阅读说明：本技术 一种马鞭草多糖的制备方法与应用 (Preparation method and application of verbena polysaccharide ) 是由 罗巅辉 于 2021-08-24 设计创作，主要内容包括：本发明公开了一种马鞭草多糖的制备方法,采用本发明的制备方法首先用2-3倍体积乙醇沉淀得到粗提物,简单有效的对马鞭草多糖进行了初步分离,使用蛋白酶对粗提物水溶液进行蛋白质降解,有效的解离了缠绕在一起的蛋白质和多糖两类大分子,然后对酶解产物直接进行特定柱层析纯化,得到本发明所述的具有抗炎症和抗氧化作用的马鞭草多糖,方法步骤简单有效。上述方法制备所得马鞭草多糖成分明确,其是均一杂多糖,重均分子量是290KDa的,单糖组成是半乳糖,葡萄糖,甘露糖,鼠李糖和阿拉伯糖组成,各单糖摩尔比为2.8:1.5:1:1:1.4。本发明制备得到的马鞭草多糖具有显著的抗炎和抗氧化作用,本发明所得马鞭草多糖可用于制备化妆品和医药用抗炎制剂。(The invention discloses a preparation method of verbena polysaccharide, which is characterized in that firstly, 2-3 times of volume of ethanol is used for precipitation to obtain a crude extract, the verbena polysaccharide is simply and effectively primarily separated, protease is used for carrying out protein degradation on an aqueous solution of the crude extract, two types of macromolecules of protein and polysaccharide which are intertwined together are effectively dissociated, then, specific column chromatography purification is directly carried out on an enzymolysis product, and the verbena polysaccharide with anti-inflammation and anti-oxidation effects is obtained. The verbena polysaccharide prepared by the method has definite components, is homogeneous heteropolysaccharide, has the weight average molecular weight of 290KDa, and consists of galactose, glucose, mannose, rhamnose and arabinose, and the molar ratio of the monosaccharides is 2.8:1.5:1:1: 1.4. The verbena polysaccharide prepared by the invention has obvious anti-inflammatory and anti-oxidation effects, and can be used for preparing cosmetics and anti-inflammatory preparations for medicine.)

1. A preparation method of verbena polysaccharide is characterized by comprising the following steps: the method comprises the following steps:

(1) taking dry verbena powder, adding distilled water according to a liquid-material ratio of 20: 1-100: 1, extracting in a water bath at 65-95 ℃ in a constant-temperature water bath kettle for 120-240 min, and centrifuging to remove precipitates to obtain a crude extract;

(2) concentrating the crude extract obtained in the step (1), adding absolute ethyl alcohol with the volume 2-3 times that of the concentrated solution, standing at 4 ℃ for a certain time, centrifuging at 5000r/min for 5min, and drying the precipitate to obtain a verbena crude extract;

(3) dissolving 500mg of the crude extract of verbena officinalis obtained in the step (2) in 20mL of distilled water, adding protease, keeping the temperature in a water bath at 37 ℃ for 48h, centrifuging at 5000r/min for 5min, and removing precipitates to obtain an enzymatic hydrolysate;

(4) and (3) putting the enzymatic hydrolysate obtained in the step (3) on a DEAE-Sepharose CL-6B chromatographic column, adjusting the flow rate to be 1mL/min, eluting with distilled water for 16-20 h, then performing gradient elution with 0.2-0.4mol/L NaCl solution, setting each automatic part collector to collect 12mL of eluent, combining 20-40 th-tube eluents according to the distribution condition of sugar and protein content, dialyzing the collected liquid with a dialysis bag with the molecular weight cutoff of 3500-5000, dialyzing the flowing water and the distilled water for 24h respectively, concentrating the dialyzate, and freeze-drying to obtain verbena polysaccharide VOP, wherein the average extraction rate of the verbena polysaccharide VOP is more than 8.3%.

2. The method of claim 1, wherein the preparation of verbena polysaccharide comprises: taking dry verbena powder in the step (1), adding distilled water according to the liquid-material ratio of 20:1, extracting in a water bath at 83 ℃ for 210min in a constant-temperature water bath kettle, and centrifuging at 5000r/min for 10min to remove precipitates to obtain a crude extract.

3. The method of claim 1, wherein the preparation of verbena polysaccharide comprises: and (2) concentrating the crude extract obtained in the step (1) by 40 times, and adding absolute ethyl alcohol with the volume 2 times that of the concentrated solution.

