阅读说明：本技术 一种炮天雄均一多糖及其制备方法及应用 (Radix aconiti carmichaeli uniform polysaccharide, and preparation method and application thereof ) 是由 覃军 邓广海 罗明超 龚又明 陈文良 林华 胡黎 郑显辉 陈艳红 于 2018-07-24 设计创作，主要内容包括：本发明申请公开了一种炮天雄均一多糖及其制备方法及应用,其分子量为1.41×10<Sup>5</Sup>Da,并由鼠李糖、甘露糖、岩藻糖和葡萄糖组成。炮天雄均一多糖的制备方法具体如下：取炮天雄饮片,Sevage法脱蛋白及XAD-16型树脂脱色素等方法去除粗多糖中的非糖杂质,并采用离子交换DEAE FAST FLOW层析柱和Superdex-200凝胶层析柱进行进一步的分离纯化得到炮天雄均一多糖。本发明方法制得的炮天雄多糖为均一多糖,得率高,具有抗氧化和神经保护活性,可以清除DPH自由基、ABST+,还原力测定强,可用于制备抗氧化剂和神经保护制剂。(The invention discloses a uniform polysaccharide of radix aconiti lateralis preparata, a preparation method and application thereof, wherein the molecular weight of the uniform polysaccharide is 1.41 multiplied by 10 5 Da and consists of rhamnose, mannose, fucose and glucose. The preparation method of the uniform polysaccharide of the radix aconiti lateralis preparata comprises the following steps: removing non-sugar impurities from crude polysaccharide by Sevage deproteinization and XAD-16 resin depigmentation, and further separating and purifying with ion exchange DEAE FAST FLOW chromatographic column and Superdex-200 gel chromatographic column to obtain radix Aconiti Preparata homogeneous polysaccharide. The prepared radix aconiti lateralis preparata polysaccharide is homogeneous polysaccharide, has high yield, antioxidant and neuroprotective activity, capacity of eliminating DPH free radical and ABST +, powerful reducing capacity measurement and other advantagesCan be used for preparing antioxidant and neuroprotective preparation.)

1. The uniform polysaccharide of the cannon eupatorium is characterized in that: its molecular weight is 1.41X 10⁵Da and consists of rhamnose, mannose, fucose and glucose.

2. The method for preparing the artillery tianxiong homopolysaccharide as claimed in claim 1, which is characterized by comprising the following steps: the method comprises the following steps:

step 1: heating, condensing and refluxing the dried stir-fried radix aconiti lateralis preparata decoction pieces for 2-3 times by using 5-8 times of ethanol, and degreasing to obtain dregs of a decoction;

step 2: extracting the dregs of a decoction with 5-8 times of water for 2-3 times at 80-100 ℃ for 1.5-2 h each time, concentrating the water extract under reduced pressure, adding absolute ethyl alcohol until the alcohol content reaches 80-85%, standing for 18-24 h, removing the supernatant, and collecting the precipitate to obtain crude polysaccharide of radix aconiti lateralis preparata;

and step 3: redissolving the crude polysaccharide of the radix aconiti lateralis preparata, adding pepsin until the content of the pepsin is 0.1-0.4%, carrying out constant-temperature water bath for 10-12 h at the temperature of 32-37 ℃, removing precipitates, repeating the steps for 2-3 times, collecting an upper solution, concentrating, adding a Sevage reagent, violently oscillating for 20-30 min, standing for 8-12 h, collecting an upper polysaccharide solution, and repeating the steps until no protein characteristic absorption peak is generated by ultraviolet scanning;

and 4, step 4: concentrating the upper-layer polysaccharide solution, adding 4-6 times of 95-100% ethanol, standing for 18-24 h, collecting precipitate, repeating for 2-3 times, and freeze-drying to obtain crude polysaccharide powder of radix aconiti lateralis preparata;

