CN113866288B - Method for analyzing ginsenoside content of ten-ingredient xianglu capsules - Google Patents

Abstract

The method for analyzing the content of ginsenoside in Shiwei Xianglu Capsules belongs to the technical field of traditional Chinese medicine formulations. The invention fully utilizes the value of various traditional Chinese medicines and has the functions of soothing the liver and relieving stagnation, softening hard masses and resolving stagnation. It has a regulatory effect on the neuroendocrine function of the pituitary-gonadal axis, can excite the pituitary gland to secrete gonadotropins, promote the biosynthesis of DNA and protein synthesis-related enzyme proteins, and increase the activity of the gonads. Through the two-way regulation of the pituitary-gonadal axis, balance The positive and negative feedback function of neuroendocrine can achieve the purpose of reducing the abnormally increased estradiol, and inhibit the proliferation of breast cells controlled by ovarian hormones; the internal components of traditional Chinese medicine are selected as internal standards by means of one test and multiple evaluations, and measured The relative correction factor RCF between other components to be tested can be used to calculate the content of components to be tested, thereby reducing the use of reference substances and saving costs.

Description

Analysis Method of Ginsenoside Content in Shiweixianglu Capsules

This case is a divisional application of 201911198034.5

Technical Field

The invention belongs to the technical field of traditional Chinese medicine formulas, and in particular relates to a traditional Chinese medicine for regulating breast hyperplasia and a method for verifying its efficacy.

Background Art

Hyperplasia of the mammary glands (HMG) belongs to the category of "breast pain" in traditional Chinese medicine. The basic cause of the occurrence and development of this disease lies in the dysfunction of the three major internal organs - liver depression, spleen deficiency, and kidney deficiency, and the external manifestations are phlegm congestion, qi stagnation, and blood stasis. The main ingredients of existing treatment prescriptions include bupleurum, thunbergii, angelica, mint, tuckahoe, green peel, atractylodes, selfheal, white peony root, curcuma and licorice, coptis, scutellaria, pangolin, turtle shell, peony bark, chuanxiong, salvia miltiorrhiza, three-ling, honeysuckle, lianchi, zedoaria, coix seed and dried ginger. The original recipe of Taohong Siwu Decoction adopts the method of promoting blood circulation and adds bupleurum, angelica, chuanxiong and other drugs on the basis of the main prescription, or a self-prepared prescription of the method of dispersing knots and relieving pain, and the main drugs are bupleurum, curcuma, white peony root, corydalis, selfheal, oyster shell and thunbergii; there are many kinds of related internal and external Chinese medicine components, but the healing effect is not obvious, or the cycle is long; therefore, in the existing technology, a new technical solution is urgently needed to solve this problem.

Summary of the invention

The technical problem to be solved by the present invention is to provide Shiwei Xianglu Capsule and its preparation method and ginsenoside content analysis method, so as to give full play to the value of each Chinese medicine, have the effects of soothing liver and relieving depression, softening and dispersing nodules, etc., can reduce the breast alveolar cavity, reduce the number of lobules and alveoli and reduce the effect of estradiol, and mainly treat breast diseases caused by liver depression and phlegm stagnation.

The Shiwei Xianglu Capsule is characterized by comprising 10-30 parts of Cyperus rotundus, 2-6 parts of Xiebai, 2-6 parts of Deer Horn Deer Horn, 2-6 parts of Fritillaria thunbergii, 2-6 parts of Curcuma zedoaria, Scolopendra subspinipes, 2-6 parts of Asparagus cochinchinensis, 2-6 parts of Trionyx turtle shell, 2-6 parts of raw sun-dried ginseng and 1-3 parts of Platycodon grandiflorum in terms of mass fraction ratio.

