CN107375503A - The preparation method of soapberry pericarp activity extract and its antimycotic application - Google Patents

The preparation method of soapberry pericarp activity extract and its antimycotic application Download PDF

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Publication number
CN107375503A
CN107375503A CN201710649920.XA CN201710649920A CN107375503A CN 107375503 A CN107375503 A CN 107375503A CN 201710649920 A CN201710649920 A CN 201710649920A CN 107375503 A CN107375503 A CN 107375503A
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ethanol
soapberry
soapberry pericarp
preparation
water
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苗得足
胡清文
焦启扬
曹桂东
曹燕
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Reyoung Pharmaceutical Co Ltd
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Reyoung Pharmaceutical Co Ltd
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/77Sapindaceae (Soapberry family), e.g. lychee or soapberry
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30Extraction of the material
    • A61K2236/33Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30Extraction of the material
    • A61K2236/33Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30Extraction of the material
    • A61K2236/39Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50Methods involving additional extraction steps
    • A61K2236/51Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50Methods involving additional extraction steps
    • A61K2236/55Liquid-liquid separation; Phase separation

Abstract

The invention belongs to the extraction process technology field of active ingredient of natural product, and in particular to a kind of preparation method of soapberry pericarp activity extract and its antimycotic application.Soapberry pericarp is crushed, handled with solvent, the extract solution that merging treatment obtains, concentration, by concentrate by D392 type weak-base anion-exchange resin posts, eluted successively with the ethanol water of water and various concentrations, collect the eluent of ethanol water, the eluent of collection is concentrated, dried, produces product.Reach more than 95% using the soapberry pericarp activity extract purity obtained by the method for the invention extraction purification, there is the characteristics of cost is low, easy to operate, purity is high, activity is good, mechanism is clear and definite, has no toxic side effect.The soapberry pericarp activity extract prepared by preparation method of the present invention, applied to the medicine for preparing the associated diseases such as Candida albicans, Trichophyton rubrum, envelope monilial infection of in antifungal drug, being particularly suitable for use in.

Description

The preparation method of soapberry pericarp activity extract and its antimycotic application
Technical field
The invention belongs to the extraction process technology field of active ingredient of natural product, and in particular to a kind of soapberry pericarp is lived The preparation method of property extract and its antimycotic application.
Background technology
Due to the development of the technologies such as the extensive use of various antibiotic, organ transplant, the illness rate of fungal infection and lethal Rate is in rising trend in the whole world.According to the difference for invading human body, fungal infectious disease can be divided into superficial fungi sense Dye, dermatophytid infection, hypodermis fungal infection and systemic fungal infection.Wherein, superficial fungal infection and dermatophyte Infection is referred to as mycotic infection of superficial part, and hypodermis fungal infection and systemic fungal infection are referred to as invasive infections with fungi. Mycotic infection of superficial part is the infection caused by the cutaneous cuticula of pathomycete, hair and deck, and Main Pathogenic Bacteria is skin Skin tinea bacterium, other common pathogens also have chlosma and candida albicans etc., and directly contact is its main mode of transmission.Dermatophyte Deng Main Pathogenic Bacteria from 3 category, i.e., Microsporon, trichophyton and Epidermophyton, colpomycosis mainly cause Germ is Mycotoruloides.Deep fungal infection is mainly by opportunistic fungus, such as Mycotoruloides, aspergillus and Cryptococcus institute Cause, account for more than the 80% of HSCT, organ transplant and other immune deficient patients' fungal infections.
Due to widely using for various high-efficiency broad spectrum antibacterials, pathogenic microorganism generates a variety of resistance mechanisms to keep The continuity of species, and it is exactly a kind of important resistance mechanism to form biofilm.Biofilm refers to that a large amount of microorganisms attach to A kind of film sample aggregation that the surface of some objects is formed, has complicated three-dimensional structure and function, it is now recognized that human body 80% is micro- Biological infection is related to the biofilm of pathogenic microorganism.Biofilm related infection can influence patient's prognosis, clinical research Show that its fatal rate of candida albicans bloodstream infection is by not producing biofilm candida albicans as caused by generation biofilm candida albicans Caused by 1.77 times of bloodstream infection, the independent hazard factor that biofilm produces and candidemia stream infected patient is dead. Zoopery also indicates that there is stronger poison from the free candida albicans of the more abiotic envelope of free candida albicans of biofilm diffusion Power, so as to which its infection can cause worse final result.Biofilm related infection is difficult control, because it has stronger resistance Property, common antifungal drug treatment is difficult fully erased biofilm.Clinic there is no preferable right to candida albicans biofilm at present Plan, how to prevent and treat and overcome biofilm resistance problems, it has also become clinically extremely challenging problem.
