Fructus amomi quality analysis method based on switchable deep eutectic solvent

Abstract

The invention discloses a fructus amomi quality analysis method based on a switchable deep eutectic solvent. The method specifically comprises the following steps: (1) collecting and crushing fructus amomi samples from different sources; (2) preparing SDES from caprylic acid and tetrabutylammonium bromide, and extracting phenolic compounds in fructus amomi; (3) determining the content of phenolic compounds; (4) digesting the fructus amomi by using SDES, and then determining the content of mineral elements in the fructus amomi; and (5) in combination with a content determination result, performing quality evaluation on the fructus amomi in different areas of Yunnan by adopting multivariate statistical analysis (correlation analysis, clustering analysis and principal component analysis). The method established in the invention has the advantages of rapidness, simplicity, convenience, high extraction efficiency, environmental protection and the like, and a comprehensive fructus amomi quality evaluation system based on SDES can provide certain reference for quality evaluation, quality control and safety analysis of fructus amomi.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and particularly relates to a quality analysis method of Amomum villosum based on a switchable deep eutectic solvent. Background Art

[0002] Amomum villosum is the dried mature fruit of Amomum villosum, an edible plant of the ginger family. It is rich in flavonoids, polysaccharides, volatile oils, terpenes and other active ingredients, and has pharmacological effects such as antioxidant, antibacterial, hypotensive and hypoglycemic. In recent years, research on Amomum villosum has mainly focused on the extraction process and pharmacological activity of volatile oils. At present, there are relatively few studies on the non-volatile components of Amomum villosum, and most of them use traditional ethanol and methanol extraction methods, which have problems such as low extraction rate, high energy consumption and long extraction time. Therefore, it is necessary to develop a new, green and sustainable extraction process.

[0003] Deep eutectic solvents (DES) are generally considered to be excellent alternative solvents for extracting natural products due to their easy synthesis, low cost, and environmental friendliness. Switchable deep eutectic solvents (SDES) are a type of DES whose properties (hydrophilicity and hydrophobicity) change when changing the system temperature, adjusting the system pH, and alternately bubbling CO2 / N2. The switching of SDES properties allows it to continuously complete the extraction and separation of target analytes in solution without the need for additional equipment assistance. The emergence of SDES simplifies the sample pretreatment steps and reduces the use of organic solvents, and has good application prospects in the extraction and separation of active components of natural products.

[0004] As the demand for medicinal materials increases year by year, their safety has received more and more attention. Therefore, the content of mineral elements has gradually become one of the important indicators for quality evaluation. Usually, the elements are determined by inductively coupled plasma optical emission spectrometry (ICP-OES) after digestion. Traditional digestion and microwave digestion have great risks in operation and are potentially harmful to operators and the environment. Neither is the ideal choice. In addition to being an extraction solvent, SDES can also be used as a digestion solvent because SDES can donate or accept electrons or protons to form hydrogen bonds, which gives SDES excellent solubility. With the assistance of ultrasound or stirring, SDES is sufficient to complete the digestion of solid samples, which not only eliminates some potential dangers, but is also more environmentally friendly and time-saving. Therefore, it is imperative to establish a comprehensive quality evaluation system for Amomum villosum based on SDES. Summary of the invention

[0005] The object of the present invention is to provide a quality analysis method of Amomum villosum based on a switchable deep eutectic solvent, which has the advantages of being rapid and simple, having high extraction efficiency and being green and environmentally friendly, and can provide a certain reference for the quality evaluation, quality control and safety analysis of Amomum villosum.

[0006] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for analyzing the quality of Amomum villosum based on a switchable deep eutectic solvent, which comprises the following steps:

[0008] (1) Collect and crush Amomum villosum samples from different sources;

[0009] (2) SDES was prepared using octanoic acid and tetrabutylammonium bromide, and phenolic compounds were extracted from Amomum villosum samples;

[0010] (3) determining the content of phenolic compounds in step (2);

[0011] (4) digesting the Amomum villosum sample using the SDES in step (2), and then digesting and determining the content of mineral elements in the Amomum villosum sample;

[0012] (5) Based on the content of phenolic compounds in step (3) and the content of mineral elements in step (4), multivariate statistical analysis is used to evaluate the quality of the Amomum villosum sample.

[0013] Furthermore, the specific steps of step (1) are: collecting Amomum villosum samples from different origins, marking the collected Amomum villosum samples, placing them in a cool and dry place, taking the dried mature fruits of the Amomum villosum medicinal material, crushing them, passing them through a 120-mesh sieve, and setting them aside.

