CN116693383A - Preparation and anti-aging application of coumaric acid - Google Patents
Preparation and anti-aging application of coumaric acid Download PDFInfo
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- CN116693383A CN116693383A CN202310689551.2A CN202310689551A CN116693383A CN 116693383 A CN116693383 A CN 116693383A CN 202310689551 A CN202310689551 A CN 202310689551A CN 116693383 A CN116693383 A CN 116693383A
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- coumaric acid
- carob
- ethanol
- ethyl acetate
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- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 230000003712 anti-aging effect Effects 0.000 title claims abstract description 6
- 239000002253 acid Substances 0.000 title description 14
- NGSWKAQJJWESNS-UHFFFAOYSA-N 4-coumaric acid Chemical compound OC(=O)C=CC1=CC=C(O)C=C1 NGSWKAQJJWESNS-UHFFFAOYSA-N 0.000 claims abstract description 67
- NGSWKAQJJWESNS-ZZXKWVIFSA-M 4-Hydroxycinnamate Natural products OC1=CC=C(\C=C\C([O-])=O)C=C1 NGSWKAQJJWESNS-ZZXKWVIFSA-M 0.000 claims abstract description 33
- DFYRUELUNQRZTB-UHFFFAOYSA-N Acetovanillone Natural products COC1=CC(C(C)=O)=CC=C1O DFYRUELUNQRZTB-UHFFFAOYSA-N 0.000 claims abstract description 32
- 240000008886 Ceratonia siliqua Species 0.000 claims abstract description 28
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- 206010051246 Photodermatosis Diseases 0.000 claims abstract description 10
- 239000002537 cosmetic Substances 0.000 claims abstract description 4
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- 230000003647 oxidation Effects 0.000 claims abstract description 3
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 59
- 239000003208 petroleum Substances 0.000 claims description 21
- 238000000605 extraction Methods 0.000 claims description 16
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 12
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- 150000003254 radicals Chemical class 0.000 abstract description 3
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- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 5
- AIJULSRZWUXGPQ-UHFFFAOYSA-N Methylglyoxal Chemical compound CC(=O)C=O AIJULSRZWUXGPQ-UHFFFAOYSA-N 0.000 description 4
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical group OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 description 4
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- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 2
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- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 description 2
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- NGSWKAQJJWESNS-ZZXKWVIFSA-N trans-4-coumaric acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C=C1 NGSWKAQJJWESNS-ZZXKWVIFSA-N 0.000 description 2
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- HAMNKKUPIHEESI-UHFFFAOYSA-N aminoguanidine Chemical compound NNC(N)=N HAMNKKUPIHEESI-UHFFFAOYSA-N 0.000 description 1
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Classifications
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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
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/36—Carboxylic acids; Salts or anhydrides thereof
- A61K8/365—Hydroxycarboxylic acids; Ketocarboxylic acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C59/40—Unsaturated compounds
- C07C59/42—Unsaturated compounds containing hydroxy or O-metal groups
- C07C59/52—Unsaturated compounds containing hydroxy or O-metal groups a hydroxy or O-metal group being bound to a carbon atom of a six-membered aromatic ring
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
- A61K2800/522—Antioxidants; Radical scavengers
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
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- Oil, Petroleum & Natural Gas (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Emergency Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
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Abstract
The invention relates to a preparation method and application of carob pod extract p-coumaric acid. The carob pod extract provided by the invention has anti-aging effect, and in particular has the effects of resisting photoaging, resisting saccharification, resisting free radical oxidation and the like. Has good development potential in the field of cosmetics and/or foods.
Description
Technical Field
The invention relates to the field of cosmetics, in particular to a preparation method and application of p-coumaric acid extracted from carob fruits.
Background
Aging is represented by a decline in the function of cells and tissues over time, and skin serves as an external organ of the body and is an intuitive reference index for judging the degree of aging. The aging of human skin is gradually accelerated along with the aging of human skin, and the aging of the skin is accelerated in the modern society due to the factors of high pressure, irregular work and rest, electric radiation and the like. Skin aging is manifested by increased wrinkles, loose skin, yellowing, shiny and smooth skin, rough texture, increased color spots, etc. Skin aging is mainly caused by ultraviolet photoaging, oxidative free radicals, skin glycation, etc.
