CN104173382A - Rana chensinensis extracted mixed liquid and application thereof in preparing medicines for treating and preventing dermatophytosis - Google Patents
Rana chensinensis extracted mixed liquid and application thereof in preparing medicines for treating and preventing dermatophytosis Download PDFInfo
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- CN104173382A CN104173382A CN201410360106.2A CN201410360106A CN104173382A CN 104173382 A CN104173382 A CN 104173382A CN 201410360106 A CN201410360106 A CN 201410360106A CN 104173382 A CN104173382 A CN 104173382A
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Abstract
The invention provides a rana chensinensis extracted mixed liquid and an application thereof in preparing medicines for treating and preventing dermatophytosis, and belongs to the technical field of medical bio-pharmacy. The mixed liquid is prepared by adopting the following steps: performing impurity-removal and dust-collection on dried rana chensinensis, crushing, adding complex enzyme hydrolysate, and centrifuging to obtain upper supernate; grinding remaining mixture, shearing, and drying by means of spray to obtain powder; adding the superfine powder into acetic acid, performing ultrasonic treatment, and filtering by virtue of a gauze element, centrifuging to obtain supernate, and concentrating at reduced pressure so as to obtain a mixture of polysaccharide, antimicrobial peptide and calcium ions; and mixing all the materials to obtain the rana chensinensis extracted mixed liquid. The mixed liquid is a pure biological preparation, and does not have side effect; micromolecular antimicrobial peptide with a remarkable antibacterial effect is combined with the calcium ions to reinforce destruction of the micromolecular antimicrobial peptide to fungal cell walls, and can enter fungal cells to achieve the antimicrobial effect; the oligopeptide collagen can enter normal cells in the action of polysaccharide to give sufficient nutrition to the cells, reinforce the defending function of the cells, helping the normal cells defend external damage factors and repairing injured cells.
Description
Technical field
The invention belongs to medical biotechnology pharmaceutical technology field, be specifically related to a kind of wood frog and extract mixed liquor and the purposes in the medicine of preparation treatment and prevention beriberi thereof.
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 in the time that 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, taking wood frog as key word, retrieve domestic and international patent, do not find the patent about wood frog abroad, 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 to infect field with spray and two kinds of forms of gel for clinical skin infection, empyrosis; 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 taking 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 adopt supersound extraction antibacterial peptide taking forest frog body as raw material, 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 is all taking 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 bacteria 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 taking dry Rana dybowskii skin as raw material; It is taking Rana sylvatica Le conte skin as raw material that CN102229972A " prepares the method for collagen peptides from tree frog skin ", and above three patents are all taking Rana sylvatica Le conte skin as raw material or primary raw material extracts contained collagen protein in Rana sylvatica Le conte skin.
Extracting method of the prior art does not all extract polysaccharide and the calcium ion that wood frog contains, and only extract merely Wood frog antibiotic peptides, or extract merely Rana chensinensis collagen, most importantly the antibacterial peptide extracting or the molecular weight ranges of collagen protein are unfixing, difference is larger, be not described in detail, effective ingredient rationally do not utilized.
Summary of the invention
One of object of the present invention is to provide a kind of wood frog and extracts mixed liquor, and it is to be prepared by following steps:
(1) will after 10～45 weight portion dried wood frog remove impurity dedustings, pulverize through 20～100 orders respectively, make the particle diameter of dried wood frog ground product below 500 μ m, add combinative enzyme hydrolysis liquid according to the ratio of 1g:1～10mL, in combinative enzyme hydrolysis liquid, contain papain 1～5wt%, trypsin 2～4wt%, flavor protease 1～3wt%, hydrolysis temperature is 50～55 DEG C, enzymolysis time is 1～2h, enzymolysis pH is 7.0～7.5, then enzyme denaturing 5～15min at 85～90 DEG C, mixture is centrifugal when 10～30 DEG C of the temperature, centrifugation time is 1～5min, centrifugal rotational speed is 2000～4000rpm, get the upper strata supernatant of centrifugal rear mixeding 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, wherein basic amino acid (arginine, 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%) and 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 dried wood frog and extract the superfine powder of the materials such as residual fiber after small peptide collagen protein, sclerotin;
(2) superfine powder of the material such as fiber, sclerotin being joined to mass concentration is in 2～10% acetic acid, and acetic acid is 10:1～40:1 with the volume mass of superfine powder than (mL:g); Ultrasonic 5～75min, ultrasonic temperature is 4～30 DEG C; Filter through 150 μ m gauzes, 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, stir 10～30min at 30～50 DEG C, mixing speed is 100～200rpm; Filtrate after stirring is centrifugal, and centrifuging temperature is 4～30 DEG C, 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 DEG C, and concentrated volume ratio is 8～40:1, now obtains the mixture of polysaccharide, antibacterial peptide and calcium ion, and 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 polysaccharide, antibacterial peptide and calcium ion mixture, acidic amino acid (aspartic acid, glutamic acid) accounts for 10.01～11.47% (mass ratioes) of polysaccharide, antibacterial peptide and calcium ion mixture;
The product that step (1) and step (2) are obtained mixes, and obtains wood frog of the present invention and extracts mixed liquor.
