Cistanche tubulosa polysaccharide, preparation method and application thereof

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to a Cistanche deserticola polysaccharide, its preparation method, and its applications. The Cistanche deserticola polysaccharide provided by this invention has a weight-average molecular weight (Mw) of 85.34 kDa, a number-average molecular weight (Mn) of 56.39 kDa, and a dispersion factor (Mw / Mn) of 1.51. This invention uses pre-column derivatization via PMP to determine the monosaccharide composition, followed by methylation-GC / MS to identify sugar residue linkage sites. Finally, it combines nuclear magnetic resonance spectroscopy to comprehensively analyze the sugar ring configuration and sequence information, providing a material basis for subsequent activity studies. The polysaccharide purified by this invention has lipid-lowering effects, increasing the added value of Cistanche deserticola. Simultaneously, it achieves the intensive processing and comprehensive utilization of this unique resource. Furthermore, the polysaccharide is derived from green and natural sources, and the product is safe and has no toxic side effects, making it promising for the development of lipid-lowering products.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a Cistanche deserticola polysaccharide, its preparation method, and its application. Background Technology

[0002] Cistanche deserticola ( Cistanche deserticola YC Ma, CD *Cynanchum paniculatum* (CD) is a plant in the Orobanchaceae family that is both medicinal and edible. Its medicinal history can be traced back to the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), and it has long been used as a traditional Chinese medicine and tonic. CD has the effects of tonifying the kidneys and strengthening yang, moistening the intestines and relieving constipation, and is used to treat sexual dysfunction, deficiency of essence and blood, and weakness of the lower back and knees. Modern pharmacological studies have shown that CD possesses various biological activities, such as anti-aging, antidepressant, hepatoprotective, hypoglycemic and hypolipidemic effects, anti-tumor and anti-inflammatory effects, anti-osteoporosis, and anti-fatigue activity. These effects are closely related to its unique phenylethanoid glycosides, iridoid glycosides, lignans, and polysaccharides.

[0003] Polysaccharides, a class of water-soluble biomacromolecules, are formed by the condensation of 10 or more monosaccharide units through glycosidic bonds and are widely distributed in animal, plant, and microbial systems. Their excellent biocompatibility and safety characteristics make them ideal candidate molecules for drug development and functional nutritional factors. However, due to factors such as linkage sites, glycan ring structures, heterostructures, and branching, the structural characterization of polysaccharides with numerous glycosidic bonds is extremely difficult. With the development of analytical techniques, the emergence of analytical instruments such as nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) has enabled precise analysis of the primary structure of polysaccharides (monosaccharide composition, linkage sequence, and glycosidic bond type). Nevertheless, given the difficulty in achieving high-purity separation of Cistanche deserticola polysaccharides and their structural complexity, significant gaps remain in the study of their structure-activity relationship. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention provides a Cistanche deserticola polysaccharide CDPS-W1.

[0005] This invention further provides a method for preparing the above-mentioned Cistanche deserticola polysaccharide. A multi-stage purification system was constructed by combining DEAE-52 cellulose anion exchange column chromatography (ion exchange type) and Sephacryl S-200 dextran gel column chromatography (molecular sieve type). After systematic separation and optimization, a homogeneous polysaccharide component CDPS-W1 was successfully prepared.

[0006] The present invention further provides the application of the above-mentioned Cistanche deserticola polysaccharide CDPS-W1.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0008] This invention provides a Cistanche deserticola polysaccharide CDPS-W1, whose repeating structural unit is:

[0009] .

[0010] The above-mentioned Cistanche deserticola polysaccharide CDPS-W1 has the following structural units:

[0011] .

[0012] Preferably, the Cistanche deserticola polysaccharide CDPS-W1 has a weight-average molecular weight (Mw) of 85.34 kDa, a number-average molecular weight (Mn) of 56.39 kDa, and a dispersion index (Mw / Mn) of 1.51.

[0013] This invention also provides a method for preparing the above-mentioned Cistanche deserticola polysaccharide, comprising the following steps:

[0014] (1) The medicinal material of Cistanche deserticola was extracted by heating with distilled water and precipitated with ethanol to obtain crude polysaccharide of Cistanche deserticola;

[0015] (2) The crude polysaccharide of Cistanche deserticola was eluted by distilled water and different concentrations of NaCl through a chromatography column. Five elution peaks were obtained by tracking and detection by phenol-sulfuric acid method and named CDPS-W, CDPS-0.15M, CDPS-0.3M, CDPS-0.45M and CDPS-0.6M respectively.

[0016] (3) Dissolve the CDPS-W component in deionized water, then separate it with Sephacryl S-200, elute with deionized water, and three elution peaks appear. Collect the three segments and name them CDPS-W1, CDPS-W2 and CDPS-W3 respectively.

[0017] Preferably, in step (1), the volume ratio of the Cistanche deserticola medicinal material to distilled water is 1:10; the heating extraction is performed at a slight boiling state for 2 hours; and the extraction is repeated three times.

[0018] Preferably, in step (2), the chromatography column is a DEAE-52 cellulose ion exchange chromatography column; the concentrations of NaCl are 0.15 M, 0.3 M, 0.45 M and 0.6 M, respectively; and the elution flow rate is 4 mL / min.

