Method for extracting erythrinine from beautiful millettia root by using eutectic solvent and purification process

Abstract

The invention discloses a method for extracting erythrinine from beautiful millettia roots by using a deep-eutectic solvent and a purification process. Wherein the optimal process for extracting the erythrinine is as follows: LA-Fruu (1: 1) with the water content of 40% is used under the conditions that the ultrasonic temperature is 80 DEG C, the ultrasonic time is 40 minutes and the liquid-material ratio is 20: 1, and the extraction amount of the erythrinine can reach 3.1495 + / -0.0690 mg / g; the optimal purification process is as follows: DM132 type macroporous resin is used, the eluent is 50% ethanol, the elution flow rate is 2.5 BV / h, the height-diameter ratio of a chromatographic column is 21: 1, the elution volume is 4BV, and the recovery rate of erythrinine is 89.53 + / -3.23%. According to the invention, a deep eutectic solvent and macroporous resin combined technology is established, the erythrinine is extracted and purified from the millettia specisoa champ in a high-efficiency, rapid, green and environment-friendly manner, and a green and efficient way is provided for the extraction and purification of the erythrinine.

Description

Technical Field

[0001] The invention belongs to the technical field of biological extraction, and particularly relates to a method for extracting erythrina alkaloids from Radix Erythrinae with a low eutectic solvent and a purification process. Background Art

[0002] With the improvement of living standards, the increase of food safety issues and the enhancement of people's health awareness, the demand for nutrition and health products has gradually increased. At the same time, people pay more attention to replacing synthetic ingredients with plant active ingredients in these food products, and emphasize the necessity of green and healthy development and utilization of plant resources. Nanhaia speciosa (Champ.ex Benth.) J. Compton & Schrire, synonyms: Millettia speciosa and Calleryaspeciosa, belongs to the genus Nanhaia of the Leguminosae family. It is a typical plant with medicinal and edible properties. Its root is called Niu Dali, which is rich in protein, vitamins, alkaloids and flavonoids. It is often used in the preparation of medicinal food, medicinal wine and soup in southern China. At the same time, Niu Dali is also a traditional medicine. It is the main component of Chinese patent medicines such as Zhuangyao Jianshen Pills, Guilong Ointment, and Huoluo Zhitong Pills. It has the effects of nourishing the lungs, strengthening tendons and activating collaterals. It is often used to treat chronic diseases such as lumbar muscle strain, rheumatoid arthritis, tuberculosis and chronic bronchitis.

[0003] Hypaphorine (HYP), the main component of Radix Codonopsis pilosulae, is an indole alkaloid with rich medicinal value and has been widely studied in health care and medical treatment. Studies have shown that erythrin has specific anti-acetylcholinesterase activity, indicating its potential application in the treatment of neurodegenerative diseases such as Alzheimer's disease. Erythrin also shows the potential to regulate sleep patterns. Studies have shown that it can increase non-rapid eye movement sleep in normal mice, indicating its potential application in sleep aid or sleep disorder management. In addition, erythrin exhibits anti-inflammatory properties and reduces inflammatory responses by inhibiting multiple signaling pathways such as NFκB, ERK, p38 and JNK. In terms of metabolic health, studies have found that erythrin can inhibit adipocyte differentiation and improve insulin sensitivity, indicating its potential application in the management of obesity and insulin resistance. In addition, it also exhibits the effect of inhibiting osteoclastogenesis, which has potential benefits in preventing inflammatory bone loss diseases such as osteoporosis. The diverse biological activities of erythrine, including neuroprotection, sleep regulation, anti-inflammatory and metabolic effects, make it a promising candidate for the development of novel therapeutic agents. Its interactions with key biochemical pathways and significant physiological effects highlight its research value and potential clinical applications.

[0004] At present, the reagents for extracting erythrinaine from plants are all volatile organic solvents, such as methanol, ethanol, etc. However, these organic reagents are usually toxic and pollute the environment. Now people are increasingly advocating green and environmentally friendly extraction methods to use non-toxic and environmentally friendly solvents to extract active ingredients from plants for food or medicine. Therefore, it is particularly necessary to find green and safe extraction solvents to extract erythrinaine from Radix Codonopsis pilosulae. In recent years, with the advocacy of green chemistry, more and more green solvents have been developed and applied, such as super / subcritical water, supercritical carbon dioxide, ionic liquids and deep eutectic solvents (DES). In the past two decades, deep eutectic solvents have been widely studied. Deep eutectic solvents were discovered by Abbott et al. in 2003. They are a uniform liquid mixed with hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA) in a certain proportion. They have the characteristics of non-toxicity, biodegradability, thermal stability, low volatility and designability, and have been widely used in the green extraction of active ingredients from medicinal plants. In recent years, some progress has been made in the materials for separation and purification of plant active ingredients. Macroporous adsorbent resin (MAR) has the advantages of good stability, good selectivity, convenient regeneration, long service life, low cost, etc., and is widely used in the separation and purification of active ingredients in plants. Due to its specific surface chemical properties and structure, macroporous resin has good selectivity for polar compounds, can reduce nonspecific adsorption, and effectively separate target compounds. Summary of the invention

[0005] The purpose of the present invention is to establish a technology for combining a low eutectic solvent with a macroporous resin to extract and purify erythrinaine from Herba Dioscoreae in an efficient, rapid and environmentally friendly manner, thereby providing a green and efficient way for the extraction and purification of erythrinaine.

