Traditional Chinese medicine combination effective part for improving diabetic peripheral neuropathy

Abstract

The invention discloses a traditional Chinese medicine combination effective part capable of improving diabetic peripheral neuropathy. The traditional Chinese medicine combination effective part is prepared from a volatile oil effective part, a water-soluble effective part and an alcohol-soluble effective part, the effective part of the volatile oil comprises alpha-ocimene, alpha-santalene, bisabolol, beta-bisabolol and nerolidol; the water-soluble effective part comprises salvianolic acid A, salvianolic acid B and tanshinol; the alcohol-soluble effective part is prepared from Z-ligustilide, alpha-masticinic acid, beta-masticinic acid, tanshinone IIA, and 2-methoxy-5-acetyl-furangermacane-1 (10)-ene-6-ketone, and the alcohol-soluble effective part is prepared from the following raw materials in parts by weight: 2-methoxy-5-acetyl-furangermacane-1 (10)-ene-6-ketone. The traditional Chinese medicine composition effective part provided by the invention can regulate blood sugar, improve the conduction speed of sciatic nerves, repair damaged tissues, inhibit oxidative stress reaction, resist inflammation and relieve pain, so that the diabetic peripheral neuropathy is improved, the curative effect is reliable, the safety is good, the adverse reaction is low, and the traditional Chinese medicine composition effective part can be used for preparing a medicinal preparation for improving the diabetic peripheral neuropathy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an effective part of a traditional Chinese medicine combination capable of improving diabetic peripheral neuropathy and a preparation method thereof. Background Art

[0002] Diabetic peripheral neuropathy (DPN) is the most common and intractable complication of diabetes. Its primary clinical manifestations include bilateral distal extremity burning, tingling, tearing pain, and hyperalgesia, accompanied by paresthesias. Without timely intervention and treatment, DPN can lead to foot ulcers, gangrene, and amputation, placing significant financial strain on patients and resulting in devastating consequences. The pathogenesis of DPN is complex and is closely linked to metabolic disorders, oxidative stress, inflammation, vascular damage, and neurotrophic disorders.

[0003] Currently, there is no specific drug for the treatment of DPN. Most of the relief is achieved by regulating blood sugar, taking neurotrophic drugs, and antioxidant drugs. Although there is a certain improvement effect, its efficacy is still insufficient. Some drugs also have unavoidable side effects such as liver and kidney damage, gastrointestinal reactions, etc., so patients cannot take them for a long time.

[0004] Traditional Chinese Medicine classifies DPN as a syndrome of "xiaoke bi syndrome." Its etiology is often due to excessive depletion of Qi and blood, leading to stagnation and internal blood stasis. This obstruction in the meridians impedes the smooth flow of Qi and blood, causing bi blockage and ultimately leading to symptoms such as limb pain and numbness. Chinese Angelica, Salvia miltiorrhiza, Frankincense, and Myrrh are commonly used clinically to dispel blood stasis and relieve pain. Chinese Angelica, for its blood-tonifying and pain-relieving properties, is the primary herb for promoting blood circulation. Salvia miltiorrhiza cools the blood, eliminates carbuncles, and dispels blood stasis and promotes new growth. Frankincense and Myrrh are often used together, promoting blood circulation, promoting muscle growth, relieving pain, and dispersing blood stasis. The combined effects of these four herbs nourish blood, activate blood circulation, relax the meridians, activate the collaterals, promote Qi circulation, relieve pain, and eliminate accumulation and symptoms. Summary of the Invention

[0005] Purpose of the invention: The purpose of the present invention is to provide an effective fraction of a traditional Chinese medicine combination with reliable efficacy, good safety, and excellent ability to improve diabetic peripheral neuropathy. Another purpose of the present invention is to provide a method for preparing and using the effective fraction of the traditional Chinese medicine combination.

[0006] Technical solution: In order to achieve the above objectives, the technical solution adopted by the present invention is: A Chinese medicine combination effective fraction having the effect of improving diabetic peripheral neuropathy, which is prepared from a volatile oil effective fraction, a water-soluble effective fraction and an alcohol-soluble effective fraction; The effective parts of the volatile oil include α-ocimene, α-santalene, bisabolene, β-bisabolene and nerolidol; The water-soluble effective parts include salvianolic acid A, salvianolic acid B and danshensu; Alcohol-soluble active ingredients include Z -ligustilide, α -Boswellic acid, β -Boswellic acid, tanshinone ⅡA, 2-methoxy-5-acetyl-furylgermazine-1(10)-en-6-one.