[0014] Preferably, in step (2), the molar ratio of octanoic acid to tetrabutylammonium bromide is 1:3.

[0015] Furthermore, the specific steps of extracting phenolic compounds in step (2) are: weighing the powder of the Amomum villosum sample and adding it to SDES to mix evenly, then adding Na2CO3 solution, extracting at high temperature, then adding HNO3 solution to undergo phase change, then removing the SDES phase, adding methanol to dilute it, filtering it with a filter membrane, and taking the filtrate for HPLC analysis.

[0016] Furthermore, the volume of the SDES is 2.0 mL; the volume of the Na2CO3 solution is 1.3 mL; the auxiliary method is microwave-assisted; the extraction time is 4 min; and the extraction temperature is 78°C.

[0017] Furthermore, the phenolic compounds in step (2) include epicatechin, vanillic acid, catechin, quercetin, isoquercetin, and protocatechuic acid.

[0018] Furthermore, in step (3), HPLC is used to determine the content of phenolic compounds, and the specific chromatographic conditions are: Agilent Extend-C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: (A) methanol-(B) phosphoric acid water (0.1%), gradient elution: 0-5 min, 6%-10% A; 5-15 min, 10%-20% A; 15-25 min, 20%-30% A; 25-35 min, 30%-40% A; 35-45 min, 40% A, flow rate: 1 mL / min, column temperature: 30°C; detection wavelength: 260 nm for protocatechuic acid and vanillic acid; 256 nm for isoquercitrin and quercetin; 280 nm for catechin and epicatechin; injection volume: 10.0 μL, acquisition time: 45 min.

[0019] Furthermore, the digestion step in step (4) is as follows: weigh the Amomum villosum sample powder, add SDES, place it in an oil bath and magnetically stir to complete the digestion of the sample, then add HNO3 solution to the solution for ultrasonic treatment, centrifuge the sample to remove the clear liquid, filter it and dilute it with deionized water for subsequent ICP-OES determination.

[0020] Furthermore, the digestion conditions are as follows: auxiliary method: oil bath auxiliary; SDES usage volume: 2.0 mL; digestion temperature: 50°C; digestion time: 5 min; HNO3 solution volume: 5.0 mL.

[0021] Furthermore, in step (4), the mineral elements include P, Mg, Ca, Mn, Zn, Cu, and Ni.

[0022] Furthermore, in step (4), the determination conditions of the content of mineral elements in Amomum villosum are as follows: ICP radio frequency power: 1300 W; plasma gas flow rate (Ar): 15 L / min; auxiliary gas flow rate (Ar): 0.2 L / min; nebulizer gas flow rate (Ar): 0.55 L / min; pump injection volume: 1.5 mL / min; reading delay time: 40 s; repetition: 3 times; analysis spectral lines: Ni 231.60 nm, Cu 327.39 nm, Mn 257.61 nm, Ca 317.93 nm, Zn 206.29 nm, Mg 285.21 nm, P 213.61 nm.

[0023] Furthermore, the limit of detection (LOD) of the Amomum villosum digestion method in step (4) was in the range of 0.29–7.94 µg / L, the limit of quantification (LOQ) was in the range of 0.97–26.46 µg / L, and the inter-day precision (n = 3) and intra-day precision (n = 3) were both less than 5%.

[0024] Furthermore, in the step (4), the average contents of mineral elements are P > Mg > Ca > Mn > Zn > Cu > Ni from high to low.

[0025] Furthermore, the multivariate statistical analysis includes correlation analysis, cluster analysis and principal component analysis.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] 1. The present invention adopts for the first time a new green solvent SDES as an extraction solvent and a digestion solvent for the extraction and digestion of Amomum villosum phenolic compounds and mineral elements, respectively, and combines them with high performance liquid chromatography (HPLC) and ICP-OES, respectively, to establish a green and efficient SDES-based extraction process of Amomum villosum phenolic compounds and a mineral element digestion method, which have higher efficiency and precision than traditional methods, and are greener and more environmentally friendly.

[0028] 2. The present invention uses multivariate statistical analysis (correlation analysis, cluster analysis and principal component analysis) based on the content determination results to evaluate the quality of different batches of Amomum villosum in Yunnan Province. At the same time, the content of phenolic compounds and mineral elements is used as multivariate evaluation indicators, and the established comprehensive evaluation system for the quality of Amomum villosum is more comprehensive and systematic. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the extraction flow chart of phenolic compounds from Amomum villosum.

[0030] Figure 2 This is the digestion flow chart of mineral elements in Amomum villosum.

