Traditional Chinese medicine for reducing uric acid and / or preventing uric acid increase as well as preparation method and application of traditional Chinese medicine

Abstract

The invention discloses a traditional Chinese medicine for reducing uric acid and / or preventing uric acid increase and a preparation method and application thereof. Specifically, the invention discloses a type of traditional Chinese medicine which comprises mistletoe traditional Chinese medicine, such as one or more of mistletoe, viscum viscum, viscum archaieri, viscum ovum, viscum coloratum or viscum coloratum. The mistletoe and the mistletoe are preferably selected, and the optimal mistletoe is selected. Research finds that after traditional Chinese medicines are extracted by adopting a water extraction method disclosed by the invention and a 0-100V / V% alcohol aqueous solution is separated by macroporous resin, active ingredients of mistletoe can be effectively extracted, and the active ingredients can reduce the content of blood uric acid, can be used for preparing medicines or health-care products for resisting hyperuricemia, and can be used for preparing medicines or health-care products for resisting hyperuricemia. The development, utilization and protection of traditional Chinese medicine resources are facilitated, and the economic value is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicines and preparation methods and applications thereof, and more particularly to the application of traditional Chinese medicines of the genus Viscum in reducing uric acid and / or preventing uric acid elevation. BACKGROUND

[0002] Uric acid is the end product of purine metabolism in the body, which is difficult to dissolve in water and will cause metabolic diseases such as gout when it is deposited in joints, soft tissues and kidneys. Hyperuricemia (HUA) is a disease caused by the blood uric acid concentration in the body exceeding the normal value. High blood uric acid level will directly or indirectly cause diseases such as gout and uric acid nephropathy, and high uric acid has also become an important risk factor for metabolic diseases such as diabetes, hyperlipidemia and chronic kidney disease, which seriously affect human health. However, the currently used first-line drugs such as colchicine, allopurinol and benzbromarone often have great toxic side effects, and the use of patients with liver and kidney dysfunction is limited, which makes it difficult to meet the clinical needs. Therefore, the development of high-efficiency and low-toxicity drugs for reducing uric acid is the current research focus.

[0003] The genus Viscum originally belongs to the Loranthaceae family, and is now incorporated into the Santalaceae family. There are currently about 70 species of plants under the genus Viscum, of which there are 14 species of plants in China, including Viscum liquidambaricola Hayata, Viscum liquidambaricola Hayata, Viscum liquidambaricola Hayata, etc. (see Background Art Appendix Figure 1 ). The applicant's previous research has proved that Viscum liquidambaricola Hayata under the genus Viscum has the efficacy of treating hyperuricemia. However, the pharmacological activity and effect of other traditional Chinese medicinal materials in the genus Viscum are not clear, and because the active components of the genus Viscum vary greatly with the species of the host, it is urgent to study and develop the pharmacological activity of other plants under the genus Viscum and improve the medicinal value and economic value of the plants under the genus Viscum. SUMMARY

[0004] The purpose of the present application is to provide a kind of traditional Chinese medicine for treating hyperuricemia and its preparation method and application in view of the deficiencies and defects of the current drugs for treating hyperuricemia in the prior art.

[0005] In one aspect of the present application, a traditional Chinese medicine for reducing uric acid and / or preventing uric acid elevation is provided, which comprises a traditional Chinese medicine of the genus Viscum, and the traditional Chinese medicine of the genus Viscum is one or more of Viscum liquidambaricola Hayata, Viscum liquidambaricola Hayata, Viscum liquidambaricola Hayata, Viscum liquidambaricola Hayata, Viscum liquidambaricola Hayata or Viscum liquidambaricola Hayata.

[0006] Preferably, the traditional Chinese medicine of the genus Viscum is Viscum liquidambaricola Hayata or Viscum liquidambaricola Hayata.

[0007] Most preferably, the Chinese medicine of the genus Viscum is Viscum coloratum (Kom.) Nakai. Viscum coloratum (Kom.) Nakai is parasitic on tea trees, pines and various plants, and is included in the Chinese Pharmacopoeia 2020 edition, and the Latin name is Viscum coloratum (Kom.) Nakai. Viscum coloratum (Kom.) Nakai is a traditional Chinese medicine and is widely distributed in China, and has different aliases in various provinces. At present, Viscum coloratum (Kom.) Nakai is commonly used for treating rheumatism and preventing miscarriage, and its efficacy and pharmacological research for reducing uric acid are not clear. Viscum coloratum (Kom.) Nakai has small toxic and side effects and a large safe dose range. At present, there is no systematic research on the use of Viscum coloratum (Kom.) Nakai for reducing uric acid, and therefore, the present application creatively proves that Viscum coloratum (Kom.) Nakai has the effects of reducing uric acid and / or preventing uric acid from rising.

