CN110317774A - Aqueous extract of Chinese herbal medicine and combinations thereof and purposes - Google Patents
Aqueous extract of Chinese herbal medicine and combinations thereof and purposes Download PDFInfo
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- 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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- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/987—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of species other than mammals or birds
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Abstract
The invention discloses aqueous extract of Chinese herbal medicine and combinations thereof and purposes, more particularly to more sequence rock astragalus (Hedysarum polybotrys Hand.-Mazz.), rhizoma alismatis (Alisma plantago-aquatica (Sam.) Juzep), balsam pear (Momordica charantia L.) or the aqueous extract of pheretima (Eisenia foetida), and their combination, the new application in cell culture.The aqueous extract of said herbal medicine can replace the partial function of animal blood serum in cell culture medium, the dosage of animal blood serum in cell culture medium can thus be reduced, thus the production cost of cell is reduced, and is reduced because using health risk brought by animal blood serum.The present invention also relates to the cosmetic formulations of the anti-wrinkle containing the aqueous extract of the Chinese herbal medicine and resisting age of skin.
Description
Technical field
The present invention relates to the new application of the aqueous extract of Chinese herbal medicine, the aqueous extract of especially single kind Chinese herbal medicine,
Or the aqueous extract combination of several Chinese herbal medicines, new compositions and new application in cell culture, and in confrontation skin
New compositions and new application on wrinkle and skin aging.
Background technique
Fibroblast is present in the intracorporal many tissues of animal, for synthesizing extracellular matrix and collagen, there is dimension
The function of the essential structure of connective tissue is held, and is played an important role in wound healing.Fibroblast as by
To the effect of cytohormone secreted by oneself or peripheral cell, and has and migrate and the reaction of hyperplasia.
The fibroblast of culture have promote tissue repairing effect, applicable range is quite extensive, including it is acute and
Chronic wounds arrive beauty and reconstruction operations, all there is good clinical effectiveness.For the repairing of wound, fibroblast can
It is incubated at bracket made by Biodegradable material, is made into Graftskin product.There are many skins currently on the market
Substitute, but its safety and quality pole need strictly to be planned and managed.For example, whether cell origin is human body, product is
It is no sterile, if there is pollution, it is whether still active after chilled, if it is non-autologous, if having repulsion or virus infection
Possibility etc., be all the part for needing strict control.In addition, how cost cutting to minimum and product to be promoted to most with property
Greatly and correlative technology field personnel wish the project solved in a hurry.
In the treatment of medical cosmetology at present, also can in a manner of injection by self fibroblast as tissue filling
Object replaces conventional injection bovine collagen albumen that may have repulsion or infected risk.Injection sodium hyaluronate is also possible to that skin can be generated
Lower excessive immune reaction, causes red and swollen phenomenon.Therefore, injecting self fibroblast has been that current medical cosmetology dividing value must be sent out
The treatment method of exhibition.Face wrinkles and scar can be improved by injecting self fibroblast, and be not required to consider to have repulsion etc. no
The risk of good reaction, and inject living cells therapeutic effect can be made to extend, reduce treatment number (Weiss, R.A.et al.,
Dermatol.Surg.33(3):263-268)。
In response to application of the fibroblast on autoinjector, reduced under the premise of effectively culture human fiber's mother cell
The dosage of fetal calf serum (fetal bovine serum, abbreviation FBS) has its necessity.Currently, fibroblast mainly with
Dahl Burke Improved Eagle Medium (DMEM) containing 10%FBS is culture solution, and wherein fetal calf serum is for providing fiber
Nutrient and growth factor required for mast cell growth.But when using fibroblast preparation, either with injection system
Or carried out in a manner of mesotherapy (mesotherapy), require to make it into patient body, and in fetal calf serum at
Dividing may enter into the cell during culture, and safety is difficult to estimate, and may cause repulsion, allergy, even disease sense
The risk of dye.Moreover fetal calf serum unit price is very high.Therefore, the use of fetal calf serum is reduced in the incubation of fibroblast
Amount, for current considerable subject under discussion.Some researchs at present are female as culture fiber with separated serum out of drawing blood self
The source of nutrition of cell culture fluid.Although this method is safer, culture fibroblast needs a large amount of culture serum.
If it is desired to patient to be treated has anaemia phenomenon, it can not just obtain a large amount of blood systems and separate out enough serum.In addition clinically, fiber
Mother cell may need multiple subculture, and the demand of serum is higher at this time.In these cases, patient may can not be successfully progress
Treatment.Therefore, industry is for can promote the fetal calf serum substitute of Fibroblast Growth, or can reduce fetal calf serum
The cell culture replenishers of dosage, there are ardent demands.
The cell culture replenishers of non-animal have low, low in cost, abundance the advantage of disease infection risk.
Fa Ruihandi et al. proposes that training of the plant protein hydrolysate as human skin fibroblast of soybean and rice can be used
It supports replenishers (Farizhandi, A.A.K.et al., Adv.Environ.Biol.8 (3 Spec.Issue), 838-843).
WO2004/083400A2 discloses a kind of for cultivating the culture medium replenishers of zooblast, it is by peanut, filbert, apricot
The vegetable seeds such as benevolence are extracted and are obtained, can be for being used as substitute or complementary goods for fetal calf serum.Japanese patent application 2-
No. 49579 are proposed basal medium of arranging in pairs or groups using the hydrolysate and saccharomycete extract of gluten, for cultivating zooblast.
Summary of the invention
Corium fabric mother cell sole duty manufactures collagen, the extracellular matrixs such as elastin, with maintain skin elasticity and
Density.There is skin corium the fibroblast of quantity abundance to secret out of enough collagens, can just smooth the wrinkle of skin
And keep the elasticity of skin.Therefore, the quantity and vigor of corium fabric mother cell, health and plump compactness with skin
It is closely bound up.Present inventor passes through mankind's corium fabric mother cell culture primary (primary human dermal
Fibroblast cell culture) a cells ex vivo model is set up, it being capable of augmentor fiber mother cell for filtering out
Survival rate, and/or promote its to secrete collagen, and/or its is promoted to secrete growth factor, and/or enhance its anti-oxidant energy
The medicinal components of power are applied in skin care products, to reach skin wrinkle resisting and anti-aging effect.
