CN106074494A - Reduce aflatoxin trap and the compound recipe nanoparticle preparation method of hepatic injury - Google Patents
Reduce aflatoxin trap and the compound recipe nanoparticle preparation method of hepatic injury Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
- A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
Abstract
The invention discloses a kind of compound recipe nanoparticle preparation method reducing aflatoxin trap and hepatic injury, comprise the following steps: 1), first by the mass mixing such as catechin and procyanidin, obtain compound；The polyphosphoric acids sodium solution 200ml mix homogeneously that mass concentration is 1.4～1.6% is added in 12g compound；2), adding water in the chitosan of 4～6g is settled to 1000ml, and after magnetic agitation, regulation pH is 3.5～4.5, obtains chitosan liquid；3), by step 1) gained solution all adds to step 2) in the chitosan liquid of gained, the reaction system of gained continues stirring 40～80min in 20～30 DEG C, obtains compound recipe nanoparticle suspension.
Description
Technical field
The present invention relates to the preparation method of a kind of compound recipe nanoparticle reducing aflatoxin intestinal absorption degree and hepatic injury.
Background technology
Tea polyphenols (Tea Polyphenols, TP) is Polyphenols and the general name of derivant thereof in Folium Camelliae sinensis, mainly has catechu
The material compositions such as element, flavonoid, anthocyanidin and phenolic acid, wherein catechin accounts for tea polyphenols total amount 60%-90%.Catechin is tea
Topmost active skull cap components in leaf, has multiple pharmacology and health active；Catechin has the strongest biological activity, in a large number
Research all shows that catechin has the plurality kinds of health care such as oxidation and removing free radicals, anti-cardiovascular disease, inhibiting bacteria and diminishing inflammation and pharmacology is made
With.Procyanidin is that the one being present in the plants such as Semen Vitis viniferae, Lycium ruthenicum Murr., Folium Ginkgo, Cortex Pini and Flos Rosae Rugosae is natural super
Powerful antioxidant, has become as in medical science and threpsology the material using the strongest natural removing free radical.Procyanidin is one
Plant the bioflavonoids having special molecular structure, be that the removing people's interior free yl generally acknowledged the most in the world is maximally effective natural
Antioxidant.Generally red-brown powder, feeble QI, puckery, be dissolved in water and most organic solvent.Generally Semen Vitis viniferae extract or
French maritime pine bark extract.Procyanidin (Semen Vitis viniferae extract, GSPE) is a kind of new and effective antioxidant, is current
Till the most potent free radical scavenger that found, there is the strongest activity in vivo.It is demonstrated experimentally that procyanidin anti-from
It is 50 times of vitamin E by base oxidability, ascorbic 20 times.Structurally, procyanidin is by the catechu of varying number
Element or epicatechin are polymerized.Simplest procyanidin is catechin or epicatechin or catechin and epicatechin shape
The dimer become, in addition with trimer, the tetramer and until ten aggressiveness.
Aflatoxin (AFT) is the secondary metabolite that the toxigenic bacterium strain such as Aspergillus flavus, aspergillus parasiticus bacterium produces, and extensively deposits
It is in the food polluted, especially most with the Semen arachidis hypogaeae gone mouldy, Semen Maydis and frumentum content.AFT is slightly soluble in water, be soluble in oils and fats,
The organic solvent such as chloroform and methanol.AFT is poor to the sensitivity of temperature, and 280 DEG C could crack, under the conditions of the most general culinary art not
The most destroyed.Within 1993, AFT is just classified as I class carcinogen by the Agency for Research on Cancer of World Health Organization (WHO) (WHO), is a kind of toxicity
Extremely strong extremely toxic substance.In the food of natural contamination, AFB1 (AFB1) is most, is to be currently known the strongest chemistry cause
One of cancer thing, therefore AFT is typically with AFB1 as representative, as pollution monitoring index in Food Monitoring.Numerous studies all show
AFT is the carcinogen that hepatocarcinoma is the strongest.