[0006] The present invention utilizes ultrasound-assisted extraction of Radix Codonopsis pilosulae, and uses UPLC-Q-Orbitrap HRMS technology to qualitatively and quantitatively analyze erythrinae alkaloids in 37 different deep eutectic solvent extracts to determine the best deep eutectic solvent for extracting erythrinae alkaloids. Subsequently, single factor optimization is performed, and the extraction process is optimized and verified using the response surface method to determine the best extraction process. In addition, the mechanism of ultrasound-assisted deep eutectic solvent extraction of erythrinae alkaloids is discussed, and the morphological changes of Radix Codonopsis pilosulae powder before and after extraction are compared using scanning electron microscopy (SEM). The interaction between hydrogen donors and hydrogen acceptors is analyzed using infrared spectroscopy, and the interaction between low eutectic solvents and erythrinae alkaloids is explored using density functional theory (DFT) calculations. Seven macroporous adsorption resins are used to study the separation and purification process of erythrinae alkaloids, and the best macroporous adsorption resin and separation process for purifying erythrinae alkaloids are determined.

[0007] On the one hand, the present invention provides a method for extracting erythrina alkaloids from Radix Glehniae, comprising the following steps: drying and crushing the Radix Glehniae roots, sieving, taking the Radix Glehniae powder, adding a low eutectic solvent, vortex mixing, ultrasonic treatment, centrifuging, and taking the supernatant to obtain a Radix Glehniae extract; the low eutectic solvent is composed of a compound of lactic acid (LA) and fructose (Fru).

[0008] The low eutectic solvent is composed of lactic acid and fructose in a molar ratio of 1:1 and has a water content of 40%.

[0009] The liquid-to-solid ratio of the Herba Lysimachiae powder and the low eutectic solvent is 20:1 mL / g.

[0010] The temperature of the ultrasonic treatment is 80° C. and the time is 40 min.

[0011] The frequency of the ultrasound is 40KHz, and the power is 600W / 300W.

[0012] The sieving is through an 80-mesh sieve.

[0013] On the other hand, the present invention provides a purification process of erythrinaine, comprising the following steps: preparing an erythrinaine extract according to the above extraction method, eluting the erythrinaine extract using a DM132 macroporous resin and an ethanol solution as an eluent, collecting the eluent, concentrating it, and freeze-drying it to obtain an erythrinaine sample. The concentration of the ethanol solution is 50% by volume.

[0014] The elution conditions are: elution flow rate 2.5 BV / h, chromatographic column height-to-diameter ratio 21:1, and elution volume 4 BV.

[0015] The present invention has the following beneficial effects:

[0016] Erythrina is an alkaloid with rich medicinal value. The present invention optimizes the parameters of extracting erythrina from Herba Lycopodii by using a low eutectic solvent of LA-Fru (1:1) with a water content of 40%. Under the conditions of ultrasonic temperature of 80°C, ultrasonic time of 40min, and liquid-to-solid ratio of 20:1, the extraction amount of erythrina can reach 3.1495±0.0690mg / g, which is 105.19% and 70.62% higher than that of water and ethanol extraction, respectively. SEM results show that the low eutectic solvent has a certain dissociation effect on Herba Lycopodii, FTIR shows the formation of hydrogen bonds between LA and Fru, and DFT calculations reveal the formation of hydrogen bonds between erythrina and LA-Fru (1:1). In addition, DM132 macroporous resin is used to purify erythrina in the low eutectic solvent under the conditions of 50% ethanol, elution flow rate of 2.5BV / h, chromatographic column height-to-diameter ratio of 21:1, and elution volume of 4BV, and the recovery rate is 89.53±3.23%. The invention proposes a new method of green and efficient low eutectic solvent extraction and macroporous resin purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Results of ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) for the detection of erythrine in the extract of Radix Codonopsis pilosulae. (A) Chemical structure of erythrine. (B) Total ion current of erythrine standard. (C) Standard curve of erythrine. (D) Total ion current of Radix Codonopsis pilosulae extracted with a low eutectic solvent.

[0018] Figure 2 The effects of different extraction conditions on the amount of erythrina alkaloids extracted. (A) The effect of the type of deep eutectic solvent. (B) The effect of the water content in the deep eutectic solvent. (C) The effect of extraction time. (D) The effect of liquid-to-solid ratio. (E) The effect of extraction temperature. Error bars represent relative standard deviations (n=3). In the same figure, different letters at the top of each column indicate significant differences (P<0.05).

[0019] Figure 3 Response surface diagram of the effects of different extraction conditions on the extraction amount of erythrina alkaloids. (A)(D) Extraction temperature and liquid-to-solid ratio, (B)(E) Extraction temperature and extraction time, (C)(F) Response surface diagram of the effects of liquid-to-solid ratio and extraction time on the extraction amount of erythrina alkaloids.

[0020] Figure 4 The extraction amount of erythrine by LA-Fru (1:1), ethanol and water. The different letter positions at the top of each column indicate significant differences (P<0.05).

[0021] Figure 5 Infrared spectra of LA, Fru and LA-Fru (1:1).

[0022] Figure 6 The following are scanning electron microscope images of the powder of Radix Codonopsis pilosulae. (A) The original powder of Radix Codonopsis pilosulae was magnified 1000 times under a scanning electron microscope. (B) The powder of Radix Codonopsis pilosulae was extracted by LA-Fru (1:1) at 80℃, 40min, liquid-to-solid ratio = 20:1 (mL / g) and magnified 1000 times. (C) The powder of Radix Codonopsis pilosulae was extracted by LA-Fru (1:1) at 80℃, 40min, liquid-to-solid ratio = 20:1 (mL / g) and magnified 2000 times.

