阅读说明：本技术 应用tlc-cms技术评价米糠中生物碱抗氧化性的方法 (Method for evaluating oxidation resistance of alkaloid in rice bran by TLC-CMS technology ) 是由 杨卫民 杜京旗 陈丹 秦永其 赵青红 张利军 于 2019-09-26 设计创作，主要内容包括：本发明方法是应用TLC-CMS技术评价米糠中生物碱的抗氧化性。以米糠为原料,以70%乙醇为提取剂,料液比为1:20,经微波超声波萃取仪辅助提取获得生物碱粗提取物。通过薄层色谱对生物碱进行分离纯化并测定其含量,运用质谱仪对其分子信息进行检测,紫外测定其清除DPPH、羟基自由基的能力,最后应用荧光光度法进一步证明其抗氧化性。薄层层析可快速从米糠中分离纯化生物碱,双波长薄层扫描法测定生物碱含量简便且重现性较好。通过紫外和荧光光度法测定其对DPPH、羟基自由基的抗氧化能力所得结论是一致的。(The method of the invention is to apply TLC-CMS technology to evaluate the oxidation resistance of the alkaloid in the rice bran. Taking rice bran as a raw material, taking 70% ethanol as an extracting agent, and obtaining an alkaloid crude extract by the auxiliary extraction of a microwave ultrasonic extractor, wherein the material-liquid ratio is 1: 20. Separating and purifying alkaloid by thin layer chromatography, measuring the content of alkaloid, detecting the molecular information by using a mass spectrometer, measuring the ability of eliminating DPPH and hydroxyl free radicals by using ultraviolet, and finally further proving the oxidation resistance by using a fluorescence photometry. The thin layer chromatography can quickly separate and purify the alkaloid from the rice bran, and the double-wavelength thin layer scanning method for measuring the alkaloid content is simple and convenient and has better reproducibility. The results of measuring the antioxidant capacity of the compound to DPPH and hydroxyl radicals by ultraviolet and fluorescence photometry are consistent.)

1. A method for evaluating the oxidation resistance of alkaloid in rice bran by using a TLC-CMS technology is characterized by comprising the following steps: the method comprises the following steps:

(1) crude extraction of alkaloid from rice bran

1.1, raw material treatment: drying testa oryzae, and pulverizing;

1.2, ethanol water extraction: weighing testa oryzae in a conical flask, adding 70% ethanol-water solution, stirring to form homogenate, and placing into a gas bath constant temperature oscillator for oscillation for 24 hr at 45 deg.C;

1.3, auxiliary treatment of an ultrasonic microwave extractor: extracting the suspension with ultrasonic microwave assisted extraction instrument for three stages, each for 10min and 30 min; the specific parameters are as follows: under the condition of constant ultrasonic waves, setting the temperature of each stage to be 40 ℃, 45 ℃ and 50 ℃, setting the ultrasonic power to be 800W, 900W and 1000W, setting the microwave power to be 150W, 200W and 250W, setting the ultrasonic frequency to be 50KHz, setting the mode to be 15:10, and setting the motor rotating speed to be 930r/min, and finally forming homogenate;

1.4, centrifugal filtration: centrifuging the suspension in a low-speed centrifuge at 4000r/min for 10min to obtain supernatant and filter residue, mixing all supernatant, filtering, and collecting and weighing all filter residues;

1.5, concentrating and drying: placing the supernatant in a round-bottom flask of a rotary evaporator, opening a condenser pipe, setting the water bath temperature at 79 ℃, performing rotary evaporation of ethanol until no liquid is evaporated during condensation, and recovering ethanol;

1.6, acidifying and extracting, namely pouring the sample liquid subjected to rotary evaporation into a conical flask, adding HCl with the mass fraction of 2% to acidify to the pH value of 2 ~ 3, pouring the acidified filtrate into a separating funnel, adding equal volume of chloroform for extraction, fully shaking up, standing for 10min, collecting a lower chloroform layer, adding equal volume of chloroform for extraction into an upper layer of solution, extracting for three times, and collecting an upper acidic aqueous solution for later use;

alkalizing and extracting, namely alkalizing an acidic aqueous solution, alkalizing the acidic aqueous solution to the pH value of 9 ~ 10 with NaOH with the mass fraction of 5%, adding chloroform, namely methanol =3:1 (v/v) into the obtained solution, extracting, taking a chloroform methanol phase, adding equal volume of chloroform and methanol solution into an upper layer of alkaline aqueous solution, extracting for three times, and collecting the upper layer of alkaline aqueous solution;

1.8, evaporation concentration and volume fixing: evaporating and concentrating the chloroform-methanol layer liquid by a rotary evaporator until the chloroform-methanol layer liquid is dried, calculating the extraction rate, and fixing the volume of the evaporated and dried substance to 25mL by using chromatographic methanol;