Soapberry (Sapindus mukorossi Gaertn.), also known as oil suffers from son, wood suffers from son, fruit of washing one's hands, soap tree etc., Belong to Sapindaceae Sapindus defoliation small arbor, be distributed widely on the south China Huaihe River and south east asia, in China's main product In Guangdong, Guangxi.Soapberry begins to be recorded in《Compendium of Materia Medica》, there is the effect such as heat-clearing eliminating the phlegm, disperse accumulations desinsection.Modern pharmacological research It was found that soapberry has a variety of physiologically actives, mainly including antibacterium, antimycotic, antitumor, activity against snails, insecticidal activity, anti- Inflammation, antiulcer, Ischemic myocardium, preventing thrombosis, spermicidal, liver protection etc..
The main chemical compositions of soapberry pericarp are soapberry active site, predominantly hederagenin type triterpene soap Glycosides, there is very strong non-ionic surface active to act on, be excellent natural detergent, so being ground at present on a large amount of of soapberry Study carefully and be concentrated mainly in its cleaning dirt-removing power.Soapberry applies more, the extraction of its antibacterial activity position in cleaning chemical products Method mainly includes water extraction or organic solvent extraction.Although researchers have done many works to the separation and Extraction of soapberry Make, but generally speaking all also in the Primary Study stage, accomplishing scale production, it is low purity to be also present, such as water extraction, water extraction The recovery rate of method is higher, but is often also extracted some monose, oligosaccharides, amino acid, protein, lymphatic temperament etc., So as to cause the saponin(e purity in extract to reduce, or the shortcomings of cost height, poor operability.There is ultrafiltration point at present From soapberry active site extraction processes such as, Microwave Extractions, but the shortcomings of cleaning suffering, solvent loss are big, and purity is not high be present, Therefore it is necessary to soapberry active site extraction process carry out deeper into research, with obtain low cost, high-purity, operation Easy, green extraction process, realize or improve the industrialized production of soapberry active site.
The content of the invention
In view of the shortcomings of the prior art, it is an object of the invention to provide a kind of preparation side of soapberry pericarp activity extract Method, the soapberry pericarp activity extract purity of preparation reach more than 95%, have it is easy to operate, be adapted to industrialized production, pure Degree is high, activity is good, mechanism clearly, the characteristics of having no toxic side effect;The present invention also provides one kind and extract is applied into antifungal Applied in thing.
A kind of preparation method of soapberry pericarp activity extract of the present invention, soapberry pericarp is crushed, use is molten Agent is handled, the extract solution that merging treatment obtains, concentration, by concentrate by D392 type weak-base anion-exchange resin posts, according to It is secondary to be eluted with the ethanol water of water and various concentrations, the eluent of ethanol water is collected, the eluent of collection is carried out dense Contracting, dry, produce product.
Wherein:
Preferably, eluted successively with the ethanol water of water and various concentrations, the eluent for collecting ethanol water is:According to It is secondary with water, 50 ± 2% ethanol, 75 ± 2% ethanol, 95% ± 2 ethanol elution, collect 50 ± 2% ethanol, 75 ± 2% ethanol and 95 ± 2% ethanol eluates.Wherein 50 ± 2% ethanol represent ethanol water of the concentration as 48-52%, and other are analogized.
Solvent is water, ethanol water, methanol aqueous solution or aqueous acetone solution.
Handled with solvent is to handle or add solvent with solvent supersonic to be heated to reflux handling.
It it is at least 3 times with solvent number of processes, solvent addition weight is 8-12 times of soapberry pericarp when per treatment, often Secondary processing time is 1.8-2.2 hours.
Soapberry fruit tare weight amount is 1 with resin column volume ratio:2, soapberry fruit tare weight amount is in terms of g, and resin column volume is with mL Meter;The blade diameter length ratio of resin column is 1:10.
Eluent flow rate is 1 column volume/hour.
It is to be crushed after soapberry pericarp is dried that soapberry pericarp, which is crushed, and the particle diameter after crushing is not specially required.
