Method for separating and purifying proanthocyanidins from lotus seed shell and application thereof

Abstract

This invention belongs to the field of analytical chemistry, specifically relating to a method and its application for separating and purifying proanthocyanidins from lotus seed shells. This invention uses mesoporous activated carbon as an adsorbent for dispersed solid-phase extraction, selectively adsorbing and removing alkaloids from lotus seed shells to purify proanthocyanidins, followed by analysis using ultrafluid chromatography-tandem triple quadrupole mass spectrometry (UPLC-QQQ-MS / MS). The mesoporous activated carbon used in this invention is biochar, rich in oxygen-containing groups on its surface, and has a large specific surface area. Adding mesoporous activated carbon to the lotus seed shell extract and performing one-step vortex dispersion adsorption to remove alkaloids achieves a removal rate of 53%-92%, and a proanthocyanidin recovery rate of 75%-90%. This method is simple, rapid, and low-cost. DPPH free radical scavenging experiments showed that the scavenging rate of proanthocyanidin extract from lotus seed shells increased from 48% to 56% after treatment with mesoporous activated carbon.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a method and its application for separating and purifying proanthocyanidins from lotus seed shells. Background Technology

[0002] Lotus (Nelumbo nucifera Gaertn.) is an aquatic herbaceous plant belonging to the genus Nelumbo in the family Nelumbo. During the processing of lotus seeds into food products, a large amount of lotus seed shells are easily produced as a byproduct. The lotus seed shells are hard and thick-walled, and most are burned by lotus farmers in paddy fields as fertilizer, resulting in low utilization and waste of lotus resources. Lotus seed shells contain various bioactive substances such as proanthocyanidins, alkaloids, and flavonoids. Proanthocyanidins (PCs), also known as condensed tannins, are a class of mixtures with different degrees of polymerization formed by the condensation of flavan-3-ol structural units through C-C bonds. They possess various pharmacological effects, including antioxidant, anti-inflammatory, anti-cancer, anti-obesity, and hypoglycemic properties.

[0003] Proanthocyanidins are typically obtained from natural products through solvent extraction and further purification. Conventional separation methods for proanthocyanidins are multi-step processes, such as macroporous resin methods, with packing materials mostly being AB-8, ZTC-1, D101, XAD-8, and D312 resins. These adsorbents adsorb and desorb proanthocyanidins to remove impurities, but this method is time-consuming, solvent-intensive, and costly.

[0004] Therefore, researching new, environmentally friendly, rapid, and simple methods for separating and purifying proanthocyanidins from lotus seed shells is of positive significance for promoting the high-value-added development of lotus seed shells. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a dispersion solid-phase extraction technique using carbon-based materials modified with oxygen-containing groups as adsorbents. The carbon-based materials modified with oxygen-containing groups can selectively adsorb and remove alkaloids and other substances from lotus seed shell extract, while effectively retaining proanthocyanidins in the extract.

[0006] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution:

[0007] A method for separating and purifying proanthocyanidins from lotus seed shells includes the following steps:

[0008] (S.1) Take lotus seed shells, freeze dry, pulverize and extract with solvent, vortex, stand, sonicate and centrifuge, and take the supernatant to obtain lotus seed shell extract;

[0009] (S.2) The lotus seed shell extract obtained in step (S.1) is diluted for the first time with the first diluent to obtain the diluted lotus seed shell extract. Then, carbon-based material modified with oxygen-containing groups is added and mixed evenly. The mixture is then vortexed and centrifuged to obtain the supernatant.

[0010] (S.3) Take the supernatant obtained in step (S.2), filter it, and add the second diluent for a second dilution to obtain the test solution.

[0011] This invention utilizes a carbon-based material with oxygen-containing groups on its surface to adsorb alkaloids and other impurities from lotus seed shells, thereby separating and purifying proanthocyanidins. This process requires only one step of vortex adsorption to remove alkaloids and other impurities, eliminating the need for elution. The operation is simple and consumes minimal organic solvents.

[0012] This invention uses a carbon-based material modified with oxygen-containing groups as an adsorbent for dispersed solid-phase extraction. The adsorbent interacts with alkaloids in lotus seed shells, adsorbing the alkaloids to achieve purification. The method is simple to operate, rapidly purifies proanthocyanidins, effectively improves the DPPH radical scavenging rate of total proanthocyanidins, and reduces matrix effects. This invention uses a carbon-based material modified with oxygen-containing groups as an adsorbent for dispersed solid-phase extraction to purify proanthocyanidins in lotus seed shells, which is environmentally friendly, simple, and efficient. Further analysis using ultrafluid chromatography-tandem triple quadrupole mass spectrometry (UPLC-QQQ-MS / MS) achieves the separation and purification of proanthocyanidins in lotus seed shells. This method is simple, rapid, non-toxic, and uses a small amount of organic solvent. This method can also be applied to other parts of the lotus, such as lotus leaves, lotus seeds, and lotus receptacles, as well as other plants such as tea leaves and pomegranate peels, which contain natural products of proanthocyanidins and alkaloids, and can purify proanthocyanidins in these plants, demonstrating strong practicality.

[0013] Preferably, the solvent used for extraction in step (S.1) is any one or a combination of aqueous solutions of ethyl lactate, aqueous solutions of chloroform, and aqueous solutions of ethanol.

