CN105213254B - Preparation method and application of compound miracle fruit leaf extract - Google Patents
Preparation method and application of compound miracle fruit leaf extract Download PDFInfo
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- CN105213254B CN105213254B CN201510638507.4A CN201510638507A CN105213254B CN 105213254 B CN105213254 B CN 105213254B CN 201510638507 A CN201510638507 A CN 201510638507A CN 105213254 B CN105213254 B CN 105213254B
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Abstract
The invention discloses a preparation method of compound miracle fruit leaf extract. The method includes: preparing Chinese herbal medicine raw materials proportionally, preprocessing, extracting, coarsely filtering, finely filtering, concentrating, redissolving and sterilizing. The invention further discloses application of the compound miracle fruit leaf extract to cosmetics. The compound miracle fruit leaf extract has both antioxidant activity and tyrosinase inhibitory activity and overcomes the defect that the miracle fruit leaf only has high antioxidant activity. Miracle fruit leaf compounds are prepared into the compound miracle fruit leaf extract through preprocessing, stirring and heat reflux extracting, coarse filtering, fine filtering, concentrating, redissolving and sterilizing, and the compound miracle fruit leaf extract is higher in flavonoid content, higher in DPPH free radical scavenging rate, superoxide anion free radical scavenging rate and tyrosinase inhibition rate, and applicable to the cosmetics as the effective antiaging component.
Description
Technical field
The present invention relates to cosmetic field, more particularly to a kind of preparation method of compound recipe Synsepalum duleificum leaf extract and its
Application in cosmetics.
Background technology
Natural Synsepalum duleificum leaf, belongs to tropical evergreen shrub, evergreen all the year round, national treasure level rare species, embargo.Originate in
West Africa, Ghana, the Congo one carry, the sixties in 20th century, when Premier Zhou Enlai to West Africa accesses, Republic of Ghana using Synsepalum duleificum as
Premier Zhou is given in state's gift.Hereafter, start in China's cultivation, rich in crude fibre, crude protein, crude fat, VC, polyphenol, polysaccharide, pyrite
Class material, is mainly used in health care of food product, though application has no report, because of its polysaccharide, polyphenol, Huang in cosmetic field
Letones content is higher, has greatly potential using value in cosmetics.Contain more nutrition in Synsepalum duleificum leaf to become
Point, such as:
Synsepalum duleificum leaf nutritional labeling and content table
Content of the invention
The technical problem to be solved in the present invention is to provide a kind of to have answering of antioxidant activity and tyrosinase inhibitory activity concurrently
Square Synsepalum duleificum leaf extract and its application in cosmetics, and the extracting method of this compound recipe Synsepalum duleificum leaf extract is provided.
A kind of preparation method of compound recipe Synsepalum duleificum leaf extract, comprises the steps:
(1) prepare herbal raw material in proportion:Described herbal raw material is made up of major ingredient and adjuvant, and described major ingredient is mystery
Fruit leaf, percentage by weight is 30%~50%, and adjuvant is Radix Paeoniae Rubra, Fructus Atriplicis Sibiricae, the Radix Astragali, Radix Rhodiolae, Radix Scutellariae, Rhizoma Curcumae Longae, and Olibanum is red
Ginseng, the combination of any three kinds in Radix Notoginseng, percentage by weight respectively 20%~30%, 10%~30% and 10%~
20%;
(2) pretreatment:Carry out dries pulverizing process after foreign body in said herbal medicine raw material is chosen, obtain herbal raw material
Powder, four kinds of herbal powder mix homogeneously make Synsepalum duleificum leaf compound;
(3) extract:Described Synsepalum duleificum leaf compound and solvent are mixed united extraction, obtains extraction solution;
(4) coarse filtration:By described extraction solution coarse filtration, obtain compound recipe Synsepalum duleificum leaf extract coarse filtration liquid;
(5) fine straining:By described compound recipe Synsepalum duleificum leaf extract coarse filtration liquid fine straining, obtain compound recipe Synsepalum duleificum leaf extract fine straining
Liquid;
(6) concentrate:By described compound recipe Synsepalum duleificum leaf extract fine straining liquid, obtain compound recipe Synsepalum duleificum leaf extract concentrated solution;
(7) redissolve:Described compound recipe Synsepalum duleificum leaf extract concentrated solution is redissolved, obtains compound recipe Synsepalum duleificum leaf extract and redissolve
Thing;
(8) sterilize, described compound recipe Synsepalum duleificum leaf extract is redissolved thing sterilizing, obtains compound recipe Synsepalum duleificum leaf extract.
The preparation method of compound recipe Synsepalum duleificum leaf extract of the present invention, wherein, Synsepalum duleificum leaf described in step (3) is multiple
Join thing and solvent by weight 1:10~40 are mixed, and extract 0.5~3 hour, must extract molten at a temperature of 25~90 DEG C
Liquid.
The preparation method of compound recipe Synsepalum duleificum leaf extract of the present invention, wherein, redissolves described in step (7) and adopts 1:
20 butanediol aqueous solution redissolves.
