WO2022016644A1 - 一种刺五加均一多糖及其制备方法及应用 - Google Patents

一种刺五加均一多糖及其制备方法及应用 Download PDF

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WO2022016644A1
WO2022016644A1 PCT/CN2020/109792 CN2020109792W WO2022016644A1 WO 2022016644 A1 WO2022016644 A1 WO 2022016644A1 CN 2020109792 W CN2020109792 W CN 2020109792W WO 2022016644 A1 WO2022016644 A1 WO 2022016644A1
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acanthopanax senticosus
polysaccharide
solution
homogeneous
collect
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PCT/CN2020/109792
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French (fr)
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王恩瀚
陈汉坤
叶木英
林虹佳
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广州青岚生物科技有限公司
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Priority to JP2021534247A priority Critical patent/JP7414303B2/ja
Priority to GB2216547.6A priority patent/GB2613696A/en
Priority to AU2020459437A priority patent/AU2020459437B2/en
Publication of WO2022016644A1 publication Critical patent/WO2022016644A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of pharmacy, in particular to a homogeneous polysaccharide of Acanthopanax senticosus and a preparation method and application thereof.
  • Acanthopanax senticosus is the dry root and rhizome or stem of Acanthopanax senticosus (Rupr.Et Maxim.) Harms. It is widely distributed in the coniferous forest belt of the Russian Far East, Northeast China, Hebei, Shanxi, Japan, North Korea, etc. Alias Wujiashen, thorn crutches, tiger shackles. In traditional Chinese medicine, the application of Acanthopanax senticosus as a medicine has a very long history, and it has been recorded in the herbal works of all dynasties.
  • Acanthopanax senticosus is warm in nature, acrid in taste, slightly bitter; it returns to the spleen, kidney, and heart meridians; it has the effects of invigorating the spleen, invigorating the spleen, invigorating the kidney and soothing the mind, mainly used for spleen and kidney yang deficiency, physical weakness, loss of appetite, waist and knee pain, insomnia Dreamy embolism. (Pan Jingzhi, Jin Sha, Cui Wenyu, etc. Research progress on chemical constituents and pharmacological activities of Acanthopanax senticosus [J].
  • Acanthopanax senticosus has immune function regulation, anticancer, It has various pharmacological activities such as protecting the liver, anti-aging, anti-oxidation, anti-inflammatory, lowering blood pressure, and anti-stress.
  • these are the experimental results of crude polysaccharides, which have poor repeatability and cannot make standardized products, so the industrialization value is limited.
  • the present invention carries out separation and purification, structural analysis and biological activity evaluation of the crude polysaccharide of Acanthopanax senticosus, in order to obtain the uniform polysaccharide of Acanthopanax senticosus with good biological activity, which not only has very important scientific significance, but also can be used for The industrialization of Wujia homogeneous polysaccharide lays the foundation.
  • the technical problem to be solved by the present invention is to provide a polysaccharide formed by condensation of a monosaccharide molecule by grading the Acanthopanax senticosus polysaccharide by molecular weight, further separating and purifying, and performing structural characterization. Homogeneous polysaccharide was added, and the antioxidative activity and anti-aging activity of the obtained Acanthopanax senticosus homogeneous polysaccharide were preliminarily studied.
  • the purpose of the present invention is to provide a kind of Acanthopanax senticosus homogeneous polysaccharide, its molecular weight is 6.83 ⁇ 10 5 Da, which is composed of arabinose, galactose, glucose, mannose and xylose, and the molar percentage of each monosaccharide is 16.42%, 32.27%, 40.38%, 7.21% and 3.72%.
  • Another object of the present invention is to provide the preparation method of this Acanthopanax senticosus homogeneous polysaccharide, comprising the steps:
  • step 2) Mix the supernatant collected in step 2) with Sevage reagent in a volume ratio of 1:1, shake vigorously for 30 minutes, let stand for 12 hours, collect the upper polysaccharide solution, and mix the upper polysaccharide solution with Sevage reagent in a volume ratio of 1:1 Repeat the above operation until the UV scan has no protein characteristic absorption peak;
  • step 3 After concentrating the upper polysaccharide solution finally collected in step 3), add 4-6 times the volume of anhydrous ethanol, let it stand at 4° C. for 48 hours, and collect the precipitate by centrifugation; add anhydrous ethanol to the precipitate and repeat the above operation 3 times and then freeze-drying to obtain Acanthopanax senticosus polysaccharide powder;
  • step 5 The Acanthopanax senticosus polysaccharide powder obtained in step 4) was completely dissolved in distilled water and separated by DEAE Fast Flow ion chromatography column, elution conditions: flow rate was 2.5mL/min, followed by pure water, 0.05mol/L , 0.1mol/L, 0.2mol/L, 0.4mol/L, 1mol/L sodium chloride solution for elution, using sulfuric acid-phenol method for tracking detection, collecting eluent;
  • step 6 After concentrating the solution of the main peak part in the elution curve collected in step 6), use a dialysis bag with a molecular weight cut-off of 3500Da to dialysis for 2d desalination, and finally the solution in the dialysis bag is concentrated and freeze-dried to obtain Acanthopanax senticosus homogeneous. Polysaccharide powder.
  • the invention provides a preparation method of Acanthopanax senticosus homogeneous polysaccharide, comprising the following steps:
  • step 2) Mix the supernatant collected in step 2) with Sevage reagent (chloroform:n-butanol volume ratio 4:1) in a volume ratio of 1:1, shake vigorously for 30min, stand for 12h, collect the upper polysaccharide solution, put the upper layer After mixing the polysaccharide solution and Sevage reagent in a volume ratio of 1:1, repeat the above operation until there is no protein characteristic absorption peak in UV scanning;
  • Sevage reagent chloroform:n-butanol volume ratio 4:1
  • step 3 After concentrating the upper polysaccharide solution finally collected in step 3), add 4-6 times the volume of anhydrous ethanol, let stand at 4° C. for 48 hours, and centrifuge at 3000 rpm/min for 10 minutes to collect the precipitate; repeat the addition of anhydrous ethanol to the precipitate After 3 times of above-mentioned operations, freeze-dry the final obtained precipitate to obtain Acanthopanax senticosus polysaccharide powder;
  • step 6 After concentrating the eluate collected in step 5), separate it again with a Sephadex G-200 Sephadex column, elute with distilled water at a flow rate of 0.5 mL/min, and collect with an automatic collector. 5.0mL, after detection by phenol-sulfuric acid method, collect the main peak part in the elution curve;
  • step 6 After concentrating the solution of the main peak part in the elution curve collected in step 6), use a dialysis bag with a molecular weight cut-off of 3500Da to dialysis for 2d desalination, and finally the solution in the dialysis bag is concentrated and freeze-dried to obtain Acanthopanax senticosus homogeneous. Polysaccharide powder.
  • the volume ratio of chloroform:n-butanol 4:1.
  • the present invention finds that extraction by water extraction and alcohol precipitation, and further separation and purification by anion exchange DEAE Fast Floe chromatography column and Superdex-200 gel chromatography column, the molecular weight of 6.83 ⁇ 10 5 Da is obtained, which is composed of Acanthopanax homogeneous polysaccharide composed of arabinose, galactose, glucose, mannose and xylose.
