CN105662941A - Immature bitter orange extract product and preparation method thereof and application of immature bitter orange extract product for delaying skin aging - Google Patents
Immature bitter orange extract product and preparation method thereof and application of immature bitter orange extract product for delaying skin aging Download PDFInfo
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- CN105662941A CN105662941A CN201610019332.3A CN201610019332A CN105662941A CN 105662941 A CN105662941 A CN 105662941A CN 201610019332 A CN201610019332 A CN 201610019332A CN 105662941 A CN105662941 A CN 105662941A
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- aurantii immaturus
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- immature bitter
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/133—Amines having hydroxy groups, e.g. sphingosine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/75—Rutaceae (Rue family)
- A61K36/752—Citrus, e.g. lime, orange or lemon
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
- A61K8/41—Amines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/60—Sugars; Derivatives thereof
- A61K8/602—Glycosides, e.g. rutin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
Abstract
The invention relates to the field of plant extraction, and especially relates to an immature bitter orange extract product, a preparation method and an application of the immature bitter orange extract product for delaying skin aging. The provided extraction method improves the extraction technological parameters, aiming at the anti-aging effect. The obtained immature bitter orange extract product can obviously reduce the injury of ultraviolet ray on fibroblast and keratinocyte, the expression of type I collagen in fibroblast can be promoted, the expression of MMP-1 is inhibited, and efficacy for delaying skin aging can be performed. The immature bitter orange extract product can reach a purpose of delaying skin aging from epidermis and dermis, and research insufficiency of health care efficacy of the immature bitter oranges can be made up. The immature bitter orange extract product provides powerful support for development of anti-aging food, health products and cosmetics.
Description
Technical field
The present invention relates to field of plant extraction, particularly relate to a kind of Furctus Aurantii Immaturus extract and preparation method thereof and apply with in delay skin aging.
Background technology
Aging, is refer to each organ dysfunction of body process general, that be gradually lowered. In body aging process, skin very easily appears old and feeble vestige owing to directly contacting with external environment. Skin aging generally has two kinds of forms of expression: natural aging and photoaging.
The skin thickness general trend of naturally-aged is thinning, and epidermal renewal slows down, and stratum corneum barrier function declines, and flattens with corium connecting portion. Epidermis cell is gradually in flat, and keratinocyte vigor declines, and horny layer hydratability declines. Natural skin aging is to be determined by inherited genetic factors and internal hormonal readiness.
Photoaging is then an independent process, and it is obvious to the infringement of collagen protein in skin and elastic fibers. The negative effect of skin dermis can be promoted the generation of senilism by irradiation. DNA in skin directly can be caused damage by UVB, and the harmful effect of skin is produced by UVA mainly by oxygen-derived free radicals, integrate the damage that can cause nucleus and mitochondrial DNA and cause the activation of some enzymes such as substrate gold fibroin enzyme, and then the 26S Proteasome Structure and Function of damaging cells epimatrix.
Since ancient times, defying age is the problem that people earnestly pay close attention to, and the product of defying age is also varied, not only includes the compositions of various Chinese medicine, also includes various active substance. The product of these defying age has the raising effect such as immunity of organisms, alleviating physical fatigue mostly, but whether epidermis cell is effectively rarely had and mentions.
Fructus Aurantii Immaturus (FructusAurantiiImmaturus) is one of tcm clinical practice common drug, bitter in the mouth warm in nature, pungent, sour, returns taste warp, has dispelling the stagnated QI removing food stagnancy, the effect of scattered painful abdominal mass of reducing phlegm. Through chemical composition analysis, the main active of Fructus Aurantii Immaturus includes flavonoid, alkaloids, volatile oil etc. Wherein, Flavone content accounts for 5%～28%, is mainly Hesperidin, hesperetin, naringin, naringenin etc. Alkaloids is mainly Neosynephrine, N-methyltyramine etc., and the content in Fructus Aurantii Immaturus is about about 10mg/g.Volatile oil mainly includes terpene compound, based on monoterpene.
Furctus Aurantii Immaturus extract purposes in defying age has not yet to see report.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is in that to provide a kind of Furctus Aurantii Immaturus extract and preparation method thereof to apply with in delay skin aging, Furctus Aurantii Immaturus extract provided by the invention can significantly reduce the ultraviolet injury to fibroblast and keratinocyte, promote NTx protein expression in fibroblast, suppress the expression of MMP-1, play effect of delay skin aging.
