CN113527529A

CN113527529A - Aleuritopteris argentea fee polysaccharide and preparation method and application thereof

Info

Publication number: CN113527529A
Application number: CN202110861220.3A
Authority: CN
Inventors: 王昭晶
Original assignee: Huaqiao University
Current assignee: Huaqiao University
Priority date: 2021-07-29
Filing date: 2021-07-29
Publication date: 2021-10-22
Anticipated expiration: 2041-07-29
Also published as: CN113527529B

Abstract

The invention discloses a polysaccharide of Tonglingcao, a preparation method and application thereof. The polysaccharide of the Aleuritopteris argentea Bunge prepared by the method has definite components, and is composed of homogeneous polysaccharide with weight average molecular weight of 480KDa, and monosaccharide is glucose, galactose, mannose, xylose and arabinose. The inventive polysaccharide has anti-inflammatory and antioxidant activity, without toxicity to cell. Through the research on the scavenging activity of superoxide radical, hydroxyl radical and DPPH radical, the antioxidant effect of the menstrual grass polysaccharide is superior to that of vitamin c, and the menstrual grass polysaccharide can be used for preparing cosmetics and anti-inflammatory and antioxidant preparations for medical use.

Description

Aleuritopteris argentea fee polysaccharide and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a menstrual grass polysaccharide, and a preparation method and application thereof.

Background

The Aleuritopteris argentea (Aleuritopteris argentea) is a whole plant of Stenoloma sinicum (Linn.) Kuntze of Pteridaceae, and is mainly distributed in provinces such as Shandong, inner Mongolia, Hebei and Yunnan of China, and has abundant resources. The tongjing herbs are widely applied to minority regions in China, such as Korean, Tibetan and Mongolian nationalities, and have extremely high medicinal value, and reports show that the tongjing herbs are used for treating irregular menstruation, cough, wound suppuration, dizziness and the like.

At present, the research on the ricepaperplant pith is less at home and abroad, the main focus is on the chemical components of the ricepaperplant pith crude extract, and the research on the ricepaperplant pith polysaccharide is only limited to the extraction of the crude polysaccharide. Zhang Xiaowen et al (Tongdingcao polysaccharide extraction process research [ J ]. Shanxi agricultural science, 2016,62(01):43-46) optimize the Tongdingcao polysaccharide extraction process by using single factor and orthogonal experiment, and the optimal extraction process is that the extraction temperature is 90 ℃, the extraction time is 100min, the liquid-material ratio is 20:1(mL/g), and the actual extraction rate of the polysaccharide under the condition is 2.329%.

The research on the polysaccharide of the Tonglingcao in the prior art is limited to the level of crude polysaccharide, and the polysaccharide component is not clear, so that the preparation method and the characteristics of the polysaccharide of the Tonglingcao need to be deeply researched so as to provide a new object for developing a substitute product of a natural medicine component without toxic or side effect.

Disclosure of Invention

In order to improve the above background art problems, an object of the present invention is a method for preparing a ricepaperplant pith polysaccharide, comprising the steps of:

(1) taking dry dahliang grass powder, adding distilled water according to a material-liquid ratio of 1: 40-1: 120, extracting in a water bath at a constant temperature of 95 ℃ for 140-180 min, and centrifuging to remove precipitates to obtain a crude extract;

(2) concentrating the crude extract obtained in the step (1) to obtain a concentrated solution, measuring the volume of the concentrated solution, adding 2 times of ethanol in the volume of the concentrated solution, storing at 4 ℃ for more than 15h, centrifuging, and drying the precipitate to obtain a primary crude extract;

(3) continuously adding the supernatant obtained by centrifuging in the step (2) into ethanol with the volume 2-3 times that of the concentrated solution obtained by the step (2), standing at 4 ℃ for more than 15h, centrifuging, and drying the precipitate to obtain a secondary crude extract;

(4) taking 300 mg of the secondary crude extract obtained in the step (3), dissolving the secondary crude extract in 15mL of distilled water, centrifuging, taking supernate, passing through a chromatographic column, adjusting the flow rate to be 0.9-1.1 mL/min, setting an automatic part collector to collect 12mL of eluent in each tube, and carrying out sectional elution in three steps: eluting with distilled water for 13-18 h, eluting with 600-1200 mL of 0.3-0.4 mol/L NaCl solution in the second step, eluting with 1200-1800 mL of 1-2 mol/L NaCl solution in the third step, combining eluates collected in the second step, dialyzing with a dialysis bag with cut-off molecular weight of 8000, dialyzing with running water and distilled water for 20-28 h respectively, concentrating the dialysate, and freeze-drying to obtain the menstrual flow grass polysaccharide.

