Application of throat lozenge extract in preparation of product for resisting beta hemolytic streptococcus infection

Abstract

The invention relates to application of a throat lozenge extract in preparation of a product for resisting beta hemolytic streptococcus infection, in particular to the field of public health. The invention relates to a novel application of a throat lozenge extract in preparation of a product for resisting beta hemolytic streptococcus infection. The throat lozenge extract comprises mesona chinensis, liquorice, chrysanthemum, honeysuckle and momordica grosvenori. It is found that the throat lozenge extract can be used for inhibiting beta hemolytic streptococcus infection, and pharyngeal injury caused by beta hemolytic streptococcus infection can be effectively relieved.

Description

Technical Field

[0001] The invention provides application of a throat lozenge extract in preparing a product for resisting beta-hemolytic streptococcal infection, and particularly relates to the field of public health. Background Art

[0002] Group B Streptococcus (GBS) is an important human pathogen that mainly colonizes the upper respiratory tract and skin surface of humans. In some cases, it can cause various infections, including pharyngitis, scarlet fever, pneumonia, skin abscesses, etc. In rare cases, GBS may also cause more serious invasive infections such as sepsis, toxic shock syndrome and necrotizing fasciitis.

[0003] Chinese patent CN115886116B discloses a method for preparing hard candy from a low-solid plant-based concentrate, which includes the composition and preparation process of throat lozenge concentrate. The throat lozenge has the effects of clearing away heat and detoxifying, clearing the throat and relieving pharyngitis. However, after years of research, it has been found that the throat lozenge concentrate also has the effect of resisting beta-hemolytic streptococcal infection.

[0004] Chinese patent CN117599100A discloses a new use of throat lozenge concentrate for treating bacterial inflammation. The throat lozenge concentrate is prepared from the following raw materials in weight percentage: 50-56% of Herba Cibotii, 19-23% of Licorice, 13-17% of Chrysanthemum, 7-11% of Monk Fruit and 1-4% of Honeysuckle. In the patent, LPS is used to induce inflammatory response, and a zebrafish bacterial inflammation model is established to verify the inflammation (LPS-induced) relief effect of throat lozenge concentrate. This patent simulates bacterial inflammation and proves that throat lozenge concentrate has potential anti-inflammatory efficacy, but there are many types of bacteria and the diseases caused are also diverse. The patent does not evaluate specific bacteria and specific diseases. Summary of the invention

[0005] In order to solve the above problems in the prior art, the present invention provides an application of a throat lozenge extract in the preparation of an anti-beta-hemolytic streptococcal infection product, comprising the following technical solutions:

[0006] In a first aspect, the present invention provides a use of a throat lozenge extract in the preparation of a product for resisting beta-hemolytic streptococcal infection, wherein the throat lozenge extract comprises grass jelly, licorice, chrysanthemum, honeysuckle and monk fruit;

[0007] Specifically, the throat lozenge extract is prepared from the following raw materials in parts by weight: 30-60 parts of grass jelly, 10-30 parts of licorice, 5-20 parts of chrysanthemum, 0.5-4 parts of honeysuckle and 3-15 parts of monk fruit;

[0008] Specifically, the throat lozenge extract is prepared from the following raw materials in parts by weight: 40-55 parts of grass jelly, 15-25 parts of licorice, 10-15 parts of chrysanthemum, 1-3 parts of honeysuckle and 5-10 parts of monk fruit;

[0009] Specifically, the throat lozenge extract contains 2-2.5 parts of honeysuckle and 11-13 parts of chrysanthemum per 100 parts;

[0010] Specifically, the throat lozenge extract is prepared by the following steps:

[0011] S1, extracting the raw material with water and filtering to obtain an extract;

[0012] S2, centrifuging the extract obtained in step S1;

[0013] S3, separating and concentrating the centrifuged extract obtained in step S2 to obtain a concentrated solution;

[0014] S4, sterilizing the concentrated solution obtained in step S3 for later use to obtain the original drug solution;

[0015] Specifically, the amount of water in step S1 is 8-16 times the weight of the raw material, the temperature of the water is 80-99° C., and the extraction time is 1-1.5 h.

