CN114591449B

CN114591449B - Geranium neutral polysaccharide and preparation method and application thereof

Info

Publication number: CN114591449B
Application number: CN202210383498.9A
Authority: CN
Inventors: 刘录; 冯佳怡; 张鹏; 周倩; 周志宏; 谭文红; 杨竹雅
Original assignee: Yunnan University of Traditional Chinese Medicine TCM
Current assignee: Yunnan University of Traditional Chinese Medicine TCM
Priority date: 2022-04-13
Filing date: 2022-04-13
Publication date: 2022-12-06
Anticipated expiration: 2042-04-13
Also published as: CN114591449A

Abstract

The invention discloses a geranium neutral polysaccharide and a preparation method and application thereof, wherein the geranium neutral polysaccharide is formed by linking L-rhamnose, D-arabinose and D-mannose residues, and the content of the L-rhamnose: d-arabinose: d-mannose molar ratio is 7.59, 0.15, molecular weight is 1663 Da. The preparation method adopts a compound extraction method of water extraction and alcohol precipitation to extract the geranium coarse polysaccharide, so that the operation is simple, the cost is low, the efficiency is higher, the practicability is higher, and the biological activity of the polysaccharide is better guaranteed. The neutral polysaccharide extracted from the geranium wilfordii has anti-inflammatory and antioxidant activity, has an inhibiting effect on the secretion level of TNF-alpha and IL-6 preliminarily when the concentration is 10 mug/mg, has a certain DPPH free radical scavenging capacity when the concentration is 0.8mg/mL, has the characteristic of small toxic and side effects, has a wide application prospect in the development of low-toxicity and high-efficiency anti-inflammatory and antioxidant medicines, and is beneficial to the further development and utilization of geranium wilfordii resources.

Description

Geranium neutral polysaccharide and preparation method and application thereof

Technical Field

The invention belongs to the technical field of natural product extraction, and particularly relates to geranium neutral polysaccharide and a preparation method and application thereof.

Background

Polysaccharides are widely found in plants, algae, fungi and animals, and are one of the most abundant biomacromolecules in nature. Research shows that the polysaccharide component has various pharmacological activities, such as immunity regulation, anti-inflammation, antioxidation, antitumor and the like, and has a plurality of researches and applications in the fields of medicines and health-care foods. At present, the common extraction technologies of polysaccharide include a hot water extraction method, an acid extraction method and an alkali extraction method, the technologies are simple to operate and have low cost, but the extraction rate of polysaccharide is generally low, and the bioactivity of polysaccharide is reduced.

Geranium wilfordii (Geranium wilfordii Maxim.) is a plant of Geranium of Geraniaceae, is one of the commonly used Chinese medicines in China, and has the effects of dispelling wind-damp, dredging channels and collaterals, and the like. At present, no report is found in the research on polysaccharide in geranium wilfordii, and in order to better develop and utilize geranium wilfordii resources, the invention aims to provide a preparation method and application of geranium wilfordii neutral polysaccharide.

Disclosure of Invention

The first purpose of the invention is to provide a geranium neutral polysaccharide, the second purpose of the invention is to provide a preparation method of the geranium neutral polysaccharide, and the third purpose of the invention is to provide an application of the geranium neutral polysaccharide.

The first purpose of the invention is realized by that the geranium neutral polysaccharide is composed of linked L-rhamnose, D-arabinose and D-mannose residues, and the ratio of L-rhamnose: d-arabinose: d-mannose molar ratio is 7.59, 0.15, molecular weight is 1663 Da.

