CN107213053A - A kind of composition for suppressing tyrosinase activity and its preparation method and application - Google Patents
A kind of composition for suppressing tyrosinase activity and its preparation method and application Download PDFInfo
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- CN107213053A CN107213053A CN201710379359.8A CN201710379359A CN107213053A CN 107213053 A CN107213053 A CN 107213053A CN 201710379359 A CN201710379359 A CN 201710379359A CN 107213053 A CN107213053 A CN 107213053A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/82—Preparation or application process involves sonication or ultrasonication
Abstract
The invention provides a kind of composition for suppressing tyrosinase activity, only it is prepared from rose, three kinds of raw materials of peony and China rose, formula is succinct.The raw material sources used are extensive, cheap, reduce manufacturing cost, while raw material is natural plants, not only sensitization is low, and effect harmless to the human body；Above-mentioned raw materials compounding use, there is the suppression to tyrosinase activity, during using L Tyr and L Dopa as substrate, 92.68% and 89.12% can be reached to the inhibiting rate of tyrosinase activity, half-inhibition concentration can reach 0.175mg/mL and 1.229mg/mL, 0.201mg/mL is reached to the active half-inhibition concentration of tyrosinase in melanocyte, to the inhibiting rate of the generation of melanin in melanocyte up to 40.21%；Also have effects that to remove free radical DPPH simultaneously, median elimination concentration reaches 0.043mg/mL.
Description
Technical field
The invention belongs to skin care whitening technical field, it is related to a kind of composition with suppression tyrosinase activity and is somebody's turn to do
The preparation method of composition.
Background technology
Whitening spot-removing is always emphasis of concern, and the principal element that research shows to determine skin color is black in skin
The amount of chromatophore and the ability of melanocyte synthesis of melanin, therefore, from the point of view of in mechanism, the fundamental way of whitening spot-removing
To reduce the amount of melanocyte or suppress the ability of its synthesis of melanin, if but melanocyte metabolism destroyed or
Suppress, some diseases, such as albinism, melanocytoma (melanoma, one kind of cancer) etc. can be produced, so that beautiful
The research emphasis of white nti-freckle falls to be formed in suppression melanin.
Tyrosinase (EC 1.14.18.1, Tyrosinase, TYR) is also known as polyphenol oxidase, catechol-oxydase, is one
The metalloenzyme of cupric is planted, is widely distributed in microorganism, animals and plants and human body, is being closed with melanin for currently the only determination
Into relevant enzyme.Tyrosinase is as the key enzyme of B16 cell, and its catalytic mechanism is：It is catalyzed the conversion of TYR hydroxylating
For L-3,4 dihydroxyphenylalanine, simultaneous oxidation L-3,4 dihydroxyphenylalanine formation DOPA quinone, DOPA quinone passes through series reaction, ultimately forms melanin again.Therefore,
Suppress the activity of tyrosinase to reduce the raw material DOPA quinone of synthesis of melanin, be one of important channel of skin whitening.
At present, tyrosinase activity inhibitor can substantially be divided into chemical synthesis class inhibitor and natural extract inhibitor
Two classes, chemical synthesis class inhibitor mainly includes quinhydrones and azelaic acid, and being used for a long time can have undesirable effect to human body, such as hydrogen
Quinone is used for a long time not only has larger side effect to human body, and can cause melanin deposition in skin, causes to be difficult to clean off, nonyl
Diacid can cause to stimulate to skin, eyes, mucous membrane and the upper respiratory tract；Natural extract inhibitor mainly include kojic acid, ursin,
Licoflavone and safflower flavone etc., although it is harmless, limited source, price is high, such as natural ursin
Exist only in black bearberry leaf, not only black bearberry quantity is few, plantation difficulty is big, and in its leaf ursin content it is also not high.
