CN116178583B - Pilose asiabell root polysaccharide acid shear segment CPP-1-1, preparation method and application thereof - Google Patents
Pilose asiabell root polysaccharide acid shear segment CPP-1-1, preparation method and application thereof Download PDFInfo
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- CN116178583B CN116178583B CN202310226970.2A CN202310226970A CN116178583B CN 116178583 B CN116178583 B CN 116178583B CN 202310226970 A CN202310226970 A CN 202310226970A CN 116178583 B CN116178583 B CN 116178583B
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- polysaccharide
- acid
- cpp
- adsorption resin
- macroporous adsorption
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61Q19/00—Preparations for care of the skin
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Abstract
The invention provides a codonopsis pilosula multi-sugar acid cut-off CPP-1-1 and a preparation method and application thereof, and relates to the technical field of codonopsis pilosula resource development. The invention improves the purity of the polysaccharide by purifying the pilose asiabell root crude polysaccharide with macroporous adsorption resin and removing lipid, protein and other impurities; the codonopsis pilosula polysaccharide acid shear fragment CPP-1-1 prepared by degrading and classifying and purifying the purified polysaccharide has relatively simple structure, small molecular weight and good water solubility, has good inhibition effect on tyrosine monophenolase and bisphenol enzyme, and can effectively remove DPPH, ABTS+, OH and O 2‑ Free radical, has good free radical oxidation resistance, aging prevention and excellent moisturizing effect, can effectively inhibit the generation of melanin, and can avoid the generation of problems such as skin lack of water, darkness, color spots and the like; has good application prospect in cosmetics and functional foods as a whitening agent, a moisturizing agent and an antioxidant.
Description
Technical Field
The invention relates to the technical field of radix codonopsis resource development, in particular to a radix codonopsis polysaccharide acid cut-off CPP-1-1, a preparation method and application thereof.
Background
The codonopsis pilosula Codonopsis spiculela is dried root of codonopsis pilosula Codonopsis pilosula (Franch.) which is perennial winding herb plant of platycodaceae, nannf, codonopsis pilosula Codonopsis spiculela Nannf. Var. Modesta (Nannf.) L.T. shen, codonopsis pilosula Codonopsis tangshen Oliv. And the like, is a plant with homology of medicine and food, and contains rich nutrient substances such as protein, organic lipid, various vitamins, mineral substances and the like and various effective components. In recent years, various types of compounds have been isolated and identified from the rhizomes of codonopsis pilosula, including polysaccharides, lignans, polyacetylenes and polyacetylenes glycosides, alkaloids, triterpenes, lignans, flavonoids, lactones and the like. Wherein, the polysaccharide is a main active ingredient and has higher medicinal value. Researches show that the codonopsis pilosula polysaccharide has the effects of regulating immune function, protecting stomach function, protecting nervous system, enhancing immunity, improving learning and memory disorder, resisting oxidation, virus, cancer, inflammation and the like.
The skin color of a person is determined by the content and distribution of melanin. Melanin is produced by melanocytes of the basal layer, is transported to the basal cells by the dendritic structure of the melanocytes, and then travels up the epidermis with the cells. Tyrosinase is an active site containing Cu 2+ Is also critical for the rate limiting effect in this process. Tyrosinase converts L-tyrosine to dopaquinone by two-step catalysis,this is the main cause of darkening of human skin. Modern pharmacological research shows that the total polysaccharide of codonopsis pilosula has an inhibition effect on the activity of tyrosinase. Other polar groups such as hydroxyl and carboxyl contained in polysaccharide molecules form hydrogen bonds with water molecules, a large amount of water in the air needs to be combined, meanwhile, polysaccharide molecular chains are mutually interwoven to form a net shape, and the water molecules are combined with the hydrogen bonds of water to play a very strong role in water retention. The polysaccharide also has good film forming performance, can form a layer of film on the surface of skin, reduces the evaporation of water on the surface of skin, ensures that the water is dispersed from basal tissues to the horny layer, induces the horny layer to be further hydrated, and preserves the water of the skin. Therefore, the highly water-absorbing and good film-forming properties of the polysaccharide are perfectly combined, and a good moisturizing effect can be provided for the skin. Research shows that the pilose asiabell root polysaccharide has the functions of eliminating in vitro and in vivo free radicals and superoxide anion, and can effectively relieve the condition of reduced activity of superoxide dismutase (SOD) and Catalase (CAT) in serum of aging model mice, and the anti-aging of the pilose asiabell root polysaccharide is related to free radical elimination and lipid peroxidation resistance. However, the reported total polysaccharide of radix codonopsis has large molecular weight and complex structure, and has undefined whitening, moisturizing and anti-aging effects, which affects the further development and utilization of radix codonopsis plants.
Disclosure of Invention
In view of the above, the invention aims to provide the pilose asiabell root polysaccharide acid cut-off CPP-1-1, and the preparation method and the application thereof, and the pilose asiabell root polysaccharide acid cut-off CPP-1-1 has low molecular weight, simple structure and excellent whitening, moisturizing and anti-aging effects.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a pilose asiabell root polysaccharide acid cut-off CPP-1-1, which comprises the following steps:
dissolving the crude radix codonopsis polysaccharide in water, and purifying the obtained crude radix codonopsis polysaccharide aqueous solution by macroporous adsorption resin to obtain purified polysaccharide;
sequentially carrying out acid shearing, neutralization and water dialysis on the purified polysaccharide to obtain a pilose asiabell root polysaccharide acid shearing fragment mixture;
subjecting the codonopsis pilosula polysaccharide acid cut-off mixture to DEAE-52 cellulose column chromatography to obtain a codonopsis pilosula polysaccharide acid cut-off CPP-1-1 crude product; the eluent adopted by the DEAE-52 cellulose column chromatography is water and NaCl solution in sequence, and the concentration of the NaCl solution is 0.01-0.5 mol/L;
subjecting the codonopsis pilosula polysaccharide acid cut-off CPP-1-1 crude product to Sephadex-G75 column chromatography to obtain codonopsis pilosula polysaccharide acid cut-off CPP-1-1; the eluent adopted by the Sephadex-G75 column chromatography is water and NaCl solution in sequence, and the concentration of the NaCl solution is 0.1-0.5 mol/L.
Preferably, the macroporous adsorption resin comprises one or more of macroporous adsorption resin S-8, macroporous adsorption resin ADS-F8, macroporous adsorption resin LSA-21, macroporous adsorption resin LSA-10, macroporous adsorption resin HP-20, macroporous adsorption resin ADS-17, macroporous adsorption resin NKA-9, macroporous adsorption resin DM130, macroporous adsorption resin AB-8, macroporous adsorption resin X-5, macroporous adsorption resin H103, macroporous adsorption resin D101, macroporous adsorption resin XAD-8 and macroporous adsorption resin DA 201.
Preferably, the conditions for purifying the macroporous adsorption resin include: the loading concentration of the codonopsis pilosula crude polysaccharide aqueous solution is 0.5-2 mg/mL, the eluent is water, the dosage of the eluent is 1-3 BV, and the flow rate of the eluent is 0.5-2 BV/h.
Preferably, the concentration of the acid solution for acid shearing is 0.04 to 0.3mol/L.
Preferably, the temperature of the acid shearing is 70-90 ℃ and the time is 1-3 h, and the acid shearing is carried out under inert atmosphere.