[0016] Specifically, step S2 is: cooling the extract to 15-25° C. and then centrifuging;

[0017] Specifically, the centrifugal speed is 5000-10000 r / min.

[0018] Specifically, the extract separation and concentration in step S3 is to separate and concentrate the extract through multiple stages of continuous membrane elements.

[0019] Specifically, the solid content of the concentrated solution in step S3 is 10%-25%.

[0020] Specifically, the sterilization temperature in step S4 is 136-140° C. and the time is 15-40 seconds.

[0021] Specifically, the anti-beta-hemolytic streptococcal infection product is any one of food, health care product and medicine.

[0022] In a second aspect, the present invention provides a product for resisting beta-hemolytic streptococcal infection, comprising the above-mentioned throat lozenge extract;

[0023] Specifically, the anti-beta-hemolytic streptococcal infection product is any one of food, health care product and medicine;

[0024] Specifically, the food also includes food additives;

[0025] Specifically, the food additive includes at least one of a leavening agent, an emulsifier, a thickener, a colorant, a sweetener and a preservative;

[0026] Specifically, the health care product also includes supplementary materials for health care products;

[0027] Specifically, the auxiliary materials for health care products include at least one of a thickener, an emulsifier, a stabilizer, a colorant, a sweetener and a preservative;

[0028] Specifically, the medicine also includes pharmaceutical excipients;

[0029] Specifically, the pharmaceutical excipients include at least one of a filler, a binder, a disintegrant, a lubricant and a stabilizer.

[0030] Beneficial Effects

[0031] (1) The throat lozenge extract of the present invention can resist infection by beta-hemolytic streptococci, and is a novel invention;

[0032] (2) The throat lozenge extract of the present invention can relieve throat discomfort caused by beta-hemolytic streptococcal infection. DETAILED DESCRIPTION

[0033] The present invention will be further described in detail below in conjunction with preferred embodiments, but those skilled in the art will appreciate that these are only for the purpose of explaining preferred embodiments and therefore should not be regarded as limiting the scope of the present invention.

[0034] 1. Preparation of throat lozenge extract

[0035] Example

[0036] Take 50 parts of grass jelly, 20 parts of licorice, 12 parts of chrysanthemum, 2 parts of honeysuckle and 8 parts of monk fruit, mix the raw materials with 85°C hot water of 15 times their mass, extract for 1.5 hours, and filter to obtain an extract. The extract is cooled to 25°C by a plate heat exchanger and then centrifuged at a rate of 10000r / min and a centrifuge flow rate of 5 tons / h. The extract after centrifugation is separated and concentrated by multi-stage continuous membrane elements to obtain a concentrate; the solid content of the concentrate is 25%, and the concentrate is sterilized at ultra-high temperature instantaneously at a temperature of 136-140°C for 15-40 seconds to obtain a throat lozenge extract original drug solution (RHTE).

[0037] 2. In vivo experiments

[0038] 1. Experimental Materials

[0039] Test drug: original drug solution (RHTE) prepared in Example;

[0040] Positive control drug: Amoxicillin capsules, a product of Zhuhai United Pharmaceutical Co., Ltd. Zhongshan Branch, batch number: 39002211, production date: 2023.09.22, valid until: 2026.03, specification: 0.25g / capsule.

[0041] Experimental animals: SD rats, 290±10g, SPF grade, 60 rats, half male and half female, all provided by Beijing Huafukang Biotechnology Co., Ltd., animal license number: SCXK(Beijing)2020-0004, certificate number: 110322241101274778.

[0042] Bacterial strain: beta-hemolytic Streptococcus, purchased from Beina Chuanglian Biotechnology Co., Ltd., passaged in the laboratory, and stored in a -80°C refrigerator for future use.

[0043] 2. Dosage design and drug solution preparation

[0044] Three dosages were set in the experiment: RHTE as a high dose, RHTE diluted 1 / 2 times with distilled water as a medium dose, and RHTE diluted 1 / 4 times with distilled water as a low dose. The dosage for rats was 10ml / kg. According to the weighing, 1ml of RHTE original solution = 1g, so the high, medium, and low dosages for rats were 10g / kg / d, 5g / kg / d, and 2.5g / kg / d, respectively. According to the body surface area coefficient of humans and rats being 6, the dosages for adults were converted to 1.8g / kg / d, 0.9g / kg / d, and 0.45g / kg / d.

