Acanthopanax trifoliatus extract as well as preparation method, application and quality detection method thereof

Abstract

The invention provides an acanthopanax trifoliatus extract, which is prepared by taking fresh acanthopanax trifoliatus as a raw material, and purifying and drying the acanthopanax trifoliatus extract by adopting an enzyme-assisted extraction technology and macroporous adsorption resin. The total amount of chlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C in the extract is not less than 30% w / w, and the mass fraction of total phenolic acid is not less than 55% w / w. The invention further provides a preparation method and application of the acanthopanax trifoliatus extract. The acanthopanax trifoliatus extract is high in content of polyphenols and good in antioxidant activity, and a theoretical basis is provided for development and utilization of acanthopanax trifoliatus.

Description

Technical Field

[0001] The invention relates to a balsam pear extract and a preparation method, application and quality detection method thereof. Background Art

[0002] The scientific name of taro is white taro (Acanthopanax trifoliatus (Linn.) Merr.), which is also known as "goose palm tendons" or "grass palm taro" in Guangdong, "three-leaf taro" in Hunan and Zhejiang, and "three-leaf taro" in Guangxi and Sichuan. It is a climbing shrub of the genus Acanthopanax of the Araliaceae family, distributed in a large area of ​​central and southern my country (Zhang Kun, Cheng Shupeng, Wu Wenjing, et al. Research progress on chemical components, biological activities and safety of taro [J]. Strait Pharmacy, 2017, 29(04): 1-5.). The roots (bark) and leaves of taro are commonly used Chinese herbal medicines. Based on life experience, people in some areas often boil the roots and stems in water for bathing, which is used to treat common skin diseases such as eczema and prickly heat, and the effect is particularly significant. The Ming Dynasty's Food Materia Medica records that "Eupatorium scabra, leaves as vegetables, can cure skin rheumatism". It is good at removing wind and dampness, relaxing muscles and activating blood circulation, reducing swelling and detoxifying. It is often used to treat colds, coughs, rheumatism, sciatica and other diseases (Chen Lanqing, Li Shuzhen, Gan Lishe, et al. Analysis of chemical components of taro based on ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap high-resolution mass spectrometry [J]. Subtropical Plant Science, 2023, 52(04): 301-309.). Modern studies have shown that the rich polyphenols contained in taro are its main active ingredients, such as chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C and other ingredients. These ingredients have shown pharmacological activities in anti-inflammatory, antibacterial, antiviral, immunomodulatory and antioxidant aspects.

[0003] The herb is grown in abundance in Enping, Guangdong. For hundreds of years, local residents have had the custom of using it as both medicine and food. Processed products made from herb have begun to gain popularity in the market. What ideas and methods can be used to quickly and effectively extract the effective substances from herb and maximize the effects of herb has become a hot issue in the development of herb products. The reports on the extraction methods of effective substances from herb in the literature mainly include alcohol extraction (Lao Jinghui, Pan Chaomei, Yu Qin, et al. Study on the inhibition of Propionibacterium acnes and whitening activity of herb extract [J]. Modern Chinese Medicine, 2016, 18(09): 1120-1124.), carbon dioxide supercritical extraction (Yu Jiani, Feng Caixia, Liu Xiangqian, et al. Protective effect of herb total polyphenols on oxidative damage and senescence of PC12 cells induced by Aβ25-35 and AAPH [J]. Journal of Zhejiang Sci-Tech University (Natural Science), 2023, 49(03): 353-358.), ultrasonic extraction (Huang Junsheng. Research on the preparation of Chaozhou cuisine special seasoning by supercritical carbon dioxide extraction of flavonoids from white taro[J]. Chinese Condiment, 2017, 42(05): 57-60.) and microwave extraction (Gao Xia, Li Yunxiang, Cai Lingyun. Research on the extraction process and content determination of total saponins from white taro leaves[J]. Spectral Laboratory, 2009, 26(04): 814-821.) and other methods. For example, the preparation method of taro extract reported in "Comparison of total polyphenol content and antioxidant activity of different varieties of taro" is: "Collect fresh tender branches and leaves of different varieties of taro produced in Enping, dry them at room temperature and then crush them, take out 100g of each, add 75% ethanol by volume for cold soaking extraction, filter every 1d, extract 3 times in total, combine the extracts, and concentrate them with a rotary evaporator to obtain extracts of different varieties of taro", which takes a long time and has a low total polyphenol content of 82.3-112.3mg. GAE / g extract; the preparation method reported in "Process Optimization of Purification of Total Polyphenols from Amur Leaf by Macroporous Adsorption Resin" is "Weigh 1kg into a conical flask, add 95% ethanol 3 times the volume of Amur leaf powder, and then ultrasonically extract, filter the extract, and concentrate under reduced pressure to obtain a paste, repeat the extraction 3 to 4 times, combine and concentrate the obtained extract, add an appropriate amount of 45°C distilled water to dissolve, add 3 times the amount of petroleum ether to repeat the extraction 3 to 4 times, combine the obtained aqueous phases, evaporate and concentrate, and freeze-dry to obtain a crude extract of total polyphenols from Amur leaf. Weigh 3g of crude extract of total polyphenols from Amur leaf, ultrasonically dissolve and fix the volume in a 500mL volumetric flask to prepare a total polyphenol loading solution with a mass concentration of 6mg / mL", and then select HPD100 resin for purification, which is a complicated process. And because the composition of polyphenols is complex, different extraction and purification processes will affect the activity of the final extract. Summary of the invention

[0004] The invention provides a balsam pear extract and a preparation method, application and quality detection method thereof.

[0005] The invention provides a leek extract, which takes fresh leek as a raw material, adopts enzyme-assisted extraction technology, macroporous adsorption resin purification and drying to obtain the leek extract; the total amount of chlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C contained in the extract is not less than 30% w / w, and the mass fraction of total phenolic acid is not less than 55% w / w.

[0006] Among them, the extract contains 12.0% to 16.0% w / w of chlorogenic acid, 0.6% to 0.9% w / w of cryptochlorogenic acid, 2.5% to 4.0% w / w of rutin, 8.0% to 12.0% w / w of isochlorogenic acid A, 0.8% to 1.5% w / w of isochlorogenic acid, and 1.5% to 2.0% w / w of isochlorogenic acid C.

[0007] Wherein, the enzyme is cellulase; the model of the macroporous resin is: HPD-450.

