CN113577187A

CN113577187A - Anti-influenza traditional Chinese medicine composition, traditional Chinese medicine extract and preparation method and application thereof

Info

Publication number: CN113577187A
Application number: CN202110862668.7A
Authority: CN
Inventors: 李卫民; 史利卿; 周欣欣; 朱贺年; 冯毅凡
Original assignee: Beijing Yishengtang Medicine Science And Technology Research Co ltd
Current assignee: Beijing Yishengtang Medicine Science And Technology Research Co ltd
Priority date: 2021-07-29
Filing date: 2021-07-29
Publication date: 2021-11-02
Anticipated expiration: 2041-07-29
Also published as: CN113577187B

Abstract

The invention relates to the technical field of anti-influenza medicines, in particular to an anti-influenza traditional Chinese medicine composition, a traditional Chinese medicine extract, and a preparation method and application thereof. The paint comprises the following components in parts by weight: 5-10 parts of notopterygium root, 5-10 parts of divaricate saposhnikovia root, 3-8 parts of schizonepeta spike, 3-8 parts of cassia twig, 3-8 parts of Chinese mosla herb, 3-8 parts of rhizoma atractylodis, 3-8 parts of pepper, 3-6 parts of wild chrysanthemum flower, 3-6 parts of sweet wormwood herb, 3-6 parts of rhizoma anemarrhenae, 3-6 parts of caulis sinomenii and 2-5 parts of liquorice. The anti-influenza traditional Chinese medicine composition provided by the invention still has a good treatment effect on influenza under the condition of not containing ephedra, achieves the purpose of quickly cooling, and can inhibit the rise of body temperature and quickly bring down fever after being administered to a patient for 1 h; in addition, the traditional Chinese medicine composition provided by the invention has obvious functions of inhibiting bacteria, diminishing inflammation, reducing fever, easing pain and regulating immunity.

Description

Anti-influenza traditional Chinese medicine composition, traditional Chinese medicine extract and preparation method and application thereof

Technical Field

The invention relates to the technical field of anti-influenza medicines, in particular to an anti-influenza traditional Chinese medicine composition, a traditional Chinese medicine extract, and a preparation method and application thereof.

Background

Influenza is an acute respiratory infectious disease caused by influenza virus, and is clinically characterized by acute onset, obvious systemic poisoning symptoms, such as high fever, headache, systemic aching pain, weakness and the like, and light respiratory symptoms. Influenza is mainly transmitted by contact and spray, the infectivity is strong, the course of disease of mild patients is short, the disease is usually self-limiting, and severe patients need to be treated by medicaments and can be generally cured.

The traditional Chinese medicine is a medical treasury in China and even in the world, and the traditional Chinese medicine has a history of thousands of years for treating viral infection diseases. The traditional Chinese medicine has obvious anti-influenza virus curative effect, wide anti-influenza virus spectrum and no drug resistance, and has more advantages than the single anti-influenza medicine for treating fever abatement and inflammation reduction and relieving upper respiratory symptoms.

However, the existing traditional Chinese medicine composition for treating cold caused by influenza virus usually comprises ephedra, for example, the patent of publication No. CN 107890503A discloses a traditional Chinese medicine composition for resisting influenza virus, wherein the traditional Chinese medicine composition is prepared by compounding Tibetan ephedra and a plurality of traditional Chinese medicines such as nutgrass galingale rhizome, corydalis tuber, rhubarb, and the like; for example, Daqinglong decoction recorded in Zhang Zhongjing Shang Han Lun is a drastic sweating prescription, which is composed of seven medicines of ephedra, cassia twig, honey-fried licorice root, bitter apricot seed, ginger, Chinese date and gypsum. The ephedra herb is also a key traditional Chinese medicine for quickly reducing fever, and the traditional Chinese medicine formula for treating cold is lack of the ephedra herb, has poor sweating effect and cannot achieve the purpose of quickly reducing the temperature.

Disclosure of Invention

In order to solve the problems, the invention provides an anti-influenza traditional Chinese medicine composition, a traditional Chinese medicine extract, and a preparation method and application thereof. The anti-influenza traditional Chinese medicine composition provided by the invention has a good treatment effect on influenza in accordance with the condition that the Chinese ephedra is not contained, and the purpose of quickly cooling is achieved.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides an anti-influenza traditional Chinese medicine composition which comprises the following components in parts by weight:

5-10 parts of notopterygium root, 5-10 parts of divaricate saposhnikovia root, 3-8 parts of schizonepeta spike, 3-8 parts of cassia twig, 3-8 parts of Chinese mosla herb, 3-8 parts of rhizoma atractylodis, 3-8 parts of pepper, 3-6 parts of wild chrysanthemum flower, 3-6 parts of sweet wormwood herb, 3-6 parts of rhizoma anemarrhenae, 3-6 parts of caulis sinomenii and 2-5 parts of liquorice.

The invention also provides a preparation method of the anti-influenza traditional Chinese medicine extract, which comprises the following steps:

1) subjecting Chinese medicinal powder corresponding to the above Chinese medicinal composition to supercritical CO₂Extracting to obtain first volatile oil and Chinese medicinal residue;

2) subjecting the Chinese medicinal residue to supercritical CO treatment in 95% ethanol water solution₂Extracting to obtain a second volatile oil;

and mixing the first volatile oil and the second volatile oil to obtain the anti-influenza traditional Chinese medicine extract.

Preferably, the supercritical CO is used in step 1) and step 2)₂The extraction pressure and the extraction temperature are respectively 25-32 Mpa and 35-45 ℃.

Preferably, the supercritical CO is used in step 1) and step 2)₂Extracted CO₂The extraction flow rates were 25L/h/kg, respectively.

Preferably, the supercritical CO in step 1)₂The extraction time of the extraction is 55-65 min, the separation pressure is 8-10 Mpa, and the separation temperature is 48-52 ℃.

Preferably, the supercritical CO in step 2)₂The extraction time of the extraction is 35-45 min, the separation pressure is 4-6 Mpa, and the separation temperature is 33-37 ℃.

Preferably, the volume-weight ratio of the ethanol aqueous solution in the step 2) to the Chinese medicine powder in the step 1) is (0.25-0.5) L:1 Kg.

The invention also provides application of the traditional Chinese medicine composition or the traditional Chinese medicine extract prepared by the preparation method in preparing a medicine for treating influenza.

The invention also provides application of the traditional Chinese medicine composition or the traditional Chinese medicine extract prepared by the preparation method in preparing medicines with various effects, wherein the various effects comprise one or more of bacteriostasis, inflammation diminishing, defervescence, pain easing and immunity regulating.

The invention also provides anti-influenza traditional Chinese medicine particles which comprise the traditional Chinese medicine extract prepared by the preparation method and particle auxiliary materials.

