Biochar-based molecularly imprinted polymer as well as preparation method and application thereof

Abstract

The invention provides a biochar-based molecularly imprinted polymer, which is prepared by taking orange peel biochar as a carrier, hesperidin as a template molecule, acrylamide as a functional monomer, ethylene glycol dimethacrylate as a cross-linking agent and azodiisobutyronitrile as an initiator, carrying out cross-linking polymerization in an N, N-dimethylformamide / methanol mixed solution, and then removing the template molecule to obtain the biochar-based molecularly imprinted polymer. And preparing. The biochar-based molecularly imprinted polymer prepared by the invention shows high adsorption selectivity to hesperidin, the selective imprinting factor is 2.49, and the biochar-based molecularly imprinted polymer can be used for specific adsorption, separation and loading of hesperidin.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of compound preparation, and particularly relates to a biochar-based molecular imprinting polymer, a preparation method and application thereof. BACKGROUND

[0002] Under the background of current industrial innovation, the utilization rate of renewable resources such as agricultural and food waste is showing a significant increase, among which fruit waste accounts for the highest proportion. Citrus fruits, as the world's major economic crops, are rich in essential nutrients such as carbohydrates and bioactive substances. Oranges, as the leading variety, account for more than 60% of the total citrus production. In the process of industrialized processing (such as juice, jam and jelly production), about 50% of fresh oranges are converted into by-products, of which orange peel accounts for 65%. Therefore, achieving efficient resource utilization of orange peel has become an important issue. The traditional disposal method of orange peel is landfill and burning, but this will have harmful effects on the environment. Therefore, using a reasonable method of reusing orange peel not only can reduce its threat to the environment, but also can turn waste into treasure, making orange peel have broad economic utilization value. The comprehensive recycling ways of orange peel include extracting active ingredients, preparing adsorption materials, etc.

[0003] Hesperidin is a flavonoid compound, widely exists in lemon, citrus, daye flower and other fruit peels, has many pharmacological activities such as antioxidant, anti-inflammatory, anticancer, antibacterial, analgesic, immunomodulatory, neuroprotective and so on. Moreover, hesperidin also has great potential in the treatment of osteoporosis, obesity and diabetes and other diseases. However, hesperidin has low solubility and bioavailability, which affects its tissue distribution in vivo and limits the exertion of its pharmacological activity. Oral hesperidin can enter the intestinal tract, and the expression of UDP-glucuronosyltransferase and sulfotransferase in the intestinal tract can metabolize flavonoid compounds such as hesperidin into glucuronide and sulfate, in addition, the intestinal tract also contains many other microbial groups, which can further metabolize hesperidin into phenolic acid or simple phenolic substances through the effects of C-ring cleavage, demethylation and dehydroxylation. Various metabolites of hesperidin also show many biological and pharmacological activities, one of which: homoeriodictyol, has the effect of promoting appetite. Another metabolite of hesperidin: eriodictyol, shows similar biological activity to hesperidin, including antioxidant, neuroprotective and anti-inflammatory properties. Hesperetin glucuronide is a phase II metabolite of hesperidin, which has significant anti-ultraviolet A damage activity. Therefore, it is crucial to develop a method that can specifically adsorb, separate and load hesperidin and its analogues. SUMMARY

[0004] Therefore, the present application aims to solve the technical problem of providing a biochar-based molecular imprinting polymer, a preparation method and application thereof, for the specific adsorption, separation and loading of hesperidin and its analogues.

[0005] The application provides a biochar-based molecularly imprinted polymer, a preparation method and application thereof, and is characterized in that: taking orange peel biochar as a carrier, hesperidin as a template molecule, acrylamide as a functional monomer, ethylene glycol dimethacrylate as a crosslinking agent, and azobisisobutyronitrile as an initiator, crosslinking polymerization is carried out in a dimethylformamide / methanol mixed solution, and then the template molecule is removed to obtain BC / MIP. N , N -If the template molecule is not added, a biochar-based non-molecularly imprinted polymer is prepared by using the same steps, and is denoted as BC / NIP.