Therefore, Chinese patent literature CN104622735A discloses a kind of tree peony compound essential oil of whitening spot-removing, its raw material
Including Rosa Damascana, Salvia japonica essential oil, Lavender, Geranium Essential, jasmine oil, fresh peony flower essential oil, peony seed oil.On
The technical scheme disclosed in patent document is stated, uses various plants essential oil to be used in mixed way to realize the suppression to tyrosinase activity
System, so as to obtain effect of whitening spot-removing.It uses plants essential oil as raw material, it is to avoid chemical synthesis inhibitor on human body
Injury, and the plants essential oil source used is also relatively broad, reduces cost to a certain extent, but plant-derived essential oil
Complexity, long-term use easily causes allergic, and nowadays environmental pollution is serious in addition, and anaphylactogen increases, and the patient of allergy occurs
It is more and more, reduce the universality of product.
The content of the invention
To solve the existing defect that tyrosinase inhibitor side effect is obvious, universality is poor, so as to improve a kind of suppression
The natural composition of tyrosinase activity.
A kind of composition for suppressing tyrosinase activity, includes the raw material of following parts by weight：
30-60 parts of rose, 20-40 parts of China rose, 10-40 parts of peony.
Preferably, in the composition of described suppression tyrosinase activity, the raw material of following parts by weight is included：
45-55 parts of rose, 30-35 parts of China rose, 15-25 parts of peony.
Preferably, in the composition of described suppression tyrosinase activity, the China rose and the matter of the peony
Amount is than being 2:1.
Preferably, in the composition of described suppression tyrosinase activity, the matter of the rose and the peony
Amount is than being 3:2.
A kind of preparation method for the composition for suppressing tyrosinase activity, comprises the following steps：
After rose, China rose and peony are soaked in ethanol solution, ultrasonic assistant ethanol is extracted, and is filtered, filter
Liquid reclaims ethanol, and the composition for suppressing tyrosinase activity is made.
Preferably, in described preparation method, the temperature that the ultrasonic assistant ethanol is extracted is 50-60 DEG C, time
For 30-60min, supersonic frequency is 53kHz.
Preferably, in described preparation method, the volumetric concentration of the ethanol solution is 40%-60%.
Preferably, in described preparation method, the consumption of the ethanol solution is that rose, China rose and peony are total
20-25 times of quality.
Preferably, in described preparation method, soak time is 2-3h, and soaking temperature is 22-27 DEG C.
A kind of application for the composition for suppressing tyrosinase activity.
Technical solution of the present invention, has the following advantages that：
The invention provides a kind of composition for suppressing tyrosinase activity, including rose, peony and China rose three
Raw material is planted, formula is succinct.And it is the wide material sources of raw material rose, China rose and the peony used, cheap, significantly
Reduce manufacturing cost, while above-mentioned raw materials are natural plants, not only sensitization is low, and effect harmless to the human body, fit
In long-term use；
Above-mentioned raw materials cooperate, and with to the significant suppression effect of tyrosinase activity, are experimentally confirmed, with L-
When Tyr (TYR) and L-Dopa (L-3,4 dihydroxyphenylalanine) is substrate, 92.68% He can be reached to the inhibiting rate of tyrosinase activity
89.12%, half-inhibition concentration can reach 0.175mg/mL and 1.229mg/mL, to tyrosinase in melanocyte
The half-inhibition concentration of activity fundamentally inhibits its inhibiting rate of the generation of melanin reachable up to 0.201mg/mL
40.21%；In addition, also with stronger removing free radical DPPH functions, median elimination concentration is bright up to 0.043mg/mL
It is aobvious to be better than contrast groups.
To sum up the composition disclosed by the invention for suppressing tyrosinase activity not only has significant whitening and light to skin
Effect of spot, and can effectively reduce the generation of wrinkle.
Embodiment
Technical scheme will be clearly and completely described below, it is clear that described embodiment is this hair
Bright a part of embodiment, rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art are not having
There is the every other embodiment made and obtained under the premise of creative work, belong to the scope of protection of the invention.In addition, below
Just can be mutual as long as the technical characteristic involved by described different embodiments of the present invention does not constitute conflict each other
Knot.
