CN110511169A - The method for preparing Determination of Astaxanthin in Haematococcus Pluvialis based on Transdermal absorption - Google Patents
The method for preparing Determination of Astaxanthin in Haematococcus Pluvialis based on Transdermal absorption Download PDFInfo
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- CN110511169A CN110511169A CN201910702974.7A CN201910702974A CN110511169A CN 110511169 A CN110511169 A CN 110511169A CN 201910702974 A CN201910702974 A CN 201910702974A CN 110511169 A CN110511169 A CN 110511169A
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- transdermal
- physiological saline
- ethyl alcohol
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- 238000000034 method Methods 0.000 title claims abstract description 44
- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 title claims abstract description 37
- MQZIGYBFDRPAKN-ZWAPEEGVSA-N astaxanthin Chemical compound C([C@H](O)C(=O)C=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-ZWAPEEGVSA-N 0.000 title claims abstract description 37
- 229940022405 astaxanthin Drugs 0.000 title claims abstract description 37
- 235000013793 astaxanthin Nutrition 0.000 title claims abstract description 37
- 239000001168 astaxanthin Substances 0.000 title claims abstract description 37
- 238000010521 absorption reaction Methods 0.000 title claims abstract description 34
- 241000168517 Haematococcus lacustris Species 0.000 title claims abstract description 26
- 239000002504 physiological saline solution Substances 0.000 claims abstract description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000007788 liquid Substances 0.000 claims abstract description 24
- 238000000605 extraction Methods 0.000 claims abstract description 23
- 239000011347 resin Substances 0.000 claims abstract description 22
- 229920005989 resin Polymers 0.000 claims abstract description 22
- 238000002425 crystallisation Methods 0.000 claims abstract description 17
- 230000008025 crystallization Effects 0.000 claims abstract description 17
- 238000012360 testing method Methods 0.000 claims abstract description 17
- 238000002137 ultrasound extraction Methods 0.000 claims abstract description 17
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims abstract description 15
- 239000000243 solution Substances 0.000 claims abstract description 14
- 238000001179 sorption measurement Methods 0.000 claims abstract description 14
- 238000000338 in vitro Methods 0.000 claims abstract description 12
- 239000012466 permeate Substances 0.000 claims abstract description 11
- 239000004615 ingredient Substances 0.000 claims abstract description 10
- 239000000047 product Substances 0.000 claims abstract description 9
- 238000007654 immersion Methods 0.000 claims abstract description 8
- 230000004888 barrier function Effects 0.000 claims abstract description 7
- 239000003480 eluent Substances 0.000 claims abstract description 6
- 239000012046 mixed solvent Substances 0.000 claims abstract description 6
- 230000007935 neutral effect Effects 0.000 claims abstract description 4
- 238000005406 washing Methods 0.000 claims abstract description 4
- 238000001514 detection method Methods 0.000 claims description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 2
- 230000001502 supplementing effect Effects 0.000 claims 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- 235000019441 ethanol Nutrition 0.000 description 8
- 239000002904 solvent Substances 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000003960 organic solvent Substances 0.000 description 6
- 230000008499 blood brain barrier function Effects 0.000 description 5
- 210000001218 blood-brain barrier Anatomy 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 230000035515 penetration Effects 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000003814 drug Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 231100000252 nontoxic Toxicity 0.000 description 4
- 230000003000 nontoxic effect Effects 0.000 description 4
- 238000000638 solvent extraction Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 239000000823 artificial membrane Substances 0.000 description 3
- 239000002537 cosmetic Substances 0.000 description 3
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- 238000004519 manufacturing process Methods 0.000 description 3
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- 239000000592 Artificial Cell Substances 0.000 description 2
- 241000195493 Cryptophyta Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
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- 229920000289 Polyquaternium Polymers 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
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- 238000003556 assay Methods 0.000 description 2
