阅读说明：本技术 基于透皮吸收制备雨生红球藻中虾青素的方法 (The method for preparing Determination of Astaxanthin in Haematococcus Pluvialis based on Transdermal absorption ) 是由 王平 刘玲 于 2019-07-31 设计创作，主要内容包括：本发明提供一种基于透皮吸收制备以及评价雨生红球藻中虾青素的方法,该方法包括以下步骤：(1)雨生红球藻的提取工艺采用正己烷-乙醇有机混合溶剂对雨生红球藻进行超声提取；(2)产物的树脂吸附将上述提取液加入大孔树脂吸附,吸附结束后,水洗中性,再用乙醇溶液洗脱,减压回收洗脱液放置结晶,滤出得粗结晶；(3)样品的透皮吸收用人造生物膜模拟人体表皮皮肤,然后于生理盐水中浸泡处理后,作为透皮屏障,以粗结晶为透皮释放液,以乙醇-生理盐水为透皮吸收液,于恒温恒速的条件下进行透皮吸收试验,间隔一段时间后移取适量透过液,再补充相同体积的乙醇-生理盐水,分离出易被皮肤吸收的成分。(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.)

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.

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.