CN101374496A - Use of a composition containing an ecdysteroid - Google Patents

Abstract

The invention concerns the use in cosmetics, pharmaceutics or as nutritional supplement, of a composition containing an ecodysteroid, an ecodysteroid derivative, or a natural plant or animal extract containing same, to stimulate the natural defences of cells, such as protein p53, against the consequences of their being exposed to ultraviolet rays in particular solar radiation.

Description

The purposes that comprises the compositions of ecdysteroid

The present invention relates to comprise ecdysteroid, ecdysteroid derivant or contain its natural plants or the purposes of the compositions of animal extracts, described purposes is the natural defence p53 albumen for example of chafe and mucomembranous cell, with antagonism owing to being exposed to the consequence that ultraviolet radiation causes.

Ecdysteroid is to carry 7-alkene-6-ketone groups and the alpha-hydroxy polyhydroxy steroid of 14-, and it is cis and connects between A ring and B ring.It is the hormone of insect molting, yet also usually finds (Lafont R., Chem Listy 97,280-281,2003) in plant.

Many effects for mammal (comprising the people) are confirmed: the growth of-irritation cell, breeding, propagation and differentiation, the metabolism of stimulating protein, saccharide and lipid, the system that excites nerve and cardiovascular system, the Detoxication of stimulation liver and kidney;

-antiinflammatory and immunomodulatory effect;

-antibiosis effect (Lafont R. and Dinan L.Journal of Insect Science3:7,30pp, 2003, can onlinely consult: insectscience.org).

Document CN-86 01 06791-A relates to the cosmetology product that comprises β-ecdysone, and it can prevent skin aging, keeps the moisture of skin and eliminate acne and brown macules.

Document WO-90/03778-A relates to and comprises for example compositions based on aquation lamellar phase or liposome of ecdysteroid, and said composition especially can be as promoting healing or anti-wrinkle composition to use.

Document WO-94/04132-A relates to and comprises for example beauty treatment or the dermatological compositions of ecdysteroid, and said composition especially is intended to make skin surface Paint Gloss and soft, strengthens the function of the water barrier of skin, and the compactness that increases epidermis cell.

Document FR-2801504-A relates to the extract of the Xinjiang Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.) plant that comprises at least a ecdysteroid and at least a iridoid, and the cosmetic composition that comprises this extract, to be used to improve the differentiation of keratinocyte, perhaps regulate the discharge in the epidermis and the heavily absorption of water, perhaps make the epidermis aquation.

Patent US-6641848 relates to the compositions of the associating that comprises triterpene saponin and ecdysteroid, to be used for the synthetic of chafe IV Collagen Type VI.

Document FR-2863378-A relates to ecdysteroid and is used to prepare the beauty treatment of anti-alopecia or the purposes of dermatological compositions.

The part that document FR-863886-A relates to the ecdysteroid receptor is used for keeping the purposes of the homeostatic compositions of skin in preparation.

In addition, ecdysterone also is widely used in sericulture, and being used to control casting off a skin of silkworm, and it has also entered the composition that very many being used for improve the food supplement of muscle quality (people such as Lafont R., 2003, quote).

On the other hand, skin is exposed to the damage that ultraviolet radiation causes the DNA that exists in skin and the mucomembranous cell repeatedly, especially in being called fibroblastic cell, this cell is responsible for upgrading the macromole (people such as Goukassian D. of the extracellular matrix that constitutes corium, FASEB J., 14:1325-34,2000).

These damages can be repaired natively.But, when failing to repair, just may produce serious consequence and even may cause photic cancer (Goukassian D.A. and GilchrestB.A., Rejuvenation Res.7 (3): 175-85,2004) by pair cell.

In addition, people think always that for a long time the cell of the skin that is exposed to sunlight can lose the ability (Gilchrest B.A., J Gerontol., 35 (4): 537-41,1980) of its breeding quickly.

No longer the cell that can breed is called old and feeble cell, it is characterized by great expression and have active beta galactosidase when pH6.With (people such as Dimri G.P. in culture through the cell of continuous passage and ageing, Proc.Nat.Acad.Sci.USA92:9363-67,1995) or experience repeatedly ultraviolet stress culture in (people such as Chainiaux F., Int.J.Biochem.Cell.Boil.34 (11): 1331-9,2002) the same, people are at the keratinocyte and the middle cell of also having found more this beta galactosidase of expression of fibroblast of old people's skin.

