Extraction method of chaff bound phenol, obtained product and application

Abstract

The invention belongs to the field of active ingredient extraction, and particularly relates to an extraction method of chaff bound phenol, an obtained product and application. The method comprises the following steps: firstly, adding crushed and sieved rice bran into a solvent, oscillating at normal temperature, then adding a NaOH solution, hydrolyzing, centrifuging, degreasing, and extracting with ethyl acetate to obtain a rice bran bound phenol crude extract; and purifying the crude bran-bound phenol extract by using the pretreated macroporous resin, then washing by using deionized water and gradient ethanol, and carrying out rotary evaporation on liquids with different gradients to obtain the bran-bound phenol. The extraction method provided by the invention is simple, impurities such as saccharides, amino acids and fat-soluble substances can be removed to the greatest extent, and the prepared chaff bound phenol is high in purity; the prepared chaff bound phenol is excellent in antioxidant activity and has good capacity of removing DPPH (1, 1-diphenyl-2-picrylhydrazyl) and superoxide free radicals.

Description

Technical Field

[0001] The present invention belongs to the field of active ingredient extraction, and particularly relates to a method for extracting bran-bound phenol, and the obtained product and application. Background Art

[0002] Millet, also known as "fowl," has a high nutritional value and is one of the most important sources of protein and carbohydrates in the human diet. It also contains a large number of other nutrients, such as dietary fiber, minerals, and vitamins. However, rice bran, a byproduct of grain milling and used medicinally as rice bran, is typically discarded as a processing waste during grain processing. Specific and effective methods for converting this agricultural waste into value-added products have yet to be developed. This not only results in significant energy and resource waste, but also leads to low ecological and social benefits.

[0003] The polyphenol compounds in rice bran mainly include flavonoids and phenolic acid compounds. Among them, phenolic acid compounds are divided into free phenolic acids and bound phenolic acids. The polyphenol compounds in rice bran are mainly bound polyphenols, and the antioxidant activity of free polyphenols is significantly weaker than that of bound polyphenols. Some studies have reported that the polyphenol content in rice bran is the bound polyphenols in the outer shell of rice bran, followed by the bound polyphenols in the inner shell. In addition, the total polyphenol content in rice bran is significantly higher than that in millet. However, the currently extracted extracts of rice bran-bound phenols have low purity and contain impurities such as sugars, amino acids and fat-soluble substances, which affect their activity. Therefore, how to separate and purify a high-purity rice bran-bound phenol has become an urgent problem to be solved. Summary of the Invention

[0004] In response to the problems existing in the prior art, the present invention provides a method for extracting bran-bound phenol. The bran-bound phenol prepared by this method has high purity, can remove impurities in the crude extract to the greatest extent, and has excellent antioxidant activity.

[0005] The present invention also provides bran-bound phenol prepared by the extraction method.

[0006] Another object of the present invention is to provide the use of the above-mentioned bran-bound phenol in the preparation of antioxidant products.

[0007] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are as follows: The present invention provides a method for extracting bran-bound phenol, comprising the following steps: (1) Adding the crushed and sieved rice bran to the solvent and mixing them thoroughly, shaking at room temperature, centrifuging, and rotary evaporating the supernatant to obtain a crude extract of rice bran free phenols, and air-drying the precipitate; (2) After drying the precipitate, add NaOH solution for hydrolysis. After centrifugation, take the supernatant. After adjusting the pH of the supernatant, centrifuge again, take the supernatant to remove fat, then extract with ethyl acetate, combine the organic phases, evaporate to dryness under reduced pressure, and finally collect the crude extract of bran-bound phenols after drying; (3) Purify the crude extract of bran-bound phenols using pretreated macroporous resin, then rinse with deionized water and gradient ethanol, and rotary evaporate the liquids with different gradients to obtain bran-bound phenols.

[0008] Preferably, in step (1), the material-liquid ratio of the bran to the solvent is 1:20; the solvent is 70% ethanol; the time for shaking at room temperature is 1 h.

[0009] Preferably, in step (2), the concentration of the NaOH solution is 2 M; the material-liquid ratio of the precipitate to the NaOH solution is 1:40; the volume ratio of the supernatant after defatting to ethyl acetate is 1:1; the number of extractions is 3 times; for the hydrolysis, first shake at room temperature for 1 h, then stir magnetically for 3 h; adjust the pH to 1.5 - 2.0.

[0010] Preferably, in step (3), the concentration of the solution of the crude extract of bran-bound phenols is 10 mg / ml.

[0011] Preferably, in step (3), the pretreatment method of the macroporous resin is: soak the macroporous resin in 95% ethanol, fully swell for 24 h, then wash with absolute ethanol until there is no white turbidity, and then wash with deionized water until there is no alcohol smell. Then soak the resin in 5% hydrochloric acid solution for 3 h, wash with deionized water until neutral, then soak the resin in 5% sodium hydroxide solution for 3 h, wash with deionized water until neutral, and dry for standby.

