CN111777689A

CN111777689A - Pseudobulbus cremastrae seu pleiones homogeneous polysaccharide and application thereof

Info

Publication number: CN111777689A
Application number: CN202010689163.0A
Authority: CN
Inventors: 李晓军; 张雪
Original assignee: Yangzhou Borui Sugar Biotechnology Co ltd
Current assignee: Yangzhou Borui Sugar Biotechnology Co ltd
Priority date: 2020-07-17
Filing date: 2020-07-17
Publication date: 2020-10-16

Abstract

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.

Description

Pseudobulbus cremastrae seu pleiones homogeneous polysaccharide and application thereof

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:

example 1

Preparation and identification of uniform polysaccharide of edible tulip

S1, taking dry edible tulip 1KG, adding 5L of water, extracting for 3 times under the boiling condition, adding ethanol with the volume being 4 times that of the mixture, carrying out alcohol precipitation for 24 hours, filtering, collecting precipitates, drying the precipitates, removing residual ethanol, and preparing an edible tulip polysaccharide crude product;

s2, taking 10g of prepared polysaccharide crude product, adding 100mL of distilled water for dissolving, loading the polysaccharide crude product on a chlorine type anion exchange resin DEAE-Fastflow chromatographic column, eluting by using distilled water, detecting sugar content by using a sulfuric acid-phenol method, detecting at 490nm of an enzyme labeling instrument to obtain an elution curve, collecting a sugar-containing solution, drying, dialyzing for 24 hours by using a dialysis bag of 8000Da, and freeze-drying to obtain the iphigenia indica water-phase polysaccharide;

s3, dissolving the obtained edible tulip water component polysaccharide with 0.2mol/L NaCl solution, centrifuging, taking supernate, filtering with a 0.22 mu m filter membrane, loading, eluting with 0.2mol/L NaCl solution sodium chloride solution, detecting by using a differential detector, collecting and concentrating according to a peak shape (shown in figure 1), and dialyzing by adopting a 1000Da dialysis bag to obtain the uniform edible tulip polysaccharide, namely SCGW for short.

Example 2

Purity and molecular weight determination

Adopting high performance liquid gel chromatography (HPGPC), wherein a chromatographic column is BRT105, 104 and 102, a detector is a differential detector, a mobile phase is 0.05mol/L sodium chloride solution, the column temperature is 40 ℃, the flow rate is 0.6mL/min, the sample injection volume is 20uL, precisely weighing a sample and a standard substance, preparing the sample into a 5mg/mL solution, centrifuging for 10min at 12000rpm, filtering supernatant by using a 0.22 mu m microporous filter membrane, transferring the sample into a 1.8mL sample injection small bottle, performing sample injection with the sample injection amount of 20 mu L, and making a standard curve by using glucans (Mw1152, 5220, 11600, 23800, 48600, 80900, 148000, 273000, 409800 and 667800) with different relative molecular masses as standard substances to determine the purity and the relative molecular mass of the polysaccharide;

calibration curves were obtained for lgMp-RT (peak molecular weight), lgMw-RT (weight average molecular weight), lgMn-RT (number average molecular weight):

the lgMp-RT calibration curve equation is: y-0.1878 x +12.155 (R)²＝0.9961)；

The lgMw-RT calibration curve equation is: y-0.2002 x +12.75 (R)²＝0.9948)；

The lgMn-RT calibration curve equation is: y-0.1855 x +11.987 (R)²＝0.9926)；

According to the standard curve, a calculation formula is obtained to calculate the molecular weight of each sample, a high performance liquid gel chromatogram (HPGPC) of the iphigenia indica polysaccharide is obtained in figure 2, a single symmetrical peak is shown in the figure and is indicated as a uniform polysaccharide, and the peak molecular weight of the SCGW polysaccharide is calculated by the formula to be 384.6kDa, the weight average molecular weight is 555.4kDa and the number average molecular weight is 314.4 kDa.

Example 3

Infrared analysis

Analyzing functional groups of polysaccharide through FT-IR, precisely weighing 2mg of sample and 200mg of potassium bromide, pressing into tablets, tabletting by adopting potassium bromide powder as blank control, and respectively placing in a Fourier transform infrared spectrometer FT-IR650 for scanning and recording;

the results are shown in FIG. 3, the infrared results for the polysaccharide fraction. The absorption band is a stretching vibration absorption peak of-OH at 3600-3200cm-1, and the absorption peak in the region is a characteristic peak of the saccharide. The method comprises the following specific steps: 3369cm-1 is the stretching vibration absorption peak of-OH, which is a characteristic peak of saccharides. The absorption peak at 2929cm-1 is weak and is attributed to C-H stretching vibration of polysaccharide, the absorption peak at 1606cm-1 is probably asymmetric stretching vibration of C ═ O, the absorption peak at 1407cm-1 is attributed to absorption peak caused by variable angle vibration of C-H, and the absorption peak in the range of 1200 and 1000cm-1 is C-O-C stretching vibration, which indicates that the component is pyranose ring.

