Semen cuscutae polysaccharide with moisturizing and anti-inflammatory effects as well as preparation method and application of semen cuscutae polysaccharide

Abstract

The invention provides semen cuscutae polysaccharide with moisturizing and anti-inflammatory effects and a preparation method and application thereof.The method is based on a high-speed shearing wall-breaking extraction technology, semen cuscutae dry powder is separated and purified, and the method mainly comprises the steps that a semen cuscutae dry powder mixed solution is subjected to flash-type auxiliary hot water stirring extraction treatment, and a semen cuscutae polysaccharide extracting solution is obtained; and then decoloring, deproteinizing and concentrating the extracting solution to obtain a crude product of the semen cuscutae polysaccharide, and finally obtaining a pure product of semen cuscutae polysaccharide crystal through alcohol precipitation and column chromatography methods. Compared with a single hot water extraction method and an ultrasonic extraction method, the method has the advantages of being high in extraction rate, easy to operate and the like, the optimal extraction method is obtained through a single factor and orthogonal design experiment, the obtained crude polysaccharide is separated and purified, the pure semen cuscutae polysaccharide is prepared, and the method has important significance on maintaining the original chemical structure and biological activity of the polysaccharide. And the semen cuscutae polysaccharide obtained by the invention has good moisturizing and / or anti-inflammatory functions.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction, and particularly relates to a dodder polysaccharide with moisturizing and anti-inflammatory effects, and a preparation method and application thereof. Background Art

[0002] Cuscuta chinensis is a parasitic plant. It obtains nutrients by wrapping around other plants and mainly parasitizes plants in the leguminous family. Cuscuta chinensis is used as a medicinal material in traditional Chinese medicine. It has the effects of nourishing the liver and kidneys, improving vision, and is often used to treat blurred vision, sore waist and knees, etc.

[0003] Cuscuta polysaccharide is a bioactive substance extracted from Cuscuta seeds. Studies have shown that it has multiple biological activities such as immunomodulation, anti-oxidation, and anti-tumor. In recent years, scientists have gradually deepened their research on Cuscuta polysaccharide and found that it has good effects in promoting cell proliferation and enhancing immune function. In addition, Cuscuta polysaccharide is also believed to have certain health value and may play a certain auxiliary therapeutic effect on diseases such as diabetes and tumors. Overall, Cuscuta seeds and their polysaccharide extracts have shown certain application potential in both traditional herbal medicine and modern medicine, and are worthy of further research and development.

[0004] At present, the extraction methods of dodder polysaccharides reported in the literature are mainly based on the traditional water extraction and alcohol precipitation method. However, this method has some obvious defects, such as high extraction temperature, long time required and low extraction rate. These factors may also destroy the structure of polysaccharides, resulting in changes in their biological activity. With the continuous advancement of technology, many advanced extraction technologies have been applied to the extraction of natural polysaccharides, such as ultrasound-assisted extraction, acid-base continuous extraction and enzyme-assisted hot water extraction. For example, patent CN116143953B discloses a kind of kelp polysaccharide and its preparation method and application, which includes mannose, rhamnose, glucuronic acid, glucose, xylose, fructose and galactose; the molecular weight is 5.98-8.41KDa. The patent uses ultrasound-assisted extraction combined with single factor and response surface experiments to obtain the optimal extraction scheme of kelp polysaccharides and their components, thereby improving the extraction efficiency of kelp polysaccharides. Patent CN101828750A provides a method of extracting and preparing a pure, safe and efficient plant natural preservative using bamboo leaves as raw materials, integrating ultrasonic assisted extraction technology, membrane separation technology, composite solvent continuous countercurrent extraction technology, and vacuum concentration technology. The preservative contains flavonoid compounds, phenolic acid compounds, bioactive polysaccharides and other ingredients, and can be widely used in fruits and vegetables, food, medicine, feed and cosmetics. Patent CN116693718B discloses a high-purity naked oat β-glucan and its preparation method and application. The high-purity naked oat β-glucan is extracted by enzyme-assisted water extraction and alcohol precipitation. The extract has the function of maintaining water stability in cereal products and improving water holding capacity. The high-purity naked oat β-glucan extraction provided by the patent is obtained by pretreatment, hot water extraction, enzyme-assisted removal of starch and protein, freeze drying to obtain crude polysaccharides, and crude polysaccharide purification. It can be used in bread, biscuits, and meal replacement products to achieve its high-value application.

[0005] The above patents involve ultrasonic assisted extraction, acid-base continuous extraction and enzyme-assisted hot water extraction, etc., which have the disadvantages of low extraction efficiency, high energy consumption per unit time, and ultrasonic extraction technology is restricted by ultrasonic attenuation factors. The effective ultrasonic action area is annular. When the diameter of the extraction container is large, an ultrasonic blank area will be formed on the wall of the container; the acid-base continuous extraction method may cause changes in the structure of the target polysaccharide itself and affect its subsequent application; enzyme-assisted extraction is affected by the activity of the enzyme itself, and the enzyme is easily affected by the external environment and loses its catalytic activity during enzymatic extraction. Therefore, we urgently need to develop more efficient and environmentally friendly extraction methods to improve the extraction rate of polysaccharides. Summary of the invention

[0006] Based on the shortcomings of the above-mentioned prior art, the purpose of the present application is to provide a dodder polysaccharide with moisturizing and anti-inflammatory effects, and a preparation method and application thereof. The method is based on a high-speed shear wall-breaking extraction process, and the obtained dodder polysaccharide extract is separated and purified. The obtained dodder polysaccharide exhibits good moisturizing and / or anti-inflammatory effects.