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- 238000001764 infiltration Methods 0.000 description 2
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- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
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- 230000001093 anti-cancer Effects 0.000 description 1
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- 239000006227 byproduct Substances 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
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- 208000014674 injury Diseases 0.000 description 1
- -1 keto-acid carotenoids Chemical class 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C403/00—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone
- C07C403/24—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone having side-chains substituted by six-membered non-aromatic rings, e.g. beta-carotene
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/08—Investigating permeability, pore-volume, or surface area of porous materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
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- Chemical & Material Sciences (AREA)
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- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Dispersion Chemistry (AREA)
- Medicinal Preparation (AREA)
Abstract
The present invention provides a kind of method for preparing based on Transdermal absorption and evaluating Determination of Astaxanthin in Haematococcus Pluvialis, method includes the following steps: the extraction process of (1) haematococcus pluvialis carries out ultrasonic extraction to haematococcus pluvialis using the organic mixed solvent of n-hexane-ethyl alcohol；(2) macroporous resin adsorption is added in said extracted liquid by the resin adsorption of product, and after absorption, washing is neutral, then is eluted with ethanol solution, and eluent is recovered under reduced pressure and places crystallization, filters out to obtain coarse crystallization；(3) the artificial biological film human epidermal skin of the Transdermal absorption of sample, then in physiological saline after immersion treatment, as transdermal barrier, using coarse crystallization as transdermal release liquid, using ethyl alcohol-physiological saline as Transdermal absorption liquid, skin permeation test in vitro is carried out under conditions of Yu Hengwen constant speed, appropriate permeate is pipetted after certain interval of time, it is supplemented ethyl alcohol-physiological saline of same volume, isolates the ingredient being easily absorbed by the skin.
Description
Technical field
The invention belongs to the fields such as biological medicine, health food, cosmetics.More particularly to be related to based on Transdermal absorption prepare
And the method for evaluation Determination of Astaxanthin in Haematococcus Pluvialis
Background technique
Astaxanthin (3,3 '-carrotene -4 dihydroxy-β, β ' -, 4 '-diketone) is a kind of orange-red keto-acid carotenoids
Element is widely present in shrimp, crab, ornamental fish, flamingo and certain algae, mushroom etc..Conjugated diene and insatiable hunger in molecular structure
With the presence of carbonyl so that astaxanthin inoxidizability with super strength, in addition to this, astaxanthin also show important biological function
It can be as slow such as antitumor, anti-inflammatory, whitening sun protection, antiatherosclerosis, strengthen immunity, reduction ultraviolet injury, anti-cancer, reduction
Property disease etc..Therefore astaxanthin is widely used in the industries such as medicine, food, cosmetics.Currently, the production technology packet of astaxanthin
Include biology preparation and chemical synthesis, due to chemically synthesized astaxanthin structure, function, in terms of be inferior to
Biology prepares astaxanthin, while natural astaxanthin is the carotenoid that unique one kind can pass through blood-brain barrier.
Therefore, the PRODUCTION TRAITS of natural astaxanthin is always an important field.The biological source of natural astaxanthin has
Three kinds: waste, phaffia rhodozyma and the haematococcus pluvialis of processing of aquatic products.Wherein, content astaxanthin in the waste of processing of aquatic products
Low, extraction cost is high, is not suitable for large-scale production.Phaffia rhodozyma conduct, one of source of natural astaxanthin are green safe, raw
Long period is short, but production technology is more complex and the average accumulation content of astaxanthin is lower than haematococcus pluvialis, therefore rain life is red
Ball algae becomes the Excellent sources of natural astaxanthin as the astaxanthin being currently known, the highest species of accumulation.
