Traditional Chinese medicine compound extract for treating hyperuricemia nephropathy as well as preparation method and application of traditional Chinese medicine compound extract

Abstract

The invention provides a traditional Chinese medicine compound extract for treating hyperuricemia nephropathy as well as a preparation method and application of the traditional Chinese medicine compound extract. The traditional Chinese medicine compound extract is prepared from the following Chinese herbal medicine raw material medicines in parts by weight: 1-10 parts of astragalus membranaceus, 1-10 parts of lonicerae flos, 1-10 parts of rhizoma smilacis glabrae and 1-10 parts of plantain herb. The traditional Chinese medicine compound extract is prepared into an aqueous solution to be used for treating a mouse with the hyperuricemia nephropathy for 6 weeks, and the result shows that the extract has a good treatment effect on the hyperuricemia nephropathy and has the potential of being developed into a medicine for treating the hyperuricemia nephropathy. In addition, in an in-vitro experiment, the traditional Chinese medicine compound extract can also inhibit uric acid induced apoptosis of normal human kidney proximal tubule cells (HK-2), and the effect is better than that of four single medicine extracts.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine, and specifically relates to a traditional Chinese medicine compound extract, a preparation method thereof and application of the extract in preparing medicines. Background Art

[0002] Hyperuricemia is a chronic metabolic disease caused by purine metabolism disorder, which can cause gout, cardiovascular disease, diabetes, hyperuricemia nephropathy and other diseases. Among them, excessive uric acid concentration in the blood can cause uric acid to crystallize and deposit in the kidneys in the form of sodium urate, leading to renal dysfunction and pathological changes, including oxidative stress damage, inflammatory response and cell apoptosis.

[0003] Currently, strategies for lowering uric acid mainly include controlling diet, inhibiting liver xanthine oxidase, supplementing exogenous uricase, and inhibiting tubular reabsorption. Although these drugs are effective in lowering blood uric acid concentration, they are not effective in treating hyperuricemia nephropathy, especially chronic hyperuricemia nephropathy, and have significant side effects.

[0004] CN109674958B discloses a Chinese medicine composition with uric acid lowering effect and its preparation method and application, which is made of 20-80 parts of Poria cocos, 20-60 parts of Coix seeds, 5-20 parts of Gardenia jasminoides, 5-20 parts of peach kernels, 5-20 parts of papaya, 5-15 parts of Polygonatum sibiricum, 5-15 parts of Lophatherum gracile, 5-15 parts of Licorice root, 2-20 parts of Lycium barbarum, 30-90 parts of Pueraria root, 20-60 parts of Morus alba, and 2-15 parts of Ginseng. However, the indication of the composition is hyperuricemia, and it is not reported whether the composition has a therapeutic effect on hyperuricemia nephropathy.

