CN1109356A - Immunologic adjuvant - Google Patents

Abstract

The present invention uses the effective component lactones (ASDE) from traditional Chinese medicinal herb Angelica sinensis (oliv) Diels ASD of umbelliferae plant as immunological adjuvant, and uses poly saccharoses (ASDP) as the carrier of vaccine and the immunological adjuvant, and composite immanological adjuvant (ASDP/ASDE) is prepred. It can enhance the immunogenicity, its toxicity is very small. It is superior to the existing vaccine adjuvnt aluminium hydroxide jel.

Description

Immunologic adjuvant

The invention belongs to the pharmaceutical preparation field that derives from plant.

The vaccine of using clinically is aluminium hydroxide gel (Alum) with immunological adjuvant (Immunological Adjuvant) at present.It is from the initial usefulness thirties, and now existing more than 60 year historical.Its advantage: (1) is better to traditional vaccine adjuvanticity effect; (2) toxicity is little; (3) production cost is not high.But along with the appearance of new generation vaccine (as DNA recombiant vaccine, synthetic peptide vaccine), Alum then exists some shortcomings that can't overcome: 1, to the no adjuvanticity effect of some new generation vaccine (as influenza vaccines); 2, Alum preparation process, quality control is difficult to markization; 3, can not induce the cell immune response of body; 4, criticize and batch between adjuvanticity effect fluctuation big, poor repeatability or the like.Therefore, surplus in the of nearly ten year over, various countries' researcher has adopted separate sources and approach, has developed a series of new immunological adjuvants, but owing to reasons such as weak effect or toxicity are big, does not still have a kind of immunologic adjuvant so far, can be used as the immunological adjuvant of human vaccine.Maximum in new generation vaccine research and exploitation, one of common problem is that immunogenicity is very low, uses separately to be difficult to make the immunne response that body produces protective effect.Immunologic adjuvant and new generation vaccine share, and are means and strategies traditional, effective, that improve vaccine immunogenicity.Therefore, immunologic adjuvant just becomes the requisite constituent of new generation vaccine.U.S. Pat 4,843,067 less important composition that proposes raise immunity is the ASD polysaccharide, be used for immunization therapy, its Main Ingredients and Appearance is that astragalus polysaccharides plays potentiation to immunologic function, treatment people's immunologic hypofunction such as old-age group etc., its separation extraction preparation method and the present invention are different fully.

The objective of the invention is to have developed that adjuvanticity is effective, toxicity is little, be better than the new immunological adjuvant of used aluminium hydroxide gel.Immunologic adjuvant combines with corresponding vaccine (being antigen), forms best prescription, promptly forms immunostimulating complex, could best its adjuvanticity effect of performance.

Main contents of the present invention are, from traditional Chinese herbal medicine samphire [Angelica Sinensis(Oliv) Diels] be the ASD separation and Extraction and preparation polysaccharide partly ASDP is as the carrier of vaccine and adjuvant, ASDP itself also has immunobiologic activity.Lactone is ASDE1-8 partly, mainly uses ASDE ₃, 8 parts can be separately with also share, as immunological adjuvant.And partly be mixed with into by a certain percentage the complex immunity adjuvant with two, ASDP/ASDE, again respectively with related antigen such as the former vaccine of hepatitis B blood, hepatitis B gene engineering vaccine (r-HBsAg) etc. share (as experimental group), with Alum in contrast, use many index, detected the adjuvanticity effect.Result of study shows ASDP/ASDE ₃In experimental animals, can obviously strengthen former vaccine of hepatitis B blood and hepatitis B gene engineering vaccine immunogenicity (Immunogenicity), its antigenic half effective dose (ED ₅₀), experimental group generally will hang down 3～4 times than matched group, in the research process, does not see toxic and side effects; Induce in addition simultaneously and immunoregulation effect widely such as the cellular immunization of enhancing body, cytokine.From the broad sense immunological adjuvant, ASDP/ASDE is immunomodulator (Immunomodulator), both can be used for immunoprophylaxis (Immunoprophylaxis) and also can be used as immunization therapy fully.（Immunotherapy）

1, immunologic adjuvant, lactone material, ASDE series (ASDE _1-8), it is characterized in that from traditional Chinese herbal medicine samphire [Angelica Sinensis(Oliv) Diels] being to separate the ASD, extract and the lactone material of preparation;

A, Umbelliferae tradition Chinese herbal medicine (Angelica Sinensis(Oliv) Diels ASD), pulverize with pulverizer.