Preferably, the dialysis further comprises the steps of sequentially concentrating, alcohol precipitating and solid-liquid separating the obtained pilose asiabell root polysaccharide acid shear fragment mixture solution, and drying the obtained solid product to obtain the pilose asiabell root polysaccharide acid shear fragment mixture.
Preferably, the preparation method of the codonopsis pilosula crude polysaccharide comprises the following steps: degreasing radix codonopsis, extracting with water, and precipitating with lower alcohol to obtain crude polysaccharide of radix codonopsis.
Preferably, the volume fraction of the lower alcohol in the system is 55-85% during the lower alcohol precipitation; the lower alcohol comprises one or more of methanol, ethanol and propanol.
The invention provides a codonopsis pilosula polysaccharide acid shear segment CPP-1-1 prepared by the preparation method in the technical scheme, which comprises galacturonic acid, glucose and galactose, wherein the molar ratio of galacturonic acid to glucose to galactose is 45:0.65:0.26; the peak molecular weight Mp of the pilose asiabell root polysaccharide acid shearing segment CPP-1-1 is 2000-5000 Da.
The invention provides application of the pilose asiabell root polysaccharide acid cut-off segment CPP-1-1 in cosmetics or functional foods.
The invention provides a preparation method of a pilose asiabell root polysaccharide acid cut-off CPP-1-1, which comprises the following steps: dissolving the crude radix codonopsis polysaccharide in water, and purifying the obtained crude radix codonopsis polysaccharide aqueous solution by macroporous adsorption resin to obtain purified polysaccharide; sequentially carrying out acid shearing, neutralization and water dialysis on the purified polysaccharide to obtain a pilose asiabell root polysaccharide acid shearing fragment mixture; subjecting the codonopsis pilosula polysaccharide acid cut-off mixture to DEAE-52 cellulose column chromatography to obtain a codonopsis pilosula polysaccharide acid cut-off CPP-1-1 crude product; the eluent adopted by the DEAE-52 cellulose column chromatography is water and NaCl solution in sequence, and the concentration of the NaCl solution is 0.01-0.5 mol/L; subjecting the codonopsis pilosula polysaccharide acid cut-off CPP-1-1 crude product to Sephadex-G75 column chromatography to obtain codonopsis pilosula polysaccharide acid cut-off CPP-1-1; the eluent adopted by the Sephadex-G75 column chromatography is water and NaCl solution in sequence, and the concentration of the NaCl solution is 0.1-0.5 mol/L. The purity of polysaccharide is improved by degreasing codonopsis pilosula, extracting with water, precipitating with lower alcohol and purifying with macroporous adsorption resin to remove impurities such as lipid, protein, pigment and the like; the prepared pilose asiabell root polysaccharide acid shear fragment CPP-1-1 has relatively simple structure, small molecular weight, good water solubility, good inhibition effect on tyrosinase and bisphenol enzyme, excellent moisturizing effect and antioxidation effect, can effectively inhibit the generation of melanin, avoids the generation of problems such as skin water shortage, darkness, color spots and the like, has good application prospect in cosmetics and functional foods as a whitening agent, a humectant and an antioxidant, solves the problems of large molecular weight, complex structure, undefined whitening and moisturizing effect components, difficult practical application and the like of the pilose asiabell root polysaccharide, and makes a contribution to the research of pharmacological effects of the pilose asiabell root polysaccharide. As shown by the test results of the examples, the inhibition rate of the pilose asiabell root polysaccharide acid cut-off fragment CPP-1-1 provided by the invention on tyrosinase is dose-dependent, and the inhibition rates on tyrosinase monophenolase and bisphenol enzyme are both greater than those of pilose asiabell root crude polysaccharide; under the condition that the air humidity is 43%, the moisture retention percentage of the codonopsis pilosula polysaccharide acid shear segment CPP-1-1 after 24 hours is still above 99.6%, and the moisture retention effect is superior to that of glycerin, so that the codonopsis pilosula polysaccharide acid shear segment CPP-1-1 has excellent moisture retention effect.
Drawings
FIG. 1 is a flow chart of a process for preparing a pilose asiabell root polysaccharide acid cut segment of the invention;
FIG. 2 is a graph of adsorption/desorption rates for different types of resins;
FIG. 3 is a graph showing the effect of different factors on the purification effect of crude Codonopsis pilosula polysaccharide;
FIG. 4 is a contour plot and response surface of a composite score for different factor interactions;
FIG. 5 is a FT-IR diagram of a pilose asiabell root polysaccharide acid cut-off CPP-1-1;
FIG. 6 is a TG pattern of codonopsis pilosula polysaccharide acid cut-off CPP-1-1;
FIG. 7 is a HPGPC chart of a pilose asiabell root polysaccharide acid cut-off CPP-1-1;
FIG. 8 is an HPLC chromatogram of a PMP derivative of Codonopsis pilosula polysaccharide acid fragment CPP-1-1;
FIG. 9 is an SEM image of a codonopsis pilosula polysaccharide acid cut-out CPP-1-1.
Detailed Description
The invention provides a preparation method of a pilose asiabell root polysaccharide acid cut-off CPP-1-1, which comprises the following steps:
dissolving the crude radix codonopsis polysaccharide in water, and purifying the obtained crude radix codonopsis polysaccharide aqueous solution by macroporous adsorption resin to obtain purified polysaccharide;
sequentially carrying out acid shearing, neutralization and water dialysis on the purified polysaccharide to obtain a pilose asiabell root polysaccharide acid shearing fragment mixture;
subjecting the codonopsis pilosula polysaccharide acid cut-off mixture to DEAE-52 cellulose column chromatography to obtain a codonopsis pilosula polysaccharide acid cut-off CPP-1-1 crude product; the eluent adopted by the DEAE-52 cellulose column chromatography is water and NaCl solution in sequence, and the concentration of the NaCl solution is 0.01-0.5 mol/L;
Subjecting the codonopsis pilosula polysaccharide acid cut-off CPP-1-1 crude product to Sephadex-G75 column chromatography to obtain codonopsis pilosula polysaccharide acid cut-off CPP-1-1; the eluent adopted by the Sephadex-G75 column chromatography is water and NaCl solution in sequence, and the concentration of the NaCl solution is 0.1-0.5 mol/L.
The raw materials adopted by the invention are all commercial products unless specified.
The invention dissolves the crude polysaccharide of radix codonopsis into water, and the obtained crude polysaccharide aqueous solution of radix codonopsis is purified by macroporous adsorption resin to obtain purified polysaccharide.
The present invention is not particularly limited, and the preparation method well known to those skilled in the art may be adopted. In a specific embodiment of the present invention, the preparation method of the Codonopsis pilosula crude polysaccharide preferably comprises the following steps: degreasing radix codonopsis, extracting with water, and precipitating with lower alcohol to obtain crude polysaccharide of radix codonopsis.
In the present invention, the dangshen is preferably washed with water, dried and pulverized before use, and the present invention is not particularly limited, and the dried dangshen powder having a particle size of 60 mesh or less can be obtained by using drying conditions and pulverizing means well known to those skilled in the art.