[0014] The volume fraction of the solute in the solvent used for extraction in step (S.1) is 10-90%.

[0015] As a further preferred embodiment, the solvent used for extraction in step (S.1) is an aqueous solution of ethyl lactate.

[0016] Ethyl lactate is a bio-based solvent produced by microbial fermentation of crops such as corn and soybeans. It has the advantages of being renewable, completely degradable, and having high extraction efficiency, and is gradually replacing traditional solvents for extracting biological components from natural products.

[0017] Preferably, the reaction conditions for ultrasound in step (S.1) are as follows:

[0018] The ultrasonic temperature is 20–60℃, the ultrasonic power is 64–462W, and the ultrasonic time is 2–20min.

[0019] Preferably, the carbon-based material modified with oxygen-containing groups in step (S.2) contains any one or more combinations of carboxyl groups, aldehyde groups, and hydroxyl groups.

[0020] Preferably, in step (S.1), the lotus seed shells are crushed and then sieved through a 100-mesh sieve to obtain lotus seed shell powder.

[0021] Preferably, the carbon-based material modified with oxygen-containing groups in step (S.2) is mesoporous activated carbon.

[0022] Mesoporous activated carbon has good adsorption capacity for alkaloids in lotus seed shells, but does not adsorb the target compound proanthocyanidins.

[0023] Preferably, the ratio of the carbon-based material modified with oxygen-containing groups added in step (S.2) to the lotus seed shell extract dilution is 3:10-35 mg / mL.

[0024] Preferably, the reaction conditions for vortexing in step (S.2) are: vortexing time of 0 to 3 minutes.

[0025] As a further preferred option, the reaction conditions for the vortex in step (S.2) are: the vortex time is 3 to 180 s.

[0026] When the vortexing time is too short or no vortexing is performed, the carbon-based material modified with oxygen-containing groups cannot be completely dispersed in the lotus seed shell extract, making it difficult for the modified carbon-based material to mix thoroughly with the lotus seed shell extract. This leads to a decrease in the adsorption performance of the modified carbon-based material, further reducing the extraction efficiency of proanthocyanidins. When the vortexing time is too long, the modified carbon-based material is prone to breakage, which in turn affects subsequent separation and purification. In addition, prolonged vortexing can easily raise the temperature of the lotus seed shell extract, further reducing the stability of proanthocyanidins and even causing degradation, thus affecting the quality and purity of the final product and increasing energy consumption.

[0027] Preferably, the first diluent in step (S.2) is any one or a combination of an aqueous solution of ethanol, an aqueous solution of ethyl lactate, and an aqueous solution of isopropanol;

[0028] The second diluent added in step (S.3) is any one or a combination of aqueous solutions of acetonitrile, ethyl lactate, and isopropanol.

[0029] Preferably, the volume fraction of the solute in the first diluent in step (S.2) is 5-90%;

[0030] The volume fraction of the solute in the second diluent added in step (S.3) is 5-50%.

[0031] As a further preferred embodiment, the volume fraction of the solute in the first diluent in (S.2) is 10%.

[0032] As a further preferred embodiment, the solute volume fraction in the second diluent added in step (S.3) is 10%.

[0033] An aqueous solution of ethanol (ethanol:water = 1:9, v / v) was chosen as the first diluent because pure water has poor solubility for lotus seed shells, requiring the addition of an appropriate amount of ethanol to aid dissolution. A high proportion of ethanol was avoided because mesoporous activated carbon is a hydrophilic material. The mobile phase system for liquid chromatography-mass spectrometry (LC-MS) is acetonitrile-water, which needs to be close to the initial gradient for accurate results. Therefore, an aqueous solution of acetonitrile (acetonitrile:water = 1:9, v / v) was chosen as the second diluent for sample dilution during injection. This helps improve the response value and further ensures the accuracy of the detection data.

[0034] The method described above for separating and purifying proanthocyanidins from lotus seed shells is applied to the separation and purification of proanthocyanidins contained in plants.

[0035] As a preferred option, the method for separating and purifying proanthocyanidins in lotus seed shells as described above is applied to the separation and purification of proanthocyanidins contained in lotus leaves, lotus seeds, lotus receptacles, tea leaves, and pomegranate peels.

[0036] Therefore, the present invention has the following beneficial effects:

[0037] (1) The dispersion solid phase extraction method provided by the present invention, which uses mesoporous activated carbon modified with oxygen-containing groups as an adsorbent, can adsorb and remove alkaloids in lotus seed shell extract dilution in just one step of vortex dispersion solid phase extraction. The removal rate of 24 alkaloids is 53%-92%.

[0038] (2) The mesoporous activated carbon used in this invention is biomass char with a specific surface area of ​​1000-1400 m². 2 / g, containing carboxyl, aldehyde, hydroxyl and other groups, can form acid-base reactions with alkalis. The interaction between the mesoporous activated carbon and the alkaloids in the lotus seed shell is utilized to adsorb the alkaloids, thereby achieving the purpose of removing impurities and purifying.

[0039] (3) The method of the present invention can also be applied to other parts of the lotus, such as lotus leaves, lotus seeds and lotus receptacles, as well as other plants such as tea leaves, pomegranate peels, etc., which contain natural products of proanthocyanidins and alkaloids. The method can purify the proanthocyanidins in these plants and has strong practicality.