The extracting method of current Astaxanthin In Haematococcus Pluvialis is mainly supercritical CO2It extracts and solvent extracts two classes.It is super
Critical CO2The activity that can preferably keep astaxanthin is extracted, but equipment is expensive, and operating cost is higher.In contrast, You Jirong
Agent extraction method is simple, easily operated amplification, later separation technology maturation, at low cost.Methylene chloride, ethyl acetate, acetone etc. are equal
It can be used for extracting the astaxanthin in haematococcus pluvialis, however these solvents more or less have certain toxicity.Select one kind
Less toxic, efficient solvent is a critical bottleneck problem of extract by solvents astaxanthin.
The features such as organic solvent extraction has treating capacity big, and low energy consumption, and speed is fast, simple to equipment requirement, and be easy to
Realize continuous operation and automation control.The more other methods of astaxanthin are extracted using organic solvent method to be more conducive to realize industrial metaplasia
It produces.
But whether cosmetics or the transdermal arrival human body environment of food must play the role of expected and effect, these are all
It is unknown.
Summary of the invention
For to the extraction process of haematococcus pluvialis, directlying adopt organic solvent extraction at present and extract, then carry out
Resin adsorption.The present invention establishes a kind of the new evaluating method of measurement sample transdermal absorption factor based on skin surface, in conjunction with tradition
Extraction process simulates human body blood-brain barrier, isolates easy absorbed ingredient, so that it is stronger to establish a kind of biological relevance,
Horizontal, the more scientific effective extracting method closer to animal experiment.
The present invention carries out Transdermal absorption experiment by simulation human epidermal skin in vitro, is easily absorbed by the skin with isolating
Ingredient, to carry out relevant detection to the content astaxanthin of sample.Specifically, the present invention is in extraction using most simple
The features such as general organic solvent extraction, this method is practical, simple to equipment requirement, and be easily achieved continuous operation and
Automation control；
Present invention firstly provides in traditional extraction technique and then further transdermal by in-vitro simulated human epidermal skin
Absorption experiment isolates the ingredient being easily absorbed by the skin, then carries out Structural Identification and assay to it.It is selected in extraction process
Selected the organic solvent extraction and resin adsorption method of simple general-purpose, this method is practical, it is simple to equipment requirement the features such as,
And it is easily achieved continuous operation and automation control.
The object of the invention is achieved through the following technical solutions:
The method for being prepared based on Transdermal absorption and evaluating Determination of Astaxanthin in Haematococcus Pluvialis, method includes the following steps:
(1) extraction process of haematococcus pluvialis
Ultrasonic extraction is carried out to haematococcus pluvialis using the organic mixed solvent of n-hexane-ethyl alcohol；
(2) resin adsorption of product
Macroporous resin adsorption is added in said extracted liquid, after absorption, washing is neutral, then is eluted with ethanol solution, subtracts
It pushes back and receives eluent placement crystallization, filter out to obtain coarse crystallization；
(3) Transdermal absorption of sample
With artificial biological film human epidermal skin, then in physiological saline after immersion treatment, as transdermal barrier,
Using coarse crystallization as transdermal release liquid, using ethyl alcohol-physiological saline as Transdermal absorption liquid, transdermal suction is carried out under conditions of Yu Hengwen constant speed
Acceptance test pipettes appropriate permeate after certain interval of time, is supplemented ethyl alcohol-physiological saline of same volume, isolates easily quilt
The ingredient that skin absorbs.
In some embodiments of the present invention, the volume ratio of step (1) n-hexane-ethyl alcohol is 1:1
In some embodiments of the present invention, step (1) solid-liquid ratio is 1:600 (g/m L).
In some embodiments of the present invention, step (1) Extracting temperature is 50 DEG C.
In some embodiments of the present invention, step (1) extraction time is 40 minutes.
In some embodiments of the present invention, step (3) the artificial biological film pretreatment refers to biomembrane with scissors
It is cut into 5cm*5cm size, then the immersion treatment 30min in physiological saline.
In some embodiments of the present invention, step (3) the Transdermal absorption instrument constant temperature constant speed refer to constant temperature in 37 DEG C, it is permanent
Skin permeation test in vitro is carried out under conditions of fast 3800r/min.