The second purpose of the invention is realized by that the preparation method of the geranium neutral polysaccharide is realized according to the following steps:

1) Drying the geranium wilfordii medicinal materials to the degree of being easily broken or powdered by hand, pulverizing into coarse powder, sieving with a 10-mesh sieve, defatting by methanol under reflux for 2-3 times, extracting with water at 80-85 deg.C for 2.5-4h for 2-3 times, mixing extractive solutions, and concentrating under reduced pressure to obtain extract;

2) Precipitating the extract obtained in the step 1 with 80-85% ethanol, centrifuging, discarding supernatant, redissolving the precipitate with distilled water, removing protein by sevege method, centrifuging, discarding the precipitate for 5-6 times, until no precipitate is formed after centrifugation, concentrating under reduced pressure, and freeze-drying to obtain a geranium coarse extract;

3) And (3) dissolving the crude extract of the geranium wilfordii obtained in the step (2) with distilled water, carrying out centrifugal filtration, purifying by DEAE Sepharose Fast Flow anion exchange chromatography, eluting with water, collecting the eluate, and dialyzing and concentrating to obtain neutral polysaccharide.

The third purpose of the invention is realized by that the geranium neutral polysaccharide is applied as an active ingredient or a medicinal carrier in the preparation of anti-inflammatory and antioxidant medicaments.

The invention has the beneficial effects that:

1. the invention provides a method for effectively and quickly extracting polysaccharide, which is characterized in that the crude polysaccharide of geranium is extracted by a compound extraction method of water extraction and alcohol precipitation, so that the method is simple to operate, low in cost, more efficient and more practical, and the biological activity of the polysaccharide is better guaranteed.

2. The neutral polysaccharide GNP extracted from Geranium wilfordii has anti-inflammatory and antioxidant activities, has a primary inhibition effect on the secretion levels of TNF-alpha and IL-6 when the concentration is 10 mug/mg, and has a certain DPPH free radical scavenging capacity when the concentration is 0.8 mg/mL. In addition, the neutral polysaccharide GNP provided by the invention has the characteristic of small toxic and side effects, so that the neutral polysaccharide GNP has wide application prospect in the development of low-toxicity and high-efficiency anti-inflammatory and antioxidant medicines, and is beneficial to the further development and utilization of Geranium wilfordii resources.

Drawings

FIG. 1 is a glucose standard curve of GNP (Geranium wilfordii neutral polysaccharide);

FIG. 2 is a cellulose anion exchange column separation spectrum of Geranium sibiricum neutral polysaccharide GNP;

FIG. 3 is a GC-MS data chart of the GNP (Geranium wilfordii neutral polysaccharide);

FIG. 4 is HPGPC chromatogram of GNP (Geranium wilfordii neutral polysaccharide);

FIG. 5 is the total ion flow diagram of the Geranium neutral polysaccharide GNP;

FIG. 6 is an infrared spectrum of the Geranium neutral polysaccharide GNP;

FIG. 7 is a schematic diagram of the third cell proliferation rate of Geranium neutral polysaccharide GNP;

FIG. 8 is a schematic diagram showing the effect of GNP (Geranium wilfordii neutral polysaccharide) on the inhibition of LPS-induced RAW264.7 macrophage production of proinflammatory cytokine IL-6;

FIG. 9 is a graph showing the effect of GNP in inhibiting the production of the pro-inflammatory cytokine TNF- α by RAW264.7 macrophages induced by lipopolysaccharide;

FIG. 10 is a schematic diagram of the detection of the IL-6 and TNF-alpha indexes of gentian polysaccharide;

FIG. 11 is a graph showing the scavenging effect of neutral polysaccharide GNP and vitamin C of Geranium sibiricum on DPPH free radicals;

FIG. 12 is a graph of DPPH free radical scavenging ability of the prior polysaccharides Caesalpinia crista neutral polysaccharide (CJP-C) and Caesalpinia crista acidic polysaccharide (CJP).

Detailed Description

The invention is further illustrated by the following figures and examples, which are not intended to limit the invention in any way, and any variations or modifications which are based on the teachings of the invention are intended to be within the scope of the invention.

The invention provides geranium neutral polysaccharide which is formed by linking L-rhamnose, D-arabinose and D-mannose residues, wherein the content of L-rhamnose: d-arabinose: d-mannose molar ratio is 7.59, 0.15, molecular weight is 1663 Da.