Has the advantages that:

the invention provides an anti-influenza traditional Chinese medicine composition which comprises the following components in parts by weight: 5-10 parts of notopterygium root, 5-10 parts of divaricate saposhnikovia root, 3-8 parts of schizonepeta spike, 3-8 parts of cassia twig, 3-8 parts of Chinese mosla herb, 3-8 parts of rhizoma atractylodis, 3-8 parts of pepper, 3-6 parts of wild chrysanthemum flower, 3-6 parts of sweet wormwood herb, 3-6 parts of rhizoma anemarrhenae, 3-6 parts of caulis sinomenii and 2-5 parts of liquorice. The formula of the traditional Chinese medicine composition provided by the invention takes Qiang prevention agent as a main component, and strengthens the products for regulating the middle warmer and resolving dampness according to the common clinical characteristics of exterior excess due to wind-cold and dampness obstruction of influenza, so that the traditional Chinese medicine composition has the effects of relieving exterior syndrome, dispelling cold, dispelling wind and eliminating dampness, and is used for treating the symptoms of wind-cold and dampness; wherein the notopterygium root and the divaricate saposhnikovia root are monarch drugs and play roles in relieving exterior syndrome, dispelling cold, expelling wind and removing dampness; herba Schizonepetae, ramulus Cinnamomi, herba Moslae, rhizoma Atractylodis and fructus Piperis are used as ministerial drugs for inducing sweat, relieving exterior syndrome, eliminating dampness, regulating the middle warmer, dispelling pathogenic wind and relieving pain; rhizoma anemarrhenae, sweet wormwood, wild chrysanthemum flower and caulis sinomenii are adjuvant drugs, wherein the sweet wormwood expels pathogenic factors to reach the outside, the rhizoma anemarrhenae purges heat and moistens lung, and the wild chrysanthemum flower clears heat and detoxifies heat and detoxicate, thereby assisting the effect of relieving fever and assisting the pungent and warm properties of various drugs of monarch and minister drugs; caulis Sinomenii has the effects of dispelling pathogenic wind, dredging collaterals, dispelling pathogenic wind, eliminating pathogenic factors and relieving pain; the liquorice is used as a guiding drug and plays the roles of clearing heat and removing toxicity and harmonizing the effects of the other drugs. Meanwhile, the pepper and the caulis sinomenii have the effects of promoting the sweating of a patient and quickly reducing fever in the formula. Experiments show that the traditional Chinese medicine composition provided by the invention can inhibit the body temperature rise and realize quick fever reduction after a patient takes the traditional Chinese medicine composition for 1 hour.

Detailed Description

If not specifically stated, the sources of the components of the Chinese medicinal composition of the invention have no special requirements, and the invention can be realized by adopting commercial products which are well known to those skilled in the art.

The traditional Chinese medicine composition provided by the invention comprises 5-10 parts by weight of notopterygium root, preferably 6-9 parts by weight, and more preferably 7 parts by weight. The notopterygium root has the effects of relieving exterior syndrome with pungent and warm natured drugs, dispelling cold, eliminating dampness and relieving pain, and is mainly used for treating wind-cold.

The traditional Chinese medicine composition provided by the invention comprises 5-10 parts by weight of divaricate saposhnikovia root, preferably 6-9 parts by weight, and more preferably 8 parts by weight. The divaricate saposhnikovia root has the effects of dispelling wind, relieving exterior syndrome, eliminating dampness, relieving pain, relieving spasm and the like; it is mainly used for treating cold headache, rheumatalgia, rubella pruritus, tetanus, etc.

The notopterygium root and the divaricate saposhnikovia root are monarch drugs and play roles of relieving exterior syndrome, dispelling cold, expelling wind and removing dampness together.

The traditional Chinese medicine composition provided by the invention comprises 3-8 parts by weight of schizonepeta spike, preferably 4-7 parts by weight, and more preferably 5 parts by weight. The schizonepeta spike has the effects of sweating, dispelling cold, resisting bacteria, diminishing inflammation, stopping bleeding and relieving exterior syndrome; is mainly applied to the conditions of cold, headache, nasal obstruction and the like.

The traditional Chinese medicine composition provided by the invention comprises 3-8 parts by weight of cassia twig, preferably 4-7 parts by weight, and more preferably 5 parts by weight. The cassia twig has warm nature, pungent and sweet taste and the effects of inducing sweat, expelling pathogenic factors from muscles, warming meridians and promoting blood circulation.

The traditional Chinese medicine composition provided by the invention comprises 3-8 parts by weight of elsholtzia, preferably 4-7 parts by weight, and more preferably 5 parts by weight. The elsholtzia herb has the functions of sweating, relieving exterior syndrome, regulating the middle warmer and promoting diuresis.

The traditional Chinese medicine composition provided by the invention comprises 3-8 parts by weight of rhizoma atractylodis, preferably 4-7 parts by weight, and more preferably 5 parts by weight. The rhizoma atractylodis is bitter in nature, pungent and warm in taste, and has the effects of drying dampness, strengthening spleen and dispelling wind and dampness.

The traditional Chinese medicine composition provided by the invention comprises 3-8 parts by weight of pepper, preferably 4-7 parts by weight, and more preferably 5 parts by weight. The pepper of the invention is pungent in taste and hot in nature; entering stomach and large intestine meridians; has effects of warming spleen and stomach for dispelling cold, dispelling cold of spleen and stomach, dispelling cold evil in stomach to stimulate appetite, and also has effects of descending qi, activating stagnancy, eliminating phlegm and relieving chest stuffiness.

The schizonepeta spike, the cassia twig, the elsholtzia, the rhizoma atractylodis and the pepper are ministerial drugs and play roles of inducing sweat, relieving exterior syndrome, eliminating dampness, regulating the middle warmer, dispelling wind and relieving pain together. In the invention, the parts by weight of schizonepeta spike, cassia twig, elsholtzia, rhizoma atractylodis and pepper in the traditional Chinese medicine composition are preferably the same.

The traditional Chinese medicine composition provided by the invention comprises 3-6 parts by weight of wild chrysanthemum flower, preferably 3.5-5 parts by weight, and more preferably 4 parts by weight. The wild chrysanthemum flower of the invention has the functions of clearing away heat and toxic material, and soothing wind and cooling liver.

The traditional Chinese medicine composition provided by the invention comprises 3-6 parts by weight of sweet wormwood, preferably 3.5-5 parts by weight, and more preferably 4 parts by weight. The sweet wormwood has the effects of cooling blood, stopping bleeding, clearing heat, detoxifying and resisting malaria.

The traditional Chinese medicine composition provided by the invention comprises 3-6 parts by weight of rhizoma anemarrhenae, preferably 3.5-5 parts by weight, and more preferably 4 parts by weight. The rhizoma anemarrhenae can nourish yin, moisten dryness and tonify kidney, is used for treating bone steaming tidal fever caused by yin deficiency and fire excess and lung and kidney deficiency, can treat night sweat and vexation, and has the effects of nourishing yin and lowering fire.

The traditional Chinese medicine composition provided by the invention comprises 3-6 parts by weight of caulis sinomenii, preferably 3.5-5 parts by weight, and more preferably 4 parts by weight. The caulis sinomenii has the effects of dispelling wind, removing dampness, inducing diuresis and reducing edema.

The anemarrhena, the sweet wormwood, the wild chrysanthemum flower and the orientvine are adjuvant drugs, wherein the sweet wormwood penetrates through pathogenic factors and reaches outside, the Zhimu purges heat and moistens lung, and the wild chrysanthemum flower clears heat and detoxifies, so that the effects of assisting in clearing heat can be achieved, and the pungent and warm properties of various drugs of monarch and minister can be assisted and processed; caulis Sinomenii has the effects of dispelling pathogenic wind, dredging collaterals, dispelling pathogenic wind, eliminating pathogenic factors and relieving pain.

The traditional Chinese medicine composition provided by the invention comprises 2-5 parts by weight of liquorice, preferably 3.5-4.5 parts by weight, and more preferably 4 parts by weight. The liquorice is a messenger drug and has the effects of tonifying spleen and qi, clearing away heat and toxic materials, eliminating phlegm and stopping cough and relieving pain.

In the invention, the weight parts of rhizoma anemarrhenae, sweet wormwood, wild chrysanthemum flower, caulis sinomenii and liquorice in the traditional Chinese medicine composition are preferably the same.

