An acidic polysaccharide from Imperata cylindrica, preparation method and application

Abstract

The present invention discloses an acidic polysaccharide extracted from Imperata cylindrica Beauv. root, and the acidic polysaccharide is polysaccharide RPS-DS 0.1 , the mass fractions of total sugar and protein are 91.22% and 0.02% respectively, the weight-average molecular weight is 29.79 kDa, and it is composed of rhamnose, arabinose, galactose, glucose, xylose, mannose and glucuronic acid, and the molar ratios are 2.48:11.76:20.08:50.92:10.25:2.08:2.12 respectively. The structural formula of polysaccharide RPS-DS 0.1 is shown in Formula I; The Imperata cylindrica Beauv. root acidic polysaccharide RPS-DS 0.1 obtained in the present invention has excellent hypoglycemic effect, filling the blank of no hypoglycemic activity in the prior art. At the same time, the present invention further analyzes the structure of the hypoglycemic-active Imperata cylindrica Beauv. root polysaccharide, providing reference and data support for the development and application of Imperata cylindrica Beauv. root and polysaccharides;#imgabs0#

Description

Technical Field

[0001] The present invention relates to the technical field of extraction of acid polysaccharides from Imperata cylindrica (L.) Beauv. roots, and more particularly to an acid polysaccharide extracted from Imperata cylindrica (L.) Beauv. roots, a preparation method and applications thereof. Background Art

[0002] Imperata cylindrica (L.) Beauv. roots are the dried rhizomes of Imperata cylindrica (L.) Beauv., also known as sweet root grass, cogongrass rhizome, silk cogongrass, etc. They can be harvested in both spring and autumn and are one of the traditional Chinese medicinal materials in China, being wild distributed throughout the country. Compendium of Materia Medica records that Imperata cylindrica (L.) Beauv. roots can treat consumptive fatigue, replenish middle qi, remove stasis, promote urination, relieve stranguria, remove pathogenic heat from the intestines and stomach, quench thirst and strengthen tendons, and treat metrorrhagia in women. Imperata cylindrica (L.) Beauv. roots contain a variety of natural compounds with medicinal value, mainly including polysaccharides, triterpenoids, flavonoids, lignans, lactones, steroids, organic acids, etc.

[0003] In China, more than 90% of the total diabetes patients are type II diabetes (T2DM) patients. Research has shown that the development of T2DM is mainly affected by pancreatic β-cell dysfunction and insulin resistance. Commonly used drugs for treating this disease often have defects such as large side effects, limited choices, and high costs. Plant polysaccharides have attracted wide attention from scholars due to their low toxicity, high safety, good biological regulatory ability and biological activity, and can be used as ideal natural drugs.

[0004] Imperata cylindrica (L.) Beauv. polysaccharides are usually used for functions such as regulating immunity, diuretic and antihypertensive, antibacterial, anti-inflammatory and analgesic, anti-tumor, antioxidant, and improving renal function. Imperata cylindrica (L.) Beauv. polysaccharides are an important active ingredient in Imperata cylindrica (L.) Beauv. roots. However, current research on Imperata cylindrica (L.) Beauv. polysaccharides focuses on the extraction process of its crude polysaccharides, and there are no reports on the structural characterization and hypoglycemic activity of its homogeneous polysaccharides.

[0005] Therefore, how to provide an acid polysaccharide extracted from Imperata cylindrica (L.) Beauv. roots is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] One object of the present invention is to provide an acid polysaccharide extracted from Imperata cylindrica (L.) Beauv. roots, and the acid polysaccharide is polysaccharide RPS-DS 0.1 , with the total sugar and protein mass fractions being 91.22% and 0.02% respectively, the weight-average molecular weight being 29.79 kDa, and being composed of rhamnose, arabinose, galactose, glucose, xylose, mannose and glucuronic acid, with the molar ratios being 2.48:11.76:20.08:50.92:10.25:2.08:2.12 respectively. The polysaccharide RPS-DS 0.1 has a structural formula as shown in Formula I:

[0007]

[0008] Preferably, the main backbone of the acidic polysaccharide is →6)-β-D-Glcp-(1→, →3,6-α-D-Gal p-(1→, β-L-Araf-(1→, and →4)-β-L-Xylp-(1→.

