Extraction method of cynanchum paniculatum polysaccharide and optimization of process conditions thereof

Abstract

The invention relates to an extraction method of cynanchum paniculatum polysaccharide and optimization of process conditions thereof, and provides a method for extracting cynanchum paniculatum polysaccharide from cynanchum paniculatum, compared with a single hot water extraction method, an ultrasonic extraction method and a flash extraction method, the efficient ultrasonic enzymolysis extraction process provided by the invention has the advantages of high extraction rate, simplicity in operation and the like; the optimal extraction method is obtained through single factor and response surface experiments, the purity of the obtained polysaccharide is high, and the method is of great significance in maintaining the original chemical structure and biological activity of the polysaccharide. And the paniculate swallowwort root polysaccharide obtained by the invention has good antioxidant and anti-inflammatory functions.

Description

Technical Field

[0001] The invention relates to the technical field of natural product extraction, and in particular to a method for extracting Cynanchum cynanchum polysaccharide and optimization of its process conditions. Background Art

[0002] The commonly used Chinese medicine Cynanchum chinense belongs to the herbaceous plant of the Asclepiadaceae family, and its medicinal parts are roots and rhizomes. "Compendium of Materia Medica" records that Cynanchum chinense has pharmacological effects such as promoting blood circulation and dispelling wind, relieving pain and swelling, regulating immunity, sedation, analgesia, improving myocardial ischemia, anti-virus, anti-inflammatory, and anti-allergic, but it should be taken with caution by the weak. The water extract of Cynanchum chinense has anti-tumor proliferation effects and has a growth inhibitory effect on transplanted ascites cancer and solid tumors. Therefore, it is also used to develop related drugs and has certain economic value. Past studies have shown that Cynanchum chinense mainly contains paeonol, amino acids, flavonoids and polysaccharides. Cynanchum chinense polysaccharide is one of the important active ingredients of Cynanchum chinense.

[0003] The extraction methods of Cynanchum cyrtonema polysaccharides reported in the literature mainly include the traditional water extraction and alcohol precipitation method; however, this method has obvious disadvantages, such as high temperature, long time, low extraction rate, etc. With the continuous development of technology, some advanced extraction technologies have been applied to the extraction of natural polysaccharides, such as ultrasound-assisted extraction and flash-assisted extraction. Ultrasound is a commonly used method for extracting medicinal materials. It uses the cavitation effect of ultrasonic vibration. During the oscillation process, the microflow around the cavitation bubble will produce tangential force on the medicinal materials in the solution, thereby accelerating the penetration of the solvent into the cells, dissolving the effective components in the medicinal material cells, and extracting the medicinal material components. However, ultrasonic power, time, etc. will affect the extraction rate of medicinal materials. At present, ultrasound has been widely used in the extraction of various chemical components, such as polysaccharides, glycosides, flavonoids and alkaloids, but the ultrasonic extraction method generally takes tens of minutes or even hours to extract about 80% of the effective components of the medicinal materials. Flash extraction, also known as tissue crushing extraction, originated from the crushing of biological tissues. Later, it was gradually developed and innovated for crushing plant tissues and used for the extraction of Chinese medicinal materials. It has the advantages of saving time, stability and energy saving, but flash extraction has high requirements on equipment.

[0004] Therefore, it is necessary to develop an extraction method that is more efficient, more environmentally friendly, has a high polysaccharide yield and does not destroy the polysaccharide structure. Summary of the invention

[0005] The purpose of the present invention is to provide a method for extracting Cynanchum chinense polysaccharide and the optimization of its process conditions. The present invention uses a high-efficiency ultrasonic enzymatic extraction process to extract Cynanchum chinense polysaccharide with significant antioxidant and anti-inflammatory effects from Cynanchum chinense. The obtained polysaccharide has a high purity, so that the extracted Cynanchum chinense polysaccharide has better antioxidant and anti-inflammatory effects, which is of great significance for maintaining the original chemical structure and biological activity of the polysaccharide.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] A method for extracting Cynanchum chinense polysaccharide, the specific steps are as follows:

[0008] S1, drying Cynanchum chinense, crushing and sieving to obtain Cynanchum chinense dry powder;

[0009] S2, adding the Cynanchum chinense dry powder obtained in step S1 into deionized water to obtain a mixed solution A;

[0010] S3, adding papain to the mixed solution A obtained in step S2 for enzymolysis, and immediately inactivating the enzyme after a period of enzymolysis to obtain a mixed solution B;

[0011] S4, subjecting the mixed solution B obtained in step S3 to ultrasonic treatment, and taking the supernatant after centrifugation to obtain a polysaccharide extract;

[0012] S5, using trichloroacetic acid method to remove protein from the polysaccharide extract obtained in step S4, and after rotary evaporation concentration, dialyzing the polysaccharide extract from which protein has been removed to obtain a dialysate;

[0013] S6. Add anhydrous ethanol to the dialysate obtained in step S5, stir and mix evenly, let stand for alcohol precipitation, centrifuge to obtain the precipitate, wash and vacuum freeze-dry to obtain Cynanchum cyrtonema polysaccharide.

[0014] Furthermore, in step S1, the Cynanchum chinense powder is sieved through a sieve with a mesh size of 50 meshes.

[0015] Furthermore, in step S2, the solid-liquid ratio of the Cynanchum chinense dry powder and deionized water is 1:15 to 1:35 g / mL.

[0016] Furthermore, the solid-liquid ratio of the Cynanchum chinense dry powder and deionized water is 1:24.064 g / mL.

[0017] Furthermore, in step S3, the amount of papain added is 1% to 3% (W / W) of the mixed solution A.

[0018] Furthermore, the amount of papain added is 1.5% (W / W) of the mixed solution A.

[0019] Furthermore, in step S3, the enzymolysis time is 0.5 to 2.5 hours, the enzymolysis temperature is 40 to 60° C., and the enzymolysis pH is 4.0 to 6.0.

[0020] In the above, further, the enzymolysis time is 1.5 hours, the enzymolysis temperature is 50° C., and the enzymolysis pH is 5.0.

