Kadsura pepper stem polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to kadsura pepper stem polysaccharide as well as a preparation process and application thereof. The kadsura pepper stem polysaccharide (KPSP) is extracted by taking kadsura pepper stem as a raw material and combining an ultrasonic-assisted extraction method with a compound enzyme enzymolysis method. The extraction process of the kadsura pepper stem polysaccharide is simple to operate, high in extraction rate and purity, time-saving, efficient and measurable in molecular weight, and the extraction system is mild. Results of the embodiment show that the extracted kadsura pepper stem polysaccharide can enhance the exercise ability and stress ability of CL4176 nematodes, and then the extracted kadsura pepper stem polysaccharide can be applied to preparation of products and / or drugs for improving the exercise ability and stress ability.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a Pieris fasciata polysaccharide and a preparation method and application thereof. Background Art

[0002] In recent years, with the continuous changes in the living environment and the accelerated pace of life, humans are generally under high pressure, which has led to the generation of negative emotions. Long-term negative emotions can easily lead to serious psychological problems. If the stressful situation persists, it is easy to experience depression, slow thinking, and decreased work and study memory. In severe cases, it can lead to problems such as decreased motor ability and stress resistance, which will have a serious impact on daily life and work. Different degrees of decreased motor and stress resistance can lead to a series of health problems, such as uncoordinated limb movements leading to falls, slow response to stimuli, and decreased memory.

[0003] Caenorhabditis elegans (C.elegans) is a tiny, transparent nematode with a thick head and a thin tail. In the laboratory, it usually uses Escherichia coli OP50 as a food source. It reproduces quickly, has a short life cycle, has high genetic homology, and is easy to culture. Researchers have found that up to 60% to 80% of the genes in CL4176 Caenorhabditis elegans have a high degree of homology with the human genome. In addition, the nematode's movement ability and stress response can be observed through a microscope, and the stress response, movement ability, and basic physiological processes of higher animals are all reflected in the CL4176 nematode model. Therefore, CL4176 nematodes have gradually become an important animal in drug screening research, which is of great significance to the research and development of medical undertakings.

[0004] Studies have shown that traditional Chinese medicine has significant advantages in the treatment of such diseases, providing a direction for the future research or development of new drugs that regulate motor and stress abilities. Kadsura pepper stem is the dried stem of Piper kadsura (Choisy) Ohwi., a plant of the Piperaceae family. It is pungent and bitter, slightly warm in nature, and belongs to the liver meridian. It has the effects of removing rheumatism, unblocking meridians, and stopping arthralgia. Therefore, it is often used by the public to treat symptoms such as wind-cold dampness and arthralgia, limb pain, etc. Chemical composition studies have shown that Kadsura pepper contains polysaccharides, lignans, alkaloids, volatile oils, flavonoids, epoxy compounds, steroids and other components, among which neolignans and amide alkaloids are its main chemical components. Modern pharmacological studies have shown that Kadsura pepper has anti-inflammatory, antioxidant, platelet activation factor inhibition and neuroprotective effects. Among them, Kadsura pepper stem polysaccharide (KPSP) is one of the main components of Kadsura pepper stem, has significant biological activity, plays an important role in physiological processes such as immune regulation and tissue protection, and is widely used in various health products and drugs. The resources of Pieris hirta should be further rationally developed and utilized, but problems such as low extraction rate and low purity have not yet been solved.

[0005] Therefore, it is urgent to develop a Pieris ovata polysaccharide using the traditional Chinese medicine raw material Pieris ovata. Summary of the invention

[0006] The object of the present invention is to provide a Pieris ovata polysaccharide and a preparation method thereof. The method provided by the present invention is not only simple to operate and has a mild extraction system, but also has a high extraction rate and high purity of the polysaccharide.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The invention provides a Pieris damicornis polysaccharide, which comprises five monosaccharides: Man, Rha, GlcA, GalA and Gal, and the molar ratio of the monosaccharides is 78.6:0.35:10.31:2.24:8.36.

[0009] Preferably, the Pieris ovata polysaccharide is extracted from Pieris ovata by combining ultrasound-assisted extraction and complex enzyme hydrolysis; the complex enzyme consists of papain and cellulose protease.

[0010] The present invention also provides a method for preparing the above-mentioned Kadzu clitoria polysaccharide, which specifically comprises the following steps:

[0011] S1. Petroleum ether is added to the Kadura dremel, and the Kadura dremel is defatted by Soxhlet extraction. Deionized water is added to the defatted Kadura dremel, and ultrasonic extraction is performed. The filtrate is hydrolyzed by a composite enzyme to obtain an enzymatic solution.

