Preparation method of horseradish tree leaf polyphenol extract with antitumor activity

Abstract

The invention provides a preparation method of a horseradish tree leaf polyphenol extract with anti-tumor activity, and relates to the field of extraction of horseradish tree leaf polyphenol. The preparation method of the anti-tumor activity moringa oleifera leaf polyphenol extract comprises the following steps: extracting and purifying the moringa oleifera leaf extract, carrying out rotary evaporation and concentration to obtain three groups of different polar parts, namely an ethyl acetate part, an n-butyl alcohol part and a water part, freezing and drying to obtain a powdery sample, and collecting the powdery sample. Standard curves of gallic acid and rutin are made, and the contents of total phenols and total flavonoids in the three groups of samples are respectively determined. The antioxidant capacity of the three groups of samples is measured by adopting three methods of DPPH clearance rate, ABTS clearance capacity and FRAP total antioxidant capacity, and meanwhile, the standard deviation of each concentration of the three groups of samples is calculated and significant difference analysis is carried out. And carrying out component analysis on compounds in the three groups of samples by adopting an HPLC (High Performance Liquid Chromatography) chromatographic analysis method on the three groups of samples and the original extracts of the moringa oleifera leaves, screening out the compounds with high antioxidant activity from the three groups of samples, and determining the corresponding content.

Description

Technical Field

[0001] The invention relates to the technical field of moringa leaf polyphenol extraction, and in particular to a method for preparing a moringa leaf polyphenol extract with anti-tumor activity. Background Art

[0002] Moringa is a perennial plant of the genus Moringa in the Moringaceae family. It is evergreen or semi-deciduous and originated in northern India. It is also known as the strange tree and the drumstick tree. It is a perennial tree plant and is widely planted in tropical regions such as Asia and Africa. The leaves, stems, roots, and pods of Moringa are naturally rich in various vitamins, minerals, proteins, lipids, trace elements and other nutrients. It also contains a variety of different types of natural pharmacologically active ingredients, mainly including polyphenols, alkaloids, terpenes, organic acids and their esters, sterols and other compounds. Among them, polyphenol compounds include flavonoids, phenolic acids, phenylpropanoids, etc.

[0003] Hongqiang Lin et al. isolated hundreds of compounds from Moringa plants in China and India, including flavonoids, phenolic acids and other compounds. Related studies have shown that flavonoids and phenolic acids are closely related to the antioxidant activity of Moringa plants. In addition, flavonoids also have physiological activities that can achieve antibacterial enhancement and anti-inflammatory effects to a good extent.

[0004] In previous experimental research methods on the antioxidant biological activity mechanism of Moringa plants, there has been little research on the antioxidant substances in different polar parts of Moringa leaves, and further detailed systematic research is still needed on some different types of antioxidant compounds actually contained in different polar parts. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for preparing a Moringa leaf polyphenol extract with anti-tumor activity, which solves the problem of effectively determining the detailed component content of Moringa leaf polyphenols.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for preparing an anti-tumor active Moringa leaf polyphenol extract, comprising the following steps: Step 1, weigh a portion of Moringa leaf extract, take out half of the total Moringa leaf extract in a -25°C to -18°C refrigerator, weigh its mass to obtain 7.5316g, dissolve it in 250mL of distilled water, add 250mL of an equal volume of ethyl acetate, fully oscillate on a constant temperature oscillator for 15min, mix and extract; Step 2: After the shaking is completed, the mixture is allowed to stand for about 30 minutes, and the upper ethyl acetate portion is taken and concentrated by rotary evaporation. When no ethyl acetate is evaporated out, the evaporation is stopped, and the ethyl acetate portion obtained is collected as M1; Step 3: Add an equal volume of anhydrous n-butanol to the lower water portion, perform layered extraction using the above method, collect the obtained n-butanol portion and the remaining water portion, perform rotary evaporation and concentration respectively, and then collect to obtain the n-butanol portion M2 and the water portion M3; Step 4: freeze-dry the three extracted components M1, M2, and M3 in a freeze dryer for 10-16 hours, and then store the samples in a -25°C refrigerator overnight for 10-12 hours. Repeat the steps 4-6 times to obtain three groups of powdered samples. Step 5. Perform total phenol content determination, total flavonoid content determination, DPPH scavenging ability determination, ABTS scavenging ability determination, FRAP total antioxidant capacity determination, and Moringa leaf polyphenol component analysis on the three groups of Moringa leaf extract samples.

[0007] Preferably, the parameters of the oscillator in step 1 are: temperature 26.6° C., rotation speed 200 r / min.

[0008] Preferably, the conditions for evaporation concentration in step 2 are: temperature 35° C., rotation speed 70 r / min, and pressure 0.09 MPa on a rotary evaporator.

[0009] Preferably, the freeze-drying conditions in step 4 are as follows: temperature below -45°C and vacuum.

[0010] Preferably, the total phenol content determination step in step 5 is: Step I: Take 0.020 g of gallic acid standard, add appropriate amount of distilled water and stir thoroughly until dissolved, stir evenly and dilute to a 10 mL standard volumetric flask, and take 0.0, 0.5, 1.0, 1.5, 2.0, and 3.0 mL of the prepared standard solution and add them to a 10 mL volumetric flask and dilute to volume; Step II, prepare a solution with a concentration gradient of 0, 10, 20, 30, 40, 50, 60 μg / mL, then pipette 1 mL of the above solution into each test tube, add 2 mL of Folin phenol reagent to each test tube, mix well, react for 6 minutes, add 3 mL of 7.5% Na2CO3 solution to each test tube, mix well, and place in a dark place to react for about 60 minutes; Step III, weigh 0.02 g of each of the M1, M2, and M3 samples, take an appropriate amount of distilled water and dilute them to a dissolved state, pour them into a volumetric flask and dilute to 100 mL, prepare a sample solution with a concentration of 0.2 mg / mL, treat it with the above gallic acid preparation method, do three replicates for each sample, measure the ultraviolet absorbance at a wavelength of 765 nm, use distilled water as a blank control, draw a gallic acid standard curve, and measure the actual ultraviolet absorption spectrophotometric value of each sample; The total phenol content was calculated as follows: Total phenol yield (%) =

[0011] Where: N: dilution factor; V: fixed volume of extract (mL); C: polyphenol content of sample solution calculated from the standard curve (g / mL); M: weight of the weighed Moringa leaf sample (g).