[0021] Furthermore, in step S3, the enzyme inactivation temperature is 90-100° C., and the enzyme inactivation time is 10-20 min.

[0022] In the above, further, the enzyme inactivation temperature is 95° C., and the enzyme inactivation time is 15 min.

[0023] Furthermore, in step S4, the ultrasonic time is 20 to 60 minutes, the ultrasonic power is 200 to 400 W, and the ultrasonic temperature is 40 to 60°C.

[0024] Furthermore, in the above, the ultrasonic time is 44.735 min, the ultrasonic power is 295.661 W, and the ultrasonic temperature is 50°C.

[0025] Furthermore, in step S5, the volume percentage of trichloroacetic acid is 5-15%.

[0026] Furthermore, in step S5, the volume ratio of trichloroacetic acid to polysaccharide solution is 1:1.

[0027] Furthermore, in step S5, the polysaccharide extract from which the protein has been removed is dialyzed using a dialysis bag, wherein the molecular weight cut-off of the dialysis bag is ≥3500Da.

[0028] Furthermore, in the above, the molecular weight cut-off of the dialysis bag is 3500Da.

[0029] Furthermore, in step S5, the dialysis time is 5 to 10 hours, preferably 8 hours.

[0030] Furthermore, in step S6, the volume ratio of the anhydrous ethanol to the dialysate is 5:1, and the alcohol precipitation time is 10 to 20 hours, preferably 12 hours.

[0031] The present invention also provides an antioxidant and anti-inflammatory product, wherein the antioxidant and anti-inflammatory product contains Cynanchum chinense polysaccharide in a pharmacologically effective concentration.

[0032] In addition, the present invention also provides an optimization process of process conditions for extracting Cynanchum cyrtonema polysaccharide, and the specific steps are as follows:

[0033] S1. Six factors affecting the extraction rate of Cynanchum cyrtonema polysaccharide were screened out: solid-liquid ratio, enzyme addition amount, enzymatic hydrolysis time, ultrasonic temperature, ultrasonic time, and ultrasonic power;

[0034] S2. Taking the extraction rate of Cynanchum chinense polysaccharide as a reference index, a single factor experiment was performed according to the six factors screened out in step S1;

[0035] S3. Through single factor experiment, three factors with significant influence on the extraction rate of polysaccharide from Cynanchum chinense were selected: solid-liquid ratio (A), ultrasonic time (B), and ultrasonic power (C) as independent variables, and the extraction rate of polysaccharide from Cynanchum chinense was taken as the response value. The above response surface experimental design data were subjected to regression fitting analysis using statistical software, and a quadratic multinomial regression equation prediction model for the extraction rate of polysaccharide from Cynanchum chinense (Y) was obtained.

[0036] S4. Use statistical software to optimize the extraction process, obtain the best extraction process conditions, and conduct verification tests based on the obtained best extraction process.

[0037] Furthermore, in steps S3 and S4, the statistical software is Design-Expert 12.

[0038] Further, in step S3, the quadratic multinomial regression equation prediction model of the extraction rate of Cynanchum chinense polysaccharide (Y) is: Y (%) = 61.57 + 3.68A-0.67B + 3.12C-0.385AB-1.45AC-0.935BC-5.04A 2 –4.96B 2 –4.8C 2 . (-1≤A≤1, -1≤B≤1, -1≤C≤1). Wherein, Y is the extraction rate of Cynanchum cyrtonema polysaccharide (%), A is the solid-liquid ratio (g / mL), B is the ultrasonic time (min), and C is the ultrasonic power (W).

[0039] Furthermore, in step S4, after the above process optimization process, the optimal extraction process conditions were obtained as follows: the solid-liquid ratio was 24.064 g / mL, the ultrasonic time was 44.735 min, and the ultrasonic power was 295.661 W; the predicted value of the polysaccharide extraction rate of Cynanchum cyrtonema was 59.584%.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] 1. The present invention provides a method for extracting Cynomorium polysaccharide from Cynomorium cynomorium. Compared with a single hot water extraction method, an ultrasonic extraction method, and a flash extraction method, the high-efficiency ultrasonic enzymatic extraction process provided by the present invention has the advantages of high extraction rate and simple operation, and the optimal extraction method is obtained through single factor and response surface experiments, and the obtained polysaccharide has a high purity, which is of great significance for maintaining the original chemical structure and biological activity of the polysaccharide. And the Cynomorium polysaccharide obtained by the present invention has good antioxidant and anti-inflammatory functions.

[0042] 2. Ultrasonic waves can produce cavitation effects, destroy the cell walls and cell membranes of plant cells, and make it easier to release polysaccharides in cells, thereby significantly improving the extraction rate of polysaccharides; the enzymatic hydrolysis process has high selectivity and can be carried out under relatively mild conditions, which helps to protect the structure and biological activity of polysaccharides. The synergistic effect of ultrasound and enzymatic hydrolysis can accelerate the dissolution rate of polysaccharides and improve the extraction rate of polysaccharides. The efficient ultrasonic enzymatic hydrolysis extraction process also reduces the use of chemical reagents, is more environmentally friendly, and meets the requirements of sustainable development.

