CN114478815A

CN114478815A - Preparation and application of curcuma zedoary polysaccharide nCKAP-2

Info

Publication number: CN114478815A
Application number: CN202210126413.9A
Authority: CN
Inventors: 董彩霞; 陶宁; 姜思亮; 秦志海
Original assignee: Tianjin Medical University
Current assignee: Tianjin Medical University
Priority date: 2022-02-10
Filing date: 2022-02-10
Publication date: 2022-05-13

Abstract

The invention discloses a zedoary turmeric polysaccharide nCKAP-2, a preparation method and an application thereof, wherein crude zedoary turmeric polysaccharide nCKCP is obtained by water extraction and alcohol precipitation from Guangxi zedoary turmeric medicinal materials, the crude zedoary turmeric polysaccharide nCKAP-2 is obtained after separation and purification, the molecular weight is 76.4-214kDa, and monosaccharide composition of the crude zedoary turmeric polysaccharide nCKAP-2 contains a large amount of Ara, Xyl and Gal and a small amount of Man, Rha, Glc, GalA and GlcA. The curcuma zedoary polysaccharide nCKAP-2 is extracted from curcuma zedoary, is a natural polysaccharide, has the characteristics of low toxicity, high efficiency, safety and easy acceptance, and provides a research basis for further developing antitumor drugs with novel action mechanism, definite curative effect and definite target; the curcuma zedoary polysaccharide nCKAP-2 prepared by the method can induce MSC2 cells to undergo apoptosis in the G0/G1 stage, so that the effect of inhibiting T cell proliferation is reduced, and the curcuma zedoary polysaccharide nCKAP-2 can be used for preparing antitumor drugs.

Description

Preparation and application of curcuma zedoary polysaccharide nCKAP-2

Technical Field

The invention belongs to the field of natural medicinal chemistry, and particularly relates to preparation and application of curcuma zedoary polysaccharide nCKAP-2.

Background

Polysaccharides are one of the most important biomacromolecules in life, and participate in processes such as cell recognition, biological information carrying and transmission, metabolism and secretion, immune response regulation, protein transfer and the like. Polysaccharides have been faced with significant challenges in terms of structural complexity, including diversity of saccharide units, various linkages between saccharide units, and different configurations of saccharide rings, as compared to nucleosomes and proteins. The biological activity of polysaccharides has been receiving much attention because the complex and diverse sugar chain structures can carry more abundant information. Curcuma kwangsiensis S.G.Lee et C.F.Liang is a Curcuma plant of angiospermaceae, widely distributed in Guangxi, Sichuan and Vietnam, and often grown in sunny, soil-wet, and thick ditch edge, forest edge and hillside land, and is fond of high temperature and high humidity environment and drought resistance; the rhizome of the curcuma zedoaria is used as the traditional Chinese medicine, and is one of three basic sources of the traditional Chinese medicine curcuma zedoary.

Myeloid-derived suppressor cells (MDSCs) are a group of immature myeloid cells, have the ability to suppress innate and adaptive immune responses, and in healthy individuals, MDSCs differentiate rapidly after being produced from bone marrow, but in pathological conditions cause immune system disorders, and MDSC differentiation is blocked, so that a large number of MDSCs accumulate in vivo to form a cell population capable of suppressing immune functions. The emergence of such immune function-suppressing cell populations represents a common feature of cancer and other non-cancerous diseases, such as sepsis, bacterial, viral and parasitic infections, autoimmune diseases, and the like. Meanwhile, MDSCs are also the cornerstone of the immunosuppressive barrier as the most important protective cells in the tumor microenvironment (full name in english, TME). Numerous preclinical experimental studies have shown that elimination or inhibition of MDSC function can improve the ability of the host immune system to attack tumor cells and improve the efficacy of immunotherapy, and thus the scientific and medical community has begun to focus on the development of methods for eliminating MDSC. There are many attempts to target MDSC against its immune mechanism, such as all-trans retinoic acid, the tyrosine kinase inhibitor sunitinib and sildenafil, but the safety of these drugs remains uncertain, so that the development of a natural, non-toxic and safe MDSC-targeting drug is imperative to replace or assist the existing drugs.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method and application of curcuma zedoary polysaccharide nCKAP-2.

The technical scheme adopted by the invention is as follows: a Curcumae rhizoma polysaccharide nCKAP-2 comprises Ara, Xyl and Gal, and has molecular weight of 76.4-214 kDa;

ara consists of t-, (1 → 3) -, (1 → 3,5) -and (1 → 2,3,5) -Araf residues;

xyl is composed of (1 → 2) -, (1 → 2,4) -and (1 → 2,3,4) -Xyl residues;

gal consists of t-, (1 → 3) -and (1 → 3,6) -Galp residues.

Preferably, one or more of Man, Rha and Glc are also included;

man consists of (1 → 3) and (1 → 4) -Manp residues;

rha consists of t-, (1 → 3) and (1 → 2) -Rhap residues;

glc consists of (1 → 4) -Glcp residues.

Preferably, the degree of branching is 0.72.

A method for preparing Curcumae rhizoma polysaccharide nCKAP-2 comprises extracting Curcumae rhizoma with water and precipitating with ethanol to obtain Curcumae rhizoma polysaccharide nCKAP-2.

Preferably, the specific steps are as follows:

reflux-extracting Curcumae rhizoma with ethanol, defatting to remove oligosaccharide, and collecting residue;

extracting the residue with water, filtering, concentrating, adding anhydrous ethanol, stirring, and collecting precipitate to obtain Curcumae rhizoma crude polysaccharide nCKCP;

separating and purifying with chromatographic column to obtain Curcumae rhizoma polysaccharide nCKAP-2.

Preferably, the crude zedoary turmeric polysaccharide nCKCP is sequentially subjected to DEAE-650M anion exchange resin column, Sepharose 6B gel chromatographic column and Sephacryl S-300HR gel chromatographic column to obtain the zedoary turmeric polysaccharide nCKAP-2.

