Preparation method and application of corn stigma polysaccharide compound with synergistic effect

Abstract

The invention discloses a preparation method and application of a corn stigma polysaccharide compound with a synergistic interaction effect. Raw materials are pretreated, and then corn stigma crude polysaccharide, ganoderma lucidum crude polysaccharide and hericium erinaceus crude polysaccharide are extracted by adopting a hot water extraction method; respectively crushing the corn stigma crude polysaccharide, the ganoderma lucidum crude polysaccharide and the hericium erinaceus crude polysaccharide, sieving with a 200-mesh sieve, and compounding to obtain a corn stigma polysaccharide compound with two components or a corn stigma polysaccharide compound with three components. The corn stigma polysaccharide compound prepared by the method has synergistic anti-oxidation and synergistic effects and the effect of reducing the use dosage of single polysaccharide under the same anti-oxidation effect. Through the compound synergistic effect, the productivity of polysaccharide products with the same antioxidant effect can be improved.

Description

Technical Field

[0001] The invention relates to a preparation method and application of a corn silk polysaccharide compound with synergistic effects, belonging to the technical field of extraction and separation of natural plant active polysaccharides. Background Art

[0002] Oxidative stress refers to the imbalance between the body's reactive oxygen species (ROS) generation and antioxidant defense system. ROS include superoxide anions, hydroxyl radicals, and ABTS+ radicals, all of which are derived from oxidative stress and can attack biological macromolecules in cells, tissues, and organs. High concentrations of ROS in cells increase the likelihood of diseases such as diabetes, atherosclerosis, cancer, neurodegeneration, and aging. Therefore, removing excess ROS from the body has an important protective effect on organisms. In recent years, polysaccharides from natural resources have received extensive attention due to their good non-cytotoxicity and important biological activities, especially the antioxidant activity of polysaccharides. Using them as natural antioxidants can prevent the harmful effects of free radicals in the human body and protect the human body from excessive ROS.

[0003] Corn silk and edible fungi are both food and medicine, and are the hot topics of current research. They also have high activity, but there are few reports on the synergistic effect between the two. In order to further determine whether corn silk polysaccharides and edible fungi polysaccharides have synergistic antioxidant effects, and to develop antioxidant products with high yield, good activity, and easier to be recognized by the public, this patent uses corn silk crude polysaccharides as the basis, adds Ganoderma lucidum crude polysaccharides and Hericium erinaceus crude polysaccharides to it to prepare a corn silk polysaccharide compound with synergistic effects.

[0004] Ganoderma lucidum has strong practical application value, very high social recognition, and strong antioxidant capacity, but the extraction rate of its polysaccharide products is not high. Therefore, the compound development of Ganoderma lucidum polysaccharides and corn silk polysaccharides can further enhance the product's popularity, while increasing the preparation rate of the corresponding products, which is more conducive to the industrialization of related products.

[0005] Hericium erinaceus is also a widely used and recognized edible mushroom variety. At the same time, Hericium erinaceus polysaccharide has the advantage of high extraction rate, which can increase the productivity of compound products. Summary of the invention

[0006] The present invention provides a preparation method and application of a corn silk polysaccharide compound with synergistic effects. The corn silk polysaccharide compound prepared by the method has synergistic antioxidant and synergistic effects and reduces the dosage of a single polysaccharide under the same antioxidant effect. The compound has a protective effect on oxidative stress in the body.

[0007] The technical solution adopted by the present invention to solve the technical problem is: a method for preparing a corn silk polysaccharide compound with synergistic effect, the steps are as follows:

[0008] 1. Pretreatment of raw materials

[0009] Corn silk, Hericium erinaceus, and Ganoderma lucidum samples were cleaned and dried after impurities removal, crushed, and passed through a 60-mesh sieve. The crushed raw materials were weighed and mixed evenly with 80% ethanol at a ratio of 1:20 (g:mL), and placed in a water bath at 60°C for 1 hour, stirring every 10 minutes. The extract was filtered under reduced pressure, and the resulting residue was dried at 60°C to constant weight after drying, which was the defatted sample after pretreatment and stored in a dry environment for later use.

[0010] 2. Preparation of polysaccharides

[0011] The crude polysaccharides from corn silk, ganoderma lucidum and hericium erinaceus were extracted by hot water extraction.

[0012] Take a certain amount of pretreated raw materials, add distilled water at a mass volume ratio of 1:20 (g / mL), stir and extract at 95-100℃ for 2h, centrifuge at 5000r / min for 10min, repeat the above operation once for the obtained precipitate, collect the two supernatants and filter under reduced pressure to obtain a combined extract. After evaporating the solution to 1 / 8 of the original volume, add 4 times the volume of anhydrous ethanol and mix evenly, let it stand at 4℃ for 24h, centrifuge at 5000r / min for 15min, collect the precipitate, and dry it to constant weight, which is the crude polysaccharide sample.