[0031] Figure 3 Characterization diagram of SDES 2 and single factor optimization diagram for extraction of phenolic compounds from Amomum villosum; (a) is the characterization diagram, (b) is the type of SDES, (c) is the molar ratio of SDES 2, (d) is the volume of SDES 2, (e) is the volume of Na2CO3 solution, (f) is the auxiliary method, (g) is the extraction time, and (h) is the extraction temperature.

[0032] Figure 4 3D diagram and contour diagram of the response surface optimization for the extraction of phenolic compounds from Amomum villosum; (a) and (b) are time-temperature; (c) and (d) are time-Na2CO3 volume; (e) and (f) are temperature-Na2CO3 volume.

[0033] Figure 5This is the single factor optimization diagram for the digestion of mineral elements in Amomum villosum; where (a) is the auxiliary method, (b) is the molar ratio of SDES 2, (c) is the volume of SDES 2, (d) is the digestion temperature, (e) is the digestion time, and (f) is the volume of HNO3 solution.

[0034] Figure 6 The figures are heat maps based on the correlation analysis of phenolic compounds and mineral element contents in Amomum villosum; (a) is the heat map for the correlation analysis of mineral elements and effective ingredients in Amomum villosum, (b) is the heat map for the correlation analysis of effective ingredients in Amomum villosum, and (c) is the heat map for the correlation analysis of mineral elements in Amomum villosum.

[0035] Figure 7 The heat map of cluster analysis based on the contents of phenolic compounds and mineral elements in Amomum villosum; (a) is the heat map of cluster analysis based on the content of phenolic compounds, and (b) is the heat map of cluster analysis based on the content of mineral elements.

[0036] Figure 8 The PCA diagrams are based on the contents of phenolic compounds and mineral elements in Amomum villosum; (a) is the PCA diagram based on phenolic compounds in Amomum villosum and (b) is the PCA diagram based on the contents of mineral elements. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described in detail with reference to the following specific examples.

[0038] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0039] Example 1: Sample collection and preparation

[0040] A total of 22 samples were collected and identified as Amomum villosum Lour., a plant of the Zingiberaceae family, mainly produced in Honghe Prefecture, Wenshan Prefecture, and Pu'er City, Yunnan Province (China). Detailed information is shown in Table 1. Amomum villosum was obtained from the dried mature fruits of Amomum villosum. Each sample was crushed after being marked, passed through a 120-mesh sieve, and set aside.

[0041] Table 1 Source information of Amomum villosum

[0042]

[0043] Example 2: Microwave-assisted SDES extraction of phenolic compounds from Amomum villosum

[0044] 1. Extraction and determination of phenolic compounds in Amomum villosum

[0045] Figure 1Schematic diagram of the extraction and determination of phenolic compounds from Amomum villosum. 0.2 g of Amomum villosum powder (SR0) was accurately weighed and mixed evenly with 2.0 mL of SDES 2 (tetrabutylammonium bromide and octanoic acid) with a molar ratio of 1:3 in a microwave tube. Then, Na2CO3 solution was added as a phase change trigger. At this time, SDES 2 was converted from hydrophobic to hydrophilic. The sample was placed in a microwave instrument for extraction at 80°C for 4 min, and then HNO3 solution was added for phase change. At this time, the fine organic droplets of SDES 2 produced quickly captured the target components and reduced to a hydrophobic form to complete the extraction. 0.6 mL of SDES phase was transferred, diluted with 0.4 mL of methanol, filtered with a 0.45 μm filter membrane, and the filtrate was sampled for HPLC analysis. The extraction rate was calculated as follows:

[0046]

[0047] Wherein, n: dilution factor; C: concentration of target component in extractant (mg / mL); V: volume of extractant (mL); M: weight of Amomum villosum powder (mg).

[0048] 2. Characterization of SDES 2

[0049] Figure 3 (a) shows the FT-IR spectra of octanoic acid, tetrabutylammonium bromide and synthesized SDES 2 (different ratios, after switching). It can be seen from the figure that after the synthesis of SDES, the stretching vibration of N–H in tetrabutylammonium bromide changes from 2958 cm -1 Blue shift to 2926cm -1 , and due to the formation of hydrogen bonds, the spectral band becomes wider; at the same time, the vibration of C–N decreases slightly compared with the original. The bending vibration of –OH in octanoic acid increases from 1412 cm -1 Blue shift to 1380 cm -1 The reason is that part of the electron cloud of the oxygen atom is transferred to the hydrogen bond; since the electron-withdrawing group –OH can increase the absorption wave number of the adjacent group, the vibration of C=O in octanoic acid increases from 1704 cm -1 Red shifted to 1727 cm -1 , and the spectral band becomes wider.

