CN107019672A - A kind of preparation method rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome - Google Patents
A kind of preparation method rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome Download PDFInfo
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Abstract
The invention discloses a kind of preparation method rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome, the present invention is using olive growing leaves as raw material, through the extraction of 30~50% ethanol, ceramic membrane and Ultra filtration membrane, NF membrane concentration prepares olive polyphenol extract.90% hydroxytyrosol and 10~40% oleuropein extracts and 90% verbascoside are compounded, prepare and be rich in hydroxytyrosol and verbascoside olive polyphenol extract, using hydroxytyrosol and verbascoside polyphenol as cast material, it is made up of lecithin, cholesterol, Tween 80 surfactant etc. and corresponding liposome is made.This liposome significantly increases the bioavilability of olive polyphenol extract, and envelop rate is high, and particle diameter distribution is uniform, and stability is good, there is slow release, inoxidizability and biocidal property effect.The preparation method simple possible of the present invention, equipment is simple, can expand the application of its field of medicaments.
Description
First, technical field
The present invention relates to the preparation method of biological, chemistry and medicine field, more particularly to hydroxytyrosol liposome.
2nd, background technology
Liposome is mainly the bilayer hollow beads being made up of lipid materials, while having hydrophily and hydrophobicity two
Water-soluble substances can be embedded again by planting property, therefore can encapsulating liposoluble substance.It is water-soluble when lipid materials are dispersed in water
Material can spontaneously be distributed in the inside of liposome, and liposoluble substance is distributed in the lipophilic end of liposome.The preparation of liposome
Method mainly has：Film dispersion method, ultrasonic dispersion, freeze-thaw method, alcohol injection, reverse phase evaporation etc..Its physicochemical property
Evaluation index mainly have：Liposome average grain diameter and its particle diameter distribution, envelop rate, physicochemical stability etc..The liposome of preparation
There is targeting, slow release, protectiveness, low dosage, long-term effect, hypotoxicity.
Long circulating liposome is also known as long-acting liposome, is that a kind of surface is contained natural or synthetic polymer-modified lipoid and spread out
Biological novel lipide.Residence time in blood, so as to extend drug treating time, with long-acting.It is conventional
Trim have polyethylene glycol (PEG) and the sweet ester of neuromere (GM1).GM1 strengthens membrane rigidity, reduces mononuclear phagocyte system
(MPS) intake, the hold-up of this long circulating liposome in blood by the ratio of MPS intakes with being higher than traditional liposomal
Tens times.But GM1 is difficult to a large amount of acquisitions, and has certain immunotoxicity.Quasi-grease derivative containing PEG, they are in liposome
Surface has the effect of height modification, can form space bit resistance layer, this sterically hindered liposome to be protected not to be identified, take the photograph
Take, slow down its elimination, extended durations of action.
Substantial amounts of polyphenol compound, such as oleuropein, hydroxytyrosol, verbascoside and tyrosol are included in olive.
Especially hydroxytyrosol (Hydroxytyrosol, Hydroxytyrosol, abbreviation HT), the ability for removing free radical is strong, table
Reveal uniqueness biology and pharmacological activity, such as anti-oxidant, antibacterial, anti-inflammatory, improve heart coronary blood flow, effectively suppress by third
Olefine aldehydr causes oxidative damage and the mitochondrial function imbalance of retinal epithelial cells, protects cartilage and anti-osteoporosis etc..And
HT can also suppress the expansion such as mankind prorubricyte leukaemia HL-60, colon cancer cell HT-29, female mammary gland cancer cell MCF-7
Dissipate, circulation through blocks tumor cells and induce its apoptosis, with good active anticancer.Although HT has many biological and medicines
Reason activity, gradually causes the concern of researcher.But, HT contains 3 phenolic hydroxyl groups, and exposure is easily oxidized in atmosphere, property
Matter is very unstable.And hydrophily is strong, it is difficult to be preferably dissolved in membrane structure both effectiveness to strengthen its.If HT is prepared as into fat
After plastid, the influence that can not only protect it from outside environmental elements improves stability, moreover it is possible to strengthen its bioavilability (anti-
The bioactivity such as cancer, antibacterial, anti-oxidant).
