White-leaf Dancong tea and application of active ingredients thereof in reducing uric acid

Abstract

The invention belongs to the technical field of medicines, and particularly relates to white-leaf Dancong tea and application of active ingredients of the white-leaf Dancong tea in reducing uric acid. Researches show that the white-leaf Dancong tea extract has strong biological activity, can inhibit the activity of xanthine oxidase (XOD), effectively reduces the activity of adenosine deaminase (ADA) in blood, reduces the generation and accumulation of uric acid, and reduces the content of uric acid in the blood from the source. Further research finds that the active ingredient 3-O methyl gallic acid in the white-leaf Dancong tea plays a key role in inhibiting XOD activity so as to reduce uric acid. In the field of treatment of hyperuricemia and gout, many traditional medicines have many adverse reactions. Compared with the prior art, the white-leaf Dancong tea and the active ingredients of the white-leaf Dancong tea bring a brand new idea for clinical treatment, and by means of the characteristics of being pure natural, safe, reliable and remarkable in effect, new hope and choice are provided for patients suffering from pain.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to application of white leaf dancong tea and active ingredients thereof in reducing uric acid. Background Art

[0002] Hyperuricemia (HUA) refers to the concentration of uric acid in the blood exceeding the normal range. Usually, hyperuricemia can be diagnosed when the blood uric acid level exceeds 7.0 mg / dL (420 μmol / L) for men and 6.0 mg / dL (360 μmol / L) for women. The cause of hyperuricemia is complex, mainly related to excessive production or reduced excretion of uric acid. Uric acid is mainly excreted through the kidneys, and renal insufficiency or abnormal renal tubular function can lead to reduced uric acid excretion. Hyperuricemia itself has no clinical symptoms, but long-term high uric acid levels can cause urate crystals to precipitate and deposit in the joint cavity, kidneys, blood or other tissues, which can induce gout, arthritis, kidney stones, chronic kidney disease and other diseases.

[0003] The treatment of hyperuricemia aims to control blood uric acid levels and prevent gout attacks and related complications. Currently, allopurinol, febuxostat, benzbromarone, etc. are commonly used drugs for the treatment of hyperuricemia. Although these drugs can effectively lower blood uric acid levels, they will bring a series of adverse reactions and potential risks, such as inducing gastrointestinal discomfort, increasing the chance of uric acid stones, liver and kidney stones, and may also cause liver damage. Therefore, the research and development of functional agricultural products that are both natural, non-toxic, and economical to replace drugs for the treatment of hyperuricemia has become a key area of ​​concern for researchers.

[0004] In recent years, the functional components in tea have become a research hotspot for the prevention and treatment of HUA due to their natural, low toxicity and multi-target regulatory properties. Among them, white leaf single-bush oolong tea is a semi-fermented tea and a traditional famous tea that combines the fragrance of green tea and the mellow taste of black tea. This tea has a unique floral, honey, fruity and tea aroma and has great potential in the market. From the perspective of health value, oolong tea has anti-cancer, antioxidant and weight loss effects. A large number of in vitro and in vivo experimental results have shown that oolong tea is also effective in alleviating metabolic diseases such as metabolic syndrome, and its positive effect on health has been widely confirmed. However, it is not clear whether white leaf single-bush tea can play a role in lowering uric acid, and there are currently no relevant research reports. Summary of the invention

[0005] In order to overcome the deficiencies of the above-mentioned prior art, the present invention has found through research that white leaf single bush oolong tea can reduce the uric acid content in the serum of hyperuricemia patients, and has good application prospects in anti-hyperuricemia. At the same time, on the basis of clarifying that white leaf single bush tea has the effect of lowering uric acid, its potential active ingredients and mechanism of action are deeply analyzed, and research is carried out with the help of the cutting-edge technology of network pharmacology. Through professional databases and network pharmacology analysis platforms, the chemical components of white leaf single bush tea are systematically screened and analyzed, and it is found that the key active ingredient that exerts the effect of lowering uric acid is 3-O-methyl gallic acid. This provides a new, safe and effective natural medicine option for the treatment of hyperuricemia and gout.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides the use of white leaf dancong tea or its active ingredient 3-O-methyl gallic acid in the preparation of uric acid lowering products.

