Traditional Chinese medicine composition for treating beriberi

Abstract

The invention belongs to the technical field of traditional Chinese medicine preparations, and relates to a traditional Chinese medicine composition for treating beriberi. The invention discloses a traditional Chinese medicine composition for treating beriberi, which is a compound nano-particle spray and comprises compound nano-particles prepared from folium artemisiae argyi, dandelion, oriental wormwood, motherwort, rhizoma nardostachyos, radix angelicae extract and lactic acid-glycolic acid copolymer (PLGA) in specific parts by weight. The traditional Chinese medicine composition can effectively inhibit and kill trichophyton rubrum and destroy a fungal biofilm so as to achieve the effect of treating beriberi.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a traditional Chinese medicine composition for treating tinea pedis. BACKGROUND

[0002] Tinea pedis (athlete's foot) is a common skin infection caused by pathogenic fungi such as Trichophyton rubrum, which is characterized by easy recurrence and easy formation of biofilm, resulting in poor therapeutic effect of traditional drugs. At present, chemical antifungal drugs such as terbinafine are mainly used clinically, but long-term use is easy to produce drug resistance, and the penetration and clearance ability of the fungi biofilm is limited. Although there is a long history of treating tinea pedis with traditional Chinese medicine, ordinary traditional Chinese medicine preparations have problems such as poor permeability of active ingredients and low bioavailability. Although there are some traditional Chinese medicine compounds in the prior art for treating tinea pedis, the curative effect is unstable, and the treatment problem related to fungal biofilm cannot be effectively solved. Therefore, it is of great significance to develop a traditional Chinese medicine composition which can effectively solve the above problems. SUMMARY

[0003] The application aims to provide a traditional Chinese medicine compound nanoparticle spray for treating tinea pedis, which can effectively inhibit and kill Trichophyton rubrum and destroy fungal biofilm, and has good safety. To achieve the above-mentioned purpose, the application provides a traditional Chinese medicine composition for treating tinea pedis, which is a compound nanoparticle spray and comprises a compound nanoparticle and a pharmaceutically acceptable excipient. The compound nanoparticle is prepared from a compound herbal extract and poly (lactic-co-glycolic acid) (PLGA); the compound herbal extract is a total extract prepared by extracting volatile oil from raw materials in the following proportions by weight: 1-5 parts of Artemisia argyi, 3-7 parts of Taraxacum mongolicum, 3-7 parts of Herba Artemisiae Scopariae, 5-9 parts of Leonurus japonicus, 1-5 parts of Nardostachys jatamansi, and 1-5 parts of Angelica dahurica, and then refluxing the residues with an ethanol aqueous solution to combine the obtained volatile oil and ethanol extract.

[0004] Preferably, the average particle size of the compound nanoparticle is 100-200 nm. Preferably, the polydispersity index (PDI) of the compound nanoparticle is 0.1-0.2. Preferably, the mass ratio of the compound herbal extract to PLGA is 1-2:1.

[0005] Preferably, the weight percentage of the compound nanoparticle in the spray is 1.5%-5.5%; and the pH value of the spray is 5.5.

[0006] The application has the following beneficial effects: 1. By combining the leaves of Artemisia argyi, dandelion, capillary, motherwort, sweet pine needles and Bai Zhi according to the specific weight ratio of the present application, and nano-encapsulating, it is determined by experiments that the MIC value is 15.6-17.2 μg / mL, and the MFC value is 31.3-34.4 μg / mL, and the experimental comparison shows that the antibacterial and bactericidal activity of the compound nanoparticle is stronger than that of single component or part of the component combination.

[0007] 2. Experiments show that the inhibition rate of the composition of the present application on Trichophyton rubrum biofilm can reach more than 80%, indicating that it can effectively destroy the fungus bio.

[0008] 3. The composition of the present application has no obvious irritation and is suitable for long-term use.

[0009] 4. In a preliminary clinical case observation, the total effective rate is 91%, the fungal clearance rate is 93%, and it shows good treatment prospect, and the effective rate is higher than that of the positive control drug terbinafine.

