CN110917313A - Ten-ingredient xianglu capsules, preparation method thereof and ginsenoside content analysis method - Google Patents
Ten-ingredient xianglu capsules, preparation method thereof and ginsenoside content analysis method Download PDFInfo
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Abstract
The invention provides a ten-ingredient deer capsule, a preparation method thereof and a ginsenoside content analysis method, belonging to the technical field of traditional Chinese medicine formulas, wherein the capsule gives full play to the value of each traditional Chinese medicine, has the efficacies of soothing liver and relieving depression, softening hardness and dissipating stagnation and the like, wherein the drug effect substance of ginseng is ginsenoside which has a regulating effect on the neuroendocrine function of pituitary-gonadal axis, can excite the pituitary to secrete gonadotropin, promote the biosynthesis of DNA and protein synthesis related enzyme protein, increase the activity of gonadal, balance the positive and negative feedback function of neuroendocrine through the bidirectional regulating effect on the pituitary-gonadal axis so as to achieve the purpose of reducing abnormally increased estradiol and inhibit the hyperplasia of mammary gland cells controlled by ovarian hormone; the internal components of the traditional Chinese medicine are selected as internal standards in a one-test and multi-evaluation mode, and the content of the components to be tested is calculated by measuring the relative correction factor RCF between the internal standard and other components to be tested, so that the use amount of a reference substance is reduced, and the cost is saved.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine formulas, and particularly relates to a traditional Chinese medicine for regulating hyperplasia of mammary glands and a verification method of the efficacy of the traditional Chinese medicine.
Background
The hyperplasia of mammary glands (Hmg) belongs to the category of breast pain in traditional Chinese medicine, and the basic reason for the occurrence and development of the hyperplasia of mammary glands is the dysfunction of three major internal organs, namely liver depression, spleen deficiency and kidney deficiency, which are externally manifested as phlegm coagulation, qi stagnation and blood stasis. The existing therapeutic prescription mainly comprises radix bupleuri, thunberg fritillary bulb, Chinese angelica, mint, tuckahoe, green tangerine peel, bighead atractylodes rhizome, selfheal, white paeony root, radix curcumae and honey-fried licorice root, coptis chinensis, scutellaria baicalensis, pangolin, turtle shell, tree peony bark, szechuan lovage rhizome, salvia miltiorrhiza bunge, mitsubishi, honeysuckle, weeping forsythia, zedoary, coix seed and dried ginger, and the original formula of the peach red four-ingredient decoction adopts a blood activating method and a self-prepared formula by a large method for dispersing knots and relieving pain, wherein the main medicines comprise radix bupleuri, radix curcumae, white paeony root, rhizoma corydalis, selfheal, forged oyster and thunberg fritillary bulb; related oral and external traditional Chinese medicine components are various, but the healing effect is not obvious, or the period is long; therefore, a new technical solution is needed to solve the problem in the prior art.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides ten-ingredient Xianglu capsules, a preparation method thereof and a ginsenoside content analysis method, fully exerts the value of each traditional Chinese medicine, has the efficacies of soothing liver and relieving depression, softening hardness and dissipating stagnation and the like, can reduce the acinar cavity of mammary gland, reduce the number of lobules and acini and reduce estradiol, and is mainly used for treating mammary gland diseases caused by liver depression and phlegm coagulation.
Ten-ingredient xianglu capsules are characterized in that: the traditional Chinese medicine composition comprises, by mass, 10-30 parts of rhizoma cyperi, 2-6 parts of allium macrostemon, 2-6 parts of antler base, 2-6 parts of thunberg fritillary bulb, 2-6 parts of curcuma zedoary, centipede, 2-6 parts of asparagus cochinchinensis, 2-6 parts of turtle shell, 2-6 parts of sun-dried ginseng and 1-3 parts of platycodon grandiflorum.