P-coumaric acid, also known as trans-p-hydroxy cinnamic acid, is one of the common ingredients of various food-borne fruits and vegetables, and has wide physiological activities of antioxidation, anti-inflammation, antibiosis, anti-apoptosis and the like. The preparation of the p-coumaric acid by chemical synthesis has the advantages of long time, large solvent consumption, low conversion rate and high cost, and limits the wide application of the p-coumaric acid. The p-coumaric acid is extracted by using natural plants through one-step alkaline hydrolysis, but the extraction effect is poor. HPLC samples are simple to prepare, do not need special treatment, but have weak separation capability, and are difficult to separate and detect hydroxy cinnamic acid dimer generated in the alkaline hydrolysis process. The coumaric acid has high activity, is easy to oxidize and light sensitive, and is easy to degrade in the extraction and processing process, so that the original activity function is greatly limited, and the coumaric acid is added and applied to foods and health-care products. The prior art is mainly applied to the protection of various diseases such as atherosclerosis, parkinson's disease, diabetes and the like. However, no report is made on the aging resistance of coumaric acid.
Disclosure of Invention
In one aspect, the invention provides a method for extracting p-coumaric acid with high purity, high content and high stability from carob pods.
The invention further provides an application of the p-coumaric acid extracted from the pod of the carob in the anti-aging field.
Specifically, the preparation method of the p-coumaric acid comprises the following steps:
providing pods of crushed carob seeds;
extracting with 80-95% ethanol, extracting with 60-75% ethanol, mixing extractive solutions, and concentrating to obtain extract;
diluting and dispersing the obtained extract with water, adsorbing with macroporous adsorbent resin D101, eluting with distilled water to remove saccharide impurities; eluting with 95% ethanol, and recovering solvent from the eluate; dispersing with methanol, extracting sequentially with petroleum ether and ethyl acetate;
recovering solvent from ethyl acetate extraction part, and separating and purifying by silica gel column, sephadex LH-20 column chromatography and semi-prepared liquid phase to obtain p-coumaric acid.
According to an embodiment of the present invention, preferably, the pod of the carob seed is crushed to 60-80 mesh, for example 60 mesh. It was found that the pod cell structure can be broken sufficiently within this particle size, facilitating sufficient dissolution of coumaric acid.
According to the embodiment of the invention, the extraction is performed by using 80-95% ethanol and then 60-75% ethanol, and researches show that the extraction rate of the coumaric acid can be increased.
According to the embodiment of the invention, when 80-95% ethanol is used for extraction for the first time, the weight ratio of 80-95% ethanol to pod of carob seeds is 5-10:1, and research shows that the method is favorable for removing part of impurities by precipitation.
According to the embodiment of the invention, 80-95% ethanol is used for extraction for 2-3 times for 1-3 hours for the first time.
According to the embodiment of the invention, when 60-75% ethanol is used for the second extraction, the weight ratio of 60-75% ethanol to pod of carob seeds is 5-10:1, and researches show that the method is beneficial to increasing the solubility of coumaric acid and improving the extraction rate.
According to the embodiment of the invention, the second extraction is performed 2-3 times with 60-75% ethanol, each time for 1-3 hours.
It was found that the ionizable substance, the saccharide substance, and the macromolecular substance can be separated by adsorption with the macroporous adsorbent resin D101.
It is found that substances with different partition coefficients can be separated by petroleum ether and ethyl acetate extraction.
According to the embodiment of the invention, preferably, when the ethyl acetate part is separated by adopting 200-300 mesh silica gel column chromatography, petroleum ether-ethyl acetate (1:1-0:1, volume ratio) is adopted for elution.
According to the embodiment of the invention, preferably, 200-300 mesh silica gel column chromatography is adopted for separation, and the eluent is petroleum ether-ethyl acetate (4:1-1:1 by volume ratio).
According to an embodiment of the invention, preferably, sephadex LH-20 column chromatography is used, and the eluent is dichloromethane-methanol (1:1 volume ratio).
According to an embodiment of the present invention, it is preferable to separate and purify the semi-preparative liquid phase, which is column i, methanol: purified water=52:48 (volume ratio), absorption wavelength: 280nm.
The invention provides the application of the p-coumaric acid in cosmetics, foods, medicines or health care products.
In particular, the p-coumaric acid is applied to anti-aging products and has at least one or more of oxidation resistance, photo-aging resistance and saccharification resistance.
