Magnolol and borneol nanogel as well as preparation method and application thereof in treating diabetic peripheral neuropathy

Abstract

The invention discloses magnolol and borneol nanogel, a preparation method thereof and application of the magnolol and borneol nanogel to treatment of diabetic peripheral neuropathy, the magnolol-borneol nanogel is prepared through experimental screening, and the prepared magnolol-borneol nanogel is beneficial to retention of magnolol in skin, and has a good application prospect in treatment of diabetic peripheral neuropathy. The bioavailability of magnolol can be obviously improved. Pharmacodynamics proves that the Mag-Bor NPs Gel prepared by the invention can be used for improving the forms of sciatic nerves and DRG and repairing damaged nerves, and has important significance on treatment of diabetic peripheral neuropathy.

Description

Technical Field

[0001] The present invention belongs to a novel delivery system, and particularly relates to honokiol and borneol nanogels, a preparation method thereof, and an application in treating diabetic peripheral neuropathy. Background Art

[0002] Among the complications of diabetes, diabetic peripheral neuropathy (DPN) caused by diffuse damage to the peripheral nervous system is currently the most common, with an incidence rate as high as half among all diabetic patients. DPN patients usually experience symmetrical pain from the lower limbs to the upper limbs, especially at the distal extremities of the limbs, showing a "glove and sock" - like distribution. The onset of DPN is usually insidious in the early stage and is easily overlooked. As the disease progresses, it gradually develops into irreversible nerve damage, resulting in loss of sensation in the lower limbs of patients, leading to diabetic foot, and even posing a risk of amputation. It not only seriously affects the quality of life of patients but also brings a huge social and economic burden.

[0003] The damage of nerve cells is usually caused by metabolic disorders, oxidative stress, and inflammatory responses. Its mechanisms mainly include the polyol pathway, the advanced glycation end - product pathway, and the mitogen - activated protein kinase pathway.

[0004] Currently, the clinical treatment methods for DPN mainly include reducing the blood glucose level of diabetic patients, drug and non - drug treatments.

[0005] Blood glucose, as a direct inducer of DPN, controlling the blood glucose level is of great significance in the subsequent treatment of DPN. Research shows that the incidence of peripheral neuropathy increases with the deterioration of blood glucose conditions, and the incidence rate of patients with diagnosed diabetes is approximately 5 times that of patients with normal blood glucose. Active blood glucose control can improve the nerve conduction velocity and perception threshold of patients, and significantly reduce the risk and progression rate of DPN in diabetic patients.

[0006] Currently, there is no drug that can reverse the progression of DPN. Clinically used drugs for treating DPN are mainly divided into symptom - improving drugs and drugs targeting the pathogenesis. For the pain symptoms of DPN, current clinical options include anticonvulsants (pregabalin, gabapentin), tricyclic antidepressants (amitriptyline), and serotonin - norepinephrine reuptake inhibitors (duloxetine). The most commonly recommended first - line drugs for treating painful DPN are gabapentin and pregabalin. Amitriptyline is restricted in use due to its potential cholinergic side effects, especially for elderly patients. For opioid drugs, although research has demonstrated their efficacy in neuropathic pain related to DPN, they should not be routinely used due to limited efficacy, long - term safety issues, and the possibility of abuse.

[0007] Other drugs for improving symptoms include drugs for improving microcirculation (prostaglandins and prostaglandin analogs, hexoprenaline, pancreatinogenase, Bactrim), neurotrophic drugs (mecobalamin), drugs for improving cellular energy metabolism, drugs for counteracting oxidative stress (α-lipoic acid), aldose reductase activity inhibitors (epalrestat), and angiotensin-converting enzyme inhibitors. Many studies have reported that these drugs, either alone or in combination with other drugs, can promote peripheral nerve regeneration and improve the clinical symptoms of DPN patients, but the magnitude of their benefits remains controversial. Despite the variety of drugs, due to the complex pathogenesis, diverse clinical manifestations, and immature staging of DPN, there is still a lack of specific drugs and treatment regimens for DPN. This has led to relatively conservative current drug regimens, with dose limitations to avoid severe side effects and ultimately affecting the treatment outcome.

[0008] Since the efficacy of existing drugs in treating DPN is not yet clear, non-drug treatments are also widely used clinically as an adjunct to drug treatment, including psychological support, acupuncture, and transcutaneous electrical nerve or muscle stimulation.

[0009] Magnolol (Mag) is a polyphenolic biphenyl compound present in the bark of Magnolia officinalis and is an isomer of honokiol. In traditional Chinese medicine, Magnolia officinalis bark is considered to have antipyretic, analgesic, sedative, laxative, anti-asthmatic, cardioprotective, vasoprotective, or antibacterial properties. A large number of scientific studies have reported the biological activities of magnolol, including antioxidant, anti-inflammatory, neuroprotective, analgesic, anticancer, anticoagulant, and smooth muscle relaxation effects. Summary of the Invention

[0010] The object of the present invention is to solve the problem of drug administration in the existing treatment of diabetic peripheral neuropathy. Through experimental screening, magnolol-borneol nanogel (Mag-Bor NPs Gel) is prepared. The prepared magnolol-borneol nanogel is beneficial to the retention of magnolol in the skin and can significantly improve the bioavailability of magnolol. Proven by pharmacodynamics, the Mag-Bor NPs Gel prepared in the present invention can improve the morphology of the sciatic nerve and DRG, repair damaged nerves, and is of great significance for the treatment of diabetic peripheral neuropathy.

[0011] Technical Solution: To achieve the above object, the technical solution adopted by the present invention is as follows:

[0012] A magnolol and borneol nanogel, which is prepared by the following method:

[0013] (1) Weigh magnolol and borneol, dissolve them in absolute ethanol as the organic phase; weigh the stabilizer and dissolve it in ultrapure water as the aqueous phase; place the aqueous phase on a magnetic stirrer, and quickly inject the organic phase into the aqueous phase according to a certain ratio. Finally, remove the absolute ethanol through a rotary evaporator to obtain a magnolol-borneol nano-suspension, and concentrate it through an ultrafiltration cup to prepare a concentrated magnolol-borneol nanoparticle solution.

[0014] (2) Preparation of carbomer gel: Weigh carbomer 940 and disodium ethylenediaminetetraacetate dihydrate, add water to make them swell fully, dropwise add triethanolamine, stir and mix evenly, adjust the pH to prepare a blank gel matrix.

[0015] (3) Preparation of magnolol-borneol nanogel: Weigh the same mass of the concentrated magnolol-borneol nanoparticle solution and the blank gel matrix, mix them, and stir evenly to prepare magnolol-borneol nanogel.

[0016] As a preferred embodiment, for the magnolol-borneol nanogel described above, in step (1): Weigh magnolol and borneol with a mass ratio of 1:0.2 - 1:1, dissolve them in absolute ethanol as the organic phase; weigh the stabilizer polyvinylpyrrolidone-K30 and dissolve it in ultrapure water as the aqueous phase. Place the aqueous phase on a magnetic stirrer, quickly inject the organic phase into the aqueous phase, and finally remove the absolute ethanol through a rotary evaporator to obtain a magnolol-borneol nano-suspension; concentrate the prepared magnolol-borneol nano-suspension through an ultrafiltration cup to prepare a concentrated magnolol-borneol nanoparticle solution.

[0017] As a preferred embodiment, for the magnolol-borneol nanogel described above, the concentration of the stabilizer polyvinylpyrrolidone-K30 is 0.05 - 2%, the stirring speed of the magnetic stirrer is 1500 - 2000 r / min, the volume ratio of the organic phase to the aqueous phase is 1:20 - 1:50, the mass ratio of magnolol to borneol is 1:0.5, and the concentration of magnolol and borneol is 20 - 30 mg / mL.

[0018] As a preferred embodiment, for the magnolol-borneol nanogel described above, the concentration of the stabilizer polyvinylpyrrolidone-K30 is 0.1%, the stirring speed of the magnetic stirrer is 1500 r / min, the volume ratio of the organic phase to the aqueous phase is 1:40, the mass ratio of magnolol to borneol is 1:0.5 - 1:1, and the concentration of magnolol and borneol is 25 mg / mL.

[0019] Beneficial effects:

[0020] Through a large number of experiments, by investigating the types and concentrations of stabilizers, stirring speed, volume ratio of organic phase to aqueous phase, and drug concentration, the optimal formulation and preparation process of magnolol nanoparticles were successfully selected as follows: the stabilizer is PVP-K30, the stabilizer concentration is 0.1%, the stirring speed is 1500 r / min, the volume ratio of organic phase to aqueous phase is 1:40, the drug concentration is 25 mg / mL, and the particle size of the prepared magnolol nanoparticles is less than 200 nm within 7 days, and the PDI is less than 0.2, showing good stability.

[0021] In addition, the optimal ratio of magnolol to borneol was further screened out, and a safe and highly transdermal permeable magnolol-borneol nanogel was successfully prepared.

[0022] The magnolol-borneol nanogel prepared by the present invention is beneficial to the retention of magnolol in the skin and can significantly improve the bioavailability of magnolol.

