CN112007107A

CN112007107A - Traditional Chinese medicine oral liquid for treating infantile rickets and detection method thereof

Info

Publication number: CN112007107A
Application number: CN202010713070.7A
Authority: CN
Inventors: 向阳; 黄志军; 肖飞; 代虹剑; 冷娇龙; 何定邦; 付浩; 王涛涛
Original assignee: Jianmin Pharmaceutical Groups Corp ltd
Current assignee: Jianmin Pharmaceutical Groups Corp ltd
Priority date: 2020-07-22
Filing date: 2020-07-22
Publication date: 2020-12-01

Abstract

The invention discloses a traditional Chinese medicine oral liquid for treating infantile rickets, and also discloses a detection method of the oral liquid, belonging to the field of medicinal preparations and analysis. Compared with the original dosage form, the oral liquid provided by the invention has the advantages of good taste, convenience in taking and quick absorption, and can improve the medication compliance of patients. The quality detection method established by the invention comprises the thin-layer qualitative identification of three traditional Chinese medicinal materials and the content measurement of the two chemical drugs, and meanwhile, the content detection of n-butanol extract is added according to the main active parts of the traditional Chinese medicines, so that the quality of the produced oral liquid is controlled qualitatively, quantitatively and comprehensively, the quality uniformity of the produced oral liquid is higher, and the curative effect can be better ensured.

Description

Traditional Chinese medicine oral liquid for treating infantile rickets and detection method thereof

Technical Field

The invention relates to a traditional Chinese medicine oral liquid for treating infantile rickets, in particular to a Longmu bone strengthening oral liquid, and a detection method of the oral liquid, belonging to the field of medicinal preparation and analysis.

Background

The calcium deficiency of 37 percent of children and 59 percent of middle-aged and elderly people in China, huge population base of children, 2000 ten thousand newborns each year, higher morbidity, unsettled nutritional status of children, unreasonable dietary structure of Chinese and other factors determine that the market potential of the calcium supplement for children is huge, the main brands of the calcium supplement medicines/health care products in China at present have cover-in-cover, sanjing, cover-in-the-air, and powerful calcium, and the dosage forms comprise tablets, oral liquid, granules, chewable tablets, capsules, effervescent tablets and the like, and various calcium supplement products try to occupy and expand own territories in imagination; calcium supplement products of various manufacturers are continuously promoted to be new, and series products are released to segment the market. Each product can clearly determine the positioning of the product through own market segment, for example, the high calcium tablet of the cap in the cap is positioned in the old, the oral liquid of the cap in the cap locks children, the powerful calcium tablet and the like are also segmented into adults and children, and the calcium glucose tri-refined calcium is also drawn close to the calcium supplement of the children.

Since the Longmu Zhuanggu granules are produced and marketed in the 80 th century, the Longmu Zhuanggu granules are widely and favorably evaluated in curative effect, are first-line medicines for treating rickets in pediatrics and are also rare pediatric Chinese and western medicine compound preparations in China. The product is prepared from radix Codonopsis, radix astragali, radix Ophiopogonis, carapax et Plastrum Testudinis, Atractylodis rhizoma, rhizoma Dioscoreae, fructus Schisandrae, Os Draconis, Concha Ostreae, Poria, fructus Jujubae, Glycyrrhrizae radix, calcium lactate, endothelium corneum Gigeriae Galli, vitamin D2, and calcium gluconate, and can be used for treating and preventing infantile rickets and osteomalacia; it can also be used for treating hyperhidrosis, night terror, anorexia, dyspepsia, and hypoevolutism in children.

The Longmu Zhuanggu granules belong to non-prescription medicines and basic medicines in China, and are collected in medical insurance catalogues, the product is a Chinese and western compound, and the product is the first brand of the calcium agent market in the 90 s. The product is suitable for children, and has the advantages of regulating spleen and stomach, multiple calcium sources, addition of vitamin D, high safety, and capability of solving the problem of poor absorption of single calcium supplement for children.

The product can effectively supplement calcium, promote the deposition and mineralization of calcium in bones, simultaneously regulate spleen and stomach, enhance digestion and absorption functions, promote the comprehensive absorption of children on dietary nutrition, prevent and treat calcium deficiency and growth retardation, and is very beneficial to the health condition. The product has long time to market, so the market penetration is sufficient, and the product has a batch of faithful consumers. However, the product is aged gradually due to long history, and gradually enters the decline stage from the maturation stage, and particularly, the dosage form of the product does not conform to the current main flow dosage form for calcium supplement of children, so that the product is inconvenient to take; the taste is inferior to the fruit taste type popular in the current market, and no product line is formed, so that the competitive advantage is gradually lost in the market.

Through the analysis, more than half of the calcium preparation in the current market is oral liquid, and the calcium preparation has the advantages of quick absorption, high bioavailability, good taste, convenience in taking and carrying and the like, so that the calcium preparation becomes the leading preparation in the current market. If the preparation is successfully developed and approved to be on the market, the health-care pharmaceutical industry group can fully utilize marketing resources in the calcium supplement market of children, exert brand advantages and create a curiosity for sale.

Disclosure of Invention

The invention aims to provide a traditional Chinese medicine oral liquid for treating infantile rickets, which is prepared from the following raw materials in parts by weight:

the preparation method comprises the following steps:

vitamin D₂Clathrating with beta-cyclodextrin; decocting radix Codonopsis, radix astragali, radix Ophiopogonis, carapax et Plastrum Testudinis, Atractylodis rhizoma, rhizoma Dioscoreae, fructus Schisandrae chinensis, Os Draconis, Concha Ostreae, Poria, fructus Jujubae, Glycyrrhrizae radix, and endothelium corneum Gigeriae Galli with water for three times, mixing decoctions, filtering, standing, concentrating the supernatant to relative density of 1.05-1.15, cooling, adding ethanol to ethanol content of 50-70% (volume), standing, collecting the supernatant, recovering ethanol under reduced pressure, concentrating to relative density of 1.15-1.25 to obtain extract; collecting calcium lactate, calcium gluconate, and vitamin D₂Boiling the clathrate in water, adding polysorbate 80 and the above extract, cooling, adding potassium sorbate, aspartame and essence, adding water to 1000 volume, filtering, bottling, sterilizing,

the weight is related to the volume in g/ml.

Preferably, the tortoise shells and the schisandra chinensis are processed with vinegar.

Preferably, the bighead atractylodes rhizome and the chicken gizzard-membrane are fried.

Preferably, the keel and the oyster are calcined.

Preferably, the water is added for decoction for three times, wherein the water is added for 2 hours for the first time and the second time and is added for 1 hour for the third time, and the water addition amount of each time is 8 times of the weight of the medicinal materials.

Preferably, the alcohol content is 60%.

Preferably, the inclusion conditions are: stirring at 60 ℃, clathrating for 3 hours, wherein the weight ratio of beta-cyclodextrin to 60% ethanol is 1: 12-1: 16, and the beta-cyclodextrin and VitD₂The molar ratio of (A) to (B) is 7: 1.

Preferably, the packaging material used for the potting is a brown finger-top bottle.

