CN110123905A - A kind of soapberry in-situ gel preparation that treating candida albicans vaginitis and preparation method - Google Patents
A kind of soapberry in-situ gel preparation that treating candida albicans vaginitis and preparation method Download PDFInfo
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- CN110123905A CN110123905A CN201910504792.9A CN201910504792A CN110123905A CN 110123905 A CN110123905 A CN 110123905A CN 201910504792 A CN201910504792 A CN 201910504792A CN 110123905 A CN110123905 A CN 110123905A
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/77—Sapindaceae (Soapberry family), e.g. lychee or soapberry
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation 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
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation 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
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction 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 or decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation 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
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
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- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Gynecology & Obstetrics (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Reproductive Health (AREA)
- Biotechnology (AREA)
- Medical Informatics (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Communicable Diseases (AREA)
- Alternative & Traditional Medicine (AREA)
- Urology & Nephrology (AREA)
- Botany (AREA)
- Oncology (AREA)
- Microbiology (AREA)
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- Endocrinology (AREA)
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Abstract
The present invention relates to a kind of soapberry in-situ gel preparation for treating candida albicans vaginitis and preparation methods, belong to Amino-Cerv technical field.The technical solution of the present invention is as follows: preparing soapberry water mentions Fermented Condensed liquid, fermentation liquid is mentioned according to the water that its saponin content is configured to suitable concentration, and the water is mentioned into fermentation liquid and is prepared into responsive to temperature type gel, guaranteed in intravaginal effective stay time.Soapberry in-situ gel preparation preparation is simple, and toxic side effect is small, anti-candida albicans significant effect, and the potential drug resistance that can be avoided azole antifungal agent threatens, and soapberry is from a wealth of sources, materials convenience.
Description
Technical field
The present invention relates to Amino-Cerv technical fields, and in particular to a kind of soapberry for treating candida albicans vaginitis
In-situ gel preparation and preparation method.
Background technique
Candida albicans is a kind of conditioned pathogen, and candidiasis is by candidal infection, it may occur however that
Skin, eyes, oral cavity, esophagus, gastrointestinal tract, vagina and vascular system.Vulvovaginal candidiasis (VVC) is common women yin
Tract disease.According to statistics, in the world, VVC is the most common fungal infection of the mankind, and the women more than 75% is their one
At least breaking-out is primary in life, and the women of 5%-8% then can recurrent exerbation.Vulvovaginitis candidiasis (VVC) clinical manifestation is
Vagina and vulva tissue itch, pain and burning heat sensation, vaginal fluid increase, to influence female life quality.
It is controlled currently, providing azole antifungal agent with intravaginal local administration or oral dosage form and being usually proposed as first choice
Treatment method.However, this antifungal agents composes narrow and biggish toxic effect, and since excessively using antifungal agent, there are potential resistance to
Pharmacological property threatens, and the factors such as low dosage or single-dose treatment cause vulvovaginitis candidiasis (VVC) recurrence rate to rise.Therefore,
Natural origin substance antibacterial therapy VVC has become the direction of more and more colpitis medicine exploitations.
Soapberry, also known as fruit of washing one's hands, You Luoshu, a kind of deciduous tree being grown on torrid areas have anti-inflammatory, analgesia,
The functions such as anti-tumor activity, root, skin, leaf, pericarp, seed etc. all have very strong medical value, are rare natures
Resource.Saponin(e rich in soapberry pericarp, existing research show that sapindoside has antibacterial activity in vitro, and without trouble
Son materials convenience, has good product development prospect.
The extracting method type of soapberry pericarp is more, including water extraction and alcohol precipitation method, n-butanol extraction, Ultra filtration membrane
Method, Amberlyst process and microbe fermentation method etc..Wherein, water extraction and alcohol precipitation method application is the most universal, but product saponin(e it is pure
It spends not high.In recent years, microbe fermentation method applied very extensive, some macromolecular complex by the more difficult removal of physical chemistry method
Matter can be decomposed by microorganism, and effect is very significant.Also, its product purity of microbe fermentation method is than conventional water extraction
Products obtained therefrom purity is high, while saponin(e can be modified during the fermentation, so that the biology for improving saponin(e effective component is living
Property.