At present due to rapid economic development, the quickening of people's rhythm of life, growth in the living standard in addition, dietary structure
Changing, the hyperuricemia caused and various cardiovascular and cerebrovascular vessel chronic disease gradually present the trend of rising, often with hypertension,
Modern endocrine, the metabolism disorder disease such as hyperlipidemia, hyperglycemia.Owing to live and work pressure is excessive, divide in often leading to
Secreting and get muddled, thus cause body function and various combined condition occur further and aggravate, most people are good for all in Asia
Health state.The most various fast food diet and food-safety problem highlight, so that people are in the most internal accumulation
Substantial amounts of free radical, may slowly grow various disease simultaneously, and especially hepar damnification risk is in high-risk status.Once drink
Having a large amount of aflatoxin to take in food, removing toxic substances and the antibody Monoclonal ability of liver cannot withstand a single blow, and more easily cause sending out of hepatocarcinoma
Raw.Therefore, exploitation meets modern's life style and can resist the novel of hepatic injury caused by aflatoxin absorption and metabolism
Health product, significant and market prospect.
Research in terms of improving catechin and procyanidin biomembrane transhipment at present is concentrated mainly on catechin-derivedization
In the preparation of thing and application, recent years also has scholar to carry out catechin/tea polyphenol nano grain preparation or injection nano-emulsion
Research, but this type of research is primarily directed to how to promote that what natural plant polyphenols transmembrane transport carried out benefits our pursuits.But close
Prepare in catechin and procyanidin compound recipe nanoparticle and play damage-retardation by reducing aspergillus flavus resisting toxin bioavailability
There is not been reported to hinder the research of aspect.Although having document to report variable concentrations green tea and AFB1 being caused by tealeaf residue
The impact of rat liver cancer effect, also studies have reported that tea polyphenols elemental abundances in tree genesis of HCC simultaneously, but
Be the studies above simply from hepar damnification angle to the protection studied tea polyphenols aflatoxin is damaged or intervention effect.About
The defencive function of aflatoxin, hepatic injury is studied and also be there is not yet report by procyanidin.From novel catechin and former cyanine
Element compound recipe nanometer solution and reduction aflatoxin bioavailability visual angle are studied aflatoxin defencive function, there is not yet
Report.
Summary of the invention
The technical problem to be solved in the present invention is to provide and a kind of reduces answering of aflatoxin intestinal absorption degree and hepatic injury
The preparation method of side's nanoparticle, uses the method to prepare and can significantly reduce aflatoxin intestinal absorption degree regulating liver-QI damage
The compound recipe nanoparticle of wound so that catechin and procyanidin work in coordination with the damage toxicity reducing aflatoxin.
In order to solve above-mentioned technical problem, the present invention provides a kind of compound recipe reducing aflatoxin trap and hepatic injury
Nanoparticle preparation method, comprises the following steps:
1), first by the mass mixing such as catechin and procyanidin, compound is obtained；Mass concentration is added in 12g compound
It it is 1.4～the polyphosphoric acids sodium solution 200ml mix homogeneously of 1.6% (most preferably 1.5%)；
2), adding water in the chitosan of 4～6g is settled to 1000ml, and after magnetic agitation, regulation pH is 3.5～4.5 (preferably
It is 4.0), obtain chitosan liquid；
3), by step 1) gained solution (that is, the polyphosphoric acids sodium solution of catechin and procyanidin) all additions extremely step
Rapid 2), in the chitosan liquid of gained, the reaction system of gained continues stirring 40～80min (examples in 20～30 DEG C (preferably 25 DEG C)
As for 60min), obtain compound recipe nanoparticle suspension.
Improvement as the compound recipe nanoparticle preparation method reducing aflatoxin trap and hepatic injury of the present invention:
Described step 3) in, by step 1) gained solution (that is, the polyphosphoric acids sodium solution of catechin and procyanidin) in
Within 60～80 minutes, in (preferably 70 minutes), uniformly drop to step 2) in the chitosan liquid of gained.
The compound recipe nanoparticle preparation method reducing aflatoxin trap and hepatic injury further as the present invention
Improve: described step 2) in, the rotating speed of magnetic agitation is 800～1200r/min.
