CN102871848B - Pentacyclic triterpenoid cyclodextrin clathrate compound proliposome and preparation method thereof - Google Patents
Pentacyclic triterpenoid cyclodextrin clathrate compound proliposome and preparation method thereof Download PDFInfo
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Abstract
The invention discloses pentacyclic triterpenoid cyclodextrin clathrate compound proliposome and a preparation method thereof. The main component comprises one or more of pentacyclic triterpenoid compound series, for example, ursolic acid, oleanolic acid, betulonic acid, beta-masticinic acid, cyclodextrin clathrate compound, phospholipid material and cholesterol. According to the pentacyclic triterpenoid cyclodextrin clathrate compound proliposome, the pentacyclic triterpenoid compound is clathrated through cyclodextrin to improve the hydrophilicity; the liposome is used as a carrier to clad, and the proliposome is obtained by freezing and drying. According to the pentacyclic triterpenoid cyclodextrin clathrate compound proliposome, the bioavailability and the stability of the pentacyclic triterpenoid compound can be improved; transdermal absorption is promoted, and the skin irritation is reduced. In addition, the cyclodextrin clathrate compound proliposome is of a powder shape, good in stability and rehydration reconstruction and convenient to use, and can be applied to a plurality of cosmetic dosage forms such as cream, emulsion, gel and essence.
Description
Technical field
The present invention relates to a kind of pentacyclic triterpenoid cyclodextrin clathrate pro-liposome and preparation method thereof and the application as cosmetic active ingredient.
Background technology
There is many biological activitys taking ursolic acid and oleanolic acid as the pentacyclic triterpenoid of representative, as antioxidation, antiinflammatory, check melanin form and collagen protein reworking.Japan patent of invention JP8165231A, JP2000302659A disclose its application as anti-light aging skin care item active component; A kind of anti-aging cosmetic of patent CN101111289A invention, its main component comprises ursolic acid and oleanolic acid.Although pentacyclic triterpenoid has extensive biologic activity mostly, but due to the particularity of pentacyclic triterpenoid mother nucleus structure, make it have strong-hydrophobicity, be unfavorable for absorbing, lack bin stability, bioavailability is lower, and these side effect have limited its application at cosmetic field to a great extent.
Cyclodextrin is the cyclic oligosaccharide that a class has hydrophobicity central cavity and water-wetted surface, can the various organic and inorganic and biomolecule formation host-guest clathrates of selectivity bonding.So-called " enclose " is exactly that subject and object passes through noncovalent intermolecular interactions, completes the process of identification each other, finally makes guest molecule partly or entirely enter the phenomenon of body interior.The molecule cavity internal diameter of beta-schardinger dextrin-is of moderate size, about 70-80nm, can with most compound formation clathrates, applied range.HP-β-CD water solublity is fabulous, nontoxic to kidney, and muscle, mucosa are almost had no stimulation.Lyophobic dust forms the solubility property that can improve medicine after clathrate as guest molecule and cyclodextrin, adjustment release speed, improves stability, covers bad smell and reduces toxic and side effects and zest.Chinese invention patent CN1785203A discloses a kind of astragaloside cyclodextrin clathrate and preparation method thereof, and after enclose, the dissolubility of astragaloside is increased to 188.4-386.8 μ g/ml from 22.2 μ g/ml, and bioavailability brings up to 11% by 3%.Chinese patent CN102342897A has invented the preparation method of the cyclodextrin clathrate of a kind of skin sunscreen, and sunscreen has reduced bad smell after forming clathrate, has improved light stability.U.S. patent of invention US6596263 discloses a kind of 'alpha '-hydroxy acids cyclodextrin clathrate and the application aspect skin preparation thereof.
Liposome is the complete totally enclosed single or multiple lift vesicle of the bilayer that formed by lipoids such as phospholipid.Its similar, in cell, can be sealed water solublity and liposoluble substance.Liposome, as topical remedy's carrier, with keratodermatitis intercellular lipid structural similarity, can see through soon horny layer and enter deep skin, makes more active substances stay epidermis between corium.The cosmetics for skin that U.S. patent of invention US20110081402A1 discloses a kind of collagen liposome component and contained this composition.Chinese patent CN1437931 has invented coenzyme Q10 pro-liposome, has improved the stability of coenzyme Q10 and has facilitated its application in cosmetics.The ceramide nano liposome preparations of Chinese patent CN101214198 invention has high stability, water dissolubility and good biocompatibility.But this type of invention is coated hydrophilic active principle, for coated strong-hydrophobicity active component, its hydrophobicity makes molecule can not directly embed liposome vesicle inner chamber, has the deficiency of the low and poor stability of envelop rate.
