AU2020459437A1

AU2020459437A1 - Acanthopanax senticosus Harms homogeneous polysaccharide, preparation method therefor and use thereof

Info

Publication number: AU2020459437A1
Application number: AU2020459437A
Authority: AU
Inventors: Hankun CHEN; Hongjia LIN; Enhan WANG; Muying YE
Original assignee: Guangzhou Qinglan Biotechnology Co Ltd
Current assignee: Guangzhou Qinglan Biotechnology Co Ltd
Priority date: 2020-07-20
Filing date: 2020-08-18
Publication date: 2022-12-08
Anticipated expiration: 2040-08-18
Also published as: CN111961141B; GB202216547D0; AU2020459437B2; CN111961141A; GB2613696A; WO2022016644A1; JP2022544723A; JP7414303B2

Abstract

Disclosed is an Acanthopanax senticosus Harms homogeneous polysaccharide, having a molecular weight of 6.83 × 10

Description

ACANTHOPANAX SENTICOSUS HOMOGENEOUS POLYSACCHARIDE, AND PREPARATION METHOD AND USE THEREOF TECHNICAL FIELD

[001] The present invention relates to the field of pharmaceutical technologies, in particular to an Acanthopanax senticosus homogeneous polysaccharide, and a preparation method and use thereof.

BACKGROUND

[002] Acanthopanax senticosus is the dried radix and rhizome or stem of Acanthopanax senticosus (Rupr Et Maxim.) Harms belonging to Acanthopanax Miq. plants in Araliaceae, which is widely distributed in the Far East coniferous forest belt of the Russia, the northeast, Hebei and Shanxi of China, the northern regions of Japan and Korea. It is also known as Wujiashen, Ciguaibang, and Laohuliaozi. In traditional Chinese medicine, the use of the Acanthopanax senticosus as a medicine has a long history and has been recorded in the herbal works of the past generations. Tao Hongjing in the Southern Dynasty (420-589) pointed out in Miscellaneous Records of Famous Physicians in China that "Acanthopanax senticosus with five-leaf were beneficial" and Acanthopanax senticosus had the effect of "tonifying the center, benefiting the essence, strengthening the bones and muscles, and enhancing the willpower". Li Shizhen in the Ming Dynasty (1368-1644) stated in the Compendium of Materia Medica that the Acanthopanax senticosus was a "superior product in the Classic" and had the effect of "tonifying the centerand benefiting Qi, strengthening the bones and muscles, and enhancing the willpower, and keeping the body fit after long-term administration". The Acanthopanax senticosus is warm in mature, pungent in smell and slightly bitter in taste, which can be used for treating diseases caused by spleen meridian, kidney meridian and heart meridian, and is mainly used for treating spleen and kidney Yang deficiency, asthenia, inappetence, aching waist and knees, insomnia and dreaminess. (Pan Jingzhi, Jin Sha, Cui Wenyu, et al. Research progress on chemical constituents and pharmacologicalactivities of Acanthopanax senticosus [J]. Food Industry Science and Technology, 2019, 40 (23): 353-360). The Acanthopanax senticosus has a variety of pharmacological activities such as immune function regulation, anticancer, liver-protecting, anti-aging, antioxidant, anti-inflammatory, hypotensive and anti-stress activities, and other pharmacological activities, and its active constituents are Acanthopanax glycosides, flavonoids, lignans, polysaccharides, etc.

However, these were experimental results of crude polysaccharides, which were poor in repeatability and cannot make standardized products, so the industrialization value was limited. Based on the previous research, the present invention carries out separation and purification, structural analysis and biological activity evaluation on Acanthopanax senticosus crude polysaccharide so as to obtain an Acanthopanax senticosus homogeneous polysaccharide with an excellent biological activity, which is not only of great scientific significance, but also can lay a foundation for the industrialization of the Acanthopanax senticosus homogeneous polysaccharide.

SUMMARY

[003] The technical problem to be solved by the present invention is to provide an Acanthopanax

senticosus homogeneous polysaccharide with a medicinal value, which is a polysaccharide

condensed by monosaccharide molecules and which is prepared by grading Acanthopanax

senticosus polysaccharides according to molecular weights, then further separating and purifying,

and carrying out structural characterization, and to perform preliminary study on an antioxidant

activity and an anti-skin aging activity of the obtained Acanthopanax senticosus homogeneous

polysaccharide.

[004] An object of the present invention is to provide an Acanthopanax senticosus homogeneous

polysaccharide, having a molecular weight of 6.83x10 5Da, and consisting of arabinose, galactose,

glucose, mannose and xylose, with molar percentages of all the monosaccharides sequentially being

16.42%, 32.27%, 40.38%, 7.21% and 3.72%.

[005] Another object of the present invention is to provide a preparation method of the

Acanthopanax senticosus homogeneous polysaccharide. The preparation method includes the

following steps:

[006] 1) crushing dried Acanthopanax senticosus decoction chips to a medicinal powder, sieving,

adding water 5 to 8 times the weight of the sieved medicinal powder, extracting for 3 times at 80°C

to 100°C for 2 h each time to obtain extract solutions, combining the extract solutions and

centrifuging the combined extract solutions, collecting a supernatant, and concentrating the

supernatant to obtain a concentrated solution;

[007] 2) after the concentrated solution is cooled, adding a a-amylase until a weight content

thereof is 0.1% to 0.4%, adjusting a pH value to 7.0, and performing enzymatic digestion in a water

bath at 60°C until a solution has no color change when the solution meets an iodine-potassium iodide reagent, quickly heating up to 100°C and keeping for 5 min for enzyme deactivation, centrifuging, and collecting a supernatant;

[008] 3) mixing the supernatant collected in step 2) with Sevage reagent according to a volume

ratio of 1:1, shaking violently for 30 min, standing for 12 h, collecting an upper polysaccharide

solution, mixing the upper polysaccharide solution with the Sevage reagent according to a volume

ratio of 1:1, and repeating the above operations until no protein characteristic absorption peak

appears in ultraviolet scanning;

[009] 4) after the upper polysaccharide solution finally collected in step 3) is concentrated,

adding absolute ethanol 4 to 6 times the volume of the concentrated upper polysaccharide solution,

standing at 4°C for precipitation for 48 h, centrifuging, and collecting a precipitate; adding

anhydrous ethanol to the precipitate, the above operations are repeated for 3 times, followed by

freeze-drying so as to obtain an Acanthopanax senticosus crude polysaccharide powder;

[0010] 5) after the Acanthopanax senticosus crude polysaccharide powder obtained in step 4) is

completely dissolved in distilled water, separating by means of a DEAE Fast Flow ion

chromatographic column, wherein elution conditions are as follows: a flow rate is 2.5 mL/min, and

pure water, sodium chloride solutions with 0.05 mol/L, 0.1 mol/L, 0.2 mol/L, 0.4 mol/L and 1

mol/L are sequentially used for elution; performing tracking detection by means of a sulfuric

acid-phenol method, collecting eluents;

[0011] 6) after the eluents collected in step 5) are concentrated, separating again by means of a

Sephadex G-200 glucan gel column chromatographic column, eluting with the distilled water at a

flow rate of 0.5 mL/min, and after detection by a phenol-sulfuric acid method, collecting a main

peak part in an elution curve;

[0012] 7) after a solution of the main peak part in the elution curve collected in step 6) is

concentrated, dialyzing for 2 days by means of a dialysis bag with a molecular weight cutoff of

3,500 Da for desalination, and finally concentrating and freeze-drying a solution in the dialysis bag

to obtain an Acanthopanax senticosus homogeneous polysaccharide powder.