[0050] 3. Single factor optimization of extraction of phenolic compounds from Amomum villosum

[0051] (1) Types of SDES

[0052] Four different SDES (SDES 1: choline chloride and oxalic acid; SDES 2: tetrabutylammonium bromide and octanoic acid; SDES 3: choline chloride and L-malic acid; SDES 4: thymol and methanol) were selected as extraction solvents to investigate the extraction effect of SDES types on phenolic compounds. The results are shown in Figure 3 (b). The switching between the hydrophilicity and hydrophobicity of SDES is the main reason for the efficient extraction of target analytes, and the speed of SDES property switching is one of the important factors affecting the extraction efficiency. The carboxyl groups in the structures of SDES 1 and 3 are more acidic than the carboxyl groups in SDES 2. The switching speed is fast, and the analyte and the extraction solvent cannot fully contact, resulting in lower extraction efficiency. SDES 2 showed better extraction ability, so SDES 2 was selected as the best extraction solvent.

[0053] (2) SDES 2 molar ratio

[0054] The solubility of the extraction solvent to the target analyte and the permeability to the raw material have a decisive influence on the extraction process, and the volume ratio of the SDES components will affect the solubility and permeability of the solvent. Therefore, the extraction effect of SDES 2 on phenolic compounds at five different molar ratios (octanoic acid: tetrabutylammonium bromide were 1:1, 1:1.5, 1:2, 1:3, 1:4, respectively) was investigated. The results are shown in Figure 3 (c) By Figure 3 (c) It can be seen that the total extraction rate increases first and then decreases with the increase of the molar ratio, and the maximum value appears when the molar ratio is 1:3. As the molar ratio increases, the viscosity of the solvent will decrease, which is conducive to the dissolution of the target component. However, as the TBAB ratio increases further, the interaction between the two components weakens, resulting in a decrease in the extraction rate. Therefore, the best extraction solvent is SDES 2 with a molar ratio of 1:3.

[0055] (3) SDES 2 volume

[0056] For a certain mass of raw materials, a larger volume of extraction solvent can appropriately reduce the concentration of the target component, promote the mass transfer rate of the solid-liquid phase and increase the extraction rate of the target component. However, excessive extraction solvent increases the load of liquid-solid separation extraction or is too wasteful. Therefore, the extraction effect of different volumes of SDES 2 on phenolic compounds was investigated, and the results are shown in Figure 3 (d). The results show that the volume of SDES 2 has a great influence on the extraction rate, and the extraction rate reaches the maximum value when the volume is 2.0 mL, reaching mass transfer equilibrium and completely extracting the target compound. Therefore, 2.0 mL is selected as the optimal extraction volume.

[0057] (4) Volume of Na2CO3

[0058] Figure 3(e) The effect of Na2CO3 volume on the extraction of phenolic compounds. The results show that the extraction rate reaches the maximum value when the Na2CO3 volume is 1.0 mL. The reason is that when the Na2CO3 volume is small, SDES 2 cannot be completely switched, and its reduced contact with the target analyte leads to a lower extraction rate. When the Na2CO3 volume is large, the imbalance of the system's pH is not conducive to the dissolution of the target analyte. Therefore, 1.0 mL was selected as the optimal volume of Na2CO3 for further study.

[0059] (5) Auxiliary methods

[0060] The effects of ultrasound, oil bath, microwave and vortex on the extraction of phenolic compounds were investigated. Figure 3 (f). The results show that under certain material-liquid ratio, temperature and time, the effect of microwave-assisted extraction is more significant. Generally, the microwave-assisted method uses high-frequency vibration to cause the molecules in the plant to collide and squeeze with each other, which helps to quickly extract the target components. Further combination with solvent extraction can also shorten the extraction time and improve the extraction rate. At the same time, the literature shows that the microwave-assisted method can promote the dissolution of the target component under appropriate conditions without damaging the structure of the target component. Therefore, the microwave method was selected as the best extraction method.

[0061] (6) Extraction time

[0062] The effect of extraction time on the extraction of phenolic compounds was investigated in the range of 1–7 min. Figure 3 As shown in (g), the extraction rate of phenolic compounds increased within a certain period of time. However, further increase in time may lead to degradation of the target components and dissolution of impurities. Therefore, the extraction time was selected as 4 min.

[0063] (7) Extraction temperature

[0064] The results of the effect of temperature on the extraction of phenolic compounds are shown in Figure 3 (h). The results show that the total extraction rate reaches a maximum value at 80°C as the extraction temperature increases. Higher extraction temperatures often cause the loss of system solvents, resulting in a significant decrease in its ability to extract target components. Therefore, 80°C was selected as the optimal extraction temperature.

