CN106420634A - EGCG (Epigallocatechin Gallate) functionalized chitosan derivative as well as EGCG functionalized chitosan derivative carried honokiol nano particle and preparation methods of EGCG functionalized chitosan derivative and EGCG functionalized chitosan derivative carried honokiol nano particle - Google Patents
EGCG (Epigallocatechin Gallate) functionalized chitosan derivative as well as EGCG functionalized chitosan derivative carried honokiol nano particle and preparation methods of EGCG functionalized chitosan derivative and EGCG functionalized chitosan derivative carried honokiol nano particle Download PDFInfo
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Abstract
The invention provides an EGCG (Epigallocatechin Gallate) functionalized chitosan derivative, an EGCG functionalized chitosan derivative carried honokiol nano particle and preparation methods of the EGCG functionalized chitosan derivative and the EGCG functionalized chitosan derivative carried honokiol nano particle. The structural formula of the EGCG functionalized chitosan derivative is as shown in the description; in the structural formula, the content of EGCG is 10 to 60 percent; the deacetylation degree is 50 to 98 percent; the content of amino is 50 to 98 percent. According to the EGCG functionalized chitosan derivative carried honokiol nano particle, the nano particle is prepared from the EGCG functionalized chitosan derivative and honokiol; the honokiol and the EGCG functionalized chitosan derivative form the nano particle through charge action; the honokiol is encapsulated inside the nano particle.
Description
Technical field
The invention belongs to biomedical materials field, it is related to chitin derivatives, its load honokiol of EGCG functionalization
Nano-particle and preparation method.
Background technology
Nano medication becomes study hotspot in recent years because having the special natures such as targeting, slow-releasing, hypotoxicity.Shell
Polysaccharide, is present in Crustacean shell in a large number, its deacetylated partially or totally based products shitosan have substantial amounts of sun from
Son, has good biocompatibility and biological degradability, and the nanoparticle based on chitin deacetylation product is in food and doctor
Medicine field is obtained for practical application.Chitosan drug-carrying nanometer particle has embodied preferable solubilising or slow release effect, but
It is that existing drug-carrying nanometer particle substantially directly adopts shitosan (chitosan) as carrier, chitin is not entered
Row structural modification, leads to not lift the biological activity being supported molecule in medicine-carried system.Therefore further based on chitin
Modify, make modified outcome while having chitin easy nanorize property, have the effect of medicament curative effect enhancement, have important
Meaning.
Epigallocatechin gallate (EGCG) (EGCG) is a kind of composition extracting from Chinese green tea, and it is green tea master
The activity wanted and water-soluble components, are content highest components in catechin, account for the 9%-13% of green tea gross weight, due to having spy
Different stereochemical structure, EGCG has very strong antioxidant activity, act as important in terms of anticancer and cardiovascular disease
Role, it has obvious anti-lung cancer and hepatocarcinoma effect and not normal cells intact.
Fei Lei etc. is prepared for a kind of covalent conjunct agent of shitosan-EGCG (see Journal of Applied
Polumer Science, 2014,131,39732), directly covalent bond is reacted with EGCG using deacetylated chitin, it is deposited
Deficiency be that the amino of chitosan is not completely retained, most amino has all been grafted some EGCG, this meeting
Have a strong impact on the nanorize performance of chitin-EGCG it is difficult to form nano-particle it is impossible to be used for nanometer medicine-carried system.
Honokiol (Honokiol) is detached lignan component from Cortex Magnoliae Officinalis.Honokiol has good
Antibacterial, antiinflammatory, antioxidation and anti-tumor activity, but due to water solublity extreme difference, bioavailability in vivo is low, so that
Increase consumption and long-term taking, this significantly limit its clinical practice.CN 104095817 A discloses a kind of directly using and takes off
Chitosan loads the nanoparticle of magnolol and honokiol, and this nanoparticle improves honokiol dissolubility and dissolution,
But its biological activity does not get a promotion.
Based on the above-mentioned state of the art, if the chitin derivatives being easy to that the EGCG of nanorize modifies can be developed, and it is based on
The derivant that this is easy to nanorize develops the nano-particle having sustained release performance and effectively improving honokiol biological activity,
To biomedicine field by positive meaning.
