CN109771481B

CN109771481B - Quality detection method and application of traditional Chinese medicine composition for tonifying kidney and relaxing spine

Info

Publication number: CN109771481B
Application number: CN201811627453.1A
Authority: CN
Inventors: 解素花; 段瑞刚; 杜菁; 张蓓
Original assignee: Beijing Zhongyan Tongrentang Chinese Medicine R & D Co ltd
Current assignee: Beijing Zhongyan Tongrentang Chinese Medicine R & D Co ltd
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2022-02-22
Anticipated expiration: 2038-12-28
Also published as: CN109771481A

Abstract

The invention provides a detection method of a traditional Chinese medicine composition, which comprises the following raw material medicines in parts by weight: 8-12 parts of rhizoma drynariae, 4-8 parts of rhizoma cibotii, 4-8 parts of eucommia bark, 4-8 parts of cassia twig, 2-6 parts of antler, 5-9 parts of corydalis tuber, 5-9 parts of teasel root, 5-9 parts of gentiana macrophylla, 7-11 parts of orientvine and 4-8 parts of notopterygium root, preparing a test solution by determining a proper extraction method or purification method through limited multiple screening, effectively separating active ingredients from other impurities in the traditional Chinese medicine composition by utilizing an HPLC (high performance liquid chromatography) method and selecting proper chromatographic conditions, and accurately determining the contents of the active ingredients of the traditional Chinese medicine composition, namely naringin and sinomenine; the identification of corydalis tuber, gentiana macrophylla and notopterygium root in the traditional Chinese medicine composition is realized by selecting a proper developing agent by a TLC method.

Description

Quality detection method and application of traditional Chinese medicine composition for tonifying kidney and relaxing spine

Technical Field

The invention relates to the field of traditional Chinese medicine preparations, in particular to a quality detection method and application of a traditional Chinese medicine composition for tonifying kidney and relaxing spine.

Background

Ankylosing Spondylitis (AS) is a chronic progressive immunoinflammatory disease of unknown cause, which mainly attacks medial joints such AS the sacroiliac joint, spinal apophysis, paravertebral soft tissues and peripheral joints and may be accompanied by extra-articular manifestations. Clinically, the pain is mainly manifested by lumbosacral pain, back pain, neck pain, hip pain and joint pain, and the serious patient may have spine deformity and joint stiffness, affecting the physical and mental health of the patient, and affecting the organs such as eyes, heart, lung and kidney, resulting in high disability rate.

The treatment principle of the national guidelines considers that no radical treatment method is available at present for the ankylosing spondylitis, if a patient can be discovered as soon as possible and diagnosed and reasonably treated in time, symptoms can be controlled and prognosis can be improved, and for patients in later period, surgical operation can be performed if necessary to correct malformed joints, so that the quality of life and the prognosis of the patient can be improved. The traditional western medicine for treating ankylosing spondylitis comprises nonsteroidal anti-inflammatory drugs (NSAIDs), disease-improving antirheumatic drugs (DMARDs), glucocorticoids, biological agents and the like. As the AS cause is unknown, the ideal specific treatment medicine is not available in Western medicine at present. A large number of clinical experiences show that the traditional Chinese medicine has unique curative effect on AS. Some patients who enter the hand treatment stage by stage, or those who emphasize syndrome differentiation and type differentiation, are treated by single therapy, while some patients are treated by acupuncture and moxibustion and external treatment, but basically, the treatment methods of tonifying liver and kidney, clearing heat and removing dampness, dispelling wind and cold, promoting blood circulation to remove blood stasis, dredging collaterals and relieving pain are adopted for the patients with the deficiency of healthy qi and excess of pathogenic factors.

Because the traditional Chinese medicine is a complex system consisting of a plurality of components, and the components of the traditional Chinese medicine are influenced by a plurality of factors, such as varieties, production areas, harvesting, processing and the like, the diversified and complex components in the traditional Chinese medicine are material bases with good curative effects, wide effects and small side effects, and the material bases are in a state of vagueness and difficult comprehensive evaluation for a long time. After the compatibility of a plurality of medicinal herbs is decocted, the quality of the compound cannot be evaluated by clear, effective and reasonable indexes, however, the treatment effect of the traditional Chinese medicine compound has a direct relation with the quality, the poor quality of the traditional Chinese medicine compound can directly cause the poor treatment effect, and the quality of the traditional Chinese medicine compound needs to be controlled to ensure the stability and controllability of the treatment effect. The quality control of the traditional Chinese medicine needs to monitor all the effective components of the medicine, and the established quality control system can ensure the effectiveness, controllability and safety of the medicine. Therefore, establishing a modern quality control system with the basic theoretical characteristics of the traditional Chinese medicine, solving the problem of traditional Chinese medicine analysis and improving the existing quality control method is a hotspot of current research.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide a quality detection method of a traditional Chinese medicine composition for tonifying kidney and relieving spinal column and application thereof, so that the defect that the quality of the traditional Chinese medicine composition cannot be comprehensively and clearly detected and monitored due to various traditional Chinese medicine medicines and complex components and mutual interference is overcome, and the stability, consistency and controllability of the quality of the traditional Chinese medicine composition are improved.

The invention provides a quality detection method of a traditional Chinese medicine composition for tonifying kidney and relaxing spine, wherein the traditional Chinese medicine composition comprises the following raw material medicines in parts by weight: 8-12 parts of rhizoma drynariae, 4-8 parts of rhizoma cibotii, 4-8 parts of eucommia bark, 4-8 parts of cassia twig, 2-6 parts of antler, 5-9 parts of corydalis tuber, 5-9 parts of teasel root, 5-9 parts of gentiana macrophylla, 7-11 parts of caulis sinomenii and 4-8 parts of notopterygium root;

the quality detection method of the traditional Chinese medicine composition comprises the following content determination steps:

A. content determination of naringin

Preparation of a test solution: adding methanol solution into the Chinese medicinal composition, ultrasonically extracting, filtering, evaporating the filtrate to dryness, dissolving in water, purifying by column chromatography, collecting eluate, evaporating to dryness, adding methanol solution, dissolving and fixing volume to obtain sample solution;

preparation of control solutions: collecting naringin reference substance, and adding organic solvent to obtain reference substance solution;

chromatographic conditions and system applicability test: measuring by high performance liquid chromatography, and using octadecylsilane chemically bonded silica as filler; acetonitrile is used as a mobile phase A, 0.1 vt% phosphoric acid solution is used as a mobile phase B, and according to the mobile phase A: the volume ratio of the mobile phase B is 10-30%: 90-70%; the detection wavelength is 260-300nm, and the theoretical plate number is not lower than 2000 calculated according to naringin; or

Measuring by high performance liquid chromatography, and using amino bonded silica gel as filler; acetonitrile is used as a mobile phase A, water is used as a mobile phase B, and the weight ratio of the acetonitrile to the water is determined according to the weight ratio of the mobile phase A: the volume ratio of the mobile phase B is 70-100%: 30-0 percent; the detection wavelength is 260-300nm, and the theoretical plate number is not lower than 2000 calculated according to naringin;

the determination method comprises the following steps: respectively sucking the reference solution and the test solution, injecting into a liquid chromatograph, measuring and calculating.

Further, in the content measurement of naringin, the specific method for preparing the test solution comprises the following steps: precisely weighing the traditional Chinese medicine composition, precisely adding 10-100ml (v t% is volume concentration) of 70-100vt% methanol solution, carrying out ultrasonic treatment for 0.1-1.0h, taking out, cooling, weighing again, complementing the loss weight by 70-100vt% methanol solution, shaking up, filtering, taking 5-50ml of filtrate, evaporating to dryness, adding 1-10ml of water into residue, dissolving, cooling, passing through a polyamide chromatographic column, eluting with 10-100ml of water solution, discarding water solution, eluting with 10-100ml of methanol, collecting eluate, evaporating to dryness, fixing the volume to 1-50ml with methanol, shaking up, and filtering to obtain the traditional Chinese medicine composition.

