A nardoo extract having blood lipid regulating, damp dispelling and chronic bronchitis relieving properties and a method of preparation thereof

Abstract

The present application belongs to the technical field of microbial fermentation, and particularly relates to a Marsilea extract with the functions of regulating blood lipid, removing dampness and relieving chronic bronchitis and a preparation method thereof. The preparation method comprises the following steps: (1) freeze-thawing: Marsilea slurry is frozen at-20 to-25 DEG C for 1 to 3 hours, and then dissolved at 25 to 35 DEG C to obtain a freeze-thawed product; (2) first enzymolysis: a composite enzyme is added to the freeze-thawed product for enzymolysis to obtain an enzymolysis liquid A; (3) second enzymolysis: Bacillus subtilis neutral protease is added to the enzymolysis liquid A for enzymolysis, and the enzyme is inactivated by high-temperature treatment after the enzymolysis to obtain an enzymolysis liquid; (4) fermentation: the enzymolysis liquid is inoculated with Lactobacillus gasseri KB24 for fermentation to obtain a fermentation product; and (5) separation: the fermentation product is separated into solid and liquid, the supernatant is concentrated and freeze-dried to obtain the Marsilea extract. The Marsilea extract prepared by the method has the functions of regulating blood lipid, removing dampness and relieving chronic bronchitis.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a herb extract that regulates blood lipids, removes dampness, and relieves chronic bronchitis, and its preparation method. Background Technology

[0002] Mesona chinensis, also known as grass jelly, is an annual or perennial herb belonging to the genus Mesona in the Lamiaceae family. It is mainly distributed in Fujian, Guangdong, and Yunnan provinces. It has a long history of medicinal and edible uses, and is considered a natural plant with both medicinal and culinary uses. The chemical components of Mesona chinensis include polysaccharides, flavonoids, terpenes, vitamins, and phenols. Flavonoids in Mesona chinensis have been shown to inhibit cancer cell growth and lower blood pressure; polysaccharides enhance immunity; and aromatic compounds have cooling, detoxifying, and diuretic effects. Its efficacy in treating sore throats was already known during the Xianfeng era of the Qing Dynasty. In addition, it possesses antioxidant, hypoglycemic, antibacterial, and anti-hypoxia properties.

[0003] Current research on the preparation process of Mesona chinensis is relatively limited, mostly relying on a single extraction system, such as single enzymatic hydrolysis, single microbial fermentation, or organic solvent extraction. For example, Chinese patent CN119591739A discloses a method for preparing Mesona chinensis polysaccharide with glucose-regulating activity. The preparation method includes micro-fermentation drying, raw material pretreatment (stirring with anhydrous ethanol), pulping, targeted polysaccharide extraction (enzymatic hydrolysis with a complex protease of cellulase, pectinase, and papain), separation, purification, and drying to obtain Mesona chinensis polysaccharide, which has the ability to regulate glucose metabolism. This patent only uses a single enzymatic extraction method, and its efficacy is only focused on blood glucose management.

[0004] For example, Chinese patent CN12156990A discloses a method for preparing insoluble dietary fiber from *Gynostemma pentaphyllum* with high bound phenol content. This method extracts insoluble dietary fiber from *Gynostemma pentaphyllum* through a "defatting-compound enzymatic hydrolysis-gradient washing" process. The enzymatic hydrolysis uses α-amylase, protease, and saccharifying enzyme sequentially. The prepared insoluble dietary fiber from *Gynostemma pentaphyllum* achieves a hypoglycemic effect through multiple mechanisms, including water absorption and swelling, glucose adsorption, delayed glucose dialysis rate, antioxidant effects, and inhibition of α-amylase activity. This patent also only uses enzymatic hydrolysis, focusing its efficacy on lowering blood sugar.

[0005] For example, Chinese patent CN104387488A discloses a method for extracting mesona chinensis polysaccharides through microbial fermentation. This method includes steps such as pulverization, initial extraction, preparation of fermentation bacterial seed liquid, inoculation, fermentation, secondary extraction, coarse filtration and centrifugation, membrane concentration, alcohol precipitation, and drying. This method combines bio-fermentation, membrane concentration, and alcohol precipitation technologies, resulting in high yield of mesona chinensis polysaccharides, environmentally friendly processes, and low production costs. However, the Aspergillus niger and Aspergillus oryzae used in the fermentation bacterial seed liquid require a high degree of sterility and are prone to contamination during production, which is detrimental to industrial-scale production.

[0006] In the field of traditional Chinese medicine fermentation technology, lactic acid bacteria are widely used in the processing of traditional Chinese medicine due to their high biocompatibility and broad market acceptance. They can promote the dissolution and transformation of effective components in traditional Chinese medicine and assist in the efficient exertion of its efficacy. However, in the existing technology, there is a lack of research reports on the combined application of enzymatic hydrolysis and lactic acid bacteria fermentation to prepare herb extracts, and there are also few reports on their efficacy in improving the inflammation of chronic bronchitis. Summary of the Invention

[0007] Therefore, this invention provides a novel method for preparing Mesona chinensis extract by sequentially using freeze-thaw, enzymatic hydrolysis, and lactic acid bacteria fermentation. The Mesona chinensis extract obtained by this method has the effects of regulating blood lipids, removing dampness, and relieving chronic bronchitis.

[0008] To achieve the above objectives, the technical content of the present invention includes:

[0009] On the one hand, the present invention provides a method for preparing a herb extract that regulates blood lipids, removes dampness, and relieves chronic bronchitis, comprising the following steps:

[0010] (1) Freeze-thaw: The herb slurry was frozen at -20℃ to -25℃ for 1-3 hours, and then thawed at 25-35℃ to obtain the freeze-thawed product;

[0011] (2) First enzymatic hydrolysis: Add a compound enzyme to the frozen-thawed material to hydrolyze it, and obtain hydrolysate A;

[0012] (3) Second enzymatic hydrolysis: Add Bacillus subtilis neutral protease to the enzymatic hydrolysate A for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, inactivate the enzyme by high temperature to obtain the enzymatic hydrolysate;

[0013] (4) Fermentation: Lactobacillus gasseri KB24 was inoculated into the enzymatic hydrolysate and fermented to obtain the fermentation product;

[0014] (5) Separation: The fermentation product is separated into solid and liquid components, and the supernatant is concentrated and freeze-dried to obtain the mesona extract;

[0015] Preferably, the Lactobacillus gasseri KB24 has the accession number CGMCC No. 35180.