[0007] As a preferred embodiment, the effective part of the above-mentioned traditional Chinese medicine combination, the effective part of the volatile oil comprises 15-30% by mass of α-ocimene, 11-25% by mass of α-santalene, 8-20% by mass of bisabolene, 8-20% by mass of β-bisabolene, and 3-10% by mass of nerolidol; The water-soluble effective parts include 10-20% of salvianolic acid A, 25-50% of salvianolic acid B, and 20-40% of danshensu in terms of mass percentage; The alcohol-soluble effective part includes 22~45% by mass Z -ligustilide, α - Boswellic acid 20~40%, β -Boswellic acid 20~40%, tanshinone ⅡA 5~15%, 2-methoxy-5-acetyl-furylgermazine-1(10)-en-6-one 10~30%.

[0008] As a particularly preferred embodiment, the effective part of the above-mentioned traditional Chinese medicine combination, the effective part of the volatile oil comprises 35.9% by mass of α-ocimene, 23.5% by mass of α-santalene, 17.2% by mass of bisabolene, 17% by mass of β-bisabolene, and 6.4% by mass of nerolidol; The water-soluble effective parts include 15% of salvianolic acid A, 45% of salvianolic acid B and 40% of danshensu by mass percentage; The alcohol-soluble effective fraction includes the mass percentage Z - Ligustilide 30%, α - Boswellic acid 27%, β -Boswellic acid 23%, Tanshinone ⅡA 7%, 2-methoxy-5-acetyl-furylgermazine-1(10)-en-6-one 13%.

[0009] The invention relates to an application of the effective fraction of the traditional Chinese medicine combination in the preparation of a drug for improving diabetic peripheral neuropathy.

[0010] According to actual needs, the effective parts of the Chinese medicine combination of the present invention and a pharmaceutically acceptable carrier can be prepared into a drug in the form of tablets, pills, powders, decoctions, granules, injections, capsules or decoctions.

[0011] Description of beneficial effects: The effective fraction of the traditional Chinese medicine combination for improving diabetic peripheral neuropathy disclosed herein is scientifically formulated through experimental screening, comprising an aromatic fraction of a volatile oil, phenolic acids in an aqueous solution, and alcohol-soluble lactones / steroids and tanshinones. Experimental studies have confirmed that the effective fraction of the traditional Chinese medicine combination provided herein can regulate blood sugar, improve sciatic nerve conduction velocity, repair damaged tissue, inhibit oxidative stress, and provide anti-inflammatory and analgesic effects, demonstrating significant efficacy in improving diabetic peripheral neuropathy. No significant toxic or side effects were observed during the experiments, demonstrating a good safety profile. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The results of the effects of the effective fraction of the Chinese herbal combination on rat body weight and fasting blood glucose levels are shown in the figure. A: Rat body weight trend chart; B: Rat fasting blood glucose trend chart; C: Rat final body weight; D: Rat final fasting blood glucose level.

[0013] Figure 2 This is a graph showing the effects of the effective parts of the Chinese herbal combination on the mechanical pain threshold of rats.

[0014] Figure 3 The results of the effect of the effective fraction of the Chinese herbal combination on the sciatic nerve conduction velocity of rats. A: Motor nerve conduction velocity of rats; B: Sensory nerve conduction velocity of rats.

[0015] Figure 4 This is a graph showing the effects of the effective parts of the Chinese herbal combination on the sciatic nerve tissue of rats.

[0016] Figure 5 This is a graph showing the effects of the effective parts of the Chinese herbal combination on rat dorsal root ganglion tissue.

[0017] Figure 6 Figure 1 shows the effects of the effective fractions of a traditional Chinese medicine combination on oxidative stress markers 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 Figure 1 shows the effects of the effective fraction of a Chinese herbal combination on oxidative stress indicators in rat sciatic nerve tissue. A: GSH level in sciatic nerve; B: SOD level in sciatic nerve.

[0019] Figure 8 The results of the effect of the effective parts of the Chinese herbal medicine combination on the levels of inflammatory factors in rat serum and sciatic nerve. A: Tumor necrosis factor- α (TNF- α ) level; B: serum interleukin-1 β(IL-1 β ) levels; C: serum interleukin-6 (IL-6) levels; D: TNF- α Level; E: IL-1 in sciatic nerve β Levels; F: IL-6 levels in sciatic nerve.

[0020] Figure 9 Figure 1 shows the effects of the effective fraction of a traditional Chinese medicine combination on pain markers in rat serum. A: Serum brain-derived neurotrophic factor (BDNF) level; B: Serum prostaglandin E2 (PGE2) level; C: Serum substance P (SP) level. DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. After reading this invention, modifications of various equivalent forms of the present invention made by those skilled in the art fall within the scope defined by the appended claims. The raw materials of the present invention are all commercially available ingredients.