Summary of the invention
The object of this invention is to provide a kind of pentacyclic triterpenoid cyclodextrin clathrate pro-liposome, to overcome the pentacyclic triterpenoid of strong-hydrophobicity as the deficiency of cosmetic active ingredient, improve hydrophilic by cyclodextrin inclusion compound, and be coated taking liposome as carrier, make pro-liposome powder, improve its bioavailability and stability.
Another object of the present invention is to provide the preparation method of above-mentioned pentacyclic triterpenoid cyclodextrin clathrate pro-liposome.
Implementation procedure of the present invention is as follows:
A kind of pentacyclic triterpenoid cyclodextrin clathrate pro-liposome, is prepared by following substances:
Pentacyclic triterpenoid 2-50 part
Cyclodextrin 30-100 part
Phospholipid material 100-220 part
Cholesterol 20-70 part
Freeze drying protectant 40-155 part.
Described pentacyclic triterpenoid is selected from one or more in ursolic acid, oleanolic acid, Betulinic Acid, beta boswellic acid.
Described cyclodextrin is beta-schardinger dextrin-or HP-β-CD.
Described phospholipid material is selected from one or more in soybean lecithin, Ovum Gallus domesticus Flavus lecithin, hydrogenation egg yolk lecithin, hydrogenated soya phosphatide, dimyristoyl phosphatidyl choline, two Semen Myristicae phosphatidyl glycerols, dipalmitoyl phosphatidyl choline, two Petiolus Trachycarpi phosphatidyl glycerols, distearoyl phosphatidylcholine, distearyl phosphatidyl glycerol.
Described freeze drying protectant is selected from one or more in trehalose, mannitol, sorbitol, xylitol, cottonseed sugar, dextran, lactose.
It is 80-600nm that above-mentioned cyclodextrin clathrate pro-liposome rehydration is rebuild rear particle diameter.
A kind of preparation method of pentacyclic triterpenoid cyclodextrin clathrate pro-liposome, the preparation of employing two step method, form cyclodextrin clathrate by pentacyclic triterpenoid after by cyclodextrin inclusion compound, be embedded in again liposome vesicle and form cyclodextrin inclusion liposome, and obtain pentacyclic triterpenoid cyclodextrin clathrate pro-liposome by lyophilization.
Specifically, preparation method comprises the following steps:
(1) pentacyclic triterpenoid is dissolved in ethanol or 95% ethanol, cyclodextrin is water-soluble, the alcoholic solution of pentacyclic triterpenoid is added in cyclodextrin aqueous solution and stirred, through centrifuging and taking supernatant, vacuum concentration, the dry pentacyclic triterpene cyclodextrin clathrate that to obtain;
(2) phospholipid material and cholesterol are dissolved in ether, pentacyclic triterpene cyclodextrin clathrate is dissolved in the phosphate buffered solution of pH 6～8, ultrasonic after two solution are mixed, evaporation is removed ether and is formed colloidal solution, adds the phosphate buffered solution of pH 6～8 to obtain inclusion liposome suspension;
(3) freeze drying protectant is added in liposome turbid liquor, dissolve postlyophilization and obtain pentacyclic triterpenoid cyclodextrin clathrate pro-liposome.
Pentacyclic triterpenoid is strengthened transdermal effect by the present invention; reduce skin irritation; in composition, cyclodextrin, phospholipid material and freeze drying protectant are the composition to skin beneficiating simultaneously; make it be applicable as cosmetic active ingredient, as multiple State of cosmetics such as emulsion, cream, gel, essences.
Above-mentioned pentacyclic triterpenoid cyclodextrin clathrate pro-liposome can be used in external preparation for skin cosmetics, is added into and in cosmetics, makes skin emulsion, nourishing cream, massage cream, facial gel, eye cream, eye essence, eye gel, cleansing cream, facial milk cleanser, foundation cream, body lotion, health frost, body gels, addition is 0.1 ~ 10%.
Pentacyclic triterpenoid cyclodextrin clathrate pro-liposome of the present invention has following remarkable advantage:
(1) solved the deficiency of hydrophobicity pentacyclic triterpenoid as cosmetic active ingredient, the preparation method of pentacyclic triterpenoid cyclodextrin clathrate pro-liposome and the application as cosmetic active ingredient thereof are provided.