Preferably, in the step (1), the dried Tongdong grass powder is taken, distilled water is added according to the material-liquid ratio of 1:40, water bath extraction is carried out at the constant temperature of 95 ℃ for 155min, centrifugation is carried out, and precipitates are removed, so as to obtain a crude extract.

Preferably, in the step (2), the crude extract obtained in the step (1) is concentrated by 50 times.

Preferably, in the step (3), the supernatant obtained by centrifuging in the step (2) is added into absolute ethyl alcohol with the volume 2 times that of the concentrated solution in the step (2).

Preferably, in step (4), the chromatographic column is a DEAE-Sepharose CL-6B chromatographic column, and has a diameter of 4.6cm and a height of 35 cm.

In a preferred mode, in the step (4), the step elution is carried out in three steps: the first step was eluted with distilled water for 13h, the second step was eluted with 1200mL of 0.35mol/L NaCl solution, and the third step was eluted with 1200mL of 1mol/L NaCl solution.

The invention also aims to provide the menstrual flow grass polysaccharide obtained by the preparation method, wherein the menstrual flow grass polysaccharide has the weight average molecular weight of 480KDa, is a homogeneous heteropolysaccharide and consists of glucose, galactose, mannose, xylose and arabinose. Further, the molar ratio of glucose, galactose, mannose, xylose and arabinose was 2.5:2.14:2.07:1: 3.88.

The invention also aims to provide the application of the menstrual flow grass polysaccharide in preparing anti-inflammatory and antioxidant preparations for cosmetics and medicines.

The invention has the beneficial effects that:

(1) the invention optimizes the extraction process of the polysaccharide of the Aleuritopteris argentea by using a response surface method, and the extraction rate is improved by about 4 times compared with the reported extraction process of the polysaccharide of the Aleuritopteris argentea.

(2) The invention firstly uses ethanol fractional precipitation to obtain a first-stage crude extract and a second-stage crude extract, wherein the two crude extracts contain different menstrual grass polysaccharides, which shows that the ethanol fractional precipitation method is used for preliminary separation of the menstrual grass polysaccharides, and the method is simple and effective. And (3) carrying out three-step elution on the secondary crude extract to obtain the ricepaper pith polysaccharide, wherein the method has simple steps, and the ricepaper pith polysaccharide can be obtained only by accurately mastering elution conditions in each step.

(3) The polysaccharide of the Aleuritopteris argentea Bunge prepared by the invention has definite components, and consists of homogeneous heteropolysaccharide with the weight-average molecular weight of 480KDa, and monosaccharide consists of glucose, galactose, mannose, xylose and arabinose with the molar ratio of 2.5:2.14:2.07:1: 3.88.

(4) The inventive polysaccharide has significant anti-inflammatory and antioxidant activity, and the addition of 75 μ g/mL or 150 μ g/mL of Aleuritopteris argentea polysaccharide has no cytotoxicity to RAW264.7 cells, and can significantly reduce the relative expression amount of mRNA of TNF-alpha, COX-2 and iNOS (P <0.05) in RAW264.7 cells induced by lipopolysaccharide, and the anti-inflammatory effect of Aleuritopteris argentea polysaccharide on TNF-alpha and iNOS has no significant difference (P >0.05) from that of positive control group. Through the research on the scavenging activity of superoxide radical, hydroxyl radical and DPPH radical, the antioxidant effect of the menstrual grass polysaccharide is superior to Vc.

Drawings

FIG. 1 is a graph showing the relationship between the extraction rate of the polysaccharides of Tonglingcao in example 1 and the liquid-to-material ratio.

FIG. 2 is a graph showing the relationship between the extraction rate and the extraction time of the Aleuritopteris argentea polysaccharide in example 1 of the present invention.

FIG. 3 is a graph showing the extraction rate of Aleuritopteris argentea polysaccharide with the variation of extraction temperature in example 1 of the present invention.

Fig. 4 is a residual normal probability distribution diagram in embodiment 1 of the present invention.

FIG. 5 is a model diagnosis graph in example 1 of the present invention, wherein A is a residual map, and B is a scatter plot of predicted values and actual values.