The invention also provides a method for detecting the traditional Chinese medicine oral liquid for treating the infantile rickets, which comprises the following steps:

[ IDENTIFICATION ] collecting 60ml of the product, extracting with water saturated n-butanol under shaking for 3 times (20 ml each time), mixing n-butanol solutions, washing with 1% sodium hydroxide solution for 3 times (20 ml each time), discarding the alkali solution, washing with n-butanol saturated water to neutrality, evaporating n-butanol solution on water bath, and dissolving the residue with 0.5ml of methanol to obtain a sample solution. Adding methanol into astragaloside IV control to obtain solution containing 0.5mg per 1ml, and making into control solution. Performing thin layer chromatography (appendix VI B of first part of the Chinese pharmacopoeia 2015 edition), sucking 10 μ l of each of the two solutions, respectively dropping on the same silica gel G thin layer plate, developing with a developing agent of lower layer solution of chloroform-ethyl acetate-methanol-water (10:20:11:5) which is placed overnight at 10 deg.C, taking out, air drying, spraying with 10% sulfuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. In the chromatogram of the test sample, spots of the same color appear in the sunlight at the positions corresponding to those of the chromatogram of the control sample; fluorescent spots of the same color appear under an ultraviolet lamp (365 nm).

(2) Collecting 40ml of the product, extracting with water saturated n-butanol under shaking for 3 times (20 ml each time), mixing n-butanol solutions, washing with water for 2 times (20 ml each time), evaporating n-butanol solution on water bath, and dissolving the residue with 1ml of acetone to obtain sample solution. Taking 0.5g of rhizoma Atractylodis Macrocephalae as reference material, and making into reference material solution by the same method. Performing thin-layer chromatography (appendix VI B of the first part of the 2015 edition of Chinese pharmacopoeia), collecting 15 μ l of the sample solution and 10 μ l of the reference solution, respectively dropping on the same silica gel G thin-layer plate, developing with chloroform-acetone (19:1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution.

(3) Extracting 60ml of the product with diethyl ether for 2 times (20 ml each time), discarding the diethyl ether solution, extracting the water solution with water saturated n-butanol under shaking for 5 times (20 ml each time), mixing n-butanol solutions, washing with water for 5 times (20 ml each time), evaporating n-butanol solution on water bath, and dissolving the residue with 1ml of methanol to obtain sample solution. And adding water 10ml into Glycyrrhrizae radix control 0.5g, treating with ultrasound for 15 min, filtering, collecting filtrate, and making into control solution by the same method. Performing thin layer chromatography (appendix VI B of the first part of the 2015 edition of Chinese pharmacopoeia), sucking 10 μ l of the test solution and 1 μ l of the control solution, respectively dropping on the same silica gel G thin layer plate prepared from 1% sodium hydroxide solution, developing with the upper solution of ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as developing agent, taking out, air drying, spraying with 10% sulfuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. In the chromatogram of the test solution, spots of the same color appear in the corresponding positions of the chromatogram of the reference solution under sunlight.

[ EXAMINATION ] the relative density should not be less than 1.03 (appendix VII A of the first part of the pharmacopoeia 2015).

The pH value should be 4.0-6.0 (appendix VII G of the first part of the Chinese pharmacopoeia 2015).

Other ingredients should meet the regulations of the mixture (appendix I J of the first part of the 2015 edition of Chinese pharmacopoeia).

[ n-butanol extract ] this product was measured accurately 20ml, extracted with water-saturated n-butanol with shaking 5 times, 20ml each time, the n-butanol extracts were combined, placed in an evaporation dish dried to constant weight, evaporated to dryness, dried at 105 ℃ for 3 hours, transferred to a desiccator, cooled for 30 minutes, weighed rapidly and accurately, and calculated to obtain the final product.

The content of n-butanol extract in each capsule should not be less than 0.06 g.

[ MEASUREMENT ] preparation of calcium control solution precisely calls about 60mg of calcium carbonate control dried at 110 ℃ to constant weight, puts into a 100ml measuring flask, adds 10ml of water for moistening, adds 5ml of dilute hydrochloric acid for dissolving, adds water to the scale, shakes uniformly, precisely measures 25ml, puts into a 100ml measuring flask, adds water for diluting to the scale, shakes uniformly, precisely measures 1.0ml, 1.5ml, 2.0ml, 2.5ml and 3.0ml, respectively puts into a 25ml measuring flask, adds 1ml of lanthanum test solution, adds water to the scale, shakes uniformly, and obtains;

preparation of a test solution, precisely measuring 1ml of the product under the condition of a measuring amount, placing the product in a 100ml measuring flask, adding 10ml of water and 5ml of dilute hydrochloric acid, shaking up, adding water to a scale, shaking up, filtering, precisely measuring 2ml of a subsequent filtrate, placing the subsequent filtrate in a 25ml measuring flask, adding 1ml of lanthanum test solution, adding water to a scale, and shaking up to obtain the test solution;

the determination method comprises taking reference substance and sample solution, measuring at 422.7nm wavelength according to atomic absorption spectrophotometry (first method of content determination method V D in appendix of first part of pharmacopoeia 2015 edition), and calculating;

the content of calcium (Ca) in each branch should not be less than 45.0 mg.

Vitamin D₂Measured according to high performance liquid chromatography (appendix VI D of the first part of the 2015 edition of Chinese pharmacopoeia).

Octadecylsilane chemically bonded silica is used as a filling agent in chromatographic condition and system adaptability tests; methanol-water (97:3) as mobile phase; the detection wavelength is 265 nm; flow rate: 1.5 ml/min. Theoretical plate number according to vitamin D₂Peak calculation should be no less than 3000;

preparation of control solution vitamin D was taken₂Accurately weighing 12.5mg of a reference substance, placing the reference substance in a 100ml brown measuring flask, adding absolute ethyl alcohol to dissolve the reference substance and fix the volume to a scale, shaking up, accurately weighing 1ml, placing the reference substance in a 25ml brown measuring flask, adding absolute ethyl alcohol to dilute the reference substance to the scale, and shaking up to obtain the product;

preparing test solution by filtering with microporous membrane (0.45 μm) to obtain filtrate;

the determination method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, and determining;

the product contains vitamin D₂(C₂₈H₄₄O) must not be less than 36. mu.g.

The invention has the beneficial effects that: compared with the original dosage form, the oral liquid provided by the invention has the advantages of good taste, convenience in taking and quick absorption, and can improve the medication compliance of patients. The invention overcomes the technical problems of complex components, poor taste and stability and the like of the Chinese and western compound preparation, the prepared oral liquid can be stored for a long time without influencing the effectiveness and safety of the medicine, and the storage period can reach more than 1.5 years.

The quality detection method established by the invention comprises the thin-layer qualitative identification of three traditional Chinese medicinal materials and the content measurement of the two chemical drugs, and meanwhile, the content detection of n-butanol extract is added according to the main active parts of the traditional Chinese medicines, so that the quality of the medicines is controlled in a qualitative and quantitative manner in all directions, the produced oral liquid has higher quality uniformity, and the curative effect can be better ensured. The established detection method has high accuracy, sensitivity and reproducibility through methodological investigation, and meanwhile, the method has the advantages of simplicity in operation, low detection cost, high speed and the like.

Drawings

FIG. 1 is a photograph of thin layer chromatography identification of Astragalus membranaceus. In the figure, 1 is a negative control; 2.3, 4 are samples to be detected; 5 is astragaloside control.

FIG. 2 is a photograph of thin layer chromatography identification of Atractylodis rhizoma. In the figure, 1 is a negative control; 2.3, 4 are samples to be detected; and 5, a white atractylodes rhizome control medicinal material.

FIG. 3 is a photograph of thin layer chromatography identification of Glycyrrhiza uralensis Fisch. In the figure, 1 is a negative control; 2.3, 4 are samples to be detected; 5 is a control drug of licorice.

FIG. 4 is a standard curve for calcium content determination.

FIG. 5 is vitamin D₂And (5) a special experimental result.

FIG. 6 is vitamin D₂Standard curve for assay.

