CN113603802B - Preparation method and application of verbena polysaccharide - Google Patents

Abstract

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, and the method steps are simple and effective. The verbena polysaccharide prepared by the method has definite components, is homogeneous heteropolysaccharide, has a weight-average molecular weight of 290KDa, consists of galactose, glucose, mannose, rhamnose and arabinose, and has a monosaccharide molar ratio of 2.8. 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.

Description

A kind of preparation method and application of verbena polysaccharide

technical field

The invention belongs to the technical field of medicine, and in particular relates to a preparation method and application of verbena polysaccharide.

Background technique

Verbena Officinalis is the whole plant of the Verbena family and the genus Verbena. It has a wide distribution range in my country and is rich in resources. Verbena, a traditional Chinese medicine, is bitter in taste and has the functions of promoting blood circulation and removing blood stasis, detoxification, diuresis and swelling.

The research on Verbena at home and abroad mainly focuses on its alcohol solubility and the chemical composition of the crude extract of decoction. Chao-Chao Jin et al. (Optimization of polysaccharides extracted from Verbena officinalis L and their inhibitory effects on invasion and metastasis of colorectal cancer cells[J].TropicalJournal of Pharmaceutical Research,2017,16(10):2387- 2394) optimized the crude polysaccharide of Verbena The optimal extraction conditions are: liquid-solid ratio of 27.5:1 (mL/g), extraction time of 92 min, and reflux extraction twice. Under these conditions, the actual extraction rate of polysaccharides is 1.48±0.06%. At the same time, the obtained crude polysaccharides have Inhibition of colon cancer cell metastasis.

The research of the above-mentioned prior art to the verbena polysaccharide is limited to the crude polysaccharide level, and the obtained crude polysaccharide composition is not clear, so it is necessary to carry out in-depth research on the preparation method of the verbena polysaccharide and its main characteristics, with a view to the natural organisms added to cosmetics. The development of active substances, as well as the development of alternative products for pharmaceutical ingredients provides new targets.

SUMMARY OF THE INVENTION

One of the objects of the present invention is to provide a kind of preparation method of verbena polysaccharide improving the extraction rate of verbena polysaccharide, comprising the steps:

(1) take the dried verbena powder, add distilled water according to the liquid-material ratio of 20:1～100:1, extract in a constant temperature water bath at 65～95 ℃ for 120～240min, and centrifuge to remove the precipitation to obtain a crude extract;

(2) Concentrating the crude extract obtained in step (1), adding absolute ethanol with 2 to 3 times the volume of the concentrated solution, placing at 4°C for a certain period of time, centrifuging at 5000 r/min for 5 min, taking the precipitate and drying to obtain the crude extract of Verbena ;

(3) Dissolving 500 mg of the crude verbena extract obtained in step (2) in 20 mL of distilled water, adding protease, keeping at 37° C. water bath for 48 h, centrifuging at 5000 r/min for 5 min, removing the precipitation, and obtaining an enzymatic hydrolysis solution;

(4) Take the enzymatic hydrolysis solution obtained in step (3) and place it on a DEAE-Sepharose CL-6B chromatography column, adjust the flow rate to 1 mL/min, elute with distilled water for 16-20 h, and then use 0.2-0.4 mol/L NaCl solution for gradient washing The automatic partial collector is set to collect 12 mL of eluate in each tube. According to the distribution of sugar and protein content, the 20th to 40th tubes are combined, and the collected solution is dialyzed with a dialysis bag with a molecular weight cut-off of 3500 to 5000. Dialyzed with distilled water for 24 hours, the dialysate was concentrated and then freeze-dried to obtain verbena polysaccharide VOP. The average extraction rate of verbena polysaccharide VOP was over 8.3%.

In a preferred manner, in step (1), take the dried verbena powder, add distilled water according to the liquid-material ratio of 20:1, extract in a constant temperature water bath at 83° C. for 210 min, and centrifuge at 5000 r/min for 10 min to remove the precipitation to obtain a crude extract.