[0010] 5. In the form of nanoparticle spray, the drug is uniformly dispersed, has strong penetration and is convenient to use, and is suitable for refractory sites such as interdigital spaces. DETAILED DESCRIPTION

[0011] The following examples are intended to further illustrate the present application, but not in any way limit the scope of the present application. Unless otherwise specified, the methods described in the present application all use conventional experimental methods in the art, and the reagents and materials used can be obtained by commercial means.

[0012] The water vapor distillation method described in the present application can be carried out according to the conventional techniques in the art, and the preferred conditions include: crushing the medicinal materials to pass through a 20-60 mesh sieve, adding 6-10 times the amount of water, soaking at room temperature for 20-60 minutes, then heating and distilling, controlling the distillation speed to be 1-4 drops per second, and collecting the distillate until the volatile oil no longer increases. The volatile oil can be collected by conventional methods such as ether extraction or using a volatile oil detector.

[0013] Raw materials and equipment Raw materials: leaves of Artemisia argyi, dandelion, capillary, motherwort, sweet pine needles, Bai Zhi, lactic acid-hydroxyacetic acid copolymer (PLGA), polyvinyl alcohol (PVA-1788), azone, menthol, hydroxypropyl methyl cellulose (HPMC), glycerol, anhydrous ethanol (pharmaceutical grade), citric acid, sodium citrate, ethylenediaminetetraacetic acid disodium (EDTA-2Na), mannitol, purified water.

[0014] Equipment: multifunctional traditional Chinese medicine extraction and concentration machine set, rotary evaporator, ultrafiltration device, magnetic stirrer, high-speed refrigerated centrifuge, freeze dryer, high-speed shearing disperser, liquid preparation tank, 0.22 μm microporous filter membrane filtration device, quantitative spray bottle filling line.

[0015] Example 1 1) Preparation of the compound herbal extract: Weigh 1 part of Artemisia argyi, 3 parts of Taraxacum mongolicum, 3 parts of Herba scurfi, 5 parts of Motherwort, 1 part of Gansong leaf, and 1 part of Bai Zhi, and mix them together and then crush them. First, extract the mixture by steam distillation for 6 hours, collect the volatile oil part, and reserve it. Then, reflux extract the residue with 10 times the amount of 70% ethanol water solution for 2 times, each for 1.5 hours. Combine the ethanol extract, filter it, and concentrate it under reduced pressure to the extract of relative density 1.20 at 60°C. Mix the obtained extract with the volatile oil, and stir them thoroughly to form a uniform mixture. Thus, the compound herbal extract is obtained, and it is stored at 4°C.

[0016] 2) Preparation of the compound nanoparticles: a. Dissolve PLGA in anhydrous ethanol, and then add the compound herbal extract, and stir them to mix them thoroughly to form a uniform organic phase; b. Dissolve 500 mg of polyvinyl alcohol (PVA-1788) in 100 mL of purified water to form an aqueous phase; c. Under the condition of magnetic stirring at 500 rpm, inject the organic phase into the aqueous phase at a rate of 2 mL / min, and continue to stir for 45 minutes after the injection is completed to obtain a nanoparticle suspension; d. Purify and concentrate the nanoparticle suspension by ultrafiltration through a 100 kDa ultrafiltration membrane to remove ethanol and free components; e. Add 5% (w / v) mannitol to the concentrated nanoparticle suspension, and then pre-freeze and freeze-dry it to obtain the compound nanoparticle lyophilized powder.

[0017] The average particle size thereof is 158.3 nm, and the polydispersity index (PDI) thereof is 0.1.

[0018] 3) Preparation of the spray: Prepare the spray according to the following weight percentages: compound nanoparticle lyophilized powder: 1.5%; azone: 1.5%; menthol: 0.5%; hydroxypropyl methylcellulose (HPMC): 1.0%; glycerol: 2.0%; disodium ethylenediaminetetraacetate (EDTA-2Na): 0.05%; anhydrous ethanol: 30%; citric acid / sodium citrate buffer system: appropriate amount, adjust the pH to 5.5; and purified water: add to 100%.