4. The method of claim 1, wherein the preparation of verbena polysaccharide comprises: the protease in the step (3) comprises 4mg of trypsin and 4mg of proteinase K.

5. The method of claim 1, wherein the preparation of verbena polysaccharide comprises: in the step (4), distilled water is used for eluting for 16h, and the molecular weight cut-off of the dialysis bag is 3500.

6. The method for preparing verbena polysaccharide of claim 1, wherein the verbena polysaccharide obtained in step (4) is a heteropolysaccharide with a weight-average molecular weight of 290KDa, and the monosaccharide components are galactose, glucose, mannose, rhamnose and arabinose, and the molar ratio of the monosaccharides is 2.8:1.5:1:1: 1.4.

7. The application of verbena polysaccharide is characterized in that: the verbena polysaccharide is prepared by the preparation method of claim 1, and is applied to cosmetics and preparation of anti-inflammatory preparations for medical use.

Technical Field

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

Background

Verbena Officinalis (Verbena Officinalis) is a whole plant of Verbenaceae and Verbena plants, and has wide distribution range and rich resources in China. The traditional Chinese medicine verbena is bitter in property and taste, has the effects of promoting blood circulation, removing blood stasis, detoxifying, inducing diuresis and reducing edema and the like, and is mainly used for treating amenorrhea and dysmenorrhea, carbuncle swelling, malaria, edema, heat stranguria and other diseases.

The research on verbena at home and abroad mainly focuses on the alcohol solubility and the chemical components of a water-decocted crude extract, and the research on water-soluble verbena polysaccharide is only limited to the level of crude polysaccharide. Chao-Chao Jin et al (Optimization of polysaccharides extracted from Verbena of pharmaceuticals L and the hair inhibition effects on invasion and metabolism of color cancer cells [ J ]. Tropical Journal of Pharmaceutical Research,2017,16(10): 2387-.

The research on verbena polysaccharide in the prior art is limited to the level of crude polysaccharide, and the obtained crude polysaccharide component is not clear, so that the preparation method and the main characteristics of the verbena polysaccharide need to be deeply researched so as to provide a new object for the research and development of natural bioactive substances added in cosmetics and the development of substitute products of medicinal components.

Disclosure of Invention

One of the purposes of the invention is to provide a preparation method of verbena polysaccharide for improving the extraction rate of the verbena polysaccharide, which comprises the following steps:

(1) taking dry verbena powder, adding distilled water according to a liquid-material ratio of 20: 1-100: 1, extracting in a water bath at 65-95 ℃ in a constant-temperature water bath kettle for 120-240 min, and centrifuging to remove precipitates to obtain a crude extract;

(2) concentrating the crude extract obtained in the step (1), adding absolute ethyl alcohol with the volume 2-3 times that of the concentrated solution, standing at 4 ℃ for a certain time, centrifuging at 5000r/min for 5min, and drying the precipitate to obtain a verbena crude extract;

(3) dissolving 500mg of the crude extract of verbena officinalis obtained in the step (2) in 20mL of distilled water, adding protease, keeping the temperature in a water bath at 37 ℃ for 48h, centrifuging at 5000r/min for 5min, and removing precipitates to obtain an enzymatic hydrolysate;

(4) and (3) putting the enzymatic hydrolysate obtained in the step (3) on a DEAE-Sepharose CL-6B chromatographic column, adjusting the flow rate to be 1mL/min, eluting with distilled water for 16-20 h, then performing gradient elution with 0.2-0.4mol/L NaCl solution, setting each automatic part collector to collect 12mL of eluent, combining 20-40 th-tube eluents according to the distribution condition of sugar and protein content, dialyzing the collected liquid with a dialysis bag with the molecular weight cutoff of 3500-5000, dialyzing the flowing water and the distilled water for 24h respectively, concentrating the dialyzate, and freeze-drying to obtain verbena polysaccharide VOP, wherein the average extraction rate of the verbena polysaccharide VOP is more than 8.3%.

Preferably, the dry verbena powder is taken in the step (1), distilled water is added according to the liquid-material ratio of 20:1, the water bath extraction is carried out for 210min at 83 ℃ in a constant temperature water bath kettle, and the centrifugation is carried out for 10min at 5000r/min to remove the precipitate, so as to obtain the crude extract.