The preparation method of the geranium neutral polysaccharide is realized according to the following steps:

1) Drying the geranium wilfordii herb to the degree that the geranium wilfordii herb is easily broken or is powdered by hand, crushing the geranium wilfordii herb into coarse powder, sieving the coarse powder with a 10-mesh sieve, defatting the powder for 2 to 3 times by methanol reflux, extracting the powder with water at the temperature of between 80 and 85 ℃ for 2.5 to 4 hours each time, combining extracting solutions, and concentrating the extracting solutions under reduced pressure to obtain extract;

3) Dissolving the crude extract of the geranium wilfordii obtained in the step 2 with distilled water, carrying out centrifugal filtration, purifying by DEAE Sepharose Fast Flow anion exchange chromatography, eluting with water, collecting eluent, and carrying out dialysis and concentration to obtain target neutral polysaccharide;

in the step 1, the temperature of methanol reflux degreasing is 60-65 ℃, the concentration of methanol is 80-90%, and the addition amount of methanol is 6-10 times of the weight of the geranium medicinal herbs (V: m = 6-10).

In the step 1, the temperature for boiling and extracting the tea leaves with hot water is 80-85 ℃.

In the step 2, the concentration of the ethanol is 80-85%, and the addition amount of the ethanol is 2-3 times of that of the extract (V: m = 2-3).

In step 3, the cut-off molecular weight during dialysis is 3400 Da.

The application of the geranium neutral polysaccharide is the application of the geranium neutral polysaccharide as an active ingredient or a medicinal carrier in preparing anti-inflammatory and antioxidant medicaments.

The geranium polysaccharide is added into pharmaceutically acceptable auxiliary materials to be prepared into tablets, hard capsules, soft capsules, powder, pills and granules.

Example 1

Pulverizing 1kg of herba Erodii seu Geranii, and defatting with 6L methanol under reflux at 60 deg.C for 3 times. Decocting the defatted herba Erodii seu Geranii with 10L 80 deg.C hot water for 3 hr for 2 times, mixing extractive solutions, and concentrating under reduced pressure. Precipitating the concentrated extract with 2 times volume of 80% ethanol, centrifuging, discarding supernatant, redissolving the precipitate with distilled water, removing protein by sevege method, centrifuging, discarding the precipitate for 5 times, and centrifuging until no precipitate is formed. And finally, decompressing and concentrating the polysaccharide after protein removal, and freeze-drying to obtain the 120 g geranium crude polysaccharide.

Purifying the crude polysaccharide of Geranium wilfordii by DEAE Sepharose Fast Flow anion exchange chromatography (separation pattern is shown in figure 2): dissolving the crude extract 10g of Geranium wilfordii in a small amount of distilled water to prepare a solution of 30mg/mL, centrifuging at 4000rpm for 12min, filtering the supernatant with a 0.45-micrometer filter head, and loading; eluting with water at flow rate of 1mL/min for 5 times of column volume, collecting eluate, and concentrating under reduced pressure to 1/11 volume; putting the concentrated eluent into a dialysis bag with the molecular weight of 3400 Da, and dialyzing in distilled water for 30h, wherein water is changed every 5 h; the obtained eluent is frozen and dried to obtain the neutral crude polysaccharide of 2.5 g geranium wilfordii, and the yield is 25 percent.

Example 2

Pulverizing 1kg of herba Erodii seu Geranii, and defatting with 8L methanol at 65 deg.C for 3 times. Decocting the defatted herba Erodii seu Geranii with 12L of 85 deg.C hot water for 2.5 hr each time for 3 times, mixing extractive solutions, and concentrating under reduced pressure. Precipitating the concentrated extract with 85% ethanol 2.5 times the volume of the extract, centrifuging, discarding supernatant, redissolving the precipitate with distilled water, removing protein by sevege method, centrifuging, discarding the precipitate for 6 times, and centrifuging until no precipitate is formed. And finally, decompressing and concentrating the polysaccharide after protein removal, and freeze-drying to obtain a 140 g geranium crude extract.

Purifying the crude geranium polysaccharide by DEAE Sepharose Fast Flow anion exchange chromatography: dissolving the crude extract 10g of Geranium wilfordii in a small amount of distilled water to prepare a solution of 30mg/mL, centrifuging at 5000rpm for 8min, filtering the supernatant with a 0.45-micrometer filter head, and loading; eluting with water at flow rate of 1mL/min for 4 times of column volume, collecting eluate, and concentrating under reduced pressure to 1/9 volume; putting the concentrated eluent into a dialysis bag with the molecular weight of 3400 Da, and dialyzing in distilled water for 24h, wherein water is changed every 3 h; the obtained eluent is frozen and dried to obtain 2.3g of geranium neutral crude polysaccharide, and the yield is 22%.

Example 3

Pulverizing 1kg of herba Erodii seu Geranii, and defatting with 10L methanol under reflux at 63 deg.C for 3 times. Decocting the defatted herba Erodii seu Geranii with 12L 83 deg.C hot water for 3 times each for 3.5 hr, mixing extractive solutions, and concentrating under reduced pressure. Precipitating the concentrated extract with 3 times volume of 85% ethanol, centrifuging, discarding supernatant, redissolving the precipitate with distilled water, removing protein by sevege method, centrifuging, discarding the precipitate for 6 times, and centrifuging until no precipitate is formed. And finally, decompressing and concentrating the polysaccharide after protein removal, and freeze-drying to obtain 138g of geranium crude polysaccharide.

Purifying the crude geranium polysaccharide by DEAE Sepharose Fast Flow anion exchange chromatography: dissolving 10g of the crude extract of Geranium wilfordii in a small amount of distilled water to prepare a solution of 35 mg/mL, centrifuging at 4500rpm for 9min, filtering the supernatant with a 0.45-micrometer filter head, and loading; eluting with water at flow rate of 1mL/min for 6 times of column volume, collecting eluate, and concentrating under reduced pressure to 1/9 volume; putting the concentrated eluent into a dialysis bag with the molecular weight of 3400 Da, dialyzing 27 h in distilled water, and changing water every 6 h; the obtained eluent is frozen and dried to obtain 2.7g of geranium neutral crude polysaccharide, and the yield is 27%.

The geranium neutral polysaccharide prepared by the invention is named as GNP, and the GNP is analyzed and detected for structure, activity and the like by taking example 1 as an example.

Test example 1 measurement of polysaccharide content in Geranium neutral polysaccharide GNP

The determination method comprises the following steps: phenol-sulfuric acid process

(1) Preparation of 6% phenol solution: 15g of phenol solid is accurately weighed, 250mL of distilled water is added to be fully dissolved in water bath at 60 ℃, and the mixture is stored in a brown ground bottle in the dark for standby.

(2) Preparation of a 0.1mg/mL GNP solution: accurately weighing 10mg of neutral polysaccharide GNP of Geranium wilfordii, adding distilled water to a constant volume of 100mL, preparing a GNP solution of 0.1mg/mL, and measuring.

(3) Preparation of 0.1mg/mL glucose standard solution: accurately weighing 10mg of glucose standard substance, adding distilled water to a constant volume of 100mL volumetric flask, and preparing into 0.1mg/mL glucose standard solution for later use.

(4) Drawing a glucose standard curve: the glucose standard solution was measured by pipette tip at 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL in a 12mL glass tube, and then added with distilled water to 1mL, each concentration was repeated 3 times. Then, 0.5mL of 6% phenol solution was added to each tube, 2.5mL of concentrated sulfuric acid was continuously and slowly added, the tubes were rapidly shaken, and after the color change was stabilized by natural cooling, the absorbance was measured at 490 nm. The mass of the glucose standard was plotted on the abscissa and the absorbance a on the ordinate to obtain a standard curve (fig. 1).

(5) Content determination of polysaccharide samples: 1mL of the 0.1mg/mL GNP solution was added with 0.5mL of a 6% phenol solution and 2.5mL of a concentrated sulfuric acid solution, and the absorbance was measured at 490nm wavelength. And calculating the content of the geranium neutral polysaccharide according to the standard curve.

(6) As a result, as shown in table 1, the content of polysaccharide in GNP obtained in example 1 was 96.38%.