The preparation method of compound recipe Synsepalum duleificum leaf extract of the present invention, wherein, described solvent be pure water, 40%~
95% ethanol solution or 40%~95% acetone soln.
The preparation method of compound recipe Synsepalum duleificum leaf extract of the present invention, wherein, is extracted as described in step (3) stirring
Reflux, extract, or supersound extraction.
Application in cosmetics for the compound recipe Synsepalum duleificum leaf extract of the present invention.
A kind of cosmetics, including described compound recipe Synsepalum duleificum leaf extract, weight shared by described compound recipe Synsepalum duleificum leaf extract
Amount percentage ratio is 0.2%~8%.
Cosmetics of the present invention, wherein, the percentage by weight of described compound recipe Synsepalum duleificum leaf extract is 0.4~6%.
Cosmetics of the present invention, wherein, also include solvent, emollient, solubilizing agent, antioxidation in described cosmetics
Agent, pH adjusting agent, penetrating agent, emulsion stabilizer, preservative, wetting agent, thickening agent, nertralizer, chelating agen, surfactant,
One or more of emulsifying agent, essence, coloring agent, sunscreen, skin conditioning agent, active matter and functional additive.
Cosmetics of the present invention, wherein, described cosmetics be astringent, bath gel, facial cleaning cream, facial film, facial cream,
Curry, emulsion, essence, makeup remover or transparent soap.
The preparation method difference from prior art of compound recipe Synsepalum duleificum leaf extract of the present invention is:
Synsepalum duleificum leaf has good antioxidant activity, but does not suppress the function of tyrosinase activity, wants to play it
Outstanding defying age, Bearberry Extract, need to consider to carry out compound recipe extraction, to give full play to the same of Synsepalum duleificum leaf antioxidant activity
When, carry more excellent skin-care effect.
With regard to the selection of compound recipe, according to theory of Chinese medical science " dialectical, entirety, synthesis " and the monarch and his subjects' principles of formulating prescriptions " monarch, minister, assistant,
Make ", compounded, wherein determined the main status that Synsepalum duleificum leaf is " monarch ", select the Chinese herbal medicine that disposition matches therewith to carry out multiple
Combo side, obtains flavone, polyphenols as basis for estimation with maximization, and centre carries out the optimization design of extraction process, to
Reach the best extracting solution of white-skinned face function, senile-resistant efficacy to be applied in all kinds of State of cosmetics.
The present invention is by Synsepalum duleificum leaf and Radix Paeoniae Rubra, Fructus Atriplicis Sibiricae, the Radix Astragali, Radix Rhodiolae, Radix Scutellariae, Rhizoma Curcumae Longae, Olibanum, Radix Salviae Miltiorrhizae, Radix Notoginseng etc.
Compounded, had concurrently antioxidant activity and tyrosinase inhibitory activity, compensate for Synsepalum duleificum leaf and only there is stronger antioxidation work
Property deficiency, the present invention by Synsepalum duleificum leaf compound pass through pretreatment, stirring circumfluence distillation, coarse filtration, fine straining, concentration, redissolution,
Compound recipe Synsepalum duleificum leaf extract is made in the process of sterilizing, and its flavones content is higher, and DPPH free radical scavenging activity, superoxide anion are certainly
Also higher by base clearance rate, tyrosinase inhibition rate, can be applied in cosmetics as whitening anti-aging functional component.
Specific embodiment
Embodiment 1
Herbal raw material is carried out pretreatment, makes herbal raw material powder;
Synsepalum duleificum leaf, Radix Paeoniae Rubra, Fructus Atriplicis Sibiricae, the Radix Astragali are made mystery according to 30%, 30%, 20%, 20% ratio respectively
Fruit leaf compound;
By Synsepalum duleificum leaf compound and pure water by weight 1:10 are mixed, and make mixture, and this mixture is heated
To 90 DEG C about, supersound extraction 30 minutes, make Synsepalum duleificum leaf compound and extract solution;
By Synsepalum duleificum leaf compound and extraction solution coarse filtration, make compound recipe Synsepalum duleificum leaf extract coarse filtration liquid;
By compound recipe Synsepalum duleificum leaf extract coarse filtration liquid fine straining, make compound recipe Synsepalum duleificum leaf extract fine straining liquid;
Compound recipe Synsepalum duleificum leaf extract fine straining liquid is concentrated, makes compound recipe Synsepalum duleificum leaf extract concentrated solution;
Compound recipe Synsepalum duleificum leaf extract concentrated solution is used 1:20 butanediol aqueous solution redissolves, and makes compound recipe Synsepalum duleificum leaf and carries
Thing is taken to redissolve thing;
Compound recipe Synsepalum duleificum leaf extract is redissolved thing sterilizing, makes compound recipe Synsepalum duleificum leaf extract.