[0022] Example 1 A Chinese medicine combination effective fraction having the effect of improving diabetic peripheral neuropathy, which is prepared from a volatile oil effective fraction, a water-soluble effective fraction and an alcohol-soluble effective fraction; The effective parts of the volatile oil include 35.9% by mass of α-ocimene, 23.5% by mass of α-santalene, 17.2% by mass of bisabolene, 17% by mass of β-bisabolene, and 6.4% by mass of nerolidol. The water-soluble effective parts include 15% by mass of salvianolic acid A, 45% by mass of salvianolic acid B, and 40% by mass of danshensu; The alcohol-soluble effective fraction includes the mass percentage Z - Ligustilide 30%, α - Boswellic acid 27%, β -Boswellic acid 23%, Tanshinone ⅡA 7%, 2-methoxy-5-acetyl-furylgermazine-1(10)-en-6-one 13%.

[0023] Example 2 Experimental study on the effect of effective fractions of Chinese herbal medicine combination on improving diabetic peripheral neuropathy 1. Experimental Materials and Drugs 1. Drugs and Reagents Lipoic acid tablets (National Medicine Standard No. H20100152, Shandong Qidu Pharmaceutical Co., Ltd.); sodium citrate buffer (0.1mmol·L -1, pH 4.5, batch number: C-1013, Beijing Solaibao); streptozotocin (STZ; batch number: S0130, Sigma, USA); high-fat feed (formula: maintenance base feed 43.6%, sucrose 12%, lard 17.5%, casein 10%, whole milk powder 10%, experimental animal premix 2%, calcium hydrogen phosphate 2%, microcrystalline cellulose 1.9%, stone powder 1%) were purchased from Shuyu Biotechnology Co., Ltd.

[0024] Test drug: the effective fraction of the Chinese medicine combination of Example 1 of the present invention.

[0025] 2. Experimental Instruments Sinocare Anwen+ blood glucose meter (Batch number: 2J01B240303, Sinocare Biosensor Co., Ltd.); Von-Frey fiber filament mechanical stimulation needle (Danmic Aesthesio, USA); Enspire multifunctional microplate reader (PerkinElmer, USA); small animal anesthesia machine (Shenzhen Ruiwode Life Science Technology Co., Ltd.); electromyography evoked potential device (Suzhou Haishen Company); Tissuelyer-48 fully automatic sample rapid grinder (Shanghai Jingxin Industrial Development Co., Ltd.).

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

[0027] 2. Experimental Methods 1. Establishment of a rat model of diabetic peripheral neuropathy After adaptive feeding, healthy rats were randomly divided into a blank group (CON) of 8 and a DPN model group of 72 rats using a stratified grouping method according to body weight. The blank group was fed a normal diet until the end of the experiment. The DPN model group was fed a high-fat diet for 3 weeks, fasted but not watered for 12 hours, and then injected unilaterally with a 1% STZ solution prepared with sodium citrate buffer at a dose of 35 mg / kg. The blank group was injected with an equal dose of sodium citrate buffer at the same location. 72 hours later, the fasting blood glucose in the tail vein of the rats was measured to be ≥11.1 mmol·L. -1 The diabetic rat model was considered successful.

[0028] After continuing to feed a high-fat diet for 3 weeks, the diabetic rats were tested using the up-and-down method. The rats were placed in a cage with a porous steel wire bottom and allowed to acclimate for 30 to 60 minutes. A 2.0 g von Frey fiber needle was first used to vertically stimulate the middle of the rat's hind limb, avoiding the paw pad to avoid influencing the experimental results. A negative response was considered when the fiber curved into an S-shape and the rat did not withdraw or avoid the paw for more than 6 seconds. A positive response was considered when the rat licked, bit, lifted its paw, or dodged. This was repeated three times, with a 2-minute interval between each stimulation. Depending on the animal's response, a larger or smaller fiber was used for the next stimulation until six valid measurements were obtained. A 10% decrease in the rat's mechanical pain threshold was considered a successful DPN model.

[0029] 2. Administering Chinese herbal medicine combinations to the effective site The rats with successfully established DPN models were divided into six groups: model group (MOD, n=11), herbal volatile oil fraction group (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 herbal combination (HLXL, 6.25 g·kg-1·d-1, n=10), and positive drug lipoic acid group (Y, 0.1 g·kg-1·d-1, n=10). The drug-treated group was orally gavaged with the corresponding drug every afternoon for 30 consecutive days, while the blank group and DPN model group were orally gavaged with normal saline. The gavage volume was 10 mL·kg-1 once daily for 30 consecutive days.

