Application of oxidized berberine and berberrubine in treatment of diabetic osteoporosis

Abstract

The invention discloses application of oxidized berberine and berberrubine in treatment of diabetic osteoporosis, and belongs to the technical field of biological medicine. Researches find that both oxidized berberine and berberrubine have the effect of treating diabetic osteoporosis, and can significantly improve the femur microstructure of a diabetic osteoporosis model animal, so that the femur microstructure recovers to a normal level; the blood glucose clearing capacity can be improved, the insulin resistance index is reduced, and the insulin signal channel function is recovered; the oxidative stress state of diabetic osteoporosis model animals can be relieved, the proinflammatory factor level is reduced, and the anti-inflammatory and anti-oxidation effects are achieved. The invention provides a new scheme and data support for the treatment of diabetic osteoporosis, has better curative effect, safety and economy than the prior art, and has important clinical transformation value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of oxidized berberine and berberine red in the treatment of diabetic osteoporosis. BACKGROUND

[0002] Diabetic osteoporosis (DOP) is a common complication of diabetes, characterized by reduced bone density, destruction of femoral microstructure, and significantly increased risk of fracture, which seriously affects the quality of life of patients and increases the medical burden, and has become one of the important causes of disability and death in the elderly diabetic population.

[0003] Current clinical treatment options for DOP have obvious shortcomings, mainly symptomatic treatment, and cannot be cured: bisphosphonates, hormone replacement therapy, etc. can only inhibit bone resorption and cannot improve the root problems of high blood sugar, insulin resistance and oxidative stress caused by diabetes, and are prone to relapse after drug withdrawal. Drug side effects: long-term use of bisphosphonates may cause mandibular osteonecrosis, and hormone therapy may cause blood glucose fluctuations and increase the risk of thrombosis. Bottleneck of application of berberine: Although the main component of traditional Chinese medicine coptis rhizome, berberine, has been proven to improve osteoporosis, its oral bioavailability is less than 1%, it is widely metabolized in the body, and the blood drug concentration is extremely low, which limits its clinical efficacy. Therefore, developing DOP treatment drugs with "hypoglycemic-anti-inflammatory-bone metabolism regulation" triple action, high safety and strong biological activity has become an urgent need in the current medical field. SUMMARY

[0004] The purpose of the present application is to provide the application of oxidized berberine and berberine red in the treatment of diabetic osteoporosis to solve the problems existing in the prior art. The present application found that both oxidized berberine and berberine red have the effect of treating diabetic osteoporosis, can significantly improve the femoral microstructure of diabetic osteoporosis model animals, and restore it to normal level; can improve the blood glucose clearance capacity, reduce the insulin resistance index, and restore the function of the insulin signaling pathway; can also alleviate the oxidative stress state of diabetic osteoporosis model animals, reduce the level of pro-inflammatory factors, and achieve the effects of anti-inflammatory and antioxidant. The present application provides a new scheme and data support for the treatment of diabetic osteoporosis, and the efficacy, safety and economy are superior to those of the prior art, and have important clinical transformation value.

[0005] To achieve the above-mentioned purpose, the present application provides the following scheme:

[0006] The present application provides the application of oxidized berberine in the preparation of a drug for treating diabetic osteoporosis.

[0007] The present application also provides the application of berberine red in the preparation of a drug for treating diabetic osteoporosis.

[0008] The application also provides application of the combination of the oxidized berberine and berberrubine in preparation of a medicine for treating diabetic osteoporosis.

[0009] Further, the treatment of diabetic osteoporosis includes reducing the fasting blood glucose level, insulin level and insulin resistance index.

[0010] Further, the treatment of diabetic osteoporosis includes improving the bone density and bone volume fraction, increasing the trabecular bone number and trabecular bone thickness, and reducing the trabecular bone separation degree.

[0011] Further, the treatment of diabetic osteoporosis includes anti-inflammation and reducing the oxidative stress level.

[0012] The application also provides a medicine for treating diabetic osteoporosis, characterized in that the medicine comprises the oxidized berberine and berberrubine as effective components.

[0013] Optionally, the medicine further comprises a pharmaceutically acceptable carrier or excipient.