Situ-gel is that one kind is administered with solution state, phase transition can occur in agents area, be converted to non-ization by liquid
Learn the preparation of crosslinking semi-solid gel.Original flavor gel has good biocompatibility, can have effectively in medicine-feeding part
Residence time is a kind of novel drug delivery system to guarantee the effect of drug.
Summary of the invention
Primary and foremost purpose of the invention is to be to provide one for the deficiency of existing treatment candida albicans vaginitis drug
Kind is prepared simply, and toxic side effect is small, and the soapberry of the effective treatment candida albicans vaginitis of anti-candida albicans is former
Position gel preparation and preparation method.
It is another object of the present invention to the limitations of administration mode existing for the product for existing treatment Candida albicans
Property, it is to provide the vaginal drug delivery system of above-mentioned in-situ gel preparation.
The purpose of the invention is achieved by the following technical solution:
A kind of preparation method of soapberry in-situ gel preparation that treating candida albicans vaginitis, is mentioned with soapberry water
Fermented Condensed liquid is main active, mentions fermentation liquid according to the water that its saponin content is configured to suitable concentration, and the water is mentioned
Fermentation liquid is prepared into responsive to temperature type gel；It is main to be realized by following steps:
Step 1: preparing soapberry water mentions Fermented Condensed liquid
(1.1) soapberry dry fruit skin is crushed, powder is mixed into extraction with water 1:3 in mass ratio under conditions of 40 DEG C
First time filtrate and filter residue is obtained by filtration in 6h.
(1.2) under conditions of 40 DEG C, filter residue is mixed with water by the mass ratio of 1:2 and extracts 4h, is obtained by filtration second
Secondary filtrate, discards filter residue；
(1.3) merge first time filtrate and second of filtrate, obtain sapindoside coarse filtration liquid.By sapindoside coarse filtration
Liquid obtains soapberry Aqueous extracts with 5000r/min centrifugation 10min；
(1.4) soapberry Aqueous extracts are concentrated to get sapindoside concentrate in evaporation equipment.
(1.5) it is inoculum concentration with 1.0%, grape wine dry ferment is inoculated with into sapindoside concentrate, is issued at 30 DEG C
Ferment 4d obtains soapberry water and mentions Fermented Condensed liquid.
Step 2: measurement soapberry water proposes the content of saponin(e in Fermented Condensed liquid
(2.1) production of standard curve: configuration 1mg/mL oleanolic acid standard solution.The test tube of dried and clean is taken, respectively
0,30,60,90,120,150 μ L oleanolic acid standard solution are added, is placed in 50 DEG C of water-bath drying, 0.4mL5% is successively added
(wt%) it is color developing agent that vanillic aldehyde-glacial acetic acid solution and 1.4mL, which analyze pure perchloric acid, and concussion mixes.It is placed in 70 DEG C of water-baths and adds
Hot 20min, ice-water bath are cooled to room temperature, and are added 10mL glacial acetic acid as solvent, are mixed well.Existed with ultraviolet specrophotometer
Absorbance is measured at 540nm；
(2.2) sample measures: soapberry water is mentioned into Fermented Condensed liquid extension rate, takes 100 μ L dilutions in test tube, weight
Multiple aforesaid operations measurement soapberry water proposes the content of saponin(e in Fermented Condensed liquid.
Step 3: preparing soapberry in-situ gel preparation
At 3-6 DEG C, taking polyethylene glycol 400 is dissolved into soapberry water and mentions in Fermented Condensed liquid, is uniformly mixed, and pool Lip river is added
Husky nurse 407 and PLURONICS F87 swelling completely, add distilled water, soapberry in-situ gel preparation are obtained after mixing.
In the step (1.1), soapberry dry fruit skin is crushed to 20-60 mesh, can be 20 mesh, 30 mesh, 40 mesh, 50 mesh
Or 60 mesh etc..
In the step (1.4), cycles of concentration is 6-7 times；
It is living by grape wine dry-pitching in four times of dilutions of sapindoside concentrate in the step (1.5)
Change medium is simple, operation is simple, and the time is short.
In the step (2.2), it is 10-100 times that soapberry water, which mentions Fermented Condensed liquid extension rate, can be 10 times, 20
Again, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times.