[0065] 4. Response surface methodology to further optimize the extraction conditions of phenolic compounds

[0066] (1) Box-Behnken (BBD) design and statistical analysis

[0067] The software Design-Expert 12 was used to further optimize the extraction time, extraction temperature and Na2CO3 volume to obtain the highest extraction rate of phenolic compounds. The experimental factors and levels are shown in Table 2, and the obtained variance analysis results are shown in Table 3. From the results, it can be seen that the regression model of the target is significant (p < 0.0001), and the lack of fit term is not significant (p > 0.05), indicating that the model has good experimental stability. The correlation coefficient of the established model R 2 The corrected determination coefficient R Adj 2 is 0.9877, which is consistent with R 2 The experimental model is close to the actual experiment. The results of the response surface test were subjected to multiple linear regression and binomial fitting analysis, and the regression equation was: Y = 0.1160 – 0.0267 A – 0.0239 B +0.0171 C + 0.0159 AB + 0.0275 AC + 0.0047 BC – 0.0227 A 2 – 0.0513 B 2 – 0.0127C 2 .

[0068] Table 2 Factors and levels of response surface experiment for the extraction of phenolic compounds from Amomum villosum

[0069]

[0070] Table 3 Results of variance analysis of response surface experiment

[0071]

[0072] Note: ** indicates extremely significant difference, p < 0.01; * indicates significant difference, p < 0.05.

[0073] (2) Analysis and verification of test results

[0074] In this work, the 3D response surface plot and contour plot based on the three-factor three-level interaction effect of A (time), B (temperature), and C (Na2CO3 volume) are shown in Figure 4As shown. The results show that the interaction between AB and BC is very significant when extracting phenolic compounds, while the interaction between AC is relatively mild. Among the three parameters, A has the greatest influence on the extraction of phenolic compounds, and the influence of the three factors is ranked as A > B > C. Under the ideal conditions given by the model (extraction time 4 min, extraction temperature 78 ° C, and added Na2CO3 volume of 1.3 mL), three repeated experiments were carried out, and the total extraction rate of phenolic compounds (0.1269%) had only a relative error of 0.71% with the predicted value (0.1260%), indicating that the experimental condition parameters are reliable.

[0075] 5. Determination results of phenolic compounds in Amomum villosum

[0076] The same mass of Amomum villosum medicinal materials from different batches were weighed, processed, measured and the content was calculated according to the same method. The measurement results are shown in Table 4.

[0077] Table 4 Determination results of phenolic compounds in Amomum villosum

[0078]

[0079] Example 3: Oil bath assisted digestion of Amomum villosum with SDES

[0080] 1. Oil bath assisted digestion of Amomum villosum with SDES

[0081] Figure 2 The following is a schematic diagram of the analytical detection procedure based on the SDES digestion method combined with ICP-OES. The digestion was carried out in a 50.0 mL round-bottom centrifuge tube. Weigh 0.1000 g of Amomum villosum sample powder, add 2.0 mL of SDES 2 with a molar ratio of 1:4, and place it in an oil bath for 5 min with magnetic stirring to complete the digestion of the sample. Then add 5.0 mL of 2 mol / L HNO3 solution to the solution and sonicate for 5 min. Finally, centrifuge the sample at 4000 rpm for 5 min, take 2.0 mL of the supernatant, filter it with a 0.45 μm membrane filter, and dilute it to 10.0 mL with deionized water for subsequent ICP-OES determination. The extraction rate calculation formula is as follows:

[0082]

[0083] Where n1: dilution factor of SDES digestion. SDES : Analyte concentration in SDES digestion (μg / mL). n2: Dilution factor in microwave digestion. MD : Analyte concentrations after microwave digestion (μg / mL).

[0084] 2. Single factor optimization

[0085] (1) Auxiliary methods

[0086] The effects of ultrasound, oil bath, microwave and vortex on the digestion of mineral elements in Amomum villosum were investigated. Figure 5 (a). Since the oil bath assist effect is relatively good, the oil bath assist is selected for further optimization.

[0087] (2) SDES 2 molar ratio

[0088] The digestion effects of 5 different molar ratios of SDES 2 on mineral elements were investigated. Figure 5 (b). As can be seen from the figure, the total extraction rate reaches its maximum value when the molar ratio is 1:4, and the extraction rates of the seven mineral elements are all above 90%. Studies have shown that the protons of the carboxyl group can act as a good receptor for metal complexes. As the molar ratio increases, the increased carboxyl groups in the SDES 2 structure improve the solubilization capacity of metal oxides in Amomum villosum. Therefore, the extraction rate increases with the increase in the molar ratio. However, the extraction rate begins to decrease at a higher molar ratio, and the final molar ratio is 1:4.

