CN111184682B - Preparation method of sea fennel cell extract with whitening and antioxidation effects - Google Patents
Preparation method of sea fennel cell extract with whitening and antioxidation effects Download PDFInfo
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- CN111184682B CN111184682B CN202010254582.1A CN202010254582A CN111184682B CN 111184682 B CN111184682 B CN 111184682B CN 202010254582 A CN202010254582 A CN 202010254582A CN 111184682 B CN111184682 B CN 111184682B
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Classifications
-
- 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
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
-
- 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
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
-
- 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
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- 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
- A61Q19/08—Anti-ageing preparations
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Birds (AREA)
- Biotechnology (AREA)
- Engineering & Computer Science (AREA)
- Dermatology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Botany (AREA)
- Tropical Medicine & Parasitology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Cosmetics (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a preparation method of a sea fennel cell extract with whitening and antioxidation effects, which is characterized by comprising the following steps of: accurately weighing the sea fennel cells, adding an extraction solvent for extraction, centrifuging or suction filtering, collecting supernatant to obtain sea fennel cell extract, detecting the contents of total flavonoids, total phenolic acids and total phenols in the sea fennel cell extract, and measuring the whitening and antioxidation effects. The sea fennel cell extract prepared by the invention has good corrosion resistance and stability due to the inclusion of butanediol or propylene glycol, and does not need to additionally add other preservatives or stabilizers; the extract has good water solubility, and is different from oil-soluble products in market.
Description
Technical Field
The invention belongs to the technical field of plant extraction biology, and relates to a preparation method of a sea fennel cell extract with whitening and antioxidation effects.
Background
Sea fennel grows at the earliest place on the pure pollution-free seashore of britagne, the britany peninsula is located in the western part of france as if one sharp corner were extending into the eastern part of the vast atlantic ocean, from where the sea wind from the atlantic ocean begins to enter the continental europe, thus creating a temperate marine climate that is matted throughout europe. Not only is warm in winter and cool in summer, but also forms various magic coastal landforms due to the combined action of the sea wind and the coastal bedrock. The land and coast of Bytani are depressed and the onion is filled with various magic special plants, the sea fennel is one of them. A unique life system has been developed because roots must absorb nutrients between coasts to face the harsh land environment. The growth season of the fennel is limited to spring, the harvesting period of one year is about from four months to six months, so the fennel is also classified by French as a national treasured plant limited to be mined, and the characteristics of manual picking and difficult limited harvesting of growth are self-evident as the most rare maintenance component of the 21 st century.
The sea fennel contains volatile terpene substances, has special fragrance, and is mainly used in the field of food cooking. In the existing market, the sea fennel extract mainly aims at the extraction of volatile terpene substances, has lower activity on most bacteria, has good inhibition effect on the growth of anaerobic clostridium perfringens, and has values of staphylococcus epidermidis, staphylococcus aureus, escherichia coli and pseudomonas aeruginosa of more than 250.
The existing product development mainly takes natural sea fennel as a main body, but the growth of the sea fennel has regional limitation, so that the development and the use of the sea fennel have limitations. The invention takes the artificially cultured sea fennel plant cells as a body, can carry out large-scale industrialized production, and has no use limitation; meanwhile, the existing sea fennel series products in the market mainly aim at the extraction of volatile matter terpenes, the products are oil-soluble, the compatibility of the products is limited, and the application of sea fennel cell extracts is limited. The invention has good water solubility and solves the problem of incompatibility with water aqua products.
Disclosure of Invention
The invention extracts the active ingredients in the sea fennel cells, increases the application range of the sea fennel cell extract, solves the problems of limited use and limited compatibility, and develops the sea fennel cell extract with the functions of whitening and resisting oxidation.
