CN114957499A - Radix Angelicae Dahuricae polysaccharide composition and its application in removing toxic substance, caring skin, whitening skin or resolving macula - Google Patents

Abstract

The application discloses an angelica dahurica polysaccharide composition and application thereof in expelling toxin, beautifying, whitening or removing freckles. The radix angelicae dahuricae polysaccharide composition comprises at least one of F1-F5, wherein F1-F5 respectively comprise rhamnose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid, and the molar ratios of the rhamnose, the arabinose, the xylose, the mannose, the glucose and the galactose are the same. The 5 angelica dahurica polysaccharides act on melanoma cells, can inhibit the tyrosinase activity of the angelica dahurica polysaccharides and reduce the melanin generation, and by detecting the mRNA level of related genes, the angelica dahurica polysaccharides are inferred to realize a regulation mechanism for inhibiting the tyrosinase activity and reducing the melanin generation by inhibiting the expression of Mitf, Tyr and Tyrp1 genes, so that the 5 angelica dahurica polysaccharides provided by the embodiment of the application have the application prospect in the fields of removing freckles, expelling toxin and beautifying or whitening.

Description

Radix Angelicae Dahuricae polysaccharide composition and its application in removing toxic substance, caring skin, whitening skin or resolving macula

Technical Field

The application relates to the technical field of radix angelicae, in particular to an radix angelicae polysaccharide composition and application thereof in expelling toxin, beautifying, whitening or removing freckles.

Background

The Angelica dahurica is the dried root of Angelica dahurica Angelica Dahurica (Fisch. ex Hoffm.) Benth.et hook.f. or Angelica dahurica Angelica Dahurica (Fisch. ex Hoffm.) Benth.et hook.f. var.Formosana (Boiss.) Shann et Yuan of Umbelliferae, and has the effects of relieving exterior syndrome and dispelling cold, dispelling wind and relieving pain, freeing nasal orifices, eliminating dampness and stopping leukorrhagia, and reducing swelling and expelling pus, and the national Weijian is a medicinal and edible traditional Chinese medicine.

The angelica dahurica mainly contains coumarins, volatile oils, glycosides, polysaccharides, alkaloids and amino acid compounds, and modern pharmacology proves that the coumarin banners have the use value of whitening when being applied to cosmetics. The externally applied Chinese medicine preparation compound dahurian angelica root is used in treating white paralysis by means of the characteristic of the photosensitive effect of the coumarin components in dahurian angelica root. Researches find that the total effective rate of the halometasone emulsifiable paste combined with the compound radix angelicae for treating the common paraplegia is obviously higher than that of the halometasone emulsifiable paste used alone. Researches show that part of coumarins components such as bergapten lactone in the angelica dahurica have the function of activating tyrosine kinase.

However, the continuous development of the active ingredients related to the angelica dahurica and the exploration of the application prospect of the active ingredients in cosmetics or health care products for whitening, detoxifying, removing freckles and the like still have very practical significance.

Disclosure of Invention

In view of the above, the present application aims to find at least one active ingredient of radix angelicae dahuricae different from the prior art, and explore the application prospects of the active ingredient in cosmetics or health care products for whitening, detoxifying, removing freckles, and the like.

In a first aspect, the embodiment of the application discloses an angelica dahurica polysaccharide composition, which comprises at least one of F1-F5, wherein F1-F5 comprise rhamnose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid, and the molar ratios of rhamnose, arabinose, xylose, mannose, glucose and galactose are the same.

In the present examples, F1 comprises a molar percentage of rhamnose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid of 7.374%, 7.374%, 4.733%, 6.187%, 50.006%, 20.676%, 1.980% and 1.670%;

f2 contains the molar percentages of rhamnose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid 7.284%, 7.284%, 4.675%, 6.111%, 49.395%, 20.423%, 2.750% and 2.078%;

f3 contained rhamnose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid in a molar percentage of 7.316%, 4.695%, 6.137%, 49.607%, 20.511%, 2.516% and 1.903%;

f4 contained rhamnose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid in mole percentages of 7.320%, 7.320%, 4.698%, 6.141%, 49.638%, 20.523%, 2.579% and 1.781%;

f5 contained rhamnose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid in mole percentages of 7.320%, 7.320%, 4.698%, 6.141%, 49.638%, 20.523%, 2.579% and 1.781%.

In the examples of the present application, the glycosidic bond in the molecular structure of F1 includes the glycosidic bond of → 1) -L-Rhap- (3 → 1) -D-Araf- (3 → 1) -D-Arap- (2 →, → 1) -D-Manp- (2,4 →, → 1) -D-Manp- (3,6 →, → 1) -D-Glcp- (2,4,6 →, → 1) -D-Glcp- (4 →, → 1) -D-Glcp- (6 →, → 1) -D-Galp- (3 → 1) -D-Galp- (6 →, → 1) -D-ylxp → (2,3 → and → 1) -D-Xylp- (3 → D), and takes Glcp and Galp as terminal sugar units; wherein, the molar content of the → 1) -D-Glcp- (2,4,6 → and → 1) -D-Galp- (3 → exceeds 10%;

glycosidic linkages in the molecular structure of F2 include the glycosidic linkages of → 1) -L-Rhap- (3 →, → 1) -D-Araf- (3 →, → 1) -D-Arap- (2 →, → 1) -D-Manp- (4 →, → 1) -D-Manp- (2,4 →, → 1) -D-Manp- (3,6 →, → 1) -D-Glcp- (2,4,6 →, → 1) -D-Galp- (3 →, → 1) -D-Xylp → (2,3 → and → 1) -D-Xylp- (3 → with Glcp and Galp as terminal saccharide units; wherein, the molar contents of → 1) -D-Glcp- (2,4,6 → and → 1) -D-Galp- (3 → are all more than 10%;

glycosidic linkages in the molecular structure of F3 include the glycosidic linkage of → 1) -L-Rhap- (3 →, → 1) -D-Arap- (2 →, → 1) -D-Manp- (3,6 →, → 1) -D-Glcp- (2,4,6 →, → 1) -D-Galp- (3 →, → 1) -D-Xylp → (2,3 → and → 1) -D-Xylp- (3 → and with Glcp and Galp as terminal saccharide units; wherein, the molar content of the → 1) -D-Glcp- (2,4,6 → and → 1) -D-Galp- (3 → exceeds 10%;

the glycosidic bond in the molecular structure of F4 includes the glycosidic bond of → 1) -L-Rhap- (3 →, → 1) -D-Araf- (3 →, → 1) -D-Manp- (2,4 →, → 1) -D-Glcp- (2,4,6 →, → 1) -D-Galp- (3 →, → 1) -D-Xylp → (2,3 → and → 1) -D-Xylp- (3 → and takes Glcp and Galp as terminal saccharide units; and wherein, the molar content of → 1) -L-Rhap- (3 →, → 1) -D-Glcp- (2,4,6 → and → 1) -D-Galp- (3 → exceeds 10%.

In a second aspect, the embodiment of the application discloses a health care product for expelling toxin, beautifying, whitening and removing freckles, which comprises the angelica dahurica polysaccharide composition in the first aspect, and the mass percentage is 0.001-20 wt%.

In embodiments of the present application, the nutraceutical further comprises a nutraceutical or cosmetically acceptable adjuvant comprising at least one of a wetting agent, a softening agent, a preservative, a pH adjusting agent, organic and inorganic pigments, a fragrance, a cooling agent, an antiperspirant, a stabilizer, a hydration or oleolysis promoter, a diluent, a binder, a salt for adjusting the osmotic pressure and/or a buffer.

