CN109078033A - Saussurea involucrata culture inhibits protein non-enzyme glycosylation to react the application in anti-aging product - Google Patents

Saussurea involucrata culture inhibits protein non-enzyme glycosylation to react the application in anti-aging product Download PDF

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CN109078033A
CN109078033A CN201810896249.3A CN201810896249A CN109078033A CN 109078033 A CN109078033 A CN 109078033A CN 201810896249 A CN201810896249 A CN 201810896249A CN 109078033 A CN109078033 A CN 109078033A
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culture
saussurea involucrata
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刘汉石
孙晓琳
刘雅萍
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PURUIKANG BIOTECHNOLOGY CO Ltd DALIAN
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    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps

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Abstract

本发明公开了雪莲培养物抑制蛋白质非酶糖基化反应在抗衰老产品中的应用，所述抗衰老包括抗皱纹以及抗老年色素形成，由于非酶糖基化反应会导致人体皮肤老化，而实验证明雪莲培养物能够有效抑制非酶糖基化反应，因此，说明雪莲培养物可实现人体皮肤的抗老化，应用于制备抗皮肤老化产品中。The invention discloses the application of snow lotus culture to inhibit protein non-enzymatic glycosylation reaction in anti-aging products, the anti-aging includes anti-wrinkle and anti-aging pigment formation, because non-enzymatic glycosylation reaction will cause human skin aging, and Experiments have proved that Saussurea sauraceae culture can effectively inhibit non-enzymatic glycosylation reaction, therefore, it shows that Saussurea sauraceae culture can realize anti-aging of human skin, and it can be used in the preparation of anti-aging skin products.

Description

Saussurea involucrata culture inhibits protein non-enzyme glycosylation reaction in anti-aging product Using
Technical field
The present invention discloses the technical field for being related to Saussurea involucrata culture application more particularly to Saussurea involucrata culture inhibits protein non- Application of the enzyme glycosylation in anti-aging product.
Background technique
Skin aging refers to that skin function aging damages, makes skin to the protective capacities of body, regulating power etc. subtracts It moves back, thus skin does not adapt to the variation of internal and external environment, changing for the appearances integral status such as color, color, form, texture occurs Become.The aging of skin is divided into: intrinsic aging and extrinsic aging.It is naturally old that intrinsic aging refers to that skin increases with the age Change, shows as whiteness of skin, fine wrinkles, flexibility decrease, cutis laxa etc. occur；Extrinsic aging is main the reason is that day Shine caused by light aging, show as wrinkle, cutis laxa, coarse, Beige color dermatodyschroia, telangiectasis, Pigmented spots are formed.
Patent of invention ZL201410635060 discloses red goosefoot extract due to containing a variety of active ingredients, can effectively facilitate The collagen secretion rate of dermal fibroblasts effectively prevents collagen saccharification and resists the injury of UV light, can resist Skin aging.
Patent of invention 2011104395270 discloses natural plant extracts with anti-sunlight function and preparation method and answers With lanatechead saussurea herb with flower is as one such ingredient, for sun-proof.
Therefore, there is presently no met related Saussurea involucrata culture by inhibiting protein non-enzyme glycosylation reaction in anti-skin The report applied in terms of aging.
Summary of the invention
In consideration of it, the present disclosure provides Saussurea involucrata cultures to inhibit protein non-enzyme glycosylation reaction in anti-aging product In application.
Technical solution provided by the invention, specifically, Saussurea involucrata culture inhibits protein non-enzyme glycosylation reaction anti-ageing Application in old product.
It is preferred that the resisting age of skin is that anti-wrinkle and anti-age pigment are formed.