The invention extracts the sea fennel cells different from the sea fennel products on the market, the invention firstly extracts the functional components by taking the sea fennel cells as the body, the components are different from the sea fennel components which are cultured naturally, secondly, the product on the market extracts and develops the product by taking the nonpolar components in the sea fennel as the functional components, the invention extracts and develops the product by taking the water-soluble components and the weak polar components in the sea fennel cells as the functional components, and thirdly, the product of the invention is mainly used in the cosmetic field with whitening and antioxidation functions.
In order to achieve the above object, the present invention discloses the following technical contents:
a preparation method of a sea fennel cell extract with whitening and antioxidation effects is characterized by comprising the following steps:
accurately weighing the sea fennel plant cells, adding the prepared multielement solvent for extraction, centrifuging or suction filtering, collecting supernatant, namely the sea fennel cell extract, and then detecting the contents of total flavonoids, total phenolic acids and total phenols in the sea fennel cell extract for whitening and antioxidation efficacy measurement;
the extraction solvent refers to: in the extraction solvent, the ratio of butanediol or propylene glycol is 20-80% respectively or in total, and the ratio refers to the mass ratio. The extraction method comprises the following steps of 3 types: high temperature extraction, leaching and ultrasonic extraction; the sea fennel cell comprises: dried, fresh and frozen sea fennel cells.
The extraction method of the invention comprises the following steps of:
the high-temperature extraction method comprises the following steps: extracting at 60-100deg.C for 0.5-2h, wherein the ratio of fresh cell or frozen cell to sea fennel stem cell is 1:20-1:100 by weight part, and the ratio of fresh cell or frozen cell to sea fennel stem cell is 1:2-1:5 by weight part;
the ultrasonic extraction method comprises the following steps: extracting at 40-60 ℃ for 30-60min under ultrasonic treatment, wherein the weight ratio of the material liquid of the sea fennel stem cells is 1:20-1:100, and the weight ratio of the material liquid of the fresh cells or the frozen cells is 1:2-1:5;
the leaching extraction method comprises the following steps: extracting at normal temperature for 4-24h, wherein the weight ratio of the sea fennel stem cell feed liquid is 1:20-1:100, and the weight ratio of the fresh cell or frozen cell feed liquid is 1:2-1:5.
The invention further discloses application of the sea fennel cell extract prepared by the preparation method of the sea fennel cell extract with the whitening and antioxidation effects in cosmetics with the whitening and antioxidation effects. Experimental results show that the sea fennel cell extract has the effects of whitening, resisting oxidation and resisting inflammation.
The invention mainly solves the problems of the preparation method of the sea fennel cell extract with better water solubility, and mainly examines the whitening and antioxidation effects of the prepared sea fennel cell extract, and has the main difficulties of screening the extraction solvent and determining the proportion in the sea fennel extraction process.
More detailed description of the invention:
1, accurately weighing 5g of dried plant cells (dry powder) of fennel, and mixing the materials according to a feed liquid ratio of 1:40 adding 200mL of 40% butanediol aqueous solution, boiling and extracting for 1h, centrifuging at 6500rpm for 10min, and collecting supernatant to obtain sea fennel cell extract liquid. Detecting the contents of total flavonoids, total phenolic acids and total phenols in the sea fennel cell extract, and measuring the antioxidant and whitening effects.
The detection method of total flavonoids comprises the following steps:
1) Drawing a standard curve: accurately weighing 10mg of rutin reference substance, dissolving in methanol, and fixing volume to 100mL to prepare 0.2mg/mL rutin reference substance solution. Precisely sucking rutin reference substance solutions 1, 2, 3, 4, 5 and 6 mL, respectively placing in 25mL measuring flasks, respectively adding water to 6 mL, adding 5% sodium nitrite solution 1mL, shaking uniformly, standing for 6 min, adding 10% aluminum nitrate solution 1mL, shaking uniformly, standing for 6 min, adding 4% sodium hydroxide test solution 10mL, adding pure water to constant volume of 25mL, shaking uniformly, standing for 15min, taking corresponding reagent as blank, and measuring absorbance at 500 nm wavelength according to spectrophotometry.
