Traditional Chinese medicine composition for freshening breath and application thereof

Abstract

The invention discloses a traditional Chinese medicine composition for freshening breath. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 10-20 parts of kadsura longepedunculata, 20-40 parts of malus spectabilis, 10-30 parts of honeysuckle, 20-40 parts of dandelion and 20-40 parts of clove. Five traditional Chinese medicines including kadsura longepedunculata, malus hupehensis, honeysuckle, dandelion and clove are combined to form the KLF, so that the KLF can relieve the symptom of the stomach heat syndrome, improve the damage of gastric mucosa tissues and regulate intestinal flora, and has a remarkable effect in the aspect of treating the ozostomia caused by the stomach heat syndrome.

Description

Technical Field

[0001] The invention specifically relates to a breath-freshening Chinese medicinal composition and application thereof. Background Art

[0002] Bad breath, also known as "fishy smell" and "foul breath" in traditional Chinese medicine books, can be clinically divided into non-pathological bad breath and pathological bad breath. The causes of non-pathological bad breath include eating irritating foods, reduced saliva secretion during sleep, which leads to a large amount of bacteria decomposing food residues, etc., resulting in temporary bad breath. Pathological bad breath is mostly caused by local or systemic diseases. According to its source, it is divided into oral bad breath, non-oral bad breath and mental bad breath. Non-oral bad breath includes respiratory diseases (nasal cavity, pharynx, lung infection and necrosis, etc.), digestive system diseases (gastritis, gastrointestinal metabolic disorders, constipation, etc.), solid organ damage (liver failure, renal failure) and bad breath caused by uremia, leukemia, etc. Regarding the etiology and pathogenesis of bad breath, doctors of all dynasties have classified it as heat syndrome. For example, Jingyue Complete Book, Miscellaneous Symptoms, Biji, Koushe, records that excessive stomach heat, or pathogenic heat invading the stomach, or overeating fatty, sweet, spicy and hot foods can lead to excessive stomach heat, which will damage body fluids, cause body fluids to be lost, and cause turbid air to fail to descend and rise up to the mouth, resulting in bad breath. Medical Introduction says: "If the spleen is hot, the mouth will be dry or smelly... Bad breath is caused by stomach heat." It is believed that stomach fire and spleen heat are the main causes of bad breath. Ming Li Shizhen's Compendium of Materia Medica, Koushe, says: "Bad breath is caused by stomach fire and food stagnation." Therefore, the main location of bad breath is the stomach and spleen, which is closely related to the heart, liver, lungs and kidneys. The dysfunction of the five internal organs, the obstruction of the spleen and stomach's transportation and ascending and descending functions, the endogenous generation of turbid air, and the rise of turbid air in the mouth cause bad breath.

[0003] Traditional Chinese medicine has a long history of treating bad breath, and its efficacy is remarkable. TCM diagnoses diseases through "differentiation and treatment", starting from the overall situation, combining the four diagnoses and symptoms, and believes that the human body is an organic whole, with coordinated functions and mutual influence in pathology. Bad breath is not only a local disease of the body, but also an external manifestation of pathological changes in the internal organs. The principle of "treating the disease to the root" is to achieve the purpose of eliminating bad breath. Although there are many studies on bad breath as a symptom category, most of them are concentrated in the direction of stomatology, and there is a lack of relevant consensus data in the direction of spleen and stomach diseases. Therefore, the Chinese Association of Traditional Chinese Medicine's spleen and stomach disease took the lead in formulating the 2023 TCM diagnosis and treatment expert consensus on bad breath based on the results of clinical research on bad breath-related diseases in my country and combined with the experience of experts. The consensus shows that the treatment goal of bad breath is to eliminate the symptoms of bad breath. It is believed that the location of bad breath focuses on the stomach. The treatment principle is that the stomach is used for communication and descending is smooth, so the treatment is mainly to eliminate evil, supplemented by communication and descending.

[0004] With the progress of pharmacological research on traditional Chinese medicine, the application of traditional Chinese medicine to bad breath will have the advantages of strong targeting and high efficiency. In addition, traditional Chinese medicine is widely used in the food and health care products industry due to its wide availability, diverse pharmacological activity and few adverse reactions, and is easily accepted by patients.

[0005] However, there is a lack of combining syndrome differentiation and treatment with the pharmacological effects of Chinese medicinal materials to treat halitosis, especially stomach-heat type halitosis, in order to improve the cure rate of halitosis. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a Chinese medicine composition for freshening breath, which is prepared from the following raw materials in weight ratio:

[0007] 10-20 parts of Schisandrae Chinensis Fruit, 20-40 parts of Malus Malus, 10-30 parts of Honeysuckle, 20-40 parts of Taraxacum Herba, and 20-40 parts of Cloves.

[0008] Furthermore, it is prepared from the following raw materials in weight ratio:

[0009] 15 parts of Schisandrae Chinensis Fruits, 30 parts of Malus Malus, 20 parts of Flos Honeysuckle, 30 parts of Herba Taraxaci, and 30 parts of Flos Caryophylli.

[0010] Furthermore, the crabapple is a Hubei crabapple.

[0011] Furthermore, it is a preparation prepared from fine powder of raw materials, or water extract of raw materials, or alcohol extract of raw materials as active ingredients, and auxiliary materials acceptable to medicines or daily chemical products.

[0012] Furthermore, the preparation is an oral preparation or an oral preparation.

[0013] Furthermore, the oral preparation includes solutions, tablets, granules, pastes, pills, capsules or powders;

[0014] The oral preparations include lozenges, mouthwashes, popping beads or toothpaste.

[0015] The present invention also provides a method for preparing the aforementioned Chinese medicine composition, which comprises the following steps:

[0016] (1) Weigh the raw materials according to the ratio;

[0017] (2) A preparation made from a fine powder of a raw material, or an aqueous extract of a raw material, or an alcohol extract of a raw material as an active ingredient, plus excipients acceptable to medicines or daily chemical products.

[0018] The present invention also provides a use of the aforementioned traditional Chinese medicine composition in preparing a medicine for treating gastric mucosal damage, improving intestinal flora disorder and / or refreshing breath.

[0019] Furthermore, the medicine is a medicine for treating gastric mucosal damage of stomach-heat syndrome type and / or bad breath of stomach-heat syndrome type.

[0020] Furthermore, the drug has the effect of inhibiting Fusobacterium nucleatum and / or Porphyromonas gingivalis.

[0021] Finally, the present invention provides a use of the aforementioned traditional Chinese medicine composition in preparing daily chemical products for refreshing breath.

[0022] Furthermore, the daily chemical product is a daily chemical product for improving gastric heat syndrome type halitosis.

[0023] Furthermore, the daily chemical product has the function of inhibiting Fusobacterium nucleatum and / or Porphyromonas gingivalis.

[0024] The Hubei crabapple of the present invention is the dried leaves of the Hubei crabapple Malus hupehensis (Pamp.) Rehd. of the Rosaceae family.

[0025] In the early stage of the present invention, halitosis caused by stomach heat was taken as the research object, and the effective ingredients and formulas of Chinese medicines with the same origin of medicine and food were screened, and then the antibacterial test was further carried out to screen the compatibility ratio, and finally it was determined that the five Chinese medicines of Schisandra chinensis, Malus hupehensis, Honeysuckle, Taraxacum officinale and Clove were used to form the formula for leaving a fragrant mouth and teeth (KLF). In the formula, Malus hupehensis is the main medicine for eliminating accumulation and stagnation, harmonizing the stomach and strengthening the spleen, Honeysuckle and Taraxacum officinale are the ministerial medicines for clearing away heat, purging fire and detoxifying, Schisandra chinensis is the adjuvant for invigorating qi and promoting body fluid, and Clove is the guiding medicine, which plays the role of antibacterial and taste correction.

[0026] Animal experiments and clinical trials have confirmed that the combination of the five Chinese medicines of the present invention can relieve the symptoms of stomach heat syndrome, improve gastric mucosal tissue damage, and regulate intestinal flora, and has a significant effect in treating bad breath caused by stomach heat syndrome.