(2) hydrophobicity pentacyclic triterpenoid has been improved to hydrophilic by cyclodextrin inclusion compound, and be coated taking liposome as carrier, be prepared as pro-liposome powder, improve its bioavailability and stability, strengthening transdermal effect, reduces skin irritation.
(3) cyclodextrin material in the present invention, phospholipid material and freeze drying protectant are the cosmetic material to skin beneficiating, can act synergistically on skin.
(4) pentacyclic triterpenoid cyclodextrin clathrate pro-liposome of the present invention is Powdered, easy to use, it is little that rehydration is rebuild rear particle diameter, and preparation method is simple, envelop rate is high, makes it can be applicable to the multiple State of cosmetics such as emulsion, cream, gel, essence.
Brief description of the drawings
Fig. 1 is pentacyclic triterpenoid cyclodextrin clathrate pro-liposome preparation technology flow chart;
Fig. 2 is pentacyclic triterpenoid cyclodextrin clathrate pro-liposome outward appearance;
Fig. 3 is pentacyclic triterpenoid cyclodextrin clathrate pro-liposome stability;
Fig. 4 is pentacyclic triterpenoid cyclodextrin inclusion liposome transmission electron microscope picture, figure (1) embodiment 1: ursolic acid Benexate Hydrochloride liposome; (2) embodiment 2: ursolic acid hydroxypropyl-beta-cyclodextrin inclusion liposome; (3) embodiment 3: ursolic acid hydroxypropyl-beta-cyclodextrin inclusion liposome; (4) embodiment 4: oleanolic acid hydroxypropyl-beta-cyclodextrin inclusion liposome;
Fig. 5 is pentacyclic triterpenoid cyclodextrin inclusion liposome particle size distribution figure, figure (A), (B), (C) and (D) liposome for preparing corresponding to embodiment 1-4.
Detailed description of the invention
Below in conjunction with concrete case, the invention will be further described.
As shown in Figure 1, more particularly, the preparation method of pentacyclic triterpenoid cyclodextrin clathrate pro-liposome of the present invention is as follows:
(1) pentacyclic triterpenoid is dissolved in 95% ethanol, cyclodextrin dissolves in deionized water, under continuing to stir, pentacyclic triterpenoid solution is slowly dropped in cyclodextrin aqueous solution, 20-60 DEG C is stirred 1-6h afterwards by clathrate dispersion liquid centrifugal 60min under 70000g, supernatant is the aqueous solution of pentacyclic triterpene cyclodextrin clathrate, and by supernatant vacuum concentration, concentrated solution obtains the pentacyclic triterpene cyclodextrin clathrate of white powder at 40 DEG C of dry 3-6h of vacuum drying oven;
(2) phospholipid material is mixed and is dissolved in ether with cholesterol, pentacyclic triterpene cyclodextrin clathrate is dissolved in the phosphate buffered solution of pH 7.4 of 0.02M.Water-bath is ultrasonic to be dropped to inclusion complex in solution in immobilized artificial membrane material solution down, continue ultrasonic 5 min, in Rotary Evaporators, ether is removed in decompression, form after colloidal state, add the phosphate buffered solution of the pH 7.4 of 0.02M, the gel being hydrated on bottle wall comes off, and obtains milky inclusion liposome suspension, and this suspension is adopted to high pressure homogenizer homogenizing 4-5 time;
(3) freeze drying protectant is added in above liposome turbid liquor; after dissolving, inclusion liposome suspension poured is carried out in culture dish to lyophilization; lyophilisation condition is: pre-freeze temperature-60 ± 2 DEG C; adopt quick-freezing mode to carry out; pre-freeze time 6h, condenser temperature-50 ~-55 DEG C, vacuum < 100mT; freeze-drying time 24h, obtains pentacyclic triterpenoid cyclodextrin clathrate pro-liposome.When use, add deionized water aquation and rebuild, light shaking is dissolved the inclusion liposome suspension completely obtaining after reconstruction.
The active component that this pentacyclic triterpenoid cyclodextrin clathrate pro-liposome can be used as in the outside cosmetics application combination of skin uses.
The addition of this pentacyclic triterpenoid cyclodextrin clathrate pro-liposome in cosmetic formulations is 0.1 ~ 10%.
Pentacyclic triterpenoid cyclodextrin clathrate pro-liposome of the present invention can be used in skin emulsion, nourishing cream, massage cream, facial gel, eye cream, eye essence, eye gel, cleansing cream, facial milk cleanser, foundation cream, body lotion, health frost, body gels etc.