In the step 3, soapberry in-situ gel preparation preparation temperature be 3-6 DEG C, can be 3 DEG C, 3.5 DEG C, 4 DEG C,
4.5 DEG C, 5 DEG C, 5.5 DEG C or 6 DEG C etc..
In the step 3, it is 10-80mg/mL that soapberry water used, which mentions in Fermented Condensed liquid saponin concentrations, such as can be with
It is 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 60mg/mL or 80mg/mL etc..
In the step 3, the additive amount of poloxamer188 is 16-22 parts, such as can be 16 parts, 17 parts, 18 parts, 19
Part, 20 parts, 21 parts or 22 parts etc..
In the step 3, the additive amount of PLURONICS F87 is 0.1-5 parts, such as can be 0 part, 0.5 part, 1 part, 1.5
Part, 2 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts, 4.5 parts or 5 parts etc..
In the step 3, the additive amount of polyethylene glycol 400 is 0.1-3 parts, such as can be 0 part, 0.5 part, 1 part, 1.5
Part, 2 parts, 2.5 parts or 3 parts etc..
In the step 3, the additive amount that soapberry water mentions Fermented Condensed liquid is 50 parts.
The present invention has the following advantages compared with the existing technology and effect:
1. the present invention directlys adopt soapberry pericarp and extracts, operating procedure is simple and easy, high-efficient, and soapberry fruit
Skin materials are convenient, simple and easy to get.
2. a kind of soapberry in-situ gel preparation for treating candida albicans vaginitis that the present invention obtains has preparation
Simply, the characteristics of toxic side effect is small, anti-candida albicans significant effect acts on the minimum inhibitory concentration of Candida albicans
It (MIC) can be down to 0.0625mg/mL (in terms of saponin content).
3. a kind of soapberry in-situ gel preparation for treating candida albicans vaginitis that the present invention obtains, can make up for it
The deficiency of antibiotics reduces the recurrence rate of candida albicans vaginitis.
4. a kind of soapberry in-situ gel preparation for treating candida albicans vaginitis that the present invention obtains, can be realized
Situ treatment extends drug effective stay time, is a kind of vaginal drug delivery system.
5. a kind of soapberry in-situ gel preparation for treating candida albicans vaginitis that the present invention obtains, pharmacological evaluation
It proves that there is Candida albicans and significantly inhibits effect, animal experiments prove that, Candida albicans mouse can be obviously improved
Illness.
Detailed description of the invention
Fig. 1 is the mouse vagina irrigating solution fungal burden figure infected after Candida albicans ATCC10231.
Fig. 2 is different immunological marker object level views.Wherein, (a) is IL-1 β, (b) is IL-6, is (c) IL-8, (d) is
LDH。
Fig. 3 is mouse vagina tissue pathological slice figure.Wherein, (A) is IL-1 β, and (B) is IL-6, and (C) is IL-8, and (D) is
LDH。
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, embodiment of the present invention will be done into one below
The detailed description of step, but these embodiments must not be interpreted as the limiting to the claimed invention in all senses.
Soapberry water proposes the preparation of Fermented Condensed liquid:
Soapberry dry fruit skin is crushed to 20-60 mesh, extracts this powder and water 1:3 in mass ratio under conditions of 40 DEG C
First time filtrate and filter residue is obtained by filtration in 6h.Under the conditions of same temperature, filter residue is extracted into 4h in the ratio of 1:2 with water again,
Second of filtrate is obtained by filtration, discards filter residue, merges filtrate twice, obtains sapindoside coarse filtration liquid.By coarse filtration liquid with
6000r/min centrifugation 10min obtains soapberry Aqueous extracts, is concentrated 6 times in evaporation equipment and obtains sapindoside concentrate.Choosing
Selecting grape wine dry ferment is fermentation strain, and with 1.0% inoculum concentration, the 4d that ferments at 30 DEG C obtains soapberry water and mentions Fermented Condensed
Liquid.
