CN104173868A - Compounded traditional Chinese medicinal rana chensinensis extract preparation and application thereof in preparation of medicine for treating and preventing dermatophytosis - Google Patents

Abstract

The invention relates to a compounded traditional Chinese medicinal rana chensinensis extract preparation and an application thereof in preparation of a medicine for treating and preventing dermatophytosis, and belongs to the technical field of traditional Chinese medicine preparations. The preparation method of the preparation comprises the following steps: removing impurities and dust from dried rana chensinensis and then smashing, adding a compound enzyme hydrolysate and centrifuging to obtain a supernate at an upper layer; grinding the rest of mixtures, then shearing and carrying out spray drying to form powder, adding the supper micropowder into an acetic acid and carrying out ultrasonic treatment, then filtering the micropowder with a gauze and centrifuging to obtain a supernate, and carrying out reduced pressure concentration on the supernate so as to obtain a mixture of polysaccharides, antibacterial peptides and calcium ions; mixing the mixtures so as to obtain a mixed rana chensinensis extracting liquid; fully mixing the mixed rana chensinensis extracting liquid with a compounded medicine extracting liquid so as to obtain the compounded traditional Chinese medicinal rana chensinensis extract preparation. The obtained preparation is a traditional Chinese medicinal preparation without any side effects, wherein short peptide collagens contained in the preparation can favorably enter normal cells under the action of polysaccharides of a fungus plant growing from the head part of an old tea tree with parasitical calanthe japonica blume so as to supply sufficient nutrition to the cells and restore the injured cells, and the antibacterial peptides can be combined with the calcium ions so as to enhance the damage of the short peptide collagens to the cytoderm of fungal cells, so that the effective bactericidal components of traditional Chinese medicines can enter the fungal cells to reach a bactericidal effect.

Description

Medicine composite preparation and the purposes in the medicine for the treatment of and prevention beriberi thereof in a kind of wood frog extraction

Technical field

The invention belongs to Chinese materia medica preparation technique field, be specifically related to medicine composite preparation and the purposes in preparation treatment and prevention beriberi thereof in a kind of wood frog extraction.

Background technology

Beriberi is a kind of extremely common dermatosis.In adult, 70-80% people suffers from beriberi, just weight difference.Be everlasting and increase the weight of summer, alleviate winter, also has people not heal all the year round.Pathogenic bacterium polyphyly trichophyton and epidermophyton kind, main bacteria seed has trichophyton, trichophyton gypseum, acrothesium floccosum, trichophyton meginii.The main cause that beriberi is difficult for curing is removed these funguses to be had outside stronger drug resistance, and in therapeutic process, moistening, the anaerobic environment at sufferer place is again the strong hotbed of its residue fungus; Patient body contains fungus simultaneously, and when external stops administration, the fungus in body can continue breeding, has caused the outbreak repeatedly of beriberi, is difficult to radical cure.

At present, domestic Rana sylvatica Le conte skin antibacterial peptide is studied, the patent of declaring is also increasing, take wood frog as key word, retrieve domestic and international patent, do not find the external patent about wood frog, CN1216768A in domestic patent " biological antibacterial peptide and preparation method thereof " relates to a kind of wood frog frog skin biological antibacterial peptide with broad spectrum antibiotic activity and preparation method thereof; CN1583166A " Chinese forest frog bactericidal peptide spray agent and preparation method thereof ", CN1583165A " Wood frog antibiotic peptides gel and preparation method thereof " relate to protect wood frog antibacterial skin peptide for clinical skin infection, empyrosis, to infect field with spray and two kinds of forms of gel; CN1679632A " aqueous extracts of analgesic from woodfrog and preparation method thereof and preparation method thereof " is a kind of aqueous extracts of analgesic from woodfrog and technology of preparing thereof with analgesic activity of extracting from Rana sylvatica Le conte skin about protection.Be all to take Rana sylvatica Le conte skin as raw material, extracted Rana sylvatica Le conte skin antibacterial peptide.CN103073628A preparation method that utilizes ultrasound wave to extract antibacterial peptide from forest frog body is to take forest frog body as raw material employing supersound extraction antibacterial peptide, the antibacterial peptide extracting is that molecular weight is that 4KDa optimum pH is 3.5, the basic polypeptide that isoelectric point, IP pI value is 8.0 1 10.5.Above patent all be take antibacterial peptide as protection content, its effect at antibiosis of application protection, described antibacterial refer to opposing antibacterial and fungus comprise the gram positive bacterias such as the gram negative bacterias such as escherichia coli, bacillus pyocyaneus and staphylococcus aureus and clinical drug-resistant bacterium klebsiella spp, Acinetobacter bauamnnii, Candida albicans, saccharomycetic one or more; And antibacterial therapy approach is topical administration.CN103432058A " Rana chensinensis collagen liquid and preparation technology thereof " is formulated with Rana sylvatica Le conte skin extract, Radix Panacis Quinquefolii Saponin, Fructus Schisandrae Chinensis leachate, Aloe extract and water; CN102492760A " a kind of method of utilizing compound enzyme to produce active Rana dybowskii collagen oligopeptide " is that to take dry Rana dybowskii skin be raw material; CN102229972A " method of preparing collagen peptides from tree frog skin " be take Rana sylvatica Le conte skin as raw material, and above three patents are all to take Rana sylvatica Le conte skin to extract contained collagen protein in Rana sylvatica Le conte skin as raw material or primary raw material.

Summary of the invention

One of object of the present invention is to provide a kind of wood frog and extracts medicine composite preparation in mixing, and it is prepared by following steps:

(1) monarch drug wood frog is extracted the preparation of mixed liquor

To after the dried wood frog remove impurity dedusting of 40～55 weight portions, through 20～100 orders, pulverize respectively, make the particle diameter of dried wood frog ground product below 500 μ m, according to the ratio of 1g:1～10mL, add combinative enzyme hydrolysis liquid, in combinative enzyme hydrolysis liquid, contain papain 1～5wt%, trypsin 2～4wt%, flavor protease 1～3wt%, hydrolysis temperature is 50～55 ℃, enzymolysis time is 1～2h, enzymolysis pH is 7.0～7.5, then 85～90 ℃, 5～15min enzyme denaturing, mixture is centrifugal when 10～30 ℃ of the temperature, centrifugation time is 1～5min, centrifugal rotational speed is 2000～4000rpm, the upper strata supernatant of extracting centrifugal liquid volume 20～40%, now in the supernatant of upper strata, contain dried wood frog small peptide collagen protein (due to the effect of centrifugal force, short chain collagen protein will concentrate in the supernatant of upper strata, long-chain protein molecular will be concentrated in the middle lower floor remaining mixture of mixed liquor), its molecular weight is 2～3KDa, isoelectric point, IP pI value is 7.5～7.8, basic amino acid (arginine wherein, lysine, histidine) account for 11.04～12.89% (mass ratioes) of upper strata supernatant, acidic amino acid (aspartic acid, glutamic acid) account for 17.99～19.13% (mass ratioes) of upper strata supernatant.

Get remaining mixture after supernatant (original volume 60～80%), put into colloid mill, grind 2～15min, make the mean diameter of mixture below 300 μ m; Mixture is put into high-shearing dispersion emulsifying machine, shear 1～15min, make mixture mean diameter between 100～200 μ m, mixture is spray dried to Powdered, now obtain the superfine powder that dried wood frog extracts the materials such as residual fiber after small peptide collagen protein, sclerotin;

It is in 2～10% acetic acid that the superfine powder of the materials such as fiber, sclerotin is joined to mass concentration, and acetic acid is 10～40mL:1g with the volume mass ratio of superfine powder; Ultrasonic 5～75min, ultrasonic temperature is 4～30 ℃; Through 150 μ m gauzes, filter, again above 150 μ m gauzes not medium to add mass volume ratio be 0.01～0.03mol/L sodium hydroxide of 1g:1～8mL, carry out microwave extraction, microwave power is 500～600W, microwave time 3～9min, filters through 150 μ m gauzes again; Merge filtrate twice, 30～50 ℃, stir 10～30min, mixing speed is 100～200rpm; Filtrate after stirring is centrifugal, and centrifuging temperature is 4～30 ℃, and centrifugation time is 2～40min, and centrifugal rotational speed is 2000～15000rpm; Get supernatant, concentrating under reduced pressure, pressure is 600～700 holders, and temperature is 65～70 ℃, and concentrated volume ratio is 8～40:1; Now obtain the mixture of polysaccharide, antibacterial peptide and calcium ion, wherein the molecular weight of polysaccharide is 1～5KDa; The molecular weight of antibacterial peptide is 1～3KDa, its isoelectric point, IP pI value is 7.5～8.0, and wherein basic amino acid (arginine, lysine, histidine) accounts for 11.28～13.05% (mass ratioes) of the mixture of polysaccharide, antibacterial peptide and calcium ion, acidic amino acid (aspartic acid, glutamic acid) accounts for 10.01～11.47% (mass ratioes) of the mixture of polysaccharide, antibacterial peptide and calcium ion;

The upper strata supernatant that contains small peptide collagen protein is mixed with polysaccharide, antibacterial peptide and calcium ion mixture, thereby obtain wood frog, extract mixed liquor.