Document report both domestic and external extracts separating oleuropein, hydroxytyrosol, feltwort from olive growing leaves or olive fruit
The polyphenol compound such as glycosides and tyrosol, but be at present 10~40% olives mostly using olive leaf extract oil olive polyphenol extract
Bitter glucoside extract, hydroxytyrosol content is less than 10%, and verbascoside is less than 5%, the Multifunction open of limitation olive polyphenol activity
Hair.
3rd, the content of the invention
In order to solve the high active polyphenol stability such as oleuropein, hydroxytyrosol, verbascoside and bioactivity, the present invention
Find one kind and be rich in hydroxytyrosol and verbascoside olive polyphenol extract complex method, and be processed into long circulating lipid
Body, therefore, the technical scheme is that being realized using following steps：
The first step：It is prepared by olive polyphenol extract
Take olive growing leaves, add 30~50% ethanol and extract, olive growing leaves and 30~50% ethanol mass volume ratios (kg:
L) 1: 5~20, extract twice, 70~85 DEG C of temperature merges extract solution, and filtrate crosses ceramic membrane (aperture 50nm) and molecular weight
3000Da milipore filters, then filtrate is concentrated with NF membrane, filtrate crosses NF membrane, olive leaf and concentration filtrate quality volume ratio (kg: L)
1: 1~3, concentrate vacuum drying is ground into powder and is made olive polyphenol extract, and HPLC analyses, wherein oleuropein 10~
40%, verbascoside 3~6%, hydroxytyrosol 2~7%, water solubility dissolves 2~8g for 100mL water；
Second step：Prepared rich in hydroxytyrosol and verbascoside olive polyphenol extract
90% hydroxytyrosol and 10~40% oleuropein extracts and 90% verbascoside are compounded, example is in mass ratio
4~8: 2~5: 1~3,3~8 times of absolute ethyl alcohols are added, 60~90 DEG C are heated to reflux 10~30min, mistake after sample fully dissolves
Filter, be concentrated under reduced pressure, prepare be rich in hydroxytyrosol and verbascoside olive polyphenol extract, wherein hydroxytyrosol be 50~
70%, oleuropein is 10~20%, and verbascoside is 5~15%；
3rd step：Film is scattered-and mechanical oscillation method prepares long circulating olive polyphenol liposome
Using rich in hydroxytyrosol and verbascoside olive polyphenol extract as encapsulating target, 40~70 DEG C of design temperature,
Lecithin is 1: 1~8: 1 with cholesterol mass ratio, claims lecithin and cholesterol 3~10g of quality, the body of surface active agent tween -80
Product 2~10mL, 200 1~5g of PEG, put in people's round-bottomed flask, add appropriate organic solvent, 5~20min of sonic oscillation, dissolving
Decompression thin film evaporation removes solvent afterwards, adds 1~4mg/mL hydroxytyrosols (HT) 1~10mL of the aqueous solution, olive polyphenol water
Film 5~50min of hydration time is washed in 1~3mL of solution, rotation, then 5~30min of ultrasonic dissolution can obtain liposome.According to single factor test
Optimize with orthogonal design, prepare uniform milky long circulating liposome, with stability, slow release, inoxidizability and antibacterial
The functions such as property；
4th step：Rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome envelop rate
Liposome is taken in beaker, deionized water is added, liposome and deionized water quality volume by volume concentration be 2~
15mg/mL, is put into bag filter, and dialyse 1~15h at room temperature, during HPLC analyses are water-soluble in HT concentration (C) and dialysis filtrate
HT concentration (C0), HT water solubility volumes (V), the cumulative volume (V for filtrate of dialysing0), calculated according to following formula and be rich in hydroxytyrosol and hair stamen
Flower glycosides olive polyphenol extract long circulating liposome envelop rate is more than 20~80%；
5th step：Rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome particle mean size and its
Distribution
1~10mg/mL liposomes are put into cuvette, a certain amount of deionized water are added, with ZetaPlus type laser
Particle size analyzer in optical source wavelength 368nm and the test of 90 ° of angle of scattering lower room temperature, the particle diameter distribution of the liposome of preparation 200~
1000nm。
6th step：Stability rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome
1. physical stability：1~8mg/mL Liposomal suspensions and the same concentrations HT aqueous solution are taken, 4 000r/ are used respectively
000r/min 10~the 20min of centrifuge of min~10, the liposome of preparation in 4 000~6000r/min without precipitation and not
Layering, emulsion-stabilizing；In being preserved at 4 DEG C~25 DEG C, hydroxytyrosol and verbascoside in 0~30d, liposome are stable, the same terms
It is lower to improve 20~40% than HT stability in the HT aqueous solution；
2. hydrolytic stability：Film it is scattered-mechanical oscillation method prepares liposome suspension, is 3.0~7.0 in pH value
Citrate buffer solution (0.1~0.5mol/L citric acid solutions and 0.2~0.5mol/L disodium phosphate solns), is determined each respectively
The envelop rate of liposome, as a result, with the rise of hydrating fluid pH value, envelop rate increase.