Embodiment 1
This implementation provides a kind of preparation method of soapberry in-situ gel preparation for treating candida albicans vaginitis,
Wherein, soapberry in-situ gel preparation includes the component of following weight: 50 parts of soapberry water mention Fermented Condensed liquid (20mg/mL with
Saponin(e note), 20 parts of poloxamer188s, 3 parts of PLURONICS F87s, 2 parts of polyethylene glycol 400s, distilled water polishing is to 100 parts, no trouble
Sapindoside concentration is 10mg/mL in sub- in-situ gel preparation.
The present embodiment soapberry in-situ gel preparation the preparation method is as follows:
At 4 DEG C, polyethylene glycol 400 is weighed in parts by weight be dissolved into 50 parts of soapberry water and mention in Fermented Condensed liquid, mix
It closes uniformly, poloxamer188 and PLURONICS F87 swelling is added completely, continues plus distilled water is to 100 parts, nothing is obtained after mixing
Suffer from sub- in-situ gel preparation.
Embodiment 2
This implementation provides a kind of preparation method of soapberry in-situ gel preparation for treating candida albicans vaginitis,
Wherein, soapberry in-situ gel preparation includes the component of following weight: soapberry water mention 50 parts of Fermented Condensed liquid (30mg/mL with
Saponin(e note), 20 parts of poloxamer188s, 3 parts of PLURONICS F87s, 2 parts of polyethylene glycol 400s, distilled water polishing is to 100 parts, no trouble
Sapindoside concentration is 15mg/mL in sub- in-situ gel preparation.
The present embodiment soapberry in-situ gel preparation the preparation method is as follows:
At 5 DEG C, polyethylene glycol 400 is weighed in parts by weight be dissolved into 50 parts of soapberry water and mention in Fermented Condensed liquid, mix
It closes uniformly, poloxamer188 and PLURONICS F87 swelling is added completely, continues plus distilled water is to 100 parts, nothing is obtained after mixing
Suffer from sub- in-situ gel preparation.
Implement 3
This implementation provides a kind of preparation method of soapberry in-situ gel preparation for treating candida albicans vaginitis,
Wherein, soapberry in-situ gel preparation includes the component of following weight: soapberry water mention 50 parts of Fermented Condensed liquid (60mg/mL with
Saponin(e note), 20 parts of poloxamer188s, 3 parts of PLURONICS F87s, 2 parts of polyethylene glycol 400s, distilled water polishing is to 100 parts, no trouble
Sapindoside concentration is 30mg/mL in sub- in-situ gel preparation.
The present embodiment soapberry in-situ gel preparation the preparation method is as follows:
At 5.5 DEG C, polyethylene glycol 400 weighed in parts by weight be dissolved into 50 parts of soapberry water and mention in Fermented Condensed liquid,
It is uniformly mixed, poloxamer188 and PLURONICS F87 swelling is added completely, continues plus distilled water is to 100 parts, obtained after mixing
Soapberry in-situ gel preparation.
Embodiment 4
This implementation provides a kind of preparation method of soapberry in-situ gel preparation for treating candida albicans vaginitis,
Wherein, soapberry in-situ gel preparation includes the component of following weight: soapberry water mention 50 parts of Fermented Condensed liquid (80mg/mL with
Saponin(e note), 20 parts of poloxamer188s, 3 parts of PLURONICS F87s, 2 parts of polyethylene glycol 400s, distilled water polishing is to 100 parts, no trouble
Sapindoside concentration is 40mg/mL in sub- in-situ gel preparation.
The present embodiment soapberry in-situ gel preparation the preparation method is as follows:
At 6 DEG C, polyethylene glycol 400 is weighed in parts by weight be dissolved into 50 parts of soapberry water and mention in Fermented Condensed liquid, mix
It closes uniformly, poloxamer188 and PLURONICS F87 swelling is added completely, continues plus distilled water is to 100 parts, nothing is obtained after mixing
Suffer from sub- in-situ gel preparation.
Embodiment 5
This implementation provides a kind of soapberry in-situ gel preparation for treating candida albicans vaginitis and preparation method,
Difference with embodiment 1 is only that soapberry water mentions 50 parts of Fermented Condensed liquid (30mg/mL is remembered with saponin(e).
Embodiment 6
This implementation provides a kind of soapberry in-situ gel preparation for treating candida albicans vaginitis and preparation method,
Difference with embodiment 1 is only that soapberry water mentions 50 parts of Fermented Condensed liquid (40mg/mL is remembered with saponin(e).