[0006] The structural formula of the hesperidin is shown in formula I. Formula I.

[0007] Preferably, the orange peel biochar is prepared by using fresh orange peel through a limited oxygen pyrolysis method.

[0008] Preferably, the fresh orange peel is washed with tap water and distilled water, cut into small pieces with a length of 1-2 cm, filtered to remove a large amount of water, and dried in a blast drying oven at 60 DEG C for 12 h. After the dried orange peel pieces are crushed into powder by using a pulverizer, the powder is sieved through a 100-mesh sieve.

[0009] In some specific embodiments of the application, the limited oxygen pyrolysis process of the orange peel is as follows: 10 g of orange peel powder is placed in a crucible, compacted, covered with a lid, and placed in a muffle furnace. The pyrolysis temperature is increased to 600 DEG C at a heating rate of 10 DEG C / min, maintained for 3 h, and then decreased to room temperature at a rate of 5 DEG C / min. The obtained orange peel biochar is uniformly ground, washed with distilled water until the pH of the filtrate is close to 7, and then dried in a blast drying oven at 80 DEG C for 12 h. Finally, the dried orange peel biochar is uniformly ground, sieved through a 100-mesh sieve again, and used.

[0010] Preferably, the use amount ratio of the orange peel biochar, the template, the acrylamide, the ethylene glycol dimethacrylate and the azobisisobutyronitrile is (80-120) mg:(40-100) mg:(2-8) mmol:(10-16) mmol:(40-100) mg.

[0011] Further preferably, the use amount ratio is 100 mg:60 mg:4 mmol:12 mmol:60 mg.

[0012] Preferably, the solvent for the polymerization reaction is a mixed solution of dimethylformamide and methanol. N , N Preferably, the solvent for the polymerization reaction is a mixed solution of dimethylformamide and methanol.

[0013] Preferably, the hesperidin template molecule is first dissolved in dimethylformamide, and then methanol is added for the polymerization reaction. N , N Preferably, the hesperidin template molecule is first dissolved in dimethylformamide, and then methanol is added for the polymerization reaction.

[0014] The N , N The volume ratio of dimethylformamide to methanol is (1-4):(2-7).

[0015] In some specific embodiments of the present invention, the volume ratio of methanol to chloroform is 1:2, 1:3, 1:4, 2:5, or 4:7; preferably 1:4. At this ratio, the prepared biochar-based molecularly imprinted polymer exhibits high adsorption performance for hesperidin.

[0016] This invention provides a method for preparing biochar-based molecularly imprinted polymers, comprising the following steps: S1) Template molecules and functional monomers in N , N The prepolymerization is carried out in a mixed solution of dimethylformamide and methanol, wherein the template molecule is hesperidin and the functional monomer is acrylamide; S2) The prepolymerized solution is mixed with biochar, ethylene glycol dimethacrylate and azobisisobutyronitrile to carry out the polymerization reaction; S3) Wash the solid material obtained after the polymerization reaction with methanol / glacial acetic acid solution to remove the template molecules and obtain the biochar-based molecularly imprinted polymer.

[0017] The biochar is preferably orange peel biochar, prepared using fresh orange peel through an oxygen-limited pyrolysis method.

[0018] The preferred temperature for prepolymerization is 30°C.

[0019] In step S2), the polymerization reaction temperature is preferably 40-80℃, more preferably 60℃, and the polymerization time is 16-28h.

[0020] In a preferred embodiment of the present invention, after the polymerization reaction, the polymerization product is obtained by filtration and washed three times each in methanol and distilled water to remove unreacted substances and byproducts.

[0021] In step S3), the volume ratio of methanol to glacial acetic acid is preferably 7 to 10:1.

[0022] Experimental results show that the molecularly imprinted polymer of the orange peel biochar prepared in this invention as a carrier has a uniform core-shell structure, good thermal stability, and excellent adsorption and selectivity for hesperidin. It can be used for the specific adsorption, separation and loading of hesperidin and its analogues.

