White hyacinth bean flower homogeneous polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a white hyacinth bean flower homogeneous polysaccharide as well as a preparation method and application thereof. The purified white hyacinth bean flower polysaccharide (BDHPE2-F) is separated and purified from the white hyacinth bean flower crude polysaccharide, and the obtained BDHPE2-F is an acidic polysaccharide which mainly contains galacturonic acid and galactose, has the molecular weight of 10.814 kDa and has very high methyl esterification degree. As a novel polysaccharide, the BDHPE2-F is low in price and wide in application, has high anti-inflammatory activity and has development and commercial value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a homogeneous polysaccharide from white hyacinth bean flowers, its preparation method, and its application. Background Technology

[0002] Hyacinth bean flowers, used both as food and medicine, possess excellent anti-inflammatory effects and are rich in polysaccharides, offering significant nutritional and medicinal value. However, no purified polysaccharides from hyacinth bean flowers have been reported in existing patent or non-patent literature.

[0003] Yin Shuhua et al. (Physicochemical Properties, Antioxidant Activity and Antibacterial Properties of Non-Starch Polysaccharides from Hyacinth Bean. Food Industry Technology 41.19(2020):6.) reported the physicochemical properties, antioxidant activity and antibacterial properties of non-starch polysaccharides from Hyacinth Bean, laying a theoretical foundation for the deep processing and further research and development of Hyacinth Bean. Liu Fugang et al. (Study on In Vitro Antioxidant Activity of Polysaccharides from Four Traditional Chinese Medicines including Hyacinth Bean. Henan Science 27.10(2009):4.) provided a method for extracting polysaccharides from Hyacinth Bean by ethanol reflux and reported on the study on the in vitro antioxidant activity of Hyacinth Bean polysaccharides, providing a basis for the further development and utilization of antioxidants from traditional Chinese medicine.

[0004] Patent ZL202310859935.4 discloses a method for extracting polysaccharides from white hyacinth bean through hot water extraction and reveals the application of white hyacinth bean polysaccharides in the preparation of drugs for the prevention and / or treatment of ulcerative colitis. This provides a scientific basis for the use of white hyacinth bean polysaccharides in the prevention and treatment of UC and also provides a reference for future related industrial transformation. However, it has disadvantages such as cumbersome preparation method and high cost. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention isolates and purifies a purified polysaccharide (BDHPE2-F) from crude polysaccharide of *Hymenthocarpus spp.* flowers. The structure of BDHPE2-F was identified based on molecular weight, monosaccharide composition, FT-IR, and NMR techniques. BDHPE2-F was characterized by molecular weight, monosaccharide composition, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. The results show that BDHPE2-F is a highly methylated acidic polysaccharide mainly containing galacturonic acid and galactose, with a weight-average molecular weight of 10.814 kDa. BDHPE2-F exhibits high anti-inflammatory activity. While there are few patent reports on the anti-inflammatory properties of floral medicinal materials, the data shows that BDHPE2-F possesses good anti-inflammatory activity. Therefore, BDHPE2-F, as a novel polysaccharide, is inexpensive, has wide applications, and possesses development and commercial value.

[0006] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows: A homogeneous polysaccharide from white hyacinth bean flowers, wherein the homogeneous polysaccharide is composed of mannose (Man), glucosamine hydrochloride (GlcN), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), xylose (Xyl) and arabinose (Ara). Furthermore, the molar ratio of each monosaccharide—mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and arabinose—is 1.000:0.287:1.73:0.764:56.416:5.674:7.899:0.236:0.910. This invention, combining the results of monosaccharide composition determination, methylation analysis, and one-dimensional and two-dimensional NMR information analysis of polysaccharide samples, indicates that BDHPE2-F is a pectin polysaccharide mainly composed of HG domains and exhibits a high degree of methylation. Its possible repeating structure model is shown below: .

[0007] A method for extracting homogeneous polysaccharides from the above-mentioned white hyacinth bean flowers, comprising the following steps: (1) The crude polysaccharide of white hyacinth bean flower was subjected to ion exchange column chromatography and eluted sequentially with different salt concentrations of NaCl single distilled aqueous solution: dH2O, 0.2 M NaCl, 0.5 M NaCl, 1.0 M NaCl. After determining the polysaccharide content of each tube by the anthrone-sulfuric acid method, the samples were collected sequentially to obtain four fractions: BDHP-E1, BDHP-E2, BDHP-E3 and BDHP-E4. (2) The component sample BDHP-E2 was selected and subjected to Sephadex LH-20 gel filtration chromatography, concentrated under reduced pressure, and freeze-dried under vacuum to obtain the isolated and purified homogeneous polysaccharide of white hyacinth bean flower.

[0008] Furthermore, the crude polysaccharide from white hyacinth bean flowers is the crude polysaccharide from white hyacinth bean flowers obtained according to Example 1 of the specification of patent CN 118812737 B.

[0009] Furthermore, the gradient elution described in (1) is carried out sequentially with different salt concentrations: dH2O, 0.2 M NaCl, 0.5 M NaCl, and 1.0 M NaCl, with each concentration eluted twice the column volume and the elution rate being 15 mL / min.

[0010] Furthermore, (2) in the Sephadex LH-20 gel filtration column chromatography, ultrapure water was used for elution and separation, the column flow rate was 2.0 mL / min, eluted 1 column volume, and the eluted fraction in the 82-122 min region was collected.

[0011] This invention also provides the application of the homogeneous polysaccharide from white hyacinth bean flowers obtained by the above extraction method in the preparation of anti-inflammatory drugs. The anti-inflammatory effects include inhibiting nitric oxide (NO) production, inhibiting the expression of inflammatory factors (such as TNF-α, IL-6, and IL-1β), and inhibiting the activation of the NF-κB signaling pathway.