TABLE 1 phenol-sulfuric acid method for determining the absorbance of GNP prepared in example 2

Test example 2 identification of GNP monosaccharide composition as neutral polysaccharide of Geranium wilfordii

The identification method comprises the following steps: accurately weighing GNP and monosaccharide standards (L-rhamnose, D-mannose, D-glucose, D-galactose, D-fucose, D-arabinose, and D-xylose) 2mg respectively, and making into serum bottles, wherein the monosaccharide standards are subjected to derivatization reaction of single standard and mixed standard. Weighing 10mg of hydroxylamine hydrochloride and 1mg of internal standard inositol, then adding 2mL of pyridine, reacting for 30min at 90 ℃, after naturally cooling, adding 2mL of acetic anhydride, screwing a bottle cap, reacting for 30min at 90 ℃, and adding 2mL of distilled water to terminate the reaction after naturally cooling after the reaction is finished. Adding 2mL of dichloromethane into the sample after the derivatization reaction, fully shaking the sample, standing the sample for layering, sucking the lower layer solution, transferring the lower layer solution into a 25mL distillation flask, repeatedly extracting the lower layer solution for 1 time by using 1mL of dichloromethane, concentrating the solution obtained by two times of extraction under reduced pressure until the solution is dried to remove excessive moisture, filtering the solution through a 0.22-micron organic filter membrane, and performing GC-MS combined chromatography detection, wherein a gas chromatogram is shown in figure 3.

As a result: comparing each retention time with a monosaccharide standard substance, determining that the monosaccharide composition contained in the monosaccharide standard substance is as follows: l-rhamnose: d-arabinose: d-mannose, L-rhamnose: d-arabinose: the molar ratio of D-mannose is 7.59.

Test example 3 measurement of molecular weight of Geranium wilfordii neutral polysaccharide GNP

Weighing 5mg of sample GNP and dextran standard substances with different molecular weights, respectively adding 0.05M NaCl solution to prepare 5mg/ml sample solution and standard substance solution, filtering with 0.22 μ M microporous membrane, transferring to sample injection vial, and refrigerating for use. By adopting an HPGPC method, a high performance liquid chromatograph, a differential detector and polymer matrix water-soluble SEC (GFC) chromatographic columns OHpak SB-803 HQ, ohpak SB-804 HQ and Ohpak SB-805 HQ (8 x 300 mm) are used for serial column detection, the mobile phase is 0.05M NaCl solution, the flow rate is 0.6mL/min, the column temperature is 40 ℃ and the sample introduction amount is 30 mu L. Using a high performance gel permeation chromatography tandem column to detect a test sample and a standard sample, adopting Waters Empower software to analyze results, taking the retention time of a corresponding chromatographic peak as a horizontal coordinate, taking the logarithm value of the relative molecular mass Mp of the standard sample as a vertical coordinate to perform linear regression, establishing a standard curve, substituting the retention time of the test sample into the standard curve, and calculating to obtain the relative molecular mass 1663 Da of the test sample (figure 4).

Test example 4 GNP methylation analysis of neutral polysaccharide of Geranium wilfordii

Weighing 10mg GNP, adding 1mL of primary water for dissolving, adding 1mL of 100mg/mL carbodiimide, reacting for 2h, continuously adding 1mL of 2M imidazole, equally dividing the sample into two parts, and respectively adding 1mL of 30mg/mL NaBH ₄ And 1mL of 30mg/ml NaBD4, and reacting for 3h, and then adding 100. Mu.L of glacial acetic acid to stop the reaction. The samples were dialyzed for 48h and freeze-dried after dialysis was complete. And (3) carrying out methylation treatment on the freeze-dried sample, adding 500 mu L of DMSO into the freeze-dried sample to dissolve the sample, adding 1mg of NaOH, incubating for 30min, continuously adding 50 mu L of iodomethane solution to react for 1h, adding 1mL of water and 2mL of dichloromethane, uniformly mixing by vortex, centrifuging, and discarding the water phase. The water wash was repeated 3 times, the lower dichloromethane phase was aspirated and evaporated to dryness. Adding 100 μ L of 2M TFA, reacting at 121 deg.C for 90 min, evaporating at 30 deg.C, adding 50 μ L of 2M ammonia water, and 50 μ L of 1M NaBD ₄ Mixing, reacting at room temperature for 2.5h, adding 20 μ L acetic acid to terminate the reaction, blowing to dry with nitrogen, washing with 250 μ L methanol twice, and blowing to dry with nitrogen. Adding 250 mu L of acetic anhydride, uniformly mixing by vortex, reacting for 2.5h at 100 ℃, continuously adding 1mL of water, standing for 10min, adding 500 mu L of dichloromethane, uniformly mixing by vortex, centrifuging, and discarding the water phase. And (5) repeatedly washing for 3 times, and taking down a dichloromethane phase at the lower layer after the reaction is finished to be tested.

