estate medicine, playing powder, over 60 mesh sieve. Weigh 100 g placed in fleece-flower root powder flask,
the volume of take 55% of ethanol by adding 700 ml volumetric
flask (the solid-liquid ratio of 1:7), the put it aside for infiltration 30 min, at 75 ? heating reflux, ethanol
extraction time for 45 min, cooled, put it aside for later allowed to fully precipitate filtration, abandoned to the residues. And then extract recovery of ethanol by rotary evaporator and concentrated, 55 ~ 60 ? vacuum concentrated
extract at room temperature, put it aside for 24 h, then centrifuged into the Round-bottom flask, and adding proper
amount of distilled water to the calibration constant volume
have oral liquor, HPLC determination of the other stilbene glucoside content, be diluted to 0.1%, adding preservatives, adjust pH to 7.0 ~ 7.5, filtration, encapsulation, sterilization, that may Shouwu Oral Solution 30 mg (containing
stilbene glucoside) / 10 ml / teams.
2.2 column chromatographic conditions for the Symmetry
C18 (3.9 mm × 250 mm, 5μm), mobile phase of acetonitrile
water (eighty-three past five p.m.), flow rate of 1.0 ml / min, detection wavelength 320 nm, column temperature 30 ? .
Theoretical plate numbers calculated by two stilbene glucoside peaks should not be less than 4500. Stilbene glycoside reference substance and Shouwu Oral Solution (for the test product) in chromatogram shown in Figure 1.
Figure 1 HPLC map (omitted)
2.3 Preparation of Solution
2.3.1 Preparation of reference substance solution precision Weigh 1.35 mg stilbene glucoside reference substance has been dried to constant temperature placed in 10 ml of the flask, add 50% ethanol to constant volume scale, shake, with
0.45 μm of the microporous membrane filter back.
2.3.2 Preparation for the test product solution precision drawing self-stilbene glucoside has been dried to 100 μl
placed in constant temperature of 10 ml of the flask, add 50% ethanol to constant volume scale, shake, with 0.45 μm micro -
pore membrane filter back.
2.3.3 Preparation of blank solution blank experiment is based on the proportion of various elements of a prescription, since with non-fleece-flower root of the oral solution,
negative control made of their craft, and then prepare for the
test materials were prepared by solution and to measure. Results blank solution of stilbene glycoside reference substance with the retention time of the same place free of chromatographic peaks, so that no interference.
2.4 linear relationship between the investigation into the reference substance solution, 1 ml sample tubes, No. 1-5,
respectively, to take 3,6,9,12,15 μl sample, recording
chromatograms and the concentrations of the corresponding peak area value to stilbene glycoside reference substance content (X) of the peak area values (Y) Draw the standard curve obtained by the statistical regression equation: Y = 4.73e 006X-2.12e 005, r = 0.999 987, results show that the diphenyl Vinyl glycosides in 0.45 ~ 1.8 μg linear relationship was
good. This Act with the external standard method content.
2.5 Stability test for the test product solution to take an interval of about 2h, respectively, 0,1,3,5,8 h sample 10μ
l, the measured levels of stilbene glucoside followed by 7.32,7.26,7.22,7.17,7.11 mg / ml, mean 7.22 mg / ml, RSD 0.7%. Measurements showed that the samples within 8 h was basically stable. Reposted elsewhere in the paper for free download
2.6 Experimental precision precision drawing the same reference substance solution of 10 μl sample 5 consecutive
times, measured peak area average of 2.999 × 106, the peak
area RSD was 0.4%.
2.7 Repeatability precision experiments that take five
copies of the same batches of samples (each about 1 ml), according to 2.3.2 item for the Preparation of test materials were prepared solution and the above chromatographic conditions were determined, the measured average concentration
of 7.53 mg / ml, RSD = 0.9% (n = 5), results showed a good repeatability of this method.
2.8 The average recovery experiments with precision, said Kayo Recovery Act to take a known concentration of 5 copies of the same batch of samples, accurate reference substance by
adding two stilbene glucoside (0.675 mg / ml), according to 2.3.2 item solution for the test product Preparation Method of Preparation and determination of the above-mentioned
chromatographic conditions. The results in Table 1.
Table 1 stilbene glucoside recoveries assay (abbreviated)
Content of the sample were 0.753 mg, dosage were 0.675 mg, n = 5
2.9 Determination of the respective sample precision drawing reference substance solution and sample test items for
the solution of 10 μl, the above-mentioned chromatographic
conditions were determined by three batches of samples. Determination of concentration in Table 2.
It is reported in the literature , two stilbene glucoside peaks of the spectral absorption curve (320 ? 1) nm
with maximum absorption; this study has used ultraviolet -
visible spectrophotometry stilbene glycoside reference substance solution at 200 ~ 420 nm wavelength scan measured the maximum absorption wavelength of 320 nm, and are therefore
choose 320 nm as detection wavelength.
On the chromatographic conditions, results show that the mobile phase of acetonitrile water (eighty-three past five
p.m.), flow rate 1.0 ml / min, the two stilbene glucoside
peaks reached a satisfactory separation, theoretical plate number of 4535, Separation degree 8.61, tailing factor was 1.31, so this chromatographic conditions were set at the best conditions for determination of oral liquid.
Table 2 different batches Determination of stilbene
It is reported in the literature , two stilbene glucoside light unstable, water-soluble in pH 7.0 ~ 7.5 at
room temperature, under the conditions of stability and, therefore, determination of Shouwu Oral Solution, it is
desirable under the conditions in the dark, and the use of brown containers; for the test materials, reference substance solution should be prepared immediately before use and adjust
the pH between 7.0 ~ 7.5, column temperature was controlled at
30 ?, in order to reduce the error.
Application of Symmetry-C18 column, use of high-purity
silica filler, has a good chromatographic peak shape, high reproducibility and long column life, and has a good user background characteristics.
In this study, HPLC measurements Shouwu Oral Solution in the stilbene glucoside content of the active ingredient, high sensitivity, quantitative and accurate method is simple, reproducible, and can effectively control the Shouwu fine quality oral liquid preparations.
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