CN103191157B - Propolis flavone liposome for improving livestock and poultry immunity and preparation method thereof - Google Patents
Propolis flavone liposome for improving livestock and poultry immunity and preparation method thereof Download PDFInfo
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- CN103191157B CN103191157B CN201210203668.7A CN201210203668A CN103191157B CN 103191157 B CN103191157 B CN 103191157B CN 201210203668 A CN201210203668 A CN 201210203668A CN 103191157 B CN103191157 B CN 103191157B
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- 241000241413 Propolis Species 0.000 title claims abstract description 73
- 229940069949 propolis Drugs 0.000 title claims abstract description 73
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 title claims abstract description 71
- 229930003944 flavone Natural products 0.000 title claims abstract description 71
- 235000011949 flavones Nutrition 0.000 title claims abstract description 71
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 title claims abstract description 71
- 150000002212 flavone derivatives Chemical class 0.000 title claims abstract description 68
- 239000002502 liposome Substances 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 230000036039 immunity Effects 0.000 title abstract description 6
- 244000144972 livestock Species 0.000 title abstract description 4
- 244000144977 poultry Species 0.000 title abstract description 3
- 239000003814 drug Substances 0.000 claims abstract description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000002347 injection Methods 0.000 claims abstract description 14
- 239000007924 injection Substances 0.000 claims abstract description 14
- 239000000463 material Substances 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 7
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 8
- 229940107161 cholesterol Drugs 0.000 claims description 4
- 235000012000 cholesterol Nutrition 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- -1 wherein Substances 0.000 claims description 4
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 claims description 3
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 3
- 229960004756 ethanol Drugs 0.000 claims description 3
- 229940067606 lecithin Drugs 0.000 claims description 3
- 235000010445 lecithin Nutrition 0.000 claims description 3
- 239000000787 lecithin Substances 0.000 claims description 3
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- 238000003756 stirring Methods 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 13
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- 241000287828 Gallus gallus Species 0.000 description 27
- 102100037850 Interferon gamma Human genes 0.000 description 13
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Abstract
The invention relates to propolis flavone liposome for improving livestock and poultry immunity and a preparation method thereof, and belongs to the field of traditional Chinese veterinary drug preparation and immunization application. The preparation method is characterized in that an ethanol injection method is adopted; and according to encapsulation efficiency as an index, a liposome-drug ratio, a membrane-material ratio and an injection rate are researched by a response surface method and are selected, and according to the selection result, the optimal liposome-drug ratio of 9.6: 1, the optimal membrane-material ratio of 8.5: 1 and the optimal injection rate of 0.8mL.min<-1> are determined as preparation conditions. The propolis flavone liposome has high encapsulation efficiency and strong immunological enhancement activity. The propolis flavone liposome obviously improves immunological enhancement activity of propolis flavone.
Description
One, technical field
The present invention relates to a kind of preparation method of propolis flavone liposome, and improve the application of immunologic function of livestock and birds, belong to herbal medicine technology of preparing and immune application.
Two, background technology
Propolis flavone (propolis flavone, PF) be one of main effective ingredient contained in propolis, there is biological function and the pharmacotoxicological effect widely such as antioxidation, resisting pathogenic microbes, antiinflammatory, infection, antiallergic, antitumor and enhancing immunologic function.Yet, the antioxidation of flavone compound is mainly to avoid other composition oxidized by the preferential oxidation of self, this just makes flavone self unstable, and the unstability of propolis flavone affects the performance of storage, use and its effect of propolis extract to a great extent.And propolis flavone is directly used in veterinary clinic as a kind of immunostimulant, exist the weak points such as internal metabolism is very fast, action time is shorter, complicated component, quality control is difficult, immunological enhancement is relatively weak, thereby limited the clinical practice of propolis flavone.In order to develop better the pharmacological action of propolis, the research of propolis novel form is become to focus gradually.