Content of the invention
Chitin derivatives, the EGCG of EGCG functionalization, it is an object of the invention to overcoming the deficiencies in the prior art, are provided
The chitin derivatives of functionalization carry honokiol nano-particle and preparation method, to improve the chitin derivatives of EGCG modification
Nanometer processability, and effectively improve the biological activity of honokiol.
The chitin derivatives of the EGCG functionalization that the present invention provides, structural formula is as follows：
In structure above, EGCG content is 10%～60%, and deacetylation is 50%～98%, and amino content is 50%
～98%.
The chitin derivatives of the EGCG functionalization that the present invention provides carry honokiol nano-particle, and this nano-particle is by upper
State the chitin derivatives of EGCG functionalization and honokiol forms, honokiol is led to the chitin derivatives of EGCG functionalization
Cross charge effect and form nano-particle, honokiol is wrapped in inside nano-particle.
In above-mentioned nano-particle, honokiol content is 10～500 μ g/mg, contains in every milligram of nano-particle and thick
Plain phenol 10～500 microgram.
The particle diameter of above-mentioned nano-particle is 20～300nm.
The monodispersity index of above-mentioned nano-particle is 0.091～0.253.
Present invention also offers the preparation method of the chitin derivatives of above-mentioned EGCG functionalization, step is as follows：
(1) chitin is dissolved in and in hydrochloric acid, forms the chitin hydrochloric acid solution that chitin concentration is 2～20mg/mL, will
Epigallocatechin gallate (EGCG) (EGCG) is dissolved in water and forms the epigallo catechin that concentration is 2～15mg/mL
Gallic acid aqueous solution of ester；
(2) chitin hydrochloric acid solution 100 weight portion is added in container, be subsequently adding 5～30 weight portions and be dissolved with oxidation
Protectant oxidant, stirs at least 10min at 20～50 DEG C, adds epigallocatechin gallate (EGCG) solution 15
～80 parts, stir 30～120min, simultaneously lyophilization obtains intermediate product by the dialysis of gained reactant liquor；
(3) step (2) gained intermediate product is dissolved in 40wt%～60wt% sodium hydroxide solution, 100～180
DEG C, 0.5～1MPa steaming and decocting 8～16h, then drain, during gained solid phase is washed, eluate is in neutrality, is drying to obtain EGCG function
The chitin derivatives changed.
In the preparation method of chitin derivatives of above-mentioned EGCG functionalization, the concentration of described hydrochloric acid be 0.5wt%～
10wt%.
In the preparation method of chitin derivatives of above-mentioned EGCG functionalization, described oxidant is H2O2Or dimethyl is sub-
Sulfone (DMSO), above-mentioned oxidation protection agent is ascorbic acid, and generally, the preparation method being dissolved with the oxidant of oxidation protection agent is：
The oxidation protection agent of 1～20 weight portion is dissolved in the oxidant of 1～40 weight portion.
Present invention also offers a kind of chitin derivatives of EGCG functionalization carry the preparation side of honokiol nano-particle
Method, step is as follows：
(1) chitin is dissolved in and in hydrochloric acid, forms the chitin hydrochloric acid solution that chitin concentration is 2～20mg/mL, will
Epigallocatechin gallate (EGCG) (EGCG) is dissolved in water and forms the epigallo catechin that concentration is 2～15mg/mL
Gallic acid aqueous solution of ester, by honokiol be dissolved in the ethanol-water solution that volume fraction of ethanol is 30%～80% shape and
Magnolol concentration is 2～20mg/mL solution；
(2) chitin hydrochloric acid solution 100 weight portion is added in container, be subsequently adding 5～30 weight portions and be dissolved with oxidation
Protectant oxidant, stirs at least 10min at 20～50 DEG C, adds epigallocatechin gallate (EGCG) solution 15
～80 parts, stir 30～120min, simultaneously lyophilization obtains intermediate product by the dialysis of gained reactant liquor；
(3) step (2) gained intermediate product is dissolved in 40wt%～60wt% sodium hydroxide solution, 100～180
DEG C, 0.5～1MPa steaming and decocting 8～16h, then drain, during gained solid phase is washed, eluate is in neutrality, is drying to obtain EGCG function
The chitin derivatives changed；
(4) chitin derivatives of step (3) gained EGCG functionalization are dissolved in formation EGCG function in acetic acid solution
The chitin derivatives changed are the chitin derivatives acetic acid solution of the EGCG functionalization of 2～10mg/mL, to 10～50 weight portions
In the chitin derivatives acetic acid solution of EGCG functionalization add honokiol solution 10～50 weight portion, adjust pH value be 2～
6.5, stir 10～40min, Deca 5～20 weight portion concentration is the sodium tripolyphosphate solution of 1～5mg/mL, stirring 30～
120min, gained suspension is centrifuged, and adds freeze drying protectant, lyophilization obtains final product the shell of EGCG functionalization after discarding supernatant
Polysaccharide derivates carry honokiol nano-particle.
