WO2022134244A1

WO2022134244A1 - Cold and cough granules and preparation method therefor

Info

Publication number: WO2022134244A1
Application number: PCT/CN2021/072048
Authority: WO
Inventors: 刘景萍; 刘全国; 陈克领; 吴育强; 郑国菊; 王家; 李党
Original assignee: 海南葫芦娃药业集团股份有限公司
Priority date: 2020-12-21
Filing date: 2021-01-15
Publication date: 2022-06-30
Also published as: CN112451633A

Abstract

Cold and cough granules and a preparation method therefor, the method comprising the following steps: taking 15-25% of trichosanthes root according to a prescription, crushing, passing same through a sieve of 70-90 meshes to obtain a fine powder of trichosanthes root; taking the remaining amount in the prescription of trichosanthes root with honeysuckle, loquat leaves, stemona root, and balloon flower root, crushing, and passing same through a sieve of 30-50 meshes, adding an ethanol solution for extraction, then filtering, and saving the resulting medicinal residue for later use, recovering ethanol from the filtrate until no smell of alcohol is present so as to obtain an extraction solution A; taking the medicinal residue, adding a sodium hydroxide solution to same for ultrasonic extraction, filtering, and adjusting the pH value of the filtrate to neutral by using a hydrochloric acid solution, so as to obtain an extraction solution B; combining the extraction solution A with the extraction solution B, concentrating same to obtain a clear paste, adding the fine powder of trichosanthes root, sucrose and dextrin to same, and mixing, granulating and drying to obtain granules; taking eucalyptus oil, encapsulating with hydroxypropyl-β-cyclodextrin to obtain an encapsulated matter; and taking the granules and the encapsulated matter, adding talc to same, and mixing well to prepare the cold and cough granules.

Description

A kind of cold and cough granule and preparation method thereof

technical field

The invention relates to the technical field of pharmaceutical preparations, in particular to a cold and cough granule and a preparation method thereof.

Background technique

The prescription of the present invention is a variety "Cold Cough Granule", which is included in the third volume of "Medicine Standards of the Ministry of Health of the People's Republic of China" for traditional Chinese medicine formulations. The medicine has the effects of clearing away heat and detoxifying, relieving cough and resolving phlegm. It is used for colds, fever, headache and cough. The original dosage form "Gao Gan Chou Granule" has a history of clinical application for many years, and has good clinical efficacy, and no toxic and side effects have been reported. In addition to the original dosage form "Cold Cough Granule", a number of companies have conducted research on its modified dosage form, and produced "Cold Cough Granules", "Cold Cough Tablets" and "Cough Cough Capsules".

The cold and cough granules can be flushed into liquid when used, the medicine is convenient to take, the patient's compliance is good, and there is a good market prospect. However, there is volatile ingredient eucalyptus oil in this prescription, which is directly added to the granules, and is volatile and unstable under the conditions of water, light and heat, and in the prior art, the extraction of various medicinal materials in this prescription is The method still uses the original method. With the extensive development of basic scientific research, it is necessary to further study the active ingredients or effective parts in the medicinal materials, so as to change the original extraction and purification method or production process, so that the product can obtain better curative effect and efficiency. stability.

SUMMARY OF THE INVENTION

In view of this, the present invention aims at the shortcomings of the original medicinal material extraction method and production process of the original cold and cough granules, and studies the extraction of the active ingredients of the medicinal materials and the preparation production process, so as to provide a cold and cough with good curative effect, stable and controllable product quality, and low production energy consumption. Preparation method of granules.

The technical scheme of the present invention is achieved as follows: a preparation method of cold and cough granules, comprising the following steps:

S1, respectively take honeysuckle, loquat leaves, Baibu, Platycodon grandiflorum, Trichosanus, cleanly select, and dry;

S2, take by weighing the components according to the prescription amount: honeysuckle, loquat leaf, Baibu, Platycodon grandiflorum, Trichosanthes, eucalyptus oil;

S3, take 15-25% of the trichosanthin of the prescription, pulverize, and pass through a 70-90 mesh sieve to obtain fine powder of trichosanthin;

S4, take the medicinal materials of Trichosanthes and honeysuckle, loquat leaves, Baibu, and Platycodon grandiflorum in the prescription balance, pulverize, pass through a 30-50 mesh sieve, add an ethanol solution for extraction, filter, and use medicinal residues for later use. Obtain extract A;

S5, get the medicinal dregs of step S4, add sodium hydroxide solution ultrasonic extraction, filter, filtrate is adjusted pH value to neutrality with hydrochloric acid solution, obtains extract B;

S6. Combine the extraction solution A of step S4 and the extraction solution B of step S5, concentrate it to a clear paste, add the fine powder of trichosanthin, sucrose, and dextrin of step S3, mix well, granulate, and dry to obtain granules;

S7, get eucalyptus oil, with hydroxypropyl-β-cyclodextrin inclusion, obtain inclusion compound;

S8, take the granules obtained in step S6, add the inclusion compound of step S7, then add talcum powder, and mix evenly to prepare cold and cough granules.

