CN111467443A - Preparation and quality control method of traditional Chinese medicine composition for preventing and treating asthma in remission stage - Google Patents

Preparation and quality control method of traditional Chinese medicine composition for preventing and treating asthma in remission stage Download PDF

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CN111467443A
CN111467443A CN202010409149.0A CN202010409149A CN111467443A CN 111467443 A CN111467443 A CN 111467443A CN 202010409149 A CN202010409149 A CN 202010409149A CN 111467443 A CN111467443 A CN 111467443A
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郑明昱
延光海
南敏伦
赫玉芳
马吉胜
丁云录
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Yanbian University
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Yanbian University
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Abstract

The invention relates to a Chinese medicinal composition for preventing and treating asthma in remission stage, and its preparation method and quality control method, this formulation is made up of eight raw materials such as cornu Cervi Pantotrichum, radix Ophiopogonis, job's tears, rhizoma Dioscoreae, Asparagus, bitter almond, fructus Schisandrae chinensis, herba Ephedrae, etc., the invention uses different routes to extract, prepare into granular formulation, through rational compatibility, preferential preparation, make its function supplement each other, tonify lung and purge liver, wash phlegm and dispel blood stasis, act on asthma in remission stage safely and effectively, the invention has increased the optional formulation for the clinical medication of this compound through formulation preparation process and quality control research, hopefully expand medical value and economic worth of this Chinese medicinal compound.

Description

Preparation and quality control method of traditional Chinese medicine composition for preventing and treating asthma in remission stage
Technical Field
The invention relates to a traditional Chinese medicine composition and a preparation method thereof, in particular to a preparation method and a quality control method of a traditional Chinese medicine composition for preventing and treating asthma in a remission stage, and belongs to the field of traditional Chinese medicines.
Background
Asthma is a chronic immune allergic disease seriously threatening human health, the incidence rate of which is rapidly increased in the world in recent years, 2/3 accounting for the total number of asthma diseases, and nearly 80% of asthma of children and adults are allergic asthma, which is the most common chronic respiratory disease. In large-scale investigation of more than 40 major and middle cities in China, the incidence rate of children asthma is increased by 2.6 times and reaches 2.05 percent compared with that before 10 years. The WHO reports that 1 hundred million new asthmatics are predicted to appear in 2025, and the asthma brings heavy burden to governments, families and patients of various countries, and becomes a public health problem concerned globally.
Asthma is a chronic inflammatory disease of the airways involving a variety of cells such as eosinophils, mast cells and T lymphocytes, and is characterized by important pathological features of airway inflammation (acute attack phase) and airway remodeling (remission phase or chronic duration). At present, more drugs, especially chemical drugs such as salbutamol, are used in the acute attack stage, and the treatment effect on the acute attack stage is very obvious. The medicines used in the asthma remission stage are relatively few, and the western medicines for treating the asthma remission stage at present mainly comprise glucocorticoid, salbutamol and the like. The treatment of chronic duration asthma by glucocorticoid is only limited to the intervention of local inflammation of airways, and no overall intervention scheme is available for the long-term existence of systemic immune dysfunction and atopic constitution of asthma patients, so that the long-term curative effect is not ideal, and particularly, the asthma attack condition is recovered to the treatment before the withdrawal of the drug. The use of glucocorticoids has been reported to partially inhibit myofibroblast accumulation and airway remodeling, with certain side effects.
The importance of traditional Chinese medicine on the treatment of asthma in the remission stage is always emphasized. The TCM branches towards the doctor, has own advantages and characteristics, and in the treatment of chronic and prolonged asthma, the general concept is mostly considered, namely, the differentiation of constitutions first, and then the differentiation of syndromes and diseases. The book of Dong Yi shou Shi Baoyuan says that the zang organs of yin and yang are short and long, and the change of yin and yang is also known. The congenital factors are determined. The different constitutions and the different syndromes will cause different selectivity of the body to the drugs, sometimes causing "dissimilarity of the drugs". Therefore, the health care product emphasizes on improving the physical condition, improving the disease prevention capability of the patient, regulating the immunity of the body, and achieving the disease treatment purposes of preventing the disease from changing, preventing the disease from happening before and mainly preventing the disease.
The traditional Chinese medicine composition consists of pilose antler, dwarf lilyturf tuber, coix seed, Chinese yam, asparagus, Chinese magnoliavine fruit, bitter apricot seed and ephedra herb. In the formula, Lu Rong is the main drug and has the action of promoting the lung to straighten and stretch; radix Ophiopogonis, Coicis semen, fructus Schisandrae, rhizoma Dioscoreae, radix asparagi, semen Armeniacae amarum, and herba Ephedrae can assist cornu Cervi Pantotrichum in regulating lung qi, tonifying lung and strengthening lung. The ephedra herb deeply accumulates phlegm and coagulates blood, activates blood circulation to dissipate blood stasis, and strengthens the efficacy of removing phlegm and removing blood stasis. The coordination relationship between the liver and the lung is the opposition and restriction between yin and yang, and the gradual strengthening of lung function can eliminate liver function, so as to regulate liver qi. The whole formula aims at dredging qi movement, embodies the efficacies of tonifying lung and purging liver, removing phlegm and removing blood stasis, and achieves the effect of treating both principal and secondary aspects of disease. The formula has the advantages that the unique administration laws of ' taking medicine according to the symptoms ' in the medicine aspect of ' taking medicine according to the symptoms ', distinguishing the symptoms and treating, adding and subtracting according to symptoms, not mixing, preventing the abnormal reaction of the medicine ' and the like are developed, the deficiency of the lung and the spleen is always protected in the attack period, the surplus of the liver is eliminated, and therefore the purpose of dispelling the unbalanced relation of the size deviation unbalance of the viscera, finally dispelling the latent phlegm and preventing and treating asthma is achieved. The traditional Chinese medicine is characterized in that the pilose antler serving as a monarch drug has unique characteristics in treating lung diseases according to the ministerial medicine theory. Has been applied to the clinical treatment of asthma in the remission stage for many years, and has obvious effect, and the total effective rate of a treatment group is 86.96 percent. A large number of clinical and experimental studies of the subject group find that the reason why the subject can treat the remission stage of asthma is that the subject can relieve bronchial smooth muscle spasm and prevent repeated attack.