[0007] One of the technical solutions of the present invention provides a method for preparing dodder polysaccharide, wherein the dodder polysaccharide is extracted from dodder seeds and separated and purified; the method mainly comprises the following steps:

[0008] 1) drying the dodder seeds, crushing and sieving to obtain dodder dry powder;

[0009] 2) taking dried dodder powder, adding deionized water according to the solid-liquid ratio, stirring and leaching in a water bath to obtain a mixed solution;

[0010] 3) using a flash extractor to treat the mixed solution obtained in step 2), centrifuging and filtering, the liquid obtained by filtering is a dodder polysaccharide extract, retaining the dodder polysaccharide extract, and calculating the polysaccharide extraction rate using a phenol-sulfuric acid method;

[0011] 4) using a macroporous resin to decolorize the dodder polysaccharide extract obtained in step 3), and using ultraviolet-visible spectroscopy to determine the decolorization rate, so that the decolorization rate is about 70%;

[0012] 5) After the protein in the decolorized dodder polysaccharide extract in step 4) is initially removed by enzymatic hydrolysis, the protein is removed by TCA method, and the protein content is measured by Coomassie Brilliant Blue method to make the protein content below 1%, thereby obtaining a dodder polysaccharide purified solution;

[0013] 6) dialyzing the purified dodder polysaccharide solution obtained in step 5) to obtain a dodder polysaccharide concentrated solution;

[0014] 7) adding anhydrous ethanol dropwise to the dodder polysaccharide concentrate obtained in step 6), stirring evenly, standing to precipitate with alcohol, taking the precipitate, washing it with anhydrous ethanol, and freeze-drying it in vacuum to obtain a crude dodder polysaccharide;

[0015] 8) The crude dodder polysaccharide was separated and purified by column chromatography: first, cellulose column chromatography was used, and pure water and sodium chloride solution were graded for elution; second, dextran gel column chromatography was used, and distilled water was used for desalination and elution; finally, white loose fibrous dodder polysaccharide crystals were obtained by vacuum freeze drying.

[0016] Furthermore, the mesh size of the sieve used for the crushing and screening in step 1) is 60 meshes.

[0017] Furthermore, in step 2), the solid-liquid ratio between the dodder seed dry powder and deionized water is 1:60 to 1:140 g / mL; preferably 1:100 to 1:140 g / mL, and more preferably 1:140 g / mL.

[0018] Furthermore, the stirring and leaching temperature in step 2) is 30 to 70° C., and the stirring and leaching time is preferably 20 to 60 minutes;

[0019] Furthermore, the temperature of the stirring and leaching in step 2) is preferably 50° C., and the time of the stirring and leaching is preferably 40 to 60 minutes, more preferably 50 minutes.

[0020] Furthermore, the flash voltage in step 3) is 80 to 120 V, and the flash time is 40 to 120 s.

[0021] Furthermore, the flash voltage in step 3) is preferably 100-120V, and the flash time is preferably 80-120s.

[0022] Furthermore, the flash voltage in step 3) is preferably 110V, and the flash time is preferably 120s.

[0023] Furthermore, through multi-factor orthogonal experiments, the most preferred combination of the solid-liquid ratio, flash voltage, flash time, and extraction time in step 2) and step 3) is: solid-liquid ratio 1:140g / mL, flash voltage 120V, flash time 120s, and extraction time 50min.

[0024] Furthermore, the macroporous resin in step 4) is preferably D101 macroporous adsorption resin; the wet weight of the D101 macroporous adsorption resin is 20 to 30 times the mass of the polysaccharide, preferably 25 times.

[0025] Furthermore, the enzyme used in the enzymolysis in step 5) is papain with a mass concentration of 1-3%, preferably papain with a mass concentration of 2%; the solvent of the papain is water; the temperature of the enzymolysis is 50-60°C, the time is 1-3h, preferably enzymolysis at 55°C for 2h.

[0026] Furthermore, in step 5), the TCA method for removing protein is to use trichloroacetic acid (TCA) with a volume concentration of 5 to 10% to remove protein, preferably trichloroacetic acid (TCA) with a volume concentration of 10%; the solvent of the trichloroacetic acid (TCA) is water; the volume ratio of the trichloroacetic acid (TCA) to the dodder polysaccharide extract is (1 to 1.5): (1 to 1.5), preferably 1:1; after adding trichloroacetic acid (TCA), the pH of the dodder polysaccharide extract is adjusted to 6.5 to 7.5, preferably 7.

[0027] Furthermore, the molecular weight cutoff of the dialysis bag used in the dialysis in step 6) is 3500Da; the dialysis time is 10 to 15 hours, preferably 12 hours.

[0028] Furthermore, in step 7), the volume of the anhydrous ethanol is 5 to 10 times, preferably 5 times, of the Cuscuta polysaccharide concentrate; and the standing alcohol precipitation time is 10 to 15 hours, preferably 12 hours.

[0029] Furthermore, in step 8), the cellulose column chromatography uses a DEAE-52 cellulose column, the principle is anion exchange, and the flow rate is 1 mL / min; the dextran gel column chromatography uses a Sephadex G-100 dextran gel column, and the flow rate is 0.2 mL / min; the sodium chloride solution is a 0.1-0.5 M sodium chloride solution, preferably a 0.1 M sodium chloride solution, a 0.3 M sodium chloride solution, or a 0.5 M sodium chloride solution.

