CN108721202A - A kind of roselle stem cell freeze-dried powder eye cream and preparation method thereof - Google Patents

Abstract

The invention belongs to cosmetic fields, and in particular to a kind of roselle stem cell freeze-dried powder eye cream and preparation method thereof.The present invention is anti-oxidant using roselle stem cell, stimulate the function of application on human skin keratinocyte proliferation and anti-aging, the present invention provides a kind of roselle stem cell culture methods that cultivation cycle is short, and roselle stem cell is lyophilized into powder, the roselle stem cell freeze-drying powder content of gained is high, in addition, the present invention by roselle stem cell freeze-dried powder with polymerize almond protein, this gold of rich ether, dimethyl siloxane, caprylic capric glyceryl ester, isopropyl palmitate, synthesize saualane, the ingredients such as the hard ester alcohol ether of spermaceti and L-PROLINE combine, it is prepared with efficient restoring ocular damaged cell, it is crease-resistant, eliminate livid ring around eye and pouch, the eye cream for eye skin of compacting.

Description

A kind of roselle stem cell freeze-dried powder eye cream and preparation method thereof

Technical field

The invention belongs to daily cosmetics technical fields, and in particular to a kind of roselle stem cell freeze-dried powder eye cream and its system Preparation Method.

Background technology

The eye problems such as livid ring around eye, pouch, temporal wrinkle are mainly by internal cause at present：Endocrine is uncoordinated, and mood swing is big, eye Fatigue, cell ageing make ocular circulation be obstructed melanin accumulation；External cause：Solarization smokes, stays up late, habitual strabismus, makes true The collagen of cortex is aoxidized, is broken.

To be directed to both of these case, many therapies and product are released on the market, and therapy has Chinese medicine conditioning and west Doctor's operation, Chinese medicine conditioning mainly by taking Chinese medicine, improve endocrine, and then improve table flesh, alleviate eye problems, the method consumption Duration, decocting for Chinese herbal medicine are troublesome and need to can be only achieved effect by diet compatibility etc.；Doctor trained in Western medicine operation is newfashioned to control this year Treatment method, by excision aging, extra skin irritatin skin regeneration function, but this method because personal eye conditions difference is big, Technology requires height, dangerous big, costly, most of to have the personnel of eye problems to be unwilling to attempt.

There are various eye creams, eye essence, eye mask etc. in product on the market, is made a return journey by adding plurality of Chinese extract The problems such as except livid ring around eye, pouch, canthus line, but most of eye creams be all by replenishing water and preserving moisture respite eye problems or because Eye part skin thin (0.3mm) can not absorb situations such as excessive Chinese medical extract forms eye fat granule, and fundamentally without Method effectively stimulates eye skin self-regeneration and regeneration.

But since 2003, Switzerland scientist Dr.Fred Zulli were found that a kind of apple stem cell has and remove wrinkle Anti-aging and the beauty special effect for extending cell survival, then, apple stem cell are done thin by first Application to cosmetics for plant Born of the same parents lay a good foundation in the research field of anti-aging.Plant stem cell beautifying technique and its Related product are just by beauty circle The extensive concern of expert and insider, after apple stem cell, Asian puccoon stem cell, Morocco nut stem cell also by Beauty product is developed into, as Chinese Patent Application No. 201610268493.6 discloses aloe stem cell freeze-dried powder and its preparation side Method and application, this method can get a large amount of aloe stem cell and will not damage the content of its main component barbaloin.Thus With the development of modern technologies, do not broken up fully using stem cell, is used with unlimited self-renewing duplication and the potential broken up In cosmetic industry be needed for trend.

Roselle (Hibiscus sabdariffa Linn.) also known as Roselle, Hong Jinmei, red plum fruit, are high mallow Comos Rose of Sharon category, the herbaceous plant that growth period is 1 year, originates in Perenniporia martius.The flavone compound that Roselle flower contains has flavones Alcohol and flavanols, they exist in the form of monomer or polymer, including hibiscitrin, gossypitrin, Quercetin, Luteolin, green Ortho acid, protocatechuic acid, eugenol, ergosterol, Gamma-Tocopherol etc..Many kinds of substance all has anti-oxidant and anti-aging biology Activity, therefore, roselle stem cell have stronger oxidation resistant effect, are a kind of pure natural, nontoxic and pollution-free anti-agings Plant stem cell plants roselle, by roselle stem cell system extensively on the Guangdong in China, Guangxi, Fujian, Yunnan and other places It at freeze-dried powder, then applies in the preparation of cosmetics especially anti-aging product, not only needs not worry about cosmetic material source And the problem of quality, cosmetics frontier has also been opened up, first domestic plant stem cell anti-aging product is developed.

