Application of chitosan functionalized mesoporous carbon in extraction of flavonoids from rose flowers

Abstract

The application discloses application of chitosan functionalized mesoporous carbon in extraction of flavonoid compounds in rose flowers, and realizes rapid enrichment of the flavonoid compounds in the rose flowers by using the chitosan functionalized mesoporous carbon as an adsorbent self-assembled solid-phase extraction column. The chitosan functionalized mesoporous carbon greatly improves dispersibility and stability of the mesoporous carbon, and performs specific adsorption on the flavonoid compounds. The functionalized mesoporous carbon composite material mixed adsorbent has excellent extraction performance. The chitosan functionalized mesoporous carbon can enhance its stability and solubility, and the strong solubility of the composite material can promote rapid enrichment of target analytes. Therefore, the application can improve extraction efficiency of the flavonoid compounds in the rose flowers, and the process will use less toxic and volatile chemical solvents, and saves operation time.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of flavonoid extraction, and relates to an application method of a chitosan functionalized mesoporous carbon composite material self-assembly extraction column in extracting flavonoids in roses. BACKGROUND

[0002] Flavonoids are polyphenolic compounds widely present in fruits, vegetables and medicinal plants. Due to their small side effects, less drug resistance, high safety index and other characteristics, they have received more and more attention in the field of human nutrition and health. Flavonoids not only play an important role in plant stress resistance, but also have various benefits for human and animal health. Modern pharmacological studies have shown that flavonoids have significant antioxidant, anti-inflammatory, anticancer, cardiovascular protection, blood glucose reduction, antibacterial and antiviral effects, and can be used for the development of drugs for the treatment of related diseases. For example, quercetin can prevent inflammation and pain, protect the human body from heart disease, reduce blood pressure, control diabetes, and improve immunity. Rutin and kaempferol also have many biochemical effects and can be used as antioxidants, anticancer agents and anti-inflammatory agents. In addition, flavonoids have antioxidant activity and are used as preservatives, colorants and antioxidants in the food industry. Therefore, in order to better understand the role of flavonoids in plant protection, medicine and food safety, it is of great academic value and research significance to develop effective and sensitive methods for the efficient enrichment, separation and rapid detection of flavonoids.

[0003] In recent years, high-performance liquid chromatography, gas chromatography, capillary electrophoresis, supercritical fluid chromatography and spectrophotometry have become common methods for determining flavonoids. However, due to different contents of flavonoids, complex matrix effects and different actual samples, direct analysis is sometimes impossible. Solid phase extraction (SPE) is a very popular sample pretreatment method, which has the advantages of simple operation, high pre-concentration coefficient and less solvent consumption, and overcomes the above obstacles. The most widely used adsorbent in SPE method is commercial amorphous silica C 18 Due to the single interaction form with the target (mainly relying on hydrophobic interaction), it is difficult to effectively extract flavonoids with multiple polar groups. Therefore, researchers have modified silica with different compounds or developed new adsorbents to efficiently extract flavonoids. So far, some SPE adsorbents such as octadecylsilane-modified wormhole-like mesoporous silica, multi-walled carbon nanotubes and carbamate-modified silica have been successfully developed and applied to the extraction of flavonoids in different actual samples. However, new adsorbents with high selectivity, stability and recyclability for flavonoids still need to be further explored.

[0004] Mesoporous carbon (CMK-3) as a new type of porous material, has high surface area, uniform pore size distribution, high mechanical strength and good electrical conductivity, showing great application potential in the field of adsorption separation, catalysis, biology, electrical conductivity material and so on. Although the research of using CMK-3 as adsorbent to enrich analyte is more and more, its application is mainly limited by poor stability and poor dispersibility, resulting in low extraction efficiency of target analyte. Chitosan is a natural polysaccharide macromolecule with biocompatibility, non-toxicity and biodegradability. Chitosan contains rich hydroxyl and amino functional groups, and is often used as a surface modification material of nano-carrier. Chitosan modified mesoporous carbon can enhance the dispersibility and biocompatibility of the material, and has been widely used in wastewater treatment and enrichment and separation of bioactive ingredients. However, there is little research on the extraction performance of chitosan functionalized mesoporous carbon composite material as adsorbent for flavonoids in the prior art, and no literature has been reported. SUMMARY

[0005] The purpose of the present application is to provide an application of chitosan functionalized mesoporous carbon composite material in extracting flavonoids from rose flowers, which can quickly enrich flavonoids in rose flowers.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is: an application of chitosan functionalized mesoporous carbon in extracting flavonoids from rose flowers, wherein the chitosan functionalized mesoporous carbon is used as an adsorbent to self-assemble a solid phase extraction column, and quickly enriches flavonoids in rose flowers.

