Dendrobium nobile lindl. flower acidic polysaccharide, preparation method and application thereof

Abstract

This invention discloses an acidic polysaccharide from Dendrobium nobile flowers, its preparation method, and its applications, belonging to the field of traditional Chinese medicine preparation technology. Using Dendrobium nobile flowers from Chishui City, Guizhou Province, this invention is the first to prepare a water-soluble pure Dendrobium nobile flower polysaccharide, DNLPS-3. Structural analysis shows that the obtained Dendrobium nobile flower polysaccharide is an acidic pectin polysaccharide, and the weight-average molecular weight of DNLPS-3 is 1.494 × 10⁻⁶. 4 The polysaccharide (Dalbergia odorifera) has a number-average molecular weight of 9.391 × 10³ Da and a polydispersity index of 1.66. It is composed of fucose, rhamnose, arabinose, galactose, glucose, xylose, galacturonic acid, and glucuronic acid in different proportions (0.23%: 2.14%: 3.67%: 3.50%: 0.43%: 14.64%: 74.06%: 1.34%). Using HepaRG cells (normal human hepatocytes) damaged by H₂O₂ as a model, its hepatoprotective activity was evaluated. The results showed that DNLPS-3 significantly alleviated oxidative stress in the body. It possesses good hepatoprotective activity, providing a theoretical basis for the development and application of Dendrobium nobile flower polysaccharides in the food and pharmaceutical fields, and is beneficial for the full utilization and development of Dendrobium nobile flower resources.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine preparation and application technology, specifically to an acidic polysaccharide from Dendrobium nobile flowers, its preparation method, and its application. Background Technology

[0002] Naturally derived plant polysaccharides are a type of active ingredient that is abundant, safe, highly effective, relatively non-toxic, biocompatible, and valuable. Due to their excellent physicochemical properties and various pharmacological activities, they have been found to have a variety of biological activities, such as enhancing the body's immunity, anti-oxidation, lowering blood sugar, anti-tumor, and anti-aging.

[0003] Dendrobium nobile flowers are a component of Dendrobium nobile resources, and are characterized by large quantities and easy accessibility. They are commonly used in Guizhou folk medicine for tea and have been included in the Guizhou Provincial Food Safety Local Standard (DBS52 / 049-2021). The polysaccharide components in the medicinal flowers play an important role in anti-tumor, antioxidant, anti-inflammatory, and hypoglycemic biological activities.

[0004] Currently, the reported Dendrobium nobile flower polysaccharides are limited to crude polysaccharides, rather than pure Dendrobium nobile flower polysaccharides (single polysaccharides). This means that the crude polysaccharides have not undergone impurity removal, separation, and purification steps. They contain a large number of protein molecules, pigments, inorganic salts, and other small molecule impurities. The low purity of the product and the complex and unknown structure of the product seriously affect the research progress on its biological activity.

[0005] Under normal physiological conditions, reactive oxygen species (ROS) generated by biological cells during oxidation can be effectively degraded by antioxidant defense molecules such as superoxide dismutase, glutathione, thioreductase, catalase, and peroxidase. However, when organisms are damaged by exogenous or endogenous oxidants, the dynamic balance between the production and scavenging of free radicals is disrupted, leading to the accumulation of ROS and cytotoxic effects. Excessive ROS in cells can induce significant cytotoxicity and damage cell membranes, organelles, and biomolecules such as lipids, proteins, and DNA, resulting in oxidative stress damage. This abnormal state is known as oxidative stress, and further research has revealed that oxidative stress is one of the major fatal mechanisms of various liver injuries. Modern pharmacological studies have found that crude extracts of Dendrobium nobile contain polysaccharides, suggesting that polysaccharides may be one of the main active ingredients for hepatoprotection. Currently, the hepatoprotective efficacy of Dendrobium nobile flower polysaccharides has not been reported. Therefore, research on the hepatoprotective effects of Dendrobium nobile flower polysaccharides is of great significance for the development and utilization of its medicinal value. Summary of the Invention

[0006] The purpose of this invention is to provide an acidic polysaccharide from Dendrobium nobile flowers that has liver-protective and cell proliferation-promoting functions, as well as a method for preparing the polysaccharide and its applications.

[0007] To achieve the above objectives, the technical solution adopted is as follows: an acidic polysaccharide from Dendrobium nobile flowers, specifically an acidic pectin polysaccharide, named Dendrobium nobile flower polysaccharide DNLPS-3, with a weight-average molecular weight of 1.494 × 10⁻⁶. 4 Da, with a number-average molecular weight of 9.391 × 10³ Da and a polydispersity index of 1.66, consists of fucose 0.1-0.3%, rhamnose 1-3%, arabinose 2-4%, galactose 2-4%, glucose 0.2-0.6%, xylose 13-15%, galacturonic acid 70-78%, and glucuronic acid 1-2% by mass. Its structure is [→4)- α -Gal( p )UA-(1→] n The main chain.

[0008] Furthermore, the acidic polysaccharides from Dendrobium nobile flowers, by mass ratio, consist of fucose 0.23%, rhamnose 2.14%, arabinose 3.67%, galactose 3.50%, glucose 0.43%, xylose 14.64%, galacturonic acid 74.06%, and glucuronic acid 1.34%.

[0009] The preparation method of the above-mentioned Dendrobium nobile flower acid polysaccharide includes the following steps: (1) Extraction Weigh out the flowers of Dendrobium nobile, soak them in 95% ethanol for 7 days, changing the 95% ethanol every 2 days during this period, filter, air dry in a cool place, and then dry in a 60 ℃ oven; then add pure water at a ratio of 1:15 w / v, heat in an 85 ℃ water bath for 1 hour each time, repeat 3 times, filter with gauze after extraction, combine the filtrates, concentrate and centrifuge, and store the supernatant in a 4 ℃ refrigerator for later use; (2) Alcohol precipitation and deproteinization The protein components in the crude extract of Dendrobium nobile flowers were removed using the Sevag method. Four times the volume of 95% ethanol was added to the concentrate obtained in step (1) above, bringing the final ethanol concentration to 80%. After standing overnight at 4 °C, the extract was collected by centrifugation and filtration. The extract was then dissolved in ultrapure water, and Sevag reagent was added at a volume ratio of 1:5 v / v. The solution was shaken on a shaker for 30 minutes, then centrifuged at 9000 g / min for 5 minutes. The supernatant was collected, and the protein removal process was repeated 10 times. The supernatant was then dialyzed using a 3500 Da dialysis bag for 3 days, with the pure water changed every 6 hours. The solution in the dialysis bag was concentrated and then freeze-dried to obtain the crude polysaccharide from Dendrobium nobile flowers. The Sevag reagent was 4 parts chloroform to 1 part n-butanol. (3) Ion exchange column chromatography The DEAE FAST FLOW column (3.5 cm × 50 cm) was equilibrated with ultrapure water for 24 hours. The polysaccharide obtained in step (2) was fully dissolved in ultrapure water and filtered through a 0.45 μm aqueous phase filter membrane. The solution was then slowly added along the column wall and allowed to stand for 30 minutes. Gradient elution was performed sequentially with ultrapure water, 0.1 mol / L NaCl solution, and 0.3 mol / L NaCl solution at a flow rate of 1.0 mL·min-1. The eluent was collected using an automated collector, 10 mL per tube. Each tube was monitored using the phenol-sulfuric acid method. The 0.3 mol / L NaCl eluent was collected, dialyzed to desalt at 3500 Da, and then freeze-dried to obtain the preliminary Dendrobium nobile flower polysaccharide, named DNLP-3. (4) Gel filtration column chromatography The polysaccharide DNLP-3 obtained in step (4) was eluted with ultrapure water using a SephacrylS-200 gel chromatography column. After further concentration and freeze-drying, a pure polysaccharide was obtained and named Dendrobium nobile flower polysaccharide DNLPS-3.

[0010] And the use of the above-mentioned polysaccharides in the preparation of drugs or health products with immunomodulatory effects.

[0011] And the use of the above-mentioned polysaccharides in the preparation of drugs or health products that promote cell proliferation.

[0012] And the use of the aforementioned polysaccharides in the preparation of drugs or health products that alleviate cellular oxidative stress.

[0013] And the use of the aforementioned polysaccharides in the preparation of drugs or health products with hepatoprotective effects.

[0014] The beneficial effects of the above-mentioned scheme are as follows: The hepatoprotective activity of this Dendrobium nobile flower acidic polysaccharide was evaluated using a H2O2-damaged human hepatocyte (HepaRG) model. The results showed that DNLPS-3 can significantly alleviate the body's oxidative stress state. It exhibits good hepatoprotective activity, providing a theoretical basis for the development and application of Dendrobium nobile flower polysaccharide in the food and pharmaceutical fields, and facilitating the full utilization and development of Dendrobium nobile flower resources. Attached Figure Description

[0015] Figure 1 This is a flowchart of the preparation process of purified polysaccharide DNLPS-3 from Dendrobium nobile flowers.

[0016] Figure 2 This is the DEAE FAST FLOW anion exchange column chromatography elution diagram of Dendrobium nobile flower polysaccharide.

[0017] Figure 3 This is the elution chromatogram of purified polysaccharide DNLPS-3 from Dendrobium nobile flowers using a SephacrylS-200 gel chromatography column.

[0018] Figure 4 This is a GPC image of the purified polysaccharide DNLPS-3 from Dendrobium nobile flowers.

[0019] Figure 5 This is a comparison chart of the monosaccharide composition of purified polysaccharide DNLPS-3 from Dendrobium nobile flowers with that of the standard.

[0020] Figure 6 This is the ultraviolet absorption spectrum of DNLPS-3, a purified polysaccharide from Dendrobium nobile flowers.

[0021] Figure 7 This is the infrared absorption spectrum of DNLPS-3, a purified polysaccharide from Dendrobium nobile flowers.

[0022] Figure 8 This is a scanning electron microscope image of DNLPS-3, a purified polysaccharide from Dendrobium nobile flowers.

[0023] Figure 9 This is a Congo red experimental diagram of the purified polysaccharide DNLPS-3 from Dendrobium nobile flowers.

[0024] Figure 10 This is the total ion current spectrum of the purified polysaccharide DNLPS-3 from Dendrobium nobile flowers, analyzed for methylation.

[0025] Figure 11 This is the nuclear magnetic resonance spectrum analysis of DNLPS-3, a purified polysaccharide from Dendrobium nobile flowers.

[0026] Figure 12 This is the nuclear magnetic resonance spectrum analysis of DNLPS-3, a purified polysaccharide from Dendrobium nobile flowers.

[0027] Figure 13 This is the nuclear magnetic resonance spectrum analysis of DNLPS-3, a purified polysaccharide from Dendrobium nobile flowers.

