CN106692177A

CN106692177A - Anti-influenza and immune polysaccharide preparation lozenges and preparation method thereof

Info

Publication number: CN106692177A
Application number: CN201610851321.1A
Authority: CN
Inventors: 张菊明; 林佩芳; 张舸
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-09-27
Filing date: 2016-09-27
Publication date: 2017-05-24

Abstract

Anti-influenza and immune polysaccharide preparation lozenges are prepared from a traditional Chinese medicine immunomodulator-ACPS (actinidia chinensis polysaccharide) which is firstly studied, named and proved to have anti-influenza and immune functions. The lozenges not only have the immuno-enhancement function, but also have double functions of attack and defense as well as prevention of influenza and are safe and effective natural preparations. The technical core is a combination of ACPS extraction and a lozenge formula, crude dry powder is obtained from Chinese medicinal material, namely, actinidia chinensis, with a water extraction and alcohol precipitation method and supplemented with a golden tea extract, and the anti-influenza and immune polysaccharide preparation lozenges are prepared. The anti-influenza and immune polysaccharide preparation lozenges are mainly used for preventing influenza through sucking and treating simple type influenza and are ensured to be safe and non-toxic through security evaluation.

Description

A kind of anti-influenza polysaccharide formulation lozenge and its preparation method

Technical field

A kind of new Chinese medicine immune active ingredient --- Chinese gooseberry polysaccharide（Actinidia Chinensis Polysaccharide, abbreviation ACPS）, show through the present inventor's research, as a kind of new polysaccharide immunomodulator, can be effective Suppress duplication and the prevention influenza of influenza virus." the anti-influenza polysaccharide buccal tablet " formulated accordingly, for preventing and treating is flowed Row sexuality is emitted there is provided a kind of new safely and effectively natural medicine.The invention belongs to medicine bioengineering technical field.

Background technology

Influenza（Abbreviation influenza）, it is the Acute respiratory infectious disease caused by influenza virus.Because virus surface is anti- It is former（HA、NA）Variation is susceptible to, vaccine prevention influenza is increased parameter, crowd is lacked immunity.So propagate rapid, It is popular extensive.Once the life of tens of millions of people had been seized when being very popular in the past.Influenza virus has first, second, third gradegrade C type.China once occurred Ten larger prevalences several times.Mainly caused by Alphavirus.New avian influenza virus H7N9 also falls within subtypes of influenza A virus.At present Still lack the medicine that special efficacy prevents and treats influenza.Though some new drugs are had been developed that, such as amantadine, ribavirin, Peramivir, Because of the medicine that these synthesize, there is different degrees of toxicity.Safely and effectively anti-current is thus found from autonomic drug susceptible Cytotoxic drug, has obtained the attention of the various countries such as America and Europe and China.Immunocompromised person and the elderly, are influenza Susceptible population. Therefore, the research work for finding the medicament of energy enhance immunity has also launched.A kind of herbal medicine Echinacea preparation released by the U.S., With the health food for making adjustments immunity, flu-prevention, China market is had been enter into recent years.But U.S. FDA has given a warning title " I Be used for the foundation of food to the Echinacea of former approval and not can determine that.", and order violation company to be corrected.In any case, Searching is new can to improve immunity of organisms, reach flu-prevention, directly can suppress influenza virus again and reach the natural for the treatment of Medicine, just seem in the urgent need to.

The content of the invention

ACPS has resisiting influenza virus and flu-prevention to act on.Between 1983 ~ 1992 years, we are in anticancer among the people is originally found The extract of herbal medicine Chinese actinidia root, with the effect of Lectin samples.And after studying for many years, it is found that its active site is many one kind Sugar, is by galactolipin（G）With arabinose（A）It is polymerized, G/A=3.05 ± 0.28.Then, we are named as China Kiwi berry polysaccharide（ACPS）.And at home and abroad prove that this is a kind of new immunomodulator first.