Before the completion of the present invention, no report on the quality control method of the present Chinese medicinal composition has been found.
Disclosure of Invention
One of the purposes of the invention is to provide a formula of a traditional Chinese medicine composition for preventing and treating asthma in a remission stage, which can provide a new traditional Chinese medicine composition for a medicine for treating asthma in the remission stage; the invention also aims to provide a preparation method which has strong operability and is suitable for industrial production; the invention also aims to solve the problem of quality control method of ephedra, schisandra, pilose antler, dwarf lilyturf tuber, Chinese yam and other medicines in the composition, and provides guarantee for the safety and effectiveness of the medicine. The other important purpose of the invention is to solve the problem that the quality standard of the traditional compound traditional Chinese medicine is difficult to establish.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, the invention relates to a traditional Chinese medicine composition for preventing and treating asthma in remission stage, which is characterized in that the traditional Chinese medicine composition is prepared from the following raw materials:
Figure BDA0002492498800000021
preferably, the traditional Chinese medicine composition for preventing and treating asthma in remission stage is characterized by being prepared from the following raw materials:
Figure BDA0002492498800000022
Figure BDA0002492498800000031
in a second aspect, a preparation method of a traditional Chinese medicine composition for preventing and treating asthma in remission stage is provided, which is characterized by comprising the following steps:
a. taking pilose antler, decocting with 10 times of water for 3 times, each time for 2 hours, filtering, combining filtrates, and concentrating to obtain thick paste with the relative density of 1.30-1.35 at 60 ℃ for later use;
b. crushing fructus schizandrae into coarse powder, performing reflux extraction for 3 times with 85% ethanol in an amount which is 6 times that of the coarse powder, performing 2 hours each time, filtering, collecting dregs in another container, combining filtrates, recovering ethanol, and concentrating to obtain thick paste with a relative density of 1.30-1.35 at 60 ℃ for later use;
c. decocting residues and six medicines of radix ophiopogonis, semen coicis, Chinese yam, radix asparagi, bitter almond, ephedra and the like with 10 times of water for 2 times, each time for 2 hours, filtering, combining filtrates, standing, taking supernate, concentrating the supernate into fluid extract with the relative density of 1.06-1.10 at 60 ℃, adding one time of ethanol, stirring uniformly, refrigerating for 12-48 hours, filtering to take supernate, recovering ethanol, concentrating into thick paste with the relative density of 1.30-1.35 at 60 ℃ for later use;
d. mixing the above three soft extracts, drying at 60 deg.C, pulverizing into fine powder, adding dextrin and sucrose powder at a ratio of 1: 4, mixing uniformly, granulating with 90% ethanol, drying, and finishing.
In a third aspect, an identification method of a traditional Chinese medicine composition for preventing and treating asthma in remission stage is provided:
a. collecting 3.0g of the product, grinding, adding water 30ml for dissolving, adding onto strong acid cation exchange resin column (inner diameter 3cm, length 10cm), eluting with 100ml of water, discarding water eluate, eluting with 80ml of 1% ammonia water, collecting eluate, evaporating to dryness, and dissolving residue with 1ml of 70% ethanol to obtain sample solution. Taking 0.5g of cornu Cervi Pantotrichum control material, adding 5ml of 70% ethanol, performing ultrasonic treatment for 15 min, and filtering to obtain filtrate as control solution. Performing thin layer chromatography (2015 version of Chinese pharmacopoeia, general rules of the four parts 0502), sucking 3 μ l of each of the two solutions, respectively dropping on the same silica gel G thin layer plate, developing with n-butanol-glacial acetic acid-water (3: 1: 1) as developing agent, taking out, air drying, spraying with 2% ninhydrin ethanol solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.
b. Grinding 5.0g of the product, adding water 30ml for dissolving, adding concentrated ammonia solution 5ml, mixing, extracting with diethyl ether for 2 times (30 ml each time), mixing diethyl ether solutions, volatilizing, and dissolving the residue with methanol 1ml to obtain test solution. Taking herba Ephedrae reference material 0.5g, adding concentrated ammonia solution 0.5ml, adding chloroform 10ml, heating and refluxing for 1 hr, filtering, evaporating filtrate, dissolving residue with methanol 1ml to obtain reference material solution. Then, the ephedrine hydrochloride control is added with methanol to obtain a solution containing 0.5mg per 1ml as a control solution. Testing by thin layer chromatography (2015 version of Chinese pharmacopoeia 0502, Ministry of the four parts of the Ministry of Japan), sucking the above three solutions 5 μ l, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-concentrated ammonia solution (10: 2: 0.3) as developing agent, taking out, air drying, spraying with 2% ninhydrin ethanol solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution and the reference solution.