Preferably, the specific method for the chromatographic condition and system suitability test is as follows: measuring by high performance liquid chromatography, and using amino bonded silica gel as filler; acetonitrile is used as a mobile phase A, water is used as a mobile phase B, and the weight ratio of the acetonitrile to the water is determined according to the weight ratio of the mobile phase A: the volume ratio of the mobile phase B is 90%: 10 percent; the detection wavelength is 283nm, and the theoretical plate number is not lower than 2000 calculated according to naringin;

or, the specific method for testing the chromatographic conditions and the system applicability comprises the following steps: measuring by high performance liquid chromatography, and using octadecylsilane chemically bonded silica as filler; acetonitrile is used as a mobile phase A, 0.1 vt% phosphoric acid solution is used as a mobile phase B, and according to the mobile phase A: the volume ratio of the mobile phase B is 20%: 80 percent; the detection wavelength is 283 nm; the theoretical plate number calculated by naringin should not be less than 2000.

Further, the quality detection method of the traditional Chinese medicine composition further comprises at least one of the following content determination methods or identification methods:

B. determination of sinomenine content

Preparation of a test solution: precisely weighing a proper amount of the traditional Chinese medicine composition, precisely adding 10-50ml of ethanol solution with volume concentration of 40-95%, ultrasonically treating for 0.1-1.0h, cooling, complementing the weight loss by using the ethanol solution with volume concentration of 40-95%, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the traditional Chinese medicine composition;

preparation of control solutions: taking a proper amount of sinomenine reference substance, precisely weighing, and adding 50-100% methanol solution by volume concentration to prepare sinomenine reference substance solution;

chromatographic conditions and system applicability test: measuring by high performance liquid chromatography, and using octadecylsilane chemically bonded silica as filler; methanol is used as a mobile phase A, phosphate buffer solution with pH of 9.0 is used as a mobile phase B, and the ratio of the mobile phase A: the volume ratio of the mobile phase B is 25-40%: 75-60%; the detection wavelength is 240-280 nm; the theoretical plate number is not less than 1500 calculated according to sinomenine;

the determination method comprises the following steps: precisely sucking 2-20 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, measuring and calculating; or

C. Identification of rhizoma corydalis:

preparation of a test solution: taking 0.5-4g of the traditional Chinese medicine composition, adding 30-80ml of methanol solution with volume concentration of 90-100%, carrying out ultrasonic treatment for 15-60 minutes, filtering, evaporating filtrate to dryness, adding 5-20ml of water into residue to dissolve the residue, adding concentrated ammonia test solution to adjust to alkalinity, shaking and extracting with diethyl ether for 1-5 times, 5-20ml each time, combining diethyl ether solutions, evaporating to dryness, and adding 1-3ml of methanol with volume concentration of 90-100% into residue to dissolve the residue to obtain the traditional Chinese medicine composition;

preparing a reference medicinal material solution and a reference solution: taking 0.2-2g of rhizoma corydalis reference material, adding 30-80ml of methanol solution with volume concentration of 90-100%, ultrasonically treating for 15-60 minutes, filtering, evaporating filtrate to dryness, adding 5-20ml of water into residue to dissolve, adding concentrated ammonia test solution to adjust to alkalinity, shaking and extracting with diethyl ether for 1-5 times, 5-20ml each time, combining ethyl ether solution, evaporating to dryness, adding 1-3ml of methanol with volume concentration of 90-100% into residue to dissolve to obtain reference material solution, taking tetrahydropalmatine reference substance, adding 90-100% methanol solution to prepare solution containing 0.1-1mg of tetrahydropalmatine reference substance per 1ml to obtain reference substance solution;

and (3) identification: performing thin layer chromatography test, precisely sucking the reference solution, the reference solution and the sample solution 1-10 μ 1 respectively, spotting on the same silica gel G thin layer plate, developing with toluene-acetone saturated with strong ammonia water at volume ratio of 5-15: 1-5 as developing agent, taking out, air drying, placing in iodine jar for 1-5 min, taking out, volatilizing iodine adsorbed on the plate, and inspecting under ultraviolet lamp (365 nm); spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution; or

D. And (3) identification of gentiana macrophylla:

preparation of a test solution: taking 0.5-4g of the traditional Chinese medicine composition, adding 5-30ml of methanol solution with volume concentration of 90-100%, carrying out ultrasonic treatment for 10-60 minutes, cooling, filtering, and taking subsequent filtrate to obtain a test solution;

preparation of control solutions: adding 90-100% methanol into gentiopicrin control to obtain 0.2-2mg gentiopicrin control solution per 1ml to obtain control solution;

and (3) identification: performing thin layer chromatography by sucking the above sample solution and control solution 1-10 μ l respectively, and spotting on the same silica gel GF₂₅₄Spreading ethyl acetate-methanol-water solution at volume ratio of 5-20: 1-5: 0.5-2 as developing agent on thin layer plate, taking out, air drying, and inspecting under ultraviolet lamp (254 nm); spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution; or

E. And (3) identification of gentiana macrophylla:

preparation of control solutions: taking a roburic acid reference substance, and adding 90-100% chloroform solution to obtain a solution containing 0.2-2mg per 1ml, to obtain a reference substance solution;

and (3) identification: performing thin-layer chromatography test, sucking the sample solution 5-20 μ 1 and the reference solution 1-5 μ 1, respectively dropping on the same silica gel G thin-layer plate, developing with dichloromethane-methanol-formic acid at volume ratio of 40-60: 0.5-2: 0.2-1 as developing agent, taking out, air drying, spraying with 8-12 vt% sulfuric acid ethanol solution, and heating at 102-; spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution; or

F. Identification of notopterygium root:

preparation of control solutions: adding 90-100 vol% methanol solution into another decursin control to obtain solution containing decursin 0.2-2mg per 1ml, to obtain control solution;

and (3) identification: performing thin layer chromatography, sucking the sample solution and the reference solution 1-10 μ 1 respectively, and respectively dropping on the same silica gel G thin layer plate at a volume ratio of 5-10: 1-3 parts of dichloromethane-methanol as developing agent, developing, taking out, air drying, and inspecting under ultraviolet lamp (365 nm); spots of the same color appear in the chromatogram of the test solution at positions corresponding to those in the chromatogram of the control solution.

Preferably, the traditional Chinese medicine composition comprises the following raw material medicines in parts by weight: 10 parts of rhizoma drynariae, 6 parts of rhizoma cibotii, 6 parts of eucommia bark, 6 parts of cassia twig, 4 parts of antler, 7 parts of corydalis tuber, 7 parts of teasel root, 7 parts of gentiana macrophylla, 9 parts of caulis sinomenii and 6 parts of notopterygium root; or

8 parts of rhizoma drynariae, 4 parts of rhizoma cibotii, 4 parts of eucommia bark, 4 parts of cassia twig, 2 parts of antler, 5 parts of corydalis tuber, 5 parts of teasel root, 5 parts of gentiana macrophylla, 7 parts of caulis sinomenii and 4 parts of notopterygium root; or

12 parts of rhizoma drynariae, 8 parts of rhizoma cibotii, 8 parts of eucommia bark, 8 parts of cassia twig, 6 parts of antler, 9 parts of corydalis tuber, 9 parts of teasel root, 9 parts of gentiana macrophylla, 11 parts of caulis sinomenii and 8 parts of notopterygium root.

Further, the pharmaceutical composition is added with conventional auxiliary materials and directly or indirectly prepared into clinically acceptable tablets, granules, capsules, oral liquid or injections according to a conventional process.

Preferably, the preparation method of the traditional Chinese medicine composition comprises the following steps:

(1) weighing cassia twig and notopterygium root according to the selected weight parts, mixing, adding a proper amount of water, performing steam distillation, respectively collecting volatile oil and water extract for later use, adding beta-cyclodextrin and water into the volatile oil, performing inclusion, and drying to obtain an inclusion compound for later use;

(2) weighing rhizoma Cibotii, Eucommiae cortex, cornu Cervi and radix Dipsaci according to selected weight parts, mixing, adding water for extraction to obtain extractive solution, mixing with the extractive solution prepared in step (1), and concentrating under reduced pressure to obtain fluid extract;

(3) weighing rhizoma Drynariae, caulis Sinomenii, radix Gentianae Marcrophyllae and rhizoma corydalis according to selected weight parts, extracting with 20-95vt% ethanol solution, concentrating, and mixing with above fluid extract; concentrating, drying, pulverizing into fine powder, adding the clathrate, and mixing.

