Dendrobium officinale polysaccharide extract, preparation method and application

Abstract

The present application provides a dendrobium officinale polysaccharide extract, a preparation method and an application, the dendrobium officinale polysaccharide extract is with dendrobium officinale stem as raw material, is extracted by purified water reflux, is precipitated by ethanol, is deproteinized by Sevag, is decolorized and is deashed and is obtained by gel / film separation and purification, the mass percentage content of polysaccharide in the extract is 50-95wt%, the average molecular weight is 10-300 kDa, and the monosaccharide component includes mannose, glucose, galactose, fucose, rhamnose and arabinose.The dendrobium officinale polysaccharide extract provided by the present application has immune activation effect on natural killer cells, can promote the NK cell CD107a degranulation reaction and the expression of IFN-gamma, perforin and granzyme B in a dose-dependent manner, up-regulates the NKG2D related pathway, thereby activating the NK cell function and enhancing the killing activity of NK cells on tumor target cells such as K562.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a Dendrobium officinale polysaccharide extract, its preparation method, and its application. Background Technology

[0002] Tumors are a class of diseases characterized by abnormal cell proliferation caused by various carcinogenic factors, and their occurrence and development are closely related to the body's immune function. The immune system plays an important defensive role in the process of tumorigenesis by recognizing and eliminating mutated cells. Natural killer (NK) cells, as key members of the innate immune system, have the ability to directly recognize and kill tumor cells without prior antigen sensitization, and are one of the important lines of defense in the body's anti-tumor immune system.

[0003] However, during tumor development and progression, NK cell function is often suppressed by the tumor microenvironment. Increased secretion of immunosuppressive cytokines, metabolic abnormalities, and upregulation of inhibitory ligands on the tumor cell surface all lead to decreased NK cell degranulation function and weakened cytotoxicity, thereby weakening the body's natural anti-tumor defense capabilities. While commonly used chemotherapy, radiotherapy, or immune checkpoint therapy can delay tumor progression, they are often accompanied by decreased NK cell function or immunosuppressive side effects. Therefore, finding a natural active substance that can safely and effectively activate NK cells and enhance their anti-tumor killing activity is of great significance.

[0004] Dendrobium officinale, a plant belonging to the genus Dendrobium in the Orchidaceae family, is one of China's traditional and precious medicinal herbs, known for its effects of "nourishing yin and clearing heat, promoting body fluid production and benefiting the stomach, and strengthening the body and prolonging life." Modern pharmacological studies have shown that polysaccharides contained in Dendrobium officinale are its main active ingredients, possessing various biological activities such as anti-oxidation, anti-tumor, immunomodulation, hypoglycemia, and anti-fatigue. 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 enhancing the body's immune function.

[0005] Existing research mainly focuses on the antioxidant, anti-inflammatory, and general immune-enhancing effects of Dendrobium officinale polysaccharides, but there is a lack of systematic research and published reports on whether they can directly enhance NK cell activation and their killing effect on tumor cells. Although traditional chemoimmunostimulants can increase NK activity in the short term, they often have problems such as large toxic side effects, poor tolerability, and limited clinical application.

[0006] Therefore, how to develop a natural polysaccharide preparation based on Dendrobium officinale that is naturally derived, has low toxicity, high safety, and can significantly activate NK cell function and enhance its killing power against tumor cells is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0007] Based on this, the present invention provides a Dendrobium officinale polysaccharide extract, its preparation method, and its application. The extract provided by the present invention is naturally derived, has low toxicity, and is highly safe. It can significantly enhance the immune activation and cytotoxic effects of NK cells, effectively improve the immunosuppressive state in the tumor microenvironment, thereby achieving the purpose of enhancing the anti-tumor effect of NK cells and inhibiting tumor growth.

[0008] A method for preparing a polysaccharide extract from Dendrobium officinale, using Dendrobium officinale stems as raw material, involves extraction by reflux extraction with purified water, ethanol precipitation, Sevag deproteinization, decolorization and deashing, and gel / membrane separation purification. The polysaccharide extract contains 50–95 wt% polysaccharides with an average molecular weight of 10–300 kDa. The monosaccharide components include mannose, glucose, galactose, fucose, rhamnose, and arabinose. The main chain of the Dendrobium officinale polysaccharide extract comprises the following two types of structural units:

[0009] .