In some embodiments of the present invention, step (3) is described separately sampled, and supplements after sampling same amount of transdermal
Absorbing liquid refer to 1h, 2h, 4h, 8h, 12h, for 24 hours, each sample point of 36h, 48h with clean syringe pipette 2mL permeate respectively, then
2mL30% ethyl alcohol-physiological saline is supplemented with clean syringe.
In some embodiments of the present invention, the permeate that step (3) is isolated is carried out using high performance liquid chromatography
Structural Identification and content detection.
It is highly preferred that the present invention uses following specific method, the extraction process of haematococcus pluvialis: (1) using safe and non-toxic
1:1 n-hexane-organic mixed solvent of ethyl alcohol, in certain solid-liquid ratio 1:600 (g/mL), 50 DEG C of Extracting temperature, extraction time
Ultrasonic extraction was carried out to haematococcus pluvialis in 40 minutes；(2) said extracted liquid, gradient are adjusted PH by the resin adsorption of product, are added
Macroporous resin adsorption, after absorption, washing is neutral, then is eluted with 6-8BV ethanol solution, and eluent is recovered under reduced pressure and places knot
Crystalline substance filters out to obtain coarse crystallization；(3) Transdermal absorption of sample tests artificial biological film human epidermal skin, with scissors by people
It makes biomembrane and is cut into 5cm*5cm size, then in physiological saline after immersion treatment 30min, as transdermal barrier, with coarse crystallization
For transdermal release liquid, using 30% ethyl alcohol-physiological saline as Transdermal absorption liquid, 37 DEG C of Yu Hengwen, under conditions of constant speed 3800r/min
Carry out skin permeation test in vitro.
In particular embodiments of the invention, using step is implemented as follows:
(1) extraction process of haematococcus pluvialis:
Using safe and non-toxic 1:1 n-hexane-organic mixed solvent of ethyl alcohol, in solid-liquid ratio 1:600 (g/mL), Extracting temperature
50 DEG C, extraction time 40 minutes to haematococcus pluvialis ultrasonic extraction；
(2) resin adsorption of product:
By macroporous resin adsorption in acquired solution, model DA201, the ratio of height to diameter of resin column is 8:1~15:1；In room temperature
Under normal pressure, with 1BV/h flow velocity, first with distillation water elution, elution volume 3-5BV；Again with the ethanol solution of 75%-95% in 1-
3BV/h flow velocity, elution volume are to be eluted under 6-8BV, and eluent is recovered under reduced pressure and places crystallization, filters out to obtain coarse crystallization；
(3) skin permeation test in vitro
Test material:
Artificial membrane is bought from Guangzhou Bao Long International Trading Company Ltd, model polyquaternium -51Lipidure PMB
Artificial cell membrane；
30% ethyl alcohol-physiological saline；Physiological saline.
Test method:
With artificial biological film human epidermal skin, artificial biological film is cut into 5cm*5cm size with scissors, then in
In physiological saline after immersion treatment 30min, as transdermal barrier, Examples 1 to 9 and reference examples 1, reference examples 2 are obtained slightly
Crystallization is dissolved in after physiological saline as transdermal release liquid, using 30% ethyl alcohol-physiological saline as Transdermal absorption liquid, 37 DEG C of Yu Hengwen,
Skin permeation test in vitro is carried out under conditions of constant speed 3800r/min.1h, 2h, 4h, 8h, 12h, for 24 hours, each sample point of 36h, 48h uses
Clean syringe pipettes 2mL permeate respectively, then supplements 2mL30% ethyl alcohol-physiological saline with clean syringe.