Further, in the inclusion process in the step (1), the dosage ratio of the volatile oil, the beta-cyclodextrin and the water is as follows: 1 ml: 4-10 g: 40-100 ml;

the inclusion time is 20-60 minutes, and the inclusion compound is refrigerated for 12-48 hours after inclusion.

Preferably, in the steam distillation process in the step (1), 8-12 times of water is added, and the steam distillation is carried out for 4-8 hours;

in the water extraction process of the step (2), adding water, decocting for 1-3 times, 1-2 hours each time, and adding 4-10 times of water each time;

in the ethanol extraction process in the step (3), heating and refluxing the ethanol solution with the volume concentration of 60-80% for 1-3 times, each time for 1-2 hours, adding 6-12 times of the ethanol solution with the volume concentration of 60-80% for each time, filtering the extracting solution, and combining the extracting solutions.

The invention also provides the application of the quality detection method in the quality detection of the traditional Chinese medicine composition or the traditional Chinese medicine preparation.

The technical scheme of the invention has the following advantages:

1. the quality detection method of the traditional Chinese medicine composition for tonifying kidney and relaxing spine realizes effective monitoring of product quality by measuring the content of the active ingredient naringin of the traditional Chinese medicine composition, in the preparation of a test solution of the naringin content test method, in combination with the characteristics of complexity and diversity of the medicinal ingredients in the traditional Chinese medicine composition, a suitable extraction method is obtained by multiple screening, a methanol solution is firstly added, ultrasonic extraction is carried out, filtration is carried out, filtrate is evaporated to dryness, water is added for dissolution, column chromatography purification is carried out, eluent is collected for evaporation to dryness, the methanol solution is added for dissolution and volume fixing to obtain the test solution, on the basis, a suitable mobile phase composition is obtained by screening, and a normal phase chromatography or a reverse phase chromatography of HPLC is utilized to select a suitable chromatographic condition, so that naringin in the test solution can be effectively separated from other impurity peaks finally, and the defects of the traditional Chinese medicine composition caused by various medicinal ingredients are effectively overcome, The method has the advantages that the chromatographic peak of the active ingredient naringin in the traditional Chinese medicine composition is obtained, the determined HPLC content determination method is not only strong in specificity, but also meets the requirement of medicine analysis on linear relation, precision and recovery rate, the purpose of controlling the content of the traditional Chinese medicine composition or traditional Chinese medicine preparation by determining the content of the active ingredient naringin is achieved, the quality of the traditional Chinese medicine composition can be comprehensively and clearly detected, the quality control of the traditional Chinese medicine composition is further achieved, and the quality detection method has the advantages of simplicity, rapidness, stability, reliability, high precision and easiness in mastering.

2. According to the quality detection method of the traditional Chinese medicine composition for tonifying the kidney and relaxing the ridges, the comprehensiveness and reliability of quality monitoring on the traditional Chinese medicine composition are improved by measuring the content of the sinomenine; the traditional Chinese medicine composition is extracted by a proper extraction method obtained by screening to prepare a test solution, a proper mobile phase is selected, and the finally determined HPLC content determination method has strong specificity, meets the requirement of pharmaceutical analysis on linear relation, precision and recovery rate, can quickly and accurately know the quality condition of the product, and can effectively monitor the quality performance of the product.

3. According to the quality detection method of the traditional Chinese medicine composition for tonifying kidney and relaxing the spine, the corydalis tuber, the gentiana macrophylla or the notopterygium root are identified by a TLC method, so that the quality detection of the traditional Chinese medicine composition for tonifying kidney and relaxing the spine is more comprehensive and clear, the effective active components and the quality of the medicine are more completely represented, the comprehensive monitoring of the active components is facilitated, the stability, the consistency and the controllability of the quality of the traditional Chinese medicine composition for tonifying kidney and relaxing the spine are further ensured, and the safety and the effectiveness of the traditional Chinese medicine composition are ensured.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a thin layer chromatogram of the Chinese medicinal composition described in example 4 of the present invention; from left to right in the figure: rhizoma corydalis negative sample, test sample 1, test sample 2, test sample 3, tetrahydropalmatine reference substance, and rhizoma corydalis reference medicinal material;

FIG. 2 is a thin layer chromatogram of the Chinese medicinal composition described in example 5 of the present invention; from left to right in the figure: a large-leaved gentian negative sample, a test sample 1, a test sample 2, a test sample 3 and a gentiopicroside reference substance;

FIG. 3 is a thin layer chromatogram of the Chinese medicinal composition described in example 6 of the present invention; from left to right in the figure: a large-leaved gentian negative sample, a test sample 1, a test sample 2, a test sample 3 and a roburic acid reference substance;

FIG. 4 is a thin layer chromatogram of the Chinese medicinal composition described in example 7 of the present invention; from left to right in the figure: notopterygium root negative sample, test sample 1, test sample 2, test sample 3 and decursin reference sample;

FIG. 5 is a content measurement high performance liquid chromatogram of test group 1 and test group 2 and comparative group 1 in Experimental example 1; a is a test sample of the test group 1; b is a test sample of the test group 2; c is a test sample of the comparative group 1;

FIG. 6 is a high performance liquid chromatogram for content measurement of comparative groups 2 to 4 in Experimental example 1; a is the sample of comparative group 2; b is a negative control solution chromatogram of the comparison group 2; c is the chromatogram of the test solution in the comparison group 3; d is a chromatogram of the test solution of the comparison group 4;

FIG. 7 is a high performance liquid chromatogram in Experimental example 2; wherein A is a chromatogram of a reference solution; b is a chromatogram of the test solution, and C is a chromatogram of the negative control solution;

FIG. 8 is a high performance liquid chromatogram in Experimental example 3; wherein A is a chromatogram of a reference solution; b is a chromatogram of the test solution, and C is a chromatogram of the negative control solution;

FIG. 9 is a high performance liquid chromatogram in Experimental example 4; wherein A is a chromatogram of a reference solution; b is a chromatogram of the test solution, and C is a chromatogram of the negative control solution;

FIG. 10 is a standard graph of naringin obtained from the investigation of the linear relationship in Experimental example 2;

FIG. 11 is a linear graph of naringin obtained from the examination of the linear relationship in Experimental example 3;

FIG. 12 is a linear graph of sinomenine obtained in the examination of the linear relationship in Experimental example 4.

Detailed Description

Example 1 granules

Prescription: 10g of rhizoma drynariae, 6g of rhizoma cibotii, 6g of eucommia bark, 6g of cassia twig, 4g of antler, 7g of rhizoma corydalis, 7g of teasel root, 7g of gentiana macrophylla, 9g of orientvine and 6g of notopterygium root;

the preparation method comprises the following steps: weighing rhizoma drynariae, rhizoma cibotii, eucommia bark, cassia twig, antler, rhizoma corydalis, teasel root, large-leaved gentian, caulis sinomenii and notopterygium root according to the selected weight parts for later use; taking cassia twig and notopterygium root, adding 10 times of water, performing steam distillation for 6 hours, extracting volatile oil, collecting the volatile oil, and filtering the water extract for later use; according to the proportion that the volatile oil, the beta-cyclodextrin and the water are 1 ml: 6 g: 60ml, adding beta-cyclodextrin and water into the volatile oil, stirring, clathrating for 30 minutes, refrigerating for 24 hours, filtering, and drying at 40 ℃ to obtain a volatile oil clathrate compound for later use; decocting rhizoma Cibotii, Eucommiae cortex, cornu Cervi and radix Dipsaci in water for 2 times, each time for 2 hr, each time adding 6 times of water, filtering decoction, mixing with above extractive solution, and concentrating under reduced pressure to obtain fluid extract with relative density of 1.15(50 deg.C); extracting rhizoma Drynariae, caulis Sinomenii, radix Gentianae Marcrophyllae and rhizoma corydalis with 70% ethanol under reflux for 3 times (2 hr for each time) and 8 times of 70% ethanol for each time, filtering the extractive solutions, mixing, recovering ethanol, concentrating to relative density of 1.15(50 deg.C), and mixing with the above fluid extract; concentrating to obtain fluid extract with relative density of 1.30(50 deg.C), drying, and pulverizing into fine powder; and (3) adding the volatile oil inclusion compound, a proper amount of dextrin and a proper amount of aspartame into the fine powder, uniformly mixing, and preparing 1000g of granules.

