CN109730952A - Lava sea water extraction process containing dendrobium candidum - Google Patents
Lava sea water extraction process containing dendrobium candidum Download PDFInfo
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- CN109730952A CN109730952A CN201910008542.6A CN201910008542A CN109730952A CN 109730952 A CN109730952 A CN 109730952A CN 201910008542 A CN201910008542 A CN 201910008542A CN 109730952 A CN109730952 A CN 109730952A
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- dendrobium candidum
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- lava seawater
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Abstract
The invention discloses the lava sea water extraction process containing dendrobium candidum, use lava seawater for extract stoste, the sterilising filter degerming of lava seawater;Dendrobium candidum stem is added in lava seawater, then refluxing extraction is used filter-cloth filtering, then filtered with filter to solvent is completely removed, dendrobium candidum lava seawater extractive powder is obtained.The present invention uses lava seawater dendrobium candidum extract for effective component, has the effect of improving wrinkle to skin, inhibits saccharification, whitening and moisturizing.
Description
Technical field
The present invention relates in relation to the lava sea water extraction process containing dendrobium candidum.
Background technique
In the organ that human endothelium's skin is with external environment close contact the most, there is the function of protection inside of human body
Can, epidermis, corium, subcutaneous fat can be divided into.Skin is answering of constituting of by the cell of various function and therewith adaptable substance
Miscellaneous organ, wherein skin corium is the combination of flexible solid matter (fiber) and sticking liquid substance, can maintain skin
Intrinsic elasticity.So skin has the reaction of unique physical property to tension, we are called viscoplasticity.Skin has such viscous
Elasticity is to constitute distinctive three-D because proteoglycans have collagen fabric and elastomer and constitute.Generally with
The reasons such as old and ultraviolet light, public hazards, pressure, lead to the decline of skin function and atrophic variation, Skin Cell quantity subtracts
Phenomena such as less or thickness of skin is thinning.For these reasons, skin elastomer deformation cause skin elasticity reduce or
Cutis laxa.
Collagen is as the main component for constituting skin, and wherein II collagen of type accounts for 80%, type, III collagen egg
It is white to account for 15%.In the case where ultraviolet light exposes for a long time, the activity amount of bringing up in skin is generated, and leads to matrix metalloprotease matter point
The expression of the enzyme MMP-1 of decomposition collagen in solution enzyme MMPs or activity are promoted, and cause the synthesis of collagen low, from
And lead to the reduction of skin elasticity.So we are in developing new material, the material in order to treat skin and prevent skin aging
Have expression MMP-1 and inhibit its activity, the effect of collagen synthesis can be promoted, this can become a kind of good strategy.
In addition, the influence of collagen and elastin laminin vulnerable to the chemical reaction for the body interior for being referred to as saccharification.This is
Free amine group containing protein combines the non-ferment reaction of bring with as the reduced sugar glucose.Energy is provided to cell
The glucose of amount can react with protein as collagen.This will cause Advaned Glycation End-
Products (AGEs) and reactive oxygen species (ReactiveOxygen Species, ROS).One in AGEs
Carboxymethyllysine (CML) especially makes the skin elasticity in photo aging process caused by UV generate variation, to skin
Elasticity and collagen accumulation AGEs have negative shadow to the intersection combination, elastic force loss, intradermal variation etc. of protein fibre
It rings.
When generating AGEs in skin, receptor site can be acted on, generates AGE (RAGE) complex.RAGE signal
Convey inflammation and the relevant intracellular processes of sequelae.These are the main originals of inflammation ageing process and many disease catalytic action
Cause, so being very important.It illustrates, the glycemic index in the blood of diabetic is higher, with the generation of internal AGEs
And the complication such as cataract, artery sclerosis occurred.Because of these reasons, diabetes are seen as causing due to the generation of AGEs
Inflammation increases and the disease of accelerated ageing.This is not limited solely to diabetes.Physical strength is low, heart disease is related to brain many
Disease all has relationship with saccharification.Scientist thinks that reducing saccharification means that slowing down ageing process and disease induces.The generation of AGEs
It is the mechanism that decide how skin goes always, our internal diseases generate, is the research field to attract most attention.
