Camellia oleifera cake extract as well as preparation method and application thereof

Abstract

The invention relates to an oil tea cake extract as well as a preparation method and application thereof, and belongs to the technical field of plant extraction. The extract comprises more than 75% of flavonoid compounds, and the flavonoid compounds at least comprise greater than or equal to 29% of kaempferol, greater than or equal to 4.5% of luteolin-7-O-glucoside, greater than or equal to 8% of luteolin-4 '-O-glucoside, greater than or equal to 4.5% of kaempferol-3-O-galactoside-7-O-rhamnoside and greater than or equal to 1% of 5, 7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromenone. The substance with the acyl-CoA oxidase 1 inhibiting capability is extracted from the oil-tea cake, a thought is provided for high-value conversion of the oil-tea cake, and the preparation method is simple and easy to industrialize.

Description

Technical Field

[0001] The invention relates to the technical field of plant extraction, and in particular to a camellia oleifera cake extract and a preparation method and application thereof. Background Art

[0002] Abnormally elevated acyl-CoA oxidase 1 activity is closely related to metabolic diseases such as non-alcoholic fatty liver disease and diabetes. Inhibiting acyl-CoA oxidase 1 activity is a potential therapeutic strategy to improve lipid metabolism disorders. Chinese patent CN104162163A reported that 10,12-pentacadiynoic acid or 10,12-pentacadiynoyl-CoA has significant acyl-CoA oxidase 1 inhibitory activity. The document "Shang, Z.; Gao, Y.; Xue, Y.; Zhang, C.; Qiu, J.; et al. Shenge Formula attenuates high-fat diet-induced obesity and fatty liver via inhibiting ACOX1. Phytomedicine 2024, 123, 155183." reported that Shenge Formula can improve obesity and liver lipid metabolism disorders by inhibiting acyl-CoA oxidase 1 activity.

[0003] Camellia oleifera is an evergreen small tree of the genus Camellia of the Theaceae family and is one of the important woody oil crops in China. Camellia oleifera cake is a byproduct of tea oil pressing and is rich in bioactive ingredients such as tea saponins, polysaccharides, proteins and polyphenols. In the prior art, polysaccharides, tea saponins and the like are usually extracted from camellia oleifera cake. Chinese patent CN105440092A discloses a method for rapidly preparing flavonoid glycosides from camellia oleifera cake, from kaempferol 3-O-[2-O-β-D-xylose-6-O-α-L-rhamnose]-β-D-glucoside and kaempferol 3-O-[2-O-β-D-galactose-6-O-α-L-rhamnose]-β-D-glucoside, which has an antioxidant effect.

[0004] At present, there is no report on the extraction of acyl-CoA oxidase 1 inhibitors from camellia oleifera cake. Summary of the invention

[0005] In view of the problems in the prior art, the present invention proposes a tea cake extract and its preparation method and use. By isolating an extract with acyl-CoA oxidase 1 inhibitory ability from tea cake, it can not only increase the added value of tea cake, but also provide a new treatment method for improving glucose and lipid metabolism disorders.

[0006] The technical solution of the present invention is as follows:

[0007] A tea oil cake extract comprises more than 75% of flavonoids, wherein the flavonoids at least include: kaempferol ≥ 29%, luteolin-7-O-glucoside ≥ 4.5%, luteolin-4′-O-glucoside ≥ 8%, kaempferol-3-O-galactoside-7-O-rhamnoside ≥ 4.5% and 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromenone ≥ 1%.

[0008] After comparing the active ingredients, it was found that luteolin-7-O-glucoside and luteolin-4′-O-glucoside had a good inhibitory effect on the activity of acyl-CoA oxidase 1. Among them, the inhibitory effect of luteolin-4′-O-glucoside was more significant.

[0009] A fatty acyl-CoA oxidase 1 inhibitor comprises the above-mentioned camellia oleifera cake extract.

[0010] Furthermore, the inhibition rate of acyl-CoA oxidase 1 was more than 83%, and the IC 50 The value was below 19 mg / mL.