c. Grinding 7.0g of the product, adding 50ml of chloroform, heating and refluxing for 1 hour, filtering, evaporating filtrate to dryness, and dissolving residue in 1ml of chloroform to obtain a sample solution. Taking 1.0g of fructus Schisandrae chinensis as reference material, and making into reference material solution by the same method. Adding methanol into Schizandrol A control to obtain solution containing 1mg of Schizandrol A per 1ml, and making into control solution. Performing thin layer chromatography (2015 version of Chinese pharmacopoeia 0502, Ministry of Japan) test, sucking 3 μ l of the above three solutions, respectively dropping on the same silica gel GF254 thin layer plate, spreading with petroleum ether (3060 deg.C) -ethyl formate-formic acid (15: 5: 1) upper layer solution as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm). In the chromatogram of the test solution, fluorescent spots of the same color appear at the positions corresponding to the chromatogram of the reference solution and the chromatogram of the reference solution.
d. Taking 6.0g of the product, grinding, adding water 30ml for dissolving, shaking and extracting with water saturated n-butanol solution for 2 times, 25ml each time, combining n-butanol solutions, evaporating to dryness, adding water 10ml for dissolving residue, adding hydrochloric acid 2ml, heating and refluxing for 1 hour, cooling, shaking and extracting with chloroform for 2 times, 25ml each time, combining chloroform solutions, evaporating to dryness, adding methanol 1ml for dissolving residue to obtain test solution. And adding water into radix Ophiopogonis 1.0g for 50ml, decocting for 30min, filtering, adding hydrochloric acid 2ml into the filtrate, and making into control solution. Performing thin layer chromatography (2015 version of Chinese pharmacopoeia 0502, Ministry of the four parts of the Ministry of Japan) test, sucking the two solutions each 8 μ l, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-acetone (4: 1) as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.
e. Taking 12.0g of the product, grinding, adding 25ml of petroleum ether (3060 ℃) for ultrasonic treatment for 2 times and 20 minutes each time, discarding petroleum ether liquid, volatilizing solvent from residue, adding 30ml of methanol, ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, adding 30ml of water and 0.5ml of hydrochloric acid into residue, heating for 1 hour, cooling, shaking and extracting for 2 times and 25ml each time by using trichloromethane, combining trichloromethane liquid, evaporating to dryness, adding 1ml of methanol into residue to dissolve, and using the residue as a sample solution. Preparing 2.0g of rhizoma Dioscoreae as reference material, and making into reference material solution by the same method. Performing thin layer chromatography (2015 version of Chinese pharmacopoeia 0502, Ministry of the four parts of the Ministry of Japan) test, sucking 5 μ l of the above two solutions, respectively dropping on the same silica gel G thin layer plate, developing with toluene-acetone (5: 1) as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.
In a fourth aspect, a content determination method of a traditional Chinese medicine composition for preventing and treating asthma in remission stage is provided:
the schizandrol A is determined by high performance liquid chromatography (0512 in the four headings of the 2015 pharmacopoeia of China).
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; methanol-water (60: 40) is used as a mobile phase; the detection wavelength was 250 nm. The number of theoretical plates is not less than 5000 calculated according to the schizandrol A peak.
Preparation of control solution A proper amount of schizandrol A control is precisely weighed, placed in brown measuring flask, and added with methanol to obtain solution containing 40 μ g per 1 ml.
Preparation of test solution the product under the condition of different loading amount is taken, ground, about 2.0g is taken, precisely weighed, placed in a conical flask with a plug, precisely added with 25ml of methanol, tightly plugged, weighed, ultrasonically treated (power 250W, frequency 20kHz) for 30 minutes, cooled, weighed again, supplemented with methanol to the loss weight, shaken evenly, filtered, and a subsequent filtrate is taken, thus obtaining the test solution.
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.
One gram of the product contains schisandra fruit and schisandrin (C)24H32O7) Calculated, the content of the active ingredient should not be less than 0.35 mg.
Ephedrine hydrochloride and pseudoephedrine hydrochloride were determined by high performance liquid chromatography (0512 in the four-part general rules of the pharmacopoeia 2015).
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-0.2% phosphoric acid water solution (5: 95) is used as a mobile phase; the detection wavelength was 207 nm. The number of theoretical plates is not less than 5000 calculated according to ephedrine hydrochloride peak.
Preparation of reference solution ephedrine hydrochloride reference and pseudoephedrine hydrochloride reference are weighed precisely, and mixed solution containing 30 μ g and 15 μ g per lml is prepared by adding methanol.
Preparation of test solution the product under the condition of different loading amount is taken, ground, about 3.0g is taken, precisely weighed, placed in a conical flask with a plug, 20ml of methanol is precisely added, the plug is sealed, the weight is weighed, ultrasonic treatment (power 250W, frequency 20kHz) is carried out for 30 minutes, the flask is cooled, the weight is weighed again, the weight loss is complemented by the methanol, shaken up and filtered, 10ml of subsequent filtrate is taken, and added into a neutral alumina column (a) (neutral alumina column) (a) (20 kHz)
Figure BDA0002492498800000051
Mesh, 2g, inner diameter of 1.5cm), eluting with methanol, collecting eluate and eluate, placing in 25ml measuring flask, adding 50% methanol to scale, shaking, filtering, and collecting 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.
