阅读说明：本技术 一种山慈菇均一多糖及其应用 (Pseudobulbus cremastrae seu pleiones homogeneous polysaccharide and application thereof ) 是由 李晓军 张雪 于 2020-07-17 设计创作，主要内容包括：本发明公开了一种山慈菇均一多糖及其应用,S1、取干燥山慈菇,先对干燥的山慈菇饮片进行乙醇脱脂处理,脱过脂的山慈菇饮片进行水提醇沉操作,低温烘干,除去其中乙醇,制得山慈菇多糖粗品,S2、对步骤S1中获得的山慈菇多糖粗品用水溶解后上氯型阴离子交换柱,本发明涉及天然药物技术领域。该山慈菇均一多糖及其应用,通过从山慈菇中提取分离得到山慈菇均一多糖,并对其结构进行详细的鉴定,同时山慈菇均一多糖能够显著性提高RAW264.7细胞的免疫活性,同时,具有很好的抗氧化活性,有望成为预防和治疗免疫低下病人的免疫调节剂,同时本发明制备方案简单,可以快速得到高纯度的均一多糖,适合于规模化生产。(The invention discloses a pseudobulb uniform polysaccharide and application thereof, S1, taking dry pseudobulb, firstly carrying out ethanol degreasing treatment on dry pseudobulb decoction pieces, carrying out water extraction and alcohol precipitation operation on the degreased pseudobulb decoction pieces, drying at low temperature, removing ethanol in the pseudobulb decoction pieces to prepare a pseudobulb polysaccharide crude product, S2, dissolving the pseudobulb polysaccharide crude product obtained in the step S1 with water, and then feeding the pseudobulb polysaccharide crude product to a chlorine type anion exchange column. The iphigenia indica homogeneous polysaccharide and the application thereof are characterized in that the iphigenia indica homogeneous polysaccharide is obtained by extracting and separating from the iphigenia indica, the structure of the iphigenia indica homogeneous polysaccharide is identified in detail, meanwhile, the iphigenia indica homogeneous polysaccharide can remarkably improve the immunocompetence of RAW264.7 cells, and meanwhile, the iphigenia indica homogeneous polysaccharide has good antioxidant activity and is expected to become an immunomodulator for preventing and treating patients with low immunity.)

1. The iphigenia indica homogeneous polysaccharide is characterized in that: the preparation method specifically comprises the following steps:

s1, taking dry edible tulip, degreasing the dry edible tulip decoction pieces with ethanol, carrying out water extraction and alcohol precipitation on the degreased edible tulip decoction pieces, drying at low temperature, and removing ethanol to obtain an edible tulip polysaccharide crude product;

s2, dissolving the crude product of the edible tulip polysaccharide obtained in the step S1 with water, then loading the solution on a chlorine-type anion exchange column, eluting with water, tracking an elution curve by a sulfuric acid-phenol method, collecting a sugar-containing solution, concentrating, dialyzing by a 8000Da dialysis bag, and drying to obtain the aqueous-phase edible tulip polysaccharide;

s3, dialyzing the water-phase polysaccharide obtained in the step S2 with a dialysis bag of 8000Da, then freeze-drying to obtain the iphigenia indica water component polysaccharide, and performing 8000Da membrane ultrafiltration on the collected water elution component to obtain the iphigenia indica water component polysaccharide;

s4, dissolving the edible tulip water component polysaccharide obtained in the step S3 with 0.1-0.3mol/L NaCl solution, centrifuging, taking supernate to pass through a 0.22 mu m filter membrane, loading, eluting with 0.1-0.3mol/L NaCl solution, detecting by using a differential detector, collecting and concentrating according to peak shapes, and dialyzing by using a 1000Da dialysis bag to obtain the uniform edible tulip polysaccharide.

