Method for extracting catalase inhibitor from rapeseed cakes

Abstract

The invention relates to a method for extracting a catalase inhibitor from rapeseed cakes, and belongs to the technical field of plant extraction. The catalase inhibitor is extracted from rapeseed cakes, and the IC50 value of the catalase inhibitor to recombinant human catalase reaches 3.10 mg / mL or below; main active components of the composition comprise ferulic acid, p-coumaric acid, pelargonisin, sinapic acid and kaempferol. The substance with catalase inhibition capability is extracted from the rapeseed cake meal, a thought is provided for high-value conversion of the rapeseed cake meal, and the preparation method is simple and easy to industrialize.

Description

Technical Field

[0001] The invention relates to the technical field of plant extraction, in particular to a method for extracting a catalase inhibitor from rapeseed cake. Background Art

[0002] Catalase (CAT) is widely present in organisms that require oxygen to survive, and is mainly distributed in the peroxisomes of plant and animal cells. Catalase catalyzes the dismutation reaction of the substrate H2O2, decomposing H2O2 into O2 and H2O, thereby reducing the content of reactive oxygen species (ROS) in cells. When catalase is inhibited, ROS in cells and even in the body will continue to accumulate and begin to attack cells. When the ROS level rises to a certain concentration, it causes cytotoxicity and eventually leads to cell apoptosis, which provides a theoretical basis for the development of potential anticancer drugs by inhibiting enzyme activity. Therefore, in recent years, the research on CAT inhibitors has become increasingly popular.

[0003] At present, inhibitors of catalase have also been found in some natural active compounds. The document "Krych, Justyna, and Lidia Gebicka. "Catalase is inhibited by flavonoids." International journal of biological macromolecules 58 (2013): 148-153." reported the inhibitory effect of flavanols such as epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin gallate (EGCG) on bovine liver catalase, but there are few reports on the inhibitory effect of phenolic acid substances on catalase.

[0004] Rapeseed meal is a byproduct left after rapeseed oil is extracted. In most cases, rapeseed meal is only used as feed, fertilizer, fuel, etc., and the value of a large number of active substances in it has not been fully utilized and effectively explored. Chinese patent CN1680220A reports a method for extracting polyphenols from rapeseed meal, and the prepared polyphenols have antioxidant function. Extracting human catalase (hCAT) inhibitors from rapeseed meal can not only increase the added value of rapeseed meal, but also find potential lead compounds for cancer treatment.

[0005] At present, there is no report on the extraction of catalase inhibitors from rapeseed cake. Summary of the invention

[0006] In view of the problems in the prior art, the present invention proposes a method for extracting a catalase inhibitor from rapeseed meal. By isolating an extract having catalase inhibitory ability from rapeseed meal, it not only provides technical support for the high-value utilization of rapeseed meal, but also opens up a new direction for the development of catalase inhibitors.

[0007] The technical solution of the present invention is as follows:

[0008] A catalase inhibitor extracted from rapeseed meal, with an IC 50 The value reached below 3.10 mg / mL.

[0009] Preferably, the active ingredients in the catalase inhibitor include ferulic acid, p-coumaric acid, pelargonidin, sinapinic acid and kaempferol.

[0010] A method for extracting a catalase inhibitor from rapeseed cake comprises the following steps:

[0011] S1. Sample pretreatment: collect rapeseed cake, crush, sieve, dry and defatted;

[0012] S2, extraction: the rapeseed cake and ethanol solution are mixed in a solid-liquid ratio of 1:2-4, and ultrasonic extraction is performed to obtain an extract;

[0013] S3, post-treatment of the extract: after the extraction, centrifuge to obtain an ethanol extract; concentrate by rotary evaporation to obtain a crude extract; extract with ethyl acetate, and rotary evaporate and freeze-dry the organic phase to obtain a powder;