In a preferred manner, in the step (2), the crude extract obtained in the step (1) is concentrated by 40 times, and anhydrous ethanol with 2 times the volume of the concentrated solution is added.

In a preferred manner, the protease in the step (3) includes 4 mg of trypsin and 4 mg of proteinase K.

In a preferred manner, in the step (4), distilled water is used for elution for 16 hours, and the molecular weight cut-off of the dialysis bag is 3500.

In a preferred manner, the verbena polysaccharide obtained in the step (4) is a homogeneous heteropolysaccharide, the weight average molecular weight is 290KDa, the monosaccharide composition is galactose, glucose, mannose, rhamnose and arabinose, and the molar ratio of each monosaccharide is galactose, glucose, mannose, rhamnose and arabinose. is 2.8:1.5:1:1:1.4.

Adopting the above-mentioned technical scheme, the beneficial effects of the present invention are:

(1) The response surface method used in the present invention optimizes the extraction process of verbena total sugar, especially the water-soluble verbena polysaccharide. Compared with the reported extraction method of verbena total sugar, the extraction rate is increased by nearly 5 times.

(2) The present invention firstly uses 2 to 3 times the volume of absolute ethanol to preliminarily separate the verbena polysaccharide, and secondly, the crude polysaccharide is enzymatically hydrolyzed. The protease can hydrolyze the macromolecular protein and can release the polysaccharide originally entangled with the protein. The hydrolyzed product can be purified by specific column chromatography to obtain the verbena polysaccharide. The method has simple steps and remarkable effect, and only needs to accurately grasp the experimental parameters of each step, and then the verbena polysaccharide with uniform composition can be obtained.

(3) The Verbena polysaccharide VOP prepared by the present invention has clear components, is composed of homogeneous heteropolysaccharides, has a weight-average molecular weight of 290KDa, and is composed of galactose, glucose, mannose, rhamnose and arabinose, and its molar composition is 290KDa. The ratio is 2.8:1.5:1:1:1.4.

(4) The verbena polysaccharide VOP prepared by the present invention can significantly reduce the inflammation-related factors TNF-α, IL-1β, IL-6, COX-2 and lipopolysaccharide-induced RAW264.7 cells at a mass concentration of 75 μg/mL. The relative mRNA expression of iNOS (P<0.05) showed significant anti-inflammatory effect and no cytotoxicity. At the same time, the polysaccharide has a significant scavenging effect on superoxide free radicals and ABTS free radicals, the scavenging effect is equivalent to vitamin C, and the antioxidant effect is remarkable.

Another object of the present invention is to provide an application of the verbena polysaccharide, the verbena polysaccharide being applied to cosmetics, and the preparation of medicinal anti-inflammatory preparations.

Description of drawings

Fig. 1 is the relationship curve of the total sugar extraction rate of Verbena and the ratio of liquid to material in Example 1 of the present invention

Fig. 2 is the relation curve of extraction rate and extraction time of total sugar of Verbena in Example 1 of the present invention

Fig. 3 is the relation curve of extraction rate and extraction temperature of total sugar of Verbena in Example 1 of the present invention

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

Fig. 5 is a model diagnosis diagram of the embodiment of the present invention 1. (a): residual diagram; (b): scatter plot of predicted value and actual value

Figure 6 is the distribution curve of sugar (a) and protein (b) after VO1 after enzymatic hydrolysis in Example 2 of the present invention is eluted with a gradient of 0.2-0.4M NaCl

Fig. 7 is the high performance liquid chromatogram of the embodiment of the present invention 2VOP

FIG. 8 is a full-wavelength scanning diagram of VOP in Embodiment 2 of the present invention

Fig. 9 is the high performance liquid chromatogram of the embodiment of the present invention 2VO2-P

Figure 10 shows the distribution curves of sugar (a) and protein (b) after the unenzymatic hydrolyzed VO1 in Example 2 of the present invention was eluted with a gradient of 0.2-0.4M NaCl

Figure 11 is the distribution curve of sugar (a) and protein (b) after 0-1M NaCl gradient elution in Example 2 of the present invention

