CN114796087B

CN114796087B - Preparation method of sea fennel stem cell extract and application of sea fennel stem cell extract in cosmetics

Info

Publication number: CN114796087B
Application number: CN202210220475.6A
Authority: CN
Inventors: 盘学平
Original assignee: Guangzhou Shangzi Chemical Technology Co ltd
Current assignee: Guangzhou Shangzi Chemical Technology Co ltd
Priority date: 2022-03-08
Filing date: 2022-03-08
Publication date: 2023-06-13
Anticipated expiration: 2042-03-08
Also published as: CN114796087A

Abstract

The invention discloses a preparation method of a sea fennel stem cell extract, which comprises the following steps: performing ultrasonic treatment on the sea fennel stem cells to obtain an ultrasonic treatment mixture; performing supercritical extraction on the ultrasonic treatment mixture to obtain an extract; and (3) carrying out enzymolysis on the extract liquid by using cellulase, trypsin and pectase to obtain the sea fennel stem cell extract. The invention also discloses a composition which comprises, by weight, 10-30 parts of the above-mentioned sea fennel stem cell extract, 6-18 parts of lime tea extract, 20-40 parts of pomegranate seed essence, 10-20 parts of milk essence, 3-7 parts of glycerol and 10-20 parts of vitamin E. The composition has good DPPH radical scavenging effect, hydroxyl radical scavenging effect and superoxide anion radical scavenging effect.

Description

Preparation method of sea fennel stem cell extract and application of sea fennel stem cell extract in cosmetics

Technical Field

The invention relates to a skin care product, in particular to a preparation method of a sea fennel stem cell extract and application thereof in cosmetics.

Background

The skin is the largest organ of the human body, which protects our body against external environmental attacks, and has an extremely important barrier function. However, as the environment changes and the age increases, the barrier function of the skin decreases, and collagen and elastin in the skin gradually decrease, thereby causing aging phenomena such as wrinkles, loss of elasticity, dullness, etc. in the skin. According to the Hayflick boundary theory, the number of stem cell divisions of adult tissues is limited, and thus depletion of stem cells limits the life span of skin tissues. While continuous renewal and repair of skin tissue is necessary to maintain youthful skin, current essence products, whether spread or facial films, do not achieve good efficacy.

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 stem cell contains the whole components of sea fennel and is rich in various lipids, phenolic compounds, saccharides and polyhydroxy substances.

Supercritical CO ₂ The principle of the fluid extraction (SFE) separation process is to use the relationship between the dissolution capacity of the supercritical fluid and its density, i.e., the influence of pressure and temperature on the dissolution capacity of the supercritical fluid. In the supercritical state, the supercritical fluid is contacted with the substance to be separated to selectively extract the components with polarity, boiling point and molecular weight in sequence. Of course, the extracts obtained corresponding to the pressure ranges cannot be single, but the conditions can be controlled to obtain the mixed components with the optimal proportion, then the supercritical fluid is changed into common gas by means of decompression and temperature rising, and the extracted substances are completely or basically separated out, thereby achieving the purposes of separation and purification, thus the supercritical CO ₂ The fluid extraction process is a combination of extraction and separation processes.

The patent application with the application publication number of CN107929220A discloses a stem cell skin care compound with the functions of repairing and resisting aging and a preparation method thereof, wherein the stem cell skin care compound comprises the following components in parts by weight: 0.5-2 parts of stem cells, 0.1-0.5 part of sodium hyaluronate, 0.1-0.5 part of hydrolyzed sclerotium rolfsii, 0.5 part of trehalose, 3-5 parts of betaine, 3-5 parts of glycerol, 0.03 part of triethanolamine, 3 parts of joba oil, gatuline Expression AF parts of Collrepair DG, and 3 parts of Collrepair DG. The invention is compounded by three stem cells, and the expression genetic factors contained in the plant stem cells can effectively provide nutrients for human adult stem cells, effectively activate skin epidermis stem cells, dermis stem cells and subcutaneous tissue stem cells, can achieve dermis, and the active ingredients in the plant stem cells can promote the regeneration capability of skin, improve epidermis and dermis from inside to outside so as to protect and maintain the functions of human adult stem cells, update and repair skin tissues, delay aging, improve skin quality and reduce the addition of other functional raw materials so as to reduce cost.

