CN103655628A - Propolis extract and application thereof - Google Patents
Propolis extract and application thereof Download PDFInfo
- Publication number
- CN103655628A CN103655628A CN201310473370.2A CN201310473370A CN103655628A CN 103655628 A CN103655628 A CN 103655628A CN 201310473370 A CN201310473370 A CN 201310473370A CN 103655628 A CN103655628 A CN 103655628A
- Authority
- CN
- China
- Prior art keywords
- extract
- propolis
- extraction
- ethanol
- supernatant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Jellies, Jams, And Syrups (AREA)
Abstract
The invention discloses an extract with the functions of inflammation prevention and liver protection, which is prepared from propolis through a great deal of process research. Strict activity experiment shows that the extract has the inhibiting effect on human leukocyte elastase and has the protection effect on liver injury caused by carbon tetrachloride. The extract can be applied to development of plant source inflammation prevention and liver protection products. At present, the propolis is utilized primitively in China. As a raw material for producing plant source inflammation prevention and liver protection products, the propolis extract which is disclosed by the invention has the advantages of high eating security and simple process. A tablet prepared from the propolis extract is simple in prescription and preparation method, stable in quality and beneficial to popularization and application.
Description
Technical field
The present invention relates to a kind of propolis extract, be specifically related to a kind of propolis extract being prepared by ad hoc approach and application thereof.
Background technology
Propolis is the natural gum of Apis herborization, and sneaks into frontal gland secretions and Cera Flava on it and chew through Apis a kind of colloid substance that modulation forms.The chemical composition of propolis mainly comprises flavonoid, terpenes, and the esters of aromatic acid and aromatic acid, phenol, alcohols, aldehyde, ketone, contain micro-protein and enzyme in addition.At present propolis be proved there is antibacterium, antifungal, antiviral, antiinflammatory, antioxidation, the pharmacologically active widely such as anticancer.The discovery of these effects directly provides foundation for developing extraordinary nourishing healthy and medicine.
Experiment showed, that propolis has good antiinflammatory action.Propolis ethanol extract and propolis aqueous extract can resist pleuritis Rat Pleural to be increased.Many experiments have all proved that propolis all has significant protective effect for various hepatic injury in addition.
Prior art discloses the preparation method of some propolis extracts, as applicant patent ZL200710111189.1 discloses a kind of have bioactive water-soluble bee glue extract and light propolis extract.Wherein disclose first propolis raw material is pulverized, added 10-60% alcoholic solution and extract 2-4 time, the consumption of each solvent is not less than four times of amounts, extracting solution merges filtration, decompression recycling ethanol obtains concentrated solution, and standing being precipitated and supernatant is dried after precipitation washing and can obtains solid light color propolis extract.
But the extract that technique scheme obtains is mainly light propolis, fail effective enrichment antiinflammatory liver-protecting activity composition, do not there is the biologic activity that good antiinflammatory protects the liver, cannot effectively be applied to take antiinflammatory, protect the liver as the development of nourishing healthy and the medicine of object.
In view of this, special proposition the present invention.
Summary of the invention
The object of this experiment is to provide a kind of propolis extract with antiinflammatory, liver-protecting activity, and its preparation method and methods for using them is further provided.
For achieving the above object, technical scheme of the present invention provides a kind of propolis extract, and its preparation process is:
(1) get propolis powder and add 3～6 times of volume 40% ethanol to extract, isolated by filtration, gets residue;
(2) residue is added to 3～6 times of volume 70% ethanol, heating is extracted, and isolated by filtration supernatant repeats to extract, and merges supernatant, 4 ℃ of refrigerator overnight, sucking filtration or centrifugal, residue is standby, and supernatant is through decompression dealcoholysis, standing 24h～48h, abandons supernatant, retains precipitation, obtains 70% alcohol soluble substance;
(3) to the residue after 70% ethanol extraction, add 3～6 times of volume 95% ethanol, heating is extracted, isolated by filtration supernatant, and residue repeats to extract 2-4 time, merges supernatant; 4 ℃ of refrigerator overnight, sucking filtration or centrifugal, gets supernatant and obtains 95% alcohol soluble substance;
(4) 95% alcohol soluble substance in 70% alcohol soluble substance obtaining in step (2) and step (3) is merged, concentrating under reduced pressure obtains propolis extract after being also dried.
Propolis powder of the present invention can adopt the disclosed multiple known propolis raw material of prior art, and as preferred technical scheme, propolis powder gel content of the present invention is 25～50%, and in this content range, the extraction effect of preparation method of the present invention is best.
Extract of the present invention, step (1) also comprises the process of propolis pulverizing sieve screening before extraction, preferred 20-120 mesh sieve excessively, so that extract better.
Extract of the present invention, in described step (1), 30～35 ℃ of heating extraction temperature, 4～12 hours extraction times, repeat extraction time 1～2 time.In described step (2), 30～50 ℃ of heating extraction temperature, 24～72 hours extraction times, repeat extraction time 2～4 times.In described step (3), 30～45 ℃ of heating extraction temperature, 12～48 hours extraction times, repeat extraction time 2～3 times.
