CN110894244B

CN110894244B - Structure of ground beetle polysaccharide and application thereof

Info

Publication number: CN110894244B
Application number: CN201910987685.6A
Authority: CN
Inventors: 周长林; 沈雯; 丁佳露; 谢鑫; 郭建; 马菱蔓
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2019-10-17
Filing date: 2019-10-17
Publication date: 2021-06-11
Anticipated expiration: 2039-10-17
Also published as: CN110894244A

Abstract

The invention relates to a ground beetle polysaccharide structure and application thereof, the structure is identified by chemical analysis and spectral technology, and the main chain in the ESPS repeating unit structure mainly consists of → 4) -alpha-D-Glcp- (1 → glucose and → 3) -Galp- (1 → galactose, and the branched chain is connected on the main chain through the O-6 bond of glucose, the O-4 bond and the O-6 bond of galactose, namely the ESPS is a heteropolysaccharide with novel structure which is separated from the ground beetle for the first time. Meanwhile, the ESPS can stimulate the proliferation of splenic lymphocytes and can also promote the killing effect of lymphocytes and NK cells on various tumor cells, which shows that the polysaccharide ESPS has the effects of promoting immunity and resisting tumors.

Description

Structure of ground beetle polysaccharide and application thereof

Technical Field

The invention relates to a compound in animal traditional Chinese medicinal materials, in particular to eupolyphaga sinensis Walker polysaccharide (ESPS) with a backbone structure of galactoglucan, which consists of → 4) -alpha-D-Glcp- (1 → glucose and → 3) -Galp- (1 → galactose, and application thereof.

Background

The Eupolyphaga Seu Steleophaga polysaccharide is a natural animal polysaccharide extracted from traditional Chinese medicinal material Eupolyphaga Seu Steleophaga. According to records in Ben Cao gang mu, the ground beetle has the functions of breaking blood stasis, dissipating hard mass, resolving coagulation, treating blood, reuniting bones and muscles, relieving swelling and pain, and the like. The results of recent years show that the ground beetle is not only a precious traditional Chinese medicine material, but also contains multiple active natural ingredients such as anticoagulation, anti-tumor and the like.

Polysaccharides are a high molecular polymer which is present in a wide range in animals, plants, and microorganisms as a main component of living bodies and has the highest content in the natural world. With the development of medicine and cell biology techniques, researchers have conducted intensive research on polysaccharides in recent years. The polysaccharide has various biological activities, but has little toxicity to cell tissues, thereby showing very wide development prospect. In recent years, the research on the polysaccharide mainly focuses on the immunocompetence and the antitumor activity of the polysaccharide, and the polysaccharide also has research and research on various biological activities such as blood sugar reduction, blood fat reduction, oxidation resistance, virus resistance and the like. The action mechanism of the polysaccharide as an immunotherapy medicament is mainly focused on that the polysaccharide can promote the secretion of related immune cell factors, activate T cells, Natural Killer (NK) cells, Dendritic Cells (DC) and other related immune cells, and improve the immunity of a host so as to achieve the indirect anti-tumor effect. The polysaccharides approved for clinical application at home and abroad at present comprise lentinan, coriolus versicolor polysaccharide and the like, and the research range of the polysaccharides mainly focuses on the structural representation and biological function of the polysaccharides and applies the polysaccharides to the pharmaceutical industry. With the gradual and intensive research on the structure-activity relationship of polysaccharides, in order to find novel polysaccharides having good biological activity, the research on the biological activity of polysaccharide compounds has been rapidly developed.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide ground beetle polysaccharide which is heteropolysaccharide separated from ground beetles for the first time. The invention also aims to provide an extraction method of the polysaccharide. The invention also aims to indicate that the polysaccharide has remarkable immune enhancement and anti-tumor activity, particularly has good treatment effect on liver cancer and pancreatic cancer, and can be prepared into related medicaments.

The technical scheme is as follows: the ground beetle polysaccharide is characterized by consisting of rhamnose, fucose, arabinose, xylose, glucose and galactose according to a molar ratio of 7.4:3.1:13.9:9.3:39.7: 26.5; relative molecular weight is 2.14X 10⁴Da; knot with repeating unit of grape galactan main chain structureThe main chain consists of → 4) -alpha-D-Glcp- (1 → glucose and → 3) -Galp- (1 → galactose, and the branch chain is connected on the main chain through the O-6 bond of glucose, the O-4 bond and the O-6 bond of galactose, and the main chain unit structure and the branch chain segment are shown in figure 12.