[0023] Furthermore, this includes daily chemical products containing specific adsorption, separation, and loading of hesperidin and its analogues, such as nutrient fortifiers, sunscreens, antibacterial agents, dispersants, and solubilizers.

[0024] Further, including orange / lemon / citrus / Day Day flower fruit peel waste adsorbent, stomach / intestine / blood / liver / kidney tissue biological sample processing agent, hesperidin metabolite adsorbent and in vivo drug delivery agent containing hesperidin and its analogs specific adsorption, separation and loading.

[0025] Compared with the prior art, the present application provides a biochar-based molecular imprinting polymer, taking orange peel biochar as the carrier, hesperidin as the template molecule, acrylamide as the functional monomer, ethylene glycol dimethacrylate as the crosslinking agent, and azobisisobutyronitrile as the initiator, which is prepared by crosslinking polymerization in a dimethylformamide / methanol mixed solution and then removing the template molecule. N , N The biochar-based molecular imprinting polymer prepared by the present application shows high adsorption capacity and selectivity (selective imprinting factor of 2.49) to hesperidin. In view of the large amount of by-product orange peel produced in the industrial processing process, the traditional treatment method will have harmful effects on the environment, and hesperidin is widely present in the peels of lemons, oranges, Day Day flowers and the like, and has many pharmacological activities such as antioxidant, anti-inflammatory, antibacterial and anticancer. Therefore, in the fields of nutritional supplements, sunscreen products, bacteriostatic agents, dispersants, cosmetology solubilizers, orange / lemon / citrus / Day Day flower fruit peel waste adsorbent, stomach / intestine / blood / liver / kidney tissue biological sample processing agent, hesperidin metabolite adsorbent and in vivo drug delivery agent, the biochar-based molecular imprinting polymer of the present application can not only reduce the threat of orange peel waste to the environment, turn waste into treasure, make the orange peel have broad utilization value, but also effectively solve the problems of low solubility / bioavailability of hesperidin and complicated traditional hesperidin extraction and separation steps, and fully exert the potential value of orange peel waste and the natural active ingredients contained therein. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 SEM and TEM images of BC / MIP and BC / NIP; Figure 2 IR spectra of orange peel biochar, BC / MIP and BC / NIP; Figure 3 TG analysis curve of BC / MIP and N2 adsorption and desorption curves of BC / MIP and BC / NIP; Figure 4 Column chart of the effects of the amounts of acrylamide, ethylene glycol dimethacrylate and hesperidin on the adsorption capacity and selectivity of BC / MIP; Figure 5 Column chart of the selective adsorption performance of BC / MIP; Figure 6 Column chart of the application of BC / MIP to separation and enrichment of hesperidin and its metabolites in tissue samples, and the column chart of the obtained tissue distribution results of hesperidin; Figure 7 BC / MIP for the separation and enrichment of hesperidine and its metabolites in tissue samples, and the resulting hesperidine metabolic pathway. DETAILED DESCRIPTION

[0027] To further illustrate the present application, the biochar-based molecularly imprinted polymer, the preparation method and the application thereof provided by the present application are described in detail below in conjunction with examples.

[0028] Example 1 Preparation of biochar-based molecularly imprinted polymer (1) Preparation of orange peel biochar The fresh orange peel was washed with tap water and distilled water in sequence, cut into small pieces of 1-2 cm, filtered to remove a large amount of water, and placed in a 60°C air oven for drying for 12 h. After the dried orange peel pieces were crushed into powder using a pulverizer, they were sieved through a 100-mesh sieve. Biochar was prepared using the limited oxygen pyrolysis method, and the pyrolysis process was as follows: 10 g of the orange peel powder was placed in a crucible, compacted, covered with a lid, and placed in a muffle furnace. The pyrolysis temperature was increased to 600°C at a heating rate of 10°C / min, maintained for 3 h, and then decreased to room temperature at a rate of 5°C / min. Since the orange peel biochar had obvious organic matter dissolved after the completion of the firing, which interfered with the subsequent experiments, the orange peel biochar was first ground uniformly, then washed with distilled water until the pH of the filtrate was close to 7, and then placed in a 80°C air oven for drying for 12 h. Finally, the dried orange peel biochar was ground uniformly, sieved through a 100-mesh sieve again, and used.