Although prior art discloses and has usingd the technical scheme that different concentration ethanol extracts propolis as medium, but owing to only having adopted the technical scheme of conventional alcohol extraction, to extracting medium and extraction conditions, do not make and optimizing and revising, extract obtained complicated component, impurity (without antiinflammatory, liver-protecting activity person) content is large, causes being effectively applied to actual preparation preparation process.Meanwhile, the requirement that extraction ratio and extraction scale do not reach commercial application, effective site (having antiinflammatory, liver-protecting activity person) extraction ratio is low to moderate 10 left and right.In order to obtain extraction effect better, and can mass production, the present invention has designed said extracted condition, for each extraction stage, select best extraction medium and extraction conditions respectively, impelled the maximum stripping of active component and impurity (without antiinflammatory, liver-protecting activity person's) removal.
Further, the present invention optimize to limit also having made on extracting mode, and the optional ultrasonic extraction of the present invention or room temperature lixiviate in the present invention have adopted temperature control lixiviate (30～50 ℃) more, to extract better and prolection composition.Step (1) is stirring extraction in step (3), mixing speed 50～100rpm in step (1), mixing speed 110～200rpm in step (2) and (3).Under this mixing speed, be conducive to accelerate the stripping of material, improve extraction efficiency.
Extract of the present invention, wherein, in preparation process (1) and (3), temperature when concentrating under reduced pressure is sloughed ethanol is 35～45 ℃.
Extract of the present invention, wherein in preparation process (4), drying mode is natural drying, vacuum drying or lyophilization.
Extract of the present invention, in step 4, concentrating under reduced pressure temperature is 40～60 ℃.
The propolis extract that the present invention obtains is separated through the extraction of 40% ethanol, hydrophilic component in propolis can be removed.70% ethanol extraction is divided into the molten part of 70% alcohol and 70% water-soluble part through concentrating under reduced pressure, and object is by active component enrichment more.70% ethanol extraction and 95% ethanol extraction are standing through 4 ℃, and object is except Cera Flava and remove impurity.Extract enrichment correlation function composition, action effect is obvious.To be inventor determine on the basis of lot of experiments research in the selection of above-mentioned solvent, and retentive activity material is substantially removed in propolis powder the impurity without antiinflammatory, liver-protecting activity.Preparation method extraction ratio of the present invention is high, and low to appointed condition requirement, method is simple, easy to implement, and cost is low, is applicable to producing.
The second object of the present invention is to protect the application of above-mentioned propolis extract in preparation treatment antiinflammatory, hepatic.
The present invention finds that resulting extract has fine antiinflammatory, liver-protecting activity, so it can be prepared into various food, medicine or the health product of can clinical practice or conveniently taking according to conventional method, and as a kind of anti-inflammatory composition, or a kind of compositions that protects the liver.
In addition the further claimed pharmaceutical composition that contains above-mentioned propolis extract of the present invention.Described compositions includes, but are not limited to tablet, capsule, injection etc.The present invention is preferably tablet or capsule.
Currently available technology has no the relevant report about the ripe preparation of propolis extract, complicated because considering the composition of extract, easily occur quality problems, and concrete stripping discharges in preservation process also unsatisfactory.Inventor is devoted for years to the research in extract formulation, and finally obtain a kind of desirable tablet, by its prescription is proposed to specificity, adjust, make stability and the stripping of the tablet that obtains can reach desirable level, really having realized it can industry and the application of the formula of popularization.
The tablet that contains propolis extract of the present invention, is prepared from by the raw material that comprises following weight portion: propolis extract 80-120 part, dextrin 2-6 part, crospolyvinylpyrrolidone 5-10 part, magnesium stearate 0.2-0.6 part.
Wherein, preferably by the raw material that comprises following weight portion, be prepared from: 100 parts of propolis extracts, 4 parts, dextrin, 8 parts of crospolyvinylpyrrolidone, 0.4 part of magnesium stearate.
Tablet of the present invention can adopt the disclosed traditional methods of prior art to be prepared from, the present invention is not particularly limited this, it will be appreciated by those skilled in the art that, for tablet, the factor of core writes out a prescription, on the clear and definite basis of writing out a prescription, selecting suitable preparation method to prepare tablet is ordinary skill in the art means, on the impact of tablet quality, can ignore.
In order to improve stability and the dissolution rate of active component, the preparation that Chinese medicine extract is prepared tablet need to add a large amount of excipient and disintegrating agent and binding agent etc. conventionally, but said method easily causes the dose of tablet to increase, cause the maximization of tablet, cost is high, and too much easily causes the potential problems such as untoward reaction because of the consumption of adjuvant.In addition, Chinese medicine extract is different from the active component of Western medicine, and the selection of adjuvant also exists interactional interactively to active ingredient group in extract, and therefore, in the preparation process of Chinese medicine extract, the selection of prescription directly determines the quality of tablet.
Tablet of the present invention has been selected being used in conjunction with of dextrin+crospolyvinylpyrrolidone by specificity, in conjunction with sodium stearate, can guarantee that tablet has desirable stability and dissolution.Through evidence, with simple tablet formulation of the present invention, adopt the known conventional preparation method of prior art, after testing, the hardness of gained tablet is greater than 21daN, disintegration time is 12-18min, at the dissolution rate of 5min, 10min, 15min, 30min and 45min, is respectively 30-40%, 60-70%, 75-85%, 92-96%, 94-99%.The present invention that hence one can see that has obtained disintegration time section and the good tablet of stripping property.
Capsule of the present invention can obtain the direct fill capsule shells of propolis extract powder, and the present invention is not particularly limited this.