The patent application with publication number of CN103040941A discloses a pollen supercritical extraction extract and preparation and application thereof, and relates to a pollen supercritical extraction extract with high safety and 5 alpha-reductase inhibition activity and preparation and application thereof. The pollen supercritical extraction extract is prepared by the following method: weighing pollen raw materials, adding into extraction kettle, screwing the cover of the extraction kettle, and passing through supercritical CO ₂ Extracting for 1-3h at 30-60 ℃ under 200-500 bar by an extraction instrument, and collecting the pollen supercritical extract after extraction is finished; the pollen raw material is from corn poppy, schisandra chinensis or corn. The beneficial effects of the invention are mainly as follows: the method can obtain 3 pollen supercritical extraction extracts with better 5 alpha-reductase inhibition activity, the sources of raw materials are safe, and the supercritical extraction process adopted by the invention does not involve toxic and harmful organic solvents, so that the finished product has fewer toxic and side effects and high safety, and is very beneficial to the development of subsequent medical and health-care foods.

The skin care has the defects of destroyed functional components, incapability of effectively utilizing active structures and the like, and has the defects of more raw material requirements, higher cost, complex preparation process and the like, does not relate to the actual application process technology and formula, and has not been reported about the application of the sea fennel stem cell extract in the whitening antioxidant cosmetics. Therefore, there is a need in the current research and practice for an effective extraction method of sea fennel stem cells, and the active extract is used as an antioxidant ingredient for skin care products or cosmetics.

Disclosure of Invention

The invention discloses a preparation method of a sea fennel stem cell extract obtained by a supercritical extraction method and application of the sea fennel stem cell extract in cosmetics. The sea fennel stem cell supercritical extraction extract is prepared by the following steps: accurately weighing the sea fennel stem cells, performing steps of ultrasonic pretreatment, supercritical extraction, enzymolysis, vacuum concentration, post-treatment and the like, obtaining the sea fennel stem cell extract, having better antioxidant and whitening activities, wherein active ingredients in the stem cells can improve the regeneration capacity of skin, improve epidermis and dermis from inside to outside so as to protect and maintain the functions of human adult stem cells, update and repair skin tissues and delay aging, further improve skin quality, and reduce the addition of other functional raw materials so as to reduce cost.

The invention aims to provide a method for preparing a sea fennel stem cell extract by utilizing supercritical extraction, which is mainly used in the field of cosmetics with whitening and antioxidation functions.

The first aspect of the present invention provides a preparation method of a sea fennel stem cell extract, comprising the steps of:

s1: performing ultrasonic treatment on the sea fennel stem cells to obtain an ultrasonic treatment mixture;

s2: performing supercritical extraction on the ultrasonic treatment mixture to obtain an extract;

s3: and (3) carrying out enzymolysis on the extract liquid by using enzymes to obtain the sea fennel stem cell extract, wherein the enzymes are any one, any two or three of cellulase, trypsin and pectase.

In some embodiments, the method of making further comprises the steps of:

s4: concentrating the sea fennel stem cell extract to obtain a concentrated sea fennel stem cell extract.

In some embodiments, the sea fennel stem cells are freeze-dried powders of sea fennel callus cultures.

In some embodiments, the sea fennel stem cell particle size is 50nm-150nm (e.g., any one of 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm or a range therebetween).

In some embodiments, in step S1, the ultrasound conditions are: the power is 100-300W (e.g., any value or range between 150W, 200W, 250W), the temperature is 20-30 ℃ (e.g., any value or range between 21 ℃,22 ℃, 23 ℃, 24 ℃,25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃) and the time is 20-40min (e.g., any value or range between 25min, 30min, 35 min).

In some embodiments, in step S2, the supercritical extraction is performed with carbon dioxide as an extraction gas.

In some embodiments, in step S2, the temperature of the extraction is 25-50 ℃ (e.g., either or both of 30 ℃,35 ℃,40 ℃, 45 ℃).

In some embodiments, in step S2, the extraction pressure is 20-50MPa (e.g., any one of 25MPa, 30MPa, 35MPa, 40MPa, 45MPa, or a range therebetween).