[0029] (2) Preparation of biochar-based molecularly imprinted polymer 60 mg of hesperidine template molecules were added to 4 mL of N,N-dimethylformamide, ultrasonically dissolved, and then 16 mL of methanol and 4 mmol of acrylamide were added and mixed uniformly. The mixture was placed in a 30°C oil bath, magnetically stirred for pre-polymerization for 4 h, and then 100 mg of biochar was added and stirred for another 2 h. 12 mmol of ethylene glycol dimethacrylate and 60 mg of azobisisobutyronitrile were added, and the reaction was carried out under a nitrogen atmosphere at 60°C for 24 h. The molecularly imprinted polymer was obtained by suction filtration, washed with methanol and distilled water for 3 times each, and then washed with a methanol / glacial acetic acid solution (9 / 1, v / v) for 12 h. During the washing, the hesperidine template molecules were removed by elution for a total of 6 times. After the elution was completed, the product was washed with methanol and distilled water for 3 times each, and then suction filtered and placed in a 50°C air oven for drying for 12 h to obtain the biochar-based molecularly imprinted polymer, which was denoted as BC / MIP. A biochar-based non-molecularly imprinted polymer was prepared by the same steps without adding the template molecules, and was denoted as BC / NIP. N , N

[0030] Example 2 Characterization of biochar-based molecularly imprinted polymer (1) Morphological characterization ​The morphology of BC / MIP and BC / NIP was studied by scanning electron microscopy and transmission electron microscopy, and the results are shown in Figure 1 .

[0031] Figure 1 In the figures, Figure A and Figure B are scanning electron micrographs of BC / NIP and BC / MIP, and Figure C and Figure D are transmission electron micrographs of BC / NIP and BC / MIP.

[0032] Figure 1 In the figures, Figure A and Figure B show that the surface of BC / MIP is rougher than that of BC / NIP, with many small pores, and Figure C and Figure D show that the molecular imprinting layer (gray) is coated on the surface of orange peel biochar (black), and because of the elution of the template molecule, the imprinting layer of BC / NIP is smoother than that of BC / MIP, and the imprinting layer of BC / MIP is uneven, proving that the preparation of core-shell structure biochar-based molecularly imprinted polymer is successful.

[0033] (2) Infrared spectrum analysis The functional groups on the surface of orange peel biochar, BC / MIP and BC / NIP were analyzed by infrared spectrum. The infrared spectrum has two regions, the functional group region 4000-1350 cm -1 and the fingerprint region 1350-400 cm -1 . The infrared spectrum of orange peel biochar, BC / MIP and BC / NIP is shown in Figure 2 .

[0034] Among them, Figure 2 In the figures, Figure a, Figure b and Figure c are the infrared spectrum of biochar, BC / MIP and BC / NIP, respectively.

[0035] Figure 2 It is shown that the absorption peak at 3445 cm -1 in curve a is the O-H stretching vibration peak, and 1059 cm -1 is the C-O stretching vibration peak of alcohol, indicating that the orange peel biochar contains alcohol organic matter. The above absorption peaks also appear in the FT-IR infrared spectrum of BC / MIP and BC / NIP (curves b and c), indicating the successful introduction of the surface orange peel biochar. In addition, the O-H stretching vibration peak is broadened, indicating that hydrogen bond interaction occurs, and the absorption peaks at 2990 cm -1 , 1729 cm -1 , 1636 cm -1 , 1160 cm -1 are attributed to C-H, C=O, C=C, C-O stretching vibration in crosslinking agent ethylene glycol dimethacrylate and functional monomer acrylamide, respectively. Based on the above results, it is proved that orange peel biochar, functional monomer and crosslinking agent participate in the synthesis of biochar-based molecularly imprinted polymer.