FIG. 7 is vitamin D₂Ultraviolet scanning graph.

Detailed Description

The present invention will be described in detail below with reference to specific examples.

Example 1 study on the production of Longmu Zhuanggu oral liquid

1. The basis of the selection of the dosage form

The Longmu bone strengthening oral liquid is prepared by modifying the formulation on the basis of Longmu bone strengthening granules, the Longmu bone strengthening granules are unique fist products of our company, belong to the first-class protection variety of Chinese medicaments in China, and gradually enter the decline stage from the maturity stage since the product enters the market, so that our company plans to develop from the formulation transversely, carries out deep secondary development on the product, gradually forms a product series, changes the current situation that the formulation and the taste of the product are single at present, and forms a monopoly technology for treating rickets of children.

The oral liquid is the mainstream preparation formulation of the existing calcium supplement medicine, has the advantages of good taste, convenient taking and quick absorption, and the calcium preparation with the largest market sale amount is the preparation formulation according to the research, such as cover-in-cover, three essences, and super calcium. The successful development of the product is believed to bring better market prospect for the dragon and oyster series products.

2. Vitamin D₂Description of the Inclusion method

VitD₂The active component is one of the active components in the prescription, has the physical property that the active component is insoluble in water and easily soluble in ethanol and ether, and has unstable chemical property and easy deterioration due to the existence of conjugated triple bonds in the chemical structure when being exposed to light, oxygen, heat and the like. Therefore, the original preparation process adopts a beta-cyclodextrin inclusion method, and the purpose is to improve the VitD₂The stability of the bone strengthening oral liquid ensures the curative effect of the bone strengthening oral liquid of the dragon and the oyster. Simultaneously due to VitD₂Inclusion with beta-cyclodextrin increases solubility in water and is therefore particularly suitable for making liquid preparations, such as oral liquids.

VitD₂The inclusion method is reported in a plurality of literatures, the subject develops the inclusion process research aiming at the Longmu bone strengthening oral liquid, and the adopted optimal inclusion conditions are as follows: stirring at 60 deg.C for 3 hr to obtain beta-cyclodextrin and 60% ethanol at a weight ratio of 1: 12-1: 16And VitD₂The ratio (molar ratio) of (A) to (B) is 7: 1. The research finds that: inclusion by the above conditions, VitD₂Has an inclusion rate of 80% or more, and has been proved to be compatible with non-included VitD by stability examination₂In contrast, VitD₂The beta-CD inclusion compound has better stability in oral liquid and is more stable to light, heat and moisture.

3. Study on the amount of polysorbate 80

Due to the presence of VitD in the product prescription₂The clarity of the liquid medicine is not good after the components with low solubility, such as the beta-CD inclusion compound, are dissolved in water, and the calcium lactate and the calcium gluconate have high solubility but are easy to precipitate after being placed for a long time, so the clarity and the stability of the product are improved by adding the solubilizer.

Tween 80(Tween 80, Polyoxylene 80 sorbate 80) is a nonionic surfactant, can be used as an emulsifier, a dispersant, a solubilizer or a stabilizer, and is widely applied to medicines, foods and the like. The Tween 80 is applied to a large number of medicinal preparations in oral routes or parenteral routes, the safety of the Tween 80 is deeply researched, and research results show that the Tween 80 has a wider safety window, has no toxic or side effect and cannot influence the safety of the product.

In order to examine the dosage of the Tween 80, the prescribed amounts of calcium lactate, calcium gluconate and vitamin D are taken₂Adding water into the inclusion compound until the volume is 950ml, boiling, adding the extractum, respectively adding 0.2g, 0.4g, 0.6g and 0.8g of Tween 80, uniformly mixing, fixing the volume to 1000ml, storing at room temperature, observing the clarity and the precipitation condition, and the test results are shown in a table 1-1.

TABLE 1-1 study of soil temp. 80

From the test results, it can be seen that the addition of proper Tween 80 to the oral liquid can not only clarify the medicine, but also improve the stability of the medicine, so that the medicine is not easy to precipitate. Meanwhile, as can be seen from the table, adding 0.6g of Tween 80 to one prescription is better.

4. Determination of the amount of preservative

The best potassium sorbate currently used is used as preservative. The potassium sorbate has obvious inhibiting effect on the growth and development of mould, yeast and aerobic bacteria, and is a broad-spectrum nontoxic preservative which is most widely used. In terms of safety, sorbic acid is an internationally recognized and safe preservative, and has high safety. It is easy to dissolve in water, and the lower the pH value, the stronger the bacteriostasis ability. But is easy to volatilize with water vapor when heated, so the water vapor is added in the later stage of heating.

The potassium sorbate has the main characteristics of (1) good mildew-proof effect. The mildew-proof ability of sorbic acid and sylvite is obviously higher than that of benzoic acid and salts, and the mildew-proof effect of potassium sorbate is 5-10 times that of sodium benzoate.

(2) The product has low toxicity and high safety. The toxic side effect of sorbate is only 1/4 for benzoate. The safe use range of sorbic acid and potassium salt in human bodies is as follows: the dosage of the composition per kilogram of body weight per day is not more than 25 mg, so the composition is particularly suitable for children.

(3) No change in food properties. Sorbic acid is an unsaturated fatty acid, and after entering a human body, the sorbic acid participates in the metabolism process of the human body, and the metabolites are carbon dioxide and water.

To determine the optimum amount of potassium sorbate, the following tests were performed: taking calcium lactate, calcium gluconate and vitamin D according to the prescription amount₂Adding water into the inclusion compound until the inclusion compound is boiled to 950ml, adding 0.6g of Tween 80 and the extract according to the prescription amount, fixing the volume to 1000ml, respectively adding 0.6 per thousand, 0.8 per thousand, 1.0 per thousand and 1.2 per thousand of potassium sorbate, filtering, placing for 10 days after steam sterilization, observing the change of microorganisms, taking the total number of moulds as an investigation index, and finding the results shown in tables 1-2.

TABLE 1-2 investigation of preservative usage

The test results show that when 1.0 per mill of potassium sorbate is added, the increase of the total number of the mould can be effectively controlled, and the product is qualified. Therefore, the dosage of the potassium sorbate is 1.0 per mill, namely 1g of potassium sorbate is added in one prescription.

5. Description of the type and amount of other flavoring agents

The sweetener adopts aspartame: aspartame is the best substitute of sucrose at present, has a taste very similar to that of sucrose, has a sweetness 200 times that of sucrose, and is widely applied to food and medicine industries. The aspartame has low calorie, no caries, no influence on blood sugar level, and is an ideal sweetener for sugar-free food. Is approved to be used as food additive in China and used as sweetener. According to the test, the sweet taste of the product is best when the dosage is 0.3 per mill of the liquid medicine amount.

6. According to the above research, the prescription and production process of the product are finally determined as follows:

first, prescription

Second, preparation method

The above sixteen ingredients, vitamin D₂Clathrating with beta-cyclodextrin; adding 8 times of water into thirteen parts of codonopsis pilosula, astragalus, dwarf lilyturf tuber, tortoise shell, largehead atractylodes rhizome, Chinese yam, schisandra chinensis, dragon bone, oyster, poria cocos, Chinese date, liquorice, chicken's gizzard-membrane and the like, decocting for three times, 2 hours for the first time and the second time and 1 hour for the third time, merging decoction, filtering, standing filtrate, taking supernate, concentrating the supernate to the relative density of about 1.08(75-85 ℃), cooling, adding ethanol to ensure that the alcohol content is 60%, standing, taking supernate, recovering ethanol under reduced pressure, and concentrating the supernate to an extract with the relative density of about 1.20(75-85 ℃) for later use. Collecting calcium lactate, calcium gluconate, and vitamin D₂Adding water into the clathrate to 950ml, boiling, adding 0.6g polysorbate 80 and the above extract, cooling, adding potassium sorbate 1g and A0.3g of aspartame, 4.5g of orange flavor, and water are added to 1000ml, and the mixture is filtered, filled, sealed and sterilized to obtain the product.

