A pipevine polysaccharide, and a preparation method and application thereof

Abstract

The application belongs to the technical field of traditional Chinese medicine, and particularly relates to a polysaccharide of kopsia and a preparation process and application thereof. The polysaccharide of kopsia (KPSP) is extracted from kopsia by means of ultrasonic-assisted extraction combined with complex enzyme enzymolysis. The extraction process of the polysaccharide of kopsia is simple in operation, high in extraction rate and purity, time-saving and efficient, and the molecular weight can be measured, and the extraction system is mild. The results of examples show that the polysaccharide of kopsia extracted by the application can enhance the movement ability and stress ability of CL4176 nematodes, and then the polysaccharide of kopsia extracted by the application can be applied to preparation of products and / or drugs for improving movement ability and stress ability.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine, and particularly relates to a polygiprazine polysaccharide and a preparation method and application thereof. BACKGROUND

[0002] In recent years, with the continuous change of human living environment and the acceleration of life rhythm, the pressure borne by human beings is generally high, which leads to the generation of negative emotions. Long-term negative emotions are easy to lead to serious psychological problems. The continuous existence of stress situations is easy to appear problems such as emotional depression, slow thinking, memory decline in work and study, and severe decline in motor ability and stress resistance, which will have a serious impact on daily life and work. Different degrees of decline in motor ability and stress resistance will lead to a series of health problems, such as uncoordinated limb movement leading to falling, slow response to stimulation, and memory decline.

[0003] Caenorhabditis elegans (C.elegans) is a kind of individual micro, transparent, head thick and tail thin nematode, which usually takes E. coli OP50 as food source in the laboratory, has fast breeding speed, short life cycle, high gene homology and is easy to culture. Researchers found that up to 60% to 80% of genes in CL4176 C.elegans have high homology with human genome. In addition, the movement ability and stress response of nematodes can be observed through a microscope, and the stress response, movement ability and basic physiological process of higher animals are embodied to a certain extent on the CL4176 nematode model, so the CL4176 nematode has gradually become an important animal in drug screening research, and has important significance for the research and development of medical career.

[0004] Existing studies have demonstrated that traditional Chinese medicine exhibits significant advantages in treating these types of diseases, providing direction for future research or development of novel drugs that regulate motor function and stress response. *Piper Kadsura* (Choisy) Ohwi., a plant in the Piperaceae family, is a dried vine stem with a pungent and bitter taste, slightly warm in nature, and enters the liver meridian. It has the effects of dispelling wind and dampness, clearing the meridians, and relieving pain. Therefore, it is commonly used in folk medicine for symptoms such as wind-cold-dampness arthralgia and joint pain. Chemical composition studies have shown that *Piper Kadsura* contains polysaccharides, lignans, alkaloids, volatile oils, flavonoids, epoxides, and steroids, among which neolignans and amide alkaloids are its main chemical components. Modern pharmacological studies have shown that *Piper Kadsura* has anti-inflammatory, antioxidant, platelet-activating factor inhibitory, and neuroprotective effects. Among these, *Piper Kadsura* polysaccharide (KPSP) is one of the main components, possessing significant biological activity and playing an important role in physiological processes such as immune regulation and tissue protection. It is widely used in various health products and medicines. The resources of *Piper kadsura* should be further developed and utilized in a more rational manner, but problems such as low extraction rate and low purity have not yet been solved.

[0005] Therefore, there is an urgent need to develop a polysaccharide from the traditional Chinese medicine ingredient, *Piper kadsura*. Summary of the Invention

[0006] The purpose of this invention is to provide a polysaccharide from *Piper kadsura* and its preparation method. The method provided by this invention is not only simple to operate and has a mild extraction system, but also has a high extraction rate and high purity of polysaccharide.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a polysaccharide of *Piper kadsura*, which comprises five monosaccharides: Man, Rha, GlcA, GalA, and Gal, with a molar ratio of 78.6:0.35:10.31:2.24:8.36.

[0009] Preferably, the polysaccharide from *Piper kadsura* is obtained by a combination of ultrasonic-assisted extraction and complex enzyme hydrolysis; the complex enzyme consists of papain and cellulase.

