Heat-clearing, dampness-removing and anti-toxic granules and preparation method thereof

Abstract

The present invention provides a heat-clearing and damp-removing anti-toxic granule and a preparation method thereof, wherein the heat-clearing and damp-removing anti-toxic granule comprises a main component, which comprises, by weight, 280-320 parts of patchouli, 280-320 parts of boiled bitter almonds, 280-320 parts of mulberry leaves, 190-210 parts of cardamom, 280-320 parts of wine scutellaria, 280-320 parts of forsythia, 280-320 parts of tuckahoe, 280-320 parts of talc, 490-510 parts of coix seeds, 190-210 parts of liquorice tablets, and 90-110 parts of cicada shells. The heat-clearing and damp-removing anti-toxic granule provided by the present invention realizes heat-clearing and damp-removing anti-toxic, can inhibit lung tissue inflammation, and alleviate lung damage.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine, and in particular to a heat-clearing, dampness-removing and toxic-resistant granule and a preparation method thereof. Background Art

[0002] For patients with pneumonia, their inflammatory factors are high, which can easily lead to lung damage, and some patients have the following symptoms: dry cough with little sputum, frequent cough, dry lips, thirst and love of drinking, fever but no obvious aversion to cold, or heaviness and pain, alternating cold and heat. White and greasy tongue coating, floating or rapid pulse. In addition, the above symptoms are also common in patients with some viral respiratory infections.

[0003] Traditional Chinese medicine has unique advantages in the face of pneumonia or viral respiratory infections. Therefore, developing a Chinese medicine composition for treating pneumonia or viral respiratory infections, inhibiting lung tissue inflammation, clearing away heat and dampness, and alleviating lung damage is a technical problem that needs to be solved by technical personnel in this field. Summary of the invention

[0004] To this end, the present invention proposes a heat-clearing, dampness-removing and toxin-resistant granule and a preparation method thereof, so as to develop a 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 heat-clearing, dampness-removing and anti-toxic granule, comprising main components, which, by weight, include: 280-320 parts of patchouli, 280-320 parts of stewed bitter almonds, 280-320 parts of mulberry leaves, 190-210 parts of cardamom, 280-320 parts of wine scutellaria, 280-320 parts of forsythia, 280-320 parts of poria, 280-320 parts of talc, 490-510 parts of coix seeds, 190-210 parts of licorice tablets, and 90-110 parts of cicada shells.

[0006] Another aspect of the present invention provides a method for preparing the above-mentioned heat-clearing, dampness-removing and anti-toxic granules, the preparation method comprising the following steps:

[0007] (1) 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 and boil it for 1.5 hours, and add 6-6.5 times the amount of water for the second time 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 1.1-1.2 at 55°C;

[0008] (2) vacuum drying the extract at 60°C, -0.09 MPa, and 10 h to obtain an extract powder with a water content of <5%;

[0009] (3) Add auxiliary materials, wetting agents and flavoring agents to the extract powder, mix and granulate, and then pass through a 14-mesh sieve to obtain wet granules;

[0010] (4) Drying the wet granules to prepare heat-clearing, dampness-removing and anti-toxic granules.

[0011] The heat-clearing, dampness-removing and anti-toxic granules provided by the present invention are explained as follows:

[0012] The heat-clearing, damp-removing and anti-toxic granules are mainly used to treat damp-heat in the three jiaos, with the upper jiao as the main treatment. The "bitter, pungent and cold method" is used to treat it. Patchouli in the prescription enters the lung, spleen and stomach meridians. It is aromatic and pungent but not harsh, slightly warm and damp-removing but not hot. It is good at dispelling cold, regulating qi and removing dampness, stopping vomiting and harmonizing the stomach, and awakening the spleen and appetizing. Boiled bitter almonds enter the lung and large intestine meridians and are good at relieving cough and relieving asthma. Patchouli and boiled bitter almonds are combined into the Taiyin lung to relieve the exterior, dispel the lung and remove dampness. They can also enter the spleen, stomach and large intestine. If there is evil, they will eliminate it. If there is no evil, they will first calm the place that has not been affected by the evil. They are the main medicine. Mulberry leaves and cardamoms descend the Taiyin and penetrate and resolve dampness and heat as the minister medicine. Scutellaria baicalensis is bitter and descends and clears heat, Forsythia suspensa is bitter and cool, dispersing and penetrating heat, Talcum, Poria cocos, and Coix seeds are sweet, cool, mild, penetrating, clearing dampness and heat, and Cicadae exuviae dissipates wind. They are all adjuvant medicines. Licorice tablets clear heat and detoxify, and harmonize all medicines. They are the guiding medicine. The whole formula can be used together to eliminate toxins, relieve heat, remove dampness and stop cough.

[0013] Main medicine: Patchouli, burnt bitter almonds;

[0014] Minister medicine: mulberry leaves, cardamom;

[0015] Auxiliary drugs: wine-soaked scutellaria, forsythia, tuckahoe, talc, coix seed, cicada shell;

[0016] Assistant drug: Licorice tablets.

[0017] The present invention studies the effect of Qingrehuashikangdu granules on the lungs of mice with lung injury induced by lipopolysaccharide (LPS) through experiments. The experiments illustrate the protective effect of Qingrehuashikangdu granules on LPS-induced lung injury in mice. Qingrehuashikangdu granules can reduce lipopolysaccharide-induced lung inflammation in mice by inhibiting the production of proinflammatory cytokines and promoting the expression of anti-inflammatory cytokines, as well as reducing the expression of immunoglobulins in the body. Qingrehuashikangdu granules can regulate Treg and TH17A cell differentiation and maintain CD4 / CD8 balance, thereby inhibiting the progression of inflammation in mice with lipopolysaccharide-induced lung injury. In addition, through anti-coronavirus experiments, it is shown that Qingrehuashikangdu granules have 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.