[0012] Preferably, the method for determining the total flavonoid content in step 5 is as follows: Take 0.01g of rutin standard, add methanol to a 10mL volumetric flask, dilute to a 1mg / mL standard solution, take the 1mg / mL standard solution to prepare 0, 0.1, 0.2, 0.5, 1.0mg / mL rutin standard solutions, then take 1.01g of AlCl3, prepare 100mL of 1% AlCl3 solution, take 0.01g of samples M1, M2, and M3 respectively, dissolve in a 10mL volumetric flask, add anhydrous methanol and dilute to 10mL, and also prepare a 1mg / mL solution. Each sample needs to be made in triplicate, then take 4mL of AlCl3 solution and add it to each test tube, mix well, then add 5mL of methanol respectively, shake and mix thoroughly, then react in a 40℃ water bath for 60min, and measure the absorbance at 410nm; The total flavonoids content was calculated as follows: Total flavonoid yield (%) =

[0013] Where: N: dilution multiple; V: fixed volume of extract (mL); C: total flavonoids content in the sample solution calculated from the standard curve (mg / mL); M: weighed mass of Moringa leaf sample (g).

[0014] Preferably, the DPPH scavenging ability determination method in step 5 is: Take 0.05g of each sample M1, M2, M3 and ascorbic acid M4, dissolve in a 50mL volumetric flask and dilute to volume. Prepare a 0.1mg / mL sample solution, dilute to 0.02, 0.04, 0.06, 0.08, 0.10mg / mL sample solution, weigh 0.045g DPPH powder, add anhydrous ethanol to a 100mL volumetric flask and dilute to volume, prepare a 0.045mg / mL DPPH solution, and prepare the solution for determination as shown in the following table;

[0015] Two parallel samples of A2 mixed solution for each gradient were prepared, and three parallel samples of A3 mixed solution were prepared. After preparation, they were placed on a microplate oscillator for mixing, and then placed in a cabinet to react in the dark for at least 30 minutes, and the absorbance was measured at 517 nm.

[0016] Preferably, the ABTS scavenging ability determination method in step 5 is: Take an appropriate amount of peroxidase, dilute it 10 times, and prepare a peroxidase working solution; mix the detection buffer, ABTS solution and 1 / 1000 hydrogen peroxide solution to prepare an ABTS working solution; The reagent addition volume and order are as follows: first add 20 μL of peroxidase working solution to the detection well, then add 10 μL of distilled water, various concentrations of Trolox standard solution and three groups of sample solutions to the blank control group, standard curve detection group and sample detection group, respectively, and measure A734 after standing for 6 minutes. Three parallels are also made at each sample concentration, and the absorbance is measured at 734 nm.

[0017] Preferably, the FRAP total antioxidant capacity determination method in step 5 is: The TPTZ diluent and TPTZ solution were first mixed, and then the detection buffer was added to prepare the FRAP working solution. The reagent addition volume and order were as follows: 180 μL of FRAP working solution was added to each detection well of the 96-well plate, and 5 μL of distilled water, various concentrations of FeSO4·H2O standard solutions and three groups of sample solutions were added to the blank control group, standard curve detection group and sample detection group, respectively. The solutions were placed at 37°C for 5 min, and three parallels were made for each sample concentration, and the absorbance was measured at 593 nm.

[0018] The present invention provides a method for preparing a Moringa leaf polyphenol extract with anti-tumor activity. The method has the following beneficial effects: The present invention will separate and extract the different polarity parts of Moringa leaf crude extract by this experiment, collect three groups of different polarity parts, remove with DPPH, remove with ABTS, FRAP method these 3 kinds of antioxidant capacity methods to measure these three groups of different polarity parts respectively, compare the difference of its antioxidant capacity. And adopt high performance liquid chromatography to carry out quantitative analysis to three groups of polarity parts and crude extract, measure the content of various polyphenol compounds, finally screen out the parts with higher polyphenol content and stronger antioxidant capacity. Meanwhile, also provide experimental basis for the antioxidant capacity and polyphenol content of different polarity parts of Moringa leaf, so as to facilitate the further development and research of Moringa plant in practical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a standard curve diagram of two sets of standards of the present invention; Figure 2 The figure is a graph showing the clearance rate of DPPH by three groups of polar parts of Moringa leaves and Vc of the present invention; Figure 3 The standard curve of the Trolox solution of the present invention and the antioxidant activity comparison diagram of three groups of samples; Figure 4The standard curve of FeSO4·7H2O of the present invention and the comparison diagram of antioxidant activity of three groups of samples; Figure 5 HPLC chromatograms of four mixed standard solutions of the present invention; Figure 6 Schematic diagram of HPLC chromatograms of four groups of samples of the present invention; Figure 7 It is a standard curve diagram of four standard products of the present invention. DETAILED DESCRIPTION

[0020] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Example

[0021] like Figure 1-7 As shown, the embodiment of the present invention provides a method for preparing a Moringa leaf polyphenol extract with anti-tumor activity, comprising the following steps: Step 1, weigh a portion of Moringa leaf extract, take out half of the total Moringa leaf extract in a -25°C to -18°C refrigerator, weigh its mass to obtain 7.5316g, dissolve it in 250mL of distilled water, add 250mL of an equal volume of ethyl acetate, fully oscillate on a constant temperature oscillator for 15min, mix and extract; Step 2: After the shaking is completed, the mixture is allowed to stand for about 30 minutes, and the upper ethyl acetate portion is taken and concentrated by rotary evaporation. When no ethyl acetate is evaporated out, the evaporation is stopped, and the ethyl acetate portion obtained is collected as M1; Step 3: Add an equal volume of anhydrous n-butanol to the lower water portion, perform layered extraction using the above method, collect the obtained n-butanol portion and the remaining water portion, perform rotary evaporation and concentration respectively, and then collect to obtain the n-butanol portion M2 and the water portion M3; Step 4: freeze-dry the three extracted components M1, M2, and M3 in a freeze dryer for 10-16 hours, and then store the samples in a -25°C refrigerator overnight for 10-12 hours. Repeat the steps 4-6 times to obtain three groups of powdered samples. Step 5. Perform total phenol content determination, total flavonoid content determination, DPPH scavenging ability determination, ABTS scavenging ability determination, FRAP total antioxidant capacity determination, and Moringa leaf polyphenol component analysis on the three groups of Moringa leaf extract samples.

