Preparation method of lotus leaf polysaccharide and acetylated lotus leaf polysaccharide as well as product and application thereof

Abstract

The invention discloses a preparation method of lotus leaf polysaccharide and acetylated lotus leaf polysaccharide as well as products and application of the acetylated lotus leaf polysaccharide and the acetylated lotus leaf polysaccharide, lotus leaves are used as raw materials, the homogeneous monosaccharide component in the lotus leaves is obtained after separation and purification, the molecular weight of the homogeneous monosaccharide component is 245612Da, monosaccharide mainly comprises arabinose, mannose, glucose and galactose according to the molar ratio of 5.23: 2.35: 1.0: 5.15, and the molecular weight of the homogeneous monosaccharide component is 2,500 Da. Meanwhile, the invention discloses a preparation method and application of the polysaccharide component acetylation product. The acetylated lotus leaf polysaccharide product disclosed by the invention has relatively strong antioxidant and antitumor activity, and can be applied to the fields of foods and medicines as an antioxidant and an antitumor drug.

Description

Technical Field

[0001] The invention relates to natural ingredient extraction technology, and in particular to a preparation method of lotus leaf polysaccharide and acetylated lotus leaf polysaccharide, and products and applications thereof. Background Art

[0002] Lotus leaf ( Nelumbinis folium ) is a perennial plant of the Nymphaeaceae family, Lotus ( Nelumbo nucifera Gairtn.) Lotus leaves contain polysaccharides, flavonoids, alkaloids, volatile oils and other bioactive compounds. Nelumbinis folium , NFP) is one of the important active ingredients in lotus leaves. Li Gen extracted lotus leaf polysaccharides using hot water extraction, ultrasonic extraction, pectinase enzymolysis and cellulase enzymolysis. Zhang Yan et al. optimized the process of ultrasonic-assisted disodium ethylenediaminetetraacetic acid extraction of lotus leaf polysaccharides by response surface. Xing Ying et al. extracted lotus leaf polysaccharides using hot water, ultrasonic, enzyme and alkaline methods, and investigated the effects of the four extraction methods on the yield, structure and antioxidant activity of lotus leaf polysaccharides. Liu Junbo et al. explored the extraction process of lotus leaf polysaccharides assisted by cellulase. Tu Zongcai et al. studied the optimal process of ultrasonic-assisted extraction of lotus leaf polysaccharides and its antioxidant activity, and compared them with lotus leaf polysaccharides obtained by traditional water extraction. Li Gen found that lotus leaf polysaccharides can regulate inflammation to a certain extent and have potential anti-inflammatory activity. Xiong Jianwen et al. found that lotus leaf polysaccharides have a certain inhibitory effect on Bacillus subtilis and Escherichia coli, but have no obvious inhibitory effect on yeast and Aspergillus niger. Xu Jinrong and others found that lotus leaf polysaccharides can improve the ability to scavenge free radicals and have significant antioxidant effects.

[0003] At present, the research on lotus leaf polysaccharides mainly focuses on the extraction, preliminary purification and biological activity of crude polysaccharides, but there are few reports on the structural modification and activity evaluation of single components of lotus leaf polysaccharides. Therefore, the present invention studies and develops an acetylated modified product of a single polysaccharide component of lotus leaves and its new use, aiming to provide a basis for the development of a new antioxidant and anti-tumor drug in the market. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a lotus leaf polysaccharide to provide a new, naturally extracted antioxidant.

[0005] Another technical problem to be solved by the present invention is to provide a method for preparing the lotus leaf polysaccharide.

[0006] The technical problem that the present invention needs to solve is to provide the acetylated modified product of the lotus leaf polysaccharide and a preparation method thereof.

[0007] The technical problem that the present invention needs to solve is to provide the application of the lotus leaf polysaccharide and acetylated lotus leaf polysaccharide as antioxidants.

[0008] The final technical problem to be solved by the present invention is to provide the application of the acetylated lotus leaf polysaccharide in the preparation of anti-tumor drugs.