The present invention in conjunction with calcium ion, strengthens its destruction to fungal cell wall by significant contained wood frog fungistatic effect micromolecule antibacterial peptide, and can enter better fungal cell and reach fungistatic effect; Small peptide collagen protein can better enter normal cell under the effect of polysaccharide, gives cell sufficient nutrition, strengthens cytophylaxis function, helps normal cell to resist extraneous destructive factor, and can repair damaged cell.
Described wood frog is extracted mixed liquor 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.
Described wood frog is extracted mixed liquor 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 mixed liquor ointment, and it is prepared by following steps:
(1) preparation of oil phase: get 5～15 weight portion paraffin, 2～10 weight portion glyceryl monostearate, 10～20 weight portion white vaseline, 5～30 weight portion liquid paraffin, 1～15 weight portion span 40,2～10 weight portion polyoxyethylene nonylphenol ethers, 1～8 weight portion ethylparaben, after mixing, be ground, obtain oil phase;
(2) wood frog is extracted the preparation of mixed liquor ointment: the oil phase that step (1) is made extracts mixed liquor with the wood frog being prepared as stated above by 10～45 weight portion dried wood frog and is heated to respectively 70～100 DEG C, wood frog is extracted to mixed liquor to be joined in described oil phase, stir while adding, mixing time is 10～15min, mixing speed is 100～300rpm, through nanometer homogenizer homogeneous 4～7 times again, 115～121 DEG C of sterilizing 10～30min; 1～5 weight portion Borneolum Syntheticum is heated to 70～100 DEG C of mistakes, 0.45 μ m film, under aseptic condition, mixs homogeneously with above-mentioned sterilizing afterproduct, obtain the wood frog extraction mixed liquor ointment that is used for the treatment of and prevents beriberi of the present invention.
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 mixed liquor spray, and it prepares by the following method:
Extract the PEG4 that adds 2～4 weight portions in mixed liquor to the wood frog being prepared as stated above by 10～45 weight portion dried wood frog
400, the sodium chloride of 1～5 weight portion, the benzalkonium bromide of 1～2 weight portion, under 100～200rpm, stir 10～25min, add water to 1000 weight portions, under 150～200rpm, stir 10～30min, above-mentioned substance is fully mixed, sterilizing 10～20min at 115～121 DEG C; Autologously be distributed into aerosol bottle after being cooled to room temperature, obtain wood frog and extract mixed liquor spray.
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 mixed liquor wet tissue, and it prepares by the following method:
Extract the PEG4 that adds 2～4 weight portions in mixed liquor to the wood frog being prepared as stated above by 10～45 weight portion dried wood frog
400, the sodium chloride of 1～5 weight portion, the benzalkonium bromide of 1～2 weight portion, under 100～200rpm, stir 10～25min, add water to 1000 weight portions, under 150～200rpm, stir 10～30min, above-mentioned substance is fully mixed, sterilizing 10～20min at 115～121 DEG C; Autologous be cooled to room temperature after every 50～60mL join in the independent package bag that is placed with the aseptic non-woven fabrics of (15～20) cm × (15～20) cm, seal and obtain wood frog and extract mixed liquor wet tissue.
