CN106466323A - Treated using isorhamnetin-3-O-β-D-glycopranoside and/or pre- antiradiation injury - Google Patents

Abstract

The present invention discloses isorhamnetin-3-O-β-D-glycopranoside and can be used to treatment and/or pre- antiradiation injury.

Description

Treated using isorhamnetin-3-O-β-D-glycopranoside and/or prevent Radiation damage

Technical field

The invention relates to using isorhamnetin-3-O-β-D-glycopranoside (isorhamnetin-3-O- β-D-glucoside) is treating and/or pre- antiradiation injury (radiation injury).

Background technology

Radiation (radiation) refers to that energy is worn in the form of ripple (waves) or particle (particles) Get over space (space) or a material medium (material medium) to launch or to transmit.Typically For, radiation can be classified as：(1) dissociate radiation (ionizing radiation), and it is to carry High-energy and can be by the electromagnetic wave of atom (atoms) or molecule (molecules) freeization (electromagnetic waves) [such as X-ray (X ray) and gamma ray (gamma Ray)] or particle [such as alpha-particle (alpha particle), beta-particle (beta particle) and Neutron (neutron)]；And (2) non-free radiation (non-ionizing radiation), it is band Have more low-yield and cannot be by electromagnetic wave [the such as visible ray of atom or dissociating of molecule (visible light), infrared ray (infrared), ultraviolet (ultraviolet, UV) and microwave (microwave)].DNA damage (DNA can be caused when organism is exposed to radiation Damage), so produce gene mutation (gene mutation) and induction apoptosis (apoptosis) with cell death (cell death), finally result in radiation damage (radiation Injury) [free radiation damage (ionizing radiation injury) and non-free spoke are included Penetrate damage (non-ionizing radiation injury)].

Free radiation damage can be according to degree (the degree of radiation of radioactive exposure Exposure) it is divided into：

(1) [be otherwise known as acute radiation syndrome (acute radiation syndrome, ARS) radiation Poisoning (radiation poisoning) and Radiation sickness (radiation sickness)], it is Because the whole body (whole body) of human individual is exposed to, to last one under free radiation short Time (for example, 24 hours) is caused, and its symptom can be divided into following 3 big class： Related symptom [the such as hypoplasia with hemopoietic system (hematopoietic system) Property anemia (aplastic anemia)] and gastronintestinal system (gastrointestinal system) Related symptom [for example nauseous (nausea) and vomiting (vomiting)] and with god Menses guard system (neurovascular system) related symptom is [for example dizzy (dizziness)]；

(2) chronic radiation syndrome (chronic radiation syndrome, CRS), it is due to people The individual systemic exposure of class last under free radiation one long-time (for example, several moons or Several years) caused, its symptom includes atrophoderma (skin atrophy), cataract And infertile (sterility) etc. (cataract)；And

(3) side effect (side effect of radiation therapy) of radiation therapy, it is due to people The individual specific part (specific part) of class is exposed to caused by under free radiation, it Symptom include anorexia (anorexia), (lassitude) tired out, diarrhoea (diarrhea), red Speckle (erythema), desquamation (desquamation), intestinal stenosis (bowel stenosis), bone Necrosis (necrosis of bone) and lung fibrosiss (fibrosis of lung) etc..

Clinically it is used for the radioprotectant for the treatment of and/or pre- antiradiation injury at present (radioprotective agent) includes：Free radical scavenger (free radical Scavenger) [such as catalase (catalase)], antioxidant (antioxidant) (such as vitamin E), cytohormone (cytokine) [be for example situated between plain -1 (interleukin-1) in vain], mercaptan (thiol) [such as amifostine (Amifostine)] and steroid (steroid) [such as 5- androstane Enediol (5-Androstenediol)], wherein amifostine is unique via U.S.'s food and medicine The radiation protection that thing management board (Food and Drug Administration, FDA) is approved Agent.Although amifostine can effectively protective tissue to Antiradiation injury, it can be right User produces serious side effect (side effect) [such as nauseous (nausea), vomiting (vomiting) and hypotension (hypotension)].Therefore, the related researcher of this area Attempt being available for find from Chinese medicine (traditional Chinese medicines, TCM) Active component (active component) for treatment and/or pre- antiradiation injury.

Fructus Hippophae (Classification system：Hippophae rhamnoides L.；English popular name： sea-buckthorn；The Chinese phonetic alphabet：sha j i；Chinese nickname：Fructus hippophae or acid thorn) it is recklessly ruined The machaka of scarabaeidae (Elaeagnaceae) Hippophae (Hippophae), producing region is distributed mainly on The areas such as Hebei China, Shanxi, Shaanxi, Gansu and Qinghai.Study and pointed out, husky Spine can be used to treat hepatic injury (liver injury), gastric ulcer (gastric ulcer) and tumor (tumor) etc. (Cheng T.J. (1992), Zhonghua Yu Fang Yi Xue Za Zhi, 26:227-229；Xing J.et al.(2002),Fitoterapia,73:644-650； Yasukawa K.et al.(2009),Fitoterapia,80:164-167).

In recent years, the extract being related to Fructus Hippophae is at radiation protection (radioprotection) aspect Research have started to receive publicity.For example, in Chawla R.et al. (2007), J.Med. Food,10:In 101-109, the Fructus Hippophae by drying and through powdered for the Chawla R. et al. Berry extracted with ethanol (ethanol), then filtered and concentrated, then By the ethanolic extract being obtained with hexane (hexane) and ether (ether) to clean in order to Remove nonpolar differentiator (nonpolar fraction), then contain acetic acid second by using one [their relative scale is 2 for ester (ethyl acetate) and methanol (methanol)：3(v/v)] Silica gel bed (bed of silica gel) to carry out purification to remaining differentiator.Then, by Contain low pole aggretion type adsorbent resin (weakly polar polymeric using one Adsorbent resin) tubing string the partially purified differentiator being obtained is carried out second Purification, is connect and is eluted with 20-80% ethanol and obtained the extract through distinguishing (fractionated extracts).Afterwards, the collected extract through distinguishing is closed And (pooling) and concentration, obtain a differentiator being rich in flavonoid whereby (flavonoid-rich fraction) (it is named as REC-1001).Then, REC-1001 Via high-effect liquid chromatography (LC) (high performance liquid chromatography, HPLC) analyze and be found to have and bear non-flavonol (kaempferol), isorhamnetin and Mongolian oak The compositions such as Pi Su (quercetin).REC-1001 is brought further carries out Analysis on Biological Activity, And experimental result confirms that it has antioxidation, removes free radical and radiation proof activity. Chawla R. et al. thinks accordingly：Contained by REC-1001 may non-flavonol, different Mus Li Su and Quercetin give it and have these biological activitys, therefore can be used as a kind of safety And effective antioxidation nutriment (antioxidant nutraceutical product).

Isorhamnetin (isorhamnetin) is that one kind is present in medicinal plants [such as halberd leaf knotweed (Persicaria thunbergii), Fructus Hippophae (Hippophae rhamnoides L.) and Semen Brassicae Campestriss (Brassica campestris L.)] in flavonoid (flavonoid), it has following chemical formula (I)：

Be currently known isorhamnetin have prevention endothelial cell damage (endothelial cell injury), Anti-tumor (anti-tumor), anti-lipogenesis (anti-adipogenesis) and treatment enteritis Effectiveness (enteritis) (Bao M.and Lou Y. (2006), Eur.J.Pharmacol., 547:22-30；Teng B.S.et al.(2006),Pharmacol.Res.,54:186-194； Lee J.et al.(2010),Life Sci.,86:416-423；CN 103462957A).