[0018] The preparation method of the heat-clearing, damp-removing and anti-toxic 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 = (4-4.5): 1; at the same time, the weight ratio of the main component to the auxiliary material is defined as: main component: auxiliary material = (1.1-1.3): 1. In conjunction with the optimized preparation process of the present invention, firstly, the main component is 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 1.1-1.2 at 55°C, and then vacuum dried under limited conditions to obtain an extract powder with a water content of less than 5%, and then auxiliary materials, a wetting agent, and a flavoring agent are added, mixed and granulated, and then passed through a 14-mesh sieve to obtain wet granules, and finally the wet granules are dried to prepare the heat-clearing, damp-removing and anti-toxic granules. Experimental results show that the granules obtained by the method have good efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the HPLC chart of amygdalin;

[0020] Figure 2 It is the linear regression equation diagram for the determination of amygdalin content;

[0021] Figure 3 It is the graph of the weight, weight change rate and weight growth rate of mice;

[0022] Figure 4 It is the change graph of lung weight, lung weight index, spleen weight, and spleen weight index of mice;

[0023] Figure 5 This is the HE staining result of the mouse lung tissue in the control group;

[0024] Figure 6 This is the HE staining result of lung tissue of mice in the LPS group;

[0025] Figure 7 This is the HE staining result of the lung tissue of mice in the QRHS1 group;

[0026] Figure 8 This is the HE staining result of the lung tissue of mice in the QRHS2 group;

[0027] Fig. 9 This is the HE staining result of the lung tissue of mice in the QRHS3 group;

[0028] Fig.10 This is the HE staining result of lung tissue of mice in the PAT group;

[0029] Fig.11 This is a graph showing changes in cytokine levels in mouse lung tissues detected by ELISA;

[0030] Fig.12This is a graph showing changes in immunoglobulin levels in mouse lung tissues detected by ELISA;

[0031] Fig.13 Mouse lung tissue CD4 + T cell level influences the result diagram;

[0032] Fig.14 Mouse lung tissue CD4 + CD25 + T cell level influences the result diagram;

[0033] Fig.15 This is the result of the in vitro efficacy evaluation of the Qingrehuashikangdu Granules;

[0034] Fig.16 This is a graph showing the results of the Qingrehuashikangdu granules inhibiting the expression level of HCOV-229E virus M protein mRNA. DETAILED DESCRIPTION

[0035] In order to facilitate understanding of the present invention, the present invention will be described more fully below with reference to various embodiments, but 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.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0037] The following is a further description of 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.

[0038] Embodiment 1:

[0039] A heat-clearing, dampness-removing and anti-toxic granule comprises a main component and auxiliary materials. The main component comprises, by weight, 300 parts of patchouli, 300 parts of stewed bitter almonds, 300 parts of mulberry leaves, 200 parts of cardamoms, 300 parts of wine-cooked scutellaria, 300 parts of forsythia, 300 parts of poria, 300 parts of talc, 500 parts of coix seeds, 200 parts of liquorice tablets and 100 parts of cicada shells.

[0040] The auxiliary material is composed of dextrin and powdered sugar, and the weight ratio of each component in the auxiliary material is:

[0041] Dextrin: powdered sugar = 4.5:1;

[0042] The weight ratio of the main ingredient to the auxiliary material is:

[0043] Main ingredient: auxiliary material = 1.1:1.

[0044] Embodiment 2:

[0045] A heat-clearing, dampness-removing and anti-toxic granule comprises a main component and auxiliary materials. The main component comprises, by weight, 310 parts of patchouli, 320 parts of stewed bitter almonds, 280 parts of mulberry leaves, 210 parts of cardamom, 320 parts of wine scutellaria, 290 parts of forsythia, 310 parts of poria, 280 parts of talc, 490 parts of coix seeds, 210 parts of liquorice tablets and 100 parts of cicada shells.

[0046] The auxiliary material is composed of dextrin and powdered sugar, and the weight ratio of each component in the auxiliary material is:

[0047] Dextrin: powdered sugar = 4:1;

[0048] The weight ratio of the main ingredient to the auxiliary material is:

[0049] Main ingredient: auxiliary material = 1.2:1.

[0050] Embodiment 3:

[0051] A heat-clearing, dampness-removing and anti-toxic granule comprises a main component and auxiliary materials. The main component comprises, by weight, 280 parts of patchouli, 280 parts of stewed bitter almonds, 320 parts of mulberry leaves, 200 parts of cardamoms, 300 parts of wine-soaked scutellaria, 280 parts of forsythia, 280 parts of poria, 320 parts of talc, 510 parts of coix seeds, 200 parts of liquorice tablets and 110 parts of cicada shells.

[0052] The auxiliary material is composed of dextrin and powdered sugar, and the weight ratio of each component in the auxiliary material is:

[0053] Dextrin: powdered sugar = 4.5:1;

[0054] The weight ratio of the main ingredient to the auxiliary material is:

[0055] Main ingredient: auxiliary material = 1.3:1.

[0056] Embodiment 4:

[0057] A heat-clearing, dampness-removing and anti-toxic granule comprises a main component and auxiliary materials. The main component comprises, by weight, 320 parts of patchouli, 310 parts of stewed bitter almonds, 310 parts of mulberry leaves, 190 parts of cardamoms, 310 parts of wine scutellaria, 300 parts of forsythia, 300 parts of poria, 310 parts of talc, 500 parts of coix seeds, 190 parts of liquorice tablets and 90 parts of cicada shells.

[0058] The auxiliary material is composed of dextrin and powdered sugar, and the weight ratio of each component in the auxiliary material is:

[0059] Dextrin: powdered sugar = 4:1;

[0060] The weight ratio of the main ingredient to the auxiliary material is:

[0061] Main ingredient: auxiliary material = 1.1:1.

[0062] Embodiment 5:

[0063] A heat-clearing, dampness-removing and anti-toxic granule comprises a main component and auxiliary materials. The main component comprises, by weight, 290 parts of patchouli, 310 parts of stewed bitter almonds, 290 parts of mulberry leaves, 200 parts of cardamoms, 280 parts of wine scutellaria, 320 parts of forsythia, 320 parts of poria, 300 parts of talc, 510 parts of coix seeds, 200 parts of liquorice tablets and 100 parts of cicada shells.

[0064] The auxiliary material is composed of dextrin and powdered sugar, and the weight ratio of each component in the auxiliary material is:

[0065] Dextrin: powdered sugar = 4.5:1;

[0066] The weight ratio of the main ingredient to the auxiliary material is:

[0067] Main ingredient: auxiliary material = 1.2:1.

[0068] Example 6

[0069] A method for preparing heat-clearing, dampness-removing and anti-toxic granules comprises the following steps:

[0070] (1) Weigh the main component and auxiliary materials according to the proportion of Example 1, add water to the main component and boil it twice, add 10 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 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 1.1 at 55°C. The conditions for reduced pressure concentration are: -0.08Mpa, 65°C, 1.5h;

[0071] (2) vacuum drying the extract at 60°C, -0.09 MPa, and 10 h to obtain an extract powder with a water content of <5%;

[0072] (3) Add auxiliary materials, wetting agents, and flavoring agents to the extract powder, mix and granulate, and then pass through a 14-mesh sieve to obtain wet granules. The wetting agent is 75% ethanol, and the weight ratio of the extract powder to the wetting agent is 7:1. The flavoring agent is sucralose, and the amount of the flavoring agent added is 0.6% of the extract powder. The conditions for mixing and granulating are: the stirring paddle speed is 250 rpm, the granulating knife speed is 1100 rpm, and the mixing is 2 minutes.