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 mixed liquor oral agents, and it is prepared by following steps:
(1) concentrating under reduced pressure: the wood frog being prepared as stated above by 10～45 weight portion dried wood frog is extracted to mixed liquor and distill under reduced pressure, pressure is 8 × 10
3～3 × 10
4handkerchief, temperature is 50～85 DEG C, cocnentration factor is 1～5:1, obtains concentrated solution;
(2) 1～5 weight portion zinc acetate is slowly joined in concentrated solution, stir while adding, mixing speed is 100～300rpm, and mixing time is 10～15min; Adding the rear adjusting of the NaOH with 0.1mol/L pH is 6.2～6.5, makes its natural subsidence, 4 DEG C of cool overnight after stirring evenly;
(3) by above-mentioned product 0.45 μ m membrane filtration, obtain after supernatant liquid, add 5～15 weight portion citric acids, 5～25 weight portion Mel, 5～30 weight portion white sugars, be heated to 40～75 DEG C, 100～300rpm stirs, mixing time 10～30min, mix homogeneously, 0.45 μ m membrane filtration while hot, then add 5～15 weight portion xanthan gum and 5～15 weight portion potassium sorbate in clear liquid, through 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 DEG C, keep 30～50min, the autologous room temperature that is cooled to is wood frog extraction mixed liquor oral agents.
Usage and dosage: for oral administration, every day sooner or later twice oral, 10mL/ time 7 days was 1 course for the treatment of.
Good effect of the present invention is: it is pure biological preparation that wood frog is extracted mixed liquor, without any side effects, the significant micromolecule antibacterial peptide of contained fungistatic effect, in conjunction with calcium ion, strengthens its destruction to fungal cell wall, and can enter better fungal cell and reach fungistatic effect; Contained small peptide collagen protein can better enter normal cell under the effect of polysaccharide, gives cell sufficient nutrition, strengthens cytophylaxis function, helps normal cell to resist extraneous destructive factor, and can repair damaged cell.The present invention to mucocutaneous non-stimulated, sterilization speed is fast, easy to use, be applicable to different age people use.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.
Brief description of the drawings
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.Blue glucosan using molecular weight as 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.Taking Ve/Vt as abscissa, 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.
Make and draw polysaccharide molecular weight standard curve with blue glucosan and different molecular weight glucose standard;
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, measure wood frog with Ultraviolet Detector under Sephadex G-25 gel filtration chromatography 254nm 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 1～5Kda.
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 DEG C of hydrolysis 16h after-blow dry liquids.Polysaccharide standard sample after methanol solution after drying, adds 2mol/L trifluoroacetic acid 2mL, and 120 DEG C are filled N
2tube sealing hydrolysis 3h, hydrolyzate is poured in evaporating dish, adds 45 DEG C 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 0.5mL0.3mol/L in evaporating dish, gets 0.1mL wherein in little centrifuge tube, 70 DEG C 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 × 4.6mm)
Detected temperatures: 35 DEG C
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 composition 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, wherein rhamnose: 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, i.e. 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.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described.It is the foot of a hill or mountain, Changbai Mountain, the Northeast of China 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, the Northeast of China, belong to medicine, eat dual-purpose precious frog kind, and aboundresources.