The invention provides a kind of Furctus Aurantii Immaturus extract, mainly include naringin that mass fraction is 17.35%～29.27%, mass fraction be 13.83%～26.82% neohesperidin and mass fraction be the Neosynephrine of 1.69%～3.03%.
The preparation method of Furctus Aurantii Immaturus extract provided by the invention is, is pulverized by Fructus Aurantii Immaturus decoction pieces, extracts with ethanol water and prepares Furctus Aurantii Immaturus extract;
In ethanol water, the volume fraction of ethanol is 30%～90%;
The temperature extracted is 20 DEG C～70 DEG C, and the time of extraction is 1h～3h.
Fructus Aurantii Immaturus decoction pieces is the medical material that Fructus Aurantii Immaturus crude drug obtains after concocting. Described Fructus Aurantii Immaturus crude drug is fructus citri aurantii immaturi, Fructus Citri Fructus Aurantii Immaturus, Fructus Ponciri Trifoliatae, Fructus Citri sinensis Fructus Aurantii Immaturus; Described concocting method is cutting or parched with bran.
Described cutting is for add watertight moistening to fully absorbing rear shave by Fructus Aurantii Immaturus crude drug, in 60 DEG C of dry 12h.
Described parched with bran be by health product Fructus Aurantii Immaturus and Testa Tritici with mass ratio for 100:5 in 180 DEG C of parch 1min.
Crude drug at the Fructus Aurantii Immaturus decoction pieces of present invention employing is fructus citri aurantii immaturi or Fructus Citri sinensis Fructus Aurantii Immaturus, and concocting method is parched with bran.
Different solvents has different polarity, and the dissolubility of various compositions is also different, and the extract of different solvent extraction gained is not virgin yet. Fructus Aurantii Immaturus decoction pieces is extracted by the present invention through ethanol water, and the extract of gained tests prove that to have, it is possible to suppresses fibroblast old and feeble, increases collagenation, it is suppressed that collagenase; Keratinocyte can also be suppressed old and feeble, to corium and epidermis combined effect to anti aging effect simultaneously. The present invention passes through Optimizing Process Parameters, makes the extract obtained anti-aging effects to skin significantly improve.
In certain embodiments, in ethanol water, the volume fraction of ethanol is 60%.
In further embodiments, in ethanol water, the volume fraction of ethanol is 30% or 90%.
The infiltration of material, dissolving, diffusivity with temperature raise and increase, the viscosity of solution with temperature raise and reduce, therefore, in extraction heating can strengthen molecular motion, again can softening tissue, improve dissolubility, accelerate diffusion thus improving extraction ratio. But too high temperature is not only returned and is caused the volatilization of solvent in extraction process, also can make some that heat sensitive active substance is degraded.
The extraction ratio of effective ingredient increases with the prolongation of extraction time, till reaching balance. Therefore, long extraction time it is not necessary that, not only lose time, and often nullified composition extend in time and proposes in a large number, affect extract quality, reduce the effect of extract.
In certain embodiments, the temperature of extraction is 50 DEG C, and the time of extraction is 2h.
In further embodiments, the temperature of extraction is 20 DEG C or 70 DEG C, and the time of extraction is 1h or 3h.
Generally, it is considered that solvent load is too low, effective ingredient can not fully leach, if but consumption is excessive, then can bring burden to enriching service.
In an embodiment of the present invention, Fructus Aurantii Immaturus decoction pieces is 1:(5～20 with the quality-volume ratio of ethanol water).
In certain embodiments, Fructus Aurantii Immaturus decoction pieces is 1:15 with the quality-volume ratio of ethanol water.
In further embodiments, the quality-volume ratio of Fructus Aurantii Immaturus decoction pieces and ethanol water is 1:5 or 1:20,
Extraction effect also can be produced impact by extraction time, and, extraction time is in close relations with Extraction solvent. In the present invention, it it is 1 time to the extraction time of Fructus Aurantii Immaturus.
Effective ingredient often includes concentration and dry step after extracting, quality and the effect of extract are had direct impact by dry or concentration method. Theoretically, the time of concentration is more long, and temperature is more high, and loss of effective components is more big.
In an embodiment of the present invention, the step dried also is included with ethanol water after extracting; Dry method is vacuum film concentration; Dry temperature is 20～60 DEG C.
In certain embodiments, dry temperature is 45 DEG C.
In further embodiments, dry temperature is 20 DEG C or 60 DEG C.
