A method for extracting polysaccharide from monorchidium

Abstract

The application discloses a kind of extraction methods for monkey ear ring polysaccharide, belong to extraction process technical field.The application carries out ultrasonic extraction to monkey ear ring using multi-frequency composite ultrasonic device, and the multi-frequency composite ultrasonic device is combined by probe type ultrasonic device and groove type ultrasonic device.Single-factor experiment result of multi-frequency composite ultrasonic-assisted water extraction method for extracting PCBPs is as follows: optimal extraction time 8 min, optimal liquid-solid ratio 30 mL / g and optimal extraction power 510 W, and the result is used as reference to design the value range of three factors in response surface model, and the optimal extraction process parameters of PCBPs are obtained as follows: extraction time 8 min, solid-liquid ratio 32 mL / g, and extraction power 510 W.The final PCBPs extraction rate is 9.90±0.16% under the verification of the process conditions.

Description

Technical Field

[0001] This invention belongs to the field of extraction technology, and particularly relates to a method for extracting polysaccharides from monkey earrings. Background Technology

[0002] Polysaccharides are key components and important bioactive substances in *Earringia stenoptera*, and selecting a suitable extraction method is a crucial step in the study of *Earringia stenoptera* polysaccharides (PCBPs). Different extraction methods result in variations in the extraction rate, structural characteristics, functional properties, and bioactivity of the polysaccharides. The dried branches and leaves of *Earringia stenoptera* are relatively hard, and traditional hot water extraction methods yield low polysaccharide yields. More importantly, these methods are time-consuming, and energy consumption is a significant concern for industrial applications. Therefore, providing a more efficient extraction method is extremely important for the industrial production of *Earringia stenoptera* polysaccharides. Summary of the Invention

[0003] In view of the problems existing in the prior art, the present invention proposes a method for extracting polysaccharides from monkey earrings.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] A method for extracting polysaccharides from monkey earrings includes the following steps: using a multi-frequency composite ultrasonic device to perform ultrasonic extraction on monkey earrings, wherein the multi-frequency composite ultrasonic device is composed of a probe-type ultrasonic device and a slot-type ultrasonic device; wherein the total ultrasonic power of the probe-type ultrasonic device and the slot-type ultrasonic device is 375-555W.

[0006] Furthermore, the multi-frequency composite ultrasound device is assembled by mounting a probe-type ultrasound device on top of a slot-type ultrasound device; wherein the total ultrasound power of the probe-type ultrasound device and the slot-type ultrasound device is 510W.

[0007] Furthermore, the specific steps of the extraction method are as follows:

[0008] Fresh monkey earring branches and leaves were crushed and sieved to obtain monkey earring powder;

[0009] The monkey earring powder was mixed with water, and ultrasonic extraction was performed by starting a probe-type ultrasonic device and a tank-type ultrasonic device. After one centrifugation, a monkey earring polysaccharide extract was obtained.

[0010] The monkey earlobe polysaccharide extract was concentrated, the concentrate was decolorized, centrifuged twice, the extract was deproteinized, and the supernatant was collected.

[0011] Anhydrous ethanol was added to the supernatant, the mixture was frozen and allowed to stand overnight, centrifuged three times, dialyzed, and then freeze-dried under vacuum to obtain monkey earring polysaccharide.

[0012] Furthermore, the pulverization and sieving process involves passing the material through a 40-mesh sieve.

[0013] Furthermore, the ratio of the monkey earring powder to water is 1g:32mL.

[0014] Furthermore, the parameters for the first centrifugation are: 5500 rpm and 10 min; the parameters for the second centrifugation are: 4500 rpm and 5 min; and the parameters for the third centrifugation are: 5500 rpm and 10 min.

[0015] Furthermore, during the ultrasonic extraction process, the frequency of the probe-type ultrasonic device is 270W, the frequency of the trough-type ultrasonic device is 240W, and the ultrasonic time is 8 minutes.

[0016] Furthermore, the decolorization treatment specifically refers to: adding activated carbon to the concentrated solution at a liquid-to-solid ratio of 30 mL: 1 g, stirring at 1000 rpm for 1 hour, and then stirring at 4500 rpm for 5 minutes to remove the activated carbon, and then combining the extracts.

[0017] Furthermore, the deproteinization process specifically refers to: adding 1 / 4 volume of Sevag reagent to the extract, shaking vigorously, centrifuging at 5500 rpm for 10 min, repeating the operation (i.e., the entire process of adding Sevag reagent, shaking, and centrifuging), and collecting the supernatant.

[0018] Furthermore, the volume ratio of the supernatant to anhydrous ethanol is 1:4.

[0019] The KQ-600DE trough-type ultrasonic device used in this invention has an ultrasonic frequency of 40 kHz. Ultrasonic waves are emitted from the bottom of the device, and the energy is transmitted upwards from the bottom of the extraction unit, creating cavitation in the solvent to extract the target analyte. It has a capacity of 22.5 L and can extract a large amount of raw material at once. The SCIENTZ-ⅡD probe-type ultrasonic device has an ultrasonic frequency of 20-25 kHz and an ultrasonic capacity of 0.5-600 mL. It emits ultrasonic waves from the bottom of the amplitude transformer and transmits them downwards. The instantaneous release of enormous energy can create cavitation in the solution, thereby fully extracting the target analyte.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] This invention employs a multi-frequency composite ultrasonic device, mounting a probe-type ultrasonic device on top of a trough-type ultrasonic device. This allows the raw material to simultaneously receive energy transmitted from the bottom up by the trough-type device and from the top down by the probe-type device, thus fully extracting the target compound. By controlling the power of the two ultrasonic devices, the time to reach the maximum extraction rate can be shortened to 8 minutes, representing reductions of 73.3% and 42.9% respectively compared to single-frequency ultrasonic extraction methods. This multi-frequency composite ultrasonic extraction method significantly improves extraction efficiency, saves production energy, and provides a promising approach for the industrial production of monkey ear fungus polysaccharide. Attached Figure Description

[0022] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0023] Figure 1 This is the standard curve for glucose.

