Traditional Chinese medicine compound preparation for treating diabetic peripheral neuropathy

Abstract

The present application relates to the technical field of traditional Chinese medicine, in particular to a traditional Chinese medicine compound preparation for treating diabetic peripheral neuropathy, which comprises the exclusive active enrichment part of eight kinds of traditional Chinese medicines such as Astragalus, Radix Rehmanniae, and Caulis Sargentodoxae, and is combined with pharmaceutical excipients. The preparation establishes the therapeutic principle of tonifying qi and nourishing yin, removing blood stasis and dredging collaterals, tonifying kidney and protecting marrow, and directly attacks the core pathogenesis of the disease. The effective components are precisely enriched by using the processes of enzymolysis, aqueous two-phase extraction, and ultrafiltration, and the pH-dependent enteric-soluble pellet capsules are optimized to improve the bioavailability and reduce the gastrointestinal irritation. The pharmacodynamics confirms that the product can regulate glucose metabolism, resist oxidative stress, inhibit inflammation, improve nerve microcirculation, repair damaged nerve myelin sheath, significantly improve the pain threshold and nerve conduction velocity, and the curative effect is better than that of conventional Chinese and western medicines. The acute and long-term toxicity tests show that the product has high safety, the process is stable and controllable, and is suitable for industrial production, thereby providing an efficient and safe treatment option for the disease.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine pharmaceutical technology, specifically to a traditional Chinese medicine compound preparation for treating diabetic peripheral neuropathy. Background Technology

[0002] Diabetic peripheral neuropathy (DPN) is one of the most common chronic complications of diabetes. Clinical epidemiological data shows that the prevalence of DPN among type 2 diabetes patients in my country exceeds 50%, with more than 20% of these patients already exhibiting varying degrees of peripheral nerve damage at the time of diabetes diagnosis. The core clinical manifestation of DPN is bilateral symmetrical distal limb sensory abnormalities, including numbness, tingling, burning, paresthesia, decreased or absent sensation. As the disease progresses, muscle weakness, muscle atrophy, and even foot ulcers and gangrene may occur, ultimately leading to amputation. It is one of the major causes of disability and death in diabetic patients, severely reducing their quality of life and imposing a heavy medical burden on families and society.

[0003] The pathogenesis of diabetic peripheral nerve distension (DPN) is not yet fully understood in modern medicine. The mainstream view is that it is related to multiple factors, including long-term hyperglycemia leading to polyol pathway hyperactivity, oxidative stress damage, excessive release of inflammatory factors, microvascular lesions causing nerve ischemia and hypoxia, and a lack of neurotrophic factors. Ultimately, this leads to peripheral nerve axonal degeneration, demyelination, and neurological dysfunction. Western medicine clinical treatment of DPN is based on strict blood glucose control, supplemented by symptomatic treatment. Commonly used drugs include neurotrophic drugs (such as mecobalamin and epalrestat), antioxidant stress drugs (such as alpha-lipoic acid), and analgesics (such as gabapentin and pregabalin). However, the above treatment options have obvious limitations: First, they can only relieve clinical symptoms and cannot reverse the existing peripheral nerve myelin damage and axonal degeneration, thus failing to fundamentally block the progression of DPN; Second, long-term use has significant adverse reactions, such as gastrointestinal irritation, dizziness, drowsiness, and liver and kidney damage, resulting in poor patient compliance; Third, single-target treatment is insufficient to address the complex pathogenesis of DPN involving multiple factors and pathways, leading to limited overall efficacy, and there is still a lack of effective drugs that can treat both the symptoms and the root cause in clinical practice.

[0004] Traditional Chinese medicine (TCM) categorizes diabetic paralysis (DPN) under the categories of "diabetes mellitus" and "muscle weakness," believing that its core pathogenesis is the depletion of qi and yin due to prolonged diabetes mellitus. Qi deficiency weakens blood circulation, while yin deficiency leads to sluggish blood vessels, ultimately resulting in blood stasis obstructing the meridians and causing numbness and weakness in the limbs. Current TCM clinical treatment of DPN primarily focuses on "tonifying qi and activating blood circulation, unblocking meridians and relieving pain," commonly using formulas such as Huangqi Guizhi Wuwu Tang, Danggui Sini Tang, and Buyang Huanwu Tang, with modifications. While these formulas can improve numbness and pain symptoms to some extent, they still have several shortcomings. First, current formulas often focus on the symptom of "meridian obstruction," neglecting the fundamental pathogenesis of kidney yin deficiency and insufficient marrow in prolonged diabetes mellitus, leading to peripheral nerves being deprived of nourishment by kidney essence. The repair of peripheral nerve myelin sheaths depends on the replenishment of kidney essence; simply activating blood circulation and unblocking meridians is insufficient to reverse nerve damage, resulting in limited clinical efficacy. Firstly, the efficacy is limited and the recurrence rate is high. Secondly, most existing Chinese medicine preparations use traditional water decoction processes, resulting in low extraction rates of effective components, high impurity content, and difficulty in precisely controlling the dosage of effective components, leading to poor efficacy stability. Thirdly, the effective components of conventional oral preparations are easily degraded and inactivated in the gastric acid environment, and some bitter, cold, and insect-derived medicinal materials can easily cause gastrointestinal irritation, resulting in poor patient tolerance and low bioavailability. In addition, there are few existing Chinese medicine compound preparations that target multiple aspects and multiple points of DPN pathogenesis while simultaneously addressing symptom relief and neural structure repair, which cannot meet the clinical treatment needs.

[0005] Therefore, developing a traditional Chinese medicine compound preparation with scientific compatibility, definite efficacy, the ability to intervene in the pathogenesis of DPN from multiple targets, reverse peripheral nerve damage, high safety, and good bioavailability has important clinical value and social significance. Summary of the Invention

[0006] To address the limitations of existing drugs for treating diabetic peripheral neuropathy (DPN), such as limited efficacy, irreversible nerve damage, low bioavailability, and significant side effects, this invention aims to provide a traditional Chinese medicine compound preparation for treating DPN. This preparation is scientifically formulated, can intervene in the pathogenesis of DPN at multiple targets, and treats both the symptoms and the root cause. Furthermore, through specific activity enrichment processes and targeted formulation technology, the bioavailability and efficacy stability of the active ingredients are significantly improved, while maintaining high safety. Another objective of this invention is to provide a method for preparing this preparation.