[0030] The second technical solution of the present invention provides dodder polysaccharide obtained by the above preparation method, wherein the dodder polysaccharide includes dodder polysaccharide extract, dodder polysaccharide purified liquid, dodder polysaccharide concentrated liquid, dodder polysaccharide crude product, and dodder polysaccharide crystalline pure product.

[0031] The third technical solution of the present invention provides the application of the above-mentioned dodder polysaccharide, wherein the dodder polysaccharide is used for preparing moisturizing and / or anti-inflammatory products.

[0032] A fourth technical solution of the present invention provides a moisturizing and / or anti-inflammatory product comprising the above-mentioned Cuscuta australis polysaccharide.

[0033] Furthermore, the moisturizing and / or anti-inflammatory products include cosmetics, medicines, and health products.

[0034] (1) The present invention provides a method for extracting dodder polysaccharide from dodder, which has the advantages of high extraction rate, simple operation, high extraction efficiency, etc. compared with single hot water extraction method, ultrasonic extraction method, enzyme-assisted hot water extraction method, and separation and purification of crude dodder polysaccharide to obtain pure dodder polysaccharide. It is of great significance to maintain the original chemical structure and biological activity of polysaccharide.

[0035] (2) Compared with the traditional hot water extraction method and ultrasonic extraction method, the flash-assisted hot water stirring extraction method of the present invention has the advantages of uniform effective extraction area, high extraction efficiency, and short flash extraction time. In addition, the unit energy consumption of the flash extraction method is significantly reduced, the energy-saving effect is obvious, and the extraction rate is significantly increased compared with the traditional extraction method.

[0036] (3) The dodder polysaccharide obtained by the present invention has good moisturizing and / or anti-inflammatory functions and can be used in medicines, health products and cosmetics for moisturizing, reducing inflammation and improving skin symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 :Effect of solid-liquid ratio on the extraction rate of polysaccharides from Cuscuta australis;

[0038] Figure 2 :Effect of extraction temperature on the extraction rate of polysaccharides from Cuscuta australis;

[0039] Figure 3 :Effect of flash voltage on the extraction rate of polysaccharides from Cuscuta australis;

[0040] Figure 4 :Effect of flash time on the extraction rate of polysaccharides from Cuscuta australis;

[0041] Figure 5 :Effect of extraction time on the extraction rate of polysaccharides from Cuscuta australis;

[0042] Figure 6 : Hyaluronidase inhibition rate of different concentrations of Cuscuta polysaccharide before and after purification;

[0043] Figure 7 :Cytotoxicity of different concentrations of Cuscuta polysaccharides before and after purification on HaCaT cells;

[0044] Figure 8 :Effects of different drying times on the survival rate of HaCaT cells;

[0045] Fig. 9 :The repair effect of different concentrations of Cuscuta polysaccharides on HaCaT cells before and after purification;

[0046] Fig.10 : Protective effects of different concentrations of Cuscuta polysaccharides before and after purification on HaCaT cells. DETAILED DESCRIPTION

[0047] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, several variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0048] All raw materials of the present invention have no particular restrictions on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art. The information of the reagents and instruments involved is shown in Tables 1-2:

[0049] Table 1 Experimental reagents and drug information

[0050]

[0051]

[0052] Table 2 Experimental equipment information

[0053]

[0054] Embodiment 1:

[0055] 1) drying the dodder seeds, crushing and sieving to obtain dodder dry powder; the dodder powder is sieved with a sieve having a mesh number of 60;

[0056] 2) Take dodder seed dry powder, add deionized water at a solid-liquid ratio of 1:100 g / mL, and extract at a temperature of 50° C. for 40 min;

[0057] 3) Treat at a flash voltage of 100V for 80s;

[0058] 4) Centrifuging and filtering, retaining the filtered liquid (i.e., the dodder polysaccharide extract), and calculating the polysaccharide extraction rate using the phenol-sulfuric acid method.

[0059] The calculation formula is:

[0060]

[0061] Among them, crude polysaccharide mass refers to the total amount of polysaccharides obtained from the raw materials through the extraction process;

[0062] Raw material mass: refers to the total amount of raw materials used for extraction; the calculation method of polysaccharide extraction rate in the following examples and comparative examples is the same.

[0063] The extraction rate of dodder polysaccharide obtained by the method is 16.96%.

[0064] Comparative Example 1:

[0065] 1) drying the dodder seeds, crushing and sieving to obtain dodder dry powder;

[0066] 2) Take dodder seed powder and add deionized water at a solid-liquid ratio of 1:100 g / mL;

[0067] 3) Extract in a 50°C water bath for 40 min;

[0068] 4) Centrifuging and filtering, retaining the filtered liquid (i.e., the dodder polysaccharide extract), and calculating the polysaccharide extraction rate using the phenol-sulfuric acid method.

[0069] The extraction rate of dodder polysaccharide obtained by the method is 6.28%.

[0070] Comparative Example 2:

[0071] 1) drying the dodder seeds, crushing and sieving to obtain dodder dry powder;

[0072] 2) Take dodder seed powder and add deionized water at a solid-liquid ratio of 1:100 g / mL;

[0073] 3) Treat at an ultrasonic power of 250 W and a temperature of 50°C for 40 min;

[0074] 4) Centrifuging and filtering, retaining the filtered liquid (i.e., the dodder polysaccharide extract), and calculating the polysaccharide extraction rate using the phenol-sulfuric acid method.

