CN104367512B - A kind of blueberry leaf flavones Essence and preparation method and application - Google Patents
A kind of blueberry leaf flavones Essence and preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of blueberry leaf flavones Essence and preparation method and application.The present invention discloses a kind of preparation method of blueberry leaf flavones Essence, comprises the following steps：(1) disodium ethylene diamine tetraacetate dissolving is added in water, carbomer U20 is added and mixes, be heated to 70 85 DEG C, glycerine mixing is added, 70 80 DEG C of insulation 5min 15min add allantoin dissolving and mixed, 45 DEG C 55 DEG C are cooled to, blueberry leaf flavones is added, A phase solution is obtained.Present invention demonstrates that blueberry leaf flavones has anti-inflammatory efficacy, the blueberry leaf flavones Essence using it as active component can apply to cosmetic field.
Description
Technical field
The present invention relates to a kind of blueberry leaf flavones Essence and preparation method and application, belong to cosmetic field.
Background technology
Flavonoid substances belong to the secondary metabolite of plant, are that a class has multiple biological activities plant polyphenol kind thing
Matter.Research shows that Flavonoid substances can reduce the adhesion effect between the endothelial cell in inflammatory reaction and immunocyte, from
And slow down inflammatory reaction.Research shows the content of Flavonoid substances in blueberry (Vaccinium uliginosum L.) leaf very
Height, and species is abundant, and with anti-aging, anti-inflammatory, antiallergy, antiviral, anticancer strengthen cell membrane, make damaged cell again
The multi-efficiency such as raw.Blueberry leaf can also be used to the chronic inflammation occurred and the urgency occurred by damage on processing skin histology
Property inflammation, it is possible to prevent and treat in oral cavity produce inflammatory reaction.
IL-10 is a kind of T h1 Cellular immunity suppression factors, thin in regulation Th1 with immunosupress and anti-inflammatory effect
Played an important role in the cell factor in born of the same parents source, thus be referred to as CSIF in early days.IL-10 belongs to cell
Interference family in the factor, research find endogenous and exogenous IL-10 can on transcriptional level strong inhibition IL-1,
IL-6, IL-8, tumor necrosis factor-alpha (TNF-α), GM-CSF, G-CSF synthesis.Nearly ten years, research thinks that IL-10 is main
It is activation by suppressing IKK or NK-k β DNA binding abilities, so that suppressing NK-k β starts related proinflammatory factor gene
Transcription.Therefore anti-inflammatory efficacy is evaluated with IL-10 expression quantity on a molecular scale, be in inflammation early period of origination even inflammation still
The effective ways that the nonevent stage is evaluated it.Some treatment psoriasis medicine for example corticosteroid, Calcipotriol,
Vitamine D3, anthraline etc. can dramatically increase IL-10 expression, and research confirms that IL-10 function of resisting psoriasis is by regulation
Caused by keratinocyte secrete cytokines.Further IL-10 is supported to be played an important role in onset of psoriasis, clinically
Through treating psoriasis using Recombinant Human IL-10.In addition, scholar generally believes that IL-10, IL-8 can be as psoriasis disease development
Early monitoring index.
At present for the research of anti-inflammatory class cosmetics, it is broadly divided into live body, structural efficacy validation, molecular level is ground
Study carefully the form for being only limitted to that zooblast is pattern, with skin progenitor cell to the rare report of flavones anti-inflammatory efficacy, and pass through mankind's table
Skin stem cell carries out the identification of blueberry flavones anti-inflammatory efficacy and also belonged to first.
The content of the invention
It is an object of the invention to provide a kind of blueberry leaf flavones Essence and preparation method and application.