In the present invention, the degreasing is preferably a reflux degreasing with anhydrous lower alcohol, preferably including anhydrous ethanol; the ratio of dry weight of the codonopsis pilosula to the feed liquid of the anhydrous lower alcohol is preferably 1g:3 to 10mL, more preferably 1g: 5-8 mL; the temperature of the reflow degreasing of the anhydrous lower alcohol is preferably 50-80 ℃, more preferably 60-65 ℃; the number of times of the reflow degreasing of the anhydrous lower alcohol is preferably 1 to 3, more preferably 2 to 3, and the time of the single reflow degreasing of the anhydrous lower alcohol is preferably 0.5 to 2 hours, more preferably 1 to 1.5 hours. In the present invention, the anhydrous lower alcohol includes one or more of ethanol, methanol and propanol.
After the degreasing is completed, the method preferably further comprises the steps of carrying out solid-liquid separation on the degreasing system, and drying the solid component to obtain the degreasing codonopsis pilosula powder. The drying conditions are not particularly limited in the present invention, and water may be completely removed. The solid-liquid separation is not particularly limited, and may be performed by a solid-liquid separation method known to those skilled in the art, such as filtration, suction filtration, or centrifugal separation. In the present invention, the drying temperature is preferably 25 to 45 ℃, more preferably 20 to 30 ℃.
In the invention, the ratio of the lipid-removed dangshen powder to water for water extraction is preferably 1g:10 to 20mL, more preferably 1g:15mL. In the present invention, the water extraction is preferably ultrasonic-assisted water extraction, more preferably comprises sequentially performing soaking, ultrasonic and water extraction; the soaking temperature is preferably room temperature, and the soaking time is preferably 0.5-2 h, more preferably 1-1.5 h; the temperature of the ultrasonic wave is preferably room temperature, and the time of the ultrasonic wave is preferably 0.5-2 h, more preferably 0.5-1 h; the temperature of the water extraction is preferably 55-75 ℃, more preferably 60-65 ℃; the number of times of water extraction is preferably 1 to 3, more preferably 2 to 3, and the time of single water extraction is preferably 0.5 to 2 hours, more preferably 0.5 to 1 hour; the water extraction is preferably carried out under stirring.
After the water extraction is completed, the invention preferably further comprises solid-liquid separation of the obtained water extract, concentration of the obtained liquid component, and cooling to room temperature to obtain a water extraction concentrate. The solid-liquid separation is not particularly limited, and a solid-liquid separation mode well known to those skilled in the art is adopted; in a specific embodiment of the present invention, the solid-liquid separation preferably includes sequentially filtering with a filter cloth, centrifuging the obtained filtrate, and filtering the obtained supernatant with an aqueous filter membrane; the rotating speed of the centrifugal separation is preferably 3000-5000 r/min, more preferably 4000r/min, and the time of the centrifugal separation is preferably 4-6 min, more preferably 5min; the pore size of the water-based membrane for suction filtration of the water-based filter membrane is preferably 0.45 μm. The concentration is not particularly limited, and a concentration method well known to those skilled in the art, such as reduced pressure distillation, may be adopted; the volume of the aqueous extract concentrate is preferably 15 to 25% of the volume of the aqueous extract, more preferably 20%. The cooling method is not particularly limited, and a cooling method well known to those skilled in the art, such as natural cooling, may be used.
In the present invention, the lower alcohol for lower alcohol precipitation preferably includes one or more of absolute ethanol, methanol and propanol. In the invention, the volume fraction of the lower alcohol in the system during the lower alcohol precipitation is preferably 55-85%, more preferably 60-80%, and even more preferably 70-75%; the temperature of the lower alcohol precipitation is preferably 0-10 ℃, more preferably 4 ℃; the time of the lower alcohol precipitation is preferably 8-15 h, more preferably 12h; the lower alcohol precipitation is preferably carried out under sealed and stationary conditions.
After the lower alcohol precipitation is completed, the invention preferably further comprises the steps of carrying out solid-liquid separation on the obtained lower alcohol precipitation liquid, and drying the obtained solid component to obtain the codonopsis pilosula crude polysaccharide. The solid-liquid separation is not particularly limited, and may be performed by a solid-liquid separation method known to those skilled in the art, such as filtration, suction filtration, or centrifugal separation. In the present invention, the drying temperature is preferably 20 to 40 ℃, more preferably 20 to 30 ℃; the drying is preferably vacuum drying, and the vacuum degree of the vacuum drying is not particularly limited in the present invention, and vacuum degree of vacuum drying well known to those skilled in the art may be adopted; the drying time is not particularly limited, and the drying time is required to be constant.
After obtaining the crude codonopsis pilosula polysaccharide, the invention dissolves the crude codonopsis pilosula polysaccharide in water, and the obtained crude codonopsis pilosula polysaccharide aqueous solution is purified to obtain purified polysaccharide.
In the present invention, the purification is preferably macroporous adsorbent resin purification; the macroporous adsorption resin preferably comprises one or more of macroporous adsorption resin S-8, macroporous adsorption resin ADS-F8, macroporous adsorption resin LSA-21, macroporous adsorption resin LSA-10, macroporous adsorption resin HP-20, macroporous adsorption resin ADS-17, macroporous adsorption resin NKA-9, macroporous adsorption resin DM130, macroporous adsorption resin AB-8, macroporous adsorption resin X-5, macroporous adsorption resin H103, macroporous adsorption resin D101, macroporous adsorption resin XAD-8 and macroporous adsorption resin DA 201.
In the present invention, the macroporous adsorbent resin is preferably pretreated before use, and the pretreatment of the macroporous adsorbent resin is not particularly limited and may be performed by a pretreatment method well known to those skilled in the art. In the invention, the macroporous adsorption resin comprises one or more of macroporous adsorption resin S-8, macroporous adsorption resin ADS-F8, macroporous adsorption resin LSA-21, macroporous adsorption resin LSA-10, macroporous adsorption resin HP-20, macroporous adsorption resin ADS-17, macroporous adsorption resin NKA-9, macroporous adsorption resin DM130, macroporous adsorption resin AB-8, macroporous adsorption resin X-5, macroporous adsorption resin H103, macroporous adsorption resin D101, macroporous adsorption resin XAD-8 and macroporous adsorption resin DA201, and is more preferably ADS-F8 macroporous adsorption resin.
In the present invention, the conditions for purifying the macroporous adsorbent resin preferably include: the loading concentration of the codonopsis pilosula crude polysaccharide aqueous solution is preferably 0.5-2 mg/mL, more preferably 1-1.5 mg/mL; the eluent is preferably water; the eluent is preferably used in an amount of 1-3 BV, more preferably 1.5-2 BV; the flow rate of the eluent is preferably 0.5-2 BV/h, more preferably 1-1.5 BV/h.
After purified polysaccharide is obtained, the purified polysaccharide is subjected to acid shearing, neutralization and water dialysis in sequence to obtain the codonopsis pilosula polysaccharide acid shearing fragment mixture.
The acid solution for acid shearing is not particularly limited, and an acid solution well known to those skilled in the art, such as a hydrochloric acid solution; the concentration of the acid solution is preferably 0.04 to 0.3mol/L, more preferably 0.05 to 0.2mol/L, and still more preferably 0.05 to 0.1mol/L. In the present invention, the feed liquid ratio of the purified polysaccharide to the hydrochloric acid solution is preferably 1g:150 to 450mL, more preferably 1g: 200-300 mL. In the acid shearing process, purified polysaccharide is hydrolyzed under the action of hydrochloric acid to obtain polysaccharide acid shearing fragments CPP-1-1, CPP-1-2, CPP-3 and CPP-4.
