Preparation method of isoquercitrin and application thereof in preparation of product for improving lung nodule and nourishing lung

Abstract

This invention discloses an enzymatic preparation process for isoquercetin and its application in the preparation of products for improving pulmonary nodules and nourishing the lungs. The method uses rutin as a raw material, hydrolyzing it under the action of rhamnosidase to obtain high-purity isoquercetin; the process is green and efficient. Experiments show that the obtained isoquercetin can significantly reduce the levels of pulmonary inflammatory factors IL6 and TNFα, alleviate pulmonary edema, and improve pathological changes in pulmonary nodules, exhibiting clear lung-nourishing and moisturizing effects. The isoquercetin of this invention can be used to prepare pharmaceutical or health care products for improving pulmonary nodules, alleviating pulmonary inflammation, and nourishing and moisturizing the lungs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and functional food technology, specifically relating to a method for preparing high-purity isoquercitrin by enzymatic method, and the use of the obtained isoquercitrin in the preparation of products for improving pulmonary nodules, relieving pulmonary inflammation, and nourishing and moisturizing the lungs. Background Technology

[0002] Isoquercetin (isoquercetin glycoside) is a glycoside derivative of the flavonoid compound quercetin, possessing various biological activities such as anti-inflammatory, antioxidant, and anti-allergic effects. Traditional preparations of isoquercetin mainly rely on chemical acid hydrolysis or plant extraction, which suffers from problems such as complex processes, low yields, significant environmental pollution, and difficulty in controlling product purity.

[0003] With increasing air pollution and rising incidence of respiratory diseases, conditions such as pulmonary nodules and chronic lung inflammation are receiving growing attention. Currently, there is a lack of safe and effective natural-source prevention and treatment products in clinical practice. Although the anti-inflammatory effects of quercetin compounds have been reported, systematic research has not yet been conducted on the specific efficacy and commercial applications of its isomer, isoquercetin, especially isoquercetin prepared via a green enzymatic method, in improving pulmonary nodules and chronic lung inflammation. Summary of the Invention

[0004] This invention aims to provide an efficient and environmentally friendly enzymatic preparation process for isoquercetin, and to clarify its application in the preparation of products that improve pulmonary nodules, alleviate pulmonary inflammation, and nourish and moisturize the lungs.

[0005] On the one hand, the present invention provides a method for preparing isoquercetin, which is prepared by hydrolyzing rutin with rhamnosidase.

[0006] Specifically, it includes the following steps: (1) Moisten rutin with methanol, add water and sodium metabisulfite, and adjust the pH to 4.0. 5.0; (2) In 40 Rhamnosidase was added at 50℃ to carry out the enzymatic hydrolysis reaction; (3) After the reaction is complete, separate the solid product, dissolve it in methanol and add activated carbon for decolorization; (4) Concentrate the filtrate, cool and crystallize it, and then separate and dry it to obtain high-purity isoquercitrin.

[0007] Secondly, the present invention provides an application of isoquercetin in the preparation of products that improve pulmonary nodules or relieve pulmonary inflammation, or in products that nourish and moisturize the lungs.

[0008] Preferably, the product for improving pulmonary nodules or relieving pulmonary inflammation, or the lung-nourishing product, includes pharmaceutically or food-grade excipients.

[0009] Preferably, the product for improving pulmonary nodules and relieving pulmonary inflammation is an oral preparation.

[0010] Preferably, the oral preparation is selected from tablets, capsules, granules, powders, oral liquids, or pills.

[0011] Beneficial effects The preparation process of this invention is mild, environmentally friendly, and produces a product with high purity (>95%). This invention is the first to demonstrate through animal experiments that isoquercetin can significantly reduce pulmonary inflammatory factors (IL-6, TNF-α), alleviate pulmonary edema, and improve pathological changes in pulmonary nodules. It has clear effects in improving pulmonary nodules, alleviating pulmonary inflammation, and nourishing and moisturizing the lungs, providing a new natural raw material and product form for the prevention and treatment of pulmonary nodules and chronic pulmonary inflammation. Attached Figure Description

[0012] Figure 1 HE-stained pathological sections of lung tissue from mice in each group. Detailed Implementation

[0013] The present invention will be further described below with reference to embodiments and experimental examples, but the scope of protection of the present invention is not limited thereto.