Further study show that ACPS has clear and definite anti influenza to act on.It is briefly described as below：

The inhibitory action that 1.ACPS is formed to virus plaque

The dog kidney passage cell MDCK of influenza infection is incubated in shrinkage pool plate, adds the ACPS of various concentrations, and control group is not Plus ACPS.After continuing to cultivate, observation calculates plaque number in per hole, is calculated by formula：Plaque reduction ratio %=（The average plaque of control group The average plaque number of number-administration group）Average plaque number × 100% of/control group.Result is as shown in table 1：

The inhibitory action of the ACPS infected by influenza plaque tests of table 1

Result shows the inhibiting rate to each strain of listed influenza virus, strengthens with the increase of ACPS concentration.Concentration is more than 0.5mg/ml is to show inhibitory action.The suppression of ACPS infected by influenza plaque tests, shows that it can suppress influenza virus Replicate；

2. ACPS is given in advance, to the preventive effect of mouse infection influenza virus

The advance abdominal cavity once daily ACPS of Kunming mouse, control group abdominal cavity gives normal saline placebo.It is small after 24 hours Mouse intravenous injection influenza virus infection A type H₃N₂The anti-75-39 plants allantoic fluid in capital.Mouse death rate in seven days is observed, with comfort Agent group compares, as a result as shown in Figure of description Fig. 1.Show that advance disposable celiac gives ACPS, mouse death rate is from virus The 90% of control group (not being administered) is down to 30% (25mg/kg groups) P ＜ 0.01 of administration group.And show that the dose-dependant of drug effect is closed System.Show that ACPS has the effect of obvious flu-prevention.In 24 hours before influenza virus infection, ACPS convection current is disposably given Feel the protecting effect of mouse.Every group of mouse 10, intraperitoneal injection（i.p）Administration.In addition, according to 240 mouse of total in four batches Secondary experiment, in disposable intraperitoneal injection before 24 hours（i.p）ACPS is given, then influenza virus infection.ACPS is measured to influenza The half effective agent of the lethal protective effect of virus（ED₅₀mg/kg）Respectively 13.70/8.17/11.67 and 8.48, average ED₅₀For 9.95±3.36；

3. different time is administered in advance, for the preventive effect that mouse influenza virus infect

Each group mouse gave ACPS 3.125mg/ in 24 hours before Intravenous Infection influenza virus and 6 hours, respectively disposable i.P Kg, 6.25 mg/kg, 12.5 mg/kg, 25 mg/kg, 50 mg/kg and placebo physiological saline.With batch intravenous injection after administration Situations such as influenza virus, observation death, extending life.Result is as shown in table 2.The first 24 hours preventive effects of administration of display infection It is administered before better than 6 hours, and shows ED₅₀The former is 6.25 mg/kg, and the latter is 8.43 mg/kg；

The different time of table 2 gives protective effects of the ACPS to mouse vein influenza virus infection in advance

Note：- 6, -24 infect first 6 hours, 24 hours once dailies（i.P）.*P<0.05 **P<0.01

Additionally, in another group of experiment, in after disposable oral 25 mg/kg ACPS before 24 hours, influenza virus infection shows The protective rate and increase in life span of mouse, suitable with the mg/kg groups of intraperitoneal administration 3.125, the former is respectively 22.22% and 42.8%, The latter is respectively 22.22% and 47.6%；

Interferon is produced in 4.ACPS inducing mouses body

Kunming mouse disposable i.P give ACPS, and blood was taken in 2,4,6,18 hours, separates serum, after 2 times of dilutions, adds L₉₂₉ In cell culture, determine serum interferon titre, and with positive anti-viral medicine poly IC Poly I:C is contrasted, and is as a result shown, The serum interferon of 2 hours reaches peak after intraperitoneal injection ACPS.When ACPS dosage is 50mg/kg, 25 mg/kg and 12.5mg/ Kg, it is 160,40 and 10 that interferon titre is measured respectively, in dose-effect relationship.The serum interferon titre of 2 hours and Poly I:C Quite, titre declines after 4 hours, is disappeared after 6 hours.The interferon that ACPS is induced, to acid and the tolerance and poly IC phase of heat Seemingly.Accordingly, it is possible to be also alpha-interferon.As shown in table 3：

Interferon and its comparing to acidproof heat-proof that the ACPS of table 3 is induced

Brief summary：1. formed to suppress to test by virus plaque in vitro and prove that ACPS can directly suppress the duplication of influenza virus；② In vivo by the experiment of administration postoperative infection influenza virus in advance, it was demonstrated that ACPS plays the role of flu-prevention.The anti influenza of ACPS Effect may be also relevant with its energy inducement interferon.