[0045] According to the information provided by the company, every 100g of RHTE contains 2.3g of honeysuckle and 12g of chrysanthemum. The high dose set in this trial is converted into an adult dose of 2.48g / person / d for honeysuckle and 12.96g / person / d for chrysanthemum, which is in line with the general clinical medication dose. Therefore, the dose design of this trial is reasonable.

[0046] Amoxicillin capsules: The clinical dosage for adults is 1g / 60kg / d. Based on the body surface area coefficient of humans and rats being 6, the rat dosage is 0.1g / kg / d, which is equivalent to the same multiple of the human clinical dosage.

[0047] 3. Experimental methods

[0048] Sixty SPF SD rats, weighing 290±10g, were randomly divided into 6 groups: normal control group, model control group, positive control group, RHTE high-dose group, RHTE medium-dose group, and RHTE low-dose group, with 10 rats in each group.

[0049] After anesthesia, the animals in the other groups except the normal control group had their mouths opened using sterile forceps and 1×10 8The pharynx was punctured with an injection needle containing a solution of beta-hemolytic streptococci at a concentration of cfu / ml, and the solution was injected, 0.2 ml per mouse, for 2 consecutive days. The normal control group was injected with nutrient broth under the same conditions.

[0050] 4 hours after the first infection, rats in the positive control group, RHTE high-dose group, RHTE medium-dose group, and RHTE low-dose group were given the drug by gavage at 10 ml / kg, once a day for 5 consecutive days. The normal control group and model control group were given distilled water under the same conditions.

[0051] 4. In vivo experimental results

[0052] (1) Blood routine test of rats

[0053] On the 6th day, blood was collected from the orbits of rats in the normal control group, model control group, positive control group, RHTE high-dose group, RHTE medium-dose group, and RHTE low-dose group. About 0.5 ml was placed in EDTA K2 anticoagulant blood collection tubes for routine blood testing. The results are shown in Table 1.

[0054] The results in Table 1 show that after infection with beta-hemolytic streptococci, the white blood cell (WBC), neutrophil percentage (NEUT%) and absolute value of neutrophils (NEUT#) in the model control group were significantly increased compared with those in the normal group (P<0.01), and the beta-hemolytic streptococcal infection was successful in the rats in the model control group.

[0055] The high-dose RHTE group can significantly reduce WBC, NEUT% and NEUT#, and there are significant differences compared with the model control group (P<0.01, P<0.05). The high-dose RHTE can reduce the white blood cell (WBC), neutrophil percentage (NEUT%), and absolute neutrophil value (NEUT#) indicators of rats after beta-hemolytic streptococcus infection.

[0056] Table 1 Blood routine results of six groups of rats

[0057] Group Number of animals Dosage (g / kg / day) WBC (10^9 / L) NEUT(%) NEUT#(10^9 / L) Normal control group 10 / 9.55±1.39 12.20±4.18 <![CDATA[1.42±0.63 ** ]]> Model control group 10 / <![CDATA[18.13±2.76 ## ]]> <![CDATA[27.57±5.74 ## ]]> 5.94±2.12 Amoxicillin group 10 0.1 <![CDATA[13.24±2.30 ** ]]> <![CDATA[13.79±3.14 ** ]]> <![CDATA[2.08±0.67 ** ]]> RHTE high dose group 10 10 <![CDATA[12.85±1.21 ** ]]> <![CDATA[15.96±5.89 ** ]]> <![CDATA[3.19±2.35 * ]]> RHTE medium dose group 10 5 18.86±4.09 22.74±6.54 5.28±2.87 RHTE low dose group 10 2.5 17.36±3.48 25.51±7.68 4.54±1.84

[0058] Note: ## Compared with the normal control group, P < 0.01; ** Compared with the model control group, P < 0.01; * Compared with the model control group, P<0.05.

[0059] (2) Rat serum inflammatory factors

[0060] On the 6th day, blood was collected from the rats' orbits, and about 1 ml was placed in an ordinary centrifuge tube, kept at 4°C overnight, and centrifuged at 1000×g for 20 min. The supernatant was collected and stored at -80°C for the detection of inflammatory factors. The results are shown in Table 2.