[0019] Preferably, in step (3), the concentration of the CDPS-W solution is 50 mg / mL.

[0020] Preferably, in step (3), the separation range of the Sephacryl S-200 is 5000-250000 Da.

[0021] Preferably, in step (3), the flow rate of the deionized water elution is 0.5 mL / min.

[0022] The present invention further provides the application of the above-mentioned Cistanche deserticola polysaccharide in the preparation of lipid-lowering drugs.

[0023] The beneficial effects of this invention are as follows:

[0024] (1) In this invention, a multi-stage purification system was first constructed using DEAE-52 cellulose anion exchange column chromatography (ion exchange type) and SephacrylS-200 dextran gel column chromatography (molecular sieve type) to separate and purify homogeneous polysaccharides from Cistanche deserticola. Then, the monosaccharide composition was determined by pre-column derivatization using PMP. Subsequently, the sugar residue linkage sites were determined by methylation-GC / MS technology. Finally, the sugar ring configuration and sequence information were comprehensively analyzed by combining nuclear magnetic resonance spectroscopy, which provided a material basis for subsequent activity studies.

[0025] (2) The polysaccharide purified by this invention has lipid-lowering effects, which increases the added value of Cistanche deserticola. At the same time, it realizes the intensive processing and comprehensive utilization of characteristic resources. Moreover, the polysaccharide is green and natural, and the product is safe and has no toxic side effects. It is expected to be applied to the development of lipid-lowering products. Attached Figure Description

[0026] Figure 1 Elution curves of total polysaccharides from Cistanche deserticola via a DEAE-52 cellulose anion exchange chromatography column;

[0027] Figure 2 Elution curves for a Sephacryl S-200 chromatography column;

[0028] Figure 3 The HPGPC chromatogram of CDPS-W1;

[0029] Figure 4 Chromatogram of mixed standards;

[0030] Figure 5 The chromatogram of CDPS-W1;

[0031] Figure 6 The GC-MS total ion chromatogram of the CDPS-W1 methylated products;

[0032] Figure 7 For CDPS-W1 1 H NMR spectrum;

[0033] Figure 8 For CDPS-W1 13 C NMR spectrum;

[0034] Figure 9 DEPT135 spectrum of CDPS-W1;

[0035] Figure 10COSY spectrum of CDPS-W1;

[0036] Figure 11 The HSQC spectrum of CDPS-W1;

[0037] Figure 12 HMBC spectrum of CDPS-W1;

[0038] Figure 13 NOESY spectrum of CDPS-W1;

[0039] Figure 14 The figure shows the lipid-lowering effect of CDPS-W1 on HepG-2 cells; in the figure, A represents the effect on TC content in HepG2 cells; and B represents the effect on TG content in HepG2 cells. Detailed Implementation

[0040] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0041] Example 1

[0042] 1. Experimental instruments and reagents

[0043] 1.1 Experimental Apparatus

[0044] Table 1

[0045]

[0046] 1.2 Experimental Reagents

[0047] Table 2

[0048]

[0049] 2. Experimental Methods

[0050] 2.1 Preparation of total polysaccharides from Cistanche deserticola

[0051] Weigh 1000 g of dried Cistanche deserticola and pulverize and sieve it. Add distilled water at a ratio of 1:10 and heat to a gentle boil for 2 hours. Filter with gauze and repeat the extraction 3 times. Combine the extracts and centrifuge at 3000 r·min. -1 (30 min), discard the precipitate, combine the supernatants and concentrate to 3 L, add 4 times the amount of anhydrous ethanol (to make the volume fraction of ethanol in the solution 80%), and stir slowly with a glass rod during the addition of anhydrous ethanol until no precipitate is formed. Place in an environment of 4 °C overnight, wash the precipitate 3 times with anhydrous ethanol and then vacuum dry to constant weight to obtain total polysaccharides of Cistanche deserticola.

[0052] 2.2 DEAE-52 cellulose ion exchange column chromatography

[0053] 2.2.1 Packing material pretreatment

[0054] Add 300 g of DEAE-52 anion exchange cellulose packing material to 2000 mL of deaerated distilled water, soak overnight to fully swell, and wash repeatedly until there is no obvious odor. Filter with a vacuum pump to remove any air bubbles that may be present in the packing material, and place the treated packing material in deaerated distilled water for later use.

[0055] 2.2.2 Column Loading and Balancing

[0056] A 5.5 cm × 30 cm borosilicate glass chromatography column was selected and vertically fixed on an iron stand. The distilled water was degassed using an ultrasonic apparatus (degassed distilled water was used throughout the process). After sealing the bottom valve, degassed water was injected to 1 / 3 of the column height. The pretreated DEAE-52 cellulose was mixed with one column volume of degassed water to form a homogeneous suspension, which was slowly added to the column using a glass rod. The outlet at the bottom of the column was opened, allowing the packing material to settle naturally under gravity. During this process, the packing material inside the column needed to be stirred slowly, and the column surface should be observed constantly to prevent it from drying out. After packing, the column was flushed with degassed distilled water overnight to allow it to equilibrate.

[0057] 2.2.3 Sample loading and elution

[0058] Dissolve 1.0 g of total polysaccharides from Cistanche deserticola in 2.0 mL of distilled water, and centrifuge at high speed to remove insoluble matter. Then, add the solution to a pre-equilibrated DEAE-52 cellulose ion-anion exchange column (5.5 cm × 30 cm, Cl). - The sample was eluted sequentially with distilled water and 0.15, 0.3, 0.45, and 0.6 mol / L NaCl solutions at a flow rate of 4 mL / min. The phenol-sulfuric acid method, combined with λ220 nm tracking detection, was used. The eluents were combined, concentrated, and the salt components were removed using a dialysis belt with a pore size of 3500 Da. Finally, the sample was filtered through a 0.45 μm filter membrane and freeze-dried to obtain five components: CDPS-W, CDPS-0.15M, CDPS-0.3M, CDPS-0.45M, and CDPS-0.6M. The elution curves are shown below. Figure 1 As shown.

[0059] 2.3 Sephacryl S-200 column chromatography

[0060] Take the distilled water eluent fraction CDPS-W from section 2.2.3, dissolve it in deionized water to a concentration of 50 mg / mL, and load it onto a Sephacryl S-200 column (1.6 cm × 80 cm) with a sample loading volume of 1 mL. Elute with deionized water at a flow rate of 0.5 mL / min, and detect using the phenol-sulfuric acid method. Collect the eluent fractions, combine and concentrate them, and freeze-dry them to obtain fractions CDPS-W1, CDPS-W2, and CDPS-W3. The elution curves are shown below. Figure 2 As shown.

[0061] 2.4 Purity and Molecular Weight Determination

[0062] 2.4.1 Preparation of standard solutions

[0063] Accurately weigh approximately 10 mg of dextran with Mw values ​​of 1000, 5000, 12000, 25000, 50000, 80000, 150000, and 410000 respectively. Dilute each solution to 1 mL with the mobile phase and dissolve thoroughly. Filter the solution through a 0.45 μm microporous membrane into a liquid chromatography vial for HPLC analysis.

[0064] 2.4.2 Preparation of the test solution

[0065] Accurately weigh approximately 10 mg of total polysaccharides from Cistanche deserticola, dilute to 1 mL with the mobile phase and dissolve thoroughly to prepare a 10 mg / mL sample solution. Filter the solution through a 0.45 μm microporous membrane into a liquid chromatography vial for molecular weight determination.

[0066] 2.4.3 Chromatographic conditions

[0067] Instrument: Agilent 1200 HPLC system with RID detector; Column: PL aqnagel-OH MIXEDM (7.5 mm × 300 mm, 8 μm); Flow rate: 1 mL·min -1 Column temperature: 30 °C; RID detector temperature: 40 °C; Mobile phase: 0.1 M NaNO3; Injection volume: 5 μL.

[0068] 2.4.4 Establishment of the Standard Curve

[0069] Plot the logarithm of the molecular weight (lg Mw) of reference solutions with known molecular weights on the ordinate, and the corresponding retention time (t) on the ordinate. R Plotting the graph on the x-axis and performing narrow calibration using GPC software, a standard curve was obtained, which was then used to determine the molecular weight of the total polysaccharide sample from Cistanche deserticola.

[0070] 2.5 Monosaccharide Composition

[0071] 2.5.1 Complete acid hydrolysis

[0072] Weigh 10 mg of total polysaccharide sample from Cistanche deserticola into a 20 mL stoppered test tube, add 5 mL of 4 mol / L trifluoroacetic acid (TFA), purge with N2, stopper the tube, and hydrolyze at 120 ℃ for 2 h. Transfer the solution, concentrate to dryness under reduced pressure at 60 ℃, add a small amount of methanol, evaporate to dryness, leave exposed for 10 min, add methanol again, evaporate to dryness, repeat 3-5 times to completely evaporate the TFA. Obtain the hydrolyzed total polysaccharide sample from Cistanche deserticola for HPLC analysis after derivative preparation.

[0073] 2.5.2 HPLC Analysis

[0074] 2.5.2.1 Preparation of mixed reference standard PMP derivatives

[0075] Accurately weigh approximately 10 mg each of mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, and arabinose, and place them in a 10 mL volumetric flask. Dilute to volume with distilled water. Transfer 200 μL of this solution to a 25 mL pear-shaped flask and add 100 μL of 0.3 mol·L⁻¹ solution sequentially. -1 NaOH solution and 100 μL 0.3 mol·L⁻¹ -1 PMP methanol solution was reacted at 70 °C for 1 h. After cooling, 100 μL of 0.3 mol·L⁻¹ was added. -1 Hydrochloric acid solution. Add 1 mL of distilled water, mix well, add 1.5 mL of chloroform, centrifuge, and discard the meta layer. Add an equal volume of chloroform and wash three times. Collect the upper aqueous phase, filter through a 0.45 μm filter membrane, and make up to volume to obtain the mixed monosaccharide reference derivative.