[0027] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0028] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1is the effect of KLF on ethanol-treated GES-1 cells; A: The effect of 0-9% ethanol concentration treatment for 3h on the survival rate of GES-1 cells; B: The effect of 0-9% ethanol concentration treatment for 4h on the survival rate of GES-1 cells; C: The effect of 0-9% ethanol concentration treatment for 5h on the survival rate of GES-1 cells; D: The effect of different concentrations of KLF treatment on the survival rate of GES-1 cells; E: The effect of KLF on the survival rate of ethanol-treated GES-1 cells; FH: The effect of KLF on the level of inflammatory factors in ethanol-treated GES-1 cells; IJ: The effect of KLF on the wound healing migration of ethanol-treated GES-1 cells; Note: & is the inhibition rate of GES-1 cells is 40%; ##p<0.01 compared with the control group, *p<0.05, **p<0.01 compared with the model group;

[0030] Figure 2 The changes in the appearance of rats are A: the redness and swelling of the gums of rats in different groups; B: the redness and swelling of the paws of rats in different groups; C: the redness and swelling of the auricles of rats in different groups; D: the feces of rats in different groups;

[0031] Figure 3 The therapeutic effect of KLF on halitosis of gastric heat syndrome induced by dried ginger decoction; A: Apparent state of rat gastric mucosa; B: Pathological changes of rat gastric mucosa (200×); CE: Effect of KLF on the levels of serum oxidative stress factors MDA, SOD and GSH in rats; FH: Effect of KLF on the levels of serum inflammatory factors IL-6, IL-8 and TNF-α in rats; IL: Effect of KLF on the levels of serum gastric mucosal repair factors GAS, TXB2 and 6-keto-PGF1α in rats and the ratio of TXB2 to 6-keto-PGF1α;

[0032] Figure 4 The effect of KLF on the intestinal flora of rats; A: ASV Venn diagram; B: Chao1 index of intestinal flora dilution curve based on ASV abundance; C: Shannon index of intestinal flora dilution curve based on ASV abundance; DE: Chao1 and Shannon index of intestinal flora based on ASV abundance; F: Principal coordinate analysis (PCoA) of intestinal flora structure; G: Bray_curtis similarity analysis (ANOISM) of inter-group differences;

[0033] Figure 5Effect of KLF on the species structure of intestinal flora in rats A: Species distribution diagram of intestinal flora among groups at the phylum level; B: Species distribution diagram of intestinal flora among groups at the genus level; C: Kruskal-Wallis H test bar graph of intestinal flora among groups at the phylum level; D: Kruskal-Wallis H test bar graph of intestinal flora among groups at the genus level; *p<0.05, **p<0.01;

[0034] Figure 6 This is the clustering heat map of PICRUSt2 function prediction of rat intestinal flora. The ordinate represents the functional annotation, and the abscissa is the sample information. Red and blue represent the higher and lower relative abundance of the function in the group, respectively;

[0035] Figure 7 is the minimum inhibitory concentration of Kouchiliuxiang Recipe and single herbs against Fusobacterium nucleatum (FN) and Porphyromonas gingivalis (PG);

[0036] Figure 8 The effect of the series of products that leave a good smell in your mouth—lozenges, toothpaste, and mouthwash in removing bad breath; A: Changes in the VSC values ​​of the subjects before, 0 hours, and 1 hour after using lozenges; B: Changes in the VSC values ​​of the subjects before, 0 hours, and 1 hour after using mouthwash; C: Changes in the VSC values ​​of the subjects before, 0 hours, and 1 hour after using toothpaste; D: Changes in the VSC values ​​of the subjects before, 7 days, 14 days, 21 days, and 28 days after using the series of products that leave a good smell in your mouth—lozenges, toothpaste, and mouthwash. **Compared with the initial value, p<0.01. DETAILED DESCRIPTION

[0037] The raw materials and equipment used in the specific embodiments of the present invention are all known products, which are obtained by purchasing commercially available products. Among them, the preparation process of modern Chinese medicine preparations made from the Chinese medicine composition of the present invention is mature and can be made by referring to the conventional methods of the prior art.

[0038] Example 1 Preparation of the composition of the present invention

[0039] Recipe: 15g Schisandra chinensis, 30g Begonia chinensis, 20g Honeysuckle, 30g Dandelion, 30g Clove

[0040] Preparation method: Weigh the above-mentioned drugs according to the ratio, mix and grind into coarse powder, add 10 times the amount of water, soak for 2 hours, decoct once, filter with double-layer gauze and collect the filtrate; add 8 times the amount of water to the residue and decoct for 1 hour, remove the residue and combine the two filtrates, concentrate at 80°C, and dry under reduced pressure to obtain dry extract powder.

[0041] Example 2 Preparation of the composition of the present invention

[0042] Formula: 10g of Schisandra chinensis, 20g of Begonia chinensis, 10g of Honeysuckle, 20g of Dandelion, 20g of Clove

[0043] Preparation method: Weigh the above-mentioned drugs according to the ratio, mix and grind into coarse powder, add 10 times the amount of water, soak for 2 hours, decoct once, filter with double-layer gauze and collect the filtrate; add 8 times the amount of water to the residue and decoct for 1 hour, remove the residue and combine the two filtrates, concentrate at 80°C, and dry under reduced pressure to form dry extract powder, add commonly used excipients in medicines to make tablets.

[0044] Example 3 Preparation of the composition of the present invention

[0045] Recipe: 20g Schisandra chinensis, 40g Begonia chinensis, 30g Honeysuckle, 40g Dandelion, 40g Clove

[0046] Preparation method: weigh the above-mentioned drugs according to the ratio, mix and grind into coarse powder, add 10 times the amount of water, soak for 2 hours, decoct once, filter with double-layer gauze and collect the filtrate; add 8 times the amount of water to the residue and decoct for 1 hour, remove the residue and combine the two filtrates, concentrate at 80°C, and dry under reduced pressure to form a dry extract powder, add the auxiliary materials commonly used in daily chemical products to make a mouthwash.

[0047] Example 4 Preparation of the composition of the present invention

[0048] Recipe: 15g Schisandra chinensis, 30g Begonia chinensis, 20g Honeysuckle, 30g Dandelion, 30g Clove

[0049] Preparation method: weigh the above drugs according to the ratio, mix and grind into coarse powder, add 10 times the amount of water, soak for 2 hours, decoct once, filter with double gauze and collect the filtrate; add 8 times the amount of water to the residue and decoct for 1 hour, remove the residue and combine the two filtrates, concentrate at 80°C, and dry under reduced pressure to form dry extract powder, add excipients commonly used in daily chemical products to make toothpaste.

[0050] The beneficial effects of the present invention are described below through test examples.

[0051] Experimental Example 1 Protective effect of the Kou Chi Liu Xiang Fang (KLF) of the present invention on GES-1 cell damage

[0052] 1 Materials and methods

[0053] 1.1 Materials

[0054] Human gastric mucosal cells (GES-1 cells, TCH-C410) were purchased from Suzhou Haixing Biotechnology Co., Ltd., inverted electron microscope (Nikon Technology Co., Ltd., Ts2-FL), carbon dioxide incubator (Thermo Fisher Scientific, 371), constant temperature water bath (Jiangsu Datang Medical Instrument Co., Ltd., HH-S), centrifuge (Jiangsu Datang Medical Instrument Co., Ltd., TD-4C), multifunctional microplate reader (Thermo Fisher Scientific, 51119670DP), trypsin (CSP087), penicillin- Streptavidin-antibody (CSP006), fetal bovine serum FBS (11011-8611), PBS buffer (PYG0021), one-step freezing solution (GUCP-R201-100), RPMI-1640 culture medium (aqueous solution, SH30809.01), MTT (1334MG250), interleukin-8 (IL-8, MM-1558H2), interleukin-6 (IL-6, MM-0049H2), and tumor necrosis factor (TNF-α, MM-0122H2).

[0055] 1.2 Preparation of Kouchiliuxiangfang (KLF) extract

[0056] According to the formula ratio of Example 1, a total of 500g of Schisandra chinensis, Malus melongena, honeysuckle, dandelion and clove were weighed, mixed and crushed into coarse powder, added with 10 times the amount of water, soaked for 2h, decocted once, filtered with double gauze and the filtrate was collected; 8 times the amount of water was added to the residue and decocted for 1h, the two filtrates were combined after removing the residue, concentrated at 80°C, and dried under reduced pressure to obtain dry extract powder for standby use.

[0057] 1.3 GES-1 cell recovery, culture and passaging

[0058] The human gastric epithelial cell line GES-1 is an immortalized cell. The revived GES-1 cells were cultured in complete medium (containing 10% FBS + 1% P / S double antibody + 89% RPMI-1640 medium) and placed at 37°C and 5% CO 2 Culture in a saturated humidity constant temperature incubator for 24 hours. When the cells grow well and the cell monolayer covers 80% to 90% of the culture flask wall, wash the cells with PBS buffer, add 1mL of 0.25% trypsin solution to each T25 culture flask to digest the cells, and add 3mL of complete culture medium after 2-3 minutes to terminate the digestion. Centrifuge at 1000r / min for 5 minutes, discard the supernatant, add 1mL of complete culture medium to resuspend the cells, subculture 2 to 3 times a week, and perform cell subculture, cryopreservation, and plate seeding operations.

[0059] 1.4 Establishment of GES-1 cell injury model

[0060] GES-1 cells were placed at 37°C with 5% CO2 Incubate in a saturated humidity constant temperature incubator until the cells grow well and then plate them. 4 Cells were plated in 96-well plates and incubated in an incubator for 24 hours. After the cells adhered to the wall, the culture medium was aspirated and washed twice with PBS. The GES-1 cells were treated with culture medium containing 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, and 9% ethanol concentrations for 3, 4, and 5 hours, respectively. The degree of GES-1 cell damage was observed and the optimal ethanol concentration and action time for modeling were screened. The experimental parameters when the inhibition rate of GES-1 cells detected by MTT method reached about 40% can be used as the optimal GES-1 cell injury model. Subsequent experiments established the GES-1 cell injury model with the optimal ethanol concentration and action time for modeling.

