Extraction and detection of caffeoylquinic acids in eisenia foetida

Abstract

The application discloses a method for extracting and detecting caffeoylquinic acids in Blumea aromatica. Nine kinds of caffeoylquinic acids (CQAs) are obtained by extracting Blumea aromatica with seven kinds of deep eutectic solvents (DESs). Compared with traditional solvents, DESs show significant advantages in extracting specific CQAs, and the antioxidant activity of DES extracts is better than that of traditional solvent extracts. The application also provides a detection method for CQAs. The content of the nine kinds of CQAs in Blumea aromatica can be determined simultaneously by using UPLC-Q-Orbitrap HRMS technology. The methodological study shows that the method has excellent precision, accuracy, repeatability and stability. The application has important value for exploring the green extraction method of CQAs and studying the chemical components and biological activity of the medicinal plant Blumea aromatica.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological extraction, and particularly relates to an extraction and detection method of caffeoylquinic acids in fargesiae. BACKGROUND

[0002] Phenolic compounds are secondary metabolites produced by plants during growth. They are abundant in vegetables, fruits, and medicinal plants. Phenolic compounds have a variety of structures and biological activities. They can act as antioxidants, antimicrobials, anticancer agents, anti-aging agents, anti-inflammatory agents, and antihypertensive agents. Therefore, plant phenolic compounds have a wide range of applications in the food and pharmaceutical fields. Caffeoylquinic acids (CQAs) are an important part of phenolic compounds, derived from the phenylpropanoid biosynthetic pathway. These compounds are acylated by quinic acid with one to four caffeic acid groups, with a total of 15 possible combinations, including four monomeric CQAs, six dimeric CQAs, four trimeric CQAs, and one tetrameric CQA. Caffeoylquinic acids have a variety of biological activities, such as antioxidant, antibacterial, anticancer, antiviral, anti-Alzheimer's disease, and neuroprotective activities. Caffeoylquinic acids are usually ingested through plant-based foods such as fruits, vegetables, and coffee beverages. Due to their diverse biological activities, caffeoylquinic acids have attracted increasing attention, and they have the potential to be developed as new drugs.

[0003] Although there have been some studies on the qualitative and quantitative methods of caffeoylquinic acids, the characterization and accurate quantitative analysis of caffeoylquinic acid isomers still present great challenges. This is mainly due to several factors, including the presence of multiple isomers with similar physicochemical properties, the lack of commercial standards, and the degradation and transformation of caffeoylquinic acids during sample processing. These challenges hinder the accurate characterization of caffeoylquinic acid isomers and the development of comprehensive characterization methods for caffeoylquinic acids. In order to overcome these challenges and promote the research on caffeoylquinic acids in various fields including food additives, health products, and pharmaceuticals, it is necessary to further study the extraction methods and qualitative and quantitative analysis techniques for caffeoylquinic acids.

[0004] Phenolic compounds are mainly extracted from plant matrices through organic solvents or physical auxiliary techniques. However, organic solvent extraction has disadvantages such as high volatility, toxicity, flammability, and low biodegradability. Deep eutectic solvents (DESs) are a new type of environmentally friendly solvent, usually composed of a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA). The melting point of DESs is lower than that of any component, so they can exist stably in liquid form at room temperature. Compared with organic solvents, DESs are easy to synthesize, low in cost, stable, non-volatile, biodegradable, and environmentally friendly. In recent years, DESs have attracted more attention and are widely used in the extraction of natural products.

[0005] Blumea aromatica DC. is a perennial herb of the genus Blumea, which grows in southwest China and Southeast Asia. It is known as "Shanfeng" in traditional Chinese medicine and is widely used to treat rheumatism, joint pain, and eczema. Despite its potential, little is known about the chemical composition and biological activity of Blumea aromatica. Notably, in previous studies, the compounds isolated from Blumea aromatica were mainly labdane and bisnor diterpenoids, which exhibited diverse biological activities, including anti-inflammatory, immunosuppressive, and adenylyl cyclase-activating properties. However, there is currently no report on the extraction method and content determination of caffeoylquinic acids in Blumea aromatica. SUMMARY

[0006] One of the purposes of the present application is to provide a method for extracting caffeoylquinic acids from Blumea aromatica. The present application shows that the use of seven DESs to extract nine caffeoylquinic acids from Blumea aromatica has good extraction efficiency compared to three traditional solvents, and for specific caffeoylquinic acid derivatives, the use of specific DESs can achieve higher extraction efficiency.

[0007] The second purpose of the present application is to provide a method for detecting caffeoylquinic acids in Blumea aromatica. The present application develops a UPLC-Q-Orbitrap HRMS method that can simultaneously determine the content of nine caffeoylquinic acids in Blumea aromatica; the methodological study shows that the method has excellent precision, accuracy, repeatability, and stability.