The present invention provides a kind of preparation method of blueberry leaf flavones Essence, comprises the following steps：
(1) disodium ethylene diamine tetraacetate dissolving is added in water, carbomer U20 is added and mixes, 70-85 DEG C is heated to, then
Add glycerine to mix, 70-80 DEG C of insulation 5min-15min adds allantoin dissolving and mixed, be cooled to 45 DEG C -55 DEG C, add
Blueberry leaf flavones, obtains A phase solution；
(2) Microcare MTI, butanediol, 1,2- pentanediols are mixed, obtains C phase solution；
(3) 1g/100ml aqueous solution of sodium hyaluronate, sea is sequentially added in temperature is 45 DEG C -55 DEG C of A phase solution
Algae sugar, often adds a kind of material and is intended to mixing, obtain A phases, the mixed liquor of B phases；
(4) A phases at 45 DEG C -50 DEG C, B mix addition C phase solution in solution, mix, obtain A phases, B phases and C phases
Mixed liquor；
(5) add water, produce blueberry leaf flavones Essence；
In the blueberry leaf flavones Essence, the water in the step (1) accounts for the quality of the blueberry leaf flavones Essence
Percentage composition is that the weight/mass percentage composition that the 60%, disodium ethylene diamine tetraacetate accounts for the blueberry leaf flavones Essence is
The weight/mass percentage composition that 0.02-0.1%, the allantoin account for the blueberry leaf flavones Essence is 0.1-0.5%, the card ripple
Nurse U20 accounts for the weight/mass percentage composition of the blueberry leaf flavones Essence and accounts for the blueberry leaf flavones for 0.2-0.8%, the glycerine
The weight/mass percentage composition of Essence is the quality hundred that 1.0-6.0%, the blueberry leaf flavones account for the blueberry leaf flavones Essence
Point content is that 0.2-4.0%, the aqueous solution of sodium hyaluronate of the 1g/100ml account for the quality of the blueberry leaf flavones Essence
Percentage composition is that the weight/mass percentage composition that 1.0-6.0%, the trehalose account for the blueberry leaf flavones Essence is 0.2-
2.0%th, the Microcare MTI account for the weight/mass percentage composition of the blueberry leaf flavones Essence for 0.05-0.1%, described
Butanediol accounts for the weight/mass percentage composition of the blueberry leaf flavones Essence and accounts for the indigo plant for 1.0-3.0%, 1, the 2- pentanediols
The weight/mass percentage composition of certain kind of berries leaf flavones Essence is that 0.5-5.0%, surplus are water added by step (5)；
The pH of the blueberry leaf flavones Essence is 5.5-6.5；
The entitled acrylic acid of INCI (ester) class of the carbomer U20/C10-30 alkylol acrylamide acid esters cross-linked polymers；
The entitled METHYLISOTHIAZOLINONE of INCI of the Microcare MTI
IODOPROPYNYLBUTYLCARBAMATE。
In the above method, after the step (5), also have and adjust the pH of the blueberry leaf flavones Essence to 5.5-
6.5 the step of；
Reagent used in the pH regulations is specially sodium hydrate aqueous solution or aqueous citric acid solution；
The sodium hydrate aqueous solution is specially 5g/100ml sodium hydrate aqueous solution；
The aqueous citric acid solution is specially 10g/100ml aqueous citric acid solution.
In any of the above-described described method, the time being incubated described in the step (1) is 10min；
The temperature of A phase solution is 50 DEG C in the step (3).
In any of the above-described described method, the preparation method of the blueberry leaf flavones is as follows：By blueberry leaf powder and volume
Percentage composition is 50%-90% ethanol water according to solid-liquid ratio 1g:20ml-1g:70ml is mixed, and is existed using homogenate extraction method
Homogenate extraction 30s-270s under 30V-110V voltage, obtains extract solution, then extract solution centrifugation is obtained into supernatant, by supernatant
Freeze-drying is produced.
In the above method, the ethanol water is the ethanol water that volumn concentration is 80%；
The solid-liquid ratio is 1g:40ml；
The voltage of the homogenate extraction method is 60V；
The time of the homogenate extraction is 90s.
The blueberry leaf flavones Essence prepared by any of the above-described described method falls within protection scope of the present invention.
Application of the blueberry leaf flavones in the product with anti-inflammatory and/or skin care effect is prepared falls within the protection of the present invention
Scope.
In above-mentioned application, the preparation method of the blueberry leaf flavones is as follows：It is by blueberry leaf powder and volumn concentration
50%-90% ethanol water is according to solid-liquid ratio 1g:20ml-1g:70ml is mixed, using homogenate extraction method 30V-110V's
Homogenate extraction 30s-270s under voltage, obtains extract solution, then extract solution centrifugation is obtained into supernatant, is by supernatant freeze-drying
.
In above-mentioned application, the ethanol water is the ethanol water that volumn concentration is 80%；
The solid-liquid ratio is 1g:40ml；
The voltage of the homogenate extraction method is 60V；
The time of the homogenate extraction is 90s.
Application of the above-mentioned blueberry leaf flavones Essence in the product with anti-inflammatory and/or skin care effect is prepared falls within this
The protection domain of invention.
Present invention demonstrates that blueberry leaf flavones has anti-inflammatory efficacy, the blueberry leaf flavones Essence using it as active component can be with
Applied to cosmetic field.
Brief description of the drawings
Fig. 1 is quantitative fluorescent PCR program.
Fig. 2 is that leaf of Fructus vaccini vitis-idaeae extract handles different time to the influence of IL-10 expression quantity.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
U20 (carbomer, INCI titles：Acrylic acid (ester) class/C10-30 alkylol acrylamide acid esters cross-linked polymer) it is purchased from road
Rich extraordinary chemical industry (Shanghai) Co., Ltd. of profit.
HA (Sodium Hyaluronate) is purchased from Bloomage Freda Biopharm Co., Ltd., cosmetics-stage.1%HA is that concentration is
1g/100ml aqueous solution of sodium hyaluronate, preparation method is：1g Hyal powders are weighed, 100ml deionizations are dissolved in
In water, clear liquid is uniformly mixed to.
Microcare MTI (INCI titles：METHYLISOTHIAZOLINONE
IODOPROPYNYLBUTYLCARBAMATE) it is purchased from the Chemical Company of the Shanghai universe one.
Hydrolite 5 (1,2- pentanediol) is purchased from fragrant (Shanghai) Co., Ltd. of moral, and catalog number is 616751.
The preparation of KGM basal mediums：90ml DMEM (Gibco is purchased from, catalog number is 11965) basal medium,
10ml FBS (being purchased from Hyclone, catalog number is SH30084.03) are added, then addition 20mM HEPES (is purchased from thereto
Gibco, catalog number be 15630106), 0.18nM Adenine (being purchased from Sigma, catalog number is A9795), 2mM
L-Glutamine (be purchased from Gibco, catalog number is 25030-081), 2nM 3,3', 5-Tri iodothyronine
(being purchased from Sigma, catalog number is T2725), 1nM Cholera toxin (are purchased from Sigma, catalog number is
C9903), finally add Penicillin Streptomycin according to 100UI/mL and (be purchased from Gibco, catalog number is
15140163) mixed dissolution is uniform.
The preparation of application on human skin epidermal stem cells culture medium (matching while using)：
Each reagent of following final concentration is added in 100ml KGM basal mediums：
The preparation of embodiment 1, blueberry leaf flavones
After blueberry leaf is crushed, 80 mesh sieves are crossed, are 1g according to solid-liquid ratio:40ml(1g:20ml-1g:70ml) to indigo plant
The ethanol water that volumn concentration is 80% (50%-90%) is added in certain kind of berries leaf powder, is existed using homogenate extraction method
Homogenate extraction 90s (30s-270s) under 60V (30V-110V) voltage conditions, obtains extract solution, then by extract solution
10000rpm centrifuges 15min, centrifuges radius 5-15cm, supernatant is obtained, according to the measure of flavones in mono- annex of Ch.P-2005
The general flavone amount that method is measured supernatant is 1.99g/100ml.
Supernatant is freeze-dried after revolving, blueberry leaf flavone extract powder, following referred to as blueberry leaves is obtained
Flavones.
The identification of embodiment 2, blueberry leaf flavones anti-inflammatory efficacy
First, the separation of application on human skin epidermal stem cells
By the ring cutting prepuce tissues of people, abundant washing by soaking 3 times in the tincture of iodine and 75% alcohol, adipose tissue is cut
Abandon, and skin abbreviation 5mm × 5mm fritters are put into the centrifuge tube equipped with the neutral proteinases of Dispase II, 4 DEG C of enzymolysis
Overnight, then by skin histology take out, be put into DPBS, separated epidermis and dermal tissue with tweezers.Epidermis is shredded, dress is put into
There is 8ml 0.125% trypsin treatment 10min, obtain ferment treatment liquid.Ferment treatment liquid is centrifuged into 8min under 360g, collected
Supernatant, adds KGM basal mediums and terminates digestion, be denoted as digestive juice 1.Sediment fraction is added to 0.125% tryptose again
Enzyme secondary digestion, collects supernatant, adds KGM basal mediums and terminates digestion, will terminate postdigestive solution and cross 100 μm successively
Filter membrane with 40 μm, obtains filtrate, and the filtrate is mixed with digestive juice 1,4 DEG C, 360g centrifugation 8min, abandons supernatant, obtains after precipitation
Cell.(BD, catalogue are purchased from the integrins of β 1 (being purchased from Santa Cruz, catalog number is sc-9970) and CD49
Number for 555736) antigenic mark cell, sorted by flow cytometer.The double positive cells of two kinds of marks of selection, be
Application on human skin epidermal stem cells.