The preparation method of Shiweixianglu Capsule is characterized in that the preparation of the Shiweixianglu Capsule comprises the following steps, and the following steps are performed in sequence:

Step 1, according to the ratio of claim 1, take centipede and 0.5 times the amount of raw sun-dried ginseng in the formula and grind them into fine powder with a particle size of 150 to 180 μm;

Step 2, according to the ratio of claim 1, take Cyperus rotundus, Curcuma zedoaria and Allium macrostemon, add 4 times the amount of medicinal materials and steam distill for 6 hours to obtain volatile oil, encapsulate the volatile oil with β-cyclodextrin for later use, and collect and retain the remaining aqueous solution and medicinal residues after distillation;

Step 3, according to the ratio of claim 1, take the deer antlers and turtle shells, add 6 times the amount of water of the medicinal materials and boil for 6 hours, and collect and retain the decoction and the residue respectively;

Step 4, according to the ratio described in claim 1, take Fritillaria thunbergii, Asparagus cochinchinensis, Platycodon grandiflorum and the remaining 0.5 times amount of raw sun-dried ingredients in the formula, combine the residues obtained in step 2 and step 3, add 4 times amount of medicinal materials in water and boil for 3 times, each time for 2 hours, and combine the decoction obtained with the aqueous solution obtained in step 2 and the decoction obtained in step 3, filter, concentrate to a relative density of 1.21-1.25 at 80°C, add ethanol to make the alcohol content reach 70%; let stand for 24 hours, filter, recover ethanol from the filtrate, and reduce to a thick paste;

Step 5: Add the ginseng centipede powder obtained in step 1 to the thick paste obtained in step 4, grind it into fine powder with a particle size of 150-180 μm after drying, mix it with the volatile oil inclusion compound prepared in step 2, and encapsulate it to prepare the finished product of Shiweixianglu Capsule.

The method for analyzing the ginsenoside content of Shiweixianglu Capsules is characterized by: determining the ginsenoside content of the prepared Shiweixianglu Capsules, comprising the following steps, and the following steps are performed in sequence:

Step 1: Prepare reference solution

Accurately weigh 2.04 mg of ginsenoside Rb ₁ reference substance, 2.04 mg of ginsenoside Rb ₃ reference substance, 2.02 mg of ginsenoside Re reference substance, 2.02 mg of ginsenoside Rf reference substance, and 2.01 mg of ginsenoside Rg ₁ reference substance, add methanol to dissolve, make up to 10 ml volumetric flask, mix and set aside;

Step 2: Prepare the test solution

Take the finished powder of Shiweixianglu Capsule and pass it through a 65-mesh sieve, accurately weigh 1.0002g into a 50mL EP tube, add 30mL methanol, and ultrasonically oscillate for 1h at a power of 250W and a frequency of 50kHz. After filtration, evaporate to dryness, dissolve the evaporated matter in 20mL water, extract it with 20mL dichloromethane and ethyl acetate three times in sequence, and then extract it with 20mL of water-saturated n-butanol for three times. Combine the upper extracts, evaporate to dryness, add appropriate amount of methanol to dissolve, and dilute to a 5mL volumetric flask for use;

Step 3: Ginseng-deficient negative sample solution

Grind centipede into fine powder, accurately weigh 1.0025g, and set aside; take 50.0028g of Cyperus rotundus, 30.0124g of Curcuma zedoaria, and 30.0113g of Allium macrostemon, add 4 times the amount of water, and extract volatile oil by steam distillation for 6h to obtain volatile oil solution, set aside, and collect the distilled aqueous solution in another container; take 3.0122g of Deer Horn Desquamation Plate, 3.0024g of Turtle Shell, add 6 times the amount of medicinal materials water and boil for 6h, and collect the decoction in another container; take 6.0241g of Fritillaria thunbergii, Combine 6.0125g of Asparagus cochinchinensis and 3.0026g of Platycodon grandiflorum with the above-mentioned medicinal residues, add 4 times the amount of medicinal materials water and boil for 3 times, each time for 2 hours, filter, combine the filtrate with the aqueous solution collected in another device and the decoction collected in another device, concentrate to a relative density of 1.21-1.25 at 80°C, add anhydrous ethanol to make its alcohol content reach 70%, let stand for 24 hours, filter, concentrate to a thick paste, add the above-mentioned centipede fine powder; dry in a ventilated place, grind into fine powder, mix with the volatile oil for use;