[0030] 3. Efficacy evaluation method (1) Mechanical pain threshold and sciatic nerve conduction velocity testing methods After drug administration, the rats' mechanical pain thresholds were measured, and their sciatic nerve conduction velocity was measured in vivo using a direct in vivo method: After anesthesia, the rats were fixed in the prone position on a blanket-covered operating table. A stimulating electrode was placed at the sciatic notch, a recording electrode was placed at the ipsilateral toe muscle, and a ground electrode was placed at the tail near the spine. Electrical stimulation was performed, and the latency (T1) was recorded. The stimulating electrode was then placed at the ankle joint, and the latency (T2) was recorded. The distance between the sciatic notch and the ankle joint was measured, recorded as L. The motor nerve conduction velocity (MNCV) = L / (T1-T2). The stimulating electrode was placed at the ankle joint, the recording electrode was placed at the sciatic notch, and the ground electrode remained in place. Electrical stimulation was performed, and the latency was recorded. The sensory nerve conduction velocity (SNCV) = the distance between the stimulating and recording electrodes / the latency.

[0031] (2) Biochemical index test During the modeling and drug administration period, the hair color and activity of the rats were observed, and the body weight and fasting blood glucose level were tested. After the last administration, blood was collected from the abdominal aorta of each group of rats, and the serum was collected to test the indicators according to the kit instructions. After the blood was collected, the skin of the rat legs was cut open, and the sciatic nerve tissue of the rat was carefully peeled off for indicator detection.

[0032] 4. Statistical Processing All experimental data were statistically analyzed using GraphPad Prism 8 software, and comparisons among groups were performed using one-way analysis of variance, two-way analysis of variance, or t Test analysis, the results are ± s express, P The difference was statistically significant when the value was <0.05.

[0033] 3. Experimental Results The rats in the blank group were in good general condition, with shiny and smooth hair and good spirits; the rats in the successful DPN model had yellow, dry hair, hair loss, polydipsia, polyphagia, polyuria, and required daily bedding changes and were listless. Figure 1 As shown in the figure, compared with the blank group, the body weight of rats in the model group was significantly reduced, and the fasting blood glucose level was significantly increased ( P <0.001); compared with the model group, there was no significant change in body weight in each drug group after intervention, and the fasting blood glucose levels decreased to varying degrees. Among them, the volatile oil group (HFY), the effective part of the Chinese herbal combination group (HLXL), and the positive drug lipoic acid group (Y) had the most significant effect in reducing fasting blood glucose levels.

[0034] like Figure 2 As shown in Figure 2, before administration, the mechanical pain threshold of rats in each modeling group was significantly decreased compared with the blank group ( P <0.001), showing hyperalgesia, indicating successful modeling. After the end of drug administration, the mechanical pain thresholds of rats in the model group and each drug group increased, and it is speculated that as the disease progresses, sensation will decrease. Compared with the model group, the mechanical pain thresholds of rats in each drug group improved to varying degrees after drug intervention, among which the improvement effect in the ST group, HLXL group, and Y group was more obvious ( P <0.001).

[0035] The results of the sciatic nerve conduction velocity test in rats were as follows Figure 3 As shown in the figure, 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 showed sensory impairment; compared with the model group, the sciatic nerve conduction velocity of the rats in each group was improved to varying degrees after the intervention of the effective part of the Chinese medicine combination, among which the HFY group, ST group, HLXL group and Y group had the most significant effect on improving the nerve conduction velocity ( P <0.001).

[0036] The results of H&E staining of rat sciatic nerve tissue are as follows Figure 4 As shown in the results, the myelinated nerve fibers of the sciatic nerves of the blank group were intact and orderly arranged. The axons in the middle of the nerve fibers were surrounded by a reticular myelin sheath, with a clear and complete structure. Many blue Schwann cell nuclei of uniform size were seen scattered around the myelin sheaths. In the model group, the myelinated nerve fibers of the sciatic nerves of the rats were loosely arranged, with axons mostly atrophied and surrounding myelin sheaths undergoing vacuolar degeneration. The myelin sheath spacing of some nerve fibers was abnormally increased or lost, and the number of Schwann cells decreased. Compared with the model group, the sciatic nerve injury in the drug-treated groups was alleviated, the myelinated nerve fibers were more neatly arranged, the axonal atrophy in the middle, the vacuolar changes in the surrounding myelin sheaths, and the abnormal spacing were all improved. The number of Schwann cell nuclei increased. The ST and Y groups showed the most significant improvement in the degree of sciatic nerve injury.