[0016] More preferably, the inoculum size of Lactobacillus gasseri KB24 is 3-10%. Specifically, the inoculum size refers to V / V, that is, the ratio of the volume of Lactobacillus gasseri KB24 bacterial suspension to the volume of the fermentation system.

[0017] More preferably, the viable count of the *Lactobacillus gasseri* KB24 is 1 × 10⁻⁶. 11 CFU / g or 1×10 11 CFU / mL or higher.

[0018] In some embodiments of the present invention, the inoculation of *Lactobacillus gasseri* KB24 includes activating the strain. The activation method involves activating the strain twice consecutively in MRS liquid medium at a 2% inoculation rate, allowing the strain to gradually regain its viability. After culturing at 37°C for 18 hours, the *Lactobacillus gasseri* KB24 culture is centrifuged at 5000 rpm for 15 minutes and the bacterial cells are collected. The bacterial cells are washed three times repeatedly with sterile physiological saline to remove the culture medium. The bacterial cells are resuspended in a final 10% sterile skim milk solution. The skim milk resuspension is aliquoted into preservation tubes and stored at -80°C for later use. One tube of skim milk resuspension is taken out and viable cell counts are performed using a serial dilution method. Before use, the bacterial solution is diluted with fresh sterile skim milk solution to achieve a final viable cell concentration of 1 × 10⁻⁶. 11 CFU / mL.

[0019] In some embodiments of the present invention, the MRS culture medium comprises 10.0 g / L peptone, 8.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 14.0 g / L agar, and 1.0 g / L Tween 80.

[0020] Preferably, the complex enzyme includes cellulase and hemicellulase; more preferably, the complex enzyme is composed of cellulase and hemicellulase.

[0021] Preferably, the amount of the compound enzyme added is 1%-5%. Specifically, the amount added refers to g / v; that is, the weight of the compound enzyme accounts for 1-5% of the volume of the fermentation system.

[0022] Preferably, the ratio of vitaminase to hemicellulase is (0.5-1):(1-3).

[0023] Preferably, the amount of Bacillus subtilis neutral protease added is 0.5%-3%. Specifically, the amount added refers to g / v; that is, the weight of the Bacillus subtilis neutral protease accounts for 0.5-3% of the fermentation system volume.

[0024] Preferably, the cellulase has an enzyme activity of 20,000 U / g to 40,000 U / g; the hemicellulase has an enzyme activity of 6,000 U / g to 10,000 U / g; and the Bacillus subtilis neutral protease has an enzyme activity of 80,000 U / g to 120,000 U / g.

[0025] Preferably, the preparation method of the herb slurry is as follows: herb and water are mixed evenly at a material-to-liquid ratio of 1:(8-10) to obtain herb slurry.

[0026] Preferably, the method for preparing the freeze-thawed product is as follows: freeze the herb slurry at -20°C to -25°C for 2 hours, and then dissolve it at 35°C until it is completely watery to obtain the freeze-thawed product.

[0027] Preferably, step (2) further includes adjusting the pH of the frozen-thawed material to 4.5-6.5 before the first enzymatic hydrolysis.

[0028] Preferably, step (3) further includes adjusting the pH of the enzymatic hydrolysate A to 8.0-9.0 before the second enzymatic hydrolysis.

[0029] Preferably, the temperature of the first enzymatic hydrolysis in step (2) is 40-50℃ and the time is 30-60min.

[0030] Preferably, the temperature of the second enzymatic hydrolysis in step (3) is 40-50℃ and the time is 60-90min.

[0031] Preferably, the fermentation temperature in step (4) is 30-45℃ and the time is 15-25h.

[0032] Preferably, the high-temperature sterilization temperature in step (3) is 90-110℃ and the time is 40-60min.

[0033] On the other hand, the present invention also provides the application of the herb extract obtained by the above preparation method in the preparation of products for regulating blood lipids, removing dampness and relieving chronic bronchitis.

[0034] Compared with the prior art, the present invention has the following advantages: 1. The present invention provides a method for preparing Mesona chinensis extract, which innovatively combines freeze-thaw, step-by-step enzymatic hydrolysis and fermentation. In this method, Mesona chinensis slurry is frozen and then thawed. During the freeze-thaw process, the mechanical action of ice crystals inside the Mesona chinensis cells on the cell wall can break the cell wall, which is conducive to the dissolution of effective substances. Then, an enzyme preparation is added to further decompose the effective components in Mesona chinensis. Finally, Lactobacillus gasseri is added for fermentation. The inventors found that Lactobacillus gasseri KB24 has a better effect on releasing active substances than other lactic acid bacteria. As a result, the obtained Mesona chinensis extract has better effects on regulating blood lipids, removing dampness and relieving chronic bronchitis.

[0035] 2. This invention provides a method for preparing Mesona chinensis extract. The enzyme preparations selected are cellulase, hemicellulase, and Bacillus subtilis neutral protease. Cellulase and hemicellulase are first used for enzymatic hydrolysis. Cellulase can destroy the fibrous skeleton in the Mesona chinensis cell wall, releasing the active ingredients, while hemicellulase can degrade hemicellulase, breaking down the fibrous structure and further releasing the active ingredients. Subsequently, the Bacillus subtilis neutral protease is used to decompose Mesona chinensis cell wall binding proteins, lipoproteins, or proteoglycan complexes, further releasing the active ingredients and degrading molecular weight. The inventors have found that the three enzyme preparations and the distributed enzymatic hydrolysis mode selected in this invention can significantly improve the effects of the obtained Mesona chinensis extract in regulating blood lipids, removing dampness, and relieving chronic bronchitis.