Further preferably, the Saussurea involucrata culture is the alcohol extract of Saussurea involucrata culture, it is specific the preparation method comprises the following steps:
Saussurea involucrata culture 5.0g is weighed, after being crushed, crosses the sieve that granularity is 30 mesh, 125mL volumetric concentration, which is added, is 75% ethyl alcohol, under the conditions of temperature is 65 DEG C, after ultrasonic extraction 30min, the condition for being 350 revs/min in revolving speed by extracting solution After lower centrifugation, filter to get filtrate, it is spare；
Filter residue is added into the ethyl alcohol that 100ml volumetric concentration is 75%, under the conditions of temperature is 60 DEG C, Microwave Extraction After 40min, extracting solution after centrifugation, is filtered to get filtrate under conditions of revolving speed is 350 revs/min；
After inciting somebody to action filtrate mixing twice, under the conditions of temperature is 70 DEG C, it is concentrated by evaporation after being 1:20 to solid-to-liquid ratio, obtains paste alcohol Extract.
Further preferably, the product is drug or health care product.
Further preferably, the administration mode of the drug is oral, injection, infusion or cutaneous penetration.
Further preferably, the product is cosmetics and skin care item.
Further preferably, the concentration of Saussurea involucrata culture is more than or equal to 12.5mg/ml in the product.
Although saussurea involucrata has been disclosed for oxidation resistant effect, Saussurea involucrata culture of the invention is to pass through plant by Herba Saussureae Involueratae (specific cultural method is detailed in ZL200510115902.0) that object cell culture technology obtains, color, shape and Herba Saussureae Involueratae It is entirely different, and the content of brass in Saussurea involucrata culture, polysaccharide, chlorogenic acid, ligustrin is significantly larger than wild Tianshan Mountains Saussurea involucrata, in addition, also without containing colchicin this small amount of toxic component in Herba Saussureae Involueratae in Saussurea involucrata culture.Therefore, saussurea involucrata Although culture derives from Herba Saussureae Involueratae, different with Herba Saussureae Involueratae.
Non-enzymatic glycation will lead to human skin aging, and the present invention is found by experiment that: Saussurea involucrata culture can Effectively inhibit non-enzymatic glycation, to illustrate that Saussurea involucrata culture can be realized the anti-aging of human skin, is applied to preparation In resisting age of skin product.
Specific embodiment
Explanation is further explained to the present invention combined with specific embodiments below, but is not intended to restrict the invention Protection scope.
Non-glycosylation (nonenzymatic glycosylation, NEG) aging theory, egg caused by non-glycosylation The main reason for white matter crosslinking damage is aging.Non-glycosylation not only results in the crosslinking denaturation of various albumen, antioxidase With the damage of the functions such as DNA repair enzyme, but also the reduction of energy supply will cause, the reduction of metabolic function, dysequilibrium etc. Ageing process.Research has shown that Advanced glycation endproducts (advanced glycationend products, AGEs) are not only Cell and function of organization can be directly affected, the generation of disease is participated in, it can also be by changing albumen in conjunction with specific receptors Matter and cell function lead to the pathological change of body.
Non-enzymatic glycation leads to being mainly shown as skin aging: wrinkle and age pigment are formed.
1) wrinkle
There are a large amount of collagen and elastin laminin in fell skin tissue, both protein metabolisms are slow, and containing more Lysine and hydroxylysine, this for occur NEG reaction provides material base.
The glucose of collagen and extracellular fluid occurs NEG and forms AGEs, and increases with advancing age.With AGEs progressive increases, and collagen forms intermolecular cross-linking, not only reduces the permeability of connective tissue, also makes nutriment and gives up The diffusion of object weakens, tissue ductility and hardness increase；It reduces the solubility of collagen and is difficult to be made by collagen enzyme hydrolysis At skin flexibility decrease, wrinkle is generated.Since the AGEs irreversibility generated causes wrinkle constantly to be deepened and is not easy to calm down.Bullet Property albumen and extracellular fluid glucose occur NEG formed AGEs, cause elastomer occur denaturation crosslinking, elasticity subside, skin Skin is thinning, water content is reduced, the further atrophy of skin, crumple.
