CN113384632B - A composition with tranquilizing effect and its preparation method - Google Patents

Abstract

The invention discloses a composition with a nerve soothing effect, which is prepared from the following raw materials in parts by mass: 10 to 20 portions of epimedium, 10 to 20 portions of malaytea scurfpea fruit, 7 to 13 portions of dragon bone, 10 to 20 portions of spina date seed, 10 to 20 portions of Chinese magnoliavine fruit and 10 to 20 portions of south dodder seed. The invention has the functions of warming kidney and replenishing vital essence, calming liver and suppressing yang, and nourishing liver and soothing nerves, and is commonly used for insomnia and amnesia caused by kidney yang deficiency, and symptoms comprise liver and kidney deficiency, dizziness and tinnitus, insomnia and dreaminess, waist soreness and neurasthenia.

Description

A composition with tranquilizing effect and its preparation method

Technical Field

The invention particularly relates to a composition with a nerve soothing effect and a preparation method thereof

Background

Modern people have fast pace of life, unhealthy eating habits, high social pressure, insufficient sleep, overstrain and illness, and easily hurt the liver and kidney, so that immunity is reduced, people mostly improve the subhealthy body through medicines or health care products with the function of regulating the human body, but the medicines are toxic in three parts, the side effect of the medicines is obvious, the medicines cannot be used for a long time, and corresponding health care products in the current market mostly have single functions of supplementing vitamins, collagen, bee products and the like, and the effect is not obvious.

The medicinal liquor has a long history of application in China, and is a health preserving method integrating treatment and health preserving and health care. The medicinal and edible wine is a medicinal and dietary beverage, and is a type of medicinal and dietary. The traditional Chinese medicine medicated diet is formed by organically combining traditional Chinese medicines and food materials according to a certain theory and principle, has the effects of food nourishing and food therapy, is not only food, but also different from common food, has the functions of promoting weight and congenital advantages on health care and disease prevention and treatment, and accords with the big health concept of eliminating sub-health, improving physical quality, and well managing and maintaining health.

At present, no medicinal liquor taking roasted epimedium and dragon bones as main medicines is used for warming kidney, replenishing vital essence, calming liver, suppressing yang hyperactivity, nourishing liver and soothing nerves.

Disclosure of Invention

In order to solve the problems, the invention provides a composition with a nerve soothing effect, which is prepared from the following raw materials in parts by weight:

10 to 20 portions of epimedium, 10 to 20 portions of malaytea scurfpea fruit, 7 to 13 portions of dragon bone, 10 to 20 portions of spina date seed, 10 to 20 portions of Chinese magnoliavine fruit and 10 to 20 portions of south dodder seed.

Further, the material is prepared from the following raw materials in proportion:

15 parts of epimedium, 15 parts of malaytea scurfpea fruit, 10 parts of dragon bone, 15 parts of spina date seed, 15 parts of Chinese magnoliavine fruit and 15 parts of south dodder seed.

Further, the keel is forged keel; the herba Epimedii is processed herba Epimedii.

Furthermore, the preparation is prepared by taking powder of the raw materials, or water or organic solvent extract of the raw materials as active ingredients and adding pharmaceutically acceptable auxiliary materials.

Further, the preparation is an oral preparation, and the oral preparation is a tincture, a paste or a wine, preferably a wine.

Further, the wine is prepared from the following raw materials in proportion:

15 parts of epimedium, 15 parts of fructus psoraleae, 10 parts of keel, 15 parts of spina date seed, 15 parts of schisandra chinensis, 15 parts of semen cuscutae, 30 parts of honey, 1 part of citric acid and 1000 parts of wine by volume.

Further, the wine is 35-52 degrees of white spirit, and 48 degrees of white spirit is preferred.

The invention also provides a preparation method of the composition, which comprises the following steps:

(1) Weighing the raw materials according to the proportion;

(2) Pulverizing fructus Psoraleae, semen Ziziphi Spinosae, fructus Schisandrae chinensis and semen Cuscutae into coarse powder, adding processed herba Epimedii and Os Draconis preparata, mixing, adding Chinese liquor, soaking for 10-20 days, and stirring once a day;

(3) Taking the soaked solution, filtering, standing, adding the rest Chinese liquor, adding Mel and citric acid, mixing, and filtering.

The invention also provides application of the composition in preparation of medicines for warming kidney, replenishing vital essence, calming liver, suppressing yang hyperactivity and/or nourishing liver and soothing nerves.

Furthermore, the medicine is used for treating kidney-yang deficiency type insomnia, amnesia, liver and kidney deficiency, dizziness, tinnitus, insomnia, dreaminess, waist soreness and neurasthenia.

In the pharmaceutical composition, the monarch drug roasted epimedium has the effects of tonifying kidney yang, strengthening muscles and bones, dispelling wind-damp, mainly contains biological activities such as flavonoid, polysaccharide, volatile oil, alkaloid and the like, and has various effects of resisting osteoporosis, tumors and oxidation, treating diseases of a blood system, a nervous system, an immune system and the like; the calcined dragon bone as a ministerial drug is an animal with blood and meat emotion, has the effects of relieving palpitation and calming the nerves, calming the liver and suppressing yang, mainly contains calcium hydroxy phosphate, and also contains a small amount of calcium carbonate and elements such as iron, aluminum, magnesium, manganese, strontium and the like, and has the pharmacological effects of regulating immunity, resisting convulsion, calming, hypnotizing and the like; the ministerial medicine fructus psoraleae has the effects of tonifying kidney and strengthening yang, and securing essence and reducing urination, mainly comprises compounds such as coumarin, flavone, monoterpene phenol, benzofuran and the like, and has the effects of resisting tumor activity, treating osteoporosis, and resisting oxidation; semen Cuscutae is used as adjuvant for nourishing liver and kidney, arresting spontaneous emission and reducing urination, and mainly comprises flavone, polysaccharide, sterols and alkaloids, and has pharmacological effects in improving immunity, resisting aging, nourishing nerve, and protecting liver. The assistant medicine schisandra fruit has five chemical components including lignin, volatile oil, polysaccharide, organic acid, etc. for astringing, benefiting vital energy, promoting the secretion of saliva or body fluid, invigorating kidney and calming heart. Has good effects of resisting inflammation and oxidation, relieving insulin resistance, and inhibiting angiopathy. The semen Ziziphi Spinosae has effects in nourishing liver, calming heart, tranquilizing mind, arresting sweating, containing flavonoids, saponins, alkaloids, and terpenoids, and has effects in tranquilizing mind, improving sleep, relieving anxiety, resisting depression, relieving inflammation, protecting cardiovascular system, protecting liver, and enhancing immunity.

The medicines are combined to play the roles of tranquilizing and allaying excitement, nourishing liver and kidney, strengthening mind and replenishing vital essence. The wine prepared by the composition of the invention has the functions of warming kidney, replenishing vital essence, calming liver, suppressing yang hyperactivity, nourishing liver, soothing nerves and regulating body functions, and is used for treating liver and kidney deficiency, dizziness, tinnitus, insomnia, dreaminess, waist soreness and neurasthenia. The test proves that: the main components of the medicinal ingredients in the composition have better dissolution rate in wine, the wine is used for extracting and dissolving coal, the effect is quicker, the effect is better, and meanwhile, the medicinal liquor has mellow taste and high public acceptance degree, and is easy to popularize and use in the market.

It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and common practice in the field.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

The specificity of the extractive solution is examined in FIG. 1 (upper left picture test solution, upper right picture control solution, lower left picture moxibustion herba Epimedii negative test solution, lower right picture diluent methanol)

FIG. 2 standard curve chart of prepared icariin as extractive solution

FIG. 3 identification chart of roasted herba Epimedii by thin layer chromatography

FIG. 4 identification chart of fructus Psoraleae by thin layer chromatography

FIG. 5 identification diagram of semen Cuscutae by thin layer chromatography

FIG. 6 identification chart of fructus Schisandrae by thin layer chromatography

FIG. 7 thin-layer chromatography identification chart for wild jujube seeds

FIG. 8 Standard Curve of fossil fragments tranquilization wine icariin

FIG. 9 investigation of specificity of fossil fragments soothing wine (upper left test solution, upper right control solution, lower left test solution, and lower right diluent methanol)

Detailed Description

Example 1 preparation of medicated wine of the invention

The formula is as follows: prepared epimedium 15g, psoralea fruit 15g, calcined dragon bone 10g, wild jujube seed 15g, schisandra fruit 15g, dodder seed 15g, honey 30g, citric acid 1g,1000ml 48 degree white spirit

The preparation method comprises the following steps:

1) Pulverizing fructus Psoraleae, semen Ziziphi Spinosae, fructus Schisandrae chinensis and semen Cuscutae into coarse powder, adding processed herba Epimedii and Os Draconis preparata, mixing, adding 48 ° Chinese liquor, soaking for 10 days, and stirring once a day;

2) Filtering the soaked solution, standing for 8h, supplementing lost Chinese liquor, adding Mel and citric acid, mixing, and filtering with 200 mesh gauze.