2nd, the preparation of blueberry leaf flavonoids solution
Blueberry leaf flavones prepared by embodiment 1 is dissolved in sterile PBS buffer, is configured to 60 μ g/mL solution, will be molten
Liquid crosses 0.45 μm of miillpore filter.
3rd, the application on human skin epidermal stem cells for separating step one are with 2 × l06The density in/hole is inoculated with 6 orifice plates and uses application on human skin
Epidermal stem cells medium culture, per hole add 5 μ L step 2 prepare blueberry leaf flavonoids solution, respectively handle cell 0,15,
30th, 60,90 and 120min, is denoted as processing 0,15,30,60,90 and 120min groups respectively.
4th, the total serum IgE and reverse transcription for extracting the cell of each treatment group are cDNA.
5th, quantitative fluorescent PCR
IL-10 is one kind in anti-inflammatory factors, is the important immunosuppressive factor of body, is mainly produced by T cell, and it is led
The biological characteristics wanted is the effect for suppressing the inflammatory factors such as IL-2.When the skin is exposed to uv rays, keratinocyte
Inflammation inhibiting factor, such as IL-10 and TGF-β can be secreted, its effect is the propagation by suppressing lymphocyte and then alleviated
Inflammatory reaction through generation.
(1) respectively to handle 0,15,30,60,90 and 120min groups cDNA as template, quantitative fluorescent PCR is prepared by table 1
Reaction system (before use, 40uL Dye I is added in 1mL MIX), uses ddH2O supplies system to 20 μ L, analyzes each processing
The expression of the IL-10 genes of group cell, while being used as internal reference using β-actin.
The quantitative fluorescent PCR reaction system of table 1
The primer of each gene is as shown in table 2 in quantitative fluorescent PCR.
The fluorescence quantification PCR primer of table 2
Experiment sample is expanded using ABI7300 quantitative real time PCR Instruments, quantitative fluorescent PCR program is as shown in figure 1,20 μ L are anti-
Answer system, 36 circulations.
(2) anti-inflammatory factors gene (IL-10) expression analysis
The IL-10 of each treatment group relative expression quantity result is as shown in Figure 2.
Fig. 2 shows that influence of the blueberry leaf flavones to IL-10 expression quantity, which changes with time, is presented the overall trend improved.
In the blueberry leaf flavones processing prolonged scopes of 0min to 60min, the change of IL-10 expression quantity is not obvious, but 60min is arrived
90min time IL-10 expression quantity starts to be significantly improved, subsequent 30min be 90min to 120min time in its
Expression quantity is declined slightly, but compared with initial value, stills remain at a relatively high level.Entirety sees that blueberry leaf flavones is to IL-10
Expression there is facilitation, reach the level of signifiance (P ＜ 0.05), therefore blueberry leaf flavones prepared by embodiment 1 can be drawn
With anti-inflammatory efficacy.
The preparation of embodiment 3, blueberry leaf flavones Essence
The formula and manufacture craft of blueberry leaf flavones Essence are as shown in table 3.
Table 3
The patch experiment of embodiment 4, blueberry leaf flavones Essence
Given the test agent title：Blueberry leaf flavones Essence prepared by embodiment 3
Test purpose：Detection sample causes human body skin adverse reaction possibility
Method of testing：Human body skin enclosed type patch test, Ministry of Health of the People's Republic of China《Cosmetics health specification》
(2007)
Sample：100 times of blueberry leaf flavones Essence distilled water diluting prepared by embodiment 3
Control：Distilled water
Volunteer's number:30 women
Volunteer's age：21-63 Sui
Amount of samples：0.025ml
Test zone：Back
Spot tries device：Patch test adhesive tape (is purchased from Shanghai Medical Instruments (Group) Co., Ltd. Heath Material Plant)
Test result：Totally 30 qualified volunteers participate in this test, and test result judges effectively.Removing tested material
Spot tries device is checked product zone and check plot by dermatologist dermoreaction after 30 minutes, 24 hours and 48 hours.As a result show
Show, 2 order reaction 2 is observed in product zone after 30 minutes；Observe 2 order reaction 1 within 24 hours；1 grade is observed after 48 hours instead
Answer 1,2 skin adverse reactions of order reaction 1；Distilled water check plot does not observe skin adverse reaction, and experiment statisticses result is such as
Shown in table 4.