[0023] The magnolol-borneol nanogel can improve the myelin sheath structure of the sciatic nerve in DPN rats, effectively reduce the expression level of NF-κB protein and the expression amount of NF-κB, improve the inflammatory reaction in the DRG tissue of DPN rats, effectively reduce the inflammatory damage of the dorsal root ganglion and sciatic nerve, promote its nerve repair, and is of great significance for the treatment of diabetic peripheral neuropathy. Description of the Drawings

[0024] Figure 1 Results of the investigation of stabilizer types, nanoparticle size (left) and nanoparticle PDI (right);

[0025] Figure 2 Results of the investigation of stabilizer concentration (solid line is particle size, dashed line is PDI);

[0026] Figure 3 Results of the investigation of stirring speed (solid line is particle size, dashed line is PDI);

[0027] Figure 4 Results of the investigation of the volume ratio of organic phase to aqueous phase (solid line is particle size, dashed line is PDI);

[0028] Figure 5 Results of the investigation of drug concentration (solid line is particle size, dashed line is PDI);

[0029] Figure 6 XRD patterns of magnolol raw drug, borneol raw drug, and magnolol-borneol nanoparticles;

[0030] Figure 7 Results of the skin permeability of magnolol-borneol nanogel;

[0031] Figure 8Effect of magnolol-borneol nanogel on the pain threshold time of photothermal stimulation on the plantar surface of rats;

[0032] Figure 9 Effect of magnolol-borneol nanogel on the mechanical withdrawal threshold of rats;

[0033] Figure 10 Effect of magnolol-borneol nanogel on the sciatic nerve conduction velocity of rats;

[0034] Figure 11 Intradermal nerve fiber density in the plantar skin of the hind paws of rats in each group (left) and statistical analysis of nerve fiber density in different groups (right);

[0035] Figure 12 Expression of MBP in the sciatic nerve of rats in each group (left) and statistical analysis of MBP in different groups (right);

[0036] Figure 13 Expression of NF-κB in the dorsal root ganglia of rats in each group (left) and statistical analysis of NF-κB in different groups (right). Specific implementation mode

[0037] Example 1 Prescription study of magnolol nanoparticles

[0038] 1. Experimental method

[0039] 1.1 Establishment of in vitro analysis method for magnolol

[0040] 1.1.1 Chromatographic conditions

[0041] Chromatographic column: Kromasil C18 column (150 mm × 4.6 mm, 5 μm)

[0042] Mobile phase: methanol-water (78:22);

[0043] Detection wavelength: 294 nm; Flow rate: 1.0 mL / min; Column temperature: 30 °C; Injection volume: 20 μL

[0044] 1.1.2 Preparation of solutions

[0045] Preparation of reference stock solution: Weigh accurately 5.00 mg of magnolol reference substance, place it in a 5 mL volumetric flask, dissolve it with methanol and make up to the scale, to prepare a magnolol reference stock solution with a concentration of 1.0 mg / mL, and store it in a refrigerator at 4 °C for later use.

[0046] 1.1.3 Establishment of standard curve

[0047] The magnolol stock solution was serially diluted with methanol to obtain magnolol reference solutions at concentrations of 400, 200, 100, 50, 25, 10, 5, and 1 μg / mL. Detection was performed under the above chromatographic conditions. A standard curve was plotted with concentration (C) on the abscissa and peak area (A) on the ordinate.

[0048] 1.1.4 Precision

[0049] Magnolol reference solutions at concentrations of 5, 50, and 100 μg / mL were prepared separately. According to the above chromatographic conditions, the samples were injected continuously 6 times, the peak areas were measured, and the RSD values of the peak areas were calculated.

[0050] 1.1.5 Repeatability

[0051] Magnolol reference solutions at concentrations of 5, 50, and 100 μg / mL were prepared separately in 6 parallel samples. Detection was performed under the above chromatographic conditions, and the RSD values of the peak areas were calculated.

[0052] 1.2 Investigation of the physicochemical properties of magnolol

[0053] 1.2.1 Determination of the equilibrium solubility of magnolol

[0054] 2 mL of ultrapure water and phosphate buffer solutions with pH values of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 were separately placed in 10-mL stoppered test tubes. 1 mg of magnolol was added to each solution. The stoppered test tubes were placed in a constant temperature water bath oscillator at 37 ± 0.5 °C with a frequency of 120 r / min and shaken for 24 h to achieve complete dissolution. The supernatant was centrifuged at 12,000 r / min for 5 min, and injection analysis was performed under the above chromatographic conditions to calculate the solubility of magnolol in ultrapure water and different pH buffer solutions.

[0055] 1.2.2 Determination of the oil-water partition coefficient of magnolol

[0056] The oil-water partition coefficient of magnolol was determined by the classical shake-flask method. Preparation of water-saturated n-octanol solution and n-octanol-saturated aqueous solution: n-Octanol and water were mixed in equal volumes, vortexed and mixed evenly, placed in a constant temperature water bath oscillator at 37 ± 0.5 °C with a frequency of 120 r / min, shaken for 24 h, and then allowed to stand for several hours to separate layers. The upper layer was the water-saturated n-octanol solution, and the lower layer was the n-octanol-saturated aqueous solution.

[0057] Take 5 mL of the water-saturated n-octanol solution and place it in a 10-mL stoppered test tube. Add an excess of magnolol until insoluble precipitate appears in the solution. Place the stoppered test tube in a constant temperature water bath oscillator at 37 ± 0.5 °C with a frequency of 120 r·min -1Shake it in a constant temperature water bath oscillator for 24 h to prepare a saturated n-octanol solution containing the drug. Precisely pipette 3 mL of the saturated n-octanol solution containing the drug and the water solution saturated with n-octanol into a 10 mL stoppered test tube. After vortex mixing, place it in a constant temperature water bath oscillator at a temperature of 37 ± 0.5 °C and a frequency of 120 r / min and shake it for 24 h. Let it stand for several hours. After stratification, take the upper and lower layers respectively: Take 1 mL of the upper layer, dilute it 1000 times with methanol, filter it through a 0.22 μm microporous filter membrane, and take the subsequent filtrate for sample injection analysis; take 1 mL of the lower layer, filter it through a 0.22 μm microporous filter membrane, and take the subsequent filtrate for sample injection analysis. Calculate the apparent oil-water partition coefficient of magnolol according to the formula P = C o / C w where C o is the drug concentration in the oil phase and Cw is the drug concentration in the water phase.

[0058] 1.3 Study on the compatibility of excipients for magnolol nanoparticles

[0059] Prepare magnolol nanoparticles by the anti-solvent precipitation method. Weigh a certain amount of magnolol and dissolve it in absolute ethanol as the organic phase; weigh another certain amount of stabilizer and dissolve it in ultrapure water as the aqueous phase. Place the aqueous phase on a magnetic stirrer and quickly inject the organic phase into the aqueous phase. Remove the organic solvent through a rotary evaporator to finally obtain a magnolol nano-suspension.

[0060] 1.3.1 Investigation of the types of stabilizers

[0061] Select poloxamer 188 (P188), sodium dodecyl sulfate (SDS), polyvinylpyrrolidone-K30 (PVP-K30), and hydroxypropyl methylcellulose (HPMC) as stabilizers. Weigh 40 mg of magnolol and dissolve it in 2 mL of absolute ethanol as the organic phase; weigh 0.1 g each of poloxamer 188 (P188), sodium dodecyl sulfate (SDS), polyvinylpyrrolidone-K30 (PVP-K30), and hydroxypropyl methylcellulose (HPMC) and dissolve them in 100 mL of ultrapure water respectively as the aqueous phase. Place the aqueous phase on a magnetic stirrer. Under the stirring condition of 1500 r / min and with the volume ratio of the organic phase to the aqueous phase being 1:40, quickly inject the organic phase into the aqueous phase. Remove the organic solvent through a rotary evaporator to finally obtain a magnolol nano-suspension, and let it stand at room temperature for later use. At the time point of standing at room temperature for 1 h, take 1 mL of the nano-particle suspension into the sample cell and place it in a Malvern particle size analyzer. In the automatic mode, measure the particle size and polydispersity index (PDI). In the same way, measure the particle size and polydispersity index (PDI) of the nano-particles at 3, 6, 9, 12, and 24 h. Take the particle size and polydispersity index (PDI) of the nano-particles as the evaluation indexes to investigate the types of stabilizers.

[0062] 1.3.2 Investigation of the concentration of stabilizers

[0063] PVP-K30 was selected as the stabilizer. 40 mg of magnolol was weighed and dissolved in 2 mL of absolute ethanol as the organic phase. 0.025 g, 0.05 g, and 0.1 g of PVP-K30 were weighed respectively and dissolved in 50 mL of ultrapure water to prepare PVP-K30 solutions with concentrations of 0.05%, 0.1%, and 0.2% as the aqueous phase. The aqueous phase was placed on a magnetic stirrer. Under the stirring condition of 1500 r / min, with the volume ratio of the organic phase to the aqueous phase being 1:40, the organic phase was quickly injected into the aqueous phase, and the organic solvent was removed by a rotary evaporator to finally obtain a magnolol nano-suspension, which was left at room temperature for standby. At the time point of standing at room temperature for 1 h, 1 mL of the nano-particle suspension was taken into a sample cell and placed in a Malvern particle size analyzer. In the automatic mode, the particle size and PDI were measured. In the same way, the particle size and polydispersity index (PDI) of the nano-particles at 3, 6, 9, 12, 24, 48, 72, 96, 120, 144, 168 h were measured. Taking the particle size and polydispersity index (PDI) of the nano-particles as the evaluation indexes, the concentration of PVP-K30 was investigated.