[0008] To achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0009] A method for extracting caffeoylquinic acids from Blumea aromatica, comprising the following steps: drying and crushing Blumea aromatica plants, sieving, taking Blumea aromatica powder, adding a eutectic solvent, oscillating and mixing, then performing ultrasonic treatment, cooling to room temperature, centrifuging, and taking the supernatant to obtain the product; the eutectic solvent is composed of any one of choline chloride and lactic acid, acetic acid, ethylene glycol, urea, glycerol, 1,4-butanediol, or oxalic acid.

[0010] Preferably, the caffeoylquinic acids include chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, 1,5-O-dicaffeoylquinic acid, 1,3-O-dicaffeoylquinic acid, 1,4-O-dicaffeoylquinic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C.

[0011] Preferably, the molar ratio of choline chloride to lactic acid, acetic acid, ethylene glycol, urea, glycerol, 1,4-butanediol, or oxalic acid in the eutectic solvent is 1:2-3.

[0012] Preferably, the eutectic solvent is composed of choline chloride and ethylene glycol or 1,4-butanediol with a molar ratio of 1:3.

[0013] Preferably, the ultrasonic treatment is performed at a power of 100-800 W, a frequency of 40 kHZ, a temperature of 30-80℃, and a time of 10-60 min.

[0014] Preferably, the ratio of the farnesyl Ainsliaea powder to the eutectic solvent is 1 g:10-200 mL.

[0015] A method for detecting coffee quinic acids in farnesyl Ainsliaea, comprising the following steps:

[0016] (1) Preparation of standard solution: accurately weigh chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, 1,5-O-dicaffeoylquinic acid, 1,3-O-dicaffeoylquinic acid, 1,4-O-dicaffeoylquinic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C reference substances, respectively, and prepare a mixed standard solution with different concentrations using methanol solution as the solvent;

[0017] (2) Preparation of sample solution: vortex mix the supernatant obtained by the above method with methanol solution, filter to obtain the sample solution;

[0018] (3) Detection: use UPLC-Q-Orbitrap HRMS to detect the mixed standard solution and the sample solution, take the concentration of the reference substance as the abscissa, and the peak area of the mass chromatogram of the quantitative daughter ion as the ordinate, draw a standard curve, and detect different coffee quinic acid components in the sample solution according to the standard curve.

[0019] Preferably, the concentration of the methanol solution is 50% by mass.

[0020] Preferably, the filtration is performed using a 0.22 μm microporous membrane.

[0021] Preferably, the chromatographic conditions of the UPLC-Q-Orbitrap HRMS are as follows:

[0022] Chromatographic column: Thermo Fisher Scientific Bremen Hypersill GOLD AQ (100 mm x 2.1 mm, 1.9 μm); mobile phase A: acetonitrile containing 0.1% formic acid by volume, mobile phase B: water containing 0.1% formic acid by volume; gradient elution: 0-15 min, B 97%; 15-20 min, B 97%-96%; 20-25 min, B 96%-90%; 25-50 min, B 90%-87%; 50-54 min, B 87%-5%; flow rate 0.3 mL / min, injection volume 2 μL; column temperature 42 °C, detection wavelength 330 nm, and detection by DAD.

[0023] The mass spectrometry conditions are as follows: in a negative ion scanning mode, the detection conditions are as follows: HESI ion source, an electrospray voltage of 3.5 kV, a capillary temperature of 325 °C, and an auxiliary gas temperature of 300 °C; a mass spectrometry scanning range of m / z 200-600; nitrogen is used as sheath gas, auxiliary gas, and purge gas; a sheath gas flow rate of 50 L / min, an auxiliary gas flow rate of 8 L / min, and a purge gas flow rate of 1 L / min; and a resolution of 60000.

[0024] The present application has the following beneficial effects:

[0025] 1. The present application uses seven kinds of deep eutectic solvents as extractants, which has significant advantages in extracting specific caffeoylquinic acids from Blumea aromatica compared with three traditional solvents (ethanol, methanol, and water). Among them, DES-6 (choline chloride: 1,4-butanediol) is the best solvent for extracting 3-O-caffeoylquinic acid and 4,5-O-dicaffeoylquinic acid, with concentrations of 4.71±0.31 and 2.28±0.19 mg / g, respectively. DES-7 (choline chloride: oxalic acid) is the best solvent for extracting 4-O-caffeoylquinic acid, with a concentration of 2.05±0.05 mg / g. DES-5 (choline chloride: glycerol) is the best solvent for extracting 5-O-caffeoylquinic acid, 1,3-O-dicaffeoylquinic acid, 1,4-O-dicaffeoylquinic acid, and 3,4-O-dicaffeoylquinic acid, with concentrations of 1.70±0.14, 2.17±0.04, 1.39±0.14, and 2.30±0.06 mg / g, respectively. DES-2 (choline chloride: acetic acid) is the best solvent for extracting 1,5-O-dicaffeoylquinic acid and 3,5-O-dicaffeoylquinic acid, with concentrations of 7.60±0.38 and 5.70±0.23 mg / g, respectively.