[0076] 2.5.2.2 Preparation of PMP derivatives for samples

[0077] Add 200 μL of distilled water to the completely hydrolyzed sample to dissolve it completely. Then add 100 μL of glucosamine hydrochloride solution (5 mg / mL) and 200 μL of 0.3 mol·L⁻¹ hydrochloride solution. -1 NaOH solution and 200 μL 0.3 mol·L⁻¹ -1 A PMP methanol solution was incubated in a water bath at 70 °C for 1 h, then cooled to room temperature. 200 μL of 0.3 mol·L⁻¹ PMP solution was added to a stoppered test tube. -1 Add HCl solution, vortex for 30 s, then add 1 mL of distilled water to neutralize, dilute and shake well, transfer to a centrifuge tube, add 1.5 mL of chloroform, vortex for 30 s, and centrifuge (6000 r·min). -1(3 min), take the supernatant, add an equal volume of chloroform, repeat the washing three times, take the upper aqueous phase, filter it through a 0.45 μm filter membrane, make up to volume, transfer it to the injection vial, and wait for analysis.

[0078] 2.5.2.3 Chromatographic conditions

[0079] Column: YMC-Pack ODS-A (4.6 mm × 250 mm, 5 μm); Mobile phase: 0.05 mol·L⁻¹ -1 Phosphate buffer (KH₂PO₄-NaOH, pH = 7.2):acetonitrile = 81:19; column temperature: 30 ℃; detection wavelength: 250 mm; injection volume: 10 μL; flow rate: 0.8 mL·min -1 .

[0080] 2.6 Methylation Analysis

[0081] 2.6.1 Sample Derivatization

[0082] Accurately weigh approximately 2 mg of the sample to be tested, dissolve it in 500 μL of DMSO, add 1 mg of NaOH, incubate for 30 min, add 50 μL of iodomethane solution and react for 1 h, add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the washing with water 3 times, collect the lower dichloromethane phase and evaporate to dryness, add 100 μL of 2 M trifluoroacetic acid (TFA) reagent, react at 120 ℃ for 90 min, evaporate to dryness at 30 ℃, add 50 μL of 2 M ammonia water and 50 mL of 1 M NaBD4, mix well, react at room temperature for 2.5 h, add 20 μL of acetic acid to terminate the reaction, and blow dry under nitrogen. Wash twice with 250 μL of methanol, blow dry under nitrogen, add 250 μL of acetic anhydride, vortex to mix, react at 100 ℃ for 2.5 h, add 1 mL of water and let stand for 10 min, add 500 μL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. The mixture was washed three times with water, and the lower layer of dichloromethane phase was collected and analyzed.

[0083] 2.6.2 Instrument Parameters

[0084] The chromatographic system used was an Agilent 7890A gas chromatograph with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm). High-purity helium (purity not less than 99.999%) was used as the carrier gas at a flow rate of 1.0 mL / min and an injection port temperature of 260 °C. The injection volume was 1 μL, split injection with a split ratio of 10:1 and a solvent delay of 2.2 min. The temperature program was: 50 °C held for 1 min, then increased to 130 °C at 50 °C / min, then to 230 °C at 3 °C / min, and held for 2 min. The mass spectrometry system used was an Agilent 5977B quadrupole mass spectrometer with an electron impact ionization (EI) source and a MassHunter workstation. Electron impact ionization (EI) source, inlet temperature: 230 ℃, quadrupole temperature: 150 ℃, electron energy: 70 eV. Full scan mode (SCAN) was used, with a mass scan range of m / z 30~600.

[0085] 2.7 Nuclear Magnetic Resonance Spectroscopy Analysis

[0086] Accurately weigh approximately 20 mg of CDPS-W1, dry it under vacuum at 60 °C for 24 h, dissolve it in 1 mL of D2O, and measure its one-dimensional nuclear magnetic resonance (NMR) on an ADVANCEⅢ HD-600MHz NMR spectrometer. 1 H-NMR, 13 C-NMR and two-dimensional nuclear magnetic resonance DEPT135, 1 H- 1 H COSY, HSQC, HMBC, NOESY.

[0087] 2.8 Study on lipid-lowering activity

[0088] 2.8.1 HepG-2 cell culture and lipid accumulation model

[0089] HepG-2 cells were cultured in high-glucose DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin under incubator conditions of 37 °C, 5% CO2, and 95% air. Cells were passaged every 1-2 days at a cell density of 80-90%. Lipid accumulation was simulated in vitro using ethanol and free fatty acid (FFA) solutions.

[0090] 2.8.2 Effect of the compound on HepG-2 cell viability as determined by the CCK-8 assay

[0091] HepG-2 cells were introduced at a rate of 1 × 10⁻⁶ per well. 5Cells were seeded at a density of 1,000 cells per well in 96-well plates. After 24 hours, once the cells had fully adhered, the complete culture medium was removed, and an incomplete culture medium containing free fatty acids (FFA) and either ethanol or CDPS-W1 was added. The plates were then incubated at 37°C and 5% CO2 for 24 hours. The modeling concentrations of ethanol and FFA, or the CDPS-W1 treatment concentration, were selected using the CCK-8 assay.

[0092] 2.8.3 Detection of HepG-2 cell biochemical indicators

[0093] HepG2 cells were introduced at a rate of 1 × 10⁻⁶ per well. 6 Cells were seeded at a density of [number] cells per well in 6-well plates and cultured for 24 hours. After 24 hours, except for the control group (Con), the modeling drug was added to the cells in each group and cultured for another 24 hours. After successful modeling, except for the control group (Con) and the Mol group, the cells in each group were treated with different drugs for 24 hours. Intracellular TC and TG levels were measured according to the kit instructions.