2. The homogeneous polysaccharide of Pseudobulbus Cremastrae Seu pleiones according to claim 1, wherein: the step S1 includes the following specific operations: the preparation method comprises the steps of carrying out ethanol reflux degreasing on dried edible tulip decoction pieces, drying residues, adding water, heating and extracting for 2-5 times, combining extracted liquid medicines, concentrating, carrying out high-speed centrifugation, collecting supernate and carrying out alcohol precipitation.

3. The homogeneous polysaccharide of Pseudobulbus Cremastrae Seu pleiones according to claim 2, wherein: the mass fraction of ethanol used for degreasing is 45-95%, the heating extraction temperature is 100 ℃, and the extraction time is 2-4 hours each time.

4. The homogeneous polysaccharide of Pseudobulbus Cremastrae Seu pleiones according to claim 1, wherein: and (3) when carrying out alcohol precipitation in the step S1, adding 90-100% ethanol in a mass fraction of 4 times of the volume of the mixture, stirring the mixture until the mass fraction of the final ethanol is 75-85%, and standing the mixture for 4-48 hours.

5. The homogeneous Pseudobulbus Cremastrae Seu pleiones polysaccharide of any one of claims 1-4, wherein: the application of the iphigenia indica homopolysaccharide as an active ingredient in cell immune regulation.

Technical Field

The invention relates to the technical field of natural medicines, in particular to a pseudobulbus cremastrae seu pleiones homogeneous polysaccharide and application thereof.

Background

Pseudobulbus Cremastrae Seu pleiones is dried pseudobulb of Pleiones bulbocodioides of Orchidaceae. The edible tulip is originally appeared in the herb Shigao and belongs to a few common medicines in Chinese pharmacopoeia. Sweet in taste, slightly pungent and cold in flavor, entering liver and spleen channels, and has good effects of clearing heat and removing toxicity, eliminating carbuncle and dissipating stagnation. As a conventional Chinese medicine, studies on the iphigenia indica polysaccharide have been reported.

The Bulbus Iphigeniae Indicae contains protocatechuic acid, succinic acid, gastrodin, carotene, and polysaccharide. The research on the polysaccharide mainly focuses on the fact that the iphigenia indica polysaccharide has the most antitumor activity on liver cancer mice H22 and breast cancer mice 4T1, and the characteristics of carbuncle elimination and stagnation elimination of the traditional Chinese medicine are met. In addition, the edible tulip polysaccharide extract has good inhibition effect on various tumors such as gastric cancer, colorectal cancer, brain cancer and the like. The content of the edible tulip polysaccharide in the total compound is the highest. However, current research is limited to only crude polysaccharides, or extracts. The content of the structural research is less, and no research report on the homogeneous polysaccharide obtained by extracting and purifying the iphigenia indica and the chemical research of the homogeneous polysaccharide is reported so far.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a pseudobulbus cremastrae seu pleiones homogeneous polysaccharide and application thereof, so as to widen the application range of the pseudobulbus cremastrae seu pleiones.

In order to achieve the purpose, the invention is realized by the following technical scheme: the preparation method of the iphigenia indica homogeneous polysaccharide specifically comprises the following steps:

s1, taking dry edible tulip, degreasing the dry edible tulip decoction pieces with ethanol, carrying out water extraction and alcohol precipitation on the degreased edible tulip decoction pieces, drying at low temperature, and removing ethanol to obtain an edible tulip polysaccharide crude product;

s2, dissolving the crude product of the edible tulip polysaccharide obtained in the step S1 with water, then loading the solution on a chlorine-type anion exchange column, eluting with water, tracking an elution curve by a sulfuric acid-phenol method, collecting a sugar-containing solution, concentrating, dialyzing by a 8000Da dialysis bag, and drying to obtain the aqueous-phase edible tulip polysaccharide;

s3, dialyzing the water-phase polysaccharide obtained in the step S2 with a dialysis bag of 8000Da, then freeze-drying to obtain the iphigenia indica water component polysaccharide, and performing 8000Da membrane ultrafiltration on the collected water elution component to obtain the iphigenia indica water component polysaccharide;

s4, dissolving the edible tulip water component polysaccharide obtained in the step S3 with 0.1-0.3mol/L NaCl solution, centrifuging, taking supernate to pass through a 0.22 mu m filter membrane, loading, eluting with 0.1-0.3mol/L NaCl solution, detecting by using a differential detector, collecting and concentrating according to peak shapes, and dialyzing by using a 1000Da dialysis bag to obtain the uniform edible tulip polysaccharide.

