CN107032994A - A kind of extraction process of Rosmarinic acid - Google Patents
A kind of extraction process of Rosmarinic acid Download PDFInfo
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- CN107032994A CN107032994A CN201710245446.4A CN201710245446A CN107032994A CN 107032994 A CN107032994 A CN 107032994A CN 201710245446 A CN201710245446 A CN 201710245446A CN 107032994 A CN107032994 A CN 107032994A
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- rosmarinic acid
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- crude extract
- rosemary
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/37—Esters of carboxylic acids
- A61K8/375—Esters of carboxylic acids the alcohol moiety containing more than one hydroxy group
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
Abstract
The invention belongs to technical field of natural product extraction, a kind of extraction process of Rosmarinic acid is specifically disclosed, is comprised the following steps：S1：After rosemary is beaten successively, dried, crushing, Rosmarinic acid crude extract is extracted to obtain；S2：Rosmarinic acid crude extract obtained by S1 is filtered, Rosmarinic acid crude extract is concentrated to give；S3：Rosmarinic acid crude extract is purified using the gel columns of Sephadex LH 20, rosmarinic acid extract is obtained；Dried in S1 as freeze-drying, temperature 70 C~80 DEG C of freeze-drying, cooling time are that 40h~60h, vacuum pressure are 10Pa~30Pa.The extraction process that the present invention is provided has higher recovery rate, and active constituent content height, the impurity extracted in obtained rosmarinic acid extract is few, with preferable antioxidation activity.
Description
Technical field
The invention belongs to technical field of natural product extraction, more particularly, to a kind of extraction process of Rosmarinic acid.
Background technology
Rosemary, Classification system (Rosmarinus officinalis) Labiatae shrub.
Property happiness temperature climate, originate in the northern Mediterranean of European Region and Africa.In Caowei Kingdom just once introduces a fine variety in period
State.
Antioxidant and Rosemary Oil with excellent anti-oxidation characteristics can be extracted from the flower and leaf of rosemary.Fan changes
Fragrant antioxidant is widely used in medicine, fried food, rich oil food and the fresh-keeping of each quasi-grease and guaranteed the quality；And rosemary essence is then used
In spices, air freshener, drive the daily-use chemical industry industry such as ant agent and sterilization, desinsection.
The extraction process of current Rosmarinic acid mainly uses macroreticular resin initial gross separation method of purification and solvent extraction method.Macropore
Time-consuming and recovery rate is low for resin initial gross separation method of purification, and miscellaneous in the substantial amounts of solvent of solvent extraction rule needs and extract
Matter content is higher；In addition, the active constituent content that existing extraction process is extracted in obtained rosmarinic acid extract is relatively low.Fan
Repeatedly fragrant extract has stronger astriction, nurses one's health greasy skin, stimulates circulation, and stimulates hair regeneration.Improve alopecia
Phenomenon.Therefore the research of the extraction technology of preparing of Rosmarinic acid is significant to fully exploitation rosemary resource.
For the deficiency of existing Rosmarinic acid extraction process, the present invention is intended to provide a kind of recovery rate is high, it is extract obtained
The extraction process for the Rosmarinic acid that middle active constituent content is high, impurity is few.
The content of the invention
It is an object of the invention to according to deficiency of the prior art, there is provided a kind of extraction process of Rosmarinic acid.
Another object of the present invention is to extract the Rosmarinic acid prepared by said extracted technique.
The purpose of the present invention is achieved through the following technical solutions：
A kind of extraction process of Rosmarinic acid, comprises the following steps：
S1：After rosemary is beaten successively, dried, crushing, Rosmarinic acid crude extract is extracted to obtain；
S2：Rosmarinic acid crude extract obtained by S1 is filtered, Rosmarinic acid crude extract is concentrated to give；
S3：Rosmarinic acid crude extract is purified using Sephadex LH-20 gel columns, rosmarinic acid extract is obtained.
Wherein, dried in S1 as freeze-drying, -70 DEG C~-80 DEG C of the temperature of freeze-drying, cooling time be 40h~
60h, vacuum pressure are 10Pa~30Pa.