In the present invention:
Select the chitosan meeting following condition: molecular weight is 110,000, deacetylation ＞ 80%, content ＞ 99%.Select
Meet the catechin of following condition: Determination of Polyphenols ＞ 98%, wherein catechin EGCG ＞ 80%.
Step 3) in stirring be 200～400r/min.
Step 1) in, according to actual needs, the hydrochloric acid of preparation 1mol/L or sodium hydroxide solution, survey pH by dropping limit, limit
Method achieves the goal pH value.
The usage of the present invention is: oral, consumption is about 10mg/kg.
The present invention, during invention, have employed following detection method:
The quantitative analysis method (using EGCG dissolution as Testing index) of catechin EGCG in method one, blood plasma
Using the method adding catechin EGCG standard substance in 0.3mL blank plasma, compound concentration scope is 0.1-
The catechin EGCG standard sample of 200mg/L, then it is added thereto to the ascorbic acid of 30 μ L 20% (mass percentage concentration), then
Adding 30 μ L 100 μ g/mL vanillin as internal standard, add 3mL ethyl acetate and extract after mixing, vibrate 1min, 6000r/
Min high speed centrifugation 5min, obtains organic facies (being positioned at upper strata) and aqueous phase (being positioned at lower floor) respectively；Aqueous phase (being positioned at lower floor) is used 3mL
Ethyl acetate repeats to extract once (extraction conditions is ibid), merges twice extraction organic facies, nitrogen stream (weak nitrogen in 45 DEG C of water-baths
Air-flow) dry up, the acetonitrile solution of residue obtained 0.1mL 20% (volume %) dissolves, after sonic oscillation, 18000r/min
High speed centrifugation 3min, takes 20 μ L of supernatant liquid HPLC sample introduction analyses.
Particularly as follows: with Japan's Shimadzu high performance liquid chromatography, two yuan of high-pressure pumps, UV-detector, Dalian Erie is special
Hypersil BDS C18Post (5 μm, 250mm × 4.6mm), flowing is acetonitrile mutually: 0.1% (quality %) aqueous citric acid solution=
10∶90；Column temperature 30 DEG C, wavelength 280nm；Flow velocity is 1.0mL min-1；Sample size is 20 μ L.
Remarks illustrate: blank plasma is the true plasma not adding EGCG.
According to HPLC testing result, with the ratio (Y) of catechin EGCG peak area and internal standard vanillin peak area as vertical coordinate,
With the mass concentration (X) of catechin EGCG concentration as abscissa, draw standard curve, obtain curvilinear equation and correlation coefficient (r).
Prepare containing high, medium and low concentrations Plasma sample in catechin EGCG curve ranges 0.2,10.0,50.0mg/L as Quality Control sample simultaneously
Product (QC), sample introduction analysis after processing according to above-mentioned sample treatment respectively, each concentration samples is repeated 5 times, with in sample
EGCG peak area and the ratio being directly dissolved in the most lower the surveyed peak area that flows, calculate the method response rate under high, medium and low 3 kinds of concentration
(that is, EGCG peak area and the ratio=EGCG response rate being directly dissolved in the most lower the surveyed peak area that flows in sample).Relatively with loading
The change of the peak area that product measure in a few days 5 times and in the daytime 5 times, calculates withinday precision and day to day precision.
Remarks illustrate: " withinday precision and day to day precision " is mainly used in evaluating said method (i.e. HPLC detection method)
Precision and accuracy, judge the reliability of final detection result with this.
Result is:
During gained processes blood plasma according to the method described above, EGCG is Y=at 0.1-200mg/L concentration range internal standard curve
0.004x+0.0002 (r ＞ 0.999), in standard curve range, the high, medium and low concentration catechin EGCG response rate is all higher than
More than 85%, withinday precision and day to day precision are respectively less than 10%, are shown in Table 1.
The response rate of catechin EGCG and precision (n=5) in table 1. blood plasma
According to the above results, we can learn: the above-mentioned detection method set by the present invention can meet the catechu of the present invention
The requirement of the detection of element intestinal absorption degree.
