CN109907317A - For adjusting the dendrobium candidum extract and preparation method thereof of skin metabolism - Google Patents
For adjusting the dendrobium candidum extract and preparation method thereof of skin metabolism Download PDFInfo
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- 230000004060 metabolic process Effects 0.000 title claims abstract description 18
- 238000000605 extraction Methods 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 102000008186 Collagen Human genes 0.000 claims abstract description 26
- 108010035532 Collagen Proteins 0.000 claims abstract description 26
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- 239000001963 growth medium Substances 0.000 claims abstract description 21
- 230000006698 induction Effects 0.000 claims abstract description 9
- 230000004913 activation Effects 0.000 claims abstract description 7
- 240000004638 Dendrobium nobile Species 0.000 claims abstract description 3
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- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 abstract description 9
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Abstract
The present invention relates to plant extracts fields, especially for adjusting the dendrobium candidum extract and preparation method thereof of skin metabolism.The skin dendrobium nobile extract is obtained again through an extraction step via after the induction of the 1/3MS culture medium of addition 1- methyl α-naphthyl acetate (NAA) and 6- aniline purine (BA) and Multiplying culture.And dendrobium candidum extract of the invention can be generated via activation grain wire body, promotion collagen and sodium hyaluronate, and then have the effect of adjusting skin metabolism.
Description
Technical field
It is the present invention relates to plant extracts field, in particular to a kind of for adjusting the dendrobium candidum extract of skin metabolism
And preparation method thereof.
Background technique
Plant tissue culture technique originates in nearly 100 years, and method is that domestication of plants body is utilized different auxin
(auxin) it is stimulated with the ratio of the basic element of cell division (cytokinin) allotment, reaches and turn out adventitious root, adventitious bud or callus group
The effect knitted.Plant stem cell is the original still undifferentiated state of plant, from the apical meristem (apical of stem and root
Meristem) or body cell (somatic cell), commonly referred to as callus (callus).
There is callus apparent gene effect (epigentic) can break up with totipotential differentiation ability (totipotent)
At plant embryos cell and then form new plant.And helpful cell metabolism is newborn, postpones aging and imparting vigor and other effects.
Modern age utilizes method for plant tissue culture, and successful development goes out a set of technology for belonging to plant meristem proliferation, however right
In different cultivars plant it is still necessary to find out suitable culture formula via systematic experiment, can output have the plant of utility value
Object separate living tissue provides industry utilization.
Dendrobium candidum (Dendrobium officinale Kimura et Migo) belongs to dendrobium perennial herb and plants
Object, alias are ribbed hedyotis herb, for Chinese precious Chinese herbal medicine, Dabie Mts, anhui originating in China, East Zhejiang province Mt. Tiantai, Ninghua County, Fujian Province, Guangxi
The ground such as Tiane, Yunnan mountain of papers and Sichuan are distributed on the dark and damp rock in mountainous region half of the nearly km of height above sea level more, are generally resistant to -5 DEG C
Low temperature.Growth and reproductive characteristic due to dendrobium candidum itself, particular/special requirement and huge city to ecological environmental condition
Reasons, the dendrobium officinales such as the artificial excessive excavation that field demand causes are endangered.Therefore, it is trained using special tissue
The method of supporting carries out dendrobium candidum tissue culture mass propagation, can effectively mitigate the pressure of wild resource acquisition, and is not only restricted to
The influence of weather and calendar variation.
Summary of the invention
In view of this, the present invention is being tieed up using special tissue culture technique induction and proliferation dendrobium candidum separate living tissue
Hold cell it is undifferentiated in the case where using specific culture formula and operation technique separate living tissue yield reached into maximum production, for
Industry utilizes.The separate living tissue that the present invention is bred using this cultural method can effectively reach via test cell line result
It activates grain wire body, promote collagen and sodium hyaluronate generation and other effects, to promote the application value of dendrobium candidum, and be industrial circle
The preparation method of dendrobium candidum preciousness raw material is provided.