  • the Acanthopanax senticosus homogeneous polysaccharide powder prepared by the invention has multiple functions such as anti-oxidation and anti-aging, and can be used for preparing anti-aging cosmetics and skin therapeutic drugs.
  • the invention also relates to a skin care cosmetic, comprising the above-mentioned homogeneous polysaccharide of Acanthopanax senticosus with anti-aging effect on skin and auxiliary materials used in the cosmetic field.
  • the present invention also relates to a therapeutic drug for skin, comprising the above-mentioned homogeneous polysaccharide of Acanthopanax senticosus with anti-aging effect on skin and a medically acceptable carrier.
  • the homogeneous polysaccharide of Acanthopanax senticosus provided by the present invention can be especially used for preparing skin care cosmetics, including creams, lotions, lotions, gels, facial masks, liniments or lotions; According to a known method in the field of cosmetics industry, the composition composed of the above-mentioned Acanthopanax senticosus homogeneous polysaccharide and auxiliary materials used in the field of cosmetics is sterilized according to a known method to prepare various external preparations.
  • the obtained Acanthopanax senticosus homogeneous polysaccharide powder can be mixed with known bases or auxiliary materials, carriers and additives for cosmetics and medicines, and the preparation is carried out according to a conventional method, wherein the Acanthopanax senticosus homogeneous polysaccharide powder is prepared.
  • the polysaccharide powder accounts for 3% to 10% of the total weight of the cosmetic.
  • Fig. 1 is the elution curve diagram of the crude polysaccharide fraction of Acanthopanax senticosus through DEAE Fast Flow ion chromatography column.
  • Figure 2 is the HPGPC gel chromatogram of Acanthopanax senticosus homogeneous polysaccharide.
  • Figure 3 is a total ion current chromatogram of six standard monosaccharides by GC-MS.
  • Figure 4 is the total ion current chromatogram of Acanthopanax senticosus homogeneous polysaccharide GC-MS.
  • Figure 5 is the infrared spectrum of the homogeneous polysaccharide of Acanthopanax senticosus.
  • Figure 6 is the 1 H spectrum of the homogeneous polysaccharide of Acanthopanax senticosus, Note: (left ⁇ right).
  • Figure 7 is the 13 C spectrum of the homogeneous polysaccharide of Acanthopanax senticosus, Note: (left ⁇ right).
  • Dried Acanthopanax senticosus decoction pieces were ground into powder, passed through a 100-mesh sieve, and the sieved medicinal powder was extracted 3 times with 5 times the weight of the medicinal powder. , and then collect the supernatant, and concentrate the supernatant to one-fifth of the original volume by rotary evaporation to obtain a concentrated solution.
  • dialysis is performed with a dialysis bag (molecular weight cut-off is 3500 Da).
  • the water in the beaker containing the dialysis bag was changed 5 times a day, and after 7 days of dialysis, the polysaccharide solution in the dialysis bag was centrifuged, and then freeze-dried to obtain the preliminarily purified Acanthopanax senticosus polysaccharide.
  • HPLC conditions Agilent 1200 HPLC, the chromatographic column is TSK GEL G3000PW XL (7.8 ⁇ 300mm, 7 ⁇ m) and TSK GEL G5000PW XL (7.8 ⁇ 300mm, 10 ⁇ m) in series, the mobile phase is 0.02mol/L KH 2 PO 4 solution, the flow rate is 0.5mL/min, the column temperature is 35°C, and the detector is a Waters 2414 refractive index detector. The homogeneous polysaccharide of Acanthopanax senticosus obtained in step 2 was dissolved with an appropriate amount of water, and the sample injection was 10 ⁇ L. The result is shown in Figure 2.
  • the chromatographic peak of the homogeneous polysaccharide of Acanthopanax senticosus in the chromatogram is a single symmetrical peak, indicating that the homogeneous polysaccharide of Acanthopanax senticosus prepared by the present invention It is indeed a homogeneous polysaccharide.
  • Acid hydrolysis was performed first, followed by acetylation for derivatization, followed by gas phase GC analysis.
  • the acid hydrolysis method is as follows: Weigh 10 mg of Acanthopanax senticosus homogeneous polysaccharide samples, place them in ampoules respectively, add 4 mL of trifluoroacetic acid with a concentration of 2 mol/L, blow out the air in the bottle with nitrogen, and seal the tube with an alcohol blowtorch. After hydrolysis at 110 °C for 6 hours, the sample was rotary evaporated to dryness, dissolved in 2 mL of methanol, evaporated to dryness, repeated 3 times, and the trifluoroacetic acid polysaccharide hydrolyzate in the sample was removed as much as possible.
  • GC analysis was performed.
  • GC detection conditions Agilent 6890N gas chromatography system, using Agilent HP-5 quartz capillary column (30m ⁇ 0.32mm ⁇ 0.25 ⁇ m); constant pressure mode is 20PSI; carrier gas is N 2 ; injection volume: 1.0 ⁇ L; flow rate is 1.0mL
  • GC results Analyzed according to the retention time of six monosaccharide standards including arabinose, galactose, glucose, mannose, xylose and fucose, and calculated the monosaccharide monosaccharide of Acanthopanax senticosus based on the area ratio of the peaks mole percent.
  • the results show that, compared with the retention time in the GC spectrum of the monosaccharide standard, the results show that the homogeneous polysaccharide of Acanthopanax senticosus is composed of arabinose, galactose, glucose, mannose and xylose.
  • Five monosaccharides were calculated according to the internal standard method. The mole percentages are 16.42%, 32.27%, 40.38%, 7.21% and 3.72%.
  • GC-MS chromatographic conditions Agilent 6890-5973N gas chromatography-mass spectrometer, chromatographic column: HP-5MS capillary column (30m ⁇ 250pm ⁇ 0.25umD); carrier gas: helium e; heater temperature: 250°C: process Temperature program: the initial temperature was raised to 200°C at 140°C/min, maintained for 5 minutes, and then raised to 240°C at 8°C/min: split ratio: 50:1; injection volume: 5 ⁇ L.
  • Table 1 The methylation analysis data of Acanthopanax senticosus homogeneous polysaccharide are shown in Table 1.
  • the methylation results of the homogeneous polysaccharide of Acanthopanax senticosus showed that the homogeneous polysaccharide of Acanthopanax senticosus was mainly composed of ⁇ ,6)- ⁇ -Galp(1 ⁇ , and the Glcp residue was the main unit of the homogeneous polysaccharide of Acanthopanax senticosus, with 1 ⁇ 4, 1 ⁇ 6, 1 ⁇ 4, 6 and 1 ⁇ linkages exist, and Galp residues exist in a 1 ⁇ 4 linkage.
  • the Acanthopanax senticosus homogeneous polysaccharide obtained in Example 1 is used to prepare the essence milk, and the weight percent of the components and the production process are as follows:
  • Production process under stirring, heat phase A and phase B to 70°C to dissolve and mix evenly, then add phase B to phase A at 70°C to form W/Q type emulsion. After stirring evenly, cool to room temperature.
