Dendrobium nobile polysaccharide component group, preparation method thereof and application thereof in immunoregulation

Abstract

The application discloses a Dendrobium nobile Lindl polysaccharide component group, a preparation method thereof and application of the Dendrobium nobile Lindl polysaccharide component group in immunoregulation. Four polysaccharide components with definite structures, namely DNP-0-1 (>100 kDa), DNP-0-2 (30-100 kDa), DNP-0-3 (<30 kDa) and DNP-1-1 (<50 kDa), are synchronously separated from Dendrobium nobile Lindl by combining ultrasonic extraction process optimized by a response surface method with DEAE-cellulose column chromatography and ultrafiltration membrane series grading technology. After structure characterization and in-vitro immunological activity evaluation, the four components all have immunoregulatory activity, and the activity of DNP-0-1 is optimal. Pearson correlation analysis shows that the content of galactose and the molecular weight are extremely significantly positively correlated with the immunological activity, and the content of glucose and mannose is significantly negatively correlated with the activity. The application establishes an integrated research system of 'preparation by grading-structure characterization-activity screening-structure-activity analysis', and provides a material basis and evaluation basis for the development of immunoregulatory drugs or functional foods.

Description

Technical Field

[0001] This invention belongs to the field of natural drug extraction and biomedicine technology, specifically involving four polysaccharide components with well-defined structural characteristics obtained by simultaneous isolation and purification from Dendrobium nobile Lindl., their preparation method, systematic structural characterization, evaluation of immunomodulatory activity, and a study on the structure-activity relationship of the polysaccharides based on Pearson correlation analysis. Background Technology

[0002] Dendrobium nobile, a plant belonging to the genus Dendrobium in the Orchidaceae family, is one of the traditional and precious Chinese medicinal herbs. The polysaccharides contained in its stems have been proven to possess various biological activities, including immunomodulation, antioxidation, and anti-inflammation. However, existing research mostly focuses on crude polysaccharides or single purified fractions from Dendrobium nobile, lacking studies on the systematic fractionation and preparation of polysaccharides from the same source. Polysaccharide fractions with different molecular weight ranges and charge properties are often mixed together, making it difficult to attribute activity evaluation results to specific structural features. Although ultrasound-assisted extraction can improve polysaccharide extraction efficiency, the lack of systematic optimization of process parameters leads to insufficient extraction rate and polysaccharide structural stability. Furthermore, crude polysaccharides contain impurities such as proteins, pigments, and starch, affecting purity and activity evaluation; existing purification methods struggle to accurately obtain structurally homogeneous fractions.

[0003] The structural characteristics of polysaccharides, such as molecular weight, monosaccharide composition, glycosidic bond type, and conformation, are closely related to their immunomodulatory activity. Although *Dendrobium nobile* polysaccharides possess immunomodulatory potential, the structures of their active components and their mechanisms of action are unclear. Therefore, it is urgent to establish an efficient, stable, and controllable extraction and purification process for *Dendrobium nobile* polysaccharides to obtain structurally defined active components and elucidate their mechanisms of action in immunomodulation, thereby promoting their application in pharmaceuticals and functional foods. Summary of the Invention

[0004] This invention aims to provide an efficient and green ultrasonic extraction process for Dendrobium nobile polysaccharides, establish a graded purification system, clarify the structural characteristics of each component, and screen out the advantageous components with immunomodulatory activity, providing a scientific basis for their application in the development of immune-related products.

[0005] This invention provides a polysaccharide component group from Dendrobium nobile, the component group comprising four polysaccharide components with clearly defined structural characteristics, namely:

[0006] (1) Neutral polysaccharide component DNP-0-1: weight average molecular weight greater than 100 kDa, galactose molar ratio ≥50%;

[0007] (2) Neutral polysaccharide component DNP-0-2: weight average molecular weight is 30~100 kDa;

[0008] (3) Neutral polysaccharide component DNP-0-3: weight average molecular weight less than 30 kDa;

[0009] (4) Weakly acidic polysaccharide component DNP-1-1: weight average molecular weight less than 50 kDa, containing uronic acid;

[0010] The four polysaccharide components were simultaneously separated from Dendrobium nobile using DEAE-cellulose column chromatography combined with ultrafiltration membrane tandem fractionation technology.

[0011] Furthermore, the structural characterization of each component revealed the following characteristics:

[0012] (1) DNP-0-1: weight average molecular weight 1.13×10^5 Da, total sugar content ≥86%, uronic acid content ≤0.7%; galactose molar ratio in monosaccharide composition ≥50%, and contains mannose; infrared spectrum shows characteristic absorption peak of β-glycosidic bond at 878 cm⁻¹; conformational parameter α=0.35, exhibiting a relaxed random coil conformation; scanning electron microscopy shows a fibrous network structure; thermal degradation temperature Tdmax=282.7℃;

[0013] (2) DNP-0-2: weight-average molecular weight is 3.06×10 4 The Da and 2.15×10³ Da bimodal distribution; the monosaccharide composition includes galactose, mannose and glucose; the conformational parameter α=0.16, exhibiting a tight coil conformation;

[0014] (3) DNP-0-3: weight-average molecular weight 1.50×10 4 Da; the monosaccharide composition includes galactose, arabinose, and mannose; the conformational parameter α=0.17, exhibiting a close globular conformation; atomic force microscopy shows an aggregate height of 2 nm;

[0015] (4) DNP-1-1: weight-average molecular weight is 3.72×10 4 The Da and 5.94×10³ Da double peak distribution; the uronic acid content is 2.7%; the mannose molar ratio in the monosaccharide composition is 62.7%, and it also contains glucose; the infrared spectrum has a characteristic absorption peak of esterification at 1738 cm⁻¹.