[0007] The technical solution adopted by this invention to solve its technical problem is: a traditional Chinese medicine compound preparation for treating diabetic peripheral neuropathy, wherein the preparation is prepared from active ingredients and pharmaceutically acceptable excipients. The active ingredients are composed of the following parts by weight of the active enriched parts of traditional Chinese medicine raw materials: Astragalus membranaceus 25-35 parts, Rehmannia glutinosa 18-25 parts, Spatholobus suberectus 15-22 parts, Siegesbeckia orientalis 12-18 parts, Drynaria fortunei 10-16 parts, Hirudo medicinalis 3-8 parts, Coptis chinensis 8-12 parts, and Cinnamomum cassia 2-5 parts. Among them, the active enriched fraction of Astragalus membranaceus is the saponin enriched fraction with an astragaloside content of ≥10wt%; the active enriched fraction of Rehmannia glutinosa is the iridoid enriched fraction with an alkaline content of ≥8wt%; the active enriched fraction of Spatholobus suberectus is the flavonoid phenolic acid enriched fraction with a total flavonoid content of ≥25wt%; the active enriched fraction of Siegesbeckia orientalis is the diterpenoid enriched fraction with an alkaline content of ≥3wt%; the active enriched fraction of Drynaria fortunei is the flavonoid enriched fraction with a naringin content of ≥15wt%; the active enriched fraction of Hirudo medicinalis is the hirudin freeze-dried powder with a molecular weight of 3000-7000Da; the active enriched fraction of Coptis chinensis is the alkaloid enriched fraction with a berberine hydrochloride content of ≥30wt%; and the active enriched fraction of Cinnamomum cassia is the volatile oil β-cyclodextrin inclusion complex with a cinnamaldehyde content of ≥20wt%.

[0008] Specifically, the active ingredients are composed of the following parts by weight of the active enriched parts of traditional Chinese medicine raw materials: Astragalus membranaceus 28-32 parts, Rehmannia glutinosa 19-22 parts, Spatholobus suberectus 16-20 parts, Siegesbeckia orientalis 13-16 parts, Drynaria fortunei 11-14 parts, Hirudo medicinalis 4-6 parts, Coptis chinensis 9-11 parts, and Cinnamomum cassia 2.5-4 parts.

[0009] Specifically, the active ingredients are composed of the following parts by weight of the active enriched parts of traditional Chinese medicine raw materials: 30 parts of Astragalus membranaceus, 20 parts of Rehmannia glutinosa, 18 parts of Spatholobus suberectus, 15 parts of Siegesbeckia orientalis, 12 parts of Drynaria fortunei, 5 parts of Hirudo medicinalis, 10 parts of Coptis chinensis, and 3 parts of Cinnamomum cassia.

[0010] Specifically, the dosage form of the preparation is an oral preparation, selected from any one of capsules, tablets, granules, and micro-pellets.

[0011] Specifically, the formulation is a pH-dependent enteric-coated microcapsule, wherein the enteric-coated microcapsule consists of a drug-containing pellet, an isolation layer, and an enteric coating layer from the inside out.

[0012] Specifically, the medicated pill is prepared from active ingredients, fillers, and binders. The filler is selected from at least one of microcrystalline cellulose, lactose, and mannitol, and the binder is selected from at least one of povidone K30 and hydroxypropyl methylcellulose. The coating material of the isolation layer is hydroxypropyl methylcellulose, with a coating weight gain of 2%-4%. The coating material of the enteric coating layer is acrylic resin L30D-55, with a coating weight gain of 6%-10%.

[0013] Specifically, the preparation method of the formulation includes the following steps: S1. Prepare the active enrichment fractions of each Chinese herbal raw material separately; S2. Mix the various active enrichment fractions prepared in step S1 evenly according to the prescription ratio to obtain a mixture of active ingredients; S3. Mix the active ingredient mixture with pharmaceutically acceptable excipients to prepare the desired formulation.

[0014] Specifically, the preparation method of the astragaloside enrichment fraction in step S1 is as follows: take astragalus slices, crush them, add purified water, add cellulase for enzymatic hydrolysis, filter after enzyme inactivation, concentrate the filtrate, precipitate with alcohol, filter, recover ethanol from the filtrate to obtain crude extract; add the crude extract to an ethanol-ammonium sulfate aqueous two-phase system, let it stand to separate into layers, take the upper phase to recover ethanol, and dry to obtain the crude extract; the composition of the aqueous two-phase system is 20-25wt% ethanol, 15-18wt% ammonium sulfate, and the remainder is crude extract.

[0015] Specifically, the preparation method of the enriched fraction of iridoid ethers from Rehmannia glutinosa in step S1 is as follows: take Rehmannia glutinosa slices, crush them, add purified water, add pectinase for enzymatic hydrolysis, filter after enzyme inactivation, concentrate the filtrate and pass it through a macroporous adsorption resin column, wash with water and then elute with 20% ethanol, collect the eluent to recover the ethanol, and dry to obtain the product.

[0016] Specifically, the preparation method of hirudin freeze-dried powder in step S1 is as follows: take leech medicinal material and homogenize it at low temperature, extract it with purified water at low temperature, centrifuge to obtain the supernatant, ultrafilter to retain the component with a molecular weight cutoff of 3000-7000 Da, and freeze-dry it to obtain the product.

