CN108721202B - Roselle stem cell freeze-dried powder eye cream and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of cosmetics, and particularly relates to roselle stem cell freeze-dried powder eye cream and a preparation method thereof. The invention utilizes the functions of oxidation resistance, stimulation of human skin keratinocyte proliferation and aging resistance of roselle stem cells, provides a culture method of the roselle stem cells with short culture period, and the roselle stem cells are freeze-dried into powder, the freeze-dried powder content of the obtained roselle stem cells is high, in addition, the freeze-dried powder of the roselle stem cells is combined with components such as polymerized almond protein, Fufang gold, dimethyl siloxane, caprylic/capric triglyceride, isopropyl palmitate, synthesized squalane, cetostearyl alcohol ether, L-proline and the like, and the eye cream with high efficiency for repairing damaged cells of eyes, resisting wrinkles, eliminating black eye circles and eye bags and tightening eye skin is prepared.

Description

Roselle stem cell freeze-dried powder eye cream and preparation method thereof

Technical Field

The invention belongs to the technical field of daily cosmetics, and particularly relates to roselle stem cell freeze-dried powder eye cream and a preparation method thereof.

Background

At present, eye problems such as black eye circles, eye bags, eye tail wrinkles and the like are mainly caused by internal reasons: endocrine is uncoordinated, mood fluctuation is large, eye fatigue and cell aging cause melanin accumulation which hinders eye blood circulation; external factors: solarization, smoking, staying up all night, habitual strabismus, and the collagen in the dermis is oxidized and broken.

Aiming at the two conditions, a plurality of treatment methods and products are provided on the market, the treatment methods comprise traditional Chinese medicine conditioning and western medicine operations, the traditional Chinese medicine conditioning is mainly used for taking traditional Chinese medicines to condition internal secretion, further improving the surface muscles and relieving the eye problems, the method is long in time consumption, the traditional Chinese medicines are troublesome to decoct, and the effects can be achieved by diet compatibility; western medicine operation is a new popular treatment method in the present year, and skin regeneration function is stimulated by removing aging and redundant skin, but the method has large difference of individual eye conditions, high technical requirements, high risk and high cost, and most people with eye problems are reluctant to try.

Various eye creams, eye essences, eye masks and the like are available on the market, the problems of black eye circles, under-eye puffiness, canthus wrinkles and the like are solved by adding a plurality of traditional Chinese medicine extracts, but most of the eye creams temporarily relieve the eye problems by moisturizing, or the eye skin cannot absorb too much traditional Chinese medicine extracts to form eye fat granules and the like due to the fact that the eye skin is thin (0.3mm), and the self-repair and regeneration of the eye skin cannot be effectively stimulated fundamentally.

Since 2003, Dr.Fred Zuli, Swiss scientist discovered that the apple stem cell has the cosmetic miraculous effects of removing wrinkles, resisting aging and prolonging the service life of cells, and then the apple stem cell is applied to cosmetics for the first time, so that a foundation is laid for the research field of plant stem cells for delaying aging. Plant stem cell beauty technology and related products thereof are receiving wide attention from experts in the beauty industry and the industry, and after apple stem cells, lithospermum stem cells and argan nut stem cells have also been developed into beauty products, for example, Chinese patent application No. 201610268493.6 discloses aloe stem cell freeze-dried powder and a preparation method and application thereof, and the method can obtain a large amount of aloe stem cells and does not damage the content of the main component aloin. For this reason, with the development of modern technologies, it is a trend to utilize stem cells that are not fully differentiated and have the potential for unlimited self-renewal replication and differentiation for use in the cosmetic industry.

Hibiscus sabdariffa Linn, also known as Hibiscus sabdariffa Linn, Hibiscus sabdariffa, HONGJINMEI, and HONGMEI fruit, is a one-year-old herb of Hibiscus in Malvaceae, and is produced in tropical and subtropical zones. The flavonoids contained in the flowers of Hibiscus Sabdariffa comprise flavonol and flavanol, and are in the form of monomer or polymer, including hibiscus glycoside, gossyposide, quercetin, luteolin, chlorogenic acid, protocatechuic acid, eugenol, ergosterol, gamma-tocopherol, etc. Various substances have biological activities of oxidation resistance and aging resistance, so the roselle stem cells have stronger oxidation resistance, are pure natural, nontoxic and pollution-free anti-aging plant stem cells, roselle is widely planted in Guangdong, Guangxi, Fujian, Yunnan and other places in China, the roselle stem cells are prepared into freeze-dried powder, and then the freeze-dried powder is applied to the preparation of cosmetics, particularly anti-aging products, so that the problems of the sources and the quality of cosmetic raw materials are not needed to be worried, the new field of the cosmetics is developed, and the first plant stem cell anti-aging products in China are developed.