[0013] The present invention provides a preparation method of the Acanthopanax senticosus

homogeneous polysaccharide. The preparation method includes the following steps:

[0014] 1) crushing dried Acanthopanax senticosus decoction chips to a medicinal powder, sieving

with a 100-mesh sieve, extracting the sieved medicinal powder with water 5 to 8 times the weight of the medicinal powder for 3 times at 80°C to 100°C for 2 h each time to obtain extract solutions, combining the extract solutions, centrifuging the combined extract solutions at 3000 rpm for 20 min, then collecting a supernatant, and concentrating the supernatant by rotary evaporation to one-fifth of an original volume of the supernatant to obtain a concentrated solution;

[0015] 2) after the concentrated solution is cooled, adding a a-amylase until a weight content thereof is 0.1% to 0.4%, adjusting a pH value to 7.0, and performing enzymatic digestion in a water

bath at 60°C for 4 h (where a solution has no color change when the solution meets an

iodine-potassium iodide reagent), quickly heating up to 100°C and keeping for 5 min for enzyme deactivation, then centrifuging at 3000 rpm for 10 min, and collecting a precipitate;

[0016] 3) mixing the supernatant collected in step 2) with Sevage reagent (wherein the Sevage

reagent is prepared by mixing chloroform and n-butanol according to a volume ratio of 4:1) according to a volume ratio of 1:1, shaking violently for 30 min, standing for 12 h, collecting an

upper polysaccharide solution, mixing the upper polysaccharide solution with the Sevage reagent according to a volume ratio of 1:1, and repeating the above operations until no protein characteristic

absorption peak appears in ultraviolet scanning;

[0017] 4) after the upper polysaccharide solution finally collected in step 3) is concentrated, adding absolute ethanol 4 to 6 times the volume of the concentrated upper polysaccharide solution,

standing at 4°C for precipitation for 48 h, centrifuging at 3000 rpm for 10 min, and collecting a

precipitate; adding anhydrous ethanol to the precipitate, repeating the above operations for 3 times to obtain a final precipitate, and freeze-drying the final precipitate to obtain an Acanthopanax senticosus crude polysaccharide powder;

[0018] 5) after the Acanthopanax senticosus crude polysaccharide powder obtained in step 4) is completely dissolved in distilled water, separating by means of a DEAE Fast Flow ion

pure water, sodium chloride solutions with 0.05 mol/L, 0.1 mol/L, 0.2 mol/L, 0.4 mol/L and 1 mol/L are sequentially used for elution; collecting eluents in gradient by means of an automatic

collector, wherein an volume of each solution in gradient elution is three times a column volume,

and 30 tubes with each of which contains 5.0 mL eluent are collected; performing tracking detection every other tube by means of a sulfuric acid-phenol method;

[0019] 6) after the eluents collected in step 5) are concentrated, separating again by means of a

flow rate of 0.5 mL/min, colleting by means of the automatic collector, each tube containing 5.0 mL eluent, and after detection by a phenol-sulfuric acid method, collecting a main peak part in an

elution curve;

[0020] 7) after a solution of the main peak part in the elution curve collected in step 6) is concentrated, dialyzing for 2 days by means of a dialysis bag with a molecular weight cutoff of

to obtain an Acanthopanax senticosus homogeneous polysaccharide powder.

[0021] Furthermore, in the Sevage reagent, the volume ratio of chloroform to n-butanol is 4: 1.

[0022] The present invention discloses that by means of the technical solution of a water

extraction and alcohol precipitation method for extraction, the DEAE Fast Flow anion exchange chromatographic column and Sephadex G-200 gel chromatographic column for further separation

and purification, an Acanthopanax senticosus homogeneous polysaccharide having a molecular weight of 6.83x10 5 Da and consisting of arabinose, galactose, glucose, mannose and xylose is

obtained.

[0023] Tests prove that the Acanthopanax senticosus homogeneous polysaccharide powder prepared by the present invention has multiple effects such as antioxidant, skin aging resistance,

and can be used for in the preparation of anti-skin aging cosmetics or a therapeutic medicine for

skin.

[0024] The present invention further relates to a skin care cosmetic, including the Acanthopanax senticosus homogeneous polysaccharide with the anti-skin aging effect, and an auxiliary material

used in a cosmetic field.

[0025] The present invention further relates to a therapeutic medicine for skin, including the

Acanthopanax senticosus homogeneous polysaccharide with the anti-skin aging effect, and a

medically-acceptable carrier.

[0026] The Acanthopanax senticosus homogeneous polysaccharide provided by the present

invention may be especially used for preparing a skin care cosmetic, including a cream, emulsion,

toner, gel, facial mask, liniment or lotion, but is not limited to the above preparation forms. According to the known methods in the industrial field of skin care products, after being sterilized

by a known method, compositions composed of the above Acanthopanax senticosus homogeneous polysaccharide and auxiliary materials used in the cosmetics field may be prepared into various external preparations.

[0027] In case of preparing a skin care cosmetic, the prepared Acanthopanax senticosus

homogeneous polysaccharide powder may be mixed with a matrix or an auxiliary material of a

known cosmetic and medicine, a carrier and an additive according to a conventional method,

wherein the Acanthopanax senticosus homogeneous polysaccharide powder accounts for 3%-10%

of a total weight of the cosmetic.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Fig. 1 shows an elution curve graph of Acanthopanax senticosus crude polysaccharide

components passing through a DEAE Fast Flow ion chromatographic column.

[0029] Fig. 2 shows a HPGPC gel chromatogram of an Acanthopanax senticosus homogeneous

polysaccharide.

[0030] Fig. 3 is a GC-MS total ion current chromatogram of six standard monosaccharides.

[0031] Fig. 4 is a GC-MS total ion current chromatogram of the Acanthopanax senticosus

homogeneous polysaccharide.