Liposome is the folliculus with multiple layer or monolayer unit membrane structure of being prepared by lipoids, and its similar biomembrane, in vivo can biodegradation.Because liposome composition is phospholipid and cholesterol, be also the main component of biological cell membrane, belong to body endogenous material, there is good biocompatibility and degradability, nontoxic, non-immunogenicity.The structure of liposome is similar with biological cell, easily by cell absorption, fusion, fat exchange, endocytosis and by cellular uptake; And there is certain elasticity and morphotropism, than the nanoparticle of the other types of same particle size, easily enter lesion tissue.Therefore, liposome has become one of content of modern medicines study on the carrier.Liposome also can be used as immunological adjuvant simultaneously, as far back as 1974, by the body fluid of the reported first lipid physical ability enhancing body such as Allison and cell-mediated immunne response, can reach decades of times even higher to the potentiation of vaccine.Utilize liposome to there is the advantages such as targeting drug release, slow release, non-immunogenicity, reduction poisonous side effect of medicine, propolis flavone is encapsulated in liposome, liposome not only can prevent that propolis flavone is oxidized, and can effectively maintain the valid density of propolis flavone in body, improved its bioavailability.Thereby liposome can be used as a kind of excellent carrier of propolis flavone.
The present invention adopts alcohol injection that propolis flavone is prepared into propolis flavone liposome (propolis flavone liposome first, PFL), and it is applied together with animal vaccine, set up the preparation method of propolis flavone liposome, optimized preparation condition, find that propolis flavone lipid physical ability significantly improves the immunological enhancement of propolis flavone, improves its bioavailability.
Three, summary of the invention
Technical problem
The present invention is directed to the problems such as propolis flavone is oxidizable, water solublity is low, be difficult for micro-absorption, bioavailability is low, a kind of preparation method of propolis flavone liposome is provided, reach and significantly improve propolis flavone immunological enhancement object, and it is clinical to be applied to poultry.
Technical scheme
Preparation method with alcohol injection and response surface method design propolis flavone liposome.After optimizing, the best preparation method of propolis flavone liposome is: take by a certain percentage lecithin, cholesterol and propolis flavone, be dissolved in the dehydrated alcohol of 10mL and make its dissolving.At the uniform velocity be injected in 40 ℃ of buffer PBS20mL (0.01mol/L), continue constant temperature and stir, remove organic solvent until without ethanol taste, the ultrasonic 20min of water-bath, cross successively 0.80 μ m, 0.45 μ m and 0.22 μ m microporous filter membrane each 1 time, obtain propolis flavone liposome.The propolis flavone liposome of according to said method preparing, average envelop rate is 91.67% ± 0.21%.
Propolis flavone liposome is applied to chicken together with newcastle disease vaccine, can significantly improve that chicken serum newcastle epidemic disease antibody is tired, lymphopoiesis and cytokine content, have good immune enhancing function, to improving fowl immunologic function, disease preventing and treating has significant application value.
Beneficial effect
Animal epidemic mainly prevents by vaccine, but a lot of vaccine exists the shortcomings such as less immunogenic, needs immunostimulant to assist and could produce powerful immunne response.Existing conventional immunostimulant as oily adjuvant, aluminium glue adjuvant etc. often exist zest strong, have shortcomings such as residual.Because Chinese medicine has green safety, from Chinese medicine, developing immunostimulant has become focus.
Propolis flavone is one of main effective ingredient contained in propolis, has biological function and the pharmacotoxicological effect widely such as antioxidation, resisting pathogenic microbes, antiinflammatory, infection, antiallergic, antitumor and enhancing immunologic function.Yet, the antioxidation of flavone compound is mainly to avoid other composition oxidized by the preferential oxidation of self, this just makes flavone self unstable, and the unstability of propolis flavone affects the performance of storage, use and its effect of propolis extract to a great extent.And propolis flavone is directly used in veterinary clinic as a kind of immunostimulant, exist that poorly water-soluble, internal metabolism are very fast, action time is shorter, complicated component, quality control has the weak points such as certain difficulty, immunological enhancement relatively a little less than, thereby limited clinical practice.Utilize liposomal encapsulated propolis flavone, not only can prevent that propolis flavone is oxidized unstable, solve the problems such as poorly water-soluble, and can effectively maintain the valid density of propolis flavone in body, improve its bioavailability.