Example 2 preparation of medicated wine of the invention

The formula is as follows: prepared epimedium 15g, psoralea fruit 15g, calcined dragon bone 10g, wild jujube seed 15g, schisandra fruit 15g, dodder seed 15g, honey 30g, citric acid 1g,1000ml 35 degree white spirit

The preparation method comprises the following steps:

1) Pulverizing fructus Psoraleae, semen Ziziphi Spinosae, fructus Schisandrae chinensis and semen Cuscutae into coarse powder, adding processed herba Epimedii and Os Draconis preparata, mixing, adding 35 ° Chinese liquor, soaking for 20 days, and stirring once a day;

2) Filtering the soaked solution, standing for 8h, supplementing lost Chinese liquor, adding Mel and citric acid, mixing, and filtering with 200 mesh gauze.

Example 3 preparation of medicated wine of the invention

The formula is as follows: herba Epimedii 15g, fructus Psoraleae 15g, os Draconis preparata 10g, semen Ziziphi Spinosae 15g, fructus Schisandrae 15g, semen Cuscutae 15g, mel 30g, citric acid 1g,1000ml 32 ° Chinese liquor

The preparation method comprises the following steps:

1) Pulverizing fructus Psoraleae, semen Ziziphi Spinosae, fructus Schisandrae chinensis and semen Cuscutae into coarse powder, adding processed herba Epimedii and Os Draconis, mixing, adding 32 ° Chinese liquor, soaking for 8 days, and stirring once a day;

2) Taking the soaked solution, filtering, standing for 8h, supplementing lost Chinese liquor, adding Mel and citric acid, mixing, and filtering with 200 mesh gauze.

Example 4 preparation of medicated wine of the invention

The formula is as follows: herba Epimedii 10g, fructus Psoraleae 10g, os Draconis preparata 7g, semen Ziziphi Spinosae 10g, fructus Schisandrae 10g, semen Cuscutae 10g, mel 21g, citric acid 0.5g,1000ml 48 degree Chinese liquor

The preparation method comprises the following steps:

1) Pulverizing fructus Psoraleae, semen Ziziphi Spinosae, fructus Schisandrae chinensis and semen Cuscutae into coarse powder, adding processed herba Epimedii and Os Draconis, mixing, adding 48 ° Chinese liquor, soaking for 8 days, and stirring once a day;

2) Taking the soaked solution, filtering, standing for 8h, supplementing lost Chinese liquor, adding Mel and citric acid, mixing, and filtering with 200 mesh gauze.

Example 5 preparation of medicated wine of the invention

The formula is as follows: 20g of roasted epimedium herb, 20g of malaytea scurfpea fruit, 13g of calcined dragon bone, 20g of spina date seed, 20g of Chinese magnoliavine fruit, 20g of south dodder seed, 39g of honey, 2g of citric acid and 1000ml of 52-degree white spirit

The preparation method comprises the following steps:

1) Pulverizing fructus Psoraleae, semen Ziziphi Spinosae, fructus Schisandrae chinensis and semen Cuscutae into coarse powder, adding processed herba Epimedii and Os Draconis, mixing, adding 52 ° Chinese liquor, soaking for 8 days, and stirring once a day;

2) Filtering the soaked solution, standing for 8h, supplementing lost Chinese liquor, adding Mel and citric acid, mixing, and filtering with 200 mesh gauze.

The advantageous effects of the present invention will be described below by way of test examples.

Test example 1 preparation Process study

1. Process route design and pre-test

1. Design of process route

Purified solution of 6 Chinese medicinal filtrates such as herba Epimedii and Os Draconis → cold soaking → filtering, removing impurities → flavoring, blending → semi-finished product → encapsulating, sterilizing → finished product

2. Preliminary test

Weighing decoction pieces according to a formula proportion, namely taking 85g of roasted epimedium herb, 10g of calcined dragon bone, 15g of fructus psoraleae, 15g of semen cuscutae, 15g of schisandra chinensis and 15g of spina date seeds, adding 1000mL of white spirit to soak for 30 days, stirring once per day, filtering, standing, adding the white spirit to 1000mL, adding 30g of honey, mixing uniformly, filtering, and subpackaging to obtain 1000mL of medicinal liquor.

As a result: the wine can be prepared according to the drawn-up process soaking route, and has good clarity condition, and basically meets the requirements of wine.

3. Feasibility analysis

The roasted epimedium herb in the composition is a monarch drug, wherein the flavonoid is a main active ingredient, and the icariin is a main active ingredient; in the 'Chinese pharmacopoeia' of 2020 edition, epimedin A, epimedin B and epimedin C are also included in the quality control indexes. The four flavonoid components are easily dissolved in wine, and have high transfer rate, so epimedin A, epimedin B, epimedin C and icariin are selected as content determination indexes of the keel soothing wine. According to the preparation condition of the wine and the content measurement result of the pre-test wine, the wine can be prepared according to the drawn process route and the curative effect of the preparation can be ensured.

2. Identification and pretreatment of decoction pieces

1. Identification of medicinal materials

Roasting epimedium: the product is dried leaf of Epimedium brevicomu Maxim belonging to berberidaceae. The product is purchased from Limited liability company of traditional Chinese medicine decoction pieces in Sichuan province, and conforms to the relevant regulation of decoction pieces, namely roasted epimedium, under the item of epimedium in 2020 edition.

Calcining keel: the product is bone fossil of ancient mammals, such as three-toe horse, rhinoceros, deer, cattle, elephant, etc. The product is purchased from Limited liability company of decoction pieces of traditional Chinese medicine in Sichuan province, and conforms to the related regulation of decoction pieces-calcined Os Draconis under the item of Os Draconis in 1977 of Chinese pharmacopoeia.

Fructus psoraleae: the product is dried mature fruit of Psoralea corylifolia L. The product is purchased from Chinese medicinal decoction pieces Limited liability company in Sichuan province, and conforms to the relevant regulation of the decoction pieces-fructus Psoraleae under the item of fructus Psoraleae in 2020 edition of Chinese pharmacopoeia.

Dodder seed: the product is dried mature seed of Cuscuta chinensis Lam of Convolvulaceae. The product is purchased from Limited liability company of Chinese medicinal decoction pieces in Sichuan province, and conforms to the relevant regulation of semen Cuscutae (decoction pieces under the term of semen Cuscutae in 2020 edition of Chinese pharmacopoeia).

Schisandra chinensis: the product is dried mature fruit of Schisandra chinensis (Thunb.) Kaltz.) Baill. The product is purchased from Chinese medicinal decoction pieces Limited liability company of Sichuan province, and conforms to the relevant regulation of decoction pieces-fructus Schisandrae chinensis under the item of fructus Schisandrae chinensis in 2020 edition of Chinese pharmacopoeia.

And (2) spina date seed: the product is dry mature seed of Ziziphus jujuba Mill.var. Spinosa (Bunge) Hu ex H.F.Chou belonging to Rhamnaceae. The product is purchased from Chinese medicinal decoction pieces Limited liability company in Sichuan province, and conforms to the related regulation of the decoction pieces, namely the spina date seed, under the item of the spina date seed in the 2020 edition of Chinese pharmacopoeia.

2. Pretreatment of medicinal materials

Roasting epimedium: removing impurities, cleaning and drying.

Psoralea fruit: removing impurities, cleaning, pulverizing, and drying.

And (3) semen cuscutae: removing impurities, cleaning and drying.

Schisandra chinensis: removing impurities, cleaning, pulverizing, and drying.

Wild jujube seed: removing impurities, cleaning, pulverizing, and drying.