The preparation method of the ten-flavor deer capsule is characterized by comprising the following steps: the preparation of the ten-flavor deer capsule comprises the following steps which are sequentially carried out,
step one, according to the mixture ratio of claim 1, pulverizing centipedes and 0.5 times of sun-dried ginseng in a formula into fine powder with the particle size of 150-180 microns;
step two, according to the mixture ratio of claim 1, adding 4 times of the amount of the medicinal materials into the rhizoma cyperi, the curcuma zedoary and the allium macrostemon, distilling for 6 hours to obtain volatile oil, including the volatile oil by β -cyclodextrin for later use, and respectively collecting and retaining residual aqueous solution and dregs after distillation;
step three, taking antler plates and turtle shells according to the mixture ratio of claim 1, adding 6 times of medicinal materials into water, decocting for 6 hours, and respectively collecting and retaining decoction and dregs;
step four, according to the mixture ratio of claim 1, taking thunberg fritillary bulb, asparagus, platycodon grandiflorum and the residual 0.5 times of raw sun-dried medicinal residues in the formula to be combined in the step two and the step three, adding 4 times of medicinal water to decoct for 3 times, and 2 hours for each time, combining the obtained decoction with the aqueous solution obtained in the step two and the decoction obtained in the step three, filtering, concentrating to the relative density of 1.21-1.25 at 80 ℃, and adding ethanol to ensure that the alcohol content reaches 70%; standing for 24 hr, filtering, recovering ethanol from the filtrate, and concentrating into soft extract;
and step five, adding the ginseng centipede fine powder obtained in the step one into the thick paste obtained in the step four, drying, crushing into fine powder with the particle size of 150-180 mu m, mixing with the volatile oil inclusion compound prepared in the step two, and encapsulating to prepare a finished product of the ten-flavor deer capsule.
The method for analyzing the content of the ginsenoside in the ten-ingredient xianglu capsules is characterized by comprising the following steps of: the method for measuring the content of the ginsenoside in the prepared ten-flavor deer capsule finished product comprises the following steps which are sequentially carried out,
step one, preparing a reference substance solution
Precisely weighing ginsenoside Rb1Reference substance 2.04mg and ginsenoside Rb3Reference substance 2.04mg, ginsenoside Re reference substance 2.02mg, ginsenoside Rf reference substance 2.02mg, and ginsenoside Rg1Adding 2.01mg of reference substance into methanol for dissolving, fixing the volume to a 10ml volumetric flask, and mixing for later use;
step two, preparing a test solution
Taking ten-flavor deer capsule finished product powder, sieving with a 65-mesh sieve, precisely weighing 1.0002g to 50mL LEP tube, adding 30mL of methanol, carrying out ultrasonic oscillation for 1h under the conditions of 250W power and 50kHz frequency, filtering, evaporating to dryness, dissolving the evaporated material in 20mL of water, sequentially extracting with 20mL of dichloromethane and ethyl acetate for 3 times, extracting with 20mL of water-saturated n-butanol for 3 times, combining upper-layer extract liquor, evaporating to dryness, adding a proper amount of methanol for dissolving, and fixing the volume to a 5mL volumetric flask for later use;
step three, ginseng deficiency negative sample solution
Grinding Scolopendra into fine powder, precisely weighing 1.0025 g; taking 50.0028g of rhizoma cyperi, 30.0124g of curcuma zedoary and 30.0113g of allium macrostemon, adding 4 times of water, extracting volatile oil for 6 hours by a steam distillation method to obtain volatile oil solution for later use, and collecting the distilled water solution in another container; adding 3.0122g of antler base and 3.0024g of turtle shell into 6 times of water, decocting for 6 hours, and collecting decoction in another container; mixing 6.0241g of thunberg fritillary bulb, 6.0125g of radix asparagi and 3.0026g of platycodon grandiflorum with the residues, adding 4 times of medicinal water, decocting for 3 times, each time for 2 hours, filtering, mixing the filtrate with the aqueous solution collected by the other container and the decoction collected by the other container, concentrating to the relative density of 1.21-1.25 at the temperature of 80 ℃, adding absolute ethyl alcohol until the alcohol content reaches 70%, standing for 24 hours, filtering, concentrating to thick paste, and adding the centipede fine powder; drying in a ventilated place, grinding into fine powder, and mixing with the volatile oil for later use;
step four, testing the adaptability of the system