[0075] The extraction rate of dodder polysaccharide obtained by the method is 11.93%.

[0076] Comparative Example 3:

[0077] 1) drying the dodder seeds, crushing and sieving to obtain dodder dry powder;

[0078] 2) Take dodder seed powder and add deionized water at a solid-liquid ratio of 1:100 g / mL;

[0079] 3) At room temperature, treat at a flash voltage of 100 V for 80 seconds;

[0080] 4) Centrifuging and filtering, retaining the filtered liquid (i.e., the dodder polysaccharide extract), and calculating the polysaccharide extraction rate using the phenol-sulfuric acid method.

[0081] The extraction rate of dodder polysaccharide obtained by the method is 14.17%.

[0082] Comparative Example 4:

[0083] 1) drying the dodder seeds, crushing and sieving to obtain dodder dry powder;

[0084] 2) Take dodder seed powder, add deionized water at a solid-liquid ratio of 1:100 g / mL, add 2% (W / W) papain, perform enzymolysis for 2 h in a hot water bath at 50°C and pH=5.0, and then immediately inactivate the enzyme at 90-100°C for 15 min;

[0085] 3) Extraction in a 50°C water bath for 40 min;

[0086] 4) Centrifuging and filtering, retaining the filtered liquid (i.e., the dodder polysaccharide extract), and calculating the polysaccharide extraction rate using the phenol-sulfuric acid method.

[0087] The extraction rate of dodder polysaccharide obtained by the method is 10.88%.

[0088] Comparative Example 5:

[0089] 1) drying the dodder seeds, crushing and sieving to obtain dodder dry powder;

[0090] 2) Take dodder seed powder, add deionized water at a solid-liquid ratio of 1:100 g / mL, add 2% (W / W) cellulase, perform enzymolysis for 2 h in a hot water bath at 50° C. and pH=5.0, and then immediately inactivate the enzyme at 90-100° C. for 15 min;

[0091] 3) Extraction in a 50°C water bath for 40 min;

[0092] 4) Centrifuging and filtering, retaining the filtered liquid (i.e., the dodder polysaccharide extract), and calculating the polysaccharide extraction rate using the phenol-sulfuric acid method.

[0093] The extraction rate of dodder polysaccharide obtained by the method is 8.34%.

[0094] Comparative Example 6:

[0095] 1) drying the dodder seeds, crushing and sieving to obtain dodder dry powder;

[0096] 2) Take dodder seed powder, add deionized water at a solid-liquid ratio of 1:100 g / mL, add a total amount of pectinase of 2% (W / W), perform enzymolysis for 2 h in a hot water bath at 50°C and pH = 5.0, and then immediately inactivate the enzyme at 90-100°C for 15 min;

[0097] 3) Extraction in a 50°C water bath for 40 min;

[0098] 4) Centrifuging and filtering, retaining the filtered liquid (i.e., the dodder polysaccharide extract), and calculating the polysaccharide extraction rate using the phenol-sulfuric acid method.

[0099] The extraction rate of dodder polysaccharide obtained by the method is 8.02%.

[0100] Comparative Example 7:

[0101] 1) drying the dodder seeds, crushing and sieving to obtain dodder dry powder;

[0102] 2) Take dodder seed powder, add deionized water at a solid-liquid ratio of 1:100 g / mL, add α-amylase in a total amount of 2% (W / W), perform enzymolysis for 2 h in a hot water bath at 50° C. and pH=5.0, and then immediately inactivate the enzyme at 90-100° C. for 15 min;

[0103] 3) Extraction in a 50°C water bath for 40 min;

[0104] 4) Centrifuging and filtering, retaining the filtered liquid (i.e., the dodder polysaccharide extract), and calculating the polysaccharide extraction rate using the phenol-sulfuric acid method.

[0105] The extraction rate of dodder polysaccharide obtained by the method is 7.07%.

[0106] The processing conditions and polysaccharide extraction rates of the above Example 1 and Comparative Examples 1 to 7 are summarized in Table 3, as follows:

[0107] Table 3 Summary of treatment conditions and polysaccharide extraction rates of Example 1 and Comparative Examples 1 to 7

[0108]

[0109]

[0110] Example 2 Optimization of process conditions for Cuscuta polysaccharide

[0111] This example screened out 5 factors that affect the polysaccharide extraction rate, including solid-liquid ratio, extraction temperature, extraction time, flash voltage, and flash time. The polysaccharide content of the dodder extract was used as a reference index to conduct a single factor experiment. The specific method is as follows:

[0112] (1) Five different solid-liquid ratios were selected for single factor experiments, the solid-liquid ratios were 1:60 g / mL, 1:80 g / mL, 1:100 g / mL, 1:120 g / mL, and 1:140 g / mL. Figure 1 As shown, it can be seen that the polysaccharide content of the dodder extract shows a continuous growth trend with the increase of the solid-liquid ratio, and the content of dodder polysaccharide is higher when the solid-liquid ratio is 1:140 g / mL. In order to avoid excessive waste of solvent, a higher solid-liquid ratio is not selected.

[0113] (2) Five different extraction temperatures were selected for single factor experiments, and the extraction temperatures were 30℃, 40℃, 50℃, 60℃, and 70℃, respectively. The effect of extraction temperature on the extraction rate of Cuscuta polysaccharides is shown in Figure 2 As shown, it can be seen that the polysaccharide content of the dodder extract increases first, then decreases, and then increases slightly with the increase of the extraction temperature. When the extraction temperature is 50°C, the content of dodder polysaccharide is higher. Therefore, the optimal extraction temperature is 50°C.