The traditional Chinese medicine composition provided by the invention can be rapidly cooled without using ephedra, and has an outstanding treatment effect on influenza.

The traditional Chinese medicine composition is preferably crushed to obtain traditional Chinese medicine powder before extraction. In the invention, the particle size of the traditional Chinese medicine powder is preferably 20-60 meshes, and more preferably 40 meshes. The method of pulverization in the present invention is not particularly limited, and pulverization methods known to those skilled in the art may be employed.

After obtaining the Chinese medicinal powder, subjecting the Chinese medicinal powder corresponding to the Chinese medicinal composition to supercritical CO₂Extracting to obtain the first volatile oil and the traditional Chinese medicine residue. In the present invention, the supercritical CO in the step 1)₂The extraction pressure of the extraction is preferably 25-32 Mpa, and more preferably 29 Mpa; supercritical CO described in step 1)₂The extraction temperature of the extraction is preferably 35-45 ℃, and more preferably 45 ℃; supercritical CO described in step 1)₂Extracted CO₂The extraction flow is preferably 25L/h/kg; supercritical CO described in step 1)₂The extraction time of the extraction is preferably 55-65 min, and more preferably 60 min; the separation pressure of the first volatile oil and the traditional Chinese medicine residues in the step 1) is preferably 8-10 Mpa, and more preferably 9 Mpa; the separation temperature of the first volatile oil and the traditional Chinese medicine residues in the step 1) is preferably 48-52 ℃, and more preferably 50 ℃. The invention is realized byMore isoimperatorin and cinnamaldehyde with the effects of relieving exterior syndrome and inducing perspiration can be prepared under the extraction conditions, and the efficacy of the traditional Chinese medicine extract for relieving exterior syndrome and inducing perspiration is improved.

After the first volatile oil and the traditional Chinese medicine residues are obtained, the invention carries out supercritical CO treatment on the traditional Chinese medicine residues in the presence of 95 percent ethanol water solution with volume concentration₂Extracting to obtain a second volatile oil; and mixing the first volatile oil and the second volatile oil to obtain the anti-influenza traditional Chinese medicine extract. In the present invention, the supercritical CO in the step 2)₂The extraction pressure of the extraction is preferably 25-32 Mpa, and more preferably 29 Mpa; supercritical CO in step 2)₂The extraction temperature of the extraction is preferably 35-45 ℃, and more preferably 45 ℃; supercritical CO in step 2)₂Extracted CO₂The extraction flow is preferably 25L/h/kg; supercritical CO in step 2)₂The extraction time of the extraction is preferably 35-45 min, and more preferably 40 min; the separation pressure of the second volatile oil and the traditional Chinese medicine residues in the step 2) is preferably 4-6 Mpa, and more preferably 5 Mpa; the separation temperature of the second volatile oil and the traditional Chinese medicine residues in the step 2) is preferably 32-37 ℃, and more preferably 35 ℃; the volume weight ratio of the ethanol aqueous solution in the step 2) to the Chinese medicinal powder in the step 1) is preferably (0.25-0.5) L:1Kg, and more preferably 0.35L:1 Kg. The invention can improve the utilization rate of the traditional Chinese medicine composition by secondary extraction, and the proper extraction condition can improve the oil yield of the volatile oil and improve the transfer rate of main substances such as pulegone, isoimperatorin, atractylodin, piperine, cinnamaldehyde and the like, thereby providing the anti-influenza effect of the traditional Chinese medicine composition.

The invention also provides application of the traditional Chinese medicine composition or the traditional Chinese medicine extract prepared by the preparation method in preparing a medicine for treating influenza. The anti-influenza traditional Chinese medicine composition or the traditional Chinese medicine extract provided by the invention still has a good treatment effect on influenza under the condition of not containing ephedra, and the aim of quickly cooling is fulfilled.

The invention also provides the application of the traditional Chinese medicine composition or the traditional Chinese medicine extract prepared by the preparation method in preparing medicines with various effects, wherein the various effects comprise one or more of bacteriostasis, inflammation diminishing, defervescence, analgesia and immunity regulation; the modulating immunity preferably comprises suppressing a delayed hypersensitivity reaction. The anti-influenza traditional Chinese medicine composition or the traditional Chinese medicine extract provided by the invention has obvious effects of inhibiting bacteria, diminishing inflammation, reducing fever, easing pain and inhibiting delayed allergy.

The invention also provides anti-influenza traditional Chinese medicine particles, which comprise the traditional Chinese medicine extract prepared by the preparation method and particle auxiliary materials. The invention has no special requirements on the components and the dosage proportion of the particle auxiliary materials, and the particle auxiliary materials which are well known by the technical personnel in the field can be adopted.

For further illustration of the present invention, the anti-influenza traditional Chinese medicine composition, the anti-influenza traditional Chinese medicine extract, the preparation method and the application thereof provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.

Example 1

An anti-influenza traditional Chinese medicine composition comprises the following components: 175g (7 parts by weight) of notopterygium root, 200g (8 parts by weight) of wind-proof herb, 125g (5 parts by weight) of schizonepeta spike, 125g (5 parts by weight) of cassia twig, 125g (5 parts by weight) of elsholtzia, 125g (5 parts by weight) of rhizoma atractylodis, 125g (5 parts by weight) of pepper, 100g (4 parts by weight) of chrysanthemum indicum, 100g (4 parts by weight) of sweet wormwood, 100g (4 parts by weight) of rhizoma anemarrhenae, 100g (4 parts by weight) of caulis sinomenii and 100g (4 parts by weight) of liquorice; the above components are purchased from Hebei Renzxin pharmaceutical Co., Ltd.

Example 2

An anti-influenza traditional Chinese medicine composition comprises the following components: 50g (5 parts by weight) of notopterygium root, 50g (5 parts by weight) of divaricate saposhnikovia root, 30g (3 parts by weight) of schizonepeta spike, 30g (3 parts by weight) of cassia twig, 30g (3 parts by weight) of Chinese mosla herb, 30g (3 parts by weight) of rhizoma atractylodis, 30g (3 parts by weight) of pepper, 30g (3 parts by weight) of wild chrysanthemum flower, 30g (3 parts by weight) of sweet wormwood herb, 30g (3 parts by weight) of rhizoma anemarrhenae, 30g (3 parts by weight) of caulis sinomenii and 20g (2 parts by weight) of liquorice; the above components were purchased from Hebei Renzhen pharmacy Co., Ltd.

Example 3

An anti-influenza traditional Chinese medicine composition comprises the following components: 100g (10 parts by weight) of notopterygium root, 100g (10 parts by weight) of wind-proof herb, 80g (8 parts by weight) of schizonepeta spike, 80g (8 parts by weight) of cassia twig, 80g (8 parts by weight) of elsholtzia, 80g (8 parts by weight) of rhizoma atractylodis, 80g (8 parts by weight) of pepper, 60g (6 parts by weight) of wild chrysanthemum flower, 60g (6 parts by weight) of sweet wormwood herb, 60g (6 parts by weight) of rhizoma anemarrhenae, 60g (6 parts by weight) of caulis sinomenii and 50g (5 parts by weight) of liquorice; the above components were purchased from Hebei Renzhen pharmacy Co., Ltd.