[0005] CN1720943A discloses a drug complex for treating hyperuricemia and uric acid nephropathy, which is made of the following raw materials in weight ratio: 5-30 grams of fenugreek seeds, 5-30 grams of white mustard seeds, 5-30 grams of plantain seeds, 5-30 grams of wintergreen seeds, 5-30 grams of clematis root, and 5-20 grams of raw rhubarb. According to the record of the patent application, the drug complex is aimed at the pathogenesis of hyperuricemia with phlegm and blood stasis, and has established the treatment principle of promoting blood circulation and unblocking collaterals, removing dampness and removing phlegm, and Chinese medicine attributes it to "gout, stranguria, and lumbago", and adopts Chinese medicinal materials such as plantain seeds to promote diuresis and remove phlegm, wintergreen seeds and fenugreek seeds to remove blood stasis and promote menstruation, and is compatible with white mustard seeds to dissipate phlegm and disperse nodules, raw rhubarb to promote blood circulation and remove blood stasis, and clematis root is added to remove rheumatism and dredge the meridians, and the phlegm and blood stasis can be removed by combining the drugs. The document also points out that 5-30 grams of Trillium or 5-30 grams of Curcuma can be used to replace Vaccaria semen to remove blood stasis and promote menstruation; 5-30 grams of Elephant Shell or 5-30 grams of Gleditsia sinensis can be used to replace White Mustard Seed to resolve phlegm and disperse nodules; 5-30 grams of Dianthus Superbus or 5-30 grams of Polygonum multiflorum can be used to replace Plantago seed to promote diuresis and eliminate phlegm; 5-30 grams of Radix Dianthus or 5-30 grams of Cynanchum wilfordii can be used to replace Clematis chinensis to remove rheumatism and dredge the meridians; 5-30 grams of Polygonum cuspidatum or 5-30 grams of Achyranthes bidentata can be used to replace Melaleuca alternifolia to remove blood stasis and promote menstruation; 5-30 grams of Cassia seed or 5-30 grams of processed Rhubarb can be used to replace raw Rhubarb to promote blood circulation and remove blood stasis. The preparation method of the compound is decoction method: soak in cold water for 30 minutes before decoction, the water volume is slightly higher than the medicine by one inch, and decoct for a long time with slow fire, decoct the first juice for 25 minutes, decoct the second juice for 15-20 minutes, and mix the first and second juices; the plantain seeds in the medicine need to be wrapped with gauze for decoction, and the raw rhubarb needs to be put in 10 minutes before the first decoction is completed. The drug compound has been confirmed by clinical trials and animal experiments to have the effects of reducing blood uric acid, improving renal function, reducing blood lipids, increasing SOD activity, and scavenging oxygen free radicals.

[0006] Given that there is currently no specific drug for hyperuricemia nephropathy, there is an urgent need to research and develop new drugs to reduce renal inflammation and cell apoptosis caused by hyperuricemia. Summary of the invention

[0007] Therefore, the purpose of the present invention is to provide a Chinese medicine compound for treating hyperuricemia nephropathy, a preparation method and application thereof, which is indicated for hyperuricemia nephropathy. It not only has the effect of lowering uric acid, but also has a good therapeutic effect on hyperuricemia nephropathy, and can reduce renal inflammatory response and cell apoptosis caused by high uric acid.

[0008] The mechanism of action of the complex provided in CN1720943A may be related to improving the activity of SOD and scavenging oxygen free radicals, etc. The mechanism described is relatively broad and not yet clear; and the prescription of the complex is complex and the cost is high.

[0009] The inventor of the present invention has formulated a Chinese medicine compound based on the diagnosis and treatment concept and clinical experience of traditional Chinese medicine for kidney diseases, which contains four Chinese herbal medicines: Astragalus, Honeysuckle, Smilax glabra and Plantago. Among them, Astragalus has the effects of strengthening the spleen and replenishing the middle, raising yang and lifting the sunken, benefiting the body and consolidating the exterior, diuresis, toxin-removing and muscle-regenerating; Honeysuckle has the effects of clearing heat and detoxifying, evacuating wind-heat; Smilax glabra has the effects of detoxification, dehumidification, and joint clearance; Plantago has the effects of clearing heat and diuresis, infiltrating dampness and relieving stranguria, clearing the liver and improving eyesight. The inventor found through experimental research that the Chinese medicine compound extract made from these four Chinese herbal medicines has a good therapeutic effect on hyperuricemia nephropathy. This discovery fills the gap that there is currently no specific medicine for the treatment of hyperuricemia nephropathy, and the Chinese medicine compound can be used for the development and application of drugs for the treatment of hyperuricemia nephropathy.

[0010] Compared with the compound provided by CN1720943A, the Chinese medicine compound of the present invention has the advantages of concise prescription and reasonable compatibility, the compound effect is better than that of single medicine, the Chinese medicinal materials used are cheap and easy to obtain, the toxic and side effects are small, and it is safe and effective. The experimental results show that the Chinese medicine compound extract of the present invention can inhibit the signaling pathway related to NF-κB and p-53, thereby inhibiting the kidney inflammation reaction and cell apoptosis caused by high uric acid, thereby improving kidney function and inhibiting kidney pathological damage. The mechanism is clear and therefore more reliable.