It was found that and prove ACPS immunoregulation effect：

1. in vivo studies shows that ACPS can strengthen the phagocytic function and mouse anti-infectious immunity power of macrophage.BALB/C mice is every Day i.P injection ACPS 32 mg/kg × 12, after drug withdrawal next day measure peritoneal macrophage phagocytosis Rhodotorula sp function it is obvious Increase, the percentage of phagocyte increases to 37.5 ± 9.0 (p of administration group from the 15.0 ± 4.8 of non-administration control group< 0.01)；Phagocytic index increases to the 0.92 ± 0.67 of administration group from the 0.28 ± 0.13 of control group（p<0.05）.In addition, mouse is used After the viable bacteria E.Coli infection of median lethal dose, its death rate is down to the 0% of administration group from the 50% of control group（p<0.0.1）, but ACPS not bacteria growing inhibitings in vitro；

2. the proliferation function of the T lymphocytes of pair mouse and people

After mouse thymus T cell is cultivated together with ACPS, add³After H-TdR, display ACPS promotes the DNA of thymus T cells to close Into.Average CPM ± SD increases to 13342 ± 2473 (p of administration group from the 3449 ± 231 of blank control group<0.01).In addition, through The human peripheral T and bone-marrow-derived lymphocyte for obtaining are separated, is cultivated together with ACPS, the proliferation and metabolism for measuring T cell with mtt assay is obvious Increase, and have medicine dose-effect relationship.But B cell is without influence.As shown in table 4：

The ACPS of table 4 is to people T, the influence of B cell proliferation

5 Chronic Hepatitis Bs volunteer application ACPS 2-4mg/ days × 10 days, take PBLC, use monoclonal antibody IIF measures T₄And T₈Cell.Result shows T₄Cell（CD₄Cell）22-30.4% from before treatment（Averagely 25.2%）Increase to the 31-37.5% after treatment（Average 36.5%）, T₄/T₈Ratio is also shown in that recovery increases；

3. the white betweenness of endogenous mouse is induced₂（1L-2）

Daily mg/kg × 4 day of I.P ACPS 6 of mouse, the 3rd, 6 days after drug withdrawal, the T cell strain ctll cell relied on 1L-2, Plus after 3H-T α R are mixed, measure in the SPL culture supernatant of mouse, it promotes the 1L-2 activity of ctll cell, with life Reason saline control group compares, and is respectively increased 243%（3 days, p<0.01）With 265%（6 days, P<0.05）.24 mg/kg dosage groups Result is similar to；

4.ACPS activates internal NK（NK）The activity of cell

Continuous 10 days of BALB/C mice i.P ACPS, SPL was respectively taken as effector cell in the 10th, 13 days.Target cell YAC-1 oncocytes are used in advance¹²⁵The radioactivity that I-UdR is marked as each cell is 1-2CPM.It is 200 by effect target cell ratio:1 With 400:1, after both are mixed, the radioactivity umber of pulse of supernatant and cell pellet is determined respectively, calculate¹²⁵I-UdR The release total CPM/ of percentage=supernatant (the total CPM of cell CPM+ supernatants) × 100%.Result is as shown in table 5：

Influences of the ACPS to spleen nk cell activity is given in the body of table 5

The result of table 5 shows, compared with placebo, the spleen NK cells to ACPS group mouse are bright to the CDCC of target cell It is aobvious to improve, show that ACPS can activate the oncolytic activity of NK cells in vivo

Brief summary；Each result proves ACPS as a kind of immunomodulator above, and the macrophage to being played an important role in immune response is thin Born of the same parents, particularly T lymphocytes, T₄Cell, NK cells etc. have facilitation, so as to be conducive to starting the multiple of immune defense chain Link, to resist the infectious disease such as tumour, virus, fungi for causing because immune surveillance function is low.

Security evaluation and test confirms ACPS safety non-toxics：

1. acute toxicity test in mice.Dosage>800mg/kg, intraperitoneal injection, without dead mouse；

2. rat, family's dog Formulations for systemic administration long term toxicity test.Dosage from -100 mg/kg/ days 5 mg/kg/ days, continuous 60 days and 180 days, have no animal dead., without significant change, blood, routine urinalysis, hepatic and renal function are showed no exception for body weight, appetite, behavior.It is right The internal organs check pathological section such as the heart, lung, liver, kidney, intestines, has no pathologic damage；

3. the long-term eye drip of rabbit（45 days）, cornea IR 0, intraocular pressure and pupil be without exception, and cornea check pathological section has no Damage.The mg/kg of rabbit vein injection ACPS dosage 4.5,13.5 mg/kg, 22.5 mg/kg, 12 experiments, do not measure altogether Electrocardiographic abnormality, blood pressure breathing also has no significant effect.