Isorhamnetin candy glycosides (isorhamnetin glycosides) is that one kind of isorhamnetin derives Thing.Common isorhamnetin candy glycosides includes isorhamnetin-3-O-β-D-glycopranoside (isorhamnetin-3-O- β-D-glucoside), isorhamnetin -3-O- β-D- rutinoside (isorhamnetin-3-O- β-D-rutinoside) and Isorhamnetin- 3- O- galactoside (isorhamnetin-3-O-galactoside) etc..Isorhamnetin-3-O-β-D-glycopranoside can It is separated from Fructus Hippophae, bend wood (Cochlospermum religiosum) and Semen Brassicae Campestriss, It is a kind of flavonoid glucoside (flavonoidal with following chemistry formula (II) glucoside)：

Study and pointed out, isorhamnetin-3-O-β-D-glycopranoside has had treatment tumor and glycosuria Disease, preventing liver injury and the effectiveness (CN delaying selenite induced catatract (selenite cataract) 1518986 A；Lee Y.S.et al.(2005),Biol.Pharm Bull.,28:916-918； Igarashi K.et al.(2008),Biosci.Biotechnol.Biochem.,72:856-860； Devi V.G.et al.(2010),Toxicol In Vitro.,24:1662-1669).

With regard to known to applicant, there is no any document or patent application once to disclose different Fructus rhamni (Rhamnus davurica Pall.) so far Element -3-O- β-D-Glucose glycosides can be used to treatment and/or pre- antiradiation injury.

Content of the invention

Then, in the first aspect, the present invention provides isorhamnetin -3-O- β-D-Glucose Glycosides for be applied to prepare one for treat and/or pre- antiradiation injury pharmaceuticals purposes.

In second aspect, the present invention provides and a kind of have for treatment one or under a cloud have The method of the individuality of radiation damage, it includes this individuality is offerd medicine with isorhamnetin - 3-O- β-D-Glucose glycosides.

At the 3rd aspect, the present invention provides isorhamnetin-3-O-β-D-glycopranoside supply It is used for the purposes of radiation proof cosmetics for preparation one.

Brief description

Below in conjunction with the accompanying drawings and embodiment comes that the present invention is described in detail, so the present invention In above-mentioned and other purposes and feature, can be by with reference to following description, with appended by literary composition Claims and accompanying drawing and become apparent, in accompanying drawing：

Fig. 1 shows that each group cell culture of BNL CL.2 cell is analyzed by population of cells Measured survival rate, wherein " * " represent：When making comparisons with X-ray matched group 3, p ＜ 0.05；

Fig. 2 shows that each group cell culture of NMuMG cell analyzes institute by population of cells The survival rate recording；

Fig. 3 shows each group cell culture of BNL CL.2 cell by Western Blot analysis The performance situation of the measured half Guang-aspartyl protease -9 through cracking；And

Fig. 4 shows each group cell culture of NMuMG cell by Western Blot analysis institute The performance situation of the half Guang-aspartyl protease -9 through cracking recording, wherein " * " table Show：When making comparisons with Normal group, p ＜ 0.05；And " # " represents：When penetrating with X Line matched group is made comparisons, p ＜ 0.05.

Specific embodiment

The above-mentioned and other purpose of the present invention, feature and advantage, with reference to greater detail below Illustrate with preferred embodiment after, will be apparent from.

Unless in addition defined, all technical and scientific terminology herein being used There is the meaning that familiar personage of the art understands jointly.One is familiar with this area Person cognitive can be described in the similar or equivalent method of person herein and material to many to those Material, they can be used for implementing the present invention.Certainly, the present invention is never subject to described side Method and the restriction of material.

On the medicine that exploitation can be used for treatment and/or pre- antiradiation injury, applicant is unexpectedly Find：Isorhamnetin-3-O-β-D-glycopranoside has the industry application potential of this respect. Then, the present invention discloses isorhamnetin-3-O-β-D-glycopranoside for being applied to prepare one Come to treat and/or pre- antiradiation injury pharmaceuticals purposes.

According to the present invention, isorhamnetin-3-O-β-D-glycopranoside is by vitro (in vitro test) and be proved effectively to protect cell (to include mouse liver cell, mice Galactophore epithelial cell and human skin horn cell) antagonism X-ray-induction death (X Ray-induced death), cell death (the X ray-induced cell of X-ray-induction Death), the apoptosis (X ray-induced apoptosis) of X-ray-induction and ultraviolet The apoptosis (ultraviolet-induced apoptosis) of line-induction.Therefore, the present invention carries For a kind of pharmaceuticals for treatment and/or pre- antiradiation injury, it includes isorhamnetin - 3-O- β-D-Glucose glycosides.

As used herein, term " treatment (treating) " or " treatment (treatment) " Mean to reduce (reducing), mitigate (alleviating), improve (ameliorating), alleviate (relieving) or control (controlling) one disease (disease) or obstacle (disorder) one Or multiple clinical symptom (clinical sign), and reduce (lowering), stop (stopping) Or reverse the patient's condition (condition) that is being treated of (reversing) one or symptom (symptom) progress (progression of severity) of seriousness.

As used herein, term " prevention (preventing) " or " prevention (prevention) " mean there is no disease incidence (disease onset) when a medicine is used in one Symptom but have disease incidence high risk individual when, stop or delay (delaying) The symptom of disease incidence.

As used herein, term " radiation damage (radiation injury) " means to give birth to Any position of object is in caused damage (injury) after radioactive exposure or infringement (damage).

According to the present invention, this radiation damage is a free radiation damage (ionizing radiation Injury) or one non-free radiation damage (non-ionizing radiation injury).

According to the present invention, this free radiation damage is following at least one：Acute radiation disease Wait group (acute radiation syndrome, ARS), chronic radiation syndrome (chronic Radiation syndrome, CRS) and radiation therapy side effect (side effect of radiation therapy).

As used herein, term " acute radiation syndrome " means the complete of organism Body (whole body) lasts a short time (for example, 24 hours) under being exposed to free radiation Caused acute symptom (acute symptoms) afterwards, this includes, but are not limited to：With hemopoietic Related symptom [for example, the hypoplastic anemia of system (hematopoietic system) Atrophy (the atrophy of of (aplastic anemia), haemolysis (hemolysis) and lymph node Lymph nodes)] and gastronintestinal system (gastrointestinal system) related symptom [for example, nauseous (nausea), vomiting (vomiting) and stomachache (abdominal pain)] with And it is [for example, dizzy with neural blood vessel system (neurovascular system) related symptom (dizziness), headache (headache) and level of consciousness reduce (decreased level of consciousness)].

As used herein, term " chronic radiation syndrome " means the complete of organism Lasted for one long-time (for example, several moons or several years) under free radiation and drawn afterwards in being exposed to The chronic sympton (chronic symptoms) rising, this includes, but are not limited to：Atrophoderma (skin Atrophy), cataract (cataract), recurrent infection (recurrent infection), slight Fever (low grade fever), anorexia (loss of appetite), tired (fatigue), Faint (fainting) and alopecia (hair loss).

As used herein, term " side effect of radiation therapy " means human individual Specific part in caused symptom after free radioactive exposure, this includes：But do not limit In：Anorexia (anorexia), (lassitude) tired out, diarrhoea (diarrhea), erythema (erythema), Desquamation (desquamation), intestinal stenosis (bowel stenosis), necrosis (the necrosis of of bone Bone) and lung fibrosiss (fibrosis of lung).

According to the present invention, this non-free radiation damage is a uv damage (ultraviolet damage).

As used herein, term " uv damage " means that human individual's is any Position is in caused disease after ultraviolet over-exposure (ultraviolet overexposure) Shape, this includes, but are not limited to：Chafing (skin inflammation), retardance are tanned Reaction (delayed tanning reaction), joint and intramuscular and circumference of eyes serious Pain (severe pains in the j oints and muscles and around the eyes), stop Gram (shock), fever (fever), nauseous (nausea), vomiting (vomiting) and whole body are no Power (generalized weakness).

According to the present invention, isorhamnetin-3-O-β-D-glycopranoside can utilize chemist institute Known to synthetic technology and be made.

