CN111961141B - Acanthopanax root uniform polysaccharide and preparation method and application thereof - Google Patents
Acanthopanax root uniform polysaccharide and preparation method and application thereof Download PDFInfo
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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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
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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Abstract
The invention discloses an acanthopanax homogeneous polysaccharide with a molecular weight of 6.83 multiplied by 10 5 Da, consisting of fucose, arabinose, galactose, glucose and xylose, with molar ratios of monosaccharides of 16.42%, 32.27%, 40.38%, 7.21% and 3.72%. The preparation method of the acanthopanax homogeneous polysaccharide comprises the following steps: pulverizing radix Acanthopanacis Senticosi decoction pieces into powder, extracting radix Acanthopanacis Senticosi crude polysaccharide with water extraction method, removing protein in crude polysaccharide with Sevage method, and further separating and purifying with anion exchange DEAE Fast Flow chromatographic column and Sephadex G-200 Sephadex column chromatographic column to obtain radix Acanthopanacis Senticosi homogeneous polysaccharide. The acanthopanax polysaccharide prepared by the method is uniform polysaccharide, has antioxidant and skin aging resisting activities, and can be used for preparing cosmetics and medicines with skin aging resisting effects.
Description
Technical Field
The invention relates to the technical field of pharmacy, in particular to acanthopanax uniform polysaccharide and a preparation method and application thereof.
Background
The dry root and rhizome or stem of acanthopanax senticosus (Acanthopanax senticosus (rupr. Et maxim.) Harms) belonging to the genus acanthopanax of the family acanthaceae is widely distributed in russia far east needle leaf forest belt, northeast China, hebei, shanxi China, japan, north korea and the like. The aliases include acanthopanax root, spiny crutch, and Tiger's body. In traditional Chinese medicine, acanthopanax has very long history of application as a medicament, and is recorded in the historical herbal works. The south-oriented pottery Hongding Jing Jiu is pointed out in Ming Yi Bie Lu, five of which is five She Zheliang, and has the effects of invigorating the middle warmer, replenishing vital essence, strengthening tendons and bones and strengthening mind. The Ming dynasty plum delicacies in the compendium of materia medica indicate that acanthopanax is the superior product of the principal meridian and has the effects of tonifying middle-jiao and Qi, strengthening bones and muscles, strengthening intention and resisting aging after long-term taking. Radix Acanthopanacis Senticosi is warm in nature, pungent in taste and slightly bitter; spleen, kidney and heart channels; has effects of invigorating qi and spleen, invigorating kidney and tranquillizing, and can be used for treating spleen and kidney yang deficiency, asthenia, anorexia, soreness of waist and knees, insomnia and dreaminess. (Pan Jingzhi, jinsha, cui Wenyu, etc. the chemical components and pharmacological activities of acanthopanax are researched and developed [ J ]. The food industry technology, 2019, 40 (23): 353-360) acanthopanax has various pharmacological activities of regulating immune function, resisting cancer, protecting liver, resisting aging, resisting oxidation, resisting inflammation, reducing blood pressure, resisting stress, etc., and the active components are eleutheroside, flavonoid, lignans, polysaccharides, etc. However, these are experimental results of crude polysaccharide, have poor repeatability, and cannot be standardized, so that the industrialization value is limited. On the basis of the previous research, the invention carries out separation and purification, structural analysis and biological activity evaluation on the acanthopanax crude polysaccharide so as to obtain the acanthopanax uniform polysaccharide with good biological activity, which not only has very important scientific significance, but also lays a foundation for industrialization of the acanthopanax uniform polysaccharide.
Disclosure of Invention
The invention aims to solve the technical problems that the acanthopanax polysaccharide is classified according to molecular weight, then is further separated and purified and is subjected to structural characterization, so that the acanthopanax polysaccharide which is formed by condensing a monosaccharide molecule and has medical value is provided, and the obtained acanthopanax polysaccharide is subjected to preliminary study on the antioxidant activity and the skin aging resistance.
The invention aims to provide acanthopanax homogeneous polysaccharide with a molecular weight of 6.83 multiplied by 10 5 Da, consisting of fucose, arabinose, galactose, glucose and xylose, with molar ratios of monosaccharides of 16.42%, 32.27%, 40.38%, 7.21% and 3.72%.
The invention also aims to provide a preparation method of the acanthopanax uniform polysaccharide, which comprises the following steps:
1) Pulverizing the dried acanthopanax decoction pieces, sieving with a 100-mesh sieve, extracting the sieved medicinal powder with 5-8 times of water for 3 times at 80-100 ℃ for 2 hours each time, mixing the extracting solutions, centrifuging the extracting solutions at 3000rpm/min for 20min, collecting supernatant, and rotationally evaporating and concentrating the supernatant to be one fifth of the original volume;
2) After the concentrated solution is cooled, adding alpha-amylase until the content of the alpha-amylase is 0.1-0.4%, regulating the pH value to 7.0, carrying out water bath enzymolysis for 4 hours at 60 ℃ (the solution has no color change when meeting iodine-potassium iodide reagent), quickly heating to 100 ℃ and keeping for 5min to inactivate the enzyme, and centrifuging at 3000rpm/min for 10min to collect supernatant.