[0010] The above preparation method, wherein the method specifically includes:

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] In the above preparation method, the molar ratio of mannose to glucose is (1.0-1.8):(1.0), and the proportion of galactose to total monosaccharides is 5-15%.

[0016] A polysaccharide extract from Dendrobium officinale was prepared by the method described above.

[0017] The above-mentioned application of Dendrobium officinale polysaccharide extract is used to prepare drugs that activate NK cell function and enhance the killing activity of NK cells against tumor cells.

[0018] 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.

[0019] According to the above applications, the drug is a lyophilized powder, oral liquid, injectable solution, or tablet.

[0020] 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.

[0021] 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.

[0022] Beneficial effects:

[0023] 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

[0024] Figure 1 The molecular weight distribution of Dendrobium officinale polysaccharide extract by gel permeation chromatography (GPC) is shown.

[0025] Figure 2 High-performance liquid chromatogram of the monosaccharide composition of Dendrobium officinale polysaccharide extract;

[0026] Figure 3 Fourier transform infrared spectrum of Dendrobium officinale polysaccharide extract;

[0027] Figure 4This is a graph showing the tumor cell count in the K562 control group.

[0028] Figure 5 This is a graph showing the results of NK cells killing K562 tumor cells in the negative control group.

[0029] Figure 6 The figure shows the results of the effect of Dendrobium officinale polysaccharide preparation in experimental group A on the killing of K562 tumor cells by NK cells;

[0030] Figure 7 The figure shows the effect of Dendrobium officinale polysaccharide preparation in experimental group B on the killing of K562 tumor cells by NK cells.

[0031] Figure 8 Flow cytometry image showing changes in CD107a expression on the surface of NK cells;

[0032] Figure 9 The image shows the Western blot results of perforin and granzyme B protein expression in NK cells after treatment with Dendrobium officinale polysaccharide preparation.

[0033] Figure 10 Western blot results of NKG2D protein expression in mouse spleen NK cells after treatment with Dendrobium officinale polysaccharide preparation;

[0034] Figure 11 Histological section of mouse spleen. Detailed Implementation

[0035] To facilitate understanding of the present invention, a more complete description will be given below with reference to various embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

[0036] 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 this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0037] Example 1: A method for preparing a polysaccharide extract from Dendrobium officinale.

[0038] 1. Preparation process

[0039] Take dried Dendrobium officinale stems, crush them through a 40-mesh sieve, weigh 1000 g, add 20 times the volume of purified water, reflux extract at 90℃ for 3 h, filter and take the supernatant; add 15 times the volume of purified water to the filter residue again, repeat the extraction twice, and combine the filtrates.

[0040] The combined extracts were concentrated under reduced pressure to 1 / 5 of their original volume. Four times the volume of 95% ethanol was slowly added, and the mixture was stirred until homogeneous. The mixture was then allowed to stand for 24 hours to allow the polysaccharides to precipitate completely. After centrifugation (4000 rpm, 10 min), the supernatant was discarded, and the precipitate was collected.

[0041] The precipitate was dissolved in an appropriate amount of distilled water, and the protein was removed using the Sevag method (chloroform: n-butanol = 4:1, volume ratio), and the treatment was repeated 3 times. The protein-removed aqueous solution was decolorized with activated carbon for 30 min, and after filtering out the activated carbon, it was dialyzed through a dialysis bag (molecular weight cutoff 8000~14000 Da) for 48 h, using deionized water as the dialysate, and the dialysate was changed every 8 h.

[0042] The dialysis liquid was concentrated under reduced pressure and then freeze-dried to obtain a light yellow powdery crude extract of Dendrobium officinale polysaccharides. Further processing was performed using DEAE-cellulose column chromatography to remove heteropolysaccharide components, followed by Sephadex G-100 gel column separation and purification. The main peak fraction was collected and freeze-dried to obtain the Dendrobium officinale polysaccharide extract.

[0043] 2. Physicochemical properties and monosaccharide composition analysis

[0044] (1) Determination of total sugar content

[0045] The total sugar content was determined using the phenol-sulfuric acid method.

