An effective part of traditional chinese medicine combination for improving diabetic peripheral neuropathy

Abstract

The application discloses a traditional Chinese medicine combined effective part for improving diabetic peripheral neuropathy, which is made of a volatile oil effective part, a water-soluble effective part and an alcohol-soluble effective part; the volatile oil effective part comprises alpha-rolene, alpha-santalene, bisabolene, beta-bisabolene and nerolidol; the water-soluble effective part comprises salvianolic acid A, salvianolic acid B and danshensu; and the alcohol-soluble effective part comprises Z ligustilide, α boswellic acid, β boswellic acid, tanshinone IIA and 2-methoxy-5-acetyl-furanojimane-1(10)-en-6-ketone. The traditional Chinese medicine combined effective part provided by the application can regulate blood sugar, improve sciatic nerve conduction velocity, repair damaged tissues, inhibit oxidative stress reaction and relieve inflammation and pain, thereby improving diabetic peripheral neuropathy, and has reliable curative effect, good safety, low adverse reaction and the like, and can be used for preparing a drug preparation for improving diabetic peripheral neuropathy.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an effective component of a traditional Chinese medicine combination that improves diabetic peripheral neuropathy and its preparation method. Background Technology

[0002] Diabetic peripheral neuropathy (DPN) is the most common refractory complication of diabetes. Its main clinical manifestations include burning, tingling, tearing pain, and hyperalgesia in both distal extremities, accompanied by sensory abnormalities. Without timely intervention, it can easily lead to foot ulcers, gangrene, and amputation, placing a heavy financial burden on patients and causing devastating consequences. The pathogenesis of DPN is complex, mainly related to metabolic disorders, oxidative stress, inflammatory responses, vascular damage, and neurotrophic disorders.

[0003] Currently, there is no specific drug for treating DPN. Most treatments rely on regulating blood sugar, taking neurotrophic drugs, and antioxidants to alleviate symptoms. While these methods offer some relief, their efficacy is still insufficient. Some medications also have unavoidable side effects such as liver and kidney damage and gastrointestinal reactions, making them unsuitable for long-term use.

[0004] Traditional Chinese medicine classifies DPN (digestive neuropathy) under the category of "Xiao Ke Bi Zheng" (wasting and thirsting arthralgia). Its etiology is often due to excessive depletion of Qi and blood, leading to stagnation and internal obstruction of Qi and blood, which in turn obstructs the meridians, causing stagnation and ultimately resulting in symptoms such as limb pain and numbness. Angelica sinensis, Salvia miltiorrhiza, frankincense, and myrrh are commonly used clinically to remove blood stasis and relieve pain. Among them, Angelica sinensis nourishes blood and relieves pain, serving as the main herb for promoting blood circulation; Salvia miltiorrhiza cools the blood, reduces swelling, removes blood stasis, and promotes new tissue growth; frankincense and myrrh are often used together to promote blood circulation, regenerate tissue, relieve pain, and disperse blood stasis. The four herbs combined exert the effects of nourishing and promoting blood circulation, soothing the meridians, regulating Qi, relieving pain, and eliminating masses and lumps. Summary of the Invention

[0005] Objective of the Invention: The objective of this invention is to provide an effective component of a traditional Chinese medicine combination that is reliable in efficacy, safe, and effectively improves diabetic peripheral neuropathy. Another objective of this invention is to provide a method for preparing and applying the effective component of the aforementioned traditional Chinese medicine combination.

[0006] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows: A combination of effective components of a traditional Chinese medicine that improves diabetic peripheral neuropathy is made from volatile oil effective components, water-soluble effective components and alcohol-soluble effective components; The effective components of the volatile oil include α-ocimene, α-santalene, bisabolene, β-bisabolene, and nerolidol; The water-soluble active components include salvianolic acid A, salvianolic acid B, and tanshinone; Alcohol-soluble effective fractions include Z -Ligustilide, α - Frankincense acid, β - Boswellic acid, tanshinone IIA, 2-methoxy-5-acetyl-furangermaline-1(10)-en-6-one.

[0007] As a preferred embodiment, the effective components of the above-mentioned traditional Chinese medicine combination include, by weight percentage, 15-30% α-ocimene, 11-25% α-santalene, 8-20% bisabolene, 8-20% β-bisabolene, and 3-10% nerolidol; The water-soluble effective fractions include, by weight percentage, 10-20% salvianolic acid A, 25-50% salvianolic acid B, and 20-40% tanshinone; The alcohol-soluble effective fraction comprises 22-45% by weight. Z -Ligustilide, α - Boswellic acid 20~40%, β - Boswellic acid 20~40%, tanshinone IIA 5~15%, 2-methoxy-5-acetyl-furangermaline-1(10)-en-6-one 10~30%.