  • the matrix components used in the preferred embodiment of the present invention are as described above, and the matrix components used in this embodiment can maximize the efficacy of the pharmaceutical composition of the present invention.
  • common substrates produced by other manufacturers that can be applied to cosmetics can also be used in the present invention, and the dosage is only required to meet the national cosmetic additive dosage standard, which will not affect the effect of the present invention, so it is not listed one by one.
  • the homogeneous emulsification equipment used in the present invention is the FV-30L FISCO vacuum homogeneous emulsifier produced by Shanghai Fluke Fluid Machinery Manufacturing Co., Ltd., which has functions such as homogenization, stirring and temperature control.
  • Homogeneous emulsification equipment for cosmetic preparation produced by other manufacturers can also be used in the present invention, as long as the operation is performed strictly in accordance with the process parameters described in the present invention, the effects described in the present invention can be achieved.
  • mice 1.1 Experimental animals Experimental animals 40 SPF female Kunming mice, weighing (20 ⁇ 2) g, were purchased from Guangdong Provincial Laboratory Animal Center, experimental animal quality license number: 44005800003406, in the Science and Technology Industrial Park of Guangzhou University of Traditional Chinese Medicine. Room (use license number: SYXK (Guangdong) 2013-0014) rearing experiment. Since the ultimate purpose of this study is to develop anti-aging cosmetics for women, all female mice were selected as experimental animals. Disposal of mice is in accordance with the principles of animal ethics.
  • the essence milk containing Acanthopanax senticosus homogeneous polysaccharide is the essence milk prepared in Example 3; D-galactose: Beijing Solaibao Biotechnology Co., Ltd.; HA assay kit: Shanghai Enzyme Link Biotechnology Co., Ltd.; HYP Assay kit, SOD assay kit, Coomassie brilliant blue: Nanjing Jiancheng Bioengineering Institute.
  • mice were reared adaptively for 7 days to ensure that the mice were adapted to the current environment and then started the experiment.
  • 30 female mice were divided into 3 groups according to the method of random number table grouping, among which 2 groups of mice were used as model groups, subcutaneously injected with D-galactose 1.0g ⁇ kg -1 ⁇ d -1 for a total of 30 days; The mice in the group were taken as the blank group, and were injected with the same volume of normal saline every day. After 30 days, the skin appearance of the modeling group and the blank group was compared, and the skin of the model group was obviously slack and fine wrinkles appeared, while the blank group was the opposite.
  • Two groups of skin aging model mice were divided into model group and Acanthopanax senticosus homogeneous polysaccharide group. The hair on the back of the mouse was cut short with scissors, and then shaved with a razor for use.