Disclosure of Invention

In order to solve the problems of long time, high risk and high cost of the prior art for treating the eye problems, the eye skin care product only has the effects of surface moisturizing and moisture retention, and the problem of eye skin aging is not fundamentally solved.

The invention provides roselle stem cell freeze-dried powder eye cream which comprises the following components in parts by weight:

8.0-20.0g of roselle stem cell freeze-dried powder, 1.0-4.0g of propylene glycol, 2.0-6.0g of glycerol, 1.0-5.0g of butanediol, 0.6g of carbomer 9400.1, 0.1-0.2g of preservative, 0.5-2.0g of dimethyl siloxane, 1.0-4.0g of caprylic/capric triglyceride, 1.0-4.0g of isopropyl palmitate, 1.0-3.0g of synthetic squalane, 2.0-6.0g of cetylstearyl alcohol ether, 1.0-3.0g of polymerized almond protein, 0.5-2.0g of rich ether platinum, 0.2-0.4g of triethanolamine and 50-100g of water.

Preferably, the roselle stem cell freeze-dried powder eye cream has the following formula:

10ng of roselle dry cell freeze-dried powder, 0.15g of preservative, 2g of propylene glycol, 4g of glycerol, 3g of butanediol, 0.35g of carbomer, 1g of dimethyl siloxane, 3g of caprylic/capric triglyceride, 3g of isopropyl palmitate, 2g of synthetic squalane, 3.5g of ceteareth, 2g of polymeric almond protein, 1g of Fufang gold, 0.35g of triethanolamine and 85g of water.

Furthermore, the roselle stem cell freeze-dried powder eye cream provided by the invention is characterized in that the cetyl stearyl alcohol ether is cetyl alcohol ether-2.

Further, the roselle stem cell freeze-dried powder eye cream provided by the invention is Euxyl K350 as a preservative.

Further, the preparation method of the roselle stem cell freeze-dried powder eye cream provided by the invention comprises the following steps:

a. taking roselle tissues, cleaning and disinfecting;

b. cutting the obtained roselle into 5mm by 5mm, inoculating to solid culture medium containing zinc sulfate 2-3mg/L, citric acid 75-80mg/L, glycine 78-82mg/L, indoleacetic acid 1.0-2.0mg/L, sucrose 10.5-11.5mg/L, active carbon 0.45-1g/L, barbaloin 3-5mg/ml and agar 2.8-3.5mg/L, culturing at 24-28 deg.C and pH 6.2-6.8 for 12-18 days to obtain roselle dry cells from cambium;

c. transferring the roselle stem cells from cambium to a liquid culture medium containing 180 mg/L magnesium sulfate, 75-80mg/L citric acid, 76.0-84.0mg/L glycine, 1.0-2.0mg/L indoleacetic acid, 32.0-34.0mg/L sucrose, 3-5mg/ml barbaloin, 65-75mg/L medical stone particles and 65-75mg/L selenium stone particles for culture;

d. c, transferring the roselle stem cells with the cambium source cultured and collected in the step c into an airlift bioreactor for culture, continuously stirring the culture solution at normal temperature by using the liquid culture solution obtained in the step c, culturing until the 7 th day, and injecting cane sugar, methyl jasmonate and sterile magnetized water into the airlift bioreactor;

e. filtering the culture solution in the airlift bioreactor in the step d by a filter, taking out the trapped cell line culture, placing the trapped cell line culture in a refrigerator with the temperature of 80 ℃ below zero for ultralow temperature freezing for 2 to 3 hours, taking out the cell line culture, thawing for 30 to 40min at room temperature, and repeating for 3 to 5 times; after the last thawing, crushing the cells by using an ultrasonic crusher, collecting filtrate, and concentrating to obtain a roselle stem cell extract;

f. and e, adding water into the roselle stem cell extract in the step e to adjust the final concentration of protein to be 50 +/-0.5 mu g/ml, adding a freeze-drying protective agent accounting for 4-6% of the total mass of the roselle stem cell extract, sterilizing, and freeze-drying at the temperature of-20 to-35 ℃ and the vacuum degree of 50-200 Pa to obtain the roselle stem cell freeze-dried powder.

