Application of effective component formula of Guiaojiaogangrene in treatment of peripheral neuropathy caused by diabetes mellitus

Abstract

The invention provides an application of an effective component formula of Guiaojiaogangrene in treating peripheral neuropathy caused by diabetes mellitus. The invention mainly comprises the composition and corresponding proportion of the effective component formula of Guiaogangrene, the optimal proportion and preparation type of each component of the effective component formula of Guiaogangrene, and the application type of the effective component formula of Guiaogangrene in treating peripheral neuropathy caused by diabetes. The behavior change condition, the weight change condition, the blood sugar change condition, the nerve conduction speed change condition and the IL-6, TNF-alpha and NA + +-K + + P-ATP enzyme content change condition in serum of rats in each test group show that the known glycomyelitis effective component formula has a good curative effect in treating peripheral neuropathy caused by the diabetes mellitus by combining the behavior change condition, the weight change condition, the blood sugar change condition, the nerve conduction speed change condition and the IL-6, TNF-alpha and NA + +-K + + P-ATP enzyme content change condition.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to an application of a formula of effective ingredients of saccharomyces cerevisiae in treating peripheral neuropathy caused by diabetes. Background Art

[0002] Diabetes has been identified as the third most serious chronic disease that endangers human health after cardiovascular disease and cancer. Diabetes is a group of lifelong metabolic diseases caused by multiple causes and characterized by chronic hyperglycemia. Long-term high blood sugar leads to damage to large blood vessels and microvessels and endangers peripheral nerves. There are more than 100 complications of diabetes, making it the disease with the most known complications. Among them, diabetic peripheral neuropathy is the most common type of diabetic neuropathy, and more than 50% of patients may have symptoms, manifested as burning pain, electric shock or tingling sensations, hyperesthesia and numbness. These symptoms usually worsen at night and affect the feet and hands. Some patients are asymptomatic and only abnormalities can be found during neurological examinations.

[0003] At present, the drugs used in the market for treating peripheral neuropathy caused by diabetes can be divided into three categories: the first category is drugs for improving microcirculation, such as pentoxifylline, anisodamine, cilostazol, etc.; the second category is drugs for nerve repair, such as methyl vitamin B12 (methylcobalamin), neurotrophic factors, etc.; the third category is drugs for anti-oxidative stress, such as a-lipoic acid, etc. The application of traditional Chinese medicine in this regard is relatively rare.

[0004] In TCM, diabetic peripheral neuropathy is called Biwei; from the perspective of etiology and pathogenesis, it is explained as poor blood circulation in the extremities and Biwei caused by qi deficiency and blood stasis, liver and kidney yin deficiency, and blood stasis blocking the collaterals. Most scholars believe that blood stasis plays an important role in diabetes, and qi, blood, essence and body fluids are blocked by tangible evil, resulting in pain, numbness and other symptoms. Therefore, blood stasis blocking the collaterals is the main cause of diabetes Biwei, and the treatment is mainly based on replenishing qi and nourishing yin, promoting blood circulation and unblocking collaterals.

[0005] The medicinal ingredients of Guizhi Tanggan are made of angelica sinensis, rhizoma anemarrhenae, honeysuckle, honeysuckle, coptis chinensis, rehmannia root, figwort, liquorice and safflower seeds as raw materials, and are mainly used to treat a series of complications caused by diabetes. Among them, angelica sinensis and safflower seeds are mainly used to promote blood circulation and dissipate blood stasis, while rhizoma anemarrhenae, honeysuckle, honeysuckle, coptis chinensis, rehmannia root, figwort and liquorice are mainly used to clear away heat and detoxify, soothe wind and dredge collaterals.

[0006] In general, there is no relevant research on the application of the effective ingredient formula of Guizhi sugar cane in the preparation of treatment for peripheral neuropathy caused by diabetes. Summary of the invention

[0007] In order to make up for the deficiencies of the prior art, the present invention provides an application of a formula of effective ingredients of Gastrodia elata in treating peripheral neuropathy caused by diabetes.

[0008] The present invention is achieved through the following technical solutions:

[0009] In a first aspect, the present invention provides an application of an effective ingredient formula of Guizhi tangnausea in treating peripheral neuropathy caused by diabetes, wherein the effective ingredient formula of Guizhi tangnausea is composed of the following ingredients: ligusticum chuanxiong lactone A, ligusticum chuanxiong lactone I, angloside, coptisine hydrochloride, cynaroside, digitalis folioside, and harpaoside;

[0010] The mass ratio of each component of the Guizhi anthrax effective ingredient formula is (5.4-6.3): (3.2-4.1): (2.9-3.5): (1.5-2.7): (1.2-3.0): (2.2-3.5): (0.9-1.6).

