CN105250236A - Preparation method for catechin preparation for fish - Google Patents

Abstract

The invention discloses a preparation method for catechin preparation for fish. The preparation method comprises the following steps: (1), dissolving catechin in an ultrapure water to prepare catechin EGCG liquid with the concentration of 9-11mg/mL; (2), adding PEG1000 to the catechin EGCG liquid till the final mass concentration of the PEG1000 is 10-30%; then adding an antioxidant and mixed amino acid; finally adding PEG4000 till the final mass concentration of the PEG4000 is 10-20%; (3), preparing a liquid-filled capsule: filling liquid obtained in step (2) in a capsule, thereby obtaining the catechin preparation for the fish. The catechin preparation for the fish can improve the bioavailability of catechin in fish bodies.

Description

The fish preparation method of catechin preparation

Technical field

Catechin is topmost active skull cap components in Folium Camelliae sinensis, has multiple pharmacology and health active.The present invention relates to a kind of preparation method and evaluation thereof of fish catechin health preparation of novel, high bioavailability.

Background technology

At present, because the fast development of industrial or agricultural and the mankind constantly ask for the impact on China's water environment, aquatic product fishery cultivation such as environmental pollution, climate change caused also manifest gradually natural, especially in the area that some economy of China are relatively flourishing, due to a large amount of pollutant emissions of chemical plant, contour contaminating enterprises of printing and dyeing mill, severe contamination is created to locality cultivation and open water supply.In recent years, in addition the deepening continuously of the intensive technology of aquaculture, produce subsequently remained by cultivation water environment feedstuff, fish disease that the factor such as eutrophication and the water pollutions that thereupon produces causes is on the rise, the disease that especially in feedstuff, high nutrient causes is given prominence to gradually.At present, poultry feeders, in order to accelerate the growth of Fish, shortens breeding cycle, a large amount of nutrients is added in feedstuff, thus cause the liver property disease that the disorder of liver lipid, nutrient metabolic causes, although this type of disease is without infectiousness, harm may considerably beyond infectious disease.

Tea polyphenols (TeaPolyphenols, TP) is the general name of Polyphenols and derivant thereof in Folium Camelliae sinensis, and mainly contain the material compositions such as catechin, flavonoid, anthocyanidin and phenolic acid, wherein catechin accounts for the 60%-90% of tea polyphenols total amount.Catechin has very strong biological activity, and large quantity research all shows that catechin has oxidation and removing free radicals, anti-cardiovascular disease, the multiple medicine healthy sofa effect such as (hyperlipidemia, hypertension and hyperglycemia), inhibiting bacteria and diminishing inflammation of falling " three-hypers ".In addition catechin compounds has the ability of poisonous and harmful substances in eliminating body (as removing heavy metal, free radical etc.), and therefore development of new fish catechin aquatic animal health product have good Prospect of R & D for the green aquaculture of development China, guarantee healthy fish, the use of reduction fisheries drug and aquatic products drug residue.

Simultaneously because China is Tea planting state maximum in the world and Tea Processing exported country, there are a large amount of high-quality tea picking, processing and production every year, in these high-quality Tea Processing processes, a large amount of substandard products, Folium Camelliae sinensis leftover bits and pieces etc. go out of use, and wherein still containing active skull cap components such as a large amount of catechins, if directly these substandard products, leftover bits and pieces abandoned, not only ample resources is wasted, but also causes substantial pollution to environment.Therefore, by various abstraction technique, a large amount of catechin is wherein prepared purification, be then applied to aquaculture field, not only reduce the wasting of resources, and to development tea industry recycling economy, there is material impact.So Novel fish catechin animal health-care product is prepared in research and development, thus improve the bioavailability in catechin stability and fish body, solid foundation will be established for the exploitation of catechin and the combination of China's fish production.

