CN111514203A

CN111514203A - Application of garden burnet charcoal in preventing and treating tinea corporis, tinea cruris, tinea manus and tinea pedis

Info

Publication number: CN111514203A
Application number: CN202010570186.XA
Authority: CN
Inventors: 顾琦; 梁莹; 陈军; 顾薇; 苏曼; 王昊
Original assignee: Nanjing University of Chinese Medicine
Current assignee: Nanjing University of Chinese Medicine
Priority date: 2020-06-21
Filing date: 2020-06-21
Publication date: 2020-08-11

Abstract

The invention provides application of garden burnet charcoal in preventing and treating tinea corporis, tinea cruris, tinea manus and tinea pedis, and application of garden burnet charcoal in preparation of medicines or daily necessities. Simultaneously provides the application of one or more components of gallic acid, pyrogallic acid, gallic acid methyl ester, gallic acid ethyl ester and protocatechuic aldehyde in preventing and treating tinea corporis and cruris, tinea manus and pedis, and the application in the preparation of medicines or daily necessities. The present invention shows that the sanguisorba root charcoal can obviously inhibit the growth of tinea corporis, tinea cruris, tinea manus and tinea pedis, and the effect of the sanguisorba root charcoal is equivalent to that of common traditional Chinese medicines of radix sophorae flavescentis and golden larch bark for treating tinea. After the sanguisorba officinalis charcoal is administrated, the growth of trichophyton rubrum is obviously inhibited by ocular observation. Clinical curative effect observation shows that the cure rate of patients after 4 weeks of treatment by 0.5 percent sanguisorba officinalis charcoal gel reaches 96.15 percent, and half of patients are cured. Meanwhile, the main components of the garden burnet, namely gallic acid, pyrogallic acid, gallic acid methyl ester, gallic acid ethyl ester and protocatechuic aldehyde, have remarkable anti-trichophyton rubrum effects, and the contents are not reported.

Description

Application of garden burnet charcoal in preventing and treating tinea corporis, tinea cruris, tinea manus and tinea pedis

Technical Field

The invention belongs to the field of external application of traditional Chinese medicines, and particularly relates to application of carbonized sanguisorba root in prevention and treatment of tinea corporis, tinea cruris, tinea manus and tinea pedis.

Background

The pathogenic fungi of tinea corporis, cruris, tinea manuum and tinea pedis are dermatophytes which are both characterized by being cutiphilic. About more than 40 kinds of dermatophytes are available, and about more than 20 kinds of dermatophytes can cause diseases in human beings or animals. The domestic circulation and regulation data of various regions and different hospitals show that pathogenic bacteria of tinea corporis, tinea cruris, tinea manus and pedis are approximately the same, the most common is trichophyton rubrum which is the main pathogenic bacteria, and the other pathogenic bacteria comprise trichophyton mentagrophytes, microsporum canis, microsporum gypseum and the like. Dermatophytes can be transmitted from person to person, animal to person, pollutant to person, and various parts of the human body. The physique and environmental factors of patients play a certain role in the pathogenesis, such as the damp-heat area and the high-temperature season are the causes of tinea corporis, tinea cruris, tinea manuum and tinea pedis, the patients wear airtight and over-tight clothes, and the obese and hyperhidrosis patients or drivers are prone to suffer from femoral diseases; the feed is used for raising pets, diabetics, close contact infected persons and pollutants, and patients suffering from tinea manus, tinea pedis or onychomycosis are infected by other fungal infections at other parts and are susceptible to tinea corporis.

The clinical treatment of tinea corporis, cruris, tinea manus and pedis generally adopts local external administration, however, patients who apply glucocorticoid and immunosuppressant locally or systemically for a long time can make skin lesions atypical and form 'indiscernible tinea'. ("Chinese guide for diagnosis and treatment of tinea corporis and tinea cruris" 2018 revised edition)