[0008] As a particularly preferred embodiment, the effective components of the above-mentioned traditional Chinese medicine combination include, by weight percentage, 35.9% α-ocimene, 23.5% α-santalene, 17.2% bisabolene, 17% β-bisabolene, and 6.4% nerolidol; The water-soluble effective fractions include, by weight percentage, 15% salvianolic acid A, 45% salvianolic acid B, and 40% tanshinone; The alcohol-soluble effective fraction includes a percentage by weight. Z -Ligustin 30%, α - Boswellic acid 27%, β - Boswellic acid 23%, tanshinone IIA 7%, 2-methoxy-5-acetyl-furangermaline-1(10)-en-6-one 13%.

[0009] The application of the effective components of the traditional Chinese medicine combination described in this invention in the preparation of drugs to improve diabetic peripheral neuropathy.

[0010] According to actual needs, the effective components of the traditional Chinese medicine combination of the present invention and a pharmaceutically acceptable carrier can be prepared into drugs in dosage forms such as tablets, pills, powders, decoctions, granules, injections, capsules or decoction pastes.

[0011] Explanation of beneficial effects: The effective components of the traditional Chinese medicine combination for improving diabetic peripheral neuropathy described in this invention, selected through experimental screening, contain volatile aromatic oils, phenolic acids and alcohol-soluble lactones / steroids and tanshinones in aqueous solution, scientifically combined. Experiments have confirmed that the effective components of the traditional Chinese medicine combination provided by this invention can regulate blood sugar, improve sciatic nerve conduction velocity, repair damaged tissue, inhibit oxidative stress, and have anti-inflammatory and analgesic effects, demonstrating significant efficacy in improving diabetic peripheral neuropathy. Furthermore, no obvious toxic side effects were observed during the experiments, indicating good safety. Attached Figure Description

[0012] Figure 1 The graph shows the effects of the effective components of the traditional Chinese medicine combination on rat body weight and fasting blood glucose levels. A: Trend graph of rat body weight; B: Trend graph of rat fasting blood glucose levels; C: Last body weight of rats; D: Last fasting blood glucose level of rats.

[0013] Figure 2 The figure shows the effect of the effective components of the traditional Chinese medicine combination on the mechanical pain threshold of rats.

[0014] Figure 3 The graph shows the effect of the effective components of the traditional Chinese medicine combination on the sciatic nerve conduction velocity in rats. A: Motor nerve conduction velocity in rats; B: Sensory nerve conduction velocity in rats.

[0015] Figure 4 The figure shows the effect of the effective components of the traditional Chinese medicine combination on the sciatic nerve tissue of rats.

[0016] Figure 5 The figure shows the effect of the effective components of the traditional Chinese medicine combination on the dorsal root ganglion tissue of rats.

[0017] Figure 6 The figure shows the effects of the effective components of the traditional Chinese medicine combination on oxidative stress indicators in rat serum. A: Serum malondialdehyde (MDA) level; B: Serum glutathione (GSH) level; C: Serum superoxide dismutase (SOD) level; D: Serum total antioxidant capacity (TAOC) level; E: Serum advanced glycation end products (AGEs) level.

[0018] Figure 7 The figure shows the effects of the effective components of the traditional Chinese medicine combination on oxidative stress indicators in the sciatic nerve tissue of rats. A: GSH level in the sciatic nerve; B: SOD level in the sciatic nerve.

[0019] Figure 8 The graph shows the effects of the effective components of a traditional Chinese medicine combination on the levels of inflammatory factors in rat serum and sciatic nerve. A: Serum tumor necrosis factor- α (TNF- α A: Serum interleukin-1 level; B: Serum interleukin-1 β(IL-1 β C: Serum interleukin-6 (IL-6) level; D: TNF-α level in the sciatic nerve. α Level; E: IL-1 in the sciatic nerve β Level; F: IL-6 level in the sciatic nerve.

[0020] Figure 9 The figure shows the effect of the effective components of the traditional Chinese medicine combination on pain indicators in rat serum. A: Serum level of brain-derived neurotrophic factor (BDNF); B: Serum level of prostaglandin E2 (PGE2); C: Serum level of substance P (SP). Detailed Implementation

[0021] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading this invention, any modifications of the invention in various equivalent forms by those skilled in the art fall within the scope defined by the appended claims. All raw materials used in this invention are commercially available.