Chinese herbal medicine is the rarity of Chinese culture long history, is contained tired for the knowledge of pharmaceutical animals and plants for thousands of years
Product.A variety of Chinese herbal medicine extract folk prescriptions or compound are examined to combine with above-mentioned cells ex vivo model, present inventor
It was found that Duo Xuyan astragalus (Hedysarum polybotrys Hand.-Mazz.), rhizoma alismatis (Alisma plantago-
Aquatica (Sam.) Juzep), balsam pear (Momordica charantia L.) and pheretima (Eisenia foetida) etc. pass
Unite Chinese herbal medicine aqueous extract, be either used alone or in combination, can only containing low amounts fetal calf serum in vitro
The growth for promoting mankind or animal cell under condition of culture can especially promote the growth of fibroblast, such as promote people
The growth of class corium fabric mother cell.Accordingly, the aqueous extract of said herbal medicine is available for reducing fetal calf serum in cell
Thus usage amount in culture medium reduces the production cost of zooblast.In particular, when the fibre produced according to the application
When tieing up mother cell importing human body, it can reduce significantly because fetal calf serum enters health risk caused by human body.
Therefore, according to the present invention in a first aspect, it provides a kind of cell culture medium replenishers, it is characterised in that: it is described
Replenishers include the aqueous extract from one or more biological species, and the biological species are selected from more sequence rock astragalus
(Hedysarum polybotrys Hand.-Mazz.), rhizoma alismatis (Alisma plantago-aquatica (Sam.) Juzep),
Balsam pear (Momordica charantia L.) and pheretima (Eisenia foetida).
In a preferred embodiment, the aqueous extract is selected from more sequence rock astragalus and the aqueous extract of pheretima
The combination of combination, rhizoma alismatis and the balsam pear and the aqueous extract of pheretima of combination, Duo Xuyan astragalus and rhizoma alismatis and the aqueous extract of balsam pear,
The combination of more sequence rock astragalus and balsam pear and the aqueous extract of pheretima and more sequence rock astragalus and rhizoma alismatis and balsam pear and the aqueous extraction of pheretima
The combination of object out.In a more preferable specific embodiment, the aqueous extract is selected from more sequence rock astragalus and the aqueous extraction of pheretima
The combination of object out.
According to the second aspect of the invention, a kind of cell culture medium is provided, it is characterised in that: before the culture medium includes
State cell culture medium replenishers；And a basal medium, it is supplemented on the basis of the total volume according to the cell culture medium
The substantially below animal blood serum of 10 volume %, the preferably substantially below animal blood serum of 5 volume %, more preferably substantially
Lower than the animal blood serum of 1 volume %, for example, about animal blood serum of 0.5 volume %.
According to the third aspect of the invention we, a kind of method for cultivating cell is provided, it is characterised in that:
The method comprises the steps of
(a) cell is made to be in contact with prior culture media；And
(b) under conditions of being suitable for that the cell is supported in vitro cultivate, the cell is cultivated.
In a preferred embodiment, the cell is mankind or animal cell.In a more preferable specific embodiment party
In case, the cell is fibroblast.In a more preferable specific embodiment, the cell is that human dermal's fiber is female thin
Born of the same parents.
Present inventor it has furthermore been found that astragalus and the aqueous extract of pheretima combination, not only can promote fiber mother
Cell Proliferation also can promote fibroblast secretion collagen, can more increase with H2O2Cell survival rate after processed.
Quantity and vigor of this Chinese herbal medicine combination for keeping corium fabric mother cell have potentiality so that anti-wrinkle to occur very much.
Therefore, fourth aspect according to the present invention provides a kind of anti-wrinkle cosmetic formulations, it is characterised in that described
Cosmetic formulations include: the aqueous extract of Duo Xuyan astragalus (Hedysarum polybotrys Hand.-Mazz.),
The aqueous extract and a cosmetic acceptability carrier of imperial (Eisenia foetida).
The 5th aspect according to the present invention, provides a kind of method prevented or improve wrinkle of skin again, it is characterised in that
The method includes: aforementioned anti-wrinkle cosmetic formulations are coated on skin surface.
Present inventor is, it was also found that the aqueous extract folk prescription of pheretima can not only promote superoxide dismutase
The activity of (superoxide dismutase, abbreviation SOD), in H2O2Cell can also be increased in the cell senescence model of induction to deposit
Motility rate.In addition, the combination of more sequence rock astragalus and the aqueous extract of pheretima, the combination of balsam pear and the aqueous extract of pheretima, and it is bitter
The combination of melon and the aqueous extract of rhizoma alismatis, can also increase with H2O2Cell survival rate after processed.The combination pair of these Chinese herbal medicines
In the survival for maintaining corium fabric mother cell and its aging is prevented, there are potentiality very much.
Therefore, the 6th aspect according to the present invention, provides the cosmetic formulations of a kind of pair of resisting age of skin, feature
It is that the cosmetic formulations include: from the aqueous extract of one or more biological species, is selected from the aqueous extraction of pheretima
Object, the combination of the aqueous extract of Duo Xuyan astragalus and the aqueous extract of pheretima, the aqueous extract of balsam pear and pheretima is aqueous extracts out out
The combination of object and the combination of the aqueous extract of balsam pear and the aqueous extract of rhizoma alismatis；An and cosmetic acceptability carrier.
The 7th aspect according to the present invention, provides a kind of method prevented or improve skin aging again, it is characterised in that
The method includes: the aforementioned cosmetic formulations to resisting age of skin are coated on skin surface.
The purpose of the present invention, feature and advantage will be described in detail by embodiment combination attached drawing.