The pigmentation in skin such as black mole, freckle results from the increase of melanin in epidermis, determines the important of the colour of skin
Reason is also melanin.The generation of melanin is the pigment granule organ in the melanocyte of basal layer of epidermis.I.e. because of purple
Outside line generates the mitosis of melanocyte, and then melanocyte is activated.Promote in the melanocyte of activation
Into the synthesis of tyrosinase tyrosinase, the generation for aggravating melanin makes it transport and be discharged to outside epidermis.
Naturally the melanin generated be alkali slightly solubility black melanin plain (new melanin) and alkali-soluble yellow to red
The mixture of color melanin (pheomelanin).Melanin polymer generally largely refers to new melanin.And according to Prota (G.
Prota., J. Invest. Dermatol., 75, p122,1980.) and Pavel (S. Pavel et al., Cancer
Detection and Prevention, 6, p311., 1983.) etc. research, new melanin not only have us to expect always
Dihydroindole 5,6-dihydroxyindole, there are also other melanin monomers (5,6-dihydroxyindole-2-
Carboxylic acid) and the melanin that is generated from various melanin mesostates.The intracorporal melanin production of melanocyte
Process is tyrosine by ferment tyrosinase oxidation (DOPA) → (dopaquinone) → (dopachrome) → (5,6-
Dihydroxyindole) → (indole-5,6-quinone) is then generated because of the polymerization of (indole-5,6-quinone)
Melanin.Wherein until tyrosine step, tyrosinase is to intervene, and several steps are autoxidations behind
Possibility is big.It is due to the UV-induced melanocyte in sunray in pigmentation, that is, melanism of the generations such as sunbath
Color generates caused by increase.
It observes the melanin production process in relation in melanocyte obvious so far and inhibits melanin raw
At the mechanism of action, two kinds can be roughly divided into.The first is with cytotoxicity to melanocyte, so that reaching final inhibits black
The generation of element is for second the generation for hindering the melanin in melanocyte, to inhibit melanin production.According to nearest hair
There are also TRP-1 and TRP-2 can also generate melanin for the paper of table, not only tyrosinase, these are also to intervene melanin life
The important ferment of object synthesis, if the substance that these ferment can be hindered to generate is developed, to preventing skin pigmentation to be also one
Good method.
Human body is constituted by sustaining life body important component-water.Water accounts in vivo in normal skin
70%, cuticula contains 20% to support the elasticity and flexibility of skin.It is stemness skin when the moisture of cuticula is below 10%
Skin, the reason of being commonly called as problem skin.The moisture equilibrium of skin is adjusted according to epidermis.Although the cuticula of epidermis is by dead
Cell accumulation forms, but maintains moisture to play very important effect for skin.Inside cuticula caused by systematicness keratinization
Water content, maintain 10 ~ 20% always.This is the sebum film mixed by sweat and sebum, prevents angle from outer protection skin
The moisture of matter layer evaporates.Natural moisturizing factor and corium lipid catch moisture, maintain moisture.Sebum film NMF and corium lipid
One reacts on maintenance moisture.NMF is generated in cornification processes, is hydrophilic hygroscopic matter, to skin moisture-keeping
Play very important effect.This is can to lock cuticula moisture by Amino acid profile.There are also the PCA in NMF is particularly important.
As the matrix filled between each cell, forming layer is to maintain internal moisture.
It was reported that showing, the amount of sodium hyaluronate is reduced with old age in the skin of people, this is considered as causing with aging
Skin elasticity decline and one of the immediate cause of water content reduction.
Summary of the invention
In order to solve the above technical problems, the present invention provides the lava sea water extraction process containing dendrobium candidum, using lava
Seawater is extract stoste, the sterilising filter degerming of lava seawater;Dendrobium candidum stem is added in lava seawater, reflux mentions
It takes, then uses filter-cloth filtering, then filtered to solvent is completely removed with filter, obtain dendrobium candidum lava seawater extractive powder.
Further, which further includes AQP-3 or HAS-2.
Further, by their entirety on the basis of 100% weight, 0.01 ~ 10% weight of lava seawater dendrobium candidum extract is added.