[0016] The present invention also provides a method for preparing the above-mentioned Dendrobium nobile polysaccharide components, which is carried out in the following steps:

[0017] (1) The raw material of Dendrobium nobile was crushed and defatted, and extracted by ultrasonic-assisted water extraction: the liquid-solid ratio was controlled at 20:1 to 25:1 mL / g, the extraction temperature was 70-75℃, the extraction time was 75-80 min, and the ultrasonic power was 450-470 W.

[0018] (2) The extract was centrifuged to collect the supernatant, concentrated, precipitated with alcohol, and centrifuged to collect the precipitate. The starch was then removed by enzymatic hydrolysis and dialysis to obtain crude polysaccharide from Dendrobium nobile.

[0019] (3) The crude polysaccharide was separated by DEAE-52 cellulose anion exchange column chromatography, and eluted with distilled water and 0.1 M NaCl solution respectively. The water elution fraction DNP-0 and the 0.1 M NaCl elution fraction DNP-1 were collected respectively.

[0020] (4) The DNP-0 components were sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 100 kDa and 30 kDa for tandem fractionation. The >100 kDa cutoff liquid, the 30-100 kDa cutoff liquid and the <30 kDa permeate were collected respectively. After freeze-drying, neutral polysaccharide components DNP-0-1, DNP-0-2 and DNP-0-3 were obtained.

[0021] (5) Pass the DNP-1 component through an ultrafiltration membrane with a molecular weight cutoff of 50 kDa, collect the permeate with a molecular weight cutoff of <50 kDa, and freeze-dry it to obtain the weakly acidic polysaccharide component DNP-1-1.

[0022] Furthermore, the optimal process parameters for ultrasonic-assisted extraction in step (1) were determined by Box-Behnken design optimization using response surface methodology. Specifically, the liquid-to-solid ratio was 21 mL / g, the extraction time was 77 min, the extraction temperature was 73℃, and the ultrasonic power was 460 W. The coefficient of determination of the regression model was R²=0.9900, the model P<0.0001, and the lack-of-fit term P=0.7122.

[0023] This invention also provides a method for analyzing the structure-activity relationship of polysaccharides from Dendrobium nobile. Using the four polysaccharide components as research objects, the method reveals the intrinsic relationship between polysaccharide structure and activity by measuring the structural characteristic parameters and immunomodulatory activity indicators of each component and employing Pearson correlation analysis. The structural characteristic parameters include: weight-average molecular weight, polydispersity index, molar ratio of monosaccharide composition, conformational parameter α, thermal degradation temperature, and microscopic morphology. The immunomodulatory activity indicators include: RAW264.7 macrophage proliferation rate, nitric oxide release, tumor necrosis factor-α release, and interleukin-6 release.

[0024] The Pearson correlation analysis results showed that galactose content was significantly positively correlated with the release of NO, TNF-α, and IL-6, with correlation coefficients r ≥ 0.94 and P < 0.01; weight-average molecular weight was significantly positively correlated with the release of IL-6, with r = 0.79 and P < 0.05; and glucose and mannose content were significantly negatively correlated with immune activity indicators, with r ≤ -0.91 and P < 0.05.

[0025] Compared with the prior art, the present invention has the following beneficial effects.

[0026] (1) Innovation in separation and purification technology

[0027] This invention is the first to use DEAE-cellulose column chromatography combined with ultrafiltration membrane tandem fractionation technology to simultaneously separate four polysaccharide components with distinct structural characteristics from Dendrobium nobile. This achieves efficient and precise separation of polysaccharide components with different charge properties and molecular weight ranges, overcoming the shortcomings of traditional methods such as mixed polysaccharide components and incomplete structural characterization.

[0028] (2) The extraction process is optimized and reliable.

[0029] The ultrasonic-assisted extraction process was systematically optimized using response surface methodology, and the optimal extraction parameters (liquid-to-solid ratio 21:1 mL / g, extraction time 77 min, extraction temperature 73℃, and ultrasonic power 460 W) were established, resulting in a polysaccharide extraction rate of 2.43%. The process was stable and reproducible, providing a reliable guarantee for the efficient preparation of Dendrobium nobile polysaccharides.

[0030] (3) Comprehensive structural characterization system

[0031] The four polysaccharide components were systematically characterized in terms of physicochemical properties and structure. For the first time, key structural features such as molecular weight distribution, monosaccharide composition, glycosidic bond configuration, chain conformation, microstructure and thermal stability of each component were revealed, laying a material basis for the study of structure-activity relationship of polysaccharides.

[0032] (4) The dominant immune-active components are clearly identified.

[0033] In vitro immunomodulatory activity evaluation confirmed that all four polysaccharide components had immunomodulatory activity, among which the high molecular weight neutral polysaccharide component DNP-0-1 had the best activity, which could significantly promote the proliferation of macrophage RAW264.7 and the release of immune factors such as NO, TNF-α, and IL-6, indicating that it has great potential in the preparation of immunomodulatory drugs or functional foods.

[0034] (5) In-depth analysis of structure-function relationship

[0035] For the first time, Pearson correlation analysis was used to systematically reveal the intrinsic relationship between the structural characteristics of Dendrobium nobile polysaccharides and their immunomodulatory activity: galactose content and molecular weight were significantly positively correlated with immunomodulatory activity (r≥0.94, P<0.01), while glucose and mannose content were significantly negatively correlated with activity (r≤-0.91, P<0.05). Based on this, a predictive index of highly active polysaccharides was established using galactose content ≥50%, molecular weight >100 kDa, and conformational parameter α≥0.35, providing a new model for the precise screening of active components based on the structure-activity relationship of polysaccharides.

[0036] (6) Innovation of integrated research system

[0037] This invention establishes an integrated research system of "graded preparation - structural characterization - activity screening - structure-activity analysis", which not only provides a scientific basis for the in-depth development of Dendrobium nobile polysaccharides, but also provides a methodological reference for the screening of active components and structure-activity relationship studies of polysaccharides from other natural sources.