[0023] Figure 7 Interactions between erythrine and LA-Fru. (A) Hydrogen bond interactions and energies between erythrine and L-LA-Fru. (B) Hydrogen bond interactions and energies between erythrine and D-LA-Fru. Color scheme: gray - carbon, white - hydrogen, red - oxygen, blue - nitrogen, dashed lines - hydrogen bonds.

[0024] Figure 8The effects of different purification conditions on the purification of erythrina alkaloids. (A) The adsorption / desorption capacity and recovery rate of 7 macroporous resins for erythrina alkaloids. (B) The effects of ethanol concentration (C) elution flow rate (D) column height-to-diameter ratio (E) elution volume on the recovery rate of erythrina alkaloids. DETAILED DESCRIPTION

[0025] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0026] Example 1

[0027] 1. Materials and Chemicals

[0028] The plant was collected in Yunfu, Guangdong, and identified as Rhizoma Cynomorium by Dr. Ji Ruifeng of Guangdong Pharmaceutical University. The roots of Rhizoma Cynomorium were dried in an oven at 50°C (WGL-230B, Tianjin Taisite Instrument Co., Ltd., China), and crushed with a high-speed pulverizer (800C, Yongkang Hongtaiyang Electromechanical Co., Ltd., China). The powder was sieved through an 80-mesh sieve and stored at 4°C until use. Glucose (99.0%), sucrose (99.0%), and tartaric acid (99.5%) were purchased from Guangdong Guanghua Technology Co., Ltd., choline chloride (98.0%) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., DL-lactic acid (85.0%) was purchased from Shanghai Yuanye Technology Co., Ltd., citric acid (99.5%) and anhydrous ethanol (99.7%) were purchased from Guangzhou Brand Chemical Reagent, and fructose (99.0%) was purchased from Guangzhou Aleshan Biotechnology Co., Ltd. Macroporous resins were purchased from Nanjing Feishier Wharton Technology Co., Ltd.

[0029] 2. Preparation of low eutectic solvent

[0030] A deep eutectic solvent (see Table 1) was prepared by heating and stirring. The corresponding hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD) of the prepared deep eutectic solvent and water were weighed in proportion and placed in a 50 mL beaker, a magnetic stirrer was added, and the mixture was sealed. The beaker containing the mixture was placed in a magnetic stirrer and stirred at 80°C until a uniform transparent liquid was formed.

[0031] Table 1 Types of deep eutectic solvents

[0032]

[0033]

[0034] 3. Extraction method of erythrine

[0035] 0.2000g of eutectic powder was accurately weighed in a 10mL centrifuge tube using a 1 / 10,000 balance (ATY124, Shimadzu Enterprise Management Co., Ltd., Japan), and the corresponding low eutectic solvent or traditional solvent (water / ethanol) was added according to the experimental design, and a vortex machine was used to vortex and mix briefly. Ultrasonic (JP-100S, Shenzhen Jiemeng Cleaning Equipment Co., Ltd., China) extraction was performed under the conditions of the single-factor experimental design, and the frequency and power of the ultrasound used were 40KHz and 600W / 300W. The single-factor extraction experiment used four important parameters: low eutectic solvent water content (10%, 20%, 30%, 40%, 50%), liquid-to-solid ratio (10:1, 20:1, 30:1, 40:1, 50:1), ultrasonic temperature (50℃, 60℃, 70℃, 80℃, 90℃), and ultrasonic time (20min, 30min, 40min, 50min, 60min) to determine the extraction amount of erythrina alkaloids and optimize the extraction process. After the ultrasonic treatment, the samples were centrifuged at 4000 r / min for 30 min using a high-speed centrifuge (L550, Hunan Xiangyi Laboratory Instrument Development Co., Ltd., China), and the supernatant was diluted 50 times and analyzed using UPLC-Q-Orbitrap HRMS with the extraction amount of erythrine as the result.

[0036] 4. Quantitative analysis of erythrine

[0037] UPLC-Q-Orbitrap HRMS analysis was performed using a Vanquish Flex UHPLC system and an Orbitrap Exploris 120 quadrupole electrostatic field orbital trap high-resolution mass spectrometer from Thermo Fisher Scientific. The mass spectrometer resolution was 15,000. N2 was used as the sheath gas at a flow rate of 50 L / min, as the auxiliary gas at a flow rate of 8 L / min, and as the purge gas at a flow rate of 1 L / min. The ion fragments with m / z between 100.0 and 300.0 were scanned using a HESI ion source and positive ion mode at a capillary voltage of 3.5 kV, a capillary temperature of 325°C, and an auxiliary gas temperature of 350°C. Separation was performed using a Thermo Fisher Scientific Bremen Hypersil GOLD AQ column (100 mm × 2.1 mm, 1.9 μm) at 35°C. The composition of the mobile phase was as follows: eluent A: acetonitrile containing 0.1% formic acid, and eluent B: water containing 0.1% formic acid. An isocratic elution of 95% A was used for 9 min, the flow rate and injection volume were 0.3 mL / min and 2 μL, respectively, and the detection wavelength was 280 nm.

[0038] Prepare different concentrations of erythrinaine standard solutions and establish erythrinaine standard curves. The samples were identified by comparing the spectra and retention times with those of the standard compounds, and the erythrinaine concentration was determined according to the standard curve using TraceFinder 5.1GeneraL Quan software, and the erythrinaine was quantitatively calculated according to formula (1).

[0039]

[0040] In formula (1), C is the concentration of erythrine in the extraction solvent (mg / mL), V is the volume of the extraction solvent (mL), and W is the weight of the Herba Lycopodii powder.