[0094] 3. Experimental Results

[0095] 3.1 Preparation of total polysaccharides from Cistanche deserticola

[0096] 10 kg of Cistanche deserticola was extracted with hot water and precipitated with ethanol to obtain 980 g of total polysaccharides, with a polysaccharide yield of 9.8%, of which the polysaccharide content was 63.00% and the protein content was 18.00%.

[0097] 3.2 Column Chromatography Separation and Purification Results

[0098] 300 g of crude polysaccharide from Cistanche deserticola was eluted using a DEAE-52 cellulose ion-exchange chromatography column with distilled water and different concentrations of NaCl (0.15 M, 0.3 M, 0.45 M, and 0.6 M). Five elution peaks were detected using the phenol-sulfuric acid method. Figure 1The fractions were named CDPS-W (10.8 g, polysaccharide content 74.96%), CDPS-0.15M (4.10 g, polysaccharide content 56.77%), CDPS-0.3M (5.9 g, polysaccharide content 39.8%), CDPS-0.45M, and CDPS-0.6M, respectively. The CDPS-W fraction was further separated using a Sephacryl S-200 (5000-250000 Da) eluted with deionized water, resulting in a broad elution peak. The fractions were collected in three segments and named CDPS-W1 (408.85 mg, polysaccharide content 96.51%), CDPS-W2 (339.65 mg, polysaccharide content 91.85%), and CDPS-W3 (97.25 mg, polysaccharide content 82.75%), respectively. CDPS-W1 and CDPS-W2 turned into white flocculent matter after freeze-drying, while CDPS-W3 turned into pale yellow flocculent matter after freeze-drying.

[0099] 3.3 Polysaccharide and protein content

[0100] A standard curve was plotted using the concentration of glucose standard as x and absorbance as y, yielding y = 14.954x + 0.0142, R0. 2 = 0.998. The total sugar content was calculated by measuring the absorbance of the sample using the phenol-sulfuric acid method. A standard curve was plotted using the concentration of bovine serum albumin standard as x and absorbance as y, yielding y = 1.2778x + 0.0075, R0. 2 = 0.999. The absorbance of the sample was measured using the Folin-Ciocalteu reagent method, and the protein content was calculated. The total sugar content of CDPS-W1 was 96.51 ± 0.82% and the protein content was 0.64 ± 0.36%.

[0101] 3.4 Purity and Molecular Weight Detection

[0102] The HPGPC analysis results are shown below. Figure 3 The results show that CDPS-W1 has a symmetrical single peak, indicating that CDPS-W1 is a homogeneous polysaccharide component. Furthermore, its weight-average molecular weight (Mw) is 85.34 kDa, its number-average molecular weight (Mn) is 56.39 kDa, and its dispersion index (Mw / Mn) is 1.51.

[0103] 3.5 Monosaccharide Composition

[0104] The HPLC chromatogram of the mixed standards is shown below. Figure 4 The HPLC chromatogram of CDPS-W1 is shown below. Figure 5The retention times are shown in Table 3. By comparing the retention times with those of monosaccharide standards, it was finally determined that CDPS-W1 is composed of four monosaccharides: glucose, arabinose, galactose, and mannose. Among them, glucose is the main monosaccharide, accounting for 98.34%, galactose accounts for 1.16%, arabinose accounts for 0.36%, and mannose accounts for 0.12%. Therefore, CDPS-W1 is more likely to be a glucan.

[0105] Table 3

[0106]

[0107] 3.6 Methylation Analysis

[0108] The sample underwent methylation, acid hydrolysis, reduction, and acetylation to obtain partially methylated sugar alcohol acetate derivatives (PMASS). All free hydroxyl groups of the sugar were converted into dimethyl ethers. Hydrolysis then released the methylated monosaccharides, which were subsequently reduced to sugar alcohols by NaBH4. Acetylation of the hydroxyl groups generated from hydrolysis yielded various partially methylated sugar alcohol acetylated derivatives. GC-MS analysis yielded four chromatographic peaks (…). Figure 6 The retention times of these four peaks were 16.796, 20.149, 20.535, and 23.319, respectively. After comparison with the secondary fragments in the CCRC standard database (https: / / glygen.ccrc.uga.edu / ccrc / specdb / ms / pmaa / pframe.html), these four peaks were determined to belong to →4)-α-D-Glc p -(1→,→4,6)-α-D-Glc p -(1→,6)-α-D-Glc p -(1→ and the ratio is approximately 13:4:1, see Table 4 for details.

[0109] Table 4. Analysis results of CDPS-W1 methylation products

[0110]

[0111] 3.7 Nuclear Magnetic Resonance Spectroscopy Analysis

[0112] To further obtain structural feature information of the sample, one-dimensional nuclear magnetic resonance was performed. 1 H-NMR, 13 C-NMR and two-dimensional nuclear magnetic resonance DEPT135, 1 H- 1 H COSY, HSQC, HMBC, and NOESY were determined, and their spectra are shown below. Figures 7 to 13 As shown, we obtain all H and C chemical shift information of each major sugar residue and infer the connection order between each sugar residue.