The present invention provides a kind of dendrobium candidum extract and is used to prepare the purposes for adjusting the medical composition of skin metabolism.
The present invention provides a kind of method induced and be proliferated dendrobium candidum separate living tissue again, includes: using addition 1- naphthalene second
The 1/3MS of sour (1-Naphthaleneacetic acid, NAA) and 6- aniline purine (6-benzyladenine, BA) culture
The step of base.
The present invention separately provides a kind of culture medium for cultivating skin dendrobium nobile separate living tissue, and wherein the culture medium includes a 1- methyl α-naphthyl acetate
(1-Naphthaleneacetic acid, NAA).
In one embodiment of this invention, wherein the culture medium further includes 6- aniline purine (6-
Benzyladenine, BA).
In one embodiment of this invention, wherein the dendrobium candidum extract is by adding 1- methyl α-naphthyl acetate (1-
Naphthaleneacetic acid, NAA) and the 1/3MS culture medium of 6- aniline purine (6-benzyladenine, BA) lure
Lead and Multiplying culture dendrobium candidum separate living tissue after obtained again through an extraction step, and the extraction step is with 75% alcoholic extract
It takes.
In one embodiment of this invention, wherein adjusting skin metabolism is activation grain wire body, promotes collagen and glass
Uric acid generates.
In one embodiment of this invention, wherein adjusting skin metabolism is to promote 1 chain (collagen of Type I collagen α
Alpha-1 (I), COL1A1), 2 chain of Type I collagen α (collagen alpha-2 (I), COL1A2), I collagen type v α, 4 chain
(collagen alpha-4 (IV), COL4A4), matrix metalloprotease tissue depressant (tissue inhibitors of
Metalloproteinase-1, TIMP-1), from amido oxidizing ferment (lysyloxidase, LOX) and hyaluronic acid synthetase
The performance of (hyaluronan synthase, HAS) gene；And inhibit MMP-2 (matrix
Metalloproteinase-2, MMP-2) and Matrix Metalloproteinase-9 (matrix metalloproteinase-9,
MMP-9) gene shows.
In one embodiment of this invention, wherein the medical composition further includes a pharmaceutically acceptable carrier.
Therefore, the present invention provides a kind of characteristic using dendrobium candidum callus regeneration, promotes Skin Cell grain wire body
Activity increases；In addition, dendrobium candidum extract of the invention, can via promote COL1A1, COL1A2, COL4A4, TIMP-1,
The performance of LOX and HAS gene；And inhibit the performance of MMP-2 and MMP-9 gene, and then promote collagen and sodium hyaluronate raw
At, be finally reached adjust skin metabolism effect.
Embodiments of the present invention are further illustrated below in conjunction with schema, and following cited embodiments are to illustrate
The present invention, the range being not intended to limit the invention is any to be familiar with this those skilled in the art, is not departing from the spirit and scope of the present invention
It is interior, when can do it is a little change and retouch, therefore protection scope of the present invention when regard appended claims institute's defender as
It is quasi-.
Detailed description of the invention
Fig. 1 is the datagram of the activation grain wire body effect of dendrobium candidum extract of the invention；Compared to control group * * * p <
0.001；
Fig. 2 be dendrobium candidum extract of the invention in promote 1 chain of Type I collagen α (collagen alpha-1 (I),
COL1A1) gene, 2 chain of Type I collagen α (collagen alpha-2 (I), COL1A2) 4 chain of gene and I collagen type v α
The datagram of (collagen alpha-4 (IV), COL4A4) gene performance amount；
Fig. 3 is dendrobium candidum extract of the invention in promotion matrix metalloprotease tissue depressant (tissue
Inhibitors of metalloproteinase-1, TIMP-1) gene performance, inhibition MMP-2 (matrix
Metalloproteinase-2, MMP-2) and Matrix Metalloproteinase-9 (matrix metalloproteinase-9,
MMP-9) the datagram of gene performance；
Fig. 4 be the promotion of dendrobium candidum extract of the invention from amido oxidizing ferment (lysyl oxidase, LOX) and
The datagram of hyaluronic acid synthetase (hyaluronan synthase, HAS) gene performance.
