A Chinese medicine composition and its preparation method and application

Abstract

The present invention belongs to the technical field of traditional Chinese medicine preparations, and specifically relates to a traditional Chinese medicine composition, a preparation method, and an application thereof. The traditional Chinese medicine composition not only has fewer medicinal flavors and a simplified prescription, but also, by combining Artemisia selengensis, black borneol, Lithospermum officinale, Myrrha mukul, Tibetan madder, Oxytropis chinensis, and Musk, has the effect of clearing away heat and detoxifying, significantly reducing the degree of plantar swelling in rats induced by LPS and lowering the body temperature of febrile rats. The traditional Chinese medicine composition can be used to prepare anti-inflammatory and / or antipyretic drugs. In addition, the traditional Chinese medicine composition provided by the present invention has a good therapeutic effect on influenza virus and has no side effects. Compared with decoctions, granules prepared by combining a specific filler with the traditional Chinese medicine composition have a better therapeutic effect on influenza A (H1N1) and influenza A (H5N1).

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a traditional Chinese medicine composition and a preparation method and application thereof. Background Art

[0002] Influenza viruses, also known as influenza viruses, can infect and cause illness in a variety of animals, including humans and pigs. Typical clinical symptoms include fever, body aches, fatigue, and respiratory symptoms such as sneezing, runny nose, sore throat, and shortness of breath. Human influenza is primarily caused by influenza A viruses and is contagious. Influenza A (H1N1) and influenza A (H5N1) are both highly contagious acute respiratory illnesses with high morbidity and rapid spread, and can be transmitted through direct or indirect contact.

[0003] Currently, common clinical treatments for influenza virus infection are neuraminidase inhibitors such as zanamivir and oseltamivir. While these drugs have strong antiviral properties, they can also cause adverse reactions such as nausea, vomiting, insomnia, dizziness, and bronchitis. Traditional Chinese medicine (TCM) offers minimal side effects and holistic treatment options. However, it suffers from a plethora of medicinal flavors and lacks a streamlined approach. Therefore, there is a continued need to develop new TCMs with fewer medicinal flavors and improved therapeutic efficacy. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problem that the traditional Chinese medicine composition prescription in the prior art has too many medicinal flavors and is not streamlined, thereby providing a traditional Chinese medicine composition with fewer medicinal flavors and significant anti-influenza virus effect.

[0005] To this end, the present invention provides a traditional Chinese medicine composition comprising the following raw materials in parts by weight: 30-50 parts of Artemisia selengensis, 20-35 parts of black borneol, 20-35 parts of Lithospermum officinale, 20-35 parts of Myrrha mukul, 20-35 parts of Tibetan madder, 20-35 parts of Oxytropis oxyphylla and 0.3-0.5 parts of musk.

[0006] In the present invention, the term "commiphora mukul" refers to the oleoresin exuded from the bark of the trunk of Commiphora mukul Eng, a plant of the Oleaceae family, and various plants of the same genus. "Musk" refers to artificial musk.

[0007] The present invention provides a method for preparing the above-mentioned traditional Chinese medicine composition, comprising the following steps:

[0008] Weigh Artemisia selengensis, black borneol, Lithospermum officinale, Myrrha mukul, Rubia cordifolia and Oxytropis oxyphylla, extract with water, filter, collect the filtrate, concentrate and / or dry, and mix with musk to obtain a traditional Chinese medicine composition.

[0009] The invention provides a pharmaceutical preparation comprising the following raw materials in parts by weight: 30-50 parts of Artemisia selengensis, 20-35 parts of black borneol, 20-35 parts of Lithospermum officinale, 20-35 parts of Myrrha mukul, 20-35 parts of Tibetan madder, 20-35 parts of Oxytropis oxytropis and 0.3-0.5 parts of musk, and also comprising pharmaceutically acceptable excipients.

[0010] Furthermore, the pharmaceutical preparation is a decoction, tablet, pill, granule or capsule.

[0011] Furthermore, the pharmaceutical preparation is a granule, and the auxiliary materials of the granule include a filler and / or a sweetener.

[0012] Furthermore, the filler is selected from one or more of sucrose, lactose, starch, microcrystalline cellulose, mannitol and dextrin; preferably, the filler comprises lactose and mannitol in a mass ratio of 4:0.8-1.2.

[0013] Furthermore, the sweetener is one or more of stevioside, aspartame, and sucralose; preferably stevioside.

[0014] The present invention also provides a method for preparing any of the above-mentioned pharmaceutical preparations, comprising the following steps:

[0015] Artemisia selengensis, black borneol, lithospermum officinale, myrrha mukul, madder cordifolia and oxytropis chinensis are weighed, extracted with water, filtered, the filtrate is collected, concentrated and / or dried to prepare an extract, and pharmaceutically acceptable excipients and musk are added to prepare a pharmaceutical preparation according to a conventional process in the art.

[0016] Furthermore, the pharmaceutically acceptable excipients include fillers, and the mass ratio of the dry matter in the extract to the filler is 1:0.5-3.

[0017] Furthermore, the pharmaceutically acceptable excipients further include a sweetener, and the mass percentage of the sweetener to the total mass of the dry matter and filler in the extract is 0.05%-6%, preferably 0.5%.

[0018] Furthermore, during the preparation of the pharmaceutical preparation, the dried extract powder or concentrated extract can be mixed with excipients and musk, and granulated to obtain granules. Alternatively, the dried extract powder or concentrated extract can be mixed with only excipients to obtain granules, and musk can be added during the granulation process. Alternatively, the excipients can be added before drying, and musk can be added after drying, and granulated to obtain granules.

[0019] Furthermore, in the water extraction process, the number of water extractions is 1-3 times, the amount of water (mass) used each time is 8-12 times the mass of the medicinal material, and the extraction time each time is 60-120 minutes.