Farthest to protect the functional component in rosemary not to be destroyed, the present invention is located from Freeze Drying Technique
Manage rosemary, inventor find cryogenic temperature and vacuum in Freeze Drying Technique to the extraction of active ingredient in rosemary and
The influence of recovery rate is larger.
When the process conditions of freeze-drying are -70 DEG C~-80 DEG C of cryogenic temperature, cooling time 40h~60h, vacuum pressure
During 10Pa~30Pa, the rosemary after freeze-drying process not only has higher recovery rate, extracts obtained Rosmarinic acid
Extract also has higher antioxidation activity.
In the present invention, vacuum is too low can make that the water sublimed in rosemary is not thorough, and effect is poor, and vacuum
Spend height then high to instrument requirements, energy consumption is big and can shorten the service life of instrument；Cryogenic temperature is too high to cause rosemary sample
Product freezing is not thorough, and drying effect is poor；Can have in addition, cryogenic temperature is too high or too low instrument compared with lossy, and it is cold
Freeze that temperature is too low to cause energy consumption to raise.
The present inventor also attempts various methods to remove the impurity in gained Rosmarinic acid crude extract, by numerous
Inventor has found after trial, can be with maximum limit when carrying out purifying rosmarinic acid crude extract from Sephadex LH-20 gel columns
The impurity removed in crude extract of degree, and the content of Rosmarinic acid is enriched with, obtained using the purifying of Sephadex LH-20 gel columns
Rosmarinic acid extract in phenolic content be up to 60~90%；And when selecting the purifying of Sephadex LH-20 gel columns
Between it is short, and select other gel columns disengaging time length and separating effect is not good.
The present invention by being freeze-dried to rosemary after extract again, extract solution is after filtering, being concentrated under reduced pressure, then uses
Sephadex LH-20 gel columns are purified, and the active constituent content in obtained Rosmarinus officinalis extract is high, impurity is few, with preferable
Antioxidant effect.
Preferably, the volume of rosemary crude extract and the volume ratio of gel column are 1 in S3：10~1：30.
Preferably, the volume of rosemary crude extract and the volume ratio of gel column are 1 in S3：20.Under this condition, rosemary
Phenolic acid compound in sour crude extract can more preferably, faster be separated with impurity.
Preferably, used in S3 volume fraction for 30~90% ethanol solution as eluent；Using ethanol as washing
De- liquid, can obtain the higher Rosmarinic acid compound of purity while avoiding other organic solvents to greatest extent to target product
Pollution, it is ensured that it uses the security of cosmetics.
It is further preferable that use volume fraction for 90% ethanol solution as eluent, elute 2~3 column volumes.
The ratio between the post footpath of Sephadex LH-20 gel columns and pillar height are essentially that higher separating effect is better, but in this hair
If ratio exceeds 1 in bright:80 flow velocitys are excessively slow, influence the efficiency of separation and Extraction, and when the ratio between post footpath and pillar height are less than 1:50
Shi Zehui causes that impurity can not be sufficiently separated；It is preferred that when being purified in S3 using Sephadex LH-20 gel columns,
The ratio between post footpath and pillar height are 1:40~1:60；
It is further preferable that the volume ratio of post footpath and pillar height is 1:50.
Preferably, when being purified in S3 using Sephadex LH-20 gel columns, flow velocity is 1.0ml/min.
Preferably, it is filtered into and is taken out under conditions of 15~20um double-layer filter papers is in -0.1MPa vacuum, aperture described in S2
Filter；Concentration is concentrated under reduced pressure to be 115rpm/min in -0.1MPa vacuum, rotary speed to be concentrated under reduced pressure described in S2, evaporation temperature
Concentrated under conditions of 45 DEG C of degree, 2 DEG C of circulating condensing temperature.
Preferably, -76 DEG C of the temperature of freeze-drying, cooling time are 46h, and vacuum pressure is 18Pa.
Preferably, the mode of the extraction in S1 is water-bath extraction, ultrasonic extraction or Microwave Extraction.
Preferably, the extracting mode in S1 is ultrasonic extraction, and the technique of the ultrasonic extraction is：Ultrasonic power 400~
800W, concentration of alcohol 50~80%, solid-liquid ratio 1:50~100,40~70 DEG C of 30~120min of extraction time, Extracting temperature.