Application of the soapberry pericarp activity extract prepared by preparation method of the present invention in antimycotic, no trouble Sub- pericarp activity extract is used to prepare treatment fungal infection associated diseases, monilial infection associated diseases, Candida albicans sense Contaminate associated diseases, envelope monilial infection associated diseases, Trichophyton rubrum infection associated diseases, colpomycosis or ringworm of the body Medicine.
Pharmaceutical dosage form is ointment, emulsion, lotion or suppository, the addition weight of soapberry pericarp activity extract in medicine For 1%~20%.
First, sample 1-12 antifungal activities are tested:
Minimal inhibitory concentration (MIC) assay method:Bacterial strain uses therefor is:Candida albicans SC5314 (wild type), red hair Tinea bacterium ATCC 28188, black-koji mould ATCC 16404.Bacterial strain is suspended in the physiological saline containing 20% glycerine, in -80 DEG C It is long-term to preserve.Before experiment, bacterial strain line is forwarded to YPD solid plates, 30 DEG C of quiescent culture 24h, the single bacterium colony grown turned YPD fluid nutrient mediums are connected to, 30 DEG C, 200rpm is incubated overnight, by the bacterium solution being incubated overnight with about 1:100 ratios are inoculated in fresh YPD culture mediums in, 30 DEG C of shaken cultivation 4h, now Candida albicans be in exponential phase, can be utilized for subsequent experimental.
The determination of MIC value is the fungi sensitivity Detection side formulated according to U.S. clinical and experimental standard association (CLSI) Method (M27-A3), is measured using the method for Microdilution.Specific experiment step is:With RPMll640 culture mediums by bacterium to be measured Strain bacteria concentration is adjusted to 0.5~2.5 × 103cells/mL:200 μ l are added in 96 orifice plate, first hole contain maximum concentration treat The bacterium solution of medicine is surveyed, remaining respectively adds the blank bacterium solution of 100 μ l not drug containing per hole;Using the method for two times of gradient dilutions by bacterium Liquid is diluted to a series of concentration gradients;By orifice plate in 35 DEG C of constant temperature quiescent culture 24h.Each concentration gradient is detected with ELIASA to exist Absorbance (OD values) at 570nm, deduct blank cultures absorbance after, using the ratio of each hole OD values divided by control group OD values as Its growth rate, it is MIC value using Cmin of the inhibiting rate (1- growth rates) more than 80% as its minimal inhibitory concentration.
The preparation of sample:
1st, soapberry pericarp 100g is taken respectively, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;With water ultrasonic extraction 3 Secondary, every time plus 10 times of amount solvents, extraction time are 2 hours, merge extract solution, are concentrated into appropriate;Concentrate is passed through into D392 types Resin, soapberry medicinal material weight are 1 with resin volume ratio:2, the blade diameter length ratio of resin column is 1:10, successively with water, 50 ± 2% second Alcohol, 75 ± 2% ethanol, 95% ± 2 ethanol elution, flow velocity are 1 column volume/hour, collect ethanol eluate, are concentrated under reduced pressure, Vacuum drying, produces product, is sample 1.
2nd, soapberry pericarp 100g is taken respectively, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;With water ultrasonic extraction 3 Secondary, every time plus 10 times of amount solvents, extraction time are 2 hours, merge extract solution, are concentrated into appropriate;Concentrate is passed through into D101 types Resin, soapberry medicinal material weight are 1 with resin volume ratio:2, the blade diameter length ratio of resin column is 1:10, successively with water, 50% ± 2% Ethanol, 75% ± 2% ethanol, 95% ± 2% ethanol elution, flow velocity are 1 column volume/hour, collect ethanol eluate, decompression Concentration, vacuum drying, produces product, is sample 2.
3rd, soapberry pericarp 100g is taken respectively, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;With water ultrasonic extraction 3 Secondary, every time plus 10 times of amount solvents, extraction time are 2 hours, merge extract solution, are concentrated into appropriate;Concentrate is passed through into ADS-2 types Resin, soapberry medicinal material weight are 1 with resin volume ratio:2, the blade diameter length ratio of resin column is 1:10, successively with water, 50% ± 2% Ethanol, 75% ± 2% ethanol, 95% ± 2% ethanol elution, flow velocity are 1 column volume/hour, collect ethanol eluate, decompression Concentration, vacuum drying, produces product, is sample 3.