Preferably, in the preparation of the roselle stem cell freeze-dried powder eye cream provided by the invention, the roselle tissue cut in the step a is roselle root tip with root caps removed.

Preferably, in the preparation of the roselle stem cell freeze-dried powder eye cream provided by the invention, the filter in the step e is a sterile microporous filter membrane with the thickness of 0.45 mu M, the ultrasonic conditions are 4 ℃, 40W, 3s ultrasonic and 3s interval, and the total time is 9 min.

Preferably, in the preparation of the roselle stem cell lyophilized powder eye cream provided by the invention, the lyophilized protectant in the step f is L-proline, which is one of components of plant proteins, is an ideal osmotic adjusting substance, can be used as a protective substance for membranes and enzymes and a free radical scavenger, and can play a role in adjusting cytoplasmic osmotic balance.

The preparation method of the roselle stem cell freeze-dried powder eye cream comprises the following steps:

s1: mixing propylene glycol, glycerol, butanediol, carbomer 940 and antiseptic, dissolving in water, heating to 75-80 deg.C, and stirring to dissolve completely to obtain mixture A;

s2: mixing dimethyl siloxane, caprylic capric triglyceride, isopropyl palmitate, synthetic squalane and ceteareth, dissolving in water, heating to 75-80 deg.C, and stirring to dissolve completely to obtain mixture B;

s3: adding the mixture B prepared in the step S2 into the mixture A prepared in the step S1, putting the mixture A into a homogenizer for 3-5 min, and cooling to 30-50 ℃ to obtain a mixture C;

s4: dissolving the roselle stem cell freeze-dried powder, the polymerized almond protein and the ether-rich Spanish gold in water, and stirring until the components are completely dissolved to obtain a mixture D;

s5: and (4) adding the mixture D prepared in the step S4 and triethanolamine into the mixture C prepared in the step S3, and homogenizing to obtain the alcohol-based fuel.

The preparation method of the freeze-dried powder of the roselle stem cells provided by the invention has no cell variation in the culture process, the proliferation and division of the cells are rapid, the purity of the roselle stem cells which are separated and cultured is high and can reach more than 95%, and the popularization and the application of the preparation method of the freeze-dried powder of the roselle stem cells are facilitated.

In the preparation of the roselle stem cell freeze-dried powder, the indoleacetic acid in the culture medium is plant auxin and promotes the proliferation and division of root tips to form layer cells, and the added barbaloin is an extract extracted from plants, so that the roselle stem cell freeze-dried powder has an antibacterial effect and reduces the addition of antibiotics.

Compared with the prior art, the roselle stem cell freeze-dried powder eye cream and the preparation method thereof provided by the invention have the following advantages:

1) according to the preparation method of the roselle stem cell freeze-dried powder, the aloin is added, so that the aloin has an antibacterial effect, is combined with indoleacetic acid, obviously shortens the cell culture period and promotes cell proliferation;

2) the roselle stem cell freeze-dried powder prepared by the invention has simple preparation method and high repeatability, utilizes the effects of repairing damaged cells, promoting cell regeneration and protecting skin of roselle stem cells, combines polymerized almond protein, rich ether platinum and synthetic canthashark alkane, and is synergistic with each other, plays a role in tightening skin, and has the effects of resisting aging and prolonging the service life of cells on human skin;

3) the roselle stem cell freeze-dried powder eye cream prepared by the invention has obvious improvement effect on fishtail lines, eye bags, black eyes and skin laxity of eyes.