[0011] The preparation method of the above-mentioned camellia oleifera cake extract comprises the following steps:

[0012] S1. Sample pretreatment: collect camellia oil cake, crush, sieve, dry and defatted;

[0013] S2, extraction: mixing the camellia oleifera cake with the ethanol solution at a solid-liquid ratio of 1:6, and performing ultrasonic extraction to obtain an extract;

[0014] S3, post-treatment of the extract: after the extraction, centrifuge to obtain an ethanol extract; and condense by rotary evaporation to obtain a crude extract;

[0015] S4, purification of target product: the crude extract is purified and eluted by macroporous resin to obtain an eluate;

[0016] S5. Post-treatment of the purified solution: the eluate is concentrated by rotary evaporation and then freeze-dried to obtain the camellia oleifera cake extract.

[0017] 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 hours.

[0018] Preferably, in step S1, the degreasing is degreasing with petroleum ether.

[0019] Preferably, in step S2, the concentration of the ethanol solution is 55-60%, the extraction temperature is 65-75°C, the extraction time is 60-80min; and the ultrasonic power is 200-400W.

[0020] Preferably, in step S4, the specific process of purification and elution is:

[0021] Weigh 20g of pretreated macroporous resin, fill the chromatographic column with wet method, and elute 1-2 column volumes with pure water; dissolve the crude oil-tea cake in ultrapure water, slowly load 0.5 column volume of extract solution with a peristaltic pump at a flow rate of 0.5 column volume / hour, and open the piston switch at the bottom; respectively elute the chromatographic column with 2 column volumes of ultrapure water and 20%, 40%, 60%, 80%, and 100% ethanol solutions at a flow rate of 1 column volume / hour, and collect the 4th tube sample of 40% ethanol eluate, which is the eluate.

[0022] Preferably, the macroporous resin is AB-8 macroporous resin.

[0023] Use of the camellia oleifera cake extract or the acyl-CoA oxidase 1 inhibitor in preparing medicines or health products for treating or alleviating diabetes.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention uses camellia oleifera cake, a natural plant by-product, as raw material, which fully realizes the resource utilization of waste and significantly reduces the production cost, meeting the requirements of green production. Camellia oleifera cake is a by-product of camellia oleifera seed oil extraction, which is abundant in resources and extremely low in cost, providing a sustainable economic and scalable basis for the present invention.

[0026] (2) The extract of the present invention has high inhibitory activity on acyl-CoA oxidase 1, with an inhibition rate of more than 83%; and has good purification effect, IC 50 It can be reduced to below 19 mg / mL. The active ingredients that inhibit acyl-CoA oxidase 1 (such as luteolin-4′-O-glucoside, luteolin-7-O-glucoside, etc.) have been identified, further improving the scientificity and accuracy of product activity research.

[0027] (3) 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 and is easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The present invention provides a method for extracting and purifying acyl-CoA oxidase 1 inhibitor from camellia oleifera cake.

[0029] Figure 2 This is a physical picture of a method for extracting and purifying an acyl-CoA oxidase 1 inhibitor from oil-tea camellia cake provided by the present invention.

[0030] Figure 3 IC of acyl-CoA oxidase 1 inhibitors against acyl-CoA oxidase 1 before and after purification50 value.

[0031] Figure 4 This is a total ion current diagram obtained by identifying the compounds in the extract in Example 1 using ultra-high performance liquid chromatography-quadrupole electrostatic field orbital trap high-resolution mass spectrometry.

[0032] Figure 5 This is a schematic diagram of the 3D structure of luteolin-4′-O-glucoside docking with acyl-CoA oxidase 1 molecule.

[0033] Figure 6 This is a schematic diagram of the 2D structure of luteolin-4′-O-glucoside docking with acyl-CoA oxidase 1 molecule.

[0034] Figure 7 It is the root mean square deviation trend of the complex formed by the first five compounds screened out from the extract and acyl-CoA oxidase 1.

[0035] Figure 8 The IC of luteolin-7-O-glucoside on acyl-CoA oxidase 1 50 value.