3. Research on extraction Process

1. Water absorption measurement

Weighing three parts of medicinal materials according to the proportion of a prescription, wherein the three parts are 42.57g, 42.51g and 42.50g respectively, adding 500mL of white wine, soaking until the mixture is permeated through the core, filtering, measuring the filtrates to be 412mL, 417mL and 410mL in sequence, calculating the water absorption rate, and the total wine absorption rates of the medicinal materials are 207.06 percent, 195.25 percent and 211.76 percent respectively, the average is 204.67 percent and is about 2 times.

2. Screening of soaking process conditions

2.1 evaluation index and measurement method

The total content (i.e. total flavone) and dry extract rate of epimedin A, epimedin B, epimedin C and icariin are used as evaluation indexes to carry out comprehensive scoring, thereby optimizing the soaking extraction process.

2.2 determination of content of epimedin A, epimedin B, epimedin C and icariin

(1) Materials: model e-2695 high performance liquid chromatograph (Voltset, inc., USA), model 2489 UV/Vis detector (Voltset, inc., USA), model BT-125D electronic analytical balance (one hundred thousandth, sadolis scientific instruments (China), inc.). Reagent: acetonitrile and phosphoric acid are chromatographically pure, water is self-made purified water, and other reagents are analytically pure. Comparison products: icariin (batch No. 110737-201516, purity 94.2%) was purchased from the Chinese food & drug testing institute.

(2) Chromatographic conditions are as follows: agilent Zorbax SB-C18 chromatography column (250 mm. Times.4.6 mm,5 μm); the mobile phase was acetonitrile (a) and water (B), and gradient elution (0-30min, 24% → 26% a, 30-35min, 26% → 26% a); the volume flow is 1.0mL/min; the detection wavelength is 270nm; the column temperature is 30 ℃; sample introduction volume: 10 μ L. According to the 2020 version of Chinese pharmacopoeia, the content of epimedin A, epimedin B, epimedin C and icariin is calculated by taking an icariin reference substance as a reference and taking the corresponding peak as an S peak and multiplying the peak by a correction factor respectively, and the relative retention time and the correction factor are shown in the following table 1.

TABLE 1 relative retention time and correction factor between the four components of moxibustion herba Epimedii

(3) Preparation of control solutions: accurately weighing icariin 15.30mg, placing in a 25mL measuring flask, adding methanol to dissolve, fixing volume, and shaking to obtain icariin control stock solution (concentration of 0.6120 mg/mL).

(4) Preparing a test solution: shaking the keel tranquilizing wine sample evenly, precisely sucking a proper amount of the keel tranquilizing wine sample, filtering the mixture by using a filter membrane of 0.45 mu m, and taking a subsequent filtrate to obtain the keel tranquilizing wine.

(5) Special attribute investigation: weighing 70g of the negative prescription of the moxibustion-deficient epimedium according to the prescription proportion, adding 1000mL of white spirit to soak for 30 days, stirring once a day, filtering, standing, adding the white spirit to 1000mL to obtain the moxibustion-deficient epimedium negative test solution, and preparing the moxibustion-deficient epimedium negative test solution according to the preparation method of the test solution. Taking reference solution, test solution, and the prepared herba Epimedii negative test solution, and diluent methanol, performing sample injection measurement under the above chromatographic conditions, and recording chromatogram, the result is shown in figure 1. According to results, the negative sample has no interference at the corresponding chromatographic peak position and has strong specificity.

(6) Linear investigation: precisely sucking 0.5ml, 1ml, 2ml, 3 ml, 4 ml and 5ml of icariin, respectively placing into 6 10ml volumetric flasks, adding methanol for dilution, fixing volume, and shaking to obtain series concentration reference solutions. And then, carrying out sample injection measurement according to the chromatographic conditions, recording peak areas, drawing a standard curve, and calculating an r value. As is clear from the results in Table 2 and FIG. 2, the icariin concentration range of 30.6 to 306.0ug/ml is in good linear relationship (r.gtoreq.0.9991).

TABLE 2 Linear relationship test results

(7) Precision investigation: taking control solution (line 3, containing icariin 122.4ug/ml per 1 ml), measuring for 6 times by continuous injection according to the above chromatographic conditions, recording chromatogram, and measuring icariin peak area of each injection, and the result is shown in Table 3. From the results, it was found that icariin showed less than 0.91% of RSD in terms of peak area and retention time, and the results indicated that the apparatus was accurate.

TABLE 3 results of precision test

(8) And (3) stability investigation: taking the extractive solution of the preliminary experiment, preparing the sample solution according to the preparation method of the sample, carrying out sample injection measurement according to the chromatographic conditions after 0, 2, 4, 6, 8, 12 and 24 hours. Taking control solution (line 3, each 1ml containing icariin 122.4 ug/ml) at 0, 2, 4, 6, 8, 12, and 24 hr, and injecting sample according to the above chromatographic conditions. The icariin peak area was recorded for each injection and the results are shown in table 4. The results show that the stability of icariin is less than 3.0 in terms of peak area, and the results show that the stability of the test solution and the control solution is good within 24 hours.

TABLE 4 stability study

(9) The determination method comprises the following steps: respectively and precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and calculating content by external standard method.

2.3 determination of Dry extract Rate

Precisely sucking 10mL of each orthogonal sample solution, respectively placing the orthogonal sample solutions into evaporation dishes which are dried to constant weight, evaporating the orthogonal sample solutions in a water bath to dryness, drying the orthogonal sample solutions for 3 hours at 105 ℃, placing the orthogonal sample solutions into a dryer to cool for 30 minutes, rapidly and precisely weighing the orthogonal sample solutions, and calculating the yield of dry paste.

2.4 Single factor investigation

(1) Examination of base liquor concentration: weighing 6 medicines of roasted epimedium herb, dragon bone and the like according to the proportion of the prescription, wherein the total amount is 42.5g and 5 parts. Adding 500mL of Chinese liquor with concentration of 35 °, 38 °, 42 °, 48 °, 52 °, respectively, soaking for 15 days, filtering, diluting to 500mL, and determining total flavone content. The determination result is shown in 5, and the content of the total flavone is gradually increased along with the increase of the concentration when the concentration of the base wine is 35-48 degrees, and the trend is obvious; the concentration of the base wine is 48-52 degrees, and the concentration is gradually reduced. Therefore, three levels of 42 degrees, 48 degrees and 52 degrees of the base wine are selected for carrying out soaking extraction process tests.

TABLE 5 base liquor concentration survey

(2) Investigation of liquid-material ratio: weighing 6 medicines of the roasted epimedium herb, the dragon bone and the like according to the proportion of the formula, wherein the total amount is 42.5g. Adding the mixture into a reactor in a volume ratio of 1: 6. 1: 8. 1: 10. 1: 12. 1: soaking in 14% (v/v) 48% (v/v) Chinese liquor for 15 days, filtering, and determining total flavone content. The measurement results are shown in 6, and it is found that when the liquid-to-material ratio is 1: 6-1: 12 hours, the content of the total flavonoids is gradually increased along with the increase of the concentration; the liquid-material ratio is 1:12 to 1:14, the content of the index component is reduced. In the aspects of saving raw materials and reducing experimental steps, the liquid-material ratio is selected to be 1: 8. 1, 10 and 1.

TABLE 6 investigation of liquid to material ratio

(3) Investigation of the number of days of soaking: weighing 6 medicines of roasted epimedium herb, dragon bone and the like according to the proportion of the prescription, wherein the total amount is 42.5g and 5 parts. Adding 600mL of 48 ° Chinese liquor, soaking for 10 days, 15 days, 20 days, 25 days, and 30 days, respectively, filtering, and determining total flavone content. The measurement results are shown in Table 7, and it is understood that the total flavone content gradually decreases with the increase of the soaking days, and the trend is slow. Therefore, three levels of 10 days, 15 days and 20 days of soaking are selected for carrying out soaking extraction process tests.

Table 7 immersion days study

(4) As a result: selecting three levels of base liquor with the concentrations of 42 degrees, 48 degrees and 52 degrees, wherein the liquid-material ratio is 1: 8. 1: 10. 1: three levels of 12, namely 10 days, 15 days and 20 days are considered in the test.

2.5 optimal Process screening

(1) Test method

Weighing 6 medicines of roasted epimedium herb, dragon bone and the like according to the proportion of the formula, wherein the total amount is 42.5g and 9 parts. Extracting according to the conditions of the experimental design table, and filtering for later use. The soaking extraction method is adopted, the levels of three factors are selected according to the single-factor test result, the test is carried out according to the table 8, and the test result is shown in the table 9.

TABLE 8 soaking extraction design factor levels

(2) Test results

The results of content determination and dry paste rate are shown in Table 9.