Method two, the envelop rate of assessment catechin+procyanidin compound recipe nanoparticle
In the present invention, by the compound recipe nanoparticle suspension of certain volume (about 2mL) with ultra-filtration centrifuge tube (10000MWCO,
Co., Ltd in Mi Libo) centrifugal 10min under 4000r/min, take filter membrane (10000MWCO aperture filter membrane, i.e. for staying
The ultrafilter membrane that compound minimum molecular weight is 10000 on filter membrane) the filtrate 1mL of lower floor, it is placed in centrifuge tube, according to method one
Middle identical chromatographic conditions, the filtrate sample introduction analysis obtained after taking the ultracentrifugation in centrifuge tube (6000r/min is centrifuged 5min).
Calculate nanoparticle envelop rate, envelop rate (%)={ (catechin EGCG content in catechin EGCG initial content-filtrate)/catechu
Element EGCG initial content } × 100%.
The quantitative analysis method of AFB1 in method three, blood plasma
Using the method adding AFB1 standard substance in 0.3mL blank plasma, compound concentration scope is 0.1-
The AFB1 standard sample of 100ng/mL, adds 3mL normal hexane and extracts after mixing, vibrate 1min, 6000r/min
High speed centrifugation 5min, obtains organic facies (being positioned at upper strata) and aqueous phase (being positioned at lower floor) respectively；Aqueous phase (being positioned at lower floor) is used 3mL dichloro
Methane repeats to extract once, obtains organic facies (being positioned at lower floor) and aqueous phase (being positioned at upper strata) respectively；By organic facies (being positioned at lower floor) with
Above-mentioned gained normal hexane organic facies merges, and in 45 DEG C of water-baths, nitrogen stream (weak nitrogen stream) dries up, and residue obtained 0.3mL flows
The acetonitrile solution of 45% (volume %) dissolves mutually, after sonic oscillation, and 18000r/min high speed centrifugation 3min, take 20 μ L of supernatant liquid
HPLC sample introduction is analyzed.
Particularly as follows: U.S.'s Waters e2695 highly effective liquid phase chromatographic system, original-pack chromatographic work station, detector is original-pack
2473 fluorescence detectors, Thermo BDS C18Post (5 μm, 250mm × 4.6mm), flowing is acetonitrile mutually: water=45: 55；Column temperature
30 DEG C, excitation wavelength 360nm, launch wavelength 450nm；Flow velocity is 1.0mL min-1；Sample size is 20 μ L.
Remarks illustrate: blank plasma is the true plasma not adding AFB1.
According to HPLC testing result, with AFB1 peak area (Y) as vertical coordinate, with AFB1 concentration
Mass concentration (X) is abscissa, draws standard curve, obtains curvilinear equation and correlation coefficient (r).Prepare containing Aspergillus flavus poison simultaneously
In element B1 curve ranges, high, medium and low concentrations Plasma sample 0.5,1.0,10.0mg/L is as quality-control sample (QC), respectively according to upper
State sample treatment process after sample introduction analysis, each concentration samples is repeated 5 times, with AFB1 peak area in sample with
It is directly dissolved in the ratio of the most lower the surveyed peak area that flows, calculates the method response rate under high, medium and low 3 kinds of concentration (that is, yellow bent in sample
Mould toxin B1 peak area and the ratio=AFB1 response rate being directly dissolved in the most lower the surveyed peak area that flows).More than Bi compare
The change of the peak area that sample measures in a few days 5 times and in the daytime 5 times, calculates withinday precision and day to day precision.
Remarks illustrate: " withinday precision and day to day precision " is mainly used in evaluating said method (i.e. HPLC detection method)
Precision and accuracy, judge the reliability of final detection result with this.
Result is:
During gained processes blood plasma according to the method described above, AFB1 is bent in 0.1-100ng/mL concentration range internal standard
Line is Y=2 × 107X 1646.4 (r ＞ 0.9999), in standard curve range, high, medium and low concentration AFB1 reclaims
Rate is all higher than more than 90%, and withinday precision and day to day precision are respectively less than 10%, are shown in Table 2.