[0032] Fig. 5 is an infrared spectrum of the Acanthopanax senticosus homogeneous

polysaccharide.

[0033] Fig. 6 is a1H spectrum of the Acanthopanax senticosus homogeneous polysaccharide. Note:

(left -> right).

[0034] Fig. 7 is a1 3 C spectrum of the Acanthopanax senticosus homogeneous polysaccharide.

Note: (left - right).

DETAILED DESCRIPTION

[0035] The present invention will be further described in detail by specific examples below.

[0036] Example1: Preparation and structural characterization of Acanthopanax senticosus

homogeneous polysaccharide

[0037] 1. Extraction of the Acanthopanax senticosus crude polysaccharide.

[0038] Dried Acanthopanax senticosus decoction chips were crushed into a medicinal powder, and

the medicinal powder was sieved with a 100-mesh sieve; the sieved medicinal powder was extracted with water 5 times the weight thereof for 3 times at 80°C for 2 h each time to obtain extract solutions; the extract solutions were combined; the combined extract solutions were centrifuged at 3000 rpm for 20 min, and then a supernatant was collected; and the supernatant was concentrated by rotary evaporation to one fifth of the original volume of the supernatant to obtain a concentrated solution. After the concentrated solution was cooled,a-amylase was added into the concentrated solution until the weight content of the a-amylase was 0.1%; the pH value was adjusted to 7.0, enzymatic reaction was performed in a water bath at 60°C for 4 h (where the solution had no color change when the solution met an iodine-potassium iodide reagent), temperature was quickly raised to 100°C and kept for 5 min to inactivate the enzyme; and then centrifugation was performed at 3000 rpm for 10 min, and a supernatant was collected. The supernatant was mixed with Sevage reagent (the Sevage reagent was prepared by mixing chloroform and n-butanol according to a volume ratio of 4:1) at a volume ratio of 1:1, shaking was performed violently for 30 min, standing for 12 h, and an upper polysaccharide solution was collected and then mixed with the Sevage reagent according to a volume ratio of 1:1, and the above operations were repeated until no protein characteristic absorption peak appeared in ultraviolet scanning. After the finally collected upper polysaccharide solution was concentrated, anhydrous ethanol 4 times the volume of the collected upper polysaccharide solution was added, standing at

4°C for precipitation was carried out for 48 h, centrifugation was performed at 3000 rpm for 10 min,

and a precipitate was collected. The anhydrous ethanol was added to the precipitate, the above operations were repeated for 3 times to obtain a final precipitate, and the final precipitate was freeze-dried to obtain an Acanthopanax senticosus crude polysaccharide powder.

[0039] 2. Purification of the Acanthopanax senticosus homogeneous polysaccharide

[0040] After the obtained Acanthopanax senticosus crude polysaccharide powder was completely

dissolved in distilled water, separation was performed by means of a DEAE Fast Flow ion

chromatographic column, wherein elution conditions were as follows: a flow rate was 2.5 mL/min, and water, sodium chloride solutions with 0.05 mol/L, 0.1 mol/L, 0.2 mol/L, 0.4 mol/L and 1 mol/L

were sequentially used for elution. Eluents were collected in gradient by an automatic collector,

wherein the volume of each solution in gradient elution was three times a column volume, and 30 tubes, each of which contained 5.0 mL eluent were collected; and tracking detection every other

tube was performed by means of a sulfuric acid-phenol method. An elution curve of the

Acanthopanax senticosus crude polysaccharide components passing through the DEAE Fast Flow

ion chromatographic column was shown in Fig. 1.

[0041] On one hand, after the collected liquids corresponding to the different eluents were

concentrated to liquids of certain volumes, the concentrated liquids were dialyzed by means of a

dialysis bag (with a molecular weight cutoff of 3,500 Da). Water in a beaker with the dialysis bag

was changed 5 times each day, and after dialysis for 7 days, a polysaccharide solution in the dialysis

bag was centrifuged, and freeze-drying was performed to obtain a preliminarily-purified

Acanthopanax senticosus polysaccharide.

[0042] On the other hand, after the collected eluents were concentrated, the concentrated eluents

were separated again by means of a Sephadex G-200 glucan gel column chromatographic column,

the separated eluents were eluted with distilled water at a flow rate of 0.5 mL/min. Eluents were

collected by the automatic collector, wherein each tube contained 5.0 mL eluent; and after detection

by means of a sulfuric acid-phenol method, a main peak part in an elution curve was collected.

Then, after a solution of the collected main peak part in the elution curve was concentrated, the

concentrated solution was dialyzed for 2 days by means of a dialysis bag with a molecular weight

cutoff of 3,500 Da for desalination, and finally, a solution in the dialysis bag was concentrated and

freeze-dried to obtain the Acanthopanax senticosus homogeneous polysaccharide powder.

[0043] 3. Purity identification of the Acanthopanax senticosus homogeneous polysaccharide

[0044] High-performance liquid chromatography conditions were as follows: an Agilent 1200

high-performance liquid chromatograph, a TSK GEL G3000PWXL (7.8x300 mm, 7 Lm)

chromatographic column and a TSK GEL G5000PWXL (7.8x300 mm, 10 m) chromatographic

column that were in series connection, a mobile phase being a 0.02 mol/L KH 2 PO4 solution, a flow

rate being 0.5 mL/min, a column temperature being 35°C, and a Waters 2414 differential refractive

index detector. The Acanthopanax senticosus homogeneous polysaccharide obtained in step 2 was

dissolved in a proper amount of water, and a sample injection volume was 10 L. Results were

shown in Fig. 2, and a chromatographic peak of the Acanthopanax senticosus homogeneous

polysaccharide in a chromatogram was a single symmetrical peak, indicating that the Acanthopanax

senticosus homogeneous polysaccharide prepared by the present invention was indeed a

homogeneouspolysaccharide.

[0045] 4. Determination of molecular weight of the Acanthopanax senticosus homogeneous polysaccharide

[0046] 1 mg of pullulans with molecular weights of 50 KDa, 80 KDa, 150 KDa, 270 KDa, 410

KDa and 670 KDa were dissolved in 1mL of water, respectively, and the chromatographic

conditions were the same as above. An Agilent 1200 high-performance liquid chromatograph was

used for analysis, a retention time was recorded, a logarithm of a relative molecular weight (ogM)

was taken as an ordinate and the retention time (t) as an abscissa to obtain a standard curve Y =

14.62-0.980X. A peak appearance time of the Acanthopanax senticosus homogeneous

polysaccharide was substituted into the curve equation, and then, the molecular weight of the

Acanthopanax senticosus homogeneous polysaccharide was 6.83x10 5 Da.