  • each group of ointments was applied on the surface of the skin area selected by the corresponding mice in each group, and 0.3 g was applied to each mouse every day. After 24 hours, the skin was cleaned and reapplied for 21 days. Also need to hand hair removal 4 times.
  • HYP skin hydroxyproline
  • HA skin hyaluronic acid
  • SOD skin superoxide dismutase
  • mice in each group were observed: compared with the animals in the blank group, the skin of the mice in the model group was loose, with a lot of folds, and the regeneration of body hair was slower. Compared with the model group, the mice smeared with 6% Acanthopanax senticosus homogeneous polysaccharide had obvious improvement in skin folds, smoothness and relaxation.
  • mice in the blank group were smooth, firm and elastic.
  • D-galactose-injected skin aging model mice had loose skin, increased wrinkles, and significantly decreased skin moisture content, hydroxyproline content, hyaluronic acid content, and superoxide dismutase activity. It was determined that after external application of 6% Acanthopanax senticosus homogeneous polysaccharide essence, the skin appearance of the mice was significantly improved, and the moisture content, hyaluronic acid content, hydroxyproline content, superoxide The indicators such as the activity of compound dismutase have also been significantly improved.

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Abstract

公开了一种刺五加均一多糖，其分子量为6.83×10 5Da，由阿拉伯糖、半乳糖、葡萄糖、甘露糖和木糖组成，各单糖的摩尔百分比依次是16.42％、32.27％、40.38％、7.21％和3.72％。刺五加均一多糖的制备方法具体如下：取刺五加饮片粉碎成粉，采用水提法提取刺五加粗多糖，然后运用Sevage法脱蛋白法去除粗多糖中的蛋白，并采用阴离子交换DEAE Fast Flow层析柱和Sephadex G-200葡聚糖凝胶柱层析柱进行进一步的分离纯化得到刺五加均一多糖。上述方法制得的刺五加多糖为均一多糖，具有抗氧化和抗皮肤衰老活性，可用于制备具有抗皮肤衰老作用的化妆品和药物。

Description

一种刺五加均一多糖及其制备方法及应用 技术领域
本发明涉及药学技术领域，具体涉及一种刺五加均一多糖及其制备方法及应用。
背景技术
刺五加为五加科五加属植物刺五加(Acanthopanax senticosus(Rupr.Et Maxim.)Harms)的干燥根和根茎或茎，广泛分布于俄罗斯远东针叶林带，中国东北、河北、山西，日本、朝鲜的北部地区等。别名五加参、刺拐棒、老虎镣子。在中医学中，刺五加作为药物的应用历史十分悠久，在历代本草著作中均有记载。南朝陶弘景在《名医别录》中指出，刺五加以“五叶者良”，具有“补中、益精、坚筋骨、强志意”的功效。明代李时珍在《本草纲目》中表明，刺五加为“本经上品”，具有“补中益气，坚筋骨，强意志，久服轻身耐老”的功效。刺五加性温，味辛，微苦；归脾、肾、心经；具有益气健脾，补肾安神的功效，主要用于脾肾阳虚，体虚乏力，食欲不振，腰膝酸痛，失眠多梦等症。(潘景芝、金莎、崔文玉等.刺五加的化学成分及药理活性研究进展[J].食品工业科技，2019，40(23)：353－360)刺五加具有免疫功能调节、抗癌、保护肝脏、抗衰老、抗氧化、抗炎、降血压、抗应激等多种药理活性，其活性成分是五加苷类、黄酮类、木脂素类、多糖类等。但这些都是粗多糖的实验结果，重复性较差，不能做出标准化产品，因此产业化价值有限。本发明在既往研究基础上，对刺五加粗多糖开展分离纯化、结构解析和生物活性评价，以期得到有良好生物活性刺五加均一多糖，这不仅具有十分重要的科学意义，且可为刺五加均一多糖的产业化奠定基础。
发明内容
本发明所要解决的技术问题是通过对刺五加多糖按分子量进行分级，然后进一步分离纯化，进行结构表征，提供一种由一种单糖分子缩合而成的多糖，有药用价值的刺五加均一多糖，并对得到的刺五加均一多糖进行抗氧化活性以及抗皮肤衰老活性的初步研究。
本发明的目的是提供一种刺五加均一多糖，其分子量为6.83×10 5Da，由阿拉伯糖、半乳糖、葡萄糖、甘露糖和木糖组成，各单糖的摩尔百分比依次是16.42％、32.27％、40.38％、7.21％和3.72％。
本发明的另一个目的是提供该刺五加均一多糖的制备方法，包括如下步骤：
1)将干燥刺五加饮片粉碎，过筛后加药粉重量5～8倍量的水提取3次，提取温度为80～100℃，每次2h，合并提取液，提取液离心取上清液，并将上清液浓缩，得浓缩液；
2)待浓缩液冷却后，加入α-淀粉酶至其重量含量为0.1～0.4％，调节pH值至7.0，60℃水浴酶解，直至溶液遇碘—碘化钾试剂无颜色变化，迅速升温至100℃保持5min灭酶，离心收集上清液；
3)将步骤2)中收集的上清液与Sevage试剂按体积比1：1混合，剧烈振荡30min，静置12h，收集上层多糖溶液，将上层多糖溶液与Sevage试剂按体积比1：1混合重复上述操作至紫外扫描无蛋白特征吸收峰时止；
4)对步骤3)中最终收集到的上层多糖溶液浓缩后加4～6倍体积量的无水乙醇，4℃静置沉淀48h，离心收集沉淀；将沉淀加无水乙醇重复上述操作3次后进行冷冻干燥，得刺五加粗多糖粉末；
5)将步骤4)中获得的刺五加粗多糖粉末用蒸馏水完全溶解后经DEAE Fast Flow离子层析柱分离，洗脱条件：流速为2.5mL/min，依次以纯水、0.05mol/L、0.1mol/L、0.2mol/L、0.4mol/L、1mol/L的氯化钠溶液进行洗脱，采用硫酸—苯酚法进行跟踪检测，收集洗脱液；
6)将步骤5)中收集的洗脱液浓缩后，用Sephadex G-200葡聚糖凝胶柱再分离，以蒸馏水按照流速0.5mL/min进行洗脱，苯酚—硫酸法检测后，收集洗脱曲线中的主峰部分；
7)将步骤6)中收集的洗脱曲线中的主峰部分的溶液浓缩后，用截留分子量为3500Da的透析袋透析2d脱盐，最后将透析袋内的溶液进行浓缩冷冻干燥，得到刺五加均一多糖粉末。