Specific embodiment
Of the invention has dendrobium candidum (the Dendrobium officinale Kimura et for adjusting skin metabolism
Migo) the extraction procedure of extract is via dendrobium candidum culture proliferation, the induction of dendrobium candidum separate living tissue, dendrobium candidum point
Raw tissue purifying culture, dendrobium candidum separate living tissue proliferation, the dry extraction of dendrobium candidum separate living tissue and etc. it is prepared.Its
In, preparation method of the invention is a set of stable dendrobium candidum separate living tissue induction, enrichment procedure, and is demonstrate,proved using cell experiment
The related efficacy of real dendrobium candidum separate living tissue extract.
The preparation method of 1 dendrobium candidum extract of embodiment
The present invention using growth hormone matched combined to induce, Multiplying culture dendrobium candidum separate living tissue, develop a set of
It is capable of the preparation flow of steady production dendrobium candidum extract.
Firstly, the auximone of 1mg/L is added using 1/3MS (Murashige and Skoog, 1962) culture medium
The basic element of cell division 6- aniline purine (6- of 1- methyl α-naphthyl acetate (1-Naphthaleneacetic acid, NAA) and 7mg/l
Benzyladenine, BA) sufficiently it is mixed evenly, after 3% sucrose and 0.8% agar are sufficiently mixed into rear 1/3MS culture medium,
It is quantitative between pH value 5.7 to 5.8 using 0.1N sodium hydroxide solution, it is trained after sterilizing for the induction of dendrobium candidum separate living tissue
It supports.
Then, using 1/3MS (Murashige and Skoog, 1962) culture medium, 3% sucrose and 0.8% agar are filled
Divide after being mixed into rear 1/3MS culture medium, it is quantitative between pH value 5.7 to 5.8 using 0.1N sodium hydroxide solution, it is used for after sterilizing
The Multiplying culture of dendrobium candidum separate living tissue.
Both the above culture medium is enough to provide the induction of dendrobium candidum separate living tissue and proliferation, and separate living tissue yield is up to 4
Times as many as, compared to dendrobium candidum plant, the extract of this case have stronger cell grain wire body activity, promote collagen with
And the effect of sodium hyaluronate hyperplasia.Via the separate living tissue product that tissue culture technique is proliferated, first with freeze drier with -40 DEG C
It is dried 16 hours, harvest product ensures moisture lower than 5% or less；Then, recycle 75% edible alcohol solution in 70 DEG C
Under the conditions of carry out extraction 30 minutes, generate the of the invention dendrobium candidum extract for adjusting skin metabolism.
The Skin Cell grain wire body activity analysis of the dendrobium candidum extract of the invention of embodiment 2
The present invention carries out the wire body activity analysis of Skin Cell grain with human skin fibroblast.Human skin fiber is female thin
Born of the same parents purchased from Taiwan living resources save and research center (Bioresource Collection and Research Center,
BCRC), number BCRC 60153.By the cell culture in addition 10% fetal calf serum (fetal bovine serum, FBS)
(GIBCO company, number 10438-026, the U.S.), 1.5g/L sodium bicarbonate (Sigma company, number S5761, the U.S.), 1mM third
Minimum required culture solution (the Minimum essential of ketone acid sodium (GIBCO company, number 11360-070, the U.S.)
Medium, MEM) (GIBCO company, number 41500-034, the U.S.).
Each hole inoculation 1.5 × 10 in the 6 hole culture plates containing the above-mentioned culture medium of 2mL5A mankind's skin fiber is female
Cell, respectively plus 4%, 2% dendrobium candidum extract or cell culture medium are incubated at 37 DEG C 24 hours as a control group, above-mentioned
Culture medium (n=3).Then remove culture medium and with 1 times of phosphate buffer (phosphate buffered saline,
PBS) (GIBCO company, number 14200-075, the U.S.) is cleaned twice；Trypsase (trypsin)/EDTA processing is added later
After 3 minutes, the cell of suspension is drawn to the centrifuge tube of 1.5mL, the cell of Shen Dian is collected within 5 minutes with the centrifugation of 400g revolving speed.