Further, in step S4, the extraction method is ultrasonic extraction, soaking for 0.5-1.5 hours before ultrasonication, the volume concentration of the ethanol solution is 55-65%, and the addition amount of the ethanol solution is 6-8% of the weight of the medicinal material. times, ultrasonic extraction for 20-40 minutes, repeated 2 to 3 times. More preferably, the volume concentration of the ethanol solution is 60%, soaked for 1 hour before ultrasonication, and ultrasonically extracted twice for 30 minutes each time.

Further, in step S5, the volume concentration of the sodium hydroxide solution is 0.008-0.012mol/L, the addition amount of the sodium hydroxide solution is 4-5 times the weight of the medicinal residue, and the ultrasonic extraction is performed for 20 to 40 minutes, Repeat 2 to 3 times.

Further, in step S5, the concentration of the hydrochloric acid solution is 0.8-1.2 mol/L.

Further, in step S6, the relative density of the clear paste at 80°C is 1.20-1.25.

Further, in step S7, the method for encapsulating eucalyptus oil with hydroxypropyl-β-cyclodextrin is as follows: take hydroxypropyl-β-cyclodextrin, add water to make a saturated solution, and stir at a constant temperature of 38-42 °C. , add eucalyptus oil, the weight-to-volume ratio of hydroxypropyl-β-cyclodextrin and eucalyptus oil kg/L is 9-12:1, continue stirring at constant temperature for 0.8-2 hours, put it in the refrigerator for 0.8-2 hours, take out, pump Filtration to obtain the inclusion compound and place it under vacuum drying at 30-40°C for 3-4 hours to obtain the volatile oil inclusion compound. More preferably, the weight-to-volume ratio kg/L of the hydroxypropyl-β-cyclodextrin and eucalyptus oil is 10:1, the temperature of the constant temperature stirring is 40°C, and the time is 1 hour.

Further, each component recipe quantity is: 750 parts by weight of honeysuckle, 1,800 parts by weight of loquat leaves, 550 parts by weight of 100 parts, 750 parts by weight of Platycodon grandiflorum, 250 parts by weight of Trichosanthes, 35 parts by weight of eucalyptus, and when the unit of measure is kg, The unit of measurement for volume is L.

A cold and cough granule is prepared by the preparation method of any one of the cold and cough granules of the present invention.

Compared with the prior art, the beneficial effects of the present invention are:

1. the present invention carries out the hydroxypropyl-β-cyclodextrin inclusion of the eucalyptus oil component in the prescription, and makes the inclusion compound, which can effectively reduce the loss of the volatile oil of the finished product in the processes such as storage and transportation, and improve the stability of the product. sex, thereby ensuring the efficacy of the drug.

2. The present invention extracts the active ingredients in the medicinal materials: such as flavonoids, saponins, triterpenes, organic acids and other anti-inflammatory, cough-relieving and phlegm-relieving ingredients, thereby increasing the content of the main active ingredients in the product. Compared with the product prepared by the original production process, the content of chlorogenic acid is increased by more than 40%.

3. The cold and cough granules prepared by the present invention have the functions of clearing away heat and detoxifying, relieving cough and resolving phlegm, and are used for cold and fever, headache and cough. The cold and cough granules of the improved extraction method and production process have improved product curative effect. The clinical test results show that the total effective rate of the product of the present invention in treating cold and cough reaches 96%, which is obviously higher than that of the cold and cough granules obtained by the original production process.

4. The present invention utilizes the characteristic of trichosanthin that is rich in starch, directly takes part of the fine powder of trichosanthin as the excipient in the granulation process, and reduces the addition of raw and auxiliary materials. The preparation method of the invention is simple and easy to operate, and compared with the original production process method, the production time is shortened, the energy consumption is reduced, and the production cost is saved.

Detailed ways

In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

The experimental methods used in the embodiments of the present invention are conventional methods unless otherwise specified.

Materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources unless otherwise specified.

1. Research on the extraction process

1. The extraction process of honeysuckle

The present invention has compared following extraction method:

(1) Method 1 (original production process method): take 500 g of honeysuckle, add 10 times the amount and 8 times the amount of water and decoct twice, 2 hours each time, filter, and concentrate the filtrate to a thick paste.

(2) Method 2: take 500 g of honeysuckle, add 8 times the weight of 60% (v/v) ethanol solution, soak for 1 h, ultrasonicate for 30 minutes, filter, recover ethanol from the filtrate, and concentrate to a thick paste.

(3) Method 3: take 500 g of honeysuckle, add 8 times the weight of 60% (v/v) ethanol solution, soak for 1 hour, heat under reflux for 60 minutes, filter, return the filtrate to ethanol, and concentrate to a thick paste.

(4) Method 4: take 500 g of honeysuckle, add 8 times the weight of water, soak for 1 h, ultrasonicate for 30 minutes, filter, and concentrate the filtrate to a thick paste.

Get the thick paste obtained by the above method respectively, measure its paste rate and chlorogenic acid content, the results are shown in Table 1.