Step 4: System suitability test

The chromatographic conditions are as follows: using an Agilent EC-C ₁₈ chromatographic column 4.6 mm × 150 mm, 2.7 μm; the mobile phase is acetonitrile A-water B, the gradient elution is 0-60 min, 19% A; 60-120 min, 30%-50% A; the flow rate is 0.5 mL/min; the column temperature is 25° C.; the detection wavelength is 203 nm; under the chromatographic conditions, 3 μL of the reference solution obtained in step 1, the test solution obtained in step 2, and the ginseng-deficient negative sample solution obtained in step 3 are injected;

Step 5: Linear relationship test

Take 1 μL, 2 μL, 4 μL, 6 μL, 8 μL, and 10 μL of the reference solution obtained in step 1, respectively, and measure under the chromatographic conditions described in step 4, and calculate the linear regression equation and linear range of each component with the mass concentration of the reference as the abscissa X and the peak area as the ordinate Y;

Step 6: Sample recovery test

Take a sample of Shiweixianglu Capsule, grind it into fine powder, accurately weigh 6 times in parallel, and the samples are 0.2004g, 0.2008g, 0.2006g, 0.2004g, 0.2003g, and 0.2002g respectively, and accurately add the reference solution prepared in step 1 at a ratio of 1:1; prepare 6 test solutions in step 2, and determine the peak areas of ginsenoside _Rg1 , ginsenoside Re, ginsenoside Rf, ginsenoside _Rb1 , and ginsenoside Rb3 in the test solution according to the chromatographic conditions in step ₄ , and calculate the average recovery value and relative mean deviation RSD value;

Step 7: Calculation of relative correction factor

The reference solution prepared in step 1 was taken and measured under the chromatographic conditions of step 4. Ginsenoside Rb ₁ was used as the internal standard. The relative correction factor RCF between ginsenosides was calculated by the slope method and multi-point calibration method.

Slope method calculation formula: fi _/s = _ki / _ks ,

Wherein, f is the relative correction factor, k is the slope, i is the component to be measured, and s is the internal standard Rb ₁ ;

Multi-point correction method calculation formula: f = _fi / _fs = ( _Ai / _Ci ) / ( _As / _Cs )

Wherein, Ai is the peak area of the component to be measured, _Ci is the concentration of the component to be measured, in μg/μL; _As is the peak area of Rb ₁ , _Cs is the concentration of Rb ₁ , in μg/μL;

Step 8: Positioning parameter determination and repeatability inspection

Taking ginsenoside Rb ₁ as the internal standard, the relative retention values RT _Ri/s of ginsenoside Rg ₁ , ginsenoside Re, ginsenoside Rf and ginsenoside Rb ₃ were calculated by the formula RT _Ri/s = t _Ri /t _Rs , where t _Ri is the retention time of the component to be measured and t _Rs is the retention time of Rb ₁ .

Through the above design scheme, the present invention can bring the following beneficial effects: Shiweixianglu Capsule and its preparation method and ginsenoside content analysis method can reduce the breast alveolar cavity, reduce the number of lobules and alveoli and reduce the effect of estradiol, wherein the medicinal material basis of ginseng is ginsenoside, which has a regulatory effect on the neuroendocrine function of the pituitary-gonad axis, can excite the pituitary to secrete gonadotropin, promote the biosynthesis of enzyme proteins related to DNA and protein synthesis, increase the activity of the gonads, balance the positive and negative feedback functions of the neuroendocrine through the bidirectional regulatory effect on the pituitary-gonad axis to achieve the purpose of reducing abnormally increased estradiol, and inhibit the proliferation of breast cells controlled by ovarian hormones; due to the scarcity and high cost of reference substances in quality control, the intrinsic components of traditional Chinese medicine are selected as internal standards by adopting a one-test-multiple-evaluation method, and the content of the components to be tested is calculated by measuring the relative correction factor RCF between the components to be tested and other components to be tested, thereby reducing the use of reference substances and saving costs.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further described below with reference to the accompanying drawings and specific embodiments:

FIG. 1 is an HPLC chromatogram of a reference solution of the Shiweixianglu capsule of the present invention and its preparation method and a ginsenoside content analysis method system adaptability test.