FIG. 6 is a graph showing the distribution of sugar (A) and protein content (B) after the second elution with DEAE-Sepharose CL-6B column of the second crude extract of example 2 of the present invention.

FIG. 7 is a graph showing the distribution of sugar (A) and protein content (B) after the second crude extract of example 2 of the present invention is eluted through a third step of DEAE-Sepharose CL-6B column chromatography.

FIG. 8 is a graph showing the distribution of sugar (A) and protein (B) contents after elution with 0.5mol/L NaCl in the second crude extract of example 2 of the present invention.

FIG. 9 is a graph showing the distribution of sugar (A) and protein content (B) after the first-stage crude extract of example 2 of the present invention is eluted through a second step of DEAE-Sepharose CL-6B column chromatography.

FIG. 10 is a high performance liquid chromatogram of the first crude extract after the second elution step in example 2 of the present invention.

FIG. 11 is a high performance liquid chromatogram of the Aleuritopteris argentea polysaccharide AAP1 in example 3 of the present invention.

FIG. 12 is the ultraviolet absorption spectrum of the Aleuritopteris argentea polysaccharide AAP1 in example 3 of the present invention.

FIG. 13 is a gas chromatogram of the Aleuritopteris argentea polysaccharide AAP1 of example 3 of the present invention (peaks 1-5 in FIG. 13 represent glucose, galactose, mannose, xylose and arabinose, respectively).

FIG. 14 is a graph showing the effect of AAP1 on LPS stimulation of mRNA expression of macrophage inflammatory factor in RAW264.7 mice in example 4 of the present invention.

FIG. 15 is a graph showing the results of the antioxidant activity test of AAP1 in example 4 of the present invention.

Detailed Description

The technical solution of the present invention is further illustrated and described by the following detailed description.

A preparation method of Aleuritopteris argentea fee polysaccharide comprises the following steps:

(2) concentrating the crude extract obtained in the step (1) to obtain a concentrated solution, measuring the volume of the concentrated solution, adding ethanol with 2 times of the volume of the concentrated solution, storing for more than 15h in a refrigerator at 4 ℃, centrifuging for 5min at 5000r/min, and drying the precipitate to obtain a primary crude extract;

(3) continuously adding the supernatant obtained by centrifuging in the step (2) into ethanol with the volume 2-3 times that of the concentrated solution obtained by the step (2), standing in a refrigerator at 4 ℃ for more than 15h, centrifuging at 5000r/min for 5min, and drying the precipitate to obtain a secondary crude extract;

(4) taking 300 mg of the secondary crude extract obtained in the step (3), dissolving the secondary crude extract in 15mL of distilled water, centrifuging the solution at 5000r/min for 5min, taking the supernatant, passing the supernatant through a DEAE-Sepharose CL-6B chromatographic column (the diameter is 4.6cm, the height is 35cm), adjusting the flow rate to be 0.9-1.1 mL/min, setting an automatic part collector to collect 12mL of eluent in each tube, and carrying out sectional elution in three steps: eluting with distilled water for 13-18 h, eluting with 600-1200 mL of 0.3-0.4 mol/L NaCl solution in the second step, eluting with 1200-1800 mL of 1-2 mol/L NaCl solution in the third step, combining eluates collected in the second step, dialyzing with a dialysis bag with cut-off molecular weight of 8000, dialyzing with running water and distilled water for 20-28 h respectively, concentrating the dialysate, and freeze-drying to obtain the menstrual flow grass polysaccharide.

Example 1 response surface method optimized Aleuritopteris argentea polysaccharide extraction process

The experimental method comprises the following steps: a single-factor experiment is adopted to study the influence of 3 factors, namely the liquid-material ratio, the extraction temperature and the extraction time, on the extraction rate of the polysaccharide of the ricepaperplant pith. In the specific experiment, under the experiment condition of fixing two factors, another 1 factor is considered, so that the influence of the factor on the extraction rate of the polysaccharide of the ricepaper plant pith under different experiment levels can be obtained.

In order to optimize the extraction conditions of the Aleuritopteris argentea polysaccharide, 1g of Aleuritopteris argentea powder is weighed by adopting an orthogonal test and a response surface test and taking the liquid-material ratio, the extraction temperature and the extraction time as experimental factors, and the test is arranged according to an orthogonal table and a Box-Behnken scheme. Extracting with distilled water at certain extraction temperature for certain time, centrifuging to obtain crude extractive solution, and detecting polysaccharide extraction rate of the obtained crude extractive solution by phenol-sulfuric acid method.