The theoretical foundation of ultrasonic wave extraction is to utilize ultrasonic wave calorifics mechanism, ultra sonic machinery mechanism and cavitation, is made
Plant cell wall ruptures and target product is discharged into extract solution.Ultrasonic wave extraction is carried with water-bath, compared with Microwave Extraction, and it is carried
Take speed faster, treating capacity is higher, and the impurity in extract solution is low compared with conventional method.
Protection extracts obtained rosmarinic acid extract to the present invention by said extracted technique simultaneously.
Compared with prior art, the present invention has advantages below and beneficial effect：
The present invention to rosemary be freeze-dried after carry out again extract can farthest protect in rosemary it is effective into
Divide and improve the recovery rate of Rosmarinic acid, recovery rate can reach 8%~15%, there is the rosmarinic acid extract finally given
Preferable antioxidation activity；
The present invention is purified using Sephadex LH-20 gel columns to rosemary crude extract, is farthest eliminated
The content of Rosmarinic acid in impurity in crude extract, Sync enrichment extract；
Compared with solvent extraction method and macroreticular resin separating and extracting process, the present invention extracts obtained rosmarinic acid extract
Middle rosmarinic acid contents are higher and active strong, and the phenolic content in gained Rosmarinus officinalis extract is up to more than 75%.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment, but embodiment does not do any type of to the present invention
Limit.Unless stated otherwise, the reagent of the invention used, method and apparatus is the art conventional reagent, methods and apparatus.
Unless stated otherwise, agents useful for same and material of the present invention are purchased in market.
Embodiment 1:
(1) fresh rosemary is beaten, freezed under the conditions of cryogenic temperature -76, cooling time 46h, vacuum pressure 18Pa dry
It is dry and crush to obtain powder from rosemary；3.00g powder from rosemary is weighed, in ultrasonic power 600W, solid-liquid ratio 1：80, concentration of alcohol
60%, extraction time is extraction under conditions of 60min, 60 DEG C of Extracting temperature, obtains Rosmarinic acid crude extract；
(2) Rosmarinic acid crude extract is filtered under the conditions of -0.1MPa vacuum, aperture 15~20um double-layer filter papers, so
Depressurized under conditions of vacuum afterwards in -0.1MPa, rotary speed 115rpm/min, 45 DEG C of evaporating temperature, 2 DEG C of circulating condensing temperature
It is concentrated to give Rosmarinic acid crude extract；
(3) Rosmarinic acid crude extract is crossed into Sephadex LH-20 gel columns, the ratio between gel column post footpath and pillar height are 1:
50, the volume of rosemary crude extract and the volume ratio of gel column are 1：20, use volume fraction for 60% ethanol solution conduct
Eluent, elution flow rate is 1.0ml/min, elutes 2~3 column volumes, produces rosmarinic acid extract, determines in extract and is confused
Repeatedly fragrant acid content and anti-oxidant value.
Embodiment 2
(1) fresh rosemary is beaten, freezed under the conditions of cryogenic temperature -70, cooling time 40h, vacuum pressure 10Pa dry
It is dry and crush to obtain powder from rosemary；3.00g powder from rosemary is weighed, in microwave power 800W, solid-liquid ratio 1：50, concentration of alcohol
50%, extraction time is extraction under conditions of 40min, 40 DEG C of Extracting temperature, obtains Rosmarinic acid crude extract；
(2) Rosmarinic acid crude extract is filtered under the conditions of -0.1MPa vacuum, aperture 15~20um double-layer filter papers, so
Depressurized under conditions of vacuum afterwards in -0.1MPa, rotary speed 115rpm/min, 45 DEG C of evaporating temperature, 2 DEG C of circulating condensing temperature
It is concentrated to give Rosmarinic acid crude extract；
(3) Rosmarinic acid crude extract is crossed into Sephadex LH-20 gel columns, the ratio between gel column post footpath and pillar height are 1:
50, the volume of rosemary crude extract and the volume ratio of gel column are 1：15, use volume fraction for 70% ethanol solution conduct
Eluent, elution flow rate is 1.0ml/min, elutes 2~3 column volumes, produces rosmarinic acid extract, determines in extract and is confused
Repeatedly fragrant acid content and anti-oxidant value.