[0043] 3. The Cynanchum chinense polysaccharide obtained by the present invention has good antioxidant and anti-inflammatory functions and can be used in medicines, foods and cosmetics for antioxidant, anti-inflammatory and skin symptom improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of the effect of different solid-liquid ratios on the extraction rate of polysaccharides from Cynanchum chinense;

[0045] Figure 2 The figure is a schematic diagram showing the effect of different enzyme addition amounts on the extraction rate of Cynanchum cyrtonema polysaccharide;

[0046] Figure 3 Schematic diagram of the effect of different enzymatic hydrolysis times on the extraction rate of Cynanchum cyrtonema polysaccharides;

[0047] Figure 4 Schematic diagram of the effect of different ultrasonic temperatures on the extraction rate of polysaccharides from Cynanchum cyrtonema;

[0048] Figure 5 Schematic diagram of the effect of different ultrasonic time on the extraction rate of polysaccharides from Cynanchum cyrtonema;

[0049] Figure 6 Schematic diagram of the effect of different ultrasonic powers on the extraction rate of polysaccharides from Cynanchum cyrtonema;

[0050] Figure 7 The response surfaces and contour plots of the interaction between different factors on the extraction rate of polysaccharides from Cynanchum chinense, wherein (a) is the response surface and contour plot of the interaction between ultrasound time and solid-liquid ratio on the extraction rate of polysaccharides from Cynanchum chinense, (b) is the response surface and contour plot of the interaction between ultrasound power and solid-liquid ratio on the extraction rate of polysaccharides from Cynanchum chinense, and (c) is the response surface and contour plot of the interaction between ultrasound power and ultrasound time on the extraction rate of polysaccharides from Cynanchum chinense;

[0051] Figure 8 is the DPPH free radical scavenging rate of Cynanchum chinense polysaccharide at different concentrations;

[0052] Fig. 9 is the ABTS free radical scavenging rate of Cynanchum chinense polysaccharide at different concentrations;

[0053] Fig.10is the hydroxyl radical scavenging rate of Cynanchum chinense polysaccharide at different concentrations;

[0054] Fig.11 is the hyaluronidase inhibition rate of different concentrations of Cynanchum chinense polysaccharide;

[0055] Fig.12 is the standard curve of glucose;

[0056] Fig.13 The standard curve is bovine serum albumin. DETAILED DESCRIPTION

[0057] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0058] In the following examples, the sources of the materials are as follows:

[0059] Purchased by Xu Changqing in Bozhou Traditional Chinese Medicine Market.

[0060] In conjunction with the accompanying drawings, some embodiments of the present invention are described in detail below. In the absence of conflict, the following embodiments and features in the embodiments may be combined with each other.

[0061] Example 1

[0062] This embodiment provides an optimization process of the process conditions of a method for extracting polysaccharides from Cynanchum chinense, and the specific steps are as follows:

[0063] S1, screening out 6 factors that affect the polysaccharide extraction rate, including solid-liquid ratio, enzyme addition amount, enzymatic hydrolysis time, ultrasonic temperature, ultrasonic time, and ultrasonic power, taking the polysaccharide extraction rate of Cynanchum chinense as a reference indicator, and conducting a single factor experiment based on the 6 factors screened out in step S1;

[0064] S1-1, 5 different material-liquid ratios were selected for single factor experiment, the material-liquid ratios were 1:15g / mL, 1:20g / mL, 1:30g / mL, 1:30g / mL, 1:35g / mL;

[0065] Effect of solid-liquid ratio on the extraction rate of Cynanchum cyrtonema polysaccharide Figure 1 As shown, it can be seen that the extraction rate of Cynanchum chinense polysaccharide first increases and then decreases with the increase of the solid-liquid ratio. When the solid-liquid ratio is 1:20 g / mL, the extraction rate of Cynanchum chinense polysaccharide is higher, so the optimal solid-liquid ratio is 1:20 g / mL.

[0066] S1-2, select 5 different enzyme addition amounts for single factor experiment, the enzyme addition amounts are 1%, 1.5%, 2%, 2.5%, and 3% respectively;

[0067] Effect of enzyme addition on the extraction rate of polysaccharides from Cynanchum chinense Figure 2 As shown, it can be seen that the extraction rate of Cynanchum chinense polysaccharide increases first and then decreases with the increase of enzyme addition amount. When the enzyme addition amount is 1.5%, the extraction rate of Cynanchum chinense polysaccharide is higher, so the optimal enzyme addition amount is 1.5%.

[0068] S1-3, select 5 different enzymatic hydrolysis times for single factor experiment, the enzymatic hydrolysis times are 0.5h, 1h, 1.5h, 2h, and 2.5h respectively;

[0069] Effect of enzymatic hydrolysis time on the extraction rate of Cynanchum cyrtonema polysaccharides Figure 3 As shown, it can be seen that the extraction rate of Cynanchum chinense polysaccharide increases first and then decreases with the increase of enzymatic hydrolysis time. When the enzymatic hydrolysis time is 1.5h, the extraction rate of Cynanchum chinense polysaccharide is higher, so the optimal enzymatic hydrolysis time is 1.5h.

[0070] S1-4, select 5 different ultrasonic temperatures for single factor experiment, the ultrasonic temperatures are 40℃, 45℃, 50℃, 55℃, and 60℃ respectively;

[0071] Effect of ultrasonic temperature on the extraction rate of polysaccharides from Cynanchum cyrtonema Figure 4 As shown, it can be seen that the extraction rate of Cynanchum chinense polysaccharide increases first and then decreases with the increase of ultrasonic temperature. When the ultrasonic temperature is 50℃, the extraction rate of Cynanchum chinense polysaccharide is higher, so the optimal ultrasonic temperature is 50℃.

[0072] S1-5, 5 different ultrasound times were selected for single factor experiment, the ultrasound times were 20min, 30min, 40min, 50min, and 60min respectively;

[0073] Effect of ultrasonic time ratio on the extraction rate of polysaccharides from Cynanchum cyrtonema Figure 5 As shown, it can be seen that the extraction rate of polysaccharides from Cynanchum chinense first increases and then decreases with the increase of ultrasonic time. When the ultrasonic time is 40 minutes, the extraction rate of polysaccharides from Cynanchum chinense is higher, so the optimal ultrasonic time is 40 minutes.

[0074] S1-6, select 5 different ultrasonic powers for single factor experiment, the ultrasonic powers are 200W, 250W, 300W, 350W, and 400W respectively;

[0075] Effect of ultrasonic power ratio on the extraction rate of polysaccharides from Cynanchum cyrtonema Figure 6 As shown, it can be seen that the extraction rate of Cynanchum chinense polysaccharide shows a trend of first increasing, then decreasing and then increasing with the increase of ultrasonic power. When the ultrasonic power is 300W, the extraction rate of Cynanchum chinense polysaccharide is relatively high, so the optimal ultrasonic power is 300W.