[0009] Technical solution: To solve the above technical problems, the present invention provides the following technical solutions: A lotus leaf polysaccharide comprises the following steps: (1) Extraction and preliminary purification of crude polysaccharides: The lotus leaves were washed, dried and crushed, and water was added according to a solid-liquid ratio (g / mL) of 1:40-1:10, preferably a solid-liquid ratio of 1:20 (g / mL). Then, a composite enzyme was added to the mixture, the mass ratio of cellulase and pectinase was (1-5):1, preferably a ratio of cellulase and pectinase was 2.5:1, and the amount of composite enzyme added was 3% of the mass of the mixture. The enzymatic hydrolysis time was 30-80 min, preferably 40 min, and the enzymatic hydrolysis temperature was 30-55°C, preferably 45°C. After the extraction was completed, the supernatant was obtained by centrifugation, and the supernatant was concentrated under reduced pressure at 50°C. After concentration, four times the volume of anhydrous ethanol was added to the concentrate, and the concentrate was allowed to stand at 4°C for 24 h, and then centrifuged (10000 r / min, 10 min) to collect the precipitate. The precipitate was freeze-dried for 48 h. The freeze-drying conditions were as follows: pre-freezing rate 0.30°C / min, pre-freezing time 4 h, pre-freezing temperature -30 ° C, maximum heating plate temperature 55 ° C, vacuum pressure 1.2 mbar, to obtain lotus leaf crude polysaccharide; the crude polysaccharide is prepared into a solution with a concentration of 15 mg / mL, Sevag reagent (n-butanol: chloroform = 1:4) is added to deproteinize 3 to 6 times, and then the first dialysis is performed, preferably 4 times, the molecular weight cutoff of the dialysis bag is 8000Da, the dialysis is performed for 12 to 72 hours, preferably 48 hours, and the dialysate is freeze-dried. The freeze-drying conditions are as follows: pre-freezing rate 0.30 ° C / min, pre-freezing time 4h, pre-freezing temperature -30 ° C, maximum heating plate temperature 55 ° C, vacuum pressure 1.2 mbar, to obtain preliminary purified lotus leaf polysaccharide; (2) Separation of lotus leaf polysaccharide components: The preliminarily purified lotus leaf polysaccharide was prepared into a solution with a concentration of 15 mg / mL, loaded onto a DEAE-Cellulose 52 ion column (2.5 cm × 45 cm), and eluted with deionized water, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, and 0.4 mol / L NaCl solutions in sequence at a flow rate of 1 mL / min. The eluate was automatically collected in 10 mL / tubes, and the OD was detected by the phenol-sulfuric acid method. 490Then draw an elution curve, merge single sugar peaks, selectively collect the eluate corresponding to the main peak with the largest peak area, that is, merge the eluate corresponding to the 0.2 mol / L NaCl solution, dialyze the eluate with deionized water for 12 to 48 hours, preferably 24 hours, the molecular weight cutoff of the dialysis bag is 8000Da, and freeze-dry to obtain the lotus leaf polysaccharide component primary purification product NFP-0.2; Weigh 2 g of lotus leaf polysaccharide component primary purification product NFP-0.2, dissolve it in 100 mL of deionized water, load it onto a Sepharose CL-6B gel column, elute with deionized water at a flow rate of 1 mL / min, automatically collect 5 mL / tube of the eluate, and detect OD by phenol-sulfuric acid method. 490 Then, an elution curve was drawn. According to the elution curve, two elution peaks were obtained. The eluate corresponding to the main peak with the largest peak area was selectively collected. The eluate was dialyzed with deionized water for 24 hours. The molecular weight cutoff of the dialysis bag was 8000Da. The lotus leaf polysaccharide uniform monosaccharide component NFP-0.2-c was obtained by freeze drying, which was used as a polysaccharide substrate for further acetylation modification.

[0010] The lotus leaf polysaccharide prepared by the above-mentioned lotus leaf polysaccharide preparation method is within the protection scope of the present invention. The molecular weight of the lotus leaf polysaccharide is 245612Da. The lotus leaf polysaccharide is composed of arabinose, mannose, glucose and galactose. The molar ratio of arabinose, mannose, glucose and galactose is 5.23:2.35:1.0:5.15.

[0011] A method for preparing acetylated lotus leaf polysaccharide comprises the following steps: Weigh 0.5 g of the above-mentioned lotus leaf polysaccharide homogeneous monosaccharide component NFP-0.2-c and dissolve it in 20 mL of distilled water, and stir and dissolve it at room temperature. After adjusting the pH of the solution to 9.0 with NaOH solution (1.0 mol / L), add 2-8 mL of acetic anhydride, preferably 5.5 mL of acetic anhydride, and react at 40-70 ° C for 2-6 hours, preferably 4.5 hours, and preferably 50.5 ° C. After the reaction is completed, adjust the pH to 7 with HCl solution (2 mol / L), place the reaction solution in a dialysis bag, and dialyze with deionized water for 24 hours. The dialyzate is concentrated, precipitated with alcohol, and freeze-dried to obtain acetylated lotus leaf polysaccharide Ac-NFP-0.2-c, i.e., the final product, whose acetylation substitution degree is 0.312-0.509, and preferably the acetylation substitution degree of the acetylated lotus leaf polysaccharide is 0.428.