Adopt technique scheme, the present invention has obtained a kind of extract with antiinflammatory, hepatoprotective effect by a large amount of technical studies from propolis.By rigorous activity experiment, prove that this extract is inhibited to human leukocyte elastase; Carbon tetrachloride is caused to hepatic injury and there is protective effect.The present invention can be applicable to the exploitation of plant source antiinflammatory, liver-protecting product.China is comparatively original for the utilization of propolis at present, and propolis extract of the present invention, as the raw material of producing plant source antiinflammatory, liver-protecting product, has edible safety, the simple advantage of technological process.Prepared tablet formulation and preparation method are simple, and steady quality, are conducive to apply.
Accompanying drawing explanation
Fig. 1 is that propolis extract causes the result schematic diagram of hepatocyte SOD content influence to carbon tetrachloride;
Fig. 2 is that propolis extract causes the result schematic diagram of hepatocyte MDA impact on carbon tetrachloride;
Fig. 3 is that propolis extract causes the result schematic diagram of hepatocyte LDH content influence to carbon tetrachloride;
Fig. 4 is that propolis extract causes the result schematic diagram of hepatocyte CYP2E1 level affects to carbon tetrachloride;
Fig. 5 is that propolis extract causes the result schematic diagram of hepatocyte Caspase-3 protein level impact on carbon tetrachloride.
The specific embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art, the raw materials used commercial goods that is.
A preparation method for propolis extract, this preparation method comprises the following steps successively:
1, take and pulverize rear propolis powder (gel content 50%) 401.3g that crosses 120 mesh sieves, be placed in conical flask, by volume for 1:6 adds 40% ethanol, at 30 ℃, under the rotating speed of 90rpm, extract 8h, for 1:4 adds 40% ethanol, under the same terms, extract 12h by volume, extracting solution merges filtration, and concentrating under reduced pressure is standby as 40% extract in test example.
2, get step 1 residue obtained, by volume for 1:6 adds 70% ethanol, at 30 ℃, under the rotating speed of 180rpm, extract 1 day, by volume for 1:4 adds 70% ethanol, under the same terms, extract 2 days, volume ratio is that 1:3 adds 70% ethanol, extracts merge extractive liquid, under the same terms 3 days, at 4 ℃ of standing nights, then carry out sucking filtration.By decompression filtrate recycling ethanol, extremely completely without alcohol, standing 2 days, precipitation separation and supernatant, be dried after precipitation washing, is 70% alcohol soluble substance; Water lotion and supernatant merge, and after being dried, are the water-soluble part of 70% ethanol, standby as 70% water extract in test example.
3. get step 2 gained 70% alcohol soluble substance, by volume for 1:6 adds 95% ethanol, at 30 ℃, under the rotating speed of 150rpmn, extract 12h, by volume for 1:4 adds 95% ethanol, under the same terms, extract 24h, volume ratio is that 1:3 adds 95% alcohol, extracts 48h, merge extractive liquid, under the same terms, at 4 ℃ of standing nights, then carry out sucking filtration.Obtain 95% alcohol soluble substance, itself and 70% alcohol soluble substance are merged, drying under reduced pressure obtains described propolis extract 165.5g, and extracting yield is 41.24%; And each extract is placed in to-20 ℃ saves backup.
A preparation method for propolis extract, this preparation method comprises the following steps successively:
1, take and pulverize rear propolis powder (gel content 50%) 100g that crosses 80 mesh sieves, be placed in conical flask, by volume for 1:4 adds 40% ethanol, at 35 ℃, under the rotating speed of 100rpm, extract 12h, for 1:3 adds 40% ethanol, under the same terms, extract 4h by volume, extracting solution merges filtration, and concentrating under reduced pressure is standby as 40% extract in test example.
2, get step 1 residue obtained, by volume for 1:6 adds 70% ethanol, at 40 ℃, under the rotating speed of 200rpm, extract 2 days, by volume for 1:5 adds 70% ethanol, under the same terms, extract 1 day, volume ratio is that 1:3 adds 70% ethanol, extracts merge extractive liquid, under the same terms 1 day, at 4 ℃ of standing nights, then carry out sucking filtration.By decompression filtrate recycling ethanol, extremely completely without alcohol, standing 2 days, precipitation separation and supernatant, be dried after precipitation washing, is 70% alcohol soluble substance;
3. get step 2 gained 70% alcohol soluble substance, by volume for 1:4 adds 95% ethanol, at 45 ℃, under the rotating speed of 110rpmn, extract 24h, for 1:3 adds 95% ethanol, under the same terms, extract 24h by volume, volume ratio is that 1:3 adds 95% ethanol, extracts 48h, merge extractive liquid, under the same terms, 4 ℃ of standing nights, then carry out sucking filtration, obtain 95% alcohol soluble substance, itself and 70% alcohol soluble substance are merged, drying under reduced pressure obtains described propolis extract 40.89g, and extracting yield is 40.89%.
A preparation method for propolis extract, this preparation method comprises the following steps successively:
1, take and pulverize rear propolis powder (gel content 50%) 500g that crosses 80 mesh sieves, be placed in conical flask, by volume for 1:3 adds 40% ethanol, at 35 ℃, under the rotating speed of 60rpm, extract 12h, for 1:4 adds 40% ethanol, under the same terms, extract 6h by volume, extracting solution merges filtration, and concentrating under reduced pressure is standby as 40% extract in test example.
