A kind of cold-dispelling, damp-removing and anti-toxic granule and its preparation method

Abstract

The present invention provides a cold-dispelling, damp-removing and anti-toxic granule and a preparation method thereof. The cold-dispelling, damp-removing and anti-toxic granule comprises a main component and auxiliary materials. The main component comprises, by weight, 150-170 parts of patchouli, 150-170 parts of boiled bitter almonds, 150-170 parts of cassia twigs, 100-120 parts of perilla leaves, 150-170 parts of peucedanum chinense, 150-170 parts of stir-fried fructus aurantii with bran, 150-170 parts of dried tangerine peel, and 150-170 parts of platycodon grandiflorum. 150-170 parts of Poria cocos, 150-170 parts of Pinellia ternata, 150-170 parts of bran-fried Atractylodes macrocephala, 150-170 parts of ginger, 150-170 parts of jujube, 100-120 parts of roasted licorice root, and 50-65 parts of cicada slough; the auxiliary ingredients are composed of dextrin and powdered sugar, with the weight ratio of the components in the auxiliary ingredients being: dextrin: powdered sugar = (3.5-4):1; the weight ratio of the main ingredient to the auxiliary ingredients is: main ingredient: auxiliary ingredient = (1-1.2):1. The cold-dispelling, dampness-removing, and toxin-removing granules provided by the present invention can dispel cold, dampness, and toxins, inhibit lung tissue inflammation, and alleviate lung damage.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine, and in particular to a cold-dispelling, dampness-removing and poison-resisting granule and a preparation method thereof. Background Art

[0002] Pneumonia patients have elevated inflammatory factors, which can easily lead to lung damage. Some patients also experience the following symptoms: aversion to cold, dampness, and dryness, with a predominant aversion to cold and fever, accompanied by body aches, heaviness, no sweating, fatigue, a dry cough, or scanty, sticky sputum. These symptoms are also common in patients with viral respiratory infections.

[0003] Traditional Chinese medicine has unique advantages in the face of pneumonia or viral respiratory infections. Therefore, developing a traditional Chinese medicine composition for treating pneumonia or viral respiratory infections, inhibiting lung tissue inflammation, removing dampness and resisting toxins, and reducing lung damage is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0004] To this end, the present invention proposes a cold-dispelling, dampness-removing and toxin-resistant granule and a preparation method thereof, so as to develop a new Chinese medicine composition for treating pneumonia or viral respiratory tract infection, inhibiting lung tissue inflammation, removing dampness and toxins, and alleviating lung damage.

[0005] One aspect of the present invention provides a cold-dispelling, dampness-removing and toxic-removing granule, comprising a main component and auxiliary materials, wherein the main components include, by weight, 150-170 parts of patchouli, 150-170 parts of boiled bitter almonds, 150-170 parts of cassia twigs, 100-120 parts of perilla leaves, 150-170 parts of peucedanum, 150-170 parts of stir-fried fructus aurantii with bran, 150-170 parts of dried tangerine peel, 150-170 parts of platycodon grandiflorum, 150-170 parts of poria, 150-170 parts of pinellia ternata, 150-170 parts of stir-fried atractylodes macrocephala with bran, 150-170 parts of ginger, 150-170 parts of jujubes, 100-120 parts of roasted licorice, and 50-65 parts of cicada sloughs;

[0006] The auxiliary material consists of dextrin and powdered sugar, and the weight ratio of each component in the auxiliary material is:

[0007] Dextrin: powdered sugar = (3.5-4): 1;

[0008] The weight ratio of the main component and the auxiliary materials is:

[0009] Main ingredient: auxiliary material = (1-1.2): 1.

[0010] Another aspect of the present invention provides a method for preparing the above-mentioned cold-dispelling, dampness-removing and anti-toxic granules, the preparation method comprising the following steps:

[0011] (1) Weigh the main ingredient and auxiliary materials in proportion, add water to the main ingredient and boil it twice. The first time, add 10-10.5 times the amount of water and boil it for 1.5 hours. The second time, add 6-6.5 times the amount of water and boil it for 1 hour. Combine the extracts, filter, and concentrate the filtrate under reduced pressure to an extract with a relative density of 1.2-1.3 at 60°C.

[0012] (2) The extract is vacuum dried at 60°C, -0.09 MPa, and 12 h to obtain an extract powder with a water content of <5%;

[0013] (3) Add excipients, wetting agents, and flavoring agents to the extract powder, mix and granulate, and then pass through an 80-mesh sieve to obtain wet granules;

[0014] (4) Dry the wet granules to make granules for dispelling cold, removing dampness and resisting poison.

[0015] The cold-dispelling, dampness-removing and toxic-resistant granules provided by the present invention have a formula as follows:

[0016] Patchouli in the formula enters the lung, spleen, and stomach meridians. Its aromatic and pungent aroma is dispersing but not harsh, and it is slightly warm and dehumidifies without being hot. It is good at dispersing cold, regulating qi, and dehumidifying, stopping vomiting and harmonizing the stomach, and invigorating the spleen and appetite. Simmered bitter almonds enter the lung and large intestine meridians. It is good at relieving cough and asthma. Patchouli and boiled bitter almonds taken together in the Taiyin Lung can play the role of dispelling exterior pathogens, promoting lung function, and dehumidifying. They can also enter the spleen, stomach, and large intestine, expelling pathogens if there are any, and calming the areas that have not been affected by pathogens if there are none. This is the monarch drug. Cinnamon twig, Perilla frutescens, and Peucedanum chinense dispel exterior pathogens, induce slight sweating, and help the monarch drug resist toxicity and reduce fever. This is the minister drug. Citrus aurantium, dried tangerine peel, and Platycodon grandiflorum regulate qi, resolve phlegm, and relieve chest tightness. Poria cocos, Pinellia ternata, and Atractylodes macrocephala remove phlegm and penetrate dampness. Ginger and jujube harmonize the Ying and Wei. Cicada slough dispels wind. This is the adjuvant drug. Licorice harmonizes all the drugs. This is the guiding drug. The whole formula can be used together to eliminate toxins, relieve exterior symptoms, reduce fever, promote smooth flow of qi, eliminate phlegm, remove dampness, and restore lung function.

[0017] Main medicine: Patchouli, burnt bitter almonds;

[0018] Assistant medicines: cinnamon twig, perilla leaf, peucedanum;

[0019] Auxiliary drugs: fried Citrus aurantium with bran, dried tangerine peel, Platycodon grandiflorum, Poria cocos, Pinellia ternata, fried Atractylodes macrocephala with bran, ginger, jujube, cicada shell;

[0020] Assistant drug: roasted licorice.

[0021] The present invention experimentally studies the effect of Sanhan Chushi Kangdu Granules on the lungs of mice with lipopolysaccharide (LPS)-induced lung injury. The experiment illustrates the protective effect of Sanhan Chushi Kangdu Granules on LPS-induced lung injury in mice. Sanhan Chushi Kangdu Granules can reduce lipopolysaccharide-induced lung inflammation in mice by inhibiting the production of proinflammatory cytokines and promoting the expression of anti-inflammatory cytokines. Sanhan Chushi Kangdu Granules can promote the increase in Treg cell levels and the expression level of the anti-inflammatory factor IL-10, effectively inhibiting the progression of inflammation in mice with lipopolysaccharide-induced lung injury. In addition, through anti-coronavirus experiments, it is shown that Sanhan Chushi Kangdu Granules has a good ability to inhibit coronavirus activity in vitro and can significantly reduce the expression level of HCoV-229E virus M protein mRNA in cell supernatant.