Each gram of the product contains ephedrine hydrochloride (C)10H15NO. HC1) and pseudoephedrine hydrochloride (C)10H15NO. HC1) of not less than 0.5 mg.
Compared with the prior art, the invention has the following beneficial effects:
(1) the traditional Chinese medicine composition comprises the raw materials of pilose antler, dwarf lilyturf tuber, coix seed, Chinese yam, asparagus, Chinese magnoliavine fruit, bitter apricot seed, ephedra herb and the like, the raw materials are scientifically matched, the respective complementary coordination effect is fully exerted, and the traditional Chinese medicine composition has the effects of tonifying lung, purging liver, removing phlegm and removing blood stasis. It is suitable for chronic bronchitis, emphysema, bronchial asthma of Taiyin people, and asthmatic asthma of the elderly.
(2) The quality control method can better control the quality of the variety, effectively supervise the production, is more favorable for controlling the product quality, ensures the stability of the product quality and ensures safer food taking.
(3) The preparation process of the invention well retains the active ingredients in the raw materials, and is suitable for industrial production.
The composition of the invention consists of eight traditional Chinese medicines such as pilose antler, ephedra herb and the like, and has the efficacies of tonifying lung, purging liver, removing phlegm and removing blood stasis. It is suitable for chronic bronchitis, emphysema, bronchial asthma of Taiyin people, and asthmatic asthma of the elderly. The inventors studied the main pharmacodynamics of the drugs according to their functions and indications. The following experimental examples further illustrate the invention.
1. The composition of the invention has the effect on the cough caused by the citric acid spraying method of guinea pigs
1.1 materials and methods
1.1.1 animal research experiment uses guinea pig, male and female half, the weight is 200 ~ 220 g. Animal certification number: SCXK- (Ji) 2016-.
1.1.2 drugs and reagents the composition of the invention, properties: the product is brown granule; a positive control drug codeine phosphate tablet, specification: 30 mg/tablet, batch number: 161018, a product of national drug group industries, ltd; citric acid, specification: analytical grade, 500 g/bottle, batch number: 201709119 product of Guang Compound technology development, Tianjin.
1.1.3 multifunctional cough-inducing asthma-inducing apparatus Y L S-8A, model Y L S-8A, manufactured by Shandong province institute of medical science and institute of technology.
1.1.4 method take 50 guinea pigs with weight of 200-220 g, the guinea pigs are used for both male and female, the guinea pigs are randomly divided into 5 groups, each group comprises 10 control groups, each group is a positive control drug codeine phosphate 20mg/kg group, the composition of the invention comprises three dose groups of 3.0, 1.5 and 0.75g/kg, the preventive administration is carried out for 4 weeks, 40min after the last administration, 17.5% citric acid solution is sprayed by a Y L S-8A multifunctional cough-inducing asthma apparatus for 1min to induce cough, the cough latency (S) and the cough frequency within 5min of the guinea pigs (the cough of the guinea pigs is loud, the latency is calculated by the heard cough sound, the latency is the time from the beginning of spraying to the generation of cough) are observed and recorded, the results are calculated by average standard deviation
Figure BDA0002492498800000073
The statistical treatment was performed using the inter-group t-test.
1.2 results
As can be seen from the test results in Table 1, the cough frequency of guinea pigs can be obviously inhibited by the three doses of the composition of the invention, namely 3.0, 1.5 and 0.75g/kg, and compared with the control group, the cough frequency is respectively P < 0.01 or P < 0.05; there was also a trend towards an increase in cough latency, but not statistically significant. The test result shows that the composition has a remarkable inhibiting effect on cough caused by citric acid stimulating respiratory tract.
TABLE 1 Effect of the compositions of the present invention on the latency to cough and the number of coughs in guinea pigs
Figure BDA0002492498800000071
Figure BDA0002492498800000072
Note: compared with the control group, the compound of the formula,**P<0.01；*P<0.05。
2. research on asthma-inducing and asthma-relieving effects of guinea pig acetylcholine
2.1 materials and methods
2.1.1 animal research experiment guinea pig, male and female half, weight 180 ~ 200 g. Animal certification number: SCXK- (Ji) 2016-.
2.1.2 drugs and reagents the composition of the invention, properties: the product is brown granule; the positive control drug aminophylline tablet has the specification: 0.1 g/piece, batch number: 1603109, Tianjin Lisheng pharmaceutical products, Inc.; acetylcholine chloride, specification: 25 g/bottle, batch number: 440837, Shanghai Aladdin Biotechnology, Inc.; the histamine phosphate specification: 5 g/bottle, batch number: 34428 Shanghai Aladdin Biotechnology Ltd.
2.1.3 multifunctional cough-inducing asthma-inducing apparatus Y L S-8A, model Y L S-8A, manufactured by Shandong province institute of medical science and institute of technology.