The specific implementation mode is as follows:

example 1: a preparation method of roselle stem cell freeze-dried powder comprises the following steps:

a. taking the cut root tips of roselle, cleaning and disinfecting;

b. cutting the obtained roselle into 5mm by 5mm, inoculating to solid culture medium containing zinc sulfate 2.5mg/L, citric acid 75.5mg/L, glycine 80.0mg/L, indoleacetic acid 1.5mg/L, sucrose 11.0mg/L, activated carbon 0.8g/L, barbaloin 4.5mg/ml and agar 3.2mg/L, culturing at 25 deg.C and pH 6.5 for 14 days to obtain roselle dry cells from cambium;

c. transferring the roselle stem cells from cambium to a liquid culture medium containing 181mg/L magnesium sulfate, 79mg/L citric acid, 78.0mg/L glycine, 1.5mg/L indoleacetic acid, 33.0mg/L sucrose, 2.5mg/ml barbaloin, 70.0mg/L medical stone particles and 70.0mg/L selenium stone particles for culture;

d. c, transferring the roselle stem cells with the cambium source cultured and collected in the step c into an airlift bioreactor for culture, continuously stirring the culture solution at normal temperature by using the liquid culture solution obtained in the step c, culturing until the 7 th day, and injecting cane sugar, methyl jasmonate and sterile magnetized water into the airlift bioreactor;

e. d, enabling the culture solution in the airlift bioreactor in the step d to pass through a sterile microporous filter membrane of 0.45 mu M, taking out the trapped cell line culture, putting the trapped cell line culture into a refrigerator at minus 80 ℃ for ultralow temperature freezing for 2h, taking out the cell line culture, unfreezing the cell line culture for 30min at room temperature, and repeating the process for 4 times; and (3) after the final thawing, crushing the cells by using an ultrasonic crusher under the ultrasonic conditions of 4 ℃, 40W, 3s of ultrasonic and 3s of interval, wherein the total time is 9 min. Collecting the filtrate, and concentrating the filtrate to obtain the extract of the roselle stem cells;

f. and e, adding water into the roselle stem cell extract in the step e to adjust the final concentration of protein to be 50 +/-0.5 mu g/ml, adding 5 percent of L-proline in the total mass of the roselle stem cell extract, sterilizing, and freeze-drying at the temperature of-20 to-35 ℃ and the vacuum degree of 50Pa to obtain the roselle stem cell freeze-dried powder.

Comparative example 1: a preparation method of roselle stem cell freeze-dried powder comprises the following steps:

a. taking the cut root tips of roselle, cleaning and disinfecting;

b. cutting the obtained roselle into 5mm by 5mm, inoculating the roselle into a solid culture medium containing 2.5mg/L of zinc sulfate, 75.5mg/L of citric acid, 80.0mg/L of glycine, 1.5mg/L of indoleacetic acid, 11.0mg/L of sucrose, 0.8g/L of active carbon and 3.2mg/L of agar, culturing at the temperature of 25 ℃ and the pH value of 6.5 for 14 days to obtain roselle dry cells from cambium;

c. transferring the roselle stem cells from cambium to a liquid culture medium containing 181mg/L magnesium sulfate, 79mg/L citric acid, 78.0mg/L glycine, 1.5mg/L indoleacetic acid, 33.0mg/L sucrose, 70.0mg/L medical stone particles and 70.0mg/L selenium stone particles for culture;

d. c, transferring the roselle stem cells with the cambium source cultured and collected in the step c into an airlift bioreactor for culture, continuously stirring the culture solution at normal temperature by using the liquid culture solution obtained in the step c, culturing until the 7 th day, and injecting cane sugar, methyl jasmonate and sterile magnetized water into the airlift bioreactor;

e. d, enabling the culture solution in the airlift bioreactor in the step d to pass through a sterile microporous filter membrane of 0.45 mu M, taking out the trapped cell line culture, putting the trapped cell line culture into a refrigerator at minus 80 ℃ for ultralow temperature freezing for 2h, taking out the cell line culture, unfreezing the cell line culture for 30min at room temperature, and repeating the process for 4 times; and (3) after the final thawing, crushing the cells by using an ultrasonic crusher under the ultrasonic conditions of 4 ℃, 40W, 3s of ultrasonic and 3s of interval, wherein the total time is 9 min. Collecting the filtrate, and concentrating the filtrate to obtain the extract of the roselle stem cells;

f. and e, adding water into the roselle stem cell extract in the step e to adjust the final concentration of protein to be 50 +/-0.5 mu g/ml, adding 5 percent of L-proline in the total mass of the roselle stem cell extract, sterilizing, and freeze-drying at the temperature of-20 to-35 ℃ and the vacuum degree of 50Pa to obtain the roselle stem cell freeze-dried powder.

Comparative example 1 differs from the medium of example 1 in that comparative example 1 does not have barbaloin added.