Figure 12 is a full-wavelength scanning diagram of the 0-1M NaCl gradient elution of sugar in Example 2 of the present invention

Fig. 13 is the gas chromatogram of Verbena polysaccharide VOP in Example 3 of the present invention. (1-5 in the figure represent glucose, galactose, mannose, rhamnose and arabinose respectively)

Figure 14 shows the inhibitory effect of VOP on the growth of RAW264.7 mouse macrophages in Example 4 of the present invention

Figure 15 shows the effect of VOP in Example 4 of the present invention on the mRNA expression of inflammatory factors in RAW264.7 mouse macrophages stimulated by LPS. (a): TNF-α; (b): IL-1β; (c): IL-6; (d): COX-2 (different letters in the figure represent significant differences, P<0.05)

Figure 16 shows the effect of VOP in Example 4 of the present invention on LPS-stimulated iNOS gene expression in RAW264.7 mouse macrophages. (Different letters in the figure represent significant differences, P<0.05)

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

Detailed ways

The technical solutions of the present invention will be further illustrated and described below through specific embodiments.

Embodiment 1 The extraction process optimization of the total sugar of Verbena

Experimental method: The single factor method and response surface experiment method were used to optimize the extraction process of total sugar in Verbena, and the extraction rate of total sugar in Verbena was optimized by studying several factors such as the ratio of liquid to material, extraction time and extraction temperature. The single factor experiment is to examine only the third experimental factor under the condition of fixing two experimental factors, so that the optimal experimental level of this factor can be obtained.

In order to optimize the extraction process of total sugar of verbena, response surface experiment and Box-Behnken design scheme were used, taking the ratio of liquid to material, extraction time and extraction temperature as experimental factors, weighing 1 g of verbena powder and adding an appropriate amount of distilled water to carry out each experiment, The phenol-sulfuric acid method was used to detect the extraction rate of total sugar of Verbena in the crude extract.

Experimental results:

The effects of solid-liquid ratio, extraction time and extraction temperature on the extraction rate of total sugar in Verbena were studied. The results of single factor experiment are shown in Figure 1, Figure 2 and Figure 3. According to the results of the single factor experiment, 3 experimental levels of each factor were selected for subsequent response surface experiments.

According to the results of the single factor experiment, the three levels of the solid-liquid ratio are determined as 20:1, 60:1, and 100:1, with 65°C, 80°C, and 95°C as the three levels of extraction temperature and the three levels of extraction time. For 30min, 120min, 210min, the code of total sugar extraction rate of verbena is set as Y. The factors and levels of the response surface test design are shown in Table 1, and the experimental design scheme and test results are shown in Table 2.

Table 1 Response surface test design factor levels and codes

Table 2 Box-Behnken experimental design and results of total sugar extraction rate of verbena

The Design-Expert.V11.1.0 software was used to statistically process the test results, and the regression equation model was calculated by data fitting as

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 the prediction of the extraction scheme, and to further determine the degree of influence of various factors on the extraction rate of verbena polysaccharide, the regression equation was analyzed by variance, and the results are shown in Table 3.

Table 3 Regression equation analysis of variance and significant results

According to the data results, the proposed model is Quadratic model, the p value of this model is 0.0224 (<0.05), the regression model is significant, the lack of fit item p is 0.2381 (>0.05), and the lack of fit item is not significant, indicating that the model has no effect on the test results. Good fit. The R ² of the equation was 0.8660, and the fitting degree of the equation was good. This model could be used to analyze and predict the extraction rate of verbena polysaccharides. Judging from the influence degree of each parameter in the regression equation on the extraction rate of total sugar of Verbena, the influence of B on the response value (extraction rate) was extremely significant (p<0.01).

Not only can the ANOVA results analyze whether the model is available, but the diagnostic plot can also diagnose the adequacy of the predictive model. The normal map of the residuals is shown in Figure 4. The residuals are distributed on the diagonal line, indicating that the residuals obey the normal distribution and the model is reliable. The diagnostic plot is shown in Figure 5, all data points are within the acceptable range (±4.82), and the linear fit plot shows a good correlation between actual and predicted returns.