The human body skin enclosed type patch test results (n=30 people) of table 4
*：" -- "=negative reaction；" ± "=suspicious reaction：Only faint erythema；"+"=(erythema is anti-for weakly positive reaction
Should)：Erythema, infiltration, oedema, there can be papule；" ++ "=strong positive reaction (bleb reaction)：Erythema, infiltration, oedema, papule, blister
Rash, reaction can exceed tested area；" +++ "=extremely strong positive reaction (reaction of amalgamation bleb)：Obvious erythema, severe infiltration, water
Swollen, amalgamation bleb, reaction exceeds tested area.
Claims (3)
1. a kind of preparation method of blueberry leaf flavones Essence, comprises the following steps：
(1) disodium ethylene diamine tetraacetate dissolving is added in water, carbomer U20 is added and mixes, be heated to 70-85 DEG C, add
Glycerine is mixed, 70-80 DEG C of insulation 10min, is added allantoin dissolving and is mixed, is cooled to 45 DEG C -55 DEG C, adds blueberry leaf yellow
Ketone, obtains A phase solution；
(2) Microcare MTI, butanediol, 1,2- pentanediols are mixed, obtains C phase solution；
(3) 1g/100ml aqueous solution of sodium hyaluronate, trehalose is sequentially added in temperature is 50 DEG C of A phase solution, is often added
Enter a kind of material and be intended to mixing, obtain A phases, the mixed liquor of B phases；
(4) C phase solution is added in A phases at 45 DEG C -50 DEG C, the mixed solution of B phases, is mixed, the mixed of A phases, B phases and C phases is obtained
Close liquid；
(5) add water, produce blueberry leaf flavones Essence；
In the blueberry leaf flavones Essence, the water in the step (1) accounts for the quality percentage of the blueberry leaf flavones Essence
Content is that the weight/mass percentage composition that the 60%, disodium ethylene diamine tetraacetate accounts for the blueberry leaf flavones Essence is 0.02-
0.1%th, the weight/mass percentage composition that the allantoin accounts for the blueberry leaf flavones Essence is 0.1-0.5%, the carbomer U20
Account for the weight/mass percentage composition of the blueberry leaf flavones Essence and account for the blueberry leaf flavones elite for 0.2-0.8%, the glycerine
The weight/mass percentage composition of liquid is that 1.0-6.0%, the blueberry leaf flavones account for the quality percentage of the blueberry leaf flavones Essence and contained
The aqueous solution of sodium hyaluronate measured as 0.2-4.0%, the 1g/100ml accounts for the quality percentage of the blueberry leaf flavones Essence
Content is that the weight/mass percentage composition that 1.0-6.0%, the trehalose account for the blueberry leaf flavones Essence is 0.2-2.0%, institute
State Microcare MTI and account for the weight/mass percentage composition of the blueberry leaf flavones Essence and accounted for for 0.05-0.1%, the butanediol
The weight/mass percentage composition of the blueberry leaf flavones Essence is 1.0-3.0%, 1, the 2- pentanediols account for the blueberry leaf flavones
The weight/mass percentage composition of Essence is that 0.5-5.0%, surplus are water added by step (5)；
The pH of the blueberry leaf flavones Essence is 5.5-6.5；
The entitled acrylic acid of INCI (ester) class of the carbomer U20/C10-30 alkylol acrylamide acid esters cross-linked polymers；
The entitled METHYLISOTHIAZOLINONE IODOPROPYNYL of INCI of the Microcare MTI
BUTYLCARBAMATE；
After the step (5), there is the step of adjusting the pH of the blueberry leaf flavones Essence to 5.5-6.5；
Reagent used in the pH regulations is sodium hydrate aqueous solution or aqueous citric acid solution；
The preparation method of the blueberry leaf flavones is as follows：By the ethanol water that blueberry leaf powder and volumn concentration are 80%
According to solid-liquid ratio 1g:40ml is mixed, using homogenate extraction method under 360V voltage homogenate extraction 90s, obtain extract solution, then will
Extract solution centrifugation obtains supernatant, and supernatant freeze-drying is produced.
2. the blueberry leaf flavones Essence prepared as the method described in claim 1.
3. the answering in the product with anti-inflammatory and/or skin care effect is prepared of the blueberry leaf flavones Essence described in claim 2
With.
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Effective date of registration: 20191227 Address after: 100123 No. 3, Gaobeidian North Road, Beijing, Chaoyang District Co-patentee after: Beijing Technology and Business University Patentee after: China Inspection and Quarantine Research Institute Co-patentee after: Beijing Sino German joint cosmetics Research Institute Co Ltd Address before: 100123 No. 3, Gaobeidian North Road, Beijing, Chaoyang District Co-patentee before: Beijing Technology and Business University Patentee before: China Inspection and Quarantine Research Institute |