The preparation that embodiment mono-wood frog is extracted mixed liquor
Get dried wood frog 20g (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, add combinative enzyme hydrolysis liquid according to the ratio of 1g:6mL, in combinative enzyme hydrolysis liquid, contain papain 3wt%, trypsin 2wt%, flavor protease 1wt%, water 94wt%, hydrolysis temperature is 52 DEG C, enzymolysis time is 1.5h, enzymolysis pH is 7.2, then 87 DEG C, 10min enzyme denaturing, mixture is centrifugal when 25 DEG C of the temperature, centrifugation time is 3min, centrifugal rotational speed is 2500rpm, get upper strata supernatant (cumulative volume 25%), now extract 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 is collected and is measured 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 (accounting for the mass percent of upper strata supernatant) of aspartic acid, glutamic acid 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 dried wood frog and extract the micronizing of the materials such as residual fiber after small peptide collagen protein, sclerotin;
It is in 4% acetic acid that the superfine powder of the material 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 DEG C; Filter, filter through 150 μ m gauzes; Above 150 μ m gauzes not medium to add mass volume ratio be the 0.02mol/L sodium hydroxide of 1:5, carry out microwave extraction, microwave power is 550W, microwave time 4min filters through 150 μ m gauzes again; Merge filtrate twice, stir 15min, mixing speed is 160rpm; Filtrate after stirring is centrifugal, and centrifuging temperature is 20 DEG C, 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 DEG C, and concentrated volume ratio is 15:1; Now obtain polysaccharide, antibacterial peptide, calcium ion mixture (detection of calcium ion adopts EDTA titrimetry to detect);
Polysaccharide in mixture (Sephadex G ?25 gel column separated and collected monosaccharide, high effective liquid chromatography for measuring monosaccharide composition) mainly formed by mannose, rhamnose, glucose, galactose, xylose, arabinose, fucose, glucuronic acid and galacturonic acid, 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 is collected and is measured 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 (accounting for the mass percent of mixture) of aspartic acid, glutamic acid is respectively 4.47%, 5.98%.
Small peptide collagen protein, polysaccharide, antibacterial peptide, calcium ion are mixed, obtain wood frog and extract mixed liquor, volume is 53mL, wherein calcium ions 42.4mg (80mg/100ml).
The preparation that embodiment bis-wood frogs are extracted mixed liquor ointment
Prescription composition (by weight): dried wood frog 20g, paraffin 8g, liquid paraffin 6g, white vaseline 12g, glyceryl monostearate 10g, span 40 9g, polyoxyethylene nonylphenol ether 3g, ethylparaben 1g, Borneolum Syntheticum 1g.
Preparation method:
(1) wood frog is extracted the preparation of mixed liquor
Take dried wood frog 20g (Rana temporaria chensinensis David Changbai Mountain subspecies, purchased from large pharmacy, Jilin), be prepared into wood frog according to the method for embodiment mono-and extract mixed liquor, obtain wood frog extracting solution 53mL
(2) preparation of oil phase: get 8g paraffin, 10g glyceryl monostearate, 12g white vaseline, 6g liquid paraffin, 9g span 40,3g polyoxyethylene nonylphenol ether, 1g ethylparaben and be ground, obtain oil phase;
(3) preparation of wood frog extracting solution ointment: the oil phase that step (2) is made and wood frog are extracted mixed liquor and be heated to respectively 90 DEG C, wood frog is extracted to mixed liquor to be joined in described oil phase, limit edged stirs, mixing time is 12min, mixing speed is 150rpm, through nanometer homogenizer homogeneous 5 times again, 115 DEG C of sterilizing 10min; Borneolum Syntheticum is heated to 90 DEG C of mistakes, 0.45 μ m film, under aseptic condition, mixs homogeneously with above-mentioned sterilizing afterproduct, obtain the wood frog extraction mixed liquor ointment that is used for the treatment of and prevents beriberi of the present invention.
The preparation that embodiment tri-wood frogs are extracted mixed liquor spray
Prescription composition (by weight): dried wood frog 20g, sodium chloride 19mg PEG
40023mg, benzalkonium bromide 6mg.
Preparation method:
Take dried wood frog 20g (Rana temporaria chensinensis David Changbai Mountain subspecies, purchased from large pharmacy, Jilin), be prepared into wood frog according to the method for embodiment mono-and extract mixed liquor, obtain wood frog extracting solution 53mL, in add the PEG4 of above-mentioned formula ratio
400, sodium chloride, benzalkonium bromide, 120rpm stirs, mixing time is 10min, adds water to 1000mL, 150rpm stirs 10min, and above-mentioned substance is fully mixed, 121 DEG C of sterilizing 10min.The autologous room temperature that is cooled to, is distributed into aerosol bottle (20mL/ bottle), to obtain final product.
The preparation that embodiment tetra-wood frogs are extracted mixed liquor wet tissue
Prescription composition (by weight): dried wood frog 20g, sodium chloride 24mg PEG
40011mg, benzalkonium bromide 2mg.