Embodiment 2
Synsepalum duleificum leaf, Radix Paeoniae Rubra, Radix Rhodiolae, Radix Scutellariae are made Synsepalum duleificum in 36%, 28%, 18%, 18% ratio respectively
Leaf compound;
Acetone soln by weight 1 by Synsepalum duleificum leaf compound and 40%:15 are mixed, and make mixture, should
Mixture is heated to 80 DEG C about, supersound extraction 50 minutes, makes Synsepalum duleificum leaf compound and extracts solution;
By Synsepalum duleificum leaf compound and extraction solution coarse filtration, make compound recipe Synsepalum duleificum leaf extract coarse filtration liquid;
By compound recipe Synsepalum duleificum leaf extract coarse filtration liquid fine straining, make compound recipe Synsepalum duleificum leaf extract fine straining liquid;
Compound recipe Synsepalum duleificum leaf extract fine straining liquid is concentrated, makes compound recipe Synsepalum duleificum leaf extract concentrated solution;
Compound recipe Synsepalum duleificum leaf extract concentrated solution is used 1:20 butanediol aqueous solution redissolves, and makes compound recipe Synsepalum duleificum leaf and carries
Thing is taken to redissolve thing;
Compound recipe Synsepalum duleificum leaf extract is redissolved thing sterilizing, makes compound recipe Synsepalum duleificum leaf extract.
Embodiment 3
Synsepalum duleificum leaf, Radix Rhodiolae, Radix Scutellariae, the Radix Astragali are made Synsepalum duleificum in 42%, 26%, 16%, 16% ratio respectively
Leaf compound;
Acetone soln by weight 1 by Synsepalum duleificum leaf compound and 80%:20 are mixed, and make mixture, should
Mixture is heated to 70 DEG C about, stirring hot reflux 2 hours, makes Synsepalum duleificum leaf compound and extracts solution;
By Synsepalum duleificum leaf compound and extraction solution coarse filtration, make compound recipe Synsepalum duleificum leaf extract coarse filtration liquid;
By compound recipe Synsepalum duleificum leaf extract coarse filtration liquid fine straining, make compound recipe Synsepalum duleificum leaf extract fine straining liquid;
Compound recipe Synsepalum duleificum leaf extract fine straining liquid is concentrated, makes compound recipe Synsepalum duleificum leaf extract concentrated solution;
Compound recipe Synsepalum duleificum leaf extract concentrated solution is used 1:20 butanediol aqueous solution redissolves, and makes compound recipe Synsepalum duleificum leaf and carries
Thing is taken to redissolve thing;
Compound recipe Synsepalum duleificum leaf extract is redissolved thing sterilizing, makes compound recipe Synsepalum duleificum leaf extract.
Embodiment 4
Synsepalum duleificum leaf, Fructus Atriplicis Sibiricae, Radix Scutellariae, the Radix Astragali are made Synsepalum duleificum in 45%, 25%, 15%, 15% ratio respectively
Leaf compound;
Ethanol solution by weight 1 by Synsepalum duleificum leaf compound and 50%:30 are mixed, and make mixture, should
Mixture is heated to 60 DEG C about, stirring hot reflux supersound extraction 2.5 hours, makes Synsepalum duleificum leaf compound and extracts solution;
By Synsepalum duleificum leaf compound and extraction solution coarse filtration, make compound recipe Synsepalum duleificum leaf extract coarse filtration liquid;
By compound recipe Synsepalum duleificum leaf extract coarse filtration liquid fine straining, make compound recipe Synsepalum duleificum leaf extract fine straining liquid;
Compound recipe Synsepalum duleificum leaf extract fine straining liquid is concentrated, makes compound recipe Synsepalum duleificum leaf extract concentrated solution;
Compound recipe Synsepalum duleificum leaf extract concentrated solution is used 1:20 butanediol aqueous solution redissolves, and makes compound recipe Synsepalum duleificum leaf and carries
Thing is taken to redissolve thing;
Compound recipe Synsepalum duleificum leaf extract is redissolved thing sterilizing, makes compound recipe Synsepalum duleificum leaf extract.
Embodiment 5
Synsepalum duleificum leaf, Radix Paeoniae Rubra, Radix Rhodiolae, the Radix Astragali are made Synsepalum duleificum in 50%, 24%, 14%, 12% ratio respectively
Leaf compound;
Ethanol solution by weight 1 by Synsepalum duleificum leaf compound and 90%:40 are mixed, and make mixture, should
Mixture is heated to 50 DEG C about, stirring hot reflux 3 hours, makes Synsepalum duleificum leaf compound and extracts solution;
By Synsepalum duleificum leaf compound and extraction solution coarse filtration, make compound recipe Synsepalum duleificum leaf extract coarse filtration liquid;
By compound recipe Synsepalum duleificum leaf extract coarse filtration liquid fine straining, make compound recipe Synsepalum duleificum leaf extract fine straining liquid;
Compound recipe Synsepalum duleificum leaf extract fine straining liquid is concentrated, makes compound recipe Synsepalum duleificum leaf extract concentrated solution;
Compound recipe Synsepalum duleificum leaf extract concentrated solution is used 1:20 butanediol aqueous solution redissolves, and makes compound recipe Synsepalum duleificum leaf and carries
Thing is taken to redissolve thing;
Compound recipe Synsepalum duleificum leaf extract is redissolved thing sterilizing, makes compound recipe Synsepalum duleificum leaf extract.