The experimental results are as follows:

(1) single factor experimental results: extracting the polysaccharide of the Aleuritopteris argentea fee by changing the feed liquid ratio, wherein the experimental result is shown in figure 1; the influence of different extraction times on the extraction rate of the polysaccharide from the radix clematidis is shown in the graph of fig. 2; the effect of extraction temperature on the extraction rate of the polysaccharides from Clerodendranthus spicatus is shown in FIG. 3. According to the single-factor experiment result, each factor selects 3 experiment levels, and then a subsequent orthogonal experiment and a response surface experiment are arranged. According to the results of the single-factor experiment, three levels of the liquid-to-feed ratio (A) such as 1, 2 and 3 in column A of Table 1 are respectively determined as 40:1, 80:1 and 120:1, three levels of the extraction time (B) such as 1, 2 and 3 in column B of Table 1 are respectively 100min, 140min and 180min, three levels of the extraction temperature (C) such as 1, 2 and 3 in column C of Table 1 are respectively 65 ℃, 80 ℃ and 95 ℃, and the tonsil pennywort herb polysaccharide extraction rate code is set as Y.

(2) Orthogonal experiment results: the resulting protocol was tested according to the orthogonal horizontal factor table and the results are shown in table 1.

TABLE 1 Aleuritopteris argentea polysaccharide extraction yield orthogonal test results

As can be seen from Table 1, the relationship between the different levels of the same factor in the test results is: a. the₁>A₂>A₃，B₁>B₃>B₂，C₃>C₁>C₂In which K is₁(A)＝A₁B₁C₁+A₁B₂C₂+A₁B₃C₃＝9.063+8.758+9.655＝27.476，K₁(A) Is A₁The ratio of liquid to material is 40:1, and so on, taking the maximum value of K in A, B, C three columns as K₁(A)、K₁(B)、K₃(C) It can be found that the optimal level combination of the orthogonal optimization experiment of the polysaccharide extraction rate of the Tongyuan grass is A₁B₁C₃The ratio of material to liquid is 1:40, the extraction time is 100min, and the extraction temperature is 95 ℃. Extracting the polysaccharide of the Indian buead according to the optimal extraction process scheme to obtain the actual extraction rate of 9.107 percent.

(3) Response surface test design and results: the design of the response surface test and the test results are shown in table 2.

TABLE 2 Box-Behnken test design and results for Aleuritopteris argentea polysaccharide extraction

Statistical processing is carried out on the test results by adopting Design-expert.V11.1.0 software, and the regression equation model is calculated by data fitting to be 9.09-0.0942A +0.2893B +0.2885C +0.0217AB +0.1288BC-0.0384A²-0.5379B²-0.1669C². In order to verify the effect of the model on the prediction of the extraction scheme, the influence degree of each factor on the extraction rate of the polysaccharide of the Aleuritopteris argentea and the variance analysis of the regression equation are carried out, and the result is shown in Table 3.

TABLE 3 regression equation analysis of variance and significance results

The model analysis result shows that: the proposed model obtained from the data results is a Quadratic model with a p-value of 0.0275 (C) ((R))<0.05), significant regression model, and mismatching term p of 0.9713 (b)>0.05), and the mismatching item is not significant, which shows that the model has better fitting performance to the test result. R of the equation²0.8108, the equation fits well, R²(pred) (0.5436) and R²The difference of (adj) (0.6216) was less than 0.2 and the coefficient of variation (C.V.) was 3.31% lower, demonstrating that the predicted values were substantially the same as the actual values, and the above data indicate that this model can be usedThe extraction rate of the polysaccharide of the ricepaperplant pith is analyzed and predicted.

Not only the anova result can analyze whether the model is available, but also whether the prediction model is sufficient can be diagnosed through the diagnostic graph. Normal graph of residuals as shown in fig. 4, the distribution of residuals along a straight line indicates that residuals follow a normal distribution. As shown in fig. 5 (in fig. 5, a is a residual map, and B is a scatter plot of predicted values versus actual values), all data points are within an acceptable range (± 3), and a linear fit indicates that there is a good correlation between actual profitability and predicted profitability.