(2) preparation of standard substance

Precisely weighing an evodiamine standard substance, placing the evodiamine standard substance in a volumetric flask which is cleaned by ultrasonic elution, adding chromatographic methanol to scale positions, dissolving under an ultrasonic condition to obtain 1mg/mL of evodiamine standard substance solution, and placing the evodiamine standard substance solution in a refrigerator at the temperature of 2 ~ 4 ℃ for storage for later use;

(3) separating and purifying by thin layer chromatography

3.1, spotting: sucking 25 mul of alkaloid reference substance solution by using a microsyringe, placing on a full-automatic sample application machine, and setting sample application parameters: the number of sample application is 3, the sample application amount is 5 muL, 7 muL, 9 muL respectively, sample application is carried out on the thin layer chromatography silica gel plate film, then 25 muL of sample solution is absorbed, the number of sample application is 3, and the sample application amount is 10 muL;

3.2, unfolding: preparing a mixed solution of methanol, chloroform, benzene and petroleum ether =1:14:2:8 (v/v) in a chromatographic cylinder as a developing agent of alkaloid in rice bran, putting the spotted silica gel plate into the chromatographic cylinder after the developing agent is saturated, and taking out the silica gel plate to dry when the front edge of the developing agent is 1cm away from the top end of the silica gel plate;

3.3, thin layer chromatography imaging system recording: placing the dried silica gel plate into a thin-layer chromatography imaging system, taking a picture under a fluorescent lamp of 254nm to observe the chromatography condition, and taking a picture for recording;

3.4, measuring the content by a thin-layer chromatography scanner: scanning the photographed and recorded thin-layer plate by using a scanner, determining spot position parameters by tracking tracks, firstly performing spectral scanning to determine the maximum and minimum absorption peak values of spots, then determining the peak area of a target spot by using reflection scanning, and determining the content of a target spot substance by using the linear relation between the peak area and the sample application amount;

(4) mass spectrometry

4.1, mass spectrum detection of the standard: chromatographic methanol and ultrapure water are used as mobile phases; opening the instrument and the software to enable the instrument and the software to be in a working state, sucking a standard solution by a microsyringe, inserting the standard solution into a sample inlet, starting sample injection when a Load file is reached, and returning to an inject file after the sample injection is finished; analyzing and storing the spectrogram by a point-opening spectrogram interface;

4.2, detecting the mass spectrum of the solution to be detected: the mobile phase is the same as that of the standard substance; placing the thin-layer plate to be detected in a TLC-mass spectrum interface, aligning laser to the center of a circled alkaloid spot, sampling by software operation, and analyzing and storing a spectrogram by a spot spectrography interface;

(5) clearance calculation

5.1, determination of DPPH free radical scavenging ability: taking several test tubes, accurately sucking 0.1mL of alkaloid sample solution with concentration of 100. mu.g/mL, 80. mu.g/mL, 60. mu.g/mL, 40. mu.g/mL and 20. mu.g/mL, respectively adding 3.50mL of 0.06mmol/L DPPH solution into 5 test tubes, strictly keeping out of light for 30min, measuring absorbance at 515nm, and recording as A_i(ii) a Taking 5 test tubes again and sucking0.1mL of sample extract of the same concentration was added to each of 3.50mL of methanol solutions, and the absorbance was measured and recorded as A_j(ii) a Then taking 1 test tube to accurately absorb 3.50mL of DPPH solution, adding 0.10mL of methanol solution, measuring the light absorption value, and recording as A₀；

Preparing the same concentration gradient V as the sample extract_CAnd V_EMethanol solution as experimental control group; taking the average value of the absorbance values after three times of measurement;

clearance S = [ 1- (a)_i－A_j)/A₀]×100%；

5.2, determination of hydroxyl radical scavenging capacity: 2.0mL of 9mmol/L FeSO was taken₄Solution, 2.0mL of 8.8mmol/L H₂O₂The solution is reacted for 15min at 37 ℃, and the absorbance of the solution is recorded as A when measured at 536nm₀Then adding 1mL of alkaloid sample solution of 100 mu g/mL, 80 mu g/mL, 60 mu g/mL, 40 mu g/mL and 20 mu g/mL respectively, and measuring the absorbance at 536nm, and recording as Ax; preparing the same concentration gradient V as the sample extract_CAnd V_EMethanol solution as experimental control group; taking the average value of the absorbance values after three times of measurement;

clearance S = (a)₀－Ax)/A₀×100%；

(6) The oxidation resistance of the product is further proved by a fluorescence photometry

Scanning the extractive solution with ultraviolet under a fluorescence photometer, setting wavelength at 515nm, determining whether new substances are generated according to the number of peaks and the position of maximum fluorescence value, and evaluating oxidation resistance of alkaloid according to the change of fluorescence value difference before and after fluorescence scanning and adding equal amount of DPPH solution.

Technical Field

The invention relates to the field of alkaloid detection, in particular to a method for evaluating the oxidation resistance of alkaloid in rice bran by applying a TLC-CMS (thin layer chromatography-sodium content management system) technology.

