A tangerine peel ferment for alleviating smoking-induced respiratory inflammation, its preparation method and application

Abstract

The present invention belongs to the technical field of pharmaceuticals, and discloses a fermented tangerine peel for relieving smoking-induced respiratory inflammation, a preparation method thereof and an application. The fermented tangerine peel is a product obtained by fermenting tangerine peel through multi-step addition of different complex enzymes and combined probiotics. The fermented tangerine peel prepared by the present invention can significantly improve the active ingredients of tangerine peel, enhance the anti-inflammatory effect on tobacco smoke-induced respiratory inflammation, and reduce the infiltration of respiratory inflammatory cells and the transcriptional level of inflammatory factors. The preparation method provided by the present invention has high operability, is easy to standardize and intelligentize, and can achieve large-scale production in industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceuticals, and particularly relates to a fermented tangerine peel product for relieving smoking-induced respiratory inflammation, a preparation method thereof, and an application thereof. Background Art

[0002] China is the largest tobacco producer and consumer in the world, ranking first in both tobacco production and consumption. The "Report on the Health Hazards of Smoking in China 2020" released by the National Health Commission pointed out that the number of smokers in China exceeds 300 million. Although the smoking rate of the population shows a downward trend, the current smoking rate is still at a relatively high level. Especially among people aged 15 and above, the smoking rate is 25.8%, and the smoking rate of men is as high as 48.8%.

[0003] The tobacco industry has an important impact on China's economy. At the same time, smoking also brings serious health problems and social burdens. Tobacco smoke contains a large number of harmful substances, such as nicotine, carbon monoxide, and various carcinogens, which pose a serious threat to the health of smokers. In addition, the problem of second-hand smoke is equally serious. In 2018, the exposure rate of non-smokers to second-hand smoke in China was 68.1%. Long-term inhalation of smoke can lead to respiratory inflammation and various diseases, such as chronic obstructive pulmonary disease, asthma, and lung cancer, bringing a heavy burden to individual health and public health.

[0004] At present, although a variety of new tobacco products have been developed to reduce the harm of smoking, such as e-cigarettes, cigarette filters, and smokeless oral tobacco, these methods cannot completely eliminate the impact of tobacco smoke on the respiratory system. Therefore, there is a need to develop drugs that can effectively relieve smoking-induced respiratory inflammation to reduce the health risks of smokers and the surrounding population. Summary of the Invention

[0005] In view of the above problems existing in the prior art, the present invention provides a fermented tangerine peel product for relieving smoking-induced respiratory inflammation, a preparation method thereof, and an application thereof by a special fermentation method to improve the effective components of tangerine peel.

[0006] The present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, a preparation method of a fermented tangerine peel product for relieving smoking-induced respiratory inflammation is provided, comprising the following steps:

[0008] S1: Uniformly disperse tangerine peel powder in a hydrochloric acid solution with a pH value of 5-6, add composite enzyme A, enzymatically hydrolyze at 45-65 °C for 1.5-3.5 h, and collect enzymatic hydrolysate A after inactivating the enzyme.

[0009] S2: Adjust the pH value of the above enzymatic hydrolysate A to 1.5 - 2, add compound enzyme B, and perform enzymatic hydrolysis at 37°C - 39°C for 2 - 4 h. Subsequently, adjust the pH value to 7.5 - 8.5, and collect enzymatic hydrolysate B after enzyme inactivation treatment;

[0010] S3: Add combined probiotics to the above enzymatic hydrolysate B for fermentation treatment, collect the fermentation broth and perform freeze-drying, and then perform microbial sterilization to obtain the fermented tangerine peel;

[0011] In the step S1, the mass ratio of the tangerine peel powder to the hydrochloric acid solution is (1 - 2):(4 - 8); the compound enzyme A is a mixture of cellulase, glucosidase, and amylase;

[0012] In the step S2, the compound enzyme B is a mixture of pepsin and trypsin;

[0013] In the step S3, the combined probiotics are Bifidobacterium, Lactobacillus rhamnosus, and Lactobacillus reuteri.

[0014] Specifically, the addition amount of the compound enzyme A accounts for 2 - 6 wt% of the tangerine peel powder amount; the mass ratio of cellulase, glucosidase, and amylase in the compound enzyme A is (1 - 3):(2 - 4):(0.5 - 1).

[0015] Specifically, the addition amount of the compound enzyme B accounts for 4 - 8 wt% of the tangerine peel powder amount; the mass ratio of pepsin and trypsin in the compound enzyme B is (0.5 - 2):(1 - 3).

[0016] Specifically, the addition amount of the combined probiotics accounts for 1 - 5 wt% of the tangerine peel powder amount; the mass ratio of Bifidobacterium, Lactobacillus rhamnosus, and Lactobacillus reuteri in the combined probiotics is (2 - 5):(4 - 7):(1 - 5).