[0024] Figure 2 The effect of trough ultrasonic power on PCBPs extraction rate;

[0025] Figure 3 The effect of trough ultrasonic time on PCBPs extraction rate;

[0026] Figure 4 The effect of liquid-to-material ratio in trough ultrasonic extraction on PCBPs extraction rate;

[0027] Figure 5 The following are response surface plots showing the interactive effects of three extraction factors on the extraction rate of monkey ear loop polysaccharide using trough ultrasound: a: Response surface and contour plot of ultrasound time and liquid-to-solid ratio; b: Response surface and contour plot of ultrasound time and ultrasound power; c: Response surface and contour plot of liquid-to-solid ratio and ultrasound power.

[0028] Figure 6 The effect of probe-type ultrasonic time on the extraction rate of PCBPs;

[0029] Figure 7 The effect of liquid-to-material ratio in probe-type ultrasonic extraction on the extraction rate of PCBPs;

[0030] Figure 8 The effect of probe-type ultrasonic power on the extraction rate of PCBPs;

[0031] Figure 9 The response surface plots show the interactive effects of three extraction factors on the extraction rate of PCBPs using a probe-based method; a: response surface and contour plot of ultrasonic time and liquid-to-material ratio; b: response surface and contour plot of ultrasonic time and ultrasonic power; c: response surface and contour plot of liquid-to-material ratio and ultrasonic power.

[0032] Figure 10 The effect of multi-frequency composite ultrasonic power on the extraction rate of monkey earrings;

[0033] Figure 11 The effect of multi-frequency composite ultrasound time on the extraction rate of PCBPs;

[0034] Figure 12 The effect of liquid-to-material ratio in multi-frequency composite ultrasonic treatment on the extraction rate of PCBPs;

[0035] Figure 13 The response surface plots show the interactive effects of three extraction factors (multi-frequency composite ultrasound) on the extraction rate of PCBPs; a: response surface and contour plot of ultrasound time and liquid-to-material ratio; b: response surface and contour plot of ultrasound time and ultrasound power; c: response surface and contour plot of liquid-to-material ratio and ultrasound power.

[0036] Figure 14 The scavenging rate curves of PCBPs and ascorbic acid on DPPH free radicals are shown.

[0037] Figure 15 The scavenging rate curves of ABTS free radicals by PCBPs and ascorbic acid are shown. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] Unless otherwise specified, "room temperature" in this invention refers to 25±2℃.

[0044] This invention uses the extraction rate of monkey ear loop polysaccharides (PCBPs) as a metric to investigate the effects of three ultrasonic extraction processes—tank-type ultrasonic extraction, probe-type ultrasonic extraction, and multi-frequency composite ultrasonic extraction—on the extraction rate of PCPPs. For each extraction process, single-factor experiments were conducted to examine the effects of extraction power, extraction time, and liquid-to-solid ratio on the extraction rate. Based on the single-factor experiments, Box-Benhnken Design response surface methodology was used to determine the optimal process conditions for ultrasonic-assisted extraction of monkey ear loop polysaccharides. Finally, the optimal process conditions for the three ultrasonic extraction processes were comprehensively analyzed.

[0045] The following embodiments are further illustrations of the technical solution of the present invention.

[0046] Example 1

[0047] 1.1 Materials and Instruments

[0048] 1.1.1 Experimental Materials

[0049] The fresh branches and leaves of the monkey earrings were harvested from Yunfu, China, and dried by natural sun-drying and oven drying. The chemical reagents used in this invention are detailed in Table 1.

[0050] Table 1 Main Reagents

[0051]

[0052] 1.1.2 Experimental Apparatus

[0053] Table 2 Main Instruments

[0054]

[0055] 1.2 Experimental Methods

[0056] 1.2.1 Pretreatment of monkey earrings

[0057] After the branches and leaves of the monkey ear ring are dried, they are pulverized using a traditional Chinese medicine pulverizer. The powder is then passed through a 40-mesh sieve and stored in a drying container for later use.

[0058] 1.2.2 Determination of polysaccharide extraction rate

[0059] 1.2.2.1 Plotting the glucose standard curve

[0060] Accurately weigh 5.0 mg of anhydrous glucose and place it in a volumetric flask. Dissolve the glucose in distilled water and bring the volume to 50 mL to prepare a glucose standard solution with a concentration of 0.1 mg / mL. Then, measure 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the glucose standard solution into test tubes, add distilled water to a final volume of 2 mL, and mix well. Using distilled water as a blank control, add 1 mL of 5% phenol solution to a test tube, followed by 5 mL of concentrated sulfuric acid, and mix well. Incubate in a boiling water bath for 20 min, then rapidly cool to room temperature. Measure the absorbance at 490 nm and plot a standard curve.

[0061] 1.2.2.2 Calculation of polysaccharide extraction rate

[0062] Weigh an appropriate amount of monkey earring powder and perform ultrasonic-assisted extraction of PCPPs under selected conditions. After extraction, centrifuge at 5500 rpm for 10 min to obtain the polysaccharide extract, and determine the polysaccharide extraction rate. The formula for calculating the extraction rate is as follows:

[0063]

[0064] Where C is the polysaccharide concentration (mg / mL) calculated from the regression equation of the glucose standard curve; V is the volume of the polysaccharide solution used for analysis (mL); D is the dilution factor; and M is the weight of the monkey earring powder (mg).

[0065] 1.2.3 Single-factor experiment of trough-type ultrasonic-assisted water lifting method

[0066] Using PCPPs extraction rate as the evaluation index, the effects of ultrasonic power (240, 300, 360, 420, 480 W), ultrasonic time (10, 20, 30, 40, 50 min), and ultrasonic liquid-to-solid ratio (10, 20, 30, 40, 50 mL / g) on ​​PCPPs extraction rate were investigated.