In addition, show (people such as Debacq-ChainauxF., J.Cell.Sci.118 (Pt4): 743-58 recently, 2005), fibroblast with the age increase can a forfeiture part its repair by the ability of the radiation-induced damage of ultraviolet (UV) (people such as GoukassianD., 2000, quote).

Therefore, obviously, making us very interested is can have the means of the natural reparation that is used to stimulate the Skin Cell damage that is caused by ultraviolet radiation, so that postpone the appearance of the senile cell of forfeiture fertility.

Therefore, one of purpose of the present invention is exactly the natural defence proteic means of p53 for example that are provided for irritation cell, with antagonism owing to be exposed to the Skin Cell that ultraviolet radiation causes and damage.

This purpose and other purposes that occur subsequently, by so reaching, promptly use natural plants or the natural defence that comes chafe and mucomembranous cell of the compositions of animal extracts comprise ecdysteroid, ecdysteroid derivant or to contain it, with antagonism owing to being exposed to the consequence that ultraviolet radiation causes.

Advantageously, be ecdysterone according to ecdysteroid of the present invention.

Preferably, described ecdysteroid is a derivative form, and for example single or many acyl groupizations are derivant, acetonide, the phosphate ester of acetyl groupization especially.

Advantageously, employed ecdysteroid is also contained in for example darkish blue Auricled Hedyotis Herb of the spider's thread (Cyanotisarachnoidea) of plant extract, blue Auricled Hedyotis Herb (Cyanotis vaga), Xinjiang Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.) (Ajuga turkestanica), her watt Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.) (Ajuga iva), ajuga decumbens (Ajuga decumbens), Radix Serratulae coronatae (Serra tulacoronata), deer grass (Leuzea (or Rhaponticum) carthamoides), Rhizoma Paridis (Parispolyphylla), South America Herba Amaranthi tricoloris (Pfaffia paniculata), Pfaffia iresinoides, Pfaffia stenophylla, adder's-fern (Polypodium vulgare), Polypodiumaureum, Polypodium lepidopteris, yellow snow wheel (Silene otites), Radix Cyathulae (Cyatula officinalis), headdress flower Herba Cyathulae Prostratae (Cyatula capitata), Radix Achyranthis (Achyrantes aspera), bidentate Radix Achyranthis Bidentatae (Achyrantes bidentata), Herba Spinaciae (Spinacia oleracea), in the extract of quinoa (Chenopodium quinoa), perhaps be included in animal extracts for example in the extract of silkworm (Bombyx mori).

According to the present invention, described compositions is also drawn together and is comprised:

-at least a antioxidant, for example vitamin E, vitamin C, carotenoid, flavone compound, isoflavonoid, polyphenol, procyanidin, resveratrol, epigallocatechin gallate (EGCG), genisteine, Daidezin, formononetin, equol, green tea extract.

-at least a anti-inflammatory substance, for example Glycyrrhiza glabra L. (Glycyrrhiza glabra) extract, enoxolone.

Preferably, be used to stimulate the reparation of the cell injury that skin is caused owing to be exposed to ultraviolet according to Dermatology of the present invention or cosmetic composition, it comprises 0.001 to 95 weight % and the ecdysterone of 0.01 to 1 weight % more accurately.

Advantageously, more particularly said composition is coated on the skin.It also can by oral route be used, for example with the form of food supplement.

Preferably, utilize said composition to come the proteic activity of p53 in chafe or the mucomembranous cell.

But, be all beyond one's expectations now and discovery surprisingly, if before shining, come the fibroblastic culture medium that experiences repeatedly uv light exposure is replenished by adding ecdysteroid, just can reduce the number of the cell that enters aging state very significantly, described aging state is characterised in that and associated betagalactosidase activity (" old and feeble relevant beta galactosidase " or " SA β-gal ") occurs.

Below do not show the description of any qualification characteristic, relate to some embodiments of the present invention especially, these embodiment make those skilled in the art can understand the present invention better.