2) Determination of total flavone content of the sample: the total flavone content of the sea fennel cell extract is detected by adopting the method, and the total flavone content is calculated.
The method for detecting the total phenolic acid comprises the following steps:
1) Drawing a standard curve: accurately weighing chlorogenic acid 2.7mg, dissolving with absolute ethanol, fixing volume to 10mL, precisely sucking reference substance solutions 40, 80, 120, 160 and 200 μL, placing into an EP tube of 1.5mL, respectively adding water to fix volume to 1.0mL,
placing in 25mL measuring flask respectively, and adding absolute ethanol to 5mL respectively; 2mL of 0.3% sodium dodecyl sulfate solution is added, the mixture is shaken uniformly, 1mL of 0.9% potassium ferricyanide and 0.6% ferric trichloride solution are added, the mixture is shaken uniformly, the mixture is placed in the dark for 5 minutes, 0.1mol/L hydrochloric acid solution is added to the scale mark, the mixture is shaken uniformly, and the mixture is placed in the dark for 20 minutes. The absorbance was measured at 760nm using a 5mL volume of solution added with absolute ethanol as a blank, and a linear regression equation, y=kx+b, was calculated from the mass of the absorbance corresponding thereto.
2) Determination of total phenolic acid content of sample: and detecting the total phenolic acid content of the sea fennel cell extract by adopting the method, and calculating the total phenolic acid content.
The method for detecting the total phenols comprises the following steps:
1) Drawing a standard curve: accurately weighing 10mg of gallic acid, fixing the volume to 100mL, precisely sucking 40, 80, 120, 160 and 200 mu L of reference substance solution, adding water to complement to 1.2mL, adding 50 mu L of Fu Lin Fen reagent, uniformly mixing, standing at room temperature for 7 minutes, adding 290 mu L of freshly prepared 20% Na2CO3 solution, uniformly vortex, standing for 1 hour, measuring the absorbance at 760nm wavelength, calculating a linear regression equation according to the absorbance and the corresponding mass, and calculating Y=kx+b.
2) Sample total phenol content determination: and detecting the total phenol content of the sea fennel cell extract by adopting the method, and calculating the total phenol content.
The total flavone, total phenolic acid and total phenol contents of the sea fennel cell extract were measured as described above, and the results are shown in table 1 below:
TABLE 1 Total flavone, total phenolic acid and Total phenolic content in sea Foeniculi cells
Antioxidant Activity assay
1) DPPH Activity assay
Preparing a DPPH ethanol solution: accurately weighing 3.94 mg DPPH, adding absolute ethyl alcohol for dissolving and fixing the volume to 100mL
In a volumetric flask, a solution of DPPH of 0.1 mM was prepared and stored at low temperature (0-4 ℃) protected from light. Measuring the water solutions of the sea fennel plant cell extracts with different volumes, adding the water solutions into 3mL of DPPH solution, uniformly mixing, adding water to supplement until the water is 4mL, measuring the absorbance at 517nm, and calculating the clearance rate, wherein the calculation formula is as follows:
wherein the A control is absorbance without adding sample solution and with adding 1.0mL purified water; sample a is the absorbance of different volumes of sample solution.
The experimental results are shown in table 2, and the experimental results show that the extractive solution of the sea fennel plant cell culture has strong DPPH free radical scavenging ability.
TABLE 2 scavenging of DPPH free radical by the extract of the plant cell culture of sea fennel
2) ABTS experiment
The required solution was prepared by diluting the extract of the culture of sea fennel plant cells obtained at 100℃in example 5 by 5-fold, and then performing an in vitro antioxidation experiment.