The radix Sangusorbae is dried root of Sanguisorbae officinalis L. or Sanguisorbae officinalis L. var. longifolia (Bert.) Kuet Li, which is commonly called "sanguisorbae officinalis". The charred radix Sangusorbae is processed by charring method, and has brown surface, brown interior, charred fragrance, and bitter taste. Sanguisorba root, charred and slightly cold in nature, bitter, sour and astringent in flavor, enters liver and large intestine meridians. Has effects in cooling blood, stopping bleeding, removing toxic substance, and healing sore, and can be used for treating hematochezia, hemorrhoidal bleeding, bloody dysentery, metrorrhagia, scald due to hot water and fire, carbuncle, swelling, and sore. The current drug effect reports of the sanguisorba officinalis charcoal are mainly for hemostasis and burn and scald treatment (in the 'Chinese pharmacopoeia' 2015 edition), the report on the antibacterial effect is few, the report on the tinea corporis and cruris pathogenic bacteria such as trichophyton rubrum and the like is not found, the report on the prevention or treatment of the tinea corporis and cruris is not found, and the description of the type is not found in ancient books.

Disclosure of Invention

The invention provides application of garden burnet root charcoal in preventing and treating tinea corporis, tinea cruris, tinea manus and tinea pedis.

Simultaneously provides the application of the garden burnet root charcoal in preparing the medicaments or daily necessities for preventing and treating tinea corporis, tinea cruris, tinea manus and pedis.

The radix Sangusorbae charcoal is radix Sangusorbae charcoal powder or extract.

As a further improvement, the extraction solvent of the garden burnet root charcoal extract is an ethanol water solution.

As a further improvement, the extraction solvent of the garden burnet root charcoal extract is 75% ethanol extract. The invention also provides application of one or more components of gallic acid, pyrogallic acid, gallic acid methyl ester, gallic acid ethyl ester and protocatechuic aldehyde in preventing and treating tinea corporis, tinea cruris and tinea manus and pedis.

And one or more components of gallic acid, pyrogallic acid, gallic acid methyl ester, gallic acid ethyl ester and protocatechuic aldehyde in the preparation of medicines or daily necessities for preventing and treating tinea corporis, tinea cruris, tinea manus and pedis.

Advantageous effects

The invention shows that the sanguisorba officinalis charcoal extract can obviously inhibit the growth of trichophyton rubrum (ATCC MYA-4438), the minimum inhibitory concentration MIC of the sanguisorba officinalis charcoal extract reaches 64 mu g/ml, the effect of the sanguisorba officinalis charcoal extract is equivalent to that of common traditional Chinese medicines of radix sophorae flavescentis and golden larch bark for tinea corporis and tinea cruris and tinea manus and pedis, the sanguisorba officinalis charcoal extract is superior to that of crude medicinal materials of rheum officinale, clove, common cnidium fruit, radix stemonae, scutellaria baicalensis, fructus kochiae, male. The flora shape of the sanguisorba officinalis after administration is observed by 10 times of an ocular lens and 40 times of an objective lens, hypha separation becomes fuzzy after administration of sanguisorba officinalis charcoal (0.5 × MIC), the medicine influences the growth of hypha, and the hypha is hardly separated after administration of sanguisorba officinalis charcoal (1 × MIC), so that the growth is obviously inhibited. Then the sanguisorba officinalis charcoal gel with the content of 0.5 percent is administrated to patients for clinical observation, and the clinical curative effect shows that the cure rate of the patients reaches 96.15 percent after the sanguisorba officinalis charcoal gel is used for treating for 4 weeks, wherein half of the patients are cured.

Drawings

The invention will be further explained with reference to the drawings, in which:

FIG. 1 is a figure of charred sanguisorba officinalis prepared by processing sanguisorba officinalis

FIG. 2 shows total ion flow, A-anion mode and B-cation mode of radix Sangusorbae charcoal extract

FIG. 3 the relative content distribution of the components in the extract of Garden Burnet root charcoal

FIG. 4 microscopic observation results of administration of carbonized sanguisorba root to Trichophyton rubrum, wherein A: normal growth control wells; b: after administration of sanguisorba officinalis charcoal (0.5 × MIC); c: after administration of Garden Burnet root charcoal (1 × MIC)

Detailed Description

The technical solution of the present invention is further described below with reference to specific examples, which are not intended to limit the technical solution.

Example 1 examination of the Activity of Garden Burnet root charcoal against Trichophyton rubrum

The method comprises measuring the inhibitory effect of Garden Burnet root charcoal on Trichophyton rubrum by micro liquid-based dilution method, comparing with various common antibacterial traditional Chinese medicines such as fructus Cnidii, Kochiae fructus, spina Gleditsiae, cortex pseudolaricis, cortex Dictamni Radicis, and radix Sophorae Flavescentis, and comparing with garden burnet root which is the original medicinal material of garden burnet root charcoal, wherein the shape change before and after processing is shown in figure 1.