[0076] S2. According to the above single factor experimental results, three factors with more significant influence on the extraction rate of polysaccharides from Cynanchum chinense were selected: solid-liquid ratio (A), ultrasonic time (B), and ultrasonic power (C) as independent variables, and the extraction rate of polysaccharides from Cynanchum chinense as the response value. Design-Expert 12 software was used, and the design scheme and results are shown in Table 1.

[0077] Table 1 Experimental plan and results

[0078]

[0079]

[0080] S3. Build model:

[0081] Through 17 different experimental combinations and response values, the extraction rate of polysaccharides from Cynanchum chinense was between 43.66% and 63.72%, which was described by multiple regression fitting analysis and response surface quadratic model; the response variable Cynanchum chinense polysaccharide extraction rate can be related by the following second-order polynomial equation:

[0082] Y(%)=61.57+3.68A-0.67B+3.12C-0.385AB–1.45AC–0.935BC–5.04A 2 –

[0083] 4.96B 2 –4.8C 2 . (-1≤A≤1, -1≤B≤1, -1≤C≤1).

[0084] Wherein, Y is the extraction rate of Cynanchum chinense polysaccharide (%), A is the solid-liquid ratio (g / mL), B is the ultrasonic time (min), and C is the ultrasonic power (W).

[0085] S4. Significance test:

[0086] As shown in Table 2, the F value of the equation model is 15.61, and the probability value P value is 0.0008 (P<0.001), indicating that the change in the response value caused by the change in the independent variable is very significant, and the established model is meaningful. At the same time, the F value of the lack of fit term is 1.86, and the P value is 0.2773, indicating that the quadratic regression model fits the actual situation well, and the model can be used for theoretical prediction of the polysaccharide extraction rate of Cynanchum cyrtonema. In this model, the linear terms A and C have significant statistical differences in the effect on the polysaccharide extraction rate of Cynanchum cyrtonema (P<0.01); the cross terms AB, BC, and AC have no statistical differences in the effect on the polysaccharide extraction rate (P>0.05); the quadratic term A 2 , B 2 , C 2The results showed that the ultrasonic power and liquid-to-solid ratio were the most important factors affecting the extraction of polysaccharides from Cynanchum chinense. According to the results of variance analysis, the influence of each factor on the extraction of polysaccharides from Cynanchum chinense was as follows: A (liquid-to-solid ratio) > C (ultrasonic power) > B (ultrasonic time).

[0087] Table 2 Response surface variance analysis results

[0088]

[0089] S5. Optimize the extraction process using the statistical software Design-Expert 12 to obtain the best extraction process conditions, and conduct verification tests based on the obtained best extraction process.

[0090] like Figure 7 As shown in the figure, the response surface contour map and response surface three-dimensional map of the extraction of Cynanchum cyrtonema polysaccharide were drawn. The three-dimensional graph can intuitively reflect the influence of the interaction of three factors, namely, solid-liquid ratio, ultrasonic time and ultrasonic power, on the response value, and find out the optimal parameters and the interaction between the parameters. For example, the influence of the interaction of ultrasonic time and ultrasonic power on the extraction rate of Cynanchum cyrtonema polysaccharide was analyzed by fixing the solid-liquid ratio; and the contour map is a way of expressing the three-dimensional graph on the two-dimensional plane.

[0091] Figure 7 (a) shows the effect of solid-liquid ratio and ultrasonic time on polysaccharide extraction rate. The polysaccharide extraction rate of Cynanchum chinense increases with the increase of solid-liquid ratio at the beginning. When the ultrasonic time reaches a certain value, the polysaccharide extraction rate of Cynanchum chinense decreases with the increase of solid-liquid ratio or ultrasonic time. Similarly, the change trend of polysaccharide extraction rate of Cynanchum chinense is shown in Figure 7 (b) and Figure 7 (c) as shown.

[0092] The optimal process conditions for extracting polysaccharides from Cynanchum chinense obtained by response surface experiment are: solid-liquid ratio of 1:24.064 g / mL, ultrasonic time of 44.735 min, and ultrasonic power of 295.661 W. The theoretical extraction rate of polysaccharides from Cynanchum chinense is 59.584%. The optimal conditions were verified, and the actual extraction rate of polysaccharides from Cynanchum chinense was 58.70%±3.10% (n=5). It is very close to the theoretical value, indicating that the model is effective and reliable.

[0093] Example 2

[0094] This embodiment provides a method for extracting polysaccharides from Cynanchum chinense, and the specific steps are as follows:

[0095] S1, drying Cynanchum chinense, crushing and sieving to obtain Cynanchum chinense dry powder;

[0096] S2, adding the Cynanchum chinense dry powder obtained in step S1 to deionized water at a solid-liquid ratio of 1:24 g / mL, stirring and mixing in a water bath to obtain a mixed solution A;

[0097] S3, adding 1.5% (W / W) papain to the mixed solution A obtained in step S2, performing enzymolysis at 50°C and pH=5.0, and immediately inactivating the enzyme at 95°C for 15 minutes after enzymolysis for 1.5 hours to obtain a mixed solution B;

[0098] S4, treating the mixed solution B obtained in step S3 in an ultrasonic instrument, with an ultrasonic power of 295 W, an ultrasonic temperature of 50° C., and an ultrasonic treatment time of 45 min. After centrifugation, the supernatant was filtered to obtain a polysaccharide extract;

[0099] S5, using trichloroacetic acid method to remove protein from the polysaccharide extract obtained in step S4, and after rotary evaporation concentration, dialyzing the polysaccharide extract from which protein has been removed using a 3500Da dialysis bag to obtain a dialysate;

[0100] S6. Add about 5 times the volume of anhydrous ethanol dropwise to the dialysate obtained in step S5, stir rapidly, and after the anhydrous ethanol and the dialysate are evenly mixed, let stand at 4°C for 12 hours, centrifuge to obtain the precipitate, wash three times with anhydrous ethanol, re-dissolve with water, concentrate by rotary evaporation, and freeze-dry in vacuum to obtain Cynanchum cyrtonema polysaccharide.