B, add 95% ethanol, equal-volume, 60 ℃, extracting 3 times, each 1 hour;

C, ethanol extraction merge, concentrating under reduced pressure removes organic solvent;

Insoluble matter behind d, the ethanol extraction dries, and is standby;

E, ethanol extraction column chromatography: (1) column chromatography: 100 * 3CM, (2) silica gel particle size range 100 orders, Haiyang Chemical Plant, Qingdao's product, 300 grams, (3) go up sample size and be 1:50(w/w) with the silica gel ratio, (4) eluent ethylacetate: thiacyclohexane 5:95～20:80(v/v), (5) flow velocity 2ml/ branch, eluent is collected by every part of 100ml, and (6) use silica GF254 (TLC), expanding system simultaneously, ethyl acetate: thiacyclohexane (1:5, V/V) survey Rf value, through ultraviolet analysis instrument for three purposed, wavelength 2537

Detect ASDE _3-8, 3650

Detect ASDE _1-2(7) according to the detection wavelength, elution time and Rf value difference, identical collection liquid, merging, concentrating under reduced pressure obtain ASDE lactone series (ASDE successively respectively _1-8): ASDE ₁Elution time 1 hour, Rf value 0.70, ASDE ₂Elution time 1-2 hour, Rf value 0.61, ASDE ₃Elution time 2.5-3 hour, Rf value 0.51, ASDE ₄Elution time 3-3.5 hour, Rf value 0.41, ASDE ₅Elution time 3.5-4.5 hour, Rf value 0.28, ASDE ₆Elution time 4.5-5.0 hour, Rf value 0.17, ASDE ₇Elution time 5-5.5 hour, Rf value 0.06, ASDE ₈Elution time 5.5-6.5 hour, Rf value 0;

F, ASDE _1-8These eight parts all have the immunological adjuvant activity, generally use ASDE in the experiment ₃;

G, ASDE _1-8Partly through the TLC spectrum, analyses such as ultraviolet and infrared spectrum meet the lactone material.

2, new generation vaccine and adjuvant carrier, polysaccharose substance, ASDP is characterized in that separation from traditional Chinese herbal medicine ASD, extraction and preparation method:

A, the ASD insoluble matter after the ethanol extracting is weighed;

B, add isopyknic distilled water PH7.0,60 ℃, extracting 1 hour;

C, water extract, concentrating under reduced pressure;

D, water are carried concentrate, add the chloroform of 1/10 volume: n-butyl alcohol (4:1), vibrate after 5 minutes, and filter;

E, filtrate, concentrating under reduced pressure;

F, 2 times of volume ethanol of adding, 5 minutes after-filtration vibrate;

G, precipitate are given a baby a bath on the third day after its birth inferior with ethanol;

After h, precipitate are washed, freezing, dry, get the ASDP sample;

I, ASDP sample are composed through TLC, and ultraviolet spectra and infrared spectrum (have significantly-the OH base-CH ₂Physicochemical properties such as base peak meet polysaccharose substance.

3, NEW TYPE OF COMPOSITE immunological adjuvant, promptly ASDP/ASDE is characterized in that, immunological adjuvant wherein is by the ASDE series for preparing among the ASD, uses ASDE usually more ₃, vaccine and immunological adjuvant carrier take by weighing ASDE and ASDP respectively for by the ASDP for preparing among the ASD, press the part by weight mixing of 1:2 after, be prepared into complex immunity adjuvant ASDP, ASDE, share by needed dosage and related antigen again.

4, the purposes of NEW TYPE OF COMPOSITE immunological adjuvant is characterized in that preparing the purposes of the immunomodulator (Immunomodulator) of immunoprophylaxis (Immunoprophylaxis) and immunization therapy (Immunotherapy).

ASDP/ASDE ₃The adjuvanticity evaluation:

1, ASDP/ASDE ₃Adjuvanticity

Use ASDP/ASDE ₃Share with hepatitis B vaccine from blood, hepatitis B gene engineering vaccine respectively, immune mouse, separation of serum after 6 weeks is with solid-phase radioimmunoassay (RIA Kli), measure mice serum sun revolution, serological conversion rate, S/N ratio and RIA reference units, experimental group all has obviously than matched group and increases.Antigen half effective dose (ED ₅₀), matched group is higher more than 3 times than experimental group.(table 1)

2, immunological adjuvant ASDE _1-8In, functional parts share, and the enhancement antigen immunogenicity is had synergism.(seeing Table 3)