Example 4

An anti-influenza traditional Chinese medicine granule, the preparation method of the traditional Chinese medicine granule comprises the following steps:

1) pulverizing the Chinese medicinal composition of example 1 to obtain Chinese medicinal powder (particle size 20 mesh);

2) subjecting the Chinese medicinal powder to twice supercritical CO₂Extraction (supercritical CO)₂The extraction equipment is produced by Hua' an scientific research instrument Co Ltd, and HAs a model of HA222-50-07), the extraction pressure is 29Mpa, the extraction temperature is 45 ℃, and CO is₂The extraction flow is 25L/h/kg; the first separation pressure is 9Mpa, and the temperature is 50 ℃; the second separation pressure is 5Mpa, and the temperature is 35 ℃; first CO₂Performing supercritical extraction for 1 hr, adding 95% ethanol (0.5L: 1Kg of ethanol solution and Chinese medicinal powder, ethanol is available from Jinan union chemical engineering Co., Ltd.) for 40min, and mixing the extractive solutions to obtain Chinese medicinal extract;

3) placing 0.23g of HPMCe50 (hydroxypropyl methylcellulose E50) and 7.44g of 80% (weight concentration) ethanol solution in a stirring tank, uniformly mixing, and preparing into 3 wt.% HPMC solution without caking and in a clear and transparent state for later use;

4) putting 5.81g of the traditional Chinese medicine extract obtained in the step 2) and 7.67kg of 3 wt.% HPMC solution into a wet granulator, and stirring for 30min at the rotation speed of 65 rpm;

5) adding 5.81g of weighed silicon dioxide into the wet granulator in the step 4), starting stirring at the rotating speed of 65rpm for 25min to obtain mixed powder;

6) placing the mixed powder prepared in the step 5) in a hot air drying oven, and drying at 45 ℃ (the water content is less than or equal to 2%);

7) sieving the dried mixed powder with a 80-mesh sieve to obtain inclusion powder;

8) putting 11g of the inclusion powder prepared in the step 7), 26.02g of lactose, 0.5g of xanthan gum, 1.88g of citric acid and 0.19g of sucralose into a wet granulator, stirring for 20min until the materials are uniformly mixed, rotating the speed to 50rpm, stirring for more than 5min, and obtaining a soft material in a state of being held into a lump and being scattered by pressing;

9) and 8) sieving the soft material prepared in the step 8) by a swing granulator, granulating by using a 10-mesh sieve, drying in a hot air drying oven (at 45-50 ℃, the moisture content is less than or equal to 7 percent), and grading by using the 10-mesh sieve to obtain the traditional Chinese medicine granules.

Example 5

An anti-influenza herbal granule similar to that of example 4, except that the extraction temperature was 40 ℃.

Example 6

An anti-influenza Chinese medicinal granule similar to that of example 4, except that the extraction temperature was 35 ℃ and the extraction pressure was 25 MPa.

Example 7

An anti-influenza Chinese medicinal granule similar to example 4, except that 95% ethanol is added for the second time, wherein the weight ratio of ethanol solution to Chinese medicinal powder is 0.2L:1 Kg.

Example 8

An anti-influenza Chinese medicinal granule similar to example 4, except that 95% ethanol is added for the second time, wherein the weight ratio of ethanol solution to Chinese medicinal powder is 0.3L:1 Kg.

Example 9

An anti-influenza traditional Chinese medicine granule similar to that in example 4 is only different in that 95% ethanol is added for the second time, wherein the volume weight ratio of ethanol solution to traditional Chinese medicine powder is 0.35L:1Kg, and the extracted traditional Chinese medicine extract is named as traditional Chinese medicine cold medicine No. two.

Application example 1

Determining the oil yield of the volatile oil and the detection amount and transfer rate of isoimperatorin, pulegone, piperine, atractyloin and cinnamaldehyde in the traditional Chinese medicine extract obtained by the step 2) in the example 4-5 by adopting a High Performance Liquid Chromatography (HPLC), wherein the oil yield is 100% of the weight of the supercritical extract/the feeding amount of the medicinal materials; the detection amount and transfer rate of effective substances are determined by high performance liquid chromatography (general rule 0512) according to Chinese pharmacopoeia (2010 version).

TABLE 1 oil yield and effective substance detection under different extraction conditions

TABLE 2 transfer rate of effective substances under different extraction conditions

As can be seen from tables 1 and 2, the oil yield, the detected amount of cinnamaldehyde and the transfer rate were high at 29Mpa/45 ℃, and the detected amount of pulegone and atractylone and the transfer rate were high at 25Mpa/35 ℃, and the extraction conditions of step 2) in example 1 were the optimum process parameters for extracting the active ingredients of the traditional Chinese medicine, since isoimperatorin and cinnamaldehyde have an important effect on relieving exterior syndrome and inducing perspiration in the present pharmaceutical composition.

Application example 2

An anti-influenza traditional Chinese medicine granule similar to example 4, the only difference is that the extraction process in the step 2) is as follows: subjecting the Chinese medicinal powder to supercritical CO for three times₂Extraction (supercritical CO)₂The extraction equipment is produced by Hua' an scientific research instruments Co Ltd, and HAs a model of HA222-50-07), the extraction pressure is 29Mpa, the extraction temperature is 45 ℃, and CO is₂The extraction flow is 25L/h/kg; the first separation pressure is 9Mpa, and the temperature is 50 ℃; the second separation pressure is 5Mpa, and the temperature is 35 ℃; the third separation pressure is 5Mpa and the temperature is 35 ℃; first CO₂Performing supercritical extraction for 1 hr, adding 95% ethanol (0.35L: 1Kg of ethanol solution and Chinese medicinal powder, ethanol is available from Jinan Co., Ltd.) for 40min, and adding 95% ethanol (ethanol solution and Chinese medicinal powder) for the third time0.15L/1 Kg, ethanol from Jinan Shunji chemical Co., Ltd.) for 40min, and mixing the three extractive solutions to obtain the Chinese medicinal extract.

Determining the oil yield of the volatile oil and the detected amount and transfer rate of peucedanum root element, pulegone, piperine, atractylodin and cinnamaldehyde in different Chinese medicinal extracts obtained by the first extraction, the second extraction and the third extraction in the step 2) in the application example 2 by adopting a High Performance Liquid Chromatography (HPLC), and determining the results in tables 3 and 4; wherein the oil yield is 100% of the weight of the supercritical extract/the material input amount; the detection amount and transfer rate of effective substances are determined by high performance liquid chromatography (general rule 0512) according to Chinese pharmacopoeia (2010 version).

TABLE 3 oil yield and effective substance detection under three extraction conditions

TABLE 4 transfer rate of effective substance under three extraction conditions

As can be seen from tables 3 and 4, after the third supercritical extraction with 95% ethanol (the volume/weight ratio of ethanol to the powder of the Chinese medicinal materials is 0.15L:1Kg), the oil yield, the detected amounts of pulegone, isoimperatorin, atractylodin, piperine and cinnamaldehyde were small, and therefore, the optimum number of extractions was determined to be 2 times in consideration of the extraction cost and the extraction time.

Application example 3

Determining the oil yield of the volatile oil and the detection amount and transfer rate of isoimperatorin, pulegone, piperine, atractyloin and cinnamaldehyde in the traditional Chinese medicine extract obtained by the two-time extraction in the step 2) in the example 7-9 by adopting a High Performance Liquid Chromatography (HPLC), comparing the difference of three-time extraction by taking the oil yield of the volatile oil (calculated according to a formula I) and the total amount of isoimperatorin, pulegone, piperine, atractyloin and cinnamaldehyde (the total amount of each effective component is calculated according to the formula I) as evaluation indexes, and determining the result shown in tables 5-7; wherein the oil yield is 100% of the weight of the supercritical extract/the material input amount; the detection amount and transfer rate of effective substances are determined by high performance liquid chromatography (general rule 0512) according to Chinese pharmacopoeia (2010 version).