Embodiment 7
This implementation provides a kind of soapberry in-situ gel preparation for treating candida albicans vaginitis and preparation method,
Difference with embodiment 1 is only that soapberry water mentions 50 parts of Fermented Condensed liquid (50mg/mL is remembered with saponin(e).
Embodiment 8
This implementation provides a kind of soapberry in-situ gel preparation for treating candida albicans vaginitis and preparation method,
Difference with embodiment 1 is only that soapberry water mentions 50 parts of Fermented Condensed liquid (60mg/mL is remembered with saponin(e).
Embodiment 9
This implementation provides a kind of soapberry in-situ gel preparation for treating candida albicans vaginitis and preparation method,
Difference with embodiment 1 is only that soapberry water mentions 50 parts of Fermented Condensed liquid (70mg/mL is remembered with saponin(e).
Embodiment 10
This implementation provides a kind of soapberry in-situ gel preparation for treating candida albicans vaginitis and preparation method,
Difference with embodiment 1 is only that soapberry water mentions 50 parts of Fermented Condensed liquid (80mg/mL is remembered with saponin(e).
Comparative example 1
This implementation provides a kind of soapberry in-situ gel preparation for treating candida albicans vaginitis and preparation method,
Difference with embodiment 4 is only that, is not added with soapberry water and is mentioned Fermented Condensed liquid.
As follows, the performance of 1-10 of the embodiment of the present invention and comparative example 1 in-situ gel preparation provided is surveyed
Examination:
Soapberry water proposes the measurement of Fermented Condensed liquid saponin content
The production of standard curve: configuration 1mg/mL oleanolic acid standard solution.The test tube for taking dried and clean, is separately added into 0,
30,60,90,120,150 μ L oleanolic acid standard solution are placed in 50 DEG C of water-bath drying, it is fragrant that 0.4mL 5% (wt%) are successively added
It is color developing agent that oxalaldehyde-glacial acetic acid solution and 1.4mL, which analyze pure perchloric acid, and concussion mixes.It is placed in 70 DEG C of water-baths and heats 20min,
Ice-water bath is cooled to room temperature, and is added 10mL glacial acetic acid as solvent, is mixed well.With ultraviolet specrophotometer at 540nm
Measure absorbance；Obtain the accounting equation of content of oleanolic acid y (μ g), y (μ g)=0.0056*OD-0.0491, R2=
0.9995。
The measurement of sapindoside content: sapindoside extracting solution is diluted 50 times, takes 100 μ L dilutions in 20mL ratio
In colour tube, the dilution is then measured into absorbance according to the method described above, calculated content of oleanolic acid y (μ g), sample repeats to survey
Determine three times.Measurement result arithmetic mean of instantaneous value will be calculated three times, and substitute into the content that following equation calculation goes out sapindoside.
The accounting equation of sapindoside content and content of oleanolic acid: the content of saponin(e=y (μ g) × 2.3.
Soapberry water proposes fermentation liquid lowest bacteria fogging-resistant concentration determining (MIC measurement)
Method: experimental strain uses reference culture Candida albicans ATCC10231, and picking colony is used using Maxwell turbidimetry
It is about 10 that sterile saline, which is diluted to bacterial concentration,6The bacterium solution of cfu/mL is stand-by.
12 test tubes are formed a line, are numbered, solid medium (SDA culture medium) 1mL of double nutrition is respectively added,
1mL sample to be tested (concentration 10mg/mL, solvent are distilled water) is added in one pipe, 1mL to the 2nd is drawn after mixing and is managed, is mixed again
Take 1mL to the 3rd to manage, and so on to the 9th pipe, discard 1mL, the 11st pipe makees blank control, the 12nd pipe plus fluorine health with physiological saline
Azoles makees positive control.121 DEG C of high pressure steam sterilizations, after culture medium solidification, above-mentioned prepared 20 μ L of bacteria suspension is added in every pipe,
It is placed in stationary culture 24-48h in 30 DEG C of incubators.Observe result judgement: media surface indicates no bacterial growth without bacterium colony, has
Bacterium colony indicates bacterial growth, the sapindoside extracting solution highest dilution of bacterial growth can be inhibited minimum antibacterial as its
Concentration (MIC).