Embodiment 3
(1) fresh rosemary is beaten, freezed under the conditions of cryogenic temperature -80, cooling time 60h, vacuum pressure 30Pa dry
It is dry and crush to obtain powder from rosemary；3.00g powder from rosemary is weighed, in microwave power 400W, solid-liquid ratio 1：100, concentration of alcohol
60%, extraction time is water-bath extraction under conditions of 1200min, 80 DEG C of Extracting temperature, obtains Rosmarinic acid crude extract；
(2) Rosmarinic acid crude extract is filtered under the conditions of -0.1MPa vacuum, aperture 15~20um double-layer filter papers, so
Depressurized under conditions of vacuum afterwards in -0.1MPa, rotary speed 115rpm/min, 45 DEG C of evaporating temperature, 2 DEG C of circulating condensing temperature
It is concentrated to give Rosmarinic acid crude extract liquid；
(3) Rosmarinic acid crude extract is crossed into Sephadex LH-20 gel columns, the ratio between gel column post footpath and pillar height are 1:
50, the volume of rosemary crude extract and the volume ratio of gel column are 1：25, use volume fraction for 90% ethanol solution conduct
Eluent, elution flow rate is 1.0ml/min, elutes 2~3 column volumes, produces rosmarinic acid extract, determines in extract and is confused
Repeatedly fragrant acid content and anti-oxidant value.
Embodiment 4
(1) fresh rosemary is beaten, freezed under the conditions of cryogenic temperature -80, cooling time 60h, vacuum pressure 30Pa dry
It is dry and crush to obtain powder from rosemary；3.29g rosemary drying sample is weighed, in ultrasonic power 500W, solid-liquid ratio 1：60, concentration of alcohol
55%, extraction time is extraction under conditions of 100min, 50 DEG C of Extracting temperature, obtains Rosmarinic acid crude extract；
(2) Rosmarinic acid crude extract is filtered under the conditions of -0.1MPa vacuum, aperture 15~20um double-layer filter papers, so
Depressurized under conditions of vacuum afterwards in -0.1MPa, rotary speed 115rpm/min, 45 DEG C of evaporating temperature, 2 DEG C of circulating condensing temperature
It is concentrated to give Rosmarinic acid crude extract；
(3) Rosmarinic acid crude extract is crossed into Sephadex LH-20 gel columns, the ratio between gel column post footpath and pillar height are 1:
50, the volume of rosemary crude extract and the volume ratio of gel column are 1：18, use volume fraction for 80% ethanol solution conduct
Eluent, elution flow rate is 1.0ml/min, elutes 2~3 column volumes, produces rosmarinic acid extract, determines in extract and is confused
Repeatedly fragrant acid content and anti-oxidant value.
Reference examples 1
In this reference examples after directly rosemary is beaten in step (1) i.e. in addition to ultrasonic extraction, other techniques and
Embodiment 1 is identical.
(1) fresh rosemary is beaten, takes 59.52g slurries, plus 95% ethanol 284.21ml, water 106.27ml, stirring is equal
It is even, in ultrasonic power 600W, solid-liquid ratio 1：80, concentration of alcohol 60% extracts under conditions of extraction time 60min, obtains rosemary
Sour crude extract；
(2) Rosmarinic acid crude extract is filtered under the conditions of -0.1MPa vacuum, aperture 15~20um double-layer filter papers, so
Depressurized under conditions of vacuum afterwards in -0.1MPa, rotary speed 115rpm/min, 45 DEG C of evaporating temperature, 2 DEG C of circulating condensing temperature
It is concentrated to give Rosmarinic acid crude extract；
(3) Rosmarinic acid crude extract is crossed into Sephadex LH-20 gel columns, the ratio between gel column post footpath and pillar height are 1:
50, the volume of rosemary crude extract and the volume ratio of gel column are 1：20, use volume fraction for 60% ethanol solution conduct
Elution flow velocity is 1.0ml/min, elutes 2~3 column volumes, produces rosmarinic acid extract, determines in extract and is confused
Repeatedly fragrant acid content and anti-oxidant value.
Reference examples 2
In this reference examples in addition to selecting oven drying rosemary in step (1), other techniques are same as Example 1.