Table 9 test design and results

Overall evaluation = total flavone content 70/18.81+ dry extract yield 30/12.68

And (4) analyzing results: as can be seen from the visual analysis of Table 9, test 5 had the highest score and the process conditions to be selected and optimized were A ₂ B ₂ D ₁ Namely adding base wine with the alcohol content of 48 degrees and the liquid-material ratio of 1.

2.6 validation test

In order to verify the screening results and to ensure the reasonability and reliability of the extraction process, the process conditions are further determined. Weighing about 425g of tablets according to the proportion of the prescription, weighing 3 parts in total, extracting according to the analytical and predicted process conditions (the base alcohol degree is 48 degrees, the liquid-material ratio is 1. The verification test result shows that: the process is stable and feasible, so that the alcohol content of the base wine is determined to be 48 degrees, the liquid-material ratio is 1.

Table 10 verification of process results

3. Study of purification Process

The extracted extract has impurities which are difficult to meet the clarity requirement of the wine and need to be purified. The liquid medicine for verification of 2.6 verification test and 3 times is taken, mixed and refrigerated for standby use, and is used as the liquid medicine for purification, concentration and examination.

The experiment mainly inspects 3 common purification modes of standing filtration, centrifugation and activated carbon adsorption. Standing and filtering the liquid medicine: taking 100mL of the verified mixed liquid medicine, standing overnight, pouring out the supernatant, filtering with 200-mesh gauze, and adding wine to a constant volume of 100mL. Centrifuging the liquid medicine: taking 100mL of the verified mixed liquid medicine, centrifuging at 6000r/min for 10mins, pouring out the supernatant, and adding wine to a constant volume of 100mL. Activated carbon adsorption: taking 100mL of verified mixed liquid medicine, adding 0.5g of active carbon, shaking uniformly, standing overnight, filtering, adding wine to a constant volume of 100mL. Taking the above verified mixed medicinal liquid (stock solution), standing, filtering, centrifuging, adsorbing with active carbon, and determining total flavone content and dry extract rate by the method under item "2.2 and 2.3", with the experimental results shown in Table 11. The results show that the stock solution has a small amount of impurities, the standing and filtering liquid medicine, the centrifugal liquid medicine and the alcohol precipitation liquid medicine are basically clear, and only a small amount of precipitates which are scattered by slight shaking are obtained, so that the requirement of purifying the wine is met. After standing, filtering or centrifuging, the content of total flavone and the dry paste rate are not obviously changed compared with the original solution, and each index is slightly reduced after the adsorption treatment of the active carbon. The active carbon adsorption does not belong to the traditional process of traditional Chinese medicine preparations, and the liquid medicine obtained by the standing filtration method has high clarity, low cost and short time consumption, so the standing filtration method is supposed to be used for purifying the extracting solution.

TABLE 11 clarification Process

4. Research on molding process

4.1 preparation of Molding Process extract

Weighing about 1700g of drinking tablets (100 daily clinical prescriptions), adding 10 times of 48-degree white spirit, soaking for 10 days, stirring once per day, filtering, standing, pouring out supernatant, filtering with 200-mesh gauze, adding white spirit to a constant volume of 20L to obtain the molding process investigation extract.

4.2 formulation design of the preparation

(1) Dose screening of finished product

The feasibility of making 200mL and 100mL of wine was investigated for each daily clinical formula (17 g). 200mL of the extract (1 daily clinical prescription containing 17.0g of crude drug) is taken for forming process investigation, and concentration is continued until the concentration reaches 100mL, so that the wine is turbid and has poor fluidity after 100mL of the liquid medicine is prepared. According to the test results, the clinical prescription amount of the preparation for one day is determined by combining the actual production efficiency and the clinical use consideration, namely 17.0g of decoction pieces are prepared into 200mL of medicinal liquor.

(2) Screening of type and dosage of corrective

In order to obtain higher transfer rate of effective components of the dragon bone tranquilizing wine, base wine with 48 percent of volume fraction is selected to extract medicinal materials in the production process conditions, and the dragon bone tranquilizing wine obtained under the conditions has higher alcohol degree and bitter taste and cannot meet the market demand. The flavor of the dragon bone soothing wine needs to be optimized, and flavoring agents such as citric acid, honey and the like are added. Through preliminary experiments, the level ranges of the design factors are determined to be 10-50 g/L of honey concentration (A) and 1.0-5.0 g/L of citric acid (B), sensory evaluation is carried out, the scores of appearance, color and aroma respectively account for 20 points, the style is 10 points, the taste is 30 points, the design scheme is shown in a table 12, and the sensory evaluation result is shown in a table 13.

Table 12 taste test design scheme

TABLE 13 sensory evaluation results of the dragon bone soothing wine

From the test results it can be seen that: the addition amount of honey is determined to be 30g/L, the addition amount of citric acid is determined to be 1g/L, at the moment, the dragon bone soothing wine is a brown yellow liquid, the color is pure and clear, the unique aroma and the rich bouquet of the roasted epimedium herb are provided, the taste is moderate, no bitter taste is generated, and no throat feeling is caused.

(3) Prescription determined

Prepared from 15g of roasted epimedium herb, 15g of malaytea scurfpea fruit, 10g of calcined dragon bone, 15g of spina date seed, 15g of Chinese magnoliavine fruit, 15g of south dodder seed, 30g of honey and 1g of citric acid into 1000mL

The preparation method comprises the following steps: pulverizing fructus Psoraleae, semen Ziziphi Spinosae, fructus Schisandrae chinensis and semen Cuscutae into coarse powder, adding processed herba Epimedii and Os Draconis preparata, mixing, adding 48 ° Chinese liquor, soaking for 10 days, stirring once per day, collecting the soaked solution, filtering, standing for 8 hr, supplementing lost Chinese liquor, adding Mel and citric acid, mixing, and filtering with 200 mesh gauze.

Test example 2 quality of medicated wine of the present invention

1. Traits

According to three batches of finished products of the medicinal liquor, namely the dragon bone tranquilizing liquor, the product is determined to be brown yellow clear liquid; fragrant smell, pungent, sweet and slightly bitter taste.

2. Authentication

Psoralen (batch No. 110739-201918, purity 99.6%), isopsoralen (batch No. 110738-202016, purity 99.4%), icariin (batch No. 110737-201516, purity 94.2%), schizandrol A (batch No. 110857-201815, purity 99.7%), spina date seed control medicinal material (121517-201604) and semen cuscutae control medicinal material (121232-201403) were purchased from China food and drug inspection institute. The dragon bone soothing wine (batch number: 20210101, 20210102, 20210103) prepared according to example 1 was provided by the second hospital for traditional Chinese medicine, formulation laboratory, sichuan province. Silica gel G (for thin-layer chromatography), silica gel GF254 (for thin-layer chromatography) are purchased from Qingdao ocean chemical engineering Co., ltd, and polyamide film (for thin-layer chromatography) is purchased from Tetramethyl Biochemical plastics factory of Taoism, tanzhou, zhejiang province, all used reagents are analytically pure, and silica gel G plates, silica gel G254 plates and purified water are self-made in laboratories.

2.1 thin-layer identification of processed herba Epimedii

(1) Preparing a test solution: taking 20ml of the keel nerve-soothing wine of the invention, evaporating to dryness, and adding 1ml of ethanol into residues for dissolving.

(2) Preparation of control solutions: dissolving icariin control in methanol to obtain icariin control solution of about 1 mg/ml.

(3) Preparing a deficient roasted epimedium negative test solution: weighing about 70g of other drink tablets of the epimedium stir-fried lacking in weight according to the proportion of the prescription, preparing the epimedium negative medicinal liquor stir-fried lacking in weight according to the method under the item of the preparation method of the embodiment 1, and preparing the epimedium negative test sample solution for stir-fried lacking in weight according to the preparation method of the test sample solution.

(4) Thin layer chromatography conditions: performing thin layer chromatography (0502 of Profibus Committee of the four departments of the 2020 edition in Chinese pharmacopoeia).

Thin layer plate: silica gel G plate (0.4% CMC-Na as binder)

Sample amount dispensing: 5uL of the control solution, 10 uL of the test solution and 10 uL of the negative sample solution.

Unfolding the system I: ethyl acetate-butanone-methanol-water (10

And (3) unfolding a system II: chloroform-methanol-water (13.

The unfolding mode is as follows: the materials are all saturated for 15min, spread upwards and have the spread distance of about 12cm.