[0017] The beneficial effects of this invention are: Scientifically formulated, addressing both the symptoms and root cause, this invention directly targets the core pathogenesis of DPN. Breaking away from the conventional treatment principle of "tonifying qi and activating blood circulation," this invention establishes a three-pronged treatment principle of "tonifying qi and nourishing yin, resolving blood stasis and unblocking collaterals, and tonifying the kidney and protecting the marrow" to address the core pathogenesis of DPN, which is "qi deficiency and yin deficiency as the root cause, blood stasis and obstruction of collaterals as the symptoms, and lack of nourishment of the marrow as the core damage." In this formula, Astragalus membranaceus tonifies Qi and raises Yang, promotes blood circulation and relieves stagnation, while Rehmannia glutinosa clears heat, cools the blood, nourishes Yin and generates fluids. Together, they serve as the principal herbs, tonifying Qi to promote blood circulation and nourishing Yin to moisten the vessels, addressing the fundamental pathogenesis of prolonged thirst that depletes Qi and damages Yin. Spatholobus suberectus invigorates blood, replenishes blood, relaxes muscles and tendons, and promotes blood circulation. Siegesbeckia orientalis dispels wind and dampness, benefits joints, and unblocks meridians. Together, they serve as the assistant herbs, invigorating blood and unblocking meridians to improve limb pain and assisting the principal herbs in promoting blood circulation and unblocking meridians. Drynaria fortunei tonifies the kidneys and strengthens bones, invigorates blood and heals injuries, replenishes kidney essence to nourish the myelin sheath, and repairs damaged peripheral nerves. Hirudo medicinalis breaks up blood stasis and promotes blood circulation. Coptis chinensis clears heat, dries dampness, drains fire and detoxifies, inhibits inflammatory responses, and improves insulin resistance. Together, they serve as the adjuvant herbs. Cinnamomum cassia tonifies fire and assists Yang, warms and unblocks meridians, guides fire back to its source, and at the same time restrains the cold and cooling properties of the herbs in the formula, serving as the guiding herb. The formula is meticulously formulated, combining cold and hot properties, tonifying and purging, and treating both the symptoms and the root cause. It not only improves patients' clinical symptoms such as numbness and pain, but also repairs damaged nerve myelin sheaths, fundamentally blocking the progression of DPN.

[0018] By employing specific active enrichment sites, this invention precisely controls the effective components and ensures stable therapeutic efficacy. Targeting the core active components of each medicinal herb in the formula, a proprietary enrichment process is used to prepare specific concentrations of active enrichment sites. This removes a large number of ineffective impurities and large-molecule sensitizing components from the medicinal materials, significantly increasing the content of effective components and achieving precise dosage control. This avoids the shortcomings of traditional decoctions, such as large fluctuations in the content of effective components and unstable therapeutic effects, while also reducing the risk of adverse reactions.

[0019] The preparation process is advanced, with a high extraction rate and is environmentally friendly. This invention targets the cell wall structure of root and rhizome medicinal materials, employing gradient enzymatic hydrolysis technology. Cellulase, pectinase, and other biological enzymes specifically degrade the plant cell wall, significantly improving the dissolution rate of active ingredients. Compared to traditional water decoction processes, the extraction rate of active ingredients is increased. Simultaneously, technologies such as aqueous two-phase extraction, macroporous resin adsorption, and ultrafiltration are used to achieve efficient enrichment of active ingredients. The process conditions are mild, avoiding the damage of active ingredients caused by high temperatures. The amount of organic solvent used is small, making it environmentally friendly and suitable for large-scale industrial production.

[0020] Targeted formulation design improves bioavailability and reduces gastrointestinal irritation. This invention preferably uses pH-dependent enteric-coated microcapsules, preparing the active ingredients into enteric-coated microcapsules. This avoids degradation and inactivation of the active ingredients in the acidic environment of the stomach, while simultaneously targeting release in the terminal ileum, significantly improving the absorption efficiency of the active ingredients. The active ingredients of bitter and cold herbs like Coptis chinensis and leeches are encapsulated within the microcapsules, avoiding direct irritation to the gastric mucosa. Furthermore, the volatile oil of cinnamon is encapsulated using β-cyclodextrin inclusion technology, solving the problems of volatility and instability of volatile oils, while also reducing gastrointestinal irritation and significantly improving patient tolerance.

[0021] The efficacy is definite, it targets multiple pathways to intervene in the pathogenesis of DPN, and it has a high safety profile. The formulation of this invention can intervene in the pathogenesis of DPN through multiple pathways: Firstly, it regulates glucose and lipid metabolism, improves insulin resistance, lowers blood glucose and glycated hemoglobin levels, and removes the initiating factors of DPN pathogenesis; Secondly, it inhibits oxidative stress response, increases serum SOD activity, reduces MDA content, and alleviates the damage of oxidative stress to nerve tissue; Third, it inhibits the inflammatory response, reduces serum levels of pro-inflammatory factors such as TNF-α and IL-6, and alleviates inflammatory infiltration of nerve tissue; Fourth, it improves nerve microcirculation, increases blood supply to nerve tissue, and alleviates nerve ischemia and hypoxia. Fifth, it repairs damaged peripheral nerve myelin sheaths, improves the motor and sensory conduction speed of the sciatic nerve, and reverses nerve dysfunction.

[0022] The efficacy of the preparation of this invention is significantly better than that of traditional Chinese medicine compound prescriptions and conventional Western medicines, and it has no obvious toxic side effects and high safety. Attached Figure Description

[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0024] Figure 1 The flowchart illustrates the preparation method of a traditional Chinese medicine compound preparation for treating diabetic peripheral neuropathy provided by this invention. Detailed Implementation

[0025] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0026] The traditional Chinese medicine compound preparation for treating diabetic peripheral neuropathy described in this invention is prepared from active ingredients and pharmaceutically acceptable excipients; the active ingredients are composed of the following active enriched parts of traditional Chinese medicine raw materials in parts by weight: Astragalus membranaceus 25-35 parts, Rehmannia glutinosa 18-25 parts, Spatholobus suberectus 15-22 parts, Siegesbeckia orientalis 12-18 parts, Drynaria fortunei 10-16 parts, Hirudo medicinalis 3-8 parts, Coptis chinensis 8-12 parts, and Cinnamomum cassia 2-5 parts.

[0027] Among them, the active enriched fraction of Astragalus membranaceus is the saponin enriched fraction with an astragaloside content of ≥10wt%; the active enriched fraction of Rehmannia glutinosa is the iridoid enriched fraction with an alkaline content of ≥8wt%; the active enriched fraction of Spatholobus suberectus is the flavonoid phenolic acid enriched fraction with a total flavonoid content of ≥25wt%; the active enriched fraction of Siegesbeckia orientalis is the diterpenoid enriched fraction with an alkaline content of ≥3wt%; the active enriched fraction of Drynaria fortunei is the flavonoid enriched fraction with a naringin content of ≥15wt%; the active enriched fraction of Hirudo medicinalis is the hirudin freeze-dried powder with a molecular weight of 3000-7000Da; the active enriched fraction of Coptis chinensis is the alkaloid enriched fraction with a berberine hydrochloride content of ≥30wt%; and the active enriched fraction of Cinnamomum cassia is the volatile oil β-cyclodextrin inclusion complex with a cinnamaldehyde content of ≥20wt%.