A. Content determination of naringin

Preparation of a test solution: taking about 1.5g of the prepared granules, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of methanol, carrying out ultrasonic treatment (power 250W and frequency 40kHz) for 0.5 hour, taking out, cooling, weighing again, supplementing the weight loss by using methanol, shaking up, filtering, taking 10ml of filtrate, drying by distillation, adding 5ml of water into residues, dissolving, cooling, passing through a polyamide chromatographic column (60-80 meshes, 2g and inner diameter of 1.8cm), eluting with 25ml of water solution, discarding water solution, eluting with 25ml of methanol, collecting the eluate, drying by distillation, fixing the volume to 5ml by using methanol, shaking up and filtering to obtain the finished product;

preparation of control solutions: taking a proper amount of naringin reference substance, precisely weighing, and adding methanol solution to obtain a solution containing 75 μ g of naringin reference substance per 1 ml;

chromatographic conditions and system applicability test: measuring by high performance liquid chromatography, and using amino bonded silica gel as filler; acetonitrile is used as a mobile phase A, water is used as a mobile phase B, and the weight ratio of the acetonitrile to the water is determined according to the weight ratio of the mobile phase A: the volume ratio of the mobile phase B is 90%: 10 percent; the detection wavelength is 283nm, and the theoretical plate number is not lower than 2000 calculated according to naringin;

the determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring and calculating;

the product contains naringin (C) for supplementing rhizoma Drynariae₂₇H₃₂O₁₄) Calculated, not less than 17.64 mg.

Example 2 granules

Prescription: 8g of rhizoma drynariae, 4g of rhizoma cibotii, 4g of eucommia bark, 4g of cassia twig, 2g of antler, 5g of rhizoma corydalis, 5g of teasel root, 5g of gentiana macrophylla, 7g of orientvine and 4g of notopterygium root;

the preparation method comprises the following steps: weighing rhizoma drynariae, rhizoma cibotii, eucommia bark, cassia twig, antler, rhizoma corydalis, teasel root, large-leaved gentian, caulis sinomenii and notopterygium root according to the selected weight parts for later use; taking cassia twig and notopterygium root, adding 12 times of water, performing steam distillation for 4 hours, extracting volatile oil, collecting the volatile oil, and filtering the water extract for later use; according to the proportion that the volatile oil, the beta-cyclodextrin and the water are 1 ml: 4 g: 80ml, adding beta-cyclodextrin and water into the volatile oil, clathrating for 60 minutes, refrigerating for 12 hours, filtering, and drying at 50 ℃ to obtain a volatile oil clathrate compound for later use; decocting rhizoma Cibotii, Eucommiae cortex, cornu Cervi and radix Dipsaci in water for 1 time, 2 hr each time, 4 times of water each time, filtering decoction, mixing with above extractive solution, and concentrating under reduced pressure to obtain fluid extract with relative density of 1.15(50 deg.C); extracting rhizoma Drynariae, caulis Sinomenii, radix Gentianae Marcrophyllae and rhizoma corydalis with 70% ethanol under reflux for 3 times (2 hr for each time) and 8 times of 60% ethanol for each time, filtering the extractive solutions, mixing, recovering ethanol, concentrating to relative density of 1.15(50 deg.C), and mixing with the above fluid extract; concentrating to obtain fluid extract with relative density of 1.30(50 deg.C), drying, and pulverizing into fine powder; and (3) adding the volatile oil inclusion compound, a proper amount of dextrin and a proper amount of aspartame into the fine powder, uniformly mixing, and preparing 1000g of granules.

A. Content determination of naringin

chromatographic conditions and system applicability test: measuring by high performance liquid chromatography, and using octadecylsilane chemically bonded silica as filler; acetonitrile is used as a mobile phase A, 0.1% phosphoric acid solution is used as a mobile phase B, and the ratio of the mobile phase A: the volume ratio of the mobile phase B is 20%: 80 percent; the detection wavelength is 283nm, and the theoretical plate number is not lower than 2000 calculated according to naringin;

Example 3 granules

Consists of the following components: 12g of rhizoma drynariae, 8g of rhizoma cibotii, 8g of eucommia bark, 8g of cassia twig, 6g of antler, 9g of rhizoma corydalis, 9g of teasel root, 9g of gentiana macrophylla, 11g of orientvine and 8g of notopterygium root;

the preparation method comprises the following steps: weighing rhizoma drynariae, rhizoma cibotii, eucommia bark, cassia twig, antler, rhizoma corydalis, teasel root, large-leaved gentian, caulis sinomenii and notopterygium root according to the selected weight parts for later use; taking cassia twig and notopterygium root, adding 8 times of water, carrying out steam distillation for 8 hours, extracting volatile oil, collecting the volatile oil, and filtering the water extract for later use; according to the proportion that the volatile oil, the beta-cyclodextrin and the water are 1 ml: 10 g: 40ml, adding beta-cyclodextrin and water into the volatile oil, clathrating for 20 minutes, refrigerating for 48 hours, filtering, and drying at 30 ℃ to obtain a volatile oil clathrate compound for later use; decocting rhizoma Cibotii, Eucommiae cortex, cornu Cervi and radix Dipsaci in water for 3 times, 1 hr each time, 10 times of water each time, filtering decoction, mixing with above extractive solution, and concentrating under reduced pressure to obtain fluid extract with relative density of 1.10(50 deg.C); extracting rhizoma Drynariae, caulis Sinomenii, radix Gentianae Marcrophyllae and rhizoma corydalis with 80% ethanol under reflux for 1 time (1 hr for each time) and 12 times of 60% ethanol for each time, filtering the extractive solutions, mixing, recovering ethanol, concentrating to relative density of 1.05(50 deg.C), and mixing with the above fluid extract; concentrating to obtain fluid extract with relative density of 1.20(50 deg.C), drying, and pulverizing into fine powder; and (3) adding the volatile oil inclusion compound, a proper amount of dextrin and a proper amount of aspartame into the fine powder, uniformly mixing, and preparing 1000g of granules.

B. Determination of sinomenine content

Preparation of a test solution: taking about 1g of the prepared granules, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of ethanol with the volume concentration of 70%, sealing the plug, weighing, carrying out ultrasonic treatment (power of 250W and frequency of 40kHz) for 20 minutes, cooling, weighing again, adding ethanol with the volume concentration of 70% to supplement the loss weight, shaking up, filtering, and taking the subsequent filtrate to obtain the product;

preparation of control solutions: taking a proper amount of sinomenine reference substance, precisely weighing, and adding methanol to obtain a solution containing 0.2mg sinomenine reference substance per 1 ml;

chromatographic conditions and system applicability test: measuring by high performance liquid chromatography, and using octadecylsilane chemically bonded silica as filler; methanol is used as a mobile phase A, a phosphate buffer solution (0.005mol/L disodium hydrogen phosphate solution, 0.005mol/L sodium dihydrogen phosphate is used for adjusting the pH value to 8.0, and 1% triethylamine is used for adjusting the pH value to 9.0) is used as a mobile phase B, and the weight ratio of the components is determined according to the following steps: the volume ratio of the mobile phase B is 32%: 68 percent; the detection wavelength is 262nm, and the theoretical plate number is not less than 1500 calculated according to sinomenine;

the determination method comprises the following steps: precisely sucking 5 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring and calculating;

the product contains sinomenine (C) in daily dose₂₇H₃₂O₁₄) Calculated, the content of the active ingredients should not be less than 18.9 mg.