[0041] 5. Experimental Design and Statistical Analysis

[0042] Response surface methodology (RSM) was performed using Design-Expert 13.0. The parameters selected from the single-factor experiments of liquid-to-solid ratio, ultrasonic temperature, and ultrasonic time were optimized by Box-Behnken design (BBD). All experiments were performed independently at least 3 times. GraphPad Prism 8.0.2 software was used to draw graphs, and SPSS26.0 software was used for one-way analysis of variance (ANOVA) to compare the extraction amount and recovery rate of erythrine. Different letters indicate statistically significant differences (P<0.05).

[0043] 6. Results

[0044] 6.1 Development and validation of analytical methods

[0045] In order to comprehensively evaluate the extraction effect, ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) was used to detect the extracted erythrinaine. Figure 1 As shown in B, it was observed that Figure 1 Peak 1 in D is erythrine, and its chemical structure is as follows Figure 1 A. A calibration curve was established based on the relationship between peak area and content. The regression equation of erythrine is Y = 94454X + 52749274 (r2 = 0.9995). Figure 1 C. The methodological validation of the developed method showed that the stability, accuracy and precision of the established method could meet the requirements of qualitative and quantitative analysis. The results of the methodological validation are shown in Table 2.

[0046] Table 2 Calibration curve, precision, repeatability and stability of erythrine

[0047]

[0048] 6.2 Evaluation of the performance of deep eutectic solvents in extracting erythrine

[0049] Under the extraction conditions of 30% water content of the deep eutectic solvent, 80℃ ultrasonic temperature, 60min ultrasonic time and 30:1 liquid-solid ratio, the extraction amount of erythrine by 37 deep eutectic solvents was as follows: Figure 2 As shown in A. Compared with the low eutectic solvents with choline chloride (ChCl) and citric acid (CA) as hydrogen acceptors, the low eutectic solvent with lactic acid (LA) as the hydrogen acceptor has a high extraction amount of erythrina alkaloids, and LA-Fru (1:1) has the highest extraction amount of erythrina alkaloids. Studies have shown that the lower the pH value is when extracting alkaloid compounds with low eutectic solvents, the more conducive it is to extract alkaloids. ChCl is a quaternary ammonium base. When ChCl is part of the low eutectic solvent, the pH value of the low eutectic solvent is relatively high, so it is not conducive to extraction. Under the same molar ratio of HBA and HBD, when LA is used as the hydrogen acceptor, the extraction amount of erythrina alkaloids by lactic acid-fructose (LA-Fru) is greater than that by lactic acid-glucose (LA-Glu) and lactic acid-sucrose (LA-Suc). Viscosity has a significant effect on the extraction amount of alkaloids extracted by low eutectic solvents. The higher the viscosity, the lower the extraction amount. Studies have shown that among glucose (Glu), fructose (Fru), and sucrose (Suc), Suc has the highest viscosity, followed by Glu, and Fru has the lowest viscosity. The LA-Fru system eutectic solvent formed under the same molar ratio of HBA and HBD has a low viscosity, so the extraction amount is large.

[0050] From the perspective of the low eutectic solvents of the LA-Fru system, the low eutectic solvent of LA-Fru (1:1) has a larger extraction amount of erythrina alkaloids than the low eutectic solvents of other molar ratios. The viscosity of Fru is greater than that of LA, and the viscosity of the low eutectic solvent increases with the increase of Fru. At the same time, as Fru increases, LA decreases, and the pH of the low eutectic solvent increases. Therefore, LA-Fru (1:1) has a higher extraction amount of erythrina alkaloids than LA-Fru (1:2), LA-Fru (1:3), and LA-Fru (1:4). Compared with LA-Fru (1:1), LA-Fru (2:1) theoretically has a lower pH and lower viscosity, but it extracts less erythrina alkaloids. This may be related to the number of hydrogen bonds and entropy changes generated during the extraction process. As we will discuss further, hydrogen bonds play a crucial role in the binding energy between low eutectic solvents and erythrina alkaloids. In conclusion, most deep eutectic solvents, especially LA as the deep eutectic solvent for HBA, showed great potential in the extraction of erythrina alkaloids.

[0051] 6.3 Optimization of extraction conditions

[0052] (1) Effect of water content of deep eutectic solvent

[0053] In this study, the effect of water content on the extraction amount of erythrina alkaloids was investigated under the condition that other extraction conditions remained unchanged, namely, ultrasonic temperature was 80℃, ultrasonic time was 60min, and liquid-to-solid ratio was 30:1. Figure 2 As shown in Figure B, as the water content increases from 10% to 40%, the extraction amount of erythrina alkaloids increases significantly. The extraction amount is the highest when the water content reaches 40%. As the water content further increases from 40% to 50%, the extraction amount decreases. Since the low eutectic solvent formed by the mixture of lactic acid and fructose has a large viscosity, it is difficult for it to penetrate into the medicinal materials. Adding an appropriate amount of water to the low eutectic solvent can reduce the viscosity of the low eutectic solvent, making it easier for the low eutectic solvent to penetrate the medicinal materials and bind more tightly with erythrina alkaloids. However, adding excessive water will destroy the hydrogen bonds between the components of the low eutectic solvent, resulting in a decrease in the interaction between erythrina alkaloids and the low eutectic solvent, thereby reducing the extraction amount. Therefore, a water content of 40% was selected for more research.