[0020] The present invention also provides the use of the above-mentioned traditional Chinese medicine composition or any pharmaceutical preparation described above or the traditional Chinese medicine composition or pharmaceutical preparation prepared by the preparation method in the preparation of anti-inflammatory and / or antipyretic drugs.

[0021] The present invention also provides use of the aforementioned traditional Chinese medicine composition or any pharmaceutical preparation described above, or the traditional Chinese medicine composition or pharmaceutical preparation prepared by the preparation method, in the preparation of an anti-influenza virus medicament. Preferably, the influenza virus is selected from H1N1 influenza virus or H5N1 influenza virus; more preferably, the H1N1 influenza virus is numbered A / GZ / GIRD07 / 09 or A / Puerto Rico / 8 / 1934.

[0022] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0023] 1. The Chinese medicine composition provided by the present invention not only has few medicinal flavors and a simple prescription, but also has the effect of clearing away heat and detoxifying by combining Artemisia selengensis, black borneol, lithospermum officinale, myrrha mukul, madder, oxtera and musk. It can significantly reduce the degree of plantar swelling in rats caused by LPS and lower the body temperature of febrile rats. It can be used to prepare anti-inflammatory and / or antipyretic drugs. In addition, the Chinese medicine composition provided by the present invention has a good therapeutic effect on influenza virus and has no side effects.

[0024] 2. The pharmaceutical preparation provided by the present invention uses lactose and mannitol as fillers in a mass ratio of 4:0.8-1.2 (particularly 4:1) compared to other excipients. The resulting granules significantly reduce moisture absorption while exhibiting excellent molding efficiency, fluidity, and solubility, achieving the highest overall rating. Furthermore, compared to decoctions, granules prepared using this filler and a traditional Chinese medicine composition demonstrate superior therapeutic efficacy for influenza A (H1N1) and influenza A (H5N1). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 These are particles made from different excipients in the filler screening experiment in Experimental Example 1;

[0027] Figure 2 The effect of the maximum dose group (15 g / kg / d) of the granules of the present invention on the spleen of 10 mice (×200);

[0028] Figure 3 The effect of the maximum dose group (15 g / kg / d) of the granules of the present invention on the kidneys of 10 mice (×200);

[0029] Figure 4 The effect of the maximum dose group of the granules of the present invention (15 g / kg / d) on the hearts of 10 mice (×200);

[0030] Figure 5 The effect of the maximum dose group (15 g / kg / d) of the granules of the present invention on the livers of 10 mice (×200);

[0031] Figure 6 The figure shows the effect of the medicine of the present invention on the body temperature of rats. DETAILED DESCRIPTION

[0032] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0033] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0034] Example 1 Extract

[0035] The raw material composition of the Chinese medicine composition provided in this embodiment is as follows:

[0036] Artemisia selengensis 50g, Myrrha mukul 35g, Black borneol 35g, Lithospermum officinale 35g, Tibetan madder 35g, Oxytropis 35g and Musk 0.5g.

[0037] The preparation method of the above-mentioned Chinese medicine composition is as follows:

[0038] According to the above weight, Artemisia selengensis, black borneol, Lithospermum officinale, Myrrha mukul, Rubia cordifolia and Oxytropis oxytropis were weighed, decocted in water for 3 times, the amount added each time was 8 times the weight of the medicinal material, the decocting time each time was 60 minutes, filtered, the filtrate was taken, dried, and musk was added to obtain a traditional Chinese medicine composition (i.e., traditional Chinese medicine extract).

[0039] Example 2 Extract

[0040] The raw material composition of the Chinese medicine composition provided in this embodiment is as follows:

[0041] Artemisia selengensis 40g, black borneol 28g, lithospermum officinale 28g, myrrh mukul 28g, madder 28g, oxtera 28g and musk 0.4g.

[0042] The preparation method of the above-mentioned Chinese medicine composition is as follows:

[0043] According to the above weight, Artemisia selengensis, black borneol, Lithospermum officinale, Myrrha mukul, Rubia cordifolia and Oxytropis oxytropis were weighed, decocted in water for 3 times, with the amount added each time being 8 times the weight of the medicinal materials, and the decocting time each time being 60 minutes. The mixture was filtered, the filtrate was taken, dried, and musk was added to obtain a traditional Chinese medicine composition.

[0044] Example 3 Granules

[0045] The raw material composition of the Chinese medicine composition provided in this embodiment is as follows:

[0046] Artemisia selengensis 50g, Myrrha mukul 35g, Black borneol 35g, Lithospermum officinale 35g, Tibetan madder 35g, Oxytropis 35g and Musk 0.5g.

[0047] The preparation method of the granules of the above-mentioned pharmaceutical composition is as follows:

[0048] ① According to the above weight, weigh Artemisia selengensis, black borneol, Lithospermum officinale, Myrrha mukul, Tibetan madder and Oxytropis oxytropis, decoct in water for 3 times, each adding 8 times the weight of the medicinal material, and each decoction time is 60 min. Filter, take the filtrate, and dry to obtain 33.75 g of extract powder.

[0049] ② Take 33.75g of extract powder, 78.75g of filler (lactose and mannitol in a mass ratio of 4:1) and 0.56g of stevia and mix them evenly. Use 75% (volume percentage) ethanol as a wetting agent to make a soft material, and then granulate it through a 14-mesh sieve. Add 0.5g of musk during the granulation process, put it in an oven, and dry it at 60°C for 2 hours to obtain granules.

[0050] Example 4 Granules

[0051] The raw material composition of the Chinese medicine composition provided in this embodiment is as follows:

[0052] Artemisia selengensis 40g, black borneol 28g, lithospermum officinale 28g, myrrh mukul 28g, madder 28g, oxtera 28g and musk 0.4g.

[0053] The preparation method of the granules of the above-mentioned pharmaceutical composition is as follows:

[0054] ① According to the above weight, weigh Artemisia selengensis, black borneol, Lithospermum officinale, Myrrha mukul, Tibetan madder and Oxytropis oxytropis, decoct in water and extract twice, each adding 8 times the weight of the medicinal materials, and each decoction time is 60 minutes. Filter, take the filtrate, and dry to obtain the extract powder.