In some embodiments, in step S2, the flow rate of carbon dioxide is 500-950 kg/hr (e.g., 550 kg/hr, 600 kg/hr, 650 kg/hr, 700 kg/hr, 750 kg/hr, 800 kg/hr, 850 kg/hr, 900 kg/hr, or a range therebetween).

In some embodiments, in step S2, the extraction time is 1-3h (e.g., any one of 1.5h, 2h, 2.5h, or a range therebetween).

In some embodiments, in step S2, the weight ratio of the sea fennel stem cells to the extract obtained is 1:1.5-4.0 (e.g., any ratio or range between 1:2.0, 1:2.5, 1:3.0, 1:3.5).

In some embodiments, in step S2, the residue obtained from the extraction is subjected to a second extraction, and a second extract obtained from the second extraction is combined with the extract.

In some embodiments, the amount of material and extraction conditions of the second extraction are the same as the extraction.

In some embodiments, in step S3, the enzyme is used in an amount by weight to the sea fennel stem cells: 2-10:1000000 (e.g., any ratio of 3:1000000, 4:1000000, 1000000, 5:1000000, 6:1000000, 7:1000000, 8:1000000, 9:1000000, or ranges therebetween).

In some embodiments, in step S3, the cellulase, the trypsin and the pectase are used in an amount by weight ratio of: 1:1-2:2-3 (e.g., any value or range between any value of 1:1.2, 1.4, 1.6, 1.8: 2.2, 2.4, 2.6, 2.8).

In some embodiments, in step S3, the enzymatic hydrolysis is performed for a period of 2-3 hours, and the enzymatic hydrolysis is performed at a temperature of 30-55 ℃ (e.g., 35 ℃,40 ℃, 45 ℃ or any value or range therebetween).

In some embodiments, after the completion of the enzymatic hydrolysis in step S3, the reaction system is inactivated at 85-95 ℃ (e.g., 86 ℃, 88 ℃, 90 ℃, 92 ℃, 94 ℃ or any value therebetween) for 3-10 minutes (e.g., 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes or any value therebetween).

In some embodiments, the concentration is a vacuum concentration with a vacuum level of 100-120Kpa (e.g., any one or range between 105Kpa, 110Kpa, 115 Kpa).

In some embodiments, the weight yield of the sea fennel stem cell extract relative to the sea fennel stem cells is 10-20% (e.g., any one of 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or a range therebetween) via the concentrating.

According to a second aspect of the present invention, there is provided a maritime fennel stem cell extract prepared according to the preparation method of the first aspect of the present invention.

In a third aspect, the invention provides a composition comprising, by weight, 10-30 parts (e.g., 12 parts, 14 parts, 16 parts, 18 parts, 20 parts, 22 parts, 24 parts, 26 parts, 28 parts, or a range therebetween) of a sea fennel stem cell extract of the second aspect of the invention, 6-18 parts of lime tea extract, 20-40 parts (e.g., 22 parts, 24 parts, 26 parts, 28 parts, 30 parts, 32 parts, 24 parts, 36 parts, 38 parts, or a range therebetween) of pomegranate seed concentrate, 10-20 parts (e.g., 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts, or a range therebetween), 3-7 parts (e.g., 4 parts, 5 parts, 6 parts, or a range therebetween) of glycerin, and 10-20 parts (e.g., 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts, or a range therebetween) of vitamin E.

In some embodiments, the dosage form of the composition is selected from: lotions, creams, masks, eye creams and essences.

In a fourth aspect, the present invention provides a process for the preparation of the composition of the third aspect of the present invention, the process comprising: mixing the sea fennel stem cell extract, the lime tea extract, the pomegranate seed extract, the milk extract, the glycerin and the vitamin E.

The fifth aspect of the invention provides the use of the preparation method according to the first aspect of the invention, the sea fennel stem cell extract according to the second aspect of the invention, the composition according to the third aspect of the invention or the preparation method according to the fourth aspect of the invention for preparing an antioxidant, whitening or anti-aging preparation.

The composition of the invention has good DPPH radical, hydroxyl radical and superoxide anion radical scavenging effect.