By optimizing through a response surface method, software analysis shows that the optimal condition for extracting the ricepaper pith polysaccharide is that A (material-liquid ratio) is 1:40, the average extraction rate of the polysaccharide from the ricepaperplant pith was found to be 9.688% which is 0.343% from the theoretical value, when 3 parallel experiments were carried out according to this protocol with a B (time) of 155min and a C (temperature) of 95 ℃.

The extraction process of the Aleuritopteris argentea polysaccharide is optimized by a response surface method, an extraction rate prediction model is established, and the optimal extraction conditions are obtained. Compared with an orthogonal experiment method, the response surface method is used for optimizing the extraction of the Aleuritopteris argentea polysaccharide, the extraction rate is high, and the effect is better.

Example 2 separation and purification of the polysaccharides from the plant Tonglingcao, comprising the following steps:

(1) taking 150g of dried Aleuritopteris argentea powder, adding into 6000mL of distilled water, extracting in water bath at 95 deg.C for 155min in a constant temperature water bath kettle, centrifuging at 5000r/min for 5min, and removing precipitate to obtain crude extract;

(2) concentrating the extracting solution obtained in the step (1) to 120mL, adding 240mL of absolute ethyl alcohol, standing in a refrigerator at 4 ℃ for more than 15h, centrifuging at 5000r/min for 5min, and drying the precipitate to obtain a primary crude extract;

(3) continuously adding 240mL of absolute ethyl alcohol into the supernatant obtained after the centrifugation in the step (2), standing for more than 15h at 4 ℃ in a refrigerator, centrifuging for 5min at 5000r/min, and drying the precipitate to obtain a secondary crude extract;

(4) taking 300 mg of the secondary crude extract obtained in the step (3), dissolving the secondary crude extract in 15mL of distilled water, centrifuging for 5min at 5000r/min, taking supernatant, putting the supernatant on a DEAE-Sepharose CL-6B chromatographic column (the diameter is 4.6cm, the height is 35cm), opening a constant flow pump, regulating the flow rate to be 0.9mL/min, and carrying out fractional elution in three steps: the method comprises the steps of eluting with distilled water for 13 hours without collecting eluent, eluting with 1200mL of 0.35mol/L NaCl solution in the second step, setting each tube of an automatic part collector to collect 12mL of eluent, eluting with 1200mL of 1mol/L NaCl solution in the third step, setting each tube of the automatic part collector to collect 12mL of eluent, respectively measuring the sugar and protein contents of the eluents in the second step and the third step by using a phenol-sulfuric acid method and an ultraviolet spectrophotometer, combining 30-50 tubes of the eluents in the second step according to the distribution conditions of the sugar and protein contents, dialyzing the collected liquid by using a dialysis bag with molecular weight cut-off of 8000, dialyzing flowing water and distilled water for 24 hours respectively, concentrating the dialyzate, and freeze-drying to obtain the menstrual flow polysaccharide AAP 1.

As a result: the second elution profile in step (4) is shown in FIG. 6, and the third elution profile is shown in FIG. 7. According to FIG. 6, it can be seen that 30-50 tubes of the eluate detected one sugar peak (as shown in the left half of FIG. 6) and 1 protein peak (as shown in the right half of FIG. 6), and the peak pattern and tube number were matched, indicating that the protein is a binding protein. As can be seen from FIG. 7, no sugars were detected (as in the left half of FIG. 7) and only proteins were detected (as in the right half of FIG. 7) using the higher ionic strength NaCl solution, indicating that the Tetrapanax papyriferus polysaccharide AAP1 had been eluted in the second step.

The concentration of NaCl solution used in the second step of elution in the step (4) is very important, the ionic strength is high when the concentration is too high, other impurity proteins can be eluted along with the elution of the Tongyuan grass polysaccharide, the Tongyuan grass polysaccharide cannot be eluted when the concentration is too low, and the second step of elution is very important when NaCl solution with the concentration of 0.35mol/L is used through experiments. In comparison with FIG. 6, FIG. 8 shows the result of the second step of step (4) eluting with 0.5mol/L NaCl solution, and it can be seen that the protein distribution curve is significantly broader than the sugar distribution curve, and the peaks of the two curves are different, indicating the presence of the hetero-protein.