Embodiment 1: the preparation of ursolic acid Benexate Hydrochloride pro-liposome
The preparation of ursolic acid Benexate Hydrochloride: the beta-schardinger dextrin-that takes 3.75g, add the distilled water of 100ml that it is fully dissolved, separately get ursolic acid 0.50g, after dissolving with 95% ethanol 20ml, slowly drip in above-mentioned beta-schardinger dextrin-aqueous solution, 30 DEG C are stirred after 6h the dispersion liquid of clathrate centrifugal 60min under 70000g, supernatant is the aqueous solution of ursolic acid Benexate Hydrochloride, by supernatant vacuum concentration, concentrated solution is in 40 DEG C, vacuum drying oven, the dry 6h of 0.08MPa obtains the ursolic acid Benexate Hydrochloride of white powder, reach 82.34% by measuring inclusion rate.
Ursolic acid content is measured: adopt high effective liquid chromatography for measuring ursolic acid, chromatographic condition be AtlantisTM C18 analytical column (4.6 mm × 250 mm, 5 μ m); Mobile phase is 0.02 mol/L sodium dihydrogen phosphate methanol solution (volume ratio is 15:85); Detecting wavelength is 215 nm; Flow rate of mobile phase is 0.8 ml/min.
The mensuration of inclusion rate: precision takes a certain amount of ursolic acid Benexate Hydrochloride and is placed in volumetric flask, adds distilled water fully to dissolve, standardize solution.Press ursolic acid content assay method and measure free ursolic acid content, calculate ursolic acid content in clathrate.Ursolic acid inclusion rate basis below formula is calculated:
Inclusion rate %=
mclathrate ×
mursolic acid in clathrate/
madd ursolic acid × 100%
10.25g soybean lecithin is mixed and is dissolved in 50ml absolute ether with 2.50g cholesterol; Ursolic acid Benexate Hydrochloride 3.00g is dissolved in the phosphate buffered solution of pH7.4 of 50ml 0.02M.After two solution are fully mixed, ultrasonic 5 min of water-bath, form stable w/o type emulsion, removal of solvent under reduced pressure in Rotary Evaporators afterwards, rotate to form after colloidal state, add the phosphate buffered solution of the pH7.4 of 50ml 0.02M, the gel that rotating water is bonded on bottle wall comes off, obtain milky inclusion liposome suspension, this suspension is adopted to high pressure homogenizer homogenizing 5 times.
5.00g trehalose is entered in above liposome turbid liquor, after fully dissolving by the lyophilization in plastic culture dish of inclusion liposome suspension poured, lyophilisation condition is: pre-freeze temperature-60 ± 2 DEG C, adopt quick-freezing mode to carry out, pre-freeze time 6h, condenser temperature-50 ~-55 DEG C, vacuum < 100mT, freeze-drying time 24h, obtain pentacyclic triterpene cyclodextrin clathrate pro-liposome (as shown in Figure 2), when use, adding deionized water aquation rebuilds, light shaking is dissolved the inclusion liposome suspension completely obtaining after reconstruction, measuring envelop rate is 56.37%.
Entrapment efficiency determination: adopt supercentrifugation, by 10mL liposome solutions centrifugal 60min under 20000 r/min, get supernatant appropriate, measure free ursolic acid concentration, computational envelope rate.
Embodiment 2: the preparation of ursolic acid hydroxypropyl-beta-cyclodextrin inclusion pro-liposome
The preparation of ursolic acid hydroxypropyl-beta-cyclodextrin inclusion: the HP-β-CD that takes 8.00g, add the distilled water of 100ml that it is fully dissolved, separately get ursolic acid 0.38g, after dissolving with 95% ethanol 20ml, slowly drip in above-mentioned beta-schardinger dextrin-aqueous solution, 20 DEG C are stirred after 3h the dispersion liquid of clathrate centrifugal 60min under 70000g, supernatant is the aqueous solution of ursolic acid hydroxypropyl-beta-cyclodextrin inclusion, by supernatant vacuum concentration, concentrated solution is in 40 DEG C, vacuum drying oven, the dry 6h of 0.08MPa obtains the ursolic acid hydroxypropyl-beta-cyclodextrin inclusion of white powder, reach 79.08% by measuring inclusion rate.