Additionally, isorhamnetin-3-O-β-D-glycopranoside is possible with being used in this area Isolation and purification method and isolated and purified out from a natural origin (natural source) Come.In this regard, may be referred to, such as Lee Y.S.et al. (2005) (with above-mentioned) and Igarashi K.et al. (2008) (with above-mentioned).

According to the present invention, this natural origin can be following at least one：Semen Brassicae Campestriss (Brassica Campestris L.), Tai Baimei flowers and plants (Callianthemum taipaicum), Fructus Foeniculi (Foeniculum vulgare Mill.), Fructus Hippophae (Hippophae rhamnoides L.), Flos Caryophylli Flos Inulae (Inula britannica), Taiwan Herba Sedi Lineariss (Sedum formosanum), Caulis et Folium Brassicae junceae (Brassica juncea), big Flos Caraganae Sinicae (Caragana arborescens Lam.), melonidum are celestial People's palm [Opuntia dillenii (Ker-Gawl) Haw.] and grass green salicornia europaeal (Salicornia herbacea).

According to the present invention, this pharmaceuticals can be utilized and is familiar with the technology that this field person knows in detail and quilt It is manufactured into one and be suitable for parenteral genuine (parenterally), oral ground (orally) or partly (topically) dosage form (dosage form) offerd medicine, this includes, but are not limited to：Injection product (inj ection) [for example, aseptic aqueous solution (sterile aqueous solution) or dispersion liquid (dispersion)], aseptic powder (sterile powder), lozenge (tablet), tablet (troche), mouth containing ingot (lozenge), capsule (capsule), Dispersible powders (dispersible ) or fine grained (granule), solution, suspension (suspension), Emulsion powder (emulsion), syrup (syrup), elixir (elixir), underflow (slurry), external preparation (external preparation) and the like.

According to the present invention, this pharmaceuticals can further include one and has been widely used in medicine Pharmaceutically acceptable supporting agent (the pharmaceutically acceptable of manufacturing technology carrier).For example, this pharmaceutically acceptable supporting agent can comprise one or more be selected from by under Arrange the reagent in constituted group：Solvent (solvent), buffer (buffer), suspending agent (suspending agent), distintegrant (decomposer), disintegrating agent (disintegrating Agent), dispersant (dispersing agent), binding agent (binding agent), excipient (excipient), tranquilizer (stabilizing agent), chelating agen (chelating agent), dilute Release agent (diluent), gellant (gelling agent), preservative (preservative), lubrication Agent (lubricant), absorption delaying agent (absorption delaying agent), liposome (liposome) and the like.Selection about these reagent is to fall to being familiar with this with quantity In the Specialized Quality of the personage of technology and routine technology category.

According to the present invention, this pharmaceuticals can one be selected from by following constituted group Parenteral approach (parenteral routes) is offeing medicine：Peritoneal injection (intraperitoneal Injection), subcutaneous injection (subcutaneous injection), intramuscular injection (intramuscular injection) and intravenous injection (intravenous injection).Relatively Goodly, this pharmaceuticals is manufactured into the dosage form being suitable to be offerd medicine with subcutaneous injection.

According to the present invention, this pharmaceuticals can be utilized and is familiar with the technology that this field person knows in detail and quilt It is manufactured into an external preparation being suitable for partly being applied on skin, this includes, but does not limit In：Emulsion (emulsion), gel (gel), ointment (ointment), cream (cream), patch Piece (patch), liniment (liniment), powder (powder), aerosol (aerosol), spraying (spray), emulsion (lotion), serum (serum), paste (paste), foam (foam), drip Agent (drop), suspension (suspension), ointment (salve) and binder (bandage).

According to the present invention, this external preparation be by by the pharmaceuticals of the present invention with one for being familiar with The substrate (base) that this those skilled in the art knows in detail mutually mixes and is produced.

According to the present invention, this substrate can include one or more and be selected from following additive (additives)：Water, alcohol (alcohols), glycol (glycol), Hydrocarbon (hydrocarbons) [such as oil glue (petroleum jelly) and white vaseline (white Petrolatum)], wax (wax) [such as paraffin (paraffin) and Cera Flava (yellow wax)], Preservative agent (preserving agents), antioxidant (antioxidants), interfacial agent (surfactants), absorption enhancer (absorption enhancers), tranquilizer (stabilizing Agents), gellant (gelling agents) [such as carbopol(941), micro- Crystalline cellulose (microcrystalline cellulose) and carboxy methyl cellulose (carboxymethylcellulose)], activating agent (active agents), wetting agent (humectants), odour absorbents (odor absorbers), spice (fragrances), pH Regulator (pH adjusting agents), chelating agen (chelating agents), emulsifying agent (emulsifiers), occlusive agent (occlusive agents), softening agent (emollients), thickening Agent (thickeners), cosolvent (solubilizing agents), penetration enhancers (penetration Enhancers), counter-stimuluss (anti-irritants), coloring agent (colorants) and propellant (propellants) etc..Selection about these additives is to fall to being familiar with technique with quantity The Specialized Quality of personage and routine technology category in.

It is preferred that this pharmaceuticals includes a pharmaceutically acceptable solvent, and this solvent It is following any one：Water, normal saline (normal saline), phosphate-buffered physiology salt Water (phosphate buffered saline, PBS), sugary soln and the aqueouss containing alcohol are molten Liquid (aqueous solution containing alcohol).

The present invention also provide for a kind of for treatment one have or under a cloud have radiation damage The method of body, it includes this individuality is offerd medicine with isorhamnetin-3-O-β-D-glycopranoside.

According to the present invention, the dosage of isorhamnetin-3-O-β-D-glycopranoside and dispensing Number of times can change depending on following factors：The seriousness of disease to be treated, dosing way, And age of individuality to be treated, health and reaction.In general, working as foundation When the pharmaceuticals of the present invention are partly to be offerd medicine, each dosage typically 0.01 to The skin area of 0.05mg/ square centimeter, about 1 to 3 time daily.And when according to this Bright pharmaceuticals be by parenteral genuine or oral offer medicine when, each dosage is typically 25 to 50mg/Kg body weight, about 1 time daily.

In the present invention, it is found by the applicant that the pre- place of isorhamnetin-3-O-β-D-glycopranoside Reason can protect the apoptosis to uvioresistant-induction for the human skin horn cell effectively. Therefore, the present invention is it is also contemplated that isorhamnetin-3-O-β-D-glycopranoside is for being applied to prepare one Purposes for the cosmetics of radiation protection (radioprotection).

As used herein, term " radiation protection " means to block or mitigate radiation cruelly Harmful clinic caused by dew, histology and immunologic effect (adverse clinical, histological and immunological effect).These effects include acute effect (example As hypoplastic anemia, erythema, stomachache and headache) and chronic effect is (for example, Atrophoderma, cataract and alopecia).

According to the present invention, this cosmetics can further include one and has been widely used in cosmetic Acceptable adjuvant (cosmetically acceptable on the cosmetics of product manufacturing technology adjuvant).For example, on this cosmetics, acceptable adjuvant can include one or more and is selected from In following reagent：Solvent, gellant, activating agent, preservative, antioxidant, masking Agent (screening agent), chelating agen, interfacial agent, staining reagent (coloring agent), Thickening agent (thickening agent), filler (filler), spice and odour absorbents.Relevant The selection of these reagent and quantity are to fall in the Specialized Quality of one skilled in the art and example In row technology category.

According to the present invention, this cosmetics can be utilized and is familiar with the technology that this field person knows in detail and quilt It is manufactured into a form being suitable for skin protection (skincare) or cosmetic (makeup), this includes, but It is not limited to：Aqueous solution (aqueous solution), water-alcohol solution (aqueous-alcohol Solution) or oily solution (oily solution), in oil-in-water type (oil-in-water type), Water-in-oil type (water-in-oil type) or compound Emulsion, gel, ointment, cream, Facial film (mask), paster, patch (pack), liniment, powder, aerosol, spraying, emulsion, Serum, paste, foam, dispersion liquid, drop, mousse (mousse), suntan lotion (sunblock), Astringent (tonic water), foundation cream (foundation), eye shadow (eyeshadow), makeup removing are produced Product (makeup remover products), soap (soap) and other body cleansing products (body cleansing products) etc..