The present invention uses lava seawater dendrobium candidum extract for effective component, and having to skin improves wrinkle, inhibits sugar
Change, the effect of whitening and moisturizing.
Detailed description of the invention
Fig. 1 shows the cytotoxicity results of embodiment 1 and comparative example 1 to fiber sprout cell Fibroblast.
Fig. 2 indicates the embodiment 1 of Human Keratinocytes HaCaT and the cytotoxicity result of comparative example 1.
Fig. 3 indicates the 1 gene expression of Collagen type of embodiment 1 and comparative example 1 in fiber sprout cell
Comparison result.
Fig. 4 indicates the comparison result that the RAGE gene of embodiment 1 and comparative example 1 are expressed in fiber sprout cell.
Fig. 5 indicates the comparison knot that the AQP-3 gene of embodiment 1 and comparative example 1 are expressed in keratinocyte
Fruit.
Fig. 6 indicates the comparison result that the HAS-2 gene of embodiment 1 and comparative example 1 are expressed in keratinocyte.
Fig. 7 is indicated to the embodiment 1 of melanoma (B16-F1) and the cytotoxicity result of comparative example 1.
Fig. 8 shows the ratios in the inner embodiment 1 of melanoma (B16-F1) and the unit cell melanin production rate of comparative example 1
Relatively result.
Specific embodiment
Production Example 1: the water intaking of lava seawater
Underground 150m is excavated in the East Jun Jiuzuo Yi Han, north Jizhou, Jizhou Island eastern region, on underground mean seal level (sea level
On the basis of) the lava seawater sterilising filter degerming taken at 44.35m.Lava seawater (the not desalination of degerming in this way
Salt water state) it is used in the manufacture of later dendrobium candidum lava seawater extractive.The minerals of degerming lava seawater form
Such as following table 1.
[table 1]
Embodiment 1: the manufacture of dendrobium candidum lava seawater extractive
Take dendrobium candidum stem 1kg to add 20 kg of lava seawater, 90 DEG C refluxing extraction 3 hours, with 250 mesh filter-cloth filterings, after
It is filtered with 1 filter.Filtered fluid reduced pressure machine (EYELA, N-1000, Japan) is completely removed into solvent, is obtained
226 g of extract powder of dendrobium candidum and lava seawater.
0.01 ~ 10% weight of lava seawater dendrobium candidum extract is added on the basis of whole 100% weight, is wrinkled to skin
Line inhibits saccharification to have good improvement, and has whitening and moisture-keeping function.
Comparative example 1: the manufacture of the pure water extract of dendrobium candidum
It is exactly to substitute lava seawater with pure water with manchining feature object under conditions of dendrobium candidum stem and 1 striking resemblances of embodiment
To manufacture.Obtain dendrobium candidum pure water extract powder 29g.
Compare: being measured using the cytotoxicity of fiber sprout cell (Fibroblast) and keratinocyte (HaCaT)
For measure embodiment 1 dendrobium candidum lava seawater extractive and comparative example 1 the pure water extract of dendrobium candidum it is anti-
Wrinkle effect, anti-saccharification result, whitening effect and moistening effect, in people fiber sprout cell Fibroblast and keratinocyte
Toxicity detection has been carried out by MTT assay in HaCaT.MTT assay is detection living cells quantity, it is common to use is increased in cell
It grows or the detection method of cytotoxicity.The cell to live in mitochondria be it is water-soluble, yellow is utilized in this detection method
Salt MTT (3- [4,5-dimethlythiazole-2-yl] -2,5-diphenyl tetrazolium bromide) is because of succinic acid
Dehydrogenase (succinate dehydrogenase or mitochondrial dehydrogenase) and restore become water not
The principle of dissolubility blue first formazan derivative.By fiber sprout cell Fibroblast and keratinocyte HaCaT cell
It is inoculated in culture dish bottom, is put into containing penicillin 100U/, streptomysin 100/, 10% cow's serum (Fetal
Bowine serum, FBS) IMDM (use Iscove ' s modified dulbecco ' s medium-Fibroblast
Cell, or using the culture solution of DMEM (Dulbecco ' s modified Eagle ' s medium-HaCaT cell), 37
It is cultivated in DEG C culture medium containing 5% carbon dioxide.By people fiber sprout cell Fibroblast and keratinocyte HaCaT
Cell is in 24 porocyte culture plates respectively with 1x105cells/ml, 1.5x105The concentration dilution of cells/ml, is inoculated with 1 respectively
It cultivates 24 hours afterwards.Culture solution is all removed after culture, culture solution not containing cow's serum is put into, by 1 He of embodiment
The extract of comparative example 1 is handled according to concentration.It 37 DEG C, after cultivating 24 hours in the culture medium containing 5% carbon dioxide, clears all
Culture solution is replaced with the culture solution that 2.5mg/ MTT solution dilutes 10 times, at culture medium culture 4 hours.After 4 hours, remove
Upper clear liquid adds 1 dissolution solvent DMSO solution, and the MTT formazan 570nm crystalline solid of generation is detected absorbance.This
When, control group uses dissolution solvent DMSO.Toxicity detection result please refers to table 1, table 2.