2, get step 1 residue obtained, by volume for 1:6 adds 70% ethanol, at 40 ℃, under the rotating speed of 180rpm, extract 2 days, by volume for 1:5 adds 70% ethanol, under the same terms, extract 1 day, volume ratio is that 1:3 adds 70% ethanol, extracts merge extractive liquid, under the same terms 1 day, at 4 ℃ of standing nights, then carry out sucking filtration.By decompression filtrate recycling ethanol, extremely completely without alcohol, standing 2 days, precipitation separation and supernatant, be dried after precipitation washing, is 70% alcohol soluble substance;
3. get step 2 gained 70% alcohol soluble substance, by volume for 1:6 adds 95% ethanol, at 45 ℃, under the rotating speed of 150rpmn, extract 24h, for 1:3 adds 95% ethanol, under the same terms, extract 24h by volume, volume ratio is that 1:4 adds 95% ethanol, extracts 24h, merge extractive liquid, under the same terms, 4 ℃ of standing nights, then carry out sucking filtration, obtain 95% alcohol soluble substance, itself and 70% alcohol soluble substance are merged, drying under reduced pressure obtains described propolis extract 202g, and extracting yield is 40.4%.
Prescription: propolis extract 1000g, DEXTRIN g, crospolyvinylpyrrolidone 80g, magnesium stearate 4g.
Preparation method: propolis extract is mixed with dextrin, crospolyvinylpyrrolidone, take the circumstances into consideration to add a small amount of 75% alcohol granulation depending on concrete condition, 60 ℃ dry; Add magnesium stearate, mix, tabletting and get final product.After testing, the hardness of the present embodiment gained tablet is 24daN, and disintegration time is 12min, at the dissolution rate of 5min, 10min, 15min, 30min and 45min, is respectively 40%, 70%, 85%, 96%, 99%.
Prescription: propolis extract 800g, dextrin 20g, crospolyvinylpyrrolidone 50g, magnesium stearate 2g.
Preparation method: propolis extract is mixed with dextrin, crospolyvinylpyrrolidone, take the circumstances into consideration to add a small amount of 75% alcohol granulation depending on concrete condition, 60 ℃ dry; Add magnesium stearate, mix, tabletting and get final product.After testing, the hardness of the present embodiment gained tablet is 21daN, and disintegration time is 14min, at the dissolution rate of 5min, 10min, 15min, 30min and 45min, is respectively 30%, 60%, 75%, 92%, 94%.
Prescription: propolis extract 1200g, dextrin 60g, crospolyvinylpyrrolidone 100g, magnesium stearate 6g.
Preparation method: propolis extract is mixed with dextrin, crospolyvinylpyrrolidone, take the circumstances into consideration to add a small amount of 75% alcohol granulation depending on concrete condition, 60 ℃ dry; Add magnesium stearate, mix, tabletting and get final product.After testing, the hardness of the present embodiment gained tablet is 25daN, and disintegration time is 18min, at the dissolution rate of 5min, 10min, 15min, 30min and 45min, is respectively 35%, 63%, 78%, 93%, 96%.
For the effect that proves that the antiinflammatory of said extracted thing protects the liver, the propolis extract that the method that inventor uses to be provided in above-described embodiment 1 prepares (also can be described as below activity extract) has carried out pharmacodynamic experiment, and result of study is as follows:
One, propolis extract antiinflammatory experiment-human elastase enzyme (HLE) suppresses experiment
In micro-sampling pipe, (250 μ L) successively adds PBS buffer 30 μ L, HLE enzyme liquid 50 μ L and sample liquid 20 μ L, 37 ℃ of balance 10min.Add again substrate solution 50 μ L.Sample and blank are all in triplicate.
37 ℃ of reactions add 20% acetum 50 μ L cessation reactions after 120min, finally each reactant liquor are carried out under 405nm wavelength to HPLC analysis, measure PNA growing amount; Blank group replaces sample liquid with DMSO.Reaction volume 150 μ L.Liquid-phase condition: chromatographic column: C18(VP-ODS, 150mm * 4.6mm, 5 μ m); Mobile phase: first alcohol and water; Condition of gradient elution: methanol volume fraction: 0min～10min is that 25%, 10min～15min is that 25%～90%, 15min～20min is that 90%～25%, 20min～25min is 25%; Flow velocity: 1mL/min; Detect wavelength: 405nm; Column temperature: 40 ℃; Sampling volume: 40 μ L.
The impact of table 1 propolis activity extract on HLE
Note: (1), in same column data, different shoulders are marked letter representation utmost point significant differences (p<0.01);
As shown in Table 1, when sample quality concentration is 50 μ g/mL and 100 μ g/mL, three extraction components all have HLE to suppress active.And suppression ratio and concentration are proportionate.The HLE suppression ratio of the water-soluble part of 40% ethanol is minimum, and significantly lower than other extracts, 100ug/ml only has 48.77%, and significant difference is extremely significantly (p<0.01); Under two concentration, all higher than the HLE suppression ratio (58.34% and 66.24%) of positive reference substance ursolic acid, statistically there is utmost point significant difference (p<0.01) in activity extract.Experimental result shows that gained propolis extract of the present invention is that activity extract has good HLE inhibition activity, is that in propolis, HLE suppresses active material base place.