Comparative example 2: a preparation method of roselle stem cell freeze-dried powder comprises the following steps:

a. taking the cut root tips of roselle, cleaning and disinfecting;

b. cutting the obtained roselle into 5mm by 5mm, inoculating the roselle into a solid culture medium containing 2.5mg/L of zinc sulfate, 75.5mg/L of citric acid, 80.0mg/L of glycine, 2.5mg/L of naphthylacetic acid, 11.0mg/L of cane sugar, 0.8g/L of active carbon and 3.2mg/L of agar, culturing at the temperature of 25 ℃ and the pH value of 6.5 for 14 days to obtain roselle dry cells from cambium;

c. transferring the roselle stem cells from cambium to a liquid culture medium containing 181mg/L magnesium sulfate, 79mg/L citric acid, 78.0mg/L glycine, 2.5mg/L naphthylacetic acid, 33.0mg/L sucrose, 70.0mg/L medical stone particles and 70.0mg/L selenium stone particles for culture;

d. c, transferring the roselle stem cells with the cambium source cultured and collected in the step c into an airlift bioreactor for culture, continuously stirring the culture solution at normal temperature by using the liquid culture solution obtained in the step c, culturing until the 7 th day, and injecting cane sugar, methyl jasmonate and sterile magnetized water into the airlift bioreactor;

e. d, enabling the culture solution in the airlift bioreactor in the step d to pass through a sterile microporous filter membrane of 0.45 mu M, taking out the trapped cell line culture, putting the trapped cell line culture into a refrigerator at minus 80 ℃ for ultralow temperature freezing for 2h, taking out the cell line culture, unfreezing the cell line culture for 30min at room temperature, and repeating the process for 4 times; and (3) after the final thawing, crushing the cells by using an ultrasonic crusher under the ultrasonic conditions of 4 ℃, 40W, 3s of ultrasonic and 3s of interval, wherein the total time is 9 min. Collecting the filtrate, and concentrating the filtrate to obtain the extract of the roselle stem cells;

f. and e, adding water into the roselle stem cell extract in the step e to adjust the final concentration of protein to be 50 +/-0.5 mu g/ml, adding 5 percent of L-proline in the total mass of the roselle stem cell extract, sterilizing, and freeze-drying at the temperature of-20 to-35 ℃ and the vacuum degree of 50Pa to obtain the roselle stem cell freeze-dried powder.

Comparative example 2 is different from example 1 in that the auxin in comparative example 2 replaces indoleacetic acid with naphthylacetic acid.

Examples 2 to 4: the roselle stem cell freeze-dried powder eye cream comprises the following components in parts by weight:

TABLE 1 EXAMPLE 2-4 ingredients and weights of Hibiscus sabdariffa Stem cell lyophilized powder eye cream

The roselle stem cell freeze-dried powder is prepared by the preparation method of the roselle stem cell freeze-dried powder in the embodiment 1.

The preparation method of the roselle stem cell freeze-dried powder eye cream comprises the following steps of respectively preparing the components and the weight thereof in examples 2-4 in table 1:

s1: mixing propylene glycol, glycerol, butanediol, carbomer 940 and a preservative, dissolving in water, heating to 75 ℃, and stirring to completely dissolve to obtain a mixture A;

s2: mixing dimethyl siloxane, caprylic capric triglyceride, isopropyl palmitate, synthetic squalane and ceteareth, dissolving in water, heating to 75 deg.C, and stirring to dissolve completely to obtain mixture B;

s3: adding the mixture B prepared in the step S2 into the mixture A prepared in the step S1, putting the mixture A into a homogenizer for 5min, and cooling to 30 ℃ to obtain a mixture C;

s4: dissolving the roselle stem cell freeze-dried powder, the polymerized almond protein and the ether-rich Spanish gold in water, and stirring until the components are completely dissolved to obtain a mixture D;

s5: and (4) adding the mixture D prepared in the step S4 and triethanolamine into the mixture C prepared in the step S3, and homogenizing to obtain the alcohol-based fuel.

The first test example: medium composition test in preparation of roselle stem cell freeze-dried powder eye cream

1. Test subjects: the freeze-dried powder of the roselle stem cells prepared in the example 1, the comparative example 1 and the comparative example 2.