[0114] (3) Five different flash voltages were selected for single factor experiments, and the flash voltages were 80 V, 90 V, 100 V, 110 V, and 120 V. The effect of flash voltage on the extraction rate of Cuscuta polysaccharides is shown in Figure 2. Figure 3 As shown, it can be seen that the polysaccharide content of the dodder extract increases first and then decreases with the increase of the flash voltage. When the flash voltage is 110V, the content of dodder polysaccharide is higher. Therefore, the optimal flash voltage is 110V.

[0115] (4) Five different flash times were selected for single factor experiment, and the flash times were 40s, 60s, 80s, 100s, and 120s. Figure 4 As shown in the figure, it can be seen that the polysaccharide content of the dodder extract shows a continuous growth trend with the increase of the flash time, and the content of dodder polysaccharide is higher when the flash time is 120s. In order to avoid the overheating of the blade caused by too long a flash time, which affects the structure of the polysaccharide, a flash time of more than 120s is not selected.

[0116] (5) Five different extraction times were selected for single factor experiment, and the extraction time was 20min, 30min, 40min, 50min, and 60min respectively. Figure 5 As shown, it can be seen that the polysaccharide content of the dodder extract increases first and then decreases with the increase of extraction time. When the extraction time is 50 minutes, the content of dodder polysaccharide is relatively high.

[0117] According to the orthogonal experiment principle and the above-mentioned single-factor experimental results, four factors with more significant influence on the polysaccharide content of Cuscuta australis extract were selected: solid-liquid ratio (A), flash voltage (B), flash time (C), and extraction time (D). The effects of the four factors and the corresponding three-level interactions on the extraction rate of Cuscuta australis polysaccharide were investigated. The orthogonal factor level table is shown in Table 4. The orthogonal design experimental results and analysis are shown in Table 5.

[0118] Table 4 Orthogonal factor level table

[0119]

[0120] Table 5 Orthogonal experimental scheme and analysis results

[0121]

[0122]

[0123] According to the factor level table, an orthogonal test was conducted, and 9 different experimental combinations were used. The test results are shown in Table 5. The solid-liquid ratio (A), flash voltage (B), flash time (C), and extraction time (D) all have an effect on the content of polysaccharide extracted from the sample, and the degree of influence is C>B>A>D, that is, flash time>flash voltage>solid-liquid ratio>extraction time. The extraction rate of polysaccharides from Cuscuta chinensis is between 16.36% and 22.62%. With the help of range analysis, the combination of A3B3C3D2 is the optimal combination, that is, solid-liquid ratio 1:140g / mL, flash voltage 120V, flash time 120s, and extraction time 50min. This optimal condition was verified, and the experimental verification showed that the polysaccharide extraction rate of the optimal combination was 25.5316%±2.98% (n=5). It is higher than the results of all orthogonal test samples, indicating that the model is effective and reliable.

[0124] Example 3 Isolation and Purification of Cuscuta Polysaccharide

[0125] 1) using a macroporous resin (D101 macroporous adsorption resin, the wet weight is 25 times the mass of the polysaccharide) to decolorize the dodder polysaccharide extract obtained in Example 1, and the decolorization rate is determined by ultraviolet-visible spectroscopy technology to be 72.45±0.69%, which meets the requirements;

[0126] The calculation formula is:

[0127]

[0128] Wherein, the absorbance of polysaccharide before decolorization refers to the absorbance A value of the polysaccharide solution before decolorization measured at an ultraviolet wavelength of 450nm;

[0129] The absorbance of polysaccharide after decolorization refers to the absorbance A value of the decolorized polysaccharide solution with the same concentration as the polysaccharide solution before decolorization measured at an ultraviolet wavelength of 450nm.

[0130] 2) using papain (commercially available to those skilled in the art) combined with TCA method (a method familiar to those skilled in the art, the details of which will not be described here) to remove protein, and measuring the protein content by Coomassie Brilliant Blue method, the measured protein content was 0.67±0.53%, which met the requirements;

[0131] The calculation formula is:

[0132]

[0133] Among them, crude polysaccharide mass refers to the total amount of polysaccharides obtained from the raw materials through the extraction process;

[0134] Raw material mass: refers to the total amount of raw materials used for extraction.

[0135] 3) After rotary evaporation and concentration, the polysaccharide was dialyzed using a dialysis bag with a molecular weight cut-off of 3500 Da;

[0136] 4) Add about 5 times the volume of anhydrous ethanol dropwise, stir rapidly, and after the ethanol and the stock solution are evenly mixed, stand at 4°C for 12 hours, centrifuge to obtain the precipitate, wash three times with ethanol, re-dissolve with water, concentrate by rotary evaporation, and freeze-dry in vacuum to obtain a crude product of Cuscuta polysaccharide.

[0137] 5) The crude dodder polysaccharide was separated and purified by column chromatography. First, a DEAEcellulose-52 column (2.6 cm×60 cm) was used for separation and purification, and pure water, 0.1 M sodium chloride solution, 0.3 M sodium chloride solution, and 0.5 M sodium chloride solution were used for graded elution at a flow rate of 1 mL / min and an elution time of 10 min / tube, and the peak fractions were concentrated. Secondly, a Sephadex G-100 polysaccharide gel chromatography column (1.6×80 cm) was used, and distilled water was used for desalting and elution at a flow rate of 0.2 mL / min for further separation and purification. Finally, white loose fibrous flocculent dodder polysaccharide crystals were obtained by vacuum freeze-drying.