[0022] The parameters of the oscillator in step 1 are: temperature 26.6° C., rotation speed 200 r / min.

[0023] The conditions for evaporation and concentration in step 2 are: temperature 35° C., rotation speed 70 r / min, and pressure 0.09 MPa on a rotary evaporator.

[0024] The freeze-drying conditions in step 4 are as follows: temperature below -45°C and vacuum.

[0025] The present invention provides an embodiment: the total phenol content determination step in step 5 is: Step I: Take 0.020 g of gallic acid standard, add appropriate amount of distilled water and stir thoroughly until dissolved, stir evenly and dilute to a 10 mL standard volumetric flask, and take 0.0, 0.5, 1.0, 1.5, 2.0, and 3.0 mL of the prepared standard solution and add them to a 10 mL volumetric flask and dilute to volume; Step II, prepare a solution with a concentration gradient of 0, 10, 20, 30, 40, 50, 60 μg / mL, then pipette 1 mL of the above solution into each test tube, add 2 mL of Folin phenol reagent to each test tube, mix well, react for 6 minutes, add 3 mL of 7.5% Na2CO3 solution to each test tube, mix well, and place in a dark place to react for about 60 minutes; Step III, weigh 0.02 g of each of the M1, M2, and M3 samples, take an appropriate amount of distilled water and dilute them to a dissolved state, pour them into a volumetric flask and dilute to 100 mL, prepare a sample solution with a concentration of 0.2 mg / mL, treat it with the above gallic acid preparation method, do three replicates for each sample, measure the ultraviolet absorbance at a wavelength of 765 nm, use distilled water as a blank control, draw a gallic acid standard curve, and measure the actual ultraviolet absorption spectrophotometric value of each sample; The total phenol content was calculated as follows: Total phenol yield (%) =

[0026] Where: N: dilution factor; V: fixed volume of extract (mL); C: polyphenol content of sample solution calculated from the standard curve (g / mL); M: weight of the weighed Moringa leaf sample (g).

[0027] The method for determining the total flavonoid content in step 5 is as follows: Take 0.01g of rutin standard, add methanol to a 10mL volumetric flask, dilute to a 1mg / mL standard solution, take the 1mg / mL standard solution to prepare 0, 0.1, 0.2, 0.5, 1.0mg / mL rutin standard solutions, then take 1.01g of AlCl3, prepare 100mL of 1% AlCl3 solution, take 0.01g of samples M1, M2, and M3 respectively, dissolve in a 10mL volumetric flask, add anhydrous methanol and dilute to 10mL, and also prepare a 1mg / mL solution. Each sample needs to be made in triplicate, then take 4mL of AlCl3 solution and add it to each test tube, mix well, then add 5mL of methanol respectively, shake and mix thoroughly, then react in a 40℃ water bath for 60min, and measure the absorbance at 410nm; The total flavonoids content was calculated as follows: Total flavonoid yield (%) =

[0028] Where: N: dilution multiple; V: fixed volume of extract (mL); C: total flavonoids content in the sample solution calculated from the standard curve (mg / mL); M: weighed mass of Moringa leaf sample (g).

[0029] Mass recovery rates of three different polar components: After freeze drying, the masses of samples M1, M2, and M3 were 0.2017 g, 0.9681 g, and 3.5670 g, respectively. The mass recovery rates of M1, M2, and M3 were 2.68%, 12.85%, and 47.36%, respectively; Analysis of total phenolic and flavonoid content: component analysis, standard curves of gallic acid and rutin Figure 1 As shown. The standard curve of gallic acid was calculated to be y=24.657x+0.0111, with a correlation coefficient of R2=0.9992, and a good linear relationship in the concentration range of 0-0.06mg / ml. The standard curve of rutin was y=3.11943x-0.03219, with a correlation coefficient of R2=0.99826, and a good linear relationship in the concentration range of 0-0.5mg / ml; In the total phenol content determination method, the absorbance values ​​of M1, M2, and M3 sample solutions were measured as 1.200, 0.593, and 0.135, respectively. In the total flavonoid content determination method, the absorbance values ​​of M1, M2, and M3 sample solutions were measured as 1.840, 1.368, and 0.073, respectively. The results of total phenol and total flavonoid content are as follows: Total phenolic and flavonoid contents in three polar parts M1, M2 and M3

[0030] Activity evaluation: From the data analysis in the above table, it can be obtained that the total phenol content of M1, M2, and M3 samples is 24.11 mg / g, 11.8 mg / g, and 2.51 mg / g, respectively. Among them, the total phenol content measured in the M1 part is the highest, about 9.6 times that of M3. It is proved that the total phenol content contained in the ethyl acetate part is the highest, followed by the n-butanol part, and finally the water part; the total flavonoid content of M1, M2, and M3 samples is 60.68 mg / g, 43.93 mg / g, and 2.59 mg / g, respectively. Among them, the total flavonoid content measured in the M1 part is the highest, about 23 times that of M3. The determination results of the total flavonoid content are consistent with the above conclusions.

[0031] In summary, the contents of total phenols and total flavonoids are in the following order: ethyl acetate part > n-butanol part > water part.