Embodiment 1
A kind of preparation method for the composition for suppressing tyrosinase activity, including：
(1) by rose 30g, China rose 20g and peony 40g, in the environment of 25 DEG C, it is 40% to be placed in volumetric concentration
Ethanol solution in, soak 2h, the consumption of ethanol solution is rose, 20 times of China rose and peony quality summation；
(2) water bath sonicator instrument is used, it is 50 DEG C to control bath temperature, (ethanol is molten by the soak after being soaked in step (1)
Liquid) and rose, China rose and peony, 60min is extracted using ultrasonic assistant ethanol, wherein ultrasonic frequency is
53kHz, power density is 0.5W/cm2；
(3) filter, filter out solid, vacuum distillation removes ethanol solution, obtains refined liquid, using distilled water that refined liquid is dilute
3g/L is released, as suppresses the composition of tyrosinase activity, while reclaiming ethanol solution.
Embodiment 2
A kind of preparation method for the composition for suppressing tyrosinase activity, including：
(1) by rose 60g, China rose 40g and peony 10g, in the environment of 22 DEG C, it is 60% to be placed in volumetric concentration
Ethanol solution in, soak 2h, the consumption of ethanol solution is rose, 20 times of China rose and peony quality summation；
(2) water bath sonicator instrument is used, it is 60 DEG C to control bath temperature, (ethanol is molten by the soak after being soaked in step (1)
Liquid) and rose, China rose and peony, 60min is extracted using ultrasonic assistant ethanol, wherein ultrasonic frequency is
53kHz, power density is 1.5W/cm2；
(3) filter, filter out solid, vacuum distillation removes ethanol solution, obtains refined liquid, using distilled water that refined liquid is dilute
3g/L is released, as suppresses the composition of tyrosinase activity, while reclaiming ethanol solution.
Embodiment 3
A kind of preparation method for the composition for suppressing tyrosinase activity, including：
(1) by rose 45g, China rose 35g and peony 25g, in the environment of 27 DEG C, it is 50% to be placed in volumetric concentration
Ethanol solution in, soak 3h, the consumption of ethanol solution is rose, 25 times of China rose and peony quality summation；
(2) water bath sonicator instrument is used, it is 50 DEG C to control bath temperature, (ethanol is molten by the soak after being soaked in step (1)
Liquid) and rose, China rose and peony, 60min is extracted using ultrasonic assistant ethanol, wherein ultrasonic frequency is
53kHz, power density is 1.0W/cm2；
(3) filter, filter out solid, vacuum distillation removes ethanol solution, obtains refined liquid, using distilled water that refined liquid is dilute
3g/L is released, as suppresses the composition of tyrosinase activity, while reclaiming ethanol solution.
Embodiment 4
A kind of preparation method for the composition for suppressing tyrosinase activity, including：
(1) by rose 55g, China rose 32g and peony 16g, in the environment of 25 DEG C, it is 40% to be placed in volumetric concentration
Ethanol solution in, soak 3h, the consumption of ethanol solution is rose, 25 times of China rose and peony quality summation；
(2) water bath sonicator instrument is used, it is 50 DEG C to control bath temperature, (ethanol is molten by the soak after being soaked in step (1)
Liquid) and rose, China rose and peony, 60min is extracted using ultrasonic assistant ethanol, wherein ultrasonic frequency is
53kHz, power density is 1.0W/cm2；
(3) filter, filter out solid, vacuum distillation removes ethanol solution, obtains refined liquid, using distilled water that refined liquid is dilute
3g/L is released, as suppresses the composition of tyrosinase activity, while reclaiming ethanol solution.