Total oil yield (%) - < pure CO >₂Oil yield (%) of the first extraction + 95% ethanol-pure CO₂Second extraction oil yield (%) formula I

The total weight of the functional components in the Chinese medicinal coarse powder is multiplied by 100% and formula II

TABLE 5 measurement results of the extract of step 2) in example 7

TABLE 6 measurement results of the extract of step 2) in example 8

TABLE 7 measurement results of the extract of step 2) in example 9

As can be seen from the results in tables 5 to 7, the oil yield, the detected amounts and the transfer rates of menthone, isoimperatorin, atractylodin and piperine of the traditional Chinese medicine extract obtained by extraction in example 9 are high, and the extraction conditions in step 2) in example 9 are considered as the optimal process parameters for extracting the active ingredients of traditional Chinese medicine.

Application example 4

The traditional Chinese medicine extract (marked as traditional Chinese medicine cold medicine II) prepared in the embodiment 9 of the invention is compared with the in-vitro antibacterial property of the antibacterial drugs on the market.

The test substance, namely the traditional Chinese medicine cold drug No. II, is examined for the antibacterial capacity against Staphylococcus aureus (Staphylococcus aureus), pseudomonas aeruginosa (p.aeruginosa), escherichia coli (escherichia coli), Klebsiella pneumoniae (Klebsiella) and haemophilus influenzae (moraxella catarrhalis) under the action of different concentrations through an in vitro minimum inhibitory concentration test (MIC) and a minimum bactericidal concentration test (MBC). The antibacterial ability and the antimicrobial spectrum screening of the test substance are analyzed, and preclinical experimental data are provided for the research of the test substance, namely the traditional Chinese medicine cold medicament II, on the medicines for treating cold and influenza.

Test method

Subject dosage design and dosing regimen

The antibacterial test against each strain had a total of 22 groups, namely: a solvent control group; a positive control group; positive drug control groups 1-10; test subjects a to j. See table 8 for details.

TABLE 8 summary of group and dosage design

Group number	Name of test substance or reference substance	Concentration of test substance	1: 1 final concentration after mixing
				1	Solvent control group	0	0
2	Positive control group	0	0
				3～12	Positive drug control groups 1-10	0.49～100(μg/mL)	0.977～50.00(μg/mL)
13～22	Group II Chinese medicine cold-treating medicine a-j	1.25～625.00(mg/mL)	0.61～312.50(mg/mL)

The method for configuring the test object comprises the following steps:

1) the second Chinese medicine cold medicine stock solution: 1g of the stock solution was put into a 10mL volumetric flask, and PBS was added thereto to prepare a 10mL volume of 625 mg/mL. The solution was taken up in a 10ml syringe and filtered through a 0.22 μm filter for sterilization. Sealing the sterile tube, filling the preparation label in detail, and storing at-20 deg.C. When in use, the temperature is taken out 30 minutes in advance, and the mixture is shaken and mixed evenly. The test substances at each concentration were prepared by a double dilution method, and the specific preparation method is shown in Table 9.

Table 9: trace twofold broth dilution method for cold medicine II

Note: the medicine of the tested matter is prepared by a two-fold dilution method.

2) The positive medicine preparation method comprises the following steps: weighing 1mg of corresponding positive drug by using a balance, placing the positive drug in a 10mL volumetric flask, and dissolving the positive drug with PBS to a constant volume to prepare a positive drug mother solution with the concentration of 0.1 mg/mL. Filtering with 0.22 μm filter membrane for sterilization, sealing with aseptic tube, filling preparation label in detail, and storing in refrigerator at 2-8 deg.C for 1 week. When in use, the temperature is taken out 30 minutes in advance, and the mixture is shaken and mixed evenly. The test substances with various concentrations are prepared by a double dilution method, and the specific preparation method is shown in tables 10 and 11.

TABLE 10 respective Positive controls and concentrations of the test bacteria

Name of bacterium	Positive control
		Staphylococcus aureus	Cefixime
Pseudomonas aeruginosa	Cefradine
		Escherichia coli	Cefuroxime sodium
Klebsiella pneumoniae	Cefradine
		Haemophilus influenzae	Cefuroxime sodium

TABLE 11 dilution of trace twofold broth of positive drugs

Preparation of bacterial liquid

Activated bacteria were streaked on a plate medium, and cultured at 37 ℃ for 24 hours. And selecting a single colony, inoculating the single colony into a test tube containing 5mL of liquid nutrient medium, and placing the test tube into a constant temperature incubator for culturing for 16-20 h at 35 +/-2 ℃ and 200 rpm. Measuring OD (OD600 ^ 0.5 concentration is 10^ when OD600 ^ 0.5) with a small amount of bacteria culture solution on a sterile operating platform⁸Per ml), diluted 100-fold with the corresponding nutrient broth liquid medium to a concentration of 1-2X 10^ s⁶cfu/ml for use.

MIC assay

100 μ l of liquid nutrient medium for the corresponding bacteria was added to each well of a 96-well plate using a line gun on a sterile super clean bench, near an alcohol burner. Add 100. mu.l of the prepared drug stock to the first well of the third row A/B/C and then double dilute the drug. That is, after adding the liquid medicine into the first hole, fully blowing (at least three times) the liquid medicine with a liquid transfer gun to fully mix the medicine with the broth, sucking 100 μ l of the liquid medicine, adding into the second hole, fully blowing and mixing the liquid medicine with the broth, repeating the above steps until the last hole is reached, sucking 100ul of the liquid medicine out of the 12 th row and throwing away the liquid medicine (each hole contains 100 μ l of the medicine-containing culture medium). Then, 100. mu.l of diluted bacterial suspension was added to each well, and three wells (three rows of A/B/C samples) were repeated. The same operation is carried out on three D/E/F rows of the same row, and positive drug control (adding bacteria liquid and positive drug liquid) is carried out. A row of negative controls (with only the blank broth without the addition of the broth) and a row of positive controls (with the addition of the broth without the addition of the liquid) were performed on G, H rows on the same plate. And (3) putting the 96-well plate into a constant-temperature incubator with 37 ℃ and 35 +/-2 ℃ and 200rpm for culturing for 16-20 hours, and observing the result. If the bacteria grow on the bottom of the long pore plate, white precipitate is generated.

And (3) determining the MIC value: the concentration of the last well without a pellet in the upper row of the 96-well plate corresponds to the MIC value of the drug.

MBC determination

And (3) preparing a 24-hole solid culture medium plate on the same day of the test, slightly blowing and uniformly mixing bacterial liquid in holes (with the symbol of-or-/+) in which bacteria can not grow by naked eyes, sucking the bacterial liquid, inoculating the bacterial liquid on the 24-hole solid culture medium plate without the antibacterial agent, uniformly coating an inoculating ring, culturing at 35 +/-2 ℃ for 18-48 hours, and counting bacterial colonies. The concentration of the drug on the agar plate where no bacterial colony growth was observed or the colony count was less than 5 was the minimum bactericidal concentration. 3 replicates per concentration.

Results of the experiment

The administration concentrations of different groups are shown in Table 12, and the measurement results of MIC and MBC of different strains cultured under different drug concentrations are shown in tables 13-17.