Preferably, the crude polysaccharide nCKCP of the curcuma zedoary is dialyzed to remove substances with the kDa of less than 7.0 before being purified by chromatography.

The crude polysaccharide nCKCP containing the polysaccharide nCKAP-2 of the zedoary is prepared by the method.

Application of Curcumae rhizoma polysaccharide nCKAP-2 or Curcumae rhizoma crude polysaccharide nCKCP in preparing antitumor drug is provided.

Preferably, the zedoary turmeric polysaccharide nCKAP-2 or zedoary turmeric crude polysaccharide nCKCP inhibits the immunosuppressive function of MDSC.

The invention has the advantages and positive effects that: the natural polysaccharide extracted from the curcuma zedoary has low toxicity, high efficiency, safety and easy acceptance, and provides a research basis for further developing antitumor drugs with novel action mechanism, definite curative effect and definite target; the structure of the polysaccharide determines the biological activity and function of the polysaccharide, and the curcuma zedoary polysaccharide nCKAP-2 prepared by the method can induce the cell apoptosis of MSC2 cells in the G0/G1 stage, thereby reducing the effect of inhibiting the proliferation of T cells.

Drawings

FIG. 1 shows the process for extracting crude polysaccharide from Curcumae rhizoma;

FIG. 2 shows the high performance liquid gel chromatography of zedoary turmeric polysaccharide;

FIG. 3 Total sugar Standard Curve;

FIG. 4 uronic acid standard curve;

FIG. 5 protein standard curve;

FIG. 6 PMP-HPLC chromatogram of monosaccharide composition;

1. mannose; 2. rhamnose; 3. glucuronic acid; 4. galacturonic acid; 5. glucose; 6. galactose; 7. xylose; 8. arabinose; 9. fucose;

FIG. 7 nCKAP-2 Infrared Spectroscopy;

FIG. 8 effect of nCKAP-2 on growth of splenocytes;

FIG. 9 the curcumene polysaccharide nCKAP-2 relieves the inhibitory effect of MSC2 on T cells;

FIG. 10 nCKAP-2 induces MDSC apoptosis;

FIG. 11 nCKAP-2 induced MSC2 cell cycle arrest at stage G0/G1;

FIG. 12 nCKAP-2 up-regulates the expression level of MSC2 apoptotic protein;

FIG. 13 nCKAP-2 induced apoptosis of MSC 2;

FIG. 14 nCKAP-2 upregulates TLR-4 mRNA levels in MSC 2;

FIG. 15 shows changes in protein levels of nCAKP-2 acting on the receptor and signaling pathway molecules of MSC 2;

figure 16 AKT inhibitors can partially block nCKAP-2 induced apoptosis of MSC 2;

FIG. 17 nCKAP-2 reduces ROS production levels by MSC 2.

Detailed Description

Embodiments of the present invention will be described below with reference to the accompanying drawings.

The method extracts crude curcuma zedoary polysaccharide (nCKCP) from curcuma kwangsiensis by a water extraction and alcohol precipitation method, and then separates and purifies the nCKCP by DEAE-650M, Sephrose 6B and Sephacryl S-300HR to obtain a main polysaccharide component nCKAP-2 with uniform molecular weight and charge. The uniformity and molecular weight of the polysaccharide are determined by High Performance Gel Permeation Chromatography (HPGPC), the total sugar content and the uronic acid content are respectively determined by a sulfuric acid-phenol method and a m-hydroxybiphenyl method, and the analysis shows that the relative molecular weight of the curcuma zedoary polysaccharide nCKAP-2 is 76.4-214kDa, and in addition, a small amount of uronic acid and protein are contained.

PMP derivatization is combined with an HPLC method to determine monosaccharide composition of nCKAP-2, and methylation, hydrolysis, acetylation, GC-MS, FT-IR and other methods are combined to characterize chemical structures of polysaccharides so as to judge glycosidic bond connection modes of the polysaccharides. The monosaccharide composition results show that all three polysaccharides contain large amounts of Ara, Xyl, Gal, and small amounts of Man, Rha, Glc, GalA, GlcA. Wherein Ara is a main component consisting of t-, (1 → 3) -, (1 → 3,5) -and (1 → 2,3,5) -Ara f residues; xyl is the residue of (1 → 2) -, (1 → 2,4) -and (1 → 2,3,4) -Xyl p; gal consists of t-, (1 → 3) -and (1 → 3,6) -Gal p residues. Man consists of (1 → 3) and (1 → 4) -Man p residues; rha consists of t-, (1 → 3) and (1 → 2) -Rha p residues; glc consists of (1 → 4) -glcp residues. The Branching Degree (BD) was calculated according to the formula BD ═ NT + NB)/(NT + NB + NL), and BD for nCKAP-2 was 0.72.

Firstly extracting effective components from curcuma zedoaria medicinal materials by water extraction and alcohol precipitation, removing substances below 7.0kDa by dialysis to obtain curcuma zedoaria crude polysaccharide nCKCP, and then separating and purifying the curcuma zedoaria crude polysaccharide nCKCP by a DEAE-650M anion exchange resin column, a Sepharose 6B gel chromatographic column and a Sephacryl S-300HR gel chromatographic column in sequence to obtain the curcuma zedoaria polysaccharide nCKAP-2

As shown in FIG. 1, the specific preparation method of the zedoary turmeric crude polysaccharide (nCKCP) in one embodiment of the present invention can be as follows:

the method comprises the following steps: weighing 2kg of Guangxi rhizoma zedoariae medicinal decoction pieces, shearing, washing with water, removing silt, reflux-extracting with 75% ethanol for 2h, repeating for three times, degreasing to remove oligosaccharide, obtaining dregs, drying in the shade to remove alcohol smell;

step two: extracting the above residue with water, placing the residue in a 30L barrel, adding 10kg pure water, heating to 100 deg.C, boiling, maintaining slight boiling for 2 hr, and repeatedly extracting for 3 times;

step three: removing residues, mixing filtrates, concentrating to 3L, adding anhydrous ethanol at a ratio of 1:4, stirring, shaking, standing overnight, removing upper clear ethanol solution, mixing the suspension, centrifuging at 5000rpm for 15min, recovering ethanol solution, scraping precipitate, dissolving in pure water again, and repeating the operation for 1 time

Step four: dialyzing the water solution (circulating running water) for 36h, removing substances below 7.0kDa, lyophilizing, and storing to obtain Curcumae rhizoma crude polysaccharide nCKCP.

Separating and purifying the obtained crude polysaccharide nCKCP of the curcuma zedoary to obtain polysaccharide nCKAP-2 of the curcuma zedoary, wherein the specific preparation method comprises the following steps:

step five: weighing 1.5g of crude polysaccharide of Curcumae rhizoma, dissolving in 50mL of pure water, stirring thoroughly to dissolve, centrifuging (6000rpm, 20min) twice, removing insoluble substances, and loading 40mL of supernatant onto DEAE-650M anion exchange resin column (4.4 × 20 cm). After the sample is completely adsorbed, the sample is eluted by 1.5L of pure water (the flow rate is about 0.5mL/min), collected by an automatic partial collector, and is positive through sulfuric acid-phenol detection, so that the sample is proved to contain neutral polysaccharide. Then eluting with 0.5 and 1.0mol/L NaCl solution in sequence, eluting with 0.2mol/L NaOH solution, detecting by a sulfuric acid-phenol method, eluting until the detection is negative, and respectively collecting eluates. Measuring protein at 280nm, measuring absorbance at 490nm by sulfuric acid-phenol method, mixing according to elution curve, concentrating, dialyzing, and lyophilizing;

step six: separating component 1.0g with DEAE-650M, adding pure water, centrifuging at 4500rpm for 2 times, each time for 15min, collecting supernatant, placing into Sepharose 6B gel chromatographic column, eluting with 0.L mol/L NaCl, collecting eluate, measuring protein at 280nm, measuring absorbance at 490nm by sulfuric acid-phenol method, mixing according to elution curve, concentrating, dialyzing, and lyophilizing;

step seven: separating sample components by Sephacryl S-300HR, respectively taking 300mg, adding pure water, centrifuging at 3500rpm for 15min, taking supernatant, loading the supernatant to a Sephacryl S-300HR gel chromatographic column which is completely filled with the supernatant, eluting by 0.l mol/L NaCl, collecting eluent, measuring protein at 280nm, detecting the absorbance at 490nm by a sulfuric acid-phenol method, combining according to an elution curve, concentrating, dialyzing, and freeze-drying to obtain the zedoary turmeric polysaccharide nCKAP-2.

MDSC is the main immunosuppressive cell in the tumor microenvironment, and polysaccharide and MDSC have close drug effect correlation, so that the elimination of MDSC immunosuppression is the research hotspot of antitumor drugs. The invention discloses a traditional anti-cancer traditional Chinese medicine with definite curative effect, which takes zedoary polysaccharide as a research object to explore MDSC maturation differentiation induction and mediated immunosuppression function and molecular action mechanism reversion. The curcuma zedoary polysaccharide nCKAP-2 or the curcuma zedoary crude polysaccharide nCKCP disclosed by the invention can be used for preparing antitumor drugs.

The following describes the scheme of the present invention with reference to the accompanying drawings, wherein experimental methods without specific description of operation steps are all performed according to corresponding commercial specifications, and instruments, reagents and consumables used in the examples can be purchased from commercial companies without specific description.

Example 1: extraction of curcuma zedoary crude polysaccharide nCKCP

Weighing 2kg of Guangxi rhizoma zedoariae medicinal decoction pieces, shearing, washing with water, removing silt, reflux-extracting with 75% ethanol for 2h, repeating for three times, degreasing to remove oligosaccharide, obtaining residue, drying in the shade to remove alcohol smell, extracting with water, placing the residue in a 30L bucket, adding 10kg of pure water, heating to 100 deg.C, boiling, keeping slight boiling for 2h, and repeatedly extracting for 3 times. Removing residues, mixing filtrates, concentrating to 3L, adding anhydrous ethanol at a ratio of 1:4, stirring, shaking, standing overnight, removing upper clear ethanol solution, mixing the suspension, centrifuging at 5000rpm for 15min, recovering ethanol solution, scraping out precipitate, adding pure water, dissolving again, and repeating for 1 time. Dialyzing the water solution (circulating running water) for 36h to remove substances below 7.0kDa, lyophilizing, and storing to obtain Curcumae rhizoma crude polysaccharide nCKCP 16.94g with extraction rate of about 0.85%.

Example 2: preparation of curcuma zedoary polysaccharide nCKAP-2

1.5g of the crude zedoary turmeric polysaccharide prepared in example 1 was weighed, dissolved in 50mL of pure water, sufficiently stirred and dissolved, centrifuged (6000rpm, 20min) twice, insoluble matter was removed, and 40mL of the supernatant was applied to a DEAE-650M anion exchange resin column (4.4X 20 cm). After the sample is completely adsorbed, the sample is eluted by 1.5L of pure water (the flow rate is about 0.5mL/min), collected by an automatic partial collector, and is positive through sulfuric acid-phenol detection, so that the sample is proved to contain neutral polysaccharide. Then eluting with 0.5 and 1.0mol/L NaCl solution in sequence, eluting with 0.2mol/L NaOH solution, detecting by a sulfuric acid-phenol method, eluting until the detection is negative, and respectively collecting eluates. Measuring protein at 280nm, measuring absorbance at 490nm by sulfuric acid-phenol method, combining according to elution curve, concentrating, dialyzing, and lyophilizing.

Separating component 1.0g with DEAE-650M, adding pure water, centrifuging at 4500rpm for 2 times, each time for 15min, collecting supernatant, placing into Sepharose 6B gel chromatographic column, eluting with 0.L mol/L NaCl, collecting eluate, measuring protein at 280nm, measuring absorbance at 490nm by sulfuric acid-phenol method, mixing according to elution curve, concentrating, dialyzing, and lyophilizing.

Separating sample components by Sephacryl S-300HR, adding 300mg of pure water, centrifuging at 3500rpm for 15min, collecting supernatant, loading to Sephacryl S-300HR gel chromatographic column with complete filling balance, eluting with 0.l mol/L NaCl, collecting eluate, measuring protein at 280nm, detecting absorbance at 490nm by sulfuric acid-phenol method, mixing according to elution curve, concentrating, dialyzing, and lyophilizing to obtain homogeneous polysaccharide nCKAP-2 with yield of 3.9%

Example 3: analysis of physicochemical Properties of Zedoariae rhizoma polysaccharide nCKAP-2

3.1 homogeneity analysis and molecular weight determination

Taking pullulan series as standard substances (with molecular weights of 642, 337, 194, 107, 47.1, 21.1, 9.6 and 6.1kDa respectively), respectively taking 5mg, dissolving with ultrapure water, preparing 5mg/mL pullulan series standard solution, and filtering with 0.22 μm filter membrane. 10 μ L of each standard solution was taken and subjected to HPGPCDetermination by CH₃COONH₄The solution is eluted by a mobile phase. With retention time a as ordinate and molecular weight C as abscissa, a standard curve a ═ 1.392C +19.516, R2 ═ 0.9976 was plotted.

Collecting separated Curcumae rhizoma polysaccharide samples 5mg, dissolving with pure water to obtain 5mg/mL sample solution, filtering with 0.22 μm filter membrane, measuring with HPGPC, and measuring with CH₃COONH₄The solution is eluted by a mobile phase. The uniformity and molecular weight of each component of the zedoary turmeric polysaccharide are determined by the retention time of each component of the zedoary turmeric polysaccharide. As shown in FIG. 2, nCKAP-2 exhibits a smooth and symmetrical single peak pattern as measured by HPGPC, indicating a homogeneous polysaccharide; calculated by a pullulan series standard curve, the relative molecular weight of the polysaccharide is as follows: 127.1 KDa.

A plurality of parts of zedoary turmeric polysaccharide nCKAP-2 are additionally extracted according to the preparation methods of examples 1 and 2, and the relative molecular weight distribution of the nCKAP-2 is respectively measured to be 76.4-214 KDa.

3.2 Total sugar content determination

And (3) measuring the total sugar content of the sample by adopting a phenol-sulfuric acid method. And (3) standard substance: α -D-galactose, reagent a: phenol, prepared as a 50g/L (5%, W/V) aqueous solution; and (3) reagent B: concentrated sulfuric acid; standard solution: completely dissolving 10mg of alpha-D-galactose in pure water, and metering to 10mL, wherein the concentration is 200 mu g/mL. The standard curve was prepared according to Table 2-1. 0.2mL of alpha-D-galactose standard substance is respectively taken, 0.2mL of 5% phenol solution is added, l mL of concentrated sulfuric acid is added into the solution, the solution is fully shaken, and the absorbance is measured at 490 nm.

TABLE 1 table for preparing standard solution for measuring total sugar content

Measuring absorbance A at 490nm as ordinate, and standard solution concentration C as abscissa, drawing standard curve, and obtaining standard curve equation of y ═ 0.0097x-0.0033, R as shown in FIG. 3²The results showed that the total sugar concentration was in a linear relationship of 0 to 200. mu.g/mL, as high as 0.9999.

3.3 measurement of uronic acid content by m-hydroxybiphenyl method

The content of uronic acid is determined by m-hydroxybiphenyl method. Preparing 1mg/mL galacturonic acid standard solution by using alpha-D-galacturonic acid as a standard, sequentially diluting to 100, 20, 10, 5, 2.5 and 1.25 mu g/mL series of concentrations, respectively sucking 200 mu L galacturonic acid standard, adding sulfuric acid-sodium tetraborate solution, carrying out ice bath for 5min, heating in water bath for 5min, cooling in ice bath, adding 20 mu L m-hydroxybiphenyl solution (20 mu L NaOH solution is added in a blank group), fully shaking, standing for 5min, and measuring absorbance at 520 nm. And drawing a standard curve.

Samples are prepared into sample solution which is diluted to 50 mu g/mL, after color development is carried out according to the method, the absorbance is measured at 520nm and is substituted into a standard curve, and the uronic acid content of the samples is calculated. Drawing a standard curve by taking the light absorption value A as an ordinate and the concentration C of the standard solution as an abscissa, and obtaining a result shown in figure 4, wherein the equation of the standard curve is that Y is 0.0104X +0.0122, and R is²The results of 0.9842 show that the linear relationship is good when the uronic acid concentration is 0-150. mu.g/mL.

3.4 protein content determination

The BCA trace protein concentration determination kit is adopted for protein content determination. Preparing a working solution according to a standard reagent A, B, C, 25, 2 and 1 of the kit; the concentration of BSA standard protein solution was 40. mu.g/mL. The sample solution was prepared as a 100. mu.g/mL solution. Setting 2 repeated groups for each concentration of the prepared BSA working solution and sample solution, setting 500 mu L of each tube, adding 500 mu L of BCA working solution into each tube, immediately mixing uniformly, incubating at 37 ℃ for 1h, cooling to room temperature, and measuring the light absorption value at 562nm by a spectrophotometer.

TABLE 2 table for preparing standard solution for measuring protein content

With A₅₆₂And nm is an ordinate, the concentration C of the standard solution is an abscissa, a standard curve is drawn, the result is shown in fig. 5, the obtained standard curve equation is that Y is 0.0152X-0.0495, and R2 is 0.999, and the result shows that when the protein content is between 0 and 20 mu g/mL, the linear relation is good, and the method can be used for determining the protein content in polysaccharide.

After the determination of the total sugar content, the uronic acid content and the BCA trace protein content by the kit, nCKAP-2 has 78.04% of total sugar, 16.82% of uronic acid and 9.11% of protein, and the results are shown in Table 3.

TABLE 3 chemical composition analysis of zedoary turmeric polysaccharide

Example 4: the structural characteristics of the zedoary turmeric polysaccharide nCKAP-2

4.1 FT-IR analysis of polysaccharides

Taking a zedoary turmeric polysaccharide sample of 2mg, adding 100mg of dry KBr, placing the mixture into a drying oven to dry, transferring the mixture into an agate mortar to grind and mix, tabletting the mixture in a hydraulic press, and using an infrared spectrometer to prepare 400cm of pressed KBr tablets at 4000-400cm^-1Infrared scanning is performed within the area.

The results are shown in FIG. 7, where the infrared spectrum of polysaccharide nCKAP-2 is 3500-3000cm^-1The absorption peak at (a) is a typical hydroxyl radical oscillation peak. nCKAP-2 at 3325.11cm^-1Has an obvious absorption peak, which is the absorption caused by the O-H stretching vibration in or between sugar molecules. 2930.60cm^-1The nearby peak is a stretching vibration absorption peak of-C-H in methine group peculiar to saccharide, 1608.20cm^-1The nearby absorption peak is a C-O stretching vibration peak; 1413.18cm^-1The nearby absorption peak indicates the presence of pyranoside. 1039.05cm^-1The absorption peak at the position is narrow and sharp, and is an asymmetric oscillation peak of glycosidic bond C-O-C.

4.2 preparation of PMP derivatives

(1) Standard preparation and derivatization

Accurately weighing monosaccharide standards (Fuc, Xyl, Rha, Ara, Man, Gal, Glc, GlcA and GalA) 1mg respectively, dissolving in deionized water, adding 0.1mL respectively into a test tube, shaking uniformly, preparing mixed standard, taking 0.3mL, adding 0.6mol/L NaOH solution and 0.6mL 0.5mol/L PMP-methanol solution with the same volume. After mixing evenly, the mixture is reacted for 30min in a metal bath at 70 ℃ in a dark place. After the reaction, it was cooled to room temperature and neutralized with HCl. Extracting with chloroform, centrifuging for 4-5 times, and discarding chloroform layer. The cells were filtered through a microfiltration membrane (0.22 μm) and transferred to an HPLC vial.

(2) Preparation of samples

A1 mg sample of polysaccharide was taken and dissolved in 0.3mL of deionized water. Adding 0.3mL of 4mol/L TFA solution, heating and hydrolyzing at 120 deg.C for 2h, water bath at 40 deg.C, and N₂And (5) drying by distillation. Dissolving with 0.3mL of deionized water, transferring to a screw cap reaction tube, adding an equal volume of 0.6mol/L NaOH solution, adding 0.6mL of 0.5mol/L PMP methanol solution, mixing uniformly, reacting in a metal bath at 70 ℃ in a dark place for 30min, and cooling to room temperature. HCl was added to adjust to neutrality. The other steps are the same as the above method.

(3) Chromatographic conditions

Isocratic elution, 83% of mobile phase A (ammonium acetate buffer solution), 17% of mobile phase B (acetonitrile), total flow rate of 1mL/min, detection wavelength of 245nm, sample injection amount of 5 mu L and column temperature of 30 ℃.

As shown in fig. 6, the elution order of the mixed standard chromatogram from left to right is: mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, fucose. The monosaccharide composition analysis results of the zedoary turmeric homopolysaccharide are shown in Table 4. As can be seen from the table, nCKAP-2 is composed of a large amount of arabinose, xylose, and galactose, and a small amount of mannose, rhamnose, glucose, galactose, and galacturonic acid, and the monosaccharide composition results of the polysaccharide are substantially identical to the uronic acid content results measured by the m-hydroxybiphenyl method.

TABLE 4 monosaccharide composition analysis of zedoary turmeric polysaccharide samples

4.3 methylation analysis

The composition of the homogeneous polysaccharide glycosidic bond is analyzed according to a series of steps of methylation, hydrolytic reduction, acetylation, etc. based on the monosaccharide composition to estimate the composition of the main chain and side chain.

Taking a dried sample of the zedoary turmeric polysaccharide 2mg in a micro reaction kettle with a stirrer, adding DMSO, sealing a bottle mouth, fully stirring and dissolving, quickly grinding dry NaOH in a mortar into fine powder, drying in an oven at 70 ℃ to ensure that the NaOH fine powder is free of moisture, and adding the fine powder into the micro reaction kettle to fully stir for 2 hours. And (3) carrying out ice water bath for 10min, slowly adding 450 mu L of methyl iodide in three times, reacting for 15min each time, and continuously stirring for 1.5h to fully react. After the reaction was completed, pure water was added under ice bath to terminate the reaction, and the reaction mixture was transferred to a reaction tube and charged with N₂Blowing off unreacted methyl iodide in the solution, adding equal volume of chloroform to extract the solution, keeping a chloroform layer, adding five times of pure water to repeatedly extract, centrifuging and discarding a water layer. Collecting chloroform layer, and placing in fume hood with N₂And (5) drying.

Hydrolysis of the fully methylated polysaccharides. 1mL of 2M TFA was added to the reaction flask, hydrolyzed at 120 ℃ for 4h in a metal bath, allowed to stand to room temperature, and added with N₂Blowing the solvent, adding methanol to wash the reaction bottle, and adding N₂The methanol was blown dry. To the reaction flask was added ammonia, and about 5mg of NaDB was added₄Reacting the powder in a metal bath at 40 ℃ for 50 min. And after the reaction is finished, adding 10% acetic acid-methanol solution to terminate the reaction, transferring the solution into a heart-shaped bottle, evaporating the solvent to dryness, adding anhydrous methanol to dissolve the sample in the bottle, and performing spin-drying on the solvent for 5 times to ensure that the acetic acid in the solution is completely removed. 1.5mL of acetic anhydride and 50. mu.L of 1-methylimidazole were added thereto, and acetylation reaction was carried out at room temperature. After 15min, the solution was transferred to a reaction tube and the reaction was terminated by adding pure water under ice bath. Adding equal amount of chloroform to extract the reaction solution, collecting and combining chloroform layers, adding pure water to repeatedly extract for five times, and discarding a water layer. The remaining water in the solution was removed with anhydrous sodium sulfate, concentrated to 0.5mL, and transferred to a liquid phase bottle for GC-MS measurement.

GC-MS sample injection conditions are as follows: the carrier gas is He₂Split ratio 40:1, detector temperature: 280 ℃ and a column temperature of 120 ℃ to 280 ℃. Determining a mass spectrum according to the retention time of the sample in the meteorological chart: and taking the peak area ratio, and calculating the content ratio of the sample fragment molecules to the total molecular weight.

The glycosidic linkage pattern of the three purified polysaccharides is summarized in Table 5.

TABLE 5 methylation analysis results of the zedoary turmeric polysaccharide nCKAP-2

From GC-MS analysis of methylated derivatives of nCKAP-2, it can be seen that Ara is the predominant component, consisting mainly of t-, (1 → 3) -, (1 → 3,5) -and (1 → 2,3,5) -Araf residues. And Xyl exists mainly in the form of (1 → 2) -, (1 → 2,4) -and (1 → 2,3,4) -Xyl residues. Gal exists in the form of t-, (1 → 3) -and (1 → 3,6) -Galp. In addition, there are very small numbers of t-, (1 → 3) and (1 → 2) -Rhap, (1 → 3) and (1 → 4) -Manp, and (1 → 4) -Glcp residues. The Branching Degree (BD) was calculated according to the formula BD ═ NT + NB)/(NT + NB + NL), with a BD of nCKAP-2 of 0.72, indicating that nCKAP-2 is highly branched.

Example 5 inhibitory Effect of zedoary turmeric polysaccharide nCKAP-2 on MDSC immune function

5.1 MTT assay

In order to determine whether the curcuma zedoary polysaccharide has a direct killing effect on T cells, an MTT method is adopted to detect the cytotoxic effect. Spleen cells were plated at 5X 10³Density per well was seeded in 96-well plates and incubated with 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 1mg/mL CKAP-2 for 24 h. mu.L of MTT reagent was added to each well and incubated for 4 h. Adding triple reagent (10% sodium dodecyl sulfate, 5% isobutanol, 0.012mol/L HCl, dissolved in distilled water) to dissolve the purple crystal for 4-6h, and measuring OD₅₇₀。

As shown in FIG. 8, the survival rate of splenocytes was not only not decreased but increased with increasing concentration of nCKAP-2 administered. This indicates that the polysaccharide of Curcuma kwangsiensis has stimulating activity to splenocytes and is dose dependent.

5.2 flow cytometry

5.2.1 Effect of nCKAP-2 on MDSC-mediated T cell suppression

T cell proliferation was measured with the intracellular dye CFSE. Splenocytes were isolated from BALB/c mice and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 ℃ in 5% CO₂Was incubated in a humidified incubator for 4 h. The suspension cells were then harvested, labeled with 1M CFSE at 25 ℃ for 2min, and then quenched in 10% FBS-RPMI 1640 medium. CFSE-labeled splenocytes were co-cultured with MSC2 cells and stimulated with 1. mu.g/mL ConA at a ratio of 100: 3. After 3 days, the CFSE signal of the gated lymphocytes was analyzed by flow cytometry. Antibodies APC-anti-CD4 and PE-anti-CD8 α, diluted in 2% PBS.

As shown in fig. 9, CD4⁺When the T cells are in a resting state, the proliferation peak ratio is low; the content of the protein is only 1.46 percent, and the proliferation peak ratio is increased to 27.5 percent after 1 mu g/mL concanavalin ConA stimulation; after adding MSC2 serving as MDSC cell line into the culture system, CD4⁺The T cell proliferation peak decreased to only 3.24%; CD4 was found after adding different concentrations (0.01, 0.05, 0.1, 0.3 and 0.5mg/mL) of curdlan nCKAP-2 to the culture system⁺The T cell proliferation peak ratio was restored to some extent and presented a dose-effect relationship (fig. 9A). CD8⁺T cells showed consistent results (fig. 9B).

5.2.2 nCKAP-2 Induction of MDSC apoptosis assay

MSC2 as 5 x 10⁵Cells/well density were seeded into 96-well plates and incubated overnight. Then, nCKAP-2(0.05, 0.1, 0.2mg/mL) with different concentrations was added for treatment. After an additional 24h incubation, cells were harvested and resuspended in 500. mu.L of 1 XPBS conjugated with Annexin V, the suspension was quickly blown up, followed by the addition of 5. mu.L Annexin V-FITC and 5. mu.L PI and incubation in the dark at 37 ℃ for 10 min. MSC2 was previously treated with 1. mu.g/mL CLI-095, TLR-2, and TLR-6 blocking agent for 6h, and then stimulated with CKAP-2. FACS Caliber measures the percentage of cell staining and samples were analyzed by Flowjo7.6 and Graph Pad Prism 5.0.

As shown in fig. 10, the results show that nCKAP-2 can induce MDSCs to undergo apoptosis, and as the concentration is increased, the inhibition effect is enhanced, the percentage of early apoptotic cells is increased from 2.49% to 14.2% (0.2mg/mL), the total apoptosis rate is increased from 8.32% to 33.8% (0.2mg/mL), and all show a dose-effect relationship. This result suggests that MDSC apoptosis is one of the cellular mechanisms by nCKAP-2 to relieve MDSC inhibition of T cell proliferation.

5.2.3 cell cycle Change in MDSC after nCKAP-2 treatment

MSC2 was treated with varying concentrations of nCKAP-2(0.05, 0.1, 0.2mg/mL) for 24h, and the cells were then slowly transferred to 1.2mL of 70% cold ethanol at 4 ℃ and vortexed and fixed overnight. After washing the cells, 100. mu.L of RNase A reagent was added, incubated in a water bath for 30min, and then stained with propidium iodide for 30min in the dark. Fluorescence was measured by FACS Caliber and samples were analyzed by flowjo7.6 and Graph board prism 5.0.

As shown in FIG. 11, the ratio of cell numbers in different cell cycles of MSC2, the cell number in G0/G1 phase, gradually increased with the increase of the administration concentration, when the administration concentration reaches 0.2mg/mL, the cell ratio in G0/G1 phase is increased from 65.9% to 85.6%, and correspondingly, the ratio in S/G2 phase gradually decreased with the increase of the administration concentration, which indicates that the nCKAP-2 induced MDSC cell cycle is blocked in G0/G1 phase and presents a dose-effect relationship.

5.3 Western blot experiment

MSC2 was treated with 0.2mg/mL nCKAP-2 for various periods of time (0,0.5,1,2,4h) and then lysed with RIPA solution to give total protein. The RIPA solution was supplemented with 100mM phenylmethylsulfonyl fluoride, 25. mu.g/mL aprotinin, 1mM sodium orthovanadate and 50nM NaF. Protein samples (30. mu.g/sample) were separated under denaturing conditions on a 10% sodium dodecyl sulfate polyacrylamide gel and then electrotransferred to a nitrocellulose membrane at 100V for 70 min. The membrane was then blocked with 3% BSA in PBS-Tween (0.1% Tween-20) for 1.5h and incubated with diluted primary antibody overnight at 4 ℃. After washing with PBS-T, horseradish peroxidase-conjugated goat anti-rabbit IgG was used as a secondary antibody at a dilution of 1: 3000. After complete washing, the membrane was incubated with the chemiluminescent substrate for 3 min. The specific bands are visualized using a chemiluminescent imaging system. GAPDH was used as an internal standard and the antibody dilution ratio was 1: 3000.

The expression level of apoptotic proteins involved in this process was studied by WB. As a result, as shown in FIG. 12, Bcl-xl gradually decreased with time, while the expression levels of clear Caspase 3, clear Caspase 9 and clear PARP proteins were up-regulated, showing time dependence. Since Caspase-9, Bax and Bcl-xl are endogenous apoptotic molecules, the type of apoptosis induced by nCKAP-2 in MDSCs is likely to be endogenous. There was no significant change in ERK levels, indicating that it was not involved in this process.

5.4 nCKAP-2 on MDSC Signal Path study

Screening the receptor and signal path protein level of nCKAP-2 acting on MSC2 by using a western blot method, and the specific operation is 3.2.5. MSC2 cells were then seeded in 96-well plates at a density of 5 × 10⁵Cells/well, incubating overnight, adding TLR-2, TLR-4 and TLR-6 receptor blocking agents into each well respectively, pretreating for 6h, then adding 0.2mg/mL nCKAP-2 to induce MSC2 cells to generate apoptosis, collecting the cells after incubating for 24h, suspending the cells in 500 muL of 1 XPBS and Annexin V binding solution, blowing the suspension, adding 5 muL of Annexin V-FITC and 5 muL of PI, and incubating for 10min at 37 ℃ in the dark. FACS Caliber measures the percentage of cell staining and samples were analyzed by Flowjo7.6 and Graph Pad Prism 5.0.

The results are shown in fig. 13, after MDSCs are pretreated by different receptor blockers for 6h, nckiap-2 is added to induce MDSCs to undergo apoptosis, and the results show that the apoptosis rate of the MDSCs is 85.9% by using only nckiap-2 components, while the apoptosis rate of the MDSCs is 65.1% by using TLR-4 blocker components, but the effect is not achieved by using TLR2 and 6 blocker components, which indicates that nckiap-2 cannot be combined with MDSCs after TLR4 is blocked, so that the apoptosis rate is reduced, and therefore, a receptor antagonism experiment indicates that TLR-4 is a receptor of nckiap-2 for inducing MDSCs to undergo apoptosis.

Further analyzing by RT-PCR, treating with nCKAP-2 for different time, extracting total RNA of MSC2 by TRIzol enzyme, centrifuging sample cells to obtain cell precipitate, discarding supernatant, suspending cells by 50 μ L PBS, adding 1mL TRIzol reagent, and pipetting for 10 min; after shaking with 200. mu.L of chloroform for 30 seconds, the mixture was allowed to stand at room temperature for 5 min. Centrifuging, removing precipitate, transferring the clear solution to an EP tube, adding 400 μ L isopropanol, slightly inverting, mixing until no refraction exists in the liquid in the tube, and standing at room temperature for 20 min; the supernatant was discarded by centrifugation at 12000rpm, and the total RNA pellet was washed three times with 75% ethanol and quantified using an ND-1000 spectrophotometer.

Total RNA was reverse transcribed with PrimeScript RT Master Mix. The levels of GAPDH, TLR-2, TLR-4, TNF-. alpha.and TGF-. beta.were determined using SYBR Green II on Applied Biosystems 7500. The specific primer is GAPDH 5'-TGTTGCCATCAATGACCCCTT-3'; 5'-CTCACCGACGTACTAGCG-3' are provided. The structure is shown in FIG. 14, only the transcription level of TLR-4 is up-regulated, and further, the TLR-4 is confirmed to be nCKAP-2 acting on MDSC receptors.

To elucidate the signaling pathway of nCAKP-2 on MDSCs, the levels of related proteins downstream following TLR4 activation were determined. After 0.2mg/mL nCKAP-2 acts on MSC2 for different times (0, 1 and 2h), the expression level of TLR-4 is up-regulated, the level of a downstream NF-kB signaling pathway molecule P-P65 is also up-regulated, and P-P38 has no significant change, as shown in FIG. 15, the TLR 4-NF-kB signaling pathway is a molecular mechanism of nCAKP-2 acting on MDSC.

In this example, AKT blocking experiments were performed to determine whether AKT is involved in the process of inducing MDSC apoptosis by nCKAP-2. As shown in FIG. 16, compared with the PBS group, the apoptosis rate of nCKAP-2 was significantly increased, while the total apoptosis rate induced by nCKAP-2 was reduced from 41.1% to 25.4% after AKT inhibitor was used, indicating that AKT is involved in the apoptosis process of nCKAP-2 induced MDSC.

From the above results, it can be seen that nCKAP-2 can relieve the inhibition of T cell proliferation by inducing MDSC apoptosis, i.e., clearing MDSCs.

5.5 ROS production assay

The generation of Reactive Oxygen Species (ROS) was detected with a dichlorofluorescein diacetate fluorescent probe. At 37 ℃ and 5% CO₂Three groups of MSC2 (1X 10 per group) were cultured under the conditions of (1)⁶cells/mL). MSC2 was treated with nCKAP-2 for 6 h. Cells were resuspended in 1mL PBS containing DCFH-DA (10. mu.M) and incubated at 37 ℃ for 20min in the absence of light. Finally, after washing the residual DCFH-DA with PBS, the ROS production levels were analyzed by flow cytometry in the FITC channel at 488nm excitation and 525nm emission, similar to the fluorescence spectrum of DCF.

As shown in FIG. 17, after nCKAP-2 treatment, nCKAP-2 acted on MDSC, and the concentration gradually increased, the ROS level showed a downward regulation trend, while Arg-1 showed an upward trend. Therefore, down-regulation of reactive oxygen species levels may be one of the major mechanisms by which nCKAP-2 reduces the inhibition of T cell proliferation by MDSCs.

nCKAP-2 can stimulate mouse splenocyte proliferation, can reverse the effect of MDSC on T cell proliferation inhibition, and has gradually strengthened effect along with the increase of concentration, thus presenting a dose-effect relationship. nCKAP-2 can induce the cell apoptosis of MSC2 cells in the G0/G1 phase, thereby reducing the inhibition effect on T cell proliferation. The nCKAP-2 can reduce the Bcl-xl expression level of the MSC2 cells, and simultaneously increase the expression levels of the Cleaved Caspase 3, the Cleaved Caspase 9 and the Cleaved PARP proteins, and is time-dependent, which indicates that the nCKAP-2 can induce the endogenous apoptosis of the MSC 2. Western Blot, receptor blocking and other experimental results show that TLR-4 is a receptor for inducing MSC2 to undergo apoptosis by nCKAP-2 after nCKAP-2 is incubated; nCAKP-2 induces apoptosis of MSC2 cells mainly through TLR 4-NF-kB signaling pathway. AKT blocking agent experiment results show that AKT is also involved in nCKAP-2 induced MSC2 apoptosis. The ROS level of nCKAP-2 acting on the MDSC is detected to be remarkably reduced after the action, and the fact that the effect on the immunosuppressive function of the MDSC is also one of the action mechanisms of nCKAP-2 for reversing MDSC-induced T cell proliferation inhibition is suggested.

The embodiments of the present invention have been described in detail, but the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.

Claims

1. A zedoary turmeric polysaccharide nCKAP-2, which is characterized in that: including Ara, Xyl and Gal, and having a molecular weight of 76.4-214 kDa;

ara consists of t-, (1 → 3) -, (1 → 3,5) -and (1 → 2,3,5) -Araf residues;

xyl is composed of (1 → 2) -, (1 → 2,4) -and (1 → 2,3,4) -Xyl residues;

gal consists of t-, (1 → 3) -and (1 → 3,6) -Galp residues.

2. The curcuma zedoary polysaccharide nCKAP-2 of claim 1, wherein: further comprising one or more of Man, Rha, and Glc;

man consists of (1 → 3) and (1 → 4) -Manp residues;

rha consists of t-, (1 → 3) and (1 → 2) -Rhap residues;

glc consists of (1 → 4) -Glcp residues.

3. The curcuma zedoary polysaccharide nCKAP-2 of claim 2, wherein: the degree of branching was 0.72.

4. A process for the preparation of the curcuma zedoary polysaccharide nCKAP-2 according to any one of claims 1 to 3, characterized in that: the zedoary turmeric polysaccharide nCKAP-2 is obtained by extracting zedoary turmeric with water and precipitating with ethanol.

5. The method for preparing zedoary turmeric polysaccharide nCKAP-2 as claimed in claim 4, wherein: the method comprises the following specific steps:

6. The method for preparing zedoary turmeric polysaccharide nCKAP-2 as claimed in claim 5, wherein: sequentially passing the crude polysaccharide nCKCP of the curcuma zedoary through a DEAE-650M anion exchange resin column, a Sepharose 6B gel chromatographic column and a Sephacryl S-300HR gel chromatographic column to obtain the polysaccharide nCKAP-2 of the curcuma zedoary.

7. The method for preparing zedoary turmeric polysaccharide nCKAP-2 according to claim 5 or 6, characterized in that: before the chromatographic purification of the crude polysaccharide nCKCP of the curcuma zedoary, substances with the kDa below 7.0kDa are removed through dialysis.

8. A crude polysaccharide nCKCP of Curcumae rhizoma prepared by the method of claim 4, comprising the polysaccharide nCKAP-2 of Curcumae rhizoma as defined in any one of claims 1 to 3.

9. Use of the curzema zedoaria polysaccharide nCKAP-2 as claimed in any one of claims 1 to 3 or curzema zedoaria polysaccharide nCKCP as claimed in claim 8 in the preparation of antitumor drugs.

10. Use according to claim 9, characterized in that: the zedoary turmeric polysaccharide nCKAP-2 or zedoary turmeric crude polysaccharide nCKCP can inhibit the immunosuppressive function of MDSC.