[0067] 1.2.4 Response Surface Methodology of Ultrasonic-Assisted Water Lifting in a Trench

[0068] Based on the results of single-factor experiments, Box-BenhnkenDesign was used as the analysis software, with extraction time, liquid-to-solid ratio and ultrasonic power as independent variables and PCBPs extraction rate as the response value, to optimize the extraction process of PCBPs by trough ultrasonic-assisted water extraction.

[0069] 1.2.5 Single-factor experiment of probe-type ultrasonic-assisted water extraction method

[0070] Using PCPPs extraction rate as the evaluation index, the effects of ultrasonic time (8, 10, 12, 14, 16 min), ultrasonic liquid-to-solid ratio (10, 20, 30, 40, 50 mL / g), and ultrasonic power (180, 270, 360, 450, 540 W) on PCPPs extraction rate were investigated.

[0071] 1.2.6 Probe-type ultrasonic-assisted water lifting response surface methodology

[0072] Based on the results of single-factor experiments, Box-Benhnken Design was used as the analysis software, with extraction time, liquid-to-solid ratio and ultrasonic power as independent variables and PCBPs extraction rate as the response value, to optimize the extraction process of PCBPs by probe-type ultrasonic-assisted water extraction.

[0073] 1.2.7 Single-factor experiment on multi-frequency composite ultrasonic-assisted water extraction method

[0074] Using PCPPs extraction rate as the evaluation index, based on the processes of trough-type ultrasonic-assisted water extraction and probe-type ultrasonic-assisted water extraction, the power of the trough-type ultrasonic was fixed at 240W, and the power of the probe-type ultrasonic was set at 135, 180, 225, 270, and 315W. The effects of total multi-frequency ultrasonic power (375, 420, 465, 510, and 555W), ultrasonic time (6, 8, 10, 12, and 14 min), and ultrasonic liquid-to-solid ratio (10, 20, 30, 40, and 50 mL / g) on ​​PCPPs extraction rate were investigated.

[0075] 1.2.8 Response Surface Methodology of Multi-Frequency Composite Ultrasonic Assisted Water Lifting

[0076] Based on the results of single-factor experiments, Box-Benhnken Design was used as the analysis software, with extraction time, liquid-to-solid ratio and ultrasonic power as independent variables and PCBP extraction rate as the response value, to optimize the extraction process of monkey ear polysaccharide by multi-frequency composite ultrasonic-assisted water extraction.

[0077] 1.2.9 Preparation and deproteinization treatment of monkey earring polysaccharide

[0078] Extract the PCPPs extract under optimal conditions, centrifuge at 5500 rpm for 10 min, and combine the extracts. Add 1 / 4 volume of Sevag reagent (chloroform and n-butanol in a 4:1 volume ratio) to the extract, shake vigorously, and centrifuge (5500 rpm, 10 min) to remove the intermediate white protein layer. Repeat several times until no white flocculent material appears, and collect the supernatant. Add an appropriate amount of Coomassie Brilliant Blue reagent to the supernatant; no obvious color change is observed. While stirring, add 4 volumes of anhydrous ethanol to the supernatant, and then let it stand overnight at 4°C. After centrifugation, collect the polysaccharide, reconstitute it with a small amount of distilled water, transfer it to a dialysis bag (Mw3500Da), dialyze for 48 h, and finally freeze-dry under vacuum to obtain PCPPs.

[0079] The specific steps for extracting monkey earwax polysaccharides using multi-frequency composite ultrasound-assisted water extraction are as follows:

[0080] 1) Fresh monkey earring branches and leaves were purchased from Yunfu. After natural sun drying and oven drying, they were pulverized using a Chinese medicine pulverizer. The powder was passed through a 40-mesh sieve and stored in a drying jar for later use.

[0081] 2) Mix the monkey earring powder at a material-to-liquid ratio of 1g:32mL, and start the multi-frequency composite ultrasonic device consisting of a probe-type ultrasonic device and a trough-type ultrasonic device for ultrasonic extraction to obtain monkey earring polysaccharide extract. The frequency of the probe-type ultrasonic device is 270W, the frequency of the trough-type ultrasonic device is 240W, and the ultrasonic time is 8min. After ultrasonic extraction, filter with a Buchner funnel, combine the filtrates, and centrifuge (5500rpm, 10min) to obtain monkey earring polysaccharide extract.

[0082] 3) After rotary evaporation and concentration of the monkey ear polysaccharide extract, a high-concentration polysaccharide solution was obtained. Activated carbon powder was added at a solid-liquid ratio of 1g:30mL for decolorization (1000rpm, 1h). After decolorization, the solution was centrifuged (4500rpm, 5min) to remove the activated carbon. The extracts were then combined.

[0083] 4) Add 1 / 4 volume of Sevag reagent (chloroform and n-butanol in a volume ratio of 4:1) to the extraction solution, shake vigorously, and centrifuge (5500 rpm, 10 min) to remove the middle white protein layer. Repeat several times until no white flocculent matter appears, and collect the supernatant. Take an appropriate amount of supernatant and add Coomassie Brilliant Blue reagent. No obvious color change should be observed.

[0084] 5) While stirring, add 4 times the volume of anhydrous ethanol to the supernatant. Polysaccharide precipitation can be observed. Let it stand overnight in a refrigerator at 4°C.

[0085] 6) After centrifugation (4500 rpm, 5 min), monkey ear loop polysaccharide was collected, reconstituted with a small amount of distilled water, transferred to a dialysis bag (Mw3500 Da) and dialyzed for 48 h. Finally, it was freeze-dried under vacuum to obtain monkey ear loop polysaccharide.

[0086] The specific steps for extracting polysaccharides from monkey earrings using a trough-type ultrasonic-assisted water extraction method are as follows:

[0087] The method for extracting monkey earwax polysaccharide using multi-frequency composite ultrasonic-assisted water extraction differs in that step 2) is as follows: monkey earwax powder is mixed at a material-to-liquid ratio of 1g:33mL, and ultrasonic extraction is performed using a trough-type ultrasonic device to obtain monkey earwax polysaccharide extract. The ultrasonic frequency is 300W and the ultrasonic time is 31min. After ultrasonic extraction, the mixture is filtered through a Buchner funnel, the filtrates are combined, and centrifuged (5500rpm, 10min) to obtain monkey earwax polysaccharide extract.