[0055] ② Take the extract powder, add the above-mentioned weight of musk, the same mass of filler (dextrin) as the extract powder, and stevia of 1% of the total mass of the extract powder and the filler, mix them evenly, use 80% (volume percentage) ethanol as a wetting agent to make a soft material, then pass it through a 14-mesh sieve to granulate it, put it in an oven, and dry it at 60°C for 2 hours to obtain granules.

[0056] Example 5 Granules

[0057] The raw material composition of the Chinese medicine composition provided in this embodiment is as follows:

[0058] Artemisia selengensis 50g, black borneol 25g, lithospermum officinale 35g, myrrh mukul 25g, madder cordifolia 35g, oxtera 25g and musk 0.3g.

[0059] The preparation method of the granules of the above-mentioned pharmaceutical composition is as follows:

[0060] ① According to the above weight, weigh Artemisia selengensis, black borneol, lithospermum officinale, myrrh, madder cordifolia and oxtera, decoct in water for 3 times, each time adding 8 times the weight of the medicinal materials, and each decoction time is 60 min. Filter, take the filtrate, and concentrate to obtain an extract (relative density at 45°C is 1.3).

[0061] ② Take the extract and add the above-mentioned weight of musk, the same mass of filler (lactose and mannitol in a mass ratio of 4:1) as the dry matter in the extract, and stevioside accounting for 1% of the total mass of the dry matter and filler in the extract powder, mix them evenly, use 80% (volume percentage) ethanol as a wetting agent to make a soft material, then granulate through a 14-mesh sieve, put it in an oven, and dry it at 60°C for 2 hours to obtain granules.

[0062] Example 6 Capsules

[0063] The raw material composition of the Chinese medicine composition provided in this embodiment is as follows:

[0064] Artemisia selengensis 30g, black borneol 35g, lithospermum officinale 25g, myrrh mukul 35g, madder cordata 25g, oxtera 35g and musk 0.5g.

[0065] The preparation method of the granules of the above-mentioned pharmaceutical composition is as follows:

[0066] ① According to the above weight, weigh Artemisia selengensis, black borneol, Lithospermum officinale, Myrrha mukul, Tibetan madder and Oxytropis oxytropis, decoct in water for 3 times, each adding amount is 8 times the weight of the medicinal material, each decoction time is 60min, filter, take the filtrate, and dry to obtain the extract powder.

[0067] ② Take the extract powder, add the above-mentioned weight of musk, the same mass of the filler (lactose and mannitol in a mass ratio of 4:1) and stevioside accounting for 0.5% of the total mass of the extract powder and the filler, mix them evenly, use 80% (volume percentage) ethanol as a wetting agent to make a soft material, then pass through a 14-mesh sieve to granulate, put into an oven, dry at 60°C for 2 hours, and put into capsules to prepare capsules.

[0068] Experimental Example 1: Investigation of the extraction process

[0069] 1. Instruments and medicinal materials

[0070] DZKW-8-8 constant temperature water bath (Beijing Yongguangming Medical Instrument Factory); electronic balance (Shanghai Precision Scientific Instrument Co., Ltd.); vertical ultrapure water purifier (Chongqing Moore Water Treatment Equipment Co., Ltd.); electric hot air drying oven (Shanghai Experimental Instrument Factory Co., Ltd.); adjustable electric heating jacket model: ZDHW (Beijing Zhongxing Weiye Instrument Co., Ltd.).

[0071] The medicinal materials used are sourced from the medicinal material markets in Tibet or Gansu. All medicinal materials have passed quality inspection and comply with the requirements of the "Drug Standards of the Ministry of Health of the People's Republic of China" Tibetan Medicine (Volume 1) and the "Qinghai Province Tibetan Medicinal Material Standards" Volume 1 (2019 edition).

[0072] 2. Orthogonal test method

[0073] (1) Experimental methods

[0074] Weigh 9 portions of each of Artemisia selengensis 4g, Black Borneol 2.8g, Lithospermum officinale 2.8g, Myrrha mukul 2.8g, Rubia cordifolia 2.8g and Oxytropis 2.8g, and decoct them in water. Orthogonal test method was used, with dry paste rate as index, to conduct L9(3 3 ) Orthogonal experiments were conducted to determine the optimal extraction process conditions by examining the amount of water added, the number of decoctions, and the decoction time. The factor levels are shown in Table 1, and the orthogonal experimental design and plan are shown in Table 2.

[0075] Table 1 Factor level table

[0076]

[0077]

[0078] Table 2 Orthogonal experimental design and results

[0079]

[0080] (2) Calculation of dry paste rate

[0081] Take 50 ml of the orthogonal test extract and place it in an evaporating dish that has been dried to a constant weight (M1). Evaporate to dryness in a water bath, dry in an oven at 75°C for 5 hours, remove, place in a desiccator, cool for 30 minutes, and quickly and accurately weigh the mass, recorded as M2 (unit: g). Calculate the dry paste yield according to the following formula, where M is the mass of the prescribed medicinal material (unit: g) and V is the volume of the extract (ml).

[0082]

[0083] 3. Experimental results

[0084] According to the range R analysis in Table 2, the dry paste rate of the Chinese medicine composition of the present invention is affected by the following factors: number of decoctions (B) > amount of water added (A) > decoction time (C), so the number of decoctions has the greatest impact on the dry paste rate of the Chinese medicine composition of the present invention. According to the K-index analysis, the amount of water added K2>K3>K1; the number of decoctions K3>K2>K1; the decoction time K3>K2>K1. The experimental results show that the optimal extraction process of the Chinese medicine composition of the present invention is A2B3C3. Since the decoction time and the amount of water added have little effect, combined with the actual production, the decoction time is selected as 60 minutes, that is, the decoction is performed 3 times, the amount of water added each time is 8 times the amount, and the decoction time is 60 minutes each time. The three extracts are combined and dried to obtain dry paste powder.