[0007] The present application realizes the rapid enrichment (12 min) of four kinds of flavonoids, rutin, auraptene, quercetin and kaempferol, in rose flowers by using chitosan functionalized mesoporous carbon composite material as an adsorbent to self-assemble a solid phase extraction column. The use of chitosan functionalized mesoporous carbon greatly improves the dispersibility and stability of mesoporous carbon, and performs specific adsorption on flavonoids. Finally, the dosage of functionalized mesoporous carbon composite material, pH, enrichment time and desorption time are optimized. The selectivity, stability and reusability are studied and discussed. Compared with the existing solid phase extraction column C18 and other methods on the market, the results show that the functionalized mesoporous carbon composite material has excellent extraction performance. Chitosan functionalized mesoporous carbon can enhance the stability and solubility of the functionalized mesoporous carbon composite material, and the strong solubility of the functionalized mesoporous carbon composite material can promote the rapid enrichment of target analyte. Therefore, the present application can improve the extraction efficiency of flavonoids in rose flowers, and the process will use less toxic and volatile chemical solvents and save operation time. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 is a schematic diagram of extracting flavonoids from rose flowers by using chitosan functionalized mesoporous carbon.

[0009] Figure 2 This is a graph showing the effect of the amount of composite material used on the recovery rate of flavonoids.

[0010] Figure 3 This is a curve showing the effect of the pH value of the test solution on the extraction rate of flavonoids by the composite material.

[0011] Figure 4 This is a graph showing the effect of phosphate content on the extraction rate of flavonoids.

[0012] Figure 5 This is a bar graph showing the effect of eluent type on the extraction rate of flavonoids.

[0013] Figure 6 This is a graph showing the effect of eluent volume on the extraction rate of flavonoids.

[0014] Figure 7 This is a comparison chart of the extraction efficiencies of chitosan, mesoporous carbon, and composite materials.

[0015] Figure 8 It is a bar graph that evaluates the stability of composite adsorbents under different experimental conditions.

[0016] Figure 9 This is a bar graph evaluating the reusability of composite adsorbents.

[0017] Figure 10 A comparison curve of the extraction performance of self-assembled extraction columns and commercial C18 columns for flavonoids. Detailed Implementation

[0018] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0019] Preparation of chitosan-functionalized mesoporous carbon composite materials

[0020] A certain mass of chitosan solid was weighed, added to double-distilled water, stirred, and then acetic acid (acetic acid to solution mass ratio of 1:100) was added dropwise. After magnetic stirring for 1 hour, the mixture was allowed to stand for several hours to prepare a pure chitosan solution with a mass percentage concentration of 1%. 20 mg of mesoporous carbon was weighed, added to 60 mL of double-distilled water, and shaken to completely dissolve the mesoporous carbon. The mesoporous carbon solution was then added dropwise to the pure chitosan solution. After stirring at room temperature for 24 hours, the mixture was centrifuged. The black product was then placed in a vacuum oven and vacuum dried to prepare a chitosan-functionalized mesoporous carbon composite material of the corresponding concentration (labeled CS / CMK-3).

[0021] 2. Solid-phase extraction procedure and chromatographic conditions

[0022] 10 mg of the synthesized chitosan-functionalized mesoporous carbon composite material was packed into a 3 mL solid-phase extraction (SPE) cartridge between two polyethylene sieve plates. The packaged SPE cartridge was then connected to the SPE device and activated sequentially with methanol (2 mL) and water (2 mL). The working solution (20 mL × 100 ng / mL) was then loaded at a flow rate of 1.2 mL / min. After sample loading, the analytes enriched on the SPE column were separated using a methanol solution containing phosphoric acid as the eluent at a flow rate of 0.2 mL / min. Finally, 20 μL of the used eluent was injected into an HPLC-UV system for analysis. Figure 1 .

[0023] The volume of phosphoric acid in 0.6 mL of eluent is 15% of the eluent volume.