[0028] Furthermore, the preferred dosage form of the preparation is a pH-dependent enteric-coated microcapsule, wherein the enteric-coated microcapsule consists of a drug-containing pellet, an isolation layer, and an enteric coating layer from the inside out.

[0029] like Figure 1 As shown, the present invention also provides a method for preparing the above-mentioned formulation, comprising the following steps: S1 prepares the active enrichment fractions of each Chinese herbal raw material; S2. Mix the various active enriched fractions prepared in step S1 evenly according to the prescription ratio to obtain a mixture of active ingredients; S3 involves mixing the active ingredient mixture with pharmaceutically acceptable excipients to prepare the desired formulation.

[0030] Example 1: Preparation and pharmacodynamic verification of a traditional Chinese medicine compound enteric-coated microcapsule for treating diabetic peripheral neuropathy: 1. Prescription composition: The active ingredients in the following proportions by weight are: Astragalus membranaceus 30 parts, Rehmannia glutinosa 20 parts, Spatholobus suberectus 18 parts, Siegesbeckia orientalis 15 parts, Drynaria fortunei 12 parts, Hirudo medicinalis 5 parts, Coptis chinensis 10 parts, and Cinnamomum cassia 3 parts.

[0031] Excipients: Microcrystalline cellulose, lactose, povidone K30, hydroxypropyl methylcellulose, acrylic resin L30D-55, β-cyclodextrin, anhydrous ethanol, purified water.

[0032] 2. Preparation of active enrichment fractions of various traditional Chinese medicine raw materials: (1) Preparation of Astragalus saponin enrichment fraction: Take Astragalus slices, crush them into 10-mesh coarse powder, add 10 times the amount of purified water, add cellulase (enzyme activity 10000U / g) at 0.8% of the weight of the medicinal material, adjust the pH to 4.5, control the temperature at 50℃, and enzymatically hydrolyze for 2 hours; after enzymatic hydrolysis, raise the temperature to 90℃ to inactivate the enzyme for 10 minutes, filter, concentrate the filtrate under reduced pressure to a clear extract with a relative density of 1.05 at 60℃, add 95% ethanol to the system alcohol concentration to 70%, and heat at 4℃. After being refrigerated and allowed to stand for 24 hours, the extract was filtered. The filtrate was then subjected to reduced pressure to recover ethanol until no alcohol odor remained, yielding a crude extract of Astragalus membranaceus. The crude extract was then added to an aqueous two-phase system of ethanol and ammonium sulfate. The final composition of the system was 22 wt% ethanol, 16 wt% ammonium sulfate, and the remainder being the crude extract of Astragalus membranaceus. After stirring evenly, the mixture was allowed to stand and separate into layers. The upper organic phase was collected, and the ethanol was recovered under reduced pressure. The mixture was then dried under vacuum at 60°C to obtain the astragaloside-enriched fraction. The astragaloside A content was determined by high performance liquid chromatography to be 12.3 wt%, which met the requirements.

[0033] (2) Preparation of the iridoid enriched fraction of Rehmannia glutinosa: Take Rehmannia glutinosa slices, crush them into 10-mesh coarse powder, add 8 times the amount of purified water, add pectinase (enzyme activity 8000U / g) at 0.6% of the weight of the medicinal material, adjust the pH to 4.0, control the temperature at 45℃, and enzymatically hydrolyze for 1.5h; after the enzymatic hydrolysis is completed, raise the temperature to 90℃ to inactivate the enzyme for 10min, filter, concentrate the filtrate under reduced pressure to a clear extract with a relative density of 1.08 at 60℃, pass the clear extract through a D101 macroporous adsorption resin column with a column volume of 5 times the volume of the clear extract, first elute with 5 column volumes of purified water, discard the water washing liquid, then elute with 10 column volumes of 20% ethanol, collect the 20% ethanol eluent, recover the ethanol under reduced pressure, and vacuum dry at 60℃ to obtain the iridoid enriched fraction of Rehmannia glutinosa. The content of catalpol was detected by high performance liquid chromatography and found to be 9.2wt%, which met the requirements.

[0034] (3) Preparation of the flavonoid-phenolic acid enriched fraction of chicken blood vine: Take chicken blood vine slices, crush them into 10-mesh coarse powder, add 12 times the amount of 60% ethanol, reflux extract twice, 1.5h each time, combine the two extracts, and recover the ethanol under reduced pressure until there is no alcohol taste to obtain the crude extract of chicken blood vine; add an equal volume of ethyl acetate to extract three times, combine the ethyl acetate extracts, recover the solvent under reduced pressure, and dry under vacuum at 60℃ to obtain the flavonoid-phenolic acid enriched fraction of chicken blood vine. The total flavonoid content was 28.7wt% by ultraviolet spectrophotometry, which meets the requirements.

[0035] (4) Preparation of the diterpenoid-enriched fraction of Siegesbeckia orientalis: Siegesbeckia orientalis slices were crushed into 10-mesh coarse powder, 10 times the amount of 75% ethanol was added, and the mixture was refluxed and extracted twice, 2 hours each time. The two extracts were combined, and the ethanol was recovered under reduced pressure until there was no alcohol odor. The extract was concentrated under reduced pressure to a clear extract with a relative density of 1.10 at 60°C. The clear extract was passed through a polyamide column with a column volume of 4 times the volume of the clear extract. First, it was eluted with 6 column volumes of purified water and the water washing liquid was discarded. Then, it was eluted with 8 column volumes of 50% ethanol and the 50% ethanol eluent was collected. The ethanol was recovered under reduced pressure and the extract was dried under vacuum at 60°C to obtain the diterpenoid-enriched fraction of Siegesbeckia orientalis. The content of kilononol was detected by high performance liquid chromatography and found to be 3.6 wt%, which met the requirements.