[0022] The preparation method of the cold-dispelling, dampness-removing and toxic-resistant granules of the present invention adopts dextrin and powdered sugar as auxiliary materials, and specifically defines the weight ratio of each component in the auxiliary materials as: dextrin: powdered sugar = (3.5-4):1; simultaneously, the weight ratio of the main component to the auxiliary material is defined as: main component: auxiliary material = (1-1.2):1. In conjunction with the optimized preparation process of the present invention, the main component is first decocted with water twice, the amount of water added and the decocting time are reasonably set, and the filtrate is decompressed and concentrated to an extract with a relative density of 1.2-1.3 at 60°C, and then vacuum dried under defined conditions to obtain an extract powder with a water content of less than 5%. Then, the auxiliary materials, a wetting agent, and a flavoring agent are added, mixed and granulated, and then passed through an 80-mesh sieve to obtain wet granules. Finally, the wet granules are dried to prepare the cold-dispelling, dampness-removing and toxic-resistant granules. Experimental results show that the granules obtained by the method have good medicinal efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the HPLC chart of amygdalin;

[0024] Figure 2 This is the linear regression equation diagram for the determination of amygdalin content;

[0025] Figure 3 This is a graph showing changes in weight, weight change rate, and weight growth rate of mice;

[0026] Figure 4 This is a graph showing changes in lung weight, lung weight index, spleen weight, and spleen weight index of mice;

[0027] Figure 5 This is the HE staining result of the lung tissue of mice in the control group;

[0028] Figure 6 This is the HE staining result of the lung tissue of mice in the LPS group;

[0029] Figure 7 This is the HE staining result of lung tissue of mice in the SHCS1 group;

[0030] Figure 8 This is the HE staining result of lung tissue of mice in the SHCS2 group;

[0031] Figure 9 This is the HE staining result of lung tissue of mice in the SHCS3 group;

[0032] Figure 10 This is the HE staining result of lung tissue of mice in the PAT group;

[0033] Figure 11 This is a graph showing changes in cytokine levels in mouse lung tissues detected by ELISA;

[0034] Figure 12 This is a graph showing changes in immunoglobulin levels in mouse lung tissues detected by ELISA;

[0035] Figure 13 This is the result diagram of the impact on the level of CD4+CD25+ (Treg cells) and its subpopulation cells in mouse lung tissue;

[0036] Figure 14 This is the result of in vitro efficacy evaluation of Sanhanchushikangdu Granules;

[0037] Figure 15 This is a graph showing the results of the inhibition of the expression level of HCOV-229E virus M protein mRNA by the Sanhan Chushi Kangdu Granules. DETAILED DESCRIPTION

[0038] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to various embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention pertains. The terms used in this specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0040] The following further describes the embodiments of the present invention in multiple embodiments. The embodiments of the present invention are not limited to the following specific embodiments. Within the scope of the unchanged main rights, appropriate changes can be made to the implementation.

[0041] Example 1:

[0042] A cold-dispelling, dampness-removing and toxic-resisting granule comprises a main component and auxiliary materials. The main components comprise, by weight, 167 parts of patchouli, 167 parts of stewed bitter almonds, 167 parts of cassia twigs, 111 parts of perilla leaves, 167 parts of peucedanum, 167 parts of stir-fried fructus aurantii with bran, 167 parts of dried tangerine peel, 167 parts of platycodon, 167 parts of poria, 167 parts of pinellia, 167 parts of stir-fried atractylodes macrocephala with bran, 167 parts of ginger, 167 parts of jujube, 111 parts of roasted liquorice, and 56 parts of cicada shells.

[0043] The auxiliary material consists of dextrin and powdered sugar, and the weight ratio of each component in the auxiliary material is:

[0044] Dextrin: powdered sugar = 4:1;

[0045] The weight ratio of the main component and the auxiliary materials is:

[0046] Main ingredient: auxiliary material = 1:1.

[0047] Example 2:

[0048] A cold-dispelling, dampness-removing and toxic-resisting granule comprises a main component and auxiliary materials. The main components comprise, by weight, 150 parts of patchouli, 165 parts of stewed bitter almonds, 170 parts of cassia twigs, 100 parts of perilla leaves, 150 parts of peucedanum, 165 parts of stir-fried fructus aurantii with bran, 160 parts of dried tangerine peel, 150 parts of platycodon, 165 parts of poria, 160 parts of pinellia, 160 parts of stir-fried atractylodes with bran, 165 parts of ginger, 170 parts of jujube, 120 parts of roasted liquorice, and 50 parts of cicada shells.

[0049] The auxiliary material consists of dextrin and powdered sugar, and the weight ratio of each component in the auxiliary material is:

[0050] Dextrin: powdered sugar = 4:1;

[0051] The weight ratio of the main component and the auxiliary materials is:

[0052] Main ingredient: auxiliary material = 1.2:1.

[0053] Example 3:

[0054] A cold-dispelling, dampness-removing and toxic-resisting granule comprises a main component and auxiliary materials. The main components comprise, by weight, 160 parts of patchouli, 160 parts of stewed bitter almonds, 150 parts of cassia twigs, 115 parts of perilla leaves, 170 parts of peucedanum, 170 parts of stir-fried fructus aurantii with bran, 165 parts of dried tangerine peel, 165 parts of platycodon grandiflorum, 150 parts of poria, 170 parts of pinellia ternata, 150 parts of stir-fried atractylodes macrocephala with bran, 170 parts of ginger, 150 parts of jujubes, 110 parts of roasted liquorice, and 55 parts of cicada shells.

[0055] The auxiliary material consists of dextrin and powdered sugar, and the weight ratio of each component in the auxiliary material is:

[0056] Dextrin: powdered sugar = 3.5:1;

[0057] The weight ratio of the main component and the auxiliary materials is:

[0058] Main ingredient: auxiliary material = 1:1.

[0059] Example 4:

[0060] A cold-dispelling, dampness-removing and toxic-resistant granule comprises a main component and auxiliary materials. The main components comprise, by weight, 170 parts of patchouli, 150 parts of stewed bitter almonds, 160 parts of cassia twigs, 120 parts of perilla leaves, 160 parts of peucedanum, 150 parts of stir-fried fructus aurantii with bran, 170 parts of dried tangerine peel, 170 parts of platycodon, 170 parts of poria, 150 parts of pinellia, 170 parts of stir-fried atractylodes macrocephala with bran, 150 parts of ginger, 165 parts of jujube, 100 parts of roasted liquorice, and 65 parts of cicada shells.

[0061] The auxiliary material consists of dextrin and powdered sugar, and the weight ratio of each component in the auxiliary material is:

[0062] Dextrin: powdered sugar = 4:1;

[0063] The weight ratio of the main component and the auxiliary materials is:

[0064] Main ingredient: auxiliary material = 1.1:1.

[0065] Example 5:

[0066] A cold-dispelling, dampness-removing and toxic-resisting granule comprises a main component and auxiliary materials. The main components comprise, by weight, 165 parts of patchouli, 170 parts of stewed bitter almonds, 165 parts of cassia twigs, 110 parts of perilla leaves, 165 parts of peucedanum, 160 parts of stir-fried fructus aurantii with bran, 150 parts of dried tangerine peel, 160 parts of platycodon, 160 parts of poria, 165 parts of pinellia, 165 parts of stir-fried atractylodes macrocephala with bran, 160 parts of ginger, 160 parts of jujube, 105 parts of roasted liquorice, and 60 parts of cicada shells.

[0067] The auxiliary material consists of dextrin and powdered sugar, and the weight ratio of each component in the auxiliary material is:

[0068] Dextrin: powdered sugar = 4:1;

[0069] The weight ratio of the main component and the auxiliary materials is:

[0070] Main ingredient: auxiliary material = 1.2:1.

[0071] Example 6

[0072] A method for preparing cold-dispelling, dampness-removing and toxic-resistant granules comprises the following steps:

[0073] (1) The main component and auxiliary materials were weighed according to the proportions of Example 1, and the main component was decocted with water twice. The first time, 10 times the amount of water was added, and the decocted for 1.5 hours, and filtered through a 100-mesh filter cloth. The second time, 6 times the amount of water was added, and the decocted for 1 hour, and filtered through a 100-mesh filter cloth. The extracts were combined, filtered, and the filtrate was concentrated under reduced pressure to an extract with a relative density of 1.2 at 60°C. The conditions for reduced pressure concentration were: -0.08 MPa, 65°C, and 2.5 hours.