[0101] Comparative Example 1

[0102] This comparative example provides a method for extracting Cynanchum chinense polysaccharide, and the specific steps are as follows:

[0103] S1, drying Cynanchum chinense, crushing and sieving to obtain Cynanchum chinense dry powder;

[0104] S2, adding the Cynanchum chinense dry powder obtained in step S1 to deionized water at a solid-liquid ratio of 1:24 g / mL, stirring and mixing in a water bath to obtain a mixed solution A;

[0105] S3. Extract the mixed solution A obtained in step S2 in a 50° C. water bath for 30 min, centrifuge and filter the supernatant to obtain a polysaccharide extract.

[0106] Comparative Example 2

[0107] This comparative example provides a method for extracting Cynanchum chinense polysaccharide, and the specific steps are as follows:

[0108] S1, drying Cynanchum chinense, crushing and sieving to obtain Cynanchum chinense dry powder;

[0109] S2, adding the Cynanchum chinense dry powder obtained in step S1 to deionized water at a solid-liquid ratio of 1:24 g / mL, stirring and mixing in a water bath to obtain a mixed solution A;

[0110] S3. The mixed solution A obtained in step S2 is treated at an ultrasonic power of 295 W and a temperature of 50° C. for 45 min, and the supernatant is filtered after centrifugation to obtain a polysaccharide extract.

[0111] Comparative Example 3

[0112] This comparative example provides a method for extracting Cynanchum chinense polysaccharide, and the specific steps are as follows:

[0113] S1, drying Cynanchum chinense, crushing and sieving to obtain Cynanchum chinense dry powder;

[0114] S2, adding the Cynanchum chinense dry powder obtained in step S1 to deionized water at a solid-liquid ratio of 1:24 g / mL, stirring and mixing in a water bath to obtain a mixed solution A;

[0115] S3. Treat the mixed solution A obtained in step S2 at a flash voltage of 100 V and room temperature for 60 seconds, centrifuge and filter the supernatant to obtain a polysaccharide extract.

[0116] Comparative Example 4

[0117] This comparative example provides a method for extracting Cynanchum chinense polysaccharide, and the specific steps are as follows:

[0118] S1, drying Cynanchum chinense, crushing and sieving to obtain Cynanchum chinense dry powder;

[0119] S2, adding the Cynanchum chinense dry powder obtained in step S1 to deionized water at a solid-liquid ratio of 1:24 g / mL, stirring and mixing in a water bath to obtain a mixed solution A;

[0120] S3, treating the mixed solution A obtained in step S2 at an ultrasonic power of 295 W and a temperature of 50° C. for 45 min to obtain a mixed solution B;

[0121] S4. Treat the mixed solution B obtained in step S3 at a flash voltage of 100 V and room temperature for 60 seconds, centrifuge and filter the supernatant to obtain a polysaccharide extract.

[0122] Comparative Example 5

[0123] This comparative example provides a method for extracting Cynanchum chinense polysaccharide, and the specific steps are as follows:

[0124] S1, drying Cynanchum chinense, crushing and sieving to obtain Cynanchum chinense dry powder;

[0125] S2, adding the Cynanchum chinense dry powder obtained in step S1 to deionized water at a solid-liquid ratio of 1:24 g / mL, stirring and mixing in a water bath to obtain a mixed solution A;

[0126] S3, adding 1.5% (W / W) cellulase to the mixed solution A obtained in step S2, performing enzymolysis at 50°C and pH=5.0, and immediately inactivating the enzyme at 95°C for 15 minutes after enzymolysis for 1.5 hours to obtain a mixed solution B;

[0127] S4. Extract the mixed solution B obtained in step S3 in a 50° C. water bath for 30 min, centrifuge and filter the supernatant to obtain a polysaccharide extract.

[0128] Comparative Example 6

[0129] This comparative example provides a method for extracting Cynanchum chinense polysaccharide, and the specific steps are as follows:

[0130] S1, drying Cynanchum chinense, crushing and sieving to obtain Cynanchum chinense dry powder;

[0131] S2, adding the Cynanchum chinense dry powder obtained in step S1 to deionized water at a solid-liquid ratio of 1:24 g / mL, stirring and mixing in a water bath to obtain a mixed solution A;

[0132] S3, adding 1.5% (W / W) papain to the mixed solution A obtained in step S2, performing enzymolysis at 50°C and pH=5.0, and immediately inactivating the enzyme at 95°C for 15 minutes after enzymolysis for 1.5 hours to obtain a mixed solution B;

[0133] S4. Extract the mixed solution B obtained in step S3 in a 50° C. water bath for 30 min, centrifuge and filter the supernatant to obtain a polysaccharide extract.

[0134] Comparative Example 7

[0135] This comparative example provides a method for extracting Cynanchum chinense polysaccharide, and the specific steps are as follows:

[0136] S1, drying Cynanchum chinense, crushing and sieving to obtain Cynanchum chinense dry powder;

[0137] S2, adding the Cynanchum chinense dry powder obtained in step S1 to deionized water at a solid-liquid ratio of 1:24 g / mL, stirring and mixing in a water bath to obtain a mixed solution A;

[0138] S3, adding 1.5% (W / W) cellulase to the mixed solution A obtained in step S2, performing enzymolysis at 50°C and pH=5.0, and immediately inactivating the enzyme at 95°C for 15 minutes after enzymolysis for 1.5 hours to obtain a mixed solution B;

[0139] S4. Treat the mixed solution B obtained in step S3 in an ultrasonic instrument with an ultrasonic power of 295 W, an ultrasonic temperature of 50° C., and an ultrasonic treatment time of 45 min. After centrifugation, filter the supernatant to obtain a polysaccharide extract.