[0017] Specifically, in the step S3, the temperature of the fermentation treatment is 35°C - 40°C, and the time is 12 - 48 h.

[0018] In the second aspect of the present invention, there is also provided a fermented tangerine peel for relieving smoking-induced respiratory inflammation prepared by the above method.

[0019] In the third aspect of the present invention, there is also provided an application of the prepared fermented tangerine peel in the preparation of a drug for relieving smoking-induced respiratory inflammation.

[0020] Specifically, the drug for relieving smoking-induced respiratory inflammation is a drug that reduces macrophage, neutrophil, and lymphocyte infiltration in the respiratory tract and inhibits the gene transcription levels of CCL2, CCL20, TNF-α, IL-1β, and IL-6.

[0021] Specifically, the fermented tangerine peel can be made into drugs such as tablets, granules, capsules or oral liquids.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The fermented product of the present invention is derived from the tangerine peel of traditional Chinese medicine, which is a natural plant component. Compared with synthetic drugs or artificial chemical substances, it is safer and non-toxic, has no side effects on human health, and meets the modern people's pursuit of health and naturalness.

[0024] (2) In the present invention, tobacco smoke-induced respiratory inflammation was induced in mice, and the fermented tangerine peel was used to treat the mice. It was found that it could significantly inhibit the infiltration of macrophages, neutrophils and lymphocytes in the bronchoalveolar lavage fluid of mice, and significantly reduce the mRNA expression levels of CCL2, CCL20, TNF-α, IL-1β and IL-6. In addition, compared with the unfermented tangerine peel, the fermented tangerine peel obtained by the specific fermentation technology of the present invention has a stronger therapeutic effect on tobacco smoke-induced respiratory inflammation, indicating that the preparation method used in the present invention is an innovative technical means and application method.

[0025] (3) The preparation method provided by the present invention has high operability and is easy to standardize and intelligentize. The fermentation process used is more environmentally friendly and efficient than the traditional extraction process, and can achieve large-scale production in industrial production. Description of the Drawings

[0026] Figure 1 Shows the effects of the fermented tangerine peel in the present invention on the transcriptional levels of CCL2, CCL20, TNF-α, IL-1β and IL-6 genes in the lung tissues of mice induced by tobacco smoke. Detailed Embodiments

[0027] The present invention will be further described in detail below with reference to the embodiments. It should be noted that the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. The present application should not be construed as being limited to the specific embodiments described. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0028] Embodiment 1

[0029] This embodiment provides a preparation method of a fermented tangerine peel for relieving smoking-induced respiratory inflammation, including the following steps:

[0030] S1: Grind 100 parts by weight of tangerine peel into powder, disperse it evenly in 5 times the amount of hydrochloric acid solution with a pH value of 5.5, and add 4 parts by weight of composite enzyme A. Among them, composite enzyme A is a mixture of cellulase, glucosidase and amylase, and the mass ratio of cellulase, glucosidase and amylase is 1:2.5:0.6. Enzymolysis is carried out at 55 °C for 2.5 h, and after enzyme inactivation treatment, enzymolysis solution A is collected;

[0031] S2: Adjust the pH value of the above enzymolysis solution A to 1.6, and add 6 parts by weight of composite enzyme B. Among them, composite enzyme B is a mixture of pepsin and trypsin, and the mass ratio of pepsin and trypsin is 0.8:1.3. Enzymolysis is carried out at 37.8 °C for 2.5 h, and then add NaOH to adjust the pH value to 8.2. After enzyme inactivation treatment, enzymolysis solution B is collected;

[0032] S3: Add 3 parts by weight of combined probiotics to the above enzymolysis solution B for fermentation treatment. The combined probiotics are Bifidobacterium, Lactobacillus rhamnosus and Lactobacillus reuteri, and their mass ratio is 3:5:2. Fermentation treatment is carried out at 37.5 °C for 24 h. Collect the fermentation broth and freeze-dry it, and then carry out microbial sterilization to obtain the finished product.

[0033] Example 2

[0034] This example provides a preparation method of tangerine peel fermented product for relieving smoking-induced respiratory inflammation, including the following steps:

[0035] S1: Grind 100 parts by weight of tangerine peel into powder, disperse it evenly in 6 times the amount of hydrochloric acid solution with a pH value of 5.5, and add 5 parts by weight of composite enzyme A. Among them, composite enzyme A is a mixture of cellulase, glucosidase and amylase, and the mass ratio of cellulase, glucosidase and amylase is 2:3:0.8. Enzymolysis is carried out at 50 °C for 3 h, and after enzyme inactivation treatment, enzymolysis solution A is collected;