[0011] The first aspect of the present invention provides a Chinese medicine compound extract for treating hyperuricemia nephropathy, which is prepared from raw materials of Chinese herbal medicines in the following weight parts: 1-10 parts of Astragalus, 1-10 parts of Lonicera japonica, 1-10 parts of Smilax glabra, and 1-10 parts of Plantago.

[0012] The Chinese herbal compound extract for treating hyperuricemia nephropathy provided by the present invention is preferably prepared from raw materials of Chinese herbal medicines in the following parts by weight: 1-5 parts of Astragalus, 1-5 parts of Lonicera japonica, 1-5 parts of Smilax glabra, and 1-5 parts of Plantago.

[0013] According to the Chinese herbal compound extract for treating hyperuricemia nephropathy provided by the present invention, in some most preferred embodiments, it is prepared from raw materials of Chinese herbal medicines comprising the following parts by weight: 1-2 parts of Astragalus, 1-2 parts of Lonicera japonica, 1-2 parts of Smilax glabra, and 1-2 parts of Plantago.

[0014] The second aspect of the present invention provides a method for preparing the above-mentioned Chinese medicine compound extract for treating hyperuricemia nephropathy, comprising:

[0015] (1) Weighing astragalus, honeysuckle, smilax glabra and plantain according to weight, crushing and decocting, and combining the extracts to obtain a crude extract;

[0016] (2) filtering the crude extract to remove the filter residue, and then concentrating by rotary evaporation to obtain a concentrated solution;

[0017] (3) freeze-drying the concentrated solution to obtain the Chinese herbal compound extract.

[0018] In some embodiments of the present invention, the decoction process in step (1) may include: adding double distilled water at a liquid-to-solid ratio of 5-20:1 (v:m, mL / g), soaking for 2-4 hours, heating under reflux, and extracting 1-3 times, each time for 1-3 hours. Preferably, the extraction is ultrasonic extraction at 70-100°C and a power of 600-900W.

[0019] In other embodiments of the present invention, the decoction process in step (1) comprises: adding 60-90 wt% ethanol at a liquid-to-solid ratio of 5-20:1 (v:m, mL / g), soaking for 2-4 hours, heating under reflux, and extracting 1-3 times, each time for 1-3 hours. Preferably, the extraction is ultrasonic extraction at 70-100°C and a power of 600-900W.

[0020] The third aspect of the present invention provides a traditional Chinese medicine composition, which comprises the traditional Chinese medicine compound extract as an active ingredient and pharmaceutically acceptable excipients.

[0021] According to the traditional Chinese medicine composition provided by the present invention, the pharmaceutically acceptable excipients include one or more of a dispersion medium, a coating, a disintegrant, a lubricant, a surfactant, an antioxidant, a diluent, a solubilizing agent, an excipient, a sweetener, a flavoring agent, an adsorption carrier or a binder.

[0022] The fourth aspect of the present invention provides use of the Chinese herbal compound extract in preparing a medicament for treating hyperuricemia nephropathy.

[0023] The present invention prepares the Chinese medicine compound extract into an aqueous solution for treating hyperuricemia nephropathy mice for 6 weeks. The results show that the extract has a good therapeutic effect on hyperuricemia nephropathy and has the potential to be developed into a drug for treating hyperuricemia nephropathy. In addition, in in vitro experiments, the Chinese medicine compound extract can also inhibit uric acid-induced apoptosis of normal human renal proximal tubular cells (HK-2), and the effect is better than that of four single herbal extracts. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0025] Example 1

[0026] Preparation of the Chinese medicine compound extract of the present invention

[0027] (1) Weigh 1 part of Astragalus, 1 part of Honeysuckle, 1 part of Smilax glabra, and 1 part of Plantago, crush and mix them, add double distilled water at a liquid-to-solid ratio of 20:1 (mL / g), soak for 4 hours, heat and reflux for extraction twice, each time for 3 hours, collect the extracts and combine them to obtain a crude extract;

[0028] (2) filtering the crude extract to remove the filter residue, and then concentrating by rotary evaporation to obtain a concentrated solution;

[0029] (3) freeze-drying the concentrated solution to obtain an extract;

[0030] (4) Mix the extracts evenly, put them into a brown narrow-necked reagent bottle, and store them at -20°C for later use.

