Green preparation method of passion fruit juice polysaccharide with anti-aging activity

Abstract

The invention belongs to the technical field of plant polysaccharide extraction and separation, and particularly relates to a green preparation method of passion fruit juice polysaccharide with anti-aging activity. The preparation process of the passion fruit juice polysaccharide is green and safe, no other auxiliaries are introduced, and compared with similar polysaccharides, the obtained passion fruit juice polysaccharide has the following advantages that 1, the passion fruit juice polysaccharide is excellent in water solubility, and the problem that similar astragalus polysaccharide is low in biological activity due to poor water solubility is solved; 2) the passion fruit polysaccharide has an excellent scavenging effect on ABTS free radicals, the free radical scavenging rate is up to 98.85%, and the median inhibitory concentration (IC50) is 1.00 mg / mL and is superior to that of reported ganoderma lucidum polysaccharide and lycium barbarum polysaccharide; in addition, when the polysaccharide concentration of the passion fruit juice prepared by the method is 1mg / mL, the survival time of the nematodes is prolonged by 16.41%. The passion fruit juice polysaccharide prepared by the method has excellent development advantages in the field of food and medicine with anti-aging activity.

Description

Technical Field

[0001] The invention belongs to the technical field of plant polysaccharide extraction and separation, and particularly relates to a green preparation method of passion fruit juice polysaccharide with anti-aging activity. Background Art

[0002] Passion fruit (Passiflora edulis), also known as passionflower, is a herbaceous vine of the genus Passiflora. Traditional medicine believes that passion fruit has the effects of clearing heat, promoting salivation, calming the nerves, and promoting sleep. Modern pharmacological research has shown that it possesses antioxidant, immunomodulatory, anti-inflammatory, and anti-tumor activities. It contains a variety of active ingredients, including amino acids, proteins, polysaccharides, flavonoids, and organic acids, with polysaccharides being the most abundant, reaching a content of 34,844.77 to 96,138.98 mg / kg. Polysaccharides are a class of biomacromolecules widely found in plants, microorganisms, and animals. Numerous pharmacological studies have demonstrated that polysaccharides possess a variety of biological activities, including antioxidant, anti-radiation, immune-enhancing, anti-tumor, anti-aging, and blood sugar-lowering properties. Numerous studies have shown that most of the biological activities and functions of polysaccharides are related to antioxidant activity. Antioxidation refers to the process by which organisms use a series of mechanisms to counteract the oxidative effects of free radicals, thereby protecting cells and tissues from damage. Free radicals are also considered a key factor in aging. Antioxidant supplementation can effectively combat free radical damage and thus slow aging. Natural plant polysaccharides have attracted attention due to their safety, effectiveness, and minimal side effects, and are widely used in the anti-aging field.

[0003] In recent years, research on passion fruit polysaccharides has mainly focused on passion fruit peel polysaccharides, while research on passion fruit juice polysaccharides has been relatively limited. Therefore, if polysaccharides with antioxidant and anti-aging activities can be extracted from passion fruit juice, it will be beneficial to improve the production value of passion fruit. Summary of the Invention

[0004] In order to overcome the deficiencies of the above-mentioned prior art, the present invention proposes a green preparation method for passion fruit juice polysaccharide with anti-aging activity, extracts and separates passion fruit juice polysaccharide, and proves that it has excellent antioxidant and anti-aging activity, laying a theoretical foundation for the further development and utilization of passion fruit juice polysaccharide.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides a green preparation method of passion fruit juice polysaccharide, specifically: filtering fresh passion fruit pulp to obtain juice, then adding ethanol to the juice to an alcohol concentration of 60% to 80%, collecting the precipitate after standing, then adding water to the precipitate for washing, and collecting the supernatant; finally, pre-cooling the supernatant at -80°C, and then drying to obtain passion fruit juice polysaccharide PFJP.