[0073] (4) Drying the wet granules at 50°C for 4 hours to prepare heat-clearing, dampness-removing and anti-toxic granules.

[0074] Example 7

[0075] A method for preparing heat-clearing, dampness-removing and anti-toxic granules comprises the following steps:

[0076] (1) Weigh the main component and auxiliary materials according to the proportion of Example 1, add water to the main component and boil it twice, add 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 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 at 55°C. The conditions for reduced pressure concentration are: -0.1Mpa, 70°C, 1.5h;

[0077] (2) vacuum drying the extract at 60°C, -0.09 MPa, and 10 h to obtain an extract powder with a water content of <5%;

[0078] (3) Add auxiliary materials, wetting agents, and flavoring agents to the extract powder, mix and granulate, and then pass through a 14-mesh sieve to obtain wet granules. The wetting agent is 75% ethanol, and the weight ratio of the extract powder to the wetting agent is 7:1. The flavoring agent is sucralose, and the amount of the flavoring agent added is 0.6% of the extract powder. The conditions for mixing and granulating are: the stirring paddle speed is 300 rpm, the granulating knife speed is 1200 rpm, and the mixing time is 2 minutes;

[0079] (4) Drying the wet granules at 60°C for 4 hours to prepare heat-clearing, dampness-removing and anti-toxic granules.

[0080] Example 8

[0081] A method for preparing heat-clearing, dampness-removing and anti-toxic granules comprises the following steps:

[0082] (1) Weigh the main component and auxiliary materials according to the proportion of Example 1, add water to the main component and boil it twice, add 10 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 6.5 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 1.1 at 55°C. The conditions for reduced pressure concentration are: -0.09Mpa, 65°C, 1.5h;

[0083] (2) vacuum drying the extract at 60°C, -0.09 MPa, and 10 h to obtain an extract powder with a water content of <5%;

[0084] (3) Add auxiliary materials, wetting agents, and flavoring agents to the extract powder, mix and granulate, and then pass through a 14-mesh sieve to obtain wet granules. The wetting agent is 75% ethanol, and the weight ratio of the extract powder to the wetting agent is 7:1. The flavoring agent is sucralose, and the amount of the flavoring agent added is 0.6% of the extract powder. The conditions for mixing and granulating are: the stirring paddle speed is 350 rpm, the granulating knife speed is 1100 rpm, and the mixing is 2 minutes;

[0085] (4) Drying the wet granules at 70°C for 4 hours to prepare heat-clearing, dampness-removing and anti-toxic granules.

[0086] Example 9

[0087] Example 9 is basically the same as Example 6, except that 6.5 times the amount of water is added during the second decoction.

[0088] Example 10

[0089] Example 10 is basically the same as Example 6, except that the reduced pressure concentration conditions are: -0.08 MPa, 70°C, 1.5 h.

[0090] Embodiment 11

[0091] Example 11 is basically the same as Example 6, except that the reduced pressure concentration conditions are: -0.1 MPa, 65°C, 1.5 h.

[0092] Example 12

[0093] Example 12 is basically the same as Example 6, except that the reduced pressure concentration conditions are: -0.1 MPa, 70°C, 1.5 h.

[0094] Embodiment 13

[0095] 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 1.2 at 55°C.

[0096] Embodiment 14

[0097] Example 14 is basically the same as Example 6, except that the weight ratio of the main component to the auxiliary material is:

[0098] Main ingredient: auxiliary material = 1.3:1.

[0099] Embodiment 15

[0100] Example 15 is basically the same as Example 6, except that the weight ratio of each component in the auxiliary material is:

[0101] Dextrin: powdered sugar = 4:1.

[0102] Example 16

[0103] 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 1200 rpm, and the mixing is 2 min.

[0104] Embodiment 17

[0105] 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 1100 rpm, and the mixing is for 2 minutes.

[0106] Embodiment 18

[0107] 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 1200 rpm, and the mixing is 2 min.

[0108] Embodiment 19

[0109] Example 19 is basically the same as Example 6, except that the drying conditions are: 60° C., 4 h.

[0110] Embodiment 20

[0111] Example 20 is basically the same as Example 6, except that the drying conditions are: 70° C., 4 h.

[0112] Comparative Example 1

[0113] Comparative Example 1 is substantially the same as Example 6, except that 9 times the amount of water is added for the first time.

[0114] Comparative Example 2

[0115] Comparative Example 2 is substantially the same as Example 6, except that 11 times the amount of water is added for the first time.

[0116] Comparative Example 3

[0117] Comparative Example 3 is substantially the same as Example 6, except that 5 times the amount of water is added the second time.

[0118] Comparative Example 4

[0119] Comparative Example 4 is substantially the same as Example 6, except that 7 times the amount of water is added the second time.

[0120] Comparative Example 5

[0121] Control Example 5 is basically the same as Example 6, except that the conditions for reduced pressure concentration are: -0.06 MPa, 65°C, 1.5 h.

[0122] Comparative Example 6

[0123] Control Example 6 is basically the same as Example 6, except that the conditions for reduced pressure concentration are: -0.12 MPa, 65°C, 1.5 h.

[0124] Comparative Example 7

[0125] Control Example 7 is basically the same as Example 6, except that the conditions for reduced pressure concentration are: -0.08 MPa, 60°C, 1.5 h.

[0126] Comparative Example 8

[0127] Control Example 8 is basically the same as Example 6, except that the conditions for reduced pressure concentration are: -0.08 MPa, 75°C, 1.5 h.

[0128] Comparative Example 9

[0129] Comparative Example 9 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 at 55°C.

[0130] Comparative Example 10

[0131] 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 1.3 at 55°C.

[0132] Comparative Example 11

[0133] 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 = 1:1.

[0134] Comparative Example 12

[0135] 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.

[0136] Comparative Example 13

[0137] Comparative Example 13 is substantially the same as Example 6, except that the auxiliary material is sucrose powder.

[0138] Comparative Example 14

[0139] Control Example 14 is basically the same as Example 6, except that the auxiliary material is soluble starch.