本发明提供了一种刺五加均一多糖的制备方法，包括如下步骤：
1)将干燥刺五加饮片粉碎，过100目筛，将过筛后的药粉用药粉重量5～8倍量的水提取3次，提取温度为80～100℃，每次2h，合并提取液，将提取液3000rpm/min离心20min，然后收集上层清液，并将上清液旋转蒸发浓缩至原来体积的五分之一，得浓缩液；
2)待浓缩液冷却后，加入α-淀粉酶至其重量含量为0.1～0.4％，调节pH值至7.0，60℃水浴酶解4h(溶液遇碘-碘化钾试剂无颜色变化)，迅速升温至100℃保持5min灭酶，之后3000rpm/min离心10min收集上清液；
3)将步骤2)收集的上清液与Sevage试剂(氯仿：正丁醇体积比4:1混合)按体积比1：1混合，剧烈振荡30min，静置12h，收集上层多糖溶液，将上层多糖溶液与Sevage试剂按体积比1：1混合后重复上述操作至紫外扫描无蛋白特征吸收峰时止；
4)对步骤3)中最终收集到的上层多糖溶液浓缩后加4～6倍体积量的无水乙 醇，4℃静置沉淀48h，3000rpm/min离心10min收集沉淀；将沉淀加无水乙醇重复上述操作3次后，将最终得到的沉淀进行冷冻干燥，得刺五加粗多糖粉末；
5)将步骤4)中获得的刺五加粗多糖粉末用蒸馏水完全溶解后经DEAE Fast Flow离子层析柱分离，洗脱条件：流速为2.5mL/min，依次以纯水、0.05mol/L、0.1mol/L、0.2mol/L、0.4mol/L、1mol/L的氯化钠溶液进行洗脱；利用全自动收集器分梯度收集洗脱液，每个溶液梯度洗脱3倍柱体积，收集30管，每管5.0mL，采用硫酸—苯酚法进行隔管跟踪检测；
6)将步骤5)中收集的洗脱液浓缩后，用Sephadex G-200葡聚糖凝胶柱再分离，以蒸馏水按照流速0.5mL/min进行洗脱，利用全自动收集器收集，每管5.0mL，苯酚—硫酸法检测后，收集洗脱曲线中的主峰部分；
7)将步骤6)中收集的洗脱曲线中的主峰部分的溶液浓缩后，用截留分子量为3500Da的透析袋透析2d脱盐，最后将透析袋内的溶液进行浓缩冷冻干燥，得到刺五加均一多糖粉末。
进一步的，所述Sevage试剂中，氯仿：正丁醇体积比＝4：1。
本发明发现了通过水提醇沉法提取，并采用阴离子交换DEAE Fast Floe层析柱和Superdex-200凝胶层析柱进行进一步的分离纯化等方案，得到了分子量为6.83×10 5Da，由阿拉伯糖、半乳糖、葡萄糖、甘露糖和木糖组成的刺五加均一多糖。
试验证明，本发明制备的刺五加均一多糖粉末具有抗氧化、抗皮肤衰老等多重作用，能够用于制备抗皮肤衰老的化妆品和皮肤的治疗性药物。
本发明还涉及一种皮肤保养化妆品，包括上述具有抗皮肤衰老作用的刺五加均一多糖和化妆品领域使用的辅料。
本发明还涉及一种皮肤的治疗性药物，包括上述的具有抗皮肤衰老作用的刺五加均一多糖和医学上可接受的载体。
本发明提供的刺五加均一多糖尤其可以用于制备皮肤护理化妆品，包括霜剂、乳液、化妆水、凝胶剂、面膜、涂搽剂或洗剂；但不局限于上述剂型，可按照护肤品工业领域中已知的方法，将上述刺五加均一多糖和化妆品领域使用的辅料组成的组合物按已知方法除菌后制成各种不同的外用制剂。
制备皮肤护理化妆品时，可将制得的刺五加均一多糖粉末与已知的化妆品以及药品的基质或者辅料、载体、添加剂相混合，按照常规的方法进行制备，其中，所述刺五加均一多糖粉末占化妆品总重量的3％～10％。
附图说明
图1为刺五加粗多糖组分过DEAE Fast Flow离子层析柱的洗脱曲线图。
图2为刺五加均一多糖的HPGPC凝胶色谱图。
图3为六种标准单糖GC-MS总离子流色谱图。
图4为刺五加均一多糖GC-MS总离子流色谱图。
图5为刺五加均一多糖的红外光谱图。
图6为刺五加均一多糖的 1H谱图，注：(左→右)。
图7为刺五加均一多糖的 13C谱图，注：(左→右)。
具体实施方式
下面通过具体实施方式对本发明作进一步详细的说明：
实施例1刺五加均一多糖的制备和结构表征
1、刺五加粗多糖的提取：
干燥刺五加饮片打粉，过100目筛，将过筛后的药粉用药粉重量5倍量水提取3次，温度为80℃，每次2h，合并提取液，将提取液3000rpm/min离心20min，然后收集上层清液，并将上清液旋转蒸发浓缩至原来体积的五分之一，得浓缩液。待浓缩液冷却后，加入α-淀粉酶至其重量含量为0.1％，调节pH值至7.0，60℃水浴酶解4h(溶液遇碘-碘化钾试剂无颜色变化)，迅速升温至100℃保持5min灭酶，之后3000rpm/min离心10min收集上清液。将上清液与Sevage试剂(氯仿：正丁醇体积比4:1混合)按体积比1：1混合，剧烈振荡30min，静置12h，收集上层多糖溶液，将上层多糖溶液与Sevage试剂按体积比1：1混合后重复上述操作至紫外扫描无蛋白特征吸收峰时止。对最终收集到的上层多糖溶液浓缩后加4倍体积量的无水乙醇，4℃静置沉淀48h，3000rpm/min离心10min收集沉淀。将沉淀加无水乙醇重复上述操作重复3次后，将最终得到的沉淀进行冷冻干燥，得刺五加粗多糖粉末。
2、刺五加均一多糖的纯化
将获得的刺五加粗多糖粉末用蒸馏水完全溶解后经DEAE Fast Flow离子层析柱分离，洗脱条件：流速为2.5mL/min，依次以水、0.05mol/L、0.1mol/L、0.2mol/L、0.3mol/L、0.5mol/L的氯化钠溶液进行洗脱。利用全自动收集器分梯度收集，每个溶液梯度洗脱3倍柱体积，收集30管，每管5.0mL，采用硫酸—苯酚法进行隔管跟踪检测。其中，刺五加粗多糖组分过DEAE Fast Flow离子层析柱的洗脱曲线图如图1所示。
一方面，将不同洗脱液对应的收集液浓缩至一定体积后，用透析袋(截留分子量为3500Da)进行透析。装透析袋的烧杯每天换水5次，透析7天后，将透析袋内多糖液，离心，并将其进行冷冻干燥，得到初步纯化的刺五加多糖。
另一方面，将收集的洗脱液浓缩后，用Sephadex G-200葡聚糖凝胶柱再分离，以蒸馏水按照流速0.5mL/min进行洗脱，利用全自动收集器收集，每管5.0mL，苯酚—硫酸法检测后，收集洗脱曲线中的主峰部分。然后将收集的洗脱曲 线中的主峰部分的溶液浓缩后，用截留分子量为3500Da的透析袋透析2d脱盐，最后将透析袋内的溶液进行浓缩冷冻干燥，得到刺五加均一多糖粉末。
3、刺五加均一多糖的纯度鉴定
高效液相色谱条件：Agilent 1200高效液相色谐仪，色谱柱为TSK GEL G3000PW XL(7.8×300mm，7μm)和TSK GEL G5000PW XL(7.8×300mm，10μm)串联，流动相为0.02mol/L KH 2PO 4溶液，流速为0.5mL/min，柱温35℃，检测器为Waters 2414示差折光检测器。将步骤2获得的刺五加均一多糖用适量水溶解，进样为10μL，结果见图2，色谱图中刺五加均一多糖色谱峰呈单一对称峰，说明本发明制备的刺五加均一多糖确实为均一多糖。
4、刺五加均一多糖分子量的确定
取1mg分子量为50K、80K、150K、270K、410K和670K Da的普鲁兰多糖分别溶于1mL水中，色谱条件同上，用Agilent 1200高效液相色谱仪分析，记录保留时间，以相对分子量的对数(1ogM)为纵坐标，保留时间(t)为横坐标，得标准曲线y＝14.62-0.980X，将刺五加均一多糖出峰时间代入曲线方程，得刺五加均一多糖的分子量为6.83×10 5Da。
5、刺五加均一多糖的结构表征
5.1多糖组分分析