(2) preparation of combination drug extracting solution

Take 25～40 weight portion Gray Root Pakistan, 1～3 weight portion Rhizoma Atractylodis, 1～2 weight portion Radix Stemonae, 1～2 weight portion Herba Pogostemonis, soak with ethanol 1～the 10h that adds mass fraction 90～100% after mixing, wherein ethanol and above-mentioned substance quality and volume mass ratio be 1～20mL:1g; 100～300 μ m gauzes filter, and centrifugal 10～20min under 15～30 ℃, 3000～6000rpm goes after precipitation at 85～100 ℃ heating concentrated, and concentrated volume ratio is 24～8:1; Obtain ethanol extract;

Take 1～3 weight portion Galla Chinensis, 1～3 weight portion Radix Sophorae Flavescentis, 1～3 weight portion Herba Portulacae, 1～2 weight portion Fructus Cnidii, 1～3 weight portion Cortex Phellodendri, 1～3 weight portion Folium Perillae, 1～3 weight portion mentha leave, 1～3 weight portion Rhizoma Et Radix Notopterygii, 1～2 weight portion Radix Stephaniae Tetrandrae, 1～3 weight portion Fructus Zanthoxyli, 1～3 weight portion favus skin, 1～2 weight portion Rhizoma Picrorhizae, 1～2 weight portion Fructus Kochiae, 1～3 weight portion Flos Carthami, after mixing, add water soaking 1～24h, wherein water and above-mentioned substance quality and volume mass ratio be 10～25mL:1g, 100～300 μ m gauzes filter, 15～30 ℃, centrifugal 10～20min under 3000～7000rpm, after going precipitation, 85～100 ℃ of heating are concentrated, concentrated volume ratio is 24～8:1, obtain aqueous extract,

Ethanol extract is mixed with aqueous extract, stir 10～30min, mixing speed is 100～400rpm, obtains combination drug extracting solution;

Wood frog is extracted to mixed liquor and fully mix with combination drug extracting solution, obtain medicine composite preparation in wood frog extraction.

In described wood frog extraction, medicine composite preparation can form pharmaceutical preparation with pharmaceutically acceptable carrier or excipient, and described preparation can be the exterior-applied formulations such as spray, aerosol, cream, ointment, gel or wet tissue.

In described wood frog extraction, medicine composite preparation can form pharmaceutical preparation with pharmaceutically acceptable carrier or excipient, and described preparation can be the peroral dosage forms such as oral liquid, granule, suspension, Emulsion.

Two of object of the present invention is to provide the wood frog of a kind for the treatment of and prevention beriberi and extracts the compound ointment of Chinese medicine, and it is prepared by following steps:

(1) preparation of oil phase: get 5～15 weight portion paraffin, 2～5 weight portion glyceryl monostearate, 5～10 weight portion white vaseline, 5～10 weight portion liquid paraffin, 1～5 weight portion span 40,1～5 weight portion polyoxyethylene nonylphenol ether, 1～3 weight portion ethylparaben and be ground, obtain oil phase;

(2) wood frog extract in the preparation of medicine composite preparation ointment: during oil phase described above and the wood frog that made as stated above by 40～55 weight portion dried wood frog are extracted, medicine composite preparation is heated to respectively 70～100 ℃, during wood frog is extracted, medicine composite preparation joins in described oil phase, stir while adding to condensation, through nanometer homogenizer homogeneous 4～7 times, 115～121 ℃ of sterilizing 10～30min; Then Borneolum Syntheticum is heated to 70～100 ℃ of mistakes, 0.45 μ m film, under aseptic condition, mixs homogeneously with above-mentioned sterilizing afterproduct, obtain wood frog and extract the compound ointment of Chinese medicine.

Usage and dosage: external, every day is twice affected part coating sooner or later, and 1～3mL/ time 7 days was 1 course for the treatment of.

Three of object of the present invention is to provide the wood frog of a kind for the treatment of and prevention beriberi and extracts the agent of Chinese medicine combined mist, and it is prepared by following steps:

In extracting, the wood frog being made as stated above by 40～55 weight portion dried wood frog adds successively 1～5 weight portion sodium chloride, 2～4 weight portion PEG in medicine composite preparation ₄₀₀, 1～2 weight portion benzalkonium bromide; Under 100～200rpm, stir 10～25min, add water to 1000mL, 150～200rpm stirs 10～30min, and above-mentioned substance is fully mixed, 115～121 ℃ of sterilizing 10～20min; Autologously be distributed into aerosol bottle (20mL/ bottle) after cooling, must treat and prevent the wood frog of beriberi to extract the agent of Chinese medicine combined mist.

Four of object of the present invention is to provide the wood frog of a kind for the treatment of and prevention beriberi and extracts the compound wet tissue of Chinese medicine, and it is prepared by following steps:

In extracting, the wood frog being made as stated above by 40～55 weight portion dried wood frog adds successively 1～5 weight portion sodium chloride, 2～4 weight portion PEG in medicine composite preparation ₄₀₀, 1～2 weight portion benzalkonium bromide; Under 100～200rpm, stir 10～25min, add water to 1000mL, 150-200rpm stirs 10-30min, and above-mentioned substance is fully mixed, 115～121 ℃ of sterilizing 10～20min; The autologous room temperature that is cooled to, every 50～60mL joins in the aseptic non-woven fabrics independent package bag that is placed with (15～20) cm * (15～20) cm, and the wood frog that seals and obtain prevention beriberi is extracted the compound wet tissue of Chinese medicine.

Five of object of the present invention is to provide the wood frog of a kind for the treatment of and prevention beriberi and extracts the compound oral formulations of Chinese medicine, and it is prepared by following steps:

(1) concentrating under reduced pressure: in the wood frog extraction that 40～55 weight portion dried wood frog are made as stated above, medicine composite preparation distills under reduced pressure, and pressure is 8 * 10 ³～3 * 10 ⁴handkerchief, temperature is 50～85 ℃, cocnentration factor is 1～5:1, obtains concentrated solution;

(2) 1～5 weight portion zinc acetate is slowly joined in concentrated solution, limit edged stirs; Adding the rear adjusting of the NaOH with 0.1mol/L pH is 6.2～6.5, makes its natural subsidence, autologous cool overnight after stirring evenly;

(3) 0.45 μ m membrane filtrations, obtain after supernatant liquid, in clear liquor, add 5～15 weight portion citric acids, 5～25 weight portion Mel, 5～30 weight portion white sugars, under 50～65 ℃, 100～200rpm, stir 10～25min; 0.45 μ m membrane filtration, then add 5～15 weight portion xanthan gum and 5～15 weight portion potassium sorbate in clear liquid, finally by 0.22 μ m filter membrane fine straining;

(4) filling-in and closing bottle by applying cap: fill under aseptic condition, gland sealing;

(5) sterilizing: be heated to 62～65 ℃, keep 30～50min, the autologous room temperature that is cooled to, obtains medical treatment and prevent the wood frog of beriberi to extract the compound oral formulations of Chinese medicine.

Usage and dosage: for oral administration, every day sooner or later twice oral, 20mL/ time 7 days was 1 course for the treatment of.