Table 1 Extraction results of honeysuckle by different extraction methods

方法method	得膏率(％)Paste rate (%)	绿原酸含量(％)Chlorogenic acid content (%)
11	12.1312.13	1.701.70
22	9.069.06	2.132.13
33	13.2013.20	2.222.22
44	8.148.14	1.561.56

As can be seen from Table 1, the paste yield of method 1 (former production process method) is higher but the chlorogenic acid content is lower, the paste yield of method 4, the chlorogenic acid content are all lower, and the method 3 yield is the highest, and the green The ortho acid content is also the highest, and the method 2 yields a lower paste, but the chlorogenic acid content is also higher, indicating that this method has fewer impurities in the extract, higher active ingredient content, and ethanol solution ultrasonic extraction is relatively safe, comprehensive consideration. , ultrasonic extraction is preferred in the present invention.

On the basis of the above comparative experimental results, a screening test for ethanol concentration was also carried out. Using method 2, 40% (v/v) ethanol, 50% (v/v) ethanol and 60% (v/v) ethanol were compared. , 70% (v/v) ethanol, respectively measure the content of chlorogenic acid in the obtained thick paste, the results are shown in Table 2.

Table 2 Extraction results of honeysuckle with different concentrations of ethanol solutions

乙醇溶液weak	绿原酸含量(％)Chlorogenic acid content (%)
40％40%	1.911.91
50％50%	2.022.02
60％60%	2.312.31
70％70%	2.292.29

As can be seen from Table 2, when 60% (v/v) ethanol and 70% (v/v) ethanol are used as the extraction solvent, the content of chlorogenic acid is equivalent. Considering the cost, the present invention preferably uses 60% (v/v) ethanol as Extract the solvent.

Considering the complex composition of Chinese medicinal materials, and various components can achieve their efficacy through multi-channel, multi-part, multi-target action. To sum up, the preferred extraction method of honeysuckle and other four medicinal materials in the present invention is as follows: add 8 times the weight of 60% (v/v) ethanol solution for 1 hour and then ultrasonically extract for 30 minutes.

3. Eucalyptus Oil Inclusion Process

Eucalyptus oil is the volatile oil extracted by steam distillation of Eucalyptus globulus Labil L., Lauraceae plant Cinnamomum camphora (L.) Presl or other plants of the same family as above. It has the effect of dispelling wind and relieving pain. Itching and neuralgia are one of the important active ingredients of the cold and cough granules of the present invention. The method of adding eucalyptus oil in the original production process is direct injection. The volatile oil is easy to volatilize during storage and transportation, the content of active ingredients is reduced, and the curative effect of the product is reduced. In order to increase its stability and reduce volatilization, the present invention adopts hydroxypropyl-beta-cyclodextrin to encapsulate it to prepare volatile oil hydroxypropyl-beta-cyclodextrin inclusion compound to ensure its stability.

In the present invention, the amount of the entrapped volatile oil is used as the investigation index, and the L ₉ (3 ⁴ ) orthogonal table is used to arrange the test, and three factors are investigated for the amount of hydroxypropyl-β-cyclodextrin, the entrapment temperature and the entrapment time. The factor level table is shown in Table 3, the orthogonal test results are shown in Table 4, and the variance analysis is shown in Table 5.

Operation method: Take hydroxypropyl-β-cyclodextrin, add water to make a saturated solution, under constant temperature stirring at 35-45 °C, add eucalyptus oil, the weight volume ratio of hydroxypropyl-β-cyclodextrin and eucalyptus oil is kg /L is 8 to 12:1, continue stirring at constant temperature for 1 to 2 hours, place in the refrigerator for 1 hour, take out, suction filtration, and vacuum dry the inclusions at 30 to 40°C for 3 to 4 hours to obtain volatile oil inclusions. Put the inclusion compound in a flask, add water, connect to a volatile oil measuring device, boil for 1-2 hours, stop heating when the amount of oil no longer increases, stand for 1 hour, read and calculate the volatile oil yield.

Table 3 Inclusion factor level table

Table 4 Orthogonal test table

Table 5 Analysis of variance table

F _1-0.05(2,2) =19.0 F _1-0.10(2,2) =9.0 ** means P<0.10

The experimental results show that the factors affecting the yield of encapsulated eucalyptus oil are A>C>B in order, and the optimal combination is A ₃ B ₁ C ₂ , namely: the amount of eucalyptus oil: the amount of hydroxypropyl-β-cyclodextrin=1 : 12(ml:g), the cladding time is 1h, and the cladding temperature is 40℃. The results of variance analysis showed that the amount of hydroxypropyl-β-cyclodextrin had a significant effect on the inclusion amount of volatile oil (P<0.10). It can be seen from the experimental results that A ₂ and A ₃ have little effect on the results. When the dosage of hydroxypropyl-β-cyclodextrin is increased to a certain extent (1:10), the yield will not be improved by increasing the dosage (1:12). Further increase, the stirring time is also the same. From the perspective of saving, choose the optimal solution A ₂ B ₁ C ₂ , that is, the amount of eucalyptus oil: the amount of hydroxypropyl-β-cyclodextrin = 1:10 (ml:g), under constant temperature stirring at 40 °C, add eucalyptus oil, and continue stirring at constant temperature for 1 hour.