According to FICK second law, the size of pulverizing medicinal materials can affect extraction effect, and by raw material pulverizing to a certain extent, more than increase solid, the contact area of solvent can make extraction efficiency be significantly improved, but the granularity of for the purpose of the present invention, extract obtained effect and pulverizing is not proportionate.
In an embodiment of the present invention, Fructus Aurantii Immaturus decoction pieces is crushed to granularity is 10 order～65 orders.
In certain embodiments, being crushed to granularity is 60 orders.
The extracting method of active substance of plant is varied, and extracting method affects composition and the extraction efficiency of extract. The extraction of Fructus Aurantii Immaturus is adopted water-bath to extract by the present invention.
Many factors influences each other, and combined effect determines the composition of extract and the content of effective ingredient. ?
In one embodiment of the invention, Furctus Aurantii Immaturus extract preparation method particularly as follows:
By Fructus Aurantii Immaturus decoction pieces, being crushed to granularity is 24 orders; Add 5 times of quality volume fraction be 30% ethanol water, extract 1h in 20 DEG C of water-baths, filter, obtain filtrate; Filtrate concentrates through vacuum film in 20 DEG C, obtains Furctus Aurantii Immaturus extract.
In one embodiment of the invention, Furctus Aurantii Immaturus extract preparation method particularly as follows:
By Fructus Aurantii Immaturus decoction pieces, being crushed to granularity is 50 orders; Add 20 times of quality volume fraction be 90% ethanol water, extract 3h in 70 DEG C of water-baths, filter, obtain filtrate; Filtrate concentrates through vacuum film in 60 DEG C, obtains Furctus Aurantii Immaturus extract.
In one embodiment of the invention, Furctus Aurantii Immaturus extract preparation method particularly as follows:
By Fructus Aurantii Immaturus decoction pieces, being crushed to granularity is 60 orders; Add 15 times of quality volume fraction be 90% ethanol water, extract 2h in 50 DEG C of water-baths, filter, obtain filtrate; Filtrate concentrates through vacuum film in 45 DEG C, obtains Furctus Aurantii Immaturus extract.
Furctus Aurantii Immaturus extract prepared by method provided by the invention application in preparing collagen protein expression accelerator.
In the present invention, Furctus Aurantii Immaturus extract can promote that the collagen protein expressed is I-type collagen. The particularly I-type collagen in fibroblast.
In the present invention, Furctus Aurantii Immaturus extract promotes that the dosage that collagen protein is expressed is 2 μ g/mL～200 μ g/mL.
As preferably, Furctus Aurantii Immaturus extract promotes that the dosage that collagen protein is expressed is 200 μ g/mL.
Collagen protein is the main component in animal connective tissue, and wherein I-type collagen is primarily present skin and the osseous tissue of remaining people. Research shows, in the skin of intrinsic aging, collagen and elastic fiber all show as minimizing. In the photoaging skin that ultraviolet radiation causes, the minimizing of collagen becomes apparent from, but a large amount of hypertrophy of elastic fiber and degeneration. Being verified by experiments, Furctus Aurantii Immaturus extract prepared by the present invention can increase the expression of I-type collagen content in fibroblast.Further, under ultra-vioket radiation effect, Furctus Aurantii Immaturus extract remains to promote the expression of I-type collagen in fibroblast. And without Furctus Aurantii Immaturus extract process cell after ultra-vioket radiation, the content of I-type collagen significantly reduces. Test shows, Furctus Aurantii Immaturus extract promotes that the effect that collagen protein is expressed is dose-dependence.
The Furctus Aurantii Immaturus extract that provider's method of the present invention prepares application in preparing MMP-1 expression inhibitor.
In the present invention, Furctus Aurantii Immaturus extract can suppress the expression of MMP-1 in fibroblast.
In the present invention, Furctus Aurantii Immaturus extract suppresses the MMP-1 dosage expressed to be 2 μ g/mL～200 μ g/mL.
As preferably, Furctus Aurantii Immaturus extract suppresses the MMP-1 dosage expressed to be 200 μ g/mL.
Fibroblast collagenase (MMP-1) is explanation I type and the topmost enzyme of type III collagen, when MMP-1 overexpression, selective degradation extracellular matrix components, destroys the normal configuration of collagen fiber and elastic fibers, causes that the senile symptoms such as shrinkage microgroove occurs in skin. Experiments show that, Furctus Aurantii Immaturus extract prepared by the present invention can significantly reduce the expression of MMP-1 in fibroblast, even if under ultraviolet radiates, the expression of MMP-1 is also had significant inhibitory action by Furctus Aurantii Immaturus extract prepared by the present invention, and without Furctus Aurantii Immaturus extract process cell after ultra-vioket radiation, the expression of MMP-1 significantly raises.