The extraction preparation method of ACPS：

1. take cleaning dries Chinese actinidia root medicine materical crude slice or dry fruit skin, after coarse crushing, plus six times of boiling water, steeped overnight at room temperature

2. leachate is collected；Medicinal material adds boiling water to impregnate once again, collects and merge two-stage leaching liquid

3. leachate filtering, filtrate decompression is concentrated into 1/10

4. concentrate under agitation, is slowly added into ethanol, makes final concentration up to 30%, stands 24 hours

5. Aspirate supernatant, adds ethanol up to 75% concentration with method, stands overnight

6. it is centrifuged or leaching sediment.Plus quintuple deionized water, insoluble matter is filtered after boiling while hot.Second is slowly added into after cooling Alcohol is up to 80% concentration, taking precipitate

7. sediment washed once once, then with ether with absolute ethanol washing

8. sediment drying under reduced pressure at room temperature, obtains ACPS Thick many candies dry amorphous powder.Hermetically drying is preserved.

Recipe ingredient of anti-influenza polysaccharide buccal tablet and preparation method thereof：

1. recipe ingredient：ACPS dry powder 50-85%, gold tea extraction 15-45%, auxiliary material：Filler, lubricant etc. are appropriate, adjust Taste agent eucalyptus oil is appropriate

2. manufacture method：Prescription drug dry powder is mixed, plus appropriate filler（Starch, dextrin, Icing Sugar and eucalyptus oil is adsorbed Calcium sulfate）With lubrication humectant（50% ethanol）, stir and evenly mix rapidly, softwood processed, mechanism particle, 60 DEG C of drying under reduced pressure（Water content 3 ~ 5%）, whole grain（Sieving）, plus collapse Jie's agent mixing, compressing tablet（0.65g/ pieces）, it is coated, quality inspection is finally packed

Remarks：Also direct powder compression can be used（See embodiment）.Make the shorthand method of gold tea extraction by oneself：Take gold Tea, coarse crushing, water decoction, after concentration, acid adding, PH=1 ~ 2 are placed 24 hours, and filtering obtains precipitate, is made dry powder as gold Tea extraction.

Embodiment：10000 anti-influenza polysaccharide buccal tablets of trial-production

Prescription：ACPS 2~5.5KG

0.8 ~ 2KG of gold tea extraction

Auxiliary material：Icing Sugar, eucalyptus oil, microcrystalline cellulose, magnesium stearate etc.

Preparation method：Using direct powder compression.Got it filled powder by prescription, cross 60 ~ 80 mesh sieves, add auxiliary materials and mixing, dry-pressing is into 10000 Piece.Specification：Every 0.65 gram of weight.Usage and consumption：Buccal, one time 2, one day 10 ~ 20, gradation buccal.This lozenge is main For：The treatment of the preventative buccal and simple form influenza of influenza.

Brief description of the drawings

Fig. 1 is ACPS to mouse infection influenza virus preventive effect figure.

Claims

1. anti-influenza polysaccharide formulation lozenge and preparation method thereof, one of its technical characteristic is to use to be studied through the present inventor to demonstrate,prove The bright Chinese gooseberry polysaccharide-ACPS with immunoregulation effect and resisiting influenza virus double action.

2. its technical characteristic two described in claim 1, is the extraction preparation method of ACPS：Take Chinese actinidia root medicine materical crude slice or its dry fruit Skin, coarse crushing it, plus steeped overnight after boiling water；Leachate is collected, repeated impregnations once, merge two-stage leaching liquid；It is concentrated into original Behind the 1/10 of volume, delay and add ethanol, make up to 30% concentration；Take supernatant after standing, then add the ethanol to make up to 75%, centrifugation or leaching Sediment, then after being boiled with water, filter insoluble matter；Add ethanol up to 80% concentration after filtrate cooling；Centrifugation or leaching precipitation after standing Thing, after being washed with absolute ethyl alcohol, ether, drying under reduced pressure obtains ACPS dry powder.

3. its technical characteristic three described in claim 1, is the reasonable compatibility by ACPS and gold tea extraction, and select eucalyptus oil As flavor enhancement, by the lozenge that wet particle method or direct compression method operation are pressed into（0.65 gram/piece）, orally use；Lozenge is matched somebody with somebody Side and the feature of weight proportion：ACPS dry powder 50 ~ 85%, gold tea extraction 15 ~ 45%, filler flavor enhancement etc. are appropriate.

4. four of its technical characteristic described in claim 1, its pharmacodynamic properties is can to adjust immunologic function, enhancing body resistance Power, can suppress the duplication of influenza virus again, therefore has to influenza and have conditions in both attack and defence, prevent and treat the effect for combining；Suitable for the pre- of influenza The treatment of anti-property buccal and simple form influenza.