The chitin derivatives of above-mentioned EGCG functionalization carry in the preparation method of honokiol nano-particle, and described lyophilizing is protected
Shield agent is Mannitol, Sorbitol, lactic acid or gelatin, generally, after the addition of freeze drying protectant should make centrifugation discard supernatant
The volume ratio of material and freeze drying protectant reach 5:3～20:1.
The chitin derivatives of above-mentioned EGCG functionalization carry in the preparation method of honokiol nano-particle, centrifugally operated
Condition be 12000～16000rpm rotating speed under be centrifuged 10～60min.
The chitin derivatives of above-mentioned EGCG functionalization carry in the preparation method of honokiol nano-particle, and described acetic acid is molten
The concentration of liquid is 0.5wt%～5wt%, and the concentration of described hydrochloric acid is 0.5wt%～10wt%.
The chitin derivatives of above-mentioned EGCG functionalization carry in the preparation method of honokiol nano-particle, described oxidant
For H2O2Or dimethyl sulfoxide (DMSO), above-mentioned oxidation protection agent is ascorbic acid, generally, is dissolved with the oxygen of oxidation protection agent
The preparation method of agent is：The oxidation protection agent of 1～20 weight portion is dissolved in the oxidant of 1～40 weight portion.
Compared with prior art, the invention has the advantages that：
1. the invention provides chitin derivatives of EGCG functionalization of a kind of new structure and preparation method thereof, due to
The method is first reacted with chitin with EGCG, then product is carried out deacetylated, thus the chitin of the EGCG functionalization of preparation
The amino in chitosan, wherein amino content up to 50%～98% is remained, high amino content is favourable in derivant
Form nano-particle in it, thus this derivant has the characteristics that to be easy to nanorize.
2. test shows, the chitin derivatives of the EGCG functionalization that the present invention provides are thin to human normal liver, kidney, lung body
Born of the same parents' avirulence, good biocompatibility.
3. test shows, the chitin derivatives of the EGCG functionalization that the present invention provides carry honokiol nano-particle to be had
Significantly sustained release performance, the biological activity of honokiol is effectively improved, the especially anti-liver cancer and anti-lung cancer of honokiol
Activity is enhanced considerably.
4. the particle diameter distribution of the chitin derivatives load honokiol nano-particle of the EGCG functionalization that the present invention provides is equal
One, monodispersity index, up to 0.091～0.253, is conducive to the quality control of nano-particle.
Brief description
Fig. 1 is the FT-IR figure of the chitin derivatives of EGCG functionalization of embodiment 3 preparation；
Fig. 2 is the TEM figure of the chitin derivatives load honokiol nano-particle of the EGCG functionalization of embodiment 3 preparation；
Fig. 3 is the chitin derivatives of EGCG functionalization in embodiment 5 to LO2, the shadow of MRC-5, HK-2 cell survival rate
Ring figure；
Fig. 4 is the LO2 in embodiment 5 after the derivant group of blank control group and 2mg/mL is processed, MRC-5, HK-2
The microphotograph of cell growth status；
In Fig. 5 embodiment 6, the chitin derivatives of the EGCG functionalization of honokiol and embodiment 3 preparation carry magnolia obovata
The release profiles of honokiol in phenol nano-particle；
Fig. 6 is the nano-particle of honokiol and embodiment 3 preparation in embodiment 7 to typeⅡ pneumocyte and human liver cancer
The impact figure of HepG2 cell survival rate；
Fig. 7 is through blank control group, the honokiol medication group of 20 μ g/mL and 20 μ g/mL embodiments in embodiment 7
TypeⅡ pneumocyte after the nano-particle medication group process of 3 preparations and the microscope of human hepatoma HepG2 cell's growing state
Photo.