Example 4

Monosaccharide composition analysis

Monosaccharide composition analysis by reduction hydrolysis: collecting homogeneous polysaccharide sample 1-2mg, adding 3mL 2M TFA solution, hydrolyzing in capped test tube at 120 deg.C for 120min, taking out, rotary steaming until no sour taste, adding 50mg NaBH₄And 1mL of water, overnight, and a small amount of glacial acetic acid was added to remove excess NaBH₄And (3) performing rotary evaporation to form a viscous state, and adding methanol: 3mL of glacial acetic acid (5: 1), 3-5 times, adding methanol, spin-drying for 2-3 times, placing in an oven at 105 ℃, drying for 10min, adding 2mL of acetic anhydride, reacting in the oven for 1.5 h, adding 2mL of water to stop the reaction, adding 2mL of chloroform for extraction, washing for 2-3 times, drying with anhydrous sodium sulfate, and analyzing by using a Shimadzu GCMS-QP2010 gas chromatography-mass spectrometer to determine an acetylation product sample; GC-MS conditions are RXI-5SIL MS chromatographic column 30 x 0.25, programmed temperature rising conditions are initial temperature of 120 ℃, temperature rising is carried out at 3 ℃/min to 250 ℃/min, and the temperature is maintained5min, the temperature of the injection port is 250 ℃, the temperature of the detector is 250 ℃/min, the carrier gas is helium, and the flow rate is 1 mL/min. Wherein the standard substance comprises rhamnose, fucose, arabinose, xylose, mannose, glucose, and galactose;

and (3) detection results: GC-MS result analysis of the iphigenia indica homopolysaccharide shows 2 peaks, and comparison with standard monosaccharide proves that the homopolysaccharide consists of mannose and glucose.

Example 5

And (3) polysaccharide methylation analysis: placing the polysaccharide sample in a reaction bottle, adding DMSO, quickly adding NaOH powder, sealing, dissolving under the action of ultrasound, and adding methyl iodide for reaction. And finally adding water into the mixture to terminate the methylation reaction, taking the methylated polysaccharide, hydrolyzing the methylated polysaccharide with 1ml of 2M trifluoroacetic acid for 90min, and evaporating to dryness by using a rotary evaporator. Adding 2mL of double distilled water into residues, reducing 60mg of sodium borohydride for 8 hours, adding glacial acetic acid for neutralization, performing rotary evaporation, drying in a 101-DEG oven, adding 1mL of acetic anhydride for acetylation at 100 ℃, reacting for 1 hour, cooling, adding 3mL of toluene, performing reduced pressure concentration and evaporation, and repeating for 4-5 times to remove redundant acetic anhydride. The acetylated product was dissolved in 3mL of chloroform and transferred to a separatory funnel, and after adding a small amount of distilled water and shaking sufficiently, the upper aqueous solution was removed, and this was repeated 4 times. The chloroform layer was dried over an appropriate amount of anhydrous sodium sulfate, the volume was adjusted to 10mL, and the acetylation product sample was analyzed by using Shimadzu GCMS-QP2010 GC-MS. GC-MS conditions: RXI-5SIL MS chromatography column 30 × 0.25; the temperature programming conditions are as follows: the initial temperature is 120 ℃, the temperature is increased to 280 ℃/min at 4 ℃/min, the temperature is kept for 5min, the temperature of a sample inlet is 250 ℃, the temperature of a detector is 250 ℃/min, carrier gas is helium, and the flow rate is 1 mL/min;

the results were analyzed in order of appearance and by methylation analysis, 5 glycosidic linkages were obtained for the Pseudobulbus Cremastrae Seu pleiones polysaccharide (see table): 2, 3, 4, 6-Me₄-Glcp；2，3，4，6-Me₄-Manp；2，3， 6-Me3-Manp；2，3，6-Me3-Glcp；2，3-Me₂Manp and 2, 3-Me₂-Glcp. Its corresponding linkage is Glcp- (1 →, Manp- (1 →, → 4) -Glcp- (1 →, → 4, 6) -Manp- (1 → and → 4, 6) -Glcp- (1 →.The molar ratio is 0.59: 0.85: 58.51: 39.48: 0.33: 0.24. the polysaccharide is shown to be probably composed of glucan with a main chain of → 4) -Glcp- (1 → 4) -Manp- (1 → and more information needs to be further determined by analytical NMR.

Example 6

Nuclear magnetic carbon spectrum analysis

A sample of polysaccharide was weighed at 50mg, dissolved in 0.5ml of heavy water and freeze-dried. And then dissolving the freeze-dried powder in 0.5ml of heavy water, continuously freezing and drying, and repeating the processes to fully exchange active hydrogen. The sample is then dissolved in 0.5ml of heavy water and subjected to Nuclear Magnetic Resonance (NMR) measurement at 500MHz at 25 ℃ at room temperature¹³C NMR spectrum. The results show that: the signals of C1, C2, C3, C4, C5 and C6 of Beta-Manp-1-4 are 102.02, 72.04, 73.51, 78.39, 77.13 and 62.30ppm respectively, while the signals of C1, C2, C3, C4, C5 and C6 of Beta-Glcp-1-4 are 104.45, 74.82, 75.94, 80.45, 77.13 and 62.30ppm respectively. From the above, it can be concluded that the Pseudobulbus Cremastrae Seu pleiones polysaccharide is mainly glucomannan composed of Beta mannose 1-4 and Beta glucose 1-4, and has a small amount of branching.