[0140] Comparative Example 15

[0141] Control Example 15 is basically the same as Example 6, except that the auxiliary material is dextrin.

[0142] Comparative Example 16

[0143] Control Example 16 is basically the same as Example 6, except that the auxiliary material is sucralose.

[0144] Comparative Example 17

[0145] Comparative Example 17 is substantially the same as Example 6, except that the weight ratio of the components in the auxiliary material is:

[0146] Dextrin: powdered sugar = 3:1.

[0147] Comparative Example 18

[0148] Comparative Example 18 is basically the same as Example 6, except that the weight ratio of each component in the auxiliary material is:

[0149] Dextrin: powdered sugar = 5:1.

[0150] Comparative Example 19

[0151] Comparative Example 19 is substantially the same as Example 6, except that the wetting agent is 70% ethanol.

[0152] Comparative Example 20

[0153] Comparative Example 20 is substantially the same as Example 6, except that the wetting agent is 80% ethanol.

[0154] Comparative Example 21

[0155] Control Example 21 is basically the same as Example 6, except that the conditions for mixing and granulating are: the stirring paddle speed is 200 rpm, the granulating knife speed is 1100 rpm, and mixing is 2 minutes.

[0156] Comparative Example 22

[0157] Control Example 22 is basically the same as Example 6, except that the conditions for mixing and granulating are: the stirring paddle speed is 400 rpm, the granulating knife speed is 1100 rpm, and mixing is 2 minutes.

[0158] Comparative Example 23

[0159] Control Example 23 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 1000 rpm, and mixing is 2 minutes.

[0160] Comparative Example 24

[0161] Control Example 24 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 1300 rpm, and the mixing is 2 min.

[0162] Comparative Example 25

[0163] Control Example 25 is basically the same as Example 6, except that the drying conditions are: 45° C., 4 h.

[0164] Comparative Example 26

[0165] Control Example 26 is basically the same as Example 6, except that the drying conditions are: 75° C., 4 h.

[0166] The present invention is verified as follows

[0167] Experiment 1 Selection of criteria for evaluating granulation effect

[0168] According to the prescription, the main medicine is patchouli and stewed bitter almonds, the assistant medicine is mulberry leaf and cardamom, the adjuvant medicine is wine scutellaria, forsythia, tuckahoe, talc, coix seed, cicada shell, and the assistant medicine is licorice tablets. Among them, stewed bitter almonds are the main medicine in the prescription, and its active ingredient amygdalin is one of the main effective ingredients. Modern pharmacological studies have shown that amygdalin has the effects of relieving cough and asthma, regulating immune function, etc., which is consistent with the treatment of diseases by this prescription. Therefore, this study uses amygdalin as the standard for evaluating the granulation effect.

[0169] The feasibility of using amygdalin as an evaluation tool for granulation effect is verified below:

[0170] 1.1 Preparation of reference solution

[0171] Take an appropriate amount of amygdalin reference substance, weigh it accurately, and add methanol to make a solution containing 40 μg per 1 mL.

[0172] 1.2 Preparation of granule test solution

[0173] Take the Qingrehuashikangdu granules obtained in Example 6 to prepare the Qingrehuashikangdu granule concentrate, shake well, accurately measure 3 mL of the filtrate, put it in 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.

[0174] 1.3 Preparation of boiled bitter almond test solution

[0175] Take 0.25 g of boiled bitter almond powder (passed through No. 2 sieve), weigh accurately, place in a stoppered conical flask, add 25 mL of methanol accurately, stopper tightly, weigh, and ultrasonically treat (power 250 W, frequency 50 kHz) for 30 minutes. Let cool, weigh again, make up the lost weight with methanol, shake well, filter, accurately measure 5 mL of the filtrate, place in a 50 mL volumetric flask, add 50% methanol to dilute to the scale, shake well, filter, and take the filtrate.

[0176] 1.4 Preparation of negative sample solution

[0177] On the basis of Example 6, the blanched 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 scale, shaken, centrifuged, the supernatant was taken, filtered, and the filtrate was taken to obtain a negative sample solution.

[0178] 1.5 Chromatographic conditions

[0179] Octadecylsilane bonded silica gel was used as filler; methanol was used as mobile phase A, water was used as mobile phase B, and elution was performed according to the gradient in Table 1. Column temperature: 40°C; flow rate: 1.0 mL / min; detection wavelength: 207 nm. The theoretical plate number was calculated based on the amygdalin peak and was not less than 1500.

[0180] Table 1

[0181]

[0182] 1.6 System suitability test

[0183] Take the negative sample solution, granule test solution, boiled bitter almond test solution and reference solution respectively, and inject and measure under the chromatographic condition of 1.5. The results are shown in Figure 1 .

[0184] from Figure 1 It can be seen that the elution positions of the granule test solution, boiled bitter almond test solution and reference solution have corresponding peaks at the same retention time, while the negative sample solution has no interference at the corresponding position, the theoretical plate number is 8390, and the separation degree is 2.8. The results preliminarily show that it is feasible to use amygdalin to evaluate the granulation effect.

[0185] 1.7 Linear relationship test

[0186] Preparation of amygdalin reference stock solution: Take an appropriate amount of amygdalin reference, weigh accurately, and add methanol to make a solution containing 200 μg of amygdalin per 1 mL.

[0187] Preparation of amygdalin reference solutions of different concentrations: Accurately measure 0.5, 1, 2, 4, 6, and 8 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.

[0188] Accurately pipette 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 standard curve: y=8604.9x-1619.6, R 2 =0.9997, indicating that amygdalin has a good linear relationship in the range of 9.7755-156.4080 μg / mL. The results are shown in Table 2 and the standard curve is shown in Figure 2 .

[0189] Table 2

[0190]

[0191] 1.8 Stability test

[0192] The granule test solution was prepared according to the method in 1.2. The samples were injected at different time points (0, 2, 4, 6, 8, 12, 24, and 48 hours) to investigate the stability of the granule test solution within 48 hours. The results are shown in Table 3.

[0193] Table 3

[0194]

[0195] The results show that the peak area of ​​the granule test solution RSD It is 1.20%, indicating that the test solution is stable within 48 hours.

[0196] 1.9 Precision test

[0197] Accurately pipette 10 μL of the granule test solution, repeat the injection 6 times according to the determined chromatographic conditions, and record the peak area values ​​respectively. The results are shown in Table 4.

[0198] Table 4

[0199]

[0200] From Table 4, we can see that the peak area of ​​amygdalin RSD The value is 0.50%, indicating good precision.