[0011] Furthermore, the ratio of ligusticum chuanxiong lactone I: angloside: coptisine hydrochloride: cynaroside: digitalis folioside: harpaoside is 5.7:3.6:3.1:1.7:2.3:3.2:1.1.

[0012] Furthermore, the formula of the active ingredient of Guizhi sugar cane can significantly reduce the blood sugar level of Wistar rats within 8 weeks; and improve the nerve conduction velocity of rats with peripheral neuropathy.

[0013] Furthermore, the formula of the effective ingredient of Guizhi saccharomyces cerevisiae can increase the expression levels of three inflammatory factors, IL-6, TNF-α, and NA++-K++P-ATP, in the blood of rats with peripheral neuropathy.

[0014] Furthermore, the peripheral neuropathy caused by diabetes mainly includes symmetrical pain, numbness, decreased or even absent pain and temperature sensation at the ends of bilateral limbs, less sweating, dry and thickened skin, muscle weakness and muscle atrophy.

[0015] In a second aspect, the present invention also provides a drug for treating peripheral neuropathy caused by diabetes, the drug comprising a formula of effective ingredients of Guizhi Cannabis Sativa, the effective ingredient formula consisting of the following ingredients: Chuanxiong lactone A: Chuanxiong lactone I: Angloside: Coptis chinensis hydrochloride: Binjioxanthin: Digitalis folioside: Hapaoside is (5.4-6.3): (3.2-4.1): (2.9-3.5): (1.5-2.7): (1.2-3.0): (2.2-3.5): (0.9-1.6).

[0016] Furthermore, the mass ratio of each component in the drug is: ligusticum chuanxiong lactone A:ligusticum chuanxiong lactone I:angoloside:

[0017] The ratio of coptisine hydrochloride: cynaroside: digitalis folioside: harpagoside is 5.7:3.6:3.1:1.7:2.3:3.2:1.1.

[0018] Furthermore, the preparation types of the drug include but are not limited to granules, capsules, and tablets.

[0019] Compared with the prior art, the present invention is beneficial in that: the present invention provides a formula of effective ingredients for treating peripheral neuropathy caused by diabetes, and a drug prepared using the formula, wherein the drug has an excellent therapeutic effect on peripheral neuropathy caused by diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described below in conjunction with the accompanying drawings.

[0021] Figure 1 Line graph of the weight of each group of rats before and after medication;

[0022] Figure 2 Line graph of changes in blood sugar in each group of rats before and after medication;

[0023] Figure 3 Comparison of mean nerve conduction velocity values ​​of rats during drug administration;

[0024] Figure 4 Graph showing serum IL-6 expression levels in each group;

[0025] Figure 5 Figure 1. Serum TNF-α expression levels in each group;

[0026] Figure 6 Expression levels of serum NA++-K++P-ATP in each group. DETAILED DESCRIPTION

[0027] The present invention is described in further detail below so that those skilled in the art can implement it according to the description.

[0028] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial sources unless otherwise specified. In addition, the pharmaceutical preparation used in the present invention is a granule.

[0029] Example 1: Formulation of the active ingredient of Gyuzhi saccharomyces

[0030] The mass ratio of each component of the effective ingredient formula of Guizhi sugar cane is (5.4-6.3): (3.2-4.1): (2.9-3.5): (1.5-2.7): (1.2-3.0): (2.2-3.5): (0.9-1.6);

[0031] In this embodiment, the mass ratio of the effective ingredient formula of Guizhi Cannabis Sativa is 5.7:3.6:3.1:1.7:2.3:3.2:1.1 for ligusticum lactone A:ligusticum lactone I:angoloside:coptisine hydrochloride:binajuncea:digitolidin:harpaoside.

[0032] Example 2: Experimental study on the treatment of diabetic peripheral neuropathy in rats with Guizhi Tangwu granules

[0033] 1. Materials

[0034] 1.1 Experimental animals

[0035] Sixty male SPF Wistar rats weighing (130±20) g were provided by Changzhou Cavens Experimental Animal Co., Ltd. All experimental animals were kept in a controlled environment with a room temperature of 18-24°C and a humidity of 40%-50%. During the experiment, the animals had free access to food and water, and their circadian rhythm was normal.

[0036] 1.2 Main drugs and reagents

[0037] Guizhi Tangu Granules (prepared into granules according to the mass ratio of Example 1); Methylcobalamin Tablets: 0.5 mg / tablet*20 tablets / box, purchased from Hongjitang Pharmacy, produced by Zhejiang Kangenbei Pharmaceutical Co., Ltd.