[0064] 1.4 Prescription and process optimization

[0065] 1.4.1 Investigation of stirring speed

[0066] To compare the effects of stirring speed on the particle size and PDI of nano-particles, 500, 1000, 1500, 2000 r·min -1 were investigated. 40 mg of magnolol was weighed and dissolved in 2 mL of absolute ethanol as the organic phase. 0.1 g of PVP-K30 was weighed and dissolved in 100 mL of ultrapure water as the aqueous phase. The aqueous phase was placed on a magnetic stirrer, and the stirring speeds were set at 500, 100, 1500, 2000 r / min respectively. With the volume ratio of the organic phase to the aqueous phase being 1:40, the organic phase was quickly injected into the aqueous phase, and the organic solvent was removed by a rotary evaporator to finally obtain a magnolol nano-suspension, which was left at room temperature for standby. At the time point of standing at room temperature for 1 h, 1 mL of the nano-particle suspension was taken into a sample cell and placed in a Malvern particle size analyzer. In the automatic mode, the particle size and polydispersity index (PDI) were measured. In the same way, the particle size and PDI of the nano-particles at 3, 6, 9, 12, 24, 48, 72, 96, 120, 144, 168 h were measured. Taking the particle size and PDI of the nano-particles as the evaluation indexes, the optimal stirring speed was screened. 1.4.2 Investigation of the volume ratio of the organic phase to the aqueous phase

[0067] To compare the effects of the volume ratio of the organic phase to the aqueous phase on the particle size and PDI of nano-particles, volume ratios of the organic phase to the aqueous phase of 1:20, 1:30, 1:40, 1:50 were investigated.

[0068] Weigh 40 mg of magnolol and dissolve it in 2 mL of absolute ethanol to obtain the organic phase. Weigh 0.1 g of PVP-K30 and dissolve it in 100 mL of ultrapure water to obtain the aqueous phase. Place the aqueous phase on a magnetic stirrer and, under the condition of 1500 r / min, quickly inject the organic phase into the aqueous phase according to the volume ratios of the organic phase to the aqueous phase of 1:20, 1:30, 1:40, and 1:50. Finally, remove the absolute ethanol through a rotary evaporator to obtain a nano-suspension. At the time point of standing at room temperature for 1 h, take 1 mL of the nano-particle suspension and place it in a sample cell, then put it into a Malvern particle size analyzer and measure the particle size and polydispersity index (PDI) in the automatic mode. Similarly, measure the particle size and PDI of the nano-particles at 3, 6, 9, 12, 24, 48, 72, 96, 120, 144, 168 h. Take the particle size and PDI of the nano-particles as the evaluation indexes to screen the optimal volume ratio of the organic phase to the aqueous phase.

[0069] 1.4.3 Investigation of drug concentration

[0070] To compare the effects of drug concentration on the particle size and PDI of nano-particles, drug concentrations of 20, 25, and 30 mg / mL were investigated. Weigh 20, 25, and 30 mg of magnolol respectively and dissolve them in 1 mL of absolute ethanol to obtain the organic phases. Weigh 0.1 g of PVP-K30 and dissolve it in 100 mL of ultrapure water to obtain the aqueous phase. Place the aqueous phase on a magnetic stirrer and, under the condition of 1500 r / min, quickly inject the organic phase into the aqueous phase according to the volume ratio of the organic phase to the aqueous phase of 1:40. Finally, remove the absolute ethanol through a rotary evaporator to obtain a nano-suspension. At the time point of standing at room temperature for 1 h, take 1 mL of the nano-particle suspension and place it in a sample cell, then put it into a Malvern particle size analyzer and measure the particle size and PDI in the automatic mode. Similarly, measure the particle size and PDI of the nano-particles at 3, 6, 9, 12, 24, 48, 72, 96, 120, 144, 168 h. Take the particle size and PDI of the nano-particles as the evaluation indexes to screen the optimal drug concentration.

[0071] 2 Experimental results

[0072] 2.1 Methodology investigation

[0073] 2.1.1 Establishment of the standard curve

[0074] Draw a standard curve with the peak area (A) of magnolol as the ordinate and the concentration (C) as the abscissa. The regression equation is A = 29046c + 13665, R 2 = 0.9999. The results show that magnolol has a good linear relationship in the range of 1 - 400 μg / mL. 2.1.2 Precision

[0075] Prepare control solutions of three concentrations, 5, 50, and 200 μg / mL, and inject samples continuously for 6 times with an injection volume of 20 μL. Measure the peak areas and calculate the RSD values of the peak areas, which are 0.458%, 0.247%, and 0.540% respectively, all less than 3%. The instrument precision is good. The results are shown in Table 1.

[0076] Table 1 Results of precision experiment

[0077] Concentration (μg / mL) Average peak area RSD (%) 5 144909±663.29 0.458 50 1503422±3718.40 0.247 200 5992341±32342.02 0.540

[0078] 2.1.3 Repeatability

[0079] Prepare magnolol control solutions with concentrations of 5, 50, and 200 μg / mL respectively, in 6 parallel portions, and detect them under the chromatographic conditions described in "2.1.1". Calculate the RSD values of the peak areas, which are 1.81%, 1.94%, and 1.57% respectively, all less than 3%. This indicates that the method has good repeatability. The results are shown in Table 2.

[0080] Table 2 Results of repeatability experiment

[0081] Concentration (μg / mL) Average peak area RSD (%) 5 145036±2619.26 1.81 50 1490972±28871.05 1.94 200 5917766±92669.50 1.57

[0082] 2.2 Investigation of the physicochemical properties of magnolol

[0083] 2.2.1 Determination of the equilibrium solubility of magnolol

[0084] The equilibrium solubilities of magnolol in water and phosphate buffer solutions with different pH values are shown in Table 3.

[0085] Table 3 Equilibrium solubilities of magnolol in different solvents

[0086] Solvent Equilibrium solubility (μg / mL) RSD (%) Water 11.17±0.19 1.72 pH 2.0 Phosphate buffer 10.86±0.14 1.31 pH 3.0 Phosphate buffer 11.13±0.18 1.61 pH 4.0 Phosphate buffer 10.74±0.20 1.87 pH 5.0 Phosphate buffer 11.25±0.18 1.63 pH 6.0 Phosphate buffer 10.11±0.19 1.89 pH 7.0 Phosphate buffer 10.78±0.10 0.911 pH 8.0 Phosphate buffer 41.32±0.52 1.25

[0087] 2.2.2 Determination of the oil-water partition coefficient of magnolol

[0088] The calculation formula for the apparent oil-water partition coefficient P is P = C o / C w where C o is the drug concentration in the oil phase and C w is the drug concentration in the aqueous phase. The results are shown in Table 2-7. The HPLC results show that the concentrations of magnolol in the oil phase and aqueous phase are 219.11 ± 10.92 mg / mL and 0.02 ± 0.0005 mg / mL respectively. The calculated results of the oil-water partition coefficient are shown in Table 4. The P value of magnolol is 10918.41 ± 304.16, and lgP is 4.04 ± 0.01, indicating that magnolol has good liposolubility.

[0089] Table 4 Oil-water partition coefficient of magnolol at 37°C (n = 3)

[0090] Solvent <![CDATA[C o (mg / mL)]]> <![CDATA[C w (mg / mL)]]> P lgP Water 219.11±10.92 0.02±0.0005 10918.41±304.16 4.04±0.01

[0091] 2.3 Study on the Compatibility of Excipients of Magnolol Nanoparticles

[0092] 2.3.1 Screening of the Types of Stabilizers

[0093] The results are as Figure 1 shown. When no stabilizer was used, within 24 h, the particle size of the prepared nanoparticles increased from 544.8 nm to 938.6 nm, and the PDI increased from 0.705 to 0.964; when P188 was selected as the stabilizer, within 24 h, the particle size of the prepared nanoparticles increased from 346.9 nm to 613.9 nm, and the PDI increased from 0.237 to 0.337; when HPMC was selected as the stabilizer, within 24 h, the particle size of the prepared nanoparticles increased from 163.3 nm to 258.4 nm, and the PDI increased from 0.211 to 0.266; when SDS was selected as the stabilizer, within 24 h, the particle size of the prepared nanoparticles increased from 403.7 nm to 775.1 nm, and the PDI increased from 0.513 to 0.681; when PVP-K30 was selected as the stabilizer, within 24 h, the particle size of the prepared nanoparticles was below 200 nm and the PDI was less than 0.2, showing the best stability. Therefore, PVP-K30 was selected as the stabilizer for magnolol nanoparticles.

[0094] 3.3.2 Screening of the Concentration of Stabilizer

[0095] The results are as Figure 2 shown. When the dosage of PVP-K30 was 0.05%, the particle size of the prepared nanoparticles increased from 165.9 nm to 257 nm, and the PDI increased from 0.161 to 0.256; when the dosage of PVP-K30 was 0.2%, the particle size of the prepared nanoparticles increased from 203.8 nm to 289.8 nm, and the PDI increased from 0.192 to 0.288; when the dosage of PVP-K30 was 0.1%, the particle size of the prepared nanoparticles increased from 117.8 nm to 170.3 nm, and the PDI increased from 0.132 to 0.136, showing the best stability. Therefore, 0.1% was selected as the concentration of the stabilizer.