The chromatographic conditions are as follows: by Agilent EC-C18Chromatographic column 4.6mm × 150mm, 2.7 μm; the mobile phase is acetonitrile A-water B, and gradient elution is carried out for 0-60 min, wherein the concentration of A is 19%; 60-120 min, 30% -50% A; the flow rate is 0.5 mL/min; the column temperature was 25 ℃; the detection wavelength is 203 nm; injecting 3 mu L of the reference substance solution obtained in the step one, the test substance solution obtained in the step two and the ginseng-deficient negative sample solution obtained in the step three under the chromatographic condition;
step five, linear relation test
Respectively taking 1 muL, 2 muL, 4 muL, 6 muL, 8 muL and 10 muL of the reference substance solution obtained in the step one, measuring under the chromatographic condition described in the step four, and calculating a linear regression equation and a linear range of each component by taking the reference mass concentration as a horizontal coordinate X and the peak area as a vertical coordinate Y;
step six, sample adding recovery rate test
Taking a ten-flavor deer capsule sample, grinding into fine powder, precisely weighing in parallel 6 times to obtain 0.2004g, 0.2008g, 0.2006g, 0.2004g, 0.2003g and 0.2002g respectively, and precisely adding the reference substance solution prepared in the step one according to a ratio of 1: 1; taking 6 parts of the sample solution prepared in the second step, and determining the ginsenoside Rg in the sample solution according to the chromatographic conditions in the fourth step1Ginsenoside Re, ginsenoside Rf and ginsenoside Rb1And ginsenoside Rb3Calculating the average sample recovery rate value and the relative average deviation RSD value;
step seven, calculating relative correction factor
Sucking the reference solution prepared in the first step, measuring with the chromatographic conditions of the fourth step, and measuring with ginsenoside Rb1As an internal standard, calculating a relative correction factor RCF between ginsenosides by a slope method and a multipoint correction method, wherein the slope method is used for calculating a formula: f. ofi/s＝ki/ks，
Wherein f is a relative correction factor, k is a slope, i is a component to be detected, and s is an internal standard substance Rb1；
The multipoint correction method calculation formula is as follows: f ═ fi/fs＝(Ai/Ci)/(As/Cs)
Wherein Ai is the peak area of the component to be measured, CiThe concentration of the component to be detected is presented in the unit of mu g/mu L; a. thesIs Rb1Area of peak of (C)sIs Rb1The concentration of (d) is in units of μ g/μ L;
step eight, positioning parameter measurement and repeatability investigation
Is prepared from ginsenoside Rb1As an internal standard, by the formula RTRi/s＝tRi/tRsCalculating ginsenoside Rg1Ginsenoside Re, ginsenoside Rf and ginsenoside Rb3Relative retention value of RTRi/sWherein t isRiRetention time of the component to be measured, tRsIs Rb1The retention time of (c).
Through the design scheme, the invention can bring the following beneficial effects: ten-flavor deer capsules, a preparation method thereof and a ginsenoside content analysis method thereof, which enable the cavum of mammary gland acinus to be reduced, the number of lobules and acinus to be reduced and the function of estradiol to be reduced, wherein the drug effect substance of ginseng is ginsenoside which has the regulation function on the neuroendocrine function of pituitary-gonadal axis, can excite the pituitary to secrete gonadotropin, promote the biosynthesis of DNA and protein synthesis related enzyme protein, increase the activity of gonadal gland, balance the positive and negative feedback function of neuroendocrine through the bidirectional regulation function of pituitary-gonadal axis to achieve the purpose of reducing abnormally increased estradiol and inhibit the hyperplasia of mammary gland cells controlled by ovarian hormone; due to the scarcity and the high cost of the reference substance in the quality control, the internal components of the traditional Chinese medicine are selected as internal standards in a one-test-and-multiple-evaluation mode, and the content of the component to be detected is calculated by measuring the relative correction factor RCF between the internal standard and other components to be detected, so that the use amount of the reference substance is reduced, and the cost is saved.
Drawings
The invention is further described with reference to the following figures and detailed description:
FIG. 1 is HPLC chromatogram of a reference solution for systematic adaptability test of the Ten-flavor Xianglu capsules, a preparation method thereof and a ginsenoside content analysis method.
FIG. 2 is HPLC chromatogram of test solution in systematic adaptability test of Ten-flavor deer capsule, its preparation method and ginsenoside content analysis method.