[0074] (2) The extract is vacuum dried at 60°C, -0.09 MPa, and 12 h to obtain an extract powder with a water content of <5%;

[0075] (3) Add excipients, wetting agents, and flavoring agents to the extract powder, mix and granulate, and then pass through an 80-mesh sieve to obtain wet granules. The wetting agent is 75% ethanol, and the weight ratio of extract powder to wetting agent is 8:1. The flavoring agent is sucralose, and the amount of the flavoring agent added is 0.3% of the extract powder. The conditions for mixing and granulating are: the stirring paddle speed is 250 rpm, the granulating knife speed is 1000 rpm, and the mixing is 2 minutes.

[0076] (4) Dry the wet granules at 50°C for 4 hours to prepare cold-dispelling, damp-removing and anti-toxic granules.

[0077] Example 7

[0078] A method for preparing cold-dispelling, dampness-removing and toxic-resistant granules comprises the following steps:

[0079] (1) The main component and auxiliary materials were weighed according to the proportions of Example 1, and the main component was decocted with water twice. The first time, 10.5 times the amount of water was added, and the decocted for 1.5 hours, and filtered through a 100-mesh filter cloth. The second time, 6 times the amount of water was added, and the decocted for 1 hour, and filtered through a 100-mesh filter cloth. The extracts were combined, filtered, and the filtrate was concentrated under reduced pressure to an extract with a relative density of 1.3 at 60°C. The conditions for reduced pressure concentration were: -0.1 MPa, 70°C, and 2.5 hours.

[0080] (2) The extract is vacuum dried at 60°C, -0.09 MPa, and 12 h to obtain an extract powder with a water content of <5%;

[0081] (3) Add excipients, wetting agents, and flavoring agents to the extract powder, mix and granulate, and then pass through an 80-mesh sieve to obtain wet granules. The wetting agent is 75% ethanol, and the weight ratio of extract powder to wetting agent is 8:1. The flavoring agent is sucralose, and the amount of the flavoring agent added is 0.3% of the extract powder. The conditions for mixing and granulating are: the stirring paddle speed is 300 rpm, the granulating knife speed is 1100 rpm, and the mixing is 2 min.

[0082] (4) Dry the wet granules at 60°C for 4 hours to prepare cold-dispelling, damp-removing and anti-toxic granules.

[0083] Example 8

[0084] A method for preparing cold-dispelling, dampness-removing and toxic-resistant granules comprises the following steps:

[0085] (1) The main component and auxiliary materials were weighed according to the proportions of Example 1, and the main component was decocted with water twice. The first time, 10 times the amount of water was added, and the decocted for 1.5 hours, and filtered through a 100-mesh filter cloth. The second time, 6.5 times the amount of water was added, and the decocted for 1 hour, and filtered through a 100-mesh filter cloth. The extracts were combined, filtered, and the filtrate was concentrated under reduced pressure to an extract with a relative density of 1.2 at 60°C. The conditions for reduced pressure concentration were: -0.09 MPa, 65°C, and 2.5 hours.

[0086] (2) The extract is vacuum dried at 60°C, -0.09 MPa, and 12 h to obtain an extract powder with a water content of <5%;

[0087] (3) Add excipients, wetting agents, and flavoring agents to the extract powder, mix and granulate, and then pass through an 80-mesh sieve to obtain wet granules. The wetting agent is 75% ethanol, and the weight ratio of extract powder to wetting agent is 8:1. The flavoring agent is sucralose, and the amount of the flavoring agent added is 0.3% of the extract powder. The conditions for mixing and granulating are: the stirring paddle speed is 350 rpm, the granulating knife speed is 1000 rpm, and the mixing is 2 minutes;

[0088] (4) Dry the wet granules at 70°C for 4 hours to prepare cold-dispelling, damp-removing and anti-toxic granules.

[0089] Example 9

[0090] Example 9 is basically the same as Example 6, except that 6.5 times the amount of water is added during the second decoction.

[0091] Example 10

[0092] Example 10 is basically the same as Example 6, except that the reduced pressure concentration conditions are: -0.08 MPa, 70° C., 2.5 h.

[0093] Example 11

[0094] Example 11 is basically the same as Example 6, except that the reduced pressure concentration conditions are: -0.1 MPa, 65°C, 2.5 h.

[0095] Example 12

[0096] Example 12 is basically the same as Example 6, except that the reduced pressure concentration conditions are: -0.1 MPa, 70°C, 2.5 h.

[0097] Example 13

[0098] Example 13 is basically the same as Example 6, except that the filtrate is concentrated under reduced pressure to an extract with a relative density of 1.3 at 60°C.

[0099] Example 14

[0100] Example 14 is basically the same as Example 6, except that the weight ratio of the main ingredient to the auxiliary material is:

[0101] Main ingredient: auxiliary material = 1.2:1.

[0102] Example 15

[0103] Example 15 is basically the same as Example 6, except that the weight ratio of each component in the auxiliary material is:

[0104] Dextrin: powdered sugar = 3.5:1.

[0105] Example 16

[0106] Example 16 is basically the same as Example 6, except that the conditions for mixing and granulating are: the stirring paddle speed is 250 rpm, the granulating knife speed is 1100 rpm, and the mixing time is 2 min.

[0107] Example 17

[0108] Example 17 is basically the same as Example 6, except that the conditions for mixing and granulating are: the stirring paddle speed is 350 rpm, the granulating knife speed is 1000 rpm, and the mixing time is 2 min.

[0109] Example 18

[0110] Example 18 is basically the same as Example 6, except that the conditions for mixing and granulating are: the stirring paddle speed is 350 rpm, the granulating knife speed is 1100 rpm, and the mixing time is 2 min.

[0111] Example 19

[0112] Example 19 is basically the same as Example 6, except that the drying conditions are: 60° C., 4 h.

[0113] Example 20

[0114] Example 20 is basically the same as Example 6, except that the drying conditions are: 70°C, 4h.

[0115] Comparative Example 1

[0116] Comparative Example 1 is substantially the same as Example 6, except that 9 times the amount of water is added for the first time.

[0117] Comparative Example 2

[0118] Comparative Example 2 is substantially the same as Example 6, except that 11 times the amount of water is added for the first time.

[0119] Comparative Example 3

[0120] Comparative Example 3 is substantially the same as Example 6, except that 5 times the amount of water is added the second time.

[0121] Comparative Example 4

[0122] Comparative Example 4 is substantially the same as Example 6, except that 7 times the amount of water is added the second time.

[0123] Comparative Example 5

[0124] Comparative Example 5 is basically the same as Example 6, except that the conditions for reduced pressure concentration are: -0.06 MPa, 65° C., and 2.5 h.

[0125] Comparative Example 6

[0126] Control Example 6 is basically the same as Example 6, except that the conditions for reduced pressure concentration are: -0.12 MPa, 65° C., and 2.5 h.

[0127] Comparative Example 7

[0128] Comparative Example 7 is basically the same as Example 6, except that the conditions for reduced pressure concentration are: -0.08 MPa, 60° C., and 2.5 h.

[0129] Comparative Example 8

[0130] Comparative Example 8 is basically the same as Example 6, except that the conditions for reduced pressure concentration are: -0.08 MPa, 75° C., and 2.5 h.

[0131] Comparative Example 9

[0132] Comparative Example 9 is basically the same as Example 6, except that the filtrate is concentrated under reduced pressure to an extract with a relative density of 1.1 at 60°C.

[0133] Comparative Example 10

[0134] Comparative Example 10 is substantially the same as Example 6, except that the filtrate is concentrated under reduced pressure to an extract having a relative density of 1.4 at 60°C.

[0135] Comparative Example 11

[0136] Control Example 11 is basically the same as Example 6, except that the weight ratio of the main component to the auxiliary material is: main component: auxiliary material = 0.8:1.