Employed ecdysteroid (in this case in embodiment below and the compositions, ecdysterone) is included in for example darkish blue Auricled Hedyotis Herb of the spider's thread of plant extract, blue Auricled Hedyotis Herb, the Xinjiang Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.), her watt Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.), ajuga decumbens, Radix Serratulae coronatae, the deer grass, Rhizoma Paridis, the South America Herba Amaranthi tricoloris, Pfaffia iresinoides, Pfaffia stenophylla, adder's-fern, Polypodiumaureum, Polypodium lepidopteris, the yellow snow wheel, Radix Cyathulae, the headdress flower Herba Cyathulae Prostratae, Radix Achyranthis, the bidentate Radix Achyranthis Bidentatae, Herba Spinaciae, in the extract of quinoa, perhaps be included in animal extracts for example in the extract of silkworm.

Embodiment 1:

Weaken owing to be exposed to fibroblastic damage that ultraviolet radiation causes repeatedly by ecdysteroid for cultivation.

Used ecdysteroid is the ecdysterone sample of purity greater than the darkish blue Auricled Hedyotis Herb extract of 90% spider's thread, is provided by Kunming plant research institute (Chinese yunnan Kunming).

Cultivate the fibroblast of skin by traditional method, its be with the used same strain of people (2002, quote) such as Chainiaux F be BJ.

Verified, in the scope of 0.4-250 μ g/ml, in 24 hours, the ecdysterone no cytotoxicity.

In 5 days, it is 500mj/cm that fibroblast is exposed to dosage once a day ²UV-B in.

After each irradiation, described culture medium is replaced with the culture medium that contains (0.4), 2,10,50 and 250 μ g/ml ecdysterones.

Before the each new irradiation, these culture medium are replaced with the culture medium that does not contain ecdysterone.

After the irradiation, allow cell contact 72 hours the last time, per 24 hours renewal one subcultures with the culture medium that is supplemented with ecdysterone.

At this when last time of contact, section finished, method (quoting) according to people such as Dimri GP, by colorimetry and by the fluorescence on BioAnalyseur, estimate the activity (" old and feeble relevant beta galactosidase " or " SA β-gal ") of beta galactosidase under the pH6, each ecdysterone concentration is surveyed 400 cells at least.

The meansigma methods of the percentage of cells of expression SA β-gal is as follows:

Condition	Colorimetry	Bioana lyseur
			Contrast (no UV)	29.1	45.7
UV+0 μ g/ml ecdysterone	41.9	52.5
			UV+0.4 μ g/ml ecdysterone	47.6	/
UV+2 μ g/ml ecdysterone	50.5	55.1
			UV+10 μ g/ml ecdysterone	37.5	49.2
UV+50 μ g/ml ecdysterone	31	39.9
			UV+250 μ g/ml ecdysterone	28	31

As can be seen:

-causing presenting the active fibroblast of SA β-gal and significantly increase owing to being exposed to damage that UV-B causes repeatedly, described SA β-gal activity is the feature (being called " aging ") of the cell that no longer can breed.

-after irradiation, handle with the ecdysterone pair cell, make it possible to weaken by UV-induced damage since the concentration of 10 μ g/ml, and from 50 μ g/ml, effect is then more remarkable, and this shows as " old and feeble " fibroblastic number and keeps identical with undosed culture or still less.

Therefore it seems,, use ecdysteroid to make it possible to weaken well owing to being exposed to the cell injury that ultraviolet radiation causes according to the present invention.

Embodiment 2:

Carried out the experiment identical, wherein only mixed ecdysterone convalescent the time at 5 times postradiation 72 hours with embodiment 1.

Measure for betagalactosidase activity and to have provided following result:

Condition	Beta galactosidase by colorimetric method for determining
		Contrast UV B+0 μ g/ml ecdysterone UV B+50 μ g/ml ecdysterone UV B+250 μ g/ml ecdysterone	13.4±144.0±634.8±346.3±4

Therefore as can be seen, a series of be exposed to the ultraviolet light beta radiation after, the ecdysterone strictness has not had recovery dynatron effect in fact.

Embodiment 3:

Carried out the experiment identical, but in cell culture medium, mixed ecdysterone during back 72 hours in preceding 24 hours (comprising pre-irradiation for the first time) of each UV-B irradiation and 5 irradiations with embodiment 1.

Measure for betagalactosidase activity in the cell and to have provided following result:

Condition	Beta galactosidase by colorimetric method for determining
		Contrast contrast+UV 50 μ g/ml ecdysterones 50 μ g/ml ecdysterone+UV 250 μ g/ml ecdysterones 250 μ g/ml ecdysterone+UV	15.8±431.6±412.1±216.5±214.0±210.2±2

Therefore as can be seen, be processed into the appearance that fibrocyte makes it possible to reduce senile cell at the UV pre-irradiation with ecdysterone.