[0040] (4) This invention achieves the separation and purification of proanthocyanidins in lotus seed shells, and achieves a recovery rate of 75% to 90% for proanthocyanidins in lotus seed shells and a removal rate of 53% to 92% for alkaloids. Through the DPPH free radical scavenging experiment, it was found that after treatment with mesoporous activated carbon, the scavenging effect increased from 48% to 56%. Attached Figure Description

[0041] Figure 1 The adsorption isotherms are for apophenes (a) to (k) and trans-N-feruloyltyramine (l).

[0042] Figure 2 The adsorption isotherms are for monobenzylisoquinoline compounds (m) to (x).

[0043] Figure 3 Adsorption isotherms of 10 proanthocyanidins.

[0044] Figure 4 Figure (a) shows the adsorption kinetics curves of four alkaloids on mesoporous activated carbon;

[0045] Figure 4 Figure (b) shows the adsorption kinetics curves of six proanthocyanidins on mesoporous activated carbon.

[0046] Figure 5 Adsorption response diagrams for 24 alkaloids with different carbon-based materials and without the addition of carbon-based materials.

[0047] Figure 6 Adsorption response diagrams of 31 proanthocyanidins by different carbon-based materials and without the addition of carbon-based materials.

[0048] Figure 7 This is a SEM image of mesoporous activated carbon.

[0049] Figure 8 Figure (a) shows the removal rates of the four alkaloids under different vortex time conditions;

[0050] Figure 8 Figure (b) shows the adsorption rates of the six proanthocyanidins under different vortexing times.

[0051] Figure 9 The removal rate of 24 alkaloids was determined by the ratio of feed to liquid.

[0052] Figure 10 The recovery rate of 31 proanthocyanidins was determined by the ratio of the raw material to the liquid.

[0053] Figure 11 The DPPH free radical scavenging rate of lotus seed shell extract diluted with or without mesoporous activated carbon was compared to that with mesoporous activated carbon. Detailed Implementation

[0054] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0055] The liquid chromatography used in the following examples and comparative examples was Waters' ACQUITY PREMIERUPLC, and the mass spectrometer was a Waters Xevo TQ-XS mass spectrometer. Reagents used: Acetonitrile, chromatographic grade, Merck, Germany; formic acid, chromatographic grade, Aladdin Biochemical Technology Co., Ltd.; ethanol, chromatographic grade, TEDIA, USA; ethyl lactate, analytical grade, Sinopharm Chemical Reagent Co., Ltd. Carbon-based materials: waste residue carbon, resin carbon, mesoporous activated carbon, from Professor Li Ying's research group at Zhejiang University of Technology.

[0056] Example 1

[0057] A method for separating and purifying proanthocyanidins from lotus seed shells includes the following steps:

[0058] (S.1) Fresh, mature lotus seeds (selected from Jinhua City, Zhejiang Province) were obtained after manual peeling. The lotus seeds were then shelled to obtain lotus seed shells. The lotus seed shells were freeze-dried at -80℃, mechanically pulverized, and then passed through a 100-mesh sieve to obtain lotus seed shell powder. 1g of lotus seed shell powder was weighed and 10mL of an aqueous solution of ethyl lactate (wherein, ethyl lactate:water = 7:3, v / v) was added for extraction. The mixture was vortexed for 1min, allowed to stand for 10min, sonicated at 262W at 40℃ for 5min, cooled to room temperature, and centrifuged at 10000rpm at 4℃ for 5min. The supernatant was collected to obtain the lotus seed shell extract.

[0059] (S.2) The lotus seed shell extract obtained in step (S.1) was diluted 10 times with an aqueous solution of ethanol (ethanol:water = 1:9, v / v) to obtain a diluted lotus seed shell extract. Then, 3 mg of mesoporous activated carbon was added to 15 mL of the diluted lotus seed shell extract, vortexed for 1 min, and centrifuged at 10,000 rpm for 20 min at 15 °C to obtain the supernatant. The ratio of the added mesoporous activated carbon to the diluted lotus seed shell extract was 3:15 mg / mL.

[0060] (S.3) Take the supernatant obtained in step (S.2) and filter it through a 0.2 μm GHP membrane. Add an aqueous solution of acetonitrile (wherein, acetonitrile:water = 1:9, v / v) for a second dilution and dilute the supernatant 10 times to obtain the test solution for later use.

[0061] (S.4) Take the test solution obtained in step (S.3) and perform liquid chromatography-mass spectrometry (UPLC-MS / MS) detection. The UPLC-MS / MS detection conditions are as follows: mobile phase: phase A is acetonitrile, phase B is 0.1% formic acid water; elution gradient: 0-0.5 min, 94% B; 0.5-4 min, 94-90% B; 4-8 min, 90% B; 8-13 min, 90-85% B; 13-20 min, 85-40% B; 20-22.1 min, 40-5% B; 22.1-25 min, 5% B; 25-25.1 min, 5-94% B; 25.1-30 min, 94% B. The flow rate is 0.3 mL / min; the chromatographic column used is a Waters ACQUITY™ Premier HSS T3 column (2.1 × 100 mm, 1.8 μm); the injection volume is 1 μL.

[0062] (S.5) Detection of matrix effect: to verify the feasibility and effectiveness of the dispersion solid phase extraction method for separating and purifying proanthocyanidins in lotus seed shells.