[0026] 2. The present application carried out DPPH and ABTS radical scavenging experiments and reducing power determination on ten solvent extracts from Blumea haridora, the results showed that: DES extract showed excellent antioxidant activity, surpassing the antioxidant activity of traditional solvents, among them, DES-3 (choline chloride: ethylene glycol) extract showed the most outstanding free radical scavenging activity, the IC 50 values of DPPH and ABTS free radicals were 197.36±1.05 and 14.86±3.33 μg / mL respectively. DES-6 (choline chloride: 1,4-butanediol) extract showed the highest reducing power. In addition, correlation analysis showed that there was a positive correlation between phenolic acid content and antioxidant activity, 3-O-caffeoylquinic acid and 5-O-caffeoylquinic acid showed significant correlation (p values were less than 0.001 and 0.05 respectively). In addition, 4-O-caffeoylquinic acid, 1,3-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid showed specific and relevant antioxidant activity.

[0027] 3. The present application developed a UPLC-Q-Orbitrap HRMS method, which can simultaneously determine the content of nine caffeoylquinic acids in Blumea haridora. The methodological study showed that the method has excellent precision, accuracy, repeatability and stability. It was determined that there were two groups of isomers in the nine caffeoylquinic acids determined in this study: three mono-caffeoylquinic acids and six di-caffeoylquinic acids. Although the components in each group have slight differences in structure, they differ in the position of the substituent group. The contents of the nine caffeoylquinic acids in Blumea haridora are significantly different, and there are trace components. Therefore, simultaneously determining the content of the nine caffeoylquinic acids constitutes a considerable challenge. In the present application, by continuously optimizing the chromatographic separation conditions and utilizing the analytical advantages of UPLC-Q-Orbitrap HRMS technology, we achieved simultaneous quantitative analysis of the nine caffeoylquinic acids. The present application provides valuable reference for the research and exploration of similar compound analysis techniques. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Total ion chromatograms of 9 caffeoylquinic acid mixed reference solution (a) and methanol extract of Blumea haridora (b) in negative ion mode. (A: 4-O-caffeoylquinic acid; B: 3-O-caffeoylquinic acid; C: 5-O-caffeoylquinic acid; D: 1,3-O-dicaffeoylquinic acid; E: 1,4-O-dicaffeoylquinic acid; F: 3,4-O-dicaffeoylquinic acid; G: 3,5-O-dicaffeoylquinic acid; H: 1,5-O-dicaffeoylquinic acid; I: 4,5-O-dicaffeoylquinic acid).

[0029] Figure 2 The contents of nine caffeoylquinic acids in Blumea aromatica extracted by different solvents.

[0030] Figure 3 The DPPH radical scavenging activities of (a) ascorbic acid (VC) control and (b) Blumea aromatica extracted by different solvents.

[0031] Figure 4 The ABTS radical scavenging activities of (a) ascorbic acid (VC) control and (b) Blumea aromatica extracted by different solvents.

[0032] Figure 5 The reducing power of (a) ascorbic acid (VC) control and (b) Blumea aromatica extracted by different solvents. DETAILED DESCRIPTION

[0033] The following examples are further illustrations of the application and are not intended to limit the present application.

[0034] Example 1

[0035] 1. Chemicals, reagents and materials

[0036] Acetonitrile, methanol and formic acid for chromatography were obtained from Thermo Fisher Scientific. The water for chromatography was purchased from Guangzhou Watsons Food & Beverage Co., Ltd. (Guangzhou, China). The reference substances of 3-O-caffeoylquinic acid (chlorogenic acid, 3-CQA), 4-O-caffeoylquinic acid (cryptochlorogenic acid, 4-CQA), 5-O-caffeoylquinic acid (neochlorogenic acid, 5-CQA), 1,5-O-dicaffeoylquinic acid (1,5-diCQA), 1,3-O-dicaffeoylquinic acid (1,3-diCQA), 1,4-O-dicaffeoylquinic acid (1,4-diCQA), 3,5-O-dicaffeoylquinic acid (isochlorogenic acid A, 3,5-diCQA), 3,4-O-dicaffeoylquinic acid (isochlorogenic acid B, 3,4-diCQA) and 4,5-O-dicaffeoylquinic acid (isochlorogenic acid C, 4,5-diCQA) were obtained from Chengdu Alpha Biotech Co., Ltd. (Chengdu, China) with batch numbers AZ22011851, AFBL221, AZ21102901, AFBG1302, AFBL1284, AFCC1605, AZ22042903, AZ21092203 and AFCB0705, respectively, and the purity was not less than 98%. Choline chloride was sourced from Aleshan (Guangzhou) Biotechnology Co., Ltd. Ascorbic acid, lactic acid, acetic acid, ethylene glycol, urea, glycerol, 1,4-butanediol and oxalic acid were purchased from Guangzhou Chemical Reagent Factory. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (>98% purity) were sourced from Shanghai Aladdin Biochem Technology Co., Ltd.

[0037] The plant material used in this experiment was Blumea aromatica DC. and was collected from Zhaoqing, China, and identified by Dr. Zheng Xilong, School of Chinese Medicinal Resources, Guangdong Pharmaceutical University.