7.4mM ABTS solution: accurately weighing 0.0384 g of ABTS, placing the ABTS in a 10mL brown volumetric flask, and fixing the volume of purified water;
2.6mM potassium persulfate solution: accurately weighing 0.0066 g of potassium persulfate, placing the potassium persulfate into a 10mL volumetric flask, and fixing the volume of purified water; ABTS working fluid: the 7.4mM ABTS solution and the 2.6mM potassium persulfate solution are mixed according to the volume ratio of 1:1, and the mixture is kept stand for 12 hours at room temperature in a dark place for standby, and is diluted to the absorbance of 0.7+/-0.02 by using 80% ethanol solution before use.
The method comprises the steps of accurately measuring sample solutions of extract solutions of the maritime fennel plant cell cultures in different volumes in an ABTS oxygen radical scavenging experiment of a sample, adding the sample solutions into 3.4 mL of ABTS working solution, adding water to a constant volume of 4.0 mL, uniformly mixing, standing at room temperature for 30min, performing spectrophotometry detection at 734nm, and calculating the scavenging rate through absorbance change, wherein the calculation formula is as follows:
wherein the A control is absorbance without adding sample solution and with adding 1.0mL purified water; sample a is the absorbance of different volumes of sample solution.
The experimental results are shown in Table 3, and the results show that the extractive solution of the sea fennel plant cell culture has good clearance rate to ABTS free radicals
TABLE 3 test of the clearance of the extract of sea fennel cells to ABTS free radical
Whitening efficacy detection
1) Tyrosinase assay
Accurately weighing 0.00362g of tyrosinase powder (1380 u/mg, sigma), fixing the volume to 50mL by using PBS phosphate buffer solution with pH of 6.8, preparing sample to be tested solutions with different concentration gradients according to requirements, adding sample detection solution according to Table 4, and then placing in a water bath kettle with the temperature of 37 ℃ for 10min; then tyrosinase is added into a A, C pipe sequentially, the enzyme adding interval is 30 s each time, and 15 s vortex is immediately carried out after enzyme adding, and the reaction is carried out for 15 min. After the reaction tube was returned to room temperature, the absorbance was measured at 475nm by an ultraviolet spectrophotometer.
The experimental results are shown in fig. 1, which show that the sea fennel cell extract has the effect of inhibiting tyrosinase, thus having the effect of whitening.
TABLE 4 tyrosine detection additive reagent table
Inhibition of tyrosinase activity (%) of sample:
wherein: a-blank sample plate has enzyme system absorbance value;
b-absorbance of blank template enzyme-free system;
c, the sample group has an enzyme system absorbance value;
absorbance of D-sample group enzyme-free system
The sea fennel plant cell culture extract liquid has the effects of human body antioxidation and whitening: the extract of the sea fennel plant cell culture was added to the blank cream base at an addition amount of 5%, while the blank base was used as a control, the sample and blank base formulations were as shown in table 5 below, and then the prepared cream was applied to 10 volunteer women of 35-45, and after 42 days of application, the application effect evaluation feedback was performed. The results are shown in Table 5.
Table 5 blank matrix cream and formulations of test Marine fennel cell extract cream
Table 6 feedback of post-use effects in female volunteers
Conclusion: besides the in vitro experiments, the sea fennel cell extract has good whitening and antioxidation effects, and the human body experiments further determine the whitening, brightening and antioxidation effects of the sea fennel cell extract.
Compared with the prior art, the preparation method of the sea fennel cell extract with the whitening and antioxidation effects has the following positive effects:
(1) The prepared sea fennel cell extract has good water solubility by adopting an aqueous solution extraction solvent of butanediol or propylene glycol with different mass ratios, and is different from oil-soluble products in the market;
(2) The sea fennel cell extract prepared by the invention has good corrosion resistance and stability due to the inclusion of butanediol or propylene glycol, and does not need to additionally add other preservatives or stabilizers;
(3) The extraction solvent of the sea fennel cell extract prepared by the invention is butanediol, propylene glycol and water, so that the sea fennel cell extract is safe and non-irritating, and the butanediol and the propylene glycol are good moisturizing and skin-protecting raw materials.