7. Pilot study

Pilot scale studies were performed at 10 times the amount prescribed and the results are shown in tables 1-3 below.

TABLE 1-3 LONGMUZHUANGGU oral liquid pilot test results

Example 2 study of stability of Longmu bone strengthening oral liquid

First test, Longmu bone strengthening oral liquid influence factor test and results

According to the requirements of a new traditional Chinese medicine stability test, the bone strengthening Longmu oral liquid is subjected to a factor test of strong light irradiation so as to know whether the quality of the medicine is influenced by the light irradiation.

First, test purpose

The chemical medicine contained in the product is vitamin D₂And is easy to deteriorate when exposed to light or air, so the test aims to investigate the quality stability of the sample under strong light irradiation, and mainly comprises vitamin D₂And preliminarily determining the packaging container and the storage condition of the medicine according to the test result of the influencing factors.

Second, test conditions

The samples are placed in a packaging container on the market, and the samples are inspected at regular time under the illumination experimental conditions (4500Ix +/-500 Ix) according to the inspection time requirement of the mixture.

The test date is as follows: 2019.12.20, 12.25, 12.30, i.e. 0, 5, 10 days.

Third, sample source

The test samples were prepared by the company according to standard manufacturing requirements. The test specimens are packaged by brown finger-mouth bottles and stored in a paper box in a centralized mode, and the test specimen batches are 20191201, 20191202 and 20191203 respectively.

Fourth, the requirements of the investigation items

(one) Properties: the product should be a brownish red clear liquid; fragrant and sweet.

(II) authentication:

identification of astragalus membranaceus thin layers: detecting astragaloside IV;

② thin-layer identification of rhizoma atractylodis macrocephalae: detecting Atractylodis rhizoma;

③ identifying the thin layer of liquorice: the licorice should be detected.

And (III) checking:

the relative density: not less than 1.03;

pH value: 4.0 to 6.0.

(IV) n-butanol extract: not less than 0.06 g/count.

(V) content determination:

calcium: not less than 45.0 mg/count;

② vitamin D₂: not less than 36 mug/count.

(VI) microbial limit inspection: the number of bacteria is less than or equal to 100/ml, the number of mould and yeast is less than or equal to 100/ml, Escherichia coli and live mites cannot be detected, and salmonella cannot be detected/10 ml.

Fifth, the experiment summary of the influencing factors

According to the requirement of a new drug stability test on mixture quality stability investigation, a light test is carried out on the Longmu bone strengthening oral liquid, and the result shows that: the examination data of the sample in the examination period, such as character, identification, relative density, pH value, n-butanol extract, calcium content determination and microorganism limit examination, are all within normal ranges, and vitamin D₂The content of (A) is slightly reduced, which shows that the quality stability of the product is better, and is shown in tables 2-1-2-3.

And (II) a brown finger-mouth bottle is used as a packaging container, and the packaging container is sealed and stored in a shade place, so that the quality stability of the product is favorable.

Experiment II, Longmu bone strengthening oral liquid normal room temperature stability test and results

According to the requirements of a new Chinese medicine stability test, the quality stability of the Longmu bone strengthening oral liquid is investigated for 6 months so as to know the quality change of the medicine in the natural storage period.

First, test purpose

The quality stability of the samples under normal room temperature conditions was investigated, and the effect of the packaging container on the stability of the drug was investigated.

Second, test conditions

The samples are put into a packaging material on the market, and the samples are inspected at normal room temperature conditions (25 ℃ plus or minus 2 ℃ and RH60 percent plus or minus 10 percent) according to the inspection time requirement of the mixture.

The test date is as follows: 2019.12-2020.6

Third, sample source

Fourthly, investigation items, methods and results

Seven items such as properties, identification, relative density, PH value, n-butanol extract, content determination, microorganism limit inspection and the like are arranged together, and the requirements are the same as those of an influence factor test.

The study time is 0, 1, 2, 3 and 6 months for 6 months.

Fifth, the study of normal room temperature stability is summarized

The investigation data of the sample in the investigation period, such as characters, identification, relative density, PH value, n-butanol extract, content measurement and microorganism limit inspection, are all in a normal range, which shows that the product has good quality stability, and is shown in tables 2-4-2-6.

And (II) a brown finger-mouth bottle is adopted as a packaging container, so that the quality stability of the product is not affected.

Experiment III, accelerated stability experiment and result of Longmu bone strengthening oral liquid

In order to investigate the rationality of the preparation process and predict the stability of the medicine, the medicine is subjected to an accelerated test under constant temperature and constant humidity conditions according to the requirements of a new medicine stability test.

First, test conditions

Supersaturated sodium chloride solution is put in a closed container, the relative humidity is 75% +/-5%, the ambient temperature is 37-40 +/-2 ℃, and the supersaturated sodium chloride solution is used for storing samples.

The test date is as follows: 2019.12-2020.3

Second, sample source

The sample lots to be examined are 20191201, 20191202 and 20191203.

The preparation method and the packaging container are the same as the normal room temperature stability test "

Third, survey items, methods and results

Seven items such as properties, identification, relative density, PH value, n-butanol extract, content determination, microorganism limit inspection and the like are arranged together, and the requirements are the same as those of a normal room temperature stability test.

The study time was 0, 1, 2, 3 months for 3 months.

Fourth, accelerated test summary

The quality stability of the sample in the investigation period is good, and the table is shown in 2-7-2-9.

And (II) the preparation process of the product is reasonable through an accelerated test, and the tentative validity period is 1.5 years.

TABLE 2-1-2-3 Longmu Zhuanggu oral liquid influence factor test report

Table 2-1 batch number: 20191201 test conditions: intensity 4500Ix ± 500Ix test date: 2019.12.20-2019.12.30

Table 2-2 batch number: 20191202 test conditions: intensity 4500Ix ± 500Ix test date: 2019.12.20-2019.12.30

Table 3 batch number: 20191203 test conditions: intensity 4500Ix ± 500Ix test date: 2019.12.20-2019.12.30

Table 2-4-2-6 Longmu Zhuanggu oral liquid Room temperature stability test report

Table 2-4. batch number: 20191201 test conditions: 25 ℃ ± 2 ℃, RH 60% ± 10% test date: 2019.12-2020.6

Table 2-5 batch number: 20191202 test conditions: 25 ℃ ± 2 ℃, RH 60% ± 10% test date: 2019.12-2020.6

Table 2-6 batch number: 20191203 test conditions: 25 ℃ ± 2 ℃, RH 60% ± 10% test date: 2019.12-2020.6

Table 2-7-2-9 accelerated stability test report of Longmu bone strengthening oral liquid

Table 2-7 batch numbers: 20191201 test conditions: 37-40 ℃ ± 2 ℃, RH 75% ± 5% test date: 2019.12-2020.3

Tables 2-8 batch numbers: 20191202 test conditions: 37-40 ℃ ± 2 ℃, RH 75% ± 5% test date: 2019.12-2020.3

Table 2-9 batch number: 20191203 test conditions: 37-40 ℃ ± 2 ℃, RH 75% ± 5% test date: 2019.12-2020.3

Example 3 Studies on quality standards of bone-strengthening Longmu oral liquid

Bone strengthening Longmu oral liquid and its quality standard (draft)