Preferably, in the step (2), the crude extract obtained in the step (1) is concentrated by 40 times, and absolute ethyl alcohol of which the volume is 2 times that of the concentrated solution is added.

Preferably, the protease in step (3) comprises 4mg of trypsin and 4mg of proteinase K.

Preferably, in the step (4), the solution is eluted with distilled water for 16h, and the molecular weight cut-off of the dialysis bag is 3500.

Preferably, the verbena polysaccharide obtained in the step (4) is a homogeneous heteropolysaccharide with a weight average molecular weight of 290KDa, and the monosaccharide components are galactose, glucose, mannose, rhamnose and arabinose, wherein the molar ratio of the monosaccharides is 2.8:1.5:1:1: 1.4.

By adopting the technical scheme, the invention has the beneficial effects that:

(1) the response surface method optimizes the extraction process of the verbena total sugar, and particularly the extraction rate of the water-soluble verbena polysaccharide is improved by about 5 times compared with the reported extraction method of the verbena total sugar.

(2) Firstly, carrying out primary separation on verbena polysaccharide by using 2-3 times of volume of absolute ethyl alcohol, secondly, carrying out enzymolysis on crude polysaccharide, hydrolyzing macromolecular protein by using protease, releasing polysaccharide originally intertwined with protein, and purifying an enzymolysis product by using specific column chromatography to obtain the verbena polysaccharide. The method has simple steps and remarkable effect, and the verbena polysaccharide with uniform components can be obtained only by accurately mastering the experimental parameters of each step.

(3) The verbena polysaccharide VOP prepared by the method has definite components, consists of homogeneous heteropolysaccharide, has the weight-average molecular weight of 290KDa, consists of galactose, glucose, mannose, rhamnose and arabinose, and has the molar ratio of 2.8:1.5:1:1: 1.4.

(4) The verbena polysaccharide VOP prepared by the invention can obviously reduce the relative expression quantity (P <0.05) of mRNA (messenger ribonucleic acid) of inflammation related factors TNF-alpha, IL-1 beta, IL-6, COX-2 and iNOS in RAW264.7 cells induced by lipopolysaccharide under the mass concentration of 75 mu g/mL, and has obvious anti-inflammatory effect and no cytotoxicity. Meanwhile, the polysaccharide has an obvious scavenging effect on superoxide radicals and ABTS free radicals, the scavenging effect is equivalent to that of vitamin C, and the antioxidation effect is obvious.

The invention also aims to provide application of the verbena polysaccharide, and the verbena polysaccharide is applied to cosmetics and preparation of anti-inflammatory preparations for medicines.

Drawings

FIG. 1 is a graph showing the relationship between the total sugar extraction rate and the liquid-to-material ratio of verbena officinalis in example 1 of the present invention

FIG. 2 is a graph showing the relationship between the extraction rate of total verbena sugar and the extraction time in example 1 of the present invention

FIG. 3 is a graph showing the relationship between the extraction rate of total verbena sugar and the 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 diagnostic map (a) of example 1 of the present invention; (b) scatter plot of predicted values and actual values

FIG. 6 is the distribution curve of sugar (a) and protein (b) after VO1 is subjected to enzymolysis in example 2 of the invention and eluted by 0.2-0.4M NaCl gradient

FIG. 7 is a high performance liquid chromatogram of VOP of example 2 of the present invention

FIG. 8 is a full wavelength scan of a VOP in accordance with embodiment 2 of the present invention

FIG. 9 is a high performance liquid chromatogram of VO2-P of example 2 of the present invention

FIG. 10 is a graph showing the distribution curves of sugar (a) and protein (b) after elution of the non-enzymolyzed VO1 with a gradient of 0.2-0.4M NaCl in example 2 of the present invention

FIG. 11 is a graph showing the distribution of sugar (a) and protein (b) after elution with a 0-1M NaCl gradient in example 2 of the present invention

FIG. 12 is a full wavelength scan of example 2 of the present invention with a gradient of 0-1M NaCl to elute sugars

FIG. 13 is a gas chromatogram of verbena polysaccharide VOP (1-5 in the figure represent glucose, galactose, mannose, rhamnose and arabinose, respectively) according to example 3 of the present invention

FIG. 14 is a graph showing the inhibition of macrophage growth in RAW264.7 mice by VOP in example 4 of the present invention