[0137] Comparative Example 12

[0138] Control Example 12 is basically the same as Example 6, except that the weight ratio of the main component to the auxiliary material is: main component: auxiliary material = 1.4:1.

[0139] Comparative Example 13

[0140] Comparative Example 13 is substantially the same as Example 6, except that the auxiliary material is sucrose powder.

[0141] Comparative Example 14

[0142] Control Example 14 is basically the same as Example 6, except that the auxiliary material is soluble starch.

[0143] Comparative Example 15

[0144] Control Example 15 is basically the same as Example 6, except that the auxiliary material is dextrin.

[0145] Comparative Example 16

[0146] Control Example 16 is basically the same as Example 6, except that the auxiliary material is sucralose.

[0147] Comparative Example 17

[0148] Comparative Example 17 is substantially the same as Example 6, except that the weight ratio of the components in the auxiliary materials is:

[0149] Dextrin: powdered sugar = 1:1.

[0150] Comparative Example 18

[0151] Comparative Example 18 is basically the same as Example 6, except that the weight ratio of the components in the auxiliary materials is:

[0152] Dextrin: powdered sugar = 4.5:1.

[0153] Comparative Example 19

[0154] Comparative Example 19 is substantially identical to Example 6, except that the wetting agent is 70% ethanol.

[0155] Comparative Example 20

[0156] Comparative Example 20 is substantially the same as Example 6, except that the wetting agent is 80% ethanol.

[0157] Comparative Example 21

[0158] Control Example 21 is basically the same as Example 6, except that the mixing and granulation conditions are: the stirring paddle speed is 200 rpm, the granulating knife speed is 1000 rpm, and the mixing is 2 minutes.

[0159] Comparative Example 22

[0160] The control example 22 is basically the same as the example 6, except that the mixing and granulation conditions are as follows: the stirring paddle speed is 400 rpm, the granulating knife speed is 1000 rpm, and the mixing is 2 min.

[0161] Comparative Example 23

[0162] Control Example 23 is basically the same as Example 6, except that the mixing and granulation conditions are: the stirring paddle speed is 250 rpm, the granulating knife speed is 900 rpm, and the mixing time is 2 min.

[0163] Comparative Example 24

[0164] Control Example 24 is basically the same as Example 6, except that the mixing and granulation conditions are: the stirring paddle speed is 250 rpm, the granulating knife speed is 1200 rpm, and the mixing time is 2 min.

[0165] Comparative Example 25

[0166] Comparative Example 25 is basically the same as Example 6, except that the drying conditions are: 45° C., 4 h.

[0167] Comparative Example 26

[0168] Comparative Example 26 is basically the same as Example 6, except that the drying conditions are: 75° C., 4 h.

[0169] The present invention is verified as follows

[0170] Experiment 1 Selection of criteria for evaluating granulation effect

[0171] According to the formula explanation, the main ingredients are patchouli and braised bitter almonds; the auxiliary ingredients are cinnamon twig, perilla leaf, and peucedanum; the adjuvant ingredients are stir-fried fructus aurantii, dried tangerine peel, platycodon grandiflorum, poria, pinellia ternata, stir-fried atractylodes macrocephala, ginger, jujube, and cicada slough; and the guiding ingredient is roasted licorice root. Braised bitter almonds, the main ingredient in this formula, contain amygdalin, one of the main active ingredients. Modern pharmacological research has shown that amygdalin has antitussive and antiasthmatic effects, as well as immune regulation, consistent with the treatment of this formula. Therefore, in this study, amygdalin was used as a criterion for evaluating granulation efficacy.

[0172] The feasibility of using amygdalin as an evaluation tool for granulation effect is verified below:

[0173] 1.1 Preparation of reference solution

[0174] Take an appropriate amount of amygdalin reference substance, accurately weigh it, and add methanol to make a solution containing 40 μg per 1 mL.

[0175] 1.2 Preparation of test solution

[0176] Take the Sanhan Chushi Kangdu Granules obtained in Example 6 to prepare the Sanhan Chushi Kangdu Granule Concentrate, shake well, accurately measure 3 mL of the filtrate, put it into a 10 mL volumetric flask, add 70% methanol to the scale, shake well, centrifuge, take the supernatant, filter, take the filtrate, and obtain the test solution.

[0177] 1.3 Preparation of negative sample solution

[0178] On the basis of Example 6, the boiled bitter almonds were removed to obtain granules, and the concentrate was prepared. The concentrate was shaken, 3 mL of the filtrate was accurately measured and placed in a 10 mL volumetric flask. 70% methanol was added to the volume, shaken, centrifuged, the supernatant was taken, filtered, and the filtrate was taken to obtain a negative sample solution.

[0179] 1.4 Chromatographic conditions

[0180] Use octadecylsilane bonded silica gel as the filler; use methanol as mobile phase A and water as mobile phase B, eluting according to the gradient shown in Table 1. Column temperature: 40°C; flow rate: 1.0 mL / min; detection wavelength: 207 nm. The theoretical plate number calculated based on the amygdalin peak should be no less than 1500.

[0181] Table 1

[0182]

[0183] 1.5 System suitability test

[0184] Take negative sample solution, test solution and reference solution respectively, and inject them into the sample under the chromatographic conditions of 1.4. The results are shown in Figure 1 .

[0185] from Figure 1 It can be seen that the test solution and the reference solution have corresponding peaks at the same retention time, and the peak time of amygdalin is about 16 minutes, while the negative sample solution has no interference at the corresponding position. The results preliminarily show that it is feasible to use amygdalin to evaluate the granulation effect.

[0186] 1.6 Linear relationship test

[0187] Preparation of amygdalin reference substance stock solution: Take an appropriate amount of amygdalin reference substance, accurately weigh it, and add methanol to make a solution containing 400 μg of amygdalin per 1 mL.

[0188] Preparation of amygdalin reference solutions of different concentrations: Accurately measure 0.05, 0.1, 0.5, 1, 2, and 5 mL of the stock solution, respectively, place them in a 10 mL volumetric flask, and dilute to the scale with methanol to obtain amygdalin reference solutions of different concentrations.

[0189] Accurately draw 10 μL of reference solution of different concentrations, inject into liquid chromatograph, measure, record peak area, and regress peak area (y) against amygdalin concentration (x) to obtain the standard curve: y=9519.6x-3580.6, R 2 =0.9999, indicating that amygdalin has a good linear relationship in the range of 1.99-198.77 μg / mL. The results are shown in Table 2, and the standard curve is shown in Figure 2 .

[0190] Table 2

[0191]

[0192] 1.7 Stability test

[0193] Prepare the test solution according to the method in 1.2. Inject samples at different time points (0, 2, 4, 6, 12, and 24 hours) to investigate the stability of the test solution over 24 hours. The results are shown in Table 3.

[0194] Table 3

[0195]

[0196] The results show that the peak area of ​​the test solution RSD It was 0.67%, indicating that the test solution was stable within 24 hours.

[0197] 1.8 Precision test

[0198] Accurately pipette 10 μL of the reference solution and repeat the injection six times according to the determined chromatographic conditions, recording the peak area values. The results are shown in Table 4.

[0199] Table 4

[0200]

[0201] From Table 4, we can see that the peak area of ​​amygdalin RSD The value was 0.61%, indicating good precision.

[0202] The above results ultimately indicate that it is feasible to use amygdalin as a test for evaluating granulation effects.

[0203] Experiment 2

[0204] In Example 6, Example 7, Comparative Example 1, and Comparative Example 2, after the first decoction, the filtrate was taken to determine the solid content of the extract and the content of amygdalin, and the transfer rate of amygdalin and the extract yield were calculated. The results are shown in Table 5.

[0205] Table 5

[0206]

[0207] As shown in Table 5, compared with the control examples 1 and 2, the amygdalin transfer rate and the extract yield of Examples 6 and 7 are increased. Considering the production cycle and energy saving, it is determined that 10 to 10.5 times the amount of water is added in the first decoction.