The response rate of AFB1 and precision in table 2. blood plasma
According to the above results, we can learn: the above-mentioned detection method set by the present invention can meet the yellow bent of the present invention
The requirement of mould toxin B1 intestinal absorption degree detection.
Method four, assessment catechin EGCG and the intestinal absorption degree of aflatoxin and hepatic injury
160 SPF level ICR male mices are purchased from Zhejiang Academy of Medical Sciences Experimental Animal Center, body weight 25 ± 2g, experiment
It is divided into 4 groups, often 40 mices of group.It is randomly divided into often organizing 40 mices in 8 little mouse cages, 5, every cage, in experiment prospective adaptation
Cultivating one week, mouth fills front 12h fasting, can't help water.
Experimental group one, catechin matched group: the concentration of catechin+procyanidin normal saline solution is 10mg/mL (both
The concentration of weight sum, and catechin: procyanidin=1:1 weight ratio), according to the filling of 100mg/kg body weight mouth, (above-mentioned 100mg is
The amount of the catechin+procyanidin in normal saline solution)；40 mices are randomly divided into 8 groups, often group 5, suitable before experiment
Answering property cultivates one week, and mouth fills front 12h fasting, can't help water.
Experimental group two, catechin+procyanidin nanometer solution group: catechin+procyanidin prepared by the invention described above
Compound recipe nanoparticle suspension (such as concentration is 10mg/mL), according to 100mg/kg body weight mouth fill (above-mentioned 100mg is that compound recipe is received
The amount of the catechin+procyanidin in grain of rice suspension)；40 mices are randomly divided into 8 groups, often group 5, in experiment prospective adaptation
Property cultivation one week, mouth fills front 12h fasting, can't help water.
Experimental group three, aflatoxin mouth filling group: fill preparation to mice mouth according to 5mg AFB1/kg body weight
Aflatoxin solution；40 mices being randomly divided into 8 groups, often group 5, cultivate one week in experiment prospective adaptation, mouth fills front 12h
Fasting, can't help water.
Experimental group four, flavacin toxin+compound recipe nanoparticle mouth filling group: according to 100mg (catechin+procyanidin)/kg
Body weight fills the compound recipe nanoparticle suspension of the catechin+procyanidin of preparation to mice mouth, and above-mentioned 100mg is that compound recipe nanoparticle mixes
The weight of the catechin+procyanidin in suspension, fills to mice mouth according to 5mg AFB1/kg body weight after 30min simultaneously
The aflatoxin solution of preparation；40 mices are randomly divided into 8 groups, often group 5, cultivate one week in experiment prospective adaptation, mouth
Fill front 12h fasting, can't help water.
By above-mentioned experimental group and matched group, respectively according to above-mentioned dosage mouth fill, all in mouth fill after 5min, 10min, 15min,
30min, 60min, 120min, 180min and 240min eyeball takes blood, often 5 mices of group blood sampling, then by taken blood in liver
In elementization centrifuge tube, after 6000r/min is centrifugal, experiment one and experiment two each time points take respectively 0.3mL blood plasma in 10mL from
In heart pipe, process laggard HPLC referring concurrently to said method one and analyze.Catechin EGCG peak area in surveyed blood plasma is substituted into
State in standard curve equation (method one gained), calculate catechin EGCG concentration in blood plasma, draw plasma concentration time bent
Line, compares compound recipe nanoparticle suspension (present invention) and the catechin+procyanidin normal saline solution of catechin+procyanidin
Blood drug level, evaluate compound recipe nanoparticle to the impact of intestinal absorption degree in body of catechin in solution with this.
Experiment three and experiment four each time points take 0.3mL blood plasma in 10mL centrifuge tube, respectively referring concurrently to above-mentioned side
Method three processes laggard HPLC and analyzes.AFB1 peak area in surveyed blood plasma is substituted into above-mentioned standard curve equation (method
Three gained) in, calculate AFB1 concentration in blood plasma, draw serum AFB1 Cot curve, than
Relatively AFB1+compound recipe nanoparticle suspension (present invention) and the blood drug level of AFB1 solution, comment with this
Valency compound recipe nanoparticle is on AFB1 impact of bioavailability in body.Use glutamate pyruvate transaminase and millet straw to turn simultaneously
Hepatic injury caused by aflatoxin in experiment three and experiment four is estimated by ammonia enzyme detection kit.
