CN103611157A - Panax japonicus saponin as well as preparation method and application thereof - Google Patents
Panax japonicus saponin as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to panax japonicus saponin used as a vaccine adjuvant as well as a preparation method and application thereof. The method comprises the following steps: extraction of the panax japonicus, extraction separation, resin adsorption and the like. The panax japonicus saponin is further separated and purified, the weaknesses of the traditional method that only the total saponins component is used and the main components of the samponin is not studied can be overcome, so that the problem that the drug ingredients are not known by people can be solved; an active part of the panax japonicus saponin has an obvious immunity adjuvant effect, and a way for developing the vaccine adjuvant is provided.
Description
[technical field]
The invention belongs to vaccine adjuvant technical field.More specifically, the present invention relates to a kind of Rhizoma Panacis Japonici saponin as vaccine adjuvant, also relate to the preparation method of described Rhizoma Panacis Japonici saponin, also relate to the purposes of described Rhizoma Panacis Japonici saponin.
[background technology]
Rhizoma Panacis Japonici is famous and expensive special local product one of conventional Chinese crude drug less that araliaceae ginseng plant's Rhizoma Panacis Japonici dry is bamboo rhizome shape rhizome ，Wei China.Rhizoma Panacis Japonici is grown in the dark and damp ground of more than height above sea level 1000m broad-leaved sparse woods or rock, Gou Jian side more, and edaphic condition is had relatively high expectations, suitablely in neutrality or slightly acidic environment, grow, and happiness humus bed thickness, loose fertile, well-drained sandy loam.In Yunnan, the province such as Guizhou, Sichuan, Hubei, Shaanxi, Gansu, Henan, Zhejiang, Anhui, Jiangxi is among the people a longer applicating history, also has a small amount of outlet Hong Kong.There is strengthening by means of tonics, eliminating stasis to stop pain, the famous ethnic drug of effect ，Ye Shi Tujia that hemostasis is eliminated the phlegm is regarded as by the ancients of enshi Tujia " king of medical herbs " always.Along with modern pharmacology research is goed deep into, the main effect of Rhizoma Panacis Japonici has been embodied in certain element of the first species step by step, even to certain composition.The main effective ingredient of Rhizoma Panacis Japonici is Rhizoma Panacis Japonici saponin and saponins.Also recorded in the prior art the preparation method of Rhizoma Panacis Japonici saponin, for example CN101732379A discloses D101 enriching and purifying macroporous resin panax japonicus total saponins technique, and it has just introduced extraction process; CN102247416A discloses the effect for reducing blood fat of panax japonicus total saponins.Have no report Rhizoma Panacis Japonici saponin for vaccine adjuvant.
At present, the adjuvant for vaccine for man is that Alum adjuvant (is mainly aluminium hydroxide and phosphate thereof, Alum).Alum adjuvant has been used decades as mankind's vaccine adjuvant, becomes adjuvant conventional in vaccine for man, has good adjuvant effect.Continuous appearance along with new generation vaccine, Alum adjuvant shows some problems, comprising: (1) is because of its physicochemical property feature, the freezing rear colloidal state of aluminum salt vaccine is destroyed, therefore can not lyophilization and transport under low temperature state, but present a lot of polypeptide vaccines must cryopreservation.(2) because salinity is high, store and with the passing of time have crystalline deposit, make the vaccine stability of preparation can not get ensureing.(3) nervous system of aluminum salt pair people, animal may be influential.(4) local aseptic abscess occurs sometimes in injection site, even can form granuloma, slight local response is comparatively common.(5) tumor vaccine is not suitable for, with Alum adjuvant, makes vaccine and be mainly applicable to take the vaccine that antibody is protective immunity.(6) speed of Alum adjuvant released antigen material is slow, and effect is also more weak, all effective not to many soluble antigens, higher to the requirement of antigen.Therefore studying new vaccine adjuvant is the important step of preparing new generation vaccine.
From plant, extract saponin and can activate mammiferous immune system, its immunological adjuvant activity is subject to extensive concern.Saponin QS-21 is the most representative material, and its research has entered clinical experimental stage.Saponin QS-21 is the main component of extract soapbark saponin (Quil A) in the plant in South America, and it is longer than Alum adjuvant that research shows that it produces antibody response sustainable protection time, so can be used as Alum adjuvant substitute.But Quil A exists serious toxicity, to the lethal dose of mice, at 100-125 μ g, except this also has serious hemolytic, in injection site, can cause granuloma and injection site symptom, so limited the use in vaccine for man.So far, from Quil A, separation obtains 23 kinds of monomeric compounds.Soap former times QS-21 is 500 μ g to the fatal dose of mice, though toxicity is less than Quil A, but it is still only for tumor vaccine.And, in saponin QS-21 equimolecular structure, exist cruel bond structure to affect its stability.Thereby people more and more pay close attention to and find tool immunological adjuvant activity and the relatively low saponin of toxicity from other plants.
Rhizoma Panacis Japonici saponin preparation method of the present invention is to the further separation and purification of panax japonicus total saponins, improve the shortcoming of in the past only its main component not being studied with total saponins composition, the present invention, just as starting point, has solved the problem that frequent puzzlement people do not know medication composition; The Rhizoma Panacis Japonici saponin active site that the inventive method prepares has very significantly immunoadjuvant function, for development of new vaccine adjuvant has indicated direction.
[summary of the invention]
[technical problem that will solve]
The object of this invention is to provide a kind of Rhizoma Panacis Japonici saponin.
Another object of the present invention is to provide the preparation method of described Rhizoma Panacis Japonici saponin.
Another object of the present invention is to provide the purposes of described Rhizoma Panacis Japonici saponin.
[technical scheme]
The present invention is achieved through the following technical solutions.
The present invention relates to a kind of preparation method of the Rhizoma Panacis Japonici saponin as vaccine adjuvant.
