Extraction method of pleurotus griseolus-stevia rebaudiana compound polysaccharide as well as product and application thereof

Abstract

The invention discloses an extraction method of a pleurotus griseolus-stevia rebaudiana compound polysaccharide as well as a product and application thereof. The preparation method is carried out according to the following steps: drying and crushing raw materials of horned mushroom and stevia rebaudiana, then mixing, adding 60-80% ethanol for degreasing and deglycosylation treatment, then drying, mixing with pure water, then putting into an ultrasonic cleaning machine, carrying out ultrasonic treatment, then centrifuging, and taking the supernatant to obtain a crude extract; carrying out rotary evaporation and concentration on the crude extract at 50-70 DEG C until the volume is 20-30% of the original volume; carrying out deproteinization, decoloration and alcohol precipitation treatment on the concentrated polysaccharide solution; and performing dialysis treatment after redissolving, and performing freeze drying to obtain the pleurotus griseolus-stevia rebaudiana compound polysaccharide. The antioxidant activity of the extracted pleurotus griseolus-stevia rebaudiana compound polysaccharide is better than that of polysaccharide extracted from a single raw material, which indicates that the antioxidant activity of the compound polysaccharide can be further improved through a molecular synergistic effect.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide extraction technology, specifically relating to an extraction method, product, and application of a compound polysaccharide from *Cephalotaxus fortunei* and *Stevia repens*. Background Technology

[0002] Grey Horn Mushroom ( Craterellus cornucopioides ) and Stevia ( Stevia rebaudiana As a high-value biological resource, stevia has attracted much attention due to its unique active ingredients. *Cephalotaxus fortunei*, an edible fungus, is rich in β-1,3 / 1,6-glucan and exhibits significant immunomodulatory and antioxidant activities. Stevia, a perennial herbaceous plant of the Asteraceae family, is rich in steviol glycosides, making it an ideal natural sweetener. In addition to glycosides, it is also rich in polysaccharides; studies have shown that stevia polysaccharides have significant antioxidant and hypoglycemic activities. Global annual stevia production exceeds 500,000 tons, but the industry has long focused on steviol glycoside extraction, resulting in the waste of 90% of the leaf residue and the ineffective utilization of its highly active polysaccharides. Complex polysaccharides may further enhance antioxidant and hypoglycemic activities through synergistic effects, showing great potential in the development of functional foods and drugs.

[0003] Currently, the extraction of polysaccharides from *Catella macrantha* and *Steviolus stevia* is mainly carried out through separate, stepwise extraction methods. *Catella macrantha* polysaccharides are primarily extracted using hot water extraction, while *Steviolus stevia* polysaccharides are extracted using ultrasound-assisted extraction. This stepwise extraction requires repeated equipment, resulting in low extraction efficiency and high costs. Furthermore, physical mixing offers no synergistic effect and limited activity enhancement. There are currently no reports on research into the functionally active polysaccharides extracted from the *Catella macrantha*-stevia complex. Summary of the Invention

[0004] The purpose of this invention is to provide a method for extracting polysaccharides from *Chorus aegyptius* and *Stevia repens*, as well as the resulting product and its applications.

[0005] A method for extracting polysaccharides from *Chorus aegyptius* and *Stevia repens* comprises the following steps: (1) Raw material pretreatment: Dry the raw materials of horn mushroom and stevia at 50-70℃ and pulverize them to 40-80 mesh. Then mix the dried horn mushroom and stevia powder and add 60-80% ethanol at a ratio of 1g:(8-12)mL for defatting and deglycosylation treatment. Then dry at 40-80℃. (2) The pretreated mixed powder was mixed with pure water at a ratio of 1g:(15-25)mL and placed in an ultrasonic cleaner. After ultrasonic treatment, the mixture was centrifuged and the supernatant was collected to obtain the crude extract. (3) Concentrate the crude extract to 20-30% of its original volume by rotary evaporation at 50-70℃; (4) The concentrated polysaccharide solution is subjected to deproteinization, decolorization, and alcohol precipitation; (5) After reconstitution, the mixture was dialyzed and then freeze-dried for 18-30 hours to obtain the Agaricus blazei-stevia complex polysaccharide.

[0006] The mass ratio of the dried horn mushroom to stevia powder in step (1) is (0.5-2):1.

[0007] The ultrasonic power in step (2) is 200-400W, and the processing time is 20-40min.

[0008] In step (2), the centrifugation speed is 4000-6000 rpm and the centrifugation time is 5-15 min.

[0009] The deproteinization process described in step (4) is as follows: Mix the polysaccharide concentrate with Sevage reagent (chloroform: n-butanol = 4:1) at a volume ratio of (2-6):1 and stir for 10-30 min. After stirring, centrifuge at 4000-6000 r / min and 4℃ for 8-12 min to extract the upper water layer (water layer-protein layer-organic layer). Repeat the deproteinization process 8-10 times, and then remove the organic reagent by rotary evaporation at 30-50℃.

[0010] The decolorization operation in step (4) is as follows: add AB-8 macroporous adsorption resin to the deproteinized polysaccharide solution, decolorize for 4-6 hours at 30-50℃ and 100-200 r / min, and filter. The amount of AB-8 macroporous adsorption resin added is 15-25% of the mass of the deproteinized polysaccharide solution.

[0011] The alcohol precipitation procedure in step (4) is as follows: the polysaccharide solution after deproteinization and decolorization is precipitated overnight with alcohol at 3-5 times its volume, then centrifuged at 5000 rpm for 8-12 min, the precipitate is collected, and the precipitate is reconstituted with distilled water.

[0012] The dialysis procedure in step (5) is as follows: the polysaccharide solution that has been redissolved after alcohol precipitation is added to a dialysis bag with a capacity of 3000-4000 Da and dialyzed with flowing water for 1-3 days, followed by dialysis with pure water for 0.5-1.5 days.

[0013] A complex polysaccharide of *Chorus aegyptium* and *Stevia repens* prepared by any of the above methods.

[0014] The application of the Agaricus blazei-Stevia complex polysaccharide in antioxidation.

[0015] The beneficial effects of this invention are as follows: This invention extracts complex polysaccharides from *Cephalotaxus fortunei* and *Stemisia scoparia* using an ultrasound-assisted extraction method. Ultrasound-assisted extraction completely dissolves the substances that make up the cell wall, making it easier to extract intracellular substances and increasing the polysaccharide yield. The antioxidant activity of the *Cephalotaxus fortunei*-stevia complex polysaccharide extracted by this invention is better than that extracted from single raw materials, indicating that the complex polysaccharide may further enhance its antioxidant activity through molecular synergy (such as hydrogen bonding and receptor complementarity). Both *Cephalotaxus fortunei* and *Stemisia scoparia* possess a variety of biological activities and pharmacological effects. The complex polysaccharide extracted by combining the two may have a wider range of biological activities and pharmacological effects, such as enhancing immune responses, anti-tumor activity, anti-aging, and antioxidant activity. This provides broad prospects for the application of this product in the fields of medicine and health food. Attached Figure Description

[0016] Figure 1 The results show the ABTS free radical scavenging rates of CC-SRP, CCP, and SRP at different mass concentrations in Test Example 1.

[0017] Figure 2 The results show the DPPH free radical scavenging rates of CC-SRP, CCP, and SRP at different mass concentrations in Test Example 1.

[0018] Figure 3 The results show the scavenging rates of hydroxyl radicals by CC-SRP, CCP, and SRP at different mass concentrations in Test Example 1. Detailed Implementation

[0019] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0020] Example 1: Preparation of *Cephalotaxus fortunei*-stevia complex polysaccharide (CC-SRP) The preparation method of Agaricus blazei-stevia complex polysaccharide (CC-SRP) is carried out according to the following steps: (1) Dry the raw materials of horn mushroom and stevia in an oven at 60℃ to constant weight, crush them with a crusher and pass them through a 60-mesh sieve; mix the dried horn mushroom and stevia powder in a 1:1 ratio, stir for 1 h at 60℃ with 70% ethanol as solvent, and dry at 60℃ to obtain the pretreated mixed powder, wherein the ratio of the mixed powder to 70% ethanol is 1:10 (g / mL). (2) Mix 100 g of the pretreated mixed powder with pure water at a ratio of 1:20 (g / mL) and place it in an ultrasonic cleaner. Sonicate at 300 W for 30 min, centrifuge (5000 rpm, 10 min) and take the supernatant to obtain the crude extract. (3) The crude extract was concentrated by rotary evaporation at 60℃ to obtain 200 mL of polysaccharide concentrate; (4) Mix the polysaccharide concentrate and Sevage reagent (chloroform: n-butanol = 4:1) in a volume ratio of 4:1 and stir for 20 min. After stirring, centrifuge at 5000 r / min and 4℃ for 10 min to extract the upper water layer (water layer-protein layer-organic layer). Repeat the operation 8 times. Remove the organic reagent by rotary evaporation at 40℃. Add 20% of the mass of the polysaccharide concentrate aqueous solution to the pretreated macroporous resin, place in a shaker at 40℃, decolorize at 200 r / min for 5 h, and filter. After collection, add 4 times the volume of anhydrous ethanol and let stand overnight at 4℃. (5) Finally, the precipitate obtained by alcohol precipitation was fully reconstituted with distilled water, dialyzed with running water in a 3500 Da dialysis bag for 2 days, dialyzed with pure water for 1 day, and then freeze-dried for 1 day to obtain the Agaricus blazei-stevia complex polysaccharide sample, named CC-SRP.

