CN108295001A - Application of the snowflake water extract in inhibiting fibroblast telomere to shorten - Google Patents
Application of the snowflake water extract in inhibiting fibroblast telomere to shorten Download PDFInfo
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- CN108295001A CN108295001A CN201710020993.2A CN201710020993A CN108295001A CN 108295001 A CN108295001 A CN 108295001A CN 201710020993 A CN201710020993 A CN 201710020993A CN 108295001 A CN108295001 A CN 108295001A
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- snowflake
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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
- A61Q19/08—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
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
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Abstract
The invention discloses application of the snowflake water extract in inhibiting fibroblast telomere to shorten.Snowflake water extract can inhibit the shortening of fibroblast telomere in the present invention, fill up the blank in the prior art to snowflake water extract in terms of inhibiting fibroblast telomere shortening.
Description
Technical field
The present invention relates to application of the snowflake water extract in inhibiting fibroblast telomere to shorten.
Background technology
In Cosmetic Manufacture, the cosmetics of anti-aging are of interest by people always, this is because old for skin is resisted
The problem of change is public's emphasis of interest.Protection skin, delay skin aging are the dreams of many people, therefore anti-aging is made up
An important research direction in the development and application cosmetic industry of product.
It is cell ageing that the aging of cutaneous manifestations, which is reflected in cellular level,.In the skin of aging, there is because at
Cell Proliferation caused by fibrocyte aging weakens, and the synthesis of the extracellular matrix components such as collagen is insufficient, and even degradation increases, and leads
Cause corium aging.Therefore, how research slows down fibroblast aging and then delay skin aging has far-reaching significance.
Snowflake (Leucojum vernum) is Amaryllidaceae Galanthus, and bulbous herbaceous plant is provided with sagging white
Flower.Snowdrop has bulb, and leaf is linear, and scape is upright, and being write on scape top has a flower, bell；Six, petal, petal
Two layers inside and outside being divided into, internal layer respectively has three pieces petal, the petal of outer layer longer than the petal of internal layer and more raised with outer layer.From suspend mode
Snowdrop in the active constituent that extracts, plant dormancy essence has the function of class creotoxin, crease-resistant plain weave；It is super by promoting
The expression of superoxide dismutase (SOD) improves skin intrinsic antioxidative ability；The extract of suitable concentration can effectively and longer
Block the contraction of muscle in muscle-nerve co-culture system.It is still very big for the research space of snowflake water extract.
Invention content
The technical problem to be solved by the present invention is in the prior art inhibit snowflake water extract at fibre to overcome
The blank in terms of cell telomere shortening is tieed up, and provides snowflake water extract and is applied in inhibiting fibroblast telomere to shorten.
The present invention provides a kind of application of snowflake water extract in inhibiting fibroblast telomere to shorten.
In the present invention, the snowflake water extract generally ensures cell activity when inhibiting fibroblast telomere to shorten
Normally.
In the present invention, the snowflake water extract shortens in inhibition fibroblast telomere can be by inhibiting telomere to shorten energy
Power reflects that the inhibition telomere shortens ability generally can be by that (can make fibroblast telomere contract rapidly with stress from outside object
Short external substance, such as hydrogen peroxide) compare to obtain, specifically, the inhibition telomere shortens ability=(B-A)/(NT-A) *
100%；Wherein, A is the fibroblast end for including stress from outside object (substance for not containing other influences cell telomere length)
Grain relative length；B is (not contain other influences cell comprising extraneous stress from outside object described in the snowflake water extract and A
The substance of telomere length) fibroblast telomere relative length；NT is the relative length of normal fibroblast telomere.
The computational methods of the relative length of fibroblast telomere are the method that those skilled in the art know.
The preferred leucojum aestivum of snowflake (Leucojum aestivum) in the snowflake water extract.