[0061] Effect of 1.5KLF on GES-1 cell viability

[0062] GES-1 cells were revived and placed at 37°C and 5% CO 2 Culture in a saturated humidity incubator until the cells grow well. 4 GES-1 cells were seeded in 96-well plates and placed in a 37°C, 5% CO 2 After the cells adhered to the wall, the culture medium was aspirated and washed twice with PBS. Complete culture medium was added to the normal group GES-1 cells, and 100 μL of culture medium containing different concentrations of KLF (0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 μg / mL) was added to the other groups. Five replicate wells were set up in each group and placed at 37°C, 5% CO 2 Continue to culture in a saturated humidity incubator for 24 hours. Then add 20 μL of 5% MTT solution and protect from light for 4 hours. Aspirate the culture medium, add 100 μL of DMSO solution to each well, shake and dissolve for 10 minutes, then use a microplate reader to detect the absorbance value (OD value), the detection wavelength is 490nm, calculate the cell survival rate, and repeat the experiment 3 times. Screen high, medium and low concentrations of the drug within the concentration range that is non-toxic to cells. Cell survival rate (%) = (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%.

[0063] Effect of 1.6KLF on the survival rate of GES-1 cells damaged by ethanol

[0064] 1.6.1 Cell Grouping

[0065] Take GES-1 cells with good growth status and add 100 μL and 1×10 4Cells were seeded into each well of a 96-well plate and placed at 37°C with 5% CO 2 The cells were cultured in a saturated humidity incubator for 24 h. The cells were divided into Control group, Model group, KLF-L group, KLF-M group, and KLF-H group, with 5 replicate wells in each group.

[0066] 1.6.2 Cell modeling and drug administration

[0067] After the cells adhered to the wall, complete medium was added to the GES-1 cells in the control group, and ethanol with the best modeling concentration and action time was added to the other experimental groups. The cells were placed at 37°C and 5% CO. 2 After incubation in a saturated humidity incubator for a certain period of time, the supernatant was aspirated and washed twice with PBS. After modeling, 100 μL of drug-free culture medium was added to the control group and the model group, and 100 μL of drug-containing culture medium of KLF-L, KLF-M, and KLF-H (100, 200, and 400 μg / mL, respectively, screened by cell activity experiments) was added to the drug-treated group, and the cells were placed in an incubator at 37°C and 5% CO. 2 The cells were cultured in a saturated humidity incubator for 24 h. The activity of GES-1 cells was determined by MTT method, and the cell survival rate was calculated. The above experiment was repeated 3 times.

[0068] 1.7 Cell scratch

[0069] GES-1 cells were seeded into six-well plates (5.0×10 5 cells) and placed at 37°C, 5% CO 2 Cultured in a saturated humidity incubator until cell fusion reached about 90%, a 200μL pipette tip was used to scratch from top to bottom to form a wound, and the GES-1 cells were washed three times with PBS. The Control group was added with RPMI-1640 medium. Except for the Control group, the other experimental groups replicated the GES-1 cell injury model according to method 1.6.2. After modeling, the Model group was added with RPMI-1640 medium, and the drug-containing culture medium of KLF-L, KLF-M, and KLF-H (100, 200, and 400μg / mL, respectively, screened by cell activity experiments) was added to the drug-containing culture medium of the drug-treated group. Images were taken at 0h and 24h using an inverted microscope (magnification: 100×) to observe the degree of cell healing. The blank part of the scratch area was evaluated using Image J software to calculate the degree of cell scratch healing. The above experiment was repeated 3 times. The scratch healing rate was calculated using the following formula:

[0070] Scratch healing rate (%) = (0-24h blank area / 0h blank area) × 100%

[0071] 1.8 Cell ELISE assay

[0072] GES-1 cells with good growth status were seeded in 6-well plates (5.0×10 5 cells) and placed at 37°C, 5% CO 2 The cells were cultured in a saturated humidity incubator for 24 h. Except for the Control group, the GES-1 cell injury model was replicated in the other experimental groups according to the method in 1.6.2. After modeling, RPMI-1640 medium was added to the Control group and the Model group, and 2 mL of KLF-L, KLF-M, and KLF-H drug-containing medium was added to each experimental group and placed at 37°C and 5% CO 2 Continue to culture in a saturated humidity incubator for 24 hours. Collect the cell supernatant, centrifuge at 3000r / min for 10 minutes, transfer the supernatant to a sterile centrifuge tube, and store in a -20℃ refrigerator. Determine the levels of IL-8, IL-6, and TNF-α in the cell supernatant according to the instructions of each kit.

[0073] 1.9 Statistical analysis

[0074] Statistical analysis was performed using IBM SPSS Statistics 26.0. All data were normally distributed after the Shapiro-Wilk test. One-way analysis of variance was used for comparison between groups, followed by Tukey's post hoc test. Results were visualized using GraphPadPrism8.3.0. A significance level of p < 0.05 was considered statistically significant.

[0075] 2 Experimental results

[0076] 2.1 Establishment of GES-1 cell injury model

[0077] The results are as follows Figure 1 As shown in Figures AC, the modeling of ten groups of ethanol concentrations from 0 to 9% and three time points from 3 to 5 hours caused different degrees of damage to GES-1 cells. Under an optical microscope, it can be seen that the cell connections are reduced, the gaps are enlarged, and the cells are flaky and fall off at some concentrations. The cells shrink and become smaller, and are oval or round. In this process, GES-1 cells can undergo nuclear condensation and gradually increase the number of dead cells. After GES-1 cells were treated with 5% ethanol concentration for 4 hours, the cell inhibition rate reached 40%, so it was determined that 5% ethanol incubation for 4 hours was the best modeling condition for the GES-1 cell injury model.

[0078] 2.2 Effects of different concentrations of KLF on the survival rate of GES-1 cells

[0079] Effects of different concentrations of KLF on the activity of normal GES-1 cells Figure 1As shown in Figure D, within the KLF concentration range of 0-500 μg / mL, the cell activity of GES-1 cells showed a certain proliferation trend with the increase of concentration, that is, with the increase of drug concentration, KLF showed a certain promoting effect on the cell activity of GES-1 cells, but there was no statistical significance. Therefore, this experiment set up three groups of drug administration doses of KLF-L, KLF-M, and KLF-H, with doses of 100, 200, and 400 μg / mL respectively.

[0080] 2.3 Effect of KLF on the survival rate of GES-1 cells treated with ethanol

[0081] The results are as follows Figure 1 As shown in E, compared with the Control group, the survival rate of GES-1 cells in the Model group was significantly decreased (p<0.01); compared with the Model group, the cell survival rates in the KLF-L, KLF-M, and KLF-H groups were significantly increased (p<0.01), among which the maximum cell viability was observed when the KLF concentration was 400 μg / mL, indicating that KLF has a good protective effect on GES-1 cell damage induced by 5% ethanol.

[0082] Effect of 2.4KLF on the inflammatory level of ethanol-induced GES-1 cell injury model

[0083] ELISA kits were used to measure the changes in the levels of inflammatory factors in GES-1 cells. Figure 1 FH showed that compared with the Control group, 5% ethanol could significantly increase the expression levels of proinflammatory cytokines IL-8, IL-6, and TNF-α in GES-1 cells (p<0.01). Compared with the Model group, KLF-M and KLF-H treatment could significantly inhibit the expression levels of proinflammatory cytokines IL-8, IL-6, and TNF-α in GES-1 cells (p<0.01). The results showed that KLF could repair ethanol-induced GES-1 cell damage through anti-inflammatory effects.

[0084] Effects of 2.5KLF on wound healing and migration of ethanol-treated GES-1 cells

[0085] The wound healing migration assay was used to evaluate the in vitro repair of gastric mucosa, and the results were as follows Figure 1 IJ. Compared with the Control group, the wound healing migration ability of cells in the Model group was significantly reduced (p<0.01). Different doses of KLF treatment enhanced wound healing migration in a dose-dependent manner, among which cells treated with KLF-M and KLF-H had stronger wound healing migration ability (p<0.01). The results showed that KLF can reduce the damage caused by ethanol to GES-1 cells.

[0086] The above experiment determined that Kouchiliuxiangfang (KLF) has a protective effect on ethanol-induced gastric mucosal injury of GES-1 cells through the effects of KLF on the wound healing migration of GES-1 cells and the levels of inflammatory factors TNF-α, IL-8, and IL-6 in the supernatant of GES-1 cells.

[0087] Experimental Example 2 The therapeutic effect of the Kou Chi Liu Xiang Fang (KLF) of the present invention on bad breath caused by stomach heat syndrome in rats

[0088] 1 Materials and methods

[0089] 1.1 Experimental animals and breeding environment

[0090] 72 SPF male SD rats weighing 180-220g were purchased from Chengdu Dashuo Co., Ltd. They were kept in the animal room of the School of Pharmacy of Shaanxi University of Chinese Medicine, with an indoor temperature of (22±2)℃, a relative humidity of 50%-60%, good ventilation, and a light-on and dark-off period of 12h. The rats were kept in separate cages and given sufficient clean feed and water. They were used in the experiment after 7 days of adaptive feeding.

[0091] 1.2 Experimental Reagents

[0092] Dried ginger, 4% paraformaldehyde, extract of Kou Chi Liu Xiang Fang (KLF), Zuo Jin Wan (20230503), glutathione (GSH, A006-2-1), malondialdehyde (MDA, A003-1-2), superoxide dismutase (SOD, A001-3-2), interleukin-8 (IL-8, MM-057001), interleukin-6 (IL-6, MM-0190R1), tumor necrosis factor (TNF-α, MM-0180R1), thromboxane B2 (TXB2, MM-0516R1), 6-keto-PGF 1α , MM-0268R1), gastrin (GAS, MM-2128R1) kit.