Two, propolis extract protects the liver experiment-carbon tetrachloride and causes hepatocyte injury Protection (comprising 2.1-2.4)
2.1 propolis extracts cause the impact of damaging hepatocyte survival rate on carbon tetrachloride
Contain the trypsinization of EDTA0.25% in exponential phase Human normal hepatocyte (L02), add 1～2ml to stop digestion containing the culture medium of serum.Adjust cell concentration to 5x10
4/ ml is laid in 96 well culture plates, every hole 120ml.
After cell culture 48h, add the RPMI1640 culture fluid that contains serum in blank group, model control group, all the other each holes add respectively the carbon tetrachloride working solution of variable concentrations.Culture plate is placed in to 37 ℃, 5%CO
2in saturated humidity incubator, cultivate.
After 4h, the culture fluid in orifice plate is siphoned away, with the aseptic PBS washing after pre-cooling three times, in blank group, add serum-free medium, in all the other each holes, add the working solution 100 μ l of propolis extract and positive reference substance (silibinin), be placed in 37 ℃, 5%CO
2in saturated humidity incubator, cultivate.
After 4h, culture fluid in 96 orifice plates is siphoned away, with after aseptic PBS washing after pre-cooling three times, add after 100 μ l serum-free mediums, every hole adds MTT solution 10 μ l (5mg/ml), continue to cultivate 4h, discard culture fluid, then add 110ul DMSO, be placed in low speed concussion 15min on shaking table, crystal is fully dissolved, use enzyme-linked immunosorbent assay instrument at 490nm, to measure the light absorption value in each hole, calculate cell proliferation rate.
Table 2 propolis extract causes and damages hepatocellular impact carbon tetrachloride
Note: * * compares p<0.01 with model group; * compare p<0.05 with model group
As shown in Table 2,40% soluble component does not have activity at 50 μ g/mL and 100 μ g/mL substantially to impaired hepatocyte, compares and not there are differences with model group.70% soluble component all has certain protective effect to impaired hepatocyte under 50 μ g/mL and two concentration of 100 μ g/mL, and cell survival rate is respectively 45.376% and 58.546%, compares have the significance difference opposite sex (p<0.05) with model group.Activity extract all has very strong protective effect to impaired hepatocyte under 50 μ g/mL and two concentration of 100 μ g/mL, compares and has utmost point significant difference (p<0.01), and be significantly higher than silibinin positive controls with model group.
2.2 propolis extracts cause the impact of damaging hepatocyte SOD and MDA content on carbon tetrachloride
The phase L-02 cell of taking the logarithm, is inoculated in 24 orifice plates with 5 * 104/mL, and every hole 0.5mL tests after cultivating 48h.Each porocyte is after the carbon tetrachloride-injured liquid damage 4h of 10mmol/L, and sample treatment group adds sample working solution to intervene, and sets up blank group, model control group and positive controls simultaneously, and each group is intervened 4h.
Abandon culture fluid, each hole adds 0.5mL1%Triton-100 cell lysis, and fully piping and druming mixes rear collection, and the centrifugal 10min of 2500rpm gets supernatant and measures by test kit explanation.Computing formula is as follows:
Experimental result is shown in Fig. 1 and Fig. 2.Note: △ △ compares p<0.01 with normal group; * compares p<0.01 with model group; * compare p<0.05 with model group.
From Fig. 1 and Fig. 2, model group MDA content extremely significantly increases (p<0.01), for 2.42 times of Normal group, and SOD activity is significantly reduced to 53.52% of normal group, the cytolipin peroxide injury that tetrachloro-methane induction damage is described is serious, has produced a large amount of lipid peroxidation product MDA.SOD exhausts in a large number as the specificity antioxidase of removing the superoxide anion in ROS free radical in body.
After each grading extraction of propolis part and positive product are intervened with 50 μ g/mL and 100 μ g/mL respectively, cell SOD activity is all improved to some extent, wherein the active Extraction parts in 50 μ g/mL dosage groups is compared the SOD content utmost point and is significantly improved (p<0.01) with model group, has improved 1.71 times.Active fraction in 100 μ g/ml dosage groups is compared the SOD content utmost point and is significantly improved (p<0.01) with model group, improved 1.72 times.
The lipid peroxidation product MDA content of respectively organizing sample and silibinin positive controls after intervention all has reduction, MDA content wherein, wherein the Active fraction in 50 μ g/ml dosage groups is compared MDA content and is extremely significantly reduced (p<0.01) with model group, is respectively 59.47% of model group.MDA reduction and the sample concentration of the Active fraction in 100 μ g/ml dosage groups are proportionate.
In sum, the cell of tetrachloro-methane induction damage is after Active fraction is intervened, and membrane structure lipid peroxidation injury alleviates, and cell membrane stability improves.Experiment shows that Active fraction can pass through to remove free radical, suppresses lipid peroxidation, improve the ability of cell clearance superoxide anion, thereby alleviates the degree of injury of cell membrane under oxygen-derived free radicals is attacked, and to damaging hepatocyte, has certain protection effect.