[0096] 3.4 Optimization of the Prescription and Process

[0097] 3.4.1 Investigation of the Stirring Speed

[0098] The results are as Figure 3As shown (the solid line represents the particle size and the dashed line represents the PDI), when the stirring speed was 500 r / min, the particle size of the prepared nanoparticles increased from 142.8 nm to 223.8 nm, and the PDI increased from 0.169 to 0.197; when the stirring speed was 1000 r / min, the particle size of the prepared nanoparticles increased from 128.4 nm to 191.5 nm, and the PDI increased from 0.16 to 0.182; when the stirring speed was 1500 r / min, the particle size of the prepared nanoparticles increased from 118.1 nm to 174.8 nm, and the PDI increased from 0.126 to 0.127; when the stirring speed was 2000 r / min, the particle size of the prepared nanoparticles increased from 110.3 nm to 172 nm, and the PDI increased from 0.117 to 0.122. When the stirring speed was 2000 r / min, the speed was too fast and caused liquid splashing. Therefore, 1500 r / min was selected as the optimal stirring speed.

[0099] 3.4.2 Investigation of the volume ratio of organic phase to aqueous phase

[0100] The results are as Figure 4 shown (the solid line represents the particle size and the dashed line represents the PDI). When the volume ratio of organic phase to aqueous phase was 1:20, the particle size of the prepared nanoparticles increased from 215.4 nm to 302.7 nm, and the PDI increased from 0.215 to 0.337; when the volume ratio of organic phase to aqueous phase was 1:30, the particle size of the prepared nanoparticles increased from 170.2 nm to 274.6 nm, and the PDI increased from 0.176 to 0.234; when the volume ratio of organic phase to aqueous phase was 1:40, the particle size of the prepared nanoparticles increased from 115.5 nm to 175.2 nm, and the PDI increased from 0.117 to 0.15; when the volume ratio of organic phase to aqueous phase was 1:20, the particle size of the prepared nanoparticles increased from 116 nm to 171.9 nm, and the PDI increased from 0.101 to 0.112. When the volume ratio of organic phase to aqueous phase was 1:40, the particle size of the nanoparticles was less than 200 nm and the PDI was less than 0.2, and the drug concentration was higher. Therefore, 1:40 was selected as the optimal volume ratio of organic phase to aqueous phase.

[0101] 3.4.3 Investigation of drug concentration

[0102] The results are as Figure 5As shown (the solid line represents the particle size and the dashed line represents the PDI), when the drug concentration was 20 mg / mL, the particle size of the prepared nanoparticles increased from 119.5 nm to 168.3 nm, and the PDI increased from 0.129 to 0.17; when the drug concentration was 25 mg / mL, the particle size of the prepared nanoparticles increased from 130.6 nm to 181.7 nm, and the PDI increased from 0.151 to 0.182; when the drug concentration was 30 mg / mL, the particle size of the prepared nanoparticles increased from 169.4 nm to 242.9 nm, and the PDI increased from 0.237 to 0.352. When the drug concentration was 25 mg / mL, the particle size of the nanoparticles was less than 200 nm and the PDI was less than 0.2. Since the drug concentration was higher, 25 mg / mL was selected as the optimal drug concentration.

[0103] In this example, the physicochemical properties of magnolol were systematically studied, and on this basis, the prescription composition and preparation process of magnolol nanoparticles were successfully optimized. First, an in vitro analytical method for magnolol was established by HPLC. The linear relationship between the concentration and the peak area was good in the range of 1 - 400 μg / mL, and the precision and repeatability met the methodological requirements. Secondly, the equilibrium solubility and oil-water partition coefficient of magnolol were determined. The results showed that the solubility of magnolol in water was 11.17 ± 0.19 μg / mL; the solubility in the oil phase was 219.11 ± 10.92 mg / mL, and the solubility in the aqueous phase was 0.002 ± 0.0005 mg / mL, and the lgP was 4.04 ± 0.01, indicating good liposolubility. Further, by investigating the type and concentration of the stabilizer, the stirring speed, the volume ratio of the organic phase to the aqueous phase, and the drug concentration, the optimal prescription and preparation process of magnolol nanoparticles were successfully optimized as follows: the stabilizer was PVP-K30, the stabilizer concentration was 0.1%, the stirring speed was 1500 r / min, the volume ratio of the organic phase to the aqueous phase was 1:40, and the drug concentration was 25 mg / mL. The particle size of the prepared magnolol nanoparticles was less than 200 nm within 7 days and the PDI was less than 0.2, showing good stability.

[0104] Preparation and Characterization of Magnolol-Borneol Nanogel in Example 2

[0105] 1 Experimental Animals

[0106] SPF-grade SD rats, male, 6 rats, weighing 180 - 220 g, provided by Hangzhou Medical College, license number: SCXK(Zhe)2024 - 0002.

[0107] New Zealand rabbits, male, 6 rabbits, weighing 3 kg, provided by Jiangsu Zhenlin Biotechnology Co., Ltd., license number: SCXK(Su)2021 - 0011.

[0108] 2 Experimental Methods

[0109] 2.1 Preparation of Magnolol-Borneol Nanoparticles

[0110] 2.1.1 Preparation of Magnolol-Borneol Nanoparticles with Different Ratios

[0111] Preparation of magnolol-borneol nanoparticle solution (Mag:Bor 1:0.2): Weigh 25 mg of magnolol and 5 mg of borneol respectively, dissolve them in 1 mL of absolute ethanol as the organic phase; weigh 0.1 g of PVP-K30, dissolve it in 100 mL of ultrapure water to prepare a 0.1% PVP-K30 solution as the aqueous phase. Place the aqueous phase on a magnetic stirrer, under the condition of 1500 r / min, and quickly inject the organic phase into the aqueous phase according to the volume ratio of the organic phase to the aqueous phase of 1:40. Finally, remove the absolute ethanol by a rotary evaporator to obtain a nano-suspension. Magnolol nanoparticle solution (Mag NPs), borneol nanoparticle solution (Bor NPs), 1:0.4 magnolol-borneol nanoparticle solution (Mag:Bor 1:0.4), 1:0.5 magnolol-borneol nanoparticle solution (Mag:Bor 1:0.5), 1:0.6 magnolol-borneol nanoparticle solution (Mag:Bor 1:0.6), 1:0.8 magnolol-borneol nanoparticle solution (Mag:Bor 1:0.8), and 1:1 magnolol-borneol nanoparticle solution (Mag:Bor 1:1) can be prepared in the same way.

[0112] 2.1.2 Morphology Investigation of Magnolol-Borneol Nanoparticles

[0113] Preparation of TEM samples: Take 10 μL of the solutions prepared in item "2.1.1" respectively, drop them on a 200-mesh copper grid, after standing for 5 min, suck dry the remaining solution with filter paper, then drop 10 μL of uranyl acetate, stand for 5 min, suck dry the remaining dye with filter paper, and slowly dry it with a baking lamp. Observe the morphology of magnolol-borneol nanoparticles with different ratios through a transmission electron microscope.

[0114] 2.1.3 Determination of the Contents of Magnolol and Borneol in Magnolol-Borneol Nanoparticles with Different Ratios

[0115] Establishment of the standard curve for borneol: The content of borneol was determined by the sulfuric acid-vanillin method. Accurately weigh 6 mg of borneol reference substance, place it in a 10 mL volumetric flask, dissolve it with ultrapure water and make up to the scale line to prepare a 0.6 mg / mL borneol reference stock solution. The prepared borneol stock solution was serially diluted with ultrapure water to prepare 300, 200, 100, 50, 25 μg / mL borneol reference solutions. Weigh 1 g of vanillin and dissolve it in 100 mL of concentrated sulfuric acid to prepare a 10 mg / mL sulfuric acid-vanillin solution. Take 4.5 mL of the 10 mg / mL sulfuric acid-vanillin solution and 0.5 mL of the 300, 200, 100, 50, 25 μg / mL borneol reference solutions in a stoppered test tube, shake well, let stand at room temperature for 10 min, and then measure the absorbance at 470 nm with ultrapure water as the reference preparation. Plot the standard curve of borneol with the borneol concentration as the abscissa and the absorbance value as the ordinate.

[0116] Determination of the content of borneol in nanoparticles: The nano-suspension prepared under "2.1.1" was centrifuged at 15000 r / min for 15 min, the supernatant was discarded, 3.5 mL of ultrapure water was added, the solution was placed in a thermostatic water bath oscillator and shaken for 24 h, then centrifuged at 10000 r / min for 10 min. Take 0.5 mL of the supernatant in a 10 mL stoppered test tube, add 4.5 mL of a 10 mg / mL vanillin sulfuric acid solution, mix well, let stand at room temperature for 10 min, and measure the absorbance of the solution at 470 nm with ultrapure water as the reference, and calculate the content of borneol in the nanoparticles by substituting into the standard curve.

[0117] Determination of the content of magnolol in nanoparticles: Add 17 mL of methanol to the remaining solution, place the solution in a thermostatic water bath oscillator and shake for 24 h, then centrifuged at 10000 r / min for 10 min. According to the chromatographic conditions under "2.1.1", inject the sample for analysis, calculate the content of magnolol in the nanoparticles, and finally calculate the ratio of the content of magnolol to borneol in the magnolol-borneol nanoparticles.

[0118] 2.1.4 UV spectral study of magnolol-borneol nanoparticles

[0119] Take free magnolol (Mag), magnolol nanoparticle solution (Mag NPs), magnolol-borneol nanoparticle (Mag-Bor NPs) solution, and dilute the magnolol concentration to 0.1 mg / mL. Use a UV-visible spectrophotometer to scan in the wavelength range of 250 - 400 nm to obtain the UV absorption spectra of each solution.