Invention content

It is long to solve the method time in the prior art to treating eye problems, dangerous big, costly and eye Skin care item have only served the effect of surface moisturizing, moisturizing, not solving the problems, such as eye part skin aging fundamentally, this product with Roselle stem cell freeze-dried powder is functional component, and stimulation human keratinocyte is proliferated, and the activity of intracellular SOD enzymes is improved, to subtract Attack and damage of few oxygen radical generated by ultraviolet radiation induction to cell, and add polymerization almond protein, rich ether this Crease-resistant, the anti-oxidant, skin that compacts such as gold and saualane, the ingredient for resisting relaxation, it is intended to which the death rate for reducing cell plays reparation Cell, the effect of protection cell and the skin that compacts.

A kind of roselle stem cell freeze-dried powder eye cream provided by the invention, including following component and its weight：

Roselle stem cell freeze-dried powder 8.0-20.0g, propylene glycol 1.0-4.0g, glycerine 2.0-6.0g, butanediol 1.0- 5.0g, card wave 940 0.1-0.6g, preservative 0.1-0.2g, dimethyl siloxane 0.5-2.0g, caprylic capric glyceryl ester 1.0-4.0g, isopropyl palmitate 1.0-4.0g synthesize saualane 1.0-3.0g, the hard ester alcohol ether 2.0-6.0g of spermaceti, polymerize apricot Benevolence albumen 1.0-3.0g, this gold of rich ether 0.5-2.0g, triethanolamine 0.2-0.4g and water 50-100g.

Preferably, a kind of roselle stem cell freeze-dried powder eye cream, the formula of the eye cream are as follows：

Roselle stem cell freeze-dried powder 10ng, preservative 0.15g, propylene glycol 2g, glycerine 4g, butanediol 3g, card wave 0.35g, dimethyl siloxane 1g, caprylic capric glyceryl ester 3g, isopropyl palmitate 3g synthesize saualane 2g, the hard ester of spermaceti Alcohol ether 3.5g polymerize almond protein 2g, this gold of rich ether 1g, triethanolamine 0.35g and water 85g.

Further, roselle stem cell freeze-dried powder eye cream provided by the invention, the hard ester alcohol ether of the spermaceti are spermaceti Hard ester alcohol ether -2.

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

Further, roselle stem cell freeze-dried powder eye cream provided by the invention, the roselle stem cell freeze-dried powder Preparation method steps are as follows：

A. it takes roselle to organize, cleans, disinfection；

B. obtained roselle is cut into 5mm*5mm*5mm sizes, be seeded to containing zinc sulfate 2-3mg/L, citric acid 75- 80mg/L, glycine 78-82mg/L, heteroauxin 1.0-2.0mg/L, sucrose 10.5-11.5mg/L, activated carbon 0.45-1g/L, On the solid medium of barbaloin 3-5mg/ml and agar 2.8-3.5mg/L, cultivation temperature is 24-28 DEG C, pH value 6.2- 6.8, it cultivates 12-18 days, obtains the roselle stem cell in forming layer source；

C. the roselle stem cell in forming layer source is transferred to containing magnesium sulfate 180-182mg/L, citric acid 75- 80mg/L, glycine 76.0-84.0mg/L, heteroauxin 1.0-2.0mg/L, sucrose 32.0-34.0mg/L, barbaloin 3-5mg/ It is cultivated in the liquid culture medium of ml, medical stone granule 65-75mg/L and selenium ore particles 65-75mg/L；

D. the roselle stem cell in the forming layer source that step c cultures are collected is moved into airlift bioreactor and is trained It supports, culture solution uses the liquid culture solution of step c, is stirred continuously at normal temperatures, cultivates to the 7th day, to gas-lifting type biological respinse Injection sucrose, methyl jasmonate and sterile magnetized water in device；

E. the culture solution in the airlift bioreactor of step d is filtered by filter, the cell line of retention is trained It supports object to take out, is put into -80 DEG C of refrigerator superfreeze 2-3h, take out thaw at RT 30-40min, repeat 3-5 times；For the last time Sonicator smudge cells are utilized after defrosting, collect filtrate, and concentration obtains roselle stem cell extract；

F. the roselle stem cell extract into step e adds final concentration of 50 ± 0.5 μ g/ml of water regulatory protein matter, The freeze drying protectant of the 4-6% of roselle stem cell extract gross mass is added, sterilizing is -20~-35 DEG C in temperature, vacuum Degree is to be lyophilized in 50~200Pa, and roselle stem cell freeze-dried powder is made.