[0046] Take 0.5 mL of the polysaccharide extract sample solution, add 0.5 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid, let stand at room temperature for 30 min, and measure the absorbance at 490 nm. Plot a standard curve using glucose as the standard.

[0047] The results showed that the polysaccharide content in the sample was (85.3±1.2) wt%, indicating that the prepared extract was a high-purity polysaccharide.

[0048] (2) Molecular weight determination

[0049] Gel permeation chromatography (GPC) was used with a TSK-GEL G3000PWxl column (7.8×300 mm), a mobile phase of 0.1 mol / L NaNO3, a flow rate of 0.6 mL / min, a temperature of 35℃, and a refractive index detector (RID).

[0050] The molecular weight distribution was calculated using the dextran standard curve, and the results are shown in [Figure number missing]. Figure 1 ,from Figure 1 It can be seen that the main peak is located at approximately 1.2 × 10⁻⁶. 5 The value at Da (120 kDa) indicates that the polysaccharide has a relatively concentrated and uniform molecular weight distribution, with a molecular weight range of approximately 10–300 kDa, indicating that the polysaccharide obtained by the extraction process has good structural uniformity.

[0051] (3) Monosaccharide composition analysis

[0052] High performance liquid chromatography (HPLC) with pre-column derivatization was employed.

[0053] The polysaccharide extract sample was hydrolyzed with 2 mol / L trifluoroacetic acid (TFA) at 120 °C for 2 h, dried under reduced pressure, and then derivatized with PMP (1-phenyl-3-methyl-5-pyrazolone) and heated under alkaline conditions for 40 min.

[0054] After removing excess PMP by ether extraction, the aqueous phase was filtered through a 0.22 μm filter membrane and loaded onto a C18 column (250 × 4.6 mm, 5 μm). The mobile phase was acetonitrile–0.1 mol / L phosphate buffer (pH 6.8) (17:83, v / v), the flow rate was 1.0 mL / min, and the detection wavelength was 245 nm.

[0055] Please see Figure 2 The results showed that the extract mainly consisted of mannose, glucose, galactose, and fucose, with small amounts of rhamnose and arabinose. The molar ratio of mannose, glucose, galactose, fucose, rhamnose, and arabinose was 3.2:2.5:1.0:0.6:0.3:0.2.

[0056] (4) FT-IR spectroscopy analysis

[0057] After the polysaccharide extract sample was ground and compressed with KBr, it was subjected to chromatography at 4000–400 cm⁻¹. -1 Scan within the wavenumber range. Figure 3 The results show:

[0058] 3400cm -1 The peak at this location corresponds to the hydroxyl stretching vibration.

[0059] 2920cm -1 The peak at this location corresponds to the C–H stretching vibration.

[0060] 1635cm -1 The peak at this location represents the absorption peak of bound water.

[0061] 1070cm -1 and 1020cm -1 The peaks at this point represent the C–O–C and C–O stretching vibrations, which are typical characteristic absorption peaks of polysaccharides.

[0062] The above results indicate that the obtained extract is a typical polysaccharide containing a pyranose ring structure.

[0063] (5) Nuclear magnetic resonance (NMR) analysis

[0064] The extract was dissolved in D2O and analyzed using Bruker 700 MHz NMR. 1 H, 13 C. Determination of COSY, HSQC, and HMBC.

[0065] According to the HSQC / HMBC cross peaks, the following can be observed:

[0066] Manp C1 / H1 signal δH 4.8–5.1 ppm (α / β mixture).

[0067] Glcp C1 / H1 signal δH 4.5–4.7 ppm.

[0068] Main chain C4 bit signal δC 79–82 ppm → Typical (1→4) connection characteristics.

[0069] Galp C6-bit branch signal δC 68–70 ppm.

[0070] No O-Ac signal (δH 2.0–2.2 ppm region is empty).

[0071] Based on the monosaccharide composition, methylation, and NMR results, the *Dendrobium officinale* polysaccharide extract obtained in this embodiment is a typical linear backbone polysaccharide composed of (1→4)-D-Manp and (1→4)-D-Glcp, with a small amount of Galp side chains linked at the O-6 positions of some Manp / Glcp. It also contains trace amounts of Fuc / Rha / Ara as end groups or side chains. The backbone mainly includes the following two types of structural units:

[0072] .