Specific embodiment
By the following examples and combine accompanying drawing to the chitin derivatives of EGCG functionalization of the present invention, it carries and thick
Plain phenol nano-particle and preparation method are described further.
Embodiment 1
In the present embodiment, the chitin derivatives of EGCG functionalization and the chitin derivatives of EGCG functionalization are provided to carry
The preparation method of honokiol nano-particle, step is as follows：
(1) chitin is dissolved in the chitin hydrochloric acid that formation chitin concentration in the hydrochloric acid of 0.5wt% is 2mg/mL molten
Liquid, epigallocatechin gallate (EGCG) (EGCG) is dissolved in water and forms the EGCG aqueous solution that concentration is 2mg/mL, will
It is 2mg/mL solution that honokiol is dissolved in shape honokiol concentration in the ethanol-water solution that volume fraction of ethanol is 50%.
(2) chitin hydrochloric acid solution 100 weight portion is added in container, be subsequently adding 5 weight portions and be dissolved with ascorbic acid
H2O2(by 1 weight portion dissolution of ascorbic acid in 8 weight portion H2O2In), magnetic agitation 10min at 20 DEG C, add EGCG
15 parts of solution, magnetic agitation 30min, gained reactant liquor is dialysed 2 times and lyophilization obtains intermediate product.
(3) step (2) gained intermediate product is dissolved in 40wt% sodium hydroxide solution, at 100 DEG C, 0.5MPa steaming and decocting
8h, then drains, and during gained solid phase is washed, eluate is in neutrality, is drying to obtain the chitin derivatives of EGCG functionalization, should
The structural formula of derivant is as follows：
In this structural formula, EGCG content is 12%, and deacetylation is 80%, and amino content is 80%.
(4) chitin derivatives of step (3) gained EGCG functionalization are dissolved in the acetic acid solution of 0.5wt% and are formed
The chitin derivatives of EGCG functionalization are the chitin derivatives acetic acid solution of the EGCG functionalization of 2mg/mL, to 10 weight portions
Honokiol solution 10 weight portion is added, adjusting pH value is 2, stirring in the chitin derivatives acetic acid solution of EGCG functionalization
10min, Deca 5 weight portion concentration is the sodium tripolyphosphate solution of 1mg/mL, stirs 30min, gained suspension is centrifuged, discards
Freeze drying protectant Mannitol is added, the sample after centrifugation discards supernatant is 20 with the volume ratio of Mannitol after supernatant:1, cold
Freeze the chitin derivatives load honokiol nano-particle being drying to obtain EGCG functionalization.
The chitin derivatives of EGCG functionalization manufactured in the present embodiment carry the mean diameter of honokiol nano-particle about
For 300nm, the monodispersity index of nano-particle is 0.253, and in this nano-particle, the content of honokiol is 140.5 μ g/mg, its
Envelop rate is 68.3 ± 3.2%.
Embodiment 2
In the present embodiment, the chitin derivatives of EGCG functionalization and the chitin derivatives of EGCG functionalization are provided to carry
The preparation method of honokiol nano-particle, step is as follows：
(1) chitin is dissolved in and in the hydrochloric acid of 5wt%, forms the chitin hydrochloric acid solution that chitin concentration is 8mg/mL,
EGCG is dissolved in water and forms the EGCG aqueous solution that concentration is 6mg/mL, honokiol is dissolved in volume fraction of ethanol is
In 30% ethanol-water solution, shape honokiol concentration is 6mg/mL solution.
(2) chitin hydrochloric acid solution 100 weight portion is added in container, be subsequently adding 10 weight portions and be dissolved with ascorbic acid
DMSO (by 1 weight portion dissolution of ascorbic acid in 15 weight portion DMSO), magnetic agitation 60min at 30 DEG C, add
30 parts of EGCG solution, magnetic agitation 60min, gained reactant liquor is dialysed 5 times and lyophilization obtains intermediate product.