[0120] 2.1.5 X-ray diffraction study of magnolol-borneol nanoparticles

[0121] Take magnolol raw drug (Mag), borneol raw drug (Bor), and freeze-dried powder of magnolol-borneol nanoparticles (Mag-Bor NPs), and use an X-ray diffractometer to characterize their crystal structures. Radiation source: Cu-Kα, radiation wavelength: 1.5406 / cm, scanning speed: 5° / min, scanning range: 0 - 40°.

[0122] 2.2 Preparation of magnolol-borneol nanogel

[0123] 2.2.1 Preparation of magnolol-borneol nanogel

[0124] Preparation of magnolol-borneol nanoparticle concentrate: Weigh 250 mg of magnolol and 125 mg of borneol, dissolve them in 10 mL of absolute ethanol as the organic phase; weigh 0.4 g of polyvinylpyrrolidone-K30 and dissolve it in 400 mL of ultrapure water as the aqueous phase. Place the aqueous phase on a magnetic stirrer, and under the condition of 1500 r / min, quickly inject the organic phase into the aqueous phase according to the volume ratio of the organic phase to the aqueous phase of 1:40. Finally, remove the absolute ethanol through a rotary evaporator to obtain a magnolol-borneol nano-suspension. Concentrate the above-prepared magnolol-borneol nano-suspension through an ultrafiltration cup to prepare a magnolol-borneol nanoparticle concentrate.

[0125] Preparation of carbomer gel: Weigh 0.6 g of carbomer 940 and 0.05 g of disodium ethylenediaminetetraacetate dihydrate, add 96.55 g of water, let it stand overnight to fully swell, and gradually add triethanolamine, stir and mix well, adjust the pH to 5.0 to prepare a blank gel (Blank Gel) matrix.

[0126] Preparation of magnolol-borneol nanogel: Weigh the same mass of magnolol-borneol nanoparticle concentrate and blank gel matrix, mix them, and stir evenly to prepare magnolol-borneol nanogel (Mag-Bor NPs Gel).

[0127] 2.2.2 Investigation of the morphology of magnolol-borneol nanogel

[0128] Take appropriate amounts of blank gel matrix and magnolol-borneol nanogel, freeze-dry them, spread them on an aluminum block with conductive glue, sputter gold on the samples, and use a scanning electron microscope to observe the microscopic morphology of Blank Gel and Mag-Bor NPs Gel.

[0129] 2.2.3 Determination of the content of magnolol-borneol nanogel

[0130] Accurately weigh 0.5000 g of Mag-Bor NPs Gel, place it in a 50 mL volumetric flask, dissolve it with methanol, and make up to the mark with methanol to obtain a sample solution. Filter it through a 0.22 μm microporous membrane, and inject the subsequent filtrate under the chromatographic conditions in Example 1 to calculate the content of Mag in Mag-Bor NPs Gel.

[0131] 2.3 Study on the skin permeability of magnolol-borneol nanogel

[0132] 2.3.1 Preparation of magnolol cream

[0133] Preparation of the oil phase: Take magnolol (1.1 g), stearyl alcohol (3 g), cetyl alcohol (3 g), myristyl alcohol (2 g), octyldodecanol (4 g), liquid paraffin (3 g), glyceryl monostearate (2 g), menthol (3 g), and span 60 (1 g) and place them in a dry beaker. Heat them in a water bath at 80 °C until completely melted.

[0134] Preparation of the aqueous phase: Take tween 60 (3.5 g) and distilled water (74.4 g) and place them in another beaker. Heat it to 83 °C.

[0135] Add the aqueous phase to the oil phase, and prepare the cream at a water bath temperature of 80 °C and a rotation speed of 300 r / min. After stirring for 20 min, cool it to room temperature to obtain magnolol cream (Mag Cream).

[0136] 2.3.2 Skin penetration test

[0137] Rats were anesthetized by intraperitoneal injection of sodium pentobarbital at a dose of 35 mg / kg. The abdominal hair of the rats was moistened with normal saline, and the hair was carefully shaved off with a blade, avoiding scratching the skin. After 12 h, the rats were sacrificed with an overdose of anesthetic, the abdominal skin was dissected, the subcutaneous adipose tissue and blood vessels were removed, washed thoroughly with normal saline, and fixed in a diffusion cell with the stratum corneum of the skin facing upwards (the effective area of the diffusion cell is 3.14 cm 2), the volume of the receiving pool was 8 mL. 30% ethanol - normal saline was added to the receiving pool, and it was placed in a transdermal diffusion tester. The stirring speed was set at 300 r / min, the temperature was set at 32 ± 0.5 °C. After equilibration for 1 h, the diffusion cell was gently tapped with an ear syringe to remove the air bubbles in the diffusion cell. 0.3000 g of Mag - Bor NPs Gel and Mag Cream (equivalent to 3.3 mg of Mag) were accurately weighed and evenly coated on the stratum corneum of rats. At 2, 4, 6, 8, 10, 12, and 24 h after drug administration, 0.2 mL of sample was taken from the receiving pool, and then an equal volume of the receiving solution at the same temperature was added to the receiving pool. The taken receiving solution was centrifuged at 10000 r / min for 10 min, and the peak area was determined under the high - performance liquid chromatography conditions in "2.1.1" of Chapter 2. The drug concentration was calculated from the drug concentration - peak area standard curve. The cumulative permeation amount per unit area Q (μg / cm 2 ) was calculated according to the following formula.

[0138]

[0139] where V represents the volume of the receiving pool (mL), A represents the diffusion area of the diffusion cell (cm 2 ), Cn represents the drug concentration at the nth sampling point (μg / mL), Ci is the drug concentration measured at the ith sampling point (μg / mL), and Vi is the sampling volume (0.2 mL). Taking the cumulative permeation amount of drug per unit area Q as the ordinate and time t as the abscissa, the cumulative permeation curve was obtained.

[0140] After the experiment, the skin was taken out from the diffusion cell, and the uncoated part was cut off with scissors. The remaining part was washed with normal saline. The skin was placed in a tissue homogenizer tube, 1 mL of methanol was added, and tissue homogenization was carried out (for 15 min). After homogenization, the homogenate was centrifuged at 10000 r / min for 10 min, and the supernatant was detected under the chromatographic conditions of Example 1.

[0141] 2.4 In vitro safety evaluation of magnolol - borneol nanogel

[0142] 2.4.1 Experimental grouping

[0143] New Zealand rabbits were weighed and numbered, and randomly divided into 2 groups with 3 rabbits in each group, namely the Blank Gel group and the Mag - Bor NPs Gel group. The animals were adaptively fed for 3 days before the experiment.

[0144] 2.4.2 Drug administration

[0145] Remove the hair on both sides of the spine of the experimental animals 12 h before the experiment. The depilated area is 3 cm × 3 cm on each side, left and right. After 12 h, select white rabbits with healthy and intact skin for the test sample experiment. Apply the drug on the intact skin of the depilated area on one side of the body surface of the experimental animal, and use the other side as the self-control. Apply the drug on the dressing. The drug dose is 5 mg / kg. Stick the dressing on the back of the rabbit, and wrap the outer circle with a medical bandage. After 4 h, wash the drug administration site with normal saline. Observe and record erythema, edema, whether there is pigmentation, bleeding points, skin roughness or skin thinning, etc. at the drug application site 1 h after removing the drug, and score the erythema and edema according to Table 5 and Table 6. Apply once a day for 7 consecutive days. After the administration is completed, sacrifice the rabbit, peel off the skin at the drug administration site, and perform HE staining.

[0146] Table 5 Skin irritation response scoring table

[0147] Stimulation response Score Erythema No erythema 0 Mild erythema (barely visible) 1 Moderate erythema (visibly obvious) 2 Severe erythema 3 Purple erythema to mild eschar formation 4 Edema No edema 0 Mild edema (barely visible) 1 Moderate edema (obviously raised) 2 Severe edema (skin raised 1 mm, clear contour) 3 Serious edema (skin raised more than 1 mm and expanding) 4 Highest total score 8

[0148] Table 6 Skin irritation intensity evaluation standard table

[0149] Integral mean Evaluation 0～0.49 Non-irritant 0.5～2.99 Mild irritant 3.0～5.99 Moderate irritant 6.0～8.0 Strong irritant

[0150] 3 Experimental results

[0151] 3.1 Morphological investigation of magnolol-borneol nanoparticles

[0152] Bor did not form nanoparticles. Mag NPs were spherical. The morphologies of Mag-Bor NPs at different ratios were all spherical, with a particle size of about 200 nm and a uniform distribution.

[0153] 3.2 Determination of magnolol and borneol contents in magnolol-borneol nanoparticles

[0154] 3.2.1 Standard curve of borneol reference substance

[0155] Draw a standard curve with the absorbance (A) of borneol as the ordinate and the concentration (C) as the abscissa. The regression equation is A = 0.0019c + 0.0088, R2 = 0.9999. The results show that borneol has a good linear relationship in the range of 25 - 300 μg / mL. The results are shown in Table 7.

[0156] Table 7 Standard curve of borneol reference substance

[0157] Concentration (μg / mL) 25 50 100 200 300 Absorbance 0.0547 0.1053 0.1963 0.3877 0.575

[0158] 3.2.2 Content determination

[0159] The sulfuric acid-vanillin method was used to determine the content of Bor in the nanoparticles, and the high-performance liquid chromatography method was used to determine the content of Mag in the nanoparticles. Finally, the ratio of Mag to Bor content in the nanoparticles was calculated. The results are shown in Table 8. When the ratio of Mag to Bor in the organic phase reached 1:0.5, the ratio of Mag to Bor content in the nanoparticles remained stable and the Mag content was the highest. Continuing to increase the addition amount of Bor, there was no significant difference in the ratio of Bor to Mag content in the nanoparticles. Therefore, Mag:Bor of 1:0.5 was selected as the preparation condition for the subsequent nanoparticles.