[0036] (5) Preparation of flavonoid-rich fraction of Drynaria fortunei: Drynaria fortunei slices were crushed into 10-mesh coarse powder, 9 times the amount of 50% ethanol was added, and ultrasonic extraction was performed 3 times, 30 min each time, with ultrasonic power of 300 W and frequency of 40 kHz. The three extracts were combined, filtered, and the ethanol in the filtrate was recovered under reduced pressure until there was no alcohol odor. The extract was concentrated under reduced pressure to a clear extract with a relative density of 1.06 at 60℃. The clear extract was passed through an AB-8 type macroporous adsorption resin column with a column volume of 5 times that of the clear extract. First, it was eluted with 5 column volumes of purified water and the water washing liquid was discarded. Then, it was eluted with 10 column volumes of 70% ethanol and the 70% ethanol eluent was collected. The ethanol was recovered under reduced pressure and the extract was dried under vacuum at 60℃ to obtain the flavonoid-rich fraction of Drynaria fortunei. The naringin content was 17.5 wt% by high performance liquid chromatography, which met the requirements.

[0037] (6) Preparation of hirudin freeze-dried powder: Take leech medicinal material, wash it and homogenize it at 4℃, add 10 times the amount of purified water at 4℃, adjust the pH of the homogenate to 7.0, let it stand at 4℃ for 12h, centrifuge at 4℃ and 8000r / min for 20min, take the supernatant, use ultrafiltration membrane for fractional ultrafiltration, retain the molecular weight of 3000-7000Da, freeze-dry the retained liquid to obtain hirudin freeze-dried powder. The molecular weight distribution meets the requirements by gel permeation chromatography. The activity of hirudin is 800ATU / g by thrombin titration, which meets the requirements.

[0038] (7) Preparation of the alkaloid-rich fraction of Coptis chinensis: Coptis chinensis slices were crushed into 10-mesh coarse powder, 10 times the amount of 0.5% sulfuric acid aqueous solution was added, and after soaking for 12 hours, percolation was performed. The percolation liquid was collected in 8 times the volume of the medicinal material. The pH of the percolation liquid was adjusted to 10 with lime milk, and the mixture was allowed to stand for 2 hours. The mixture was then filtered, and the filtrate was extracted three times with an equal volume of chloroform. The chloroform extracts were combined, the solvent was recovered under reduced pressure, and the mixture was dried under vacuum at 60°C to obtain the alkaloid-rich fraction of Coptis chinensis. The berberine hydrochloride content was 35.2 wt% as determined by high performance liquid chromatography, which met the requirements.

[0039] (8) Preparation of cinnamon volatile oil β-cyclodextrin inclusion complex: Cinnamon slices were crushed into coarse powder, and volatile oil was extracted by steam distillation. The volatile oil was collected and dehydrated with anhydrous sodium sulfate for later use. According to the mass ratio of volatile oil to β-cyclodextrin = 1:8, β-cyclodextrin was taken and 10 times the amount of purified water was added. The mixture was stirred at 60°C until completely dissolved. Anhydrous ethanol solution of volatile oil (volume ratio of volatile oil to anhydrous ethanol 1:1) was slowly added dropwise. After the addition was completed, stirring was continued for 2 hours. After stirring was stopped, the mixture was refrigerated at 4°C and allowed to stand for 24 hours. The mixture was filtered, and the precipitate was washed three times with a small amount of anhydrous ethanol. The mixture was then vacuum dried at 40°C to obtain cinnamon volatile oil β-cyclodextrin inclusion complex. The cinnamaldehyde content was 22.6 wt% as determined by high performance liquid chromatography, which met the requirements.

[0040] 3. Preparation of enteric-coated microcapsules: (1) Mixing: Mix the above 8 active enrichment parts evenly according to the prescription ratio to obtain a mixture of active ingredients, and pass it through an 80-mesh sieve for later use.

[0041] (2) Preparation of vegetarian pellets: According to the mass ratio of active ingredient mixture: filler = 1:2, microcrystalline cellulose and lactose (mass ratio 1:1) are used as fillers and mixed evenly with the active ingredient mixture. 5% of 80% ethanol solution of povidone K30 is added as a binder to make soft material. Vegetarian pellets are prepared by extrusion spheroidization method. The extrusion orifice diameter is 0.3 mm, the spheroidization frequency is 50 Hz, and the spheroidization time is 5 min. The prepared vegetarian pellets are dried in a forced air at 40℃ for 4 h, granulated, and vegetarian pellets with a particle size of 0.3-0.5 mm are collected.

[0042] (3) Coating preparation: The fluidized bed bottom spray coating method was adopted. First, the pellets were coated with an isolation layer. The isolation layer coating solution was 80% ethanol solution of 5% hydroxypropyl methylcellulose. The coating air inlet temperature was 45℃, the atomization pressure was 0.2MPa, and the coating weight gain was 3%. After the isolation layer coating was completed, the enteric coating was carried out. The enteric coating solution was an aqueous dispersion of acrylic resin L30D-55 (containing 10% triethyl citrate as plasticizer). The coating air inlet temperature was 40℃, the atomization pressure was 0.2MPa, and the coating weight gain was 8%. After the coating was completed, it was cured at 40℃ for 2h to obtain enteric pellets.

[0043] (4) Capsule filling: The prepared enteric microspheres are filled into No. 3 hard capsules. Each capsule contains 0.3g of active ingredient, thus obtaining the Chinese medicine compound enteric microsphere capsule for the treatment of diabetic peripheral neuropathy.

[0044] 4. Pharmacodynamic verification experiments: (1) Experimental animals and model establishment: Seventy-two SPF-grade male SD rats, weighing 200±20g, were acclimatized for one week. Twelve rats were randomly selected as the normal group and fed a normal diet, while the remaining 60 rats were fed a high-sugar, high-fat diet for four weeks. After the high-sugar, high-fat diet ended, the rats were fasted for 12 hours and injected intraperitoneally with streptozotocin (STZ) 35mg / kg. The normal group was injected intraperitoneally with an equal volume of citrate-sodium citrate buffer. Seventy-two hours after the injection, fasting blood glucose was measured by blood collection from the tail vein. A fasting blood glucose level ≥16.7mmol / L was considered a successful establishment of the diabetic model. The rats with successful model establishment continued to be fed a high-sugar, high-fat diet for eight weeks. The mechanical pain threshold, thermal pain threshold, and sciatic nerve conduction velocity of the rats were measured. There were significant differences compared with the normal group (P<0.01), which was considered a successful establishment of the DPN model.