[0012] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) Novel purified polysaccharide: This invention isolates and purifies a purified polysaccharide (BDHPE2-F) from white hyacinth bean flowers. This is a novel natural polysaccharide with a unique molecular structure and activity. There are relatively few studies on the anti-inflammatory activity of this type of flower medicinal material, thus providing a new research direction for this field.

[0013] (2) Unique molecular characteristics: Through comprehensive analysis of molecular weight, monosaccharide composition, FT-IR and NMR techniques, BDHPE2-F was confirmed to be a methyl esterified acidic polysaccharide, with galacturonic acid and galactose as its main components and a molecular weight of 10.814 kDa. The characterization of these molecular characteristics has provided a deeper understanding of the structure of this polysaccharide and provided important scientific basis for subsequent application research.

[0014] (3) Significant anti-inflammatory activity: The BDHPE2-F of the present invention exhibits high anti-inflammatory activity. There are few existing patent reports on the anti-inflammatory activity of floral medicinal materials, therefore, this polysaccharide has great potential for application in the field of anti-inflammation and can provide a novel natural drug candidate for the treatment of related diseases.

[0015] (4) Environmental protection and sustainability: The polysaccharides of this invention are derived from natural plants and are extracted and purified using green and environmentally friendly processes. Compared with synthetic chemical drugs, they are more in line with the modern drug development requirements for natural, green and harmless substances and conform to the trend of sustainable development. Attached Figure Description

[0016] Figure 1 This is a flowchart illustrating the preparation, separation, and purification of crude polysaccharide from white hyacinth bean flowers according to the present invention. Figure 2 The structural characteristics of BDHPE2-F obtained in Example 1 are shown in the following figures: (A. HPGPC chromatogram; B. HPLC chromatogram; C. Fourier transform infrared spectrum). Figure 3 The total ion chromatogram of BDHPE2-F obtained in Example 1; Figure 4 The image shows the one-dimensional and two-dimensional NMR spectra of BDHPE2-F obtained in Example 1 (A). 1 H NMR spectrum; B. 13C10 NMR spectrum; C20 HSQC spectrum; D30 COSY spectrum; E40 HMBC spectrum; F50 NOESY spectrum); Figure 5 The effect of different concentrations of BDHPE2-F obtained in Example 1 on the survival rate of RAW 264.7 cells (A); the effect of BDHPE2-F on the production of NO, TNF-α, IL-6 and IL-1β in RAW 264.7 macrophages stimulated by LPS (Figure BE). Significant differences: *P<0.05 vs. blank group, ^P<0.05 vs. LPS group.

[0017] Figure 6 The effect of different concentrations of BDHPE2-F obtained in Example 1 on the expression of NF-κB signaling pathway proteins. Detailed Implementation

[0018] The technical solution of the present invention will be described in detail below with reference to specific embodiments. However, the described embodiments are only some embodiments of the present invention, and the content of the present invention is not limited to the following embodiments.

[0019] The relevant measurement methods are as follows in the following embodiments: Polysaccharide content determination method (1) In the formula: m1 - Mass (g) of crude polysaccharide from freeze-dried white hyacinth bean flowers; m2 - The mass (g) of white hyacinth bean flowers.

[0020] Method for determination of uronic acid content (2) In the formula, C is the sample mass concentration, mg / mL; V is the final volume of the sample, in mL; m is the sample mass, in mg.

[0021] Total sugar content was determined using the phenol-sulfuric acid colorimetric method (anthrone-sulfuric acid method). Using 0.1 mg / mL glucose solution as the standard solution, prepare standard curves (0, 0.02, 0.04, 0.08, 0.10 mg / mL). Take 0.5 mL of the 0.5 mg / mL sample solution, add 1 mL of distilled water, 1 mL of 5% phenol solution, and 5 mL of concentrated sulfuric acid, react for 30 min, and measure the absorbance at 490 nm using an ELISA reader. Record the absorbance and calculate the total sugar content (%).

[0022] Example 1: A homogeneous polysaccharide from white hyacinth bean flowers was extracted using the following method: S1. Extraction of crude polysaccharides from white hyacinth bean flowers Crude polysaccharides were extracted from white hyacinth bean flowers according to Example 1 of the specification of patent CN 118812737 B, specifically as follows: S1-1: Take 10g of white hyacinth bean flowers, wash them, dry and crush them, and pass the powder through a 60-mesh sieve; at room temperature, add 95% ethanol (liquid-to-solid ratio 10mL / g), shake on a shaker at 200rpm for 6h, then centrifuge the sample at 6000rpm for 10min, collect the precipitate, and dry it at 70℃ to obtain crude polysaccharide from white hyacinth bean flowers. S1-2. Hot water extraction: Add distilled water (liquid-to-solid ratio 20 mL / g) to the crude polysaccharide of white hyacinth bean flowers, extract at 95℃ for 6 h, cool and centrifuge at 6000 rpm for 10 min, and collect the supernatant I; S1-3. Protein removal: Add an equal volume of 3% trichloroacetic acid to the supernatant I, mix well, and let stand at 4°C overnight (12h, the same below). Centrifuge at 8000rpm for 10min and collect the supernatant II. S1-4. Crude polysaccharide from white hyacinth bean flowers: An equal volume of anhydrous ethanol was added to the supernatant II, mixed thoroughly, and allowed to stand overnight at 4°C. The mixture was then centrifuged at 8000 rpm for 10 min, the precipitate was collected, and freeze-dried to obtain crude polysaccharide from white hyacinth bean flowers. The content of crude polysaccharide, total sugar content, and uronic acid content of white hyacinth bean flowers were 7.05% ± 0.74%, 38.44% ± 1.43%, and 3.09 ± 0.80%, respectively.