and 5: dissolving the crude polysaccharide powder of the radix aconiti lateralis preparata by using distilled water completely, and separating by using a DEAE FAST FLOW ion chromatographic column under the elution condition: eluting with water, 0.05mol/L sodium chloride, 0.1mol/L sodium chloride, 0.2mol/L sodium chloride, 0.3mol/L sodium chloride and 0.1mol/L sodium hydroxide in sequence at a flow rate of 2-2.5 ml/min; gradient collection is carried out by using a full-automatic collector, each solution is subjected to gradient elution with 2-3 times of column volume, 25-30 tubes of each tube with 3-5 ml of each tube are collected, tube separation tracking detection is carried out by adopting a sulfuric acid-phenol method, the collected solution of the radix aconiti lateralis preparata polysaccharide is concentrated to a certain volume, and then dialysis is carried out by using a 3500Da molecular weight dialysis belt; changing water for 3-5 times every day in a beaker filled with a dialysis belt, dialyzing for one week, centrifuging polysaccharide liquid in the dialysis belt, and freeze-drying to obtain primarily purified stir-fried radix aconiti carmichaeli polysaccharide;

step 6: concentrating the collected eluent, separating by using a Superdex G-200 gel column, eluting by using distilled water at a flow rate of 0.15-0.2 ml/min, collecting by using a full-automatic collector, wherein each tube contains 3-5 ml of the eluent, and collecting a main peak part in an elution curve after detecting by using a phenol-sulfuric acid method;

and 7: concentrating the solution of main peak part in the collected elution curve, dialyzing with 3500Da dialysis bag for 2d for desalination, and finally concentrating, freeze drying the solution in the dialysis bag to obtain the radix Aconiti Preparata homogeneous polysaccharide.

3. The method for preparing the artillery tianxiong homopolysaccharide as claimed in claim 2, wherein the method comprises the following steps: in the Sevage reagent, the ratio of chloroform to n-butanol is 5: 1.

4. Use of the artillery tianxiong homopolysaccharide according to claim 1 in the preparation of an antioxidant product or in the preparation of a neuroprotective product.

5. The use of the uniform polysaccharide of artillery tianxiong as claimed in claim 4 in the preparation of antioxidant products or neuroprotective active products, wherein: mixing hydroxypropyl methylcellulose, carboxymethyl starch sodium, microcrystalline cellulose, lactose, medicinal starch, magnesium stearate and rhizoma Gastrodiae polysaccharide, stirring, and making into tablet.

6. The use of the uniform polysaccharide of artillery tianxiong as claimed in claim 4 in the preparation of antioxidant products or neuroprotective active products, wherein: mixing microcrystalline cellulose, medicinal starch and rhizoma Gastrodiae polysaccharide, stirring, and making into tablet.

7. The use of the uniform polysaccharide of artillery tianxiong as claimed in claim 4 in the preparation of antioxidant products or neuroprotective active products, wherein: mixing microcrystalline cellulose, medicinal starch and rhizoma Gastrodiae polysaccharide, stirring, and filling into hard capsule to obtain capsule.

Technical Field

The invention relates to the technical field of medicine and pharmacology, in particular to a uniform polysaccharide of radix aconiti lateralis preparata and a preparation method and application thereof.

Background

Chinese traditional medicine journal, 1998, 23 (6): 348-350; the national pharmacopoeia committee. The processing history of toxic traditional Chinese medicine Tianxiong is a revolutionary study [ J ] of Tianxiong (Aconitum carmichaeli Debx.) which is a processed product of Tianxiong (processed product of Tianxiong) processed at high temperature and is one of the commercial specifications of monkshood decoction pieces.

Chinese pharmacopoeia 2015 year edition: one part [ M ]. beijing: chinese medical science and technology press, 2015: 192) recorded in Shen nong Ben Cao Jing, the effects of making the drug into a big powder mainly include warming yang, relieving pain and dispelling wind.

Chinese traditional medicine journal, 2001, 26 (6): 369-; the response surface method optimizes the Ringnan cannon Tianxiong alkaloid extraction process [ J ] (Caohui, King Shi peach, Wulianying and the like.