[0008] The present invention also provides a method for preparing the taro extract, which comprises the following steps:

[0009] a. Rinse the freshly harvested arachnoides with clean water, dry it in an oven at 50°C, and then grind it and pass it through a 60-mesh sieve to obtain arachnoides powder;

[0010] b. Weigh the fine powder of leucophylla, add 35-55 times the amount of distilled water, adjust the pH to 4.5-5.5 with 1 mol / L hydrochloric acid, then add 0.4-0.8% cellulase or pectinase, mix well, perform enzymolysis in a 50°C water bath for 60-120 min, and finally inactivate in a 95°C water bath for 10 min, filter to obtain the leucophylla extract; and dry the extract to obtain the leucophylla crude extract;

[0011] b. Take 450 mL of the extract, adjust the pH to 4.0 with 1 mol / L hydrochloric acid, pass it through a chromatography column equipped with 45 mL of HPD-450 macroporous resin at a loading flow rate of 90 mL / h, and then use 60% ethanol for elution in an amount of 180 mL; collect the ethanol elution portion, add anhydrous ethanol to adjust the ethanol concentration to 75%, put it in a 4°C refrigerator for refrigeration, filter it after 16 hours, recover the ethanol under reduced pressure until there is no alcohol taste, and dry it to obtain the taro extract.

[0012] Further preferably, the pH in step b is set to 5.0; and the enzyme is 0.6% cellulase.

[0013] The invention provides use of the balsam pear extract in preparing health food with anti-oxidation effect.

[0014] The present invention provides a method for detecting the quality of the leek extract, which uses HPLC to determine the index components chlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C in the leek extract, and the chromatographic conditions are:

[0015] Japan Shimadzu Shim-pack GTS chromatographic column (4.6×250mm (HSS), 5μm C18); mobile phase: A is 0.2% phosphoric acid aqueous solution, B is acetonitrile; gradient elution program: 0-20min, 10% B; 20-45min, 10%-20% B; 45-70min, 20% B; 70-80min, 20%-10% B; column temperature: 30°C; flow rate: 1mL / min; the mixed reference solution and the test solution were filtered through a 0.22μm microporous filter membrane and injected into the chromatograph for analysis, with an injection volume of 5μL each; the detection wavelength of CA, 4DA, IAB, IAA, and IAC was 325nm, and the detection wavelength of Rut was 254nm.

[0016] Further, it comprises the following steps:

[0017] a. Prepare mixed reference solution:

[0018] Preparation of reference substance stock solution: Accurately weigh appropriate amount of CA, 4DA, Rut, IAB, IAA, and IAC reference substances, dissolve them in 60% ethanol, transfer to a 10 mL volumetric flask, add 60% ethanol to make up to volume, and prepare reference substance stock solutions with a mass concentration of 1 mg / mL respectively;

[0019] Preparation of mixed reference substance stock solution: Accurately pipette the above reference substance stock solution into a 10mL volumetric flask, of which CA, 4DA, Rut, IAB, IAA, and IAC are 3mL, 0.3mL, 0.9mL, 0.5mL, 4mL, and 0.6mL respectively. Add 60% ethanol to dilute to the mark, shake well, and the solution is ready;

[0020] Preparation of mixed reference solution: Accurately pipette 0.5 mL of mixed reference stock solution into a 2 mL volumetric flask and dilute to the mark with 60% ethanol.

[0021] b. Preparation of test solution:

[0022] Weigh 12 mg of taro extract accurately, add 60% ethanol to dissolve by ultrasonication, transfer quantitatively to a 25 mL volumetric flask, dilute to the scale with 60% ethanol, shake well, and obtain.

[0023] The present invention treats the leek with cellulase and purifies it with a macroporous adsorption resin to prepare a leek extract. In order to study the content of active ingredients in the leek extract and evaluate the in vitro antioxidant activity of the leek extract, the present invention uses high performance liquid chromatography to measure polyphenols such as chlorogenic acid (CA), cryptochlorogenic acid (4-Dicaffeoylquinic Acid, 4DA), rutin (Rutin, Rut), isochlorogenic acid A (IAA), isochlorogenic acid B (IAB), and isochlorogenic acid C (IAC) in the leek extract, and evaluates the leek extract's effects on DPPH and ABTS. + The results showed that the content of the six components in the extract of leucophylla was 31.76%, among which chlorogenic acid, isochlorogenic acid A and rutin had the highest content; DPPH and ABTS of the extract of leucophylla + Free radical scavenging ability of IC 50 The values ​​are 13.91μg / mL and 26.22μg / mL respectively. The polyphenol content in the balsam pear extract is high and has good antioxidant activity. The invention provides a theoretical basis for the development and utilization of balsam pear. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 HPLC chromatograms of the test sample (a) and the mixed reference sample (b) of the lycopodiella extract (wherein, 1. chlorogenic acid; 2. isochlorogenic acid; 3. rutin; 4. isochlorogenic acid B; 5. isochlorogenic acid A; 6. isochlorogenic acid C);

[0025] Figure 2 DPPH free radical scavenging ability of balsam pear extract and L-ascorbic acid;

[0026] Figure 3 ABTS+ free radical scavenging capacity of balsam pear extract and L-ascorbic acid. DETAILED DESCRIPTION

[0027] 1 Materials

[0028] 1.1 Materials and reagents

[0029] Acanthopanax trifoliatus, produced in Enping City, Jiangmen City, Guangdong Province, was identified by Associate Professor Wu Weihong of the Department of Traditional Chinese Medicine of Guangdong Jiangmen Vocational College of Traditional Chinese Medicine as the stems and leaves of Acanthopanax trifoliatus (Linn.) Merr. of the Araliaceae family. Chlorogenic acid (CA), 4-Dicaffeoylquinic Acid (4DA), Rutin (Rut), Isochlorogenic acid A (IAA), Isochlorogenic acid B (IAB), Isochlorogenic acid C (IAC) C, IAC), purity of 98%, Shanghai Shifeng Biotechnology Co., Ltd.; anhydrous ethanol of analytical grade, Taishan Yueqiao Reagent Plastic Co., Ltd.; hydrochloric acid and phosphoric acid of analytical grade, Guangzhou Chemical Reagent Factory; HPD-450 macroporous resin, Jiangmen Yichenghua Glass Instrument Co., Ltd.; 0.22 μm microporous filter membrane, Jinlong Company; acetonitrile of chromatographic grade, Germany MREDA Company, water is distilled water, Guangzhou Watsons Food and Beverage Co., Ltd.; 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS), 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH), Shanghai MacLean Biochemical Technology Co., Ltd. Cellulase, enzyme activity 100 000U / g, Shandong Longkote Enzyme Preparation Co., Ltd.; L-ascorbic acid, Xilong Science Co., Ltd.

