Blood particles Guiqi Quality Standards
【Abstract】 Objective To establish Guiqi Blood particles (astragalus, wolfberry fruit, hemp seed, angelica, Schisandra, etc.) quality standards. Methods TLC particles in the astragalus, wolfberry fruit, hemp seed, angelica, Schisandra carried out a qualitative identification; using high-
performance liquid chromatography - evaporative light
scattering detection (HPLC-ELSD) was determined by preparation of astragaloside content. The results of TLC spots were clear
astragaloside 0.500 1 ~ 10.002 μg in the context of linear
relations, the average recovery was 98.83%, RSD = 1.13%. Conclusion The method is simple, accurate and available to the product quality control.
Key words Blood-particle Guiqi; thin-layer chromatography;
high-performance liquid chromatography - evaporative light
scattering detector; Astragaloside
Study on Quality Standard for Guiqishengxue Granules
Abstract: ObjectiveTo establish the quality control standard of Guiqishengxue Granules (Radix Astragali, Fructus Lycii, Fructus Cannabis, Radix Angelicae Sinenis, Fructus Schisandra-e, etc.). Methods Radix Astragali, Fructus Lycii, Fructus Cannabis, Radix Angelicae Si-nenis, Fructus
Schisandrae were identified by TLC. The content of
Astragaloside IV was determined by HPLC-ELSD. ResultsThe TLC
spots developed was fairly clear. The Astragaloside IV showed good linear relationship at a range of 0.500 1 ~ 10.002 μg.
The average recovery was 98.83% with RSD was 1.13%. ConclusionThe method is simple, accurate and can be applied to the control of the products effectively.
Key words: Guiqishengxue Granules; TLC; HPLC-ELSD;
Astragaloside IV
Guiqi Blood particles mainly by the astragalus, wolfberry fruit, habitat, winter days, hemp seed, angelica, peach kernel, safflower, Schisandra, etc. composed of nine Chinese herbal medicines. With spleen and kidney, Regulating the role of blood for less than spleen and kidney, qi and blood deficiency, facial skin dystrophy caused by dry skin, yellow,
flexibility to reduce skin relaxation embolism. In order to effectively control the drug quality of the particles in the blood Guiqi Health Astragalus, wolfberry fruit, hemp seed, angelica, Schisandra qualitative study using HPLC-ELSD method,
the main component of Astragalus Astragaloside for determination, with a view to better control of the drug quality.
An instrument and reagent
Instrument: Shimadzu LC-10AT, 7725i quantitative sample
injection valve, Shimadzu C-R7Ae digital processor, the
French-type SEDEX 55 evaporative light scattering detector (ELSD); reference substance Astragaloside (content was determined with), Chinese wolfberry, hemp seed, Angelica sinensis, Schisandra control medicines (The above reference
substance and control medicines are purchased in China's pharmaceutical and biological products); use of chemical reagents were analytically pure, silica gel G (Qingdao Marine Chemical Plant), self-made silica gel plate (10 cm × 10 cm ×
0.6 mm); test sample Guiqi Blood-particles (homemade);
negative samples of prescription medicines to remove the seizure is made according to Preparation Process.
2 Methods and Results
2.1 Qualitative Identification of
2.1.1 Identification of Astragalus take this product 20
g, small study, heating water, 50 ml allows the dissolved, let cool, centrifuged and the supernatant, add ether extract three
times, 20 ml / times, combined ether solution, and the other device to collect ; Hui to make the water layer of ether,
adding water-saturated n-butanol extract three times, 20 ml /
times, combined n-butanol extract, n-butanol saturated with 1%
NaOH solution, washed three times, 20 ml / times, then n-
butanol saturated with water, washed three times, 30 ml /
second, n-butanol extract of the evaporated water bath set, allows the dissolved residue add 1 ml of methanol, as a solution for the test items. Another take astragalus negative samples 20 g, with the rule of law into the negative control solution. And then take Astragaloside reference substance, add methanol made from 1 mg per ml of solution, as a reference substance solution. According to thin-layer chromatography
"Chinese Pharmacopoeia" in 2005 edition of the Ministry of
Appendix ? VIB test, to learn for the test product solution, negative control solution of 10 μl, reference substance
solution, 5 μl, respectively, points on the same silica gel G thin-layer plate in order to chloroform - ethyl acetate -
methanol - water (10:20:11:5) 10 ? for the launch of the
lower liquid agent, started out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105 ? to spot
obvious Color clear. Test products for chromatography, in the corresponding position with the reference substance chromatography, significantly the same color spots. Negative control samples in the corresponding position there is no such spots. Figure 1.