The snowflake water extract refers to the extract that snowflake is extracted according to the water extracting method of this field routine, this
Invention particularly preferably following extracting method：By the snowflake (can be complete stool or bulb), crush in water, 10 DEG C~50 DEG C
40~60min (such as 45min) is extracted at a temperature of (such as 15 DEG C~45 DEG C), removes solid, dilute filtration；The snowflake
Lotus carries out cleaning and/or peeling before the crushing.
The snowflake can be at rest period or non-sleep phase, further preferred rest period；Rest period refer to plant or
For its organ during development, there is the period of pause in growth and metabolism, and the rest period of the snowflake is in winter.
The conventional comminution processes of this field can be used in described crush, it is preferred to use pulverizer, pulverizer rotating speed is 25000~
30000 revs/min, more preferably 29000 revs/min；The dosage of the water or pure water is the snowflake matter when crushing
5-7 times of amount；The grinding time is determined by crushing for homogenate state.
Preferably use water and/or glycerine as solvent when the dilution.
The filtering preferably uses 0.2 μm of filter to be filtered.
Without prejudice to the field on the basis of common sense, above-mentioned each optimum condition can be combined arbitrarily each preferably to get the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that：
(1) snowflake water extract can inhibit the shortening of fibroblast telomere；
(2) without thin within the scope of a concentration of 3~0.5mg/ml of the snowflake water extract in cell, more preferably 1.5mg/ml
Cellular toxicity.
Specific implementation mode
It is further illustrated the present invention below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
Product specification selects.
In the present invention, by adding the snowflake water extract of various concentration in fibroblastic incubation, survey
Whether its fixed cell activity, observation cell are damaged.
In the present invention, hydrogen peroxide and hydrogen peroxide and snowflake water extract are added respectively in Fibroblast cell-culture,
Real-time fluorescence quantitative PCR measure DNA telomere lengths, can effecting reaction snowflake water extract inhibit fibroblast telomere shorten
Effect.
1, the preparation of leucojum aestivum water extract
The leucojum aestivum plant being in hibernation is cleaned, and peeling is carried out to it.The powder in pure water (6 times of snowflake quality)
(pulverizer rotating speed is 29000 turns/min, time 3h to broken bulb, until forming homogenate state.At a temperature of 25 DEG C, spiral
Stirring extraction 45min, thick solid is dispelled by decantation.Carry out thin solid again dispels (100 DEG C or so heating slurries, decantation liquid
Phase extract is centrifuged by the disc centrifuge of flow velocity 5kg/min and is obtained to new cleaning container.It is diluted in water to obtain
The quality of 10mg/L (i.e. the solid bulb containing 10mg in every liter of water), extract are filtered using 0.2 μm of filter, final
The color of extract is light yellow.
2, cell culture and sample-adding processing
Cell culture：Human fibroblasts AL15012LF-P2 uses complete medium (DMEM, 10%FCS, 1%P/S)
In 37 DEG C of 5%CO2Cell incubator culture, until 90% when converging, a digestion kind plate.
Cytotoxicity experiment：AL15012LF-P3
5000/ hole
5%CO of 96 orifice plates at 37 DEG C2It is cultivated 48 hours in cell incubator
Sample and concentration：
* the quality containing solid bulb in every milliliter of water
Sample-adding：(aqueous solution of the light yellow extract obtained by above-mentioned 1, difference lies in concentration, store liquid for stock sample solution
Concentration is seen the above table) with culture medium dilute 100 times be added in hole；
Cell culture：Cell culture 24 hours in the culture medium (DMEM, 2%FCS, 1%P/S) containing sample to be tested
3, cytotoxicity experiment
After cell co-cultures 24 hours with sample to be tested, culture medium is removed, MTT 0.5mg/ml are added, it is careful after 3 hours
Supernatant is sucked, DMSO is added, is detected under 550nm；Using 0.25% lauryl sodium sulfate (SDS) as reference, definition
Sample well is T, and untreated hole is NT, and with the ratio (T/NT) of T and NT, i.e. Fb cell activity >=90% is considered as no cytotoxicity,
Ratio ＜ 90%, which is considered as, cytotoxicity, and experimental result is as shown in table 1 below：
Table 1
Fb cell activity (% is relative to untreated hole NT)
Compared with control SDS (0.25%) obviously has cytotoxicity, sample (SF) is within the scope of 3~0.5mg/ml of final concentration
There is no cytotoxicity, this section concentration can be used to continue subsequent experimental.