CO ₂ Too large a flow of water can cause CO in the extractor ₂ Flow rate is increased, CO ₂ The residence time is shortened, the contact time with the extracted matter is reduced, and the improvement of the extraction rate is not facilitated. On the other hand, however, CO ₂ The flow rate of the extraction process is increased, the mass transfer driving force in the extraction process can be increased, the mass transfer coefficient is correspondingly increased, and the mass transfer rate is accelerated, so that the extraction capacity of the SFE is improved. Thus, the CO is reasonably selected ₂ Is of considerable importance in SFE.

Drawings

FIG. 1 is a statistical plot of individual compositions versus three radical scavengers, three columns from left to right representing DPPH scavenger, OH scavenger, ABTS, respectively, for each composition ⁺ Clearance, in% on the ordinate.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the embodiments of the present invention will be described in further detail with reference to the accompanying drawings.

In the present invention, all the equipment and materials are commercially available or commonly used in the industry, and the methods in the examples described below are conventional in the art unless otherwise specified.

Material

Extracting the lime tea: the lime tea is prepared by fermenting fresh lime skin, and the lime skin has a great amount of skin beautifying components, so that the lime tea is rich in ascorbic acid and tannic acid from hydroxy acid capable of smoothing horny layer. The lime tea has an antioxidant effect, and the essence components extracted by the molecular distillation technology can help skin to repair and maintain the skin state stably and uniformly. The dried lime tea powder is crushed and sieved to obtain 60-mesh (particle size is 0.3 mm) powder, three extraction solvents of dichloromethane, normal hexane and ethyl acetate are used, the ratio of feed liquid is 1:20 (g/mL), after ultrasonic treatment is carried out for 15min at 40 ℃ and 90W, the extraction is carried out for 2h by low-temperature distillation, and the refined lime tea extract used by the invention is obtained.

Pomegranate seed essence: the main active ingredient is punica granatum seed polyphenol with the content of 8wt%. Purchased from shanxi Hao biotechnology limited.

Milk essence is produced from Symrise

Milk: contains 3 major proteins, wherein casein is present in an amount of up to about 83% of total protein, lactalbumin is present in an amount of about 13% and lactoglobulin and a small amount of fat globule membrane protein are present in an amount of about 4%; there are various enzymes such as peroxidases, reductases, lipolytic enzymes, lactases.

The sea fennel stem cells are undifferentiated natural cells in sea fennel plant tissues, can differentiate into various types of cells of plants and potentially contain all components of the plants, are freeze-dried powder of sea fennel callus cultures obtained by a plant tissue culture mode, are sea fennel root stem cells, are brand name of agaricus, and are C-34 sea fennel stem cells.

Example 1

A preparation method of a sea fennel stem cell extract comprises the following steps:

(1) Accurately weighing 20g of sea fennel root stem cells (agar collecting), and carrying out ultrasonic pretreatment for 30min under the conditions of power of 200W and temperature of 25 ℃.

The sonication breaks up the cells, allowing for a more complete release of the active ingredient.

(2) Supercritical extraction stage:

first supercritical extraction: placing pretreated sea fennel stem cells into an extraction tank (HA 220-50-06 type supercritical extraction device; nantong Hua' an supercritical extraction Co., ltd.) for supercritical extraction for 2 hours at 30 ℃ under 30MPa at 600 kg/h of carbon dioxide flow rate to obtain 40ml of extract A1 and residue B1;

the residue B1 was subjected to a second supercritical extraction for 2 hours at 30℃under 30MPa and at a carbon dioxide flow rate of 600 kg/h to give 25ml of extract C1.

(3) Enzymolysis: to the extract (65 ml of mixture of extracts A1 and C1) 1 x 10 was added ^-4 And (3) carrying out enzymolysis on g hydrolase (cellulase, trypsin and pectase in a weight ratio of 2:3:5), wherein the enzymolysis time is 2 hours, the enzymolysis temperature is 55 ℃, the temperature is increased to 90 ℃ after the enzymolysis is finished, the enzymolysis is kept for 5 minutes for inactivation, and the vacuum concentration is carried out under the vacuum degree of 80KPa, so as to finally obtain 2.5g of the sea fennel stem cell extract D1.