[0088] The specific steps for extracting monkey earring polysaccharides using probe-type ultrasound-assisted water extraction are as follows:

[0089] The method for extracting monkey earwax polysaccharide using multi-frequency composite ultrasonic-assisted water extraction differs in that step 2) is as follows: monkey earwax powder is mixed at a material-to-liquid ratio of 1g:32mL, and ultrasonic extraction is performed using a trough-type ultrasonic device to obtain monkey earwax polysaccharide extract. The ultrasonic frequency is 360W and the ultrasonic time is 14min. After ultrasonic extraction, the mixture is filtered through a Buchner funnel, the filtrates are combined, and centrifuged (5500rpm, 10min) to obtain monkey earwax polysaccharide extract.

[0090] When conducting single-factor analysis, each single factor is adjusted based on the above steps.

[0091] 1.3 Results and Discussion

[0092] 1.3.1 Glucose Standard Curve

[0093] A glucose standard curve was plotted with the concentration of each glucose standard solution on the x-axis and the measured absorbance on the y-axis, as shown below. Figure 1 As shown. The linear regression equation is y = 12.45x - 0.0444 (R²). 2 =0.9992), indicating that glucose concentration and absorbance have a good linear relationship in the range of 0.01-0.05 mg / mL.

[0094] 1.3.2 Single-factor test results of trough-type ultrasonic-assisted water lifting method

[0095] 1.3.2.1 Effect of ultrasonic power on monkey earring extraction rate

[0096] The liquid-to-solid ratio was set at 20 mL / g, and the ultrasonic time was 20 min. The effect of ultrasonic power on the extraction rate of PCBPs was investigated. The results are as follows: Figure 2 As shown. From Figure 2As can be seen, when the ultrasonic power increased from 240W to 300W, the extraction rate of PCBPs increased to 11.59%, but when it increased from 300W to 480W, the extraction rate gradually decreased. This may be because higher ultrasonic power generates stronger cavitation, resulting in greater damage to the cell walls of monkey ear rings, which is beneficial for polysaccharide dissolution. However, excessively high ultrasonic power can also degrade polysaccharides to some extent. Therefore, an ultrasonic power of 300W is more suitable.

[0097] 1.3.2.2 Effect of ultrasound time on monkey earring extraction rate

[0098] The liquid-to-solid ratio was set at 20 mL / g, and the ultrasonic power was 300 W. The effect of ultrasonic time on polysaccharide extraction rate was studied. Figure 3 As shown. From Figure 3 As can be seen, the extraction rate of PCBPs gradually increased with the extension of ultrasonic time, reaching a maximum of 13.99% at 30 min. After 30 min, the extraction rate began to decrease. This indicates that the polysaccharides had almost fully diffused into the solvent by ultrasonication for 30 min, while prolonged ultrasonication caused some polysaccharide degradation, resulting in a decrease in the polysaccharide extraction rate. Therefore, this study selected an ultrasonic time of 30 min.

[0099] 1.3.2.3 Effect of ultrasonic liquid-to-solid ratio on monkey earring extraction rate

[0100] The ultrasonic power was set to 300W and the ultrasonic time to 30min to study the effect of the liquid-to-solid ratio on the polysaccharide extraction rate. Figure 4 It can be seen that as the liquid-to-solid ratio increases, the polysaccharide extraction rate gradually increases and then decreases, reaching a maximum of 15.41% at 30 mL / g. The effect of the liquid-to-solid ratio on ultrasonic extraction of polysaccharides may be related to the viscosity of the extract. Within a certain range, increasing the solvent volume results in a lower solution concentration and lower viscosity, which increases the diffusion rate of polysaccharides into the surrounding solvent, thereby improving the extraction rate. However, higher polysaccharide concentrations lead to higher extract viscosity, which inhibits the formation of ultrasonic cavities and is detrimental to polysaccharide dissolution. Therefore, a liquid-to-solid ratio of 30 mL / g was chosen.

[0101] 1.3.3 Optimization of Polysaccharide Extraction Process Using Tank-Type Ultrasonic Response Surface Therapy

[0102] Based on the results of the single-factor experiments, the ultrasonic time (A, 20, 30, 40 min), liquid-to-solid ratio (B, 20, 30, 40 mL / g), ultrasonic power (C, 240, 300, 360 W) were selected as independent variables, and the PCBPs extraction rate was selected as the response value. Response surface methodology was conducted, and the experimental design and results are shown in Tables 3-5.

[0103] Table 3 Experimental Factors and Levels

[0104]

[0105] Table 4. Results of the Box-Behnken test

[0106]

[0107] Table 5. Results of Analysis of Variance

[0108]

[0109]

[0110] Note: p<0.01 indicates a highly significant difference; p<0.05 indicates a significant difference; p>0.05 indicates a non-significant difference.

[0111] Table 5 shows that the regression model's P-value is <0.0001 (<0.05), indicating that the experimental model has statistical significance. The P-value for the lack-of-fit term is 0.3419 (>0.05), indicating that the lack-of-fit term is not significant compared to the pure error, further demonstrating that the model can be used for data analysis in this experiment. Furthermore, the model R... 2 =0.9964, indicating that 99.64% of the variation can be explained by this model. Therefore, it can be used as an experimental model for optimizing the PCBPs extraction process. The table shows that among the three factors A, B, and C, A (extraction time), B (liquid-to-solid ratio), and C (ultrasonic power) all have a highly significant impact on the extraction rate (p<0.01). Furthermore, the interaction between AB and AC is not significant, while the interaction between BC is significantly different (p<0.05). In summary, the order of influence of the three factors on the extraction rate is: liquid-to-solid ratio > extraction power > extraction time. 3D response surface and 2D contour plots can visually express the interaction between factors in the extraction process. The more convex the 3D response surface and the more curved the contour curve, the stronger the interaction between the two factors. The 3D response surface and 2D contour plots between the factors are shown below. Figure 5 As shown. By Figure 5 As shown by 'c', the interaction between the material-to-liquid ratio and extraction power is the strongest, corresponding to the most curved contour lines. Figure 5 As can be seen from 'a', the interaction between extraction time and liquid-to-solid ratio is secondary, while... Figure 5 The value of b in the table shows that the interaction between extraction time and power is the weakest, which corresponds to the p-values ​​of AB, AC, and BC in Table 5.