[0023] S2. Isolation and purification of crude polysaccharides from white hyacinth bean flowers Separation and purification process such as Figure 1 As shown. The crude polysaccharide sample obtained from S1 was subjected to ion exchange column chromatography, with gradient elution using different salt concentrations of NaCl single-distilled aqueous solutions (dH2O, 0.2 M NaCl, 0.5 M NaCl, and 1.0 M NaCl solutions, respectively). After determining the polysaccharide content of each tube using the anthrone-sulfuric acid method, the following four fractions were collected sequentially: BDHP-E1 (eluted by dH2O), BDHP-E2 (eluted by 0.2 M NaCl), BDHP-E3 (eluted by 0.5 M NaCl), and BDHP-E4 (eluted by 1.0 M NaCl). After gradient elution, the eluent was analyzed using the anthrone-sulfuric acid method. No obvious color reaction was observed, indicating that the crude polysaccharide was completely eluted from the ion exchange column. Fraction BDHP-E2 was selected and subjected to Sephadex LH-20 gel filtration column chromatography. The eluted fraction from the 82-122 min zone was collected, concentrated under reduced pressure, and freeze-dried under vacuum to obtain the purified polysaccharide, named BDHPE2-F.

[0024] The specific operation of the above ion exchange column chromatography is as follows: Pretreatment of ion exchange chromatography media: Pour the DEAE sepharose FF gel suspension into a Buchner funnel, remove the liquid, and wash with about 3 times the volume of ultrapure water. Repeat the above operation several times until the packing material has no alcohol odor. Transfer it to a beaker, add half to one-half to one-time the volume of distilled water to the precipitated gel, and stir well in preparation for column packing.

[0025] Column packing and equilibration: After stirring the pretreated DEAE sepharose FF gel suspension, slowly add it to the XK chromatography column (φ3.0×50 cm). After complete sedimentation, connect the upper column head to the peristaltic pump and start the pump at a flow rate of 250 cm / h to flush the column with ultrapure water until the gel surface is stable, thus completing the column packing.

[0026] Sample loading and elution: The polysaccharide sample was dissolved in an appropriate amount of dH2O, centrifuged at 8000 rpm for 10 min to remove the precipitate, filtered through a 0.45 μm microporous membrane, and the filtrate was loaded onto a pre-equilibrated ion exchange chromatography column at a volume of 30% of the column volume. After all the sample solution had entered the column, elution was performed sequentially at different salt concentrations (dH2O, 0.2 M NaCl, 0.5 M NaCl, 1.0 M NaCl), eluting twice the column volume at each concentration at a rate of 15 mL / min. The eluent was collected using an automatic fraction collector, with 100 tubes collected for each eluent gradient, and 10 mL collected from each tube. The anthrone-sulfuric acid method was used, and the sample was read using a 630 microplate reader. The polysaccharide content in the eluent was detected by nm tracking. A polysaccharide elution curve was obtained by plotting the number of tubes on the x-axis and absorbance on the y-axis. Eluent from each tube corresponding to different elution peaks was collected, and ion-exchange column chromatography was repeated until the target substance was completely eluted and no target ions or impurities were found in the effluent. The combined eluent fractions were concentrated under reduced pressure, dialyzed through a dialysis bag with a 3.5 kDa cutoff, and then freeze-dried under vacuum to obtain the individual fractions.

[0027] The specific procedures for gel filtration column chromatography are as follows: Pretreatment of gel filtration chromatography media: Pour the Chromdex 200PG gel suspension into a Buchner funnel, remove the liquid, and wash with about 3 times the volume of ultrapure water. Repeat the above operation several times until the packing material has no alcohol odor. Transfer to a beaker, add half to one-half to one-time the volume of distilled water to the sediment, and stir well in preparation for column packing.

[0028] Column packing and equilibration: After stirring the pretreated gel filtration chromatography medium suspension, slowly add it to an XK chromatography column (φ2.6×100 cm). After complete sedimentation, connect the upper column head to the chromatography system and flush the column with ultrapure water at a flow rate of 30 cm / h until the gel surface is stable. The column packing is now complete.

[0029] Sample loading and elution: BDHP-E2 prepared by ion exchange column chromatography was dissolved in an appropriate amount of dH2O, centrifuged at 8000 rpm for 10 min to remove the precipitate, filtered through a 0.45 μm microporous membrane, and the filtrate was loaded onto a pre-equilibrated gel filtration chromatography column at a volume of 1% of the column volume. After all the sample solution had entered the column, it was eluted with ultrapure water at a flow rate of 2.0 mL / min (φ2.6×100 cm). An automatic fraction collector was used to collect the eluent, 5 mL per tube, eluting one column volume. Online detection was used to collect the eluent from the same elution peak region with high peak height and good symmetry. This process was repeated to enrich and purify the polysaccharides. The collected eluent fractions were combined, concentrated under reduced pressure, and freeze-dried under vacuum to obtain the polysaccharides purified by gel filtration column chromatography.

[0030] Structural Analysis of BDHPE2-F Structural analysis was performed according to the structural analysis method described in Example 1 of patent CN 118812737 B. Figure 2 The structural characteristic spectra of BDHPE2-F are shown in A. HPGPC chromatogram; B. HPLC chromatogram; C. Fourier transform infrared spectrum. Figure 2 As shown in A, BDHPE2-F is a homogeneous polysaccharide with a weight-average molecular weight of 10.814 kDa. Table 1 shows the monosaccharide composition of BDHPE2-F, consisting of... Figure 2 As shown in B and Table 1, BDHPE2-F is composed of mannose (Man), glucosamine hydrochloride (GlcN), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), xylose (Xyl), and arabinose (Ara), with a molar ratio of 1.000:0.287:1.73:0.764:56.416:5.674:7.899:0.236:0.910. The monosaccharide composition ratio reflects the structural characteristics of pectin polysaccharides. R1 reflects the content of homogalacturonic acid (HG) in the polysaccharide, R2 reflects the content of rhamnose-galacturonic acid-I (RG-I) domain, and R3 reflects the proportion of side chains within the RG-I domain. The HG content of BDHPE2-F is as high as 72.976%. Furthermore, the relatively large R1 value (5.349) of BDHPE2-F indicates that its main chain is dominated by HG-type domains, with a high proportion of linear regions; the relatively small R2 value (0.031) indicates a low RG-I content; and the relatively large R3 value (5.080) indicates a high proportion of side chains within the RG-I domain. These results suggest that BDHPE2-F is a pectin polysaccharide dominated by HG.