先进行酸水解，然后乙酰化进行衍生，再进行气相GC分析。酸水解的方法是：称取10mg刺五加均一多糖样品，分别置于安瓿瓶中，加入浓度为2mol/L的4mL三氟乙酸，氮气吹跑瓶内空气，用酒精喷灯封管。110℃水解6小时后，旋蒸干样品，加入2mL的甲醇溶解，蒸干，反复3次，尽量去除样品中的三氟乙酸得多糖水解物。加入1mL的甲醇将样品转移至血清瓶内，氮气吹干，然后往多糖水解物中加入1.0mL吡啶、10mg盐酸羟胺和1.0mg内标肌醇，于90℃下恒温震荡反应0.5h，冷却后加入1mL醋酸酐，于90℃下进行乙酰化反应0.5h。冷却后加水终止反应。再加入2.0mL氯仿萃取3次，用无水硫酸钠除去多余的水分后，用0.22μm有机相滤膜过滤。各单糖标准品也按照上述步骤进行衍生化。
按照上述方法衍生化后，进行GC分析。GC检测条件：Aglient6890N气相色谱系统，使用Agilent HP-5石英毛细管柱(30m×0.32mm×0.25μm)；恒压模式为20PSI；载气为N 2；进样量：1.0μL；流速是1.0mL/min；进样口温度：250℃；FID检测器的温度设置为250℃；进样口采用不分流模式；程序升温：初始温度100℃，保持30s，以3℃/min升至160℃，改变升温速度，继续升温。以10℃/min的速度升到250℃，并保持5min。阿拉伯糖(Arabinose)、半乳糖(Galactose)、葡萄糖(Glucose)、甘露糖(Mannose)、木糖(Xylose)和岩藻糖(Fucose)六种单糖标准品的GC-MS总离子流色谱图如图3所示，刺五加均一多糖GC-MS总离子流色谱图如图4所示。
GC结果：根据阿拉伯糖、半乳糖、葡萄糖、甘露糖、木糖和岩藻糖等六种单糖标准品的保留时间进行分析，并通过出峰的面积比计算出刺五加均一多糖单糖摩尔百分比。结果显示，同单糖标准品的GC图谱中的保留时间对照，结果表明刺五加均一多糖由阿拉伯糖、半乳糖、葡萄糖、甘露糖和木糖组成，根据内标法计算出五种单糖的摩尔百分比是16.42％、32.27％、40.38％、7.21％和3.72％。
5.2刺五加均一多糖红外光谱扫描
称取刺五加均一多糖样品2.0mg，与溴化钾粉末混和后研磨均匀后进行压片处理。之后将压片置于傅里叶变换红外光谱仪进行红外扫描(400～4000cm -1)，记录样品的红外吸收图谱，结果见图5。从IR图中可以看出刺五加均一多糖呈现典型的多糖吸收特征，其中3456cm －1和2929cm －1为O-H键的伸缩振动峰，1744cm －1为CH 2中C-H的伸缩振动峰；1635cm －1为CO 2或共生水引起的伸缩振动峰，1411cm －1为C-O键的变角振动峰，1242cm －1是伯醇β-OH引起的伸缩振动峰；1021cm －1处的吸收峰说明刺五加均均一多糖中含吡喃环，而在893cm －1处出现的吸收峰说明刺五加均一多糖中存在β-糖苷键，而835cm －1处出现的吸收峰又说明刺五加均一多糖中存在α-糖苷键。
5.3刺五加均一多糖甲基化分析
称取刺五加均一多糖样品20mg(样品保持充分的干燥)置于带塞试管中，用6mL的二甲基亚砜DMSO试剂，并用氮气封口，进行加热，磁力搅拌混匀，加入氢氧化钠(6mL的DMSO含有240mg的氢氧化钠)形成氢氧化钠悬浊液，过夜。隔日向试管中加入3.6mL的碘甲烷，搅拌8min，用氮气吹走碘甲烷，再次甲基化，如此重复3次后，加入6mL的蒸馏水中止反应。流水和去离子水各透析24h后，用氯仿萃取3次后，用无水亚硫酸钠干燥24h，然后氮气吹干，溶液剩下约1mL。采用三氟乙酸水解的方法对其进行水解，再加入70mg的硼氢化钠NaBH4搅拌24h，再加强酸性阳阴离子交换树脂搅拌混匀10min，抽滤，收集滤液加甲醇，氮气吹干，然后加乙酸酐、无水吡啶各0.5mL，100℃下乙酰化2h。反应结束后反复加入无水乙醇以除去乙酸酐，再进行GC-MS分析。
GC-MS色谱条件：Agilent6890-5973N型气相色谱—质谱联用仪，色谱柱：HP-5MS毛细管柱(30m×250pm×0.25umD)；载气：氦气e；加热器温度：250℃：程程序升温：初始温度140℃/min升至200℃，保持5min，再以8℃/min升至240℃：分流比：50：1；进样量：5μL。刺五加均一多糖甲基化分析数据如表1所示。刺五加均一多糖甲基化的结果表明，刺五加均一多糖主要是→,6)-β-Galp(1→组成，Glcp残基是刺五加均一多糖的主要单元，以1→4、1→6、1→4,6和1→连接方式存在，Galp残基以1→4连接方式存在。
表1刺五加均一多糖甲基化分析数据
序号 甲基化残基 糖苷键的类型 摩尔百分比(mol.％)
1 2,4-Me3-Glcp →4)-β-Glcp(1→ 10.8
2 2,3-Me2-Glcp →4,6)-β-Glcp(1→ 9.6
3 2,4,6-Me3-Galp →,6)-β-Galp(1→ 71.5
4 2,3,6-Me3-Glcp →4)-β-Glcp(1→ 5.2
5 2,3,4,6-Me4-Glcp β-Glcp(1→ 2.9
5.4刺五加均一多糖NMR分析
取30mg干燥的刺五加均一多糖，溶于0.5ml D 2O中，60℃加热1h使其完全溶解后转移至核磁管中，于德国Brucker公司AV300核磁共振仪上测定 1H谱和 13C谱，结果见图6和图7。从谱图可以看出，刺五加均一多糖 1H谱的信号分布范围窄，主要集中在δ2.0-6.0ppm(4.19，4.09，4.07，4.00，3.95，3.90，3.70，3.68，3.65，3.64，3.62，3.61，3.60，3.59，3.58，3.55，3.54和3.52ppm)范围内。而其C谱信号范围为δ60-110ppm(107.41，106.83，106.36，103.62，95.72，92.11，91.85，84.00,81.31，76.81，76.64，76.31，75.50，71.24，71.14，71.00，69.89，69.21，69.14，62.30,61.26,61.05，60.68，和60.42ppm)。刺五加均一多糖中各个糖残基的异头碳领化学位移归属见表2。
表2刺五加均一多碳氢化学位移归属
序号 糖残基 CI HI
A →5)-α-D-GalAp 90.3 7.37
B →4)-β-L-Araf-(1→ 93.2 12.50
C α-D-Rhap-(1→ 91.06 8.81
D →3,4)-α-L-GalAp-(1→ 108.2 4.27
E →3,4)-β-D-Galp-(1→ 107.3 8.58
F →4)-β-L-GalAp-(1→ 96.7 7.86
实施例2刺五加均一多糖抗氧化活性评价
现代医学认为，引起皮肤老化的关键原因是各种因素导致的真皮层和表皮层的氧化应激，因此有强大抗氧化功效的物质具有较好的抗皮肤衰老作用，本实验将实施例1所获得的刺五加均一多糖用于抗氧化活性评价。
1、清除DPPH自由基活性测定
取2mL不同质量浓度的样品溶液(12.5，25，50，100μg·ml -1)，加入2mL DPPH溶液(100μg·ml -1)，充分混匀后，避光反应30min。于517nm处测定反应体系的吸光度值(Am)。同时设定溶剂空白组(An，DPPH溶液用等体积甲醇代替)和样品空白组(Ao，样品溶液用等体积甲醇代替)。VC作为阳性对照组。实验平行操作3次，根据下列公式计算提取液对DPPH的清除率。
Figure PCTCN2020109792-appb-000001
2、清除ABTS +活性测定