Through the optimization of the response surface method, the software analysis gives the optimal conditions for extracting the total sugar of Verbena: A (liquid to material ratio) = 20:1, B (time) = 210min, C (temperature) = 83 °C, and the predicted Y value = 8.72829. Three parallel experiments were carried out according to this scheme, and the actual average extraction rate of total sugar of verbena was 8.37±0.04%. The extraction process of verbena total sugar was optimized by response surface method, the extraction rate prediction model was established, and the optimal extraction conditions were obtained.

The separation and purification of embodiment 2 Verbena polysaccharide, comprises the steps:

(1) Sun-drying Verbena, taking 200g of dried Verbena powder, adding 4000mL of distilled water, extracting in a constant temperature water bath at 83°C for 210min, centrifuging at 5000r/min for 10min, removing the precipitation, and obtaining a crude extract;

(2) the crude extract obtained in step (1) was concentrated to 100 mL using a rotary evaporator, 200 mL of absolute ethanol was added, placed in a refrigerator at 4 °C overnight, centrifuged at 5000 r/min for 5 min, and the crude extract of Verbena was obtained after the precipitate was dried ( named VO1);

(3) 500 mg of the crude verbena extract obtained in step (2) was dissolved in 20 mL of distilled water, 4 mg of trypsin and 4 mg of proteinase K were added, the temperature was kept in a water bath at 37° C. for 48 h, and centrifuged at 5000 r/min for 5 min to remove the precipitate to obtain an enzymatic hydrolyzate;

(4) Take the enzymolysis solution obtained in step (3) and place it on a DEAE-Sepharose CL-6B chromatographic column (4.6 cm in diameter, 35 cm in height), adjust the flow rate to 1 mL/min, elute with distilled water for 16 h, and do not collect the eluent, then Use 0.2-0.4mol/L NaCl solution for gradient elution, set the automatic partial collector to collect 12 mL of eluate in each tube, and combine the 20-40 tubes according to the distribution of sugar and protein content, and use a molecular weight cut-off of 3500. The collected liquid was dialyzed with a dialysis bag, and dialyzed with running water and distilled water for 24 hours each. The dialysate was concentrated and then freeze-dried to obtain the verbena polysaccharide VOP.

Results: In step (4), 0.2-0.4mol/L NaCl solution was used for gradient elution, and the elution curve was shown in Figure 6. According to Figure 6, it can be seen that 20-40 tubes of eluent detected a sugar peak, and at the same time Protein peaks were also detected at 280 nm, among which 20-40 tubes of protein distribution were consistent with the peak shape of the sugar distribution curve and the number of tubes, indicating that it was bound protein, and 20-40 tubes of eluate were collected to obtain the verbena polysaccharide VOP of the present invention. Figure 7 is the high performance liquid chromatogram of VOP, it can be seen that the peak shape is single and symmetrical, indicating that the uniformity of VOP is good. Figure 8 is the UV absorption spectrum of VOP, it can be seen that there is an absorption peak at 280 nm, which is the binding protein of VOP, and there are no other impurities.

Step (2) uses 2 times the volume of anhydrous ethanol for preliminary separation to obtain the crude extract of Verbena, wherein the use multiple of ethanol is more important. For comparison, the experiment also used 4 times the volume of absolute ethanol for separation, and the obtained crude polysaccharide was named VO2. The crude extract VO2 was chromatographed by the same method, and the eluted main sugars were collected according to the sugar distribution curve, dialyzed, concentrated and dried to obtain a polysaccharide named VO2-P. The results of VO2-P HPLC analysis are shown in Figure 9. It can be seen that VO2-P has two peaks after 5 minutes, indicating that it may contain other components or impurities. The HPLC analysis results of VO2-P and VOP are different , indicating that the two components contain different components. It is further explained that the obtained polysaccharide contains other components after the crude extract separated by 4 times the volume of ethanol is purified by the same method.