The present invention adopts alcohol injection and response surface method that propolis flavone is prepared into propolis flavone liposome, gained liposome encapsulation is high, not only can solve that propolis flavone composition is oxidizable, water solublity is low, be difficult for the problems such as preparation, and be convenient to the absorption of medicine, and can make the release on drug slow ground, effectively extend drug effect, can significantly improve Immune Function In Animals, significant to opposing epidemic disease.The present invention, as a kind of novel herbal medicine, is free from side effects.
Compared with prior art, advantage of the present invention is as follows:
1. the preparation of propolis flavone liposome has no report both at home and abroad, the invention provides a kind of preparation method of propolis flavone liposome and the condition of optimization thereof, has filled up the blank of domestic and international research.
2. the immune-enhancing activity of propolis flavone liposome has no report.By enforcement of the present invention, proof propolis flavone can significantly improve its immune-enhancing activity after being prepared into propolis flavone liposome, show as the content of obvious raising antibody titer, lymphopoiesis, IgG and IgM content, cytokine IL-2, IFN-γ, with propolis flavone ratio, in immune middle and late stage better effects if, there is stronger slow releasing function, thereby strengthen Immune Function In Animals.Therefore, propolis flavone liposome can be used as the material of development of new immunostimulant, for development of new immunostimulant provides material and demonstration.
Four, accompanying drawing explanation
Fig. 1 is the variation diagram (Log2) of the antibody titer of each group, and note: a-e marks on the same day not containing same letter person's significant difference (P < 0.05).H: high dose group, M: middle dosage group, L: low dose group.Figure below is same;
Fig. 2 is concentration change figure (the μ gmL of IgG in each group
-1);
Fig. 3 is concentration change figure (the μ gmL of IgM in each group
-1).
Five, the specific embodiment
1. the preparation of propolis flavone liposome
Adopt alcohol injection to prepare propolis flavone liposome.Take by a certain percentage lecithin, cholesterol and propolis flavone, be dissolved in the dehydrated alcohol of 10mL and make its dissolving.At the uniform velocity be injected in 40 ℃ of buffer PBS20mL (0.01mol/L), continue constant temperature and stir, remove organic solvent until without ethanol taste, the ultrasonic 20min of water-bath, cross successively 0.80 μ m, 0.45 μ m and 0.22 μ m microporous filter membrane each 1 time, obtain propolis flavone liposome.
On the basis of investigating in single factors, determine 3 major influence factors that affect propolis flavone liposome encapsulation, be fat medicine than (A), film material than (B), 3 factors of injection rate (C), take these 3 factors is independent variable, each factor is selected again 3 levels, take liposome encapsulation as response value, do totally 17 testing sites (5 central points) the quadratic regression Orthogonal Composite test of 3 factor 3 levels.EXPERIMENTAL DESIGN and the results are shown in Table 1.
In his-and-hers watches 1, result of the test is carried out response surface analysis, through secondary, reach the same goal after matching, the envelop rate that obtains propolis flavone liposome to fat medicine than (A), film material than the multinomial regression equation of secondary of (B), injection rate (C) is: Y=91.30-0.91A+2.21B-0.029C-0.64AB-2.36AC+0.36BC-7.32A
2-4.87B
2-7.57C
2.