FIG. 15 is a graph of the effect of VOP on LPS stimulation of mRNA expression of macrophage inflammatory factor in RAW264.7 mice according to example 4 of the present invention (a) TNF- α; (b) IL-1 beta; (c) IL-6; (d) COX-2 (different letters in the figure represent significant differences, P <0.05)

FIG. 16 shows the effect of VOP on the expression of iNOS gene in LPS-stimulated RAW264.7 mouse macrophage cells in example 4 (the difference between different letters in the figure is significant, P <0.05)

FIG. 17 is the antioxidant activity of VOP of example 5 of the present invention (a) superoxide radical; (b) hydroxyl radical; (c) ABTS

Detailed Description

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

Example 1 extraction Process optimization of Total verbena sugar

The experimental method comprises the following steps: a single-factor method and a response surface experiment method are adopted to optimize the extraction process of the total verbena sugar, and the extraction rate of the total verbena sugar is optimized by researching several factors of liquid-material ratio, extraction time and extraction temperature. The single-factor experiment only considers the 3 rd experiment factor under the condition of fixing two experiment factors, thereby obtaining the optimal experiment level of the factor.

In order to optimize the extraction process of the verbena total sugar, a response curved surface experiment and a Box-Behnken design scheme are adopted, the liquid-material ratio, the extraction time and the extraction temperature are taken as experimental factors, 1g of verbena powder is weighed, a proper amount of distilled water is added, each experiment is carried out, and the extraction rate of the verbena total sugar in the crude extract is detected by adopting a phenol-sulfuric acid method.

The experimental results are as follows:

the influence of the feed-liquid ratio, the extraction time and the extraction temperature on the total sugar extraction rate of verbena is researched, and the results of single-factor experiments are shown in fig. 1, fig. 2 and fig. 3. And selecting 3 experimental levels of each factor according to the single-factor experimental result so as to carry out subsequent response surface experiments.

According to the result of single-factor experiment, the three levels of the material-liquid ratio are determined to be 20:1, 60:1 and 100:1, the three levels of the extraction temperature are 65 ℃, 80 ℃ and 95 ℃, the three levels of the extraction time are 30min, 120min and 210min, and the code of the extraction rate of the total verbena sugar is Y. The response surface test design factors and levels are shown in table 1, and the test design protocol and test results are shown in table 2.

TABLE 1 response surface test design factor levels and encodings

TABLE 2 Box-Behnken test design and results for the extraction of Total sugars from Verbena officinalis

Adopting Design-expert.V11.1.0 software to carry out statistical processing on the test result, and utilizing data fitting to calculate a regression equation model

Y＝7.45-0.4662A+1.68B+0.6100C+0.0125AB-0.4450AC-0.7700BC-2.2153A²-0.6702B²-0.7178C². In order to verify the effect of the model on predicting the extraction scheme, the influence degree of each factor on the extraction rate of the verbena polysaccharide is further determined, the regression equation is subjected to variance analysis, and the result is shown in table 3.

TABLE 3 regression equation analysis of variance and significance results

The proposed model obtained according to the data result is a Quadratic model, and the p value of the model is 0.0224: (<0.05), significant regression model, and mismatching term p of 0.2381 (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 equation²0.8660, the equation fits well, and the extraction rate of verbena polysaccharide can be analyzed and predicted by using the model. From the influence degree of each parameter in the regression equation on the extraction rate of the total sugar in the verbena, the influence of B on the response value (extraction rate) is extremely obvious (p)<0.01)。

Not only the anova results can analyze whether the model is available, but also the diagnostic graph can diagnose whether the predictive model is sufficient. The normal graph of the residual error is shown in fig. 4, and the distribution of the residual error on the diagonal line shows that the residual error follows normal distribution, and the model is reliable. The diagnostic plot is shown in fig. 5, with all data points within the acceptable range (± 4.82), and a linear fit indicates that there is a good correlation between actual and predicted profitability.

By optimizing through a response surface method, software analysis shows that the optimal condition for extracting the total sugar of the verbena is A (liquid-material ratio) 20:1, B (time) 210min, C (temperature) 83 ℃, when the predicted Y value is 8.72829. The average extraction rate of the total sugar of the verbena is actually measured to be 8.37 +/-0.04 percent by performing 3 parallel tests according to the scheme. The extraction process of the total sugar of the verbena is optimized by a response surface method, an extraction rate prediction model is established, and the optimal extraction conditions are obtained.