[0012] S2, precipitating the enzymatic hydrolyzate with alcohol, concentrating under reduced pressure, and freeze-drying to obtain crude polysaccharide of Pieris fasciata;

[0013] S3, dissolving the crude Kadura dremel polysaccharide and centrifuging it, taking the supernatant for separation and purification to obtain Kadura dremel polysaccharide;

[0014] The complex enzyme consists of papain and cellulose protease.

[0015] Preferably, in step S1, deionized water is added to the defatted Kadura rapa at a solid-liquid ratio of 1:15 to 25 g / mL.

[0016] Preferably, the added amount of the composite enzyme in step S1 is 3-7%; the volume ratio of papain and cellulose protease in the composite enzyme is 6:(4-9).

[0017] Preferably, the alcohol precipitation process in step S2 is: adding anhydrous ethanol to the enzymatic hydrolysate until the ethanol concentration reaches 60%, stirring evenly, standing at 4°C for 12 hours for alcohol precipitation, and centrifuging at 5000r / min for 15-25min.

[0018] Preferably, the separation and purification process in step S3 is to use distilled water to pass through a DEAE-650M cellulose chromatography column for separation and purification.

[0019] The present invention also provides the use of the above-mentioned Pieris d'Arcticum polysaccharide or the Pieris d'Arcticum polysaccharide prepared by the above-mentioned preparation method, and the Pieris d'Arcticum polysaccharide has the function of improving the movement ability and / or stress ability of CL4176 nematodes.

[0020] The present invention also provides the use of the above-mentioned Pieris ovata polysaccharide or the Pieris ovata polysaccharide prepared by the above-mentioned preparation method in the preparation of products and / or medicines for improving athletic ability.

[0021] The present invention also provides the use of the above-mentioned Pieris d'Arcticum polysaccharide or the Pieris d'Arcticum polysaccharide prepared by the above-mentioned preparation method in the preparation of products and / or medicines for improving stress resistance.

[0022] Beneficial effects of the present invention:

[0023] (1) The present invention adopts ultrasound-assisted composite enzymatic hydrolysis to extract polysaccharides, which solves the problems of low extraction rate and purity of Pieris ovata polysaccharides. At the same time, the extraction temperature is low, and the reaction conditions are mild, fast and efficient.

[0024] (2) The preparation method disclosed in the present invention has the advantages of simple operation, high extraction rate and purity, time saving and high efficiency, measurable molecular weight and mild extraction system.

[0025] (3) The DKPSP prepared by the present invention can enhance the movement ability and stress resistance of CL4176 Caenorhabditis elegans. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0027] Figure 1 The HPGPC spectrum of DKPSP prepared by the present invention;

[0028] Figure 2 This is a HPLC chart of the monosaccharide composition analysis of DKPSP prepared by the present invention, wherein: 1. Man, 2. Rha, 3. GlcA, 4. GalA, 5. Glc, 6. Gal, 7. Xyl, 8. Ara;

[0029] Figure 3 HPLC chart of the DKPSP monosaccharide standard derivative prepared by the present invention;

[0030] Figure 4 This is a graph of the number of head swings and body bends of Caenorhabditis elegans within 2 minutes;

[0031] Figure 5 This is a graph showing the heat stress response of Caenorhabditis elegans;

[0032] Figure 6 This is a graph showing the oxidative stress response of Caenorhabditis elegans. DETAILED DESCRIPTION

[0033] The invention provides a radix kadzusii polysaccharide, which is extracted from the traditional Chinese medicine radix kadzusii. The radix kadzusii polysaccharide prepared by the invention is composed of five monosaccharides, namely Man, Rha, GlcA, GalA and Gal, and the molar ratio of the monosaccharides is 78.6:0.35:10.31:2.24:8.36. According to the molecular weight and homogeneity measurements, the average molecular weight of the radix kadzusii polysaccharide prepared by the invention is 31796Da.