Longmu bone strengthening oral liquid

Longmu Zhuanggu Koufuye

[ prescription ]

[ PREPARATION METHOD ] the above sixteen ingredients, vitamin D₂Clathrating with beta-cyclodextrin; decocting thirteen Chinese medicinal materials of codonopsis root, astragalus root, ophiopogon root, tortoise shell, ovate atractylodes root, Chinese yam, schisandra berry, dragon's bone, oyster shell, poria, jujube, licorice and gizzard lining with 8 times of water for three times, first time, second time and third time for 2 hr, combining the decoctions, filtering, standing filtrate, concentrating supernatant liquor to obtain concentrated liquor whose relative density is about 1.08(75-85 deg.C), cooling, adding the concentrated liquorMaking alcohol content be 60%, standing, collecting supernatant, recovering ethanol under reduced pressure, and concentrating to obtain extract with relative density of 1.20(75-85 deg.C). Collecting calcium lactate, calcium gluconate, and vitamin D₂Adding water into the clathrate to 950ml, boiling, adding 0.6g of polysorbate 80 and the above extract, cooling, adding 1g of potassium sorbate, 0.3g of aspartame and 4.5g of orange flavor essence, adding water to 1000ml, filtering, bottling, and sterilizing.

[ PROPERTIES ] the product is a brownish red clear liquid; fragrant and sweet.

[ MEASUREMENT ] preparation of calcium control solution precisely calls about 60mg of calcium carbonate control dried at 110 ℃ to constant weight, puts into a 100ml measuring flask, adds 10ml of water to moisten, adds 5ml of dilute hydrochloric acid to dissolve, adds water to the scale, shakes evenly, precisely measures 25ml, puts into a 100ml measuring flask, adds water to dilute to the scale, shakes evenly, precisely measures 1.0ml, 1.5ml, 2.0ml, 2.5ml and 3.0ml, respectively puts into a 25ml measuring flask, adds 1ml of lanthanum test solution, adds water to the scale, shakes evenly, and obtains.

Preparation of test solution 1ml of the product under the condition of measuring accurately is measured, placed in a 100ml measuring flask, added with 10ml of water and 5ml of dilute hydrochloric acid, shaken up, added with water to the scale, shaken up, filtered, 2ml of continuous filtrate is measured accurately, placed in a 25ml measuring flask, added with 1ml of lanthanum test solution, added with water to the scale, and shaken up, thus obtaining the test solution.

The determination method comprises measuring control and sample solution at 422.7nm wavelength by atomic absorption spectrophotometry (first method of content determination method V D in appendix of first part of pharmacopoeia 2015 edition), and calculating.

The content of calcium (Ca) in each branch should not be less than 45.0 mg.

Octadecylsilane chemically bonded silica is used as a filling agent in chromatographic condition and system adaptability tests; methanol-water (97:3) as mobile phase; the detection wavelength is 265 nm; flow rate: 1.5 ml/min. Theoretical plate number according to vitamin D₂The peak calculation should be not less than 3000.

Preparation of control solution vitamin D was taken₂Precisely weighing 12.5mg of the reference substance, placing in a 100ml brown measuring flask, adding anhydrous ethanol to dissolve and fix the volume to scale, shaking up, precisely weighing 1ml, placing in a 25ml brown measuring flask, adding anhydrous ethanol to dilute to scale, and shaking up to obtain the final product.

Preparation of test solution the product is filtered with microporous membrane (0.45 μm) to obtain filtrate.

The determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

[ FUNCTIONS AND INDICATIONS ] can strengthen tendons and bones, harmonize stomach and invigorate spleen. Can be used for treating and preventing malnutritional rickets in children; it also has therapeutic effect on hyperhidrosis, night terror, anorexia, dyspepsia, and hypoevolutism of children.

[ DOSAGE AND ADMINISTRATION ] administered orally. 10ml for the second year, 15ml for the second to seventh years, and 20ml for the seventh year; the preparation is administered 3 times a day.

[ Specification ] 10ml per pack

[ STORAGE ] sealing and standing in the shade.

Second, draft explanation

(one), authentication

1. Thin layer identification of astragalus

The astragalus root is the monarch drug of the prescription, the main component of the astragalus root is the astragaloside IV, and the astragalus root has pharmacological activity and stable chemical property. In the quality research, the content of the astragaloside in the product is measured by using a high performance liquid chromatography and an evaporative light scattering detector by taking the astragaloside as a reference substance, but the astragaloside is large in prescription amount and has more interfering components, so that the astragaloside is not successfully obtained. And thin-layer chromatography identification of radix astragali is established by referring to Chinese pharmacopoeia and thin-layer chromatography identification in Chinese patent medicine. In the test, a astragaloside IV reference substance is used as a control, three batches of samples and negative samples are taken for carrying out the test, and the specific operation is as follows:

(1) instruments and reagents:

model 939 thin layer plate maker (Chongqing);

n-butanol, sodium hydroxide, methanol, chloroform, ethyl acetate, sulfuric acid and ethanol are analytically pure;

astragaloside IV control: for assay, purchased by the institute for testing biological products of Chinese drugs, lot number: 0781-9706;

silica gel G, provided by Qingdao ocean factories;

the Longmu bone strengthening oral liquid is provided by Jianmin pharmaceutical industry group GmbH.

(2) Preparation of a test solution: reference is made to quality standards [ identification 1 ].

(3) Preparation of control solutions: reference is made to quality standards [ identification 1 ].

(4) Preparation of negative control solution: taking the prescription amount of each medicine except the astragalus root to prepare a negative reference substance according to the preparation method of a finished product, and taking 60ml to prepare a astragalus root negative reference substance solution according to the preparation method of a test solution.

(5) Thin layer chromatography:

respectively dispensing 10 μ l of test sample solution, negative sample solution and control solution on the same silica gel G thin layer plate, spreading with lower layer solution of chloroform-ethyl acetate-methanol-water (10:20:11:5) at 10 deg.C or below overnight as developing agent, taking out, air drying, spraying with 10% ethanol sulfate solution, and heating at 105 deg.C until the spots are clearly developed. In the chromatogram of the test solution, spots with the same color appear in the corresponding positions of the chromatogram of the reference solution under sunlight; fluorescent spots with the same color appear under an ultraviolet lamp (365nm), and the negative spot is free of interference (see figure 1), so the fluorescent spots are imported into the text.

2. TLC identification of Atractylodes macrocephala

The sampling amount is 40ml, shaking and extracting with water saturated n-butanol, washing the n-butanol extract with water, evaporating to dryness, dissolving the residue with acetone, and performing a negative control test with Atractylodis rhizoma as control, specifically:

(1) instruments and reagents:

model 939 thin layer plate maker (Chongqing);

the n-butyl alcohol, the acetone and the trichloromethane are analytically pure;

the rhizoma atractylodis macrocephalae contrast medicinal materials: china pharmaceutical biological products certification institute, batch number: 0725-;

silica gel G, provided by Qingdao ocean factories;

(2) Preparation of a test solution: reference is made to quality standards [ identification 2 ].

(3) Preparation of control solutions: reference is made to quality standards [ identification 2 ].

(4) Preparation of negative control solution: taking the prescription amount of each medicine except the largehead atractylodes rhizome to prepare a negative control product according to the preparation method of a finished product, and then taking 40ml of the negative control product solution of the largehead atractylodes rhizome according to the preparation method of a test solution.