[0036] Fig. 9 The IC of luteolin-4′-O-glucoside on acyl-CoA oxidase 1 50 value.

[0037] Fig.10 The IC of kaempferol on acyl-CoA oxidase 1 50 value.

[0038] Fig.11 The IC of kaempferol-3-O-galactoside-7-O-rhamnoside on acyl-CoA oxidase 1 50 value.

[0039] Fig.12 The IC of 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromenone for acyl-CoA oxidase 1 50 value. DETAILED DESCRIPTION

[0040] 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.

[0041] 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.

[0042] Example 1

[0043] like Figure 1 As shown, 200g of crushed camellia seed cake was weighed, passed through a 60-mesh sieve, and dried at 50℃ for 20 hours. According to the material-liquid ratio of 1:3, the camellia seed cake and 600mL of petroleum ether were placed in an ultrasonic-assisted extraction device for extraction, and the ultrasonic power was 300W for the fixed power of the machine. After 30 minutes of extraction, centrifuged at 25℃ and 3000rpm for 10 minutes, and the precipitate was taken out. The defatting step was repeated three times. The defatted camellia seed cake was dried at 50℃ for 24 hours for use. 100g of defatted camellia seed cake was taken, mixed with 600mL of 58% ethanol solution according to the material-liquid ratio of 1:6, and extracted in an ultrasonic-assisted extraction device for 62 minutes, the extraction temperature was 70℃, and the ultrasonic power was 300W for the fixed power of the machine. After the extraction, the sample was centrifuged at 10000rpm for 10 minutes, the ethanol extract was collected and concentrated by rotary evaporation to obtain a crude extract. The acyl-CoA oxidase 1 inhibition rate of the crude extract was measured to be 83.2%. The crude extract was freeze-dried. Weigh 20 g of the crude extract powder, dissolve it in ultrapure water (concentration 30 mg / mL), and adjust the pH of the solution to 3. The pretreatment of AB-8 resin includes: soaking in 95% ethanol for 24 hours, repeatedly washing until the filtrate is clear; then soaking in 4% sodium hydroxide and 4% hydrochloric acid solutions for 24 hours, washing with distilled water to neutrality after each soaking, and finally soaking the resin in ultrapure water for later use. Load the solution onto a 20 g pretreated AB-8 macroporous resin column using a peristaltic pump at a flow rate of 0.5 column volume / hour. First wash with 2 column volumes of ultrapure water at a flow rate of 1 column volume / hour, then elute with 2 column volumes of 20%, 40%, 60%, 80%, and 100% ethanol solutions, collecting 50 mL in each tube, and collecting the 40% ethanol eluate as the fourth tube sample. The collected eluate was concentrated by rotary evaporation and freeze-dried to obtain the final extract (acyl-CoA oxidase 1 inhibitor), see Figure 2 , stored at -80°C. The crude extract and acyl-CoA oxidase 1 inhibitor were prepared into sample solutions with concentration gradients of 1, 2, 4, 8, 16, 32, 64, 80, 96, 112, and 128 mg / mL using ultrapure water, and the inhibitory activity of each concentration solution on acyl-CoA oxidase 1 was determined. According to the inhibition rate-concentration curve, IC was calculated using GraphPad Prism 8.0.250 IC value of crude extract 50 The value is 36.88 mg / mL, and the IC of the purified acyl-CoA oxidase 1 inhibitor is 50 The value was 19.01 mg / mL, a decrease of 48.46%. Figure 3 .

[0044] Example 2

[0045] The extract obtained in Example 1 was ground into powder, 50 mg was accurately weighed, 500 μL of methanol and acetonitrile (1:1 / V) was added to dissolve, ultrasonicated for 30 minutes (with ice packs), and then centrifuged (20°C, 17000g, 15min), the supernatant was placed in a new 1.5mL EP tube, vacuum centrifuged at 35°C to dry, and then 100 μL of 50% methanol aqueous solution (v:v) was added, vortexed until completely dissolved, and then centrifuged (4°C, 17000g, 15min), and 60 μL of supernatant was loaded. 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°C, the injection volume was 5 μL, the flow rate was 0.4mL / min, and the sample was placed in an automatic sampler at 4°C 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.