Each sample point solution detects absorbance at ultraviolet specrophotometer 475nm, according to standard curve and normalized form
The transdermal concentration and the transdermal amount Q value of corresponding unit area average accumulated for calculating each sample point sample will accumulate infiltration capacity Q clock synchronization
Between t returned, the slope of gained linear equation is the Steady penetration rate J (mgcm of drug under this condition2·h-1)；
(4) Structural Identification and content detection of sample
Structural Identification is carried out to permeate using performance liquid chromatographic column method, C18 chromatography post separation, mobile phase is methanol
: water (98: 2, v/v)；Structure is C40H52O4.Ultraviolet detection wavelength 475nm, quantified by external standard method, the yield of astaxanthin are pure up to 95%
It spends up to 90.7%.
Compared with the existing technology, the invention has the advantages that and effect:
(1) present invention carries out Transdermal absorption experiment using with artificial biological film blood-brain barrier after extraction process,
The new evaluating method for establishing a kind of measurement sample transdermal absorption factor based on artificial membrane, isolates and is easily absorbed by blood-brain barrier
Ingredient, so that it is stronger to establish a kind of biological relevance, more scientific effective preparative separation side horizontal closer to animal experiment
Method.
(2) present invention is first separated using Transdermal absorption on Structural Identification and assay, then passes through high performance liquid chromatography
Separation detection, to prepare the astaxanthin of the haematococcus pluvialis of the high-content easily absorbed by blood-brain barrier.
(3) operation of the present invention is easy, is not necessarily to expensive device, and belong to green in whole preparation process without toxic organic solvents
Color environment-protective process；The yield of astaxanthin is high during the preparation process, and by-product is few, can be applied to industrialized large-scale production.
Specific embodiment:
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment
Only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
Astaxanthin method is extracted from haematococcus pluvialis:
(1) extraction process of haematococcus pluvialis:
Using safe and non-toxic 1:1 n-hexane-organic mixed solvent of ethyl alcohol, in solid-liquid ratio 1:600 (g/mL), Extracting temperature
50 DEG C, extraction time 40 minutes to haematococcus pluvialis ultrasonic extraction；
(2) resin adsorption of product:
By macroporous resin adsorption in acquired solution, model DA201, the ratio of height to diameter of resin column is 10:1；In normal temperature and pressure
Under, with 1BV/h flow velocity, first with distillation water elution, elution volume 3BV；Again with 75% ethanol solution in 3BV/h flow velocity, wash
Lift-off product is to be eluted under 6BV, and eluent is recovered under reduced pressure and places crystallization, filters out to obtain coarse crystallization；
(3) skin permeation test in vitro
Test material:
Artificial membrane is bought from Guangzhou Bao Long International Trading Company Ltd, model polyquaternium -51Lipidure PMB
Artificial cell membrane；
30% ethyl alcohol-physiological saline；Physiological saline.
Test method:
With artificial biological film human epidermal skin, artificial biological film is cut into 5cm*5cm size with scissors, then in
In physiological saline after immersion treatment 30min, as transdermal barrier, coarse crystallization will be obtained and be dissolved in after physiological saline as transdermal release
Liquid 37 DEG C of Yu Hengwen, carries out transdermal suction under conditions of constant speed 3800r/min using 30% ethyl alcohol-physiological saline as Transdermal absorption liquid
Acceptance test.1h, 2h, 4h, 8h, 12h, for 24 hours, each sample point of 36h, 48h with clean syringe pipettes 2mL permeate respectively, then uses
Clean syringe supplements 2mL30% ethyl alcohol-physiological saline；
Each sample point solution detects absorbance at ultraviolet specrophotometer 475nm, according to standard curve and normalized form
The transdermal concentration and the transdermal amount Q value of corresponding unit area average accumulated for calculating each sample point sample will accumulate infiltration capacity Q clock synchronization
Between t returned, the slope of gained linear equation is the Steady penetration rate J (mgcm of drug under this condition2·h-1)；
(4) Structural Identification and content detection of sample
Structural Identification is carried out to permeate using performance liquid chromatographic column method, C18 chromatography post separation, mobile phase is methanol
: water (98: 2, v/v)；Structure is C40H52O4.Ultraviolet detection wavelength 475nm, quantified by external standard method, the yield of astaxanthin are pure up to 95%
It spends up to 90.7%.