[0201] The above results finally showed that it was feasible to use amygdalin as a test material for evaluating granulation effect.

[0202] Experiment 2

[0203] In Example 6, Example 7, Control Example 1 and Control 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.

[0204] Table 5

[0205]

[0206] As can be seen from Table 5, compared with Control Examples 1 and 2, the amygdalin transfer rate and 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.

[0207] Experiment 3

[0208] In Example 6, Example 9, Control Example 3, and Control 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.

[0209] Table 6

[0210]

[0211] As can be seen from Table 6, compared with Control Examples 3 and 4, the amygdalin transfer rate and extract yield of Examples 6 and 9 are increased. Considering the production cycle and energy saving, it is determined that 6 to 6.5 times the amount of water is added in the second decoction.

[0212] Experiment 4

[0213] 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.

[0214] Table 7

[0215]

[0216] It can be seen from Table 7 that 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 1.5 h.

[0217] Experiment 5

[0218] 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 yield was calculated. The results are shown in Table 8.

[0219] Table 8

[0220]

[0221] It can be seen from Table 8 that 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 liquid medicine is determined to be controlled at 1.1-1.2 (60°C).

[0222] Experiment 6

[0223] 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.

[0224] Table 9

[0225]

[0226] It can be seen from Table 9 that the granulation difficulty of Example 6 and Example 14 is easier and the particle yield is higher. Therefore, the weight ratio of the main component to the auxiliary material is determined to be: main component: auxiliary material = (1.1~1.3): 1.

[0227] Experiment 7

[0228] 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.

[0229] Table 10

[0230]

[0231] As can be seen from Table 10, the granulation difficulty of Example 6 is the easiest and the granule yield is higher, so it is determined that the auxiliary materials are composed of dextrin and powdered sugar.

[0232] Experiment 8

[0233] 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.

[0234] Table 11

[0235]

[0236] It can be seen from Table 11 that 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 = (4-4.5): 1.

[0237] Experiment 9

[0238] 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.

[0239] Table 12

[0240]

[0241] As shown in Table 12, the granulation difficulty of Example 6 is the easiest and the granule yield is the highest. The change of the wetting agent ethanol concentration will affect the 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.

[0242] Experiment 10

[0243] 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.

[0244] Table 13

[0245]

[0246] It can be seen from Table 13 that 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: the stirring paddle speed is 250-350 rpm, the granulating knife speed is 1100-1200 rpm, and the mixing time is 2 min.

[0247] Experiment 11

[0248] The moisture content of the particles of Example 6, Example 19, Example 20, Control Example 25, and Control Example 26 was calculated. The results are shown in Table 14.

[0249] Table 14

[0250]

[0251] It can be seen from Table 14 that 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 consideration of energy saving, the drying conditions are determined to be: 50-70°C, 4h.

[0252] Experiment 12

[0253] The effect of Qingrehuashikangdu granules on the lungs of mice with lipopolysaccharide (LPS)-induced lung injury was studied experimentally.

[0254] (1) Experimental animals

[0255] Sixty SPF-grade male C57BL / 6J mice (weight: 22 g ± 2 g, 6 weeks) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. [License No.: (SCXK Su) 2023-0009]. Animal husbandry conditions were in accordance with the provisions of the National Animal Care and Use Committee, and were kept in a pathogen-free environment with a temperature of 24 ± 2 °C, a humidity of 75 ± 5%, a 12-h day and night cycle, free access to drinking water and food, and were adaptively raised in the SPF-grade animal breeding room of the Experimental Animal Science and Technology Center of Jiangxi University of Traditional Chinese Medicine for 7 days.

[0256] (2) Animal grouping

[0257] The 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), Qingrehuashikangdu Granule 1 group (hereinafter referred to as QRHS1 group), Qingrehuashikangdu Granule 2 group (hereinafter referred to as QRHS2 group), Qingrehuashikangdu Granule 3 group (hereinafter referred to as QRHS3 group), and PAT group (prednisolone acetate group).

[0258] (3) Drug preparation

[0259] The granules used in group 3 of Qingrehuashikangdu Granules were prepared by the method of Example 6, the granules used in group 2 of Qingrehuashikangdu Granules were prepared by the method of Example 7, and the granules used in group 1 of Qingrehuashikangdu Granules were prepared by the method of Example 8.

[0260] (4) Modeling and drug administration

[0261] All mice were adaptively fed for 3 days. Except for the control group, mice in the LPS group, QRHS1 group, QRHS2 group, QRHS3 group, and PAT group were naturally inhaled into the lungs of mice through the nasal passage on the 4th day using 30 μL LPS (1 mg / mL). The mice in the control group were only given 30 μL of normal saline. From the 5th to the 8th day, the mice in the QRHS3, QRHS2, and QRHS1 groups were gavaged with the Qingre Huashi Kangdu Granules prepared in Example 6, Example 7, and Example 8, respectively, and the gavage volume was 20 g / kg / d. The mice in the PAT group were gavaged with 10 mg / kg / d PAT. At the same time, the mice in the control group and the LPS group were gavaged with the same amount of normal saline. On the 9th day, the mice in each group were intraperitoneally injected with 1.5% sodium pentobarbital, 0.05 mL / 10 g, and then anesthetized and killed.

[0262] (5) Test content

[0263] (5.1) General conditions of mice in each group

[0264] After anesthesia, the mice in each group were weighed and peripheral blood samples were collected. After the mice were killed, the lungs and spleens of the mice were quickly separated on an ice table, and feces were collected from the ileocecal part of the mice. The lungs and spleens of each group were weighed, and the lung weight index (ILW) and spleen weight index (ISW) were calculated. The calculation formula was: ILW (%) = lung weight / body weight × 100; ISW (%) = spleen weight / body weight × 100.

[0265] (5.2) Hematoxylin-eosin (HE) staining of mouse lungs

[0266] The staining steps are as follows:

[0267] 1. Tissue Embedding

[0268] 1) Mouse lung tissue was fixed in 4% paraformaldehyde for 48 h and then taken out from a 4°C refrigerator for later use;

[0269] 2) Cut the lung tissue into small pieces so that they can be placed in the sample box;

[0270] 3) Dehydration and transparency: 75% ethanol (1h) → 95% ethanol I (1h) → 95% ethanol II (1h) → 100% anhydrous ethanol I (30min) → 100% anhydrous ethanol II (30min) → xylene (20min);

[0271] 4) Melt the paraffin block: Place the sample box in the paraffin block and place in a constant temperature oven at 60°C (60 min);

[0272] 5) Label the sample boxes in sequence, put the tissue blocks in for embedding, and put them in the refrigerator after cooling and fixation;

[0273] 2. Paraffin Sections

[0274] Before paraffin sectioning, the water temperature is maintained at 40-45℃, and 30% ethanol solution is ready for use. After preparation, the embedded tissue wax block is placed on the slicer, the slice thickness is 4μm, and the wax strip cut by the slicer is placed on the surface of the ethanol solution to unfold the slice. The unfolded slice is then moved to the warm water of the water bath to fully unfold the slice. The fully unfolded slice is placed on a slide, and a mark is written on the ground surface, and it is placed in an oven at 60℃ to dry overnight.