[0012] The acetylated lotus leaf polysaccharide prepared by the above-mentioned preparation method of acetylated lotus leaf polysaccharide is within the protection scope of the present invention.

[0013] The use of the lotus leaf polysaccharide and acetylated lotus leaf polysaccharide as antioxidants is within the protection scope of the present invention.

[0014] The use of the above-mentioned acetylated lotus leaf polysaccharide in the preparation of anti-tumor drugs is within the protection scope of the invention.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The lotus leaf polysaccharide component product of the present invention has significantly enhanced antioxidant activity after acetylation modification. The prepared acetylated lotus leaf polysaccharide has significant anti-tumor physiological activity, achieves significant technical effects, and can be used as an antioxidant and anti-tumor drug in the food and medical fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The DEAE-Cellulose 52 elution curve of the lotus leaf purified polysaccharide of the present invention is shown in FIG.

[0017] Figure 2 This is the Sepharose CL-6B elution curve of the lotus leaf polysaccharide component NFP-0.2-c of the present invention.

[0018] Figure 3 The high performance liquid chromatogram of the monosaccharide composition of the lotus leaf polysaccharide component NFP-0.2-c of the present invention.

[0019] Figure 4 This is the molecular weight distribution diagram of the lotus leaf polysaccharide component NFP-0.2-c of the present invention.

[0020] Figure 5 This is an ultraviolet scanning image of the lotus leaf polysaccharide component NFP-0.2-c of the present invention.

[0021] Figure 6 This is the infrared spectrum of the lotus leaf polysaccharide component NFP-0.2-c of the present invention.

[0022] Figure 7 This is a diagram showing the scavenging effect of the lotus leaf polysaccharide component NFP-0.2-c and the acetylated lotus leaf polysaccharide Ac-NFP-0.2-c on hydroxyl radicals.

[0023] Figure 8 This is a diagram showing the scavenging effect of the lotus leaf polysaccharide component NFP-0.2-c and the acetylated lotus leaf polysaccharide Ac-NFP-0.2-c on DPPH free radicals.

[0024] Fig. 9 This is a diagram showing the scavenging effect of the lotus leaf polysaccharide component NFP-0.2-c and the acetylated lotus leaf polysaccharide Ac-NFP-0.2-c on superoxide anion free radicals. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods.

[0026] The present invention adopts fresh lotus leaves.

[0027] The reagents used in the present invention: acetic anhydride, 1,1-diphenyl-2-picrylhydrazyl / DPPH, tris(hydroxymethylaminomethane) / Tris (Shanghai Kaiyang Biotechnology Co., Ltd.); anhydrous ethanol, sodium hydroxide, methanol, n-butanol, hydrogen peroxide, ferrous sulfate, hydrochloric acid, salicylic acid, pyrogallol, etc. are all domestic analytical reagents.

[0028] Instruments and equipment used in the present invention: DJ-04 pulverizer, HHS-11-2 water bath, CR22GII high-speed refrigerated centrifuge, Alpha 1-2 LD freeze dryer, RE-52A rotary evaporator, SHB-B95 circulating water multi-purpose vacuum pump, EL-800 enzyme-linked detector, DF-101S constant temperature heating magnetic stirrer, Nicolet Nexus Fourier transform infrared spectrometer, 752 UV-visible spectrophotometer, UV2102 PCS UV scanner, GC-14A gas chromatograph, Waters 600 high pressure liquid gas spectrometer, DG3022 microplate reader, Nu-3500 CO2 incubator. All experiments were performed in triplicate, and the data were expressed as mean ± SD. The statistical analysis of the data was performed using t-test or ANOVA analysis. p <0.05 and p <0.01 was considered statistically significant.

[0029] In addition, in the following examples, unless otherwise specified, all reagents, raw materials and instruments used in the present invention are well known in the art, but do not limit the implementation of the present invention. Other reagents and equipment well known in the art can be applied to the implementation of the following embodiments of the present invention.