2.3 propolis extracts cause the impact of damaging hepatocyte LDH content on carbon tetrachloride
Collecting cell supernatant culture fluid, each hole adds 0.5mL1%Triton-100 cell lysis, and fully piping and druming mixes rear collection, and the centrifugal 10min of 2500rpm gets lysate supernatant and cell conditioned medium culture fluid and measures by test kit explanation.Computing formula is as follows:
Experimental result is shown in Fig. 3.Note: △ △ compares p<0.01 with normal group; * compares p<0.01 with model group; * compare p<0.05 with model group.
As shown in Figure 3, it is only 19.84% that normal group LDH spills rate, and model group LDH spills rate, be 65.36%, compare and risen 45.52% with normal group, there is statistically utmost point significant difference (p<0.01), the cell film that tetrachloro-methane induction damage is described is subject to major injury, and the LDH in born of the same parents is discharged into extracellular, makes LHD spill rate and significantly rises.After each grading extraction component of propolis and positive product silymarin are intervened with 50 μ g/mL and 100 μ g/mL respectively, cell LDH spills the equal decrease to some degree of rate, wherein the Active fraction in 50 μ g/ml dosage groups is compared LDH and is spilt rate and extremely significantly reduce (p<0.01) with model group, is 51.35% of model group.Active fraction in 100 μ g/ml dosage groups is compared LDH and is spilt rate and extremely significantly reduce (p<0.01) with model group, and shows that mediating recipe measurer has certain dependency.Explanation is after sample is intervened, and the stability of cell membrane strengthens, and degree of injury alleviates, and cell survival rate is increased.
2.4 propolis extracts cause the impact of damaging hepatocyte CYP2E1 and Caspase-3 protein level on carbon tetrachloride
Get the PBS rinsing 2 times that adds 2ml pre-cooling in the L-02 of exponential phase cell (six orifice plates, cell density 1*105/mL), exhaustion PBS(substantially dry), every hole adds the RIPA buffer150 μ l of pre-cooling.With cell sleaker scraping attached cell, cell and lysate are leniently transferred in Eppendorf pipe.In each pipe, add 1ml4* albumen sample solution (containing DTT protease inhibitor), buckle lid, put into boiling water and boil 5min.Boiled room temperature cooling, then concussion mixes, standby, and remaining sample can be placed in-20 ℃ and preserve.
Every hole applied sample amount is 40 μ l albumen, and Marker applied sample amount is 10ul, uses Special gun head or injection needle, does not overflow well.Electrophoresis adopts constant voltage electrophoresis, and first 80V treats that band goes to separation gel place (about 45min) and change the about 1h of 120V(into), when dyestuff arrives the bottom of glue, powered-down stops electrophoresis, and glue can not be deposited, and should carry out at once next step transferring film.
In half dry type transferring film, being arranged as of sandwich :/paper/glue/film/paper/, electricity consumption turns buffer soak after, be directly placed between the both positive and negative polarity of electroporation, the transferring film time is 7min.
Prepare 5% defatted milk powder: in every 100ml TBST, add 5g defatted milk powder, mix rear filtration.During sealing, shake sealing 1h, then wash once with TBST, enter hatching of next step antibody.
Hatch Buffer: the extension rate by the suggestion of antibody description, with TBST, dilute primary antibodie, the extension rate of this experimental selection 1:2000 is hatched 2h in membrane closure liquid.
Primary antibodie is hatched after end, with TBST, shakes and washes film three times, and each 5min, removes residual primary antibodie.
Two anti-hatching after end, shake and wash film three times with TBST, and each 5min removes residual two anti-.In TBST solution, 4 ℃ are spent the night.
HRP chemical luminous substrate Luminol(ECL method chemiluminescence is selected in this experiment) film is fixed in exposure box, add X-ray egative film above, compacting 1min.Film is put into developer solution 1min, and 1s shakes and makes a movement.Then film is put into fixative solution 1min again, 1s shakes and makes a movement.
Result of the test is shown in Fig. 4, Fig. 5, note: △ △ compares p<0.01 with normal group; * compares p<0.01 with model group.
Before the poison of micromolecule amount, before material and cancer, material can make the content of multiple P450 enzyme in liver and activity change as aniline, ether, carbon tetrachloride etc., and wherein more representational enzyme is CYP2E1.Because the main catalysis exogenous material of CYP2E1 generates Toxic or carcinogen, therefore, the active rising of CYP2E1 may increase the danger of tumor regulating liver-QI toxic damages.
As shown in Figure 4, model group is with respect to matched group, and protein content has increased by 49.5%.Each classification component of propolis and positive controls all have certain inhibitory action to CYP2E1 protein level in L-02 cell.The concentration of take acts on cell as 50 each samples of μ g/mL, and Active fraction is compared expressing quantity and extremely significantly reduced (p<0.01) with model group, reduced by 18.33%.Concentration is that 100 each samples of μ g/mL act on cell, and Active fraction is compared expressing quantity and extremely significantly reduced (p<0.01) with model group, reduced by 19.80%.
In sum, propolis Active fraction can suppress the effect of CYP2EI by antioxidant activity, has finally alleviated the oxidative stress damage that carbon tetrachloride causes.
Caspase-3 is the very important execution molecule of on mitochondrion apoptosis pathway, and it plays a role in many approach of apoptosis signal transduction.Caspase-3 under normal condition is present in endochylema with the form of proenzyme, and at the commitment of apoptosis, it can be activated, the corresponding endochylema karyon of cracking substrate, thus cause apoptosis.