[0036] 3. The preparation method of the herb extract provided by the present invention, by constructing a novel preparation method, consists of freeze-thaw, step-by-step enzymatic hydrolysis and fermentation. There is a synergistic effect between the above steps and between the selected enzyme preparation and fermentation agent, so that the prepared herb extract can have better effects in regulating blood lipids, removing dampness and relieving chronic bronchitis.

[0037] 4 The herbal extract provided by this invention is the first to demonstrate its efficacy in improving and relieving chronic bronchitis, and it also shows more prominent effects in regulating blood lipids and removing dampness. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products. Products from different manufacturers do not have a significant impact on the effect.

[0039] In the following examples, the source information for some products is as follows:

[0040] Lactobacillus gasseri KB24 was deposited on July 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35180, and has been published in Chinese patent CN121294257A.

[0041] Lactobacillus gasseri JCM1131 was purchased from the China General Microbiological Culture Collection Center (CGMCC), CGMCC number CGMCC 1.3396.

[0042] Lactiplantibacillus plantarum 124-2 was purchased from the China General Microbiological Culture Collection Center (CGMCC), catalog number CGMCC 1.124.

[0043] Cellulase was purchased from Shandong Longket Enzyme Preparation Co., Ltd., with an enzyme activity of 20,000 U / g; hemicellulase was purchased from Anhui Yuanzheng Bioengineering Co., Ltd., with an enzyme activity of 6,000 U / g; Bacillus subtilis neutral protease was purchased from Jiangsu Yihaotian Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g; pectinase was purchased from Shandong Longket Enzyme Preparation Co., Ltd., with an enzyme activity of 30,000 U / g; acidic protease was purchased from Shandong Longket Enzyme Preparation Co., Ltd., with an enzyme activity of 100,000 U / g; alkaline protease was purchased from Nanjing Pangbo Bioengineering Co., Ltd., with an enzyme activity of 100,000 U / g; and β-glucanase was purchased from Jiangsu Weizhirun Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g.

[0044] Experimental Example 1: Microbial Activation and Preparation of Bacterial Suspension

[0045] 1. Experimental strains

[0046] In this invention, Lactobacillus gasseri KB24 was isolated from pickled vegetable brine and deposited at the China General Microbiological Culture Collection Center on July 10, 2025, with accession number CGMCC No. 35180.

[0047] Lactobacillus gasseri JCM1131 was purchased from the China General Microbiological Culture Collection Center (CGMCC), CGMCC number 1.3396.

[0048] Lactobacillus plantarum was purchased from the China General Microbiological Culture Collection Center (CGMCC), CGMCC number 1.12974.

[0049] 2. Activation and preparation of bacterial suspension

[0050] Lactobacillus gasseri KB24, Lactobacillus gasseri (CGMCC 1.3396), or Lactobacillus plantarum (CGMCC 1.12974) were activated twice consecutively in MRS liquid medium at a 2% inoculation rate, allowing the strains to gradually regain viability. After incubation at 37°C for 18 hours, the culture medium was centrifuged at 5000 rpm for 15 minutes, and the bacterial cells were collected. The bacterial cells were washed three times with sterile physiological saline to remove the culture medium from the cells. The bacterial cells were resuspended in the final 10% sterile skim milk solution, and the resuspended skim milk solution was aliquoted into preservation tubes and stored at -80°C for later use. One tube of skim milk resuspended was used for viable cell counting using a serial dilution method. Before use, the bacterial suspension was diluted with fresh sterile skim milk solution to achieve a final viable cell concentration of 1 × 10⁻⁶. 11 CFU / mL.

[0051] MRS agar medium was purchased from Qingdao Haibo Biotechnology Co., Ltd., catalog number HB0384. The composition was: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 14.0 g / L, Tween 80 1.0 g / L, pH 6.5 ± 0.2, 25℃.

[0052] Preparation of liquid culture medium: Accurately weigh 54g of MRS agar medium, dissolve it in 1000mL of distilled water, dispense it into 50mL Erlenmeyer flasks, and autoclave at 121℃ for 15 min for later use.

[0053] Example 1: Preparation of Mesona chinensis extract

[0054] This embodiment provides a method for preparing Mesona chinensis extract, including the following steps:

[0055] (1) Preparation of Mesona chinensis slurry: Mesona chinensis powder and water are mixed at a ratio of 1:10 (w / v) to obtain Mesona chinensis slurry; (2) Freeze-thaw: The Mesona chinensis slurry is frozen at -20℃ for 2 hours, and then placed in a 35℃ water bath to thaw until it is completely dissolved into a watery state, thus obtaining the freeze-thawed product; (3) First enzymatic hydrolysis: The pH of the freeze-thawed product is adjusted to 5.0, and 1% cellulase and 1% hemicellulase are added and enzymatically hydrolyzed in a 45℃ water bath for 40 minutes to obtain enzymatic hydrolysate A; (4) Second enzymatic hydrolysis Secondary enzymatic hydrolysis: Adjust the pH of the enzymatic hydrolysate A to 8.5, add 2.5% Bacillus subtilis neutral protease and enzymatically hydrolyze in a 45℃ water bath for 80 min, then adjust the temperature to 105℃ for high-temperature enzyme inactivation sterilization for 50 min to obtain the enzymatic hydrolysate; (5) Fermentation: Inoculate the enzymatic hydrolysate with 5% Lactobacillus gasseri KB24 (prepared in Experimental Example 1), and ferment at 35℃ for 20 h to obtain the fermentation product; (6) Separate the solid and liquid of the fermentation product, and freeze-dry the supernatant to obtain the herb extract.