(1) fresh rosemary is put into 60 DEG C of baking ovens and dries 72h, obtain water content for 8.75% rosemary dry sample, high speed powder
Broken machine is crushed, and 3.29g rosemary dry sample powder is weighed, in ultrasonic power 600W, solid-liquid ratio 1：80, concentration of alcohol 60% is carried
Extraction under conditions of time 60min is taken, Rosmarinic acid crude extract is obtained；
(2) Rosmarinic acid crude extract is filtered under the conditions of -0.1MPa vacuum, aperture 15~20um double-layer filter papers, so
Depressurized under conditions of vacuum afterwards in -0.1MPa, rotary speed 115rpm/min, 45 DEG C of evaporating temperature, 2 DEG C of circulating condensing temperature
It is concentrated to give Rosmarinic acid crude extract；
(3) Rosmarinic acid crude extract is crossed into Sephadex LH-20 gel columns, the ratio between gel column post footpath and pillar height are 1:
50, the volume of rosemary crude extract and the volume ratio of gel column are 1：20, use volume fraction for 60% ethanol solution conduct
Eluent, elution flow rate is 1.0ml/min, elutes 2~3 column volumes, produces rosmarinic acid extract, determines in extract and is confused
Repeatedly fragrant acid content and anti-oxidant value.
Reference examples 3
In this reference examples except the cryogenic temperature in step (1) and vacuum pressure with the difference of embodiment 1 in addition to, other techniques with
Embodiment 1 is identical.
(1) fresh rosemary is beaten, freezed under the conditions of cryogenic temperature -58, cooling time 48h, vacuum pressure 10Pa dry
It is dry and crush to obtain powder from rosemary；3.00g powder from rosemary is weighed, in ultrasonic power 600W, solid-liquid ratio 1：80, concentration of alcohol
60%, extraction time is extraction under conditions of 60min, 60 DEG C of Extracting temperature, obtains Rosmarinic acid crude extract；
(2) Rosmarinic acid crude extract is filtered under the conditions of -0.1MPa vacuum, aperture 15~20um double-layer filter papers, so
Depressurized under conditions of vacuum afterwards in -0.1MPa, rotary speed 115rpm/min, 45 DEG C of evaporating temperature, 2 DEG C of circulating condensing temperature
Concentrate Rosmarinic acid crude extract；
(3) Rosmarinic acid crude extract is crossed into Sephadex LH-20 gel columns, the ratio between gel column post footpath and pillar height are 1:
50, the volume of rosemary crude extract and the volume ratio of gel column are 1：20, use volume fraction for 60% ethanol solution conduct
Eluent, elution flow rate is 1.0ml/min, elutes 2~3 column volumes, produces rosmarinic acid extract, determines in extract and is confused
Repeatedly fragrant acid content and anti-oxidant value.
Reference examples 4
In this reference examples except the cryogenic temperature in step (1) and vacuum pressure with the difference of embodiment 1 in addition to, other techniques with
Embodiment 1 is identical.
(1) fresh rosemary is beaten, freezed under the conditions of cryogenic temperature -88, cooling time 48h, vacuum pressure 30Pa dry
It is dry and crush to obtain powder from rosemary；3.00g powder from rosemary is weighed, in ultrasonic power 600W, solid-liquid ratio 1：80, concentration of alcohol
60%, extraction time is extraction under conditions of 60min, 60 DEG C of Extracting temperature, obtains Rosmarinic acid crude extract；
(2) Rosmarinic acid crude extract is filtered under the conditions of -0.1MPa vacuum, aperture 15~20um double-layer filter papers, so
Depressurized under conditions of vacuum afterwards in -0.1MPa, rotary speed 115rpm/min, 45 DEG C of evaporating temperature, 2 DEG C of circulating condensing temperature
It is concentrated to give Rosmarinic acid crude extract；
(3) Rosmarinic acid crude extract is crossed into Sephadex LH-20 gel columns, the ratio between gel column post footpath and pillar height are 1:
50, the volume of rosemary crude extract and the volume ratio of gel column are 1：20, use volume fraction for 60% ethanol solution conduct
Eluent, elution flow rate is 1.0ml/min, elutes 2~3 column volumes, produces rosmarinic acid extract, determines in extract and is confused
Repeatedly fragrant acid content and anti-oxidant value.