[0014] S4, purification of the target product: dissolving the powder in water, purifying and eluting with a macroporous resin to obtain an eluate, and subjecting the eluate to rotary evaporation and freeze-drying to obtain a powder;

[0015] S5. Purification of the target product by dextran gel LH-20: Take the powder obtained in step S4, dissolve it in alcohol, pass it through a 0.45 μm filter membrane, load it with a constant flow peristaltic pump at a flow rate of 500-1000 μL / min, purify it with dextran gel LH-20, and then elute it with 1.25BV double distilled water and ethanol solutions with volume fractions of 0%, 25%, 50%, 75% and 100% at 1-3 mL / min in sequence; select samples in tubes 5 to 13 in the 25% ethanol eluate for rotary evaporation and freeze-drying to obtain the catalase inhibitor.

[0016] Preferably, in step S1, the powder is sieved through a 60-80 mesh sieve after being crushed; and the drying temperature is 45° C.-55° C. and the drying time is 20-30 h.

[0017] Preferably, in step S1, the degreasing is degreasing with petroleum ether.

[0018] Preferably, in step S2, the concentration of the ethanol solution is 65-75%; the extraction time is 20-40 min, the number of extractions is 2-4 times; the extraction temperature is 20-40° C.; and the ultrasonic extraction power is 280-320W.

[0019] Preferably, in step S4, the specific process of purification and elution is:

[0020] Weigh 20g of pretreated AB-8 macroporous resin, fill the chromatographic column with wet method, and elute 1-2 column volumes with pure water; dissolve the crude rapeseed cake in ultrapure water, use a peristaltic pump at a flow rate of 0.5 column volume / hour to slowly load 0.5 column volume of extract solution, and open the piston switch below. Then use 1.25BV double distilled water and 0%, 20%, 40%, 60%, 80%, 100% ethanol solution with a volume fraction of 1-3mL / min to elute in sequence; collect samples in all tubes of 40% and 60% ethanol eluates, which are the eluates.

[0021] Preferably, the pretreatment method of Sephadex LH-20 comprises: taking Sephadex LH-20 dry powder, adding deionized water, and swelling at room temperature for more than 3 hours.

[0022] Preferably, the main active ingredients in the catalase inhibitor include ferulic acid, p-coumaric acid, pelargonidin, sinapinic acid and kaempferol.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention uses rapeseed meal, a natural plant byproduct, as raw material, fully realizing the resource utilization of waste, while significantly reducing production costs, meeting the requirements of green production, and providing a new idea for the high-value transformation of rapeseed meal.

[0025] (2) The rapeseed meal extract provided by the present invention has high catalase inhibition activity. After purification, IC 50 Can be reduced to below 3.10mg / mL.

[0026] (3) The present invention clarifies the active ingredients that inhibit catalase (such as ferulic acid, p-coumaric acid, sinapinic acid, pelargonidin, etc.), further improving the scientificity and accuracy of product activity research.

[0027] (4) The present invention provides a green, environmentally friendly, simple and efficient extraction and purification method, which further improves the economy and operability of the production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1The present invention provides a schematic diagram of a method for extracting and purifying a catalase inhibitor from rapeseed cake.

[0029] Figure 2 The effect of different elution components of AB-8 macroporous resin on CAT inhibitory activity.

[0030] Figure 3 is the elution curve of the rapeseed meal extract in Sephadex LH-20 of the present invention;

[0031] Among them, A is a 25% ethanol elution component, B is a 50% ethanol elution component, C is a 75% ethanol elution component, and D is a 100% ethanol elution component.

[0032] Figure 4 This is the effect of the rapeseed meal extract on the CAT inhibitory activity before and after purification in the present invention.

[0033] Figure 5 This is the purification result of recombinant h CAT.

[0034] Figure 6 It is the total ion current diagram of the rapeseed meal extract in the present invention.

[0035] Figure 7 This is a schematic diagram of the binding mode of ferulic acid, p-coumaric acid, sinapinic acid, pelargonidin, kaempferol, etc. with h CAT.