[0038] In summary, this invention has significant innovation and practical value in the extraction, separation and purification, structural analysis, activity evaluation and application of Dendrobium nobile polysaccharides, and is expected to promote the industrial application of Dendrobium nobile polysaccharides in the fields of medicine and functional food. Attached Figure Description

[0039] Figure 1 This is a single-factor experimental analysis diagram of the effect of ultrasonic extraction on the yield of polysaccharides from Dendrobium nobile;

[0040] Figure 2 This is a response surface methodology diagram showing the effect of ultrasonic extraction on the yield of polysaccharides from Dendrobium nobile.

[0041] Figure 3 This is the elution curve of crude polysaccharide from Dendrobium nobile on a DEAE-52 column;

[0042] Figure 4 These are the UV-Vis absorption spectra of polysaccharides from different components of Dendrobium nobile;

[0043] Figure 5 These are Fourier transform infrared spectra of polysaccharides from different components of Dendrobium nobile;

[0044] Figure 6 This is a monosaccharide composition ion chromatogram of different polysaccharide components of Dendrobium nobile;

[0045] Figure 7 These are SEC-MALLS chromatograms of different components of Dendrobium nobile polysaccharides;

[0046] Figure 8 These are electron microscopy scan results of polysaccharides from different components of Dendrobium nobile;

[0047] Figure 9 Thermogravimetric analysis of polysaccharides from different components of Dendrobium nobile;

[0048] Figure 10 The effect of different polysaccharide components of Dendrobium nobile on the viability of RAW264.7 cells;

[0049] Figure 11 The effect of different polysaccharide components of Dendrobium nobile on NO release in RAW264.7 cells;

[0050] Figure 12 The effect of different polysaccharide components of Dendrobium nobile on TNF-α release in RAW264.7 cells;

[0051] Figure 13 The effect of different polysaccharide components of Dendrobium nobile on IL-6 release in RAW264.7 cells;

[0052] Figure 14 This is a heatmap of Pearson correlation analysis between the structural characteristics of Dendrobium nobile polysaccharides and their immunomodulatory activities. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the invention.

[0054] Example 1: Single-factor optimization of ultrasonic-assisted hot water extraction process of Dendrobium nobile polysaccharides

[0055] (1) Effect of material-liquid ratio on the extraction rate of Dendrobium nobile polysaccharides: Degreased and dried Dendrobium nobile powder was weighed, extracted for 2 h at 60℃, and ultrasonically at 240 W. The effects of different material-liquid ratios (1:10, 1:15, 1:20, 1:25, 1:30 (g / mL)) on the extraction rate were investigated. The extraction rate of Dendrobium nobile polysaccharides was calculated according to the following formula (1) using the phenol-sulfuric acid method. Figure 1 A. The results showed that the polysaccharide yield first increased and then decreased with the increase of the material-to-liquid ratio, reaching a peak at 1:20 (i.e., 20 mL / g). Subsequent optimization selected a range of 15-25 mL / g.

[0056] (1)

[0057] Where: X: Extraction rate of Dendrobium nobile flower polysaccharides (%); v: Volume of the test solution (mL); c: Polysaccharide content of the Dendrobium nobile sample solution (μg / mL); D: Solution dilution factor; M: Sample mass (g)

[0058] (2) Effect of ultrasonic time on the extraction rate of polysaccharides from Dendrobium nobile: The material-to-liquid ratio was 1:20, the extraction temperature was 60℃, the extraction was performed once, and the ultrasonic power was 240 W. The effect of different ultrasonic times of 30, 60, 90, 120, and 150 min on the extraction rate was investigated. Figure 1 B. The results showed that the yield continued to increase within 90 minutes, but decreased significantly after 90 minutes. Further optimization will focus on a range of 60-120 minutes.

[0059] (3) Effect of extraction temperature on the extraction rate of Dendrobium nobile polysaccharides: The extraction time was 90 min, extraction was performed once, the material-to-liquid ratio was 1:20, and the ultrasonic power was 240 W. The effect of different extraction temperatures (40, 50, 60, 70, and 80℃) on the extraction rate was investigated. Figure 1C. The results showed that the yield continued to increase within 70℃, but the increase slowed down or even slightly decreased after exceeding 70℃. Further optimization will focus on a temperature range of 60-80℃.

[0060] (4) Effect of ultrasonic power on the extraction rate of Dendrobium nobile polysaccharides: The extraction temperature was 70℃, the extraction was performed once, the material-to-liquid ratio was 1:20, and the extraction time was 90 min. The effects of different ultrasonic powers (240, 300, 360, 420, and 480 W) on the extraction rate were investigated. Figure 1 D. The results showed that the yield continuously increased within 420 W, but declined after exceeding 420 W. Further optimization was performed within the range of 360-480 W.

[0061] Example 2: Optimization and Validation of the Extraction Process of Dendrobium nobile Polysaccharides Based on Response Surface Methodology

[0062] (1) A four-factor, three-level experiment was conducted using a Box-Behnken design, with a total of 29 experimental groups (including 5 central point replicates). The factor and level codes are shown in Table 1, and the experimental design and data results are shown in Table 2. The experimental results were subjected to multivariate quadratic fitting analysis, and the regression equations (2) for the polysaccharide extraction rate (Y) of Dendrobium nobile and the influencing factors of extraction material-liquid ratio (A), extraction time (B), extraction temperature (C), and ultrasonic power (D) are as follows:

[0063] Y=2.49+0.04 A-0.0192 B+0.0475 C+ 0.05 D-0.04 AB-0.05 AC+0.035 AD-0.03BC-0.0375 BD +0.0175 CD-0.1672 A 2 -0.0734 B 2 -0.1209 C 2 -0.0672 D 2 (R) 2 =0.99) (2)

[0065] Table 1. Response Surface Design Experimental Factors and Level Coding

[0066]

[0067] Table 2 Response Surface Experiment Scheme and Data Results

[0068]

[0069] (2) Analysis of variance: Statistical analysis of variance was performed on the quadratic regression model, and the results are as follows: Figure 2As shown in Table 3, the p-value of this model is < 0.0001, and the F-value is 98.68 > 0.05, indicating that the model has a certain degree of significance. Furthermore, the lack-of-fit term is not significant (P = 0.7122, P > 0.05). The order of influence of each factor on the extraction rate is: ultrasonic power (D) > temperature (C) > material-to-liquid ratio (A) > ultrasonic time (B). The quadratic terms A², B², C², D² and the interaction terms AB, AC, AD, BC, BD all have a highly significant effect on the extraction rate (P < 0.01).