[0028] Figure 14 This is an in vitro cell proliferation diagram of the purified polysaccharide DNLPS-3 from Dendrobium nobile flowers.

[0029] Figure 15 This is a diagram showing the protective effect of purified polysaccharide DNLPS-3 from Dendrobium nobile flowers against H2O2-induced damage to HepaRG cells.

[0030] Figure 16 This is a graph showing the effect of purified polysaccharide DNLPS-3 from Dendrobium nobile flowers on liver function enzyme indicators induced by H2O2 in HepaRG.

[0031] Figure 17 This is a graph showing the effect of purified polysaccharide DNLPS-3 from Dendrobium nobile flowers on oxidative stress indices induced by HepaRG.

[0032] Figure 18This is a diagram showing the expression of proteins related to H2O2-induced damage in HepaRG cells by the purified polysaccharide DNLPS-3 from Dendrobium nobile flowers.

[0033] In the attached figures: Control group, Model group (H2O2), Comparative group 1 (10 µg / mL), Comparative group 2 (25 µg / mL), Comparative group 3 (50 µg / mL), Comparative group 4 (100 µg / mL), and Comparative group 5 (250 µg / mL); Data are expressed as mean ± standard deviation (n=3), and significant differences between groups are not considered statistically significant. P <0.05) is indicated by * or #, indicating a highly significant difference ( P <0.01) is represented by ** or ##. Detailed Implementation

[0034] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. The described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0035] Example 1: Extraction, preparation, and purification of the polysaccharide DNLPS-3 from Dendrobium nobile flowers, as follows: Figure 1 .

[0036] (1) Weigh Dendrobium nobile flowers and soak them in 95% ethanol at a ratio of 1:15 (w / v) for 7 days. Replace the 95% ethanol every 2 days during this period. Filter the solution, air dry it in a cool place, and then dry it in an oven at 60 ℃. Add pure water at a ratio of 1:15 (w / v) and heat it in an 85℃ water bath for 1 hour each time. Repeat the process 3 times. After extraction, filter the solution with gauze, combine the filtrates, concentrate the solution, centrifuge and filter the solution. Store the supernatant in a refrigerator at 4 ℃ for later use.

[0037] (2) Removal of protein components from crude extract of Dendrobium nobile flowers by Sevag method. Add 4 times the volume of 95% ethanol to the above concentrate to make the final ethanol concentration reach 80%. After standing overnight at 4 ℃, centrifuge and filter to collect the extract. Dissolve the extract in ultrapure water and add Sevag reagent (chloroform: n-butanol = 4:1, v / v) at a volume ratio of 1:5 (v / v). Shake on a shaker for 30 minutes. Then centrifuge at 9000 g / min for 5 minutes. Take the supernatant and repeat the above protein removal process 10 times. Dialyze the supernatant with a 3500 Da dialysis bag for 3 days, changing the pure water every 6 hours. Concentrate the solution in the dialysis bag and freeze dry to obtain crude polysaccharide of Dendrobium nobile flowers.

[0038] Example 2: Isolation and purification of DNLPS-3, a polysaccharide from Dendrobium nobile flowers. (1) First, DEAE FAST FLOW packing material was packed into a chromatography column (3.5 cm × 50 cm). The constant flow pump was used to equilibrate with pure water for 24 hours at a flow rate of 1.0 mL / min. Approximately 3.0 g of Dendrobium nobile flower crude polysaccharide DNLP was fully dissolved in 300 mL of ultrapure water. After centrifugation at 9000 g / min for 5 minutes, the supernatant was filtered through a 0.45 μm aqueous phase filter membrane and then slowly added to the column for elution. The elution conditions were as follows: gradient elution with ultrapure water, 0.1 mol / L NaCl solution, and 0.3 mol / L solution, at a flow rate of 1.0 mL·min⁻¹. The eluent was collected using an automated collector, 10 mL per tube. The phenol-sulfuric acid method was used to monitor each tube, and the elution curve was plotted (see [link to table]). Figure 2 According to the elution curve, the collected 0.3 mol / L NaCl elution solutions were combined and concentrated, and then dialyzed with a 3500 Da dialysis bag for 3 days, with pure water replaced every 6 hours. The Dendrobium nobile flower polysaccharide solution in the dialysis bag was freeze-dried to obtain a preliminarily purified Dendrobium nobile flower polysaccharide sample, named DNLPS-3.

[0039] (2) Gel filtration chromatography: The polysaccharide in (4) was eluted with ultrapure water using a SephacrylS-200 gel chromatography column. After further concentration and freeze drying, a pure polysaccharide was obtained and named Dendrobium nobile flower polysaccharide DNLPS-3 (see Figure 3 ).

[0040] Example 3: Homogeneity analysis and molecular weight determination of purified polysaccharide DNLPS-3 from Dendrobium nobile flowers.

[0041] (1) The homogeneity of the purified polysaccharide DNLPS-3 from Dendrobium nobile flowers was determined by high performance gel permeation chromatography (HPGPC). Test conditions: The polysaccharide sample was dissolved in a 0.2 mol / L KNO3 solution to prepare a polysaccharide sample solution with a final concentration of approximately 2 mg / mL. The solution was filtered through a 0.45 μm microporous membrane and injected for analysis. Chromatographic conditions were as follows: mobile phase: 0.2 mol / L KNO3 solution; column: Ultrahydrogel™ 500 (7.8 mm × 300 mm); flow rate: 0.6 mL / min; column temperature: 35℃; injection volume: 40 μL; running time: 25 min; chromatograms were recorded; test results are shown below. Figure 4 .