[0036] (3) Thermal gravimetric analysis Thermal gravimetric analysis was used to characterize the thermal stability of BC / MIP, the temperature was set to 30-800℃, and the temperature was raised at a rate of 10℃ / min under nitrogen atmosphere. Figure 3 Figure A shows the thermal gravimetric analysis curve of BC / MIP.

[0037] From Figure 3 As can be seen from Figure A, the mass of BC / MIP decreased by 1.42% in the temperature range of 30-60℃, which was mainly due to the loss of water. In the temperature range of 60-210℃, the mass loss of BC / MIP was very small, which indicated that BC / MIP was relatively stable and not easy to decompose under this temperature condition. In the temperature range of 210-450℃, the mass of BC / MIP lost 86.96%, which was attributed to the decomposition of the organic imprint layer on the surface of biochar. The experimental results showed that the thermal stability of BC / MIP was good within 210℃.

[0038] (4) Specific surface area and pore size analysis Specific surface area and porosity analysis was used to characterize the specific surface area and pore structure of BC / MIP and BC / NIP. Figure 3 Figure B shows the thermal gravimetric analysis curves of BC / MIP and BC / NIP.

[0039] Figure 3 As shown in Figure B, in the middle pressure section, the curve rose faster due to the occurrence of capillary condensation, and an adsorption hysteresis loop appeared, so the nitrogen adsorption-desorption isotherm curves of BC / MIP and BC / NIP belonged to type IV adsorption curve, indicating that there were mesoporous structures in the materials. The specific surface area, pore volume and average pore size of BC / MIP and BC / NIP are shown in Table 1, the specific surface area of BC / MIP and BC / NIP was 4.46m 2 / g and 1.60m 2 / g, respectively, the pore volume was 0.0059cm 3 / g and 0.0015cm 3 / g, respectively, and the average pore size was 5.31nm and 3.79nm, respectively. Compared with BC / NIP, BC / MIP had larger specific surface area, pore volume and average pore size, which was due to the formation of imprint cavities in the process of eluting the template molecules.

[0040] Table 1 Specific surface area, pore volume and average pore size of BC / MIP and BC / NIP Specific surface area (m 2 / g) Pore volume (cm 3 / g) Average pore diameter (nm) BC / MIP 4.46 0.0059 5.31 BC / NIP 1.60 0.0015 3.79 Example 3 Influence of the amount of functional monomer, crosslinking agent and template molecule BC / MIP and BC / NIP were prepared following the same steps as in Example 1, with the amount of the functional monomer acrylamide used being 2, 4, 6, and 8 mmol, respectively. The adsorption amount and selectivity of hesperidin by the products were tested. Each experiment was repeated in triplicate. The imprinting factor (IF) was used to evaluate the specific adsorption capacity of BC / MIP. The results are as follows: Figure 4 As shown in Figure A.

[0041] IF= Q BC / MIP / Q BC / NIP (4.1) in, Q BC / MIP (μg / g) and Q BC / NIP (μg / g) represents the adsorption capacity of hesperidin by BC / MIP and BC / NIP, respectively.

[0042] Figure 4 Figure A shows the bar chart of the effect of acrylamide dosage on the synthesis of BC / MIP and BC / NIP. It can be seen that with increasing acrylamide dosage, the imprinting factor (IF) value first increases and then decreases. This is because too little functional monomer will affect the stability of the prepolymer formed with the template molecule, thus affecting the subsequent generation of imprinted sites. Too much functional monomer will generate redundant non-imprinted sites. When the functional monomer dosage is 4 mmol, the material selectivity is the best; therefore, 4 mmol is the optimal dosage of functional monomer.