4th, soapberry pericarp 100g is taken respectively, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;With water ultrasonic extraction 3 Secondary, every time plus 10 times of amount solvents, extraction time are 2 hours, merge extract solution, are concentrated into appropriate;Concentrate is passed through into AB-8 types Resin, soapberry medicinal material weight are 1 with resin volume ratio:2, the blade diameter length ratio of resin column is 1:10, successively with water, 50% ± 2% Ethanol, 75% ± 2% ethanol, 95% ± 2% ethanol elution, flow velocity are 1 column volume/hour, collect ethanol eluate, decompression Concentration, vacuum drying, produces product, is sample 4.
5th, soapberry pericarp 100g is taken respectively, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;With water ultrasonic extraction 3 Secondary, every time plus 10 times of amount solvents, extraction time are 2 hours, merge extract solution, are concentrated into appropriate;Concentrate is passed through into HP-20 types Resin, soapberry medicinal material weight are 1 with resin volume ratio:2, the blade diameter length ratio of resin column is 1:10, successively with water, 50% ± 2% Ethanol, 75% ± 2% ethanol, 95% ± 2% ethanol elution, flow velocity are 1 column volume/hour, collect ethanol eluate, decompression Concentration, vacuum drying, produces product, is sample 5.
6th, soapberry pericarp 100g is taken respectively, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;With water ultrasonic extraction 3 Secondary, every time plus 10 times of amount solvents, extraction time are 2 hours, merge extract solution, are concentrated into appropriate;Concentrate is passed through into D152 types Resin, soapberry medicinal material weight are 1 with resin volume ratio:2, the blade diameter length ratio of resin column is 1:10, successively with water, 50% ± 2% Ethanol, 75% ± 2% ethanol, 95% ± 2% ethanol elution, flow velocity are 1 column volume/hour, collect ethanol eluate, decompression Concentration, vacuum drying, produces product, is sample 6.
7th, soapberry pericarp 100g is taken respectively, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;With water ultrasonic extraction 3 Secondary, every time plus 10 times of amount solvents, extraction time are 2 hours, merge extract solution, are concentrated into appropriate;Concentrate is passed through into DM130 types Resin, soapberry medicinal material weight are 1 with resin volume ratio:2, the blade diameter length ratio of resin column is 1:10, successively with water, 50% ± 2% Ethanol, 75% ± 2% ethanol, 95% ± 2% ethanol elution, flow velocity are 1 column volume/hour, collect ethanol eluate, decompression Concentration, vacuum drying, produces product, is sample 7.
8th, soapberry pericarp 100g is taken respectively, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;With water ultrasonic extraction 3 Secondary, every time plus 10 times of amount solvents, extraction time are 2 hours, merge extract solution, are concentrated into appropriate;Concentrate is passed through into HZ-101 Type resin, soapberry medicinal material weight are 1 with resin volume ratio:2, the blade diameter length ratio of resin column is 1:10, successively with water, 50% ± 2% ethanol, 75% ± 2% ethanol, 95% ± 2% ethanol elution, flow velocity are 1 column volume/hour, collect ethanol eluate, It is concentrated under reduced pressure, is dried in vacuo, produces product, be sample 8.
9th, soapberry pericarp 100g is taken, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;With water ultrasonic extraction 3 times, often Secondary plus 10 times of amount solvents, extraction time are 2 hours, merge extract solution, are concentrated into right amount, vacuum drying, produce product, be sample 9。
10th, soapberry pericarp 100g is taken, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;Added with 50% ± 2% ethanol Circumfluence distillation 3 times, each plus 10 times of amount solvents, extraction time are 2 hours, merge extract solution, are concentrated into right amount, vacuum is done It is dry, product is produced, is sample 10.
11st, soapberry pericarp 100g is taken, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;It is heated to reflux with 50% methanol Extraction 3 times, each plus 10 times of amount solvents, extraction time are 2 hours, merge extract solution, are concentrated into right amount, vacuum drying, produce Product, it is sample 11.
12nd, soapberry pericarp 100g is taken, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;It is heated to reflux with 20% acetone Extraction 3 times, each plus 10 times of amount solvents, extraction time are 2 hours, merge extract solution, are concentrated into right amount, vacuum drying, produce Product, it is sample 12.