[0010] This invention also provides a method for preparing the above-mentioned *Piper kadsura* polysaccharide, specifically including the following steps:

[0011] S1. Add petroleum ether to the sea breeze vine, defatt it by Soxhlet extraction, add deionized water to the defatted sea breeze vine, extract by ultrasonication, filter, and the filtrate is hydrolyzed by a compound enzyme to obtain the enzymatic hydrolysate.

[0012] S2. The enzymatic hydrolysate was precipitated with alcohol, concentrated under reduced pressure, and freeze-dried to obtain crude polysaccharide from *Piper kadsura*.

[0013] S3, dissolving the crude polysaccharide of kadsurae caulitis, centrifuging, and taking supernatant to separate and purify to obtain kadsurae caulitis polysaccharide;

[0014] The complex enzyme is composed of papain and cellulase.

[0015] Preferably, the defatted kadsurae caulitis in step S1 is added into deionized water at a ratio of 1:15-25 g / mL.

[0016] Preferably, the addition amount of the complex enzyme in step S1 is 3-7%, and the volume ratio of papain to cellulase in the complex enzyme is 6:(4-9).

[0017] Preferably, the alcohol precipitation process in step S2 is: adding anhydrous ethanol into the enzymolysis solution until the ethanol concentration reaches 60%, stirring uniformly, standing at 4 DEG C for 12 h for alcohol precipitation, and centrifuging at 5000 r / min for 15-25 min.

[0018] Preferably, the separation and purification process in step S3 is separation and purification by DEAE-650M cellulose chromatographic column with distilled water.

[0019] The application further provides application of the kadsurae caulitis polysaccharide or the kadsurae caulitis polysaccharide prepared by the preparation method in improving the movement ability and / or stress ability of CL4176 nematodes.

[0020] The application further provides application of the kadsurae caulitis polysaccharide or the kadsurae caulitis polysaccharide prepared by the preparation method in preparing products and / or drugs for improving the movement ability.

[0021] The application further provides application of the kadsurae caulitis polysaccharide or the kadsurae caulitis polysaccharide prepared by the preparation method in preparing products and / or drugs for improving the stress ability.

[0022] The application has the following beneficial effects:

[0023] (1) The polysaccharide is extracted by the ultrasonic wave and complex enzyme method, which solves the problems of low extraction rate and purity of kadsurae caulitis polysaccharide, and the extraction temperature is low, the reaction condition is mild, fast and efficient.

[0024] (2) The preparation method 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 application can enhance the movement ability and stress ability of CL4176 Caenorhabditis elegans. BRIEF DESCRIPTION OF DRAWINGS

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 HPGPC spectrum of DKPSP prepared according to the present invention;

[0028] Figure 2 The HPLC chromatogram for the composition analysis of DKPSP monosaccharides prepared in this invention includes the following components: 1. Man, 2. Rha, 3. GlcA, 4. GalA, 5. Glc, 6. Gal, 7. Xyl, and 8. Ara.

[0029] Figure 3 The HPLC chromatogram is shown for the DKPSP monosaccharide standard derivative prepared in this invention.

[0030] Figure 4 A graph showing the number of head swings and body bends of a Caenorhabditis elegans nematode within 2 minutes.

[0031] Figure 5 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 Implementation

[0033] This invention provides a polysaccharide derived from the traditional Chinese medicine *Piper kadsura*. The polysaccharide prepared by this invention is composed of five monosaccharides: Man, Rha, GlcA, GalA, and Gal, with a molar ratio of 78.6:0.35:10.31:2.24:8.36. Molecular weight and uniformity were determined, and the average molecular weight of the polysaccharide prepared by this invention is 31796 Da.

[0034] This invention involves defatting *Piper kadsura* (sea pine) with petroleum ether using Soxhlet extraction. The defatted *Piper kadsura* is then added to deionized water at a material-to-liquid ratio of 1:15–25 g / mL, preferably 1:20 g / mL. Simultaneously, ultrasonic-assisted extraction and a compound enzyme hydrolysis method are combined to fully extract the effective components of *Piper kadsura*. The compound enzyme consists of papain and cellulase, with a volume ratio of papain to cellulase of (4-6):(6-4), preferably 6:4. The amount of compound enzyme added is 3-7%, preferably 5%.

[0035] The enzyme solution after enzymolysis is subjected to alcohol precipitation, reduced pressure concentration and freeze drying to obtain crude polysaccharide of Caulis Tetrapanacis, and then is subjected to DEAE-650M cellulose chromatography column separation and purification with distilled water to obtain DKPSP.

[0036] The present application sets up a plurality of comparative examples to study the preparation process of polysaccharide of Caulis Tetrapanacis, and the results show that the preparation process of polysaccharide of Caulis Tetrapanacis is simple and efficient, and the extraction rate and sugar content of polysaccharide of Caulis Tetrapanacis are higher than those of the comparative examples.

[0037] The present application studies the effect of polysaccharide of Caulis Tetrapanacis on the movement ability and stress ability of CL4176 Caenorhabditis elegans, and the results of the examples show that the DKPSP extracted by the present application can significantly improve the movement ability, heat adaptation ability and antioxidant stress ability 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 body to the thermal environment in a high environmental temperature. The physiological response of the human body under heat stress environment destroys the normal heat balance, and the human body will produce a series of complex physiological and psychological changes. Whether it is a human or an animal, the body function will be affected by heat stress.

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

[0040] The present application studies the effect of polysaccharide of Caulis Tetrapanacis on the movement ability, heat adaptation ability and antioxidant stress ability of the nematodes, and provides a new direction for future research or development of new drugs and / or products for regulating movement ability and stress ability.

[0041] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0042] The production process, experimental method or detection method involved in the examples of the present application, if not specifically stated, are all conventional methods in the prior art, and the name and / or abbreviation thereof all belong to the conventional name in the art, which is very clear and explicit in the related application field, and the person skilled in the art can understand the conventional process steps and apply the corresponding equipment according to the name, and implement it according to the 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 application are not particularly limited in origin, and are conventional products that can be purchased through normal commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0044] Example 1 Preparation method of polysaccharide in Litseae Folium

[0045] A preparation method of polysaccharide in Litseae Folium, comprising the following steps:

[0046] Take 2 kg of Litseae Folium into a round-bottom flask, add 30 L of petroleum ether, and defat for 2 h with a Soxhlet extractor, repeat 3 times, filter the residue, and dry the residue in the sun. Add deionized water at a solid-liquid ratio of 1:20 g / mL, ultrasonic extraction at 200 W and a temperature of 65 ℃ for 50 min, filter the filtrate, extract 3 times, combine the filtrate, concentrate the filtrate to 5 L, add papain and cellulase to the filtrate at a ratio of 3:2, the addition amount is 5%, stir uniformly, and enzymolysis at a temperature of 65 ℃ for 55 min, then heat to 95 ℃ to inactivate the enzyme for 15 min, centrifuge at 4000 r / min for 10 min, and then filter to obtain the enzyme solution.

[0047] Add anhydrous ethanol to the enzyme solution to a concentration of 60% by volume, stir uniformly, and stand at 4 ℃ for 12 h for alcohol precipitation, centrifuge at 5000 r / min for 20 min, redissolve the precipitate, reduce pressure to concentrate, and freeze-dry to obtain the crude polysaccharide of Litseae Folium.

[0048] Take 5 g of the crude polysaccharide of Litseae Folium, dissolve it with distilled water, centrifuge, take the supernatant, and adsorb for 3 h. Separate and purify the DEAE-650M cellulose chromatographic column with distilled water to obtain DKPSP.

[0049] The polysaccharide extraction rate formula is:

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

[0051] Prepare a 1 mg / mL DKPSP solution, perform 3 parallel experiments according to the above method, take the average value, measure the absorbance A, and calculate the sugar content by substituting the standard curve.

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

[0053] Furthermore, to further illustrate the non-obviousness of the process parameters in the technical solution disclosed in this application, the inventors have optimized the ratio of defatted sea bream to deionized water, the amount of compound enzyme added, and the proportion of compound enzyme, as detailed below:

[0054] Example 2

[0055] A method for preparing Piper kadsura polysaccharide includes the following steps:

[0056] Weigh out three 100g portions of defatted Piper kadsura and add 1.5L, 2.0L, and 2.5L of deionized water respectively. Sonicate at 200W and 65℃ for 50min, filter to obtain filtrate, and repeat the extraction three times. Combine the filtrates and concentrate the filtrate to 5L. Add papain and cellulase at a ratio of 3:2, with an addition amount of 5%, and stir well. Enzymatically hydrolyze at 65℃ for 55min, then heat to 95℃ to inactivate the enzyme for 15min. After centrifugation at 4000r / min for 10min, filter to obtain the enzymatic hydrolysate.

[0057] Anhydrous ethanol was added to the enzymatic hydrolysate until the ethanol volume concentration reached 60%. The mixture was stirred evenly and allowed to stand at 4°C for 12 hours for alcohol precipitation. The precipitate was then centrifuged at 5000 r / min for 20 min, redissolved, concentrated under reduced pressure, and freeze-dried to obtain crude polysaccharide of Piper kadsura.

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

[0059] The extraction ratio of defatted sea bream to deionized water was optimized: DKPSP1 had an extraction rate of 66.46% and a sugar content of 73.81%; DKPSP2 had an extraction rate of 68.74% and a sugar content of 74.85%; and DKPSP3 had an extraction rate of 67.26% and a sugar content of 73.15%.

[0060] Therefore, the optimal ratio of defatted sea bream to deionized water is 1:20 g / mL for preparation.

[0061] Example 3

[0062] A method for preparing Piper kadsura polysaccharide includes the following steps:

[0063] Take three 100g of defatted sea wind, add 2.0L of deionized water, ultrasonic wave at 200W, temperature at 65℃, ultrasonic extraction for 50min, filter to get filtrate, extract 3 times, combine the filtrate, concentrate the filtrate to 5L, add papain and cellulase to the filtrate according to 3:2, the adding amount is 3%, 5%, 7%, stir evenly, enzymolysis at 65℃ for 55min, then heat to 95℃ for 15min to kill the enzyme, centrifuge at 4000r / min for 10min, then filter to get the enzyme solution.

[0064] Add anhydrous ethanol to the enzyme solution to reach 60% ethanol volume concentration, stir evenly, stand at 4℃ for 12h alcohol precipitation, centrifuge at 5000r / min for 20min, redissolve the precipitate, reduce pressure concentration, freeze-drying to get sea wind crude polysaccharide.

[0065] Take 5g of sea wind crude polysaccharide, dissolve with distilled water, centrifuge, take the supernatant, adsorb for 3h, separate and purify with DEAE-650M cellulose chromatography column with distilled water, get DKPSP4, DKPSP5, DKPSP6.

[0066] According to the different adding amount of compound enzyme, the DKPSP4 extraction rate is 65.92%, the sugar content is 72.44%. The DKPSP5 extraction rate is 68.29%, the sugar content is 74.51%; The DKPSP6 extraction rate is 66.75%, the sugar content is 72.32%.

[0067] Therefore, the preferred adding amount of compound enzyme is 5% for preparation.

[0068] Example 4

[0069] A sea wind polysaccharide preparation method, comprising the following steps:

[0070] Take three 100g of defatted sea wind, add 2.0L of deionized water, ultrasonic wave at 200W, temperature at 65℃, ultrasonic extraction for 50min, filter to get filtrate, extract 3 times, combine the filtrate, concentrate the filtrate to 5L, add papain and cellulase to the filtrate according to 3:2, 1:1, 2:3, the adding amount is 5%, stir evenly, enzymolysis at 65℃ for 55min, then heat to 95℃ for 15min to kill the enzyme, centrifuge at 4000r / min for 10min, then filter to get the enzyme solution.

[0071] Add anhydrous ethanol to the enzyme solution to reach 60% ethanol volume concentration, stir evenly, stand at 4℃ for 12h alcohol precipitation, centrifuge at 5000r / min for 20min, redissolve the precipitate, reduce pressure concentration, freeze-drying to get sea wind crude polysaccharide.

[0072] Take 5g of crude polysaccharide of Litsea cubeba, dissolve it with distilled water, centrifuge, take the supernatant, adsorb for 3h, and separate and purify it with a DEAE-650M cellulose chromatographic column with distilled water, to obtain DKPSP7, DKPSP8 and DKPSP9.

[0073] The proportion of the complex enzyme is optimized according to different ratios: the extraction rate of DKPSP7 is 68.59%, the sugar content is 74.24%; the extraction rate of DKPSP8 is 67.67%, the sugar content is 73.18%; and the extraction rate of DKPSP9 is 66.15%, the sugar content is 72.83%.

[0074] Therefore, the proportion of the complex enzyme is preferably 3:2 for preparation.

[0075] In addition, in order to highlight the excellent effect of the technical scheme disclosed in the present application compared with the prior art, the following comparative experiments are carried out:

[0076] Comparative Example 1

[0077] Take 2kg of Litsea cubeba into a round-bottom flask, add deionized water according to a solid-liquid ratio of 1:20g / mL, ultrasonic extraction at 200W and a temperature of 65℃ for 50min, filter to obtain a filtrate, extract for 3 times, combine the filtrates, concentrate the filtrates to 5L, add papain and cellulase to the filtrates according to a ratio of 3:2, the addition amount is 5%, stir uniformly, enzymolysis at a temperature of 65℃ for 55min, then heat to 95℃ to inactivate the enzyme for 15min, centrifuge at 4000r / min for 10min, and then suction filter to obtain an enzymolysis liquid.

[0078] Add anhydrous ethanol to the enzymolysis liquid to a volume concentration of 60%, stir uniformly, stand still at 4℃ for 12h for alcohol precipitation, centrifuge at 5000r / min for 20min, redissolve the precipitate, reduce pressure to concentrate, freeze-dry, and obtain crude polysaccharide of Litsea cubeba.

[0079] Take 5g of crude polysaccharide of Litsea cubeba, dissolve it with distilled water, centrifuge, take the supernatant, adsorb for 3h, and separate and purify it with a DEAE-650M cellulose chromatographic column with distilled water, to obtain DKPSP7, DKPSP8 and DKPSP9.

[0080] The polysaccharide extraction rate formula is:

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

[0082] DKPSP solution of 1 mg / mL was prepared, and the average value was taken for three parallel experiments according to the above method. The absorbance A was measured, and the sugar content was calculated by substituting the standard curve.

[0083] The DKPSP-A extraction rate was 66.84%, and the sugar content was 72.11% by the method of the comparative example 1. It can be seen that the extraction rate and sugar content of the defatted DKPSP are obviously higher than those of the non-defatted DKPSP.

[0084] Comparative example 2

[0085] 2 kg of Litsea rotundifolia was weighed into a round-bottom flask, 30 L of petroleum ether was added, and the defatting was performed for 2 h by using a Soxhlet extractor. The operation was repeated for three times. After filtration, the filter residue was dried in the shade. Deionized water was added at a solid-liquid ratio of 1:20 g / mL. The liquid was concentrated to 5 L. Papain and cellulase were added to the liquid at a ratio of 3:2, and the addition amount was 5%. After uniform stirring, the enzymolysis was performed at 65°C for 55 min. Then, the enzyme was inactivated by heating to 95°C for 15 min. After centrifugation at 4000 r / min for 10 min, the filtrate was obtained by suction filtration.

[0086] Anhydrous ethanol was added to the filtrate until the ethanol volume concentration reached 60%. After uniform stirring, alcohol precipitation was performed at 4°C for 12 h. After centrifugation at 5000 r / min for 20 min, the precipitate was redissolved. After concentration under reduced pressure, freeze-drying was performed to obtain the Litsea rotundifolia crude polysaccharide.

[0087] 5 g of the Litsea rotundifolia crude polysaccharide was weighed, dissolved in distilled water, and centrifuged. The supernatant was sampled, adsorbed for 3 h, and separated and purified by using a DEAE-650M cellulose chromatographic column with distilled water. DKPSP-B was obtained.

[0088] The polysaccharide extraction rate formula is:

[0089] The sugar content was determined by the phenol-sulfuric acid method. The glucose concentration (mg / mL) was taken as the x-axis, and the A value was taken as the y-axis to draw a standard curve. The glucose standard curve regression equation was y=3.436x+0.09359, R 2 =0.9921. The glucose concentration had a good linear relationship in the range of 0.01-0.1 mg / mL. DKPSP solution of 1 mg / mL was prepared, and the average value was taken for three parallel experiments according to the above method. The absorbance A was measured, and the sugar content was calculated by substituting the standard curve.

[0090] The DKPSP-B extraction rate was 66.37%, and the sugar content was 72.98% by the method of the comparative example 2. It can be seen that the extraction rate and sugar content of the DKPSP extracted by ultrasonic-assisted extraction are obviously higher than those of the DKPSP without ultrasonic-assisted extraction.

[0091] Comparative Example 3

[0092] A method for preparing Piper kadsura polysaccharide includes the following steps:

[0093] Weigh 2 kg of *Hedyotis diffusa* into round-bottom flasks, add 30 L of petroleum ether to each flask, defatt the mixture with a Soxhlet extractor for 2 h, repeat 3 times, filter and air-dry the residue, add deionized water at a material-to-liquid ratio of 1:20 g / mL, ultrasonically extract at 200 W and 65 °C for 50 min, filter to obtain 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 each added at 5%. The mixture was stirred evenly and enzymatically hydrolyzed at 65°C for 55 min. Then, the enzyme was inactivated by heating to 95°C for 15 min. After centrifugation at 4000 r / min for 10 min, the enzymatic hydrolysate was obtained by filtration.

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

[0096] Weigh out 5g of crude polysaccharide from Piper kadsura, dissolve it in distilled water, centrifuge, take the supernatant, load it onto the sample, adsorb for 3h, and then 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 formula for polysaccharide extraction rate is:

[0098] Sugar content was determined using the phenol-sulfuric acid method. A standard curve was plotted with glucose concentration (mg / mL) on the x-axis and A value on the y-axis. The regression equation for the glucose standard curve was: y = 3.436x + 0.09359, R0 2 =0.9921, indicating a good linear relationship in the glucose concentration range of 0.01–0.1 mg / mL. Prepare a 1 mg / mL DKPSP solution, perform three parallel experiments as described above, take the average value, measure the absorbance A, and substitute it into the standard curve to calculate the sugar content.

[0099] 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] This shows that the extraction rate and sugar content of DKPSP obtained by the compound enzyme hydrolysis method are significantly higher than those obtained by the single enzyme hydrolysis method.

[0101] Determination of molecular weight and homogeneity of DKPSP

[0102] The molecular weight and homogeneity of DKPSP obtained in Example 1 were determined by high performance gel permeation chromatography (HPGPC). Instrument: Agilent 1260II HPLC; Detector: UM4800 evaporative light detector; Column: TSKgel G6000WXL; Column temperature: 40℃; Mobile phase: distilled water; Flow rate: 0.8 mL / min; Injection volume: 20 μL.

[0103] DKPSP was prepared into a 2 mg / mL solution, filtered with a 0.22 μm filter membrane, and then injected. The detection results are shown in Figure 1 The HPGPC spectrum of DKPSP showed a relatively single and symmetrical single peak, and the average molecular weight was calculated to be 31796 Da.

[0104] Determination of monosaccharide composition of DKPSP

[0105] Instrument: high performance liquid chromatograph Agilent 1260II; Column: Diamonsil C18 (250 mm x 4.6 mm, 5 μm); Mobile phase: 0.05 mol / L phosphate buffer (pH = 6.72)-acetonitrile; Volume ratio: 83:17; Detector: ultraviolet detector; Detection wavelength: 250 nm; Flow rate: 1.0 mL / min; Injection volume: 10 μL.

[0106] Precisely weigh 2 mg of glucose (Glc), galacturonic acid (GalA), arabinose (Ara), rhamnose (Rha), xylose (Xyl), mannose (Man), glucuronic acid (GlcA), and galactose (Gal) into 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℃ 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, centrifuge, repeat three times, take the aqueous solution through a 0.22 μm filter membrane, and then perform liquid phase detection.

[0107] Weigh 5 mg of DKPSP into a hydrolysis tube, add 2 mL of trifluoroacetic acid (TFA) solution, and react at 120℃ for 5 h for complete hydrolysis. Then, dry under reduced pressure at 60℃ to remove TFA. Subsequently, redissolve with deionized water, take 100 μL of sample solution, and perform derivatization according to the above experimental method of monosaccharide standard solution, extraction, centrifugation, filtration through a 0.22 μm filter membrane, and then liquid phase detection. By comparing with the peak time of the mixed monosaccharide standard, it can be seen that DKPSP is composed of Man, Rha, GlcA, GalA, and Gal five kinds of monosaccharides, and the results are shown in Figure 2 ,Figure 3 The area normalization method was used to calculate the molar ratio of each monosaccharide composition, which was 78.6:0.35:10.31:2.24:8.36.

[0108] Experiment 7 Effect of DKPSP on the movement ability of CL4176 C. elegans

[0109] A blank group (Con) and a DKPSP group prepared in Example 1 were set up. After synchronization, L4 stage CL4176 nematodes 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. After 2 days of culture, the nematodes were transferred to NGM medium with M9 solution added, and the number of head swings and body bends of the nematodes within 2 minutes was recorded as an indicator of the movement ability of the nematodes. Under a microscope, the number of head swings and body bends of C. elegans within 2 minutes was recorded. Compared with the Con group, the movement ability of the nematodes was improved after being given the DKPSP prepared in Example 1, among which the movement ability of the DKPSP-2 group was significantly improved (P<0.05), and the results are shown in Figure 4 .

[0110] Experiment 8 Effect of DKPSP on the stress ability of CL4176 C. elegans

[0111] For the heat stress experiment, a blank group (Con) and a DKPSP group prepared in Example 1 were set up. After synchronization to the L4 stage, CL4176 nematodes 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. Then the nematodes were transferred to new corresponding culture dishes and placed in a 37°C constant temperature incubator, and the survival status of the nematodes was counted every hour until all the nematodes died.

[0112] As shown in Figure 5 Compared with the Con group, the survival curves of the nematodes were all shifted to the right after being given the DKPSP prepared in Example 1, indicating that the adaptability of the nematodes to the heat environment was improved, and the survival state of the nematodes was improved, among which the heat stress resistance of the DKPSP-2 group was significantly improved.

[0113] For the oxidative stress experiment, a blank group (Con) and a DKPSP group prepared in Example 1 were set up. After synchronization to the L4 stage, CL4176 nematodes 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. Then the nematodes were transferred to NGM plates containing 5% H2O2 solution, and the survival status of the nematodes was counted every hour until all the nematodes died.

[0114] As shown in Figure 6As shown, compared with the Con group, after DKPSP prepared in Example 1 is given, the survival curves are all right-shifted, which indicates that the antioxidant stress resistance of the nematodes is improved, and the survival state of the nematodes is improved, wherein the antioxidant stress resistance of the DKPSP-2 group is significantly improved.

[0115] According to the above experimental results, it is shown that the DKPSP prepared in the application 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 application first discloses that the polysaccharide of Piper hancei can improve the movement ability or stress resistance, and provides a new direction for preparing a new type of medicine for improving the movement ability or stress resistance in the future.

[0117] Although the above embodiment has made a detailed description of the application, it is only a part of the embodiment of the application, but not all the embodiments, and other embodiments can be obtained according to the embodiment without creativity, and these embodiments all belong to the protection scope of the application.

Claims

1. The use of polysaccharides from Piper methysticum in the preparation of a medicament for improving athletic performance, characterized in that, The *Hypericum perforatum* polysaccharide comprises five monosaccharides: Man, Rha, GlcA, GalA, and Gal, with a molar ratio of 78.6:0.35:10.31:2.24:8.36; the average molecular weight of the *Hypericum perforatum* polysaccharide is 31796 Da.

2. Use according to claim 1, characterized in that, The preparation method of the *Hippophae rhamnoides* polysaccharide specifically includes the following steps: S1. Add petroleum ether to the sea breeze vine, defatt it by Soxhlet extraction, add deionized water to the defatted sea breeze vine, extract by ultrasonication, filter, and the filtrate is hydrolyzed by a compound enzyme to obtain the enzymatic hydrolysate. S2. The enzymatic hydrolysate was precipitated with alcohol, concentrated under reduced pressure, and freeze-dried to obtain crude polysaccharide from *Piper kadsura*. S3. After dissolving the crude polysaccharide of Piper kadsura, centrifuge and take the supernatant for separation and purification to obtain Piper kadsura polysaccharide; The complex enzyme consists of papain and cellulase.

3. Use according to claim 2, characterized in that, In step S1, defatted Piper kadsura is added to deionized water at a material-to-liquid ratio of 1:15~25 g / mL.

4. Use according to claim 2, characterized in that, In step S1, the amount of the compound enzyme added is 3-7%; the volume ratio of papain to cellulase in the compound enzyme is 6:(4-9).

5. Use according to claim 2, characterized in that, The alcohol precipitation process in step S2 is as follows: add anhydrous ethanol to the enzyme hydrolysate until the ethanol concentration reaches 60%, stir evenly, let stand at 4°C for 12 h for alcohol precipitation, and centrifuge at 5000 r / min for 15~25 min.

6. The use according to claim 2, characterized in that, In step S3, the separation and purification process involves passing distilled water through a DEAE-650M cellulose chromatography column for separation and purification.

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