2. The test method comprises the following steps:

taking cut roselle root tips, cleaning, sterilizing, cutting the obtained roselle into 5mm by 5mm, inoculating the roselle into a solid culture medium containing 2.5mg/L of zinc sulfate, 75.5mg/L of citric acid, 80.0mg/L of glycine, 1.5mg/L of indoleacetic acid, 11.0mg/L of sucrose, 0.8g/L of active carbon and 3.2mg/L of agar, culturing at the temperature of 25 ℃, with the pH value of 6.5, and culturing for 14 days to obtain roselle dry cells from cambium; transferring the roselle stem cells from cambium to a liquid culture medium containing 181mg/L magnesium sulfate, 79mg/L citric acid, 78.0mg/L glycine, 1.5mg/L indoleacetic acid, 33.0mg/L sucrose, 70.0mg/L medical stone particles and 70.0mg/L selenium stone particles for culture to obtain a cell suspension A;

the cell suspension A obtained above was divided into 3 parts on average, centrifuged at low speed to precipitate, the cells were collected, transferred to a 5L large-scale incubator to be cultured, and cultured with the liquid media of example 1, comparative example 1 and comparative example 2, respectively, 4L of fresh liquid media of example 1, comparative example 1 and comparative example 2 were added after 5 days of culture, the culture was continued without medium addition in the middle, and the cells were collected after 15 days and weighed. 3. And (3) test results:

the test results are shown in Table 2, and the results show that the cell weight of the medium added with the indoleacetic acid and the barbaloin is obviously higher than that of the comparative example 1 without the barbaloin and that of the comparative example 2 with the naphthylacetic acid and the barbaloin, which indicates that the indoleacetic acid and the barbaloin can promote the cell proliferation and division and shorten the culture time.

TABLE 2 cell weights

Detecting the index	Example 1	Comparative example 1	Comparative example 2
				Cell sap weight (mg)	687.8	345.7	350.7

Test example two: culture time of roselle tissue extraction part in preparation of roselle stem cell freeze-dried powder

1. Test variables: roselle has its root tip removed from the root cap, roselle stem (vascular cambium), and roselle storage root (root cap).

2. The test method comprises the following steps: according to the preparation method and the steps of the roselle stem cell instant dry powder, the specific operation is as follows:

a. respectively taking roselle, removing the root tip of the root cap, the roselle stem (vascular cambium) and the roselle storage root (root cap);

b. the resulting roselle tissues were cut into 5mm by 5mm in size, 6 parts of each tissue were inoculated into the medium of step b in example 1, and the time for the roselle root tip cells to proliferate and differentiate into cambium was observed.

3. And (3) test results:

as shown in Table 3, the time of the roselle cambium stem cells cultured from the root tips of roselle is the shortest.

TABLE 3 Hibiscus sabdariffa cambium Stem cell formation time

Test example three: roselle stem cell freeze-dried powder eye cream ultraviolet injury resistance test

1. Test subjects: healthy guinea pigs weighing 220-.

2. Test materials: the roselle stem cell freeze-dried powder eye cream prepared in the examples 2-4 is a commercially available anti-ultraviolet injury eye cream.

3. The test method comprises the following steps:

grouping: dividing guinea pigs into a blank control group, a positive control group (commercial eye cream), a negative control group, a test group 1, a test group 2 and a test group 3, wherein each group comprises 6 male and female halves, removing hairs on the back of the guinea pigs, normally raising the blank control group, carrying out ultraviolet irradiation on the negative control group for 16h, smearing physiological saline, smearing the commercial eye cream on the positive control group after carrying out ultraviolet irradiation for 16h, and smearing the eye creams prepared in the embodiment 2, the embodiment 3 and the embodiment 4 after carrying out ultraviolet irradiation on the test group 1, the test group 2 and the test group 3 for 16h respectively; the preparation is applied once every 6h, after 24h, the eye cream is washed off by using normal saline, and the skin moisture of shaved hair of each guinea pig group is measured at 0 h, 1h, 24h and 48h respectively. 4. And (3) test results:

the test results are shown in table 4, and the ultraviolet resistance effect of the roselle stem cell lyophilized powder eye cream is far higher than that of the commercially available eye cream.

TABLE 4 Hibiscus sabdariffa stem cell lyophilized powder eye cream anti-ultraviolet injury test

Wherein P < 0.05 and P < 0.01, compared to the positive control group.