Preferably, the preparation of roselle stem cell freeze-dried powder eye cream provided by the invention, the rose cut in the step a Rare eggplant is organized as the roselle tip of a root of removal root cap.

Preferably, the preparation of roselle stem cell freeze-dried powder eye cream provided by the invention, the filter in the step e are 0.45 μM of sterile miillpore filter, ultrasound condition are 4 DEG C, 40W, ultrasound 3s, are spaced 3s, total time-consuming 9min.

Preferably, the preparation of roselle stem cell freeze-dried powder eye cream provided by the invention, the frozen-dried protective in the step f Agent is L-PROLINE, and L-PROLINE is one of component of phytoprotein, is ideal osmotic adjustment, can be used as film and enzyme Protective substances and free radical scavenger, the adjustment effect to cytoplasm osmotic equilibrium can be played.

The preparation process of roselle stem cell freeze-dried powder eye cream of the present invention is as follows：

S1：By propylene glycol, glycerine, butanediol, card wave 940 and preservative mixing are soluble in water, are heated to 75-80 DEG C, Stirring obtains mixture A to being completely dissolved；

S2：By dimethyl siloxane, caprylic capric glyceryl ester, isopropyl palmitate synthesizes saualane, the hard ester alcohol of spermaceti Ether mixing is soluble in water, is heated to 75-80 DEG C, stirs to being completely dissolved, obtains mixture B；

S3：By mixture B obtained in step S2, it is added in step S1 in mixture A obtained, is put into homogenizer 3~5min of value is cooled to 30~50 DEG C, obtains mixture C；

S4：By roselle stem cell freeze-dried powder, polymerization almond protein and this gold of rich ether are soluble in water, stir to completely molten Solution, obtains mixture D；

S5：Mixture D obtained in step S4 and triethanolamine are added in step S3 in mixture C obtained, homogeneous To obtain the final product.

The acellular variation in incubation of the preparation method of roselle stem cell freeze-dried powder provided by the invention, cell increase The roselle stem cell purity height that division is quick, is separately cultured is grown, 95% or more can be reached, be conducive to the roselle stem cell The promotion and application of the preparation method of freeze-dried powder.

In prepared by the roselle stem cell freeze-dried powder of the present invention, the heteroauxin in culture medium is auximone, is promoted Tip of a root multiple fission is shaped as confluent monolayer cells, and the barbaloin of addition is the extract of plant extract, has bacteriostasis, reduces anti- The addition of raw element, in addition it was also found that heteroauxin and barbaloin, which is added, can advantageously promote cell simultaneously in culture medium Proliferation growth, shortens incubation time.

The prior art is compared, roselle stem cell freeze-dried powder eye cream provided by the invention and preparation method thereof has following Advantage：

1) it is added to barbaloin in the preparation method of roselle stem cell freeze-dried powder provided by the invention, barbaloin not only has There is fungistatic effect, be combined with heteroauxin, hence it is evident that it is short to shorten cell culture period, promotes cell Proliferation；

2) the simple repeatability of roselle stem cell method for preparing freeze-dried powder produced by the present invention is high, and rose dried eggplant is utilized Cell, which has, to be repaired damaged cell, promotes cytothesis, protection skin effect, in conjunction with polymerization almond protein, rich ether this gold, conjunction It at saualane, mutually cooperates with, plays the role of the skin that compacts, there is effect that is anti-aging and extending cell survival to human skin Fruit；

3) roselle stem cell freeze-dried powder eye cream produced by the present invention is to the crow's feet of eye, pouch, livid ring around eye, skin pine Relaxing has significant improvement result.