The Furctus Aurantii Immaturus extract that provider's method of the present invention prepares is used for reducing ultraviolet to cell injury.
The Furctus Aurantii Immaturus extract that provider's method of the present invention prepares can reduce the ultraviolet damage to fibroblast or epidermis cell.
It is 2 μ g/mL～200 μ g/mL that this Furctus Aurantii Immaturus extract can reduce the ultraviolet dosage to fibroblast or keratinocyte.
As preferably, it is 200 μ g/mL that this Furctus Aurantii Immaturus extract can reduce the ultraviolet dosage to fibroblast or keratinocyte.
Fibroblast and keratinocyte are the main cells constituting human skin, and wherein fibroblast is positioned at corium, and keratinocyte is positioned at epidermis. Experiments show that, under ultra-vioket radiation, Furctus Aurantii Immaturus extract provided by the invention can make the gray value of fibroblast aging model be effectively controlled, and this effect is dose-dependence. Under ultra-vioket radiation, the gray value of this extract Human Keratinocytes has significant inhibitory action, and this effect is obvious dose-dependence.
The present invention provides the Furctus Aurantii Immaturus extract application in the product preparing defying age that preparation method prepares.
The preparation method that the invention provides a kind of Furctus Aurantii Immaturus extract, and study discovery, this extract can promote the expression of collagen protein, it is suppressed that the expression of MMP-1, and can reduce the ultra-vioket radiation damage to fibroblast or keratinocyte. Therefore, the Furctus Aurantii Immaturus extract that this extract method prepares can be used in preparing the product of defying age. The product of described defying age refers to comprise the food of Furctus Aurantii Immaturus extract, medicine, health product or the cosmetics that provider's method of the present invention prepares. In the present invention, the product of described defying age is preferably cosmetics. Effect of described anti-aging cosmetics is mainly: removal wrinkle, desalination microgroove, improvement relax, and increase elastic.
The extracting method provided in the present invention improves Extraction technique for anti-aging effects, obtained Furctus Aurantii Immaturus extract can significantly reduce the ultraviolet injury to fibroblast and keratinocyte, promote NTx protein expression in fibroblast, suppress the expression of MMP-1, play effect of delay skin aging.Furctus Aurantii Immaturus extract in the present invention is worked along both lines from epidermis and corium and is reached the purpose of delay skin aging, compensate for the research to Fructus Aurantii Immaturus health-care effect not enough. For antisenility cistanche food, health product, cosmetics exploitation, it is provided that strong support.
Detailed description of the invention
The invention provides a kind of Furctus Aurantii Immaturus extract and preparation method thereof to apply with in delay skin aging, those skilled in the art can use for reference present disclosure, is suitably modified technological parameter and realizes. Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are considered as including in the present invention. Method and the application of the present invention are described already by preferred embodiment, methods herein and application substantially can be modified or suitably change and combination by related personnel in without departing from present invention, spirit and scope, realize and apply the technology of the present invention.
Material, reagent or instrument that the present invention adopts are all common commercially available product, all can buy in market.
Below in conjunction with embodiment, the present invention is expanded on further:
Embodiment 1
Take Fructus Aurantii Immaturus decoction pieces, pulverize, cross No. 2 screen clothes, obtain 24 order granularity raw materials. Weigh 30% ethanol water that above-mentioned Immature Orange Fruit adds 5 times, extract 1h in 20 DEG C of water-baths, filter, obtain filtrate. Filtrate concentrates in 20 DEG C of vacuum films, obtains extract. HPLC detects the content of the naringin in Furctus Aurantii Immaturus extract and neohesperidin respectively 20.78% and 13.83%, and the content of Neosynephrine is 2.12%.
Embodiment 2
Take Fructus Aurantii Immaturus decoction pieces, pulverize, cross No. 3 screen clothes, obtain 50 order granularity raw materials. Weigh 90% ethanol water that above-mentioned Immature Orange Fruit adds 20 times, extract 3h in 70 DEG C of water-baths, filter, obtain filtrate. Filtrate concentrates in 60 DEG C of vacuum films, obtains extract. HPLC detects the content of the naringin in Furctus Aurantii Immaturus extract and neohesperidin respectively 17.35% and 22.65%, and the content of Neosynephrine is 1.69%.