[0009] Another object of the present invention is to provide a method for extracting the acidic polysaccharide, and the process includes:

[0010] 1) Using the powder of Imperata cylindrica Beauv. root as raw material, adding distilled water according to a liquid-to-solid ratio of 10:1 to 30:1 mL / g, performing high-temperature extraction, ultrasonic extraction, collecting the filtrate, concentrating it, precipitating with alcohol, redissolving with ultrapure water, filtering, and then freeze-drying to obtain a light brown crude polysaccharide PRS;

[0011] 2) Removing proteins with Sevage reagent, then dialyzing for 24 - 48 h. After dialysis, concentrating and freeze-drying the polysaccharide solution to obtain refined Imperata cylindrica Beauv. root polysaccharide;

[0012] 3) Preparing the refined Imperata cylindrica Beauv. root polysaccharide into a polysaccharide solution with a mass concentration of 10 mg / mL, performing preliminary purification with a DE AE-52 cellulose chromatography column, eluting successively with 0, 0.1, 0.2, 0.3, 0.4, 0.5 mol / L NaCl solutions, collecting the eluate, then dialyzing against running water for 24 - 48 h and freeze-drying;

[0013] 4) Further separating and purifying the obtained polysaccharide component with a SephadexG-100 gel chromatography column, eluting, collecting the eluate, concentrating and freeze-drying to obtain the acidic polysaccharide RPS-DS 0.1 。

[0014] Preferably, in step 1), the high-temperature extraction is carried out in an autoclave at 100 - 120 °C for 5 - 30 min to obtain an extract.

[0015] Preferably, in step 1), the process of ultrasonic extraction, collecting the filtrate, concentrating it, and precipitating with alcohol is as follows: extracting for 15 - 35 min under the condition of an ultrasonic power of 300 - 500 W, collecting the filtrate, concentrating at 50 - 60 °C, then adding 4 volumes of absolute ethanol, standing at 4 °C for 12 - 24 h, centrifuging, and collecting the precipitate.

[0016] Preferably, in step 2), removing proteins with Sevage reagent is to mix the RPS solution with Sevage reagent and stir for 20 - 50 min to remove the precipitated proteins, and repeat the operation 3 - 5 times.

[0017] Preferably, in step 2), the dialysis is carried out with a 1000 Da dialysis bag against running water for 24 - 48 h.

[0018] Preferably, in step 3), the elution flow rate is 1 mL / min, and then the eluate is collected at 4 mL / tube. The phenol-sulfuric acid method is used to detect the polysaccharide content. With the number of collected tubes as the abscissa and the absorbance value at 490 nm as the ordinate, an elution curve is plotted. The eluate from the 38th tube to the 65th tube is collected, concentrated under reduced pressure, dialyzed with a 1000 Da dialysis bag against running water for 24 - 48 h, and then freeze-dried.

[0019] Preferably, in step 4), the elution is carried out using deionized water at a flow rate of 0.5 mL / min, and the eluate is collected at 4 mL / tube. The phenol-sulfuric acid method is used to measure the absorbance value of the eluate at 490 nm, and an elution curve is plotted. The eluate is collected according to the peak section, and the collected eluate is concentrated and then freeze-dried.

[0020] The object of the present invention also lies in providing the application of the acidic polysaccharide described above or the acidic polysaccharide prepared by the method in the preparation of hypoglycemic drugs. As can be seen from the above technical solutions, compared with the prior art, the technical effects achieved by the present invention are as follows:

[0021] (1) According to the extraction and separation process of the present invention, a homogenous white grass root acidic polysaccharide RPS-DS0.1 with a molecular weight of 29.79 kDa can be prepared.

[0022] (2) The obtained white grass root acidic polysaccharide RPS-DS0.1 of the present invention has excellent hypoglycemic effects, filling the blank of no hypoglycemic activity in the prior art.