[0160] Table 8 Results of the ratio of magnolol to borneol content in magnolol-borneol nanoparticles

[0161] Mag:Bor ratio Mag content (mg) Bor content (mg) Ratio of Bor to Mag content in nanoparticles 1:0 3.78 1:0.2 4.20 0.27 0.064 1:0.4 4.57 0.38 0.082 1:0.5 5.30 0.53 0.100 1:0.6 5.54 0.54 0.098 1:0.8 5.79 0.59 0.102 1:1 6.09 0.65 0.106

[0162] 3.3 UV spectrum of magnolol-borneol nanoparticles

[0163] The maximum absorption peak of the Mag solution group was at 294 nm, and the absorbance value was 0.6734. The maximum absorption peak of the Mag NPs group was at 284 nm, and the absorbance value was 0.2428. The maximum absorption peak of the Mag-Bor NPs group was at 284 nm, and the absorbance value was 0.3464. Compared with the Mag solution group, the maximum UV absorption peak of the Mag NPs group shifted 10 nm to the left and showed a hypochromic effect, which might be due to the molecules changing from disorder to order after assembly, resulting in a decrease in absorption intensity. Compared with the Mag NPs group, the Mag-Bor NPs group showed a hyperchromic effect, which might be due to the addition of borneol, disturbing the order of the magnolol assembly and increasing the absorption intensity.

[0164] 3.4 XRD of magnolol-borneol nanoparticles

[0165] The results are as Figure 6 shown. Mag had narrow and sharp diffraction peaks at diffraction angles 2θ of 11.3°, 13.09°, 19.66°, 21.68°, 23.08°, and 26.37°. Bor had obvious crystal diffraction peaks at diffraction angles 2θ of 7.17°, 15.94°, and 17.66°. There were no characteristic diffraction peaks in the prepared Mag-Bor NPs, indicating that the two existed in an amorphous form in the nanoparticles.

[0166] 3.5 Morphology investigation of magnolol-borneol nanogels. The Blank Gel showed a three-dimensional network structure. In the structure of Mag-Bor NPsGel, the nanoparticles were evenly distributed in the three-dimensional network, and the particle size was about 200 nm, which was consistent with the TEM results.

[0167] 3.6 Content determination of magnolol-borneol nanogels

[0168] The results are shown in Table 9. The content of Mag in Mag-Bor NPs Gel is 11.09 ± 0.39 mg / g.

[0169] Table 9 Determination Results of the Content of Magnolol in the Gel

[0170] Batch number Content (mg / g) 1 11.06 2 10.73 3 11.50 Mean ± SD 11.09±0.39

[0171] 3.7 Study on the Skin Permeability of Magnolol-Borneol Nanogel

[0172] As Figure 7 shown, the cumulative amount of drug permeated through the skin per unit area of Mag-Bor NPs Gel in 24 h is 412.83 μg / cm 2 , the amount retained in the skin is 3.38 mg / g, the cumulative amount of drug permeated through the skin per unit area of Mag Cream in 24 h is 248.76 μg / cm 2 , the amount retained in the skin is 1.97 mg / g. The cumulative skin permeation amount of Mag-Bor NPs Gel is 1.66 times that of Mag Cream, and the amount retained in the skin is 1.72 times that of Mag Cream. Mag-Bor NPs Gel improves the transdermal absorption of the drug.

[0173] 3.8 In Vitro Safety Evaluation of Magnolol-Borneol Nanogel

[0174] As shown in Table 10, no obvious erythema or edema reaction was observed in the local skin. According to the skin irritation intensity scoring standard, the irritation index of the test results is < 0.5, indicating that the prepared Mag-Bor NPs Gel has no irritation to the skin. After applying Mag-Bor NPs Gel and Blank Gel on the back skin of New Zealand white rabbits for 7 days, the section results showed that the stratum corneum was intact, and collagen fibers, fibroblasts, capillaries, hair follicles, and lymphocytes were clearly visible, with no obvious inflammatory infiltration.

[0175] The results of the skin irritation test showed that no irritation reaction occurred after applying the prepared magnolol-borneol nanogel to the rabbit skin. It indicates that the prepared magnolol-borneol nanogel has no irritation and is safe for topical skin use.

[0176] Table 10 In Vitro Safety Scoring Results of Magnolol-Borneol Nanogel

[0177]

[0178]

[0179] In this example, magnolol-borneol nanogel with safety and high transdermal permeability was successfully prepared. First, the microscopic morphology of magnolol-borneol nanoparticles was characterized by TEM, UV, and XRD. The TEM results showed that magnolol-borneol nanoparticles with different ratios were all spherical, and the particle size was about 200 nm. The ultraviolet spectrum results showed that compared with the magnolol solution group, the maximum ultraviolet absorption peak of the magnolol nanoparticle group shifted 10 nm to the left and showed a hypochromic effect. Compared with the magnolol nanoparticle group, the magnolol-borneol co-assembled nanoparticle group showed a hyperchromic effect. The XRD results showed that magnolol and borneol assembled to form an amorphous co-precipitate. Secondly, magnolol-borneol nanoparticles were mixed with blank gel to prepare magnolol-borneol nanogel, and the morphology of the gel was observed by SEM. The results showed that in the prepared gel, the nanoparticles were evenly distributed in the formed three-dimensional network structure. Further research on the permeability of the nanogel on the abdominal skin of rats showed that the cumulative skin penetration amount of magnolol-borneol nanogel was 1.66 times that of the cream, and the intradermal retention amount was 1.72 times that of the cream group. Magnolol-borneol nanogel improved the transdermal absorption of drugs. Finally, the results of the skin irritation experiment on New Zealand white rabbits showed that the prepared magnolol-borneol nanogel was non-irritating and had good safety.

[0180] Example 4 Pharmacodynamic Study of Magnolol-Borneol Nanogel

[0181] 1 Experimental Animals

[0182] SPF-grade SD rats, male, 24 in number, weighing 180 - 220 g, provided by Hangzhou Medical College, license number: SCXK(Zhe)2019 - 0002.

[0183] 2 Experimental Methods

[0184] 2.1 Establishment of Diabetic Peripheral Neuropathy Model

[0185] 2.1.1 Establishment of Type I Diabetes Model

[0186] After 24 rats were adaptively fed in the experimental animal center for 2 weeks, 6 rats were randomly selected as the normal group, and the remaining 18 rats were used to establish a model of diabetic peripheral neuropathy. The modeling steps were as follows: Prepare a citric acid-sodium citrate buffer solution with a concentration of 0.1 mol / L (pH 4.5). Weigh 0.5 g of STZ and dissolve it in 50 mL of the citric acid-sodium citrate buffer solution to a concentration of 1%. Prepare it on ice, use it immediately after preparation, and store it in the dark. Before modeling, the rats were fasted for 12 h, and then a 1% STZ solution was intraperitoneally injected at a dose of 60 mg / kg (the injection volume was 0.6 mL / 100 g) at one time. The rats in the normal group were intraperitoneally injected with an equal amount of citric acid-sodium citrate buffer solution, and the injection was completed within 10 min. After the injection, comfort the rats, check for bleeding, and disinfect and stop bleeding. Immediately after modeling, provide the rats with sufficient water to prevent them from dying due to hyperglycemic hyperosmolarity. At the same time, change the bedding every day to keep the living environment of the rats clean and tidy. Seven days after the injection, blood was taken from the tail vein of each rat to measure blood glucose. If the blood glucose value was ≥16.7 mmol / L and the "three more and one less" symptoms (polydipsia, polyphagia, polyuria, and weight loss) appeared, it indicated that the type I diabetes model was successfully established.

[0187] 2.1.2 Establishment of the peripheral neuropathy model

[0188] Eighteen type I diabetic rats were continuously fed normally to establish a model of diabetic peripheral neuropathy. Water and feed were provided daily, and the clean bedding was changed. Blood was taken from the tail vein of the rats every two weeks to measure the blood glucose level. Four weeks later, the thermal pain threshold, mechanical pain threshold, and sciatic nerve motor conduction velocity of the rats were measured. The standard for successful modeling: When the sciatic nerve conduction velocity of the diabetic rats slowed down by more than 11%, it was considered that the model of diabetic peripheral neuropathy was successfully established.

[0189] 2.2 Animal grouping and drug administration

[0190] 2.2.1 Animal grouping

[0191] Normal group (NC group), model group (DPN group), magnolol-borneol nanogel group (Mag-Bor NPs Gel group), mecobalamin group (Meco group), with 6 rats in each group.

[0192] 2.2.2 Drug administration

[0193] (1) Meco group: Intragastric administration with 0.17 mg / kg mecobalamin saline solution every day.

[0194] (2) Mag-Bor NPs Gel group: According to the drug administration site of DPN clinical patients, the gel was evenly applied to the bilateral hind limbs of the shaved rats. The treatment dose was 5 mg / kg, once a day, for 6 consecutive weeks.

[0195] 2.3 Detection indexes and methods

[0196] 2.3.1 Blood Glucose and Body Weight Monitoring

[0197] Weigh the rats and measure their fasting blood glucose every two weeks. Disinfect the rat tails with alcohol wipes before and after blood collection. Use a disposable capillary blood collection needle to collect blood from the rat tails, and detect it with the test strips of a SanNuo blood glucose meter. Plot the curves of body weight and blood glucose changes.