The results of the primary and secondary crude extracts obtained by steps (2) and (3) and the purification by step (4) using the primary crude extract are shown in FIG. 9, and it can be seen from FIG. 9 that the distribution of sugars and proteins is disordered, which is completely different from the results of FIG. 6 after the secondary crude extract is purified by step (4). The analysis result of the homogeneity of the polysaccharide after the first-stage crude extract is purified is shown in fig. 10, and fig. 10 is a high performance liquid chromatogram of the first-stage crude extract after the second-step elution. FIG. 10 shows that instead of a single peak, it further illustrates that the primary crude extract was purified in the same manner to obtain a polysaccharide that is not a homogeneous polysaccharide, unlike the Tonglingcao polysaccharide of the present invention. Further, ethanol is used for fractional precipitation to obtain a first-stage crude extract and a second-stage crude extract, different ricepaperplant pith polysaccharides are primarily separated, and the second-stage crude extract is further purified to obtain the ricepaperplant pith polysaccharides with the anti-inflammatory and anti-oxidation effects.

Example 3 characterization of the Aleuritopteris argentea polysaccharide

The experimental method comprises the following steps: the uniformity of the sample AAP1 was identified by High Performance Size Exclusion Chromatography (HPSEC) with supersar ks-804 as column, ultrapure water as mobile phase, column temperature 50 deg.C, flow rate 1mL/min, differential refractometer detector. According to the peak time of the dextran standard on the sugar ks-804 chromatographic column, a molecular weight standard curve is prepared, and the weight average molecular mass of AAP1 is calculated. To check the purity of the sample, 0.5mg/mL AAP1 aqueous solution was subjected to UV full spectrum scanning (190-800 nm). The main functional groups of the sample were analyzed using infrared spectroscopy (FTIR) to confirm the compound type.

In order to analyze the monosaccharide composition of AAP1, 20-30mg of AAP1 is hydrolyzed by 1mol/L sulfuric acid at 100 ℃ for 8h, and the hydrolysate is obtained by barium carbonate neutralization and freeze drying. Derivatization of hydrolysate by trimethylsilylation, gas chromatography analysis of the derivative on hp-5 capillary chromatographic column, temperature programming 160 ℃ → 180 ℃ (20 ℃/min) → 220 ℃ (8 ℃/min, holding for 2min) → 250 ℃ (2min), and detector temperature 280 ℃.

As a result: the high performance size exclusion chromatography results of the menstrual grass polysaccharide AAP1 are shown in FIG. 11. The result shows that only 1 peak appears in AAP1 at 6.082min, and the peak has sharp and symmetrical peak shape, which shows that AAP1 has good uniformity and single component. Through 190-800nm ultraviolet full spectrum scanning (FIG. 12), AAP1 does not contain nucleic acid and other impurities, and has high purity. According to the molecular mass standard curve (y ═ 4x +6.31, R²0.99), HPSEC peak time of AAP1, calculated to give AAP1 with a weight average molecular mass of 480 kDa. From the results of gas chromatography of AAP1 (FIG. 13), with reference to the results of GC of standard monosaccharide derivatives, it was found that AAP1 was composed of glucose and galactoseMannose, xylose and arabinose in a molar ratio of 2.5:2.14:2.07:1: 3.88. In FIG. 13, the peaks 1 to 5 represent glucose, galactose, mannose, xylose and arabinose, respectively.

Example 4 anti-inflammatory and antioxidant Effect of Aleuritopteris argentea polysaccharide AAP1

The experimental method comprises the following steps:

anti-inflammatory action studies: RAW264.7 cells were cultured under the conditions of DMEM complete medium (containing 10% serum and 1% diabody), 37 ℃, 5% carbon dioxide incubator, and 48h for one passage. The MTT method was used to test the cytotoxicity of the tomatillo-fortunei polysaccharide to RAW264.7, setting six mass concentrations of 600, 300, 150, 75, 25 and 1 μ g/mL. Inducing RAW264.7 cell to produce inflammation by using 1 microgram/mL Lipopolysaccharide (LPS), setting blank group, negative and positive control group, AAP1 sample group, extracting total RNA in each group of cells, carrying out reverse transcription to obtain cDNA, taking housekeeping gene GAPDH as internal reference gene, carrying out real-time fluorescence quantitative PCR determination on CT value, 2^-ΔΔCTThe relative expression level of mRNA was calculated according to the kit instructions of Promega corporation.