[0007] Compared with the existing technology (such as the preparation method of jujube polysaccharide with antioxidant activity disclosed in Chinese invention patent CN119409847A), the entire preparation process of the present invention is green and environmentally friendly, without the introduction of other auxiliary agents or additives, and is safer. Compared with astragalus polysaccharides (such as Chinese invention patent CN 117700575 A, a highly water-soluble and antioxidant carboxymethyl astragalus polysaccharide and its preparation method; Chinese invention patent CN 117510664A, a glutamic acid-modified astragalus polysaccharide and its preparation method and antioxidant application), the passion fruit juice polysaccharide obtained by the present invention not only has excellent performance but also has a significant water solubility advantage compared with similar plant polysaccharides with anti-aging activity. However, astragalus polysaccharide has poor water solubility, resulting in low biological activity, which greatly limits its application. In order to enhance the water solubility of modified astragalus polysaccharides (such as carboxymethyl astragalus polysaccharide and glutamic acid astragalus polysaccharide), its preparation process requires the addition of large amounts of acid-base regulators and organic solvents such as isopropyl alcohol and epichlorohydrin. Compared with Ganoderma lucidum polysaccharide (such as Ren Hongfei. Extraction, purification, structural analysis and antioxidant activity of Ganoderma lucidum polysaccharide [D]. Liaocheng University.), in vitro antioxidant experiments show that the passion fruit juice polysaccharide prepared by the present invention has an excellent scavenging effect on ABTS free radicals, and its half inhibitory concentration (IC 50 ) was 1.00 mg / mL, which was superior to the ion chromatography Ganoderma lucidum polysaccharide (its ABTS free radical half-inhibition concentration (IC 50 ) is 2.0-2.07 mg / mL); compared with wolfberry polysaccharides (such as Lu Genglin. Study on the extraction, separation, purification and functional properties of wolfberry polysaccharides [D]. South China University of Technology, 2023.), the passion fruit juice polysaccharide prepared by the present invention has a scavenging rate of ABTS free radicals of up to 98.85% at 8 mg / mL, and its antioxidant capacity is significantly better than that of wolfberry polysaccharides (its scavenging rate at 20 mg / mL is 73.39%). In addition, the passion fruit juice polysaccharide prepared by the present invention can significantly improve the survival rate of Caenorhabditis elegans. When the concentration of passion fruit juice polysaccharide is 1 mg / mL, the survival time of nematodes is extended by 16.51%. It shows that the passion fruit juice polysaccharide prepared by the present invention has excellent antioxidant and anti-aging activity, and has excellent development advantages in the field of health products and medicines.

[0008] Preferably, the passion fruit includes yellow-skin passion fruit and purple-skin passion fruit.

[0009] Preferably, the standing temperature is 4 to 10° C., and the standing time is 40 to 48 hours.

[0010] Preferably, adding water to the precipitate for washing is specifically as follows: dispersing the precipitate in 10-30 times the volume of water, shaking the dispersion for washing, and centrifuging to collect the supernatant, and repeating this process 3-5 times in total.

[0011] Preferably, the drying is vacuum freeze drying, the cold trap temperature of the vacuum freeze dryer is set to -50 to -60°C, the vacuum degree is set to 0.3 to 0.5 mbar, and the drying time is 20 to 30 hours.

[0012] The second aspect of the present invention provides passion fruit juice polysaccharide prepared by the preparation method described in the first aspect.

[0013] The third aspect of the present invention provides the use of the passion fruit juice polysaccharide described in the second aspect in the preparation of anti-aging products.

[0014] Preferably, the anti-aging products include anti-aging health products and anti-aging medicines.

[0015] Preferably, the anti-aging effect is achieved by scavenging ABTS free radicals to exert antioxidant effects.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The entire polysaccharide preparation process of the present invention is green and safe, without the introduction of other additives, resulting in green and safe passion fruit juice polysaccharides. In vitro antioxidant experiments (ABTS free radical scavenging experiments) demonstrated that the passion fruit juice polysaccharide prepared by the present invention had an excellent scavenging effect on ABTS free radicals. Animal experiments also demonstrated that the passion fruit juice polysaccharide prepared by the present invention significantly improved the survival rate of Caenorhabditis elegans, extending the nematode survival time by 16.51% at a passion fruit juice polysaccharide concentration of 1 mg / mL. This demonstrates that the passion fruit juice polysaccharide prepared by the present invention has excellent antioxidant and anti-aging activities, and has excellent development advantages in the fields of health products and pharmaceuticals. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the appearance of the passion fruit juice polysaccharide PFJP prepared in Example 1;

[0019] Figure 2 This is the infrared spectrum of PFJP prepared in Example 1;

[0020] Figure 3 This is a graph showing the in vitro antioxidant activity of PFJP prepared in Example 1 in scavenging ABTS free radicals;

[0021] Figure 4 This is the survival curve of the PFJP lifespan test prepared in Example 1. DETAILED DESCRIPTION

[0022] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0023] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0024] Example 1: A green preparation method of passion fruit juice polysaccharide (PFJP) with anti-aging activity

[0025] (1) Take fresh yellow-skin passion fruit pulp and filter it through 200-mesh gauze to obtain juice.

[0026] (2) Anhydrous ethanol was quickly added to the passion fruit juice until the alcohol concentration (volume concentration) was 70%, and the mixture was allowed to stand at 4°C for 48 hours. The standing liquid was centrifuged at 8000 r / min for 10 minutes, and the precipitate was collected. The precipitate was then dispersed in 10 times the volume of water, washed by oscillation and centrifuged. After the washing and centrifugation process was repeated 3 times, the washed supernatant was collected and the three supernatants were combined. The supernatant was placed at -80°C for pre-cooling for more than 2 hours, and then freeze-dried (the vacuum freeze dryer was set to a cold trap temperature of -57°C, a vacuum setting value of 0.370 mbar, and a drying time of 24 hours) to obtain passion fruit juice polysaccharide ( Figure 1 ).