[table 1]
[table 2]
The pure water extract of dendrobium candidum of the dendrobium candidum lava seawater extractive and comparative example 1 of embodiment 1,1000
/ concentration below does not show as toxicity.
The investigation of experimental example 2:Collagen type I mRNA expression quantity
For the glue of the pure water extract of dendrobium candidum of the dendrobium candidum lava seawater extractive and comparative example 1 of measurement embodiment 1
Former albumen synthesizes facilitation effect, and on the people fiber sprout cell of commerciality buying, processing sample has carried out Collagen type I
Gene expression experiment.In 60mm dish with contain 10% cow's serum IMDM (Iscove's Modified
Dulbesco's Media) culture solution together, ATCC (Americal Type Culture Collection) purchase people
Fiber sprout cell Fibroblast is with 1x106Cells/well concentration dispensing is at 37 DEG C, 5% CO2 Under the conditions of cultivate 24 hours.
After 24 hours, by sample be free of cow's serum IMDM culture solution, according to concentration dilution handle 24 hours, by cell aggregation in
TRIzol (invitrogen, USA) separates RNA.Isolated RNA is utilized into Qubitfluoremeter with RNA BR
After Assay kit is quantitative, Real-Time PCR:7500 Fast with Power SYBR Green PCR master is utilized
Mix (Applied Biosystems, USA) amplification gene, quantitative analysis increase product.Using PCR's
Collagen type I and β-Actin primer (Korea), primer sequence please refer to table 3.Control group uses
The processed DMSO of solvent (dimethhyl sulfoxide), positive control group has used well-known antioxidant
Vitamin C.Experimental result please refers to table 4, Fig. 3.
[table 3]
[table 4]
The pure water extract of dendrobium candidum of the dendrobium candidum lava seawater extractive and comparative example 1 of embodiment 1 has determined
The genetic expression of Collagen type I.Experimental example 1 expresses further amounts of Collagen type I than control group,
It confirmed it with anti-wrinkle effect.It confirms compared with comparative example 1, the anti-wrinkle effect of embodiment 1 is more preferable.
The investigation of 3. RAGE mRNA expression quantity of experimental example
For detect embodiment 1 dendrobium candidum lava seawater extractive and comparative example 1 the pure water extract of dendrobium candidum it is anti-
Saccharification result, on the people fiber sprout cell of commerciality buying, processing sample has carried out the expression experiment of RAGE gene.?
In 60mm dish with contain 10% cow's serum IMDM (Iscove's Modified Dulbesco's Media) culture solution one
It rises, by people's fiber sprout cell (Fibroblast) with 1X106Cell/dish concentration dispensing, at 37 DEG C, 5% CO2Under the conditions of train
It supports 24 hours.Culture solution is removed after 24 hours, adds HBSS (Hank ' s Balanced salt solution) 3, into
Row UVA 6J/cm2After processing, sample is being free of into cow's serum IMDM culture solution, is being handled 48 hours according to concentration dilution, by cell
Collection separates RNA together in TRIzol (invitrogen, USA).Isolated RNA is utilized into Qubitfluoremeter with
After RNA BR Assay kit is quantitative, synthesis cDNA (complementary DNA) carries out Real-time PCR.CDNA is closed
At used cDNA synthesis Kit (High Capacity RNA to-cDNA Kit, Applied Biosystems,
USA), tested according to the method for Kit.Utilize Real-Time PCR:7500 Fast with Power SYBR
Green PCR master mix (Applied Biosystems, USA) amplification gene, quantitative analysis increase product.