Good effect of the present invention is: in wood frog extraction, medicine composite preparation is Chinese medicine preparation, and without any side effects, contained small peptide collagen protein can better enter normal cell under the effect of Gray Root Pakistan polysaccharide, gives cell sufficient nutrition, and can repair damaged cell; Antibacterial peptide can be in conjunction with calcium ion, strengthen its destruction to fungal cell wall, make effective sterilization component of the Chinese medicines such as Galla Chinensis, Radix Sophorae Flavescentis, Herba Portulacae, Fructus Cnidii, Cortex Phellodendri, Folium Perillae, mentha leave, Rhizoma Et Radix Notopterygii, Radix Stephaniae Tetrandrae, Fructus Zanthoxyli, Rhizoma Atractylodis, favus skin, Rhizoma Picrorhizae, the Fructus Kochiae, the Radix Stemonae, Flos Carthami, Herba Pogostemonis enter fungal cell and reach bactericidal effect; The effective ingredient of invigorating blood circulation such as Gray Root Pakistan, Flos Carthami can together be repaired damaged cell with wood frog small peptide collagen protein.The present invention is fast to mucocutaneous non-stimulated, sterilization speed, fast, easy to use, the colorless and odorless of lesions position healing, is applicable to different age people and uses.The present invention has protective effect to pedal skin, improves pedal skin defence capability, the sickness rate of prevention beriberi.Toxic and side effects is little, and treating both the principal and secondary aspects of a disease is evident in efficacy, and clinical symptom disappearance is rapid, also can play preventive effect.

Accompanying drawing explanation

Fig. 1: wood frog is extracted mixed liquor contained short chain collagen gel electrophoretogram in supernatant at the middle and upper levels.

Adopt Tricine electrophoresis method to survey small peptide collagen molecules amount, using the Ultra-low molecular weight protein marker of Suo Laibao company as standard, show that contained small peptide collagen molecules amount is about 2～3KDa.

In figure, left side be the marker of standard, i.e. the electrophoretogram of standard molecular weight protein is followed successively by embodiment mono-wood frog extraction mixed liquor contained short chain collagen gel electrophoretogram in supernatant at the middle and upper levels from left to right; In embodiment bis-, wood frog is extracted mixed liquor contained short chain collagen gel electrophoretogram in supernatant at the middle and upper levels; In embodiment tri-, wood frog is extracted mixed liquor contained short chain collagen gel electrophoretogram in supernatant at the middle and upper levels; In embodiment tetra-, wood frog is extracted mixed liquor contained short chain collagen gel electrophoretogram in supernatant at the middle and upper levels; In embodiment five, wood frog is extracted mixed liquor contained short chain collagen gel electrophoretogram in supernatant at the middle and upper levels.

Fig. 2: polysaccharide molecular weight standard curve.

With standard glucosan T1 (molecular weight 1 000), T3 (molecular weight 3 000), T5 (molecular weight 5 000), T10 (molecular weight 10 000), blue glucosan (molecular weight 2 000 000) is standard substance, through Sephadex G ?25 gel columns (100cm * 1.5cm) with ultra-pure water eluting, flow velocity is 1.2mL/min, 254nm detects polysaccharide molecular weight and distributes, and calculates every kind of standard glucosan sugar peak elution volume.The blue glucosan that the molecular weight of usining is 2,000,000 and glucose are determined void volume Vo and the cumulative volume Vt of chromatographic column as standard sugar, Ve is that cumulative volume deducts void volume.Take Ve/Vt as abscissa, and lg (Mw) makes standard curve for vertical coordinate, and the formula that calculates polysaccharide molecular weight is:

lg(Mw)＝(K ₁‐K ₂)Ve/Vt

Mw: polysaccharide molecular weight;

Ve: the sugared peak elution volume of testing sample;

Vo: the void volume of molecular sieve exclusion chromatography post;

Vt: the cumulative volume of molecular sieve exclusion chromatography post;

K ₁, K ₂for constant.

With blue glucosan and different molecular weight glucose standard, make and draw polysaccharide molecular weight standard curve;

Fig. 3: wood frog is extracted the elution curve that the contained polysaccharide of mixed liquor obtains through Sephadex G-25 gel filtration chromatography.

Adopt gel chromatography preparation method to detect, with Ultraviolet Detector under Sephadex G-25 gel filtration chromatography 254nm, measure wood frog and extract mixed liquor elution curve, draw elution volume Ve.By elution volume Ve, bring formula lg (Mw)=(K into ₁?K ₂) Ve/Vt, according to standard glucosan curve (curve shown in Fig. 2), calculate the contained polysaccharide molecular weight Mw of wood frog extraction mixed liquor and be about 1～5 Kda.

Fig. 4: mixing polysaccharide standard RP-HPLC analyzes collection of illustrative plates.

Adopt high effective liquid chromatography for measuring to draw monosaccharide standard curve, take polysaccharide standard sample, add 1mL anhydrous hydrochloric acid-methanol, fill N ₂after tube sealing, 100 ℃ of hydrolysis 16h after-blow dry liquids.Polysaccharide standard sample after methanol solution after drying, adds 2mol/L trifluoroacetic acid 2mL, and 120 ℃ are filled N ₂tube sealing hydrolysis 3h, hydrolyzate is poured in evaporating dish, adds 45 ℃ of water-baths of appropriate dehydrated alcohol to steam except trifluoroacetic acid.PMP pre-column derivatization and processing thereof, get the NaOH solution of 0.5mL PMP reagent and 0.5mL 0.3mol/L in evaporating dish, gets 0.1mL wherein in little centrifuge tube, 70 ℃ of water-bath 30min after dry thing is dissolved.Carry out high performance liquid chromatogram detection.

Efficient liquid phase chromatographic analysis condition

Instrument: Shimadzu LC-14ATvp chromatograph of liquid, attached SPD-10AVD UV-detector

Chromatographic column: DIKMA Inertsil ODS-3 μ m (150 * 4.6 mm)

Detected temperatures: 35 ℃

Detect wavelength: 245nm

Eluent: 82%PBS (0.1M, pH7.0), 18%Acetonitrile (v/v)

Flow velocity: 1.0mL/min

Fig. 5: the RP-HPLC of the contained polysaccharide component of wood frog extracting solution analyzes collection of illustrative plates.

Adopt high-efficient liquid phase technique to analyze its monosaccharide and form situation, utilize the standard specimen of mixing polysaccharide to determine the ratio of monosaccharide in component.Wood frog is extracted the monosaccharide containing in mixed liquor mannose, rhamnose, glucose, galactose, xylose, arabinose, fucose, glucuronic acid and galacturonic acid, rhamnose wherein: mannose: galactose: glucose: xylose: arabinose: the ratio of fucose is about 1:4:6:12:7:5:3 (volume ratio), glucuronic acid in glucuronic acid content: galacturonic acid=1:1 (volume ratio);

Fig. 6: wood frog is extracted contained antibacterial peptide molecular gel electrophoretogram in mixed liquor.

Adopt Tricine electrophoresis method to survey antibacterial peptide molecular weight, using the Ultra-low molecular weight protein marker of Suo Laibao company as standard, show that contained antibacterial peptide molecular weight is about 1～3Kda.

In figure, left side is the marker of standard, and the electrophoretogram of standard molecular weight protein, is followed successively by from left to right wood frog in embodiment mono-and extracts contained antibacterial peptide molecular gel electrophoretogram in mixed liquor; In embodiment bis-, wood frog is extracted contained antibacterial peptide molecular gel electrophoretogram in mixed liquor; In embodiment tri-, wood frog is extracted upper contained antibacterial peptide molecular gel electrophoretogram in mixed liquor; In embodiment tetra-, wood frog is extracted contained antibacterial peptide molecular gel electrophoretogram in mixed liquor; In embodiment five, wood frog is extracted contained antibacterial peptide molecular gel electrophoretogram in mixed liquor.

The specific embodiment

Below in conjunction with embodiment, the invention will be further described.It is the foot of a hill or mountain, Changbai Mountain, China the Northeast that the present invention's dried wood frog used originates from.Rana temporaria chensinensis David (Rana temporaria chensinensis) is commonly called as Rana nigromaculata, Oviductus Ranae, snow Kazakhstan etc., on animal classification, belong to Amphibia, Anura, Ranidae, Rana, it is the peculiar kind at the foot of a hill or mountain, Changbai Mountain, China the Northeast, belong to medicine, eat dual-purpose precious frog kind, and aboundresources.