20.00g hydrogenated soya phosphatide is mixed and is dissolved in 50ml absolute ether with 5.50g cholesterol; Ursolic acid hydroxypropyl-beta-cyclodextrin inclusion 4.20g is dissolved in pH 7.4 phosphate buffered solution of 80ml 0.02M.After two solution are fully mixed, ultrasonic 5 min of water-bath, form stable w/o type emulsion, removal of solvent under reduced pressure in Rotary Evaporators afterwards, rotate to form after colloidal state, add the phosphate buffered solution of the pH7.4 of 100ml 0.02M, the gel that rotating water is bonded on bottle wall comes off, obtain milky inclusion liposome suspension, this suspension is adopted to high pressure homogenizer homogenizing 4 times.
The mannitol of 5.00g and 2.00g dextran are added in above liposome turbid liquor, after fully dissolving by the lyophilization in plastic culture dish of inclusion liposome suspension poured, lyophilisation condition is: pre-freeze temperature-60 ± 2 DEG C, adopt quick-freezing mode to carry out, pre-freeze time 6h, condenser temperature-50 ~-55 DEG C, vacuum < 100mT, freeze-drying time 24h, obtain ursolic acid hydroxypropyl-beta-cyclodextrin inclusion pro-liposome (as shown in Figure 2), when use, adding deionized water aquation rebuilds, light shaking is dissolved the inclusion liposome suspension completely obtaining after reconstruction, measuring envelop rate is 48.98%.
Embodiment 3: the preparation of oleanolic acid Benexate Hydrochloride pro-liposome
The preparation of oleanolic acid Benexate Hydrochloride: the beta-schardinger dextrin-that takes 4.80g, add the distilled water of 100ml that it is fully dissolved, separately get oleanolic acid 0.70g, after dissolving with 95% ethanol 20ml, slowly drip in above-mentioned beta-schardinger dextrin-aqueous solution, 60 DEG C are stirred after 4h the dispersion liquid of clathrate centrifugal 60min under 70000g, supernatant is the aqueous solution of ursolic acid Benexate Hydrochloride, by supernatant vacuum concentration, concentrated solution is in 40 DEG C, vacuum drying oven, the dry 6h of 0.08MPa obtains the ursolic acid Benexate Hydrochloride of white powder, reach 86.27% by measuring inclusion rate.
Content of oleanolic acid is measured: adopt high effective liquid chromatography for measuring ursolic acid, chromatographic condition C18 analytical column (250 × 3.0mm, 10 μ are m); Mobile phase is methanol-0.1% acetum (volume ratio is 88:12); Detecting wavelength is 215 nm; Flow rate of mobile phase is 1.0 ml/min.
10.50g Ovum Gallus domesticus Flavus lecithin is mixed and is dissolved in 50ml absolute ether with 3.50g cholesterol; Oleanolic acid Benexate Hydrochloride 4.00g is dissolved in the phosphate buffered solution of pH7.4 of 50ml 0.02M.After two solution are fully mixed, ultrasonic 5 min of water-bath, form stable w/o type emulsion, removal of solvent under reduced pressure in Rotary Evaporators afterwards, rotate to form after colloidal state, add the phosphate buffered solution of 50ml 0.02M pH7.4, the gel that rotating water is bonded on bottle wall comes off, obtain milky inclusion liposome suspension, this suspension is adopted to high pressure homogenizer homogenizing 5 times.
By the sorbitol of 4.00g, 4.00g trehalose, 2.00g xylitol adds in above liposome turbid liquor, after fully dissolving by the lyophilization in plastic culture dish of inclusion liposome suspension poured, lyophilisation condition is: pre-freeze temperature-60 ± 2 DEG C, adopt quick-freezing mode to carry out, pre-freeze time 6h, condenser temperature-50 ~-55 DEG C, vacuum < 100mT, freeze-drying time 24h, obtain oleanolic acid Benexate Hydrochloride pro-liposome (as shown in Figure 2), when use, adding deionized water aquation rebuilds, light shaking is dissolved the inclusion liposome suspension completely obtaining after reconstruction, measuring envelop rate is 52.49%.
Embodiment 4: the preparation of oleanolic acid hydroxypropyl-beta-cyclodextrin inclusion pro-liposome
The preparation of oleanolic acid hydroxypropyl-beta-cyclodextrin inclusion: the HP-β-CD that takes 4.00g, add the distilled water of 100ml that it is fully dissolved, separately get oleanolic acid 0.50g, after dissolving with 95% ethanol 20ml, slowly drip in above-mentioned HP-β-CD aqueous solution, 50 DEG C are stirred after 3h the dispersion liquid of clathrate centrifugal 60min under 70000g, supernatant is the aqueous solution of oleanolic acid HP-β-CD, by supernatant vacuum concentration, concentrated solution is in 40 DEG C, vacuum drying oven, the dry 6h of 0.08MPa obtains the oleanolic acid hydroxypropyl-beta-cyclodextrin inclusion of white powder, reach 82.34% by measuring inclusion rate.