[0063] Example 2

[0064] This embodiment provides a method for separating and purifying proanthocyanidins from lotus seed shells. In step (S.1), the extraction solvent is an aqueous solution of ethyl lactate (ethyl lactate:water = 1:9, v / v); the extraction is performed at 20°C with ultrasonication at 64W for 2 minutes. In step (S.2), the first diluent is an aqueous solution of ethanol (ethanol:water = 1:20, v / v); the ratio of added mesoporous activated carbon to the lotus seed shell extract dilution is 3:10 mg / mL; the vortexing time is 2 minutes. In step (S.3), the second diluent is an aqueous solution of acetonitrile (acetonitrile:water = 1:20, v / v). All other steps are the same as in Example 1.

[0065] Example 3

[0066] This embodiment provides a method for separating and purifying proanthocyanidins from lotus seed shells. In step (S.1), the extraction solvent is an aqueous solution of ethyl lactate (ethyl lactate:water = 9:1, v / v); the extraction is performed at 60°C and 462W for 20 min. In step (S.2), the first diluent is an aqueous solution of ethanol (ethanol:water = 9:1, v / v); the ratio of the added mesoporous activated carbon to the lotus seed shell extract dilution is 3:35 mg / mL; the vortexing time is 140 s. In step (S.3), the second diluent is an aqueous solution of acetonitrile (acetonitrile:water = 1:2, v / v). All other steps are the same as in Example 1.

[0067] Example 4

[0068] This embodiment provides a method for separating and purifying proanthocyanidins from lotus leaves. In step (S.1), 1g of lotus leaf powder stored at -20℃ is thawed and extracted with 10mL of an aqueous solution of ethyl lactate (wherein, ethyl lactate:water = 7:3, v / v) to further obtain a lotus receptacle extract. In step (S.2), 3mg of mesoporous activated carbon is added to 15mL of the diluted lotus leaf extract. All other steps are the same as in Example 1.

[0069] Example 5

[0070] The difference between this embodiment and Embodiment 1 is that:

[0071] This embodiment provides a method for separating and purifying proanthocyanidins from lotus seed shells, wherein the vortexing time in step (S.2) is 10 s. All other steps are the same as in Embodiment 1.

[0072] Example 6

[0073] The difference between this embodiment and Embodiment 1 is that:

[0074] This embodiment provides a method for separating and purifying proanthocyanidins from lotus seed shells, wherein the vortexing time in step (S.2) is 30 seconds. All other steps are the same as in Example 1.

[0075] Example 7

[0076] The difference between this embodiment and Embodiment 1 is that:

[0077] This embodiment provides a method for separating and purifying proanthocyanidins from lotus seed shells, wherein the vortexing time in step (S.2) is 180 s. All other steps are the same as in Example 1.

[0078] Example 8

[0079] The difference between this embodiment and Embodiment 1 is that:

[0080] This embodiment provides a method for separating and purifying proanthocyanidins from lotus seed shells, wherein the ratio of the mesoporous activated carbon added in step (S.2) to the diluted lotus seed shell extract is 3:10 mg / mL. All other steps are the same as in Example 1.

[0081] Example 9

[0082] The difference between this embodiment and Embodiment 1 is that:

[0083] This embodiment provides a method for separating and purifying proanthocyanidins from lotus seed shells, wherein the ratio of the mesoporous activated carbon added in step (S.2) to the diluted lotus seed shell extract is 3:20 mg / mL. All other steps are the same as in Example 1.

[0084] Example 10

[0085] The difference between this embodiment and Embodiment 1 is that:

[0086] This embodiment provides a method for separating and purifying proanthocyanidins from lotus seed shells, wherein the ratio of the mesoporous activated carbon added in step (S.2) to the diluted lotus seed shell extract is 3:25 mg / mL. All other steps are the same as in Example 1.

[0087] Example 11

[0088] The difference between this embodiment and Embodiment 1 is that:

[0089] This embodiment provides a method for separating and purifying proanthocyanidins from lotus seed shells, wherein the ratio of the mesoporous activated carbon added in step (S.2) to the diluted lotus seed shell extract is 3:30 mg / mL. Everything else is the same as in Example 1.

[0090] Example 12

[0091] The difference between this embodiment and Embodiment 1 is that:

[0092] This embodiment provides a method for separating and purifying proanthocyanidins from lotus seed shells, wherein the ratio of the mesoporous activated carbon added in step (S.2) to the diluted lotus seed shell extract is 3:35 mg / mL. All other steps are the same as in Example 1.

[0093] Comparative Example 1

[0094] The difference between this comparative example and Example 1 is as follows:

[0095] This comparative example provides a method for separating and purifying proanthocyanidins from lotus seed shells, wherein in step (S.2), no carbon-based material (i.e., no mesoporous activated carbon modified with oxygen-containing groups) is added to the lotus seed shell extract dilution. Everything else is the same as in Example 1.

[0096] Comparative Example 2

[0097] The difference between this comparative example and Example 1 is as follows:

[0098] This comparative example provides a method for separating and purifying proanthocyanidins from lotus seed shells, wherein in step (S.2), the mesoporous activated carbon is replaced with waste carbon residue. Everything else is the same as in Example 1.