Embodiment 3
Take Fructus Aurantii Immaturus decoction pieces, pulverize, cross No. 4 screen clothes, obtain 60 order granularity raw materials. Weigh 60% ethanol water that above-mentioned Immature Orange Fruit adds 15 times, extract 2h in 50 DEG C of water-baths, filter, obtain filtrate. Filtrate concentrates in 45 DEG C of vacuum films, obtains extract. HPLC detects the content of the naringin in Furctus Aurantii Immaturus extract and neohesperidin respectively 29.27% and 26.82%, and the content of Neosynephrine is 3.03%.
Embodiment 4 fibroblast NTx protein expression and matrix metalloproteinase (MMP-1) are expressed
With the birth Sprague-Dawley rat neonatal rat (Shanghai western pul-Bi Kai laboratory animal company limited) of latter 1 day. Put in iodophor solution and put to death. Under aseptic condition, take back full thickness skin. Prune subcutaneous tissue, fat, blood vessel etc. Skin is cut into the strip of 0.5cm*2cm. 3 times (also can rinsing) is washed with D-hank ' the s liquid containing gentamycin 800U/ml with the DMEM (CorningCellgro) containing 10% hyclone. Put in the dispase (Sigma) of 0.5% containing 1-2% hyclone DMEM configuration, 4 DEG C of refrigerator digestion 14h. D-hank ' s (Wuhan Boster Bioisystech Co., Ltd) liquid rinses 2 times. With ophthalmic tweezers, epidermis is separated with corium. After separation, corium is used for fibroblastic cultivation. The corium tissue shear of separation is fully shredded, about 0.1mm3Size, is placed in 37 DEG C, 5%CO2Incubator in 0.5%～0.04% trypsinization 30min, repeatedly blow and beat gently with Pasteur's pipe, dispel as far as possible, the DMEM of 10%FBS terminates digestion, by 150 order stainless steel sift net filtrations, centrifugal 1200rpm, 5min, add 5ml culture medium, move in 75ml culture bottle, insert 37 DEG C, 5%CO2Incubator in original cuiture.Change liquid after Fibroblast cell-culture 3d first, go down to posterity with the trypsinization of 0.25%～0.1% when cell reaches 70%～80% fusion, digest by the above process, inoculation of going down to posterity. How radially, braided or swirling traveling after fibroblast inoculation, about 4h is visible near-wall air curtain, and cell stretches out the attachment of pseudopodium initial stage, and after inoculation, 3d visible cell becomes flat, increasing number, and in fusiformis, after going down to posterity, Growth of Cells arranges relatively rule.
Utilize the fibroblast in 2-4 generation, with 12-16h after the trypsinization paving dish of 0.125%, give 60mJ/cm2UVB irradiate, use 40 μ g/ml, 4 μ g/ml, the naringin of 0.4 μ g/ml, neohesperidin, 4 μ g/ml, 0.4 μ g/ml, 0.04 μ g/ml Neosynephrine and 200 μ g/ml, 20 μ g/ml, 2 μ g/ml embodiments 1～3 process 48h. Collect cell, and crack with Trizol, extracted total RNA, use TaKaRaRNAPCR test kit to carry out RT-PCR reaction, reverse transcription reaction condition: 55 DEG C of 20min, 99 DEG C of 5min, 5 DEG C of 5min. PCR reaction condition: 94 DEG C of 5min, 94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 30sec, circulate 30 times, product is electrophoresis in 1%agarose. Product band and marker band are compared, and for internal reference, amplified production are carried out sxemiquantitative with GAPDH.
The fibroblast aging model cell that Example 1～3 and middle extract-treated are crossed carries out total serum IgE and carries out PCR, and result is such as shown in table 1～2. After ultra-vioket radiation, in fibroblast, NTx protein content reduces, and MMP-1 expresses rising. After naringin, neohesperidin, Neosynephrine, Furctus Aurantii Immaturus extract process, all increasing collagen protein and express, reduce MMP-1 and express, and there is dose-dependant, the anti-aging effects of Furctus Aurantii Immaturus extract is better than naringin, Hesperidin list, Neosynephrine one-component simultaneously.