[0056] Example 2

[0057] This embodiment provides a method for preparing Mesona chinensis extract, including the following steps:

[0058] (1) Preparation of Mesona chinensis slurry: Mesona chinensis powder and water are mixed at a ratio of 1:10 (w / v) to obtain Mesona chinensis slurry; (2) Freeze-thaw: The Mesona chinensis slurry is frozen at -25℃ for 3 hours, and then placed in a 35℃ water bath to thaw until it is completely dissolved into a watery state, thus obtaining the freeze-thawed product; (3) First enzymatic hydrolysis: The pH of the freeze-thawed product is adjusted to 5.0, and 0.5% cellulase and 2% hemicellulase are added and enzymatically hydrolyzed in a 50℃ water bath for 60 minutes to obtain enzymatic hydrolysate A; (4) (5) Second enzymatic hydrolysis: Adjust the pH of the enzymatic hydrolysate A to 8.5, add 1% Bacillus subtilis neutral protease and enzymatically hydrolyze in a 50℃ water bath for 60 min, then adjust the temperature to 105℃ for high-temperature enzyme inactivation sterilization for 60 min to obtain the enzymatic hydrolysate; (6) Fermentation: Inoculate the enzymatic hydrolysate with 8% Lactobacillus gasseri KB24 (prepared in Experiment Example 1), and ferment at 35℃ for 18 h to obtain the fermentation product; (7) Separate the solid and liquid of the fermentation product, and freeze-dry the supernatant to obtain the herb extract.

[0059] Comparative Example 1

[0060] This comparative example provides a herbal extract, the preparation method of which differs from that of Example 1 only in that hemicellulase is replaced with pectinase, otherwise it is no different from Example 1.

[0061] Comparative Example 2

[0062] This comparative example provides a herbal extract, the preparation method of which differs from that of Example 1 only in that the neutral protease of Bacillus subtilis is replaced with pectinase; specifically: step (4) second enzymatic hydrolysis: adjust the pH of the enzymatic hydrolysate A to 3.5, add 2% pectinase and enzymatically hydrolyze in a 45℃ water bath for 80 min, and then adjust the temperature to 105℃ for high-temperature enzyme inactivation sterilization for 50 min to obtain the enzymatic hydrolysate; the remaining steps are the same as in Example 1.

[0063] Comparative Example 3

[0064] This comparative example provides a herbal extract, the preparation method of which differs from that of Example 1 only in that the neutral protease of Bacillus subtilis is replaced with alkaline protease. Specifically, step (4) second enzymatic hydrolysis: adjust the pH of the hydrolysate A to 10, add 2% alkaline protease and hydrolyze in a 45°C water bath for 80 min, then adjust the temperature to 105°C for high-temperature enzyme inactivation and sterilization for 50 min to obtain the hydrolysate; the remaining steps are the same as in Example 1.

[0065] Comparative Example 4

[0066] This comparative example provides a herbal extract, the preparation method of which differs from that of Example 1 only in that the neutral protease of Bacillus subtilis is replaced with acidic protease. Specifically, step (4) second enzymatic hydrolysis: adjust the pH of the hydrolysate A to 3.0, add 2% acidic protease and hydrolyze in a 45°C water bath for 80 min, then adjust the temperature to 105°C for high-temperature enzyme inactivation and sterilization for 50 min to obtain the hydrolysate; the remaining steps are the same as in Example 1.

[0067] Comparative Example 5

[0068] This comparative example provides a mesona chinensis extract, the preparation method of which includes the following steps:

[0069] (1) Preparation of Mesona chinensis slurry: Mesona chinensis powder and water are mixed at a ratio of 1:10 (w / v) to obtain Mesona chinensis slurry; (2) Decoction: The Mesona chinensis slurry is decocted for 1.5 hours and then cooled to 45°C to obtain a mixed liquid; (3) Enzymatic hydrolysis: The pH of the mixed liquid is adjusted to 5.0, and 1% cellulase, 1% pectinase and 2.5% β-glucanase are added and enzymatically hydrolyzed in a water bath at 45°C for 120 min. Then the temperature is adjusted to 105°C for high-temperature enzyme inactivation and sterilization for 50 min to obtain the enzymatic hydrolysate; (4) Fermentation: 5% Lactobacillus gasseri KB24 is inoculated into the enzymatic hydrolysate and fermented at 35°C for 20 h to obtain the fermentation product; (5) The fermentation product is separated into solid and liquid, and the supernatant is freeze-dried to obtain Mesona chinensis extract.

[0070] Comparative Example 6

[0071] This comparative example provides a mesona chinensis extract, the preparation method of which includes the following steps:

[0072] (1) Preparation of Mesona chinensis slurry: Mesona chinensis powder and water are mixed at a ratio of 1:10 (w / v) to obtain Mesona chinensis slurry; (2) Freeze-thaw: The Mesona chinensis slurry is frozen at -20℃ for 2 hours, and then placed in a 35℃ water bath to thaw. When it is completely dissolved into water, the freeze-thawed product is obtained; (3) Enzymatic hydrolysis: The pH of the freeze-thawed product is adjusted to 6.5, and 1% cellulase, 1% hemicellulase and 2.5% Bacillus subtilis neutral protease are added and enzymatically hydrolyzed in a 45℃ water bath for 120 minutes. Then the temperature is adjusted to 105℃ for high-temperature enzyme inactivation and sterilization for 50 minutes to obtain the enzymatic hydrolysate; (4) Fermentation: 5% Lactobacillus gasseri KB24 is inoculated into the enzymatic hydrolysate and fermented at 35℃ for 20 hours to obtain the fermented product; (5) The fermented product is separated into solid and liquid, and the supernatant is freeze-dried to obtain Mesona chinensis extract.

[0073] Comparative Example 7

[0074] This comparative example provides a herbal extract, the preparation method of which differs from that of Example 1 only in that Lactobacillus gasseri KB24 is replaced with Lactobacillus plantarum (CGMCC 1.12974), otherwise it is no different from Example 1.

[0075] Comparative Example 8

[0076] This comparative example provides a herbal extract, the preparation method of which differs from that of Example 1 only in that Lactobacillus gasseri KB24 is replaced with Lactobacillus gasseri JCM1131, otherwise there is no difference from Example 1.