FIG. 3 is a HPLC chromatogram of a system adaptability test of a ginseng-deficient sample solution of the ten-flavor deer capsule, the preparation method thereof and the ginsenoside content analysis method.
Detailed Description
The ten-flavor deer capsule comprises, by mass, 10-30 parts of rhizoma cyperi, 2-6 parts of allium macrostemon, 2-6 parts of antler base, 2-6 parts of thunberg fritillary bulb, 2-6 parts of curcuma zedoary, centipede, 2-6 parts of asparagus cochinchinensis, 2-6 parts of turtle shell (roasted), 2-6 parts of sun-dried ginseng and 1-3 parts of platycodon grandiflorum. The optimal proportion is 10 portions of nutgrass galingale rhizome, 6 portions of longstamen onion bulb, 6 portions of antler base, 6 portions of thunberg fritillary bulb, 6 portions of zedoary, 1 portion of centipede, 6 portions of asparagus, 6 portions of turtle shell (roasted), 6 portions of ginseng and 3 portions of platycodon root.
The preparation method of the ten-flavor deer capsule comprises the following steps which are sequentially carried out,
step one, according to the mixture ratio of claim 1, pulverizing centipedes and 0.5 times of sun-dried ginseng in a formula into fine powder with the particle size of 150-180 microns;
step two, according to the mixture ratio of claim 1, adding 4 times of the amount of the medicinal materials into the rhizoma cyperi, the curcuma zedoary and the allium macrostemon, distilling for 6 hours to obtain volatile oil, including the volatile oil by β -cyclodextrin for later use, and respectively collecting and retaining residual aqueous solution and dregs after distillation;
step three, taking antler plates and turtle shells according to the mixture ratio of claim 1, adding 6 times of medicinal materials into water, decocting for 6 hours, and respectively collecting and retaining decoction and dregs;
step four, according to the mixture ratio of claim 1, mixing thunberg fritillary bulb, asparagus, platycodon grandiflorum and the left 0.5 times of raw sun-dried medicinal residues in the formula, adding 4 times of medicinal water, decocting for 3 times, and decocting for 2 hours each time to obtain a decoction, mixing the decoction with the aqueous solution obtained in the step two and the decoction obtained in the step three, filtering, concentrating to a relative density of 1.21-1.25 at 80 ℃, and adding ethanol to ensure that the alcohol content reaches 70%; standing for 24 hr, filtering, recovering ethanol from the filtrate, and concentrating into soft extract;
and step five, adding the ginseng centipede fine powder obtained in the step one into the thick paste obtained in the step four, drying, crushing into fine powder with the particle size of 150-180 mu m, mixing with the volatile oil inclusion compound prepared in the step two, and encapsulating to prepare a finished product of the ten-flavor deer capsule.
The drug effect substance of the ginseng in the formula is ginsenoside which has a regulating effect on the neuroendocrine function of the pituitary-gonadal axis, can excite the pituitary to secrete gonadotropin, promote the biosynthesis of DNA and protein synthesis related enzyme protein, increase the gonadal activity, balance the positive and negative feedback functions of neuroendocrine through the bidirectional regulating effect on the pituitary-gonadal axis so as to achieve the purpose of reducing abnormally increased estradiol and inhibit the hyperplasia of mammary glands controlled by ovarian hormone. Therefore, establishing the quality control method of the ginsenoside related to the drug effect of the product has important significance. The ten-ingredient Xianglu capsule standard establishes the thin-layer chromatography identification of ginseng, zedoary, centipede and Zhejiang fritillaria, the content determination of peiminine A in Zhejiang fritillaria, and the ginsenoside Rb in ginseng1、Re、Rg1But not assessed more than once. The following ginsenoside Rb1Ginsenoside Rb3Ginsenoside Re, ginsenoside Rf and ginsenoside Rg1For determining indexes, the quality control and item increasing analysis is carried out on the ten-flavor Xianglu capsules by one test and multiple evaluations.