Purity detecting:Precision weighs hederagenin reference substance 10mg, is placed in beaker, is dissolved and be settled to methanol In 100ml volumetric flask, shake up, reference substance solutions of every 1ml containing 0.1mg is made, refrigeration is standby.
The preparation of standard curve:Precision draws μ l of reference substance solution 20,40 μ l, 80 μ l, 120 μ l, 160 μ l, 200 μ l, point It is not placed in tool plug test tube, low temperature flings to solvent, adds 5% vanillic aldehyde-glacial acetic acid solution 0.2ml, puts in 60 DEG C of waters bath with thermostatic control Heat after fully mixing 15 minutes, cooled down 2 minutes with frozen water immediately, add glacial acetic acid 5ml, shake up;Blank is made with corresponding reagent, According to UV-VIS spectrophotometry, absorbance is determined at 539nm wavelength, using absorbance as ordinate, concentration is painted for abscissa Standard curve processed, it is y=20.421x-0.0266, R to obtain regression equation2=0.9999, see Fig. 1.
Assay method:Active site of the present invention about 50mg is taken, it is accurately weighed, put in 25ml measuring bottles, add methanol to fit Amount makes dissolving and is diluted to scale, shakes up, and precision measures 50 μ l, the method measure absorbance under sighting target directrix curve preparation, so Substitute into afterwards and return the mass concentration that agenda calculates total saposins in sample solution, finally calculating total saposins in sample according to formula contains Amount.The amount of hederagenin in need testing solution is read from standard curve.
Purity and antimycotic it the results are shown in Table 1.
The different preparation method antifungal activity position results of table 1
From experimental result, the active component that soapberry extraction is obtained using D392 type macroporous resin purifications, purity And anti-candida albicanses, Trichophyton rubrum, black-koji mould activity are substantially better than solvent extraction and D101, ADS-2, AB-8, HP- 20th, position obtained by D152, DM130, HZ-101 type macroporous resin purification.
2nd, effect of the heretofore described active site to envelope candida albicans
Candida albicans SC5314 bacterial strains are suspended in the physiological saline containing 20% glycerine, are preserved in -80 DEG C long-term.Experiment Before, bacterial strain line is forwarded to YPD solid plates, 30 DEG C of quiescent culture 24h, the single bacterium colony grown is forwarded to the training of YPD liquid Base is supported, 30 DEG C, 200rpm is incubated overnight, by the bacterium solution being incubated overnight with about 1:100 ratios are inoculated in fresh YPD culture mediums In, 30 DEG C of shaken cultivation 4h, now Candida albicans be in exponential phase, can be utilized for subsequent experimental.
The fungi sensitive detection method (M27-A3) formulated according to U.S. clinical and experimental standard association (CLSI), is adopted It is measured with the method for Microdilution.
Specific experiment step is:
(1) strain to be tested bacteria concentration is adjusted to l × 10 with the culture mediums of RPM II 6406cells/mL;
(2) bacterium solution diluted is added in 96 orifice plates with 100 μ l per hole, the quiescent culture 24h at 37 DEG C, now in hole Ripe envelope has been formed on plate bottom;
(3) supernatant is abandoned, is washed twice with sterile PBS buffer, adds the culture mediums of RPM II 640 containing compound, is cultivated Base adds 200 μ l and contains the bacterium solution of maximum concentration medicine to be measured, and remaining respectively adds the blank bacterium solution of 100 μ l not drug containing per hole;
(4) bacterium solution is diluted to by a series of concentration gradients using the method for two times of gradient dilutions;
(5) by orifice plate in 35 DEG C of constant temperature quiescent culture 24h;
Sampling, dilution, point plate count, and set three repeating groups, by YPD flat boards in more than 300C quiescent cultures 24h, according to The clump count grown, the viable count of original bacteria liquid is calculated, compared with viable count in 0 time point bacterium solution, calculate depositing for Candida albicans Motility rate.
Drug concentration of the Candida albicans survival rate that bacterium colony counts to get less than 10% is read envelope as active site The minimum activity of pearl bacterium.It the results are shown in Table 2.
Inhibitory action of the sample 1-12 active sites of table 2 to envelope fungi
From experimental result, the active component that soapberry extraction is obtained using D392 type macroporous resin purifications is to envelope Candida albicans activity is substantially better than D101, ADS-2, AB-8, HP-20, D152, DM130, HZ-101 type macroporous resin purification and solvent Extract the active component of gained.