2) age pigment
Another appearance for being mainly characterized by cholesteroderma and old color spot of skin aging, this variation mainly with old color The formation of element is related.Age pigment then refers to all ageing-related fluorescent material and its composite parts of intraor extracellular, such as eye The aging collagen tissue etc. of yellow fluorescence, is additionally comprised in biochemical research in ball lens cataract, cytoplasm From the fluorchrome etc. of ageing tissues.The formation of age pigment is dynamic consecutive variations process in organism, in different biologies The all ages and classes stage of individual, different tissues position are all likely to form the age pigment with different biological natures.Age pigment Including the intracorporal AGEs of biology, lipofuscin etc..Wherein lipofuscin is a kind of brown color pigment granule, mainly includes two aspects that one It is lipid peroxidation, it is another, it is NEG.The starting reaction of lipid peroxidation is unsaturated fatty acid and oxygen in aerobic situation Free radical or excessive metallic compound reaction lose a hydrogen atom, form peroxy radical, peroxy radical is subdivided solution shape At various dimethyl carbonates (DMCs).From peroxidatic reaction of lipid and the NEG DMCs reacted and different biological molecules to be crosslinked Or substance i.e. lipofuscin and AGEs that the mode of polymerization generates brown, has fluorescence, with the increase lipofuscin and AGEs at age It can build up on Skin Cell and chromatophore, lead to cholesteroderma and the appearance of foxiness eventually.The product of AGEs and lipofuscin Tire out and crosslink rear collagen and elastin laminin and forms skin splash in the accumulation of certain point.
To sum up, the advanced glycation end products that non-enzymatic glycation generates are the main reason for leading to human skin aging, Therefore, as long as effectively inhibiting the progress of non-enzymatic glycation, the production of advanced glycation end products is prevented or is reduced, can be realized Skin it is anti-aging.
Therefore, as long as being experimentally confirmed Saussurea involucrata culture has the function of inhibiting non-enzymatic glycation, can illustrate It has the function of skin anti-aging, has the function of inhibiting non-enzymatic glycation, inventor to verify Saussurea involucrata culture Devise following test.
Embodiment
It measures Saussurea involucrata culture and inhibits non-enzymatic glycation
Detection content: in pH7.4 phosphate buffer, it is with bovine serum albumin (BSA)-glucose under the conditions of 37 DEG C Model under the conditions of excitation wavelength 370nm, slit 3nm, launch wavelength 440nm, slit 3nm, uses fluorescence after being incubated for 15 days Spectrophotometric determination each group fluorescent value studies the alcohol of same batch various concentration and different batches various concentration Saussurea involucrata culture Inhibiting effect of the extract to non-enzymatic glycation dead end product (AGEs).
Experimental principle: it is primarily due to BSA and the significant structural homology of human serum albumins (HSA), is chosen as external sugar The protein of base model, D-Glucose are external glycosylated reduced sugar.It is slow in the phosphate that pH7.4 concentration is 0.2mol/L In fliud flushing system, between the free amino of protein terminal and reduced sugar carbonyl group, by condensation, rearrangement, cracking, oxidative modification Formed afterwards it is a series of structure is complicated and stable irreversible polymer, with fluorescence advanced glycation end products (AGEs).In reality It tests to be added in system and there is the substance for inhibiting non-enzymatic glycation, the amount that AGEs is generated can be reduced with delayed response.
Experimental material and instrument: experiment is mentioned with all Saussurea involucrata cultures by Dalian Puruikang Biotechnology Co For every bag of 10g dress.PH=7.4, concentration are that the phosphate-buffered salt of 0.2mol/L is configured according to States Pharmacopoeia specifications.96% cow's serum Albumin (the magnificent Bioisystech Co., Ltd in Luoyang), sodium azide (analysis is pure), glucose (analysis is pure), volumetric concentration is 75% ethyl alcohol (Tianjin great Mao) is purchased from Dalian Fei Te company.Amino orphan (AG, positive control) is purchased from the examination of Chinese medicines group chemistry Agent Co., Ltd.