TABLE 12 concentrations administered in different groups

TABLE 13 MIC determination results after Staphylococcus aureus culture at different drug concentrations

TABLE 14 MIC and MBC determination results after P.aeruginosa culture at different drug concentrations

TABLE 15 MIC and MBC determination results of Klebsiella pneumoniae after incubation at different drug concentrations

TABLE 16 MIC determination results after cultivation of E.coli at different drug concentrations

TABLE 17 measurement results of MIC and MBC after culturing Haemophilus influenzae at different drug concentrations

Note: "-" in tables 13 to 17 means clarification; "+" indicates turbidity with precipitate; ' -/+ denotes a very slight amount of precipitation

From tables 13 to 17, it can be seen that no antibacterial activity of cold drug No. two was detected against staphylococcus aureus (gram-positive bacteria) and enterobacter coli (enterobacter), and that cold drug No. two had antibacterial activity against pseudomonas aeruginosa (non-fermented gram-negative bacteria) at a concentration of ≧ 156.25 mg/kg; has antibacterial activity against Klebsiella pneumoniae (gram-negative bacillus) at a concentration of 78.13mg/kg and has antibacterial activity against Haemophilus influenzae (gram-negative bacillus) at a concentration of 78.13 mg/kg. Under the laboratory conditions, no bactericidal activity was detected for cold drug No. two against the above bacteria.

Application example 5

The effect of the traditional Chinese medicine extract (traditional Chinese medicine cold medicine II) prepared in the embodiment 9 of the invention is compared with the effect of the common cold medicine on an endotoxin fever rat model in the market.

Purpose of the experiment

By injecting Lipopolysaccharide (LPS) into animal body to generate heat to form endogenous pyrogen, inducing animal to generate heat to establish animal model with fever, observing antipyretic effect of test substance, and providing preclinical experimental data for research on medicine for treating common cold and influenza

Test protocol

Subject dose design and dosing regimens are set forth in Table 18

Table 18: summary of antipyretic test groups and dosage design

Reason for dose design

The dosage and concentration of the drug in the special test are designed by referring to information related to the tested substance and related literature data provided by pharmacology experimental methodology, pharmacology research methodology of traditional Chinese medicine (third edition) and consignment formula:

(1) the clinical application and dosage of the traditional Chinese medicine cold drug II are as follows: is administered orally. The adult dose is 17.772g daily, the body weight is calculated according to 70kg, and the rat dose is 1.60g/kg according to conversion of body weight-body surface area. The dose of the second Chinese medicine cold drug in the antipyretic test in the research is 1/4, 1/2 and 1 time of the clinical equivalent dose, namely 0.40, 0.80 and 1.60 g/kg.

(2) The clinical application and dosage of CGSN-01 are as follows: is administered orally. The adult dose is 20.22g daily, the body weight is calculated according to 70kg, and the rat dose is 1.827g/kg according to conversion of body weight-body surface area. The CGSN-01 freeze-dried powder is used as an extraction process control group in the research heat-resolving test, and the dosage is set to be the equivalent dosage of clinical dosage, namely 1.827 g/kg.

(3) Lian Hua Qing Wen granules: the clinical dosage of the Lianhua qingwen granules in adults is 18g/70kg, and the term is converted by the administration of clinically equivalent dose, and is converted into 1.62g/kg of rats according to the body surface area ratio.

(4) Paracetamol tablets: the clinical dosage of the acetaminophen tablet for adults is 0.5g/70kg, and the dosage of the acetaminophen tablet is converted into the dosage of the acetaminophen tablet according to the clinical equivalent dosage, and the dosage is converted into the dosage of about 0.045 g/kg for rats according to the body surface area ratio.

Animal screening and grouping

Before the experiment, the selected rats with qualified body temperature are randomly divided into a normal control group, a model control group, a low, medium and high dose group of a traditional Chinese medicine cold drug II, a positive control group (a honeysuckle antipyretic granule), a positive control group (a p-acetaminopher tablet) and a CGSN-01 control group according to the anal temperature and sex balance, wherein each group comprises 10 rats and is half of a male rat and a female rat. Before the anal temperature is measured each time, the thermometer is lubricated by vaseline, and the depth of the thermometer inserted each time is 2 cm.

Test method and observation index

The SD rats in each group of the test day are subjected to anal temperature measurement, before the anal temperature is measured, the rats are emptied of excrement, the body temperature of each SD rat is measured for 2 times, and the average value is taken as the basic body temperature. After completion of the measurement, the LPS solution was injected subcutaneously (dose: 150. mu.g/kg, injection volume: 2ml/kg) into the back of the neck of the rat in each of the groups except the normal control group, which was injected with an equal volume of physiological saline. After injection molding, the injection is immediately carried out on each group according to the volume of 10ml/kg for intragastric administration, and the normal control group and the model control group are administered with the same amount of pure water for intragastric administration. Specific observation and inspection indexes in the test comprise the following contents:

body temperature measurement

The anal temperature of the rats was measured 1, 2, 3, 4, 5, 6, 7, 8 hours after the administration.

The results are shown in Table 19.

TABLE 19 measurement results of anal temperature change before different groups of rats and model building

Note: "#" indicates P <0.05, "###" indicates P <0.01, as compared to a normal control group; compared to the model control group, "+" indicates P <0.05 and "+" indicates P < 0.01.

As can be seen from Table 19, the cold drug II prepared by the invention has obvious antipyretic effect and is positively correlated with concentration, and the antipyretic effect is superior to that of the antipyretic granule of lotus flower.

Application example 6

The effect of the traditional Chinese medicine extract (traditional Chinese medicine cold medicine II) prepared in the embodiment 9 of the invention on a rat foot swelling model caused by carrageenan is compared with the effect of common cold medicines in the market.

Purpose of the experiment

Through injecting carrageenan solution into sole of foot, establishing rat inflammation model caused by carrageenan, observing the effect of the tested substance on the animal model of inflammation, and providing preclinical experimental data for the research of the tested substance, namely the Chinese medicine cold medicament II for treating cold and influenza.

Test protocol

Subject dose design and dosing regimen are shown in table 20.

Table 20: anti-inflammatory test set, dosage design summary

The reason for dose design is similar to that in application example 5, the only difference is that (4) the acetaminophen tablet in application example 5 is replaced with an indomethacin capsule, and the dose design is as follows:

(4) the indometacin capsule comprises: the clinical dosage of the indometacin enteric-coated tablet for adults is 75mg/70kg, and the dose of the indometacin enteric-coated tablet is converted according to the clinical equivalent dosage and is converted into that of a rat according to the body surface area ratio, and the dose of the indometacin enteric-coated tablet is about 6.75 mg/kg. The control indomethacin positive in the literature was used in a rat carrageenan-induced plantar swelling model at a dose of 7 mg/kg. And 7mg/kg is selected as the administration dosage of the item by combining the conversion result with the reference.

Animal screening and grouping

Qualified animals screened by the foot volume are selected one day before the test and are randomly divided into a normal control group, a model control group, a low, medium and high dose group of a traditional Chinese medicine cold drug II, a positive control group (Lianhua qingwen granules), a positive control group (indometacin capsules) and a CGSN-01 control group according to the foot volume balance, wherein each group comprises 9 male rats.

Test method and observation index

On the first day of the test, the volume of the right hind foot is measured by using a plantar volume measuring instrument, the volume is measured twice continuously, and the average value is taken as the basic volume. Then injecting 1% carrageenan suspension under tendon and tendon membranes of the right hind foot sole of animals of the model control group and the tested object group, wherein each suspension is 0.1 mL; injecting equal volume of normal saline into the blank control group animals; immediately after carrageenan injection, each group was intragastrically administered to the corresponding test subjects in a volume of 10 ml/kg. The blank and model control groups were dosed with 10ml/kg of gavage. The following criteria were observed and examined after dosing:

plantar volume determination

The volume of the foot sole of the right hind foot of each group of animals was measured 1, 2, 3, 4, 5, 6h after injection, 2 times continuously, and the average value was taken as the volume of the foot sole after molding. And calculating the swelling rate and the swelling inhibition rate of the foot sole according to the volume of the foot sole at different time points after inflammation, comparing the swelling rate and the swelling inhibition rate with the model group, and performing statistical analysis. When the toe swelling rate of rats in the positive control group and the test object different dose groups is obviously lower than that of the model group, the medicine has an anti-inflammatory effect on a carrageenan-induced toe swelling model of the rats.