[0030] Example 1: Extraction and preliminary purification of crude lotus leaf polysaccharides The lotus leaves were washed, dried and crushed, and water was added according to a solid-liquid ratio (g / mL) of 1:10, and then complex enzymes (cellulase and pectinase) were added, the ratio of cellulase to pectinase was 1:1, the amount of complex enzyme added was 3%, the enzymatic hydrolysis time was 40 minutes, and the enzymatic hydrolysis temperature was 45°C. After the extraction was completed, the supernatant was obtained by centrifugation, and it was concentrated under reduced pressure at 50°C. After concentration, four times the volume of anhydrous ethanol was added to the concentrate, and it was allowed to stand at 4°C for 24 hours, and then centrifuged (10000r / min, 10min) to collect the precipitate. The precipitate was freeze-dried for 48 hours to obtain crude lotus leaf polysaccharide. The crude polysaccharide was prepared into a solution with a concentration of 15 mg / mL, and Sevag reagent (n-butanol: chloroform = 1:4) was added to deproteinize 4 times, and then dialyzed through a dialysis bag for 48 hours. The dialyzate was freeze-dried to obtain preliminary purified lotus leaf polysaccharide.

[0031] Example 2: Extraction and preliminary purification of crude lotus leaf polysaccharides The lotus leaves were washed, dried and crushed, and water was added according to a solid-liquid ratio (g / mL) of 1:20, and then complex enzymes (cellulase and pectinase) were added, the ratio of cellulase to pectinase was 2:1, the amount of complex enzyme added was 3%, the enzymatic hydrolysis time was 50 min, and the enzymatic hydrolysis temperature was 45°C. After the extraction was completed, the supernatant was obtained by centrifugation, and it was concentrated under reduced pressure at 50°C. After concentration, four times the volume of anhydrous ethanol was added to the concentrate, and it was allowed to stand at 4°C for 24 hours, and then centrifuged (10000r / min, 10min) to collect the precipitate. The precipitate was freeze-dried for 48 hours to obtain crude lotus leaf polysaccharide. The crude polysaccharide was prepared into a solution with a concentration of 15 mg / mL, and Sevag reagent (n-butanol: chloroform = 1:4) was added to deproteinize 4 times, and then dialyzed through a dialysis bag for 48 hours. The dialyzate was freeze-dried to obtain preliminary purified lotus leaf polysaccharide.

[0032] Example 3: Extraction and preliminary purification of crude lotus leaf polysaccharides The lotus leaves were washed, dried and crushed, and water was added according to a solid-liquid ratio (g / mL) of 1:30, and then complex enzymes (cellulase and pectinase) were added, the ratio of cellulase to pectinase was 3:1, the amount of complex enzyme added was 3%, the enzymatic hydrolysis time was 60 min, and the enzymatic hydrolysis temperature was 45°C. After the extraction was completed, the supernatant was obtained by centrifugation, and it was concentrated under reduced pressure at 50°C. After concentration, four times the volume of anhydrous ethanol was added to the concentrate, and it was allowed to stand at 4°C for 24 hours, and then centrifuged (10000r / min, 10min) to collect the precipitate. The precipitate was freeze-dried for 48 hours to obtain crude lotus leaf polysaccharide. The crude polysaccharide was prepared into a solution with a concentration of 15 mg / mL, and Sevag reagent (n-butanol: chloroform = 1:4) was added to deproteinize 4 times, and then dialyzed through a dialysis bag for 48 hours. The dialyzate was freeze-dried to obtain preliminary purified lotus leaf polysaccharide.

[0033] Example 4: Extraction and preliminary purification of crude lotus leaf polysaccharides The lotus leaves were washed, dried and crushed, and water was added according to a solid-liquid ratio (g / mL) of 1:40, and then complex enzymes (cellulase and pectinase) were added, the ratio of cellulase to pectinase was 4:1, the amount of complex enzyme added was 3%, the enzymatic hydrolysis time was 70 min, and the enzymatic hydrolysis temperature was 45°C. After the extraction was completed, the supernatant was obtained by centrifugation, and it was concentrated under reduced pressure at 50°C. After concentration, four times the volume of anhydrous ethanol was added to the concentrate, and it was allowed to stand at 4°C for 24 hours, and then centrifuged (10000r / min, 10min) to collect the precipitate. The precipitate was freeze-dried for 48 hours to obtain crude lotus leaf polysaccharide. The crude polysaccharide was prepared into a solution with a concentration of 15 mg / mL, and Sevag reagent (n-butanol: chloroform = 1:4) was added to deproteinize 4 times, and then dialyzed through a dialysis bag for 48 hours. The dialyzate was freeze-dried to obtain preliminary purified lotus leaf polysaccharide.