Research of antioxidant experiment: samples were prepared in an aqueous solution at a concentration of 30mg/mL to 0.0075mg/mL, and the superoxide anion scavenging ability was measured by the method of Robak, wherein 0.1mL of the sample solution was added with 1mL of 16mM Tris-HCl (pH8.0) containing 557uM NADH, 1mL of 16mM Tris-HCl (pH8.0) containing 45uM PMS, and 1mL of 16mM Tris-HCl (pH8.0) containing 108uM NBT, and incubated at 25 ℃ for 5min, and the absorbance was measured at 560nm, and the inhibition was calculated as the clearance ═ 1-sample tube/control tube). times.100%. Measurement of hydroxyl radical scavenging Effect with reference to the method of Ghiselli et al and improvement, 0.6mL of a reaction buffer (containing 2.67mM deoxyribose and 0.13mM EDTA), 0.2mL of 0.4mM ferrous sulfate, 0.05mL of 2.0mM ascorbic acid, and 20mM H were added to 0.1mL of the sample solution₂O₂0.05mL, warm bath at 37 ℃ for 15min, taking out, adding 1mL of 1% thiobarbituric acid and 1mL of 2% trichloroacetic acid, boiling in a water bath for 15min, immediately cooling on ice, measuring the absorbance at 532nm, and calculating the inhibition rate according to the clearance rate (1-sample tube/control tube) × 100%. Measuring DPPH free radical, adding 50ul sample solution into 100ul 0.3mM DPPH solution, water bathing at 25 deg.C for 20min,OD was measured at 517nm and the inhibition was calculated as the clearance (1-sample tube/control tube) × 100%.

The experimental results are as follows: through a cytotoxicity experiment, the addition of 150 mu g/mL or 75 mu g/mL of the Aleuritopteris argentea polysaccharide AAP1 is confirmed to have no toxicity on the growth of RAW264.7 cells. As shown in fig. 14, the relative expression levels of TNF- α, COX-2 and iNOS mrnas, which are inflammatory factors, were significantly increased in the negative control group (LPS) compared to the blank group (P <0.05), indicating that an inflammation model was successfully established after 1 μ g/mL Lipopolysaccharide (LPS) was added to RAW264.7 cells. In FIG. 14, panel A is a graph showing the effect of each group (blank Control, negative LPS Control, positive DXMS Control, and AAP1 concentration sample groups) on the expression level of TNF-. alpha.and panel B is a graph showing the effect of each group on the expression level of COX-2 and panel C is a graph showing the effect of each group on the expression level of iNOS. APP1-75 represents 75 μ g/mL APP1, and APP1-150 represents 150 μ g/mL APP 1.

Meanwhile, 1 mug/mL lipopolysaccharide and 1 mug/mL menstrual grass polysaccharide AAP1(75 mug/mL or 150 mug/mL) are added in RAW264.7 cells, compared with a negative control group, AAP1 with two concentrations can obviously reduce the relative expression quantity of mRNA (P <0.05) of an inflammatory factor TNF-alpha, and through significance analysis, the inhibition rate of the menstrual grass polysaccharide AAP1 to the TNF-alpha expression of a positive control group (dexamethasone and DXMS) has no significant difference (P >0.05), which shows that the anti-inflammatory effect of AAP1 to the TNF-alpha is equivalent to that of DXMS. AAP1 can also significantly reduce the relative mRNA expression level (P <0.05) of inflammatory factor COX-2 in RAW264.7 cells, and the anti-inflammatory effect of 75 mu g/mL of the Aleuritopteris argentea polysaccharide AAP1 on COX-2 is the best and better than 150 mu g/mL of Aleuritopteris polysaccharide. Compared with the negative control group, the addition of AAP1 of 75 μ g/mL or 150 μ g/mL can significantly reduce the relative expression level of mRNA of iNOS gene in RAW264.7 cells induced by lipopolysaccharide (P <0.05), and the effect is not significantly different from that of the positive control group (P > 0.05).

The antioxidant effect of the Aleuritopteris argentea polysaccharide AAP1 is shown in FIG. 15, and with vitamin c (Vc) as a positive control group, the scavenging effect of AAP1 and Vc on superoxide radical, hydroxyl radical and DPPH radical increases with the increase of concentration, and shows dose-effect relationship. EC of Aleuritopteris argentea polysaccharide AAP1 for scavenging superoxide radical, hydroxyl radical and DPPH radical₅₀Respectively 307 mug/mL, 227 mug/mL and 1.35 mug/mL, and Vc is applied to the above 3 kinds of medicinesRadical scavenging EC₅₀463. mu.g/mL, 2513. mu.g/mL and 4.62. mu.g/mL, respectively. In fig. 15, panel a shows the increasing scavenging effect of AAP1 and Vc on superoxide radicals with increasing concentration, panel B shows the increasing scavenging effect of AAP1 and Vc on hydroxyl radicals with increasing concentration, panel C shows the trend of corresponding concentration of AAP1 on DPPH scavenging effect, and panel D shows the trend of corresponding concentration of Vc on DPPH scavenging effect.