[0047] 5. Structural characterization of the Acanthopanax senticosus homogeneous polysaccharide

[0048] 5.1 Analysis of polysaccharide components

[0049] Firstly, acid hydrolysis was performed, then acetylation was performed for derivation, and

gas phase GC analysis was performed. A method for acid hydrolysis was: weighing 10 mg of

Acanthopanax senticosus homogeneous polysaccharide samples, placing them in ampoules,

respectively, 4 mL trifluoroacetic acid with a concentration of 2 mol/L was added into each ampoule,

air in the ampoule was blown off with nitrogen, and the ampoule was sealed with an alcohol

blowtorch. After hydrolysis at 110C for 6 h, each sample was dried by rotary evaporation, 2 mL of

methanol was added into the dried sample for dissolution, drying by evaporation, and the operations

were repeated for 3 times, so as to remove trifluoroacetic acid from the sample as much as possible

to obtain a polysaccharide hydrolysate. 1 mL of methanol was added into the sample, and the

sample was transferred to a serum bottle, blow-drying with nitrogen, 1.0 mL pyridine, 10 mg

hydroxylamine hydrochloride and 1.0 mg internal-standard inositol were added into the

polysaccharide hydrolysate, reaction was performed under shocking for 0.5 h at a constant

temperature of 90°C, after cooling, 1 mL acetic anhydride was added, and acetylation was

performed for 0.5 h at 90°C. After cooling, water was added to stop the reaction. Then, 2.0 mL

chloroform was added for extraction for 3 times, after excess water was removed with anhydrous

sodium sulfate, filtration was performed with a 0.22 m organic phase filter membrane. Each

monosaccharide standard substance was also derivatized according to the above steps.

[0050] After derivatization according to the above method, GC analysis was performed. GC

detection conditions were as follows: an Aglient 6890N gas chromatography system using an

Agilent HP-5 silica capillary column (30 mx 0.32 mmx0.25 tm); a constant pressure mode being 20

PSI; a carrier gas being N 2; a sample injection volume being 1.0 L; a flow rate being 1.0 ml/min; a temperature at a sample injection port being 250°C; a temperature of an FID detector being set to

250°C; the sample injection port adopting a splitlessmode; and temperature programmed: an initial

temperature being 100°C kept for 30 seconds, raising the temperate to 160°C at 3C/ min, changing a temperature raising rate, and continuing to raise the temperature. The temperature was raised to

250°C at the rate of 10°C/min and kept for 5 min. A GC-MS total ion current chromatogram of six

monosaccharide standard substances, namely, arabinose, galactose, glucose, mannose, xylose and fucose, was shown in Fig. 3, and a GC-MS total ion current chromatogram of the Acanthopanax

senticosus homogeneous polysaccharide was shown in Fig. 4.

[0051] GC results were as follows. Analysis was performed according to retention times of the six monosaccharide standard substances such as arabinose, galactose, glucose, mannose, xylose and

fucose, molar percentages of monosaccharides in the Acanthopanax senticosus homogeneous polysaccharide were calculated by a peak appearance area ratio. Results showed that compared with

the retention times in a GC chromatogram of the monosaccharide standard substances, the

Acanthopanax senticosus homogeneous polysaccharide was composed of arabinose, galactose, glucose, mannose and xylose, and the molar percentages of the five monosaccharides calculated by

an internal standard method were 16.42%, 32.27%, 40.38%, 7.21% and 3.72%.

[0052] 5.2 Infrared spectrum scanning of the Acanthopanax senticosus homogeneous polysaccharide

[0053] 2.0 mg of an Acanthopanax senticosus homogeneous polysaccharide sample was weighed,

and mixed with a potassium bromide powder; a mixture was ground evenly, and the evenly ground mixture was compressed into a tablet. Then, the tablet was placed in a Fourier transform infrared

spectrometer for infrared scanning (400 cm-1 to 4000 cm-1), and an infrared absorption spectrum of

the sample was recorded. Results were shown in Fig. 5. It could be seen from an IR image that the Acanthopanax senticosus homogeneous polysaccharide had typical polysaccharide absorption

characteristics. 3456 cm-1 and 2929 cm-1 were stretching vibration peaks of an O-H bond, and 1744

cm-1 was a stretching vibration peak of a C-H in CH 2 ; 1635 cm-1 was a stretching vibration peak caused by CO2 or associated water, 1411 cm-1 was a variable angle vibration peak of aC-O bond,

and 1242 cm-1 was a stretching vibration peak caused by a primary alcohol -OH; an absorption peak at 1021 cm-1 indicated that the Acanthopanax senticosus homogeneous polysaccharide contained a pyran ring, while an absorption peak at 893 cm-1 indicated that the Acanthopanax senticosus homogeneous polysaccharide had a -glycosidic bond, while an absorption peak at 835 cm-1 indicated that the Acanthopanax senticosus homogeneous polysaccharide had a a-glycosidic bond.

[0054] 5.3 Methylation analysis of the Acanthopanax senticosus homogeneous polysaccharide

[0055] 20 mg of an Acanthopanax senticosus homogeneous polysaccharide sample (keeping the

sample sufficiently dry) was weighed and put in a test tube with a plug; 6 mL of a dimethyl

sulfoxide DMSO reagent was added in the test tube, and an opening of the test tube was sealed with

nitrogen; heating; even stirring by means of magnetic force; and sodium hydroxide (6 mL of DMSO

contains 240 mg of sodium hydroxide) was added to form a sodium hydroxide turbid liquid left

overnight. 3.6 mL of methyl iodide was added to the test tube until the next day and stirring for 8

min, methyl iodide was blown off with nitrogen, and methylation was performed again; and after

the above operations were repeated for 3 times, 6 mL of distilled water was added to stop the

reaction. After dialyzing with running water and deionized water for 24 h, respectively, extraction

with chloroform was performed for 3 times, drying was performed with anhydrous sodium sulfite

for 24 h, then, blow-drying was performed with nitrogen, and about 1 mL of a solution was left.

Trifluoroacetic acid was used to hydrolyze the solution, 70 mg of sodium borohydride NaBH 4 was

added, stirring was performed for 24 h; then, highly-acidic cation-anion exchange resin was added,

a mixture was stirred evenly for 10 min, and extraction filtration was performed; a filtrate was

collected and added with methanol; blow-drying with the nitrogen; and then, 0.5 mL of acetic

anhydride and 0.5 ml of anhydrous pyridine were added, and acetylation was performed at 100°C

for 2 h. After the reaction, absolute ethanol was repeatedly added to remove acetic anhydride, and

then, GC-MS analysis was performed.