Embodiment 2
The extracting method of the present embodiment is substantially the same manner as Example 1, and difference is that the Extracting temperature of step (1) is 60 DEG C.
Embodiment 3
The extracting method of the present embodiment is substantially the same manner as Example 1, and difference is that the Extracting temperature of step (1) is 70 DEG C.
Embodiment 4
The extracting method of the present embodiment is substantially the same manner as Example 1, and difference is that the Extracting temperature of step (1) is 40 DEG C.
Embodiment 5
The extracting method of the present embodiment is substantially the same manner as Example 1, and difference is that the Extracting temperature of step (1) is 30 DEG C.
Embodiment 6
The extracting method of the present embodiment is substantially the same manner as Example 2, and difference is the macroreticular resin DA201 of step (2)
Replace with D101.
Embodiment 7
The extracting method of the present embodiment is substantially the same manner as Example 3, and difference is the macroreticular resin DA201 of step (2)
Replace with D101.
Embodiment 8
The extracting method of the present embodiment is substantially the same manner as Example 4, and difference is the macroreticular resin DA201 of step (2)
Replace with D101.
Embodiment 9
The extracting method of the present embodiment is substantially the same manner as Example 5, and difference is the macroreticular resin DA201 of step (2)
Replace with D101.
Reference examples 1
The extracting method of the present embodiment is substantially the same manner as Example 1, and difference is that the Extraction solvent by step (1) replaces with
Methylene chloride.
Reference examples 2
The extracting method of the present embodiment is substantially the same manner as Example 1, and difference is that the ultrasonic extraction by step (1) replaces with
Stirring is extracted.
Test example
The Steady penetration rate for the Transdermal absorption ingredient that testing example 1~9 and reference examples 1, reference examples 2 obtain, in detail
It is shown in Table 1.
Table 1
| Sample | Extraction solvent | Extracting temperature | Adsorb resin | It extracts | J value |
| Embodiment 1 | N-hexane-ethyl alcohol | 50℃ | DA201 | Ultrasonic extraction | 0.1005 |
| Embodiment 2 | N-hexane-ethyl alcohol | 60℃ | DA201 | Ultrasonic extraction | 0.0998 |
| Embodiment 3 | N-hexane-ethyl alcohol | 70℃ | DA201 | Ultrasonic extraction | 0.0829 |
| Embodiment 4 | N-hexane-ethyl alcohol | 40℃ | DA201 | Ultrasonic extraction | 0.0893 |
| Embodiment 5 | N-hexane-ethyl alcohol | 30℃ | DA201 | Ultrasonic extraction | 0.0765 |
| Embodiment 6 | N-hexane-ethyl alcohol | 60℃ | D101 | Ultrasonic extraction | 0.0888 |
| Embodiment 7 | N-hexane-ethyl alcohol | 70℃ | D101 | Ultrasonic extraction | 0.0842 |
| Embodiment 8 | N-hexane-ethyl alcohol | 40℃ | D101 | Ultrasonic extraction | 0.0739 |
| Embodiment 9 | N-hexane-ethyl alcohol | 30℃ | D101 | Ultrasonic extraction | 0.0819 |
| Reference examples 1 | Methylene chloride | 50℃ | DA201 | Ultrasonic extraction | 0.0789 |
| Reference examples 2 | N-hexane-ethyl alcohol | 50℃ | DA201 | Stirring replaces | 0.0715 |
From the above, it can be seen that being tested by in-vitro simulated human epidermal skin Transdermal absorption, using safe and non-toxic molten
Agent n-hexane and ethyl alcohol are as Extraction solvent compared to methylene chloride Extraction solvent, and Steady penetration rate is advantageously；
Secondly, we can be found that Extracting temperature is 50 DEG C by testing above, when absorption resin is DA201, obtain
Astaxanthin Steady penetration rate advantageously.