[0014] Optionally, the dosage form of the medicine comprises tablets, capsules, granules, oral liquids, pills, powders, drops and chewable tablets.

[0015] The application discloses the following technical effects:

[0016] The application researches and finds that the oxidized berberine and berberrubine both have the effect of treating diabetic osteoporosis, can significantly improve the femur microstructure of a diabetic osteoporosis model animal and restore the femur microstructure to the normal level, can improve the blood glucose clearance capacity, reduce the insulin resistance index and restore the insulin signal pathway function, and can also relieve the oxidative stress state of the diabetic osteoporosis model animal and reduce the pro-inflammatory factor level, thereby achieving the anti-inflammatory effect.

[0017] The application takes the oxidized berberine and berberrubine as effective components, proposes an integrated treatment scheme of “blood glucose reduction-bone protection-anti-inflammation” for diabetic osteoporosis, can reduce the fracture risk of a diabetic osteoporosis patient, significantly improves the life quality and reduces the disability rate. The existing diabetic osteoporosis treatment has a high annual cost, the oxidized berberine and berberrubine are used as the effective components of the medicine, the production cost is lower than that of the existing bisphosphonate medicine, and the medical expenditure can be greatly saved. Meanwhile, the oxidized berberine and berberrubine can be extracted from the natural plant Coptis chinensis and derived, can drive the development of the Chinese herbal medicine planting and deep processing industry, form an “agriculture-pharmaceutical” industrial chain, and has remarkable social and economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative effort.

[0019] Figure 1 The changes of body weight, blood glucose level and water consumption of each group of rats during the experiment were observed, and the results were as follows: ## P < 0.01 vs. Con; & P < 0.05, && P < 0.01 vs. OBB+BRB; ** P < 0.01 vs. DOP;

[0020] Figure 2 The determination results of fasting blood glucose, serum insulin and HOMA-IR of each group of rats were as follows: ## P < 0.01 vs. Con; && P < 0.01 vs. OBB+BRB; * P < 0.05, ** P < 0.01 vs. DOP;

[0021] Figure 3 The quantitative analysis results of bone microstructure (BV / TV, Tb.Sp, Tb.N, Tb.Th and BMD) of each group of rats were as follows: ## P < 0.01 vs. Con; & P < 0.05, && P < 0.01 vs. OBB+BRB; * P < 0.05, ** P < 0.01 vs. DOP;

[0022] Figure 4 The determination results of inflammatory cytokines (IL-1β, IL-6, TNF-α and IL-10) of each group of rats were as follows: ## P < 0.01 vs. Con; & P < 0.05, && P < 0.01 vs. OBB+BRB; * P < 0.05, ** P < 0.01 vs. DOP;

[0023] Figure 5The determination results of oxidative stress indicators (CAT, SOD, GSH-Px, MDA) of rats in each group; wherein, ## P < 0.01 vs. Con; & P < 0.05, && P < 0.01 vs. OBB+BRB; * P < 0.05, ** P < 0.01 vs. DOP;

[0024] Figure 6 The mRNA expression levels of RUNX2, BMP2, RANKL and OPG in bone tissues of rats in each group; wherein, ## P < 0.01 vs. Con; & P < 0.05, && P < 0.01 vs. OBB+BRB; * P < 0.05, ** P < 0.01 vs. DOP. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of certain aspects, features and embodiments of the present application, but not a limitation thereof.

[0026] It is to be understood that the terms used in the present application are merely descriptive, rather than limiting, of the particular embodiments. In addition, for numerical ranges in the present application, it is to be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value between any stated value or stated range, as well as any other stated value or intermediate value in the stated range, is also included in the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, the content of the present specification will control.

[0028] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are exemplary only.

[0029] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" or variants thereof are open-ended, and specifically do not exclude additional, unrecited elements or method steps.

[0030] The experimental methods in the following examples are all routine methods unless otherwise specified. The instruments and equipment used in the following examples are all routine laboratory instruments and equipment unless otherwise specified. The test materials used in the following examples are all purchased from routine biochemical reagent stores unless otherwise specified.