[0093] 1.3 Extraction of drug solution

[0094] 1.3.1 Preparation of dried ginger decoction

[0095] Take 1 kg of dried ginger, add 10 times the amount of water and soak for 30 minutes. After boiling, continue to decoct for 30 minutes, pour out the supernatant, filter, add 8 times the amount of water and continue to decoct. After boiling, continue to decoct for 30 minutes, filter, combine the two filtrates, and the concentration of the concentrated medicinal solution is 1g crude drug / mL. Store in the refrigerator for later use.

[0096] 1.3.2 Preparation of KLF decoction

[0097] According to the formula ratio of Example 1, a total of 500g of Schisandra chinensis, Malus melongena, honeysuckle, dandelion and clove were weighed, mixed and crushed into coarse powder, added with 10 times the amount of water, soaked for 2h, decocted for 1h, filtered with double gauze and the filtrate was collected; 8 times the amount of water was added to the residue and continued to be decocted for 1h, the two filtrates were combined after removing the residue, concentrated at 80°C, and dried under reduced pressure to obtain dry extract powder for standby use.

[0098] 1.4 Establishment of rat gastric heat syndrome model and drug administration

[0099] 72 rats were randomly divided into Control group, Model group, Positive group (Zuojinwan group), KLF-L, KLF-M, and KLF-H group, with 12 rats in each group. Except for the Control group, the other groups were gavaged twice a day with 1kg / L of dried ginger decoction at a dose of 5mL / kg for modeling, and 1mL of the solution was injected into the rat's mouth after gavage to give taste stimulation, with an interval of 4h between each gavage for 14 consecutive days. The clinical dosage of KLF is calculated based on a body weight of 60kg. According to the body surface area method, the equivalent dose of KLF in rats is 4g / kg as the medium-dose group. The high-dose concentration is doubled to 8g / kg, and the low-dose concentration is reduced by half to 2g / kg. The administration volume is 10mL / kg body weight. After the modeling, the drug administration group was given the corresponding drug by gavage, and the Control group and Model group were given an equal volume of normal saline by gavage once a day for 14 consecutive days.

[0100] 1.5 Sample collection

[0101] 1.5.1 Collection of serum samples

[0102] After the rats were anesthetized, the abdomen was opened longitudinally and blood was collected from the abdominal aorta of the rats in each group. About 4-5 mL of arterial blood was taken from each rat and placed in a test tube without anticoagulant. After standing at room temperature for 1 hour, the blood was centrifuged at 3000 r / min for 10 minutes. After centrifugation, the supernatant was aspirated with a pipette and divided into 1.5 mL EP tubes, which were numbered one by one and stored in a -20°C refrigerator for subsequent determination and analysis of related indicators.

[0103] 1.5.2 Collection of gastric tissue

[0104] After blood collection from the rats, the stomach was cut open along the greater curvature with surgical scissors after disinfection and sterilization on a sterile operating table, the inner side of the stomach was turned out, and the stomach was placed in a 0.9% sodium chloride solution for cleaning. After the contents in the stomach were cleaned, the gastric mucosal tissue was observed with the naked eye to evaluate the damage of the gastric mucosal tissue, and then part of the gastric mucosal tissue (size: 1.0 cm×0.5 cm×1.0 cm) from the damaged part of the gastric antrum was cut and immersed in 4% paraformaldehyde for 24-48 h for fixation for subsequent pathological detection. The remaining tissue was placed in a plastic bag and stored in a -80°C refrigerator for subsequent analysis.

[0105] 1.6 Detection indicators

[0106] 1.6.1 Observation of rat physical signs and general behavior

[0107] During the experiment, the following physical signs of rats in each group were observed and recorded every day:

[0108] (1) Activity status, hair, gums, auricle, front and rear paws.

[0109] (2) Color and nature of urine and feces.

[0110] (3) Body weight, food intake, and water intake.

[0111] 1.6.2 Morphology of gastric mucosa in rats

[0112] 1.6.2.1 Gastric mucosal damage

[0113] After the gastric mucosal tissue was rinsed with ice saline, the water was absorbed with filter paper, and it was placed on clean filter paper and photographed with a mobile phone to observe the damage of the gastric mucosal surface of the rat with the naked eye.

[0114] 1.6.2.2 Gastric mucosal pathology examination

[0115] After the gastric mucosal tissue was removed, it was fixed in 4% formaldehyde solution for more than 24-48 hours, dehydrated by gradient ethanol, transparentized twice in xylene, embedded in wax and then sectioned, and stained with H&E after dewaxing. The pathological changes of gastric tissue of rats in each group were observed under an optical microscope and photographed.

[0116] 1.6.3 Determination of oxidative stress indicators in rat serum

[0117] Glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) were determined using GSH, MDA, and SOD kits, respectively, and all operating steps were carried out strictly in accordance with the manufacturer's instructions.

[0118] 1.6.4 Determination of serum inflammatory factor levels in rats

[0119] Tumor necrosis factor (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8) were determined using TNF-α, IL-6, and IL-8 kits, respectively, and the determination was performed strictly in accordance with the instructions of the ELISA kits.

[0120] 1.6.5 Determination of factors related to gastric mucosal repair in rat serum

[0121] Gastrin (GAS), thromboxane B2 (TXB2), 6-keto-PGF 1α ) using GAS, TXB2, 6-keto-PGF 1α The assay was performed strictly in accordance with the ELISA kit instructions.

[0122] 1.7 Statistical analysis

[0123] Statistical analysis was performed using IBM SPSS Statistics 26.0. All data were normally distributed after the Shapiro-Wilk test. One-way analysis of variance was used for comparison between groups, followed by Tukey's post hoc test. Results were visualized using GraphPadPrism8.3.0. A significance level of p < 0.05 was considered statistically significant.

[0124] 2 Experimental Results

[0125] 2.1 Physical changes of rats

[0126] 2.1.1 Appearance changes of rats

[0127] The evaluation criteria for the success of model establishment were based on the diagnostic criteria for stomach heat syndrome in the "Guidelines for Clinical Research of New Chinese Medicines". Figure 2 As shown in the figure, when the rats were modeled with stomach heat syndrome using dried ginger decoction, the rats' claws and auricles turned red. A closer look showed that the auricles were densely covered with blood vessels, which were obvious and thick, and the gums were red and swollen. The rats' temperament became more irritable day by day, and the difficulty of gavage increased significantly after one week of gavage, and the struggles and screams were more intense. The feces of the modeled rats were significantly reduced, and the feces were mostly dry and hard, which was significantly different from the Control group.

[0128] After successful modeling, each drug-administered group was gavaged according to the dosage, and the Control and Model groups were gavaged with equal volumes of normal saline. The rats in the Control group had normal diet and water intake, good mental state, normal activities, and soft feces during the experiment. Compared with the Model group, the symptoms of stomach heat syndrome gradually eased in each drug-administered group from the beginning of drug administration to the end of gavage, among which the KLF-H group and the Positive group had the most obvious improvement.

[0129] 2.1.2 Changes in rats’ food intake, water intake, and urine

[0130] The results are shown in Table 1. After the modeling of the rats with dried ginger decoction, the amount of water and food consumed by the rats showed a trend of continuous increase compared with that before the modeling, the amount of urine showed a trend of obvious decrease, and the color of the urine turned yellow (the amount of food and water consumed were both in 24h units, and the observation was carried out for 3 consecutive days before, after and after the modeling, and then the average value was taken as the measured value of each index). The amount of water consumed (p<0.05), the amount of feces (p<0.01) and the color of urine in the Model group were significantly different from those in the Control group. Compared with the Model group, after drug intervention, the amount of food consumed by the rats in each drug-treated group showed a trend of decreasing, the feces showed a trend of obvious softening, and the feces of the Positive group and the KLF-H group showed a significant increase (p<0.01). The color of urine in each drug-treated group will be one level lower than that of the Model group.

[0131] Table 1 Changes in rat diet, water intake, feces and urine ( n=12)

[0132]

[0133] Note: ## p<0.01 compared with the control group, * p<0.05, ** p<0.01 compared with the model group.

[0134] 2.2 Effect of KLF on gastric mucosal injury in rats with gastric heat syndrome

[0135] 2.2.1 Apparent state of gastric mucosa

[0136] The results are as follows Figure 3 As shown in A, the gastric tissue structure of rats in the Control group was intact, the gastric mucosal tissue was light red, and covered with more mucus, the mucosal surface was intact, and there were no abnormal phenomena such as ulcers, edema and congestion. In the Model group, the gastric mucosal tissue was red, and oval ulcers were visible, which were concave in the middle and bulged around. The bottom of the ulcer was flat and covered with light white moss, and the surface mucus secretion was less, and there was no perforation, indicating that the gastric heat syndrome model of rats was successfully established. After administration, the redness of the gastric mucosal tissue of the rats was relieved, and the gastric mucosa in the KLF-L group had mild erosion; the gastric mucosal ulcer area in the KLF-M group was reduced, and a small number of congestion points were visible; the surface mucus secretion of the gastric mucosa in the KLF-H group increased, and the congestion was significantly relieved. The healing degree of some gastric mucosal tissues was good; the healing degree of the Positive group was relatively obvious; among them, the improvement of the gastric mucosal tissue in the KLF-H and Positive groups was the most obvious.