This preparation method comprises the steps:
A, Rhizoma Panacis Japonici extract
By Rhizoma Panacis Japonici raw material pulverizing, according to weight ratio 1:5～15 of Rhizoma Panacis Japonici and ethanol water, it is in 55～85% ethanol waters that smashing bamboo JIESHEN is immersed in to volume fraction, soak 2～5h, heating and refluxing extraction is 2～5 times again, each 1～5h, merge extractive liquid,, be evaporated to again extracting solution without alcohol taste, obtain a kind of concentrated solution;
B, extract and separate
The concentrated solution that steps A obtains repeatedly extracts with two and is separated according to the volume ratio 1:1～4 use n-butyl alcohol of concentrated solution and n-butyl alcohol, until n-butyl alcohol is mutually colourless, the butanol extraction liquid obtaining merges, then concentrating under reduced pressure, vacuum drying, so obtain n-butyl alcohol extract;
C, resin absorption
In resin column, macroporous adsorbent resin is immersed in to 20～24h in ethanol, then use ethanol water eluting not muddy to effluent, wash with water again to without ethanol taste, then macroporous adsorbent resin is immersed in by weight to 2～4h in 4～6%HCL aqueous solution, wash with water to neutrality, then macroporous adsorbent resin is immersed in by weight to 2～4h in 1～3%NaOH aqueous solution, wash with water to neutrality standby;
The n-butyl alcohol extract adsorption conditions that step B obtains is as follows: the blade diameter length ratio of resin column is 1:8～12, applied sample amount is 0.05～0.15g crude drug/milliliter column volume, standing over night after loading, after D101 absorption with macroporous adsorbent resin is saturated, is used ethanol water to carry out eluting;
Elution requirement is as follows: first water rinses to effluent colourless, then the ethanol water eluting that is 80～95% by volume fraction, and elution volume is 2～4 times of column volumes, eluting flow 0.8～1.2mL/min; Concentrating under reduced pressure is carried out in the eluate obtaining, vacuum drying then, and its product is described Rhizoma Panacis Japonici saponin.
A preferred embodiment of the invention, in steps A, described Rhizoma Panacis Japonici grinding particle size is 20-40 order.
According to another kind of preferred implementation of the present invention, in steps A, the weight ratio of Rhizoma Panacis Japonici and ethanol water is 1:8～12.
According to another kind of preferred implementation of the present invention, in steps A, the volume fraction of described ethanol water is 62～75%.
According to another kind of preferred implementation of the present invention, in step B, described concentrating under reduced pressure carries out under pressure 0.001～0.01MPa, described dry be to carry out under the condition of 30～50 ℃ of pressure 0.01～0.1MPa and temperature.
According to another kind of preferred implementation of the present invention, in step B, the volume ratio of described concentrated solution and n-butyl alcohol is 1:1.8～3.2.
According to another kind of preferred implementation of the present invention, in step C, described macroporous adsorbent resin is D101 macroporous adsorbent resin.
According to another kind of preferred implementation of the present invention, in step C, described concentrating under reduced pressure carries out under pressure 0.001～0.01MPa, described dry be to be dried under the condition of 30～50 ℃ of pressure 0.01～0.1MPa and temperature.
The invention still further relates to the Rhizoma Panacis Japonici saponin that adopts described preparation method to obtain.
The invention still further relates to the purposes of described Rhizoma Panacis Japonici saponin in preparing vaccine adjuvant.
The present invention will be described in more detail below.
The present invention relates to a kind of preparation method of the Rhizoma Panacis Japonici saponin as vaccine adjuvant.
This preparation method comprises the steps:
A, Rhizoma Panacis Japonici extract
By Rhizoma Panacis Japonici raw material pulverizing, according to weight ratio 1:5～15 of Rhizoma Panacis Japonici and ethanol water, it is in 55～85% ethanol waters that smashing bamboo JIESHEN is immersed in to volume fraction, soak 2～5h, heating and refluxing extraction is 2～5 times again, each 1～5h, merge extractive liquid,, be evaporated to again extracting solution without alcohol taste, obtain a kind of concentrated solution;
Described Rhizoma Panacis Japonici raw material is the Rhizoma Panacis Japonici Chinese crude drug of selling in current China market, for example the Rhizoma Panacis Japonici of Enshi State of Hubei Province Xuanen Chinese toon wood battalion Rhizoma Panacis Japonici planting base plantation.Conventionally, after the pretreatment such as Rhizoma Panacis Japonici Chinese crude drug need to carry out that routine is selected, cleans, is dried, re-use normally used pulverizer pulverizing dry Rhizoma Panacis Japonici in Chinese crude drug processing technique field, the Rhizoma Panacis Japonici of pulverizing sieves with standard screen again, collects 20-40 order Rhizoma Panacis Japonici powder as the processing raw material of the inventive method.
According to the present invention, with the effect of Ethanol Treatment Rhizoma Panacis Japonici, be by the effective ingredient saponin extraction of Rhizoma Panacis Japonici out.
In the present invention, using volume fraction is that 55～85% ethanol waters soak Rhizoma Panacis Japonici powder, if the concentration of ethanol water surpasses 85%, can make saponin content reduce, and the impurity content such as fat-soluble pigment raises; If the concentration of ethanol water lower than 55%, can make saponin impurity increase; Therefore, the concentration of ethanol water is 55～85%, suitable, the concentration of ethanol water preferably 60～80%, more preferably 62～75%.
When with Ethanol Treatment Rhizoma Panacis Japonici, the weight ratio of Rhizoma Panacis Japonici and ethanol water is 1:6～14 preferably, soak 2.6～4.5h, and more preferably 1:8～12, soak 3.2～3.8h.
In this step, each heating and refluxing extraction time is 1.8～4.2h, more preferably 2.5～3.6h preferably.
With the equipment that Ethanol Treatment Rhizoma Panacis Japonici is used, be the round-bottomed flask (or conventional chemical processing chemical industry equipment) of generally selling in the market, for example You Shuniu glass apparatus company is with trade name round-bottomed flask product sold.
The heating and refluxing extraction equipment using with Ethanol Treatment Rhizoma Panacis Japonici is the heating jacket (or conventional heating reflux, extract, chemical industry equipment) of selling in the market, the electric jacket product of for example being sold with trade name by Yuhua Instrument Co., Ltd., Gongyi City.
According to the present invention, under the condition of pressure 0.001～0.01MPa and room temperature, carry out concentrating under reduced pressure.The equipment using when concentrating under reduced pressure is the Rotary Evaporators of selling in the market, for example by company of Shanghai Yarong Biochemical Instrument Plant with trade name RE-52AA type Rotary Evaporators product sold.
B, extract and separate
The concentrated solution that steps A obtains repeatedly extracts with two and is separated according to the volume ratio 1:1～4 use n-butyl alcohol of concentrated solution and n-butyl alcohol, until n-butyl alcohol is mutually colourless, the butanol extraction liquid obtaining merges, then concentrating under reduced pressure, vacuum drying, so obtain n-butyl alcohol extract;
According to the present invention, use the object of n-butanol extraction to be to make the saponin that is dissolved in organic solvent n-butyl alcohol to extract.
In the present invention, the volume ratio of described concentrated solution and n-butyl alcohol is 1:1.8～3.2 preferably, more preferably 1:2.2～2.8.
In the present invention, when using n-butanol extraction, allow the n-butyl alcohol in extraction equipment fully mix with described concentrated solution, in this mixed process, the effective ingredient of described concentrated solution distributes between biphase, from described concentrated solution, transfer to n-butyl alcohol, thereby realize the separation of effective ingredient.