[0022] Example 1 A combination of effective components of a traditional Chinese medicine that improves diabetic peripheral neuropathy is made from volatile oil effective components, water-soluble effective components and alcohol-soluble effective components; The effective components of the volatile oil include, by weight percentage: 35.9% α-ocimene, 23.5% α-santalene, 17.2% bisabolene, 17% β-bisabolene, and 6.4% nerolidol; The water-soluble active components include, by weight percentage, 15% salvianolic acid A, 45% salvianolic acid B, and 40% tanshinone; The alcohol-soluble effective fraction includes a percentage by weight. Z -Ligustin 30%, α - Boswellic acid 27%, β - Boswellic acid 23%, tanshinone IIA 7%, 2-methoxy-5-acetyl-furangermaline-1(10)-en-6-one 13%.

[0023] Example 2: Experimental study on the improvement of diabetic peripheral neuropathy by effective components of traditional Chinese medicine combination I. Experimental Materials and Drugs 1. Drugs and reagents Thioctic acid tablets (National Drug Approval Number H20100152, Shandong Qidu Pharmaceutical Co., Ltd.); Sodium citrate buffer (0.1 mmol·L⁻¹) -1, pH 4.5, Batch No.: C-1013, Beijing Solarbio); streptozotocin (STZ; Batch No.: S0130, Sigma, USA); high-fat diet (formula: 43.6% maintenance basal diet, 12% sucrose, 17.5% lard, 10% casein, 10% whole milk powder, 2% laboratory animal premix, 2% calcium hydrogen phosphate, 1.9% microcrystalline cellulose, 1% stone powder) was purchased from Shuyu Biotechnology Co., Ltd.

[0024] Test drug: the effective part of the traditional Chinese medicine combination according to Example 1 of the present invention.

[0025] 2. Experimental instruments Sannuo Anwen+ blood glucose meter (Batch No.: 2J01B240303, Sinocare Inc.); Von-Frey filament mechanical stimulation needle (Danmic Aesthesio, USA); Enspire multifunctional microplate reader (PerkinElmer, USA); small animal anesthesia machine (Shenzhen RWD Life Technology Co., Ltd.); electromyography evoked potential instrument (Suzhou Haishen Company); Tissuelyer-48 full-automatic rapid sample grinder (Shanghai Jingxin Industrial Development Co., Ltd.).

[0026] 3. Laboratory animals SPF grade male SD rats, weighing 180~220 g, were purchased from Shanghai Slack Laboratory Animal Co., Ltd., with the animal license number SCXK (Shanghai) 2022-0004.

[0027] II. Experimental methods 1. Establishment of rat model of diabetic peripheral neuropathy After adaptive feeding, healthy rats were randomly divided into a blank control group (CON) of 8 rats and a DPN model group of 72 rats by stratified grouping according to body weight. The blank group was fed with normal diet until the end of the experiment. After the DPN model group rats were fed with high-fat diet for 3 weeks, they were fasted with free access to water for 12 h, and a 1% STZ solution prepared in sodium citrate buffer was injected intraperitoneally on one side at a dose of 35 mg / kg. The blank group was injected with an equal volume of sodium citrate buffer at the same site. 72 h later, the fasting tail vein blood glucose of rats was measured to be ≥11.1 mmol·L -1 which was regarded as successful modeling of diabetic rats.

[0028] After continuing to feed diabetic rats a high-fat diet for 3 weeks, the up-and-down method was used to detect the mechanical pain threshold. Rats were placed in cages with porous wire bottoms and allowed to move freely for 30–60 minutes to acclimatize. First, a 2.0 g von Frey fiber needle was used to vertically stimulate the middle of the rat's hind paw, avoiding the paw pads to prevent interference with the results. A negative response was indicated when the fiber bent into an S-shape and the rat did not withdraw its paw or try to avoid it for more than 6 seconds. A positive response was indicated when the rat licked, bit, lifted its paw, or tried to avoid the stimulation. This was repeated 3 times, with approximately 2 minutes between each stimulation. Based on the animal's response, a fiber with a larger or smaller weight was used for the next stimulation until 6 valid data points were obtained. A 10% decrease in the rat's mechanical pain threshold was considered a successful establishment of the DPN rat model.

[0029] 2. Administration of the effective components of traditional Chinese medicine combinations Rats that successfully developed the model were divided into 6 groups: model group (MOD, n=11), volatile oil fraction group of traditional Chinese medicine (HFY, 1.25 g·kg⁻¹·d⁻¹, n=10), water-soluble fraction group (ST, 6.25 g·kg⁻¹·d⁻¹, n=10), alcohol-soluble fraction group (CT, 6.25 g·kg⁻¹·d⁻¹, n=10), effective fraction group of traditional Chinese medicine combination (HLXL, 6.25 g·kg⁻¹·d⁻¹, n=10), and positive control drug lipoic acid group (Y, 0.1 g·kg⁻¹·d⁻¹, n=10). The treatment groups were administered the corresponding drugs by gavage in the afternoon each day, while the blank control group and the DPN model group were administered physiological saline by gavage. The gavage volume for all groups was 10 mL·kg⁻¹, once daily for 30 consecutive days.