[0073] 3. Conclusion

[0074] Physicochemical analysis results show that the polysaccharide content of the Dendrobium officinale polysaccharide extract obtained in this embodiment is 50-95 wt%, the average molecular weight is distributed between 10 and 300 kDa, and the monosaccharide composition includes mannose, glucose, galactose, fucose, rhamnose and arabinose, proving that the extract prepared in this embodiment has a clear chemical composition and reproducibility.

[0075] Example 2

[0076] A pharmaceutically acceptable pharmaceutical formulation comprising Dendrobium officinale polysaccharide extract as described in Example 1 and pharmaceutically acceptable excipients.

[0077] The Dendrobium officinale polysaccharide extract was prepared by mixing it with excipients in the following weight ratios: 50 parts Dendrobium officinale polysaccharide extract, 10 parts mannitol, 5 parts trehalose, 5 parts sorbitol, and 30 parts phosphate buffer.

[0078] Mix all components thoroughly, adjust the pH to 7.2±0.1, filter sterile through a 0.22μm microporous membrane, and dispense into lyophilized bottles under sterile conditions, with each bottle containing 2 mL of liquid.

[0079] The dispensed samples were frozen at -40℃ for 4 hours, then transferred to a vacuum freeze dryer and freeze-dried at -60℃ and 20 Pa for 24 hours to obtain a light yellow, loose freeze-dried powder. The obtained freeze-dried powder was stored at 25℃ and 60% relative humidity for 6 months without clumping or discoloration, and its appearance and solubility remained stable.

[0080] When using, reconstitute with sterile water for injection or 0.9% NaCl solution; it dissolves rapidly to form a clear solution. Stability studies have shown that the pH, viscosity, and polysaccharide content remain stable after reconstitution, making it suitable for in vitro pre-activation culture of NK cells or in vivo animal immune enhancement experiments.

[0081] Example 3

[0082] This embodiment aims to verify the enhancing effect of the preparation obtained in Example 2 (hereinafter referred to as: Dendrobium officinale polysaccharide preparation) on the anti-tumor activity of natural killer (NK) cells. The human leukemia cell line K562 was used as the target cell to evaluate the in vitro killing efficiency of NK cells stimulated by Dendrobium officinale polysaccharide preparation.

[0083] 1. Experimental Materials and Methods

[0084] (1) Cell source and culture conditions

[0085] K562 cells were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in RPMI-1640 medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) at 37°C and 5% CO2.

[0086] Human peripheral blood mononuclear cells (PBMCs) were separated into NK cells (CD56) using magnetic bead sorting. + CD3 - They used the same culture system to maintain growth.

[0087] (2) Experimental grouping

[0088] Control group: K562 cells were inoculated only, without the addition of NK cells;

[0089] Negative control group: K562 cells were co-cultured with untreated NK cells;

[0090] Experimental group A: NK cells were pretreated with 250 μg / mL Dendrobium officinale polysaccharide preparation for 24 h, and then co-cultured with K562 cells.

[0091] Experimental group B: NK cells were pretreated with 500 μg / mL Dendrobium officinale polysaccharide preparation for 24 h, and then co-cultured with K562 cells.

[0092] (3) Co-culture and effect-target ratio setting

[0093] NK cells and K562 cells in each group were co-cultured for 4 h at effector-to-target ratios (E:T) of 4:1, 8:1, and 20:1. Each group was set up in triplicate.

[0094] (4) Flow cytometry detection

[0095] After culture, the apoptosis rate of K562 cells was detected by Annexin V-FITC / PI double staining combined with flow cytometry, and the NK cell-mediated cytotoxic killing rate was calculated. The expression of CD107a on the surface of NK cells was also detected simultaneously to reflect the level of degranulation activation.

[0096] 2. Experimental Results

[0097] Experimental results are as follows Figures 4 to 7 The results showed:

[0098] Under the condition of low target-to-effect ratio (4:1), Dendrobium officinale polysaccharide preparation has a promoting effect on NK cell killing of K562, but it is not statistically significant (p-value>0.05).