[0032] HPLC quantitative analysis showed that two polyphenolic compounds, rutin and ferulic acid, were separated and identified in the above two parts, confirming that these two compounds may be one of the sources of polyphenolic substances in the ethyl acetate part and the n-butanol part, and therefore exhibit high antioxidant pharmacological activity.

[0033] The present invention provides an embodiment: Analysis of polyphenol components in Moringa leaves, HPLC chromatographic parameter conditions Chromatographic column: Shim-packGISTC18-AQ chromatographic column (5μm, 4.6x250) Mobile part A: 1% acetic acid solution; mobile part B: acetonitrile solution, filtered through a 0.22 μm filter membrane before use, and the gradient elution procedure was based on the chromatographic conditions for the determination of major phenolic substances in apples in the Chinese Agricultural Industry Standard

[19] .

[0034] Elution mode: linear gradient; Flow rate: 0.8ml / min; Detection wavelength: 280nm; Injection volume: 10 μl.

[0035] Single standard solution preparation According to the method of preliminary treatment before sample extraction by Hao Junguang et al.

[20] , 10 mg of each of rutin, chlorogenic acid, gallic acid, caffeic acid, ferulic acid, and catechin were taken in a 10 mL volumetric flask to prepare a single phenolic substance standard solution of about 1000 mg / L, and then diluted 100 times to prepare a 10 mg / L standard solution. Take 100 mg / L cyanidin-3-O-glucoside solution, dilute 10 times, and prepare a 10 mg / L standard solution according to the above method. Take 11.3 mg, 10.8 mg, and 12.4 mg of M1, M2, and M3 samples, add methanol to a 10 mL volumetric flask and dilute to volume to prepare the sample solution. Take 1 mL of the above prepared standard solution and sample solution, mix them and pass through a 0.22 μm filter membrane.

[0036] Preparation of mixed standard solution Liquid chromatography analysis was performed on the M1, M2 and M3 samples and the original Moringa extract sample. 9.7 mg of rutin, 30.4 mg of chlorogenic acid, 5.4 mg of gallic acid, 4.9 mg of caffeic acid and 5.3 mg of catechins were weighed respectively, and the volume was made up to 100 mL volumetric flask with methanol to prepare a mixed standard solution; 6.3 mg of cyanidin-3-O-glucoside was weighed in a test tube, 1 mL of methanol was added, and the mixture was mixed to prepare a 6300 mg / L standard solution.

[0037] Establishment of standard curve Take 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 mL of the mixed standard solution and 5, 10, 20, 40, 80, 160 μL of cyanidin-3-O-glucoside solution in a volumetric flask, dilute to 10 mL with methanol to obtain mixed standard solutions with different concentration gradients. Take the prepared 1000 mg / L ferulic acid standard solution and prepare 2 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 40 mg / L standard gradient solutions.

[0038] The present invention provides an embodiment, wherein the method for determining the DPPH scavenging ability in step 5 is: Take 0.05g of each sample M1, M2, M3 and ascorbic acid M4, dissolve in a 50mL volumetric flask and dilute to volume. Prepare a 0.1mg / mL sample solution, dilute to 0.02, 0.04, 0.06, 0.08, 0.10mg / mL sample solution, weigh 0.045g DPPH powder, add anhydrous ethanol to a 100mL volumetric flask and dilute to volume, prepare a 0.045mg / mL DPPH solution, and prepare the solution for determination as shown in the following table;

[0039] Two parallel samples of A2 mixture of each gradient were prepared, and three parallel samples of A3 mixture were prepared. After preparation, the mixture was placed on a microplate oscillator for mixing, and then placed in a cabinet to avoid light for at least 30 minutes, and the absorbance was measured at 517nm. Reference Figure 2 , Figure 2 In the above, M1 is the ethyl acetate part, M2 is the n-butanol part, M3 is the water part, and M4 is the Vc reference substance; The DPPH clearance rates and standard deviations of M1, M2, M3, and M4 are shown in Figure 3As shown,. It can be seen that: except for the water part, the average DPPH scavenging rates of the ethyl acetate part and the n-butanol part under the concentration conditions of 0.04-0.10 mg / mL are close to the scavenging rate of Vc. The DPPH scavenging rate of the ethyl acetate part at a concentration of 0.08 mg / mL reached 95.04±0.027%, while the DPPH scavenging rate of the n-butanol part at a concentration of 0.02 mg / mL reached 92.94±0.0181%. This shows that these two parts have high antioxidant activity. The DPPH scavenging rate of the water part at a concentration of 0.02 mg / mL was only 53.05±0.027%, and the scavenging rate fluctuated around 78% under the concentration conditions of 0.04-0.08 mg / mL. When the concentration was 0.1 mg / mL, the DPPH scavenging rate was 93.99±0.003%, which also reached more than 90%. In general, the order of DPPH scavenging ability is: M4>M2>M1>M3.

[0040] The present invention provides an embodiment, wherein the method for determining the ABTS scavenging ability in step 5 is: Take an appropriate amount of peroxidase, dilute it 10 times, and prepare a peroxidase working solution; mix the detection buffer, ABTS solution and 1 / 1000 hydrogen peroxide solution to prepare an ABTS working solution; The reagent addition volume and order are as follows: first add 20 μL of peroxidase working solution to the detection well, then add 10 μL of distilled water, various concentrations of Trolox standard solution and three groups of sample solutions to the blank control group, standard curve detection group and sample detection group, respectively, and measure A734 after standing for 6 minutes. Three parallels are also made at each sample concentration, and the absorbance is measured at 734 nm; The calculation method is as follows: Calculate the △734 of the standard and the sample. The △734 of the standard = A734 blank control - A734 standard, and the same is true for the sample. Make a standard curve with △734 and Trolox standard solution concentration, expressed by the formula y = ax + b (y is △734, x is the concentration of Trolox standard solution, a is the slope, and b is the intercept). According to the standard curve formula, calculate how many mmol / L concentration of Trolox standard the sample is equivalent to, and you can get the total antioxidant capacity of the sample; The results of the ABTS scavenging ability assay are shown below.