[0137] 2.2.2 Histopathological observation

[0138] By observing the gastric mucosal pathological tissue sections, the changes in the pathological state of the gastric mucosal tissue can be intuitively displayed. The pathological observation results of the gastric mucosal tissue of each group of rats are as follows: Figure 3 As shown in B, the gastric mucosal epithelial cells of rats in the Control group were arranged neatly and in a single-layer columnar shape, with basically no defects or shedding, the fundic glands were regular in shape, the cell density of the lamina propria was normal, there was no inflammatory cell infiltration in the lamina propria of the mucosa, and there was no significant change in the number of chief cells and parietal cells. The gastric mucosa of rats in the Model group showed obvious congestion and edema, less surface mucus secretion, neutrophils and large monocytes were infiltrated in the mucus lamina propria, and neutrophils were the main ones. The epithelial cells were damaged or lost to varying degrees, some glands were irregular in shape, and the number of chief cells and parietal cells was significantly reduced. The gastric mucosal epithelial cells of rats in the KLF-H group were intact, basically without congestion and edema, the mucus secretion increased, no neutrophil infiltration was observed, and the number of chief cells and parietal cells increased significantly. The gastric mucosa of rats in the KLF-L and KLF-M groups also improved after treatment, but the degree was not as significant as that of the KLF-H treatment group.

[0139] 2.3 Effect of KLF on oxidative stress in rats with gastric heat syndrome

[0140] The results are as follows Figure 3 As shown in CE, compared with the Control group, the activities of SOD and GSH in the serum of rats in the Model group were significantly reduced (p<0.01), and the MDA content was significantly increased (p<0.01). Compared with the Model group, the levels of GSH and SOD in the serum of each drug-treated group increased to varying degrees, the levels of SOD and GSH in the KLF-H and Positive groups increased significantly (p<0.01), the GSH level of KLF-M increased to a certain extent (p<0.05), and the MDA content of each drug-treated group decreased to varying degrees, and the MDA levels of the KLF-M, KLF-H and Positive groups decreased significantly (p<0.01), indicating that KLF can reduce the gastric mucosal damage of rats caused by dried ginger decoction through anti-oxidation.

[0141] 2.4 Effect of KLF on the levels of inflammatory factors in rats with gastric heat syndrome

[0142] like Figure 3 As shown in FH, compared with the Contro group, the levels of proinflammatory cytokines IL-6, IL-8, and TNF-α in the serum of the Model group were significantly increased (p<0.01). Compared with the Model group, the levels of proinflammatory cytokines IL-6, IL-8, and TNF-α in the serum of the KLF-M, KLF-H, and Positive groups were significantly decreased (p<0.01), indicating that KLF can alleviate the gastric mucosal damage of rats caused by dried ginger decoction by inhibiting inflammation.

[0143] Effects of 2.5KLF on factors related to gastric mucosal repair in rats with gastric heat syndrome

[0144] like Figure 3 As shown in IL, compared with the Control group, the levels of GAS and TXB2 in the serum of rats in the Model group were significantly increased (p<0.01), and 6-keto-PGF 1α Compared with the Model group, the levels of GAS and TXB2 in each medication group decreased to varying degrees. The levels of GAS and TXB2 in the KLF-H group and the Positive group were significantly different from those in the Model group (p<0.01), and the levels of GAS in the KLF-M group were significantly different from those in the Model group (p<0.05). 1α The levels of 6-keto-PGF increased to varying degrees in the KLF-H group and the Positive group. 1α Compared with the Control group, the level of TXB2 / 6-keto-PGF in the Model group was significantly increased (p<0.01). 1α The ratio was significantly increased (p<0.01). Compared with the Model group, the TXB2 / 6-keto-PGF 1α The ratio was significantly decreased (p<0.01), indicating that KLF can alleviate the gastric mucosal injury of rats caused by dried ginger decoction by regulating the expression of gastric mucosal repair factors.

[0145] The above experiment confirmed that KLF has a protective effect on the gastric mucosa of rats with gastric heat syndrome by treating them with Kou Chi Liu Xiang Fang (KLF) to examine the changes in the external signs of rats, the changes in the amount of food, water, feces and urine, the pathological changes in the gastric mucosa of rats, the changes in oxidative stress indicators (GSH, SOD, MDA), inflammatory factor levels (TNF-α, IL-8, IL-6), and gastric mucosal repair-related factors (GAS, TXB2, 6-keto-PGF1α) in the body's serum. In general, food accumulation can cause gastric heat, which can lead to gastric mucosal damage, which in turn can aggravate the symptoms of gastric heat and food accumulation. If food stays in the gastrointestinal tract for too long, it can cause bad breath and foul breath. KLF can play a positive therapeutic role in treating bad breath by improving the changes in physical signs caused by gastric heat, gastric mucosal damage, and its antioxidant and anti-inflammatory effects.

[0146] Experimental Example 3 Effect of Kouchiliuxiang Recipe on Intestinal Flora of Rats with Stomach-heat Syndrome

[0147] 1 Materials and methods

[0148] 1.1 Materials

[0149] 1.5mL sterile centrifuge tube, tweezers, PF Mag-Bind Stool DNA Kit extraction kit (Omega Bio-tek, Georgia, USA); FastPfu Polymerase (Beijing Quanshijin Biotechnology Co., Ltd.); ABI Gene 9700 PCR instrument (ABI, USA); DYY-6C electrophoresis instrument (Beijing Liuyi Biotechnology Co., Ltd.). High-throughput sequencing was completed by Shanghai Meiji Biotechnology Co., Ltd.

[0150] 1.2 Collection of feces

[0151] The rats in the Control, Model and KLF-H groups in Experimental Example 2 were divided into cages, with one rat in each cage. After the rat defecated, the feces were quickly taken into a cryopreservation tube with sterile tweezers and quickly frozen in liquid nitrogen. After all the feces of the rats were collected, all samples were placed in a -80°C refrigerator for future use.

[0152] 1.3 DNA extraction

[0153] Total microbial genomic DNA was extracted from rat fecal samples using the PF Mag-Bind Stool DNA Kit (Omega Bio-tek, Georgia, USA) according to the manufacturer's instructions. The quality and concentration of DNA were determined by ND-2000 spectrophotometer (Thermo Scientific Inc., USA) and stored at -80°C for further use. Primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) were used to amplify the V3-V4 hypervariable region of bacterial 16S rRNA gene. 4 μL 5×Fast Pfu buffer, 2 μL 2.5 mM dntp, 0.8 μL of each 5 μM primer, 0.4 μL Fast Pfu polymerase, 10 ng template DNA and ddH 2 O PCR reaction mixture, the final volume was 20 μL. PCR amplification cycle conditions were: 95 ° C initial denaturation for 3 min, 95 ° C denaturation for 30 s, 55 ° C annealing for 30 s, 72 ° C extension for 45 s, 72 ° C single extension for 10 min, 27 cycles, 4 ° C termination. All samples were amplified 3 times. PCR products were extracted and purified using 2% agarose gel. Then Quantus TM The quantification was performed using a fluorometer (Promega, USA).

[0154] 1.4 Illumina MiSeq sequencing

[0155] The sequencing library was generated by the TruSeqTM DNA Sample Prep Kit for Illumina, the corresponding index code was added, and the library was sequenced on the Illumina platform. The PEreads obtained by sequencing were quality controlled and filtered, and the sequence noise reduction method (DADA2 / Deblur, etc.) was further used to process and optimize the data, and the ASV (Amplicon Sequence Variant) representative sequence and abundance information were obtained to perform bacterial diversity, species difference analysis, and correlation analysis. The differential properties of bacteria were identified based on the linear discriminant analysis effect size (LEfSe) analysis.

[0156] 1.5 Statistical analysis

[0157] All data analyses were performed on the Majorbio Cloud Platform (https: / / cloud.majorbio.com), as follows: mothur software (http: / / www.mothur.org / wiki / Calculators, version 1.30.2) was used to calculate the alpha diversity index Chao index, Shannon index, etc., and the Wilcoxon rank sum test was used to analyze the inter-group differences in alpha diversity. PCoA analysis (principal coordinate analysis) based on the bray-curtis distance algorithm was used to test the similarity of the microbial community structure between samples, and the Adonis non-parametric test was combined to analyze whether the differences in the microbial community structure between sample groups were significant. The types and relative abundances of microorganisms in the Control, Model, and KLF-H groups were displayed by community bar and pie plots, and the changing trends of species in different groups can be intuitively seen to reflect the similarity and difference relationship of the community structure of different groups, and the inter-group difference test method was further used to evaluate the species information with significant differences between multiple groups. Finally, PICR USt2 functional prediction was used to understand the functions of the microbial communities in the samples, and possible metabolic pathways (KEGG, Meta Cyc) and orthologous groups (COG) and other information were predicted. A heat map was used to display the distribution of KEGG functional abundance in different samples, intuitively showing the distribution of the main dominant functions in different samples.