[0014] Example 1: Preparation of isoquercetin 10.0 g of rutin was placed in a 500 mL three-necked flask and moistened with 10 mL of methanol. 1.0 g of sodium metabisulfite and 120 mL of pure water were added sequentially, and the pH of the system was adjusted to 4.5 with 0.5 mol / L dilute sulfuric acid solution while stirring. The reaction system was heated to 45 °C and stirred for 10 minutes. Then, 2.0 g of rhamnosidase was added, and the reaction was carried out at 45 °C ± 1 °C and a stirring speed of 200 rpm for 5 hours. After the reaction, the mixture was filtered, and the filter cake was washed with a small amount of pure water. The wet filter cake was transferred to a beaker, and 150 mL of methanol and 1.0 g of powdered activated carbon were added. The mixture was stirred at 65 °C and 100 rpm for 1 hour to decolorize. After hot filtration, the filtrate was concentrated under reduced pressure at 50 °C to approximately 1 / 5 of its original volume, and then placed in a refrigerator at 4 °C for overnight crystallization. The sample was filtered again, and the resulting crystals were washed with a small amount of cold methanol. Finally, the sample was dried in a vacuum drying oven at 40°C for 6 hours to obtain 7.2 g of off-white needle-like crystals of isoquercetin. The purity of the product was greater than 95% as determined by high-performance liquid chromatography (HPLC).

[0015] Example 2: Preparation of isoquercetin tablets Take 100.0 g of isoquercetin obtained in Example 1, add 150.0 g of pregelatinized starch, 50.0 g of microcrystalline cellulose, and 2.0 g of magnesium stearate, and mix well. Compress the mixture into tablets containing 100 mg of isoquercetin each using wet granulation or direct tableting processes, and a total of 1000 tablets are produced.

[0016] Evaluation of the efficacy of isoquercetin in improving pulmonary nodules and nourishing the lungs. Laboratory animals: Balb / c mice, half male and half female, 18-22g, provided by Spiford (Suzhou) Biotechnology Co., Ltd., production license: SCXK(Su)2022-0006.

[0017] Instruments and reagents: Instruments: Micropipette (Eppendorf, Germany); Electronic balance (FA2004N, Shanghai Jinghai Instrument Co., Ltd.); Sand Eagle electric thermostatic water bath (HWS-24, Ningguo Sand Eagle Scientific Instrument Co., Ltd.); Paraffin embedding machine (YD-6L, Yidi Medical Equipment Co., Ltd.); Oven (DHG-9070A, Shanghai Jinghong Experimental Equipment Co., Ltd.); Rotary paraffin microtome (Finesse 325, Thermo Fisher Scientific, USA); 96-well microplate reader (CMax Plus, Molecular Devices, Shanghai); UV-Vis spectrophotometer (Lingxi UV-3300, Shanghai Lingxi Instrument Co., Ltd.).

[0018] Reagents: Mouse interleukin-6 (IL-6) ELISA kit (Catalog No. JL20268-96T, Shanghai Jianglai Biotechnology Co., Ltd.); Mouse tumor necrosis factor-α (TNF-α) ELISA kit (Catalog No. JL10484-96T, Shanghai Jianglai Biotechnology Co., Ltd.); Hematoxylin-eosin staining solution (Catalog No. E489517-2×500mL, Shanghai Aladdin Biochemical Technology Co., Ltd.); Ovalbumin (OVA) (Catalog No. E6337-25g, Shanghai Maclean Biochemical Technology Co., Ltd.); Lipopolysaccharide (LPS) (Catalog No. L118716-100mg, Shanghai Aladdin Biochemical Technology Co., Ltd.); Aluminum hydroxide (Catalog No. A110531-1kg, Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0019] Experimental grouping and sample distribution After acclimatization, the mice were randomly divided into 6 groups (n=10 per group): a normal control group and a model control group. The study included a positive control group (dexamethasone, 2 mg / kg), a low-dose isoquercitrin tablet group (100 mg / kg), a medium-dose isoquercitrin tablet group (200 mg / kg), and a high-dose isoquercitrin tablet group (400 mg / kg).

[0020] The administration method was oral gavage, with a gavage volume of 10 mL / kg BW. The normal control group and the model control group were given the same volume of purified water; the positive control group was given dexamethasone in week 6 of the experiment and purified water in the first 5 weeks; each isoquercetin tablet group was given the corresponding dose of sample (prepared using the tablets prepared in Example 2) daily for 6 consecutive weeks.

[0021] Model establishment: Except for the normal control group, the other groups were given 10 μL of saline solution containing 20 μg LPS via nasal drops once a week for 6 consecutive weeks. In addition, during the first and fourth weeks of the experiment, 10 μL of saline solution containing 20 μg OVA and 40 μg aluminum hydroxide adjuvant were given via nasal drops to establish a chronic lung inflammation and pulmonary nodule model.

[0022] Detection indicators and results 1. Serum inflammatory factor levels: Two hours after the last administration, mice were sacrificed, blood was collected, and serum was separated. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were detected by ELISA. The results are shown in Table 1. Isoquercetin tablets significantly reduced the elevated levels of TNF-α and IL-6 in the serum of model mice in a dose-dependent manner, and the high-dose group was more effective than the positive control drug dexamethasone.