[0041] The average absorbance value of M1 is A

[0042] Note: M1 is the ethyl acetate part, M2 is the n-butanol part, and M3 is the water part.

[0043] Standard curve of Trolox and comparison of antioxidant activities of three groups of samples Figure 3 As shown, Figure 3 Among them, M1 is the ethyl acetate part, M2 is the n-butanol part, and M3 is the water part. The standard curve of Trolox was calculated as y = 0.5858x + 0.0226, with a correlation coefficient R2 = 0.9941, showing a good linear relationship under the concentration condition of 0 - 1.5 mmol / g; Through the significant difference analysis, it can be obtained that the significant differences P of the Trolox standard solution concentrations corresponding to M1 and M2 at the concentrations of 0.02 mg / mL and 0.08 mg / mL are 0.0055 and 0.0076 respectively, P < 0.01, and the differences are extremely significant; the significant difference P at the concentration of 0.04 mg / mL is 0.0326, 0.01 < P < 0.05, and the difference is significant; the significant differences P at the concentrations of 0.06 mg / mL and 0.10 mg / mL are 0.1144 and 0.1191 respectively, P > 0.05, and the differences are not significant. At the concentration of 0.10 mg / mL, the antioxidant activity of the M2 sample is slightly lower than that of the M1 sample, and the difference is also not significant; the significant difference P between the M1 sample and the M3 sample under the five concentration conditions is < 0.01, and the difference is extremely significant; except for the significant difference at the concentration of 0.06 mg / mL, the significant difference P between the M2 sample and the M3 sample at the other concentrations is < 0.01, and the difference is extremely significant. It shows that the antioxidant ability of M1 and M2 is significantly higher than that of M3.

[0044] The total antioxidant capacity of M1 samples at the concentrations of 0.02mg / ml, 0.04mg / ml, 0.06mg / ml, 0.08mg / ml and 0.10mg / mL were 0.7±0.071mmol / L, 1.19±0.090mmol / L, 1.77±0.064mmol / L, 2.28±0.058mmol / L, 2.45±0.250mmol / L, and the average total antioxidant capacity was 29.45mmol / g; correspondingly, the total antioxidant capacity of M2 samples was 0.52±0.061mmol / L, 0 .88±0.034mmol / L, 1.38±0.221mmol / g, 1.68±0.149mmol / L, 2.31±0.165mmol / L, and the average total antioxidant capacity was 23.02mmol / g; the total antioxidant capacity of the M3 sample was 0.09±0.045mmol / L, 0.06±0.027mmol / L, 0.27±0.152mmol / L, 0.33±0.188mmol / L, 0.27±0.028mmol / L, and the average total antioxidant capacity was 3.47mmol / g. It can be seen that, except for the water part, the antioxidant capacity of the ethyl acetate part and the n-butanol part generally increases with the increase of their concentration. Compared with the other two parts, the total antioxidant capacity of the water part is lower and does not increase significantly with the increase of concentration. Under the same conditions, the scavenging capacity of ABTS is: ethyl acetate part>n-butanol part>water part.

[0045] The present invention provides an embodiment, wherein the FRAP total antioxidant capacity determination method in step 5 is: TPTZ diluent and TPTZ solution were first mixed, and then the detection buffer was added to prepare FRAP working solution. The reagent addition volume and order were as follows: 180 μL FRAP working solution was added to each detection well of the 96-well plate, and 5 μL distilled water, various concentrations of FeSO4·H2O standard solutions and three groups of sample solutions were added to the blank control group, standard curve detection group and sample detection group respectively. The samples were placed at 37°C for 5 min, and three parallels were made at each sample concentration. The absorbance was measured at 593 nm. The calculation method is as follows: Determine the A539 of the FeSO4·H2O standard solution and the sample solution, use A539 and the concentration of the FeSO4·H2O standard solution to make a standard curve, expressed by the formula y=ax+b (y is A539, ​​x is the concentration of the FeSO4·H2O standard solution, a is the slope, and b is the intercept). According to the standard curve formula, calculate how many mmol / L concentration of the FeSO4·H2O standard solution the sample is equivalent to, and you can get the total antioxidant capacity of the sample; Comparison of FRAP total antioxidant capacity: The results of FRAP total antioxidant capacity assay are as follows.

[0046] The average absorbance value of the three groups of samples is A

[0047] Standard curve of FeSO4·7H2O and comparison of antioxidant activities of three groups of samples Figure 4 As shown. The standard curve of FeSO4·7H2O is y=0.27881x-0.00925, and the correlation coefficient R2=0.98863; Figure 4 Middle: M1 is the ethyl acetate part, M2 is the n-butanol part, and M3 is the water part; Under the conditions of 0.01, 0.02, 0.03, and 0.04 mg / mL concentrations, the concentrations of FeSO4·7H2O standard solution corresponding to the M1 sample were 0.325±0.018mmol / L, 0.731±0.063mmol / L, 0.89±0.035mmol / L, and 1.653±0.043mmol / L, respectively. Since the spectrophotometric values ​​of the sample at four different concentrations were measured, the average total antioxidant capacity of the M1 sample was calculated to be equivalent to the concentration of FeSO4·7H2O solution at 35.01mmol / g; Under the conditions of 0.01, 0.02, 0.04, and 0.06 mg / mL concentrations, the concentrations of FeSO4·7H2O standard solution corresponding to the M2 sample were 0.221±0.015mmol / L, 0.681±0.171mmol / L, 1.134±0.041mmol / L, and 1.654±0.081mmol / L, respectively. The average total antioxidant capacity of the M2 sample was calculated to be equivalent to the concentration of FeSO4·7H2O solution at 28.02mmol / g.