[0099] Comparative Example 3

[0100] The difference between this comparative example and Example 1 is as follows:

[0101] This comparative example provides a method for separating and purifying proanthocyanidins from lotus seed shells, wherein in step (S.2), resin-bonded activated carbon is used instead of mesoporous activated carbon. Everything else is the same as in Example 1.

[0102] Comparative Example 4

[0103] The difference between this comparative example and Example 1 is as follows:

[0104] This comparative example provides a method for separating and purifying proanthocyanidins from lotus seed shells, wherein the vortexing time in step (S.2) is 3 seconds. All other steps are the same as in Example 1.

[0105] Detection of matrix effect

[0106] Lotus seed shells were pretreated according to the methods described in Example 1 and Comparative Example 1, respectively. Then, six proanthocyanidin standards at different concentrations (5, 10, 50, 100, 200, 400, and 800 ng / ml) were added, and UPLC-MS / MS analysis was performed. The slopes of standard curves established at the seven matrix spike concentrations and those in pure solvent were compared. The results are shown in Table 1.

[0107] Table 1: Slope Ratio of Dispersed Solid Phase Extraction

[0108]

[0109] Notes: Ⅰ: Matrix spiking curve after adding oxygen-containing modified mesoporous activated carbon to lotus seed shells; Ⅱ: Matrix spiking curve after not adding oxygen-containing modified mesoporous activated carbon to lotus seed shells.

[0110] Analysis of the data in Table 1 shows that the slope ratio of lotus seed shells treated with mesoporous activated carbon (Example 1) is better than that of the untreated lotus seed shells. The slope ratio of the treated lotus seed shells is between 107.36% and 117.72%, while the slope ratio of the untreated lotus seed shells is between 113.93% and 123.07%. This indicates that the dispersive solid-phase extraction method of the present invention can be used for the separation and purification of proanthocyanidins in lotus seed shells, and can slightly reduce the matrix effect in lotus seed shells.

[0111] Lotus seed shell test solutions and lotus leaf test solutions were obtained according to the methods in Examples 1 to 4, and then detected by liquid chromatography-mass spectrometry. The recovery rate of proanthocyanidins and the removal rate of alkaloids were calculated based on the peak areas detected.

[0112] The analytical results show that, using the dispersed solid-phase extraction method based on mesoporous activated carbon as described in this invention, the recovery rate of proanthocyanidins in the lotus seed shell test solution reached 75%–90%, and the removal rate of alkaloids reached 53%–92%. The recovery rate of proanthocyanidins in the lotus leaf test solution was between 81% and 93%, and the removal rate of alkaloids was between 9% and 53%. However, for the tested lotus leaves, the researchers found that the alkaloid content in lotus leaves was about 100 times higher than that in lotus seed shells. The proanthocyanidin content in lotus leaves was not significantly different from that in lotus seed shells, and the adsorption capacity of the material is limited, leading to a lower removal rate of alkaloids in lotus leaves. Increasing the amount of carbon-based material could be considered to remove alkaloids from lotus leaves. Furthermore, the above results for lotus leaves further demonstrate that the mesoporous activated carbon used as an adsorbent for dispersed solid-phase extraction provided by this invention can be used for the separation and purification of proanthocyanidins in lotus plants, and has good application and promotion value in analytical chemistry. Therefore, the method of this invention is practical.

[0113] [Adsorption Performance - Adsorption Isotherm Curve]

[0114] Lotus seed shell extract was obtained according to the method in Example 1. Using the equilibrium concentration of the lotus seed shell extract as the x-axis and the adsorption amount of the target analyte at the corresponding concentration as the y-axis, adsorption isotherms for 24 alkaloids and 10 proanthocyanidins were obtained at room temperature. The adsorption isotherms for apocynine (a) to (k) and trans-N-feruloyltyramine (l) are shown below. Figure 1 As shown. The adsorption isotherms of monobenzylisoquinoline derivatives (m) to (x) are shown in the figure. Figure 2 As shown in the figure. The adsorption isotherms of 10 proanthocyanidins are as follows: Figure 3 As shown.

[0115] from Figures 1-3 Analysis revealed that 25 mL of lotus seed shell extract contained 24 alkaloids and 10 proanthocyanidins. When the concentration of the 10 proanthocyanidins was between 0.01 mg / mL and 0.5 mg / mL, the mesoporous activated carbon rapidly adsorbed the compounds. As the concentration of the 10 proanthocyanidins increased from the initial concentration to 0.5 mg / mL to 1 mg / mL, the adsorption by the mesoporous activated carbon gradually slowed down. When the concentration of the 10 proanthocyanidins increased from the initial concentration to 1 mg / mL to 2 mg / mL, the adsorption by the mesoporous activated carbon reached a near-equilibrium state.

[0116] Simulated adsorption experiments can further understand the adsorption behavior of alkaloids and proanthocyanidins on mesoporous activated carbon, and adsorption isotherms reveal relevant information about the interaction between adsorbate and adsorbent. The experimental data were fitted using both Freundlich and Langmuir adsorption isotherms. The Langmuir isotherm is suitable for monolayer adsorption at homogeneous sites. Unlike the Langmuir isotherm, the Freundlich isotherm is suitable for multilayer adsorption at non-homogeneous sites.