Second, the preparation method of cold and cough granules

Example 1

A preparation method of cold and cough granules, the method comprises the following steps:

S2, take by weighing the components according to the following prescription amounts: honeysuckle 75kg, loquat leaf 180kg, 100 parts 55kg, platycodon grandiflorum 75kg, trichosandra 25kg, eucalyptus oil 3.5L;

S3, get 5kg of trichosanthin, pulverize, pass 80 mesh sieves, obtain trichosanthin fine powder;

S4. Take the remaining 20kg of Trichosanthes and honeysuckle, loquat leaves, Baibu, and Platycodon grandiflorum, pulverize, pass through a 40-mesh sieve, add 6 times the weight of 60% (v/v) ethanol solution to soak for 1 hour, and ultrasonically extract 2 times. For 30 minutes, filter, use the residues for later use, and combine the filtrates to recover ethanol until there is no alcohol smell to obtain extract A;

S5, take the medicinal residues under step S4, add 4 times the weight of 0.01mol/L sodium hydroxide solution for ultrasonic extraction 2 times, each time for 30 minutes, filter, combine the filtrates, adjust the pH value to medium with 1.0mol/L hydrochloric acid solution properties, get extract B;

S6. Combine the extract A in step S4 and the extract B in step S5, concentrate to a clear paste with a relative density of 1.20-1.25 at 80°C, add the fine powder of trichosanthin in S3, an appropriate amount of sucrose, and an appropriate amount of dextrin , mixing, granulating, drying to obtain granules;

S7, take 35kg of hydroxypropyl-β-cyclodextrin, add water to make a saturated solution, add eucalyptus oil under constant temperature stirring at 40°C, continue stirring at constant temperature for 1 hour, put it in the refrigerator for 1 hour, take out, suction filtration, and encapsulate The material was dried under vacuum at 30°C for 4 hours to obtain volatile oil inclusions;

S8, take the granules obtained in step S6, add the inclusion compound of step S7, and then add an appropriate amount of talc powder, and mix evenly to obtain the final product.

Example 2

S4. Take the remaining 20kg of Trichosanthes and honeysuckle, loquat leaves, Baibu, and Platycodon grandiflorum, pulverize, pass through a 40-mesh sieve, add 7 times the weight of 60% (v/v) ethanol solution to soak for 1 hour, ultrasonically extract 2 times, each time For 30 minutes, filter, use the residues for later use, and combine the filtrates to recover ethanol until there is no alcohol smell to obtain extract A;

S5, take the medicinal residues under step S4, add 5 times the amount of 0.01mol/L sodium hydroxide solution for ultrasonic extraction 2 times, each time for 30 minutes, filter, combine the filtrates, adjust the pH value to medium with 1.0mol/L hydrochloric acid solution properties, get extract B;

S7, take 35kg of hydroxypropyl-β-cyclodextrin, add water to make a saturated solution, add eucalyptus oil under constant temperature stirring at 40°C, continue stirring at constant temperature for 1 hour, put it in the refrigerator for 1 hour, take out, suction filtration, and encapsulate The material was dried under vacuum at 40°C for 3 hours to obtain volatile oil inclusions;

Example 3

S4. Take the remaining 20kg of Trichosanthophylla medicinal materials, honeysuckle, loquat leaves, Baibu, and Platycodon grandiflorum, pulverize, pass through a 40-mesh sieve, add 8 times the weight of 60% (v/v) ethanol solution to soak for 1 hour, ultrasonically extract twice, each time For 30 minutes, filter, use the residues for later use, and combine the filtrates to recover ethanol until there is no alcohol smell to obtain extract A;

S5, take the medicinal residues under step S4, add 4 times the amount of 0.01mol/L sodium hydroxide solution for ultrasonic extraction twice, each time for 30 minutes, filter, combine the filtrates, adjust the pH value to medium with 1.0mol/L hydrochloric acid solution properties, get extract B;

S6. Combine the extraction solution A in step S4 and the extraction solution B in step S5, concentrate to a clear paste with a relative density of 1.20-1.25 at 80°C, add the fine powder of trichosanthin in S3, an appropriate amount of sucrose, and an appropriate amount of dextrin , mixing, granulating, drying to obtain granules;

S7, take 35kg of hydroxypropyl-β-cyclodextrin, add water to make a saturated solution, add eucalyptus oil under constant temperature stirring at 40°C, continue stirring at constant temperature for 1 hour, put it in the refrigerator for 1 hour, take out, suction filtration, and encapsulate The material was dried under vacuum at 40°C for 4 hours to obtain volatile oil inclusions;

Comparative example 1 (preparation method of original production process)

S3. get the other five flavors such as honeysuckle except eucalyptus oil and decoct with water for 2 times, each time for 2 hours, combine the decoction, filter, and concentrate the filtrate to a thick paste with a relative density of 1.23 at 80 ° C, add 3 times the amount of ethanol , stirred, stood for 24 hours, filtered, and the filtrate was concentrated to a thick paste with a relative density of 1.25 at 80°C;

S4. Take the thick paste, add an appropriate amount of powdered sugar, mix well, make into granules, dry, spray into eucalyptus oil, mix well, and that's it.