[0140] Comparative Example 8

[0141] This comparative example provides a method for extracting Cynanchum chinense polysaccharide, and the specific steps are as follows:

[0142] S1, drying Cynanchum chinense, crushing and sieving to obtain Cynanchum chinense dry powder;

[0143] S2, adding the Cynanchum chinense dry powder obtained in step S1 to deionized water at a solid-liquid ratio of 1:24 g / mL, stirring and mixing in a water bath to obtain a mixed solution A;

[0144] S3, adding 1.5% (W / W) cellulase to the mixed solution A obtained in step S2, performing enzymolysis at 50°C and pH=5.0, and immediately inactivating the enzyme at 95°C for 15 minutes after enzymolysis for 1.5 hours to obtain a mixed solution B;

[0145] S4. Treat the mixed solution B obtained in step S3 at a flash voltage of 100 V and room temperature for 60 seconds, centrifuge and filter the supernatant to obtain a polysaccharide extract.

[0146] Comparative Example 9

[0147] This comparative example provides a method for extracting Cynanchum chinense polysaccharide, and the specific steps are as follows:

[0148] S1, drying Cynanchum chinense, crushing and sieving to obtain Cynanchum chinense dry powder;

[0149] S2, adding the Cynanchum chinense dry powder obtained in step S1 to deionized water at a solid-liquid ratio of 1:24 g / mL, stirring and mixing in a water bath to obtain a mixed solution A;

[0150] S3, adding 1.5% (W / W) papain to the mixed solution A obtained in step S2, performing enzymolysis at 50°C and pH=5.0, and immediately inactivating the enzyme at 95°C for 15 minutes after enzymolysis for 1.5 hours to obtain a mixed solution B;

[0151] S4. Treat the mixed solution B obtained in step S3 at a flash voltage of 100 V and room temperature for 60 seconds, centrifuge and filter the supernatant to obtain a polysaccharide extract.

[0152] Test Example 1

[0153] This test example uses the phenol-sulfuric acid method to calculate the extraction rate of polysaccharides in the polysaccharide extracts in Example 1 and Comparative Examples 1 to 9, as follows:

[0154] As shown in Table 3, the polysaccharide extraction rate of Cynanchum chinense calculated in Example 1 is 55.92%, the polysaccharide extraction rate of Cynanchum chinense calculated in Comparative Example 1 is 29.87%, the polysaccharide extraction rate of Cynanchum chinense calculated in Comparative Example 2 is 33.96%, the polysaccharide extraction rate of Cynanchum chinense calculated in Comparative Example 3 is 28.56%, the polysaccharide extraction rate of Cynanchum chinense calculated in Comparative Example 4 is 32.10%, the polysaccharide extraction rate of Cynanchum chinense calculated in Comparative Example 5 is 45.49%, the polysaccharide extraction rate of Cynanchum chinense calculated in Comparative Example 6 is 50.03%, the polysaccharide extraction rate of Cynanchum chinense calculated in Comparative Example 7 is 49.67%, the polysaccharide extraction rate of Cynanchum chinense calculated in Comparative Example 8 is 39.40%, and the polysaccharide extraction rate of Cynanchum chinense calculated in Comparative Example 9 is 43.04%.

[0155] Table 3 Extraction rate of Cynanchum chinense polysaccharide in polysaccharide extracts in Example 1 and Comparative Examples 1 to 9

[0156]

[0157]

[0158] Test Example 2

[0159] This test example uses the phenol-sulfuric acid method to determine the polysaccharide purity of the polysaccharide extract in Example 1, as follows:

[0160] Prepare the mother solution of glucose standard, dilute the mother solution of glucose standard step by step to the concentration range of 0.02-0.14 (mg / mL), take 0.5mL of glucose standard dilution, add 0.25mL of 6% phenol solution, mix well and quickly add 1.25mL of concentrated sulfuric acid, let it stand for 20min, boil in boiling water bath for 15min, let it stand for another 1h, adjust to zero with reagent blank at a wavelength of 490nm, and measure the absorbance value.

[0161] like Fig.12 As shown, the concentration range of glucose standard is 0.02~0.14 (mg / mL), and the curve equation is y=6.97014x+0.02121, R 2 =0.9994, indicating that there is a good linear relationship between glucose concentration and absorbance. This polysaccharide content determination method has high accuracy and can be used for the determination of polysaccharide content in Cynanchum cyrtonema.

[0162] Take 0.5mL of polysaccharide extract, add 0.25mL of 6% phenol solution, mix well and quickly add 1.25mL of concentrated sulfuric acid, let stand for 20min, boil in water for 15min, let stand for 1h, adjust to zero with reagent blank at 490nm wavelength, measure absorbance value. Compare with standard curve, calculate sample polysaccharide purity is 85.45%.

[0163] Test Example 3

[0164] This test example uses the Coomassie Brilliant Blue method to measure the protein content of the polysaccharide extract in step S5 of Example 1, as follows:

[0165] Prepare the mother solution of bovine serum albumin standard, dilute the mother solution of bovine serum albumin standard step by step to a concentration range of 0 to 100 (μg / mL), mix the bovine serum albumin standard dilution with 2.5 mL of Coomassie Brilliant Blue solution, let it stand for 5 minutes, and measure the absorbance at 595 nm.

[0166] like Fig.13 As shown, the concentration range of the bovine serum albumin standard is 0-100 (μg / mL), the curve equation is y=0.0054x+0.0059, R2=0.9995, indicating that the bovine serum albumin concentration has a good linear relationship with the absorbance. This protein content determination method has a high accuracy and can be used to determine the protein content in Cynanchum cyrtonema polysaccharide.

[0167] 0.5 mL of the polysaccharide extract obtained in step S5 of Example 1 was mixed with 2.5 mL of Coomassie Brilliant Blue solution, and the mixture was allowed to stand for 5 min. The absorbance was measured at 595 nm and compared with the standard curve. The protein content of the polysaccharide extract was 1.01%.

[0168] Test Example 4

[0169] This test example measures the antioxidant activity of the Cynanchum chinense polysaccharide in Example 1, as follows:

[0170] 1. DPPH free radical scavenging experiment:

[0171] 1 mL of sample solution was mixed with 1 mL of DPPH ethanol solution at room temperature and protected from light for 30 min. The absorbance was measured at 517 nm and the DPPH free radical scavenging rate was calculated according to the following formula. VC was used as the positive control.