[0042] (2) The molecular weight of DNLPS-3 was determined by high-performance gel size exclusion chromatography coupled with multi-angle laser light scattering and differential detection (HPSEC-MALLS-RI). The test conditions were as follows: the chromatographic columns were TSK Gel G6000pwxl (5μm, 7.8 mm × 300 mm) and TSK Gel G3000pwxl (5μm, 7.8 mm × 300 mm), respectively. The mobile phase was 0.9 g / 100 mL sodium chloride solution, the flow rate was 0.5 mL / min, and the column temperature was 35℃. The polysaccharide sample was prepared into a 2.0 mg / mL solution, the injection volume was 100 μL, the column temperature was 35℃, and the flow rate was 0.5 mL / min. The weight-average molecular weight of DNLPS-3 was found to be 1.494 × 10⁻⁶. 4 The number-average molecular weight is 9.391 × 10³ Da, and the polydispersity index is 1.66.

[0043] Example 5: Ion chromatography (IC) analysis of the monosaccharide composition of DNLPS-3. Approximately 2 mg of purified polysaccharide samples were weighed and added to 1 mL of 2 mol / L TFA acid solution. The samples were heated at 121 °C for 2 h and dried under nitrogen. Anhydrous methanol was added for washing, followed by drying. This methanol washing process was repeated 2-3 times. The solutions were dissolved in ultrapure water and diluted 20-fold with an appropriate amount of polysaccharide hydrolysate. The solutions were then transferred to chromatographic vials for later use. Appropriate amounts of each of the 13 standard monosaccharide reference standards (fucose, L-rhamnose, L-arabinose, D-galactose, D-glucose, xylose, D-mannose, fructose, ribose, D-galacturonic acid, D-glucuronic acid, mannuronic acid, and guluronic acid) were accurately weighed and dissolved in ultrapure water to prepare 10 mg / mL standard stock solutions. Then, an appropriate amount of each stock solution was used to prepare a 40 μg / mL monosaccharide mixed standard solution. Test conditions: Dionex column TM CarboPac TM The injection volume of the PA20 (10 μm, 150 mm × 3.0 mm) HPLC column was 5 μL; the flow rate was 0.5 mL / min; the column temperature was 30 ℃; and the gradient elution conditions are shown in Table 1. The results showed that DNLPS-3 is an acidic polysaccharide with the following composition: fucose (0.23%), rhamnose (2.14%), arabinose (3.67%), galactose (3.50%), glucose (0.43%), xylose (14.64%), galacturonic acid (74.06%), and glucuronic acid (1.34%). (See Table 1 for details.) Figure 5 .

[0044] Table 1 0 95% 5% 0% 26 85% 5% 10% 42 85% 5% 10% 42.1 60% 0% 40% 52 60% 40% 0 52.1 95% 5% 0 60 95% 5% 0 Example 6: Approximately 2 mg of purified polysaccharide sample from Dendrobium nobile flowers was weighed and placed in a test tube. 5 mL of deionized water was added to dissolve it completely, bringing the concentration to 0.5 mg / mL. A full-wavelength scan was performed using a UV spectrophotometer, with a scanning range of 200-400 nm. The results are shown below. Figure 6 This indicates that DNLPS-3 contains protein, and its protein content was determined to be 2.70%.

[0045] Example 7: Weigh approximately 2 mg of the purified polysaccharide sample from dried Dendrobium nobile flowers, mix thoroughly with potassium bromide (KBr) at a ratio of 1:100 (w / w), compress to obtain uniform, transparent thin slices, and then scan the sample 32 times using a Fourier transform infrared spectroscopy scanner, with a wavenumber range of 4000-500 cm⁻¹. -1 The results are shown Figure 7 The strong absorption peak of DNPLPS3 at 3414 cm⁻¹ is due to the OH stretching vibration, and at 2939 cm⁻¹... -1 The absorption peak at 1745 cm⁻¹ corresponds to the CH angle vibration, which preliminarily confirms that the substance is a polysaccharide. -1 1615cm -1 and 1421cm -1 The presence of distinct absorption peaks at 1400-1200 cm⁻¹ corresponds to the stretching vibrations of C=O, COO-, and CHO, indicating the presence of -COOH groups. -1 The peaks between 1000-1200 cm⁻¹ are caused by CH angle vibrations; while those between 1000-1200 cm⁻¹ are caused by CH angle vibrations. -1 The three peaks between (1016, 1099, 1146 cm) -1 ) and 921cm -1 and 765cm -1 The absorption peak at 831 cm⁻¹ indicates that the sugar may be a pyranose. -1 The absorption peak indicates that DNLPS-3 is in the α configuration.

[0046] Example 8: Appropriate amounts of purified polysaccharide samples from *Dendrobium nobile* flowers were taken and adhered to a sample stage with copper tape. The sample stage was then placed in an ion sputtering apparatus to deposit a layer of conductive gold powder. The samples were then observed under a scanning electron microscope. Operating conditions: accelerating voltage 20 kV; magnifications of 200x, 500x, 2000x, 5000x, and 10000x were used, and appropriate fields of view were selected for photographic recording. Results are shown below. Figure 8 DNLPS-3 has a relatively smooth surface and is irregularly sheet-like, possibly due to the mutual repulsion between polysaccharide molecules and the weak intermolecular attraction, which leads to the breakage of the sheet-like structure.

