阅读说明：本技术 人参皂苷compound K在制备用于治疗糖尿病肾脏病的药物中的应用 (Application of ginsenoside compound K in preparing medicine for treating diabetic nephropathy ) 是由 陈倩 王涛 张祎 刘梦扬 吴昱铮 王丹 任东文 刘洛坤 于 2021-08-26 设计创作，主要内容包括：本发明涉及人参皂苷compound K或其药学上可接受的盐在制备用于治疗糖尿病肾脏病的药物中的应用。(The invention relates to application of ginsenoside compound K or pharmaceutically acceptable salt thereof in preparing a medicament for treating diabetic nephropathy.)

1. The application of the ginsenoside compound K or the pharmaceutically acceptable salt thereof in preparing the medicine for treating diabetic nephropathy.

2. The use according to claim 1, wherein the pharmaceutically acceptable salt is any one of the following salts:

1) salt of ginsenoside compound K with sodium, potassium, magnesium, or calcium cation,

2) salt formed by ginsenoside compound K and organic alkali,

3) salt formed by ginsenoside compound K and amino acid,

4) an ammonium salt.

3. The use according to claim 1 or 2, wherein the ginsenoside compound K or pharmaceutically acceptable salt thereof is used in the form of a pharmaceutical composition in combination with pharmaceutically acceptable additives.

Technical Field

The invention belongs to the field of medicines, and particularly relates to an application of a ginsenoside compound K in preparation of a medicine for treating diabetic nephropathy.

Background

Diabetic nephropathy (DKD) is one of the most serious chronic complications of diabetes. Clinically, patients with type 1 diabetes and type 2 diabetes have microalbuminuria (urinary albumin creatinine ratio 30 to E ℃; E.C.)<300mg/g) or large albuminuria (urinary albumin creatinine ratio ≥ 300mg/g) and/or estimation of glomerular permeability (eGFR)<60ml·min^-1And lasted for more than 3 months, DKD was diagnosed. Epidemic disease survey shows that the DKD prevalence rate in China is 1.23%, the number of DKD patients is about 0.24 hundred million, and the number of new uremia patients caused by DKD reaches 99 million every year. DKD has become a leading cause of Chronic Kidney Disease (CKD) and is also an important risk factor for diabetic patients to develop cardiovascular disease and premature death.

At present, the treatment of DKD mainly comprises life style change, blood pressure and blood sugar control enhancement, proteinuria reduction, cardiovascular and cerebrovascular complications prevention, end-stage renal replacement treatment and the like. The diabetic kidney disease still has the problems of high prevalence rate, lack of means for effectively delaying the development of the kidney disease and the like. In recent years, with the intensive research on the pathogenesis of DKD, novel hypoglycemic agents, renin-angiotensin-aldosterone system (RAAS) inhibitors, and agents directed to the pathogenesis of diabetic renal disease provide new options for the drug treatment of DKD. However, in general, there is still a lack of clinically effective drugs for the targeted treatment of DKD.

DKD belongs to the category of diabetes combined with kidney-yang deficiency, edema and consumptive disease in traditional Chinese medicine according to the clinical manifestations. The traditional Chinese medicine generally considers that DKD is one of the symptoms caused by long-term diabetes, on the basis of deficiency of both qi and yin, because of long-term consumption of qi and yin by diabetes, qi deficiency can promote blood and body fluid to move and is weak, yin deficiency and blood deficiency can cause unsmooth blood circulation, blood stasis can be accumulated for a long time, the blood stasis is cemented with turbid phlegm, dryness-heat can be generated due to long-term accumulation, kidney collaterals can be blocked, essence can not be stored in the kidney, the essence can be discharged downwards to cause albuminuria, and long-term loss of essence can cause deficiency of both qi and yin, and yin and yang can cause deficiency of both yin and yang. Therefore, according to the therapeutic principle of the traditional Chinese medicine, the deficiency tonifying medicinal herbs in the effective prescription for clinically treating the DKD account for the highest proportion, and further analysis on the administration frequency of the deficiency tonifying herbs can find that the administration ratio of the qi tonifying herbs in the treatment of the DKD accounts for more than 60%.

Among the most commonly used qi tonics in an effective formulation for the treatment of DKD is ginseng (Panax ginseng). The Ginseng radix has remarkable therapeutic effect in treating DKD in clinical practice, and multiple saponin components (such as Rb1, Rb2, Rg1, Re, and Rd) have effects of resisting oxidation stress, resisting inflammation, resisting fibrosis, and antagonizing Ca to kidney²⁺Improving the expression of matrix metalloproteinase of kidney tissue, relieving urinary albumin excretion, and protecting DKD renal function.