Detailed description of the invention
Fig. 1 is histogram, and display is deposited with 3 days obtained cells of each Chinese herbal medicine extract folk prescription culture fibroblast
Motility rate；
Fig. 2 is histogram, and display is deposited with 6 days obtained cells of each Chinese herbal medicine extract folk prescription culture fibroblast
Motility rate；
Fig. 3 is histogram, and display is with collagen obtained by each Chinese herbal medicine extract folk prescription culture fibroblast 3 days
Secretory volume；
Fig. 4 is histogram, and display is with collagen obtained by each Chinese herbal medicine extract folk prescription culture fibroblast 6 days
Secretory volume；
Fig. 5 is histogram, and display is with blood vessel endothelium obtained by each Chinese herbal medicine extract folk prescription culture fibroblast 3 days
Growth factor (vascular endothelial growth factor；VEGF) secretory volume；
Fig. 6 is histogram, and display is with blood vessel endothelium obtained by each Chinese herbal medicine extract folk prescription culture fibroblast 6 days
Growth factor secretory volume；
Fig. 7 is histogram, and 3 days obtained super oxygens of each Chinese herbal medicine extract folk prescription culture fibroblast are used in display
Object mutase (SOD) activity；
Fig. 8 is histogram, and display is deposited with 3 days obtained cells of each Chinese herbal medicine extract compound culture fibroblast
Motility rate；
Fig. 9 is histogram, and display is deposited with 6 days obtained cells of each Chinese herbal medicine extract compound culture fibroblast
Motility rate；
Figure 10 is histogram, and display is with collagen egg obtained by each Chinese herbal medicine extract compound culture fibroblast 3 days
White secretory volume；
Figure 11 is histogram, and display is with collagen egg obtained by each Chinese herbal medicine extract compound culture fibroblast 6 days
White secretory volume；
Figure 12 is histogram, and display is with intravascular obtained by each Chinese herbal medicine extract compound culture fibroblast 3 days
Skin growth factor secretory volume；
Figure 13 is histogram, and display is with intravascular obtained by each Chinese herbal medicine extract compound culture fibroblast 6 days
Skin growth factor secretory volume；
Figure 14 is histogram, and 3 days obtained super oxygens of each Chinese herbal medicine extract compound culture fibroblast are used in display
Compound mutase (SOD) activity；
Figure 15 is histogram, shows each Chinese herbal medicine extract folk prescription and compound for by H2O2The shadow of treated cell
It rings；
Figure 16 is fluorescence microscope image, after display is with each Chinese herbal medicine extract folk prescription processing fibroblast, expression
The cell image of FSP-1；And
Figure 17 is histogram, after display is with each Chinese herbal medicine extract folk prescription and compound processing fibroblast, in cell
Express the percentage of FSP-1.
Specific embodiment
Unless otherwise stated, otherwise following term used in present specification and claims have hereafter give
The definition given.It note that singular shape term " one " used in present specification and claims is intended to cover one
A and more than one recorded item, for example, at least one, at least two or at least three, rather than mean only have
Single recorded item.In addition, the open conjunction such as "comprising" used in claims, " having " is to indicate right
In it is required that in the combination of assembly described in that or ingredient, however not excluded that the other assemblies or ingredient that claim does not state clearly.It should also infuse
Anticipate it is general in the sense to term "or" also include " and/or ", except non-content is separately clearly shown.Present specification and right
Term used in claim " about " or " substantial ", be to modify any error being varied somewhat, but it is this slightly
Variation can't change its essence.
This application involves the Chinese traditional herbs such as more sequence rock astragalus, rhizoma alismatis, balsam pear and pheretima in cell culture and skin care
On purposes.
In the present specification, term " Duo Xuyan astragalus " and " astragalus " can be used interchangeably, and cover Hedysarum
Recently the harvest of polybotrys Hand.-Mazz. (referred to as H.polybotrys) this plant species, without processing and passing through
The intact plant or plant part processed, the under ground portion of the espespecially described plant are crossed, such as is bought to resell in Chinese traditional herbs shop
The dried roots for the plant sold.It should be noted that common astragalus includes Mongolian astragalus (Astragalus on the market
Membranaceus (Fisch.) Bge.var.mongholicus (Bge.) Hsiao) and film pod astragalus (Astragalus
Membranaceus (Fisch.) Bge.), they and " Duo Xuyan astragalus " used in this application belong to different plant species.This Shen
Please used in " Duo Xuyan astragalus " by TaiWan, China Industrial Technology Research Institute carry out plant-origin identification, really
Recognize and belongs to the species.
Term " rhizoma alismatis (Alisma plantago-aquatica (Sam.) Juzep as used in this specification；Or letter
Referred to as A.plantago-aquatica) ", cover the harvest recently of this plant species, without processing and by the complete of processing
Whole plant or plant part, the under ground portion of the espespecially described plant, such as the plant peddled in Chinese traditional herbs shop
Object stem tuber part.The stem tuber part that the plant is usually excavated when winter cauline leaf starts withered, is dried with small fire after cleaning, is removed
Fibrous root and rough bark are removed, slice is moisturized with water, then dried.Rhizoma alismatis peddling in Chinese traditional herbs shop be typically in the form of through
Dry tuber part after crossing the processing means processing such as bran is fried or salt water is fried.
Term " balsam pear (Momordica charantia L. as used in this specification；Or referred to as
M.charantia) ", cover the harvest, intact plant or plant without processing and by processing recently of this plant species
Object part, the fruit parts of the espespecially described plant.Balsam pear is fruit and vegetable common fruit type vegetables in the market.It is used in this application
Balsam pear by TaiWan, China Industrial Technology Research Institute carry out plant-origin identification, confirmation belong to the species.
Term " pheretima (Eisenia foetida as used in this specification；Or referred to as E.foetida) ", also referred to as
For Eisenia Foetida, cover the harvest recently of this animal species, without processing and by the complete polypide of processing or worm
Body portion, such as the complete polypide without processing as bait is peddled in fishing articles shop, or in Chinese traditional herbs shop
That is peddled passes through the full polypide processed or polypide segment.In traditional concocting process, pheretima is usually captured in summer, autumn, is cutd open
Internal organ and silt are washed away after opening abdomen, then carries out exposure or low temperature drying.Dry polypide can direct hyoscine, or further
12.5 kilograms of yellow rice wine are added in every 100 kilograms of pheretimas, stirs to surface in brown, cools spare.