In a third aspect, the embodiment of the application discloses an oral preparation for expelling toxin, beautifying, whitening and removing freckles, which comprises 0.001-10 parts by mass of the radix angelicae dahuricae polysaccharide composition, 100-500 parts by mass of a diluent and 20-50 parts by mass of a binder.

In a fourth aspect, the embodiment of the application discloses a cream for expelling toxin, beautifying, whitening and removing freckles, which comprises 0.001-20 wt% of the angelica dahurica polysaccharide composition, 0.5-1.75 wt% of sodium stearate, 1.5-2.5 wt% of polysorbitol 60, 2.0-4.0 wt% of polyethylene glycol 60 hydrogenated castor oil, 1.5-5.0 wt% of squalane, 1.5-5.0 wt% of caprylic/capric triglyceride, 15-35.0 wt% of glycerol, 0.5-3.0 wt% of propylene glycol, 0.02-0.2 wt% of triethanolamine, 0.2-2 wt% of preservative, 0.1-0.25 wt% of perfume and the balance of water.

In a fifth aspect, the present application discloses a preparation method of the angelica dahurica polysaccharide composition of the first aspect, comprising the following steps:

obtaining degreased radix angelicae dahuricae filter residues, refluxing with ethanol, treating with glycosidase to obtain an enzymolysis product, and extracting the enzymolysis product to obtain a radix angelicae dahuricae polysaccharide crude product;

and purifying the radix angelicae dahuricae polysaccharide crude product by gel chromatography to obtain the radix angelicae dahuricae polysaccharide composition.

In the examples of the present application, an aqueous solution containing α -mannosidase, ammonium sulfate and zinc acetate was used as an enzymatic treatment agent.

In a sixth aspect, the embodiment of the application discloses an application of the radix angelicae dahuricae polysaccharide composition in preparing health care products or cosmetics related to toxin expelling, skin beautifying, whitening or freckle removing.

Compared with the prior art, the application has at least the following beneficial effects:

according to the embodiment of the application, the resource of the conventional Chinese medicinal material angelica dahurica with homology of medicine and food is fully utilized, 5 polysaccharide components are developed and obtained from the resource, and a certain research is carried out on the primary structure of the polysaccharide components. Therefore, the five angelica dahurica polysaccharides act on melanoma cells, the tyrosinase activity of the angelica dahurica polysaccharides can be inhibited, the melanin generation can be reduced, and the angelica dahurica polysaccharides are inferred to realize a regulation mechanism for inhibiting the tyrosinase activity and reducing the melanin generation by inhibiting the expression of Mitf, Tyr and Tyrp1 genes through detecting the mRNA level of related genes. Animal experiments finally prove that the cream and the tablet prepared from the 5 angelica polysaccharide components have the effect of reducing the synthesis of melanin of a model mouse, and the 5 angelica polysaccharides provided by the embodiment of the application have application prospects in the fields of removing freckles, expelling toxin and beautifying or whitening.

Drawings

FIG. 1 is an elution profile of SephadexG-50 purification provided in example 1 of the present application.

FIG. 2 is an elution profile of SephadexG-25 purification provided in example 1 of the present application.

FIG. 3 is an elution profile of SephadexG-50 purification provided in comparative example 1 of the present application.

FIG. 4 is an infrared spectrum of F1-F5 provided by the present application.

FIG. 5 is a total ion flow chromatogram of the methylation product of F1 provided herein.

Fig. 6 is a microscopic image of melanoma cells provided herein after passage.

FIG. 7 is a photograph of melanoma cells provided herein after being administered 8-MOP for 56 h.

Fig. 8 is a microscopic image of a melanoma cell model set provided herein.

FIG. 9 is a graph showing HE staining of skin tissue sections of normal mice in the animal experiments of the present application.

FIG. 10 is a graph showing HE staining of skin tissue sections of mice in a model group provided in an animal experiment of the present application.

FIG. 11 is HE staining chart of skin tissue section of positive group 1 mice provided in the animal experiments of the present application.

FIG. 12 is a graph showing HE staining of positive 2 groups of mouse skin tissue sections in an animal experiment according to the present application.

FIG. 13 is a graph showing HE staining of skin tissue sections of mice in the emulsion group (example 2) provided in the animal experiments of the present application.

FIG. 14 is a graph showing HE staining of mouse skin tissue sections from the tablet group (example 7) provided in the animal experiments of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

Angelica polysaccharide

Materials and methods

1. Radix angelicae: powder, available from national treasures pharmaceutical ltd, mn.

2. Extraction of

The extraction implementation process of a specific embodiment 1 is as follows:

(1) weighing 250g of radix angelicae powder, and adding into 2L of petroleum ether for reflux degreasing treatment for 4 h;

(2) filtering to obtain about 230g of filter residue, adding 100% ethanol aqueous solution, performing reflux extraction for 2h, filtering to obtain filter residue, adding 95% ethanol aqueous solution again, performing reflux treatment for 2h, filtering to obtain filter residue, adding 85% ethanol aqueous solution again, performing reflux treatment for 2h, filtering, and drying to obtain 210g of filter residue;

(3) taking 210g of filter residue, fully dispersing the filter residue in 2L of PBS (phosphate buffer solution) with the pH value of 4.5, simultaneously adding 200mL of alpha-mannosidase containing 23.5g/L, 3.0M ammonium sulfate and 0.1mM zinc acetate aqueous solution (M7257, Sigma-Aldrich), fully stirring for 2h, transferring to water bath at about 90-105 ℃ for stewing for 2h after overnight so as to fully inactivate enzyme and denature related protein, filtering, and concentrating the filtrate under reduced pressure to obtain 150mL of concentrated solution;

(4) adding 40mL of chloroform n-butanol (the volume ratio of chloroform to n-butanol is 4:1) extraction solvent into 150mL of concentrated solution, fully mixing, standing, taking supernatant, centrifuging at 3500rpm for 15min, taking supernatant, adding 4 times of anhydrous ethanol, fully mixing, standing overnight, centrifuging at 4000rpm for 15min, taking precipitate, and sequentially washing with 95% ethanol solution, anhydrous ethanol, diethyl ether and acetone for 2 times respectively to obtain 15.3g of radix angelicae polysaccharide crude product.

A specific extraction procedure of comparative example 1 was carried out in substantially the same manner as in example 1, except for the step (3) described above, specifically:

(3) taking about 200g of filter residue, adding 2L of distilled water, fully and uniformly mixing, transferring to water bath at about 90-105 ℃, cooking for 2h, filtering to obtain the filter residue, adding water again, cooking, combining the filtrate, and concentrating the filtrate under reduced pressure.

3. Purification of

And purifying the obtained radix angelicae dahuricae polysaccharide crude product by adopting a gel chromatographic column.

(1) Column mounting

SephadexG-50 and SephadexG-25 are purchased from GE Healthcare, 50g of the Sephadex G-50 and the SephadexG-25 are respectively and accurately weighed, are respectively and fully soaked for 24 hours at room temperature by 1L of deionized water, are repeatedly washed until the Sephadex G-25 and the SephadexG-25 are fully swelled, are respectively and finely washed for 0.5 hour by 0.5M alkali liquor and 0.5M sodium chloride solution in sequence, are washed to be neutral by deionized water, and are filled into an AKTA protein purification column by a wet method.