Swelling ratio E (%) ═ Vt-Vn)/Vn X100%

Wherein Vn and Vt represent the toe volume before and after application of an inflammatory agent

The results are shown in Table 21.

TABLE 21 results of specific swelling ratio measurement before model building for rats of different groups

Note: model control group comparison, "#" indicates P <0.05 and "##" indicates P < 0.01.

As can be seen from table 21, cold drug No. two prepared by the present invention has significant anti-inflammatory efficacy on the cross-carrageenan induced foot swelling model under clinical dosing equivalence, 1/2 and 1/4 dosing concentrations. Compared with a positive control drug, the anti-inflammatory drug effect has obvious advantages compared with the traditional Chinese medicine cold drug Lianhua antipyretic granule on the market, and the onset time is rapid.

Application example 7

The effect of the traditional Chinese medicine extract (traditional Chinese medicine cold medicine II) prepared in the embodiment 9 of the invention is compared with that of a mouse ear swelling model caused by paraxylene of a common cold medicine in the market.

Purpose of the experiment

Xylene is coated on auricles of mice to generate stimulation to skin mucous membranes, an acute inflammation model is induced, a test substance, namely a traditional Chinese medicine cold drug II is given, and the influence of the traditional Chinese medicine cold drug II on auricles swelling and edema of the mice is observed. Provides preclinical experimental data for the research of the tested traditional Chinese medicine cold medicament II for resisting inflammation and reducing swelling and treating local acute inflammatory reaction caused by cold and influenza.

Test protocol

Subject dose design and dosing regimen are shown in table 22.

Table 22: test set and dosage design summary

Reason for dose design

(1) the clinical application and dosage of the traditional Chinese medicine cold drug II are as follows: is administered orally. The adult dose is 17.772g daily, the body weight is calculated according to 70kg, and the mouse dose is 2.31g/kg according to conversion of body weight-body surface area. The dose of the traditional Chinese medicine cold drug II in the heat clearing test in the research is 1/4, 1/2 and 1 time of the clinical equivalent dose, namely 0.58, 1.16 and 2.31 g/kg.

2) The clinical application and dosage of CGSN-01 are as follows: is administered orally. The adult dose is 20.22g daily, the body weight is calculated according to 70kg, and the mouse dose is 2.63g/kg according to the conversion of body weight-body surface area. The CGSN-01 freeze-dried powder in the antipyretic test of the research is used as an extraction process control group, and the dosage is set to be the equivalent dosage of clinical dosage, namely 2.63 g/kg.

(3) Lian Hua Qing Wen granules: the clinical dosage of the Lianhua qingwen granules for adults is 18g/70kg, and the dosage of the Lianhua qingwen granules is converted according to clinical equivalent dosage, and the dosage is converted into 2.34g/kg of mice according to the body surface area ratio.

(4) Hydrocortisone tablets: the clinical dosage of the hydrocortisone tablet for adults is 30mg/70kg, and the dosage of the hydrocortisone tablet is converted according to the clinical equivalent dosage and is converted into about 3.9mg/kg by the conversion of the body surface area ratio.

Animal screening and grouping

Selecting qualified mice after observation, and randomly dividing the mice into a solvent control group, a model control group, a low, medium and high dose group of a traditional Chinese medicine cold drug II, a positive control group (Lianhua qingwen granules), a positive control group (hydrocortisone tablets) and a CGSN-01 control group according to the balance of weight and sex, wherein each group comprises 10 mice.

Test method and observation index

Selecting qualified mice after observation, and randomly dividing the mice into a blank control group, a model control group and a first low, medium and high dosage group of the traditional Chinese medicine cold drug according to the balance of weight and sex, wherein each group comprises 10 mice. After grouping, each test group of animals is subjected to intragastric administration for 1 time per day, and the test substance is continuously administered for 3 days. After 1h of administration on day 3, each mouse was evenly smeared with 0.1ml of xylene on both sides of the midpoint of the auricle of the right ear, and the left ear was left untreated. The mice are killed by a cervical dislocation method after being smeared for 1h, round ear pieces are respectively punched on the same parts (in the range of coating with dimethylbenzene) of the left ear and the right ear of the mice by a puncher with the diameter of 8mm, the mice are weighed by an electronic balance, and the swelling degree of the auricles of the mice and the inhibition rate of the drugs are calculated according to a calculation formula.

Swelling degree of auricle

Shearing off double ears along the base line of the auricle of the mouse, respectively punching off round ear sheets at the same positions of the left ear and the right ear of the mouse by using a puncher with the diameter of 8mm, weighing by using an electronic balance, and calculating the swelling degree of the auricle of the mouse and the inhibition rate of the medicament according to a calculation formula:

swelling degree-right ear quality-left ear quality

The swelling inhibition rate (%) of the drug was (degree of swelling in saline-degree of swelling in administered group)/degree of swelling in saline × 100%.

The results are shown in Table 23.

TABLE 23 measurement results of swelling degree and swelling degree inhibition ratio of mice of different groups

Note: compared to the model control group, "+" indicates P <0.05 and "+" indicates P < 0.01.

As can be seen from Table 23, the high dose of the cold drug II prepared by the invention has the effect of obviously inhibiting the sudden acute inflammation model of mice; the drug effect is not obviously inhibited in low and medium dosage statistics. After the high dose of the cold medicine II is continuously administrated for 3 days, the drug effect on sudden acute inflammation of mice has no obvious difference with a positive medicine hydrocortisone tablet.

Application example 8

The effect of the traditional Chinese medicine extract (traditional Chinese medicine cold medicine II) prepared in the embodiment 9 of the invention on the acetic acid induced pain model of the mice is compared with the effect of the common cold medicine on the market.

Purpose of the experiment

The experimental study on the effect of the traditional Chinese medicine cold drug II on an acetic acid-induced pain model provides preclinical experimental data for the study on the drugs of the tested traditional Chinese medicine cold drug II for easing pain and treating cold and pain reaction caused by influenza.

The protocol is shown in Table 24.

Subject dosage design and dosing regimen

Table 24: summary of analgesic test groups and dosage design

Reason for dose design

(3) Lian Hua Qing Wen granules: the clinical dosage of the Lianhua qingwen granules for adults is 18g/70kg, and the term is converted by the administration of clinical equivalent dose, and is converted to about 2.34g/kg of mice according to the body surface area ratio.

(4) The indometacin capsule comprises: the clinical dosage of the indometacin capsule in an adult is 75mg/70kg/d, and the indometacin capsule is converted into a mouse by the conversion of the body surface area ratio according to the clinical equivalent dosage, wherein the mouse is about 9.73 mg/kg.

Animal screening and grouping

Selecting mice qualified after observation, and randomly dividing the mice into a blank control group, a model control group, a low, medium and high dose group of a traditional Chinese medicine cold drug II, a positive control group (Lianhua qingwen granules), a positive control group (indomethacin capsules) and a CGSN-01 control group according to weight and gender balance, wherein each group comprises 12 mice and half of the mice.