[0117] Langmuir fitting equation: Q e =k L Q m C e / (1+k L C e (1)

[0118] Freundlich fitting equation: Q e =k F C e n (2)

[0119] In the above formula, C e Q is the concentration (μg / L) of the target analyte at adsorption equilibrium; e Q is the equilibrium adsorption capacity (μg / g) of the target analyte; m It is the saturated adsorption capacity of the target analyte (μg / g); k L It is the Langmuir equilibrium constant; k F is the Freundlich equilibrium constant; n is the concentration exponent.

[0120] The fitting results are shown in Tables 2, 3, and 4. The correlation coefficient r of the Langmuir fitting equation is... L A correlation coefficient greater than 0.98, higher than that of the Freundlich model, indicates that the adsorption of 24 alkaloids and 10 proanthocyanidins by mesoporous activated carbon conforms to the Langmuir adsorption model, exhibiting monolayer adsorption behavior with no lateral interactions between adsorbed molecules. The Langmuir adsorption isotherm fitting results in Tables 2, 3, and 4 show that the adsorption equilibrium constant (k0) of mesoporous activated carbon for alkaloids is... L The adsorption capacity of the alkaloids is much greater than that of the proanthocyanidins, indicating that the adsorption capacity of the alkaloids is stronger than that of the proanthocyanidins.

[0121] Table 2: Fitting results of adsorption isotherm curves of mesoporous activated carbon for 11 apophenes and trans-N-feruloyltyramine

[0122]

[0123] Table 3: Fitting results of adsorption isotherm curves of 12 monobenzylisoquinolines for mesoporous activated carbon

[0124]

[0125] Table 4: Fitting results of adsorption isotherm curves of 10 proanthocyanidins by mesoporous activated carbon

[0126]

[0127]

[0128] [Adsorption Performance - Adsorption Kinetics]

[0129] The lotus seed shells were treated according to the method described in Example 1. The adsorption kinetics curves of the four alkaloids and six proanthocyanidins are shown below. Figure 4 As shown. Among them, Figure 4 Figure (a) shows the adsorption kinetics curves of four alkaloids on mesoporous activated carbon; Figure 4 Figure (b) shows the adsorption kinetics curves of six proanthocyanidins by mesoporous activated carbon. The fitting results of the adsorption kinetics of four alkaloids by mesoporous activated carbon are shown in Table 5. The fitting results of the adsorption kinetics of six proanthocyanidins by mesoporous activated carbon are shown in Table 6.

[0130] from Figure 4 Analysis revealed that between 0 and 10 seconds, the adsorption capacity of mesoporous activated carbon for basiline, N-demethylnecholine, linderine, nuciferine, catechin, epigallocatechin, proanthocyanidins B1, B2, B3, and B4 increased rapidly. This is likely because, under the external influence of the vortex, the target substances quickly occupied the adsorption sites on the material, saturating them. When the adsorption and desorption processes of the target substances on the material reached a dynamic equilibrium (after 60 seconds), the adsorption rate gradually decreased, while the adsorption capacity remained almost constant, indicating that adsorption had essentially reached equilibrium.

[0131] The two most commonly studied kinetic models in adsorption process kinetics are pseudo-first-order kinetics and pseudo-second-order kinetics. To further understand the adsorption process of mesoporous activated carbon, pseudo-first-order and pseudo-second-order kinetics were used for fitting, and the pseudo-first-order and pseudo-second-order kinetic equations are shown in equations (3) and (4), respectively:

[0132] Ln / (Q e - Q t )=lnQ e - k1t (3)

[0133] t / Q t =1 / (k2Q) e 2) + t / Q e (4)

[0134] In the above formula: Q e Q is the amount of adsorption that reaches equilibrium (μg / g); t K is the adsorption amount at time t (μg / g); k1 is the pseudo-first-order kinetic adsorption rate constant (s). -1 k2 is the pseudo-second-order kinetic adsorption rate constant (g·μg); -1 ·s -1 ); t is the adsorption time (s).

[0135] Combining the adsorption kinetic results in Tables 5 and 6, the correlation coefficient r1 of the pseudo-first-order kinetics is greater than 0.8768, and the correlation coefficient r2 of the pseudo-second-order kinetics is greater than 0.999, indicating a very good fit. This suggests that the adsorption process of these four alkaloids and six proanthocyanidins by mesoporous activated carbon conforms to the pseudo-second-order kinetic model. (Q...) e,2 The theoretical simulation values ​​and actual measurements further demonstrate that the adsorption process conforms to pseudo-second-order kinetics.

[0136] Table 5: Adsorption kinetics fitting results of four alkaloids on mesoporous activated carbon

[0137]

[0138] Table 6: Adsorption kinetics fitting results of six proanthocyanidins by mesoporous activated carbon

[0139]

[0140] [Optimization of Dispersive Solid Phase Extraction Conditions - Selection of Adsorbents]

[0141] Lotus seed shells were treated according to the methods described in Example 1 and Comparative Examples 1-3, respectively. The adsorption performance of different carbon-based materials—mesoporous activated carbon (Example 1), resin-based activated carbon (Comparative Example 3), waste residue activated carbon (Comparative Example 2), and no carbon-based material added (Comparative Example 1)—on proanthocyanidins and alkaloids in lotus seed shell extract was investigated. The adsorption responses of different carbon-based materials and no carbon-based material added to 24 alkaloids are shown below. Figure 5 As shown. The adsorption responses of different carbon-based materials and those without carbon-based materials to 31 proanthocyanidins are as follows. Figure 6 As shown in the figure. The morphology of the mesoporous activated carbon was characterized by scanning electron microscopy (SEM). The SEM images of the mesoporous activated carbon are shown below. Figure 7As shown in Table 7, the content of functional groups on the surface of the mesoporous activated carbon was determined by the alkali titration method reported in the literature [Boehm, HPCarbon 1994, 32, 759]. The pH value of the mesoporous activated carbon was determined using a PHS-25 digital pH meter according to the method described in the literature [Hou Chunyan, Feng Liangrong, Li Zijian, Wang Zheng, Qiu Fali, Acta Chimica Sinica, 2009, 67(13): 1528]. The surface structural parameters of the mesoporous activated carbon are shown in Table 7 below.