The preparation that embodiment mono-wood frog is extracted Compound Chinese Herbs for Fatigue-Resistance of Animal

(1) wood frog is extracted the preparation of mixed liquor

Get dried wood frog 40g (Rana temporaria chensinensis David Changbai Mountain subspecies, purchased from large pharmacy, Jilin), after remove impurity dedusting respectively through 20 orders, 50 orders, 100 orders are pulverized, make the particle diameter of dried wood frog ground product below 500 μ m, according to the ratio of 1g:6mL, add combinative enzyme hydrolysis liquid, in combinative enzyme hydrolysis liquid, contain papain 3wt%, trypsin 2wt%, flavor protease 1wt%, hydrolysis temperature is 52 ℃, enzymolysis time is 1.5h, enzymolysis pH is 7.2, then 87 ℃, 10min enzyme denaturing, mixture is centrifugal when 25 ℃ of the temperature, centrifugation time is 3min, centrifugal rotational speed is 2500rpm, get the upper strata supernatant of cumulative volume 25%, now extracted the small peptide collagen protein containing in dried wood frog,

(method of UV spectral analysis analysis of amino acid classification in the contained collagen protein of upper strata supernatant, automatic amino acid analyzer collection mensuration amino acid masses mark) content (accounting for the mass percent of upper strata supernatant) of arginine, lysine, histidine is respectively 3.12%, 7.43%, 0.73%, and the content of aspartic acid, glutamic acid (accounting for the mass percent of upper strata supernatant) is respectively 7.86%, 11.19%.

Get remaining mixture after supernatant (original volume 75%), put into colloid mill, grind 3min, make the mean diameter of mixture below 300 μ m; Mixture is put into high-shearing dispersion emulsifying machine, shear 2min, make mixture mean diameter between 100～200 μ m, mixture is spray dried to Powdered, now obtain the superfine powder that dried wood frog extracts the materials such as residual fiber after small peptide collagen protein, sclerotin;

It is in 4% acetic acid that the superfine powder of the materials such as fiber, sclerotin is joined to mass concentration, and acetic acid is 17:1 with the volume mass of superfine powder than (mL:g).Ultrasonic, ultrasonic time is: 30min, and ultrasonic temperature is 20 ℃; Filter, through 150 μ m gauzes, filter; Above 150 μ m gauzes, medium does not add mass volume ratio (g:mL) for the 0.02mol/L sodium hydroxide of 1:5, carries out microwave extraction, and microwave power is 550W, and microwave time 4min filters through 150 μ m gauzes again; Merge filtrate twice, stir 15min, mixing speed is 150rpm; Filtrate after stirring is centrifugal, and centrifuging temperature is 20 ℃, and centrifugation time is 10min, and centrifugal rotational speed is 5000rpm; Get supernatant, concentrating under reduced pressure, pressure is 600 holders, and temperature is 68 ℃, and concentrated volume ratio is 15:1; Now obtain the mixture (detection of calcium ion adopts EDTA titrimetry to detect) of polysaccharide, antibacterial peptide, calcium ion;

Polysaccharide in mixture (Sephadex G ?25 gel column separated and collected monosaccharide, high effective liquid chromatography for measuring monosaccharide forms) mainly by mannose, rhamnose, glucose, galactose, xylose, arabinose, fucose, glucuronic acid and galacturonic acid, formed, wherein containing monosaccharides rhamnose: mannose: galactose: glucose: xylose: arabinose: fucose is about 1:4:6:12:1:0.5:1 (volume ratio), glucuronic acid in glucuronic acid content: galacturonic acid=1:1 (volume ratio);

(method of UV spectral analysis analysis of amino acid classification in the contained antibacterial peptide of mixture, automatic amino acid analyzer collection mensuration amino acid masses mark) content (accounting for the mass percent of mixture) of arginine, lysine, histidine is respectively 2.73%, 8.16%, 0.69%, and the content of aspartic acid, glutamic acid (accounting for the mass percent of mixture) is respectively 4.47%, 5.98%.

Small peptide collagen protein, polysaccharide, antibacterial peptide, calcium ion are mixed, obtain wood frog and extract mixed liquor 95.6ml, wherein calcium ions 76.5mg (80mg/100ml).

(2) preparation of combination drug extracting solution

Take Gray Root Pakistan 29g, Rhizoma Atractylodis 3g, Radix Stemonae 1g, Herba Pogostemonis 1g, the soak with ethanol 2h that adds the mass fraction 95% of 17 times of volumes (mL:g), 200 μ m gauzes filter, 25 ℃, the centrifugal 15min of 5000rpm, go after precipitation, 98 ℃ of heating are concentrated, and concentrated volume ratio is 15:1, obtains ethanol extract; Take Galla Chinensis 2g, Radix Sophorae Flavescentis 1g, Herba Portulacae 1g, Fructus Cnidii 2g, Cortex Phellodendri 1g, Folium Perillae 1g, mentha leave 2g, Rhizoma Et Radix Notopterygii 1g, Radix Stephaniae Tetrandrae 1g, Fructus Zanthoxyli 2g, favus skin 1g, Rhizoma Picrorhizae 1g, Fructus Kochiae 1g, Flos Carthami 1g, the water soaking 4h that adds 15 times of volumes (mL:g), 200 μ m gauzes filter, 25 ℃, the centrifugal 10min of 5000rpm, go after precipitation, 98 ℃ of heating are concentrated, and concentrated volume ratio is 10:1, obtains aqueous extract; Ethanol extract is mixed with aqueous extract, stir 15min, mixing speed is 150rpm, obtains combination drug extracting solution;

Wood frog is extracted to mixed liquor and fully mix with combination drug extracting solution, obtain medicine composite preparation in wood frog extracting solution, its volume is 175mL.

The preparation that embodiment bis-wood frogs are extracted the compound ointment of Chinese medicine

(1) preparation of medicine composite preparation in wood frog extraction

Take dried wood frog 40g (Rana temporaria chensinensis David Changbai Mountain subspecies, purchased from large pharmacy, Jilin), Gray Root Pakistan 29g (purchased from pharmacy, Chengdu), Galla Chinensis 2g, Radix Sophorae Flavescentis 1g, Herba Portulacae 1g, Fructus Cnidii 2g, Cortex Phellodendri 1g, Folium Perillae 1g, mentha leave 2g, Rhizoma Et Radix Notopterygii 1g, Radix Stephaniae Tetrandrae 1g, Fructus Zanthoxyli 2g, Rhizoma Atractylodis 3g, favus skin 1g, Rhizoma Picrorhizae 1g, Fructus Kochiae 1g, Radix Stemonae 1g, Flos Carthami 1g, Herba Pogostemonis 1g (above 17 taste Chinese medicines are all purchased from large pharmacy, Jilin), is prepared into medicine composite preparation in wood frog extraction according to the method for embodiment mono-, and volume is 175mL.

(2) preparation of oil phase: get paraffin 8g, liquid paraffin 6g, white vaseline 12g, glyceryl monostearate 10g, span 40 9g, polyoxyethylene nonylphenol ether 3g, ethylparaben 1g is ground, and obtains oil phase;

(3) wood frog is extracted the preparation of the compound ointment of Chinese medicine: in the oil phase that step (2) is made and wood frog extraction, medicine composite preparation is heated to respectively 90 ℃, during wood frog is extracted, medicine composite preparation joins in described oil phase, limit edged is stirred to condensation, 115 ℃ of sterilizing 10min, Borneolum Syntheticum 1g is heated to 90 ℃ and crosses film, mix homogeneously under aseptic condition, subpackage.

Embodiment tri-wood frogs are extracted the preparation of Chinese medicine combined mist agent

(1) preparation of medicine composite preparation in wood frog extraction

Get dried wood frog 40g (Rana temporaria chensinensis David Changbai Mountain subspecies, purchased from large pharmacy, Jilin), Gray Root Pakistan 29g (purchased from pharmacy, Chengdu), Galla Chinensis 2g, Radix Sophorae Flavescentis 1g, Herba Portulacae 1g, Fructus Cnidii 2g, Cortex Phellodendri 1g, Folium Perillae 1g, mentha leave 2g, Rhizoma Et Radix Notopterygii 1g, Radix Stephaniae Tetrandrae 1g, Fructus Zanthoxyli 2g, Rhizoma Atractylodis 3g, favus skin 1g, Rhizoma Picrorhizae 1g, Fructus Kochiae 1g, Radix Stemonae 1g, Flos Carthami 1g, Herba Pogostemonis 1g (above 17 taste Chinese medicines are all purchased from large pharmacy, Jilin), is prepared into medicine composite preparation in wood frog extraction according to the method for embodiment mono-, and volume is 175mL.

(2) wood frog is extracted the preparation of Chinese medicine combined mist agent

In extracting, wood frog adds PEG4 in medicine composite preparation ₄₀₀4g, sodium chloride 5g, benzalkonium bromide 1g, mix, and 150rpm stirs, and mixing time is 15min, adds water to 1000mL, and 200rpm stirs 20min, and above-mentioned substance is fully mixed, 121 ℃ of sterilizing 10min.The autologous room temperature that is cooled to, is distributed into aerosol bottle (20mL/ bottle), obtains.