2.1.4 taking guinea pigs with the weight of 180-200 g, pre-selecting qualified (asthma induction latency is less than 100S) 50 guinea pigs, using male and female, randomly dividing into 5 groups, each group comprising 10 positive control drugs, respectively control group, positive control drug aminophylline 50mg/kg group, adding three dosage groups of the composition of the invention 3.0, 1.5 and 0.75g/kg 1 time/d, continuously taking medicine for 4 weeks, 40min after the last administration, respectively putting into a Y L S-8A multifunctional cough induction asthma apparatus, spraying an aqueous solution of 0.1% histamine phosphate and 2% acetylcholine chloride mixed in a ratio of 1: 1, recording the time from the beginning of spraying to the fall of the guinea pigs as the asthma induction latency plus-minus period
Figure BDA0002492498800000083
The statistical treatment was performed using the inter-group t-test.
2.2 results
As can be seen from the test results in Table 2, the three doses of the composition of the present invention, 3.0, 1.5 and 0.75g/kg, were all able to significantly suppress the asthma latency in guinea pigs, and compared with the control group, P was < 0.01 or P < 0.05, respectively. The test result shows that the composition has obvious inhibition effect on asthma caused by acetylcholine.
TABLE 2 Effect of the compositions of the present invention on the latent phase of asthma in guinea pigs
Figure BDA0002492498800000081
Figure BDA0002492498800000082
Note: compared with the control group, the compound of the formula,**P<0.01；*P<0.05。
3. effect of the composition of the present invention on ear swelling in mice
3.1 materials and methods
3.1.1 animal ICR mouse, male, weight 18 ~ 22 g. Animal certification number: SCXK- (Ji) 2016-0003, available from Yinshi laboratory animal technology, Inc., Changchun city.
3.1.2 medicine and reagent the composition of the invention has the characteristics of brown granules, a positive control medicine dexamethasone tablet with the specification of 0.75 mg/tablet, the batch number of 171106, a product of Guangdong south China pharmaceutical industry group, a bottle with the specification of 500m L, analytically pure, the batch number of 20170327 and a product of Tianjin horizontal reagent, Inc.
3.1.3 method take 60 SPF grade ICR mice, male, with the body mass of 18-22 g, randomly divided into 5 groups, namely a control group and a positive control dexamethasone tablet 1.8mg/kg group, the composition of the invention has three dose groups of 6.0g/kg, 3.0g/kg and 1.5g/kg, and 5 groups, wherein each group has 10 administration groups, the administration groups are administrated according to the body mass (20m L/kg) ig of the mice, the control groups are administrated with distilled water with the same volume according to the body mass, 1 time per day and 4 weeks continuously, the mice are fasted for 12 hours at night on the 27 th day of the experiment, after administration for 40min on the 28 th day, the mice are evenly coated with a inflammatory agent xylene (20 mu L/mouse) before and after the right ear, the mice are killed after 30min of cervical dislocation, two ears are cut along the auricles, the same ear part is cut by a double-ear instrument with the diameter of 6mm, the weight difference of the ears is analyzed, and the swelling degree (mg) of the two ears is calculated, and the swelling rate is calculated.
Swelling inhibition rate (average swelling degree of control group-average swelling degree of administration group)/average swelling degree of control group
3.2 results
Results of experiments on auricle swelling of mice caused by xylene show that the auricle swelling degree of mice in a control group (11.6 +/-3.9) mg and the auricle swelling degree of mice in three dose groups of 6.0g/kg, 3.0g/kg and 1.5g/kg of the composition of the invention are respectively (7.5 +/-2.9) mg, (8.5 +/-2.1) mg and (8.7 +/-2.2) mg. Compared with a control group, the composition of the invention can obviously reduce the ear swelling degree of mice at the doses of 6.0g/kg and 3.0g/kg (P is less than 0.05). The composition can inhibit acute inflammation caused by xylene. See table 3.
TABLE 3 Effect of the compositions of the present invention on swelling of mouse auricles by xylene
Figure BDA0002492498800000091
Figure BDA0002492498800000092
Note: compared with the control group, the compound of the formula,**P<0.01；*P<0.05。
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.
EXAMPLE 1 Process for the preparation of the composition of the invention
Figure BDA0002492498800000101
Decocting the pilose antler with 10 times of water for 3 times, 2 hours each time, filtering, combining the filtrates, and concentrating to obtain thick paste with the relative density of 1.30-1.35 (60 ℃) for later use; pulverizing fructus schizandrae into coarse powder, extracting with 6 times of 85% ethanol under reflux for 3 times (2 hours each time), filtering, collecting the residues in another container, mixing the filtrates, recovering ethanol, and concentrating to obtain soft extract with relative density of 1.30-1.35 (60 deg.C); decocting the residue with six medicines of radix ophiopogonis, semen coicis, Chinese yam, asparagus cochinchinensis, bitter apricot seed, ephedra herb and the like by adding 10 times of water for 2 times, each time for 2 hours, filtering, combining the filtrates, standing, taking the supernatant, concentrating the supernatant into a fluid extract with the relative density of 1.06-1.10 (60 ℃), adding one time of ethanol, stirring uniformly, refrigerating for 12-48 hours, filtering to take the supernatant, recovering the ethanol, concentrating the concentrate into a thick paste with the relative density of 1.30-1.35 (60 ℃), combining the two thick pastes, drying (60 ℃), crushing into fine powder, adding dextrin-sucrose powder (1: 4), mixing uniformly, granulating with 90% ethanol, drying, finishing granules, and preparing 1000 g.