Fibroblast aging model NTx protein expression is affected by table 1 extract
Fibroblast aging model matrix metalloproteinase (MMP-1) is expressed impact by table 2 extract
The embodiment 5 impact on fibroblast aging model
Utilize the fibroblast in 2-4 generation, with 12-16h after the trypsinization paving dish of 0.125%, give 60mJ/cm2UVB irradiate, use 40 μ g/ml, 4 μ g/ml, the naringin of 0.4 μ g/ml, neohesperidin, 4 μ g/ml, 0.4 μ g/ml, 0.04 μ g/ml Neosynephrine and 200 μ g/ml, 20 μ g/ml, 2 μ g/ml embodiments 1～3 process 48h. Old and feeble staining kit is utilized to carry out BgaA dyeing. Test arranges undressed normal control, and does not only adopt the model group of extraction process through ultra-vioket radiation.
In embodiment 1～3, extract is as shown in table 3 to the action effect of ultra-vioket radiation fibroblast aging model. After ultra-vioket radiation, old and feeble fibroblast increases. 2～200 μ g/ml Furctus Aurantii Immaturus extracts process, and all can significantly reduce fibroblastic aging (P < 0.01), there is dose-dependant, and the anti-aging effects of Furctus Aurantii Immaturus extract is better than naringin, Hesperidin, Neosynephrine.
The impact on fibroblast aging model of table 3 extract
* compare with model group, P < 0.05; * compares with model group, P < 0.01.
Embodiment 6: the effect to HaCaT cell ageing model
HaCaT (offer of Fu Xiang bio tech ltd, Shanghai) cell, through the trypsinization of 0.125%, terminates digestion with the DMEM containing 10%FBS, is centrifuged (1200rpm, 10min), resuspended with DMEM, counts and inoculates paving dish.The HaCaT cell utilizing 2-4 generation carries out old and feeble experiment. The UVB that the HaCaT cell in 2-4 generation gives 120mJ is irradiated, and is subsequently adding corresponding tested material and processes, and utilizes old and feeble staining kit to carry out BgaA dyeing, count positive cell number after dyeing after irradiation after 48h.
Embodiment 1～3 extract is as shown in table 4 to the action effect of ultra-vioket radiation HaCaT aging model. After ultra-vioket radiation, old and feeble stained positive keratinocyte increases. Process through Furctus Aurantii Immaturus extract, all can significantly reduce the aging (P < 0.01) of keratinocyte, the anti-aging effects of Furctus Aurantii Immaturus extract is better than naringin, Hesperidin, Neosynephrine simultaneously, and the inhibitory action of Furctus Aurantii Immaturus extract has dose-dependant.
The impact on HaCaT aging model of table 4 extract
*, compare with model group, P < 0.05; *, compares with model group, P < 0.01.
Below it is only the preferred embodiment of the present invention; it should be pointed out that, for those skilled in the art, under the premise without departing from the principles of the invention; can also making some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (10)
1. a Furctus Aurantii Immaturus extract, it is characterised in that mainly include naringin that mass fraction is 17.35%～29.27%, mass fraction be 13.83%～26.82% neohesperidin and mass fraction be the Neosynephrine of 1.69%～3.03%.
2. the preparation method of Furctus Aurantii Immaturus extract described in claim 1, pulverizes including by Fructus Aurantii Immaturus decoction pieces, extracts with ethanol water and prepares Furctus Aurantii Immaturus extract;
In described ethanol water, the volume fraction of ethanol is 30%～90%;
The temperature of described extraction is 20 DEG C～70 DEG C, and the time of extraction is 1h～3h.
3. preparation method according to claim 2, it is characterised in that in described ethanol water, the volume fraction of ethanol is 60%; Quality-the volume ratio of described Fructus Aurantii Immaturus decoction pieces and ethanol water is 1:(5～20).
4. preparation method according to claim 2, it is characterised in that the temperature of described extraction is 50 DEG C, the time of extraction is 2h.
5. preparation method according to claim 2, it is characterised in that the described step also to include drying after ethanol water extraction; Described dry method is vacuum film concentration; Described dry temperature is 20～60 DEG C.
6. the application in preparing collagen protein expression accelerator of the Furctus Aurantii Immaturus extract described in claim 1.
7. the application in preparing MMP-1 expression inhibitor of the Furctus Aurantii Immaturus extract described in claim 1.
8. Furctus Aurantii Immaturus extract described in claim 1 is used for reducing ultraviolet to cell injury.
9. the application in the product preparing defying age of the Furctus Aurantii Immaturus extract described in claim 1.
10. the cosmetics of a defying age, it is characterised in that include Furctus Aurantii Immaturus extract described in claim 1.
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| CN115813838A (en) * | 2022-12-07 | 2023-03-21 | 广州兰蝶生物科技有限公司 | Cosmetic with anti-aging composition and preparation method and application thereof |
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