As shown in Figure 5, model group is with respect to matched group, and protein content has increased by 42.0%.Illustrate that carbon tetrachloride has activated Caspase-3 albumen in hepatocyte, apoptosis pathway is unlocked.Each classification component of propolis and positive controls all have certain inhibitory action to Caspase-3 protein level in impaired L-02 cell.The concentration of take acts on cell as 50 each samples of μ g/mL, and Active fraction is compared expressing quantity and extremely significantly reduced (p<0.01) with model group, reduced by 18.03%.Concentration is that 100 each samples of μ g/mL act on cell, and Active fraction is compared expressing quantity and extremely significantly reduced (p<0.01) with model group, reduced by 19.01%.
In sum, the mitochondrial swelling that propolis Active fraction can stress cause by inhibited oxidation, and then suppressed the activation of PROTEIN C aspase-3, the apoptosis pathway of blocking-up cell.
So that other embodiment disclosed by the invention are extract obtained, repeat above-mentioned test, obtain identical conclusion (of pressure testing), repeat no more herein.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a propolis extract, is characterized in that, adopts with the following method and is prepared from:
(1) get propolis powder and add 3～6 times of volume 40% ethanol to extract, isolated by filtration, gets residue;
(2) residue is added to 3～6 times of volume 70% ethanol, heating is extracted, and isolated by filtration supernatant repeats to extract, and merges supernatant, 4 ℃ of refrigerator overnight, sucking filtration or centrifugal, residue is standby, and supernatant is through decompression dealcoholysis, standing 24h～48h, abandons supernatant, retains precipitation, obtains 70% alcohol soluble substance;
(3) to the residue after 70% ethanol extraction, add 3～6 times of volume 95% ethanol, heating is extracted, isolated by filtration supernatant, and residue repeats to extract 2-4 time, merges supernatant; 4 ℃ of refrigerator overnight, sucking filtration or centrifugal, gets supernatant and obtains 95% alcohol soluble substance;
(4) 95% alcohol soluble substance in 70% alcohol soluble substance obtaining in step (2) and step (3) is merged, concentrating under reduced pressure obtains propolis extract after being also dried.
2. propolis extract as claimed in claim 1, is characterized in that, described propolis powder gel content is 25～50%.
3. extract as claimed in claim 1 or 2, is characterized in that, in described step (1), 30～35 ℃ of heating extraction temperature, 4～12 hours extraction times, repeat extraction time 1～2 time.
4. extract as claimed in claim 1 or 2, is characterized in that, in described step (2), 30～50 ℃ of heating extraction temperature, 24～72 hours extraction times, repeat extraction time 2～4 times.
5. extract as claimed in claim 1 or 2, is characterized in that, in described step (3), 30～45 ℃ of heating extraction temperature, 12～48 hours extraction times, repeat extraction time 2～3 times.
6. extract as claimed in claim 1 or 2, is characterized in that, described step (1) is stirring extraction in step (3), mixing speed 50～100rpm in step (1), mixing speed 110～200rpm in step (2) and (3).
7. the application of propolis extract in preparation treatment antiinflammatory, hepatic described in claim 1-6 any one.
8. the pharmaceutical composition that contains propolis extract described in claim 1-6 any one.
9. pharmaceutical composition according to claim 8, it is characterized in that: described pharmaceutical composition is tablet, by the raw material that comprises following weight portion, is prepared from: propolis extract 80-120 part, dextrin 2-6 part, crospolyvinylpyrrolidone 5-10 part, magnesium stearate 0.2-0.6 part.
10. pharmaceutical composition according to claim 9, it is characterized in that: described pharmaceutical composition is tablet, by the raw material that comprises following weight portion, is prepared from: 100 parts of propolis extracts, 4 parts, dextrin, 8 parts of crospolyvinylpyrrolidone, 0.4 part of magnesium stearate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310473370.2A CN103655628B (en) | 2013-10-11 | 2013-10-11 | A kind of propolis extract and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310473370.2A CN103655628B (en) | 2013-10-11 | 2013-10-11 | A kind of propolis extract and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103655628A true CN103655628A (en) | 2014-03-26 |
| CN103655628B CN103655628B (en) | 2015-12-09 |
Family
ID=50295189
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310473370.2A Active CN103655628B (en) | 2013-10-11 | 2013-10-11 | A kind of propolis extract and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103655628B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108464965A (en) * | 2018-06-15 | 2018-08-31 | 沈阳药科大学 | A kind of propolis mouthwash and preparation method thereof containing active skull cap components |
| CN110237100A (en) * | 2019-07-01 | 2019-09-17 | 北京葆年堂(菏泽)药业有限公司 | A kind of preparation method of propolis medicine materical crude slice and propolis medicine materical crude slice |
| CN112485421A (en) * | 2020-11-09 | 2021-03-12 | 江苏江大源生态生物科技股份有限公司 | Test method for propolis anti-tumor effect |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1360897A (en) * | 2000-12-28 | 2002-07-31 | 上海大承生物制品有限公司 | Bee glue purifying technological process |