2 is a HPLC chromatogram of the test sample solution of the Shiweixianglu Capsule and its preparation method and the ginsenoside content analysis method system adaptability test of the present invention.

3 is a HPLC chromatogram of a ginseng sample solution missing from a system adaptability test of the Shiweixianglu capsule of the present invention and its preparation method and ginsenoside content analysis method.

DETAILED DESCRIPTION

Shiweixianglu capsules, according to the mass ratio, include 10-30 parts of cyperus, 2-6 parts of Xiebai, 2-6 parts of antlers, 2-6 parts of Fritillaria thunbergii, 2-6 parts of Curcuma zedoaria, centipede, 2-6 parts of asparagus, 2-6 parts of turtle shell (roasted), 2-6 parts of raw sun-dried ginseng and 1-3 parts of Platycodon grandiflorum. The best ratio is 10 parts of cyperus, 6 parts of Xiebai, 6 parts of antlers, 6 parts of Fritillaria thunbergii, 6 parts of Curcuma zedoaria, 1 part of centipede, 6 parts of asparagus, 6 parts of turtle shell (roasted), 6 parts of ginseng and 3 parts of Platycodon grandiflorum.

The preparation method of Shiweixianglu Capsule comprises the following steps, and the following steps are performed in sequence:

Step 1, according to the ratio of claim 1, take centipede and 0.5 times the amount of raw sun-dried ginseng in the formula and grind them into fine powder with a particle size of 150 to 180 μm;

Step 2, according to the ratio of claim 1, take Cyperus rotundus, Curcuma zedoaria and Allium macrostemon, add 4 times the amount of medicinal materials and steam distill for 6 hours to obtain volatile oil, encapsulate the volatile oil with β-cyclodextrin for later use, and collect and retain the remaining aqueous solution and medicinal residues after distillation;

Step 3, according to the ratio of claim 1, take the deer antlers and turtle shells, add 6 times the amount of water of the medicinal materials and boil for 6 hours, and collect and retain the decoction and the residue respectively;

Step 4, according to the ratio described in claim 1, take Fritillaria thunbergii, Asparagus cochinchinensis, Platycodon grandiflorum and the remaining 0.5 times amount of raw sun-dried ginseng in the formula, combine the residues obtained in step 2 and step 3, add 4 times amount of medicinal materials in water and boil for 3 times, each time for 2 hours, combine the decoction obtained with the aqueous solution obtained in step 2 and the decoction obtained in step 3, filter, concentrate to a relative density of 1.21-1.25 at 80°C, add ethanol to make the alcohol content reach 70%; let stand for 24 hours, filter, recover ethanol from the filtrate, and reduce to a thick paste;

Step 5: Add the ginseng centipede powder obtained in step 1 to the thick paste obtained in step 4, grind it into fine powder with a particle size of 150-180 μm after drying, mix it with the volatile oil inclusion compound prepared in step 2, and encapsulate it to prepare the finished product of Shiweixianglu Capsule.