[0038] 1.3 Instruments, equipment and reagents and consumables required for animal modeling and experimental operation (see Table 1)

[0039] Table 1

[0040]

[0041]

[0042] 1.4 Main instruments and equipment required for animal testing (see Table 2)

[0043] Table 2

[0044]

[0045] 2 Experimental methods

[0046] 2.1 Animal model preparation

[0047] After two weeks of adaptive feeding, 60 male Wistar rats were randomly selected from the normal group and the remaining 50 rats were used to model diabetic peripheral neuropathy. The model was established by intraperitoneal injection of 2% streptozotocin to induce diabetic peripheral neuropathy. Streptozotocin was diluted to a 2% concentration solution with 0.1mol / L sodium citrate buffer. After fasting for 12h, the rats were intraperitoneally injected with drugs, and according to the dosage standard of 50mg / kg, random blood sugar was tested by tail tip vein blood sampling after 72 hours. Rats with random blood sugar test meeting ≥16.5mmol / L were included in the experimental subjects, and 45 models were successfully established.

[0048] 2.2 Experimental Animal Grouping

[0049] There were 15 rats in the normal group, and the 45 DPN rats with modeling were divided into 3 groups using the random number table method, namely, model group, Chinese medicine group, and Western medicine group, with 15 rats in each group.

[0050] 2.3 Administration

[0051] The Chinese medicine group was given Guizhi Tangju Granules aqueous solution (concentration 15 mg / ml) by intragastric administration.

[0052] Western medicine group: Methylcobalamin tablets were added to distilled water to prepare a 15ug / ml suspension for oral administration.

[0053] The model group and the normal group were intragastrically administered with an equal volume of distilled water.

[0054] Gavage cycle: 8 consecutive weeks, once a day.

[0055] 2.4 Observation indicators and methods

[0056] 2.4.1 General

[0057] During the drug treatment, the rats' diet and water intake, urine volume, coat color, body weight, mental state, and daily activities were continuously observed daily. The rats were given sufficient food and water, the bedding was changed in time, and corresponding daily records were kept.

[0058] 2.4.2 Blood glucose measurement

[0059] The blood glucose levels of the rats were measured at 0, 4, and 8 weeks after administration.

[0060] 2.4.3 Body weight measurement

[0061] The body weights of the rats were measured at five time points: 0 week, 2 weeks, 4 weeks, 6 weeks, and 8 weeks after administration.

[0062] 2.4.4 Determination of motor nerve conduction velocity in experimental animals

[0063] The stimulating electrode and the recording electrode are placed on the sciatic notch on one side of the rat and the ankle joint on the same side of the rat, respectively. The reference electrode is placed between the two electrodes, and the width of the single pulse square wave is adjusted to 0.1ms. The stimulating recording electrode is placed at 1cm, and the stimulation threshold is determined. During the determination, the stimulation interval is greater than 5s, and the stimulation intensity is set to 1.5 times the pain threshold. The time from the stimulation of the stimulating electrode to the recording electrode showing the stimulation is recorded. Multiple measurements, about 9-11 times, are taken and recorded with the average value. The precise distance between the stimulating electrode and the recording electrode on the leg of the rat is measured, and the direction of the rat's distribution along the sciatic nerve is determined. Substitute the conduction time and the precise distance of conduction into the equation: motor nerve conduction velocity (m / s) = distance between the stimulating electrode and the recording electrode / conduction time, thereby calculating the motor nerve conduction velocity of the rat's sciatic nerve.

[0064] 2.4.5 Enzyme-linked immunosorbent assay to determine serum IL-6, TNF-α, and NA+-K+-ATPase levels

[0065] Rats were anesthetized by intraperitoneal injection of 4% urethane 4 ml / kg, fixed to expose the abdominal aorta fully, cut the abdominal cavity along the midline of the abdomen with surgical scissors, and 2 ml of blood was collected from a blood collection tube and marked. The collected blood was placed in a test tube and centrifuged at 3000 r / min for 10 min, and the supernatant was aspirated and stored for later use.

[0066] (1) Dilution of Streptavidin Standard

[0067]

[0068] (2) Sample loading: Mark the blank wells, standard wells, and sample wells on the ELISA plate in order. First, add 50ul of sample.

[0069] The standard is placed in the standard well, followed by 40ul of sample diluent in the sample well, and then 10ul of the sample is added.