Disclosure of Invention

The invention aims to provide a medicament capable of effectively treating diabetic nephropathy. After intensive research, the inventor discovers that a ginsenoside Compound K (CK) which is a main in-vivo metabolite of the panaxadiol type saponin can improve the kidney injury caused by hyperglycemia and provides a scientific basis for clinical treatment of DKD.

Specifically, the present invention provides:

1. the application of the ginsenoside compound K or the pharmaceutically acceptable salt thereof in preparing the medicine for treating diabetic nephropathy.

2. The use according to item 1 above, wherein the pharmaceutically acceptable salt is any one of the following salts: 1) a salt of ginsenoside compound K with sodium, potassium, magnesium, or calcium cations, 2) a salt of ginsenoside compound K with an organic base, 3) a salt of ginsenoside compound K with an amino acid, 4) an ammonium salt.

3. The use according to item 1 or 2 above, wherein the ginsenoside compound K or pharmaceutically acceptable salt thereof is used in the form of a pharmaceutical composition in combination with pharmaceutically acceptable additives.

Effects of the invention

According to the present invention, ginsenoside compound K can improve kidney damage caused by hyperglycemia, and thus is expected to be a candidate drug for the treatment of diabetic nephropathy.

Drawings

FIG. 1 shows the pathological morphology of the kidney after H & E, PAS and Masson staining of mouse kidney tissue. In FIG. 1, the magnification is 400 times and the scale bar is 20 μm.

FIG. 2 is a bar graph of kidney mesangial matrix index statistics for each group of mice to evaluate the effect of compound K on kidney mesangial matrix proliferation.

FIG. 3 is a bar graph showing statistics of mesangial expansion of kidney tissues of various groups of mice to evaluate the effect of compound K on mesangial proliferation.

FIG. 4 is a bar graph showing statistics of collagen deposition in glomeruli of kidney tissue of each group of mice to evaluate the effect of compound K on collagen deposition.

It should be noted that, in fig. 2 to 4,^###indicates comparison with the Normal group, p<0.001；^***Representation and model set comparison, p<0.001。

Detailed Description

The present invention is described in detail below by way of the description of the specific embodiments, but this is not a limitation of the present invention, and various modifications or improvements can be made by those skilled in the art according to the basic idea of the present invention, but they are within the scope of the present invention as long as they do not depart from the basic idea of the present invention.

Ginsenoside Compound K is the main metabolite and final absorption form of diol type ginsenoside in human intestinal tract. The chemical name is 20-O-beta-D-glucopyranosyl-20 (S) -protopanaxadiol, and the molecular formula is C₃₆H₆₂O₈Molecular weight is 622.87, and the structural formula is shown in the following chemical formula 1.

[ chemical formula 1]

Ginsenoside Compound K is a non-natural ginsenoside and can be converted from other diol type ginsenosides. At present, the biotransformation process generally includes an enzymatic transformation process and a microbial fermentation process. Ginsenoside Compound K can be prepared by reference to prior art literature (e.g., CN101921304A, CN105255970A, CN101139562A, CN1570133A, etc.). Commercially available products, for example, from Ningbo gold Ainong Biotech limited (product name: ginsenoside Compound K, packaging Specification 80% HPLC), may also be used.

In the present invention, salts of ginsenoside Compound K can also be used. The salts may be pharmaceutically acceptable salts. Specifically, there may be mentioned: acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, etc., or with organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, mandelic acid, tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, aspartic acid, glutamic acid, etc.; salts with metal cations such as sodium, potassium, magnesium, calcium, and aluminum, salts with organic bases such as methylamine, ethylamine, and ethanolamine, salts with various amino acids and amino acid derivatives such as acetylleucine, lysine, and ornithine, or ammonium salts.

In the present invention, the ginsenoside compound K or a pharmaceutically acceptable salt thereof may be used in the form of a pharmaceutical composition in combination with a pharmaceutically acceptable additive.

In the above pharmaceutical composition, the ginsenoside compound K or a pharmaceutically acceptable salt thereof is mixed with a pharmaceutically acceptable additive to obtain a pharmaceutical composition that can be orally or parenterally administered. The pharmaceutical compositions may be formulated by conventional methods.

Administration can be carried out in a form of oral administration (e.g., tablets, capsules, granules, powders, liquid preparations, etc.) or in a form of parenteral administration (e.g., injections, etc.).

As the solid composition for oral administration, tablets, pills, powders, granules, capsules and the like can be used. In such a solid composition, the ginsenoside compound K or a pharmaceutically acceptable salt thereof is mixed with at least one additive. The additives include excipient, disintegrant, lubricant, etc.