[0047] Example 9: Analysis of Congo Red in DNLPS-3 1 mol / L NaOH was diluted to prepare a series of NaOH solutions of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L, and mixed with 2 mg / mL DNLPS-3 solution and 0.01% Congo red reagent, respectively (see Table 2). A UV-Vis spectrophotometer was used to perform full-wavelength scanning, and the maximum absorption wavelength of each sample solution at different NaOH concentrations was recorded. The results showed that DNLPS-3—Congo red exhibited a red shift in UV absorption in 0–0.1 mol / L NaOH solutions, indicating a complexation reaction between the sample and Congo red. With further increases in NaOH concentration, it showed metastable behavior in the 0.2–0.5 mol / L range, suggesting that both DNLP1 and DNLPS-3 possess a triple-helix conformation. (See Table 2 for more details.) Figure 9 .

[0048] Table 2 NaOH (mol / L) 0 0.1 0.2 0.3 0.4 0.5 NaOH (mL) 0 0.4 0.8 1.2 1.6 2 Distilled water (mL) 2 1.6 1.2 0.6 0.4 0 Polysaccharide solution (mL) 1 1 1 1 1 1 Congo red solution (mL) 1 1 1 1 1 1 Total volume (mL) 4 4 4 4 4 4 Example 10: Methylation Analysis of DNLPS-3 (1) Reduction of uronic acid: Weigh approximately 10 mg of purified polysaccharide sample from Dendrobium nobile flower, dissolve in 1 ml of pure water, add 1 mL of 100 mg / mL 1-cyclohexyl-2-morpholinoethyl carbodiimide methyl p-toluenesulfonate, and react for 2 h. Add 1 mL of 2 mol / L imidazole, divide the sample into two equal portions, add 1 mL of 30 mg / mL NaBH4, and react for 3 h. Terminate the reaction by adding 100 μL of glacial acetic acid. Dialyze the reaction solution through a 3500 Da dialysis bag for 48 h, then freeze-dry the sample. Prepare the freeze-dried sample for methylation analysis.

[0049] (2) Methylation experiment: Dissolve the dried sample in 500 μL LDMSO, then add 1 mg NaOH, incubate for 30 min, then add 50 μL iodomethane solution and react for 1 h, add 1 mL ultrapure water and 2 mL dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water washing 3 times. The lower dichloromethane phase was collected and dried under nitrogen. 100 μL of 2 mol / L LTFA was added, and the mixture was reacted at 121 °C for 90 min. The mixture was then evaporated to dryness at 30 °C. 50 μL of 2 mol / L ammonia and 50 μL of 1 mol / L NaBD4 were added, and the mixture was mixed. The mixture was reacted at room temperature for 2.5 h. The reaction was terminated by adding 20 μL of acetic acid. The mixture was dried under nitrogen, washed twice with 250 μL of methanol, and dried under nitrogen. 250 μL of acetic anhydride was added, and the mixture was vortexed. The mixture was reacted at 100 °C for 2.5 h. 1 mL of water was added, and the mixture was allowed to stand for 10 min. 500 μL of dichloromethane was added, and the mixture was vortexed. The mixture was centrifuged, and the aqueous phase was discarded. The washing was repeated three times with water. Finally, the lower dichloromethane phase was collected and analyzed by GC-MS. The test conditions were: Agilent 7890A, BPX70 column (30 m × 0.25 mm × 0.25 µm), injection volume 1 μL, split ratio 10:1, high-purity helium, initial temperature 140 °C and hold for 2 min, temperature ramped at 3 °C / min, final temperature 230 °C and hold for 3 min. The results are shown in Table 3, and the total ion chromatogram is shown below. Figure 10 .

[0050] Table 3 6.037 T-Ara() 1,4-di-O-acetyl-2,3,5-tri-O-methyl arabinitol 2.08 7.368 T-Xyl() 1,5-di-O-acetyl-2,3,4-tri-O-methyl xylitol 9.20 10.006 T-Gal()-UA 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol 6.36 12.935 3-Gal()-UA 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl galactitol 1.12 13.882 4-Gal()-UA 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol 56.76 15.841 3,4-Gal()-UA 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl galactitol 10.18 16.319 3,4-Glc() 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl glucitol 2.76 17.051 2,4-Gal()-UA 1,2,4,5-tetra-O-acetyl-3,6-di-O-methyl galactitol 1.39 17.75 2,3,4-Gal() 1,2,3,4,5-penta-O-acetyl-6-O-methyl galactitol 4.79 18.838 4,6-Gal() 1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl galactitol 4.28 20.478 3,4,6-Gal() 1,3,4,5,6-penta-O-acetyl-2-O-methyl galactitol 1.08 Example 11: Nuclear Magnetic Resonance Spectroscopy Analysis of DNLPS-3 Weigh 50 mg of each purified polysaccharide sample from *Dendrobium nobile* flowers, add D2O to prepare a 50 mg / mL solution, vortex the solution, centrifuge, collect the supernatant, freeze, and perform HD exchange overnight. Transfer 0.5 mL of the solution to an NMR tube and perform NMR scanning using a Bruker AVANCENEO 500M NMR spectrometer at 25℃. Analyze the purified polysaccharides. 1 H, 13 C 1 H-HCOSY, HSQC, and HMBC spectra. Results are shown in [link to results]. Figure 11-13 The obvious signal peak at 170.79 ppm is a typical uronic acid signal. Figure 11 -B); the methylation results show that DNLPS-3 is mainly 4-Gal ( pThe composition of DNLPS-3 includes sugar residues and other sugar residues in smaller proportions. Due to the low proportion of other sugar residues, the response on NMR scans may be too low to detect them. One-dimensional NMR analysis only confirmed that DNLPS-3 is an acidic polysaccharide and verified its monosaccharide composition, infrared spectroscopy, and methylation results. The COSY spectrum of DNLPS-3 (…) Figure 12 -D) δ 1.19, δ A signal was observed at 16.33, which is a rhamnose signal, indicating the presence of rhamnose in DNLPS-3, consistent with the monosaccharide composition analysis results. This was observed in the 1H-NMR spectrum. δ There is a distinct signal peak at 3.74 ppm, and a distinct carbon signal is also present at 53.59 ppm in the 13C-NMR spectrum. These two signals are consistent with the HSQC spectrum ( Figure 12 -C) δ 3.74, δ The signals from 53.59) mutually verified each other, proving the presence of a methyl ester group in the DNLPS-3 structure, and simultaneously in δ The absorption peaks at 1.19 and 1.99 ppm are the chemical shifts of the methyl group and the O-acetyl group. In the 13C-NMR spectrum, there are two types of carbonyl carbon signals. δ 174.72 ppm is the concentration of unesterified Gal ( p The free carboxyl group at the C-6 position of A, while δ 170.79 is carboxyl esterified. α- Gal ( p The C-6 carbonyl ester of A may be methylated with galacturonic acid. It is speculated that the polysaccharide DNLPS-3 mainly adopts the [→4)- α -Gal( p )UA-(1→] n It is a pectin polysaccharide.