(2) Loading and elution

Accurately weighing 200mg of the radix angelicae dahuricae polysaccharide crude product prepared in the embodiment 1 and the comparative example 1, dissolving the crude product with 2mL of deionized water, filtering the solution with a 0.45-micron filter membrane to prepare a sample loading solution, loading the sample loading solution on SephadexG-50 and SephadexG-25 chromatographic columns respectively, standing the chromatographic columns for 30min, eluting the chromatographic columns with deionized water at the flow rate of 0.25mL/min, collecting the eluent in parts, collecting 5mL of each chromatographic column, collecting 50 chromatographic columns in total, detecting the light absorption value of each chromatographic column by a phenol-sulfuric acid method, drawing an elution curve, determining a combined collecting tube according to the elution curve, and freeze-drying to obtain the radix angelicae dahuricae polysaccharide refined freeze-dried powder.

4. Detection of molecular weight of refined dahurian angelica polysaccharide freeze-dried powder

A chromatographic column: an Ultrahydrogel water-soluble gel chromatography column; the mobile phase was 0.1N sodium nitrate, column temperature 30 ℃, differential refractometer detection (Waters 2424), sample size 20. mu.L, and all test results were integrated by Breeze software (Waters Chromatography, Inc).

Standard curve: taking standard glucan (molecular weight is 1000,5000,12000,80,150,270,670kDa, Nanjing Tolyre Biotechnology Limited) to prepare a 2% solution, centrifuging for 10min at 13000 r/min, preparing a standard curve by using supernate, feeding 20 mu L of the solution to obtain a chromatogram, drawing a standard curve by using the elution volume (mL) of eluent with a peak as a horizontal coordinate and the logarithm 1gMw of the weight average molecular weight of the glucan as a vertical coordinate, and calculating the molecular weight of a sample according to the molecular weight standard curve.

And (3) sample determination: dissolving the lyophilized powder purified by gel chromatography with mobile phase to obtain 10mg/mL solution, filtering with 0.45 μm filter membrane, detecting by sampling under the above conditions, substituting the retention time into standard curve, and calculating to obtain corresponding molecular weight.

5. Ultraviolet (UV) full scan detection

Weighing 2mg of purified angelica polysaccharide freeze-dried powder, preparing 1mg/mL solution by using distilled water, and carrying out ultraviolet full scanning at 200-400 nm.

6. Infrared Spectroscopy (IR)

Taking 2mg of purified angelica polysaccharide freeze-dried powder, tabletting by using KB, and then carrying out IR scanning, wherein the scanning range is 4000-400 cm ^-1 。

7. Monosaccharide composition analysis

And (3) carrying out qualitative and quantitative analysis on the composition types and concentrations of monosaccharides from F1 to F5 by adopting ion chromatography:

pretreatment: accurately preparing 2M trifluoroacetic acid (trifluoroacetic acid TFA) containing 1mg/mL of F1, F2, F3, F4 and F5 samples respectively, and treating 2mL of the trifluoroacetic acid TFA at 110 ℃ for 6 hours to hydrolyze the trifluoroacetic acid; and then, the hydrolysate is dried by rotary evaporation at 40 ℃, 3mL of TriSil reagent (pyridine: hexamethyldisilazane: trimethylchlorosilane: 10:2:1 volume ratio) is added, heating treatment is carried out for 1h in a water bath at 80 ℃, the mixture is subjected to silicon etherification, the mixture is dried by nitrogen after the reaction is finished, the volume of 1mL of n-hexane is constant, and the detection is carried out by GC-MS/MS.

And (3) chromatographic detection conditions: TRACE 1310 GC, TSQ 8000 Evo triple quadrupole mass spectrometer, TG-5MS capillary column, available from Thermo Fisher Scientific, USA; an Organomation N-EV AP nitrogen blower available from Organomation Inc., USA. Chromatographic parameters: mass spectrum scanning mode, positive ion mode; the scanning range is 50-500; the gas-phase carrier gas is nitrogen, and the flow rate is 1.5 mL/min; the sample injection mode is that the sample is not divided and the sample injection amount is 1 mu L. The temperature raising procedure of the gas phase is that the temperature is raised from 80 ℃ to 150 ℃ at a speed of 10 ℃/min, the temperature is maintained at 150 ℃ for 2min, then the temperature is raised to 180 ℃ at a speed of 1.5 ℃/min, and then the temperature is raised to 210 ℃ at a speed of 10 ℃ for 20min, and the temperature is maintained at 210 ℃ for 20 min.

And (3) standard substance: respectively preparing 100 mu g/mL of aqueous solutions of rhamnose, fucose, arabinose, galactose, glucose, xylose, mannose, fructose and glucosamine; preparing a mixed standard solution with the concentration of 5 mu g/mL; taking 10 mu g inositol as internal standard from 5, 1, 0.5, 0.1 and 0.05mL solution respectively, and drying by a nitrogen blower. The silicon etherification derivatization was performed in the same manner as the samples, and the GC-MS/MS detection was performed under the above conditions.

8. Periodic acid oxidation-Smith degradation

Because the continuous hydroxyl or continuous trihydroxyl bond in the sugar molecule can be specifically broken under the oxidation action of sodium periodate; in general, every 2 molecules of periodic acid can oxidize 1 sugar residue containing a vicinal triradical and generate 1 molecule of formic acid; 1 molecule of periodic acid consumed can oxidize 1 sugar residue containing a vicinal dihydroxy group without generating formic acid; if the sugar chain contains neither a diradical nor a diradical residue, it cannot be oxidized with periodic acid and does not produce formic acid. Therefore, the type of glycosidic bond and the structural information such as the form of linkage, the position of the branch, the self-assembly, etc. are determined by measuring the consumption of periodic acid and the amount of formic acid produced.

The specific method for quantifying the consumption of sodium iodate is as follows:

standard curve: respectively preparing 50.0mL of 0.015M sodium periodate solution and 50mL of 0.015M sodium periodate solution, mixing the two solutions according to the volume ratio of 5:0, 4:1, 3:2, 2:3, 1:4 and 0:5, respectively taking 0.4mL of mixed solution in each proportion, using distilled water to fix the volume to 50mL, and then using a spectrophotometer to measure the light absorption value at 223 nm. A standard curve of the consumption of sodium periodate is drawn by taking the concentration (mol/L) of sodium periodate in the mixed solution of each proportion as an abscissa and taking an Absorbance (Absorbance) at 223nm as an ordinate.

Determination of the sodium periodate consuming capacity of the polysaccharide samples: 20mg of polysaccharide sample is added into a 0.015M sodium periodate solution containing 60mL and placed in a dark brown ground bottle, after the solution is shaken and dissolved, the mixture is placed in a dark place of a refrigerator at 4 ℃ for oxidation, and periodic shaking and sampling are carried out. After 0.4mL of sodium periodate was taken out from the flask each time, the volume was adjusted to 50mL with distilled water, and then the absorbance was measured at a wavelength of 223nm, and the amount of sodium periodate consumed was calculated from a standard curve.

The products obtained are different due to the different way of linkage between the periodate-oxidized sugar residues. Further by Smith degradation, periodate oxidation products can be reduced to more stable polyols such as glycerol, erythritol and non-oxidized monosaccharide residues. Among them, the types of the sugar bond which can produce glycerol are (1 →), (1 → 6), (1 → 2) and (1 → 2,6), and the types of the sugar bond which can produce erythritol are (1 → 4) and (1 → 4, 6). The type and branching location of glycosidic linkages can therefore be inferred by identifying the hydrolysis products.

Smith degradation was performed on the basis of periodic acid oxidation. After the periodic acid oxidation reaction is finished, excessive NaBH4 is added for reduction for 24h, and then glacial acetic acid is used for removing excessive NaBH ₄ Then, the mixture was evaporated to dryness under reduced pressure. NaBH treated with 1M TFA ₄ The reduced matter is subjected to acid hydrolysis, and the reaction time is 2 h. Methanol was continuously added to the reaction system and distilled under reduced pressure to remove excess TFA. Then adding a certain amount of water to dissolve the sample, adjusting the system to be neutral or alkalescent by NaOH, removing water by reduced pressure distillation, and obtaining the oligosaccharide by freeze drying. And finally, carrying out methylation analysis on the obtained oligosaccharide.

9. Methylation analysis

(1) Reduction of carboxyl groups

Weighing 20.0mg of F1-F5 freeze-dried powder, dissolving in 5.0mL of deionized water, adding 0.01mol/L HCl to adjust the pH value to 4.75, then slowly adding 100.0mg of CMC, continuously stirring for reaction for 1-2h, and after the reaction is finished, slowly adding 4mL of 2.0mol/L NaBH ₄ And 4mol/L HCL is added dropwise to maintain the pH value at 7.0 until NaBH is added ₄ After the addition, the mixture is kept standing for reaction for 1h at room temperature. And after the reaction is finished, transferring the mixture into a dialysis bag, dialyzing for 24 hours by running water, dialyzing for 24 hours by deionized water, and freeze-drying after dialysis to obtain a reduction product of F1-F5.

(2) Methylation reaction

Weighing 10.0mgF 1-F5 reduction products, placing the reduction products in a dry 25mL reaction bottle, sealing a sealing film, then puncturing by using an injector, placing the reaction bottle in a P20 drier for drying for 24h, adding a magnetic stirrer, adding 10mL anhydrous DMSO under the condition of nitrogen filling, sealing the reaction bottle in a dark place, using ultrasonic wave to assist and fully dissolve the reduction products, quickly adding 20.0mg NaOH powder, placing the reaction bottle in an ice bath after the ultrasonic-assisted reaction is finished, slowly adding 0.5mL methyl iodide, and reacting the reaction bottle in a dark place for 1 h; after the reaction is finished, 3.0mL of deionized water is added to quench the methylation reaction, then the mixture is transferred into a dialysis bag (3500Da), the deionized water is dialyzed until the reaction solution is clear, and the dialyzed solution is concentrated and freeze-dried. Repeating the above steps for 1-2 times, and detecting whether the methylated polysaccharide sample is completely methylated by adopting Fourier infrared spectrum.

(3) Hydrolysis and derivatization of methylated polysaccharides

Weighing 5.0mg of methylated F1-F5 samples, placing the samples in an ampere bottle, respectively adding 5.0mL of formic acid solution, sealing the opening of an alcohol blast lamp, reacting for 3 hours at 100 ℃, transferring the samples to a nitrogen blowing instrument for blowing nitrogen to dry (60 ℃) after the reaction is finished, adding 3.0mL of methanol to remove residual formic acid, and repeating the steps for 3 times to obtain the polysaccharide depolymerized product. Dissolving the depolymerized product in 4.0mL of 3.0M trifluoroacetic acid, transferring the solution into an ampere bottle, sealing the bottle by using an alcohol burner, reacting for 2 hours at 121 ℃, transferring the bottle into a nitrogen blowing instrument to blow the bottle with nitrogen (60 ℃) after the reaction is finished, adding 3.0mL of methanol to remove residual trifluoroacetic acid, and repeating for 3 times to obtain a hydrolysate of methylated F1-F5;

dissolving the hydrolysis products of methylated F1-F5 in 2.0% sodium borohydride solution, standing at room temperature for reaction for 2.0h, adding 20.0% (v/v) acetic acid to neutralize the sodium borohydride which is not reacted until no bubbles are generated, then transferring the solution into a nitrogen blowing instrument for nitrogen blow drying (60 ℃), adding 3.0mL of methanol to remove the residual borate, and repeating the steps for 3 times to obtain the hydrolysis reduction products of methylated F1-F5.

Adding 1.0mL of acetic anhydride and 1.0mL of pyridine into the reduction product in sequence, carrying out vortex mixing, transferring the mixture into an ampere bottle, sealing the mixture by using an alcohol blast lamp, reacting for 2 hours at 100 ℃, adding 4.0mL of deionized water to remove unreacted acetic anhydride after the reaction is finished, adding 5.0mL of chloroform for extraction, repeatedly extracting for three times, collecting a chloroform layer, adding deionized water with the same volume to wash the chloroform layer for 3 times, collecting the chloroform layer, concentrating by using a nitrogen blowing method to about 200 mu L to obtain a partial methylated sugar alcohol acetylation Product (PMAAs), filtering by using a filter membrane, and carrying out GC-MS separation.

GC-MS chromatographic conditions, namely an Agilent DB-SMS capillary column (30m \0.25mm \0.25 mu m); carrier gas is high-purity He; the flow rate of the column is 1.0 mL/min; the sample injection amount is 1. mu.L; the split ratio is 10: 1; the temperature of a sample inlet is 260 ℃; the temperature raising program is that the initial temperature is 60 ℃, the temperature is raised to 80 ℃ at the speed of 5.0 ℃/min and kept for 1.0min, the temperature is raised to 220 ℃ at the speed of 10 ℃/min and kept for 1.0min, and finally the temperature is raised to 300 ℃ at the speed of 20 ℃/min and kept for 1.0 min.

Mass spectrometry conditions Electron Impact (EI) ion source; electron energy 70 eV; the ion source temperature is 300 ℃; the interface temperature is 300 ℃; the solvent delay time is 3.0 min; the mass scanning range m/z is 30-450 amu.

Second, result in

1. Gel chromatography purification results and relative molecular weight detection

As can be seen from FIG. 1, in the elution curve purified by SephadexG-50 in example 1, elution peaks appeared in the 29 th to 31 th tubes and the 40 th to 50 th tubes, but the elution peaks in the 40 th to 50 th tubes were somewhat flat; further, the concentrated solution obtained by eluting and combining the fractions was purified by further using SephadexG-25, and as shown in FIG. 2, elution peaks appeared in the elution curves of SephadexG-25 in the 7 th to 10 th tubes, 14 th to 18 th tubes and 21 th to 24 th tubes.

As shown in FIG. 3, it is clear that only one peak eluted in the 6 th to 13 th tubes was obtained in comparative example 1 after SephadexG-50 purification.

Thus, eluents 29 to 31 (named F1) of the eluent purified and eluted by Sephadex G-50, eluents 7 to 10 (named F2), eluents 14 to 18 (named F3) and eluents 21 to 24 (named F4) of the eluent purified and eluted by Sephadex G-50 in example 1, and eluents 6 to 13 (named F5) of the eluent purified by Sephadex G-50 in comparative example 1 were collected, respectively concentrated and freeze-dried to obtain freeze-dried powders of F1 to F5. Ultraviolet spectrum (UV) full-scan detection is carried out on the F1-F5 freeze-dried powder, no obvious characteristic absorption peak exists at 260-280 nm, and the F1-F5 freeze-dried powder prepared in the embodiment 1 and the comparative example 1 are polysaccharide components.

Further, relative molecular weights of lyophilized powders F1 to F5 were measured, and a standard curve fitting equation obtained using dextran was-1.489X +13.65 (R) ² With-0.9564), the elution volumes of F1 to F5 were 6.625mL, 6.745mL, 6.828mL, 6.867mL and 6.205mL in this order, and by substituting the above standard curve fitting equation, the relative molecular weights of F1 to F5 were calculated to be 6095, 4045, 3040, 2660 and 25763, respectively.