" aqueous extract " alleged by the application refers to one kind by making one or more aforementioned medicinal materials connect with an aqueous solvent
It touches and follows the composition come out prepared by standard extraction program.Term " from " as used in this specification is for referring to
The crude drug source of the aqueous extract is prepared out.The example of the aqueous solvent includes but is not limited to water, C1-6Alkanol (C1- 6alkanols)、C1-4Aklylene glycol (C1-4Alkylene glycols), aqueous saccharide solution, their saline solution, with
And their mixture.In a preferred embodiment, the aqueous solvent is selected from water and its saline solution.To promote
Into the efficiency of extraction, first medicinal material can be ground, and improve temperature and/or pressure when extraction.In a preferred specific implementation
In scheme, the aqueous extract be with deionized water is extracted at high temperature and is obtained, such as under normal pressure in about 100 DEG C into
Row extraction.Duration is extracted depending on the yield of extraction, it will usually continue 30 minutes to 1 day, preferably 1 to 12 hour,
Such as 1 to 3 hour." aqueous extract " this term is covered through thick extraction object obtained by single aqueous extraction process, and
Further receive the thick extraction object of one or more separation and/or purification procedures, including receives one or more additionally by making slightly to extract object
Extraction, concentration, filtering, distillation or other purification procedures and the active constituent or fraction (fractions) that are derived.Work as water
Property extract from two or more medicinal materials when, can be first extracted respectively for individual medicinal materials, then by individual extracts
It is mixed, can also first be mixed these medicinal materials, then extracted jointly.The aqueous extract liquid can be presented
Body form, such as solution, concentrate or distillate, can also be presented semi-liquid form, such as gel.If it is desirable, can be with
Aqueous extract is freeze-dried, and is stored in sterile chamber.Before use, adding again aqueous such as deionized water
Solvent carries out rehydration.
According to the application, Duo Xuyan astragalus, rhizoma alismatis, balsam pear and the aqueous extract of pheretima and these aqueous extracts
Compound combination, the life of mankind or animal cell can be promoted under the in vitro condition of culture only containing low amounts fetal calf serum
It is long, it can especially promote the growth of fibroblast, such as promote the growth of human dermal's fibroblast.This points out above-mentioned
The aqueous extract of Chinese herbal medicine is for the replenishers as Zooblast culture medium, to reduce animal blood serum in cell culture medium
In usage amount.In general, Zooblast culture medium is all supplemented with 10% to 20% animal blood serum, to provide cell growth institute
Outside the protein that needs, inorganic salts, growth factor, hormone and cell attachment fac etc.." animal blood serum " referred to herein means to come
From the liquid product of the blood in non-human animal, wherein blood cell, fibrinogen (fibrinogen) and fibrin
(fibrin) it is all removed, for making an addition to culture medium to provide nutriment needed for cell is grown comprising but it is not limited to fetal calf serum
(FBS), calf serum, bovine serum or the serum of other animals, such as the serum product from horse and camel.
The application is using mankind's corium fabric mother cell primary as the Screening Platform of the aqueous extract of Chinese herbal medicine.As hereinafter
Shown in embodiment 1 and 7, from the perspective of just promoting hyperplasia, compared to using the basal medium for being supplemented with 0.5%FBS
Control group, separately added other than 0.5%FBS more sequence rock astragalus, rhizoma alismatis, balsam pear or the aqueous extract folk prescription of pheretima or
The basal medium of their compound combination can all be such that the quantity increase of fibroblast reaches when cultivating fibroblast 3 days
It to 1.5 times, cultivates 3 days Shi Gengke and is higher by 1.4 to 2.4 times, show that the aqueous extract of aforementioned Chinese herbal medicine all has promotion fiber
The effect of mother cell growth.Embodiment 7 further displays, compared with 5% and 10%FBS, the aqueous extract folk prescription of Chinese herbal medicine or
Compound collocation 0.5%FBS make the degree of hyperplasia also can achieve using FBS conventional amount used (10%) about 50% to
66.7%.While not wishing to be restrained by any theory, but it is believed that not only contain in the aqueous extract of aforementioned Chinese herbal medicine
Zooblast grow required for nutrition, it is also possible to containing can stimulate according to fibroblast release growth factor at
Part, and then promote cell fast breeding.Embodiment 12 also confirms that the fiber handled by the aqueous extract of aforementioned Chinese herbal medicine is female
Cell can't change its original cell characteristic, will not break up as mature cell while rapid multiplication.
Be worth it is specifically intended that as described in Example 7, when with more sequence rock astragalus and the aqueous extract of pheretima group
It closes, is the combination of combination, rhizoma alismatis and the balsam pear and the aqueous extract of pheretima of Duo Xuyan astragalus and rhizoma alismatis and the aqueous extract of balsam pear, more
The combination of sequence rock astragalus and balsam pear and the aqueous extract of pheretima and more sequence rock astragalus and rhizoma alismatis and balsam pear and pheretima is aqueous extracts out
Fibroblast is cultivated in the combination of object, and can not only to make the hyperplasia multiple of cell be more than 2 times, but also these compounds combination gained
To hyperplasia effect it is also more preferable compared with the obtained effects of folk prescription individual in combination.While not wishing to be arrested by any theory
Beam, but it is believed that the composition in this five kinds of compound combinations produces cooperative effect on promoting hyperplasia.Therefore, excellent one
It selects in specific embodiment, it is one of that the aqueous extract comes from aforementioned five kinds of compounds combination.More preferably have one
In body embodiment, the aqueous extract comes from the combination of more sequence rock astragalus and the aqueous extract of pheretima.
Accordingly, the application provides a kind of cell culture medium replenishers, it includes: from the water of one or more biological species
Property extract, the biological species be selected from more sequence rock astragalus, rhizoma alismatis, balsam pear and pheretima." cell culture medium supplement referred to herein
Agent ", which refers to, to be made an addition in cell culture medium to assist the survival for maintaining the cell being inoculated in the culture medium and/or promote it
The composition of proliferation.The cell culture medium replenishers are suitable for secondarily supporting the in vitro training of the mankind or zooblast
It supports, preferably secondarily supports the in vitro culture of the mankind or animal origin mother cell, such as secondarily support the mankind true
The in vitro culture of hide fiber mother cell.The dosage of the aqueous extract of said herbal medicine in the medium, can be by the application
The those of ordinary skill of involved technical field determines via the conventional means of this field.Aqueous due to said herbal medicine extracts out
Object can replace animal blood serum some or all of in the medium function, so the dosage of animal blood serum in cell culture medium
It can be greatly reduced by the conventional amount used on the basis of the total volume according to the cell culture medium for 10% to according to described in
The animal blood serum of 10 volume %, preferably substantially below 5 volume % are substantially below on the basis of the total volume of cell culture medium
Animal blood serum, the more preferably substantially below animal blood serum of 1 volume %, for example, about animal blood serum of 0.5 volume %.
Therefore, the application also provides a kind of cell culture medium, it includes: aforementioned cells culture medium replenishers；An and base
Basal culture medium is supplemented on the basis of the total volume according to the cell culture medium the substantially below animal blood of 10 volume %
Clearly, the preferably substantially below animal blood serum of 5 volume %, the more preferably substantially below animal blood serum of 1 volume %, such as
The animal blood serum of about 0.5 volume %.Liquid or semi-liquid form is generally presented in the culture medium.
