Extraction method and application of sea grape polysaccharide

Abstract

The invention relates to an extraction method and application of sea grape polysaccharide, and the extraction method comprises the following steps: 1) selecting fresh and mature sea grapes, drying and crushing to obtain light green sea grape powder for later use; 2) adding hot water into the sea grape powder, extracting, centrifuging, and taking supernate; 3) concentrating the supernate, adding 4 times of absolute ethyl alcohol in volume, carrying out alcohol precipitation overnight at 4 DEG C, collecting precipitate, re-dissolving the precipitate in water, and centrifuging to remove water-insoluble impurities; and 4) fully dissolving the polysaccharide after impurity removal in ultrapure water, deproteinizing, centrifuging, dialyzing the polysaccharide solution, concentrating and freeze-drying to obtain the sea grape crude polysaccharide. By setting the extraction mode of the sea grape polysaccharide and determining the parameters, the optimal extraction of the sea grape polysaccharide is realized, the extraction process is simple, the extraction efficiency is high, and the sea grape polysaccharide extracted by the method has better effects in neuroprotective ability and intestinal inflammation determination, and is suitable for popularization and application. Therefore, a new direction is provided for modernization of traditional Chinese medicines and deep application of polysaccharide products.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and specifically relates to the extraction and application of Caulerpa lentillifera polysaccharide. Background Art

[0002] Currently, the methods for extracting natural plant polysaccharides include: water extraction and alcohol precipitation method, acid (alkali) solution extraction method, ultrasonic-assisted extraction method, etc. Water extraction and alcohol precipitation is a traditional method for extracting plant polysaccharides, which separates polysaccharides based on the principle that polysaccharides are polar molecules soluble in water and insoluble in alcohols because they are difficult to form hydrogen bonds with alcohol solvents; acid solutions can break the glycosidic bonds of polysaccharides, and alkali solutions can extract polysaccharides bound to proteins; ultrasonic-assisted extraction uses the mechanical, cavitation, and thermal effects of ultrasonic vibration to destroy the cell wall structure, thereby rapidly releasing intracellular polysaccharides.

[0003] Caulerpa lentillifera is a green alga, known as sea grapes because of its appearance, belonging to the family Caulerpaceae, order Bryopsidales, phylum Chlorophyta. It contains high polysaccharides, dietary fiber, protein, and several essential unsaturated fatty acids. In recent years, studies on its biological activities have shown that it contains antioxidants and has biological activities such as anticoagulation, anti-cancer activity, immune regulation, and anti-inflammatory effects. More and more studies have confirmed that polysaccharides from natural sources, especially marine polysaccharides, have significant biological activities, such as antioxidant, antiviral, anti-lipid, and immune regulation, and relatively low toxicity and side effects. The diversity of their structures and biological activities and the large population determine their great development and utilization value. Caulerpa lentillifera polysaccharide is a potential seaweed polysaccharide, but the related research is relatively lacking.

[0004] Aiming at the problems existing in the prior art, the purpose of the design of the present invention is to provide a method for extracting and applying Caulerpa lentillifera polysaccharide, which is specifically realized through the following technical solutions:

[0005] The method for extracting Caulerpa lentillifera polysaccharide includes the following steps:

[0006] 1) Select fresh and mature Caulerpa lentillifera, soak and wash it, then dry and crush it in an oven to obtain light green Caulerpa lentillifera powder for standby;

[0007] 2) Heat and extract the Caulerpa lentillifera powder obtained in step 1) with hot water, and take the supernatant after centrifugation;

[0008] 3) Concentrate the supernatant obtained in step 2), add 4 times the volume of absolute ethanol, and perform alcohol precipitation overnight at 4°C. Collect the precipitate, redissolve the precipitate in water, and remove water-insoluble impurities by centrifugation;

[0009] 4) Dissolve the polysaccharide after impurity removal in step 3) in ultrapure water, remove proteins using the Sevage method. After centrifugation, dialyze the protein-free polysaccharide solution against running water using a 3500 Da dialysis bag for 48 h, and concentrate and lyophilize to obtain crude Caulerpa lentillifera polysaccharide.

[0010] Preferably, in step 1), the oven temperature is 45 °C.

[0011] Preferably, in step 2), the solid-liquid ratio is 1 g: 200 mL, and the hot water temperature is maintained at 80 °C for extraction for 2 - 4 h.

[0012] Preferably, in step 2), the centrifugation conditions are centrifugation at 4000 r / min for 15 min.

[0013] Preferably, in step 4), the protein removal by the Sevage method is specifically as follows: Prepare the protein removal solution in advance, add 1 / 4 volume of the protein removal solution to the aqueous solution of the polysaccharide, stir vigorously for 30 min, then centrifuge at 4000 r / min for 10 min. The protein removal solution is a mixed solution of chloroform and n-butanol, and the volume ratio of the two is chloroform: n-butanol = 5: 1.

[0014] Use of the above-prepared Caulerpa lentillifera polysaccharide in the preparation of a product for preventing and / or treating a product with neuroprotective effects.

[0015] Use of the above-prepared Caulerpa lentillifera polysaccharide in the preparation of a product for relieving intestinal inflammation.

[0016] A product with neuroprotective effects, the product is a drug or a functional food, and the product contains the active ingredient Caulerpa lentillifera polysaccharide.

[0017] By setting the extraction method of Caulerpa lentillifera polysaccharide and determining the parameters, the present invention realizes the optimal extraction of Caulerpa lentillifera polysaccharide. The extraction process is simple and the extraction efficiency is high. The Caulerpa lentillifera polysaccharide extracted by the method of the present invention has good effects in both neuroprotection ability and intestinal inflammation determination, thus providing a new direction for the modernization of traditional Chinese medicine and the in-depth application of polysaccharide products. Description of the Drawings

[0018] Figure 1 Comparison of the COP antioxidant capacity of four extraction methods;

[0019] Figure 2 COP ability to reduce NO;

[0020] Figure 3 COP ability to reduce TNF-α;

[0021] Figure 4 COP ability to reduce DAO;

[0022] Figure 5It is a typical graph of the fluorescence intensity of the zebrafish intestine after COP treatment;

[0023] Figure 6 It is the fluorescence intensity of the zebrafish intestine after COP treatment;

[0024] Figure 7 It is a typical graph of the number of neutrophils in the zebrafish intestine after COP treatment;

[0025] Figure 8 It is the number of neutrophils in the zebrafish intestine after COP treatment. Specific embodiments

[0026] The following further illustrates the present invention in conjunction with specific embodiments to better understand the technical solution.

[0027] Select fresh and mature Caulerpa lentillifera, soak and wash it, dry it in an oven at 45°C and then crush it to obtain light green Caulerpa lentillifera powder for standby.

[0028] Example 1: Extraction of Caulerpa lentillifera polysaccharide

[0029] Hot water extraction: Using 2L of ultrapure water as the solvent, mixing with the Caulerpa lentillifera powder at a solid-liquid ratio of 1g:200mL, extracting at 80°C for 3 hours. After extraction, centrifuge at a speed of 4000r / min for 15min using a centrifuge and collect the supernatant.

[0030] Ultrasonic extraction: Using 2L of ultrapure water as the solvent, mixing with the Caulerpa lentillifera powder at a solid-liquid ratio of 1g:200mL, extracting at 8°C for 1 hour. After extraction, centrifuge at a speed of 4000r / min for 15min using a centrifuge and collect the supernatant.

[0031] Acid extraction: Using 2L of ultrapure water as the solvent and adding 0.1mol / L HCl, mixing with the Caulerpa lentillifera powder at a solid-liquid ratio of 1g:200mL, extracting at 60°C for 3 hours. After extraction, centrifuge at a speed of 4000r / min for 15min using a centrifuge and collect the supernatant.

[0032] Alkali extraction: Using 2L of ultrapure water as the solvent and adding 0.1mol / L NaOH, mixing with the Caulerpa lentillifera powder at a solid-liquid ratio of 1g:200mL, extracting at 60°C for 3 hours. After extraction, centrifuge at a speed of 4000r / min for 15min using a centrifuge and collect the supernatant.

[0033] Purification and freeze-drying: The extracts obtained by the above four extraction methods were concentrated separately using a rotary evaporator. Four volumes of absolute ethanol were added, and the mixture was allowed to precipitate overnight at 4°C. The precipitate was redissolved in water, and water-insoluble impurities were removed by centrifugation. The polysaccharide obtained was fully dissolved in ultrapure water, and the Sevage method was used for protein removal: A protein removal solution (chloroform: n-butanol = 5:1) was prepared in advance. One-fourth volume of the protein removal solution was added to the aqueous solution of the polysaccharide, and the mixture was vigorously stirred for 30 min and then centrifuged at 4000 r / min for 10 min. The protein-free polysaccharide solution was dialyzed against running water using a 3500 Da dialysis bag for 48 h, and then concentrated and freeze-dried to obtain crude Caulerpa lentillifera polysaccharide.

[0034] Among them, the polysaccharide of Caulerpa lentillifera extracted by hot water extraction is denoted as COP-H, the polysaccharide of Caulerpa lentillifera extracted by ultrasonic extraction is denoted as COP-C, the polysaccharide of Caulerpa lentillifera extracted by acid extraction is denoted as COP-A, and the polysaccharide of Caulerpa lentillifera extracted by alkali extraction is denoted as COP-U.

[0035] Example 2: Antioxidant assay

[0036] Antioxidant assay: Hydroxyl radical scavenging ability assay, DPPH radical scavenging ability assay, reducing power assay, and ABTS radical scavenging ability assay.

[0037] (1) DPPH radical scavenging ability assay

[0038] Sample solutions of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL were prepared with distilled water. Then, 1.0 mL of the sample solution was mixed with 1.0 mL of 0.2 mM DPPH solution, and the mixture was incubated in a water bath at 37°C for 30 min (protected from light). The absorbance value Ai was measured at 517 nm after zeroing with absolute ethanol. The operation was repeated using absolute ethanol instead of DPPH to obtain Aj. Three parallel measurements were made for each concentration, and A0 was measured using distilled water instead of the sample.

[0039] DPPH radical scavenging rate (%) = [1 - (Ai - Aj) / A0] × 100%

[0040] (2) Reducing power assay

[0041] 0.5 mL of the sample solution, 0.5 mL of PBS (2 M, pH = 6.6), and 0.5 mL of 1% potassium ferricyanide were mixed and incubated in a water bath at 50°C for 20 min. After rapid cooling, 0.5 mL of 10% trichloroacetic acid was added to stop the reaction, and the mixture was centrifuged at 3000 rpm for 10 min. 1.0 mL of the supernatant was taken, 1.0 mL of distilled water and 0.2 mL of 0.1% ferric chloride were added, and the mixture was mixed well and allowed to stand for 10 min. The absorbance was measured at 700 nm. Three parallel measurements were made for each concentration, and zeroing was done with distilled water.

[0042] (3) ABTS radical scavenging ability assay

[0043] Dissolve 7 mmol / L ABTS ammonium salt in 2.45 mmol / L K2S2O8, and prepare a stable radical cation ABTS stock solution with a volume ratio of 2:1. Let it stand overnight at room temperature before use. After dilution with ethanol, the absorbance at 734 nm is 0.700 ± 0.02. Mix 0.1 mL of the polysaccharide solution with 1 mL of the ABTS dilution solution, vortex, and measure the absorbance at 734 nm after 6 min.

[0044] 3. Determination of neuroprotective ability

[0045] Studies have shown that sulfated polysaccharides can directly act on Aβ protein to inhibit and remove the deposition of Aβ protein, maintain the plasticity of mitochondria, and thus alleviate the symptoms of AD. Use LPS to model mouse microglial cells (BV-2), measure various inflammatory indicators, and examine the protective effect of COP on nerve cells.

[0046] 4. Determination of the ability to reduce intestinal inflammation

[0047] Studies have shown that many polysaccharides have the function of protecting the intestine, specifically reflected in promoting the proliferation of beneficial bacteria, improving the intestinal environment, enhancing immune function, and regulating blood sugar. Use LPS to model c57 mice, measure the content of DAO in their serum, and examine the protective effect of COP on intestinal inflammation.

[0048] 5. The total sugar content and crude yield of the COP crude polysaccharide extracted by different extraction methods are shown in Table 1. It can be seen from Table 1 that the sugar content of hot water extraction is the highest, reaching 59.7%, and the crude yield of hot water extraction is also relatively high. Generally speaking, hot water extraction is relatively excellent, and both the sugar content and the crude yield are the highest among the four methods.

[0049] The results of antioxidant ability are as Figure 1 shown. COP extracted by four different methods all showed different degrees of antioxidant ability, and showed a dose-dependent relationship. This result not only verified the potential of COP as an antioxidant, but also showed that its antioxidant activity was affected by the extraction process. In the DPPH radical and ferric ion reducing ability tests, the antioxidant effect of hot water extraction was significantly better than the other three; in the ABTS radical test, it maintained a high scavenging rate, and the antioxidant effect of alkaline extraction was the best, and hot water extraction was better. Considering the antioxidant ability, sugar content, extraction rate, simplicity of operation of the polysaccharides extracted by the four methods, and possible environmental pollution problems of some extraction methods, we finally selected hot water extraction for the optimization operation of subsequent experiments.

[0050] Example 3: Determination of the neuroprotective ability of COP

[0051] During the inflammatory response, inflammatory mediators such as cytokines and chemokines activate endogenous nitric oxide synthase (iNOS), which in turn promotes the massive synthesis of nitric oxide (NO). This increase in synthesis is highly correlated with the severity and duration of inflammation. BV-2 cells were modeled with LPS, and the increase or decrease of inflammation was judged by the NO level. Cells in the logarithmic growth phase were used for the experiment and divided into 3 groups: control group, model group, and drug administration group, which were seeded in 96-well plates. After they grew confluent, the cells in the model group were treated with 1 μg / mL LPS for 24 h, and the cells in the drug administration group were pretreated with different concentrations of COP for 2 h and then treated with 1 μg / mL LPS for 24 h. After 24 h, the NO content in the cell supernatant was measured.

[0052] As Figure 2 shown, under the action of different concentrations of COP, the NO content was reduced to a certain extent, and the NO levels were significantly reduced at the doses of 50 and 10 μg / mL (P<0.001).

[0053] In various inflammatory diseases, the level of TNF-α will increase abnormally. This increase is closely related to the severity and duration of inflammation, so it can be used as an index to evaluate the degree of inflammation.

[0054] As Figure 3 shown, under the action of different concentrations of COP, the TNF-α content was significantly reduced (P<0.05). Cells in the logarithmic growth phase were used for the experiment and divided into 3 groups: control group, model group, and drug administration group, which were seeded in 96-well plates. After they grew confluent, the cells in the model group were treated with 1 μg / mL LPS for 24 h, and the cells in the drug administration group were pretreated with different concentrations of COP for 2 h and then treated with 1 μg / mL LPS for 24 h. After 24 h, the TNF-α content in the cell supernatant was measured using an ELISA kit.

[0055] Therefore, COP can reduce neuroinflammation in BV-2 cells, thus achieving the effect of neuroprotection.

[0056] Example 4: Determination of the anti-intestinal inflammation ability of COP

[0057] 1. Effect of COP on the DAO level of intestinal inflammation in mice In gastrointestinal diseases, the serum DAO will increase in small intestinal inflammation. As Figure 4As shown, in the LPS-induced mouse neuroinflammation model, C57 BL / 6 male mice were randomly divided into 3 groups: control group (Control), LPS model group (LPS), and COP treatment group (LPS+COP). Mice in the control group and the LPS group were continuously intragastrically administered normal saline for 21 days, and mice in the COP group were intragastrically administered COP at 300-600 mg / kg for 21 days. From the 14th day, after intragastric administration for 30 minutes each day, mice in the control group were intraperitoneally injected with sterile normal saline for 7 days, and mice in the LPS group and the COP group received an intraperitoneal injection of 2.0 mg / kg of LPS for 7 days. Serum was collected after the mice were sacrificed. The DAO content in the serum was measured using a DAO kit.

[0058] The DAO activity in the mouse serum was significantly increased, and the DAO content decreased after COP treatment. The DAO activity in the serum was significantly reduced in the COP600 dose group (P<0.05).

[0059] 2. Effects of COP on zebrafish intestinal inflammation and diarrhea

[0060] The intestinal components of zebrafish (Danio rerio) are similar to those of humans, such as connective tissue, outer longitudinal muscle, and circular muscle. Here, LPS was used to induce intestinal inflammation and diarrhea in zebrafish. Inflammation assay: Randomly selected 5-dpf wild-type AB strain zebrafish were given 10.0 ng / mL nile red in water as a fluorescent indicator of intestinal contents. After feeding for 2 hours, the nile red was eluted, and the zebrafish were randomly assigned to 6-well plates, with 30 zebrafish in each well (experimental group). Samples were given in water, and a normal control group and a model control group were set up at the same time, with a volume of 3 mL per well. Except for the normal control group, LPS was injected into the intestine of the remaining experimental groups to establish a zebrafish intestinal inflammation model. After treatment at 28°C for 1 day, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. The NIS-Elements D 3.20 advanced image processing software was used to collect data and analyze the fluorescence intensity of the zebrafish intestine. The statistical analysis results of this index were used to evaluate the efficacy of the sample in improving diarrhea.

[0061] Diarrhea assay: Randomly selected 5-dpf transgenic neutrophil green fluorescent zebrafish (MPX) in 6-well plates, with 30 zebrafish in each well (experimental group). Samples were given in water, and a normal control group and a model control group were set up at the same time, with a volume of 3 mL per well. Except for the normal control group, LPS was injected into the intestine of the remaining experimental groups to establish a zebrafish intestinal inflammation model. After treatment at 28°C for 1 day, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. The NIS-Elements D 3.20 advanced image processing software was used to collect data and analyze the number of neutrophils in the zebrafish intestine. The statistical analysis results of this index were used to evaluate the efficacy of the sample in improving intestinal inflammation.

[0062] The results are shown in Table 1 and Figures 5 - 6 as follows. In the model group, inflammation and diarrhea were severe, while COP significantly alleviated intestinal inflammation and diarrhea. Therefore, COP has the efficacy of improving diarrhea to varying degrees.

[0063] Table 1: Evaluation experimental results of the efficacy of samples in improving diarrhea

[0064]

[0065] Compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001

[0066] The evaluation experimental results of the efficacy of samples in improving intestinal inflammation are shown in Table 2 and Figures 7 - 8 as follows.

[0067] Table 2: Evaluation experimental results of the efficacy of samples in improving intestinal inflammation (n = 10)

[0068]

[0069] Compared with the model control group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0070] Therefore, COP has the efficacy of improving intestinal inflammation to varying degrees, specifically manifested as a reduction in the number of intestinal neutrophils.

[0071] In summary, COP has the ability to alleviate intestinal inflammation and the effect of protecting nerves.

Claims

1. A method for extracting sea grape polysaccharide, characterized in that: The extraction method comprises the following steps: 1) Select fresh and ripe sea grapes, soak and clean them, and then dry and crush them in an oven to obtain light green sea grape powder for later use; 2) extracting the sea grape powder obtained in step 1) with heated water, and taking the supernatant after centrifugation; 3) After concentrating the supernatant of step 2), 4 volumes of anhydrous ethanol were added, and the mixture was precipitated overnight at 4° C., and the precipitate was collected. After the precipitate was redissolved in water, water-insoluble impurities were removed by centrifugation; 4) The polysaccharide removed from step 3) is fully dissolved in ultrapure water, and the protein is removed by Sevage method. After centrifugation, the deproteinized polysaccharide solution is dialyzed for 48 hours using 3500Da dialysis bag flowing water, and concentrated and freeze-dried to obtain the crude sea grape polysaccharide.

2. The method for extracting sea grape polysaccharide according to claim 1, characterized in that: In step 1), the oven temperature is 45°C.

3. The method for extracting sea grape polysaccharide according to claim 1, characterized in that: In step 2), the solid-liquid ratio is 1 g:200 mL, and the hot water temperature is maintained at 80° C. for extraction for 2-4 hours.

4. The method for extracting sea grape polysaccharide according to claim 1, characterized in that: In step 2), the centrifugation condition is 4000r / min for 15min.

5. The method for extracting sea grape polysaccharide according to claim 1, characterized in that: In step 4), the Sevage method for deproteinization is specifically as follows: prepare a deproteinization solution in advance, add 1 / 4 volume of the deproteinization solution to the polysaccharide aqueous solution, stir vigorously for 30 minutes, and then centrifuge at 4000r / min for 10 minutes. The deproteinization solution is a mixture of chloroform and n-butanol, and the volume ratio of the two is chloroform: n-butanol = 5:

1.

6. Use of the sea grape polysaccharide according to any one of claims 1 to 5 in the preparation of a product having a neuroprotective effect for prevention and / or treatment.

7. Use of the sea grape polysaccharide according to any one of claims 1 to 5 in the preparation of a product for alleviating intestinal inflammation.

8. A product with neuroprotective effect, characterized in that The product is a medicine or a functional food, and contains the active ingredient sea grape polysaccharide.

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