The formula of the present invention is not limited to embodiment 1~embodiment 5, and research finds, using other in the scope of the invention
Combination also there is similar effect, such as by Synsepalum duleificum leaf, Rhizoma Curcumae Longae, Olibanum, Radix Salviae Miltiorrhizae respectively according to 34%, 20%, 29%,
17% ratio makes Synsepalum duleificum leaf compound;Or by Synsepalum duleificum leaf, Olibanum, Radix Notoginseng, Fructus Atriplicis Sibiricae respectively press 43%, 22%,
16%th, 19% ratio makes Synsepalum duleificum leaf compound;Ultimately form compound recipe Synsepalum duleificum leaf extract, also can play the present invention's
Effect.
Embodiment 6:The preparation of compound recipe Synsepalum duleificum leaf extract astringent
Above-mentioned compound recipe Synsepalum duleificum leaf extract is the arbitrary described compound recipe Synsepalum duleificum leaf extract of embodiment 1~embodiment 5.
Embodiment 7:The preparation of compound recipe Synsepalum duleificum leaf extract bath gel
Above-mentioned compound recipe Synsepalum duleificum leaf extract is the arbitrary described compound recipe Synsepalum duleificum leaf extract of embodiment 1~embodiment 5.
Embodiment 8:The preparation of compound recipe Synsepalum duleificum leaf extract essence
Above-mentioned compound recipe Synsepalum duleificum leaf extract is the arbitrary described compound recipe Synsepalum duleificum leaf extract of embodiment 1~embodiment 5.
Embodiment 9:The preparation of compound recipe Synsepalum duleificum leaf extract facial cream
Above-mentioned compound recipe Synsepalum duleificum leaf extract is the arbitrary described compound recipe Synsepalum duleificum leaf extract of embodiment 1~embodiment 5.
Embodiment 10:The preparation of compound recipe Synsepalum duleificum leaf extract facial film
Above-mentioned compound recipe Synsepalum duleificum leaf extract is the arbitrary described compound recipe Synsepalum duleificum leaf extract of embodiment 1~embodiment 5.
Embodiment 11:The preparation of compound recipe Synsepalum duleificum leaf extract curry
Above-mentioned compound recipe Synsepalum duleificum leaf extract is the arbitrary described compound recipe Synsepalum duleificum leaf extract of embodiment 1~embodiment 5.
Embodiment 12:The preparation of compound recipe Synsepalum duleificum leaf extract emulsion
Above-mentioned compound recipe Synsepalum duleificum leaf extract is the arbitrary described compound recipe Synsepalum duleificum leaf extract of embodiment 1~embodiment 4.
Embodiment 13:Preparation in compound recipe Synsepalum duleificum leaf extract facial cleaning cream
Above-mentioned compound recipe Synsepalum duleificum leaf extract is the arbitrary described compound recipe Synsepalum duleificum leaf extract of embodiment 1~embodiment 5.
Index test, statistical experiment result are carried out to the compound recipe Synsepalum duleificum leaf extract of embodiment 1- embodiment 5 gained 5%
As shown in the table:
Testing index | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 |
Flavones content/mg mL-1 | 0.39 | 0.41 | 0.43 | 0.40 | 0.39 |
DPPH clearance rate/% | 88 | 81 | 87 | 84 | 93 |
Tyrosinase inhibition rate/% | 91 | 87 | 89 | 93 | 84 |
Hyaluronic acid suppression ratio/% | 65 | 57 | 65 | 68 | 67 |
Using compounding above and process program beneficial effect:The present invention by Synsepalum duleificum leaf and Radix Paeoniae Rubra, Fructus Atriplicis Sibiricae, the Radix Astragali,
Radix Rhodiolae, Radix Scutellariae, Rhizoma Curcumae Longae, Olibanum, Radix Salviae Miltiorrhizae, any three kinds in Radix Notoginseng are compounded, and have antioxidant activity and tryrosinase concurrently
Inhibitory activity, compensate for the deficiency that Synsepalum duleificum leaf only has stronger antioxidant activity, and Synsepalum duleificum leaf compound is led to by the present invention
Cross pretreatment, stirring circumfluence distillation, coarse filtration, fine straining, concentration, redissolution, sterilizing process make compound recipe Synsepalum duleificum leaf extract,
Its flavones content is higher, and DPPH free radical scavenging activity, ultra-oxygen anion free radical clearance rate, tyrosinase inhibition rate also compare
Height, can be applied in cosmetics as whitening anti-aging functional component.
The active matter flavones content detection of compound recipe Synsepalum duleificum leaf extract product of the present invention
Compound recipe Synsepalum duleificum leaf extract crude drug mass concentration is 0.05g/mL, and flavones content testing result is as shown in table 1.
Table 1 compound recipe Synsepalum duleificum leaf extract flavones content
Remarks:Flavones content detection adopts NaNO2-Al(NO3)3Colorimetry.