[0019] Preparation method: HPMC was first dispersed in anhydrous ethanol, and after swelling, part of purified water was added and stirred until completely dissolved. Glycerol, azone, menthol, EDTA-2Na and pH buffer were added in turn and stirred uniformly. Under high-speed shearing at 10,000 rpm, the compound nanoparticle freeze-dried powder was slowly added and dispersed uniformly, and then purified water was added to the full amount. Finally, it was filtered through a 0.22 μm microporous filter and filled into a container with a quantitative spray pump to obtain the compound nanoparticle spray for treating beriberi.

[0020] Example 2 1) Preparation of compound herbal extract: Weigh the following ingredients by weight: 3 parts of Artemisia argyi, 5 parts of dandelion, 5 parts of capillary, 7 parts of motherwort, 3 parts of sweet pine needles, and 3 parts of white peony. Mix and crush. First, extract the volatile oil for 6 hours by steam distillation, and collect the volatile oil for later use. The residue is extracted twice with 10 times the amount of 70% ethanol solution by refluxing for 1.5 hours each time. Combine the ethanol extracts, filter, and concentrate under reduced pressure to a relative density of 1.20 at 60°C. Mix the resulting extract with the volatile oil, stir thoroughly to form a uniform mixture, and obtain the total extract of the compound herbal medicine, which is stored at 4°C.

[0021] 2) Preparation of compound nanoparticles: a. Dissolve PLGA in anhydrous ethanol, then add the compound herbal extract, stir to disperse and mix thoroughly, and form a uniform organic phase; b. Dissolve 500 mg of polyvinyl alcohol (PVA-1788) in 100 mL of purified water to form an aqueous phase; c. Under the condition of magnetic stirring at 300 rpm, inject the organic phase into the aqueous phase at a rate of 0.5 mL / min, continue stirring for 30 minutes after injection is complete, and obtain a nanoparticle suspension; d. Purify and concentrate the nanoparticle suspension by ultrafiltration through a 100 kDa ultrafiltration membrane to remove ethanol and free components; e. Add 5% (w / v) mannitol to the concentrated nanoparticle suspension, pre-freeze and freeze-dry to obtain compound nanoparticle freeze-dried powder.

[0022] The average particle size is 200 nm and the PDI is 0.15.

[0023] 3) Preparation of spray: Prepare the spray with the following weight percentages: compound nanoparticle freeze-dried powder: 3.5%; azone: 2.5%; Menthol: 0.5%; Hydroxypropyl methylcellulose (HPMC): 2.0%; Glycerin: 2.0%; Disodium EDTA-2Na: 0.05%; Anhydrous ethanol: 30%; Citric acid / sodium citrate buffer system: appropriate amount, adjust pH to 5.5; Purified water: add to 100%.

[0024] Preparation method: First, disperse HPMC in anhydrous ethanol, and after swelling, add some purified water and stir until completely dissolved. Then, add glycerol, azone, menthol, EDTA-2Na, and pH buffer sequentially, and stir until homogeneous. Under high-speed shearing at 10000 rpm, slowly add the compound nanoparticle lyophilized powder, disperse evenly, and then add purified water to the final volume. Finally, filter through a 0.22 μm microporous membrane and fill into a container equipped with a metered-dose spray pump to obtain the compound nanoparticle spray for treating athlete's foot.

[0025] Example 3 1) Preparation of Compound Herbal Extract: Weigh out 5 parts by weight of Artemisia argyi, 7 parts by weight of Taraxacum mongolicum, 7 parts by weight of Artemisia capillaris, 9 parts by weight of Leonurus japonicus, 5 parts by weight of Nardostachys jatamansi, and 5 parts by weight of Angelica dahurica. Mix and pulverize. First, extract by steam distillation for 6 hours, collecting the volatile oil fraction for later use. Reflux the residue twice with 10 times the volume of 70% ethanol aqueous solution, 1.5 hours each time. Combine the ethanol extracts, filter, and concentrate under reduced pressure to an extract with a relative density of 1.20 at 60℃. Mix the obtained extract with the aforementioned volatile oil, stirring thoroughly until a homogeneous mixture is formed, thus obtaining the total compound herbal extract, which is stored at 4℃.