[0275] 3. HE staining method

[0276] 1) Dewaxing: Dewaxing the dried slices in xylene twice (20 min each time) → 100% ethanol (5 min) → 95% ethanol (5 min) → 85% ethanol (5 min) → 75% ethanol (5 min) → rinsing with tap water (3 min);

[0277] 2) Staining: Place the slices in hematoxylin for staining (30 minutes), and rinse with running water (15 minutes). During the process, the water flow should not be too large to prevent the slices from falling off. Stop rinsing when the slices turn blue.

[0278] 3) Differentiation: Put the slices into 1% hydrochloric acid ethanol solution to fade, wait until the tissue slices turn red and the color becomes lighter, the time is about tens of seconds. After fading, put them into tap water to rinse again until the slices return to blue again;

[0279] 4) Dehydration: Take out the slices and place them in 50% ethanol (5 min) → 70% ethanol (5 min) → 80% ethanol (5 min) for gradient dehydration in different concentrations of ethanol solutions;

[0280] 5) Restaining: Take out the slices and stain them in 0.5% eosin ethanol solution for 3 minutes;

[0281] 6) Dehydration: Prepare 95% ethanol to absorb the excess red in the slices, then place in 100% ethanol solution for 3 minutes. Finally, take out the slices and wipe off the excess ethanol solution;

[0282] 7) Transparency: Place the slices in xylene for 5 minutes;

[0283] 8) Sealing: Seal the slide with neutral resin and observe under a microscope.

[0284] (5.3) Mouse ELISA test

[0285] ELISA method 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 in lung tissues as well as immunoglobulins IgA, IgG, and IgM among the groups.

[0286] The detection steps are as follows: 1) Prepare multiple wells for blank, standard, and test samples. Add 0.1 ml of sample diluent to the blank well, and add 0.1 ml 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 0.1 ml 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 0.35 ml per well, gently shake dry the liquid in the well, and the remaining liquid in the well can be blotted with absorbent paper. 4) Add 0.1 ml of enzyme conjugate working solution to each well. The working solution should be prepared within 20 minutes before use and stored in a dark place, 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 the plate washing 5 times. 6) Add 90 μL of colorimetric reagent (TMB) to each well, cover the plate with a film, and incubate at 37°C in the dark for 15 minutes. 7) Add 50 μL of stop solution to each well to stop the reaction. The blue color in the well will turn yellow immediately. 8) Use an ELISA reader to measure the optical density (OD value) of each well at a wavelength of 450 nm. 9) Use the horizontal axis as the concentration of the standard and the vertical axis as the OD value to draw a standard curve and formula with the help of software. Substitute the measured sample OD value into the standard curve formula, and then divide the calculated value by the corresponding protein concentration of the sample to obtain the actual concentration of the indicator to be measured.

[0287] (5.4) Mouse flow cytometry experiment

[0288] 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 + The IL-10 cell subset was also evaluated in this group. In these cell suspensions, the following steps were performed:

[0289] 1) Take 80 μL of anticoagulant, add 100 μL of 1640 medium, add 1 mL of 1X hemolysin, incubate for 15 min, centrifuge at 500 g for 5 min, and remove the supernatant;

[0290] 2) Wash the cells twice with 1 mL of staining buffer, centrifuge at 500 g for 5 min, and remove the supernatant;

[0291] 3) Resuspend the cells with 100 μL staining buffer. Add 1 μg of Fc blocking agent (553141), incubate at 4°C for 8 min, add 2.5 μL of CD4-BV510, CD25-Percp-CY5.5, Foxp3-EF450 and IL-10-FITC antibodies for surface staining, and incubate at room temperature for 15 min;

[0292] 4) Wash twice with 1 mL of staining buffer, remove the supernatant, and add 500 μL of staining buffer to resuspend;

[0293] 5) For flow cytometry, quadrant labeling restrictions are always based on negative populations and isotype controls;

[0294] 6) The data were analyzed using FlowJo software10, and inactive cells were excluded by gating.

[0295] (5.5) Statistical methods

[0296] GraphPad Prism 9.0 software was used for statistical analysis. Data were expressed as mean ± standard deviation (SD). Statistical tests among multiple groups of data were performed by one-way analysis of variance, with the "," "and" "" indicates P<0.05, P<0.01 and P<0.001 compared with the control group. "##" indicates P<0.01 compared with the LPS group. P<0.05 was considered statistically significant.

[0297] (6) Experimental results

[0298] (6.1) Qingrehuashikangdu Granules Improve Lung Injury in Mice

[0299] Before modeling: During the 3-day adaptive feeding period, the rats in each group were generally in good condition, and the rats could freely eat and drink water, had shiny fur, were agile, and had a gentle personality. There were no significant differences in the spirit and body weight of the rats in each group.

[0300] After modeling: The rats in the control group were generally in good condition, with no significant changes in food and water intake, smooth and clean fur, agile reactions, normal limb strength, and good mental state in daily observation, with no significant changes in defecation. The rats in the remaining groups showed significant changes, including increased food intake, rough fur, increased daily activity, hyperactivity, increased defecation, intense resistance during gavage, and a significant decrease in body weight compared with the control group.

[0301] After administration: the above conditions in the QRHS1, QRHS2, and QRHS3 groups improved, and the rats' food intake gradually recovered, their hair gradually became shiny, their activity decreased, their mental state improved, their resistance to oral administration weakened, and their body weight slowly recovered, while there was no significant change in the control group. Figure 3 .

[0302] like Figure 4 As shown in the results, when measuring lung weight, lung weight index, spleen weight, and spleen weight index, it was observed that the lung weight of the LPS group was significantly higher than that of the other groups, and there was a significant difference compared with the other groups (p<0.05). This shows that in the lung injury LPS group, the lung tissue was significantly affected and the lung weight increased. In the QRHS1 group, QRHS2 group, and QRHS3 group, the lung weight was significantly lower than that of the LPS group, showing an inhibitory effect on lung injury, and the lung weight of the QRHS1 group, QRHS2 group, and QRHS3 group was lighter than that of the PAT group.