[0077] Comparative Example 9

[0078] This comparative example provides a method for preparing Mesona chinensis extract, comprising the following steps:

[0079] (1) Preparation of Mesona chinensis slurry: Mesona chinensis powder and water are mixed at a ratio of 1:10 (w / v) to obtain Mesona chinensis slurry; (2) Freeze-thaw: The Mesona chinensis slurry is frozen at -20℃ for 2 hours, and then placed in a 35℃ water bath to thaw until it is completely dissolved into a watery state, thus obtaining the freeze-thawed product; (3) First enzymatic hydrolysis: The pH of the freeze-thawed product is adjusted to 5.0, and 1% cellulase and 1% hemicellulase are added and enzymatically hydrolyzed in a 45℃ water bath for 40 minutes to obtain the enzymatic hydrolysate. Liquid A; (4) Second enzymatic hydrolysis: Adjust the pH of the enzymatic hydrolysate A to 8.5, add 2.5% Bacillus subtilis neutral protease and enzymatically hydrolyze in a 45℃ water bath for 80 min, then adjust the temperature to 105℃ for high-temperature enzyme inactivation sterilization for 50 min to obtain the enzymatic hydrolysate; (5) Fermentation: Inoculate the enzymatic hydrolysate with 5% sterile water and ferment at 35℃ for 20 h to obtain the fermentation product; (6) Separate the solid and liquid of the fermentation product, and freeze-dry the supernatant to obtain the herb extract.

[0080] Comparative Example 10

[0081] This comparative example provides a method for preparing Mesona chinensis extract, comprising the following steps:

[0082] (1) Preparation of Mesona chinensis slurry: Mesona chinensis powder and water are mixed at a ratio of 1:10 (w / v) to obtain Mesona chinensis slurry; (2) First enzymatic hydrolysis: The pH of Mesona chinensis slurry is adjusted to 5.0, 1% cellulase and 1% hemicellulase are added and enzymatically hydrolyzed in a 45℃ water bath for 40 min to obtain hydrolysate A; (3) Second enzymatic hydrolysis: The pH of hydrolysate A is adjusted to 8.5, 2.5% Bacillus subtilis neutral protease is added and enzymatically hydrolyzed in a 45℃ water bath for 80 min, and then the temperature is adjusted to 105℃ for high-temperature enzyme inactivation and sterilization for 50 min to obtain hydrolysate; (4) Fermentation: 5% Lactobacillus gasseri KB24 is inoculated into the hydrolysate and fermented at 35℃ for 20 h to obtain fermentation product; (5) The fermentation product is separated into solid and liquid, and the supernatant is freeze-dried to obtain Mesona chinensis extract.

[0083] Experimental Example 2: Rats - Blood Lipid Regulation and Dampness Removal

[0084] Samples to be tested: Herba Mesona chinensis extracts obtained in Examples 1-2 and Comparative Examples 1-10.

[0085] Healthy adult male SPF-grade rats, 8 weeks old and weighing (210±20) g, were randomly divided into 15 groups of 8 rats each after 1 week of acclimatization: model group, blank control group, experimental group (Example 1 group, Example 2 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group, Comparative Example 5 group, Comparative Example 6 group, Comparative Example 7 group, Comparative Example 8 group, Comparative Example 9 group, Comparative Example 10 group) and positive drug control group.

[0086] A rat model of hyperlipidemia with spleen deficiency and dampness was established by feeding the rats with a high-fat diet and using a moist bedding environment.

[0087] The blank control group was fed a normal maintenance diet daily; the model group, experimental group, and positive drug control group were all fed a high-lipid atherosclerosis (AS) diet [composed of 83.75% basal diet + 15% lard + 1.25% cholesterol, purchased from Spiefol (Beijing) Biotechnology Co., Ltd.]. The blank control group was fed normal rat wood shavings bedding, with approximately 70g of wood shavings added each time, and the bedding was changed every other day; the model group, experimental group, and positive drug control group were fed moist bedding, which was prepared by watering the normal rat wood shavings bedding, with approximately 350mL of water added per 70g of bedding to maintain the humidity in the cage at (60±5)%, and the bedding was changed every other day. The model was established for 21 consecutive days. Starting from the 7th day of modeling, the blank control group and the model group were administered distilled water by gavage at 18:00 every day. The experimental group was administered the corresponding herb extract by gavage. The positive drug control group was administered 0.4mg / mL simvastatin solution by gavage for 14 consecutive days.

[0088] The gavage dosage for the experimental group was 2g per day for a human (70kg). The gavage dose for rats was calculated to be 180mg / kg based on the equivalent dose method converted from body surface area ratio. This dose was administered by gavage.

[0089] After modeling, rats were fasted for 12 hours and then anesthetized with 3% sodium pentobarbital solution. Following successful anesthesia, the rats were placed on a board, and their abdomens were disinfected with iodine-soaked cotton balls before dissection. Approximately 5 mL of whole blood was collected from the abdominal aorta into a blood collection tube without anticoagulant. The collected blood was allowed to stand in the blood collection tube for at least 2 hours, allowing for natural stratification. The blood was then centrifuged at 3000×g for 10 min in a high-speed refrigerated centrifuge to extract serum. The levels of total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) in the serum of each group of rats were measured using a fully automated biochemical analyzer. Serum ALD and ADH levels were detected according to the ELISA kit instructions. The ELISA kits for aldosterone (ALD) and antidiuretic hormone (ADH) were used (ELISA kits from ELISA-Linked Biotechnology, batch numbers: ml002876, ml059245).

[0090] The results of blood lipid indicators in each group of rats are shown in Table 1. In the model group, HDL-C levels were significantly decreased, while TG and TC levels were significantly increased, indicating a disorder in lipid metabolism or transport function, thus the model was successfully established. In contrast, groups 1-2 (Examples 1-2) showed significantly increased HDL-C levels and significantly decreased TG and TC levels compared to the model group, with no significant difference from the positive drug control group. This indicates that the *Mesona chinensis* extract provided in this invention can effectively regulate lipid metabolism or transport function in rats, exerting a lipid-lowering effect similar to simvastatin and maintaining normal blood lipid levels.