2. Monosaccharide compositions of F1-F5 and infrared spectra

The results of the IR spectroscopy analysis F1-F5 are shown in FIG. 4. 3600 to 320 DEG c m ^-1 The broad peak appeared as stretching vibration of O-H, C-H bond, and the samples were all polysaccharides. 1665-1635 cm ^-1 The absorption peaks of (A) indicate the presence of an acetamido group (-NHCOCH) in F1 to F5 ₃ ) The C ═ O stretching vibration is acidic polysaccharide; 1200 to 100 ℃ m ^-1 The absorption peak with larger interval is C-O-H or C-O-C of a sugar ring caused by the stretching vibration of two large absorption peaks, and the two large absorption peaks are both characteristic peaks of pyranose; 830cm ^-1 And 897cm ^-1 Spectral band absorberThe peak is shown that F1-F5 contain glycosidic bonds in alpha-and beta-configurations. In addition, infrared analysis showed no 1730cm detected ^-1 And an obvious absorption peak is nearby, which shows that uronic acid in the three polysaccharides of hovenia dulcis is unesterified uronic acid.

The statistics of monosaccharide composition measurements for F1-F5 are shown in Table 1.

Table 1 mol% w/w

Components	F1	F2	F3	F4	F5
						Rhamnose	7.374	7.284	7.316	7.320	7.462
Arabinose	7.374	7.284	7.316	7.320	7.462
						Xylose	4.733	4.675	4.695	4.698	4.789
Mannose	6.187	6.111	6.137	6.141	6.260
						Glucose	50.006	49.395	49.607	49.638	50.601
Galactose	20.676	20.423	20.511	20.523	20.921
						Glucuronic acid	1.980	2.750	2.516	2.579	1.628
Galacturonic acid	1.670	2.078	1.903	1.781	0.876

3. Periodic acid oxidation, Smith degradation and methylation analysis

As shown in Table 2, the periodic acid oxidation results of F1-F5 showed that the consumption of periodic acid and the amount of formic acid generated after periodic acid oxidation were greater than 2, indicating that the glucan bond types contained therein all included type I: 1 →,1 → 2 or 1 → 6 linkage type; type II: 1 → 4, 1 → 2,6 or 1 → 4,6 linkage type; type III: 1 → 3,4,6, 1 → 2,3,6, 1 → 2,4 or 1 → 2, 46. Gas chromatography detection of the Smith degradation products revealed that glycerol was detected in large amounts, but no erythritol was detected, indicating that F1-F5 contained a large amount of type I glycosidic linkages and did not contain type II 1 → 4 and 1 → 4,6 glycosidic linkages.

Table 2 mol% w/w

Components	Consumption of periodic acid (mol/mol)	Amount of formic acid formed (mol/mol)	Bond type of dextran
				F1	0.685	0.327	I. Forms II and III
F2	0.677	0.3165	I. Forms II and III
				F3	0.658	0.3038	I. Forms II and III
F4	0.643	0.3142	I. Forms II and III
				F5	0.768	0.3738	I. Forms II and III

Since F1-F5 all contain uronic acid, the infrared spectrum of reduced products is 3390cm ^-1 There is no obvious characteristic peak, which indicates complete reduction of uronic acid. Completely methylating completely reduced F1-F5 to obtain sugar alcohol acetate derivatives (PMAAs), determining by GS-MS to obtain a total ion flow diagram and a corresponding mass spectrogram of The PMAAs of F1-F5, comparing The mass spectrogram corresponding to The peak of The obtained PMAAs with a standard Spectral library ((The CCRC Spectral Database for PMAA') summarized by sugar complex research of The university of Georgia and chemical data of The Chinese academy of sciences) to determine The type of methylated sugar residues, and calculating The relative mole percentage of each methylated sugar residue according to The peak area of The chromatographic peak to finally obtain The type and The mole percentage of The glycosidic bond of The two polysaccharide components, wherein The specific statistical result is shown in tables 3,4 and 5.

TABLE 3

TABLE 4 PMAAs, glycosidic linkages and Mass Spectrometry fragments thereof

The molar percentages of glycosidic linkages in each molecule in tables 5F 1-F5

In Table 3, "-" indicates no detection. As is clear from Table 3, the glycosidic bond distribution of F5 is the widest, and the bond distributions of F1 to F4 are the most concentrated. F4 is most concentrated and includes the glycosidic bond of → 1) -L-Rhap- (3 →, → 1) -D-Araf- (3 →, → 1) -D-Manp- (2,4 →, → 1) -D-Glcp- (2,4,6 →, → 1) -D-Galp- (3 →, → 1) -D-Xylp → (2,3 → and → 1) -D-Xylp- (3 → with Glcp and Galp as terminal saccharide units; and wherein, the content of → 1) -L-Rhap- (3 →, → 1) -D-Glcp- (2,4,6 → and → 1) -D-Galp- (3 → exceeds 10%.

Whereas F1 mainly includes → 1) -L-Rhap- (3 →, → 1) -D-Araf- (3 →, → 1) -D-Arap- (2 →, → 1) -D-Manp- (2,4 →, → 1) -D-Manp- (3,6 →, a glycosidic bond of → 1) -D-Glcp- (2,4,6 →, → 1) -D-Glcp- (4 →, → 1) -D-Glcp- (6 →, → 1) -D-Galp- (3 →, → 1) -D-Galp- (6 →, → 1) -D-Xylp → (2,3 → and → 1) -D-Xylp- (3 → and with Glcp and Galp as terminal saccharide units; wherein, the content of → 1) -D-Glcp- (2,4,6 → and → 1) -D-Galp- (3 → is more than 10%.

And F2 mainly includes a glycosidic bond consisting of → 1) -L-Rhap- (3 →, → 1) -D-Araf- (3 →, → 1) -D-Arap- (2 →, → 1) -D-Manp- (4 →, → 1) -D-Manp- (2,4 →, → 1) -D-Manp- (3,6 →, → 1) -D-Glcp- (2,4,6 →, → 1) -D-Galp- (3 →, → 1) -D-Xylp → (2,3 → and → 1) -D-Xylp- (3 → and with Glcp and Galp as terminal saccharide units; wherein, the content of the → 1) -D-Glcp- (2,4,6 → and → 1) -D-Galp- (3 → exceeds 10 percent.

And F3 mainly includes a glycosidic bond consisting of → 1) -L-Rhap- (3 →, → 1) -D-Arap- (2 →, → 1) -D-Manp- (3,6 →, → 1) -D-Glcp- (2,4,6 →, → 1) -D-Galp- (3 →, → 1) -D-Xylp → (2,3 → and → 1) -D-Xylp- (3 → and with Glcp and Galp as terminal saccharide units; wherein, the content of the → 1) -D-Glcp- (2,4,6 → and → 1) -D-Galp- (3 → exceeds 10 percent.

Cell experiments

Materials and methods

1. Cell and sample solution

Mouse melanoma cells (B16), beijing solibao science ltd, cat # v: SCC-211111.

Test solution: the angelica dahurica polysaccharide freeze-dried powders of F1-F5 are respectively prepared into aqueous solutions by distilled water to be used as test solution.

2. B16 model cell

Thawing 1mL of cell suspension in 37 deg.C water bath, adding into 4mL of 10% RPMI-1640 medium, and culturing at 37 deg.C with 5% CO ₂ Culturing until the cell density reaches more than 80%, and performing subculture. Suspension subculture: collecting cells, centrifuging at 100rpm for 5min, discarding supernatant, adding 1-2 mL of culture solution, blowing uniformly, and distributing the cell suspension into a new dish or bottle containing 8mL of culture medium according to the proportion of 1:2 to 1:5 to serve as model cells.