[0303] like Figures 5 to 10 As shown in the figure, after HE staining, the lung tissue structure of mice in the control group was clear, the alveolar septa were normal, and there was no obvious inflammatory cell infiltration. However, the lung tissue structure of mice in the LPS group after lipopolysaccharide induction was obviously disordered, the lung septa were abnormal, and a large number of inflammatory cell infiltrations were visible to the naked eye. For the QRHS1 group, QRHS2 group, and QRHS3 group, after the intervention of the Qingrehuashikangdu granules, the inflammation of the mouse lung tissue was alleviated, the inflammatory cells were reduced compared with the LPS group, and the lung structure was also restored. In addition, compared with the QRHS1 group and the QRHS2 group, the HE staining effect of the QRHS3 group was closer to the control group, the alveolar structure was clearly visible, and the inflammatory cell infiltration almost disappeared.

[0304] In summary, these results indicate that the Qingrehuashikangdu granules significantly alleviated lung injury in mice, and the Qingrehuashikangdu granules prepared in Example 6 corresponding to the QRHS3 group showed a more significant therapeutic effect.

[0305] (6.2) Effect of Qingrehuashikangdu Granules on Inhibition of Lung Tissue Inflammation

[0306] In the mouse ELISA test, relevant inflammatory factors and immunoglobulins were detected in lung tissue specimens of different treatment groups to evaluate the effects of different treatments on lung injury-related indicators.

[0307] See also Fig.11 The results showed that the levels of pro-inflammatory cytokines (IL-2, IL-6, IL-12, IL-17A, IL-21, IL-23, TNF-α, IFN-γ) in the lung tissue of the LPS group were significantly higher than those in the other treatment groups, showing a significant inflammatory response. In the QRHS1 group, QRHS2 group, and QRHS3 group, the levels of related inflammatory factors were relatively low, showing an inhibitory effect on inflammation, and there were significant differences compared with the LPS group (p<0.05); the levels of anti-inflammatory cytokines (IL-10, IL-4) in the lung tissue of the LPS group were significantly lower than those in the other treatment groups, and the anti-inflammatory effect was significantly inhibited. In the QRHS1 group, QRHS2 group, and QRHS3 group, the levels of anti-inflammatory factors were relatively high, showing an inhibitory effect on inflammation, and there were significant differences compared with the LPS group (p<0.05); in terms of immunoglobulins, please refer to Fig.12 After LPS induction, the levels of IgG, IgM, and IgA in the serum of mice increased, while they showed a downward trend after being treated with Qingrehuashikangdu Granules. The results showed that Qingrehuashikangdu Granules can improve the expression of inflammatory factors in mice, regulate the balance between pro-inflammatory cytokines and anti-inflammatory cytokines, and reduce the expression of immunoglobulins in the body, thereby alleviating LPS-induced lung inflammation in mice.

[0308] (6.3) Flow cytometry results

[0309] See also Fig.13 and Fig.14 Compared with the control group, the CD4 + IL17A + (Th17A) cell expression increased significantly, CD4 + CD25 + Foxp3 + (Treg), CD4 + IL10 + After treatment with Qingrehuashikangdu Granules and prednisone acetate, the CD4 + CD25 + Foxp3 + (Treg), CD4 + IL10 +(Th10) cell levels are upregulated, CD4 + IL17A + The expression of Th17A decreased (P<0.05), indicating that the Qingre Huashi Kangdu Granules can regulate the differentiation of Treg and TH17A cells and maintain the balance of CD4 / CD8.

[0310] Experiment 13

[0311] The inhibitory effect of Qingrehuashikangdu granules on coronavirus was studied experimentally.

[0312] (1) Experimental materials

[0313] (1.1) Cells

[0314] MRC-5 was purchased from China Center for Type Culture Collection and cultured, passaged, and cryopreserved in vitro according to the instructions.

[0315] (1.2) Coronavirus

[0316] The HCoV-229E virus was provided by the Institute of Virology, Chinese Center for Disease Control and Prevention, and was cultured according to the instructions. It has been effectively amplified and stored in the P2 laboratory of the Cancer Research Center of Jiangxi University of Traditional Chinese Medicine.

[0317] (2) Experimental methods

[0318] (2.1) In vitro efficacy evaluation method

[0319] (2.1.1) Preparation of monolayer cells:

[0320] Add 100uL of sterile triple-distilled water to all wells around the 96-well plate used for the assay (36 wells in total); then digest the cells with trypsin, count the cells with a cell counter under a microscope, and then dilute the cell suspension with complete culture medium to adjust the cell density to 1×10 4 Cells / ml. Add 100uL / well of the diluted cell suspension to the wells in the central area of ​​the 96-well plate; place it in an incubator and culture it overnight at 37°C and 5% CO2 to form a cell monolayer with 80-90% confluence.

[0321] (2.1.2) Virus-infected cells:

[0322] ①Discard the monolayer cell culture supernatant, rinse once with 1×PBS, and once with DMEM medium.

[0323] ② Add virus, where TCID50 of HCoV-229E virus is 10 -5.35 / 0.1mL, MOI=0.1;

[0324] ③ Place the cells with virus back into the cell culture incubator and allow the virus to infect for 2 hours. During this period, gently mix the virus dilution every 30 minutes to allow the virus to fully contact and infect the cells.

[0325] (2.1.3) Drug intervention

[0326] ① Remove the virus liquid and rinse the cells twice with serum-free DMEM.

[0327] ② Experimental grouping and drug administration: A maintenance medium without drugs was set as a control group. The granules of Example 6 were dissolved and diluted with a DMEM complete medium containing only 2% FBS to obtain drugs of different concentrations.

[0328] (2.1.4) The Reed-Muench method and CCK8 method were used to calculate the half inhibitory concentration IC50 and therapeutic index SI (SI=TC50 / IC50) of the drug for viral inhibition, and the viral inhibition rate was calculated.

[0329] (2.2) Fluorescence quantitative PCR detection of coronavirus M protein expression in each group of cells

[0330] (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;

[0331] (2.2.2) Drug preparation: After the granules of Example 6 were dissolved, they were diluted with DMEM complete medium containing only 2% FBS to obtain drugs with concentrations of 8 mg / mL, 2 mg / mL, and 0.5 mg / mL, respectively.