Term " basal medium " refers to any without containing animal blood serum but containing for supporting the mankind or zooblast
The fluid nutrient medium of the basic nutritions composition such as inorganic salts, amino acid and vitamin in vitro cultivating.It is preferably specific real one
It applies in scheme, the basal medium does not contain growth factor.It includes unlimited for being suitable for the invention the example of basal medium
In basal medium of Eagle (Basal Medium Eagles；BME), minimum essential medium (Minimum Essential
Medium；MEM), Dahl Burke Improved Eagle Medium (Dulbecco's Modified Eagle's Medium；
DMEM), nutrition mixed culture medium F-10 (HAM's F-10) and nutrition mixed culture medium F-12 (HAM's F-12).It is more excellent one
It selects in specific embodiment, the basal medium is DMEM.
The application provides a kind of method that cell is cultivated using prior culture media again, and it includes the following steps: (a) making institute
Cell is stated to be in contact with prior culture media；And (b) under conditions of being suitable for that the cell is supported in vitro cultivate, culture
The cell.Term used herein " contact ", which refers to, is loaded with cell merging in the culture vessel of culture medium.Make cell with
The means that culture medium is in contact belong to the conventional customary means of technical field involved by the application comprising but be not limited to make cell with
Culture medium is mixed, moves into the culture vessel for being loaded with cell culture medium with pipette, is dipped in cell Shen in culture medium.This
Locate used in term " culture " refer to be conducive to cell growth, survival and/or differentiation in vitro under the conditions of maintain cell.
In a preferred embodiment, the cell is mankind or animal cell.It is described in a more preferable specific embodiment
Cell is fibroblast.In a more preferable specific embodiment, the cell is human dermal's fibroblast.In vitro
The means of culture cell belong to the conventional technical means of technical field involved by the application.In general, the most thermophilic of zooblast
About 35 to 37 DEG C of degree.Deviate this temperature range, the eubolism of cell and growth will be affected, and even result in dead
It dies.For temperature at 43 DEG C or more, cell mostly can be dead.Low temperature is smaller to impact cell, cell is placed in 25 to 35 DEG C, cell is still
It can survive and grow, but speed is slow.General cell is incubated under constant temperature aerobic environment, and gives certain density dioxy
Change carbon, maintains the pH-value of culture medium.In a preferred embodiment, cell is trained in carbon dioxide incubator
It supports.The carbon dioxide incubator is normally set up in constant temperature (such as 37 DEG C), stable CO2Horizontal (such as 5%), constant acid
Basicity (such as pH7.2-7.4) and higher relative saturation humidity (such as 95%), to simulate the growth of cell in vivo
Environment.
The application by mankind's corium fabric mother cell primary as Screening Platform, wrinkle with anti-skin by further screening
The aqueous extract of the Chinese herbal medicine of line effect.It is generally believed that the generation of wrinkle is inducement (such as UV sunlight, perfume (or spice) in environment
The smog of cigarette and air pollution) free radical and active oxygen destroy point of collagen and elastin in the skin corium of skin
It is coupled between son, and causes the peroxidating of Cell membrane lipids.Therefore, proliferation of fibroblast can be promoted and promote its secretion
The aqueous extract of the Chinese herbal medicine of collagen has the potentiality that anti-wrinkle occurs.As shown in following Example 8, Duo Xuyan
The combination of astragalus and the aqueous extract of pheretima is cultivated fibroblast 6 days, and cell can be promoted to secrete collagen significantly,
Its effect even up to uses 2 to 3 times of secretory volume obtained by 10%FBS, to facilitate the consumption of collagen in reduction skin
Damage.It is worth it is specifically intended that the combination of more sequence rock astragalus and the aqueous extract of pheretima promotes the effect of cell secretion collagen
Fruit, obtained effect when being used alone much higher than more sequence rock astragalus and pheretima.This experimental result points out, Duo Xuyan astragalus water
Property extract and the aqueous extract of pheretima promote fibroblast secretion collagen on produce cooperative effect.Embodiment 7
It also shows, the combination of this compound has the effect of promoting Fibroblast Growth.Embodiment 11 further displays, this compound group
Conjunction can increase with H2O2Cell survival rate after processed.
Accordingly, the application also provides a kind of anti-wrinkle cosmetic formulations, it includes: the aqueous of Duo Xuyan astragalus extracts out
Object, the aqueous extract of pheretima and a cosmetic acceptability carrier." anti-wrinkle " referred to herein means pre- anti-wrinkle in skin
Occur and/or improve the wrinkle occurred on the skin on skin.It should be noted that " prevention " and " improvement " is not meant as described in application
The skin of anti-wrinkle cosmetic formulations never gives birth to wrinkle, and refers to compared to the skin for not applying the cosmetic formulations
It can have the less and or less obvious wrinkle of appearance.
On the other hand, spontaneous antioxidant molecule and ferment can gradually subtract as the age is gradually long in human skin
It is few, cause skin oxidation resistance can not fully erased environment free radical and active oxygen.In addition, superoxide dismutase (SOD)
It is that one kind can be catalyzed the enzyme that superoxides is converted into oxygen and hydrogen peroxide by disproportionated reaction, it is in fibroblast
A kind of primary anti-oxidant.Excessive environmental stimuli (such as UV sunlight), which not only may cause, generated polyradical,
May SOD be lost activity.These factors all lead to skin aging.The application is also by mankind's corium fabric mother cell primary
As Screening Platform, the aqueous extract of Chinese herbal medicine with resisting age of skin effect is filtered out.Firstly, the application is referring to Sha's support etc.
The data in literature of people and Qiu et al. form oxidative pressure for cell using hydrogen peroxide-induced, to set up cell senescence
Vitro model (Satoh, A.et al., J.Pharm.Pharmacol.57 (10): 1335-1343；And Choi,
M.J.et al.,Biol.Pharm.Bull.33(3):421-426).As shown in following Example 11, the aqueous extract list of pheretima
The combination of side and more sequence rock astragalus and the aqueous extract of pheretima, the combination of balsam pear and the aqueous extract of pheretima and balsam pear and
The combination of the aqueous extract of rhizoma alismatis, in H2O2The survival rate of cell can all be increased in the cell senescence model of induction.Embodiment 5 into
One step shows that the aqueous extract folk prescription of pheretima can also promote the activity of superoxide dismutase.These Chinese herbal medicines combination for
It maintains the survival of corium fabric mother cell and prevents its aging, there are potentiality very much.