Twice with 1 times of phosphate buffer cleaning by the cell, then with 100 μ LJC-1 stain (BD company, number RUO-
551302, U.S.) the settling flux cell, it is being protected from light lower culture 15 minutes after mixing；5 points are centrifuged later with 400g revolving speed
Clock, then twice with cleaning buffer solution cleaning.With the cell of 1 times of the phosphate buffer settling flux containing 2%FBS, stream is utilized
Grain wire body film potential changes when formula cell instrument (BD Accuri C6Plus) analysis and observation Apoptosis, and carries out t- with Excel
Examine the statistical significance of difference between (student t-test) statistical analysis sample populations.
The experimental result of dendrobium candidum extract Skin Cell grain wire body activity analysis of the invention, as shown in Figure 1, by
The processing of dendrobium candidum extract, grain wire body activity obviously increase, and 2% dendrobium candidum extract obviously increases than control group
11.9%.Therefore, this case dendrobium candidum extract is more excellent than activation grain wire body effect of control group, shows this case iron sheet stone
Dry measure used in former times extract has effects that excellent activation grain wire body.
The dendrobium candidum extract of the invention of embodiment 3 is in promote collagen and sodium hyaluronate to generate the effect of
Due to known 1 chain of promotion Type I collagen α (collagen alpha-1 (I), COL1A1) gene and Type I collagen α 2
The performance of chain (collagen alpha-2 (I), COL1A2) gene, can promote the generation of the first collagen type；Promote I V-type
The performance of 4 chain of collagen α (collagen alpha-4 (IV), COL4A4) gene, can promote the generation of the 4th collagen type；Promote
Into matrix metalloprotease tissue depressant (tissue inhibitors of metalloproteinase-1, TIMP-1) base
Because of performance, MMP-2 (matrix metalloproteinase-2, MMP-2) and matrix metal can be inhibited
The gene table of the ferment relevant to collagen is decomposed such as protease -9 (matrix metalloproteinase-9, MMP-9)
It is existing；Promote to show from amido oxidizing ferment (lysyl oxidase, LOX) gene, the generation of collagen can be promoted；Promote saturating
Bright matter acid enzyme (hyaluronan synthase, HAS) gene performance, can promote sodium hyaluronate (hyaluronic
Acid), uronic acid, higher polysaccharides class generate effect, therefore the present invention further detect dendrobium candidum extract to COL1A1,
The influence of COL1A2, COL4A4, TIMP-1, LOX, MMP-2 and MMP-9 gene performance amount.
The method as described in previous embodiment 2 obtains the dermal fibroblasts of the Shen Dian, each in 6 hole culture plates
Hole inoculation 1.5 × 105A cell, to add 2% dendrobium candidum extract or not add any ingredient (as control group)
Above-mentioned culture medium is incubated at 37 DEG C 24 hours；Dermal fibroblasts are collected later.Use RNA extraction agent group
The RNA of the above-mentioned cell of (Genemark company, the U.S.) extraction, recycling reverse transcriptase (III Reverse
Transcriptase) RNA reverse transcription is cDNA by (Invitrogen company, the U.S.), then uses KAPAFAST
QPCR reagent set (KAPABiosystems company, the U.S.) measure target gene (COL1A1, COL1A2, COL4A4, TIMP-1,
MMP-2, MMP-9, LOX and HAS) transcription amount, analyzed with quantitative PCR apparatus.