1. Bacterial strains

Trichophyton rubrum ATCC MYA-4438 was purchased from American Type Culture Collection (ATCC).

2. Preparation of samples to be tested

Respectively taking 10g of medicinal powder of fructus cnidii, fructus kochiae, spina gleditsiae, cortex pseudolaricis, cortex dictamni, radix sophorae flavescentis, radix sanguisorbae and carbonized sanguisorba root, adding 10 times of ethanol aqueous solution, soaking at 50 ℃ overnight, carrying out ultrasonic treatment at 50 ℃ for 1h, filtering, carrying out rotary evaporation drying at 50 ℃ until no liquid flows, and drying in an oven at the same temperature for 48h to obtain a sample to be detected.

Wherein the ethanol aqueous solution is preferably 75% ethanol solution.

3. Activation of bacterial strains

The frozen trichophyton rubrum glycerin tube is streaked and inoculated to a PDA plate, and the PDA plate is cultured for 6 days under the conditions of 35 ℃ and 80% of humidity.

4. Inoculation preparation

The plate was covered with 5ml of 0.9% NaCl solution, and the surface of the plate was gently scraped with an applicator to obtain a spore suspension. After diluting the suspension 100-fold, counting was performed using a microscope and a red blood cell counter. The counting results were as follows: 47, 57, 44, 54. The concentration of the stostespores is about 505,000 spores/ml. Mu.l of this culture was added to 250ml of a test medium (RPMI 1640 supplemented with 0.165M MOPS, pH 7.0) to obtain an inoculum.

5. Compound assay plate preparation

On the day of experiment, a sample to be detected is firstly dissolved in DMSO and then prepared into a mother solution of 51.2 mg/ml; the control Fluconazole was dissolved in DMSO and then prepared into 12.8mg/ml mother liquor, wherein the DMSO concentration was controlled at 0.05%.

1) Preparation of compound master

Compounds were diluted 11 times in 2-fold gradients in 96-V well plates to give 100-fold working solution: 51.2, 25.6, 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05, 0 mg/ml.

Fluconazole performed 11 2-fold gradient dilutions in 96-V well plates to give 100-fold working solution: 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625, 0 mg/ml.

2) Compound daughter board preparation

Transfer 2. mu.l of compound working solution from the compound master plate to a 96-U well plate to which a medium was added to obtain a compound detection plate. Three in parallel.

6. Compound assay plate inoculation

198. mu.l of each fungal inoculum was added to the compound assay plate to obtain the MIC assay plate.

MIC detection

And (3) placing the MIC detection plate under the conditions of 35 ℃ and 80% humidity, and reading the MIC value after standing and culturing for 4 days. MIC (minimal Inhibitory concentration) is the minimum Inhibitory concentration, and refers to the minimum drug concentration that can inhibit the growth of pathogenic bacteria in a culture medium after 18 to 24 hours of in vitro bacteria culture, and MIC is an index for measuring the antibacterial activity of antibacterial drugs. MIC read criteria are referenced in table 1 and results are shown in table 2.

TABLE 1 MIC judgement of antibiotics for test fungi (cf. CLSI M38)

The reference MIC value for CLSI to Trichophyton rubrum ATCC MYA-4438 was 0.5-4. mu.g/ml (4 days of culture).

TABLE 2 determination of Minimal Inhibitory Concentration (MIC) of extracts of herbs on Trichophyton rubrum

The results show that the sanguisorba charcoal has obvious inhibiting effect on trichophyton rubrum, and has the effect equivalent to that of common traditional Chinese medicines of radix sophorae flavescentis and golden larch bark for preventing and treating tinea corporis, tinea cruris and tinea manus and pedis, and the radix sophorae flavescentis and the golden larch bark are developed into traditional Chinese medicine preparations for preventing and treating tinea corporis, tinea cruris and tinea manus and pedis as monarch medicines. The bacteriostatic effect of the garden burnet root charcoal is superior to that of other clinically common bacteriostatic traditional Chinese medicines of common cnidium fruit, broom cypress fruit, spina gleditsiae and cortex dictamni, and is also superior to that of the medicinal material garden burnet root before processing, and the bacteriostatic effect of the garden burnet root charcoal is not reported.