That is, the present invention utilizes laboratory animal entirety intestinal absorption model, is received by the compound recipe of detection by quantitative gained of the present invention
The grain of rice and AFB1 are at intestinal absorption degree, and preparation one can play enhancing catechin intestinal absorption degree, reduce simultaneously
The new formulation of aflatoxin intestinal absorption degree, reduces hepatic injury caused by aflatoxin the most simultaneously, thus plays Aspergillus flavus
The protective effect of toxin damage.
In sum, present invention employs RP-HPLC methods analyst and have detected catechin EGCG in the biological samples such as blood plasma
With accurate, the detection by quantitative means of AFB1, detection limit respectively reaches Gamma Magnitude and nanogram level, is fully able to meet little
Catechin EGCG and the detection of AFB1 in Mus blood sample.
That is, the present invention is on the basis of the compound recipe nanoparticle of preparation catechin and procyanidin, explores this compound recipe nanoparticle pair
Synergistic protective effect in terms of AFB1 intestinal absorption degree, thus expand catechin and the novel effect of procyanidin and answer
With field, give full play to the biological activity of the natural polyphenol such as catechin and procyanidin, serve clinic.
Accompanying drawing explanation
Below in conjunction with the accompanying drawings the 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 to take mice plasma chromatogram after the compound recipe nanoparticle suspension described in embodiment 1；
Wherein peak 1 is catechin EGCG chromatographic peak；Peak 2 is internal standard；
Fig. 4 is that the compound recipe nanoparticle described in embodiment 1 contrasts with generic physiological solution group EGCG Cot curve
Figure；
Fig. 5 is the reference colour spectrogram of AFB1；
Fig. 6 is the chromatogram of blank plasma；
Fig. 7 is the blood plasma chromatogram after mouth fills AFB1；
Wherein peak 1 is AFB1；
Fig. 8 is the AFB1 after mouth fills AFB1 and AFB1+embodiment 1 compound recipe nanoparticle
Cot curve comparison diagram；
Fig. 9 is the hepatic injury contrast after mouth fills AFB1 and AFB1+embodiment 1 compound recipe nanoparticle
Figure.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This:
Embodiment 1, the preparation method of a kind of compound recipe nanoparticle reducing aflatoxin intestinal absorption degree and hepatic injury, depend on
Secondary follow the steps below:
1) catechin and the procyanidin that, accurately weigh mixed in equal amounts amount to 12g (that is, catechin and the matter of procyanidin
Amount ratio is 1:1), it is added thereto to the polyphosphoric acids sodium solution that 200mL mass concentration is 1.5%, mix homogeneously；
2), adding water in the chitosan of 5g is settled to 1000ml, in 5000mL round-bottomed flask, and (rotating speed after magnetic agitation
1000r/min), regulate pH 4.0, obtain chitosan liquid；
3), by step 1) in preparation solution (that is, the polyphosphoric acids sodium solution of catechin and procyanidin) uniformly by
Drip be slowly added dropwise to 2) in round-bottomed flask in, within 70 minutes, complete drip (that is, rate of addition is about 50 droplets/minute)；
The reaction system of gained continues to stir 60min (rotating speed 400r/min) at a temperature of 25 DEG C, obtains catechin and former flower
The compound recipe nanoparticle suspension of blue or green element.
Embodiment 2, the preparation method of a kind of compound recipe nanoparticle reducing aflatoxin intestinal absorption degree and hepatic injury:
By step 1) in the mass ratio of catechin and procyanidin made into 1:3 by 1:1；Gross weight is constant, is still 12g；
By step 2) in the consumption of chitosan made into 2g by 5g；
Remaining is equal to embodiment 1.
Embodiment 3, the preparation method of a kind of compound recipe nanoparticle reducing aflatoxin intestinal absorption degree and hepatic injury:
By step 1) in the mass ratio of catechin and procyanidin made into 3:1 by 1:1；Gross weight is constant, is still 12g；
By step 2) in the consumption of chitosan made into 1.5g by 5g；
Remaining is equal to embodiment 1.