In the present invention, the temperature of the acid shearing is preferably 70 to 90 ℃, more preferably 75 to 85 ℃, still more preferably 80 ℃; the acid shearing time is preferably 1 to 3 hours, more preferably 1 to 2 hours, and still more preferably 1 to 1.5 hours; the acid shearing is preferably carried out under an inert atmosphere; the inert atmosphere preferably comprises helium or argon. In the acid shearing process, purified polysaccharide is hydrolyzed under the action of acid to obtain a mixed solution of polysaccharide acid shearing fragments.
The neutralizing alkali is not particularly limited, and the pH of the system may be neutralized to 7 by using alkali known to those skilled in the art.
In a specific embodiment of the present invention, the neutralizing base preferably comprises sodium hydroxide; the base is preferably used in the form of an alkaline solution, the mass concentration of which is preferably 20%.
The dialysis is not particularly limited, and dialysis conditions for removing water-soluble polysaccharide impurities, which are well known to those skilled in the art, may be employed. In a specific embodiment of the invention, the water dialysis is preferably: after the long-flow tap water is dialyzed for 24 hours, the tap water is changed into ultrapure water for dialysis for 3 times every 3 hours, and the tap water is dialyzed until no chloride ions are detected; the molecular weight cut-off of the dialysis bag for dialysis is preferably 1000 to 4000Da, more preferably 1500 to 2000Da. In the present invention, the purpose of the dialysis is to remove small molecule impurities.
After the dialysis is completed, the method preferably further comprises the steps of sequentially concentrating, alcohol precipitating and solid-liquid separating the obtained pilose asiabell root polysaccharide acid shear fragment mixture solution, and drying the obtained solid product to obtain the pilose asiabell root polysaccharide acid shear fragment mixture. The concentration of the present invention is not particularly limited, and may be performed by any concentration means known to those skilled in the art, such as distillation under reduced pressure. In the present invention, the volume fraction of the alcohol in the alcohol precipitation system is preferably 55 to 85%, more preferably 60 to 85%, still more preferably 75 to 80%; the alcohol is preferably a lower alcohol, more preferably includes one or more of ethanol, methanol, and propanol. The solid-liquid separation method is not particularly limited, and may be any solid-liquid separation method known to those skilled in the art, such as filtration, suction filtration, or centrifugal separation. In the present invention, the drying temperature is preferably 20 to 40 ℃, more preferably 20 to 30 ℃; the drying is preferably vacuum drying, and the vacuum degree of the vacuum drying is not particularly limited in the present invention, and vacuum degree of vacuum drying well known to those skilled in the art may be adopted; the drying time is not particularly limited, and the drying time is required to be constant.
After obtaining a codonopsis pilosula polysaccharide acid cut-off mixture, carrying out DEAE-52 cellulose column chromatography on the codonopsis pilosula polysaccharide acid cut-off mixture to obtain a codonopsis pilosula polysaccharide acid cut-off CPP-1-1 crude product; the eluent adopted by the DEAE-52 cellulose column chromatography is water and NaCl solution in sequence; the water is preferably ultrapure water; the concentration of the NaCl solution is 0.01-0.5 mol/L. In the present invention, the concentration of the NaCl solution is preferably 0.1 to 0.4mol/L, more preferably 0.15 to 0.35mol/L. In the invention, the elution mode of DEAE-52 cellulose column chromatography is preferably gradient elution, and the eluent adopted by the gradient elution is preferably water, 0.15mol/LNaCl solution, 0.25mol/LNaCl solution and 0.35mol/LNaCl solution in sequence.
After obtaining a crude product of the codonopsis pilosula polysaccharide acid cut-off CPP-1-1, carrying out Sephadex-G75 column chromatography on the crude product of the codonopsis pilosula polysaccharide acid cut-off CPP-1-1 to obtain the codonopsis pilosula polysaccharide acid cut-off CPP-1-1; the eluent adopted by the Sephadex-G75 column chromatography is water and NaCl solution in sequence; the water is preferably ultrapure water; the concentration of the NaCl solution is 0.1-0.5 mol/L. In the present invention, the concentration of the NaCl solution is preferably 0.1 to 0.4mol/L, more preferably 0.15 to 0.35mol/L. In the invention, the elution mode of the Sephadex-G75 column chromatography is preferably gradient elution, and the eluent adopted by the gradient elution is preferably water, 0.15mol/LNaCl solution, 0.25mol/LNaCl solution and 0.35mol/LNaCl solution in sequence.
The invention provides a codonopsis pilosula polysaccharide acid shear fragment CPP-1-1 prepared by the preparation method in the technical scheme, which comprises galacturonic acid (GalA), glucose (Glu) and galactose, wherein the molar ratio of the galacturonic acid to the glucose to the galactose is 45:0.65:0.26; the peak molecular weight Mp of the pilose asiabell root polysaccharide acid shearing segment CPP-1-1 is 2000-5000 Da, preferably 2000-3000 Da.
The invention provides application of the pilose asiabell root polysaccharide acid cut-off segment CPP-1-1 in cosmetics. The codonopsis pilosula polysaccharide tablet CPP-1-1 provided by the invention has the advantages of relatively simple structure, small molecular weight, good water solubility, good inhibition effect on tyrosine monophenolase and bisphenol enzyme, good free radical oxidation resistance, excellent moisturizing effect, capability of effectively inhibiting melanin from generating, capability of avoiding the problems of skin water shortage, darkness, color spots and the like, and good application prospect in cosmetics and functional foods as a whitening agent, a moisturizing agent and an antioxidant.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Macroporous adsorbent resin purification condition screening
(1) Pretreatment of macroporous adsorption resin: resin types S-8, ADS-F8, LSA-21, LSA-10, HP-20, ADS-17, NKA-9, DM130, AB-8, X-5, H103, D101, XAD-8 and DA201 are selected to be 14 kinds of resins with different polarities, 95% ethanol which is 2-3 cm higher than the surface of the resins is added for soaking for 24 hours, purified water is used for washing till no alcohol taste, naOH aqueous solution with the mass concentration of 4% is added for soaking, after the NaOH aqueous solution is removed, HCl aqueous solution with the mass concentration of 4% is added for soaking, purified water is used for washing till neutrality, and the pretreated macroporous adsorption resin is obtained for standby.
(2) Static adsorption of macroporous adsorption resin: 2g of each pretreated macroporous adsorbent resin is weighed, respectively added into 100mL conical flasks filled with 50mL of crude polysaccharide aqueous solution with the concentration of 1mg/mL, sealed by preservative film, oscillated for 12h in a shaking table at 30 ℃ and 150r/min, filtered, desorbed by 50mL of ultrapure water, and each macroporous adsorbent resin is performed in parallel for 3 times. Calculating the adsorption rate R of each macroporous adsorbent resin according to the formula (1) and the formula (2) A Desorption rate R D (see fig. 2 for results, each representing the mean ± RSD (n=3)), a preferred macroporous adsorbent resin model for crude polysaccharide purification of codonopsis pilosula was initially identified as ADS-F8.