[0054] (2) Influence of ultrasonic temperature

[0055] The effect of ultrasonic temperature on the extraction amount of erythrina alkaloids was investigated with low eutectic solvent (water content 40%), ultrasonic time 60 min, and liquid-to-solid ratio 30:1 as constants. Figure 2 As shown in Figure E, the extraction amount of erythrina alkaloids increases with the increase of temperature. The extraction amount is the highest when the temperature is 80℃, but if the temperature is further increased to 90℃, the extraction amount of erythrina alkaloids decreases. The viscosity of the low eutectic solvent decreases with the increase of temperature, which helps the low eutectic solvent to penetrate the medicinal materials, and the molecular thermal motion will become more intense with the increase of temperature, further increasing the solubility of the low eutectic solvent and erythrina alkaloids. However, high temperature can cause some erythrina alkaloids to react with lactic acid and lactic acid dehydration condensation products, so the extraction amount decreases. In summary, 80℃ is selected as the optimal extraction temperature.

[0056] (3) Effect of ultrasound time

[0057] The water content of the low eutectic solvent was 40%, the ultrasonic temperature was 80°C, and the liquid-to-solid ratio was 30:1 as constants, and the time gradient was set for extraction. Figure 2 As shown in C, the amount of erythrina alkaloids extracted increases with the increase of extraction time and reaches the maximum when the ultrasonic time is 40 minutes. However, as the time continues to extend, the amount of erythrina alkaloids extracted decreases. Generally, long-term extraction is not suitable for bioactive compounds because it will change the chemical structure of the target compound. In this work, the extraction time is relatively short, which helps to stabilize the low eutectic solvent for effective extraction. In summary, 40 minutes was selected as the optimal extraction time.

[0058] (4) Influence of liquid-to-solid ratio

[0059] The effect of liquid-to-solid ratio on the extraction amount of erythrina alkaloids was investigated by taking the water content of the deep eutectic solvent as 40%, the ultrasonic temperature as 80℃ and the ultrasonic time as 40min as constants. Figure 2 As shown in D, when the liquid-to-solid ratio is between 10:1 and 20:1, the increase in the liquid-to-solid ratio will lead to an increase in the amount of erythrina alkaloids extracted. When the liquid-to-solid ratio is 20:1, the amount of erythrina alkaloids extracted is the highest, but the amount of extraction decreases as the liquid-to-solid ratio continues to increase. According to the literature, it is speculated that the mass transfer driving force will affect the extraction of erythrina alkaloids and the mass transfer driving force is positively correlated with the mass transfer distance. The increase in the liquid-to-solid ratio can increase the mass transfer distance between the low eutectic solvent and erythrina alkaloids, thereby increasing the mass transfer driving force between the low eutectic solvent and erythrina alkaloids, which is conducive to the increase in the amount of erythrina alkaloids extracted. However, with the increase in the liquid-to-solid ratio, the low eutectic solvent has fully extracted the erythrina alkaloids in Radix Codonopsis pilosulae. Increasing the liquid-to-solid ratio may cause the extracted erythrina alkaloids to desorb and return to the extract to be extracted, resulting in a decrease in the amount of erythrina alkaloids extracted. In summary, 20:1 is selected as the optimal liquid-to-solid ratio.

[0060] 6.4 Optimization of extraction conditions using response surface methodology (RSM)

[0061] In this work, LA-Fru (1:1) with a water content of 40% was used for extraction, and a response surface experiment was conducted on three factors: ultrasonic temperature (A), liquid-to-solid ratio (B), and ultrasonic time (C) (Table 3). The fitted second-order model of the extraction amount (Y) and the coding factors (A, B, and C) is expressed as follows:

[0062] Y=3.14-0.1318A+0.0819B-0.1254C-0.0792AB-0.0846AC+0.0217BC-0.4130A 2 -0.2090B 2 -0.2935C 2

[0063] In the regression model, the obtained model F value was 63.55, and the missing fit F value was 0.4369, which was much larger than 0.05, indicating that the model fitting was effective and successful (Table 4). In addition, the P value was <0.0001, indicating that the model difference was significant and the experimental result error was small, reflecting the relationship between various factors and the response value. The adjusted R 2 This value (0.9879) is close to 1, indicating that the model can significantly predict the experimental values. Figure 3The three-dimensional (3D) and two-dimensional (2D) diagrams represent the interaction between ultrasonic temperature of 80℃, ultrasonic time of 40min, and liquid-to-solid ratio. The optimal extraction conditions obtained by Design-Expert 13.0 software are ultrasonic temperature of 78.38℃, liquid-to-solid ratio of 22.17:1, and extraction time of 38.18min. The predicted extraction amount of erythrina alkaloids can reach 3.17593mg / g. Considering the practical feasibility, the extraction factors are determined as follows: ultrasonic temperature of 80℃, ultrasonic time of 40min, and liquid-to-solid ratio of 20:1.

[0064] Table 3. Response surface optimization experiment factor level table

[0065]

[0066] Table 4. Response surface optimization experiment factor level table

[0067]

[0068]

[0069] 6.5 Extraction of the best process verification experimental results

[0070] Three parallel validation experiments were conducted using LA-Fru (1:1) with a water content of 40% under the conditions of ultrasonic temperature of 80°C, ultrasonic time of 40 min, and liquid-to-solid ratio of 20:1, and the results were compared with those of traditional solvent extraction of erythrinain from Radix Glehniae. Figure 4 .Depend on Figure 4 Under these conditions, the extraction amount of erythrina alkaloids was 3.1495 mg / g, which was close to the extraction amount of erythrina alkaloids predicted by the response surface method, with an error of 0.83%, proving that the optimal process parameters were reliable. Compared with water and ethanol as extraction solvents, the extraction amount increased by 105.19% and 70.62% respectively, so the extraction process optimized in this experiment was highly efficient compared with traditional solvents.