In conjunction with this laboratory early-stage Study accumulation and amount of literature data known, because catechin compounds polarity is larger, water solublity is higher, biomembrane permeability is poor, in addition transport protein (p-glycoprotein in intestinal epithelial cell film, multidrug-associated protein etc.) " arranging " effect may be played outward to catechin, thus be difficult to enter systemic blood circulation by intestinal epithelial cell after making this compounds oral, so that the bioavailability of catechin compounds in body is lower, major part in intestinal by various enzyme or Institute of Micro-biology's metabolism and degraded, or with defecate to external, thus make this compounds be difficult to play useful effect in vivo.Therefore, how improving catechin compounds biomembrane transport velocity, thus improve its bioavailability and strengthen the enrichment of catechin compounds at effect target site, is the crucial common problem solving restriction catechin large-scale application this important " bottleneck ".Research at present in the transhipment of raising catechin biomembrane mainly concentrates on the Synthesis and applications of catechin-derived compound, the research of recent years also had scholar to carry out catechin/tea polyphenol nano grain preparation or injection nano-emulsion, but what this type of research was mainly carried out for the exploitation of human health care's product benefits our pursuits, and this type of is studied and is not suitable for the application on aquaculture.

Specifically, at present in order to improve biomembrane transport velocity or the bioavailability of catechin natural component, following several dosage form and preparation is mainly contained:

1, the microcapsule formulation of tea polyphenols: this dosage form take ethyl cellulose as capsule material, solvent volatilization technology is adopted to prepare tea polyphenols (TP) slow-releasing microcapsule, this dosage form can improve TP stability and slow releasing function, and envelop rate is higher, but the content of TP main active catechin reduces, may be contain polyphenol hydroxyl in ethyl cellulose structure, be combined with catechin phenolic hydroxyl group and make it to be difficult to stripping.

2, the microball preparation of tea polyphenols: this dosage form with Biodegradable polymer material hydroxypropylmethyl cellulose phthalate (HPMCP) for framework material, prepare TP microsphere, result display adds the envelop rate that a small amount of emulsifying agent can improve TP, has certain slow release effect.

3, the liposome of tea polyphenols: adopt reverse phase evaporation to prepare TP-vitamin E liposome, can increase the stability of TP, and have long-acting feature after encapsulating, also can significantly improve the transhipment of its biomembrane and bioavailability simultaneously.

But above-mentioned these can significantly improve the preparation of catechin compounds biomembrane transport velocity or bioavailability, and be prepared by the occupation mode for the mankind, these preparations are difficult to use in fish species.

Summary of the invention

The technical problem to be solved in the present invention is to provide the semi-solid capsule preparations of a kind of fish improving catechin bioavailability---the preparation method of fish catechin preparation.

In order to solve the problems of the technologies described above, the invention provides the preparation method of a kind of fish catechin preparation, comprising the following steps:

1), the preparation of catechin solution:

Catechin is dissolved in ultra-pure water, is mixed with the catechin EGCG liquid that concentration is 9 ~ 11mg/mL (being preferably 10mg/mL);

2), in catechin EGCG liquid, PEG1000 (cetomacrogol 1000, as absorption enhancer) is added, until the whole mass concentration of PEG1000 is 10% ~ 30%;

And then add antioxidant and kilnitamin, finally add PEG4000 (as semi-solid gellant) until the whole mass concentration of PEG4000 is 10% ~ 20%;

The mass ratio of described antioxidant and catechin EGCG liquid is 0.25 ~ 0.5%, and the mass ratio of kilnitamin and catechin EGCG liquid is 0.5 ~ 1.0%;

3), the preparation of filling liquid capsule:

By step 2) gained liquid pours in capsule, obtains fish catechin preparation.

Remarks illustrate: aforesaid liquid natural coagulation can form semi-solid state in capsule.

As the improvement of fish of the present invention by the preparation method of catechin preparation:

Described antioxidant is sodium sulfite;

Described kilnitamin is made up of theanine and glycine, and the weight content of described theanine is 8 ~ 12% (better weight content is 10%, that is, kilnitamin is obtained by mixing according to the part by weight of 1:9 by theanine and glycine).