[0112] The optimal extraction parameters predicted by the analysis software were: extraction time 30.8 min, liquid-to-solid ratio 32.6 mL / g, and extraction power 296.4 W, with a predicted value of 15.45%. Considering practical conditions, the final process parameters were revised to: extraction time 31 min, liquid-to-solid ratio 33 mL / g, and extraction power 300 W. The results showed that the actual extraction rate of PCBPs under these process conditions was 15.20 ± 0.36%, which is close to the predicted value, indicating that the extraction process of monkey ear loop polysaccharides optimized using this response surface model is scientifically feasible.

[0113] 1.3.4 Single-factor experimental results of probe-type ultrasonic-assisted water extraction method

[0114] 1.3.4.1 Effect of ultrasound time on the extraction rate of monkey earrings

[0115] The liquid-to-solid ratio was set at 30 mL / g, the ultrasonic power at 450 W, and the ratio of probe working time to rest time at 1:1 to study the effect of ultrasonic time on polysaccharide extraction rate. Figure 6 As shown, the polysaccharide extraction rate gradually increased with the extension of ultrasonic time, reaching a maximum of 12.97% at 14 minutes. After 14 minutes, the extraction rate began to decrease. This indicates that the cell walls of the raw material were completely destroyed by ultrasonication at 14 minutes, and the polysaccharides diffused fully into the solvent. However, the energy of the probe-type ultrasonic device is concentrated entirely within the extraction container. Excessive ultrasonication will release a large amount of heat, and the combined effect of the heat energy and ultrasonic pulses will cause some polysaccharides to degrade, resulting in a decrease in the polysaccharide extraction rate. Therefore, this invention selected an ultrasonic time of 14 minutes.

[0116] 1.3.4.2 Effect of ultrasonic liquid-to-solid ratio on monkey earring extraction rate

[0117] The ultrasonic power was set at 450W, and the ultrasonic time was 14 min to study the effect of the liquid-to-solid ratio on the polysaccharide extraction rate. Figure 7 It can be seen that as the liquid-to-solid ratio increases, the polysaccharide extraction rate gradually increases and then decreases, reaching a maximum of 12.97% at 30 mL / g. Within a certain range of the liquid-to-solid ratio, as the amount of solvent increases, the solution concentration is lower and the viscosity is lower, increasing the diffusion rate of polysaccharides into the surrounding solvent, thereby improving the extraction rate. However, as the polysaccharide concentration increases, the viscosity of the extract increases, inhibiting ultrasonic cavitation and thus reducing polysaccharide dissolution. Therefore, a liquid-to-solid ratio of 30 mL / g was chosen.

[0118] 1.3.4.3 Effect of ultrasonic power on monkey earring extraction rate

[0119] The liquid-to-solid ratio was set at 30 mL / g, and the ultrasonic time was 14 min. The effect of ultrasonic power on the polysaccharide extraction rate was investigated. The results are as follows: Figure 8As shown, when the ultrasonic power increased from 180W to 360W, the extraction rate of PCBPs increased to 13.43%, but when it increased from 360W to 540W, the extraction rate gradually decreased. This may be because within a certain range, the higher the ultrasonic power, the stronger the cavitation effect, and the greater the damage to the cell wall of monkey ear rings, which is conducive to polysaccharide dissolution. However, with further increases in ultrasonic power, the strong pulse energy will have a certain degree of degradation effect on polysaccharides. Therefore, an ultrasonic power of 360W was chosen.

[0120] 1.3.5 Probe-based ultrasonic response surface methodology for optimizing polysaccharide extraction process

[0121] Based on the results of the single-factor experiments, the ultrasonic time (A, 12, 14, 16 min), liquid-to-solid ratio (B, 20, 30, 40 mL / g), and ultrasonic power (C, 270, 360, 450 W) were selected as independent variables, and the PCBPs extraction rate was selected as the response value. Response surface methodology was conducted, and the experimental design and results are shown in Tables 6-8.

[0122] Table 6 Experimental Factors and Levels

[0123]

[0124] Table 7. Box-Behnken Test Results

[0125]

[0126]

[0127] Table 8 Results of Analysis of Variance

[0128]

[0129] Note: p<0.01 indicates a highly significant difference; p<0.05 indicates a significant difference; p>0.05 indicates a non-significant difference.

[0130] Table 8 shows that the regression model's p-value is <0.0001 (<0.05), indicating that the experimental model has statistical significance. The p-value for the lack-of-fit term is 0.1331 (>0.05), indicating that the lack-of-fit term is not significant compared to the pure error, further demonstrating that the model can be used for data analysis in this experiment. Furthermore, the model R... 2=0.9959, indicating that 99.59% of the variation can be explained by this model. Therefore, it can be used as an experimental model for optimizing the PCBPs extraction process. The table shows that among the three factors A, B, and C, A (extraction time) has a significant impact on the extraction rate (p<0.05), while B (liquid-to-solid ratio) and C (extraction power) both have extremely significant impacts on the extraction rate (p<0.01). Furthermore, the interaction between A and B is significantly different (p<0.05), while the interaction between AC and BC is not significant. In summary, in the probe-type ultrasonic-assisted water extraction method, the order of influence of the three factors on the extraction rate is: liquid-to-solid ratio > extraction time > extraction power. The 3D response surface and two-dimensional contour plots of each factor are shown below. Figure 9 As shown. By Figure 9 As shown in 'a', the interaction between the material-to-liquid ratio and extraction power is the strongest, corresponding to the most curved contour lines. From... Figure 9 As can be seen from 'c', the interaction between extraction time and liquid-to-solid ratio is secondary, while... Figure 9 The value of b in the table shows that the interaction between extraction time and power is the weakest, which corresponds to the P values ​​of AB, AC, and BC in Table 8.