[0061] The results in Table 2 showed that after infection with beta-hemolytic streptococcus, the levels of IL-1β, IL-6, and TNF-α in the serum of rats in the model control group were significantly increased compared with those in the normal control group (P<0.01), and the beta-hemolytic streptococcus infection was successful in the rats in the model control group.

[0062] Both the high-dose and medium-dose RHTE groups could reduce the levels of IL-1β, IL-6, and TNF-α in the serum of rats after infection with beta-hemolytic streptococcus, and there were significant differences compared with the model control group (P<0.01).

[0063] The low-dose RHTE group could reduce the serum content of TNF-α, which was significantly different from the model control group (P<0.01).

[0064] Table 2 Serum inflammatory factors in six groups of rats

[0065]

[0066] Note: ## Compared with the normal control group, P < 0.01; ** Compared with the model control group, P<0.01.

[0067] (3) Pathological changes in rat pharyngeal tissue

[0068] The rats were weighed after blood was drawn, and then killed by CO2 anesthesia. The pharyngeal mucosal tissues were obtained and fixed for pathological examination.

[0069] The pathological changes in the pharynx of rats were graded and scored according to the standards in Table 3, and the results were subjected to rank sum test using SPSS 20.0 statistical software, with "-" representing 0 points, "+" representing 1 point, "++" representing 2 points, "+++" representing 3 points, and "++++" representing 4 points for one-way analysis of variance; P < 0.05 was used as the test standard for significant difference.

[0070] Table 3 Grading score table

[0071]

[0072]

[0073] The grading and scoring results of the histopathological changes in the pharynx of the six groups of rats are shown in Table 4 , where mean±SD is the mean plus minus the variance.

[0074] Table 4 Grading and scoring results of pharyngeal histopathological changes in six groups of rats

[0075]

[0076]

[0077] Note: ## Compared with the normal control group, P < 0.01; ** Compared with the model control group, P < 0.01; * Compared with the model control group, P<0.05.

[0078] Normal control group: The rat pharyngeal mucosal epithelial structure was intact, without degeneration, necrosis or hyperplasia, no inflammatory exudate on the surface, no edema and inflammatory cell infiltration in the submucosal layer, no glandular hyperplasia, and normal tissue structure.

[0079] Model control group: Microscopically, the pharyngeal mucosal epithelium was significantly hyperplastic, the submucosal connective tissue was loose, the interstitium was mild to severe congestion and edema, and a large number of inflammatory cells infiltrated, mainly lymphocytes and mononuclear macrophages, neutrophils and eosinophils were also seen, granulomatous tissue hyperplasia was seen in some areas, the glands were significantly hypertrophic and hyperplastic, the secretion was increased, and local necrosis or cystic changes occurred, which were significantly different from the normal control group (P < 0.01).

[0080] Positive drug group: Most rats had no obvious hyperplasia of pharyngeal mucosal epithelium, the submucosal connective tissue was tight, the interstitium was slightly or mildly edematous with a small amount of inflammatory cell infiltration, and no obvious abnormal changes in the glands, which were significantly alleviated compared with the model control group (P < 0.01).

[0081] In the medium and high dose RHTE groups, the pharyngeal mucosal epithelium of rats was thinner, with segmental hyperplasia, and the submucosal connective tissue was relatively tight, with mild edema and a small amount of inflammatory cell infiltration. The degree of tissue hyperplasia and inflammatory edema was significantly reduced compared with the model control group (P < 0.05).

[0082] RHTE low-dose group: The scope and degree of pharyngeal tissue damage in rats were not significantly different from those in the model control group.

[0083] Moderate and high doses of RHTE can alleviate the histopathological damage caused by beta-hemolytic streptococcal infection.

[0084] 3. In vitro experiments

[0085] 1. Experimental Materials

[0086] Test drug: throat lozenge extract stock solution (RHTE) prepared in Example.

[0087] Positive control drug: Amoxicillin capsules, a product of Zhuhai United Pharmaceutical Co., Ltd. Zhongshan Branch, batch number: 39002211, production date: 2023.09.22, valid until: 2026.03, specification: 0.25g / capsule.