As the further improvement of fish of the present invention by the preparation method of catechin preparation: described step 2) carry out (magnetic agitation) under the speed of agitator of 100 ~ 200 revs/min.

As the further improvement of fish of the present invention by the preparation method of catechin preparation:

Described step 1) in: the concentration of catechin EGCG liquid is 10mg/mL;

Described step 2) in: the whole mass concentration of PEG1000 is 20%, the mass ratio of sodium sulfite and catechin EGCG liquid is 0.4%, the mass ratio of kilnitamin and catechin EGCG liquid is 1.0%, described kilnitamin is obtained by mixing according to the part by weight of 1:9 by theanine and glycine, and the whole mass concentration of PEG4000 is 20%.

In the present invention:

Step 1) can supersonic vibration be utilized, thus make catechin be dissolved in ultra-pure water;

Step 2) under the stirring condition of 100 ~ 200 revs/min, carry out (such as magnetic agitation), step 2) gained liquid is in low viscous catechin mixed solution under magnetic agitation;

Step 3) in, the liquid of general every capsules fill 0.5-1.0mL.

Fish of the present invention preparation (fish catechin preparation) belongs to the semi-solid capsule preparations of a kind of bioavailability enhancement mode catechin fish.

The present invention is in invention process, according to fish intestinal absorption model, by the different absorption enhancer of detection by quantitative to the absorbtivity in catechin EGCG body, thus filter out the absorption enhancer that can improve catechin bioavailability, and make it to mix with proper proportion with catechin, thus make semi-solid capsule preparations.Fish of the present invention can improve the catechin bioavailability in fish body with preparation.Namely, the present invention mainly considers fish intestines feature and life habit, adopt PEG1000 (for absorption enhancer) and PEG4000 (for semi-solid gellant) as pharmaceutical adjunct, thus significantly can reach the rapid transport feature absorbed in the integrity and intestinal meeting catechin dosage form.

The present invention adopts fish plasma bioavailability method evaluation, is specifically implemented as follows:

1), the foundation of the quantitative detecting method of catechin EGCG in Experimental fish plasma sample: adopt the method adding catechin EGCG standard substance in 0.3mL blank plasma, compound concentration scope is the catechin EGCG standard sample of 0.5-500mg/L, add the ascorbic acid of 30 μ L20% wherein, 2-3mL ethyl acetate is added after mixing, vibration 1min, 6000r/min high speed centrifugation 5min, take out whole organic facies, residue with 2mL ethyl acetate re-extract once, merge twice extracted organic phase, in 45 DEG C of water-baths, nitrogen current dries up, the acetonitrile solution of residue 0.1mL20% dissolves, after sonic oscillation, 12000r/min high speed centrifugation 3min, get 20 μ L supernatant HPLC sample introduction analyses.

According to HPLC testing result, with peak area (Y) for vertical coordinate, mass concentration (X) is abscissa, and drawing standard curve obtains curvilinear equation and correlation coefficient (r).Preparation is containing high, medium and low concentrations Plasma sample 0.5 in catechin EGCG curve ranges simultaneously, 5.0,50.0mg/L is as quality-control sample (QC), respectively according to sample introduction analysis after above-mentioned sample treatment process, each concentration samples repeats 5 times, with EGCG peak area in sample be directly dissolved in mobile phase lower the ratio of survey peak area, calculate the method response rate under high, medium and low 3 kinds of concentration.Relatively above sample, in a few days 5 times and the change of peak area that measures for 5 times in the daytime, calculates withinday precision and day to day precision.

2), the selection of Experimental fish: test and be purchased from Hangzhou Xiaoshan Yue Teng aquaculture company limited with healthy Carassius auratus, body weight 150 ± 20g, experimental group 200 healthy Carassius auratuss are divided into 4 groups at random, capsule is fed group (the present invention), common catechin EGCG particulate material group, lumbar injection group and blank group, often organize 50, often organize and put into 2 1m × 1m × 1m aquarium adaptability respectively and cultivate 3 days, circulating water cultivation.Above-mentioned 4 experimental grouies formally test and feed before the equal fasting of 12h.Feed group, common catechin particulate material group dosage of capsule is 200mg/kg. days, and lumbar injection group dosage is 100mg/kg. days; Remarks illustrate: above-mentioned 200mg, 100mg all refer to the consumption of effective ingredient catechin.