The chromatographic system adopts an Agilent gas chromatographic system (Agilent 7890A, agilent technologies, USA), the sample injection amount is 1 mu L, the split ratio is 10, and the carrier gas is high-purity helium gas; the initial temperature of the column oven is 140 ℃ and is kept for 2.0min, the temperature is increased to 230 ℃ by a program of 3 ℃/min and is kept for 3min. The mass spectrometry system used was a quadrupole mass spectrometry detection system (Agilent 5977b, agilent technologies, usa) from Aiglent corporation, usa, equipped with an electron impact ion source (EI) and a MassHunter workstation. The analytes are detected in a full SCAN (SCAN) mode using electron impact ion sources (EI), with a mass SCAN range (m/z) of 30-600. And (3) carrying out sample injection detection on the sample solution according to the chromatographic and mass spectrum conditions to obtain characteristic fragments after polysaccharide methylation, wherein the GC/MS reconstructed total ion current spectrum of the methylated fragments is shown in figure 5. The results are shown in Table 2, which were obtained by comparing the existing databases and confirming the bonding method.

TABLE 2 GNP derivatives and bonding modes

The analysis shows that the main derivative of GNP is 2,3,6-Me3-Galp、3,4,6-Me3-Glcp、2,3-Me2-ArafAnd 2,3,4,6-Me4-GlcpThe main link mode is → 1) -Galp-(4→。

Test example 5 GNP Infrared Spectroscopy of Geranium neutral polysaccharide

Accurately weighing 2mg of GNP and KBr, fully mixing and grinding the GNP and the KBr to prepare a potassium bromide tablet, placing the potassium bromide tablet in an FT-IR instrument for scanning for three times, and repeating the steps until the detection wavelength is 4000-400 nm.

The results are shown in the infrared spectrum of FIG. 6, the GNP values are 3400cm ^-1 A strong-OH stretching vibration absorption peak exists nearby; at 2930cm ^-1 A weaker C-H stretching vibration absorption peak is nearby; at 1600cm ^-1 Has a slightly weaker-OH bending vibration absorption peak; 1215-1220 cm ^-1 An absorption peak is caused by C-O stretching vibration; 1032-1034 cm ^-1 An absorption peak of-OH variable angle vibration is generated nearby; GNP at 937cm ^-1 A characteristic peak at which a β -type glycosidic bond is presumed; at 832cm ^-1 Should be the characteristic absorption peak of the alpha pyranose; furthermore, GNP is at 1420cm ^-1 A deformation absorption peak with = CH2 nearby; 1368cm ^-1 A C-H bending vibration absorption peak is arranged nearby; 1270cm ^-1 The vicinity may be an absorption peak caused by stretching vibration of O-H in COOH.