[0158] 2 Experimental results

[0159] 2.1 Analysis of ASV distribution in rat intestinal flora by KLF

[0160] 16S rRNA high-throughput sequencing technology was used to determine the composition and community structure of the intestinal flora. Fecal samples of rats in the Control, Model, and KLF-H groups were analyzed using the Illumina platform. A total of 3,109,422 reads were obtained, and the amplified fragment was 468 bp. After quality control screening and noise reduction, the data was flattened according to the minimum number of sequences, and 30,201 reads were obtained. Using the Venn diagram, the common and unique ASVs between groups can be intuitively understood. The results are shown in Figure 2. Figure 4 As shown in A, all groups shared 333 of the total richness of 6253 ASVs, among which the number of species in the Model group increased significantly, and the number of species in the KLF-H group decreased and was similar to that in the Control group.

[0161] 2.2 Effect of KLF on Alpha Diversity of Rat Intestinal Microbiota

[0162] The dilution curve can directly reflect the rationality of the sequencing data volume. As the sample sequencing volume increases, the dilution curve reaches a platform and tends to saturation, and the number of species no longer increases linearly or rapidly, indicating that the sequencing data volume is reasonable ( Figure 4 BC). The slopes and rank sum richness of the three groups showed consistent trends, indicating that the sequencing depth was sufficient to reflect the microbial diversity of the community. Alpha diversity is mainly used to study the community diversity in samples. By evaluating a series of Alpha diversity indices, information such as the richness and diversity of species in the environmental community can be obtained. The Chao index is used to reflect the community richness at the species ASV level, and the Shannon index is used to reflect the degree of difference in community diversity at the species ASV level. The results are shown in Figure 4 DE shows that compared with the Control group, the Chao and Shannon indexes of the Model group increased significantly (p<0.01), and compared with the Model group, the Chao (p<0.05) and Shannon indexes of the KLF-H group decreased, and the Chao and Shannon indexes of the KLF-H group tended to decrease towards the Control group. In summary, the decoction of dried ginger can cause intestinal flora disturbance in rats, resulting in an increase in the richness and diversity of intestinal flora in the Model group rats. KLF treatment can regulate the intestinal flora disturbance caused by the decoction of dried ginger and restore the intestinal flora of rats to a balanced state.

[0163] 2.3 Effect of KLF on Beta diversity of intestinal flora in rats

[0164] Beta diversity analysis further revealed the effects of dried ginger decoction and KLF treatment on the richness and diversity of rat intestinal microbiota. PCoA analysis (principal co-ordinates analysis) and NMDS non-metric multidimensional scaling analysis are often used to reflect the Beta diversity of intestinal microbial flora between different groups. The PCoA distribution results of different groups are shown in Figure 2. Figure 4 As shown in F, the Model group was significantly separated from the Control group. After KLF treatment, the KLF-H group was significantly separated from the Model group and tended to shift toward the Cotrol group. NMDS analysis also showed similar results, such as Figure 4 As shown in G, the Model group was significantly separated from the Control group, and the intestinal microorganisms of the samples of KLF-H after KLF treatment were significantly separated from the Model group, and were close to those of the Control group. The above results indicate that the dried ginger decoction has a significant effect on the intestinal flora of rats, and KLF can improve the disorder of intestinal flora microorganisms in rats with stomach heat syndrome and tend to be close to the Control group.

[0165] Effects of 2.4KLF on intestinal flora species in rats

[0166] In order to further analyze the effect of KLF on the composition and structure of rat intestinal flora, the relative abundance of intestinal flora at different levels was statistically analyzed. Figure 5 As shown in AB, different ecosystems were dominated by two major phyla, Firmicutes and Bacteroidetes, accounting for more than 93%. Compared with the Control group, the level of Firmicutes in the Model group increased, and the level of Bacteroidetes decreased. After KL FH treatment, the level of Firmicutes decreased, and the level of Bacteroidetes increased. At the genus level, the relative abundance of Lactobacillus, norank_f__Muribaculaceae, and Bifidobacterium in the Model group was lower than that in the Control group, and the relative abundance of norank_f__norank_o__Clostridia_UCG-014 and unclassified_f__Lachnospiraceae was higher than that in the Control group. After KLF-H treatment, the relative abundance of Lactobacillus, norank_f__Muribaculaceae, and Bifidobacterium increased, and the relative abundance of norank_f__norank_o__Clostridia_UCG-014 and unclassified_f__Lachnospiraceae decreased. The relative abundances of Lactobacillus in the Control group, Model group and MH-H group were 57%, 11% and 31%, respectively, and the relative abundances of Bifidobacterium were 36%, 2% and 62%, respectively.

[0167] The bar graphs further show the differences in intestinal flora at the phylum and genus levels. Figure 5 CD shows that at the phylum level, KLF-H treatment can significantly reduce the abundance of Proteobacteria, Cyanobacteria and Verrucomicrobia (p<0.05). At the genus level, KLF-H treatment can significantly increase the abundance of Lactobacillus (p<0.01) and Bifidobacterium (p<0.05), and significantly reduce the abundance of norank_f__norank_o__Clostridia_UCG-014 (p<0.05), Ruminococcus (p<0.01), NK4A214_group (p<0.01), etc. The above results indicate that the stomach heat syndrome induced by dried ginger decoction leads to an increase in harmful bacteria in the intestinal flora of rats. KLF-H treatment induces a potentially beneficial intestinal microbial response, reduces the relative abundance of harmful bacteria in the intestinal flora, and increases the relative abundance of probiotics, which promotes the intestinal flora to return to normal.

[0168] 2.5 PICRUSt2 function prediction

[0169] PICRUSt2 function prediction is used to predict the functional information of microbial communities in different samples, and to further understand some potential microbial functional characteristics in the disease process through functional composition and abundance. PICRUST2 can predict information such as metabolic pathways (KEGG, MetaCyc) and orthologous groups (COG) in the disease process. Functional prediction of the intestinal flora of the Control, Model and KLF-H groups was performed, and the KEGG function heat map at level 3 was drawn, and clustering was performed according to the degree of functional difference. The results are shown in Figure 2. Figure 6 As shown, after modeling with dried ginger decoction, the PI3K-Akt signaling pathway, phenylalanine, tyrosine and tryptophan biosynthesis, 2-oxocarboxylic acid metabolism, and bile metabolism pathways showed a significant upward trend. After KLF treatment, the above pathways in the KLF-H group showed an opposite trend, indicating that KLF may play a role in treating gastric heat syndrome halitosis by regulating intestinal microbiota with different functions and abundances.

[0170] The above experiment collected rat feces for 16S rRNA sequencing to observe the changes in intestinal microbial abundance and diversity after dry ginger modeling and KLF administration, thereby determining that Kouchi Liuxiang Fang (KLF) has the function of regulating the balance of intestinal microecology, improving stomach heat symptoms by maintaining the balance of intestinal microecology, and thus achieving the effect of freshening breath.

[0171] Experimental Example 4 Effect of Kou Chi Liu Xiang Fang on Oral Microorganisms Causing Bad Breath

[0172] There are at least 80 kinds of microorganisms in the oral cavity that can produce sulfide during metabolism and cause bad breath. These microorganisms mainly include Fusobacterium subspnucleatum (Fn) and Porphyromonas gingivalis (Pg). Therefore, this experiment measured the minimum inhibitory concentration of the single herb of Kou Chi Liu Xiang Fang and the compound (prepared according to Example 1) against Fusobacterium subspnucleatum and Porphyromonas gingivalis to evaluate its effect in removing bad breath.

[0173] 1 Inspection basis

[0174] According to the antibacterial test method in 2.1.8 of "Technical Specifications for Disinfection (2022)", the minimum inhibitory concentration (MIC) of Fn and Pg was determined for the Kouchiliuxiang recipe and single herbs: Schisandra chinensis, Malus malus, honeysuckle, dandelion, and clove.

[0175] 2. Strain information: Test strains: Fusobacterium nucleatum (CMCC1.1113), Porphyromonas gingivalis (ATCC33277)

[0176] Preparation of bacterial suspension: Aseptically open the strain preservation tube in a biosafety cabinet, use a sterile pipette to absorb an appropriate amount of nutrient broth, blow and suck several times to melt and disperse the strain. Take a test tube containing 5.0mL~10.0mL nutrient broth medium, drop a small amount of bacterial suspension, and culture at 36℃ for 18h~24h. Use a sterile inoculation loop to take the bacterial suspension of the first generation culture, streak inoculate on the nutrient agar medium plate, and culture at 36℃ for 18h~24h. Pick the typical colonies in the above second generation culture, inoculate on the nutrient agar slant, and culture at 36℃ for 18h~24h. This is the third generation culture.

[0177] Take the 3rd to 14th generation culture, use a 5.0mL sterile pipette to draw 3.0mL to 5.0mL of diluent into the slant test tube, blow and draw repeatedly to wash off the bacterial moss. Then use a 5.0mL sterile pipette to transfer the washing liquid to another sterile test tube, mix well with a mixer, or tap on the palm of your hand 80 times to make the bacterial suspension uniform.

[0178] The bacterial suspension prepared initially should first be roughly measured for its bacterial concentration using the bacterial concentration turbidimetric method, and then diluted with diluent to the desired concentration (the bacterial suspension should be stored in a 4°C refrigerator for future use and should not be used overnight on the same day).

[0179] 3. Reagent Information

[0180] Nutrient broth medium, nutrient agar medium, PBS diluent (phosphate buffer) 0.03 mol / L: 2.83 g of anhydrous disodium hydrogen phosphate, 1.36 g of potassium dihydrogen phosphate, add distilled water to 1000 mL, adjust the pH to 7.2-7.4, sterilize at 121°C for 20 min for use.