[0024] The analysis of flavonoids was performed using an Agilent 1100 modular high-performance liquid chromatography system (Agilent Technologies, USA) equipped with a UV-Vis detector. Flavonoids were separated at 25 °C on a C18 column (Hedera ODS-2, 250 mm × 4.6 mm id, 5 μm). The mobile phase was a mixture of methanol and 0.25% (v / v) aqueous phosphoric acid (70 / 30, v / v), with a flow rate of 0.8 mL / min. UV detection was performed at 360 nm.

[0025] Selective research:

[0026] The selectivity of functionalized mesoporous carbon composites for flavonoids was investigated using five bisphenol compounds and six polycyclic aromatic hydrocarbons as interfering agents. 20 mL each of a flavonoid standard solution (100.0 μg / L), a bisphenol compound solution (100.0 μg / L), and a polycyclic aromatic hydrocarbon solution (100.0 μg / L) were prepared. After SPE treatment, the selectivity of the functionalized mesoporous carbon composite adsorbent for flavonoids was examined by monitoring changes in recovery rate.

[0027] Sample preparation

[0028] Rose petals were purchased from a local supermarket in Lanzhou as actual samples for analysis. The specific processing procedure was as follows: the rose petal sample was pulverized using a sample pulverizer, and 1 g of sample powder was dissolved in 10 mL of 60% ethanol solution by shaking until the powder was completely dissolved. Then, it was sonicated for 45 min at 40℃; after cooling to room temperature, the sample was diluted to a 50 mL volumetric flask, filtered through a 0.45 μm membrane, and stored at 4℃ for further SPE treatment.

[0029] To obtain optimal solid-phase extraction performance, key parameters such as adsorbent dosage, solution pH, phosphoric acid content, eluent solvent type, and eluent volume were optimized. Extraction efficiency under different conditions was evaluated using the extraction recovery rate (R%).

[0030] First, the extraction efficiency of functionalized mesoporous carbon composite adsorbents for flavonoids in the mass range of 2–16 mg was investigated, such as... Figure 2 The extraction recovery rate of flavonoids gradually increased with increasing adsorbent dosage. However, when the adsorbent dosage reached 8 mg, the recovery rate of flavonoids no longer changed significantly with further increases in adsorbent dosage. Therefore, to achieve optimal extraction efficiency while conserving adsorbent dosage, subsequent experiments used 8 mg of functionalized mesoporous carbon composite material.

[0031] The pH value of the sample not only affects the surface state of the functionalized mesoporous carbon composite material, but also the form in which flavonoids exist, thus affecting the enrichment capacity. Therefore, Figure 3 This is a schematic diagram illustrating the effect of test solutions with different pH values ​​(2.0 ~ 9.0) on flavonoid compounds on the functionalized mesoporous carbon composite adsorbent. From... Figure 3 It can be seen that the recovery rate (R%) is low at pH 2. This may be because the amino protonation of the functionalized mesoporous carbon composite material weakens its interaction with flavonoids in the low pH medium. When the pH is between 5.0 and 9.0, the recovery rate of flavonoids remains above 90%. This is mainly due to the enhanced hydrogen bonding interaction between the functionalized mesoporous carbon composite material and flavonoids as the pH increases, thus improving the extraction performance. Since the pH of the samples used in this experiment is between 5 and 7, subsequent experiments do not require pH adjustment.

[0032] Experiments showed that pure methanol could not completely elute flavonoids, such as... Figure 4 Considering that pH affects the form in which flavonoids exist, adding acid during extraction can weaken hydrogen bonding and electrostatic interactions, thus facilitating desorption. Therefore, the effect of adding different volumes of phosphoric acid (5–25%, v / v) to methanol solution on elution efficiency was tested. The results showed that when phosphoric acid (15% of the total volume of methanol and sulfuric acid) was added to methanol solution, all adsorbed flavonoids could be eluted; therefore, a methanol solution containing 15% phosphoric acid was chosen as the eluent. Furthermore, the elution efficiency of flavonoids was evaluated using three eluents containing 15% phosphoric acid (methanol, acetonitrile, and acetone). Figure 5 The results showed that methanol provided the best elution effect; therefore, acidic methanol solution was chosen as the desorption solvent. The effect of eluent volume (1.00–5.00 mL) was also investigated. Figure 6It can be seen that flavonoids can be completely eluted with a volume of 2.50 mL. Considering the dilution effect, the smaller the volume of the eluent, the higher the enrichment coefficient, i.e., the larger the peak area, thus improving the sensitivity. Therefore, to obtain higher sensitivity, a 2.50 mL methanol / phosphoric acid mixture was chosen as the eluent.