3), the calculating of bioavailability: by above-mentioned different experiments group and matched group, respectively at process rear 0.083,0.17,0.5,1.0,2.0,4.0,8.0,12.0,24.0 and 48.0h tail venous blood sampling, each time point takes a blood sample 5, then by got blood in heparinization centrifuge tube, after 6000r/min is centrifugal, getting 1.5mL blood plasma in 10mL centrifuge tube, simultaneously according to 1) the laggard HPLC of disposal methods under item analyzes.Catechin EGCG peak area in surveyed blood plasma is substituted in above-mentioned standard curve equation, calculate catechin EGCG concentration in blood plasma, draw blood drug level---time graph, utilize area (AUC) under 3P97 pharmacokinetics computed in software drug level-time graph, group of being fed by capsule and common catechin EGCG pellet group compare with lumbar injection group gained AUC numerical value respectively, evaluate new formulation to the impact of catechin at little enteral organism-absorbing availability with this.

The present invention to catechin on the basis that fish intestinal absorption potentiation is studied, filter out the good new formula of catechin health product, thus prepare fish catechin new type of health goods (i.e. fish preparation of the present invention), the preparation of gained of the present invention is applicable to aquaculture and uses, the present invention can give full play to the biological activity of catechin, serves the sound development of China's culture fishery.

Accompanying drawing explanation

Below in conjunction with accompanying drawing, result of implementation of the present invention is described in further detail.

Fig. 1 is the reference colour spectrogram of catechin EGCG;

Fig. 2 is the chromatogram of blank plasma;

Fig. 3 is Carassius auratus blood plasma chromatogram after Novel capsule of the present invention is fed;

Fig. 4 is the catechin Novel capsule group EGCG concentration-time plot of embodiment 1;

Fig. 5 is common catechin particulate material group EGCG concentration-time plot;

Fig. 6 is lumbar injection group EGCG concentration-time plot;

Wherein peak 1 is catechin EGCG chromatographic peak.

Detailed description of the invention

Embodiment 1, a kind of fish preparation method of catechin preparation, carry out following steps successively:

1), the preparation of catechin solution: accurately take catechin EGCG in clean volumetric flask, add a certain amount of ultra-pure water wherein, supersonic vibration (supersonic frequency of 50Hz), make it dissolve, then use ultra-pure water standardize solution, thus be mixed with the catechin EGCG liquid that concentration is 10mg/mL.

2), adopt PEG1000 as absorption enhancer, accurately measure a certain amount of PEG1000 and add in above-mentioned catechin EGCG liquid, make the final concentration of PEG1000 be 20% (quality %).

Then add account for catechin EGCG liquid 0.4% (quality %) sodium sulfite as antioxidant, add the kilnitamin accounting for catechin EGCG liquid 1.0% (quality %) again, finally add PEG4000 (as semi-solid gellant) until the whole mass concentration of PEG4000 is 20%;

Above-mentioned kilnitamin is obtained by mixing according to the part by weight of 1:9 by theanine and glycine.

Above-mentioned whole step 2) carry out under magnetic agitation (200 revs/min).

3), the preparation of filling liquid capsule: by step 2) under the magnetic agitation (200 revs/min) prepared in low viscous catechin solution according to every capsules 1.0mL fill in capsule shells, aforesaid liquid natural coagulation can form semi-solid state in capsule, can prevent intravenous extravasation.

Embodiment 2, a kind of fish preparation method of catechin preparation, carry out following steps successively:

1), with embodiment 1;

2), adopt PEG400 as absorption enhancer, accurately measure a certain amount of PEG400 and add in above-mentioned catechin EGCG liquid, make the final concentration of PEG400 be 20% (quality %).