Embodiment 5
A kind of preparation method for the composition for suppressing tyrosinase activity, including：
(1) by rose 45g, China rose 30g and peony 15g, in the environment of 25 DEG C, it is 40% to be placed in volumetric concentration
Ethanol solution in, soak 3h, the consumption of ethanol solution is rose, 22 times of China rose and peony quality summation；
(2) water bath sonicator instrument is used, it is 60 DEG C to control bath temperature, (ethanol is molten by the soak after being soaked in step (1)
Liquid) and rose, China rose and peony, 30min is extracted using ultrasonic assistant ethanol, wherein ultrasonic frequency is
53kHz, power density is 1.5W/cm2；
(3) filter, filter out solid, vacuum distillation removes ethanol solution, obtains refined liquid, using distilled water that refined liquid is dilute
3g/L is released, as suppresses the composition of tyrosinase activity, while reclaiming ethanol solution.
Embodiment 6
A kind of preparation method for the composition for suppressing tyrosinase activity, including：
(1) by rose 50g, China rose 33g and peony 20g, in the environment of 25 DEG C, it is 50% to be placed in volumetric concentration
Ethanol solution in, soak 3h, the consumption of ethanol solution is rose, 22 times of China rose and peony quality summation；
(2) water bath sonicator instrument is used, it is 50 DEG C to control bath temperature, (ethanol is molten by the soak after being soaked in step (1)
Liquid) and rose, China rose and peony, 45min is extracted using ultrasonic assistant ethanol, wherein ultrasonic frequency is
53kHz, power density is 1.0W/cm2；
(3) filter, filter out solid, vacuum distillation removes ethanol solution, obtains refined liquid, using distilled water that refined liquid is dilute
3g/L is released, as suppresses the composition of tyrosinase activity, while reclaiming ethanol solution.
Comparative example 1
A kind of preparation method for the composition for suppressing tyrosinase activity, including：
(1) by rose 70g, China rose 50g and peony 45g, in the environment of 25 DEG C, it is 40% to be placed in volumetric concentration
Ethanol solution in, soak 3h, the consumption of ethanol solution is rose, 22 times of China rose and peony quality summation；
(2) water bath sonicator instrument is used, it is 60 DEG C to control bath temperature, (ethanol is molten by the soak after being soaked in step (1)
Liquid) and rose, China rose and peony, 30min is extracted using ultrasonic assistant ethanol, wherein ultrasonic frequency is
53kHz, power density is 1.5W/cm2；
(3) filter, filter out solid, vacuum distillation removes ethanol solution, obtains refined liquid, using distilled water that refined liquid is dilute
3g/L is released, as suppresses the composition of tyrosinase activity, while reclaiming ethanol solution.
Comparative example 2
A kind of preparation method for the composition for suppressing tyrosinase activity, including：
(1) rose 90g is placed in the ethanol solution that volumetric concentration is 40%, in the environment of 25 DEG C, soaks 3h, second
The consumption of alcoholic solution is 22 times of rose quality；
(2) water bath sonicator instrument is used, it is 60 DEG C to control bath temperature, (ethanol is molten by the soak after being soaked in step (1)
Liquid) and rose, 30min is extracted using ultrasonic assistant ethanol, wherein ultrasonic frequency is 53kHz, and power density is 1.5W/
cm2；
(3) filter, filter out solid, vacuum distillation removes ethanol solution, obtains refined liquid, using distilled water that refined liquid is dilute
3g/L is released, as suppresses the composition of tyrosinase activity, while reclaiming ethanol solution.
Comparative example 3
A kind of preparation method for the composition for suppressing tyrosinase activity, including：
(1) China rose 90g is placed in the ethanol solution that volumetric concentration is 40%, in the environment of 25 DEG C, soaks 3h, second
The consumption of alcoholic solution is 22 times of China rose quality；
(2) water bath sonicator instrument is used, it is 60 DEG C to control bath temperature, (ethanol is molten by the soak after being soaked in step (1)
Liquid) and China rose, 30min is extracted using ultrasonic assistant ethanol, wherein ultrasonic frequency is 53kHz, and power density is 1.5W/
cm2；
(3) filter, filter out solid, vacuum distillation removes ethanol solution, obtains refined liquid, using distilled water that refined liquid is dilute
3g/L is released, as suppresses the composition of tyrosinase activity, while reclaiming ethanol solution.