(5) Thin layer chromatography:

taking 15 μ l of each sample and negative sample, and 10 μ l of Atractylodis rhizoma control solution, respectively spotting on the same silica gel G thin layer plate, developing with chloroform-acetone (19:1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material, and the negative color is not interfered (see figure 2), so the chromatogram of the test solution is imported into the text.

3. Thin layer identification of licorice

Operating according to pharmacopeia standard, removing the step of water-adding ultrasonic treatment, and obtaining clear result spots, wherein the test is as follows:

(1) instruments and reagents:

model 939 thin layer plate maker (Chongqing);

the ethyl ether, the n-butanol, the methanol, the ethyl acetate, the formic acid, the glacial acetic acid, the sodium hydroxide, the sulfuric acid and the ethanol are analytically pure;

and (3) liquorice as reference medicinal materials: china pharmaceutical biological products certification institute, batch number: 0904-;

silica gel G, provided by Qingdao ocean factories;

(2) Preparation of a test solution: reference is made to quality standards [ identification 3 ].

(3) Preparation of control solutions: reference is made to quality standards [ identification 3 ].

(4) Preparation of negative control solution: taking the medicines except the liquorice according to the prescription amount, preparing a negative control product according to the preparation method of the finished product, and taking 60ml to prepare a liquorice negative control product solution according to the preparation method of the test solution.

(5) Thin layer chromatography:

respectively dropping 10 μ l of test sample, negative sample and control solution on the same silica gel G thin layer plate prepared from 1% sodium hydroxide solution, developing with the upper solution of ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as developing agent, taking out, air drying, spraying with 10% sulfuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. In the chromatogram of the test solution, spots of the same color appear at the corresponding positions of the chromatogram of the reference drug, and the negative color is not interfered (see figure 3), so the test solution is imported into the text.

(II) inspection

Six batches of Longmu bone-strengthening oral liquid are prepared according to the general rule of the combination of the drugs (appendix I J of the first part of the national pharmacopoeia 2015), the properties, the relative density, the pH value, the loading and the microbial limit are respectively checked, and the heavy metal (IXE in the appendix IXE of the first part of the national pharmacopoeia 2015 (second method)) and the arsenic salt (I) in the appendix IXF of the first part of the national pharmacopoeia 2015 (first method)) of the product are checked, wherein the heavy metal is less than 10ppm, and the arsenic salt is less than 2 ppm. The results are shown in Table 3-1.

TABLE 3-1 results of six sample runs

[ n-butanol extract ] n-butanol extract studies were performed.

(1) The extraction times of the same batch of samples (20191002 batches) were compared by using water-saturated n-butanol as the extraction solvent. The results show that the results of 5 times and 6 times of extraction are basically consistent, so that 5 times of extraction of water saturated n-butanol is selected. (see Table 3-2) Table 3-2 examination results of extraction frequency

Number of times	3	4	5	6
					Results (g/count)	0.0330	0.0485	0.0627	0.0623

(2) N-butanol extract assay was performed on ten samples of the Longmu Zhuanggu oral liquid, and the results are shown in tables 3-3.

TABLE 3-3 table of n-butanol extract content measurement results

According to the above determination results, the content of n-butanol extract in each temporary product should not be less than 0.06 g.

[ CONTENT DETERMINATION ] 1 is the determination of the content of the effective ingredient calcium in the product. The experiment adopts atomic absorption spectrophotometry to carry out feasibility research on the determination of the content of calcium in the preparation so as to achieve the purpose of controlling the quality of the product. A series of methodological investigation tests prove that the method is simple, convenient, rapid and accurate, and is really feasible for controlling the quality of the product.

(1) Instruments and reagents

A PE3110 type atomic absorption spectrophotometer;

calcium carbonate (baseline);

the other reagents are analytically pure.

(2) Detection conditions

Light source: calcium hollow cathode lamp

Wavelength: 422.7nm

Gas combustion: acetylene-air

Gas flow rate: 2.0

Slit: 0.5nm

Lamp current: 6mA

(3) Preparation of a test solution: reference is made to quality standards [ content determination ] calcium.

(4) Preparation of control solutions: reference is made to quality standards [ content determination ] calcium.

(5) Specificity test

Preparing a negative sample without adding the extract and the calcium gluconate and the calcium lactate, preparing according to the preparation method of the test solution, and determining. And the result negative sample has no absorption value, which indicates that the calcium content is not interfered.

(6) Investigation of Linear relationships

Precisely weighing 59.9mg of calcium carbonate reference substance, placing the calcium carbonate reference substance in a 100ml measuring flask, adding 10ml of water for wetting, adding 5ml of dilute hydrochloric acid for dissolving, adding water to the scale, shaking up, precisely measuring 25ml, placing the calcium carbonate reference substance in the 100ml measuring flask, adding water for diluting to the scale, shaking up, precisely measuring 1.0ml, 1.5ml, 2.0ml, 2.5ml and 3.0ml, respectively placing the calcium carbonate reference substance in the 25ml measuring flask, adding 1ml of lanthanum test solution, adding water to the scale, shaking up, measuring according to the conditions, obtaining results shown in tables 3-4, performing linear regression by taking the absorbance (Abs) as a vertical coordinate (Y), the calcium concentration (mu g/ml, and the calcium content in calcium carbonate calculated according to 40%) as a horizontal coordinate (X), and obtaining a regression equation: y is 0.0551X +0.1488 and r is 0.9993. The results show that the calcium concentration is in the range of 2.396-7.188 mu g/ml with good linear relation. (see FIG. 4)

Table 3-4 results of linear relationship examination (n ═ 3)

(7) Precision test

Six samples (20191002 batches) of the same batch are repeatedly taken, operated according to a text content determination method and calculated. The results show that the method has good repeatability, and the relative standard deviation is 1.08%. (see tables 3-5)

Tables 3-5 results of precision examination

Secondly, the intermediate precision is calculated by operating three batches of samples in the same laboratory on different dates and different personnel according to the text content measuring method. The results show that the method herein has good intermediate precision. (see tables 3-6)

Tables 3-6 intermediate precision results (n ═ 3)

And thirdly, the reproducibility is verified by operating three batches of samples according to the text content measuring method through two laboratories. The measurement result shows that the method has good repeatability. (see tables 3-7)

Tables 3-7 reproducibility test results (n ═ 3)

(8) Accuracy test

Precisely weighing a proper amount of calcium carbonate reference substances, and preparing according to a preparation method of a reference substance solution, wherein the concentrations are respectively a, 2.30208mg/ml, b, 2.92992mg/ml, c and 3.488mg/ml for later use; precisely measuring 0.5ml of Os Draconis and Concha Ostreae oral liquid (20191002 batches) with known content, placing into a 100ml measuring flask, dividing into three groups of A, B, C, correspondingly and precisely adding 1ml of the reference substance solution, adding 10ml of water and 5ml of dilute hydrochloric acid, shaking up, adding water to scale, shaking up, filtering, precisely measuring 2ml of subsequent filtrate, placing into a 25ml measuring flask, adding 1ml of lanthanum test solution, adding water to scale, and shaking up to obtain sample solution for determining recovery rate; the recovery rate was calculated according to the text sample content measurement method and is shown in tables 3-8. The results show that: the method has good recovery rate, the average recovery rate is 100.49%, and the relative standard deviation is 1.25%.

Table 3-8 calcium recovery assay results (n ═ 3)

(9) Range of

Ten samples of the Longmu Zhuanggu oral liquid were measured, and the results are shown in tables 3-9.

Table 3-9 table of calcium content measurement results (n ═ 3)

According to the above determination results, the calcium content of each unit of the product should not be less than 45.0 mg.