[0070] Table 3. Results of Analysis of Variance for Response Surface

[0071]

[0072] Note: * indicates a significant difference (P < 0.05), ** indicates an extremely significant difference (P < 0.01).

[0073] (3) Process Optimization and Verification: Based on the above regression model, optimization analysis was performed using Design-Expert 13 software. With the goal of maximizing the polysaccharide extraction rate, the theoretically optimal process parameters for ultrasonic-assisted hot water extraction of Dendrobium nobile polysaccharides were obtained (Table 4). To facilitate practical operation, the theoretical parameters were rounded to: material-to-liquid ratio 1:21, time 77 min, temperature 73°C, and power 460 W. Under these conditions, three parallel experiments were conducted, and the polysaccharide extraction rate was 2.43 ± 0.05%, with a relative error of 3.18% (< 5%) compared to the model prediction (2.51%). This indicates that the model prediction is accurate, the optimized process is reliable, and the reproducibility is good. All subsequent extraction experiments were strictly conducted under these conditions.

[0074] Table 4 Predictive Experiment Results

[0075]

[0076] Example 3: Preparation of Dendrobium nobile polysaccharides

[0077] Extraction was performed under optimal conditions based on response surface methodology: a solid-liquid ratio of 1:23 (w / v), an ultrasonic temperature of 73℃, an ultrasonic power of 420 W, and an extraction time of 73 min. The mixture was manually stirred every 10 min during extraction. Centrifugation (3500 rpm, 10 min) was used to remove insoluble impurities, and the supernatant was collected. This yielded an ultrasonic-assisted hot water extract of Dendrobium nobile polysaccharides.

[0078] Example 4: Isolation and purification of Dendrobium nobile polysaccharides

[0079] (1) First, pack the packing material into a 2.5 cm × 55 cm chromatography column and equilibrate with distilled water at a flow rate of 1.5 mL / min for 12 h. Accurately weigh 300 mg of crude polysaccharide from Dendrobium nobile, dissolve it in 30 mL of distilled water, remove impurities through a 0.45 μm aqueous filter membrane, and then load the sample. Elute sequentially with distilled water, 0.1 mol / L, 0.3 mol / L, and 0.5 mol / L NaCl solutions at a flow rate of 1.5 mL / min, collecting one tube every 8 min with an automatic collector. Determine the total sugar content of each tube using the phenol-sulfuric acid method, and measure the absorbance at 490 nm using an ELISA reader to plot the elution curve (e.g., ...). Figure 3 Based on the elution curve, the corresponding polysaccharide solutions were collected, concentrated by rotary evaporation, dialyzed with distilled water for 3 days (using a 3500 Da dialysis bag), and finally freeze-dried to obtain polysaccharide powders with different charge properties.

[0080] (2) The neutral water-washed fraction DNP-0 was sequentially fractionated using polyethersulfone (PES) ultrafiltration membranes with molecular weight cutoffs of 100 kDa and 30 kDa. The weakly acidic fraction DNP-1, eluted with 0.1 mol / L NaCl, was separated using a PES ultrafiltration membrane with a molecular weight cutoff of 50 kDa. The ultrafiltration process was carried out at room temperature, with the operating pressure kept constant at 0.2 MPa. A cross-flow filtration mode was used to reduce membrane fouling and improve separation efficiency. The retentate and permeate from each separation stage were collected separately, dialyzed through a dialysis bag with a molecular weight cutoff of 3500 Da for desalination, concentrated, and freeze-dried to finally obtain polysaccharide subfractions with different molecular weight ranges.

[0081] Example 5: Ultraviolet and Infrared Spectroscopic Analysis of Polysaccharides from Different Components of Dendrobium nobile

[0082] (1) Dissolve 5 mg of the sample in distilled water to a concentration of 0.5 mg / mL, using the distilled water as a blank to prepare the test solution. The UV-Vis absorption spectrum of DNP was recorded in the wavelength range of 200-400 nm using a UV-spectrophotometer. The results are as follows: Figure 4 The spectral curves of the polysaccharide samples tend to be smooth, indicating that the impurities in the purified samples have been basically removed.

[0083] (2) Weigh 40 mg of dry potassium bromide powder and grind it thoroughly into a fine powder in an agate mortar. Add 2 mg of polysaccharide sample powder, mix well, and grind thoroughly. Use a tablet press to form transparent sheets at 4000-400 cm⁻¹. -1 Infrared spectral scanning was performed within the range. Results are as follows: Figure 5The infrared spectra of the four components were basically consistent. The broad and strong absorption band at 3389 cm⁻¹ was attributed to the stretching vibration of the hydroxyl (OH) groups in the polysaccharide molecule. The absorption signal at 2933 cm⁻¹ was characteristic of the stretching vibration of the CH bonds in the CH, CH2, and CH3 groups (symmetric stretching of the CH bonds in the pyranose ring). Compared with the other three components, the DNP-1-1 polysaccharide sample showed a stronger absorption band at 1738 cm⁻¹. -1 A relatively weak signal peak is observed, indicating trace amounts of acetylation or esterification. The spectral profile shows a weak absorption at 1649 cm⁻¹, indicating the presence of both asymmetric and symmetric stretching vibration absorption peaks at C=O. (1416 cm⁻¹) -1 The band at this point is attributed to CH bending vibration. 1000-1200 cm -1 The stretching vibrations of the COC and COH of the pyranose ring are present. The FT-IR absorption peak at 878 cm⁻¹ indicates the presence of β-configured glycosidic bonds in different polysaccharide components.