3. Stability test

The samples of Examples 1-3 and Comparative Example 1 were packaged in composite film bags, respectively, and placed in an accelerated stability test box to test: temperature: 40±2° C., relative humidity: 75±5%, and accelerated test for 3 months. Results The appearance properties, volatile oil content, main component content of the samples in Examples 1-3 were compared with the measurement results of the samples in 0 months, and there was no significant change, while the appearance properties, volatile oil content, etc. of the sample in Comparative Example 1 were stable. Compared with the measurement results of the samples in 0 months, there are obvious changes in the key performance indicators and the sample measurement results, indicating that the quality of the samples in Examples 1-3 of the present invention is relatively stable, which can meet the stability requirements of storage, transportation and use. Eucalyptus oil is not easily volatile after encapsulation, which increases its stability. In addition, it can also be seen from Table 6 that the measurement results of the main active ingredient chlorogenic acid content of the samples of Examples 1-3 are all higher than those of the Comparative Example 1 sample, indicating that the preparation method of the present invention improves the content of the main active ingredient, thereby improving the Product efficacy. The test results are shown in Table 6.

Table 6 Stability test result table of Ganmao cough granules

4. Pharmacological tests

(1) Animal acute toxicity test

The samples of Example 1-3 of the present invention were administered to mice, administered 3 times within 12h, the total dose was 15g/kg, and BW (adult (according to 60kg) taking the dose was 30mg/kg.d), within 7d of administration All animals survived, with normal appetite and feces, and no significant changes in body weight. Limited to drug concentration and volume, the LD50 could not be measured, so the LD50 must be greater than 15 and/kg, BW (500 times the adult dose). It shows that the samples of Examples 1-3 of the present invention are very safe for oral administration.

(2) Animal long-term toxicity test

The samples of Examples 1-3 of the present invention were continuously fed to the rats for 28 days by 60mg/kg, 1500mg/kg and 3000mg/kg (respectively being 2 times, 50 times and 100 times the daily doses intended for clinical use), and the results showed that the animals appeared , behavior, weight gain normal, blood routine, blood biochemistry, liver and kidney function, and histological examination of heart, lung, liver, spleen, kidney, adrenal gland, testis, ovary, etc. were not abnormal. It is shown that taking the samples of Examples 1-3 of the present invention according to the proposed dose, route and course of treatment is safe and has no toxic and side effects.

(3) The main pharmacodynamic test

Objective: To provide experimental basis for clinical medication by investigating the antitussive, expectorant, anti-inflammatory and antibacterial effects of the product of the present invention.

1 Test material

Samples: Samples of Example 2 of the present invention and Comparative Example 1.

Experimental animals: Kunming mice, clean grade, male and female, body weight 20±2g, provided by the animal room of Guangxi Medical University.

2 Methods and results

2.1 Xylene-induced ear swelling experiment in mice

Take 50 Kunming mice and randomly divide them into 5 groups: Example 2 sample high (480mg/kg), medium (240mg/kg), low (120mg/kg) three dose groups, blank control group (with the same volume of 0.9 % physiological saline), the sample of Comparative Example 1 (240 ml/kg). Oral administration, 1 time/d, for 7 days. 30 min after the last administration, the mice in each group were evenly coated with 50 μl of xylene in front of and behind the right ear of the mice to induce inflammation. After 1 hour, the mice were killed by cervical dislocation, and the left and right ears were cut along the baseline of the auricle, and holes were punched with a diameter of 9 mm. The round ear pieces were made on the same part of the two ears by the instrument, and the ear swelling degree and swelling inhibition rate were calculated according to the following formula:

Ear swelling degree (mg) = right ear piece weight - left ear piece weight;

Immediately after the inflammation, the right ear of the mice in each group appeared highly red and swollen. Compared with the blank control group, the difference in the ear swelling between the high, medium and low dose groups of the samples of Example 2 of the present invention and the sample group of Comparative Example 1 was statistically significant (P<0.01 or P<0.05); The swelling degree and swelling inhibition rate of the three different doses of the sample administration group of Example 2 of the present invention showed an obvious dose-dependent relationship. The results show that in the range of 120-480 mg/kg, the sample of Invention Example 2 can significantly reduce the swelling of the auricle in mice caused by xylene, and has an anti-inflammatory effect, and the effect is better than that of the sample of Comparative Example 1 (prepared by the original production method). See Table 7 for details.