[0045] (2) Grouping and administration: Sixty rats that successfully developed the DPN model were randomly divided into 5 groups of 12 rats each, namely the model group, Example 1 group, Control 1 group, Control 2 group, and Control 3 group. The normal group and the model group were given an equal volume of physiological saline by gavage. Example 1 group was given the capsule contents suspension prepared in this example at a dose of 2g crude drug / kg body weight. Control 1 group was given the decoction of Astragalus and Cinnamon Twig Decoction (Astragalus 30g, Cinnamon Twig 10g, White Peony Root 15g, Ginger 6g, Jujube 10g, decocted and concentrated in water in a conventional manner) at a dose of 2g crude drug / kg body weight. Control 2 group was given the conventional decoction of all the medicinal materials in the prescription of this example at a dose of 2g crude drug / kg body weight. Control 3 group was given 100μg / kg of mecobalamin suspension + 60mg / kg of α-lipoic acid suspension by gavage once a day for 8 consecutive weeks.

[0046] (3) Detection Indicators and Results: After drug administration, rats were fasted for 12 hours, anesthetized by intraperitoneal injection of 3% sodium pentobarbital, and blood was collected from the abdominal aorta. Serum was separated, and fasting blood glucose (FBG), glycated hemoglobin (HbA1c), serum superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) levels were detected. The mechanical pain threshold and thermal pain threshold of the rat hind limbs were measured. The sciatic nerve of the rats was exposed, and the sciatic nerve motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) were measured. The experimental results are shown in the table below: Table 1 Comparison of blood glucose and glycated hemoglobin levels in rats after drug administration (x±s, n=12):

[0047] Table 2 Comparison of pain threshold and nerve conduction velocity in rats after drug administration (x±s, n=12):

[0048] Table 3 Comparison of serum oxidative stress and inflammatory factor levels in rats of different groups (x±s, n=12):

[0049] (4) Experimental conclusion: The traditional Chinese medicine compound preparation prepared in this embodiment can significantly reduce blood glucose and glycated hemoglobin levels in DPN model rats, increase mechanical pain threshold and thermal pain threshold, improve sciatic nerve motor and sensory conduction velocity, and significantly increase serum SOD activity and reduce MDA, TNF-α and IL-6 levels. The efficacy is significantly better than traditional Chinese medicine compound, whole herbal decoction and conventional Western medicine. It can intervene in the pathogenesis of DPN at multiple targets and effectively improve the pathological damage of DPN.

[0050] Example 2: Pilot-scale preparation and quality stability study of a traditional Chinese medicine compound preparation for treating diabetic peripheral neuropathy: 1. Prescription composition: The active ingredients in the following proportions by weight are: Astragalus membranaceus 32 parts, Rehmannia glutinosa 22 parts, Spatholobus suberectus 20 parts, Siegesbeckia orientalis 16 parts, Drynaria fortunei 14 parts, Hirudo medicinalis 6 parts, Coptis chinensis 11 parts, and Cinnamomum cassia 4 parts.

[0051] Excipients: Microcrystalline cellulose, lactose, povidone K30, hydroxypropyl methylcellulose, acrylic resin L30D-55, β-cyclodextrin, anhydrous ethanol, and purified water, all of pharmaceutical grade and in compliance with the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0052] Pilot-scale production: Each batch of raw materials consists of 100 kg of medicinal materials, producing 100,000 capsules. A total of 3 batches were produced.

[0053] 2. Pilot-scale preparation process: (1) Pilot-scale preparation of active enriched fractions: The preparation method of each active enriched fraction of medicinal materials is the same as that in Example 1. A 500L enzymatic hydrolysis reaction tank, a 1000L extraction tank, a two-phase extraction equipment, a macroporous resin column unit, an ultrafiltration unit, a vacuum drying oven, and a freeze dryer were used for pilot-scale production. Each process parameter was strictly controlled to ensure that the content of effective components in each active enriched fraction met the requirements.

[0054] (2) Pilot-scale preparation of enteric-coated microcapsules: The materials were mixed using a high-efficiency mixing granulator, the capsules were prepared using an extrusion spheronizer, the isolation layer and enteric layer were coated using a fluidized bed coating machine, and the capsules were filled using a fully automatic capsule filling machine. Each capsule contained 0.3g of active ingredient. The temperature (18-26℃) and relative humidity (45%-65%) of the production environment were strictly controlled during the preparation process, which met the GMP production requirements.

[0055] 3. Pilot-scale product quality testing: Three batches of pilot-scale products underwent full testing according to pre-established quality standards. The test results are shown in the table below: Table 4. Results of full-item quality testing for three batches of pilot-scale samples:

[0056] The test results showed that all test indicators of the three batches of pilot products met the quality standard requirements, and the quality difference between batches was minimal, indicating that the preparation process of the present invention is stable and controllable and suitable for large-scale industrial production.

[0057] 4. Stability assessment: Three batches of pilot-scale products were packaged in commercially available packaging (aluminum-plastic blister packaging, sealed with an outer aluminum foil bag) and subjected to accelerated stability testing at a temperature of 40℃±2℃ and a relative humidity of 75%±5%. Samples were taken at 0 months, 1 month, 2 months, 3 months, and 6 months for comprehensive quality testing. The test results are shown in the table below: Table 5. Results of accelerated stability study of pilot-scale samples after 6 months (taking the first batch as an example):

[0058] Accelerated stability testing results showed that during the 6-month accelerated stability period, all three batches of samples exhibited no significant changes in any of the tested indicators and met the quality standard requirements, indicating that the formulation of this invention has good stability and its quality is controllable within its shelf life. Simultaneously, long-term stability testing was conducted on the samples under conditions of 25℃±2℃ and 60%±10% relative humidity. The 12-month period has now been completed, and all indicators of the samples meet the quality standard requirements, further verifying the stability of the formulation.