Drawings
FIG. 1 is a graph showing the effect of carob source on coumaric acid on cell viability.
Figure 2 is the effect of carob source on coumaric acid on ROS levels.
FIG. 3 is a graph showing the effect of carob source on coumaric acid on superoxide anion radical scavenging.
FIG. 4 is a graph showing the effect of carob source on the inhibition of AGEs by coumaric acid.
Fig. 5 is a p-coumaric acid chemical formula.
Detailed Description
The following examples are illustrative of the invention but are not intended to limit the scope of the invention.
According to an embodiment of the present invention, the carob is a leguminous plant carob (academic name: ceratonia siliquaLinn.).
Example 1 extraction of p-coumaric acid from carob pods
Pulverizing dried carob pod into 60 mesh coarse powder, reflux-extracting with 90% ethanol 7 times the weight of the coarse powder for 3 times (1.5 hr each time), reflux-extracting with 70% ethanol 8 times the weight of the coarse powder for 1 time (1.5 hr), mixing the extractive solutions, and concentrating to obtain extract. Dispersing the extract with 5 times of water, adsorbing with macroporous adsorbent resin D101, eluting with distilled water for 3 column volumes to remove impurities such as saccharide, eluting with 95% ethanol for 3 column volumes, and recovering solvent to obtain non-saccharide small molecule part. Dispersing the obtained non-saccharide small molecule part with 4 times of 50% methanol, sequentially extracting with petroleum ether and ethyl acetate for 5 times, and recovering solvent to obtain petroleum ether part, ethyl acetate part and water part.
Recovering solvent from ethyl acetate part, separating by 200 mesh silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate, detecting by TLC thin layer with eluent petroleum ether-ethyl acetate volume ratio of 1:1, 1:3, 0:1, mixing similar fractions, and recording the fractions as A-E according to polarity from small to big.
And (3) performing gradient elution on the fraction B by adopting a 300-mesh silica gel column chromatography, wherein the eluent is petroleum ether-ethyl acetate, the volume ratio is 4:1, 3:1, 2:1 and 1:1, TLC thin layer detection is performed, and similar fractions are combined to obtain 4 fractions B1-B4.
Eluting the fraction B2 with Sephadex LH-20 and dichloromethane-methanol (1:1) to obtain 5 fractions B2a-B2e, and separating the fraction B2e with semi-prepared liquid phase (column I, methanol: purified water=52:48 (volume ratio), absorption wavelength: 280 nm) to obtain p-coumaric acid (t) R =11.84 min), purity reaches 98.5%.
Example 2
Pulverizing dried carob pod into 80 mesh coarse powder, reflux-extracting with 80% ethanol 10 times the weight of the coarse powder for 3 times (1.5 hr each time), reflux-extracting with 60% ethanol 5 times the weight of the coarse powder for 1 time (1.5 hr), mixing the extractive solutions, and concentrating to obtain extract. Dispersing the extract with 6 times of water, adsorbing with macroporous adsorbent resin D101, eluting with distilled water for 3 column volumes to remove impurities such as saccharide, eluting with 95% ethanol for 3 column volumes, and recovering solvent to obtain non-saccharide small molecule part. Dispersing the obtained non-saccharide small molecule part with 50% methanol 5 times of the weight, extracting with petroleum ether and ethyl acetate respectively for 5 times, and recovering solvent to obtain petroleum ether part, ethyl acetate part and water part.
Recovering solvent from ethyl acetate part, separating with 300 mesh silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate, detecting with TLC thin layer with eluent petroleum ether-ethyl acetate volume ratio of 1:1, 1:3, 0:1, mixing similar fractions, and recording the fractions as A-E according to polarity from small to big.
And (3) performing gradient elution on the fraction B by adopting a 200-mesh silica gel column chromatography, wherein the eluent is petroleum ether-ethyl acetate, the volume ratio is 4:1, 3:1, 2:1 and 1:1, TLC thin layer detection is performed, and similar fractions are combined to obtain 4 fractions B1-B4.
Eluting the fraction B2 with Sephadex LH-20 and dichloromethane-methanol (1:1) to obtain 5 fractions B2a-B2e, and separating the fraction B2e with semi-prepared liquid phase (column I, methanol: purified water=52:48 (volume ratio), absorption wavelength: 280 nm) to obtain p-coumaric acid (t) R =11.84 min), purity reaches 97.5%.