[0023] Table 1. Effects of isoquercitrin tablets on serum IL-6 and TNF-α levels in mice (n = 10)

[0024] Table 2. Compared with the model control group, *p<0.05, ***p<0.001 2. Lung Index After sacrifice, intact lung tissue was harvested and weighed, and the lung index (lung weight / body weight × 100%) was calculated. As shown in Table 2, there were no statistically significant differences in the lung index between the drug-treated groups and the model control group, indicating that isoquercitrin improved inflammation without causing abnormal lung tissue weight gain.

[0025] Table 3. Effect of isoquercitrin tablets on lung coefficient in mice (n = 10)

[0026] Table 4. Compared with the model control group, *p<0.05 3. Lung tissue dry-to-wet weight ratio After weighing the wet weight of the left lung, it was dehydrated in a 70℃ oven for 24 hours until constant weight, and then weighed dry and the wet-dry weight ratio was calculated. As shown in Table 3, the medium and high dose groups of isoquercetin tablets significantly increased the wet-dry weight ratio of lung tissue, indicating that it reduced inflammatory edema of lung tissue.

[0027] Table 5. Effect of isoquercitrin tablets on the dry-to-wet weight ratio of mouse lung tissue (n = 10)

[0028] Table 6. Compared with the model control group, *p<0.05 4. Pathological staining of lung tissue Right lung tissue was fixed, dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). Microscopic observation showed (e.g.) Figure 1 (A. Normal control group; B. Model control group; C. Dexamethasone group; D. Low-dose isoquercitrin group; E. Medium-dose isoquercitrin group; F. High-dose isoquercitrin group): Blue arrows indicate inflammatory nodules. In the normal control group, the lung tissue structure of mice was intact, the alveolar walls were thin and uniform, and there was no inflammatory exudation. In the model control group, a large number of inflammatory cells were observed to aggregate and infiltrate, forming nodular lesions. Compared with the model control group, the inflammatory infiltration in the dexamethasone group basically disappeared, but the alveolar structure was not fully restored; while the isoquercitrin tablets at various doses, especially the medium and high dose groups, showed a dose-dependent improvement effect, with a significant reduction in inflammatory cell infiltration, better repair of alveolar structure, and a significant reduction in lung nodules.

[0029] Under the experimental conditions described above, a mouse model of chronic lung inflammation and pulmonary nodules was successfully established using OVA combined with LPS nasal drops. The results showed that the isoquercetin tablets prepared in this invention dose-dependently: significantly reduced the levels of key pro-inflammatory factors (IL-6, TNF-α) in the serum of model mice; effectively alleviated inflammatory edema in lung tissue (increasing the wet-to-dry weight ratio); and significantly improved pathological damage to lung tissue, reducing inflammatory cell infiltration and the size of pulmonary nodules. These combined effects confirm that the isoquercetin and its preparations provided by this invention have significant effects in improving pulmonary nodules and nourishing and moisturizing the lungs, providing a solid experimental basis for the development of related functional products.

[0030] The basic principles, main features, and advantages of this invention in the explored field have been described in detail above, and some usage examples have been detailed. Finally, it should be noted that the examples given above are only for illustrative purposes and are not intended to limit the invention. Although we have described this invention in detail with reference to the examples, those skilled in the art can still modify the described examples and solutions, or replace related technical parts. Therefore, any modifications or equivalent substitutions made within the spirit and principles of this invention are within the protection scope of the claims of this patent.

Claims

1. The use of isoquercetin in the preparation of products for improving pulmonary nodules or relieving pulmonary inflammation, characterized in that, Isoquercetin is prepared by hydrolyzing rutin with rhamnosidase.

2. The application of isoquercetin in the preparation of lung-nourishing and moisturizing products, characterized in that, Isoquercetin is prepared by hydrolyzing rutin with rhamnosidase.

3. The application according to claim 1 or 2, characterized in that, The product used to improve pulmonary nodules or relieve lung inflammation, or the lung-nourishing product, includes pharmaceutically or food-grade excipients.

4. The application according to claim 1 or 2, characterized in that, The product used to improve pulmonary nodules and relieve lung inflammation is an oral preparation.

5. The application according to claim 4, characterized in that, The oral preparations are selected from tablets, capsules, granules, powders, oral liquids, or pills.

6. The application according to claim 1 or 2, characterized in that, The preparation method of isoquercetin includes the following steps: rutin is moistened with methanol, then water and sodium metabisulfite are added, and the pH is adjusted to 4.

0. 5.0; at 40 Rhamnosidase was added at 50°C for enzymatic hydrolysis; the obtained product was separated, dissolved in methanol and decolorized with activated carbon; concentrated, cooled and crystallized, separated and dried to obtain isoquercetin.

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