[0031] The structural characteristics of polysaccharides were analyzed using FT-IR at 4000-400 cm⁻¹. -1Infrared spectral scanning was performed within the range, and the results are as follows: Figure 2 As shown in Figure C, the infrared spectrum reveals the typical characteristics of polysaccharides. It can be seen from the figure that at 3443 cm⁻¹... -1 The broad and strong absorption peak at 2935 cm⁻¹ is due to the stretching vibration of the OH groups in sugars. -1 The smaller absorption peaks appearing at 1400–1200 cm⁻¹ are due to the stretching vibrations of methyl or methylene CH₄. -1 The peaks between these points are likely due to the CH angle vibrations of sugars, indicating that the sample is a polysaccharide. (At 1743 cm⁻¹) -1 The absorption peak at 1642 cm⁻¹ can be attributed to the symmetric stretching vibration of the C=O group of the esterified carboxyl group in galacturonic acid, while the peak at 1642 cm⁻¹ is... -1 The peak at 1743 cm⁻¹ is a characteristic absorption peak of the asymmetric stretching vibration of the free carboxyl group COO- in galacturonic acid. -1 1642 cm -1 The absorption peak area at 1743 cm⁻¹ is directly proportional to the number of esterified carboxyl groups and free carboxyl groups in the polysaccharide. Based on this principle, the degree of methyl esterification of pectin can be inferred from the area of ​​these two absorption peaks. The figure shows that 1743 cm⁻¹ is the optimal absorption peak area. -1 The absorption peak at 1642 cm⁻¹ is significantly greater than that at 1642 cm⁻¹. -1 The absorption peak at A1743 indicates a high degree of esterification. The calculated degree of esterification, DE = A1743 / (A1743+A1642), is 0.659, indicating a high degree of methyl esterification in the polysaccharide sample. The above infrared spectroscopy results show that the polysaccharide sample is an acidic polysaccharide with a high degree of methyl esterification.

[0032] Table 1 Monosaccharide composition of BDHPE2-F Methylation analysis of BDHPE2-F Sample pretreatment: Weigh 5 mg of sample, dissolve in 1 mL of primary water, add 200 µL of 0.2 M MES, then add 500 µL of 200 mg / mL carbodiimide, and react at room temperature for 2 h; add 1 mL of 2 M imidazole, then add 1 mL of freshly prepared 70 mg / mL NaBD4 aqueous solution, and react for 3 h; add 300 μL of glacial acetic acid to terminate the reaction, dialyze the sample for 48 h (with a cutoff value of 1 kDa), freeze-dry the sample after dialysis, and perform methylation treatment.

[0033] Dissolve 1 mg of lyophilized sample in 1 mL of DMSO, add 30 mg of NaOH, and incubate for 30 min; add 250 μL of iodomethane, purge with nitrogen, and react in the dark for 1 h, then add another 250 μL of iodomethane and react for 1 h; add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Wash three times with water; aspirate the lower dichloromethane phase and dry under nitrogen; add 1 mL of 2M TFA and react at 121℃ for 120 min; dry under nitrogen at 30℃; add 1 mL of freshly prepared 1 M NaBD4 (ammonia water) and incubate with magnetic stirring at room temperature for 2.5 h; add 300 μL of acetic acid to terminate the reaction and dry under nitrogen; add 2 mL of 5% (vol / vol) acetic acid methanol and dry under nitrogen twice at 40℃, then add another 2 mL of 5% (vol / vol) acetic acid methanol and dry under nitrogen twice at 40℃; add 1.5 mL of acetic anhydride, vortex to mix, and react at 100℃ for 2.5 h; add 2 mL of water and let stand for 10 min; add 1 mL of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and wash three times with water; take the lower dichloromethane phase and analyze it.

[0034] Instrument parameters (a) Chromatographic parameters The chromatographic system used was an Agilent gas chromatograph (Agilent 7890A; Agilent Technologies, USA), with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA). High-purity helium (purity not less than 99.999%) was used as the carrier gas. The flow rate was 1.0 mL / min, and the injection port temperature was 260℃. The injection volume was 1 μL, using split injection at a split ratio of 10:1, with a solvent delay of 2.2 min.

[0035] Temperature program: Hold at 50℃ for 1.0 min, increase to 130℃ at 50℃ / min, increase to 230℃ at 3℃ / min, and hold for 2 min.

[0036] (b) Mass spectrometry parameters The mass spectrometry system used was an Agilent 5977B quadrupole mass spectrometer from Agilent Technologies, USA, equipped with an electron impact ionization (EI) source and a MassHunter workstation. The EI source had an inlet temperature of 230°C, a quadrupole temperature of 150°C, and an electron energy of 70 eV. The scanning mode was full scan (SCAN), with a mass scan range (m / z) of 30-600.

[0037] The methylation analysis results of BDHPE2-F are as follows: Figure 3 As shown in Table 2, BDHPE2-F is a complex pectin polysaccharide containing 10 types of sugar residues. The 10 sugar residue types present in BDHPE2-F are as follows: t-Rhap, 1,2-Rhap, t-Glcp, t-GalpA, t-Galp, 1,4-GalpA, 1,4-Galp, 1,4-Glcp, 1,3,4-GalpA, and 1,4,6-GalpA, with a relative molar ratio of 1.517:0.949:1.143:9.754:2.172:78.173:2.356:1.902:1.058:0.975. Among them, t-GalpA and 1,4-GalpA are the major sugar residues, accounting for 9.754% and 78.173% respectively, while the remaining sugar residues account for a smaller proportion. The results showed that BDHPE2-F is mainly composed of GalA and Gal, which is consistent with the results of monosaccharide composition analysis.