[0013]

[0014] Polysaccharide extraction rate (%) = crude polysaccharide extraction rate × polysaccharide content

[0015] 3. Preparation of corn silk polysaccharide compound

[0016] Corn silk crude polysaccharide, Ganoderma lucidum crude polysaccharide and Hericium erinaceus crude polysaccharide are respectively crushed and passed through a 200-mesh sieve for compounding.

[0017] A corn silk polysaccharide complex with two components: add Hericium erinaceus crude polysaccharide to corn silk crude polysaccharide in proportion, and after mixing, the Hericium erinaceus corn silk polysaccharide complex with the Hericium erinaceus crude polysaccharide accounting for 10%-30%, hereinafter referred to as Hericium erinaceus complex polysaccharide; add Ganoderma lucidum crude polysaccharide to corn silk crude polysaccharide in proportion, and after mixing, the Ganoderma lucidum corn silk polysaccharide complex with the Ganoderma lucidum crude polysaccharide accounting for 20%-50%, hereinafter referred to as Ganoderma lucidum complex polysaccharide.

[0018] A three-ingredient corn silk polysaccharide complex: first, prepare a Hericium erinaceus corn silk polysaccharide complex in which Hericium erinaceus crude polysaccharide accounts for 30%, and then add Ganoderma lucidum crude polysaccharide to the polysaccharide complex in proportion to prepare a Ganoderma lucidum Hericium erinaceus corn silk polysaccharide complex in which Ganoderma lucidum crude polysaccharide accounts for 15%-30%, hereinafter referred to as Ganoderma lucidum Hericium erinaceus complex polysaccharide.

[0019] The beneficial effects of the present invention are as follows: the corn silk polysaccharide compound prepared by the method has synergistic antioxidant and synergistic effects and the effect of reducing the dosage of a single polysaccharide under the same antioxidant effect. The compound synergistic effect will increase the productivity of polysaccharide products with the same antioxidant effect. Hericium erinaceus polysaccharide has the advantage of high extraction rate (15-17%), and its biological activity is highly recognized by the public. Adding Hericium erinaceus polysaccharide can increase the productivity and popularity of the corn silk polysaccharide compound. Ganoderma lucidum has a broad consumer base and is known as fairy grass. The active application of Ganoderma lucidum has a long history. At the same time, Ganoderma lucidum polysaccharide has good biological activity. The corn silk polysaccharide compound prepared by adding Ganoderma lucidum polysaccharide can increase the popularity of the product and facilitate subsequent promotion. The corn silk polysaccharide complex obtained by the present invention has an in vivo oxidative stress protection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram showing the effect of five types of Hericium erinaceus complex polysaccharides in scavenging DPPH free radicals.

[0021] Figure 2 This is a diagram showing the effect of five types of Ganoderma lucidum polysaccharides in scavenging DPPH free radicals.

[0022] Figure 3 It is the composite polysaccharide of six kinds of Ganoderma lucidum and Hericium erinaceus that has the effect of scavenging DPPH free radicals.

[0023] Figure 4 The effect of TCSP-15 on the mortality of zebrafish larvae with oxidative damage.

[0024] Figure 5 The effect of TCSP-15 on the heart rate of zebrafish larvae with oxidative damage.

[0025] Figure 6 The effect of TCSP-15 on the body length of zebrafish larvae with oxidative damage.

[0026] Figure 7 The effect of TCSP-15 on the morphological development of zebrafish larvae with oxidative damage.

[0027] Figure 8 The effect of TCSP-15 on the spontaneous movement of zebrafish larvae with oxidative damage.

[0028] Fig. 9 The effect of TCSP-15 on the movement of oxidatively damaged zebrafish larvae under light and dark stimulation.

[0029] Fig.10 TCSP-15 is H 2 O 2 Effect of induced oxidative damage on T-SOD activity in zebrafish.

[0030] Fig.11 TCSP-15 is H2 O 2 Effects of induced oxidative damage on GSH-PX activity in zebrafish.

[0031] Fig.12 Is TCSP-15 to H 2 O 2 Effects of induced oxidative damage on CAT activity in zebrafish. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0033] Example 1

[0034] A method for preparing a corn silk polysaccharide compound with synergistic effects, comprising the following steps:

[0035] 1. Pretreatment of raw materials

[0036] Corn silk, Hericium erinaceus, and Ganoderma lucidum samples were cleaned and dried after impurities removal, crushed, and passed through a 60-mesh sieve (diameter 0.25 mm). The crushed raw materials were weighed and mixed evenly with 80% ethanol at a ratio of 1:20 (g:mL), and placed in a water bath at 60°C for 1 hour, stirring every 10 minutes. The extract was filtered under reduced pressure, and the resulting residue was dried at 60°C to constant weight after drying, which was the defatted sample after pretreatment and stored in a dry environment for later use.

[0037] 2. Preparation of polysaccharides

[0038] The crude polysaccharides from corn silk, ganoderma lucidum and hericium erinaceus were extracted by hot water extraction.