[0008] Further, the Viscum coloratum (Kom.) Nakai is parasitic on tea trees, pines and various plants. Viscum coloratum (Kom.) Nakai grows faster on tea trees, and from the economic point of view, the Viscum coloratum (Kom.) Nakai parasitic on tea trees is preferably used in actual use.

[0009] Further, the active ingredients of the Chinese medicine of the genus Viscum are extracted by a solvent, and the extraction solvent is any one of water, a hydrophilic organic solvent and a lipophilic organic solvent. The hydrophilic organic solvent includes but is not limited to methanol, ethanol or acetone, and the lipophilic organic solvent includes but is not limited to trichloromethane, diethyl ether, ethyl acetate or petroleum ether.

[0010] Further, the active ingredients of the Chinese medicine of the genus Viscum are extracted by a solvent, and the extraction solvent is any one of water, a hydrophilic organic solvent and a lipophilic organic solvent. The hydrophilic organic solvent includes but is not limited to methanol, ethanol or acetone, and the lipophilic organic solvent includes but is not limited to trichloromethane, diethyl ether, ethyl acetate or petroleum ether.

[0011] Further, the extraction method of the water extraction method is any one of decoction method, immersion method, percolation method, reflux extraction method, steam distillation method, supercritical fluid extraction method, microwave-assisted extraction method and ultrasonic-assisted extraction method.

[0012] The extraction method further includes a repeated extraction step: collecting dregs, adding the extraction solvent again in the dregs, collecting the medicinal liquid after extraction; the dregs are repeatedly collected and extracted for several times, and the medicinal liquids extracted for several times are combined. The medicinal liquid can be concentrated as needed to prepare a medicinal paste.

[0013] Further, for the convenience of administration, the medicinal liquid can be concentrated and the Chinese medicine can be dried, and the drying method includes but is not limited to any one of normal pressure drying, reduced pressure drying, boiling drying, spray drying, freeze drying, infrared drying and microwave drying.

[0014] On the other hand, the present application also provides a component separation method of the active extract of the Chinese medicine of the genus Viscum and an application of the active extract in resisting hyperuricemia.

[0015] Furthermore, after extracting the active ingredients of the *Mistletoe* herbal medicine using water extraction, the active ingredients are eluted and separated using an elution solvent. The elution solvent is an aqueous solution of an alcohol with a volume fraction of 0-100 V / V%, wherein the alcohol is either methanol or ethanol.

[0016] Further, the elution solvent is an aqueous solution of an alcohol with a volume fraction of 0-70 V / V%, wherein the alcohol is methanol or ethanol. Preferably, the elution solvent is an aqueous solution of methanol with a volume fraction of 70 V / V.

[0017] Furthermore, the separation method employs macroporous resin extraction and separation.

[0018] Furthermore, the active ingredient can reduce serum uric acid levels in hyperuricemia.

[0019] Furthermore, after elution with a 70% methanol aqueous solution, the obtained active component showed the best reduction in serum uric acid levels in hyperuricemia.

[0020] Furthermore, the preferred traditional Chinese medicine of the mistletoe genus is *Mallotus argentea*.

[0021] On the other hand, the present invention provides a traditional Chinese medicine preparation, characterized in that: the traditional Chinese medicine preparation includes the above-mentioned traditional Chinese medicine and pharmaceutically acceptable excipients.

[0022] Furthermore, the traditional Chinese medicine preparation is one of the following: liquid dosage form, gas dosage form, solid dosage form, and semi-solid dosage form.

[0023] Furthermore, the solid dosage forms include, but are not limited to: powders, granules, tablets, capsules, pellets, films, etc.

[0024] The liquid dosage forms include, but are not limited to: fragrances, solutions, injections, lotions, mixtures, liniments, etc.

[0025] The gaseous dosage forms include, but are not limited to: aerosols, powder sprays, and sprays.

[0026] The semi-solid dosage forms include, but are not limited to, ointments, suppositories, and films.

[0027] On the other hand, the present invention provides an application of the above-mentioned traditional Chinese medicine in the preparation of anti-hyperuric acid drugs or health products, characterized in that the drug or the health product is a drug or health product for reducing uric acid and / or preventing uric acid elevation.