UV-3000 double beam ultraviolet-visible spectrophotometer (Shimadzu, Japan)；F-7000 fluophotometer (Hitachi, Japan)；PHS-3C type acidometer (Shanghai Lei Ci instrument plant)；Electronic balance, water bath with thermostatic control.
Experimental method:
(1) alcohol extract of various concentration Saussurea involucrata culture inhibits non-enzymatic glycation
Sample solution configuration: precision weighs Saussurea involucrata culture dry product (H180104) 10.0g, and after being crushed, crossing granularity is The ethyl alcohol that 200mL volumetric concentration is 75% is added in the sieve of 30 mesh, under the conditions of temperature is 65 DEG C, after ultrasonic extraction 30min, Extracting solution after centrifugation, is filtered to get filtrate under conditions of revolving speed is 350 revs/min, it is spare；Filter residue is added into 200ml volume Extracting solution in revolving speed is 350 revs/min after Microwave Extraction 40min under the conditions of temperature is 60 DEG C by the ethyl alcohol that concentration is 75% Under conditions of be centrifuged after, filter to get filtrate；After inciting somebody to action filtrate mixing twice, under the conditions of temperature is 70 DEG C, it is concentrated by evaporation to solid-liquid After for 1:20, weighing obtains cream solid 4.010g, with the dissolution of 10mL water, is configured to 400mg/mL mother liquor.Successively it is diluted to The sample solution of 50mg/mL, 25mg/mL, 12.5mg/mL, 6.25mg/mL, 3.125mg/mL.
Bovine serum albumin solution configuration: weighing 1g bovine serum albumin(BSA), and 200mg sodium azide mixed solution is added It is dissolved in 7.4 phosphate buffer solution of 100mL pH.
The configuration of glucose solution: weighing glucose 7.45g and be added in 7.4 phosphate buffer solution of 100mL pH and dissolve, 74.5mg/mL solution.
Taking concentration respectively is the sample solution of 50mg/ml, 25mg/ml, 12.5mg/ml, 6.25mg/ml, 3.125mg/ml And 1mL glucose solution and 1mL bovine serum albumin solution is added in blank group (water) 1mL, places 15 under 37 DEG C of aseptic conditions It, 3 groups of operation repetitive.After 15 days, fluorescent spectrophotometer assay each group fluorescent value, location parameter are as follows: excitation wavelength are used 370nm, slit 3nm, launch wavelength 440nm, slit 3nm.
Measurement result see the table below:
Wherein, inhibiting rate %:I=(a-b)/a × 100% (a is blank group fluorescent value, and b is sample-adding group fluorescent value)
As seen from the above table, various concentration Saussurea involucrata culture alcohol extract is inhibited to non-enzymatic glycation, and Increase as concentration increases its inhibiting effect.
(2) alcohol extract of different batches Saussurea involucrata culture inhibits non-enzymatic glycation
Sample solution configuration: precision weighs different batches Saussurea involucrata culture 5.0g, and after being crushed, crossing granularity is 30 purposes The ethyl alcohol that 125mL volumetric concentration is 75% is added in sieve, under the conditions of temperature is 65 DEG C, after ultrasonic extraction 30min, will extract Liquid after centrifugation, filters to get filtrate under conditions of revolving speed is 350 revs/min, spare；Filter residue, which is added 100ml volumetric concentration, is 75% ethyl alcohol, under the conditions of temperature is 60 DEG C, after Microwave Extraction 40min, the condition for being 350 revs/min in revolving speed by extracting solution After lower centrifugation, filter to get filtrate；After inciting somebody to action filtrate mixing twice, under the conditions of temperature is 70 DEG C, being concentrated by evaporation to solid-to-liquid ratio is 1: After 20, weighing obtains cream solid, is configured to the sample solution that concentration is 6.25mg/mL, 12.5mg/mL.