7th step：Slow release rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome
It is 1~8mg/mL Liposomal suspensions to prepare HT concentration, pipettes 1~5mL of suspension and is put into bag filter, and it is soaked completely
Enter in the beaker containing 100mL physiological saline；In the water-bath that beaker is put into 37 DEG C of constant temperature, analyzed in 24h at interval of 2h
HT concentration in dialysis filtrate.Compared with the same concentrations HT aqueous solution, (43.66 ± 1.82) % released in HT aqueous solution 2h,
Liposome releases (28.62 ± 1.87) %；After 24h, HT solution and liposome release be respectively (73.41 ± 1.72) % and
Liposome improves 30~65% sustained release performance under (55.72 ± 1.88) %, the same terms.
8th step：Inoxidizability rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome
The absolute ethyl alcohol that Liposomal suspensions and HT aqueous samples are made into 0.25,0.5,1,1.5,2 μ g/mL respectively is to be measured
Solution.1mL testing samples add 40mg/L DPPH alcoholic solution 3mL, after fully mixing, at room temperature lucifuge reaction 30min,
Absorbance (A is determined under 517nm wavelengthi).Each sampleParallel 3 times, average.It is computed learningThe IC of HT liposomes50For
0.5~2 μ g/mL, are that the HT aqueous solution and liposome are unanimous on the whole to DPPH radicals scavenging effects.Illustrate to utilize liposome technology
HT is prepared into after corresponding liposome, its elimination effect to DPPH free radicals is not affected.
Sample solution is as follows to the calculation formula of DPPH free radical scavenging activities (η)：
In formula：
A0--- 1mL testing samples and the light absorption value (blank value) after the mixed liquor 30min of 3mL absolute ethyl alcohols；
A1--- 1mL absolute ethyl alcohols and the light absorption value (free radical total value) after 3mLDPPH alcoholic solution hybrid reactions 30min；
Ai--- 1mL testing samples and the light absorption value after 3mL DPPH alcoholic solution hybrid reactions 30min.
9th step：Biocidal property rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome
Cell concentration is used for 105Cfu/mL bacteria suspension prepare Liposomal suspensions and HT aqueous samples concentration according to
Secondary is 1024,512,256,128,64,32,16,8,4,2,1 μ g/mL sample solution.The above-mentioned solution prepared is placed into constant temperature
In constant humidity incubator, observed after 37 DEG C of culture 24h in result, test tube almost without the corresponding sample concentration of turbid phenomenon as this sample
The MIC value of product.Every kind of sample does 3 parallel testings.On the basis of MIC measurement results, each pipe for having no bacterial growth is chosen
Nutrient solution, pipettes 0.1mL and is coated on solid medium, places in 37 DEG C of incubators, result is observed after 24h, is still given birth to without bacterium
The corresponding sample concentration of long phenomenon as this sample MBC values.Relative to the HT aqueous solution, the fungistatic effect of liposome is suitable, and
MIC to staphylococcus aureus is that 4~16 μ g/mL, MBC values are 16~64 μ g/mL.MIC to Escherichia coli is 16~64 μ
G/mL, MBC value are 32~128 μ g/mL.
The polyphenol compounds such as oleuropein, hydroxytyrosol, verbascoside have preferable alcohol-soluble, existing market production
Not only the polyphenol compound content such as oleuropein, hydroxytyrosol, verbascoside is low for olive extract, and water-soluble bad,
It is water-soluble in order to improve oleuropein extract, be conducive to liposome preparation and activity, in the present invention, using 30~50% second
Alcohol extracting olive growing leaves, are extracted twice, 70~85 DEG C of temperature, using ceramic membrane (aperture 50nm) and molecular weight 3000Da milipore filters
Filtering, then filtrate is concentrated with NF membrane, remove high molecular weight protein, polysaccharide, gel, tannin etc., concentrate is water-soluble small point
The polyphenol compounds such as sub- oleuropein, hydroxytyrosol, verbascoside, vacuum drying, powder, which is broken into powder olive polyphenol is made, to be carried
Thing is taken, water solubility dissolves 1~6g for 100ml water, with good water solubility.
In order to reach the effect of olive polyphenol extract long circulating liposome, the present invention using film it is scattered-machinery shakes
Swing method.Revolving decompression film pressure -0.05~-0.1Mpa, 50~70 DEG C of temperature, vacuum freeze drying film pressure 2~
10Mpa, temperature -10~-50 DEG C, ultrasound machinery hunting power 100~200W, 20~40min of time, high speed shear power 500
~1000W, 5~10min of time, the scattered effect of film is reached using one of which or several method.
In order to improve the stability of olive polyphenol extract long circulating liposome, phosphatide is used to be emulsified for surface-active
One kind or compound in agent, preferably soya lecithin, egg yolk lecithin and hydrogenated soya phosphatide.In order to preferably scattered, also add
Plus cholesterol 3~10g of quality, the volume of surface active agent tween -80 200 1~5g of 2~10mL, PEG, form stable homogeneous
Newborn phase.To form homogeneous in order to which olive polyphenol extract and emulsion adjuvant fully dissolve, using absolute ethyl alcohol, acetone, isopropyl acetone
Organic solvent is used as with one kind in chloroform.
Beneficial effects of the present invention are shown as：
Prepared by novelty is rich in hydroxytyrosol and verbascoside olive polyphenol extract, and hydroxytyrosol is 50~70%,
Oleuropein is 10~20%, and verbascoside is hydroxytyrosol, oleuropein and verbascoside ratio in 5~15%, active polyphenol
Rationally, water solubility is up to 100ml water dissolving 6g polyphenol extracts to example, and concentration is high, and antioxidation activity is higher.
2. using film it is scattered-mechanical oscillation method is prepared for olive polyphenol extract liposome, envelop rate is high, particle diameter point
Cloth is uniform, stability is good；
3. what is prepared has slow release rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome,
Times for spraying can be reduced；
4. what is prepared has necessarily rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome
Inoxidizability and biocidal property.
4th, illustrate：
The temperature of accompanying drawing 1 is to rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome envelop rate
Influence；
The ovum courage of accompanying drawing 2, which is compared, is rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome envelop rate
Influence；
The Tween-80 volume of accompanying drawing 3 is to rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome
The influence of envelop rate；
Accompanying drawing 4HT mass is to rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome envelop rate
Influence.
5th, embodiment
Following examples are some citings of the present invention, should not be seen as limitation of the invention.
Embodiment 1
It is prepared by olive polyphenol extract
1kg olive growing leaves are taken, 30% ethanol 8L is added and extracts, 85 DEG C of temperature, is extracted twice, merged at 2 hours extraction times
Extract solution, is respectively adopted ceramic membrane (aperture 50nm) and molecular weight 3000Da ultrafiltration membrance filters, then concentrate filtrate extremely with NF membrane
1.5L, concentrate vacuum drying, powder is broken into powder and olive polyphenol extract is made, and HPLC is analyzed, wherein oleuropein 36.8%,
Verbascoside 5.6%, hydroxytyrosol 3.4%, water solubility dissolves 5.2g for 100ml water.