[0071] Example 2

[0072] 1. Characterization of the powder morphology of Niu Da Li

[0073] (1) Method: The best extraction process was used to extract the powder of Radix Codonopsis pilosulae. After extraction, the powder was centrifuged. The residue after centrifugation was washed with water and freeze-dried for 24 hours using a freeze dryer (Alpha1-4LSC basic, Marin Christ, Germany). The sample surface was sprayed with gold, and the powder of Radix Codonopsis pilosulae before and after extraction was scanned using a scanning electron microscope (JSM-7610FPlus, Shimadzu Enterprise Management Co., Ltd., Japan) at magnifications of 1000 and 2000 and an accelerating voltage of 15KV.

[0074] (2) Results: The morphology of the powder of Radix Angelicae Pubescentis before and after extraction with the deep eutectic solvent under scanning electron microscopy is as follows: Figure 6 A, 6B, 6C. Before extraction, the surface of the Herba Cynoglossi powder was smooth and flat without obvious wrinkles and cracks. Compared with the sample morphology before extraction, the surface of the Herba Cynoglossi powder after extraction with a low eutectic solvent showed obvious wrinkles and cracks, and the surface structure was severely damaged with many irregular pore structures. This may be because the ultrasound damaged the sample surface, accelerating the dissolution and release of intracellular erythrina alkali, which is beneficial to the extraction of erythrina alkali by a low eutectic solvent. By comparing the morphological analysis of Herba Cynoglossi powder before and after extraction with a low eutectic solvent, the microstructure was significantly different, indicating that ultrasound-assisted low eutectic solvent extraction of erythrina alkali in Herba Cynoglossi has a better effect.

[0075] 2. Infrared spectroscopy (FTIR) analysis

[0076] (1) Method: The infrared spectra of the best deep eutectic solvent and its raw materials were measured using an FTIR spectrometer (IRAffinity-1S, Shimadzu Enterprise Management Co., Ltd., Japan). The measurement range was 4000-500 cm -1 , resolution 4cm -1 Solid samples were analyzed by KBr compressed tablets, and liquid samples were analyzed by infrared spectroscopy scanning using the liquid cell method.

[0077] (2) Results: Infrared spectra can provide the vibration changes of atomic groups (chemical bonds), which is an important characteristic of low eutectic solvents. LA, Fru and LA-Fru (1:1) were selected for FTIR characterization and analysis of molecular interactions such as Figure 5 The FT-IR spectrum of LA shows that CH -1 There is a stretching vibration peak at 3568cm -1 There is a stretching vibration peak at 1749cm -1 There is a stretching vibration peak at 3407 cm -1 There is a stretching vibration peak at 1645cm -1 There is a stretching vibration peak at 2944 cm -1 There is a stretching vibration peak at 3407 cm-1. Compared with the single compound, the FT-IR absorption peak of LA-Fru (1:1) has shifted significantly. Specifically, the absorption peak of Fru-OH in LA-Fru (1:1) shifted from 3407 cm-1 to 3407 cm-1. -1 Displacement to 3200cm -1 In addition, the absorption peaks of C=O and -OH of LA in LA-Fru (1:1) increased from 1749 cm -1 and 3568cm -1 Move to 1731cm-1 and 3529cm -1 ( Figure 5 ). There was no destruction of functional groups and generation of new functional groups between LA and Fru, which indicated that LA and Fru may form a low eutectic solvent through the hydrogen bonding interaction between -OH on Fru and -COOH on LA.

[0078] 3. Density functional theory (DFT) calculations

[0079] (1) Methods: To further understand the interaction between LA-Fru (1:1) and erythrinaine at the molecular level. This study used the PM6-DH+ precision method to merge the Molclus program with MOPAC v22.0.4 to conduct a collaborative exploration of the molecular conformations of LA-Fru (1:1) and erythrinaine. The Gaussian 09 software package was used in combination with the B3LYP functional and D3BJ dispersion correction for structural optimization, vibration analysis, and energy calculations. The 6-31G* basis set was used to determine the optimized molecular structure and its corresponding vibrational frequency. The binding energy was calculated using the M06-2X functional and the def2-TZVP basis set according to formula (2).

[0080] ΔE DES-HYP =E DES-HYP -(E DES +E HYP ) (2)

[0081] In formula (2), E DES-HYP represents the energy of the deep eutectic solvent and erythrine, E DES represents the energy of the deep eutectic solvent, E HYP Indicates the energy of erythrine.

[0082] (2) Results: By simulating the interaction between LA-Fru (1:1) and erythrinaine, we understand and explore the mechanism of LA-Fru (1:1) in the extraction of erythrinaine. The optimized geometric configuration of LA-Fru (1:1) is shown in Figure 2. Figure 7 . It was found that the O of C=O of L-LA interacted with the H of -OH of Fru to form a hydrogen bond, namely OH…O. The H of -COOH on D-LA interacted with the O of -OH of Fru to form a hydrogen bond, namely OH…O. This is consistent with the infrared verification results, that is, there is a hydrogen bond interaction between -COOH of DL-LA and -OH of Fru, forming a low eutectic solvent. At the same time, the number and distance of hydrogen bonds formed between DL-LA-Fru (1:1) and erythrinaine are as follows Figure 7As shown in the figure, there are three hydrogen bonds between L-LA-Fru (1:1) and erythrinaine, namely OH…O, OH…O, and CH…O. The binding energy between L-LA-Fru (1:1) and erythrinaine is stable at -56.27 kcal / mol. There are three hydrogen bonds between D-LA-Fru (1:1) and erythrinaine, namely CH…O, CH…O, and OH…O. The binding energy between D-LA-Fru (1:1) and erythrinaine is stable at -40.87 kcal / mol. Since DL-lactic acid was used in this experiment, the average value of the two was taken when calculating its binding energy, that is, the binding energy between DL-LA-Fru (1:1) and erythrinaine is approximately stable at -48.57 kcal / mol. Studies have shown that the lower the binding energy, the stronger the solvent's ability to extract the extract. Compared with the literature, the binding energy of DL-LA-Fru (1:1) for extracting erythrinaine is lower, indicating that DL-LA-Fru (1:1) has a better extraction effect on erythrinaine. At the same time, compared with the extraction of erythrinaine by L-LA-Fru (1:1) and D-LA-Fru (1:1), the binding energy of L-LA-Fru (1:1) for extracting erythrinaine is lower. If L-LA-Fru (1:1) is used to extract erythrinaine, the extraction amount may be further increased, but further verification is needed.