[0036] Among them, A is ferulic acid, B is p-coumaric acid, C is sinapinic acid, D is pelargonidin, and E is kaempferol. DETAILED DESCRIPTION

[0037] In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and easy to understand, the specific implementation of the present invention is described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In the embodiments, if specific conditions are not specified, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0039] Example 1

[0040] A method for extracting a catalase inhibitor from rapeseed cake comprises the following steps:

[0041] The extraction process is as follows Figure 1 As shown, 200g of crushed rapeseed cake was weighed, passed through a 100-mesh sieve, and dried at 50°C for 20 hours. According to the material-liquid ratio of 1:3, the rapeseed cake and 600mL of petroleum ether were placed in an ultrasonic-assisted extraction device for extraction, and the ultrasonic power was 300W, which was the fixed power of the machine. After 30 minutes of extraction, centrifuged at 25°C and 5000rpm for 15 minutes, and the precipitate was taken out. The defatting step was repeated three times. The defatted rapeseed cake was dried at 50°C for 24 hours for use. 200g of defatted rapeseed cake was taken, mixed with 600mL of 70% ethanol solution at a material-liquid ratio of 1:3, and extracted in an ultrasonic-assisted extraction device for 30 minutes, the extraction temperature was 30°C, and the ultrasonic power was 300W, which was the fixed power of the machine. After the extraction, the sample was centrifuged at 5000rpm for 15 minutes. All ethanol extracts were combined and rotary evaporated to remove all ethanol to obtain an aqueous phase. Add 3 volumes (relative to the volume of the aqueous phase) of ethyl acetate to the aqueous phase for extraction, separate the organic phase and the aqueous phase, and continue to extract twice with 3 volumes of ethyl acetate. Combine all ethyl acetate extracts, remove all ethyl acetate by rotary evaporation, and freeze-dry to obtain an extract.

[0042] Weigh 10g of the crude extract powder and dissolve it in ultrapure water (concentration 5mg / mL). The pretreatment of AB-8 resin includes: soaking in 95% ethanol for 24h, washing repeatedly until the filtrate is clear; then soaking in 4% sodium hydroxide and 4% hydrochloric acid solutions for 24h, washing with distilled water until neutral after each soaking, and finally soaking the resin in ultrapure water for later use. Load 100mL of sample solution into a 20g pretreated AB-8 macroporous resin column using a peristaltic pump at a flow rate of 0.5 column volume / hour. First elute with 2 column volumes of ultrapure water at a flow rate of 1 column volume / hour, and then elute with 20%, 40%, 60%, 80%, and 100% ethanol solutions. Figure 2 As shown, the inhibitor activity was higher in the 40% and 60% ethanol eluates, so the samples in all tubes of the 40% and 60% ethanol eluates were combined, and the collected eluates were concentrated by rotary evaporation and then freeze-dried.

[0043] Accurately weigh 0.5g of rapeseed extract sample purified by AB-8 macroporous resin, dissolve it with a small amount of 95% ethanol, filter the dissolved sample through a 0.45μm filter membrane, and load it with a constant flow peristaltic pump at a flow rate of 500-1000μL / min; then use 1.25BV double distilled water and ethanol solutions with volume fractions of 25%, 50%, 75%, and 100% at 1-3mL / min to elute in sequence, and collect the eluate in separate tubes, collecting 4mL in each tube, and determine the inhibitory activity of each tube of collected liquid on CAT. Figure 3As shown, the inhibitor activity in the samples of tubes 5-13 in the 25% ethanol eluate was higher, so the samples of tubes 5-13 in the 25% ethanol eluate were selected for rotary evaporation and freeze-dried storage to obtain the final catalase inhibitor, which was stored at -80°C.