[0085] Experimental Example 2: Screening experiment of fillers

[0086] 1. Instruments and test drugs

[0087] (1) Instruments

[0088] Electric hot air drying oven (Shanghai Experimental Instrument Factory Co., Ltd.); Sartorius (Beijing Saiduosi Balance Co., Ltd.); vertical ultrapure water device (Chongqing Moore Water Treatment Equipment Co., Ltd.); electronic balance (produced by Shanghai Precision Scientific Instrument Co., Ltd.); pharmaceutical stability test chamber (Shanghai Longyue Instrument Equipment Co., Ltd.); TG20G desktop high-speed centrifuge (Jiangsu Gaoke Centrifuge); BT-1000 powder comprehensive characteristics tester (Dandong Baxter Instrument Co., Ltd.).

[0089] (2) Drug testing

[0090] The effects of different excipients (sucrose, lactose, starch, microcrystalline cellulose, mannitol, and dextrin) on the granules were investigated. The API composition and preparation method of each granule were essentially the same as those in Example 3, with the only difference being the type of filler added in step ②.

[0091] Granules were prepared by using single excipients, namely sucrose, lactose, starch, microcrystalline cellulose, mannitol or dextrin, as fillers, or composite excipients, namely sucrose and dextrin in a mass ratio of 1:1 or mannitol and dextrin in a mass ratio of 1:3, as fillers instead of the fillers in Example 3, and compared with the granules prepared in Example 3 using lactose and mannitol in a mass ratio of 4:1 as fillers.

[0092] 2. Test methods and results

[0093] (1) Properties

[0094] The properties of the granules made with different excipients are shown in the table below.

[0095] Table 3 Description of properties of single excipients

[0096]

[0097] Table 4 Description of properties of mixed excipients

[0098]

[0099] (2) Determination of formability

[0100] The prepared granules were weighed and passed through a No. 1 sieve, then a No. 4 sieve. Pellets that passed through the No. 1 sieve but not the No. 4 sieve were collected and weighed. Formability = granule mass after sieving / granule mass before sieving * 100%. Formability score was calculated using the formula: (15 / maximum formability value) × formability value (see the Chinese Pharmacopoeia). The results are shown in the table below.

[0101] Table 5 Determination of formability of single auxiliary material

[0102]

[0103] According to Table 5, the mixed granules of extract powder and mannitol have the highest molding rate, which is 83.17%, and the highest score.

[0104] Table 6 Determination of formability of composite auxiliary materials

[0105]

[0106] According to Table 6, it can be seen that the granules formed by using lactose:mannitol with a mass ratio of 4:1 as fillers have the highest forming rate, but the forming rate is lower than that of the granules formed by mixing the extract powder and mannitol as a single auxiliary material.

[0107] (3) Determination of bulk density

[0108] Bulk density, also known as apparent density or loose density, refers to the mass of particles per unit volume. The volume used in bulk density refers to the total volume of the particles, including the voids within them and the spaces between them. A high bulk density indicates a low bulk volume, which can indicate the particle's level of confidentiality and determine the volumetric dosage.

[0109] Experimental Procedure: Place the sieved particles into a dry graduated cylinder and gently vibrate. Read the volume (V) near the scale mark. Use the sieved mass as M (g). The ratio of the two is the bulk density: Bulk density = M / V. The bulk density score is calculated using the formula: (15 / maximum bulk density) × bulk density. Results are shown in the table below.

[0110] Table 7 Determination of bulk density of single auxiliary material

[0111]

[0112] According to the above table, the granules made from sucrose as a single auxiliary material have the largest bulk density and the highest score.

[0113] Table 8 Determination of bulk density of composite excipients

[0114]

[0115] According to the above table, it can be seen that the granules prepared with a mass ratio of lactose to mannitol of 4:1 in the composite excipients have the largest bulk density and the highest score.

[0116] (4) Determination of the angle of repose

[0117] Flowability is a key property of granules. It is related to granule quality and dosing accuracy. In pharmacy, flowability is often expressed in terms of flow rate and angle of repose. The smaller the angle of repose, the better the flowability. Generally, smaller particles or particles with a wider size distribution have a larger angle of repose. Particles with round, large, and uniform particle sizes flow easily and have a smaller angle of repose.

[0118] The angle of repose of the particles was measured using a BT-1000 powder comprehensive property tester. Three replicates were performed and the average value was taken. The angle of repose score was calculated using the formula: (minimum angle of repose value × 15) / angle of repose value.

[0119] Table 9 Determination of the angle of repose of a single excipient

[0120]

[0121] According to the above table, among the single excipients, the granules made with mannitol have the lowest angle of repose and the highest score.

[0122] Table 10 Determination of angle of repose of composite excipients

[0123]

[0124] According to the above table, the granules prepared with a lactose:mannitol mass ratio of 4:1 in the composite excipients have the lowest angle of repose and the highest score.

[0125] (5) Determination of dissolution rate

[0126] Add precisely weighed particles to a dry, constant-weight 5ml centrifuge tube (minimum graduation 0.1ml). Add 5ml of boiling water, stir and shake for 5 minutes, and centrifuge at 3000 rpm for 15 minutes. Discard the supernatant and dry the residue at 80°C to a constant weight. Weigh accurately and calculate the solubility. Solubility = mass of dissolved particles / mass of particles * 100%. The solubility score is calculated using the formula: (20 / maximum solubility value) × solubility value.

[0127] Table 11 Determination of the dissolution rate of a single excipient

[0128]

[0129] According to the table above, the granules made from sucrose as a single auxiliary material have the highest solubility rate, which is 90.29%, and the highest score.

[0130] Table 12 Determination of dissolution rate of composite excipients

[0131]

[0132] According to the above table, it can be seen that the granules prepared with a mass ratio of lactose to mannitol of 4:1 in the composite excipients have the highest solubility rate, which is 84.96%, and the highest score.