Test example four: roselle stem cell freeze-dried powder eye cream anaphylaxis test

1. Test subjects: healthy guinea pigs weighing 220-.

2. Test materials: the roselle stem cell freeze-dried powder eye cream prepared in example 2.

3. The test method comprises the following steps:

grouping: dividing guinea pigs into 4 groups at random, including a blank control group, a positive sensitization group, a test group 1 and a test group 2, wherein the blank control group comprises 10 male and female halves, depilating the back of the guinea pig body, and smearing 0.2-0.25ml of physiological saline on the depilated part; the positive sensitization group is smeared on the depilation position with 0.2-0.25ml of sensitization cream and is evenly smeared on the depilation position of the guinea pig; the test group 1 and the test group 2 were applied with 0.2-0.25ml of the roselle stem cell lyophilized powder eye cream prepared in example 2 at the unhairing place, and the skin allergy was observed at 0, 1, 24, 48, and 72 hours, respectively.

4. And (3) test results:

the test results show that 10 guinea pigs in the blank control group, the test group 1 and the test group 2 have no erythema and edema, 10 guinea pigs in the sensitized group have obvious erythema, the erythema appears in the sensitized group after the administration for 1h, the erythema appears in the sensitized group after the administration for 6h, the erythema is most obvious, the erythema gradually subsides after 24h, and the erythema basically subsides after 72 h.

Test example 5: anti-aging effect of roselle stem cell freeze-dried powder eye cream

1. Test products: the roselle stem cell freeze-dried powder eye cream prepared in the example 3 is a commercially available eye cream with anti-wrinkle and black eye circles.

2. The test population:

number: 100 persons

Age: between 18 and 48 years of age

Sex: woman

Health condition: the skin of the subject is healthy, has no allergic history of the skin disease, and meets the voluntary selection standard of the subject.

3. The test time is 1 month, and the test time is 1 time in the morning and at night each day.

4. The test method comprises the following steps:

after the face of the subject is cleaned, the control group is coated with a commercially available eye cream around the eyes, and the test group coats the roselle stem cell freeze-dried powder eye cream prepared in example 3 around the eyes to observe and feel the using effect.

5. The test evaluation results are as follows:

table 5 summary of trial feedback for eye creams prepared in example 3

TABLE 6 summary of trial feedback for commercial eye creams

The testers all show that compared with the commercially available eye cream for resisting wrinkles, resisting black eyes and pouches, the roselle stem cell freeze-dried powder eye cream prepared in example 3 can obviously improve the water content of skin, enhance the moisture retention capacity and skin elasticity of the skin, keep the moisture feeling for a long time, and simultaneously has the effects of lightening fishtail lines and black eyes of eyes and eliminating pouches after being used for a long time. And no allergy phenomenon appears in users, and the eye cream prepared in the example 3 is safe and good-effect eye cream for improving and repairing eye skin.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (8)

1. The roselle stem cell freeze-dried powder eye cream is characterized by comprising the following components in parts by weight:

8.0-20.0ng of roselle stem cell freeze-dried powder, 1.0-3.0g of polymerized almond protein, 0.5-2.0g of Fuyesijin, 0.1-0.2g of preservative, 1.0-4.0g of propylene glycol, 2.0-6.0g of glycerol, 1.0-5.0g of butanediol, 78-0.6 g of carbomer 9400.1, 0.5-2.0g of dimethyl siloxane, 1.0-4.0g of caprylic/capric triglyceride, 1.0-4.0g of isopropyl palmitate, 1.0-3.0g of synthetic squalane, 2.0-6.0g of cetylstearyl alcohol ether, 0.2-0.4g of triethanolamine and 50-100g of water;

the preparation method of the roselle stem cell freeze-dried powder comprises the following steps:

a. taking roselle tissues, cleaning and disinfecting;

b. cutting the obtained roselle tissue into 5mm by 5mm, inoculating to solid culture medium containing zinc sulfate 2-3mg/L, citric acid 75-80mg/L, glycine 78-82mg/L, indoleacetic acid 1.0-2.0mg/L, sucrose 10.5-11.5mg/L, active carbon 0.45-1g/L, barbaloin 3-5mg/ml and agar 2.8-3.5mg/L, culturing at 24-28 deg.C and pH 6.2-6.8 for 14-18 days to obtain roselle dry cell from cambium;