3) The supernatant was combined with Sevage reagent (chloroform: n-butanol volume ratio 4:1) in a volume ratio of 1:1 mixing, vigorously oscillating for 30min, standing for 12h, collecting upper polysaccharide solution, and repeating until no protein characteristic absorption peak exists in ultraviolet scanning;
4) Concentrating the upper layer polysaccharide solution in the step 3), adding 4-6 times of absolute ethyl alcohol, standing at 4 ℃ for precipitation for 48h, centrifuging at 3000rpm/min for 10min, and collecting precipitate. Repeating for 3 times, and lyophilizing to obtain radix Acanthopanacis Senticosi crude polysaccharide powder;
5) Completely dissolving the acanthopanax crude polysaccharide powder obtained in the step 4) by distilled water, and separating by a DEAE Fast Flow ion chromatographic column under the elution conditions: the flow rate was 2.5mL/min, and elution was carried out with pure water, 0.05mol/L, 0.1mol/L, 0.2mol/L, 0.4mol/L, and 1mol/L of sodium chloride solution in this order. And (3) carrying out gradient collection by using a full-automatic collector, eluting 3 times of column volume by using each solution gradient, collecting 30 tubes, and carrying out tube separation tracking detection by using a sulfuric acid-phenol method for 5.0mL of each tube. The collected solutions corresponding to the different eluents are concentrated to a certain volume, and then dialyzed by a dialysis bag (3500 Da). Changing water for 5 times per day in a beaker filled with a dialysis bag, dialyzing for 7 days, centrifuging polysaccharide liquid in the dialysis bag, and freeze-drying to obtain primarily purified acanthopanax polysaccharide;
6) Concentrating the eluent collected in the step 5), then separating by using a Sephadex G-200 Sephadex column, eluting by using distilled water according to the flow rate of 0.5mL/min, collecting by using a full-automatic collector, detecting by using a phenol-sulfuric acid method, and collecting a main peak part in an elution curve;
7) Concentrating the solution of the main peak part in the elution curve collected in the step 6), dialyzing with 3500Da dialysis bag for 2d for desalination, and concentrating, freeze drying the solution in the dialysis bag to obtain radix Acanthopanacis Senticosi uniform polysaccharide powder.
Further, in the Sevage reagent, chloroform: n-butanol volume ratio = 4:1
The invention discovers that the molecular weight is 6.83 multiplied by 10 by adopting the schemes of water extraction and alcohol precipitation, further separation and purification by adopting an anion exchange DEAE Fast Floe chromatographic column and a Superdex-200 gel chromatographic column and the like 5 Da, an acanthopanax homogeneous polysaccharide consisting of fucose, arabinose, galactose, glucose and xylose.
Experiments prove that the acanthopanax uniform polysaccharide powder prepared by the invention has multiple functions of resisting oxidization, resisting skin aging and the like, and can be used for preparing cosmetics for resisting skin aging and therapeutic medicaments for skin;
the invention also relates to a skin care cosmetic, which comprises the pharmaceutical composition with the skin aging resistance and auxiliary materials used in the cosmetic field;
the invention also relates to a skin therapeutic drug comprising the pharmaceutical composition with skin aging resistance and a pharmaceutically acceptable carrier.
In particular, it can be used for preparing skin care cosmetics, including creams, lotions, gels, masks, liniments or lotions; but not limited to the above dosage forms, the above pharmaceutical composition may be sterilized according to a known method in the skin care product industry to prepare various external preparations.
When the skin care cosmetic is prepared, the prepared acanthopanax root uniform polysaccharide powder can be mixed with known matrixes or auxiliary materials, carriers and additives of cosmetics and medicines, and the mixture is prepared according to a conventional method, wherein the acanthopanax root uniform polysaccharide powder accounts for 3% -10% of the total weight of the cosmetics.
Description of the drawings
FIG. 1 is a graph showing elution of crude polysaccharide fraction of Acanthopanax senticosus by DEAE Fast Flow ion chromatography column
FIG. 2 is a HPGPC gel chromatogram of Acanthopanax senticosus homogeneous polysaccharide
FIG. 3 is a GC-MS total ion flow chromatogram of six standard monosaccharides
FIG. 4 shows a GC-MS total ion flow chromatogram of acanthopanax homogeneous polysaccharide (bottom)
Fig. 5 is an infrared spectrum of acanthopanax uniform polysaccharide.