The medicinal material basis of ginseng in the formula of the present invention is ginsenoside, which has a regulatory effect on the neuroendocrine function of the pituitary-gonad axis, can excite the pituitary to secrete gonadotropin, promote the biosynthesis of enzyme proteins related to DNA and protein synthesis, increase the activity of the gonads, and balance the positive and negative feedback functions of neuroendocrine through the bidirectional regulatory effect on the pituitary-gonad axis to achieve the purpose of reducing abnormally increased estradiol, and inhibit the proliferation of breast cells controlled by ovarian hormones. Therefore, it is of great significance to establish a quality control method for ginsenosides related to the medicinal effect of this product. The standard of Shiwei Xianglu Capsule has established the thin layer chromatography identification of ginseng, zedoaria, centipede, and thunbergii, the content determination of thunbergine in thunbergii, and the content of ginsenoside Rb ₁ , Re, and Rg ₁ in ginseng, but no one-test-multiple-evaluation has been carried out. In the following, ginsenoside Rb ₁ , ginsenoside Rb ₃ , ginsenoside Re, ginsenoside Rf and ginsenoside Rg ₁ were used as determination indicators to conduct one-test-multiple-evaluation quality control additional item analysis on Shiweixianglu Capsules.

The method for analyzing the content of ginsenosides in Shiweixianglu capsules comprises the following steps, and the following steps are performed in sequence:

Step 1: Prepare reference solution

Accurately weigh 2.04 mg of ginsenoside Rb ₁ reference substance, 2.04 mg of ginsenoside Rb ₃ reference substance, 2.02 mg of ginsenoside Re reference substance, 2.02 mg of ginsenoside Rf reference substance, and 2.01 mg of ginsenoside Rg ₁ reference substance, add methanol to dissolve, make up to 10 ml volumetric flask, mix and set aside;

Step 2: Prepare the test solution

Take the finished powder of Shiweixianglu Capsule and pass it through a 65-mesh sieve, accurately weigh 1.0002g into a 50mL EP tube, add 30mL methanol, and ultrasonically oscillate for 1h at a power of 250W and a frequency of 50kHz. After filtration, evaporate to dryness, dissolve the evaporated matter in 20ml water, extract it with 20mL dichloromethane and ethyl acetate three times in sequence, and then extract it with 20mL water-saturated n-butanol for three times. Combine the upper extracts, evaporate to dryness, add appropriate amount of methanol to dissolve, and dilute to a 5mL volumetric flask for use;

Step 3: Negative sample solution lacking ginseng

Grind centipede into fine powder, accurately weigh 1.0025g, and set aside; take 50.0028g of Cyperus rotundus, 30.0124g of Curcuma zedoaria, and 30.0113g of Allium macrostemon, add 4 times the amount of water, and extract volatile oil by steam distillation for 6h to obtain volatile oil solution, set aside, and collect the distilled aqueous solution in another container; take 3.0122g of Deer Horn Desquamation Plate, 3.0024g of Turtle Shell, add 6 times the amount of medicinal materials water and boil for 6h, and collect the decoction in another container; take 6.0241g of Fritillaria thunbergii, Combine 6.0125g of Asparagus cochinchinensis and 3.0026g of Platycodon grandiflorum with the above-mentioned medicinal residues, add 4 times the amount of medicinal materials water and boil for 3 times, each time for 2 hours, filter, combine the filtrate with the aqueous solution collected in another device and the decoction collected in another device, concentrate to a relative density of 1.21-1.25 at 80°C, add anhydrous ethanol to make its alcohol content reach 70%, let stand for 24 hours, filter, concentrate to a thick paste, add the above-mentioned centipede fine powder; dry in a ventilated place, grind into fine powder, mix with the volatile oil for use;

Step 4: System suitability test

The chromatographic conditions are as follows: using an Agilent EC-C ₁₈ chromatographic column of 4.6 mm×150 mm, 2.7 μm; the mobile phase is acetonitrile A-water B, the gradient elution is 0-60 min, 19% A; 60-120 min, 30%-50% A; the flow rate is 0.5 mL/min; the column temperature is 25° C.; the detection wavelength is 203 nm; under the chromatographic conditions, 3 μL of the reference solution obtained in step 1, the test solution obtained in step 2, and the ginseng-deficient negative sample solution obtained in step 3 are injected; the HPLC chromatograms of the results are shown in FIGS. 1 to 3, wherein 1 is ginsenoside Rg ₁ , 2 is ginsenoside Re, 3 is ginsenoside Rf, 4 is ginsenoside Rb ₁ , and 5 is ginsenoside Rb ₃ ;