(10) Durability test

Instrument precision test

The control solution was taken and measured continuously and repeatedly for 6 times according to the measurement conditions, and the absorbance is shown in tables 3-10. The results show that the fine density of the instrument is good, and the relative standard deviation is 0.09%.

Tables 3-10 results of precision examination

② test of stability of test solution

The bone strengthening Longmu oral liquid sample (20191002 batches) is stored at room temperature, the absorption degree is measured at regular intervals, and the relative standard deviation is 0.12%. The results show that: the sample solution remained stable for 60 min. The results are shown in tables 3 to 11.

Tables 3-11 stability test results

[ CONTENT DETERMINATION ] 2 is the chemical vitamin D contained in the product₂The content of (3) is measured. Domestic vitamin D₂The content determination of (2) has been reported in the literature. The experiment aims at the traditional Chinese medicine taste and the process characteristics of the prescription, and adopts the high performance liquid chromatography to measure the vitamin D in the preparation₂Content measurement of (2)Feasibility study is carried out to achieve the purpose of controlling the quality of the product. A series of methodological investigation tests prove that the method is really feasible.

(1) Instruments and reagents

An HP-Agilent1100 high performance liquid chromatograph (comprising a vacuum degasser, a quaternary pump, an autosampler, an MWD detector and an HP 1100 chemical workstation);

dionex hplc (including quaternary pump, autosampler, UVD170U detector, Chromeleon chemical workstation);

Kromasil C₁₈(10nm 5 μm, 4.6X 250mm) chromatographic column, theoretical plate number is vitamin D₂Peak calculation should not be below 3000;

Hypersil BDS C₁₈(10 mu, ID 4.6X 250mm) chromatography column, theoretical plate number according to vitamin D₂Peak calculation should not be below 3000;

Diamonsil C₁₈(5 mu, 250 multiplied by 4.6mm) chromatography column, theoretical plate number according to vitamin D₂Peak calculation should be no less than 3000;

vitamin D₂A reference substance (for containing detection, purchased from China pharmaceutical biological product verification, with the batch number of 10155-;

methanol (chromatographically pure, purchased from Honeywell, usa); re-steaming water for the second time; the other reagents are analytically pure.

(2) Chromatographic conditions

A chromatographic column: octadecylsilane chemically bonded silica gel column

Mobile phase: methanol-water (97:3)

Detection wavelength: 265nm

Column temperature: at room temperature

Flow rate: 1.5ml/min

Sample introduction amount: 10 μ l

(3) Preparation of test solution

Operating according to a pharmacopoeia method: precisely measuring 50ml of the product, placing the product in a 150ml flask, adding 2.5g of potassium hydroxide and 0.03g of vitamin C, adding 25ml of absolute ethyl alcohol, heating and refluxing for 45 minutes in a 70 ℃ water bath, cooling, extracting for 3 times with 50ml of petroleum ether (30-60 ℃) by shaking, combining extracting solutions obtained in three times, recovering the petroleum ether in a 40 ℃ water bath under reduced pressure until the petroleum ether is dry, dissolving residues with the absolute ethyl alcohol, transferring the residues into a 25ml brown measuring flask, adding the absolute ethyl alcohol to scale, shaking up, filtering, and taking a subsequent filtrate to obtain the product.

Secondly, operating according to the text: filtering with microporous membrane (0.45 μm), and collecting the filtrate.

The test result shows that the sample solution prepared by the second method has more impurities, but does not influence the separation of the main peak, and the content is higher than that of the first method. Therefore, the method II is adopted to prepare the test solution, which is not only accurate, but also rapid, simple and convenient.

(4) Preparation of control solutions: the concentration of the control solution was adjusted to 0.005mg/ml based on the peak area of the test solution.

(5) Specificity test

Preparation of vitamin D deficiency₂The negative sample is prepared according to the preparation method of the test solution, 10 mu l of the negative sample solution is absorbed and injected into a liquid chromatograph for determination. The results show that: negative samples were not perturbed. (see FIG. 5)

(6) Investigation of Linear relationships

Precisely weighing vitamin D₂12.6mg of a reference substance is put into a 500ml brown measuring flask, absolute ethyl alcohol is added to dissolve the reference substance, the volume is fixed to the scale, and the reference substance is shaken up. Precisely sucking 1, 3, 5, 7 and 9ml of the reference substance solution respectively, placing the reference substance solution in a 25ml brown measuring flask, adding absolute ethyl alcohol to dilute the solution to a scale, shaking the solution uniformly, measuring according to the chromatographic conditions, and performing linear regression on the sample volume by peak integral area to obtain a regression equation, wherein the results are shown in tables 3 to 12: Y1.6206X +0.9332, r 0.99996. The results show that vitamin D₂The linear relation is good within the range of sample injection amount of 10.08-90.72 ng. (see FIG. 6)

Table 3-12 results of linear relationship examination (n ═ 3)

Sample volume (ng)	10.08	30.24	50.40	70.56	90.72
						Peak area (mAU)	17.671	49.477	82.194	115.909	147.815

(7) Precision test

Six samples (20191001 batches) of the same batch are repeatedly taken, operated according to a text content determination method and calculated. The results show that the method has good repeatability, and the relative standard deviation is 1.28%. (see tables 3-13)

Tables 3-13 results of precision examination

And operating and calculating three batches of samples according to the text content measuring method by using intermediate precision in the same laboratory on different dates and different instruments. The results show that the method herein has good intermediate precision. (see tables 3-14)

Tables 3-14 intermediate precision results (n ═ 3)

And thirdly, the reproducibility is verified by operating three batches of samples according to the text content measuring method through two laboratories. The measurement result shows that the method has good repeatability. (see tables 3-15)

Tables 3-15 reproducibility test results (n ═ 3)

(8) Accuracy test

Precisely weighing appropriate amount of vitamin D2 reference substance, adding anhydrous ethanol to obtain three reference substance solutions with different concentrations of a, 3.92 μ g/ml, b, 4.9 μ g/ml, c, and 5.88 μ g/ml respectively; precisely measuring 5ml of Os Draconis/Concha Ostreae oral liquid (20191001 batches) with known content, placing into 10ml measuring bottles, dividing into three groups of A, B, C, adding corresponding 5ml of the above reference solutions, adding anhydrous ethanol to desired volume, shaking, filtering with microporous membrane (0.45 μm), and collecting filtrate to obtain sample solution for determining recovery rate; the content of the sample is determined according to the text sample content determination method, and the recovery rate is calculated and shown in tables 3-16. The results show that: the method has good recovery rate, the average recovery rate is 97.81 percent, and the relative standard deviation is 1.19 percent.

Table 3-16 vitamin D2 recovery assay results (n ═ 3)

(9) Range of

Ten samples of the Longmu Zhuanggu oral liquid were measured, and the results are shown in tables 3-17.

Tables 3-17 vitamin D₂Table of contents measurement results (n ═ 3)

Determining the vitamin D content of each unit according to the above determination results₂(C₂₈ H₄₄ O)Not less than 36. mu.g.

(10) Durability test

Selection of detection wavelength

By the action of vitamin D₂The control solution was UV scanned and had an absorption maximum at 265. + -.2 nm (see FIG. 7), so 265nm was chosen as the measurement wavelength.

② comparison of different columns

The content of the same batch of samples (20191001 batches) is measured by adopting different chromatographic columns according to the text content measurement method, and the relative standard deviation of the result is 0.82 percent, which shows that the content measurement of the product has no special selectivity to the chromatographic columns. (see tables 3-18) comparison of different columns in tables 3-18 (n ═ 3)

③ comparison of different flow rates

The same column (Kromasil C) was used₁₈) The same batch of samples (20191001 batches) was subjected to assay at different flow rates according to the text assay method with a relative standard deviation of 0.51% (see tables 3-19), indicating that flow rates had no significant effect on assay results and that 1.5ml/min flow rates were used for analysis in order to save assay time.