Background

Currently, Lvliang, Xinzhou and Changzhi are major millet producing areas in Shanxi province, the millet growing area is about 150 ten thousand mu, the by-product after husking the millet in the millet production process is rice bran which is 5% ~ 7% of the quality of the millet, the rice bran contains a plurality of physiologically active substances such as vitamin B, tocopherol, tocotrienol, polyphenol compounds, oryzanol, flavonoids compounds, sterol, ceramide, alkaloid and the like, and the substances have certain diagnosis and treatment effects on certain diseases.

Alkaloids are a class of basic organic compounds containing nitrogen, have properties similar to those of bases, and are bitter and astringent. The rice bran is known as a "Tiancio nutrient source", but the rice bran is used as feed and even burned in a large scale, which causes resource waste and environmental pollution, so the rice bran should be developed and utilized. The rice bran contains alkaloid, and the alkaloid has pharmacological effect. Some alkaloids have strong practicability in the aspects of biological pharmacy, sanitation and the like, and have the effects of resisting oxidation, viruses and tumors, so that the alkaloids are gradually the objects of intensive research of people.

Many data and researches show that the prior methods for extracting the alkaloid are commonly used such as a methanol cold-soaking extraction method, an ethanol reflux extraction method, an ultrasonic-assisted extraction method, a macroporous resin adsorption method and the like. However, with the development of technology in recent years, many scholars have made intensive studies on the extraction and content determination of alkaloids, such as: thin layer chromatography is used by Baimai Muslim et al to analyze and separate alkaloid components in the lima beans; lixueli et al identified the alkaloid active ingredients in Sophora alopecuroides extract by thin layer chromatography. Many reports have been made on the research of alkaloids, most of them are the research of extraction process, the physiological activities of alkaloids are various, and the antioxidant effect of alkaloids shows wide application prospect, but the research on the separation and purification of alkaloids in rice bran and the antioxidant effect thereof has not been seen.

Disclosure of Invention

On the basis of the previous research, the invention tries to adopt an ultrasonic microwave-assisted extraction method to carry out crude extraction on the alkaloid in the rice bran, uses a TLC (thin layer chromatography) technology to separate and purify the alkaloid in the rice bran, uses a mass spectrometer to detect the molecular information of the alkaloid and evaluates the oxidation resistance of the alkaloid.

The invention is realized by adopting the following technical scheme:

a method for evaluating oxidation resistance of alkaloid in rice bran by TLC-CMS technology comprises the following steps:

(1) crude extraction of alkaloid from rice bran

1.1, raw material treatment: drying testa oryzae, and pulverizing.

1.2, ethanol water extraction: weighing testa oryzae in a conical flask, adding 70% ethanol-water solution, stirring to form homogenate, and placing into a gas bath constant temperature oscillator for oscillation for 24 hr at 45 deg.C.

1.3, auxiliary treatment of an ultrasonic microwave extractor: extracting the suspension with ultrasonic microwave assisted extraction instrument for three stages, each for 10min and 30 min; the specific parameters are as follows: under the condition of constant ultrasonic waves, the temperature of each stage is respectively set to be 40 ℃, 45 ℃ and 50 ℃, the ultrasonic power is set to be 800W, 900W and 1000W, the microwave power is set to be 150W, 200W and 250W, the ultrasonic frequency is 50KHz, the mode is 15:10, and the motor rotating speed is 930r/min, and finally homogenate is formed.

1.4, centrifugal filtration: centrifuging the suspension in a low-speed centrifuge at 4000r/min for 10min, repeating for three times to obtain supernatant and filter residue, mixing all supernatant, filtering, collecting and weighing all filter residues.

1.5, concentrating and drying: and (3) placing the supernatant in a round-bottom flask of a rotary evaporator, opening a condensation pipe, setting the water bath temperature to be 79 ℃, performing rotary evaporation on the ethanol until no liquid is evaporated during condensation, and recovering the ethanol in a receiving flask for reuse.

And 1.6, acidifying and extracting, namely pouring the sample liquid subjected to rotary evaporation into a conical flask, adding 2 mass percent of HCl to acidify to the pH value of 2 ~ 3, pouring the acidified filtrate into a separating funnel, adding equal volume of chloroform for extraction, fully shaking up, standing for 10min, collecting a lower chloroform layer, adding equal volume of chloroform for extraction into an upper layer of solution, extracting for three times, and collecting an upper layer of acidic aqueous solution for later use.

Alkalizing and extracting, namely alkalizing an acidic aqueous solution, alkalizing the acidic aqueous solution to the pH value of 9 ~ 10 with 5% NaOH by mass fraction, adding chloroform to methanol =3:1 (v/v) into the obtained solution, extracting, taking a chloroform methanol phase, adding equal volume of chloroform and methanol solution into an upper layer of alkaline aqueous solution, extracting for three times, and collecting the upper layer of alkaline aqueous solution.

1.8, evaporation concentration and volume fixing: and (3) evaporating and concentrating the chloroform-methanol layer liquid by a rotary evaporator until the chloroform-methanol layer liquid is dried, calculating the extraction rate, and fixing the volume of the evaporated and dried substance to 25mL by using chromatographic methanol.