[0031] Example 1

[0032] 1. Experimental materials

[0033] Experimental animals: 5-week-old female SD rats (body weight 140-160 g), SPF level environment feeding (temperature 23±2℃, humidity 50±10%, 12h light-dark cycle).

[0034] Experimental drugs: Oxyberberine (OBB): CAS number 549-21-3, purity: HPLC≥98%;

[0035] Berberrubine (BRB): CAS number 15401-69-1, purity: HPLC≥98%.

[0036] 2. Experimental methods

[0037] 2.1 Grouping and administration

[0038] DOP model was constructed. After bilateral ovariectomy (OVX) of rats for 6 weeks, high-fat diet (HFD, fat content 34.9%) was given for 4 weeks, and then streptozotocin (STZ, 30 mg / kg, pH=4.5 sodium citrate buffer) was injected intraperitoneally for 2 consecutive days. 72 h later, if the fasting blood glucose of the tail vein was ≥11.1 mmol / L, it was considered that the model was successfully constructed. The successfully modeled rats were randomly divided into 5 groups, including DOP model group, metformin group (Met) group, BRB group, OBB group and OBB+BRB group, 6 rats in each group. In addition, a sham operation control group (Control) was set up, in which the bilateral ovaries were not removed (OVX) and the rats were fed with ordinary feed. After normal feeding for 10 weeks, the rats were given 0.5% CMC-Na by gavage, the same as the modeling group. Each group was given drug for 12 weeks in succession. The water intake, fasting blood glucose and body weight changes of rats during the experiment were continuously monitored.

[0039] Table 1 Grouping and drug administration

[0040]

[0041] 2.2 Index detection

[0042] Glycometabolism index: fasting blood glucose (Fasting Blood glucose, FBG), insulin level (Insulin), insulin resistance index (HOMA-IR); FBG was detected by blood glucose meter, fasting insulin was detected by ELISA and HOMA-IR was calculated.

[0043] Femur microstructure index: bone volume fraction (BV / TV), trabecular separation (Tb.Sp), trabecular number (Tb.N), trabecular thickness (Tb.Th), bone mineral density (BMD) and the like; Micro-CT scan of rat femur and quantitative analysis.

[0044] Serum factor index: inflammatory factors (IL-1β, IL-6, TNF-α, IL-10); detected by ELISA kit.

[0045] Oxidative stress index: superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), catalase (CAT); detected by biochemical kit.

[0046] Molecular mechanism related index: RANKL, BMP2, OPG, RUNX2 gene expression level; detected by qPCR.

[0047] 3、Experimental results

[0048] 3.1 Effect of oxoberberine and berberine red on water intake, fasting blood glucose and body weight of rats

[0049] like Figure 1 As shown, compared with the normal group of rats, after successful modeling, the rats in the model group experienced a continuous decrease in body weight, but a significant increase in water intake and blood glucose levels, which did not recover until the end of the experiment. Oral administration of oxidized berberine and berberine significantly reduced the weight loss, water intake, and fasting blood glucose levels in the rats during the experiment.

[0050] 3.2 Effects of oxidized berberine and berberine on fasting blood glucose, insulin levels, and insulin resistance index in rats

[0051] like Figure 2 As shown in Table 2, compared with the model group rats, the fasting blood glucose of the OBB, BRB, and OBB+BRB groups was significantly reduced (P<0.01), insulin levels were reduced, and the HOMA-IR index was decreased (P<0.05).

[0052] Table 2. Results (average) of fasting blood glucose, insulin levels, and insulin resistance index in rats of each group.

[0053]

[0054] 3.3 Effects of oxidized berberine and berberine on the fine structure and biomechanics of rat femur

[0055] like Figure 3 As shown in Table 3, Micro-CT results revealed that in the model group rats, femoral BMD, Tb.Th, BV / TV decreased, Tb.N decreased, and Tb.Sp increased. OBB, BRB, and OBB+BRB all increased BMD and BV / TV, increased Tb.Th and Tb.N, and decreased Tb.Sp. Furthermore, the data in Table 3 indicate that the OBB+BRB group showed a significantly better effect on improving the fine structure of the rat femur than the OBB and BRB groups, demonstrating a clear synergistic effect.