The preparation that embodiment tetra-wood frogs are extracted the compound wet tissue of Chinese medicine

(1) preparation of medicine composite preparation in wood frog extraction

Get dried wood frog 40g (Rana temporaria chensinensis David Changbai Mountain subspecies, purchased from large pharmacy, Jilin), Gray Root Pakistan 29g (purchased from pharmacy, Chengdu), Galla Chinensis 2g, Radix Sophorae Flavescentis 1g, Herba Portulacae 1g, Fructus Cnidii 2g, Cortex Phellodendri 1g, Folium Perillae 1g, mentha leave 2g, Rhizoma Et Radix Notopterygii 1g, Radix Stephaniae Tetrandrae 1g, Fructus Zanthoxyli 2g, Rhizoma Atractylodis 3g, favus skin 1g, Rhizoma Picrorhizae 1g, Fructus Kochiae 1g, Radix Stemonae 1g, Flos Carthami 1g, Herba Pogostemonis 1g (above 17 taste Chinese medicines are all purchased from large pharmacy, Jilin), is prepared into medicine composite preparation in wood frog extraction according to the method for embodiment mono-, and volume is 175mL.

(2) wood frog is extracted the preparation of the compound wet tissue of Chinese medicine

In extracting, wood frog adds PEG in medicine composite preparation ₄₀₀3g, sodium chloride 2g, benzalkonium bromide 1g, mix, and 120rpm stirs, and mixing time is 14min, adds water to 1000mL, and 180rpm stirs 19min, and above-mentioned substance is fully mixed, 121 ℃ of sterilizing 10min.The autologous room temperature that is cooled to, every 50mL joins and is placed with in aseptic non-woven fabrics (15cm * 15cm) independent package bag, seals and get final product.

The preparation that embodiment five wood frogs are extracted mixed liquor peroral dosage form

(1) wood frog is extracted the preparation of mixed liquor

Get dried wood frog 40g (Rana temporaria chensinensis David Changbai Mountain subspecies, purchased from large pharmacy, Jilin), Gray Root Pakistan 29g (purchased from pharmacy, Chengdu), Galla Chinensis 2g, Radix Sophorae Flavescentis 1g, Herba Portulacae 1g, Fructus Cnidii 2g, Cortex Phellodendri 1g, Folium Perillae 1g, mentha leave 2g, Rhizoma Et Radix Notopterygii 1g, Radix Stephaniae Tetrandrae 1g, Fructus Zanthoxyli 2g, Rhizoma Atractylodis 3g, favus skin 1g, Rhizoma Picrorhizae 1g, Fructus Kochiae 1g, Radix Stemonae 1g, Flos Carthami 1g, Herba Pogostemonis 1g (above 17 taste Chinese medicines are all purchased from large pharmacy, Jilin), is prepared into medicine composite preparation in wood frog extraction according to the method for embodiment mono-, and volume is 175mL.

(2) concentrating under reduced pressure: during wood frog is extracted, medicine composite preparation distills under reduced pressure, and pressure is 9.2 * 10 ³handkerchief, temperature is 74 ℃, cocnentration factor is 1.09:1, obtains concentrated solution 160ml;

(3) regulate mixed liquor

Slowly add wood frog to extract in mixed liquor the zinc acetate of porphyrize, limit edged stirs; Adding rear is 6.4 ± 0.1 with 0.1mol/LNaOH adjusting pH, makes its natural subsidence, autologous cool overnight after stirring evenly;

(4) filter

Through 0.45 μ m membrane filtration, obtain after supernatant liquid, in clear liquor, directly add 5g citric acid, 10g Mel, 18g white sugar, temperature is in the time of 55 ℃, and 150rpm stirs, mixing time is 15min, filter, then add 6g xanthan gum and 5g part potassium sorbate in clear liquid, through 0.22 μ m filter membrane fine straining;

(5) filling-in and closing bottle by applying cap

Fill under aseptic condition, every bottle of 20mL, gland sealing;

(6) sterilizing

The good wood frog of fill is extracted to mixed liquor oral formulations and be heated to 65 ℃, keep 30min, the autologous room temperature that is cooled to is finished product.

Following test example shows the purposes of the present invention in treatment beriberi

Test example 1

The method that adopts completely random, is assigned to treatment group (300 examples, totally 5 groups) and matched group (480 examples, 8 groups) by 780 routine study subjects.Treatment group adopts respectively embodiment bis-, embodiment tri-, embodiment tetra-, embodiment five, the wood frog that embodiment bis-+embodiment five makes is extracted Compound Chinese Herbs for Fatigue-Resistance of Animal, matched group adopts new ointment for tinea pedis (the Jiu Futang bio-pharmaceuticals of commercially available Guangzhou), Rana sylvatica Le conte skin antibacterial peptide (adopting the preparation of CN1216768A method), forest frog body antibacterial peptide (adopting the preparation of CN103073628A method), middle medicine composite preparation A group (consisting of the external preparation that removes dried wood frog in the present invention), middle medicine composite preparation A1 group (consisting of the oral formulations that removes dried wood frog in the present invention), middle medicine composite preparation B group (consists of and in the present invention, removes dried wood frog, the external preparation of Gray Root Pakistan), middle medicine composite preparation C group (consists of and in the present invention, removes dried wood frog, Galla Chinensis, Radix Sophorae Flavescentis, Fructus Cnidii, Radix Stephaniae Tetrandrae, the external preparation of Rhizoma Atractylodis), medicine composite preparation A1 group in middle medicine composite preparation A+.According to criterion of therapeutical effect judgement curative effect, the results are shown in Table 1.

Bis-groups of usage: embodiment, tri-groups of embodiment, tetra-groups of embodiment, new ointment for tinea pedis group, Rana sylvatica Le conte skin antibacterial peptide group, forest frog body antibacterial peptide group, middle medicine composite preparation A group, middle medicine composite preparation B group, middle medicine composite preparation C group are external group, sooner or later used medicine after cleaning foot twice every day, bis-groups of each dosages of embodiment are 2g, application method is for smearing foot, 2 times/d; During tri-groups of each medications of embodiment, spray foot 5 times, be approximately 2mL medicinal liquid, 2 times/d; During tetra-groups of each medications of embodiment more than wiping foot 1min, 2 times/d; The each dosage of new ointment for tinea pedis group is 2g, and application method is for smearing foot, 2 times/d; Rana sylvatica Le conte skin antibacterial peptide group, forest frog body antibacterial peptide group, middle medicine composite preparation A group, middle medicine composite preparation B group, middle medicine composite preparation C group, each dosage is 2mL, application method is for smearing foot, 2 times/d;

Five groups of embodiment, middle medicine composite preparation A1 group is for oral group; In five groups of embodiment bis-+embodiment, middle medicine composite preparation A+, medicine composite preparation A1 group is external+oral group.Five groups of embodiment, middle medicine composite preparation A1 group is sooner or later oral every day, each 20mL, and period in a medicine is without food prohibited; Embodiment bis-+embodiment five groups of every days are oral embodiment five sooner or later, each 20mL, and sooner or later clean external embodiment bis-after foot for twice, and each dosage is 2g, application method is for smearing foot, 2 times/d; In middle medicine composite preparation A+ medicine composite preparation A1 group every day oral Chinese medicine compound formulation A1 sooner or later, each 20mL, and sooner or later clean external application Chinese medicine compound formulation A after foot for twice, each dosage is 2g, application method is for smearing foot, 2 times/d.

Each group is inactive other correlation means and medicine simultaneously, all take 7 days as 1 course for the treatment of.

Clinical classification mainly contains: erosive beriberi, blister type beriberi, squama keratinization type beriberi.Clinical cardinal symptom shows as:

Erosive type: moist between toe, dipping turns white, after dry desquamation, peels off scurf and is the rotten to the corn face of moistening, flushing or with breach, odd itching, easily secondary infection.

Vesicle: be apt to occur in sufficient edge, vola portion, from the beginning of being the full phlysis of wall thickness, the be fused into bulla having, bleb liquid is transparent, around without blush.Consciously very itch, after scratching often because secondary infection causes erysipelas, lymphangitis etc.