And (6) inspection: after development, the material is taken out, dried, sprayed with an aluminum trichloride test solution, heated at 105 ℃ for several minutes, and inspected under an ultraviolet lamp (365 nm).

(5) As a result: the test solution chromatogram shows fluorescent spots with the same color at the corresponding positions of the control solution chromatogram, and the negative control solution chromatogram has no corresponding spots. Wherein, the developing agent I is better, the spots are clear and round, the Rf value is moderate, and the income standard is shown in figure 3 for verifying the reproducibility of three batches of samples.

2.2 thin layer identification of Psoralea corylifolia

(1) Preparation of a test solution: 20ml of fossil fragments nerve calming wine is taken, 20ml of petroleum ether (30-60 ℃) is added for shaking extraction, the petroleum ether is taken separately and evaporated to dryness, and 1ml of ethyl acetate is added into residues for dissolving to be used as a test solution.

(2) Preparation of control solutions: collecting psoralen reference substance and isopsoralen reference substance, and adding ethyl acetate to obtain psoralen reference substance solution and isopsoralen reference substance solution containing 1mg per 1 ml.

(3) Preparing a fructus psoraleae deficiency negative test sample solution: weighing about 70g of other tablets lacking fructus Psoraleae according to the proportion of the prescription, preparing fructus Psoraleae-deficient negative medicated liquor according to the method of the preparation method of example 1, and preparing fructus Psoraleae-deficient negative test solution according to the preparation method of the test solution.

(4) Thin layer chromatography conditions: performing thin layer chromatography (0502 of the four ministry of the general rules of the design of 2020 in Chinese pharmacopoeia).

Thin-layer plate: silica gel G plate (0.4% CMC-Na as binder)

Sample amount of spotting: 5uL of the control solution, 10 uL of the test solution and 10 uL of the negative sample solution.

Unfolding the system I: n-hexane-ethyl acetate (4

And (3) unfolding a system II: petroleum ether (60-90 ℃) ethyl ether-chloroform (5.

The unfolding mode is as follows: the mixture is saturated for 15min, and is unfolded upwards with the spreading distance of 16cm.

And (3) inspecting: taking out, air drying, spraying 10% potassium hydroxide methanol solution, and inspecting under ultraviolet lamp (365 nm).

(5) As a result: in the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the control solution, and no corresponding spots exist in the chromatogram of the negative control solution. Wherein, the developing agent I is better, the spots are clear and round, the Rf value is moderate, the separation degree of psoralen and isopsoralen is better, and the developing agent is used for verifying the reproducibility of three batches of samples, so the income standard is shown in figure 4.

2.3 thin layer identification of semen Cuscutae

(1) Preparation of a test solution: 20ml of fossil fragments nerve calming wine is taken, 20ml of petroleum ether (30-60 ℃) is added for shaking extraction, the petroleum ether layer is removed, ethyl acetate is added for shaking extraction for 2 times, 20ml of ethyl acetate extracting solution is added each time, the ethyl acetate extracting solution is combined, evaporation is carried out, and 1ml of ethyl acetate is added to residues for dissolution to obtain a sample solution.

(2) Preparation of reference drug solution: taking 0.5g of semen Cuscutae reference material, adding 30ml of water, decocting for 30min, cooling, filtering, and preparing semen Cuscutae reference material solution according to the preparation method of the test solution.

(3) Preparing semen cuscutae negative test sample solution: weighing about 70g of the other drinking tablets without the semen cuscutae according to the proportion of the prescription, preparing the semen cuscutae negative medicinal liquor according to the method under the item of the preparation method of the embodiment 1, and preparing the semen cuscutae negative test solution according to the preparation method of the test solution.

(4) Thin layer chromatography conditions: performing thin layer chromatography (0502 of Profibus Committee of the four departments of the 2020 edition in Chinese pharmacopoeia).

Thin-layer plate: silica gel G plate (0.4% CMC-Na as binder)

Sample amount of spotting: the control solution, the sample solution and the negative sample solution are 10 μ L.

Unfolding the system I: ethyl acetate-formic acid-water (20

And (3) unfolding a system II: toluene-ethyl acetate-carboxylic acid (5.

The unfolding mode is as follows: the cells are saturated for 15min, spread upwards and spread by 16cm.

And (3) inspecting: taking out, air drying, and inspecting under ultraviolet lamp (365 nm).

(5) As a result: in the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the control solution, and no corresponding spots exist in the chromatogram of the negative control solution. Wherein, the developing agent I is better, the spots are clear and round, the Rf value is moderate, and the developing agent I is used for verifying the reproducibility of three batches of samples, so the income standard is shown in figure 5.

2.4 thin layer identification of Schisandra chinensis

(1) Preparing a test solution: collecting 20ml Os Draconis tranquilization wine, extracting with chloroform for 2 times, each time 20ml, mixing chloroform extractive solutions, recovering solvent to dry, and dissolving residue with 0.5ml methanol to obtain test solution.

(2) Preparation of control solutions: collecting Schizandrol A control, adding methanol to obtain solution containing 1mg per 1ml, and making into control solution.

(3) Preparing a schisandra chinensis deficiency negative test sample solution: weighing about 70g of the other tablets without fructus Schisandrae chinensis according to the formula proportion, preparing fructus Schisandrae chinensis negative medicated liquor according to the method of "preparation method of example 1", and preparing fructus Schisandrae chinensis negative test solution according to the preparation method of test solution.

(4) Thin layer chromatography conditions: performing thin layer chromatography (0502 of the four ministry of the general rules of the design of 2020 in Chinese pharmacopoeia).

Thin-layer plate: silica gel GF254 plate (0.4% CMC-Na as binder).

Sample amount of spotting: 5uL of the control solution, 10 uL of the test solution and 10 uL of the negative sample solution.

Unfolding the system I: cyclohexane-acetic acid ethyl ester (3

And (3) unfolding a system II: petroleum ether (30 to 60 ℃) -methyl formate-formic acid (15)

The unfolding mode is as follows: the solution is saturated for 10min, spread upwards and spread by 17cm.

And (6) inspection: taking out, air drying, and inspecting under ultraviolet lamp (254 nm).

(5) As a result: the test solution chromatogram shows fluorescent spots with the same color at the corresponding positions of the control solution chromatogram, and the negative control solution chromatogram has no corresponding spots. Wherein, the developing agent I is better, the spots are clear and round, the Rf value is moderate, and the income standard is shown in figure 6 for verifying the reproducibility of three batches of samples.

2.5 thin-layer identification of wild jujube seed

(1) Preparation of a test solution: taking 20ml of Os Draconis tranquilization wine, evaporating to dryness, adding 15ml of water into residue for dissolving, extracting with saturated n-butanol under shaking for 2 times, 15ml each time, evaporating n-butanol solution to dryness, and adding 1ml of methanol into residue for dissolving to obtain sample solution.

(2) Preparation of control solutions: taking jujuboside A and jujuboside B as reference substances, adding methanol to obtain mixed solutions each containing 1mg per 1ml as reference substance solutions.

(3) Preparing a jujube kernel negative test sample solution: weighing about 70g of other tablets of the jujube kernel lacking in sour according to the proportion of the prescription, preparing the jujube kernel negative medicinal liquor according to the method under the item of the preparation method of the example 1, and preparing the jujube kernel negative test sample solution according to the preparation method of the test sample solution.

(4) Thin layer chromatography conditions: performing thin layer chromatography (0502 of Profibus Committee of the four departments of the 2020 edition in Chinese pharmacopoeia).

Thin layer plate: silica gel G plate (0.4% CMC-Na as binder).

Sample amount of spotting: 5uL of the control solution, 10 uL of the test solution and 10 uL of the negative sample solution.

Unfolding the system I: water saturated n-butanol solution

And (3) unfolding a system II: chloroform-methanol-water-formic acid (13.

The unfolding mode is as follows: the mixture is saturated for 15min, and is unfolded upwards with a spreading distance of 17cm.

And (3) inspecting: taking out, air drying, spraying 2% vanillin solution, heating at 105 deg.C for several minutes, and inspecting in sunlight.

(5) As a result: the test samples in the two development systems fail to detect spots corresponding to the reference medicinal material solution or the detected spots are not clear or the negative interference is strong, so that the identification is difficult, and the wild jujube seeds are not included temporarily as a thin-layer identification item, which is shown in figure 7.