[0039] Take a certain amount of pretreated raw materials, add distilled water at a mass volume ratio of 1:20 (g / mL), stir and extract at 95-100℃ for 2h, centrifuge at 5000r / min for 10min, repeat the above operation once for the obtained precipitate, collect the two supernatants and filter under reduced pressure to obtain a combined extract. After evaporating the solution to 1 / 8 of the original volume, add 4 times the volume of anhydrous ethanol and mix evenly, let it stand at 4℃ for 24h, centrifuge at 5000r / min for 15min, collect the precipitate, and dry it to constant weight, which is the crude polysaccharide sample.

[0040]

[0041] Polysaccharide extraction rate (%) = crude polysaccharide extraction rate × polysaccharide content

[0042] 3. Preparation of corn silk polysaccharide compound

[0043] Corn silk crude polysaccharide, Ganoderma lucidum crude polysaccharide and Hericium erinaceus crude polysaccharide were respectively ground and passed through a 200-mesh sieve (diameter 0.0750 mm) for compounding.

[0044] A corn silk polysaccharide complex with two components: add Hericium erinaceus crude polysaccharide to corn silk crude polysaccharide in proportion, and after mixing, the Hericium erinaceus corn silk polysaccharide complex with the Hericium erinaceus crude polysaccharide accounting for 10%-30%, hereinafter referred to as Hericium erinaceus complex polysaccharide; add Ganoderma lucidum crude polysaccharide to corn silk crude polysaccharide in proportion, and after mixing, the Ganoderma lucidum corn silk polysaccharide complex with the Ganoderma lucidum crude polysaccharide accounting for 20%-50%, hereinafter referred to as Ganoderma lucidum complex polysaccharide.

[0045] A three-ingredient corn silk polysaccharide complex: first, prepare a Hericium erinaceus corn silk polysaccharide complex in which Hericium erinaceus crude polysaccharide accounts for 30%, and then add Ganoderma lucidum crude polysaccharide to the polysaccharide complex in proportion to prepare a Ganoderma lucidum Hericium erinaceus corn silk polysaccharide complex in which Ganoderma lucidum crude polysaccharide accounts for 15%-30%, hereinafter referred to as Ganoderma lucidum Hericium erinaceus complex polysaccharide.

[0046] Example 2

[0047] DPPH free radical scavenging ability determination:

[0048] Take 2mL of polysaccharide sample solution of different concentrations and mix it with 2mL of DPPH reagent. Replace the sample solution with distilled water as a blank control. After standing at room temperature and away from light for 30 minutes, measure the absorbance value at a wavelength of 517nm. Ascorbic acid is used as a positive control. The calculation formula of DPPH free radical scavenging rate is as follows:

[0049]

[0050] A 0 :2mL DPPH free radical reagent + 2mL distilled water;

[0051] A 1 :2mL DPPH free radical reagent + 2mL sample;

[0052] A 2 :2mL sample + 2mL anhydrous ethanol;

[0053] Synergy analysis method:

[0054] (1) Chou-Talalay method

[0055] ①Combination Index (CI)

[0056] The combined index (CI) formula proposed by Chou and Talalay in 1938 is:

[0057]

[0058] D 1 , D 2 D is the dose required for each of the two drugs when used together to produce effect X. x1, D x2 It is the dose of each drug when the two drugs are used alone to produce effect X. If CI<1, the two drugs have a synergistic effect, and the smaller the value, the stronger the synergistic effect; CI=1 is an additive effect; CI>1 is an antagonistic effect, and the larger the value, the stronger the antagonistic effect.

[0059] ②Dose reduction index (DRI)

[0060] The dose change before and after the combined use of drugs is also an important indicator for evaluating the combined effect. In 1988, Chou proposed the Dose-Reduction Index (DRI), which is used to measure the multiple reduction in the dose of each substance when used synergistically compared to when used alone at the same level of action. It refers to the ratio of the dose of a single sample required to achieve a certain inhibition rate to the dose of the sample when used in combination. The larger the ratio, the more significant the reduction in the dose of the sample when used in combination is to achieve the same therapeutic effect as a single sample.

[0061]

[0062] D 1 , D 2 D is the dose required for each of the two drugs when used together to produce effect X. x1 , D x2 The doses of the two drugs when used alone produce the X effect.

[0063] (2) Isobologram analysis

[0064] The samples are mixed in pairs at a fixed ratio, the ratio depends on the IC of a single sample 50 Compare the theoretical IC values ​​of the compounds 50add The IC value obtained after the actual experiment 50mix If the actual value is smaller than the theoretical value, it means that there is a synergistic effect after compounding.

[0065]

[0066] In the formula, R = IC 50A / IC 50B , A and B are the ratio of half inhibition rate when used alone, K 1 With K 2 , respectively, are the ratios of samples A and B in the compound, K 1 +K 2 =1.