(3) step (2) gained intermediate product is dissolved in 50wt% sodium hydroxide solution, at 130 DEG C, 0.7MPa steaming and decocting
10h, then drains, and during gained solid phase is washed, eluate is in neutrality, is drying to obtain the chitin derivatives of EGCG functionalization, should
The structural formula of derivant is as follows：
In this structural formula, EGCG content is 23%, and deacetylation is 90%, and amino content is 90%.
(4) chitin derivatives of step (3) gained EGCG functionalization are dissolved in the acetic acid solution of 3wt% and are formed
The chitin derivatives of EGCG functionalization are the chitin derivatives acetic acid solution of the EGCG functionalization of 6mg/mL, to 20 weight portions
Honokiol solution 10 weight portion is added, adjusting pH value is 4, stirring in the chitin derivatives acetic acid solution of EGCG functionalization
20min, Deca 10 weight portion concentration is the sodium tripolyphosphate solution of 3mg/mL, stirs 60min, gained suspension is existed
Be centrifuged 20min under 13000rpm, discard and add freeze drying protectant Mannitol after supernatant, centrifugation discard supernatant after sample with
The volume ratio of Mannitol is 5:3, the chitin derivatives that lyophilization obtains final product EGCG functionalization carry honokiol nano-particle.
The chitin derivatives of EGCG functionalization manufactured in the present embodiment carry the mean diameter of honokiol nano-particle about
For 90nm, the monodispersity index of nano-particle is 0.205, and in this nano-particle, the content of honokiol is 130.5 μ g/mg, its
Envelop rate is 75.6 ± 5.5%.
Embodiment 3
In the present embodiment, the chitin derivatives of EGCG functionalization and the chitin derivatives of EGCG functionalization are provided to carry
The preparation method of honokiol nano-particle, step is as follows：
(1) chitin is dissolved in the chitin hydrochloric acid that formation chitin concentration in the hydrochloric acid of 10wt% is 15mg/mL molten
Liquid, EGCG is dissolved in water and forms the EGCG aqueous solution that concentration is 10mg/mL, honokiol is dissolved in volume fraction of ethanol
It is 12mg/mL solution for shape honokiol concentration in 80% ethanol-water solution.
(2) chitin hydrochloric acid solution 100 weight portion is added in container, be subsequently adding 20 weight portions and be dissolved with ascorbic acid
H2O2(by 10 weight portion dissolution of ascorbic acid in 30 weight portion H2O2In), magnetic agitation 50min at 40 DEG C, add
60 parts of EGCG solution, magnetic agitation 90min, gained reactant liquor is dialysed 8 times and lyophilization obtains intermediate product.
(3) step (2) gained intermediate product is dissolved in 55wt% sodium hydroxide solution, at 160 DEG C, 0.9MPa steaming and decocting
13h, then drains, and during gained solid phase is washed, eluate is in neutrality, is drying to obtain the chitin derivatives of EGCG functionalization, should
The FT-IR of derivant schemes as shown in figure 1, the structural formula of this derivant is as follows：
In this structural formula, EGCG content is 40%, and deacetylation is 95%, and amino content is 95%.
(4) chitin derivatives of step (3) gained EGCG functionalization are dissolved in the acetic acid solution of 5wt% and are formed
The chitin derivatives of EGCG functionalization are the chitin derivatives acetic acid solution of the EGCG functionalization of 10mg/mL, to 40 weight
Honokiol solution 30 weight portion is added, adjusting pH value is 4.5, stirs in the chitin derivatives acetic acid solution of part EGCG functionalization
Mix 30min, Deca 15 weight portion concentration is the sodium tripolyphosphate solution of 5mg/mL, stirs 90min, gained suspension is existed
Be centrifuged 40min under 15000rpm, discard and add freeze drying protectant Mannitol after supernatant, centrifugation discard supernatant after sample with
The volume ratio of Mannitol is 10:1, the chitin derivatives that lyophilization obtains final product EGCG functionalization carry honokiol nano-particle.
The chitin derivatives of EGCG functionalization manufactured in the present embodiment carry the mean diameter of honokiol nano-particle about
For 60nm, the monodispersity index of nano-particle is 0.091, and the TEM photo of this nano-particle is as shown in Fig. 2 in this nano-particle
The content of honokiol is 190.5 μ g/mg, and its envelop rate is 88.3 ± 5.1%.