Example 2 isolation and purification of verbena polysaccharide, comprising the following steps:

(1) drying herba Verbenae, taking 200g of dried herba Verbenae powder, adding 4000mL of distilled water, extracting in water bath at 83 ℃ for 210min in a constant-temperature water bath kettle, centrifuging at 5000r/min for 10min, and removing precipitate to obtain a crude extract;

(2) concentrating the crude extract obtained in the step (1) to 100mL by using a rotary evaporator, adding 200mL of absolute ethyl alcohol, standing overnight in a refrigerator at 4 ℃, centrifuging for 5min at 5000r/min, and drying the precipitate to obtain a verbena crude extract (named as VO 1);

(3) dissolving 500mg of the crude extract of verbena officinalis obtained in the step (2) in 20mL of distilled water, adding 4mg of trypsin and 4mg of proteinase K, preserving heat in a water bath at 37 ℃ for 48 hours, centrifuging at 5000r/min for 5min, and removing precipitates to obtain an enzymolysis liquid;

(4) and (3) putting the enzymatic hydrolysate obtained in the step (3) on a DEAE-Sepharose CL-6B chromatographic column (diameter is 4.6cm, height is 35cm), adjusting the flow rate to be 1mL/min, eluting with distilled water for 16h, not collecting the eluent, then performing gradient elution by using 0.2-0.4mol/L NaCl solution, setting an automatic part collector to collect 12mL of eluent in each tube, combining 20-40 tubes of eluents according to the distribution condition of sugar and protein content, dialyzing the collected liquid by using a dialysis bag with molecular weight cut-off of 3500, dialyzing the collected liquid by using running water and distilled water for 24h respectively, concentrating the dialyzate, and performing freeze drying to obtain verbena polysaccharide VOP.

As a result: and (3) performing gradient elution by using 0.2-0.4mol/L NaCl solution in the step (4), wherein an elution curve is shown in figure 6, a sugar peak can be detected by 20-40 tubes of eluent according to figure 6, a protein peak is also detected at 280nm, the protein distribution condition of 20-40 tubes is matched with the peak type and the tube number of the sugar distribution curve, which indicates that the protein is combined, and 20-40 tubes of eluent are collected to obtain the verbena polysaccharide VOP. FIG. 7 is a high performance liquid chromatogram of VOP showing that the peak pattern is single and symmetrical, indicating that the VOP has good uniformity. FIG. 8 is a graph of the UV absorption spectrum of VOP, and it can be seen that 280nm has an absorption peak, which is a binding protein of VOP, and other impurities are not present.

And (2) carrying out primary separation by using 2 times of volume of absolute ethyl alcohol to obtain a verbena crude extract, wherein the use times of the ethyl alcohol are important. For comparison, the experiment was also isolated using 4 volumes of absolute ethanol and the resulting crude polysaccharide was named VO 2. Performing chromatography on the crude extract VO2 by the same method, collecting main eluted sugar according to a sugar distribution curve, dialyzing, concentrating, and drying to obtain polysaccharide named as VO 2-P. The analysis result of VO2-P by high performance liquid chromatography is shown in FIG. 9, it can be seen that VO2-P has 2 peaks after 5min, which indicates that other components or impurities may be contained, and that the analysis result of VO2-P is different from that of VOP, which indicates that the two components are different. Further, the crude extract obtained by separation with 4 volumes of ethanol was purified by the same method as described later, and the obtained polysaccharide contained other components.

And (3) performing chromatography in the step (4) by using the sugar sample subjected to enzymolysis, and the effect is obvious. For comparison, the results of the subsequent chromatography without using the enzymolysis solution are shown in FIG. 10, and compared with the elution after enzymolysis in FIG. 6, it can be seen that the sugar content distribution curves of the two are unchanged, and only 1 sugar peak is present. However, the protein distribution in FIG. 10 is disordered, the peak pattern is broad, indicating that the protein is abundant, while the protein distribution in FIG. 6 clearly distinguishes more than 2 peaks. The protein and the sugar belong to macromolecules, are difficult to separate, are often polymerized and intertwined together, are subjected to enzymolysis, are degraded, release the originally intertwined polysaccharide, and are eluted after being put on a chromatographic column, the protein with usually more charge content can be easily left behind by the ion exchange function of the chromatographic column, so that the aim of removing protein impurities is simply achieved.