[0131] The optimal extraction parameters predicted by the analysis software were: extraction time 14.06 min, liquid-to-solid ratio 32.2 mL / g, and extraction power 369 W, with a predicted value of 13.78%. Considering practical conditions, the final extraction parameters were revised to: extraction time 14 min, liquid-to-solid ratio 32 mL / g, and extraction power 360 W. The results show that the actual extraction rate of PCBPs under these conditions was 13.70 ± 0.19%, which is close to the predicted value, indicating that the extraction process of monkey ear loop polysaccharides optimized using this response surface model is scientifically feasible.

[0132] 1.3.6 Results of Single-Factor Experiments on Multi-Frequency Composite Ultrasonic Assisted Water Extraction Method

[0133] 1.3.6.1 Effect of ultrasonic power on monkey earring extraction rate

[0134] The power of the fixed-tank ultrasonic device was 240W, the liquid-to-solid ratio was 30mL / g, and the ultrasonic time was 10min. The power of the probe-type ultrasonic device was varied to investigate the effect of the total power of multi-frequency ultrasonication on the polysaccharide extraction rate. The results are as follows: Figure 10As shown, when the total power of the multi-frequency ultrasound increased from 375W to 510W, the polysaccharide extraction rate increased to 9.84%, but the extraction rate gradually decreased when increasing from 510W to 555W. The raw material in the extraction container is simultaneously subjected to energy transmitted from the bottom up by the trough-type ultrasound device and from the top down by the probe-type ultrasound device. These two types of ultrasound energy synergistically break down cells, producing cavitation. With a certain increase in total power, the synergistic effect of the two types of ultrasound energy may be further enhanced, which is beneficial for polysaccharide dissolution. However, when the total power is too high, the two types of ultrasound energy may cancel each other out while degrading some polysaccharides, reducing the PCBPs extraction rate. Therefore, an ultrasound power of 510W is more suitable.

[0135] 1.3.6.2 Effect of ultrasound time on monkey earring extraction rate

[0136] The power of the trough-type ultrasonic device was set at 240W, and the power of the probe-type ultrasonic device was set at 270W, resulting in a total multi-frequency composite ultrasonic power of 510W. The liquid-to-solid ratio was 30mL / g. The effect of ultrasonic time on the extraction rate of PCBPs was studied. Figure 11 As shown, the extraction rate of PCBPs gradually increased with the extension of ultrasonic time, reaching a maximum of 10.28% at 8 minutes. After 8 minutes, the extraction rate began to decrease. This indicates that at 8 minutes of ultrasonication, the raw material cells were almost completely pulverized by the synergistic effect of the two ultrasonic energies, and the polysaccharides were fully diffused into the solvent. However, excessively long ultrasonication caused some polysaccharides to degrade, resulting in a decrease in the polysaccharide extraction rate. Therefore, this invention selected an ultrasonic time of 8 minutes.

[0137] 1.3.6.3 Effect of ultrasonic liquid-to-solid ratio on monkey earring extraction rate

[0138] The power of the trough-type ultrasonic device was set at 240W, and the power of the probe-type ultrasonic device was set at 270W, resulting in a total multi-frequency composite ultrasonic power of 510W. The ultrasonic treatment time was 8 minutes to study the effect of the liquid-to-solid ratio on the polysaccharide extraction rate. Figure 12 It can be seen that as the liquid-to-solid ratio increases, the polysaccharide extraction rate gradually increases and then decreases, reaching a maximum of 9.98% at 30 mL / g. This is consistent with the results of both tank-type and probe-type ultrasonic devices; regardless of whether it is single-frequency or multi-frequency, the optimal liquid-to-solid ratio for PCBP extraction is 30 mL / g. Therefore, a liquid-to-solid ratio of 30 mL / g was chosen.

[0139] 1.3.7 Optimization of Polysaccharide Extraction Process Using Multi-Frequency Composite Ultrasonic Response Surface

[0140] Based on the results of the single-factor experiments, the ultrasonic time (A, 6, 8, 10 min), liquid-to-solid ratio (B, 20, 30, 40 mL / g), and ultrasonic power (C, 465, 510, 555 W) were selected as independent variables, and the PCBPs extraction rate was selected as the response value. Response surface methodology was conducted, and the experimental design and results are shown in Tables 9-11.

[0141] Table 9 Experimental Factors and Levels

[0142]

[0143]

[0144] Table 10 Results of the Box-Behnken Test

[0145]

[0146] Table 11 Results of Analysis of Variance

[0147]

[0148] Note: p<0.01 indicates a highly significant difference; p<0.05 indicates a significant difference; p>0.05 indicates a non-significant difference.