R A ＝(C 0 -C 1 )/C 0 X 100% formula (1);
R D ＝C 2 V 2 /(C 0 -C 1 )V 1 x 100% formula (2);
in the formulas (1) to (2), C 0 Represents the initial concentration of polysaccharide (mg/mL); c (C) 1 Represents the polysaccharide concentration (mg/mL) after adsorption; c (C) 2 Represents the concentration of polysaccharide after desorption (mg/mL); v (V) 1 Represents the volume (mL) of the Codonopsis pilosula polysaccharide solution; v (V) 2 Representing the desorption volume (mL).
(3) Dynamic adsorption of macroporous adsorption resin: and loading the macroporous adsorption resin screened by static adsorption into a column by a wet method. Through a single factor experiment, the comprehensive scores of polysaccharide recovery rate (Cr,%), deproteinization rate (Pr,%) and decoloration rate (Dr,%) are taken as Y values, and the calculation formulas are shown in formulas (3) to (6). When the single factor variable of the loading concentration is studied, the loading amount of the aqueous solution of the crude polysaccharide of dangshen is fixed to be 1BV, the flow rate of the eluent (ultrapure water) is 1BV/h, the volume of the eluent is 1.5BV, and the influence of the loading concentration (0.5 mg/mL, 1mg/mL, 1.5mg/mL, 2mg/mL and 2.5mg/mL respectively) on the purification effect of the crude polysaccharide of dangshen is changed. The loading amount is changed, the loading concentration of the aqueous solution of the immobilized codonopsis pilosula crude polysaccharide is 1mg/mL, the flow rate of the eluent is 1BV/h, the volume of the eluent is 1.5BV, and the influence of the loading amount (0.5 BV, 1BV, 1.5BV, 2BV and 2.5BV respectively) on the purification effect of the codonopsis pilosula crude polysaccharide is studied. The flow rate of the eluent is changed, the loading concentration of the aqueous solution of the immobilized codonopsis pilosula crude polysaccharide is 1mg/mL, the volume of an upper column is 1.5BV, the volume of the eluent is 1.5BV, and the influence of the flow rates of the eluent (0.5 BV/h, 1BV/h, 1.5BV/h, 2BV/h and 2.5BV/h respectively) on the purification effect of the codonopsis pilosula crude polysaccharide is studied. The volume of the eluent is changed, the loading concentration of the aqueous solution of the crude polysaccharide of the codonopsis pilosula is fixed to be 1mg/mL, the volume of an upper column is 1.5BV, the flow rate of the eluent is 1BV/h, the influence of the volumes of the eluent (1 BV, 1.5BV, 2BV, 2.5BV and 3BV respectively) on the purification effect of the crude polysaccharide of the codonopsis pilosula is studied, and the variable range is determined.
Dr＝(D 0 -D 1 )/D 0 X 100% formula (3);
Pr＝(P 0 -P 1 )/P 0 x 100% formula (4);
Cr＝C 1 /C 0 x 100% formula (5);
y=0.4×cr+0.3×dr+0.3×pr formula (6);
in the formulas (3) to (6), D 0 Represents absorbance before decolorization, D 1 Represents absorbance after decolorization; p (P) 0 Represents the protein content, P, in the polysaccharide solution prior to purification 1 Represents the protein content of the purified polysaccharide solution; c (C) 1 Represents the content of purified polysaccharide, C 0 Represents the polysaccharide content prior to purification; y represents the comprehensive score of polysaccharide purification effect, and the result is shown in FIG. 3.
Optimizing macroporous adsorption resin to purify the codonopsis pilosula crude polysaccharide by a response surface curve method: according to static experiments, screening out proper resin types, loading the column by a wet method, screening out three factors with the largest influence through a single factor experiment, carrying out three-factor and three-level response surface experimental design, and optimizing the effect of purifying the codonopsis pilosula crude polysaccharide by using a Box-Behnken center combined experimental design of 3 variables. Three factors with great influence on polysaccharide purification effect are selected: loading concentration (X) 1 ) Volume of eluent (X 2 ) And eluent flow rate (X 3 ) And (3) carrying out a response surface experiment of a 3-factor 3 level, taking the comprehensive score Y as a response value for investigation, wherein the factor level design is shown in a table 1, the result is shown in a table 2, and the contour diagram and the response curve of the comprehensive score are shown in a figure 4.
TABLE 1 response surface Experimental factors level design
TABLE 2Box-Behnken response surface Experimental design and results
As can be seen from table 2, the results of the response surface show that the optimal purification process for macroporous adsorption resin purification is: the loading concentration of the codonopsis pilosula crude polysaccharide aqueous solution is 1mg/mL, the volume of the eluent is 1.5BV, the flow rate of the eluent is 1BV/h, and the comprehensive score under the condition is 73.02%. Three parallel verification experiments are carried out under the condition, and the predicted value of the optimized process condition is finely adjusted to an integer value according to the operability of the experiments: the loading concentration of the codonopsis pilosula crude polysaccharide aqueous solution is 1mg/mL, the volume of the eluent is 1.5BV, the flow rate of the eluent is 1BV/h, and the comprehensive score is 73.12+/-0.06% (n=3).
Example 2
The preparation method of the pilose asiabell root polysaccharide acid cut-off CPP-1-1 according to the process flow chart shown in FIG. 1 comprises the following specific steps:
pulverizing the washed and dried radix Codonopsis (particle size is less than or equal to 60 mesh) to obtain radix Codonopsis powder. Mixing the radix codonopsis powder (1000 g) with absolute ethanol according to a feed liquid ratio of 1g: mixing in proportion of 5mL, refluxing for degreasing for 3 times (1.5 h, 1h and 0.5h respectively) at 65deg.C, suction filtering, and drying the obtained solid component to obtain degreasing radix Codonopsis powder (780 g). Mixing the degreasing radix codonopsis powder (200 g) with ultrapure water according to a feed liquid ratio of 1g: mixing 15mL, standing at room temperature (25deg.C) for 1 hr, performing ultrasonic treatment for 0.5 hr, reflux-extracting at 65deg.C for 3 times (1.5 hr, 1 hr, and 0.5 hr respectively), filtering with filter cloth, centrifuging the obtained filtrate at 4000r/min for 5min, vacuum-filtering the obtained supernatant with 0.45 μm water-based filter membrane, vacuum-distilling the obtained filtrate to 1/5 of the total volume, and cooling to room temperature to obtain water-concentrated solution. Absolute ethyl alcohol is added into the water extraction concentrated solution until the volume fraction of the ethyl alcohol is 75%, glass rods are added and stirred at the same time, alcohol precipitation is carried out for 12 hours under the conditions of 4 ℃ and sealing and standing, suction filtration is carried out, and the obtained precipitate is dried under vacuum at 30 ℃ until the constant weight is obtained, thus obtaining the codonopsis pilosula crude polysaccharide (74.18 g).
The pretreated macroporous adsorption resin ADS-B8 prepared in example 1 was packed by wet method, and the crude dangshen polysaccharide was dissolved in ultrapure water to obtain a crude dangshen polysaccharide aqueous solution with a concentration of 1 mg/mL. Loading the codonopsis pilosula crude polysaccharide aqueous solution, and purifying by using a macroporous adsorption resin column by taking ultrapure water as an eluent to obtain purified polysaccharide (54.17 g); wherein the volume of the eluent is 1.5BV and the flow rate of the eluent is 1.0BV/h.