According to the present invention, this cosmetics also can be selected from following known activity with one or more External preparation (external use agents) merge use together：Whitening agent (whitening Agents) [such as retinoic acid (tretinoin), catechin (catechin), kojic acid (kojic Acid), arbutin (arbutin) and vitamin C (vitamin C)], wetting agent, anti-inflammatory Agent (anti-inflammatory agents), antibacterial (bactericides), UV absorbent (ultraviolet absorbers), plant extract (plant extracts) [such as aloe extract (aloe extract)], skin-nourishing agent (skin nutrients), anesthetis (anesthetics), Anti-acne agent (anti-acne agent), prurituss (antipruritic), analgesic (analgesic), Anti- dermatitises agent (antidermatitis agents), anti-keratinization agent (antihyperkeratolytic excessively Agents), anti-dry skin agent (anti-dry skin agents), anti-diaphoretic (antipsoriatic Agents), age resister (antiager), anti-wrinkle agent (antiwrinkle agents), seborrhea Go out agent (antiseborrheic agents), Wound healing and bone regeneration agent (wound-healing agents), skin Matter steroid (corticosteroids), hormone (hormones), free radical scavenger are (for example tactile Enzyme), antioxidant (such as Vitamin E), cytohormone (be for example situated between in vain element -1), mercaptan (example As amifostine) and steroid (such as 5- androstenediol).Choosing about these external preparations With being the Specialized Quality falling in one skilled in the art and routine technology category with quantity Interior.

According to the present invention, this cosmetics is in a form supplying local application.In general, Each application dosage according to the cosmetics of the present invention is typically 1 to 5 μ g/ square centimeter Skin area, about 2 to 5 times daily.

The present invention will be described further with regard to the following examples, however, it should be noted that this A little embodiments are simply for illustrating, and are not necessarily to be construed as the limit in the enforcement of the present invention System.

<Embodiment>

General experiment material：

1. the source of cell strain and culture：

Mouse liver cell strain (mouse liver cell line) used in embodiment below BNL CL.2 (BCRC 60180) and mammary gland of mouse epithelial cell strain (mouse mammary Gland epithelial cell line) NMuMG (BCRC 60087) is all available from Chinese platform Foodstuff Industrial and Development Inst. (Food Industry Research and Development in gulf Institute, FIRDI) living resources preserve and research center (Biosource Collection and Research Center,BCRC)；And human skin horn cell cell strain (human Skin keratinocyte cell line) HaCaT is by China Medical University's basic medical research Huang Zhiyang professor provided.

This 3 kinds of cells are incubated at 6-cm respectively in accordance with the culture medium shown in table 1 below In culture dish (6-cm petri dish), and in incubator (37 DEG C, 5%CO₂) in cultivated. When cell density reaches about 80-90% and converges (confluence), remove culture medium and with phosphorus Hydrochlorate buffer saline (phosphate buffered saline, PBS) (pH 7.4) (Gibco) To clean cell 1 time, to be subsequently added into trypsin-EDTA (trypsin-EDTA) so that thin Born of the same parents depart from from the bottom of culture dish.Afterwards, fresh culture medium is added to neutralize trypsin Activity and repeatedly inhale and rush culture medium fully to break up cell to measure suction pipe (pipette), so Afterwards the cell suspending liquid being formed is assigned in new culture bottle, and carries out in incubator Culture.

Table 1. is used for the culture medium of 3 kinds of cell strains of culture

2. the preparation of stock solution (stock solution)：

The preparation method of the 2 kinds of stock solutions being used in the following embodiments is respectively such as Lower described：

(1) stock solution of isorhamnetin-3-O-β-D-glycopranoside：

Isorhamnetin-3-O-β-D-glycopranoside (is had purchased from the tall and erect biotechnology in Shanghai times Limit company, Cat.No.5041-82-7) it is dissolved in dimethyl sulfoxide (dimethylsulfoxide, DMSO) In, and obtain the stock solution that a concentration is 10mM.

(2) stock solution of isorhamnetin：

By isorhamnetin (purchased from Shanghai Yuan Ye bio tech ltd, Cat.No. 480-19-3) it is dissolved in DMSO, and obtain the stock solution that a concentration is 10mM.

General experimental technique：

1. statistical analysis (statistical analysis)：

In the following embodiments, the experiment of each group is repeated 3 times, and experimental data be with The standard error (standard error of the mean, SEM) of meansigma methodss ± meansigma methodss carrys out table Show.All of data is by paired history apprentice Deng Shi t- test (paired Student ' s T-test) performing an analysis, in order to assess the diversity between each group.If obtained statistical analysiss Result is p ＜ 0.05, and this indicates significance,statistical (statistical significance).

Embodiment 1. isorhamnetin-3-O-β-D-glycopranoside The pretreatment of (isorhamnetin-3-O- β-D-glucoside) (pretreatment) for cell in antagonism X-ray-induction Effect assessment on dead (X ray-induced death)

Experimental technique：

A, to process mouse liver cell strain BNL with isorhamnetin-3-O-β-D-glycopranoside CL.2 and mammary gland of mouse epithelial cell strain NMuMG：

First, will according to above " general experiment material " the 1st " source of cell strain With culture " carrying out the BNL CL.2 cell of successive transfer culture and NMuMG cell each It is divided into 8 groups, including 1 Normal group (normal control), 1 different Fructus rhamni (Rhamnus davurica Pall.) Element -3-O- β-D-Glucose glycosides group, 3 X-ray matched groups (X ray control) are (namely X-ray matched group 1,2 and 3) and 3 experimental grouies (namely experimental group 1,2 and 3).By the BNL CL.2 cell of each group with one for 2 × 10⁵The quantity of cell and by each group NMuMG cell is with one for 2.5 × 10⁵The quantity of cell is incubated at thin containing 5mL respectively Born of the same parents' culture medium (culture medium being used about each cell be as table 1 above when described in) In 6-cm culture dish, and in incubator (37 DEG C, 5%CO₂) in carry out culture to last 24 little When.

Then, the cell culture of each group is changed respectively with fresh culture medium, then will The stock solution of appropriate isorhamnetin-3-O-β-D-glycopranoside is added separately to different Fructus rhamni (Rhamnus davurica Pall.) In element -3-O- β-D-Glucose glycosides group and each experimental group, and make isorhamnetin - 3-O- β-D-Glucose glycosides group and each experimental group all have a ultimate density and are 30 μM Isorhamnetin-3-O-β-D-glycopranoside.As for Normal group and each X-ray The cell culture of matched group does not then make any process.

Each group cell culture is in incubator (37 DEG C, 5%CO₂) in carry out culture to last 2 little Shi Hou, obtained cell culture is analyzed by bringing the population of cells carrying out following B item (clonogenic assay).

B, population of cells's analysis：

First, using a linear accelerator (linear accelerator) (ELEKTA) and under foundation Dosage shown in face table 2 carrys out the A item to " experimental technique " according to the present embodiment respectively Central obtained experimental group 1 to 3 and the cell culture of X-ray matched group 1 to 3 Carry out x-ray bombardment (X ray irradiation) (energy is 6MeV).As for normal control The cell culture of group and isorhamnetin-3-O-β-D-glycopranoside group does not then make any place Reason.