Table 7 Effects of xylene-induced ear swelling in mice (x±s)

组别group	nn	剂量(mg/kg)Dosage (mg/kg)	耳肿胀度(mg)Ear swelling (mg)	肿胀抑制率(％)Swelling inhibition rate (%)
高剂量组high dose group	1010	480mg/kg480mg/kg	5.02±4.68 ^ 5.02±4.68 ^	25.7225.72
中剂量组medium dose group	1010	240mg/kg240mg/kg	6.45±4.86 ^ 6.45±4.86 ^	19.7519.75
低剂量组low dose group	1010	120mg/kg120mg/kg	8.91±5.75 ^* 8.91±5.75 ^*	13.4713.47
空白对照组Blank control group	1010	——	12.04±5.3412.04±5.34	——
对比例1Comparative Example 1	1010	240mg/kg240mg/kg	7.53±5.22 ^* 7.53±5.22 ^*	15.7515.75

Note: Compared with blank control group: ^* P＜0.05, ^** P＜0.01

2.2 Cough induced by concentrated ammonia water in mice

Take 50 Kunming mice and randomly divide them into 5 groups: Example 2 sample high (480mg/kg), medium (240mg/kg), low (120mg/kg) three dose groups, blank control group (with the same volume of 0.9 % physiological saline), the sample of Comparative Example 1 (240 ml/kg). Oral administration, 1 time/d, for 7 days. 1h after the last administration, the mice were put into a 4L airtight dry container one by one, sprayed with 25% concentrated ammonia water aerosol at constant pressure, sprayed for 5s to induce cough, and observed the cough incubation period and the number of coughs (every 2min).

After the cough was induced, the mice in each group showed coughing phenomena such as mouth opening and abdominal muscle contraction to varying degrees. The incubation period of cough in mice was significantly prolonged (P<0.01 or P<0.05), and the number of coughs was reduced (P<0.01 or P<0.05). The results showed that with the increase of the dose of the sample of Example 2 of the present invention, the incubation period of coughing in each group of mice was prolonged, and the number of coughs was reduced. The sample of Example 2 of the present invention had a certain antitussive effect, and the effect was better than that of the comparative example. 1 (prepared by the original production process) sample. See Table 8 for details.

Table 8. Cough response of mice induced by concentrated ammonia water

group

Dosage (mg/kg)

Cough incubation period(s)

Number of coughs (times/2min)

高剂量组high dose group	480mg/kg480mg/kg	27.08±2.13 ^ 27.08±2.13 ^	6.13±3.67 ^ 6.13±3.67 ^
中剂量组medium dose group	240mg/kg240mg/kg	21.17±2.26 ^* 21.17±2.26 ^*	9.68±4.73 ^ 9.68±4.73 ^
低剂量组low dose group	120mg/kg120mg/kg	15.85±2.61 ^* 15.85±2.61 ^*	11.42±4.16 ^* 11.42±4.16 ^*
空白对照组Blank control group	——	3.28±1.953.28±1.95	17.13±4.7217.13±4.72
对比例1Comparative Example 1	240mg/kg240mg/kg	17.48±2.55 ^* 17.48±2.55 ^*	10.59±4.67 ^* 10.59±4.67 ^*

Note: Compared with blank control group: ^* P＜0.05, ^** P＜0.01

2.3 Phenol red excretion experiment in mouse trachea

Fifty Kunming mice were randomly divided into 5 groups: high (480mg/kg), medium (240mg/kg), and low (120mg/kg) three-dose groups, blank control group (with the same volume of 0.9% saline) ), the sample of Comparative Example 1 (240ml/kg). Oral administration, 1 time/d, for 7 days. 30min after the last administration, each mouse was intraperitoneally injected with 0.02ml/g body weight of 2.5% phenol red NaHCO ₃ solution. After 30min, the mice were sacrificed, the trachea was dissected and separated, and each tracheal segment was placed in a pre-filled 1.5ml 5% NaHCO solution. _3. In the test tube of the solution, ultrasonically clean it to completely release the phenol red in the trachea section, centrifuge the cleaning solution, take the supernatant liquid at 546nm of the full wavelength microplate reader for colorimetry, measure the absorbance (A) value, and substitute it into the phenol red standard curve , to calculate the phenol red content.

Compared with the blank control group, the secretion of phenol red in the high and medium dose groups of the samples of Example 2 of the present invention and the sample group of Comparative Example 1 (prepared by the original production process method) increased significantly (P<0.05 or P<0.01). Although the secretion of phenol red in the low-dose group of the sample in Example 2 increased to a certain extent, the difference was not statistically significant (P>0.05). The results show that the high and medium dose groups of the samples of Example 2 of the present invention have a certain expectorant effect. See Table 9 for details.

Table 9 Effects on the excretion of phenol red in the tracheal segment of mice

组别group	nn	剂量(ml/kg)Dosage (ml/kg)	酚红含量(μg/ml)Phenol red content (μg/ml)
高剂量组high dose group	1010	480mg/kg480mg/kg	0.351±0.11 ^ 0.351±0.11 ^
中剂量组medium dose group	1010	240mg/kg240mg/kg	0.285±0.10 ^* 0.285±0.10 ^*
低剂量组low dose group	1010	120mg/kg120mg/kg	0.182±0.110.182±0.11
空白对照组Blank control group	1010	——	0.104±0.110.104±0.11
对比例1Comparative Example 1	1010	240mg/kg240mg/kg	0.237±0.10 ^* 0.237±0.10 ^*