[0038] Table 2. Analysis of sugar residue data of BDHPE2-F NMR analysis of BDHPE2-F To further obtain structural characteristics of the polysaccharide sample BDHPE2-F, one-dimensional nuclear magnetic resonance was performed. 1 H-NMR, 13 C-NMR and two-dimensional NMR measurements using HSQC, COSY, HMBC, and NOESY spectra are shown below. Figure 4 As shown in AF, the chemical shift information of all H and C of each major sugar residue is obtained, and the connection order between each sugar residue is inferred.

[0039] One-dimensional nuclear magnetic resonance hydrogen spectroscopy (NMR) was used 1 H-NMR and carbon spectroscopy (H-NMR) 13 Further analysis using C-NMR revealed the glycosidic bond configuration of BDHPE2-F. The proton NMR signals of the polysaccharide were mostly in the δ 3.0–5.5 ppm range, with the δ 4.5–5.5 ppm range typically representing the anomeric proton (H-1) resonance region. 1The characteristic signals of H-NMR can identify certain sugar residues or groups. For example, the methyl proton signal of the 6-position deoxy sugar appears in the high field region of δ 0.8~1.4 ppm; the methyl proton signal of the acetyl group (CH3COO- or -OAc) appears in the low field region of δ 1.9~2.2 ppm; and the methyl proton signal of the methyl ester (-COOCH3 or -COOMe) is located in δ 3.0~3.8 ppm. The chemical shifts of the anomeric carbon (C-1) signal in polysaccharides are generally between δ 90 and 110 ppm. Specifically, the α-configuration anomeric carbon signal typically appears between δ 95 and 103 ppm, while the β-configuration anomeric carbon signal usually appears above δ 101 ppm. The signal concentration region for C-2 to C-5 is located in the δ 65–85 ppm region. The chemical shifts of the substituted carbons exhibit glycosylation shifts, moving downfield. The unsubstituted C-6 signal is located near δ 60 ppm, while the substituted C-6 signal shifts downfield. According to... 13 The characteristic signals of C-NMR can identify certain sugar residues or groups. For example, the methyl carbon signal of deoxygenated sugars at position 6 appears in the high field region of δ 15–20 ppm; the carbonyl signal of uronic acid (-COOH) and its methyl ester or acetyl group appears in the low field region of δ 170–180 ppm; the methyl carbon signal of methyl ester is located in the region of δ 50–60 ppm; and the methyl carbon signal of acetyl group appears in the higher field region of δ 20–25 ppm.

[0040] from 1 H-NMR and 13 On the C-NMR spectrum, some characteristic signal peaks were found: (1) 1The δ 1.90~2.20ppm region in the H-NMR spectrum is the methyl proton signal of the O-acetyl group, with multiple signal peaks, indicating that the substitution of the acetyl group occurs at different positions of the sugar residues in the sugar chain. The methyl signal near δ 20.00ppm in the carbon spectrum is not obvious within the acetyl group, and the peak signal intensity is very small. Based on this, it can be inferred that there is a very low degree of acetylation in this polysaccharide sample; (2) The monosaccharide composition of the polysaccharide sample contains a large amount of galacturonic acid (79.101%). The characteristic absorption peak at δ 52.83ppm represents the methyl carbon connected at the C-6 position in the methyl esterified galacturonic acid residue. The cross peak of these two signals δ 3.71 / 52.81ppm is found in the HSQC spectrum. δ 3.71ppm is the methyl proton signal of the methyl ester (-COOMe). The methyl proton signal of the methyl ester δ 3.71ppm is found to have a cross peak δ 3.71 / 170.69 in the HMBC spectrum. The ppm value indicates that the signal at δ 170.69 ppm belongs to the C-6 position of esterified galacturonic acid residues, while the characteristic absorption peak at the C-6 position of unesterified galacturonic acid residues is located near δ 172.10 ppm. The C-6 signal of esterified galacturonic acid residues is significantly stronger than that of unesterified galacturonic acid residues. These characteristic signals indicate the presence of highly methylated galacturonic acid. The ratio of the peak heights of the two can be used to estimate the degree of esterification, DE = 58.9 / (58.9+29.5) = 0.666. Similar to the results of infrared spectroscopy, it can be inferred that the ratio of esterified to unesterified galacturonic acid residues is approximately 2:1.

[0041] In the monosaccharide composition test and methylation analysis results, the monosaccharide composition was mainly GalA, and the methylation analysis results showed that it mainly contained 1,4-GalpA sugar residues, as well as 1,4-Galp, t-Galp, and other sugar residues, suggesting that the polysaccharide sample may contain a large number of HG-type structural domains. Based on the monosaccharide composition and methylation analysis results of BDHPE2-F, the analysis... 1 H-NMR, 13 C-NMR, HSQC, and COSY NMR spectra revealed multiple anodic signals. The specific NMR analysis of the main sugar residue structures is as follows: Sugar residue GE 1,4 The anodic signals of this sugar residue, determined by HSQC and COSY, are δ 4.88 ppm (H-1) and δ 100.27 ppm (C-1), indicating that the sugar residue is in the α configuration. Based on the monosaccharide composition test and methylation analysis results, it is inferred that this residue is an α-GalpA sugar residue. The H-1 chemical shift of this sugar residue was determined to be δ 4.88 ppm using HSQC and COSY, and further analysis using COSY spectra... Figure 4D) The cross peaks can be used to deduce the H-2, H-3, and H-4 signals. The H-2, H-3, and H-4 chemical shifts of this sugar residue are assigned to δ 3.63 ppm, δ 3.92 ppm, and δ 4.38 ppm, respectively. After assigning the chemical shifts of hydrogen atoms on the sugar ring, the chemical shifts of C-1 to C-4 on the sugar ring can be assigned using HSQC correlation spectroscopy, which are δ 100.27 ppm, δ 67.43 ppm, δ 67.73 ppm, and δ 78.45 ppm, respectively. Furthermore, a cross-peak between H-5 and C-5 is found in the HSQC spectrum at δ 5.04~4.99 / 70.23 ppm, and a cross-peak at δ 3.71 / 52.81 ppm is found in the HSQC spectrum. δ 3.71 ppm represents the methyl proton signal of the methyl ester (-COOMe). The methyl proton signal and carbon signal of the methyl ester are δ 3.71 ppm and δ 52.81 ppm, respectively. In the HMBC spectrum (… Figure 4 A signal cross-peak of δ 3.71 / 170.69 ppm was found on E). Furthermore, an H-5 / C-6 signal cross-peak of δ 5.04~4.99 / 170.69 ppm was found on the HMBC spectrum. It can be determined that δ 170.69 ppm belongs to the C-6 position signal of the esterified galacturonic acid residue. The chemical shift of C-6 is δ 170.69 ppm, indicating that this sugar residue is methylated. The chemical shifts of C-1 and C-4 shift to a lower field indicate that the residue has undergone substitution at the C-1 and C-4 positions of the sugar ring. A large lower field shift of H-5 is due to the introduction of the methoxy group, which shifts the chemical shifts of the protons in the sugar ring. Combined with the methylation results, the sugar residue is deduced to be →4)-α-D-GalpA-6-OMe-(1→, labeled GE 1,4 The chemical shifts are assigned in Table 3.