[0089] (3) SDES 2 volume

[0090] The effect of SDES 2 volume on sample digestion was investigated. Figure 5 (c) When the volume of SDES 2 was 2.0 mL, it was found that the extraction rates of the studied mineral elements were close to 100%; when the volume of SDES was greater than 2.0 mL, it was found that the recovery rate gradually decreased and more solvent was consumed. Therefore, 2.0 mL was selected as the optimal volume of SDES 2.

[0091] (4) Digestion temperature

[0092] The results of the effect of temperature on the digestion of mineral elements are shown in Figure 5 (d). When the temperature is 50℃, the extraction rate is close to 100%. At lower temperatures, the sample digestion is incomplete, resulting in relatively low digestion efficiency; above 50℃, the extraction rate of mineral elements decreases to varying degrees. Therefore, 50℃ is selected as the optimal digestion temperature.

[0093] (5) Digestion time

[0094] The results of the effect of time on the digestion of mineral elements are shown in Figure 5 (e). As can be seen from the figure, the extraction rate of mineral elements reaches the maximum at 5 min, but gradually decreases with the further increase of digestion time. Therefore, 5 min is selected as the optimal digestion time.

[0095] (6) HNO3 volume

[0096] The results of the investigation on the effect of HNO3 volume on the digestion of mineral elements are shown in Figure 5 (f). Acidic environment is conducive to the dissociation of metal ions from organic compounds. Therefore, adding HNO3 solution after SDES digestion can make the mineral elements dissolve more completely. When the volume of HNO3 is 5.0 mL, the extraction rate of the studied mineral elements is greater than 90%; and as the volume of HNO3 increases further, the extraction rate begins to decrease. Therefore, 5.0 mL is selected as the optimal volume for adding HNO3 solution.

[0097] 3. Analyze performance indicators

[0098] Under the optimized conditions, the analytical performance of the established digestion method was studied, and the results are shown in Table 5. The LODs of Ni, Cu, Mn, Ca, Zn, Mg, and P were in the range of 0.29–7.94 μg / L, and the LOQs were in the range of 0.97–26.46 μg / L. The inter-day precision (n = 3) defined by the relative standard deviation (RSD, %) was in the range of 1.37%–2.51%, the intra-day precision (n = 3) was in the range of 0.58–3.75%, and the R 2 Both are better than 0.9995.

[0099] Table 5 Analytical performance based on SDES digestion

[0100]

[0101] 4. Method Validation

[0102] The accuracy of the method was verified by adding 0.5, 1.0, and 1.5 times the actual content of the studied mineral elements to the sample (SR0), and the results are shown in Table 6. The recoveries of all samples were within the acceptable range of 91.6–108.5%, demonstrating the reliability of the developed method.

[0103] Table 6 SDES digestion actual sample spike recovery results

[0104]

[0105] 5. Determination results of mineral elements in Amomum villosum

[0106] Weigh different batches of Amomum villosum of the same mass, process and measure the samples in the same way, and calculate the content. The results are shown in Table 7. It can be seen from the table that there are differences in the content of mineral elements in different batches of Amomum villosum, and the average content is P > Mg > Ca > Mn > Zn > Cu > Ni. At the same time, referring to the limit standard of heavy metals in the 2020 edition of the Chinese Pharmacopoeia, the Cu content in batches SR13 and SR19 exceeds the prescribed content of the Chinese Pharmacopoeia (not exceeding 20 mg / kg), and measures should be taken to control it to reduce the pollution introduced during the planting process. The limit standards for other elements are not clearly specified.

[0107] Table 7 Determination results of mineral element contents in Amomum villosum

[0108]

[0109] a ND, not detected.

[0110] Example 4: Correlation analysis based on determination of effective components and mineral element contents in Amomum villosum

[0111] 1. Correlation analysis

[0112] Correlation analysis was used to evaluate the relationship between all quantified analytes, and the results were as follows Figure 6 As shown. The color bar on the right side of the figure represents the correlation coefficient. A correlation coefficient greater than zero indicates a positive correlation, a correlation coefficient less than zero indicates a negative correlation, and an absolute value of the correlation coefficient greater than 0.8 indicates a strong correlation, between 0.5–0.8 indicates a moderate correlation, between 0.3–0.5 indicates a general correlation, and less than 0.3 indicates a weak correlation or no correlation. The "*" in the figure indicates the degree of significant difference, and when the significance p < 0.05, it means that the correlation between the two is statistically significant.