The extract D1 contains sea fennel essential oil, CIC-2, anisole, alpha-anisole, methyl piperonyl alcohol (methyl havicol), anisaldehyde, vitamin C, etc.

(4) Preparation of the composition: the composition comprises, by weight, 1 part of a sea fennel stem cell extract D, 12 parts of a lime tea extract, 32 parts of a pomegranate seed essence, 16 parts of a milk essence, 5 parts of glycerol and 15 parts of vitamin E.

The preparation method comprises the following steps: the materials are stirred and mixed uniformly to obtain the composition E1.

Example 2

(2) Supercritical extraction stage:

first supercritical extraction: placing pretreated sea fennel stem cells into an extraction tank (HA 220-50-06 type supercritical extraction device; nantong Hua' an supercritical extraction Co., ltd.) for supercritical extraction for 2 hours at 30 ℃ under 35MPa at 600 kg/h of carbon dioxide flow rate to obtain 45ml of extract A2 and residue B2;

and carrying out second supercritical extraction on the residue B2 for 2 hours at the temperature of 30 ℃, the extraction pressure of 30MPa and the flow rate of carbon dioxide of 600 kg/h, and obtaining 25ml of extract C2 after extraction.

(3) Enzymolysis: to the extract (70 ml of mixture of extracts A2 and C2) 1 x 10 was added ^-4 And (3) carrying out enzymolysis on g hydrolase (cellulase, trypsin and pectase in a weight ratio of 2:3:5), wherein the enzymolysis time is 2 hours, the enzymolysis temperature is 55 ℃, the temperature is increased to 90 ℃ after the enzymolysis is finished, the enzymolysis is kept for 5 minutes for inactivation, and the vacuum concentration is carried out under the vacuum degree of 80KPa, so as to finally obtain 3.0g of the sea fennel stem cell extract D2.

(4) Preparation of the composition: the composition comprises, by weight, 20 parts of a sea fennel stem cell extract D, 12 parts of a lime tea extract, 32 parts of a pomegranate seed essence, 16 parts of a milk essence, 5 parts of glycerol and 15 parts of vitamin E.

The preparation method comprises the following steps: the materials are stirred and mixed uniformly to obtain the composition E2.

Example 3

(2) Supercritical extraction stage:

first supercritical extraction: placing pretreated sea fennel stem cells into an extraction tank (HA 220-50-06 type supercritical extraction device; nantong Hua' an supercritical extraction Co., ltd.) for supercritical extraction for 2 hours at 50 ℃ under 45MPa at a flow rate of 800 kg/h of carbon dioxide to obtain 42ml of extract A3 and residue B3;

and carrying out second supercritical extraction on the residue B3 for 2 hours at 50 ℃, wherein the extraction pressure is 45MPa, the flow rate of carbon dioxide is 800 kg/h, and 23ml of extract C3 is obtained after extraction.

(3) Enzymolysis: to the extract (65 ml of mixture of extracts A3 and C3) 1 x 10 was added ^-4 And (3) carrying out enzymolysis on g hydrolase (cellulase, trypsin and pectase in a weight ratio of 2:3:5), wherein the enzymolysis time is 2 hours, the enzymolysis temperature is 55 ℃, the temperature is increased to 90 ℃ after the enzymolysis is finished, the enzymolysis is kept for 5 minutes for inactivation, and the vacuum concentration is carried out under the vacuum degree of 80KPa, so as to finally obtain 2.5g of the sea fennel stem cell extract D3.

(4) Preparation of the composition: the composition comprises, by weight, 3 parts of a sea fennel stem cell extract D, 12 parts of a lime tea extract, 32 parts of a pomegranate seed essence, 16 parts of a milk essence, 5 parts of glycerol and 15 parts of vitamin E.

The preparation method comprises the following steps: the materials are stirred and mixed uniformly to obtain the composition E3.