[0030] 3. Methods for evaluating drug efficacy (1) Methods for testing mechanical pain threshold and sciatic nerve conduction velocity After drug administration, the mechanical pain threshold of rats was measured, and the sciatic nerve conduction velocity was measured in vivo using a direct in vivo method: After anesthesia, rats were fixed in a prone position on an operating table covered with a blanket. The stimulating electrode was placed at the sciatic notch, the recording electrode at the ipsilateral toe muscle, and the ground electrode at the tail near the spine. Electrical stimulation was performed, and the latency (T1) was recorded. Then, the stimulating electrode was placed at the ankle joint, and the latency (T2) was recorded. The distance between the sciatic notch and the ankle joint was measured and recorded as L. The motor nerve conduction velocity (MNCV) was calculated as L / (T1-T2). Alternatively, the stimulating electrode was placed at the ankle joint, the recording electrode at the sciatic notch, and the ground electrode position remained unchanged. Electrical stimulation was performed, and the latency was recorded. The sensory nerve conduction velocity (SNCV) was calculated as the distance between the stimulating and recording electrodes divided by the latency.

[0031] (2) Biochemical index testing During modeling and drug administration, the fur color and activity of rats were observed, and their weight and fasting blood glucose levels were measured. After the last administration, blood was collected from the abdominal aorta of each group of rats, and serum was collected to detect indicators according to the kit instructions. After blood collection, the skin of the rat's leg was cut open, and the sciatic nerve tissue of the rat was carefully dissected for indicator detection.

[0032] 4. Statistical processing All experimental data were statistically processed using GraphPad Prism 8 software. One-way ANOVA, two-way ANOVA, or other comparative methods were used for comparisons between groups. t Test analysis, results are as follows ± s express, P <0.05 indicates a statistically significant difference.

[0033] III. Experimental Results The control group rats were generally in good condition, with shiny and smooth fur and good mental state. Rats with successfully established DPN models had yellow, dry, and brittle fur, hair loss, increased thirst, appetite, and urination, requiring daily bedding changes and exhibiting lethargy. Body weight and fasting blood glucose test results are as follows: Figure 1 As shown, compared with the blank group, the rats in the model group had significantly reduced body weight and significantly increased fasting blood glucose levels. P <0.001); Compared with the model group, there was no significant change in body weight after drug administration intervention in each drug administration group, and the fasting blood glucose level decreased to varying degrees. Among them, the volatile oil group (HFY), the effective part group of traditional Chinese medicine combination (HLXL), and the positive drug lipoic acid group (Y) had the most significant effect on reducing fasting blood glucose level.

[0034] like Figure 2 As shown, compared with the control group, the mechanical pain threshold of rats in each modeling group was significantly reduced before drug administration. P <0.001), indicating hyperalgesia, suggesting successful model establishment. After drug administration, the mechanical pain threshold increased in both the model group and all drug-treated groups, suggesting that sensory impairment may occur in the later stages of disease progression. Compared to the model group, the mechanical pain threshold improved to varying degrees in all drug-treated groups after intervention, with the ST, HLXL, and Y groups showing the most significant improvement. P <0.001).

[0035] Results of rat sciatic nerve conduction velocity measurement: Figure 3 As shown, compared with the blank group, the motor nerve conduction velocity and sensory nerve conduction velocity of rats in each modeling group were significantly decreased. P<0.001), indicating that the rats exhibited sensory impairment; compared with the model group, the sciatic nerve conduction velocity of rats in all groups was improved to varying degrees after intervention with the effective parts of the traditional Chinese medicine combination, among which the HFY group, ST group, HLXL group and Y group showed the most significant effect in improving nerve conduction velocity. P <0.001).