[0051] Example 13: Effects of Dendrobium nobile flower acidic pectin polysaccharide DNLPS-3 on human liver HepaRG cells.

[0052] (1) Preparation of solution of Dendrobium nobile flower polysaccharide DNLPS-3: Weigh 5.0 mg DNLPS-3, add 2 mL of basal culture medium to prepare a stock solution of 2500 µg / mL, and then dilute with basal culture medium to the working concentration for cell treatment.

[0053] (2) Preparation of complete culture medium: Take a 50 mL centrifuge tube, add 45 mL of basal culture medium, 5 mL of LFBS and 1 mL of double antibiotics to the 50 mL centrifuge tube respectively, mix well, filter to remove impurities, seal and store at 4℃.

[0054] (3) CCK-8 dilution: CCK-8 and basal culture medium are diluted at a ratio of 1:9.

[0055] (4) Human liver HepaRG cell culture: HepaRG cells were placed in complete culture medium and cultured statically in an incubator at 37°C, 5% CO2, and 95% relative humidity. When the cell confluence reached 80%–90%, the cells were passaged, and cells in good growth condition and in the logarithmic growth phase were used for experiments.

[0056] (5) To determine the effect of different concentrations of DNLPS-3 on cell proliferation, cells in the logarithmic growth phase were adjusted to a concentration of 1×10⁵ cells / mL with complete culture medium and seeded in 96-well plates. Concentrations of 10µg / mL, 25µg / mL, 50µg / mL, 100µg / mL, 250µg / mL, and 500µg / mL were added to the culture medium. The blank control was added only with complete culture medium. After 12 h of intervention, the cells were gently washed three times with PBS buffer, and 90µL of basal culture medium and 10µL of LCK-8 were added to each well (in the dark). The plates were then incubated for another 2 h. The absorbance (OD) value at 450 nm was measured using a microplate reader, and cell viability was calculated. See [link to microplate]. Figure 14 Compared with the control group (CON), the survival rate of HepaRG cells was significantly reduced when the concentration of polysaccharide DNLPS-3 exceeded 250 µg / mL. This indicates that high concentrations of polysaccharide have a certain inhibitory effect on HepaRG cells. To eliminate the influence of polysaccharide on HepaRG cells, concentrations with a survival rate of over 100% were selected for subsequent experiments, namely 10 µg / mL, 25 µg / mL, 50 µg / mL, 100 µg / mL, and 250 µg / mL.

[0057] Example 13: Effect of Dendrobium nobile flower acidic pectin polysaccharide DNLPS-3 on H2O2-induced human liver HepaRG cells. 1×10⁻⁶ DNLPS-3 was added to a 96-well plate. 5 HepaRG cells in logarithmic growth phase (cells / mL) were incubated at 37°C in a 5% CO2 incubator for 24 h. 100 µL of different concentrations of DNP-3 were added to each well in the experimental groups, and the cells were incubated for 12 h. The supernatant was discarded, and the cells were washed twice with PBS. 100 µL of 250 µM H2O2 was added, and the cells were incubated for another 12 h. Then, prepared CCK-8 was added, and the cells were incubated at 37°C for 2 h. The absorbance (OD) at 450 nm was measured using a microplate reader. The results are shown below. Figure 15 The results showed that, compared with the blank control group, the cell survival rate in the H2O2 model group was significantly reduced. P<0.01); compared with the H2O2 model group, the cell survival rate of the DNLPS-3 intervention group was increased ( P <0.01), pretreatment of HepaRG cells with different concentrations of DNLPS-3, at concentrations of 10 µg / mL, 25 µg / mL, 50 µg / mL, 100 µg / mL, and 250 µg / mL, all significantly improved cell viability compared to the model group. P <0.05 or P <0.01).