Drawings
The inhibition of tyrosinase by the sample of FIG. 1;
FIG. 2 shows a process flow of the extraction of sea fennel cells.
Detailed Description
The invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The raw materials and reagents used in the invention are all commercially available.
Example 1
A preparation method of a sea fennel cell extract with whitening and antioxidation effects comprises the following steps:
accurately weighing 5g of dried plant cells of the fennel, and mixing the materials according to a feed liquid ratio of 1:40 adding 200mL of 40% butanediol aqueous solution, ultrasonic extracting for 1h at 60deg.C, centrifuging at 6500rpm for 10min, and collecting supernatant to obtain sea fennel cell extract.
Example 2
Accurately weighing 10g of sea fennel stem cells, adding 40% butanediol or propylene glycol water solution and pure water according to a feed liquid ratio of 1:60, boiling and extracting for 1h, centrifuging at 6500rpm for 10min, and detecting the contents of flavone, phenolic acid and total phenol in the supernatant, wherein the detection result is as follows:
TABLE 7 Total flavone, total phenolic acid and Total phenolic content in sea Foeniculum vulgare cell extract
Example 3
Accurately weighing 10g of sea fennel stem cells, respectively adding 40% butanediol according to the feed liquid ratio of 1:20,1:40, 1:60,1:80 and 1:100, leaching for 24 hours at normal temperature, centrifuging at 6500rpm for 10min, and detecting the content of flavone, phenolic acid and total phenol in the supernatant, wherein the detection result is as follows:
TABLE 8 Total flavone, total phenolic acid and Total phenolic content in sea Foeniculum vulgare cell extract
Example 4
Accurately weighing 10g of sea fennel stem cells, adding 60% propylene glycol aqueous solution according to a feed liquid ratio of 1:80, respectively carrying out ultrasonic extraction for 15min, 30min, 45min and 60min at 40 ℃, centrifuging at 6500rpm for 10min, and detecting the content of flavone, phenolic acid and total phenol in the supernatant, wherein the detection result is as follows:
TABLE 9 Total flavone, total phenolic acid and Total phenolic content in sea Foeniculum vulgare cell extract
Example 5
Accurately weighing 50g of fresh sea fennel cells and frozen cells, adding 40% butanediol water solution according to a feed liquid ratio of 1:2, performing ultrasonic extraction at 50 ℃ for 30min, centrifuging at 6500rpm for 10min, and detecting the contents of flavone, phenolic acid and total phenol in the supernatant, wherein the detection result is as follows:
TABLE 10 Total flavone, total phenolic acid and Total phenolic content in sea Foeniculum vulgare cell extract
Example 6
Accurately weighing 50g of fresh sea fennel cells and frozen cells, adding 40% butanediol water solution according to a feed liquid ratio of 1:2, leaching for 12 hours at normal temperature, centrifuging at 6500rpm for 10 minutes, and detecting the contents of flavone, phenolic acid and total phenol in supernatant, wherein the detection result is as follows:
TABLE 11 Total flavone, total phenolic acid and Total phenolic content in sea Foeniculum vulgare cell extract
Claims (1)
1. The application of the sea fennel cell extract in preparing the cosmetics with the whitening and antioxidation effects is characterized in that the sea fennel cell extract is extracted by taking artificially cultured sea fennel plant cells as a body and taking water-soluble components and weak polar components in the sea fennel plant cells as functional components, wherein the components are different from natural cultured sea fennel components, and the whitening and antioxidation effects are tested;
the preparation of the sea fennel cell extract comprises the following steps: accurately weighing 10g of sea fennel stem cells, adding 40% butanediol according to a feed-liquid ratio of 1:20, leaching for 24 hours at normal temperature, centrifuging at 6500rpm for 10 minutes, and detecting the contents of flavone, phenolic acid and total phenol in supernatant.
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