[0033] Figure 7 This image shows the enrichment effect of different composite materials as adsorbents on flavonoid yellow compounds under the same extraction conditions. Figure 7 The results show that, compared with chitosan and mesoporous carbon, chitosan-functionalized mesoporous carbon composites have a better enrichment effect on flavonoids. This is mainly due to the synergistic effect among the functionalized mesoporous carbon composites (covalent bonding, hydrogen bonding, and electrostatic interactions). Functionalized mesoporous carbon composites endow them with abundant porous structures, large specific surface areas, and active functional groups.

[0034] After the above-mentioned experimental optimization, under the optimized conditions, a solid-phase extraction column self-assembled by functionalized mesoporous carbon composite material was used in combination with high performance liquid chromatography to rapidly enrich and detect flavonoid spikes in the range of mass-volume concentration of 1–300 μg / L, and a quantitative analysis of the corresponding flavonoids was established, as shown in Table 1.

[0035] Table 1 Performance analysis of functionalized mesoporous carbon composites for determining flavonoids

[0036]

[0037] As shown in Table 1, the measured flavonoids exhibited good linearity (R0) within the mass-volume concentration range of 1–300 μg / L. 2 (≥0.9992). Meanwhile, the limits of detection and quantitation, based on signal-to-noise ratios of 3 and 10, were 0.12–0.35 μg / L and 0.40–1.20 μg / L, respectively, further demonstrating the high sensitivity of the functionalized mesoporous carbon composite material for detecting flavonoids. The intra-day and inter-day relative standard deviations (RSDs) were 1.88–4.66% and 2.56–4.70%, respectively, indicating good repeatability. These results preliminarily demonstrate that the functionalized mesoporous carbon composite material, as an adsorbent, exhibits excellent extraction performance in the self-assembled solid-phase extraction method for detecting flavonoids.

[0038] To further investigate the performance of functionalized mesoporous carbon composites as extraction materials, the stability of extraction columns self-assembled from synthesized functionalized mesoporous carbon composites in different environmental media was tested. First, the functionalized mesoporous carbon composite extraction columns were immersed in 1 mol / L HCl, pure water, and 1 mol / L NaOH for 3 h, respectively. Flavonoids were then extracted using the treated solid-phase extraction columns, and the stability was evaluated by changes in extraction recovery.Figure 8 As can be seen, the R% did not change significantly, indicating that the composite material has good chemical stability. The reproducibility of five batches of self-assembled solid-phase extraction columns was also investigated. The experimental results showed that the recovery rates of different batches did not differ significantly. Figure 9 This indicates that the functionalized mesoporous carbon composite material has good reproducibility. Furthermore, from... Figure 9 It can be seen that after seven repeated extraction experiments, the same extraction column still has a good R% (above 80%), which further demonstrates that the functionalized mesoporous carbon composite material has good reusability.

[0039] Further comparisons were made between the extraction performance of a functionalized mesoporous carbon composite self-assembled extraction column and a commercial C18 column for flavonoids, using rutin as a representative sample in spiking experiments (concentrations of 20 μg / L, 50 μg / L, and 100 μg / L). Figure 10 It was found that the extraction recovery rate of flavonoids using commercial C18 columns was lower than that using extraction columns self-assembled from functionalized mesoporous carbon composites. This further confirms the superior extraction performance of functionalized mesoporous carbon composites, as C18 columns primarily rely on hydrophobic interactions and cannot effectively extract flavonoids containing multipolar functional groups. Furthermore, the established solid-phase extraction method using functionalized mesoporous carbon composites was compared with existing methods for determining flavonoids, as shown in Table 2.

[0040] Table 2 Comparison with existing methods for determining flavonoid content

[0041]

[0042] Table 2 shows that using functionalized mesoporous carbon composite materials for the extraction of flavonoids has advantages such as good linear range, low detection limit, low dosage, and good extraction recovery.

[0043] The applicability of the established method was evaluated by determining the content of four flavonoids in actual rose samples, as shown in Table 3.