[0043] BC / MIP and BC / NIP were prepared following the same steps as in Example 1, with ethylene glycol dimethacrylate amounts of 10, 12, 14, and 16 mmol, respectively. The adsorption amount and selectivity of hesperidin by the products were tested. Each experiment was repeated three times in parallel, and the results are as follows: Figure 4 Figure B shows the effect of ethylene glycol dimethacrylate dosage on the synthesis of BC / MIP and BC / NIP. The results indicate that with increasing ethylene glycol dimethacrylate dosage, the IF (internal crosslinking agent) initially increases and then decreases. This is because a lower amount of crosslinking agent leads to poor mechanical stability of the subsequently formed molecularly imprinted polymer, while a higher amount results in excessive polymer rigidity due to over-crosslinking, making it difficult to elute the template molecules. The best material selectivity is observed when the ethylene glycol dimethacrylate dosage is 12 mmol; therefore, 12 mmol is the optimal amount of crosslinking agent.

[0044] BC / MIP and BC / NIP were prepared following the same steps as in Example 1, with the amount of hesperidin template molecule used being 40, 60, 80, and 100 mg, respectively. The amount and selectivity of hesperidin adsorbed by the product were tested. Each experiment was repeated in triplicate, and the results are as follows: Figure 4Figure C is a bar graph showing the effect of hesperidin dosage on the synthesis of BC / MIP and BC / NIP. The results show that as the dosage of hesperidin increases, the IF first increases and then decreases, and when the dosage of hesperidin is 60 mg, the IF reaches the highest value. This is because too much or too little dosage of hesperidin will affect the number of imprint sites, so 60 mg is the optimal dosage of template molecules.

[0045] Example 4 Selectivity Investigation Naringin and rutin were selected as structural analogues of hesperidin to perform specific adsorption experiments, and their structures are shown below, where a is the structure of hesperidin, b is the structure of naringin, and c is the structure of rutin.

[0046] a: b: c: Take 20 mg of BC / MIP or BC / NIP, add 5 mL of 100 mg / L hesperidin, naringin or rutin solution (solvent is dimethylformamide / methanol / water, 2 / 4 / 4), and place in a constant temperature shaker at 30°C for 12 h. Take the supernatant, then analyze the concentration of hesperidin, naringin and rutin in the supernatant by high performance liquid chromatography, and each group of experiments is repeated three times in parallel. The calculation method of the imprint factor IF value of BC / MIP is the same as equation (4.1), and the adsorption capacity of BC / MIP and BC / NIP is calculated by the following formula: N , N Q ( C 0- C e ) V / m (4.2) wherein, Q (μg / g) is the adsorption capacity, C 0(μg / mL) and C e (μg / mL) are the concentrations of hesperidin, naringin or rutin solution before and after adsorption, respectively, V (mL) is the volume of the adsorption solution; m (g) is the mass of BC / MIP and BC / NIP.

[0047] The experimental results are shown in Figure 5 , which are bar graphs of the selective adsorption of BC / MIP and BC / NIP. Figure 5

[0048] ​​The results show that the adsorption capacity and imprint factor of BC / MIP for hesperidin are higher than those of naringin and rutin, indicating that the imprint polymer has good imprint effect. Meanwhile, the adsorption effect of BC / NIP on hesperidin, naringin and rutin is equivalent, indicating that BC / NIP is a non-specific adsorption material. It can be predicted that the prepared biochar-based molecular imprinting polymer BC / MIP can be used for simultaneous enrichment of structurally similar molecules in complex matrix without the influence of other substances.

[0049] Further, the selective factor (β) is used to evaluate the specific adsorption capacity of BC / MIP.

[0050] β IF 模板 IF 类似物 wherein, IF 模板 and IF 类似物 are the imprint factors of BC / MIP for template molecules and structurally similar molecules, respectively.