The equipment using during in the present invention with n-butanol extraction is the separatory funnel sold in the market (or conventional extraction chemical industry equipment), for example by Sichuan Shu Niu glass apparatus company with trade name separatory funnel product sold.
Described n-butyl alcohol is mutually colourless should be appreciated that it is with after concentrated solution described in n-butanol extraction, and the n-butyl alcohol after extraction is not have colouredly with an eye observation, or does not almost have coloured.
In this step, described concentrating under reduced pressure as previously described, does not repeat them here.
Described dry be to carry out under the condition of 30～50 ℃ of pressure 0.01～0.1MPa and temperature.The drying equipment that the present invention uses is sold in the market, for example by Shanghai Yiheng Scientific Instruments Co., Ltd with trade name vacuum drying oven product sold.
C, resin absorption
Macroporous adsorbent resin pretreatment: in resin column, macroporous adsorbent resin is immersed in to 20～24h in ethanol, then use ethanol water eluting not muddy to effluent, wash with water again to without ethanol taste, then macroporous adsorbent resin is immersed in by weight to 2～4h in 4～6%HCL aqueous solution, wash with water to neutrality, then macroporous adsorbent resin is immersed in by weight to 2～4h in 1～3%NaOH aqueous solution, wash with water to neutrality standby;
The n-butyl alcohol extract adsorption conditions that step B obtains is as follows: the blade diameter length ratio of resin column is 1:8～12, applied sample amount is 0.05～0.15g crude drug/milliliter column volume, standing over night after loading, after D101 absorption with macroporous adsorbent resin is saturated, is used ethanol water to carry out eluting;
Elution requirement is as follows: first water rinses to effluent colourless, then the ethanol water eluting that is 80～95% by volume fraction, and elution volume is 2～4 times of column volumes, eluting flow 0.8～1.2mL/min;
Concentrating under reduced pressure is carried out in the eluate obtaining, vacuum drying then, and its product is described Rhizoma Panacis Japonici saponin.
Macroporous resin is to be prepared from through polyreaction by additives such as polymerization single polymerization monomer and cross-linking agent, porogen, dispersants.After polymer formation, porogen is removed, and has left a large amount of holes in resin.Therefore, macroporous resin its inside under drying regime has higher porosity, and aperture large (100～1000nm).Macroporous adsorbent resin be a class containing cation exchange groups and there is the preparation of macroporous structure, there is good macroporous netlike structure and larger specific surface area, to be adsorbed as feature.It is a kind of organic high molecular polymer that is insoluble to acid, alkali and various organic solvents.
The macroporous adsorbent resin that the present invention uses is D101 macroporous adsorbent resin, for example, be with trade name D101 macroporous adsorbent resin product sold by Shanghai Zi Yi reagent company.
Because D101 macroporous adsorbent resin contains some impurity, therefore need to carry out following pretreatment:
D101 macroporous adsorbent resin pretreatment: D101 macroporous adsorbent resin is used after soak with ethanol 24h, not muddy to effluent with ethanol elution, wash with water again to without alcohol taste, then with the resin column of 5%HCL immersion by weight 2-4h, wash with water to neutrality, then with the resin column of 2%NaOH immersion by weight 2-4h, be washed to neutral standby.
In the present invention, using soak with ethanol D101 macroporous adsorbent resin object is to remove the alcohol dissolubility impurity adsorbing on resin; The object of using 5%HCL to soak resin is in order to allow the alkaline components adsorbing on resin become salt and to rinse away; The object of using 2%NaOH to soak resin is in order to allow the acid ingredient adsorbing on resin become salt and to rinse away; Be washed to neutral object and be and remove that to rinse be the salt constituents of residual acid, alkali and generation.
D101 absorption with macroporous adsorbent resin condition is as follows:
Post blade diameter length ratio is 1:10, and applied sample amount is 0.1g crude drug/milliliter column volume, standing over night after loading.In the present invention, the blade diameter length ratio of filling macroporous adsorbent resin is 1:10, is in order to keep suitable flow velocity.Applied sample amount is chosen as 0.1g crude drug/milliliter column volume, if too low, dead absorption is serious, affects saponin yield; If applied sample amount is excessive, can cause overload, affect disintegrate-quality.Standing over night after loading is in order to allow saponin and macroporous adsorbent resin fully adsorb.
D101 macroporous adsorbent resin elution requirement is as follows:
Elution requirement is: first water rinses to effluent colourless, then the ethanol water that is 80-95% by volume fraction, and elution volume is 3 times of column volumes, and eluting flow is 1mL/min.
In the present invention, first make water rinse to effluent colourless, object is to wash away the saponins impurity weak with absorption with macroporous adsorbent resin ability.The ethanol water eluting that is 80-95% by volume fraction is again more suitable, because if use ethanol water to be less than 80%, polarity is less, eluting power a little less than, incomplete for the eluting of saponin; If select the concentration of ethanol to be greater than 95%, effect and 95% ethanol do not have notable difference, and preparation is more difficult.More preferably for volume fraction of ethanol is 88-92%.
In this step, described concentrating under reduced pressure is identical with step B with vacuum drying, repeats no more.
The Rhizoma Panacis Japonici saponin that adopts the inventive method to prepare has carried out HPLC analysis, and its HPLC analysis condition is as follows:
Chromatographic column: YMC-pack ODS-AQ post (250 * 4.6mm, particle diameter 5 μ m);
Mobile phase: acetonitrile (A)-0.4% phosphate aqueous solution (B);
Column temperature: 30 ℃;
Type of elution: gradient elution (0～5min, 5～5%A; 5～20min, 5～30%A; 20～30min, 30～30%A; 30～50min, 30～85%A; 50～60min, 85～85%A);
Detect wavelength: 203nm; Sample size: 10 μ l; Flow velocity: 1.0mlmin-1.
UV-detector; Nitrogen pressure is 33Ps; 40 ℃ of drift tube temperatures.
The HPLC collection of illustrative plates of the Rhizoma Panacis Japonici saponin that employing the inventive method prepares is listed in accompanying drawing 1.As can be seen from the figure, in retention time, be that four larger chromatographic peaks appear in 37min-39min altogether, the inventor has carried out Structural Identification to above four chromatographic peaks, four corresponding compositions of chromatographic peak are defined as respectively to compound 1, compound 2, compound 3 and compound 4 according to vertical order, adopt
1h-NMR,
13c-NMR method identifies described four kinds of compounds, and its qualification result is as follows:
Compound 1: white powder, ESI-MS m/z:955[M-H]
-.