FIG. 6 shows the homogeneous polysaccharide of acanthopanax 1 H spectrogram, note: (left- & gt right)
FIG. 7 shows the uniform polysaccharide of acanthopanax 13 C spectrogram, note: (left- & gt right)
Detailed Description
The invention is described in further detail below by way of specific embodiments:
example 1 preparation and Structure characterization of Acanthopanax senticosus Uniform polysaccharide
1 extraction of acanthopanax crude polysaccharide:
pulverizing dried radix Acanthopanacis Senticosi decoction pieces, sieving with 100 mesh sieve, extracting the sieved powder with 5 times of water for 3 times at 80deg.C for 2 hr each time, mixing extractive solutions, centrifuging at 3000rpm/min for 20min, collecting supernatant, concentrating the supernatant by rotary evaporation to one fifth of the original volume, cooling the concentrated solution, adding alpha-amylase to 0.1%, regulating pH to 7.0, performing enzymolysis in water bath at 60deg.C for 4 hr (the solution has no color change when meeting iodine-potassium iodide reagent), rapidly heating to 100deg.C, maintaining for 5min, inactivating enzyme, centrifuging at 3000rpm/min for 10min, and collecting supernatant. The supernatant was mixed with Sevage reagent (chloroform: n-butanol volume ratio 4:1) at a volume ratio of 1:1, mixing, vigorously shaking for 30min, standing for 12h, collecting upper polysaccharide solution, and repeating until no protein characteristic absorption peak exists in ultraviolet scanning. Concentrating the upper polysaccharide solution, adding 4 times of absolute ethanol, standing at 4deg.C for precipitation for 48 hr, centrifuging at 3000rpm/min for 10min, and collecting precipitate. Repeating for 3 times, and lyophilizing to obtain radix Acanthopanacis Senticosi crude polysaccharide powder.
2 purification of acanthopanax homogeneous polysaccharide
Completely dissolving the obtained acanthopanax crude polysaccharide powder with distilled water, and separating by using a DEAE Fast Flow ion chromatographic column, wherein the elution conditions are as follows: the flow rate was 2.5mL/min, and elution was performed sequentially with water, 0.05mol/L, 0.1mol/L, 0.2mol/L, 0.3mol/L, and 0.5mol/L of sodium chloride solution. And (3) carrying out gradient collection by using a full-automatic collector, eluting 3 times of column volume by using each solution gradient, collecting 30 tubes, and carrying out tube separation tracking detection by using a sulfuric acid-phenol method for 5.0mL of each tube. The collected solutions corresponding to the different eluents are concentrated to a certain volume, and then dialyzed by a dialysis bag (3500 Da). Changing water 5 times per day in a beaker filled with a dialysis bag, dialyzing for 7 days, centrifuging polysaccharide liquid in the dialysis bag, and freeze-drying to obtain primarily purified acanthopanax polysaccharide. Concentrating the collected eluent, separating by using a Sephadex G-200 Sephadex gel column, eluting with distilled water according to the flow rate of 0.5mL/min, collecting by using a full-automatic collector, detecting by using a phenol-sulfuric acid method at the concentration of 5.0 mL/pipe, and collecting the main peak part in an elution curve. And concentrating the solution of the main peak part in the collected elution curve, dialyzing for 2d by using a 3500Da dialysis bag for desalination, and concentrating, freeze-drying the solution in the dialysis bag to obtain the acanthopanax uniform polysaccharide powder.
3 purity identification of acanthopanax homogeneous polysaccharide
High performance liquid chromatography conditions: agilent 1200 high performance liquid chromatograph, chromatographic column TSK GEL G3000PW XL (7.8X300 mm,7 μm) and TSK GEL G5000PW XL (7.8X105 mm,10 μm) in series, mobile phase 0.02mol/L KH 2 PO 4 The flow rate of the solution was 0.5mL/min, the column temperature was 35℃and the detector was a Waters 2414 differential refraction detector. Dissolving the acanthopanax uniform polysaccharide obtained in the step 2 with a proper amount of water, sampling the mixture to 10 mu l, and obtaining a result shown in a figure 2, wherein chromatographic peaks of the acanthopanax uniform polysaccharide in a chromatogram show that the acanthopanax uniform polysaccharide is a single symmetrical peak, which indicates that the acanthopanax uniform polysaccharide prepared by the invention is really a uniform polysaccharide.