[0059] Example 3: Optimization and safety evaluation of a traditional Chinese medicine compound preparation for treating diabetic peripheral neuropathy: 1. Prescription composition: The active ingredient raw materials by weight are: Astragalus membranaceus 28 parts, Rehmannia glutinosa 19 parts, Spatholobus suberectus 16 parts, Siegesbeckia orientalis 13 parts, Drynaria fortunei 11 parts, Hirudo medicinalis 4 parts, Coptis chinensis 9 parts, and Cinnamomum cassia 2.5 parts.

[0060] Excipients and preparation method: Same as in Example 1, enteric-coated microcapsules were prepared, each containing 0.3g of active ingredient.

[0061] 2. Pharmacodynamic comparative verification: Using the same DPN rat model and experimental methods as in Example 1, the efficacy differences between the formulation in this example and the formulation in Example 1 were compared. The experimental results are shown in the table below: Table 6 Comparison of efficacy results of the formulations in Example 3 and Example 1 (x±s, n=12):

[0062] Experimental results show that the formulation prepared in this embodiment can significantly improve blood glucose, nerve conduction velocity, oxidative stress and inflammatory factor levels in DPN model rats. The efficacy is not significantly different from that of the formulation in Example 1 (P>0.05), and is significantly better than that of the model group (P<0.01). This indicates that the prescription ratio of the present invention has good therapeutic effect within the scope of the claims and has wide applicability.

[0063] 3. Acute toxicity test: Forty SPF-grade Kunming mice (half male and half female, weighing 20±2g) were randomly divided into an experimental group and a control group (n=20 per group) after 3 days of acclimatization. The experimental group mice were administered the suspension of the preparation described in this embodiment at the maximum dosing concentration (0.5g active ingredient / mL) and maximum gavage volume (0.4mL / 10g body weight), twice daily, for a cumulative dose of 200g crude drug / kg body weight, equivalent to 200 times the recommended daily dose for adults. The control group mice were administered an equal volume of physiological saline by gavage. The mice were observed for 14 consecutive days after administration, with daily records of their diet, water intake, activity, mental state, weight changes, and mortality. After the observation period, the mice were euthanized, and morphological changes in major organs such as the heart, liver, spleen, lungs, kidneys, stomach, and intestines were observed. Pathological histological examinations were performed on abnormal organs.

[0064] The experimental results showed that during the 14-day observation period, no mice in the experimental group died, and their diet, water intake, activity, and mental state were all normal. Their weight gain was not significantly different from the control group (P > 0.05). Post-mortem examination revealed no obvious morphological abnormalities in the major organs of the experimental group mice, and no significant pathological changes were observed compared to the control group. This indicates that the preparation of this invention has extremely low acute toxicity and high safety for clinical application.

[0065] 4. Long-term toxicity testing: Eighty SPF-grade SD rats (half male and half female, weighing 180±20g) were randomly divided into four groups of 20 rats each after one week of acclimatization: control group, low-dose group, medium-dose group, and high-dose group. The low-, medium-, and high-dose groups were administered the suspension of the formulation described in this example at doses of 2g crude drug / kg, 4g crude drug / kg, and 10g crude drug / kg body weight, respectively (equivalent to 20, 40, and 100 times the clinical dose, respectively). The control group received an equal volume of physiological saline. The administration was performed once daily by gavage for six consecutive months. During the administration period, the general condition of the rats was observed daily, and their body weight and food intake were recorded weekly. At 3 months, 6 months, and 2 weeks after drug withdrawal, blood was collected from the abdominal aorta of some rats in each group. Complete blood count and blood biochemical indicators were measured, major organs were dissected and observed, organ coefficients were calculated, and histopathological examination of the major organs was performed.

[0066] Experimental results showed that during the 6-month continuous administration period, the rats in each dosage group were in good general condition, with normal diet and activity, and no significant difference in weight gain compared to the control group; all blood routine and blood biochemical indicators were within the normal range and showed no significant difference compared to the control group; the coefficients of major organs showed no significant difference compared to the control group, and histopathological examination revealed no drug-related toxic pathological changes; no delayed toxic reactions were observed 2 weeks after drug withdrawal. This indicates that the preparation of this invention has no obvious toxic side effects with long-term use and is safe and reliable for long-term clinical application.

[0067] Preparation of Huangqi Guizhi Wuwu Decoction (as in Example 1): Prescription: Astragalus membranaceus 30g, Cinnamomum cassia 10g, Paeonia lactiflora 15g, Zingiber officinale 6g, Ziziphus jujuba 10g.

[0068] Preparation method: Take the above-mentioned medicinal materials, add 10 times the amount of purified water and soak for 30 minutes, bring to a boil over high heat and then simmer over low heat for 30 minutes, filter, add 8 times the amount of purified water to the dregs and continue to simmer for 20 minutes, filter, combine the two decoctions, concentrate under reduced pressure to a crude drug concentration of 1 g / mL, refrigerate at 4℃ for later use in the pharmacodynamic control experiment of Example 1.

[0069] Comparative Example 2: Preparation of the conventional decoction of all medicinal materials in the prescription of this invention: Prescription: Astragalus membranaceus 30g, Rehmannia glutinosa 20g, Spatholobus suberectus 18g, Siegesbeckia orientalis 15g, Drynaria fortunei 12g, Hirudo medicinalis 5g, Coptis chinensis 10g, Cinnamomum cassia 3g.

[0070] Preparation method: Take the above-mentioned medicinal materials, except for cinnamon, add 10 times the amount of purified water and soak for 30 minutes. After boiling over high heat, simmer over low heat for 30 minutes. Add cinnamon and continue to simmer for 10 minutes. Filter. Add 8 times the amount of purified water to the dregs and continue to simmer for 20 minutes. Filter. Combine the two decoctions and concentrate under reduced pressure to a crude drug concentration of 1 g / mL. Refrigerate at 4℃ for later use in the pharmacodynamic control experiment of Example 1.