[0113] One-dimensional nuclear magnetic resonance hydrogen spectroscopy (NMR) was used 1 Further analysis using 1H-NMR revealed the glycosidic bond configuration of the polysaccharide sample. 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. Methylation results showed that the main sugar residues contained in this polysaccharide sample were t-Glc(p), 1,4-Glc(p), 1,4,6-Glc(p), and 1,6-Glc(p). 1 H-NMR, 1 D-NOESY 13 C-NMR, 1 H- 1 H COSY and HSQC were used to assign signals to major sugar residues. NMR analysis revealed five different anodic signals for these sugar residues: δ 5.31 ppm / δ 99.68 ppm, δ 4.88 ppm / δ 97.76 ppm, δ 5.32 ppm / δ 99.67 ppm, δ 5.25 ppm / δ 99.97 ppm, and δ 4.87 ppm / δ 98.14 ppm. Based on these anodic signal peaks, and following COSY, HSQC, HMBC, and references, signals corresponding to other positions were identified, such as H-2 / C-2, H-3 / C-3, H-4 / C-4, H-5 / C-5, and H-6 / C-6. Combining these signal characteristics with methylation results, the sugar residues corresponding to these anodic signals were assigned to different positions of →4)-α-D-Glc. p -(1→, and →4,6)-α-D-Glc p -(1→、→6)-α-D-Glc p -(1→、α-D-Glc p -(1→, marked as G4, G4', G 46 G6, G t .

[0114] Table 5. Sugar residues in CDPS-W1 1 H and 13 C chemical shift assignment

[0115]

[0116] The main types of sugar residues in the sample 1 H and 13 The C chemical shift signal assignment results are shown in Table 5. The structural analysis of each sugar residue is as follows:

[0117] Sugar residue G4: Anomaly signals δ 5.31 ppm (H-1) and δ 99.68 ppm (C-1) indicate that the sugar residue is in the α configuration. Its H-2 and C-2 chemical shift signals are δ 3.54 ppm (H-2) and δ 71.46 ppm (C-2), respectively. Combined with methylation results, this confirms that the sugar residue is in the α configuration of Glc. COSY spectra (…) Figure 10 The cross peaks clearly identified the H-3, H-4, and H-5 signals. The chemical shifts of H-3, H-4, and H-5 for this sugar residue were assigned to δ 3.88 ppm, δ 3.56 ppm, and δ 3.74 ppm, respectively. (COSY spectrum) Figure 10 ) and HSQC related spectrum ( Figure 11 The values ​​were δ 3.76 ppm and δ 3.67 ppm. After assigning the chemical shifts of the hydrogen atoms on the sugar ring, the results could be obtained through HSQC correlation spectroscopy (…). Figure 11 The chemical shifts of the carbons on the sugar ring are assigned as δ 99.48 ppm, δ 71.59 ppm, δ 73.09 ppm, δ 76.70 ppm, δ 71.09 ppm, and δ 60.45 ppm, respectively (see Table 5). The chemical shifts of C-1 and C-4 shift to lower field indicate that these residues have undergone substitution at positions C-1 and C-4 of the sugar ring. Combined with the methylation results, the sugar residue is inferred to be →4)-α-D-Glc. p -(1→).

[0118] Sugar residue G4': Anomaly signals δ 4.88 ppm (H-1) and δ 97.76 ppm (C-1) indicate that the sugar residue is in the α configuration. Its H-2 and C-2 chemical shift signals are δ 3.46 ppm (H-2) and δ 71.45 ppm (C-2), respectively. Combined with methylation results, this confirms that the sugar residue is in the α configuration of Glc. COSY spectra (…) Figure 10 The cross peaks clearly identified the H-3, H-4, and H-5 signals. The chemical shifts of H-3, H-4, and H-5 for this sugar residue were assigned to δ 3.94 ppm, δ 3.56 ppm, and δ 3.75 ppm, respectively. Its H-6a and H-6b signals can be determined using COSY spectra (…). Figure 10 ) and HSQC related spectrum ( Figure 11 The chemical shifts of the hydrogen atoms on the sugar ring were assigned as δ 3.67 ppm and δ 3.75 ppm. After assigning the chemical shifts of the hydrogen atoms on the sugar ring, the results can be obtained through HSQC correlation spectroscopy (HSQC). Figure 11The chemical shifts of the carbons on the sugar ring are assigned as δ 97.76 ppm, δ 71.45 ppm, δ 73.28 ppm, δ 76.78 ppm, δ 71.10 ppm, and δ 60.27 ppm, respectively (see Table 5). The chemical shifts of C-1 and C-4 shift to lower field indicate that these residues have undergone substitution at positions C-1 and C-4 of the sugar ring. Combined with the methylation results, the sugar residue is inferred to be →4)-α-D-Glc. p -(1→).