取0.4mL不同质量浓度的样品溶液(12.5，25，50，100μg·ml -1)，加入ABTS +溶液4mL，反应6min，于734nm处测定其吸光度(Ai)。同时设定溶剂空白组(Aj，ABTS +溶液用等体积甲醇代替)，样品空白组(Ah，样品溶液用等体积甲醇代替)和阳性对照组。实验平行操作3次，根据下列公式计算提取液对ABTS +的清除率：
Figure PCTCN2020109792-appb-000002
3、还原力测定
取0.8mL不同质量浓度的样品溶液(12.5，25，50，100μg·ml -1)，加入2mL磷酸盐缓冲液(PH＝6.6)和2mL 1％铁氢化钾，50℃水浴20min，加2mL 10％三氯乙酸，3000rpm，离心10min，取2mL上清加2mL去离子水，0.4mL 0.1％三氯化铁，反应5min，于700nm处测吸光度值，以VC为阳性对照，实验平行操作3次，测得的吸光度值越大表示还原能力越强。
结果：如表3-5所示，刺五加均一多糖清除DPPH自由基和ABTS +的IC 50值分别为3.17±0.57μg·mL -1和15.26±5.79μg·mL -1，还原力EC 50值为为36.02±10.89μg·mL -1，其抗氧化能力强于阳性药维生素C，差异有统计学意义(P＜0.01)，这说明实施例1制备的刺五加均一多糖具有较强的抗氧化活性。
表3刺五加均一多糖清除DPPH自由基活性(n＝6，
Figure PCTCN2020109792-appb-000003
)
组别 回归方程 r IC 50/μg·mL -1
刺五加均一多糖 Y＝3.572X+3.876 0.999 3.17±0.57 ﹡﹡
维生素C Y＝5.872X+1.483 0.999 6.78±0.96
与维生素C组比较， ﹡P＜0.05， ﹡﹡P＜0.01。
表4刺五加均一多糖清除ABST +活性(n＝6，
Figure PCTCN2020109792-appb-000004
)
组别 回归方程 r IC 50/μg·mL -1
维生素C Y＝2.157X+7.624 0.997 34.21±6.08
刺五加均一多糖 Y＝1.024X+4.275 0.999 15.26±5.79 ﹡﹡
与维生素C组比较， ﹡P＜0.05， ﹡﹡P＜0.01。
表5刺五加均一多糖还原力测定(n＝6，
Figure PCTCN2020109792-appb-000005
)
组别 回归方程 r EC 50/μg·mL -1
维生素C Y＝0.0068X+0.0047 0.999 68.47±11.22
刺五加均一多糖 Y＝0.0028X+0.0043 0.998 36.02±10.89 ﹡﹡
与维生素C组比较， ﹡P＜0.05， ﹡﹡P＜0.01。
实施例3含刺五加均一多糖精华乳的制备
本实验将实施例1所获得的刺五加均一多糖用于制备精华乳，组分的重量百分比和生产工艺如下：
Figure PCTCN2020109792-appb-000006
生产工艺：搅拌下将A和B相分别加热至70℃，至全部溶解混合均匀，然后在70℃条件下将B相加入A相中，至形成W/Q型乳剂。搅拌均匀后，冷却至室温即得。
本发明较佳实施例所用的基质成分如上所述，本实施例所用的基质成分可以将本发明的药物组合物的功效发挥到最佳。但其它厂家生产的可应用于化妆品的常用基质也可用于本发明，用量只要符合国家化妆品添加剂用量标准即可，不会影响本发明效果，因此不一一列举。
本发明所用的均质乳化设备为上海弗鲁克流体机械制造有限公司生产的FV-30L FISCO真空均质乳化机，其有均质、搅拌和温控等功能。其它厂家生产的用于化妆品制备的均质乳化设备也可以用于本发明，只要严格按照本发明所述的工艺参数进行操作，均可达到本发明所述的效果。
实施例4含刺五加均一多糖精华乳抗皮肤衰老活性评价
本实验将实施例3所获得的含刺五加均一多糖的精华乳用于抗皮肤衰老活性评价。
1材料和方法
1.1实验动物实验动物SPF级雌性昆明种小鼠40只，体质量(20±2)g，购自广东省实验动物中心，实验动物质量许可证号：44005800003406，在广州中医药大学科技产业园动物房(使用许可证号：SYXK(粤)2013-0014)饲养 实验。由于本研究最终目的是为了研制适用于女性的抗皮肤衰老化妆品，所以全部选用雌性小鼠作为实验动物。小鼠的处置符合动物伦理学原则。
1.2材料和试剂含刺五加均一多糖的精华乳为实施例3制备的精华乳；D-半乳糖：北京索莱宝生物科技有限公司；HA测定试剂盒：上海酶联生物科技有限公司；HYP测定试剂盒、SOD测定试剂盒、考马斯亮兰：南京建成生物工程研究所。
1.3分组与造模首先将雌性小鼠适应性饲养7d，保证小鼠适应现环境后开始进行实验。将30只雌性小鼠按照随机数字表分组的方法分成3组，其中2组小鼠作为造模组，皮下注射D-半乳糖1.0g·kg -1·d -1，一共注射30d；余下一组小鼠作为空白组，每天注射同体积生理盐水。30d后造模组和空白组皮肤外观比对，模型组明显皮肤松弛，出现细密皱纹，而空白组则相反。将2组皮肤衰老模型小鼠分为：模型组、刺五加均一多糖组。用剪刀将小鼠背部毛发剪短，再用剃须刀剃毛备用。
1.4小鼠皮肤外用精华乳刺五加均一多糖组小鼠使用实施例3所获得的含6％刺五加均一多糖的精华乳，模型组则使用不含刺五加多糖的精华乳，具体方法为选取小鼠背部中央位置4cm×7cm的区域，将各组膏剂涂抹在对应各组小鼠选取的皮肤区域表面，每只每天涂抹0.3g，24h后清洁皮肤并再次涂抹，连续涂抹21d，期间还需手工脱毛4次。
1.5小鼠皮肤的表观特征观察比对各组小鼠用药部位皮肤的色泽、光滑程度、皱纹等表观特征并拍照记录。然后脱颈法处死实验小鼠，迅速剥下用药部位皮肤，去除皮下脂肪和其他结缔组织，铺平后用直径2cm打孔器在正中位置切取小鼠皮肤用于含水率测定，余下的皮肤于-20℃冷冻保存，用于测定皮肤羟脯氨酸等成分。
1.6皮肤含水率的测定精确称量打孔器切取的皮肤湿重，随即将其放入烘箱中，于50℃烘干12h，然后称取干重，计算出各实验组皮肤含水率。公式如下：皮肤含水率＝(湿重-干重)/湿重×100％。
1.7皮肤羟脯氨酸(HYP)含量测定取涂药部位皮肤组织0.5g，用冰浴预冷的生理盐水进行漂洗，滤纸擦干，加入冰浴预冷的生理盐水，使用玻璃匀浆器研磨成浓度为10％的匀浆液。将得到的匀浆液在0℃下3 000r/min离心10min，取上清液。按照HYP试剂盒说明书所示方法，使用超微量微孔板分光光度计测定OD值，计算各实验组小鼠皮肤HYP含量。
1.8皮肤透明质酸(HA)含量测定取涂药部位皮肤组织0.5g，加入冰浴预冷的PBS缓冲溶液，使用玻璃匀浆器研磨成浓度为10％的匀浆液。将得到的匀浆液在0℃下1000r/min离心4min，取上清液。按照HA试剂盒说明书所示方法，使用超微量微孔板分光光度计测定OD值，计算各实验组小鼠皮肤HA的含量。
1.9皮肤超氧化物歧化酶(SOD)活力的测定取涂药部位皮肤组织0.5g，用冰浴预冷的生理盐水进行漂洗，之后加入冰浴预冷后的生理盐水，使用玻璃匀浆器研磨成浓度为10％的匀浆液。将得到的匀浆液在0℃下3 000r/min离心10min，取上清液。按照SOD试剂盒说明书所示方法，使用超微量微孔板分光光度计测定OD值，计算各实验组小鼠皮肤SOD活力。
1.10统计学方法运用SPSS18.0统计软件进行数据分析，计量数据采用
Figure PCTCN2020109792-appb-000007
表示；组间比较采用单因素方差分析；方差齐性检验结果显著，两两比较采用Dunnett T3检验；方差齐性检验结果不显著，两两比较采用LSD检验，以P＜0.05为差异有统计学意义。