[0084] Example 6: Determination of polysaccharide and monosaccharide composition of different components of Dendrobium nobile

[0085] Monosaccharide composition was determined using high-performance anion exchange chromatography (HPAEC). Accurately weigh 10.00 mg of standards for fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), galacturonic acid (GalA), glucuronic acid (GlcA), glucosamine (GlcN), and galactosamine (GalN). Dissolve each standard in water to prepare a 10 mg / mL standard stock solution. Then, take an appropriate amount of the stock solution to prepare a 50 µg / mL standard mixture. HPAEC analysis was performed. 5 mg of the polysaccharide sample was added to 2 mL of 3 mol / L trifluoroacetic acid (TFA) and hydrolyzed in a sealed container at 120 °C for 3 h to obtain the sample hydrolysate. To thoroughly remove acid solution and other impurities, residual acid solution was removed by adding nitrogen gas. The sample was then washed with methanol solution and dried with nitrogen gas, a process repeated three times to ensure cleaning effectiveness. Finally, 5 mL of sterile water was added to dissolve the sample, and 200 µL of the solution was added to 800 µL of ultrapure water. The mixture was centrifuged at 12000 rpm for 5 min, and the supernatant was filtered through a 0.22 µm microporous membrane for HPAEC analysis.

[0086] Chromatographic column: Dionex Carbopac™ PA20 (3 mm × 150 mm, 5 μm) column, column temperature 30℃, injection volume: 25 µL; flow rate: 0.3 mL / min; mobile phase A: H2O; B: 15 mM NaOH; C: 15 mM NaOH and 100 mM NaOAc; elution gradient as follows: 0 min A / B / C (98.8:1.2:0, v / v / v), 18 min A / B / C (98.8:1.2:0, v / v / v), 20 min A / B / C (30:70:0, v / v / v), 30 min A / B / C (30:70:0, v / v / v), 30.1 min A / B / C (0:0:100, v / v / v), 46 min A / B / C (0:0:100, v / v / v), 46.1 min A / B / C (0:100:0, v / v / v), 50 min A / B / C (0:100:0, v / v / v), 50.1 min A / B / C (98.8:1.2:0, v / v / v), 80 min A / B / C (98.8:1.2:0, v / v / v). Detector: Electrochemical detector.

[0087] The results showed that the different components exhibited significant differences in the molar ratio of monosaccharides. For example... Figure 6 Glucose (Glc), mannose (Man), and galactose (Gla) are stably present in all components, collectively forming the basic framework of Dendrobium nobile polysaccharides. Notably, DNP-0-1 and DNP-0-3 have the highest galactose (Gal) molar ratios, at 0.515 and 0.530 respectively, the most prominent among all components, resulting in a galactose-enriched structure. Conversely, DNP-1-1 is dominated by mannose (0.627) and glucose (0.306) monosaccharides, with a significantly low galactose content.

[0088] Table 2-5 Analysis of polysaccharide and monosaccharide composition of different components of Dendrobium nobile

[0089]

[0090] Note: "Fuc, GalN, Rha, Ara, GlcN, Gal, Glc, Xyl, Man, GalA, and GlcA" represent fucose, galactosamine, rhamnose, arabinose, glucosamine, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid, respectively. The symbol " / " indicates that the value was not detected.

[0091] Example 7: Determination of molecular weight and conformation of polysaccharides from different components of Dendrobium nobile

[0092] The molecular weight and distribution of various components of Dendrobium nobile polysaccharides were determined using a high-performance gel permeation chromatography-differential refractive index-multi-angle laser light scattering (HPSEC-RID-MALLS) system. The instrument setup included a high-performance liquid chromatograph (2695e, Waters), a guard column (OHpak SB-G, Shodex, Japan), an SB-806M analytical column (300 mm × 7.8 mm, Shodex, Japan), a multi-angle laser light scattering detector, and a differential refractive index detector (DAWN HELEOS II, Wyatt Technology, USA). The laser light scattering system coupled with the SEC column accurately determined the weight-average molecular weight (Mw), number-average molecular weight (Mn), and their distribution of Dendrobium nobile polysaccharides. The results are shown in the table. The test conditions were as follows: 0.05 mol / L NaCl solution was prepared using ultrapure water as the mobile phase; the column temperature was 35℃; the injection volume was 100 μL; isocratic elution was performed at a flow rate of 0.5 mL / min; data collection time was 60 min; the differential refractive index increment (dn / dc) was 0.138 mL / g; and the average molecular weight and polydispersity (Mw / Mn) of the polysaccharide were characterized using ASTRA 8.2.2 software. Based on the simultaneously acquired LS and RI chromatograms and molecular weight distribution data, such as... Figure 7 .

[0093] Table 2-6 Molecular weight and conformation of polysaccharides from different components of Dendrobium nobile

[0094]

[0095] Example 8: Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) Analysis

[0096] (1) After passing a certain amount of dried polysaccharide powder through a 100-mesh sieve, the sample is adhered to a sample tray using conductive adhesive. Excess powder is blown away with a bulb syringe, followed by gold sputtering in a vacuum sputtering machine. The sample is then scanned under a scanning electron microscope at 500x magnification, with adjustments made to the contrast and brightness. Figure 8 A, DNP-0-1 exhibits an interlinked fibrous network structure, consistent with theoretical predictions of the extended random coil conformation exhibited by high molecular weights. DNP-0-2 is a dense, irregular, blocky aggregate, while DNP-0-3 and DNP-1-1 are smaller fragments or short fibrous morphologies.