[0091] Analysis of the effect of enzyme selection on blood lipid indicators in rats: Comparative Example 1 differs from Example 1 in that cellulase is replaced with pectinase; Comparative Example 2 differs from Example 1 in that Bacillus subtilis neutral protease is replaced with pectinase; Comparative Example 3 differs from Example 1 in that Bacillus subtilis neutral protease is replaced with alkaline protease; Comparative Example 4 differs from Example 1 in that Bacillus subtilis neutral protease is replaced with acidic protease; Comparative Example 6 differs from Example 1 in that distributed enzymatic hydrolysis is replaced with one-step enzymatic hydrolysis. As shown in Table 1, the HDL-C level in Comparative Examples 1-4 and Comparative Example 6 is significantly lower than that in Example 1, while the TG and TC levels are significantly higher than those in Example 1. This indicates that the step-by-step enzymatic hydrolysis treatment using cellulase, hemicellulase, and Bacillus subtilis used in this invention can fully release the active ingredients in Mesona chinensis, thereby enabling the Mesona chinensis extract to exert a good effect on regulating blood lipid levels.

[0092] Analysis of the effect of freeze-thaw technology selection on the regulation of blood lipids in rats: Comparative Example 10 differs from Example 1 only in that it does not include the freeze-thaw step, while Comparative Example 5 differs from Example 1 in that it replaces freeze-thaw with hot decoction, and the enzyme preparation is also replaced. As shown in Table 2, Comparative Examples 10 and 5 are significantly less effective than Example 1 in regulating TC, TG, and HDL-C levels, demonstrating that the freeze-thaw pretreatment step provided by this invention can promote the release of active ingredients in Mesona chinensis to a certain extent.

[0093] Analysis of the effects of fermentation and fermentation inoculum selection on the regulation of blood lipids in rats: Comparative Example 9 differed from Example 1 in that it did not involve a fermentation step; Comparative Examples 7-8 differed from Example 1 in that Lactobacillus gasseri KB24 was replaced. As shown in Table 1, there was no significant difference in TC levels between Comparative Example 9 and the model group. In Comparative Examples 7-9, TG and TC levels were significantly higher than in Example 1, while HLD-C levels were significantly lower than in Example 1. This indicates that the fermentation process used in this invention can further enhance the release of active ingredients in Mesona chinensis, and the selected Lactobacillus gasseri KB24 as the fermentation inoculum can significantly enhance the lipid-regulating effect of Mesona chinensis extract.

[0094] In summary, the present invention, through the combined use of freeze-thaw, segmented enzymatic hydrolysis, and fermentation processes, produces a mesona chinensis extract that can lower TC and TG levels and increase HDL-C levels, exhibiting excellent lipid-regulating activity. Furthermore, the synergistic enzymatic hydrolysis by cellulase, pectinase, and Bacillus subtilis neutral protease, combined with fermentation using Lactobacillus gasseri KB24, maximizes the release of active substances and fully leverages the lipid-regulating effects of the mesona chinensis extract.

[0095] Table 1 Comparison of blood lipid levels in rats of different groups (mean ± standard deviation, n=8)

[0096]

[0097] Note: Compared with the same model group, # indicates P < 0.05, ## indicates P < 0.01, and ### indicates P < 0.001; compared with Example 1, & indicates P < 0.05, && indicates P < 0.01, and &&& indicates P < 0.001.

[0098] The comparison results of ADH and ALD levels in each group of rats are shown in Table 2. ADH is a 9-peptide hormone secreted by nerve cells in the supraoptic and paraventricular nuclei of the hypothalamus. After reaching the neurohypophysis via the hypothalamic-pituitary tract, it is released. After binding to V2 receptors on the peritubular membrane of distal convoluted tubules and collecting duct epithelial cells, ADH activates the cAMP-PKA pathway, increasing AQP2 phosphorylation activity, thereby promoting water reabsorption. It is a key regulatory hormone for urine concentration and dilution. Aldosterone is a steroid hormone synthesized and secreted by the zona glomerulosa of the adrenal gland. It has sodium and water retention functions and is a hormone that affects the regulation of extracellular fluid volume and electrolytes, playing an important role in maintaining homeostasis. In this application, the serum ADH and ALD levels of rats in the model group were elevated, indicating the presence of water retention and decreased water metabolism in the rats, confirming the successful establishment of the spleen deficiency and water retention model.

[0099] In Examples 1-2, the levels of ADH and ALD were significantly lower than those in the model group, indicating that the herb extract provided by this invention can improve water metabolism disorders in the body with spleen deficiency and water retention, and has the function of regulating water metabolism.

[0100] Compared with Example 1, Comparative Examples 1-4 showed significantly increased ADH and ALD levels, indicating that the type of enzyme preparation affects the ability of Mesona chinensis extract to regulate water metabolism. The Mesona chinensis extract obtained by using cellulase, hemicellulase and Bacillus subtilis neutral protease as enzyme preparations selected in this invention has a better ability to regulate water metabolism in cases of spleen deficiency and dampness retention.

[0101] The ADH and ALD levels in Comparative Examples 7-9 were significantly higher than those in Example 1, indicating that the choice of fermentation steps and fermentation agents are also key factors in determining the regulatory ability of the water metabolism function of the Mesona chinensis extract. The Mesona chinensis extract obtained by using Lactobacillus gasseri KB24 as the fermentation agent in this invention has a better regulatory effect on water metabolism.

[0102] The levels of ADH and ALD in Comparative Examples 10, 5, and 6 were significantly higher than those in Example 1, indicating that the freeze-thaw pretreatment and the step-by-step enzymatic hydrolysis process of the specific enzyme preparation provided by the present invention both affect the ability of Mesona chinensis extract to regulate water metabolism. The present invention combines freeze-thaw pretreatment, step-by-step enzymatic hydrolysis, and fermentation processes to prepare Mesona chinensis extract with the best effect in improving spleen deficiency and water retention and regulating body fluid metabolism.