[0138] Polysaccharide purity test: Phenol-sulfuric acid method was used for determination. 0.5 mL of polysaccharide extract was taken, 0.25 mL of 6% phenol solution was added, 1.25 mL of concentrated sulfuric acid was quickly added after mixing, and the mixture was allowed to stand for 20 minutes, and then placed in a boiling water bath for 15 minutes, and then allowed to stand for another hour. At a wavelength of 490 nm, the reagent blank was used to adjust the zero value, and the absorbance value was measured. Compared with the standard curve, the experiment was repeated three times, and the polysaccharide purity was calculated to be 90.09 ± 3.50%.

[0139] Example 4: Hyaluronidase inhibition assay to evaluate the anti-inflammatory activity of Cuscuta polysaccharide

[0140] The specific method of this embodiment is as follows:

[0141] 1) Mix 0.1 mL of 12.5 mmol / L calcium chloride solution and 0.05 mL of 2.5 mg / mL hyaluronidase (prepared with 0.1 M acetate buffer) to prepare a mixed solution, and divide it into 4 groups, with 3 parallel samples in each group;

[0142] 2) Add 0.1 mL of sample solution (T group: sample group; T0 group: sample control group (0.1 M acetate buffer replaced hyaluronidase)) or 0.1 mL of deionized water (C group: blank group; C0: blank control group (0.1 M acetate buffer replaced hyaluronidase)) to each of the mixed solutions above;

[0143] 3) After the mixed solutions of the above groups were treated at 37°C for 40 min, 0.2 mL of 2 mg / mL sodium hyaluronate was added respectively and treated at 37°C for 60 min; 0.1 mL of 0.4 mol / L sodium hydroxide solution and 0.1 mL of acetylacetone solution were added respectively and treated at 100°C for 15 min; then cooled to room temperature, 0.5 mL of p-dimethylaminobenzaldehyde was added and treated at 37°C for 10 min, and the absorbance was measured at 585 nm.

[0144]

[0145] T: absorbance of the sample group at 585 nm; T0: absorbance of the sample control group (0.1 M acetate buffer instead of hyaluronidase) at 585 nm; C: absorbance of the blank group at 585 nm; C0: absorbance of the blank control group (0.1 M acetate buffer instead of hyaluronidase) at 585 nm.

[0146] The anti-inflammatory activity of 1-40 mg / mL of Cuscuta polysaccharide (CCP) before purification (in Cuscuta polysaccharide extract), 0.25-1.5 mg / mL of purified Cuscuta polysaccharide (CCP-1-A), and 0.025-0.5 mg / mL of positive control dipotassium glycyrrhizinate (Dg) was determined by hyaluronidase assay. The results are as follows: Figure 6 As shown:

[0147] With the increase of concentration, the inhibitory effect of dodder polysaccharide on hyaluronidase increased. The inhibition rate of hyaluronidase by 40 mg / mL CCP was 96.67±2.96%, that by 1.5 mg / mL CCP-1-A was 96.55±2.53%, and that by 0.5 mg / mL Dg was 96.67±2.55%. The half-clearance concentration (IC50) of CCP, CCP-1-A and Dg was calculated by linear fitting. 50 ) are 12.78mg / mL, 0.804mg / mL and 0.112mg / mL respectively, indicating that CCP is weaker than CCP-1-A and Dg in inhibiting hyaluronidase, but can reach more than 90% for hyaluronidase inhibition rate. Meanwhile, CCP-1-A has an inhibitory ability for hyaluronidase close to the positive control Dg, indicating that CCP-1-A has a significant anti-inflammatory effect. Therefore, it is shown that Cuscuta polysaccharide can significantly inhibit the activity of hyaluronidase, has an anti-inflammatory effect, and the purified Cuscuta polysaccharide has a greatly improved inhibitory ability for hyaluronidase.

[0148] Example 5 Moisturizing activity evaluation:

[0149] (1) Cytotoxicity of polysaccharides on HaCaT cells

[0150] 1×104 The number of cells per well HaCaT cells (a type of human immortalized keratinocytes, which exhibit a typical epithelial cell-like morphology under a microscope and have the characteristics of adherent growth; the cells form a tight monolayer of cells in a culture flask and do not have tumor characteristics) were inoculated in a 96-well plate. After 24 hours of adherence, different concentrations (0, 1, 2, 4, 8, 16, 32 mg / mL) of purified Cuscuta polysaccharide solutions were added to culture the cells for 24 hours. After 24 hours, the supernatant was discarded, and CCK-8 solution was added to incubate the cells for 50 minutes. The OD value was measured at 450nm. The blank control was DMEM complete culture medium. The data were recorded and the effects of different concentrations of sample solutions on the activity of HaCaT cells were analyzed. The formula for calculating cell activity is as follows:

[0151]

[0152] Among them, OD 样品 Indicates the absorbance value of the sample group at 450nm; OD 空白 Indicates the absorbance value of the blank group at 450nm; OD 对照 It represents the absorbance value of the control group at 450 nm.

[0153] The following cell activities were calculated in the same way.

[0154] The experimental results are as follows Figure 7 As shown, the results showed that at the concentrations of 1, 2, and 4 mg / mL, the cell activity of the Cuscuta polysaccharide CCP before purification and the Cuscuta polysaccharide CCP-1-A after purification were both >85%. Therefore, 1, 2, and 4 mg / mL were selected as the sample concentrations for subsequent efficacy determination, and as shown in the figure, at the same concentration, the cell activity of the purified Cuscuta polysaccharide was significantly better than that of the Cuscuta polysaccharide before purification.