In conclusion, the addition of 75 μ g/mL or 150 μ g/mL of AAP1 can significantly reduce the relative expression levels of TNF- α, COX-2 and iNOS mRNA (P <0.05) in RAW264.7 cells induced by lipopolysaccharide, and the anti-inflammatory effects of AAP1 on TNF- α and iNOS are not significantly different from those of the positive control group (P > 0.05). Through the research on the scavenging action of superoxide radical, hydroxyl radical and DPPH radical, the antioxidant effect of the menstrual grass polysaccharide AAP1 is superior to that of Vc.

The human body can continuously generate free radicals in vivo due to contact with the outside, including factors such as respiration, radioactive rays and air pollution, and scientific research shows that the excessive free radicals are closely related to aging, cardiovascular diseases, arthritis, cancers and the like, so that the development of the antioxidant preparation is listed as one of the main research and development directions by cosmetics and pharmaceutical enterprises.

Excessive expression of inflammatory factors can cause various diseases, such as central nervous system diseases, atherosclerosis, chronic obstructive pulmonary diseases, rheumatoid arthritis and the like. The anti-inflammatory and the antioxidant supplement each other, and because free radicals are one of the 'genuine' which causes skin inflammation, many cosmetics in the market contain anti-inflammatory and antioxidant components, and the search for natural non-toxic anti-inflammatory and antioxidant products is one of the most important functional requirements in the market.

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents and modifications within the scope of the description.

Claims

1. A preparation method of Aleuritopteris argentea fee polysaccharide is characterized in that: the method comprises the following steps:

2. The method for preparing the Tonglingcao polysaccharide as claimed in claim 1, wherein the polysaccharide is prepared from the following raw materials: in the step (1), the dried Tongdong grass powder is taken, distilled water is added according to the material-liquid ratio of 1:40, the mixture is extracted in a water bath at the constant temperature of 95 ℃ for 155min, and the coarse extract is obtained after centrifugation and sediment removal.

3. The method for preparing the Tonglingcao polysaccharide as claimed in claim 1, wherein the polysaccharide is prepared from the following raw materials: in the step (2), the crude extract obtained in the step (1) is concentrated by 50 times.

4. The method for preparing the Tonglingcao polysaccharide as claimed in claim 1, wherein the polysaccharide is prepared from the following raw materials: in the step (3), the supernatant obtained by centrifugation in the step (2) is continuously added into absolute ethyl alcohol with the volume 2 times that of the concentrated solution in the step (2).

5. The method for preparing the Tonglingcao polysaccharide as claimed in claim 1, wherein the polysaccharide is prepared from the following raw materials: in the step (4), the chromatographic column is a DEAE-Sepharose CL-6B chromatographic column, the diameter of which is 4.6cm, and the height of which is 35 cm.

6. The method for preparing the Tonglingcao polysaccharide as claimed in claim 1, wherein the polysaccharide is prepared from the following raw materials: in the step (4), the step of stepwise elution is carried out in three steps: the first step was eluted with distilled water for 13h, the second step was eluted with 1200mL of 0.35mol/L NaCl solution, and the third step was eluted with 1200mL of 1mol/L NaCl solution.

7. The polysaccharide of the ricepaperplant pith, which is obtained by the preparation method of any one of claims 1 to 6, is a homogeneous heteropolysaccharide comprising glucose, galactose, mannose, xylose and arabinose, and the weight-average molecular weight of the polysaccharide of the ricepaperplant pith is 480 KDa.

8. The Aleuritopteris argentea polysaccharide of claim 7, wherein the molar ratio of glucose, galactose, mannose, xylose and arabinose is 2.5:2.14:2.07:1: 3.88.

9. Use of the polysaccharides of Andrographis paniculata according to claims 7 or 8 for the preparation of anti-inflammatory and antioxidant preparations for cosmetic and pharmaceutical use.