[0103] Table 2 Comparison of ADH and ALD levels in rats of different groups (mean ± standard deviation, n=8)

[0104]

[0105] Note: Compared with the same model group, # indicates P < 0.05, ## indicates P < 0.01, and ### indicates P < 0.001; compared with Example 1, & indicates P < 0.05, && indicates P < 0.01, and &&& indicates P < 0.001.

[0106] Experiment Example 3: Rat Experiment - Relief of Chronic Bronchitis

[0107] SPF-grade male SD rats (6 weeks old, weighing 180-220g) were randomly divided into 15 groups of 8 rats each after one week of acclimatization: model group, blank control group, experimental group (Example 1 group, Example 2 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group, Comparative Example 5 group, Comparative Example 6 group, Comparative Example 7 group, Comparative Example 8 group, Comparative Example 9 group, Comparative Example 10 group) and positive drug control group.

[0108] A rat model of chronic bronchitis was established using lipopolysaccharide intratracheal instillation and fumigation.

[0109] The specific method is as follows: (1) After anesthetizing the rats on the 1st and 14th days, cut open the neck and drip 200 μg of lipopolysaccharide solution (1 μg / uL, dissolved in physiological saline) into the trachea. After the drip is finished, suture the wound and disinfect the sutured wound with povidone-iodine. The rats are fasted but not allowed to drink water for 12 hours.

[0110] (2) On days 2-13 and 15-28, rats in each modeling group were placed in a 60cm×60cm×60cm smoke chamber. Burned Hongqiqu cigarettes were placed in the smoke chamber, with a total of 10 cigarettes used each time for 30 minutes, twice daily. Except for the blank control group, all other groups underwent chronic bronchitis rat modeling. The blank control group underwent sham surgery, with the same volume of physiological saline instilled into the trachea, and did not undergo cigarette smoke inhalation.

[0111] (3) Drug intervention was started after the second infusion of lipopolysaccharide (D14), i.e., simultaneous gavage administration from D15 to D28. The experimental group was administered gavage at a dose of 180 mg / kg, while the positive control was administered dexamethasone tablets (0.2 mg / kg) by gavage. The administration lasted for 14 days.

[0112] (4) After the last administration, the rats were anesthetized, and blood was collected from the abdominal aorta. After resting for 30 min, the blood was centrifuged at 1500 r / min for 30 min, and the serum was collected. The concentrations of IL-1β, IL-6, and TNF-α in the serum were measured sequentially according to the instructions of the ELISA kit; the concentration of MDA in the serum was measured according to the instructions of the MDA kit (the IL-1β, IL-6, and TNF-α kits were purchased from Andygene, and the MDA kit was purchased from Nanjing Jiancheng Biotechnology Institute). The experimental results are shown in Table 3.

[0113] Table 3. Serum levels of IL-1β, IL-6, TNF-α, and MDA in rats of each group (n=8, mean ± standard deviation)

[0114]

[0115] Note: Compared with the same model group, # indicates P < 0.05, ## indicates P < 0.01, and ### indicates P < 0.001; compared with Example 1, & indicates P < 0.05, && indicates P < 0.01, and &&& indicates P < 0.001.

[0116] Interleukin-1β (IL-1β) is a mediator of inflammatory responses, and abnormally elevated IL-1β levels can exacerbate various types of chronic diseases. IL-6 is a pleiotropic pro-inflammatory cytokine that can regulate the differentiation of immune cells (such as macrophages, T cells, and neutrophils) and thus control the body's inflammation levels. TNF-α is a potent pro-inflammatory cytokine that plays an important role in inflammation, cell proliferation, differentiation, apoptosis, and the immune system. LPS can bind to TLR4 and activate NF-κB, Jun, Fos, and IRFs, inducing the secretion of cytokines such as IL-1β, IL-6, and TNF-α. Cigarette smoke can also induce the production of pro-inflammatory cytokines (such as TNF-α, IL-1β, and IL-6) and increase the accumulation of immune cells in the airways, promoting inflammation.

[0117] MDA content is an important parameter reflecting the body's antioxidant potential and can indirectly reflect the degree of tissue peroxidation damage. Cigarette smoke contains a large number of free radicals and oxidants, which can disrupt the body's oxidation and antioxidant balance and induce oxidative stress.

[0118] As shown in Table 3, the levels of TNF-α, IL-1β, IL-6, and MDA in the model group were significantly higher than those in the blank control group, indicating that LPS combined with cigarette smoke inhalation can induce a significant increase in inflammation and oxidative stress in rats, and that the chronic bronchitis model was successfully established.

[0119] As shown in Table 3, the levels of TNF-α, IL-1β, IL-6, and MDA in the Example 1-2 groups were significantly lower than those in the model group, and there were no significant differences between the indicators and the positive drug control group. This indicates that the herb extract provided by the present invention can effectively reduce the inflammation level of rats with chronic bronchitis and alleviate oxidative stress damage, and can be applied to products for the treatment of chronic bronchitis.

[0120] The difference between Comparative Examples 1-4 and Example 1 is that the hemicellulase and Bacillus subtilis neutral protease used in this invention were replaced with pectinase, alkaline protease, or acidic protease. As shown in Table 3, the levels of TNF-α, IL-1β, and IL-6 in Comparative Examples 1-4 were significantly higher than those in Example 1, indicating that the three enzymes selected in this invention—cellulase, pectinase, and Bacillus subtilis neutral protease—can work synergistically to form a good enzymatic synergistic effect, thereby more effectively reducing the inflammation level in rats with chronic bronchitis.

[0121] The difference between Comparative Example 6 and Example 1 is that the step-by-step enzymatic hydrolysis was changed to a one-step enzymatic hydrolysis process. As shown in Table 3, the levels of TNF-α, IL-1β, and IL-6 in Comparative Example 6 were significantly higher than those in Example 1. This indicates that the distributed enzymatic hydrolysis process selected in this invention can give full play to the advantages of enzyme preparations, maximize the enzymatic hydrolysis effect, and thus achieve a better effect in reducing the inflammation level of rats with chronic bronchitis.