[0021] Comparative Example 1: Preparation of Polysaccharide from *Chorium argenteum* (CCP) The preparation method of *Chorus agaricus* polysaccharide (CCP) is carried out according to the following steps: (1) Dry the raw material of horn mushroom in an oven at 60℃ to constant weight, crush it with a crusher and pass it through a 60-mesh sieve; stir the dried horn mushroom powder at 60℃ with 70% ethanol as solvent for 1 h, and dry it at 60℃ to obtain the pretreated powder, wherein the ratio of powder to 70% ethanol is 1:10 (g / mL). (2) Mix 100 g of the pretreated powder with pure water at a ratio of 1:20 (g / mL) and place it in an ultrasonic cleaner. Ultrasonic treatment is performed at 300 W for 30 min. The supernatant is collected by centrifugation (5000 rpm, 10 min) to obtain the crude extract. (3) The crude extract was concentrated by rotary evaporation at 60℃ to obtain 200 mL of polysaccharide concentrate; (4) Mix the polysaccharide concentrate and Sevage reagent (chloroform: n-butanol = 4:1) in a volume ratio of 4:1 and stir for 20 min. After stirring, centrifuge at 5000 r / min and 4℃ for 10 min to extract the upper water layer (water layer-protein layer-organic layer). Repeat the operation 8 times. Remove the organic reagent by rotary evaporation at 40℃. Add 20% of the mass of the polysaccharide concentrate aqueous solution to the pretreated macroporous resin, place in a shaker at 40℃, decolorize at 200 r / min for 5 h, and filter. After collection, add 4 times the volume of anhydrous ethanol and let stand overnight at 4℃. (5) Finally, the precipitate obtained by alcohol precipitation was fully reconstituted with distilled water, dialyzed with running water in a 3500 Da dialysis bag for 2 days, dialyzed with pure water for 1 day, and then freeze-dried for 1 day to obtain the polysaccharide sample of Agaricus blazei, which was named CCP.

[0022] Comparative Example 2: Preparation of Stevia polysaccharide (SRP) The preparation method of stevia polysaccharide (SRP) is carried out according to the following steps: (1) The stevia raw material was dried in an oven at 60°C to constant weight, crushed by a crusher and passed through a 60-mesh sieve; the dried stevia powder was stirred at 60°C with 70% ethanol as solvent for 1 h, and dried at 60°C to obtain the pretreated powder, wherein the ratio of powder to 70% ethanol was 1:10 (g / mL). (2) Mix 100 g of the pretreated powder with pure water at a ratio of 1:20 (g / mL) and place it in an ultrasonic cleaner. Ultrasonic treatment is performed at 300 W for 30 min. The supernatant is collected by centrifugation (5000 rpm, 10 min) to obtain the crude extract. (3) The crude extract was concentrated by rotary evaporation at 60℃ to obtain 200 mL of polysaccharide concentrate; (4) Mix the polysaccharide concentrate and Sevage reagent (chloroform: n-butanol = 4:1) in a volume ratio of 4:1 and stir for 20 min. After stirring, centrifuge at 5000 r / min and 4℃ for 10 min to extract the upper water layer (water layer-protein layer-organic layer). Repeat the operation 8 times. Remove the organic reagent by rotary evaporation at 40℃. Add 20% of the mass of the polysaccharide concentrate aqueous solution to the pretreated macroporous resin, place in a shaker at 40℃, decolorize at 200 r / min for 5 h, and filter. After collection, add 4 times the volume of anhydrous ethanol and let stand overnight at 4℃. (5) Finally, the precipitate obtained by alcohol precipitation was fully reconstituted with distilled water, dialyzed with running water in a 3500 Da dialysis bag for 2 days, dialyzed with pure water for 1 day, and then freeze-dried for 1 day to obtain the stevia polysaccharide sample, which was named SRP.