Based on the absorption ingredient that the above skin permeation test in vitro obtains, the yield and purity of astaxanthin are measured, such as 2 institute of table
Show,
Table 2
| Sample | Yield | Purity |
| Embodiment 1 | 95% | 90.7% |
| Embodiment 2 | 88% | 88.6% |
| Embodiment 3 | 80% | 83.9% |
| Embodiment 4 | 88% | 87.3% |
| Embodiment 5 | 85% | 82.6% |
| Embodiment 6 | 83% | 86.2% |
| Embodiment 7 | 78% | 81.9% |
| Embodiment 8 | 85% | 89.7% |
| Embodiment 9 | 81% | 85.2% |
| Reference examples 1 | 78% | 80.9% |
| Reference examples 2 | 73% | 85.1% |
Structural content and purity detecting after Transdermal absorption, it is found that the yield of the acquisition astaxanthin of embodiment 1
Highest, and its purity has reached 90.7%；And since the ingredient origin is via Transdermal absorption, obtain herein easily by blood
The astaxanthin of the haematococcus pluvialis for the high-content that brain barrier absorbs.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (10)
1. the method for preparing Determination of Astaxanthin in Haematococcus Pluvialis based on Transdermal absorption, it is characterised in that the following steps are included:
(1) extraction process of haematococcus pluvialis
Ultrasonic extraction is carried out to haematococcus pluvialis using n-hexane-alcohol mixed solvent；
(2) resin adsorption of product
Macroporous resin adsorption is added in the extracting solution that step (1) is obtained, and after absorption, washing is neutral, then is washed with ethanol solution
It is de-, eluent is recovered under reduced pressure and places crystallization, filters out to obtain coarse crystallization；
(3) Transdermal absorption of sample
By artificial biological film mould in physiological saline after immersion treatment, as transdermal barrier, using coarse crystallization as transdermal release liquid, with
Ethyl alcohol-physiological saline is Transdermal absorption liquid, carries out skin permeation test in vitro under conditions of Yu Hengwen constant speed, certain interval of time moves back
Appropriate permeate is taken, ethyl alcohol-physiological saline of same volume is supplemented, to isolate the ingredient being easily absorbed by the skin.
2. the method according to claim 1, wherein the volume ratio of step (1) n-hexane-ethyl alcohol is 1:1.
3. the method according to claim 1, wherein step (1) solid-liquid ratio is 1:600 (g/mL).
4. the method according to claim 1, wherein step (1) Extracting temperature is 50 DEG C.
5. the method according to claim 1, wherein step (1) extraction time is 40 minutes.
6. the method according to claim 1, wherein step (3) artificial biological film pretreatment refers to cutting
Biomembrane is cut into 5cm*5cm size by knife, then the immersion treatment 30min in physiological saline.
7. extracting method according to claim 1, which is characterized in that step (3) the Transdermal absorption instrument constant temperature constant speed is
Refer to that constant temperature carries out skin permeation test in vitro under conditions of 37 DEG C, constant speed 3800r/min.
8. the method according to claim 1, wherein step (3) is described separately sampled, and supplementing phase after sampling
The Transdermal absorption liquid of same amount refer to 1h, 2h, 4h, 8h, 12h, for 24 hours, each sample point of 36h, 48h pipettes with clean syringe respectively
2mL permeate, then 2mL30% ethyl alcohol-physiological saline is supplemented with clean syringe.
9. the method according to claim 1, wherein being isolated to step (3) using high performance liquid chromatography
Permeate carries out Structural Identification and content detection.
10. the astaxanthin that method described in any one obtains according to claim 1~9.
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| CN111714537A (en) * | 2020-08-10 | 2020-09-29 | 黑龙江天龙药业有限公司 | Film agent capable of relieving chest drop and atrophy and preparation method thereof |
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