[0023] (3) The present invention further analyzes the structure of the white grass root polysaccharide with hypoglycemic activity, providing reference and data support for the development and application of white grass root and polysaccharides. Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0025] Figure 1 The drawing is the elution curve map of purifying RPS by DEAE-52 cellulose anion exchange column;

[0026] Figure 2 The drawing is the elution curve map of purifying RPS-DS0.1 by SephadexG-100 gel column;

[0027] Figure 3The attached figure is the ultraviolet spectrum scanning result diagram of RPS-DS0.1; UV spectrum analysis (A), infrared spectrum diagram (B), molecular weight determination result (C), monosaccharide composition (D), thermogravimetric analysis (E);

[0028] Figure 4 The attached figure is the spectrum analysis of polysaccharide RPS-DS0.1; (A) 1H-nuclear magnetic resonance spectrum; (B) 13C-nuclear magnetic resonance spectrum; (C) 1H-1H two-dimensional nuclear magnetic spectroscopy; (D) HSQC spectrum; (E) HMBC spectrum; (F) Congo red analysis curve.

[0029] Figure 5 The attached figure is the inhibition rate result of polysaccharide RPS-DS0.1 on α-amylase;

[0030] Figure 6 The attached figure is the effects of RPS-DS0.1 at different mass concentrations on cell viability (a), glucose consumption (b), glycogen content (c), hexokinase activity (d), pyruvate kinase activity (e);

[0031] Figure 7 The attached figure is the ROS accumulation result of polysaccharide RPS-DS0.1 in IR-HepG2 cells. Detailed implementation manners

[0032] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0033] Example 1: Preparation of Imperata cylindrica polysaccharide

[0034] (1) Accurately weigh 20 g of Imperata cylindrica powder into a conical flask, add 500 mL of ultrapure water, extract in an autoclave at 112 °C for 15 min, take it out and cool to room temperature, continue to extract at a ultrasonic power of 410 W for 25 min, collect the filtrate, concentrate it under reduced pressure to 30 mL at 60 °C, then add 120 mL of absolute ethanol, let it stand at 4 °C for 24 h, centrifuge at 8000 r / min for 5 min, collect the precipitate, dissolve it in 20 mL of ultrapure water, filter and freeze-dry to obtain a light brown crude polysaccharide, denoted as RPS;

[0035] (2) Protein was removed using Sevage reagent [V(chloroform):V(n-butyl alcohol) = 4:1]. 500 mg of RPS was dissolved in 30 mL of distilled water, 6 mL of Sevage reagent was added, and the mixture was stirred magnetically for 30 min, centrifuged, and the precipitated protein was removed. The operation was repeated 4 times until no white precipitate appeared. The polysaccharide solution with removed protein was evaporated by rotary evaporation to remove the residual Sevage reagent, the RPS solution was collected, and dialyzed against running water for 24 h using a 1000 Da dialysis bag. After dialysis, the polysaccharide solution was concentrated and freeze-dried to obtain refined Imperata cylindrica polysaccharide.

[0036] Example 2: Isolation and purification of Imperata cylindrica polysaccharide

[0037] (1) 100 mg of deproteinized RPS was weighed and dissolved in 10 mL of deionized water, filtered through a 0.45 μm water-based membrane, and reserved for use. The polysaccharide solution was slowly injected into a DEAE-52 cellulose chromatography column, and eluted successively with 0, 0.1, 0.2, 0.3, 0.4, 0.5 mol / L NaCl solutions at a flow rate of 1 mL / min, 4 mL per tube. The polysaccharide content was detected by the phenol-sulfuric acid method. With the number of collection tubes as the abscissa and the absorbance at 490 nm as the ordinate, an elution curve was plotted, as Figure 1 shown.

[0038] (2) According to Figure 1 the elution curve, the eluates of the 38th to 65th tubes were collected, concentrated under reduced pressure to 10 mL, dialyzed against running water for 24 h using a 1000 Da dialysis bag, and then freeze-dried to obtain polysaccharide fraction RPS-D 0.1 .