[0172]

[0173] Where:

[0174] A1: absorbance of the sample solution;

[0175] A0: absorbance of sample solution (excluding free radicals);

[0176] A: DPPH absorbance;

[0177] The antioxidant activity of 0.1-10 mg / mL of Cynanchum chinense polysaccharide and 3.125-50 μg / mL of VC was determined by DPPH free radical scavenging experiment. Figure 8As shown, Cynanchum chinense polysaccharide exhibits good DPPH free radical scavenging activity, and the scavenging rate of Cynanchum chinense polysaccharide on DPPH free radicals is positively correlated with its concentration. Through linear fitting, it is deduced that the half inhibition concentration (IC 50) of Cynanchum chinense polysaccharide and VC are 0.81mg / mL and 25.65μg / mL, respectively. The scavenging rate of 10mg / mL Cynanchum chinense polysaccharide reaches 83.46%, and the scavenging rate of 50μg / mL VC reaches 95.06%. The effect of Cynanchum chinense polysaccharide in scavenging DPPH free radicals is lower than that of VC, but it can also reach a free radical scavenging rate of more than 80% at a higher concentration, indicating that Cynanchum chinense polysaccharide has antioxidant properties.

[0178] 2. ABTS free radical scavenging experiment

[0179] 1 mL of sample solution was mixed with 1 mL of ABTS ethanol solution at room temperature and protected from light for 30 min. The absorbance was measured at 734 nm and the ABTS free radical scavenging rate was calculated according to the following formula. VC was used as the positive control.

[0180]

[0181] Where:

[0182] A1: absorbance of the sample solution;

[0183] A0: absorbance of sample solution (excluding free radicals);

[0184] A: ABTS absorbance;

[0185] The antioxidant activity of 0.1-10 mg / mL Cynanchum chinense polysaccharide and 2.5-20 μg / mL VC was determined by ABTS free radical scavenging experiment. Fig. 9 As shown, Cynanchum cynanchum polysaccharide exhibits good ABTS free radical scavenging activity, and the scavenging rate of Cynanchum cynanchum polysaccharide on ABTS free radicals is positively correlated with its concentration. Through linear fitting, it is deduced that the half inhibition concentration (IC 50) of Cynanchum cynanchum polysaccharide and VC are 0.81mg / mL and 4.86μg / mL, respectively. The scavenging rate of 10mg / mL Cynanchum cynanchum polysaccharide reaches 99.70%, and the scavenging rate of 20μg / mL VC reaches 98.72%. The effect of Cynanchum cynanchum polysaccharide in scavenging ABTS free radicals is lower than that of VC, but it can also reach a free radical scavenging rate of more than 90% at a higher concentration, indicating that Cynanchum cynanchum polysaccharide has antioxidant properties.

[0186] 3. Hydroxyl free radical scavenging experiment

[0187] First add 1 mL of FeSO4 and 1 mL of salicylic acid-ethanol solution, then add 2 mL of sample solution, and then add 1 mL of H2O2 to start the reaction. Keep the temperature in a 37°C water bath for 30 minutes, measure the absorbance at 510 nm, and calculate the hydroxyl radical scavenging rate according to the following formula, with VC as the positive control.

[0188]

[0189] Where:

[0190] A1: absorbance of the sample solution;

[0191] A0: absorbance of sample solution (excluding free radicals);

[0192] A: absorbance of negative control group;

[0193] The antioxidant activity of 0.1-10 mg / mL Cynanchum chinense polysaccharide and 0.05-0.35 mg / mL VC was determined by hydroxyl radical scavenging experiment. Fig.10 As shown, Cynanchum cynanchum polysaccharide exhibits good hydroxyl radical scavenging activity, and the scavenging rate of Cynanchum cynanchum polysaccharide on hydroxyl radicals is positively correlated with its concentration. Through linear fitting, it is deduced that the half inhibition concentration (IC50) of Cynanchum cynanchum polysaccharide and VC are 3.76mg / mL and 0.17mg / mL, respectively. The scavenging rate of 10mg / mL Cynanchum cynanchum polysaccharide reaches 87.49%, and the scavenging rate of 0.35mg / mL VC reaches 95.98%. The effect of Cynanchum cynanchum polysaccharide in scavenging hydroxyl radicals is lower than that of VC, but it can also reach a free radical scavenging rate of more than 80% at a higher concentration, indicating that Cynanchum cynanchum polysaccharide has antioxidant properties.

[0194] Test Example 4

[0195] This test example determines the anti-inflammatory activity of the Cynanchum chinense polysaccharide in Example 1 by a hyaluronidase inhibition experiment, as follows:

[0196] 0.1mL 12.5mmol / L calcium chloride solution was mixed with 0.05mL 2.5mg / mL hyaluronidase, and then 0.1mL sample solution (T group) or 0.1mL distilled water (C group) was added, and the mixture was treated at 37℃ for 40min. 0.2mL 2mg / mL sodium hyaluronate was added, and the mixture was treated at 37℃ for 60min. 0.1mL 0.4mol / L sodium hydroxide solution and 0.1mL acetylacetone solution were added, and the mixture was treated at 100℃ for 15min. The mixture was then cooled to room temperature, 0.5mL p-dimethylaminobenzaldehyde was added, and the mixture was treated at 37℃ for 10min. The absorbance was measured at 585nm, and the hyaluronidase inhibition rate was calculated according to the following formula.

[0197]

[0198] Where:

[0199] T: sample solution group;

[0200] T0: sample solution control group;

[0201] C: blank group;

[0202] C0: blank control group.