[0046] An 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.

[0047] 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. Component identification was first confirmed based on the precise molecular weight (molecular weight error ≤ 30ppm), 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.).

[0048] Based on accurate molecular weight, fragment ion information and database comparison, a total of 138 chemical components were identified, covering multiple chemical categories. The total ion current is shown in Figure 4 The main components are flavonoids, totaling 57 species, including kaempferol, kaempferol-3-O-rutinoside, luteolin-4′-O-glucoside, etc. In addition, a variety of compounds such as triterpenoid saponins, anthocyanins, phenolic acids, and alkaloids were also identified.

[0049] Example 3

[0050] The crystal structure of mouse acyl-CoA oxidase 1 was established by AlphaFold (https: / / www.alphafold.ebi.ac.uk). The two-dimensional structure of the active substance was obtained from the PubChem database (http: / / pubchem.ncbi.nlm.nih.gov / ). The top 20 compounds in the oil-tea cake extract analyzed by ultra-high performance liquid chromatography-quadrupole electrostatic field orbital trap high-resolution mass spectrometry were converted into energy-minimized three-dimensional structures by Chem 3D for molecular docking with acyl-CoA oxidase 1. Acyl-CoA oxidase 1 was imported into Maestro, and the protein preparation wizard program in Maestro was used to convert the protein into the best docking conformation. The Glide program was used with the force field set to OPLS4, and the other parameters remained unchanged for molecular docking. After the molecular docking score was initially screened, the binding free energy of the ligand-receptor binding mode was predicted and evaluated by the molecular force field generalized Boltzmann surface area binding free energy calculation. The molecular docking effect was evaluated and substances with strong interactions with acyl-CoA oxidase 1 were screened out.

[0051] The complex was simulated by molecular dynamics for 100 ns using Gromacs 2023. The protein was simulated using the CHARMM 36 force field parameters, and the ligand topology was constructed using the GAFF2 force field parameters. The protein-ligand complex was placed in a cubic box using periodic boundary conditions. The TIP3P water model was used to fill the box with water molecules. Electrostatic interactions were treated using the particle mesh Ewald (PME) and Verlet algorithms, respectively. This was followed by 100,000 steps of isothermal isochoric ensemble equilibrium and isothermal isobaric ensemble equilibrium with a coupling constant of 0.1 ps and a simulation duration of 100 ps. Both van der Waals and Coulomb interactions were calculated using a cutoff of 1.0 nm. Finally, the system was simulated by molecular dynamics using Gromacs 2023 at constant temperature (300 K) and pressure (1 bar) for a total of 100 ns.

[0052] From the 20 high-content compounds identified in the tea cake extract, five compounds with strong binding ability to acyl-CoA oxidase 1 were screened out through docking score initial screening and binding free energy, including luteolin-7-O-glucoside, luteolin-4′-O-glucoside, kaempferol, kaempferol-3-O-galactoside-7-O-rhamnoside and 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromenone. The peak area data of the compounds obtained by mass spectrometry analysis in Example 2 are as follows: Figure 4 As shown, the relative content of the compound in the sample is calculated, and the relative content of the compound = the peak area of ​​the compound / the peak area of ​​the total compound * 100%. After calculation, the relative content of flavonoids is above 75%, among which the relative contents of active ingredients luteolin-7-O-glucoside, luteolin-4′-O-glucoside, kaempferol, kaempferol-3-O-galactoside-7-O-rhamnoside and 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromenone in the sample are 4.83%, 8.07%, 29.61%, 4.86% and 1.06%, respectively. The molecular docking scores and binding free energy calculation results of the top 20 compounds with acyl-CoA oxidase 1 are shown in Table 1. The smaller the value of the binding free energy and the molecular docking score, the stronger the molecular interaction ability. The results of molecular dynamics simulation showed that the root mean square deviation of the complex formed by luteolin-4′-O-glucoside and acyl-CoA oxidase 1 reached a stable state the fastest, indicating that its binding effect was the best. The 3D binding diagram of luteolin-4′-O-glucoside and acyl-CoA oxidase 1 is shown in Figure 5 , 2D combined diagram see Figure 6 The root mean square deviation of the complexes formed by the five compounds with acyl-CoA oxidase 1 is shown in Figure 7The five compounds were prepared into sample solutions with a concentration gradient of 2.5, 5, 10, 15, 20, 30, 40, and 50 mg / mL, and the inhibitory activity of each concentration solution on acyl-CoA oxidase 1 was determined. According to the inhibition rate-concentration curve, GraphPad Prism 8.0.2 was used to calculate the IC 50 Value. Figure 8-12 As shown, the IC values ​​of luteolin-7-O-glucoside, luteolin-4′-O-glucoside, kaempferol, kaempferol-3-O-galactoside-7-O-rhamnoside, and 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromenone 50 They are 14.15 mg / mL, 8.09 mg / mL, 17.96 mg / mL, 19.15 mg / mL and 26.07 mg / mL respectively.