[0044] Table 3. Determination results and spiked recoveries of flavonoids in actual samples (n = 3)

[0045]

[0046] Table 3 shows that flavonoids were detected in different blank food samples. The content of quercetin was 35.6 μg / g, rutin and kaempferol were 68.5 and 14.3 μg / g, respectively, and hesperidin was 15.6 μg / g. Standard solutions of flavonoids with mass concentrations of 50.0 μg / g and 100.0 μg / g were added to actual samples, and the recoveries were analyzed by solid-phase extraction. The average recoveries of the four flavonoids ranged from 92.4% to 104.3%, with RSDs ranging from 0.72% to 6.35% (n=3). These results indicate that the solid-phase extraction method based on functionalized mesoporous carbon composites is least affected by matrix effects and exhibits high selective enrichment of flavonoids. Therefore, functionalized mesoporous carbon composites are also suitable for the rapid enrichment and extraction determination of flavonoids in other complex samples.

[0047] This invention, for the first time, utilizes a composite material synthesized from chitosan-functionalized mesoporous carbon as an excellent solid-phase extraction adsorbent. Combined with high-performance liquid chromatography (HPLC), a highly efficient method with good linearity, low detection limits, and low organic solvent consumption was established for the analysis of flavonoids in rose petals. Experimental results show that the functionalized mesoporous carbon composite material, as an adsorbent, achieved satisfactory results in terms of selectivity, stability, and reusability. Finally, the high-efficiency detection of flavonoids in rose petals validated its potential application value in food samples. Results indicate a good linear relationship within a concentration range of 1–300 μg / L, with an average recovery rate of 92.4%–104.3%, a relative standard deviation (RSD) <10, and minimal matrix influence on the detection results. In conclusion, the functionalized mesoporous carbon composite material is a promising adsorbent for extracting flavonoids from food. By optimizing parameters such as eluent type, solution pH, composite material dosage, and eluent volume, the experimental results showed that the average recovery rate of the spiked samples was 92.4%–104.3%, with a relative standard deviation (RSD) of <10%. The limits of detection and quantitation based on signal-to-noise ratios of 3 and 10 were 0.12–0.35 μg / L and 0.40–1.20 μg / L, respectively. Therefore, this invention provides a new strategy for the rapid separation and enrichment of flavonoids and has important practical value for the study of effective components of traditional Chinese medicine.

Claims

1. The application of chitosan-functionalized mesoporous carbon in the extraction of flavonoids from rose, wherein the chitosan-functionalized mesoporous carbon is used as an adsorbent to self-assemble a solid-phase extraction column for rapid enrichment of flavonoids from rose. The specific application is as follows: 10 mg of synthesized chitosan-functionalized mesoporous carbon composite material is filled into a 3 mL solid-phase extraction (SPE) cartridge between two polyethylene sieve plates. Then, the packaged SPE cartridge is connected to the SPE device and activated sequentially with methanol and water, respectively. Next, the working solution is loaded at a flow rate of 1.2 mL / min. After sample loading, a methanol solution containing phosphoric acid is used as the eluent, and the analytes enriched on the SPE column are separated at a flow rate of 0.2 mL / min. Finally, 20 μL of the used eluent is injected into an HPLC-UV system for analysis. Preparation of the test solution for loading the sample: The rose sample was crushed using a sample press. 1 g of sample powder was dissolved in 10 mL of 60% ethanol solution and shaken to completely dissolve the sample powder. Then, it was sonicated for 45 min at 40℃. After cooling to room temperature, it was diluted to a volumetric flask and filtered through a 0.45 μm membrane to obtain the sample. The test solution with a pH value of 5.0 to 9.0 was prepared using this sample.

2. The application of chitosan-functionalized mesoporous carbon as described in claim 1 in the extraction of flavonoids from rose, characterized in that, The amount of methanol and water used is 2 mL each.

3. The application of chitosan-functionalized mesoporous carbon as described in claim 1 in the extraction of flavonoids from rose, characterized in that, The working solution has a mass-volume concentration of 100 ng / mL and a volume of 20 mL.

4. The application of chitosan-functionalized mesoporous carbon as described in claim 1 in the extraction of rose flavonoids, characterized in that, The volume of phosphoric acid in 0.6 mL of eluent is 15% of the eluent volume.

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