EXAMPLE 4 tablets

Consists of the following components: 12g of rhizoma drynariae, 8g of rhizoma cibotii, 8g of eucommia bark, 8g of cassia twig, 2g of antler, 5g of rhizoma corydalis, 5g of teasel root, 9g of gentiana macrophylla, 11g of orientvine and 8g of notopterygium root;

the preparation method comprises the following steps: weighing rhizoma drynariae, rhizoma cibotii, eucommia bark, cassia twig, antler, rhizoma corydalis, teasel root, large-leaved gentian, caulis sinomenii and notopterygium root according to the selected weight parts for later use; taking cassia twig and notopterygium root, adding 8 times of water, carrying out steam distillation for 8 hours, extracting volatile oil, collecting the volatile oil, and filtering the water extract for later use; according to the proportion that the volatile oil, the beta-cyclodextrin and the water are 1 ml: 10 g: 40ml, adding beta-cyclodextrin and water into the volatile oil, clathrating for 20 minutes, refrigerating for 48 hours, filtering, and drying at 30 ℃ to obtain a volatile oil clathrate compound for later use; decocting rhizoma Cibotii, Eucommiae cortex, cornu Cervi and radix Dipsaci in water for 3 times, 1 hr each time, 10 times of water each time, filtering decoction, mixing with above extractive solution, and concentrating under reduced pressure to obtain fluid extract with relative density of 1.10(50 deg.C); extracting rhizoma Drynariae, caulis Sinomenii, radix Gentianae Marcrophyllae and rhizoma corydalis with 80% ethanol under reflux for 1 time (1 hr for each time) and 12 times of 60% ethanol for each time, filtering the extractive solutions, mixing, recovering ethanol, concentrating to relative density of 1.05(50 deg.C), and mixing with the above fluid extract; concentrating to obtain fluid extract with relative density of 1.20(50 deg.C), drying, and pulverizing into fine powder; mixing the above fine powder with the volatile oil clathrate, dextrin and aspartame, mixing, and making into tablet.

C. Identification of corydalis tuber

Preparation of a test solution: preparation of a test solution: taking 2g of the prepared tablet, adding 50ml of methanol solution, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, adding 10ml of water into residue to dissolve the residue, adding concentrated ammonia test solution to adjust to alkalinity, shaking and extracting with diethyl ether for 3 times, 10ml each time, combining diethyl ether solutions, evaporating to dryness, and adding 1ml of methanol to the residue to dissolve the residue to obtain the product;

preparing a reference medicinal material solution and a reference solution: taking 1g of rhizoma corydalis control medicinal material, adding 50ml of methanol solution, performing ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, dissolving residue with 10ml of water, adding concentrated ammonia solution to adjust to alkalinity, shaking and extracting with diethyl ether for 3 times, 10ml each time, mixing ethyl ether solution, evaporating to dryness, and dissolving residue with 1ml of methanol to obtain rhizoma corydalis control medicinal material solution. Adding methanol into tetrahydropalmatine control to obtain solution containing 0.5mg tetrahydropalmatine per 1ml to obtain tetrahydropalmatine control solution;

rhizoma corydalis negative sample solution: prepared according to the preparation method of a test solution, which is only different from the preparation method that the corydalis tuber is not contained in the prescription;

and (3) identification: and (3) performing thin-layer chromatography test, precisely sucking the reference substance solution, the test substance solution and the rhizoma corydalis negative sample solution by 3 mu 1 respectively, respectively dropping the reference substance solution, the test substance solution and the rhizoma corydalis negative sample solution on the same silica gel G thin-layer plate, and respectively saturating the silica gel G thin-layer plate with concentrated ammonia water in a volume ratio of 9: 2, developing the mixture by using toluene-acetone as a developing agent, taking out the mixture, airing the mixture, putting the mixture into an iodine jar for about 3 minutes, taking out the mixture, volatilizing the iodine adsorbed on the board, and then placing the board under an ultraviolet lamp (365nm) for inspection; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution; the chromatogram is shown in figure 1, wherein 1 is rhizoma corydalis negative sample, 2, 3, and 4 are samples to be tested, 5 is tetrahydropalmatine control, and 6 is rhizoma corydalis control.

EXAMPLE 5 capsules

Consists of the following components: 8g of rhizoma drynariae, 8g of rhizoma cibotii, 8g of eucommia bark, 8g of cassia twig, 2g of antler, 5g of rhizoma corydalis, 5g of teasel root, 5g of gentiana macrophylla, 7g of orientvine and 4g of notopterygium root;

the preparation method comprises the following steps: weighing rhizoma drynariae, rhizoma cibotii, eucommia bark, cassia twig, antler, rhizoma corydalis, teasel root, large-leaved gentian, caulis sinomenii and notopterygium root according to the selected weight parts for later use; taking cassia twig and notopterygium root, adding 12 times of water, performing steam distillation for 4 hours, extracting volatile oil, collecting the volatile oil, and filtering the water extract for later use; according to the proportion that the volatile oil, the beta-cyclodextrin and the water are 1 ml: 8 g: 80ml, adding beta-cyclodextrin and water into the volatile oil, clathrating for 60 minutes, refrigerating for 12 hours, filtering, and drying at 50 ℃ to obtain a volatile oil clathrate compound for later use; decocting rhizoma Cibotii, Eucommiae cortex, cornu Cervi and radix Dipsaci in water for 1 time, 2 hr each time, 4 times of water each time, filtering decoction, mixing with above extractive solution, and concentrating under reduced pressure to obtain fluid extract with relative density of 1.15(50 deg.C); extracting rhizoma Drynariae, caulis Sinomenii, radix Gentianae Marcrophyllae and rhizoma corydalis with 70% ethanol under reflux for 3 times (2 hr for each time) and 8 times of 60% ethanol for each time, filtering the extractive solutions, mixing, recovering ethanol, concentrating to relative density of 1.15(50 deg.C), and mixing with the above fluid extract; concentrating to obtain fluid extract with relative density of 1.30(50 deg.C), drying, and pulverizing into fine powder; mixing the above fine powder with the volatile oil clathrate, dextrin and aspartame, granulating, and making into capsule.

D. Identification of Gentiana macrophylla

Preparation of a test solution: taking 2g of the capsules, removing capsule shells, taking granules, adding 10ml of methanol solution, carrying out ultrasonic treatment for 15 minutes, cooling, filtering, and taking subsequent filtrate to obtain a test solution;

preparation of control solutions: adding methanol into gentiopicroside reference substance to obtain a solution containing 1mg gentiopicroside reference substance per 1ml to obtain reference substance solution;

gentiana macrophylla negative sample solution: the traditional Chinese medicine composition is prepared according to a preparation method of a test solution, and is only different in that the traditional Chinese medicine composition does not contain gentiana macrophylla;

and (3) identification: performing thin layer chromatography by sucking 3 μ l of the sample solution and the reference solution, respectively dropping on the same silica gel GF₂₅₄Spreading on thin layer plate with ethyl acetate-methanol-water solution at volume ratio of 10: 2: 1 as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm); spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution; the negative sample is free of interference by virtue of the control of the gentiana macrophylla negative sample, and the spectrogram is shown in figure 2, wherein 1 is the gentiana macrophylla negative sample, 2, 3 and 4 are the test samples, and 5 is the gentiopicroside control sample.