[0051] The calculated imprint factors and selective factors are listed in Table 2. The selective factor β of BC / MIP for naringin is 1.441, and for rutin is 1.912. The selective factor for naringin is smaller than that for rutin, because compared with rutin, the molecular structure and groups of naringin are more similar to those of hesperidin. The double sugar units of both hesperidin and naringin are connected to the aglycone through the 7-O group, while the double sugar units in the structure of rutin are connected to quercetin through the 3-O group. The results prove that BC / MIP has high selectivity and adsorption capacity, and can be used for simultaneous separation and enrichment of hesperidin and its analogues from complex matrix.

[0052] Table 2. Selective adsorption related parameters of BC / MIP and BC / NIP Parameter Hesperidin Naringin Rutin Q BC / MIP (μg / g)]]> ​ 568.88 415.39 337.37 Q BC / NIP (μg / g) ​ 228.57 240.47 259.15 2.49 1.73 1.30 - 1.44 1.91 Example 5 Application (1) BC / MIP is used to study the tissue distribution of hesperidin in mice of different dose groups Dosing design: healthy male mice, body weight about 25 g each, fasting for 12 h before test, free water. Take BC / MIP loaded with hesperidin, add 0.3% carboxymethylcellulose sodium aqueous solution to prepare gavage liquid, and randomly divide the mice into low (10 mg / kg), medium (50 mg / kg), high (100 mg / kg) dose groups and blank control group, 3 mice in each group, and perform gavage.

[0053] ​​After 3h of intragastric administration, liver, kidney, stomach, and intestinal tissue samples were taken, cleaned with normal saline, and residual liquid was absorbed with filter paper. The samples were weighed, 2 times the amount of normal saline was added, and homogenized using a homogenizer. The homogenate was aliquoted and stored frozen. Blood was collected in a centrifuge tube containing sodium heparin, centrifuged at 5000r / min for 2min, and the upper light yellow plasma was stored frozen.

[0054] 0.5mL of each tissue homogenate was taken, 1mL of N , N dimethylformamide / methanol / water (2 / 4 / 4, v / v / v) solution was added, 0.1mL of plasma was taken, and 0.2mL of N , N dimethylformamide / methanol / water solution was added. After sealing, vortex for 5min, ultrasonic for 10min, centrifuge for 2min, and take the supernatant to a blank centrifuge tube (the above extraction operation was repeated twice). 2mL of tissue sample extract or 0.4mL of plasma sample extract was taken, 20mg of BC / MIP was added, and adsorbed on a 30℃, 105rpm shaker for 12h. Centrifugation obtained BC / MIP, added 250μL of methanol / glacial acetic acid (9 / 1, v / v) eluent, ultrasonic for 5min, centrifugation for 2min, obtained the upper eluent, repeated 4 times, combined the eluent, dried under nitrogen at 40℃ water bath, obtained the residue, added 1mL of N , N dimethylformamide, and detected by high performance liquid chromatography.

[0055] The experimental results are shown in Figure 6 , which show that in the three low, medium, and high dose groups, the distribution of hesperidin in the tissues shows the same trend, and the concentration of hesperidin in the stomach and intestine of mice is the highest, followed by blood, liver, and kidney, and the concentration of hesperidin in each tissue gradually increases with the increase of dose. The content of hesperidin in the stomach is the most abundant, and the concentration of hesperidin in the stomach and intestine is much higher than that in the blood, liver, and kidney, which is due to the low solubility and bioavailability of hesperidin, which affects its tissue distribution in vivo. In addition, this result also provides a side basis for the statement that "hesperidin can be used to prevent or improve intestinal inflammation, intestinal cancer, and other diseases".

[0056] (2) BC / MIP for studying the intestinal metabolic pathway of hesperidin The intestinal tissue sample extract of the high-dose group was detected using an ultra-high performance liquid chromatograph-mass spectrometer. The experimental conditions were as follows: the chromatographic column was Hypersil GOLDTM(100 mm x 2.1 mm x 3 µm), the flow rate was 0.3 mL / min, and the column temperature was 30 ℃. The mobile phase was 0.1% formic acid aqueous solution / acetonitrile (v / v), and gradient elution was performed (0-10 min, 95 / 5-80 / 20; 10-20 min, 80 / 20-65 / 35; 20-30 min, 65 / 35-40 / 60; 30-40 min, 40 / 60-5 / 95; 40-45 min, 5 / 95-95 / 5; 45-50 min, 95 / 5). The mass spectrometry ion mode was set to ESI + and ESI - , the scanning range was set to 50-750 m / z, the positive ion spray voltage was set to 3.5 KV, and the negative ion spray voltage was set to 3.0 KV.