[0099] Under medium-to-high target ratios (8:1 and 20:1), NK cells pre-stimulated with Dendrobium officinale polysaccharide significantly increased their killing efficiency against K562 cells (p-value < 0.01), showing a dose-dependent increasing trend. Specifically:

[0100] At E:T = 8:1, the apoptosis rate of K562 cells in the 250 μg / mL treatment group increased from 32.5% ± 2.1% in the control group to 47.8% ± 2.6%, and further increased to 58.9% ± 3.3% in the 500 μg / mL treatment group.

[0101] At E:T = 20:1, the apoptosis rates of K562 were 62.7%±3.0% (250 μg / mL) and 74.2%±2.8% (500 μg / mL), respectively, which were significantly higher than those of the negative control group (approximately 45.1%±2.4%).

[0102] Meanwhile, flow cytometry results showed that the positivity rate of CD107a on the surface of NK cells increased by approximately 1.4-fold and 2.1-fold in the 250 μg / mL and 500 μg / mL treatment groups, respectively. Figure 8 As shown.

[0103] 3. Results and Conclusions

[0104] Experimental results showed that Dendrobium officinale polysaccharide preparations can significantly enhance the cytotoxic function of NK cells, and its effects are manifested as follows:

[0105] (1) Promotes NK cell degranulation response (CD107a upregulation);

[0106] (2) Under conditions of high efficiency target ratio, it significantly improves the killing efficiency of NK cells against K562 tumor cells, and the effect is more significant with increasing concentration of the preparation.

[0107] Therefore, the Dendrobium officinale polysaccharide preparation provided by the present invention can effectively activate the immune function of NK cells and enhance their killing activity against tumor cells, providing a safe, natural candidate preparation for tumor immunotherapy that significantly enhances NK function.

[0108] Example 4

[0109] To further verify the effect of Dendrobium officinale polysaccharide preparation on the cytokine secretion function of NK cells, this example tested the change in the proportion of IFN-γ positive cells after co-incubation with tumor target cells.

[0110] 1. Experimental Materials and Methods

[0111] (1) Cells and grouping

[0112] The NK cell source was the same as in Example 3. K562 cells were used as the target cells. The experiment was divided into:

[0113] Control group: K562 cells only;

[0114] Negative control group: K562+ untreated NK cells;

[0115] Experimental group A: NK cells were pre-stimulated with 250 μg / mL Dendrobium officinale polysaccharide preparation for 24 h;

[0116] Experimental group B: NK cells were pre-stimulated with 500 μg / mL Dendrobium officinale polysaccharide preparation for 24 h.

[0117] Subsequently, each group was incubated for 4 hours at effective-to-target ratios (E:T) of 4:1, 8:1, and 20:1.

[0118] (2) Golgi blockade and fixed permeability

[0119] After co-incubation for 30 min, Brefeldin A (final concentration 10 μg / mL) was added to block cytokine efflux. After incubation, cells were collected, washed once with PBS, fixed with 4% paraformaldehyde for 10 min, and then treated with commercial permeabilization buffer (Perm / Washbuffer) for 10 min.

[0120] (3) Antibody staining and flow cytometry detection

[0121] Surface markers CD3-FITC and CD56-PE were used, and intracellular staining was performed with IFN-γ-APC. The live / dead stain was 7-AAD. The staining steps were as follows: live / dead staining, surface staining, fixation and permeabilization, and intracellular staining.

[0122] Data were acquired using flow cytometry (BD FACSCanto II) after staining, with ≥30,000 lymphocyte events collected per sample. The gating strategy was: FSC / SSC → single cell → live cell → CD3 - CD56 + (NK cells) → IFN-γ + .

[0123] The positive threshold was determined using the FMO-IFN-γ control.

[0124] 2. Experimental Results

[0125] NK cells IFN-γ in the negative control group + The proportions are as follows:

[0126] 8%±2% (E:T = 4:1)

[0127] 12%±3% (E:T = 8:1)

[0128] 17%±4% (E:T = 20:1)

[0129] After stimulation by Dendrobium officinale polysaccharide preparations, IFN-γ + The proportion increased significantly and showed a concentration-dependent increasing trend, as shown in Table 1:

[0130] Table 1. IFN-γ after stimulation by Dendrobium officinale polysaccharide preparation + Proportion

[0131]

[0132] Statistical analysis showed that, compared with the negative control, the p-values ​​of the two treatment groups were <0.01 at E:T = 8:1 and 20:1, and <0.05 at E:T = 4:1.