Excipients include, for example, lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, and/or magnesium aluminum silicate, and the like.

Disintegrants include cellulose (e.g., calcium carboxymethylcellulose, low-substituted hydroxypropylcellulose, carboxymethylcellulose, croscarmellose sodium or crystalline cellulose), starches (e.g., corn starch, pregelatinized starch or sodium carboxymethyl starch), crospovidone, and the like.

The lubricant comprises magnesium stearate, calcium stearate, stearic acid, sucrose fatty acid ester, talc, polyethylene glycol, etc.

The solid preparation of the present invention may optionally contain appropriate amounts of other additives according to the purpose. These additives include binders, plasticizers, coating agents, deflocculants, solubilizers, sweeteners, acidulants, flavorants, pH adjusters, co-solvents, colorants, flavorants, and the like. The binder includes sodium alginate, gelatin, dextrin, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, etc. The plasticizer comprises triethyl citrate, glycerin fatty acid ester, polyethylene glycol, etc. The coating agent comprises ethyl cellulose, hydroxypropyl methyl cellulose, etc. Deflocculants include talc, calcium stearate, and the like. The solubilizer comprises sucrose fatty acid ester, sorbitan monostearate, sodium dodecyl sulfate, etc. The sweetener comprises saccharin, dipotassium glycyrrhizinate, and sweet stevia. Acidulants include citric acid, malic acid, ascorbic acid, fumaric acid, and the like. Flavoring agents include 1-menthol, sodium chloride, sucrose, and the like. The pH regulator includes citrate, phosphate, carbonate, acetate, etc. The cosolvent comprises cyclodextrin, arginine, lysine, triaminomethane, etc. The colorant comprises ferric oxide yellow, ferric oxide, sodium copper chlorophyllin, etc. Flavoring agents include orange oil, lemon oil, peppermint oil, eucalyptus oil, and the like.

The tablets of the invention may be prepared according to conventional methods in the manufacture of tablets, for example, by: i) preparing a mixture comprising ginsenoside compound K or a pharmaceutically acceptable salt thereof, and one or more additives selected from the group consisting of excipients, disintegrants, lubricants, binders, plasticizers, coating agents, deflocculants, solubilizers, sweeteners, acidifiers, flavors, pH adjusters, cosolvents, colorants, and flavoring agents, and then ii) compression-molding the mixture obtained in the above i).

Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of the aqueous solvent include distilled water for injection and physiological saline. Examples of the nonaqueous solvent include vegetable oils such as propylene glycol, polyethylene glycol, and olive oil, alcohols such as ethanol, and tween 80. The preparation may also contain isotonic agent, antiseptic, wetting agent, emulsifier, dispersant, stabilizer, or dissolving adjuvant. For example, it may be sterilized by filtration through a filter that retains bacteria, by addition of a bactericide, or by irradiation. Alternatively, the composition may be prepared as a sterile solid, and may be used after being dissolved or suspended in sterile water or a sterile solvent for injection before use.

In the case of oral administration, the dose is preferably 1 day to about 1 to 1000mg/kg, preferably 10 to 500mg/kg, per body weight, and the dose is administered once or in 2 to 4 divided doses. The dose is appropriately selected depending on the condition, age, sex, and the like.

Examples

The present invention will be further explained or illustrated by the following examples, which should not be construed as limiting the scope of the present invention.

It should be noted that various reagents and the like used in the following examples are known in the art and are commercially available.

Grouping and administration of laboratory animals

db/db diabetic model mice have been shown to exhibit many symptoms similar to those of DKD patients, with marked elevation of blood glucose at 10 weeks of age, glomerular hypertrophy at 10 to 16 weeks of age, severe dilation of mesangial at 16 weeks of age, and increased proteinuria from 8 weeks of age to 25 weeks of age, as animal models for DKD studies (see references 1 and 2 below).

Reference 1: the number of the Breyer MD is small,E,Brosius FC,Coffman TM,Harris RC,Heilig CW,Sharma K.Mouse models of diabetic nephropathy.J Am Soc Nephrol 2005；16(1): 27-45。

reference 2: brosius F, Alpers C, Bottinger E, Breyer M, Coffman T, Gurley S, Harris R, Kakoki M, Kretzler M, Leiter E, Levi M, McIndo R, Sharma K, Smithies O, Susztak K, Takahashi N, Takahashi T. mouse models of diabatic neuropathopy.J. Am Soc Nephrol 2009; 20(12):2503-2512.

