CN113956369A - Preparation method and application of Caryopteris clandonensis polysaccharide - Google Patents

Preparation method and application of Caryopteris clandonensis polysaccharide Download PDF

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CN113956369A
CN113956369A CN202111226468.9A CN202111226468A CN113956369A CN 113956369 A CN113956369 A CN 113956369A CN 202111226468 A CN202111226468 A CN 202111226468A CN 113956369 A CN113956369 A CN 113956369A
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caryopteris
polysaccharide
precipitate
ethanol
deionized water
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CN113956369B (en
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赵莹
刘懿娴
陈慧平
周火祥
王卫东
谢巍
许芳
赵倩伟
章金涛
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Henan Academy of Medical and Pharmaceutical Sciences
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • 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
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • 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
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • 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
    • 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
    • A61K2800/74—Biological properties of particular ingredients
    • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
    • A61K2800/782—Enzyme inhibitors; Enzyme antagonists

Abstract

The invention discloses a preparation method of Caryopteris clandonensis polysaccharide, which comprises the following steps: taking cleaned and dried Caryopteris clathrata leaves, crushing, sieving with a 40-mesh sieve, adding deionized water according to a material-liquid ratio of 1:20, soaking at normal temperature for 2h, heating, boiling and extracting for 6h, collecting an extracting solution, filtering, concentrating under reduced pressure, precipitating a concentrated solution with 4 times of 95% ethanol for 12h, redissolving a precipitate with deionized water to the volume of an original supernatant, repeatedly precipitating with 4 times of 95% ethanol for one time, washing the precipitate with 95% ethanol, absolute ethanol, acetone and diethyl ether according to 1g of precipitate and 50mL of solvent for 3 times alternately, volatilizing the solvent, redissolving the precipitate in deionized water, deproteinizing by a Sevag method, and freeze-drying to obtain the Caryopteris cla polysaccharide.