[0198] 2.3.2 Determination of the Threshold Time of Photothermal Stimulation Pain in Rat Plantar

[0199] Perform a hot plate experiment on SD rats using an intelligent hot plate tester to determine the latency of the thermal stimulation-induced paw withdrawal response. Six hours before the test, the rats are fasted and water-deprived to avoid the influence of urine on the test results. Before the test, carefully wipe the operation table clean. After drying, place the rats on the operation table and restrain them with an acrylic cover to make them adapt for 15 - 30 minutes to keep the rats in a stable state. Set the temperature in the hot plate to 52°C, and record the time when the rats start to show pain sensations such as licking their paws and jumping. A total of 3 tests are performed, with a 20-minute interval between each test. The thermal paw withdrawal latency is the average of the 3 results. Test before and after drug intervention respectively.

[0200] 2.3.3 Detection of Mechanical Stimulation Paw Withdrawal Threshold

[0201] The mechanical pain threshold is measured using the Von Frey filament method. Before the experiment starts, first place the rats on a special metal grid for a period of time to adapt. When the rats are adapted to the environment and no longer move around randomly, use Von Frey filaments of different specifications to stimulate the plantar surface for testing. When the stimulation reaches the threshold, the rats will respond by flicking their paws. At the beginning of the test, start with a 2.0 g Von Frey filament. Gently press the filament on the plantar surface of the hind paw, apply enough force to bend the filament, and keep it in contact for 1 - 2 seconds. Select the size of the next filament according to the response to the previous filament. If no withdrawal response is observed, choose a higher-level filament; if a withdrawal response occurs, choose a lower-level filament. After the rats show a straddling response, continue to stimulate with the filament four more times, and record the value of the last Von Frey filament. Calculate the mechanical pain threshold of the rats according to the following formula. Test before and after drug intervention respectively.

[0202]

[0203] 2.3.4 Determination of the Motor Conduction Velocity of the Sciatic Nerve in Rats

[0204] The in-vivo direct measurement method was used to measure the sciatic nerve conduction velocity of rats. First, the rats were anesthetized with a respiratory anesthesia machine, placed in the prone position and fixed on the operating table, and a heating blanket was placed under them to maintain a constant temperature of 37°C. The leg hair of the rats was shaved off with a razor, and the legs and the surrounding area were fully exposed. Then, using a double electrode single stimulation, a stimulation with a duration of 0.05 ms and an intensity of 3 V was applied, and the instrument automatically recorded the curve. The nerve conduction velocity of the rats was expressed in m / s. The measurements were taken before and after drug intervention respectively.

[0205] 2.4 Animal material collection

[0206] After the end of the drug administration period and the completion of the detection of the pain threshold of plantar photothermal stimulation, the mechanical withdrawal threshold of the rats, and the sciatic nerve conduction velocity, the rats were over-anesthetized and sacrificed. The skin and muscles of the hind limbs were cut open to expose the sciatic nerve, and the sciatic nerve was removed; the spine was cut open to remove the dorsal root ganglion (DRG); the feet of the rats were cut off to remove the skin of the hind paw sole. The tissue specimens were fixed with 4% paraformaldehyde. Dehydration was carried out successively with gradient ethanol (75%, 85%, 95%, 100% ethanol), made transparent with xylene, infiltrated with paraffin wax, and then embedded into wax blocks. The wax blocks were placed on a paraffin slicer for sectioning. The thickness of the sciatic nerve sections was 2 μm, the thickness of the footpad sections was 6 μm, and the thickness of the DRG sections was 6 μm.

[0207] 2.5 Histological staining

[0208] 2.5.1 HE staining

[0209] Deparaffinization and hydration of paraffin sections: The sections were successively placed in xylene I for 20 min, xylene II for 20 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 5% alcohol for 5 min, and washed with tap water.

[0210] Hematoxylin staining: The sections were immersed in hematoxylin staining solution for 3 - 5 min, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with bluing solution, and rinsed with running water.

[0211] Eosin staining: The sections were dehydrated successively in 85% and 95% gradient ethanol for 5 min each, and then stained in eosin staining solution for 5 min.

[0212] Dehydration and mounting: The sections were successively placed in absolute ethanol I for 5 min, absolute ethanol II for 5 min, absolute ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min for transparency, and then mounted with neutral gum.

[0213] Microscopic examination and image acquisition and analysis.

[0214] 2.5.2 Toluidine blue staining

[0215] Deparaffinize the paraffin sections to water: sequentially place the sections in xylene I for 20 min, xylene II for 20 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 95% alcohol for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and wash with tap water.

[0216] Nissl staining: Immerse the sections in the staining solution for 2 - 5 min, wash with water, slightly differentiate with 1% glacial acetic acid, wash with tap water to terminate the reaction, control the degree of differentiation under the microscope, and after washing with tap water, dry the sections in an oven.

[0217] Clear and mount: Immerse the sections in clean xylene for clearing for 15 min, and mount with neutral gum.

[0218] Examine under a microscope and collect and analyze the images.

[0219] 2.5.3 Immunofluorescence staining

[0220] Slice the specimens and stain them overnight at 4°C with PGP9.5 (1:1000), MBP (1:500), and NF-κB (1:500) antibodies respectively, and incubate with the corresponding secondary antibody at 37°C for 1 h. Collect images using a fluorescence microscope and perform quantification with Image J.

[0221] 3 Experimental results

[0222] 3.1 Blood glucose and body weight monitoring

[0223] The rats in the NC group had good mental states and their body weights continuously increased. The diabetic rats showed obvious symptoms of "polyphagia, polydipsia, polyuria, and weight loss", and the average blood glucose exceeded 20 mmol / L, meeting the diabetes modeling criteria. Throughout the experimental period, the diabetic rats maintained the diabetic state of "hyperglycemia and low body weight".

[0224] 3.2 Effects of honokiol-borneol nanogel on the sciatic nerve function of DPN rats

[0225] 3.2.1 Effects of honokiol-borneol nanogel on the pain threshold time of plantar photothermal stimulation in rats

[0226] The results are as Figure 8As shown in the figure, before treatment, the thermal pain threshold (TWL) of rats in each group was measured. The TWL of the NC group was 10.3 s, that of the DPN group was 17 s, that of the Meco group was 16.6 s, and that of the Mag-Bor NPs Gel group was 16.4 s. The latency of the paw withdrawal response to thermal stimulation in the model rats was significantly increased (P < 0.001), indicating that the model of diabetic peripheral neuropathy in rats was successfully established. After drug treatment, the TWL of the NC group was 10.5 s, that of the DPN group was 19.9 s, that of the Meco group was 11.6 s, and that of the Mag-Bor NPs Gel group was 12.5 s. Compared with the DPN group, the TWL of the Meco group was reduced by 0.71 times, and that of the Mag-Bor NPs Gel group was reduced by 0.59 times. The TWL of the Meco group and the Mag-Bor NPs Gel group was significantly reduced (P < 0.01). It shows that Mag-Bor NPs Gel has an improving effect on the TWL of DPN rats.

[0227] 3.2.2 Effect of honokiol-borneol nanogel on the mechanical withdrawal threshold of rats

[0228] The results are as Figure 9 shown in the figure. Before treatment, the mechanical withdrawal threshold (MWT) of rats in each group was measured. The MWT of the NC group was 2.15 g, that of the DPN group was 4.49 g, that of the Meco group was 4.52 g, and that of the Mag-Boe Gel group was 4.48 g. The mechanical withdrawal threshold of the model rats was significantly increased (P < 0.01), indicating that the model of diabetic peripheral neuropathy in rats was successfully established. After drug treatment, the MWT of the NC group was 1.96 g, that of the DPN group was 4.89 g, that of the Meco group was 2.31 g, and that of the Mag-Bor NPs Gel group was 2.58 g. Compared with the DPN group, the MWT of the Meco group was reduced by 1.1 times, and that of the Mag-Bor NPs Gel group was reduced by 0.9 times. The MWT of the Meco group and the Mag-Bor NPs Gel group was significantly reduced (P < 0.001). It shows that Mag-Bor NPs Gel has an improving effect on the MWT of DPN rats.

[0229] 3.2.3 Effect of honokiol-borneol nanogel on the sciatic nerve conduction velocity of rats

[0230] The results are as Figure 10As shown, before treatment, the motor nerve conduction velocity (MNCV) of the sciatic nerve in each group of rats was measured. The MNCV of the NC group was 60.58 m / s, that of the DPN group was 41.70 m / s, that of the Meco group was 42.80 m / s, and that of the Mag-Bor NPs Gel group was 41.47 m / s. The MNCV of the model group rats was significantly decreased (P < 0.01), indicating that the diabetic peripheral neuropathy model in rats was successfully established. After drug treatment, the MNCV of the NC group was 62.71 m / s, that of the DPN group was 39.38 m / s, that of the Meco group was 56.72 m / s, and that of the Mag-Bor NPs Gel group was 53.03 m / s. Compared with the DPN group, the MNCV of the Meco group increased by 0.44 times, and that of the Mag-Bor NPs Gel group increased by 0.35 times. The MNCV of the Meco group and the Mag-Bor NPs Gel group increased significantly (P < 0.001). It is indicated that Mag-Bor NPs Gel has an improving effect on the MNCV of DPN rats.