(2) Preparation of standard substance

Precisely weighing an evodiamine standard substance, placing the evodiamine standard substance in a volumetric flask which is cleaned by ultrasonic elution, adding chromatographic methanol to scale positions, dissolving under an ultrasonic condition to obtain a 1mg/mL evodiamine standard substance solution, and placing the solution in a refrigerator at 2 ~ 4 ℃ for storage for later use.

(3) Separating and purifying by thin layer chromatography

3.1, spotting: sucking 25 mul of alkaloid reference substance solution by using a microsyringe, placing on a full-automatic sample application machine, and setting sample application parameters: the number of sample application is 3, the sample application amount is 5 muL, 7 muL, 9 muL, sample application is carried out on thin layer chromatography silica gel plate film, then 25 muL of sample solution is absorbed, the number of sample application is 3, and the sample application amount is 10 muL.

3.2, unfolding: preparing a mixed solution of methanol, chloroform, benzene and petroleum ether =1:14:2:8 (v/v) in a chromatographic cylinder as a developing agent of alkaloid in rice bran, putting the spotted silica gel plate into the chromatographic cylinder after the developing agent is saturated, and taking out the silica gel plate to dry when the front edge of the developing agent is 1cm away from the top end of the silica gel plate.

3.3, thin layer chromatography imaging system recording: and (4) placing the dried silica gel plate into a thin-layer chromatography imaging system, taking a picture under a fluorescent lamp of 254nm to observe the chromatography condition, and taking a picture for recording.

3.4, measuring the content by a thin-layer chromatography scanner: scanning the photographed and recorded thin-layer plate by using a scanner, determining spot position parameters by tracking tracks, firstly performing spectral scanning to determine the maximum and minimum absorption peak values of spots, then determining the peak area of a target spot by using reflection scanning, and determining the content of a target spot substance by using the linear relation between the peak area and the sample application amount.

(4) Mass spectrometry

4.1, mass spectrum detection of the standard: chromatographic methanol and ultrapure water are used as mobile phases; opening the instrument and the software to enable the instrument and the software to be in a working state, sucking a standard solution by a microsyringe, inserting the standard solution into a sample inlet, starting sample injection when a Load file is reached, and returning to an inject file after the sample injection is finished; and analyzing and storing the spectrogram by a point-opening spectrogram interface.

4.2, detecting the mass spectrum of the solution to be detected: the mobile phase is the same as that of the standard substance; and (3) enabling the instrument and the software to be in a working state, placing the thin-layer plate of the product to be detected on a TLC-mass spectrum interface, aligning laser to the center of the circled alkaloid spot, sampling by software operation, and analyzing and storing the spectrogram by the spot spectrography interface.

(5) Clearance calculation

5.1, determination of DPPH free radical scavenging ability: taking several test tubes, accurately sucking 0.1mL of alkaloid sample solution with concentration of 100. mu.g/mL, 80. mu.g/mL, 60. mu.g/mL, 40. mu.g/mL and 20. mu.g/mL, respectively adding 3.50mL of 0.06mmol/L DPPH solution into 5 test tubes, strictly keeping out of light for 30min, measuring absorbance at 515nm, and recording as A_i(ii) a Taking 5 test tubes to absorb 0.1mL of sample extracting solution with different concentrations, adding 3.50mL of methanol solution respectively, measuring light absorption value, and recording as A_j(ii) a Then taking 1 test tube to accurately absorb 3.50mL of DPPH solution, adding 0.10mL of methanol solution, measuring the light absorption value, and recording as A₀. Equipment and sampleV of the same concentration gradient of the extract_CAnd V_EMethanol solution as experimental control group; taking the average value of the absorbance values after three times of measurement;

clearance S = [ 1- (a)_i–A_j)/A₀]×100%。

5.2, determination of hydroxyl radical scavenging capacity: 2.0mL of 9mmol/L FeSO was taken₄Solution, 2.0mL of 8.8mmol/L H₂O₂The solution is reacted for 15min at 37 ℃, and the absorbance of the solution is recorded as A when measured at 536nm₀Then adding 1mL of alkaloid sample solution of 100 mu g/mL, 80 mu g/mL, 60 mu g/mL, 40 mu g/mL and 20 mu g/mL respectively, and measuring the absorbance at 536nm, and recording as Ax; preparing the same concentration gradient V as the sample extract_CAnd V_EMethanol solution as experimental control group; taking the average value of the absorbance values after three times of measurement;

clearance S = (a)₀–Ax)/A₀×100%。

(6) The oxidation resistance of the product is further proved by a fluorescence photometry

Scanning the extractive solution with ultraviolet under a fluorescence photometer, setting wavelength at 515nm, determining whether new substances are generated according to the number of peaks and the position of maximum fluorescence value, and evaluating oxidation resistance of alkaloid according to the change of fluorescence value difference before and after fluorescence scanning and adding equal amount of DPPH solution.