Preparation method:
Take dried wood frog 20g (Rana temporaria chensinensis David Changbai Mountain subspecies, purchased from large pharmacy, Jilin), be prepared into wood frog according to the method for embodiment mono-and extract mixed liquor, obtain wood frog extracting solution 53mL, in add the PEG4 of above-mentioned formula ratio
400, sodium chloride, benzalkonium bromide, 130rpm stirs, mixing time is 13min, adds water to 1500mL, 130rpm stirs 10min, and above-mentioned substance is fully mixed, 121 DEG C 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 oral agents
Prescription composition (by weight): dried wood frog 20g, citric acid 5g, Mel 21g, white sugar 22g, xanthan gum 6g part, potassium sorbate 8g, zinc acetate 1g.
Preparation method:
(1) take dried wood frog 20g (Rana temporaria chensinensis David Changbai Mountain subspecies, purchased from large pharmacy, Jilin), be prepared into wood frog according to the method for embodiment mono-and extract mixed liquor, obtain wood frog extracting solution 53mL,
(2) concentrating under reduced pressure: wood frog is extracted to mixed liquor and distill under reduced pressure, pressure is 9.1 × 10
3handkerchief, temperature is 75 DEG C, cocnentration factor is 1.06:1, obtains concentrated solution 50mL;
(2) regulate mixed liquor
Slowly add wood frog to extract in mixed liquor the zinc acetate of porphyrize, limit edged stirs, and 130rpm stirs, and mixing time is 10min; Add rear with 0.1mol/L NaOH regulate pH be 6.4 ± 0.1, after stirring evenly, make its natural subsidence, 4 DEG C are spent the night;
(3) filter
Through 0.45 μ m membrane filtration, obtain after supernatant liquid, add citric acid, Mel, the white sugar of above-mentioned formula ratio, heat 45 DEG C, 150rpm stirs, and mixing time is 12min, stirs; 0.45 μ m membrane filtration, then to the xanthan gum, the potassium sorbate that add above-mentioned formula ratio in clear liquid, 120rpm stirs, mixing time is 10min, stirs; Through 0.22 μ m filter membrane fine straining;
(4) filling-in and closing bottle by applying cap
Fill under aseptic condition, every bottle of 10mL, gland sealing;
(5) sterilizing
Wood frog good fill is extracted to mixed liquor oral formulations and be heated to 65 DEG C, 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 (420 examples, totally 7 groups) and matched group (180 examples, 3 groups) by 600 routine study subjects.Treatment group adopts respectively the wood frog that embodiment bis-, embodiment tri-, embodiment tetra-, embodiment five, embodiment bis-+embodiment five, embodiment tri-+embodiment five, embodiment tetra-+embodiment five make to extract mixed liquor preparation, and 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).Judge curative effect according to criterion of therapeutical 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 are external group, sooner or later used medicine after cleaning foot twice every day, bis-groups of each dosages of embodiment are 2g, and application method is for smearing foot, 2 times/d; When tri-groups of each medications of embodiment, spray foot 5 times, be approximately 2mL medicinal liquid, 2 times/d; When 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; The each dosage of Rana sylvatica Le conte skin antibacterial peptide group is 2mL, and application method is for smearing foot, 2 times/d; The each dosage of forest frog body antibacterial peptide group is 2mL, and application method is for smearing foot, 2 times/d;
Five groups of embodiment are oral group; Five groups of five groups of embodiment bis-+embodiment, five groups of embodiment tri-+embodiment, embodiment tetra-+embodiment are external+oral group.Embodiment is sooner or later oral five groups of every days, each 10mL, and period in a medicine is without food prohibited; Embodiment bis-+embodiment five groups of every days oral embodiment five sooner or later, each 10mL, and sooner or later clean external embodiment bis-after foot for twice, each dosage is 2g, application method is for smearing foot, 2 times/d; Embodiment tri-+embodiment five groups of every days oral embodiment five sooner or later, each 10mL, and sooner or later clean external embodiment tri-after foot for twice, sprays foot 5 times when each medication, is approximately 2mL medicinal liquid, 2 times/d; Embodiment tetra-+embodiment five groups of every days oral embodiment five sooner or later, each 10mL, and sooner or later clean external embodiment tetra-after foot for twice, when each medication more than wiping foot 1min, 2 times/d.