[0031] Example 2

[0032] Preparation of the Chinese medicine compound extract of the present invention

[0033] (1) Weigh 2 parts of Astragalus, 2 parts of Honeysuckle, 1 part of Smilax glabra, and 1 part of Plantago, crush and mix them, add 75% ethanol at a liquid-to-solid ratio of 20:1 (mL / g), soak overnight, heat and reflux for extraction twice, each time for 2 hours, collect the extracts and combine them to obtain a crude extract;

[0034] (2) filtering the crude extract to remove the residue, and then rotary evaporating until there is no alcohol smell to obtain a concentrated solution;

[0035] (3) freeze-drying the concentrated solution to obtain an extract;

[0036] (4) Mix the extracts evenly, put them into a brown narrow-necked reagent bottle, and store them at -20°C for later use.

[0037] Example 3

[0038] Preparation of the Chinese medicine compound extract of the present invention

[0039] (1) Weigh 2 parts of Astragalus, 2 parts of Honeysuckle, 1 part of Smilax glabra, and 1 part of Plantago, crush and mix them, add 75% ethanol at a liquid-to-solid ratio of 20:1 (mL / g), soak overnight, and extract twice at 100°C and 600W ultrasonically, each time for 3 hours, collect the extracts and combine them to obtain a crude extract;

[0040] (2) filtering the crude extract to remove the residue, and then rotary evaporating until there is no alcohol smell to obtain a concentrated solution;

[0041] (3) freeze-drying the concentrated solution to obtain a crude extract;

[0042] (4) Mix the extracts evenly, put them into a brown narrow-necked reagent bottle, and store them at -20°C for later use.

[0043] Example 4

[0044] Animal experiment on treating hyperuricemia nephropathy with the extract of the Chinese herbal compound of the present invention

[0045] After the mice were adaptively fed for one week, they began to enter this experiment. The mice were randomly divided into six groups, namely normal control group (CON), model control group (MOD), low-dose treatment group (LDT), medium-dose treatment group (MDT), high-dose treatment group (HDT), and positive drug control group (allopurinol, ALL), with 6 mice in each group.

[0046] The specific groups and their interventions are as follows:

[0047] CON: intraperitoneal injection of normal saline and oral administration of deionized water;

[0048] MOD: intraperitoneal injection of potassium oxonate (250 mg / kg) and oral administration of deionized water;

[0049] LDT: intraperitoneal injection of potassium oxonate (250 mg / kg), oral administration of the Chinese herbal extract prepared in Example 3 (0.4 g / kg);

[0050] MDT: intraperitoneal injection of potassium oxonate (250 mg / kg), and oral administration of the Chinese herbal extract prepared in Example 3 (1.2 g / kg);

[0051] HDT: intraperitoneal injection of potassium oxonate (250 mg / kg), oral administration of the Chinese herbal extract prepared in Example 3 (3.6 g / kg);

[0052] ALL: potassium oxonate (250 mg / kg) was injected intraperitoneally and allopurinol (10 mg / kg) was given by oral gavage.

[0053] Example 5

[0054] Effect of the Chinese herbal compound extract of the present invention on renal function of mice with hyperuricemia nephropathy

[0055] After the intervention as described in Example 4, urine samples were collected from mice using metabolic cages, blood samples were drawn from mice using the cardiac blood sampling method, and serum was separated by centrifugation and stored in a -80°C refrigerator for subsequent detection of renal function-related indicators.