[0058] Example 14: Effect of Dendrobium nobile flower acidic pectin polysaccharide DNLPS-3 on H2O2-induced oxidative stress index levels in human liver HepaRG cells. HepaRG cells in logarithmic growth phase were used at 3.0 × 10⁻⁶ cells / year. 6 Cells were seeded per 100 mm culture dish and divided into three groups: Control group, H2O2 intervention group (250 µmol / L), and H2O2 + DNLPS-3 group, with 5 replicates per group. Cells were cultured for 24 h. 7 mL of different concentrations of DNLPS-3 (10, 25, 50, 100, and 250 µg / mL) were added to each well of the experimental groups. Cells were incubated at 37°C and 5% CO2 for 12 h. The supernatant was discarded, and the cells were washed twice with PBS. 250 µmol / L H2O2 was added, and the cells were incubated at 37°C and 5% CO2 for another 12 h. HepaRG cells from each group were then collected, centrifuged at 1200 r / min for 10 min, and the supernatant was discarded. AST, ALT, LDH, SOD, MDA, and GSH were measured according to the kit instructions. Results are shown below. Figure 16 and Figure 17 , Figure 16 The results showed that, compared with the control group, the levels of ALT, AST, and LDH in the H2O2 group were significantly increased. P <0.01, demonstrating that H2O2 severely damages HepaRG, and a model was successfully established. Compared with the H2O2 group, DNLPS-3 concentrations in the range of 10µg / mL to 100µg / mL reduced the levels of ALT, AST, and LDH in a dose-dependent manner. DNLPS-3 concentrations of 50µg / mL and 100µg / mL significantly reduced AST release. P <0.01); a DNLPS-3 concentration of 25 µg / mL reduced ALT release ( P <0.05), concentrations of 50 µg / mL and 100 µg / mL significantly reduced ALT levels; DNLPS-3 concentrations of 25, 50, 100, and 250 µg / mL significantly reduced LDH levels ( P <0.01). Figure 17 -A and Figure 17 As shown in Figure -B, compared with the Control group, the H2O2 group significantly reduced the levels of SOD and GSH ( P <0.01), compared with the H2O2 group, the concentration of DNLPS-3 in the range of 10µg / mL to 100µg / mL increased the levels of SOD and GSH. When the concentration of DNLPS-3 was 100µg / mL, it significantly increased the levels of SOD and GSH. P <0.01, polysaccharide concentrations in the 100 µg / mL range only increased GSH levels ( P <0.01). Figure 17 As shown in Figure -C, compared with the normal group, HepaRG cells in a solution of 250 µmol / L showed a significantly increased MDA level ( P <0.01), confirming H2O2-induced cytotoxicity, and at concentrations of 50 µg / mL and 100 µg / mL, significantly reduced MDA content ( P <0.01), and the concentration was dose-dependent in the range of 10 µg / mL to 100 µg / mL. In summary, the results indicate that DNLPS-3 can effectively mitigate H2O2-induced cytotoxicity in HepaRG cells and has a good protective effect on HepaRG cells. Since the above experimental results show that concentrations of 50 µg / mL and 100 µg / mL can significantly promote HepaRG cell proliferation, combined with these results, the optimal concentration of DNLPS-3 for protecting against H2O2-induced HepaRG is 100 µg / mL.

[0059] Example 15: Western blot detection of DNP-3 expression of H2O2-induced HepaRG cell damage-related proteins.

[0060] (1) Take HepaRG cells in good growth condition and use 3.0 × 10⁻⁶ cells. 6Cells were seeded per 100 mm culture dish, and the cells were divided into three groups: Control group, H2O2 intervention group, and H2O2+DNLPS-3 group. After 24 h of culture, 7 mL of basal medium was added to the blank control group, 7 mL of 250 µM H2O2 was added to the H2O2 intervention group, and the cells were cultured in a cell culture incubator for 12 h. 7 mL of DNLPS-3 at concentrations of 25 µg / ml, 50 µg / ml, and 100 µg / ml were added to the H2O2+DNP-3 groups, and the cells were cultured in an incubator for 12 h. The supernatant was discarded, the cells were washed twice with PBS, and 7 mL of 250 µM H2O2 was added, and the cells were cultured in an incubator for 12 h. After the specified time, the culture medium was aspirated, and the cells were gently washed once with pre-cooled PBS. 400 μL of cell lysis buffer (RIPA lysis buffer: PMSF protease inhibitor: phosphatase inhibitor = 100:1:1) was added to each dish and lysed on ice for 30 min to obtain total cell protein. The protein concentration was detected according to the BCA kit instructions. The protein samples were diluted to the same concentration with PBS buffer, and the corresponding volume of loading buffer was added. The samples were then boiled in a water bath for denaturation for 5-10 min. After aliquoting into EP tubes, the samples were stored at -20°C.

[0061] (2) An SDS-PAGE electrophoresis apparatus was used, with a 4% stacking gel on top and a 12.5% ​​separating gel on the bottom. The protein loading amount per well was 10 μg. The initial electrophoresis voltage was 80 V, and electrophoresis was stopped when the bromophenol blue reached the bottom of the glass. Transfer was performed at 250 mA for 100 min. After blocking with freshly prepared 5% skim milk powder at room temperature for 2 h, the PVDF membrane was washed three times with TBST, then incubated with primary antibody overnight at 4°C on a shaker. The primary antibody dilution ratios were as follows: HO-1 (1:1000), Nrf2 (1:1000), and GAPDH (1:3000). After overnight incubation at 4°C on a shaker, the membrane was washed with TBST. Wash three times, 10 min each time. Incubate the secondary antibody on a shaker at room temperature for 1 h, then wash three times with TBST, 10 min each time. Add ECL developer to saturate the bands, develop and image using a BIO-RAD chemiluminescence imager, and analyze the grayscale values ​​of the protein bands using ImageLab software.