3rd, the antifungic action Mechanism Study of heretofore described active site
Detect permeability of cell membrane:We are with shadows of the fluorescent dye PI come detection compound to permeability of cell membrane.Specifically Experimental procedure is:
(1) final concentration of 0,32,64,128 μ g/ml active site is added in the SC5314 bacterium solutions diluted, 30 DEG C It is incubated 3h;
(2) final concentration of 5 μ g/ml PI dyestuffs are added, dark is incubated 30min;
(3) after PBS washing, flow cytomery is transferred to be colored cells ratio.Fluidic cell instrument parameter is adjusted Adjusting method:Undyed normal white candida albicans is adjusted within 101, keeps parameter constant to detect remaining sample, it is strong with fluorescence The cell more than 101 is spent to be colored cell, as a result sees Fig. 2.
PI is film selective penetrated property dyestuff, and it can only pass through to receive and damage and the increased cell membrane of permeability.Experimental result Show that the addition of active site of the present invention can result in by the cell quantity of PI dyeing to be increased, this result shows higher dose The active site of the present invention of amount more easily causes the damage of cell membrane.Above test result indicates that, it is of the present invention Active site can interfere with the integrality that membrane structure causes the increase of its permeability to destroy cell membrane.
In summary, because the antifungal mechanisms of active site of the present invention are increase membrane passages, so as to The integrality of cell membrane is destroyed, therefore is applied to a variety of fungal infections.It is particularly suitable for use in and mainstream medicine in the market is produced Raw drug resistance and the infection as caused by envelope candida albicans, such as the vaginitis as caused by candida albicans infection and by Trichophyton rubrum Ringworm of the body caused by infection etc. is by fungus-caused disease.
4th, toxicity of the heretofore described active site to normal cell
1. material
Cell line:HUVEC human umbilical vein endothelial cell lines.
1.2 reagents and culture medium
1.2.1 reagent and source
It is shown in Table 3.
Table 3 is to normal cytotoxicity experiment reagent
Compound accurate weighing is dissolved in DMSO, and AMB accurate weighings are dissolved in aseptic deionized water, and mother liquid concentration is 10mg/ml, be stored in -20 DEG C it is standby.
1.2.2 instrument and equipment
It is shown in Table 4.
Table 4 is to normal cytotoxicity experiment instrument and equipment
2. method
Cell line culture containing 10% hyclone, 100U/ml penicillin, 100 μ g/ml streptomysins DMEM culture mediums In, 5%CO2, 37 DEG C of constant temperature and humidity quiescent cultures.
The influence bred using MTT method come detection compound to human cell line HUVEC cells.Specific experiment step For:
(1) cell is diluted to 1 × 104Cells/Ml, it is inoculated in 96 orifice plates, 37 DEG C, 5%CO2Cultivate 24h;
(2) compound of the present invention and AMB (positive control) of each concentration gradient are added, each gradient sets 3 Repeating hole, similarity condition culture 24h;
(3) the 10 final concentration of 0.5mg/ml of μ l MTT solution is added per hole, continues 37 DEG C of lucifuges and is incubated 4h, now bottom hole There is bluish violet crystallization;
(4) add 100 μ l DMSO in every hole to be completely dissolved crystallization, per hole extinction under ELIASA detection 570nm wavelength Degree;
(5) IC is calculated according to each hole drug concentration and absorbance50Value.
3rd, observation index
It is shown in Table 5.
Table 5 is to normal cytotoxicity
From experimental result, the active site of the embodiment of the present invention 1 toxicity of normal cell is less than D101, ADS-2, The active site of AB-8, HP-20, D152, DM130, HZ-101 type macroporous resin purification, much smaller than anphotericin, Clinical practice It is safer.
In summary, generally developed immunity to drugs problem for antifungal drug clinical conventional at present, it is of the present invention The antifungal mechanisms of soapberry active site are clear and definite, hence it is evident that better than solvent extraction, D101, ADS-2, AB-8, HP-20, D152, The active component of DM130, HZ-101 type macroporous resin purification purifying gained;The toxicity of normal cell is less than D101, ADS-2, Active component obtained by AB-8, HP-20, D152, DM130, HZ-101 type macroporous resin purification, hence it is evident that less than anphotericin.Tool Have that mechanism is clear and definite, curative for effect, the advantages of being easy to industrial production, being particularly suitable for use in, it is resistance to that mainstream medicine in the market is produced The property of medicine and the infection as caused by envelope candida albicans.