[0042] Sugar residue GA 1,4The anodic signals of this sugar residue were determined to be δ 4.99 ppm (H-1) and δ 99.80 ppm (C-1) using HSQC and COSY, indicating that the sugar residue is in the α configuration. Based on the monosaccharide composition test and methylation analysis results, it is inferred that this residue is an α-GalpA sugar residue. The H-1 chemical shift of this sugar residue was determined to be δ 4.99 ppm using HSQC and COSY. Then, the H-2, H-3, and H-4 signals can be deduced from the cross peaks in the COSY spectrum. The H-2, H-3, and H-4 chemical shifts of this sugar residue were assigned to δ 3.66 ppm, δ 3.94 ppm, and δ 4.37 ppm, respectively. After assigning the chemical shifts of the hydrogen atoms on the sugar ring, the chemical shifts of C-1 to C-4 on the sugar ring can be assigned using HSQC correlation spectroscopy, which are δ 99.80 ppm, δ 67.43 ppm, δ 67.73 ppm, and δ 78.44 ppm, respectively. Furthermore, a cross-peak between H-5 and C-5 at δ 4.93~4.97 / 69.99 ppm was found on the HSQC spectrum. Additionally, a cross-peak between H-5 and C-6 at δ 4.93~4.97 / 172.10 ppm was found on the HMBC spectrum. It can be determined that δ 172.10 ppm belongs to the C-6 position signal of the unesterified galacturonic acid residue. The chemical shifts of C-1 and C-4 shift to a lower field indicate that this residue has undergone substitution at the C-1 and C-4 positions on the sugar ring. Combined with the methylation results, the sugar residue is inferred to be →4)-α-D-GalpA-(1→, labeled GA 1,4 The chemical shifts are assigned in Table 3.

[0043] Sugar residue G 1,4 The anodic signals of this sugar residue, determined by HSQC and COSY, are δ 4.54 ppm (H-1) and δ 104.26 ppm (C-1), indicating that the sugar residue is in the β configuration. The H-1 chemical shift of this sugar residue, determined by HSQC, is δ 4.54 ppm, and the anodic signal is also determined by COSY. Figure 4D) The cross-peaks in the spectrum lead to the H-2, H-3, H-4, and H-5 signals. The chemical shifts of H-2, H-3, H-4, and H-5 for this sugar residue are assigned to δ 3.59 ppm, δ 3.78 ppm, δ 4.07 ppm, and δ 3.62 ppm, respectively. The H-6a and H-6b signals can be assigned using HSQC correlation spectroscopy, to δ 3.63 ppm and δ 3.71 ppm, respectively. After assigning the chemical shifts of the hydrogens on the sugar ring, the chemical shifts of the carbons on the sugar ring can be assigned using HSQC correlation spectroscopy, to δ 104.26 ppm, δ 71.87 ppm, δ 69.01 ppm, δ 77.50 ppm, δ 74.45 ppm, and δ 60.61 ppm, respectively. The chemical shifts of C-1 and C-4 towards the lower field indicate that these residues have undergone substitution at positions C-1 and C-4 of the sugar ring. Combined with the monosaccharide composition and methylation analysis results, the sugar residue is inferred to be →4)-β-D-Galp-(1→, labeled G 1,4 The chemical shifts are assigned in Table 3.

[0044] Two reducing end-group signals appeared at δ 92.15 ppm and δ 95.93 ppm. Cross-peaks of these two signals were found on the HSQC spectrum at δ 5.26 / 92.15 ppm and δ 4.57 / 95.93 ppm. Further analysis of the cross-peaks in the COSY spectrum at δ 5.26 / 3.74 ppm and δ 4.57 / 3.40 ppm led to the deduced H-2 chemical shifts of δ 3.74 ppm and δ 3.40 ppm for these two sugar residues. Based on the monosaccharide composition determination and methylation analysis results, and in conjunction with literature, the C-1 signal peaks are inferred to belong to the reducing end-groups α-GalpA and β-GalpA, and are labeled as R... α and R β Using COSY and HSQC, the chemical shifts were derived and assigned to them, as shown in Table 3.

[0045] Following a similar method, combining monosaccharide composition determination, methylation analysis results, and literature reports, the hydrogen and carbon signals of other residues were derived to infer GE. 1,3,4 For →3,4)-α-D-GalpA-6-OMe-(1→, infer GA t Given α-D-GalpA-(1→), infer G t The chemical shifts of the main sugar residues H and C in the polysaccharide samples are summarized in Table 3.