Each experimental group of table 2. and X-ray matched group are carried out roentgenogram The dosage penetrated

Group	Dosage (Gy)
		X-ray matched group 1	2
X-ray matched group 2	4
		X-ray matched group 3	6
Experimental group 1	2
		Experimental group 2	4
Experimental group 3	6

Each group cell culture is in incubator (37 DEG C, 5%CO₂) in carry out culture to last 2 little Shi Hou, is separately added into 0.5mL trypsin-EDTA to each group cell culture so that thin Born of the same parents depart from from the bottom of culture dish, then add the fresh culture of 2mL (thin about each Born of the same parents using culture medium be as table 1 above when described in) neutralizing tryptic activity And repeatedly inhale and rush culture medium fully to break up cell to measure suction pipe, then thin by formed (plating) is cultivated in new 6-cm culture dish in born of the same parents' suspension square position, wherein isorhamnetin - 3-O- β-D-Glucose glycosides group and Normal group are with one for 2.0 × 10²The quantity of cell To cultivate, experimental group 1 and X-ray matched group 1 are with one for 4.0 × 10²The quantity of cell To cultivate, experimental group 2 and X-ray matched group 2 are with one for 8.0 × 10²The quantity of cell To cultivate, and experimental group 3 is with one for 1.6 × 10 with X-ray matched group 3³Cell Quantity is cultivating.

Each group cell is in incubator (37 DEG C, 5%CO₂) in carry out after culture lasts 2 weeks, with 4% (w/v) formaldehyde (formaldehyde) of 5mL is fixing formed population of cells (cell Colony), connect and add 5mL 0.005% (w/v) crystal violet (crystal violet) and in At 25 DEG C, reaction lasts 30 minutes.Afterwards, using an inverted microscope (Olympus CH40) And carry out observing the counting with population of cells under an amplification being 100 times.

Square position culture efficiency (plating efficiency, PE) (%) is by by measured by each group Population of cells's quantity and initially by square position culture (plated) cell quantity substitute into respectively Following equation (1) and be calculated：

Formula (1)：A=(B/C) × 100

Wherein：A=square position culture efficiency (%)

The quantity of B=population of cells

The cell quantity that C=is cultivated by square position

Experimental group 1 to 3 and the survival rate of isorhamnetin-3-O-β-D-glycopranoside group (survival fraction) is by by experimental group 1 to 3 and isorhamnetin -3-O- β-D- The square position culture efficiency of glucoside group is respectively divided by isorhamnetin-3-O-β-D-glycopranoside The square position of group is cultivated efficiency and is calculated.In addition, X-ray matched group 1 to 3 and just Often the survival rate of matched group is by by X-ray matched group 1 to 3 and Normal group Square position is cultivated efficiency and is respectively divided by the square position culture efficiency of Normal group and is calculated.It Afterwards, according to above " general experimental technique " the 1st " statistical analysis " when described in Method come the experimental data obtained by analyzing.

Result：

Fig. 1 and Fig. 2 shows each group of BNL CL.2 cell and NMuMG cell respectively Cell culture is by the measured survival rate of population of cells's analysis.From Fig. 1 and Fig. 2, Either BNL CL.2 cell or NMuMG cell, X-ray matched group 1 to 3 Survival rate is all significantly reduced compared to Normal group institute tool person, and experimental group 1 to 3 Survival rate also have significantly compared to isorhamnetin-3-O-β-D-glycopranoside group institute tool person Reduce, and can more they tend to the increase of the dosage of x-ray bombardment substantially, this expression X-ray can suppress the growth of BNL CL.2 cell and NMuMG cell.In addition, working as During using identical cell strain and with identical dosage to carry out x-ray bombardment, experimental group Survival rate can be higher than X-ray matched group institute tool person.This experimental result shows：Cell is entered The pretreatment of row isorhamnetin-3-O-β-D-glycopranoside can protect cells against X-ray- The death of induction.

The pretreatment of embodiment 2. isorhamnetin-3-O-β-D-glycopranoside is for X Cell death (the X ray-induced cell of ray-induction Death impact)

Experimental technique：

A, to process mammary gland of mouse epithelial cell strain with isorhamnetin-3-O-β-D-glycopranoside NMuMG：

First, will according to above " general experiment material " the 1st " source of cell strain With culture " it is divided into 4 groups carrying out the NMuMG cell of successive transfer culture, including 1 Normal group, 1 X-ray matched group and 2 experimental grouies (namely experimental group 1 with 2).By the NMuMG cell of each group with one for 2.5 × 10⁵The quantity of cell be incubated at containing (culture medium being used about NMuMG cell is as table 1 above to the cell culture medium of 5mL When described in) 6-cm culture dish in, and in incubator (37 DEG C, 5%CO₂) in trained Support and last 24 hours.

Then, the cell culture of each group is changed respectively with fresh culture medium, then will The stock solution of appropriate isorhamnetin-3-O-β-D-glycopranoside is added separately to experimental group In 1 and 2, and make experimental group 1 and 2 be respectively provided with a ultimate density be 15 μM and 30 μM of isorhamnetin-3-O-β-D-glycopranoside.Penetrate as Normal group and X The cell culture of line matched group does not then make any process.

Each group cell culture is in incubator (37 DEG C, 5%CO₂) in carry out culture to last 2 little Shi Hou, obtained cell culture is brought the cell cycle analysis carrying out following B item (cell cycle analysis).

B, cell cycle analysis：

First, using a linear accelerator and one be 20Gy dosage under to according to this reality Apply obtained X-ray matched group in the middle of the A item of " experimental technique " of example, experimental group 1 and the cell culture of experimental group 2 carry out x-ray bombardment.As for Normal group Cell culture does not then make any process.Each group cell culture incubator (37 DEG C, 5% CO₂) in carry out after culture lasts 96 hours, by each group cell culture at 25 DEG C with 1200rpm carries out centrifugation and lasts 3 minutes.After removing upper clear liquid, with ice-cold PBS (pH 7.4) come the precipitate obtained by cleaning, then come with the ice-cold methanol of 1mL (methanol) Fixing cell, then will stand overnight through fixing cell at -20 DEG C.Then, with ice-cold PBS (pH 7.4) to clean through fixing cell, then add the ice-cold DNA of 250 μ L Staining solution [is assigned in secondary water, the RNase solution A containing 200 μ g/mL (Sigma-Aldrich), 50 μ g/mL propidium iodide (propidium iodide, PI) (Sigma-Aldrich) and 0.1% (v/v) Triton X-100] to dissipate floating cell, then Carry out lucifuge effect and last 30 minutes at 37 DEG C.

Afterwards, with BD Accuri^TMC6 stream type cell analyzer (BD Accuri^TMC6flow Cytometer) (BD Biosciences) to carry out cell week to obtained dyed cell Phase is analyzed, and analyzes 1.0 × 10 every time⁶Individual cell.Cell is with the laser beam 488nm of argon ion Excite and send fluorescence, and fluorescence intensity under a wavelength being 585nm.Institute The data obtaining is with BD Accuri^TMC6 software (BD Accuri^TMC6 software)(BD Biosciences) analyzing the percentage ratio of the cell being in each cell cycle phase.

Finally, according to above " general experimental technique " the 1st " statistical analysis " when Described in method come the experimental data obtained by analyzing.

Result：

Table 3 below shows that each group cell culture of NMuMG cell divides by cell cycle The measured cell cycle distribution of analysis.As seen from Table 3, compared with Normal group relatively under, Higher ratio is had to be in Sub-G1 phase (Sub-G1 in the middle of the cell of X-ray matched group Phase), this represents that X-ray can promote NMuMG cell to produce cell death.In addition, Compared with X-ray matched group relatively under, have relatively low ratio in the middle of the cell of experimental group 1 and 2 It is in the Sub-G1 phase, and can be with the concentration of isorhamnetin-3-O-β-D-glycopranoside Increase and more they tend to substantially.This experimental result shows：Isorhamnetin is carried out to cell The pretreatment of -3-O- β-D-Glucose glycosides can protect cells against the cell of X-ray-induction Dead.

The each group cell culture of table 3.NMuMG cell is by measured by cell cycle analysis Cell cycle distribution

^*：When making comparisons with Normal group, p ＜ 0.05.

^**：When making comparisons with Normal group, p ＜ 0.01.

^#：When making comparisons with X-ray matched group, p ＜ 0.05.