Step 5: Linear relationship test

1 μL, 2 μL, 4 μL, 6 μL, 8 μL, and 10 μL of the reference solution obtained in step 1 were taken respectively, and measured under the chromatographic conditions described in step 4. The linear regression equation and linear range of each component were calculated with the mass concentration of the reference as the abscissa X and the peak area as the ordinate Y. The results are shown in Table 1 below;

Table 1 Linear relationship of ginsenosides

Step 6: Precision test

Take the reference solution prepared in step 1, and inject it continuously for 6 times according to the chromatographic conditions in step 4, and determine the peak area values of ginsenoside Rg ₁ , ginsenoside Re, ginsenoside Rf, ginsenoside Rb ₁ , and ginsenoside Rb _3. The relative average deviations (RSDs) are calculated to be 1.23%, 2.46%, 1.42%, 1.83%, and 1.79%, respectively, which proves that the precision of the instrument is good;

Step 7: Stability test

Take the same sample solution prepared in step 2, and inject it at 0h, 3h, 6h, 9h, 12h, and 24h according to the chromatographic conditions in step 4, and determine the peak areas of ginsenoside Rg ₁ , ginsenoside Re, ginsenoside Rf, ginsenoside Rb ₁ , and ginsenoside Rb _3. The relative average deviations (RSDs) were calculated to be 1.49%, 1.04%, 1.39%, 1.36%, and 1.75%, respectively, proving that the sample solution of Shiwei Xianglu Capsule is relatively stable within 24h;

Step 8: Repeatability test

Take the Shiweixianglu capsule sample, prepare 6 copies in parallel according to the method in step 2, and determine the contents of ginsenoside Rg ₁ , ginsenoside Re, ginsenoside Rf, ginsenoside Rb ₁ , and ginsenoside Rb ₃ according to the chromatographic conditions in step 4. The calculated relative mean deviations (RSDs) are 1.82%, 1.43%, 1.89%, 2.00%, and 1.92%, respectively, which proves that this method has good repeatability.

Step 9: Sample recovery test

Take Shiweixianglu Capsule sample, grind it into fine powder, and accurately weigh 0.2004g, 0.2008g, 0.2006g, 0.2004g, 0.2003g, and 0.2002g in parallel 6 times, and accurately add the reference solution prepared in step 1 at a ratio of 1:1; take step 2 to prepare 6 test solutions, and determine the peak areas of ginsenoside _Rg1 , ginsenoside Re, ginsenoside Rf, ginsenoside _Rb1 and ginsenoside _Rb3 in the test solution according to the chromatographic conditions in step 4, calculate the average sample recovery value and relative average deviation RSD value, and the results are shown in Table 2 below.

Table 2 Results of the recovery test of ginsenosides (n=6)

Step 10: Calculation of relative correction factor

The reference solution prepared in step 1 was taken and measured under the chromatographic conditions of step 4. Ginsenoside Rb ₁ was used as the internal standard. The relative correction factor RCF between ginsenosides was calculated by the slope method and multi-point calibration method.

Slope method calculation formula: fi _/s = _ki / _ks ,

Wherein, f is the relative correction factor, k is the slope, i is the component to be measured, and s is the internal standard Rb ₁ ;

Multi-point correction method calculation formula: f = _fi / _fs = ( _Ai / _Ci ) / ( _As / _Cs )

Wherein, Ai is the peak area of the component to be measured, _Ci is the concentration of the component to be measured, in μg/μL; _As is the peak area of Rb ₁ , _Cs is the concentration of Rb ₁ , in μg/μL;

The relative errors RE of the above two methods are both less than 3.0%, and the results are shown in Table 3 below.