[0034] The present invention adds petroleum ether to the Pieris d'Arcticum and uses the Soxhlet extraction method to perform defatting. After defatting, deionized water is added to the Pieris d'Arcticum according to a solid-liquid ratio of 1:15-25 g / mL, preferably 1:20 g / mL. At the same time, an ultrasonic-assisted extraction method and a composite enzyme enzymolysis method are combined to fully extract the effective components of the Pieris d'Arcticum. The composite enzyme is composed of papain and cellulase, and the volume ratio of papain and cellulase is (4-6): (6-4), preferably 6:4; the addition amount of the composite enzyme is 3-7%, preferably 5%.

[0035] The enzymatic hydrolyzate was subjected to alcohol precipitation, reduced pressure concentration and freeze-drying to obtain crude polysaccharide of Pieris ovata, and then separated and purified by DEAE-650M cellulose chromatography column with distilled water to obtain DKPSP.

[0036] The present invention sets up multiple comparative examples to study the preparation process of Pieris ovata polysaccharide, and the results show that the preparation process of Pieris ovata polysaccharide of the present invention is simple and efficient, and the extraction rate and sugar content of Pieris ovata polysaccharide are higher than those of the comparative example.

[0037] The present invention studies the effect of Pieris damicornis polysaccharide on the locomotion and stress resistance of CL4176 Caenorhabditis elegans. The results of the examples show that the DKPSP extracted by the present invention can significantly improve the locomotion, heat adaptability and antioxidant stress resistance of the nematodes, and improve the survival state of the nematodes.

[0038] Heat stress refers to the sum of non-specific physiological responses of the body to any requirements of the thermal environment in high ambient temperature. Human physiological response to heat stress Under temperature stress environment, the normal thermal balance is disrupted, and the human body will produce a series of complex physiological and psychological changes. Whether it is human or animal, its body function will be affected by heat stress.

[0039] Oxidative stress (OS) refers to a state of imbalance between oxidation and anti-oxidation in the body, which tends to be oxidized, leading to inflammatory infiltration of neutrophils, increased secretion of proteases, and the production of a large number of oxidative intermediates. Oxidative stress is a negative effect produced by free radicals in the body and is considered an important factor leading to aging and disease.

[0040] Studying the effects of the effective substances of the traditional Chinese medicine Piper melongena on the motility, heat adaptation and anti-oxidative stress ability of nematodes will provide new directions for future research or development of new drugs and / or products that regulate motility and stress resistance.

[0041] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0043] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0044] Example 1 Preparation method of Pieris ovata polysaccharide

[0045] A method for preparing Kadzu clitoria polysaccharide comprises the following steps:

[0046] Weigh 2 kg of Pieris hirta into a round-bottom flask, add 30 L of petroleum ether, degrease with a Soxhlet extractor for 2 h, repeat 3 times, filter and dry the residue in the shade, add deionized water at a solid-liquid ratio of 1:20 g / mL, ultrasonically extract for 50 min at 200 W and 65 °C, filter to obtain the filtrate, extract 3 times, combine the filtrate, concentrate the filtrate to 5 L, add papain and cellulase at a ratio of 3:2 to the filtrate, add 5%, stir evenly, enzymolyze at 65 °C for 55 min, then heat to 95 °C to inactivate the enzyme for 15 min, centrifuge at 4000 r / min for 10 min, and filter to obtain the enzymatic solution.

[0047] Add anhydrous ethanol to the enzymatic hydrolyzate until the ethanol volume concentration reaches 60%, stir evenly, let it stand at 4°C for 12 hours for alcohol precipitation, centrifuge at 5000r / min for 20 minutes, re-dissolve the precipitate, concentrate under reduced pressure, and freeze-dry to obtain crude Kadura vine polysaccharide.

[0048] Weigh 5 g of crude polysaccharide from Pieris ovata, dissolve it in distilled water and centrifuge it, take the supernatant and apply it to the sample, adsorb it for 3 hours, and separate and purify it through a DEAE-650M cellulose column with distilled water to obtain DKPSP.

[0049] The polysaccharide extraction rate formula is:

[0050] The sugar content was determined by the phenol-sulfuric acid method. The standard curve was drawn with the glucose concentration (mg / mL) as the x-axis and the A value as the y-axis. The regression equation of the glucose standard curve was: y = 3.436x + 0.09359, R 2 =0.9921, and the linear relationship was good in the range of glucose concentration from 0.01 to 0.1 mg / mL.

[0051] Prepare 1 mg / mL DKPSP solution, perform the experiment three times in parallel according to the above method, take the average value, measure its absorbance A, and substitute it into the standard curve to calculate the sugar content.

[0052] The extraction rate of DKPSP obtained in Example 1 was 68.43% and the sugar content was 74.97%.

[0053] In addition, in order to further illustrate the non-obviousness of the process parameter conditions in the technical solution disclosed in the present application, the inventors have optimized the material-liquid ratio of defatted Kadura dwindlingii and deionized water, the amount of compound enzyme added, and the ratio of compound enzyme, and the specific contents are as follows:

[0054] Example 2

[0055] A method for preparing Kadzu clitoria polysaccharide comprises the following steps:

[0056] Weigh three portions of 100g of defatted Pieris ovata, add 1.5L, 2.0L, and 2.5L of deionized water respectively, extract for 50min at 200W ultrasonic wave and 65℃, filter the filtrate, extract 3 times, combine the filtrate, concentrate the filtrate to 5L, add papain and cellulase in a ratio of 3:2 to the filtrate, add 5%, stir evenly, hydrolyze at 65℃ for 55min, then heat to 95℃ to inactivate the enzyme for 15min, centrifuge at 4000r / min for 10min, and filter to obtain the enzymatic solution.

[0057] Add anhydrous ethanol to the enzymatic hydrolyzate until the ethanol volume concentration reaches 60%, stir evenly, let it stand at 4°C for 12 hours for alcohol precipitation, centrifuge at 5000r / min for 20 minutes, re-dissolve the precipitate, concentrate under reduced pressure, and freeze-dry to obtain crude Kadura vine polysaccharide.

[0058] Weigh 5 g of crude polysaccharide from Pieris ovata, dissolve it in distilled water and centrifuge it, take the supernatant and apply it to the sample, adsorb it for 3 hours, and separate and purify it by passing it through a DEAE-650M cellulose column with distilled water to obtain DKPSP1, DKPSP2 and DKPSP3.

[0059] Optimization was performed according to different solid-liquid ratios of defatted Pieris ovata and deionized water: the extraction rate of DKPSP1 was 66.46% and the sugar content was 73.81%; the extraction rate of DKPSP2 was 68.74% and the sugar content was 74.85%; the extraction rate of DKPSP3 was 67.26% and the sugar content was 73.15%.

[0060] Therefore, the preferred solid-liquid ratio of defatted Pieris ovata and deionized water is 1:20 g / mL for preparation.

[0061] Example 3

[0062] A method for preparing Kadzu clitoria polysaccharide comprises the following steps:

[0063] Weigh three portions of 100g of defatted Pieris odorifera, add 2.0L of deionized water, extract for 50min at 200W ultrasonic wave and 65℃, filter the filtrate, extract three times, combine the filtrate, concentrate the filtrate to 5L, add papain and cellulase to the filtrate in a ratio of 3:2, with the addition amounts of 3%, 5% and 7% respectively, stir evenly, hydrolyze at 65℃ for 55min, then heat to 95℃ to inactivate the enzyme for 15min, centrifuge at 4000r / min for 10min, and filter to obtain the enzymatic solution.

[0064] Add anhydrous ethanol to the enzymatic hydrolyzate until the ethanol volume concentration reaches 60%, stir evenly, let it stand at 4°C for 12 hours for alcohol precipitation, centrifuge at 5000r / min for 20 minutes, re-dissolve the precipitate, concentrate under reduced pressure, and freeze-dry to obtain crude Kadura vine polysaccharide.

[0065] Weigh 5 g of crude polysaccharide from Pieris ovata, dissolve it in distilled water and centrifuge it, take the supernatant and apply it to the sample, adsorb it for 3 hours, and separate and purify it by passing it through a DEAE-650M cellulose column with distilled water to obtain DKPSP4, DKPSP5 and DKPSP6.

[0066] According to the different addition amounts of the complex enzyme, the extraction rate of DKPSP4 was 65.92% and the sugar content was 72.44%. The extraction rate of DKPSP5 was 68.29% and the sugar content was 74.51%. The extraction rate of DKPSP6 was 66.75% and the sugar content was 72.32%.

[0067] Therefore, it is preferred that the complex enzyme be prepared with an addition amount of 5%.

[0068] Example 4

[0069] A method for preparing Kadzu clitoria polysaccharide comprises the following steps:

[0070] Weigh three portions of 100 g of defatted Pieris odorifera, add 2.0 L of deionized water, extract for 50 min at 200 W ultrasonic wave and 65 ° C, filter the filtrate, extract 3 times, combine the filtrate, concentrate the filtrate to 5 L, add papain and cellulase to the filtrate in a ratio of 3:2, 1:1, and 2:3, respectively, with an addition amount of 5%, stir evenly, enzymatically hydrolyze at 65 ° C for 55 min, then heat to 95 ° C for 15 min to inactivate the enzyme, centrifuge at 4000 r / min for 10 min, and filter to obtain the enzymatic solution.

[0071] Add anhydrous ethanol to the enzymatic hydrolyzate until the ethanol volume concentration reaches 60%, stir evenly, let it stand at 4°C for 12 hours for alcohol precipitation, centrifuge at 5000r / min for 20 minutes, re-dissolve the precipitate, concentrate under reduced pressure, and freeze-dry to obtain crude Kadura vine polysaccharide.

[0072] Weigh 5 g of crude polysaccharide from Pieris ovata, dissolve it in distilled water and centrifuge it, take the supernatant and apply it for sample, adsorb it for 3 h, and separate and purify it by passing it through a DEAE-650M cellulose column with distilled water to obtain DKPSP7, DKPSP8 and DKPSP9.

[0073] Optimization was performed according to different ratios of the complex enzymes: the extraction rate of DKPSP7 was 68.59% and the sugar content was 74.24%; the extraction rate of DKPSP8 was 67.67% and the sugar content was 73.18%; the extraction rate of DKPSP9 was 66.15% and the sugar content was 72.83%.

[0074] Therefore, it is preferred that the ratio of the complex enzyme be 3:2 for preparation.

[0075] Furthermore, in order to highlight the superior effect of the technical solution disclosed in the present invention application compared with the prior art, the following comparative experiments were conducted:

[0076] Comparative Example 1

[0077] Weigh 2 kg of Pieris ovata into a round-bottom flask, add deionized water at a solid-liquid ratio of 1:20 g / mL, perform ultrasonic extraction at 200 W and 65 °C for 50 min, filter to obtain the filtrate, extract 3 times, combine the filtrates, concentrate the filtrate to 5 L, add papain and cellulase at a ratio of 3:2 to the filtrate, add 5%, stir evenly, perform enzymolysis at 65 °C for 55 min, then heat to 95 °C to inactivate the enzyme for 15 min, centrifuge at 4000 r / min for 10 min, and filter to obtain the enzymatic solution.

[0078] Add anhydrous ethanol to the enzymatic hydrolyzate until the ethanol volume concentration reaches 60%, stir evenly, let it stand at 4°C for 12 hours for alcohol precipitation, centrifuge at 5000r / min for 20 minutes, re-dissolve the precipitate, concentrate under reduced pressure, and freeze-dry to obtain crude Kadura vine polysaccharide.

[0079] Weigh 5 g of crude polysaccharide from Pieris ovata, dissolve it in distilled water and centrifuge it, take the supernatant and apply it to the sample, adsorb it for 3 hours, and separate and purify it through a DEAE-650M cellulose column with distilled water to obtain DKPSP-A.

[0080] The polysaccharide extraction rate formula is:

[0081] The sugar content was determined by the phenol-sulfuric acid method. The standard curve was drawn with the glucose concentration (mg / mL) as the x-axis and the A value as the y-axis. The regression equation of the glucose standard curve was: y = 3.436x + 0.09359, R 2 =0.9921, and the linear relationship was good in the range of glucose concentration from 0.01 to 0.1 mg / mL.

[0082] Prepare 1 mg / mL DKPSP solution, perform the experiment three times in parallel according to the above method, take the average value, measure its absorbance A, and substitute it into the standard curve to calculate the sugar content.

[0083] By adopting the method of comparative example 1, the DKPSP-A prepared had an extraction rate of 66.84% and a sugar content of 72.11%. It can be seen that the extraction rate and sugar content of the defatted DKPSP were significantly higher than those of the non-defatted DKPSP.

[0084] Comparative Example 2

[0085] Weigh 2 kg of Piper mesona var. mongolica into a round-bottom flask, add 30 L of petroleum ether, degrease with a Soxhlet extractor for 2 h, repeat 3 times, filter and dry the residue in the shade, add deionized water at a solid-liquid ratio of 1:20 g / mL, concentrate the liquid to 5 L, add papain and cellulase at a ratio of 3:2 to the liquid, add 5%, stir evenly, hydrolyze at 65 ° C for 55 min, then heat to 95 ° C for 15 min to inactivate the enzyme, centrifuge at 4000 r / min for 10 min, and filter to obtain the enzymatic solution.

[0086] Add anhydrous ethanol to the enzymatic hydrolyzate until the ethanol volume concentration reaches 60%, stir evenly, let it stand at 4°C for 12 hours for alcohol precipitation, centrifuge at 5000r / min for 20 minutes, re-dissolve the precipitate, concentrate under reduced pressure, and freeze-dry to obtain crude Kadura vine polysaccharide.

[0087] Weigh 5 g of crude polysaccharide from Pieris ovata, dissolve it in distilled water and centrifuge it, take the supernatant and apply it to the sample, adsorb it for 3 hours, and separate and purify it through a DEAE-650M cellulose column with distilled water to obtain DKPSP-B.

[0088] The polysaccharide extraction rate formula is:

[0089] The sugar content was determined by the phenol-sulfuric acid method. The standard curve was drawn with the glucose concentration (mg / mL) as the x-axis and the A value as the y-axis. The regression equation of the glucose standard curve was: y = 3.436x + 0.09359, R 2 =0.9921, the linear relationship is good in the range of glucose concentration from 0.01 to 0.1 mg / mL. Prepare 1 mg / mL DKPSP solution, perform the experiment three times in parallel according to the above method, take the average value, measure its absorbance A, and substitute it into the standard curve to calculate the sugar content.

[0090] By adopting the method of comparative example 2, the extraction rate of DKPSP-B prepared was 66.37% and the sugar content was 72.98%. It can be seen that the extraction rate and sugar content of DKPSP after ultrasonic assisted extraction were significantly higher than those of DKPSP without ultrasonic assisted extraction.

[0091] Comparative Example 3

[0092] A method for preparing Kadzu clitoria polysaccharide comprises the following steps:

[0093] Weigh 2 kg of Piper mesona var. mongolica and put them into round-bottom flasks, add 30 L of petroleum ether, degrease with a Soxhlet extractor for 2 h, repeat 3 times, filter and dry the residue in the shade, add deionized water at a solid-liquid ratio of 1:20 g / mL, extract for 50 min with ultrasound at 200 W and 65 °C, filter to obtain the filtrate, extract 3 times, combine the filtrates, and concentrate the filtrate to 5 L.

[0094] Papain and cellulase were added to the two filtrates at a ratio of 3:2, with 5% added each. The mixture was stirred evenly and enzymatically hydrolyzed at 65°C for 55 min. The mixture was then heated to 95°C for 15 min to inactivate the enzyme. After centrifugation at 4000 r / min for 10 min, the enzymatic hydrolyzate was filtered out.

[0095] Anhydrous ethanol was added to the enzymatic hydrolysate until the ethanol volume concentration reached 60%, stirred evenly, allowed to stand at 4°C for 12 h for alcohol precipitation, centrifuged at 5000 r / min for 20 min, the precipitate was redissolved, concentrated under reduced pressure, and freeze-dried to obtain two portions of Pieris ovata crude polysaccharides.

[0096] Weigh 5 g of crude polysaccharide from Pieris ovata, dissolve it in distilled water and centrifuge it, take the supernatant and apply it to the sample, adsorb it for 3 hours, and separate and purify it by passing it through a DEAE-650M cellulose column with distilled water to obtain DKPSP-D and DKPSP-E.

[0097] The polysaccharide extraction rate formula is:

[0098] The sugar content was determined by the phenol-sulfuric acid method. The standard curve was drawn with the glucose concentration (mg / mL) as the x-axis and the A value as the y-axis. The regression equation of the glucose standard curve was: y = 3.436x + 0.09359, R 2 =0.9921, the linear relationship is good in the range of glucose concentration from 0.01 to 0.1 mg / mL. Prepare 1 mg / mL DKPSP solution, perform the experiment three times in parallel according to the above method, take the average value, measure its absorbance A, and substitute it into the standard curve to calculate the sugar content.

[0099] By using the method of Comparative Example 3, the extraction rate of KPSP-D was 67.11%, and the sugar content was 73.49%; the extraction rate of KPSP-E was 67.63%, and the sugar content was 73.53%.

[0100] It can be seen that the extraction rate and sugar content of DKPSP obtained by the composite enzyme hydrolysis method are significantly higher than those obtained by the single enzyme hydrolysis method.

[0101] Example 5 Determination of molecular weight and homogeneity of DKPSP

[0102] The molecular weight and homogeneity of the DKPSP obtained in Example 1 were detected by high performance gel permeation chromatography (HPGPC), instrument: Agilent 1260II HPLC; detector: UM4800 evaporative light detector; chromatographic column: TSKgelG6000WXL; column temperature: 40°C; mobile phase: distilled water; flow rate: 0.8 mL / min; injection volume: 20 μL.

[0103] DKPSP was prepared into a 2 mg / mL solution and filtered through a 0.22 μm filter membrane before injection. Figure 1 The HPGPC spectrum of DKPSP showed a relatively single and symmetrical single peak, and the calculated average molecular weight was 31796Da.

[0104] Example 6 Determination of Monosaccharide Composition of DKPSP

[0105] Instrument: High performance liquid chromatograph Agilent 1260II; chromatographic column: Diamonsil C18 (250 mm×4.6 mm, 5 μm); mobile phase: 0.05 mol / L phosphate buffer (pH=6.72)-acetonitrile; volume ratio: 83:17; detector: UV detector; detection wavelength: 250 nm; flow rate: 1.0 mL / min; injection volume: 10 μL.

[0106] Accurately weigh 2 mg each of glucose (Glc), galacturonic acid (GalA), arabinose (Ara), rhamnose (Rha), xylose (Xyl), mannose (Man), glucuronic acid (GlcA), and galactose (Gal) and mix them in a hydrolysis tube, add 400 μL of 0.5 mol / L PMP solution and 0.3 mol / L NaOH solution, shake well and react in a 70°C water bath for 0.5 h. After the reaction is completed, add 400 μL of 0.3 mol / L HCl solution for neutralization. Finally, add 2 mL of chloroform for extraction and centrifuge. Repeat three times. Take the water layer solution and pass it through a 0.22 μm filter membrane for liquid phase detection.

[0107] Weigh 5 mg of DKPSP into a hydrolysis tube, add 2 mL of trifluoroacetic acid (TFA) solution, react at 120 ° C for 5 hours for complete hydrolysis, and then decompress and spin dry at 60 ° C to remove TFA. Then add deionized water to dissolve, take 100 μL of sample solution and derivatize according to the above-mentioned monosaccharide standard solution experimental method, extract, centrifuge, and pass through a 0.22 μm filter membrane for liquid phase detection. By comparing the peak time with the mixed monosaccharide standard, it can be seen that DKPSP is composed of five monosaccharides: Man, Rha, GlcA, GalA, and Gal. The results are as follows Figure 2 , Figure 3 As shown, the molar ratio of each monosaccharide composition calculated by area normalization method is 78.6:0.35:10.31:2.24:8.36.

[0108] Experiment 7 Effect of DKPSP on the motility of CL4176 Caenorhabditis elegans

[0109] A blank group (Con) and a DKPSP group prepared in Example 1 were set up. The synchronized L4 stage CL4176 nematodes were randomly transferred to the culture medium of the blank group and the DKPSP group with different concentrations (0.5, 1, and 2 mg / mL), with 20 nematodes per dish, and cultured in a 16°C incubator. After culturing for 2 days, the nematodes were transferred to a blank NGM culture medium and M9 solution was added. The number of times the nematodes' heads swung and the number of times the nematodes' bodies bent within 2 minutes were recorded, which were used as indicators of the nematodes' motor ability. The number of times the heads of the Caenorhabditis elegans nematodes swung and the number of times the bodies bent within 2 minutes were recorded under a microscope. Compared with the Con group, the motor ability of the nematodes was improved after being given the DKPSP prepared in Example 1. Among them, the motor ability of the DKPSP-2 group was significantly improved (P<0.05). The results are shown in Table 1. Figure 4 .

[0110] Experiment 8 Effect of DKPSP on stress tolerance of CL4176 Caenorhabditis elegans

[0111] The heat stress experiment was set up with a blank group (Con) and a DKPSP group prepared in Example 1. The CL4176 nematodes synchronized to the L4 stage were randomly transferred to the blank group and the DKPSP groups (0.5, 1, 2 mg / mL) culture medium, 20 per dish, and cultured in a 16°C incubator for 2 days. The nematodes were transferred to new corresponding culture dishes and cultured at 37°C. After that, the survival status of the nematodes was counted every hour until all the nematodes died.

[0112] The results are as follows Figure 5 As shown, compared with the Con group, after administration of the DKPSP prepared in Example 1, the survival curves all shifted to the right, indicating that the adaptability of the nematodes to the thermal environment was enhanced and the survival status of the nematodes was improved, among which the resistance to heat stress of the DKPSP-2 group was significantly improved.

[0113] The oxidative stress experiment was set up with a blank group (Con) and a DKPSP group prepared in Example 1. The CL4176 nematodes synchronized to the L4 stage were randomly transferred to the blank group and the DKPSP group (0.5, 1, 2 mg / mL) culture medium, 20 per dish, and cultured in a 16°C incubator for 2 days. The nematodes were then transferred to an NGM plate containing 5% H2O2 solution, and the survival status of the nematodes was counted every hour until all the nematodes died.

[0114] The results are as follows Figure 6As shown, compared with the Con group, after administration of DKPSP prepared in Example 1, the survival curves all shifted to the right, indicating that the ability of nematodes to resist oxidative stress was improved, and the survival state of nematodes was improved, among which the ability of DKPSP-2 group to resist oxidative stress was significantly improved.

[0115] In summary, the experimental results show that the DKPSP prepared by the present invention has high extraction rate and sugar content, and can enhance the movement ability and stress resistance of CL4176 nematodes to a certain extent.

[0116] The present invention discloses for the first time that Pieris damicornis polysaccharide can improve athletic ability or stress resistance, and provides a new direction for preparing new drugs for improving athletic ability or stress resistance in the future.

[0117] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Kadzu clitoria polysaccharide, characterized in that: The Kadzus rapa polysaccharide comprises five monosaccharides: Man, Rha, GlcA, GalA and Gal, and the molar ratio of each monosaccharide is 78.6:0.35:10.31:2.24:8.

36.

2. The Kadzu clitoria polysaccharide according to claim 1, characterized in that The Pieris d'Arcticum polysaccharide is extracted from Pieris d'Arcticum by combining ultrasonic-assisted extraction and complex enzyme hydrolysis. The complex enzyme consists of papain and cellulose protease.

3. A method for preparing the Kadzu clitoria polysaccharide according to claim 1, characterized in that: The specific steps include: S1. Petroleum ether is added to the Kadura dremel, and the Kadura dremel is defatted by Soxhlet extraction. Deionized water is added to the defatted Kadura dremel, and ultrasonic extraction is performed. The filtrate is hydrolyzed by a composite enzyme to obtain an enzymatic solution. S2, precipitating the enzymatic hydrolyzate with alcohol, concentrating under reduced pressure, and freeze-drying to obtain crude polysaccharide of Pieris fasciata; S3, dissolving the crude Kadura dremel polysaccharide and centrifuging it, taking the supernatant for separation and purification to obtain Kadura dremel polysaccharide; The complex enzyme consists of papain and cellulose protease.

4. The preparation method according to claim 3, characterized in that: In the step S1, deionized water is added to the defatted Kadura rapa according to a solid-liquid ratio of 1:15-25 g / mL.

5. The preparation method according to claim 3, characterized in that: The added amount of the composite enzyme in step S1 is 3-7%; the volume ratio of papain and cellulose protease in the composite enzyme is 6:(4-9).

6. The preparation method according to claim 3, characterized in that: The alcohol precipitation process in step S2 is as follows: adding anhydrous ethanol to the enzymatic hydrolysate until the ethanol concentration reaches 60%, stirring evenly, standing at 4° C. for 12 hours for alcohol precipitation, and centrifuging at 5000 r / min for 15 to 25 minutes.

7. The preparation method according to claim 3, characterized in that: The separation and purification process in step S3 is to use distilled water to pass through a DEAE-650M cellulose chromatography column for separation and purification.

8. Use of the Pieris d'Arcana polysaccharide according to any one of claims 1 to 2 or the Pieris d'Arcana polysaccharide prepared by the preparation method according to any one of claims 3 to 7, characterized in that: The Kadzus rapa polysaccharide can improve the movement ability and / or stress ability of CL4176 nematodes.

9. Use of the Pieris d'Arcticum polysaccharide described in any one of claims 1 to 2 or the Pieris d'Arcticum polysaccharide prepared by the preparation method of any one of claims 3 to 7 in the preparation of products and / or drugs for improving athletic ability.

10. Use of the Pieris d'Arcticum polysaccharide according to any one of claims 1 to 2 or the Pieris d'Arcticum polysaccharide prepared by the preparation method according to any one of claims 3 to 7 in the preparation of products and / or drugs that improve stress resistance.

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