Due to COL1A1, COL1A2, COL4A4, TIMP-1, MMP-2, MMP-9, LOX and HAS gene and collagen
Or sodium hyaluronate forms correlation, as a result as shown in Figures 2 to 4, dendrobium candidum extract of the invention can promote COL1A1,
The performance of COL1A2, COL4A4, TIMP-1, LOX and HAS gene inhibits the performance of MMP-2 and MMP-9 gene.It confirms to promote
The generation of collagen；Dendrobium candidum extract of the invention, which can reach, promotes collagen and sodium hyaluronate to generate, reduce collagen
The effect of protein breakdown, so as to reduce wrinkle, prevent skin aging, protection bone, pre- preventing bone rarefaction, Saving cortilage,
Improve degenerative arthritis, promote wound healing, reduce scar, reduce skin wrinkle, promoted it is compact with it is elastic, additionally it is possible to promoted
Skin moisturizing reduces dry skin.
In conclusion dendrobium candidum extract of the invention is via addition 1- methyl α-naphthyl acetate (NAA) and 6- aniline purine
(BA) 1/3MS culture medium induction and Multiplying culture after again through an extraction step obtain；Furthermore dendrobium candidum extraction of the invention
Object can be generated via activation grain wire body, promotion collagen and sodium hyaluronate, and then have the effect of adjusting skin metabolism.Therefore,
Dendrobium candidum extract of the present invention is a natural component, will not generate side effect brought by traditional artificial synthetic ingredient, can answer
With adjust skin metabolism medical composition, the composition be with powdered, graininess, liquid, glue or paste presence, and its
Dosage form is provided in the form of food, drink, drug, reagent or nutritional supplement.
Claims (11)
1. a kind of dendrobium candidum extract is used to prepare the purposes for adjusting the medical composition of skin metabolism.
2. purposes according to claim 1, which is characterized in that the dendrobium candidum extract is by adding 1- methyl α-naphthyl acetate
The 1/3MS culture medium of (1-Naphthaleneacetic acid, NAA) and 6- aniline purine (6-benzyladenine, BA)
It is obtained again through an extraction step after induction and Multiplying culture dendrobium candidum separate living tissue.
3. purposes according to claim 2, which is characterized in that the extraction step is with 75% alcoholic extract.
4. purposes according to claim 1, which is characterized in that the adjusting skin metabolism is activation grain wire body.
5. purposes according to claim 1, which is characterized in that the adjusting skin metabolism is to promote collagen and glass urine
Acid generates.
6. purposes according to claim 1, which is characterized in that the adjusting skin metabolism is to promote Type I collagen α 1
Chain (collagen alpha-1 (I), COL1A1), 2 chain of Type I collagen α (collagen alpha-2 (I), COL1A2), I V-type glue
Former 4 chain of α (collagen alpha-4 (IV), COL4A4), matrix metalloprotease tissue depressant (tissue
Inhibitors of metalloproteinase-1, TIMP-1), from amido oxidizing ferment (lysyl oxidase, LOX) and
The performance of hyaluronic acid synthetase (hyaluronan synthase, HAS) gene.
7. purposes according to claim 1, which is characterized in that the adjusting skin metabolism is to inhibit matrix metalloprotease
Enzyme -2 (matrix metalloproteinase-2, MMP-2) and Matrix Metalloproteinase-9 (matrix
Metalloproteinase-9, MMP-9) gene performance.
8. purposes according to claim 1, which is characterized in that the medical composition further includes one and can pharmaceutically connect
The carrier received.
9. a kind of method of induction and proliferation dendrobium candidum separate living tissue, includes: using addition 1- methyl α-naphthyl acetate (1-
Naphthaleneacetic acid, NAA) and 6- aniline purine (6-benzyladenine, BA) 1/3MS culture medium
Step.
10. a kind of culture medium for cultivating skin dendrobium nobile separate living tissue, which is characterized in that the culture medium includes a 1- methyl α-naphthyl acetate (1-
Naphthaleneacetic acid, NAA).
11. culture medium according to claim 10, which is characterized in that the culture medium further includes 6- aniline purine
(6-benzyladenine, BA).
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| CN106667869A (en) * | 2017-01-10 | 2017-05-17 | 皖西学院 | Anti-allergic dendrobium facial mask liquid preparation technology |
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| CN106667869A (en) * | 2017-01-10 | 2017-05-17 | 皖西学院 | Anti-allergic dendrobium facial mask liquid preparation technology |
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