[0039] (3) 100 mg of the polysaccharide RPS-D 0.1 obtained in (2) was dissolved in 10 mL of deionized water, filtered through a 0.45 μm filter membrane, and slowly injected into a Sephadex G-100 gel chromatography column. Elution was carried out with deionized water at a flow rate of 0.5 mL / min, 4 mL per tube. The absorbance of the eluate at 490 nm was measured by the phenol-sulfuric acid method, and an elution curve was plotted, as Figure 2 shown. The eluates of the 4th to 25th tubes were collected and combined, the collected eluates were concentrated and freeze-dried to obtain a polysaccharide fraction RPS-DS with a uniform molecular weight 0.1 .

[0040] Example 3: Structural characterization of Imperata cylindrica polysaccharide

[0041] (1) Ultraviolet spectroscopy was used to further analyze nucleic acids and proteins in the purified polysaccharide. The Imperata cylindrica polysaccharide RPS-DS 0.1 was prepared into a polysaccharide solution with a mass concentration of 1 mg / mL using deionized water. Using deionized water as the blank control, ultraviolet spectral scanning was carried out at a wavelength of 200 - 400 nm. The results were asFigure 3 As shown in (A), RPS-DS0.1 has no obvious absorption peaks at 260 nm and 280 nm, indicating that the purified polysaccharide contains almost no nucleic acids and proteins.

[0042] (2) Use KBr tablets to remove background interference, and then weigh 2 mg of polysaccharide sample RPS-DS 0.1 , grind it thoroughly with 200 mg of KBr, and press it into a thin film with a tablet press. Perform infrared spectroscopy scanning in the range of 4000 - 500 cm -1 at a resolution of 4 cm-1. The results are as Figure 3 shown in (B). Strong absorption peaks near 3385 cm -1 and 2929 cm -1 represent the stretching vibrations of O-H and C-H, which are characteristic absorption peaks of polysaccharide substances. The absorption peaks at 1647 cm -1 and 1417 cm -1 are the asymmetric and symmetric stretching vibration absorption peaks of carboxyl groups, indicating the presence of uronic acid in RPS-DS 0.1 . The absorption peak at 1340 cm -1 is the out-of-plane bending vibration absorption peak of alkyl C-H; the absorption peak at 1014 cm -1 indicates the presence of pyranose rings in RPS-DS 0.1 ; the absorption peaks at 866 cm -1 and 846 cm -1 represent the presence of β-glycosidic bonds and α-glycosidic bonds in the polysaccharide; the absorption peak at 763 cm -1 is caused by the symmetric stretching vibration of D-xylose.

[0043] (3) According to the method described in the literature, use high performance gel chromatography to determine the relative molecular weight of the sample. Conditions are as follows: use a BRT105-103-101 series gel column (8×300 mm); mobile phase (0.2 mol / L NaCl solution); flow rate is 0.8 mL / min; column temperature is set at 40 °C; injection volume is 25 μL; detector is a differential refractive index detector RID-10A. The chromatogram of RPS-DS 0.1 is as Figure 3 shown in (C). RPS-DS 0.1 is a single peak, indicating a polysaccharide with a uniform molecular weight, and the weight average molecular weight is 29.79 kDa.

[0044] (4) According to the method described in the literature, compare the retention times of the test sample and monosaccharide standards in the chromatogram to determine the monosaccharide composition. Conditions are as follows: use Dionex TM CarboPac TMPA20 (150×3.0 mm, 10 μm) liquid chromatography column; injection volume is 5 μL; mobile phase A (H2O), mobile phase B (0.1 mol / L NaOH), mobile phase C (0.1 mol / L NaOH, 0.2 mol / L NaAc), flow rate 0.5 mL / min; column temperature is 30 °C. RPS-DS 0.1 The monosaccharide composition of Figure 3 (D) is shown as follows. RPS-DS 0.1 is composed of rhamnose, arabinose, galactose, glucose, xylose, mannose and glucuronic acid, and their molar ratios are 2.48:11.76:20.08:50.92:10.25:2.08:2.12 respectively.

[0045] (5) Synchronous thermal analysis was used to analyze the thermal stability of the purified polysaccharide. Weighed 5 mg of the polysaccharide sample and RPS-DS 0.1 , placed in a crucible, and thermogravimetric analysis was carried out in the range of 50 - 800 °C at a heating rate of 10 °C / min. As shown in Figure 3 (E), it has good thermal stability.