3. Cell grouping experiment

The cell concentration is regulated to 5 × 10 ⁴ After the concentration of the cells, 96-well culture plates were added, 100. mu.L per well, and the plates were divided into a model group, an experimental group, and a positive group. Wherein, 100 mu L of culture medium is added into the model group cells; the positive group was added with 100. mu.L of 100. mu.M 8-methoxyAqueous psoralen (8-MOP, Vickqi); the experimental components are respectively added with 100mg/L of F1-F5, and each concentration is provided with 3 compound holes; at 37 ℃ with 5% CO ₂ Culturing for 56h, and observing the morphological change of the cells by an inverted microscope. And the viability of each group of cells was examined using the CCK-8 kit (holy next).

4. Determination of melanin synthesis:

after the experimental group, the model group, and the positive group 56, the supernatant was removed, washed three times with PBS having a pH of 7.4, 200 μ L of an alkaline solution containing 10% DMSO was added, and incubated in a constant temperature water bath at 80 ℃ for 2 hours, to sufficiently lyse the cells and the melanin granules, and the OD value was measured at 490nm using a microplate reader, the black synthesis rate (%) was (a-B)/B) × 100%, where a represents the OD value of each group of cells after the above lysis, and B represents the OD value of the primary melanocytes after the above lysis.

5. Detection of tyrosinase activation rate

By adopting the same grouping experiment, after the cells are cultured for 56h, the culture solution is discarded, PBS (pH is 7.4) is used for washing for 2 times, 90 mu L of 1% Triton X-100 solution is added into each hole, the cells are quickly placed into a refrigerator at minus 80 ℃ for 30min, then the cells are completely cracked after being melted at room temperature, 10g/L L-DOPA solution (100 mu L/hole, levodopa, CAS No.59-92-7, MedChemExpress) is added after the cells are pre-warmed at 37 ℃, an enzyme reader is incubated for 30min to detect the absorbance value (OD) at 490nm, the tyrosinase activation rate (%) (A-B)/B) X100% is calculated according to the following formula, wherein A represents the OD value of each group of the cells after being cracked, and B is the OD value of the primary melanocytes after being cracked.

6. And qRT-PCR is used for detecting the expression quantity of the genes Tyr, Tyr-1 and Mitf.

Total RNA of each cell group in the above grouping experiment was extracted using RNA Synthesis Assay kit (ab228561), and the total RNA was inverted into cDNA using reverse transcription kit (QIAGEN, Germany), and PCR was performed using the template according to SYBR Premix Ex Taq II with 10. mu.L of template DNA with 1. mu.L of template DNA, 0.4. mu.L of each of the upstream and downstream primers, and 20. mu.L of DEPC water, according to the reaction scheme: pre-denaturation at 95 ℃ for 10min, at 95 ℃ for 15s, at 56 ℃ or at 60 ℃ for 30s, at 70 ℃ for 20s, for 40 cycles. Each group was set to 3 replicates, samples were sequentially added on ice as follows, after the reaction was completed, the melting curve was observed,determining the specificity of the reaction between the target gene and the reference gene, and using the relative expression amounts of mRNA in the experimental group and the control group 2 ^-ΔΔCT And (4) calculating.

See table 6 for primer sequences, among others.

TABLE 6

7. Statistical analysis

All test data are expressed as mean and standard deviation, data were processed using SPSS13.0 software, and multiple comparisons and marked for significant differences for each column of data.

Second, result in

As shown in fig. 6, the morphology of the melanoma cells after passage was a long spindle type, and the melanocytes which are dividing and proliferating were observed under a microscope. As can be seen in FIG. 7, the division of melanoma cells was evident 56h after 8-MOP administration, indicating that the proliferation of positive group cells was accelerated; whereas the model group cells showed the same trend (fig. 8).

As can be seen from table 7, although the survival rates of the experimental group cells are not as good as those of the positive group and the model group, the survival rates of the experimental group cells and the model group cells are both over 90%, which indicates that the angelica dahurica polysaccharide provided in the embodiments of the present application has no obvious toxicity to melanoma cells.

As can be seen from table 7, the melanin synthesis rate and the tyrosinase activation rate of the melanocytes of the model group and the positive group are significantly higher than those of the experimental group, which indicates that the angelica dahurica polysaccharide provided by the embodiment of the present application can significantly inhibit the tyrosinase activity of the melanoma cells after acting on the melanoma cells, thereby reducing the melanin synthesis.

TABLE 7

TABLE 8 mRNA levels relative to beta-actin

As can be seen from table 8, the levels of Mitf, Tyr and Tyrp1 in melanoma cells of the model group and the positive group were significantly increased relative to the mRNA levels of the normal group, indicating that the tyrosinase activity was increased and the melanin synthesis rate was increased in the above experiments, and that the expression of Mitf, Tyr and Tyrp1 genes was up-regulated in the model group and the positive group. In the experimental group, after F1-F5 intervene in the model cells, the expression of related genes is down-regulated to be equivalent to that of the normal group, and the expression is inhibited. It is presumed that the angelica dahurica polysaccharides of F1-F5 provided in the embodiments of the present application may realize molecular regulation mechanisms on tyrosinase activity and melanin synthesis of melanoma cells by regulating Mitf, Tyr and Tyrp1 gene expression.

Animal experiments

Materials and methods

1. Laboratory animal

The Kunming female mice, which weigh about 20g, have the cargo number of CS-003 and live Liaoning organisms, are fed with water and water freely at the room temperature of 20-25 ℃ and the humidity of 20-30% under the same natural conditions for 10 days and then are adapted to the environment, and then the experiment is started.

2. Test article

Based on the angelica dahurica polysaccharide provided by the embodiment of the application, the embodiment of the application also discloses a health care product for expelling toxin, beautifying, whitening and removing freckles, which comprises at least one of F1-F5 polysaccharide, wherein the mass percentage is 0.001-20 wt%, and the term of wt% refers to the mass percentage.

Specifically, the health care product provided by the embodiment of the application can be an internal preparation or an external skin care product, can be prepared into a cosmetic composition dosage form, and can contain a cosmetically or dermatologically allowable medium or base agent to prepare the dosage form.

In particular, the topical skin care or cosmetic compositions provided herein can provide all dosage forms suitable for topical use, such as solutions, emulsions of an oil phase dispersed in an aqueous phase, emulsions of an aqueous phase dispersed in an oil phase, suspensions, solids, gels, powders, pastes, foams (foam), or aerosol compositions. Specifically, the composition of the present invention can be provided in the form of a cream, lotion, milky lotion, dusting powder, ointment, spray, concealer stick, or the like. The compositions of these dosage forms may be prepared according to methods common in the art.

Further, the skin care product for external use provided in the examples of the present application may further include a moisturizer, a softener, a preservative, a pH adjuster, organic and inorganic pigments, a perfume, a cooling agent, an antiperspirant, a stabilizer, a hydration or oleolysis promoter, a salt for adjusting osmotic pressure, and/or a buffer, which are cosmetically acceptable excipients.

The skin care product for external use provided in the embodiment of the present application is not particularly limited in formulation form, and may be formulated into cosmetics such as softening lotion, tightening lotion, nourishing cream, massage cream, essence, eye cream, eye essence, mask, loose powder, skin lotion, skin cream, skin oil, and skin essence.

Specifically, the embodiment of the application provides a toxin-expelling, beauty-maintaining, whitening and freckle-removing cream which comprises 0.001-20 wt% of F1-F5 freeze-dried powder, 0.5-1.75 wt% of sodium stearate, 1.5-2.5 wt% of polysorbitol 60, 2.0-4.0 wt% of polyethylene glycol 60 hydrogenated castor oil, 1.5-5.0 wt% of squalane, 1.5-5.0 wt% of caprylic/capric triglyceride, 15-35.0 wt% of glycerol, 0.5-3.0 wt% of propylene glycol, 0.02-0.2 wt% of triethanolamine, 0.2-2 wt% of preservative, 0.1-0.25 wt% of perfume and the balance of water.