[0026] 2) Preparation of compound nanoparticles: a. Dissolve PLGA in anhydrous ethanol, then add the compound herbal extract, and stir to fully disperse and mix it to form a homogeneous organic phase; b. Dissolve 500 mg of polyvinyl alcohol (PVA-1788) in 100 mL of purified water to form an aqueous phase; c. Under magnetic stirring at 800 rpm, the organic phase is injected into the aqueous phase at a rate of 5 mL / min. After the injection is completed, stirring is continued for 60 minutes to obtain a nanoparticle suspension. d. The nanoparticle suspension is purified and concentrated by ultrafiltration through a 100 kDa ultrafiltration membrane to remove ethanol and free components; e. Add 5% (w / v) mannitol to the concentrated nanoparticle suspension, and then pre-freeze and freeze-dry to obtain compound nanoparticle lyophilized powder.

[0027] The average particle size was measured to be 100 nm, and the PDI was 0.2.

[0028] 3) Preparation of the spray: The spray is prepared according to the following weight percentage: compound nanoparticle lyophilized powder: 5.5%; azone: 3.5%; menthol: 0.5%; hydroxypropyl methylcellulose (HPMC): 3.0%; glycerol: 2.0%; ethylenediaminetetraacetic acid disodium salt (EDTA-2Na): 0.05%; anhydrous ethanol: 30%; citric acid / sodium citrate buffer system: appropriate amount, adjust pH to 5.5; purified water: add to 100%.

[0029] Preparation method: first, disperse HPMC in anhydrous ethanol, swell, then add part of purified water and stir until completely dissolved. Add glycerol, azone, menthol, EDTA-2Na and pH buffer in turn, and stir uniformly. Under high-speed shearing at 10,000 rpm, slowly add the compound nanoparticle lyophilized powder, disperse uniformly, then add purified water to the full amount. Finally, filter through a 0.22 μm microporous filter membrane, fill into a container with a quantitative spray pump, and the compound nanoparticle spray for treating beriberi is obtained.

[0030] Comparative Example According to the methods described in the preparation of compound herbal extract and the preparation of compound nanoparticles in Example 1, the corresponding nanoparticle lyophilized powder of each group was prepared. Except for the herbal components and proportions, other process parameters were completely consistent. The specific parameters are shown in Table 1 below.

[0031] Table 1: Compound ratio design of comparative example (by weight) Experimental Example 1: Evaluation of Antifungal Activity 1) Experimental materials and methods: Test samples: The following nanoparticle lyophilized powders were prepared according to the method of Example 1 and uniformly prepared into 10 mg / mL suspensions for use: Group 1: prepared by Example 1; Group 2: prepared by Example 2; Group 3: prepared by Example 3; Groups 4-7: prepared by Comparative Examples 1-4, respectively; Groups 8-10: prepared by Comparative Examples 5-7, respectively; Group 11: prepared by Comparative Example 8; Group 12: terbinafine solution (1 mg / mL).

[0032] Experimental strain: clinically isolated Trichophyton rubrum.

[0033] Experimental method: According to the M38-A2 scheme of the American Clinical and Laboratory Standards Association (CLSI), the micro-droplet dilution method was used. After the test samples were diluted by 2 times, the standardized fungal spore suspension was inoculated, and after 7 days of culture at 28℃, the minimum concentration without fungal growth was observed by naked eye as the minimum inhibitory concentration (MIC). The liquid in the well without sterile growth was transferred to a fresh sabouraud glucose agar plate, and after culture, the minimum concentration without fungal growth was the minimum fungicidal concentration (MFC). All experiments were repeated three times independently.

[0034] 2) Experimental results: Table 2 In vitro antifungal activity of each group against Trichophyton rubrum The experimental results show that the three embodiments provided by the present application all show significant and bacteriostatic and bactericidal activity against Trichophyton rubrum, with MIC values in the range of 15.6-17.2 μg / mL and MFC values in the range of 31.3-34.4 μg / mL. The activity is higher than that of any single component, which confirms the necessity of the combination of the six medicines claimed in the present application in the present proportion range and the excellent antifungal effect brought by it.