1H-NMR(600MHz，C
5D
5N)δ6.34（1H，d，J=7.8Hz，Glc-H-1），5.42（1H，br?s，H-12），5.41（1H，s，Glc-H-1），5.02(1H，d，J=6.6Hz，GlcA-H-1)，3.28（1H，dd，J=11.4，4.2Hz，H-3），3.18（1H，dd，J=13.2，2.4Hz，H-18），1.28，1.26，1.11，1.09，0.91，0.88，0.83（each3H，s，7×CH
3)。
According to compound 1
1h-NMR,
13c-NMR data, authenticating compound 1 is Rhizoma Panacis Japonici saponin V.
Compound 2: white powder, ESI-MS m/z:925[M-H]
-.
1H-NMR(600MHz，C
5D
5N)δ6.33（1H，d，J=8.4Hz，Glc-H-1），5.42（1H，br?s，H-12），5.28（1H，d，J=6.0Hz，xyl-H-1），4.99(1H，d，J=3.0Hz，GlcA-H-1)，3.29（1H，br?d，J=8.4Hz，H-3），3.20(1H，br?d，J=10.8Hz，H-18），1.29(6H，s，2×CH
3)，1.10，1.10，0.92，0.89，0.85（each3H，s，5×CH
3)。
According to compound 2
1h-NMR,
13c-NMR data, authenticating compound 2 is pjs-2.
Compound 3: white powder, ESI-MS m/z:925[M-H]
-.
1H-NMR(600MHz，C
5D
5N)δ6.33（1H，d，J=8.4Hz，Glc-H-1），6.17（1H，br?s，1H，Araf-H-1），5.42（1H，s，H-12），5.02(1H，d，J=3.6Hz，GlcA-H-1)，3.33（1H，dd，J=10.8，3.6Hz，H-3），3.19（1H，dd，J=13.8，3.6Hz，H-18），1.29(6H，s，2×CH
3)，1.09，0.98，0.92，0.89，0.83（each3H，s，5×CH
3）。
According to the ESI-MS of compound 3,
1h-NMR,
13c-NMR data, authenticating compound 3 is Rhizoma Panacis Japonici saponin IV.
Compound 4: white powder, ESI-MS m/z:793[M-H]
-.
1H-NMR(600MHz，C
5D
5N)δ6.34（1H，d，J=7.8Hz，Glc-H-1），5.42（1H，br?s，H-12），5.04(1H，d，J=7.8Hz，GlcA-H-1)，3.37（1H，br?d，J=9.9Hz，H-3），3.19（1H，dd，J=10.2，2.4Hz，H-18），1.31，1.28，1.06，1.00，0.91，0.89，0.83（each3H，s，7×CH
3）。
According to compound 4
1h-NMR,
13c-NMR data, authenticating compound 4 is Rhizoma Panacis Japonici saponin IV a.
Therefore, adopting the main component of the resulting product of the inventive method is Rhizoma Panacis Japonici saponin V, pjs-2, Rhizoma Panacis Japonici saponin IV and Rhizoma Panacis Japonici saponin IV a, four kinds of saponin are oleanane glycoside, therefore, adopt method of the present invention can realize enrichment and the purification of oleanane glycoside in Rhizoma Panacis Japonici.
The invention still further relates to and adopt the resulting Rhizoma Panacis Japonici saponin of described preparation method.Described Rhizoma Panacis Japonici saponin content assaying method is as follows:
Vanillin-perchloric acid step is as follows:
A, reference substance solution preparation: it is appropriate that precision takes ginsenoside Re's reference substance, adds the reference substance solution that methanol is made into 1.32mg/ml, standby.
B, need testing solution preparation: precision takes the dry Rhizoma Panacis Japonici saponin total extract 0.2g of the present invention, puts in 25ml measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up filtration and obtains need testing solution.
Determining of C, detection wavelength: the accurate A50 μ l of absorption and B40 μ l, be placed in respectively 10ml test tube, add successively 5% vanillin-glacial acetic acid solution 0.2ml by weight, perchloric acid 0.8ml, shake up, in temperature 60 C water-bath, heat 15min, take out, cooling, add glacial acetic acid and be diluted to scale, shake up, by blank reagent, in the scanning of wavelength 400～700nm place, determine that wavelength is wavelength to be measured at 552nm.
The drafting of D, standard curve: precision measures reference substance solution 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml in steps A, is placed in respectively 10ml measuring bottle, adds methanol and is diluted to scale, shakes up; Draw 1.0ml and put in 10ml tool plug test tube, by step C operation, at 552nm place, survey absorbance, take absorbance A as vertical coordinate, reference substance concentration C is abscissa drawing standard curve, by this standard curve, can obtain regression equation A=0.07141C-0.00623, r=0.9993.
E assay: prepare each section of saponin sample liquid according to step B, respectively get 40 μ l and put in 10ml tool plug test tube, according to step C operation, each sample is surveyed 3 parallel samples, surveys absorbance, then record each section of saponin content according to standard curve with 552nm place.
The present invention has following characteristic and advantage as the Rhizoma Panacis Japonici saponin preparation method of vaccine adjuvant:
A, preparation method is simple, reagent is cheap, simple to operate, be easy to a large amount of become to produce.
B, with different determining alcohol eluting D101 macroporous adsorbent resins, make the saponin of different sections study, more fully demonstrate the different activities of each saponin in total saponins, for further research lays the foundation.
The invention still further relates to the purposes of described Rhizoma Panacis Japonici saponin in preparing vaccine adjuvant.
Adjuvant is nonspecific immunity strengthening agent, when injecting together with antigen or inject body in advance, it can enhancing body to the immunne response of antigen or change type of immune response.
Compare with vaccine adjuvant of the prior art, Rhizoma Panacis Japonici saponin vaccine adjuvant tool of the present invention has the following advantages:
The side effect of A Rhizoma Panacis Japonici saponin vaccine adjuvant is little, wide material sources, be easy to produce.
B Rhizoma Panacis Japonici saponin adjuvant has stronger adjuvanticity, and auxiliary vaccine that can be larger plays a role.
C Rhizoma Panacis Japonici saponin vaccine adjuvant is to belong to Chinese medicine vaccine adjuvant, for development of new vaccine adjuvant is laid a good foundation.
[beneficial effect]
The invention has the beneficial effects as follows: the present invention is that Rhizoma Panacis Japonici saponin has obvious adjuvanticity, and without the toxic and side effects of haemolysis, can assist vaccine performance gain potentiation.Rhizoma Panacis Japonici saponin has the Th1 cellular immunization of promotion and antibody produces activity, and being obviously better than traditional alum adjuvant can only stimulate the antibody of Th2 type to produce ability.Therefore Rhizoma Panacis Japonici saponin can be used for the adjuvant of protein subunit vaccine and tumor vaccine.