[0023] The three polysaccharides (CC-SRP, CCP, and SRP) were compared in terms of polysaccharide yield and basic components. The results are shown in Table 1.

[0024] Table 1 Comparison of yield and purity of leaf polysaccharides from three types of stevia

[0025] By comparing the three polysaccharides, Table 1 shows that the yields of CC-SRP, CCP, and SRP are not significantly different, and the purity of the three polysaccharides is also similar.

[0026] Test Example 1 This test example uses DPPH free radical scavenging activity, ABTS free radical scavenging ability and hydroxyl free radical scavenging ability as indicators to evaluate the antioxidant properties of the three polysaccharides extracted in Example 1, Comparative Example 1 and Comparative Example 2.

[0027] CC-SRP, CCP, and SRP polysaccharides were dissolved in water to prepare sample solutions of different concentrations (1, 2, 3, 4, and 5 mg / mL) and vitamin C, with vitamin C serving as a positive control group.

[0028] (1) ABTS free radical scavenging ability A mixture of 7.4 mmol / L ABTS solution and 2.6 mmol / L potassium persulfate solution at a 1:1 (v / v) ratio was pre-reacted in the dark for 12 h. The mixture was then adjusted with anhydrous ethanol to an absorbance of 0.70 ± 0.20 (734 nm) to obtain the ABTS radical working solution. 0.2 mL of polysaccharide solutions of different concentrations and 3.8 mL of the ABTS radical working solution were mixed thoroughly and allowed to stand at room temperature in the dark for 10 min. The absorbance was measured at 734 nm. The ABTS radical scavenging rate was calculated using the following formula: ABTS radical scavenging rate (%) = (1 - A1−A2 / A0) × 100%. Where: A1 - sample absorbance; A2 - absorbance measured with anhydrous ethanol instead of the ABTS working solution; A0 - absorbance measured with anhydrous ethanol instead of the polysaccharide solution.

[0029] result Figure 1 As shown, within the range of 1-5 mg / mL, the ABTS radical scavenging rate of the three polysaccharides was dose-dependent with respect to their concentration. The ABTS radical scavenging abilities of the different polysaccharides differed significantly (p < 0.05). At a concentration of 5 mg / mL, CC-SRP exhibited the strongest ABTS radical scavenging ability, at 75.63%, significantly higher than the other two polysaccharides.

[0030] (2) DPPH free radical scavenging ability Prepare 0.2 mmol / L DPPH-anhydrous ethanol solution and polysaccharide solutions of different concentrations (2, 4, 6, 8, 10 mg / mL). Vortex mix 1 mL of each polysaccharide solution with 1 mL of DPPH-anhydrous ethanol solution, then incubate in the dark for 30 min. The absorbance at 517 nm is then measured using a microplate reader. The DPPH free radical scavenging rate is calculated using the following formula: DPPH free radical scavenging rate (%) = (1 - A1−A2 / A0)×100%. Where: A1 - absorbance of 1 mL polysaccharide solution + 1 mL DPPH-anhydrous ethanol solution; A2 - absorbance of 1 mL sample solution + 1 mL anhydrous ethanol solution; A0 - absorbance of 1 mL DPPH-anhydrous ethanol solution + 1 mL distilled water.

[0031] The results are as follows Figure 2 As shown, within the range of 1-5 mg / mL, the DPPH radical scavenging rates of the three polysaccharides were dose-dependent with respect to their concentration. At a polysaccharide concentration of 5 mg / mL, the DPPH radical scavenging rates of CC-SRP and SRP were 92.52% and 90.31%, respectively, with no significant difference (p < 0.05), and were very close to the DPPH radical scavenging capacity of VC.

[0032] (3) Hydroxyl radical scavenging ability Take 1 mL each of polysaccharide solution of different concentrations, 10 mmol / L FeSO4 solution, 10 mmol / L salicylic acid-ethanol solution, and 10 mmol / L H2O2 solution, mix well, and incubate at 37℃ for 30 min. Then measure the absorbance at 510 nm. Calculate the hydroxyl radical scavenging rate using the following formula: Hydroxyl radical scavenging rate (%) = (1 - A1−A2 / A0) × 100%. Where: A1 - sample absorbance; A2 - absorbance measured with distilled water instead of polysaccharide solution; A0 - absorbance measured with distilled water instead of H2O2 solution.