[0046] (6) Congo red method was used to analyze the triple helix structure of the purified polysaccharide. Took 2 mL of the RPS-DS 0.1 polysaccharide solution with a concentration of 2 mg / mL and mixed it with 2 mL of the Congo red solution with a concentration of 160 μmol / L. Then added 0, 0.1, 0.2, 0.5, 0.6, 0.8 mol / L of NaOH solution in sequence, left it standing at room temperature for 15 min, and carried out full wavelength scanning in the range of 400 - 600 nm to measure the maximum absorption wavelength of the solution. Taking the NaOH concentration as the abscissa and the maximum absorption wavelength as the ordinate, and plotted the curve as shown in Figure 3 (F). As the NaOH concentration increases, the maximum absorption wavelength of RPS-DS 0.1 continually decreases. Compared with the Congo red solution, there is no red shift. It is inferred that RPS-DS 0.1 does not have a triple helix structure.

[0047] (8) The methylation analysis method of the existing literature was referred to for RPS-DS 0.1 to characterize the glycosidic bond. Mixed 3 mg of the sample with 1 mL of anhydrous DMSO. Then added methylation reagent A and dissolved it by sealed ultrasound, and then added methylation reagent B in a water bath at 30 °C for 60 minutes. Then terminated the methylation reaction by adding 2 mL of ultrapure water.

[0048] After methylation, the polysaccharide was hydrolyzed with 2 mol / L TFA (1 mL) for 90 minutes and then rotary evaporated. Further reaction was carried out by adding 2 mL of double-distilled water and 60 mg of NaBH4 for 8 hours. The reaction was neutralized by adding glacial acetic acid and drying at 101 °C. Subsequently, 1 mL of acetic anhydride was added and the reaction was carried out at 100 °C for 1 h for acetylation. To remove the excess acetic anhydride, 3 mL of toluene was added and then centrifugal distillation was carried out, repeating 4 times.

[0049] After acetylation was completed, the acetylated product sample was analyzed by GC-MS. The GC-MS conditions were as follows:

[0050] An RXI-5SILMS (30 mm × 0.25 mm × 0.25 μm) chromatographic column was used. The initial temperature was 120 °C, and it was heated to 250 °C at a rate of 3 °C / min and held for 5 minutes. The inlet temperature of the injection port was 250 °C, while the detector temperature and the carrier gas were both set to 250 °C. The carrier gas used was helium, and the flow rate was 1 mL / min.

[0051] Combining the peak retention times and main fragments of the GC-MS mass spectrum, as well as the composition and proportion of monosaccharides in RSP-DS 0.1 to determine that RSP-DS 0.1 is composed of 11 glycosidic bonds. The results of methylation analysis are shown in Table 1.

[0052] Table 1 Methylation analysis of RSP-DS 0.1

[0053]

[0054] (8) Carefully weigh 20 mg of the polysaccharide sample and then dissolve it in D 2 O. The resulting solution was filtered through a 0.45 μm filter membrane and then transferred to an NMR tube. Various nuclear magnetic resonance spectra were recorded using a Bruker AM-500 NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). These included 1H NMR, 13C NMR, homonuclear chemical shift correlation (1H-1H COSY), heteronuclear multiple quantum coherence (1H-13C HMQC), and heteronuclear multiple bond correlation (1H-13C HMBC) spectra.