Example 7

Immunity regulation experiment of Pseudobulbus Cremastrae Seu pleiones homogeneous polysaccharide

1. The immune activity of the iphigenia indica polysaccharide is screened through a mouse abdominal cavity macrophage RAW264.7 cell experiment. The detailed steps are as follows: recovering macrophage in abdominal cavity of mouse, culturing and passaging. Then, the cells were seeded in DMEM medium at a cell concentration of 5x 10⁴Cell mass/mL of cell fluid was cultured in 96-well plates for 8 hours, and then the cells were treated with various concentrations of Pseudobulbus Cremastrae Seu pleiones polysaccharide solution (0-200. mu.g/mL), incubated for 24 hours, and then 20ul of MTT solution (5mg/mL) was added to each well. After 4 hours of incubation, the supernatant was discarded, and then 100ul of dimethylsulfoxide solution was added. And shaking the mixture on a shaking table for 15 minutes in a dark condition, measuring the absorbance at 570nm by using a microplate reader, and calculating the content of the nitric oxide. The result obviously improves the nitric oxide release amount of RAW264.7 cells;

2. screening of Pseudobulbus Cremastrae Seu pleiones by mouse abdominal cavity macrophage RAW264.7 cell experimentImmunological activity of the polysaccharide. The detailed steps are as follows: resuscitating macrophage in abdominal cavity of mouse, culturing, subculturing, and then planting cells with DMEM medium at cell concentration of 5x 10⁴2mL of cell solution is taken and placed in a 6-well plate to be cultured for 8 hours, then the tulip polysaccharide solution with different concentrations is adopted to treat the cells (0-200 mug/mL), the cells are incubated for 24 hours, and a TNF α kit is adopted to measure RAW264.7 cells, so that the release amount of a tumor necrosis factor TNF α of the RAW264.7 cells is obviously improved.

Example 8

Experiment of antioxidant activity of Pseudobulbus Cremastrae Seu pleiones homogeneous polysaccharide

1. Determination of DPPH radical scavenging Capacity: preparing polysaccharide sample solution (0.25-2.0mg/ml) with deionized water, placing 30 μ L sample solution in 96-well plate, taking solvent water as blank control, adding 180 μ L DPPH solution (methanol is prepared to be 0.05mM) respectively, mixing uniformly at room temperature in dark for 30min, measuring absorbance value at 517nm wavelength, and preparing ascorbic acid solution as positive control;

the clearance calculation formula is:

A_sis the absorbance value of the sample, A₀Is a blank control group, A_jIs a background control group;

the result shows that the iphigenia indica homogeneous polysaccharide can remove DPPH free radicals, has an antioxidant effect, and has a removal rate of about 40% when the concentration of the polysaccharide reaches 8 mg/ml;

2. determination of iron ion reducing ability (FRAP)

Preparing a polysaccharide sample solution (0.25-2.0mg/ml) by using a 0.1M PBS (pH6.6) mixed solution, taking 200 mu L of the sample solution, adding 200 mu L of potassium ferricyanide (water is prepared to be 1% w/v), carrying out a water bath reaction at 50 ℃ for 20min, cooling, adding 200 mu L of trifluoroacetic acid (water is prepared to be 10% w/v), mixing, centrifuging at 3000rpm for 10min, taking 100 mu L of supernatant, placing the supernatant into a 96-well plate, adding 10 mu L of ferric trichloride solution (water is prepared to be 0.1% w/v), carrying out a reaction for 2min, and carrying out absorbance determination on the obtained Prussian blue solution under the condition of 700nm, wherein FRAP activity (%) is calculated as follows:

wherein A is_sampleIs the absorbance of the sample, A_AA(plateau)Is the maximum value at which the absorbance of ascorbic acid reaches equilibrium. Aj is background control;

the result shows that the polysaccharide has iron ion reducing capacity, and when the concentration reaches 4mg/ml, the reduction rate of the polysaccharide on iron ions reaches about 50 percent.

In summary, the following steps: the edible tulip uniform polysaccharide provided by the invention has the advantages of well promoting NO and TNF alpha secretion of RAW264.7 cells, and simultaneously has the effects of eliminating DPPH free radical scavenging capacity and reducing iron ions. Is expected to become a good immunomodulator, and the development of a new preparation is particularly important for expanding the clinical application of the edible tulip polysaccharide. The preparation method is simple, can obtain the high-purity uniform Pseudobulbus Cremastrae Seu pleiones polysaccharide, is high in purity, is suitable for large-scale production, and is good in practicability.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

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

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.