Soapberry in-situ gel preparation lowest bacteria fogging-resistant concentration determining (MIC measurement)
Method: experimental strain uses reference culture Candida albicans ATCC10231, and picking colony is used using Maxwell turbidimetry
It is about 10 that sterile saline, which is diluted to bacterial concentration,6The bacterium solution of cfu/mL is stand-by.
12 test tubes are formed a line, are numbered, solid medium (SDA culture medium) 1mL of double nutrition is respectively added,
1mL facial cleanser (concentration 10mg/mL, solvent are distilled water) is added in one pipe, 1mL to the 2nd is drawn after mixing and is managed, mixing takes again
1mL to the 3rd is managed, and so on to the 9th pipe, discard 1mL, the 11st pipe makees blank control, the 12nd pipe plus Fluconazole with physiological saline
Make positive control.121 DEG C of high pressure steam sterilizations, after culture medium solidification, above-mentioned prepared 20 μ L of bacteria suspension is added in every pipe, is set
The stationary culture 24-48h in 30 DEG C of incubators.Observe result judgement: media surface indicates no bacterial growth without bacterium colony, there is bacterium
Falling indicates bacterial growth, the sapindoside extracting solution highest dilution of bacterial growth can be inhibited minimum antibacterial dense as its
It spends (MIC).
Fungistatic effect evaluation in soapberry in-situ gel preparation body (by taking case study on implementation 1 as an example)
Soapberry in-situ gel preparation interior animal experiment
(1) mouse beads bacterial type vaginitis model is established
26 eight week old ICR mouse are taken, chooses 5 at random and is only used as blank control group, remaining 21 are subcutaneously injected benzene every other day
Formic acid estradiol (0.1mg/ is only) stimulation false heat, continuous injection one week.After last time stimulates, mouse infection is buffered by PBS
The Candida albicans bacteria suspension (10 of liquid configuration8CFU/mL), infected in such a way that liquid-transfering gun is close to introitus, every 20 μ L,
Consecutive infection 2d.Then, mouse restores 48h so that the infection of Candida albicans is colonized.With PBS buffer solution lavation yin after 48h
Road takes vaginal douche coating SDA agar plate to determine fungal burden, mouse is divided into 3 groups at random according to plate count result,
That is model control group, positive control (Fluconazole) group, soapberry situ-gel group.
(2) grouping administration
In such a way that vagina is perfused, 50 μ L PBS buffer solution are perfused in blank control group and model control group, and soapberry is former
Fluconazole in-situ gel preparation is perfused in the soapberry in-situ gel preparation of position gel group perfusion 10mg/mL, positive controls, daily
Once, continue 11d, and the 0th, 5,10,11d with 50 μ L PBS buffer solution lavation vaginas three times, it is true to be coated on SDA agar plate
Determine fungal burden.
(3) immunological hallmark object and pathological section
It takes treatment 13d to take vaginal douche, cell factor (IL-1 β, IL- is measured by Enzyme-linked Immunosorbent Assay (ELISA)
6, IL-8) and lactic dehydrogenase [LDH].Disconnected cervical approach puts to death mouse, takes vagina tissue, paraffin embedding, HE dyeing, microscopic observation disease
Reason variation, to judge soapberry in-situ gel preparation effect.
The result of above-mentioned performance test is as follows:
1 embodiment 1-14 minimum inhibitory concentration MIC of table
Fungal burden
In the 11d treated after VVC mouse infection, the fungal burden 0,5,10,11d is determined, Fig. 1 is specifically shown in.Mould
The fungal burden of type group, all without significant difference (P < 0.05), illustrates Candida albicans colonisation success in 11d；Control group
(Fluconazole) fungal burden starts the conspicuousness decline (P < 0.05) compared with model group in treatment 10d, illustrates VVC mouse mould
Type models successfully.
Significant difference (P < 0.05) is presented between soapberry situ-gel group and control group and model group, table in 11d
Bright soapberry situ-gel can effectively inhibit mouse body white candida albicans to grow as control group.Soapberry situ-gel
Fungal burden persistently reduce, and there is also significant difference (P < 0.05), explanations between 11d and the fungal burden of 10d
Soapberry situ-gel therapeutic effect is gradually shown since 10d.