[0028] Furthermore, conditions associated with elevated uric acid include one or more of the following: hyperuricemia, gout, renal dysfunction, and kidney damage.

[0029] Furthermore, the hyperuricemia mentioned is either primary hyperuricemia or secondary hyperuricemia.

[0030] On the other hand, the traditional Chinese medicine provided by this invention can be used to lower uric acid or prevent uric acid elevation. Diseases that cause uric acid elevation include, but are not limited to, hyperuricemia, gout, renal dysfunction, and kidney damage.

[0031] Furthermore, the traditional Chinese medicine can lower uric acid levels, and further reduce serum uric acid levels.

[0032] Furthermore, the traditional Chinese medicine can lower serum creatinine levels, lower plasma urea nitrogen levels, improve renal tubular dilation, alleviate pathological conditions such as glomerular atrophy, and improve renal function.

[0033] Furthermore, the traditional Chinese medicine can inhibit the activity of hepatic xanthine oxidase and reduce uric acid production.

[0034] The present invention has the following beneficial effects:

[0035] 1) This invention systematically studies the pharmaceutical properties of traditional Chinese medicines of the genus *Mistletoe*, proving that they have the effect of resisting hyperuricemia, can reduce and / or prevent the rise of uric acid, and can be used to treat diseases such as hyperuricemia, gout, renal dysfunction, and kidney damage.

[0036] 2) This invention develops a new and highly effective uric acid-lowering drug, which includes traditional Chinese medicines of the genus *Mistletoe*. This solves the long-standing problem of unclear mechanisms of action and unclear actual efficacy of this type of traditional Chinese medicine, which is conducive to the development, utilization and protection of ethnic medicinal resources and improves their economic value. Attached Figure Description

[0037] Figure 1 The background art refers to plant species under the genus *Mistletoe*.

[0038] Figure 2 Image of H&E staining in mouse kidney tissue.

[0039] Figure 3 To investigate the effects of eluted and separated active components of Mistletoe on serum uric acid (UA) levels in mice from different experimental groups. Detailed Implementation

[0040] The present invention will now be clearly and thoroughly described in conjunction with specific embodiments and experimental results. Obviously, the specific embodiments described are only some embodiments and are used only to illustrate the present invention, not to limit the scope of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional experimental methods; the experimental materials and reagents used, unless otherwise specified, are commercially available reagents and materials.

[0041] Example 1: Preparation of mistletoe extract and its efficacy in treating hyperuricemia

[0042] (I) Preparation of Traditional Chinese Medicine

[0043] 1. Medicinal materials: Mistletoe

[0044] 2. Extraction method: Take 500 g of mistletoe, pulverize it with a pulverizer, and obtain 486 g of medicinal material powder. Add extraction solvent water or 75% ethanol to the medicinal material powder, stir and soak it充分. Subsequently, use ultrasonic extraction method to ultrasonically extract for 30 min to fully extract the active substances. Filter and retain the filtrate for standby. Add the extraction solvent to soak the filter residue again, repeat the ultrasonic extraction operation, and extract a total of three times. Combine the three filtrates, concentrate and then perform freeze-drying under vacuum. The obtained particles after freeze-drying are the water extract of mistletoe.

[0045] (2) Pharmacodynamic experiments

[0046] 1. Experimental materials

[0047] 40 SPF-grade Kunming mice aged 4 - 5 weeks, weighing 18 - 22 g, male, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (License number: SCXK(Beijing)2021 - 0006). Potassium oxonate (Aladdin reagent), hypoxanthine (Aladdin reagent), febuxostat (Aladdin reagent), uric acid assay kit, urea nitrogen assay kit, xanthine oxidase assay kit were all purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., and the specific operation steps were carried out according to the kit instructions.

[0048] 2. Experimental methods

[0049] Animal grouping: Take 40 male Kunming mice. After taking blood from the submandibular vein, use the uric acid assay kit to detect the serum uric acid content, and randomly group them into 5 groups according to the uric acid level: control group, model group, febuxostat administration group (5 mg / kg), mistletoe administration group (500 mg / kg), with 10 mice in each group.

[0050] Among them, the mice in the control group are normal mice, fed with conventional food and not treated otherwise.

[0051] The mice in the model group, febuxostat administration group (5 mg / kg), and mistletoe administration group (500 mg / kg) are model animals. The specific modeling process is as follows: Inject 300 mg / kg of potassium oxonate intraperitoneally and gavage 300 mg / kg of hypoxanthine daily to establish a high uric acid model in mice.