Note: Compared with blank control group: ^* P＜0.05, ^** P＜0.01

2.4 Bacteriostatic effect

A certain number of small test tubes were taken and divided into 10 groups, 10 tubes in each group, and 1 ml of nutrient broth medium was added to each tube of each group. 1mL of the sample solution (0.5g/ml) of Example 2 of the present invention was added to the first small test tube of groups 1-5, and 1mL of the sample solution of Comparative Example 1 (0.5g/ml) was added to the first test tube of groups 6-10, and each group was mixed well Then aspirate 1mL of each and add it to the second small test tube, and repeat the decreasing dilution to the ninth tube. After the ninth tube is mixed, aspirate 1mL of the mixture and discard it. The bacterial suspension diluted to 10 ^-3 (the number of bacteria per 1mL is about 100 million cfu), each group is added 0.1mL from the first tube to the eighth tube, and 0.1mL of bacterial solution is also added to the tenth tube, and the ninth tube is added. The tube with drug and no bacteria was used as a negative control, and the tenth tube with bacteria solution and no drug was used as a positive control. The small test tubes prepared above were placed in an incubator at 36° C. for 24-48 hours, and the presence or absence of bacterial growth in each tube was observed with the naked eye. The results are shown in Table 10.

Table 10 Antibacterial titers to experimental strains

The results in Table 10 show that the samples of Example 2 of the present invention have inhibitory effects on Staphylococcus aureus, beta-hemolytic streptococcus, pneumococcus, Haemophilus influenzae and Pseudomonas aeruginosa in vitro, but in vitro With regard to the inhibitory effects of hemolytic streptococcus, Haemophilus influenzae and pneumococcus, the antibacterial results of the samples of Example 2 were better than those of the samples of Comparative Example 1. It is shown that the sample of Example 2 of the present invention has a strong bacteriostatic effect on common pathogenic bacteria in the respiratory tract.

The above pharmacodynamic test results show that the sample of Example 2 of the present invention has a certain inhibitory effect on the ear swelling of mice caused by xylene after oral administration, indicating that it has a certain anti-inflammatory effect; It is significantly reduced, and has a certain dose-effect relationship, indicating that it has a certain antitussive effect; it has an increasing effect on the excretion of phenol red in the lungs of mice, indicating that it has a certain expectorant effect; in vitro antibacterial experiments have proved that the embodiment of the present invention 2 samples have certain inhibitory effects on Staphylococcus aureus, beta-hemolytic streptococcus, Haemophilus influenzae, pneumococcus and Pseudomonas aeruginosa. To sum up, the sample of Example 2 of the present invention has antitussive, expectorant, anti-inflammatory and bacteriostatic effects, and the effect is better than the cold cough granules produced by the original process (sample 1 of Comparative Example), indicating that the preparation method of the product of the present invention is effective. Improved efficacy.

5. Clinical trials

1. Materials and methods

1.1 General information

100 cases of cold and cough patients were selected and randomly divided into 2 groups, namely the experimental group and the control group, with 50 cases in each group. Among them, there were 26 males and 24 females in the experimental group, aged 18 to 60 years, with an average age of (35.60±4.45) years, and a disease duration of 5 to 25 days, with an average of (13.24±2.85) days; the control group had 24 males and 26 females, aged 18 ～60 years old, mean (38.12±4.82) years old, disease duration 6～23 days, mean (12.52±2.82) days. There was no significant difference in general data between the two groups (P>0.5), which was comparable.

1.2 Case selection

The above cases all met the relevant standards for colds and coughs in the Guidelines for Diagnosis and Treatment of Cough (2015 Edition), Guidelines for Diagnosis and Treatment of Common Colds (2015 Edition), and Guidelines for Diagnosis and Treatment of Cough (2015 Edition). Patients with lower respiratory tract infection, signs of bacterial infection, asthma and other diseases were excluded; patients with body temperature >38.5°C were excluded; pregnant women and lactating patients were excluded.

1.3. Treatment

The experimental group was treated with the sample of Example 2 of the present invention, orally, 1 bag at a time, 2-3 times a day. The control group was treated with the sample of Comparative Example 1 of the present invention, orally, 1 bag at a time, 2-3 times a day.

1.4 Efficacy evaluation

The therapeutic effects and adverse reactions of the two groups were observed. The efficacy evaluation criteria are:

(1) Recovery: After treatment, the patient's body temperature returned to normal, and symptoms such as cough were eliminated by more than 95%;

(2) Significantly effective: body temperature returns to normal, and symptoms such as cough are eliminated by more than 70%;

(3) Ineffective: body temperature did not recover or rise, and symptoms such as cough were eliminated <70%.

1.5 Statistical processing

SPSS 22.0 statistical software was used for data processing and analysis, and t-test was used at the same time. When P<0.05, the difference was statistically significant.

2. Results

2.1 Adverse reactions and safety evaluation

During the treatment, there were no adverse reactions in the two groups.