Each group is inactive other correlation means and medicine simultaneously, all taking 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 inspection, P<0.05 is that difference has statistical significance.
Efficacy assessment standard:
With reference in the guideline of clinical investigations of beriberi " new Chinese medicine treatment " 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
Description of test extracts under the treatment of mixed liquor wood frog of the present invention, and patient cures soon, and curative effect is obvious, and relapse rate is extremely low.Illustrate after wood frog extraction mixed liquor small peptide collagen protein, polysaccharide, antibacterial peptide, the calcium ion contained by dried wood frog fully extracts, mixing treatment beriberi, than using, the curative effect of forest frog body antibacterial peptide or Rana sylvatica Le conte skin antibacterial peptide is obvious; Most important is that the relapse rate that uses wood frog to extract mixed liquor treatment beriberi is 1.7～5.7%, and the utmost point is lower than the relapse rate 80% and the relapse rate 82% that uses forest frog body antibacterial peptide that use Rana sylvatica Le conte skin antibacterial peptide.
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 320 examples is newly entered to duty is assigned to prevention group (280 examples, totally 7 groups) and matched group (40 example).Prevention group adopts respectively the wood frog that embodiment bis-, embodiment tri-, embodiment tetra-, embodiment five, embodiment bis-+embodiment five, embodiment tri-+embodiment five, embodiment tetra-+embodiment five make to extract mixed liquor preparation, not administration of matched group.According to beriberi, standard test judges 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; When tri-groups of each medications of embodiment, spray foot 5 times, be approximately 2mL medicinal liquid, 2 times/d; When 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 five groups of embodiment bis-+embodiment, five groups of embodiment tri-+embodiment, embodiment tetra-+embodiment are external+oral group.Embodiment is sooner or later oral five groups of every days, each 10mL, and period in a medicine is without food prohibited; Embodiment bis-+embodiment five groups of every days oral embodiment five sooner or later, each 10mL, and sooner or later clean external embodiment bis-after foot for twice, each dosage is 2g, application method is for smearing foot, 2 times/d; Embodiment tri-+embodiment five groups of every days oral embodiment five sooner or later, each 10mL, and sooner or later clean external embodiment tri-after foot for twice, sprays foot 5 times when each medication, is approximately 2mL medicinal liquid, 2 times/d; Embodiment tetra-+embodiment five groups of every days oral embodiment five sooner or later, each 10mL, and sooner or later clean external embodiment tetra-after foot for twice, when each medication more than wiping foot 1min, 2 times/d;
Not administration of matched group, taking 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 inspection, 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 has effect positive, that be better than prior art aspect prevention beriberi.
Test example 3: further prove curative effect of the present invention by following model case
Case 1
Zhao, man, 27 years old, infect beriberi 5 years, cardinal symptom performance has: moist rotten to the corn between toe, pruritus, stream yellow fluid, festers and casts off a skin.Adopt externally used paste treatment of the present invention (prepared by embodiment bis-), 4 days take effect, and no longer fester, and do not flow yellow fluid, and decortication obviously reduces, and after 16 days, transference cure, no longer casts off a skin, and no longer pruritus is cured.Within 1 year, pay a return visit afterwards, have no recurrence.
Case 2
Lee, female, 46 years old, infect beriberi 8 years, cardinal symptom performance has: vola portion is moist rotten to the corn, and pruritus, festers and cast off a skin.Adopt peroral dosage form treatment of the present invention (prepared by embodiment five), 7 days take effect, and no longer fester, and decortication reduces, and after 20 days, transference cure, no longer casts off a skin, and no longer pruritus is cured.Within 1 year, pay a return visit afterwards, have no recurrence.
Case 3
Zhang, man, 18 years old, infect beriberi 1 year, cardinal symptom performance has: skin whitening between toe, peeling then, desquamation, exposes red soft textive, and with serious pruritus.Adopt spray treatment of the present invention (prepared by embodiment tri-), 2 days take effect, and pruritus is obviously alleviated, and uses continuously 7 days, and between toe, skin color restoration is normal, and no longer peeling desquamation is cured.Within 1 year, pay a return visit afterwards, have no recurrence.