The oxidative pressure that the aqueous extract of aforementioned Chinese herbal medicine can be formed resistive to hydrogen peroxide, it might even be possible to promote super oxygen
The activity of compound mutase has the potentiality of confrontation Skin Cell aging.Accordingly, it is old to provide a kind of confrontation skin again by the application
The cosmetic formulations of change, it includes: from the aqueous extract of one or more biological species, extract out selected from pheretima is aqueous
Object, the combination of the aqueous extract of Duo Xuyan astragalus and the aqueous extract of pheretima, the aqueous extract of balsam pear and the aqueous extract of pheretima
Combination and the aqueous extract of balsam pear and the aqueous extract of rhizoma alismatis combination；An and cosmetic acceptability carrier.
Aforementioned anti-wrinkle cosmetic formulations and to the cosmetic formulations of resisting age of skin can in it is any it is suitable in the form of,
Without specific limitation.In a preferred embodiment, the cosmetic formulations are that the solution for local application, cream is presented
The shape of the skin care products such as liquid, creams, gel, ointment, foaming agent, soap, eye shadow agent, lipstick, bath oil, shaving cream or cosmetics
Formula can be applied via coated on skin surface.In a preferred embodiment, the cosmetic formulations are to be in
Now for being injected in subcutaneous or intravenous transfusion form, such as suspension agent, venoclysis liquid, dry powder injection, suspension injection
Agent and dry powder suspension injection etc..
" cosmetic acceptability carrier " alleged by the application means to be used load as the aqueous extract of the Chinese herbal medicine
The inert substance of body does not have toxicity, irritation, Pyrogenicity, antigenic and haemolysis for the position (such as skin) of application
Property, and unsubstantial pharmacological activity, the performance of the beneficial effect of the aqueous extract of the Chinese herbal medicine will not be interfered.It is general next
Say, the dosage of cosmetic acceptability carrier be according to cosmetic formulations total weight on the basis of be about 1% to about 99.9%,
Preferably from about 50% to about 99%.Cosmetic acceptability carrier is applicable in type depending on the form of cosmetic formulations.
The following example is only for of the invention, the range being not intended to limit the present invention for example.Shown by each embodiment
Experimental result is to test (Student ' s t test) by Situ Deng Shi t, is made comparisons with control group, #:0.05 < p < 0.1；*: p
<0.05。
Preparation example 1: the Initial culture of human dermal's fibroblast
Dermatological specimens are obtained under the agreement of subject.The skin that dermatological specimens are cut into 0.5 to 1 mm cube size is broken
Block.Then, the merging of skin fragment is contained in the DMEM of FBS, Collagenase and neutral proteinase (dispase), in 37 DEG C
It is lower to cultivate 16 to 18 hours.Centrifugation 5 minutes is carried out with 1,500rpm.Then, supernatant is removed, it is thin to be supplemented with breaing up for FBS
Born of the same parents and skin chunk, and the skin chunk broken up and cell are uniformly distributed in conical flask, it is cultivated at 37 DEG C.Cell growth
Density then carries out cell subculture up to 80%.Aftermentioned experiment is carried out using the cell of the third generation to the 6th generation.
Preparation example 2: the preparation of Chinese herbal medicine extract
More sequence rock astragalus and rhizoma alismatis are purchased from traditional Chinese medicine shop.Pheretima is purchased from regional fishing articles shop.Balsam pear is purchased from
In regional fruit and vegetable market.
Sequence rock astragalus more than 100 grams is added in 500 grams of secondary deionized waters, is then boiled under normal pressure 2 hours.Weighing obtains
Sequence rock astragalus suspension more than 210 grams.
100 grams of rhizoma alismatis are added in 500 grams of secondary deionized waters, are then boiled under normal pressure 2 hours.Weighing obtains 242 grams
Rhizoma alismatis suspension.
100 grams of balsam pear fruits are added in 500 grams of secondary deionized waters, are then boiled under normal pressure 2 hours.Weighing obtains
150 grams of balsam pear suspension.
The complete polypide of 100 grams of pheretimas without processing is added in 500 grams of secondary deionized waters, is then boiled under normal pressure
2 hours.Weighing obtains 170 grams of pheretima suspension.
Above-mentioned various suspension are made to pass through gauze respectively to filter off impurity.Filter liquor is collected, is centrifuged 30 under 6000rpm
Minute.Supernatant is taken, makes it through the filter of 0.2 micron pore size, and weighing records.Filter liquor is collected, as each Chinese herbal medicine
The test stoste of extract, and with sterile serum bottle bottle cover for locking, it is placed in -20 DEG C of refrigerators and saves.In order to avoid the medium-height grass
Experimental result is influenced containing plurality of heavy metal in medicine extract, the content of beary metal of each Chinese herbal medicine extract is all after testing.
Testing result discovery, lead, mercury, the cadmium content of four kinds of Chinese herbal medicine extracts are all lower than detection lowest limit 2ppm (mg/kg).
In the examples described below, each Chinese herbal medicine extract stoste will dilute several times with control group culture medium, reuse dilute
Liquid is released to handle cell.For example, astragalus 100X refers to more sequence rock astragalus extract stostes by 100 times of control group culture medium
Obtained dilution after dilution.
Embodiment 1: cell is handled using extract folk prescription
Quantitative 1 gained fibroblast of preparation example is taken, is planted in 96 hole square position culture dishes, and made in preparation example 2
In the presence of the individual Chinese herbal medicine extracts obtained, it is incubated at containing 0.5% heat deactivation calf serum (FBS；New York, United States
Gibco company) Du Shi modified Eagle medium (DMEM；Missouri, USA Saint Louis city Sigma Chemical
Co.), 3 to 6 days are lasted.Control group is that preparation example 1 fiber cultivated using the DMEM containing 0.5% or 1.0%FBS is female thin
Born of the same parents.Supernatant and cell are collected, is analyzed respectively.