[0008] Preferably, the uric acid lowering is to inhibit the activity of xanthine oxidase (XOD), reduce the activity of adenosine deaminase (ADA) in the blood, and thus reduce the generation and accumulation of uric acid.

[0009] The present invention has been found through research that white leaf single bush tea can significantly inhibit the activity of serum ADA and XOD in the liver, and reduce the production of uric acid, thereby reducing the level of uric acid in the serum of individuals with hyperuricemia, achieving the effect of treating hyperuricemia and gout. Further based on network pharmacology, it is predicted that 3-O-methyl gallic acid, the active ingredient of white leaf single bush tea, plays a key role in inhibiting XOD activity and thus reducing uric acid production, further revealing the potential application of active ingredients of white leaf single bush tea in lowering uric acid. Therefore, white leaf single bush tea, as a traditional medicinal tea dual-purpose beverage, has a highly efficient uric acid-lowering effect and provides a safe and effective new natural medicine for the treatment of hyperuricemia and gout.

[0010] Preferably, the white leaf single bush tea is a white leaf single bush tea water extract.

[0011] Preferably, the preparation method of the white leaf tea water extract is:

[0012] With water as solvent and a tea-water ratio of 1:15-25, the white-leaf Dancong tea is soaked at 90-110° C. for 40-60 minutes, and the extract is collected by filtration.

[0013] More preferably, the number of extractions is 2-4 times.

[0014] More preferably, the extract is collected and then prepared into freeze-dried powder.

[0015] More preferably, the white-leaf Dancong tea is processed into oolong tea by an oolong tea processing process before steeping.

[0016] More preferably, the uric acid lowering product includes a uric acid lowering drug or a uric acid lowering health product.

[0017] A second aspect of the present invention provides a pharmaceutical preparation for lowering uric acid, the pharmaceutical preparation comprising a water extract of Camellia dasyphylla or 3-O-methylgallic acid.

[0018] Preferably, the pharmaceutical preparation further comprises a pharmaceutically acceptable excipient.

[0019] More preferably, the excipients include fillers (lactose, microcrystalline cellulose, mannitol, etc.), binders (hydroxypropyl methylcellulose, polyvinyl alcohol, gelatin, etc.), disintegrants (cross-linked sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, effervescent disintegrants, etc.), lubricants (stearic acid, polyethylene glycol, sodium lauryl sulfate, etc.), solubilizers (polysorbate 80, bile salts, sodium benzoate, etc.), preservatives (parabens, sorbic acid, benzalkonium bromide, etc.).

[0020] Preferably, the dosage form of the pharmaceutical preparation includes pills, powders, granules, tablets, capsules, injections, syrups and the like.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] White leaf single bush tea can be used as a drink in daily life, and it has extremely high safety due to its dual-purpose characteristics of medicine and tea. Studies have shown that the white leaf single bush tea extract has strong biological activity, which can inhibit the activity of xanthine oxidase (XOD), effectively reduce the activity of adenosine deaminase (ADA) in the blood, reduce the generation and accumulation of uric acid, and reduce the uric acid content in the blood from the source. Further studies have found that the active ingredient 3-O methyl gallic acid in white leaf single bush tea plays a key role in inhibiting XOD activity and thus reducing uric acid. In the field of hyperuricemia and gout treatment, many traditional drugs have many adverse reactions. In comparison, the white leaf single bush tea and its active ingredients of the present invention bring a new idea to clinical treatment, and provide new hope and choice for patients suffering from illness by virtue of its pure natural, safe and reliable, and significant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results of the analysis of the inhibitory effect of white leaf tea on xanthine oxidase Note: ( *** p<0.001vs Congroup);

[0024] Figure 2 The uric acid-lowering effect of white leaf Dancong tea on hyperuricemia rats (** p<0.01, *** p<0.001vs Congroup, ## p<0.01vs Mod group);

[0025] Figure 3 The inhibitory effect of white leaf Dancong tea on ADA in hyperuricemia rats ( ** p<0.01, *** p<0.001vsCon group, ## p<0.01vs Mod group);

[0026] Figure 4 The Venn diagram of white leaf Dancong tea and hyperuricemia targets;

[0027] Figure 5 This is the core target map of white leaf Dancong tea for lowering uric acid;

[0028] Figure 6 This is the gene network diagram of white leaf Dancong tea components and hyperuricemia targets;