Squama keratinization type: be apt to occur in heel, sufficient edge.Because fungal infection causes slightly thick, desquamation of xerosis cutis, cutin, easily chap.This type is without vesicle and suppuration, and the course of disease is slow, how not to heal throughout the year, and Most patients is transformed by erosive type, vesicle.

Statistical method: measurement data is checked with t, numeration data X ₂and Rdiit check, P<0.05 is that difference has statistical significance.

Efficacy assessment standard:

With reference in the guideline of clinical investigations > > of < < new Chinese medicine treatment beriberi about curative effect of disease criterion.Recovery from illness: symptom, sign disappears or substantially disappears, and integrated value reduces I>95%; Effective: symptom, sign is obviously improved, and integrated value reduces >=70%; Effective: symptom, sign take a favorable turn, and integrated value reduces >=30%; Invalid: symptom, sign has no improvement, and integrated value reduces <30%.

Table 1 the present invention treats the situation (example) of beriberi

Under the treatment of description of test medicine composite preparation in wood frog of the present invention is extracted, patient cures soon, and curative effect is obvious, and relapse rate is extremely low.Illustrate and use medicine composite preparation treatment beriberi in wood frog extraction, than using, the curative effect of medicine composite preparation A1 in forest frog body antibacterial peptide or Rana sylvatica Le conte skin antibacterial peptide, middle medicine composite preparation A, middle medicine composite preparation B, middle medicine composite preparation C, middle medicine composite preparation A1, middle medicine composite preparation A+ is obvious; Most important is that the relapse rate of using wood frog to extract compound formulation treatment beriberi is 1.7～5.7%, the utmost point is lower than the relapse rate 80%, 82%, 91.7%, 100% of using Rana sylvatica Le conte skin antibacterial peptide, forest frog body antibacterial peptide, middle medicine composite preparation A, middle medicine composite preparation A1, lower than the relapse rate 60%, 56.7%, 52.6% of medicine composite preparation A1 in medicine composite preparation B, middle medicine composite preparation C, middle medicine composite preparation A+ in using.

Test example 2

Beriberi is at the many susceptible diseases in humid area in summer, and bathing staff is due to for a long time environmental work moist, improper ventilation, and volume of the flow of passengers great Yi causes infection simultaneously, so bathing staff is high-risk susceptible personnel.The method that adopts completely random, the bathing staff who 240 examples is newly entered to duty is assigned to prevention group (200 examples, totally 5 groups) and matched group (40 example).Prevention group adopt respectively wood frog that embodiment bis-, embodiment tri-, embodiment tetra-, embodiment five, embodiment bis-+embodiment five make extract in medicine composite preparation, not administration of matched group.According to beriberi standard test judgement preventive effect.The results are shown in Table 2.

Tetra-groups of bis-groups of usage: embodiment, tri-groups of embodiment, embodiment are external group, use medicine after sooner or later cleaning foot twice every day, and bis-groups of each dosages of embodiment are 2g, and application method is for smearing foot, 2 times/d; During tri-groups of each medications of embodiment, spray foot 5 times, be approximately 2mL medicinal liquid, 2 times/d; During tetra-groups of each medications of embodiment more than wiping foot 1min, 2 times/d;

Five groups of embodiment are oral group; Five groups of embodiment bis-+embodiment are external+oral group.Embodiment is sooner or later oral five groups of every days, each 20mL, and period in a medicine is without food prohibited; Embodiment bis-+embodiment five groups of every days are oral embodiment five sooner or later, each 20mL, and sooner or later clean external embodiment bis-after foot for twice, and each dosage is 2g, application method is for smearing foot, 2 times/d.

Not administration of matched group, take 30 days as 1 course for the treatment of.

Clinical cardinal symptom shows as, erosive type: moist between toe, dipping turns white, and after dry desquamation, peels off scurf and be the rotten to the corn face of moistening, flushing or with breach, odd itching, easily secondary infection.Vesicle: be apt to occur in sufficient edge, vola portion, from the beginning of being the full phlysis of wall thickness, the be fused into bulla having, bleb liquid is transparent, around without blush.Consciously very itch, after scratching often because secondary infection causes erysipelas, lymphangitis etc.Squama keratinization type: be apt to occur in heel, sufficient edge.Because fungal infection causes slightly thick, desquamation of xerosis cutis, cutin, easily chap.This type is without vesicle and suppuration, and the course of disease is slow, how not to heal throughout the year, and Most patients is transformed by erosive type, vesicle.

Statistical method: measurement data is checked with t, numeration data X ²and Rdiit check, P<0.05 is that difference has statistical significance.

Prevention evaluation criteria: be further divided into for three phases according to severity, only foot odor sweaty foot is the first phase; Occur that moist between toe, dipping turns white, the full phlysis of protuberance wall thickness is the second phase; Very itch hard to bear, occur erosion be obvious beriberi symptom be the third phase.

Table 2 the present invention prevents the situation (example) of beriberi

Description of test the present invention is having certain effect aspect prevention beriberi.

Test example 3: further prove curative effect of the present invention by following model case

Case 1

Xue, man, 39 years old, infect beriberi 6 years, cardinal symptom performance has: moist rotten to the corn between toe, pruritus, festers and cast off a skin, stream yellow fluid.Adopt externally used paste treatment of the present invention (embodiment bis-preparations), 3 days take effect, and do not flow yellow fluid, after 5 days, no longer fester, and obviously minimizing of decortication, after 15 days, transference cure, no longer festers, and no longer pruritus, cures.Within 1 year, pay a return visit afterwards, have no recurrence.

Case 2

Feng, man, 52 years old, infect beriberi 14 years, cardinal symptom performance has: vola portion pruritus is hard to bear, moist rotten to the corn between toe, thick the casting off a skin of bilateral heel skin keratin.Adopt externally applied spray treatment of the present invention (embodiment tri-preparations), 7 days take effect, and between toe, no longer fester, and heel decortication reduces, and after 18 days, transference cure, no longer casts off a skin, and no longer pruritus, cures.Within 1 year, pay a return visit afterwards, have no recurrence.

Case 3

Soup, man, 34 years old, infect beriberi 2 years, cardinal symptom performance has: heel skin keratin is firmly thick, yellow skin, and illing skin is coarse a large amount of desquamations, the symptom of chapping.Adopt external wet tissue treatment of the present invention (embodiment tetra-preparations), 6 days take effect, and heel xerosis cutis is alleviated, and uses continuously 14 days, mouthful healing of chapping, and heel desquamation reduces, uses continuously 25 days, local skin deliquescing, color restoration is normal, cures.Within 1 year, pay a return visit afterwards, have no recurrence.

Case 4

Zhang, man, 28 years old, suffer from beriberi 4 years, cardinal symptom performance has: pruritus, cast off a skin, tear, pain, adopt external wet tissue treatment of the present invention (embodiment tetra-preparations), sooner or later use wet tissue wiping 1 time, the same day pain relief, 6 days take effect, pain, the transference cure of casting off a skin, use 24 days continuously, tearing port healing, pruritus disappears, and cures.Within 1 year, pay a return visit afterwards, have no recurrence.

Case 5

Lin, female, 42 years old, infect beriberi 4 years, cardinal symptom performance has: foot interphalangeal erosion, pruritus, festers and cast off a skin.Adopt oral formulations treatment of the present invention (embodiment five), 3 days take effect, and the phenomenon minimizing of casting off a skin of festering, after 5 days, no longer festers and cast off a skin, and after 15 days, transference cure, no longer festers, and no longer pruritus, cures.Within 1 year, pay a return visit afterwards, have no recurrence.

Case 6

Jiang, man, 32 years old, suffer from beriberi 12 years, cardinal symptom performance has: pain, pruritus, cast off a skin, tear, adopt external wet tissue of the present invention and oral formulations therapeutic alliance (embodiment tetra-preparations and embodiment five preparations), used wet tissue (embodiment tetra-) on every 4 hours of daytime, take oral formulations (embodiment five) sooner or later for twice, the same day pain relief, 8 days take effect, pain, pruritus, the transference cure of casting off a skin, used 28 days continuously, tearing port recovery from illness, local skin color restoration is normal, cures.Within 1 year, pay a return visit afterwards, have no recurrence.