Embodiment 2: extract folk prescription promotes cell growth
With commercially available XTT assay kit (Missouri, USA Saint Louis city Sigma Chemical Co.) and reference
Kit guide carrys out 1 gained cell of Processing Example, and the reading in each hole in the culture dish of square position is then obtained in 450nm, obtains thin
Born of the same parents' survival rate.When Fig. 1 and Fig. 2 is shown respectively with astragalus, rhizoma alismatis and pheretima extract culture fibroblast 3 days and 6 days, all
It can be observed that the number of cell has significant increase compared to 0.5%FBS control group, wherein hyperplasia multiplying power is about at culture 3 days
1.2 times of 0.5%FBS control group are only reached, more may be up to 1.5 times or more of control group when cultivating 6 days.Using balsam pear extract
Example in, then culture 6 days after just remarkably promote proliferation of fibroblast.The present embodiment shows, used in this application 4 kinds
Chinese herbal medicine extract folk prescription all has the ability for promoting human dermal's proliferation of fibroblast.
Embodiment 3: effect of the extract folk prescription for fibroblast secretion collagen
With commercially available human recombinant I collagen type test kit (New York, United States Gibco company；Kit is compiled
Number PRO-479A) and take 1 gained supernatant of Processing Example referring to kit guide, obtain fibroblast in each experimental group
Collagen secretion amount.
Fig. 3 is shown with collagen secretion amount obtained by individual Chinese herbal medicine extract culture fibroblast 3 days.Compared to
0.5%FBS control group, astragalus extract all can significantly stimulate the collagen point of fibroblast under three experimental concentrations
The amount of secreting, and the obtained effect of 200X dilution is best.Rhizoma alismatis extract can significantly promote collagen when 4800X dilutes
Secretory volume.Pheretima extract can also significantly stimulate fibroblast to secrete collagen when 4000X and 2000X dilutes.It is bitter
Melon extract does not have the ability for stimulating fibroblast to secrete collagen under experimental concentration.Fig. 4 is shown with individual Chinese herbal medicines
Collagen secretion amount obtained by folk prescription culture fibroblast 6 days, wherein the only group of astragalus 200X and pheretima 8000X can
Remarkably promote the secretion of collagen.The present embodiment shows that the extract folk prescription collocation 0.5%FBS of astragalus and pheretima can promote
Collagen is secreted into fibroblast, facilitation effect is even more than 1.0%FBS effect achieved.
Embodiment 4: effect of the extract folk prescription for fibroblast secretion growth factor
With commercially available VEGF test kit (Minn. R&D Systems company；VEGF
ELISA Kit, number DY293B) and take 1 gained supernatant of Processing Example referring to kit guide, it obtains in each experimental group
The VEGF secretory volume of fibroblast.
Fig. 5 and Fig. 6 is shown respectively with VEGF obtained by individual Chinese herbal medicine extract culture fibroblasts 3 days and 6 days points
The amount of secreting.Compared to the influence that 0.5%FBS control group, Chinese herbal medicine extract folk prescription secrete fibroblast VEGF, whether
Culture 3 days or 6 days, is all not significantly different.
In the folk prescription test experiments of embodiment 1 to 4, astragalus extract effect when diluting 400X is best, and pheretima extracts out
Object effect when diluting 2000X is best, and rhizoma alismatis extract effect when diluting 4800X is best, and balsam pear extract is diluting
Effect is best when 2500X.Therefore, in subsequent embodiment, astragalus 400X, pheretima 2000X, rhizoma alismatis 4800X and balsam pear are used
2500X is tested.
Embodiment 5: the antioxidant activity of extract folk prescription
Using commercial reagent kit (Cayman chemical company, Michigan, United States Ann Arbor city (Cayman Chemical),
Kit number #706002) and take 1 gained supernatant of Processing Example referring to kit guide, obtain the super of each experimental group
Superoxide dismutase (SOD) activity.Promote SOD active as shown in fig. 7, pheretima 2000X has compared to control group significantly
Ability, this indicates that pheretima extract has the function of promoting cellular anti-oxidant capacity.
Embodiment 6: cell is handled using extract compound
Repeat embodiment 1 described in process, but by astragalus 400X, pheretima 2000X, rhizoma alismatis 4800X and balsam pear 2500X this four
Kind extract dilution, which is used alone, or combinations of pairs is for cultivating fibroblast lasts 3 to 6 days.Control group is to make
1 fibroblast of preparation example cultivated with the DMEM containing 0.5% or 1.0%FBS.Collect supernatant and cell, respectively into
Row analysis.
Embodiment 7: extract compound promotes cell growth
It handles and implements with commercially available XTT assay kit (Missouri, USA Saint Louis city Sigma Chemical Co.)
6 gained cell of example then obtains the reading in each hole in the culture dish of square position in 450nm, obtains cell survival rate.Fig. 8 and Fig. 9 are shown
With above-mentioned four kinds of extracts dilution culture fibroblast 3 days and 6 days, whether folk prescription or compound, can promote fibre
Tie up mother cell hyperplasia.When cultivating 3 days, each group all can promote about 1.5 times of proliferation of fibroblast or more, more up to 2 when cultivating 6 days
Times.The present embodiment is also shown, these four extract dilutions when cultivating fibroblast, will not be interfering with each other and inhibit cell
It generates.It is worth noting that, as shown in figure 9, when using astragalus+pheretima, astragalus+rhizoma alismatis+balsam pear, rhizoma alismatis+balsam pear+pheretima, Huang
The compounds such as over sixty years of age+balsam pear+pheretima and astragalus+rhizoma alismatis+balsam pear+pheretima combine can more make when cultivating fibroblast 6 days
The hyperplasia multiple of cell is more than 2 times, and compound combines obtained hyperplasia effect also compared with obtained by folk prescriptions individual in combination
The effect arrived is more preferable.Compared to 5% and 10%FBS is obtained as a result, the aqueous extract folk prescription of various Chinese herbal medicines or compound are taken
It also can achieve about 50% to 66.7% hyperplasia effect with 0.5%FBS.
Embodiment 8: effect of the extract compound for fibroblast secretion collagen
With commercially available collagen test kit (New York, United States Gibco company；Kit number PRO-479A) simultaneously
And take 6 gained supernatant of Processing Example referring to kit guide, obtain the collagen point of fibroblast in each experimental group
The amount of secreting.
Figure 10 and Figure 11 display be used alone or four kinds of Chinese herbal medicine extracts of combinations of pairs come cultivate fibroblast 3 days and
Collagen secretion amount obtained by 6 days.Compared to 0.5%FBS control group, the compound combination of astragalus and pheretima can make collagen egg
White secretory volume increases nearly 3 times, and facilitation effect is also substantially more than 1.0%FBS effect achieved.