[0133] (6) Determination of hygroscopicity

[0134] Take a certain amount of granules prepared with different excipients and place them in a 30°C oven to constant weight for 48 hours. Place NaCl in a glass desiccator containing a saturated NaCl solution at the bottom, periodically adding NaCl until a supersaturated NaCl solution forms. The relative humidity in the desiccator should be 75%. Place granules approximately 2 mm thick at the bottom of a flat weighing bottle that has been held constant weight, accurately weigh them, and place them in the desiccator (with the flat weighing bottle open). Weigh the granules after 24 hours and calculate the percentage of moisture absorption. Perform two sets of tests and calculate the average.

[0135]

[0136] The moisture absorption score is calculated using the formula: (minimum moisture absorption value × 35) / moisture absorption value.

[0137] Table 13 Hygroscopicity determination of single excipients

[0138]

[0139] According to the above table, it can be seen that the granules made from mannitol as a single excipient have the lowest moisture absorption rate, with a moisture absorption percentage of 6.86%, which is the highest score.

[0140] Table 14 Determination of hygroscopicity of composite excipients

[0141]

[0142] According to the hygroscopicity test results of the composite excipients in the above table, it can be seen that the granules prepared with a mass ratio of lactose to mannitol of 4:1 in the composite excipient have the lowest hygroscopicity, with a hygroscopicity percentage of 9.05%, which is the highest score.

[0143] (7) Comprehensive score

[0144] The above five test results are comprehensively evaluated, and the comprehensive index = (15 / maximum formability value) × formability value + (15 / maximum bulk density value) × bulk density value + (minimum angle of repose value × 15) / angle of repose value + (20 / maximum solubility value) × solubility value + (minimum moisture absorption value × 35) / moisture absorption value.

[0145] Table 15 Comprehensive score of single excipient

[0146]

[0147] According to the comprehensive scoring results of the above table 1 excipients, it can be seen that the granules made of mannitol have the highest comprehensive score.

[0148] Table 16 Comprehensive score of composite excipients

[0149]

[0150] According to the comprehensive scoring results of the composite excipients in the above table, it can be seen that when the excipients for preparing granules are selected with a mass ratio of lactose:mannitol of 4:1, the comprehensive score of granule performance is the highest. Therefore, lactose:mannitol with a mass ratio of 4:1 is selected as the preferred filler for the granules of the Chinese medicine composition of the present invention.

[0151] Experimental Example 3: Investigation of sweetener dosage

[0152] The effect of the sweetener dosage on the granules was investigated. The raw material composition and preparation method of each granule were basically the same as those in Example 3, with the only difference being the dosage of the sweetener in step ②.

[0153] Granules were prepared by adding stevia at 0.05%, 0.1%, 0.5%, 1%, 3% and 6% of the total weight of the extract powder and the filler. Each group of granules was dissolved in 250 ml of hot water and the taste was evaluated orally as shown in the table below.

[0154] Table 17 Stevia dosage screening results

[0155]

[0156] According to the results of the investigation on the dosage of sweeteners, the granules obtained by using 0.5% stevia have moderate sweetness and good taste.

[0157] Experimental Example 4 Acute Toxicity Experimental Investigation of the Granules of the Present Invention

[0158] 1. Experimental instruments and test drugs

[0159] 1.1 Instrument

[0160] Epoch microplate reader (Bio-Tek, USA), TGL-16K desktop high-speed refrigerated centrifuge (Hunan Xiangyi Centrifuge Factory), Olympus BX51 microscope (Olympus Corporation, Japan), 4% tissue fixative (Solarbio Company), automatic dehydrator (Jinhua Kedi Instrument Equipment Co., Ltd., Zhejiang Province), KD-BMⅡ computer biological tissue embedding machine (Jinhua Kedi Instrument Equipment Co., Ltd., Zhejiang Province), KD-BL freezing table (Jinhua Kedi Instrument Equipment Co., Ltd., Zhejiang Province), microtome (MICROM), manual pipette (Eppendorf Company, Germany).

[0161] 1.2 Experimental Reagents

[0162] Aspirin: (Aojina Pharmaceutical Co., Ltd., batch number: 191202), ALT and AST kits were purchased from Nanjing Jiancheng Bioengineering Institute, batch numbers: 20211028 and 20210420.

[0163] 1.3 Experimental drugs

[0164] The drug of the present invention (granules prepared in Example 3) was dissolved in an appropriate amount of water to prepare a drug solution of the desired concentration before the experiment.

[0165] 2 Methods and Results

[0166] 2.1 Observe the pathological changes of transaminases and major organs after 14 days of the maximum daily dose

[0167] Twenty mice were randomly divided into a normal group and a drug group. Prior to the experiment, mice were fasted for 12 hours but not water. Within 24 hours of the experiment, mice in the drug group were gavaged with the aforementioned granules, administered orally every 5 hours at a concentration of 0.5 g / mL and a volume of 10 mL / kg. The mice were observed for 14 consecutive days for mental status, fur changes, and mortality. Any deaths were recorded and dissected. After 14 days, the mice were sacrificed by cervical dislocation, and the hearts, livers, spleens, and kidneys were removed and fixed in 4% paraformaldehyde. After 48 hours, the tissues were dehydrated in varying degrees of alcohol, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) for microscopic observation.

[0168] 2.2 Data Processing

[0169] All data were expressed as mean ± standard deviation (X ± s). The differences between the two groups were compared using the t test.

[0170] 2.3 Results

[0171] Compared with the normal group, the mice in the drug group did not lose weight significantly, there was no death, and there was no significant change in mental state, fur changes, etc. From the statistical data of serum ALT and AST transaminase in Table 18, there was no effect on the two (p>0.05), indicating that no liver toxicity side effects were produced. Figure 2-Figure 5 Pathological section analysis showed no significant effects on the structures of the heart, liver, spleen, and kidneys.