[0029] Figure 7 The inhibitory effect of the active ingredients of white leaf Dancong tea on xanthine oxidase. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0031] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0032] Example 1: Preparation of White Leaf Single Bush Oolong Tea Water Extract

[0033] (1) Raw material preparation: All white-leaf single-bush tea samples were collected in the "Hometown of Lingtou Single-bush Tea in China" in Raoping County, Chaozhou City, Guangdong Province in the autumn of 2023. Standard fresh tea leaves (paired leaves) were processed according to the oolong tea processing process. The main processing procedures are as follows: ① After the fresh tea leaves are picked, they are fully mixed, and first spread until the fresh leaves have no grass smell and emit a fresh fragrance; ② After withering, the fresh tea leaves are shaken three times, then withered at 230℃, and then rolled for 30 minutes, and finally dried at high temperature to make oolong tea.

[0034] (2) Extraction of active ingredients: Extract in a water bath at 100°C for 45 min at a tea-water ratio of 1:20 (g:mL). Each sample was extracted twice, and the two extracts were combined. The extract was filtered to obtain the extract, which was then subjected to rotary evaporation to obtain the concentrate. The concentrate was then placed in a -80°C refrigerator to freeze into blocks, and finally subjected to vacuum freeze drying to obtain freeze-dried powder.

[0035] Example 2: Inhibitory effect of Baiye Dancong Oolong tea water extract on xanthine oxidase

[0036] 1. Experimental Materials

[0037] (1) Experimental drugs and reagents

[0038] 4-aminoantipyrine, horseradish peroxidase, phenol, Tris-HCl, Camellia dasyphylla, and positive drug allopurinol.

[0039] (2) Experimental instruments

[0040] Multifunctional microplate reader.

[0041] 2. Experimental methods

[0042] The detection of xanthine oxidase activity was carried out by double enzyme coupling method (experimental grouping is shown in Table 1): first, 0.0619 g of 4-aminoantipyrine, 0.006 g of horseradish peroxidase, and 0.168 g of phenol were diluted in 300 mL of 0.05 mol / L Tris-HCl buffer (pH=8.0) to prepare a color development solution, which was stored at 4° C. Then, 0.2 mL of sample solution (500 ug / mL, 1000 ug / mL, 2000 ug / mL of white leaf single-bush tea water extract or 64 ug / mL of positive drug allopurinol) and 0.05 mL of 0.52 U / mL xanthine oxidase solution were mixed, incubated at 37° C. for 10 min, and then 0.4 mL of 0.22 mmol / L xanthine solution and 3.05 mL of color development solution were added. The resulting mixture was then incubated at 37°C for 20 min, and then 0.1 mL of 1 mol / L NaOH solution was used to inactivate the enzyme and terminate the reaction. After cooling to room temperature, the absorbance was measured at 508 nm, and the system without the extracted sample and enzyme was used as the blank to adjust the zero. Finally, the inhibition rate was calculated according to the following formula:

[0043] XOD inhibitoryrate(%)=(0-(2-1)) / 0×100%;

[0044] Among them, A0 represents the absorbance of the control group, A2 represents the absorbance of the experimental group, and A1 represents the absorbance of the sample control group.

[0045] Table 1 Grouping of xanthine oxidase activity experiment

[0046]

[0047] 3. Experimental results

[0048] Figure 1 The results of the analysis of the inhibitory effect of the extract of Baiyedancong tea on xanthine oxidase are shown in Figure 2. Figure 1 The results showed that the inhibition rates of 500ug / mL, 1000ug / mL, and 2000ug / mL of the extracts of white leaf tea were 59.89%, 76.75%, and 99.73% respectively. Compared with the Con group, the extracts of different concentrations of tea showed significant XOD inhibition, and were concentration-dependent.

[0049] Example 3: Therapeutic effect of Baiye Dancong Oolong tea water extract on hyperuricemia rats

[0050] 1. Experimental Materials

[0051] (1) Experimental animals

[0052] SPF male SD rats, weighing 200 ± 2 g, were purchased from Guangzhou Chashi Ruihua Biotechnology Co., Ltd. (Lot: SHD-2024-06-18-1828) and approved by the Animal Care and Welfare Committee of the Tea Research Institute of Guangdong Academy of Agricultural Sciences. All rats were adaptively fed and raised at 23-27°C and 50-70% humidity for one week, with free access to water and food.