[0038] 2. Instruments

[0039] The chromatographic separation system was a Vanquish Flex ultra-performance liquid chromatograph from Thermo Fisher Scientific. The detection and quantification of chemical constituents were performed on a Thermo Fisher Scientific Orbitrap Exploris 120 quadrupole-Orbitrap high-resolution mass spectrometer. The sample weighing was performed on an ATY124 one-millionth balance from Shimadzu (China) Co., Ltd.

[0040] 3. Determination of the content of caffeoylquinic acids

[0041] (1) Detection conditions

[0042] Chromatographic conditions: Chromatographic separation was performed using a Thermo Fisher Scientific Bremen Hypersill GOLDAQ column (100 mm x 2.1 mm, 1.9 pm). The mobile phase consisted of two parts: A, acetonitrile with 0.1% formic acid (v / v), B, water with 0.1% formic acid (v / v). A gradient elution was applied with the following conditions: 0-15 min, B 97%; 15-20 min, B 97%-96%; 20-25 min, B 96%-90%; 25-50 min, B 90%-87%; 50-54 min, B 87%-5%. The flow rate was set to 0.3 mL / min and the injection volume was 2 pL. The column temperature was kept at 42 °C and the detection wavelength was 330 nm. Detection was performed using DAD.

[0043] Mass spectrometry conditions: The study was performed in negative ion mode. The detection conditions were as follows: HESI ion source, the electrospray voltage was set to 3.5 kV, the capillary temperature was maintained at 325 °C, and the auxiliary gas temperature was set to 300 °C. The mass spectrometry scan range was set to m / z 200-600. Nitrogen was used for sheath gas, auxiliary gas, and sweep gas; the sheath gas flow rate was 50 L / min, the auxiliary gas flow rate was 8 L / min, and the sweep gas flow rate was 1 L / min; the resolution was 60,000.

[0044] (2) Preparation of deep eutectic solvents (DESs)

[0045] Deep eutectic solvents were prepared by heating a mixture of a hydrogen bond acceptor (HBA, choline chloride) with seven different hydrogen bond donors (HBD) in a specific molar ratio. A modified method was used, as described in the reference (Ivanovic, M., Islamcevic Razborsek, M., Kolar, M., 2020. Innovative extraction techniques using deep eutectic solvents and analytical methods for the isolation and characterization of natural bioactive compounds from plant material. Plants 9(11), 1428.). The specified amount of reactants was placed in a 150 mL beaker, and a magnetic stirrer bar was added. Then the mixture was covered with cling film and parafilm, heated and stirred on a magnetic stirrer at 60 °C for 40 min until a homogeneous transparent liquid was obtained. After cooling to room temperature, 30% water was added and mixed well. The resulting mixture was transferred to a 50 mL centrifuge tube and stored in a refrigerator at 4 °C for later use. The composition of different types of DESs is shown in Table 1.

[0046] Table 1. Composition of seven different deep eutectic solvents (DESs)

[0047]

[0048] (3) Preparation of sample solution and standard solution

[0049] Preparation of sample solution: The whole plant of Blumea aromatica was dried, crushed and sieved through a 50 mesh sieve. Accurately weighed Blumea aromatica powder (0.1 g, three parallel groups) was placed in a 15 mL centrifuge tube. Then, 5 mL of ethanol, methanol, water and seven different deep eutectic solvents were added separately. The centrifuge tube was sealed, mixed by shaking, and weighed. Ultrasonic treatment was carried out under specific conditions, including power 400 W, frequency 40 kHZ, temperature 50 °C, for 20 min. After cooling to room temperature, reweighed, and the corresponding extraction solvent was added to compensate for the weight loss. Centrifugation was carried out at a speed of 4000 rpm, and 2 mL of supernatant was collected and stored in a separate centrifuge tube. This process was repeated three times in parallel, and the supernatant obtained was the extract. From the supernatant, a total of 200 μL of sample was transferred to a 2 mL centrifuge tube. Subsequently, 800 μL of 50% methanol aqueous solution was added and mixed uniformly by vortex. Then, the mixture was filtered through a 0.22 μm microporous membrane to obtain the test solution.

[0050] Preparation of standard solution: Accurately weighed 1.00 mg of each of the nine caffeoylquinic acid reference substances, and placed in a 5 mL volumetric flask. The names and structures of the nine caffeoylquinic acids are shown in Table 2. These reference substances were dissolved in 50% methanol aqueous solution and diluted to the calibration line to obtain a mother liquor with a concentration of 0.2 mg / mL. Next, 0.4 mL of the mother liquor of isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C, and 0.625 mL of the mother liquor of the other six caffeoylquinic acids were transferred to separate 5 mL volumetric flasks. Adjust to the calibration line with 50% methanol aqueous solution, shake to mix to obtain mixed reference solutions with different concentrations. Finally, all samples were stored in a refrigerator at 4 °C.