Specific implementation mode：

Embodiment 1：A kind of preparation of roselle stem cell freeze-dried powder：

A. it takes and cuts the roselle tip of a root, clean, disinfection；

B. obtained roselle is cut into 5mm*5mm*5mm sizes, be seeded to containing zinc sulfate 2.5mg/L, citric acid 75.5mg/L, glycine 80.0mg/L, heteroauxin 1.5mg/L, sucrose 11.0mg/L, activated carbon 0.8g/L, barbaloin On the solid medium of 4.5mg/ml and agar 3.2mg/L, cultivation temperature is 25 DEG C, and pH value 6.5 is cultivated 14 days, must be formed The roselle stem cell in layer source；

C. the roselle stem cell in forming layer source is transferred to containing magnesium sulfate 181mg/L, citric acid 79mg/L, sweet ammonia Sour 78.0mg/L, heteroauxin 1.5mg/L, sucrose 33.0mg/L, barbaloin 2.5mg/ml, medical stone granule 70.0mg/L and selenium It is cultivated in the liquid culture medium of ore particles 70.0mg/L；

D. the roselle stem cell in the forming layer source that step c cultures are collected is moved into airlift bioreactor and is trained It supports, culture solution uses the liquid culture solution of step c, is stirred continuously at normal temperatures, cultivates to the 7th day, to gas-lifting type biological respinse Injection sucrose, methyl jasmonate and sterile magnetized water in device；

E. the culture solution in the airlift bioreactor of step d will be retained by 0.45 μM of sterile miillpore filter Cell line culture take out, be put into -80 DEG C of refrigerator superfreeze 2h, take out thaw at RT 30min, be repeated 4 times；Last Sonicator smudge cells, ultrasound condition are utilized to be 4 DEG C, 40W, ultrasound 3s, be spaced 3s, total time-consuming 9min after secondary defrosting. Filtrate is collected, filtrate is concentrated, roselle stem cell extract is obtained；

F. the roselle stem cell extract into step e adds final concentration of 50 ± 0.5 μ g/ml of water regulatory protein matter, 5% L-PROLINE of roselle stem cell extract gross mass is added, sterilizing is -20~-35 DEG C in temperature, and vacuum degree is It is lyophilized in 50Pa, roselle stem cell freeze-dried powder is made.

Comparative example 1：A kind of preparation of roselle stem cell freeze-dried powder：

A. it takes and cuts the roselle tip of a root, clean, disinfection；

B. obtained roselle is cut into 5mm*5mm*5mm sizes, be seeded to containing zinc sulfate 2.5mg/L, citric acid 75.5mg/L, glycine 80.0mg/L, heteroauxin 1.5mg/L, sucrose 11.0mg/L, activated carbon 0.8g/L and agar 3.2mg/ On the solid medium of L, cultivation temperature is 25 DEG C, and pH value 6.5 cultivates 14 days, obtains the roselle stem cell in forming layer source；

C. the roselle stem cell in forming layer source is transferred to containing magnesium sulfate 181mg/L, citric acid 79mg/L, sweet ammonia Sour 78.0mg/L, heteroauxin 1.5mg/L, sucrose 33.0mg/L, medical stone granule 70.0mg/L and selenium ore particles 70.0mg/ It is cultivated in the liquid culture medium of L；

D. the roselle stem cell in the forming layer source that step c cultures are collected is moved into airlift bioreactor and is trained It supports, culture solution uses the liquid culture solution of step c, is stirred continuously at normal temperatures, cultivates to the 7th day, to gas-lifting type biological respinse Injection sucrose, methyl jasmonate and sterile magnetized water in device；

E. the culture solution in the airlift bioreactor of step d will be retained by 0.45 μM of sterile miillpore filter Cell line culture take out, be put into -80 DEG C of refrigerator superfreeze 2h, take out thaw at RT 30min, be repeated 4 times；Last Sonicator smudge cells, ultrasound condition are utilized to be 4 DEG C, 40W, ultrasound 3s, be spaced 3s, total time-consuming 9min after secondary defrosting. Filtrate is collected, filtrate is concentrated, roselle stem cell extract is obtained；

F. the roselle stem cell extract into step e adds final concentration of 50 ± 0.5 μ g/ml of water regulatory protein matter, 5% L-PROLINE of roselle stem cell extract gross mass is added, sterilizing is -20~-35 DEG C in temperature, and vacuum degree is It is lyophilized in 50Pa, roselle stem cell freeze-dried powder is made.