2.2 Comparison of clinical efficacy

Table 11 Comparison of clinical efficacy between the two groups (cases)

组别group	nn	痊愈get well	显效effective	无效invalid	总有效always valid
试验组test group	5050	30(60.00％)30 (60.00%)	18(36.00％)18 (36.00%)	2(4.00％)2 (4.00%)	48(96.00％)48 (96.00%)
对照组control group	5050	19(38.00％)19 (38.00%)	16(32.00％)16 (32.00%)	15(30.00％)15 (30.00%)	35(70.00％)35 (70.00%)

See Table 11. The results show that: the total effective rate and recovery rate of the sample in Example 2 of the present invention are significantly greater than those of the control group in the original production process.

3. Conclusion

The above clinical test results show that the sample of Example 2 produced by the preparation method of the present invention is superior to the cold and cough granules produced by the original production method (the sample of Comparative Example 1) in the treatment of cold and cough, indicating that it improves the curative effect of the product, and takes Safe and reliable, no adverse reactions and side effects.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the present invention. within the scope of protection.

Claims

A preparation method of cold and cough granules, characterized in that, comprising the following steps:

S1, respectively take honeysuckle, loquat leaves, Baibu, Platycodon grandiflorum, Trichosanthinae, cleanly select, and dry;

S2, take by weighing the components according to the prescription amount: honeysuckle, loquat leaf, Baibu, Platycodon grandiflorum, Trichosanthes, eucalyptus oil;

S3, take 15-25% trichosanthin of the prescription, pulverize, pass through a 70-90 mesh sieve, and obtain trichosanthin fine powder;

S4, take the medicinal materials of Trichosanthes and honeysuckle, loquat leaves, Baibu, and Platycodon grandiflorum in the prescription balance, pulverize, pass through a 30-50 mesh sieve, add an ethanol solution for extraction, filter, and use the medicinal residues for later use. Obtain extract A;

S5, take the medicinal residues of step S4, add sodium hydroxide solution to ultrasonically extract, filter, adjust the pH value of the filtrate to neutrality with hydrochloric acid solution to obtain extract B;

S6. Combine the extraction solution A of step S4 and the extraction solution B of step S5, concentrate it to a clear paste, add the fine powder of trichosandra, sucrose, and dextrin of step S3, mix, granulate, and dry to obtain granules;

S7, get eucalyptus oil, use hydroxypropyl-β-cyclodextrin inclusion, obtain inclusion compound;

S8, take the granules obtained in step S6, add the inclusion compound of step S7, then add talc, and mix evenly to prepare cold and cough granules.
The preparation method of cold and cough granules according to claim 1, characterized in that, in step S4, the extraction method is ultrasonic extraction, soaking for 0.5-1.5 hours before ultrasonication, and the volume concentration of the ethanol solution is 55-65% , the added amount of the ethanol solution is 6-8 times the weight of the medicinal material, and the ultrasonic extraction is performed for 20-40 minutes and repeated 2-3 times.
The preparation method of cold and cough granules according to claim 1, is characterized in that, in step S5, the volume concentration of described sodium hydroxide solution is 0.008-0.012mol/L, and the addition amount of described sodium hydroxide solution is medicine 4-5 times the weight of the slag, ultrasonically extract for 20-40 minutes, repeat 2-3 times.
The preparation method of the cold and cough granules according to claim 1, characterized in that, in step S5, the concentration of the hydrochloric acid solution is 0.8-1.2 mol/L.
The preparation method of cold and cough granules according to claim 1, characterized in that, in step S6, the relative density of the clear paste at 80°C is 1.20-1.25.
The preparation method of Ganmao cough granules according to claim 1, is characterized in that, in step S7, the method for encapsulating eucalyptus oil with hydroxypropyl-β-cyclodextrin is: take hydroxypropyl-β-cyclodextrin , add water to make a saturated solution, under constant temperature stirring at 38-42 ℃, add eucalyptus oil, the weight-volume ratio of hydroxypropyl-β-cyclodextrin and eucalyptus oil kg/L is 9-12:1, and continue to stir at constant temperature for 0.8 -2 hours, refrigerate for 0.8-2 hours, take out, and filter with suction to obtain inclusions and place them under vacuum at 30-40°C for 3-4 hours to obtain volatile oil inclusions.
The preparation method of cold and cough granules according to claim 2, wherein in step S4, the volume concentration of the ethanol solution is 60%, soaked for 1 hour before ultrasonication, and ultrasonically extracted twice, 30 minutes each time.
The preparation method of Ganmao cough granules according to claim 7, wherein in step S7, the weight-to-volume ratio kg/L of the hydroxypropyl-β-cyclodextrin and eucalyptus oil is 10:1, and the The temperature of constant temperature stirring was 40°C, and the time was 1 hour.
The preparation method of cold and cough granules according to claim 1, characterized in that, the recipe quantities of each component are: 750 parts by weight of honeysuckle, 1,800 parts by weight of loquat leaves, 550 parts by weight of 100 parts, 750 parts by weight of Platycodon grandiflorum, 250 parts by weight of Trichosanthes parts, 35 parts by volume of eucalyptus oil, when the weight measurement unit is kg, the volume measurement unit is L.
A cold and cough granule, characterized in that, it is prepared by the preparation method of the cold and cough granule according to any one of claims 1-9.