Experiment 1, detection nanoparticle envelop rate:
Embodiment 1, embodiment 2, the compound recipe nanoparticle suspension of embodiment 3 gained are detected according to the method described above,
Result is as follows:
The envelop rate (%)=75.57% of the compound recipe nanoparticle suspension of embodiment 1 gained；
The envelop rate (%)=53.84% of the compound recipe nanoparticle suspension of embodiment 2 gained；
The envelop rate (%)=41.94% of the compound recipe nanoparticle suspension of embodiment 3 gained.
Test 2, embodiment 1, embodiment 2, the compound recipe nanoparticle suspension of embodiment 3 gained are carried out according to the method described above
Detection.
Particularly as follows: fill the compound recipe nanoparticle suspension of preparation to mice mouth according to 100mg/kg body weight；100mg is that compound recipe is received
The weight sum of the catechin+procyanidin in grain of rice suspension.
The above results shows: compound recipe nanoparticle prepared in the present invention is EGCG concentration in different blood sampling time point blood
It is all remarkably higher than Normal Saline solution group, illustrates that the intestinal absorption degree of compound recipe nanoparticles oral solution of the present invention is significantly higher than
Normal Saline solution group.
Final result such as table 3 below (for meansigma methods).
Compound recipe nanoparticle EGCG plasma concentration time data (mg/L) in table 3, different embodiment
Experiment 3, by embodiment 1, embodiment 2, the compound recipe nanoparticle of embodiment 3 gained, detect according to the method described above.
Particularly as follows:
Fill the compound recipe nanoparticle suspension+Aspergillus flavus of embodiment 1 gained of preparation to mice mouth according to 100mg/kg body weight
Toxin mouth fills,
Fill the compound recipe nanoparticle suspension+Aspergillus flavus of embodiment 2 gained of preparation to mice mouth according to 100mg/kg body weight
Toxin mouth fills,
Fill the compound recipe nanoparticle suspension+Aspergillus flavus of embodiment 3 gained of preparation to mice mouth according to 100mg/kg body weight
Toxin mouth fills,
Above-mentioned 100mg is the weight sum of the catechin+procyanidin in compound recipe nanoparticle suspension.
The above results shows: compound recipe nanoparticle prepared in the present invention+aflatoxin mouth fills, at different blood sampling times
In some blood, AFB1 concentration is substantially less than independent aflatoxin mouth filling group, and compound recipe nanometer prepared by the present invention is described
Grain is oral can significantly reduce aflatoxin intestinal absorption degree.
Final result such as table 4 below (meansigma methods).
AFB1 Concentration Time Data (ng/mL) in table 4, different embodiment
Experiment 4, by embodiment 1, embodiment 2, the compound recipe nanoparticle of embodiment 3 gained, assess this according to the method described above
The protective effect to hepatic injury caused by aflatoxin of the compound recipe nanoparticle of bright preparation.
Particularly as follows:
Fill the compound recipe nanoparticle suspension+Aspergillus flavus of embodiment 1 gained of preparation to mice mouth according to 100mg/kg body weight
Toxin mouth fills,
Fill the compound recipe nanoparticle suspension+Aspergillus flavus of embodiment 2 gained of preparation to mice mouth according to 100mg/kg body weight
Toxin mouth fills,
Fill the compound recipe nanoparticle suspension+Aspergillus flavus of embodiment 3 gained of preparation to mice mouth according to 100mg/kg body weight
Toxin mouth fills,
Above-mentioned 100mg is the weight sum of the catechin+procyanidin in compound recipe nanoparticle suspension.
Compound recipe nanoparticle prepared in the present invention+aflatoxin mouth fills, and gathers blood separation at 240min time point
Serum, utilizes serum transaminase test kit to detect glutamate pyruvate transaminase and glutamic oxaloacetic transaminase, GOT activity, result table in mice serum respectively
Compound recipe nanoparticle suspension prepared by the bright present invention is oral can significantly reduce the hepatic injury caused by aflatoxin.