Comparative example 4
A kind of preparation method for the composition for suppressing tyrosinase activity, including：
(1) peony 90g is placed in the ethanol solution that volumetric concentration is 40%, in the environment of 25 DEG C, soaks 3h, second
The consumption of alcoholic solution is 22 times of peony quality；
(2) water bath sonicator instrument is used, it is 60 DEG C to control bath temperature, (ethanol is molten by the soak after being soaked in step (1)
Liquid) and peony, 30min is extracted using ultrasonic assistant ethanol, wherein ultrasonic frequency is 53kHz, and power density is 1.5W/
cm2；
(3) filter, filter out solid, vacuum distillation removes ethanol solution, obtains refined liquid, using distilled water that refined liquid is dilute
3g/L is released, as suppresses the composition of tyrosinase activity, while reclaiming ethanol solution.
Comparative example 5
By Rosa Damascana 5.0g, Salvia japonica essential oil 3.6g, Lavender 6.5g, Geranium Essential 7.0g, refined jasmine oil
8.0g and fresh peony flower essential oil 5.0g, is put into stirring container, normal temperature stirring at low speed, speed of agitator is 30-50r/min, makes each
Raw material is sufficiently mixed uniformly, adds peony seed oil 364g, lucid asparagus extract 2g, normal temperature stirring at low speed, speed of agitator is 30-
50r/min, fully dilution fusion, is made composite essential oil.
Comparative example 6
(1) by rose 45g, Chinese rose leaf 30g, peony 15g, honeysuckle 3g and feverfew 5g, in 25 DEG C of environment
Under, it is placed in the ethanol solution that volumetric concentration is 40%, soaks 3h, the consumption of ethanol solution is rose, Chinese rose leaf, tree peony
22 times of flower, honeysuckle and feverfew quality summation；
(2) water bath sonicator instrument is used, it is 60 DEG C to control bath temperature, (ethanol is molten by the soak after being soaked in step (1)
Liquid) and rose, Chinese rose leaf, peony, honeysuckle and feverfew, 30min is extracted using ultrasonic assistant ethanol, wherein super
Frequency of sound wave is 53kHz, and power density is 1.5W/cm2；
(3) filter, filter out solid, vacuum distillation removes ethanol solution, obtains refined liquid, using distilled water that refined liquid is dilute
3g/L is released, the plant composition with white-skinned face function, while reclaiming ethanol solution.
Compliance test result
Composition no prepared by embodiment 1-6 and comparative example 1-6 is 1-12.
(1) DPPH radicals scavengings are tested
Free radical scavenging activity (2,2-diphenyl-1-picrylhydrazyl, DPPH) is a kind of stable
Organic free radical, its stability and makes to be clipped in wherein nitrogen essentially from the spatial obstacle of Resonance Stabilization action and 3 phenyl ring
Unpaired electron on atom can not play its due electronics and act in pairs.Pass through the clear of detectable substance confrontation DPPH free radicals
Removing solid capacity can represent the power of its inoxidizability.
Precision weighs DPPH 20mg, is dissolved with 95% ethanol and is settled to 1000mL, obtains the DPPH that concentration is 20mg/L
Standard liquid, the composition test solution that compound concentration is 1mg/mL respectively for being 1-10 and 12 with numbering；
90 μ L DPPH standard liquids are pipetted respectively into 10 reactors with pipettor, successively into each reaction vessel
10 μ L test solution is added, places after 30min, using MD ELIASAs, absorbance is determined under 517nm, using VC as positive control.
The IC for removing DPPH is calculated according to testing result50Value, the results are shown in Table 1.