^##：When making comparisons with X-ray matched group, p ＜ 0.01.

The pretreatment of embodiment 3. isorhamnetin-3-O-β-D-glycopranoside is for X Apoptosis (the X ray-induced of ray-induction Apoptosis impact)

In the present embodiment, applicant is respectively using isorhamnetin-3-O-β-D-glycopranoside And isorhamnetin to carry out pretreatment to cell, and inquire into them for X-ray-induction Apoptotic impact.

Experimental technique：

A, mouse liver cell strain BNL CL.2 and mammary gland of mouse epithelial cell strain NMuMG Pretreatment：

First, will according to above " general experiment material " the 1st " source of cell strain With culture " it is divided into 4 groups carrying out the BNL CL.2 cell of successive transfer culture, including 1 Individual Normal group, 1 X-ray matched group and 2 experimental group (namely experimental group 1 With 2).In addition, will according to above " general experiment material " the 1st " cell strain come Source and culture " is divided into 6 groups carrying out the NMuMG cell of successive transfer culture, including 1 Individual Normal group, 1 X-ray matched group, 2 isorhamnetin group (namely different Fructus rhamni (Rhamnus davurica Pall.)s Element group 1 and 2) and 2 experimental grouies (namely experimental group 1 and 2).By each group BNL CL.2 Cell is with one for 2 × 10⁵The quantity of cell and by each group NMuMG cell with one for 2.5 ×10⁵It is (thin about each that the quantity of cell is incubated at the cell culture medium containing 5mL respectively The culture medium that born of the same parents are used be as table 1 above when described in) 6-cm culture dish in, and Incubator (37 DEG C, 5%CO₂) in carry out culture and last 24 hours.

Then, the cell culture of each group is changed respectively with fresh culture medium, then will The stock solution of isorhamnetin-3-O-β-D-glycopranoside and isorhamnetin bring respectively with The cell culture of each group carries out pretreatment, about the pretreatment bar of the cell culture of each group Part is shown in table 4 below.

The pretreatment condition of table 4. each group cell culture

Each group cell culture is in incubator (37 DEG C, 5%CO₂) in carry out culture to last 2 little Shi Hou, obtained cell culture is brought the experiment carrying out following B to D item respectively.

B, Apoptosis assay：

First, using a linear accelerator and one be 15Gy dosage under to according to this reality The X applying obtained BNL CL.2 cell in the middle of the A item of " experimental technique " of example penetrates The cell culture of line matched group, experimental group 1 and experimental group 2 and NMuMG cell X-ray matched group, isorhamnetin group 1, isorhamnetin group 2, experimental group 1 and experimental group 2 Cell culture carry out x-ray bombardment.Normal group as this 2 kinds of cell strain Cell culture does not then make any process.Afterwards, by each group cell of BNL CL.2 cell Culture is placed in incubator (37 DEG C, 5%CO₂) in carry out culture and last 96 hours, and will The each group cell culture of NMuMG cell is placed in incubator (37 DEG C, 5%CO₂) in carry out Culture lasts 72 hours.Then, each group cell culture of this 2 kinds of cell strains is added respectively Enter 1mL trypsin-EDTA so that cell departs from from the bottom of culture dish, then add (culture medium being used about each cell is that table 1 above such as is worked as to the fresh culture medium of 1mL Described in) neutralizing tryptic activity, then to measure suction pipe by the cell suspension being formed Liquid is drawn to centrifuge tube and is given centrifugation under 1200rpm and lasts 3 minutes.On removing After clear liquid, cell 1 time is cleaned with ice-cold PBS (pH 7.4), then in 1200rpm Under carry out centrifugation and last 3 minutes.Then, remove upper clear liquid and add the ice-cold film of 100 μ L Connection albumen V combination buffer (Annexin V binding buffer) (BD Pharmingen^TM) Fully to dissipate floating cell pellet (cell pellet), obtaining a cell concentration whereby is 1.0 ×10⁶The cell suspending liquid of cell/mL, is then separately added into obtained cell suspending liquid FITC annexin V (FITC Annexin V) (the BD Pharmingen of 5 μ L^TM) and 5 Propidium iodide (PI) staining solution [propidium iodide (PI) staining of μ L solution](BD Pharmingen^TM) and be sufficiently mixed uniformly, then enter at 4 DEG C The effect of row lucifuge lasts 30 minutes.

Afterwards, obtained dyed cell is with BD Accuri^TMC6 fluidic cell divides Analyzer, to be analyzed, wherein has and is dyeed but cannot be dyeed by PI by FITC annexin V [namely FITC annexin V positive (FITC Annexin V positive) and PI are cloudy Property (PI negative)] cell represent and be induced early stage apoptosis (early apoptosis) Cell, have and [namely FITC annexin dyeed by FITC annexin V and PI V is positive and the PI positive (PI positive)] cell represent and be induced late cell apoptosis The cell of (late apoptosis), and cannot be dyeed by FITC annexin V and PI [ It is exactly FITC annexin V feminine gender (FITC Annexin V negative) and PI feminine gender] Cell then represent living cells (viable cell).Relevant by FITC annexin V and PI The cell number of dyeing is by using BD Accuri^TMC6 software and be calculated.

The percentage of cerebral apoptosis (apoptosis percentage) (%) of each group is by will be surveyed The cell number obtaining substitutes into following equation (2) and is calculated：

Formula (2)：D=[(E+F)/G] × 100

Wherein：D=percentage of cerebral apoptosis (%)

E=is dyeed but cannot be contaminated by PI by FITC annexin V The cell number of color

F=is by the cell of FITC annexin V and PI dyeing Number

The whole cell number of G=

Afterwards, according to above " general experimental technique " the 1st " statistical analysis " when Described in method come the experimental data obtained by analyzing.

C, the analysis of grain wire body transmembrane potential：

The depolarization (depolarization) of known grain wire body transmembrane potential is in apoptosis process One of early stage markup phenomenon (early mark event), therefore, in order to inquire into different Mus Whether Li Su -3-O- β-D-Glucose glycosides can affect BNL CL.2 cell and NMuMG is thin The grain wire body transmembrane potential of born of the same parents, and then protect the cell that these cells resist X-ray-induction to wither Die, following experiment is carried out.

First, using a linear accelerator and one be 15Gy dosage under to according to this reality The X applying obtained BNL CL.2 cell in the middle of the A item of " experimental technique " of example penetrates Line matched group carries out x-ray bombardment with the cell culture of experimental group 1, and is 10 one Under the dosage of Gy, the X-ray matched group of NMuMG cell is trained with the cell of experimental group 1 Foster thing carries out x-ray bombardment.Cell culture as the Normal group of this 2 kinds of cell strains Thing does not then make any process.

Afterwards, by each group cell culture of BNL CL.2 cell be placed in incubator (37 DEG C, 5%CO₂) in carry out culture and last 120 hours, and will be thin for each group of NMuMG cell Born of the same parents' culture is placed in incubator (37 DEG C, 5%CO₂) in carry out culture and last 48 hours.Then, The each group cell culture of this 2 kinds of cell strains is cleaned 1 time with PB S, then to this The each group cell culture of 2 kinds of cell strains be separately added into 1mL trypsin-EDTA so that Cell departs from from the bottom of culture dish.Then, the fresh culture medium of 1mL is added to neutralize Tryptic activity, is drawn the mixed solution being formed to centrifuge tube with measuring suction pipe then In, under 400 × g, then give centrifugation last 5 minutes.After removing upper clear liquid, add The 1X JC-1 dye solution (staining buffer) of 500 μ L simultaneously carries out lucifuge at 37 DEG C Effect lasts 15 minutes.Then, add 1mL JC-1 analysis buffer (assay buffer) To clean dyed cell, under 400 × g, then to carry out centrifugation last 5 minutes.Moving After upper clear liquid, add 500 μ L JC-1 dye solution come to dissipate float cell, then with BD Accuri^TMC6 stream type cell analyzer carrying out the analysis of a wire body transmembrane potential, then with BD Accuri^TMC6 software is calculating the grain wire body film potential depolarising percentage ratio of cell (mitochondrial membrane potential depolarization percentage) (%).