In step (3), the enzymatically hydrolyzed sugar sample is used to perform the chromatography in step (4), and the effect is remarkable. For comparison, the results of subsequent chromatography without using the enzymatic hydrolysis solution are shown in Figure 10. Compared with Figure 6 of the elution after enzymatic hydrolysis, it can be seen that the sugar content distribution curve of the two has not changed, and there is only one sugar peak. However, the protein distribution in Figure 10 is chaotic and the peak shape is broad, indicating that there are many proteins, while the protein distribution in Figure 6 clearly distinguishes more than two peaks. Proteins and sugars are macromolecules and are difficult to separate. They are often polymerized and entangled together. The sugar samples are enzymatically hydrolyzed, the protein is degraded, and the originally entangled polysaccharides are released. After being applied to the chromatography column, the ion exchange of the chromatography column The function can easily leave the protein with more charge content behind to elute out, so as to simply achieve the purpose of removing protein impurities.

The 0.2-0.4 mol/L NaCl solution used in step (4) is subjected to gradient elution, and the concentration used is very important. If the concentration is too large, the ionic strength will be high. With the elution of verbena polysaccharide, other impurities will be eluted. If the concentration is too small, the target polysaccharide will not be eluted. After experiments, it is found that the NaCl concentration is 0.2-0.4mol/L. . Figure 11 shows the results after elution with a wider range of 0-1 mol/L NaCl solution. Compared with Figure 6, it can be seen that the protein distribution is chaotic and effective separation is not carried out. The eluate was further collected, concentrated and lyophilized to obtain polysaccharide, and the obtained polysaccharide was subjected to ultraviolet spectrum scanning. The results are shown in Figure 12. Two absorption peaks were detected at 280 nm and 350 nm, indicating that it contained proteins and carbonyl compounds. Compared with the UV absorption spectrum of VOP obtained by elution with 0.2-0.4 mol/L NaCl (Fig. 8), the polysaccharide obtained by elution with 0-1 mol/L NaCl solution contains impurities, mainly substances at 350 nm, such as carbonyl compounds.

Example 3 Homogeneity and characteristic analysis of Verbena polysaccharide

Experimental method: The homogeneity and molecular weight of Verbena polysaccharide VOP were identified by high performance liquid chromatography. The specific method is: using ultrapure water as the mobile phase, sugar ks-804 chromatographic column, the column temperature is 50 ° C, the flow rate is 1 mL/min, and the differential refraction detector is used for detection. According to the peak time of the dextran standard, a molecular weight standard curve was prepared, and the weight-average molecular mass of VOP was calculated. Take 0.5 mg/mL VOP aqueous solution for UV-Vis full-wavelength scanning (190-800 nm). The main functional groups of the samples were analyzed by infrared spectroscopy (FTIR) to confirm the compound type.

To analyze the monosaccharide composition of VOP, 20-30 mg of VOP was hydrolyzed with 1 mol/L sulfuric acid at 100 °C for 8 h, the sample was neutralized with barium carbonate, and the supernatant was centrifuged and lyophilized to obtain a dry hydrolyzate. The thoroughly dried VOP hydrolyzate was derivatized with trimethylsilyl ether, and the derivative was analyzed by gas chromatography using a hp-5 capillary column with a programmed temperature of 160 °C → 180 °C (20 °C/min) → 220 °C (8 °C /min, keep for 2min)→250℃(2min).

Results: The high performance liquid chromatographic results of Verbena polysaccharide VOP are shown in Figure 7. The results show that VOP only has a single peak at 6.349min, and the peak shape is sharp and symmetrical, indicating that the VOP has good uniformity. The VOP aqueous solution was scanned at the full wavelength of 190-800 nm (Fig. 8), and the results showed that it did not contain impurities and had high purity. According to the molecular mass standard curve (y=-4x+6.31, R ² =0.99) and the peak time of VOP, the weight-average molecular weight of VOP was calculated to be 290 kDa. The infrared spectrum of VOP showed that characteristic absorption peaks of sugars appeared at 3400cm ^-1 , 2900cm ^-1 , 1100cm ^-1 and 1650cm ^-1 . The VOP hydrolyzate was derivatized, and the gas chromatography results of the derivatized products are shown in Figure 13. Referring to the gas chromatography results of standard monosaccharide derivatives, it was concluded that VOP was composed of galactose, glucose, mannose, rhamnose and arabinose, Its molar ratio is 2.8:1.5:1:1:1.4.