Examination of

3.1 Total solids

The total solid content is checked according to the first method of total solid content check under the item of 0185 liquor in the general rule of the four departments in the 2020 edition of Chinese pharmacopoeia. Taking three bottles of the product, precisely measuring 50ml, placing the bottles in an evaporation dish, steaming the bottles in water bath to be thick paste, adding absolute ethyl alcohol, stirring and extracting for 4 times, 10ml each time, filtering, combining filtrates, placing the combined filtrates in the evaporation dish dried to constant weight, steaming the dried product to be nearly dry, precisely adding 1g of diatomite (dried at 105 ℃ for 3 hours, moved to a dryer for cooling for 30 minutes), uniformly stirring, drying at 105 ℃ for 3 hours, moved to the dryer for cooling for 30 minutes, rapidly and precisely weighing the weight, and deducting the added diatomite amount. The results are shown in Table 14, and it is concluded from the results that the total solid content of the tranquilizing dragon's bones is not less than 2.00%.

3.2 amount of methanol

According to the first method of gas chromatography (capillary column method) in 0871, the four ministry of communications in 2020 edition of Chinese pharmacopoeia. Adopting Agilent 7890B gas chromatograph (Agilent technologies, inc.), DB-624 capillary column (30m 0.25mm 0.25 μm), FID detector, sample inlet temperature 220 deg.C, detector temperature 220 deg.C, column temperature 38 deg.C as initial temperature, maintaining for 3 min, heating to 65 deg.C at a heating rate of 3 deg.C/min, heating to 220 deg.C at a heating rate of 25 deg.C/min, and maintaining for 10min; the sample injection amount is 1.0 mu L, the flow rate of the carrier gas is 3.0mL/min, the split ratio is 10:1. the number of theoretical plates is not less than 10000 calculated according to a methanol peak, and the separation degree of the methanol peak and other chromatographic peaks is more than 1.5. Taking the test solution as a test solution. Precisely measuring 1ml of methanol, placing in a 100ml measuring flask, adding water to dilute to scale, shaking, precisely measuring 5ml, placing in a 100ml measuring flask, adding water to dilute to scale, and shaking to obtain a reference solution. The test sample is obtained by calculating the peak area according to an external standard method, the calculation result is shown in a table 14, and the result shows that the methanol content of three batches of the test samples is less than 0.1 percent.

3.3 amount of ethanol

According to the first method (capillary column method) of gas chromatography in the general regulation 0711 of the four departments in the 2020 edition of Chinese pharmacopoeia. Adopting Agilent 7890B gas chromatograph (Agilent technologies, inc.), DB-624 capillary column (30m 0.53mm 0.30 μm), FID detector, sample inlet temperature 200 deg.C, detector temperature 220 deg.C, column temperature 40 deg.C as initial temperature, maintaining for 3 min, heating to 65 deg.C at a heating rate of 3 deg.C/min, heating to 200 deg.C at a heating rate of 25 deg.C/min, and maintaining for 10min; the sample injection amount is 1.0 mu L, the flow rate of the carrier gas is 3.0mL/min, the split ratio is 20:1. the number of theoretical plates is not less than 10000 calculated according to the ethanol peak, and the separation degree of the ethanol peak and the n-propanol peak is more than 2.0. Precisely measuring 5ml of absolute ethyl alcohol with the constant temperature of 20 ℃, and paralleling the two parts; placing in a 100ml measuring flask, adding 5ml of n-propanol (internal standard substance) with constant temperature of 20 deg.C, diluting with water to scale, shaking, precisely measuring 1ml of the solution, placing in a 100ml measuring flask, diluting with water to scale, and shaking to obtain reference solution. And (3) injecting each control solution for 3 times, measuring peak areas, and calculating an average correction factor, wherein the relative standard deviation of the obtained correction factor is not more than 2.0%. The determination method comprises precisely measuring a proper amount of sample (corresponding to about 5ml of ethanol) with constant temperature of 20 deg.C, placing in a 100ml measuring flask, precisely adding 5ml of n-propanol with constant temperature of 20 deg.C, diluting with water to scale, shaking, precisely measuring 1ml of the solution, placing in a 100ml measuring flask, diluting with water to scale, and shaking to obtain sample solution. Calculating according to the peak area by an internal standard method to obtain the product. The calculation results are shown in Table 14, and the results show that the ethanol content of the three batches of pilot samples is 48-52%, so the ethanol content is temporarily 45-55%.

3.4 heavy Metal examination

The examination is carried out according to the relevant regulations under the second Law of the four-part general rules 0821 of the Chinese pharmacopoeia 2020 edition. Taking 2mL of the product, slowly burning till complete carbonization, cooling, adding 0.5mL of sulfuric acid to ensure proper wetting, heating at low temperature till the sulfuric acid is removed, adding 0.5mL of nitric acid, evaporating till the nitrogen oxide vapor is removed, cooling, burning at 500-600 ℃ till complete ashing, cooling, adding 2mL of hydrochloric acid, placing on a water bath, evaporating till the water is dried till the water is 15mL, dropwise adding an ammonia test solution till the indicator is pink, adding 2mL of acetate buffer solution (pH3.5), dissolving by micro-heating, moving to a Nashi colorimetric tube, and adding water to dilute into 25mL; taking another reagent for preparing a test sample solution, placing the reagent in a porcelain dish, drying by distillation, adding 2ml of acetate buffer solution (pH3.5) and 15ml of water, dissolving by micro-heating, transferring the solution into a Nashy colorimetric tube, adding 1ml of standard lead solution (10 ug/ml), and adding water to dilute the solution into 25ml; adding 2ml of thioacetamide test solution into each tube, shaking, standing for 2 min, placing on white paper, and observing from top to bottom, wherein the color of the test solution tube is not darker than that of the tube containing standard lead solution. See table 14. The results show that the heavy metals contained in the three batches of pilot samples do not exceed 10PPm.

3.5 arsenic salt

The examination was carried out according to the relevant regulations under the item of the ancient Chua's method of 0822, the four ministry of general rules of the 2020 edition of Chinese pharmacopoeia. Taking 1mL of the product, adding 1.0g of calcium hydroxide, uniformly mixing, adding water for wetting, drying at low temperature, completely carbonizing by small fire, burning to ash at 500-600 ℃, cooling, transferring into an arsenic measuring bottle, adding 5mL of hydrochloric acid and 21mL of water, adding 5mL of potassium iodide test solution and 5 drops of acidic stannous chloride test solution, placing for 10 minutes at room temperature, adding 2g of zinc particles, measuring by an Easy method, simultaneously making positive standard arsenic spots, and comparing the generated arsenic spots with the standard arsenic spots to be not deeper. As a result, the color of the arsenic spots formed by the test article was lighter than that of the positive standard arsenic spots (2 PPm), and the results are shown in Table 14. The results show that all three batches of pilot samples contained less than 2PPm of arsenic salt, so this item is not included in the text at this time.

TABLE 14 examination results of Total solids, methanol amount, ethanol amount, heavy metals, and arsenic salt

3.6 filling difference

According to the item of 0185 'wine agent' in the four-part general rule of 2020 edition of Chinese pharmacopoeia, the wine agent filled in multiple doses is checked by the lowest filling amount checking method (general rule 0942). The contents of the dragon bone soothing wine 3 bottles were taken and transferred to a pre-calibrated dry measuring cylinder, the containers were inverted for 15 minutes and poured out as clean as possible, and the contents of each container were read. The average filling amount should not be less than the labeled filling amount, and the filling amount of each container should not be less than 97% of the labeled filling amount. The test results are shown in Table 15. The result shows that the difference of the loading of the product meets the regulations.

TABLE 15 table of results of the load variation inspection

3.7 microbial Limit

According to the inspection of the general rules 1105 and 1106 and the general rules 1107 in the version 2020 of Chinese pharmacopoeia, the total number of aerobic bacteria in the product is less than or equal to 500cfu/ml, the total number of mould and yeast is less than or equal to 100cfu/ml, and staphylococcus aureus and pseudomonas aeruginosa (1 ml) are not detected. The results show that all three samples meet the specifications and the results are shown in Table 16. The results show that the three batches of samples meet the regulations under the four-part general rules 1105, 1106 and 1107 in the 2020 version of Chinese pharmacopoeia.