[0030] 1.2 Instruments and Equipment

[0031] SECURA125-1CN electronic balance, Sartorius Scientific Instruments (Beijing) Co., Ltd.;

[0032] NEXERA XR high performance liquid chromatograph, diode array detector, UV-2600i ultraviolet visible spectrophotometer, Shimadzu Corporation, Japan; HH-600 digital electric constant temperature water bath, Changzhou Aohua Instrument Co., Ltd.; KQ-500DE ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; RV 10auto rotary evaporator, Guangzhou Yike Laboratory Technology Co., Ltd.; BCD-521WKM(E) refrigerator, Midea Group Co., Ltd.

[0033] Example 1 Preparation of balsam pear extract

[0034] After freshly harvested taro is rinsed with clean water, it is placed in an oven at 50°C for drying, then crushed and passed through a 60-mesh sieve to obtain taro powder. Weigh a certain mass of taro fine powder, add 45 times the amount of distilled water, adjust the pH to 5.0 with 1 mol / L hydrochloric acid, then add 0.6% cellulase, mix well, enzymolyze in a 50°C water bath for 60 minutes, and finally inactivate in a 95°C water bath for 10 minutes, and filter to obtain taro extract. After the extract is dried, the crude extract of taro is obtained.

[0035] 450mL of the extract was measured, the pH was adjusted to 4.0 with 1mol / L hydrochloric acid, and the sample was passed through a chromatography column equipped with 45mL HPD-450 macroporous resin at a flow rate of 90mL / h, and then eluted with 60% ethanol in an amount of 180mL. The ethanol elution portion was collected, anhydrous ethanol was added to adjust the ethanol concentration to 75%, and the mixture was placed in a 4°C refrigerator for refrigeration. After 16h, the extraction was performed by suction filtration, and the ethanol was recovered under reduced pressure until there was no alcohol taste, and dried to obtain the araucaria extract. Three batches of araucaria extracts were prepared according to this process, and were marked as No. 202405, No. 202406, and No. 202407.

[0036] Example 2 Parameter screening test of the balsam pear extract of the present invention

[0037] 1. Instrument

[0038] UV-visible spectrophotometer, analytical balance, low-speed centrifuge, constant temperature water bath, pulverizer, pH meter, 10mL brown volumetric flask, 25mL stoppered Erlenmeyer flask, 200mL brown volumetric flask, 50mL beaker.

[0039] 2. Medicines

[0040] Gallic acid standard 100mg, anhydrous sodium carbonate 500g, Folin phenol reagent 100mL, macroporous resin; 0.22μm microporous filter membrane; water is distilled water. Cellulase, enzyme activity 100 000U / g, pectinase, cellulase.

[0041] 3. Experimental steps

[0042] 3.1 Determination of Gallic Acid Content

[0043] 3.1.1 Standard curve drawing

[0044] Accurately weigh 22.3 mg of gallic acid standard, dissolve it in water and dilute it in a 200 mL volumetric flask to obtain a 0.1115 mg / mL standard stock solution. Accurately pipette 0.00, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.8 mL of gallic acid reference solution into 10 mL brown volumetric flasks, add water to 2 mL each, add 0.5 mL of forlin phenol reagent, shake well and let stand for 2 minutes, add 1.5 ml of 20% sodium carbonate solution, add water to the scale, shake well, place in a 75°C water bath to avoid light for 10 minutes, take out, quickly cool to room temperature, use the corresponding blank solvent as a blank, and measure the absorbance (A) at a wavelength of 765 nm. Use the A value as the ordinate and the mass concentration of the gallic acid reference solution as the abscissa to draw a standard curve. The regression equation is: Y = 0.1001X + 0.0232, R 2 =0.9992.

[0045] 3.1.2 Extraction of total phenols from samples

[0046] Take freshly harvested arachnoides, rinse it with clean water, place it in an oven at 55°C to dry, then crush it and pass it through a 60-mesh sieve to obtain white arachnoides leaf powder. Accurately weigh 1.00g of arachnoides powder and place it in a 50mL stoppered conical flask, add distilled water according to the solid-liquid ratio, adjust the pH with 0.5mol / L hydrochloric acid solution or 1mol / L NaOH solution, add a certain amount of enzyme to enzymolyze at a certain temperature, heat it at 95°C for 10min to inactivate the enzyme, centrifuge the obtained extract at 3000r / min for 5min, take the supernatant, and determine the total polyphenol content. The total phenolic acid extraction rate of arachnoides / %=CV / (m×1000)×100, where: C is the concentration of total phenolic acid in the extract, mg / mL; V is the volume of the extract, mL; m is the mass of the arachnoides sample, g.

[0047] 3.1.3 Determination of total phenol content in samples

[0048] Pipette 0.05mL of total polyphenol extract, add water to 2mL, add 0.5mL of Folin-phenol reagent respectively, shake well and let stand for 2min, add 1.5mL of 20% sodium carbonate solution, add water to the scale, shake well, place in a 75℃ water bath to react for 10min in the dark, take out, quickly cool to room temperature, use the corresponding blank solvent as blank, measure the absorbance (A) at a wavelength of 765nm, and calculate the total polyphenol content in the extract according to the working curve.

[0049] 3.2 Single Factor Experiment

[0050] 3.2.1 Effect of single enzyme on the extraction rate of total phenolic acids from baicalensis

[0051] Weigh 1.0g of lansonia truncatum powder, add 0.6% (mass fraction, the same below) of protease, cellulase and pectinase respectively under the conditions of solid-liquid ratio of 1:35 (g / mL) and pH 5, and perform enzymolysis at 50℃ for 90min, and calculate the extraction rate respectively. As shown in Table 1, cellulase and protease have the best effect, followed by pectinase, while protease has basically no effect. (Extraction rate = total phenolic acid mass / lansonia truncatum mass*100%, with gallic acid as the standard substance)

[0052] Table 1 Effects of different enzymes on the extraction rate of total phenolic acids from taro

[0053] Types of enzymes Extraction rate (%) Protease 4.26 Cellulase 5.59 Pectinase 4.83 No enzyme added 4.22

[0054] 3.2.2 Effect of cellulase dosage on the extraction rate of total phenolic acids from taro

[0055] Weigh 1.0g of raphania oleifera powder, add 0.2%, 0.4%, 0.6%, 0.8%, 1.0% (mass fraction) of cellulase at a solid-liquid ratio of 1:35 (g / mL) and pH 5, perform enzymolysis at 50°C for 90 minutes, and calculate the extraction rate. As shown in Table 2, when the amount of cellulase is 0.6%, the extraction rate of total phenolic acids in raphania oleifera is the highest.