The method for analyzing the content of the ginsenoside in the ten-flavor deer capsules comprises the following steps which are sequentially carried out,
step one, preparing a reference substance solution
Precisely weighing ginsenoside Rb1Reference substance 2.04mg and ginsenoside Rb3Reference substance 2.04mg, ginsenoside Re reference substance 2.02mg, ginsenoside Rf reference substance 2.02mg, and ginsenoside Rg1Adding 2.01mg of reference substance into methanol for dissolving, fixing the volume to a 10ml volumetric flask, and mixing for later use;
step two, preparing a test solution
Taking ten-flavor deer capsule finished product powder, sieving with a 65-mesh sieve, precisely weighing 1.0002g to 50mL LEP tube, adding 30mL of methanol, carrying out ultrasonic oscillation for 1h under the conditions of 250W power and 50kHz frequency, filtering, evaporating to dryness, dissolving the evaporated material in 20mL of water, sequentially extracting with 20mL of dichloromethane and ethyl acetate for 3 times, extracting with 20mL of water-saturated n-butanol for 3 times, combining upper-layer extract liquor, evaporating to dryness, adding a proper amount of methanol for dissolving, and fixing the volume to a 5mL volumetric flask for later use;
step three, ginseng deficiency negative sample solution
Grinding Scolopendra into fine powder, precisely weighing 1.0025 g; taking 50.0028g of rhizoma cyperi, 30.0124g of curcuma zedoary and 30.0113g of allium macrostemon, adding 4 times of water, extracting volatile oil for 6 hours by a steam distillation method to obtain volatile oil solution for later use, and collecting the distilled water solution in another container; adding 3.0122g of antler base and 3.0024g of turtle shell into 6 times of water, decocting for 6 hours, and collecting decoction in another container; mixing 6.0241g of thunberg fritillary bulb, 6.0125g of radix asparagi and 3.0026g of platycodon grandiflorum with the residues, adding 4 times of medicinal water, decocting for 3 times, each time for 2 hours, filtering, mixing the filtrate with the aqueous solution collected by the other container and the decoction collected by the other container, concentrating to the relative density of 1.21-1.25 at the temperature of 80 ℃, adding absolute ethyl alcohol until the alcohol content reaches 70%, standing for 24 hours, filtering, concentrating to thick paste, and adding the centipede fine powder; drying in a ventilated place, grinding into fine powder, and mixing with the volatile oil for later use;
step four, testing the adaptability of the system
The chromatographic conditions are as follows: by Agilent EC-C18Chromatographic column 4.6mm × 150mm, 2.7 μm; the mobile phase is acetonitrile A-water B, and gradient elution is carried out for 0-60 min, wherein the concentration of A is 19%; 60-120 min, 30% -50% A; flow velocity of 0.5mL/min; the column temperature was 25 ℃; the detection wavelength is 203 nm; injecting 3 mu L of the reference substance solution obtained in the step one, the test substance solution obtained in the step two and the ginseng-deficient negative sample solution obtained in the step three under the chromatographic condition; the HPLC chromatograms of the results are shown in fig. 1-3, wherein 1 is ginsenoside Rg12 is ginsenoside Re, 3 is ginsenoside Rf, and 4 is ginsenoside Rb1And 5 is ginsenoside Rb3；
Step five, linear relation test
Respectively taking 1 muL, 2 muL, 4 muL, 6 muL, 8 muL and 10 muL of the reference substance solution obtained in the first step, measuring under the chromatographic conditions described in the fourth step, and calculating a linear regression equation and a linear range of each component by taking the reference mass concentration as a horizontal coordinate X and the peak area as a vertical coordinate Y, wherein the results are shown in the following table 1;
TABLE 1 Linear relationship of each ginsenoside
Step six, precision test
Taking the reference solution prepared in the step one, continuously injecting sample for 6 times according to the chromatographic condition in the step four, and determining the ginsenoside Rg1Ginsenoside Re, ginsenoside Rf and ginsenoside Rb1Ginsenoside Rb3The calculated relative average deviation RSD is 1.23%, 2.46%, 1.42%, 1.83% and 1.79% respectively, which proves that the precision of the instrument is good;
step seven, stability test
Sampling the same sample solution prepared in step two for 0h, 3h, 6h, 9h, 12h and 24h according to the chromatographic conditions in step four, and determining ginsenoside Rg1Ginsenoside Re, ginsenoside Rf and ginsenoside Rb1Ginsenoside Rb3The calculated relative average deviation RSD is respectively 1.49%, 1.04%, 1.39%, 1.36% and 1.75%, which proves that the test solution of the ten-flavor deer capsule is more stable within 24 h;
step eight, repeatability test