Embodiment 4
In the present embodiment, the chitin derivatives of EGCG functionalization and the chitin derivatives of EGCG functionalization are provided to carry
The preparation method of honokiol nano-particle, step is as follows：
(1) chitin is dissolved in the chitin hydrochloric acid that formation chitin concentration in the hydrochloric acid of 10wt% is 20mg/mL molten
Liquid, EGCG is dissolved in water and forms the EGCG aqueous solution that concentration is 15mg/mL, honokiol is dissolved in volume fraction of ethanol
It is 20mg/mL solution for shape honokiol concentration in 50% ethanol-water solution.
(2) chitin hydrochloric acid solution 100 weight portion is added in container, be subsequently adding 30 weight portions and be dissolved with ascorbic acid
H2O2(by 1 weight portion dissolution of ascorbic acid in 40 weight portion H2O2In), magnetic agitation 60min at 50 DEG C, add EGCG
80 parts of solution, magnetic agitation 90min, gained reactant liquor is dialysed 10 times and lyophilization obtains intermediate product.
(3) step (2) gained intermediate product is dissolved in 60wt% sodium hydroxide solution, at 180 DEG C, 1MPa steaming and decocting
16h, then drains, and during gained solid phase is washed, eluate is in neutrality, is drying to obtain the chitin derivatives of EGCG functionalization, should
The structural formula of derivant is as follows：
In this structural formula, EGCG content is 55%, and deacetylation is 96%, and amino content is 96%.
(4) chitin derivatives of step (3) gained EGCG functionalization are dissolved in the acetic acid solution of 5wt% and are formed
The chitin derivatives of EGCG functionalization are the chitin derivatives acetic acid solution of the EGCG functionalization of 10mg/mL, to 50 weight
Honokiol solution 10 weight portion is added, adjusting pH value is 6.5, stirs in the chitin derivatives acetic acid solution of part EGCG functionalization
Mix 40min, Deca 20 weight portion concentration is the sodium tripolyphosphate solution of 3mg/mL, stirs 120min, gained suspension is existed
Be centrifuged 60min under 16000rpm, discard and add freeze drying protectant Mannitol after supernatant, centrifugation discard supernatant after sample with
The volume ratio of Mannitol is 20:1, the chitin derivatives that lyophilization obtains final product EGCG functionalization carry honokiol nano-particle.
The chitin derivatives of EGCG functionalization manufactured in the present embodiment carry the mean diameter of honokiol nano-particle about
For 130nm, the monodispersity index of nano-particle is 0.195, and in this nano-particle, the content of honokiol is 110.5 μ g/mg, its
Envelop rate is 56.2 ± 4.2%.
Embodiment 5
In the present embodiment, the chitin derivatives of EGCG functionalization are to normal human liver, kidney, the somatic toxicity of lung.
The chitin derivatives of EGCG functionalization prepared by embodiment 3 are disperseed with DMSO, and are diluted with DMEM culture fluid
Form derivative solution to finite concentration.Take the logarithm trophophase LO2 respectively, MRC-5, HK-2 cell strain, preparation density be about 4 ×
104The cell suspension of/L, is inoculated on 96 well culture plates with every pore volume 100 μ L, in CO224h is cultivated, cell pastes in incubator
After wall, add aforementioned derivative solution, make the final concentration of 0mg/mL (blank of the chitin derivatives of EGCG functionalization
Group), 0.5,1,2mg/mL (derivant group), in each hole, DMSO content is respectively less than 0.5%, and each concentration sets 6 parallel holes,
CO2After culture 48h in incubator, the amount according to every hole 50 μ L adds the MTT of 2mg/mL, in CO2Continue culture in incubator
Terminate culture after 4h.In the hole culture supernatant is abandoned in suction, and every hole adds 150 μ LDMSO liquid, fully vibrates, and so that bluish violet is precipitated fully
Dissolving, measures the absorbance value at 490nm wavelength with microplate reader in 30min, calculates cell survival rate, cell survival rate (%)
=(A-A0) × 100%, A0For the absorbance of blank control group, A is the absorbance of derivant group, result as shown in figure 3,
From the figure 3, it may be seen that the chitin derivatives of EGCG functionalization are to LO2, MRC-5, HK-2 cell all no growth inhibition effects.Through sky
LO2, microphotograph such as Fig. 4 of MRC-5, HK-2 cell growth status after the derivant group process of white matched group and 2mg/mL
Shown, as shown in Figure 4, the chitin derivatives of EGCG functionalization to LO2, MRC-5, HK-2 cell all no growth inhibition effects.