Description

Preparation method and application of Caryopteris clandonensis polysaccharide
Technical Field
The invention relates to the technical field of natural extracts in cosmetics, and particularly relates to a preparation method and application of Caryopteris clandonensis polysaccharide.
Background
Caryopteris terniflora Maxim is a Caryopteris plant of Verbenaceae, subshrubes, also called Liuyue Han, herba Hydnocarpi, flos Carthami, wild sesame, roadside tips, HUAGUDAN, wild sesame, Potentilla chinensis, wild mint, herba Schizonepetae, and HUACIZHONG bubble. The Caryopteris santalina resource is rich, is mainly distributed in places such as Sichuan, Henan, Hebei, Shanxi, Gansu and the like, is mostly a positive plant, is pleased with light, is pleased with dryness, is extremely drought-resistant, low-temperature resistant, barren-resistant, strong in adaptability and has high ecological benefit and greening value. In addition, the Caryopteris clando has pungent and slightly bitter taste and mild nature, has the efficacies of dispelling wind, relieving exterior syndrome, diffusing lung, relieving cough, promoting blood circulation, regulating menstruation and the like, is a common herbal medicine for folk, is mainly used for treating cold, cough, whooping cough, nebula, postpartum abdominal pain, water and fire scald and the like, and has higher medicinal value.
Tyrosinase inhibitors are currently recognized whitening additives for whitening cosmetics, such as the whitening spot-lightening ingredient kojic acid. Therefore, the strengthening of the development of tyrosinase inhibitors can enrich the variety of whitening and spot-fading additives in cosmetics.
The plant polysaccharide is a gift for human beings, and many plant polysaccharides have biological activities, such as health promotion effects of regulating immunity, resisting tumor, reducing blood sugar, reducing blood lipid, resisting radiation, resisting bacteria and virus, and protecting liver. Therefore, the plant polysaccharide has been widely applied to the fields of medical science, catering and other public lives. The application of the plant polysaccharide in cosmetics has the advantages of safety and no irritation.
Disclosure of Invention
In view of the fact that the research on the polysaccharide in the Caryopteris triquetrum in the prior art is few and no report on the antioxidant activity and the tyrosinase inhibitory activity is available, the invention provides a preparation method and application of the Caryopteris triquetrum polysaccharide.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for preparing Caryopteris clandonensis polysaccharide, the method comprises: taking cleaned and dried Caryopteris clathrata leaves, crushing, sieving with a 40-mesh sieve, adding deionized water according to a material-liquid ratio of 1:20, soaking at normal temperature for 2h, heating, boiling and extracting for 6h, collecting an extracting solution, filtering, concentrating under reduced pressure, precipitating a concentrated solution with 4 times of 95% ethanol for 12h, redissolving a precipitate with deionized water to the volume of an original supernatant, repeatedly precipitating with 4 times of 95% ethanol for one time, washing the precipitate with 95% ethanol, absolute ethanol, acetone and diethyl ether according to 1g of precipitate and 50mL of solvent for 3 times alternately, volatilizing the solvent, redissolving the precipitate in deionized water, deproteinizing by a Sevag method, and freeze-drying to obtain the Caryopteris cla polysaccharide.
Further, based on the basis of the invention, the invention also provides an application of the Caryopteris clandonensis polysaccharide as a natural antioxidant.
Furthermore, based on the characteristic of the Caryopteris clando polysaccharide which can be used as a natural antioxidant, the invention further provides a cosmetic with antioxidant, skin whitening and speckle lightening effects, wherein the cosmetic contains an additive Caryopteris clando polysaccharide, and the mass percent of the additive Caryopteris clando polysaccharide is 2-10%.
The invention has the beneficial effects that: the test result shows that the Caryopteris triquetrum polysaccharide has high antioxidant activity and high tyrosinase inhibition effect, and can be used for developing natural antioxidants and cosmetic additives with antioxidant, whitening and spot-lightening effects.
Detailed Description
The present invention will be further described below, it should be noted that the present embodiment is based on the technical solution, and a detailed implementation manner and a specific operation process are provided, but the protection scope of the present invention is not limited to the present embodiment.
Example 1
A method for preparing Caryopteris clandonensis polysaccharide, the method comprises: taking cleaned and dried Caryopteris clathrata leaves, crushing, sieving with a 40-mesh sieve, adding deionized water according to a material-liquid ratio of 1:20, soaking at normal temperature for 2h, heating, boiling and extracting for 6h, collecting an extracting solution, filtering, concentrating under reduced pressure, precipitating a concentrated solution with 4 times of 95% ethanol for 12h, redissolving a precipitate with deionized water to the volume of an original supernatant, repeatedly precipitating with 4 times of 95% ethanol for one time, washing the precipitate with 95% ethanol, absolute ethanol, acetone and diethyl ether according to 1g of precipitate and 50mL of solvent for 3 times alternately, volatilizing the solvent, redissolving the precipitate in deionized water, deproteinizing by a Sevag method, and freeze-drying to obtain the Caryopteris cla polysaccharide.
The content of the polysaccharide is 87.23% and the content of the polysaccharide is higher by the phenol-sulfuric acid method. The protein content in polysaccharide is 0.76% and the protein content is very little, which is determined by Coomassie brilliant blue method.
Example 2
Anti-oxidation effect of Caryopteris clando polysaccharide
1. DPPH-scavenging activity of Caryopteris sanhuanensis polysaccharide
(1) Preparation of a DPPH test solution: 8.0mg of DPPH is accurately weighed and dissolved by 100mL of methanol to prepare 0.08mg/mL DPPH solution which is kept away from light for later use.
(2) The experimental steps are as follows: preparing the Caryopteris clandonensis polysaccharide prepared in the example 1 and a positive drug Butylated Hydroxytoluene (BHT) into solutions to be detected with five series concentrations of 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL and 0.06mg/mL, and mixing a 2.0mL series concentration Caryopteris clandonensis polysaccharide solution and 1.0mL DPPH solution to be recorded as a solution s; mixing polysaccharide solution with 2.0mL of series concentration and 1.0mL of methanol to obtain solution b; 2.0mL of methanol and 1.0mL of DPPH solution were combined and designated as solution c. Placing the above solution in a shaking table at room temperature in a dark place for 30min, measuring absorbance values As, Ab and Ac at 517nm, setting 3 multiple holes in each group, and measuring for 3 times of repeated experiments. According to the formula: (iii) clearance (%) [1- (As-Ab)/Ac]Calculating the DPPH free radical clearance rate by x 100%, and plotting the inhibition rate against the inhibitor concentration to obtain IC of the polysaccharide and the positive antioxidant BHT according to the inhibition curve50The value is obtained. The results are shown in Table 1.
2. Oenothera trilobata polysaccharide activity in eliminating OH
A sample solution of 2.0mg/mL was prepared from the Caryopteris clandonensis polysaccharide prepared in example 1 and the positive drug VC. Taking 5 pieces of the test tube, adding 2.0mmol/L FeSO into the test tube41.0mL and 6.0mmol/L salicylic acid-ethanol solution 1.0mL, shaking, adding polysaccharide solution 0, 0.2, 0.4, 0.6, 0.8, and 1.0mL, respectively, adding distilled water 1, 0.8, 0.6, 0.4, 0.2, and 0.0mL, respectively, adding 1.0mmol/L H2O21.0mL, shaken, heated in a 37 ℃ water bath for 30min, zeroed with distilled water, and the absorbance measured at 510nm using an ultraviolet spectrophotometer. The OH clearance was calculated As follows, and (%) - ([ 1- (As-Ab)/Ac)]X 100% where AcAbsorbance value for 1mL of distilled water, AsTo add absorbance values of samples of different concentrations, AbTo add no H2O2The absorbance value of (a). At the same time, the inhibition rate is used to plot the concentration of the inhibitor, and the IC of the Caryopteris clando polysaccharide and the positive antioxidant VC are obtained according to the inhibition curve50The value is obtained. The results are shown in Table 1.
Figure BDA0003314280800000051
Example 3
Inhibitory Activity of Caryopteris triquetrum polysaccharides against tyrosinase
1. Inhibitory Effect of Caryopteridis Incanae polysaccharide on tyrosinase monophenolase
The Caryopteris clandonensis polysaccharide prepared in example 1 and the positive drug kojic acid are respectively prepared into sample solutions with different concentrations, the reaction is carried out in a 96-well culture plate, and the total reaction system is 200 mu L: sample solutions of different concentrations were 40 μ L; 80 mu L of 67mmol/L phosphate buffer solution; 40 mu L of 100U/mL tyrosinase solution; 2.5mmol/L L-tyrosine 40. mu.L. Zero set well add only 67mmol/L phosphate buffer 200. mu.L. After the sample is added, mixing uniformly, placing the mixture into a water bath incubator at 37 ℃ for incubation for 20min, detecting the specific light absorption value of the reaction product at 490nm by using an enzyme linked immunosorbent assay detector, and repeating the test for 3 times.
The samples and kojic acid were calculated for tyrosinase monophenol according to the following formulaInhibition of enzymes. Inhibition rate (%) ([ 1- (A) ]1-A2)/(A3-A4)]X 100%, wherein: a. the1、A2、A3、A4The absorbance of the sample group, the sample background group (PBS instead of tyrosinase), the blank group (distilled water instead of sample), and the blank background group (distilled water instead of sample, PBS instead of tyrosinase), respectively. At the same time, the inhibition rate is used to plot the concentration of the inhibitor, and the IC of the Caryopteris clando polysaccharide and the positive drug kojic acid is obtained according to the inhibition curve50The value is obtained. The results are shown in Table 2.
2. Inhibitor of tyrosinase-diphenolase by Caryopteris clando polysaccharide
Referring to the monophenolase inhibition step, 2.5mmol/L L-dopa solution was used instead of 2.5mmol/L L-tyrosine solution, incubation was continued at 37 ℃ for 20min, and absorbance was measured at 490nm immediately after shaking. Calculating the inhibition rate of the sample and kojic acid on tyrosinase and bisphenol enzyme according to the formula in formula 3.1, and calculating IC50The value is obtained. The results are shown in Table 2.
Figure BDA0003314280800000061
In conclusion, the test results of the invention show that the Caryopteris triquetrum polysaccharide has high antioxidant activity and high tyrosinase inhibition effect, and can be used for developing natural antioxidants and cosmetic additives with antioxidant, whitening and spot-lightening effects.
Various other changes and modifications to the above-described embodiments and concepts will become apparent to those skilled in the art from the above description, and all such changes and modifications are intended to be included within the scope of the present invention as defined in the appended claims.