Case 4
Wang, man, 35 years old, infect beriberi 3 years, cardinal symptom performance has: thick hard, the yellow skin of bilateral heel skin keratin, illing skin is coarse desquamation, the symptom of chapping.Adopt externally used paste of the present invention treatment (prepared by embodiment bis-), 5 days take effect, and heel xerosis cutis is alleviated, and uses continuously 14 days, mouthful recovery from illness of chapping, and desquamation no longer, and the obvious deliquescing of local skin, color is normal, cures.Within 1 year, pay a return visit afterwards, have no recurrence.
Case 5
Lin, man, 67 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-is prepared and embodiment five preparations), used wet tissue (embodiment tetra-) on every 4 hours of daytime, sooner or later take oral formulations (embodiment five) for twice, the same day pain relief, 8 days take effect, pain, pruritus, the transference cure of casting off a skin, use 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: wood frog is extracted pharmaceutical preparation extracorporeal bacteria inhibitor test prepared by mixed liquor
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.Bacterial strain: trichophyton, trichophyton gypseum, acrothesium floccosum provide by Disease Control and Prevention Center of Jilin Province.
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, select different culture medium according to different strains, experimental technique is consistent.) be cooled to 50 DEG C 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).Directly vertically put cup (the circular tubule of internal diameter 6mm, external diameter 8mm, high 10mm in Oxford with sterile working in media surface, 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) 100 μ l, do not make it excessive.Fill it up with rearmounted 37 DEG C 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, the forest frog body antibacterial peptide bacteriostasis rate for trichophyton, trichophyton gypseum, cotton-shaped epidermis tinea.
Test example 5: safety testing---the irritation test of pharmaceutical preparation prepared by wood frog extraction mixed liquor
Test material: rabbit 1.8～2.2kg.
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-, the each 2mL of embodiment tetra-, 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 4, table 5.
Table 4: 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 5: 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 |
It is all good that comprehensive conclusions can know that wood frog is extracted mixed liquor effect aspect minimizing zest.
Test example 6: the safety testing of pharmaceutical preparation prepared by wood frog extraction mixed liquor---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-, the each 1mL of embodiment tetra-, 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 pharmaceutical preparation prepared by wood frog extraction mixed liquor
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 that uses 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-, the each 1mL of embodiment tetra-, 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, respectively organizes mice and carries out copulation, checks the every physical signs of the cub of institute Mus, with Normal group comparison, respectively organizes filial mice physical signs normal, 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, respectively organizes mice and carries out copulation, checks the every physical signs of the cub of institute Mus, with Normal group comparison, respectively organizes filial mice physical signs normal, 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 mixed liquor, and it is prepared by following steps:
(1) will after 10～45 weight portion dried wood frog remove impurity dedustings, pulverize through 20～100 orders respectively, make the particle diameter of dried wood frog ground product below 500 μ m, add combinative enzyme hydrolysis liquid according to the ratio of 1g:1～10mL, in combinative enzyme hydrolysis liquid, contain papain 1～5wt%, trypsin 2～4wt%, flavor protease 1～3wt%, hydrolysis temperature is 50～55 DEG C, enzymolysis time is 1～2h, enzymolysis pH is 7.0～7.5, then enzyme denaturing 5～15min at 85～90 DEG C, mixture is centrifugal when 10～30 DEG C of the temperature, centrifugation time is 1～5min, centrifugal rotational speed is 2000～4000rpm, get the upper strata supernatant of centrifugal rear mixeding liquid volume 20～40%,
(2) will get supernatant after remaining mixture 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 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 DEG C; Filter through 150 μ m gauzes, 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, stir 10～30min at 30～50 DEG C, mixing speed is 100～200rpm; Filtrate after stirring is centrifugal, and centrifuging temperature is 4～30 DEG C, 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 DEG C, and concentrated volume ratio is 8～40:1;
(3) product step (1) and step (2) being obtained mixes, and obtains wood frog and extracts mixed liquor.
2. wood frog claimed in claim 1 is extracted the purposes of mixed liquor in the medicine of preparation treatment and prevention beriberi.