First, the efficacy assessment of compound recipe Synsepalum duleificum leaf extract product of the present invention
1st, DPPH free radical scavenging activity
DPPH is a kind of stable organic free radical, is 517nm in visible region maximum absorption band, in ethanol solution,
Each DPPH molecule can generate in the solution one stable containing nitrogen free radical, there is typical purple, when it with 1 electronics is provided
Free radical scavenger effect when, generate colourless product, so that the typical purple of solution is shoaled, its fading extent and sharing electron number
Become stoichiometric relationship.Therefore, available spectrophotography carries out quantitative analyses, to detect free radical scavenging situation, thus evaluating
The ability of the removing free radical of sample.Research shows, DPPH clearance rate is higher, and antioxidant activity is stronger.Under different tested concentration,
The DPPH clearance rate experimental result of compound recipe Synsepalum duleificum leaf extract is as shown in table 2.
Table 2 compound recipe Synsepalum duleificum leaf extract DPPH free radical scavenging experimental result
As shown in Table 2, compound recipe Synsepalum duleificum leaf extract has good DPPH free radical scavenging effect, and tested concentration exists
When 2.0%, DPPH free radical scavenging activity reaches 88% about, shows that compound recipe Synsepalum duleificum leaf extract has in biochemistry level good
Good antioxidant activity, and the antioxidant activity of compound recipe Synsepalum duleificum leaf extract is better than commercially available competing product.
2nd, tyrosinase inhibition rate
Participating in B16 cell mainly has three kinds of enzymes:Tryrosinase, dopachrome interconvertible enzyme and dihydroxy indole carboxylic acid oxygen
Change enzyme.Tryrosinase belongs to oxidoreductase, is the major rate-limiting enzyme of B16 cell, and tryrosinase and its substrate L-Tyrosine can
There is catalytic reaction.
After with the addition of the reagent of tyrosinase activity inhibitory action in experimental system, catalytic reaction can be produced
Inhibitory action, by the light absorption value at 475nm before and after mensure interpolation reagent, to evaluate the suppression to tyrosinase activity for the reagent
Rate.Under different tested concentration, the tyrosinase inhibition rate experimental result of compound recipe Synsepalum duleificum leaf extract is as shown in table 3.
Table 3 compound recipe Synsepalum duleificum leaf extract tyrosinase inhibition rate experimental result
As shown in Table 3, compound recipe Synsepalum duleificum leaf extract has good tyrosinase inhibition rate, when tested concentration is
When 1.0%, tyrosinase inhibition rate reaches 80% about, shows that compound recipe Synsepalum duleificum leaf extract has in biochemistry level good
White-skinned face function.And under identical concentration, the suppression ratio of compound recipe Synsepalum duleificum leaf extract tryrosinase is less than commercially available competing product, is being subject to
In examination concentration range, the tyrosinase inhibition rate of commercially available competing product all reaches 100% about.
3rd, hyaluronic acid enzyme inhibition rate
Hyaluronic acid enzyme inhibition rate size has reacted the antiinflammatory vigor of sample to a certain extent, and suppression ratio is higher, antiinflammatory
Activity is stronger.By building vitro inhibition hyaluronidase experimental system, assessment compound recipe Synsepalum duleificum leaf extract is to hyaluronic acid
The suppression ratio of enzyme.Under different tested concentration, compound recipe Synsepalum duleificum leaf extract suppression hyaluronidase experimental result is as shown in table 4.
Table 4 compound recipe Synsepalum duleificum leaf extract hyaluronic acid enzyme inhibition rate experimental result
As shown in Table 4, compound recipe Synsepalum duleificum leaf extract has certain hyaluronic acid enzyme inhibition rate, shows that it has necessarily
Anti-inflammatory activity.And under the tested concentration of identical, compound recipe Synsepalum duleificum leaf extract hyaluronidase inhibitor is superior to city
Sell competing product.4th, improve skin microcirculation experiment
Skin microcirculation blood flow is medically widely used.Within the specific limits, skin microcirculation blood flow is got over
Show that greatly Skin Cell has good self renewal and regeneration capacity.The compound recipe preparing Synsepalum duleificum leaf extract is added to
In cream substrate, add concentration 5%, tested volunteer is analyzed using " laser-Doppler blood flow tester " and uses compound recipe Synsepalum duleificum
Improve the situation of change of skin microcirculation blood flow before and after the cream of leaf extract.
Tested volunteer 15 people, age 25-35 year, in experimentation control relative humidity (30 ± 5%) and temperature (25 ±
1℃).Result shows, compound recipe Synsepalum duleificum leaf extract has functions that good to improve skin microcirculation blood flow.Participate in experiment
In 15 volunteers, the skin microcirculation blood flow having 11 tested volunteers has obtained effective raising, and effective percentage reaches
73%.In experiment, all 15 tested volunteer's skins all have no adverse reaction.Accordingly, compound recipe Synsepalum duleificum leaf extract can
Apply to ruddy skin, improve black eye.
5th, melanocyte test
Inquire into the propagation to melanocyte for the compound recipe Synsepalum duleificum leaf extract sample and the impact of whitening function.Using B16
Melanoma cell, the impact to melanocyte proliferation function for the determination sample, and to melanocyte synthesis of melanin
Impact.