[0056] Table 2 Effect of cellulase dosage on the extraction rate of total phenolic acids from taro

[0057] Amount of cellulase (%) Extraction rate (%) 0.2 4.42 0.4 4.75 0.6 5.53 0.8 4.84 1.0 4.53

[0058] 3.2.3 Effect of pH on the extraction rate of total phenolic acids from taro

[0059] Weigh 1.0g of lansonia truncatum powder, the solid-liquid ratio is 1:35 (g / mL), adjust the pH to 4, 4.5, 5.0, 5.5, 6.0, add 0.6% cellulase, and enzymolyze at 50°C for 90 minutes. Calculate the extraction rate. As shown in Table 3, when the pH is 5.0, the extraction rate of total phenolic acids in lansonia truncatum is the highest.

[0060] Table 3 Effect of pH on the extraction rate of total phenolic acids from taro

[0061] pH Extraction rate (%) 4 4.23 4.5 5.06 5.0 5.58 5.5 4.94 6.0 4.69

[0062] 3.2.4 Effect of enzymatic hydrolysis time on the extraction rate of total phenolic acids from taro

[0063] Weigh 1.0g of arachis powder, add 0.6% (mass fraction) of cellulase at a solid-liquid ratio of 1:35 (g / mL) and pH 5, and perform enzymolysis at 50°C for 30, 60, 90, 120, and 150 min, and calculate the extraction rate. As shown in Table 4, when the enzymolysis time is 60 min, the extraction rate of total phenolic acids in arachis is the highest.

[0064] Table 4 Effect of enzymatic hydrolysis time on the extraction rate of total phenolic acids from taro

[0065] Enzymatic hydrolysis time (min) Extraction rate (%) 30 3.42 60 5.67 90 5.56 120 5.24 150 4.83

[0066] 3.2.5 Effect of solid-liquid ratio on the extraction rate of total phenolic acids from basil

[0067] Weigh 1.0g of arachis powder, add 0.6% (mass fraction) of cellulase at a solid-liquid ratio of 1:15, 1:25, 1:35, 1:45, 1:55 (g / mL), pH 5, and perform enzymolysis at 50°C for 60 minutes, and calculate the extraction rate. As shown in Table 5, when the solid-liquid ratio is 1:45 (g / mL), the extraction rate of total phenolic acids in arachis is the highest.

[0068] Table 5 Effect of solid-liquid ratio on the extraction rate of total phenolic acids from taro

[0069] Solid-liquid ratio (g / mL) Extraction rate (%) 1：15 4.02 1：25 4.85 1：35 5.48 1：45 5.68 1：55 5.15

[0070] 3.3 Optimization and verification experiments of extraction process

[0071] According to the results of the single factor test, the optimal extraction process is as follows: freshly harvested taro leaves are rinsed with clean water, dried in an oven at 50°C, then crushed and sieved through a 60-mesh sieve to obtain taro leaves powder. Weigh a certain mass of taro leaves powder, add 45 times the amount of distilled water, adjust the pH to 5.0, then add 0.6% cellulase, mix thoroughly, enzymolyze in a 50°C water bath for 60 minutes, and finally inactivate in a 95°C water bath for 10 minutes, and centrifuge to obtain the taro leaves extract. Three parallel tests were conducted, and the extraction rates were 5.77%, 5.82%, and 5.65%, respectively.

[0072] Characteristics of the extraction method: the solvent is water, the extraction rate is high, the extraction time is short, and the conditions are mild.

[0073] 4. Macroporous adsorption resin purification process

[0074] 4.1 Static adsorption test and desorption test

[0075] (1) Preparation of crude extract

[0076] After freshly harvested taro leaves are rinsed with clean water, placed in an oven at 50°C for drying, then crushed and passed through a 60-mesh sieve to obtain taro leaves powder. Weigh a certain mass of taro leaves powder, add 45 times the amount of distilled water, adjust the pH to 5.0 with 1mol / L hydrochloric acid, then add 0.6% cellulase, mix thoroughly, enzymolyze in a 50°C water bath for 60 minutes, and finally inactivate in a 95°C water bath for 10 minutes, and filter to obtain taro leaves extract. Measure 450mL of the extract, adjust the pH to 4.0 with 1mol / L hydrochloric acid, and set aside.

[0077] (1) Static adsorption: Weigh 1.0 g of each of the 13 pre-treated macroporous resins, place them in a 100 mL conical flask with a stopper, add 25 mL of the total polyphenol crude extract, and plug the flask. Place it in a constant temperature oscillator at 30°C and 100 r / min for full oscillation and adsorption for 24 hours, then filter to obtain the adsorbed solution. Add 25 mL of 70% ethanol to the resin from which the adsorption liquid has been filtered out, fully desorb for 24 hours, and then filter to obtain the desorbed solution. Determine the A value according to the Folin-Ciocalteu method. Then, the total polyphenol content is obtained from the gallic acid standard equation, and according to the formula: adsorption amount = (CV-C1V1) / W, desorption rate = C2V2 /

[0078] (CV-C1V1)×100%, respectively calculate the specific adsorption capacity and desorption rate of different types of resins for total polyphenols, where: C, V are the concentration (mg / mL) and volume (mL) of the solution before adsorption, C1, V1 are the concentration (mg / mL) and volume (mL) of the solution after adsorption, W is the mass (g) of the resin, C2, V2 are the concentration (mg / mL) and volume (mL) of the solution after desorption. As shown in Table 6, the specific adsorption capacity and desorption rate of HPD-450 macroporous resin are the best, and it is preferred for the purification study of total polyphenols in taro.