Determination of uniform polysaccharide molecular weight of acanthopanax senticosus
Dissolving 1mg of pullulan with molecular weights of 50K, 80K, 150K, 270K, 410K and 670K Da in 1mL of water respectively, analyzing by Agilent 1200 high performance liquid chromatograph under the same chromatographic conditions, recording retention time, taking logarithm of relative molecular weight (1 ogM) as ordinate and retention time (t) as abscissa to obtain a standard curve y=14.62-0.980X, substituting peak-out time of acanthopanax uniform polysaccharide into a curve equation to obtain acanthopanaxThe molecular weight of the acanthopanax homogeneous polysaccharide is 6.83×10 5 Da。
Structural characterization of acanthopanax homogeneous polysaccharide
5.1 polysaccharide component analysis
Acid hydrolysis is carried out, then acetylation is carried out for derivatization, and gas-phase GC analysis is carried out. The acid hydrolysis method comprises the following steps: 10mg of acanthopanax uniform polysaccharide samples are weighed, placed in ampoule bottles respectively, 4mL of trifluoroacetic acid with the concentration of 2mol/L is added, air in the bottle is blown out by nitrogen, and a tube is sealed by an alcohol burner. After hydrolysis for 6 hours at 110 ℃, the sample is evaporated to dryness by spin, 2mL of methanol is added for dissolution, the evaporation is repeated for 3 times, and trifluoroacetic acid in the sample is removed as much as possible to obtain polysaccharide hydrolysate. 1mL of methanol was added to transfer the sample into a serum bottle, and the mixture was blown dry with nitrogen, and then
1.0mL of pyridine, 10mg of hydroxylamine hydrochloride and 1.0mg of internal standard inositol are added into the polysaccharide hydrolysate, the mixture is subjected to shaking reaction at a constant temperature of 90 ℃ for 0.5h, 1mL of acetic anhydride is added after cooling, and the acetylation reaction is performed at 90 ℃ for 0.5h. After cooling, water was added to terminate the reaction. 2.0mL of chloroform was added thereto and the mixture was extracted 3 times, and after removing excess water with anhydrous sodium sulfate, the mixture was filtered through a 0.22 μm organic phase filter. Each monosaccharide standard was also derivatized as described above.
After derivatization as described above, GC analysis was performed. GC detection conditions: agilent 6890N gas chromatography system using an Agilent HP-5 quartz capillary column (30 m 0.32mm 0.25 μm); the constant pressure mode is 20PSI; the carrier gas being N 2 The method comprises the steps of carrying out a first treatment on the surface of the Sample injection amount: 1.0. Mu.L; the flow rate was 1.0m L/min; sample inlet temperature: 250 ℃; the temperature of the FID detector was set to 250 ℃; the sample inlet adopts a non-split mode; programming temperature: the initial temperature is 100 ℃, the temperature is kept for 30 seconds, the temperature is increased to 160 ℃ at 3 ℃/min, the temperature increasing speed is changed, and the temperature is continuously increased. The temperature was raised to 250℃at a rate of 10℃per minute and maintained for 5 minutes.
GC results: analysis is carried out according to the retention time of six monosaccharide standards such as arabinose, galactose, glucose, mannose, xylose and fucose, and the mole percentage of the acanthopanax uniform polysaccharide monosaccharides is calculated through the area ratio of the peaks. The results showed that the acanthopanax homogeneous polysaccharide consisted of fucose, arabinose, galactose, glucose and xylose, and the molar ratios of the five monosaccharides were 16.42%, 32.27%, 40.38%, 7.21% and 3.72% calculated according to the internal standard method, as compared with the retention time in GC profile of the monosaccharide standard.
5.2 Infrared Spectroscopy scanning of Acanthopanax senticosus Uniform polysaccharide
Weighing 2.0mg of acanthopanax uniform polysaccharide sample, mixing with potassium bromide powder, grinding uniformly, and tabletting. Then the pressed tablet is placed in a Fourier transform infrared spectrometer to carry out infrared scanning (400-4000 cm) -1 ) The infrared absorption spectrum of the sample was recorded. From the 1R chart, it can be seen that the uniform polysaccharide of acanthopanax senticosus exhibits typical polysaccharide absorption characteristics, wherein 3456cm －1 And 2929cm －1 Is the stretching vibration peak of O-H bond, 1744cm －1 Is CH 2 A stretching vibration peak of C-H; 1635cm －1 Is CO 2 Or a stretching vibration peak caused by the co-water, 1411cm －1 Angle-variable vibration peak of C-O bond, 1242cm －1 Is a telescopic vibration peak caused by primary alcohol beta-OH; 1021 －1 cm －1 The absorption peak at the place shows that the acanthopanax uniform polysaccharide contains pyran ring and the average polysaccharide is 893cm －1 The absorption peak shows that the acanthopanax uniform polysaccharide has beta-glycosidic bond and 835cm －1 The absorption peak appears again to indicate the presence of alpha-glycosidic bond in the acanthopanax homogeneous polysaccharide.