[0053] (2) Experimental drugs

[0054] White leaf Dancong tea water extract, benzbromarone (dosage is 10mg / kg / d), normal saline, potassium oxonate + adenosine (used for modeling, usually the dose of potassium oxonate is 100-300mg / kg body weight per day, and the dose of adenosine is 50-200mg / kg body weight per day).

[0055] (3) Experimental reagents

[0056] Uric acid (UA) test kit (enzyme colorimetric method) and adenosine deaminase (ADA) assay kit were purchased from Nanjing Jiancheng Bioengineering Institute.

[0057] (4) Experimental instruments

[0058] Multifunctional microplate reader.

[0059] 2. Experimental methods

[0060] As shown in Table 2, SD rats were randomly divided into 6 groups, with a total of 48 rats: Con group; Mod group; Yang medicine-benzbromarone group (Pos); low-dose group of water extract of white leaf single bush tea (DCl, 200mg / kg); medium-dose group of water extract of white leaf single bush tea (DCm, 400mg / kg); high-dose group of water extract of white leaf single bush tea (DCh, 800mg / kg). Except for the Con group, the other groups were gavaged with corresponding drugs 1 hour after modeling, once a day, for 2 consecutive weeks. The healthy Con group was given the same volume of 0.5% sodium carboxymethyl cellulose. The mice were weighed every day, and their water intake and food intake were measured every three days.

[0061] Table 2 Grouping of animal model experiments

[0062]

[0063]

[0064] After fasting for 12 h, rats were anesthetized with 40 mg / kg pentobarbital, and whole blood was obtained by cardiac puncture and added to a test tube without anticoagulant (to obtain serum), which was then separated by centrifugation at 2500 rpm for 20 min. The obtained supernatant was stored at -80°C for the detection of uric acid and adenosine deaminase.

[0065] 3. Experimental results

[0066] Figure 2 The uric acid-lowering effect of the extract of the white leaf tea on hyperuricemia rats. Figure 2 The results showed that the serum UA content in the Con group was significantly lower than that in the Mod group by 2.01 times (P < 0.01). After the intervention with the extract of Albizia dasyphylla tea, the serum UA content in rats was significantly lower than that in the Mod group (P < 0.01), among which DCl, DCm and DCh decreased by 1.43 times, 1.63 times and 1.70 times, respectively.

[0067] Figure 3 The inhibitory effect of the water extract of white leaf tea on ADA in hyperuricemia rats. Figure 3 The results showed that compared with the Con group, the ADA level in the serum of hyperuricemia rats was significantly increased by 12.84 times (P < 0.01). After drug intervention, the levels of allopurinol in the Yang medicine group and the white leaf Dancong tea water extract group were significantly lower than those in the Mod group (P < 0.001), among which DCl, DCm and DCh decreased by 4.44, 5.18 and 5.64 times, respectively.

[0068] Example 4: Prediction and verification of the core active ingredients of white leaf dancong tea for lowering uric acid based on network pharmacology

[0069] 1. Experimental Materials

[0070] (1) Experimental drugs and reagents

[0071] 3-O-methylgallic acid, choline, sesamin, 4-aminoantipyrine, horseradish peroxidase, phenol, Tris-HCl, white leaf tea, and positive drug allopurinol.

[0072] (2) Experimental instruments

[0073] High performance liquid chromatography, liquid chromatography-mass spectrometry, multifunctional ELISA reader.

[0074] 2. Experimental methods

[0075] (1) The compounds of white leaf tea were detected based on the UPLC-MS / MS platform (commissioned by Wuhan Maiwei Metabolism Company) and their composition was identified.

[0076] (2) Prediction of potential targets: Use the PubChem database to search for the SMILES number of the identified compound. Please use the SIWSS platform to use the SMILES number to screen compounds in the SIWSS database. The screening must meet the following conditions: the GI absorption score of the compound is "high", and at least two results in its druglikeness evaluation are "YES", and then obtain the target of the compound that meets the conditions. Then predict in the SWILES Target on the SWILES website, merge all targets and remove duplicates, identify the target gene of Baiye Dancong tea, and determine its potential target.