Taking 2g of purified polysaccharide and hydrochloric acid solution with the concentration of 0.05mol/L according to the feed-liquid ratio of 1g:200mL mix, at N 2 After protection and hydrolysis for 1h under the water bath condition of 80 ℃, neutralizing with 20% sodium hydroxide solution, dialyzing with long-flow tap water for 24h, changing into ultrapure water for dialyzing for 3 times every 3h, dialyzing until no chloride ions are detected, concentrating the obtained pilose asiabell root polysaccharide acid fragment mixture solution, and precipitating to obtain a pilose asiabell root polysaccharide acid fragment mixture (1.18 g).
Sequentially adopting ultrapure water, 0.15mol/L NaCl aqueous solution, 0.25mol/L NaCl aqueous solution and 0.35mol/L NaCl aqueous solution as eluent to carry out DEAE-52 cellulose column chromatography on the codonopsis pilosula polysaccharide acid shear fragment mixture, collecting eluent of 0.35mol/LNaCl aqueous solution, and concentrating to constant weight to obtain a codonopsis pilosula polysaccharide acid shear fragment CPP-1-1 crude product.
Sequentially adopting ultrapure water, 0.15mol/L NaCl aqueous solution, 0.25mol/L NaCl aqueous solution and 0.35mol/LNaCl aqueous solution as eluent to carry out Sephadex-G75 column chromatography on the crude product of the pilose asiabell root polysaccharide acid shearing fragment CPP-1-1, collecting eluent of the 0.35mol/LNaCl aqueous solution, concentrating, and drying to constant weight to obtain the pilose asiabell root polysaccharide acid shearing fragment CPP-1-1 (0.42G, total yield is 4.42%).
FIG. 5 is a FT-IR view of a pilose asiabell root-polysaccharide acid-cutting fragment CPP-1-1, as can be seen from FIG. 5, the pilose asiabell root-polysaccharide acid-cutting fragment CPP-1-1 is 3419.61cm -1 The absorption peak at 2927.29cm is caused by stretching vibration of-OH -1 The absorption peak at this point is the C-H stretching vibration peak belonging to methylene. The above is the characteristic absorption peak of the saccharide in the infrared spectrum, and the peak area is wide because the saccharide molecule contains a large amount of hydroxyl groups and forms intramolecular hydrogen bonds.
FIG. 6 is a TG plot of Codonopsis pilosula polysaccharide acid cut CPP-1-1. As shown in FIG. 6, the weight of Codonopsis pilosula polysaccharide acid cut CPP-1-1 becomes smaller as the temperature increases, and there is a mass loss stage, the mass loss rate at 41.96 ℃to 136.96 ℃is 9.28%, the mass loss rate at 170.18 ℃to 468.58 ℃is 59.76%, and the final residual mass is 21.91%. The mass loss rate of CPP-1-2 at 230.00-406.20 ℃ is 47.37%, and the final residual mass is 26.75%.
FIG. 7 is a HPGPC chart of a pilose asiabell root polysaccharide acid cut-off CPP1-1 with a single symmetrical peak CPP-1-1 as a chromatographic peak, a retention time of 20.467 on a high performance gel permeation chromatographic column, and a peak molecular weight Mp of 2.02X10 3 Da。
FIG. 8 is an HPLC chromatogram of a PMP derivative of the pilose asiabell root polysaccharide acid fragment CPP-1-1, as can be seen from FIG. 8, CPP-1-1 is mainly prepared by the following steps: galacturonic acid (Gala), glucose (Glu) and galactose at 0.26.
FIG. 9 is a scanning electron microscope image of a pilose asiabell root polysaccharide acid cut-off CPP-1-1. As can be seen from FIG. 9, the pilose asiabell root polysaccharide acid cut-off CPP-1-1 has rough and bumpy surface, has raised fibrous shape, and is agglomerated in block shape.
Test example 1
Whitening Activity test of Codonopsis pilosula polysaccharide cut-out CPP-1-1 and Codonopsis pilosula crude polysaccharide prepared in example 1
The test principle is as follows: when the activity of tyrosinase is enhanced by external stimulus, tyrosine generates 3, 4-dihydroxyphenylalanine, namely L-dopa under the catalysis of tyrosinase, and L-dopa is dehydrogenated to generate dopaquinone under the further catalysis of diphenolase, and the dopaquinone accumulates in the body and forms melanin through a series of reactions. According to the principle, a whitening activity experiment mainly inhibiting tyrosinase activity is designed. And testing the inhibition condition of aqueous solutions of the codonopsis pilosula polysaccharide acid cut fragments CPP-1-1 with different concentrations on tyrosinase monophenolase and diphenolase activities. The inhibition rate of tyrosinase monophenolase is measured by taking L-tyrosine as a reaction substrate and catalytic enzyme as tyrosinase, and the inhibition rate of tyrosinase diphenolase is measured by taking L-tyrosine as a reaction substrate.
Sample solution: the final concentrations of the codonopsis pilosula polysaccharide acid shear fragments CPP-1-1 and codonopsis pilosula crude polysaccharide, and phenethyl resorcinol (control) in the sample to be tested are 20 mug/mL, 40 mug/mL, 60 mug/mL, 80 mug/mL and 100 mug/mL respectively.
TABLE 3Z 1 ～Z 4 Group test sample composition
| Composition of the components | Z 1 Group of | Z 2 Group of | Z 3 Group of | Z 4 Group of |
| Sample solution/mL | 1 | 1 | 0 | 0 |
| PBS/mL | 2 | 3 | 3 | 4 |
| Tyrosinase solution/mL | 1 | 0 | 1 | 0 |
| L-tyrosine solution/mL | 1 | 1 | 1 | 1 |
| L-dopa solution/mL | 1 | 1 | 1 | 1 |
According to the composition shown in Table 3, at Z 1 、Z 2 、Z 3 、Z 4 Four groups (composition shown in Table 1) of sample solutions, PBS and tyrosinase solutions, of which the concentrations are 20. Mu.g/mL, 40. Mu.g/mL, 60. Mu.g/mL, 80. Mu.g/mL and 100. Mu.g/mL, respectively, were removed from the test tubes, heated at low temperature for 10min, 1.5mmol/mL of L-tyrosine (monophenolase) solution-1 mmol/mL of L-dopa (diphenolase) solution were added to the four test tubes, and reacted at 37℃for 15min, and the absorbance A of the four groups was measured at 475nm, respectively 1 、A 2 、A 3 And A 4 . The calculation formula of the inhibition rate of the sample on monophenolase and diphenolase is shown as formula (7):
inhibition ratio% = 1- [ (a 1 -A 2 )/(A 3 -A 4 )]X 100% formula (7);
in the formula (7), A 1 Represents Z 1 Absorbance of the group; a is that 2 Represents Z 2 Absorbance of the group; a is that 3 Represents Z 3 Absorbance of the group; a is that 4 Represents Z 4 Absorbance of the group.
The results of the whitening activity test of phenethyl resorcinol, the pilose asiabell polysaccharide shear fragment CPP-1-1 prepared in example 1 and the pilose asiabell polysaccharide are shown in tables 4 to 5.