[0046] Based on the polysaccharide methylation analysis results, the polysaccharide sample also contained 1,4-Glcp, 1,2-Rhap, and t-Rhap sugar residue linkages, but the content was very low (less than 2%), and the signals in NMR were very weak, making it impossible to determine their exact composition. 1 H and 13 C is assigned to a specific category.

[0047] By analyzing the coupling signals between anomeric hydrogens and carbons on each sugar residue in the HMBC long-range correlation spectrum, or the coupling signals between anomeric carbons and hydrogens on each sugar residue, and considering that two protons at adjacent sugar residue linkage sites tend to generate strong NOE signals due to their spatial proximity, the linkage order between sugar residues can be further inferred using HMBC long-range correlation and NOESY spectra. HMBC correlation spectra of polysaccharide samples ( Figure 4 E) and NOESY spectrum ( Figure 4 As shown in F), the following coupling signals can be found in the figure: (1) Glycosyl residues GE in HMBC map 1,4 H-1 (δ 4.88 ppm) and sugar residues GE 1,4 C-4 (δ 78.45ppm) has a related signal peak (GE 1,4 H-1 / GE 1,4 C-4), GE sugar residues in the NOESY spectrum 1,4 H-1 (δ 4.88 ppm) and sugar residues GE 1,4 H-4 (δ 4.38 ppm) showed a cross peak (GE). 1,4 H-1 / GE 1,4 H-4), indicating the presence of →4)-α-D-GalpA-6-OMe-(1→4)-α-D-GalpA-6-OMe-(1→connection; (2) Glycosyl ester residues in HMBC spectrum 1,4 H-1 (δ 4.88 ppm) and sugar residues GA 1,4 C-4 (δ 78.44 ppm) exhibited a cross peak (GE). 1,4 H-1 / GA 1,4 C-4), indicating the existence of →4)-α-D-GalpA-6-OMe-(1→4)-α-D-GalpA-(1→connection; (3) Glycosyl residues GA in HMBC map 1,4 H-1 (δ 4.99 ppm) and sugar residues GE 1,4 C-4 (δ 78.45ppm) has a related signal peak (GA) 1,4 H-1 / GE 1,4 C-4), GA sugar residues in the NOESY spectrum 1,4H-1 (δ 4.99 ppm) and sugar residues GE 1,4 The H-4 (δ 4.38 ppm) exhibited a cross peak (GA). 1,4 H-1 / GE 1,4 H-4), indicating the presence of →4)-α-D-GalpA- (1→4)-α-D-GalpA-6-OMe-(1→connection; (4) G sugar residues in the HMBC map 1,4 H-1 (δ 4.54 ppm) and sugar residues GE 1,3,4 C-3 (δ 77.92ppm) has a related signal peak (G 1,4 H-1 / GE 1,3,4 C-3) indicates the presence of a connection between →4)-β-D-Galp-(1→ and →3,4)-α-D-GalpA-6-OMe-(1→, with the connection site located at position O-3.

[0048] Table 3 Sugar residues in BDHPE2-F 1 H and 13 C chemical shift distribution "-" indicates undetermined or not detected. Based on the determination of monosaccharide composition, methylation analysis, and one-dimensional and two-dimensional NMR information of the polysaccharide sample, it is indicated that BDHPE2-F is a pectin polysaccharide mainly composed of HG domains and exhibits a high degree of methylation. Its possible repeating structure model is shown below: Example 2: Anti-inflammatory activity analysis of BDHPE2-F 1. Cytotoxicity assay The BDHPE2-F obtained in Example 1 was dissolved in sterile water to obtain different concentrations of BDHPE2-F. The effect of BDHPE2-F on the survival rate of RAW264.7 cells (purchased from ATCC) was determined using a CCK-8 assay kit (CCK-8, Beyotime, China) (this step was performed according to the kit instructions). RAW264.7 cells were cultured at 5 × 10⁶ cells per well. 3Cells were seeded at a density of 100 μL in 96-well plates and cultured for 24 h. Subsequently, cells were treated with 100 μL of different concentrations of BDHPE2-F (0, 6.25, 12.5, 25, 50, 100, and 200 μg / mL) and co-incubated with 1 μg / mL LPS for 24 h. Afterward, 10 μL of CCK-8 solution was added to each well, and incubation was continued for 1 h in the dark. Finally, the absorbance (OD) value at 450 nm was measured.

[0049] like Figure 5 As shown in Figure A, BDHPE2-F at concentrations of 6.25, 12.5, 25, 50, 100, and 200 μg / mL showed no toxicity to RAW264.7 cells. However, compared to other dosage groups, 200 μg / mL BDHPE2-F showed a trend of inhibiting RAW264.7 cells. Therefore, subsequent experiments selected BDHPE2-F concentrations of 25, 50, and 100 μg / mL. ELISA results more clearly demonstrated the anti-inflammatory effect.

[0050] 2. NO inhibition rate experiment (1) Cell culture: First, RAW264.7 macrophages were cultured in DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin antibiotics at a constant temperature of 37℃ and 5% CO2. Cells in good growth condition were selected and the cell density was adjusted to 1×10⁶ cells / year. 5 100 μL of cells per well were seeded into 96-well plates, with 100 μL of sterile PBS buffer (pH 7.4, 0.01 M) added to the outer wells. The plates were incubated at 37°C and 5% CO2 for 12 hours.

[0051] (2) Sample preparation: First, weigh the compound obtained in Example 1, and prepare it with a certain amount of sterile water to 10 mg / mL. Then, use serum-free DMEM medium to prepare test compound solutions with different concentration gradients, with concentrations of 25, 50 and 100 μg / mL.

[0052] (3) Drug treatment: After cell adhesion, the DMEM medium in the wells was aspirated. For the drug group, 1 μg / mL LPS and different concentrations of the test compound solution were added, 100 μL per well; for the blank group, 100 μL of serum-free DMEM medium was added per well; for the model group, 1 μg / mL LPS solution was added, 100 μL per well; each group had 3 replicates. The 96-well plate was returned to the incubator and cultured for 24 hours, then the NO assay kit was used for detection.