Compared with prior art, the present invention has advantages below:
(1) use the soapberry pericarp activity extract purity obtained by the method for the invention extraction purification reach 95% with On, there is the characteristics of purity is high, activity is good, mechanism is clear and definite, has no toxic side effect.
(2) complicated for prior art soapberry method for extraction and purification, efficiency is low, the actual application problem such as cost height, this Invention provides a kind of low cost, high-purity, easy to operate, suitable industrialized production, green soapberry active site and carried The new method of purifying is taken, is generally developed immunity to drugs problem for antifungal drug clinical conventional at present, the invention provides one The new sources of kind antifungal drug.
(3) the soapberry pericarp activity extract prepared by preparation method of the present invention, applied to antifungal drug In, it is particularly suitable for use in and prepares Candida albicans, Trichophyton rubrum, the medicine of envelope monilial infection associated diseases.
Brief description of the drawings
Fig. 1 is the canonical plotting of hederagenin;
Fig. 2 is active site permeability of cell membrane figure.
Embodiment
With reference to embodiment, the present invention will be further described.
Embodiment 1
Soapberry pericarp 10kg is taken, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;With water ultrasonic extraction 3 times, every time Add 10 times of amount solvents, extraction time is 2 hours, merges extract solution, is concentrated into appropriate;Concentrate is passed through into D392 type resins, nothing It is 1 to suffer from sub- medicinal material weight with resin volume ratio:2, the blade diameter length ratio of resin column is 1:10, successively with water, 50% ± 2% ethanol, 75% ± 2% ethanol, 95% ± 2% ethanol elution, 50% ± 2% ethanol, 75% ± 2% ethanol eluate are collected, will be collected Eluent be concentrated under reduced pressure, be dried in vacuo, produce.
Embodiment 2
Soapberry pericarp 10kg is taken, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;Boiled 3 times with decocting, add 10 every time Amount solvent again, extraction time are 2 hours, merge extract solution, are concentrated into appropriate;Concentrate is passed through into D392 type resins, soapberry Medicinal material weight is 1 with resin volume ratio:2, the blade diameter length ratio of resin column is 1:10, successively with water, 50% ± 2% ethanol, 75% ± 2% ethanol, 95% ± 2% ethanol elution, 75% ± 2% ethanol, 95% ± 2% ethanol eluate are collected, by the elution of collection Liquid is concentrated under reduced pressure, and vacuum drying, is produced.
Embodiment 3
Soapberry pericarp 10kg is taken, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;Heated back with 50% ± 2% ethanol Stream extraction 3 times, every time plus 10 times of amount solvents, extraction time are 2 hours, merge extract solution, are concentrated into appropriate;Concentrate is passed through D392 type resins, soapberry medicinal material weight are 1 with resin volume ratio:2, the blade diameter length ratio of resin column is 1:10, successively with water, 50% ± 2% ethanol, 75% ± 2% ethanol, 95% ± 2% ethanol elution, collect 50% ± 2% ethanol, 75% ± 2% ethanol, 95% ± 2% ethanol eluate, the eluent of collection is concentrated under reduced pressure, is dried in vacuo, produces.
Embodiment 4
Soapberry pericarp 10kg is taken, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;It is heated to reflux carrying with 50% methanol Take 3 times, every time plus 10 times of amount solvents, extraction time are 2 hours, merge extract solution, are concentrated into appropriate;Concentrate is passed through into D392 Type resin, soapberry medicinal material weight are 1 with resin volume ratio:2, the blade diameter length ratio of resin column is 1:10, successively with water, 50% ± 2% ethanol, 75% ± 2% ethanol, 95% ± 2% ethanol elution, collect 50% ± 2% ethanol, 75% ± 2% ethanol, 95% ± 2% ethanol eluate, the eluent of collection is concentrated under reduced pressure, is dried in vacuo, produces.