[0142] Table 7: Surface structure parameters of mesoporous activated carbon

[0143] name <![CDATA[Specific surface area (m 2 / g)]]> COOH (mmol / g) COR (mmol / g) OH (mmol / g) pH Mesoporous activated carbon 1000-1400 0.06-0.81 0.02-0.52 0.1-0.22 3-7 .

[0144] Depend on Figure 7 It can be seen that the average pore size of mesoporous activated carbon is approximately 8-9 nm. Table 7 shows that the specific surface area of ​​mesoporous activated carbon is 1000 m². 2 / g-1400m 2 / g, its surface is rich in oxygen-containing groups (mostly carboxyl and hydroxyl groups), and the mesoporous activated carbon is slightly acidic. From Figures 5-6 Analysis shows that resin-based carbon and waste residue carbon, these two carbon-based materials, hardly adsorb the alkaloids and proanthocyanidins contained in lotus seed shell extract. From... Figures 5-6 As can be seen, mesoporous activated carbon can effectively adsorb alkaloids and a small amount of proanthocyanidins. Therefore, mesoporous activated carbon can be optimally selected as the adsorbent to remove alkaloids from lotus seed shells while retaining proanthocyanidins.

[0145] [Optimization of Dispersion Solid Phase Extraction Conditions - Selection of Vortex Time]

[0146] Vortexing time is a factor affecting the adsorption equilibrium of alkaloids and proanthocyanidins in lotus seed shells. Lotus seed shells were treated according to the methods described in Examples 1, 5-7, and Comparative Example 4. The removal rates of the four alkaloids and the adsorption rates of the six proanthocyanidins under different vortexing time conditions are shown below. Figure 8 As shown. Among them, Figure 8 Figure (a) shows the removal rates of the four alkaloids under different vortex time conditions; Figure 8 Figure (b) shows the adsorption rates of the six proanthocyanidins under different vortexing times.

[0147] from Figure 8It can be seen that when the vortexing time is 3-30 seconds, the removal rate of alkaloids and the adsorption rate of proanthocyanidins increase rapidly. During this time period, the mesoporous activated carbon dispersed in the lotus seed shell extract promotes the full adsorption of alkaloids by the mesoporous activated carbon. When the vortexing time changes to 60 seconds, the removal of alkaloids and the adsorption of proanthocyanidins gradually slow down. When the vortexing time changes to 180 seconds, the removal of alkaloids and the adsorption of proanthocyanidins remain almost unchanged. This indicates that the present invention only requires a vortexing time of 60 seconds to reach extraction equilibrium, saving operation time. Therefore, a vortexing time of 60 seconds is selected as the optimal adsorption time.

[0148] [Optimization of Dispersion Solid Phase Extraction Conditions - Selection of Feed-to-Liquid Ratio]

[0149] The material-to-liquid ratio is also a factor affecting the recovery rate of proanthocyanidins and the removal rate of alkaloids in lotus seed shells. Lotus seed shells were treated according to the methods described in Examples 1 and 8-12, respectively. The recovery rate of proanthocyanidins and the removal rate of alkaloids in different loading volumes (10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL) of the diluted lotus seed shell extract (i.e., the extract after the first dilution) were investigated using mesoporous activated carbon. The effect of the material-to-liquid ratio on the removal rate of 24 alkaloids is shown in the figure below. Figure 9 As shown in the figure. The recovery rates of 31 proanthocyanidins were compared between the solid-liquid ratio. Figure 10 As shown.

[0150] from Figures 9-10 Analysis showed that when the amount of mesoporous activated carbon was 3 mg and the volume of the lotus seed shell extract dilution was 15 mL (Example 1), the recovery rate of proanthocyanidins reached over 70%, while the removal rate of alkaloids was the highest. With increasing sample volume, the recovery rate of proanthocyanidins gradually increased, but the removal rate of alkaloids gradually decreased. To ensure that the recovery rate of proanthocyanidins was as high as possible (over 80%) and that the removal rate of alkaloids was also high, a sample volume of 15 mL for the lotus seed shell extract dilution was ultimately selected, with an optimal material-to-liquid ratio of 3:15 mg / mL.

[0151] Extraction recovery rate

[0152] The lotus seed shells were processed according to the method in Example 1. The extraction recoveries of 31 proanthocyanidins by mesoporous activated carbon are shown in Table 8. The removal rates of 11 apophene alkaloids and trans-N-feruloyltyramine by mesoporous activated carbon are shown in Table 9. The removal rates of 12 monobenzylisoquinoline alkaloids by mesoporous activated carbon are shown in Table 10.