The ground beetle polysaccharide contains pyranose ring.

The ground beetle polysaccharide is prepared by the following method:

1. a separation step: extracting Eupolyphaga Seu Steleophaga powder with water, removing protein, and precipitating with ethanol;

2. a purification step:

(1) taking a ground beetle polysaccharide crude product prepared by water extraction, deproteinization by combining Sevag reagent with papain and alcohol precipitation, fully dissolving the ground beetle polysaccharide crude product in deionized water, and eluting the polysaccharide crude product by using a well-balanced ion exchange column Cellulose DE-52;

(2) performing gel chromatography on the ground beetle polysaccharide water washing component obtained by separating the ion exchange column by using a Sephacryl S-300 molecular sieve column, eluting by using deionized water, collecting in parts, performing tracking detection by using a sulfuric acid phenol method, and combining the same components; and (5) freezing and drying to obtain the ground beetle polysaccharide.

The extraction method of the ground beetle polysaccharide comprises separation and purification, wherein the separation comprises the steps of water extraction, protein removal and ethanol precipitation of ground beetle powder, and is characterized in that the purification uses a column chromatography method, and specifically comprises the following steps:

(2) performing Sephacryl S-300 molecular sieve gel chromatography on the ground beetle polysaccharide water washing component obtained by separating the ion exchange column, eluting with deionized water, collecting fractions, performing tracking detection by a sulfuric acid phenol method, and mixing the same components; and (5) freezing and drying to obtain the ground beetle polysaccharide.

The application of the ground beetle polysaccharide in preparing an immunopotentiator.

The ground beetle polysaccharide is applied to the preparation of immune antitumor drugs.

The ground beetle polysaccharide is applied to the preparation of the medicine for treating liver cancer and pancreatic cancer.

The ESPS can obviously promote the proliferation of spleen lymphocytes under different concentrations (25-800 mu g/ml), and the effect is in certain dose dependence; and has no obvious cytotoxic effect on various normal cells at the concentration of 3.1-800 mu g/ml. The result of the effect-target ratio experiment shows that the ESPS can promote the killing effect of lymphocytes and NK cells on various tumors. In vivo experiments show that the ESPS has the effect of resisting H22 and Panc02 tumor, the spleen index and the thymus index are obviously increased compared with chemotherapy positive medicines, and Elisa experiments show that the content of IL-2 and IFN-gamma in the serum of a mouse can be obviously improved after the ESPS is administrated.

Has the advantages that: the Eupolyphaga sinensis polysaccharide ESPS is heteropolysaccharide with novel structure separated from Eupolyphaga sinensis. Meanwhile, the ESPS can stimulate the proliferation of splenic lymphocytes and can also promote the killing effect of the lymphocytes and NK cells on various tumors, which shows that the ESPS has the effects of promoting immunity and resisting tumors.

Drawings

FIG. 1 is a flow chart of the process for extracting Eupolyphaga Seu Steleophaga polysaccharide (ESPS) in the present invention;

FIG. 2 is a standard curve of ESPS molecular weight and a liquid phase spectrum according to the present invention;

FIG. 3 is a GC-MS spectrum of ESPS monosaccharide composition in accordance with the present invention;

FIG. 4 is an ESPS methylation analysis GC-MS profile of the present invention;

FIG. 5 is a UV spectroscopy spectrum of an ESPS in the present invention;

FIG. 6 is an infrared spectroscopic spectrum of an ESPS in the present invention;

FIG. 7 shows the ESPS of the present invention¹H NMR spectrum;

FIG. 8 shows the ESPS of the present invention¹³A C NMR spectrum;

FIG. 9 shows the ESPS of the present invention¹H-¹H COSY map;

FIG. 10 is an HSQC map of ESPS of the present invention;

FIG. 11 is a NOESY map of ESPS in the present invention;

FIG. 12 shows backbone repeating units and branched segments of ESPS according to the present invention;

FIG. 13 is a schematic representation of ESPS's promotion of splenic lymphocyte proliferation in accordance with the present invention;

FIG. 14 is a schematic representation of the in vitro growth of ESPS in accordance with the present invention on a variety of tumor cells as well as normal cells;

FIG. 15 is a schematic diagram showing the killing effect of ESPS-activated murine splenic lymphocytes on Yac-1, CT-26, Panc02 tumor cells in accordance with the present invention;

FIG. 16 is a graph showing the killing effect of ESPS-activated NK-92 on Panc-1, HepG2, A375, MCF-7 tumor cells in accordance with the present invention;

FIG. 17 is a graph showing the in vivo anti-tumor effect of ESPS in the present invention on H22 tumor-bearing mice;

FIG. 18 is a graph showing the in vivo anti-tumor effect of ESPS in the present invention on Panc02 tumor-bearing mice;

FIG. 19 is a schematic diagram showing that ESPS promotes secretion of IL-2 and IFN-gamma cytokines in serum of Panc02 tumor-bearing mice in the present invention.

Detailed Description

Example 1

1. Separation and purification process

Separation: (1) pulverizing Eupolyphaga Seu Steleophaga with a Chinese medicinal pulverizer, sieving with 40 mesh sieve, placing 500g of the collected Eupolyphaga Seu Steleophaga powder in 5L distilled water according to a ratio of 1:10(w/v), extracting at 90 deg.C for 3 hr, repeating for 3 times, mixing water extractive solutions for 15L, and concentrating under reduced pressure at 40 deg.C to 1L.

(2) Adding 10g of papain into the concentrated solution, and carrying out water bath at 60 ℃ for 6 h. Protein was removed by Sevag method 10-15 times. 1/5 volumes of a pre-prepared Sevag reagent (chloroform: n-butanol: 5:1) were added, the mixture was shaken on a shaker for 15min, centrifuged at 8000rpm for 15min, the supernatant was removed, and the above procedure was repeated until no protein precipitated at the interface between water and chloroform. The organic reagent was removed by concentration at 40 ℃ under reduced pressure and the volume of the solution was concentrated to 200 ml.

(3) Adding 1L of anhydrous ethanol precooled to 4 deg.C slowly into the concentrated solution without protein, standing overnight at 4 deg.C to precipitate polysaccharide, filtering with Buchner funnel, washing the precipitate with anhydrous ethanol for 2 times. The crude polysaccharide was dissolved in 50ml of distilled water, and freeze-dried to obtain 10.4g of crude polysaccharide.

And (3) purification: preparing 3g of crude polysaccharide into 50mg/mL aqueous solution, centrifuging, and separating supernatant with Cellulose DE-52 Cellulose ion exchange column (phi 4.5cm × 30 cm). Eluting with distilled water at flow rate of 2ml/min, collecting 10ml fractions in each tube, measuring sugar content tube by phenol-sulfuric acid method, drawing elution curve, collecting 32 nd-48 th tubes, mixing, and freeze drying to obtain 993mg of water-washed sugar. Dissolving 200mg water washing sugar in 4ml distilled water, filtering with 0.22 μm filter membrane, separating by Sephacryl S-300 column chromatography (phi 1.6cm × 120cm), eluting with distilled water at flow rate of 0.5ml/min, collecting 3ml fractions in each tube, detecting sugar content by sulfuric acid-phenol method tube by tube, drawing elution curve, collecting elution peak, freeze drying to obtain Eupolyphaga Seu Steleophaga polysaccharide, and freeze drying to obtain 23.4mg Eupolyphaga Seu Steleophaga polysaccharide (ESPS). The specific process is shown in figure 1.

2. Chemical structure identification

(1) Molecular weight

The ESPS and the standard glucan (Mw1152, 5200, 11600, 23800, 148000, 273000 and 410000) in the invention are precisely weighed, and a standard curve is made through high performance gel column chromatography (HPGPC) analysis, so that the purity and the relative molecular mass of the polysaccharide are determined. As can be seen from FIG. 2, the ESPS showed a peak at 30.299min, the uniformity was good, and the molecular weight of the ESPS was calculated to be 2.14X 10 in accordance with the standard curve in FIG. 2⁴。

(2) Monosaccharide composition

After ESPS in the invention is hydrolyzed into monosaccharide, corresponding sugar alcohol is reduced under the action of sodium borohydride, and corresponding sugar alcohol acetate derivative is obtained through acetylation. The hydrolysate of the ESPS contains seven major peaks by GC-MS analysis, the retention times of which are shown in FIG. 3, indicating that the ESPS consists of six monosaccharides. And (3) carrying out parallel operation on the monosaccharide standard, wherein the peak output time of the standard is consistent with the retention time of a main peak in a map of the sugar alcohol acetate, which indicates that the ESPS consists of rhamnose, fucose, arabinose, xylose, glucose and galactose according to the ratio of 7.4:3.1:13.9:9.3:39.7: 26.5. The results are shown in Table 1:

TABLE 1ESPS and 7 monosaccharide standards monosaccharide composition determination of retention time and ratio

(3) Methylation analysis

Weighing 8mg of ESPS in the invention into a reaction bottle, drying in vacuum, adding 3ml of DMSO into the dried sample, stirring until the polysaccharide sample is completely dissolved, adding dried 20mg of NaOH powder, sealing, dissolving under the action of ultrasound, dropwise adding 0.6ml of methyl iodide for reaction, gradually clarifying the solution, and finally adding water into the mixture to terminate the methylation reaction.

Dissolving the completely methylated sample in formic acid solution, sealing, hydrolyzing at 100 deg.C for 6 hr, evaporating to dryness under reduced pressure, adding methanol, evaporating to dryness, and repeating for three times to remove excessive formic acid. 4ml of 2mol/L TFA was then added to the hydrolysed sample, after sealing and hydrolysis at 110 ℃ for 3h, the solution in the reaction flask was evaporated to dryness under reduced pressure, methanol was added, evaporation to dryness was carried out and this was repeated three times to remove excess TFA. The hydrolyzed sample is dissolved by 2ml of water, 20mg of NaBH4 is added to react for 2h at room temperature, then glacial acetic acid is used for adjusting the pH value to about 5, methanol and a drop of glacial acetic acid are added, the pressure is reduced and the evaporation is carried out, and the steps are repeated three times to remove excessive acetic acid. Drying at 100 deg.C for 10-15min, adding 3ml acetic anhydride, reacting at 100 deg.C for 1 hr, evaporating under reduced pressure to remove unreacted acetic anhydride, adding 2ml toluene, evaporating under reduced pressure, and repeating the above steps for three times to remove acetic anhydride. Finally, the acetylated samples were dissolved in chloroform for gas chromatography-mass spectrometry (GC-MS) analysis, GC-MS conditions: RXI-5SIL MS chromatography column 30 × 0.25; the temperature programming conditions are as follows: the initial temperature is 120 ℃, and the temperature is increased to 250 ℃/min at the speed of 3 ℃/min; keeping for 5 min; the temperature of the sample inlet is 250 ℃, the temperature of the detector is 250 ℃/min, the carrier gas is helium, and the flow rate is 1 mL/min.

Referring to fig. 4, fifteen ion peaks of the methylated derivatives of the Eupolyphaga Seu Steleophaga polysaccharide appear, mass spectrograms are searched online, attribution of each mass spectrogram is preliminarily determined, primary fragments and secondary fragments in the mass spectrograms are attributed according to the cracking rule of the partially methylated sugar alcohol acetate derivatives, and the result is shown in table 2 by analysis:

TABLE 2ESPS methylated derivatives ion Peak assignment

(4) Ultraviolet spectrum

The ESPS in the present invention was prepared as a 1mg/ml aqueous solution, and scanned with an ultraviolet spectrophotometer in the range of 200-700 nm. See FIG. 5, which shows no characteristic peaks at 260nm and 280nm, indicating that the ESPS contains no nucleic acids and proteins.

(5) Infrared spectroscopy

Taking 1mg of the above-mentioned dried ESPS, tabletting with KBr at 400-4000cm^-1Infrared spectral scanning is performed in the range of (1). Referring to FIG. 6, the infrared analysis result shows that the absorption band is 3600-3200cm^-1Is a stretching vibration absorption peak of-OH, and the absorption peak in this region is a characteristic peak of the glucide. The method comprises the following specific steps: 3392cm^-1Is a stretching vibration absorption peak of-OH and is a characteristic peak of the saccharide. At 2925cm^-1There is a weak absorption peak, which is the C-H stretching vibration of the polysaccharide. At 1410-1200 cm^-1Is an absorption peak caused by the variable angle vibration of C-H. At 1610cm^-1And the absorption peaks at the position and the position of 1427cm < -1 >, which are respectively the absorption peaks caused by the asymmetric stretching vibration of C ═ O and the variable angle vibration of C-OH. 1200-1000 cm^-1Caused by two C-O stretching vibrations, the absorption band is mainly the vibration of the ring overlapping the stretching vibration of the side branch of C-OH and the vibration of the C-O-C glycosidic bond. The absorption peaks of the ESPS component in this region were 1099cm^-1、1004cm^-1And 1149cm^-1The absorption peak shows that the component is pyranose ring.

(6) Nuclear Magnetic Resonance (NMR) spectrum

About 20mg of the ESPS of the present invention was taken after freeze-drying, and dissolved in 0.5ml of D after vacuum-drying at room temperature for one day₂In O, it was measured on an AVANCE 500 NMR spectrometer¹H NMR and¹³c NMR spectrum, measurement temperature 25 ℃. In addition, 2D NMR spectra such as 1H-1H COSY, HSQC, NOESY and the like of the ESPS were measured. Referring to FIG. 7, the hydrogen spectrum signal is mainly concentrated between 0.0 ppm and 5.5 ppm. Delta 3.2-4.0ppm is sugar ring proton signal, main terminal proton peaks delta 4.45, 4.58, 4.76,The signal peaks of 5.03, 5.09, 5.12, 5.16 and 5.32 are distributed in the region of 4.3-5.5 ppm.

Referring to FIG. 8, the carbon spectrum signal is mainly concentrated between 60-120 ppm. By observing the carbon spectrum, the main anomeric carbon signal peaks delta 97.14, 100.97, 101.14 and 107.78 can be seen, and the anomeric carbon regions are mainly between delta 93 and 110. And the main signal peaks of delta 61.74, 61.89, 62.36, 63.76, 69.24, 69.73, 70.73, 71.32, 72.13, 72.56, 73.12, 74.25, 74.66, 75.37, 75.93, 77.56, 78.05 and 82.64 are distributed in the area of 60-85 ppm. The polysaccharide is composed of rhamnose, fucose, arabinose, xylose, glucose, and galactose according to monosaccharide composition. And the ratio of glucose and galactose is the largest, which indicates that the polysaccharide is mainly the galactoglucan.

Thus is paired with¹D NMR and²a complete analysis of the D NMR spectra and complete assignment of the C, H signals of the ESPS are shown in Table 3

TABLE 3 chemical shift assignment of ESPS NMR hydrogen and carbon spectra

The following results can be obtained by combining the above analyses:

1. the results of molecular weight determination showed that the ESPS of the present invention is a homogeneous polysaccharide with a molecular weight of about 2.14X 10⁴；

2. Monosaccharide composition analysis shows that ESPS is heteropolysaccharide composed of rhamnose, fucose, arabinose, xylose, glucose and galactose;

3. methylation analysis shows that ESPS glycosidic bonds are complex and mainly consist of 1 → 4 glycosidic bonds and non-reducing ends 1 →;

UV analysis showed no proteins and nucleic acids in the ESPS. The IR results showed that the ESPS absorption peaks in this region were 1099cm each^-1、1004cm^-1And 1149cm^-1Is provided with an absorption peakIndicating that the component is a pyranose ring;

NMR analysis shows that the ESPS main chain is the polygalactose;

therefore, the derivation result of the repeat unit structure of Eupolyphaga Seu Steleophaga polysaccharide (ESPS) in the present invention is shown in FIG. 12, which is a novel heteropolysaccharide isolated from Eupolyphaga Seu Steleophaga.

Example 2

Materials and reagents: eupolyphaga Seu Steleophaga polysaccharide ESPS (homemade, homogeneous polysaccharide by HPLC detection), LPS (Sigma), MTT (Sigma), RPMI Medium 1640(Gibco), spleen lymphocyte separation Medium (GE)

Animals: clean Balb/c mouse (Chinese university of medicine laboratory animal center)

The instrument comprises the following steps: clean bench (Suzhou clean plant), CO₂Incubator (U.S. ThermoForma corporation), microplate reader (U.S. Bio-Rad corporation)

Proliferative Effect of ESPS on mouse spleen lymphocytes

Collecting 6-8 week old Balb/c mouse, killing by dislocation of cervical vertebra, aseptically preparing spleen lymphocyte suspension, and adjusting mouse spleen lymphocyte concentration to 1 × 10 with 1640 culture medium containing fetal calf serum⁶Adding 100 mul of cell suspension into each well of a 96-well plate, adding culture solution of ESPS with different concentrations, wherein the final concentrations are respectively 25, 50, 100, 200, 400 and 800 mul/ml, the final volume of each well is 200 mul, and simultaneously setting a blank control well and a positive control group. Each group was provided with 4 multiple holes and placed at 37 ℃ with 5% CO₂After culturing for 44h in the incubator, 20 μ l of MTT solution is added to each well, the culture is continued for 4h, and the OD value is measured at 570nm of the microplate reader. The results are shown in FIG. 13, which shows that ESPS has a significant proliferative effect on splenic lymphocytes and is dose-dependent at 25-800. mu.g/ml.

Effect of ESPS on growth of tumor cells and Normal cells in vitro

Taking tumor cells HepG2, B16F10, Panc1, HCT116 and MDA in logarithmic growth phase and normal cells MDCK, L-02, Hacat, GES-1 and HPDE, and adjusting the cell concentration to 1 × 10 by using corresponding culture medium⁵One cell per ml, 100. mu.l of cell suspension was added to a 96-well plate, and 100. mu.l/well of ESPS (final concentration: min) diluted with the culture medium at various concentrations was addedRespectively, the following steps: 3.1, 6.25, 12.5, 25, 50, 100, 200, 400, 800. mu.g/ml), culture medium was used as blank control. After culturing at 37 ℃ for 44 hours with 5% CO2, 20. mu.l of MTT solution was added to each well, and the culture was continued for 4 hours, and the absorbance at a wavelength of 570nm was measured with a microplate reader. The results are shown in FIG. 14, which indicates that ESPS has no significant cytotoxic effect on the above-mentioned various tumor cells and normal cells.

ESPS activates killing effect of mouse spleen lymphocytes on Yac-1, CT-26 and Panc02 tumor cells

Collecting 6-8 week old Balb/c mice, killing by dislocation of neck, aseptically preparing spleen lymphocyte suspension, and adjusting cell concentration to 1 × 10 with RPMI-1640 culture solution containing 10% calf serum⁶Each/ml of the effector cells was cultured for 48 hours by adding ESPS (final concentrations: 100, 200, and 400, respectively) diluted with a culture medium at different concentrations, and the number of the target cells was adjusted to 40:1, 20:1, and 10:1, respectively, by taking Yac-1, CT-26, and Panc02 cells cultured for 24 to 48 hours as the target cells. Spreading the effect and target cells in 96-well culture plate at the adjusted concentration, and 100 μ l each of the effect and target cells in the experimental wells; adding 100 mul of effector cells and culture solution into effector cell control wells; target cells and culture medium were added to control wells of target cells in an amount of 100. mu.l each, and 3 wells were prepared for each group. After incubation of the target cells for 6h, 20. mu.l of MTT was added to each well and incubation was continued for 4h at 37 ℃ in a 5% CO2 incubator. And (3) after incubation, discarding the supernatant, adding 150 mu l of DMSO into each hole, slightly oscillating for 10min to dissolve the formazan, and measuring a light absorption value at 570nm of an microplate reader. The results are shown in FIG. 15, which shows that ESPS can obviously improve the killing activity of mouse spleen lymphocytes on Yac-1, CT-26 and Panc 02.

Killing effect of ESPS activated NK-92 on Panc-1, HepG2, A375 and MCF-7 tumor cells

NK-92 cells were adjusted to a cell concentration of 1X 10⁶Each/ml of the cells was used as effector cells, different concentrations of ESPS (final concentrations: 50, 100, 200, respectively) diluted with culture medium were added and cultured for 48h, and the number of cells was adjusted by taking Panc-1, HepG2, A375, MCF-7 cells cultured for 24-48h as target cells, respectively, at an effective-to-target ratio of 40:1, 20:1, 10: 1. Spreading the effect and target cells in 96-well culture plate at the adjusted concentration, and 100 μ l each of the effect and target cells in the experimental wells; control wells for effector cells plus effector cellsAnd 100. mu.l of each culture solution; target cells and culture medium were added to control wells of target cells in an amount of 100. mu.l each, and 3 wells were prepared for each group. After incubation of the target cells for 6h, 20. mu.l of MTT was added to each well and incubation was continued for 4h at 37 ℃ in a 5% CO2 incubator. And (3) after incubation, discarding the supernatant, adding 150 mu l of DMSO into each hole, slightly oscillating for 10min to dissolve the formazan, and measuring a light absorption value at 570nm of an microplate reader. The results are shown in FIG. 16, which shows that ESPS can obviously improve the killing activity of NK-92 on Panc-1, HepG2, B16F10 and MCF-7.

Therefore, it can be seen from example 2 that the woodlouse polysaccharide (ESPS) of the present invention is an immunopotentiator, which can significantly activate the relevant immune cells and enhance the immune activity, and lays a good foundation for the subsequent anti-tumor experiments.

Example 3

1. In vivo antitumor effect of Eupolyphaga Seu Steleophaga polysaccharide (ESPS) on H22 and Panc02 tumor-bearing mice

(1) Establishment and administration of mouse liver cancer H22 tumor-bearing mouse model

Selecting ascites mouse inoculated with H22 cells for 7 days, sterilizing abdominal skin, sucking ascites with sterile syringe, diluting with normal saline, staining with trypan blue, counting with blood counting plate under inverted microscope to obtain viable cells with number of more than 98%, and adjusting cell concentration to 5 × 10⁶One per ml. 40 mice, 18-22g Balb/c, were inoculated subcutaneously with 0.1ml of cell suspension in the right anterior axilla.

(2) Influence of ESPS on tumor inhibition rate, spleen index and thymus index of H22 tumor-bearing mice

Mice with over-or under-sized tumors were culled about 12 days after inoculation and randomly divided into 5 groups of six mice each. The drug is respectively a model group, 5-Fu, a low, medium and high dose administration group (10, 20 and 40mg/kg groups), the drug is administered once every other day for two weeks, the administration mode is tail vein injection, each administration is 0.2ml, and the model group is tail vein injection with 0.2ml of physiological saline each time. Two weeks later, mice were sacrificed in the short neck and tumors, spleen and thymus were weighed. The results are shown in FIG. 17

As can be seen from FIG. 17, the ESPS administration groups significantly inhibited tumor growth compared to the blank group: (^***P<0.001) and protects spleen and thymusIn effect, the thymus index and spleen index showed a dose-dependent increase compared to the positive group of drugs.

(3) Establishment and administration of mouse pancreatic cancer Panc02 tumor-bearing mouse model

Establishing a mouse model according to a transplantation tumor research method. Selection of subcutaneous inoculation 5X 10⁶Sterilizing oxter skin of tumor-bearing mice 9-11 days after each/ml Panc02 cell, aseptically peeling tumor mass of mice, gently grinding with needle core, sieving with 200 mesh sieve, washing with normal saline, collecting separated tumor cell suspension, centrifuging at 1500r/min for 10min, and discarding supernatant. Diluting with normal saline, staining with trypan blue, counting with blood counting plate under inverted microscope to obtain viable cell with number of more than 98%, and adjusting cell concentration to 5 × 10⁶One per ml. 40 mice, 18-22g Balb/c, were inoculated subcutaneously with 0.1ml of cell suspension in the right anterior axilla.

(4) ESPS influences the tumor inhibition rate, spleen index and thymus index of Panc02 tumor-bearing mice

The tumor volume of the mouse is 50-100mm³On the left and right, mice with too large or too small tumors were culled and randomly divided into 5 groups of six mice each. The drug is administered once every other day for two weeks in a mode of tail vein injection, wherein the drug is 0.2ml for each administration, and the drug is 0.2ml for each tail vein injection in the model group. Two weeks later, mice were sacrificed in the short neck and tumors, spleen and thymus were weighed. The results are shown in FIG. 18

As can be seen from fig. 18, each administration group of ESPS significantly inhibited tumor growth, and protected spleen and thymus compared to the blank group, and the thymus index and spleen index showed an increase in dose dependence compared to the positive drug group.

ESPS promotes secretion of IL-2 and IFN-gamma cytokines in serum of Panc02 tumor-bearing mice

Serum from each group of experiments was collected in sterile EP tubes. The experiment was performed as indicated in the ELISA kit instructions for mouse IFN-. gamma./IL-2 monoclonal antibodies. The method comprises the following specific steps:

(1) the desired panel was removed from the sealed bag which had been equilibrated to room temperature.

(2) Dilution and sample loading of standard: samples or standards of different concentrations (100. mu.l/well) were added to the corresponding wells, except for blank wells.

(3) And (3) incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 30 min.

(4) Washing: the plate was washed 5 times.

(5) Adding an enzyme: 50 μ l of enzyme-labeled reagent was added to each well, except for blank wells.

(6) The plate is sealed by a sealing plate membrane and then incubated at 37 ℃ for 30min, and the plate is washed for 5 times.

(7) Color development: adding color developing agent (100 μ l/well) into each well, shaking, mixing, and developing at 37 deg.C in dark for 15 min.

(8) And (4) terminating: stop solution (50. mu.l) was added to each well to terminate the reaction.

(9) And (3) determination: the blank wells were zeroed and the absorbance (OD value) of each well was measured at a wavelength of 450 nm.

The concentration of the standard substance is taken as the abscissa and the OD value is taken as the ordinate, and a standard curve is drawn by each group of OD 450 values. The IFN-gamma and IL-2 concentrations in each test well can be calculated from the corresponding OD 450 values according to the standard curve.

As can be seen from FIG. 19, ESPS can significantly increase the secretion of IL-2 and IFN-gamma in the serum of Panc02 tumor-bearing mice. The stimulation effect is continuously enhanced along with the increase of the administration dosage, and when the ESPS dosage is 40mg/kg, the secretion of IL-2 and IFN-gamma is obviously different from that of a model group (the dosage is not less than the standard dosage of the ESPS)^**P<0.01), indicating that the ESPS activity of promoting the secretion of IL-2 and IFN-gamma presents obvious dose dependence.

Therefore, it can be seen from example 3 that the woodlouse polysaccharide (ESPS) of the present invention exhibits excellent antitumor activity in vivo, and can exert indirect antitumor effect by enhancing host immunological activity.

Claims

1. A Eupolyphaga sinensis Walker polysaccharide is characterized by consisting of rhamnose, fucose, arabinose, xylose, glucose and galactose, wherein the molar ratio is 7.4:3.1:13.9:9.3:39.7: 26.5; relative molecular weight is 2.14X 10⁴D_a(ii) a Has a backbone structure of a repeating unit of a glucosyl galactan, wherein the backbone structure is represented by → 4) -alpha-D-Glcp- (1 → glucose and → 3) -Galp- (1 → galactose, the branch chain is connected with the main chain through the O-6 bond of glucose, the O-4 bond of galactose and the O-6 bond, the main chain unit structure and the branch chain segment are shown as follows:

main chain structure

Branched chain 1 alpha-L-Araf- (1 → C →)

Branched chain 2Xyl p- (1 → 4) -Xyl p- (1 → 3) -Xyl p- (1 →

Branched 3 α -L-Araf- (1 → (5) - α -L-Araf- (1)3 →

Branched chain 4

Branch 5

。

2. The Eupolyphaga Seu Steleophaga polysaccharide of claim 1, which comprises a pyranose ring.

3. The Eupolyphaga Seu Steleophaga polysaccharide according to claim 1, wherein the polysaccharide is prepared by:

(1) a separation step: extracting Eupolyphaga Seu Steleophaga powder with water, removing protein, and precipitating with ethanol;

(2) a purification step:

firstly, taking a ground beetle polysaccharide crude product prepared by water extraction, protein removal by a Sevag reagent in combination with papain and ethanol precipitation, fully dissolving the ground beetle polysaccharide crude product in deionized water, and eluting the polysaccharide crude product by using a well-balanced ion exchange column Cellulose DE-52;

secondly, performing gel chromatography on the ground beetle polysaccharide water washing component obtained by separating the ion exchange column by using a Sephacryl S-300 molecular sieve column, eluting by using deionized water, collecting in parts, tracking and detecting by using a phenol sulfate method, and combining the same components; and (5) freezing and drying to obtain the ground beetle polysaccharide.

4. The method for extracting the ground beetle polysaccharide of claim 1, which comprises the steps of separation and purification, wherein the separation comprises the steps of water extraction, protein removal and ethanol precipitation of ground beetle powder, and the method is characterized in that the purification uses a column chromatography method, and specifically comprises the following steps:

5. Use of the woodlouse polysaccharide of claim 1 in the preparation of an immunopotentiator.

6. The use of Eupolyphaga Seu Steleophaga polysaccharide as claimed in claim 1 in the preparation of antitumor drugs for immunity.

7. The use of Eupolyphaga Seu Steleophaga polysaccharide according to claim 1 in the preparation of a medicament for treating liver cancer and pancreatic cancer.