[0035] Experimental example 2 Anti-biofilm experiment The biofilm formed by Trichophyton rubrum is a key factor leading to the persistence of tinea pedis and drug resistance. Traditional antifungal drugs (such as terbinafine) have significant effects on planktonic bacteria, but often lack the ability to penetrate and clear mature biofilms.

[0036] 1) Experimental materials and methods: Test sample: The same as the in vitro antifungal experiment, the nanoparticle suspension (10 mg / mL) of each group.

[0037] Strain: Clinically isolated Trichophyton rubrum.

[0038] Reagent: RPMI-1640 culture medium, XTT detection kit, crystal violet dye.

[0039] Experimental method: a. The Trichophyton rubrum spore suspension was inoculated in a 96-well cell culture plate at a concentration of 1×10 6 CFU / mL, 200 μL per well. It was placed at 28℃ and incubated for 48-72 hours to form mature biofilm at the bottom of the well. The supernatant was discarded, and PBS was gently washed twice to remove unattached planktonic bacteria.

[0040] b.Each test sample was diluted to 16.0 μg / mL with RPMI-1640 medium. 200 μL of the corresponding drug solution was added to the wells in which the biofilm had been formed. The medium without drug was used as a blank control. Six replicates were set up for each group.

[0041] c.The culture plates with the added drug were further incubated at 28°C for 48 hours.

[0042] d.The metabolic activity of the biofilm was detected by XTT method. After the incubation, the drug solution was discarded and the wells were washed with PBS. 150 μL of a mixture containing XTT and phenazine methosulfate (PMS) was added to each well, and the mixture was incubated at 37°C in the dark for 2-3 hours. The absorbance (OD value) of each well was measured at 490 nm by using a microplate reader. The biofilm inhibition rate (%) was calculated according to the formula: [1 - (OD 给药组 / OD 空白对照组 )] x 100%.

[0043] 2) Experimental results: Table 3 Inhibition rate of each group on Trichophyton rubrum biofilm The experimental results show that the three compound example groups (groups 1-3) all exhibit an inhibition rate of more than 80% on Trichophyton rubrum biofilm. This shows that the compound compatibility and nanometer preparation technology used in the present application can effectively overcome the barrier of fungal biofilm and play an important role in addressing this key difficulty in the treatment of tinea pedis.

[0044] Experimental Example 3 Safety evaluation experiment 1) Experimental materials and methods: Test sample: compound nanoparticle spray of the present application: Example 1 formula; blank matrix spray: nanoparticle without drug.

[0045] Experimental animals: healthy white mice were used for skin irritation test and skin allergy test.

[0046] a. Acute skin irritation test: reference to the "Cosmetic Safety Technical Specification". Six mice were selected, and the back was depilated 24 hours before administration. The left depilated area was sprayed with the test sample (the present application), and the right depilated area was sprayed with the blank matrix. Each administration was 0.5 mL, covered with gauze, and fixed. After 24 hours, the test sample was removed with warm water, and the skin erythema and edema reaction was observed and recorded at 1, 24, 48, and 72 hours after removal, and scored.

[0047] b.Skin allergy test: 30 mice were selected and randomly divided into test product group, blank matrix control group and positive control group (1% 2,4-dinitrochlorobenzene). The mice were depilated on the interscapular region, 10 mice in each group. The test product group was applied with 0.5 mL of the spray of the application, and the control group was applied with the corresponding substance. After covering and fixing for 6 hours, the skin was removed, and the induction was continuously performed for 3 days, and then rested for 2 weeks. After the rest, the mice were depilated on the new area of the abdomen which was not tested, and 0.5 mL of the corresponding test product or control product was applied. After covering and fixing for 6 hours, the skin was removed, and the skin allergy reaction was observed and scored at 24 and 48 hours after removal.

[0048] 2) Experimental results: Table 4: Skin acute irritation test score results The experimental results show that the average integral mean of the compound nanoparticle spray of the application on the intact skin of mice is <0.5, and according to the judgment standard of the specification, the composition of the application belongs to non-irritating.