Taking a ten-flavor deer capsule sample, preparing 6 parts in parallel under the method of the step two, and determining ginsenoside Rg according to the chromatographic conditions in the step four1Ginsenoside Re, ginsenoside Rf and ginsenoside Rb1Ginsenoside Rb3The calculated relative average deviation RSD is 1.82%, 1.43%, 1.89%, 2.00% and 1.92% respectively, which proves that the method has good repeatability;
step nine, sample adding recovery rate test
Taking a ten-flavor deer capsule sample, grinding into fine powder, precisely weighing in parallel 6 times to obtain 0.2004g, 0.2008g, 0.2006g, 0.2004g, 0.2003g and 0.2002g respectively, and precisely adding the reference substance solution prepared in the step one according to a ratio of 1: 1; taking 6 parts of the sample solution prepared in the second step, and determining the ginsenoside Rg in the sample solution according to the chromatographic conditions in the fourth step1Ginsenoside Re, ginsenoside Rf and ginsenoside Rb1And ginsenoside Rb3The average sample recovery rate value and the relative average deviation RSD value are calculated, and the results are shown in the following table 2,
table 2 sample recovery rate test results of each ginsenoside (n ═ 6)
Step ten, calculating relative correction factor
Sucking the reference solution prepared in the first step, measuring with the chromatographic conditions of the fourth step, and measuring with ginsenoside Rb1As an internal standard, calculating a relative correction factor RCF between ginsenosides by a slope method and a multipoint correction method,
the slope method has the calculation formula: f. ofi/s＝ki/ks，
Wherein f is a relative correction factor, k is a slope, i is a component to be detected, and s is an internal standard substance Rb1；
Multi-point calibration method meterCalculating a formula: f ═ fi/fs＝(Ai/Ci)/(As/Cs)
Wherein Ai is the peak area of the component to be measured, CiThe concentration of the component to be detected is presented in the unit of mu g/mu L; a. thesIs Rb1Area of peak of (C)sIs Rb1The concentration of (d) is in units of μ g/μ L;
the relative error RE of both methods is less than 3.0%, and the results are shown in table 3 below,
TABLE 3 relative correction factor for each ginsenoside
Eleven steps of positioning parameter measurement and repeatability investigation
Is prepared from ginsenoside Rb1As an internal standard, by the formula RTRi/s＝tRi/tRsCalculating ginsenoside Rg1Ginsenoside Re, ginsenoside Rf and ginsenoside Rb3Relative retention value of RTRi/sWherein t isRiRetention time of the component to be measured, tRsIs Rb1The results are shown in table 4 below,
table 4 relative retention of each ginsenoside (n ═ 6)
Meanwhile, the ten-flavor deer capsule, the preparation method thereof and the ginsenoside content analysis method are also subjected to durability examination, a proper amount of the reference substance solution prepared in the step one is absorbed, sample injection measurement is carried out under the spectrum condition described in the step four, and an Agilent 1260 chromatograph, an Agilent 1100Series chromatograph and an Agilent EC-C are examined by adopting a multipoint correction method18Chromatographic column, Zorbax SB-C18Chromatography column, Extend-C18The results of the effect of the chromatographic column on the relative correction factor are shown in table 5 below, and it is clear that there is no significant effect;
TABLE 5 Effect of different instruments, chromatography columns on relative correction factors
Locating chromatographic peaks of the component to be detected, verifying relative retention values of other 4 components under different chromatographs and chromatographic columns through the algorithm in the step eleven, and determining that the results are as shown in the following table 6 and have no obvious influence;
comparing the QAMS (one-test-multiple-evaluation method) and ESM (external standard method) measuring results, respectively measuring 5 components of the ten-flavor deer capsule by adopting QAMS and ESM according to the chromatographic conditions described in the step four. The results are shown in the following table 7,
TABLE 7 comparison of the results obtained by the one-test-multiple-evaluation method with the external standard method (n ═ 6)
The ten-flavor deer capsule, the preparation method thereof and the ginsenoside content analysis method establish a one-test-multiple-evaluation method with simple operation and reliable result by measuring the content of various ginsenosides in the ten-flavor deer capsule, and solve the problems of deficiency and high price of a ginsenoside reference substance and the like. Ginsenoside Rb is used for the test1As internal standard, by ginsenoside Rg1Ginsenoside Re, ginsenoside Rf and ginsenoside Rb3The content thereof is calculated with respect to the correction factor. As a result, the result obtained by the method has no significant difference compared with the external standard method. The analysis method supplements the quality evaluation method of the multi-drug effective components of the ten-flavor Xianglu capsules, and more effectively controls the product quality.