3. the wood frog for the treatment of and prevention beriberi is extracted a mixed liquor ointment, and it is prepared by following steps:
(1) preparation of oil phase: get 5～15 weight portion paraffin, 2～10 weight portion glyceryl monostearate, 10～20 weight portion white vaseline, 5～30 weight portion liquid paraffin, 1～15 weight portion span 40,2～10 weight portion polyoxyethylene nonylphenol ethers, 1～8 weight portion ethylparaben, after mixing, be ground, obtain oil phase;
(2) wood frog is extracted the preparation of mixed liquor ointment: the oil phase that step (1) is made and the wood frog being made by 10～45 weight portion dried wood frog claimed in claim 1 are extracted mixed liquor and be heated to respectively 70～100 DEG C, wood frog is extracted to mixed liquor to be joined in described oil phase, stir while adding, mixing time is 10～15min, mixing speed is 100～300rpm, through nanometer homogenizer homogeneous 4～7 times again, 115～121 DEG C of sterilizing 10～30min; Then 1～5 weight portion Borneolum Syntheticum is heated to 70～100 DEG C of mistakes, 0.45 μ m film, under aseptic condition, mixs homogeneously with above-mentioned sterilizing afterproduct, be used for the treatment of and prevent the wood frog of beriberi to extract mixed liquor ointment.
4. the wood frog for the treatment of and prevention beriberi is extracted a mixed liquor spray, and it prepares by the following method: extract to the wood frog being made by 10～45 weight portion dried wood frog claimed in claim 1 the PEG4 that adds 2～4 weight portions in mixed liquor
400, the sodium chloride of 1～5 weight portion, the benzalkonium bromide of 1～2 weight portion, under 100～200rpm, stir 10～25min, add water to 1000 weight portions, under 150～200rpm, stir 10～30min, above-mentioned substance is fully mixed, sterilizing 10～20min at 115～121 DEG C; Autologously be distributed into aerosol bottle after being cooled to room temperature, obtain medical treatment and prevent the wood frog of beriberi to extract mixed liquor spray.
5. the wood frog for the treatment of and prevention beriberi is extracted a mixed liquor wet tissue, and it prepares by the following method: extract to the wood frog being made by 10～45 weight portion dried wood frog claimed in claim 1 the PEG4 that adds 2～4 weight portions in mixed liquor
400, the sodium chloride of 1～5 weight portion, the benzalkonium bromide of 1～2 weight portion, under 100～200rpm, stir 10～25min, add water to 1000 weight portions, under 150～200rpm, stir 10～30min, above-mentioned substance is fully mixed, sterilizing 10～20min at 115～121 DEG C; Autologous be cooled to room temperature after every 50～60mL join in the independent package bag that is placed with the aseptic non-woven fabrics of (15～20) cm × (15～20) cm, sealing obtains medical treatment and prevents the wood frog of beriberi to extract mixed liquor wet tissue.
6. the wood frog for the treatment of and prevention beriberi is extracted a mixed liquor oral agents, and it is prepared by following steps:
(1) concentrating under reduced pressure: the wood frog being made by 10～45 weight portion dried wood frog claimed in claim 1 is extracted to mixed liquor and distill under reduced pressure, pressure is 8 × 10
3～3 × 10
4handkerchief, temperature is 50～85 DEG C, cocnentration factor is 1～5:1, obtains concentrated solution;
(2) 1～5 weight portion zinc acetate of porphyrize is slowly joined in concentrated solution, stir while adding, mixing speed is 100～300rpm, and mixing time is 10～15min; Add rear with NaOH regulate pH be 6.2～6.5, after stirring evenly, make its natural subsidence, 4 DEG C of cool overnight;
(3) by above-mentioned product 0.45 μ m membrane filtration, obtain after supernatant liquid, add 5～15 weight portion citric acids, 5～25 weight portion Mel, 5～30 weight portion white sugars, be heated to 40～75 DEG C, 100～300rpm stirs, mixing time 10～30min, mix homogeneously, 0.45 μ m membrane filtration again while hot, 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 DEG C, keep 30～50min, the autologous room temperature that is cooled to obtains medical treatment and prevent the wood frog of beriberi to extract mixed liquor oral agents.
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| CN113854550A (en) * | 2021-09-13 | 2021-12-31 | 穆棱市蛙宝生物科技发展有限公司 | Production process of active snow clam ultrafine freeze-dried powder |
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