Embodiment 9: effect of the extract compound for fibroblast secretion growth factor
With commercially available VEGF test kit (Minn. R&D Systems company；VEGFELISA
Kit, number DY293B) and take 6 gained supernatant of Processing Example referring to kit guide, it is female to obtain fiber in each experimental group
The VEGF secretory volume of cell.
Figure 12 and Figure 13 shows exclusive use or four kinds of Chinese herbal medicine extracts of combinations of pairs respectively to cultivate fibroblast 3
It and VEGF secretory volume obtained by 6 days.Compared to 0.5%FBS control group, Chinese herbal medicine extract compound is for fibroblast
The effect of VEGF secretion, whether cultivates 3 days or 6 days, is all not significantly different.
Embodiment 10: the antioxidant activity of extract folk prescription
Experiment flow described in embodiment 5 is repeated, but is used for 6 gained supernatant of Processing Example, obtains the SOD of each experimental group
Activity.Figure 14 display is used alone or four kinds of Chinese herbal medicine extracts of combinations of pairs are living come SOD obtained by cultivating fibroblast 3 days
Property.Compared to 0.5%FBS control group, the compound combination of each Chinese herbal medicine extract does not make significant difference for SOD activity.
Embodiment 11: anti-cell aging aptitude tests
Referring to the data in literature of Sha's support et al. and Qiu et al., establish test ageing resistance cell model (Satoh,
A.et al.,J.Pharm.Pharmacol.57(10):1335-1343；And Choi, M.J.et al.,
Biol.Pharm.Bull.33(3):421-426).First by 1 gained fibroblast kind of preparation example in 12 hole square position culture dishes
Overnight.After cell attachment is in culture dish, each Chinese herbal medicine extract dilution is added.After processing 72 hours, with 300 μM of H2O2Thorn
Swash 24 hours, induces cell and do sth. in advance aging.
It is observed in the present embodiment, in H2O2Under the influence of, the form of fibroblast can change, and attached situation of lying prone is poor, hair
Raw early hair aging (stress-induced premature senescence) of pressure induction property, and Apoptosis occurs
(apoptosis).Calculated cell number, which is dyed, with trypan blue (trypan blue) is shown in Figure 15.H is added2O2Before,
H is significantly relatively added in the cell number of 0.5%FBS control group2O2It is more afterwards.In advance with pheretima, astragalus+pheretima, rhizoma alismatis+balsam pear or hardship
Melon+pheretima extract is handled cell 72 hours, can significantly improve H2O2Caused phenomena of apoptosis.
Embodiment 12: effect of the extract to fibroblast characteristic
In order to confirm that Chinese herbal medicine will not generate change to the property of fibroblast, the present embodiment is with antibody Dyeing Example
6 gained fibroblasts, examine whether it can still give expression to -1 (fibroblast of fibroblast specific proteins
Specific protein 1, abbreviation FSP-1).As shown in figure 16, after with Chinese herbal medicine extract processing 3 days, each group fiber is female thin
Born of the same parents can all express this index albumen of FSP-1.Each Chinese herbal medicine extract folk prescription and compound combined treatment fiber are used in Figure 17 display
After mother cell, the percentage of FSP-1, the i.e. purity of fibroblast are expressed in cell.The results show that the processed fiber of each group
Mother cell can express the cell of FSP-1 all 95% or more, indicate that Chinese herbal medicine extract folk prescription or compound combination all will not influence
The characteristic of corium fabric mother cell expression FSP-1 primary.This points out that the fundamental characteristics of fibroblast will not be extracted out by Chinese herbal medicine
The influence of object and change.
The above various embodiments is only for illustrating the present invention, and not limitation of the present invention, those skilled in the relevant art,
It does not depart from various transformation that technical scope of the invention is made or variation also should belong to protection category of the invention.
Claims (10)
1. a kind of cell culture medium replenishers, it is characterised in that:
The replenishers include the aqueous extract from one or more biological species, and it is yellow that the biological species are selected from more sequence rocks
Over sixty years of age (Hedysarum polybotrys Hand.-Mazz.), rhizoma alismatis (Alisma plantago-aquatica (Sam.)
Juzep), balsam pear (Momordica charantia L.) and pheretima (Eisenia foetida).
2. cell culture medium replenishers according to claim 1, wherein the aqueous extract be selected from more sequence rock astragalus and
Combination, Duo Xuyan astragalus and the rhizoma alismatis of the aqueous extract of pheretima and combination, rhizoma alismatis and balsam pear and the pheretima of the aqueous extract of balsam pear
The combination of combination, Duo Xuyan astragalus and the balsam pear and the aqueous extract of pheretima of aqueous extract and more sequence rock astragalus and rhizoma alismatis
With the combination of balsam pear and the aqueous extract of pheretima.
3. cell culture medium replenishers according to claim 2, wherein the aqueous extract be selected from more sequence rock astragalus and
The combination of the aqueous extract of pheretima.
4. a kind of cell culture medium, it is characterised in that:
The culture medium includes the cell culture medium replenishers such as any one of claim 1-3；And a basal medium,
The substantially below animal blood serum of 10 volume % is supplemented on the basis of the total volume according to the cell culture medium.
5. cell culture medium according to claim 4, wherein the basal medium is supplemented with according to the cell culture
The animal blood serum of 5 volume % is substantially below on the basis of the total volume of base.
6. cell culture medium according to claim 5, wherein the basal medium is supplemented with according to the cell culture
The animal blood serum of 1 volume % is substantially below on the basis of the total volume of base.
7. cell culture medium according to claim 6, the basal medium is supplemented with according to the cell culture medium
It is the animal blood serum of about 0.5 volume % on the basis of total volume.
8. cell culture medium according to claim 4, wherein the cell is fibroblast.
9. cell culture medium according to claim 8, wherein the cell is human dermal's fibroblast.
10. a kind of anti-wrinkle cosmetic formulations, it is characterised in that:
The cosmetic formulations include the aqueous extract and a cosmetics of the aqueous extract of more sequence rock astragalus, pheretima
Acceptable carrier.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021133309A1 (en) * | 2019-12-26 | 2021-07-01 | Gazi Universitesi | Red californian worm extract and the use thereof |
| CN112569158A (en) * | 2021-01-06 | 2021-03-30 | 湖南秋之韵生物技术有限公司 | Traditional Chinese medicine mask for removing freckles and whitening |
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