[accompanying drawing explanation]
Fig. 1 is the HPLC collection of illustrative plates of alcohol eluting Rhizoma Panacis Japonici saponin of the present invention;
Fig. 2 represents the impact that adjuvant produces the lymphocyte IL-2 of activation.Compare * p<0.05, * * p<0.01 with OVA group;
Fig. 3 represents the impact that adjuvant produces the lymphocyte IFN-γ of activation.Compare * p<0.05, * * p<0.01 with OVA group;
Fig. 4 represents the impact that adjuvant antagonist IgG and hypotype thereof produce.Compare * p<0.05 with OVA group.
[specific embodiment]
By following embodiment, can understand better the present invention.In the present invention, do not having under the prerequisite of specified otherwise, the percentage ratio of the component in liquid phase mixture should be understood to percent by volume, and the percentage ratio of the component of solid-phase mixture is to be understood that and is weight percentage.
Embodiment 1: the preparation of precipitate with ethanol Rhizoma Panacis Japonici saponin of the present invention
The implementation step of this embodiment is as follows:
A, Rhizoma Panacis Japonici extract
By Rhizoma Panacis Japonici raw material pulverizing, according to the weight ratio 1:5 of Rhizoma Panacis Japonici and ethanol water, smashing bamboo JIESHEN is immersed in to concentration by weight in 55% ethanol water, soak 2h, heating and refluxing extraction is 3 times again, each 1h, merge extractive liquid,, then concentrating under reduced pressure obtains a kind of concentrated solution under pressure 0.006MPa;
B, extract and separate
The concentrated solution that steps A obtains repeatedly extracts with two and is separated with n-butyl alcohol according to the volume ratio 1:1 of concentrated solution and n-butyl alcohol, until n-butyl alcohol is mutually colourless, the butanol extraction liquid obtaining merges, and follows concentrating under reduced pressure under pressure 0.006MPa, so obtain n-butyl alcohol extract;
C, resin absorption
The pretreatment of D101 macroporous adsorbent resin:
D101 macroporous adsorbent resin is with after soak with ethanol 24h, not muddy to effluent with ethanol elution, then wash with water to without alcohol taste, then with the resin column of 5%HCL immersion by weight 3h, wash with water to neutrality, then, with the resin column of 2%NaOH immersion by weight 3h, be washed to neutral standby.
D101 absorption with macroporous adsorbent resin condition is as follows:
Post blade diameter length ratio is 1:10, and applied sample amount is 0.1g crude drug/milliliter column volume, standing over night after loading.
D101 macroporous adsorbent resin elution requirement is as follows:
It is colourless to effluent that first water rinses resin column, then the ethanol water eluting that is 90% by volume fraction, and elution volume is 3 times of column volumes, and eluting flow is 1mL/min.
Concentrating under reduced pressure is carried out in the eluate obtaining under pressure 0.006MPa, then under the condition of 35 ℃ of pressure 0.06MPa and temperature, is dried, so obtain described Rhizoma Panacis Japonici saponin.
The Rhizoma Panacis Japonici saponin product for preparing of the present embodiment that adopted methods analyst that this description describes, accompanying drawing 1 is shown in by its HPLC collection of illustrative plates, determines that thus the main component of Rhizoma Panacis Japonici saponin product is Rhizoma Panacis Japonici saponin V, pjs-2, Rhizoma Panacis Japonici saponin IV and Rhizoma Panacis Japonici saponin IV a.Adopting method that this description is described to measure the present embodiment, to prepare the Rhizoma Panacis Japonici saponin content of product be 68.9%.
Embodiment 2: the preparation of precipitate with ethanol Rhizoma Panacis Japonici saponin of the present invention
The implementation step of this embodiment is as follows:
A, Rhizoma Panacis Japonici extract
By Rhizoma Panacis Japonici raw material pulverizing, according to the weight ratio 1:15 of Rhizoma Panacis Japonici and ethanol water, smashing bamboo JIESHEN is immersed in to concentration by weight in 85% ethanol water, soak 5h, heating and refluxing extraction is 2 times again, each 5h, merge extractive liquid,, then concentrating under reduced pressure obtains a kind of concentrated solution under pressure 0.008MPa;
B, extract and separate
The concentrated solution that steps A obtains repeatedly extracts with two and is separated with n-butyl alcohol according to the volume ratio 1:4 of concentrated solution and n-butyl alcohol, until n-butyl alcohol is mutually colourless, the butanol extraction liquid obtaining merges, and follows concentrating under reduced pressure under pressure 0.008MPa, so obtain n-butyl alcohol extract;
C, resin absorption
According to the mode identical with embodiment 1, carry out the pretreatment of D101 macroporous adsorbent resin, absorption and gradient elution, concentrating under reduced pressure is carried out in the eluate obtaining under pressure 0.008MPa, then under the condition of 40 ℃ of pressure 0.08MPa and temperature, be dried, so obtain described Rhizoma Panacis Japonici saponin.
Adopting method that this description is described to measure the present embodiment, to prepare the Rhizoma Panacis Japonici saponin content of product be 70.1%.
Embodiment 3: the preparation of precipitate with ethanol Rhizoma Panacis Japonici saponin of the present invention
The implementation step of this embodiment is as follows:
A, Rhizoma Panacis Japonici extract
By Rhizoma Panacis Japonici raw material pulverizing, according to the weight ratio 1:6 of Rhizoma Panacis Japonici and ethanol water, smashing bamboo JIESHEN is immersed in to concentration by weight in 60% ethanol water, soak 2.6h, heating and refluxing extraction is 3 times again, each 1.8h, merge extractive liquid,, then concentrating under reduced pressure obtains a kind of concentrated solution under pressure 0.001MPa;
B, extract and separate
The concentrated solution that steps A obtains repeatedly extracts with two and is separated with n-butyl alcohol according to the volume ratio 1:1.8 of concentrated solution and n-butyl alcohol, until n-butyl alcohol is mutually colourless, the butanol extraction liquid obtaining merges, and follows concentrating under reduced pressure under pressure 0.001MPa, so obtain n-butyl alcohol extract;
C, resin absorption
According to the mode identical with embodiment 1, carry out the pretreatment of D101 macroporous adsorbent resin, absorption and gradient elution, concentrating under reduced pressure is carried out in the eluate obtaining under pressure 0.001MPa, then under the condition of 30 ℃ of pressure 0.01MPa and temperature, be dried, so obtain described Rhizoma Panacis Japonici saponin.
Adopting method that this description is described to measure the present embodiment, to prepare the Rhizoma Panacis Japonici saponin content of product be 61.5%.