[0079] Table 6 Adsorption and desorption performance of different types of macroporous resins on total polyphenols of taro

[0080] Serial number Resin Model Resin polarity Specific adsorption capacity (mg / g) Desorption rate (%) 1 HPD-400 Intermediate 13.16 81.87 2 XAD-16N Weak polarity 13.62 84.14 3 HPD-BJQH Intermediate 12.14 82.51 4 DM301 Intermediate 12.01 86.90 5 HPD-826 polarity 13.51 84.11 6 HPD-100 Non-class 11.36 83.11 7 LX-8 polarity 8.96 81.27 8 LSA-10 Weak polarity 15.44 77.91 9 HPD-300 Non-polar 12.59 79.24 10 NKA-II polarity 6.78 71.90 11 HP-20 Non-class 9.67 75.33 12 H103 Non-polar 12.14 86.68 13 HPD-450 Intermediate 16.11 90.02

[0081] 4.2 Single Factor Investigation

[0082] (1) Determination of sample loading amount

[0083] Take the pretreated macroporous resin column (diameter-to-height ratio of 1:9), take the crude extract of taro, adjust the pH to 4.0 with 1 mol / L hydrochloric acid, and -1 (column volume / hour) flow rate was used for dynamic adsorption, and the effluent was collected in sections, with each 2BV (column volume) as one portion. The total polyphenol content in each portion of the effluent was determined and the leakage was calculated. The results are shown in Table 7. It can be seen that when the sample volume reached 10BV, 8.27% of the total polyphenols in the taro leaf had leaked. After comprehensive consideration, the sample volume was determined to be 10BV. The leakage was calculated according to formula (2):

[0084] Calculate. Leakage rate / % = C1V1) / CV×100(2)

[0085] Where: C——mass concentration of total polyphenols in the sample solution, mg / mL;

[0086] V——volume of total polyphenols in the sample solution, mL;

[0087] C1——mass concentration of total polyphenols in the solution after adsorption, mg / mL;

[0088] V1——Volume of total polyphenols in solution after adsorption, mL.

[0089] Table 7 Effect of sample volume on adsorption

[0090]

[0091] (2) Investigation of sample adsorption flow rate

[0092] Take a pretreated macroporous resin column (diameter-to-height ratio of 1:9), measure 10BV of the crude extract of taro, adjust the pH to 4.0 with 1mol / L hydrochloric acid, and load the sample through the resin column at a flow rate of 1BV / h, 2BV / h, 3BV / h, 4BV / h, and 5BV / h, respectively, collect the effluent, determine the content of total polyphenols in each effluent, calculate the adsorption rate, and optimize the sample loading adsorption flow rate. The results are shown in Table 8. It can be seen that when the sample loading flow rate is too fast, the total phenolic acid is not completely adsorbed and the loss increases. Considering the actual production, the preferred sample loading flow rate is 2BV / h. The adsorption rate is calculated according to formula (1).

[0093] Adsorption rate / % = (CV-C1V1) / CV×100(1)

[0094] Where: C——mass concentration of total flavonoids in the sample solution, mg / mL;

[0095] V——the volume of total flavonoids in the sample solution, mL;

[0096] C1——Mass concentration of total flavonoids in the solution after adsorption, mg / mL;

[0097] V1——Volume of total flavonoids in the solution after adsorption, mL.

[0098] Table 8 Effect of sample loading adsorption flow rate on adsorption

[0099]

[0100] (3) Investigation of eluents

[0101] Take 10BV of the extract of arachnoides japonica, adjust the pH to 4.0 with 1mol / L hydrochloric acid, pass through a glass column (diameter-to-height ratio of 1:9) equipped with a macroporous resin at a loading flow rate of 2BV / h, and elute with 2BV of distilled water and 60% ethanol at a flow rate of 2BV / h. Collect the eluate according to the concentration of the ethanol eluent used, and determine the concentration of total polyphenols in the eluate. The results are shown in Table 9. It can be seen that the total polyphenols are mainly concentrated in the 60% ethanol eluent, while water has a weak elution ability for total polyphenols and can remove other impurities. Therefore, it is determined to use water to remove impurities and 60% ethanol as the elution solvent for total polyphenols.

[0102] Table 9 Effect of different elution solvents on the process

[0103]

[0104] (4) Investigation of water washing amount

[0105] Take 10BV of the extract of taro, adjust the pH to 4.0 with 1mol / L hydrochloric acid, pass through a macroporous resin column (diameter-to-height ratio is 1:9) at a loading flow rate of 2BV / h, and elute with distilled water at a flow rate of 2BV / h. Collect 1 portion of the eluate for every 1BV, and detect the carbohydrate compounds in the fraction with molish reagent. The results show that the molish reaction in the 2BV eluate is negative, so the water elution volume is selected as 2BV.

[0106] (5) Investigation of the amount of 60% elution solvent

[0107] Take the pretreated macroporous resin filler and load it into the resin column (diameter-to-height ratio is 1:9), measure 10BV of taro extract, adjust the pH to 4.0 with 1mol / L hydrochloric acid, load the sample at a flow rate of 2BV / h, first use 2BV of distilled water to elute to remove sugars, then elute with 60% ethanol at a flow rate of 2BV / h, collect one portion of eluate per 1BV, determine the concentration of total polyphenols in the eluate, and determine the amount of 60% ethanol eluent. The results show that 4BV of 60% ethanol can basically elute total polyphenols from the resin column.

[0108] Table 10 Effect of 60% ethanol elution dosage on process

[0109]

[0110] 4.3 Process Validation

[0111] The pretreated macroporous resin filler was loaded into a resin column (diameter-to-height ratio of 1:9), 10 BV of the extract of the white balsam pear leaf was measured, the pH was adjusted to 4.0 with 1 mol / L hydrochloric acid, the sample was loaded at a flow rate of 2 BV / h, 2 BV of distilled water was used to elute the sugar first, and then 4 BV of 60% ethanol was used to elute at a flow rate of 2 BV / h. The 60% ethanol eluate was recovered, concentrated and dried to obtain an extract, the mass was weighed, and the content of total phenolic acid in the extract was determined. As a result, the content of total phenolic acid was 55.97%, achieving the purpose of increasing the relative content of effective ingredients.

[0112] Table 11 Process verification results

[0113] batch Mass of balsam pear extract (g) Total phenolic acid content (%) 1 1.33 55.71 2 1.34 56.42 3 1.31 55.78

[0114] Example 3 Determination of the content of active ingredients in the extract of the present invention

[0115] 1. Prepare mixed reference solution

[0116] Preparation of reference stock solution: Accurately weigh appropriate amount of chlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C reference substances, dissolve them in 60% ethanol, transfer them to a 10 mL volumetric flask, add 60% ethanol to make up to volume, and prepare reference stock solutions with a mass concentration of approximately 1 mg / mL.