[0036] S2: Adjust the pH value of the above enzymolysis solution A to 1.6, and add 7 parts by weight of composite enzyme B. Among them, composite enzyme B is a mixture of pepsin and trypsin, and the mass ratio of pepsin and trypsin is 1:1.5. Enzymolysis is carried out at 37.8 °C for 3 h, and then add NaOH to adjust the pH value to 8.2. After enzyme inactivation treatment, enzymolysis solution B is collected;

[0037] S3: Add 2 parts by weight of combined probiotics to the above enzymolysis solution B for fermentation treatment. The combined probiotics are Bifidobacterium, Lactobacillus rhamnosus and Lactobacillus reuteri, and their mass ratio is 3:4:3. Fermentation treatment is carried out at 37.5 °C for 18 h. Collect the fermentation broth and freeze-dry it, and then carry out microbial sterilization to obtain the finished product.

[0038] Example 3

[0039] This embodiment provides a method for preparing a tangerine peel fermented product for alleviating smoking-induced respiratory inflammation, comprising the following steps:

[0040] S1: Grind 100 parts by weight of tangerine peel into powder, uniformly disperse it in 8 times the amount of hydrochloric acid solution with a pH value of 5.5, add 6 parts by weight of complex enzyme A, wherein complex enzyme A is a mixture of cellulase, glucosidase and amylase, and the mass ratio of cellulase, glucosidase and amylase is 1:4:0.7. Enzymatically hydrolyze at 60 °C for 2 h, and collect the enzymatic hydrolysate A after inactivating the enzyme;

[0041] S2: Adjust the pH value of the above enzymatic hydrolysate A to 1.6, add 6.5 parts by weight of complex enzyme B, wherein complex enzyme B is a mixture of pepsin and trypsin, and the mass ratio of pepsin and trypsin is 1:1.2. Enzymatically hydrolyze at 37.8 °C for 2.5 h, then add NaOH to adjust the pH value to 8.2, and collect the enzymatic hydrolysate B after inactivating the enzyme;

[0042] S3: Add 2.5 parts by weight of a combined probiotic to the above enzymatic hydrolysate B for fermentation treatment. The combined probiotic is Bifidobacterium, Lactobacillus rhamnosus and Lactobacillus reuteri, and their mass ratio is 2:3:3. Ferment at 37.5 °C for 30 h. Collect the fermentation broth and freeze-dry it, and then perform microbial sterilization to obtain the finished product.

[0043] Comparative Example 1

[0044] The specific implementation method is the same as that of Example 1, except that complex enzyme A is not added.

[0045] Comparative Example 2

[0046] The specific implementation method is the same as that of Example 1, except that complex enzyme B is not added.

[0047] Comparative Example 3

[0048] The specific implementation method is the same as that of Example 1, except that the combined probiotic is not added.

[0049] Efficacy test

[0050] To verify the efficacy of the tangerine peel fermented product of the present invention in alleviating smoking-induced respiratory inflammation, the following verification tests were designed.

[0051] The animals used in this experiment were male C57BL / 6 mice, 8 weeks old, with a body weight of 18 - 22 g. All mice were adaptively fed for 7 days before the experiment. Sixty - four mice were divided into 8 groups, namely the blank control group, the model group, the groups of Example 1 - 3, and the groups of Comparative Example 1 - 3. Except for the blank control group, mice in other groups were induced with a respiratory inflammation model using tobacco smoke. The modeling method was as follows: 8 mice were placed in a smoking exposure test chamber, and tobacco smoke was introduced 4 times a day, 5 cigarettes each time. The modeling period was 2 weeks. Starting from the first day of modeling, the treatment groups were respectively intragastrically administered with the finished products prepared from Example 1 - 3 and Comparative Example 1 - 3 at a dose of 50 mg / kg per day for 2 weeks. The blank control group and the model group were intragastrically administered with an equal volume of normal saline.

[0052] (1)Respiratory inflammatory cell infiltration assay

[0053] On the 15th day after modeling, the mice were sacrificed and tracheal intubation was inserted. 1 mL of HBSS buffer was perfused through the tracheal intubation, a total of 3 times. The 3 lavage fluids were combined and centrifuged, washed 2 times, and finally resuspended with 1 mL of HBSS buffer. A fully automatic cell analyzer was used to count macrophages, neutrophils, and lymphocytes. The detailed results are shown in Table 1.