[0172] Table 18 Effects of the maximum daily dose of the granules of the present invention on serum ALT and AST (n=10, )

[0173]

[0174] Experimental Example 5 Study on the Effect of the Granules of the Present Invention on Lipopolysaccharide (LPS)-Induced Fever in Rats

[0175] 1. Experimental instruments and test drugs

[0176] 1.1 Instrument

[0177] Epoch microplate reader (Bio-Tek, USA), TGL-16K desktop high-speed refrigerated centrifuge (Hunan Xiangyi Centrifuge Factory), TS0-B electronic thermometer (Zhengzhou Maiston Medical Technology), and manual pipette (Eppendorf, Germany).

[0178] 1.2 Reagents

[0179] Aspirin: (Aojina Pharmaceutical Co., Ltd., batch number: 191202), rat tumor necrosis factor α (TNF-α) ELISA kit (Jiangsu Enzyme Immunity Industrial Co., Ltd., batch number: MM-0180R2), rat interleukin 1β (IL-1β) ELISA kit (Jiangsu Enzyme Immunity Industrial Co., Ltd., batch number: MM-0047R2), rat cyclic adenosine monophosphate (cAMP) ELISA kit (Jiangsu Enzyme Immunity Industrial Co., Ltd., batch number: MM-0549R2), rat prostaglandin E2 (PGE2) ELISA kit (Jiangsu Enzyme Immunity Industrial Co., Ltd., batch number: MM-0068R2), lipopolysaccharide (LPS) (Shanghai Yuanye Biotechnology Co., Ltd., batch number: J01GS150052).

[0180] 1.3 Test drugs

[0181] Before the experiment, the drug of the present invention (granules prepared in Example 3) was added to an appropriate amount of water to prepare a test drug solution of the required concentration.

[0182] 2 Experimental methods and data processing methods

[0183] 2.1 Experimental methods

[0184] A rat fever model was established using 120 μg / kg of LPS (lipopolysaccharide). After screening, rats with normal body temperature were divided into three groups: a blank group, a model group, a group with the granules of the present invention (three dose groups: high 0.36 g / kg, medium 0.18 g / kg, and low 0.09 g / kg), and an aspirin (0.1 g / kg) positive group, with six rats in each group. Except for the model and blank groups, all groups received the drug once. Rectal temperatures were measured every 15 minutes for a total of 3 hours, and temperature curves were plotted. Serum cytokine levels (TNF-α, IL-1β, cAMP, and PGE2) and hypothalamic thermoregulatory mediators (cAMP and PGE2) were measured using ELISA.

[0185] 2.2 Data processing methods

[0186] All data are expressed as mean ± standard deviation (X ± s). The antipyretic effect of the two groups was compared using the t-test method.

[0187] 2.3 Experimental Results

[0188] (1) Effect of the granules of the present invention on body temperature changes in LPS-induced febrile rats

[0189] See the results Figure 6 As shown in the figure, compared with the blank group, the body temperature of the model group was significantly higher than that of the blank group, and the body temperature of the rats after drug administration was significantly lower than that of the model group, indicating that each drug-treated group had a significant inhibitory effect on the increase in body temperature of rats induced by LPS.

[0190] (2) Effects of the granules of the present invention on TNF-α, IL-1β, PGE2 and cAMP in the serum of rats with LPS-induced fever

[0191] The results are shown in the table below. Compared with the normal control group, the levels of TNF-α, IL-1β, PGE2 and cAMP in the serum of the rats in the model control group were significantly increased. Compared with the model group, except that the low dose of the granules of the present invention had no statistically significant effect on cAMP, the other aspirin groups and the granules of the present invention at each dose group could significantly reduce the levels of TNF-α, IL-1β, PGE2 and cAMP in the serum of the rats.

[0192] Table 19 Effects of the granules of the present invention on TNF-α, IL-1β, PGE2 and cAMP in serum of rats with LPS-induced fever ( n=6)

[0193]

[0194] Note: Compared with the blank group and the model group, # P<0.05, ## P<0.01, each drug-treated group was compared with the model group, * P<0.05, ** P<0.01.

[0195] (3) Effect of the granules of the present invention on PGE2 and cAMP in brain tissue homogenates of rats with LPS-induced fever

[0196] The results are shown in the table below. Compared with the normal control group, the PGE2 and cAMP levels in the brain tissue homogenate of the rats in the model control group were significantly increased. Compared with the model group, except for the low dose of the granules of the present invention which had no statistically significant effect on cAMP, the other aspirin groups and the granules of the present invention at all doses could reduce the PGE2 and cAMP levels in the rat brain tissue.

[0197] Table 20 Effects of the granules of the present invention on PGE2 and cAMP in brain tissue homogenates of LPS-induced fever rats ( n=6)

[0198]

[0199] Note: Compared with the blank group and the model group, # P<0.05, ## P<0.01, each drug-treated group was compared with the model group, * P<0.05, ** P<0.01.

[0200] 3. Experimental Conclusion

[0201] (1) The granules of the present invention have a good antipyretic effect on LPS fever model rats. The antipyretic effect may be related to its inhibition of the increase of TNF-α, IL-1β, PGE2 and cAMP and the release of cAMP and PGE2 in the hypothalamus.

[0202] (2) Based on the results of this experiment, the medium dose has a better overall effect. The high dose may cause the temperature to rise initially due to the large dose, then decrease and stabilize. The antipyretic effect of the granules of the present invention lasts for about 30 minutes, and its long duration is its characteristic.

[0203] Experimental Example 6 Experimental study on antipyretic and anti-inflammatory effects of the granules of the present invention

[0204] 1. Experimental samples

[0205] Before the experiment, the drug of the present invention (granules prepared in Example 3) was added to an appropriate amount of water to prepare a test drug solution with a concentration of 0.20 g / mL. Positive drug: Aspirin was added to an appropriate amount of water to prepare a drug solution with a concentration of 0.05 g / mL.