[0133] 3. Results Analysis and Conclusions

[0134] Experimental results show that the Dendrobium officinale polysaccharide preparation can significantly enhance the ability of NK cells to produce IFN-γ after interacting with tumor target cells, and its effect increases with increasing dose and effector-to-target ratio. This result is consistent with the trend of upregulation of CD107a in NK cells in Example 3, further demonstrating that the preparation described in this invention can effectively enhance the activation level and anti-tumor immune function of NK cells.

[0135] Example 5

[0136] To further verify the molecular mechanism by which Dendrobium officinale polysaccharide preparations enhance the cytotoxic activity of NK cells, this embodiment uses Western blot to detect changes in the protein expression levels of perforin and granzyme B in NK cells.

[0137] 1. Experimental Materials and Methods

[0138] (1) Cells and grouping

[0139] The source and processing method of NK cells were the same as in Example 3. Three groups were set up:

[0140] Negative control group: No Dendrobium officinale polysaccharide preparation added;

[0141] Experimental group A: Stimulated with 250 μg / mL Dendrobium officinale polysaccharide preparation for 24 h;

[0142] Experimental group B: Stimulated with 500 μg / mL Dendrobium officinale polysaccharide preparation for 24 h.

[0143] Collect 3 × 10⁶ NK cells after stimulation ends 6 ~5×10 6 One sample per unit is used for protein extraction.

[0144] (2) Protein extraction and quantification

[0145] Cells were lysed on ice for 30 min using RIPA lysis buffer containing 1 mM PMSF, with shaking to mix every 10 min.

[0146] Centrifuge at 12,000×g for 15 min at 4℃ and collect the supernatant. Determine the protein concentration using the BCA method and adjust to 2 μg / μL with lysis buffer.

[0147] (3) SDS-PAGE and transfer

[0148] Take an equal amount of protein (20 μg / well) and mix it with 4× loading buffer at a ratio of 3:1, and denature at 95℃ for 5 min.

[0149] After electrophoresis on a 10% separating gel (120 V, 60-90 min), the samples were transferred to a PVDF membrane (300 mA, 60 min, 4 °C).

[0150] After transfer, the membrane was blocked with 5% skim milk powder TBST solution for 1 h.

[0151] (4) Antibody incubation

[0152] The PVDF membranes were incubated overnight at 4°C with the following primary antibodies:

[0153] Perforin (PRF1, approximately 67 kDa), diluted 1:1000;

[0154] Anti-granzyme B (GZMB, approx. 32 kDa), diluted 1:1000;

[0155] Internal control against β-actin (approximately 42 kDa), diluted 1:3000.

[0156] The next day, wash the membrane three times with TBST for 5 minutes each time. Add HRP-labeled secondary antibody (1:5000) and incubate at room temperature for 1 hour, then wash the membrane three more times.

[0157] (5) Color development and imaging

[0158] ECL chemiluminescence substrate was used for color development, and a gel imaging system was used to acquire band images. Gray-scale analysis was performed on the bands of perforin, granzyme B, and β-actin to calculate the relative expression levels (target protein gray value / β-actin gray value).

[0159] 2. Experimental Results

[0160] Please see Figure 9 Western blot results showed that, compared with the negative control group, NK cells treated with Dendrobium officinale polysaccharide preparations exhibited significantly deeper bands for perforin and granzyme B, with a dose-dependent increase. It should be noted that... Figure 9 In the diagram, negative control group-1 and negative control group-2 represent two experiments performed on the negative control group, experimental group A-1 and experimental group A-2 represent two experiments performed on experimental group A, and experimental group B-1 and experimental group B-2 represent two experiments performed on experimental group B.

[0161] In the 250 μg / mL treatment group, the relative expression levels of perforin and granzyme B increased by approximately 1.5-fold and 1.4-fold, respectively, compared to the control group.

[0162] In the 500 μg / mL treatment group, the relative expression levels of the two increased by approximately 2.2-fold and 2.0-fold, respectively.

[0163] The difference was statistically significant (p-value < 0.01).