Test example 4: the extracorporeal bacteria inhibitor test of medicine composite preparation in wood frog extraction

1, experiment material

Get embodiment bis-1g, add normal saline and make 2mL medicinal liquid; Get embodiment tri-, embodiment tetra-medicinal liquid 1mL, add normal saline and make 2mL medicinal liquid; Embodiment five gets 2mL medicinal liquid; New ointment for tinea pedis (the Jiu Futang bio-pharmaceuticals of commercially available Guangzhou) is prepared into 20mg/mL solution (pH6.5); Rana sylvatica Le conte skin antibacterial peptide (adopting the preparation of CN1216768A method) 2mL, forest frog body antibacterial peptide (adopting the preparation of CN103073628A method) 2mL; Middle medicine composite preparation A group (consisting of the external preparation that removes dried wood frog in the present invention) 2mL, middle medicine composite preparation A1 group (consisting of the oral formulations that removes dried wood frog in the present invention) 2mL, middle medicine composite preparation B group (consisting of the external preparation that removes dried wood frog, Gray Root Pakistan in the present invention) 2mL, middle medicine composite preparation C group (consisting of the external preparation that removes dried wood frog, Galla Chinensis, Radix Sophorae Flavescentis, Fructus Cnidii, Radix Stephaniae Tetrandrae, Rhizoma Atractylodis in the present invention) 2mL.Bacterial strain: trichophyton, trichophyton gypseum, acrothesium floccosum provide by Jilin Province Disease Control and Prevention Center.

2, experimental technique

Sterilized agar culture medium is heated to completely and is melted, be poured in culture dish, every ware 18mL (lower floor), treats that it solidifies.In addition, by the LB culture medium (dextrose bouillon, ordinary nutrient agar culture medium, husky Bao Shi agar culture medium, bile salt lactose fermentation tube, hexadecane trimethyl ammonium bromide agar culture medium, the blood agar culture-medium that melt, according to different strains, select different culture medium, experimental technique is consistent.) be cooled to 50 ℃ of left and right to sneak into test organisms, the culture medium 5mL that is mixed with bacterium is added to and in the culture medium of having solidified, waits to solidify (upper strata).With sterile working, in media surface, directly vertically put Oxford cup (the circular tubule of internal diameter 6mm, external diameter 8mm, high 10mm, the two ends of pipe are smooth, also useable glass pipe, porcelain tube), pressurization gently, make it contact tight with culture medium, in cup, add sample to be checked (embodiment bis-, embodiment tri-, embodiment tetra-, embodiment five, new ointment for tinea pedis, Rana sylvatica Le conte skin antibacterial peptide, forest frog body antibacterial peptide, middle medicine composite preparation A, middle medicine composite preparation B, middle medicine composite preparation C) 100 μ l, do not make it excessive.Fill it up with rearmounted 37 ℃ of cultivations, observed result.The results are shown in Table 3:

Table 3: Antibacterial Activity result of the present invention

Description of test the present invention is higher for the trichophyton, trichophyton gypseum, the equal bacteriostasis rate of cotton-shaped epidermis tinea that cause beriberi, is significantly better than Rana sylvatica Le conte skin antibacterial peptide, forest frog body antibacterial peptide, middle medicine composite preparation A, middle medicine composite preparation B, middle medicine composite preparation C, middle medicine composite preparation A1 for the bacteriostasis rate of trichophyton, trichophyton gypseum, cotton-shaped epidermis tinea.

Test example 5: safety testing---the irritation test of medicine composite preparation in wood frog extraction

Test material: rabbit 1.8～2.2kg, male and female half and half.

Getting every 10 of healthy rabbits is one group, and many group experiments are set, and uses embodiment bis-, embodiment tri-, embodiment tetra-, embodiment five to make pharmaceutical preparation and new ointment for tinea pedis and makes medicinal liquid and carry out irritation test; First 24 hours of the percutaneous drug delivery of rabbit is by 10% of the about surface area of family's rabbit back two side skin unhairing.Embodiment bis-gets 2g and adds normal saline and make 20mL medicinal liquid; Embodiment tri-, embodiment tetra-each 2mL, add normal saline and make 20mL medicinal liquid; Embodiment five medicinal liquid 20mL; Get new ointment for tinea pedis 2g and make 20mL medicinal liquid; Be coated in respectively on the skin of rabbit of each group, 1 hour, 24 hours, within 48 hours, check the irritant reaction of medicine to rabbit skin, the results are shown in Table 5, table 6.

Table 5: irritation test erythema of the present invention bears results

Erythematous response progression	Bis-groups of embodiment	Tri-groups of embodiment	Tetra-groups of embodiment	Five groups of embodiment	New ointment for tinea pedis group	Normal group
							0 intact skin without erythema	10	10	10	10	9	10
1 slight erythema	0	0	0	0	1	0
							2 moderate erythema	0	0	0	0	0	0
3 severe erythema	0	0	0	0	0	0
							4 severe erythema are to slight eschar	0	0	0	0	0	0

Table 6: irritation test edema of the present invention bears results

The edema order of reaction	Bis-groups of embodiment	Tri-groups of embodiment	Tetra-groups of embodiment	Five groups of embodiment	New ointment for tinea pedis group	Normal group
							0 intact skin without edema	10	10	10	10	9	10
1 Mild edema (visible reluctantly)	0	0	0	0	1	0
							2 slight edema (profile raised panel is clear)	0	0	0	0	0	0
3 intermediate edema	0	0	0	0	0	0
							4 severe edemas	0	0	0	0	0	0

Comprehensive conclusions can know that in wood frog extraction, medicine composite preparation effect aspect minimizing zest is all good.

Test example 6: the safety testing of medicine composite preparation in wood frog extraction---allergy test

Test material: mice 15～20g, male and female half and half.

Getting every 20 of healthy mice is one group, and many group experiments are set, and the pharmaceutical preparation of embodiment bis-, embodiment tri-, embodiment tetra-is made to medicinal liquid; First 24 hours of the percutaneous drug delivery of mice is 10% of the about surface area of mouse back two side skin unhairing, and embodiment bis-gets 1g and adds normal saline and make 2mL medicinal liquid; Embodiment tri-, embodiment tetra-each 1mL, add normal saline and make 2mL medicinal liquid; Be coated in respectively on the mouse skin of each group, administration every day 3 times, successive administration 60 days, checks that medicine is to mouse skin allergy.With Normal group comparison, each group all occurs without skin abnormal phenomena.Get embodiment five medicinal liquid gastric infusion every day 2 times, each 0.5mL, successive administration 60 days, checks that medicine is to the every physical signs allergy of mice, with Normal group comparison, each group all occurs without every physical signs abnormal phenomena.

Test example 7: safety testing---the tertogenicity test of medicine composite preparation in wood frog extraction

Test material: mice 15～20g, male and female half and half.

Getting every 20 of healthy mice is one group, and many group experiments are set, and the pharmaceutical preparation of using embodiment bis-, embodiment tri-, embodiment tetra-, embodiment five to make, carries out tertogenicity test.First 24 hours of the percutaneous drug delivery of mice is 10% of the about surface area of mouse back two side skin unhairing, and embodiment bis-gets 1g and adds normal saline and make 2mL medicinal liquid; Embodiment tri-, embodiment tetra-each 1mL, add normal saline and make 2mL medicinal liquid; Be coated in respectively on the mouse skin of each group, administration every day 3 times, successive administration 90 days, each is organized mice and carries out copulation, checks the every physical signs of the cub of institute Mus, and with Normal group comparison, it is normal that each organizes filial mice physical signs, and medicine affects without teratogenesis laboratory animal.Get embodiment five medicinal liquid gastric infusion every day 2 times, each 0.5mL, successive administration 90 days, each is organized mice and carries out copulation, checks the every physical signs of the cub of institute Mus, and with Normal group comparison, it is normal that each organizes filial mice physical signs, and medicine affects without teratogenesis laboratory animal.

The above; it is only the specific embodiment of the present invention; but protection scope of the present invention is not limited to this; any those of ordinary skill in the art are in the disclosed technical scope of the present invention; the variation that can expect without creative work or replacement, within all should being encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claims were limited.