Experimental example 3
Pulverizing dried carob pod into 70 mesh coarse powder, reflux-extracting with 85% ethanol for 3 times (1.5 hr each time) and 75% ethanol for 1 time (1.5 hr) and 10 times, mixing extractive solutions, and concentrating to obtain extract. Dispersing the extract with 5 times of water, adsorbing with macroporous adsorbent resin D101, eluting with distilled water for 3 column volumes to remove impurities such as saccharide, eluting with 95% ethanol for 3 column volumes, and recovering solvent to obtain non-saccharide small molecule part. Dispersing the obtained non-saccharide small molecule part with 2 times of 50% methanol, sequentially extracting with petroleum ether and ethyl acetate for 5 times, and recovering solvent to obtain petroleum ether part, ethyl acetate part and water part.
Recovering solvent from ethyl acetate part, separating by 200 mesh silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate, detecting by TLC thin layer with eluent petroleum ether-ethyl acetate volume ratio of 1:1, 1:3, 0:1, mixing similar fractions, and recording the fractions as A-E according to polarity from small to big.
And (3) performing gradient elution on the fraction B by adopting a 300-mesh silica gel column chromatography, wherein the eluent is petroleum ether-ethyl acetate, the volume ratio is 4:1, 3:1, 2:1 and 1:1, TLC thin layer detection is performed, and similar fractions are combined to obtain 4 fractions B1-B4.
Eluting the fraction B2 with Sephadex LH-20 and dichloromethane-methanol (1:1) to obtain 5 fractions B2a-B2e, and separating the fraction B2e with semi-prepared liquid phase (column I, methanol: purified water=52:48 (volume ratio), absorption wavelength: 280 nm) to obtain p-coumaric acid (t) R =11.84 min), purity reached 97%.
Comparative example 1
The only difference compared to example 1 is that the ethanol concentration of the first ethanol reflux extraction was 70%.
The p-coumaric acid is obtained, and the purity reaches 62.4 percent.
Comparative example 2
The only difference compared to example 1 is that the ethanol concentration of the second ethanol reflux extraction was 50%.
The p-coumaric acid is obtained, and the purity reaches 64.1%.
Comparative example 3
The only difference compared to example 1 is that the comminution gives a 50 mesh coarse powder.
The p-coumaric acid is obtained, and the purity reaches 60.5 percent.
Comparative example 4
The only difference compared to example 1 is that p-coumaric acid was obtained in 40.1% purity by separation with semi-preparative liquid phase (column i, methanol: purified water=50:50, absorption wavelength: 280 nm).
Comparative example 5
The only difference compared to example 1 is that p-coumaric acid was obtained in a purity of 32.5% by separation with semi-preparative liquid phase (column i, methanol: purified water=48:52, absorption wavelength: 280 nm).
Experimental example
1. Effect of photo-aging
Selecting HaCaT cells, culturing under standard conditions of 37deg.C, saturated humidity and 5% CO with DMEM high sugar medium containing 100g/L calf serum and 10g/L penicillin-streptomycin 2 Concentration. Changing the culture medium every 2 days to give birth to cellsPassaging was performed until 90% of the cells were grown, passaging was performed using a medium containing 0.25% of pancreatin-EDTA, digestion was stopped immediately after digestion was completed, the supernatant was removed by centrifugation, and the cells were inoculated into a desired cell culture dish after the cell density was adjusted using a high-sugar DMEM medium.
The experiments were divided into a normal control group, a photoaging model group and a high (10. Mu.g/ml), medium (5. Mu.g/ml), low (2. Mu.g/ml) dose group (p-coumaric acid prepared in example 1). Adding DMEM culture medium into normal control group and photoaging model group, adding DMEM culture medium containing the products prepared in example 1 at different doses into the examples, sucking out the culture medium after cell culture until cell adhesion is completed, adding 1mL (6-well plate) PBS (phosphate buffer saline) into each well, performing ultraviolet irradiation UVA (ultraviolet irradiation) on the other groups except the blank group for 10cm, wherein the irradiation time is 30min, and the irradiation dose is 5J/cm 2 PBS was removed. Adding DMEM culture medium, culturing at 37deg.C, saturated humidity and 5% CO 2 After 24 hours of incubation at concentration, cell viability (MTT method), ROS levels in the cells were measured.