SPF male BKS-Lepr was selected for this study^em2Cd47930/Gpt (db/db) diabetic mice, 7 weeks old (30 + -2 g), 10 SPF-class male wild-type (db/m) mice (24 + -2 g) of the same week old were purchased from Nanjing university model animal institute. After adaptive breeding for 1 week, db/db mice were divided into a model group, a positive control group, and a CK administration group according to blood glucose levels, and db/m mice of the same week age were used as a wild type control group (normal group).

The wild type control group (normal group) and the model group were fed with conventional feeds, the positive control group was fed with 200mg/kg of a feed containing 0.2% of metformin (> 98.0%, HPLC, shanghai shieiyi chemical industry development limited), and the CK administration group was fed with 40mg/kg of a feed containing 0.03% of CK (> 98.0%, HPLC, shanghai-sourced leaf biotechnology limited).

Renal function and histological examination

After 16 weeks of continuous feeding, groups of mice were placed in metabolic cages fasted without water deprivation, and 24 hour urine was collected and volume recorded. Then, the supernatant was centrifuged at 3500g at 4 ℃ for 10min, and the supernatant was collected and stored at-20 ℃. According to the determination method of the specification of a biochemical kit (Zhongsheng Bei-accu Biotechnology GmbH), the contents of albumin (MALB) and creatinine (Cre) in a mouse urine sample are detected, and the ratio (UACR, mu g/mg) of urine protein creatinine is calculated. Isoflurane was respiratory anesthetized and dissected, and kidney tissue was removed intact from the mice for H & E, PAS and Masson staining to evaluate the effect of CK on mouse kidney function comprehensively. These operations are conventional techniques well known to those skilled in the art (see references 3 and 4 below).

Reference 3: zhang YY, Tang PMK, Tang PCT, Xiao J, Huang XR, Yu C, Ma RCW, Lan hy. lrna9884, a novel smad 3-dependentlncrna, proteins diabetic kit in our db/db microorganism via enhancing mcp-1-dependentresidual in errors 2019; 68(7):1485-1498.

Reference 4: liang G, Song LT, Chen ZL, Qian YY, Xie JJ, Zhao LW, Lin Q, Zhu GH, Tan Y, Li XK, Mohammadi M, huangg zf. fiber growth factor 1 animals diabetes neuropathology by an anti-inflammation mechanism. kidney Int 2018; 93(1), 95-109.

Statistical treatment

The experimental results were analyzed using SPSS 26.0 statistical software, and the data were measured as mean. + -. standard deviation: () The sample comparison was performed using a separate sample t test, and the test level α was 0.05.

Results of the experiment

TABLE 1 Change in the major metabolic parameters of the groups of mice after feeding

Note: n-7, compared to the normal group,^##p<0.01，^###p<0.001; in comparison with the set of models,^*p<0.05

the results in Table 1 show that the body weight average of db/m mice and db/db mice increases with increasing week age, and that the body weight of the model group is significantly higher than that of the normal group. Compared with the normal group, the kidney weight, 24h urine volume, urine MALB and UACR levels of db/db mice are all obviously increased. After CK is administered, the urine volume of 24h, the urine MALB and UACR levels can be obviously reduced, and CK can be shown to improve the kidney function of db/db mice.

In addition, fig. 1 shows the pathological morphology of the kidney after H & E, PAS and Masson staining of mouse kidney tissue. As shown in FIG. 1, based on the pathological morphological observation under the mirror, the glomerular mesangial region in the kidney tissue of the model db/db mouse was significantly amplified, the inflammatory infiltration was observed locally in H & E staining and PAS staining, PAS staining positive substance (purple color) specifically labeled glycogen deposition was significantly increased, and Masson staining specifically labeled collagen deposition showed heavier blue collagen deposition, compared with the normal db/m mouse. After the administration is continued for 4 months, as shown in fig. 2 and fig. 3, statistics are carried out on the mesangial matrix index and the mesangial expansion of the kidney tissues of each group of mice, and it can be seen that in the CK administration group, the mesangial matrix index and the mesangial expansion of db/db mice are obviously reduced, and meanwhile, the inflammatory infiltration is relieved; and as shown in FIG. 1, PAS staining positive material (purple) is reduced; meanwhile, as shown in fig. 4, the collagen deposition in glomeruli of kidney tissue of each group of mice was counted, and it was found that the collagen deposition (blue color) was significantly reduced in the CK administration group. Further shows that CK can improve kidney injury of db/db mice and relieve the pathogenesis of DKD.

The above results indicate that CK can improve DKD kidney damage and thus is expected to be a candidate drug for the treatment of diabetic nephropathy.