Table 1 removes DPPH IC50Value
The removing DPPH of the composition of suppression tyrosinase activity prepared by the embodiment 1-6 IC from table 150Value is substantially low
In the removing DPPH of the composition of the suppression tyrosinase activities prepared of comparative example 1-4 and 6 IC50Value, it was demonstrated that the present embodiment 1-6
The ability of the composition removing DPPH free radicals of the suppression tyrosinase activity of preparation is better than suppression prepared by comparative example 1-4 and 6
The composition of tyrosinase activity.
(2) tyrosinase activity suppresses experiment
2.1 tyrosinase inhibition rate (I) detection using L-Tyr and L-Dopa as substrate
During tyrosine is converted into melanin, the catalytic action of tyrosinase occurs mainly in tyrosine and is converted into L-
DOPA, and during L-3,4 dihydroxyphenylalanine is converted into DOPA quinone, DOPA quinone is a kind of coloring matter, has absorption in 475nm, available
Spectrophotometric determination content.
The composition test solution that compound concentration is 3mg/mL respectively for being 1-10 with numbering；
Respectively using L-Tyr and L-Dopa as substrate, the composition that numbering is 1-10 prepares test solution group, profit according to table 2
Absorbance is detected with spectrophotometer, absorbance is detected in the case where wavelength is 475nm, according to the absorbance detected, according to
Formula (1), which is calculated, numbers the composition for being 1-10 to the inhibiting rate (I) of tyrosinase activity, and result of the test is shown in Table 3.
The testing sample of table 2 constitutes (mL)
The calculation formula of tyrosinase inhibition rate (I) is as follows：
Wherein,
Aa：For the absorbance of the mixed liquor of a groups；
Ab：For the absorbance of the mixed liquor of b groups；
Ac：For the absorbance of the mixed liquor of c groups；
Ad：For the absorbance of the mixed liquor of d groups.
2.2 IC suppressed to tyrosinase by substrate of L-Tyr and L-Dopa50Detection：
The each composition numbered in the composition for being 1-10 is prepared to a series of standard test solution of gradient concentrations,
Corresponding one group of standard test solution is each numbered according to the method in 2.1 respectively using L-Tyr and L-Dopa as substrate, standard is molten
The inhibiting rate of each concentration in liquid, using concentration as abscissa, inhibiting rate is ordinate, show that the dose-effect of concentration and inhibiting rate is closed
System, it can thus be concluded that the IC suppressed to composition to tyrosinase activity50Value, testing result is shown in Table 4.
The inhibitory activity against tyrosinase of table 3 (I)
As can be seen from Table 3 either using L-Tyr be substrate still using L-Dopa as substrate, embodiment 1-6 prepare
Suppress the composition of tyrosinase activity to inhibitory activity against tyrosinase, the suppression junket prepared obviously higher than comparative example 1-4
The composition of propylhomoserin enzymatic activity, it was demonstrated that the composition of suppression tyrosinase activity prepared by embodiment 1-6 is to tyrosinase activity
Rejection ability be substantially better than comparative example 1-4 preparation suppression tyrosinase activity composition.
Table 4 suppresses the IC of tyrosinase activity50Value
As can be seen from Table 4 either using L-Tyr be substrate still using L-Dopa as substrate, embodiment 1-6 prepare
The composition for suppressing tyrosinase activity suppresses the IC of tyrosinase activity50Value, is significantly less than the suppression of comparative example 1-4 preparations
The composition of tyrosinase activity, it was demonstrated that the composition of suppression tyrosinase activity prepared by embodiment 1-6 suppresses tyrosinase
The ability of activity is substantially better than the composition of the suppression tyrosinase activity of comparative example 1-4 preparations.