[0057] The experimental results are shown in Table 1. Figure 7 The results show that the intestinal metabolic pathway of hesperidin mainly includes nine hesperidin metabolites in addition to hesperidin. First, hesperidin is hydrolyzed to remove one molecule of rhamnose to obtain compound N1, and then one molecule of glucose is removed to obtain compound N2, i.e., hesperetin. Subsequently, hesperetin is metabolized to obtain seven compounds N3-N9, mainly including phenolic substances and sulfates. The de-glycosylated compounds, de-methylated compounds, phenolic compounds, and sulfate compounds detected in the intestinal tissue may have an important contribution to the intestinal bioactivity of hesperidin.

[0058] The present application can be industrialized as a daily chemical product with the functions of specific adsorption, separation, and loading of hesperidin and its analogues, as a nutritional fortifier, sunscreen, bacteriostatic agent, dispersant, and cosolvent, or as an orange / lemon / citrus / daya flower peel waste adsorbent, a stomach / intestine / blood / liver / kidney tissue biological sample processing agent, a hesperidin metabolite adsorbent, and an in vivo drug delivery agent with the functions of specific adsorption, separation, and loading of hesperidin and its analogues.

[0059] The above examples are only used to help understand the method of the present application and its core idea. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A biochar-based molecularly imprinted polymer, which is prepared by cross-linking polymerization using biochar as a carrier, hesperidin as a template molecule, acrylamide as a functional monomer, ethylene glycol dimethacrylate as a cross-linking agent, and azobisisobutyronitrile as an initiator, followed by removal of the template molecule; The biochar is orange peel biochar prepared by oxygen-limited pyrolysis of fresh orange peel. The ratio of the carrier, template, functional monomer, azobisisobutyronitrile, and ethylene glycol dimethacrylate is (80-120) mg:(40-100) mg:(2-8) mmol:(10-16) mmol:(40-100) mg; The solvent for the polymerization reaction is N , N -A mixed solution of dimethylformamide and methanol; The N , N The volume ratio of dimethylformamide to methanol is 1:

4.

2. The method for preparing the biochar-based molecularly imprinted polymer according to claim 1, comprising the following steps: S1) Template molecules and functional monomers in N , N The prepolymerization is carried out in a mixed solution of dimethylformamide and methanol, wherein the template molecule is hesperidin and the functional monomer is acrylamide; S2) The prepolymerized solution is mixed with biochar, ethylene glycol dimethacrylate and azobisisobutyronitrile to carry out the polymerization reaction; S3) Wash the solid material obtained after the polymerization reaction with methanol / glacial acetic acid solution to remove the template molecules and obtain the biochar-based molecularly imprinted polymer.

3. The application of the biochar-based molecularly imprinted polymer according to claim 1 or the biochar-based molecularly imprinted polymer prepared by the preparation method according to claim 2 in the specific adsorption, separation and loading of hesperidin and its analogues.

4. The application according to claim 3, characterized in that: This includes daily chemical products containing specific adsorption, separation and loading of hesperidin and its analogues, such as nutrient fortifiers, sunscreens, antibacterial agents, dispersants and solubilizers.

5. The application according to claim 3, characterized in that, These include adsorbents for orange / lemon / citrus / bitter orange peel waste containing specific adsorption, separation and loading of hesperidin and its analogues; biological sample processing agents for gastric / intestinal / blood / liver / kidney tissues; adsorbents for hesperidin metabolites; and in vivo drug delivery agents.

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