9.00g dimyristoyl phosphatidyl choline is mixed and is dissolved in 50ml ether with 2.50g cholesterol; Oleanolic acid HP-β-CD 3.00g is dissolved in the phosphate buffered solution of pH 7.4 of 80ml 0.02M.After two solution are fully mixed, ultrasonic 5 min of water-bath, form stable w/o type emulsion, removal of solvent under reduced pressure in Rotary Evaporators afterwards, rotate to form after colloidal state, add the phosphate buffered solution of the pH7.4 of 80ml 0.02M, the gel that rotating water is bonded on bottle wall comes off, obtain milky inclusion liposome suspension, this suspension is adopted to high pressure homogenizer homogenizing 4 times.
By the mannitol of 8.00g, 1.00g trehalose, 1.00g cottonseed sugar adds in above liposome turbid liquor, after fully dissolving by the lyophilization in plastic culture dish of inclusion liposome suspension poured, lyophilisation condition is: pre-freeze temperature-60 ± 2 DEG C, adopt quick-freezing mode to carry out, pre-freeze time 6h, condenser temperature-50 ~-55 DEG C, vacuum < 100mT, freeze-drying time 24h, obtain oleanolic acid HP-β-CD pro-liposome (as shown in Figure 2), when use, adding deionized water aquation rebuilds, light shaking is dissolved the inclusion liposome suspension completely obtaining after reconstruction, measuring envelop rate is 50.16%.
Embodiment 5: the mensuration of pentacyclic triterpenoid cyclodextrin inclusion compound properties
1, the mensuration of dissolubility: get cyclodextrin clathrate and ursolic acid (oleanolic acid) prepared by above-described embodiment 1-4 appropriate, respectively at 25 ± 1 DEG C of magnetic agitation 5h, make water saturation solution, with micropore filter, (0.45 μ m) samples, with external standard two-point method mensuration ursolic acid (oleanolic acid) dissolubility, measurement result is that the dissolubility of ursolic acid in ursolic acid Benexate Hydrochloride is 32.82mg/100ml, in ursolic acid hydroxypropyl-beta-cyclodextrin inclusion, the dissolubility of ursolic acid is 39.36mg/100ml, and the dissolubility of ursolic acid is 2.54mg/100ml.In oleanolic acid Benexate Hydrochloride, the dissolubility of ursolic acid is 36.78mg/100ml, and in oleanolic acid hydroxypropyl-beta-cyclodextrin inclusion, the dissolubility of ursolic acid is 40.03mg/100ml, and the dissolubility of oleanolic acid is 2.86mg/100ml.
2, temperature stability experiment: by being placed in respectively at 4 DEG C, 25 DEG C, 45 DEG C after ursolic acid Benexate Hydrochloride pro-liposome aluminium foil bag vacuum sealed package, keep in Dark Place; In 60d, sample respectively every 10d, and measure the retention rate of ursolic acid.
Light stability experiment: will irradiate (intensity 300LX) under room temperature natural lighting condition and under high light with transparent bag vacuum packaging ursolic acid Benexate Hydrochloride pro-liposome, in 60d, sample respectively every 10d, and measure the retention rate of ursolic acid.
As shown in Figure 3, result shows, ursolic acid Benexate Hydrochloride pro-liposome is good in room temperature and lucifuge condition stability inferior.
3, pentacyclic triterpenoid cyclodextrin inclusion liposome transmission electron microscope and particle size distribution
Suitable concn is rebuild and be diluted to the cyclodextrin clathrate pro-liposome rehydration of with deionized water being prepared by embodiment 1-4, measures mean diameter and the distribution thereof of liposome with Ma Erwen dynamic light scattering (Zetasizernanos, Ma Erwen company of Britain).Adopt transmission electron microscope (JEM-2100, NEC company) to observe liposome form, see Fig. 4, Fig. 5.