[0044] Take out the crude extract and the sample powder separated and collected by the last dextran gel LH-20, and prepare them into sample solutions with concentration gradients of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 mg / mL with double distilled water, respectively, measure the activity of CAT in each tube, and then calculate the inhibition rate of CAT activity of each concentration sample. Figure 4 As shown, IC was calculated using GraphPad Prism 8.0.2 based on the inhibition rate-concentration curve. 50 IC values ​​of purified catalase inhibitors 50 The value was 3.10 mg / mL. Compared with the crude extract, the purification process significantly improved the activity of the inhibitor.

[0045] Example 2

[0046] The human catalase gene (hCAT) GenBank ID is 101093891, which was synthesized by General (Anhui) Biotechnology Co., Ltd. and inserted into the expression vector pET-15b through the restriction sites NdeI and XhoI to obtain the human catalase recombinant expression vector pET-15b-hCAT.

[0047] After the BL21 competent cells are thawed, take out the catalase recombinant expression plasmid (pET-15b-hCAT) DNA powder, dissolve it with sterile water after thawing, draw 3μL and add it to the centrifuge tube containing BL21 competent cells, mix gently, and let it stand on ice for 30min. Incubate in a 42℃ water bath for 90s, then quickly ice bath for 2min. Add 967μL LB liquid culture medium to the centrifuge tube, and form a 1mL system with plasmid and competent cells, and then place the centrifuge tube in a constant temperature shaker, 37℃, 160rpm / min shake for 60min. Centrifuge the sample at 4000rpm / min for 2min, discard most of the supernatant in the clean bench, and leave about 100aL to mix with the precipitate with a gun tip. In a culture dish containing LB solid culture medium containing ampicillin (AMP), gently spread it evenly with a coating tool. Place the culture dish in a constant temperature incubator and culture it overnight at 37℃.

[0048] Use a gun to pick up a single colony with good growth and inoculate it into a test tube containing LB medium containing AMP (100 mg / L). After the operation is completed, fix the test tube in a constant temperature shaker and culture it at 220 rpm / min and 37°C for 12-16 hours. Shake the bacterial solution in LB liquid medium with AMP added according to the ratio of bacterial solution: medium = 1:100, and then place the medium in a shaker and expand it at 220 rpm / min and 37°C for 4 hours. Add IPTG inducer with a final concentration of 0.5 mmol / L to the bacterial solution that has been expanded to A600 of about 0.8, and continue to culture at 30°C and 220 rpm / min for about 18 hours. Divide all the bacterial solution into 50 mL centrifuge tubes, centrifuge at 4°C and 12000 rpm / min for 10 minutes, and divide the supernatant and precipitate into aliquots. The precipitate was rinsed with 10 mM PBS buffer for 3 times, and then about 20 mL of PBS buffer was added, and then the bacteria were ultrasonically disrupted in an ice bath (400 W, 5 s ultrasonic / 5 s interval, total time 30 min). The disrupted bacterial solution was placed in a centrifuge and centrifuged at 4°C, 12000 rpm / min for 10-15 min.

[0049] First, wash the nickel column twice with 2 column volumes (Bed volume, BV) of ultrapure water, and then balance the column with 50mM imidazole. Load the supernatant collected after crushing and centrifugation into the nickel column. After the sample protein is hung on the nickel column, it is gradient eluted with 2BV of different concentrations of imidazole (50mM, 100mM, 250mM, 500mM). Collect the liquid eluted with 250mM imidazole, and the enzyme solution purified by the nickel column is loaded into the dialysis bag, and the dialysis bag is suspended and immersed in 1L dialysis solution (10mM PBS, pH 7.4) and stirred at 4°C for 8h. Dialyze 4 times in total, and the dialysis solution needs to be replaced each time. Store the solution in a refrigerator at -20 degrees for later use.

[0050] The results are as follows Figure 5 As shown, it was shown that 250 mM imidazole elution resulted in a relatively pure recombinant human catalase with a specific activity of 93.21 U / mg.