[0067] Synergistic effect of Hericium erinaceus and corn silk polysaccharide compound (Hericium erinaceus complex polysaccharide)

[0068] ① Scavenging effect of Hericium erinaceus polysaccharide on DPPH free radicals

[0069] See also Figure 1 , Figure 1 This is a diagram showing the effect of five types of Hericium erinaceus complex polysaccharides in scavenging DPPH free radicals.

[0070] Table 1 IC of five Hericium erinaceus polysaccharides in scavenging DPPH free radicals 50 value

[0071]

[0072]

[0073] Depend on Figure 1 It can be seen that the scavenging effect of different proportions of compound polysaccharides on DPPH is positively correlated with the concentration. All five Hericium erinaceus compound polysaccharides showed good scavenging ability, and the scavenging ability increased with the increase of polysaccharide concentration, showing an obvious dose-effect relationship; among them, the IC 50 The lowest was 121.15±2.18μg / mL, followed by 20% Hericium erinaceus polysaccharide and 30% Hericium erinaceus polysaccharide. 50 The values ​​were 140.00±0.87μg / mL and 156.73±1.73μg / mL respectively, indicating that the three compound ratios had the best DPPH scavenging effect, and there was no significant difference among the three. 50 The value gradually decreases with the increase of the proportion of corn silk crude polysaccharides, and gradually increases with the increase of the proportion of Hericium erinaceus crude polysaccharides.

[0074] ②Synergy analysis

[0075] Table 2 Synergistic analysis of scavenging DPPH free radicals from five Hericium erinaceus polysaccharides

[0076]

[0077] As shown in Table 2, the IC values ​​of 10%, 20%, and 30% Hericium erinaceus polysaccharide complex are 50mix The values ​​are all less than IC 50add The CI values ​​of 40% and 50% Hericium erinaceus polysaccharides were less than 1, which confirmed this view. 50mix The values ​​are greater than IC 50addThe value and CI value are both greater than 1, proving that the two ratios of composite polysaccharides have no synergistic effect; according to the synergistic effect ranking of CI value: 10% composite polysaccharide> 20% composite polysaccharide = 30% composite polysaccharide, it can be seen that with the decrease of the proportion of Hericium erinaceus polysaccharide in the composite, the synergistic effect is enhanced, and when the proportion of Hericium erinaceus polysaccharide is greater than 40%, there is no synergistic effect, indicating that the main contributor to the synergistic effect of the two may be corn silk polysaccharide. Given that Hericium erinaceus polysaccharide has a higher extraction rate (15-17%) among the three polysaccharides, increasing its addition amount is beneficial to improving the productivity of the polysaccharide complex, so the optimal addition amount of Hericium erinaceus polysaccharide is 30%.

[0078] Table 3 Five Composite Polysaccharides for Clearing DPPH Free Radical DRI

[0079]

[0080]

[0081] As shown in Table 3, the DRI values ​​of corn silk polysaccharide and Hericium erinaceus polysaccharide in various compounding ratios are all greater than 1, indicating that when the two are used in combination, they can play a role in reducing the dosage of each. Compared with the two substances, the DRI value of Hericium erinaceus polysaccharide is significantly greater than the DRI value of corn silk polysaccharide at five different compounding ratios, indicating that the dosage reduction effect of Hericium erinaceus polysaccharide is more significant than that of corn silk polysaccharide when used in combination.

[0082] Ganoderma lucidum corn silk polysaccharide compound (Ganoderma lucidum complex polysaccharide) synergistic effect:

[0083] ① The scavenging effect of Ganoderma lucidum polysaccharide on DPPH free radicals

[0084] See also Figure 2 , Figure 2 This is a diagram showing the effect of five types of Ganoderma lucidum polysaccharides in scavenging DPPH free radicals.

[0085] Table 4 IC values ​​of five Ganoderma lucidum polysaccharides for scavenging DPPH free radicals 50 value

[0086]

[0087] Depend on Figure 2 It can be seen that the different proportions of compound polysaccharides showed a concentration-dependent effect on scavenging DPPH free radicals. 50All of them are lower than Ganoderma lucidum polysaccharide, indicating that the scavenging ability of the compound is stronger than Ganoderma lucidum polysaccharide. Among the compounds, 20% Ganoderma lucidum polysaccharide has the strongest scavenging ability, and the scavenging rate of DPPH free radicals reaches 84.23% at a concentration of 300μg / mL, which is slightly higher than the corn silk polysaccharide of the same concentration (80.24%), and significantly stronger than the Ganoderma lucidum polysaccharide of the same concentration (65.75%). As shown in Table 4, the IC 50 The scavenging ability of the five compounds was 103.00±3.37μg / mL, which was stronger than that of corn silk polysaccharide (IC 50 =119.44±4.58μg / mL) of complex polysaccharide.

[0088] ②Interaction analysis

[0089] Table 5 Synergistic analysis of scavenging DPPH free radicals of five Ganoderma lucidum polysaccharides

[0090]

[0091]

[0092] As shown in Table 5, the IC values ​​of 20%, 30%, 40%, and 50% Ganoderma lucidum polysaccharide are 50mix The values ​​are all less than IC 50add The values ​​of 10% Ganoderma lucidum polysaccharide IC 50mix Value greater than IC 50add The CI values ​​are all greater than 1, which proves that the composite polysaccharides in this ratio have no synergistic effect. From the CI values ​​in the table, it can be seen that the CI value of 20% Ganoderma lucidum composite polysaccharide is the smallest (0.78), indicating that the synergistic effect of Ganoderma lucidum and corn silk at this ratio is the best.

[0093] Table 6 DRI values ​​of five Ganoderma lucidum polysaccharides for scavenging DPPH free radicals

[0094]

[0095] As shown in Table 6, except for 10% DRI of Ganoderma lucidum polysaccharide 玉米须The value is less than 1, and the DRI values ​​of corn silk polysaccharide and Ganoderma lucidum polysaccharide in other proportions of composite polysaccharides are all greater than 1, indicating that when the proportion of Ganoderma lucidum polysaccharide is 20%, 30%, 40%, and 50%, the combined use of the two can reduce the dosage of each substance. Compared with the two substances, the DRI value of Ganoderma lucidum polysaccharide is significantly greater than the DRI value of corn silk polysaccharide at each composite ratio, indicating that the dosage reduction effect of Ganoderma lucidum polysaccharide is better than that of corn silk polysaccharide when used in combination. With the gradual decrease in the proportion of Ganoderma lucidum polysaccharide in combined use, the DRI value of Ganoderma lucidum polysaccharide increases from 3.55 to 14.76, and the dosage reduction effect gradually increases, indicating that corn silk polysaccharide may play a major role in synergy when used in combination, that is, the higher the content of corn silk polysaccharide, the better the synergistic effect. This conclusion is consistent with the results of CI value analysis.

[0096] The synergistic effect of three polysaccharide compounds (Ganoderma lucidum and Hericium erinaceus complex polysaccharides):

[0097] ① Ganoderma lucidum and Hericium erinaceus complex polysaccharide synergistically scavenging DPPH free radical activity

[0098] See also Figure 3 , Figure 3 This is a diagram showing the effect of six types of Ganoderma lucidum and Hericium erinaceus complex polysaccharides in scavenging DPPH free radicals.

[0099] Table 7 IC values ​​of six kinds of Ganoderma lucidum and Hericium erinaceus polysaccharides in scavenging DPPH free radicals 50 value

[0100]

[0101]

[0102] Depend on Figure 3 It can be seen that the scavenging ability of composite polysaccharides at different concentrations on DPPH is concentration-dependent. At a concentration of 100 μg / mL, the scavenging rate of 15% composite polysaccharide reached 40%, and the scavenging effect was the best among polysaccharides of the same concentration. As shown in Table 7, when the addition amount of Ganoderma lucidum polysaccharide was 15% or above, the IC 50 The values ​​were all lower than those of the respective polysaccharide samples before compounding, indicating that the DPPH free radical scavenging activity of the composite polysaccharides with Ganoderma lucidum polysaccharide addition amounts of 15%, 20%, 25% and 30% was better than that before compounding, showing a synergistic effect between the three polysaccharides.

[0103] ②Interaction analysis

[0104] Table 8 Synergistic analysis of scavenging DPPH free radicals from six kinds of Ganoderma lucidum and Hericium erinaceus polysaccharides

[0105]

[0106] As shown in Table 8, the IC values ​​of 15%, 20%, 25%, and 30% composite polysaccharides are50mix The values ​​are all less than IC 50add The CI values ​​of 5% and 10% composite polysaccharides were less than 1, which confirmed this view. 50mix Value greater than IC 50add The CI values ​​were all greater than 1, indicating that the two ratios of composite polysaccharides did not have a synergistic effect.

[0107] Table 9 DRI values ​​of six kinds of Ganoderma lucidum and Hericium erinaceus polysaccharides in scavenging DPPH free radicals

[0108]

[0109] It can be seen from Table 9 that in the complex polysaccharides with a Ganoderma lucidum polysaccharide content of 15% or more, the DRI values ​​of Hericium erinaceus complex polysaccharides and Ganoderma lucidum polysaccharides are both greater than 1, indicating that when the Ganoderma lucidum polysaccharide content is 15%, 20%, 25%, and 30%, the combined use of the two can reduce the dosage of each, while the 5% and 10% addition levels do not have this effect.

[0110] Example 3

[0111] Protective effect of corn silk polysaccharide compound on oxidative stress in zebrafish embryos:

[0112] 1. Experimental Methods

[0113] 1. Zebrafish Embryo Collection

[0114] At 17:00 the day before the experiment, adult zebrafish were randomly selected and placed in a mating tank with a transparent baffle in a ratio of 2:2 between female and male. The baffle was removed at 9:00 the next day and light was provided. After the zebrafish spawned, the normal fertilized eggs were washed with culture water under a stereomicroscope and collected for the experiment.

[0115] 2. Zebrafish oxidative damage model

[0116] Using H 2 O 2 Induce zebrafish embryos to establish a zebrafish oxidative damage model. According to the results of previous experiments, 5 mM H 2 O 2 As an oxidative stress induction, two control groups and three treatment groups were set up. The control group was a blank control of aquaculture water and 5mMH 2 O 2 Oxidative damage control; treatment groups were protected and incubated with polysaccharide solutions at concentrations of 50, 150, and 300 μg / mL, and 5 mM H 2 O 2 Oxidative damage was performed to 24 hpf, the cells were washed with fresh culture water, and cultured to 96 hpf.

[0117] 3. Embryonic development indicators

[0118] (1) Impact on mortality

[0119] Zebrafish embryos were divided into groups and administrated with drugs. The embryonic development was observed from 7 hpf to 96 hpf. During the process, the embryonic mortality was recorded and the mortality rate was calculated according to the following formula.

[0120] Mortality rate / % = number of dead embryos / total number of embryos × 100

[0121] (2) Impact on heart rate

[0122] When the embryos developed to 96 hpf, 10 fry were randomly selected from each group and their heart rates were observed under a stereomicroscope for 15 s each time. The heart rates were counted 3 times for each fry and the average value was taken.

[0123] (3) Impact on morphological development

[0124] During embryonic development from 48 hpf to 96 hpf, the development of zebrafish in each group was observed and recorded using a stereomicroscope every 24 hours, and the deformity rate was calculated according to the following formula.

[0125] Deformity rate / % = number of deformed embryos / total number of embryos × 100

[0126] 4. Neurodevelopmental indicators

[0127] (1) Assessment of spontaneous movement level

[0128] The zebrafish behavioral system was used to detect the spontaneous movement level of embryos at 120 hpf. The total acquisition time was 120 min, and the total movement distance of the zebrafish was calculated.

[0129] (2) Assessment of exercise level by light and dark stimulation

[0130] The zebrafish behavior system was used to monitor and evaluate the behavior of zebrafish. The behavioral effects produced by light / dark stimulation of young fish were used to analyze the changes in the movement speed of each group of zebrafish, obtain the response ability of zebrafish, and evaluate the neurodevelopment of zebrafish.

[0131] 5. Antioxidant enzyme activity index

[0132] (1) Protein quantification (TP) determination

[0133] Twenty juveniles were taken from each group and placed in ice water for freezing and death. After being washed three times with saline, they were placed in a 1.5 mL centrifuge tube. The liquid in the tube was aspirated with a syringe, and 0.5 mL of saline was added. The zebrafish were ground evenly using a handheld electric tissue grinder in an ice water bath, and centrifuged at 2500 r / min for 10 min. The supernatant was collected for the determination of protein concentration. The determination method was carried out according to the detailed steps of the TP assay kit.

[0134] (2) T-SOD activity assay

[0135] Take an appropriate amount of zebrafish homogenate supernatant, measure the absorbance at 550 nm using an ELISA reader according to the method described in the kit, and calculate the T-SOD activity in zebrafish according to the formula.

[0136] (3) CAT activity assay

[0137] Take an appropriate amount of zebrafish homogenate supernatant, measure the absorbance at 405 nm using an ELISA reader according to the method described in the kit, and calculate the CAT activity in zebrafish according to the formula.

[0138] (4) GSH-PX activity assay

[0139] Take an appropriate amount of zebrafish homogenate supernatant, measure the absorbance at 405 nm using an ELISA reader according to the method described in the kit, and calculate the GSH-PX activity in zebrafish according to the formula.

[0140] 2. Experimental Results

[0141] 1. Protective effect of TCSP-15 on the development of zebrafish induced by oxidative stress

[0142] TCSP-15 is a three-component corn silk polysaccharide compound. The specific preparation method is to first prepare a Hericium erinaceus corn silk polysaccharide complex with 30% of Hericium erinaceus crude polysaccharide, and then add 15% of Ganoderma lucidum crude polysaccharide to the polysaccharide complex to obtain a Ganoderma lucidum Hericium erinaceus corn silk polysaccharide complex with 15% of Ganoderma lucidum crude polysaccharide.

[0143] ①Effects on embryonic mortality during zebrafish hatching

[0144] Depend on Figure 4 It can be seen that the mortality rate of zebrafish in the model group was significantly higher than that in the blank group (P<0.05), reaching 26.67%, indicating that 5 mM H 2 O 2 It has developmental toxicity to zebrafish embryos and affects their survival rate. In the medium and high concentration protection groups of TCSP-15, the mortality rate of zebrafish dropped to 13.33% and 5.00%, respectively, which was significantly lower than that of the model group. This shows that TCSP-15 has a positive effect on H 2 O 2 The induced zebrafish embryo damage has a protective effect. In the figure, different letters from a to c indicate that there are significant differences among different groups (P<0.05).

[0145] ②Effects on zebrafish heart rate

[0146] Depend on Figure 5It can be seen that the heart rate of zebrafish in the blank group was 51 beats / 15s, and the heart rate of the model group was 29.4% lower than that of the blank group, showing a significant inhibitory effect, and the difference was statistically significant (P<0.05). Under the protection of TCSP-15, the heart rate of zebrafish embryos in the medium and high concentration groups increased significantly, showing a dose-dependent manner, indicating that TCSP-15 can improve H 2 O 2 The induced abnormal heart rate in zebrafish showed a potential protective effect in alleviating cardiac dysfunction caused by oxidative damage.

[0147] ③Effects on zebrafish body length

[0148] In ecotoxicology experiments, the body weight and length of the test organisms are often used as indicators to determine whether growth is delayed. Figure 6 It can be seen that compared with the average body length of zebrafish in the blank group (3.93 mm), the body length of the juveniles in other groups was shortened, and the body length of zebrafish in the model group (2.54 mm) was shortened by nearly 35.4%, indicating that H 2 O 2 The growth and development of zebrafish were inhibited. After TCSP-15 treatment, the body length of zebrafish in each protection group increased significantly, showing a dose-dependent manner. This result shows that TCSP-15 can alleviate H 2 O 2 Caused by abnormal development of zebrafish.

[0149] ④Effects on zebrafish morphological development

[0150] See also Figure 7 In this experiment, zebrafish that still showed symptoms such as yolk cyst (YSE), pericardial edema (PE), and swim bladder deficiency (SBD) at 96 hpf were identified as deformed. The experimental results showed that zebrafish in the blank group developed normally, with complete head shape, fully inflated swim bladder and straight spine, and the deformity rate was only 3.33%. In contrast, zebrafish exposed to 5mM H 2 O 2 The deformity rate of zebrafish in the model group increased significantly to 36.67%, which was about 11 times that of the blank group (P<0.05). Figure 7 The results showed that the deformity rate of zebrafish in the medium and high concentration polysaccharide treatment groups decreased significantly (P<0.05), and the morphological development of the juveniles was improved. Among them, the deformity rate of the juveniles in the high concentration treatment group was not significantly different from that in the blank group, indicating that its protective effect reached the best effect.

[0151] In summary, medium and high concentrations of TCSP-15 have an important effect on H 2 O2 The induced oxidative stress damage in zebrafish has a significant protective effect, not only reducing the deformity rate of young fish, but also effectively improving developmental abnormalities caused by oxidative damage.

[0152] 2. Effects of TCSP-15 on neural development in zebrafish induced by oxidative stress

[0153] ① Effects on zebrafish spontaneous movement

[0154] Depend on Figure 8 It can be seen that the total movement distance of zebrafish in the model group was (1514.40±138.02) cm, which was significantly reduced by 60.69% (P<0.05) compared with the blank group (3852.16±190.70) cm, indicating that 5 mM H 2 O 2 The movement behavior of zebrafish was significantly inhibited. After treatment with medium and high concentrations of TCSP-15, the movement distance of zebrafish reached 1.75 times and 2.09 times that of the model group, respectively, significantly improving the slow movement caused by oxidative damage, showing the protective effect of TCSP-15.

[0155] ②Effects on zebrafish movement under light and dark stimulation conditions

[0156] See also Fig. 9 The response of zebrafish to alternating light and dark stimulation can indicate whether the structure of its spinal motor neurons or neuromuscular junctions is intact. When normal larvae enter light from darkness, their movement speed will drop significantly and then stabilize. When going from light to darkness, the movement speed of zebrafish larvae will increase rapidly. Fig. 9 It can be seen that the overall movement speed of zebrafish in each experimental group under light-dark alternation conditions has a similar trend of change, that is, the movement speed of zebrafish in the dark state is higher than that in the light state. Compared with the blank group, the average speed of zebrafish in the model group decreased by 34.24% (P<0.05), of which the dark state decreased by 37.14% and the light state decreased by 29.38%. This result shows that H 2 O 2 Oxidative damage may affect the development of the zebrafish nervous system. After treatment with medium and high concentrations of polysaccharides, the average movement speed of zebrafish was significantly improved, increasing by 20.48% and 38.19% respectively compared with the model group. These results show that under the stimulation of alternating light and dark, oxidative stress can cause the reaction of zebrafish to decrease, but after polysaccharide protection treatment, its movement ability and reaction ability are improved.

[0157] 3. Effects of TCSP-15 on antioxidant enzymes in zebrafish with oxidative stress

[0158] ①Effects on T-SOD enzyme activity in zebrafish

[0159] See also Fig.10 SOD enzyme is the first line of defense for cells in the body to resist free radical damage. The level of SOD activity indirectly reflects the ability of cells to clear ROS. Fig.10 As shown in the figure, compared with the blank group (51.67U / mg protein), the T-SOD activity of zebrafish in the model group (21.08U / mg protein) decreased significantly, by about 59.2%, indicating that the oxidative damage model caused damage to the zebrafish body, resulting in a decrease in T-SOD activity. Compared with the model group, the T-SOD activity of the medium and high concentration treatment groups was significantly increased, and in a dose-dependent manner. Among them, the T-SOD activity of the high concentration treatment group increased by about 2.05 times compared with the model group, showing a protective effect on the body, thereby inhibiting the body's oxidative damage.

[0160] ②Effects on GSH-PX activity in zebrafish

[0161] See also Fig.11 GSH-PX is a GSH-based enzyme widely present in organisms that can reduce hydrogen peroxide to hydroxyl compounds by promoting H 2 O 2 Decomposition of free radicals protects cell membranes from damage by free radicals. Fig.11 It can be seen that H 2 O 2 The results showed that H induced oxidative stress in zebrafish embryos and significantly reduced the enzyme activity of GSH-PX (P<0.05). Compared with the blank group, the enzyme activity in the model group decreased by about 75.41%, indicating that H 2 O 2 The antioxidant system in zebrafish was damaged, resulting in a decrease in GSH-PX synthesis. After treatment with TCSP-15, the enzyme activity of zebrafish embryos was significantly increased in both the medium and high concentration treatment groups (P<0.05), showing a dose-dependent manner, indicating that TCSP-15 plays an important role in alleviating H 2 O 2 It has a protective effect against oxidative damage induced by

[0162] ③Effects on CAT activity in zebrafish

[0163] See also Fig.12 CAT is an important enzyme that uses iron porphyrin as a cofactor. It can catalyze the decomposition of hydrogen peroxide into water and oxygen, continuously remove oxidative metabolites produced in the body, and protect cells from damage. Fig.12 It can be seen that compared with the blank group, the 2 O 2The CAT activity in zebrafish after treatment decreased significantly (P<0.05), indicating that it caused damage to the zebrafish body. Compared with the model group, the CAT activity of zebrafish increased to varying degrees after treatment with medium and high concentrations of TCSP-15, showing a dose-dependent manner. This shows that TCSP-15 has a certain recovery effect on the decrease in CAT activity caused by oxidative damage in zebrafish.

Claims

1. A method for preparing a corn silk polysaccharide compound with synergistic effect, characterized in that: Here are the steps: (1) Pretreatment of raw materials Corn silk, Hericium erinaceus, and Ganoderma lucidum samples were cleaned and dried after impurities removal, crushed, and passed through a 60-mesh sieve. The crushed raw materials were weighed and mixed evenly with 80% ethanol at a ratio of 1:20 (g:mL). The samples were placed in a water bath at 60°C for 1 hour, stirred every 10 minutes, and the extract was filtered under reduced pressure. The obtained residue was dried at 60°C to constant weight, which was the defatted sample after pretreatment and stored in a dry environment for later use. (2) Preparation of polysaccharides The crude polysaccharides of corn silk, ganoderma lucidum and hericium erinaceus were extracted by hot water extraction. Take a certain amount of pretreated raw materials respectively, add distilled water at a mass volume ratio of 1:20 (g / mL), extract at 95-100℃ with stirring for 2 h, centrifuge at 5000 r / min for 10 min, repeat the above operation once for the obtained precipitate, collect the two supernatants and filter under reduced pressure to obtain a combined extract, evaporate the solution to 1 / 8 of the original volume, add 4 times the volume of anhydrous ethanol and mix evenly, let stand at 4℃ for 24 h, centrifuge at 5000 r / min for 15 min, collect the precipitate, and dry to constant weight to obtain the crude polysaccharide sample; (3) Preparation of corn silk polysaccharide compound Corn silk crude polysaccharide, Ganoderma lucidum crude polysaccharide and Hericium erinaceus crude polysaccharide are respectively crushed and passed through a 200-mesh sieve for compounding.

2. The method for preparing a corn silk polysaccharide compound with synergistic effect as claimed in claim 1, characterized in that: Corn silk polysaccharide complex with two components: adding Hericium erinaceus crude polysaccharide to corn silk crude polysaccharide in proportion, and mixing well to obtain Hericium erinaceus corn silk polysaccharide complex with Hericium erinaceus crude polysaccharide accounting for 10%-30%; Adding Ganoderma lucidum crude polysaccharide to corn silk crude polysaccharide in proportion, and mixing well to obtain a Ganoderma lucidum corn silk polysaccharide complex in which the Ganoderma lucidum crude polysaccharide accounts for 20%-50%; A three-ingredient corn silk polysaccharide complex: first, prepare a Hericium erinaceus corn silk polysaccharide complex in which the crude Hericium erinaceus polysaccharide accounts for 30%, and then add Ganoderma lucidum crude polysaccharide to the polysaccharide complex in proportion to prepare a Ganoderma lucidum Hericium erinaceus corn silk polysaccharide complex in which the crude Ganoderma lucidum polysaccharide accounts for 15%-30%.

3. An application of a corn silk polysaccharide compound with synergistic effect, characterized in that: It has a protective effect against oxidative stress in the body.

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