Difference lies in comparative example 1 is not added with barbaloin to the culture medium of comparative example 1 and embodiment 1.

Comparative example 2：A kind of preparation of roselle stem cell freeze-dried powder：

A. it takes and cuts the roselle tip of a root, clean, disinfection；

B. obtained roselle is cut into 5mm*5mm*5mm sizes, be seeded to containing zinc sulfate 2.5mg/L, citric acid 75.5mg/L, glycine 80.0mg/L, methyl α-naphthyl acetate 2.5mg/L, sucrose 11.0mg/L, activated carbon 0.8g/L and agar 3.2mg/L Solid medium on, cultivation temperature be 25 DEG C, pH value 6.5, cultivate 14 days, obtain the roselle stem cell in forming layer source；

C. the roselle stem cell in forming layer source is transferred to containing magnesium sulfate 181mg/L, citric acid 79mg/L, sweet ammonia Sour 78.0mg/L, methyl α-naphthyl acetate 2.5mg/L, sucrose 33.0mg/L, medical stone granule 70.0mg/L and selenium ore particles 70.0mg/L Liquid culture medium in cultivate；

D. the roselle stem cell in the forming layer source that step c cultures are collected is moved into airlift bioreactor and is trained It supports, culture solution uses the liquid culture solution of step c, is stirred continuously at normal temperatures, cultivates to the 7th day, to gas-lifting type biological respinse Injection sucrose, methyl jasmonate and sterile magnetized water in device；

E. the culture solution in the airlift bioreactor of step d will be retained by 0.45 μM of sterile miillpore filter Cell line culture take out, be put into -80 DEG C of refrigerator superfreeze 2h, take out thaw at RT 30min, be repeated 4 times；Last Sonicator smudge cells, ultrasound condition are utilized to be 4 DEG C, 40W, ultrasound 3s, be spaced 3s, total time-consuming 9min after secondary defrosting. Filtrate is collected, filtrate is concentrated, roselle stem cell extract is obtained；

F. the roselle stem cell extract into step e adds final concentration of 50 ± 0.5 μ g/ml of water regulatory protein matter, 5% L-PROLINE of roselle stem cell extract gross mass is added, sterilizing is -20~-35 DEG C in temperature, and vacuum degree is It is lyophilized in 50Pa, roselle stem cell freeze-dried powder is made.

Difference lies in the auxin methyl α-naphthyl acetate in comparative example 2 is instead of heteroauxin with embodiment 1 for comparative example 2.

Embodiment 2-4：A kind of roselle stem cell freeze-dried powder eye cream, including following component and its weight：

The ingredient and its weight of 1 embodiment 2-4 roselle stem cell freeze-dried powder eye creams of table

The roselle stem cell freeze-dried powder is made by the preparation method of roselle stem cell freeze-dried powder described in embodiment 1 's.

The preparation method of roselle stem cell freeze-dried powder eye cream, respectively presses the ingredient of embodiment 2-4 and its weight in table 1 It is prepared by following steps：

S1：By propylene glycol, glycerine, butanediol, card wave 940 and preservative mixing are soluble in water, are heated to 75 DEG C, stirring To being completely dissolved, mixture A is obtained；

S2：By dimethyl siloxane, caprylic capric glyceryl ester, isopropyl palmitate synthesizes saualane, the hard ester alcohol of spermaceti Ether mixing is soluble in water, is heated to 75 DEG C, stirs to being completely dissolved, obtains mixture B；

S3：By mixture B obtained in step S2, it is added in step S1 in mixture A obtained, is put into homogenizer Value 5min is cooled to 30 DEG C, obtains mixture C；

S4：By roselle stem cell freeze-dried powder, polymerization almond protein and this gold of rich ether are soluble in water, stir to completely molten Solution, obtains mixture D；

S5：Mixture D obtained in step S4 and triethanolamine are added together in step S3 in mixture C obtained, Homogeneous to obtain the final product.

Test example one：Medium component composition experiment in the preparation of roselle stem cell freeze-dried powder eye cream

1. subjects：The roselle stem cell freeze-dried powder that embodiment 1, comparative example 1 and comparative example 2 are prepared.