[0206] 2. Experimental reagents and instruments

[0207] Fresh egg white (Guilin Medical College Fresh Supermarket), aspirin (Shenyang Aojina Pharmaceutical Co., Ltd., batch number: 191202), vernier caliper (Menite, Germany).

[0208] 3. Experimental methods

[0209] Fifty-four male rats were randomly divided into nine groups, with six rats in each group. Groups and doses are shown in the table below. Three toe width measurements were taken before the experiment, and the average value was used as the normal toe width. All groups except the blank group were injected with 0.1 mL of 10% (v / v) fresh egg white into the right hind paw. All groups except the model and blank groups were gavage-administered 10 mL / kg of the drug 0.5 h before modeling; the model group was given 0.5% saline solution. Vernier calipers (Menite, Germany) were used to measure right hind paw swelling 5 min, 0.5 h, 2 h, and 4 h after drug administration. The change in right hind paw swelling before and after inflammation was calculated for each group, and the paw swelling rate was used to represent the anti-inflammatory effect of the drug.

[0210] Foot swelling rate = (foot swelling after modeling - foot width before modeling) / foot width before modeling.

[0211] 4. Experimental results

[0212] The plantar swelling of the rats in the model group was obvious after modeling, and the swelling quickly reached a peak about 30 minutes after modeling; compared with the model group, the positive group showed significant anti-inflammatory effects at all time periods, and the high dose of the granules of the present invention significantly reduced the degree of plantar swelling of the rats 30 minutes after modeling (P < 0.05), which was statistically significant. The changes at other doses and time periods were not obvious and had no statistical significance.

[0213] Table 21 Effects of the granules of the present invention on egg white-induced toe swelling in rats (n=6, )

[0214]

[0215]

[0216] Note: Comparison between blank group and model group, #P<0.05, ##P<0.01, comparison between drug-treated group and model group, *P<0.05, **P<0.01

[0217] 5. Experimental Conclusion

[0218] In this experiment, since the swelling quickly reached a peak around 30 minutes after modeling, the high dose of the granules of the present invention significantly reduced the swelling of the paws of rats 30 minutes after modeling (P < 0.05), which was statistically significant, indicating that the high dose of the granules of the present invention had an anti-inflammatory effect on extremely significant inflammation.

[0219] Experimental Example 7: Efficacy against different viruses

[0220] 1. Preparation of test drug solution and virus solution

[0221] (1) Granular test drug solution: The granules prepared in Example 3 were used as the test drug, dissolved in water to obtain a stock solution with a concentration of 0.2 g / mL, and diluted with cell culture medium to obtain drug solutions with concentrations of 1000 μg / mL, 500 μg / mL, and 250 μg / mL, respectively;

[0222] Extract drug solution: The Chinese herbal extract prepared in Example 1 was used as the test drug, dissolved in water to obtain a mother solution with a concentration of 0.1 g / mL, and then added to cell culture medium to dilute the solution to concentrations of 500 μg / ml, 250 μg / ml, and 125 μg / ml, respectively.

[0223] (2) Virus solution

[0224] Gaussia luciferase recombinant virus H1N1, Gaussia luciferase recombinant virus H5N1.

[0225] 2. Experimental methods

[0226] The following methods were used to test the inhibitory effects of the extract and granules of the Chinese medicine composition of the present invention on H1N1 and H5N1, respectively: a sterile 96-well culture plate was prepared, 100 μL of a 1×10 5cells / mLBEAS-2B cells were cultured at 37°C, 5% CO2 for 24 hours and randomly divided into a cell control group, a granule experimental group, an extract experimental group, and a virus control (negative control) group. The granule experimental group, the extract experimental group control group, and the virus control group were each added with 100 TCID50 virus solution at 100 μL / well. After 2 hours, the cell culture medium in the 96-well culture plate was discarded. The granule experimental group and the extract experimental group were respectively added with different concentrations of the granule test drug solution and the extract drug solution at a dose of 100 μL / well, with three replicates for each concentration. A blank control (solvent control) group was also established; the blank control group received 100 μL / well of cell culture medium without cells.

[0227] Each group of cells was incubated in a 37°C, 5% CO2 incubator for 36 hours. 50 μl of supernatant was taken from each well of the 96-well cell culture plate and placed on a clean 96-well black plate. After setting up the microplate reader program, a 20 μg / ml coelenterazine solution (prepare in the dark and use immediately) was prepared and added to the 96-well black plate, 25 μl per well, and the chemiluminescence value was immediately measured (measured 3 times). Calculate the IC50. Inhibition rate (%) = [(average luminescence intensity of the virus control group - average luminescence intensity of the experimental group) / (average luminescence intensity of the virus control group - average luminescence intensity of the blank control group] × 100%. The 50% effective concentration (IC50) was calculated by regression analysis.

[0228] 3. Experimental results

[0229] Table 22 IC50μg / ml of the drugs of the present invention against different viruses

[0230] project H1N1 H5N1 Extract of the Chinese medicine composition of the present invention 12.15 μg / ml 13.54 μg / ml Granules of the present invention 10.56 μg / ml 12.42 μg / ml

[0231] The above experimental results show that the extract and granules of the Chinese herbal composition of the present invention can significantly inhibit the replication of influenza viruses H1N1 and H5N1 in vitro. The granules are more effective than the Chinese herbal extract of the present invention. The effect on H1N1 is better than that on H5N1.

[0232] Experimental Example 8: Efficacy against different viruses

[0233] 1. Preparation of test drug solution and virus solution

[0234] (1) Granular test drug solution: The granules prepared in Example 3 were used as the test drug, dissolved in water to obtain a stock solution with a concentration of 0.2 g / mL, and diluted with cell culture medium to obtain drug solutions with concentrations of 1000 μg / mL, 500 μg / mL, and 250 μg / mL, respectively;

[0235] Extract drug solution: The Chinese herbal extract prepared in Example 1 was used as the test drug, dissolved in water to obtain a mother solution with a concentration of 0.1 g / mL, and then added to cell culture medium to dilute the solution to concentrations of 500 μg / ml, 250 μg / ml, and 125 μg / ml, respectively.