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

[0038] The results of the detection of oxidative stress indicators in rat serum and sciatic nerve tissue are shown in Figure 6 and Figure 7 Compared with the blank group, the SOD activity and GSH levels in the serum and sciatic nerve of the model group rats were significantly reduced, the TAOC activity in the serum was significantly reduced, and the MDA and AGEs contents were significantly increased ( P<0.001), suggesting that the sciatic nerve and the entire body of DPN rats were damaged by oxidative stress. Compared with the model group, all treatment groups reduced MDA and AGE levels and increased SOD activity, GSH levels, and TAOC activity to varying degrees. The HLXL and Y groups showed significant effects in reducing GSH levels, lowering AGE levels, and enhancing total antioxidant capacity, indicating that the effective fraction of the traditional Chinese medicine combination effectively inhibited oxidative stress in rats and protected the sciatic nerve from damage.

[0039] The results of inflammatory factor determination are shown in Figure 8 Compared with the blank group, the levels of IL-1 β , IL-6, TNF- α The content increased significantly ( P <0.001), suggesting that DPN rats are in a systemic inflammatory state. Compared with the model group, inflammatory factors in the serum and sciatic nerve of rats in all treatment groups were downregulated to varying degrees. The ST and HLXL groups showed the most significant effect in downregulating inflammatory factor levels in the serum and tissues of DPN rats, indicating that the effective fraction of the Chinese herbal combination can effectively inhibit the production of inflammatory factors, thereby exerting an anti-inflammatory effect.

[0040] The levels of PGE2, SP and BDNF in rat serum were detected by ELISA. Figure 9 As shown in Figure 2, compared with the blank group, the levels of PGE2 and SP in the serum of rats in the model group were significantly increased, and the content of BDNF was significantly decreased ( P <0.001); compared with the model group, the pain conditions of the rats in each group improved to varying degrees after drug intervention. Among them, the HFY group, HLXL group, and Y group could significantly downregulate the levels of PGE2 and SP in the serum of DPN rats; the HLXL group and Y group could significantly restore the BDNF content, suggesting that the effective part of the Chinese medicine combination can exert analgesic effects by regulating pain indicators such as PGE2 and SP in the body.

[0041] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An effective part of a Chinese medicine combination having the effect of improving diabetic peripheral neuropathy, characterized in that: It is made of volatile oil effective parts, water-soluble effective parts and alcohol-soluble effective parts; The effective parts of the volatile oil include α-ocimene, α-santalene, bisabolene, β-bisabolene and nerolidol; The water-soluble effective parts include salvianolic acid A, salvianolic acid B and danshensu; Alcohol-soluble active ingredients include Z -ligustilide, α -Boswellic acid, β -Boswellic acid.

2. The effective part of the Chinese medicine combination according to claim 1, characterized in that The effective parts of the volatile oil include 15-30% by mass of α-ocimene, 11-25% by mass of α-santalene, 8-20% by mass of bisabolene, 8-20% by mass of β-bisabolene, and 3-10% by mass of nerolidol. The water-soluble effective parts include 10-20% of salvianolic acid A, 25-50% of salvianolic acid B, and 20-40% of danshensu in terms of mass percentage; The alcohol-soluble effective part includes 22~45% by mass Z -ligustilide, α - Boswellic acid 20~40%, β -Boswellic acid 20~40%, tanshinone ⅡA 5~15%, 2-methoxy-5-acetyl-furylgermazine-1(10)-en-6-one 10~30%.

3. The effective part of the Chinese medicine combination according to claim 2, characterized in that: The effective parts of the volatile oil include 35.9% by mass of α-ocimene, 23.5% by mass of α-santalene, 17.2% by mass of bisabolene, 17% by mass of β-bisabolene, and 6.4% by mass of nerolidol. The water-soluble effective parts include 15% of salvianolic acid A, 45% of salvianolic acid B and 40% of danshensu by mass percentage; The alcohol-soluble effective fraction includes the mass percentage Z - Ligustilide 30%, α - Boswellic acid 27%, β -Boswellic acid 23%, Tanshinone ⅡA 7%, 2-methoxy-5-acetyl-furylgermazine-1(10)-en-6-one 13%.

4. Use of the effective fraction of the Chinese medicine combination according to any one of claims 1 to 3 in the preparation of a medicament for improving diabetic peripheral neuropathy.

5. The use according to claim 4, characterized in that The dosage form of the drug is tablets, pills, powders, decoctions, granules, injections, capsules or decoctions.

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