Table 1 Box-Behnken EXPERIMENTAL DESIGN table and result
Utilize the result of Design expert7.0 software his-and-hers watches 1 to carry out multiple linear regression matching, model is carried out to variance analysis, the results are shown in Table 2, as shown in Table 2, model P < 0.0001, represents that model is extremely remarkable; This model establishment is described, this experimental technique is reliable.Lose and intend a P=0.2150 > 0.05, not remarkable; Show that regression equation exists without losing pseudo-factor, regression equation matching better.Further illustrate and in trial stretch, can be used for explaining and prediction experiment result.A, B, AC, A in model
2, B
2, C
2p value be respectively 0.0387,0.0005, < 0.0001, < 0.0001, < 0.0001 be all less than 0.05, shows that they have significant impact to PF liposome encapsulation., according to the large I of F value, know by inference, having the greatest impact of film material comparison PF liposome encapsulation, is secondly fat medicine ratio and injection rate meanwhile.Be film material than > fat medicine than > injection rate.
The variance analysis of table 2 response surface
Note: P represents to be greater than the probability of F value.P < 0.05, illustrates the remarkable on the impact of response value because of rope of correspondence, with *, represents; P < 0.01, illustrates that corresponding factor is remarkable on the impact of response value, represents with * *; P < 0.0001, illustrates that the impact that corresponding factor subtend should be worth is extremely remarkable, with * * *, represents.
The optimum preparating condition that obtains preparing propolis flavone liposome by response surface design method optimization is: fat medicine than 9.6: 1, film material than 8.5: 1, injection rate 0.8mLmin
-1.The average envelop rate of propolis flavone liposome of preparing under optimum preparating condition is 91.67% ± 0.21%,
2. propolis flavone liposome immune-enhancing activity comparison
The propolis flavone liposome (PFL) of preparing according to optimum preparating condition of take is object of study, with propolis flavone (PF) and blank nanometer liposome (BL) in contrast, measured they on newcastle disease (ND) vaccine immunity after the impact of body antibody titer, lymphopoiesis, IgG and IgM content, cytokine IL-2, IFN-γ content.
(1) animal grouping and processing
350 14 Japanese instar chicklings are divided into 7 groups at random, are respectively the height (0.500mgmL of PFL
-1), in (0.375mgmL
-1), low (0.250mgmL
-1) 3 dosage groups, PF group (0.500mgmL
-1), BL group, vaccine group (VC) and blank group (BC).During 14 age in days, except blank group, with NDV-IV, be all vaccine immunity, 28 ages in days two are exempted from.In first immunisation, 1～5 group of difference intramuscular injection relative medicine, only, vaccine group and blank group are injected equivalent normal saline to 0.5mL/, every day 1 time, continuously 3d.Before exempting from respectively at head and after first exempting from 7,14,21,28,35,42d detects the variation of dynamic change, serum HI antibody titer, IgG and the IgM content of periphery blood T lymphocyte propagation and cytokine IL-2, IFN-γ content.
(2) impact that propolis flavone liposome antagonist is tired
Head exempts from latter the 7th day, and the antibody titer of PF group is the highest in all groups, and is significantly higher than other each groups (P < 0.05), PFL
h, PFL
mand PFL
lantibody titer be significantly higher than BL group, VC group and BC group (P < 0.05); Head exempts from latter the 14th day, PFL
hthe antibody titer of group is the highest in all groups, and and PFL
m, PF group is significantly higher than PFL
lgroup, BL group, VC group and BC group (P < 0.05); Head exempts from latter the 21st day, PFL
hthe antibody titer of group is the highest in all groups, and is significantly higher than PFL
lgroup, PF group, BL group, VC group and BC group (P < 0.05); Head exempts from latter the 28th, 35,42 days, PFL
hthe antibody titer of group is the highest in all groups, and and PFL
mthe antibody titer of group is significantly higher than other each groups (P < 0.05) (Fig. 1).