Claims (3)
1. Ten-ingredient xianglu capsules are characterized in that: the traditional Chinese medicine composition comprises, by mass, 10-30 parts of rhizoma cyperi, 2-6 parts of allium macrostemon, 2-6 parts of antler base, 2-6 parts of thunberg fritillary bulb, 2-6 parts of curcuma zedoary, centipede, 2-6 parts of asparagus cochinchinensis, 2-6 parts of turtle shell, 2-6 parts of sun-dried ginseng and 1-3 parts of platycodon grandiflorum.
2. The preparation method of the ten-flavor deer capsule is characterized by comprising the following steps: the preparation of the ten-flavor deer capsule of claim 1, comprising the following steps, and the following steps are performed sequentially,
step one, according to the mixture ratio of claim 1, pulverizing centipedes and 0.5 times of sun-dried ginseng in a formula into fine powder with the particle size of 150-180 microns;
step two, according to the mixture ratio of claim 1, adding 4 times of the amount of the medicinal materials into the rhizoma cyperi, the curcuma zedoary and the allium macrostemon, distilling for 6 hours to obtain volatile oil, including the volatile oil by β -cyclodextrin for later use, and respectively collecting and retaining residual aqueous solution and dregs after distillation;
step three, taking antler plates and turtle shells according to the mixture ratio of claim 1, adding 6 times of medicinal materials into water, decocting for 6 hours, and respectively collecting and retaining decoction and dregs;
step four, according to the mixture ratio of claim 1, taking thunberg fritillary bulb, asparagus, platycodon grandiflorum and the residual 0.5 times of raw sun-dried medicinal residues in the formula to be combined in the step two and the step three, adding 4 times of medicinal water to decoct for 3 times, and 2 hours for each time, combining the obtained decoction with the aqueous solution obtained in the step two and the decoction obtained in the step three, filtering, concentrating to the relative density of 1.21-1.25 at 80 ℃, and adding ethanol to ensure that the alcohol content reaches 70%; standing for 24 hr, filtering, recovering ethanol from the filtrate, and concentrating into soft extract;
and step five, adding the ginseng centipede fine powder obtained in the step one into the thick paste obtained in the step four, drying, crushing into fine powder with the particle size of 150-180 mu m, mixing with the volatile oil inclusion compound prepared in the step two, and encapsulating to prepare a finished product of the ten-flavor deer capsule.
3. The method for analyzing the content of the ginsenoside in the ten-ingredient xianglu capsules is characterized by comprising the following steps of: the method for measuring the content of ginsenoside in the finished product of the ten-flavor deer capsule prepared by the method in claim 2 comprises the following steps in sequence,
step one, preparing a reference substance solution
Precisely weighing ginsenoside Rb1Reference substance 2.04mg and ginsenoside Rb3Reference substance 2.04mg, ginsenoside Re reference substance 2.02mg, ginsenoside Rf reference substance 2.02mg, and ginsenoside Rg1Adding 2.01mg of reference substance into methanol for dissolving, fixing the volume to a 10ml volumetric flask, and mixing for later use;
step two, preparing a test solution
Taking ten-flavor deer capsule finished product powder, sieving with a 65-mesh sieve, precisely weighing 1.0002g to 50mL LEP tube, adding 30mL of methanol, carrying out ultrasonic oscillation for 1h under the conditions of 250W power and 50kHz frequency, filtering, evaporating to dryness, dissolving the evaporated material in 20mL of water, sequentially extracting with 20mL of dichloromethane and ethyl acetate for 3 times, extracting with 20mL of water-saturated n-butanol for 3 times, combining upper-layer extract liquor, evaporating to dryness, adding a proper amount of methanol for dissolving, and fixing the volume to a 5mL volumetric flask for later use;
step three, ginseng deficiency negative sample solution