[0056] GC-MS chromatographic conditions were as follows: Agilent 6890-5973N gas

chromatography-mass spectrometer, and a chromatographic column: an HP-5 MS capillary column

(30 mx250 pmx0.25 umD); a carrier gas: helium e; a temperature of a heater: 250°C; temperature

programmed: raising an initial temperature of 140°C to 200°C, keeping for 5 min, and then, raising

the temperature to 240°C at 8°C/min; a split ratio: 50: 1; and a sample injection volume: 5 L.

Methylation analysis data of the Acanthopanax senticosus homogeneous polysaccharide was shown in Table 1. Methylation results of the Acanthopanax senticosus homogeneous polysaccharide showed that the Acanthopanax senticosus homogeneous polysaccharide was mainly composed of

-- ,6)--Galp(1--, and Glep residues were the main units of the Acanthopanax senticosus

homogeneous polysaccharide, which existed in 1-4, 1--6, 1->4,6 and 1-> connection modes,

while the Galp residues existed in the 1->4 connection mode.

Table 1: Methylation analysis data of the Acanthopanax senticosus homogeneous polysaccharide

No. Methylation residue Type of glycosidic bond Molar percentage (mol.%)

1 2,4-Me3-Glcp ->4)-p-Glcp(1-> 10.8

2 2,3-Me2-Glcp ->4,6)-p-Glcp(1-> 9.6

3 2,4,6-Me3-Galp -- ,6)-p-Galp(1-> 71.5

4 2,3,6-Me3-Glcp ->4)-p-Glcp(1-> 5.2

5 2,3,4,6-Me4-Glcp p-Glcp(1-> 2.9

[0057] 5.4 NMR analysis of the Acanthopanax senticosus homogeneous polysaccharide

[0058] 30 mg of the dried Acanthopanax senticosus homogeneous polysaccharide was dissolve in

0.5 ml D 20, heating at 60°C was performed for 1 h to enable the dried Acanthopanax senticosus

homogeneous polysaccharide to be completely dissolved, then, a solution was transferred to a 1 13 nuclear magnetic tube, H spectrum and C spectrum were measured on an AV300 nuclear

magnetic resonance spectrometer from Brucker Company, Germany. Results were shown in Fig. 6

and Fig. 7. It could be seen from the spectrogram that a signal distribution range of the1 H spectrum

of the Acanthopanax senticosus homogeneous polysaccharide was narrow, mainly concentrated in

62.0-6.0 ppm (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). While a C spectrum signal range of the Acanthopanax senticosus

homogeneous polysaccharide is 660-110 ppm (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). See Table 2 for a chemical shift attribution of anomeric carbon of

each monosaccharide residue in the Acanthopanax senticosus homogeneous polysaccharide.

Table 2: Hydrocarbon chemical shift attribution of the Acanthopanax senticosus homogeneous

polysaccharide

No. Monosaccharide residue CI HI

A -- 5)-a-D-GalAp 90.3 7.37

B ->4)-P-L-Araf-(1-> 93.2 12.50

C a-D-Rhap-(1-> 91.06 8.81

D -- 3,4)-a-L-GalAp -(1-> 108.2 4.27

E -- 3,4)--D-Galp -(1-> 107.3 8.58

F ->4)-P-L-GalAp-(1-> 96.7 7.86

[0059] Example 2: Evaluation of antioxidant activity of the Acanthopanax senticosus homogeneous polysaccharide

[0060] Modern medicine believes that a key of skin aging is the oxidative stress of dermis and

epidermis caused by various factors, so substances with a strong antioxidant effect have a better

anti-skin aging effect. This experiment aimed to evaluate an antioxidant activity of the Acanthopanax senticosus homogeneous polysaccharide obtained in Example 1.

[0061] 1. Determination of activity of scavenging DPPH free radical

[0062] 2 mL of sample solutions with different mass concentrations (12.5, 25, 50, 100 g.ml-1

) were taken and respectively added with 2 mL of DPPH solution (100 g ml- 1); and after sufficient

mixing, reaction in the dark was performed for 30 min. An absorbance (Am) of the reaction system

was measured at 517 nm. Meanwhile, a solvent blank group (An, the DPPH solution was replaced by an equal volume of methanol) and a sample blank group (Ao, a sample solution was replaced by

an equal volume of methanol) were set. VC was used as a positive control group. The experiment was operated in parallel for 3 times, and a scavenging rate of the DPPH by the extract was

calculated according to the following formula:

scavenging rate % = (1 - Am-An) Ao x 100%.

[0063] 2. Determination of activity of scavenging ABTS*

[0064] 0.4 mL of sample solutions with different mass concentrations (12.5, 25, 50, 100 tg.ml-) were taken and respectively added with 4 mL of ABTS* solution, reaction was performed for 6 min.

An absorbance (Ai) of the sample was measured at 734 nm. Meanwhile, a solvent blank group (Aj,

the ABTS* solution was replaced by an equal volume of methanol), a sample blank group (Ah, a sample solution was replaced by an equal volume of methanol), and a positive control group were

set. The experiment was operated in parallel for 3 times, and a scavenging rate of the ABTS* by the

extract was calculated according to the following formula: scavenging rate % = (1 Ai-Aj ) x 100%.

[0065] 3. Determination of reducing power

[0066] 0.8 mL of sample solutions with different mass concentrations (12.5, 25, 50, 100 g-ml-)

were taken and added with 2 mL of a phosphate buffer solution (PH=6.6) and 2 mL of 1% potassium potassium ferricyanide; water bath was performed at 50°C for 20 min; 2 mL of 10%

trichloroacetic acid was added; centrifugation was performed at 3000 rpm for 10 min; 2 mL of a supernatant was collected, and added with 2 mL of deionized water and 0.4 ml of 0.1% ferric

chloride; reaction was performed for 5 min, and an absorbance was measured at 700 nm; and VC

was taken as a positive control, and the experiment was operated in parallel for 3 times. The greater the measured absorbance, the stronger the reducing power.

[0067] Results were as follows. As shown in Tables 3 to 5, IC5 0 values of the scavenging rate of

the DPPH radical and the scavenging rate of the ABTS* by the Acanthopanax senticosus homogeneous polysaccharide were 3.17±0.57 tg-mL-' and 15.26±5.79 tg-mL-1, respectively, and

an EC5 o value of the reducing power was 36.02±10.89 tg-mL-1, which showed that its antioxidant

ability was stronger than that of positive medicine vitamin C. The difference was statistically significant (P< 0.01), indicating that the Acanthopanax senticosus homogeneous polysaccharide

prepared in Example1 had a relatively strong antioxidant activity.

Table 3: Activity of scavenging DPPH free radical by the Acanthopanax senticosus homogeneous

polysaccharide (n=6, i is)

Group Regression equation r IC5o0/g-mL-1

Acanthopanax

senticosus Y=3.572X+3.876 0.999 3.17±0.57* homogeneous polysaccharide

Vitamin C Y=5.872 X +1.483 0.999 6.78±0.96

[0068] Compared with vitamin C group, *p < 0.05, and * *p < 0.01.

Table 4: Activity of scavenging ABST+ by the Acanthopanax senticosus homogeneous

polysaccharide (n=6, i is)

Group Regression equation r IC5o0/g-mL-a

Vitamin C Y=2.157X+7.624 0.997 34.21±6.08

Acanthopanax

senticosus Y=1.024X+4.275 0.999 15.26±5.79 homogeneous polysaccharide

[0069] Compared with the vitamin C group, *P< 0.05, and P< 0.01.

Table 5: Determination of reducing power of the Acanthopanax senticosus homogeneous

polysaccharide (n=6, . is)

Group Regression equation r EC5o0/g- mL-1

Vitamin C Y=0.0068X+0.0047 0.999 68.47±11.22

Acanthopanax senticosus Y=0.0028X+0.0043 0.998 36.02±10.89 homogeneous

polysaccharide

[0070] Compared with the vitamin C group, *P< 0.05, and P< 0.01.

[0071] Example 3: Preparation of essence lotion containing the Acanthopanax senticosus homogeneous polysaccharide

[0072] In this experiment, the Acanthopanax senticosus homogeneous polysaccharide obtained in Example was used to prepare an essence lotion. Weight percentages of components and a

production process are as follows:

Raw materials Percentages(%) Suppliers

Phase A

Glyceryl monostearate 6.0 Guangzhou Huahua Chemical Industry Co., Ltd., China

Propylene glycol 5.5 Guangzhou Huahua Chemical Industry Co.,

Ltd., China

Stearic acid 4.0 Guangzhou Huahua Chemical Industry Co., Ltd., China

Triethanolamine 0.7 Changzhou Wuming Chemical Industry

Co., Ltd., China Mineral oil 10.0 Guangzhou Huahua Chemical Industry Co.,

Ltd., China

Methyl 4-hydroxybenzoate 1.5 Shanghai Ruigu Biotechnology Co., Ltd.,

China

Isopropyl myristate 10.0 Zhejiang Wumart Chemicals Co., Ltd.,

China

Dimethyl siloxane 2.0 Changzhou Wuming Chemical Industry

Co., Ltd., China

B phase

Acanthopanax senticosus 6.0 Self-made

homogeneous polysaccharide

Deionized water 54.3 Self-made

[0073] A production process was as follows. The phase A and the phase B were respectively

heated to 70°C under stirring until they were completely dissolved and mixed evenly, and then, the

phase B was added to the phase A at 70°C to form a W/Q emulsion; after uniform stirring, cooling

to room temperature to obtain the essence lotion.

[0074] Matrix components used in a preferred example of the present invention are as described

above, and matrix components used in this example can exert the efficacy of a pharmaceutical

composition of the present invention to the best. However, common matrixes produced by other

manufacturers applicable to cosmetics may also be used in the present invention, so long as dosages

thereof meets a national standard of cosmetic additives, which will not affect the effect of the

present invention. Therefore, they are not enumerated.

[0075] A homogenizing emulsifying device used in the present invention is an FV-30L FISCO

vacuum homogenizing emulsifying machine produced by Shanghai Fluko Fluid Machinery

Manufacturing Co., Ltd., which has functions of homogenization, stirring, temperature control.

Homogenizing emulsifying devices for cosmetic preparation produced by other manufacturers may

also be used in the present invention. As long as these devices are strictly operated according to

process parameters of the present invention, effects of the present invention can be achieved.

[0076] Example 4: Evaluation of anti-skin aging activity of essence lotion containing the

Acanthopanax senticosus homogeneous polysaccharide

[0077] In this experiment, an anti-skin aging activity of the essence lotion containing the

Acanthopanax senticosus homogeneous polysaccharide obtained in Example3 was evaluated.

[0078] 1 Material and method

[0079] 1.1 Experimental animals: 40 SPF female Kunming mice weighing (20±2) g each were

purchased from Guangdong Medical Laboratory Animal Center, and had an experimental animal

quality license No. 44005800003406; and the mice were bred and experiments on the mice were

performed in an animal room of Sci-tech Industrial Park, Guangzhou University of Chinese

Medicine (license No. SYXK (Guangdong) 2013-0014). Since an ultimate goal of this study was to

develop anti-skin aging cosmetics suitable for women, all female mice were selected as

experimental animals. The treatment of mice conformed to the principles of animal ethics.

[0080] 1.2 Material and reagent: an essence lotion containing the Acanthopanax senticosus

homogeneous polysaccharide was the essence lotion prepared in Example3; D-galactose was from

Beijing Solarbio Biotechnology Co., Ltd.; an HA assay kit was from Shanghai Enzyme linked

Biotechnology Co., Ltd.; and an HYP assay kit, a SOD assay kit, and coomassie brilliant blue were

from Nanjing Jiancheng Bioengineering Institute.

[0081] 1.3 Grouping and modeling: firstly, the female mice were bred adaptively for 7 days to

ensure that the mice had adapted to a current environment and then the experiment began. The 30

female mice were divided into 3 groups according to a random number table. Two groups of the

mice were used as a modeling group, and were injected with D-galactose subcutaneously at 1.0 g

kg-1 d-1 for 30 days totally. The remaining group of the mice as a blank group was injected with the

same volume of normal saline every day. After 30 days, skin appearances of the mice in the

modeling group were compared with those in the blank group. The modeling group showed obvious

sagging skin and fine wrinkles, while the blank group showed opposite phenomena. Two groups of

skin-aging model mice were divided into a model group and an Acanthopanax senticosus

homogeneous polysaccharide group. Hair on backs of the mice was cut with scissors, and then the

backs of the mice were shaved with a razor for later use.

[0082] 1.4 Mice skins externally applied with essence lotions: the mice in the Acanthopanax

senticosus homogeneous polysaccharide group were applied with the essence lotion containing 6% of the Acanthopanax senticosus homogeneous polysaccharide obtained in Example3, while the mice in the model group were applied with an essence lotion without the Acanthopanax senticosus homogeneous polysaccharide. The specific method was as follows: an area of 4 cmx7 cm in the center of the back of each mouse was selected, and each essence lotion was applied onto the surface of the skin area selected from each mouse of the corresponding group; 0.3 g of the essence lotion was applied to each mouse per day; and the skin areas were cleaned after 24 h, and the essence lotions were applied again the same as the above; and these operations were last for 21 days, during which it was necessary to manually depilate 4 times.