The gradient elution is carried out by using 0.2-0.4mol/L NaCl solution in the step (4), and the using concentration is very important. If the concentration is too high, the ionic strength is high, other impurities can be eluted along with the elution of the verbena polysaccharide, if the concentration is too low, the target polysaccharide can not be eluted, and the NaCl concentration is preferably 0.2-0.4mol/L through experiments. FIG. 11 shows the results after elution with a wider range of 0-1mol/L NaCl solution, and comparison with FIG. 6 shows that the protein distribution is disordered and no effective separation is performed. Further collecting eluent, concentrating, lyophilizing to obtain polysaccharide, scanning the obtained polysaccharide with ultraviolet spectrum, and detecting two absorption peaks at 280nm and 350nm as shown in figure 12, which indicates that the polysaccharide contains protein and carbonyl compounds. Comparing the UV absorption spectrum of VOP eluted with 0.2-0.4mol/L NaCl (FIG. 8), the polysaccharide eluted with 0-1mol/L NaCl solution contains impurities, mainly 350nm substances, such as carbonyl compounds.

Example 3 homogeneity and characterization of Verbena officinalis polysaccharide

The experimental method comprises the following steps: and identifying the uniformity and molecular weight of verbena officinalis polysaccharide VOP by adopting a high performance liquid chromatography. The specific method comprises the following steps: ultrapure water was used as a mobile phase, a supersar ks-804 column chromatography was used, the column temperature was 50 ℃ and the flow rate was 1mL/min, and a differential refractometer was used for detection. And (4) preparing a molecular weight standard curve according to the peak-appearing time of the glucan standard product, and calculating the weight-average molecular mass of the VOP. And taking 0.5mg/mL VOP aqueous solution to perform ultraviolet-visible light full-wavelength scanning (190-800 nm). The main functional groups of the sample were analyzed using infrared spectroscopy (FTIR) to confirm the compound type.

To analyze the monosaccharide composition of VOP, 20-30mg VOP was hydrolyzed with 1mol/L sulfuric acid at 100 ℃ for 8h, the sample was neutralized with barium carbonate, centrifuged, and the supernatant was freeze-dried to obtain a dried hydrolysate. The fully dried VOP hydrolysate was subjected to trimethylsilyl derivatization and the derivatives were subjected to gas chromatography using an hp-5 capillary chromatography column programmed with a temperature rise of 160 ℃→ 180 ℃ (20 ℃/min) → 220 ℃ (8 ℃/min, hold for 2min) → 250 ℃ (2 min).

As a result: the high performance liquid chromatography result of verbena polysaccharide VOP is shown in FIG. 7, and the result shows that only 1 single peak appears in 6.349min, the peak shape is sharp and symmetrical, and the VOP uniformity is good. The VOP aqueous solution is scanned at the full wavelength of 190-800nm (figure 8), and the result shows that the VOP aqueous solution contains no impurities and has high purity. According to the molecular mass standard curve (y ═ 4x +6.31, R²0.99) and the time to peak of the VOP, the weight average molecular weight of the VOP was calculated to be 290 kDa. The IR spectrum of the VOP showed it to be at 3400cm^-1、2900cm^-1、1100cm^-1And 1650cm^-1Characteristic absorption peaks of the saccharides appear. The VOP hydrolysate was derivatized and the gas chromatography results of the derivatized product are shown in fig. 13, referring to the gas chromatography results of standard monosaccharide derivatives, resulting in VOP consisting of galactose, glucose, mannose, rhamnose and arabinose in a molar ratio of 2.8:1.5:1:1: 1.4.

Example 4 anti-inflammatory Effect study of Verbena officinalis polysaccharide VOP

The experimental method comprises the following steps:

culture of RAW264.7 cells: DMEM complete medium (containing 10% serum and 1% diabody), 37 ℃, 5% carbon dioxide incubator, was used for 48h passages.

2. And (3) carrying out cytotoxic test: the cytotoxicity of verbena polysaccharide VOP on RAW264.7 was tested by MTT method, and the experiment was set to five mass concentrations (600, 300, 150, 75 and 25. mu.g/mL).