(1) mensure (mtt assay) of cell proliferation rate
Culture Murine melanoma B16 cells, treat that cell growth, to logarithmic (log) phase, is digested with 0.25% pancreatin, adjusts cell
Concentration is 5 × 104Individual/mL, is inoculated in 96 orifice plates, every hole 100 μ L.After being placed in culture 24h in incubator, abandon supernatant, add
Plus pastille culture medium, three concentration groups of each sample.Each concentration sets 3 multiple holes, and matched group is not added with medicine, instead accordingly
Solvent.After incubation 72h, every hole adds the MTT solution 20 μ L of 5g/L, abandons supernatant after 4h, and every hole adds cell pyrolysis liquid 100 μ
L, continues overnight incubation, detects each hole absorbance in microplate reader, and wavelength is 570nm.
Cell proliferation inhibition rate %=(1- medicine group average absorbance value/matched group average absorbance value) × 100
(2) mensure of B16 cell suppression ratio
By B16 melanocyte with 20 × 104The density of individual/mL is inoculated in 24 plates.Every hole 1mL, after incubation 24h, abandons
Clear liquid, adds medicine to be measured (sample effect final concentration is with aforementioned), after medicine effect 72h, abandons supernatant, PBS washs, and every hole adds
Enter the 1mol/L NaOH solution 500 μ L containing 10%DMSO, shake 5min.Each hole light absorption value under measurement 490nm.
B16 cell suppression ratio %=[1- (medicine hole 490nm absorbance/medicine hole 570nm absorbance)/(comparison
Hole 490nm absorbance/control wells 570nm absorbance)] × 100
After adding variable concentrations compound recipe Synsepalum duleificum leaf extract tested material, sample synthesizes suppression to melanocyte melanin
Rate and cell proliferation inhibition rate experimental result are as shown in table 5.
Table 5 variable concentrations compound recipe Synsepalum duleificum leaf extract sample generates to the inhibited proliferation of B16 cell and melanin
Effect
Conclusion:After adding the tested material of variable concentrations, compound recipe Synsepalum duleificum leaf extract sample has preferable suppression black
The effect of plain cell synthesis of melanin, in tested concentration range (0.5%-2.0%), B16 cell suppression ratio is in 30%-
40% about, show that compound recipe Synsepalum duleificum leaf extract has good white-skinned face function, and the shadow of cell proliferation in cellular level
Ring and be less than commercially available competing product.
2nd, the safety evaluation of compound recipe Synsepalum duleificum leaf extract product of the present invention
1st, erythrocyte hemolysis test
Erythrocyte hemolysis rate to a certain extent can be higher with the zest of response sample, hemolysis rate, potential zest
Bigger.Compound recipe Synsepalum duleificum leaf extract extracting solution erythrocyte hemolysis rate experimental result is as shown in table 6 below.
Table 6 compound recipe Synsepalum duleificum leaf extract erythrocyte hemolysis rate experimental result
As shown in Table 6, compound recipe Synsepalum duleificum leaf extract extracting solution has extremely low erythrocyte in 10% concentration range
Hemolysis rate, shows that it has preferable safety.
2nd, the multiple Skin Irritation Test of mice
By the multiple Skin Irritation Test of mice, determine and evaluationization compound recipe Synsepalum duleificum leaf extract is to mammal skin
Whether local has stimulation or corrosiveness and its degree.
Skin irritation/corrosion test method that method specifies with reference to 2007 editions cosmetics health specifications.By tested material
Once (or repeatedly) be coated on the skin of animal subject, in predetermined time interval, observe animal skin local excitation and make
With degree and scored.Using own control, to evaluate the stimulation to skin for the tested material.Acute skin irritation tries
Testing the observation time limit should be enough to evaluate reversibility or the irreversibility of this effect.
Result of the test shows, continuously smears 14 days given the test agent and (adds compound recipe Synsepalum duleificum leaf extract extracting solution 5% respectively
And 10%), all erythema and edema phenomenon in mouse skin.
Result illustrates, compound recipe Synsepalum duleificum leaf extract safety is good, nonirritant.
3rd, human closed formula patch test
Human body patch test method reference《Cosmetic hygienic practice (2007 editions)》, prepare the compound recipe god containing 1.0% and 5.0%
The cream of secret fruit leaf extract extracting solution carries out closed patch, investigates the probability that sample causes untoward reaction to human body.
Skin adverse reaction classification is as shown in table 7 below.
Table 7 skin adverse reaction is classified
According to table 7, observe experimenter and remove dermoreaction situation during speckle examination device 30min, 24h and 48h.
Result of the test is as shown in following table 8- table 10.
Table 8 removes speckle examination device 30min observed result
Table 9 removes speckle examination device 24h observed result
Table 10 removes speckle examination device 48h observed result
According to 2007《Cosmetics health specification》Requirement, judge that sample 1, sample 2 have no adverse reaction to human body skin.
(wherein, sample 1 is the cream of the compound recipe Synsepalum duleificum leaf extract extracting solution adding 1.0%, and sample 2 is the compound recipe of interpolation 5.0%
The cream of Synsepalum duleificum leaf extract extracting solution.)
Conclusion:Add the cream of the compound recipe Synsepalum duleificum leaf extract of 1.0%, 5.0% two concentration, to human body skin invariably
Good reaction occurs, and safety is good.