Table 4L-tyrosine monophenolase inhibitory activity test results (n=3)
Table 5L-dopa solution (diphenolase) inhibitory activity test results (n=3)
As shown in tables 4 to 5, the inhibition rate of the pilose asiabell root polysaccharide acid cut-off section CPP-1-1 to tyrosinase is dose dependent, the inhibition rate of the pilose asiabell root polysaccharide acid cut-off section CPP-1-1 to tyrosinase monophenolase and bisphenolase both increase with the increase of the concentration, the inhibition rate of the pilose asiabell root polysaccharide to tyrosinase monophenolase and bisphenolase is far higher than that of the pilose asiabell root total polysaccharide, and the inhibition effect of the pilose asiabell root polysaccharide to tyrosinase monophenolase is higher than that of the reference substance phenethyl resorcinol when the concentration of the CPP-1-1 aqueous solution is 100 mu g/mL; when the concentration of the CPP-1-1 aqueous solution is 100 mug/mL, the inhibition effect on tyrosinase bisphenol enzyme is similar to that of the reference substance phenethyl resorcinol. The codonopsis pilosula polysaccharide acid cut-off CPP-1-1 prepared by the invention has better inhibition effect on tyrosine monophenolase and bisphenol enzyme.
Test example 2
Moisturizing test of Codonopsis pilosula polysaccharide cut-out CPP-1-1 and Codonopsis pilosula polysaccharide prepared in example 1
Sample solution: the aqueous solution of the codonopsis pilosula polysaccharide acid shear fragment CPP-1-1 and the codonopsis pilosula crude polysaccharide are prepared into 500 mug/mL aqueous solution. As a control, an aqueous glycerol solution was used. The preparation method of the glycerol aqueous solution comprises the steps of drying glycerol at 60 ℃ for 24 hours, and preparing the glycerol aqueous solution with the concentration of 500 mug/mL.
The relative humidity of saturated potassium carbonate solution in a closed environment at 20 ℃ is 43%, and the moisture retention rate of the sample solution in the environment with 43% air humidity is tested, and the specific steps are as follows: the dry weight of the samples is respectively weighed and recorded as M 1 Placing in a closed container with constant temperature and humidity (25deg.C, and relative humidity of 43%) for 1 hr, 2 hr, 4 hr, 6 hr, and 8 hrWeigh at 12h, 16h and 24h, recorded as M 2 The formula of the moisture retention rate is shown as formula (8):
moisture retention% 2 /M 1 X 100% formula (8).
The results of the moisture retention test are shown in table 6.
Table 6 moisturizing test results (n=3)
As is clear from Table 6, the pilose asiabell root polysaccharide cut-off CPP-1-1 has a good moisturizing effect at an air relative humidity of 43%, and the moisturizing effect is equivalent to that of glycerin and slightly higher than that of total polysaccharide for 24 hours.
Test example 3
Test of anti-radical Oxidation Activity of Codonopsis pilosula polysaccharide cut-off CPP-1-1 and Codonopsis pilosula crude polysaccharide prepared in example 1
Scientific research has shown that antioxidant is an important step in preventing aging, because free radicals or oxidants break down cells and tissues, affect metabolic functions, and cause different health problems. If it is capable of eliminating excessive oxidative free radicals, it is possible to prevent many diseases associated with aging caused by free radicals, such as common cancers, arteriosclerosis, diabetes, cataract, cardiovascular diseases, senile dementia, arthritis, etc., which are considered to be associated with free radicals. And the skin of the human body is overmuch in free radicals, attacks skin cells, causes aging of the body, skin aging and immunity reduction, causes relaxation of collagen and elastic fibers, slows down the regeneration of epidermal cells, is dark in facial color, has a great amount of water loss and the like. The invention tests the antioxidant activity of the codonopsis pilosula polysaccharide acid cut-off fragment CPP-1-1 based on a Single Electron Transfer (SET) method, and the antioxidant can block free radicals to damage normal cells by providing electrons to the free radicals.
Reference substance V C And party toThe ginseng polysaccharide acid-sheared fragments CPP-1-1 were prepared into aqueous solutions (concentrations of 20. Mu.g/mL, 40. Mu.g/mL, 60. Mu.g/mL, 80. Mu.g/mL and 100. Mu.g/mL, respectively) and tested for V C Purified polysaccharide fragment pair DPPH. ABTS + OH and O 2 - Clearance, evaluation of the antioxidant properties of the pilose asiabell polysaccharide acid cut-off CPP-1-1 in vitro.
(1) DPPH clearance test
2mL of purified acid-sheared codonopsis pilosula polysaccharide acid-sheared fragments CPP-1-1 with different concentrations are respectively taken, added with 2mL of DPPH aqueous solution with the concentration of 0.1mmol/L, evenly mixed, reacted for 30min at room temperature in a dark environment, and the absorbance is measured at 517 nm. Experiments were performed in triplicate. The DPPH clearance calculation formula is shown in formula (9):
DPPH clearance (%) = [ (a) 0 -A 1 )/A 0 ]X 100% formula (9);
in the formula (9), A 0 Is the absorbance of distilled water (negative control), A 1 Is the absorbance of the sample. The DPPH radical scavenging results are shown in Table 7.
(2)ABTS + Clearance test
ABST aqueous solution (10 mL,7 mmol/L) was mixed with potassium persulfate aqueous solution (176. Mu.L, 140 mmol/L), and reacted at room temperature under dark conditions for 24 hours to obtain ABTS radical solution. The ABTS radical solution was diluted with PBS buffer (10 mmol/L, ph=7.4) to a working solution with absorbance at 734nm of 0.70±0.02 for use. 2mL of codonopsis pilosula multi-sugar acid shear fragments CPP-1-1 aqueous solution with different concentrations are respectively taken, 6 mLABSS free radical solution is respectively added, the reaction is carried out for 2 hours at room temperature and in dark condition, and the absorbance at 734nm is measured, namely the experimental group. The blank was replaced with deionized water and the experiments were run in triplicate. ABTS + The clearance calculation formula is shown in formula (10):
ABTS + clearance (%) = [ (a) 0 -A 1 )/A 0 ]X 100% formula (10);
in the formula (10), A 0 Is the absorbance of distilled water (negative control), A 1 Is the absorbance of the sample. ABTS + The clearance test results are shown in Table 8.
(3) OH clearance test
Respectively taking 1mL of pilose asiabell root polysaccharide acid shear fragments CPP-1-1 with different concentrations into test tubes, respectively and sequentially adding 1mL of FeSO with the concentration of 9mmol/L 4 The solution and 1mL of salicylic acid solution with the concentration of 9mmol/L are added with 1mL of hydrogen peroxide solution (9 mmol/L) to start the reaction, the mixture is fully and uniformly mixed, the reaction is carried out for 30min in a water bath at 37 ℃, and the absorbance at 510nm is measured. Experiments were performed in triplicate. The formula of the clearance rate of the hydroxyl radical is shown as formula (11):
OH clearance (%) = [ (A) 0 -A 1 )/A 0 ]X 100% formula (11);
in the formula (11), A 0 Is the absorbance of distilled water (negative control), A 1 Is the absorbance of the sample. The OH clearance test results are shown in Table 9.
(4)O 2 - Clearance test
2mL of PBS buffer solution with the concentration of 50mmol/L is taken in each test tube, 1mL of purified pilose asiabell root polysaccharide acid shear fragment CPP-1-1 is sequentially taken, then 250 mu L of pyrogallol solution with the concentration of 35mmol/L is respectively added, and the mixture is uniformly shaken and subjected to water bath at 25 ℃ for 5min. Finally, the reaction was terminated by adding 0.2mL of 8mmol/L HCl solution, reacted at room temperature for 5min, and the absorbance of the mixture was measured at 560 nm. Experiments were performed in triplicate. The formula of the scavenging rate of the superoxide anion free radical is shown as a formula (12):
O 2 - Clearance%o= [ (a) 0 -A 1 )/A 0 ]X 100% formula (12);
in the formula (12), A 0 Is the absorbance of distilled water (negative control), A 1 Is the absorbance of the sample. O (O) 2 - The clearance test results are shown in Table 10.