[0155] (2) Establishment of HaCaT cell dryness injury model

[0156] According to the method of Example 5(1), a HaCaT cell suspension was prepared at 2×10 4 The number of cells per well was inoculated in a 96-well plate. After 12 hours of adherence, the culture medium was aspirated and the culture plate was placed on a clean bench with a dry wind speed of 0.4 m / s for 5 minutes to 45 minutes. DMEM culture medium was then added and cultured for 24 hours. The wells with only DMEM culture medium were used as the control group in the experiment. The data were recorded and the cell survival rate was calculated according to the formula.

[0157]

[0158] OD 样品 OD 空白 OD 对照 The content shown is the same as above.

[0159] The drying time corresponding to 50% HaCaT cell activity was used as the standard for subsequent experiments, indicating that the creation of this drying injury model is reasonable.

[0160] Depend on Figure 8 It can be seen that the activity of HaCaT cells with drying time of 5, 10, 15, 20, 25, 30, 35, 40, and 45 minutes was investigated. As the drying time increased, the survival rate of the cells decreased. When the drying stimulation time of HaCaT cells was 15 minutes, the survival rate of the cells was 55.46±2.78% (P<0.001), which was close to the cell half lethality. Therefore, the drying time of 15 minutes was selected to establish the cell drying injury model.

[0161] (3) HaCaT cell repair experiment

[0162] 2×10 4 HaCaT cells were inoculated into 96-well plates. After 24 hours of attachment, they were dried for a certain period of time under a wind speed of 0.4 m / s. Then 100 μL of culture medium containing different concentrations (0, 1, 2, 4 mg / mL) of sample solution (CCP, CCP-1-A) or positive control glycerol (Gl) was added and cultured for 24 hours. The OD value of the cells was measured at 450 nm, and the cell survival rate was calculated according to the cell activity calculation formula. Glycerol (Gl) was used as a positive control. The cell activity calculation formula is as follows:

[0163]

[0164] OD 样品 OD 空白 OD 对照 The content shown is the same as above.

[0165] The repair ability of 1-4 mg / mL Cuscuta polysaccharide (CCP, CCP-1-A) before and after purification and 1-4 mg / mL (1, 2, 4 mg / mL) of positive control glycerol (Gl) on HaCaT cells was determined by CCK-8 assay. Fig. 9As shown, the cell survival rates of 4 mg / mL CCP, CCP-1-A and Gl were 72.87±3.03%, 85.58±1.41% and 93.39±2.52% (P<0.05), all of which had the effect of repairing the dryness damage of HaCaT cells, and within the same experimental concentration range, the protective effect of CCP-1-A on cells was stronger than that of CCP. With the increase of concentration of CCP and CCP-1-A, the dryness death rate of HaCaT cells decreased, and the effect of resisting the dryness damage of HaCaT cells became stronger, and the ability of CCP-1-A to repair the dryness damage of HaCaT cells was close to the positive control Gl. Therefore, it is shown that CCP and CCP-1-A can repair the dryness damage of HaCaT cells, have moisturizing effect, and CCP-1-A has a very obvious ability to repair the dryness damage of HaCaT cells.

[0166] (4) HaCaT cell protection experiment

[0167] 2×10 4 HaCaT cells were inoculated in a 96-well plate. After 24 hours of attachment, the culture medium was removed except for the control group. 100 μL of culture medium containing different concentrations of sample solution (CCP, CCP-1-A) was added and incubated for 24 hours. Then, the cells were dried for a certain period of time under a wind speed of 0.4 m / s. The cell survival rate was calculated according to the cell activity calculation formula. Glycerol (Gl) was used as a positive control. The OD value of the cells was measured at 450 nm, and the cell survival rate was calculated according to the cell activity calculation formula. The sample concentration in the experiment was a concentration that was non-toxic to the cells or had a proliferative effect on the cells. The cell activity calculation formula is as follows:

[0168]

[0169] OD 样品 OD 空白 OD 对照 The content shown is the same as above.

[0170] The protective ability of 1-4 mg / mL Cuscuta polysaccharide (CCP, CCP-1-A) before and after purification and 1-4 mg / mL (1, 2, 4 mg / mL) of positive control glycerol (Gl) on HaCaT cells was determined by CCK-8 assay. Fig.10As shown, the cell survival rates of 4 mg / mL CCP, CCP-1-A and Gl were 67.49±4.45%, 91.96±4.48% and 94.75±1.81% (P<0.05), all of which had the effect of resisting the drying injury of HaCaT cells, and within the same experimental concentration range, the protective effect of CCP-1-A on cells was stronger than that of CCP. As the concentration of CCP and CCP-1-A increased, the drying death rate of HaCaT cells decreased, and the effect of resisting the drying injury of HaCaT cells became stronger, and the ability of CCP-1-A to resist the drying death of HaCaT cells was close to that of the positive control Gl. Therefore, it is shown that CCP and CCP-1-A can resist cell drying injury, have moisturizing effect, and CCP-1-A has the ability to significantly resist the drying death of HaCaT cells.