[0119] Sugar residue G 46 The anodic signals δ 5.32 ppm (H-1) and δ 99.67 ppm (C-1) indicate that the sugar residue is in the α configuration. Its H-2 and C-2 chemical shift signals are δ 3.54 ppm (H-2) and δ 71.52 ppm (C-2), respectively. Combined with the methylation results, this confirms that the sugar residue is in the α configuration of Glc. This was confirmed by COSY mapping (…). Figure 10 The cross peaks clearly identified the H-3, H-4, and H-5 signals. The chemical shifts of H-3, H-4, and H-5 for this sugar residue were assigned to δ 3.89 ppm, δ 3.74 ppm, and δ 3.97 ppm, respectively. (COSY spectrum) Figure 10 ) and HSQC related spectrum ( Figure 11 The values ​​were δ 3.65 ppm and δ 3.89 ppm. After assigning the chemical shifts of the hydrogen atoms on the sugar ring, the results could be obtained through HSQC correlation spectroscopy (…). Figure 11 The chemical shifts of the carbons on the sugar ring are assigned as δ 99.67 ppm, δ 71.52 ppm, δ 73.3 ppm, δ 76.56 ppm, δ 70.4 ppm, and δ 65.5 ppm, respectively (see Table 5). The chemical shifts of C-1, C-4, and C-6 shift to a lower field indicate that these residues have undergone substitution at positions C-1, C-4, and C-6 of the sugar ring. Combined with the methylation results, the sugar residue is inferred to be →4,6)-α-D-Glc p -(1→).

[0120] Sugar residue G6: Anomaly signals δ 5.25 ppm (H-1) and δ 99.97 ppm (C-1) indicate that the sugar residue is in the α configuration. Its H-2 and C-2 chemical shift signals are δ 3.55 ppm (H-2) and δ 71.47 ppm (C-2), respectively. Combined with methylation results, this confirms that the sugar residue is in the α configuration of Glc. COSY spectra (…) Figure 10 The cross peaks clearly identified the H-3, H-4, and H-5 signals. The chemical shifts of H-3, H-4, and H-5 for this sugar residue were assigned to δ 3.75 ppm, δ 3.43 ppm, and δ 3.81 ppm, respectively. (COSY spectrum) Figure 10 ) and HSQC related spectrum ( Figure 11 The values ​​were δ 3.66 ppm and δ 3.89 ppm. After assigning the chemical shifts of the hydrogen atoms on the sugar ring, the results could be obtained through HSQC correlation spectroscopy (…). Figure 11 The chemical shifts of the carbons on the sugar ring are assigned as δ 99.97 ppm, δ 71.47 ppm, δ 72.76 ppm, δ 69.78 ppm, δ 70.4 ppm, and δ 65.54 ppm, respectively (see Table 5). The chemical shifts of C-1 and C-6 shift to lower field indicate that these residues have undergone substitution at positions C-1 and C-6 of the sugar ring. Combined with the methylation results, the sugar residue is inferred to be →6)-α-D-Glc. p -(1→).

[0121] Sugar residue G t The anodic signals δ 4.87 ppm (H-1) and δ 98.14 ppm (C-1) indicate that the sugar residue is in the α configuration. Its H-2 and C-2 chemical shift signals are δ 3.48 ppm (H-2) and δ 71.49 ppm (C-2), respectively. Combined with the methylation results, this confirms that the sugar residue is in the α configuration of Glc. The COSY spectrum (…) Figure 10 The cross peaks clearly identified the H-3, H-4, and H-5 signals. The chemical shifts of H-3, H-4, and H-5 for this sugar residue were assigned to δ 3.62 ppm, δ 3.34 ppm, and δ 3.63 ppm, respectively. Its H-6a and H-6b signals can be determined using COSY spectra (…). Figure 10 ) and HSQC related spectrum ( Figure 11 Assignments were performed, yielding δ 3.73 ppm and δ 3.63 ppm. After assigning the chemical shifts of the hydrogen atoms on the sugar ring, correlation spectra (HSQC) were obtained. Figure 11 The chemical shifts of the carbons on the sugar ring are assigned as δ 98.14 ppm, δ 71.49 ppm, δ 72.90 ppm, δ 69.21 ppm, δ 72.40 ppm, and δ 60.46 ppm, respectively (see Table 5). The chemical shift at C-1 shifts to a lower field, indicating that this residue has undergone substitution at the C-1 position of the sugar ring. Combined with the methylation results, the sugar residue is inferred to be α-D-Glc. p -(1→).