Example 2 thin layer identification of the compositions of the invention
(1) Collecting 3.0g of the product, grinding, adding water 30ml for dissolving, adding onto strong acid cation exchange resin column (inner diameter 3cm, length 10cm), eluting with 100ml of water, discarding water eluate, eluting with 80ml of 1% ammonia water, collecting eluate, evaporating to dryness, and dissolving residue with 1ml of 70% ethanol to obtain sample solution. Taking 0.5g of cornu Cervi Pantotrichum control material, adding 5ml of 70% ethanol, performing ultrasonic treatment for 15 min, and filtering to obtain filtrate as control solution. Performing thin layer chromatography (2015 version of Chinese pharmacopoeia, general rules of the four parts 0502), sucking 3 μ l of each of the two solutions, respectively dropping on the same silica gel G thin layer plate, developing with n-butanol-glacial acetic acid-water (3: 1: 1) as developing agent, taking out, air drying, spraying with 2% ninhydrin ethanol solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.
(2) Grinding 5.0g of the product, adding water 30ml for dissolving, adding concentrated ammonia solution 5ml, mixing, extracting with diethyl ether for 2 times (30 ml each time), mixing diethyl ether solutions, volatilizing, and dissolving the residue with methanol 1ml to obtain test solution. Taking herba Ephedrae reference material 0.5g, adding concentrated ammonia solution 0.5ml, adding chloroform 10ml, heating and refluxing for 1 hr, filtering, evaporating filtrate, dissolving residue with methanol 1ml to obtain reference material solution. Then, the ephedrine hydrochloride control is added with methanol to obtain a solution containing 0.5mg per 1ml as a control solution. Testing by thin layer chromatography (2015 version of Chinese pharmacopoeia 0502, Ministry of the four parts of the Ministry of Japan), sucking the above three solutions 5 μ l, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-concentrated ammonia solution (10: 2: 0.3) as developing agent, taking out, air drying, spraying with 2% ninhydrin ethanol solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution and the reference solution.
(3) Grinding 7.0g of the product, adding 50ml of chloroform, heating and refluxing for 1 hour, filtering, evaporating filtrate to dryness, and dissolving residue in 1ml of chloroform to obtain a sample solution. Taking 1.0g of fructus Schisandrae chinensis as reference material, and making into reference material solution by the same method. Adding methanol into Schizandrol A control to obtain solution containing 1mg of Schizandrol A per 1ml, and making into control solution. Performing thin layer chromatography (0502 of the four parts of the pharmacopoeia 2015 edition) by sucking 3 μ l of each of the three solutions, and spotting on the same silica gel GF254On the thin layer plate, petroleum ether is added
Figure BDA0002492498800000112
Developing the upper solution of ethyl formate-formic acid (15: 5: 1) as developing agent, taking out, air drying, and inspecting under an ultraviolet lamp (254 nm). In the chromatogram of the test solution, fluorescent spots of the same color appear at the positions corresponding to the chromatogram of the reference solution and the chromatogram of the reference solution.
(4) Taking 6.0g of the product, grinding, adding water 30ml for dissolving, shaking and extracting with water saturated n-butanol solution for 2 times, 25ml each time, combining n-butanol solutions, evaporating to dryness, adding water 10ml for dissolving residue, adding hydrochloric acid 2ml, heating and refluxing for 1 hour, cooling, shaking and extracting with chloroform for 2 times, 25ml each time, combining chloroform solutions, evaporating to dryness, adding methanol 1ml for dissolving residue to obtain test solution. And adding water into radix Ophiopogonis 1.0g for 50ml, decocting for 30min, filtering, adding hydrochloric acid 2ml into the filtrate, and making into control solution. Performing thin layer chromatography (2015 version of Chinese pharmacopoeia 0502, Ministry of the four parts of the Ministry of Japan) test, sucking the two solutions each 8 μ l, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-acetone (4: 1) as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.
(5) Taking 12.0g of the product, grinding, adding petroleum ether
Figure BDA0002492498800000111
Ultrasonic treating 25ml for 2 times, each time for 20 minutes, discarding petroleum ether solution,volatilizing solvent from residue, adding methanol 30ml, ultrasonic treating for 30min, filtering, evaporating filtrate, adding water 30ml and hydrochloric acid 0.5ml into residue, heating for 1 hr, cooling, extracting with chloroform under shaking for 2 times, each time 25ml, mixing chloroform solutions, evaporating, and dissolving residue with methanol 1ml to obtain sample solution. Preparing 2.0g of rhizoma Dioscoreae as reference material, and making into reference material solution by the same method. Performing thin layer chromatography (2015 version of Chinese pharmacopoeia 0502, Ministry of the four parts of the Ministry of Japan) test, sucking 5 μ l of the above two solutions, respectively dropping on the same silica gel G thin layer plate, developing with toluene-acetone (5: 1) as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.
EXAMPLE 3 method for measuring the content of the composition of the present invention
The schizandrol A is determined by high performance liquid chromatography (0512 in the four headings of the 2015 pharmacopoeia of China).
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; methanol-water (60: 40) is used as a mobile phase; the detection wavelength was 250 nm. The number of theoretical plates is not less than 5000 calculated according to the schizandrol A peak.
Preparation of control solution A proper amount of schizandrol A control is precisely weighed, placed in brown measuring flask, and added with methanol to obtain solution containing 40 μ g per 1 ml.