[0117] Preparation of mixed reference substance stock solution: Accurately pipette the above reference substance stock solution into a 10mL volumetric flask, where chlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C are 3mL, 0.3mL, 0.9mL, 0.5mL, 4mL, and 0.6mL, respectively. Add 60% ethanol to dilute to volume, shake well, and the mixture is ready.

[0118] Preparation of mixed reference solution: Accurately pipette 0.5 mL of mixed reference stock solution into a 2 mL volumetric flask and dilute to the mark with 60% ethanol.

[0119] 2. Preparation of test solution

[0120] Weigh 12 mg of taro extract accurately, add 60% ethanol to dissolve by ultrasonication, transfer quantitatively to a 25 mL volumetric flask, dilute to the scale with 60% ethanol, shake well, and obtain.

[0121] 3. Liquid chromatography conditions

[0122] Japan Shimadzu Shim-pack GTS chromatographic column (4.6×250mm (HSS), 5μm C18); mobile phase: A is 0.2% phosphoric acid aqueous solution, B is acetonitrile; gradient elution program: 0-20min, 10% B; 20-45min, 10%-20% B; 45-70min, 20% B; 70-80min, 20%-10% B; column temperature: 30°C; flow rate: 1mL / min; the mixed reference solution and the test solution were filtered through a 0.22μm microporous filter membrane and injected into the chromatograph for analysis, with an injection volume of 5μL each; the detection wavelength of CA, 4DA, IAB, IAA, and IAC was 325nm, and the detection wavelength of Rut was 254nm.

[0123] 4. Calculation formula

[0124] The mixed standard solution and the test solution were subjected to chromatographic analysis. The mass concentration of each target component was obtained by liquid chromatography software analysis, and the content of each component was calculated according to formula (1).

[0125]

[0126] Where: w i——Contents of the six target ingredients in the sample, %;

[0127] i——corresponds to 6 target components respectively;

[0128] ci——mass concentrations of the six target components calculated by the external standard method, μg / mL;

[0129] v——sample fixed volume, mL;

[0130] m——sample mass, mg.

[0131] 5. Analysis of content in balsam pear extract

[0132] 5.1 Specificity test

[0133] Under the chromatographic conditions of item "3", the mixed reference solution and the extract test solution were measured respectively to obtain the HPLC chromatogram. Figure 1 Under this chromatographic condition, the theoretical plate numbers of the corresponding absorption peaks of chlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C were not less than 10 000, and the separation of two adjacent chromatographic peaks was greater than 1.5.

[0134] 5.2 Investigation of linear relationship

[0135] Accurately aspirate the mixed reference solution of each concentration under item "1", and perform analysis under the chromatographic conditions under item "3". Perform regression analysis with the mixed reference concentration as the horizontal axis (x, μg / mL) and the peak area of ​​the target compound as the vertical axis (y) to obtain the linear regression equations of the six components. The results are shown in Table 12.

[0136] Table 12 Linear relationship of 6 components

[0137]

[0138] 5.3 Precision test

[0139] The same mixed reference solution was taken and injected continuously for 6 times according to the chromatographic conditions under item "3". The peak areas of chlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C were measured. The RSDs were 0.24%, 0.83%, 0.21%, 0.48%, 0.23% and 0.64%, respectively, indicating that the instrument had good precision.

[0140] 5.4 Repeatability test

[0141] Take the same batch (N0.202405) of extracts, prepare 6 test solutions in parallel according to the method under "2", and determine them under the chromatographic conditions of "3". The results show that the average contents of chlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C are 14.53%, 0.78%, 3.38%, 1.21%, 10.07%, and 1.76%, respectively, and the RSDs are 1.24%, 1.74%, 1.26%, 1.68%, 1.85%, and 1.92%, respectively, indicating that the method has good repeatability.

[0142] 5.5 Stability test

[0143] The same sample solution was taken and injected at 0, 2, 4, 8, 16 and 24 h according to the chromatographic conditions under item "3". The peak areas of chlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C were measured. The RSDs were 0.41%, 0.37%, 0.25%, 0.52%, 0.43% and 0.65%, respectively, indicating that the sample solution had good stability within 24 h.

[0144] 5.6 Sample recovery test

[0145] Accurately weigh 6 portions of 12 mg each of the taro extracts with known contents of each component, add appropriate amount of 60% ethanol, ultrasonically dissolve and make up to 25 mL, transfer 12.5 mL respectively into a 25 mL volumetric flask, accurately add a mixed standard solution containing chlorogenic acid (72.8 μg / mL), cryptochlorogenic acid (4 μg / mL), rutin (16.8 μg / mL), isochlorogenic acid B (6.4 μg / mL), isochlorogenic acid A (50.4 μg / mL), and isochlorogenic acid C (8.8 μg / mL) to each portion, dilute to the scale, shake well, and determine according to the chromatographic conditions under item "3" to calculate the average recovery rate. Results The average recoveries of chlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C were 100.44%, 99.58%, 100.91%, 96.11%, 100.37% and 100.46%, respectively, and the RSDs were 1.57%, 2.17%, 2.19%, 1.62%, 1.58% and 1.95%, respectively.

[0146] 5.7 Sample content determination

[0147] Take 3 different batches of lycopodiella extract, prepare the test solution according to the method under "2", determine the peak area under the chromatographic conditions of "3", and calculate the contents of chlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C by the external standard method. The results are shown in Table 13.

[0148] Table 13 Results of determination of the contents of six components in the extract of taro (%, n=3)

[0149]

[0150] The beneficial effects of the present invention are demonstrated by the following efficacy tests.

[0151] Test Example 1 Evaluation of the antioxidant activity of extracts in vitro

[0152] 1. Determination of DPPH free radical scavenging ability

[0153] Weigh 19.84 mg of DPPH powder, add 250 mL of anhydrous ethanol to dissolve, prepare 0.1 mmol / L DPPH working solution, and adjust the absorbance to 1.000 ± 0.002 with anhydrous ethanol before use. Prepare the extract of taro, crude extract of taro, and L-ascorbic acid test sample according to the method under "Example 1", and prepare sample solutions with mass concentrations of 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 32.5, and 35 μg / mL with 60% ethanol before use. During the reaction: add 3 mL of sample group solutions of different concentrations to 10 mL stoppered test tubes, then add 3 mL of DPPH working solution, vortex for 10 seconds to mix evenly, and place in the dark to react for 30 minutes. Anhydrous ethanol was used as the reference solution, and three parallels were set up in each group. The absorbance A2 was measured at 517 nm. At the same time, the blank absorbance of the sample was A0 and the absorbance of the sample itself was A1. The DPPH free radical scavenging rate of each sample group was calculated according to formula (2). The regression equation was fitted with the elimination rate as the ordinate (y) and the sample solution concentration as the abscissa (x), and the IC was calculated according to the fitted equation. 50 .