TABLE 16 microbial Limit test results

4 content determination

The monarch drugs in the recipe of roasted epimedium mainly contain flavones, and icariin, epimedin A, epimedin B and epimedin C are the main active ingredients. The four flavonoid components are easily dissolved in wine, have high transfer rate, and refer to part of "Chinese pharmacopoeia" of 2020 edition and related documents, and a method for determining contents of epimedin A, epimedin B, epimedin C and icariin in the product by high performance liquid chromatography is established.

4.1 instruments and reagents

The instrument comprises the following steps: model e2695 high performance liquid chromatograph (watet limited, usa); model 2489 UV/Vis Detector (Watts Inc., USA); the BT-125D type analytical balance [ one hundred thousand, sedoli 20210102, 20210103) was provided by the second hospital for traditional Chinese medicine, preparation laboratory, of sichuan province. Icariin (batch No. 110737-201516, purity 94.2%) was purchased from the Chinese food & drug testing institute. Reagent: acetonitrile, methanol and phosphoric acid are used as chromatographic purities, water is purified water, and other reagents are analytical purities.

4.2 chromatographic conditions

4.2.1 selection of flow Rate, column temperature

In the experiment, 3 flow rates of 0.8, 0.9 and 1.0ml/min are mainly considered at the column temperature of 30 ℃, and 3 column temperatures of 25, 30 and 35 ℃ are considered at the flow rate of 1.0 ml/min. According to experimental results, except that the components to be detected, namely the epimedin A, the epimedin B, the epimedin C and the icariin, are not completely separated at the flow rate of 0.8ml/min and the column temperature of 25 ℃, chromatographic peaks and adjacent peaks to be detected under other conditions are not interfered, the separation degree is good, the peak symmetry is good, and no obvious difference exists. Therefore, the flow rate is selected to be 1.0ml/min, and the column temperature is 30 ℃.

4.2.2 defined chromatographic conditions

A chromatographic column: agilent ZORBAX SB-C18 (4.6X 250mm, 5um)

Mobile phase: acetonitrile (A) and water (B), gradient elution procedure 0-30min, 24% → 26%; 30 to 35min,26% → 26% of A;

column temperature: 30 ℃;

detection wavelength: 270nm.

4.2.3 assay

Precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

4.3 preparation of the solution

4.3.1 preparation of control solutions

Accurately weighing icariin 15.30mg, placing in a 25mL measuring flask, adding methanol to dissolve, fixing volume, and shaking to obtain icariin control stock solution (concentration of 0.6120 mg/mL).

4.3.2 preparation of test solutions

Precisely sucking 50ml of the dragon bone soothing wine, drying 3 parts of the dragon bone soothing wine in an evaporating dish by distillation, respectively adding methanol and ethanol to dissolve the dragon bone soothing wine in a 50ml volumetric flask, fixing the volume, shaking up, and filtering through a 0.45-micron microporous membrane to obtain a subsequent filtrate; and the other part is directly filtered through a 0.45um microporous filter membrane, and a subsequent filtrate is taken, thus obtaining the product. The icariin content was calculated by recording peak area measured under the above chromatographic conditions, and the results are shown in Table 17. As can be seen from the results, the difference in icariin content between the methanol dissolution after evaporation to dryness and the direct filtration was small, while the difference in icariin content between the ethanol dissolution after evaporation to dryness and the direct filtration was large, and the direct filtration was attempted.

TABLE 17 different extraction methods and solvent types icariin content

4.4 inspection of Linear Range

Precisely sucking 0.5ml, 1ml, 2ml, 3 ml, 4 ml and 5ml of icariin, respectively placing into 6 10ml volumetric flasks, adding methanol for dilution, fixing volume, and shaking to obtain series concentration reference solutions. And then, carrying out sample injection measurement according to the chromatographic conditions, recording peak areas, drawing a standard curve, and calculating an r value. The results are shown in Table 18 and FIG. 8, and it is understood from the results that icariin is in a good linear relationship (r.gtoreq.0.9991) in the concentration range of 30.6-306.0 ug/ml.

TABLE 18 results of the Linear relationship test

4.5 specialization examination

Weighing the prescription amount decoction pieces containing the processed herba Epimedii according to the prescription proportion, and preparing the negative wine of the processed herba Epimedii according to the preparation process. Taking 10 μ l of methanol, reference solution, test solution, and processed herba Epimedii test solution, respectively, performing sample injection determination under chromatographic condition, and recording chromatogram. As can be seen from the figure, the chromatogram of the test solution has corresponding chromatographic peaks at the positions of epimedin A, epimedin B, epimedin C and icariin, while the chromatogram of the non-roasted epimedin negative test solution has no corresponding chromatographic peaks at the positions of epimedin A, epimedin B, epimedin C and icariin under the same chromatographic condition, which shows that the detecting specificity of the epimedin A, epimedin B, epimedin C and icariin under the chromatographic condition is good and the negative is free from interference. The chromatogram is shown in FIG. 9.

4.6 precision test

Taking control solution (linear 3, containing icariin 122.4ug/ml per 1 ml), continuously injecting sample according to the above chromatographic conditions, measuring for 6 times, recording chromatogram, and measuring icariin peak area of each injection, the results are shown in Table 19. From the results, it was found that icariin was less than 1.35% in terms of RSD% calculated from the peak area and retention time, and the results showed that the instrument was excellent in precision.

TABLE 19 results of precision test

4.7 stability test

Taking Os Draconis tranquilization liquor (batch No. 20210101), preparing the sample solution according to the sample preparation method, adding into the sample solution for 0, 2, 4, 6, 8, 12, and 24h, and measuring according to the above chromatographic conditions. The retention time and peak area of icariin were recorded for each injection and the results are shown in table 20. As can be seen from the results, the stability of icariin, both in terms of peak area and retention time, was less than 3.0% at RSD, and the results indicate that the test solution had good stability within 24 hours.

TABLE 20 stability of test articles

4.8 repeatability test

Taking Os Draconis tranquilization liquor (batch No. 20210101), preparing 6 test samples in parallel according to the test sample preparation method, and carrying out sample injection determination according to the chromatographic conditions. The icariin peak area was recorded for each injection and the results are shown in Table 21. According to the results, the concentration of icariin is less than 3.0, and the results show that the preparation method of the test solution has good repeatability.

TABLE 21 results of repeated experiments

4.9 sample recovery test

Precisely sucking 50ml of fossil fragments tranquilization wine (batch number: 20210101, each 1ml contains icariin 0.0757 mg) for 9 parts, precisely adding appropriate amount of icariin reference substance, shaking, and preparing 9 parts of sample test solution according to the preparation method of the test solution. The icariin content was calculated by measuring according to the above chromatographic conditions and recording the peak area, and the results are shown in Table 22. The results show that the average recovery rate of icariin is 99.73%, which indicates that the method has better sample recovery rate and better accuracy.

TABLE 22 recovery test results

4.10 durability test

The keel soothing wine (batch No. 20210101) is prepared into a test sample solution according to the preparation method of the test sample solution, and different brands of chromatographic columns are used for carrying out durability tests, and the results are shown in Table 23. The results show that: the content determination results of different chromatographic columns adopted by the product are similar, which shows that the established content determination method has better durability.

TABLE 23 durability test results

4.11 sample determination

Taking appropriate amount of three batches of Os Draconis tranquilization liquor samples (batch Nos. 20210101, 20210102, 20210103), and preparing the test solution according to the preparation method of the test solution. Taking the reference solution and the sample solution, performing sample injection measurement according to the chromatographic conditions, recording peak area, and calculating glycoside content, wherein the results are shown in Table 24. The results show that the total flavone content of the three batches of pilot samples containing the epimedin A, the epimedin B, the epimedin C and the icariin is 0.2742, 0.2483 and 0.2556mg/ml in sequence, and the average value is 0.2594mg/ml.

The concentration of the crude drugs of the processed epimedium contained in the finished product of the dragon bone tranquilizing wine is 0.015g/mL, namely the total amount of the epimedin A, the epimedin B, the epimedin C and the icariin contained in three batches of samples is 18.28mg/g, 16.55mg/g and 17.04mg/g in sequence according to the crude drugs of the epimedium, and the average value is 17.29mg/g. The epimedium stir-fried decoction piece prescription under the item of epimedium in the first part of 2020 edition of Chinese pharmacopoeia is that the total content of epimedin A, epimedin B, epimedin C and icariin is not less than 12.00mg/g by dry product, and the total content of the epimedin A, the epimedin B, the epimedin C and the icariin in the decoction piece stir-fried epimedium used in the three batches of samples is 37.50mg/g according to the requirement, thereby meeting the content requirement. The transfer rate of the three preparations is 46.10 percent by calculation, and the total content of the epimedin A, the epimedin B, the epimedin C and the icariin in the sample is 5.53mg/g based on the crude drug of the roasted epimedium according to the lower limit requirement of pharmacopeia, and the total content of the epimedin A, the epimedin B, the epimedin C and the icariin the dragon bone tranquilizer is 0.0830mg/ml temporarily.