5.3 methylation analysis of Acanthopanax senticosus homogeneous polysaccharide
20mg of acanthopanax uniform polysaccharide sample (the sample is kept sufficiently dry) is weighed and placed in a test tube with a plug, 6mL of dimethyl sulfoxide DMSO reagent is used for sealing, heating is carried out, magnetic stirring and mixing are carried out uniformly, sodium hydroxide (6 mL of DMSO contains 240mg of sodium hydroxide) is added to form sodium hydroxide suspension, and the suspension is kept overnight. 3.6mL of methyl iodide was added to the tube every other day, stirred for 8min, methyl iodide was purged with nitrogen, and the reaction was stopped by adding 6mL of distilled water after repeating this 3 times. After 24h dialysis with each of running water and deionized water, extraction with chloroform was performed 3 times, and dried with anhydrous sodium sulfite for 24h, followed by drying with nitrogen, leaving about 1mL of solution. Hydrolyzing with trifluoroacetic acid, adding 70mg sodium borohydride NaBH4, stirring for 24 hr, adding acidic cation exchange resin, stirring, mixing for 10min, suction filtering, collecting filtrate, adding methanol, blow drying with nitrogen, adding acetic anhydride and anhydrous pyridine, respectively, and acetylating at 100deg.C for 2 hr. After the reaction, absolute ethanol was repeatedly added to remove acetic anhydride, followed by GC-MS analysis. GC-MS chromatographic conditions: agilent6890-5973N gas chromatography-mass spectrometer, chromatographic column: HP-5MS capillary column (30 m.times.250 pm.times.0.25. 0.25 umD); carrier gas: helium e; heater temperature: 250 ℃ C: programming temperature: the initial temperature is 140 ℃/min to 200 ℃, kept for 5min, and then is increased to 240 ℃ at 8 ℃/min: split ratio: 50:1, a step of; sample injection amount: 5 μl. The methylation result of the acanthopanax homogeneous polysaccharide shows that the acanthopanax homogeneous polysaccharide is mainly formed by 1-4, 1-6, and the Glcp residue is the main unit of the acanthopanax homogeneous polysaccharide, and exists in a 1-4, 1-6, 1-4, 6 and 1-connection mode, and the Galp residue exists in a 1-4 connection mode.
TABLE 1 methylation analysis data of Acanthopanax senticosus homogeneous polysaccharide
5.4 NMR analysis of Acanthopanax senticosus Uniform polysaccharide
Dissolving 30mg of dried radix Acanthopanacis Senticosi homogeneous polysaccharide in 0.5ml D 2 O, heating at 60deg.C for 1 hr to dissolve completely, transferring into nuclear magnetic tube, and measuring on AV300 nuclear magnetic resonance apparatus of Brucker company 1 H spectrum 13 C spectrum. From the spectrogram, the acanthopanax root uniform polysaccharide 1 The signal distribution range of the H spectrum is narrow and mainly concentrated in the delta 2.0-6.0ppm (4.19,4.09,4.07,4.00,3.95,3.90,3.70,3.68,3.65,3.64,3.62,3.61,3.60,3.59,3.58,3.55,3.54 and 3.52 ppm) range. And its C spectrum signal range is delta 60-110ppm (107.41, 106.83, 106.36, 103.62, 95.72, 92.11, 91.85, 84.00,81.31, 76.81, 76.64, 76.31, 75.50, 71.24, 71.14, 71.00, 69.89, 69.21, 69.14, 62.30,61.26,61.05, 60.68, and 60.42 ppm). The chemical shift assignment of the anomeric carbon-collar of each sugar residue in the acanthopanax homogeneous polysaccharide is shown in table 2.
TABLE 2 acanthopanax root uniform multi-hydrocarbon chemical shift assignment
Example 2 evaluation of antioxidant Activity of Acanthopanax senticosus polysaccharide
Modern medicine holds that the key cause of skin aging is the oxidative stress of the dermis and epidermis caused by various factors, so that substances with strong antioxidant effect have better skin aging resistance, and the acanthopanax uniform polysaccharide obtained in the example 1 is used for evaluating the antioxidant activity.
1 determination of DPPH free radical scavenging Activity
2mL of sample solutions of different mass concentrations (12.5, 25, 50, 100. Mu.g.ml -1 ) 2mL of DPPH solution (100. Mu.g.ml) -1 ) After fully mixing, the mixture reacts for 30min in a dark place. The absorbance (Am) of the reaction system was measured at 517 nm. A solvent blank (An, DPPH solution replaced with equal volume of methanol) and a sample blank (Ao, sample solution replaced with equal volume of methanol) were set simultaneously. VC was used as positive control. Experiments were run 3 times in parallel and the clearance of DPPH from the extract was calculated according to the following formula.
2 clear ABTS + Activity determination
0.4mL of sample solutions of different mass concentrations (12.5, 25, 50, 100. Mu.g.ml -1 ) Adding ABTS + 4mL of the solution was reacted for 6 minutes, and the absorbance Ai was measured at 734 nm. At the same time, solvent blank group (Aj, ABTS) + The solution was replaced with equal volume of methanol), a sample blank (Ah, the sample solution was replaced with equal volume of methanol) and a positive control. The experiments were run 3 times in parallel and the extract pair ABTS was calculated according to the following formula + Is a clearance rate of (2):
3 determination of reducing force
0.8mL of sample solutions of different mass concentrations (12.5, 25, 50, 100. Mu.g.ml -1 ) 2mL of phosphate buffer (PH=6.6) and 2mL of 1% potassium iron hydride are added, water bath is carried out at 50 ℃ for 20min, 2mL of 10% trichloroacetic acid is added, centrifugation is carried out at 3000rpm for 10min, 2mL of supernatant is taken, 2mL of deionized water is added, 0.4mL of 0.1% ferric trichloride is taken for reaction for 5min, absorbance value is measured at 700nm, VC is used as positive control, experiment is carried out for 3 times in parallel, and the larger measured absorbance value indicates stronger reducing capability.