Final result is as shown in Figure 9.
Comparative example 1-1, by embodiment 1 step 1) in the polyphosphoric acids sodium solution that mass concentration is 1.5% make water, body into
Accumulated amount is constant, is still 200mL, and remaining is equal to embodiment 1.
Comparative example 1-2, by embodiment 1 step 1) in the mass concentration of polyphosphoric acids sodium solution made into 3% by 1.5%, body
Accumulated amount is constant, is still 200mL, and remaining is equal to embodiment 1.
Comparative example 1-3, by embodiment 1 step 2) in polyphosphoric acids sodium solution make sodium alginate soln, mass concentration into
Constant, it is still 1.5%, volume is constant, is still 200mL, and remaining is equal to embodiment 1.
Comparative example 1-4, by embodiment 1 step 2) in polyphosphoric acids sodium solution make sodium tripolyphosphate solution into, quality is dense
Spending constant, be still 1.5%, volume is constant, is still 20mL, and remaining is equal to embodiment 1.
Contrast experiment 1, the envelop rate of detection compound recipe nanoparticle:
Being detected according to the method described above by the compound recipe nanoparticle of above-mentioned comparative example gained, result is as follows:
The envelop rate (%)=16.28% of the compound recipe nanoparticle of comparative example 1 gained；
The envelop rate (%)=56.37% of the compound recipe nanoparticle of comparative example 2 gained；
The envelop rate (%)=39.66% of the compound recipe nanoparticle of comparative example 3 gained；
The envelop rate (%)=45.75% of the compound recipe nanoparticle of comparative example 4 gained.
Contrast experiment 2, that the compound recipe nanoparticle of above-mentioned comparative example gained is carried out AFB1 according to the method described above is dense
The detection of degree, result is following (meansigma methods):
AFB1 Concentration Time Data (ng/mL) in the different comparative example of table 5
| Time (min) | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 |
| 5 | 1.05 | 0.91 | 1.03 | 0.96 |
| 15 | 3.95 | 2.51 | 3.19 | 3.02 |
| 120 | 1.20 | 0.69 | 0.80 | 0.70 |
| 240 | 0.40 | 0.17 | 0.22 | 0.20 |
Contrast experiment 3, assess the compound recipe nanoparticle of above-mentioned comparative example gained according to the method described above to caused by aflatoxin
The protective effect of hepatic injury；Result such as table 6 below.
Table 6
| Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | |
| AST | 505 | 390 | 475 | 420 |
| ALT | 515 | 280 | 420 | 390 |
Finally, in addition it is also necessary to it is noted that listed above is only catechin in the present invention+procyanidin compound recipe nanoparticle mouth
Take the specific embodiment of solution.It is clear that the invention is not restricted to above example, it is also possible to there are many deformation.This area common
All deformation that technical staff can directly derive from present disclosure or associate, are all considered as the protection of the present invention
Scope.
Claims (3)
1. reduce aflatoxin trap and the compound recipe nanoparticle preparation method of hepatic injury, it is characterized in that comprising the following steps:
1), first by the mass mixing such as catechin and procyanidin, compound is obtained；Adding mass concentration in 12g compound is 1.4
～the polyphosphoric acids sodium solution 200ml mix homogeneously of 1.6%；
2), adding water in the chitosan of 4～6g is settled to 1000ml, and after magnetic agitation, regulation pH is 3.5～4.5, obtains chitosan
Liquid；
3), by step 1) gained solution all adds to step 2) in the chitosan liquid of gained, the reaction system of gained in 20～
30 DEG C are continued stirring 40～80min, obtain compound recipe nanoparticle suspension.
Reduction aflatoxin trap the most according to claim 1 and the compound recipe nanoparticle preparation method of hepatic injury, its
Feature is:
Described step 3) in, by step 1) gained solution uniformly dropped to step 2 in 60～80 minutes) the chitosan liquid of gained
In.
Reduction aflatoxin trap the most according to claim 1 and 2 and the compound recipe nanoparticle preparation method of hepatic injury,
It is characterized in that:
Described step 2) in, the rotating speed of magnetic agitation is 800～1200r/min.
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