3, anti-HB ₅The mice serum of antibody positive, experimental group and matched group be the two-fold dilution simultaneously, surveys the antibody horizontal of each dilute serum respectively, and the result shows, the anti-HB of experimental group serum ₅Antibody horizontal is high more than 20 times than matched group.(Fig. 1)

4, ASDP/ASDE ₃Reach Alum respectively as adjuvant, do the test of dose-effect response curve, the result shows, ASDP/ASDE ₃Wider range of effective dose, mice is from 128/64mg/kg to 2/1mg/kg.(Fig. 2)

5, ASDP/ASDE ₃To the function of the mononuclear phagocyte system of mice, the activity of the cellular immunization and the various kinds of cell factor all has immunoregulation effect widely.This is ASDP/ASDE ₃As the immunobiology basis of immunological adjuvant, also be simultaneously immunobiology basis as immunotherapeutic agent.

6, ASDP/ASDE ₃Popularity hemorrhagic fever inactivated vaccine, hepatitis B vaccine from blood etc. can both improve their immunogenicity.

7, ASDP/ASDE ₃Toxicologic study result shows that toxicity is very low, and with mice and rat, 4 kinds of adjuvant samples are measured its acute toxicity (LD ₅₀), do not see acute toxic reaction (table 2); ASDP/ASDE ₃The irritation test of local application; Use number of ways, repeatedly after the medication,, there is no pathologic reaction and histologic lesion's (table 4), show ASDP/ASDE with the anaphylaxis test of mice and Cavia porcellus again through naked eyes and histological observation ₃It or not the anaphylactogen (table 5 and table 6) of anaphylaxis.ASDP and ASDE series (are ASDE _1-8Effective ingredient) physicochemical property and preliminary the discriminating: (1) ASDP is through thin layer chromatography, and the analysis of characteristics such as ultraviolet spectra and infrared spectrum thinks that polysaccharose substance (Fig. 3, Fig. 4, Fig. 5) (2) ASDE series is ASDE _1-8Totally 8 effective ingredient, through thin layer chromatography, the analysis of ultraviolet spectra and infrared spectral characteristic thinks that they all are lactone material (Fig. 3, Fig. 6, Fig. 7).

Advantage of the present invention and good effect: major advantage is to make carrier by polysaccharide (ASDP), and HBsAg adjuvanticity effect obviously is better than used aluminium hydroxide gel (Alum), and toxic and side effects is very little, ASDP and ASDE ₃To the laboratory animal mononuclear phagocyte system, the T lymphocyte, cytokine, the IA of bone-marrow-derived lymphocyte all has immunoregulation effect widely.This is ASDP/ASDE ₃As the immunobiology basis of adjuvant multi-purpose, ASDP/ASDE ₃Tentatively try out in other vaccine, good immunological adjuvant effect is also arranged.Simultaneously also can be used as new immunomodulator and be used for immunization therapy.Medicine of the present invention source abundance, the stable good reproducibility of preparation flow method, feasibility is strong.ASDP/ASDE ₃Being in the nature immunomodulator (Immuno modulators) share with antigen (vaccine), be used for immunoprophylaxis (Immunoprophylaxis), can obviously reduce antigenic effective dose, improve the prophylactic immunization effect, especially concerning the new generation vaccine of costliness, ASDP/ASDE ₃If can promote the use of the large tracts of land crowd, can obtain considerable social benefit and economic benefit surely.

Description of drawings:

Fig. 1 is new immunological adjuvant ASDP/ASDE ₃With positive control adjuvant aluminium hydroxide gel (Alum), share with Hepatitis B From Human Plasma and hepatitis B gene engineering vaccine respectively, dilute the titre level of more anti-HBs antibody behind the separation mice serum, abscissa is a dilution factor, and ordinate is a cpm average (* 10 ³), curve 1 is r-HBsAg/ASDP/ASDE ₃Curve 2 known anti-HBs antibody positive control serums, curve 3 are the positive adjuvant contrast of r-HBsAg/Alum(), curve 4 is known anti-HBs negative antibody control serum.Fig. 1 shows the anti-HB of NEW TYPE OF COMPOSITE immunological adjuvant _SAntibody horizontal is high more than 20 times than positive control adjuvant Alum.