EXAMPLE 6 Chinese medicinal composition

the preparation method comprises the following steps: weighing rhizoma drynariae, rhizoma cibotii, eucommia bark, cassia twig, antler, rhizoma corydalis, teasel root, large-leaved gentian, caulis sinomenii and notopterygium root according to the selected weight parts for later use; taking cassia twig and notopterygium root, adding 10 times of water, performing steam distillation for 6 hours, extracting volatile oil, collecting the volatile oil, and filtering the water extract for later use; according to the proportion that the volatile oil, the beta-cyclodextrin and the water are 1 ml: 4 g: 80ml, adding beta-cyclodextrin and water into the volatile oil, clathrating for 30 minutes, refrigerating for 24 hours, filtering, and drying at 40 ℃ for later use; decocting rhizoma Cibotii, Eucommiae cortex, cornu Cervi and radix Dipsaci in water for 2 times, each time for 2 hr, each time adding 6 times of water, filtering decoction, mixing with above extractive solution, and concentrating under reduced pressure to obtain fluid extract with relative density of 1.15(50 deg.C) to obtain volatile oil clathrate; extracting rhizoma Drynariae, caulis Sinomenii, radix Gentianae Marcrophyllae and rhizoma corydalis with 70% ethanol under reflux for 3 times (2 hr for each time) and 8 times of 70% ethanol for each time, filtering the extractive solutions, mixing, recovering ethanol, concentrating to relative density of 1.15(50 deg.C), and mixing with the above fluid extract; concentrating to obtain fluid extract with relative density of 1.30(50 deg.C), drying, and pulverizing into fine powder; mixing the above fine powder with the clathrate, and making into Chinese medicinal composition.

E. Identification of Gentiana macrophylla

Preparation of a test solution: adding 10ml of methanol solution into 2g of the traditional Chinese medicine composition, carrying out ultrasonic treatment for 15 minutes, cooling, filtering, and taking a subsequent filtrate to obtain a test solution;

preparation of control solutions: taking a roburic acid reference substance, and adding chloroform solution to obtain a solution containing 0.5mg roburic acid reference substance per 1ml, to obtain a reference substance solution;

gentiana macrophylla negative sample solution: is prepared according to the preparation method of a test solution, and is only different from the preparation method that the prescription does not contain gentiana macrophylla;

and (3) identification: and (3) performing thin-layer chromatography, namely sucking the test solution 10 mu 1 and the reference solution 1 mu 1, respectively dropping the two solutions on the same silica gel G thin-layer plate, and mixing the solutions in a volume ratio of 50: 1: 0.5 of dichloromethane-methanol-formic acid is used as a developing agent, developed, taken out, dried, sprayed with 10vt percent of sulfuric acid ethanol solution, and heated at 105 ℃ until the spots are clearly developed; spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution; and (3) carrying out negative sample control, wherein the negative sample is free of interference, and the spectrogram is shown in a picture 3, wherein 1 is a gentiana macrophylla negative sample, 2, 3 and 4 are test samples, and 5 is a roburic acid control sample.

Example 7

Prescription: 10g of rhizoma drynariae, 4g of rhizoma cibotii, 4g of eucommia bark, 4g of cassia twig, 4g of antler, 7g of rhizoma corydalis, 7g of teasel root, 7g of gentiana macrophylla, 11g of orientvine and 8g of notopterygium root;

the preparation method comprises the following steps: weighing rhizoma drynariae, rhizoma cibotii, eucommia bark, cassia twig, antler, rhizoma corydalis, teasel root, large-leaved gentian, caulis sinomenii and notopterygium root according to the selected weight parts for later use; taking cassia twig and notopterygium root, adding 10 times of water, performing steam distillation for 6 hours, extracting volatile oil, collecting the volatile oil, and filtering the water extract for later use; according to the proportion that the volatile oil, the beta-cyclodextrin and the water are 1 ml: 6 g: 60ml, adding beta-cyclodextrin and water into the volatile oil, performing inclusion for 30 minutes, refrigerating for 24 hours, performing suction filtration, and drying at 40 ℃ to obtain a volatile oil inclusion compound for later use; decocting rhizoma Cibotii, Eucommiae cortex, cornu Cervi and radix Dipsaci in water for 2 times, each time for 2 hr, each time adding 6 times of water, filtering decoction, mixing with above extractive solution, and concentrating under reduced pressure to obtain fluid extract with relative density of 1.10(50 deg.C); extracting rhizoma Drynariae, caulis Sinomenii, radix Gentianae Marcrophyllae and rhizoma corydalis with 70% ethanol under reflux for 3 times (2 hr for each time) and 8 times of 70% ethanol for each time, filtering the extractive solutions, mixing, recovering ethanol, concentrating to relative density of 1.10(50 deg.C), and mixing with the above fluid extract; concentrating to obtain fluid extract with relative density of 1.30(50 deg.C), drying, and pulverizing into fine powder; mixing with the volatile oil clathrate, and making into Chinese medicinal composition.

F. Identification of Notopterygium incisum

preparation of control solutions: adding methanol solution into another decursin reference substance to obtain solution containing decursin 0.5mg per 1ml to obtain reference substance solution;

a negative sample solution which is prepared according to the preparation method of the test sample solution and is only characterized in that the prescription of the traditional Chinese medicine composition does not contain notopterygium root;

and (3) identification: performing thin layer chromatography test by taking the sample solution and the reference solution 2 μ 1 respectively, respectively dropping on the same silica gel G thin layer plate, developing with dichloromethane-methanol at volume ratio of 8: 2 as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm); spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution; the negative sample is free of interference by negative sample control, and the spectrogram is shown in FIG. 4, wherein 1 is Notopterygii rhizoma negative sample, 2, 3, and 4 are samples, and 5 is decursin control.

Experimental example 1 examination of method for measuring naringin content

The granules prepared in example 1 were divided into 6 groups in parallel, test group 1, test group 2, comparative group 1, comparative group 2, comparative group 3 and comparative group 4, and naringin content was measured in accordance with the following methods, respectively.

(1) Test group 1: taking the granules, preparing a test solution and a reference solution according to the preparation method of the test solution and the preparation method of the reference solution in example 1, preparing the granules according to the formula and the preparation method described in example 1, except that rhizoma drynariae is not added to obtain a rhizoma drynariae negative reference substance, preparing the rhizoma drynariae negative reference substance solution according to the preparation method of the test solution described in example 1, and performing atlas measurement according to the chromatographic conditions and the measuring methods disclosed in example 1, wherein the results are shown in figure 5-A, the separation degrees of the naringin peak and other impurity peaks in the test solution are both greater than 1.5, and the separation degrees meet the requirements.

(2) Test group 2: taking the granules, preparing a test solution and a reference solution according to the preparation method of the test solution and the preparation method of the reference solution in example 2, preparing the granules according to the formula and the preparation method described in example 2, except that rhizoma drynariae is not added to obtain a rhizoma drynariae negative reference substance, preparing the rhizoma drynariae negative reference substance solution according to the preparation method of the test solution described in example 2, and performing atlas measurement according to the chromatographic conditions and the measuring methods disclosed in example 2, wherein the results are shown in figure 5-B, the separation degrees of the naringin peak and other impurity peaks in the test solution are both greater than 1.5, and the separation degrees meet the requirements.

(3) Comparison group 1 (pharmacopoeia method)

Preparation of a test solution: precisely weighing about 1.5g of the prepared granules, placing the granules into a conical flask with a plug, precisely adding 180ml of methanol, heating and refluxing for 3 hours, taking out the granules, cooling, filtering, placing the filtrate into a 200ml measuring flask, washing the container with methanol for multiple times, adding the washing liquid into the same measuring flask, adding methanol to the scale, and shaking up to obtain the product;

preparation of control solutions: the same procedure as in example 2 was used to prepare a control solution.

The rhizoma drynariae negative reference substance is prepared into granules according to the prescription and the preparation method described in the example 2, except that the rhizoma drynariae is not added, so that the rhizoma drynariae negative reference substance is obtained, and then the rhizoma drynariae negative reference substance solution is prepared according to the preparation method of the test solution described in the comparison group.

Chromatographic conditions and system applicability test: measuring by high performance liquid chromatography, and using octadecylsilane chemically bonded silica as filler; methanol-acetic acid-water (35:4:65) is used as a mobile phase; the detection wavelength is 283nm, and the theoretical plate number is not lower than 2000 calculated according to naringin;

the determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the test solution, injecting into a liquid chromatograph, measuring, and calculating to obtain a sample solution with naringin peak having shoulder peak, incomplete separation, and obviously unqualified separation degree, as shown in FIG. 5-C.