[0052] Administration: One hour after mouse modeling, administration began by gavage. The control and model groups received the same volume of solvent, the febuxostat group received 5 mg / kg febuxostat, and the mistletoe group received 500 mg / kg mistletoe water extract. Administered orally once daily for 28 consecutive days. After 28 days, blood samples were collected to test serum uric acid, creatinine, and blood urea nitrogen levels in each group. Subsequently, liver and kidney tissues were dissected, weighed, and the left kidney was fixed in 4% paraformaldehyde and prepared into 4 μm paraffin sections for pathological examination and H&E staining. The right kidney tissue was preserved in liquid nitrogen for subsequent testing.

[0053] Method for detecting XOD enzyme activity in the liver: Fresh mouse liver was homogenized with physiological saline, and the supernatant was obtained. The protein content of the supernatant was determined using the BCA method. XOD enzyme activity was detected in the supernatant according to the method described in the kit instructions.

[0054] H&E staining was used to observe pathological changes in renal tissue: routine H&E staining was performed to assess the degree of pathological changes in renal tissue.

[0055] (III) Experimental Results and Discussion

[0056] 1. Mistletoe reduces uric acid levels in a mouse model of hyperuricemia induced by potassium oxonate and hypoxanthine.

[0057] As shown in Table 1, the serum urinary tract infection rate in the model group mice was significantly higher than that in the normal group, indicating successful model establishment. After 28 days of treatment with febuxostat and mistletoe, the serum uric acid level in the treated mice was significantly reduced. These experimental results demonstrate that the traditional Chinese medicine mistletoe described in this application has the efficacy of lowering uric acid levels.

[0058] 2. Mistletoe can restore renal function and alleviate renal damage in a mouse model of hyperuricemia induced by potassium oxonate and hypoxanthine.

[0059] Long-term high levels of uric acid can cause kidney damage and affect renal function. As shown in Table 1, the serum creatinine and blood urea nitrogen levels in the model group mice were significantly increased compared with the control group, indicating that the model was successful and the mice in the model group developed renal function damage. However, after administration of febuxostat and mistletoe, the serum creatinine and blood urea nitrogen levels in the mice were significantly decreased, showing statistical differences compared with the model group.

[0060] At the same time, such as Figure 2 As shown in the H&E staining images, after modeling, mice exhibited renal edema, extensive tubular hydropic degeneration, tubular dilation, and tubular atrophy, among other pathological conditions. However, after intervention with mistletoe, the mice in the mistletoe-treated group showed reduced renal edema and improved renal damage, including tubular dilation and glomerular atrophy. In summary, the experimental data indicate that mistletoe treatment restored renal function in mice, and that mistletoe can improve renal function damage in mice.

[0061] Table 1. Uric acid, creatinine, and blood urea nitrogen levels in mice of each group

[0062]

[0063] Note: All values ​​are expressed as mean ± SEM, compared to the normal group. #### P < 0.0001; compared with the model group, **** P < 0.0001.

[0064] 3. Mistletoe reduces liver XOD levels in a mouse model of hyperuricemia induced by potassium oxonate and hypoxanthine.

[0065] Xanthine oxidase (XOD) is a crucial catalytic enzyme in the production of uric acid in the body. It can catalyze the formation of xanthine from hypoxanthine, which in turn produces uric acid, and it can also directly catalyze the formation of uric acid from xanthine. The marketed drug febuxostat inhibits uric acid production by suppressing XOD activity. As shown in Table 2, xanthine oxidase activity was significantly upregulated in the liver of mice with hyperuricemia, and both the febuxostat and mistletoe treatments significantly inhibited XOD activity in the mouse liver. This indicates that mistletoe administration can inhibit uric acid production.

[0066] Table 2. Liver XOD enzyme activity in mice of each group

[0067]

[0068] Note: All values ​​are expressed as mean ± SEM, compared to the normal group. ### P < 0.001; compared with the model group, *** P < 0.001.

[0069] Example 2: Isolation of active components from mistletoe extract

[0070] 1. Medicinal material: Water extract of mistletoe from Example 1

[0071] 2. Separation method:

[0072] (1) Take 20g of mistletoe water extract from Example 1, add a small amount of pure water to dissolve the sample and set aside for use.

[0073] (2) After balancing the macroporous D-101 packing material with pure water, the dissolved sample is loaded onto the packing material.