Claims (3)

1. A method for preparing Caryopteris clandonensis polysaccharide, which comprises the following steps: taking cleaned and dried Caryopteris clathrata leaves, crushing, sieving with a 40-mesh sieve, adding deionized water according to a material-liquid ratio of 1:20, soaking at normal temperature for 2h, heating, boiling and extracting for 6h, collecting an extracting solution, filtering, concentrating under reduced pressure, precipitating a concentrated solution with 4 times of 95% ethanol for 12h, redissolving a precipitate with deionized water to the volume of an original supernatant, repeatedly precipitating with 4 times of 95% ethanol for one time, washing the precipitate with 95% ethanol, absolute ethanol, acetone and diethyl ether according to 1g of precipitate and 50mL of solvent for 3 times alternately, volatilizing the solvent, redissolving the precipitate in deionized water, deproteinizing by a Sevag method, and freeze-drying to obtain the Caryopteris cla polysaccharide.
2. Use of the polysaccharide of Caryopteris clathrata as claimed in claim 1, as a natural antioxidant.
3. The cosmetic with the effects of resisting oxidation, whitening and fading spots is characterized by comprising an additive of Caryopteris clathrata polysaccharide, wherein the mass percent of the Caryopteris clathrata polysaccharide is 2-10%.
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CN114262642A (en) * 2021-12-23 2022-04-01 河南省医药科学研究院 Method for extracting and purifying volatile oil of Caryopteris clandonensis and application thereof

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