[0036] H&E staining results of rat sciatic nerve tissue are as follows Figure 4 As shown, in the control group, the myelinated nerve fibers of the sciatic nerve in rats were intact and orderly arranged. The axons in the middle of the nerve fibers were surrounded by a myelin sheath in a net-like pattern, with a clear and complete structure. Numerous uniform-sized, blue Schwann cell nuclei were scattered around the myelin sheath. In the model group, the myelinated nerve fibers of the sciatic nerve in rats were loosely arranged, the axons were mostly atrophied, and the myelin sheath around them showed vacuolar degeneration. Some nerve fibers had abnormally increased or lost myelin sheath spacing, and the number of Schwann cells was reduced. Compared with the model group, the sciatic nerve damage in the drug-treated rats was reduced, the myelinated nerve fibers were more orderly arranged, and the axonal atrophy, vacuolar changes in the myelin sheath, and abnormal spacing were all improved. The number of Schwann cell nuclei increased, with the ST group and Y group showing the most significant improvement in the degree of sciatic nerve damage.

[0037] H&E staining results of rat dorsal root ganglion tissue are as follows Figure 5 As shown, in the normal group rats, the dorsal root ganglion cells were tightly arranged, with large, round nuclei and clearly visible nucleoli. An outer layer of flattened cells were satellite cells. The gaps between ganglion cells and satellite cells were small, and Nissl bodies, representing neuronal functional status, were evenly distributed. In the model group rats, the gaps between dorsal root ganglion cells were widened, the arrangement was loose and disordered, the nuclei were smaller and of varying diameters, many ganglion cells separated from satellite cells, and Nissl bodies were sparsely and randomly distributed. Compared with the model group, the treated rats showed varying degrees of improvement after treatment. The dorsal root ganglion cells were more neatly arranged, some ganglion cells separated from satellite cells, and the number of Nissl bodies increased and their distribution was more uniform. The ST, HLXL, and Y groups showed the most significant improvement.

[0038] The results of oxidative stress marker detection in rat serum and sciatic nerve tissue are shown in the figure. Figure 6 and Figure 7 Compared with the control group, the model group rats showed significantly decreased SOD activity and GSH levels in serum and sciatic nerve, significantly decreased TAOC activity in serum, and significantly increased MDA and AGEs content. PThe result was <0.001, indicating that the sciatic nerve and the whole body of DPN rats were damaged by oxidative stress. Compared with the model group, all treatment groups could reduce the content of MDA and AGEs, and also increase SOD activity, GSH level and TAOC activity to varying degrees. Among them, the HLXL group and Y group showed better effects in restoring GSH level, reducing AGE content and increasing total antioxidant capacity, indicating that the effective parts of the Chinese medicine combination can effectively inhibit the level of oxidative stress in rats to protect the sciatic nerve from damage.

[0039] Results of inflammatory factor assays are shown below Figure 8 Compared with the blank group, the model group rats had higher levels of IL-1 in serum and sciatic nerve. β IL-6, TNF-α α The content increased significantly ( P The value <0.001 indicates that the DPN rats are in a state of systemic inflammation. Compared with the model group, the inflammatory factors in the serum and sciatic nerve of rats in all groups were downregulated to varying degrees after drug treatment. Among them, the ST group and HLXL group showed the most significant downregulation of the content of inflammatory factors in the serum and tissues of DPN rats, indicating that the effective parts of the traditional Chinese medicine combination can effectively inhibit the production of inflammatory factors and thus exert an anti-inflammatory effect.

[0040] The levels of PGE2, SP, and BDNF in rat serum were detected by ELISA. Figure 9 As shown, compared with the blank group, the serum levels of PGE2 and SP in the model group rats were significantly increased, and the BDNF content was significantly decreased. P <0.001); Compared with the model group, the pain of rats in each group was improved to varying degrees after drug intervention. Among them, the HFY group, HLXL group and Y group significantly downregulated the PGE2 and SP levels in the serum of DPN rats; the HLXL group and Y group significantly restored the BDNF content, suggesting that the effective parts of the Chinese medicine combination can exert analgesic effects by regulating pain indicators such as PGE2 and SP in the body.

[0041] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of the effective components of a traditional Chinese medicine combination in the preparation of drugs to improve diabetic peripheral neuropathy; the effective components of the traditional Chinese medicine combination are made from volatile oil effective components, water-soluble effective components, and alcohol-soluble effective components; The effective components of the volatile oil include, by weight percentage: 35.9% α-ocimene, 23.5% α-santalene, 17.2% bisabolene, 17% β-bisabolene, and 6.4% nerolidol; The water-soluble effective fractions include, by weight percentage, 15% salvianolic acid A, 45% salvianolic acid B, and 40% tanshinone; The alcohol-soluble effective fraction includes a percentage by weight. Z -Ligustin 30%, α - Boswellic acid 27%, β - Boswellic acid 23%, tanshinone IIA 7%, 2-methoxy-5-acetyl-furangermaline-1(10)-en-6-one 13%.

2. The application according to claim 1, characterized in that, The dosage forms of the drug are tablets, pills, powders, granules, injections, or capsules.

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