[0070] (3) Incubation: Use a water bath to adjust the water temperature to 36°C, seal the ELISA plate with a sealing film, and incubate for 30 min.

[0071] (4) Dilute the cleaning solution: Dilute the concentrated cleaning solution with distilled water to one-thirtieth of the original concentration and set aside.

[0072] (5) Washing: After the incubation is completed, remove the ELISA plate and remove the sealing film. Pour out the liquid inside and spin dry. Add the washing solution to each well and wait for 30 seconds before pouring it out. Repeat this process five times and drain until dry.

[0073] (6) Add enzyme: Add 50ul of enzyme-labeled reagent to the standard well and the sample well.

[0074] (7) Incubation and washing: The operation is the same as before.

[0075] (8) Color development: 50 μl of TMB One-Step substrate reagent and 50 μl of quenching buffer were added to each well, mixed evenly, and then the ambient temperature was adjusted to 37°C for color development, which took about 10 min (the entire color development process was protected from light).

[0076] (9) Termination: After color development is completed, add 50 μl of stop solution to each well to terminate the reaction.

[0077] (10) Measurement: Use a 450 nm wavelength instrument to accurately measure the OD value of each well, record the value, and calculate the OD value of each well minus the blank well value. The measurement conditions need to be carried out within 15 minutes after adding the stop solution.

[0078] (11) Calculation: Draw a standard curve for the sample reagent with the concentration of the standard as the horizontal axis and the absorbance OD value as the vertical axis. Substitute the absorbance OD value of the sample into the equation of the drawn standard curve to obtain the corresponding concentration of the sample. Multiply it by the dilution factor of the sample to finally calculate the actual concentration of the measured indicator.

[0079] 2.5 Test results

[0080] 2.5.1 Behavioral observation of rats in each group

[0081] Rats in the normal group: normal diet, normal bowel movements, moist fur, not prone to hair loss, and quick reactions.

[0082] Rats in the model group: drank and ate more, urinated more frequently and had loose stools, had dull and yellow fur, hair that fell off easily, had loose skin and soft flesh, were listless, lay still and had slow reactions.

[0083] Rats in the Chinese medicine group: their appetite was OK, their stools were slightly loose, their fur was relatively shiny and the amount of fur was OK, their spirits were better than before, their activities increased, and their reactions were OK.

[0084] Rats in the western medicine group: their appetite was OK, their stools were loose, their fur was dry and yellow, their hair fell out easily, they were lazy and often lay still, and their reactions were relatively slow.

[0085] 2.5.2 Body weight of rats in each group

[0086] There was no significant difference in the weight data of the experimental rats in each group before medication. From 2 weeks to 8 weeks after modeling, the weight data of the rats in the model group decreased significantly, and the weight data of the rats in the normal group increased significantly more than those in other groups; after medication intervention, the weight data of the rats in the western medicine group and the traditional Chinese medicine group increased (P<0.05). The weight data of the western medicine group was greater than that of the model group, and the weight data of the traditional Chinese medicine group was higher than that of the western medicine group (P<0.05). See Table 3.

[0087] Table 3 Body weight changes of rats in each group (g)

[0088]

[0089] Note: Compared with the western medicine group, # represents P < 0.05

[0090] 2.5.3 Changes in blood glucose in rats in each group

[0091] After modeling, except for the normal group, the blood glucose levels of the other three groups of rats increased significantly (P < 0.01). At the end of the 8th week of drug administration, the mean blood glucose level of the Chinese medicine group was lower than that of the Western medicine group (P < 0.05).

[0092] There was no significant difference in the mean blood glucose level between the model group and the western medicine group (P>0.05). The results showed that Guizhi Tangju Granules may have a certain hypoglycemic effect.

[0093] Table 4 Changes in blood glucose in rats in each group (mmol / L)

[0094]

[0095] Note: Compared with the model group, # represents P < 0.05, ## represents P < 0.01; compared with the western medicine group, * represents P < 0.05, ** represents P < 0.01

[0096] 2.5.4 Nerve conduction velocity of experimental animals in each group

[0097] The sciatic nerve conduction velocity of rats in each group was observed at 4 and 8 weeks after medication. The results showed that 4 weeks after modeling, the motor nerve conduction velocity of rats with peripheral neuropathy was significantly reduced (P<0.05). At 8 weeks after medication, the nerve conduction velocity of the western medicine group and the traditional Chinese medicine group was significantly improved (P<0.01). Compared with the western medicine group, the nerve conduction velocity of the traditional Chinese medicine group was significantly improved (P<0.01).