Embodiment 4: the preparation of precipitate with ethanol Rhizoma Panacis Japonici saponin of the present invention
The implementation step of this embodiment is as follows:
A, Rhizoma Panacis Japonici extract
By Rhizoma Panacis Japonici raw material pulverizing, according to the weight ratio 1:14 of Rhizoma Panacis Japonici and ethanol water, smashing bamboo JIESHEN is immersed in to concentration by weight in 80% ethanol water, soak 4.5h, heating and refluxing extraction is 4 times again, each 4.2h, merge extractive liquid,, then concentrating under reduced pressure obtains a kind of concentrated solution under pressure 0.002MPa;
B, extract and separate
The concentrated solution that steps A obtains repeatedly extracts with two and is separated with n-butyl alcohol according to the volume ratio 1:3.2 of concentrated solution and n-butyl alcohol, until n-butyl alcohol is mutually colourless, the butanol extraction liquid obtaining merges, and follows concentrating under reduced pressure under pressure 0.002MPa, so obtain n-butyl alcohol extract;
C, resin absorption
According to the mode identical with embodiment 1, carry out the pretreatment of D101 macroporous adsorbent resin, absorption and gradient elution, concentrating under reduced pressure is carried out in the eluate obtaining under pressure 0.002MPa, then under the condition of 46 ℃ of pressure 0.02MPa and temperature, be dried, so obtain described Rhizoma Panacis Japonici saponin.
Adopting method that this description is described to measure the present embodiment, to prepare the Rhizoma Panacis Japonici saponin content of product be 64.8%.
Embodiment 5: the preparation of precipitate with ethanol Rhizoma Panacis Japonici saponin of the present invention
The implementation step of this embodiment is as follows:
A, Rhizoma Panacis Japonici extract
By Rhizoma Panacis Japonici raw material pulverizing, according to the weight ratio 1:8 of Rhizoma Panacis Japonici and ethanol water, smashing bamboo JIESHEN is immersed in to concentration by weight in 62% ethanol water, soak 3.2h, heating and refluxing extraction is 4 times again, each 2.5h, merge extractive liquid,, then concentrating under reduced pressure obtains a kind of concentrated solution under pressure 0.004MPa;
B, extract and separate
The concentrated solution that steps A obtains repeatedly extracts with two and is separated with n-butyl alcohol according to the volume ratio 1:2.2 of concentrated solution and n-butyl alcohol, until n-butyl alcohol is mutually colourless, the butanol extraction liquid obtaining merges, and follows concentrating under reduced pressure under pressure 0.004MPa, so obtain n-butyl alcohol extract;
C, resin absorption
According to the mode identical with embodiment 1, carry out the pretreatment of D101 macroporous adsorbent resin, absorption and gradient elution, concentrating under reduced pressure is carried out in the eluate obtaining under pressure 0.004MPa, then under the condition of pressure 0.04MPa and temperature 50 C, be dried, so obtain described Rhizoma Panacis Japonici saponin.
Adopting method that this description is described to measure the present embodiment, to prepare the Rhizoma Panacis Japonici saponin content of product be 61.9%.
Embodiment 6: the preparation of precipitate with ethanol Rhizoma Panacis Japonici saponin of the present invention
The implementation step of this embodiment is as follows:
A, Rhizoma Panacis Japonici extract
By Rhizoma Panacis Japonici raw material pulverizing, according to the weight ratio 1:12 of Rhizoma Panacis Japonici and ethanol water, smashing bamboo JIESHEN is immersed in to concentration by weight in 75% ethanol water, soak 3.8h, heating and refluxing extraction is 5 times again, each 3.6h, merge extractive liquid,, then concentrating under reduced pressure obtains a kind of concentrated solution under pressure 0.01MPa;
B, extract and separate
The concentrated solution that steps A obtains repeatedly extracts with two and is separated with n-butyl alcohol according to the volume ratio 1:2.8 of concentrated solution and n-butyl alcohol, until n-butyl alcohol is mutually colourless, the butanol extraction liquid obtaining merges, and follows concentrating under reduced pressure under pressure 0.01MPa, so obtain n-butyl alcohol extract;
C, resin absorption
According to the mode identical with embodiment 1, carry out the pretreatment of D101 macroporous adsorbent resin, absorption and gradient elution, concentrating under reduced pressure is carried out in the eluate obtaining under pressure 0.01MPa, then dry under the condition of pressure 0.04MPa and temperature 50 C, so obtain described Rhizoma Panacis Japonici saponin.
Adopting method that this description is described to measure the present embodiment, to prepare the Rhizoma Panacis Japonici saponin content of product be 59.9%.
Test example
One, hemolytic experiment:
With anticoagulant vacuum test tube, from rabbit ear vein, gather peripheral blood, add normal saline, mix, 2000rpm, centrifugal 10min, repeated washing 3 times, makes 0.5%v/v with normal saline dilution) red cell suspension.Accurately take medicine, with normal saline preparation, through 0.22 μ m filtering with microporous membrane, then be diluted to successively with normal saline the diluent that concentration is 1280,640,320,160,80,40,20,10 μ g/mL respectively.
In 96 orifice plates, every hole adds 0.5% red cell suspension 100 μ L, then adds the oleanane glycoside diluent 100 μ L of the variable concentrations of preparing according to the inventive method, mixes, and a concentration repeats 3 holes.Establish again negative and positive haemolysis contrast, be respectively normal saline and distilled water.In 37 ℃ of incubators of temperature, place 30min, with the centrifugal l0min of 1000rpm.Get each hole supernatant 100 μ L, use microplate reader at wavelength 405nm place, to detect absorbance.Experiment repeats 3 times, and institute surveys the absorption value that absorption value of respectively organizing deducts normal saline matched group just can represent hemolytic, according to formula below, calculates its hemolysis rate:
Hemolysis rate (%)=100* (experimental group OD-negative control OD)/(positive control OD-negative control OD)
The Rhizoma Panacis Japonici oleanane glycoside of preparing according to the inventive method is listed in table 1 rabbit erythrocyte hemolytic activity result of the test.
Table 1: the hemolytic activity of saponin is measured
Note: normal saline and distilled water are respectively feminine gender and positive control, and measured value of experiment represents by mean+SD.
From table 1, Quil A starts there is haemolysis at 10 μ g/ml, and at 20 μ g/ml, just up to 97% haemolysis, and Rhizoma Panacis Japonici saponin all has no haemolysis at 640 μ g/ml.Presentation of results Rhizoma Panacis Japonici oleanane glycoside of the present invention and Quil A comparison, do not have hemolytic toxicity.