[0051] Table 2. Compound names and structures of nine caffeoylquinic acids

[0052]

[0053]

[0054] (4) Optimization of chromatographic conditions

[0055] To improve the resolution and peak shape of the sample, we considered two kinds of chromatographic columns: Thermo Fisher Hypersill GOLD AQ column (100 mm x 2.1 mm, 1.9 pm) and Fisher Hypersil GOLD C 18 column (100 mm x 2.1 mm, 1.9 pm). Both columns were subjected to the same column temperature, mobile phase, and gradient elution conditions. Comparative analysis showed that the Thermo Fisher Hypersill GOLD AQ column (100 mm x 2.1 mm, 1.9 pm) exhibited higher separation efficiency, improved peak shape, and shorter retention time. Therefore, we selected the Thermo Fisher Hypersill GOLD AQ column (100 mm x 2.1 mm, 1.9 pm) for sample separation, and the chromatogram is shown in Figure 1

[0056] (5) Method validation

[0057] Calibration curves, limits of detection (LODs) and quantification (LOQs), precision (including intra-day and inter-day precision), stability, and recovery were studied in this research. Calibration curves for nine compounds were generated by diluting stock solutions to appropriate concentrations using a 50% methanol-water mixture. Limits of detection (LODs) and quantification (LOQs) were determined using signal-to-noise (S / N) ratios of 3 and 10, respectively. Intra-day precision was evaluated by calculating the relative standard deviation (RSD%) for 6 injections performed within a single day. Inter-day precision was evaluated by calculating the average RSD% for 6 injections performed each day over 3 consecutive days. Stability was evaluated by calculating the RSD% for 6 injections performed at 0, 2, 4, 8, 12, 24, and 48 hours. The accuracy of the method was verified using 3 sample levels (80%, 100%, 120%). Recovery was calculated using the following equation (1).

[0058] Recovery (%) = (C - A) / B x 100% (1)

[0059] where A represents the amount of the tested component in the sample; B represents the amount of pure substance added; and C represents the actual measured value.

[0060] In this experiment, calibration curves for nine caffeoyl quinic acids were generated within a specific concentration range using the above method, as shown in Table 3. The obtained results showed good linear relationships, with correlation coefficients R 2 ​All were greater than 0.9990. The limit of detection (LOD) and limit of quantification (LOQ) were determined by diluting the lowest point on the standard curve to a signal-to-noise ratio of 3 and 10. The ranges of LOD and LOQ were 0.0040-0.0149 pg / mL and 0.0130-0.0498 pg / mL, respectively. Good intra-day and inter-day precision, and stability within 48 hours were shown. The relative standard deviation (RSD %) values were between 0.69-2.45%, 0.52-3.04% and 0.85-3.21%, respectively. In addition, Table 4 shows the recovery rates, ranging from 78.18% to 113.90%.

[0061] Table 3. Calibration curves, linear ranges, limits of detection (LOD), limits of quantification (LOQ), precision and repeatability of nine coffee acylquinic acids

[0062]

[0063]

[0064] Table 4. Recovery rates and relative standard deviation (RSD) of nine coffee acylquinic acids at different concentrations (80%, 100% and 120%)

[0065]

[0066]

[0067] (6) Quantitative analysis

[0068] Each batch of sample was prepared in triplicate, and the compound content in each batch of sample was determined using the calibration curve, and the results are shown in Table 5 and Figure 2As shown, the content of nine CQAs extracted from E. odoratum using different solvents revealed significant differences (P < 0.05). The concentration of CQAs ranged from 0.46 to 7.60 mg / g. The nine caffeoylquinic acids were ranked in decreasing order as follows: 1,5-diCQA > 3,5-diCQA > 3-CQA > 3,4-diCQA > 4,5-diCQA > 1,3-diCQA > 5-CQA > 4-CQA > 1,4-diCQA. In addition, the total phenolic acid content in the samples ranged from 15.35 to 27.08 mg / g. Among the solvents tested, the extraction solvent consisting of choline chloride + 1,4-butanediol (DES-6) exhibited the highest efficiency in extracting phenolic acids. The monomeric CQAs present in DESs extraction showed significantly higher contents compared to traditional extraction solvents (p < 0.05). Notably, choline chloride + 1,4-butanediol (DES-6) showed the highest concentrations of 3-CQA and 4,5-diCQA, which were 4.71 ± 0.31 and 2.28 ± 0.19 mg / g, respectively. Choline chloride + oxalic acid (DES-7) had the highest 4-CQA content, which was 2.05 ± 0.05 mg / g. Choline chloride + glycerol (DES-5) showed the highest 5-CQA, 1,3-diCQA, 1,4-diCQA, and 3,4-diCQA contents, which were 1.70 ± 0.14, 2.17 ± 0.04, 1.39 ± 0.14, and 2.30 ± 0.06 mg / g, respectively. In addition, choline chloride + acetic acid (DES-2) showed the highest 1,5-CQA and 3,5-diCQA contents, which were 7.60 ± 0.38 and 5.70 ± 0.23 mg / g, respectively.