[0097] (2) Dissolve the polysaccharide in distilled water to prepare a 5 μg / mL polysaccharide solution. After complete dissolution, centrifuge at 5000 rpm for 5 min, collect the supernatant, filter through a microporous membrane (0.45 pm), and drop an appropriate amount of solution onto a clean mica sheet. Immediately fix the solution with a glass slide, air dry at room temperature, and then perform the measurement. Measurement conditions: relative humidity 65%, room temperature, cantilever elasticity coefficient 1.6 N / m, cantilever length 85 pm, image scanning speed 600 pm / s, resonance frequency 1 Hz. Figure 8 B, 8C. The results show the particle diameter range and average height data measured by AFM images, listed in the table. Among them, DNP-0-1 and DNP-1-1 exhibited a larger average height (5 nm) and a wider diameter distribution, possibly due to the molecular chains not being extended on the substrate or the presence of multilayer stacking. DNP-0-3 had the lowest height (2 nm) and the narrowest diameter distribution (29.0-52.2 nm, spanning 23.2 nm), consistent with the compact spherical conformation and the small fragment morphology under SEM.

[0098] Table 2-7 AFM diameter range and height data of different polysaccharide components from Dendrobium nobile

[0099]

[0100] Example 9: Thermogravimetric Analysis

[0101] Thermogravimetric analysis (TGA) and differential thermogravimetric analysis (DTG) of different polysaccharide components from *Dendrobium nobile* were performed using a thermal analyzer to observe their thermal properties. Approximately 5 mg of polysaccharide sample was weighed, and the weight loss curve was recorded using an empty aluminum pan as a control. The analytical temperature range was set from 30 to 800 °C, with nitrogen as the experimental gas flow and a heating rate of 10 °C per minute. Figure 9 The results show that the TG curves indicate that DNP-0-1 has a higher maximum thermogravimetric rate temperature and a maximum degradation temperature (Tdmax) of approximately 282.7°C, exhibiting the most severe weight loss (53.65%). DNP-0-3, on the other hand, has the lowest Tdmax (approximately 142.7°C), with a slower main chain degradation (21.07% mass loss). The micro-quotient thermogravimetric (DTG) curves show that most components exhibit multi-peak characteristics. The higher first weight loss peak temperatures of DNP-0-1 and DNP-1-1 suggest a relatively stronger ability to bind water, possibly related to their fibrous morphology. The shoulder peaks or small peaks appearing near 374°C and 726°C in DNP-0-3 indicate that the aggregated structure has better thermal stability in the corresponding temperature ranges.

[0102] Example 10: Analysis of the immunomodulatory activity of different polysaccharide components (DNPs) of Dendrobium nobile against RAW264.7 macrophages

[0103] (1) Preparation of polysaccharide solution: Accurately weigh 5 mg of polysaccharide sample, add it to the basic culture medium to dissolve and make up to 5 mL to obtain a polysaccharide stock solution with a concentration of 1 mg / mL. After filtration and sterilization through a 0.22 μm microporous membrane, it is serially diluted with DMEM complete culture medium to obtain six working concentrations of 25, 50, 100, 200, 400 and 800 μg / mL. Prepare and use immediately.

[0104] (2) Grouping: Discard the old culture medium and add fresh culture medium containing different concentrations of polysaccharides. Set up a blank group (culture medium only), a negative control group (cell suspension only), a positive control group (LPS), and an experimental group (polysaccharides of different concentrations). Set up 6 replicates for each group and continue culturing for 24 hours (determine the time according to the detection indicators).

[0105] (3) Resuscitation and culture of RAW264.7 cells: Cells were immediately removed from cryopreservation tubes at -80℃ and placed in a preheated 37℃ water bath, shaken until thawed. The thawed cell suspension was transferred to a sterile centrifuge tube containing 3 mL of DMEM complete culture medium (basal medium: serum: antibiotics = 100:10:1), centrifuged at 1000 rpm for 5 min, and the supernatant was carefully removed. 3 mL of DMEM complete culture medium was added, and the cells were gently pipetted to redisperse the precipitate until resuspended. The cell suspension was then transferred to a culture flask, and 8 mL of DMEM complete culture medium was added. The flask was then placed in a cell culture incubator (37℃, 5% CO2) for culture. When the cell density reached 80-90%, passage was performed, and cells in good growth condition and in the logarithmic growth phase were used for experiments.

[0106] (4) The effect of different polysaccharide components (DNPs) of Dendrobium nobile on the growth of RAW264.7 cells was detected using the CCK-8 method. RAW264.7 cells in the logarithmic growth phase were counted and their density adjusted to 10 × 10⁴ cells / well in 96-well plates (200 μL / well). 100 μL of cell suspension was added to each well, and PBS was used to replace the edge wells. Cells were cultured at 37℃ in a 5% CO₂ incubator for 24 hours to allow cell adhesion. After removing the original culture medium, 100 μL of polysaccharide solutions of different concentrations (25, 100, 200, 400, 800 μg / mL) were added to each well in the experimental groups. A negative control group (cell suspension only), a blank group (culture medium only), and a positive control group (1 μg / mL, LPS) were also set up, with three replicates per group. The cells were cultured for another 24 h. The CCK-8 working solution was prepared by mixing the CCK-8 solution with serum-free culture medium at a volume ratio of 1:10. Add the same amount of CCK-8 working solution to each well according to the instructions. Incubate at 37℃ for 2 h. Measure the absorbance at 450 nm using a microplate reader, and calculate the cell proliferation activity of different components of Dendrobium nobile polysaccharides against RAW 264.7 cells according to the formula.

[0107] (3)

[0108] In the formula, A represents the absorbance of the sample group and the positive group; B represents the absorbance of the negative group; and C represents the absorbance of the blank group.

[0109] like Figure 10 The results showed that cell viability was above 100% in all groups at concentrations of 25–100 μg / mL. Notably, DNP-0-3 showed a tendency to inhibit cell proliferation at concentrations of 400 μg / mL and 800 μg / mL, with cell viability decreasing to below 96%, respectively. Therefore, to ensure cell condition and avoid potential inhibitory effects, 25–100 μg / mL was selected as the working concentration for subsequent immunomodulatory experiments.