Embodiment 2
Prepared rich in hydroxytyrosol and verbascoside olive polyphenol extract
90% hydroxytyrosol and 36.8% oleuropein extract and 90% verbascoside are compounded, example is 4 in mass ratio
~8: 2~5: 1~3, preferably 4~5: 2~3: 1~2,4 times of absolute ethyl alcohols are added, 60 DEG C are heated to reflux 20min, and sample is fully molten
Filter, be concentrated under reduced pressure after solution, prepare and be rich in hydroxytyrosol and verbascoside olive polyphenol extract, HPLC analyses, wherein hydroxyl
Base tyrosol is 56%, and oleuropein is 18%, and verbascoside is 7%；
In order to reach the effect of olive polyphenol extract long circulating liposome, the present invention using film it is scattered-machinery shakes
Swing method.Revolving decompression film pressure -0.05~-0.1Mpa, 50~70 DEG C of temperature, vacuum freeze drying film pressure 2~
10Mpa, temperature -10~-50 DEG C, ultrasound machinery hunting power 100~200W, 20~40min of time, high speed shear power 500
~1000W, 5~10min of time, the scattered effect of film is reached using one of which or several method.
In order to improve the stability of olive polyphenol extract long circulating liposome, phosphatide is used to be emulsified for surface-active
One kind or compound in agent, preferably soya lecithin, egg yolk lecithin and hydrogenated soya phosphatide.In order to preferably scattered, also add
Plus cholesterol 3~10g of quality, the volume of surface active agent tween -80 200 1~5g of 2~10mL, PEG, form stable homogeneous
Newborn phase.To form homogeneous in order to which olive polyphenol extract and emulsion adjuvant fully dissolve, using absolute ethyl alcohol, acetone, isopropyl acetone
Organic solvent is used as with one kind in chloroform.
Embodiment 3
Preparation rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome
Using rich in hydroxytyrosol and verbascoside olive polyphenol extract as encapsulating target, 40~70 DEG C of design temperature,
It is preferred that 55 DEG C, lecithin is 3: 1 title lecithin and cholesterol quality 4g, the body of surface active agent tween -80 with cholesterol mass ratio
Product 5mL, the 3.5g of PEG 200, are put into round-bottomed flask, add acetone, and sonic oscillation 10min depressurizes thin film evaporation and removed after dissolving
Solvent is removed, rotates and depressurizes film pressure -0.05~-0.1Mpa, 50~70 DEG C of temperature, vacuum freeze drying film pressure 2~
10Mpa, temperature -10~-50 DEG C, ultrasound machinery hunting power 100~200W, 20~40min of time, high speed shear power 500
~1000W, 5~10min of time.Add 3mg/mL hydroxytyrosols (HT) aqueous solution 6mL, the olive polyphenol extract aqueous solution
Film hydration time 15min is washed in 2mL, rotation, then ultrasonic dissolution 20min can obtain liposome.
Embodiment 4
Envelop rate rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome
0.05g liposomes are taken in beaker, 10mL deionized waters is added, places into treated bag filter, in room temperature
Wherein HT contents are determined after lower dialysis 9h, and calculate HT envelop rate.The calculation formula of liposome encapsulation：
In formula：
C --- the concentration of the HT aqueous solution, mg/mL；
V --- the volume of the HT aqueous solution, mL；
C0--- HT concentration, mg/mL in dialysis filtrate；
V0--- the cumulative volume of dialysis filtrate, mL.
When HT concentration of aqueous solution is 2mg/mL, HT aqueous solution volume is 2mL, and ovum courage ratio is that 4: 1 (lecithin 80mg, courage is solid
Alcohol 20mg, similarly hereinafter), Tween-80 is 6mL, and preparation temperature is 65 DEG C, and the envelop rate that can obtain HT liposomes is 37.51%.
Embodiment 4
Slow release rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome
It is 1mg/mL Liposomal suspensions to prepare HT concentration, pipettes suspension 2mL and is put into bag filter, be completely immersed in containing
In the beaker of 100mL physiological saline；In the water-bath that beaker is put into 37 DEG C of constant temperature, at interval of 2h analysis dialysis filters in 24h
HT concentration in liquid.Compared with the same concentrations HT aqueous solution, (43.66 ± 1.82) %, liposome are released in HT aqueous solution 2h
Release (28.62 ± 1.87) %；After 24h, HT solution and liposome release be respectively (73.41 ± 1.72) % and (55.72 ±
1.88) liposome improves 30~55% sustained release performance under %, the same terms.