Immunological hallmark object
Compared to the blank group, there is significant difference (P in three cell factors (IL-1 β, IL-6, IL-8) of model group
< 0.05), illustrate that infect Candida albicans will cause cytokine levels up-regulation really, show apparent inflammatory reaction；With
Model group is compared, and three cell factors (IL-1 β, IL-6, IL-8) of control group are said there is also significant difference (P < 0.05)
Fluconazole inflammatory reaction caused by Candida albicans that bright research uses has therapeutic effect, further illustrates this research institute
The VVC of foundation is modeled on cytokine levels can be with further investigated.
Fig. 2 is shown and model group ratio, water mention group proinflammatory factor IL-1 β there was no significant difference, proinflammatory factor IL-6 with become
Change factor IL-8 there are significant difference (P < 0.05), illustrates that soapberry situ-gel can reduce two inflammation of IL-6 and IL-8
Disease index；And proinflammatory factor IL-6 and chemotactic factor (CF) IL-8 level (the P < suitable with positive controls of soapberry situ-gel
0.05), illustrate after treating 11d, the soapberry situ-gel for the 10mg/mL that this research uses refers to two inflammation of IL-6 and IL-8
Target inhibiting effect is suitable with 2mg/mL Fluconazole.
Lactic dehydrogenase (LDH) is tissue damage index, can be used to differentiate by the microbial vagina tissue of Candida albicans
Degree of injury, LDH numerical value is higher, shows that vagina tissue is bigger by Candida albicans degree of injury.Compared to the blank group, model group
There is significant difference (P < 0.05) between control group, illustrates the VVC model of this research institute foundation in LDH index upper value
Obtain further investigated.Compared with model, the LDH level of soapberry situ-gel is remarkably decreased (P < 0.05), illustrates soapberry original position
Gel can improve vagina tissue degree of injury, have therapeutic effect.Compared with the control, the LDH level of soapberry situ-gel with
It is quite (P < 0.05), illustrate treat 11d after, 10mg/mL soapberry situ-gel improve vagina tissue degree of injury on
2mg/mL Fluconazole is suitable.
Pathological section
Vagina tissue pathological section can intuitively show the case where vagina tissue.Vagina tissue is by lumen (L), mucous membrane
(M) it is formed with submucosa (SM).As shown in figure 3, naive mice tissue line is clear, it is well arranged；The mucous membrane of model group
Layer (M) thickens, and part epithelial cell severe necrosis falls off, and illustrating candida albicans infection mainly influences mucous layer (M), makes its change
It is thick.Studies have reported that vagina mucosa is generated inflammatory reaction after candida albicans infection, vaginal mucosa oedema even inflammatory is caused
Hyperplasia thickens mucous layer.Compared with model group, control group, the mucous layer (M) of soapberry situ-gel are thinning, on
Chrotoplast only has slight obscission, shows that the two can mitigate the disease of candida albicans infection vagina tissue to some extent
Shape.
In conclusion a kind of soapberry in-situ gel preparation for treating candida albicans vaginitis of the invention, in body
Have outside to Candida albicans and significantly inhibit effect, can be effectively reduced the inflammatory reaction of VVC in vivo, is a kind of excellent
VVC therapeutic agent, and prepare simply, toxic side effect is small, and have no drug resistance threat.