[0110] (5) Determination of nitric oxide (NO) secreted by RAW264.7 macrophages: nitrite ions (NO2) -Standard curve plotting: Take 8 μL of 100 mmol / L sodium nitrite (NaNO2) solution and add 992 μL of ultrapure water to obtain 1000 μL of 800 μM standard tubes; take 200 μL from the 800 μM tubes and add 200 μL of ultrapure water to obtain 400 μM tubes, then serially dilute to obtain gradient standard tubes of 200, 100, 50, 25, 12.5, 6.25, 3.13, and 1.56 μM, and also set up a 0 concentration tube without standards. Take 50 μL from each gradient standard tube and add it to a 96-well microplate, and simultaneously add 100 μL of the polysaccharide concentration solution of each of the four components to the corresponding wells of the microplate. Follow the instructions of the Griess kit and detect the absorbance at 540 nm using a microplate reader. Plot a standard curve with NaNO2 concentration on the x-axis and absorbance on the y-axis.

[0111] RAW264.7 cells in the logarithmic growth phase were collected, counted, and their density adjusted to 1×10⁻⁶ cells. 5 Cells were seeded at a density of 200 μL / well in 96-well plates, with 100 μL of cell suspension added to each well. Margin wells were replaced with PBS. Cells were incubated at 37°C, 5% CO2 for 24 h to allow cell adhesion. After removing the original culture medium, 100 μL of different concentrations of polysaccharide solution (25, 100, 200, 400, 800 μg / mL) were added to each well in the experimental groups. A negative control group (cell suspension only), a blank group (culture medium only), and a positive control group (1 μg / mL, LPS) were also established, with five replicates per group. The absorbance was measured at 540 nm using a microplate reader according to the Griess kit instructions. The NO secretion in the cell culture medium was calculated by substituting the values ​​into the NO standard curve. Figure 11 The results showed that all tested polysaccharide components promoted NO release from RAW264.7 cells in a concentration-dependent manner within the concentration range of 25-100 μg / mL (P < 0.01 compared with the control group). Among them, the high molecular weight neutral component DNP-0-1 had the most significant promoting effect. At a concentration of 100 μg / mL, it induced NO release of 22.74 ± 1.13 μmol / mL, which was approximately 2.15 times that of the LPS group. In contrast, the acidic component DNP-1-1 showed a lower NO release under the same conditions, at 8.02 ± 0.51 μmol / mL.

[0112] (6) Determination of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6): RAW264.7 cells in logarithmic growth phase were counted and their density adjusted, and seeded at a density of 1×10⁵ cells / well in 96-well plates (200 μL / well). 100 μL of cell suspension was added to each well, and PBS was used to replace the edge wells. Cells were cultured at 37°C and 5% CO₂ for 24 h to allow cell adhesion. After removing the original culture medium, 100 μL of polysaccharide solutions of different concentrations (25, 100, 200, 400, 800 μg / mL) were added to each well in the experimental groups. A negative control group (cell suspension only), a blank group (culture medium only), and a positive control group (1 μg / mL, LPS) were set up, with 5 replicates per group. After culture, the supernatant was centrifuged at 4°C and 1000 rpm for 10 min. The levels of TNF-α and IL-6 in the supernatant were detected strictly following the ELISA kit instructions. Figure 12 The results showed that the most active component was DNP-0-1, which induced a TNF-α release of 7649.05 pg / mL at 100 μg / mL, approximately 2.98 times that of the LPS group. DNP-0-3 and DNP-0-2 also showed strong promoting effects, with TNF-α secretion levels of 6519.87 pg / mL and 4843.44 pg / mL, respectively, at the same concentration. DNP-1-1, however, showed relatively weak activity, with a TNF-α secretion level of 2903.65 pg / mL at 100 μg / mL. The activity of crude polysaccharide DNP increased with increasing concentration, but remained lower than that of DNP-0-1 at all concentrations. Figure 13 Similarly, DNP-0-1 exhibited the strongest IL-6 induction ability, with a release of 6181.22 pg / mL at 100 μg / mL, approximately 2.25 times that of the LPS group. Under the same conditions, DNP-0-3 and DNP-0-2 secreted 4429.89 pg / mL and 4249.33 pg / mL, respectively. DNP-1-1 released 2846.00 pg / mL of IL-6. Crude polysaccharide DNP secreted 1566.00 pg / mL of IL-6 at 100 μg / mL.

[0113] Example 11: Correlation analysis between polysaccharide structural characteristics and immunomodulatory activity

[0114] To investigate the potential structural factors leading to the aforementioned differences in activity, key structural parameters of each polysaccharide component, including molecular weight M, were analyzed. w Molar ratio of main monosaccharide composition, conformational parameter α, aggregate height (hight), and maximum degradation temperature T. dmaxEtc. Pearson correlation analysis was performed with the immune activity indicators (NO, TNF-α, and IL-6 release at 100 μg / mL) measured in this chapter. For example... Figure 14 The results showed that the molecular weight (M) w Galactose (Gal) content showed a significant positive correlation with IL-6 secretion (r = 0.79) and a similar trend towards positive correlation with TNF-α and NO release. Galactose content exhibited a very strong positive correlation with all three immunomodulatory indicators (r ≥ 0.94). Conversely, glucose (Glc) and mannose (Man) content showed a significant negative correlation with immunomodulatory activity (r ≤ -0.91). Among higher-order structural parameters, conformational parameters (α) were highly positively correlated with molecular weight (r = 0.94), while aggregate height and thermal stability (T) were significantly positively correlated. dmax The value of α shows a negative correlation with immune activity.