Other are measured according to (one) method.
For AG as positive control, concentration is 1.0 × 10-4, 2.0 × 10-4, 5.0 × 10-4, 1.0 × 10-3, 2.0 × 10- 3mol/L。
Wherein, inhibiting rate %:I=(a-b)/a × 100% (a is blank group fluorescent value, and b is sample-adding group fluorescent value)
As seen from the above table, each batch various concentration Saussurea involucrata culture alcohol extract has non-enzymatic glycation and inhibits to make With when extract concentrations are 6.25mg/ml, inhibiting rate is higher than 60%, and when concentration is 12.5mg/m, inhibiting rate is up to 90% or more, compared with positive control AG, it is equivalent to 2.0 × 10-3The inhibiting effect of mol/L.
Separately to above-mentioned five batches, the external non-enzymatic glycation of the alcohol extract of two concentration Saussurea involucrata cultures inhibit into Row statistics, specifically see the table below:
As seen from the above table, the alcohol extract of the Saussurea involucrata culture of same concentration different batches is to non-outside BSA-D glucose body The inhibiting effect of enzyme glycosylation does not have significant difference.
(3) the different alcohol extracts of Saussurea involucrata culture inhibit non-enzymatic glycation
Following solution is carried out according to different alcohol extracting methods respectively to prepare and test, specific as follows:
Method 1: precision weighs part Saussurea involucrata culture dry product (H180104) 5.0g, and after being crushed, crossing granularity is 30 purposes The ethyl alcohol that 125mL volumetric concentration is 75% is added in sieve, and under the conditions of temperature is 65 DEG C, alcohol reflux is extracted, and extracting solution is existed After revolving speed is centrifuged under conditions of being 350 revs/min, filter to get filtrate, it is spare；It is 75% that filter residue, which is added 100ml volumetric concentration, Ethyl alcohol in revolving speed be 350 revs/min of condition by extracting solution after alcohol reflux extracts 40min under the conditions of temperature is 60 DEG C After lower centrifugation, filter to get filtrate；After inciting somebody to action filtrate mixing twice, under the conditions of temperature is 70 DEG C, being concentrated by evaporation to solid-to-liquid ratio is 1: After 30, weighing obtains 3 parts of cream solids, and it is 6.25mg/mL, 12.5mg/mL that 3 parts of cream solids, which are each configured to concentration, Sample solution.
Method 2: precision weighs part Saussurea involucrata culture dry product (H180104) 5.0g, and after being crushed, crossing granularity is 30 purposes The ethyl alcohol that 125mL volumetric concentration is 75% is added in sieve, under the conditions of temperature is 65 DEG C, after ultrasonic extraction 30min, will extract Liquid after centrifugation, filters to get filtrate under conditions of revolving speed is 350 revs/min, spare；Filter residue, which is added 100ml volumetric concentration, is 75% ethyl alcohol, under the conditions of temperature is 60 DEG C, after Microwave Extraction 40min, the condition for being 350 revs/min in revolving speed by extracting solution After lower centrifugation, filter to get filtrate；；After inciting somebody to action filtrate mixing twice, under the conditions of temperature is 70 DEG C, being concentrated by evaporation to solid-to-liquid ratio is 1: After 20, weighing obtains cream solid, is configured to the sample solution that concentration is 6.25mg/mL, 12.5mg/mL.
Method 3: precision weighs part Saussurea involucrata culture dry product (H180104) 5.0g, and after being crushed, crossing granularity is 15 purposes The ethyl alcohol that 125mL volumetric concentration is 60% is added in sieve, under the conditions of temperature is 40 DEG C, after ultrasonic extraction 30min, will extract Liquid after centrifugation, filters to get filtrate under conditions of revolving speed is 350 revs/min, under the conditions of temperature is 45 DEG C, is concentrated by evaporation to solid-liquid After for 1:20, weighing obtains cream solid, is configured to the sample solution that concentration is 6.25mg/mL, 12.5mg/mL.