Table 3 Relative correction factors of ginsenosides

Step 11: Positioning parameter determination and repeatability inspection

Taking ginsenoside Rb ₁ as the internal standard, the relative retention values RT _Ri/s of ginsenoside Rg ₁ , ginsenoside Re, ginsenoside Rf and ginsenoside Rb ₃ were calculated by the formula RT _Ri/s = t _Ri /t _Rs , where t _Ri is the retention time of the component to be tested and t _Rs is the retention time of Rb _1. The results are shown in Table 4 below.

Table 4 Relative retention values of ginsenosides (n=6)

At the same time, the ten-flavor fragrant deer capsule and its preparation method and the ginsenoside content analysis method of the present invention are also subjected to durability inspection. An appropriate amount of the reference solution prepared in step one is taken, and the sample is injected and measured under the spectrum conditions described in step four. The multi-point calibration method is used to inspect the influence of Agilent 1260 chromatograph, Agilent 1100 Series chromatograph, Agilent EC-C ₁₈ chromatographic column, Zorbax SB-C ₁₈ chromatographic column, and Extend-C ₁₈ chromatographic column on the relative correction factor. The results are shown in Table 5 below, and it can be seen that there is no obvious influence;

Table 5 Effects of different instruments and chromatographic columns on relative correction factors

The chromatographic peak of the component to be tested is located, and the relative retention values of the other four components under different chromatographs and chromatographic columns are verified by the algorithm in step 11. The results are shown in Table 6 below, and it can be seen that there is no obvious effect;

Comparison of the results of QAMS (one measurement multiple evaluation method) and ESM (external standard method) According to the chromatographic conditions described in step 4, the five components of Shiweixianglu Capsule were determined by QAMS and ESM respectively. The results are shown in Table 7 below.

Table 7 Comparison of results obtained by one-test-multiple-evaluation method and external standard method (n=6)

The Shiweixianglu Capsule and its preparation method and ginsenoside content analysis method of the present invention establish a one-test-multiple-evaluation method with simple operation and reliable results by determining the contents of multiple ginsenosides in the Shiweixianglu Capsule, thereby solving the problems of lack and high cost of ginsenoside reference substances. The test uses ginsenoside Rb ₁ as the internal standard, and calculates its content by the relative correction factors of ginsenoside Rg ₁ , ginsenoside Re, ginsenoside Rf, and ginsenoside Rb _3. The results show that there is no significant difference between the results obtained by the method and the external standard method. This analysis method supplements the quality evaluation method of multiple pharmacological ingredients of Shiweixianglu Capsule, and more effectively controls product quality.

Claims (1)