(3) measure of intracellular tyrosine enzymatic activity
1. culture melanocyte grows into exponential phase, washed twice using PBS (phosphate buffer), use quality
Concentration digests for 0.25% pancreatin, is counted with blood counting chamber, is adjusted in every milliliter have 2 × 104Individual melanocyte, with
100 μ L amount is taken to be linked into 96 orifice plates per hole；
2. the difference numbered in the composition for being 1-12 is prepared a series of as solvent with DMSO (dimethyl sulfoxide (DMSO))
The standard test solution of gradient concentration, each numbers corresponding one group of standard test solution；
96 orifice plates are cultivated in 37 DEG C of incubators adds standard test solution respectively after 24h, each concentration sets 3 holes,
Control group is to be not added with standard test solution, in CO2Content is in 5% incubator, 37 DEG C is incubated 72h；
3. in incubator cultivating process, supernatant discarding after a pharmaceutical culture medium (96 orifice plate), 72h is changed in centre, is swung and washed with PBS
2 times, per the Kong Jiahan 0.1%Triton X-100 μ L of PBS cushioning liquid 100, vibrate and cultivated at 10min, 0 DEG C after 30min, risen
Temperature is to 37 DEG C, and addition mass concentration is the μ L of 0.1%L-Dopa solution 10, is incubated after 30min, and absorbance is surveyed at 475nm wavelength；
According to the absorbance of detection, calculate the composition that numbering is 1-12 using formula (2) and press down in melanocyte
The inhibiting rate of tyrosinase activity processed；Using concentration as abscissa, inhibiting rate is ordinate, draws curve, is calculated according to curve
Composition suppresses the IC of tyrosinase activity in melanocyte50Value, the results are shown in Table 5.
Inhibitory activity against tyrosinase (the %)=(2) of (1-A dosing groups/A control groups) × 100% ...
A dosing groups are the absorbance added with standard test solution；
A control groups are the absorbance for not adding standard test solution.
The composition of table 5 suppresses the IC of tyrosinase activity in melanocyte50Value
The composition of suppression tyrosinase activity prepared by embodiment 1-6 suppresses tyrosine enzyme activity as can be seen from Table 5
The IC of property50Value, is significantly less than the composition of the suppression tyrosinase activity of comparative example 1-6 preparations, it was demonstrated that prepared by embodiment 1-6
The composition of suppression tyrosinase activity suppress the ability of tyrosinase activity in melanocyte and be substantially better than comparative example
The composition of suppression tyrosinase activity prepared by 1-6.
The composition of suppression tyrosinase activity prepared by comparative example 6 suppresses tyrosinase activity in melanocyte
Ability is than single rose and the ability of the suppression tyrosinase activity of peony, it may be possible to which raw material compounding produces antagonism and made
With caused.
(5) composition suppresses to determine to melanin in melanocyte
1. from the melanoma tumors B16 cells in exponential phase, concentration of cell suspension is adjusted, by 2 × 104It is individual thin
Born of the same parents/mL concentration is inoculated in 96 well culture plates, wherein, the volume in each hole in 96 well culture plates is 100 μ L, is not added with thin
Filled using sterile PBS in the hole of born of the same parents；
2. with DMSO as solvent, will number in the composition for being 1-10 is respectively that 150 μ g/mL standards are surveyed with concentration
Try solution；
96 orifice plates cultivate 24h in 37 DEG C of incubators, cell monolayer is paved with 96 well culture plate bottom holes, standard is added respectively
Solution is tested, each standard liquid sets 3 holes, and control group is to be not added with standard test solution, in CO2Content is 5% incubator
In, 37 DEG C of incubation 72h；
3. in incubator cultivating process, supernatant discarding after a pharmaceutical culture medium (96 orifice plate), 72h is changed in centre, is swung and washed with PBS
2 times, digested with mass concentration for 0.25% pancreatin, harvesting is put into centrifuge tube；
4. harvesting the centrifugation that obtained cell passes through 1000r/min, supernatant discarding adds 1mL PBS solution centrifuge washings
Once, supernatant is abandoned；
5. adding 100 μ L PBS solutions, vibration adds 200 μ L of the 80 DEG C of preheatings NaOH containing 10%DMSO after mixing
(1mol/L), heats 3h at 80 DEG C, and per half an hour, vibration once, takes 100 μ L to be transferred in 96 new orifice plates, in 405nm wavelength
Lower detection absorbance.