Claims (6)

1. wood frog is extracted a medicine composite preparation in mixing, and it is prepared by following steps:

(1) monarch drug wood frog is extracted the preparation of mixed liquor

To after the dried wood frog remove impurity dedusting of 40～55 weight portions, through 20～100 orders, pulverize respectively, make the particle diameter of dried wood frog ground product below 500 μ m, according to the ratio of 1g:1～10mL, add combinative enzyme hydrolysis liquid, in combinative enzyme hydrolysis liquid, contain papain 1～5wt%, trypsin 2～4wt%, flavor protease 1～3wt%, hydrolysis temperature is 50～55 ℃, enzymolysis time is 1～2h, enzymolysis pH is 7.0～7.5, then 85～90 ℃, 5～15min enzyme denaturing, mixture is centrifugal when 10～30 ℃ of the temperature, centrifugation time is 1～5min, centrifugal rotational speed is 2000～4000rpm, the upper strata supernatant of extracting centrifugal liquid volume 20～40%,

After getting supernatant, remaining mixture is put into colloid mill, grinds 2～15min, makes the mean diameter of mixture below 300 μ m; Mixture is put into high-shearing dispersion emulsifying machine, shear 1～15min, make mixture mean diameter between 100～200 μ m, mixture is spray dried to Powdered, now obtain the superfine powder of remaining mixture; It is in 2～10% acetic acid that above-mentioned superfine powder is joined to mass concentration, and acetic acid is 10～40mL:1g with the volume mass ratio of superfine powder; Ultrasonic 5～75min, ultrasonic temperature is 4～30 ℃; Through 150 μ m gauzes, filter, again above 150 μ m gauzes not medium to add mass volume ratio be 0.01～0.03mol/L sodium hydroxide of 1g:1～8mL, carry out microwave extraction, microwave power is 500～600W, microwave time 3～9min, filters through 150 μ m gauzes again; Merge filtrate twice, 30～50 ℃, stir 10～30min, mixing speed is 100～200rpm; Filtrate after stirring is centrifugal, and centrifuging temperature is 4～30 ℃, and centrifugation time is 2～40min, and centrifugal rotational speed is 2000～15000rpm; Get supernatant, concentrating under reduced pressure, pressure is 600～700 holders, and temperature is 65～70 ℃, and concentrated volume ratio is 8～40:1; Now obtain the mixture of polysaccharide, antibacterial peptide and calcium ion;

The upper strata supernatant that contains small peptide collagen protein is mixed with polysaccharide, antibacterial peptide and calcium ion mixture, obtain wood frog and extract mixed liquor;

(2) preparation of combination drug extracting solution

Take 25～40 weight portion Gray Root Pakistan, 1～3 weight portion Rhizoma Atractylodis, 1～2 weight portion Radix Stemonae, 1～2 weight portion Herba Pogostemonis, soak with ethanol 1～the 10h that adds mass fraction 90～100% after mixing, wherein ethanol and above-mentioned substance quality and volume mass ratio be 1～20mL:1g; 100～300 μ m gauzes filter, and centrifugal 10～20min under 15～30 ℃, 3000～6000rpm goes after precipitation at 85～100 ℃ heating concentrated, and concentrated volume ratio is 24～8:1; Obtain ethanol extract;

Take 1～3 weight portion Galla Chinensis, 1～3 weight portion Radix Sophorae Flavescentis, 1～3 weight portion Herba Portulacae, 1～2 weight portion Fructus Cnidii, 1～3 weight portion Cortex Phellodendri, 1～3 weight portion Folium Perillae, 1～3 weight portion mentha leave, 1～3 weight portion Rhizoma Et Radix Notopterygii, 1～2 weight portion Radix Stephaniae Tetrandrae, 1～3 weight portion Fructus Zanthoxyli, 1～3 weight portion favus skin, 1～2 weight portion Rhizoma Picrorhizae, 1～2 weight portion Fructus Kochiae, 1～3 weight portion Flos Carthami, after mixing, add water soaking 1～24h, wherein water and above-mentioned substance quality and volume mass ratio be 10～25mL:1g, 100～300 μ m gauzes filter, 15～30 ℃, centrifugal 10～20min under 3000～7000rpm, after going precipitation, 85～100 ℃ of heating are concentrated, concentrated volume ratio is 24～8:1, obtain aqueous extract,

Ethanol extract is mixed with aqueous extract, stir 10～30min, mixing speed is 100～400rpm, obtains combination drug extracting solution;

(3) wood frog is extracted to mixed liquor and fully mix with combination drug extracting solution, obtain medicine composite preparation in wood frog extraction.

2. in a kind of wood frog extraction claimed in claim 1, the purposes in beriberi is treated and prevented to medicine composite preparation in preparation.

3. the wood frog for the treatment of and prevention beriberi is extracted the compound ointment of Chinese medicine, and it is prepared by following steps:

(1) preparation of oil phase: get 5～15 weight portion paraffin, 2～5 weight portion glyceryl monostearate, 5～10 weight portion white vaseline, 5～10 weight portion liquid paraffin, 1～5 weight portion span 40,1～5 weight portion polyoxyethylene nonylphenol ether, 1～3 weight portion ethylparaben and be ground, obtain oil phase;

(2) preparation of medicine composite preparation ointment in wood frog extraction: medicine composite preparation in above-mentioned oil phase and the wood frog extraction being made by 40～55 weight portion dried wood frog claimed in claim 1 is heated to respectively to 70～100 ℃, during wood frog is extracted, medicine composite preparation joins in described oil phase, stir while adding to condensation, through nanometer homogenizer homogeneous 4～7 times, 115～121 ℃ of sterilizing 10～30min; Then Borneolum Syntheticum is heated to 70～100 ℃ of mistakes, 0.45 μ m film, under aseptic condition, mixs homogeneously with above-mentioned sterilizing afterproduct, obtain medical treatment and prevent the wood frog of beriberi to extract the compound ointment of Chinese medicine.

4. the wood frog for the treatment of and prevention beriberi is extracted the agent of Chinese medicine combined mist, and it is prepared by following steps:

In extracting, the wood frog being made by 40～55 weight portion dried wood frog claimed in claim 1 adds successively 1～5 weight portion sodium chloride, 2～4 weight portion PEG in medicine composite preparation ₄₀₀, 1～2 weight portion benzalkonium bromide; Under 100～200rpm, stir 10～25min, add water to 1000mL, 150～200rpm stirs 10～30min, and above-mentioned substance is fully mixed, 115～121 ℃ of sterilizing 10～20min; Autologously be distributed into aerosol bottle after being cooled to room temperature, obtain medical treatment and prevent the wood frog of beriberi to extract the agent of Chinese medicine combined mist.

5. the wood frog for the treatment of and prevention beriberi is extracted the compound wet tissue of Chinese medicine, and it is prepared by following steps:

In extracting, the wood frog being made by 40～55 weight portion dried wood frog claimed in claim 1 adds successively 1～5 weight portion sodium chloride, 2～4 weight portion PEG in medicine composite preparation ₄₀₀, 1～2 weight portion benzalkonium bromide; Under 100～200rpm, stir 10～25min, add water to 1000mL, 150-200rpm stirs 10-30min, and above-mentioned substance is fully mixed, 115～121 ℃ of sterilizing 10～20min; The autologous room temperature that is cooled to, every 50～60mL joins in the independent package bag of the aseptic non-woven fabrics that is placed with (15～20) cm * (15～20) cm, and the wood frog that seals and obtain prevention beriberi is extracted the compound wet tissue of Chinese medicine.

6. the wood frog for the treatment of and prevention beriberi is extracted the compound oral formulations of Chinese medicine, and it is prepared by following steps:

(1) concentrating under reduced pressure: during the wood frog being made by 40～55 weight portion dried wood frog claimed in claim 1 is extracted, medicine composite preparation distills under reduced pressure, and pressure is 8 * 10 ³～3 * 10 ⁴handkerchief, temperature is 50～85 ℃, cocnentration factor is 1～5:1, obtains concentrated solution;

(2) 1～5 weight portion zinc acetate is slowly joined in concentrated solution, limit edged stirs; Adding rear is 6.2～6.5 with NaOH adjusting pH, makes its natural subsidence, autologous cool overnight after stirring evenly;

(3) 0.45 μ m membrane filtrations, obtain after supernatant liquid, in clear liquor, add 5～15 weight portion citric acids, 5～25 weight portion Mel, 5～30 weight portion white sugars, under 50～65 ℃, 100～200rpm, stir 10～25min; 0.45 μ m membrane filtration then adds 5～15 weight portion xanthan gum and 5～15 weight portion potassium sorbate in clear liquid again; Finally by 0.22 μ m filter membrane fine straining;

(4) filling-in and closing bottle by applying cap: fill under aseptic condition, gland sealing;

(5) sterilizing: be heated to 62～65 ℃, keep 30～50min, the autologous room temperature that is cooled to, obtains medical treatment and prevent the wood frog of beriberi to extract the compound oral formulations of Chinese medicine.