[0069] Table 5. Nine caffeoylquinic acid contents (mg / g, mean ± standard deviation, n = 3) from E. odoratum extracted by different solvents

[0070]

[0071]

[0072] Example 2

[0073] The extracts of 10 different solvents (ethanol, methanol, water, and 7 DESs) were prepared according to the method of Example 1, and the antioxidant activity of the different solvent extracts was evaluated.

[0074] 1. DPPH radical scavenging test

[0075] 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay is a simple, effective, economical and reliable method for determining the antioxidant activity of a substance. Referring to the method (Li, Z., Li, Q., 2022. Ultrasonic-Assisted Efficient Extraction of Coumarins from Peucedanum decursivum (Miq.) Maxim Using Deep Eutectic Solvents Combined with an Enzyme Pretreatment. Molecules. 27(17), 5715.), 10 different solvents (ethanol, methanol, water and 7 DESs) were used to dilute the extracts obtained from the corresponding solvents to different concentrations (3.33, 166.67, 333.33, 500, 666.66, 1000 and 1666.67 pg / mL), 1 mL of different concentrations of extract was taken into a 5 mL tube, then 1 mL of DPPH solution (2 mM) was added. After shaking well, it was placed in the dark for 30 minutes, and then the absorbance was measured at 517 nm. The scavenging rate was calculated according to the following equation (2).

[0076] DPPH scavenging activity (%) = (1 - (A1-A2) / A0) x 100% (2)

[0077] Where A0 is the absorbance of the blank control; A1 is the absorbance of the sample to be tested; A2 is the absorbance of the control sample.

[0078] The DPPH radical scavenging activity of the extracts of Blumea Figure 3 jussieui obtained using different solvents is shown in Table 2. The extracts extracted from different solvents showed certain DPPH radical scavenging ability. However, they were all relatively low, lower than that of ascorbic acid. In the concentration range of 0 to 500 pg / mL, the scavenging rate showed significant changes with the concentration, showing a dose-dependent relationship. When the concentration was more than 500 pg / mL, the increase rate of the scavenging rate slowed down. It is worth noting that when the concentration was more than 1666.7 pg / mL, ascorbic acid reached the maximum scavenging rate of 96.88% ± 0.002%. Among the extracts extracted using different solvents, DES1-6 showed a scavenging rate of more than 90%, while DES-7 showed a scavenging rate of 84.69% ± 0.005%. But compared with traditional solvents such as water, ethanol and methanol, it is still higher.

[0079] The strength of antioxidant activity increases with the decrease of IC 50 value. Ascorbic acid and various solvent extracts have IC50 Values, from low to high, are as follows: ascorbic acid (7.42 ± 0.70 μg / mL) < DES-4 (185.63 ± 1.88 μg / mL) < DES-3 (197.36 ± 1.05 μg / mL) < DES-6 (210.90 ± 4.88 μg / mL) < DES-5 (211.75 ± 4.26 μg / mL) < DES-1 (214.24 ± 12.29 μg / mL) < DES-7 (294.44 ± 10.43 μg / mL) < water (314.67 ± 12.17 μg / mL) < DES-2 (355.60 ± 3.71 μg / mL) < ethanol (585.41 ± 28.85 μg / mL) < methanol (1065.32 ± 32.53 μg / mL), as shown in Table 6.

[0080] 2. ABTS Radical Scavenging Test

[0081] 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) is green in solution and is decolorized by antioxidants that remove free radicals. Similar to ABTS, it is commonly used in in vitro tests to characterize the antioxidant capacity of substances. The method in the reference was slightly modified (Jo, Y., Cho, H., Chun, J., 2021. Antioxidant activity of β-cyclodextrin inclusion complexes containing trans-cinnamaldehyde by DPPH, ABTS and FRAP. Food Sci. Biotechnol. 30(6), 807–814.). A mixture of ABTS solution (7 mM) and K2S2O8 solution (2.45 mM) was used as the ABTS reaction solution, with a wavelength of 734 nm. The mixture was left to stand in a dark room for 12 to 16 hours and diluted with absolute ethanol until the absorbance of the diluted solution was (0.70 ± 0.20). Different concentrations of the extract (3.33, 66.67, 100, 133.33, 250, 333.33 and 500 μg / mL) were aspirated, then 1 mL of the ABTS reaction solution was added, and the absorbance was measured at 734 nm for 30 minutes. Ascorbic acid solution was used as a positive control, and each group of tests was performed three times. The scavenging rate was calculated according to equation (3).

[0082] ABTS Scavenging Activity (%) = (1 - (A1 - A2) / A\(_0\)) × 100% (3)

[0083] Where, A\(_0\) is the absorbance of the blank control; A1 is the absorbance of the sample to be measured; A2 is the absorbance of the control sample.