Afterwards, according to above " general experimental technique " the 1st " statistical analysis " when Described in method come the experimental data obtained by analyzing.

D, through cracking half Guang-aspartyl protease -9 performance situation：

First, using a linear accelerator and one be 15Gy dosage under to according to this reality Apply obtained BNL CL.2 cell in the middle of the A item of " experimental technique " of example and The cell culture of the X-ray matched group of NMuMG cell, experimental group 1 and experimental group 2 Carry out x-ray bombardment.As for this 2 kinds of cell strains Normal group cell culture then Do not make any process.

Afterwards, by each group cell culture of BNL CL.2 cell and NMuMG cell It is placed in incubator (37 DEG C, 5%CO₂) in carry out culture and last 48 hours, then to this 2 The each group cell culture planting cell strain is separately added into the dissolving buffer (lysis of 120 μ L Buffer) [containing CelLytic^TMM protein extraction reagent (CelLytic^TMM protein Extraction reagent) (Sigma-Aldrich) and protease inhibitor cocktail (proteinase inhibitor cocktail) (Sigma-Aldrich)] and give mix homogeneously.Connect , the cell mixture being formed is placed in microcentrifugal tube, and with 13000 at 4 DEG C Rpm carries out centrifugation and lasts 10 minutes, then collects upper clear liquid and in this, as gross protein sample Product, then by Bio-Rad protein analyses set group (Bio-Rad Protein Assay Kit) To measure total protein concentration.

Afterwards, the gross protein sample of each group cell is to be known in detail using being familiar with skilled person And usual technology is carrying out SDS- polyacrylamide gel electrophoresis (sodium dodecyl Sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) analyze and west Square Blot analysis (Western Blotting), the gross protein sample of wherein each group cell is pin Western Blot analysis are carried out to the half Guang-aspartyl protease -9 through cracking.In addition, β - actin (β-actin) is used as internal contrast group (internal control).

The instrument that relevant SDS-PAGE analyzes and Western Blot analysis are used is divided with reagent Not as described below：

(1) SDS-PAGE analysis is using an electrophoresis system (electrophoresis System) (Bio-Rad) is carrying out.

(2) protein transfer (protein transfer) is using half dry type electrophoresis transfer groove (semi-dry Electrophoretic transfer cell) (Bio-Rad) and poly- difluoroethylene (PVDF) film [polyvinylidene difluoride (PVDF) membrane] is carrying out.

(3) in Western Blot analysis, an antibody (primary being used for each protein Antibody) and secondary antibodies (secondary antibody) be shown in respectively following In table 5.

Table 5. is used for carrying out an antibody and the secondary antibodies of Western Blot analysis

(4) chemiluminescence dyeing (chemiluminescence staining) is using chemiluminescence HRP By matter (chemiluminescent HRP substrate) (Millipore, Cat.No. WBKLS0500) carrying out, and using an ImageScanner imaging software (ImageScanner Imaging Software)(GE Healthcare Life Sciences) To detect signal.

Afterwards, it is analyzed using ImageScanner imaging software, protein belt whereby (protein band) can by half-quantitatively calculate corresponding protein expression amount, each group Through cracking half Guang-aspartyl protease -9 performance amount connect and with its corresponding β - Actin protein expression amount giving standardization (normalized), then by normal control Group normalised through cracking half Guang-aspartyl protease -9 performance amount as 100%, and other each groups with respect to Normal group through cracking half Guang-aspartic acid egg The performance amount percentage ratio of white enzyme -9 can be computed.Afterwards, " the general experiment according to above 1st " statistical analysis " of method " when described in method to analyze obtained by reality Test data.

Result：

A, Apoptosis assay：

Table 6 below shows each group cell training of BNL CL.2 cell and NMuMG cell Foster thing is by the percentage of cerebral apoptosis measured by Apoptosis assay.As seen from Table 6, no By being BNL CL.2 cell or NMuMG cell, the apoptosis of X-ray matched group Percentage ratio has significant rising compared to Normal group institute tool person, and this represents X-ray meeting Induction BNL CL.2 cell and NMuMG cells for apoptosis.In addition, experiment The percentage of cerebral apoptosis of group 1 and 2 has significantly compared to X-ray matched group institute tool person Decline, and can with the increase of the concentration of isorhamnetin-3-O-β-D-glycopranoside more Tend to obvious.And for NMuMG cell, the apoptosis percentage of experimental group 1 and 2 Than all less than isorhamnetin group institute tool person.This experimental result shows：Different Mus are carried out to cell The pretreatment of Li Su -3-O- β-D-Glucose glycosides more can effectively protect cells against X and penetrate The apoptosis of line-induction.

The each group cell culture of table 6.BNL CL.2 cell and NMuMG cell by Percentage of cerebral apoptosis measured by Apoptosis assay

^**：When making comparisons with Normal group, p ＜ 0.01.

^#：When making comparisons with X-ray matched group, p ＜ 0.05.

^##：When making comparisons with X-ray matched group, p ＜ 0.01.

B, the analysis of grain wire body transmembrane potential：

Table 7 below shows each group cell training of BNL CL.2 cell and NMuMG cell Foster thing is by the measured grain wire body film potential depolarising percentage ratio of grain wire body transmembrane potential analysis. As seen from Table 7, either BNL CL.2 cell or NMuMG cell, X-ray pair Have aobvious compared to Normal group institute tool person according to the grain wire body film potential depolarising percentage ratio organized The rising writing, this represents that X-ray can promote BNL CL.2 cell and NMuMG cell Produce apoptosis.In addition, the grain wire body film potential depolarising percentage ratio of experimental group 1 is compared In X-ray matched group institute, tool person is significantly reduced.This experimental result shows：To cell The pretreatment carrying out isorhamnetin-3-O-β-D-glycopranoside can suppress cell being carried out The depolarization of caused grain wire body transmembrane potential after x-ray bombardment, so reach antagonism X penetrate The apoptotic effectiveness of line-induction.

The each group cell culture of table 7.BNL CL.2 cell and NMuMG cell by The measured grain wire body film potential depolarising percentage ratio of grain wire body transmembrane potential analysis

^*：When making comparisons with Normal group, p ＜ 0.05.

^**：When making comparisons with Normal group, p ＜ 0.01.

^#：When making comparisons with X-ray matched group, p ＜ 0.05.

C, through cracking half Guang-aspartyl protease -9 performance situation：

Fig. 3 and Fig. 4 shows each group of BNL CL.2 cell and NMuMG cell respectively Cell culture is by the half Guang-aspartic acid egg through cracking measured by Western Blot analysis The performance situation of white enzyme -9.From Fig. 3 and Fig. 4, either BNL CL.2 cell or It is NMuMG cell, the half Guang-aspartyl protease -9 through cracking of X-ray matched group Performance amount compared to Normal group institute tool person (by Normal group through cracking half Guang- The performance amount of aspartyl protease -9 is as 100%) there is significant rising, this represents X Ray can induce BNL CL.2 cell and NMuMG cells for apoptosis.In addition, The performance amount of the half Guang-aspartyl protease -9 through cracking of experimental group 1 and 2 is compared to X Ray contrast group institute tool person is significantly reduced, and can be with isorhamnetin The increase of concentration of -3-O- β-D-Glucose glycosides and more they tend to substantially.This experimental result shows Show：The pretreatment that cell is carried out with isorhamnetin-3-O-β-D-glycopranoside can be protected carefully Born of the same parents avoid the half Guang-aspartyl protease -9 through cracking because caused by x-ray bombardment Excessive performance, and then reach the apoptotic effectiveness of antagonism X-ray-induction.