[0056] Commercially available kits were used to detect the levels of urine protein (mg / dL), uric acid (mg / dL), and urine creatinine (mg / dL) in urine samples, and the levels of blood uric acid (mg / dL), blood creatinine (mg / dL), and blood urea nitrogen (mg / dL) in blood samples. Relevant experimental operations were performed according to the instructions of the kits to evaluate the effect of the Chinese herbal compound extract of the present invention on the renal function of mice with hyperuricemia nephropathy. The results are shown in Table 1.

[0057] Table 1

[0058]

[0059] Note: *, compared with the model control group, p<0.05.

[0060] It can be seen from the data in Table 1 that the Chinese herbal compound extract of the present invention can reduce the concentrations of urine protein, uric acid, urine creatinine, blood uric acid, blood creatinine and blood urea nitrogen in mice with hyperuricemia nephropathy and promote kidney function.

[0061] Example 6

[0062] Effect of the Chinese herbal compound extract of the present invention on renal inflammatory response in mice with hyperuricemia nephropathy

[0063] After the intervention described in Example 4, the mouse kidneys were removed and placed in a -80°C refrigerator for subsequent analysis of signaling pathways related to renal inflammatory response.

[0064] Western blotting (WB) was used to detect the protein expressions of inflammatory response-related signaling pathway molecules such as phosphorylated nuclear factor kappa-light-chain-enhancer of activated Bcells (p-NF-κB, p-p65), cleaved caspase-1 (c-Cas-1), interleukin-18 (IL-18), interleukin-1β (IL-1β), and NLR family pyrin domain containing 3 (NLRP3) in the kidneys of mice to evaluate the effect and potential mechanism of the Chinese medicine compound extract on the renal inflammatory response of hyperuricemia nephropathy mice. The results are shown in Table 2.

[0065] Table 2

[0066]

[0067] Note: *, compared with the model control group, p<0.05.

[0068] From the data in Table 2, it can be seen that although the Chinese herbal compound extract of the present invention did not change the expression of NLRP3, it could reduce the expression of p-p65, c-Cas-1, IL-1β and IL-18, and inhibit renal inflammatory response.

[0069] Example 7

[0070] Effect of the Chinese herbal compound extract of the present invention on apoptosis of renal cells in mice with hyperuricemia nephropathy

[0071] After the intervention described in Example 4, the mouse kidneys were removed and placed in a -80°C refrigerator for subsequent analysis of renal cell apoptosis-related signaling pathways.

[0072] Western blotting (WB) was used to detect the protein expressions of apoptosis-related signaling pathway molecules such as Bcl2, Bax, phosphorylated p53 (p-p53), cleaved caspase-9 (c-Cas-9), and cleaved caspase-3 (c-Cas-3) in the mouse kidneys to evaluate the potential mechanism of the Chinese medicine compound extract on renal cell apoptosis in hyperuricemia nephropathy mice. The results are shown in Table 3.

[0073] Table 3

[0074]

[0075] Note: *, compared with the model control group, p<0.05.

[0076] From the data in Table 3, it can be seen that the Chinese herbal compound extract of the present invention can increase the expression of Bcl2, reduce the expression of Bax, p-p53, c-Cas-9 and c-Cas-3, and inhibit renal cell apoptosis.

[0077] Example 8

[0078] Effect of the Chinese herbal compound extract of the present invention on apoptosis of HK-2 cells induced by uric acid

[0079] Referring to the method of Example 3, four kinds of medicinal materials were extracted separately to obtain four single-flavor medicinal extracts.

[0080] HK-2 (Human kidney 2, derived from normal human kidney proximal tubules) cells in logarithmic growth phase were seeded in 12-well plates and divided into 7 groups. The cells were stimulated with uric acid and treated with various drug extracts as shown in Table 4. After 24 hours, the cells were collected and the apoptosis of each group was analyzed by flow cytometry.