[0027] Example 2: A green preparation method of passion fruit juice polysaccharide (PFJP) with anti-aging activity

[0028] (1) Take fresh yellow-skin passion fruit pulp and filter it through 200-mesh gauze to obtain juice.

[0029] (2) Anhydrous ethanol was quickly added to the passion fruit juice until the alcohol concentration was 70%, and the mixture was allowed to stand at 4°C for 48 hours. The standing liquid was centrifuged at 8000 r / min for 20 minutes, and the precipitate was collected. The precipitate was then dispersed in 10 times the volume of water, washed by oscillation and centrifuged. After the washing and centrifugation process was repeated three times, the washed supernatant was collected, the three supernatants were combined, and then freeze-dried (the vacuum freeze dryer was set to a cold trap temperature of -57°C, a vacuum setting value of 0.370 mbar, and a drying time of 24 hours) to obtain passion fruit juice polysaccharide.

[0030] Example 3: A green preparation method of passion fruit juice polysaccharide (PFJP) with anti-aging activity

[0031] (1) Take fresh yellow-skin passion fruit pulp and filter it through 200-mesh gauze to obtain juice.

[0032] (2) Anhydrous ethanol was quickly added to the passion fruit juice until the alcohol concentration was 70%, and the mixture was allowed to stand at 4°C for 48 hours. The standing liquid was centrifuged at 8000 r / min for 20 minutes, and the precipitate was collected. The precipitate was then dispersed in 20 times the volume of water, washed by oscillation dispersion, and centrifuged. After the washing and centrifugation process was repeated three times, the washed supernatant was collected, the three supernatants were combined, and then freeze-dried (the vacuum freeze dryer was set to a cold trap temperature of -57°C, a vacuum setting value of 0.370 mbar, and a drying time of 24 hours) to obtain passion fruit juice polysaccharide.

[0033] Example 4: A green preparation method of passion fruit juice polysaccharide (PFJP) with anti-aging activity

[0034] (1) Take fresh yellow-skin passion fruit pulp and filter it through 200-mesh gauze to obtain juice.

[0035] (2) Anhydrous ethanol was quickly added to the passion fruit juice until the alcohol concentration was 70%, and the mixture was allowed to stand at 4°C for 48 hours. The standing liquid was centrifuged at 8000 r / min for 10 minutes, and the precipitate was collected. The precipitate was then dispersed in 20 times the volume of water, washed by oscillation and centrifuged. After the washing and centrifugation process was repeated three times, the washed supernatant was collected, the three supernatants were combined, and then freeze-dried (the vacuum freeze dryer was set to a cold trap temperature of -57°C, a vacuum setting value of 0.370 mbar, and a drying time of 24 hours) to obtain passion fruit juice polysaccharide.

[0036] Experimental Example 1: Infrared Spectral Analysis of PFJP

[0037] (1) Experimental method: The functional group structure of polysaccharide was analyzed by Fourier infrared spectroscopy. The polysaccharide and dried potassium bromide were fully ground and pressed into tablets. -1 Scan within the wave number to obtain the infrared spectrum.

[0038] (2) Experimental results: 3280cm -1 OH stretching vibration absorption peak, 2924 cm -1 is the stretching vibration peak of CH, 1635cm -1 is the stretching vibration of -OOC, 1024 cm -1 The above are typical characteristic peaks of polysaccharides. 860cm -1 This may be due to the vibration of the α-glycosidic bond ( Figure 2 ).

[0039] Experimental Example 2: ABTS free radical scavenging ability of PFJP

[0040] (1) Experimental method: Preparation of ABTS+ working solution: Prepare 4.9mM potassium persulfate solution and 7mM ABTS solution, mix them in a volume ratio of 1:1, and place them in the dark at room temperature for 12 hours to obtain ABTS mother solution. Dilute it with anhydrous ethanol so that its absorbance at 734nm wavelength is 0.70±0.02 to prepare the working solution, and keep it in the dark for later use. Take 2mL of the working solution and mix it with 0.4mL of a series of gradient concentrations of PFJP polysaccharide solution, react at room temperature in the dark for 30 minutes, and measure the absorbance at 734nm. Deionized water is used as the blank control, V C As a positive control, calculate the ABTS free radical scavenging rate according to formula (1-1).

[0041] ABTS free radical scavenging rate (%) = (A0-A1+A2) / A0×100% (1-1)

[0042] A1 is sample + ABTS + ;

[0043] A2 is sample + water;

[0044] A0 is water + ABTS + .