[0083] Example 3

[0084] 1. Static adsorption and desorption experiments of macroporous resin

[0085] (1) Method: Pre-treat the macroporous resin (Table 5), weigh 2 g of the pre-treated macroporous resin and add it to a 100 mL conical flask, extract erythrinaine from Herba Dioscoreae using a low eutectic solvent, detect the erythrinaine concentration C0 in the low eutectic solvent extract, take 20 mL of the low eutectic solvent extract and pour it into a conical flask, oscillate it for 24 h using a constant temperature oscillator at 5°C and 120 r / min, and filter it. Detect the erythrinaine concentration C1 in the filtrate, pour the filter residue into a conical flask, add 50 mL of 70% ethanol and oscillate it for 24 h using a constant temperature oscillator, filter it, and measure the erythrinaine concentration C2 in the filtrate, calculate the erythrinaine adsorption amount according to formula (3), calculate the desorption amount according to formula (4), and calculate the recovery rate according to formula (5).

[0086]

[0087] In formulas (3), (4) and (5), C0 is the concentration of erythrinaine in the extract after extraction with a low eutectic solvent (mg / g), C1 is the concentration of erythrinaine in the filtrate after adsorption with a macroporous resin (mg / g), C2 is the concentration of erythrinaine in the filtrate after desorption of the macroporous resin with 70% ethanol (mg / g), V1 is the volume of the filtrate after adsorption with the macroporous resin (mL), V2 is the volume of the filtrate after desorption of the macroporous resin with 70% ethanol (mL), and M is the weight of the macroporous resin (g).

[0088] Table 5 Physical properties of macroporous resin

[0089]

[0090]

[0091] (2) Results: The adsorption and desorption capacity of the target compound is a key indicator for evaluating macroporous resins. In this separation and purification process, macroporous resins were used to purify erythrina alkaloids under the optimal extraction process conditions of erythrina alkaloids with low eutectic solvents. The adsorption amount, desorption amount and recovery rate of erythrina alkaloids were used as indicators to compare and evaluate seven different macroporous resins. The physical properties of the macroporous resins are shown in Table 5. The experimental results of the adsorption amount, desorption amount and recovery rate of erythrina alkaloids by the seven macroporous resins are shown in Table 5. Figure 8 A. Under the same conditions, the adsorption, desorption and recovery rates of erythrina alkaloids by DM132 macroporous resin were the highest, while those by D101 and HP-20 were lower. Generally, the adsorption and desorption capacity of macroporous resins are related to their polarity and physical properties. The physical properties of resins include average pore size, pore volume and surface area, and macroporous resins tend to adsorb more similar substances. Studies have shown that the smaller the pore size of a macroporous resin, the larger the surface area, which allows effective components to be adsorbed on the surface, thus having better absorption capacity. The reason why DM132 macroporous resin exhibits stronger adsorption and desorption capacity may be that among the seven macroporous resins, DM132 has a small average pore size and the largest specific surface area. The adsorption area per unit mass of macroporous resin is large, so it can more fully adsorb erythrina alkaloids in low eutectic solvents than other macroporous resins. D101 and HP-20 had poor adsorption and desorption capabilities for erythrinaine, which may be due to their average pore size being much larger than that of other macroporous resins, resulting in the release of erythrinaine after adsorption by macroporous resins. Therefore, DM132 macroporous resin was selected for separation and purification in subsequent experiments.

[0092] 2. Purification of erythrine

[0093] (1) Method: Weigh 92g of the best pre-treated macroporous resin and wet-load it into the column (1BV=50mL), and wet-load 15mL of the low eutectic solvent extract to purify the column. Investigate the effects of the purification single factor ethanol elution concentration (30%, 40%, 50%, 60%, 70%, 80%), elution flow rate (1.5BV / h, 2BV / h, 2.5BV / h, 3BV / h, 3.5BV / h), column height-to-diameter ratio (21:1, 12:1, 7:1, 4:1), and elution volume (2BV, 3BV, 4BV, 5BV, 6BV) on the recovery rate of erythrina alkaloids, and optimize the purification process. The recovery rate is calculated according to formula (5).

[0094] (2) Results:

[0095] 1) Influence of eluent concentration

[0096] The effect of ethanol concentration on the recovery rate of erythrina alkaloids was investigated with an elution flow rate of 3 BV / h, a column height-to-diameter ratio of 21:1, and an elution volume of 5 BV as constants. Figure 8 As shown in B, when the ethanol concentration is between 30% and 50%, it is observed that the recovery rate of erythrine increases significantly, and the recovery rate increases from 51.92% to 82.24%. However, when the ethanol concentration exceeds 50%, the recovery rate continues to increase, but the rate of increase gradually tends to be stable. This may be due to the fact that 50% ethanol can fully desorb erythrine, and increasing the ethanol concentration cannot significantly improve the recovery rate. At the same time, due to the excessively high ethanol concentration, a part of the impurities will be washed out. Therefore, 50% ethanol was used to wash out the erythrine in Herba Dioscoreae.