Using RAGE the and β-Actin primer (Korea) in PCR, primer sequence please refers to [table 5].Control group uses
The processed DMSO of solvent (dimethhyl sulfoxide), UVA processing group have used UVA 6J/cm2It is processed with solvent
DMSO (dimethhyl sulfoxide).Positive control group has used well-known antioxidant Resveratrol.
Experimental result please refers to table 6 and Fig. 4.
[table 5]
[table 6]
The pure water extract of dendrobium candidum of the dendrobium candidum lava seawater extractive and comparative example 1 of embodiment 1 has determined RAGE
Genetic expression.Embodiment 1 and comparative example 1 have significantly all to the genetic expression of RAGE is increased caused by UVA
Inhibitory effect.It can confirm that compared with comparative example 1, the anti-wrinkle effect of embodiment 1 is more preferable.
Experimental example 4:AQP-3, HAS-2 mRNA expression investigation
For the guarantor of the pure water extract of dendrobium candidum of the dendrobium candidum lava seawater extractive and comparative example 1 of measurement embodiment 1
Wet effect, on the keratinocyte of people, processing sample is tested.In 60mm dish and containing 10% cow's serum
DMEM (Dulbecco ' s modified Eagle ' s medium) culture solution together, by keratinocyte HaCaT with
1.5X106Cell/dish concentration dispensing, at 37 DEG C, 5% CO2Under the conditions of cultivate 24 hours.After 24 hours, sample is being free of
In the DMEM culture solution of cow's serum, according to concentration dilution handle 24 hours, by cell aggregation in TRIzol (invitrogen,
USA RNA) is separated.Isolated RNA is utilized into QubitAfter fluoremeter with RNA BR Assay kit is quantitative, close
Implement Real-time PCR at cDNA (complementary DNA).CDNA synthesis has used cDNA to synthesize Kit (High
Capacity RNA to-cDNA Kit, Applied Biosystems, USA), experiment is performed according to the method for Kit.Benefit
With Real-Time PCR:7500 Fast with Power SYBR Green PCR master mix (Applied
Biosystems, USA) amplification gene after, quantitative analysis increase product.Use AQP-3, HAS-2 and the β-in PCR
Actin primer (Korea), primer sequence please refer to [table 7].Control group has used the processed DMSO of solvent
(dimethhyl sulfoxide), positive control group have used the Retinoic acid of moistening effect.Experimental result is asked
Reference table 8, table 9 and Fig. 5, Fig. 6.
[table 7]
[table 8]
It confirmed the pure water extract of dendrobium candidum of the dendrobium candidum lava seawater extractive and comparative example 1 of embodiment 1
The genetic expression of AQP-3.It shows that experimental example 1 expresses further amounts of AQP-3 than control group, can confirm that it has moisturizing effect
Fruit.It can also confirm that, compared with comparative example 1, embodiment 1 has better moistening effect.
[table 9]
It confirmed the pure water extract of dendrobium candidum of the dendrobium candidum lava seawater extractive and comparative example 1 of embodiment 1
The genetic expression of HAS-2.It shows that experimental example 1 and comparative example 1 express further amounts of HAS-2 than control group, can confirm it
There is moistening effect.It can also confirm that, compared with comparative example 1, embodiment 1 has better moistening effect.