Preparation of test solution the product under the condition of different loading amount is taken, ground, about 2.0g is taken, precisely weighed, placed in a conical flask with a plug, precisely added with 25ml of methanol, tightly plugged, weighed, ultrasonically treated (power 250W, frequency 20kHz) for 30 minutes, cooled, weighed again, supplemented with methanol to the loss weight, shaken evenly, filtered, and a subsequent filtrate is taken, thus obtaining the test solution.
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.
The product contains Schisandra chinensis fruit and schizandrol A (C) per g24H32O7) Calculated, the content of the active ingredient should not be less than 0.35 mg.
Ephedrine hydrochloride and pseudoephedrine hydrochloride were determined by high performance liquid chromatography (0512 in the four-part general rules of the pharmacopoeia 2015).
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-0.2% phosphoric acid water solution (5: 95) is used as a mobile phase; the detection wavelength was 207 nm. The number of theoretical plates is not less than 5000 calculated according to ephedrine hydrochloride peak.
Preparation of reference solution ephedrine hydrochloride reference and pseudoephedrine hydrochloride reference are weighed precisely, and mixed solution containing 30 μ g and 15 μ g per lml is prepared by adding methanol.
Preparation of test solution the product under the condition of different loading amount is taken, ground, about 3.0g is taken, precisely weighed, placed in a conical flask with a plug, 20ml of methanol is precisely added, the plug is sealed, the weight is weighed, ultrasonic treatment (power 250W, frequency 20kHz) is carried out for 30 minutes, the flask is cooled, the weight is weighed again, the weight loss is complemented by the methanol, shaken up and filtered, 10ml of subsequent filtrate is taken, and added into a neutral alumina column (a) (neutral alumina column) (a) (20 kHz)
Figure BDA0002492498800000121
Mesh, 2g, inner diameter of 1.5cm), eluting with methanol, collecting eluate and eluate, placing in 25ml measuring flask, adding 50% methanol to scale, shaking, filtering, and collecting 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.
Each g of the product contains ephedrine hydrochloride (C) to herba Ephedrae10H15NO. HC1) and pseudoephedrine hydrochloride (C)10H15NO. HC1) of not less than 0.5 mg.

Claims (5)

1. A traditional Chinese medicine composition for preventing and treating asthma in remission stage is characterized by being prepared from the following raw materials:
Figure FDA0002492498790000011
2. the traditional Chinese medicine composition for preventing and treating asthma in remission stage according to claim 1, which is characterized by being prepared from the following raw materials:
Figure FDA0002492498790000012
3. the preparation method of the traditional Chinese medicine composition for preventing and treating asthma in remission according to claim 1 or 2, which is characterized by comprising the following steps:
a. taking pilose antler, decocting with 10 times of water for 3 times, each time for 2 hours, filtering, combining filtrates, and concentrating to obtain thick paste with the relative density of 1.30-1.35 at 60 ℃ for later use;
b. crushing fructus schizandrae into coarse powder, performing reflux extraction for 3 times with 85% ethanol in an amount which is 6 times that of the coarse powder, performing 2 hours each time, filtering, collecting dregs in another container, combining filtrates, recovering ethanol, and concentrating to obtain thick paste with a relative density of 1.30-1.35 at 60 ℃ for later use;
c. decocting residues and six medicines of radix ophiopogonis, semen coicis, Chinese yam, radix asparagi, bitter almond, ephedra and the like with 10 times of water for 2 times, each time for 2 hours, filtering, combining filtrates, standing, taking supernate, concentrating the supernate into fluid extract with the relative density of 1.06-1.10 at 60 ℃, adding one time of ethanol, stirring uniformly, refrigerating for 12-48 hours, filtering to take supernate, recovering ethanol, concentrating into thick paste with the relative density of 1.30-1.35 at 60 ℃ for later use;
d. mixing the above three soft extracts, drying at 60 deg.C, pulverizing into fine powder, adding dextrin and sucrose powder at a ratio of 1: 4, mixing uniformly, granulating with 90% ethanol, drying, and finishing.