Two, acute toxicology
With PBS, sample Quil A, the Rhizoma Panacis Japonici oleanane glycoside prepared according to the inventive method being diluted is respectively the corresponding dosage of table 2, and take normal saline as blank group, amounts to 11 groups, female babl/c mice is divided into 11 groups at random, 5 every group.Each organizes mice in the subcutaneous disposable injection corresponding dosage of cervical region, conventional raising 72 hours, and toxic reaction (depilation, pain, swelling etc.) and the death condition of observing mice, and the results are shown in Table 2 in its experiment.
The acute toxicity testing of table 2 saponin
As can be seen from Table 2, Quil A is in the time of 100 μ g/, and dead 2 of mices, demonstrate very strong toxicity, and median lethal dose(LD 50) (LD50) is 125 μ g/.And it is obvious to observe toxic reaction in 72 hours, in injection site, have depilation and with swelling, lethargy, inappetence.And Rhizoma Panacis Japonici oleanane glycoside of the present invention has no overt toxicity index, a 6400 μ g/ mice, all survive.Therefore the present invention can utilize feature that Rhizoma Panacis Japonici saponin safety range is large for improving the effect of immunological adjuvant.
Three, animal grouping and immunization method
To after Rhizoma Panacis Japonici oleanane glycoside normal saline solution and the mixing of OVA normal saline solution, at room temperature with per minute 50 rotating speeds that turn, shake 30 minutes, the present invention prepares the mixed liquor of Rhizoma Panacis Japonici saponin normal saline solution and OVA normal saline solution.The mixed liquor of other adjuvant and OVA is all with same condition and method preparation.By 35 Balb/c female mice random packet, 5 every group.Normal saline matched group: every subcutaneous injection normal saline 0.2mL; Other is respectively organized dosage listed in equal according to the form below 3 and carries out corresponding immunity.Each organizes subcutaneous injection immunity 2 times, and immunity for the first time and for the second time interval 14 days between immunity, at immune latter 14 days for the second time, get eyeball of mouse blood-letting, prepares corresponding serum, and get mouse spleen under aseptic condition, carries out following research.
Table 3: animal grouping and immune condition
Four, specific lymphoproliferation assay
1, the preparation of splenocyte suspension
The disconnected neck of mice is put to death to the alcohol-pickled sterilization with 75%; Aseptic condition is dissected mice and is got spleen; With shears, reject connective tissue and fat, spleen tissue is placed in the plate that is placed with gauze, add 3m11640 culture fluid, grind spleen tissue, by filtered through gauze, draw 1640 liquid and obtain splenocyte suspension.Suspension is collected aseptic centrifuge tube, with the centrifugal 5min of 1800rpm/min, abandons supernatant, adds erythrocyte cracked liquid (Tris-NH
4cl), after erythrocyte splitting, with the centrifugal 5min of 1800rpm/min, with 1640 liquid washed cells twice, with RPMI1640 culture fluid, cell dilution is become to 1x10
6individual/ml, makes single cell suspension, for specificity lymphocyte proliferation assay.
2, specificity lymphocyte proliferation assay
In 96 well culture plates, every hole adds 100 μ l splenocyte suspension (1x10
6individual/ml), each hole adds equal-volume OVA solution (final concentration is 10 μ g/ml), repeats 3 holes, with RPMI1640 (100IU/ml penicillin, 100 μ g/ml streptomycins, 10% calf serum), mends to 200 μ l, separately establishes blank group.In 37 ℃ of temperature, 5%CO2 incubator, cultivate after 68h, each hole adds respectively 20 μ l MTT solution (5mg/ml), continue to cultivate 4 hours, suck supernatant, add acid dimethyl sulfoxide (DMSO) solution of 200 μ l, vibration, places 15min in room temperature, by microplate reader, in 570nm place, measure OD value, and be calculated as follows stimulation index (SI):
The OD value of SI (stimulation index)=be added with OVA antigen/the do not add OD value of OVA antigen
Table 3: specificity lymphocyte proliferation assay
Note: measured value of experiment represents that by mean+SD ((n=5), each group compares * P<0.05, * * P<0.01 with aluminium adjuvant group
Rhizoma Panacis Japonici oleanane glycoside of the present invention (Panax japonicus oleanane type saponins PJOS) the results are shown in Figure 3 to the impact of the OVA immune mouse splenic lymphocytes of OVA induction.
These experimental results show, compare the PJOS of the various dose mice specific lymphoproliferation assay P<0.01 that can extremely significantly promote OVA induction equally equal to QuilA with OVA matched group; PJOS and QuilA have same promotion lymphocyte proliferation activity, and Alum compares with simple OVA, significantly do not promote OVA specificity lymphocyte proliferation activity.
3, the content of IL-2 and IFN-γ in ELISA detection lymphocyte culture supernatant
Collect above-mentioned cultivation lymphocyte supernatant, the detection method illustrating according to test kit (U.S. R & D company) description detects, and concrete operations are as follows:
1) capture antibody after coated PBS dilution, every hole 100 μ l, 4 ℃ of coated spending the night.
2) ELISA cleaning mixture is washed after three times, gets rid of liquid in ELISA Plate, then facing to absorbent paper pat several under.
3) repeating step 2 after 1%BSA room temperature sealing 90min.
4) the cell conditioned medium liquid obtaining is directly added to the elisa plate that has been coated with antibody, every hole 100 μ l, room temperature 2h.
5) repeating step 2.
6) biotin labeling is detected to (final concentration is 2000pg/mL) after antibody dilution and add elisa plate, every hole 100 μ L, room temperature 2h.
7) repeating step 2.
8) will after 100 times of dilutions of ABC working solution, add elisa plate, every hole 100 μ l, put 37 ℃ of incubator reaction 30min, and 1 * PBS washes after 3 times, adds TMB nitrite ion 100 μ l, again puts 37 ℃ of lucifuge reaction 10-15min.
9) directly add TMB stop buffer, after every hole 50 μ l, in the microplate reader of 450nm wavelength, measure.
10), according to test kit explanation, drawing standard curve, calculates through OD value the cytokine concentrations that each is organized.
4, in ELISA method mensuration serum, OVA specific antibody and subclass are tired
In 96 hole ELISA Plate, every hole adds the coating buffer (carbonate buffer solutions of 50 μ L mL OVA) of 100 μ l OVA, and put 4 ℃ of temperature and hatch 24h, with cleaning mixture washing 3 times, each 3min.Every hole adds confining liquid 200 μ L, hatches 2h in 37 ℃, with cleaning mixture washing 3 times, and each 3min.Add 100 μ L test serums, and carry out doubling dilution with serum dilution.Hatch 2h for 37 ℃, with cleaning mixture washing 3 times, each 3min.Every hole adds the anti-mouse IgG antibody diluent of horseradish peroxidase-labeled rabbit (1:20000), goat anti-mouse igg 1 antibody diluent (1:16000) and goat anti-mouse igg 2b antibody diluent (1:8000) 100 μ L, hatch 2h for 37 ℃, wash each 3min 3 times.Every hole adds 100 μ L tmb substrate solution (each 50 μ L of A liquid and B liquid), and 10min is placed in 37 ℃ of dark places, and every hole adds 50 μ L/ hole 2N H2SO4 solution cessation reactions.Put microplate reader and in wavelength 450nm place, measure OD value, according to formula, calculate and tire.The maximum dilution multiple of the sample of OD value (A450nm)/negative control OD value >=2.1 of tire=sample of sample o'clock.