The method provided by the invention adopts a TLC-CMS technology to evaluate the oxidation resistance of the alkaloid in the rice bran. Taking rice bran as a raw material, taking 70% ethanol as an extracting agent, and obtaining an alkaloid crude extract by the auxiliary extraction of a microwave ultrasonic extractor, wherein the material-liquid ratio is 1: 20. Separating and purifying alkaloid by thin layer chromatography, measuring the content of alkaloid, detecting the molecular information by using a mass spectrometer, measuring the ability of eliminating DPPH and hydroxyl free radicals by using ultraviolet, and finally further proving the oxidation resistance by using a fluorescence photometry. The results obtained were examined for R of the sample and the alkaloid control by developing on a thin layer chromatography silica gel plate using methanol, chloroform, benzene, petroleum ether =1:14:2:8 (v/v) as the optimum developing agent and detecting the result at 254nm_fThe values are all around 0.8, and all appear as blue-purple spots. Reflective sawtoothType scanning display, lambda_SIs 224nm, lambda_RThe peak area integral value and the content have good linear relation (r = 0.9986) at 700nm, and the average content of the test sample measured by an external standard two-point method is 0.5509%. And through mass spectrum detection, the test sample and the standard product both contain the same ion fragment peak. The test sample liquid has the effect of removing DPPH and hydroxyl free radicals, wherein the removing capacity of the hydroxyl free radicals is relatively high, and the removing capacity is as follows: sample liquid>V_C>V_E. And (4) conclusion: the thin layer chromatography can quickly separate and purify the alkaloid from the rice bran, and the double-wavelength thin layer scanning method for measuring the alkaloid content is simple and convenient and has better reproducibility. The results of measuring the antioxidant capacity of the compound to DPPH and hydroxyl radicals by ultraviolet and fluorescence photometry are consistent.

The method is reasonable in design, and through experimental research, the method for extracting the alkaloid from the rice bran can be determined, the oxidation resistance of the alkaloid is mainly evaluated by using a TLC-CMS (thin layer chromatography-mass spectrometry) technology, a theoretical basis is provided for industrially extracting the alkaloid antioxidant from the rice bran, and the local economic development can be promoted.

Drawings

FIG. 1 shows the structural formula of evodiamine.

FIG. 2 shows thin layer plate imaging at 254 nm.

FIG. 3 shows a TLC scan of the standard.

FIG. 4 shows a TLC scan of the sample.

FIG. 5 shows the standard curve for evodiamine.

FIG. 6 shows the measurement results of the alkaloid content in the test sample.

Figure 7 shows a standard quality spectrum scan of evodiamine.

FIG. 8 shows a mass spectrometry scan curve of a test solution.

FIG. 9 shows rice bran alkaloids, V_C、V_ERelative DPPH radical clearance rate.

FIG. 10 shows alkaloid, V_C、V_ERelative hydroxyl radical clearance rate.

Detailed Description

The following detailed description of specific embodiments of the invention refers to the accompanying drawings.

A method for evaluating oxidation resistance of alkaloid in rice bran by TLC-CMS technology mainly uses rice bran as experimental material, and performs coarse extraction on the alkaloid in the rice bran through steps of ethanol extraction, microwave ultrasonic wave auxiliary treatment, centrifugation, concentration drying, extraction and the like; then, TLC-CMS technology is adopted to analyze the antioxidant activity of the alkaloid extract. The method comprises the following specific steps:

1. experimental Material

1.1 materials

The raw material is rice bran (jin Gu 8311 red millet bran collected in the county of the same city).

1.2 Primary reagents and instruments

TABLE 1 Main reagents and drugs

TABLE 2 Main instruments and leaves factory

2. Experimental methods

2.1 crude extraction of alkaloid substances from Rice bran

Raw material treatment: drying rice bran in a constant temperature cabinet, crushing the rice bran by a high-speed multifunctional crusher, and filling the crushed rice bran into a glass bottle container for later use.

Ethanol water solution leaching: weighing 10g rice bran in an erlenmeyer flask, adding 70% ethanol-water solution, stirring with a constant temperature magnetic stirrer until homogenate is formed, placing the prepared suspension in a gas bath constant temperature oscillator, oscillating for 24h, and setting the temperature at 45 deg.C (constant temperature).

Auxiliary treatment of an ultrasonic microwave extractor: extracting the suspension with ultrasonic microwave assisted extraction apparatus for three stages, each for 10min and 30 min. Under the condition of constant ultrasonic waves, the temperature of each stage is respectively set to be 40 ℃, 45 ℃ and 50 ℃, the ultrasonic power is set to be 800W, 900W and 1000W, the microwave power is set to be 150W, 200W and 250W, the ultrasonic frequency is 50KHz, the mode is 15:10, and the motor rotating speed is 930r/min, and finally homogenate is formed.

Centrifugal filtration: centrifuging the suspension in a low-speed centrifuge at 4000r/min for 10min, repeating for three times to obtain supernatant and filter residue, mixing all supernatant, filtering, collecting and weighing all filter residues.