Example 4 Study on the anti-inflammatory effect of Verbena polysaccharide VOP

experimental method:

1. Cultivation of RAW264.7 cells: use DMEM complete medium (containing 10% serum and 1% double antibody), 37°C, 5% carbon dioxide incubator, passage once 48h.

2. Cytotoxicity test: MTT method was used to detect the cytotoxicity of Verbena polysaccharide VOP to RAW264.7. Five mass concentrations (600, 300, 150, 75 and 25 μg/mL) were set in the experiment.

3. Anti-inflammatory experiment: 1μg/mL lipopolysaccharide (LPS) was used to induce RAW264.7 cells to express various inflammatory factors to establish an inflammation model. The experiment set blank group, negative control group, positive control group (dexamethasone group, DXMS), sample group (VOP75). First, the total RNA in each group was extracted, and the corresponding cDNA was obtained after reverse transcription. The CT value was determined by real-time fluorescence quantitative PCR, and the relative mRNA expression was calculated by the 2 ^-ΔΔCT method (with the housekeeping gene GAPDH as the internal reference gene). The above specific experimental steps were all Follow the kit instructions.

Experimental results: The results of the cytotoxicity experiment are shown in Figure 14. The growth inhibition rate of RAW264.7 cells by adding 75 μg/mL of Verbena polysaccharide VOP is below 20%, indicating that there is basically no cytotoxicity. The results of the anti-inflammatory experiment are shown in Figure 15. Compared with the blank group, the relative expressions of TNF-α, IL-1β, IL-6 and COX-2 in the negative control group (LPS) mRNA were significantly increased ( P<0.05), indicating that four inflammatory models have been successfully established. When adding 1 μg/mL lipopolysaccharide and 75 μg/mL mass concentration of verbena polysaccharide to RAW264.7 cells, compared with the negative control group, verbena polysaccharide can significantly reduce TNF-α, IL-1β, IL-6 and The relative mRNA expression levels of the four inflammatory factors of COX-2 (P<0.05) indicated that the verbena polysaccharide VOP had a better anti-inflammatory effect.

As shown in Figure 16, compared with the negative control group and the positive control group, the addition of 75 μg/mL verbena polysaccharide VOP can significantly reduce the relative mRNA expression of iNOS gene in LPS-induced RAW264.7 cells (P<0.05). And the effect was not significantly different from the positive control group (P>0.05). The overexpression of iNOS gene can stimulate the body to produce excessive nitric oxide, thereby damaging neurons and causing cell damage.

Example 5 Study on the Antioxidative Effect of Verbena Polysaccharide VOP

Antioxidant experimental research: The sample was prepared into an aqueous solution with a concentration of 30 mg/mL to 0.0075 mg/mL.

1. Determination of superoxide anion scavenging ability: according to Robak's method, take 0.1 mL of sample solution and add 1 mL of 16 mM Tris-HCl (pH 8.0) containing 557 uM NADH, 1 mL of 16 mM Tris-HCl (pH 8.0) containing 45 uM PMS, And 1mL of 16mM Tris-HCl (pH 8.0) containing 108uM NBT, incubate at 25℃ for 5min, take out and measure the absorbance at 560nm, and calculate the inhibition rate according to the clearance rate=(1-sample tube/control tube)×100%.

2. Determination of hydroxyl radical scavenging: refer to the method of Ghiselli et al. and improve it, take 0.1 mL of sample solution and add 0.6 mL of reaction buffer (containing 2.67 mM deoxyribose and 0.13 mM EDTA), 0.4 mM ferrous sulfate 0.2 mL, 0.05 mL of 2.0 mM ascorbic acid and 0.05 mL of 20 mM H ₂ O ₂ , incubate at 37°C for 15 min, take out, add 1 mL of 1% thiobarbituric acid and 1 mL of 2% trichloroacetic acid, bath in boiling water for 15 min, and place on ice immediately After cooling, the absorbance was measured at 532 nm, and the inhibition rate was calculated according to the clearance rate=(1-sample tube/control tube)×100%.