The results of the test are shown in fig. 1 and 2, and the cell viability and ROS level of the photoaging model group are significantly different from those of the blank control group, indicating that the modeling was successful. The cell viability of the high dose group and the medium dose group were significantly different, and the cell number was significantly higher than that of the photoaging model group, indicating that p-coumaric acid extracted from carob pods helped to increase cell viability. The ROS levels in the example groups are extremely significantly different, and particularly the high-dose group is significantly lower than that in the photoaging model group, which shows that p-coumaric acid extracted from carob pods can inhibit the increase of ROS content in cells and effectively reduce the damage caused by ultraviolet rays.
2. Determination of free radical scavenging Effect
100mg of p-coumaric acid prepared in example 1 was accurately weighed, and a 60% ethanol solution was added to prepare a 10mg/ml sample solution, and the sample solutions were diluted to different concentrations of 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml and 1 mg/ml.
Taking 4mL of LTris-HCI (pH 8.2), 2mL of pyrogallol solution (10 mmol/L) into a test tube, adding 2mL of sample solutions with different concentrations into an a-e tube, adding 2mL of 60% ethanol solution into an o tube as a control group, mixingAfter shaking, the mixture was reacted in a water bath at 37℃for 10 minutes, and the change in absorbance was measured at 325 nm. Calculation of superoxide anion (O) 2- ) Radical scavenging rate. The results are shown in FIG. 3.
The calculation formula is as follows: clearance = (Ao-a Sample )x 100%/Ao
The results show that p-coumaric acid obtained by separation and extraction from carob pods can remove superoxide anion free radicals, and the higher the concentration, the higher the removal rate.
3. Anti-glycation damage
100mg of p-coumaric acid prepared in example 1 was accurately weighed, a 60% ethanol solution was added to prepare a 10mg/ml sample solution, and the sample solutions (samples 1 to 4) were diluted to 1mg/ml, 2mg/ml, 4mg/ml and 8mg/ml at different concentrations.
1ml of 60mmol/L MGO (methylglyoxal) solution and 1ml of each component sample solution are taken and mixed uniformly in a test tube, after incubation for 2 hours at 37 ℃, 1ml of 30mg/ml BSA (bovine serum albumin) solution is added, 0.20mol/L phosphate buffer containing L% NaN3 (pH=7.4) is added for uniform dissolution, and incubation is carried out for 6 days at 37 ℃. Under the same conditions, 0.1mg/mLAG (aminoguanidine) solution was used as a positive control group, and a glycated protein solution was used as a blank control group. And detecting the fluorescence intensity of the sample under the condition of 370nm/440nm emission wavelength, and calculating the AGEs inhibition rate. The calculation formula is as follows:
the results of the inhibition ratio (%) = (blank fluorescence intensity-sample fluorescence intensity)/blank fluorescence intensity×100% are shown in fig. 4, and the inhibition ratio of AGEs of the sample group containing carob source to coumaric acid shows concentration dependence, and when the concentration is 8mg/ml, the inhibition ratio is higher than that of the positive control group, which indicates that the carob source has anti-glycation capability to coumaric acid.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (8)
1. P-coumaric acid is characterized by being extracted and separated from carob pods.
2. The process according to claim 1, characterized in that it is carried out as follows: alcohol extraction, macroporous adsorption resin D101 adsorption, petroleum ether extraction, ethyl acetate extraction, silica gel column chromatography, sephadexLH-20 column chromatography and semi-prepared liquid phase separation and purification.
3. The process according to claim 2, wherein the first extraction is carried out with 80-95% ethanol for 2-3 hours and then with 60-75% ethanol for 1-3 hours.
4. The preparation method according to claim 2, wherein the gradient elution is performed by using a 200-300 mesh silica gel column chromatography, the eluent is petroleum ether-ethyl acetate, the volume ratio of the first separation eluent is 1:1-0:1, and the volume ratio of the second separation eluent is 4:1-1:1.
5. P-coumaric acid from carob, characterized in that it is obtained by the preparation process according to claims 1-4.
6. Use of p-coumaric acid from carob as claimed in claim 5 in the cosmetic field.
7. Use of p-coumaric acid from carob as defined in claim 5 in the food field.
8. Use according to claims 6-7, characterized by anti-aging, anti-oxidation, anti-glycation, anti-photoaging, anti-free radical oxidation, at least with one or more of the above-mentioned characteristics.
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