Results: as shown in tables 3-5, acanthopanax uniform polysaccharide eliminates DPPH free radical and ABTS + Is of (2) 50 The values are 3.17+/-0.57 mug.mL respectively -1 And 15.26.+ -. 5.79. Mu.g.mL -1 The reduction force EC50 value is 36.02 + -10.89 μg.mL -1 The antioxidant capacity of the acanthopanax is stronger than that of vitamin C which is a positive medicine, and the difference is statistically significant (P is less than 0.01), which indicates that the acanthopanax uniform polysaccharide prepared in the example 1 has stronger antioxidant activity.
TABLE 3 Wilsoniana polysaccharide with DPPH radical scavenging Activity
P < 0.05, P < 0.01 compared to vitamin C group.
TABST removal of acanthopanax homogeneous polysaccharide + Activity(s)
P < 0.05, P < 0.01 compared to vitamin C group.
TABLE 5 determination of reducing force of Acanthopanax Senticosus homogeneous polysaccharide
P < 0.05, P < 0.01 compared to vitamin C group.
Example 3 preparation of an essence emulsion containing Acanthopanax Senticosus Uniform polysaccharide
The acanthopanax uniform polysaccharide obtained in the example 1 is used for preparing the essence milk in the experiment, and the weight percentages of the components and the production process are as follows:
raw material ratio (%) supplier
Phase A
Phase B
Radix Acanthopanacis Senticosi uniform polysaccharide 6.0 homemade
54.3 self-made deionized water
The production process comprises the following steps: the phase A and the phase B are heated to 70 ℃ respectively under stirring until all the phases are dissolved and mixed uniformly, and then the phase B is added into the phase A under the condition of 70 ℃ until the W/Q type emulsion is formed. And (5) uniformly stirring, and cooling to room temperature.
The matrix components used in the preferred embodiments of the present invention are as described above, and the matrix components used in the present embodiment can exert the effects of the pharmaceutical composition of the present invention to the best. However, the conventional cosmetic-applicable matrix produced by other manufacturers can be used in the present invention, and the dosage of the conventional cosmetic-applicable matrix meets the national cosmetic additive dosage standard, and the effect of the present invention is not affected, so that the present invention is not limited.
The homogenizing and emulsifying equipment used in the invention is a FV-30 LFLISCO vacuum homogenizing and emulsifying machine manufactured by Shanghai Froude fluid machinery manufacturing company, which has the functions of homogenizing, stirring, temperature control and the like. Other manufacturers produce homogenizing and emulsifying equipment for cosmetic preparations which can be used in the present invention to achieve the effects described herein, provided that the equipment is operated in strict accordance with the process parameters described herein.
Example 4 evaluation of skin aging-resisting Activity of milk containing Acanthopanax Senticosus Uniform polysaccharide essence
The essence milk containing acanthopanax uniform polysaccharide obtained in example 3 was used for evaluation of anti-skin aging activity.
1 materials and methods
1.1 laboratory animals SPF-grade female Kunming mice 40, body mass (20+ -2) g, purchased from laboratory animal center, guangdong province, laboratory animal mass license number: 44005800003406 the experiments were carried out in Guangzhou university technical industry garden animal house (use license number: SYXK (Guangdong) 2013-0014). Since the final objective of this study was to develop cosmetics suitable for female use against skin aging, female mice were all selected as experimental animals. The treatment of mice is in accordance with animal ethics.
1.2 materials and reagents the essence milk containing acanthopanax uniform polysaccharide is the essence milk prepared in the example 3; d-galactose: beijing Soy Biotechnology Co., ltd; HA assay kit: shanghai enzyme-linked biotechnology limited company; HYP determination kit, SOD determination kit and Coomassie Brilliant blue: nanjing builds the institute of bioengineering.
1.3 grouping and modeling female mice were first adaptively bred for 7d, ensuring that the mice began to experiment after adaptation to the current environment. 30 female mice were divided into 3 groups according to a random number table grouping method, wherein 2 groups of mice were used as a model group, and D-galactose was injected subcutaneously in 1.0 g.kg -1 ·d -1 A total injection of 30d; the remaining group of mice served as a blank group, and were injected with the same volume of physiological saline every day. After 30 days, the skin appearance of the model group and the blank group were compared, the skin of the model group was obviously relaxed, fine wrinkles were generated, and the blank group was opposite. The 2 groups of skin aging model mice were divided into: model group and acanthopanax root homogeneous polysaccharide group. Shearing toolThe knife cuts short the hair on the back of the mouse and shaves Mao Beiyong with a razor.