[0088] Experimental bacteria: beta-hemolytic Streptococcus, purchased from Beina Chuanglian Biotechnology Research Institute; Staphylococcus aureus, purchased from General Microbiology Center of China Microbiological Culture Collection Administration.

[0089] 2. Experimental methods

[0090] Before the test, 200 μL of each of beta-hemolytic streptococcus and Staphylococcus aureus were inoculated into test tubes containing 30 ml of broth medium and cultured in a 37°C incubator for 18 h. The turbidimeter was used to adjust the concentration to 1×10 7 / mL.

[0091] Normal control tubes, bacterial control tubes, positive drug control tubes, and RHTE control tubes with different dilution ratios were set up for beta-hemolytic streptococci and Staphylococcus aureus, respectively. 2 mL of broth medium was added to the normal control tube and bacterial control tube, and 2 mL of broth medium and amoxicillin were added to the positive drug control tube, with a configuration of amoxicillin 25 mg / ml. Nine centrifuge tubes were taken, and RHTE was diluted 2-512 times with nutrient broth medium, with a final volume of 2 mL in each tube, as RHTE control tubes with different dilution ratios.

[0092] 0.2 mL of cultured beta-hemolytic Streptococcus and Staphylococcus aureus were added to each tube of their respective groups except the normal control tube. The normal control tube was added with an equal amount of nutrient broth medium and placed in a 36°C constant temperature shaking incubator. After 24 hours, the bacterial growth in each tube was observed to determine the minimum drug concentration (MIC) without bacterial growth.

[0093] 3. Experimental results

[0094] The results of in vitro antibacterial test are shown in Table 5.

[0095] Table 5 In vitro antibacterial test results

[0096]

[0097] Note: “-” indicates no bacterial growth and “+” indicates bacterial growth

[0098] The results showed that after 24 hours of culture, there was significant bacterial growth in the bacterial control group, while the liquid in the normal control group was clear and transparent with no bacterial growth.

[0099] RHTE had no significant inhibitory effect on Staphylococcus aureus within the tested concentration range; RHTE diluted 2 and 4 times had a certain inhibitory effect on beta-hemolytic Streptococcus, so the minimum inhibitory concentration was RHTE diluted 4 times.

[0100] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. An application of a throat lozenge extract in the preparation of a product for resisting beta-hemolytic streptococcal infection, wherein the throat lozenge extract comprises grass jelly, liquorice, chrysanthemum, honeysuckle and monk fruit.

2. The use according to claim 1, characterized in that The throat lozenge extract is prepared from the following raw materials in parts by weight: 30-60 parts of grass jelly, 10-30 parts of liquorice, 5-20 parts of chrysanthemum, 0.5-4 parts of honeysuckle and 3-15 parts of monk fruit.

3. The use according to claim 2, characterized in that The throat lozenge extract contains 2-2.5 parts of honeysuckle and 11-13 parts of chrysanthemum per 100 parts.

4. The use according to claim 3, characterized in that The throat lozenge extract is prepared by the following steps: S1, extracting the raw material with water and filtering to obtain an extract; S2, centrifuging the extract obtained in step S1; S3, separating and concentrating the centrifuged extract obtained in step S2 to obtain a concentrated solution; S4, sterilizing the concentrated solution obtained in step S3 and setting it aside to obtain the original drug solution.

5. The use according to claim 4, characterized in that In step S1, the amount of water is 8-16 times the weight of the raw material, the temperature of the water is 80-99° C., and the extraction time is 1-1.5 h.

6. The use according to claim 5, characterized in that Step S2 is: cooling the extract to 15-25°C and then centrifuging; The centrifugal speed is 5000-10000 r / min.

7. The use according to claim 6, characterized in that The extract separation and concentration in step S3 is to separate and concentrate the extract through multiple stages of continuous membrane elements; The solid content of the concentrated solution in step S3 is 10%-25%.

8. The use according to claim 7, characterized in that The sterilization temperature in step S4 is 136-140° C. and the time is 15-40 seconds.

9. The use according to any one of claims 1 to 8, characterized in that: The product for resisting beta-hemolytic streptococcal infection is any one of food, health product and medicine.

10. A product for resisting beta-hemolytic streptococcal infection, characterized in that: The invention comprises the throat lozenge extract according to any one of claims 1 to 9.

Images