[0208] Experiment 3

[0209] In Example 6, Example 9, Comparative Example 3, and Comparative Example 4, after the second decoction, the filtrate was taken to determine the solid content of the extract and the content of amygdalin, and the transfer rate of amygdalin and the extract yield were calculated. The results are shown in Table 6.

[0210] Table 6

[0211]

[0212] As shown in Table 6, compared with the control examples 3 and 4, the amygdalin transfer rate and extract yield of Examples 6 and 9 were increased. Considering the production cycle and energy saving, 6 to 6.5 times the amount of water was added in the second decoction.

[0213] Experiment 4

[0214] For the extracts obtained in Example 6, Example 10, Example 11, Example 12, Control Example 5, Control Example 6, Control Example 7, and Control Example 8, the amygdalin content was measured and the amygdalin transfer rate was calculated. The results are shown in Table 7.

[0215] Table 7

[0216]

[0217] As shown in Table 7, the amygdalin transfer rates of Examples 6, 10, 11, and 12 are better than those of Control Examples 5, 6, 7, and 8. Therefore, the conditions for reduced pressure concentration are determined to be: -0.08 to -0.1 MPa, 65 to 70°C, and 2.5 h.

[0218] Experiment 5

[0219] The extract powders after vacuum drying in Example 6, Example 7, Example 8, Example 13, Comparative Example 9, and Comparative Example 10 were collected, and the extract powder yields were calculated. The results are shown in Table 8.

[0220] Table 8

[0221]

[0222] As shown in Table 8, the extract powder yields of Examples 6, 7, 8, and 13 are better than those of Control Examples 9 and 10. Therefore, the relative density of the medicinal solution is determined to be controlled at 1.2-1.3 (60°C).

[0223] Experiment 6

[0224] The particle yields of Example 6, Example 14, Comparative Example 11, and Comparative Example 12 were calculated, and the solubility of the particles was tested. The results are shown in Table 9.

[0225] Table 9

[0226]

[0227] As shown in Table 9, the granulation difficulty of Example 6 and Example 14 is easier and the granule yield is higher. Therefore, the weight ratio of the main component to the auxiliary material is determined to be: main component: auxiliary material = (1~1.2): 1.

[0228] Experiment 7

[0229] The particle yields of Example 6, Control Example 13, Control Example 14, Control Example 15, and Control Example 16 were calculated, and the solubility of the particles was tested. The results are shown in Table 10.

[0230] Table 10

[0231]

[0232] As can be seen from Table 10, Example 6 has the easiest granulation difficulty and the highest granule yield. Therefore, it is determined that the auxiliary materials are composed of dextrin and powdered sugar.

[0233] Experiment 8

[0234] The particle yields of Example 6, Example 15, Comparative Example 17, and Comparative Example 18 were calculated, and the solubility of the particles was tested. The results are shown in Table 11.

[0235] Table 11

[0236]

[0237] As shown in Table 11, the granulation difficulty and granule yield of Example 6 and Example 15 are better than those of Control Examples 17 and 18. Therefore, the weight ratio of each component in the auxiliary material is determined to be: dextrin: powdered sugar = (3.5-4): 1.

[0238] Experiment 9

[0239] The particle yields of Example 6, Control Example 19, and Control Example 20 were calculated, and the solubility of the particles was tested. The results are shown in Table 12.

[0240] Table 12

[0241]

[0242] As shown in Table 12, Example 6 has the easiest granulation difficulty and the highest granule yield. The change in wetting agent ethanol concentration has an impact on granule properties. When the ethanol concentration is 80%, the wetting agent is easy to disperse and the soft material is moderate. When the ethanol concentration is 70%, the wetting agent is not easy to disperse, the soft material is easy to agglomerate, and granulation is difficult. Therefore, it is determined that the wetting agent is 75% ethanol.

[0243] Experiment 10

[0244] The particle yields of Example 6, Example 16, Example 17, Example 18, Control Example 21, Control Example 22, Control Example 23, and Control Example 24 were calculated, and the solubility of the particles was tested. The results are shown in Table 13.

[0245] Table 13

[0246]

[0247] As shown in Table 13, Examples 6, 16, 17, and 18 are easy to granulate and have a high granule yield. Therefore, the conditions for mixed granulation are determined as follows: a stirring paddle speed of 250-350 rpm, a granulating knife speed of 1000-1100 rpm, and mixing for 2 min.

[0248] Experiment 11

[0249] The moisture content of the particles of Example 6, Example 19, Example 20, Comparative Example 25, and Comparative Example 26 was calculated. The results are shown in Table 14.

[0250] Table 14

[0251]

[0252] As shown in Table 14, the moisture content of the particles of Examples 6, 19, and 20 is lower than that of Control Example 25, and the difference with Control Example 26 is small. In order to save energy, the drying conditions are determined to be: 50-70°C, 4h.

[0253] Experiment 12

[0254] The effects of Sanhanchushikangdu granules on the lungs of mice with lipopolysaccharide (LPS)-induced lung injury were studied experimentally.

[0255] (1) Experimental animals

[0256] Sixty specific pathogen-free male C57BL / 6J mice, weighing 22 ± 2 g and aged 6–7 weeks, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. [License No. SCXK-Su)2023-0009]. Animal care conditions were in accordance with the National Animal Care and Use Committee regulations. They were maintained in a pathogen-free environment (22 ± 2°C, temperature 20–26°C, humidity 75 ± 5%, 12-h day / night cycle) with tap water and a standard diet. They were acclimated for 3 days in the SPF-rated animal housing at the Experimental Animal Science and Technology Center of Jiangxi University of Traditional Chinese Medicine. All animal experiments were strictly planned and executed in accordance with the guidelines of the Animal Research Committee of Jiangxi University of Traditional Chinese Medicine (Animal Use Permit No. SYXK(Gan)2017-0004).

[0257] (2) Animal grouping

[0258] Mice were randomly divided into 6 groups, with 10 experimental animals in each group, namely: control group (hereinafter referred to as Ctrl group), LPS group (lipopolysaccharide group), Sanhan Chushi Kangdu Granule 1 group (hereinafter referred to as SHCS1 group), Sanhan Chushi Kangdu Granule 2 group (hereinafter referred to as SHCS2 group), Sanhan Chushi Kangdu Granule 3 group (hereinafter referred to as SHCS3 group), and PAT group (prednisolone acetate group).

[0259] (3) Drug preparation

[0260] The granules used in group 3 of the Dispelling Cold, Dehumidifying and Anti-toxic Granules were prepared by the method of Example 6, the granules used in group 2 of the Dispelling Cold, Dehumidifying and Anti-toxic Granules were prepared by the method of Example 7, and the granules used in group 1 of the Dispelling Cold, Dehumidifying and Anti-toxic Granules were prepared by the method of Example 8.

[0261] (4) Modeling and drug administration

[0262] All mice were adaptively fed for 3 days. On day 4, mice in the LPS, SHCS1, SHCS2, SHCS3, and PAT groups, except the control group, received 30 μL of LPS (1 mg / mL) naturally inhaled into the lungs through the nasal passages. The control group received only 30 μL of normal saline. From days 5 to 8, mice in the SHCS3, SHCS2, and SHCS1 groups were gavaged with 50 g / kg / day of the Sanhan Chushi Kangdu Granules prepared in Examples 6, 7, and 8, respectively. Mice in the PAT group were gavaged with 10 mg / kg / day of PAT. Simultaneously, mice in the control and LPS groups received an equal volume of normal saline. On day 9, mice in each group were anesthetized by an intraperitoneal injection of 1.5% sodium pentobarbital (0.05 mL / 10 g) and then sacrificed.

[0263] (5) Test content

[0264] (5.1) General conditions of mice in each group

[0265] After anesthesia, mice in each group were weighed and peripheral blood samples were collected. Mice were sacrificed, their lungs and spleens were quickly isolated on ice, and feces were collected from the ileocecal region. Lungs and spleens were weighed in each group, and the lung weight index (ILW) and spleen weight index (ISW) were calculated using the following formulas: ILW (%) = lung weight / body weight × 100; ISW (%) = spleen weight / body weight × 100.