Chinese medicine guide, 2017, 14 (19): 31-34) (Gao Jingdong, plum blossom, Chenjialuo, etc.. the influence of the compound aconite polysaccharide on the glycosylation of tumor cells [ J ] at present, the research on chemical components of the cannon-day andra is mainly alkaloid, and the research on other components is not deep yet. Polysaccharides are polymers formed by connecting aldoses or ketoses together through glycosidic bonds, widely exist in plants, animals and microorganisms, and are one of four main substances forming life, and the aconite polysaccharide has been reported to have antitumor and antioxidant effects.

Journal of chinese traditional and western medicine, 2016, 36 (9): 1103-; ZhangAQ, Li XQ, XingC, equivalent.Antiodant activity of polysaccharide extracted from Plueurotus erectus stress surface method [ J ]. Im J Bio macro.2014, 65 (5): 28-32) this has some research value. The radix aconiti lateralis preparata is one specification of radix aconiti lateralis preparata decoction pieces, but the efficacy and the property of the radix aconiti lateralis preparata are different from those of the radix aconiti lateralis preparata due to different material selection and processing processes of the radix aconiti lateralis preparata, the radix aconiti lateralis preparata is selected to be large and good in quality, so that the contained effective ingredients are high, slicing is not needed during processing, the loss of the effective ingredients is reduced, the efficacy of tonifying yang and tonifying deficiency of the radix aconiti lateralis preparata is stronger than that of the radix aconiti lateralis preparata, whether the radix aconiti lateralis preparata polysaccharide has similar functions or not is unclear, and the data relate to crude polysaccharide and have limited industrialization value.

The invention finds that the polysaccharide is one of the main components of the radix aconiti lateralis preparata, and has very important scientific significance for the separation and purification, the structural analysis and the biological activity evaluation of the polysaccharide.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: the prepared radix aconiti lateralis preparata homogeneous polysaccharide is classified according to molecular weight, further separated and purified, and subjected to structural characterization, so that the prepared radix aconiti lateralis preparata homogeneous polysaccharide with medicinal value and polysaccharide prepared by condensing monosaccharide molecules is provided, and preliminary research on antioxidant activity and neuroprotective activity is performed on the prepared radix aconiti lateralis preparata homogeneous polysaccharide.

The technical scheme adopted by the invention for solving the technical problems is as follows: a radix Aconiti Szechenyiani homogeneous polysaccharide with molecular weight of 1.41 × 10⁵Da and consists of rhamnose, mannose, fucose and glucose.

A preparation method of uniform polysaccharide of radix aconiti lateralis preparata comprises the following steps:

step 1: heating, condensing and refluxing the dried stir-fried radix aconiti lateralis preparata decoction pieces for 2-3 times by using 5-8 times of ethanol, and degreasing to obtain dregs of a decoction;

step 2: extracting the dregs of a decoction with 5-8 times of water for 2-3 times at 80-100 ℃ for 1.5-2 h each time, concentrating the water extract under reduced pressure, adding absolute ethyl alcohol until the alcohol content reaches 80-85%, standing for 18-24 h, removing the supernatant, and collecting the precipitate to obtain crude polysaccharide of radix aconiti lateralis preparata;

and step 3: redissolving the crude polysaccharide of the radix aconiti lateralis preparata, adding pepsin until the content of the pepsin is 0.1-0.4%, carrying out constant-temperature water bath for 10-12 h at the temperature of 32-37 ℃, removing precipitates, repeating the steps for 2-3 times, collecting an upper solution, concentrating, adding a Sevage reagent, violently oscillating for 20-30 min, standing for 8-12 h, collecting an upper polysaccharide solution, and repeating the steps until no protein characteristic absorption peak is generated by ultraviolet scanning;

and 4, step 4: concentrating the upper-layer polysaccharide solution, adding 4-6 times of 95-100% ethanol, standing for 18-24 h, collecting precipitate, repeating for 2-3 times, and freeze-drying to obtain crude polysaccharide powder of radix aconiti lateralis preparata;