Embodiment 5
Inoxidizability rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome
The absolute ethyl alcohol that Liposomal suspensions and HT aqueous samples are made into 0.25,0.5,1,1.5,2 μ g/mL respectively is to be measured
Solution.1mL testing samples add 40mg/L DPPH alcoholic solution 3mL, after fully mixing, at room temperature lucifuge reaction 30min,
Absorbance (A is determined under 517nm wavelengthi).Each sample is parallel 3 times, averages.It is computed learning the IC of HT liposomes50For
0.5~2 μ g/mL, are that the HT aqueous solution and liposome are unanimous on the whole to DPPH radicals scavenging effects.Illustrate to utilize liposome technology
HT is prepared into after corresponding liposome, its elimination effect to DPPH free radicals is not affected.
Sample solution is as follows to the calculation formula of DPPH free radical scavenging activities (η)：
In formula：
A0--- 1mL testing samples and the light absorption value (blank value) after the mixed liquor 30min of 3mL absolute ethyl alcohols；
A1--- 1mL absolute ethyl alcohols and the light absorption value (free radical total value) after 3mL DPPH alcoholic solution hybrid reactions 30min；
Ai--- 1mL testing samples and the light absorption value after 3mL DPPH alcoholic solution hybrid reactions 30min.
Embodiment 6
Biocidal property rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome
Cell concentration is used for 105Cfu/mL bacteria suspension prepares HT Liposomal suspensions and HT aqueous sample concentrations
It is followed successively by 1024,512,256,128,64,32,16,8,4,2,1 μ g/mL sample solution.The above-mentioned solution prepared is placed permanent
In constant temperature and humidity incubator, observed after 37 DEG C of culture 24h in result, test tube and be used as this almost without the corresponding sample concentration of turbid phenomenon
The MIC value of sample.Every kind of sample does 3 parallel testings.On the basis of MIC measurement results, selection has no each of bacterial growth
Pipe nutrient solution, pipettes 0.1mL and is coated on solid medium, places in 37 DEG C of incubators, result is observed after 24h, still without bacterium
The corresponding sample concentration of phenomenon is grown as the MBC values of this sample.Relative to the HT aqueous solution, the fungistatic effect of liposome is suitable,
And be respectively 8 and 32 μ g/mL to MIC the and MBC values of staphylococcus aureus.MIC and MBC values to Escherichia coli are respectively 32
With 64 μ g/mL.
Claims (4)
1. a kind of preparation method rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome, its feature
It is to comprise the following steps：
The first step：It is prepared by olive polyphenol extract
Olive growing leaves are taken, 30~50% ethanol is added and extracts, olive growing leaves and 30~50% ethanol mass volume ratios (kg: L) 1: 5
~20, extract twice, 70~85 DEG C of temperature, merge extract solution, filtrate crosses ceramic membrane (aperture 50nm) and molecular weight 3000Da is super
Filter membrane, then filtrate is concentrated with NF membrane, filtrate crosses NF membrane, olive leaf and concentration filtrate quality volume ratio (kg: L) 1: 1~3,
Concentrate is dried in vacuo, and is ground into powder and olive polyphenol extract, wherein HPLC analyses, oleuropein 10~40%, hair is made
Stamen spends glycosides 3~6%, and hydroxytyrosol 2~7%, water solubility dissolves 2~8g for 100mL water；
Second step：Prepared rich in hydroxytyrosol and verbascoside olive polyphenol extract
90% hydroxytyrosol and 10~40% oleuropein extracts and 90% verbascoside are compounded, example is 4~8 in mass ratio
: 2~5: 1~3,3~8 times of absolute ethyl alcohols are added, 60~90 DEG C are heated to reflux 10~30min, and sample is filtered after fully dissolving, subtracted
Pressure concentration, prepares and is rich in hydroxytyrosol and verbascoside olive polyphenol extract, and wherein hydroxytyrosol is 50~70%, olive
Bitter glycosides is 10~20%, and verbascoside is 5~15%；
3rd step：Film is scattered-and mechanical oscillation method prepares long circulating olive polyphenol liposome
Using rich in hydroxytyrosol and verbascoside olive polyphenol extract as encapsulating target, 40~70 DEG C of design temperature, lecithin
Fat is 1: 1~8: 1 with cholesterol mass ratio, claims lecithin and cholesterol 3~10g of quality, the volume of surface active agent tween -80 2
1~5g of~10mL, PEG200, is put into round-bottomed flask, adds appropriate organic solvent, and 5~20min of sonic oscillation subtracts after dissolving
Ironed film evaporation of solvent, adds 1~4mg/mL hydroxytyrosols (HT) 1~10mL of the aqueous solution, the olive polyphenol aqueous solution 1
Film 5~50min of hydration time is washed in~3mL, rotation, then 5~30min of ultrasonic dissolution can obtain liposome.According to single factor test and just
Design optimization is handed over, uniform milky long circulating liposome is prepared, with stability, slow release, inoxidizability and biocidal property etc.