[0084] As shown in Figure 19, all the different solvent extracts of Blumea Figure 4 The ABTS radical scavenging activity of the different solvent extracts of Blumea 50 The IC50values from the lowest to the highest were as follows: ascorbic acid (3.07 ± 0.03 μg / mL) < DES-3 (14.86 ± 3.33 μg / mL) < DES-6 (21.29 ± 1.77 μg / mL) < DES-4 (73.91 ± 1.35 μg / mL) < DES-5 (76.19 ± 3.94 μg / mL) < water (81.57 ± 1.91 μg / mL) < DES-1 (84.45 ± 20.80 μg / mL) < DES-2 (103.10 ± 3.42 μg / mL) < DES-7 (103.33 ± 25.42 μg / mL) < ethanol (420.03 ± 19.57 μg / mL) < methanol (762.24 ± 44.17 μg / mL), as shown in Table 6. The IC50value of DES-3 was 14.86 ± 3.33 μg / mL, which was very close to that of ascorbic acid (3.07 ± 0.03 μg / mL), indicating that DES-3 had a high ABTS radical scavenging activity. 50 The ABTS radical scavenging activity of the different solvent extracts of Blumea

[0085] Table 6. Antioxidant activities of Blumea 50 The IC50values from the lowest to the highest were as follows: ascorbic acid (3.07 ± 0.03 μg / mL) < DES-3 (14.86 ± 3.33 μg / mL) < DES-6 (21.29 ± 1.77 μg / mL) < DES-4 (73.91 ± 1.35 μg / mL) < DES-5 (76.19 ± 3.94 μg / mL) < water (81.57 ± 1.91 μg / mL) < DES-1 (84.45 ± 20.80 μg / mL) < DES-2 (103.10 ± 3.42 μg / mL) < DES-7 (103.33 ± 25.42 μg / mL) < ethanol (420.03 ± 19.57 μg / mL) < methanol (762.24 ± 44.17 μg / mL), as shown in Table 6. The IC50value of DES-3 was 14.86 ± 3.33 μg / mL, which was very close to that of ascorbic acid (3.07 ± 0.03 μg / mL), indicating that DES-3 had a high ABTS radical scavenging activity.

[0086]

[0087] a-g: Different lower case letters indicate significant differences in the contents of the nine caffeoylquinic acids in the Blumea

[0088] 3. Reducing power assay

[0089] In this experiment, the antioxidant activity of the sample solution was determined by measuring the amount of Fe4[Fe(CN)6]3produced, with higher absorbance values indicating stronger antioxidant activity. This method was slightly modified based on a reference (Oyaizu, M., 1986. Studies on product of browning reaction antioxidative activities of products of browning reaction prepared from glucosamine. J. Nutr. 44(6), 307-315.). Different concentrations of extract (0.2, 2, 4, 10, 16, and 20 mg / mL) were taken into 3 mL, followed by the addition of 1 mL of phosphate buffer solution (PBS, pH 6.0), 2 mL of 1% potassium ferricyanide solution, mixed well, and then incubated in a constant temperature water bath at 50°C for 20 minutes. After rapid removal and cooling, 1 mL of 10% trichloroacetic acid solution was added, mixed well, and then centrifuged at 3000 r / min for 10 minutes. 2.5 mL of supernatant was taken and 1 mL of distilled water was added, and the absorbance was measured at 700 nm. Ascorbic acid was used as a positive control. The greater the absorbance value, the stronger the reducing power of the sample.

[0090] Figure 5 The reducing power of different solvent extracts of E. odorata and ascorbic acid was demonstrated. In particular, both ascorbic acid and methanol extract showed a clear dose-response relationship, i.e., linear reducing power. Ascorbic acid reached saturation at a concentration of 500 μg / mL, showing a maximum absorbance of 1.412 ± 0.003. Different extraction solvents reached saturation at a concentration of 20 mg / mL, with DES-6 showing the strongest reducing power, with a maximum absorbance of 1.593 ± 0.006. The maximum absorbance values of the remaining six DES and water extracts ranged from 1.161 to 1.555. The reducing power of E. odorata extracted by different solvents in descending order was as follows: DES-6 > DES-3 > DES-4 > DES-5 > DES-1 > DES-2 > water > DES-7 > ethanol > methanol.

[0091] Example 3

[0092] Correlation analysis between caffeic acid derivatives and antioxidant activity:

[0093] The relationship between phenolic acid content and antioxidant activity of DESs extracts of E. odorata was analyzed using Pearson correlation coefficient. The results are shown in Table 7. The total phenolic acid content in DESs extracts was positively correlated with reducing power, DPPH radical scavenging capacity and ABTS radical scavenging capacity (p < 0.01). This indicates that higher total phenolic acid content in DESs extracts corresponds to stronger antioxidant activity, suggesting that total phenolic acids make a significant contribution to in vitro antioxidant activity. In addition, 3-CQA was significantly positively correlated with total phenolic acid content, reducing power, DPPH radical scavenging capacity and ABTS radical scavenging capacity (p < 0.001). These results suggest that 3-CQA is one of the main antioxidant components of total phenolic acids in DESs extracts of E. odorata. Higher 3-CQA content in DESs extracts corresponds to stronger antioxidant activity. Similarly, 5-CQA was positively correlated with total phenolic acids, reducing power, DPPH radical scavenging capacity and ABTS radical scavenging capacity (p < 0.05), highlighting its importance in E. odorata. 4-CQA, 1,3-diCQA, 3,4-diCQA, 4,5-diCQA and DPPH radical scavenging capacity were significantly positively correlated with ABTS radical scavenging capacity (p < 0.05). In addition, the reducing power of 1,3-diCQA and 4,5-diCQA was significantly positively correlated with ABTS radical scavenging capacity (p < 0.05). These findings suggest that 4-CQA, 1,3-diCQA, 3,4-diCQA and 4,5-diCQA have certain antioxidant activity.