[0332] (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, cultured in DMEM+2% FBS maintenance medium 2 hours after virus infection), positive control group (referred to as RBV group, cultured in DMEM+2% FBS maintenance medium containing 50ug / ml ribavirin 2 hours after virus infection), preventive drug administration group (referred to as Pre group, cells were intervened with DMEM+10% FBS growth medium containing 2mg / mL drug 24 hours before infection, and cultured in DMEM+2% FBS maintenance medium 2 hours after normal virus infection), drug administration group 1 (2 hours after virus infection, cells were intervened with 8mg / mL drug), drug administration group 2 (2 hours after virus infection, cells were intervened with 2mg / mL drug), drug administration group 3 (2 hours after virus infection, cells were intervened with 0.5mg / mL drug).

[0333] (2.2.4) Virus infection of cells: The infection process is the same as (2.1.2).

[0334] (2.2.5) Extract RNA from cell supernatant 48 hours after infection. Perform fluorescence quantitative PCR detection according to the instructions, and calculate the ratio of HCoV-229E virus M protein mRNA compared with the negative control group, that is, the number of nucleic acid copies in the RBV group / the number of nucleic acid copies in the Con group, the number of nucleic acid copies in the Pre group / the number of nucleic acid copies in the Con group, the number of nucleic acid copies in the medication 1 group / the number of nucleic acid copies in the Con group, the number of nucleic acid copies in the medication 2 group / the number of nucleic acid copies in the Con group, and the number of nucleic acid copies in the medication 3 group / the number of nucleic acid copies in the Con group.

[0335] (3) Statistical analysis

[0336] GraphPad Prism9.0 software was used for experimental data analysis, and one-way analysis of variance was used for statistical tests among multiple groups of data. "," "and" ” indicates P < 0.05, P < 0.01, and P < 0.001.

[0337] (4) Experimental results

[0338] (4.1) Results of in vitro efficacy evaluation

[0339] See also Fig.15 IC50 was calculated using GraphPad Prism9.0. The IC50 of Qingre Huashi Kangdu Granules in this experiment was 1.561 mg / mL, and the therapeutic index was 5.848. This indicates that Qingre Huashi Kangdu Granules has good in vitro coronavirus inhibition activity. The SI index is greater than 2, indicating that Qingre Huashi Kangdu Granules has good therapeutic safety.

[0340] (4.2) Results of fluorescence quantitative PCR detection of coronavirus M protein expression in each group of cells

[0341] See also Fig.16 The results showed that compared with the Con group, the drug group 1, drug group 2, drug group 3, and Pre group could significantly reduce the expression level of HCoV-229E virus M protein mRNA in the cell supernatant; and for drug group 1, drug group 2, and drug group 3, the therapeutic effect was positively correlated with the dose. In addition, the efficacy of the preventive drug group was particularly significant, which was equivalent to the therapeutic effect of ribavirin 50ug / mL.

[0342] In summary, the present invention experimentally studies the effect of Qingre Huashi Kangdu Granules on the lungs of mice with lung injury induced by lipopolysaccharide (LPS). The experiment illustrates the protective effect of Qingre Huashi Kangdu Granules on LPS-induced lung injury in mice. Qingre Huashi 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, as well as reducing the expression of immunoglobulins in the body. Qingre Huashi Kangdu Granules can regulate Treg and TH17A cell differentiation and maintain CD4 / CD8 balance, thereby inhibiting the progression of inflammation in mice with lipopolysaccharide-induced lung injury. In addition, through anti-coronavirus experiments, it is shown that Qingre Huashi 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.

[0343] The preparation method of the heat-clearing, damp-removing and anti-toxic 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 = (4-4.5): 1; at the same time, the weight ratio of the main component to the auxiliary material is defined as: main component: auxiliary material = (1.1-1.3): 1. In conjunction with the optimized preparation process of the present invention, firstly, the main component is 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 1.1-1.2 at 55°C, and then vacuum dried under limited conditions to obtain an extract powder with a water content of less than 5%, and then auxiliary materials, a wetting agent, and a flavoring agent are added, mixed and granulated, and then passed through a 14-mesh sieve to obtain wet granules, and finally the wet granules are dried to prepare the heat-clearing, damp-removing and anti-toxic granules. Experimental results show that the granules obtained by the method have good efficacy.

[0344] Although the 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 present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for preparing a heat-clearing, dampness-removing and anti-toxic granule, characterized in that: The heat-clearing, dampness-removing and anti-toxic granules include a main component, which is composed of the following components by weight: 280-320 parts of patchouli, 280-320 parts of boiled bitter almonds, 280-320 parts of mulberry leaves, 190-210 parts of cardamom, 280-320 parts of wine scutellaria, 280-320 parts of forsythia, 280-320 parts of poria, 280-320 parts of talc, 490-510 parts of coix seeds, 190-210 parts of liquorice tablets, and 90-110 parts of cicada shells; The heat-clearing, dampness-removing and anti-toxic granules further include auxiliary materials, which are composed of dextrin and powdered sugar; The weight ratio of each component in the auxiliary material is: Dextrin: powdered sugar = (4-4.5): 1; The weight ratio of the main ingredient to the auxiliary material is: Main ingredient: auxiliary material = (1.1-1.3): 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. For the first time, add 10 to 10.5 times the amount of water, boil it for 1.5 hours, and filter it through a 100-mesh filter cloth. For the second time, add 6 to 6.5 times the amount of water, boil it for 1 hour, and filter it through a 100-mesh filter cloth. Combine the extracts, filter, and concentrate the filtrate under reduced pressure to an extract with a relative density of 1.1 to 1.2 at 55°C; (2) vacuum drying the extract at 60°C, -0.09 MPa, and 10 h to obtain an extract powder with a water content of <5%; (3) Add auxiliary materials, wetting agents and flavoring agents to the extract powder, mix and granulate, and then pass through a 14-mesh sieve to obtain wet granules; (4) drying the wet granules to prepare heat-clearing, dampness-removing and anti-toxic granules; In step (1), the conditions for reduced pressure concentration are: -0.08~-0.1Mpa, 65~70°C, 1.5h; 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 = 7:1; In step (3), the flavoring agent is sucralose, and the amount of the flavoring agent added is 0.6% of the extract powder; In step (3), the conditions for mixing and granulating are: stirring paddle speed is 250-350 rpm, granulating knife speed is 1100-1200 rpm, and mixing is 2 min; The drying conditions in step (4) are: 50-70°C, 4h.

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