1.4 mice skin external-use essence milk of acanthopanax root polysaccharide group mice use the essence milk containing 6% of acanthopanax root polysaccharide obtained in the example 3, and a model group uses the essence milk without acanthopanax root polysaccharide, specifically, the method comprises selecting an area of 4cm multiplied by 7cm of the central position of the back of the mice, smearing each group of ointment on the surface of the skin area selected by each group of mice, cleaning the skin after each day for 0.3g and 24h, smearing again, continuously smearing for 21d, and manually dehairing for 4 times.
1.5 apparent characteristics of the skin of the mice are observed and compared with the apparent characteristics of the skin of the drug application part, such as color, smoothness, wrinkles and the like of each group of mice, and photographed and recorded. Then killing the experimental mice by cervical spondylosis, rapidly peeling off skin at the application part, removing subcutaneous fat and other connective tissues, spreading, cutting the skin of the mice at the center position by using a puncher with the diameter of 2cm for measuring the water content, and freezing and preserving the rest skin at the temperature of-20 ℃ for measuring the components such as skin hydroxyproline.
1.6 measurement of skin moisture content the skin moisture content cut by the punch was accurately weighed, then placed in an oven, dried at 50 ℃ for 12 hours, then the dry weight was weighed, and the skin moisture content of each experimental group was calculated. The formula is as follows: skin moisture = (wet weight-dry weight)/wet weight x 100%.
1.7 skin Hydroxyproline (HYP) content measurement skin tissue of the applied part was taken in an amount of 0.5g, rinsed with ice-bath pre-chilled saline, wiped dry with filter paper, added with ice-bath pre-chilled saline, and ground to a 10% strength homogenate using a glass homogenizer. The resulting homogenate was centrifuged at 3,000 r/min at 0℃for 10min, and the supernatant was taken. According to the method shown in the specification of the HYP kit, an ultra-micro-pore plate spectrophotometer is used for measuring an OD value, and the skin HYP content of each experimental group of mice is calculated.
1.8 measurement of skin Hyaluronic Acid (HA) content 0.5g of skin tissue at the site of application was taken, added to ice-bath pre-chilled PBS buffer solution, and ground to a 10% strength homogenate using a glass homogenizer. The resulting homogenate was centrifuged at 1000r/min for 4min at 0deg.C and the supernatant was taken. According to the method shown in the HA kit specification, an ultra-micro microplate spectrophotometer is used for measuring an OD value, and the content of the mouse skin HA of each experimental group is calculated.
1.9 measurement of skin superoxide dismutase (SOD) Activity 0.5g of skin tissue of the coated part is rinsed with ice-bath pre-cooled physiological saline, and then the ice-bath pre-cooled physiological saline is added and ground into a homogenate with a concentration of 10% by using a glass homogenizer. The resulting homogenate was centrifuged at 3,000 r/min at 0℃for 10min, and the supernatant was taken. According to the method shown in the SOD kit specification, an ultra-micro-pore plate spectrophotometer is used for measuring the OD value, and the skin SOD activity of the mice in each experimental group is calculated.
1.10 statistical methods data analysis was performed using SPSS18.0 statistical software, metering data was performed using
A representation; the comparison between groups adopts single factor analysis of variance; the variance alignment test results are obvious, and Dunnett T3 test is adopted for comparison every two; the variance alignment test results are not obvious, the LSD test is adopted for comparison, and the difference of P < 0.05 is statistically significant.
2 results
2.1 comparison of skin appearance skin findings for each group of mice: compared with the animals in the blank group, the skin of the mice in the model group is loose, a large number of wrinkles are formed, and the body hair is regenerated slowly. Compared with the model group, the mice coated with the polysaccharide containing 6% of acanthopanax root uniform polysaccharide have obvious improvement in the aspects of skin wrinkling, smoothness, relaxation and the like.
2.2 influence of acanthopanax uniform polysaccharide on skin moisture content, HYP content, HA content and SOD activity of mice three groups of mice skin tissue moisture content, HYP content, HA content and SOD activity measurement results are shown in Table 6. The skin tissue water content, HYP content, HA content and SOD activity of the mice in the model group are all obviously lower than those of the mice in the blank group (P is less than 0.01), which indicates that the skin aging model of the mice is successfully molded. Compared with the model group, the skin moisture content, HYP content, HA content and SOD activity of the acanthopanax homogeneous polysaccharide group mice are obviously improved, and the difference HAs statistical significance (P is less than 0.01). Compared with the blank group, the skin moisture content, HYP content, HA content, SOD activity and other indexes of the acanthopanax uniform polysaccharide group mice have no significant difference (P is more than 0.01).
Influence of acanthopanax root polysaccharide on water content, HYP content, HA content and SOD activity of skin tissue of mice
Compared with the model group, deltaP is less than 0.05, deltaP is less than 0.01.