2. test method：

Take and cut the roselle tip of a root, clean, disinfection, obtained roselle is cut into 5mm*5mm*5mm sizes, be seeded to containing Zinc sulfate 2.5mg/L, citric acid 75.5mg/L, glycine 80.0mg/L, heteroauxin 1.5mg/L, sucrose 11.0mg/L, activity On the solid medium of charcoal 0.8g/L and agar 3.2mg/L, cultivation temperature is 25 DEG C, and pH value 6.5 is cultivated 14 days, must be formed The roselle stem cell in layer source；The roselle stem cell in forming layer source is transferred to containing magnesium sulfate 181mg/L, citric acid 79mg/L, glycine 78.0mg/L, heteroauxin 1.5mg/L, sucrose 33.0mg/L, medical stone granule 70.0mg/L and selenium ore It is cultivated in the liquid culture medium of particle 70.0mg/L, obtains cell suspension A；

Cell suspension A obtained above is equally divided into 3 parts, low-speed centrifugal precipitation collects cell, it is big to be transferred to 5L respectively It is cultivated in scale evaluation device, uses fluid nutrient medium culture in embodiment 1, comparative example 1 and comparative example 2 respectively, after culture the 5th day, It is separately added into the fresh embodiment 1 of 4L, the fluid nutrient medium of comparative example 1 and comparative example 2, continues to cultivate, centre does not add training Base is supported, cell is collected after the 15th day, weighs.3. test result：

Test result such as table 2, the results showed that, embodiment 1, the interior cell weight that heteroauxin and barbaloin is added of culture medium It is apparently higher than the cell weight that comparative example 1 is not added with barbaloin and methyl α-naphthyl acetate and barbaloin is added in comparative example 2, illustrates heteroauxin Cell Proliferation can be promoted to divide with barbaloin, shorten incubation time.

2 cell weight of table

Testing index	Embodiment 1	Comparative example 1	Comparative example 2
				Cell liquid weight (mg)	687.8	345.7	350.7

Test example two：The incubation time at roselle tissue extraction position in prepared by roselle stem cell freeze-dried powder

1. testing variable：Roselle removes the tip of a root of root cap, roselle stem (vascular cambium), roselle storage root (root Hat).

2. test method：Work as the preparation method and step of dry powder according to roselle stem cell of the present invention, concrete operations are as follows：

A. the tip of a root of roselle removal root cap, roselle stem (vascular cambium), roselle storage root (root cap) are taken respectively；

B. obtained roselle tissue is cut into 5mm*5mm*5mm sizes respectively, each 6 parts of tissue is seeded to reality respectively It applies in example 1 and is cultivated in b step culture medium, the time of observation roselle root-tip cells Proliferation, Differentiation forming stratification.

3. test result：

Test result is as shown in table 3, and the roselle forming layer stem cell time that roselle root tip culture comes out is most short.

3 roselle forming layer stem cell of table forms the time

Test example three：Roselle stem cell freeze-dried powder eye cream uvioresistant damage test

1. subjects：Healthy guinea pig, weight 220-300g, male and female are half-and-half.

2. test material：Roselle stem cell freeze-dried powder eye cream prepared by embodiment 2-4, commercially available uvioresistant damage eye cream.

3. test method：

Grouping：Cavy is divided into blank control group, positive controls (commercially available eye cream), negative control group, test group 1, examination Group 2 and test group 3 are tested, every group 6, half-and-half, the hair of guinea pig back is shaved off for male and female, and blank control group is normally raised, negative right According to a group ultraviolet irradiation 16h, physiological saline is smeared, commercially available eye cream, test group 1, experiment are smeared after positive controls ultraviolet irradiation 16h After 2 and 3 groups of ultraviolet irradiation 16h of test group of group, eye cream prepared by embodiment 2, embodiment 3 and embodiment 4 is smeared respectively；Every 6h It smears once, washes away eye cream with physiological saline afterwards for 24 hours, each group cavy shaving moisture of skin feelings are measured respectively at 0,1,24,48h Condition.4. test result：

Test result is as shown in table 4, and the UVResistant effect of roselle stem cell freeze-dried powder eye cream is far above commercially available eye cream.

4 roselle stem cell freeze-dried powder eye cream uvioresistant damage test of table

Wherein compared with positive controls, * P < 0.05, * * P < 0.01.

Test example four：Roselle stem cell freeze-dried powder eye cream sensitivity test

1. subjects：Healthy guinea pig, weight 220-300g, male and female are half-and-half.