Test method and observation index

Selecting mice qualified after observation, and randomly dividing the mice into a blank control group, a model control group, a low, medium and high dose group of a traditional Chinese medicine cold drug II, a positive control group (Lianhua qingwen granules), a positive control group (indomethacin capsules) and a CGSN-01 control group according to weight and gender balance, wherein each group comprises 12 mice and half of the mice. After grouping, the animals of each test group are subjected to intragastric administration for 1 time/day and 2 days continuously, and the intragastric volume is 20 mL/kg. Mice in the blank control group and the model control group are subjected to intragastric perfusion by using the same volume of the solvent. On the day before the test, the mice in each test group were injected with 0.9% glacial acetic acid to cause pain, the injection volume was 0.1ml/10g, and the mice in the blank control group were injected with physiological saline of the same volume about 2 hours after the administration on the day after the test. The time to first writhing response (latency) and number of writhing were recorded for each group of mice within 20min after glacial acetic acid injection. With the abdomen depressed, the trunk and hind limbs were elongated 1 time by stimulation to define 1 writhing response. And (3) calculating the inhibition rate according to the times of writhing of each group of test animals, and analyzing the analgesic effect of the test substance with each concentration of the intragastric administration in the acetic acid induced pain model of the mouse.

(1) Animal writhing latency recording time of initial writhing response (latency) within 20min after glacial acetic acid injection

(2) Animal writhing frequency records total writhing frequency of mice after initial writhing within 20min after glacial acetic acid injection

(3) Inhibition rate

Calculating inhibition rate according to the writhing times of each group of test animals, and analyzing the analgesic effect of the test object with each concentration in the acetic acid writhing pain model of the mouse after gastric lavage:

the inhibition ratio (%) - (control group average number of writhing-administration group average number of writhing)/control group average number of writhing × 100%

The results are shown in Table 25.

TABLE 25 analgesic Effect of different groups of drugs

As can be seen from table 25, the second three dosage amounts of the traditional Chinese medicine cold drug prepared by the present invention all have significant analgesic effects, and statistically, the three dosage amounts do not have significant differences. The analgesic effect of the second three traditional Chinese medicine cold-treating medicines is better than that of the Lianhua antipyretic granules, and has no obvious difference with the positive medicine indometacin.

Application example 9

The traditional Chinese medicine extract (traditional Chinese medicine cold medicine II) prepared in the embodiment 9 of the invention is compared with the influence of common cold medicines on the delayed allergy of the sheep erythrocytes to the mice in the market.

Purpose of the experiment

Carrying out sensitization by injecting sheep red blood cell suspension into mouse tail vein, giving a tested substance of a traditional Chinese medicine cold medicine II, and observing the influence of the traditional Chinese medicine cold medicine II on delayed anaphylactic reaction of the mouse. Provides preclinical experimental data for the research of the tested traditional Chinese medicine cold medicament II for regulating the immunologic function and treating cold and influenza.

Test protocol

Subject dose design and dosing regimens are set forth in Table 26

Table 26: test set and dosage design summary

Reason for dose design

(4) And (3) Keratan tablets: the dosage of an adult is 10mg/70kg/d, and the dosage of the project is converted according to the clinical equivalent dosage, and the conversion is converted into about 1.3mg/kg according to the body surface area ratio to the mouse.

Animal screening and grouping

Selecting qualified mice after observation, and randomly dividing the mice into a solvent control group, a model control group, a low, medium and high dose group of a traditional Chinese medicine cold drug II, a positive control group (Lianhua qingwen granules), a positive control group (Keruitan) and a CGSN-01 control group according to the balance of weight and sex, wherein each group comprises 14 mice and half of the mice.

Test method and observation index

Selecting mice qualified after observation, and randomly dividing the mice into a blank control group, a model control group, a traditional Chinese medicine cold drug No. two low, medium and high dose groups according to weight and sex balance, wherein each group comprises 14 mice and each half of the mice are male and female. After grouping, each test group of animals is subjected to intragastric administration for 1 time per day, the test substance is continuously administered for 10 days, and the intragastric volume is 20 mL/kg. On day 5 of administration, mice of model control and each administration group were sensitized by intraperitoneal injection of 0.2mL of 10% (V/V) physiological saline sheep blood red cell suspension (about 4X 10 per mouse⁸One SRBC), mice in the blank control group were given an equal volume of saline injection. On day 9, 0.1mL of sheep red blood cell suspension was injected into the left paw of the mouse after completion of the administration, and 0.1mL of physiological saline was injected into the right paw of the mouse. 12 hours after injection (day 10), each group was administered by gavage in groups, and the difference in volume between the two soles was measured 2 hours after administration.

Volume difference of two side soles

On day 9 of administration, 0.1mL of sheep red blood cell suspension was injected into the left sole of the mouse after completion of administration, and 0.1mL of physiological saline was injected into the right sole of the mouse. 12 hours after injection (day 10 of administration), the administration was carried out in groups, and the difference in volume between the two soles was measured 2 hours after administration.

The results are shown in Table 27.

TABLE 27 Effect of different groups of drugs on delayed hypersensitivity results

As can be seen from Table 27, the cold drug II prepared by the invention can obviously inhibit delayed anaphylaxis caused by injection of sheep erythrocyte suspension into left sole of mouse, and has regulating effect on allergic stress of immune system function.

In conclusion, the anti-influenza traditional Chinese medicine composition or the traditional Chinese medicine extract provided by the invention still has a good treatment effect on influenza under the condition of not containing ephedra herb, and achieves the purpose of quickly cooling, and the traditional Chinese medicine extract prepared by the invention has obvious effects of inhibiting bacteria, diminishing inflammation, reducing fever, relieving pain and inhibiting delayed allergy.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The anti-influenza traditional Chinese medicine composition is characterized by comprising the following components in parts by weight:

2. The preparation method of the anti-influenza traditional Chinese medicine extract is characterized by comprising the following steps:

1) shall correspond to claim 1Supercritical CO treatment of Chinese medicinal powder of Chinese medicinal composition₂Extracting to obtain first volatile oil and Chinese medicinal residue;

3. The method of claim 2, wherein the supercritical CO is obtained in step 1) and step 2)₂The extraction pressure and the extraction temperature are respectively 25-32 Mpa and 35-45 ℃.

4. The method of claim 2 or 3, wherein the supercritical CO is obtained in step 1) and step 2)₂Extracted CO₂The extraction flow rates were 25L/h/kg, respectively.

5. The method according to claim 2 or 3, wherein the supercritical CO is used in step 1)₂The extraction time of the extraction is 55-65 min, the separation pressure is 8-10 Mpa, and the separation temperature is 48-52 ℃.

6. The method according to claim 2 or 3, wherein the supercritical CO is used in step 2)₂The extraction time of the extraction is 35-45 min, the separation pressure is 4-6 Mpa, and the separation temperature is 33-37 ℃.

7. The preparation method of claim 2, wherein the volume-to-weight ratio of the ethanol aqueous solution in the step 2) to the Chinese medicinal powder in the step 1) is (0.25-0.5) L:1 Kg.

8. The use of the Chinese medicinal composition of claim 1 or the Chinese medicinal extract prepared by the preparation method of any one of claims 2 to 7 in the preparation of a medicament for treating influenza.

9. The use of the composition of claim 1 or the extract of a Chinese herbal medicine prepared by the method of any one of claims 2 to 7 in the preparation of a medicament with multiple effects, wherein the multiple effects include one or more of bacteriostasis, anti-inflammation, antipyresis, analgesia and immunoregulation.

10. An anti-influenza traditional Chinese medicine granule, which is characterized by comprising a traditional Chinese medicine extract prepared by the preparation method of any one of claims 2 to 7 and a granule auxiliary material.