Embodiment 4:

In having experienced by cell the damage of contained DNA caused stress after, cell-stimulating p53 albumen (being called as " genome bodyguard "), it induces the repair process (referring to people such as de Magal haes J.P., Expert Gerontol.39 (9): 1379-89,2004) of initial damage.

Estimated the stimulation of expressing at p53 in the fibroblast of the embodiment 1 that embodiment 1, experienced uv light exposure repeatedly: each expose preceding 24 hours and last the exposure after, in culture medium, add 250 μ g/ml ecdysterones.

The processing of the use 250mg/l ecdysterone that first " contrast " culture experience is identical, but do not have ultraviolet radiation.

The uv light exposure that second " contrast " culture experience is identical with treated culture, but do not have ecdysterone fully.

Irradiation is 6 hours afterwards the last time, is extracted into fibrocellular protein and measures its content with system of Pierce company " Micro BCA test kit ".Then, in microtest plate, hatch 5 μ g nucleoproteins with oligonucleotide bag quilt, described oligonucleotide carry can with the bonded specific DNA sequences of transcription factor p53.

At last, by this proteinic specific antibody and be conjugated with the antibody of peroxidase (TransAM de Active Motif test, Rixensart Belgique), measures the proteic content of combined p53 by colorimetry.

The gained result is as follows:

Condition	P53 is last to be shone back 6 hours (absorbance unit with the 450nm place is represented)
		0 μ g/ml ecdysterone, 250 μ g/ml ecdysterones, 0 μ g/ml ecdysterone+UV, 250 μ g/ml ecdysterone+UV	1 6 9 16

Therefore, find surprisingly that when not having UV, ecdysterone is handled and can be made fibroblastic p53 protein content increase to 6 times.

Therefore also proved conclusively, when not having ecdysterone, be exposed to ultraviolet and make fibroblastic p53 protein content increase to 9 times.

At last, show, in the presence of ecdysterone, be exposed to the expressed p53 albumen of ultraviolet fibroblast 16 times for both unprocessed also unexposed " contrast " cells.

This embodiment makes it possible to prove surprisingly that ecdysterone has stimulated the p53 Buchner's bodies, and this protein is to prevent that the damage that is caused by ultraviolet radiation is necessary, for example prevents to lose the appearance of the senile cell of fertility.

Compositions 1:

Stimulate the beauty treatment cream of natural defence.

In order to prepare traditional O/w emulsion, so carry out, promptly under agitation mix hydrophobic phase and water in 85 ℃, wherein contain the various raw materials of this product with conventional operating characteristic that are used to give, these characteristics for example are stability, safety, easily coating, happy final sense of touch or the like.

Under agitation cool off, and when temperature is reduced to 45 ℃, add the ecdysterone of 0.1 weight %.

Preferably in the morning this cream is applied to face, cervical region and hands, needs the time,, be among the contact so that at noon when sun UV exposure rate reaches maximum because stimulate the composition of natural defence to infiltrate through in the living cells.

Compositions 2:

Stimulate natural defence and have the beauty treatment cream of anti-oxidation protection.

Be prepared as follows the O/w emulsion that comprises alpha-tocopherol and alpha-tocopherol acetate; And when being cooled to 45 ℃, add ecdysterone and phosphoric acid vitamin C, present in an amount at least sufficient to reach following concentration by weight in final composition:

-ecdysterone 0.1%

-alpha-tocopherol 0.05%

-alpha-tocopherol acetate 0.2%

-phosphoric acid vitamin C, magnesium salt 0.2%.

Said preparation is a day cream.

Compositions 3:

Stimulate natural defence and have the beauty treatment cream that filterability is protected.

As prepare compositions 1, preparation is added with the O/w emulsion of titanium oxide, then in 45 ℃ under agitation, add the absolute alcohol extract of deer grass by 2 weight %.Protection coefficient S PF equals 6.

This product is used for being applied in unsheltered skin part every morning, and if possible, should use again at noon.

Compositions 4:

The beauty-care gel that is used for the natural defence of stimulation of responsive type skin.