Function；
4th step：Rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome envelop rate
Liposome is taken in beaker, deionized water is added, liposome is 2~15mg/mL with deionized water quality volume by volume concentration,
It is put into bag filter, dialyse 1~15h at room temperature, HT concentration in HT concentration (C) and filtrate of dialysing during HPLC analyses are water-soluble
(C0), HT water solubility volumes (V), the cumulative volume (V for filtrate of dialysing0), calculated according to following formula rich in hydroxytyrosol and verbascoside oil
Olive polyphenol extract long circulating liposome envelop rate is more than 20~80%；
5th step：Rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome particle mean size and its distribution
1~10mg/mL liposomes are put into cuvette, a certain amount of deionized water are added, with ZetaPlus type laser particle sizes
Instrument is in optical source wavelength 368nm and the lower room temperature test of 90 ° of angle of scattering, and the particle diameter distribution of the liposome of preparation is in 200~1000nm.
6th step：Stability rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome
1. physical stability：Take 1~8mg/mL Liposomal suspensions and the same concentrations HT aqueous solution, respectively with 4 000r/min~
10 000r/min 10~20min of centrifuge, the liposome of preparation in 4 000~6000r/min without precipitation and not stratified,
Emulsion-stabilizing；In being preserved at 4 DEG C~25 DEG C in 0~30d, liposome, hydroxytyrosol and verbascoside are stable, compare under the same terms
HT stability improves 20~40% in the HT aqueous solution；
2. hydrolytic stability：Film it is scattered-mechanical oscillation method prepares liposome suspension, in the lemon that pH value is 3.0~7.0
Acid buffer (0.1~0.5mol/L citric acid solutions and 0.2~0.5mol/L disodium phosphate solns), determines each lipid respectively
The envelop rate of body, as a result, with the rise of hydrating fluid pH value, envelop rate increase.
7th step：Slow release rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome
It is 1~8mg/mL Liposomal suspensions to prepare HT concentration, pipettes 1~5mL of suspension and is put into bag filter, is completely immersed in and contained
In the beaker for having 100mL physiological saline；In the water-bath that beaker is put into 37 DEG C of constant temperature, analyze and dialyse at interval of 2h in 24h
HT concentration in filtrate.Compared with the same concentrations HT aqueous solution, (43.66 ± 1.82) %, lipid are released in HT aqueous solution 2h
Body releases (28.62 ± 1.87) %；After 24h, HT solution and liposome release are respectively (73.41 ± 1.72) % and (55.72
Liposome improves 30~65% sustained release performance under ± 1.88) %, the same terms.
8th step：Inoxidizability rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome
The absolute ethyl alcohol that Liposomal suspensions and HT aqueous samples are made into 0.25,0.5,1,1.5,2 μ g/mL respectively is to be measured molten
Liquid.1mL testing samples add 40mg/L DPPH alcoholic solution 3mL, after fully mixing, at room temperature lucifuge reaction 30min, in 517nm
Absorbance (A is determined under wavelengthi).Each sample is parallel 3 times, averages.It is computed learning the IC of HT liposomes50For 0.5~2
μ g/mL, are that the HT aqueous solution and liposome are unanimous on the whole to DPPH radicals scavenging effects.Illustrate using liposome technology HT systems
It is standby into after corresponding liposome, its elimination effect to DPPH free radicals is not affected.