Concentrating and drying, namely placing the supernatant liquid in a round-bottom flask of a rotary evaporator, opening a condensation pipe, setting the water bath temperature to be 79 ℃ (the boiling point of ethanol is about 78 ~ 79 ℃, and setting the temperature to be 79 ℃ due to the water in the solution), performing rotary evaporation on the ethanol until no liquid is evaporated during condensation, wherein the volume of the liquid is about 1/4 (the volume of the ethanol: water =3:1, and the ethanol is evaporated to 1/4 of the original volume, namely the ethanol is completely evaporated), and recovering the ethanol in a receiving flask for reuse.

Acidifying and extracting, namely pouring the sample liquid subjected to rotary evaporation into a conical flask, adding 2% by mass of HCl (hydrochloric acid) to acidify to the pH value of 2 ~ 3, pouring the acidified filtrate into a separating funnel, adding equal volume of chloroform for extraction, fully shaking up, standing for 10min, collecting the lower chloroform layer, adding equal volume of chloroform for extraction into the upper layer of solution, extracting for three times, and collecting the upper acidic aqueous solution for later use.

Alkalizing and extracting, namely alkalizing the acidic aqueous solution, alkalizing the acidic aqueous solution to the pH value of 9 ~ 10 by using 5% NaOH by mass fraction, adding chloroform to methanol =3:1 (v/v) into the obtained solution, extracting, taking a chloroform methanol phase, adding equal volume of chloroform and methanol solution into the upper layer of alkaline aqueous solution, extracting for three times, and collecting the upper layer of alkaline aqueous solution.

Evaporation concentration and constant volume: and (4) evaporating and concentrating the chloroform-methanol layer liquid by a rotary evaporator until the chloroform-methanol layer liquid is dried, and calculating the extraction rate. The evaporated material was brought to a volume of 25mL with chromatographic methanol (the extract at this time contained the alkaloid monomer).

The standard substance is prepared by precisely weighing 25mg of evodiamine standard substance, placing in 25mL volumetric flask cleaned by ultrasonic elution, adding chromatographic methanol to scale, dissolving under ultrasonic condition to obtain 1mg/mL of evodiamine standard substance solution, and storing in refrigerator at 2 ~ 4 deg.C for use.

TABLE 3 optimized selection of optimal microwave ultrasound conditions

2.2, separation and purification by thin-layer chromatography

Sample application: slowly sucking 25 μ L of alkaloid reference solution with a microsyringe, placing on a full-automatic sample application machine, setting sample application parameters (sample application number 3, sample application amount of 5 μ L, 7 μ L, and 9 μ L respectively), applying sample on a thin layer chromatography silica gel plate film, sucking 25 μ L of sample solution, with sample application number of 3, and sample application amount of 10 μ L.

Unfolding: preparing a mixed solution of methanol, chloroform, benzene and petroleum ether =1:14:2:8 (v/v) in a chromatographic cylinder as a developing agent of alkaloid in rice bran, putting the spotted silica gel plate into the chromatographic cylinder after the developing agent is saturated, and taking out the silica gel plate to dry when the front edge of the developing agent is about 1cm away from the top end of the silica gel plate.

Thin layer chromatography imaging system record: and (4) placing the dried silica gel plate into a thin-layer chromatography imaging system, taking a picture under a fluorescent lamp of 254nm to observe the chromatography condition, and taking a picture for recording.

Measuring the content by a thin-layer chromatography scanner: scanning the photographed and recorded thin-layer plate by using a scanner, determining spot position parameters by tracking tracks, firstly performing spectral scanning to determine the maximum and minimum absorption peak values of spots, then determining the peak area of a target spot by using reflection scanning, and determining the content of a target spot substance by using the linear relation between the peak area and the sample application amount.

2.3 Mass spectrometric analysis

Mass spectrometric detection of the standard: chromatographic methanol and ultrapure water were used as mobile phases. Opening the instrument and the software to enable the instrument and the software to be in a working state, sucking the standard solution by using a microsyringe, inserting the standard solution into a sample inlet, starting sample injection when the sample is injected into a Load file, and returning the sample to an inject file after the sample is injected. And analyzing and storing the spectrogram by a point-opening spectrogram interface.

Mass spectrum detection of the liquid to be detected: the mobile phase is the same as that of the standard sample. And (3) enabling the instrument and the software to be in a working state, placing the thin-layer plate of the product to be detected on a TLC-mass spectrum interface, and aligning laser to the center of the circled alkaloid spot. Sampling by software operation, and analyzing and storing the spectrogram by a point spectrogram interface.