5, the impact of Rhizoma Panacis Japonici oleanane glycoside on IL-2, IFN-γ content in the lymphocyte of cultivating
The lymphocyte that above-mentioned stimulation is cultivated, collected supernatant in 72 hours, IL-2, IFN-γ content in application ELISA kit measurement supernatant, and it the results are shown in Fig. 2 and Fig. 3.These results show, IL-2, IFN-γ content in the lymphocyte supernatant of cultivation all can significantly promote the secretion of IL-2, IFN-γ in PJOS and the QuilA group of various dose, with antigen OVA group comparison P<0.01; And Alum compares with simple OVA, the secretion of its IL-2, IFN-γ does not have notable difference.Its result shows, PJOS and QuilA have the effect of good promotion lymphocytic emiocytosis IL-2 and IFN-γ, points out them to have the lymphopoietic activity of the Th1 of promotion.
By ELISA, detecting antibody titer Fig. 4 can find out: various adjuvants all have enhancing antibody and generate effect, and wherein PJOS and QuilA effect are the most obvious, can significantly improve OVA specific IgG in immune serum, IgGl and IgG2b antibody titer.And Alum only has the IgG of stimulation and IgG1 antibody generation effect, IgG2b is not had to obvious facilitation (with OVA comparison).These results suggest Alum is mainly for the immunological effect of Th2 type, to Th1 type cellular immunization activation a little less than, consistent with bibliographical information.PJOS and QuilA have the Th1 of promotion and Th2 type cellular immunization activation.
In sum, Rhizoma Panacis Japonici oleanane glycoside prepared by the present invention has obvious adjuvanticity, compare with traditional Alum adjuvant, Rhizoma Panacis Japonici oleanane glycoside can activating Th 1 and Th2 type cellular immunization, produce the cellular immunization of each hypotype antibody and Th1 type, may be significant in antiviral and anti-tumor vaccine application.With novel adjuvant QuilA comparison, Rhizoma Panacis Japonici oleanane glycoside has good safety, all do not produce haemolysis, and QuilA almost produces full haemolysis at 20 μ g/mL in the concentration of 640ug/mL.Therefore Rhizoma Panacis Japonici oleanane glycoside is to have safe and effective novel adjuvant.
Claims (10)
1. be used as a preparation method for the Rhizoma Panacis Japonici saponin of vaccine adjuvant, it is characterized in that this preparation method comprises the steps:
A, Rhizoma Panacis Japonici extract
By Rhizoma Panacis Japonici raw material pulverizing, according to weight ratio 1:5～15 of Rhizoma Panacis Japonici and ethanol water, smashing bamboo JIESHEN is immersed in volume fraction 55～85% ethanol waters, soak 2～5h, heating and refluxing extraction is 2～5 times again, each 1～5h, merge extractive liquid,, be evaporated to again extracting solution without alcohol taste, obtain a kind of concentrated solution;
B, extract and separate
The concentrated solution that steps A obtains repeatedly extracts with two and is separated according to the volume ratio 1:1～4 use n-butyl alcohol of concentrated solution and n-butyl alcohol, until n-butyl alcohol is mutually colourless, the butanol extraction liquid obtaining merges, then concentrating under reduced pressure, vacuum drying, so obtain n-butyl alcohol extract;
C, resin absorption
Macroporous adsorbent resin pretreatment: in resin column, macroporous adsorbent resin is immersed in to 20～24h in ethanol, then use ethanol water eluting not muddy to effluent, wash with water again to without ethanol taste, then macroporous adsorbent resin is immersed in by weight to 2～4h in 4～6%HCL aqueous solution, wash with water to neutrality, then macroporous adsorbent resin is immersed in by weight to 2～4h in 1～3%NaOH aqueous solution, wash with water to neutrality standby;
The n-butyl alcohol extract adsorption conditions that step B obtains is as follows: the blade diameter length ratio of resin column is 1:8～12, applied sample amount is 0.05～0.15g crude drug/milliliter column volume, standing over night after loading, after D101 absorption with macroporous adsorbent resin is saturated, is used ethanol water to carry out eluting;
Elution requirement is as follows: first water rinses to effluent colourless, then the ethanol water eluting that is 80～95% by volume fraction, and elution volume is 2～4 times of column volumes, eluting flow 0.8～1.2mL/min;
Concentrating under reduced pressure is carried out in the eluate obtaining, vacuum drying then, and its product is described Rhizoma Panacis Japonici saponin.
2. preparation method according to claim 1, is characterized in that in steps A, and described Rhizoma Panacis Japonici grinding particle size is 20～40 orders.
3. preparation method according to claim 1, is characterized in that in steps A, and the weight ratio of Rhizoma Panacis Japonici and ethanol water is 1:8～12.
4. preparation method according to claim 1, is characterized in that in steps A, and the volume fraction of described ethanol water is 62～75%.
5. preparation method according to claim 1, is characterized in that, in step B, described concentrating under reduced pressure carries out under pressure 0.001～0.01MPa, described dry be to carry out under the condition of 30～50 ℃ of pressure 0.01～0.1MPa and temperature.
6. preparation method according to claim 1, is characterized in that, in step B, the volume ratio of described concentrated solution and n-butyl alcohol is 1:1.8～3.2.
7. preparation method according to claim 1, is characterized in that in step C, and described macroporous adsorbent resin is D101 macroporous adsorbent resin.
8. preparation method according to claim 1, is characterized in that, in step D, described concentrating under reduced pressure carries out under pressure 0.001～0.01MPa, described dry be to be dried under the condition of 30～50 ℃ of pressure 0.01～0.1MPa and temperature.
9. the Rhizoma Panacis Japonici saponin obtaining according to preparation method described in claim 1-7.
10. the purposes of Rhizoma Panacis Japonici saponin according to claim 8 in preparing vaccine adjuvant.
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| CN115166066A (en) * | 2022-05-27 | 2022-10-11 | 陕西中医药大学 | Quality evaluation method of Qizhuibai oral liquid |
| CN115166066B (en) * | 2022-05-27 | 2023-11-03 | 陕西中医药大学 | Quality evaluation method of Qizhu oral liquid for improving white blood cells |
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