Fig. 2 is ASDP/ASDE ₃Adjuvanticity dose-effect curve figure.Abscissa is ASDP/ASDE ₃Concentration mg/kg, vertical coordinate A is cpm average (* 10 ³) the results are shown in curve 5, (contrast Al(OH) ₃Be 2.5 * 10 ³).Ordinate B for serological conversion rate (%), the results are shown in curve 6, (Al(OH) ₃Be 67%).Ordinate C for sample cpm value and known negative serum cpm value ratio (S/N), the results are shown in curve 7(AL(OH) ₃Be 28).

Fig. 3 is ASDE for ASDE adjuvant series _1-8And thin layer chromatography (TLC) collection of illustrative plates of ASDP, the expansion solvent is an ethyl acetate: (20:80, v/v) tracing are to use ultraviolet analyzer to thiacyclohexane.

Fig. 4 is ASDP ultraviolet spectrogram (UV), and curve 8 meets the polysaccharide ultraviolet spectra for ASDP is end absorption, and curve 9 has absworption peak for bSA 280mm.

Fig. 5 is ASDP infrared spectrogram (IR), and curve 10 is saccharide typical case hydroxyl (OH) peak.

Fig. 6 is ASDE series (ASDE _1-8) ultraviolet spectrogram, curve 11-18, ASDE _{1_2}UV is similar.

Fig. 7 is that curve 19,20 is respectively ASDE ₂, ASDE ₃Infrared spectrogram, typical carbonyl is all arranged, and (o=o) absworption peak (1200-1800nm) meets the lactone chemical substance.

Fig. 8 is ASDE _1-8And separation and Extraction and the preparation flow figure of ASDP.

Most preferred embodiment of the present invention

1, ASDE series (ASDE ₁- ₈Lactone) separation, extraction, preparation:

A, ASD100 gram adds 95% ethanol 100ml, and 60 ℃ of vibrations are extracted three times.

B, ethanol extraction merge, and concentrating under reduced pressure becomes 20ml.

C, silica gel column chromatography, chromatographic column 100cm * 3.0cm.

D, above-mentioned sample are 1:50(W/W with the silica gel ratio).

E, priority ethyl acetate-thiacyclohexane 5:95～20:80(v/v), eluting, flow velocity 2ml/ branch.

F, gradient elution, the eluent of fraction collection, through thin layer chromatography, expansion solvent system, ethyl acetate: thiacyclohexane (1:5, v/v), ultraviolet detection, wavelength 2537

Detect ASDE _3-8Part, 3650

Detect ASDE _1-2Part, and measure each several part Rf value (accompanying drawing 3).

G, eluent that physicochemical property is identical merge respectively, and decompression concentrates.

H, can obtain ASDE _1-8, ASDE wherein ₃It is 0.3 gram.

2, ASDP(polysaccharide) separate, extract, prepare;

A, the ASD alcohol-insoluble substance after extracting ASDE series dries 100 grams of weighing,

B, add the distilled water 100ml of PH7,

C, 60 ℃ carry and take out three times,

D, water extract are collected, merge, and concentrating under reduced pressure,

E, concentrated solution add chloroform: n-butyl alcohol 4:1, and precipitation is filtered,

F, get supernatant and add three times of ethanol precipitations and filter,

I, get precipitation and give a baby a bath on the third day after its birth time with ethanol,

J, precipitate through lyophilization, can get the ASDP3 gram.

3, immunostimulating complex is antigen+carrier (ASDP)+adjuvant (ASDE _1-8) preparation:

Mix in 2:1 ratio (w/w), vibrated 5 minutes, be prepared into the complex immunity adjuvant, and then mix with the antigen that is tried (vaccine), vibrated gently 30 minutes, after 2～3 hours, it is compound to be prepared into immunostimulation in room temperature, can the immunization experiment animal.

The new immunological adjuvant ASDP/ASDE of table 1. ₃Adjuvanticity

Hepatitis B surface is anti-

Immunological adjuvant dosage former (HBsAg) serum sun serum sun S/N RIA ED ₅₀The P value

Mg/kg microgram/Mus revolution rate of rotation %

AL(OH) ₃100 1.25 7/7 100 13.7 560 0.363

(contrast) 0.3125 3/7 43 0.8＜8

0.078 0/7 0 0 0

ASDP 60 30 1.25 7/7 100 32.9 1820 0.118 <0.01

ASDE ₃0.3125 7/7 100 12.7 551

0.078 2/6 33 6.0 22

* mice BALB/C male three ages in week, 8-10 gram

Sample dilutes with 50% calf serum 1:5

Acute toxicity (the LD of table 2.ASDP/ASDE ₅₀)

Mice (mg/kg) rat (mg/kg)

Immunological adjuvant S.C. I.M. I.V. S.C.