Claims (10)
1. a kind of preparation method for the soapberry in-situ gel preparation for treating candida albicans vaginitis, which is characterized in that step
It is rapid as follows:
Step 1: preparing soapberry water mentions Fermented Condensed liquid
(1.1) soapberry dry fruit skin is crushed, powder is mixed with water 1:3 in mass ratio under conditions of 40 DEG C and extracts 6h, mistake
Filter obtains first time filtrate and filter residue；
(1.2) under conditions of 40 DEG C, filter residue is mixed with water by the mass ratio of 1:2 and extracts 4h, second of filter is obtained by filtration
Liquid discards filter residue；
(1.3) merge first time filtrate and second of filtrate, obtain sapindoside coarse filtration liquid；By sapindoside coarse filtration liquid with
5000r/min centrifugation 10min obtains soapberry Aqueous extracts；
(1.4) soapberry Aqueous extracts are concentrated to get sapindoside concentrate in evaporation equipment；
(1.5) it is inoculum concentration with 1.0%, grape wine dry ferment is inoculated with into the dilution of sapindoside concentrate, at 30 DEG C
Lower fermentation 4d obtains soapberry water and mentions Fermented Condensed liquid；
Step 2: measurement soapberry water proposes the content of saponin(e in Fermented Condensed liquid
(2.1) production of standard curve: configuration 1mg/mL oleanolic acid standard solution；The test tube for taking dried and clean, is separately added into
0,30,60,90,120,150 μ L oleanolic acid standard solution is placed in 50 DEG C of water-bath drying, and 0.4mL mass fraction is successively added and is
It is color developing agent that 5% vanillic aldehyde-glacial acetic acid solution and 1.4mL, which analyzes pure perchloric acid, and concussion mixes；It is placed in 70 DEG C of water-baths and heats
20min, ice-water bath are cooled to room temperature, and are added 10mL glacial acetic acid as solvent, are mixed well；Existed with ultraviolet specrophotometer
Absorbance is measured at 540nm；
(2.2) sample measures: soapberry water being proposed the dilution of Fermented Condensed liquid, takes 100 μ L dilutions in test tube, repeats above-mentioned behaviour
Make the content that measurement soapberry water mentions saponin(e in Fermented Condensed liquid；
Step 3: preparing soapberry in-situ gel preparation
At 3-6 DEG C, taking polyethylene glycol 400 is dissolved into soapberry water and mentions in Fermented Condensed liquid, is uniformly mixed, and poloxamer is added
407 and PLURONICS F87 swelling completely, add distilled water, soapberry in-situ gel preparation is obtained after mixing.
2. preparation method according to claim 1, which is characterized in that in the step (1.1), by the dry fruit peel powder of soapberry
It is broken to 20-60 mesh.
3. preparation method according to claim 1 or 2, which is characterized in that in the step (2.2), soapberry water mentions hair
Ferment concentrate extension rate is 10-100 times.
4. preparation method according to claim 1 or 2, which is characterized in that in the step 3, soapberry water used mentions hair
Saponin concentrations are 10-80mg/mL in ferment concentrate.
5. preparation method according to claim 3, which is characterized in that in the step 3, soapberry water used proposes fermentation
Saponin concentrations are 10-80mg/mL in concentrate.
6. according to claim 1, preparation method described in 2 or 5, which is characterized in that in the step 3, based on parts by weight,
The additive amount of poloxamer188 is 16-22 parts, and the additive amount of PLURONICS F87 is 0.1-5 parts, the additive amount of polyethylene glycol 400
It is 0.1-3 parts, the additive amount that soapberry water mentions Fermented Condensed liquid is 50 parts, remaining as distilled water.
7. preparation method according to claim 3, which is characterized in that in the step 3, based on parts by weight, Bo Luosha
The additive amount of nurse 407 is 16-22 parts, and the additive amount of PLURONICS F87 is 0.1-5 parts, and the additive amount of polyethylene glycol 400 is 0.1-
3 parts, the additive amount that soapberry water mentions Fermented Condensed liquid is 50 parts, remaining as distilled water.
8. the preparation method according to claim 4, which is characterized in that in the step 3, based on parts by weight, Bo Luosha
The additive amount of nurse 407 is 16-22 parts, and the additive amount of PLURONICS F87 is 0.1-5 parts, and the additive amount of polyethylene glycol 400 is 0.1-
3 parts, the additive amount that soapberry water mentions Fermented Condensed liquid is 50 parts, remaining as distilled water.
9. according to claim 1, preparation method described in 2,5,7 or 8, which is characterized in that in the step (1.4), concentration times
Number is 6-7 times；In the step (1.5), by grape wine dry-pitching in four times of dilutions of sapindoside concentrate.
10. a kind of soapberry in-situ gel preparation for treating candida albicans vaginitis, which is characterized in that soapberry is solidifying in situ
Glue preparation is prepared by any preparation method of claim 1-9.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110812412A (en) * | 2019-11-15 | 2020-02-21 | 江南大学 | Sapindus temperature-control type gel preparation for treating candida albicans vaginitis and preparation method thereof |
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