[0033] The results are as follows Figure 3 As shown, within the range of 1-5 mg / mL, the hydroxyl radical scavenging rate of the three polysaccharides was dose-dependent with respect to their concentration. At a concentration of 5 mg / mL, CC-SRP exhibited a hydroxyl radical scavenging rate of 92.59%, which was significantly higher than that of SRP and CCP by approximately 12.05%.

[0034] In conclusion, the antioxidant activity of polysaccharides extracted from the Agaricus blazei-stevia complex was significantly enhanced, confirming that the antioxidant activity of polysaccharides can be improved through synergistic effects.

[0035] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for extracting polysaccharides from *Cephalotaxus fortunei* and *Stevia repens*, characterized in that... Follow these steps: (1) Raw material pretreatment: Dry the raw materials of horn mushroom and stevia at 50-70℃ and pulverize them to 40-80 mesh. Then mix the dried horn mushroom and stevia powder and add 60-80% ethanol at a ratio of 1g:(8-12)mL for defatting and deglycosylation treatment. Then dry at 40-80℃. (2) The pretreated mixed powder was mixed with pure water at a ratio of 1g:(15-25)mL and placed in an ultrasonic cleaner. After ultrasonic treatment, the mixture was centrifuged and the supernatant was collected to obtain the crude extract. (3) Concentrate the crude extract to 20-30% of its original volume by rotary evaporation at 50-70℃; (4) The concentrated polysaccharide solution is subjected to deproteinization, decolorization, and alcohol precipitation; (5) After reconstitution, the mixture was dialyzed and then freeze-dried for 18-30 hours to obtain the Agaricus blazei-stevia complex polysaccharide.

2. The extraction method of the *Cephalotaxus fortunei*-stevia complex polysaccharide according to claim 1, characterized in that, The mass ratio of the dried horn mushroom to stevia powder in step (1) is (0.5-2):

1.

3. The extraction method of the *Cephalotaxus fortunei*-stevia complex polysaccharide according to claim 1, characterized in that, The ultrasonic power in step (2) is 200-400W, and the processing time is 20-40min.

4. The extraction method of the *Cephalotaxus fortunei*-stevia complex polysaccharide according to claim 1, characterized in that, In step (2), the centrifugation speed is 4000-6000 rpm and the centrifugation time is 5-15 min.

5. The extraction method of the *Cephalotaxus fortunei*-stevia complex polysaccharide according to claim 1, characterized in that, The deproteinization process described in step (4) is as follows: after mixing polysaccharide concentrate and Sevage reagent in a volume ratio of (2-6):1, stir for 10-30 min. After stirring, centrifuge at 4000-6000 r / min and 4℃ for 8-12 min, extract the upper water layer, repeat the deproteinization process 8-10 times, and then remove the organic reagent by rotary evaporation at 30-50℃.

6. The extraction method of the *Cephalotaxus fortunei*-stevia complex polysaccharide according to claim 1, characterized in that, The decolorization operation in step (4) is as follows: add AB-8 macroporous adsorption resin to the deproteinized polysaccharide solution, decolorize for 4-6 hours at 30-50℃ and 100-200r / min, and filter. The amount of AB-8 macroporous adsorption resin added is 15-25% of the mass of the deproteinized polysaccharide solution.

7. The extraction method of the *Cephalotaxus fortunei*-stevia complex polysaccharide according to claim 1, characterized in that, The alcohol precipitation procedure in step (4) is as follows: the polysaccharide solution after deproteinization and decolorization is precipitated overnight with alcohol at 3-5 times its volume, then centrifuged at 5000 rpm for 8-12 min, the precipitate is collected, and the precipitate is reconstituted with distilled water.

8. The extraction method of the *Cephalotaxus fortunei*-stevia complex polysaccharide according to claim 1, characterized in that, The dialysis procedure in step (5) is as follows: the polysaccharide solution that has been redissolved after alcohol precipitation is added to a dialysis bag with a capacity of 3000-4000 Da and dialyzed with flowing water for 1-3 days, followed by dialysis with pure water for 0.5-1.5 days.

9. A compound polysaccharide of *Chorus aegyptium* and *Stevia repens* prepared by the preparation method of any one of claims 1-8.

10. The application of the Agaricus blazei-Stevia complex polysaccharide according to claim 9 in antioxidant activity.

Images