[0149] Table 11 shows that the regression model's p-value is <0.0001 (<0.05), indicating that the experimental model has statistical significance. The p-value for the lack-of-fit term is 0.2751 (>0.05), indicating that the lack-of-fit term is not significant compared to the pure error, further demonstrating that the model can be used for data analysis in this experiment. Model R 2 =0.9962, indicating that 99.62% of the variation can be explained by this model. Therefore, it can be used as an experimental model for optimizing the extraction process of monkey earring polysaccharide. The table shows that among the three factors A, B, and C, A (extraction time) and B (liquid-to-solid ratio) have extremely significant effects on the extraction rate (p<0.01), while C (extraction power) has no significant effect. Furthermore, the interaction between A and B is extremely significant (p<0.01), while the interaction between AC and BC is not significant. The lack of significant effect of C (extraction power) on the extraction rate may be because the two ultrasonic energies exhibit synergistic enhancement at lower power levels, while canceling each other out at higher power levels, leading to unstable cavitation. Moreover, the liquid-to-solid ratio in the response surface also significantly affects the cavitation formed by ultrasound. When the total power is constant and the liquid-to-solid ratio is high, the energy of the two ultrasounds will be further dispersed in the solvent, at which point the ultrasonic cavitation may be synergistically enhanced. The 3D response surface and two-dimensional contour plots of each factor are shown below. Figure 13 As shown. By Figure 13 As can be seen from 'a', the interaction between the material-liquid ratio and the extraction power is the strongest, and the corresponding contour lines are also the most curved. Figure 13b in Figure 13 In Figure c, the 3D response surface convexity and contour line curvature are both weaker than those in Figure a.

[0150] The optimal extraction parameters predicted by the analysis software were: extraction time 8.3 min, liquid-to-solid ratio 31.6 mL / g, and extraction power 511.35 W, with a predicted value of 9.99%. Considering practical conditions, the final extraction parameters were revised to: extraction time 8 min, liquid-to-solid ratio 32 mL / g, and extraction power 510 W. The results show that the actual extraction rate of PCBPs under these conditions was 9.90 ± 0.16%, which is close to the predicted value, indicating that the extraction process of monkey ear loop polysaccharides optimized using this response surface model is scientifically feasible.

[0151] 1.4 Summary

[0152] This invention investigates the effects of different process conditions (extraction time, material-to-liquid ratio, and extraction power) on the extraction rate of PCBPs using single-factor experiments in three methods: trough-type ultrasonic-assisted water extraction, probe-type ultrasonic-assisted water extraction, and multi-frequency composite ultrasonic water extraction. Response surface methodology is then used to optimize the extraction factors for the three ultrasonic-assisted water extraction methods. The experimental results of this invention are as follows:

[0153] (1) The single-factor experimental results of the ultrasonic-assisted water extraction method for PCPPs were as follows: the optimal extraction time was 30 min, the optimal liquid-to-solid ratio was 30 mL / g, and the optimal extraction power was 300 W. Using these results as a reference, the range of values ​​for the three factors in the response surface model was designed, and the optimal extraction process parameters for PCPPs were optimized to be: extraction time 31 min, liquid-to-solid ratio 33 mL / g, and extraction power 300 W. Verification of these process conditions showed that the final PCPPs extraction rate was 15.20 ± 0.36%.

[0154] (2) The single-factor experimental results of probe-type ultrasonic-assisted water extraction of PCPPs were as follows: optimal extraction time 14 min, optimal liquid-to-solid ratio 30 mL / g, and optimal extraction power 360 W. Using these results as a reference, the value ranges of the three factors in the response surface model were designed, and the optimal extraction process parameters for PCPPs were optimized to be: extraction time 14 min, liquid-to-solid ratio 32 mL / g, and extraction power 360 W. Verification of these process conditions showed that the final PCPPs extraction rate was 13.70 ± 0.19%.

[0155] (3) The single-factor experimental results of multi-frequency composite ultrasonic-assisted water extraction of PCPPs were as follows: the optimal extraction time was 8 min, the optimal liquid-to-solid ratio was 30 mL / g, and the optimal extraction power was 510 W. Using these results as a reference, the value range of the three factors in the response surface model was designed, and the optimal extraction process parameters for PCPPs were optimized to be: extraction time 8 min, liquid-to-solid ratio 32 mL / g, and extraction power 510 W. Verification of these process conditions showed that the final PCPPs extraction rate was 9.90 ± 0.16%.

[0156] (4) By comparing three different ultrasound-assisted water extraction methods, it can be concluded that: the trough-type ultrasound-assisted water extraction method has the highest extraction rate of PCPPs, but it takes the longest time and consumes the most energy; the probe-type ultrasound-assisted water extraction method has a slightly lower extraction rate than the trough-type method, but takes less time and consumes the least energy, about 1 / 4 of the trough-type method; the multi-frequency composite ultrasound-assisted water extraction method has a slightly lower extraction rate than the probe-type method, but it takes the least time, extracting a large amount of polysaccharides in just 8 minutes, and consumes about 1 / 3 of the energy of the trough-type method. Considering both energy consumption and time, the multi-frequency composite ultrasound-assisted water extraction method shows a significant synergistic enhancement effect of two different frequencies of ultrasound, and has a significant advantage over the trough-type and probe-type ultrasound-assisted water extraction methods for extracting PCPPs.

[0157] Example 2

[0158] Study on the in vitro antioxidant activity of monkey earring polysaccharide

[0159] Chemical substances formed when electrons are unpaired at the boundary orbitals of atoms or molecules are called free radicals. Free radicals with two or more unpaired valence electrons are called diradicals and multiradicals. Free radicals play an important role in life processes, participating in reactions such as substitution, addition, elimination, isomerization, recombination, and disproportionation in human cells. However, excessive free radicals can cause cellular oxidative stress, leading to cell damage. Oxidative stress is associated with various diseases of the nervous system, such as stroke, Parkinson's disease, and Alzheimer's disease. Therefore, developing a safe and effective free radical scavenger is urgently needed to reduce cellular oxidative stress caused by excess free radicals. Antioxidant activity is one of the representative activities of polysaccharides from monkey ear rings (PCBs). Even trace amounts of phytochemicals exhibit strong antioxidant activity, making the study and analysis of the free radical scavenging ability of PCBPs a necessary part of the research. This invention uses the scavenging ability against DPPH and ABTS free radicals as indicators to examine the antioxidant activity of PCBPs.