[0051] Example 3

[0052] After grinding the sample into powder, accurately weigh 50 mg, add 500 μL methanol and acetonitrile (1:1 / V) to dissolve, ultrasonicate for 30 minutes (with ice pack), centrifuge (20℃, 17000g, 15min), take the supernatant into a new 1.5mL EP tube, vacuum centrifuge at 35℃ to dry, add 100 μL 50% methanol aqueous solution (v:V), vortex until completely dissolved, centrifuge (4℃, 17000g, 15min), take 60 μL supernatant for sample loading. Thermo UltiMate 3000 ultra-high performance liquid phase was used, the chromatographic column was Waters HSS T3 (100*2.1mm, 1.8um), the column temperature was 40℃, the injection volume was 5 μL, the flow rate was 0.4mL / min, and the sample was placed in the 4℃ autosampler during the entire analysis process. Gradient elution was performed with 0.1% formic acid aqueous solution (A)-0.1% formic acid acetonitrile solution (B) as the mobile phase. The following gradient elution conditions were used: 0-1.5 min, 95% A; 1.5-2.5 min, 95%-90% A; 2.5-14 min, 90%-60%; 14-22 min, 60%-5% A; 22-23 min, 5% A; 23-23.1 min, 5%-95% A; 23.1-25 min, 95% A.

[0053] AB X500R Triple T0F mass spectrometer was used for primary and secondary mass spectrometry data acquisition. In each data acquisition cycle, the molecular ions with the strongest intensity greater than 100 were screened out to collect the corresponding secondary mass spectrometry data. The primary acquisition range was 50-1200, bombardment energy: 30eV, 10 secondary spectra every 50ms. The ESI ion source parameters were set as follows: nebulizer pressure (GS1): 60Psi, auxiliary gas pressure: 60Psi, air curtain pressure: 35Psi, temperature: 65℃, spray voltage: 5000V.

[0054] The collected data were converted into mzML format using Proteowizard software; peak identification, peak filtering and peak alignment were performed using MS-DIAL software; a data matrix containing information such as mass-to-charge ratio, retention time and signal intensity was obtained. The results are shown in the figure. Figure 6 Component identification was first confirmed based on the precise molecular weight (molecular weight error ≤ 30 ppm), and the collected MS / MS data were analyzed for substance identification using secondary mass spectrometry databases (such as MassBank, GNPS, RIKEN PlaSMA, BMDMS-NP, mzCloud, etc.).

[0055] Based on precise molecular weight, fragment ion information and database comparison, 16 chemical components with higher content were identified, including ferulic acid, p-coumaric acid, kaempferol, etc.

[0056] Example 4

[0057] The crystal structure of h CAT was obtained from the RCSB Protein Data Bank database (http: / / www.rcsb.org), PDB ID: 1DGG. h CAT was imported into Maestro, and the protein preparation wizard in Schrödinger was used with default parameters for protein preparation (to restore its crystallization to its native conformation). The Glide module was used, the force field was set to OPLS4, and the rest were default parameters for molecular docking. The binding mode of the ligand and receptor was predicted and scored by MM / GBSA for binding free energy prediction. The smaller the binding score, the better the binding effect. Finally, the molecular docking was performed using PyMOL software (www.pymol.org) to draw a diagram to analyze the binding mode and interaction between the ligand and the receptor. The binding modes of ferulic acid, p-coumaric acid, pelargonate, sinapinic acid, kaempferol, etc. with h CAT are shown in the figure. Figure 7 shown.

[0058] Among the 16 high-content compounds identified in rapeseed meal extract, five compounds with strong binding ability to catalase were screened out through docking score initial screening and binding free energy, including ferulic acid, p-coumaric acid, pelargonidin, sinapinic acid, and kaempferol. The docking scores and binding free energy calculation results of the top 16 compounds with catalase molecules are shown in Table 1. The smaller the value of binding free energy and molecular docking score, the stronger the intermolecular interaction ability. As can be seen from Table 1, the docking scores of ferulic acid, p-coumaric acid, pelargonidin, sinapinic acid, and kaempferol with catalase molecules are better than those of other compounds, and their binding free energies are all lower than -40 kJ / mol, indicating that they are the main inhibitory components.