Specifically, the embodiment of the application provides an oral preparation for expelling toxin, beautifying, whitening and removing freckles, which comprises 0.001-10 parts by mass of at least one of F1-F5, 100-500 parts by mass of a diluent and 20-50 parts by mass of a binder. Where "part" to any mass is used only to distinguish relative masses between different components and does not refer to absolute masses thereof.

Specifically, the diluent is selected from at least one of starch, dextrin, lactose, microcrystalline cellulose, mannitol, sorbitol and aerosil.

Specifically, the binding agent is selected from at least one of magnesium stearate, glucose, sucrose, hydroxypropyl methylcellulose, sodium hydroxymethyl cellulose, starch and dextrin.

Specifically, the oral preparation can be granules, and the preparation process can be as follows: adding 100 parts of mannitol into a high-speed mixing granulator, dissolving 10 parts of F1 freeze-dried powder in 50mL of water, adding into the mannitol while stirring, stirring for 12min, sieving with a 20-mesh sieve, granulating, drying granules in a drying oven, adding 5 parts of magnesium stearate, uniformly mixing, transferring to a tablet machine for tabletting, adjusting the weight of the tablets to 0.1g, and preparing into 1000 tablets, namely the oral tablet of F1.

Specifically, the formulations of the test samples in this experiment are shown in table 9.

TABLE 9

3. Grouping experiment

The Kunming mice were randomly divided into normal group, model group, cream group, tablet group, positive 1 group and positive 2 group, each group containing 20 mice.

Establishing a chloasma mouse model: removing hair-free region 2cm2 on both sides of mouse spine with a shaver, irradiating with a spectrum value of 320nm with medium-wave ultraviolet lamp for 60min every day 20cm away from the back for 4 weeks.

Normal group: the feed is normally fed, the model is not made, and the treatment is not carried out.

Cream group: after molding was completed, 1g of the cream shown in Table 9 was applied to the mouse molding area every day and fixed with a film and gauze. The smearing was continued for 2 weeks.

Tablet group: after molding, the mice were orally administered tablets shown in Table 9 continuously for 4 weeks at a daily dose of 10mg/20g body weight.

Positive 1 group: after the molding is finished, 1g of the Jing Xiu Tang speckle removing cream (Guang Yao Jing Xiu Bai Hua Fang) is smeared on a mouse molding area every day and fixed by a film and gauze; the smearing was continued for 2 weeks.

Positive 2 group: after the model building is finished, the mice continuously take the sedum aizoon spot-removing tablet (Furuitang) for 4 weeks, and 10mg/20g of body weight is taken every day.

4. Material drawing and index determination

After 4 weeks of intervention treatment, blood was collected from the eye sockets of each group of mice, all the mice were sacrificed by dislocation of cervical vertebrae, and skin tissue specimens after depilation were rapidly taken.

The blood sample was centrifuged at 2500r/min for 15min, and the supernatant was collected and subjected to index measurement according to the instructions of the tyrosinase assay kit (cat # CS0730, Sigma-Aldrich).

Pathological morphological detection of skin melanocytes: 1 mouse unhaired skin block (1.0 multiplied by 1.0cm) is respectively taken and fixed by 10 percent formalin, a section is embedded by normal paraffin, the melanin marking adopts an immunohistochemical technology, dewaxing is carried out until water is reached, after antigen restoration, HIVB45 is used as a first antibody, the immunohistochemical two-step method, DAB color development and hematoxylin counterstaining are carried out. PBS was used as a negative control instead of primary antibody, and the number of melanocytes in each group was counted under an optical microscope.

5. Analysis of melanin synthesis by microscopic image

The melanin was image analyzed using a CMOS computer image processing system and dedicated software, observing 5 different fields of view per case. After the melanocytes in the section were determined, the target image was stained with a multifunctional true color cell image analysis management system and immunohistochemical scoring (HIS) was performed. The scoring method is as follows: a is the positive cell percentage, A < 5% is 0, 5% -25% is 1, 25% -50% is 2, 50% -75% is 3, 75% -100% is 4; b is the staining degree of positive cells, B is negative 0, weak positive 1, moderate positive 2, strong positive 3. A. The product of the two terms B is the HIS of the tissue, and the average optical density and the integral optical density are calculated according to an image analysis management system.

6. Statistical analysis

All test data are expressed as mean and standard deviation, data were processed using SPSS13.0 software, and multiple comparisons and marked for significant differences for each column of data.

Second, result in

Watch 10

As can be seen from Table 10, the tyrosinase activity in the serum sample of the mouse in the model group is significantly higher than that in the normal group relative to the normal group, which indicates that the molding is successful; the tyrosinase activity in the serum samples of the mice of the positive group 1 and the positive group 2 is slightly lower than that of the mice of the model group, and the tyrosinase activity has certain intervention effect on the mice of the model group.

In the cream group, after the cream provided by the embodiments 2 to 6 is smeared on the model mouse, the tyrosinase activity is obviously lower than that of the model group and is also obviously lower than that of the positive group 1, and similarly, after the cream provided by the embodiments 7 and 8 is smeared on the model mouse, the tyrosinase activity is obviously lower than that of the model group and is also obviously lower than that of the positive group 2. The cream prepared based on radix angelicae F1-F5 provided by the embodiment of the application has the effect of inhibiting tyrosinase activity after being used for intervening in chloasma mice.

In order to further explore the melanin generation condition of the skin tissue of each group of mice, the experiment also performs section staining and observation on the skin tissue of each group of mice, and the result is shown in figure 9, the squamous epithelium of the skin tissue of the normal group of mice is thinner and has no hyperplasia, the squamous epithelium consists of regularly arranged basal cells and spine cells, and a small amount of cutin is arranged on the surface; normal hair follicle and sebaceous gland are found in the dermis, and sweat gland, blood vessel and inflammatory cell are not found; connective tissue, subcutaneous fat and muscle tissue are not particularly visible.

As shown in FIG. 10, the epithelial layer of the skin of the model group mice was significantly proliferated, the stratum corneum was thickened, the number of epidermal cells was increased, the proliferation of hair follicles and sebaceous glands was significant, and the number of blood vessels and inflammatory cells was increased, as compared with that of the blank group.

As shown in fig. 11 and 12, the number of cell layers in the positive 1 and positive 2 groups was smaller than that in the model group, the epithelial layer was still significantly proliferated, and the inflammatory cells were not significantly attenuated.

As shown in figure 13, the skin of the mice in the emulsion group has 2-3 epithelial layers, which are close to the normal group, the partial epithelia are slightly hyperplastic, and the secretion of hair follicles and sebaceous glands is obviously reduced compared with the secretion of the model group, basically has no hyperplasia and is close to the normal control group.

As shown in FIG. 14, the mouse with the tablet group had a thin skin epithelium layer, loose cell arrangement, less proliferation of appendages, and a lower number of hair follicles and sebaceous glands than the model group.

In addition, as can be seen from table 10, compared with the normal group, the HIS score of the skin tissue of the model group mice is significantly higher than that of the normal group, indicating that the model of chloasma model mice is successfully modeled; the HIS scores of the positive 1 and 2 groups are slightly lower than those of the model group, and the HIS scores have certain intervention effect on mice in the model group. In the cream group, after the cream provided by the embodiment 2-6 is smeared on a model mouse, the HIS score of the cream is obviously lower than that of the model group and is also obviously lower than that of the positive 1 group; similarly, the HIS scores of the cream-applied model mice provided in examples 7 and 8 were significantly lower than those of the model group and also significantly lower than those of the positive 2 group. The cream prepared based on radix angelicae F1-F5 provided by the embodiment of the application has the effects of inhibiting tyrosinase activity and reducing melanin synthesis after being applied to the intervention of chloasma mice.