[0055] RPS-DS 0.1 1H-NMR spectrum of Figure 4 (A) Seven signal peaks were shown at 5.38, 5.26, 5.28, 5.02, 4.92, 4.55, and 4.40 ppm, indicating the simultaneous presence of α and β conformations. In addition, the HSQC spectrum of RP S-DS 0.1 ( Figure 4D) reveals four sugar heterotopic H / C signals. Based on the monosaccharide composition analysis and the signal intensities in the spectrum, it can be inferred that these signals originate from the H1 / C1 residues of four sugars: glucose (A), galactose (B), arabinose (C), and xylose (D). From Figure 4 (B) In the 13C-NMR spectrum of RPS-DS 0.1 , a clear signal can be observed at 97.67 ppm. The positions of the cross-peaks shown in the HS QC spectrum confirm the assignment of H1 / C1 as 4.92 / 97.67 ppm. These signals are found to be 3.50, 3.67, 3.45, 3.83, and 3.91 ppm respectively. The correlations between the hydrogen signals and the carbon signals are obtained: H2 / C2 (3.50 / 71.36 ppm), H3 / C3 (3.67 / 73.37 ppm), H4 / C4 (3.45 / 69.49 ppm), H5 / C5 (3.83 / 70.14 ppm), and H6 / C6 (3.91 / 65.49 ppm). In addition, the peak of residue A at 3.91 / 65.49 ppm is identified as residue AH6 / C6. Compared with the unsubstituted residue C6, the C6 of residue A shows a downfield chemical shift, indicating substitution at C6. The connections between the residues are further inferred using the HMBC spectrum and the methylation results. As Figure 4 (E) shows, there are overlapping peaks between the residues: H1 of residue A is correlated with C6 of residue A (AH1 / C6A); H1 of residue A is correlated with C6 of residue B (AH1 / C6B). By combining these findings with the methylation results of the three bonds of the trisaccharide-bonded residue and the main residue →6)-β-D-Glcp-(1→, it is inferred that the branching site is at the 3rd position of →3,6-α-D-Galp-(1→. Based on the monosaccharide composition and methylation analysis results, combined with Figure 4 (A)–(C) 1D (1H, 13C) and 2D (COSY, HSQC, HMBC) NMR, the chemical shifts of all sugar residues are assigned (Table 2), and the relationships between the residues are obtained. Finally, the polysaccharide chain structure is as Figure 4 (G) shown:

[0056]

[0057] Table 2 RPS-DS 0.1 of 1 H and 13 1H NMR chemical shift analysis

[0058]

[0059] Example 4: Determination of the inhibition rate of α-amylase by Imperata cylindrica polysaccharide

[0060] According to the literature description method, for the polysaccharide RPS-DS 0.1 Prepare solutions of the polysaccharide with different mass concentrations using deionized water. Using acarbose as the positive control, take 250 μL of the polysaccharide solution, mix it thoroughly with 250 μL of α-amylase (1 U), after a 10-minute water bath at 37 °C, add 250 μL of a 1% soluble starch solution, continue the 37 °C water bath for 10 minutes, after taking it out, add 500 μL of DNS reagent to terminate the reaction, and heat it in a boiling water bath for 5 minutes, cool it to room temperature, add deionized water to 5 mL, and measure the absorbance value at 540 nm. Calculate the inhibition rate of α-amylase according to the following formula.

[0061]

[0062] Note: Y, the inhibition rate of α-amylase, %; A 0 , the absorbance value with deionized water replacing the sample; A 1 , the absorbance value after the reaction of the sample to be measured; A 2 , the absorbance value after mixing with deionized water replacing the α-amylase solution.

[0063] It can be seen from Figure 5 that RPS-DS 0.1 exhibits good α-amylase inhibitory activity, and its activity is close to that of acarbose.

[0064] Example 5: Detection of the hypoglycemic activity of Imperata cylindrica polysaccharide

[0065] (1) Under the culture conditions of 37 °C and 5% CO 2 , culture HepG2 cells with complete medium (89% DMEM medium, 10% FBS, and 1% penicillin-streptomycin). When 80% of the cells are adherent, digest and passage them with 0.25% trypsin. Take HepG2 cells in the logarithmic growth phase, add complete medium to adjust to a cell suspension of 3×10 5 cells / mL, and inoculate 100 μL into each well of a 96-well plate. Then add 10 -7 mol / L insulin solution 100 μL to each well, and establish an insulin-resistant cell model after inducing for 36 h.

[0066] (2) Divide the cells into six groups: blank group (serum-free medium), model group (10-7mol / L insulin), positive control group (10-7mol / L insulin + 1 mg / mL metformin), RPS-DS 0.1 L group (10-7mol / L insulin + 2.5 mg / mL RPS-DS 0.1 ), RPS-DS0.1M group (10-7mol / L insulin + 5 mg / mL RPS-DS 0.1 ), RPS-DS0.1 Group H (10⁻⁷ mol / L insulin + 10 mg / mL RPS-DS 0.1 ).