Grinding Scolopendra into fine powder, precisely weighing 1.0025 g; taking 50.0028g of rhizoma cyperi, 30.0124g of curcuma zedoary and 30.0113g of allium macrostemon, adding 4 times of water, extracting volatile oil for 6 hours by a steam distillation method to obtain volatile oil solution for later use, and collecting the distilled water solution in another container; adding 3.0122g of antler base and 3.0024g of turtle shell into 6 times of water, decocting for 6 hours, and collecting decoction in another container; mixing 6.0241g of thunberg fritillary bulb, 6.0125g of radix asparagi and 3.0026g of platycodon grandiflorum with the residues, adding 4 times of medicinal water, decocting for 3 times, each time for 2 hours, filtering, mixing the filtrate with the aqueous solution collected by the other container and the decoction collected by the other container, concentrating to the relative density of 1.21-1.25 at the temperature of 80 ℃, adding absolute ethyl alcohol until the alcohol content reaches 70%, standing for 24 hours, filtering, concentrating to thick paste, and adding the centipede fine powder; drying in a ventilated place, grinding into fine powder, and mixing with the volatile oil for later use;
step four, testing the adaptability of the system
The chromatographic conditions are as follows: miningWith Agilent EC-C18Chromatographic column 4.6mm × 150mm, 2.7 μm; the mobile phase is acetonitrile A-water B, and gradient elution is carried out for 0-60 min, wherein the concentration of A is 19%; 60-120 min, 30% -50% A; the flow rate is 0.5 mL/min; the column temperature was 25 ℃; the detection wavelength is 203 nm; injecting 3 mu L of the reference substance solution obtained in the step one, the test substance solution obtained in the step two and the ginseng-deficient negative sample solution obtained in the step three under the chromatographic condition;
step five, linear relation test
Respectively taking 1 muL, 2 muL, 4 muL, 6 muL, 8 muL and 10 muL of the reference substance solution obtained in the step one, measuring under the chromatographic condition described in the step four, and calculating a linear regression equation and a linear range of each component by taking the reference mass concentration as a horizontal coordinate X and the peak area as a vertical coordinate Y;
step six, sample adding recovery rate test
Taking a ten-flavor deer capsule sample, grinding into fine powder, precisely weighing in parallel 6 times to obtain 0.2004g, 0.2008g, 0.2006g, 0.2004g, 0.2003g and 0.2002g respectively, and precisely adding the reference substance solution prepared in the step one according to a ratio of 1: 1; taking 6 parts of the sample solution prepared in the second step, and determining the ginsenoside Rg in the sample solution according to the chromatographic conditions in the fourth step1Ginsenoside Re, ginsenoside Rf and ginsenoside Rb1And ginsenoside Rb3Calculating the average sample recovery rate value and the relative average deviation RSD value;
step seven, calculating relative correction factor
Sucking the reference solution prepared in the first step, measuring with the chromatographic conditions of the fourth step, and measuring with ginsenoside Rb1As an internal standard, calculating a relative correction factor RCF between ginsenosides by a slope method and a multipoint correction method,
the slope method has the calculation formula: f. ofi/s＝ki/ks，
Wherein f is a relative correction factor, k is a slope, i is a component to be detected, and s is an internal standard substance Rb1；
The multipoint correction method calculation formula is as follows: f ═ fi/fs＝(Ai/Ci)/(As/Cs)
Wherein Ai is the peak area of the component to be measured, CiThe concentration of the component to be detected is presented in the unit of mu g/mu L; a. thesIs Rb1Area of peak of (C)sIs Rb1The concentration of (d) is in units of μ g/μ L;
step eight, positioning parameter measurement and repeatability investigation
Is prepared from ginsenoside Rb1As an internal standard, by the formula RTRi/s＝tRi/tRsCalculating ginsenoside Rg1Ginsenoside Re, ginsenoside Rf and ginsenoside Rb3Relative retention value of RTRi/sWherein t isRiRetention time of the component to be measured, tRsIs Rb1The retention time of (c).
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| CN116350736A (en) * | 2023-05-26 | 2023-06-30 | 吉林华康药业股份有限公司 | Traditional Chinese medicine composition for preventing and treating breast nodules |
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