[0081] Table 4

[0082]

[0083] Note: *, compared with the uric acid-induced group, p<0.05; # , compared with the Chinese medicine compound group, p<0.05.

[0084] As shown in Table 4, compared with the normal control group, uric acid can induce apoptosis of HK-2 cells and significantly increase the apoptosis rate. Astragalus and plantain extracts have no significant effect on the apoptosis rate of HK-2 cells, while the Chinese herbal compound, Smilax glabra and honeysuckle extracts can significantly reduce the apoptosis rate of HK-2 cells, and the Chinese herbal compound extract has better effect than the single herbal extract.

[0085] The terms "a" or "an" herein are used to describe the components and ingredients of the present invention, only for the convenience of description and to give the basic concept of the present invention. This description should be understood to include one or at least one, unless otherwise explicitly indicated, and the singular also includes the plural. The term "or" herein means the same as "and / or".

[0086] The embodiments given herein are only preferred embodiments of the present invention and do not have any limiting effect on the present invention. Without departing from the scope of the technical solution of the present invention, any technical personnel in the relevant technical field may make any equivalent replacement or modification to the technical solution and technical content disclosed in the present invention in any form without departing from the technical solution of the present invention and still fall within the protection scope of the present invention.

Claims

1. A Chinese medicine compound extract for treating hyperuricemia nephropathy, which is prepared from raw materials of Chinese herbal medicines in the following weight parts: 1-10 parts of astragalus, 1-10 parts of honeysuckle, 1-10 parts of smilax glabra, and 1-10 parts of plantain.

2. The Chinese medicine compound extract according to claim 1 is prepared from raw materials of Chinese herbal medicines in the following weight parts: 1-5 parts of Astragalus, 1-5 parts of Honeysuckle, 1-5 parts of Smilax glabra, and 1-5 parts of Plantago.

3. The Chinese herbal compound extract according to claim 1 or 2, which is prepared from raw materials of Chinese herbal medicines in the following parts by weight: 1-2 parts of Astragalus, 1-2 parts of Honeysuckle, 1-2 parts of Smilax glabra, and 1-2 parts of Plantago.

4. A method for preparing a Chinese medicinal compound extract for treating hyperuricemia nephropathy according to any one of claims 1 to 3, the method comprising: (1) Weighing astragalus, honeysuckle, smilax glabra and plantain according to weight, crushing and decocting, and combining the extracts to obtain a crude extract; (2) filtering the crude extract to remove the filter residue, and then concentrating by rotary evaporation to obtain a concentrated solution; (3) freeze-drying the concentrated solution to obtain the Chinese herbal compound extract.

5. The preparation method according to claim 4, wherein The decoction in step (1) comprises: adding double distilled water according to a liquid-to-solid ratio of 5-20 mL:1 g, soaking for 2-4 hours, heating under reflux, and extracting 1-3 times, each time for 1-3 hours.

6. The preparation method according to claim 5, wherein: The extraction is performed by ultrasonic extraction at 70-100°C and a power of 600-900W.

7. The preparation method according to claim 4, wherein The decoction in step (1) comprises: adding 60-90wt% ethanol according to a liquid-to-solid ratio of 5-20mL:1g), soaking for 2-4 hours, heating under reflux, and extracting 1-3 times, each time for 1-3 hours.

8. The preparation method according to claim 7, wherein: The extraction is performed by ultrasonic extraction at 70-100°C and a power of 600-900W.

9. A Chinese medicine composition, comprising as an active ingredient the Chinese medicine compound extract according to any one of claims 1 to 3 and pharmaceutically acceptable excipients, wherein: The pharmaceutically acceptable excipients include one or more of a dispersion medium, a coating, a disintegrant, a lubricant, a surfactant, an antioxidant, a diluent, a cosolvent, an excipient, a sweetener, a flavoring agent, an adsorption carrier or a binder.

10. Use of the Chinese herbal compound extract according to any one of claims 1 to 3 in the preparation of a medicament for treating hyperuricemia nephropathy.

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