[0236] (2) Virus solution

[0237] A / Puerto Rico / 8 / 1934(H1N1), A / GZ / GIRD07 / 09(H1N1).

[0238] 2. Experimental methods

[0239] The following methods were used to test the inhibitory effects of the extract and granules of the Chinese medicine composition of the present invention on A / PuertoRico / 8 / 1934 (H1N1) and A / GZ / GIRD07 / 09 (H1N1), respectively. Specifically, a sterile 96-well culture plate was prepared, and 100 μL of a 1×10 5 cells / mLBEAS-2B cells were cultured at 37°C, 5% CO2 for 24 hours and randomly divided into a cell control group, a granule experimental group, an extract experimental group, and a virus control (negative control) group. The granule experimental group, the extract experimental group, and the virus control group were each added with 100 μL / well of 100 TCID50 virus solution. After 2 hours, the cell culture medium in the 96-well culture plate was discarded. The granule experimental group and the extract experimental group were respectively added with different concentrations of the above-mentioned granule test drug solution and extract drug solution, with a dose of 100 μL / well, and three replicates for each concentration. A blank control (solvent control) group was also established; the blank control group received 100 μL / well of cell culture medium without cells.

[0240] Each group of cells was incubated in a 37°C, 5% CO2 incubator for 36 hours. 50 μl of supernatant was taken from each well of the 96-well cell culture plate and placed on a clean 96-well black plate. After setting up the microplate reader program, a 20 μg / ml coelenterazine solution (prepare in the dark and use immediately) was prepared and added to the 96-well black plate, 25 μl per well, and the chemiluminescence value was immediately measured (measured 3 times). Calculate the IC50. Inhibition rate (%) = [(average luminescence intensity of the virus control group - average luminescence intensity of the experimental group) / (average luminescence intensity of the virus control group - average luminescence intensity of the blank control group] × 100%. The 50% effective concentration (IC50) was calculated by regression analysis.

[0241] 3. Experimental results

[0242] Table 23 IC50μg / ml of the drugs of the present invention against different viruses

[0243]

[0244] The above experimental results show that, using lung epithelial BEAS-2B cells as target cells, both the extract and the granules of the present invention significantly inhibited influenza virus-induced cytopathic effects. The granules were more effective than the extract of the present invention. The effect against A / GZ / GIRD07 / 09 (H1N1) was superior to that against A / Puerto Rico / 8 / 1934 (H1N1).

[0245] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of a Chinese medicine composition in the preparation of an anti-influenza virus drug, characterized in that: The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 30-50 parts of Artemisia selengensis, 20-35 parts of black borneol, 20-35 parts of Lithospermum officinale, 20-35 parts of Myrrha mukul, 20-35 parts of Tibetan madder, 20-35 parts of Oxytropis oxyphylla, and 0.3-0.5 parts of musk. The preparation method of the traditional Chinese medicine composition comprises the following steps: Weigh Artemisia selengensis, black borneol, lithospermum officinale, myrrha mukul, madder cordifolia and oxtera przewalskii, extract with water, filter, collect the filtrate, concentrate and / or dry, and mix with musk to obtain a traditional Chinese medicine composition. The influenza virus is selected from H1N1 influenza virus or H5N1 influenza virus.

2. Use of a pharmaceutical preparation in the preparation of an anti-influenza virus drug, characterized in that: The pharmaceutical preparation is prepared from the following raw materials in parts by weight: 30-50 parts of Artemisia selengensis, 20-35 parts of Black Borneol, 20-35 parts of Lithospermum officinale, 20-35 parts of Myrrha mukul, 20-35 parts of Tibetan Rubia cordifolia, 20-35 parts of Oxytropis oxyphylla, and 0.3-0.5 parts of Musk, and further includes pharmaceutically acceptable excipients. The influenza virus is selected from H1N1 influenza virus or H5N1 influenza virus. The preparation method of the pharmaceutical preparation comprises the following steps: Artemisia selengensis, black borneol, lithospermum officinale, myrrha mukul, madder cordifolia and oxytropis chinensis are weighed, extracted with water, filtered, the filtrate is collected, concentrated and / or dried to prepare an extract, and pharmaceutically acceptable excipients and musk are added to prepare a pharmaceutical preparation according to a conventional process in the art.

3. The use according to claim 2, characterized in that The pharmaceutical preparation is a decoction, tablet, pill, granule or capsule.

4. The use according to claim 2, characterized in that The pharmaceutical preparation is a granule, and the auxiliary materials of the granule include a filler and / or a sweetener.

5. The use according to claim 4, characterized in that The filler is selected from one or more of sucrose, lactose, starch, microcrystalline cellulose, mannitol and dextrin.

6. The use according to claim 5, characterized in that The filler comprises lactose and mannitol in a mass ratio of 4:0.8-1.

2.

7. The use according to claim 4, characterized in that The sweetener is one or more of stevia, aspartame, and sucralose.

8. The use according to claim 4, characterized in that The sweetener is stevia.

9. The use according to claim 2, characterized in that The pharmaceutically acceptable excipients include fillers, and the mass ratio of the dry matter in the extract to the filler is 1:0.5-3.

10. The use according to claim 2, characterized in that The pharmaceutically acceptable excipients further include a sweetener, and the mass percentage of the sweetener to the total mass of the dry matter and filler in the extract is 0.05%-6%.

11. The use according to claim 10, characterized in that The mass percentage of the sweetener to the total mass of the dry matter and filler in the extract is 0.5%.

12. The use according to claim 1 or 2, characterized in that: During the water extraction process, the number of water extractions is 1-3 times, the amount of water used each time is 8-12 times the weight of the medicinal material, and the extraction time each time is 60-120 minutes.

13. The use according to claim 1 or 2, characterized in that: The H1N1 influenza virus is numbered A / PuertoRico / 8 / 1934.

Images