Test example 6 detection of cell viability of Geranium neutral polysaccharide GNP

The detection method comprises the following steps: RAW264.7 cells were cultured in DMEM (containing 10% FBS,1% penicillin-streptomycin) at 37 ℃ with 5% CO ₂ The incubator of (1). The experimental groups were as follows: normal control group (NC), 0.1 mug/mL LPS treatment group (positive control group) and Geranium neutral polysaccharide homogeneous component GNP (10-1000 mug/mL) treatment group with different concentrations, wherein each group is provided with 6 multiple wells. Cells were seeded in 96-well culture plates at a concentration of 1X 10 ⁵ And each well is 100 mu L, after culturing for 24 hours, the original culture medium is absorbed, the DMEM culture medium is added into the normal group, the medicines with corresponding concentrations are added into the other groups, after culturing for 24 hours, 10 mu L of CCK8 reagent is added, after culturing for 1.5 hours in an incubator, the absorbance is detected by a microplate reader under 450nm, and the experiment is repeated for 3 times. The cell viability calculation formula is as follows:

cell viability = (As-Ab)/(Ac-Ab). Times.100%

(As: experimental wells, ac: control wells, ab: blank wells).

As shown in FIG. 7, the cell viability of the GNP (10-1000. Mu.g/mL) treated group was increased compared to the NC group cells, and the GNP dose was most significant between 50. Mu.g/mL and 250. Mu.g/mL (P < 0.05).

And (4) analyzing results: the test results show that GNP can promote the proliferation of macrophage RAW264.7 and has no toxic or side effect.

Test example 7 GNP anti-inflammatory Activity assay

The detection method comprises the following steps: the in vitro anti-inflammatory activity of the LPS activated macrophage RAW264.7 inflammatory reaction model is evaluated by detecting proinflammatory cytokine indexes IL-6 and TNF-alpha.

RAW264.7 was plated in 96-well plates at a concentration of 1X 105/mL at 100uL per well. After 24 hours of culture, a normal control group (NC), a 1 mu m/L dexamethasone treatment group (positive control group), 0.1ug/mL lps (model group) and GNP (50-500 mu g/mL) with different concentrations are set, 4 multiple wells are set for each concentration, complete culture medium is given to the normal group and the model group, and 100uL of drug-containing culture medium is given to each well of each drug group for 24 hours of culture. After 24 hours, the cell supernatants were aspirated, DMEM medium was added to the normal group, LPS (0.1. Mu.g/mL) was added to each of the other groups at a concentration of 0.1mL per well, and after 12 hours of culture, cell supernatants were collected and assayed for TNF-. Alpha.and IL-6 levels in the cell supernatants by ELISA.

The results are shown in fig. 8 and 9, where the data are mean ± SD of three independent experiments. Lipopolysaccharide alone can significantly induce RAW264.7 macrophage to produce TNF-alpha and IL-6 (P < 0.0001). In contrast, GNP treatment inhibited the secretion of these LPS-stimulated pro-inflammatory cytokines in a dose-dependent manner (fig. 8-9, p-t <0.001).

Note: in comparison to the NC group, "#####" indicates that P <0.0001; compared to the MC group, "+") "

P < 0.01, "x" indicates P <0.001, "x" indicates P <0.0001.

And (4) analyzing results:

as can be seen from FIG. 8, the model group shows that macrophages stimulated by lipopolysaccharide produce a large amount of proinflammatory cytokine IL-6, the secretion of IL-6 is remarkably reduced after the positive drug dexamethasone with the concentration of 1 μm/L is dried, GNP has an inhibitory effect on the secretion level of IL-6 preliminarily when the concentration is 10 μ g/mg, and the secretion level of IL-6 stimulated by LPS can be remarkably inhibited when the concentration is 250 μ g/mg.

As can be seen from FIG. 9, the model group shows that macrophages stimulated by lipopolysaccharide produce a large amount of proinflammatory cytokine TNF-alpha, the secretion of TNF-alpha is remarkably reduced after the intervention of dexamethasone which is a positive drug with the concentration of 1 μm/L, GNP has an inhibition effect on the secretion level of TNF-alpha preliminarily when the concentration is 10 μ g/mg, and the secretion level of TNF-alpha caused by LPS stimulation can be remarkably inhibited when the concentration is 100 μ g/mg. Compared with the existing medicine gentian polysaccharide, the gentian polysaccharide starts to have the effect of inhibiting proinflammatory factors at the concentration of 25 mu g/mg, dose dependence does not exist (figure 10), and the GNP has more remarkable anti-inflammatory effect compared with the gentian polysaccharide.