(3) propolis flavone liposome is on lymphopoietic impact
Head exempts from latter the 7th day, and the A570 value of PF group is the highest, is significantly higher than immune matched group (VC group) and blank group (BC group) (P < 0.05); Head exempts from latter the 14th, 21,28,35 days, PFL
hand PFL
mgroup is significantly higher than PF group, blank liposome group (BL group), VC group and BC group (P < 0.05); Head exempts from latter the 42nd day, PFL
mthe A570 value of group is the highest, is significantly higher than PF group, BL group, VC group and BC group (P < 0.05) (table 3).
The dynamic change of table 3 periphery blood T lymphocyte propagation
Note:
a-fwith column data mark, do not contain same letter person's significant difference (P < 0.05).H: high dose group, M: middle dosage group, L: low dose group.Following table is same.
(3) impact of propolis flavone on IgG in serum and IgM content
The chicken serum IgG content of all PFL groups all has rising in various degree with respect to matched group, and PFL presents dose-dependence to the impact of IgG content in chicken serum.Within each experimental period, head exempts from latter the 7th day, PFL
hthe chicken serum IgG content of group is the highest, and is significantly higher than other each groups (P < 0.05) except PF group; Head exempts from latter 14 days, PFL
hthe chicken serum IgG content of group is the highest, and is significantly higher than other each groups (P < 0.05); Head exempts from latter the 21st, 28,35 and 42 days, PFL
hand PFL
mthe chicken serum IgG content of group is significantly higher than other each groups (P < 0.05) (Fig. 2).
The chicken serum IgM content of all PFL group all has rising in various degree with respect to matched group, and PFL to the shadow of IgM content in chicken serum to presenting dose-dependence.Within each experimental period, after head exempts from the 7th day, PF group chicken serum IgM content be the highest, and be significantly higher than other each groups (P < 0.05), PFL
hand PFL
mthe chicken serum IgM content of group is significantly higher than PFL
lgroup, BL group, VC group and BC group; Head exempts from latter 14 days, PFL
hthe chicken serum IgM content of group is the highest, and PFL
hand PFL
mthe chicken serum IgM content of group and PF group is significantly higher than other each groups (P < 0.05); Head exempts from latter the 21st, 35 and 42 days, PFL
hand PFL
mthe chicken serum IgM content of group is the highest, and is significantly higher than other each groups (P < 0.05); Head exempts from latter the 28th day, PFL
hthe chicken serum IgM content of group is the highest, and is significantly higher than other each groups (P < 0.05) (Fig. 3).
(4) impact of propolis flavone on serum IL-2 and IFN-γ content
PFL all has rising in various degree to IL-2 content in chicken serum with respect to matched group, and PFL presents dose-dependence to the impact of 1L-2 content in chicken serum.Head exempts from latter the 7th day, PFL
h, PFL
mbe significantly higher than PFL with the chicken serum IL-2 content of PF group
l, BL, VC and BC group (P < 0.05); Head exempts from the 14th day, PFL
mthe chicken serum IL-2 content of group is the highest, and is significantly higher than PF, BL, VC and BC group (P < 0.05); After head exempts from the 21st and 35 days, PFL
hand PFL
mthe chicken serum IL-2 content of group is the highest, and is significantly higher than other each groups (P < 0.05); Head exempts from latter the 28th day, PFL
hthe chicken serum IL-2 content of group is the highest, and is significantly higher than PF, BL, VC and BC group (P < 0.05); Head exempts from latter the 42nd day, PFL
mthe chicken serum IL-2 content of group is the highest, and is significantly higher than other each groups (P < 0.05) (table 4).