[0053] Table 1. The docking results of the top 20 compounds of acyl-CoA oxidase 1 inhibitors extracted and purified from camellia oleifera cake with acyl-CoA oxidase 1

[0054]

[0055]

[0056] 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 tea cake extract, characterized in that The extract comprises more than 75% of flavonoids, wherein the flavonoids at least comprise: kaempferol ≥ 29%, luteolin-7-O-glucoside ≥ 4.5%, luteolin-4'-O-glucoside ≥ 8%, kaempferol-3-O-galactoside-7-O-rhamnoside ≥ 4.5% and 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromenone ≥ 1%.

2. An acyl-CoA oxidase 1 inhibitor, characterized in that: The invention comprises the camellia oil cake extract according to claim 1.

3. The acyl-CoA oxidase 1 inhibitor according to claim 2, characterized in that The inhibition rate of acyl-CoA oxidase 1 is more than 83%, IC 50 The value was below 19 mg / mL.

4. The method for preparing the camellia oleifera cake extract according to claim 1, characterized in that: The following steps are involved: S 1. Sample pretreatment: collect camellia oil cake, crush, sieve, dry and defatted; S2, extraction: mixing the camellia oleifera cake with the ethanol solution at a solid-liquid ratio of 1:6, and performing ultrasonic extraction to obtain an extract; S3, post-treatment of the extract: after the extraction, centrifuge to obtain an ethanol extract; and condense by rotary evaporation to obtain a crude extract; S4, purification of target product: the crude extract is purified and eluted by macroporous resin to obtain an eluate; S5. Post-treatment of the purified solution: the eluate is concentrated by rotary evaporation and then freeze-dried to obtain the camellia oleifera cake extract.

5. The preparation method according to claim 4, 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.

6. The preparation method according to claim 4, characterized in that: In step S1, the degreasing is degreasing with petroleum ether.

7. The preparation method according to claim 4, characterized in that: In step S2, the concentration of the ethanol solution is 55-60%, the extraction temperature is 65-75°C, the extraction time is 60-80 minutes, and the ultrasonic power is 200-400W.

8. The preparation method according to claim 4, characterized in that: In step S4, the specific process of purification and elution is: Weigh 20g of pretreated macroporous resin, fill the chromatographic column with wet method, and elute with pure water for 1-2 column volumes; dissolve the crude oil-tea cake 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 at the bottom; Use 2 column volumes of ultrapure water and 20%, 40%, 60%, 80% and 100% ethanol solutions to elute the column in sequence at a flow rate of 1 column volume / hour. Collect the 4th tube of sample containing 40% ethanol eluate, which is the eluate.

9. The preparation method according to claim 8, characterized in that: The macroporous resin is AB-8 macroporous resin.

10. Use of the camellia oleifera cake extract according to claim 1 or the acyl-CoA oxidase 1 inhibitor according to any one of claims 2 to 3 in the preparation of a drug or health product for treating or alleviating diabetes.

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