[0181] 4 Equipment information: incubator (36°C); high pressure steam sterilizer.

[0182] 5 Method steps

[0183] 5.1 Preparation of bacterial suspension: Before the experiment, the working strain was streaked onto an agar slant and cultured at 36°C for 24 h. The cultured test bacterial slant was washed with PBS and prepared into 5×10 5 CFU / mL~5×10 6 CFU / mL bacterial suspension is ready for use.

[0184] 5.2 Sample preparation: Weigh 1.0 g (accurate to 0.0001 g) and add it to 9.0 mL of sterile water to dissolve and prepare a sample diluent.

[0185] 5.3 Detection method:

[0186] (1) Preparation of culture medium containing antimicrobial agent: dilute the antibacterial solution with distilled water in equal multiples to prepare test solutions of different concentrations. Take 2.5 mL of the test solution of each dilution and add it into a test tube containing 2.5 mL of double-concentration nutrient broth.

[0187] (2) Take 0.1mL of the sample and the bacterial content is 10 8 The CFU / mL bacterial suspension was inoculated into a test tube containing nutrient broth containing antibacterial agent as the test group sample. When inoculating, the inoculation should be carried out from low antibacterial agent concentration to high concentration.

[0188] (3) Inoculate a test tube containing nutrient broth without antibacterial agent in the same way as the positive control sample.

[0189] (4) Take two test tubes containing nutrient broth as negative control group samples.

[0190] (5) Incubate the test group samples, positive control group samples, and negative control group samples at 36°C for 48 hours and observe the results.

[0191] (6) The concentration of bacterial suspension used in the test should be sufficient to count viable bacteria, and the effective concentration should be 5×10 5 CFU / mL~5×10 6 CFU / mL.

[0192] 5.4 Evaluation Basis

[0193] When the positive control tube showed bacterial growth (turbidity) and the negative control tube showed sterile growth (clear and transparent), the concentration of the test bacterial suspension was 5×10 5 CFU / mL~5×10 6 CFU / mL, the antibacterial agent concentration corresponding to the highest dilution of sterile growth in the test group is the MIC of the sample against the test bacteria.

[0194] 6. Experimental Results

[0195] The minimum inhibitory concentrations of different drugs against Fusobacterium nucleatum and Porphyromonas gingivalis Figure 7 As shown, compared with the single herbs of honeysuckle, dandelion, schisandra chinensis, malus hupehensis and clove, the minimum inhibitory concentration of Kouchi Liuxiang recipe against Fusobacterium nucleatum (Fn) and Porphyromonas gingivalis (Pg) was significantly decreased (p<0.01), indicating that the inhibitory effect of honeysuckle, dandelion, schisandra chinensis, malus hupehensis and clove on oral microorganisms that cause halitosis is limited and cannot effectively freshen the breath. However, after these herbs are combined according to the traditional Chinese medicine syndrome differentiation formula, the Kouchi Liuxiang recipe has a more significant antibacterial effect and can play a role in treating halitosis.

[0196] Test Example 5: Volunteer Evaluation of Products with a Leaving a Scent of Mouth

[0197] 1. Determination of breath value before and after use of the series of products that leave a lasting fragrance in your mouth

[0198] 1.1 Breath Fragrance - Measurement of Breath Using Lozenges

[0199] 1.1.1 Methods

[0200] Use Halimeter PLUS breath detector to measure hydrogen sulfide (H 2 S) content was measured, and the breath value was repeated 3 times before chewing the lozenge (the extract powder prepared in Example 1 was made by adding the commonly used auxiliary materials in medicines). There was no statistical difference in the results, indicating that the instrument performance was stable, the subjects mastered the blowing method well, and the measurement results were reliable. According to literature reports, a breath value of <110ppb is fresh breath, a breath value of 110-240ppb is mild halitosis, and a breath value of 240-500ppb is moderate halitosis. The measurement method is as follows: during the test, close your mouth for 2min, debug the machine, hold your breath in the round tube in the volunteer's mouth, and record the data when the machine value begins to steadily decrease. Repeat 3 times, observe the measurement stability, and take the average value as the baseline value of the breath before the lozenge. Then each person takes 2 lozenges, eats them within the specified time of 1-2min, tests the breath immediately, records the immediate value, and repeats the above method after 1h.

[0201] 1.1.2 Subject selection

[0202] 18 subjects were selected, aged 18-65 years old, in good health, regardless of gender; good oral health environment; willing to sign the informed consent and complete all research procedures; no eating habits such as garlic, leeks, stinky tofu, etc. that are prone to cause bad breath.

[0203] 1.1.3 Exclusion criteria

[0204] Patients with severe gingivitis, periodontitis or oral ulcers; those who have used antihistamines in the past week or immunosuppressants in the past month; patients with insulin-dependent diabetes; patients with asthma or other chronic respiratory diseases being treated; those who have received anti-cancer chemotherapy in the past 6 months; patients with immunodeficiency or autoimmune diseases; breastfeeding or pregnant women; participants in other clinical trials; those who have taken oral care products to reduce bad breath (including but not limited to toothpaste, mouthwash, etc.) within three months; those who are intolerant to the products used; and those whose other circumstances affect the test results or who cannot complete the specified content as required by the test requirements.

[0205] 1.1.4 Judgment criteria

[0206] After using the lozenges for leaving mouth fragrance, the halitosis index values ​​of the subjects were all lower than their baseline values, and the differences between the halitosis index values ​​of the subjects and their baseline values ​​were significantly different, so it is believed that the lozenges for leaving mouth fragrance has the effect of reducing halitosis.

[0207] 1.2 Odor in the mouth - measurement of breath using mouthwash

[0208] 1.2.1 Methods

[0209] Use Halimeter PLUS breath detector to measure hydrogen sulfide (H 2 S) content was measured, and the breath value was measured 3 times before using the mouthwash (the extract powder prepared in Example 1, made by adding the commonly used auxiliary materials in medicines). The results showed no statistical difference, indicating that the instrument performance was stable, the subjects mastered the blowing method well, and the measurement results were reliable. The measurement method is as follows: during the test, the mouth was closed for 2 minutes, the machine was debugged, the volunteer held the round tube in the mouth, held his breath, and the data was recorded when the machine value began to steadily decrease. Repeat 3 times, observe the measurement stability, and take the average value as the baseline value of the breath before the mouthwash was used. Then each person used 12mL (12mL / bag) of mouthwash, and finished using it within the specified time of 1 to 2 minutes, tested the breath, recorded the immediate value, and repeated the above method after 1 hour.

[0210] 1.2.2 Subject selection

[0211] 18 subjects were selected, aged 18-65 years old, in good health, regardless of gender; good oral health environment; willing to sign the informed consent and complete all research procedures; no eating habits such as garlic, leeks, stinky tofu, etc. that are prone to cause bad breath.

[0212] 1.2.3 Exclusion criteria

[0213] Patients with severe gingivitis, periodontitis or oral ulcers; those who have used antihistamines in the past week or immunosuppressants in the past month; patients with insulin-dependent diabetes; patients with asthma or other chronic respiratory diseases being treated; those who have received anti-cancer chemotherapy in the past 6 months; patients with immunodeficiency or autoimmune diseases; breastfeeding or pregnant women; participants in other clinical trials; those who have taken oral care products to reduce bad breath (including but not limited to toothpaste, mouthwash, etc.) within three months; those who are intolerant to the products used; and those whose other circumstances affect the test results or who cannot complete the specified content as required by the test requirements.

[0214] 1.2.4 Judgment criteria

[0215] After using the mouth-scenting mouthwash, the subjects' bad breath index values ​​were lower than their baseline values, and the difference between the subjects' bad breath index values ​​and their baseline values ​​was significantly different, so it is believed that the mouth-scenting mouthwash has the effect of reducing bad breath.

[0216] 1.3 Breath smell - Determination of breath smell after using toothpaste

[0217] 1.3.1 Methods

[0218] Use Halimeter PLUS breath detector to measure hydrogen sulfide (H 2 S) content was measured, and the breath value was measured 3 times before using toothpaste (the extract powder prepared in Example 1, made by adding excipients commonly used in medicines). There was no statistical difference in the results, indicating that the instrument performance was stable, the subjects had a good grasp of the blowing method, and the measurement results were reliable. The measurement method is as follows: during the test, the mouth was closed for 2 minutes, the machine was debugged, and the volunteer held the round tube in his mouth and held his breath. When the machine value began to steadily decrease, the data was recorded, and the measurement was repeated 3 times. The stability of the measurement was observed, and the average value was taken as the baseline value of the breath before the toothpaste was used. Then each person used toothpaste to brush his teeth, and finished within the specified time of 1-3 minutes, tested the breath, recorded the immediate value, and repeated the above method after 1 hour.

[0219] 1.3.2 Subject selection

[0220] 18 subjects were selected, aged 18-65 years old, in good health, regardless of gender; good oral health environment; willing to sign the informed consent and complete all research procedures; no eating habits such as garlic, leeks, stinky tofu, etc. that are prone to cause bad breath.