Example 2 analysis of the composition of Garden Burnet root charcoal extract

In example 1, the garden burnet root charcoal extract was prepared under item "2" and subjected to compositional analysis by hplc-ms under the following analysis conditions:

chromatographic conditions are as follows: gradient elution was performed using an Agilent Poroshell 120EC-C18(2.1 mm. times.50 mm,2.7 μm) column at 30 ℃ and acetonitrile as mobile phase A and water (containing 0.1% formic acid) as mobile phase B as in Table 3 at a flow rate of 0.30mL/min and a sample size of 1 μ L.

TABLE 3 procedure for mobile phase gradient elution

Primary mass spectrum conditions: an electrospray ionization (ESI) positive and negative ion mode is adopted, the temperature of dry gas is 300 ℃, the pressure of atomizing gas is 241.3kPa, the outlet voltage of a capillary tube is 100V, the flow rate of dry gas is 3L/min, the voltage of the capillary tube is 3.5k V, and the mass scanning range m/z is 100-600.

Secondary mass spectrum conditions: the method adopts a multi-reaction monitoring (MRM) mode, the capillary voltage is 5500V (positive ion mode), -4500V (negative ion mode), the desolventizing temperature is 550 ℃, the air curtain pressure is 241.3kPa, the pressure of an atomizer and a drying gas is 379.2kPa, the purity of nitrogen used by an ion source is more than or equal to 95%, and the purity of a collision gas is more than or equal to 99.999.

The experimental results are as follows: the total ion flow diagram under positive and negative ions of the garden burnet root charcoal extract is shown in figure 2. 140 chemical component databases are established by collecting relevant documents at home and abroad and databases at home and abroad. According to the high-resolution relative molecular mass of the compound, secondary mass spectrum fragment information and references, HPLC-Q/TOF-MS result analysis shows that 54 components are obtained by identifying the garden burnet root charcoal, 20 gallic acid compounds, 9 ellagic acid compounds, 7 catechin compounds, 10 triterpenoid saponins and aglycon compounds thereof and 8 other compounds, the relative content distribution of which is shown in figure 3, and it can be seen that 4 main components in the garden burnet root charcoal are respectively gallic acid, ellagic acid compounds, catechin compounds, triterpenoid saponins and aglycon compounds thereof; the content of gallic acid is highest, and the identified contents include gallic acid, methyl gallate, methoxy methyl gallate-5-O-sulfate, galloyl-methyl glucoside, and ethyl gallate. Ellagic acid and gallic acid are both hydrolyzed tannin, and relatively high content of ellagic acid, 3,4' -O-dimethyl ellagic acid, and galloylellagic acid.

Example 4 mechanism study of Garden Burnet root charcoal against Trichophyton rubrum

1. Research on substance basis of sanguisorba charcoal for resisting trichophyton rubrum

The anti-trichophyton rubrum effect of the garden burnet root charcoal active ingredients gallic acid, pyrogallic acid, methyl gallate, ethyl gallate, catechin, ellagic acid, ursolic acid and protocatechuic aldehyde was determined by a trace liquid-based dilution method, the sample preparation and the determination method were the same as example 1, and the experimental results are shown in table 4.

TABLE 4 minimum inhibitory concentration of sanguisorba officinalis active ingredient against Trichophyton rubrum

The results show that the main substance basis of the sanguisorba officinalis charcoal for resisting trichophyton rubrum can be gallic acid, pyrogallic acid, methyl gallate, ethyl gallate and protocatechuic aldehyde; the content of 3 ingredients of pyrogallic acid, methyl gallate and protocatechuic aldehyde in the garden burnet root charcoal is higher than that in the garden burnet root, which is probably an inherent mechanism for enhancing the antibacterial effect after the garden burnet root is processed.

2. Microscopic observation of administration of trichophyton rubrum by charred sanguisorba officinalis

The 1 × MIC and 0.5 × MIC wells of the 96-well plate completed with the test were photographed 10 times with an eyepiece and 40 times with an objective lens. The results are shown in FIG. 4, where A is a control well for normal growth, and hyphae are clearly separated, and after administration of Garden burnet root charcoal (0.5 × MIC), as shown in B, hyphae separation begins to become fuzzy, with a tendency to fuse, and the drug influences the hyphae growth. Charred sanguisorba officinalis was administered at MIC (panel C), hyphae were hardly separated and growth was significantly inhibited.