[0122] Comparative Example 5 used a partial processing method of Mesona chinensis from Chinese Patent CN105685903A, which involved enzymatic hydrolysis with cellulase, pectinase, and β-glucanase after decoction, followed by fermentation. Comparative Example 10 differed from Example 1 in that it did not include the freeze-thaw step. As shown in Table 3, compared to Example 1, Comparative Examples 5 and 10 showed significantly increased levels of TNF-α, IL-1β, and IL-6. Furthermore, in Comparative Example 5, IL-β and IL-6 did not show a significant decrease compared to the model group. This indicates that the freeze-thaw step before enzymatic hydrolysis and the screening of specific enzyme preparations affect the reduction of inflammation levels by Mesona chinensis extract. In this invention, freeze-thaw and specific enzyme preparations (cellulase, pectinase, and Bacillus subtilis neutral protease) can better release the active ingredients of Mesona chinensis, thereby achieving the optimal effect of reducing inflammation in rats with chronic bronchitis.

[0123] The difference between Comparative Example 9 and Example 1 is that Comparative Example 9 only underwent enzymatic hydrolysis without fermentation. As shown in Table 3, IL-β and IL-6 levels in Comparative Example 9 were not significantly lower than those in the model group, indicating that Comparative Example 9 could not reduce IL-β and IL-6 inflammation levels. This also means that fermentation can significantly improve the inhibitory effect of *Mesona chinensis* extract on inflammatory factors and is an important process step for improving anti-inflammatory activity. The difference between Comparative Examples 7-8 and Example 1 is that *Lactobacillus gasseri* KB24 was replaced. As shown in Table 3, the levels of TNF-α, IL-1β, and IL-6 in Example 1 were significantly lower than those in Comparative Examples 7-8. This means that *Lactobacillus gasseri* KB24 selected in this invention is more suitable as a fermentation substrate after freeze-thaw, cellulase, pectinase, and *Bacillus subtilis* neutral protease hydrolysis, which can further improve the effect of *Mesona chinensis* extract in reducing inflammation in rats with chronic bronchitis.

[0124] Compared with Example 1, Comparative Examples 1-10 showed significantly higher levels of the oxidative stress index MDA, indicating that the Herba Mesona chinensis extract obtained by the present invention through the complete preparation method of freeze-thaw, compound stepwise enzymatic hydrolysis (cellulase, hemicellulase and Bacillus subtilis neutral protease), and fermentation (Lactobacillus KB24) can significantly alleviate the oxidative stress level in rats with chronic bronchitis.

[0125] In summary, this invention provides a novel method for preparing Mesona chinensis extract. Through a synergistic process involving freeze-thaw cycles, a first enzymatic hydrolysis (cellulase and hemicellulase), a second enzymatic hydrolysis (Bacillus subtilis neutral protease), and fermentation (Lactobacillus gasseri KB24), the prepared Mesona chinensis extract not only reduces the levels of TNF-α, IL-1β, IL-6, and MDA in rats with chronic bronchitis, thus lowering their inflammatory and oxidative stress levels, but also regulates TG, TC, HLD-C, ADH, and ALD in rats with hyperlipidemia and spleen deficiency with dampness, achieving both lipid and water metabolism regulation effects.

[0126] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a herb extract of *Mesona chinensis* that regulates blood lipids, removes dampness, and relieves chronic bronchitis, characterized in that, Includes the following steps: (1) Freeze-thaw: The herb slurry was frozen at -20℃ to -25℃ for 1-3 hours, and then thawed at 25-35℃ to obtain the freeze-thawed product; (2) First enzymatic hydrolysis: Add a compound enzyme to the frozen-thawed material to hydrolyze it, and obtain hydrolysate A; (3) Second enzymatic hydrolysis: Add Bacillus subtilis neutral protease to the enzymatic hydrolysate A for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, inactivate the enzyme by high temperature to obtain the enzymatic hydrolysate; (4) Fermentation: Lactobacillus gasseri KB24 was inoculated into the enzymatic hydrolysate and fermented to obtain the fermentation product; (5) Separation: The fermentation product is separated into solid and liquid components, and the supernatant is concentrated and freeze-dried to obtain the mesona extract; The Lactobacillus gasseri KB24 has the accession number CGMCC No. 35180; the complex enzyme includes cellulase and hemicellulase.

2. The method for preparing the herb extract according to claim 1, characterized in that, The inoculation amount of Lactobacillus gasseri KB24 is 3-10%.

3. The method for preparing the herb extract according to claim 1, characterized in that, The amount of the compound enzyme added is 1%-5%; the amount of the Bacillus subtilis neutral protease added is 0.5%-3%.

4. The method for preparing the herb extract according to claim 1, characterized in that, The ratio of vitamin C to hemicellulase is (0.5-1):(1-3).

5. The method for preparing the herb extract according to claim 1, characterized in that, In step (2), the temperature of the first enzymatic hydrolysis is 40-50℃ and the time is 30-60 min; in step (3), the temperature of the second enzymatic hydrolysis is 40-50℃ and the time is 60-90 min.

6. The method for preparing the herb extract according to claim 1, characterized in that, The fermentation temperature in step (4) is 30-45℃ and the time is 15-25h.

7. The method for preparing the herb extract according to claim 1, characterized in that, In step (3), the temperature for high-temperature enzyme inactivation is 90-110℃ and the time is 40-60min.

8. The method for preparing the herb extract according to claim 1, characterized in that, The viable count of the Lactobacillus gasseri KB24 was 1×10⁻⁶. 11 CFU / g or 1×10 11 CFU / mL or higher.

9. The method for preparing the herb extract according to claim 1, characterized in that, The preparation method of the herb slurry is as follows: Herbs and water are mixed evenly at a material-to-liquid ratio of 1:(8-10) to obtain herb slurry.

10. The use of the herb extract prepared by any one of the methods described in claims 1-9 in the preparation of products for regulating blood lipids, removing dampness and relieving chronic bronchitis.

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