(4) Comparative group 2: taking the granules, preparing a test solution and a reference solution according to the preparation method of the test solution and the preparation method of the reference solution in example 2, preparing the granules according to the formula and the preparation method described in example 2, except that rhizoma drynariae is not added to obtain a rhizoma drynariae negative reference substance, preparing the rhizoma drynariae negative reference substance solution according to the preparation method of the test solution described in example 2, and then performing spectrum measurement according to the following chromatographic conditions.

Chromatographic conditions are as follows: measuring by high performance liquid chromatography, and using octadecylsilane chemically bonded silica as filler; methanol-water (32:68) is used as a mobile phase; the detection wavelength is 283nm, and the theoretical plate number is not lower than 2000 calculated according to naringin.

As shown in FIGS. 6-A and 6-B, which are chromatograms of the test solution and the rhizoma Drynariae negative control solution, the rhizoma Drynariae negative control has significant interference at the retention time (29min) corresponding to naringin.

(5) Comparative group 3: taking the granules, preparing a test solution and a reference solution according to the preparation method of the test solution and the preparation method of the reference solution in example 2, preparing the granules according to the formula and the preparation method described in example 2, except that rhizoma drynariae is not added to obtain a rhizoma drynariae negative reference substance, preparing the rhizoma drynariae negative reference substance solution according to the preparation method of the test solution described in example 2, and then performing spectrum measurement according to the following chromatographic conditions.

Chromatographic conditions are as follows: measuring by high performance liquid chromatography, and using octadecylsilane chemically bonded silica as filler; acetonitrile-water (15:85) is used as a mobile phase; the detection wavelength is 283nm, and the theoretical plate number is not lower than 2000 calculated according to naringin.

As shown in FIG. 6-C, the naringin peak in the test solution had a shoulder, which was not completely separated, and the degree of separation was significantly unacceptable.

(6) Comparative group 4 the above granules were prepared into a test solution and a control solution according to the "preparation of test solution" and "preparation of control solution" in example 2, and granules were prepared according to the "recipe" and "preparation method" described in example 2, except that drynaria rhizome was not added to obtain a drynaria negative control, and then a drynaria negative control solution was prepared according to the preparation of test solution described in example 2, and then subjected to chromatography measurement according to the following chromatographic conditions.

Chromatographic conditions are as follows: measuring by high performance liquid chromatography, and using octadecylsilane chemically bonded silica as filler; acetonitrile-water solution (17:83) containing 0.1vt percent of phosphoric acid is used as a mobile phase; the detection wavelength is 283nm, and the theoretical plate number is not lower than 2000 calculated according to naringin.

As shown in FIG. 6-D, which is an enlarged view of the sample solution spectrum, the naringin peak in the sample solution is disturbed, and the degree of separation is obviously unqualified.

(7) And (4) analyzing results: the chromatographic analysis results of the test group 1, the test group 2 and the comparison groups 1 to 4 show that because the traditional Chinese medicine composition has various medicinal flavors and complex components and is mutually interfered, when the traditional Chinese medicine composition is detected by using the method in the comparison groups 1 to 4, the separation degrees of the naringin peak and other impurity peaks are both less than 1.5, the separation effect is poor, and the chromatographic analysis requirement cannot be met, and in the test group 1 and the test group 2, the separation degrees of the naringin peak and other impurity peaks are both more than 1.5, wherein the separation degree of the test group 1 is 9.964, the separation degree of the test group 2 is 2.718, and the chromatographic analysis requirement is met, and compared with the test group 2, the separation degrees of the naringin peak and other impurity peaks in the normal phase chromatographic analysis of the test group 1 are obviously improved, the durability of the test group 1 is higher, and the separation effect is obviously improved.

Experimental example 2 methodological investigation of naringin normal phase chromatography assay

1 specialization examination

Preparing granules according to the prescription and the preparation method described in example 1, and then preparing a test solution and a reference solution according to the preparation method of the test solution and the preparation method of the reference solution in example 1; ② according to the prescription proportion and the preparation method recorded in the example 1, granules without drynaria, namely drynaria negative reference substance, is prepared, and then according to the preparation method of the test solution recorded in the example 1, drynaria negative reference substance solution is prepared.

According to the chromatographic conditions in the example 1, the sample solution, the reference solution and the rhizoma drynariae negative reference solution are respectively taken and injected into a chromatographic column, the result is shown in figure 7, the separation degree of the naringin peak and other impurity peaks in the sample solution is more than 1.5, the separation condition is better, and the rhizoma drynariae negative reference solution does not develop a chromatographic peak at the same retention time with the naringin, so the method is considered to be non-interference and strong in specificity.

2 examination of the Linear relationship

Precisely sucking the reference substance solution, diluting to obtain a series of standard substance solutions, precisely sucking 10 μ l of the reference substance solutions, respectively injecting into a liquid chromatograph, measuring peak area according to the chromatographic conditions, and drawing a standard curve of naringin by taking peak area integral value as ordinate and sample amount of the reference substance as abscissa, as shown in FIG. 10, regression equation: 1889107.22x-38106.42, R²1.00 percent; the result shows that the detection method disclosed in the embodiment 1 can effectively detect naringin, and the sampling amount and the peak area of the naringin are in a good linear relation within the range of 0.1518 mu g-7.5890 mu g.

3 precision test

The above control solution was precisely measured, and sample introduction was repeated 6 times under the chromatographic conditions in example 1, and the peak area of naringin was measured to calculate the RSD value of the peak area. Wherein the RSD value of the naringin peak area is 0.89%. The result shows that the instrument precision is good under the condition of the chromatogram.

4 repeatability test

Six test solutions were prepared from the granules in parallel by the method described in example 1, and the peak areas of naringin were measured to calculate the content and RSD value, wherein the RSD value of naringin content was 1.25%. The result shows that the method has good repeatability.

5 recovery test

The method of 100% sample recovery was used. About 1.5g of the above granules were weighed precisely, and 6 parts in total were weighed out, six parts of the test sample solutions were prepared according to the method for preparing the test sample solutions of example 1, and a certain amount of the control sample solution was added, and the recovery rate was calculated according to the following formula (measured value-test sample content × sample amount)/addition amount × 100%, where the average recovery rate of naringin was 100.89% and the RSD value was 1.44%. The results show that the recovery rate of the method meets the requirements.

Experimental example 3 methodological investigation of naringin reversed phase chromatography content determination

1 specialization examination

Preparing granules according to the prescription and the preparation method described in the example 2, and then preparing a test solution and a reference solution respectively according to the preparation method of the test solution and the preparation method of the reference solution in the example 2; ② according to the prescription proportion and the preparation method recorded in the example 2, the granule without drynaria, namely the drynaria negative reference substance, is prepared, and then the drynaria negative reference substance solution is prepared according to the preparation method of the test solution recorded in the example 2.

According to the chromatographic conditions in the embodiment 2, the sample solution, the reference solution and the rhizoma drynariae negative reference solution are respectively taken and injected into a chromatographic column, the result is shown in figure 8, the separation degree of the naringin peak and other impurity peaks in the sample solution is more than 1.5, the separation condition is better, and the rhizoma drynariae negative reference solution does not develop a chromatographic peak at the same retention time with the naringin, so the method is considered to be non-interference and strong in specificity.

2 examination of the Linear relationship

Precisely sucking the reference substance solution, diluting to obtain a series of standard substance solutions, precisely sucking 10 μ l, respectively injecting into a liquid chromatograph, measuring peak area according to the chromatographic conditions, and drawing a standard curve of naringin by taking peak area integral value as ordinate and sample amount of the reference substance as abscissa, as shown in FIG. 11, regression equation: 1651822.5190x +154941.2281, R²0.9998; the result shows that the detection method of the embodiment 2 can effectively detect naringin, and the sampling amount and the peak area of the naringin are in a good linear relation within the range of 0.261 mu g-20.88 mu g.

3 precision test

The above control solution was precisely measured, and sample introduction was repeated 6 times under the chromatographic conditions in example 2, and the peak area of naringin was measured to calculate the RSD value of the peak area. Wherein the RSD value of the naringin peak area is 0.49%. The result shows that the instrument precision is good under the condition of the chromatogram.