[0067] (3) The MTT method was used to determine the effect of polysaccharide RPS-DS 0.1 on the survival rate of HepG2 cells. RPS-DS 0.1 was prepared into different mass concentrations with DMEM medium for standby. 3×10 5 cells / mL of HepG2 cell suspension was added to a 96-well plate, 100 μL per well. After culturing in an incubator at 37 °C and 5% CO 2 for 24 h, 100 μL of RPS-DS 0.1 solutions with different mass concentrations (0, 1.25, 2.5, 5, 10, 20, 40, 80 mg / mL) were added and cultured for another 24 h. Then 20 μL of 5 mg / mL MTT solution was added and the reaction continued in the incubator for 4 h. The supernatant was discarded, 100 μL of DMSO solution was added, and it was shaken thoroughly for 10 min. The absorbance was measured at 570 nm. And the survival rate of HepG2 cells was calculated according to the following formula.

[0068]

[0069] Note: Y, cell survival rate, %; A 0 , absorbance of the well without cells; A 1 , absorbance of the well containing HepG2 cells and the sample to be tested; A 2 , absorbance of the well containing HepG2 cells.

[0070] From Figure 6 (a), it can be seen that when the mass concentration of RPS-DS 0.1 reaches 20 mg / mL, the survival rate of HepG2 cells decreases. It can be seen that when the mass concentration of RPS-DS 0.1 exceeds a certain range, it has an inhibitory effect on cell survival. Therefore, RPS-DS 0.1 with mass concentrations of 2.5, 5, 10 mg / mL was selected for the in vitro hypoglycemic experiment on HepG2 cells (i.e., RPS-DS 0.1 L, RPS-DS 0.1 M and RPS-DS 0.1 H).

[0071] (4) Carry out cell grouping and culture according to the methods in (1) and (2) above. Digest with 0.25% trypsin, collect the cells, centrifuge to discard the supernatant, wash the cells with isotonic solution 1 - 2 times, and centrifuge to retain the precipitated cells. Add phosphate buffer solution with a pH of 7.4 and a concentration of 0.1 mol / L to the precipitated cells, homogenize, and then ultrasonically disrupt in an ice - water bath. Detect according to the instructions of the glucose, glycogen, and total protein quantitative test kits.

[0072] (5) Similarly, carry out cell grouping and culture according to the methods in (1) and (2) above. After digesting with 0.25% trypsin, collect, centrifuge, and retain the precipitated cells for later use. The subsequent operations are detected according to the instructions of the hexokinase (HK), pyruvate kinase (PK), and total protein quantitative test kits.

[0073] The results are as Figure 6 (b) - (e) show that RPS - DS 0.1 can effectively increase the glucose consumption of IR - HepG2 cells, showing a hypoglycemic effect. RPS - DS 0.1 can significantly increase the glycogen content of IR - HepG2 cells and significantly increase the activities of HK and PK in the cells. This indicates that RPS - DS 0.1 has the effect of promoting glucose metabolism.

[0074] (6) Similarly, carry out cell grouping and culture according to the methods in (1) and (2) above. Add 1 mL of DCFH - DA fluorescent probe (0.01 mM / L) to each well, and incubate the wells in an incubator for 30 minutes. Subsequently, wash the well plate with isotonic solution multiple times. Finally, measure the cell fluorescence intensity using a cell imaging microplate detection system. The results are as Figure 7 shown. The fluorescence intensity of the cells in the model group increased significantly, indicating a significant ROS accumulation in the successfully established insulin resistance model group compared with the normal group. In addition, the fluorescence intensities of the positive control group, RPS - DS 0.1 group, RPS - DS 0.1 M group, and RPS - DS 0.1 H group all decreased to varying degrees compared with the model group. The order of fluorescence intensity is as follows: model group > RPS - DS 0.1 L group > RPS - DS 0.1 M group > RPS - DS 0.1 H group > positive control group > normal group. This indicates that high - concentration RPS - DS 0.1 can effectively inhibit and improve the ROS production of IR - HepG2 cells.