[0266] (5.2) Hematoxylin-eosin (HE) staining of mouse lungs

[0267] The staining steps are as follows:

[0268] 1. Tissue Embedding

[0269] 1) Mouse lung tissue was fixed in 4% paraformaldehyde for 48 h and then removed from a 4°C refrigerator for later use.

[0270] 2) Cut the lung tissue into small pieces so that they can be placed in the sample box;

[0271] 3) Dehydration and transparency: 75% ethanol (1 h) → 95% ethanol I (1 h) → 95% ethanol II (1 h) → 100% anhydrous ethanol I (30 min) → 100% anhydrous ethanol II (30 min) → xylene (20 min);

[0272] 4) Melt the paraffin block: Place the sample box into the paraffin block and incubate at 60°C for 60 minutes.

[0273] 5) Label the sample boxes in sequence, place the tissue blocks in the boxes for embedding, cool and fix them, and place them in the refrigerator;

[0274] 2. Paraffin Sections

[0275] Before paraffin sectioning, maintain the water temperature at 40-45°C and prepare a 30% ethanol solution. Once prepared, place the embedded tissue block on a microtome and slice to a thickness of 4 μm. Place the wax strip cut by the microtome in the ethanol solution to unfold the slice. Place the unfolded slice in warm water in a water bath to fully unfold it. Place the fully unfolded slice on a glass slide, mark the polished surface, and bake in an oven at 60°C overnight.

[0276] 3. HE staining method

[0277] 1) Dewaxing: Dewax the oven-dried sections in xylene twice (20 min each time) → 100% ethanol (5 min) → 95% ethanol (5 min) → 85% ethanol (5 min) → 75% ethanol (5 min) → rinse with tap water (3 min);

[0278] 2) Staining: Place the sections in hematoxylin for staining (30 minutes), then rinse with running water (15 minutes). Do not use too much running water to prevent the sections from falling off. Stop rinsing when the sections turn blue.

[0279] 3) Differentiation: Place the slices in 1% hydrochloric acid ethanol solution to fade. Wait for the tissue slices to turn red and then lighten in color for about ten seconds. After fading, rinse again in tap water until the slices return to blue.

[0280] 4) Dehydration: Remove the sections and place them in 50% ethanol (5 min) → 70% ethanol (5 min) → 80% ethanol (5 min) to perform gradient dehydration in different ethanol concentrations.

[0281] 5) Counterstaining: Remove the sections and stain in 0.5% eosin-ethanol solution for 3 minutes.

[0282] 6) Dehydration: Prepare 95% ethanol to absorb excess red from the sections, then place in 100% ethanol for 3 minutes. Remove the sections and wipe off any excess ethanol.

[0283] 7) Transparency: Place the sections in xylene for 5 minutes;

[0284] 8) Sealing: Seal the slide with neutral resin and observe under a microscope.

[0285] (5.3) Mouse ELISA test

[0286] ELISA was used to detect the expression of IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IL-23, IL-1β, TGF-β, TNF-α, IFN-γ and immunoglobulins IgA, IgG and IgM in lung tissue among the different groups.

[0287] The detection steps are: 1) Prepare multiple wells for blank, standard, and test samples. Add 0.1ml of sample diluent to the blank well, and add 0.1ml of standard or test sample to the remaining wells. Cover the ELISA plate with a film and incubate at 37°C for 90 minutes. 2) Discard the liquid in the well, shake dry, do not wash the plate, add 0.1ml of biotinylated antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 60 minutes. 3) Pour out the liquid in the well, wash the plate 3 times, each soaking time is 30 seconds, about 0.35ml per well, gently shake dry the liquid in the well, and the remaining liquid in the well can be absorbed with absorbent paper. 4) Add 0.1ml of enzyme conjugate working solution to each well. The working solution should be prepared within 20 minutes before use and stored in the dark. Then cover with a film and incubate at 37°C for 30 minutes. 5) Pour out the liquid in the well, then shake dry, and repeat washing the plate 5 times. 6) Add 90 μl of chromogen (TMB) to each well, cover the plate with film, and incubate at 37°C in the dark for 15 minutes. 7) Add 50 μl of stop solution to each well to terminate the reaction. The blue color in the well will immediately turn yellow. 8) Measure the optical density (OD) of each well at a wavelength of 450 nm using a microplate reader. 9) Use software to plot a standard curve and formula, with the standard concentration on the horizontal axis and the OD value on the vertical axis. Substitute the measured sample OD value into the standard curve formula. Then, divide the calculated value by the corresponding protein concentration of the sample to obtain the actual concentration of the target protein.

[0288] (5.4) Mouse flow cytometry experiments

[0289] The obtained lymphocytes were incubated with fluorescently labeled monoclonal antibodies in staining buffer. Eight-color flow cytometry analysis (n=10) was performed on a FACS Calibur instrument. Treg cells were identified using CD4 + CD25 + Marked, CD4 + CD25 + Foxp3 + It is considered to be a more rigorous and accurate Treg cell labeling method. + CD25 + In this group, the IL-10 cell subset was also evaluated. In these cell suspensions, the following steps were performed:

[0290] 1) Take 80 μL of anticoagulant, add 100 μL of 1640 medium, add 1 mL of 1X hemolysin, incubate for 15 minutes, centrifuge at 500g for 5 minutes, and remove the supernatant;

[0291] 2) Rinse the cells twice with 1 mL of staining buffer (554656), centrifuge at 500 g for 5 min, and remove the supernatant.

[0292] 3) Resuspend the cells in 100 μL of staining buffer. Add 1 μg of Fc blocking agent (553141) and incubate at 4°C for 8 minutes. Add 2.5 μL of each of CD4-BV510, CD25-Percp-CY5.5, Foxp3-EF450, and IL-10-FITC antibodies for staining and incubate at room temperature for 15 minutes.

[0293] 4) Wash twice with 1 mL of staining buffer, remove the supernatant, and add 500 μL of staining buffer to resuspend;

[0294] 5) For flow cytometry, quadrant labeling limits are always based on negative populations and isotype controls;

[0295] 6) Data were analyzed using FlowJo software 10, and inactive cells were excluded by gating.

[0296] (5.5) Statistical methods

[0297] Statistical analysis was performed using GraphPad Prism 9.0 software. Data were expressed as mean ± standard deviation (SD). Statistical tests among multiple groups of data were performed using one-way analysis of variance, with the use of “ "," "and" "" indicates P < 0.05, P < 0.01, and P < 0.001. P < 0.05 was considered statistically significant.

[0298] (6) Experimental results

[0299] (6.1) Sanhanchushikangdu Granules Improve Lung Injury in Mice

[0300] Before modeling: During the 3-day adaptive feeding period, the rats in each group were in good general condition, and they could eat and drink water freely. Their fur was shiny, they were active, and they had a gentle personality. There were no significant differences in the spirit and body weight of the rats in each group.

[0301] After modeling, the rats in the control group were generally in good condition, with no significant changes in food and water intake. Their fur was smooth and clean, their reactions were agile, and their limb strength was normal. Daily observations revealed a good mental state, with no significant changes in defecation. Significant changes were observed in the remaining groups, including increased food intake, coarse fur, increased daily activity, increased mental agitation, increased defecation, intense resistance to oral gavage, and a significant decrease in body weight compared to the control group.

[0302] After administration: the above conditions in the SHCS1, SHCS2 and SHCS3 groups improved. The rats' food intake gradually recovered, their hair gradually became shiny, their activity level decreased, their mental state improved, their resistance to oral administration weakened, and their body weight slowly recovered. However, there was no significant change in the control group. The body weight, body weight change rate and body weight growth rate of each group are shown in Table 1. Figure 3 .