c. transferring the roselle stem cells from cambium to a liquid culture medium containing 180 mg/L magnesium sulfate, 75-80mg/L citric acid, 76.0-84.0mg/L glycine, 1.0-2.0mg/L indoleacetic acid, 32.0-34.0mg/L sucrose, 3-5mg/ml barbaloin, 65-75mg/L medical stone particles and 65-75mg/L selenium stone particles for culture;

d. c, transferring the roselle stem cells with the cambium source cultured and collected in the step c into an airlift bioreactor for culture, continuously stirring the culture solution at normal temperature by using the liquid culture solution obtained in the step c, culturing until the 7 th day, and injecting cane sugar, methyl jasmonate and sterile magnetized water into the airlift bioreactor;

e. filtering the culture solution in the airlift bioreactor in the step d by a filter, taking out the trapped cell line culture, placing the trapped cell line culture in a refrigerator at the temperature of between 85 ℃ below zero and 75 ℃ below zero, freezing for 2 to 3 hours, taking out the cell line culture, thawing for 30 to 40min at room temperature, and repeating for 3 to 5 times; after the last thawing, crushing the cells by using an ultrasonic crusher, collecting filtrate, and concentrating the filtrate to obtain a roselle stem cell extract;

f. and e, adding water into the roselle stem cell extract in the step e to adjust the final concentration of protein to be 50 +/-0.5 mu g/ml, adding a freeze-drying protective agent accounting for 4-6% of the total mass of the roselle stem cell extract, sterilizing, and freeze-drying at the temperature of-35 to-20 ℃ and the vacuum degree of 50-200 Pa to obtain the roselle stem cell freeze-dried powder.

2. The roselle stem cell freeze-dried powder eye cream of claim 1, which is characterized by comprising the following components in parts by weight:

10ng of roselle dry cell freeze-dried powder, 2g of polymerized almond protein, 1g of Fufang gold, 0.15g of preservative, 2g of propylene glycol, 4g of glycerol, 3g of butanediol, 9400.35 g of carbomer, 1g of dimethyl siloxane, 3g of caprylic/capric triglyceride, 3g of isopropyl palmitate, 2g of synthetic squalane, 3.5g of cetyl stearyl ether, 0.35g of triethanolamine and 85g of water.

3. The roselle stem cell freeze-dried powder eye cream as claimed in claim 1 or 2, wherein the cetyl alcohol ether is cetyl alcohol ether-2.

4. The roselle stem cell freeze-dried powder eye cream as claimed in claim 1 or 2, wherein the preservative is Euxyl K350.

5. The roselle stem cell freeze-dried powder eye cream of claim 1, wherein the roselle stem cell freeze-dried powder preparation method comprises the step a of cutting roselle tissue into roselle root tips with root caps removed.

6. The roselle stem cell freeze-dried powder eye cream as claimed in claim 1, wherein the filter in step e of the preparation method of roselle stem cell freeze-dried powder is a sterile microporous filter membrane with the diameter of 0.45- μm, the ultrasonic condition is 4 ℃, 80W, 3s ultrasonic, 3s interval, and the total time is 9 min.

7. The roselle stem cell freeze-dried powder eye cream according to claim 1, wherein the freeze-drying protective agent in step f of the preparation method of roselle stem cell freeze-dried powder is L-proline.

8. The method for preparing roselle stem cell freeze-dried powder eye cream according to any one of claims 1 to 7, which is characterized by comprising the following steps:

s1: mixing propylene glycol, glycerol, butanediol and carbomer 940, dissolving in water, heating to 75-80 deg.C, and stirring to dissolve completely to obtain mixture A;

s2: mixing dimethyl siloxane, caprylic capric triglyceride, isopropyl palmitate, synthetic squalane and ceteareth, dissolving in water, heating to 75-80 deg.C, and stirring to dissolve completely to obtain mixture B;

s3: adding the mixture B prepared in the step S2 into the mixture A prepared in the step S1, putting the mixture A into a homogenizer for 3-5 min, and cooling to 30-50 ℃ to obtain a mixture C;

s4: dissolving the roselle stem cell freeze-dried powder, the polymerized almond protein and the ether-rich Spanish gold in water, and stirring until the components are completely dissolved to obtain a mixture D;

s5: and (4) adding the mixture D prepared in the step S4 and triethanolamine into the mixture C prepared in the step S3, and homogenizing to obtain the alcohol-based fuel.