[0098] Table 5 Nerve conduction velocity of rats in each group during 8 weeks of medication (m / s)

[0099]

[0100] Note: Compared with the normal group: #p<0.05, ##p<0.01; compared with the model group: *p<0.05, **p<0.01; compared with western medicine: ^p<0.05, ^^p<0.01.

[0101] 2.5.5 Serum IL-6, TNF-α, and NA++-K++P-ATPase levels in rats of each group

[0102] Compared with the normal group, the serum inflammatory factor levels of the three groups of rats with peripheral neuropathy were all higher than those of the control group (P<0.01); the serum TNF-α and IL-6 of the rats with peripheral neuropathy in the Chinese medicine group and the Western medicine group were significantly lower than those in the model group (P<0.01), indicating that the treatment was effective; the improvement of the inflammatory factor level in the Chinese medicine group was more obvious than that in the Western medicine group (P<0.01). Compared with the normal group, the serum NA+-K+-ATPase level of the rats in the model group was significantly reduced (P<0.01), and the serum NA+-K+-ATPase level of the Western medicine group and the Chinese medicine group was significantly increased compared with the model group, and the serum NA+-K-ATPase content of the Chinese medicine group was higher than that of the Western medicine group (P<0.01). This shows that the Chinese medicine group has a stronger therapeutic effect on rats with diabetic peripheral neuropathy.

[0103] Table 6 Serum IL-6 expression levels of rats in each group

[0104]

[0105] Note: Compared with the normal group: #p<0.05, ##p<0.01; Compared with the model group: *p<0.05, **p<0.01; Compared with western medicine: ^p<0.05, ^^p<0.01

[0106] Table 7 Serum TNF-α expression levels of rats in each group

[0107]

[0108] Note: Compared with the normal group: #p<0.05, ##p<0.01; Compared with the model group: *p<0.05, **p<0.01; Compared with the western medicine group: ^p<0.05, ^^p<0.01

[0109] Table 8 Serum NA++-K++P-ATP expression levels of rats in each group

[0110]

[0111] Note: Compared with the normal group: #p<0.05, ##p<0.01; compared with the model group: *p<0.05, **p<0.01; compared with the western medicine group: ^p<0.05, ^^p<0.01.

Claims

1. An application of a formula of effective ingredients of Gyuzhi saccharomyces in treating peripheral neuropathy caused by diabetes, characterized in that: The effective ingredient formula of Guizhi anthrax is composed of the following ingredients: ligusticum chuanxiong lactone A, ligusticum chuanxiong lactone I, angloside, coptisine hydrochloride, cynaroside, digitalis folioside, and harpaoside; The mass ratio of each component of the Guizhi anthrax effective ingredient formula is (5.4-6.3): (3.2-4.1): (2.9-3.5): (1.5-2.7): (1.2-3.0): (2.2-3.5): (0.9-1.6).

2. The use according to claim 1, characterized in that: The formula of the effective ingredients of Guizhi Cannabis Sativa is composed of: ligusticum chuanxiong lactone I: angloside: coptisine hydrochloride: cynaroside: digitalis folioside: harpaoside in a ratio of 5.7:3.6:3.1:1.7:2.3:3.2:1.

1.

3. The use according to claim 1, characterized in that: The formula of the active ingredient of Guizhi sugar cane can significantly reduce the blood sugar level of Wistar rats within 8 weeks and improve the nerve conduction velocity of rats with peripheral neuropathy.

4. The use according to claim 1, characterized in that: The formula of effective ingredients of saccharomyces cerevisiae can improve the expression levels of three inflammatory factors, IL-6, TNF-α, and NA++-K++P-ATP, in the blood of rats with peripheral neuropathy.

5. A drug for treating peripheral neuropathy caused by diabetes, characterized in that: The medicine includes an effective ingredient formula of Guizhi anthrax, and the effective ingredient formula is composed of the following ingredients: ligusticum chuanxiong lactone A: ligusticum chuanxiong lactone I: angloside: coptis chinensis hydrochloride: cynaroside: digitalis folioside: harpaoside is (5.4-6.3): (3.2-4.1): (2.9-3.5): (1.5-2.7): (1.2-3.0): (2.2-3.5): (0.9-1.6).

6. The drug according to claim 5, characterized in that The mass ratio of each component in the drug is 5.7:3.6:3.1:1.7:2.3:3.2:1.1 for ligusticum lactone A:ligusticum lactone I:angoloside:coptisine hydrochloride:binajuncea:digitolidin:harpaoside.

7. The drug according to claim 5, characterized in that The preparation types of the drug include but are not limited to granules, capsules, and tablets.

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