[0056] Table 3. Results of the measurement of fine structure of the femur in each group of rats (mean value)

[0057]

[0058] 3.4 Effects of oxidized berberine and berberine on serum inflammatory factors in rats

[0059] like Figure 4As shown in Table 4, the serum IL-1β, IL-6, TNF-α levels of the OBB+BRB group were lower than those of the model group (P<0.05); the anti-inflammatory factor IL-10 was higher than that of the model group. According to the data in Table 3, the OBB+BRB group showed a significantly better anti-inflammatory effect than the OBB group and the BRB group, especially in terms of IL-1β and TNF-α, indicating that the combination of OBB and BRB has a synergistic effect.

[0060] Table 4 Determination results of serum inflammatory factors of rats in each group (average value)

[0061]

[0062] 3.5 Regulation of oxidative stress indicators by OBB and BRB in rats

[0063] As Figure 5 As shown in Table 5, the CAT, SOD, and GSH-Px activities of the OBB+BRB group were significantly higher than those of the model group, and the MDA level was lower (P<0.01), indicating that OBB can effectively reduce oxidative stress. According to the data in Table 5, the OBB+BRB group showed a significantly better oxidative stress reduction effect than the OBB group and the BRB group, especially in terms of GSH-Px and MDA, indicating that the combination of OBB and BRB has a synergistic effect.

[0064] Table 5 Determination results of serum oxidative stress indicators of rats in each group (average value)

[0065]

[0066] 3.6 Effects of OBB and BRB on the expression of osteogenesis-related genes in rats

[0067] As Figure 6 shown, in bone tissue, OBB+BRB can up-regulate the mRNA expression of osteogenesis-related genes RUNX2 and BMP2, down-regulate the mRNA expression of osteoclast-related gene RANKL, and increase the mRNA expression level of OPG, confirming that OBB and BRB can promote osteogenesis by regulating the RANKL-OPG signaling axis.

[0068] The above results show that after the intervention of berberine and berberrubine, the bone volume fraction of the femur of the DOP model rats is significantly improved, the trabecular bone number is significantly increased, the trabecular bone spacing is reduced, the femur microstructure is close to the normal control group level, and the bone loss is effectively inhibited. In terms of glucose metabolism and insulin resistance regulation, after continuous administration, the fasting blood glucose of rats is significantly reduced, the blood glucose clearance ability is significantly improved. The fasting insulin level decreases, the insulin resistance index decreases, and the insulin signal pathway function recovers. In view of the vicious cycle of DOP "oxidative stress-chronic inflammation", after the intervention of berberine and berberrubine, the superoxide dismutase activity in the serum of rats increases, the glutathione peroxidase activity increases, the malondialdehyde level of lipid peroxidation product decreases, and the oxidative stress state is effectively relieved. The level of serum pro-inflammatory factors is reduced, the level of serum anti-inflammatory factors is improved, and its role in blocking the "inflammation-bone resorption" cascade reaction is significant.

[0069] The above-described embodiments are only to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. Use of oxidized berberine in the preparation of a drug for treating diabetic osteoporosis.

2. Use of berberine red in the preparation of a drug for treating diabetic osteoporosis.

3. Use of oxidized berberine combined with berberine red in the preparation of a drug for treating diabetic osteoporosis.

4. Use according to any one of claims 1 to 3, characterized in that, The treatment of diabetic osteoporosis includes reducing fasting blood glucose, insulin level and insulin resistance index.

5. Use according to any one of claims 1 to 3, characterized in that, The treatment of diabetic osteoporosis includes improving bone density and bone volume fraction, increasing bone trabecula number and bone trabecula thickness, and reducing bone trabecula separation degree.

6. Use according to any one of claims 1 to 3, characterized in that, The treatment of diabetic osteoporosis includes anti-inflammatory and reducing oxidative stress level.

7. A medicament for treating diabetic osteoporosis, characterized by, The drug has oxidized berberine and berberine red as effective components.

8. The medicament according to claim 7, characterized in that, The drug also includes a pharmaceutically acceptable carrier or excipient.

9. The medicament according to claim 7, characterized in that, The dosage form of the drug includes tablets, capsules, granules, oral liquids, pills, powders, drops, chewable tablets.

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