[0074] (3) Different active components were separated by eluting macroporous resin columns with 100%, 70%, 50%, 20%, and 0% methanol aqueous solutions respectively. The amount of each eluent was about 3L. The eluent was collected and concentrated to obtain an extract. The five components obtained were named component 1, component 2, component 3, component 4, and component 5 respectively.

[0075] Table 3. Separation and content of active components of Mistletoe

[0076]

[0077] Example 3: Efficacy experiment of active components of mistletoe extract against acute hyperuricemia in mice.

[0078] 1. Experimental Methods:

[0079] Animal grouping: Seventy male Kunming mice were used. After blood was collected from the submandibular vein, they were randomly divided into 7 groups according to their uric acid levels: control group, model group, and groups with different activities of mistletoe (group 1, group 2, group 3, group 4, and group 5), for a total of 7 groups, with 10 mice in each group.

[0080] The control group consisted of normal mice, fed with conventional food, and without any other treatment.

[0081] Mice in the model group and groups with different active ingredients of mistletoe (groups 1, 2, 3, 4, and 5) were used as model animals. An acute mouse model of hyperuricemia was established by intraperitoneal injection of 300 mg / kg potassium oxonate and gavage administration of 600 mg / kg hypoxanthine. Following model establishment, mice were administered medication via gavage. The control and model groups received the same volume of solvent, while the groups with different active ingredients of mistletoe (groups 1, 2, 3, 4, and 5) were administered 250 mg / kg of the respective active ingredient via gavage. Blood samples were collected two hours after administration, serum was separated, and serum uric acid levels were measured.

[0082] 2. Experimental Results and Discussion

[0083] Depend on Figure 1 As shown, two hours after modeling, the serum uric acid level in the model group mice increased significantly, showing a significant difference compared to the normal group, indicating successful modeling. After treatment with different active components of *Mistletoe*, the serum uric acid level in the treated mice was significantly reduced. The serum uric acid levels in mice eluted with 0%, 20%, and 70% methanol-water fractions (fractions 5, 4, and 2) were significantly different from those in the model group, with fraction 2 eluted with 70% methanol-water showing the best effect. These results demonstrate that the solvent extraction and elution methods described in this application can effectively extract and separate the active ingredients from *Mistletoe*, which possess anti-hyperuricemia efficacy and can reduce and / or prevent uric acid elevation.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, the implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered as equivalent substitutions and are included within the scope of protection of the present invention.

Claims

1. A traditional Chinese medicine for lowering uric acid and / or preventing elevated uric acid levels, characterized in that, The Chinese medicines mentioned include those belonging to the genus *Mistletoe*, specifically one or more of the following: *Mistletoe simonii*, *Mistletoe var. semperflorens*, *Mistletoe serrata*, *Mistletoe ovoides*, *Mistletoe linearis*, or *Mistletoe*.

2. The traditional Chinese medicine according to claim 1, characterized in that, The Chinese medicinal herbs mentioned are mistletoe or flat-branch mistletoe.

3. The traditional Chinese medicine according to claim 1 or 2, characterized in that, The active components of the *Mistletoe* genus are extracted using a solvent extraction method, wherein the solvent for the solvent extraction method includes one or more of water, methanol, ethanol, or acetone.

4. The traditional Chinese medicine according to claim 3, characterized in that, After the active ingredients of the genus *Mistletoe* are extracted by solvent extraction, the active ingredients are eluted and separated by an elution solvent, wherein the elution solvent is an aqueous solution of an alcohol with a volume fraction of 0-100 V / V%; the alcohol is either methanol or ethanol.

5. The traditional Chinese medicine according to claim 4, characterized in that, The elution solvent is an aqueous solution of an alcohol with a volume fraction of 0-70 V / V%, wherein the alcohol is methanol or ethanol.

6. The traditional Chinese medicine according to claim 5, characterized in that, The elution solvent is an aqueous methanol solution with a volume fraction of 70 V / V.

7. A traditional Chinese medicine preparation, characterized in that: The traditional Chinese medicine preparation comprises the traditional Chinese medicine as described in any one of claims 1-6 and pharmaceutically acceptable excipients.

8. The use of a traditional Chinese medicine as described in any one of claims 1-6 in the preparation of a medicine or health product, characterized in that, The drug or health product mentioned is a drug or health product for treating hyperuricemia.

9. The application according to claim 8, characterized in that, Diseases associated with elevated uric acid include one or more of the following: hyperuricemia, gout, kidney dysfunction, and kidney damage.

10. The application according to claim 9, characterized in that, The hyperuricemia mentioned includes primary hyperuricemia or secondary hyperuricemia.

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