[0303] like Figure 4 As shown, lung weight, lung weight index, spleen weight, and spleen weight index were measured, and the LPS group showed significantly higher lung weight than the other groups, with significant differences (p < 0.05). This indicates that lung tissue was significantly affected in the LPS group, resulting in increased lung weight. In contrast, lung weight was significantly reduced in the SHCS1, SHCS2, and SHCS3 groups compared to the LPS group, demonstrating an inhibitory effect on lung injury. Overall, lung weight was significantly reduced in the SHCS1, SHCS2, and SHCS3 groups compared to the PAT group.

[0304] like Figures 5 to 10 As shown in the figure, after HE staining, the lung tissue structure of the mice in the control group was clear, the alveolar septa were normal, and there was no obvious inflammatory cell infiltration. However, after lipopolysaccharide induction, the lung tissue structure of the mice in the LPS group was obviously disordered, the lung septa were abnormal, and a large number of inflammatory cells were visible to the naked eye. For the SHCS1 group, SHCS2 group, and SHCS3 group, after the intervention of Sanhan Chushi Kangdu Granules, the inflammation of the mouse lung tissue was alleviated, the number of inflammatory cells was reduced compared with the LPS group, and the lung structure was also restored. In addition, the HE staining effect of the SHCS3 group was closer to the control group than that of the SHCS1 group and SHCS2 group. The alveolar structure of the medium-dose group of Sanhan Chushi Kangdu Granules was clearly visible, and the inflammatory cell infiltration almost disappeared.

[0305] In summary, these results indicate that the Sanhan Chushi Kangdu Granules significantly alleviated lung injury in mice, and the Sanhan Chushi Kangdu Granules prepared in Example 6 corresponding to the SHCS3 group exhibited a more significant therapeutic effect.

[0306] (6.2) Effect of Sanhan Chushi Kangdu Granules on Inhibiting Lung Tissue Inflammation

[0307] In the mouse ELISA test, relevant inflammatory factors and immunoglobulins were detected in lung tissue specimens from different treatment groups to evaluate the effects of different treatments on lung injury-related indicators.

[0308] See also Figure 11The results showed that the levels of pro-inflammatory cytokines (IL-2, IL-4, IL-6, IL-12, IL-17, IL-23, IL-1β, TGF-β, TNF-α, IFN-γ) in the lung tissue of the LPS group were significantly higher than those in the other treatment groups, indicating a significant inflammatory response. In the SHCS1, SHCS2, and SHCS3 groups, the levels of related inflammatory factors were relatively low, indicating an effect of inhibiting inflammation, and there were significant differences compared with the LPS group (p<0.05); the level of anti-inflammatory cytokine (IL-10) in the lung tissue of the LPS group was significantly lower than that in the other treatment groups, and the anti-inflammatory effect was significantly inhibited. In the SHCS1, SHCS2, and SHCS3 groups, the levels of anti-inflammatory factors were relatively high, indicating an effect of inhibiting inflammation, and there were significant differences compared with the LPS group (p<0.05); in terms of immunoglobulins, please refer to Figure 12 The LPS group had lower IgA and IgG levels, while the Sanhan Chushi Kangdu Granules had a significant effect on IgA and IgG. The LPS group had higher IgM levels than the other treatment groups, indicating a more active immune response. After treatment with SHCS1, SHCS2, and SHCS3, IgM levels significantly decreased, indicating a less active immune response compared to the LPS group.

[0309] Based on the ELISA results, it can be concluded that the expression levels of relevant proinflammatory cytokines were significantly higher in the LPS group than in the other groups, while the expression levels in the SHCS1, SHCS2, and SHCS3 groups were significantly lower, showing significant differences from the LPS group. The expression levels of anti-inflammatory cytokines were significantly lower in the LPS group than in the other groups, while the SHCS1, SHCS2, and SHCS3 groups showed relatively good anti-inflammatory effects. Furthermore, in addition to its significant regulatory effects on relevant inflammatory factors, Sanhan Chushi Kangdu Granules also had a certain effect on immunoglobulins. Therefore, Sanhan Chushi Kangdu Granules alleviated LPS-induced lung inflammation in mice by inhibiting the production of proinflammatory cytokines and promoting the expression of anti-inflammatory cytokines.

[0310] (6.3) Flow cytometry results

[0311] See also Figure 13 Compared with the control group, the CD4 + CD25 + cells, CD4 + CD25 + Foxp3 + cells, and CD4 + CD25 + IL-10 + Compared with the LPS group, the CD4+ CD25 + cells, CD4 + CD25 + Foxp3 + cells, and CD4 + CD25 + IL-10 + The expression levels of cells were significantly increased (P<0.05), indicating that Han Chushi Kangdu Granules can effectively promote the increase of Treg cell levels and the expression level of the anti-inflammatory factor IL-10.

[0312] Experiment 13

[0313] The inhibitory effect of the cold-dispelling, dampness-removing and toxic-resistant granules on coronavirus was studied experimentally.

[0314] (1) Experimental materials

[0315] (1.1) Cells

[0316] MRC-5 was purchased from China Center for Type Culture Collection and cultured, passaged, and cryopreserved in vitro according to the instructions.

[0317] (1.2) Coronavirus

[0318] The HCoV-229E virus was provided by the Institute of Virology, Chinese Center for Disease Control and Prevention, a collaborative partner. Viral amplification and culture were performed according to the manufacturer's instructions. The virus has been effectively amplified and is currently stored in the P2 laboratory of the Cancer Research Center at Jiangxi University of Traditional Chinese Medicine.

[0319] (2) Experimental methods

[0320] (2.1) In vitro efficacy evaluation method

[0321] (2.1.1) Preparation of cell monolayer:

[0322] MRC-5 cells in the logarithmic growth phase were cultured at a rate of 1×10 4 Each well was inoculated into a 96-well plate and cultured overnight. The cell abundance reached about 80% the next day.

[0323] (2.1.2) Virus-infected cells:

[0324] ①Discard the monolayer cell culture supernatant, rinse once with 1×PBS, and rinse once with DMEM medium.

[0325] ② Add virus, where TCID50 of HCoV-229E virus is 10 -5.35 / 0.1mL, MOI=0.1;

[0326] ③ Place the virus-infected cells back into the cell culture incubator and allow the virus to infect for 2 hours. Gently mix the virus dilution solution every 30 minutes to allow the virus to fully contact and infect the cells.

[0327] (2.1.3) Drug intervention

[0328] ① Remove the viral liquid and rinse the cells twice with serum-free DMEM.

[0329] ② Experimental Grouping and Dosing: A control group containing maintenance medium without drug was set up. The granules of Example 6 were dissolved and diluted with DMEM complete medium containing only 2% FBS to obtain different concentrations of drug.

[0330] (2.1.4) The Reed-Muench method and CCK8 method were used to calculate the half-maximal inhibitory concentration (IC50) and therapeutic index (SI) of the drug for viral inhibition (SI = TC50 / IC50), and the viral inhibition rate was calculated.

[0331] (2.2) Fluorescence quantitative PCR detection of coronavirus M protein expression in each group of cells

[0332] (2.2.1) Preparation of monolayer cells: MRC-5 cells in the logarithmic growth phase were plated at 1.2×10 5 Cells / well were seeded into 12-well culture plates;

[0333] (2.2.2) Drug Preparation: The granules of Example 6 were dissolved and diluted with DMEM complete medium containing only 2% FBS to obtain drug concentrations of 16 mg / mL, 4 mg / mL, and 1 mg / mL, respectively.

[0334] (2.2.3) Experimental grouping and drug administration: normal group (not infected with virus, cultured in DMEM + 2% FBS maintenance medium), negative control group (referred to as Con group, 2 hours after virus infection, cultured in DMEM + 2% FBS maintenance medium), positive control group (referred to as RBV group, 2 hours after virus infection, cultured in DMEM + 2% FBS maintenance medium containing 50 μg / ml ribavirin), preventive drug administration group (referred to as Pre group, 24 hours before infection, cells were treated with 4 mg / mL drug in DMEM + 10% FBS growth medium, and 2 hours after normal virus infection, cultured in DMEM + 2% FBS maintenance medium), drug administration group 1 (2 hours after virus infection, cells were treated with 16 mg / mL drug), drug administration group 2 (2 hours after virus infection, cells were treated with 4 mg / mL drug), and drug administration group 3 (2 hours after virus infection, cells were treated with 1 mg / mL drug).