Example 5 Observation of therapeutic Effect of Garden Burnet root charcoal on treating tinea pedis

According to the revised edition of 'Chinese guidelines for diagnosing and treating tinea corporis and tinea cruris' 2018, the aim of treating tinea corporis and tinea cruris is to eliminate pathogenic bacteria, quickly relieve symptoms, eliminate skin lesions and prevent relapse. The evaluation criteria of the curative effect are that the erythema, pimple and scale are completely removed, the fungus microscopic examination and/or culture is negative, and the recovery can be considered. After the skin lesion of some patients is removed, temporary pigmentation can be left on the local part.

1. Preparation of sanguisorba officinalis charcoal gel

Carbomer 940 gel 1g was sprinkled in 100mL water overnight and triethanolamine adjusted the gel pH to 5. Taking 0.5g of dried powder of 75% ethanol extract of sanguisorba officinalis charcoal (the preparation method is shown in item 2 in example 1), redissolving the dried powder by using 75% ethanol until no precipitate exists, adding the redissolved powder into 100g of prepared gel (carbomer 940), uniformly stirring, carrying out rotary evaporation by using a rotary evaporator until no alcohol smell exists, supplementing the gel to 100g with water, and uniformly mixing to obtain the sanguisorba officinalis charcoal gel with the drug content of 0.5%.

2. General data

52 patients with tinea pedis, 23 men and 29 women, age 18-48 years, mean (26.32 +/-8.13) years and mean course (28.75 +/-21.62) months are selected.

3. Method of treatment

The sanguisorba officinalis charcoal gel is applied to the affected part for external use. The use method comprises the following steps: taking appropriate amount of Garden Burnet root charcoal gel before sleeping at night every day, directly applying on affected part skin, and continuously administering for 4 weeks.

4. Criteria for efficacy assessment

And (3) healing: clinical symptoms and physical symptoms disappear, and the direct microscopic examination result of the fungi is negative;

the effect is shown: the subjective symptoms and the skin damage condition are improved, and the direct microscopic examination result of the fungi is negative;

improvement: the subjective symptom and the skin damage are improved by 30 to 70 percent, and the direct microscopic examination result of the fungi is negative or positive;

and (4) invalidation: the subjective symptoms and the skin damage are not improved or aggravated, and the direct microscopic examination and culture results of the fungi are positive.

The apparent cure rate is (number of cure cases + number of effective cases + number of improvement cases)/total number of cases × 100%.

5. Results of clinical effects

The clinical curative effect results are shown in Table 5, and it can be seen that the cure rate reaches 96.15% after the sanguisorba officinalis charcoal gel is used for treating for 4 weeks, and half of patients are cured.

TABLE 5 clinical curative effect (example) of patients

Claims

1. The sanguisorba officinalis charcoal is applied to prevention and treatment of tinea corporis, tinea cruris, tinea manus and tinea pedis.

2. The garden burnet root charcoal is applied to the preparation of medicaments or daily necessities for preventing and treating tinea corporis, tinea cruris, tinea manus and pedis.

3. A medicine or daily necessities capable of preventing and treating tinea corporis, tinea cruris, tinea manus and tinea pedis is characterized in that: the prescription contains charred radix Sangusorbae.

4. The medicament or daily item according to claim 3, wherein: the radix Sangusorbae charcoal is radix Sangusorbae charcoal powder or extract.

5. The medicament or daily item according to claim 4, wherein: the extraction solvent of the garden burnet root charcoal extract is ethanol water solution.

6. The medicament or daily item according to claim 5, wherein: the extraction solvent of the garden burnet root charcoal extract is a 75% ethanol extract.

7. Application of one or more of gallic acid, pyrogallic acid, gallic acid methyl ester, gallic acid ethyl ester, and protocatechuic aldehyde in preventing and treating tinea corporis, tinea cruris, tinea manus, and tinea pedis.

8. A medicine or daily necessities capable of preventing and treating tinea corporis, tinea cruris, tinea manus and tinea pedis is characterized in that: the prescription contains one or more of gallic acid, pyrogallic acid, gallic acid methyl ester, gallic acid ethyl ester, and protocatechuic aldehyde.