and 5: dissolving the crude polysaccharide powder of the radix aconiti lateralis preparata by using distilled water completely, and separating by using a DEAE FAST FLOW ion chromatographic column under the elution condition: eluting with water, 0.05mol/L sodium chloride, 0.1mol/L sodium chloride, 0.2mol/L sodium chloride, 0.3mol/L sodium chloride and 0.1mol/L sodium hydroxide in sequence at a flow rate of 2-2.5 ml/min; gradient collection is carried out by using a full-automatic collector, each solution is subjected to gradient elution with 2-3 times of column volume, 25-30 tubes are collected, 3-5 ml of each tube is subjected to tube partitioning tracking detection by adopting a sulfuric acid-phenol method, the collected solution of the radix aconiti lateralis preparata polysaccharide is concentrated to a certain volume, dialysis is carried out by using a 3500Da molecular weight dialysis belt, the dialysis belt needs to be pretreated, a beaker filled with the dialysis belt is changed with water 3-5 times per day, after one week of dialysis, polysaccharide liquid in the dialysis belt is centrifuged, and the polysaccharide liquid is freeze-dried, so that primarily purified radix aconiti preparata polysaccharide is obtained;

step 6: concentrating the collected eluent, separating by using a Superdex G-200 gel column, eluting by using distilled water at a flow rate of 0.15-0.2 ml/min, collecting by using a full-automatic collector, wherein each tube is 3-5 m1, and collecting a main peak part in an elution curve after detecting by using a phenol-sulfuric acid method;

and 7: concentrating the solution of main peak part in the collected elution curve, dialyzing with 3500Da dialysis bag for 2d for desalination, and finally concentrating, freeze drying the solution in the dialysis bag to obtain the radix Aconiti Preparata homogeneous polysaccharide.

Furthermore, in the Sevage reagent, the ratio of chloroform to n-butanol is 5: 1.

The application of the prepared radix aconiti lateralis preparata homogeneous polysaccharide in preparing an antioxidant product or a neuroprotective active product.

Further, hydroxypropyl methylcellulose, carboxymethyl starch sodium, microcrystalline cellulose, lactose, medicinal starch, magnesium stearate and the processed radix aconiti lateralis uniform polysaccharide are mixed and stirred to prepare a tablet which is an antioxidant product or a nerve protection active product.

Further, mixing microcrystalline cellulose, medicinal starch and the processed radix Aconiti Szechenyiani homogeneous polysaccharide, stirring, and making into tablet, which is antioxidant crystal-producing or neuroprotective active product.

Further, mixing microcrystalline cellulose, medicinal starch and radix Aconiti Preparata polysaccharide, stirring, and filling into hard capsule to obtain capsule, which is antioxidant product or neuroprotective product.

The invention has the beneficial effects that: the prepared radix aconiti lateralis preparata homogeneous polysaccharide with the molecular weight of 1.41 multiplied by 105Da and consisting of rhamnose, mannose, fucose and glucose is obtained by extracting by a water extraction and alcohol precipitation method and adopting schemes of further separating and purifying by an ion exchange DEAE FASTFLOW chromatographic column and a Superdex-200 gel chromatographic column, the yield is high, the obtained radix aconiti lateralis preparata homogeneous polysaccharide has antioxidant activity and neuroprotective activity, can remove DPH free radicals and ABST +, has strong reducing power determination, can be used for preparing products with antioxidant activity and neuroprotective activity, and has a certain application prospect.

Drawings

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the elution of a crude Palmaria inquinans polysaccharide fraction through a DEAE FAST FLOW ion chromatography column;

FIG. 2 is a HPGPC gel chromatogram of Pacific Sagittaria homogeneous polysaccharide;

FIG. 3 is a GC-MS. total ion chromatogram of radix Aconiti Szechenyiani homopolysaccharide; note: (left → right)

FIG. 4 is a 1R diagram of Pao Tian Xiong homopolysaccharide;

FIG. 5 is a 1H spectrum of Pao Tian Xiong homopolysaccharide; note: (left → right)

FIG. 6 is a 13C spectrum of Palmaria kawakamii homopolysaccharide. Note: (left → right)

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Radix Aconiti lateralis Preparata homogeneous polysaccharide with molecular weight of 1.41 × 10⁵Da and consists of rhamnose, mannose, fucose and glucose.