4 repeatability test

Six test solutions were prepared from the granules in parallel by the method described in example 2, and the peak areas of human naringin were measured to calculate the content and RSD value, wherein RSD value of naringin content was 0.85%. The result shows that the method has good repeatability.

5 recovery test

The method of 100% sample recovery was used. About 1.5g of the above granules were weighed precisely, and 6 parts in total were weighed out, six parts of the test sample solutions were prepared according to the method for preparing the test sample solutions in example 2, and a certain amount of the control sample solution was added, and the measurement was performed under the chromatographic conditions described in example 2, and the recovery rate was calculated according to the following formula (measured value-test sample content × sample amount)/addition amount × 100%, wherein the average recovery rate of naringin was 98.8% and the RSD value was 1.80%. The results show that the recovery rate of the method meets the requirements.

Experimental example 4 methodological investigation of the determination of the Sinomenine content

1 specialization examination

Preparing granules according to the prescription and the preparation method described in example 3, and then preparing a test solution and a reference solution according to the preparation method of the test solution and the preparation method of the reference solution in example 3; ② preparing granules without orientvine, namely orientvine negative reference substances according to the prescription proportion and the preparation method recorded in the example 3, and preparing the orientvine negative reference substance solution according to the preparation method of the test solution recorded in the example 3.

According to the chromatographic conditions in the example 3, the sample solution, the reference solution and the caulis sinomenii negative reference solution are respectively taken and injected into a chromatographic column, the result is shown in figure 9, the separation degree of sinomenine and other impurity peaks in the sample solution is more than 1.5, which indicates that the separation condition is better, and the caulis sinomenii negative reference solution does not develop a chromatographic peak at the same retention time as sinomenine, so that the caulis sinomenii negative reference solution is considered to be free of interference and strong in specificity.

2 investigation of Linear relationship

Precisely sucking the sinomenine reference substance solution to dilute the sinomenine reference substance solution to obtain a series of standard substance solutions, taking 10 mu l of the standard substance solutions, respectively injecting the standard substance solutions into a liquid chromatograph, measuring peak areas according to the chromatographic conditions, taking peak area integral values as vertical coordinates and sinomenine amount as horizontal coordinates, drawing a standard curve, and as shown in figure 12, the result shows that sinomenine has a good linear relation in the range of 0.108 mu g-15.05 mu g, the obtained regression equation is that y is 995781.4686x +43146.1334, R2 is 0.9998, and the calculation can be carried out by an external standard point method.

3 precision test

Precisely measuring the reference substance solution, repeatedly injecting sample for 6 times according to the chromatographic conditions in example 3, measuring the peak area of sinomenine, and calculating the RSD value of the peak area. The RSD value was 0.74%. The result shows that the instrument precision is good under the condition of the chromatogram.

4 repeatability test

Six test solutions were prepared from the granules in parallel by the method described in example 3, and the peak areas of sinomenine were measured to calculate the content and RSD value, wherein RSD value of sinomenine content was 0.24%. The result shows that the method has good repeatability.

5 recovery test

The method of 100% sample recovery was used. About 1g of the above-mentioned granules were weighed precisely, 6 parts in total, six parts of the test solution were prepared by the method for preparing the test solution in example 3, a certain amount of the control solution was added precisely, and the recovery rate was calculated as follows (measured value-test content × added amount)/added amount × 100% under the chromatographic conditions described in example 3, the average recovery rate of 6 samples was 99.9%, and the RSD% was 1.08%. The result shows that the recovery rate of the method meets the requirement.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. A quality detection method of a traditional Chinese medicine composition for tonifying kidney and relaxing spine is characterized in that the traditional Chinese medicine composition comprises the following raw material medicines in parts by weight: 8-12 parts of rhizoma drynariae, 4-8 parts of rhizoma cibotii, 4-8 parts of eucommia bark, 4-8 parts of cassia twig, 2-6 parts of antler, 5-9 parts of corydalis tuber, 5-9 parts of teasel root, 5-9 parts of gentiana macrophylla, 7-11 parts of caulis sinomenii and 4-8 parts of notopterygium root;

A. content determination of naringin

chromatographic conditions and system applicability test:

the specific method for the chromatographic condition and system applicability test comprises the following steps: measuring by high performance liquid chromatography, and using amino bonded silica gel as filler; acetonitrile is used as a mobile phase A, water is used as a mobile phase B, and the weight ratio of the acetonitrile to the water is determined according to the weight ratio of the mobile phase A: the volume ratio of the mobile phase B is 90%: 10 percent; the detection wavelength is 283 nm; the theoretical plate number is not less than 2000 calculated by naringin;

2. The quality detection method of the traditional Chinese medicine composition according to claim 1, wherein in the content measurement of naringin, the specific method for preparing the test solution is as follows: precisely weighing the traditional Chinese medicine composition, precisely adding 10-100ml of 70-100vt% methanol solution, carrying out ultrasonic treatment for 0.1-1.0h, taking out, cooling, weighing again, complementing the loss weight by 70-100vt% methanol solution, shaking up, filtering, taking 5-50ml of filtrate, evaporating to dryness, adding 1-10ml of water into residue, dissolving, cooling, passing through a polyamide chromatographic column, eluting by 10-100ml of water solution, discarding the water solution, eluting by 10-100ml of methanol, collecting the eluent, evaporating to dryness, fixing the volume to 1-50ml by methanol, shaking up, filtering, and obtaining the traditional Chinese medicine composition.

3. The method for detecting the quality of the traditional Chinese medicine composition according to claim 1 or 2, wherein the method for detecting the quality of the traditional Chinese medicine composition further comprises at least one of the following content determination methods or identification methods:

B. determination of sinomenine content

C. Identification of rhizoma corydalis:

D. And (3) identification of gentiana macrophylla:

and (3) identification: performing thin layer chromatography test by taking 1-10 μ l of the sample solution and the reference solution, respectively dropping on the same silica gel GF254 thin layer plate, developing with ethyl acetate-methanol-water solution at volume ratio of 5-20: 1-5: 0.5-2 as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm); spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution; or

E. And (3) identification of gentiana macrophylla:

F. Identification of notopterygium root:

4. The quality detection method of the traditional Chinese medicine composition according to claim 1 or 2, wherein the traditional Chinese medicine composition comprises the following raw material medicines in parts by weight: 10 parts of rhizoma drynariae, 6 parts of rhizoma cibotii, 6 parts of eucommia bark, 6 parts of cassia twig, 4 parts of antler, 7 parts of corydalis tuber, 7 parts of teasel root, 7 parts of gentiana macrophylla, 9 parts of caulis sinomenii and 6 parts of notopterygium root; or

5. The quality detection method of the traditional Chinese medicine composition according to claim 1 or 2, characterized in that the traditional Chinese medicine composition is directly or indirectly prepared into clinically acceptable tablets, granules, capsules, oral liquid or injections by adding conventional auxiliary materials according to a conventional process.

6. The quality detection method of the traditional Chinese medicine composition according to claim 1 or 2, wherein the preparation method of the traditional Chinese medicine composition comprises the following steps:

(1) weighing cassia twig and notopterygium root according to the selected weight parts, mixing, adding a proper amount of water, performing steam distillation, respectively collecting volatile oil and water extract for later use, taking the volatile oil, adding beta-cyclodextrin and water, performing inclusion, and drying to obtain an inclusion compound for later use;

7. The quality detection method of the traditional Chinese medicine composition according to claim 6, wherein in the inclusion process in the step (1), the dosage ratio of the volatile oil, the beta-cyclodextrin and the water is as follows: 1 ml: 4-10 g: 40-100 ml;

8. The quality detection method of a Chinese medicinal composition according to claim 6,

adding 8-12 times of water in the steam distillation process in the step (1), and performing steam distillation for 4-8 hours;

in the water extraction process in the step (2), adding water, decocting for 1-3 times, 1-2 hours each time, and adding 4-10 times of water each time;

9. The use of the quality control method of any one of claims 1-8 in quality control of a Chinese medicinal composition or a Chinese medicinal preparation.