[0154]

[0155] Where: A0——absorbance after reaction of 3mL 60% ethanol + 3mL DPPH working solution;

[0156] A1——Absorbance after reaction of 3mL sample solution + 3mL DPPH working solution;

[0157] A2——Absorbance after reaction of 3mL sample solution + 3mL anhydrous ethanol.

[0158] 2. ABTS + Determination of free radical scavenging ability

[0159] Preparation of ABTS + Working solution: absorb 7mmol / L ABTS + The solution and 10 mL of 2.45 mmol / L potassium persulfate were mixed, vortexed for 1 min, and placed at room temperature in the dark for 16 h as ABTS +Stock solution. When used, dilute the stock solution with 50 times the amount of anhydrous ethanol to make its absorbance at 734nm 0.800±0.002, which is used as ABTS + Working solution. Prepare the extract of arachnoides, crude extract of arachnoides and L-ascorbic acid as the test sample according to the method in "Example 1". Prepare the sample groups with mass concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg / mL respectively with 60% ethanol before use. During the reaction: add 4 mL ABTS to 10 mL stoppered test tubes respectively. + Solution, then add 1mL of sample group solution of different concentrations, vortex for 10s to mix thoroughly, and place in the dark for 10min. Anhydrous ethanol is used as the reference solution, and 3 parallels are set up for each group. The absorbance value is measured at 734nm. At the same time, the blank absorbance of the sample is A0 and the absorbance of the sample itself is A1. Calculate ABTS according to formula (3) + Clearance rate, with clearance rate as the ordinate (y) and sample solution concentration as the abscissa (x), a regression equation was fitted, and IC was calculated based on the fitted equation 50 .

[0160]

[0161] Where: A0——1mL 60% ethanol + 4mL ABTS + The absorbance of the working solution after reaction;

[0162] A1——1mL sample solution + 4mL ABTS + The absorbance of the working solution after reaction;

[0163] A2——Absorbance after reaction of 1mL sample solution + 4mL anhydrous ethanol.

[0164] 3. Results and Discussion

[0165] 3.1 Analysis of the antioxidant capacity of balsam pear extract in vitro

[0166] 3.1.1 DPPH free radical scavenging ability

[0167] like Figure 2 As shown in the figure, when the mass concentration was 2.5-27.5 μg / mL, the DPPH free radical scavenging rate of the extract of taro leaf increased with the increase of sample mass concentration, from 10.87% to 92.28%, and the fitted linear regression equation was y=3.277 7x+4.101 4, R 2=0.996 4. In addition, at the same concentration, the DPPH radical scavenging ability of the arachnoides extract was higher than that of the arachnoides crude extract, but weaker than that of L-ascorbic acid. Under the experimental conditions, when the concentration of L-ascorbic acid reached 7.50 μg / mL, it was observed that the DPPH radical scavenging rate had significantly increased to more than 96%, and then the overall trend tended to be stable with the further increase of concentration. By fitting the linear regression equation, the relationship between the mass concentration of the arachnoides crude extract and the scavenging rate was obtained as y=1.656 1x+1.980 5(2.5~35 μg / mL, R 2 =0.996 3), the relationship between the mass concentration of L-ascorbic acid and the clearance rate is y = 13.909x-8.4018 (2.5-7.5 μg / mL, R 2 =0.998 1), after calculation, the IC values ​​of DPPH free radical scavenging rate of balsam pear extract, crude extract and L-ascorbic acid were obtained. 50 The values ​​were 13.91, 28.99, and 4.20 μg / mL, respectively. 50 The IC 50 The value was significantly lower than that of the crude extract, indicating that some inactive or low-active components were removed during the purification process, thereby improving its scavenging efficiency for DPPH free radicals, which fully illustrates the importance of the purification step in improving the antioxidant properties of natural products.

[0168] 3.1.2ABTS + Free radical scavenging ability

[0169] like Figure 3 As shown in Figure 2, at the same mass concentration, the extract of balsam pear had an effect on ABTS. + The free radical scavenging ability of the extract was significantly stronger than that of the crude extract. The scavenging rates of both increased steadily with the increase of mass concentration, and the growth trend was similar. This finding highlights the importance of the purification process in improving antioxidant activity. In addition, when the mass concentration of the taro extract was 50 μg / mL, its ABTS + The clearance rate exceeded 90%, which fully proved the excellent efficacy of the leucophylla extract as a potential antioxidant. By fitting the linear regression equation, the relationship between the mass concentration of the leucophylla extract and the clearance rate was obtained as y = 1.858 5x + 1.263 9 (5 ~ 50 μg / mL, R 2 =0.995 3), the relationship between L-ascorbic acid mass concentration and clearance rate is y = 4.6092x + 8.3791 (5-20 μg / mL, R 2 =0.9927). Under this test system, the ABTS of the oleander extract + Free radical scavenging rate IC 50 is 26.22 μg / mL, while the IC50 Although the scavenging ability of balsam pear extract is lower than that of L-ascorbic acid, it is still worthy of further research and development considering that it comes from natural plants and has higher biosafety.

[0170] 4. Conclusion

[0171] The present invention prepares a leucophylla extract, and determines six active ingredients in the extract by high performance liquid chromatography, including chlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C. It is found that the total content of the six active ingredients in the leucophylla extract is 31.76%, among which chlorogenic acid, isochlorogenic acid A and rutin have the highest contents, which are 14.53%, 10.06% and 3.39% respectively, indicating that these ingredients occupy an important position in the leucophylla extract. In addition, the leucophylla extract has high DPPH and ABTS + In free radical scavenging tests, they showed low IC 50 The value further confirms its potential as a natural potential antioxidant. Future research can further explore other biological activities and mechanisms of action of taro extract to promote its wider and deeper development and utilization.