4, cell telomere length test experiments
4.1 cell activity are tested
According to cytotoxicity experiment concrete operations described in above-mentioned 3, only sample is changed.
Sample number into spectrum H2O2+ snowflake (1.5ml/mL) represents the H of 40uM2O2(1.5ml/mL is avenged+1.5ml/mL snowflakes
Piece lotus refers to that gained light yellow mass is diluted with water to 1.5ml/mL in above-mentioned 1)；
Sample number into spectrum H2O2+ snowflake (0.75ml/mL) represents the H of 40uM2O2+ 0.75ml/mL snowflakes (0.75ml/mL
Snowflake refers to that gained light yellow mass is diluted with water to 0.75ml/mL in above-mentioned 1)；
Sample number into spectrum H2O2+ snowflake (0.5ml/mL) represents the H of 40uM2O2(0.5ml/mL is avenged+0.5ml/mL snowflakes
Piece lotus refers to that gained light yellow mass is diluted with water to 0.5ml/mL in above-mentioned 1)；
Remaining number meaning is similar.
It is normal that Fb cell activity (%) >=90% is considered as cell activity, it is seen that above-mentioned sample can be such that fibroblast keeps
Normal activity.
4.2 cell telomere lengths test sample-adding processing
Using hydrogen peroxide (H2O2) etc. oxidative pressures can cause cell telomere shorten, carry out related experiment.
People's primary fibroblast AL15012LF (P5) is inoculated in 6 orifice plates, using culture medium (DMEM+10%FCS) in
37 degree of CO248h is cultivated in incubator；
NT (untreated fish group)：Media alone (DMEM+10%FCS) is added and handles 2h, the culture medium (DMEM renewed later
+ 10%FCS)；
H2O2(pressurization aging group)：H containing 40uM is added2O2Medium treatment 2h, the culture medium (DMEM+ renewed later
10%FCS)；
Sample sets：H containing 40uM is added2O2And the culture medium of sample snowdrop 1.5mg/mL, 0.75mg/mL or 0.5mg/mL
Handle 2h, the culture medium renewed later (DMEM+10%FCS)；
Above-mentioned processing procedure continuous processing three days.
4.3 real-time fluorescence quantitative PCRs measure DNA telomere lengths
Telomere (T) repeats copy number and the ratio of single copy gene (S) and the length of telomere is proportional, i.e. T/S ratios
Rate can obtain the relative length of telomere, shown in the following formula of computational methods：
T/S=[2CT(tel)/2CT(hbg)]=2-ΔCT
It according to telomere relative length, calculates and telomere is inhibited to shorten ability, inhibiting into fibre to react snowflake water extract
Tie up the effect in terms of cell telomere shortening.
Telomere is inhibited to shorten ability=(sample telomere relative length-H2O2Group telomere relative length)/(NT telomeres are relatively long
Degree-H2O2Group telomere relative length).
3 Duplicate Samples (n=3) under the conditions of each, statistical data is as follows：
As can be seen from the above table, the shortening of fibroblast telomere is can inhibit when snowflake water extract is added, and with dense
The increase of degree, inhibiting effect are enhanced.
Claims (10)
1. application of the snowflake water extract in inhibiting fibroblast telomere to shorten.
2. application as described in claim 1, which is characterized in that the snowflake in the snowflake water extract is leucojum aestivum
(Leucojum aestivum)。
3. application as described in claim 1, which is characterized in that the snowflake water extract extracts as follows：It will
Snowflake crushes in pure water, at a temperature of 10 DEG C~50 DEG C, extracts 40~60min, removes solid, dilute filtration；It is described
Snowdrop carries out cleaning and/or peeling before the crushing.
4. application as claimed in claim 3, which is characterized in that the snowflake raw material in the snowflake water extract is snowflake
Bulb.