[0034] Example 5: Separation of lotus leaf polysaccharide components The preliminarily purified lotus leaf polysaccharide was prepared into a solution with a concentration of 15 mg / mL, loaded onto a DEAE-Cellulose 52 ion column (2.5 cm × 45 cm), and eluted with deionized water, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, and 0.4 mol / L NaCl solutions in sequence at a flow rate of 1 mL / min. 10 mL / tube of the eluate was automatically collected, and the OD was detected by the phenol-sulfuric acid method. 490 Then draw the elution curve, merge the single sugar peaks, and selectively collect the eluate corresponding to the main peak with the largest peak area, that is, merge the eluate corresponding to the 0.2 mol / L NaCl solution, dialyze the eluate with deionized water for 24 hours, and freeze-dry to obtain the lotus leaf polysaccharide component primary purification product NFP-0.2 ( Figure 1 ).

[0035] Weigh 2 g of lotus leaf polysaccharide component primary purification product NFP-0.2, dissolve it in 100 mL of deionized water, load it onto a Sepharose CL-6B gel column, elute with deionized water at a flow rate of 1 mL / min, automatically collect 5 mL / tube of the eluate, and detect OD by phenol-sulfuric acid method. 490 Then, an elution curve was drawn. According to the elution curve, two elution peaks were obtained. The eluate corresponding to the main peak with the largest peak area was selectively collected, and the eluate was dialyzed with deionized water for 24 hours. The lotus leaf polysaccharide uniform monosaccharide component NFP-0.2-c was freeze-dried to obtain the polysaccharide substrate for further acetylation modification.

[0036] Example 6: Structural characteristics of lotus leaf polysaccharide component NFP-0.2-c Determination of monosaccharide composition: 10 mg of lotus leaf polysaccharide component NFP-0.2-c sample was placed in a stoppered tube, 2 mL of 2 mol / L trifluoroacetic acid (TFA) solution was added, the tube was sealed in vacuum, and then hydrolyzed at 121°C for 1 h. After removing excess TFA from the hydrolyzate, it was vacuum dried. The sugar nitrile acetate derivatization method was used, 10 mg of hydroxylamine hydrochloride, an appropriate amount of inositol (internal standard) and 0.5 mL of pyridine were added, heated at 90°C for 30 min, then taken out and cooled to room temperature, 0.5 mL of acetic anhydride was added, and the reaction continued at 90°C for 30 min for acetylation. The reaction product was directly analyzed by gas chromatography (GC), and the type of monosaccharide was qualitatively determined according to the retention time of the monosaccharide, and the proportional relationship between the monosaccharides was determined according to the ratio of the peak areas.

[0037] Chromatographic conditions: OV1701 elastic quartz capillary column (Φ0.32mm×30m), N2 carrier gas, flow rate 1.5mL / min, FID hydrogen flame detector, vaporization chamber temperature 260℃, detector temperature 250℃, programmed temperature rise: starting temperature 150℃, dwell for 1min, increase to 190℃ at 10℃ / min, dwell for 1min, increase to 240℃ at 3℃ / min, dwell for 20min.

[0038] Relative molecular mass determination: Standard Dextran with relative molecular mass of 6100, 26290, 84000, 158000 and 291000 were injected successively, and the retention time TR was recorded by HPLC. The standard curve was drawn with TR as the horizontal axis and LgM as the vertical axis to obtain the regression equation. 20μL of the sample VBLP-3b to be tested was injected, and the relative molecular mass of the polysaccharide was calculated by the regression equation based on the obtained TR. Chromatographic column: Waters UllrallydrogelTM Linear (Φ7.8mm×300mm), detector: Waters2410 differential refractometer, 0.1mol / L NaNO3 as the mobile phase, flow rate 0.9mL / min, column temperature 45℃.

[0039] Ultraviolet spectroscopic determination: The ultraviolet spectroscopic determination of the lotus leaf polysaccharide component NFP-0.2-c was carried out using a UV2102 PCS ultraviolet scanner from Shanghai Precision Instrument Co., Ltd., with a scanning range of 190nm-900nm.

[0040] Infrared spectroscopic determination: 1 mg of lotus leaf polysaccharide component NFP-0.2-c and 100 mg of KBr were mixed, ground and pressed into tablets. Thermo Electron's Nicolet Nexus Fourier transform infrared spectrometer was used for determination with a scanning range of 4000-400 cm -1 , resolution 4cm -1 .

[0041] like Figure 3 As shown, the monosaccharides in the lotus leaf polysaccharide component NFP-0.2-c are mainly composed of arabinose, mannose, glucose and galactose, with a molar ratio of 5.23:2.35:1.0:5.15.

[0042] like Figure 4 As shown, the relative molecular mass of lotus leaf polysaccharide component NFP-0.2-c is 245612Da.