The above illustrates that the chitin derivatives of the EGCG functionalization that the present invention provides have good biocompatibility.
Embodiment 6
Weigh 5mg honokiol and the nano-particle being loaded with 5mg honokiol of embodiment 3 preparation respectively, they are divided
It is not scattered in the dissolution medium (being prepared according to Chinese Pharmacopoeia) for 6.8 for the 5mLpH value, being then transferred into molecular cut off is
In 12000～14000 bag filter, bag filter is placed in 50mL dissolution medium, stirs at 37 DEG C, the 0.5th, 1,2,3,
Take out 2mL solution from 50mL dissolution medium within 4,8,12,16,20,24 hours, measure its ultraviolet light absorption angle value and calculate magnolia obovata
The concentration of phenol, draws honokiol release profiles, as shown in figure 5, as shown in Figure 5, single honokiol has very low result
Dissolution rate, and after being prepared into nano-particle, honokiol dissolution substantially improves, nano-particle shows significantly simultaneously
Honokiol slow releasing function.
Embodiment 7
In the present embodiment, the chitin derivatives of test EGCG functionalization carry honokiol nano-particle hepatocarcinoma and pulmonary carcinoma is thin
Born of the same parents' proliferation inhibition activity.
The chitin derivatives of EGCG functionalization prepared by honokiol and embodiment 3 carry honokiol nano-particle and divide
Dissipate in DMSO, be configured to certain density storing solution.Take the logarithm trophophase typeⅡ pneumocyte strain and human liver cancer HepG2 thin
Born of the same parents' strain, adds 25g/L trypsinization, and individual cells suspension is made in piping and druming, and cell density is adjusted about 4 × 104/ L, with every
Pore volume 100 μ L is inoculated on 96 well culture plates, is placed in CO224h is cultivated, after cell attachment, to each hole respectively in incubator
Add the honokiol of variable concentrations and the nanoparticles solution of embodiment 3 preparation, so that honokiol and load and embodiment 3 is made
The final concentration of standby nano-particle is respectively 0 μ g/mL (blank control group), and 5,10,15,20,30,40 μ g/mL (use by honokiol
Medicine group, nano-particle medication group), each concentration sets 6 parallel holes, and after culture 48h, the amount according to every hole 50 μ L adds 2mg/mL
MTT, be placed in CO2Terminate culture after continuing culture 4h in incubator.In the hole culture supernatant is abandoned in suction, and every hole adds DMSO, fills
Point vibration, makes bluish violet precipitate and fully dissolves, measure the absorbance at 490nm wavelength with microplate reader in 30min.Different dense
The nano-particle of the honokiol of degree and embodiment 3 preparation presses down to the propagation of typeⅡ pneumocyte and human hepatoma HepG2 cell
Rate processed=(matched group absorbance-medication group absorbance)/matched group absorbance × 100%, result is respectively as Fig. 6 (A)
(B) shown in, it will be appreciated from fig. 6 that compared with honokiol, the nano-particle of embodiment 3 preparation is to typeⅡ pneumocyte and people
Hepatoma Hep G 2 cells have higher growth of cancer cells suppression ratio.Honokiol medication through blank control group, 20 μ g/mL
TypeⅡ pneumocyte after the nano-particle medication group process of group and 20 μ g/mL embodiment 3 preparation and human liver cancer HepG2 are thin
The microphotograph of intracellular growth situation as shown in fig. 7, as shown in Figure 7, the life to typeⅡ pneumocyte for the nano-particle medication group
Long inhibitory action is significantly stronger than honokiol, and nano-particle medication group is also strong to the growth inhibition effect of human hepatoma HepG2 cell
In honokiol.Honokiol is supported on shape in the chitin derivatives of EGCG functionalization of the present invention by above description of contents
Become nano-particle, it is possible to increase the biological activity of honokiol.