[0203] The anti-inflammatory activity of 1-20 mg / mL of Cynanchum chinense polysaccharide and 0.025-0.4 mg / mL of Dg (dipotassium glycyrrhizinate) was determined by hyaluronidase assay. Fig.11 As shown in the figure, with the increase of concentration, the inhibitory effect of Cynanchum chinense polysaccharide on hyaluronidase increased. The inhibition rate of 20 mg / mL Cynanchum chinense polysaccharide on hyaluronidase was 81.17 mg / mL, and the inhibition rate of 0.4 mg / mL Dg on hyaluronidase was 86.88 mg / mL. The half clearance concentration (IC50) of Cynanchum chinense polysaccharide and Dg was calculated by linear fitting. 50 ) were 10.81mg / mL and 0.135mg / mL respectively, indicating that the inhibitory effect of Cynanchum cynanchum polysaccharide on hyaluronidase was weaker than that of Dg, but the inhibition rate of hyaluronidase could reach more than 80%. Therefore, this test example shows that Cynanchum cynanchum polysaccharide can significantly inhibit the activity of hyaluronidase and has an anti-inflammatory effect.

[0204] 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 extracting Cynanchum chinense polysaccharide, characterized in that: The specific steps are as follows: S1, drying Cynanchum chinense, crushing and sieving to obtain Cynanchum chinense dry powder; S2, adding the Cynanchum chinense dry powder obtained in step S1 into deionized water to obtain a mixed solution A; S3, adding papain to the mixed solution A obtained in step S2 for enzymolysis, and immediately inactivating the enzyme after a period of enzymolysis to obtain a mixed solution B; S4, subjecting the mixed solution B obtained in step S3 to ultrasonic treatment, centrifuging and collecting the supernatant to obtain a polysaccharide extract; S5, using trichloroacetic acid method to remove protein from the polysaccharide extract obtained in step S4, and after rotary evaporation concentration, dialyzing the polysaccharide extract from which protein has been removed to obtain a dialysate; S6. Add anhydrous ethanol to the dialysate obtained in step S5, stir and mix evenly, let stand for alcohol precipitation, centrifuge to obtain the precipitate, wash and vacuum freeze-dry to obtain Cynanchum cyrtonema polysaccharide.

2. The method for extracting Cynanchum chinense polysaccharide according to claim 1, characterized in that: In step S1, the Cynanchum cyrtonema powder is sieved through a sieve with a mesh size of 50.

3. The method for extracting Cynanchum chinense polysaccharide according to claim 1, characterized in that: In step S2, the solid-liquid ratio of the Cynanchum chinense dry powder and deionized water is 1:15 to 1:35 g / mL.

4. The method for extracting Cynanchum chinense polysaccharide according to claim 1, characterized in that: In step S3, the amount of papain added is 1% to 3% (W / W) of the mixed solution A; The enzymolysis time is 0.5 to 2.5 hours, the enzymolysis temperature is 40 to 60° C., and the enzymolysis pH is 4.0 to 6.0; The enzyme inactivation temperature is 90-100° C., and the enzyme inactivation time is 10-20 minutes.

5. The method for extracting Cynanchum chinense polysaccharide according to claim 1, characterized in that: In step S4, the ultrasonic time is 20 to 60 minutes, the ultrasonic power is 200 to 400 W, and the ultrasonic temperature is 40 to 60°C.

6. The method for extracting Cynanchum chinense polysaccharide according to claim 1, characterized in that: In step S5, the volume percentage of trichloroacetic acid is 5 to 15%; The volume ratio of trichloroacetic acid to polysaccharide solution is 1:1; The protein-removed polysaccharide extract is dialyzed using a dialysis bag, wherein the molecular weight cut-off of the dialysis bag is ≥3500Da; The dialysis time is 5 to 10 hours.

7. The method for extracting Cynanchum chinense polysaccharide according to claim 1, characterized in that: In step S6, the volume ratio of the anhydrous ethanol to the dialysate is 5:1, and the alcohol precipitation time is 10 to 20 hours.

8. A process for optimizing the process conditions of the method for extracting Cynanchum chinense polysaccharide as claimed in any one of claims 1 to 7, characterized in that: The specific steps are as follows: S1. Six factors affecting the extraction rate of Cynanchum cyrtonema polysaccharides were screened out: solid-liquid ratio, enzyme addition, enzymatic hydrolysis time, ultrasonic temperature, ultrasonic time, and ultrasonic power; S2. Taking the extraction rate of Cynanchum chinense polysaccharide as a reference index, a single factor experiment was performed according to the six factors screened out in step S1; S3. Through single factor experiment, three factors with significant influence on the extraction rate of polysaccharide from Cynanchum chinense were selected: solid-liquid ratio A, ultrasonic time B, and ultrasonic power C as independent variables, and the extraction rate of polysaccharide from Cynanchum chinense was taken as the response value. The above response surface experimental design data were subjected to regression fitting analysis using statistical software, and a quadratic multinomial regression equation prediction model for the extraction rate of polysaccharide from Cynanchum chinense Y was obtained. S4. Use statistical software to optimize the extraction process, obtain the best extraction process conditions, and conduct verification tests based on the obtained best extraction process.

9. The process for optimizing the process conditions of the method for extracting Cynanchum cyrtonema polysaccharide according to claim 8, characterized in that: In step S3, the quadratic multinomial regression equation prediction model of the extraction rate Y of Cynanchum cyrtonema polysaccharide is: Y(%)=61.57+3.68A-0.67B+3.12C-0.385AB-1.45AC-0.935BC-5.04A 2 –4.96B 2 –4.8C 2 ; Where, -1≤A≤1, -1≤B≤1, -1≤C≤1, Y is the extraction rate of Cynanchum chinense polysaccharide (%), A is the solid-liquid ratio (g / mL), B is the ultrasonic time (min), and C is the ultrasonic power (W).

10. The process for optimizing the process conditions of the method for extracting Cynanchum cyrtonema polysaccharide according to claim 8, characterized in that: In step S4, after the above process optimization process, the best extraction process conditions are obtained as follows: the solid-liquid ratio is 24.064 g / mL, the ultrasonic time is 44.735 min, and the ultrasonic power is 295.661 W; the predicted value of the polysaccharide extraction rate of Cynanchum cyrtonema is 59.584%.

Images