1. A method for analyzing the content of ginsenosides in Shiweixianglu capsules, characterized in that the content of ginsenosides in the finished product of Shiweixianglu capsules is determined, comprising the following steps, and the following steps are performed in sequence: Step 1: Prepare reference solution Accurately weigh 2.04 mg of ginsenoside Rb ₁ reference substance, 2.04 mg of ginsenoside Rb ₃ reference substance, 2.02 mg of ginsenoside Re reference substance, 2.02 mg of ginsenoside Rf reference substance, and 2.01 mg of ginsenoside Rg ₁ reference substance, add methanol to dissolve, make up to 10 ml volumetric flask, mix and set aside; Step 2: Prepare the test solution Take the finished powder of Shiweixianglu Capsule and pass it through a 65-mesh sieve, accurately weigh 1.0002g into a 50mL EP tube, add 30mL methanol, and ultrasonically oscillate for 1h at a power of 250W and a frequency of 50kHz. After filtration, evaporate to dryness, dissolve the evaporated matter in 20mL water, extract it with 20mL dichloromethane and ethyl acetate three times in sequence, and then extract it with 20mL of water-saturated n-butanol for three times. Combine the upper extracts, evaporate to dryness, add appropriate amount of methanol to dissolve, and dilute to a 5mL volumetric flask for use; Step 3: Ginseng-deficient negative sample solution Grind centipede into fine powder, accurately weigh 1.0025g, and set aside; take 50.0028g of Cyperus rotundus, 30.0124g of Curcuma zedoaria, and 30.0113g of Allium macrostemon, add 4 times the amount of water, and extract volatile oil by steam distillation for 6h to obtain volatile oil solution, set aside, and collect the distilled aqueous solution in another container; take 3.0122g of Deer Horn Desquamation Plate, 3.0024g of Turtle Shell, add 6 times the amount of medicinal materials water and boil for 6h, and collect the decoction in another container; take 6.0241g of Fritillaria thunbergii, Combine 6.0125g of Asparagus cochinchinensis and 3.0026g of Platycodon grandiflorum with the above-mentioned medicinal residues, add 4 times the amount of medicinal materials water and boil for 3 times, each time for 2 hours, filter, combine the filtrate with the aqueous solution collected in another device and the decoction collected in another device, concentrate to a relative density of 1.21-1.25 at 80°C, add anhydrous ethanol to make its alcohol content reach 70%, let stand for 24 hours, filter, concentrate to a thick paste, add the above-mentioned centipede fine powder; dry in a ventilated place, grind into fine powder, mix with the volatile oil for use; Step 4: System suitability test The chromatographic conditions are as follows: using an Agilent EC-C ₁₈ chromatographic column 4.6 mm × 150 mm, 2.7 μm; the mobile phase is acetonitrile A-water B, the gradient elution is 0-60 min, 19% A; 60-120 min, 30%-50% A; the flow rate is 0.5 mL/min; the column temperature is 25° C.; the detection wavelength is 203 nm; under the chromatographic conditions, 3 μL of the reference solution obtained in step 1, the test solution obtained in step 2, and the ginseng-deficient negative sample solution obtained in step 3 are injected; Step 5: Linear relationship test Take 1 μL, 2 μL, 4 μL, 6 μL, 8 μL, and 10 μL of the reference solution obtained in step 1, respectively, and measure under the chromatographic conditions described in step 4, and calculate the linear regression equation and linear range of each component with the mass concentration of the reference as the abscissa X and the peak area as the ordinate Y; Step 6: Sample recovery test Take a sample of Shiweixianglu Capsule, grind it into fine powder, accurately weigh 6 times in parallel, and the samples are 0.2004g, 0.2008g, 0.2006g, 0.2004g, 0.2003g, and 0.2002g respectively, and accurately add the reference solution prepared in step 1 at a ratio of 1:1; prepare 6 test solutions in step 2, and determine the peak areas of ginsenoside _Rg1 , ginsenoside Re, ginsenoside Rf, ginsenoside _Rb1 , and ginsenoside Rb3 in the test solution according to the chromatographic conditions in step ₄ , and calculate the average recovery value and relative mean deviation RSD value; Step 7: Calculation of relative correction factor The reference solution prepared in step 1 was taken and measured under the chromatographic conditions of step 4. Ginsenoside Rb ₁ was used as the internal standard. The relative correction factor RCF between ginsenosides was calculated by the slope method and multi-point calibration method. Slope method calculation formula: fi _/s = _ki / _ks , Wherein, f is the relative correction factor, k is the slope, i is the component to be measured, and s is the internal standard Rb ₁ ; Multi-point correction method calculation formula: f = _fi / _fs = ( _Ai / _Ci ) / ( _As / _Cs ) Wherein, Ai is the peak area of the component to be measured, _Ci is the concentration of the component to be measured, in μg/μL; _As is the peak area of Rb ₁ , _Cs is the concentration of Rb ₁ , in μg/μL; Step 8: Positioning parameter determination and repeatability inspection Taking ginsenoside Rb ₁ as the internal standard, the relative retention values RT _Ri/s of ginsenoside Rg ₁ , ginsenoside Re, ginsenoside Rf and ginsenoside Rb ₃ were calculated by the formula RT _Ri/s = t _Ri /t _Rs , where t _Ri is the retention time of the component to be measured and t _Rs is the retention time of Rb ₁ .

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