[0049] Table 5: Skin allergy test reaction The sensitization rate of the compound nanoparticle spray of the application is 0%, while the sensitization rate of the positive control group is as high as 90%, and the modeling is successful. According to the judgment standard of the specification, the composition of the application belongs to non-sensitizing.

[0050] Experimental Example 4: Preliminary clinical treatment case observation 1) Experimental method: Subjects: 200 outpatients diagnosed with interdigital athlete's foot were recruited and randomly divided into two groups, 100 people in each group: Treatment group: The compound nanoparticle spray of the application was used.

[0051] Positive control group: 1% terbinafine spray was used.

[0052] Treatment plan: The patients in both groups sprayed the affected area and the surrounding skin once a day in the morning and evening, and the treatment was continuously performed for 4 weeks. After treatment, the patients were followed up for 2 weeks, and the recurrence was observed.

[0053] Evaluation index: Table 6: Evaluation index 2) Experimental results: Table 7: Clinical efficacy evaluation results (at the end of 4 weeks of treatment) The experimental results show that after 4 weeks of treatment, the total effective rate of the treatment group using the compound nanoparticle spray of the application is 91.0%, and the total effective rate of the control group is 77.0%. It shows that the overall efficacy of the composition of the application in the clinical treatment of interdigital type beriberi is better than that of the control group.

[0054] Mycological clearance: At the end of treatment, 93 cases were cleared in the treatment group, with a clearance rate of 93.0%, and 83 cases were cleared in the control group, with a clearance rate of 83.0%.

[0055] Recurrence rate: At the 6th week of follow-up, 91 cases in the treatment group and 77 cases in the control group were followed up. 2 cases in the treatment group relapsed, with a recurrence rate of 2.2%; 3 cases in the control group relapsed, with a recurrence rate of 3.9%.

[0056] Safety: 2 patients in the treatment group complained of mild and transient cooling sensation at the initial stage of medication, which disappeared without treatment, and there was no other adverse reaction. 1 patient in the control group reported mild local dryness, which also relieved itself.

[0057] In summary, the compound nanoparticle spray of the application shows good efficacy in preliminary clinical observation, and the total effective rate and fungal clearance rate are better than those of the positive control drug terbinafine, and the safety is good. We speculate that terbinafine is difficult to effectively penetrate and clear the fungal biofilm that causes infection to persist, and the compound nanoparticles of the application can effectively destroy the biofilm by virtue of its small size and multi-component synergistic effect, thereby more thoroughly clearing the fungus, which may be the reason for achieving a higher fungal clearance rate in clinical observation.

[0058] The above examples are only used to understand the method of the application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the application, the application can be improved and modified in several ways, and these improvements and modifications will also fall within the scope of protection of the claims of the application.

Claims

1. A traditional Chinese medicine composition for treating athlete's foot, characterized in that, The compound nanoparticle spray contains compound nanoparticles and pharmaceutically acceptable adjuvants; the compound nanoparticles are prepared from compound herbal extracts and lactic acid-glycolic acid copolymer (PLGA); the compound herbal extracts are total extracts prepared from raw materials in the following proportions by weight: 1-5 parts of Artemisia argyi, 3-7 parts of Taraxacum mongolicum, 3-7 parts of Herba scurfi, 5-9 parts of Leonurus japonicus, 1-5 parts of Nardostachys chinensis, and 1-5 parts of Angelica dahurica, by steam distillation to extract volatile oil, and then by reflux extraction of the residue with an ethanol aqueous solution, and by combining the obtained volatile oil and ethanol extract.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The average particle size of the compound nanoparticles is 100-200 nm, and the polydispersity index (PDI) is 0.1-0.

2.

3. The traditional Chinese medicine composition according to claim 1, characterized in that, The mass ratio of the compound herbal extracts to PLGA is 1-2:

1.

4. The traditional Chinese medicine composition according to claim 1, characterized in that, The weight percentage of the compound nanoparticles in the spray is 1.5%-5.5%, and the pH value of the spray is 5.5.

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