[0164] The band analysis results were consistent with the increasing trends of CD107a degranulation rate and IFN-γ positivity rate in Examples 3 and 4, indicating that Dendrobium officinale polysaccharide preparation can enhance the killing function of NK cells by upregulating the protein expression of cytotoxic molecules perforin and granzyme B.

[0165] 3. Results and Conclusions

[0166] This embodiment demonstrates that the Dendrobium officinale polysaccharide preparation can significantly increase the protein levels of perforin and granzyme B in NK cells, and its effect increases with increasing concentration of the preparation. This further verifies that the Dendrobium officinale polysaccharide extract of the present invention can enhance the killing power of NK cells against tumor cells by promoting the expression of cytotoxic molecules, providing protein-level evidence for its anti-tumor immune enhancement mechanism.

[0167] Example 6

[0168] To verify the regulatory effect of Dendrobium officinale polysaccharide preparation on the expression level of NK cell activation receptor NKG2D in vivo, this embodiment uses a tumor-bearing mouse model to detect the effect of Dendrobium officinale polysaccharide preparation on NK cell NKG2D protein expression, so as to evaluate its mechanism of enhancing NK cell immune activity.

[0169] 1. Experimental Materials and Methods

[0170] (1) Laboratory animals and grouping

[0171] SPF-grade BALB / c nude mice (6–8 weeks old, weighing 18–22 g) were selected and housed under standard conditions. Logarithmically growing K562 human leukemia cells were collected, resuspended in PBS, and subcutaneously injected into the right axilla at a dose of 1 × 10⁻⁶ cells per mouse. 7 Each cell.

[0172] When the tumor volume was approximately 100 mm³, the tumors were randomly assigned to groups (n = 6 / group):

[0173] Control group: normal saline (intraperitoneal injection);

[0174] Low-dose group: Dendrobium officinale polysaccharide preparation 100 mg / kg (intraperitoneal injection);

[0175] High-dose group: Dendrobium officinale polysaccharide preparation 200 mg / kg (intraperitoneal injection).

[0176] The drug was administered once daily for 10 consecutive days. On the 10th day, the animals were sacrificed, and the spleen and tumor tissue were separated.

[0177] (2) NK cell isolation and protein extraction

[0178] Spleen was ground to prepare a single-cell suspension, and CD3 was obtained by magnetic bead sorting. - CD49b + NK cells.

[0179] After collecting NK cells, they were lysed on ice for 30 min using RIPA lysis buffer containing 1 mM PMSF, centrifuged (12,000×g, 15 min, 4℃) and the supernatant was collected. The protein concentration was quantified by BCA method and adjusted to 2 μg / μL.

[0180] (3) Western blot detection of NKG2D expression

[0181] SDS-PAGE and PVDF transfer were performed according to the method described in Example 5 above.

[0182] The anti-NKG2D antibody (1:1000) was incubated overnight on a shaker at 4°C, with β-actin as an internal control (1:3000).

[0183] After washing the membrane with TBST, add HRP-labeled secondary antibody (1:5000) and incubate at room temperature for 1 h.

[0184] ECL chemiluminescence was used for color development and image acquisition.

[0185] The grayscale values ​​were analyzed using ImageJ software to calculate the relative expression levels of NKG2D / β-actin.

[0186] (4) Immunohistochemical (IHC) detection

[0187] Spleen tissue was collected from sacrificed mice and fixed in 10% neutral formaldehyde solution for 24 h. After dehydration with graded ethanol, clearing with xylene, and embedding in paraffin, sections were prepared (4 μm thickness). The sections were baked at 60 °C for 2 h and then dewaxed to water. Endogenous peroxidase was blocked with 3% hydrogen peroxide solution at room temperature for 10 min. Antigen retrieval was then performed using citrate buffer (pH 6.0) (microwave heating for 10 min, followed by natural cooling), and the sections were washed three times with PBS.

[0188] Add 5% bovine serum blocking solution to the slides and incubate at room temperature for 30 min. Then add primary antibody: anti-NKG2D antibody (1:200, incubate overnight at 4°C). The next day, wash the membrane three times with PBS, add HRP-labeled secondary antibody (1:500), and incubate at room temperature for 1 h. Develop the membrane with DAB chromogenic solution, counterstain the cell nuclei with hematoxylin, and then dehydrate with ethanol, clear with xylene, and mount with neutral resin.