Example 4

(2) Supercritical extraction stage:

first supercritical extraction: placing pretreated sea fennel stem cells into an extraction tank (HA 220-50-06 type supercritical extraction device; nantong Hua' an supercritical extraction Co., ltd.) for supercritical extraction for 2 hours at 30 ℃ under 30MPa at 800 kg/h of carbon dioxide flow rate to obtain 40ml of extract A4 and residue B4;

and carrying out second supercritical extraction on the residue B4 for 2 hours at the temperature of 30 ℃, the extraction pressure of 30MPa and the flow rate of carbon dioxide of 800 kg/h, and obtaining 25ml of extract C4 after extraction.

(3) Enzymolysis: to the extract (65 ml of mixture of extracts A4 and C4) 1 x 10 was added ^-4 And (3) carrying out enzymolysis on g hydrolase (cellulase, trypsin and pectase in a weight ratio of 2:3:5), wherein the enzymolysis time is 2 hours, the enzymolysis temperature is 55 ℃, the temperature is increased to 90 ℃ after the enzymolysis is finished, the enzymolysis is kept for 5 minutes for inactivation, and the vacuum concentration is carried out under the vacuum degree of 80KPa, so as to finally obtain 2.5g of the sea fennel stem cell extract D4.

(4) Preparation of the composition: the composition comprises, by weight, 4 parts of a sea fennel stem cell extract D4, 12 parts of a lime tea extract, 32 parts of a pomegranate seed essence, 16 parts of a milk essence, 5 parts of glycerol and 15 parts of vitamin E.

The preparation method comprises the following steps: the materials are stirred and mixed uniformly to obtain the composition E4.

Comparative example 1

(2) Supercritical extraction stage:

first supercritical extraction: placing pretreated sea fennel stem cells into an extraction tank (HA 220-50-06 type supercritical extraction device; nantong Hua' an supercritical extraction Co., ltd.) for supercritical extraction for 2 hours at 30 ℃ under 30MPa at 600 kg/h of carbon dioxide flow rate to obtain 40ml of extract A5 and residue B5;

and carrying out second supercritical extraction on the residue B5 for 2 hours at the temperature of 30 ℃, the extraction pressure of 30MPa and the flow rate of carbon dioxide of 600 kg/h, and obtaining 25ml of extract C5 after extraction.

(3) Mixing: the extract A1 and C1 were mixed to obtain 65ml of a mixture, and concentrated under vacuum at 80KPa to obtain 3.7g of a sea fennel stem cell extract D5.

(4) Preparation of the composition: the composition comprises, by weight, 5 parts of a sea fennel stem cell extract D5, 12 parts of a lime tea extract, 32 parts of a pomegranate seed essence, 16 parts of a milk essence, 5 parts of glycerol and 15 parts of vitamin E.

The preparation method comprises the following steps: the materials are stirred and mixed uniformly to obtain the composition E5.

Comparative example 2

(1) Supercritical extraction stage:

first supercritical extraction: accurately weighing 20g of sea fennel stem cells (agar collecting), placing in an extraction tank (HA 220-50-06 type supercritical extraction device; nantong Hua' an supercritical extraction Co., ltd.) for supercritical extraction at 30deg.C for 2 hr to obtain 31ml of extract A6 and residue B6;

the residue B6 is subjected to a second supercritical extraction for 2 hours at 30 ℃ under 30MPa at a flow rate of 600 kg/h of carbon dioxide to obtain 22ml of extract C6.

(3) Enzymolysis: to the extract (54 ml of mixture of extracts A6 and C6) 1 x 10 was added ^-4 And (3) carrying out enzymolysis on g hydrolase (cellulase, trypsin and pectase in a weight ratio of 2:3:5), wherein the enzymolysis time is 2 hours, the enzymolysis temperature is 55 ℃, the temperature is increased to 90 ℃ after the enzymolysis is finished, the enzymolysis is kept for 5 minutes for inactivation, and the vacuum concentration is carried out under the vacuum degree of 80KPa, so as to finally obtain 1.7g of the sea fennel stem cell extract D6.

(4) Preparation of the composition: the composition comprises, by weight, 6 parts of a sea fennel stem cell extract D6, 12 parts of a lime tea extract, 32 parts of a pomegranate seed essence, 16 parts of a milk essence, 5 parts of glycerol and 15 parts of vitamin E.