[0043] like Figure 5 As shown, from the ultraviolet spectrum of the lotus leaf polysaccharide component NFP-0.2-c, it can be observed that there is no absorption peak around 280nm, indicating that NFP-0.2-c does not contain protein after purification.

[0044] like Figure 6 As shown in the infrared spectrum of lotus leaf polysaccharide component NFP-0.2-c, it can be observed that 3413.89 cm -1 There is a broad peak near 3000cm, which is caused by the stretching vibration of -OH; -1 The peaks near the bottom of the graph are respectively assigned to the asymmetric stretching vibration of -CH2-; 1627.37cm -1 The strong peak nearby is a typical absorption peak of carbonyl, 882.72 cm -1 The absorption peak indicated that NFP-0.2-c contained α-glycosidic bonds.

[0045] Example 7: Acetylation modification of the uniform monosaccharide component NFP-0.2-c of lotus leaf polysaccharide 1. Determination of acetylation substitution degree: Determine by acid-base titration. Accurately weigh 20 mg of acetylated polysaccharide sample, then add 20 mL of NaOH solution (0.01 mol / L), use phenolphthalein as an indicator, and titrate with standard HCI solution (0.01 mol / L) until the red color disappears, and record the volume of HCI solution added. The degree of substitution is calculated as follows:

[0046] In the formula: A is the acetyl content / %; is the volume of NaOH solution added / mL; is the concentration of NaOH solution (0.01 mol / L); is the volume of HCl solution consumed / mL; is the concentration of HCl solution (0.01 mol / L); m is the sample mass / g.

[0047] 2. Acetylation modification of NFP-0.2-c Weigh 0.2g of lotus leaf polysaccharide uniform monosaccharide group NFP-0.2-c, add it to 10mL distilled water, and stir to dissolve at room temperature. After adjusting the pH of the solution to 9.0 with NaOH solution (1mol / L), add 5.5mL of acetic anhydride, react at 50.5℃ for 4.5h, and after the reaction is completed, adjust the pH to 7 with 2mol / L HCl solution, place the reaction solution in a dialysis bag, and dialyze with deionized water for 24h. The dialyzate is concentrated, precipitated with alcohol, and freeze-dried to obtain acetylated lotus leaf polysaccharide Ac-NFP-0.2-c, whose acetylation degree of substitution is 0.428.

[0048] Example 8: Antioxidant effect of acetylated lotus leaf polysaccharide Ac-NFP-0.2-c Determination of hydroxyl radical scavenging rate: Take 2 mL of lotus leaf polysaccharide NFP-0.2-c and acetylated lotus leaf polysaccharide Ac-NFP-0.2-c solutions of different concentrations, add 1 mL FeSO4 (9.0 mmol / L) and 1 mL 9.0 mmol / L salicylic acid-ethanol solution (9.0 mmol / L) respectively, mix thoroughly, then add 1 mL H2O2, place in a 37°C water bath for 30 min, and measure the absorbance A2 at a wavelength of 510 nm with distilled water as a reference. The blank control group uses the same volume of distilled water instead of the sample. Calculate the scavenging rate according to the following formula: Clearance rate (%) = [A0 - (A2 - A1)] / A0 × 100 In the formula: A2 is the absorbance measured by samples of different concentrations; A1 is the absorbance measured by using distilled water instead of salicylic acid; A0 is the absorbance measured by using distilled water instead of samples of different concentrations.

[0049] Determination of DPPH free radical scavenging rate: Take 2 mL of lotus leaf polysaccharide NFP-0.2-c and acetylated lotus leaf polysaccharide Ac-NFP-0.2-c sample solutions of different concentrations, place them in different test tubes, add 2 mL of DPPH solution (0.1 mmol / L), mix well, react at 25°C in the dark for 30 min, and measure the absorbance value A2 at 517 nm. The blank control group uses distilled water instead of polysaccharide sample, and the absorbance is measured as A1. Calculate the scavenging rate of DPPH free radicals according to the following formula: Clearance rate (%) = [A0 - (A2 - A1)] / A0 × 100 In the formula: A2 is the absorbance measured by samples of different concentrations; A1 is the absorbance measured by using distilled water instead of salicylic acid; A0 is the absorbance measured by using distilled water instead of samples of different concentrations.