Embodiment 6: the in-vitro percutaneous permeability evaluation of pentacyclic triterpenoid cyclodextrin inclusion liposome
Be that 5ml, pond mouth diffusion area are 0.785cm with receiving chamber volume
2franz transdermal diffusion instrument carry out transdermal test, by the skin corium of rat skin in vitro, towards receiving chamber, stratum corneum side is to supply chamber, controlling bath temperature is 37 ± 0.1 DEG C, rotor mixing speed is 600r.min
-1taking phosphate buffer (pH 7.4) as receiving liquid, add the cyclodextrin inclusion liposome solution of the embodiment 1-4 of 2ml rehydration reconstruction to supply chamber, respectively 0,0.25,0.5,1,1.5,2,3,4,6,8,10 and 12h get 2ml receiving liquid by sample tap, simultaneously supplement 2ml receiving liquid.Adopt HPLC to carry out ursolic acid (oleanolic acid) concentration determination in the receiving liquid sample of taking-up, when mensuration, breakdown of emulsion is measured after ursolic acid (oleanolic acid) coated in liposome is all discharged, and calculates its Steady penetration rate
js(μ gcm
-2h
-1).Compare with ursolic acid (oleanolic acid) the carbomer substrate hydrogel of same concentrations, repeat to test 5 times simultaneously.Result shows, the Steady penetration rate of cyclodextrin inclusion liposome is all greater than ursolic acid (oleanolic acid) hydrogel (P < 0.05), can promote significantly infiltration, is increased in the hold-up in skin.Specifically in table 1.
Embodiment 7: pentacyclic triterpenoid cyclodextrin inclusion liposome skin irritation is evaluated
Get 20 of Female rabbits, adopt shaver before experiment 24h by the processing of lose hair or feathers of family's rabbit back both sides, and with scraper by hair removal totally, unhairing place skin is completely exposed, unhairing scope left and right Ge Yue 3.0 × 5.0cm.Before coating, check that skin of unhairing is completely harmless.The left side evenly rehydration of coating 2ml embodiment 1-4 rebuilds liposome turbid liquor, and right side is taking ursolic acid (oleanolic acid) hydrogel 2ml as contrast, and left and right sides ursolic acid (oleanolic acid) concentration is identical.After coating, rabbit is put in rabbit box to ensure that be coated with application has enough time of staying on skin.After 4h, remove and be coated with application with warm water, perusal record coating part and have or not the situation such as erythema and edema after 30min, same steps continuously coating 7 days, last is removed and is coated with 24h after application, 48h, 72h is observed and recorded respectively.Result shows that the zest of cyclodextrin inclusion liposome is little compared with hydrogel zest, and concrete outcome is in table 2.
Embodiment 8: add the preparation of pentacyclic triterpenoid cyclodextrin inclusion liposome protective skin cream
Product is write out a prescription in table 3:
Adopt deionized water aquation to rebuild the clathrate pro-liposome in E component, slight concussion forms liposome turbid liquor.A and B component are heated to respectively to 75 ° of C.B is slowly added in A, stir 20min.When after AB emulsifying evenly, C is added, after high speed homogenization 5min, be cooled to 65 ° of C, middling speed stirs, cool to room temperature rapidly, keeps middling speed to stir 45 ° of C and adds D, and mastic can become smooth uniform state, in the time of 30 ° of C of temperature, add E component, discharging after stirring at low speed 10min.
Embodiment 9: add the preparation of pentacyclic triterpenoid cyclodextrin inclusion liposome skin-care gel
Product is write out a prescription in table 4:
Adopt deionized water aquation to rebuild the clathrate pro-liposome in E component, slight concussion forms liposome turbid liquor.A stirs mutually and is heated to 40 DEG C, after B heat phase to 40 DEG C, slowly adds A phase, stirs 10min, adds C to stir afterwards mutually 30min, adds afterwards D to stir mutually 10min, is cooled to 30 DEG C and adds the E discharging after stirring at low speed 10min of lowering the temperature mutually.
Claims (9)
1. a preparation method for pentacyclic triterpenoid cyclodextrin clathrate pro-liposome, is characterized in that comprising the following steps:
(1) pentacyclic triterpenoid is dissolved in ethanol or 95% ethanol, cyclodextrin is water-soluble, the alcoholic solution of pentacyclic triterpenoid is added in cyclodextrin aqueous solution and stirred, through centrifuging and taking supernatant, vacuum concentration, the dry pentacyclic triterpene cyclodextrin clathrate that to obtain;
(2) phospholipid material and cholesterol are dissolved in ether, pentacyclic triterpene cyclodextrin clathrate is dissolved in the phosphate buffered solution of pH 6～8, ultrasonic after two solution are mixed, evaporation is removed ether and is formed colloidal solution, adds the phosphate buffered solution of pH 6～8 to obtain inclusion liposome suspension;
(3) freeze drying protectant is added in liposome turbid liquor, dissolves postlyophilization and obtain pentacyclic triterpenoid cyclodextrin clathrate pro-liposome,
Each component and content thereof are:
Pentacyclic triterpenoid 2-50 part
Cyclodextrin 30-100 part
Phospholipid material 100-220 part
Cholesterol 20-70 part
Freeze drying protectant 40-155 part.