Reference examples 5
Except the gel column in step (3) is Sephadex G-25 gel columns, other techniques and embodiment in this reference examples
1 is identical.
(1) fresh rosemary is beaten, freezed under the conditions of cryogenic temperature -76, cooling time 46h, vacuum pressure 18Pa dry
It is dry and crush to obtain powder from rosemary；3.00g powder from rosemary is weighed, in ultrasonic power 600W, solid-liquid ratio 1：80, concentration of alcohol
60%, extraction time is extraction under conditions of 60min, 60 DEG C of Extracting temperature, obtains Rosmarinic acid crude extract；
(2) Rosmarinic acid crude extract is filtered under the conditions of -0.1MPa vacuum, aperture 15~20um double-layer filter papers, so
Depressurized under conditions of vacuum afterwards in -0.1MPa, rotary speed 115rpm/min, 45 DEG C of evaporating temperature, 2 DEG C of circulating condensing temperature
It is concentrated to give Rosmarinic acid crude extract；
(3) Rosmarinic acid crude extract is crossed into Sephadex G-25 gel columns, the ratio between gel column post footpath and pillar height are 1:
50, the volume of rosemary crude extract and the volume ratio of gel column are 1：20, use volume fraction for 60% ethanol solution conduct
Eluent, elution flow rate is 1.0ml/min, elutes 2~3 column volumes, produces rosmarinic acid extract, determines in extract and is confused
Repeatedly fragrant acid content and anti-oxidant value.
Freeze-drying obtains Rosmarinic acid in rosmarinic acid extract in the step of embodiment 1~4 and reference examples 1~5 (3)
Content and antioxidation activity test result.
(1) recovery rate
Recovery rate refers to that freeze-dried rear middle its Rosmarinic acid that determines of the rosmarinic acid extract obtained in step (3) contains
The ratio of amount, rosmarinic acid contents and 3.00g powder from rosemary is the recovery rate of rosemary.
(2) rosmarinic acid contents
HPLC methods (with reference to 2010 editions pharmacopeia)：
Chromatographic condition C18 (250mm*4.6mm, 5 μm)
Mobile phase：The phosphoric acid (B) of acetonitrile (A) -0.1%
0~10min of gradient elution 25%A；10~15min, 25%~75%A；15~25min 75%A
Flow velocity：1mL/min
Detection wavelength：317nm
Sample size：10μL
Column temperature：30℃
Rosmarinic acid recovery rate Y=C*V*100%/M
In formula：Y, yield, %；C, the total phenol content of extract solution, mg/mL；V, volume, mL；M, material quality, g.
(3) antioxidation activity
1. DPPH clearance rates are determined
20mgDPPH accurately is weighed, with methanol constant volume to 500ml, 0.04mg/ml mother liquors are obtained, 0.2ml debita spissitudo samples are taken
Product liquid adds the above-mentioned DPPH solution of 7.8ml in 20ml test tubes, after reaction 30min under the conditions of lucifuge, under the conditions of 517nm
Survey light absorption value.The DPPH gradient solutions of another configuration various concentrations, take 8ml reaction assays under these conditions in 20ml test tubes,
Obtain using sample introduction concentration (c) to the equation of linear regression of absorbance (A) as A=20.536c-0.0106 (r=0.999 9).Root
According to DPPH concentration in sample after standard curve calculating reaction.The removing of DPPH in sample is calculated according to formula Y=(S-N)/S*100
Rate.(S is DPP concentration in blank sample, and N is DPPH concentration in sample.)
2. FRAP methods survey total antioxidation value
FRAP working solution matching while using：By 25ml 300mmol/L PH3.6 acetate buffer, 2.5ml10mmol/
LTPTZ solution, 2.5ml 20mmol/LFeCl3Solution is mixed.
The FeSO of certain gradient concentration is drawn respectively4Titer 0.1ml, adds 3mlFRAP working solutions, adds 0.3ml
Ultra-pure water, is mixed, accurate response 5min, and its light absorption value is determined at 593nm, is returned to zero with ultra-pure water, standard curve is drawn, by upper
State method using sample introduction concentration (c) to the equation of linear regression of absorbance (A) as A=0.5975c+0.0311 (r=0.999 9).