2.1.2 Identification of medlar to take this product 10 g, small study, allows the dissolved 50 ml hot water, put the cold, centrifuged and the supernatant with 40 ml ethyl acetate Zhenyao extraction, extract concentration to about 1.5 ml , as a solution for the test items. An alternative medlar negative samples, with the rule of law into the negative control solution. And then take control medicines medlar 0.5 g, water 35 ml, boil 15 min, put cold, filtration, the filtrate 15 ml Zhenyao with ethyl acetate extraction, extract concentrated to about 1 ml, as control medicine solution. According to thin-
layer chromatography "Chinese Pharmacopoeia" in 2005 edition of the Ministry of Appendix ? VIB test, to learn for the test
product solution, negative control solution, the control of medicinal solution 5 μl, respectively, points on the same
silica gel G thin-layer board, with toluene - ethyl acetate -
formic acid (3:2:0.1) as the agent, started out, drying, home ultra-violet light (365 nm) under review. Test products for
chromatography, in the corresponding position control on the
medicinal chromatography, significantly the same color of the fluorescent spots. Negative control samples in the corresponding position there is no such spots. Figure 2.
Figure 1 Identification of Astragalus (omitted)
Figure 2 Identification of medlar (omitted)
2.1.3 Identification of hemp seed to make fire control medicinal hemp seed 0.5 g, ethyl vinegar 20ml, ultrasonic extraction 30 min, filtration, filtrate Hui dry ethyl 1 ml vinegar residue allows the dissolved, as the control medicine
solution. According to thin-layer chromatography "Chinese
Pharmacopoeia" in 2005 edition of the Ministry of Appendix ?
VIB test, drawing a qualitative analysis for the test materials under 2.2 solution 20 μl, control medicinal
solution, negative control solution of 5 μl, respectively, at
the same point with carboxymethyl sodium cellulose adhesive thin-layer silica gel GF-254 board with cyclohexane - acetone
(23:3) as the agent, started out, dried, sprayed with 10% sulfuric acid ethanol solution, at 105 ? heated to a clear
spot color. Test products for chromatography, in the corresponding position with the control on the medicinal chromatography, significantly the same color spots. Negative control samples in the corresponding position there is no such
spots. Figure 3.
2.1.4 Identification of Angelica qualitative analysis of 2.1 obtained under the other device to collect the ether solution, natural Hui dry residue add 1 ml of ethanol allows the dissolved, as a solution for the test items. Another take
Angelica negative samples, with the rule of law into the negative control solution. And then take control herbs Angelica 1 g, add ethyl ether 20 ml, ultrasonic extraction 15 min, filtration, the filtrate natural Hui dry residue add 1 ml of ethanol allows the dissolved, as a medicinal solution of the control. According to thin-layer chromatography "Chinese
Pharmacopoeia" in 2005 edition of the Ministry of Appendix ?
VIB test, to learn for the test product solution, negative control solution, control solution of medicinal 5μl,
respectively, points on the same silica gel G thin-layer board
with n-hexane -- ethyl acetate (9:1) as the agent, started out, drying, home ultra-violet light (365 nm) under review.
Test products for chromatography, in the corresponding
position control on the medicinal chromatography, significantly the same color of the fluorescent spots. Negative control samples in the corresponding position there is no such spots. Figure 4.