[0071] Control Example 3, Preparation of Positive Drug Suspension: Take methylcobalamin tablets, grind them into a fine powder, and prepare a suspension with a concentration of 10 μg / mL using 0.5% sodium carboxymethyl cellulose solution; take α-lipoic acid raw material, prepare a suspension with a concentration of 6 mg / mL using 0.5% sodium carboxymethyl cellulose solution, refrigerate at 4℃ for later use in the pharmacodynamic control experiment of Example 1.

[0072] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A traditional Chinese medicine compound preparation for treating diabetic peripheral neuropathy, characterized in that, The formulation is prepared from an active ingredient and pharmaceutically acceptable excipients: The active ingredients are composed of the following parts by weight of the active enriched parts of traditional Chinese medicine raw materials: Astragalus membranaceus 25-35 parts, Rehmannia glutinosa 18-25 parts, Spatholobus suberectus 15-22 parts, Siegesbeckia orientalis 12-18 parts, Drynaria fortunei 10-16 parts, Hirudo medicinalis 3-8 parts, Coptis chinensis 8-12 parts, and Cinnamomum cassia 2-5 parts. The active enriched fraction of Astragalus membranaceus is the saponin-rich fraction with an astragaloside A content ≥10wt%. The active enriched fraction of Rehmannia glutinosa is the iridoid enriched fraction with a catalpol content ≥8wt%. The active enriched fraction of *Spatholobus suberectus* is the flavonoid phenolic acid enriched fraction with a total flavonoid content ≥25wt%. The active enriched fraction of Siegesbeckia orientalis is the diterpenoid enriched fraction with a content of ≥3wt% kilonol; The active enriched part of Drynaria fortunei is the flavonoid-rich part with a naringin content ≥15wt%; The active enriched fraction of leeches is hirudin lyophilized powder with a molecular weight of 3000-7000 Da; The active enriched fraction of Coptis chinensis is the alkaloid enriched fraction with berberine hydrochloride content ≥30wt%. The active enriched fraction of cinnamon is the volatile oil β-cyclodextrin inclusion complex with a cinnamaldehyde content ≥20wt%.

2. The traditional Chinese medicine compound preparation for treating diabetic peripheral neuropathy according to claim 1, characterized in that: The active ingredients are composed of the following parts by weight of the active enriched parts of traditional Chinese medicine raw materials: Astragalus membranaceus 28-32 parts, Rehmannia glutinosa 19-22 parts, Spatholobus suberectus 16-20 parts, Siegesbeckia orientalis 13-16 parts, Drynaria fortunei 11-14 parts, Hirudo medicinalis 4-6 parts, Coptis chinensis 9-11 parts, and Cinnamomum cassia 2.5-4 parts.

3. The traditional Chinese medicine compound preparation for treating diabetic peripheral neuropathy according to claim 1, characterized in that: The active ingredients are composed of the following parts by weight of the active enriched parts of traditional Chinese medicine raw materials: Astragalus membranaceus 30 parts, Rehmannia glutinosa 20 parts, Spatholobus suberectus 18 parts, Siegesbeckia orientalis 15 parts, Drynaria fortunei 12 parts, Hirudo medicinalis 5 parts, Coptis chinensis 10 parts, and Cinnamomum cassia 3 parts.

4. The traditional Chinese medicine compound preparation for treating diabetic peripheral neuropathy according to any one of claims 1-3, characterized in that: The dosage form of the preparation is an oral preparation, selected from any one of capsules, tablets, granules, and micro-pellets.

5. The traditional Chinese medicine compound preparation for treating diabetic peripheral neuropathy according to claim 4, characterized in that: The formulation is a pH-dependent enteric-coated microcapsule, wherein the enteric-coated microcapsule consists of a drug-containing pellet, an isolation layer, and an enteric coating layer from the inside out.

6. The traditional Chinese medicine compound preparation for treating diabetic peripheral neuropathy according to claim 5, characterized in that: The medicated pills are prepared from active ingredients, fillers, and binders. The fillers are selected from at least one of microcrystalline cellulose, lactose, and mannitol, and the binders are selected from at least one of povidone K30 and hydroxypropyl methylcellulose. The coating material of the isolation layer is hydroxypropyl methylcellulose, and the coating weight gain is 2%-4%. The enteric coating layer is coated with acrylic resin L30D-55, and the coating weight gain is 6%-10%.

7. The method for preparing the traditional Chinese medicine compound preparation for treating diabetic peripheral neuropathy according to claim 1, characterized in that, Includes the following steps: S1. Prepare the active enrichment fractions of each Chinese herbal raw material separately; S2. Mix the various active enrichment fractions prepared in step S1 evenly according to the prescription ratio to obtain a mixture of active ingredients; S3. Mix the active ingredient mixture with pharmaceutically acceptable excipients to prepare the desired formulation.

8. The preparation method according to claim 7, characterized in that: The preparation method of the astragaloside enrichment fraction in step S1 is as follows: Take Astragalus membranaceus slices, crush them, add purified water, add cellulase for enzymatic hydrolysis, filter after enzyme inactivation, concentrate the filtrate, precipitate with alcohol, filter, and recover ethanol from the filtrate to obtain crude extract. Add the crude extract to an aqueous two-phase system of ethanol-ammonium sulfate, allow it to stand and separate into layers, take the upper phase to recover the ethanol, and dry it to obtain the final product. The aqueous two-phase system consists of 20-25 wt% ethanol, 15-18 wt% ammonium sulfate, and the remainder is crude extract.

9. The preparation method according to claim 7, characterized in that: The preparation method of the iridoid enrichment fraction of Rehmannia glutinosa in step S1 is as follows: take Rehmannia glutinosa slices, crush them, add purified water, add pectinase for enzymatic hydrolysis, filter after enzyme inactivation, concentrate the filtrate and pass it through a macroporous adsorption resin column, wash with water and then elute with 20% ethanol, collect the eluent to recover the ethanol, and dry to obtain the product.

10. The preparation method according to claim 7, characterized in that: The preparation method of hirudin freeze-dried powder in step S1 is as follows: take leech medicinal material and homogenize it at low temperature, extract it with purified water at low temperature, centrifuge to obtain the supernatant, ultrafilter to retain the component with a molecular weight cutoff of 3000-7000 Da, and freeze-dry it to obtain the product.

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