Experimental example 8: cytotoxicity is detected using melanocytes (B16-F1)
For the poison of the pure water extract of dendrobium candidum of the dendrobium candidum lava seawater extractive and comparative example 1 of measurement embodiment 1
Property, on melanoma (B16-F1) cell of commerciality buying, processing sample is tested.In 6 well plate and containing 10%
DMEM (Dulbecco ' s modified Eagle ' s medium) culture solution of cow's serum is together, thin by melanoma (B16-F1)
Born of the same parents are with 1X105Cell/well concentration dispensing, at 37 DEG C, 5% CO2 Under the conditions of cultivate 24 hours.After 24 hours, sample is being contained
In the new DMEM culture solution for having α-Melanocyte stimulate hormone (α-MSH) and 10% cow's serum, according to concentration
Dilution culture 72 hours.After culture 72 hours, all culture solutions are removed, alternately dilute 10 times with the MTT solution of 2.5mg/
Culture solution, at culture medium culture 4 hours.Upper clear liquid is removed after 4 hours, is added 2 DMSO (dimethhyl sulfoxide)
The MTT formazan crystalline solid of formation is detected absorbance in 570nm by solution.Non-process group has used solvent processed
DMSO (dimethhyl sulfoxide), α-MSH processing group have used α-MSH and the processed DMSO (dimethhyl of solvent
sulfoxide).Positive control group has used Arbutin (SK Bioland), and toxicity detection result please refers to table 10 and Fig. 7.
[table 10]
The pure water extract of dendrobium candidum of the dendrobium candidum lava seawater extractive and comparative example 1 of embodiment 1, all 50/
There is no toxicity under concentration below.
Experimental example 9: melanoma (B16-F1) cell detection melanin biosynthesis inhibitory effect is utilized
For the beauty of the pure water extract of dendrobium candidum of the dendrobium candidum lava seawater extractive and comparative example 1 of measurement embodiment 1
White effect, on the melanoma B16-F1 cell of commerciality buying, processing sample is tested.Utilize the obstruction black of melanocytes
Element generate (melanogenesis) effect utilization (Fukuda et al., J. Soc. Cosmetic Chem., 42
(361), 1991) method deforms its method and is detected.In 6 well plate with contain 10% cow's serum DMEM
(Dulbecco ' s modified Eagle ' s medium) culture solution together, by melanoma B16-F1 cell with 1X105Cell/
Well concentration dispensing, at 37 DEG C, 5% CO2 Under the conditions of cultivate 24 hours.After 24 hours, by sample in α-Melanocyte
It is small according to concentration dilution culture 72 in stimulate hormone (α-MSH) and new DMEM culture solution containing 10% cow's serum
When.After culture in 72 hours, the cell of all culture solutions will be removed, washed with PBS (phosphate buffer, saline) 2
It washs, carries out 1N NaOH 0.5 and handle, 1.5 tube of cell are recycled, intracellular melanin is obtained.By recycling
Cell is moved to 96 well plate, detects absorbance in 450nm, obtains a certain amount of protein sugar melanin.Non-process group
It has used the processed DMSO of solvent (dimethhyl sulfoxide), α-MSH processing group has used α-MSH and solvent to handle
The DMSO (dimethhyl sulfoxide) crossed.Positive control group has used Arbutin (SK Bioland).Melanin biology
Synthesis hinders testing result, please refers to table 11 and Fig. 8.
[table 11]
Claims (2)
1. the lava sea water extraction process containing dendrobium candidum, it is characterised in that: use lava seawater for extract stoste, lava sea
Water sterilising filter degerming;Dendrobium candidum stem is added in lava seawater, then refluxing extraction uses filter-cloth filtering, then used
Filter is filtered to solvent is completely removed, and obtains dendrobium candidum lava seawater extractive powder.
2. the lava sea water extraction process described in accordance with the claim 1 containing dendrobium candidum, it is characterised in that: 100% weight by their entirety
On the basis of amount, 0.01 ~ 10% weight of lava seawater dendrobium candidum extract is added.
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CN108464960A (en) * | 2018-04-17 | 2018-08-31 | 凯瑞欧(天津)生物技术有限公司 | A kind of dendrobium candidum beauty-care gel |
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CN106692692A (en) * | 2015-08-21 | 2017-05-24 | 中国科学院昆明植物研究所 | Dendrobium officinale extract and preparation method thereof |
KR20170059651A (en) * | 2015-11-23 | 2017-05-31 | 대봉엘에스 주식회사 | Method of preparing green tea extract by using magma seawater, carbonated water, or bedrock water, and functional cosmetic composition comprising the same |
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