[0122] 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 the strong NOESY signal generated by two protons at adjacent sugar residue linkage sites 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 12 ) and NOESY spectrum ( Figure 13 As shown in the figure, the following coupling signal can be found: there is a coupling signal (G4H-1 / G4C-4) between H-1 (δ 5.31 ppm) of sugar residue G4 and C-4 (δ 76.8 ppm) of sugar residue G4, indicating the presence of →4)-α-D-Glc p -(1→and→4)-α-D-Glc p -(1→ Linkage between sugar residues, the linking site is located at position O-4; H-1 (δ 5.31 ppm) of sugar residue G4 and sugar residue G 46 C-4 (δ 76.78 ppm) has a coupling signal (G4H-1 / G 46 C-4), indicating the existence of →4)-α-D-Glc p -(1→and→4,6)-α-D-Glc p -(1→ The linkage between sugar residues is located at position O-4; there is a coupling signal between the H-1 (δ 4.88 ppm) of sugar residue G4' and the C-6 (δ 65.54 ppm) of sugar residue G6 (G4' H-1 / G6C-6), indicating the presence of →4)-α-D-Glc p -(1→and→6)-α-D-Glc p -(1→ Linkage between sugar residues, the linking site is located at position O-6; sugar residue G t H-1 (δ 4.87 ppm) and sugar residue G 46 C-6 (δ 65.5ppm) has a coupling signal (G t H-1 / G 46 C-6) indicates the presence of α-D-Glc p -(1→and→4,6)-α-D-Glc p -(1→ The linkage between sugar residues is located at position O-6; there is a coupling signal between H-1 (δ 5.25 ppm) of sugar residue G6 and C-4 (δ 76.8 ppm) of sugar residue G4 (G6H-1 / G4C-4), indicating the presence of →6)-α-D-Glc p -(1→and→4)-α-D-Glc p -(1→ The link between sugar residues, with the linking site located at position O-4.

[0123] In the NOESY spectrum, there is a coupling signal (G4H-1 / G4H-4) between H-1 (δ 5.31 ppm) and H-4 (δ 3.56 ppm) of sugar residue G4, indicating the presence of →4)-α-D-Glc p -(1→and→4)-α-D-Glc p-(1→ Linkage between sugar residues, the linking site is located at position O-4; H-1 (δ 5.31 ppm) of sugar residue G4 and sugar residue G 46 H-4 (δ 3.56 ppm) has a coupling signal (G4H-1 / G 46 H-6), indicating the presence of →4)-α-D-Glc p -(1→and→4,6)-α-D-Glc p -(1→ Linkage between sugar residues, the linking site is located at position O-4; sugar residue G t H-1 (δ 4.87 ppm) and sugar residue G 46 H-6 (δ 3.65ppm) has a coupling signal (G t H-1 / G 46 H-6) indicates the presence of α-D-Glc p -(1→and→4,6)-α-D-Glc p -(1→ The link between sugar residues, with the linking site located at position O-6.

[0124] In summary, based on the one-dimensional and two-dimensional NMR analysis, the preliminary structure of the sample is deduced to be a →4)-α-D-Glc p -(1→and→6)-α-D-Glc p -(1→glucan that forms the main chain, in →4)-α-D-Glc p -(1→ The 6th position of the residue forms a branch, and the main sugar residue at the branch site is α-D-Glc. p -(1→。Methylation analysis results showed that →4)-α-D-Glc p -(1→,→4,6)-α-D-Glc p -(1→,6)-α-D-Glc p The ratio of -(1→ is approximately 13:4:1. The possible structural units of the polysaccharide are speculated as follows:

[0125] .

[0126] 3.8 Lipid-lowering effect of CDPS-W1 on HepG-2 cells

[0127] like Figure 14 As shown: Compared with the CON group, the TC content in the MOL group ( Figure 14 A) and TG content ( Figure 14 B in the middle was significantly increased ( P (<0.01) Compared with the MOL group, the drug-treated group could reverse this phenomenon, indicating that CDPS-W1 has the effect of reducing lipid accumulation.

Claims

1. The application of a Cistanche deserticola polysaccharide in the preparation of lipid-lowering drugs, characterized in that, The repeating structural units of Cistanche deserticola polysaccharide are: ； The Cistanche deserticola polysaccharide has a weight-average molecular weight of 85.34 kDa, a number-average molecular weight of 56.39 kDa, and a dispersion index of 1.

51.

2. The application according to claim 1, characterized in that, Cistanche deserticola polysaccharide was prepared using the following method: (1) The medicinal material of Cistanche deserticola was extracted by heating with distilled water and precipitated with ethanol to obtain crude polysaccharide of Cistanche deserticola; (2) The crude polysaccharide of Cistanche deserticola was eluted by distilled water and different concentrations of NaCl through a chromatography column. Five elution peaks were obtained by tracking and detection by phenol-sulfuric acid method and named CDPS-W, CDPS-0.15M, CDPS-0.3M, CDPS-0.45M and CDPS-0.6M respectively. (3) Dissolve the CDPS-W component in deionized water, then separate it with Sephacryl S-200, elute with deionized water, and three elution peaks appear. Collect the three segments and name them CDPS-W1, CDPS-W2 and CDPS-W3 respectively.

3. The application according to claim 2, characterized in that, In step (1), the volume ratio of the Cistanche deserticola medicinal material to distilled water is 1:10; the heating extraction is performed at a gentle boil for 2 hours; the extraction is repeated three times.

4. The application according to claim 2, characterized in that, In step (2), the chromatography column is a DEAE-52 cellulose ion exchange chromatography column; the concentrations of NaCl are 0.15 M, 0.3 M, 0.45 M and 0.6 M, respectively; and the elution flow rate is 4 mL / min.

5. The application according to claim 2, characterized in that, In step (3), the concentration of the CDPS-W solution is 50 mg / mL.

6. The application according to claim 2, characterized in that, In step (3), the separation range of Sephacryl S-200 is 5000-250000 Da.

7. The application according to claim 5 or 6, characterized in that, In step (3), the flow rate of the deionized water elution is 0.5 mL / min.

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