2结果
2.1皮肤外观比较观察各组小鼠皮肤发现：与空白组动物比较，模型组小鼠皮肤松弛，有大量褶皱，体毛再生较慢。涂抹了含6％刺五加均一多糖的小鼠与模型组相比，在皮肤褶皱、光滑、松弛等方面都有较为明显的改善。
2.2刺五加均一多糖对小鼠皮肤含水率、HYP含量、HA含量、SOD活力的影响三组小鼠皮肤组织含水率、HYP含量、HA含量、SOD活力测定结果见表6。模型组小鼠皮肤组织含水率、HYP含量、HA含量、SOD活力均显著低于空白组(P＜0.01)，说明小鼠的皮肤衰老模型造模成功。与模型组相比，刺五加均一多糖组小鼠的皮肤含水率、HYP含量、HA含量、SOD活力显著提高，差异有统计学意义(P＜0.01)。与空白组相比，刺五加均一多糖组小鼠的皮肤含水率、HYP含量、HA含量、SOD活力等各项指标无显著性差异(P＞0.01)。
表6刺五加均一多糖对小鼠皮肤组织含水量、HYP含量、HA含量和SOD活力的影响(n＝6，
Figure PCTCN2020109792-appb-000008
)
组别 皮肤含水量(％) HYP含量(μg/mg) HA含量(μg/mL) SOD活力(U/mg)
空白组 78.21±1.53 △△ 4.31±0.21 △△ 10.24±0.86 △△ 386.12±21.47 △△
模型组 50.83±1.21 3.65±0.19 6.77±0.93 302.55±17.32
刺五加均一多糖组 76.33±1.24 △△ 4.20±0.28 △△ 10.14±0.91 △△ 374.08±26.64 △△
F值 374.12 14.86 263.86 41.28
P值 0.000 0.000 0.000 0.000
事后检验(两两比较) LSD Dunnett T3 Dunnett T3 LSD
与模型组比较， △P＜0.05， △△P＜0.01。
综上所述，本实验的结果发现空白组小鼠的皮肤光滑，较为紧致，富有弹性。注射D-半乳糖的皮肤衰老模型小鼠皮肤松弛，皱褶大量增多，在皮肤含水率、羟脯氨酸含量、透明质酸含量、超氧化物歧化酶活力等指标明显下降。经测定，模型小鼠在外用含6％刺五加均一多糖精华乳后，小鼠皮肤外观改善的效果明显，且小鼠皮肤的含水率、透明质酸含量、羟脯氨酸含量、超氧化物歧化 酶活力等指标也得到了显著提高。
最后有必要在此说明的是：以上实施例只用于对本发明的技术方案作进一步地说明，不能理解为对本发明保护范围的限制，本领域的技术人员根据本发明的上述作出的一些非本质的改进和调整均属于本发明的保护范围。

Claims (7)

  1. 一种刺五加均一多糖，其分子量为6.83×10 5Da，由阿拉伯糖、半乳糖、葡萄糖、甘露糖和木糖组成，各单糖的摩尔百分比依次是16.42％、32.27％、40.38％、7.21％和3.72％。
  2. 根据权利要求1所述的刺五加均一多糖的制备方法，其特征在于：包括如下步骤：
    1)将干燥刺五加饮片粉碎，过筛后加药粉重量5～8倍量的水提取3次，提取温度为80～100℃，每次2h，合并提取液，提取液离心取上清液，并将上清液浓缩，得浓缩液；
    2)待浓缩液冷却后，加入α-淀粉酶至其重量含量为0.1～0.4％，调节pH值至7.0，60℃水浴酶解，直至溶液遇碘—碘化钾试剂无颜色变化，迅速升温至100℃保持5min灭酶，离心收集上清液；
    3)将步骤2)中收集的上清液与Sevage试剂按体积比1：1混合，剧烈振荡30min，静置12h，收集上层多糖溶液，将上层多糖溶液与Sevage试剂按体积比1：1混合重复上述操作至紫外扫描无蛋白特征吸收峰时止；
    4)对步骤3)中最终收集到的上层多糖溶液浓缩后加4～6倍体积量的无水乙醇，4℃静置沉淀48h，离心收集沉淀；将沉淀加无水乙醇重复上述操作3次后进行冷冻干燥，得刺五加粗多糖粉末；
    5)将步骤4)中获得的刺五加粗多糖粉末用蒸馏水完全溶解后经DEAE Fast Flow离子层析柱分离，洗脱条件：流速为2.5mL/min，依次以纯水、0.05mol/L、0.1mol/L、0.2mol/L、0.4mol/L、1mol/L的氯化钠溶液进行洗脱，采用硫酸—苯酚法进行跟踪检测，收集洗脱液；
    6)将步骤5)中收集的洗脱液浓缩后，用Sephadex G-200葡聚糖凝胶柱再分离，以蒸馏水按照流速0.5mL/min进行洗脱，苯酚—硫酸法检测后，收集洗脱曲线中的主峰部分；
    7)将步骤6)中收集的洗脱曲线中的主峰部分的溶液浓缩后，用截留分子量为3500Da的透析袋透析2d脱盐，最后将透析袋内的溶液进行浓缩冷冻干燥，得到刺五加均一多糖粉末。
  3. 根据权利要求2所述的刺五加均一多糖的制备方法，其特征在于：所述Sevage试剂中，氯仿：正丁醇＝4：1。
  4. 根据权利要求1所述的刺五加均一多糖的制备方法，其特征在在于：包括 如下步骤：
    1)将干燥刺五加饮片粉碎，过100目筛，将过筛后的药粉用药粉重量5～8倍量的水提取3次，提取温度为80～100℃，每次2h，合并提取液，将提取液3000rpm/min离心20min，然后收集上层清液，并将上清液旋转蒸发浓缩至原来体积的五分之一，得浓缩液；
    2)待浓缩液冷却后，加入α-淀粉酶至其重量含量为0.1～0.4％，调节pH值至7.0，60℃水浴酶解4h，此时溶液遇碘—碘化钾试剂无颜色变化，迅速升温至100℃保持5min灭酶，之后3000rpm/min离心10min收集上清液；
    3)将步骤2)中收集的上清液与Sevage试剂按体积比1：1混合，剧烈振荡30min，静置12h，收集上层多糖溶液，将上层多糖溶液与Sevage试剂按体积比1：1混合后重复上述操作至紫外扫描无蛋白特征吸收峰时止，其中，所述Sevage试剂由氯仿和正丁醇按体积比4:1混合制得；
    4)对步骤3)中最终收集到的上层多糖溶液浓缩后加4～6倍体积量的无水乙醇，4℃静置沉淀48h，3000rpm/min离心10min收集沉淀；将沉淀加无水乙醇重复上述操作3次后，将最终得到的沉淀进行冷冻干燥，得刺五加粗多糖粉末；
    5)将步骤4)中获得的刺五加粗多糖粉末用蒸馏水完全溶解后经DEAE Fast Flow离子层析柱分离，洗脱条件：流速为2.5mL/min，依次以纯水、0.05mol/L、0.1mol/L、0.2mol/L、0.4mol/L、1mol/L的氯化钠溶液进行洗脱；利用全自动收集器分梯度收集洗脱液，每个溶液梯度洗脱3倍柱体积，收集30管，每管5.0mL，采用硫酸—苯酚法进行隔管跟踪检测；
    6)将步骤5)中收集的洗脱液浓缩后，用Sephadex G-200葡聚糖凝胶柱再分离，以蒸馏水按照流速0.5mL/min进行洗脱，利用全自动收集器收集，每管5.0mL，苯酚—硫酸法检测后，收集洗脱曲线中的主峰部分；
    7)将步骤6)中收集的洗脱曲线中的主峰部分的溶液浓缩后，用截留分子量为3500Da的透析袋透析2d脱盐，最后将透析袋内的溶液进行浓缩冷冻干燥，得到刺五加均一多糖粉末。
  5. 根据权利要求1所述的刺五加均一多糖粉末的用途，其特征在于：用于制备具有抗皮肤衰老作用的皮肤保养化妆品或皮肤的治疗性药物。
  6. 皮肤保养化妆品，包括权利要求1所述的刺五加均一多糖粉末和化妆品领域使用的辅料。
  7. 皮肤的治疗性药物，包括权利要求1所述的刺五加均一多糖粉末和医学上可接受的载体。
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