[0048] Under the conditions of 0.04, 0.06, 0.08, and 0.10 mg / mL concentrations, the concentrations of FeSO4·7H2O standard solution corresponding to the M3 sample were 0.180±0.047mmol / L, 0.272±0.072mmol / L, 0.379±0.024mmol / L, and 0.449±0.010mmol / L, respectively. The average total antioxidant capacity of the M3 sample was calculated to be equivalent to the concentration of 4.57mmol / g of FeSO4·7H2O solution.

[0049] The significant difference analysis is shown in the following table:

[0050] From the above table, we can see that at a concentration of 0.04 mg / mL, the significant difference P of the Trolox standard solution concentrations between the ethyl acetate and n-butanol samples is 0.0022, the significant difference P of the Trolox standard solution concentrations between the ethyl acetate and water samples is 0.0004, and the significant difference P of the Trolox standard solution concentrations between the n-butanol and water samples is 0.0021, all of which are less than 0.01, and the differences are extremely significant. This shows that under the same concentration condition, the total antioxidant capacity of the ethyl acetate, n-butanol and water samples is very significantly different.

[0051] In summary, the total antioxidant capacity is: ethyl acetate part > n-butanol part > water part. The average total antioxidant capacity is equivalent to the concentration of FeSO4·7H2O solution at 35.01mmol / g, 28.02mmol / g, and 4.57mmol / g, respectively. And with the increase of concentration, the antioxidant activity of the three groups of samples also increases. The antioxidant capacity of the ethyl acetate part, n-butanol part, and water part samples is significantly different through significant difference analysis.

[0052] The present invention provides an embodiment: chromatographic analysis and content determination of four groups of samples, such as Figure 5 , HPLC chromatographic analysis results of the mixed standard and four groups of samples, the chromatograms of M1, M2, M3 and the original extract samples are shown in Figure 6 As shown, Figure 6 In the figure, A is the ethyl acetate part, B is the n-butanol part, C is the water part, and D is the original extract. In the figure, 1 is chlorogenic acid, 2 is caffeic acid, 3 is rutin, and 4 is ferulic acid; contrast Figure 6 The mixed standard solution can clearly identify that the four samples contain chlorogenic acid, caffeic acid, rutin and ferulic acid. The peak times of the four groups of standards are: 18.545min, 21.608min, 29.282min, 31.288min; The standard curves of four standards were prepared, and the results are as follows Figure 7 ,like Figure 7As shown, the standard curve of chlorogenic acid is: y=17197.04288x-3750.85075, correlation coefficient R2=0.99988; the standard curve of caffeic acid is: y=46196.20469x-4391.40796, correlation coefficient R2=0.99978; the standard curve of rutin is: y=7383.97015x-1647.68657, correlation coefficient R2=0.99988; the standard curve of ferulic acid is: y=39615.24491x-11561.77163, correlation coefficient R2=0.99969. The R2 values ​​of the four groups of curves are very close to 1, with good linear relationship, and can be used as references for the four compounds in the sample; Compound concentration determination and content analysis. Based on the above data, the concentration determination results of each compound in the four groups of samples are as follows: Concentration determination results of different compounds in four groups of samples

[0053] By analyzing the above table and calculating, it is obtained that the contents of the four compounds in the original extract are 5.17 mg / g, 3.19 mg / g, 31.27 mg / g and 10.03 mg / g respectively; the contents of the four compounds in the ethyl acetate part are 6.90 mg / g, 2.35 mg / g, 81.99 mg / g and 57.45 mg / g respectively; the contents of the four compounds in the n-butanol part are 7.64 mg / g, 2.53 mg / g, 136.60 mg / g and 21.72 mg / g respectively; the contents of the four compounds in the original extract are 5.17 mg / g, 3.19 mg / g, 31.27 mg / g and 10.03 mg / g respectively; the proportions of the four compounds in the water part are 4.64 mg / g, 1.26 mg / g, 6.64 mg / g and 3.47 mg / g. In general, the amount of rutin and ferulic acid in the four groups of samples was significantly higher than that of chlorogenic acid and caffeic acid. The content of rutin and ferulic acid in the ethyl acetate and n-butanol parts was significantly higher than that in the original extract and water part, while the caffeic acid content in the original extract was higher than that in the other three groups of samples. The chlorogenic acid content of the four groups of samples was similar, and the ethyl acetate and n-butanol parts were slightly higher than that in the original extract and water part. Regarding the determination of total phenol content and total flavonoid content, the total phenol content of M1, M2, and M3 samples were 24.11 mg / g, 11.8 mg / g, and 2.51 mg / g, respectively, and the total flavonoid content was 60.68 mg / g, 43.93 mg / g, and 2.59 mg / g, respectively. The results of the determination showed that the total phenol and total flavonoid contents of the ethyl acetate fraction were the highest. This is consistent with the conclusion of Lv Xiaoya

[26] , who used the 220°C subcritical water method to extract Moringa leaves and obtained extracts from different polar parts, confirming that the total phenol and total flavonoid contents in the ethyl acetate organic solvent were the highest, reaching 15.44% and 13.42%, respectively. According to the principle of like dissolves like, most phenolic and flavonoid compounds can be well dissolved in organic solvents during the extraction process. Therefore, both the ethyl acetate and n-butanol fractions contain high levels of total phenol and total flavonoid contents. According to the research progress on the antioxidant capacity of Moringa oleifera by Putianlei et al.