According to the absorbance detected, melanin is generated in melanocyte using formula (3) calculation composition and pressed down
Rate processed, the results are shown in Table 6.
B16 cell inhibiting rate=[1- (medicine hole absorbance ÷ medicines hole cell density) ÷ (control wells absorbances
Value ÷ control wells cell density)] × 100% ... .. (3)
Medicine hole absorbance is the absorbance added with standard test solution；
Medicine hole cell density is the concentration of the B16 cells added with standard test solution；
Control wells absorbance is the absorbance for the control group for being not added with standard test solution；
Control wells cell density is the concentration of the B16 cells for the control group for being not added with standard test solution.
Inhibiting rate of the composition of table 6 in melanocyte to melanin formation
As known from Table 6, when concentration is 250 μ g/mL, the composition of suppression tyrosinase activity prepared by embodiment 1-6
The composition for suppressing tyrosinase activity prepared to melanin generating suppression in melanocyte apparently higher than comparative example 1-4；
Prove that the composition of suppression tyrosinase activity prepared by embodiment 1-6 is obvious to melanin generation rejection ability in melanocyte
Better than the composition of the comparative example 1-4 suppression tyrosinase activities prepared.
Obviously, above-described embodiment is only intended to clearly illustrate example, and the not restriction to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or
Change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change thus extended out or
Among changing still in the protection domain of the invention.
Claims (10)
1. a kind of composition for suppressing tyrosinase activity, it is characterised in that include the raw material of following parts by weight：
30-60 parts of rose, 20-40 parts of China rose, 10-40 parts of peony.
2. the composition according to claim 1 for suppressing tyrosinase activity, it is characterised in that including following parts by weight
Raw material：
45-55 parts of rose, 30-35 parts of China rose, 15-25 parts of peony.
3. it is according to claim 1 or 2 suppress tyrosinase activity composition, it is characterised in that the China rose with
The mass ratio of the peony is 2:1.
4. according to the composition of any described suppression tyrosinase activities of claim 1-3, it is characterised in that the rose
Mass ratio with the peony is 3:2.
5. a kind of method of the composition of the suppression tyrosinase activity prepared described in claim any one of 1-4, including it is as follows
Step：
After rose, China rose and peony are soaked in ethanol solution, ultrasonic assistant ethanol is extracted, and filtration, filtrate is returned
Ethanol is received, the composition for suppressing tyrosinase activity is made.
6. preparation method according to claim 5, it is characterised in that the temperature that the ultrasonic assistant ethanol is extracted is
50-60 DEG C, the time is 30-60min, and supersonic frequency is 53kHz.
7. the preparation method according to claim 5 or 6, it is characterised in that the volumetric concentration of the ethanol solution is 40%-
60%.
8. according to any described preparation methods of claim 5-7, it is characterised in that the consumption of the ethanol solution is rose
20-25 times of colored, China rose and peony gross mass.
9. according to any described preparation methods of claim 5-8, it is characterised in that soak time is 2-3h, and soaking temperature is
22-27℃。
10. a kind of application of the composition of any described suppression tyrosinase activities of claim 1-4.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108721203A (en) * | 2018-08-09 | 2018-11-02 | 广州暨南生物医药研究开发基地有限公司 | Skin care item and preparation method thereof are repaired in a kind of whitening of the extract of stem cell containing tree peony |
| CN114894923A (en) * | 2022-04-20 | 2022-08-12 | 广州大学 | Method for inhibiting melanin in organism based on active oxygen free radical scavenging by antioxidant |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108721203A (en) * | 2018-08-09 | 2018-11-02 | 广州暨南生物医药研究开发基地有限公司 | Skin care item and preparation method thereof are repaired in a kind of whitening of the extract of stem cell containing tree peony |
| CN114894923A (en) * | 2022-04-20 | 2022-08-12 | 广州大学 | Method for inhibiting melanin in organism based on active oxygen free radical scavenging by antioxidant |
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| CN107213053B (en) | 2020-07-14 |
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