[0083] 1.5 Observation of apparent characteristics of mice skins: apparent characteristics of the

skins of the mice in each group, such as color, smoothness and wrinkles were compared,

photographed and recorded. Then, the experimental mice were killed by a neck-removing method,

and the skins of the application sites were quickly peeled off to remove subcutaneous fat and other

connective tissues. After paving, the mice skins were cut at the middle positions with a hole punch

having a diameter of 2 cm, so as to measure moisture contents, and the remaining skin of each

mouse was frozen at -20°C for measuring skin hydroxyproline and other components.

[0084] 1.6 Determination of skin moisture content: a wet weight of the skin cut by the hole punch

was weighed accurately, and then the skin was put into an oven and dried at 50°C for 12 h; and then,

a dry weight of the skin was weighed; and a skin moisture content in each experimental group was

calculated according to the following formula: a skin moisture content = (a wet weight-a dry

weight)/the wet weight x 100%.

[0085] 1.7 Determination of skin hydroxyproline (HYP) content: 0.5 g of a skin tissue at the

application site was taken, rinsing was performed on the skin tissue with physiological saline

precooled by an ice bath, and the skin tissue was dried with filter paper; and the physiological saline

precooled by the ice bath was added, and the skin tissue was ground by a glass homogenizer into a

homogenate with a concentration of 10%. The obtained homogenate was centrifuged at 3,000 r/min

for 10 min at 0°C, and a supernatant was collected. According to a method shown in instructions of

an HYP kit, an OD value was measured by ultra-micro microplate spectrophotometer, and a HYP

content in the skin of the mouse in each experimental group was calculated.

[0086] 1.8 Determination of skin hyaluronic acid (HA) content: 0.5 g of a skin tissue at the

application site was taken, and a PBS buffer solution precooled by ice bath was added; and the skin tissue was ground by the glass homogenizer into a homogenate with a concentration of 10%. The obtained homogenate was centrifuged at 1,000 r/min for 4 min at 0°C, and a supernatant was collected. According to a method shown in instructions of an HA kit, an OD value was measured by the ultra-micro microplate spectrophotometer, and an HA content in the skin of the mouse in each experimental group was calculated.

[0087] 1.9 Determination of skin superoxide dismutase (SOD) activity: 0.5 g of a skin tissue at the

application site was taken, and was rinsed by the physiological saline precooled by an ice bath, and

then, the physiological saline precooled by the ice bath was added; and the skin tissue was ground

by the glass homogenizer into a homogenate with a concentration of 10%. The obtained

homogenate was centrifuged at 3,000 r/min for 10 min at 0°C, and a supernatant was collected.

According to a method shown in instructions of an SOD kit, an OD value was measured by the

ultra-micro microplate spectrophotometer, and an SOD activity in the skin of the mouse in each

experimental group was calculated.

[0088] 1.10 Statistical method: SPSS18.0 statistical software was used to analyze data, and

measurement data was represented by is; single factor analysis of variance was used for

comparison between groups; and Dunnett T3 test was used for pairwise comparison if results of

homogeneity test for variance were significant, and LSD test was used for pairwise comparison if

the results of homogeneity test for variance were insignificant, with the difference of P < 0.05 being

statistically significant.

[0089] 2 Results

[0090] 2.1 Comparative observation on skin appearances of mice in each group: compared with

the blank group, the mice in the model group had sagging skin and a large number of wrinkles, and

were slower in hair regeneration; and compared with the model group, the mice applied with the

essence lotion containing 6% of the Acanthopanax senticosus homogeneous polysaccharide had

obvious reduction in skin wrinkles and obvious improvement in smoothness and elasticity.

[0091] 2.2 Effects of the Acanthopanax senticosus homogeneous polysaccharide on skin moisture

content, HYP content, HA content and SOD activity of mice: see Table 6 for the measurement

results of the skin tissue moisture contents, the HYP contents, the HA contents and the SOD

activities of the mice in three groups. The skin tissue moisture content, HYP content, HA content

and SOD activity of the mice in the model group were significantly lower than those in the blank group (P < 0.01), indicating that the skin aging model of the mice was successfully established.

Compared with the model group, the skin moisture content, HYP content, HA content and SOD

activity of the mice in the Acanthopanax senticosus homogeneous polysaccharide group were

increased significantly, with the difference being statistically significant (P < 0.01). Compared with

the blank group, there were no significant differences (P > 0.01) in the skin moisture content, HYP

content, HA content and SOD activity and other indexes of the mice in the Acanthopanax

senticosus homogeneous polysaccharide group.

Table 6: Effects of the Acanthopanax senticosus homogeneous polysaccharide on skin tissue

moisture content, HYP content, HA content and SOD activity of mice

(n=6, . is)

Group Skin moisture HYP content HA content SOD activity

content(%) (Lg/mg) ( Lg/ml) (U/mg)

Blank group 78.21±1.53." 4.31±0.21^ 10.24±0.86^^ 386.12±21.47-^

Model group 50.83±1.21 3.65±0.19 6.77±0.93 302.55±17.32

Acanthopanax 76.33±1.24` 4.20±0.28-" 10.14±0.91-' 374.08±26.64^ senticosus

homogeneous

polysaccharide

group

F value 374.12 14.86 263.86 41.28

P value 0.000 0.000 0.000 0.000

Back test (pairwise LSD Dunnett T3 Dunnett T3 LSD

comparison)

[0092] Compared with the model group, "P< 0.05, and ^^P< 0.01.

[0093] In summary, the results of this experiment showed that the skins of the mice in the blank group were smooth, relatively firm and elastic. The mice injected with D-galactose in the skin aging model had saggingskins, greatly increased wrinkles, and the decreased skin moisture content, hydroxyproline content, hyaluronic acid content, superoxide dismutase activity and other indexes. Through determination, the effect of the improvement of the skin appearances of the model mice externally applied with the essence lotion containing 6% of the Acanthopanax senticosus homogeneous polysaccharide was obvious, and the indexes such as the skin moisture content, hyaluronic acid content, hydroxyproline content and superoxide dismutase activity of each mouse were also significantly improved.

[0094] Finally, it is necessary to explain herein that the above Example sare only used to further describe the technical solutions of the present invention, and should not be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art according to the above of the present invention are all within the scope of protection of the present invention.

Claims

What is claimed is: 1. An Acanthopanax senticosus homogeneous polysaccharide, having a molecular weight of

6.83x10 5Da, and consisting of arabinose, galactose, glucose, mannose and xylose, with molar

percentages of all the monosaccharides sequentially being 16.42%, 32.27%, 40.38%, 7.21% and

3.72%.