4th, Phototoxicity experiment
Potential light be whether there is using external 3T3 red untake Phototoxicity experiment assessment compound recipe Synsepalum duleificum leaf extract
Toxicity.
Whether there is phototoxicity by calculating photostimulation factor PIF and average photo effect MPE comprehensive assessment sample, pass judgment on mark
Accurate as follows:Work as PIF<2.0 or MPE<0.1:Expected no phototoxicity;Work as PIF>2.0 and<5.0 or MPE>0.1 and<0.15:Expection can
Can there is phototoxicity;Work as PIF>5.0 or MPE>0.15:There is phototoxicity in expection.
Test is carried out using 96- well culture plate.Balb/c 3T3 cell good for growth conditions is inoculated in 96 hole cells
Culture plate (by each 2 blocks of plates of test product and positive reference substance), makes the cell density in every hole be about 1.0 × 104Individual/hole.Cultivate 24 little
Shi Hou, adds the HBSS solution being subject to test product drug-delivery preparation and positive control hydrochloric acid chlorine piperazine (CPZ) containing variable concentrations, arranges simultaneously
Blank and excipient/Vehicle controls, each process 6 multiple holes, every kind of by 2 blocks of plates of test product.After humidification culture 1 hour, point
Do not carry out illumination and non-photo-irradiation treatment, then change fresh culture fluid and continue culture 18-22 hour.Before culture terminates, about 3 is little
When, add the DMEM culture fluid containing dimethyl diaminophenazine chloride serum-free to continue culture.After culture terminates, every hole adds dimethyl diaminophenazine chloride eluent, puts
In agitator quick oscillation until forming uniform solution.The OD value in each hole is measured with spectrophotometer under 540nm
(OD540).
With the Phototox computed in software photostimulation factor (PIF) value.In test, the OD of excipient or solvent control group540Number
Value is all higher than 0.4 and positive reference substance hydrochloric acid chlorobenzene piperazine IC50(+UV) within the scope of 0.1-3.0 μ g/ml, IC50(- UV) exists
Within the scope of 7.0 90.0 μ g/ml, and PIF 6, show that pilot system is set up.
In test, during sample-adding and after process terminates, sample each concentration group is not all observed by test product Precipitation and cell
Morphological abnormalities;With Phototox computed in software result of the test, the PIF value of sample is * 1, that is,:Tested material is reaching permission
High concentration value (1000.0 μ g/mL) does not show cytotoxicity yet;Thus the no potential phototoxicity of tested material sample;The MPE of sample
Value is also respectively less than 0.1, further illustrates the expected no phototoxicity of tested material sample.
Table 11 compound recipe Synsepalum duleificum leaf extract Phototoxicity experiment result
Remarks:*1:Do not show cytotoxicity in the maximum concentration value (1000.0 μ g/mL) reaching permission yet;+:Expection is deposited
In phototoxicity;-:Expected no phototoxicity.
Therefore, under this experimental condition, the external 3T3 red untake Phototoxicity experiment of given the test agent is negative reaction,
Expected no phototoxicity.
5th, Skin allergy test
Skin allergy test, with reference to cosmetics health specification (2007 editions), applies skin test using partial closure and tests
(Buehler Test, BT) method, whether determine compound recipe Synsepalum duleificum leaf extract causes skin allergic reaction and degree.Abnormal
The scoring of reaction test dermoreaction is as shown in table 12.
The test dermoreaction scoring of table 12 allergy
Evaluation of result:When dermoreaction integration >=2 in tested material group animal, it is judged to this animal and skin metamorphosis occurs instead
Should be positive, the sensitization intensity of tested material is judged by table 13.
Table 13 sensitization rate is classified
Compound recipe Synsepalum duleificum leaf extract (under 5%, 10% two concentration) is tied to the provocative test of guinea pig skin allergy
Fruit is as shown in table 14.Result shows, guinea pig skin has no allergy, and sensitization rate is 0, animal activity and Normal-weight.
The provocative test result to guinea pig skin allergy for the table 14 compound recipe Synsepalum duleificum leaf extract
Remarks:
Vegetable oil is negative control;
2,4 dinitro-chloro-benzenes are positive control;
Sample 1 is 5% compound recipe Synsepalum duleificum leaf extract solution;
Sample 2 is 10% compound recipe Synsepalum duleificum leaf extract solution.
3rd, conclusion
1st, efficacy assessment conclusion
Through the both effectiveness assessment experiment of system, effect of compound recipe Synsepalum duleificum leaf extract extracting solution is summarized as follows:
(1) compound recipe Synsepalum duleificum leaf extract extracting solution has good antioxidant activity, tested concentration when 2.0%,
DPPH clearance rate reaches 88% about, and the antioxidant activity of Synsepalum duleificum leaf is better than commercially available competing product.
(2) compound recipe Synsepalum duleificum leaf extract extracting solution has good suppression tyrosinase activity, and tested concentration is 1.0%
When, tyrosinase inhibition rate reaches 80% about.