Then add account for catechin EGCG liquid 0.4% (quality %) sodium sulfite as antioxidant, add the kilnitamin accounting for catechin EGCG liquid 0.5% (quality %) again, finally add PEG4000 (as semi-solid gellant) until the whole mass concentration of PEG4000 is 5%;

Above-mentioned kilnitamin is obtained by mixing according to the part by weight of 1:9 by theanine and glycine.

3), the preparation of filling liquid capsule: by step 2) under the magnetic agitation (100 revs/min) prepared in low viscous catechin solution according to every capsules 1.0mL fill in capsule shells, can intravenous extravasation be prevented after solidifying.

Embodiment 3, a kind of fish preparation method of catechin preparation, carry out following steps successively:

1), with embodiment 1;

2), adopt PEG3000 as absorption enhancer, accurately measure a certain amount of PEG3000 and add in above-mentioned catechin EGCG liquid, make the final concentration of PEG3000 be 20% (quality %).

Then add account for catechin EGCG liquid 0.4% (quality %) sodium sulfite as antioxidant, add the kilnitamin accounting for catechin EGCG liquid 2.0% (quality %) again, finally add PEG4000 (as semi-solid gellant) until the whole mass concentration of PEG4000 is 30%;

Above-mentioned kilnitamin is obtained by mixing according to the part by weight of 1:9 by theanine and glycine.

3), the preparation of filling liquid capsule: by step 2) in preparation magnetic agitation (400 revs/min) under in low viscous catechin solution according to every capsules 1.0mL fill in capsule shells, can intravenous extravasation be prevented after solidifying.

Detected according to above-mentioned evaluation methodology by the Novel fish preparation of above-mentioned case gained (for embodiment 1), its corresponding result is as follows:

With Japanese Shimadzu high performance liquid chromatography, two yuan of high-pressure pumps, ultraviolet/visible detection device, the special HypersilBDSC of Dalian Erie ₁₈post (5 μm, 250mm × 4.6mm), mobile phase is acetonitrile: 0.1% aqueous citric acid solution=10: 90; Column temperature 30 DEG C, wavelength 280nm; Flow velocity is 1.0mLmin ^-1; Sample size is 20 μ L.

Step 1) EGCG is Y=12037x+560.3 (r=0.9999) at 0.5-500mg/L concentration range internal standard curve in gained process blood plasma, in standard curve range, the high, medium and low concentration catechin EGCG response rate is all greater than more than 85%, withinday precision and day to day precision are all less than 8%, in table 1.

The response rate of EGCG and precision (n=5) in table 1. blood plasma

According to oral liquid adjuvant and the formula of above-mentioned screening gained, the Novel fish of preparation is with after capsule preparations gavage, take a blood sample in different time points, detect catechin EGCG content in blood plasma, and draw blood plasma EGCG concentration-time curve, utilize 3P97 pharmacokinetics computed in software blood plasma EGCG area under the concentration-time curve (AUC).Embodiment 1 gained Novel fish capsule preparations different time points blood plasma catechin EGCG concentration v. time data is shown in Fig. 4, is 39.31mgh/L according to 3P97 pharmacokinetics computed in software its AUC known.In like manner, the AUC of embodiment 2 gained Novel fish capsule preparations is 21.38mgh/L.The AUC of embodiment 3 gained Novel fish capsule preparations is 12.03mgh/L.

And common catechin particulate material fed control group, calculating gained AUC is 11.89mgmin/L; AUC after catechin aqueous solution lumbar injection is 5182.25mgmin/L.

Comparative example 1-1, only by embodiment 1 step 2) in PEG1000 make PEG1500 into, consumption is constant, and all the other are equal to embodiment 1.

Comparative example 1-2, only by embodiment 1 step 2) in PEG4000 make PEG6000 into, consumption is constant, and all the other are equal to embodiment 1.

Comparative example 1-3, by embodiment 1 step 2) in the final concentration of PEG1000 make 5% into, all the other are equal to embodiment 1.