[0113] Figure 6 (a) is the correlation analysis of mineral elements and effective components in Amomum villosum. T, I, Q, PA, VA, C and E on the left side of the figure represent the total extraction rate, isocarpide, quercetin, protocatechuic acid, vanillic acid, catechin and epicatechin, respectively. As can be seen from the figure, in the collected Amomum villosum, the content of isocarpide has a significant positive correlation with the content of Mn, Zn, Mg and P, and the content of protocatechuic acid has a very significant positive correlation with the content of Ni, Cu, Ca and Mg.

[0114] Figure 6 (b) is a heat map of correlation analysis of effective components in Amomum villosum. As can be seen from the figure, the total extraction rate of the six phenolic compounds in Amomum villosum has a very significant positive correlation with the content of catechin and epicatechin. At the same time, there is a very significant correlation between catechin and epicatechin, and between isoquercetin and quercetin.

[0115] Figure 6 (c) is the heat map of correlation analysis of mineral elements in Amomum villosum. It can be seen from the figure that the 7 mineral elements in Amomum villosum have different positive correlations, among which 15 pairs of elements show extremely significant (p < 0.01) positive correlations, and 9 pairs of elements show significant positive correlations (p < 0.05), indicating that the elements are not independent of each other and the enrichment has a synergistic effect.

[0116] 2. Cluster analysis

[0117] Cluster analysis can structure data into objects with common attributes for easy analysis and evaluation, while cluster heat maps can intuitively show the differences between data. Therefore, after normalizing the data on the content of phenolic compounds and mineral elements in Amomum villosum, hierarchical clustering was used as the clustering method, and Euclidean distance was used as the distance measurement method, and a cluster heat map was drawn, as shown in Figure 2. Figure 7 shown.

[0118] The differences in the content of phenolic compounds can be seen in Figure 7 (a), 22 batches of Amomum villosum samples were divided into three groups. SR2, SR5, SR8, SR9 and SR17–18 were grouped into the first group, and the contents of catechin and epicatechin in these six batches were relatively high; SR3, SR13 and SR22 were grouped into the second group, and the contents of isocarpide and quercetin in these three batches were relatively high; SR1, SR4, SR6–7, SR10–12, SR14–16 and SR19–21 were grouped into the third group, and the contents of six phenolic compounds in these 13 batches were all in the middle. The results showed that there were certain differences between different batches of Amomum villosum, but there was no significant geographical difference.

[0119] The difference in mineral element content can be seen in Figure 7 (b), 22 batches of Amomum villosum samples were divided into three groups. SR2, SR5–7, SR9, SR12, SR14–17 and SR20–21 were grouped into the first group, and the overall content of mineral elements in this group of samples was relatively low; SR13 was grouped into the second group alone. As can be seen from the figure, the contents of 4 mineral elements in this batch of samples were all at high levels, and the Cu content exceeded the standard, indicating that SR13 was quite different from other batches of Amomum villosum. SR1, SR3–4, SR8, SR10~11, SR18–19 and SR22 were grouped into the third group, and the overall content of mineral elements in this group of samples was relatively high. The results show that the content of mineral elements in Amomum villosum varies greatly.

[0120] 3. Principal Component Analysis

[0121] The principal component analysis method can transform multiple indicators into several comprehensive indicators, simplifying complex data while retaining trends and patterns. In this study, the contents of phenolic compounds and mineral elements were used for principal component analysis. The analysis results are shown in Figure 8 As can be seen from the figure, all sample points are within the confidence ellipse, indicating that the measured samples are statistically significant.

[0122] The six phenolic compounds measured were mainly divided into two principal components, with a cumulative variance contribution rate of 71.8%, of which principal component 1 (PC1) contributed 51.2% and principal component 2 (PC2) contributed 20.6%. The shorter the distance between the sample points, the closer the similarity between the samples; as can be seen from the figure, only three batches of samples have relatively obvious differences. The arrows represent the original variables. The closer the original variables are, the stronger the positive correlation is. As can be seen from the figure, the correlation between isobarbital and quercetin, and between catechin and epicatechin is strong. The values ​​of the original variables corresponding to the arrows in the horizontal and vertical directions can respectively reflect the correlation of the variable with PC1 and PC2 (positive and negative correlation and its size). As can be seen from the figure, isocarpide, quercetin, catechin, and epicatechin have a greater contribution to PC1 and show a greater positive correlation, while protocatechuic acid has a smaller negative correlation with PC1; protocatechuic acid, isocarpide and quercetin have a greater positive correlation with PC2, while catechin, epicatechin and vanillic acid are negatively correlated with PC2.