[0335] (2.2.4) Virus-infected cells: The infection process is the same as (2.1.2).

[0336] (2.2.5) 48 hours after infection, extract RNA from cell supernatants. Perform quantitative PCR according to the manufacturer's instructions and calculate the ratio of HCoV-229E viral M protein mRNA compared to the negative control group: the nucleic acid copy number of the RBV group / the nucleic acid copy number of the Con group, the nucleic acid copy number of the Pre group / the nucleic acid copy number of the Con group, the nucleic acid copy number of the Dosage 1 group / the nucleic acid copy number of the Con group, the nucleic acid copy number of the Dosage 2 group / the nucleic acid copy number of the Con group, and the nucleic acid copy number of the Dosage 3 group / the nucleic acid copy number of the Con group.

[0337] (3) Statistical analysis

[0338] GraphPad Prism 9.0 software was used to analyze the experimental data, and the statistical tests among multiple groups of data were performed by one-way analysis of variance. "," "and" ” indicates P < 0.05, P < 0.01, and P < 0.001.

[0339] (4) Experimental results

[0340] (4.1) In vitro efficacy evaluation results

[0341] See also Figure 14 The IC50 value for Sanhan Chushi Kangdu Granules in this experiment was calculated using GraphPad Prism 9.0. The IC50 was 2.402 mg / mL, and the therapeutic index was 12.46. This indicates that Sanhan Chushi Kangdu Granules has good in vitro anti-coronavirus activity. An SI index greater than 2 indicates that Sanhan Chushi Kangdu Granules has good therapeutic safety.

[0342] (4.2) Fluorescence quantitative PCR detection of coronavirus M protein expression in each group of cells

[0343] See also Figure 15 The results showed that compared with the Con group, the 1st, 2nd, 3rd, and Pre groups all significantly reduced HCoV-229E viral M protein mRNA expression levels in the cell supernatant. Furthermore, the efficacy of the 1st, 2nd, and 3rd groups was positively correlated with the dose. Furthermore, the efficacy of the preventive treatment group was particularly significant, comparable to that of ribavirin 50 μg / mL.

[0344] In summary, the present invention experimentally studies the effect of Sanhan Chushi Kangdu Granules on the lungs of mice with lipopolysaccharide (LPS)-induced lung injury. The experiment illustrates the protective effect of Sanhan Chushi Kangdu Granules on LPS-induced lung injury in mice. Sanhan Chushi Kangdu Granules can reduce lipopolysaccharide-induced lung inflammation in mice by inhibiting the production of proinflammatory cytokines and promoting the expression of anti-inflammatory cytokines. Sanhan Chushi Kangdu Granules can promote the increase in Treg cell levels and the expression level of the anti-inflammatory factor IL-10, effectively inhibiting the progression of inflammation in mice with lipopolysaccharide-induced lung injury. In addition, through anti-coronavirus experiments, it was shown that Sanhan Chushi Kangdu Granules has a good ability to inhibit the activity of coronavirus in vitro, and can significantly reduce the expression level of HCoV-229E virus M protein mRNA in cell supernatant.

[0345] The preparation method of the cold-dispelling, dampness-removing and toxic-resistant granules of the present invention adopts dextrin and powdered sugar as auxiliary materials, and specifically defines the weight ratio of each component in the auxiliary materials as: dextrin: powdered sugar = (3.5-4):1; simultaneously, the weight ratio of the main component to the auxiliary material is defined as: main component: auxiliary material = (1-1.2):1. In conjunction with the optimized preparation process of the present invention, the main component is first decocted with water twice, the amount of water added and the decocting time are reasonably set, and the filtrate is decompressed and concentrated to an extract with a relative density of 1.2-1.3 at 60°C, and then vacuum dried under defined conditions to obtain an extract powder with a water content of less than 5%. Then, the auxiliary materials, a wetting agent, and a flavoring agent are added, mixed and granulated, and then passed through an 80-mesh sieve to obtain wet granules. Finally, the wet granules are dried to prepare the cold-dispelling, dampness-removing and toxic-resistant granules. Experimental results show that the granules obtained by the method have good medicinal efficacy.

[0346] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to the embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the claims and their equivalents.

Claims

1. A method for preparing a cold-dispelling, damp-removing and anti-toxic granule, characterized in that: The cold-dispelling, dampness-removing and toxic-removing granules are prepared from main ingredients and auxiliary materials, and the main ingredients are, by weight, 150-170 parts of patchouli, 150-170 parts of braised bitter almonds, 150-170 parts of cassia twigs, 100-120 parts of perilla leaves, 150-170 parts of peucedanum, 150-170 parts of stir-fried fructus aurantii with bran, 150-170 parts of dried tangerine peel, 150-170 parts of platycodon grandiflorum, 150-170 parts of poria, 150-170 parts of pinellia ternata, 150-170 parts of stir-fried atractylodes macrocephala with bran, 150-170 parts of ginger, 150-170 parts of jujubes, 100-120 parts of roasted liquorice, and 50-65 parts of cicada sloughs; The auxiliary material consists of dextrin and powdered sugar, and the weight ratio of each component in the auxiliary material is: Dextrin: powdered sugar = (3.5-4): 1; The weight ratio of the main component and the auxiliary materials is: Main ingredient: auxiliary material = (1-1.2): 1; The preparation method comprises the following steps: (1) Weigh the main ingredient and auxiliary materials in proportion, add water to the main ingredient and boil it twice. The first time, add 10-10.5 times the amount of water and boil it for 1.5 hours. The second time, add 6-6.5 times the amount of water and boil it for 1 hour. Combine the extracts, filter, and concentrate the filtrate under reduced pressure to an extract with a relative density of 1.2-1.3 at 60°C. (2) The extract is vacuum dried at 60°C, -0.09 MPa, and 12 h to obtain an extract powder with a water content of <5%; (3) Add excipients, wetting agents, and flavoring agents to the extract powder, mix and granulate, and then pass through an 80-mesh sieve to obtain wet granules; (4) Dry the wet granules to make granules for dispelling cold, removing dampness and resisting poison.

2. The method for preparing the cold-dispelling, dampness-removing and poison-removing granules according to claim 1, wherein: In step (1), the conditions for reduced pressure concentration are: -0.08~-0.1Mpa, 65~70℃, and 2.5h.

3. The method for preparing the cold-dispelling, dampness-removing and poison-removing granules according to claim 1, wherein: Step (1) specifically includes: Weigh the main ingredient and auxiliary materials in proportion, add water to the main ingredient and boil it twice, add 10-10.5 times the amount of water for the first time, boil it for 1.5 hours, and filter it with a 100-mesh filter cloth, add 6-6.5 times the amount of water for the second time, boil it for 1 hour, and filter it with a 100-mesh filter cloth, combine the extracts, filter, and concentrate the filtrate under reduced pressure to an extract with a relative density of 1.2-1.3 at 60°C.

4. The method for preparing the cold-dispelling, dampness-removing and poison-removing granules according to claim 1, wherein: In step (3), the wetting agent is 75% ethanol, and the weight ratio of the extract powder to the wetting agent is: Extract powder: wetting agent = 8:

1.

5. The method for preparing the cold-dispelling, dampness-removing and poison-removing granules according to claim 1, characterized in that: In step (3), the flavoring agent is sucralose, and the amount of the flavoring agent added is 0.3% of the extract powder.

6. The method for preparing the cold-dispelling, dampness-removing and poison-removing granules according to claim 1, characterized in that: In step (3), the conditions for mixing and granulating are: stirring paddle speed of 250-350 rpm, granulating knife speed of 1000-1100 rpm, and mixing for 2 minutes.

7. The method for preparing the cold-dispelling, dampness-removing and poison-removing granules according to claim 1, characterized in that: The drying conditions in step (4) are: 50-70°C, 4h.

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