Preferably, the step S1 includes the following specific operations: the preparation method comprises the steps of carrying out ethanol reflux degreasing on dried edible tulip decoction pieces, drying residues, adding water, heating and extracting for 2-5 times, combining extracted liquid medicines, concentrating, carrying out high-speed centrifugation, collecting supernate, and carrying out alcohol precipitation to obtain the edible tulip polysaccharide crude product.

Preferably, the mass fraction of ethanol used for degreasing is 45-95%, and the material proportion (g/mL) during degreasing is 1: 4-1: 25, the reflux degreasing time is 1-24 hours, the heating extraction temperature is 100 ℃, and each extraction time is 2-4 hours.

Preferably, in the alcohol precipitation in the step S1, 90-100% by mass of ethanol is added in 4 times of volume, and after stirring, the mixture is left standing for 4-48 hours until the final mass fraction of ethanol is 75-85%.

Preferably, the use of the iphigenia indica homopolysaccharide as an active ingredient in cellular immune modulation.

The homogeneous edible tulip polysaccharide can be various, and can be prepared into dosage forms which can enable the effective activity of the edible tulip polysaccharide to reach the in vivo, such as oral liquid, tablets, capsules, granules and the like.

Advantageous effects

The invention provides a homogeneous edible tulip polysaccharide and application thereof. Compared with the prior art, the method has the following beneficial effects: the iphigenia indica homogeneous polysaccharide and the application thereof are characterized in that the iphigenia indica homogeneous polysaccharide is obtained by extracting and separating from the iphigenia indica, the structure of the iphigenia indica homogeneous polysaccharide is identified in detail, meanwhile, the iphigenia indica homogeneous polysaccharide can remarkably improve the immunocompetence of RAW264.7 cells, and meanwhile, the iphigenia indica homogeneous polysaccharide has good antioxidant activity and is expected to become an immunomodulator for preventing and treating patients with low immunity.

Drawings

FIG. 1 is a chromatogram of a gel separation column of water-eluted Pseudobulbus Cremastrae Seu pleiones polysaccharide of example 1;

FIG. 2 is a high performance gel chromatogram of the Pseudobulbus Cremastrae seu pleiones homopolysaccharide of example 2;

FIG. 3 is an IR spectrum of the homogeneous polysaccharide of Pseudobulbus Cremastrae Seu pleiones of example 3;

FIG. 4 is a monosaccharide composition spectrum of the Pseudobulbus Cremastrae Seu pleiones homopolysaccharide of example 4;

FIG. 5 is a methylation diagram of the homogeneous polysaccharide of Pseudobulbus Cremastrae Seu pleiones of example 5;

FIG. 6 is a carbon spectrum analysis chart of the homogeneous polysaccharide of Pseudobulbus Cremastrae Seu pleiones of example 6;

FIG. 7 is a graph showing that the homogenous polysaccharide of Pseudobulbus Cremastrae seu pleiones of example 7 increases the nitric oxide release in RAW264.7 cells;

FIG. 8 is a graph showing that the homopolysaccharide of Pseudobulbus Cremastrae Seu pleiones of example 7 increases the amount of TNF α released from RAW264.7 cells;

FIG. 9 is a graph showing the DPPH radical scavenging ability of the Pseudobulbus Cremastrae seu pleiones homopolysaccharide of example 8;

FIG. 10 is a graph showing the measurement of the iron ion reducing ability of the homogeneous polysaccharide of Pseudobulbus Cremastrae Seu pleiones of example 8.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1-10, a homogeneous polysaccharide of edible tulip and the application thereof specifically include the following embodiments: