Illicium verum luffa polysaccharide with anti-oxidation and anti-inflammatory effects as well as preparation method and application thereof

Abstract

The invention provides anise loofah polysaccharide with anti-oxidation and anti-inflammatory effects and a preparation method and application thereof.The preparation method comprises the steps that dried anise loofah is taken and added into distilled water, centrifugation is performed after water bath, and after absolute ethyl alcohol is added into an obtained extracting solution for precipitation, an anise loofah crude extract is obtained; and dissolving the crude extract in distilled water, loading a sample in a chromatographic column, eluting with distilled water to obtain the anise-luffa polysaccharide LAP1, and then carrying out segmented elution with a NaCl solution to obtain the anise-luffa polysaccharide LAP2. The preparation method comprises the following steps: firstly, carrying out hot water boiling extraction and absolute ethyl alcohol precipitation on anise loofah, and purifying a crude extract by using column chromatography, so as to obtain the anise loofah polysaccharide LAP1 and the anise loofah polysaccharide LAP2 with remarkable antioxidant and anti-inflammatory effects. The two kinds of star anise and towel gourd polysaccharides can be prepared at the same time through one method, can effectively remove free radicals such as active oxygen and can inhibit overexpression of inflammatory factors, the active ingredients are clear, no toxic or side effect exists, and the star anise and towel gourd polysaccharides can be used as medical anti-oxidation and anti-inflammatory preparations.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to a loofah polysaccharide with antioxidant and anti-inflammatory effects, its preparation method, and application. Background Art

[0002] Oxidative stress is a characteristic of many neurological and neurodegenerative diseases. Highly reactive oxygen and nitrogen species can directly oxidatively damage macromolecules such as DNA, proteins, and lipids, ultimately leading to neurodegeneration in the central nervous system. In addition, oxidative stress also activates glial cells associated with many central nervous system injuries. Activated glial cells are histopathological markers of neurodegenerative diseases, and these activated glial cells release immune mediators (such as nitric oxide and reactive oxygen species, pro-inflammatory cytokines, and chemokines), and currently these immune mediators are considered candidate neurotoxins. Therefore, oxidative stress can also cause secondary neuronal damage by activating cell-mediated inflammatory mechanisms and releasing immune mediators.

[0003] During the neurodegenerative process, a mixture of antioxidants and anti-inflammatory agents may be more beneficial for preventing neurodegenerative diseases and can produce better neuroprotective effects. Therefore, finding natural products with both antioxidant and anti-inflammatory effects is of great significance for the treatment of neurodegenerative-related diseases. Summary of the Invention

[0004] The purpose of the present invention is to provide a loofah polysaccharide with antioxidant and anti-inflammatory effects. The loofah polysaccharide is loofah polysaccharide LAP1 or loofah polysaccharide LAP2, and both loofah polysaccharide LAP1 and loofah polysaccharide LAP2 have good antioxidant activity and significant anti-inflammatory effects.

[0005] Another purpose of the present invention is to provide a method for simultaneously preparing two loofah polysaccharides with antioxidant and anti-inflammatory effects. The crude loofah extract is purified using a DEAE-Sepharose CL-6B chromatography column, and then loofah polysaccharide LAP1 is obtained by eluting with distilled water, and loofah polysaccharide LAP2 is obtained by using a stepwise elution method. The loofah polysaccharides prepared by this method have clear components, no impurities, and two loofah polysaccharides can be simultaneously prepared using one method.

[0006] The third purpose of the present invention is to provide the application of the loofah polysaccharide with antioxidant and anti-inflammatory effects in the preparation of pharmaceutical antioxidant and anti-inflammatory preparations.

[0007] The present invention solves its technical problems by adopting the following technical solutions.

[0008] The present invention provides a method for simultaneously preparing two Luffa acutangula polysaccharides with antioxidant and anti-inflammatory effects, comprising the following steps: S1. Take dried Luffa acutangula, add distilled water, carry out centrifugation after constant temperature water bath to obtain an extract; S2. Concentrate the extract, add absolute ethanol thereto, then cover with a plastic wrap and react at 3 - 5°C for 15 - 24 h. The precipitate obtained after centrifugation is dried to obtain a crude extract of Luffa acutangula; S3. Dissolve the crude extract of Luffa acutangula in distilled water, load it onto a chromatography column and elute with distilled water. After concentration and freeze-drying, Luffa acutangula polysaccharide LAP1 is obtained. Then, it is fractionally eluted with a NaCl solution, and after dialysis, concentration and freeze-drying, Luffa acutangula polysaccharide LAP2 is obtained.

[0009] The present invention provides a Luffa acutangula polysaccharide with antioxidant and anti-inflammatory effects, which is prepared according to the above method. The Luffa acutangula polysaccharide with antioxidant and anti-inflammatory effects is the Luffa acutangula polysaccharide LAP1 or the Luffa acutangula polysaccharide LAP2.

[0010] The present invention provides the application of the Luffa acutangula polysaccharide with antioxidant and anti-inflammatory effects in the preparation of pharmaceutical antioxidant and anti-inflammatory preparations.

[0011] The beneficial effects of the Luffa acutangula polysaccharide with antioxidant and anti-inflammatory effects, its preparation method and application in the embodiments of the present invention are as follows: 1. The present invention uses a hot water extraction method to extract the total sugar of Luffa acutangula, and obtains the optimal extraction scheme through response surface optimization process research. The liquid-to-material ratio used in the present invention is low, which reduces the later concentration cost. Moreover, under the optimized extraction conditions used, the extraction rate of the total sugar of Luffa acutangula can be up to 2.3 times higher than that under the non-optimized conditions.

[0012] 2. The present invention first uses ethanol for precipitation to obtain a crude extract from the extract. Then, DEAE-Sepharose CL-6B column chromatography is used to purify the crude extract, and distilled water is used for elution to obtain the neutral heteropolysaccharide LAP1 of Luffa acutangula, and the second acidic heteropolysaccharide LAP2 of Luffa acutangula is obtained by using a fractional elution method. The Luffa acutangula polysaccharide prepared by the above method has a clear composition, no impurities, and two Luffa acutangula polysaccharides can be simultaneously prepared by using one method.

[0013] 3. The two loofah polysaccharide components prepared by the method of the present invention have clear compositions. Both loofah polysaccharide LAP1 and loofah polysaccharide LAP2 contain the characteristic functional groups of saccharide substances. Loofah polysaccharide LAP1 is a homogeneous neutral heteropolysaccharide with a molecular weight of 325 kDa, which is composed of glucose and galactose with a molar ratio of 3.35:1. Its molecular morphology is a non-intertwined linear shape, the surface morphology is a flat block, and the degree of intermolecular polymerization is low. Loofah polysaccharide LAP2 is a homogeneous acidic heteropolysaccharide with a molecular weight of 354 KDa, which is composed of glucose, galactose, mannose, rhamnose and uronic acid with a molar ratio of 1:3.20:1.9:1.1:1.21. Its molecular morphology is a leaf shape, the surface morphology is a loose block, and the degree of intermolecular polymerization is high. Both loofah polysaccharide LAP1 and loofah polysaccharide LAP2 are loofah polysaccharides isolated for the first time.

[0014] 4. The two loofah polysaccharides prepared by the present invention both have good antioxidant activity and significant anti-inflammatory effects. The scavenging effects of LAP1 and LAP2 on hydroxyl radicals are significantly higher than those of the positive control group Vc (P<0.05). The scavenging effect of LAP1 on ABTS radicals has no significant difference from that of the Vc group (P>0.05), and the scavenging effect of LAP2 on ABTS radicals is significantly higher than that of the Vc group (P<0.05). LAP1 and LAP2 with a mass concentration of 25 μg / mL can significantly reduce the relative mRNA expression levels of five inflammatory factors or inflammation-related genes, namely TNF-α, IL-1β, IL-6, COX-2 and iNOS, in LPS-induced RAW264.7 cells (P<0.05). Loofah polysaccharide can effectively scavenge free radicals such as reactive oxygen species, and can inhibit the overexpression of inflammatory factors. The active ingredients are clear and there are no toxic and side effects. It can be used as a medical antioxidant and anti-inflammatory preparation, such as an adjuvant drug for the secondary prevention of neurodegenerative diseases such as cerebral hemorrhage. Description of the Drawings

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0016] Figure 1 It is the fractional elution curve of the crude loofah extract of Example 1 of the present invention after elution with distilled water of DEAE-Sepharose CL-6B; Figure 2 It is the elution curve and linear gradient elution curve of the crude loofah extract of Comparative Example 1 of the present invention after elution with distilled water of DEAE-Sepharose CL-6B; Figure 3 It is a curve graph showing the relationship between the total sugar extraction rate of Luffa acutangula Roxb. and the liquid-to-material ratio; Figure 4 It is a curve graph showing the relationship between the total sugar extraction rate of Luffa acutangula Roxb. and the extraction time; Figure 5 It is a curve graph showing the change of the total sugar extraction rate of Luffa acutangula Roxb. with the extraction temperature; Figure 6 It is a residual normal probability distribution graph; Figure 7 It is a model diagnosis graph; Figure 8 It is a high performance liquid chromatography graph of Luffa acutangula Roxb. polysaccharide LAP1 and Luffa acutangula Roxb. polysaccharide LAPGE in Example 1; Figure 9 It is a high performance liquid chromatography graph for comparing Luffa acutangula Roxb. polysaccharide LAP2 in Example 1, Luffa acutangula Roxb. polysaccharide gradient elution sample LAPGE in Comparative Example 1 and Luffa acutangula Roxb. polysaccharide LAP2; Figure 10 It is an infrared spectrum graph of Luffa acutangula Roxb. polysaccharide LAP1 and Luffa acutangula Roxb. polysaccharide LAP2 in Example 1; Figure 11 It is a gas chromatography graph of Luffa acutangula Roxb. polysaccharide LAP1 in Example 1; Figure 12 It is a gas chromatography graph of Luffa acutangula Roxb. polysaccharide LAP2 in Example 1; Figure 13 It is a transmission electron microscope graph of Luffa acutangula Roxb. polysaccharide LAP1 and Luffa acutangula Roxb. polysaccharide LAP2 in Example 1; Figure 14 It is a scanning electron microscope graph of Luffa acutangula Roxb. polysaccharide LAP1 and Luffa acutangula Roxb. polysaccharide LAP2 in Example 1; Figure 15 It is a graph of the results of the free radical scavenging activity of Luffa acutangula Roxb. polysaccharide LAP1 and Luffa acutangula Roxb. polysaccharide LAP2; Figure 16 It is an amplification curve and melting curve graph of Luffa acutangula Roxb. polysaccharide LAP1 and Luffa acutangula Roxb. polysaccharide LAP2; Figure 17 It is a graph showing the effect of Luffa acutangula Roxb. polysaccharide LAP1 and Luffa acutangula Roxb. polysaccharide LAP2 on the mRNA expression of inflammatory factors in LPS-stimulated RAW264.7 mouse macrophages. Detailed implementation manners

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0018] The following specifically describes the Luffa acutangula polysaccharide with antioxidant and anti-inflammatory effects, its preparation method, and its application in the embodiments of the present invention.

[0019] The embodiments of the present invention provide a method for simultaneously preparing two Luffa acutangula polysaccharides with antioxidant and anti-inflammatory effects, including the following steps: S1. Take dry Luffa acutangula, add distilled water, centrifuge to remove impurities after constant temperature water bath to obtain an extract. The present invention uses the hot water extraction method to extract the total sugar of Luffa acutangula and optimize its extraction conditions, which can not only improve the extraction rate of the total sugar of Luffa acutangula but also reduce the later concentration cost.

[0020] Further, in a preferred embodiment of the present invention, the mass-volume ratio of the Luffa acutangula to the extract is 1:20 (g / mL), the temperature of the constant temperature water bath is 95°C, and the constant temperature water bath time is 210 min. Preferably, the centrifugation speed is 3500 - 5000 r / min, and the centrifugation time is 5 - 10 min.

[0021] S2. After the extract is concentrated, add absolute ethanol, then cover it with a plastic wrap and react at 3 - 5°C for 15 - 24 h. The precipitate obtained after centrifugation is dried to obtain the crude Luffa acutangula extract. Preferably, after the extract is concentrated and absolute ethanol is added, cover it with a plastic wrap and place it in a 4°C refrigerator for 15 - 24 h, then centrifuge at 5000 r / min for 5 - 10 min, and the precipitate is dried to obtain the crude Luffa acutangula extract.

[0022] Further, in a preferred embodiment of the present invention, the step of adding absolute ethanol after the extract is concentrated is as follows: Concentrate the extract with a rotary evaporator to obtain a concentrated solution, and then slowly add the absolute ethanol while stirring, where the volume ratio of the concentrated solution to the absolute ethanol is 50 - 100:100 - 200.

[0023] S3. Dissolve the crude extract of Luffa acutangula in distilled water, load it onto a chromatography column and elute with distilled water. After concentration and freeze-drying, Luffa acutangula polysaccharide LAP1 is obtained. Then, perform fractional elution with an NaCl solution, and after dialysis, concentration and freeze-drying, Luffa acutangula polysaccharide LAP2 is obtained. Preferably, the mass-volume ratio of the crude extract of Luffa acutangula to distilled water is 15:1 (mg / mL). In the present invention, DEAE-Sepharose CL-6B column chromatography is used to purify the crude extract. Then, Luffa acutangula polysaccharide LAP1 is obtained by eluting with distilled water, and Luffa acutangula polysaccharide LAP2 is obtained by using the fractional elution method. This method can simultaneously prepare two kinds of Luffa acutangula polysaccharides, and the components of the two kinds of Luffa acutangula polysaccharides are clear and there are no impurities.

[0024] Furthermore, in a preferred embodiment of the present invention, the chromatography column is a DEAE-Sepharose CL-6B gel column, and its preparation steps are as follows: Take the degassed DEAE-Sepharose CL-6B gel and load it into a glass chromatography column. Turn on the constant flow pump, and after equilibrating the glass chromatography column with 2000 mL of distilled water, the DEAE-Sepharose CL-6B gel column is obtained. Preferably, the diameter of the DEAE-Sepharose CL-6B gel column is 4.6 cm and the height is 35 cm.

[0025] Furthermore, in a preferred embodiment of the present invention, the usage conditions of the DEAE-Sepharose CL-6B gel column are as follows: The flow rate of the constant flow pump is 1 mL / min, and it is collected by an automatic fraction collector, and it is set to collect 11 mL per tube.

[0026] Furthermore, in a preferred embodiment of the present invention, the steps of loading onto the chromatography column, eluting with distilled water, concentrating and freeze-drying to obtain Luffa acutangula polysaccharide LAP1, and then performing fractional elution with an NaCl solution, dialysis, concentration and freeze-drying to obtain Luffa acutangula polysaccharide LAP2 are as follows: Elute with the distilled water for 17 - 19 h, combine the collected solutions in tubes 20 - 50, and after concentration and freeze-drying, obtain the Luffa acutangula polysaccharide LAP1; then perform fractional elution with a 0.33 mol / L NaCl solution, combine the collected solutions in tubes 36 - 50 for dialysis, and then dialyze with a dialysis bag with a molecular weight cut-off of 3500 against running water and distilled water for 19 - 21 h respectively. After the obtained dialysate is concentrated and freeze-dried, the Luffa acutangula polysaccharide LAP2 is obtained. Preferably, the elution time with distilled water is 18 h and the dialysis time is 20 h.

[0027] The fresh fruit of Luffa acutangular, an annual climbing herbaceous plant belonging to the genus Luffa in the family Cucurbitaceae. Traditional Chinese medicine believes that loofah tastes sweet and has the effects of clearing heat and cooling blood, regulating qi and strengthening the spleen, expelling wind and resolving phlegm, and moisturizing the muscles and beautifying the skin. Eating loofah frequently in summer can relieve summer heat and annoyance, promote the production of body fluid and quench thirst. Therefore, it can be used as a vegetable food with good health care effects. At present, there are few reports on the research of polysaccharides from Luffa acutangular at home and abroad. Chemical composition is the basis for substances to exert their medicinal effects. The present invention conducts in-depth research on the medicinal component polysaccharide of Luffa acutangular and establishes a method for preparing polysaccharides from Luffa acutangular. The established method has strong specificity and can simultaneously prepare two polysaccharides from Luffa acutangular with significant antioxidant and anti-inflammatory effects, which can be used for the adjuvant treatment of neurodegenerative diseases related to oxidative stress and overexpression of inflammatory factors.

[0028] The present invention also provides a polysaccharide from Luffa acutangular with antioxidant and anti-inflammatory effects, which is prepared according to the above method. The polysaccharide from Luffa acutangular with antioxidant and anti-inflammatory effects is the polysaccharide LAP1 from Luffa acutangular or the polysaccharide LAP2 from Luffa acutangular. The polysaccharide from Luffa acutangular can effectively scavenge free radicals such as reactive oxygen species and can inhibit the overexpression of inflammatory factors. The active ingredients are clear and there are no toxic and side effects. It can be used as a medicinal antioxidant and anti-inflammatory preparation, such as an adjuvant drug for the secondary prevention of neurodegenerative diseases such as cerebral hemorrhage.

[0029] Furthermore, in a preferred embodiment of the present invention, the polysaccharide LAP1 from Luffa acutangular is a homogeneous neutral heteropolysaccharide with a molecular weight of 325 kDa, composed of glucose and galactose with a molar ratio of 3.35:1. Its molecular form is a non-entangled linear shape, the surface morphology is a flat block, and the degree of intermolecular polymerization is low.

[0030] Furthermore, in a preferred embodiment of the present invention, the polysaccharide LAP2 from Luffa acutangular is a homogeneous acidic heteropolysaccharide with a molecular weight of 354 KDa, composed of glucose, galactose, mannose, rhamnose, and uronic acid with a molar ratio of 1:3.20:1.9:1.1:1.21. Its molecular form is leaf-shaped, the surface morphology is a loose block, and the degree of intermolecular polymerization is high.

[0031] The present invention also provides the application of the polysaccharide from Luffa acutangular with antioxidant and anti-inflammatory effects in the preparation of medicinal antioxidant and anti-inflammatory preparations. The two polysaccharides from Luffa acutangular prepared by the present invention can be used for the adjuvant treatment of neurodegenerative diseases related to oxidative stress and overexpression of inflammatory factors.

[0032] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0033] Example 1 This example provides two kinds of loofah polysaccharides with antioxidant and anti-inflammatory effects (i.e., loofah polysaccharide LAP1 and loofah polysaccharide LAP2), which are prepared according to the following steps: (1) Take 100 g of dry loofah powder and put it into 2000 mL of distilled water. After extracting in a constant temperature water bath at 95 °C for 210 min, centrifuge at 5000 r / min for 5 min to remove impurities, and obtain the extract.

[0034] (2) Concentrate the extract obtained in step (1) to 50 mL with a rotary evaporator. While stirring, slowly add 100 mL of absolute ethanol to the concentrated solution, cover it with plastic wrap and place it in the refrigerator at 4 °C for 24 h. Then centrifuge at 5000 r / min for 5 min, and dry the precipitate to obtain the crude loofah extract.

[0035] (3) Take the degassed DEAE-Sepharose CL-6B gel and load it into a glass chromatography column. Turn on the constant flow pump and equilibrate the chromatography column with 2000 mL of distilled water. After the equilibration is completed, a chromatography column with a diameter of 4.6 cm and a height of 35 cm is prepared. The use conditions are: the flow rate of the constant flow pump is 1 mL / min, and the automatic fraction collector is used for collection, and it is set to collect 11 mL per tube.

[0036] (4) Take 300 mg of the first-stage crude extract obtained in step (2), dissolve it in 20 mL of distilled water, load it onto the chromatography column prepared in step (3), elute with distilled water for 18 h, and combine the collected solutions in tubes 20 - 50, concentrate and freeze-dry to obtain LAP1. Then use a 0.33 mol / L NaCl solution for stepwise elution, combine the collected solutions in tubes 36 - 50, and dialyze with a dialysis bag with a molecular weight cut-off of 3500 against running water and distilled water for 20 h respectively. After the dialysate is concentrated and freeze-dried, the loofah polysaccharide LAP2 is obtained. Among them, the stepwise elution curve diagram of the loofah crude extract after elution with distilled water on DEAE-Sepharose CL-6B in this example is as Figure 1 shown.

[0037] Comparative Example 1 This comparative example provides two kinds of loofah polysaccharides (i.e., loofah polysaccharide LAP1 and gradient elution sample of loofah polysaccharide LAPGE), which are prepared according to the following steps: (1) Take 100 g of dry loofah powder and put it into 2000 mL of distilled water. After extracting in a constant temperature water bath at 95 °C for 210 min, centrifuge at 5000 r / min for 5 min to remove impurities, and obtain the extract.

[0038] (2) Concentrate the extract obtained in step (1) to 50 mL using a rotary evaporator. While stirring, slowly add 100 mL of absolute ethanol to the concentrated solution, cover it with plastic wrap, and place it in a refrigerator at 4 °C for 24 h. Then centrifuge at 5000 r / min for 5 min. After drying the precipitate, the crude extract of Luffa acutangula is obtained.

[0039] (3) Take the degassed DEAE-Sepharose CL-6B gel and load it into a glass chromatography column. Turn on the constant flow pump and equilibrate the chromatography column with 2000 mL of distilled water. After equilibration, a chromatography column with a diameter of 4.6 cm and a height of 35 cm is prepared. The operating conditions are as follows: the flow rate of the constant flow pump is 1 mL / min, and the automatic fraction collector is used for collection, with each tube set to collect 11 mL.

[0040] (4) Take 300 mg of the crude extract obtained in step (2), dissolve it in 20 mL of distilled water, load it onto the chromatography column prepared in step (3), elute with distilled water for 18 h, collect the eluate, and determine the polysaccharide distribution by the phenol-sulfuric acid method and the protein distribution by ultraviolet spectrophotometer. Combine the collected fractions from tubes 20 - 50, concentrate and freeze-dry to obtain Luffa acutangula polysaccharide LAP1. Then perform a linear gradient elution with 500 mL each of 0 - 1 mol / L NaCl solutions, detect the sugar and protein distributions, combine the collected fractions from tubes 33 - 73, and dialyze with a dialysis bag with a molecular weight cut-off of 3500 against running water and distilled water for 20 h each. After concentrating and freeze-drying the dialysate, the gradient elution sample of Luffa acutangula polysaccharide LAPGE is obtained. The elution curves of the crude extract of Luffa acutangula with distilled water and the linear gradient elution curve by DEAE-Sepharose CL-6B in this comparative example are as Figure 2 shown. Among them, Figure 2 (A) is the elution curve of the crude extract of Luffa acutangula with distilled water by DEAE-Sepharose CL-6B; Figure 2 (B) is the gradient elution curve of the crude extract of Luffa acutangula after elution with distilled water.

[0041] Experimental Example 1 In this experimental example, the response surface method was used to optimize the extraction process of total sugar from Luffa acutangula.

[0042] Experiment 1: Design single-factor experiments to study the effects of the solid-liquid ratio of Luffa acutangula powder to distilled water, extraction time, and extraction temperature on the extraction rate of total sugar from Luffa acutangula. The specific experimental scheme is as follows: Under the experimental conditions of fixing two factors, change the experimental conditions of the other 1 factor, so as to obtain the effect of this factor on the extraction rate of total sugar from Luffa acutangula at different experimental levels.

[0043] As Figure 3 shown is the curve of the extraction rate of total sugar from Luffa acutangula versus the liquid-solid ratio. Figure 4It is a curve graph showing the relationship between the total sugar extraction rate of Luffa acutangula Roxb. and the extraction time. Figure 5 It is a curve graph showing the change of the total sugar extraction rate of Luffa acutangula Roxb. with the extraction temperature. According to the results of single-factor experiments, with the liquid-solid ratio (20:1, 110:1, 200:1), extraction time (30, 120, 210 min), and extraction temperature (45, 70, 95 °C) as the three independent variables, a response surface experiment was arranged. The specific experimental levels of each factor are shown in Table 1.

[0044] Table 1 Factor levels and coding of response surface test design

[0045] Experiment 2: Using the Box-Behnken model, 17 groups of experiments were arranged. With the liquid-solid ratio, extraction temperature, and extraction time as the main experimental factors, the extraction conditions of the total sugar in Luffa acutangula Roxb. were optimized. The specific steps were as follows: Weigh 1 g of dry Luffa acutangula Roxb. powder, add the corresponding distilled water according to factor A (liquid-solid ratio) in the experimental design of Table 2, and perform water-boiling extraction at a specific extraction temperature (factor C) for a specific extraction time (factor B) to obtain the crude extract. The total sugar extraction rate of the obtained crude extract was detected by the phenol-sulfuric acid method.

[0046] Table 2 Box-Behnken design scheme and extraction rate for the extraction of total sugar from Luffa acutangula Roxb.

[0047] In order to achieve the purpose of maximizing the extraction of the total sugar in Luffa acutangula Roxb., the response surface analysis method was used to optimize the extraction process of the total sugar in Luffa acutangula Roxb. The extraction rate results are shown in Table 2. Using Design-Expert.V11.1.0 software, a quadratic polynomial mathematical fitting regression analysis was performed on the data in Table 2, and the quadratic polynomial regression equation of the total sugar extraction rate of Luffa acutangula Roxb. with respect to the extraction factors was obtained as follows: Y = 36.17 + 1.57A + 5.77B + 3.31C - 1.90AB - 0.8825AC + 1.22BC - 0.3180A 2 - 2.69B 2 + 1.46C 2 .

[0048] To verify the prediction effect of the model on the extraction scheme and further determine the influence degree of each factor on the total sugar extraction rate of Luffa acutangula Roxb., an analysis of variance was performed on the experimental results. The significance test results of the model coefficients are shown in Table 3.

[0049] Table 3 Analysis of variance and significance results of the regression equation

[0050] As can be seen from Table 3, the p-value of this model is 0.0333 (<0.05), and the p-value of the lack-of-fit term of the model is 0.0568 (>0.05). Therefore, the model is relatively appropriate and the experimental relative error is small. The order of the influence degree of the three factors on the total sugar extraction rate of Luffa acutangula Roxb. is extraction time (B), extraction temperature (C), and liquid-to-solid ratio (A).

[0051] The R of the equation 2 is 0.8475, and the equation has a good fitting degree, which can be used for preliminary analysis and prediction of the process study of the total sugar extraction rate of Luffa acutangula Roxb. As Figure 6 shown is the residual normal probability distribution diagram. Figure 6 The distribution of the residuals along the straight line in it indicates that the residuals follow a normal distribution. Figure 7 is the model diagnostic diagram, in which Figure 7 (a) is the residual diagram; Figure 7 (b) is the scatter plot of the predicted value and the actual value. From Figure 7 it can be seen that all data points are within the acceptable range (±4.819), and the results of the model diagnostic diagram prove the applicability and accuracy of the model.

[0052] In summary, combined with the mathematical analysis of the regression model, the optimal extraction process conditions for the total sugar of Luffa acutangula Roxb. are obtained as follows: A (liquid-to-solid ratio) = 20:1, B (time) = 210 min, C (temperature) = 95°C, and the predicted Y value at this time = 46.13%. According to this scheme, 3 parallel experiments were carried out, and the average extraction rate of the total sugar of Luffa acutangula Roxb. was measured to be (44.536±0.859)%, with a difference of 1.59% from the theoretical value. It is proved that the method of using the response surface method to optimize the extraction process of the total sugar of Luffa acutangula Roxb. is successful, and the best extraction scheme is obtained through model prediction.

[0053] Experimental Example 2 In this experimental example, the characteristics of the two Luffa acutangula Roxb. polysaccharides with antioxidant and anti-inflammatory effects in Example 1 were analyzed respectively. Among them, the specific steps of the homogeneity analysis were as follows: The component homogeneity of the samples LAP1 and LAP2 in Example 1 was identified by high performance size exclusion chromatography (HPSEC). The sample concentration was 1 mg / mL, the chromatographic column was sugar ks-804, the mobile phase was ultrapure water, the flow rate was 1 mL / min, the column temperature was 50°C, and the detector was a refractive index detector (RIU).

[0054] As Figure 8 shown are the high performance liquid chromatograms of Luffa acutangula Roxb. polysaccharide LAP1 (A) and Luffa acutangula Roxb. polysaccharide LAPGE (B) in Example 1. Figure 9 is the high performance liquid chromatogram of Luffa acutangula Roxb. polysaccharide LAP2 (A) in Example 1 and the comparison (B) of the gradient elution sample LAPGE of Luffa acutangula Roxb. polysaccharide and Luffa acutangula Roxb. polysaccharide LAP2 in Comparative Example 1. FromFigure 8 and Figure 9 It can be seen that there is only one peak for LAP1 at 6.283 min and for LAP2 at 6.230 min, indicating that both LAP1 and LAP2 have good homogeneity and are samples with a single component.

[0055] This test example also identified the molecular weights of the loofah polysaccharide LAP1 and loofah polysaccharide LAP2 of Example 1. The specific steps were as follows: Using the above method for homogeneity analysis, according to the peak elution time of the dextran standard on the sugar ks-804 chromatographic column, a molecular weight standard curve was made to calculate the relative molecular weights of LAP1 and LAP2.

[0056] According to the standard dextran, a molecular weight standard curve was made, and the equation y = -3.92x + 6.314 (R 2 = 0.99) was obtained. According to the HPSEC peak elution times of LAP1 and LAP2, the molecular weights of LAP1 and LAP2 were calculated to be 325 and 354 kDa respectively.

[0057] Test Example 3 Infrared spectroscopy can identify the characteristic functional groups of saccharide substances. In this test example, infrared spectroscopy was used to identify the characteristic functional groups of the loofah polysaccharide LAP1 and loofah polysaccharide LAP2 of Example 1 to analyze their compound types. The specific steps were as follows: 2 mg of the sample was pressed into a KBr tablet and measured with a Nicolet Nexus 470 Fourier transform infrared spectrometer.

[0058] As Figure 10 shown are the infrared spectra of the loofah polysaccharide LAP1 and loofah polysaccharide LAP2 of Example 1. From Figure 10 it can be seen that both LAP1 and LAP2 contain the characteristic functional groups of saccharide compounds.

[0059] Test Example 4 This test example respectively performed monosaccharide composition analysis on the loofah polysaccharide LAP1 and loofah polysaccharide LAP2 of Example 1. The specific steps were as follows: Respectively, 25 - 30 mg of the sample was hydrolyzed with 1 mol / L sulfuric acid at 100 °C for 8 h. After the hydrolyzate was neutralized with barium carbonate, the supernatant was taken for freeze-drying. The dried sample was the monosaccharide hydrolysis product. The hydrolysis product was subjected to trimethylsilylation derivatization, and the derivative was subjected to gas chromatography analysis. The chromatographic column used was an hp-5 capillary chromatographic column, and the temperature programming conditions were 160 °C → 180 °C (20 °C / min) → 220 °C (8 °C / min, held for 2 min) → 250 °C (2 min).

[0060] AsFigure 11 The gas chromatogram of Luffa custardii polysaccharide LAP1 of Example 1 is shown; wherein 1-2 represent isomers of glucose and galactose, respectively. Figure 12 The gas chromatogram of LAP2 of Luffa octagonalis polysaccharide of Example 1 is shown in Figure 1; wherein 1-5 represent the isomers of glucose, galactose, mannose, rhamnose and uronic acid, respectively. According to the GC results of the standard monosaccharide derivatives and the gas chromatography results of LAP1 and LAP2 ( Figure 11 and Figure 12 ) It can be seen that LAP1 is composed of glucose and galactose with a molar ratio of 3.35:1. LAP2 is composed of glucose, galactose, mannose, rhamnose and uronic acid with a molar ratio of 1:3.20:1.9:1.1:1.21.

[0061] Test Example 5 In this test example, the microstructures of the Luffa octagonal polysaccharide LAP1 and the Luffa octagonal polysaccharide LAP2 of Example 1 were observed by transmission electron microscopy and scanning electron microscopy, respectively. The specific steps are as follows: Transmission electron microscopy observation: Take equal volumes of 1 mg / mL SDS and 1 mg / mL sugar solution and mix them, heat them at 80℃ for 2 hours, dilute them to 5 µg / mL with deionized water and continue heating them at 80℃ for 2 hours to stabilize the structure. Take a 200 mesh carbon support film and add the sample, dry it at room temperature for 24 hours, and then observe it under a transmission electron microscope.

[0062] Scanning electron microscope observation: Take the dried sample powder and place it on the conductive glue on the metal stage. After spraying platinum, observe it under a scanning electron microscope at 5 KV.

[0063] like Figure 13 The transmission electron micrographs (2 k×~10 k×) of Luffa octagonal polysaccharide LAP1 and Luffa octagonal polysaccharide LAP2 of Example 1 are shown. Polysaccharide macromolecules often exist in a polymer state. After dispersing the sugar polymer with SDS, the spatial morphology of the macromolecule in the unpolymerized state can be observed. Figure 13 It can be seen that LAP1 is linear in the SDS dispersion state, and the lines are not entangled with each other. LAP2 is leaf-shaped, and fine structures can be observed under 5 k× magnification, with a leaf-like dispersed morphology.

[0064] Figure 14 The scanning electron microscope images (300 k×~20 k×) of the Luffa octagonal polysaccharide LAP1 and Luffa octagonal polysaccharide LAP2 of Example 1 are shown. The scanning electron microscope can observe the surface morphology of solid materials, provide their three-dimensional morphology information, and also help to understand the aggregation state of such materials. Figure 14It can be seen that the surface morphology of LAP1 is blocky, and its surface is relatively smooth when observed at a magnification of 10 k×, indicating that the aggregation state of the polymer is not obvious. LAP2 is loosely lumpy, and its surface is uneven when observed at a magnification, indicating that the degree of polymerization of LAP2 molecules is high and they mostly exist in an aggregated state.

[0065] Test Example 6 This test example uses superoxide free radical, hydroxyl free radical and ABTS free radical scavenging experiments to study the antioxidant activity of LAP1 and LAP2 of Example 1. The specific steps are as follows: Superoxide radical scavenging experiment: The PMS / NADH system reduction NBT experimental method was used to determine the scavenging of superoxide radicals. The specific steps were: the polysaccharide was prepared into sugar solutions of different concentrations, and then solutions containing NADH, PMS and NBT were added in sequence. After standing at room temperature for 5 min, the absorbance was measured at 560 nm.

[0066] Hydroxyl free radical scavenging experiment: Fenton reagent (hydrogen peroxide and ferrous sulfate) method was used, with hydrogen peroxide as the oxidant and Fe 2＋ The catalytic system, the generated hydroxyl radicals react with crystal violet to reduce the absorbance of the system, and the change in absorbance is used to indirectly determine the generated hydroxyl radicals. The specific steps are: First, prepare a mixed solution containing 2 mL 0.4 mM EDTA, 4 mL 4uM deoxyribose and 0.5 mL 1mM ascorbic acid. Take sample solutions of different concentrations, add the mixed solution, FeSO4 and H2O2, incubate at 37°C in a water bath for 15 min, add 2% trichloroacetic acid and 1% thiobarbituric acid, and measure the absorbance at 532 nm after boiling in a water bath for 15 min.

[0067] ABTS free radical scavenging experiment: Prepare ABTS+ solution (7 mmol / L ABTS aqueous solution and 2.45 mmol / L potassium persulfate in equal volumes), and dilute it to an appropriate multiple to make ABTS working solution before the experiment. Take sugar solutions of different concentrations and add equal volumes of ABTS working solution, place them in the dark at room temperature for 10 minutes, and then measure the absorbance at 734 nm.

[0068] The scavenging rate of each free radical was calculated according to the following formula: Clearance rate (%) = (1-A 样品组 / A 对照组 ) ×100% like Figure 15 The results of free radical scavenging activity of LAP1 and LAP2 of LAP1 polysaccharide are shown. Figure 15 (A) is the result of superoxide radical scavenging activity.Figure 15 (B) is the result graph of hydroxyl radical scavenging activity, Figure 15 (C) is the result graph of ABTS radical scavenging activity, Figure 15 (D) is the comparison graph of EC 50 values. It can be seen from Figure 15 that LAP1 has scavenging effects on both hydroxyl radicals and ABTS radicals, and LAP2 has scavenging effects on all three radicals. Within the experimental concentration range, the scavenging rates of various radicals show a dose-effect relationship, and the scavenging rate increases with the increase of concentration. The EC 50 values of LAP1 for scavenging hydroxyl radicals and ABTS radicals are (11.22 ± 1.96) μg / mL and (348.67 ± 14.73) μg / mL respectively, and the EC 50 values of LAP2 for scavenging superoxide radicals, hydroxyl radicals and ABTS radicals are (1049.43 ± 159.76) μg / mL, (8.60 ± 0.91) μg / mL and (22.91 ± 0.82) μg / mL respectively. Compared with the positive control group Vc, the scavenging effects of LAP1 and LAP2 on hydroxyl radicals are significantly higher than those of the positive control group Vc (P < 0.0001). There is no significant difference in the scavenging effect of LAP1 on ABTS radicals compared with the Vc group (P > 0.05), and the scavenging effect of LAP2 on ABTS radicals is significantly higher than that of the Vc group (P < 0.0001).

[0069] Test Example 7 In this test example, the anti-inflammatory effects of the loofah polysaccharide LAP1 and loofah polysaccharide LAP2 of Example 1 were studied respectively. The specific steps are as follows: RAW264.7 cells were cultured in a 37 °C and 5% carbon dioxide incubator and could be passaged after 48 h. The MTT method was used to detect the cytotoxicity of the polysaccharide on RAW264.7. After culturing the cells in a 96-well plate, six polysaccharide mass concentrations of 600, 300, 150, 75, 25 and 1 μg / mL were set. RAW264.7 cells were induced to produce inflammation with 1 μg / mL lipopolysaccharide (LPS) to establish an inflammation model. A blank group, a negative control group, a positive control group, an LAP1 polysaccharide sample group and an LAP2 polysaccharide sample group were arranged, and each group was set with 3 parallel samples. Total RNA in the cells was extracted according to the instructions of the total RNA extraction kit (Promega), cDNA was obtained using the reverse transcription kit (Promega), and real-time fluorescence quantitative PCR was performed using the qPCR Master Mix kit (Promega). The housekeeping gene GAPDH was used as the internal reference gene, and the relative mRNA expression levels of each inflammatory factor were calculated by the 2 -ΔΔCT - method.

[0070] As Figure 16The amplification curves and melting curves of Luffa acutangula polysaccharide LAP1 and Luffa acutangula polysaccharide LAP2 are shown. Among them, Figure 16 (A) is the amplification curve graph; Figure 16 (B) is the melting curve graph. Through cytotoxicity tests, it was confirmed that adding 25 μg / mL of Luffa acutangula polysaccharide LAP1 or LAP2 had no cytotoxicity on the growth of RAW264.7 cells. Therefore, the above concentrations were selected for subsequent anti-inflammatory experimental studies. The results of real-time quantitative PCR are as Figure 16 shown. The amplification curve shows an S shape, indicating that the amplification results are reliable. The melting curves of each gene are all single peaks, indicating good singleness of each amplified gene.

[0071] To establish an inflammation model, 1 μg / mL lipopolysaccharide was added to RAW264.7 cells as a negative control group (LPS). As Figure 17 shown is the graph of the effects of Luffa acutangula polysaccharide LAP1 and Luffa acutangula polysaccharide LAP2 on the mRNA expression of inflammatory factors in LPS-stimulated RAW264.7 mouse macrophages. Among them, A: TNF-α, B: IL-1β, C: IL-6, D: COX-2, E: iNOS (different letters a-c represent significant differences, P<0.05). From Figure 17 it can be seen that compared with the blank group, the relative mRNA expression levels of each inflammatory factor TNF-α, IL-1β, IL-6, COX-2, and iNOS genes in the negative control group were all significantly increased (P<0.05), indicating that the inflammation model was successfully constructed. Both 25 μg / mL of Luffa acutangula polysaccharide LAP1 or LAP2 could significantly reduce the relative mRNA expression levels of inflammatory factors TNF-α, IL-1β, IL-6, COX-2, and iNOS (P<0.05), and the inhibition rates all exceeded 90%. Through significant analysis, it was obtained that there was no significant difference in the inhibitory effects of LAP1 or LAP2 and the positive control group on the expression of each inflammatory factor (P>0.05), indicating that the anti-inflammatory effects of LAP1 or LAP2 were comparable to those of dexamethasone.

[0072] The embodiments described above are some embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the present invention claimed, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

Claims

1. A method for simultaneously preparing two Luffa acutangula polysaccharides with antioxidant and anti-inflammatory effects, characterized in that, It includes the following steps: S1. Take dry Luffa acutangula, add distilled water, perform constant-temperature water bath and then centrifuge to obtain an extract; S2. After concentrating the extract, add absolute ethanol, then cover it with plastic wrap and react at 3 - 5 °C for 15 - 24 h. The precipitate obtained after centrifugation is dried to obtain the crude extract of Luffa acutangula; S3. Dissolve the crude extract of Luffa acutangula in distilled water, load it onto a chromatography column and elute with distilled water. After concentration and freeze-drying, Luffa acutangula polysaccharide LAP1 is obtained. Then, perform fractional elution with NaCl solution, and after dialysis, concentration and freeze-drying, Luffa acutangula polysaccharide LAP2 is obtained.

2. The method according to claim 1, wherein In step S1, the mass-volume ratio of the Luffa acutangula to the extract is 1:20 (g / mL), the temperature of the constant-temperature water bath is 95 °C, and the time of the constant-temperature water bath is 210 min.

3. The method according to claim 1, wherein In step S2, the step of adding absolute ethanol after concentrating the extract is as follows: Concentrate the extract with a rotary evaporator to obtain a concentrated solution, and then slowly add the absolute ethanol while stirring. Among them, the volume ratio of the concentrated solution to the absolute ethanol is 50 - 100:100 - 200.

4. The method according to claim 1, characterized in that, In step S3, the chromatography column is a DEAE-Sepharose CL-6B gel column, and its preparation steps are as follows: Take the degassed DEAE-Sepharose CL-6B gel and load it into a glass chromatography column. Open the constant flow pump, and after balancing the glass chromatography column with 2000 mL of distilled water, the DEAE-Sepharose CL-6B gel column is obtained.

5. The method according to claim 4, wherein In step S3, the usage conditions of the DEAE-Sepharose CL-6B gel column are: the flow rate of the constant flow pump is 1 mL / min, collect with an automatic fraction collector, and set to collect 11 mL per tube.

6. The method according to claim 4, wherein In step S3, the step of loading onto the chromatography column, eluting with distilled water, concentrating and freeze-drying to obtain Luffa acutangula polysaccharide LAP1, and then performing fractional elution with NaCl solution, dialysis, concentration and freeze-drying to obtain Luffa acutangula polysaccharide LAP2 is as follows: Elute with the distilled water for 17 - 19 h, combine the collected liquid in tubes 20 - 50, and after concentration and freeze-drying, Luffa acutangula polysaccharide LAP1 is obtained; then perform fractional elution with 0.33 mol / L NaCl solution, combine the collected liquid in tubes 36 - 50 for dialysis, and then dialyze with a dialysis bag with a molecular weight cut-off of 3500 for 19 - 21 h with running water and distilled water respectively. The obtained dialysate is concentrated and freeze-dried to obtain Luffa acutangula polysaccharide LAP2.

7. An octagonal towel gourd polysaccharide with antioxidant and anti-inflammatory effects, characterized in that, Prepared according to the method described in any one of claims 1 - 6, the Luffa acutangula polysaccharide with antioxidant and anti-inflammatory effects is the Luffa acutangula polysaccharide LAP1 or the Luffa acutangula polysaccharide LAP2.

8. The loofah polysaccharide with antioxidant and anti-inflammatory effects according to claim 7, characterized in that, The Luffa acutangula polysaccharide LAP1 is a homogeneous neutral heteropolysaccharide, its molecular weight is 325 kDa, it is composed of glucose and galactose with a molar ratio of 3.35:1, its molecular morphology is a non-intertwined linear shape, its surface morphology is a flat block, and the degree of intermolecular polymerization is low.

9. The loofah polysaccharide with antioxidant and anti-inflammatory effects according to claim 7, characterized in that, The Luffa octagonal polysaccharide LAP2 is a uniform acidic heteropolysaccharide with a molecular weight of 354 KDa. It is composed of glucose, galactose, mannose, rhamnose and uronic acid in a molar ratio of 1:3.20:1.9:1.1:1.

21. Its molecular form is leaf-shaped, its surface morphology is loose and block-like, and its intermolecular polymerization degree is high.

10. Use of the Luffa scoparia polysaccharide with antioxidant and anti-inflammatory effects as claimed in any one of claims 7 to 9 in the preparation of medical antioxidant and anti-inflammatory preparations.

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