The measure of sample：0.1ml sample solution is measured, in the above conditions the total antioxidation value of determination sample.Sample
Antioxidation activity (FRAP values) to reach FeSO needed for identical absorbance4MM number represent.
3. the measure of reducing power
Using the reducing power of Oyaiaz method determination samples：
0.2mol/L phosphate buffers (pH6.6) 2.5ml and 1% potassium ferricyanide are separately added into 0.5ml sample solutions
Solution 2.5ml, is placed in 50 DEG C of water after being well mixed and reacts 10% solution of trichloroacetic acid 2.5ml, 3000r/ of 20min and then addition
Min centrifuges 10min, takes 2.5ml supernatants, sequentially adds 2.5ml distilled water and 0.5ml0.1% liquor ferri trichloridis, fully mixed
Close, the light absorption value of determination sample at 700nm, light absorption value is higher, and reducing power is stronger.
The performance test of the rosmarinic acid extract obtained in the embodiment 1~4 of table 1 and reference examples 1~5
Application Example one
This application test example gives the emulsion of two kinds of formulas (wt%)：
Formula 1：Jojoba seed oil 8.0g, saualane 2.0g, glycerine 6.0g, propane diols 3.0g, hydrolecithin 3.0g, it is real
Apply example 1 and extract obtained rosmarinic acid extract 2.0g, card ripple U21 0.1g, triethanolamine 0.1g, add water to 100g；
Formula 2：Jojoba seed oil 8.0g, saualane 2.0g, glycerine 6.0g, propane diols 3.0g, hydrolecithin 3.0g, card
Ripple U21 0.1g, triethanolamine 0.1g, add water to 100g.
From 40 trial volunteers respectively using after 2 months, while to be formulated 2 as control, entering to 1 using effect of formula
Row moisturizing marking is evaluated；Full marks is very, more than 8 points are excellent, and 6~8 points are good, and 3~6 points are general, and less than 3 points are invalid
Really, evaluation result such as following table 2.1 and table 2.2.
The moisturizing experiment of 1 gained rosmarinic acid extract is formulated in the embodiment 1 of table 2.1
| Effectiveness indicator | Evaluation number | Accounting (%) |
| It is excellent | 5 | 12.5 |
| Well | 22 | 55 |
| Typically | 10 | 25 |
| To no effect | 3 | 7.5 |
The moisturizing experiment of the gained of formula 2 in the embodiment 1 of table 2.2
| Effectiveness indicator | Evaluation number | Accounting (%) |
| It is excellent | 1 | 2.5 |
| Well | 11 | 27.5 |
| Typically | 20 | 50 |
| To no effect | 8 | 20 |
Application Example two
The performance of keeping humidity that embodiment 1~4 and reference examples 1~5 extract obtained rosemary crude extract is tested, and with
Glycerine and Sodium Hyaluronate are respectively as reference examples 6 and reference examples 7, and method of testing is as follows, and test result see the table below 3.
(1) hygroscopicity is determined
Sample is placed in baking oven, 3h is dried at 105 DEG C, then places into and room temperature is cooled in silica gel drier.It is accurate
1.0g really is weighed, is respectively placed in and maintains relative humidity (RH) to be 81% with the saturated ammonium sulfate aqueous solution.Saturation calcium chloride water
Claim the quality of each sample after moisture absorption in the drier that maintenance RH is 32%, 24h, 72h.
Hydroscopicity (Ra)=(Wn-W0)/W0× 100%
W0For sample quality before placing, Wn is the sample quality after placing.
(2) moisture retention is determined
The water that mass fraction is 10% is added in the sample after drying constant weight, it is 81%, 32% to be then placed on RH
And in silica gel drier.Claim the quality of each sample after 24h, 72h.
Moisturizing rate (Rr)=Hn/H0× 100%
H0For biodiversity before placing, Hn is biodiversity after placing.
Test condition in table 3 is：25 DEG C, relative humidity 32%；Test condition in table 4 is：25 DEG C, relative humidity
81%.