Sample solution is as follows to the calculation formula of DPPH free radical scavenging activities (η)：
In formula：
A0--- 1mL testing samples and the light absorption value (blank value) after the mixed liquor 30min of 3mL absolute ethyl alcohols；
A1--- 1mL absolute ethyl alcohols and the light absorption value (free radical total value) after 3mL DPPH alcoholic solution hybrid reactions 30min；
Ai--- 1mL testing samples and the light absorption value after 3mLDPPH alcoholic solution hybrid reactions 30min.
9th step：Biocidal property rich in hydroxytyrosol and verbascoside olive polyphenol extract long circulating liposome
Cell concentration is used for 105Cfu/mL bacteria suspension prepares Liposomal suspensions and HT aqueous sample concentrations are followed successively by
1024th, 512,256,128,64,32,16,8,4,2,1 μ g/mL sample solution.The above-mentioned solution prepared is placed into constant temperature and humidity
In incubator, observed after 37 DEG C of culture 24h in result, test tube and be used as this sample almost without the corresponding sample concentration of turbid phenomenon
MIC value.Every kind of sample does 3 parallel testings.On the basis of MIC measurement results, each pipe culture for having no bacterial growth is chosen
Liquid, pipettes 0.1mL and is coated on solid medium, places in 37 DEG C of incubators, result is observed after 24h, still existing without bacterial growth
As MBC value of the corresponding sample concentration as this sample.Relative to the HT aqueous solution, the fungistatic effect of liposome is suitable, and to gold
The MIC of staphylococcus aureus is that 4~16 μ g/mL, MBC values are 16~64 μ g/mL.MIC to Escherichia coli is 16~64 μ g/
ML, MBC value are 32~128 μ g/mL.
2. one kind according to claims 1 is rich in hydroxytyrosol and verbascoside olive polyphenol extract long-circulation fat
The preparation method of plastid, it is characterised in that film described in step one is scattered-and mechanical oscillation method includes：Revolving decompression film forming (pressure
Power -0.05~-0.1Mpa, 50~70 DEG C of temperature), vacuum freeze drying film forming (2~10Mpa of pressure, temperature -10~-50 DEG C)
With ultrasonic mechanical oscillation (100~200W of power, 20~40min of time), high speed shear (500~1000W of power, the time 5~
One or several kinds 10min).
3. one kind according to claims 1 is rich in hydroxytyrosol and verbascoside olive polyphenol extract long-circulation fat
The preparation method of plastid, it is characterised in that phosphatide described in step one is soybean lecithin, egg yolk lecithin and hydrogenated soybean phosphorus
One kind in fat.
4. one kind according to claims 1 is rich in hydroxytyrosol and verbascoside olive polyphenol extract long-circulation fat
The preparation method of plastid, it is characterised in that organic solvent is one in absolute ethyl alcohol, acetone, isopropyl acetone and chloroform in step one
Kind.
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| CN107929324A (en) * | 2017-12-08 | 2018-04-20 | 大连工业大学 | Load sea cucumber boiling liquid extract liposome and preparation method thereof |
| CN107998183A (en) * | 2017-12-26 | 2018-05-08 | 浙江大学 | Sweet osmanthus benzyl carbinol glycosides liposome of Chitosan-coated and preparation method thereof |
| CN108314607A (en) * | 2018-05-08 | 2018-07-24 | 广西大学 | A kind of purification process of high-purity solid hydroxytyrosol |
| CN109400658A (en) * | 2018-11-26 | 2019-03-01 | 中国科学院兰州化学物理研究所 | The method of separated in synchronization purifying oleuropein and hydroxytyrosol from olive growing leaves |
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| CN111991353A (en) * | 2020-09-13 | 2020-11-27 | 仲恺农业工程学院 | Preparation method and application of multi-vesicle dihydromyricetin liposome |
| CN112198269A (en) * | 2020-04-22 | 2021-01-08 | 山东省药学科学院 | Method for determining hydroxytyrosol in Beagle dog plasma |
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