[0171] In summary, the present application provides a method for extracting, separating and purifying dodder polysaccharides, and optimizes the extraction conditions thereof, and finds that the dodder polysaccharide extraction rate is the highest when the solid-liquid ratio is 1:140, the flash voltage is 120V, the flash time is 120s, and the extraction time is 50min; In addition, the purity of the obtained polysaccharide is 90.09±3.50%. The anti-inflammatory activity and moisturizing activity of the obtained dodder polysaccharide are measured, and it is found that dodder polysaccharides can significantly inhibit the activity of hyaluronidase, have an anti-inflammatory effect, and the inhibitory ability of the purified dodder polysaccharide on hyaluronidase is greatly improved; In addition, it is explained that the unpurified dodder polysaccharide CCP and the purified dodder polysaccharide CCP-1-A can resist cell drying damage, have moisturizing effect, and CCP-1-A has the ability to significantly resist the drying death of HaCaT cells. The dodder polysaccharide obtained by the invention has good moisturizing and / or anti-inflammatory functions and can be applied to medicines, foods and cosmetics for moisturizing, reducing inflammation and improving skin symptoms.

[0172] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing dodder polysaccharide, characterized in that: The preparation method mainly comprises the following steps: 1) drying the dodder seeds, crushing and sieving to obtain dodder dry powder; 2) taking dried dodder powder, adding deionized water according to the solid-liquid ratio, stirring and leaching in a water bath to obtain a mixed solution; 3) using a flash extractor to treat the mixed solution obtained in step 2), centrifuging and filtering, the liquid obtained by filtering is a dodder polysaccharide extract, and the dodder polysaccharide extract is retained; 4) decolorizing the dodder polysaccharide extract obtained in step 3) using a macroporous resin; 5) After the protein in the decolorized dodder polysaccharide extract in step 4) is initially removed by enzymatic hydrolysis, the protein is removed by TCA method to obtain a dodder polysaccharide purified solution; 6) dialyzing the purified dodder polysaccharide solution obtained in step 5) to obtain a dodder polysaccharide concentrated solution; 7) adding anhydrous ethanol dropwise to the dodder polysaccharide concentrate obtained in step 6), stirring evenly, standing to precipitate with alcohol, taking the precipitate, washing it with anhydrous ethanol, and freeze-drying it in vacuum to obtain a crude dodder polysaccharide; 8) The crude dodder polysaccharide was separated and purified by column chromatography: first, cellulose column chromatography was used, and pure water and sodium chloride solution were graded for elution; second, dextran gel column chromatography was used, and distilled water was used for desalination and elution; finally, vacuum freeze-drying was performed to obtain the pure crystalline dodder polysaccharide.

2. The method for preparing a dodder polysaccharide according to claim 1, characterized in that: The solid-liquid ratio between the dodder seed dry powder and deionized water in step 2) is 1:60 to 1:140 g / mL; The stirring and leaching temperature in step 2) is 30-70°C, and the stirring and leaching time is 20-60 minutes; The flash voltage described in step 3) is 80-120V, and the flash time is 40-120s.

3. The method for preparing a dodder polysaccharide according to claim 1, characterized in that: The combination of the solid-liquid ratio, flash voltage, flash time, and extraction time in step 2) and step 3) is: solid-liquid ratio 1:140g / mL, flash voltage 120V, flash time 120s, and extraction time 50min.

4. The method for preparing a dodder polysaccharide according to claim 1, characterized in that: The macroporous resin in step 4) is D101 macroporous adsorption resin; the wet weight of the D101 macroporous adsorption resin is 20 to 30 times the mass of the polysaccharide.

5. The method for preparing a dodder polysaccharide according to claim 1, characterized in that: The enzyme used in the enzymatic hydrolysis in step 5) is papain with a mass concentration of 1 to 3%, and the solvent of the papain is water; the temperature of the enzymatic hydrolysis is 50 to 60° C., and the time is 1 to 3 hours; In step 5), the TCA method for removing proteins is to use trichloroacetic acid with a volume concentration of 5 to 10% to remove proteins, and the solvent of the trichloroacetic acid is water; the volume ratio of the trichloroacetic acid to the dodder polysaccharide extract is (1 to 1.5): (1 to 1.5); In step 5), after adding trichloroacetic acid, the pH of the dodder polysaccharide extract is adjusted to 6.5-7.

5.

6. The method for preparing dodder polysaccharide according to claim 1, characterized in that: The molecular weight cutoff of the dialysis bag used in step 6) is 3500Da; the dialysis time is 10 to 15 hours; In step 7), the volume of the anhydrous ethanol is 5 to 10 times that of the Cuscuta polysaccharide concentrate, and the standing alcohol precipitation time is 10 to 15 hours.

7. The method for preparing dodder polysaccharide according to claim 1, characterized in that: In step 8), the cellulose column chromatography uses a DEAE-52 cellulose column with a flow rate of 1 mL / min; the dextran gel column chromatography uses a Sephadex G-100 dextran gel column with a flow rate of 0.2 mL / min; and the sodium chloride solution is a 0.1-0.5 M sodium chloride solution.

8. A dodder polysaccharide, characterized in that: Obtained by the preparation method according to any one of claims 1 to 7; the dodder polysaccharide includes dodder polysaccharide extract, dodder polysaccharide purified liquid, dodder polysaccharide concentrated liquid, dodder polysaccharide crude product, and dodder polysaccharide crystalline pure product.

9. The use of a dodder polysaccharide according to claim 8, characterized in that: The dodder polysaccharide is used for preparing moisturizing and / or anti-inflammatory products.

10. A moisturizing and / or anti-inflammatory product, characterized in that: It comprises the dodder polysaccharide as claimed in claim 8; the moisturizing and / or anti-inflammatory products include cosmetics, medicines, and health products.

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