TABLE 24 Pilot plant assay

5. Stability investigation test

Through stability test investigation, the wine is stored for 0, 1, 2, 3 and 6 months under an accelerated test and stored for 0, 3, 6, 9, 12 and 18 months under a long-term stability test condition, and as a result, all indexes of 3 batches of pilot samples meet the regulations, and the product is basically stable, which indicates that the wine is stable within 18 months.

6. Influence of fossil fragments tranquilization wine on sleep of suprathreshold dose pentobarbital sodium mice

50 KM mice, all males, were divided into 5 groups at random according to weight, each group had 10 mice, which were respectively a model control group (distilled water), a positive control group (estazolam tablet, 1 mg/kg), a keels soothing wine (lot number: 20210101) high dose (8.22 ml/kg), a keels soothing wine medium dose (4.11 ml/kg), and a keels soothing wine low dose (2.05 ml/kg). The drug was administered by gavage 1 time per day for 3 consecutive days. 40min after the last drug, pentobarbital sodium (0.1 ml/10 g. Wt) is intraperitoneally injected according to the suprathreshold dose of 36mg/kg, the disappearance of the righting reflex is taken as the sleep start, and the sleep latency and the sleep time of the animals within 180min are recorded. SPSS23.0 was used for analysis (using one-way anova if normal distribution was met, and non-parametric testing if normal distribution was not met).

Table 25 effect of fossil fragments tranquilization wine on sleep in suprathreshold dose pentobarbital sodium mice (n =10,

)

according to p <0.01, p <0.05 has statistical significance

And (4) conclusion: the results of the experiment (data were normally distributed using the one-way variance test) are shown in table 25: the high and medium dosage of the dragon bone tranquilizing liquor can obviously prolong the sleep time (P is less than 0.01 or P is less than 0.05), and has dose-effect relationship, and the high dosage of the dragon bone tranquilizing liquor has different shortening of the incubation period, so that the dragon bone tranquilizing liquor has the effect of promoting sleep under the high and medium dosages.

7. Influence of dragon bone soothing wine on sleep of mice with sub-threshold dose of pentobarbital sodium

50 KM mice, all male mice, were divided into 5 groups at random according to body weight, each group containing 10 mice, including model control group (distilled water), positive control group (estazolam tablet, 1 mg/kg), os Draconis tranquilization wine (lot number: 20210101) high dose (8.22 ml/kg), os Draconis tranquilization wine medium dose (4.11 ml/kg), and Os Draconis tranquilization wine low dose (2.05 ml/kg). The drug was administered by gavage 1 time a day for 3 consecutive days. 40min after the last medicine, pentobarbital sodium (0.1 ml/10 g. Wt) is injected intraperitoneally at a dose of 30mg/kg below threshold, sleep is taken when righting reflex disappears for more than 1min, and the number of sleeping animals in 30min is recorded. Chi-square test was performed using SPSS 23.0.

Table 26 effect of fossil fragments soothing wine on sleep in mice with subthreshold dose of sodium pentobarbital (n =10,

)

p <0.01 x, p <0.05 x according to chi-square test; has statistical significance

And (4) conclusion: the test results are shown in table 26: the sleep-promoting rate (P <0.05 or P < 0.01) of mice with the sub-threshold dose of pentobarbital sodium can be obviously increased within 30min by the high and medium dose groups of the keel tranquilizing wine, and a certain dose-effect relationship is provided, so that the keel tranquilizing wine has the sleep-promoting effect under the high and medium doses; because the influence of a pure liver drug enzyme inhibitor on the test result is small, the experiment shows that the sleep promoting effect of the keel nerve soothing wine is irrelevant to the inhibition of liver drug enzyme.

8. Influence of dragon bone soothing wine on spontaneous activity test of mice

50 KM mice, half of male and female, were divided into 5 groups at random according to body weight, each group containing 10 mice, including model control group (distilled water), positive control group (estazolam tablet, 0.6 mg/kg), os Draconis tranquilization wine (batch No.: 20210101) high dose (8.22 ml/kg), os Draconis tranquilization wine medium dose (4.11 ml/kg), and Os Draconis tranquilization wine low dose (2.05 ml/kg). The drug was administered by gavage 1 time per day for 3 consecutive days. 40min after the last dose, the mice were placed in an automatic activity apparatus and the activity times and standing times within 10min were recorded. Statistical analysis was performed using SPSS23.0 (single-factor analysis of variance if normal distribution was met, non-parametric tests if normal distribution was not met).

Influence of Os Draconis tranquilizing liquor on spontaneous activity of mouse (TABLE 27)

n＝10)

Note: p <0.05, P <0.01, compared to suspension control group

The results of the experiment (data were normally distributed using the one-way variance test) are shown in table 27: the high and medium dosage of the dragon bone tranquilizing wine can reduce the activity frequency (P < 0.05) of mice, can obviously reduce the standing frequency (P <0.01 or P < 0.05) of the mice, has obvious dose-effect relationship, and prompts that the dragon bone tranquilizing wine has the function of inhibiting the spontaneous activity of the mice.

In conclusion, the wine prepared by the composition of the invention warms the kidney, benefits the essence, calms the liver, subdues yang, nourishes the liver, soothes nerves and can regulate the body function, and is used for treating liver and kidney deficiency, dizziness, tinnitus, insomnia, dreaminess, waist soreness and neurasthenia. The test proves that: the main components of the medicinal ingredients in the composition have better dissolution rate in wine, the wine is used for extracting and dissolving coal, the effect is quicker, and meanwhile, the wine has mellow taste and high public acceptance degree, and is easy to popularize and use in the market.

Claims (8)

1. A composition with a nerve soothing effect is characterized in that: the composition is prepared from the following raw materials in parts by weight: 10 to 20 portions of epimedium, 10 to 20 portions of malaytea scurfpea fruit, 7 to 13 portions of keel, 10 to 20 portions of spina date seed, 10 to 20 portions of Chinese magnoliavine fruit and 10 to 20 portions of south dodder seed; the keel is forged keel; the herba Epimedii is processed herba Epimedii.

2. The composition of claim 1, wherein: the traditional Chinese medicine is prepared from the following raw materials in parts by weight: 15 parts of epimedium, 15 parts of fructus psoraleae, 10 parts of keel, 15 parts of spina date seed, 15 parts of schisandra chinensis and 15 parts of semen cuscutae.

3. The composition of claim 1, wherein: the composition is an oral preparation, and the oral preparation is a tincture, an ointment or a wine.

4. The composition of claim 3, wherein: the wine is prepared from the following raw materials in parts by weight: 15 parts of epimedium, 15 parts of fructus psoraleae, 10 parts of keel, 15 parts of spina date seed, 15 parts of schisandra chinensis, 15 parts of semen cuscutae, 30 parts of honey, 1 part of citric acid and 1000 parts of wine by volume; the keel is forged keel; the herba Epimedii is processed herba Epimedii.

5. The composition of claim 4, wherein: the wine is 35-52 degrees white spirit.

6. The composition of claim 5, wherein: the wine is 48-degree white spirit.

7. A process for preparing a composition according to any one of claims 1 to 6, characterized in that: it comprises the following steps:

(1) Weighing the raw materials according to the proportion;

(2) Pulverizing fructus Psoraleae, semen Ziziphi Spinosae, fructus Schisandrae chinensis and semen Cuscutae into coarse powder, adding processed herba Epimedii and Os Draconis preparata, mixing, adding Chinese liquor, soaking for 10-20 days, and stirring once a day;

(3) Taking the soaked solution, filtering, standing, adding the rest Chinese liquor, adding Mel and citric acid, mixing, and filtering.

8. Use of the composition of any one of claims 1 to 6 for preparing a medicament for warming kidney, replenishing vital essence, calming liver, suppressing yang hyperactivity and/or nourishing liver, and tranquilizing mind; the medicine is used for treating kidney-yang deficiency type insomnia, amnesia, liver and kidney deficiency, dizziness, tinnitus, insomnia, dreaminess, waist soreness, and neurasthenia.

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