2.4 clearance calculation

Measurement of DPPH radical scavenging ability: the single electron exists according to DPPH free radical, the absorption is strong near 515nm, and the alcoholic solution is purple. Taking several test tubes, accurately sucking 0.1mL of alkaloid sample solution with concentration of 100. mu.g/mL, 80. mu.g/mL, 60. mu.g/mL, 40. mu.g/mL and 20. mu.g/mL, respectively adding 3.50mL of 0.06mmol/L DPPH solution into 5 test tubes, strictly keeping out of light for 30min, measuring absorbance at 515nm, and recording as A_i. Taking 5 test tubes to absorb 0.1mL of sample extracting solution with different concentrations, adding 3.50mL of methanol solution respectively, measuring light absorption value, and recording as A_j. Then taking 1 test tube to accurately absorb 3.50mL of DPPH solution, adding 0.10mL of methanol solution, measuring the light absorption value, and recording as A₀. Preparing the same concentration gradient V as the sample extract_CAnd V_EMethanol solution as experimental control group. The absorbance values were averaged over three measurements.

Clearance S = [ 1- (a)_i–A_j)/A₀]×100%。

Determination of hydroxyl radical scavenging Capacity: by means of H₂O₂And Fe²⁺The mixture undergoes Fenton reaction to generate highly reactive OH, which has maximum absorption at 536 nm. 2.0mL of 9mmol/L FeSO was taken₄Solution, 2.0mL of 8.8mmol/L H ₂0₂The solution is reacted for 15min at 37 ℃, and the absorbance of the solution is recorded as A when measured at 536nm₀Then adding 1mL of alkaloid sample solution of 100. mu.g/mL, 80. mu.g/mL, 60. mu.g/mL, 40. mu.g/mL and 20. mu.g/mL respectively, measuring the absorbance at 536nm, and recording as A_x. Preparing the same concentration gradient V as the sample extract_CAnd V_EMethanol solution as experimental control group. The absorbance values were averaged over three measurements.

Clearance S = (a)₀–A_x)/A₀×100%。

2.5 fluorescence photometry further proves the oxidation resistance

Scanning the extractive solution with ultraviolet under a fluorescence photometer, setting wavelength at 515nm, determining whether new substances are generated according to the number of peaks and the position of maximum fluorescence value, and evaluating oxidation resistance of alkaloid according to the change of fluorescence value difference before and after fluorescence scanning and adding equal amount of DPPH solution.

3. Results and analysis

3.1 thin layer chromatography

3.1.1 analysis of Rice bran alkaloid thin layer imaging results

After performing multiple attempts of developing solvent by thin layer chromatography, the optimum developing solvent for the alkaloid in rice bran is methanol: chloroform: benzene: petroleum ether =1:14:2:8 (v/v), and the development of the standard and test sample observed under thin layer fluorescent lamp is shown in FIG. 2.

The number of samples spotted on the silica gel plate was 6, the right three spots were developed after spotting of the standard, and the left three spots were developed after spotting of the test sample, and examined under a 254nm fluorescent lamp. The observation from fig. 2 shows that: the sample and the standard product show blue-purple fluorescent spots at corresponding positions, and the positions of the fluorescent spots are consistent, wherein R is_fThe values were all around 0.8, and it was judged that the rice bran contained alkaloids having the same properties as the standard.

3.1.2 detection of alkaloid content by dual-wavelength thin-layer scanning method

Performing spectrum scanning on the spot at 200 ~ 850nm wavelength with thin layer chromatography scanner, wherein the result shows that the test sample and evodiamine control have absorption peak at 224nm, almost no absorption at 700nm, so λ is selected_S=224nm，λ_R=700 nm. See fig. 3 and 4.

And (3) observing a linear range, namely precisely sucking 5 mu L, 7 mu L and 9 mu L of the prepared evodiamine reference substance solution, respectively pointing the solution on the same thin-layer chromatography silica gel plate film (100 multiplied by 100 mm), scanning after unfolding, drawing a standard curve by taking the reference substance amount as a horizontal coordinate (X) and the absorption peak area value as a vertical coordinate (Y), and performing linear regression on test data to obtain a regression equation Y = 0.5789X-0.8861 (r = 0.9982). The result shows that the evodiamine is in the range of 5 ~ 9 mu g, the peak area and the reference substance amount form a good linear relation, and the content of alkaloid in the test solution is measured by adopting an external standard two-point method because the standard curve equation does not exceed the coordinate origin.

And (3) sample content determination: sucking 25 μ L of evodiamine standard solution, spotting 5 μ L, 7 μ L and 9 μ L on silica gel G plate, sucking 25 μ L of test solution, spotting 10 μ L of test solution on the same silica gel G plate, developing according to the method, and measuring content by reflection scanning method, as shown in FIG. 6.

TABLE 4 alkaloid content in test samples

As can be seen from Table 4, the mean alkaloid content in the test samples was 0.5509%.

3.2, Mass Spectrometry detection

The standard scan is shown in FIG. 7; a scan of the sample is shown in figure 8.

The prepared evodiamine standard substance and the test solution are subjected to mass spectrometry, the relative molecular mass of the evodiamine is 303.37, and a strong peak of 303 should appear in a mass spectrogram theoretically, but the comparison and observation of a figure 7 and a figure 8 show that the standard substance and the test solution respectively appear strong peaks at 304.2 and 304.4, and the evodiamine standard substance and the substance are presumed to increase an H ion under the bombardment of electrons, so that the substance is considered to be a reasonable increase, and the substance is judged to be an alkaloid compound.