2. the preparation method of pentacyclic triterpenoid cyclodextrin clathrate pro-liposome according to claim 1, is characterized in that: pentacyclic triterpenoid is selected from one or more in ursolic acid, oleanolic acid, Betulinic Acid, beta boswellic acid.
3. the preparation method of pentacyclic triterpenoid cyclodextrin clathrate pro-liposome according to claim 1, is characterized in that: described cyclodextrin is beta-schardinger dextrin-or HP-β-CD.
4. the preparation method of pentacyclic triterpenoid cyclodextrin clathrate pro-liposome according to claim 1, it is characterized in that, described phospholipid material is selected from one or more in soybean lecithin, Ovum Gallus domesticus Flavus lecithin, hydrogenation egg yolk lecithin, hydrogenated soya phosphatide, dimyristoyl phosphatidyl choline, two Semen Myristicae phosphatidyl glycerols, dipalmitoyl phosphatidyl choline, two Petiolus Trachycarpi phosphatidyl glycerols, distearoyl phosphatidylcholine, distearyl phosphatidyl glycerol.
5. the preparation method of pentacyclic triterpenoid cyclodextrin clathrate pro-liposome according to claim 1; it is characterized in that, described freeze drying protectant is selected from one or more in trehalose, mannitol, sorbitol, xylitol, cottonseed sugar, dextran, lactose.
6. the preparation method of pentacyclic triterpenoid cyclodextrin clathrate pro-liposome according to claim 1, is characterized in that: it is 80-600nm that cyclodextrin clathrate pro-liposome rehydration is rebuild rear particle diameter.
7. the pentacyclic triterpenoid cyclodextrin clathrate pro-liposome that described in claim 1, preparation method obtains is in the application of preparing in external preparation for skin cosmetics.
8. application according to claim 7, is added in cosmetics and makes skin emulsion, nourishing cream, and massage cream, facial gel, eye cream, eye essence, eye gel, cleansing cream, foundation cream, health frost, body gels, addition is 0.1 ~ 10%.
9. application according to claim 8, is added into and in cosmetics, makes facial milk cleanser or body lotion.
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| CN104306269B (en) * | 2014-11-04 | 2017-02-01 | 珀莱雅化妆品股份有限公司 | Preparation method of cyclodextrin inclusion compound liposome with whitening effect |
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| CN104826123B (en) * | 2015-04-10 | 2017-11-10 | 昆明理工大学 | A kind of inclusion compound of oleanolic acid and amine cyclodextrin |
| CN104857003A (en) * | 2015-04-10 | 2015-08-26 | 昆明理工大学 | Ursolic acid and amine cyclodextrin clathrate compound |
| CN107789207A (en) * | 2017-11-03 | 2018-03-13 | 烟台康达尔药业有限公司 | A kind of EGF/ vitamin e acetates cyclodextrin liposome and preparation method thereof, application method, application |
| CN109157515B (en) * | 2018-09-05 | 2020-11-03 | 辽宁万嘉医药科技有限公司 | Coenzyme Q10Clathrate self-assembly liposome precursor and preparation method thereof |
| CN109260057A (en) * | 2018-11-08 | 2019-01-25 | 妃瑶生物科技（上海）有限公司 | A kind of extracting method of prinsepia utilis royle oil composition |
| CN109106657A (en) * | 2018-11-08 | 2019-01-01 | 妃瑶生物科技（上海）有限公司 | A kind of prinsepia utilis royle oil liposome and preparation method thereof |
| JP7156931B2 (en) * | 2018-12-18 | 2022-10-19 | 株式会社ファンケル | Ursolic acid-containing powder composition |
| CN112168785B (en) * | 2019-07-03 | 2022-08-30 | 上海交通大学 | Ursolic acid liposome preparation, and preparation method and application thereof |
| CN110559688A (en) * | 2019-08-30 | 2019-12-13 | 浙江工业大学 | Method for separating isomers of oleanolic acid and ursolic acid |
| CN114223896A (en) * | 2021-11-18 | 2022-03-25 | 西北农林科技大学 | Red date triterpenic acid chewable tablet and preparation method thereof |
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