Tables 3-19 comparison of different flow rates (n ═ 3)

Instrument precision test

Precisely absorbing the reference substance solution, continuously and repeatedly injecting sample for 6 times according to the chromatographic condition, wherein the integral area is shown in a table 3-20. The results show that the precision of the instrument (HP-Agilent1100) is good with a relative standard deviation of 0.46%.

Tables 3-20 results of precision examination

Test of stability of test solution

The bone-strengthening Longmu oral liquid sample (20191001 batches) solution was stored at room temperature, and peak areas were measured every 2 hours or more, with a relative standard deviation of 0.52%. The results show that: the sample solution remained stable for 24 hours. The results are shown in tables 3 to 21.

Tables 3-21 stability test results

(11) Vitamin D₂The content determination of (A): vitamin D according to the text₂The method for measuring the content of (1).

(III) indications of function

Is consistent with the standard of Longmu Zhuanggu granules.

(IV) administration and dosage

Is administered orally. 10ml for the first time under the second year of age, 14ml for the second to seventh years of age, and 20ml for the first time above the seventh year of age; the preparation is administered 3 times a day. The medicine amount taken every day is the same as the Longmu Zhuanggu granules.

(V) specification

The specification of the product is set to 10ml per bag according to the pilot test results.

(VI) storage

Because the product is oral liquid, the product does not need to be placed in a dry place, namely: sealing, and placing in shade.

Claims

1. A traditional Chinese medicine oral liquid for treating infantile rickets is characterized by being prepared from the following raw materials in parts by weight:

the preparation method comprises the following steps:

vitamin D₂Clathrating with beta-cyclodextrin; decocting radix Codonopsis, radix astragali, radix Ophiopogonis, carapax et Plastrum Testudinis, Atractylodis rhizoma, rhizoma Dioscoreae, fructus Schisandrae chinensis, Os Draconis, Concha Ostreae, Poria, fructus Jujubae, Glycyrrhrizae radix, and endothelium corneum Gigeriae Galli with water for three times, mixing decoctions, filtering, standing, concentrating the supernatant to relative density of 1.05-1.15, cooling, adding ethanol to ethanol content of 50-70%, standing, collecting the supernatant, recovering ethanol under reduced pressure, concentrating to relative density of 1.15-1.25 to obtain extract; collecting calcium lactate, calcium gluconate, and vitamin D₂Boiling the clathrate in water, adding polysorbate 80 and the above extract, cooling, adding potassium sorbate, aspartame, essence, adding water to 1000 volume, filtering, bottling, sterilizing,

the weight is related to the volume in g/ml.

2. The traditional Chinese medicine oral liquid for treating rickets in children of claim 1, which is characterized in that: the carapax et Plastrum Testudinis and fructus Schisandrae are processed with vinegar.

3. The traditional Chinese medicine oral liquid for treating rickets in children of claim 1, which is characterized in that: the rhizoma Atractylodis Macrocephalae and endothelium corneum Gigeriae Galli are parched.

4. The traditional Chinese medicine oral liquid for treating rickets in children of claim 1, which is characterized in that: the Os Draconis and Concha Ostreae are calcined.

5. The traditional Chinese medicine oral liquid for treating rickets in children of claim 1, which is characterized in that: the water is added for decoction for three times, wherein the water is added for 2 hours for the first time and the second time and is added for 1 hour for the third time, and the water adding amount for each time is 8 times of the weight of the medicinal materials.

6. The traditional Chinese medicine oral liquid for treating rickets in children of claim 1, which is characterized in that: the alcohol content is 60%.

7. The treatment child of claim 1The traditional Chinese medicine oral liquid for treating the rachitis is characterized in that: the inclusion conditions are: stirring at 60 ℃, clathrating for 3 hours, wherein the weight ratio of beta-cyclodextrin to 60% ethanol is 1: 12-1: 16, and the beta-cyclodextrin and VitD₂The molar ratio of (A) to (B) is 7: 1.

8. The traditional Chinese medicine oral liquid for treating rickets in children of claim 1, which is characterized in that: the packaging material used for the potting was a brown finger-top bottle.

9. A method for detecting the Chinese medicinal oral liquid for treating rickets in children according to any one of claims 1 to 8, which is characterized by comprising the following steps:

[ IDENTIFICATION ] collecting 60ml of the product, extracting with water saturated n-butanol under shaking for 3 times (20 ml each time), mixing n-butanol solutions, washing with 1% sodium hydroxide solution for 3 times (20 ml each time), discarding the alkali solution, washing with n-butanol saturated water to neutrality, evaporating n-butanol solution on water bath, and dissolving the residue with 0.5ml of methanol to obtain a sample solution. Adding methanol into astragaloside IV control to obtain solution containing 0.5mg per 1ml, and making into control solution. According to a test of thin layer chromatography (appendix VIB of the first part of the national pharmacopoeia 2015 edition), sucking 10 mu l of each of the two solutions, respectively dropping the two solutions on the same silica gel G thin layer plate, taking a lower layer solution of chloroform-ethyl acetate-methanol-water (10:20:11:5) which is placed at the temperature of below 10 ℃ overnight as a developing agent, developing, taking out, airing, spraying with a 10% sulfuric acid ethanol solution, and heating at 105 ℃ until spots are clearly developed. In the chromatogram of the test solution, spots with the same color appear in the corresponding positions of the chromatogram of the reference solution under sunlight; fluorescent spots with the same color appear under an ultraviolet lamp (365 nm).

(2) Collecting 40ml of the product, extracting with water saturated n-butanol under shaking for 3 times (20 ml each time), mixing n-butanol solutions, washing with water for 2 times (20 ml each time), evaporating n-butanol solution on water bath, and dissolving the residue with 1ml of acetone to obtain sample solution. Taking 0.5g of rhizoma Atractylodis Macrocephalae as reference material, and making into reference material solution by the same method. Performing thin-layer chromatography (appendix VI B of the first part of the national pharmacopoeia 2015 edition), collecting 15 μ l of the sample solution and 10 μ l of the reference solution, respectively dropping on the same silica gel G thin-layer plate, developing with chloroform-acetone (19:1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution.

(3) Extracting 60ml of the product with diethyl ether for 2 times (20 ml each time), discarding diethyl ether solution, extracting water solution with water saturated n-butanol under shaking for 5 times (20 ml each time), mixing n-butanol solutions, washing with water for 5 times (20 ml each time), evaporating n-butanol solution on water bath, and dissolving residue with 1ml of methanol to obtain sample solution. And adding water 10ml into Glycyrrhrizae radix control 0.5g, ultrasonic treating for 15 min, filtering, collecting filtrate, and making into control solution by the same method. Performing thin layer chromatography (appendix VI B of the first part of the 2015 edition of Chinese pharmacopoeia), sucking 10 μ l of the test solution and 1 μ l of the control solution, respectively dropping on the same silica gel G thin layer plate prepared from 1% sodium hydroxide solution, developing with the upper solution of ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2) as developing agent, taking out, air drying, spraying with 10% sulfuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. In the chromatogram of the test solution, spots of the same color appear in the corresponding positions of the chromatogram of the reference solution under sunlight.

the content of calcium (Ca) in each branch should not be less than 45.0 mg.

Vitamin D₂Measured according to high performance liquid chromatography (appendix VID of the first part of the 2015 edition of Chinese pharmacopoeia).