[0153] Table 8: Extraction recovery rate of 31 proanthocyanidins by mesoporous activated carbon

[0154]

[0155]

[0156] Table 9: Removal rates of 11 apophene alkaloids and trans-N-feruloyltyramine by mesoporous activated carbon

[0157] compound Removal rate (%) compound Removal rate (%) Bapokaline 81.60±0.90 North American tulip tree alkaloid 90.61±0.28 N-Demethylhexaenoic acid 81.71±0.48 N-methyl-asimilobine-N-oxide 78.77±2.01 lotus leaf alkaloid 82.02±0.39 Graziven 53.76±1.77 Anthocarmine 90.66±0.89 Nuciferine-N-methanol 78.49±0.40 Lotus alkaloids 92.90±0.17 Pro-Nephrine 60.62±0.10 O-Demethylhexaenoic acid 82.12±1.46 trans-N-ferulotyramine 74.11±0.77

[0158] Table 10: Removal rate of 12 monobenzylisoquinoline alkaloids by mesoporous activated carbon

[0159]

[0160] Table 8 shows that the extraction recovery rates of 31 proanthocyanidins ranged from 75% to 90%, with 24 proanthocyanidins having a recovery rate greater than 80%. Tables 9 and 10 show that the removal rates of 24 alkaloids ranged from 53% to 92%, with 9 alkaloids having a removal rate greater than 80%. Tables 9 and 10 also indicate that mesoporous activated carbon showed good removal efficiency for most apophene alkaloids, especially for lotusine, anechoic acid, and tulip betaine, achieving removal rates exceeding 90%. However, mesoporous activated carbon showed poor removal efficiency for monobenzylisoquinoline alkaloids.

[0161] Analysis of the antioxidant activity of mesoporous activated carbon in adsorbing metabolites from lotus seed shells

[0162] The lotus seed shell extract dilutions were treated according to the methods described in Example 1 and Comparative Example 1, respectively. The DPPH free radical scavenging rates of the lotus seed shell extract dilutions treated with and without mesoporous activated carbon were compared as follows: Figure 11 As shown.

[0163] from Figure 11 It was found that the DPPH radical scavenging rate was 48% without the addition of mesoporous activated carbon (Comparative Example 1), while the DPPH radical scavenging rate was 56% after adsorption with mesoporous activated carbon (Example 1), significantly improving the DPPH radical scavenging rate. This indicates that the DPPH radical scavenging rate is enhanced after alkaloid removal by mesoporous activated carbon, and proanthocyanidins may be a potential antioxidant in lotus seed shell extract.

[0164] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.

Claims

1. A method for separating and purifying proanthocyanidins from lotus seed shells, characterized in that, Includes the following steps: (S.1) Take lotus seed shells, freeze dry, pulverize and extract with solvent, vortex, let stand, sonicate, centrifuge and take the supernatant to obtain lotus seed shell extract; (S.2) The lotus seed shell extract obtained in step (S.1) is diluted for the first time with the first diluent to obtain a diluted lotus seed shell extract. Then, carbon-based material modified with oxygen-containing groups is added and mixed evenly. The mixture is then vortexed and centrifuged to obtain a supernatant. The carbon-based material modified with oxygen-containing groups contains any one or more combinations of carboxyl groups, aldehyde groups, and hydroxyl groups. The carbon-based material modified with oxygen-containing groups is mesoporous activated carbon. (S.3) Take the supernatant obtained in step (S.2), filter it, and add the second diluent for a second dilution to obtain the test solution.

2. The method for separating and purifying proanthocyanidins from lotus seed shells according to claim 1, characterized in that, The solvent used for extraction in step (S.1) is any one or a combination of aqueous solutions of ethyl lactate, aqueous solutions of chloroform, and aqueous solutions of ethanol. The volume fraction of the solute in the solvent used for extraction in step (S.1) is 10-90%.

3. The method for separating and purifying proanthocyanidins in lotus seed shells according to claim 1, characterized in that, The reaction conditions for ultrasound in step (S.1) are as follows: The ultrasonic temperature is 20~60℃, the ultrasonic power is 64~462W, and the ultrasonic time is 2~20min.

4. The method for separating and purifying proanthocyanidins in lotus seed shells according to claim 1, characterized in that, In step (S.2), the ratio of the carbon-based material modified with oxygen-containing groups to the lotus seed shell extract dilution is 3:10~35 mg / mL.

5. The method for separating and purifying proanthocyanidins in lotus seed shells according to claim 1, characterized in that, The reaction conditions for the vortex in step (S.2) are: vortex time of 0~3min.

6. The method for separating and purifying proanthocyanidins in lotus seed shells according to claim 1, characterized in that, In step (S.2), the first diluent is any one or a combination of an aqueous solution of ethanol, an aqueous solution of ethyl lactate, and an aqueous solution of isopropanol. The second diluent added in step (S.3) is any one or a combination of aqueous solutions of acetonitrile, ethyl lactate, and isopropanol.

7. A method for separating and purifying proanthocyanidins from lotus seed shells according to claim 1 or 6, characterized in that, In step (S.2), the volume fraction of the solute in the first diluent is 5-90%; The volume fraction of the solute in the second diluent added in step (S.3) is 5-50%.

8. The method for separating and purifying proanthocyanidins in lotus seed shells as described in any one of claims 1 to 6, and its application in separating and purifying proanthocyanidins contained in plants.

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