2.1.5 Identification of Schisandra to take this product
20 g, plus chloroform 100 ml, reflux extraction 1.5 h, filtration, the filtrate concentrated to about 1 ml, as a solution for the test items. Another take Schisandra negative samples 20 g, with the rule of law into the negative control solution. And then take control medicinal Schisandra 0.5 g, plus chloroform 100 ml, reflux extraction 1.5h, filtration, the filtrate concentrated to about 1 ml, as control medicine solution. According to thin-layer chromatography "Chinese
Pharmacopoeia" in 2005 edition of the Ministry of Appendix ?
VIB test, to learn for the test product solution, negative control solution, control solution of medicinal 5μl,
respectively, at the same point with sodium carboxymethyl cellulose as a binder of silicon GF-254 gel thin-layer plate
in order to petroleum ether (30 ~ 60 ?) - ethyl - formic acid
(15:5:1) for the launch of the upper liquid agent, started out, dried, sprayed with 5 % phosphomolybdic acid ethanol solution, heated at 105 ? to spot color definition. Test
products for chromatography, in the corresponding position with the control on the medicinal chromatography, significantly the same color spots. Negative control samples in the corresponding position there is no such spots. Figure 5.
Figure 3 Identification of hemp seed (omitted)
Figure 4 Identification of Angelica sinensis (omitted)
Figure 5 Identification of Schisandra (omitted)
2.2 Quantitative Analysis
2.2.1 Chromatographic conditions the U.S. Phenomenex Inc. LUNA 5u C18 (2) column (4.6 mm × 250 mm, 5μm), mobile phase
methanol - water (75:25), flow rate 1.0 ml / min, ELSD detector drift tube temperature of 40 ?, carrier gas
(nitrogen) flow rate of 2.0bar. Reposted elsewhere in the paper for free download http://
2.2.2 Preparation of sophisticated solution that will Astragaloside appropriate reference substance (identified by the Chinese biological products offered, batch 0781-9908),
plus preparation of methanol concentration of 0.5001 mg / ml solution of the reference substance.
Preparation of test items for the solution: Take this product is about 5 g, precision that set, add water, 50 ml, dissolved, centrifugation, filtration, the filtrate set points in the funnel, using chloroform - n-butanol (2:1) to extract
four times, the first 1 50 ml, the other 3 times 30 ml / second (if there emulsification phenomenon, the emulsion layer of centrifuged supernatants obtained lower), the merger extract, evaporated and the residue plus 2% potassium hydroxide methanol solution, 20 ml, and transferred to a
conical bottle, water bath, reflux 1 h, extracts evaporated and the residue dissolved in 20 ml water, go to the sub-
funnel, the chloroform - n-butanol (2:1) to extract four
times, 20 ml / second, the merger extract, evaporated and the
residue with methanol solution allows the dissolved and transferred to 5 ml Liang Ping, add methanol to the scale, shake.
Preparation of negative reference substance solution: Take according to prescribed ratio and preparation process of
the preparation of the non-negative samples of Astragalus,
according to 3.3 to operate with the legal system into the negative control solution.
2.2.3 blank experiment will Astragaloside reference substance solution for the test product solutions and negative
control solution were injected into liquid chromatography, according to the above conditions were determined, the results shown in Figure 6 ~ 8. From the chromatogram can be seen that the sample in the other components of the Astragaloside will
not interfere with the determination.
2.2.4 linear relationship with the study sample were taking reference substance solution 1,5,10,15,20 μl, in order
to sample volume X (μg) against the number of ? X as
abscissa, the logarithm of peak area Y ? Y for the vertical
axis, drawing the standard curve. Accordance with the principle of least-squares regression method, astragaloside in the range of 0.500 1 ~ 10.002 μg good linear relationship.
The regression equation is logY = 5.840 9 2.214 4 log X (r =
0.999 5). Figure 9.
Figure 6 astragaloside reference substance for HPLC map (omitted)
Figure 7 Guiqi Blood samples particles HPLC map (omitted)
Figure 8 lack of negative control of the HPLC map of Astragalus (omitted)
Figure 9 standard curve (omitted)
2.2.5 Stability of test items for test taking solution, respectively, 0,1,2,3,4 h sample, 10 μl / times, recording
chromatography Peaks area, results showed that the particles in the blood Guiqi Health Astragaloside Determination of the 4
h to stabilize, RSD = 0.005% (n = 5).