[0053] (4) Measurement: According to the kit instructions, the amount of NO released by macrophages under the action of the above compounds was detected. The absorbance of each well was measured at a wavelength of 540 nm using an ELISA reader. The concentration of NO was calculated based on the absorbance value and the NO standard curve.

[0054] 3. ELISA detection of pro-inflammatory factors IL-6, IL-1β, and TNF-α RAW264.7 macrophages in good growth condition during the logarithmic growth phase were selected and subjected to a 1×10⁻⁶ PCR. 5 Cells were seeded per well into 96-well plates and cultured at 5% CO2 and 37°C for 12 h. The culture medium was then aspirated. Following the method in step (3) of the NO inhibition rate experiment, blank group, model group, and drug group were set up, with 6 replicates per group. After incubation at 5% CO2 and 37°C for 24 h, the supernatant was collected (without aspirating the cells). The results were analyzed according to the instructions of the enzyme-linked immunosorbent assay kit, a standard curve was constructed, and the levels of relevant inflammatory factors were calculated.

[0055] 4. Western blotting analysis of the expression levels of TLR-4, p65, p-p65, IκB, p-IκB, and β-actin. To investigate the anti-inflammatory mechanism of BDHPE2-F (0, 50, or 100 μg / mL), the activation of the NF-κB pathway was detected by Western blotting. The expression levels of TLR-4, p65, p-p65, IκB, p-IκB, and β-actin were analyzed by Western blotting.

[0056] RAW264.7 cells were treated according to step 3 above, and the culture medium was discarded after 24 hours. Cells were then washed twice with PBS and lysed with RIPA lysis buffer at 4°C. The lysis buffer was collected and centrifuged at 10,000 r / min for 5 min. The protein content in the supernatant was determined using a BCA kit. Proteins were denatured in a boiling water bath and separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to a 0.45 μm PVDF membrane. The membrane was then blocked with skim milk for 2 h and incubated overnight with primary antibody at 4°C. It was washed three times with TBST, incubated with the corresponding secondary antibody at room temperature for 1 h, and finally washed three times with TBST. Finally, antibody-specific proteins were visualized using an ECL enhancement kit, and images were taken using a chemiluminescence imaging system.

[0057] Experimental results: like Figure 5As shown in the BE study, LPS stimulation led to the overexpression of NO, TNF-α, IL-6, and IL-1β-related proteins in RAW264.7 cells, while BDHPE2-F intervention significantly inhibited this overexpression. At concentrations of 25, 50, and 100 μg / mL, BDHPE2-F significantly inhibited LPS-induced protein overexpression (P<0.05 compared to the NC group) and significantly reduced the expression levels in the LPS group (P<0.05). Notably, BDHPE2-F exhibited the strongest inhibitory effect at a concentration of 100 μg / mL.

[0058] NF-κB is a widely distributed eukaryotic transcription factor in various cells, playing a key regulatory role in innate immunity, adaptive immunity, inflammation, and stress responses. Furthermore, activated NF-κB pathways can induce the expression of genes related to NO, TNF-α, IL-6, and IL-1β. p65, p-p65, IκB, and p-IκB are key proteins in the NF-κB pathway, and its activation is influenced by the expression levels of these four proteins.

[0059] The results showed that, compared with the other three groups, the p65, p-p65, IκB, and p-IκB bands in the LPS group were significantly wider and darker, indicating that the NF-κB signaling pathway was successfully activated. Figure 6 Compared with the NC group, the brightness of the p65, p-p65, IκB and p-IκB bands in the 50 and 100 μg / mL groups showed concentration-dependent changes, and the bands were lighter than those in the LPS group, indicating that BDHPE2-F can reduce inflammation in vitro by inhibiting the NF-κB pathway.

Claims

1. A homogeneous polysaccharide from white hyacinth bean flowers, wherein the homogeneous polysaccharide is composed of mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and arabinose.

2. The homogeneous polysaccharide from white hyacinth bean flowers according to claim 1, characterized in that, The molar ratio of each monosaccharide in the mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and arabinose is 1.000:0.287:1.73:0.764:56.416:5.674:7.899:0.236:0.

910.

3. The homogeneous polysaccharide from white hyacinth bean flowers according to claim 1, characterized in that, The repeating structure model of the homogeneous polysaccharide from white hyacinth bean flowers is shown below: 。 4. The method for extracting homogeneous polysaccharides from white hyacinth bean flowers according to any one of claims 1-3, comprising the following steps: (1) The crude polysaccharide of white hyacinth bean flower was subjected to ion exchange column chromatography and eluted sequentially with different salt concentrations of NaCl single distilled aqueous solution: dH2O, 0.2 M NaCl, 0.5 M NaCl, 1.0 M NaCl. The samples were collected sequentially to obtain four fractions: BDHP-E1, BDHP-E2, BDHP-E3 and BDHP-E4. (2) The component sample BDHP-E2 was selected and subjected to Sephadex LH-20 gel filtration chromatography, concentrated under reduced pressure, and freeze-dried under vacuum to obtain the isolated and purified homogeneous polysaccharide of white hyacinth bean flower.

5. The extraction method according to claim 4, characterized in that, (1) During gradient elution, each concentration is eluted twice the column volume, and the elution rate is 15 mL / min.

6. The extraction method according to claim 4, characterized in that, In the Sephadex LH-20 gel filtration column chromatography described in (2), ultrapure water was used for elution and separation. The column flow rate was 2.0 mL / min, and 1 column volume was eluted. The eluted fraction in the 82-122 min region was collected.

7. The use of the homogeneous polysaccharide from white hyacinth bean flowers according to any one of claims 1-3 in the preparation of anti-inflammatory drugs.

8. The use of the homogeneous polysaccharide of white hyacinth bean flower obtained by the extraction method according to any one of claims 4-6 in the preparation of anti-inflammatory drugs.

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