In summary, the embodiment of the application makes full use of the resource of the traditional Chinese medicinal material angelica dahurica with homology of medicine and food, develops 5 polysaccharide components from the resource, and performs certain research on the primary structure of the polysaccharide components. Therefore, the five angelica dahurica polysaccharides act on melanoma cells, the tyrosinase activity of the angelica dahurica polysaccharides can be inhibited, the melanin generation can be reduced, and the angelica dahurica polysaccharides are inferred to realize a regulation mechanism for inhibiting the tyrosinase activity and reducing the melanin generation by inhibiting the expression of Mitf, Tyr and Tyrp1 genes through detecting the mRNA level of related genes. Animal experiments finally prove that the cream and the tablet prepared from the 5 angelica polysaccharide components have the effect of reducing the synthesis of melanin of a model mouse, and the 5 angelica polysaccharides provided by the embodiment of the application have application prospects in the fields of removing freckles, expelling toxin and beautifying or whitening.

The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.

Claims (10)

1. An angelica polysaccharide composition comprises at least one of F1-F5, wherein F1-F5 all contain rhamnose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid, and the molar ratios of rhamnose, arabinose, xylose, mannose, glucose and galactose are the same.

2. The radix angelicae polysaccharide composition according to claim 1, wherein F1 comprises the molar percentages of rhamnose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid being 7.374%, 7.374%, 4.733%, 6.187%, 50.006%, 20.676%, 1.980% and 1.670%;

f2 contains the molar percentages of rhamnose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid 7.284%, 7.284%, 4.675%, 6.111%, 49.395%, 20.423%, 2.750% and 2.078%;

f3 contained rhamnose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid in a molar percentage of 7.316%, 4.695%, 6.137%, 49.607%, 20.511%, 2.516% and 1.903%;

f4 contained rhamnose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid in mole percentages of 7.320%, 7.320%, 4.698%, 6.141%, 49.638%, 20.523%, 2.579% and 1.781%;

f5 contained rhamnose, arabinose, xylose, mannose, glucose, galactose, glucuronic acid and galacturonic acid in mole percentages of 7.320%, 7.320%, 4.698%, 6.141%, 49.638%, 20.523%, 2.579% and 1.781%.

3. The radix angelicae dahuricae polysaccharide composition according to claim 1, wherein the glycosidic bond in the molecular structure of F1 comprises: → 1) -L-Rhap- (3 → 1) -D-Araf- (3 → 1) -D-Arap- (2 → 1) -D-Manp- (2 →, → 1) -D-Manp- (2,4 →, → 1) -D-Manp- (3,6 →, a glycosidic bond of → 1) -D-Glcp- (2,4,6 →, → 1) -D-Glcp- (4 →, → 1) -D-Glcp- (6 →, → 1) -D-Galp- (3 →, → 1) -D-Galp- (6 →, → 1) -D-Xylp → (2,3 → and → 1) -D-Xylp- (3 → and with Glcp and Galp as terminal saccharide units; wherein, the molar contents of → 1) -D-Glcp- (2,4,6 → and → 1) -D-Galp- (3 → are all more than 10%;

glycosidic linkages in the molecular structure of F2 include the glycosidic linkages of → 1) -L-Rhap- (3 →, → 1) -D-Araf- (3 →, → 1) -D-Arap- (2 →, → 1) -D-Manp- (4 →, → 1) -D-Manp- (2,4 →, → 1) -D-Manp- (3,6 →, → 1) -D-Glcp- (2,4,6 →, → 1) -D-Galp- (3 →, → 1) -D-Xylp → (2,3 → and → 1) -D-Xylp- (3 → with Glcp and Galp as terminal saccharide units; wherein, the molar content of the → 1) -D-Glcp- (2,4,6 → and → 1) -D-Galp- (3 → exceeds 10%;

glycosidic linkages in the molecular structure of F3 include the glycosidic linkage of → 1) -L-Rhap- (3 →, → 1) -D-Arap- (2 →, → 1) -D-Manp- (3,6 →, → 1) -D-Glcp- (2,4,6 →, → 1) -D-Galp- (3 →, → 1) -D-Xylp → (2,3 → and → 1) -D-Xylp- (3 → and with Glcp and Galp as terminal saccharide units; wherein, the molar content of the → 1) -D-Glcp- (2,4,6 → and → 1) -D-Galp- (3 → exceeds 10%;

the glycosidic bond in the molecular structure of F4 includes the glycosidic bond of → 1) -L-Rhap- (3 →, → 1) -D-Araf- (3 →, → 1) -D-Manp- (2,4 →, → 1) -D-Glcp- (2,4,6 →, → 1) -D-Galp- (3 →, → 1) -D-Xylp → (2,3 → and → 1) -D-Xylp- (3 → and takes Glcp and Galp as terminal saccharide units; and wherein, the molar content of → 1) -L-Rhap- (3 →, → 1) -D-Glcp- (2,4,6 → and → 1) -D-Galp- (3 → exceeds 10%.

4. A health product for expelling toxin, beautifying, whitening and removing freckles comprises the angelica dahurica polysaccharide composition of claim 1, and the mass percentage is 0.001-20 wt%.

5. The nutraceutical of claim 4, further comprising a nutraceutical or cosmetically acceptable adjuvant comprising at least one of a wetting agent, a softening agent, a preservative, a pH adjusting agent, organic and inorganic pigments, a fragrance, a cooling agent, an antiperspirant, a stabilizer, a hydration or oleolysis enhancer, a diluent, a binder, a salt for adjusting osmotic pressure, and/or a buffer.

6. An oral preparation for expelling toxin, beautifying, whitening and removing freckles, which comprises 0.001-10 parts by mass of the angelica dahurica polysaccharide composition as claimed in any one of claims 1-5, 100-500 parts by mass of a diluent and 20-50 parts by mass of a binder.

7. A detoxifying, beautifying, whitening and freckle removing cream comprises 0.001-20 wt% of the angelica dahurica polysaccharide composition as defined in any one of claims 1-5, 0.5-1.75 wt% of sodium stearate, 1.5-2.5 wt% of polysorbitol 60, 2.0-4.0 wt% of polyethylene glycol 60 hydrogenated castor oil, 1.5-5.0 wt% of squalane, 1.5-5.0 wt% of caprylic/capric triglyceride, 15-35.0 wt% of glycerol, 0.5-3.0 wt% of propylene glycol, 0.02-0.2 wt% of triethanolamine, 0.2-2 wt% of preservative, 0.1-0.25 wt% of perfume and the balance of water.

8. A method of preparing the Angelica dahurica polysaccharide composition of any one of claims 1 to 5, comprising the steps of:

obtaining degreased radix angelicae dahuricae filter residues, refluxing with ethanol, treating with glycosidase to obtain an enzymolysis product, and extracting the enzymolysis product to obtain a radix angelicae dahuricae polysaccharide crude product;

and purifying the radix angelicae dahuricae polysaccharide crude product by gel chromatography to obtain the radix angelicae dahuricae polysaccharide composition.

9. The method according to claim 8, wherein an aqueous solution containing α -mannosidase, ammonium sulfate and zinc acetate is used as the enzymatic treatment agent.

10. The use of the radix angelicae dahuricae polysaccharide composition according to any one of claims 1-5 in the preparation of health care products or cosmetics related to toxin expelling, skin beautifying, whitening or freckle removing.

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