[0059] Table 1. The docking results of the top 16 compounds extracted and purified from rapeseed meal with catalase

[0060]

[0061]

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The various technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, all possible combinations of the various technical features in the above-described embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. A catalase inhibitor, characterized in that Extracted from rapeseed meal, IC of recombinant human catalase 50 The value reached below 3.10 mg / mL.

2. The catalase inhibitor according to claim 1, characterized in that The active ingredients in the catalase inhibitor include ferulic acid, p-coumaric acid, pelargonidin, sinapinic acid and kaempferol.

3. A method for extracting a catalase inhibitor from rapeseed meal, characterized in that: The following steps are involved: S1. Sample pretreatment: collect rapeseed cake, crush, sieve, dry and defatted; S2, extraction: the rapeseed cake and ethanol solution are mixed in a solid-liquid ratio of 1:2-4, and ultrasonic extraction is performed to obtain an extract; S3, post-treatment of the extract: after the extraction, centrifuge to obtain an ethanol extract; concentrate by rotary evaporation to obtain a crude extract; extract with ethyl acetate, and rotary evaporate and freeze-dry the organic phase to obtain a powder; S4, purification of the target product: dissolving the powder in water, purifying and eluting with a macroporous resin to obtain an eluate, and subjecting the eluate to rotary evaporation and freeze-drying to obtain a powder; S5. Purification of the target product by Sephadex LH-20: Take the powder obtained in step S4, dissolve it in alcohol, filter it through a 0.45 μm filter membrane, load it through a constant flow peristaltic pump at a flow rate of 500-1000 aL / min, purify it through Sephadex LH-20, and then elute it with 1.25BV double distilled water and ethanol solutions with volume fractions of 0%, 25%, 50%, 75% and 100% at 1-3mL / min in sequence; select samples in tubes 5 to 13 in the 25% ethanol eluate for rotary evaporation and freeze-drying to obtain the catalase inhibitor.

4. The method according to claim 3, characterized in that In step S1, the powder is crushed and passed through a 60-80 mesh sieve; the drying temperature is 45° C.-55° C. and the drying time is 20-30 hours.

5. The method according to claim 3, characterized in that: In step S1, the degreasing is degreasing with petroleum ether.

6. The method according to claim 3, characterized in that: In step S2, the concentration of the ethanol solution is 65-75%; the extraction time is 20-40 minutes, the number of extractions is 2-4 times; the extraction temperature is 20-40° C.; and the ultrasonic extraction power is 280-320W.

7. The method according to claim 3, characterized in that In step S4, the specific process of purification and elution is: Weigh 20g of pretreated AB-8 macroporous resin, wet fill the chromatographic column, and elute with pure water for 1-2 column volumes; dissolve the crude rapeseed meal in ultrapure water, use a peristaltic pump at a flow rate of 0.5 column volume / hour, slowly load 0.5 column volume of extract solution, and open the piston switch below. Then use 1.25BV double distilled water and ethanol solutions with volume fractions of 0%, 20%, 40%, 60%, 80%, and 100% at 1-3mL / min to elute in sequence; Collect samples from all tubes of the 40% and 60% ethanol eluates to form the eluate.

8. The method according to claim 1, characterized in that: The pretreatment method of the Sephadex LH-20 comprises: taking Sephadex LH-20 dry powder, adding deionized water, and swelling at room temperature for more than 3 hours.

9. The method according to claim 1, characterized in that: The active ingredients in the catalase inhibitor include ferulic acid, p-coumaric acid, pelargonidin, sinapinic acid and kaempferol.

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