4. The identification method of the traditional Chinese medicine composition for preventing and treating asthma in remission according to claim 1, 2 or 3, which is characterized in that:
a. collecting 3.0g of the product, grinding, adding water 30ml for dissolving, loading on strong acid cation exchange resin column with inner diameter of 3cm and length of 10cm, eluting with water 100ml, discarding water eluate, eluting with 1% ammonia water 80ml, collecting eluate, evaporating to dryness, and dissolving residue with 70% ethanol 1ml to obtain sample solution; taking 0.5g of cornu Cervi Pantotrichum reference material, adding 5ml of 70% ethanol, performing ultrasonic treatment for 15 min, and filtering to obtain filtrate as reference material solution; pipetting 3 mul of each of the two solutions, spotting the solution on the same silica gel G thin layer plate, and mixing the solution with n-butanol-glacial acetic acid-water (3: 1: 1 is a developing agent, is developed, is taken out, is dried in the air, is sprayed with 2 percent ninhydrin ethanol solution, and is heated at 105 ℃ until the spots are developed clearly; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the reference solution;
b. grinding 5.0g of the product, adding water 30ml for dissolving, adding concentrated ammonia solution 5ml, mixing, extracting with diethyl ether for 2 times (30 ml each time), mixing diethyl ether solutions, volatilizing, and dissolving the residue with methanol 1ml to obtain test solution; taking herba Ephedrae reference material 0.5g, adding concentrated ammonia solution 0.5ml, adding chloroform 10ml, heating and refluxing for 1 hr, filtering, evaporating filtrate, dissolving residue with methanol 1ml to obtain reference material solution; adding methanol into ephedrine hydrochloride reference to obtain 0.5mg solution per 1ml as reference solution; the three solutions were pipetted at 5 μ l each, spotted on the same silica gel G thin layer plate, and mixed with chloroform-methanol-concentrated ammonia solution at 10: 2: 0.3 is developing agent, developing, taking out, air drying, spraying 2% ninhydrin ethanol solution, heating at 105 deg.C until the color of the spot is clear; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution;
c. grinding 7.0g of the product, adding 50ml of chloroform, heating and refluxing for 1 hour, filtering, evaporating filtrate to dryness, and dissolving residue with 1ml of chloroform to obtain sample solution; preparing 1.0g of fructus Schisandrae reference medicinal material, and preparing reference medicinal solution by the same method; adding methanol into schisandrin control to obtain 1mg solution per 1ml as control solution; sucking the three solutions each 3. mu.l, dropping on the same silica gel GF254On the thin layer plate, the weight ratio of petroleum ether-ethyl formate-formic acid is 15: 5: 1, taking the upper solution as a developing agent, developing, taking out, airing, and placing under an ultraviolet lamp at 254nm for inspection; in the chromatogram of the test solution, at the position corresponding to the chromatogram of the reference solution and the chromatogram of the reference solution,fluorescent spots showing the same color;
d. taking 6.0g of the product, grinding, adding water 30ml for dissolving, shaking and extracting with water saturated n-butanol solution for 2 times, 25ml each time, combining n-butanol solution, evaporating to dryness, adding water 10ml to the residue for dissolving, adding hydrochloric acid 2ml, heating and refluxing for 1 hr, cooling, shaking and extracting with chloroform for 2 times, 25ml each time, combining chloroform solution, evaporating to dryness, adding methanol 1ml to the residue for dissolving to obtain a sample solution; decocting radix Ophiopogonis 1.0g with water 50ml for 30min, filtering, adding hydrochloric acid 2ml into the filtrate, and making into control solution; pipette 8 μ l of each of the two solutions, spot on the same silica gel G thin layer plate, and mix with chloroform-acetone ═ 4: 1 is developing agent, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until the spots are clearly developed; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the reference solution;
e. taking 12.0g of the product, grinding, adding 25ml of petroleum ether, carrying out ultrasonic treatment for 2 times, each time for 20 minutes, removing petroleum ether liquid, volatilizing solvent from residue, adding 30ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, drying filtrate by distillation, adding 30ml of water and 0.5ml of hydrochloric acid into residue, heating for 1 hour, cooling, shaking and extracting for 2 times by using trichloromethane, each time for 25ml, combining trichloromethane liquid, drying by distillation, adding 1ml of methanol into residue to dissolve the residue to obtain a sample solution; preparing 2.0g of rhizoma Dioscoreae as reference material, and making into reference material solution by the same method; pipette 5 μ l of each of the two solutions, spot on the same silica gel G thin layer plate, and mix with 5: 1 is developing agent, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until the spots are clearly developed; spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.
5. The method for measuring the content of the traditional Chinese medicine composition for preventing and treating the remission stage of asthma as claimed in claim 1, 2 or 3, which is characterized in that:
a. determination of schizandrol A content
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; methanol-water ═ 60: 40 is a mobile phase; the detection wavelength is 250nm, and the number of theoretical plates is not less than 5000 according to the peak of schizandrol A;
preparing a reference solution by precisely weighing a proper amount of schizandrol A reference, placing into a brown measuring flask, and adding methanol to obtain a solution containing 40 μ g of methanol per 1 ml;
preparing a test solution, grinding the product with different loading amounts, precisely weighing about 2.0g, placing into a conical flask with a plug, precisely adding 25ml of methanol, sealing the plug, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, supplementing the weight loss with methanol, shaking up, filtering, and taking the subsequent filtrate;
the determination method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, and determining;
b. determination of ephedrine hydrochloride and pseudoephedrine hydrochloride content
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; mixing acetonitrile-0.2% phosphoric acid water solution: 95 is a mobile phase; the detection wavelength is 207nm, and the number of theoretical plates is not less than 5000 calculated according to ephedrine hydrochloride peak;
preparing reference solution by precisely weighing appropriate amount of ephedrine hydrochloride reference and pseudoephedrine hydrochloride reference, and adding methanol to obtain mixed solution containing 30 μ g and 15 μ g per lml;
preparing a test solution, taking a product with different loading amounts, grinding, taking about 3.0g, precisely weighing, placing in a conical flask with a plug, precisely adding 20ml of methanol, sealing the plug, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, supplementing the weight loss by using methanol, shaking up, filtering, taking 10ml of subsequent filtrate, adding on a neutral alumina column with 100-200 meshes, 2g and an inner diameter of 1.5cm, eluting by using methanol, collecting effluent liquid and eluent, placing in a 25ml measuring flask, adding 50% of methanol to scale, shaking up, filtering, and taking the subsequent 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.
CN202010409149.0A 2020-05-14 2020-05-14 Preparation and quality control method of traditional Chinese medicine composition for preventing and treating asthma in remission stage Pending CN111467443A (en)

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