The preparation method comprises the following steps: the materials are stirred and mixed uniformly to obtain the composition E6.

Comparative example 3

(1) Supercritical extraction stage:

first supercritical extraction: accurately weighing 20g of sea fennel stem cells (agar collecting device), placing in an extraction tank (HA 220-50-06 type supercritical extraction device; nanto Hua Ann supercritical extraction Co., ltd.) for supercritical extraction at 30deg.C for 2 hours under the conditions of 30MPa and 600 kg/hr carbon dioxide flow rate to obtain 40ml of extract A7 and residue B7;

the residue B7 was subjected to a second supercritical extraction for 2 hours at 30℃under 30MPa and at a carbon dioxide flow rate of 600 kg/h to give 25ml of extract C7.

(3) Mixing: the extract A7 and C7 were mixed to obtain 65ml of a mixture, and concentrated under vacuum at 80KPa to obtain 2.0g of a sea fennel stem cell extract D7.

(4) Preparation of the composition: the composition comprises, by weight, 7 parts of a sea fennel stem cell extract D, 12 parts of a lime tea extract, 32 parts of a pomegranate seed essence, 16 parts of a milk essence, 5 parts of glycerol and 15 parts of vitamin E.

The preparation method comprises the following steps: the materials are stirred and mixed uniformly to obtain the composition E7.

E1-E7 are all emulsions.

Test example 1: in vitro antioxidant effect experiment

(1) Measurement of DPPH radical removal Rate:

according to the method described in the literature (Hu Weiwei et al, food industry, volume 35, 2 nd, pages 144-119) of the enzymatic extraction process of chicken-derived collagen peptide and the study of its free radical scavenging ability, 0.3g of each of the compositions E1-E7 was taken, placed in 50ml of absolute ethanol, and stirred at room temperature for 12 hours to obtain a sample solution.

Taking 2ml of sample solution and DPPH solution as Ai (sample inhibition absorption value) respectively; the sample solution and absolute ethanol were Ac (sample control); DPPH solution and absolute ethyl alcohol are Aj (no inhibition absorption value); absolute ethanol is a control with no inhibition of absorption values. The mixture was thoroughly mixed by shaking, left standing at room temperature for 30min, and the absorbance was measured at 517nm using a 1cm cuvette (two tubes were measured simultaneously for each value).

DPPH radical scavenging rate was calculated according to the method described in the above document and the results are shown in Table 1.

Wherein A is ₀ Representing the absorption at 0min.

A ₁ Representing the absorption value at 30min.

(2) Hydroxyl radical clearance:

according to the method described in the literature (Chen Guanhua et al, spectroscopy and spectral analysis, 2002,22 (4), pages 634-636), the above-mentioned aqueous solutions of samples were prepared into solutions of a certain mass concentration (the concentrations are 6mg/m L, respectively), 1.0mL of each solution was taken, and 1.4mL of crystal violet solution (2X 10) was added to a series of 10mL capped cuvettes, respectively ^-5 mol·L ^-1 )、1.0mL Fe ²⁺ Solution (5X 10) ^-3 mol·L ^-1 )、0.4mL H ₂ O ₂ (1%) solution, 1.0mL Tris-HCl solution (pH 5.5), diluted to 10mL with double distilled water and shaken well, and after 5min of standing, the absorbance was measured at a wavelength of 588 nm. Not adding H ₂ O ₂ As background tube. VC was used as a positive control.

The hydroxyl radical scavenging rate was calculated according to the method described in the foregoing document by the following formula, and the results are shown in table 1. Representing:

D＝[(A _S -A ₀ )/(A-A ₀ )×100％]

d represents: hydroxyl radical scavenging rate.

A _S Representing: absorbance of the system to which the composition is added;

a represents: absorbance of the system without Fenton reagent and composition;

A ₀ representing: absorbance of the system without the composition.

(3) Superoxide anion radical (ABTS) ⁺ ) Clearance rate:

according to the method described in the literature (Hu Weiwei et al, food industry, volume 35, 2 nd, pages 144-119) of the enzymatic extraction process of chicken-derived collagen peptide and the study of its free radical scavenging ability, the measurement of the scavenging rate of superoxide anions was carried out by the pyrogallol method. The corresponding absorbance was measured at a wavelength of 325nm, and as a result, VC was used as a positive control.