[0094] Table 7. Correlation analysis between phenolic acids and antioxidant activity in different solvent extracts of E. odorata

[0095]

[0096]

[0097] The above merely preferred embodiments of the present application, it should be noted that the above preferred embodiments should not be regarded as a limitation of the present application, the protection scope of the present application should be limited by the scope defined by the claims. For ordinary skilled in the art, without departing from the spirit and scope of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A method of extracting cafeariquininic acid from Blumea aromatica (L.) DC. characterized by, The method comprises the following steps: The plant of Blumea Jamiacica is dried, crushed, sieved, and the Blumea Jamiacica powder is obtained, and then a eutectic solvent is added, and the mixture is oscillated and ultrasonically treated, and then cooled to room temperature, centrifuged, and the supernatant is obtained; the eutectic solvent is composed of choline chloride and any one of lactic acid, acetic acid, ethylene glycol, urea, glycerol, 1,4-butanediol, and oxalic acid; The caffeoylquinic acids include chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, 1,5-O-dicaffeoylquinic acid, 1,3-O-dicaffeoylquinic acid, 1,4-O-dicaffeoylquinic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C; The molar ratio of choline chloride to lactic acid, acetic acid, ethylene glycol, urea, glycerol, 1,4-butanediol, or oxalic acid in the eutectic solvent is 1:2-3; The method further comprises the following steps: (1) preparing a standard solution: accurately weighing chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, 1,5-O-dicaffeoylquinic acid, 1,3-O-dicaffeoylquinic acid, 1,4-O-dicaffeoylquinic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C, respectively, and dissolving them in methanol to prepare a mixed standard solution with different concentrations; (2) preparing a sample solution to be tested: vortex-mixing the supernatant with methanol, filtering, and obtaining a sample solution to be tested; (3) detection: detecting the mixed standard solution and the sample solution to be tested by UPLC-Q-Orbitrap HRMS, taking the concentration of the control as the abscissa, and the peak area of the mass chromatogram of the quantitative daughter ion as the ordinate, drawing a standard curve, and detecting different caffeoylquinic acid components in the sample solution to be tested according to the standard curve; The chromatographic conditions of the UPLC-Q-Orbitrap HRMS are as follows: The chromatographic column is Thermo Fisher Scientific Bremen Hypersill GOLD AQ 100mm×2.1mm, 1.9μm; the mobile phase A is acetonitrile containing 0.1% formic acid by volume fraction, and the mobile phase B is water containing 0.1% formic acid by volume fraction; the gradient elution is 0-15min, B 97%; 15-20min, B 97%-96%; 20-25min, B 96%-90%; 25-50min, B 90%-87%; 50-54min, B 87%-5%; the flow rate is 0.3mL / min, the injection volume is 2μL; the column temperature is 42℃, the detection wavelength is 330nm, and DAD is used for detection; The mass spectrometric conditions are as follows: the negative ion scanning mode is used, and the detection conditions are as follows: the HESI ion source, the electrospray voltage is 3.5kV, the capillary temperature is 325℃, and the auxiliary gas temperature is 300℃; the mass spectrometric scanning range is m / z 200-600; nitrogen is used for sheath gas, auxiliary gas, and purge gas; the sheath gas flow rate is 50L / min, the auxiliary gas flow rate is 8L / min, and the purge gas flow rate is 1L / min; the resolution is 60000.

2. The method of claim 1, wherein the coffeein quinic acid is extracted from Blumea fimbriata by the following steps of: The eutectic solvent is composed of choline chloride and ethylene glycol or 1,4-butanediol with a molar ratio of 1:

3.

3. The method of claim 1, wherein the coffeein quinic acid is extracted from Blumea fimbriata by the following steps of: The ultrasonic treatment is performed at a power of 100-800 W, a frequency of 40 kHZ, a temperature of 30-80 DEG C, and a time of 10-60 min.

4. The method of claim 1, wherein the coffeein quinic acid is extracted from Blumea fimbriata by the following steps of: The ratio of the mixture of the farnesyl eupatorium fortune powder and the eutectic solvent is 1 g:10-200 mL.

5. The method of claim 1, wherein the coffeein quinic acid is extracted from Blumea fimbriata by the following steps of: The concentration of the methanol solution is 50% by mass.

6. The method of claim 1, wherein the coffeein quinic acid is extracted from Blumea fimbriata by the following steps of: The filtration is performed using a 0.22 mu m microporous membrane.

Images