(3) compound recipe Synsepalum duleificum leaf extract extracting solution has certain anti-inflammatory activity, tested concentration when 5.0%, hyalomitome
Sour enzyme inhibition rate is about 65%, and the anti-inflammatory activity of compound recipe Synsepalum duleificum leaf extract is better than commercially available competing product.
(4) improve microcirculation experimental result to show, compound recipe Synsepalum duleificum leaf extract has good improvement skin microcirculation
Effect of blood flow.
(5) melanocyte result of the test shows, compound recipe Synsepalum duleificum leaf extract is in tested concentration 0.5%, 1% and 2%
There is under three tested concentration certain B16 cell suppression ratio, can ensure that the relative motility rate of melanocyte exists simultaneously
More than 80%.Show, compound recipe Synsepalum duleificum leaf extract has certain white-skinned face function in cellular level.
2nd, safety evaluation conclusion
By erythrocyte hemolysis experiment, the multiple Skin Irritation Test of mice, human body patch test, Phototoxicity experiment and skin
Allergy is tested, the safety of assessment compound recipe Synsepalum duleificum leaf extract extracting solution, and five safety experiment results unanimously show,
Compound recipe Synsepalum duleificum leaf extract extracting solution is safe, non-stimulated, and have no adverse reaction generation to human body.
Embodiment described above is only that the preferred embodiment of the present invention is described, the not model to the present invention
Enclose and be defined, on the premise of without departing from design spirit of the present invention, the technical side to the present invention for the those of ordinary skill in the art
Various modifications and improvement that case is made, all should fall in the protection domain of claims of the present invention determination.
Claims (8)
1. a kind of compound recipe Synsepalum duleificum leaf extract preparation there is whitening effect cosmetics in application it is characterised in that:Institute
State compound recipe Synsepalum duleificum leaf extract to be prepared via a method which:
(1) prepare herbal raw material in proportion:Described herbal raw material is made up of major ingredient and adjuvant, and described major ingredient is Synsepalum duleificum
Leaf, percentage by weight is 30%~50%, and adjuvant is Radix Paeoniae Rubra, Fructus Atriplicis Sibiricae, the Radix Astragali, Radix Rhodiolae, Radix Scutellariae, Rhizoma Curcumae Longae, Olibanum, Radix Salviae Miltiorrhizae,
The combination of any three kinds in Radix Notoginseng, percentage by weight is respectively 20%~30%, 10%~30% and 10%~20%;
(2) pretreatment:Carry out dries pulverizing process after foreign body in said herbal medicine raw material is chosen, obtain herbal raw material powder
End, four kinds of herbal powder mix homogeneously make Synsepalum duleificum leaf compound;
(3) extract:Described Synsepalum duleificum leaf compound and solvent are mixed united extraction, obtains extraction solution;
(4) coarse filtration:By described extraction solution coarse filtration, obtain compound recipe Synsepalum duleificum leaf extract coarse filtration liquid;
(5) fine straining:By described compound recipe Synsepalum duleificum leaf extract coarse filtration liquid fine straining, obtain compound recipe Synsepalum duleificum leaf extract fine straining liquid;
(6) concentrate:By described compound recipe Synsepalum duleificum leaf extract fine straining liquid, obtain compound recipe Synsepalum duleificum leaf extract concentrated solution;
(7) redissolve:Described compound recipe Synsepalum duleificum leaf extract concentrated solution is redissolved, obtains compound recipe Synsepalum duleificum leaf extract and redissolve thing;
(8) sterilize, described compound recipe Synsepalum duleificum leaf extract is redissolved thing sterilizing, obtains compound recipe Synsepalum duleificum leaf extract.
2. according to claim 1 application it is characterised in that:Described in step (3), Synsepalum duleificum leaf compound and solvent are pressed
Weight compares 1:10~40 are mixed, and extract 0.5~3 hour, obtain extraction solution at a temperature of 25~90 DEG C.
3. according to claim 1 application it is characterised in that:Redissolve described in step (7) and adopt 1:20 butanediol water
Solution redissolves.
4. according to claim 1 application it is characterised in that:Described solvent is pure water, 40%~95% ethanol solution
Or 40%~95% acetone soln.
5. according to claim 1 application it is characterised in that:Be extracted as described in step (3) being stirred at reflux extraction or
Supersound extraction.
6. the application according to claim 1-5 any one it is characterised in that:Shared by described compound recipe Synsepalum duleificum leaf extract
Percentage by weight is the 0.2%~8% of cosmetics.
7. according to claim 6 application it is characterised in that:The percentage by weight of described compound recipe Synsepalum duleificum leaf extract is
The 0.4~6% of cosmetics.
8. according to claim 6 application it is characterised in that:Described cosmetics are astringent, bath gel, facial cleaning cream, face
Film, facial cream, curry, emulsion, essence, makeup remover or transparent soap.
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Effective date of registration: 20170915 Address after: 10, Shanghai, Fengxian District, Pu Pu Industrial Zone, 201499 Pu Pu Avenue, building 1104, room 111 Patentee after: Shanghai Yi Chemical Co. Ltd. Address before: 200000 Wuning Road, Shanghai, China, No. I square, No. 705, No. 99, Patentee before: SHANGHAI SHANGMEI COSMETICS CO., LTD. |