ASDE ₂>4000 >800 >800

ASDP/ASDE ₂>2000 >200 100

ASDE ₃>4000 >1600 >640

ASDP/ASDE ₃>2000 >800 200

Claims (4)

1, immunologic adjuvant, lactone material, ASDE series (ASDE _1-8), it is characterized in that from traditional Chinese herbal medicine samphire [Angelica Sinensis (Oliv) Diels] being to separate the ASD, extract and preparation lactone material:

(Angelica Sinensis (Oliv) Diels ASD), pulverizes with pulverizer a, Umbelliferae tradition Chinese herbal medicine;

B, add 95% ethanol, equal-volume, 60 ℃, extracting 3 times, each 1 hour;

C, ethanol extraction merge, concentrating under reduced pressure removes organic solvent;

Insoluble matter behind d, the ethanol extraction dries, and is standby;

E, ethanol extraction column chromatography: (1) column chromatography: 100 * 3CM, (2) silica gel particle size range 100 orders, Haiyang Chemical Plant, Qingdao's product, 300 grams, (3) going up sample size is 1: 50 (W/W) with the silica gel ratio, (4) eluent ethylacetate: cyclohexane extraction 5: 95～20: 80 (V/V), (5) flow velocity 2ml/ branch, eluent is collected by every part of 100ml, and (6) use silica GF254 (TLC), expanding system simultaneously, ethyl acetate: cyclohexane extraction (1: 5, V/V) survey Rf value, through ultraviolet analysis instrument for three purposed, wavelength 2537A detects ASDE _3-8, 3650A detects ASDE _1-2

(7), according to detecting wavelength, elution time and Rf value difference, identical collection liquid merges, concentrating under reduced pressure, obtains ASDE lactone series (ASDE successively respectively _1-8): ASDE ₁Elution time 1 hour, Rf value 0.70, ASDE ₂Elution time 1-2 hour, Rf value 0.61, ASDE ₃Elution time 2.5-3 hour, Rf value 0.51, ASDE ₄Elution time 3-3.5 hour, Rf value 0.41, ASDE ₅Elution time 3.5-4.5 hour, Rf value 0.28, ASDE ₆Elution time 4.5-5.0 hour, Rf value 0.17, ASDE ₇Elution time 5-5.5 hour, Rf value 0.06, ASDE ₈Elution time 5.5-6.5 hour, Rf value 0;

F, ASDE _1-8These eight parts all have the immunological adjuvant activity, generally use ASDE in the experiment ₃

G, ASDE _1-8Partly through the TLC spectrum, analyses such as ultraviolet and infrared spectrum meet the lactone material;

2, new generation vaccine and adjuvant carrier, polysaccharose substance, ASDP is characterized in that separation from traditional Chinese herbal medicine ASD, extraction and preparation method:

A, the ASD insoluble matter after the ethanol extracting is weighed;

B, add isopyknic distilled water PH7.0,60 ℃, extracting 1 hour;

C, water extract, concentrating under reduced pressure;

D, water are carried concentrate, add the chloroform of 1/10 volume: n-butyl alcohol (4:1), vibrate after 5 minutes, and filter;

E, filtrate, concentrating under reduced pressure;

F, 2 times of volume ethanol of adding, 5 minutes after-filtration vibrate;

G, precipitate are given a baby a bath on the third day after its birth inferior with ethanol;

After h, precipitate are washed, freezing, dry, get the ASDP sample;

I, ASDP sample are composed through TLC, and ultraviolet spectra and infrared spectrum (have significantly-the OH base-CH ₂Physicochemical properties such as base peak meet polysaccharose substance;

3, NEW TYPE OF COMPOSITE immunological adjuvant, promptly ASDP/ASDE is characterized in that, immunological adjuvant wherein is by the ASDE series for preparing among the ASD, uses ASDE usually more ₃, vaccine and immunological adjuvant carrier take by weighing ASDE and ASDP respectively for by the ASDP for preparing among the ASD, are prepared into complex immunity adjuvant ASDP/ASDE after the part by weight mixing by 1:2, share by needed dosage and related antigen again;

4, the purposes of NEW TYPE OF COMPOSITE immunological adjuvant is characterized in that preparing the purposes of the immunomodulator (Immunomodulator) of immunoprophylaxis (Immunoprophylaxis) and immunization therapy (Immunotherapy).

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