[0045] (2) Experimental results: The scavenging rate of PFJP polysaccharide on ABTS free radicals was dose-dependent. With the increase of polysaccharide concentration, the scavenging ability gradually increased. The half inhibitory concentration (IC 50 ) is 1.00 mg / mL. When the polysaccharide content reaches 8 mg / mL, the clearance rate increases to 96.78±2.93%, which is basically consistent with the experimental results of Vc. This shows that PFJP has good free radical scavenging ability ( Figure 3 )

[0046] Experimental Example 3: Nematode lifespan test

[0047] 1. Experimental methods:

[0048] (1) Cultivation of Escherichia coli OP50

[0049] After thawing at room temperature, take out E. coli OP50 uracil-deficient strain (purchased from Caenorhabditis Genetics Center, CGC) stored at -80°C and culture it in LB liquid medium overnight until it grows to OD 600A value of 0.4-0.6 is sufficient. Then, apply 400 μL to a 90 mm dish and 200 μL to a 60 mm dish. When applying, ensure that the bacterial solution is approximately 0.5 cm from the edge of the dish. This prevents nematodes from adhering to the wall and being lost, while also reducing the risk of contamination of the culture medium. Wait until a biofilm forms on the culture medium. Store the NGM plate containing E. coli OP50 at 4°C for one week.

[0050] (2) Synchronization of nematodes

[0051] Select the period when most nematodes are in the process of laying eggs, wash the nematodes with M9 buffer solution into a 2 mL centrifuge tube, centrifuge at 5000 rpm / min for 5 minutes, aspirate the supernatant, wash the worm bodies at the bottom with M9 buffer solution twice to wash away impurities such as the culture medium, then add an appropriate amount of nematode lysis solution (2.5 mL 2M sodium hydroxide, 2 mL sodium hypochlorite, 5.5 mL pure water; prepared immediately before use), vortex for 1 minute and let it stand for 5 minutes. After the worm bodies lyse and turn yellow, centrifuge at the same speed (5000 rpm / min) for 3 minutes, then wash them twice with M9 solution to wash away the nematode lysis solution, and finally place the egg mixture on an NGM culture dish containing E. coli OP50 and culture at 20°C.

[0052] (3) Life test

[0053] In the drug administration group, 10 mg / mL PFJP polysaccharide solution was prepared with pure water, filtered and sterilized, and diluted to 1 mg / mL with OP50 bacterial solution (OD=0.4-0.6). In the control group, M9 buffer solution was used instead of polysaccharide solution.

[0054] After synchronization, L4 nematodes (30 per plate, at least 90 per group) were transferred to the control and treatment groups. The next day, the number of living and dead nematodes was counted until no more nematodes were left on the plates.

[0055] 2. Experimental results: PFJP polysaccharide can significantly improve the survival rate of Caenorhabditis elegans. When the concentration of passion fruit juice polysaccharide is 1 mg / mL, the survival time of nematodes is extended by 16.51% ( Figure 4 ).

[0056] In summary, the passion fruit juice polysaccharide PFJP prepared by the present invention not only has a green and harmless preparation process, but also has excellent antioxidant and anti-aging activities, and has excellent development advantages in the fields of health products and medicines.

[0057] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. A green preparation method of passion fruit juice polysaccharide, characterized in that: Juice is obtained by filtering fresh passion fruit pulp, and then ethanol is added to the juice to an alcohol concentration of 60% to 80%. After standing, a precipitate is collected, and then water is added to the precipitate for washing, and a supernatant is collected. Finally, the supernatant is pre-cooled at -80°C and dried to obtain passion fruit juice polysaccharide PFJP.

2. A green preparation method of passion fruit juice polysaccharide according to claim 1, characterized in that, The passion fruit includes yellow-skin passion fruit and purple-skin passion fruit.

3. A green preparation method of passion fruit juice polysaccharide according to claim 1, characterized in that, The temperature of the standing state is 4-10° C., and the time is 40-48 hours.

4. A green preparation method of passion fruit juice polysaccharide according to claim 1, characterized in that, The specific steps of adding water to the precipitate for washing are as follows: dispersing the precipitate in 10-30 times the volume of water, shaking the dispersion for washing, and centrifuging to collect the supernatant, and repeating the process 3-5 times.

5. A green preparation method of passion fruit juice polysaccharide according to claim 1, characterized in that, The drying is vacuum freeze drying, the cold trap temperature of the vacuum freeze dryer is set to -50 to -60°C, the vacuum degree is set to 0.3 to 0.5 mbar, and the drying time is 20 to 30 hours.

6. Passion fruit juice polysaccharide prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the passion fruit juice polysaccharide according to claim 6 in the preparation of anti-aging products.

8. The use according to claim 7, characterized in that The anti-aging products include anti-aging health products and anti-aging medicines.

9. The use according to claim 7, characterized in that The anti-aging effect is achieved by removing ABTS free radicals to exert antioxidant effects.

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