2. A preparation method of the Acanthopanax senticosus homogeneous polysaccharide

according to claim 1, comprising the following steps:

1) crushing dried Acanthopanax senticosus decoction chips to a medicinal powder, sieving,

adding water 5 to 8 times the weight of the sieved medicinal powder, extracting for 3 times at 80°C

to 100°C for 2 h each time to obtain extract solutions, combining the extract solutions and

centrifuging the combined extract solutions, collecting a supernatant, and concentrating the

supernatant to obtain a concentrated solution;

2) after the concentrated solution is cooled, adding a a-amylase until a weight content thereof

is 0.1% to 0.4%, adjusting a pH value to 7.0, and performing enzymolysis in a water bath at 60°C

until a solution has no color change when the solution meets an iodine-potassium iodide reagent,

quickly heating up to 100°C and keeping for 5 min for enzyme deactivation, centrifuging, and

collecting a supernatant;

3) mixing the supernatant collected in step 2) with Sevage reagent according to a volume ratio

of 1: 1, shaking violently for 30min, standing for 12 h, collecting an upper polysaccharide solution,

mixing the upper polysaccharide solution with the Sevage reagent according to a volume ratio of 1:

1, and repeating the above operations until no protein characteristic absorption peak appears in

ultraviolet scanning;

4) after the upper polysaccharide solution finally collected in step 3) is concentrated, adding

absolute ethanol 4 to 6 times the volume of the concentrated upper polysaccharide solution,

standing at 4°C for precipitation for 48 h, centrifuging, and collecting a precipitate; adding

anhydrous ethanol to the precipitate, the above operations are repeated for 3 times, followed by

freeze-drying so as to obtain an Acanthopanax senticosus crude polysaccharide powder;

5) after the Acanthopanax senticosus crude polysaccharide powder obtained in step 4) is completely dissolved in distilled water, separating by means of a DEAE Fast Flow ion chromatographic column, wherein elution conditions are as follows: a flow rate is 2.5 mL/min, and pure water, sodium chloride solutions with.05 mol/L, 0.1 mol/L, 0.2 mol/L, 0.4 mol/L and 1 mol/L are sequentially used for elution; performing tracking detection by means of a sulfuric acid-phenol method; collecting eluents;

6) after the eluents collected in step 5) are concentrated, separating again by means of a

Sephadex G-200 glucan gel column chromatographic column, eluting with the distilled water at a

flow rate of 0.5 mL/min, and after detection by a phenol-sulfuric acid method, collecting a main

peak part in an elution curve;

7) after a solution of the main peak part in the elution curve collected in step 6) is concentrated,

dialyzing for 2 days by means of a dialysis bag with a molecular weight cutoff of 3,500 Da for

desalination, and finally concentrating and freeze-drying a solution in the dialysis bag to obtain

anAcanthopanaxsenticosus homogeneous polysaccharide powder.

3. The preparation method of the Acanthopanax senticosus homogeneous polysaccharide

according to claim 2, wherein in the Sevage reagent, chloroform: n-butanol is equal to 4: 1.

4. The preparation method of the Acanthopanax senticosus homogeneous polysaccharide

according to claim 1, comprising the following steps:

1) crushing dried Acanthopanax senticosus decoction chips to a medicinal powder, sieving

with a 100-mesh sieve, extracting the sieved medicinal powder with water 5 to 8 times the weight

of the medicinal powder for 3 times at 80°C to 100°C for 2 h each time to obtain extract solutions,

combining the extract solutions, centrifuging the combined extract solutions at 3000 rpm for 20 min,

then collecting a supernatant, and concentrating the supernatant by rotary evaporation to one-fifth of

an original volume of the supernatant to obtain a concentrated solution;

2) after the concentrated solution is cooled, adding a a-amylase until a weight content thereof

is 0.1% to 0.4%, adjusting a pH value to 7.0, and performing enzymolysis in a water bath at 60°C

for 4 h where a solution has no color change when the solution meets an iodine-potassium iodide

reagent; quickly heating up to 100°C and keeping for 5 min for enzyme deactivation, then

centrifuging at 3000 rpm for 10 min, and collecting a precipitate;

3) mixing the supernatant collected in step 2) with Sevage reagent according to a volume ratio

of 1: 1, shaking violently for 30min, standing for 12 h, collecting an upper polysaccharide solution, mixing the upper polysaccharide solution with the Sevage reagent according to a volume ratio of 1:

1, and repeating the above operations until no protein characteristic absorption peak appears in

ultraviolet scanning, wherein the Sevage reagent is prepared by mixing chloroform and n-butanol according to a volume ratio of 4:1;

4) after the upper polysaccharide solution finally collected in step 3) is concentrated, adding

absolute ethanol 4 to 6 times the volume of the concentrated upper polysaccharide solution, standing at 4°C for precipitation for 48 h, centrifuging at 3000 rpm for 10 min, and collecting a

precipitate; adding anhydrous ethanol to the precipitate; repeating the above operations for 3 times

to obtain a final precipitate, and freeze-drying the final precipitate to obtain an Acanthopanax senticosus crude polysaccharide powder;

5) after the Acanthopanax senticosus crude polysaccharide powder obtained in step 4) is completely dissolved in distilled water, separating by means of a DEAE Fast Flow ion

chromatographic column, wherein elution conditions are as follows: a flow rate is 2.5 mL/min, and

pure water, sodium chloride solutions with 0.05 mol/L, 0.1 mol/L, 0.2 mol/L, 0.4 mol/L and 1 mol/L are sequentially used for elution; collecting eluents in gradient by means of an automatic

collector, wherein an volume of each solution in gradient elution is three times a column volume,

and 30 tubes, each of which contains 5.0 mL eluent are collected; performing tracking detection every other tubeby means of a sulfuric acid-phenol method; 6) after the eluents collected in step 5) are concentrated, separating again by means of a

Sephadex G-200 glucan gel column chromatographic column, eluting with the distilled water at a flow rate of 0.5 mL/min, colleting by means of an automatic collector, each tube containing 5.0 mL

eluent, and after detection by a phenol-sulfuric acid method, collecting a main peak part in an

elution curve; 7) after a solution of the main peak part in the elution curve collected in step 6) is concentrated,

dialyzing for 2 days by means of a dialysis bag with a molecular weight cutoff of 3,500 Da for

desalination, and finally concentrating and freeze-drying a solution in the dialysis bag to obtain anAcanthopanaxsenticosus homogeneous polysaccharide powder.

5. Use the Acanthopanax senticosus homogeneous polysaccharide powder according to claim

1, wherein the Acanthopanax senticosus homogeneous polysaccharide powder is used for preparing

a skin care cosmetic with an anti-skin aging effect or a therapeutic medicine for skin.

6. A skin care cosmetic, comprising: the Acanthopanax senticosus homogeneous

polysaccharide powder according to claim 1, and an auxiliary material used in a cosmetic field.

7. A therapeutic medicine for skin, comprising: the Acanthopanax senticosus homogeneous

polysaccharide powder according to claim 1, and a medically-acceptable carrier.