[0231] 3.3 Pathological and morphological evaluation

[0232] 3.3.1 Improvement of magnolol-borneol nanogel on the morphology of the sciatic nerve

[0233] In the NC group, the nerve fiber bundles of the sciatic nerve in rats were regular in morphology, arranged uniformly, distributed densely and evenly. The myelin sheath surrounding the axons of the nerve was intact and clear, the axons in the myelin sheath were clear, and there was no obvious swelling or atrophy of the axons. In the DPN group, the sciatic nerve fibers in rats were loose, arranged disorderly, distributed unevenly, and there were multiple degenerations and fractures. The space between nerve fibers became larger; adjacent axons atrophied and degenerated; the density distribution of the myelin sheath was uneven, the myelin sheath became thinner, and there were manifestations such as collapse, loss, and vacuolization of the myelin sheath in some nerve fibers. In the Meco group and the Mag-Bor NPs Gel group, the sciatic nerve fibers in rats were relatively more neatly arranged compared with the DPN model group, with vacuoles and demyelination manifestations, but the degree was significantly lighter than that of the DPN model group, which was basically consistent with the results of nerve electrophysiology and others. It is indicated that Mag-Bor NPs Gel can reduce the degree of damage to the sciatic nerve to a certain extent, improve its morphology and protect the function of nerve cell fibers.

[0234] 3.3.2 Improvement of magnolol-borneol nanogel on the morphology of the dorsal root ganglion (DRG)

[0235] In the NC group of rats, the cell gaps in the dorsal root ganglion were arranged densely, compactly, and orderly. There were relatively many ganglion cells, with large and round cell nuclei, clear nucleoli, a large number of dense Nissl bodies in the cell bodies, deep staining, showing large granular or sheet-like shapes. There was a layer of flat cells, namely satellite cells, around the cell bodies, and a large number of nerve fibers could be seen in the cell population. In the DPN group of rats, the cell gaps in the dorsal root ganglion were widened, arranged loosely and disorderly, and a large number of ganglion cells were separated from satellite glial cells. The Nissl bodies in the neuron cell bodies were significantly reduced, the staining became lighter, and the granules became smaller. In the Meco group and the Mag-Bor NPs Gel group of rats, the arrangement of dorsal root ganglion cells was more orderly than that in the model group. Some cell bodies showed atrophy, the structure was relatively complete, the cell nuclei were centered, and no obvious nuclear fragmentation or disappearance was seen. Some ganglion cells were separated from satellite glial cells, indicating that Mag-Bor NPs Gel could reduce the degree of DRG damage to a certain extent.

[0236] 3.4 Immunofluorescence staining

[0237] 3.4.1 Improvement of magnolol-borneol nanogel on the density of intraepidermal nerve fibers (IENF) in DPN rats

[0238] The density of IENF in the hind paws of rats was counted by PGP 9.5 immunofluorescence staining (red fluorescence represents PGP9.5). The results were as Figure 11 shown. Compared with the rats in the NC group, the number of PGP 9.5 positive fibers in the DPN group of rats decreased by 2.6 times; compared with the DPN group of rats. The number of PGP 9.5 positive fibers in the Meco group of rats increased by 1.9 times, and the number of PGP9.5 positive fibers in the Mag-Bor NPs Gel group of rats increased by 1.6 times. The number of PGP 9.5 positive fibers in the Meco group and the Mag-Bor NPs Gel group of rats increased significantly, indicating that Mag-Bor NPs Gel could significantly improve the IENF density in DPN rats.

[0239] 3.4.2 Improvement of magnolol-borneol nanogel on the myelin sheath structure of the sciatic nerve in DPN rats

[0240] The structure of the sciatic nerve myelin sheath was evaluated by staining with myelin basic protein (MBP) (red fluorescence represents MBP). As Figure 12 shown, compared with the rats in the NC group, the expression level of MBP in the DPN group of rats decreased by 1.6 times; compared with the DPN group of rats, the expression level of MBP in the Meco group of rats increased by 1.2 times, and the expression level of MBP in the Mag-Bor NPs Gel group of rats increased by 0.96 times. The expression level of MBP in the Meco group and the Mag-Bor NPs Gel group of rats increased significantly, indicating that Mag-Bor NPs Gel could improve the myelin sheath structure of the sciatic nerve in DPN rats.

[0241] 3.4.3 Improvement of Magnolol-Borneol Nanogel on Inflammation of Dorsal Root Ganglion Tissue in DPN Rats

[0242] It is reported in the literature that NF-κB plays an important role in the process of inflammation of dorsal root ganglion tissue in DPN rats. The severity of inflammation of dorsal root ganglion tissue in rats is evaluated by staining NF-κB (red fluorescence represents NF-κB, green fluorescence represents β-tubulin isotype III, and brown is the superposition of the two). The results are as Figure 13 shown. Compared with the rats in the NC group, the expression level of NF-κB in the DPN group increased by 1.3 times; compared with the rats in the DPN group, the expression level of NF-κB in the Meco group decreased by 0.69 times, and the expression level of NF-κB in the Mag-Bor NPs Gel group decreased by 0.41 times. The expression levels of NF-κB in the Meco group and the Mag-Bor NPs Gel group were significantly reduced, indicating that Mag-Bor NPs Gel can effectively reduce the protein expression level of NF-κB and improve the inflammatory response in the DRG tissue of DPN rats.

[0243] In this example, a diabetic peripheral neuropathy model was successfully established. Magnolol-Borneol nanogel can effectively reduce the inflammatory damage of the dorsal root ganglion and sciatic nerve, and promote nerve repair. The results of behavioral tests showed that after drug treatment, the TWL of the DPN group was 19.9 s, and the TWL of the Mag-Bor NPs Gel group was 12.5 s, a decrease of 0.6 times; the MWT of the DPN group was 4.47 g, and the MWT of the Mag-Bor NPs Gel group was 2.58 g, a decrease of 0.9 times; the MNCV of the DPN group was 39.38 m / s, and the MNCV of the Mag-Bor NPs Gel group was 53.03 m / s, an increase of 0.35 times. HE staining and Nissl staining showed that after treatment with Mag-Bor NPs Gel, the histological morphology of the sciatic nerve and DRG was close to that of normal rats. Immunofluorescence staining showed that compared with the rats in the DPN group, the IENF density of the Mag-Bor NPs Gel rats increased by 1.6 times; compared with the rats in the DPN group, the MBP expression level of the Mag-Bor NPs Gel rats increased by 0.96 times, protecting the myelin sheath structure of the sciatic nerve in DPN rats; compared with the rats in the DPN group, the expression level of NF-κB in the Mag-Bor NPs Gel rats decreased by 0.41 times, inhibiting the inflammation of DRG tissue. In summary, the research in this chapter proves that Mag-Bor NPs Gel can improve the morphology of the sciatic nerve and DRG, repair damaged nerves, and is of great significance for the treatment of diabetic peripheral neuropathy.

Claims

1. A honokiol and borneol nanogel, characterized in that, It is prepared by the following method: (1) Weigh magnolol and borneol, dissolve them in absolute ethanol as the organic phase; weigh the stabilizer and dissolve it in ultrapure water as the aqueous phase; place the aqueous phase on a magnetic stirrer, and quickly inject the organic phase into the aqueous phase according to a certain ratio. Finally, remove the absolute ethanol through a rotary evaporator to obtain a magnolol-borneol nano-suspension, and concentrate it through an ultrafiltration cup to prepare a magnolol-borneol nanoparticle concentrate; (2) Preparation of carbomer gel: Weigh carbomer 940 and disodium ethylenediaminetetraacetate dihydrate, add water to make it fully swell, dropwise add triethanolamine, stir and mix evenly, adjust the pH to prepare a blank gel matrix; (3) Preparation of magnolol-borneol nanogel: Weigh the same mass of magnolol-borneol nanoparticle concentrate and the blank gel matrix, mix and stir evenly to prepare magnolol-borneol nanogel.

2. The magnolol and borneol nanogel according to claim 1, wherein (1) Weigh magnolol and borneol with a mass ratio of 1:0.2 to 1:1, dissolve them in absolute ethanol as the organic phase; weigh the stabilizer polyvinylpyrrolidone-K30 and dissolve it in ultrapure water as the aqueous phase. Place the aqueous phase on a magnetic stirrer and quickly inject the organic phase into the aqueous phase. Finally, remove the absolute ethanol through a rotary evaporator to obtain a magnolol-borneol nano-suspension; concentrate the prepared magnolol-borneol nano-suspension through an ultrafiltration cup to prepare a magnolol-borneol nanoparticle concentrate.

3. The magnolol and borneol nanogel according to claim 2, characterized in that, The concentration of the stabilizer polyvinylpyrrolidone-K30 is 0.05-2%, the stirring speed of the magnetic stirrer is 1500-2000 r / min, the volume ratio of the organic phase to the aqueous phase is 1:20-1:50, the mass ratio of magnolol and borneol is 1:0.5, and the concentration of magnolol and borneol is 20-30 mg / mL.

4. The magnolol and borneol nanogel according to claim 3, characterized in that, The concentration of the stabilizer polyvinylpyrrolidone-K30 is 0.1%, the stirring speed of the magnetic stirrer is 1500 r / min, the volume ratio of the organic phase to the aqueous phase is 1:40, the mass ratio of magnolol and borneol is 1:0.5-1:1, and the concentration of magnolol and borneol is 25 mg / mL.

5. Use of the magnolol and borneol nanogel according to any one of claims 1 to 4 in the preparation of a drug for treating diabetic peripheral neuropathy.

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