3. Determination of ABTS free radicals, take 100ul sample solution, add 100ul ABTS working solution, react in the dark at room temperature for 10min, measure OD at 734nm, and calculate the inhibition rate according to scavenging rate=(1-sample tube/control tube)×100%.

The antioxidant results of verbena polysaccharide VOP are shown in Figure 17. Taking vitamin c (Vc) as a positive control, the scavenging effects of VOP and Vc on superoxide radicals, hydroxyl radicals and ABTS radicals increased with the increase of the concentration. Both showed a dose-response relationship. Verbena polysaccharide VOP has a significant scavenging effect on superoxide free radicals and ABTS free radicals. The calculated EC ₅₀ is 287μg/mL and 390μg/mL, respectively, and the scavenging effect is comparable to that of vitamin C.

In conclusion, the addition of 75 μg/mL verbena polysaccharide VOP can significantly reduce the relative mRNA expression of inflammatory-related factors TNF-α, IL-1β, IL-6, COX-2 and iNOS in LPS-induced RAW264.7 cells. (P<0.05), the anti-inflammatory effect was significant, and there was no cytotoxicity. The scavenging effect of verbena polysaccharide VOP on superoxide free radicals and ABTS free radicals is equivalent to Vc, and the antioxidant effect is significant.

Excessive expression of inflammatory factors can cause a variety of diseases, such as central nervous system diseases, atherosclerosis, chronic obstructive pulmonary disease, rheumatoid arthritis, etc. Therefore, the development of natural non-toxic anti-inflammatory preparations is listed as a pharmaceutical company. One of the main research and development directions.

The human body will continuously generate free radicals due to contact with the outside world, including breathing, radiation, air pollution and other factors. Scientific research shows that excessive free radicals are closely related to aging, cardiovascular disease, arthritis, cancer, etc. Anti-inflammatory and antioxidant complement each other, because free radicals are also one of the "culprits" that cause skin inflammation, so there are many on the market. Cosmetics all contain anti-inflammatory and antioxidant ingredients, and finding natural and non-toxic anti-inflammatory and antioxidant products is also one of the most important functional demands on the market.

The above are only the preferred embodiments of the present invention, so the scope of implementation of the present invention cannot be limited accordingly, that is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the description should still be covered by the present invention. In the range.

Claims (3)

1. The application of verbena polysaccharide is characterized in that: the verbena polysaccharide is applied to cosmetics and preparation of anti-inflammatory preparations for medicine, and in the application, the verbena polysaccharide reduces the relative expression quantity of mRNA of inflammatory factors TNF-alpha and iNOS;

the preparation method of the verbena polysaccharide comprises the following steps:

(1) Taking dry verbena powder, adding distilled water according to a liquid-material ratio of 20;

(2) Concentrating the crude extract obtained in the step (1) by 40 times, adding absolute ethyl alcohol with the volume 2 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, carrying out water bath at 37 ℃ for 48h, carrying out centrifugation at 5000r/min for 5min, and removing precipitates to obtain an enzymatic hydrolysate;

(4) Taking the enzymatic hydrolysate obtained in the step (3) to pass through a DEAE-Sepharose CL-6B chromatographic column, adjusting the flow rate to be 1mL/min, eluting with distilled water for 16-20h, then performing gradient elution by using 0.2-0.4mol/L NaCl solution, setting each automatic partial collector to collect 12mL of eluent, combining 20-40 pipes of eluent according to the distribution condition of sugar and protein content, dialyzing the collected liquid by using dialysis bags with molecular weight cut-off 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%;

the verbena polysaccharide obtained in the step (4) is a homogeneous heteropolysaccharide, the weight average molecular weight is 290KDa, the monosaccharide components are galactose, glucose, mannose, rhamnose and arabinose, and the molar ratio of each monosaccharide is 2.8.

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

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

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