[0077] (3) Prediction of potential hyperuricemia disease targets

[0078] Using "hyperuricemia disease" and "uric acid lowering" as keywords, we searched for "hyperuricemia" related targets through GeneCards; OMIM; DisGeNET databases. Then, the predicted potential targets of Baiye Dancong tea compounds and "hyperuricemia" candidate targets were imported into the Venny2.1.0 online website to draw a Venn diagram. Through the intersection of the two, a direct active ingredient-target interaction network of the disease was constructed, and then the potential target set of Baiye Dancong tea in the treatment of hyperuricemia was identified.

[0079] (4) Protein-protein interaction (PPI) network construction

[0080] The crossover genes were uploaded to the String database, and protein-protein interaction (PPI) analysis was performed on their potential targets to obtain the relationship between the target proteins. The Cytoscape3.9.0 software was then used for visualization, and topological parameters were used to screen the key targets and key active ingredients of Baiye Dancong tea for lowering uric acid.

[0081] (5) The predicted key active ingredients for lowering uric acid were subjected to in vitro enzyme activity assay using the same method as in Example 2.

[0082] 3. Experimental results

[0083] Figure 4 This is a Venn diagram of white leaf Dancong tea and hyperuricemia targets. The genes of active substances in white leaf Dancong tea were matched with disease genes, and 136 cross-targets were obtained based on Venn diagram analysis.

[0084] Figure 5 To map the core targets of Baiye Dancong tea for lowering uric acid, 136 cross-targets were imported into the String database, the species was selected as human, the interaction threshold was set to ≥0.7, and the PPI network diagram was constructed using Cytoscape 3.9.1 software. Then, the Degree, Betweenness, and Closeness values ​​were greater than 2 times the median, and a total of two screenings were performed, and the core targets obtained were TNF, IL-1β, PPARG, MMP9, TLR4, STAT3, etc.

[0085] Figure 6 The network diagram of the components of white leaf tea and the target gene network of hyperuricemia was constructed. Based on 136 intersection targets and 64 compounds, the degree values ​​of the targets were calculated with the help of Cytoscape and NetworkAnalyzer software. The calculated degree value was used as the key basis to determine the importance of the target and construct the component-target gene network diagram of hyperuricemia. Further analysis found that when the degree value was ≥8, the main active ingredients were predicted to be quercetin, luteolin, sesamin, epigallocatechin, kaempferol, naringenin, pinoresinol, ellagic acid, choline, 3-O-methylgallic acid, etc.

[0086] Figure 7 The active ingredients of white leaf tea inhibit xanthine oxidase. This study selected substances that have not been reported to have uric acid-lowering activity [such as sesamin, choline, and 3-O-methyl gallic acid (3MGA)] for in vitro enzyme activity verification, and used GA (gallic acid) as the positive drug group, and the drug concentration was uniformly set to 120ug / mL. Figure 7 As shown, compared with the Con group, the XOD inhibitory activity of the GA group and 3-O-methylgallic acid (3MGA) was significantly increased, while the inhibitory activity of sesamin and choline was almost below 0.

[0087] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. Application of white leaf dancong tea or its active ingredient 3-O-methyl gallic acid in the preparation of uric acid lowering products.

2. The use according to claim 1, characterized in that: The white leaf single bush tea is a water extract of white leaf single bush tea.

3. The use according to claim 2, characterized in that: The preparation method of the white leaf single bush tea water extract is: using water as a solvent, using a tea-to-water ratio of 1:15-25, placing the white leaf single bush tea at 90-110° C. for 40-60 minutes, and filtering and collecting the extract.

4. The use according to claim 3, characterized in that: The number of extractions is 2-4 times.

5. The use according to claim 3, characterized in that: The extract was collected and made into freeze-dried powder.

6. The use according to claim 3, characterized in that: The white leaf single bush tea is processed into oolong tea by using oolong tea processing technology before steeping.

7. The use according to claim 1, characterized in that: The uric acid lowering method is to inhibit the activity of xanthine oxidase, reduce the activity of adenosine deaminase in the blood, and thus reduce the generation and accumulation of uric acid.

8. The use according to claim 1, characterized in that: The uric acid lowering products include uric acid lowering drugs or uric acid lowering health products.

9. A pharmaceutical preparation for lowering uric acid, characterized in that: The pharmaceutical preparation comprises a water extract of Camellia dasyphylla or 3-O-methyl gallic acid.

10. A pharmaceutical preparation for lowering uric acid according to claim 9, characterized in that: The pharmaceutical preparation also includes pharmaceutically acceptable excipients.

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