5. application as claimed in claim 3, which is characterized in that the snowflake in the snowflake water extract is the snow of rest period
Piece lotus.
6. application as claimed in claim 3, which is characterized in that the temperature is 15 DEG C~45 DEG C；The time of the extraction is
45min。
7. application as claimed in claim 3, which is characterized in that it is 25000 to use pulverizer, pulverizer rotating speed when the crushing
~30000 revs/min.
8. application as claimed in claim 3, which is characterized in that the dosage of the pure water is the 5~7 of the snowflake quality
Times.
9. application as claimed in claim 3, which is characterized in that the diluted solvent is water and/or glycerine.
10. application as claimed in claim 3, which is characterized in that the filtering is filtered using 0.2 μm of filter.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710020993.2A CN108295001A (en) | 2017-01-11 | 2017-01-11 | Application of the snowflake water extract in inhibiting fibroblast telomere to shorten |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710020993.2A CN108295001A (en) | 2017-01-11 | 2017-01-11 | Application of the snowflake water extract in inhibiting fibroblast telomere to shorten |
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| Publication Number | Publication Date |
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| CN108295001A true CN108295001A (en) | 2018-07-20 |
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| CN201710020993.2A Pending CN108295001A (en) | 2017-01-11 | 2017-01-11 | Application of the snowflake water extract in inhibiting fibroblast telomere to shorten |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020071878A1 (en) * | 1997-02-23 | 2002-06-13 | I.B.R. Israeli Biotechnology Research Ltd. | Anti proliferative preparations |
| US20090264291A1 (en) * | 1998-02-23 | 2009-10-22 | Etienne Soudant | Compositions comprising anti-proliferative agents and use thereof |
| CN102149398A (en) * | 2008-09-11 | 2011-08-10 | I·B·R·以色列生物科技研究有限公司 | Leucojum bulb extracts and use thereof |
| KR20160036121A (en) * | 2014-09-24 | 2016-04-04 | 장문식 | Cosmetic composition containing extracts consisting of Leucojum aestivum, Leontopodium alpinum, Cordyceps sinensis (Berk.) Sacc, Royal Jelly Extract and Saussureae Involucratae with nano emulsion process |
| CN106176479A (en) * | 2016-08-31 | 2016-12-07 | 广东芭薇生物科技股份有限公司 | For balancing human body inflammatory reaction the compositions of delaying skin aging disease and application thereof |
-
2017
- 2017-01-11 CN CN201710020993.2A patent/CN108295001A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020071878A1 (en) * | 1997-02-23 | 2002-06-13 | I.B.R. Israeli Biotechnology Research Ltd. | Anti proliferative preparations |
| US20090264291A1 (en) * | 1998-02-23 | 2009-10-22 | Etienne Soudant | Compositions comprising anti-proliferative agents and use thereof |
| CN102149398A (en) * | 2008-09-11 | 2011-08-10 | I·B·R·以色列生物科技研究有限公司 | Leucojum bulb extracts and use thereof |
| KR20160036121A (en) * | 2014-09-24 | 2016-04-04 | 장문식 | Cosmetic composition containing extracts consisting of Leucojum aestivum, Leontopodium alpinum, Cordyceps sinensis (Berk.) Sacc, Royal Jelly Extract and Saussureae Involucratae with nano emulsion process |
| CN106176479A (en) * | 2016-08-31 | 2016-12-07 | 广东芭薇生物科技股份有限公司 | For balancing human body inflammatory reaction the compositions of delaying skin aging disease and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| AGATA PTAK ET AL: "LCMS and GCMS for the Screening of Alkaloids in Natural and in Vitro Extracts of Leucojum aestivum", 《JOURNAL OF NATURAL PRODUCTS》 * |
| 周礼娟: "感受大自然神奇的馈赠，储存肌肤年轻的资本", 《中国化妆品》 * |
| 左伋: "《医学细胞生物学》", 31 January 2016, 复旦大学出版社 * |
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Application publication date: 20180720 |
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