2. test material：Roselle stem cell freeze-dried powder eye cream prepared by embodiment 2.

3. test method：

Grouping：Cavy is randomly divided into 4 groups, blank control group, positive sensitization group, test group 1 and test group 2, blank pair According to group, every group 10, half-and-half, cavy body back is lost hair or feathers for male and female, and blank control group smears 0.2-0.25ml lifes at depilation Manage brine；Positive sensitization group smears 0.2-0.25ml sensitization emulsifiable pastes at depilation, is uniformly applied at cavy depilation；Test group 1 Smear roselle stem cell freeze-dried powder eye cream prepared by 0.2-0.25ml embodiments 2 at depilation respectively with test group 2, respectively at 0,1,24,48,72h observes phenomena of cutaneous irritation.

4. test result：

Test result shows that 2 each 10 blank control group, test group 1 and test group cavys do not occur erythema and oedema, causes There is apparent erythema in quick group of 10 cavys, occur erythema after sensitization group cavy self administration of medication 1h, and when 6h is most apparent, for 24 hours gradually Subside, erythema subsides substantially after 72h.

Test example 5：Roselle stem cell freeze-dried powder eye cream anti-aging effects

1. trial target：Roselle stem cell freeze-dried powder eye cream prepared by embodiment 3, commercially available crease-resistant, the black-eyed eye of tool Frost.

2. tested crowd：

Number：100 people

Age：Between 18-48 Sui

Gender：Female

Health status：Subjects skin's health, no skin disease allergies meet subject and volunteer inclusion criteria.

3. 1 month tested time, daily each 1 time of morning and evening.

4. test method：

Control group and test group are set, after the cleaning of subject face, control group smears commercially available eye cream and is coated to circumference of eyes, Roselle stem cell freeze-dried powder eye cream prepared by embodiment 3 is coated to circumference of eyes by test group, observes and experience using effect.

5. it is as follows to test assessment result：

The feedback conclusive table on probation of eye cream prepared by 5 embodiment 3 of table

The feedback conclusive table on probation of 6 commercially available eye cream of table

Tester indicates that roselle stem cell freeze-dried powder eye cream prepared by embodiment 3 compares commercially available crease-resistant, anti-black eye The eye cream of circle, pouch after long-time service, can significantly improve skin water content, enhance skin moisturizing power and skin elasticity, can grow When keep water moisture feeling, while having the effect of desalinating eye crow's feet, livid ring around eye and eliminating pouch.And user does not occur Allergic phenomena, eye cream prepared by embodiment 3 are the good eye creams for improving and rehabilitating eye skin of a kind of safety, effect.

The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto, Any one skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.

Claims (9)

1. a kind of roselle stem cell freeze-dried powder eye cream, which is characterized in that including following component and its weight：

Roselle stem cell freeze-dried powder 8.0-20.0ng polymerize almond protein 1.0-3.0g, rich ether this gold 0.5-2.0g, preservative 0.1-0.2g, propylene glycol 1.0-4.0g, glycerine 2.0-6.0g, butanediol 1.0-5.0g, card wave 9400.1-0.6g, dimethyl Siloxanes 0.5-2.0g, caprylic capric glyceryl ester 1.0-4.0g, isopropyl palmitate 1.0-4.0g, synthesis saualane 1.0- 3.0g, spermaceti hard ester alcohol ether 2.0-6.0g, triethanolamine 0.2-0.4g and water 50-100g.

2. a kind of roselle stem cell freeze-dried powder eye cream according to claim 1, which is characterized in that the ingredient of the eye cream And its weight is as follows：

Roselle stem cell freeze-dried powder 10ng, polymerize almond protein 2g, this gold of rich ether 1g, preservative 0.15g, propylene glycol 2g, and third Triol 4g, butanediol 3g, card wave 940 0.35g, dimethyl siloxane 1g, caprylic capric glyceryl ester 3g, isopropyl palmitate 3g synthesizes saualane 2g, spermaceti hard ester alcohol ether 3.5g, triethanolamine 0.35g and water 85g.

3. a kind of roselle stem cell freeze-dried powder eye cream according to claim 1 or 2, which is characterized in that the spermaceti Hard ester alcohol ether is the hard ester alcohol ether of spermaceti -2.