[0189] The images were observed and photographed under a microscope (×400). Five fields of view were randomly selected, and the area and average optical density (IOD) of the brownish-yellow positive signal were measured using Image-Pro Plus software. The results were expressed as IOD values ​​to reflect the expression level of NKG2D.

[0190] 2. Experimental Results

[0191] Please see Figure 10 Western blot results showed that the Dendrobium officinale polysaccharide preparation significantly upregulated the expression level of NKG2D protein in mouse spleen NK cells. It should be noted that... Figure 10 In the above, control group-1 and control group-2 represent two experiments performed on the control group, low-dose group-1 and low-dose group-2 represent two experiments performed on the low-dose group, and high-dose group-1 and high-dose group-2 represent two experiments performed on the high-dose group.

[0192] The NKG2D expression ratio (NKG2D / β-actin) in the control group was set to 1.

[0193] The low-dose group increased by 1.6 ± 0.2 times;

[0194] The high-dose group increased by 2.4 ± 0.3 times, and the difference was statistically significant (p-value < 0.01).

[0195] Please also see Figure 11 Histological analysis showed that the infiltration of K562 tumor cells in the spleen of mice treated with Dendrobium officinale polysaccharide preparation was significantly reduced, the tissue structure was relatively intact, and cell necrosis and apoptosis were significantly increased, suggesting that Dendrobium officinale polysaccharide preparation enhances the in vivo anti-tumor immune effect by upregulating NKG2D to mediate NK cell activation.

[0196] 3. Results and Conclusions

[0197] This embodiment demonstrates that Dendrobium officinale polysaccharide preparations can significantly upregulate the protein expression level of NKG2D, the NK cell surface activation receptor, in vivo, thereby activating NK cell function and enhancing the ability of NK cells to recognize and kill tumor cells.

[0198] This result is consistent with the in vitro functional verification results of Examples 3-5, further confirming that Dendrobium officinale polysaccharide achieves NK cell-mediated anti-tumor effects by promoting NKG2D pathway activation, providing molecular-level in vivo evidence for its application in immune enhancement and anti-tumor drug development.

[0199] The experimental results above demonstrate that the *Dendrobium officinale* polysaccharide extract provided by this invention exhibits significant immune activation and antitumor effects, as verified by in vitro and in vivo experiments. This extract is naturally derived, highly safe, and can dose-dependently promote the degranulation response of natural killer (NK) cells (CD107a upregulation) under conditions without significant 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 the NK cell surface activation receptor NKG2D. In vivo experiments further demonstrate that treatment with the *Dendrobium officinale* polysaccharide preparation can reduce the infiltration of K562 tumor cells in the spleen, improve tissue structural integrity, and increase tumor cell necrosis and apoptosis, exhibiting a significant immune-dependent antitumor effect.

[0200] In summary, 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 a natural source, which is safe and non-toxic, and the extraction process is controllable and highly safe.

[0201] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing Dendrobium candidum polysaccharide extract, characterized in that, The polysaccharide extract from *Dendrobium officinale* contains 50–95 wt% polysaccharides with an average molecular weight of 10–300 kDa. The monosaccharide components include mannose, glucose, galactose, fucose, rhamnose, and arabinose. The main chain of the *Dendrobium officinale* polysaccharide extract comprises the following two types of structural units: ； The method 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.

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

3. A Dendrobium candidum polysaccharide extract, characterized in that, Prepared by the method according to claim 1 or 2.

4. The use of the Dendrobium candidum polysaccharide extract according to claim 3, characterized in that, The Dendrobium officinale polysaccharide extract is used to prepare a drug that, in a dose-dependent manner, promotes the degranulation of natural killer cells CD107a and increases the expression of IFN-γ, perforin, and granzyme B, upregulates the NKG2D pathway, thereby activating NK cell function and enhancing the killing activity of NK cells against K562 tumor cells.

5. Use according to claim 4, characterized in that, The drug is a lyophilized powder, oral liquid, injectable solution, or tablet.

6. Use according to claim 4, 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.

7. Use according to claim 6, characterized in that, The freeze-drying protectant is mannitol and / or trehalose, the buffer is phosphate buffer, and the stabilizer is sorbitol or fructooligosaccharide.

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