In conclusion, the results of this experiment found that the skin of the mice in the blank group was smooth, compact and elastic. The skin aging model mice injected with D-galactose have loose skin, greatly increased wrinkles, and obviously reduced indexes such as skin moisture content, hydroxyproline content, hyaluronic acid content, superoxide dismutase activity and the like. According to the measurement, after the model mice are externally applied with the acanthopanax uniform polysaccharide essence emulsion with the content of 6%, the effect of improving the appearance of the skin of the mice is obvious, and indexes such as the water content, the hyaluronic acid content, the hydroxyproline content, the superoxide dismutase activity and the like of the skin of the mice are also obviously improved.
Finally, what is necessary here is: the above embodiments are only for further illustrating the technical solution of the present invention, and should not be construed as limiting the scope of the present invention, and some insubstantial modifications and adjustments made by those skilled in the art from the above description of the present invention are all within the scope of the present invention.
Claims (5)
1. A preparation method of acanthopanax uniform polysaccharide with skin aging resisting effect is characterized by comprising the following steps: the method comprises the following steps:
1) Pulverizing the dried acanthopanax decoction pieces, sieving with a 100-mesh sieve, extracting the sieved medicinal powder with 5-8 times of water for 3 times at 80-100 ℃ for 2 hours each time, mixing the extracting solutions, centrifuging the extracting solutions at 3000rpm/min for 20min, collecting supernatant, and rotationally evaporating and concentrating the supernatant to be one fifth of the original volume;
2) After the concentrated solution is cooled, adding alpha-amylase until the content of the alpha-amylase is 0.1-0.4%, regulating the pH value to 7.0, carrying out water bath enzymolysis at 60 ℃ for 4h (the solution has no color change when meeting iodine-potassium iodide reagent), quickly heating to 100 ℃ and keeping for 5min for inactivating enzyme, and centrifuging at 3000rpm/min for 10min to collect supernatant;
3) Supernatant and Sevage reagent are mixed according to the volume ratio of 1:1 mixing, vigorously oscillating for 30min, standing for 12h, collecting upper polysaccharide solution, and repeating until no protein characteristic absorption peak exists in ultraviolet scanning; the Sevage reagent is obtained by mixing chloroform and n-butanol according to a volume ratio of 4:1;
4) Concentrating the upper polysaccharide solution in the step 3), adding 4-6 times of absolute ethyl alcohol, standing at 4 ℃ for precipitation for 48h, centrifuging at 3000rpm/min for 10min, collecting precipitate, repeating for 3 times, and freeze-drying to obtain acanthopanax crude polysaccharide powder;
5) Completely dissolving the acanthopanax crude polysaccharide powder obtained in the step 4) by distilled water, and separating by a DEAE Fast Flow ion chromatographic column under the elution conditions: eluting with pure water, 0.05mol/L, 0.1mol/L, 0.2mol/L, 0.4mol/L, and 1mol/L sodium chloride solution at flow rate of 2.5 mL/min; gradient collecting by using a full-automatic collector, gradient eluting each solution for 3 times of column volume, collecting 30 tubes, and 5.0 per tube mL, and performing tube separation tracking detection by using a sulfuric acid-phenol method; concentrating the collected solutions corresponding to different eluents to a certain volume, and dialyzing with a dialysis bag (3500 Da); changing water for 5 times per day in a beaker filled with a dialysis bag, dialyzing for 7 days, centrifuging polysaccharide liquid in the dialysis bag, and freeze-drying to obtain primarily purified acanthopanax polysaccharide;
6) Concentrating the eluent collected in the step 5), then separating by using a Sephadex G-200 Sephadex column, eluting by using distilled water according to the flow rate of 0.5mL/min, collecting by using a full-automatic collector, detecting by using a phenol-sulfuric acid method, and collecting a main peak part in an elution curve;
7) Concentrating the solution of the main peak part in the elution curve collected in the step 6), dialyzing with 3500Da dialysis bag for 2d for desalination, and concentrating, freeze drying the solution in the dialysis bag to obtain radix Acanthopanacis Senticosi uniform polysaccharide powder.
2. The acanthopanax root polysaccharide with skin aging resistance according to claim 1.
3. The use of acanthopanax uniform polysaccharide with skin aging resisting effect according to claim 2, characterized in that: can be used for preparing skin care cosmetics or therapeutic drugs for skin.
4. A skin care cosmetic comprising the acanthopanax homogeneous polysaccharide having an anti-skin aging effect of claim 2 and an auxiliary material used in the cosmetic field.
5. A therapeutic agent for skin comprising the acanthopanax homogeneous polysaccharide having an anti-skin aging effect according to claim 2 and a pharmaceutically acceptable carrier.
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| JP2021534247A JP7414303B2 (en) | 2020-07-20 | 2020-08-18 | Stir-fried homogeneous polysaccharide and its preparation method and application |
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