[0062] See results Figure 18 Western blot results showed that, compared with the control group, the expression of HO-1 protein in cells of the H2O2 group was significantly decreased. P <0.05; Compared with the H2O2 group, HO-1 protein expression increased when DNP-3 concentration was 50 µg / ml and 100 µg / ml. P <0.01), Nrf2 protein expression increased when the DNLPS-3 concentration was 100 µg / ml. P<0.05). DNP-3 can exert a therapeutic effect on oxidative damage in HpeaRG cells by upregulating the expression of Nrf2 / HO-1 pathway proteins.

Claims

1. An acidic polysaccharide from Dendrobium nobile flowers, characterized by: This polysaccharide is an acidic pectin polysaccharide, named Dendrobium nobile flower polysaccharide DNLPS-3, with a weight-average molecular weight of 1.494 × 10⁻⁶. 4 Da, with a number-average molecular weight of 9.391 × 10⁻⁶. 3 Da, with a polydispersity index of 1.66, comprises, by mass ratio, fucose 0.1-0.3%, rhamnose 1-3%, arabinose 2-4%, galactose 2-4%, glucose 0.2-0.6%, xylose 13-15%, galacturonic acid 70-78%, and glucuronic acid 1-2%, with a structure of [→4)- α -Gal( p )UA-(1→] is the main chain.

2. The acidic polysaccharide from Dendrobium nobile flowers according to claim 1, characterized in that: The composition by mass percentage is as follows: fucose 0.23%, rhamnose 2.14%, arabinose 3.67%, galactose 3.50%, glucose 0.43%, xylose 14.64%, galacturonic acid 74.06%, and glucuronic acid 1.34%.

3. The method for preparing acidic polysaccharides from Dendrobium nobile flowers according to claim 1, characterized in that, Includes the following steps: (1) Extraction Weigh out the flowers of Dendrobium nobile, soak them in 95% ethanol for 7 days, changing the 95% ethanol every 2 days during this period, filter, air dry in a cool place, and then dry in a 60 ℃ oven; then add pure water at a ratio of 1:15 w / v, heat in an 85 ℃ water bath for 1 hour each time, repeat 3 times, filter with gauze after extraction, combine the filtrates, concentrate and centrifuge, and store the supernatant in a 4 ℃ refrigerator for later use; (2) Alcohol precipitation and deproteinization The protein components in the crude extract of Dendrobium nobile flowers were removed using the Sevag method. Four times the volume of 95% ethanol was added to the concentrate obtained in step (1) above, bringing the final ethanol concentration to 80%. After standing overnight at 4 °C, the extract was collected by centrifugation and filtration. The extract was then dissolved in ultrapure water, and Sevag reagent was added at a volume ratio of 1:5 v / v. The solution was shaken on a shaker for 30 minutes, then centrifuged at 9000 g / min for 5 minutes. The supernatant was collected, and the protein removal process was repeated 10 times. The supernatant was then dialyzed using a 3500 Da dialysis bag for 3 days, with the pure water changed every 6 hours. The solution in the dialysis bag was concentrated and then freeze-dried to obtain the crude polysaccharide from Dendrobium nobile flowers. The Sevag reagent was 4 parts chloroform to 1 part n-butanol. (3) Ion exchange column chromatography The DEAE FAST FLOW column (3.5 cm × 50 cm) was equilibrated with ultrapure water for 24 hours. The polysaccharide obtained in step (2) was fully dissolved in ultrapure water and filtered through a 0.45 μm aqueous phase filter membrane. The solution was then slowly added along the column wall and allowed to stand for 30 minutes. Gradient elution was performed sequentially with ultrapure water, 0.1 mol / L NaCl solution, and 0.3 mol / L NaCl solution at a flow rate of 1.0 mL·min-1. The eluent was collected using an automated collector, 10 mL per tube. Each tube was monitored using the phenol-sulfuric acid method. The 0.3 mol / L NaCl eluent was collected, dialyzed to desalt at 3500 Da, and then freeze-dried to obtain the preliminary Dendrobium nobile flower polysaccharide, named DNLP-3. (4) Gel filtration column chromatography The polysaccharide DNLP-3 obtained in step (3) was eluted with ultrapure water using a SephacrylS-200 gel chromatography column. After further concentration and freeze-drying, a pure polysaccharide was obtained and named Dendrobium nobile flower polysaccharide DNLPS-3.

4. The application of the acidic polysaccharide from Dendrobium nobile flowers according to claim 1, characterized in that, The polysaccharide is used in the preparation of drugs or health products with immunomodulatory effects.

5. The application of the acidic polysaccharide from Dendrobium nobile flowers according to claim 1, characterized in that, The polysaccharide is used in the preparation of drugs that promote hepatocyte proliferation.

6. The application of the acidic polysaccharide from Dendrobium nobile flowers according to claim 1, characterized in that, The polysaccharide is intended for use in the preparation of drugs that alleviate oxidative stress in hepatocytes.

7. The application of the acidic polysaccharide from Dendrobium nobile flowers according to claim 1, characterized in that, The polysaccharide is used in the preparation of drugs or health products with hepatoprotective effects.

Images