[27] , the high antioxidant capacity of Moringa oleifera is mainly dominated by flavonoids and phenolic acids, which also provides sufficient basis for the high antioxidant capacity of ethyl acetate and n-butanol. In this experiment, the DPPH free radical scavenging rate of the 0.1 mg / mL ethyl acetate sample reached 94.27%, which is roughly consistent with the above experimental results, and also shows that the antioxidant activity of the ethyl acetate part is strong. Since the DPPH scavenging rate of the initial concentration of 0.02 mg / mL is already high, the DPPH scavenging rate of the concentration gradient of 0.02-0.1 mg / mL is not significantly different, and it should be diluted to an appropriate multiple before measurement; In the ABTS scavenging ability experiment, the average total antioxidant capacity of M1, M2, and M3 parts was 29.45mmol / g, 23.02mmol / g, and 3.47mmol / g, respectively, with the Trolox standard sample as the control. Except for the M3 part, the antioxidant capacity of the M1 part and the M2 part increased with the continuous increase of the concentration. Through the analysis of significant differences, it was found that the significant difference between the M1 and M2 samples under the concentration conditions of 0.06mg / mL and 0.10mg / mL was greater than 0.05, and the difference was not significant, while the difference under the other concentration conditions was significant or extremely significant. The spectrophotometric part of some sample solutions in the M1 part was low during the determination, which may be mainly caused by the error factors of the experiment such as reagent contamination and wrong addition of well plate reagent. The significant differences between the M1 part, the M2 part and the M3 part were all significant or extremely significant. In general, the antioxidant capacity of the ethyl acetate samples is higher than that of the n-butanol samples. Zhou Kai

[30] determined in related experiments that the total antioxidant capacity of the ethyl acetate, chloroform, n-butanol and aqueous phases of longan seeds was 3.5, 2.5, 0.9 and 0.3 mmol / g, respectively, confirming the high antioxidant capacity of the ethyl acetate. Yao Zhiren

[31] et al. conducted a study on the antioxidant activity of different polar parts of the yellow water lily, and found that at a concentration of 0.8 mg / mL, the total antioxidant capacity of the ethyl acetate reached 1.284±0.039 mmol / g. This is also much higher than the other polar parts. In this experiment, the total antioxidant capacity of the ethyl acetate part can reach 29.45 mmol / g, which is much higher than the antioxidant capacity of the ethyl acetate part in the above two groups of studies, proving that the antioxidant activity of Moringa leaves is good. In the FRAP total antioxidant capacity experiment, the total antioxidant capacity of the M1, M2, and M3 parts were 35.01mmol / g, 28.02mmol / g, and 4.57mmol / g, respectively. The experimental method requires that the spectrophotometric values ​​of the three groups of samples must be within the range of the Trolox standard curve. Due to the differences in the antioxidant capacity of the three groups of samples, the concentration ranges of the spectrophotometric values ​​of the three groups of samples are different. The three groups of samples at a concentration of 0.04mg / mL were selected for significant difference analysis. The significant difference analysis showed that the differences in the Trolox standard solution concentrations corresponding to the horizontal comparison of the three groups of samples were extremely significant, P<0.01, which fully confirmed that the antioxidant capacity of the ethyl acetate part was significantly higher than that of the n-butanol part, and the antioxidant activity of the two was significantly higher than that of the water part. When Li Kunlong et al.

[32] studied the sea moth extract, the experimental results showed that the antioxidant activity of the ethyl acetate part was slightly higher than that of the n-butanol part. As the active extraction part of the sea moth extract, its antioxidant active components are medium-to-high polar compounds. This conclusion is generally consistent with the results obtained in this experiment, confirming that the antioxidant activities of the ethyl acetate and n-butanol parts are both high, and that compounds with medium and high polarity are easily soluble in these two organic solvents, which is conducive to the further study of polyphenols in Moringa plants. In this experiment, the extract of Moringa leaves was first extracted and purified, and then concentrated by rotary evaporation to obtain three groups of different polar parts, namely ethyl acetate part, n-butanol part and water part, and then freeze-dried into powdered samples for collection. Standard curves of gallic acid and rutin were made, and the contents of total phenols and total flavonoids in the three groups of samples were determined respectively. The antioxidant capacity of the three groups of samples was determined by three methods: DPPH scavenging rate, ABTS scavenging capacity and FRAP total antioxidant capacity. At the same time, the standard deviation of each concentration of the three groups of samples was calculated and a significant difference analysis was performed. The three groups of samples and the original extract of Moringa leaves were analyzed by HPLC chromatography, and the compounds in the three groups of samples were screened out with high antioxidant activity, and the corresponding contents were determined.

[0054] The main conclusions are as follows: 1. The contents of total phenols and total flavonoids in the ethyl acetate part were 24.11 mg / g and 60.68 mg / g, respectively, and those in the n-butanol part were 11.8 mg / g and 43.93 mg / g, which were significantly higher than those in the water part (2.51 mg / g and 2.59 mg / g).

[0055] 2. The order of antioxidant capacity of each polar part is: ethyl acetate part > n-butanol part > water part. Among them, the DPPH scavenging rate of ethyl acetate part can reach 95.04% when the concentration is 0.08 mg / mL. The average total antioxidant capacity determined by ABTS method and FRAP method is 29.45mmol / g and 35.01mmol / g respectively. Through the comparison of significant differences, it can be seen that there is a significant difference between ethyl acetate part and n-butanol part, and there is a very significant difference between ethyl acetate part and water part, and between n-butanol part and water part.

[0056] 3. Chromatographic analysis showed that the ethyl acetate and n-butanol fractions contained high levels of rutin and ferulic acid, reaching 8.199 mg / g, 5.745 mg / g and 13.660 mg / g, 2.172 mg / g, respectively, which were significantly higher than the original extract and water fraction. The polarity of the ethyl acetate fraction is lower than that of the n-butanol fraction, so the solubility of ferulic acid is relatively higher and the solubility of rutin is relatively lower. The contents of chlorogenic acid and caffeic acid in the four groups of samples are relatively low. The caffeic acid content in the organic solvent fraction is lower than that in the original extract, while the chlorogenic acid content is roughly similar.