The pretreatment of embodiment 4. isorhamnetin-3-O-β-D-glycopranoside is for purple The apoptotic impact of outside line-induction

Experimental technique：

A, to process mammary gland of mouse epithelial cell strain with isorhamnetin-3-O-β-D-glycopranoside NMuMG and human skin horn cell cell strain HaCaT：

First, will according to above " general experiment material " the 1st " source of cell strain With culture " NMuMG cell and HaCaT cell to carry out successive transfer culture be each separated into 4 groups, including 1 Normal group, 1 ultraviolet matched group and 2 experimental grouies (namely experimental group 1 and 2).By each group cell with one for 2.5 × 10⁵The quantity culture of cell In the cell culture medium containing 5mL, (culture medium being used about each cell is as above Table 1 when described in) 6-cm culture dish in, and in incubator (37 DEG C, 5%CO₂) in enter Row culture lasts 24 hours.

Then, the cell culture of each group is changed respectively with fresh culture medium, then will The stock solution of appropriate isorhamnetin-3-O-β-D-glycopranoside is added separately to 2 kinds thin In the cell culture of experimental group 1 and 2 of born of the same parents' strain, and make the experiment of this 2 kinds of cell strains Group 1 and 2 cell culture are respectively provided with the different of a ultimate density as shown in Table 8 below Rhamnetin -3-O- β-D-Glucose glycosides.Normal group as this 2 kinds of cell strains and The cell culture of ultraviolet matched group does not then make any process.

The different Fructus rhamni (Rhamnus davurica Pall.) that the cell culture of the experimental group 1 and 2 of 8. 2 kinds of cell strains of table has The ultimate density of element -3-O- β-D-Glucose glycosides

Each group cell culture is in incubator (37 DEG C, 5%CO₂) in carry out culture to last 2 little Shi Hou, obtained cell culture is brought the Apoptosis assay carrying out following B item.

B, Apoptosis assay：

First, using a CL-1000 ultraviolet-crosslinkable instrument (CL-1000ultraviolet Crosslinker) (UVP) and one be 30mJ/cm²Dosage under to according to the present embodiment The ultraviolet matched group of obtained NMuMG cell in the middle of the A item of " experimental technique ", Experimental group 1 carries out ultraviolet irradiation with the cell culture of experimental group 2, and is 50 one mJ/cm²Dosage under to the ultraviolet matched group of HaCaT cell, experimental group 1 and experiment The cell culture of group 2 carries out ultraviolet irradiation.Normal control as this 2 kinds of cell strains The cell culture of group does not then make any process.

Afterwards, by each group cell culture of NMuMG cell be placed in incubator (37 DEG C, 5% CO₂) in carry out culture and last 24 hours, and each group cell culture by HaCaT cell Thing is placed in incubator (37 DEG C, 5%CO₂) in carry out culture and last 48 hours.Then, to this The each group cell culture of 2 kinds of cell strains be separately added into 1mL trypsin-EDTA so that Cell departs from from the bottom of culture dish, then adds the fresh culture medium of 1mL (about each Cell using culture medium be as table 1 above when described in) neutralizing tryptic work Property, then the cell suspending liquid being formed is drawn to centrifuge tube and in 1200 with measuring suction pipe Give centrifugation under rpm and last 3 minutes.After removing upper clear liquid, with ice-cold PBS (pH 7.4) To clean cell 1 time, under 1200rpm, then to carry out centrifugation last 3 minutes.Then, Remove upper clear liquid and add the ice-cold annexin V combination buffer of 100 μ L with fully scattered Floating cell pellet, obtaining a cell concentration whereby is 1.0 × 10⁶The cell of cell/mL Suspension, is then separately added into the FITC film connection egg of 5 μ L to obtained cell suspending liquid Propidium iodide (PI) staining solution of white V and 5 μ L is simultaneously sufficiently mixed uniformly, then Carry out lucifuge effect and last 30 minutes at 4 DEG C.

Afterwards, obtained dyed cell is with BD Accuri^TMC6 fluidic cell divides Analyzer, to be analyzed, wherein has and is dyeed but cannot be dyeed by PI by FITC annexin V The cell of (namely FITC annexin V positive and PI negative) represents and is induced morning Phase apoptotic cell, has and is dyeed (namely FITC by FITC annexin V and PI Annexin V positive and PI are positive) cell represent and be induced late cell apoptosis Cell, and (namely FITC film connection egg cannot be dyeed by FITC annexin V and PI White V is negative and PI is negative) cell then represent living cells.Relevant by FITC film join egg The cell number of white V and PI dyeing is by using BD Accuri^TMC6 software and quilt Calculate.

The percentage of cerebral apoptosis (%) of each group is to substitute into by by measured cell number Face formula (2) and be calculated.Afterwards, the 1st of " general experimental technique " according to above " statistical analysis " when described in method to analyze obtained by experimental data.

Result：

Table 9 below shows each group cell culture of NMuMG cell and HaCaT cell By the percentage of cerebral apoptosis measured by Apoptosis assay.As seen from Table 9, either NMuMG cell or HaCaT cell, the percentage of cerebral apoptosis phase of ultraviolet matched group There is significant rising compared with Normal group institute tool person, this represents that ultraviolet can induce NMuMG cell and HaCaT cells for apoptosis.In addition, experimental group 1 and 2 Percentage of cerebral apoptosis have significant decline compared to ultraviolet matched group institute tool person, and And can more they tend to bright with the increase of the concentration of isorhamnetin-3-O-β-D-glycopranoside Aobvious.This experimental result shows：Isorhamnetin-3-O-β-D-glycopranoside is carried out to cell Pretreatment can effectively protect cells against the apoptosis of ultraviolet-induction.

The each group cell culture of table 9.NMuMG cell and HaCaT cell is by cell The measured percentage of cerebral apoptosis of apoptosis analysis

^*：When making comparisons with Normal group, p ＜ 0.05.

^#：When making comparisons with ultraviolet matched group, p ＜ 0.05.

Experimental result more than synthesis, applicant thinks：Isorhamnetin -3-O- β-D- Fructus Vitis viniferae Glucosides is available for preventing and/or treat radiation damage (the free radiation damage of inclusion and non-trip From radiation damage).

The all patents being quoted in this manual and document are integrally merged in this case with it and make For reference material.If conflicted, this case describes (comprise be defined in) in detail and will account for Wind.

Although the present invention be described with reference to above-mentioned specific concrete example it will be apparent that without departing substantially from A lot of modifications and variations can be made under scope and spirit of the present invention.Therefore it is intended that The present invention is only limited by as shown in civilian appending claims.

Claims (11)

1. isorhamnetin-3-O-β-D-glycopranoside is for being applied to prepare one for treating and/or in advance The purposes of the pharmaceuticals of antiradiation injury.

2. purposes as claimed in claim 1 it is characterised in that：This radiation damage is a free spoke Penetrate damage or a non-free radiation damage.

3. purposes as claimed in claim 2 it is characterised in that：This free radiation damage is following At least one：Acute radiation syndrome, chronic radiation syndrome and radiation therapy Side effect.

4. purposes as claimed in claim 2 it is characterised in that：This non-free radiation damage is one Uv damage.

5. purposes as claimed in claim 1 it is characterised in that：This pharmaceuticals further includes One pharmaceutically acceptable supporting agent.

6. purposes as claimed in claim 1 it is characterised in that：This pharmaceuticals is to supply non-warp in one The dosage form of intestinal dispensing.

7. purposes as claimed in claim 1 it is characterised in that：This pharmaceuticals is for oral in one The dosage form of dispensing.

8. purposes as claimed in claim 1 it is characterised in that：This pharmaceuticals is for local in one The dosage form of dispensing.

9. isorhamnetin-3-O-β-D-glycopranoside is for being applied to prepare one for radiation proof The purposes of cosmetics.

10. purposes as claimed in claim 9 it is characterised in that：This cosmetics further includes Acceptable adjuvant on one cosmetics.

11. purposes as claimed in claim 9 it is characterised in that：This cosmetics is for local in one The form applied.