[0054] Table 1 Counts of macrophages, neutrophils, and lymphocytes in bronchoalveolar lavage fluid of mice

[0055]

[0056] As can be seen from Table 1, after induction with tobacco smoke, the numbers of macrophages, neutrophils, and lymphocytes in the bronchoalveolar lavage fluid of mice increased significantly. After treatment with the finished products in Example 1 - 3 and Comparative Example 1 - 3, the numbers of macrophages, neutrophils, and lymphocytes in the bronchoalveolar lavage fluid of mice decreased to varying degrees. Among them, the effects of the groups of Example 1 - 3 were significantly better than those of the groups of Comparative Example 1 - 3. Moreover, in combination with the comparative examples, it can be seen that after the treatment with the compound enzymes A and B and probiotics in the examples of the present invention, the effect of the fermented tangerine peel obtained is significantly higher than that of the fermented products obtained with a single compound enzyme or probiotic. This shows that there is a synergistic effect between the compound enzymes and probiotics of the present invention, and they can cooperate with and support each other to achieve better effects.

[0057] (2)Determination of mRNA expression levels of inflammatory factors

[0058] The therapeutic effects of the finished products in Example 1 - 3 and Comparative Example 1 - 3 on tobacco - smoke - induced respiratory inflammation were further evaluated by detecting the mRNA expression levels of inflammatory factors in lung tissues. The test results are as Figure 1As shown, after the finished products in Examples 1 to 3 and Comparative Examples 1 to 3 were processed, the mRNA expression levels of inflammatory factors induced by tobacco smoke were reduced to varying degrees, including CCL2, CCL20, TNF-α, IL-1β, and IL-6. Similarly, the effects of the groups of Examples 1 to 3 were significantly better than those of the groups of Comparative Examples 1 to 3.

[0059] Based on the above test results, the fermented tangerine peel provided by the present invention can significantly improve the therapeutic effect on respiratory inflammation induced by tobacco smoke, thereby achieving the effects of inhibiting the infiltration of inflammatory cells and reducing the transcriptional level of inflammatory factors.

[0060] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a fermented product of Citrus aurantium wilfordii for relieving respiratory inflammation induced by smoking, characterized in that: The following steps are involved: S1: Disperse the tangerine peel powder evenly in a hydrochloric acid solution with a pH value of 5-6, add complex enzyme A, perform enzymolysis at 45-65°C for 1.5-3.5h, and collect the enzymolysis solution A after enzyme inactivation; S2: The pH value of the above enzymatic hydrolysate A is adjusted to 1.5-2, and the compound enzyme B is added, and the enzymatic hydrolysis is carried out at 37°C-39°C for 2-4 hours, and then the pH value is adjusted to 7.5-8.5, and the enzymatic hydrolysate B is collected after the enzyme is inactivated; S3: adding combined probiotics to the above enzymatic hydrolysate B for fermentation, collecting the fermentation liquid and freeze-drying it, and then sterilizing the microorganisms to obtain the fermented tangerine peel; In the step S1, the mass ratio of the tangerine peel powder to the hydrochloric acid solution is 1-2: 4-8; the complex enzyme A is a mixture of cellulase, glucosidase and amylase; In step S2, the compound enzyme B is a mixture of pepsin and trypsin; In step S3, the combination of probiotics is bifidobacterium, Lactobacillus rhamnosus and Lactobacillus reuteri.

2. The method for preparing the fermented product of Citrus aurantium according to claim 1, characterized in that: The added amount of the complex enzyme A accounts for 2-6 wt % of the amount of the tangerine peel powder; the mass ratio of cellulase, glucosidase and amylase in the complex enzyme A is 1-3:2-4:0.5-1.

3. The method for preparing the fermented product of Citrus aurantium according to claim 1, characterized in that: The added amount of the complex enzyme B accounts for 4-8 wt % of the amount of the tangerine peel powder; the mass ratio of pepsin to trypsin in the complex enzyme B is 0.5-2:1-3.

4. The method for preparing the fermented product of Citrus aurantium according to claim 1, characterized in that: The added amount of the combined probiotics accounts for 1-5 wt% of the amount of the tangerine peel powder; the mass ratio of the bifidobacterium, the rhamnosus lactobacillus and the reuteri lactobacillus of the combined probiotics is 2-5:4-7:1-5.

5. The method for preparing the fermented product of Citrus aurantium according to claim 1, characterized in that: In step S3, the fermentation treatment is carried out at a temperature of 35° C. to 40° C. and for a time of 12 to 48 hours.

6. A fermented product of Citrus aurantium wilfordii for relieving smoking-induced respiratory inflammation prepared by the method according to any one of claims 1 to 5.

7. Use of the fermented product of Citrus aurantium according to claim 6 in the preparation of medicines for relieving respiratory inflammation induced by smoking.

8. The use according to claim 7, characterized in that: The medicine for relieving respiratory inflammation induced by smoking has the following effects: reducing the infiltration of macrophages, neutrophils and lymphocytes in the respiratory tract, and inhibiting the transcription levels of CCL2, CCL20, TNF-α, IL-1β and IL-6 genes.

9. The use according to claim 7, characterized in that: The medicine is in the form of tablets, granules, capsules or oral liquid.

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