Embodiment 5
Soapberry pericarp 10kg is taken, picks up the removal of impurity, by raw material stoving, is ground into coarse powder;It is heated to reflux carrying with 20% acetone Take 3 times, every time plus 10 times of amount solvents, extraction time are 2 hours, merge extract solution, are concentrated into appropriate;Concentrate is passed through into D392 Type resin, soapberry medicinal material weight are 1 with resin volume ratio:2, the blade diameter length ratio of resin column is 1:10, successively with water, 50% ± 2% ethanol, 75% ± 2% ethanol, 95% ± 2% ethanol elution, collect 50% ± 2% ethanol, 75% ± 2% ethanol, 95% ± 2% ethanol eluate, the eluent of collection is concentrated under reduced pressure, is dried in vacuo, produces.
Embodiment 6
It is prepared by soapberry ointment
Vaseline 940g and lanolin 50g is taken, is heated, filtration, is let cool to about 50 DEG C, adds soapberry activity extract 10g, stir evenly to half solidification shape, 1000g is made, produces.
Embodiment 7
It is prepared by soapberry ointment
Vaseline 750g and lanolin 50g is taken, is heated, filtration, is let cool to about 50 DEG C, adds soapberry activity extract 200g, stir evenly to half solidification shape, 1000g is made, produces.
Embodiment 8
It is prepared by soapberry emulsion
Take glycerine 80g, magnesium stearate 120g, triethanolamine 30g, liquid paraffin 180g, paraffin 80g, ethyl hydroxy benzoate 1.5g, Distilled water adds to 1000g, is placed in a container, is heated to 85~90 DEG C, treats to dissolve completely, stops heating, stirring is to condensing, i.e., Obtain emulsion-type matrix.Soapberry activity extract 200g is taken, emulsion-type matrix 800g is added, stirs, 1000g is made, i.e., .

Claims (10)

  1. A kind of 1. preparation method of soapberry pericarp activity extract, it is characterised in that:Soapberry pericarp is crushed, at solvent Reason, the extract solution that merging treatment obtains, concentration, by concentrate by D392 type weak-base anion-exchange resin posts, use successively The ethanol water of water and various concentrations elutes, and collects the eluent of ethanol water, the eluent of collection is concentrated, done It is dry, produce product.
  2. 2. the preparation method of soapberry pericarp activity extract according to claim 1, it is characterised in that:Successively with water and The ethanol water elution of various concentrations, the eluent for collecting ethanol water are:Successively with water, 50 ± 2% ethanol, 75 ± 2% ethanol, 95% ± 2 ethanol elution, collect 50 ± 2% ethanol, 75 ± 2% ethanol and 95 ± 2% ethanol eluates.
  3. 3. the preparation method of soapberry pericarp activity extract according to claim 1, it is characterised in that:Solvent be water, Ethanol water, methanol aqueous solution or aqueous acetone solution.
  4. 4. the preparation method of soapberry pericarp activity extract according to claim 3, it is characterised in that:Handled with solvent It is heated to reflux handling for solvent is handled or added with solvent supersonic.
  5. 5. the preparation method of soapberry pericarp activity extract according to claim 4, it is characterised in that:Handled with solvent Number is at least 3 times, and solvent addition weight is 8-12 times of soapberry pericarp when per treatment, and the time per treatment is 1.8- 2.2 hour.
  6. 6. the preparation method of soapberry pericarp activity extract according to claim 1, it is characterised in that:Soapberry pericarp Weight is 1 with resin column volume ratio:2, soapberry fruit tare weight amount is in terms of g, and resin column volume is in terms of mL;The blade diameter length ratio of resin column is 1:10。
  7. 7. the preparation method of soapberry pericarp activity extract according to claim 1, it is characterised in that:Eluent flow rate For 1 column volume/hour.
  8. 8. the preparation method of soapberry pericarp activity extract according to claim 1, it is characterised in that:By soapberry fruit It is to be crushed after soapberry pericarp is dried that skin, which crushes,.
  9. 9. a kind of soapberry pericarp activity extract prepared by any described preparation methods of claim 1-8 is in antimycotic Application, it is characterised in that:For preparing treatment fungal infection associated diseases, monilial infection associated diseases, Candida albicans Infect associated diseases, envelope monilial infection associated diseases, Trichophyton rubrum infection associated diseases, colpomycosis or ringworm of the body Medicine.
  10. 10. application according to claim 9, it is characterised in that:Pharmaceutical dosage form is ointment, emulsion, lotion or suppository.
CN201710649920.XA 2017-08-02 2017-08-02 The preparation method of soapberry pericarp activity extract and its antimycotic application Pending CN107375503A (en)

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