TABLE 7DPPH . Radical scavenging test results (n=3)
TABTS of Table 8ABTS + . Radical scavenging test results (n=3)
Table 9-OH clean out test results (n=3)
TABLE 10O 2 -. Clearance test results (n=3)
As is clear from tables 7 to 10, the rate of scavenging four free radicals by the codonopsis pilosula polysaccharide acid cut-off CPP-1-1 gradually increased in a concentration range of 20 to 100. Mu.g/mL as the concentration of the aqueous solution of the codonopsis pilosula polysaccharide acid cut-off CPP-1-1 increased, and the codonopsis pilosula polysaccharide acid cut-off CPP-1-1 showed a dose dependency. The highest DPPH clearance rate of the codonopsis pilosula polysaccharide acid cut-off segment CPP-1-1 can reach 52.47 percent, and the IC thereof 50 82.88. Mu.g/mL. When the concentration is 100 mug/mL, the clearance rate of the codonopsis pilosula polysaccharide acid shear fragment CPP-1-1 to DPPH is higher than that of a control product V at the same concentration C (43.29%) and higher than the content of the pilose asiabell root crude polysaccharide (37.65%) in the same concentration, the effect of the pilose asiabell root polysaccharide acid fragment CPP-1-1 on DPPH is better than V C The DPPH and free radical oxidation resistance is high. When the concentration is 100 mug/mL, the clearance rate of the pilose asiabell root polysaccharide acid shear fragment CPP-1-1 to ABTS+ is 30.10 percent, which is far higher than the clearance rate (13.62 percent) of the pilose asiabell root crude polysaccharide, but is weaker than the control substance V under the same concentration C (35.20%) shows that the pilose asiabell root polysaccharide acid cut-off segment CPP-1-1 has a certain antioxidation effect on ABTS+ free radical, but is weaker than V C . When the concentration is 100 mug/mL, the clearance rate of the codonopsis pilosula polysaccharide acid shear fragment CPP-1-1 to OH is 26.22 percent, which is higher than the clearance rate (20.12 percent) of the codonopsis pilosula crude polysaccharide, and the codonopsis pilosula polysaccharide acid shear fragment is compared with the reference substance V C The clearance (26.44%) of the (E) is similar, and the following table showsThe changium root polysaccharide acid cut segment CPP-1-1 has stronger antioxidation effect on OH free radical. At a concentration of 100 mug/mL, the codonopsis pilosula polysaccharide acid shear fragment CPP-1-1 pair O 2 - The clearance is 24.07%, which is higher than 21.52% of the crude polysaccharide of codonopsis pilosula and higher than the reference substance V C The clearance rate (23.91%) of the codonopsis pilosula polysaccharide acid cut-off fragment CPP-1-1 to O 2 - The antioxidation effect is strong. In conclusion, the pilose asiabell root polysaccharide acid shear fragment CPP-1-1 prepared by the method shows higher antioxidant activity in a smaller concentration range, and the antioxidant effect is enhanced along with the increase of the concentration, so that the pilose asiabell root polysaccharide acid shear fragment CPP-1 can be developed into an environment-friendly antioxidant and is widely applied to cosmetics and foods.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. A preparation method of a codonopsis pilosula polysaccharide acid cut-off CPP-1-1 comprises the following steps:
dissolving the crude radix codonopsis polysaccharide in water, and purifying the obtained crude radix codonopsis polysaccharide aqueous solution by macroporous adsorption resin to obtain purified polysaccharide;
sequentially carrying out acid shearing, neutralization and water dialysis on the purified polysaccharide to obtain a pilose asiabell root polysaccharide acid shearing fragment mixture; the acid solution for acid shearing is hydrochloric acid solution; the concentration of the acid solution for acid shearing is 0.04-0.3 mol/L; the feed liquid ratio of the purified polysaccharide to the hydrochloric acid solution is 1g: 150-450 mL; the temperature of the acid shearing is 70-90 ℃ and the time is 1-3 hours, and the acid shearing is carried out in an inert atmosphere;
subjecting the codonopsis pilosula polysaccharide acid cut-off mixture to DEAE-52 cellulose column chromatography to obtain a codonopsis pilosula polysaccharide acid cut-off CPP-1-1 crude product; the eluent adopted by the DEAE-52 cellulose column chromatography is water and NaCl solution in sequence, and the concentration of the NaCl solution is 0.01-0.5 mol/L;
subjecting the codonopsis pilosula polysaccharide acid cut-off CPP-1-1 crude product to Sephadex-G75 column chromatography to obtain codonopsis pilosula polysaccharide acid cut-off CPP-1-1; the eluent adopted by the Sephadex-G75 column chromatography is water and NaCl solution in sequence, and the concentration of the NaCl solution is 0.1-0.5 mol/L.
2. The preparation method according to claim 1, wherein the macroporous adsorption resin comprises one or more of macroporous adsorption resin S-8, macroporous adsorption resin ADS-F8, macroporous adsorption resin LSA-21, macroporous adsorption resin LSA-10, macroporous adsorption resin HP-20, macroporous adsorption resin ADS-17, macroporous adsorption resin NKA-9, macroporous adsorption resin DM130, macroporous adsorption resin AB-8, macroporous adsorption resin X-5, macroporous adsorption resin H103, macroporous adsorption resin D101, macroporous adsorption resin XAD-8 and macroporous adsorption resin DA 201.
3. The method according to claim 1 or 2, wherein the conditions for purification of the macroporous adsorbent resin include: the loading concentration of the codonopsis pilosula crude polysaccharide aqueous solution is 0.5-2 mg/mL, the eluent is water, the dosage of the eluent is 1-3 BV, and the flow rate of the eluent is 0.5-2 BV/h.
4. The method according to claim 1, wherein the dialysis further comprises sequentially concentrating the obtained mixture solution of the pilose asiabell root polysaccharide acid fragments, precipitating with ethanol, and separating solid from liquid, and drying the obtained solid product to obtain the mixture of the pilose asiabell root polysaccharide acid fragments.
5. The preparation method according to claim 1, wherein the preparation method of the crude codonopsis pilosula polysaccharide comprises the following steps: degreasing radix codonopsis, extracting with water, and precipitating with lower alcohol to obtain crude polysaccharide of radix codonopsis.
6. The preparation method of claim 5, wherein the volume fraction of lower alcohol in the lower alcohol precipitation system is 55-85%; the lower alcohol comprises one or more of methanol, ethanol and propanol.
7. The pilose asiabell root polysaccharide acid cut segment CPP-1-1 prepared by the preparation method of any one of claims 1-6 comprises galacturonic acid, glucose and galactose, wherein the molar ratio of galacturonic acid to glucose to galactose is 45:0.65:0.26; the peak molecular weight Mp of the pilose asiabell root polysaccharide acid shearing segment CPP-1-1 is 2000-5000 Da.
8. Use of the codonopsis pilosula polysaccharide acid cut-off CPP-1-1 according to claim 7 in cosmetics or functional foods.
Priority Applications (1)
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