[0123] The 7 mineral elements measured are mainly divided into 2 principal components, among which the contribution rate of principal component 1 (PC1) is 59.4%, the contribution rate of principal component 2 (PC2) is 18.5%, and the cumulative variance contribution rate is 77.9%, indicating that the first two principal components can represent 77.9% of the original data information of the elements. As can be seen from the figure, there is a strong correlation between Mg and P, Mn and P, and the 7 mineral elements show a large positive correlation with PC1, Ni, Cu, Ca are positively correlated with PC2, and the positive correlation between Ni and Cu and PC2 is large; Zn, Mn, Mg, P and PC2 are negatively correlated.

[0124] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A method for quality analysis of Amomum villosum based on switchable deep eutectic solvents, characterized in that: The steps include: (1) Collect and crush Amomum villosum samples from different sources; (2) SDES was prepared using octanoic acid and tetrabutylammonium bromide, and phenolic compounds were extracted from Amomum villosum samples; (3) determining the content of phenolic compounds in step (2); (4) digesting the Amomum villosum sample using the SDES in step (2), and then digesting and determining the content of mineral elements in the Amomum villosum sample; (5) Based on the content of phenolic compounds in step (3) and the content of mineral elements in step (4), multivariate statistical analysis is used to evaluate the quality of the Amomum villosum sample.

2. The method according to claim 1, characterized in that In the step (2), the molar ratio of octanoic acid to tetrabutylammonium bromide is 1:

3.

3. The method according to claim 1, characterized in that The specific steps of extracting phenolic compounds in step (2) are: weighing the powder of Amomum villosum sample and adding it to SDES to mix evenly, then adding Na2CO3 solution, extracting at high temperature, then adding HNO3 solution to carry out phase change, then removing the SDES phase, adding methanol to dilute it and filtering it with a filter membrane, and taking the filtrate to sample HPLC for analysis.

4. The method according to claim 3, characterized in that The volume of the SDES: 2.0 mL; the volume of the Na2CO3 solution: 1.3 mL; the auxiliary method: microwave-assisted; the extraction time: 4 min; the extraction temperature: 78°C.

5. The method according to claim 1, characterized in that The phenolic compounds in step (2) include epicatechin, vanillic acid, catechin, quercetin, isoquercetin, and protocatechuic acid.

6. The method according to claim 5, characterized in that In the step (3), HPLC is used to determine the content of phenolic compounds. The specific chromatographic conditions are: Agilent Extend-C18 chromatographic column; mobile phase: methanol A-0.1% phosphoric acid water B, gradient elution: 0-5 min, 6%-10% A; 5-15 min, 10%-20% A; 15-25 min, 20%-30% A; 25-35 min, 30%-40% A; 35-45 min, 40% A, flow rate: 1 mL / min, column temperature: 30°C; detection wavelength: 260 nm for protocatechuic acid and vanillic acid; 256 nm for isoquercetin and quercetin; 280 nm for catechin and epicatechin; injection volume: 10.0 μL, acquisition time: 45 min.

7. The method according to claim 1, characterized in that The digestion step in step (4) is as follows: weigh the Amomum villosum sample powder, add SDES, place it in an oil bath and magnetically stir to complete the digestion of the sample, then add HNO3 solution to the solution for ultrasonic treatment, centrifuge the sample to remove the clear liquid, filter it and dilute it with deionized water for subsequent ICP-OES determination.

8. The method according to claim 7, characterized in that The digestion conditions are as follows: auxiliary method: oil bath auxiliary; SDES usage volume: 2.0 mL; digestion temperature: 50°C; digestion time: 5 min; HNO3 solution volume: 5.0 mL.

9. The method according to claim 1, characterized in that: In the step (4), the mineral elements include P, Mg, Ca, Mn, Zn, Cu, and Ni; the determination conditions of the content of mineral elements in Amomum villosum are as follows: ICP radio frequency power: 1300 W; plasma gas flow rate (Ar): 15 L / min; auxiliary gas flow rate (Ar): 0.2 L / min; nebulizer gas flow rate (Ar): 0.55 L / min; pump injection volume: 1.5 mL / min; reading delay time: 40 s; repetition: 3 times; analysis spectral lines: Ni 231.60 nm, Cu 327.39 nm, Mn 257.61 nm, Ca 317.93 nm, Zn 206.29 nm, Mg 285.21 nm, P 213.61 nm.

10. The method according to claim 1, characterized in that The multivariate statistical analysis includes correlation analysis, cluster analysis and principal component analysis.

Images