Preparation aqueous acrylamide acid gel, wherein ecdysterone content is 0.1 weight %, and comprises water glycol (hydroglycolique) extract of the Radix Scutellariae (Scutellaria baicalensis) of 2 weight %.

Compositions 5:

Stimulate the Dermatology gel of natural defence.

Preparation contains the aqueous acrylamide acid gel of 0.2 weight % ecdysterone.

The skin part that this gel was applied to expose in morning or daytime.

Compositions 6:

Food supplement, it is used for the natural defence of chafe before being exposed to sunlight fully.

Adopt conventional art, prepare the tablet that dosage is 15mg ecdysterone/sheet from the powder formulation that comprises 20 weight % ecdysterones.

Take with low amounts of water when described tablet is had meal with noon in the morning, wherein, take 1 to 2 at every turn according to the intensity of exposing to sunlight and the persistent period of expection.

Compositions 7:

Food supplement, it is used for the natural defence of chafe before being exposed to sunlight fully.

The preparation capsule, the solid (promptly about 10 weight %) that it comprises the darkish blue Auricled Hedyotis Herb extract of the 15mg spider's thread (ecdysterone that contains about 90 weight %) and is made of gelatin shell.

Described capsule is taken as the tablet of compositions 6.

Compositions 8:

Food supplement, it is used to make skin ready for being exposed to sunlight.

Preparation comprises the capsule of powder formulation, and it contains 20 weight % ecdysterones (dosage is the 10mg ecdysterone), 15mg green tea extract, 25mg vitamin C/capsule.

The daily dose of suggestion is 2-4 capsules every day, takes at twice, takes with low amounts of water when morning and noon have meal.

Compositions 9:

Beverage, it is used for the natural defence of chafe before being exposed to sunlight fully.

Preparation comprises the beverage based on bean milk of 20 weight % Herba Spinaciae concentrated juice and 0.05 weight % ecdysterone.

This beverage gives with the dosage of 100ml.

Potion is respectively drunk in suggestion when having meal with noon in the morning.

Claims (10)

1. comprise ecdysteroid, ecdysteroid derivant or contain its plant or the purposes of the compositions of animal extracts, described purposes is to be used for the consequence that the natural defence of irritation cell causes in ultraviolet radiation owing to cellular exposure with antagonism.

2. the purposes of claim 1 is characterized in that, described ecdysteroid is an ecdysterone.

3. the purposes of claim 1 is characterized in that, described ecdysteroid is a derivative form, and for example single or many acyl groupizations are derivant, acetonide, the phosphate ester of acetyl groupization especially.

4. the purposes of claim 1, it is characterized in that, employed ecdysteroid is included in for example darkish blue Auricled Hedyotis Herb of the spider's thread of plant extract, blue Auricled Hedyotis Herb, the Xinjiang Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.), her watt Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.), ajuga decumbens, Radix Serratulae coronatae, the deer grass, Rhizoma Paridis, the South America Herba Amaranthi tricoloris, Pfaffiairesinoides, Pfaffia stenophylla, adder's-fern, Polypodium aureum, Polypodium lepidopteris, the yellow snow wheel, Radix Cyathulae, the headdress flower Herba Cyathulae Prostratae, Radix Achyranthis, the bidentate Radix Achyranthis Bidentatae, Herba Spinaciae, in the extract of quinoa, perhaps be included in animal extracts for example in the extract of silkworm.

5. each purposes in the claim 1 to 4 is characterized in that, described compositions also comprises:

-at least a antioxidant, for example vitamin E, vitamin C, carotenoid, flavone compound, isoflavonoid, polyphenol, procyanidin, resveratrol, epigallocatechin gallate (EGCG), genisteine, Daidezin, formononetin, equol, green tea extract.

-at least a anti-inflammatory substance, for example Glycyrrhiza glabra L. extract, enoxolone.

6. the purposes of claim 2 is characterized in that, described compositions comprises the ecdysterone of 0.001-95 weight %.

7. the purposes of claim 6 is characterized in that, described compositions comprises the ecdysterone of 0.01-1 weight %.

8. each purposes in the claim 1 to 7 is characterized in that, described compositions is used for topical application.

9. each purposes in the claim 1 to 7 is characterized in that, described compositions is used to eat.

10. each purposes of claim 1 to 7 is characterized in that the proteic activity of p53 in described compositions chafe or the mucomembranous cell.