3. Anti-inflammatory experiments: lipopolysaccharide (LPS) with the concentration of 1 mu g/mL is used for inducing RAW264.7 cells to express various inflammatory factors, and an inflammation model is established. The experiment was set up for a blank group, a negative control group and a positive control group (dexamethasone group, DXMS), a sample group (VOP 75). Firstly, extracting total RNA in each group of cells, obtaining corresponding cDNA after reverse transcription, and measuring CT value by real-time fluorescence quantitative PCR (polymerase chain reaction), 2^-ΔΔCTThe relative expression level of mRNA (with housekeeping gene GAPDH as reference gene) was calculated by the method, and the above-mentioned specific experimental procedures were performed according to the kit instructions.

The experimental results are as follows: the result of the cytotoxicity test is shown in FIG. 14, and the inhibition rate of the addition of 75. mu.g/mL verbena polysaccharide VOP on the growth of RAW264.7 cells is below 20%, which indicates that there is no cytotoxicity basically. The anti-inflammatory experimental results are shown in FIG. 15, compared with the blank group, the relative expression amount of mRNA of negative control group (LPS) of TNF-alpha, IL-1 beta, IL-6 and COX-2 inflammatory factors is remarkably increased (P <0.05), which indicates that four inflammation models are successfully established. Compared with a negative control group, the verbena polysaccharide can obviously reduce the mRNA relative expression level (P is less than 0.05) of four inflammatory factors of TNF-alpha, IL-1 beta, IL-6 and COX-2, and shows that the verbena polysaccharide VOP has a better anti-inflammatory effect.

As shown in fig. 16, the addition of verbena polysaccharide VOP at 75 μ g/mL significantly reduced the relative mRNA expression level of iNOS gene in RAW264.7 cells induced by lipopolysaccharide (P <0.05), compared to the negative control and the positive control, and the effect was not significantly different from the positive control (P > 0.05). The over-expression of iNOS gene can stimulate the body to produce excessive nitric oxide, thereby damaging neurons and causing cell damage.

Example 5 Studies on antioxidant Effect of Verbena officinalis polysaccharide VOP

Research of antioxidant experiment: the sample is prepared into water solution with the concentration of 30 mg/mL-0.0075 mg/mL.

1. Determination of superoxide anion scavenging ability: referring to Robak, 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, incubated at 25 ℃ for 5min, and absorbance was measured at 560nm, and the inhibition was calculated as the clearance (1-sample tube/control tube). times.100%.

2. Measurement of hydroxyl radical scavenging action: referring to the method of Ghiselli et al and modifications, 0.6mL of 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%.

And 3, measuring the ABTS free radicals, namely taking 100ul of sample liquid, adding 100ul of ABTS working solution, reacting for 10min at room temperature in the dark, measuring OD at 734nm, and calculating the inhibition rate according to the clearance rate (1-sample tube/control tube) multiplied by 100%.

The antioxidant effect of verbena polysaccharide VOP is shown in figure 17, vitamin c (Vc) is used as a positive control, and the scavenging effect of VOP and Vc on superoxide radical, hydroxyl radical and ABTS free radical is increased along with the increase of concentration, and both show dose-effect relationship. The verbena polysaccharide VOP has obvious effect of eliminating superoxide radical and ABTS free radical, and EC is calculated₅₀Is 287 mug/mL and 390 mug/mL respectively, and the cleaning effect is equivalent to that of the vitamin C.

In conclusion, the addition of 75 mug/mL verbena polysaccharide VOP can obviously reduce the relative expression quantity (P <0.05) of mRNA of inflammation related factors TNF-alpha, IL-1 beta, IL-6, COX-2 and iNOS in RAW264.7 cells induced by lipopolysaccharide, and has obvious anti-inflammatory effect and no cytotoxicity. The vervain VOP has the effect of eliminating superoxide radical and ABTS free radical equivalent to Vc, and has obvious antioxidant effect.

The excessive expression of the inflammatory factors can cause various diseases, such as central nervous system diseases, atherosclerosis, chronic obstructive pulmonary diseases, rheumatoid arthritis and the like, so the development of natural nontoxic anti-inflammatory preparations is listed as one of the main research and development directions by pharmaceutical enterprises.

The human body can generate free radicals continuously due to contact with the outside, including factors such as respiration, radioactive rays and air pollution. Scientific research shows that the excessive free radicals are closely related to aging, cardiovascular diseases, arthritis, cancer and the like, and anti-inflammation and anti-oxidation supplement each other, and because the free radicals are one of the 'fierce' which cause skin inflammation, many cosmetics in the market contain anti-inflammation and anti-oxidation components, and the search for natural non-toxic anti-inflammation and anti-oxidation 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.