Claims (10)
1. a kind of chitin derivatives of EGCG functionalization are it is characterised in that structural formula is as follows：
In structure above, EGCG content be 10%～60%, deacetylation be 50%～98%, amino content be 50%～
98%.
2. a kind of chitin derivatives of EGCG functionalization carry honokiol nano-particle it is characterised in that this nano-particle is by weighing
Profit requires the chitin derivatives of EGCG functionalization described in 1 to form with honokiol, and honokiol is many with the shell of EGCG functionalization
Sugar derivativess form nano-particle by charge effect, and honokiol is wrapped in inside nano-particle.
3. according to claim 2 EGCG functionalization chitin derivatives carry honokiol nano-particle it is characterised in that
In this nano-particle, honokiol content is 10～500 μ g/mg.
4. the chitin derivatives of EGCG functionalization according to Claims 2 or 3 carry honokiol nano-particle, and its feature exists
In nano-particle particle diameter be 20～300nm.
5. a kind of preparation method of the chitin derivatives of EGCG functionalization is it is characterised in that step is as follows：
(1) chitin is dissolved in and in hydrochloric acid, forms the chitin hydrochloric acid solution that chitin concentration is 2～20mg/mL, table is not had
Infanticide catechin and gallate is dissolved in water and forms the epigallocatechin gallate (EGCG) that concentration is 2～15mg/mL
Aqueous solution；
(2) chitin hydrochloric acid solution 100 weight portion is added in container, be subsequently adding 5～30 weight portions and be dissolved with oxidation protection
The oxidant of agent, stirs at least 10min at 20～50 DEG C, adds epigallocatechin gallate (EGCG) solution 15～80
Part, stir 30～120min, simultaneously lyophilization obtains intermediate product by the dialysis of gained reactant liquor；
(3) step (2) gained intermediate product is dissolved in 40wt%～60wt% sodium hydroxide solution, at 100～180 DEG C,
0.5～1MPa steaming and decocting 8～16h, then drains, and during gained solid phase is washed, eluate is in neutrality, is drying to obtain EGCG functionalization
Chitin derivatives.
6. according to claim 5 the preparation method of the chitin derivatives of EGCG functionalization it is characterised in that described hydrochloric acid
Concentration be 0.5wt%～10wt%.
7. a kind of chitin derivatives of EGCG functionalization carry the preparation method of honokiol nano-particle it is characterised in that step
As follows：
It is 2 that honokiol is dissolved in shape honokiol concentration in the ethanol-water solution that volume fraction of ethanol is 30%～80%
～20mg/mL solution, the chitin derivatives of the EGCG functionalization of claim 5 or 6 preparation are dissolved in shape in acetic acid solution
The chitin derivatives of one-tenth EGCG functionalization are the chitin derivatives acetic acid solution of the EGCG functionalization of 2～10mg/mL, to 10
Add honokiol solution 10～50 weight portion in the chitin derivatives acetic acid solution of～50 weight portion EGCG functionalization, adjust
PH value is 2～6.5, stirs 10～40min, Deca 5～20 weight portion concentration is the sodium tripolyphosphate solution of 1～5mg/mL, stirs
Mix 30～120min, gained suspension is centrifuged, add freeze drying protectant after discarding supernatant, lyophilization obtains final product EGCG function
The chitin derivatives changed carry honokiol nano-particle.
8. the chitin derivatives of EGCG functionalization carry the preparation method of honokiol nano-particle according to claim 7,
It is characterized in that described freeze drying protectant is Mannitol, Sorbitol, lactic acid or gelatin.
9. the chitin derivatives of EGCG functionalization according to claim 7 or 8 carry the preparation side of honokiol nano-particle
Method it is characterised in that the condition of centrifugally operated be 12000～16000rpm rotating speed under be centrifuged 10～60min.
10. the chitin derivatives of EGCG functionalization according to claim 7 or 8 carry the preparation side of honokiol nano-particle
Method is it is characterised in that the concentration of described acetic acid solution is 0.5wt%～5wt%.
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| CN110801442A (en) * | 2018-07-20 | 2020-02-18 | 复旦大学 | Slow-release microsphere coated with honokiol and pharmaceutical application thereof |
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