4. a kind of roselle stem cell freeze-dried powder eye cream according to claim 1 or 2, which is characterized in that the anti-corrosion Agent is Euxyl K350.

5. according to a kind of any roselle stem cell freeze-dried powder eye creams of claim 1-4, which is characterized in that rose dried eggplant Steps are as follows for the preparation method of cell freeze-dried powder：

A. it takes roselle to organize, cleans, disinfection；

B. obtained roselle tissue is cut into 5mm*5mm*5mm sizes, be seeded to containing zinc sulfate 2-3mg/L, citric acid 75- 80mg/L, glycine 78-82mg/L, heteroauxin 1.0-2.0mg/L, sucrose 10.5-11.5mg/L, activated carbon 0.45-1g/L, On the solid medium of barbaloin 3-5mg/ml and agar 2.8-3.5mg/L, cultivation temperature is 24-28 DEG C, pH value 6.2- 6.8, it cultivates 14-18 days, obtains the roselle stem cell in forming layer source；

C. the roselle stem cell in forming layer source is transferred to containing magnesium sulfate 180-182mg/L, citric acid 75-80mg/L, Glycine 76.0-84.0mg/L, heteroauxin 1.0-2.0mg/L, sucrose 32.0-34.0mg/L, barbaloin 3-5mg/ml, wheat meal It is cultivated in the liquid culture medium of stone particle 65-75mg/L and selenium ore particles 65-75mg/L；

D. the roselle stem cell in the forming layer source that step c cultures are collected is moved into airlift bioreactor and is cultivated, trained Nutrient solution uses the liquid culture solution of step c, is stirred continuously at normal temperatures, cultivates to the 7th day, is noted into airlift bioreactor Enter sucrose, methyl jasmonate and sterile magnetized water；

E. the culture solution in the airlift bioreactor of step d is filtered by filter, by the cell line culture of retention It takes out, is put into -75--85 DEG C of refrigerator superfreeze 2-3h, take out thaw at RT 30-40min, repeat 3-5 times；For the last time Sonicator smudge cells are utilized after defrosting, collect filtrate, filtrate is concentrated, roselle stem cell extract is obtained；

F. the roselle stem cell extract into step e adds final concentration of 50 ± 0.5 μ g/ml of water regulatory protein matter, is added The freeze drying protectant of the 4-6% of roselle stem cell extract gross mass, sterilizing are -20~-35 DEG C in temperature, and vacuum degree is It is lyophilized in 50~200Pa, roselle stem cell freeze-dried powder is made.

6. roselle stem cell freeze-dried powder eye cream according to claim 5, which is characterized in that the roselle stem cell is frozen The roselle cut in process for preparation of dry powder step a is organized as the roselle tip of a root of removal root cap.

7. roselle stem cell freeze-dried powder eye cream according to claim 5, which is characterized in that the roselle stem cell is frozen The sterile miillpore filter that filter in process for preparation of dry powder step e is 0.45 μM, ultrasound condition is 4 DEG C, 80W, ultrasound 3s, Every 3s, total time-consuming 9min.

8. roselle stem cell freeze-dried powder eye cream according to claim 5, which is characterized in that the roselle stem cell is frozen Freeze drying protectant in process for preparation of dry powder step f is L-PROLINE.

9. according to any roselle stem cell freeze-dried powder eye cream preparation methods of claim 1-8, which is characterized in that prepare step It is rapid as follows：

S1：By propylene glycol, glycerine, butanediol, card wave 940 mixes soluble in water, is heated to 75-80 DEG C, and stirring is to completely molten Solution, obtains mixture A；

S2：By dimethyl siloxane, caprylic capric glyceryl ester, isopropyl palmitate synthesizes saualane, and the hard ester alcohol ether of spermaceti is mixed It closes soluble in water, is heated to 75-80 DEG C, stirs to being completely dissolved, obtain mixture B；

S3：By mixture B obtained in step S2, it is added in step S1 in mixture A obtained, is put into mean value 3 in homogenizer ~5min is cooled to 30~50 DEG C, obtains mixture C；

S4：By roselle stem cell freeze-dried powder, polymerization almond protein and this gold of rich ether are soluble in water, stir to being completely dissolved, obtain Mixture D；

S5：Mixture D obtained in step S4 and triethanolamine are added in step S3 in mixture C obtained, homogeneous to obtain the final product.