[0172] Experimental Example 2 Comparison of in vitro antioxidant activity of the extract obtained by the present invention and the extract reported in the literature

[0173] Literature reports extract preparation: weigh 100g of white balsam leaf powder in a conical flask, add 3 times the volume of 95% ethanol and ultrasonically extract, filter the extract, and concentrate under reduced pressure to obtain a paste. Repeat the extraction 3 to 4 times, combine and concentrate the extract, add an appropriate amount of 45℃ distilled water to dissolve, add 3 times the amount of petroleum ether (30~60℃) and repeat the extraction 3 to 4 times, combine the obtained aqueous phases, concentrate under reduced pressure, and freeze-dry to obtain the crude extract of total polyphenols from white balsam leaf. Take 300mg of the crude extract of total polyphenols from white balsam leaf, add water to dissolve into a 1.0mg / mL solution, adjust the pH value to 3.0, and apply it to the treated HPD100 resin bed (resin dosage 40g), and load the sample volume flow at a volume flow of 20mL / min. The column is 300mL. Then rinse with water and then elute the macroporous resin with 400 mL of 50% ethanol solution at pH 6.0. The elution volume flow is also 20 mL / min. The 60% ethanol eluate is collected, concentrated under reduced pressure, and freeze-dried to obtain the polyphenol extract of white balsam pear leaves. (Cen Yesheng, Li Xiaolong, Chen Shujuan, et al. Process optimization of purification of total polyphenols of white balsam pear leaves by macroporous adsorption resin [J]. Chinese Herbal Medicine, 2019, 50(13): 3071-3076.)

[0174] 1. DPPH free radical scavenging ability

[0175] The experimental results are shown in Table 14.

[0176] Table 14 Comparison of DPPH free radical scavenging ability

[0177] Extract of the present invention Literature reference extract <![CDATA[IC 50 ]]> 13.91 μg / mL 19.06 μg / mL

[0178] 2. ABTS + Free radical scavenging ability

[0179] The experimental results are shown in Table 15.

[0180] Table 15 ABTS + Comparison of free radical scavenging ability

[0181] Extract of the present invention Literature reference extract <![CDATA[IC 50 ]]> 26.22 μg / mL 33.15 μg / mL

[0182] Conclusion: The in vitro antioxidant effect of the taro extract prepared by the present invention is significantly stronger than that of the reference extract in the literature.

Claims

1. A balsam pear extract, characterized in that: The invention uses fresh leek as raw material, adopts enzyme-assisted extraction technology, macroporous adsorption resin purification and drying to obtain leek extract; the total amount of chlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C contained in the extract is not less than 30% w / w, and the mass fraction of total phenolic acid is not less than 55% w / w.

2. The oleander extract according to claim 1, characterized in that: The extract contains 12.0% to 16.0% w / w of chlorogenic acid, 0.6% to 0.9% w / w of cryptochlorogenic acid, 2.5% to 4.0% w / w of rutin, 8.0% to 12.0% w / w of isochlorogenic acid A, 0.8% to 1.5% w / w of isochlorogenic acid, and 1.5% to 2.0% w / w of isochlorogenic acid C.

3. The oleander extract according to claim 1 or 2, characterized in that: The enzyme is cellulase; the model of the macroporous resin is HPD-450.

4. A method for preparing the oleander extract according to any one of claims 1 to 3, characterized in that: It includes the following steps: a. Rinse the freshly harvested arachnoides with clean water, dry it in an oven at 50°C, and then grind it and pass it through a 60-mesh sieve to obtain arachnoides powder; b. Weigh the fine powder of leucophylla, add 35-55 times the amount of distilled water, adjust the pH to 4.5-5.5 with 1 mol / L hydrochloric acid, then add 0.4%-0.8% cellulase or pectinase, mix well, perform enzymolysis in a 50°C water bath for 60-120 min, and finally inactivate in a 95°C water bath for 10 min, filter to obtain the leucophylla extract; dry the extract to obtain the leucophylla crude extract; c. Take 450 mL of the extract, adjust the pH to 4.0 with 1 mol / L hydrochloric acid, pass it through a chromatography column equipped with 45 mL of HPD-450 macroporous resin at a loading flow rate of 90 mL / h, and then use 60% ethanol for elution in an amount of 180 mL; collect the ethanol elution portion, add anhydrous ethanol to adjust the ethanol concentration to 75%, put it in a 4°C refrigerator for refrigeration, filter it after 16 hours, recover the ethanol under reduced pressure until there is no alcohol taste, and dry it to obtain the taro extract.

5. The method for preparing the oleander extract according to claim 4, characterized in that: The pH in step b is adjusted to 5.0; the enzyme is 0.6% cellulase.

6. Use of the oleander extract according to any one of claims 1 to 3 in the preparation of health food having an antioxidant effect.

7. A quality detection method for the balsam pear extract according to any one of claims 1 to 3, characterized in that: It uses HPLC to determine the index components chlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C in the extract of taro. The chromatographic conditions are: Shimadzu Shim-pack GTS chromatographic column (4.6×250 mm (HSS), 5 μm C18); mobile phase: A is 0.2% phosphoric acid aqueous solution, B is acetonitrile; gradient elution program: 0-20 min, 10% B; 20-45 min, 10%-20% B; 45-70 min, 20% B; 70-80 min, 20%-10% B; Column temperature: 30°C; flow rate: 1 mL / min; the mixed reference solution and test solution were filtered through a 0.22 μm microporous filter membrane and injected into the chromatograph for analysis, with an injection volume of 5 μL each; the detection wavelength of chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C was 325 nm, and the detection wavelength of rutin was 254 nm.

8. The quality detection method of the balsam pear extract according to claim 7, characterized in that: It includes the following steps: a. Prepare mixed reference solution: Preparation of reference stock solution: accurately weigh appropriate amount of chlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C reference substances, dissolve them in 60% ethanol, transfer to a 10 mL volumetric flask, add 60% ethanol to make up to volume, and prepare reference stock solutions with a mass concentration of 1 mg / mL respectively; Preparation of mixed reference stock solution: Accurately pipette the above reference stock solution into a 10mL volumetric flask, of which chlorogenic acid, cryptochlorogenic acid, rutin, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C are 3mL, 0.3mL, 0.9mL, 0.5mL, 4mL, and 0.6mL, respectively; add 60% ethanol to dilute to the mark, shake well, and obtain; Preparation of mixed reference solution: Accurately pipette 0.5 mL of mixed reference stock solution into a 2 mL volumetric flask and dilute to the mark with 60% ethanol. b. Preparation of test solution: Weigh 12 mg of arachis japonica extract, accurately weigh it, add 60% ethanol to dissolve it by ultrasonication, transfer it quantitatively to a 25 mL volumetric flask, dilute it to the scale with 60% ethanol, shake well, and obtain it.

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