[0160] 2.1 Experimental Methods

[0161] 2.1.1 DPPH free radical scavenging experiment

[0162] Two mL of sample solutions at different concentrations (0.02, 0.04, 0.06, 0.08, and 0.1 mg / mL) were mixed with an equal volume of DPPH-ethanol solution (0.1 mmol / L). The mixtures were reacted in the dark for 30 min, and the absorbance of each group was measured at 517 nm. Ascorbic acid was used as a positive control. The sample groups consisted of 2 mL of DPPH and an equal volume of sample solution; the control group consisted of 2 mL of anhydrous ethanol and an equal volume of sample solution; and the blank group consisted of 2 mL of DPPH and an equal volume of anhydrous ethanol. The DPPH free radical scavenging rate was calculated using the following formula:

[0163]

[0164] Among them: A 样品组 A represents the absorbance value of the sample group at 517 nm. 对照组 A represents the absorbance value of the control group at 517 nm. 空白组 The absorbance value of the blank group at 517nm is given.

[0165] 2.1.2 ABTS Free Radical Scavenging Experiment

[0166] An equal volume of ABTS solution (7.0 mM) and potassium persulfate (2.45 mM) was mixed, and the mixture was reacted at room temperature in the dark for 12-16 h to obtain ABTS free radical cations (ABTS· + Then dilute ABTS with PBS buffer (pH 7.2). + The solution was prepared so that its absorbance at 734 nm was 0.70 ± 0.02. 1.0 mL of samples of different concentrations were mixed with 3.0 mL of ABTS· + The solutions were mixed and reacted in the dark at room temperature for 6 minutes. The absorbance was then measured at 734 nm. Ascorbic acid was used as a positive control. A1: Sample + ABTS + A2: Sample + PBS buffer, A0: PBS + ABTS + The ABTS radical scavenging rate is calculated using the following formula:

[0167]

[0168] 2.2 Results and Discussion

[0169] 2.2.1 DPPH free radical scavenging experiment

[0170] Depend on Figure 14 It can be seen that PCBPs have a strong scavenging ability for DPPH· in the range of 0.02-0.10 mg / mL, and the scavenging rate increases with the increase of concentration. At 0.10 mg / mL, the scavenging rate reaches 87.96%.

[0171] 2.3.2 ABTS Free Radical Scavenging Experiment

[0172] Depend on Figure 15 It can be seen that within the range of 0.04-0.08 mg / mL, PCBPs have a significant effect on ABTS. + It exhibits strong scavenging activity, and this activity shows a positive correlation with concentration. When the concentration of monkey earring polysaccharide is 0.08 mg / mL, its scavenging effect on ABTS... + The scavenging ability is almost 100%. The scavenging effect of monkey earring polysaccharide on ABTS·+ is stronger than that on DPPH·, which may be because the scavenging of DPPH· targets the transfer of H atoms, while the scavenging of ABTS·+ focuses on the transfer of electrons.

[0173] 2.3 Summary

[0174] This invention uses vitamin C as a positive control and examines the antioxidant activity of PCBPs through a free radical scavenging experiment. DPPH·, ABTS· + Scavenging experiments showed that PCBPs exhibited strong scavenging ability against free radicals, and within the designed concentration range, their scavenging rate showed a positive correlation with concentration. PCBPs also showed a strong scavenging ability against ABTS· + Its scavenging effect is stronger than its scavenging effect on DPPH·.

[0175] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for extracting polysaccharides from monkey earrings, characterized in that, Includes the following steps: A multi-frequency composite ultrasound device was used to extract monkey earrings using ultrasound. The multi-frequency composite ultrasound device was composed of a probe-type ultrasound device and a slot-type ultrasound device. The total ultrasound power of the probe-type ultrasound device and the slot-type ultrasound device was 375-555W.

2. The extraction method according to claim 1, characterized in that, The multi-frequency composite ultrasound device is assembled by mounting a probe-type ultrasound device on top of a trough-type ultrasound device; wherein the total ultrasound power of the probe-type ultrasound device and the trough-type ultrasound device is 510W.

3. The extraction method according to claim 1 or 2, characterized in that, The specific steps of the extraction method are as follows: Fresh monkey earring branches and leaves were crushed and sieved to obtain monkey earring powder; The monkey earring powder was mixed with water, and ultrasonic extraction was performed by starting a probe-type ultrasonic device and a tank-type ultrasonic device. After one centrifugation, a monkey earring polysaccharide extract was obtained. The monkey earlobe polysaccharide extract was concentrated, the concentrate was decolorized, centrifuged twice, the extract was deproteinized, and the supernatant was collected. Anhydrous ethanol was added to the supernatant, the mixture was frozen and allowed to stand overnight, centrifuged three times, dialyzed, and then freeze-dried under vacuum to obtain monkey earring polysaccharide.

4. The extraction method according to claim 3, characterized in that, The pulverization and sieving process involves passing the material through a 40-mesh sieve.

5. The extraction method according to claim 3, characterized in that, The ratio of monkey earring powder to water is 1g:32mL.

6. The extraction method according to claim 3, characterized in that, The parameters for the first centrifugation are: 5500 rpm for 10 min; the parameters for the second centrifugation are: 4500 rpm for 5 min; and the parameters for the third centrifugation are: 5500 rpm for 10 min.

7. The extraction method according to claim 2, characterized in that, During the ultrasonic extraction process, the power of the probe-type ultrasonic device is 270W, the power of the trough-type ultrasonic device is 240W, and the ultrasonic time is 8 minutes.

8. The extraction method according to claim 3, characterized in that, The decolorization process specifically refers to: adding activated carbon to the concentrated solution at a liquid-to-solid ratio of 30 mL: 1 g, stirring at 1000 rpm for 1 hour, and then stirring at 4500 rpm for 5 minutes to remove the activated carbon, and then combining the extracts.

9. The extraction method according to claim 3, characterized in that, The deproteinization process specifically refers to: adding 1 / 4 volume of Sevag reagent to the extract, shaking vigorously, centrifuging at 5500 rpm for 10 min, repeating the operation, and collecting the supernatant.

10. The extraction method according to claim 3, characterized in that, The volume ratio of the supernatant to anhydrous ethanol is 1:4.

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