2.2.6 precision experiment to take the above-mentioned
reference substance solution, a continuous sample injection 5 times, 10 μl / times, astragaloside peak area relative standard deviations were 0.22%.
2.2.7 Repeatability test particles obtained Guiqi Blood samples (batch number 000,601), precision, said to take five copies, according to the sample were measured by determination of the content of their astragaloside, RSD = 0.22% (n = 5).
2.2.8 Experimental recoveries obtained Guiqi Blood particles (000,701 grant has been determined content astragaloside content 0.100 49 mg / g) 6 copies will be placed with precision, said plug tapered bottle, followed by adding precision Astragaloside reference substance solution (0.100 6 mg / ml) in the above-mentioned cone-shaped bottle with plugs
were dissolved by adding 50 ml of water, centrifugation, filtration, the filtrate set points in the funnel, using chloroform - n-butanol (2:1) extracted four times, the first 1
50 ml, the other 3 times 30 ml / second (if there
emulsification phenomenon, the emulsion layer of centrifuged supernatants obtained lower), combined extract, evaporated. Residue plus 2% potassium hydroxide in methanol 20 ml, and
transferred to a conical bottle, water bath, reflux 1 h, extracts evaporated and the residue dissolved in 20 ml water, go to the sub-funnel, the chloroform -- n-butanol (2:1) to
extract four times, 20 ml / second, the merger extract, evaporated and the residue with methanol allows the dissolved and transferred to 10 ml Liang Ping, add methanol to the scale, shake, after aperture 0.45 μm microporous membrane
filtration, abandoned to the beginning of the filtrate, the filtrate added as a solution for the test items. Accordance
with the development of chromatographic conditions, depicting 10μl injected into liquid chromatography to measure, each measured twice, and averaged to calculate the recovery rate, the results in Table 1.
Table 1 The average recovery experiment (omitted)
2.2.9 Determination of the sample in accordance with proposed method, a total of seven batches of samples for determination of astragaloside, each consisting of two parallel determination of the results in Table 2.
Table 2 Blood-particles in Guiqi Astragaloside assay (omitted)
3 Discussion
Astragaloside poor UV absorption components, maximum absorption wavelength of 200.8 nm. As with the UV detector influenced by the reagent, the analysis difficult, the quality
standards are currently more quantitative by TLC-scanning. TLC
separation result is not satisfactory, the background noise large and cumbersome operation. ELSD detector detector for quality-oriented, free from interference with the external
environment, reagents in the detection of all the volatile, non-interference detection, is a kind of test components Astragaloside effective way [1].
Because prescriptions are complicated, in the sample pre-
treatment process, was found with 5% sodium carbonate solution
and ammonia test solution was washed, received a lot of impurities, the content of astragaloside low, compared to 5% by the sodium carbonate solution and the ammonia test solution was replaced with 2% potassium hydroxide in methanol solution
to deal with chloroform - n-butanol extract, and then
chloroform - n-butanol extraction, the measured astragaloside improved significantly.
Law by high performance liquid chromatography method for rapid separation and determination of Astragaloside with the
2000 edition of "Chinese Pharmacopoeia" method [2] compared with no significant difference in the results, but the analysis time significantly shorter, Comparison of two methods in Table 3.
Table 3 of this Act and the "Chinese Pharmacopoeia"
Determination of Radix Astragalus astragaloside comparison (omitted)
This product is provided by comparing the Radix Astragali containing Pharmacopoeia test methods and test methods of Astragalus medicines containing the results measured by
basically the same. Therefore, this product can be used to ensure medicines meet the pharmacopoeia requirements astragalus.
【References】
[1] Bao-Qin Wang, Su, Lu Jing. Astragaloside test the application of quality control in Chinese medicine [J].
Chinese Journal of Traditional Chinese Medicine, 1996,21 (3): 161.
[2] The People's Republic of China Pharmacopoeia Committee of the Ministry of Health. Chinese Pharmacopoeia, ?
Department of [S]. Beijing: Industrial Press, 2000:249.
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