[0097] 2) Influence of elution flow rate

[0098] The effect of elution flow rate on the recovery rate of erythrina alkaloids was investigated with elution concentration of 50% ethanol, column height-to-diameter ratio of 21:1, and elution volume of 5 BV as constants. Figure 8 As shown in Figure C, when the flow velocity is between 1.5-2.5BV / h, the recovery of erythrina alkaloid by the eluent increases with the increase of flow velocity. When the flow velocity is 2.5BV / h, the recovery of erythrina alkaloid reaches a maximum value of 87.08%. When the flow velocity is greater than 2.5BV / h, the recovery of erythrina alkaloid also decreases. It may be due to the fact that the flushing effect cannot completely elute the erythrina alkaloid in the resin at a relatively low flow velocity. At a high flow velocity, the eluent is not in contact with the erythrina alkaloid on the resin for a long time, resulting in incomplete elution. Therefore, it is determined that the column chromatography elution flow rate is 2.5BV / h for subsequent experiments.

[0099] 3) Influence of chromatography column

[0100] The effect of the height-to-diameter ratio of the column on the recovery of erythrina alkaloids was investigated with an elution concentration of 50% ethanol, an elution flow rate of 2.5 BV / h, and an elution volume of 5 BV as constants. Figure 8 As shown in D, as the height-to-diameter ratio of the chromatographic column increases, the recovery rate of erythrina alkaloids also increases. When the height-to-diameter ratio of the chromatographic column is 21:1, the recovery rate of erythrina alkaloids is as high as 86.35%. This phenomenon can be explained by the theory of chromatographic columns. The longer the chromatographic column, the higher the theoretical plate number and the stronger the separation ability of the chromatographic column. Therefore, a chromatographic column with a height-to-diameter ratio of 21:1 was selected for elution of erythrina alkaloids in subsequent experiments.

[0101] 4) Influence of elution volume

[0102] The effect of elution volume on the recovery rate of erythrina alkaloids was investigated with elution concentration of 50% ethanol, elution flow rate of 2.5 BV / h, and column height-to-diameter ratio of 21:1 as constants. Figure 8 As shown in Figure E, the recovery rate of erythrina alkaloids increases with the increase of elution volume. When the elution volume increases to 4BV, the recovery rate of erythrina alkaloids is the highest. When the elution volume exceeds 4BV, the recovery rate of erythrina alkaloids tends to be stable. This may be because when the elution dose is low, the adsorption of erythrina alkaloids by the resin makes it difficult to completely desorb, resulting in incomplete elution. When erythrina alkaloids have been fully desorbed, increasing the elution volume does not significantly improve the recovery rate. Therefore, it is determined that the use of 4BV eluent is the optimal eluent amount for the purification process.

[0103] 3. Experimental results of separation and purification optimal process verification

[0104] In summary, the adsorption-desorption performance of DM132 macroporous resin is the best; the best separation method is 50% ethanol as eluent, 2.5BV / h as elution flow rate, 21:1 as column height-to-diameter ratio, and 4BV as elution volume. Three groups of parallel experiments were carried out according to this separation and purification scheme. The results showed that the average recovery rate of erythrina alkaloids in the three groups of parallel experiments was 89.53±3.23%, and the relative standard deviation was 3.61%. The experimental results had good repeatability, which verified the accuracy of this separation and purification method.

[0105] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for extracting erythrinain, characterized in that: The following steps are involved: The root of Herba Lysimachiae is dried and crushed, sieved, Herba Lysimachiae powder is taken, a low eutectic solvent is added, vortex mixed, ultrasonic treatment is performed, centrifuged, and the supernatant is taken to obtain the Herba Lysimachiae extract; the low eutectic solvent is composed of lactic acid and fructose.

2. The method for extracting erythrinain according to claim 1, characterized in that: The low eutectic solvent is composed of lactic acid and fructose in a molar ratio of 1:1 and has a water content of 40%.

3. The method for extracting erythrinain according to claim 1, characterized in that: The liquid-to-solid ratio of the Herba Lysimachiae powder and the low eutectic solvent is 20:1 mL / g.

4. The method for extracting erythrinain according to claim 1, characterized in that: The temperature of the ultrasonic treatment is 80° C. and the time is 40 min.

5. The method for extracting erythrinain according to claim 1, characterized in that: The frequency of the ultrasound is 40KHz, and the power is 600W / 300W.

6. The method for extracting erythrinain according to claim 1, characterized in that: The sieving is through an 80-mesh sieve.

7. A purification process of erythrinain, characterized in that: The method comprises the following steps: preparing a Herba Lycopodii extract according to the extraction method described in any one of claims 1 to 6, eluting the Herba Lycopodii extract using a DM132 macroporous resin and an ethanol solution as an eluent, collecting the eluent, concentrating it, and freeze-drying it to obtain a sample of erythrina alkaloids.

8. The purification process of erythrinain according to claim 7, characterized in that: The concentration of the ethanol solution is 50% by volume.

9. The purification process of erythrinain according to claim 7, characterized in that: The elution conditions are: elution flow rate 2.5 BV / h, chromatographic column height-to-diameter ratio 21:1, and elution volume 4 BV.

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