Other are measured according to (one) method, as a result see the table below
Wherein, inhibiting rate %:I=(a-b)/a × 100% (a is blank group fluorescent value, and b is sample-adding group fluorescent value)
As seen from the above table, inhibitory effect and extraction alcohol of the alcohol extracting thing of Saussurea involucrata culture for non-enzymatic glycation The relevant property of concentration.
Those skilled in the art after considering the specification and implementing the invention disclosed here, will readily occur to of the invention its Its embodiment.This application is intended to cover any variations, uses, or adaptations of the invention, these modifications, purposes or Person's adaptive change follows general principle of the invention and including the undocumented common knowledge in the art of the present invention Or conventional techniques.The description and examples are only to be considered as illustrative, and true scope and spirit of the invention are wanted by right It asks and points out.

Claims (7)

1.雪莲培养物抑制蛋白质非酶糖基化反应在抗衰老产品中的应用。1. The application of snow lotus culture to inhibit protein non-enzymatic glycosylation reaction in anti-aging products. 2.根据权利要求1所述的应用，其特征在于，所述抗皮肤老化为抗皱纹以及抗老年色素形成。2. The application according to claim 1, characterized in that the anti-aging skin is anti-wrinkle and anti-aging pigmentation. 3.根据权利要求1所述的应用，其特征在于，所述雪莲培养物为雪莲培养物的醇提物，其具体制备方法为：3. application according to claim 1, is characterized in that, described snow lotus culture is the ethanol extract of snow lotus culture, and its specific preparation method is: 称取雪莲培养物5.0g，将其粉碎后，过粒度为30目的筛子，加入125mL体积浓度为75％的乙醇，在温度为65℃条件下，超声提取30min后，将提取液在转速为350转/分的条件下离心后，过滤得滤液，备用；Weigh 5.0 g of snow lotus culture, pulverize it, pass through a sieve with a particle size of 30 mesh, add 125 mL of ethanol with a volume concentration of 75%, and ultrasonically extract for 30 min at a temperature of 65 ° C. After centrifugation under the condition of rpm, filter to obtain the filtrate for subsequent use; 将滤渣再加入100ml体积浓度为75％的乙醇，在温度为60℃条件下，微波提取40min后，将提取液在转速为350转/分的条件下离心后，过滤得滤液；Add 100 ml of ethanol with a volume concentration of 75% to the filter residue, and microwave extraction at a temperature of 60° C. for 40 minutes, centrifuge the extract at a speed of 350 rpm, and filter to obtain the filtrate; 将两次滤液混合后，在温度为70℃条件下，蒸发浓缩至固液比为1:20后，得膏状醇提物。After mixing the two filtrates, at a temperature of 70°C, evaporate and concentrate to a solid-to-liquid ratio of 1:20 to obtain a paste-like ethanol extract. 4.根据权利要求1或2所述的应用，其特征在于，所述产品为药品或保健品。4. The application according to claim 1 or 2, characterized in that the product is a medicine or a health care product. 5.根据权利要求4所述的应用，其特征在于，所述药品的给药方式为口服、注射、输液或透皮给药。5. The application according to claim 4, characterized in that the drug is administered in oral, injection, infusion or transdermal administration. 6.根据权利要求1或2所述的应用，其特征在于，所述产品为化妆品和护肤品。6. The application according to claim 1 or 2, characterized in that the product is cosmetics and skin care products. 7.根据权利要求4或6所述的应用，其特征在于，所述产品中雪莲培养物的浓度大于等于12.5mg/ml。7. The application according to claim 4 or 6, characterized in that the concentration of Saussurea edulis culture in the product is greater than or equal to 12.5 mg/ml.
CN201810896249.3A 2018-08-08 2018-08-08 Saussurea involucrata culture inhibits protein non-enzyme glycosylation to react the application in anti-aging product Pending CN109078033A (en)

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Application publication date: 20181225