3.3 determination of Oxidation resistance of alkaloid in Rice bran

3.3.1 measurement of DPPH radical scavenging ability

TABLE 5 Rice bran alkaloid DPPH clearance

TABLE 6 clearance of Vc vs DPPH

TABLE 7 clearance of Ve versus DPPH

From tables 5 to 7, it can be seen from the data in FIG. 9 that the clearance of the alkaloids, Vc and Ve relative to DPPH is in a slow rising trend and the clearance is Vc > alkaloids > Ve when the concentration is lower than 0.06mg/mL, the clearance of Vc and Ve relative to DPPH is increased in a whole rising range when the concentration is between 0.06mg/mL ~ 0.1.1 mg/mL, and the clearance of the alkaloids relative to DPPH is in a falling trend and the clearance is Vc > Ve > alkaloids when the concentration is between 0.08mg/mL ~ 0.1.1 mg/mL, and the clearance of the 3 substances relative to DPPH is about 70% in a whole, which proves that the 3 substances have the clearance relative to DPPH, but the clearance effect is not good.

3.3.2 measurement of hydroxyl radical scavenging ability

TABLE 8 clearance of hydroxyl radicals by alkaloids

TABLE 9 clearance of Ve for hydroxyl radicals

TABLE 10 clearance of Vc for hydroxyl radicals

From tables 8 to 10, the data in fig. 10 show that when the concentration is lower than 0.02mg/mL, the clearance rate of alkaloid, Vc and Ve relative to hydroxyl radicals is greatly increased, and the clearance capacity is that the alkaloid is > Vc > Ve, when the concentration is between 0.02mg/mL ~ 0.1.1 mg/mL, the clearance rate of the 3 substances relative to the hydroxyl radicals on the whole is in a slow rising trend, and the clearance capacity is still that the alkaloid is > Vc > Ve., the clearance capacity of the alkaloid is obviously higher than that of Ve and Vc, the clearance rate of the alkaloid relative to the hydroxyl radicals reaches more than 93% when the concentration is 0.02mg/mL, and the clearance rate of Vc relative to the hydroxyl radicals is about 80%, which proves that the alkaloid and Vc have stronger clearance capacity relative to the hydroxyl radicals.

3.3.3 further demonstration of the antioxidant Properties by fluorescence photometry

The oxidation resistance of the alkaloid is further analyzed by a fluorescence spectrophotometer according to the change of the fluorescence value difference, and the results show that the alkaloid has stronger oxidation resistance by taking Vc and Ve as controls, and the conclusion is consistent with that obtained by ultraviolet analysis of the oxidation resistance.

4. Conclusion

Taking rice bran as a raw material and 70% ethanol as an extractant, wherein the material-liquid ratio is 1:20, performing auxiliary treatment by a microwave ultrasonic extractor, and finally determining the optimal process conditions through multiple experiments as follows: extracting in three stages, each for 10min and 30 min. Under the condition of constant ultrasonic waves, the temperature is set to be 40 ℃, 45 ℃ and 50 ℃, the ultrasonic power is 800W, 900W and 1000W, the microwave power is 150W, 200W and 250W, the ultrasonic frequency is 50KHz, the mode is 15:10, and the motor rotating speed is 930r/min, so that the method has better extraction performance on alkaloid substances in the rice bran. The alkaloid crude extract is obtained by adopting a multi-stage extraction method and evaporation concentration.

After multiple attempts, methanol, chloroform, benzene and petroleum ether =1:14:2:8 (v/v) are determined to be the most suitable developing agent for the rice bran alkaloid, and when the sample is inspected under 254nm of a thin-layer imaging system, spots in the sample are approximately consistent with the positions of the evodiamine standard products and are blue-purple spots, so that the chromatographic condition can effectively separate and purify the alkaloid.

The dual wavelength thin layer scan results are: lambda [ alpha ]_S=224nm，λ_R=700 nm; under the condition, the linear relation between the integral value of the scanning peak area of the thin layer and the content is good (r = 0.9982), and the average content of the alkaloid in the test sample is 0.5509% by adopting an external standard two-point method.

The mass of the relative molecules was determined by mass spectrometry, and the results were consistent with those of the standards, roughly concluding that the substance in question was the desired substance. The ultraviolet spectrophotometer detects the capability of alkaloid, Vc and Ve extracting solutions with different concentrations for removing DPPH and hydroxyl free radicals, and the extracting solutions have certain capability of removing DPPH and hydroxyl free radicals, wherein the capability of removing DPPH is Vc > alkaloid > Ve, the capability of removing hydroxyl free radicals is alkaloid > Vc > Ve, and therefore the capability of removing hydroxyl free radicals is the strongest, and the conclusion is consistent with the result detected by the fluorescence photometry.

Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the detailed description is made with reference to the embodiments of the present invention, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the protection scope of the claims of the present invention.