[0115] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0116] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Polysaccharides of different components from Dendrobium nobile, characterized in that, The group of components consists of four structurally distinct polysaccharide components simultaneously isolated from Dendrobium nobile using DEAE-cellulose column chromatography combined with ultrafiltration membrane fractionation. These components are: (1) Neutral polysaccharide component DNP-0-1: molecular weight greater than 100 kDa; (2) Neutral polysaccharide component DNP-0-2: molecular weight 30~100 kDa; (3) Neutral polysaccharide component DNP-0-3: molecular weight less than 30 kDa; (4) Weakly acidic polysaccharide component DNP-1-1: molecular weight less than 50 kDa.

2. The Dendrobium nobile polysaccharide component group according to claim 1, characterized in that, Each component, after structural characterization, exhibits the following characteristics: (1) DNP-0-1: weight-average molecular weight 1.13×10 5 The total sugar content is ≥86%, and the uronic acid content is ≤0.7%. The monosaccharide composition is mainly galactose (molar ratio ≥50%) and mannose. The infrared spectrum shows a characteristic absorption peak of β-glycosidic bonds at 878 cm⁻¹. The conformational parameter α=0.35, exhibiting a relaxed random coil conformation. Scanning electron microscopy reveals a fibrous network structure. The thermal degradation temperature T0 is... dmax =282.7℃; (2) DNP-0-2: weight-average molecular weight 3.06×10 4 The Da and 2.15×10³ Da bimodal distribution; the monosaccharide composition is mainly galactose, mannose and glucose; the conformational parameter α=0.16, exhibiting a tight coil conformation; (3) DNP-0-3: weight-average molecular weight 1.50×10 4 Da; the monosaccharide composition is mainly galactose, arabinose, and mannose; the conformational parameter α=0.17, exhibiting a close globular conformation; atomic force microscopy shows that the aggregate height is 2 nm; (4) DNP-1-1: weight-average molecular weight 3.72×10 4 The Da and 5.94×10³ Da bimodal distribution; contains trace amounts of uronic acid (approximately 2.7%); the monosaccharide composition is mainly composed of mannose (62.7% molar ratio) and glucose; the infrared spectrum shows a characteristic absorption peak of esterification at 1738 cm⁻¹.

3. A method for preparing the Dendrobium nobile polysaccharide component group according to claim 1 or 2, characterized in that, Follow these steps in sequence: (1) The raw material of Dendrobium nobile was crushed and defatted, and extracted by ultrasonic-assisted water extraction: the liquid-solid ratio was controlled at 20:1 to 25:1 (mL / g), the extraction temperature was 70-75℃, the extraction time was 75-80 min, and the ultrasonic power was 450-470 W; (2) The extract was centrifuged to collect the supernatant, concentrated, precipitated with alcohol, and centrifuged to collect the precipitate. The starch was then removed by enzymatic hydrolysis and dialysis to obtain crude polysaccharide from Dendrobium nobile. (3) Crude polysaccharides were separated by DEAE-52 cellulose anion exchange column chromatography, and eluted with distilled water and 0.1 M NaCl solution respectively. The water elution fraction DNP-0 and the 0.1 M NaCl elution fraction DNP-1 were collected respectively. (4) The DNP-0 components were sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 100 kDa and 30 kDa for tandem fractionation. The >100 kDa cutoff liquid, the 30-100 kDa cutoff liquid and the <30 kDa permeate were collected respectively. After freeze-drying, neutral polysaccharide components DNP-0-1, DNP-0-2 and DNP-0-3 were obtained. (5) Pass the DNP-1 component through an ultrafiltration membrane with a molecular weight cutoff of 50 kDa, collect the permeate with a molecular weight cutoff of <50 kDa, and freeze-dry it to obtain the weakly acidic polysaccharide component DNP-1-1.

4. The preparation method according to claim 3, characterized in that, The optimal process parameters for ultrasonic-assisted extraction in step (1) were determined by Box-Behnken design optimization using response surface methodology. Specifically, the liquid-to-solid ratio was 21 w / v, the extraction time was 77 min, the extraction temperature was 73℃, and the ultrasonic power was 460 W. The regression model was highly significant (P < 0.0001), the lack-of-fit term was not significant (P = 0.7122), and the model determination coefficient R² = 0.9900.

5. A method for analyzing the structure-activity relationship of polysaccharides from Dendrobium nobile, characterized in that, Using the four polysaccharide components described in claim 1 or 2 as the research object, Pearson correlation analysis was used to reveal the intrinsic relationship between polysaccharide structure and activity by measuring the structural characteristic parameters and immunomodulatory activity indicators of each component. The structural characteristic parameters include: weight-average molecular weight (M). w Polydispersity index, monosaccharide molar ratio, conformational parameter (α), thermal degradation temperature (T) dmax Microscopic morphological characteristics; the immunomodulatory activity indicators include: RAW264.7 macrophage proliferation rate, nitric oxide (NO) release, tumor necrosis factor-α (TNF-α) release, and interleukin-6 (IL-6) release.

6. The analytical method according to claim 5, characterized in that, The Pearson correlation analysis results showed that galactose (Gal) content was significantly positively correlated with the release of NO, TNF-α, and IL-6 (r≥0.94, P<0.01); molecular weight (M) w The levels of IL-6 and IL-6 release were significantly positively correlated (r=0.79, P<0.05). The contents of glucose (Glc) and mannose (Man) were significantly negatively correlated with the immune activity indicators (r≤-0.91, P<0.05); the conformational parameter α and thermal stability showed a positive correlation with the immune activity.

7. The use of the Dendrobium nobile polysaccharide component DNP-0-1 as described in claim 1 or 2 in the preparation of a drug or health product for promoting macrophage immune activity.

8. The application of the Dendrobium nobile polysaccharide component group according to claim 1 or 2 in the preparation of structural markers for screening highly immunologically active polysaccharides, characterized in that, Galactose content ≥50%, molecular weight >100 kDa, and conformational parameter α ≥0.35 were used as predictive indicators for highly active polysaccharides.

9. The application of the Dendrobium nobile polysaccharide component group as described in claim 1 or 2 in the construction of a polysaccharide structure-activity relationship research model.

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