Comparative example 1-4, by embodiment 1 step 2) in the final concentration of PEG1000 make 40% into, all the other are equal to embodiment 1.

Comparative example 1-5, by embodiment 1 step 2) in the final concentration of PEG4000 make 5% into, all the other are equal to embodiment 1.

Comparative example 1-6, by embodiment 1 step 2) in the final concentration of PEG4000 make 30% into, all the other are equal to embodiment 1.

Comparative example 1-7, by embodiment 1 step 2) in the final concentration of PEG1000 make 30% into, make the final concentration of PEG4000 into 10%, all the other are equal to embodiment 1.

Comparative example 2-1, by embodiment 1 step 2) in theanine and the mixture of glycine make simple theanine into, weight is constant, and all the other are equal to embodiment 1.

Comparative example 2-2, by embodiment 1 step 2) in theanine and the mixture of glycine make simple glycine into, weight is constant, and all the other are equal to embodiment 1.

Comparative example 2-2, by embodiment 1 step 2) in theanine and the mixture of glycine make into be obtained by mixing according to the part by weight of 1:1 by theanine and glycine, all the other are equal to embodiment 1.

Comparative example 3-1, by embodiment 1 step 2) preparation time magnetic agitation rotating speed make 50 revs/min into, all the other are equal to embodiment 1.

Comparative example 3-2, by embodiment 1 step 2) preparation time magnetic agitation rotating speed make 600 revs/min into, all the other are equal to embodiment 1.

Contrast test, the fish of above-mentioned comparative example gained preparation to be detected according to the test method(s) corresponding to above-described embodiment 1, acquired results as in the table below:

Table 2

	AUC(mg·min/L)
		Comparative example 1-1	30.25
Comparative example 1-2	27.58
		Comparative example 1-3	31.63
Comparative example 1-4	35.01
		Comparative example 1-5	19.08
Comparative example 1-6	33.19
		Comparative example 1-7	35.23
Comparative example 2-1	29.88
		Comparative example 2-2	35.16
Comparative example 2-3	35.33
		Comparative example 3-1	31.08
Comparative example 3-2	28.09

Finally, it is also to be noted that what enumerate above is only PEG1000 sorbefacient specific embodiment in 10%-30% concentration range in the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.

Claims (4)

1. the fish preparation method of catechin preparation, is characterized in that comprising the following steps:

1), the preparation of catechin solution:

Catechin is dissolved in ultra-pure water, is mixed with the catechin EGCG liquid that concentration is 9 ~ 11mg/mL;

2), in catechin EGCG liquid PEG1000 is added, until the whole mass concentration of PEG1000 is 10% ~ 30%;

And then add antioxidant and kilnitamin, finally add PEG4000 until the whole mass concentration of PEG4000 is 10% ~ 20%;

The mass ratio of described antioxidant and catechin EGCG liquid is 0.25 ~ 0.5%, and the mass ratio of kilnitamin and catechin EGCG liquid is 0.5 ~ 1.0%;

3), the preparation of filling liquid capsule:

By step 2) gained liquid pours in capsule, obtains fish catechin preparation.

2. the fish according to claim 1 preparation method of catechin preparation, is characterized in that:

Described antioxidant is sodium sulfite;

Described kilnitamin is made up of theanine and glycine, and the weight content of described theanine is 8 ~ 12%.

3. the fish according to claim 2 preparation method of catechin preparation, is characterized in that: described step 2) carry out under the speed of agitator of 100 ~ 200 revs/min.

4. the preparation method of catechin preparation of the fish according to Claims 2 or 3, is characterized in that:

Described step 1) in: the concentration of catechin EGCG liquid is 10mg/mL;

Described step 2) in: the whole mass concentration of PEG1000 is 20%, the mass ratio of sodium sulfite and catechin EGCG liquid is 0.4%, the mass ratio of kilnitamin and catechin EGCG liquid is 1.0%, described kilnitamin is obtained by mixing according to the part by weight of 1:9 by theanine and glycine, and the whole mass concentration of PEG4000 is 20%.