The embodiment 1~4 of table 3 and reference examples 1~7 extract the performance of keeping humidity of obtained rosemary crude extract
The embodiment 1~4 of table 4 and reference examples 1~7 extract the performance of keeping humidity of obtained rosemary crude extract
| Sample | Hygroscopicity (24h) | Moisture retention (24h) | Hygroscopicity (72h) | Moisture retention (72h) |
| Embodiment 1 | 37.21% | 3.78% | 49.56% | 4.51% |
| Embodiment 2 | 36.63% | 3.34% | 44.56% | 4.26% |
| Embodiment 3 | 35.89% | 3.42% | 45.12% | 4.31% |
| Embodiment 4 | 35.59% | 3.54% | 48.32% | 4.26% |
| Reference examples 1 | 33.56% | 3.10% | 41.25% | 3.92% |
| Reference examples 2 | 30.56% | 2.58% | 40.65% | 3.45% |
| Reference examples 3 | 31.25% | 3.14% | 43.25% | 3.79% |
| Reference examples 4 | 32.14% | 3.06% | 42.78% | 4.06% |
| Reference examples 5 | 28.22% | 2.11% | 33.59% | 2.89% |
| Reference examples 6 | 15.12% | 1.7% | 25.77% | 2.34% |
| Reference examples 7 | 33.74% | 2.73% | 42.09% | 3.86% |
By table 3,4 it can be seen that various embodiments of the present invention extract the obtained synthesis performance of keeping humidity of rosmarinic acid extract and
Moisture pick-up properties is better than each reference examples, and rosmarinic acid extract can be used in cosmetics as a kind of NMF.
Claims (10)
1. a kind of extraction process of Rosmarinic acid, it is characterised in that comprise the following steps：
S1：After rosemary is beaten successively, dried, crushing, Rosmarinic acid crude extract is extracted to obtain；
S2：Rosmarinic acid crude extract obtained by S1 is filtered, Rosmarinic acid crude extract is concentrated to give；
S3：Rosmarinic acid crude extract is purified using Sephadex LH-20 gel columns, rosmarinic acid extract is obtained；
Dried in S1 as freeze-drying, -70 DEG C~-80 DEG C of the temperature of freeze-drying, cooling time are that 40h~60h, vacuum pressure are
10Pa~30Pa.
2. the extraction process of Rosmarinic acid according to claim 1, it is characterised in that in S3 the volume of rosemary crude extract with
The volume ratio of gel column is 1：10~1：30, it regard volume fraction as eluent for 30~90% ethanol solution.
3. the extraction process of Rosmarinic acid according to claim 1 or claim 2, it is characterised in that the body of rosemary crude extract in S3
Product and the volume ratio of gel column are 1：20, it regard volume fraction as eluent, 2~3 cylinders of elution for 90% ethanol solution
Product, flow velocity is 1.0ml/min.
4. the extraction process of Rosmarinic acid according to claim 1, it is characterised in that Sephadex LH-20 gel columns in S3
The ratio between post footpath and pillar height be 1:40~1:60.
5. the extraction process of Rosmarinic acid according to claim 1, it is characterised in that the temperature of freeze-drying is -76 DEG C, cold
The jelly time is 46h, and vacuum pressure is 18Pa.
6. the extraction process of Rosmarinic acid according to claim 1, it is characterised in that be filtered into described in S2 in -0.1MPa
Vacuum, aperture are suction filtration under conditions of 15~20um double-layer filter papers；Concentration described in S2 to be concentrated under reduced pressure, be concentrated under reduced pressure for-
0.1MPa vacuum, rotary speed are 115rpm/min, and 45 DEG C of evaporating temperature is concentrated under conditions of 2 DEG C of circulating condensing temperature.
7. the extraction process of Rosmarinic acid according to claim 1, it is characterised in that the mode of the extraction in S1 carries for water-bath
Take, ultrasonic extraction or Microwave Extraction.
8. the extraction process of Rosmarinic acid according to claim 8, it is characterised in that the extracting mode in S1 carries for ultrasound
Take, the technique of the ultrasonic extraction is：400~800W of ultrasonic power, concentration of alcohol 50~80%, solid-liquid ratio 1:50~100, carry
Take 40~70 DEG C of 30~120min of time, Extracting temperature.
9. any extraction process of claim 1 to 8 extracts obtained rosmarinic acid extract.
10. application of the rosmarinic acid extract described in claim 9 in skin-protection product is prepared.
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