In conclusion, the geranium neutral polysaccharide GNP can obviously reduce the level of proinflammatory cytokines and has better anti-inflammatory activity.

Test example 8 GNP antioxidant Activity measurement

The detection method comprises the following steps: DPPH radical scavenging Capacity determination

2mg of DPPH was weighed, dissolved in 10mL of absolute ethanol, and then the solution was taken up in a 50mL volumetric flask (0.04 mg/mL) and stored at 4 ℃ in the dark for later use. 1mL of GNP with a concentration of 0.05mg/mL, 0.1mg/mL, 0.2mg/mL, 0.4mg/mL, 0.8mg/mL, 1.6mg/mL was added to each of 6 tubes, an equal volume of distilled water was used as a blank, 1mL of DPPH solution was added to each tube, and the reaction was carried out in the dark for 30 minutes, and absorbance was measured at 517nm using vitamin C as a positive control, and the experimental results are shown in FIG. 11. The free radical clearance is calculated as follows:

and (4) analyzing results:

the Vc free radical clearance rate is 81 percent according to the calculation of the absorbance. As can be seen from FIG. 11, the GNP has a certain clearance already at a concentration of 0.8mg/mL, and the clearance reaches 43.9%, and the clearance reaches 52.1% at a concentration of 1.6mg/mL, indicating that the GNP has good antioxidant capacity. The contrast drugs of the white clouded ginseng neutral and acidic polysaccharide have certain removal capacity initially when the concentration reaches 2mg/mL (figure 12), and when the concentration reaches 6.4mg/mL, the removal rates of the white clouded ginseng neutral and acidic polysaccharide reach 59.7% and 65.7%, obviously, the free radical removal capacity of the geranium wilfordii neutral polysaccharide is stronger, and the potential of developing antioxidant drugs is better.

Claims

1. The geranium neutral polysaccharide is characterized by mainly comprising L-rhamnose, D-arabinose and D-mannose, wherein the content of the L-rhamnose: d-arabinose: the molar ratio of D-mannose is 7.59, the molar ratio is 1, the ratio of Mp is 1663 Da, the methylation analysis of the geranium neutral polysaccharide obtains residues of 2,3,6-Me3-Galp, 3,4,6-Me3-Glcp, 2,3-Me2-Araf and 2,3,4,6-Me4-Glcp, the ratio of 2,3,6-Me3-Galp is 16.7%, the ratio of 3,4,6-Me3-Glcp is 13.8%, the ratio of 2,3-Me2-Araf is 14.1%, and the ratio of 2,3,4,6-Me4-Glcp is 13.1%; wherein 2,3,6-Me3-Galp is a 1 → 4 linkage.

2. The method for preparing the geranium neutral polysaccharide as claimed in claim 1, which is characterized by comprising the following steps:

1) Drying the herba Erodii seu Geranii to the extent of easily breaking into powder, pulverizing, sieving with 10 mesh sieve, defatting with methanol under reflux for 2-3 times, extracting with water at 80-85 deg.C for 2.5-4h for 2-3 times, mixing extractive solutions, and concentrating under reduced pressure to obtain extract;

3. The preparation method according to claim 2, wherein in the step 1, the temperature of methanol reflux degreasing is 60-65 ℃, the concentration of methanol is 80-90%, and the volume weight ratio of methanol to the geranium wilfordii herb is 6-10:1L/Kg.

4. The preparation method according to claim 2, wherein in the step 2, the addition amount of ethanol is 2-3 times of the extract.

5. The method of claim 2, wherein the molecular weight cut-off in step 3 is 3400 Da on dialysis.

6. The use of the neutral polysaccharide of Geranium wilfordii in claim 1 as an active ingredient or a pharmaceutically acceptable carrier in the preparation of anti-inflammatory and antioxidant medicaments.

7. The use of claim 6, wherein the neutral polysaccharide of Geranium wilfordii is added with pharmaceutically acceptable adjuvants to make into tablet, hard capsule, soft capsule, powder, pill, and granule.