The superoxide anion radical scavenging rate was calculated according to the method described in the foregoing document, and the results are shown in table 1 and fig. 1.

Table 1 scavenging ability of compositions to free radicals

From this, it can be seen that E3 has a strong clear ability for the three aforementioned radicals.

It will be appreciated by those skilled in the art that the present invention can be carried out in other embodiments without departing from the spirit or essential characteristics thereof. Accordingly, the above disclosed embodiments are illustrative in all respects, and not exclusive. All changes that come within the scope of the invention or equivalents thereto are intended to be embraced therein.

Claims

1. A method for preparing a sea fennel stem cell extract, the method comprising the steps of:

s3: and (3) carrying out enzymolysis on the extract liquid by using enzymes to obtain the sea fennel stem cell extract, wherein the enzymes comprise cellulase, trypsin and pectase.

2. The method of manufacturing according to claim 1, further comprising the steps of:

3. The method of claim 1, wherein the sea fennel stem cells are freeze-dried powders of sea fennel callus cultures.

4. The method of claim 1, wherein the particles of the sea fennel stem cells have a size of 50nm to 150nm.

5. The method of claim 1, wherein in step S1, the ultrasound conditions are: the power is 100-300W, the temperature is 20-30 ℃ and the time is 20-40min.

6. The method according to claim 1, wherein in step S2, the supercritical extraction is performed using carbon dioxide as an extraction gas.

7. The process according to claim 1, wherein in step S2, the extraction temperature is 25-50 ℃.

8. The method according to claim 1, wherein in step S2, the extraction pressure is 20 to 50MPa.

9. The method according to claim 6, wherein the flow rate of the carbon dioxide is 500 to 950 kg/hr in step S2.

10. The process according to claim 1, wherein in step S2, the extraction time is 1 to 3 hours.

11. The method according to claim 1, wherein in step S2, the weight ratio of the sea fennel stem cells to the obtained extract is 1:1.5-4.0.

12. The process according to claim 1, wherein in step S2, the residue obtained from the extraction is subjected to a second extraction, and a second extract obtained from the second extraction is used in combination with the extract.

13. The method of claim 12, wherein the material used in the second extraction and the extraction conditions are the same as the extraction.

14. The method of claim 1, wherein in step S3, the enzyme is used in an amount by weight ratio to the sea fennel stem cells: 2-10:1000000.

15. The method according to claim 1, wherein in step S3, the cellulase, trypsin and pectase are used in the following weight ratio: 1:1-2:2-3.

16. The method according to claim 1, wherein in step S3, the time of the enzymolysis is 2 to 3 hours, and the temperature of the enzymolysis is 30 to 55 ℃.

17. The preparation method according to claim 1, wherein in step S3, after the completion of the enzymolysis, the reaction system is inactivated at 85 to 95 ℃ for 3 to 10 minutes.

18. The method of claim 2, wherein the concentration is vacuum concentration with a vacuum of 100 to 120KPa.

19. The method of claim 2, wherein the yield of the extract of sea fennel stem cells relative to the weight of sea fennel stem cells is 10-20% by concentration.

20. A maritime fennel stem cell extract prepared according to the preparation method of any one of claims 1-19.

21. A composition comprising, by weight, 10-30 parts of the sea fennel stem cell extract of claim 20, 6-18 parts of lime tea extract, 20-40 parts of pomegranate seed extract, 10-20 parts of milk extract, 3-7 parts of glycerin and 10-20 parts of vitamin E.

22. The composition of claim 21, wherein the composition is in a dosage form selected from the group consisting of: lotions, creams, masks, eye creams and essences.

23. A process for preparing the composition of claim 21 or 22, said process comprising: mixing the sea fennel stem cell extract, the lime tea extract, the pomegranate seed extract, the milk extract, the glycerin and the vitamin E.

24. Use of the method of preparation of any one of claims 1-19, the sea fennel stem cell extract of claim 20, the composition of claim 21 or 22, or the method of preparation of claim 23 for the preparation of an antioxidant, whitening or anti-aging formulation.