[0075] In this specification, each embodiment is described in a progressive manner. The key point of each embodiment is to illustrate the differences from other embodiments. For the same and similar parts among the embodiments, reference can be made to each other.

[0076] The foregoing description of the disclosed embodiments enables those skilled in the art to practice or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An acidic polysaccharide extracted from Imperata cylindrica Beauv. It is characterized in that The acidic polysaccharide is polysaccharide RPS-DS 0.1 , and the mass fractions of total sugar and protein are 91.22% and 0.02% respectively. The weight-average molecular weight is 29.79 kDa. It is composed of rhamnose, arabinose, galactose, glucose, xylose, mannose and glucuronic acid, and the molar ratios are 2.48:11.76:20.08:50.92:10.25:2.08:2.12 respectively. The polysaccharide RPS-DS 0.1 has a structural formula as shown in Formula I: the process includes: 1) Using the powder of Imperata cylindrica Beauv. as raw material, adding distilled water according to the liquid-solid ratio of 10:1 - 30:1 mL / g, performing high-temperature extraction, ultrasonic extraction, collecting the filtrate, concentrating, alcohol precipitation, re-dissolving with ultrapure water, filtering and then freeze-drying to obtain light brown crude polysaccharide PRS; 2) Removing proteins with Sevage reagent, then dialyzing for 24 - 48 h. After dialysis, concentrating and freeze-drying the polysaccharide solution to obtain refined Imperata cylindrica Beauv. polysaccharide; 3) Preparing the refined Imperata cylindrica Beauv. polysaccharide into a polysaccharide solution with a mass concentration of 10 mg / mL, performing preliminary purification with a DE AE-52 cellulose chromatography column, eluting successively with 0, 0.1, 0.2, 0.3, 0.4, 0.5 mol / L NaCl solutions, collecting the eluate, then performing running water dialysis for 24 - 48 h and freeze-drying; 4) Further separate and purify the obtained polysaccharide component using a Sephadex G-100 gel chromatography column, elute, collect the eluate, concentrate and lyophilize to obtain the acidic polysaccharide RPS-DS 0.1 ; In step 1), the high-temperature extraction is to extract in an autoclave at 100 - 120 °C for 5 - 30 min to obtain the extract; In step 1), the process of ultrasonic extraction, collecting the filtrate, concentrating and alcohol precipitation is: extracting under the condition of ultrasonic power of 300 - 500 W for 15 - 35 min, collecting the filtrate, concentrating at 50 - 60 °C, then adding 4 times the volume of absolute ethanol, standing at 4 °C for 12 - 24 h, centrifuging and collecting the precipitate; In step 2), removing proteins with Sevage reagent is to mix and stir the RPS solution with Sevage reagent for 20 - 50 min to remove the precipitated proteins, and repeating the operation 3 - 5 times; In step 2), the dialysis is to perform running water dialysis with a 1000 Da dialysis bag for 24 - 48 h; In step 3), the elution flow rate is 1 mL / min, then collecting 4 mL of eluate per tube, detecting the polysaccharide content by the phenol-sulfuric acid method, taking the number of collecting tubes as the abscissa and the absorbance value at 490 nm as the ordinate to draw the elution curve, collecting the eluate from the 38th tube to the 65th tube, concentrating under reduced pressure, performing running water dialysis with a 1000 Da dialysis bag for 24 - 48 h and then freeze-drying; In step 4), the elution is to elute with deionized water at a flow rate of 0.5 mL / min, collecting 4 mL of eluate per tube, measuring the absorbance value of the eluate at 490 nm by the phenol-sulfuric acid method and drawing the elution curve, collecting the eluate according to the peak segment, concentrating the collected eluate and then freeze-drying.

2. The acidic polysaccharide extracted from Imperata cylindrica Beauv. according to claim 1, it is characterized in that its main backbone is →6)-β-D-Glcp-(1→,→3,6-α-D-Galp-(1→,β-L-Araf-(1→,and→4)-β-L-Xylp-(1→.

3. Use of the acidic polysaccharide according to claim 2 in the preparation of hypoglycemic drugs.

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