[0221] 1.3.3 Exclusion criteria

[0222] Patients with severe gingivitis, periodontitis or oral ulcers; those who have used antihistamines in the past week or immunosuppressants in the past month; patients with insulin-dependent diabetes; patients with asthma or other chronic respiratory diseases being treated; those who have received anti-cancer chemotherapy in the past 6 months; patients with immunodeficiency or autoimmune diseases; breastfeeding or pregnant women; participants in other clinical trials; those who have taken oral care products to reduce bad breath (including but not limited to toothpaste, mouthwash, etc.) within three months; those who are intolerant to the products used; and those whose other circumstances affect the test results or who cannot complete the specified content as required by the test requirements.

[0223] 1.3.4 Judgment criteria

[0224] After using the toothpaste that leaves a good smell in your mouth, the bad breath index values ​​of the subjects are all lower than their baseline values, and the difference between the bad breath index values ​​of the subjects and their baseline values ​​is significantly different, so it is believed that the toothpaste that leaves a good smell in your mouth has the effect of reducing bad breath.

[0225] 1.4 Statistical methods

[0226] Descriptive statistics were performed for breath values ​​at each study time point. For diagnostic testing, changes in breath values ​​were calculated before use and 0 and 1 h after use of the Mouth Fragrance Series products. Statistical analysis was performed using IBM SPSS Statistics 26.0. All data were normally distributed by the Shapiro-Wilk test. One-way analysis of variance was used for inter-group comparisons with Tukey's post hoc test. Results were visualized using GraphPad Prism 8.3.0. A significance level of p < 0.05 was considered statistically significant.

[0227] 2. Determination of breath value of periodic use of products that leave a good taste in the mouth (lozenges, mouthwash, toothpaste)

[0228] 2.1 Methods

[0229] Use Halimeter PLUS breath detector to measure hydrogen sulfide (H 2 The breath value was determined by measuring the content of S) in the mouth. The breath value was repeated 3 times before using the series of products for mouth fragrance. There was no statistical difference in the results, indicating that the performance of the instrument was stable, the subjects had a good grasp of the blowing method, and the measurement results were reliable. After the preparation stage, there was a one-month experimental stage. The subjects used toothpaste every morning and evening, and lozenges and mouthwash after meals. The breath values ​​of the subjects were measured at the baseline and at the same time point of 1, 2, 3, and 4 weeks of use. The measurement method is as follows: during the test, close your mouth for 1 minute, debug the machine, and the volunteer holds the round tube in his mouth and holds his breath. Record the data when the machine value begins to steadily decrease. Repeat 3 times, observe the measurement stability, and take the average value as the breath value after using the series of products.

[0230] 2.2 Subject selection

[0231] 18 subjects were selected, aged 18-65 years old, in good health, regardless of gender; good oral health environment; willing to sign the informed consent and complete all research procedures; no eating habits such as garlic, leeks, stinky tofu, etc. that are prone to cause bad breath.

[0232] 2.3 Exclusion criteria

[0233] Patients with severe gingivitis, periodontitis or oral ulcers; those who have used antihistamines in the past week or immunosuppressants in the past month; patients with insulin-dependent diabetes; patients with asthma or other chronic respiratory diseases being treated; those who have received anti-cancer chemotherapy in the past 6 months; patients with immunodeficiency or autoimmune diseases; breastfeeding or pregnant women; participants in other clinical trials; those who have taken oral care products to reduce bad breath (including but not limited to toothpaste, mouthwash, etc.) within three months; those who are intolerant to the products used; and those whose other circumstances affect the test results or who cannot complete the specified content as required by the test requirements.

[0234] 2.4 Judgment criteria

[0235] After using the series of products that leave a good breath, the subjects' bad breath index values ​​are all lower than their baseline values, and the differences between the subjects' bad breath index values ​​and their baseline values ​​are significantly different. It is believed that the series of products that leave a good breath (lozenges, mouthwash, lozenges) have the effect of reducing bad breath.

[0236] 2.5 Statistical methods

[0237] Descriptive statistics were performed for the breath values ​​at each study time point. For diagnostic tests, changes relative to baseline, 1, 2, 3, and 4 weeks were calculated. Statistical analysis was performed using IBM SPSS Statistics 26.0. All data were normally distributed by the Shapiro-Wilk test. One-way analysis of variance was used for comparison between groups with Tukey's post hoc test. Results were visualized using GraphPad Prism 8.3.0. A significance level of p < 0.05 was considered statistically significant.

[0238] 3 Results

[0239] 3.1 Leaving a good taste in the mouth - Changes in VSC values ​​before and after lozenge use and 1 hour after use

[0240] The results are as follows Figure 8As shown in A, the VSC value decreased significantly after using the lozenges for leaving mouth fragrance, which was significantly different from the initial value (p<0.01). The VSC value increased partially after 1 hour of use, but was still significantly different from the initial value. Therefore, the results showed that the lozenges for leaving mouth fragrance had the effect of reducing bad breath and had a certain maintenance effect.

[0241] 3.2 Leaves a good taste in your mouth - Changes in VSC values ​​before and after using mouthwash and 1 hour after use

[0242] The results are as follows Figure 8 As shown in B, the VSC value decreased significantly after using the mouthwash, which was significantly different from the initial value (p<0.01). The VSC value increased partially after 1 hour of use, but was still significantly different from the initial value. Therefore, the results showed that the mouthwash has the effect of reducing bad breath and has a certain maintenance effect.

[0243] 3.3 Leaves a good taste in your mouth - Changes in VSC value before and after use of toothpaste and 1 hour after use

[0244] The results are as follows Figure 8 As shown in C, the VSC value decreased significantly after using the toothpaste with a good smell, which was significantly different from the initial value (p<0.01). The VSC value increased partially after 1 hour of use, but was still significantly different from the initial value. Therefore, the results show that the toothpaste with a good smell has the effect of reducing bad breath and has a certain maintenance effect.

[0245] 3.4 Changes in VSC values ​​of products that leave a lasting taste in your mouth—lozenges, toothpaste, and mouthwash after periodic use

[0246] The results are as follows Figure 8 As shown in Figure D, the VSC value decreased significantly after periodic use of the series of products that leave a sweet taste in your mouth - lozenges, toothpaste, and mouthwash. After 7 days of use, the VSC value of the subject showed a downward trend, but there was no significant difference compared with the initial value. After 14 days, 21 days, and 28 days of use, the VSC value of the subject decreased significantly, with a significant difference compared with the initial value (p<0.01), indicating that the series of products that leave a sweet taste in your mouth - lozenges, toothpaste, and mouthwash can reduce the VSC value of the subject as the use cycle increases.

[0247] The above experiments show that the series of products that leave a good taste in your mouth - lozenges, toothpaste, and mouthwash - have the effect of reducing bad breath and have a certain maintaining effect.

[0248] In summary, the present invention can relieve the symptoms of stomach heat syndrome, improve gastric mucosal tissue damage, and regulate intestinal flora through the use of five traditional Chinese medicines in combination, and has a significant effect in treating bad breath caused by stomach heat syndrome.

Claims

1. A Chinese medicine composition for freshening breath, characterized in that: It is prepared from the following raw materials in weight ratio: 10-20 parts of Schisandrae Chinensis Fruit, 20-40 parts of Malus Malus, 10-30 parts of Honeysuckle, 20-40 parts of Taraxacum Herba, and 20-40 parts of Cloves.

2. The Chinese medicine composition according to claim 1, characterized in that: It is prepared from the following raw materials in weight ratio: 15 parts of Schisandrae Chinensis Fruits, 30 parts of Malus Malus, 20 parts of Flos Honeysuckle, 30 parts of Herba Taraxaci, and 30 parts of Flos Caryophylli.

3. The Chinese medicine composition according to claim 1 or 2, characterized in that: The crabapple is the Hubei crabapple.

4. The Chinese medicine composition according to claim 1, characterized in that: It is a preparation made from fine powder of raw materials, or water extract of raw materials, or alcohol extract of raw materials as active ingredients, plus excipients acceptable to medicines or daily chemical products; The preparation is an oral preparation or an oral preparation; The oral preparations include solutions, tablets, granules, ointments, pills, capsules or powders; The oral preparations include lozenges, mouthwashes, popping beads or toothpaste.

5. A method for preparing the Chinese medicine composition according to any one of claims 1 to 4, characterized in that: It includes the following steps: (1) Weigh the raw materials according to the ratio; (2) A preparation made from a fine powder of a raw material, or an aqueous extract of a raw material, or an alcohol extract of a raw material as an active ingredient, plus excipients acceptable to medicines or daily chemical products.

6. Use of the Chinese medicine composition according to any one of claims 1 to 4 in the preparation of a medicine for treating gastric mucosal damage, improving intestinal flora disorder and / or refreshing breath.

7. The use according to claim 6, characterized in that: The medicine is a medicine for treating gastric mucosal damage of stomach-heat syndrome type and / or gastric-heat syndrome type halitosis.

8. The use according to claim 6 or 7, characterized in that: The drug has the effect of inhibiting Fusobacterium nucleatum and / or Porphyromonas gingivalis.

9. Use of the Chinese medicine composition according to any one of claims 1 to 4 in the preparation of daily chemical products for freshening breath.

10. The use according to claim 9, characterized in that: The daily chemical product is a daily chemical product for improving gastric heat syndrome type halitosis and has the function of inhibiting Fusobacterium nucleatum and / or Porphyromonas gingivalis.

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