[0057] This experiment also compared the antioxidant activity of different polar parts of Moringa leaves. By analyzing the components of the ethyl acetate part and the n-butanol part, four polyphenolic compounds, chlorogenic acid, caffeic acid, rutin and ferulic acid, were identified, which will help further study the antioxidant pharmacological activity of Moringa plants. In addition to these four polyphenolic compounds, Moringa leaves also contain a variety of other polyphenolic compounds, such as kaempferol, rhamnetin, quercetin, isoquercetin, vitexin, etc. The antioxidant activity of these polyphenolic compounds needs further analysis and identification. In addition to being a good antioxidant, Moringa plants also have a variety of pharmacological effects such as antibacterial, anti-inflammatory, anti-tumor, lipid-lowering, and blood sugar-lowering. As a plant with both medicinal and edible properties, Moringa plants have great potential in many fields such as food, medicine, cosmetics, and health products, and need further development and research.

[0058] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a Moringa leaf polyphenol extract with anti-tumor activity, characterized in that: The following steps are involved: Step 1, weigh a portion of Moringa leaf extract, take out half of the total Moringa leaf extract in a -25°C to -18°C refrigerator, weigh its mass to obtain 7.5316g, dissolve it in 250mL of distilled water, add 250mL of an equal volume of ethyl acetate, fully oscillate on a constant temperature oscillator for 15min, mix and extract; Step 2: After the shaking is completed, the mixture is allowed to stand for about 30 minutes, and the upper ethyl acetate portion is taken and concentrated by rotary evaporation. When no ethyl acetate is evaporated out, the evaporation is stopped, and the ethyl acetate portion obtained is collected as M1; Step 3: Add an equal volume of anhydrous n-butanol to the lower water portion, perform layered extraction using the above method, collect the obtained n-butanol portion and the remaining water portion, perform rotary evaporation and concentration respectively, and then collect to obtain the n-butanol portion M2 and the water portion M3; Step 4: freeze-dry the three extracted components M1, M2, and M3 in a freeze dryer for 10-16 hours, and then store the samples in a -25°C refrigerator overnight for 10-12 hours. Repeat the steps 4-6 times to obtain three groups of powdered samples. Step 5. Perform total phenol content determination, total flavonoid content determination, DPPH scavenging ability determination, ABTS scavenging ability determination, FRAP total antioxidant capacity determination, and Moringa leaf polyphenol component analysis on the three groups of Moringa leaf extract samples.

2. The method for preparing a Moringa leaf polyphenol extract with anti-tumor activity according to claim 1, characterized in that: The parameters of the oscillator in step 1 are: temperature 26.6° C., rotation speed 200 r / min.

3. The method for preparing a Moringa leaf polyphenol extract with anti-tumor activity according to claim 1, characterized in that: The conditions for evaporation and concentration in step 2 are: temperature 35° C., rotation speed 70 r / min, and pressure 0.09 MPa on a rotary evaporator.

4. The method for preparing a Moringa leaf polyphenol extract with anti-tumor activity according to claim 1, characterized in that: The freeze-drying conditions in step 4 are as follows: temperature below -45°C and vacuum.

5. The method for preparing a Moringa leaf polyphenol extract with anti-tumor activity according to claim 1, characterized in that: The total phenol content determination step in step 5 is: Step I: Take 0.020 g of gallic acid standard, add appropriate amount of distilled water and stir thoroughly until dissolved, stir evenly and dilute to a 10 mL standard volumetric flask, and take 0.0, 0.5, 1.0, 1.5, 2.0, and 3.0 mL of the prepared standard solution and add them to a 10 mL volumetric flask and dilute to volume; Step II, prepare a solution with a concentration gradient of 0, 10, 20, 30, 40, 50, 60 μg / mL, then pipette 1 mL of the above solution into each test tube, add 2 mL of Folin phenol reagent to each test tube, mix well, react for 6 minutes, add 3 mL of 7.5% Na2CO3 solution to each test tube, mix well, and place in a dark place to react for about 60 minutes; Step III, weigh 0.02 g of each of the M1, M2, and M3 samples, take an appropriate amount of distilled water and dilute them to a dissolved state, pour them into a volumetric flask and dilute to 100 mL, prepare a sample solution with a concentration of 0.2 mg / mL, treat it with the above gallic acid preparation method, do three replicates for each sample, measure the ultraviolet absorbance at a wavelength of 765 nm, use distilled water as a blank control, draw a gallic acid standard curve, and measure the actual ultraviolet absorption spectrophotometric value of each sample; The total phenol content was calculated as follows: Where: N: dilution multiple; V: fixed volume of extract (mL); C: polyphenol content of sample solution calculated from standard curve (g / mL); M: weighed mass of Moringa leaf sample (g).

6. The method for preparing a Moringa leaf polyphenol extract with anti-tumor activity according to claim 1, characterized in that: The DPPH scavenging ability determination method in step 5 is: Take 0.05g of each sample M1, M2, M3 and ascorbic acid M4, dissolve in a 50mL volumetric flask and dilute to volume. Prepare a 0.1mg / mL sample solution, dilute to 0.02, 0.04, 0.06, 0.08, 0.10mg / mL sample solution, weigh 0.045g DPPH powder, add anhydrous ethanol to a 100mL volumetric flask and dilute to volume, prepare a 0.045mg / mL DPPH solution, and prepare the solution for determination as shown in the following table; Two parallel samples of A2 mixed solution for each gradient were prepared, and three parallel samples of A3 mixed solution were prepared. After preparation, they were placed on a microplate oscillator for mixing, and then placed in a cabinet to react in the dark for at least 30 minutes, and the absorbance was measured at 517 nm.

7. The method for preparing a Moringa leaf polyphenol extract with anti-tumor activity according to claim 1, characterized in that: The FRAP total antioxidant capacity determination method in step 5 is: The TPTZ diluent and TPTZ solution were first mixed, and then the detection buffer was added to prepare the FRAP working solution. The reagent addition volume and order were as follows: 180 μL of FRAP working solution was added to each detection well of the 96-well plate, and 5 μL of distilled water, various concentrations of FeSO4·H2O standard solutions and three groups of sample solutions were added to the blank control group, standard curve detection group and sample detection group, respectively. The solutions were placed at 37°C for 5 min, and three parallels were made for each sample concentration, and the absorbance was measured at 593 nm.

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