Concentration change (the pgmL of IL-2 in each group of table 4
-1)
The chicken serum IFN-γ content of all PFL groups all has rising in various degree with respect to matched group, and PFL is also doses dependence to the impact of IFN-γ content in serum.Head exempts from latter the 7th day, and the chicken serum IFN-γ content of PF group is the highest, and is significantly higher than PFL
l, BL, VC and BC group (P < 0.05); Head exempts from latter the 14th day, PFL
hthe IFN-γ concentration of group is the highest, and and PFL
mand PFL
lgroup is significantly higher than BL, VC and BC group (P < 0.05); Head exempt from after the 21st, PFL
hthe IFN-γ concentration of group is the highest, removes and PFL
mgroup difference significantly outside (P > 0.05), is not significantly higher than other each groups (P < 0.05); After head exempts from the 28th and 35 days, PFL
hand PFL
mthe chicken serum IFN-γ content of group, removes and PFL
lgroup difference is not significantly outside (P > 0.05); Be significantly higher than other each groups (P < 0.05); Head exempts from latter the 42nd day, PFL
hthe IFN-γ concentration of group is the highest, removes and PFL
mgroup difference significantly outside (P > 0.05), is not significantly higher than other each groups (P < 0.05) (table 5).
Concentration change (the pgmL of IFN-γ in each group of table 5
-1)
According to above-mentioned animal test results, can show that propolis flavone liposome can improve IgG in chicken serum antibody titer, lymphopoiesis, serum, IgM content and cytokine IL-2, IFN-γ content, thereby raising body's immunity, illustrates that the propolis flavone liposome the present invention relates to can be used for improving fowl body's immunity to reach the object of resist the disease.Overall merit is with high dose (0.500mgmL
-1) and middle dosage (0.375mgmL
-1) effect is best, from clinical production cost, considers that every chicken consumption is for injection 0.187mg.
Claims (1)
1. the preparation method of a propolis flavone liposome, it is characterized in that: take in proportion lecithin, cholesterol and propolis flavone, be dissolved in the dehydrated alcohol of 10mL, at the uniform velocity be injected in the 0.01mol/L buffer PBS20mL of 40 ℃, continuing constant temperature stirs, remove organic solvent until without ethanol taste, the ultrasonic 20min of water-bath, cross successively 0.80 μ m, 0.45 μ m and 0.22 μ m microporous filter membrane each 1 time, obtain propolis flavone liposome, wherein, fat medicine than 9.6: 1, film material than 8.5: 1, injection rate 0.8mLmin
-1, envelop rate meansigma methods is 91.67% ± 0.21%.
Priority Applications (1)
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017089842A1 (en) * | 2015-11-23 | 2017-06-01 | Apivita S.A. | Method for preparing a stable controlled-release propolis colloidal dispersion system for various uses |
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| CN110613843A (en) * | 2019-10-22 | 2019-12-27 | 武汉轻工大学 | Preparation method of water-soluble chitosan liposome adjuvant for livestock |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102133237A (en) * | 2011-03-31 | 2011-07-27 | 射阳县庆缘康蜂产品厂 | Propolis flavone soft capsule and preparation method thereof |
| CN102138941A (en) * | 2011-03-31 | 2011-08-03 | 射阳县庆缘康蜂产品厂 | Precursor liposome of propolis flavone and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102133237A (en) * | 2011-03-31 | 2011-07-27 | 射阳县庆缘康蜂产品厂 | Propolis flavone soft capsule and preparation method thereof |
| CN102138941A (en) * | 2011-03-31 | 2011-08-03 | 射阳县庆缘康蜂产品厂 | Precursor liposome of propolis flavone and preparation method thereof |
Non-Patent Citations (3)
| Title |
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| 单良等.蜂胶黄酮萃取物脂质体制备工艺的优化及体外缓释特性.《食品科学》.2008,第29卷(第8期),第232-237页. * |
| 汤志勇.蜂胶的超临界萃取及萃取物脂质体制备.《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》.2007,(第2期),第16页倒数第5-7行，摘要. * |
| 蜂胶黄酮萃取物脂质体制备工艺的优化及体外缓释特性;单良等;《食品科学》;20080831;第29卷(第8期);第232-237页 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017089842A1 (en) * | 2015-11-23 | 2017-06-01 | Apivita S.A. | Method for preparing a stable controlled-release propolis colloidal dispersion system for various uses |
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