[0050] Determination of superoxide anion free radical scavenging rate: 4.5 mL of Tris-HCl buffer (0.05 mol / L, pH 8.2), 1 mL of lotus leaf polysaccharide NFP-0.2-c and acetylated lotus leaf polysaccharide Ac-NFP-0.2-c sample solutions of different concentrations and 3.2 mL of distilled water were placed in a test tube, mixed and preheated in a 25°C water bath for 20 min, and 0.3 mL of 7 mmol / L pyrogallol solution was immediately added. After the liquid was fully shaken, it was placed in a 25°C water bath for 3 min, and the reaction was immediately terminated with 1 drop of concentrated hydrochloric acid (10 mol / L). The absorbance at a wavelength of 325 nm was determined as A. The superoxide anion scavenging rate was calculated according to the following formula, and the determination was repeated 3 times.

[0051] Clearance rate (%) = (A0-A) / A0×100 In the formula: A is the absorbance measured by samples of different concentrations; A0 is the absorbance measured by using distilled water instead of the sample.

[0052] The scavenging effects of NFP-0.2-c and Ac-NFP-0.2-c on hydroxyl radicals, DPPH radicals and superoxide anion radicals are shown in Figure 2. Figure 7 , 8 As shown in Figures 9 and 10, within the selected mass concentration range, the scavenging effects of NFP-0.2-c and Ac-NFP-0.2-c on hydroxyl radicals, DPPH radicals and superoxide anion radicals increased with increasing concentrations. After acetylation modification, the antioxidant activity of Ac-NFP-0.2-c was significantly higher than that of NFP-0.2-c, indicating that acetylation modification can enhance the antioxidant activity of NFP-0.2-c.

[0053] Example 9: Inhibitory effect of acetylated lotus leaf polysaccharide Ac-NFP-0.2-c on tumor cells 1. Cell culture 10% calf serum was added to RPMI1640 medium, and Jurkat tumor cells were routinely cultured at 37°C in a 5% CO2 incubator. The cells were passaged every other day, and cells in the logarithmic growth phase were used for experiments.

[0054] 2. MTT method to measure cell proliferation inhibition rate Jurkat cells in the logarithmic growth phase were digested with trypsin, washed, and centrifuged to prepare a cell suspension (3.0×10 4 / mL), 100μL was inoculated in a 96-well plastic culture plate. After static culture for 12h, no polysaccharide sample was added to the control group, and acetylated lotus leaf polysaccharide Ac-NFP-0.2-c (concentrations were 2.0, 4.0, 6.0, 8.0, and 10.0μg / mL) was added to each well of the sample group, with 3 parallel wells for each concentration. Then the culture plate was moved into a CO2 incubator and cultured at 37℃, 5% CO2 and saturated humidity for 48h. Then 20μL of MTT reagent was added to each well, and the culture was continued at 37℃ for 4h. Then the culture was terminated, the supernatant was discarded, 200μLDMSO was added to each well, and the MTT reduction product was gently shaken for 10min to completely dissolve the MTT reduction product. The absorbance (OD) value of each well of the control group and the sample group was measured at 570nm using an enzyme marker. The average value of 3 wells in each group was taken as the average OD value of each group. The inhibition rate of tumor cells Jurkat was calculated according to the following formula.

[0055] Cell inhibition rate (%) = (1-sample group OD / control group OD) × 100 The effect of acetylated lotus leaf polysaccharide Ac-NFP-0.2-c on the proliferation of Jurkat cells is shown in Table 1. It was found that Ac-NFP-0.2-c had a significant inhibitory effect on the proliferation of tumor cells Jurkat. With the increase of polysaccharide concentration, the effect showed an increasing trend, which was dose-dependent and significantly different from the control group (p<0.05). The difference was statistically significant.

[0056] The above results show that acetylated lotus leaf polysaccharide Ac-NFP-0.2-c has a strong antioxidant effect and a strong inhibitory effect on tumor cell proliferation, and can be used as an antioxidant and anti-tumor drug in the field of food and medicine.

[0057] Table 1 Effect of acetylated lotus leaf polysaccharide Ac-NFP-0.2-c on the proliferation of Jurkat cells (x ±s, n=3)

[0058] Note: Different lowercase letters indicate significant differences among different groups (p < 0.05).

[0059] The above-mentioned embodiments are merely descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the present invention.

Claims

1. A method for preparing lotus leaf polysaccharide, characterized in that: The steps include: (1) Extraction and preliminary purification of crude polysaccharides: Wash, dry and crush the lotus leaves, add water according to the solid-liquid ratio of (1g:40 mL)-(1g:10mL), and then add a composite enzyme to the mixture, wherein the composite enzyme is a mixture of cellulase and pectinase. The enzymolysis time is 30-80min and the enzymolysis temperature is 30-55°C. After the enzymolysis is completed, centrifuge to obtain the supernatant, and reduce the pressure and concentrate at 50°C. After concentration, add four times the volume of anhydrous ethanol to the concentrate, let it stand, and then centrifuge to collect the precipitate. After freeze-drying the precipitate, obtain crude lotus leaf polysaccharide. The crude polysaccharide is prepared into a solution with a concentration of 15 mg / mL, and Sevag reagent is added to deproteinize 3-6 times, and then the first dialysis is performed through a dialysis bag. The dialysis time is 12-72h. The dialyzate is freeze-dried to obtain preliminary purified lotus leaf polysaccharide. (2) Separation of lotus leaf polysaccharide components: The preliminarily purified lotus leaf polysaccharide was prepared into a solution with a concentration of 15 mg / mL, loaded onto a DEAE-Cellulose 52 ion column, and eluted with deionized water, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, and 0.4 mol / L NaCl solutions in sequence at a flow rate of 1 mL / min. 10 mL of the eluate was automatically collected per tube, and the OD was detected by the phenol-sulfuric acid method. 490 Then, the elution curve was drawn, the single sugar peaks were merged, and the eluate corresponding to the main peak with the largest peak area was selectively collected, that is, the eluate corresponding to the 0.2 mol / L NaCl solution was merged, and the eluate was dialyzed for a second time with deionized water for 12 to 48 hours, and freeze-dried to obtain the lotus leaf polysaccharide component primary purification product NFP-0.2; Weigh 2 g of lotus leaf polysaccharide component primary purification product NFP-0.2, dissolve it in 100 mL of deionized water, load it onto a Sepharose CL-6B gel column, elute with deionized water at a flow rate of 1 mL / min, automatically collect 5 mL / tube of the eluate, and detect OD by phenol-sulfuric acid method. 490 Then, an elution curve was drawn. According to the elution curve, two elution peaks were obtained. The eluate corresponding to the main peak with the largest peak area was selectively collected. The eluate was dialyzed for a third time with deionized water for 12 to 48 hours. The lotus leaf polysaccharide uniform monosaccharide component NFP-0.2-c was obtained by freeze-drying.

2. The method for preparing lotus leaf polysaccharide according to claim 1, characterized in that: In step (1), the mass ratio of the cellulase to the pectinase is (1-5):1, and the added amount of the complex enzyme is 3% of the mass fraction of the mixture.

3. The method for preparing lotus leaf polysaccharide according to claim 1, characterized in that: In step (1), the molecular weight cutoff of the dialysis bag in the first dialysis is 8000Da.

4. The method for preparing lotus leaf polysaccharide according to claim 1, characterized in that: In step (2), the molecular weight cutoff of the dialysis bag for the second dialysis and the third dialysis is 5000Da.

5. The lotus leaf polysaccharide prepared by the method for preparing lotus leaf polysaccharide according to any one of claims 1 to 4, characterized in that: The molecular weight of the lotus leaf polysaccharide is 245612 Da. The lotus leaf polysaccharide is composed of arabinose, mannose, glucose and galactose. The molar ratio of arabinose, mannose, glucose and galactose is 5.23:2.35:1.0:5.

15.

6. A method for preparing acetylated lotus leaf polysaccharide, characterized in that: Weigh 0.5 g of the lotus leaf polysaccharide homogeneous monosaccharide component NFP-0.2-c described in claim 1 and dissolve it in 20 mL of distilled water, stir and dissolve it at room temperature; adjust the solution pH to 9.0 with NaOH solution, add 2-8 mL of acetic anhydride, react at 40-70°C for 2-6 hours, and after the reaction is completed, adjust the pH to 7 with HCl solution, place the reaction solution in a dialysis bag, and dialyze it with deionized water for 24 hours. The dialyzate is concentrated, precipitated with alcohol, and freeze-dried to obtain acetylated lotus leaf polysaccharide Ac-NFP-0.2-c, i.e., the final product, with an acetylation degree of substitution of 0.312-0.

509.

7. The method for preparing acetylated lotus leaf polysaccharide according to claim 6, characterized in that: The acetylation substitution degree of the acetylated lotus leaf polysaccharide Ac-NFP-0.2-c is 0.

428.

8. Acetylated lotus leaf polysaccharide prepared by the method for preparing acetylated lotus leaf polysaccharide according to claim 6.

9. Use of the lotus leaf polysaccharide according to claim 5 or the acetylated lotus leaf polysaccharide according to claim 8 as an antioxidant.

10. Use of the acetylated lotus leaf polysaccharide according to claim 8 in the preparation of anti-tumor drugs.

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