| CN101099547A (en) * | 2007-06-18 | 2008-01-09 | 中国农业科学院蜜蜂研究所 | Water soluble propolis extract with bioactive and light-colored propolis extract |
| CN102321699A (en) * | 2011-06-24 | 2012-01-18 | 江苏江大源生态生物科技有限公司 | Method for composite enzymolysis-assisted extraction of active ingredient from propolis |
| CN102764274A (en) * | 2012-07-03 | 2012-11-07 | 江苏江大源生态生物科技有限公司 | Method for extracting propolis |
| CN102973609A (en) * | 2012-11-30 | 2013-03-20 | 扬州大学 | Propolis ethanol extract for treating alcoholic fatty liver, preparation method and applications in production of enteric-coated tablets |
-
2013
- 2013-10-11 CN CN201310473370.2A patent/CN103655628B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1360897A (en) * | 2000-12-28 | 2002-07-31 | 上海大承生物制品有限公司 | Bee glue purifying technological process |
| CN101099547A (en) * | 2007-06-18 | 2008-01-09 | 中国农业科学院蜜蜂研究所 | Water soluble propolis extract with bioactive and light-colored propolis extract |
| CN102321699A (en) * | 2011-06-24 | 2012-01-18 | 江苏江大源生态生物科技有限公司 | Method for composite enzymolysis-assisted extraction of active ingredient from propolis |
| CN102764274A (en) * | 2012-07-03 | 2012-11-07 | 江苏江大源生态生物科技有限公司 | Method for extracting propolis |
| CN102973609A (en) * | 2012-11-30 | 2013-03-20 | 扬州大学 | Propolis ethanol extract for treating alcoholic fatty liver, preparation method and applications in production of enteric-coated tablets |
Non-Patent Citations (2)
| Title |
|---|
| 林贤统等: "不同溶剂提取蜂胶的得率及其提取物的抗氧化性", 《蜜蜂杂志》 * |
| 胡福良: "蜂胶的提取方法", 《养蜂科技》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108464965A (en) * | 2018-06-15 | 2018-08-31 | 沈阳药科大学 | A kind of propolis mouthwash and preparation method thereof containing active skull cap components |
| CN108464965B (en) * | 2018-06-15 | 2021-07-23 | 沈阳药科大学 | Propolis mouthwash containing natural active ingredients and preparation method thereof |
| CN110237100A (en) * | 2019-07-01 | 2019-09-17 | 北京葆年堂(菏泽)药业有限公司 | A kind of preparation method of propolis medicine materical crude slice and propolis medicine materical crude slice |
| CN112485421A (en) * | 2020-11-09 | 2021-03-12 | 江苏江大源生态生物科技股份有限公司 | Test method for propolis anti-tumor effect |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103655628B (en) | 2015-12-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Rameshrad et al. | Saffron and its derivatives, crocin, crocetin and safranal: a patent review | |
| Gao et al. | Hypoglycemic effect of Gynostemma pentaphyllum saponins by enhancing the Nrf2 signaling pathway in STZ-inducing diabetic rats | |
| Wang et al. | Investigation on the morphological protective effect of 5-hydroxymethylfurfural extracted from wine-processed Fructus corni on human L02 hepatocytes | |
| Ma et al. | Effect of pomegranate peel polyphenols on human prostate cancer PC-3 cells in vivo | |
| CN103342687B (en) | Compounds with tyrosinase inhibitory activity and preparation and application thereof | |
| CN106008172A (en) | Preparation method and application of effective part with tyrosinase inhibition effect in mulberry twigs | |
| CN103655628B (en) | A kind of propolis extract and application thereof | |
| CN103961379B (en) | Laetiporus sulphureus extract and application thereof | |
| CN108403723B (en) | A kind of American-cockroach-extract that intervening AD and its extracting method and application | |
| CN104474135B (en) | A kind of pharmaceutical composition to chemical damage with assistant protection function | |
| CN106631804A (en) | Compound separated out from labiatae isodon plant, and preparation method and application thereof | |
| CN105963342A (en) | An antiallergic compound flavone composition, and a preparing method and applications thereof | |
| CN108434399A (en) | A kind of Chinese medicine composition and preparation method of anti-curing oncoma | |
| CN104382801A (en) | Kandelia candel leaf total flavonoid nano-emulsion for whitening skin and removing freckles and gel thereof | |
| CN103497091B (en) | Compound with tyrosinase inhibitory activity and preparation method thereof | |
| CN105168520A (en) | Preparing method for kadsura coccinea extract and application of kadsura coccinea extract to treating non-alcoholic fatty liver disease | |
| CN101433564B (en) | Use of water chestnut extract in preparing medicament for treating leukaemia | |
| CN105148124B (en) | A kind of composition and its application with antitumor action | |
| CN105287474B (en) | The application of anisic acid para hydroxybenzene ethyl ester | |
| CN103800386B (en) | A kind of Cordyceps cicadae Shing extract and preparing the application in neuroprotective and antiaging agent | |
| Lee et al. | Hibiscus syriacus L. Extract by ultrasonic assistance displays anti-inflammatory and pro-apoptotic activity in LPS-stimulated Raw 264.7 cells | |
| CN103800384B (en) | Cordyceps cicadae Shing active site and preparing the application in neuroprotective and antiaging agent | |
| CN106474322A (en) | Black garlic polyphenol oral liquid containing Eurycoma longifolia extract product and preparation method thereof | |
| Zhang et al. | Effects of the Coreopsis tinctoria extracts on anti-aging in the aging model mice | |
| CN111704535A (en) | 3-carbonyl malus spectabilis ketone, extraction method and application in preparing antitumor drugs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |