Dendrobium officinale polysaccharide extract as well as preparation method and application thereof

Abstract

The invention provides a dendrobium officinale polysaccharide extract as well as a preparation method and application thereof.The dendrobium officinale polysaccharide extract is prepared from dendrobium officinale stems as a raw material through purified water reflux extraction, ethanol precipitation, Sevag deproteinization, decoloration and deliming and gel / membrane separation and purification, the mass percentage content of polysaccharide in the extract is 50-95 wt%, the average molecular weight is 10-300 kDa, and the content of polysaccharide in the extract is 50-95%. The monosaccharide components comprise mannose, glucose, galactose, fucose, rhamnose and arabinose. The dendrobium officinale polysaccharide extract provided by the invention has an immune activation effect on natural killer cells, can dose-dependently promote the degranulation reaction of NK cells CD107a and the expression of IFN-gamma, perforin and granzyme B, and up-regulate NKG2D related pathways, so that the functions of the NK cells are activated, and the killing activity of the NK cells on tumor target cells such as K562 is enhanced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a Dendrobium officinale polysaccharide extract, a preparation method and applications. BACKGROUND

[0002] Tumor is a kind of cell abnormal proliferation disease caused by various carcinogenic factors, and its occurrence and development are closely related to the immune function of the body. The immune system plays an important defense role in the process of tumor occurrence by recognizing and removing mutant cells, and the natural killer (NK) cell, as a key member of the innate immune system, has the ability to directly recognize and kill tumor cells without antigen pre-sensitization, and is one of the important defenses of the body's anti-tumor immunity.

[0003] However, during the occurrence and development of tumors, the function of NK cells is often inhibited by the tumor microenvironment, such as increased secretion of immunosuppressive cytokines, abnormal metabolism, and up-regulation of inhibitory ligands on the surface of tumor cells, all of which can lead to decreased degranulation function and reduced cytotoxicity of NK cells, thereby weakening the body's natural anti-tumor defense ability. Although common clinical treatments such as chemotherapy, radiotherapy or immune checkpoint therapy can delay tumor progression, they often accompany with decreased NK cell function or immunosuppressive side effects. Therefore, it is of great significance to find a natural active substance that can safely and effectively activate NK cells and enhance their anti-tumor killing activity.

[0004] Dendrobium officinale, a plant of the Orchidaceae family, is one of the traditional Chinese precious medicinal materials, and has the effects of "tonifying yin and clearing heat, generating fluid and benefiting stomach, strengthening body and prolonging life". Modern pharmacological studies have shown that the polysaccharides contained in Dendrobium officinale are the main active ingredients, which have antioxidant, anti-tumor, immunomodulatory, hypoglycemic and anti-fatigue activities. Dendrobium officinale polysaccharides can regulate the phagocytic function of macrophages, promote the proliferation of T and B cells and the secretion of cytokines, and have a significant effect on improving the immune function of the body.

[0005] Existing researches have focused on the antioxidant, anti-inflammatory and general immune-enhancing effects of Dendrobium officinale polysaccharides, but there is still a lack of systematic research and public reports on whether they can directly enhance the activation of NK cells and the killing effect on tumor cells. Traditional chemical immunostimulants can improve NK activity in the short term, but they often have problems such as high toxicity, poor tolerance, limited clinical application, etc.

[0006] Therefore, how to develop a natural polysaccharide preparation based on Dendrobium officinale, which is natural in origin, low in toxicity, high in safety, can significantly activate NK cell function and enhance its killing effect on tumor cells, is a technical problem that needs to be solved by those skilled in the art. SUMMARY

[0007] Based on this, the present application provides a dendrobium officinale polysaccharide extract, a preparation method and application, the extract provided by the present application is natural, low in toxicity, high in safety, can significantly enhance the immune activation and cytotoxicity of NK cells, effectively improve the immune suppression state in the tumor microenvironment, thereby realizing the purposes of enhancing the anti-tumor effect of NK cells and inhibiting tumor growth.

[0008] A preparation method of a dendrobium officinale polysaccharide extract, which takes dendrobium officinale stems as raw materials, is obtained through reflux extraction with purified water, ethanol precipitation, Sevag deproteinization, decolorization and deashing, and gel / membrane separation and purification, the mass percentage content of polysaccharides in the dendrobium officinale polysaccharide extract is 50-95 wt%, the average molecular weight is 10-300 kDa, the monosaccharide components include mannose, glucose, galactose, fucose, rhamnose and arabinose, and the main chain of the dendrobium officinale polysaccharide extract includes the following two types of structural units: .

[0009] The preparation method described above, wherein the method specifically comprises: The dried dendrobium officinale stems are crushed through a 40-mesh sieve, purified water is added, reflux extraction is carried out at 90 DEG C, and the supernatant is taken after filtration; the filter residue is added with purified water again, and the extraction is repeated twice, and the filtrates are combined; The combined extract is concentrated under reduced pressure to 1 / 5 of the original volume, 95% ethanol is added, stirring is uniformly carried out, and then standing is carried out for 24 h, the supernatant is discarded after centrifugation, and the precipitate is collected; The precipitate is dissolved in appropriate distilled water, the Sevag method is used to remove the protein, the volume ratio of chloroform and n-butanol is 4:1, the deproteinization is repeated for 3 times, the water solution after deproteinization is decolorized with activated carbon, the activated carbon is filtered out, and the dialysis bag is dialyzed for 48 h, the molecular weight cut-off is 8000-14000 Da, and the dialysis liquid is deionized water, which is replaced every 8 h; The liquid after dialysis is concentrated under reduced pressure, and then freeze-dried to obtain the dendrobium officinale polysaccharide crude extract in the form of light yellow powder, the impurity polysaccharide components are removed through DEAE-cellulose column chromatography, and then the Sephadex G-100 gel column separation and purification are carried out, the main peak components are collected, and freeze-drying is carried out to obtain the dendrobium officinale polysaccharide extract.

[0010] The preparation method described above, wherein the molar ratio of mannose to glucose is (1.0-1.8):(1.0), and the proportion of galactose in the total monosaccharide is 5-15%.

[0011] A dendrobium officinale polysaccharide extract prepared by the method described above.

[0012] The application of the dendrobium officinale polysaccharide extract described above, the dendrobium officinale polysaccharide extract is used for preparing a medicine for activating the function of NK cells and enhancing the killing activity of NK cells on tumor cells.

[0013] According to the above application, the drug is used to dose-dependently promote the degranulation of natural killer cells CD107a and the expression of IFN-γ, perforin, and granzyme B, upregulate the NKG2D pathway, thereby activating NK cell function and enhancing the killing activity of NK cells against K562 tumor cells.

[0014] According to the above applications, the drug is a lyophilized powder, oral liquid, injectable solution, or tablet.

[0015] According to the above application, the drug comprises the following raw materials in parts by weight: 5-80 parts of Dendrobium officinale polysaccharide extract, 5-20 parts of freeze-drying protectant, 20-30 parts of buffer, and 5-15 parts of stabilizer.

[0016] According to the above applications, the freeze-drying protectant is mannitol and / or trehalose, the forming agent is microcrystalline cellulose or sodium carboxymethyl starch, the buffer is phosphate buffer, and the stabilizer is sorbitol or fructooligosaccharide.

[0017] Beneficial effects: This invention uses Dendrobium officinale stems as raw material, and obtains Dendrobium officinale polysaccharide extract through purified water reflux extraction, ethanol precipitation, Sevag deproteinization, decolorization and deashing, and gel / membrane separation purification. Experimental results show that the extract has significant immune-activating effects, and can promote the CD107a degranulation reaction of NK cells and the expression of IFN-γ, perforin, and granzyme B in a dose-dependent manner, upregulate the expression level of NKG2D pathway-related proteins, thereby activating NK cell function and enhancing the killing activity of NK cells against K562 tumor cells. It can effectively inhibit tumor growth, improve the immune microenvironment, and reduce the risk of recurrence. In addition, the Dendrobium officinale polysaccharide extract of this invention is a high-purity polysaccharide component from natural sources, which is safe and non-toxic, and the extraction process is controllable and highly safe. Attached Figure Description

[0018] Figure 1 The molecular weight distribution of Dendrobium officinale polysaccharide extract by gel permeation chromatography (GPC) is shown. Figure 2 High-performance liquid chromatogram of the monosaccharide composition of Dendrobium officinale polysaccharide extract; Figure 3 Fourier transform infrared spectrum of Dendrobium officinale polysaccharide extract; Figure 4 This is a graph showing the tumor cell count in the K562 control group. Figure 5 This is a graph showing the results of NK cells killing K562 tumor cells in the negative control group. Figure 6Figure for the results of the effect of the Dendrobium candidum polysaccharide preparation in the experimental group A on the killing of K562 tumor cells by NK cells; Figure 7 Figure for the results of the effect of the Dendrobium candidum polysaccharide preparation in the experimental group B on the killing of K562 tumor cells by NK cells; Figure 8 Flow cytometry graph of the change in CD107a expression on the surface of NK cells; Figure 9 Figure of the Western blot detection results of the expression of perforin and granzyme B proteins in NK cells after treatment with the Dendrobium candidum polysaccharide preparation; Figure 10 Figure of the Western blot results of the expression of NKG2D protein in the spleen NK cells of mice treated with the Dendrobium candidum polysaccharide preparation; Figure 11 Histological section of the spleen of a mouse. DETAILED DESCRIPTION

[0019] For the purpose of facilitating the understanding of the present application, a more comprehensive description of the present application will be provided below with reference to various embodiments, but the present application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the description of the present application herein is only for the purpose of describing the specific embodiments and is not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0021] Example 1: A preparation method of a Dendrobium candidum polysaccharide extract 1. Preparation process The dried Dendrobium candidum stems were ground to pass through a 40-mesh sieve, 1000 g was weighed, 20 times the volume of purified water was added, and reflux extraction was performed at 90°C for 3 h, and the supernatant was collected by filtration. The filter residue was added with 15 times the volume of purified water again, and repeated extraction was performed twice, and the filtrates were combined.

[0022] The combined extract was concentrated under reduced pressure to 1 / 5 of the original volume, 4 times the volume of 95% ethanol was slowly added, and after stirring uniformly, it was allowed to stand for 24 h to allow the polysaccharides to fully precipitate. After centrifugation (4000 rpm, 10 min), the supernatant was discarded, and the precipitate was collected.

[0023] The precipitate was dissolved in an appropriate amount of distilled water, and the protein was removed by the Sevag method (chloroform: n-butanol = 4:1, by volume), and the treatment was repeated 3 times. The deproteinized aqueous solution was decolorized with activated carbon for 30 min, and then filtered to remove the activated carbon. The filtrate was dialyzed in a dialysis bag (molecular weight cutoff 8000-14000 Da) for 48 h, with deionized water as the dialysis liquid, and the dialysis liquid was replaced every 8 h.

[0024] The dialyzed liquid was concentrated under reduced pressure and then freeze-dried to obtain a light yellow powder of crude Dendrobium officinale polysaccharide extract. Further purification was performed by DEAE-cellulose column chromatography to remove impure polysaccharide components, followed by Sephadex G-100 gel column chromatography to separate and purify the main peak components, and freeze-drying to obtain the Dendrobium officinale polysaccharide extract.

[0025] 2. Physicochemical properties and monosaccharide composition analysis (1) Determination of total sugar content The total sugar content was determined by the phenol-sulfuric acid method.

[0026] 0.5 mL of polysaccharide extract sample solution was taken, 0.5 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid were added, and it was allowed to stand at room temperature for 30 min. The absorbance was measured at 490 nm. A standard curve was prepared using glucose as the standard.

[0027] The results showed that the mass percentage of polysaccharide in the sample was (85.3 ± 1.2) wt%, indicating that the prepared extract was a high-purity polysaccharide.

[0028] (2) Determination of molecular weight Gel permeation chromatography (GPC) was used, with a TSK-GEL G3000PWxl column (7.8 x 300 mm), a mobile phase of 0.1 mol / L NaNO3, a flow rate of 0.6 mL / min, a temperature of 35°C, and a differential refractive index detector (RID).

[0029] The molecular weight distribution was calculated using a dextran standard curve, and the results are shown in Figure 1 From Figure 1 It can be seen that the main peak is located at about 1.2 x 10 5 Da (120 kDa), indicating that the polysaccharide has a relatively concentrated and uniform molecular weight distribution, with a molecular weight range of about 10-300 kDa, indicating that the polysaccharide obtained by the extraction process has good structural uniformity.

[0030] (3) Monosaccharide composition analysis High performance liquid chromatography (HPLC) with pre-column derivatization was used.

[0031] The polysaccharide extract sample was hydrolyzed by 2 mol / L trifluoroacetic acid (TFA) at 120°C for 2 h, dried under reduced pressure, and then reacted with PMP (1-phenyl-3-methyl-5-pyrazolone) to be derivatized, and heated for 40 min under alkaline conditions.

[0032] After removing the excess PMP by ether extraction, the water phase was filtered through a 0.22 μm filter membrane, and then loaded onto a C18 chromatographic column (250×4.6 mm, 5 μm) using acetonitrile-0.1 mol / L phosphate buffer (pH 6.8) (17:83, v / v) as the mobile phase, a flow rate of 1.0 mL / min, and a detection wavelength of 245 nm.

[0033] See Figure 2 , and the results showed that the extract mainly included mannose, glucose, galactose, fucose, a small amount of rhamnose and arabinose, and the molar ratio of mannose, glucose, galactose, fucose, rhamnose, and arabinose was 3.2:2.5:1.0:0.6:0.3:0.2.

[0034] (4) Infrared spectroscopy (FT-IR) analysis After grinding and pressing the polysaccharide extract sample with KBr into a tablet, scanning was performed in the range of 4000-400 cm -1 . Figure 3 The results showed that: the peak at 3400 cm -1 was a hydroxyl stretching vibration peak; the peak at 2920 cm -1 was a C-H stretching vibration peak; the peak at 1635 cm -1 was a bound water absorption peak; the peaks at 1070 cm -1 and 1020 cm -1 were C-O-C and C-O stretching vibration peaks, which were typical polysaccharide characteristic absorption peaks.

[0035] The above results showed that the obtained extract was a typical polysaccharide containing pyranose ring structure.

[0036] (5) Nuclear magnetic resonance (NMR) analysis The extract was dissolved in D2O, and 1 H、 13 C, COSY, HSQC, and HMBC were determined by Bruker 700 MHz NMR.

[0037] According to the HSQC / HMBC cross peaks, it can be observed that: Manp C1 / H1 signal δH 4.8-5.1 ppm (α / β mixed).

[0038] Glcp C1 / H1 signal δH 4.5-4.7 ppm.

[0039] Main chain C4 position signal δC 79-82 ppm → typical (1→4) linkage characteristics.

[0040] Galp C6 position branched chain signal δC 68-70 ppm.

[0041] No O-Ac signal (the region of δH 2.0-2.2 ppm is empty).

[0042] According to the monosaccharide composition, methylation and NMR results, the Dendrobium candidum polysaccharide extract obtained in this example is a typical linear main chain polysaccharide composed of (1→4)-D-Manp and (1→4)-D-Glcp, and a small amount of Galp branched chain is connected at the O-6 position of part of Manp / Glcp, in addition, a small amount of Fuc / Rha / Ara is contained as a terminal group or a branched chain, and the main chain mainly includes the following two types of structural units: .

[0043] 3. Conclusion The physicochemical analysis results show that the mass percentage content of polysaccharide in the Dendrobium candidum polysaccharide extract obtained in this example is 50-95wt%, the average molecular weight distribution is between 10-300 kDa, and the monosaccharide composition includes mannose, glucose, galactose, fucose, rhamnose and arabinose, proving that the extract prepared in this example has a clear chemical composition and repeatability.

[0044] Example 2 A pharmaceutically acceptable pharmaceutical preparation, the preparation comprising the Dendrobium candidum polysaccharide extract as described in Example 1 and a pharmaceutically acceptable excipient.

[0045] The Dendrobium candidum polysaccharide extract and the excipient are mixed according to the following weight proportions: Dendrobium candidum polysaccharide extract 50 parts, mannitol 10 parts, trehalose 5 parts, sorbitol 5 parts, phosphate buffer 30 parts.

[0046] The components are thoroughly mixed, the pH is adjusted to 7.2±0.1, and after sterilization filtration through a 0.22μm microporous filter, the sample is aseptically filled into freeze-drying bottles, with a liquid volume of 2 mL per bottle.

[0047] The filled sample is frozen at -40℃ for 4 h, then transferred to a vacuum freeze-drying machine, and freeze-dried at -60℃ and 20 Pa for 24 h to obtain a light yellow loose freeze-dried powder. The obtained freeze-dried powder is placed at 25℃ and a relative humidity of 60% for 6 months, and no caking or discoloration occurs, and the appearance and solubility are stable.

[0048] The preparation is reconstituted with sterile water for injection or 0.9% NaCl solution, and can be quickly dissolved to form a clear solution. The preparation is stable in pH, viscosity and polysaccharide content after reconstitution, and is suitable for in vitro pre-activation culture of NK cells or in vivo immune enhancement experiments in animals.

[0049] Example 3 This example aims to verify the enhancement effect of the preparation obtained in Example 2 (hereinafter referred to as Dendrobium polysaccharide preparation) on the anti-tumor activity of natural killer (NK) cells, using human leukemia cell line K562 as target cells to evaluate the in vitro killing efficiency of NK cells stimulated by Dendrobium polysaccharide preparation.

[0050] 1. Experimental materials and methods (1) Cell source and culture conditions K562 cells were purchased from the Chinese Academy of Sciences Cell Bank and cultured in RPMI-1640 medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin) at 37°C, 5% CO2 constant temperature.

[0051] Human peripheral blood mononuclear cells (PBMC) were separated into NK cells (CD56 + CD3 - ) by magnetic bead sorting method, and maintained growth using the same culture system.

[0052] (2) Experimental grouping Blank group: only K562 cells were inoculated, without adding NK cells; Negative control group: K562 cells were co-cultured with untreated NK cells; Experimental group A: NK cells were pretreated with 250 μg / mL of Dendrobium polysaccharide preparation for 24 h, and then co-cultured with K562 cells.

[0053] Experimental group B: NK cells were pretreated with 500 μg / mL of Dendrobium polysaccharide preparation for 24 h, and then co-cultured with K562 cells.

[0054] (3) Co-culture and effector-to-target ratio setting Each group of NK cells and K562 cells were co-cultured at an effector-to-target ratio (E:T) of 4:1, 8:1, and 20:1 for 4 h. Each group had three replicate wells.

[0055] (4) Flow cytometry detection After the culture, Annexin V-FITC / PI double staining combined with flow cytometry was used to detect the apoptosis rate of K562 cells, and the cytotoxic killing rate mediated by NK cells was calculated. The expression of CD107a on the surface of NK cells was also detected simultaneously to reflect the level of degranulation activation.

[0056] 2. Experimental results The experimental results are shown in Figure 4 to Figure 7 The results show that: At a low target ratio (4:1), the Dendrobium polysaccharide preparation promotes the killing of K562 by NK cells, but is not statistically significant (p-value>0.05); At a medium-high target ratio (8:1 and 20:1), the killing efficiency of K562 cells by NK cells is significantly improved (p-value<0.01) after pre-stimulation with the Dendrobium polysaccharide preparation, and shows a dose-dependent enhancement trend. Among them: At E:T = 8:1, the K562 apoptosis rate of the 250 μg / mL treatment group increased from 32.5% ± 2.1% of the control group to 47.8% ± 2.6%, and the 500 μg / mL treatment group further increased to 58.9% ± 3.3%; At E:T = 20:1, the K562 apoptosis rate was 62.7% ± 3.0% (250 μg / mL) and 74.2% ± 2.8% (500 μg / mL), respectively, which was significantly higher than that of the negative control group (about 45.1% ± 2.4%).

[0057] At the same time, the flow detection results show that the CD107a positive rate of NK cell surface in the 250 μg / mL and 500 μg / mL treatment groups increased by about 1.4 times and 2.1 times, respectively, as shown in Figure 8 .

[0058] 3. Results and conclusions The experimental results show that the Dendrobium polysaccharide preparation can significantly enhance the cytotoxicity of NK cells, and its effect is as follows: (1) Promote NK cell degranulation reaction (CD107a up-regulation); (2) Significantly improve the killing efficiency of K562 tumor cells by NK cells under high target ratio conditions, and the effect is more significant as the concentration of the preparation increases.

[0059] Therefore, the Dendrobium polysaccharide preparation provided by the present application can effectively activate the immune function of NK cells and enhance their killing activity on tumor cells, providing a safe, natural, and significantly enhanced NK function candidate preparation for tumor immunotherapy.

[0060] Example 4 To further verify the effect of the Dendrobium polysaccharide preparation on the cytokine secretion function of NK cells, this embodiment detects the change in the proportion of IFN-γ positive cells of NK cells after co-incubation with tumor target cells.

[0061] 1. Experimental materials and methods (1) Cell and grouping NK cells were the same as in Example 3. K562 cells were target cells. The experiment was divided into: Blank group: only K562 cells; Negative control group: K562 + untreated NK cells; Experimental group A: NK cells were pre-stimulated for 24 h with 250 μg / mL Dendrobium officinale polysaccharide preparation; Experimental group B: NK cells were pre-stimulated for 24 h with 500 μg / mL Dendrobium officinale polysaccharide preparation.

[0062] Each group was then incubated at an effector-to-target ratio (E:T) of 4:1, 8:1, and 20:1 for 4 h.

[0063] (2) Golgi blocking and fixation permeation After 30 min of co-incubation, Brefeldin A (final concentration 10 μg / mL) was added to block cytokine excretion; after incubation, the cells were collected, washed once with PBS, fixed with 4% paraformaldehyde for 10 min, and then treated with commercial permeation buffer (Perm / Wash buffer) for 10 min.

[0064] (3) Antibody staining and flow detection Surface markers CD3-FITC and CD56-PE were used, intracellular staining was performed with IFN-γ-APC, and live / dead staining was performed with 7-AAD. The staining steps were live / dead staining, surface staining, fixation permeation, and intracellular staining.

[0065] After staining, data were collected by flow cytometry (BD FACSCanto II), and ≥30,000 lymphocyte events were collected per sample. The gating strategy was: FSC / SSC→single cells→live cells→CD3 - CD56 + (NK cells)→IFN-γ + .

[0066] The FMO-IFN-γ control was used to determine the positive threshold.

[0067] 2. Experimental results The proportion of NK cells producing IFN-γ + in the negative control group was: 8%±2% (E:T = 4:1) 12%±3% (E:T = 8:1) 17%±4% (E:T = 20:1) After stimulation with Dendrobium officinale polysaccharide preparation, the proportion of NK cells producing IFN-γ +The proportion was significantly increased and showed a concentration-dependent enhancement trend, as shown in Table 1: Table 1. IFN-γ stimulated by D. candidum polysaccharide preparation + Proportion

[0068] Statistical analysis showed that, compared with the negative control, the p-value of the two treatment groups was <0.01 at E:T = 8:1 and 20:1, and <0.05 at E:T = 4:1.

[0069] 3. Result analysis and conclusion The experimental results show that the D. candidum polysaccharide preparation can significantly improve the ability of NK cells to produce IFN-γ after acting on tumor target cells, and the effect is enhanced with the increase of dose and effector-target ratio. The results are consistent with the trend of up-regulation of NK cell CD107a in Example 3, further proving that the preparation described in the application can effectively enhance the activation level and anti-tumor immune function of NK cells.

[0070] Example 5 To further verify the molecular mechanism of the D. candidum polysaccharide preparation in enhancing the cytotoxic activity of NK cells, the Western blot method was used to detect the changes in the protein expression levels of perforin and granzyme B in NK cells.

[0071] 1. Experimental materials and methods (1) Cells and grouping The source and treatment method of NK cells were the same as in Example 3. Three groups were set up: Negative control group: no D. candidum polysaccharide preparation was added; Experimental group A: 250 μg / mL of D. candidum polysaccharide preparation was added for stimulation for 24 h; Experimental group B: 500 μg / mL of D. candidum polysaccharide preparation was added for stimulation for 24 h.

[0072] After stimulation, 3×10 6 ~ 5×10 6 cells / sample were collected for protein extraction.

[0073] (2) Protein extraction and quantification The cells were lysed with RIPA lysis buffer containing 1 mM PMSF on ice for 30 min, and mixed evenly every 10 min.

[0074] Centrifuged at 12,000×g for 15 min at 4°C, and the supernatant was collected. The protein concentration was determined by BCA method, and the lysis buffer was adjusted to 2 μg / μL.

[0075] (3) SDS-PAGE and membrane transfer Equal amount of protein (20 μg / well) was mixed with 4x loading buffer at 3:1, denatured at 95℃ for 5 min.

[0076] After electrophoresis in 10% separating gel (120 V, 60-90 min), the sample was transferred to PVDF membrane (300 mA, 60 min, 4℃).

[0077] After membrane transfer, 5% skim milk powder TBST solution was used for blocking for 1 h.

[0078] (4) Antibody incubation The following primary antibodies were added to the PVDF membrane respectively and incubated overnight at 4℃ on a shaker: anti-perforin (PRF1, about 67 kDa), diluted 1:1000; anti-granzyme B (GZMB, about 32 kDa), diluted 1:1000; internal reference anti-β-actin (about 42 kDa), diluted 1:3000.

[0079] The next day, the membrane was washed with TBST for 3 times, 5 min each time. HRP-labeled secondary antibody (1:5000) was added and incubated at room temperature for 1 h, and then the membrane was washed for 3 times.

[0080] (5) Color development and imaging ECL chemiluminescent substrate was used for color development, and a gel imaging system was used to collect the band images. Gray scale analysis was performed on the perforin, granzyme B and β-actin bands, and the relative expression amount was calculated (target protein gray value / β-actin gray value).

[0081] 2. Experimental results Please refer to Figure 9 , the Western blot results showed that compared with the negative control group, the perforin and granzyme B bands in the NK cells treated with Dendrocalamus latiflorus polysaccharide preparation were significantly deepened, and showed dose-dependent enhancement. It should be noted that Figure 9 , negative control group-1 and negative control group-2 represent two experiments on the negative control group, experimental group A-1 and experimental group A-2 represent two experiments on the experimental group A, and experimental group B-1 and experimental group B-2 represent two experiments on the experimental group B.

[0082] In the 250 μg / mL treatment group, the relative expression amounts of perforin and granzyme B were increased by about 1.5 times and 1.4 times respectively compared with the control group; In the 500 μg / mL treatment group, the relative expression amounts of the two were increased by about 2.2 times and 2.0 times respectively.

[0083] The difference is statistically significant (p-value < 0.01).

[0084] The band analysis results are consistent with the change trend of the CD107a degranulation rate and the increase in the proportion of IFN-γ positive in Examples 3 and 4, indicating that the Dendrocalamus latiflorus polysaccharide preparation can enhance the killing function of NK cells by up-regulating the protein expression of cytotoxic molecules perforin and granzyme B.

[0085] 3. Results and conclusions This example shows that the Dendrocalamus latiflorus polysaccharide preparation can significantly increase the protein level of perforin and granzyme B in NK cells, and the effect is enhanced with the increase of the concentration of the preparation, further verifying that the Dendrocalamus latiflorus polysaccharide extract of the present application can enhance the killing power of NK cells on tumor cells by promoting the expression of cytotoxic molecules, and providing protein level evidence for its anti-tumor immune enhancement mechanism.

[0086] Example 6 To verify the regulatory effect of the Dendrocalamus latiflorus polysaccharide preparation on the expression level of the NK cell activation receptor NKG2D in vivo, this example uses a tumor-bearing mouse model to detect the effect of the Dendrocalamus latiflorus polysaccharide preparation on the NKG2D protein expression of NK cells, in order to evaluate the mechanism of enhancing the immune activity of NK cells.

[0087] 1. Experimental materials and methods (1) Experimental animals and grouping SPF grade BALB / c nude mice (6-8 weeks old, body weight 18-22 g) were selected and raised under standard conditions. K562 human leukemia cells in the logarithmic growth phase were resuspended with PBS and subcutaneously inoculated into the right armpit, with an inoculation amount of 1×10 7 cells per mouse.

[0088] When the tumor volume was about 100 mm³, the mice were randomly divided into groups (n = 6 / group): Control group: normal saline (intraperitoneal injection); Low-dose group: Dendrocalamus latiflorus polysaccharide preparation 100 mg / kg (intraperitoneal injection); High-dose group: Dendrocalamus latiflorus polysaccharide preparation 200 mg / kg (intraperitoneal injection).

[0089] Continuous administration for 10 days, once a day. The animals were sacrificed on the 10th day, and the spleen and tumor tissues were isolated.

[0090] (2) NK cell separation and protein extraction The spleen was ground to prepare a single cell suspension, and CD3 - CD49b + NK cells were obtained by magnetic bead sorting method.

[0091] After collection of NK cells, RIPA lysis buffer containing 1 mM PMSF was used to lyse the cells on ice for 30 min, and the supernatant was obtained by centrifugation (12,000 x g, 15 min, 4°C). The protein concentration was quantified by BCA method and adjusted to 2 μg / μL.

[0092] (3) Western blot detection of NKG2D expression SDS-PAGE and PVDF membrane transfer were performed according to the method of Example 5.

[0093] The anti-NKG2D antibody (1:1000) was used for incubation at 4°C overnight, and β-actin was used as an internal reference (1:3000).

[0094] After washing the membrane with TBST, HRP-labeled secondary antibody (1:5000) was added and incubated at room temperature for 1 h.

[0095] ECL chemiluminescence color development was used to collect images.

[0096] The gray value was analyzed by ImageJ software, and the relative expression amount of NKG2D / β-actin was calculated.

[0097] (4) Immunohistochemical (IHC) detection The spleen tissue of the sacrificed mouse was fixed in 10% neutral formaldehyde solution for 24 h, dehydrated with gradient ethanol, transparentized with xylene, and embedded in paraffin to obtain a section (thickness 4 μm). After baking at 60°C for 2 h, the section was dewaxed to water, and 3% hydrogen peroxide solution was used to block endogenous peroxidase at room temperature for 10 min. Then, citric acid buffer (pH 6.0) was used for antigen repair (microwave heating for 10 min, natural cooling), and PBS was washed for 3 times.

[0098] After adding 5% bovine serum blocking solution to the section and incubating at room temperature for 30 min, the primary antibody, anti-NKG2D antibody (1:200), was added and incubated at 4°C overnight. The next day, after washing the membrane with PBS for 3 times, HRP-labeled secondary antibody (1:500) was added and incubated at room temperature for 1 h. DAB color developing solution was used for color development, and hematoxylin was used for nuclear restaining. After dehydration with ethanol and transparentization with xylene, the section was mounted with neutral resin.

[0099] Observation and photography were performed under a microscope (x400). Five fields were randomly selected, and the area and average optical density (IOD) of the brown-yellow positive signal were measured using Image-Pro Plus software. The expression level of NKG2D was reflected by the IOD value.

[0100] 2. Experimental results See Figure 10, Western blot results show that the Dendrobium polysaccharide preparation can significantly up-regulate the expression level of NKG2D protein in the mouse spleen NK cells. It is pointed out that, Figure 10 In the above experiments, control group-1 and control group-2 represent two experiments on the control group, low-dose group-1 and low-dose group-2 represent two experiments on the low-dose group, and high-dose group-1 and high-dose group-2 represent two experiments on the high-dose group.

[0101] The NKG2D expression ratio (NKG2D / β-actin) of the control group is set to 1; The low-dose group rises to 1.6±0.2 times; The high-dose group rises to 2.4±0.3 times, and the difference is statistically significant (p-value<0.01).

[0102] Meanwhile, please refer to Figure 11 , the histological analysis results show that the K562 tumor cell infiltration in the spleen of the Dendrobium polysaccharide preparation treated mice is significantly reduced, the tissue structure is relatively complete, and the cell necrosis and apoptosis are significantly increased, indicating that the Dendrobium polysaccharide preparation activates NK cells mediated by NKG2D, thereby enhancing the in vivo anti-tumor immune effect.

[0103] 3. Results and conclusions This embodiment shows that the Dendrobium polysaccharide preparation can significantly up-regulate the protein expression level of the NK cell surface activation receptor NKG2D in vivo, activate NK cell function, and enhance the ability of NK cells to recognize and kill tumor cells.

[0104] The results are consistent with the in vitro functional verification results of Examples 3-5, further confirming that the Dendrobium polysaccharide activates the NKG2D pathway to achieve NK cell mediated anti-tumor effect, and providing molecular level in vivo evidence for its application in immune enhancement and anti-tumor drug development.

[0105] From the above experimental results, it can be seen that the Dendrobium polysaccharide extract provided by the present application is verified by systematic in vitro and in vivo experiments, and has significant immune activation and anti-tumor effect. The extract is natural and safe, and can dose-dependently promote the degranulation reaction (up-regulation of CD107a) of natural killer (NK) cells without obvious cytotoxicity, enhance the expression of cytotoxic molecules (perforin and granzyme B), and significantly increase the proportion of IFN-γ positive cells and the expression level of NK cell surface activation receptor NKG2D. In vivo experiments further prove that the Dendrobium polysaccharide preparation treatment can reduce the infiltration of K562 tumor cells in the spleen, improve the integrity of the tissue structure and increase the necrosis and apoptosis of tumor cells, and exhibit a significant immune-dependent tumor inhibition effect.

[0106] In conclusion, the Dendrobium candidum stem is used as raw material, and the Dendrobium candidum polysaccharide extract is obtained through the purification water reflux extraction, ethanol precipitation, Sevag deproteinization, decolorization and deashing, and gel / film separation and purification.

[0107] The above-mentioned examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as the limitation of the patent scope of the present application. It should be pointed out that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for preparing a polysaccharide extract from Dendrobium officinale, characterized in that, The polysaccharide extract of *Dendrobium officinale* stems was obtained through purified water reflux extraction, ethanol precipitation, Sevag deproteinization, decolorization and deashing, and gel / membrane separation purification. The polysaccharide content in the extract was 50–95 wt%, with an average molecular weight of 10–300 kDa. The monosaccharide components included mannose, glucose, galactose, fucose, rhamnose, and arabinose. The main chain of the polysaccharide extract comprised the following two types of structural units: 。 2. The preparation method according to claim 1, characterized in that, The method specifically includes: Take dried Dendrobium officinale stems, crush them through a 40-mesh sieve, add purified water, and reflux extract at 90℃. Filter and collect the supernatant. Add purified water to the filter residue again and repeat the extraction twice. Combine the filtrates. The combined extracts were concentrated under reduced pressure to 1 / 5 of the original volume, 95% ethanol was added, the mixture was stirred evenly and allowed to stand for 24 h, the supernatant was discarded after centrifugation, and the precipitate was collected. The precipitate was dissolved in an appropriate amount of distilled water, and the protein was removed by the Sevag method. The volume ratio of chloroform to n-butanol was 4:

1. The treatment was repeated 3 times. The protein-removed aqueous solution was decolorized by activated carbon. After filtering out the activated carbon, the solution was dialyzed through a dialysis bag for 48 hours. The molecular weight cutoff was 8000~14000 Da. Deionized water was used as the dialysate and was changed every 8 hours. The dialysis liquid was concentrated under reduced pressure and then freeze-dried to obtain a light yellow powdery crude extract of Dendrobium officinale polysaccharides. The extract was further purified by DEAE-cellulose column chromatography to remove heteropolysaccharide components, followed by Sephadex G-100 gel column separation and purification. The main peak component was collected and freeze-dried to obtain Dendrobium officinale polysaccharide extract.

3. The preparation method according to claim 1, characterized in that, The molar ratio of mannose to glucose is (1.0–1.8):(1.0), and galactose accounts for 5–15% of the total monosaccharides.

4. A polysaccharide extract from Dendrobium officinale, characterized in that, Prepared by the method according to any one of claims 1 to 3.

5. The application of the Dendrobium officinale polysaccharide extract according to claim 4, characterized in that, The Dendrobium officinale polysaccharide extract is used to prepare a drug that activates NK cell function and enhances the killing activity of NK cells against tumor cells.

6. The application according to claim 5, characterized in that, The drug is used to dose-dependently promote the degranulation of CD107a natural killer cells and the expression of IFN-γ, perforin, and granzyme B, upregulate the NKG2D pathway, thereby activating NK cell function and enhancing the killing activity of NK cells against K562 tumor cells.

7. The application according to claim 5, characterized in that, The drug is a lyophilized powder, oral liquid, injectable solution, or tablet.

8. The application according to claim 5, characterized in that, The drug comprises the following raw materials in parts by weight: 5-80 parts of Dendrobium officinale polysaccharide extract, 5-20 parts of freeze-drying protectant, 20-30 parts of buffer, and 5-15 parts of stabilizer.

9. The application according to claim 8, characterized in that, The freeze-drying protectant is mannitol and / or trehalose, the forming agent is microcrystalline cellulose or sodium carboxymethyl starch, the buffer is phosphate buffer, and the stabilizer is sorbitol or fructooligosaccharide.

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