[0012] Preferably, the macroporous resin is AB-8 or D101; more preferably, the macroporous resin is AB-8.

[0013] Preferably, the volume fractions of the gradient ethanol are successively: 30%, 50%, 70%, 95%; the bran-bound phenols are obtained by rotary evaporation of the eluate with 50% ethanol.

[0014] The present invention also provides a kind of bran-bound phenols prepared by the above extraction method.

[0015] The present invention also provides the application of the above bran-bound phenols in the preparation of antioxidant and anti-inflammatory products.

[0016] Another object of the present invention is to provide the application of the above bran-bound phenols in the preparation of drugs for treating sepsis.

[0017] The beneficial effects of the present invention are: (1) The extraction method provided by the present invention is simple, which can remove impurities such as sugars, amino acids and fat-soluble substances to the greatest extent, and the purity of the bran-bound phenols prepared is high; (2) The bran-bound phenols prepared by the present invention have excellent antioxidant activity and good ability to scavenge DPPH and superoxide radicals. Description of the Drawings

[0018] Figure 1 Total phenol content obtained by extracting different milling components; Figure 2 Free polyphenols, bound polyphenols and total polyphenol content of bran obtained by extracting with different solvents (80% methanol and 70% ethanol); Figure 3 Polyphenol purity of the crude extract after separation by AB-8 and D101 macroporous resins; Figure 4 Adsorption rate of AB-8 and D101 macroporous resins at different times; Figure 5 Polyphenol purity obtained by purifying with macroporous resins; Figure 6 DPPH radical scavenging ability of different fractions; Figure 7 ABTS radical scavenging ability of different fractions; Figure 8 FRAP reducing ability of different fractions; Figure 9 Flow chart of animal experiments; Figure 10 Body weight change of mice after modeling; Figure 11 HE staining (A is the blank group, B is the model group, C is the low-dose group, D is the medium-dose group, E is the high-dose group); Figure 12 Levels of inflammatory factors in mouse serum (A is IL-1β, B is IL-6); Figure 13 Levels of inflammatory factors in mouse liver homogenate (A is IL-1β, B is IL-6); Figure 14 Levels of alanine aminotransferase (A) and aspartate aminotransferase (B) in mouse serum; Figure 15 Western blot detection of the expression of iNOS and COX-2 in the liver of septic mice; Figure 16 Western blot detection of the expression of NF-κB pathway proteins in the liver of septic mice. Specific Embodiments

[0019] The technical solution of the present invention will be further explained and illustrated through specific embodiments below.

[0020] Example 1 Screening of Samples The unhulled millet with husk is denoted as BF0. Use a stone mill to mill the unhulled millet (500 g) (hand-mill with a stone mill, after milling once, sieve out the husk, then mill again and sieve to obtain the second layer of husk; follow the conventional millet milling process). To obtain bran fraction 1 (BF1) and milled rice 1. Then, mill the milled rice 1 to obtain BF2 and milled rice 2. In turn, mill the milled rice in the previous step to obtain BF3 and BF4, and BF4 belongs to dehulled millet. Grind all bran fractions and the unhulled millet BF0 into fine powders so that they can completely pass through an 80-mesh sieve, and then extract the bran polyphenols.

[0021] Example 2 Extraction of Bound Phenols in Bran (1) Put the bran into a high-speed multi-functional pulverizer and pulverize for 1 min, then sieve through an 80-mesh sieve. After collection, add 70% ethanol to the pulverized bran sample in a ratio of 1:20 and mix well. After shaking at room temperature for 1 h, centrifuge at high speed (9000 rpm) in a high-speed centrifuge for 11 min, pour out the supernatant, and collect the precipitate.

[0022] (2) Dry the precipitate in a fume hood, collect the dried bran powder, add 2M NaOH solution in a ratio of 1:40 for hydrolysis, shake at room temperature for 1 h, then stir magnetically for 3 h. After centrifuging (4000 rpm) for 10 min, take the supernatant, adjust the pH to 1.5 - 2.0 with 6M HCl solution and then centrifuge (4000 rpm) for 10 min. Take the supernatant and defat with n-hexane in a ratio of 1:1, and then extract with ethyl acetate in the same ratio three times and combine the organic phases. Evaporate under reduced pressure, and finally collect the dried crude bran extract.

[0023] (3) Immerse the macroporous resins AB-8 and D101 in 95% ethanol, fully swell for 24 h, then wash with absolute ethanol until there is no white turbidity, and then rinse with deionized water until there is no alcohol smell. Then immerse the resin in 5% hydrochloric acid solution, after 3 h, rinse with deionized water until neutral, then immerse the resin in 5% sodium hydroxide solution, after 3 h, rinse with deionized water until neutral, and dry for standby.

[0024] Table 1 Parameter Specifications of Macroporous Adsorption Resins (4) Purify the phenolic compounds bound to rice bran with the pretreated macroporous resin. Add 4 kg of macroporous resin AB-8, and the pretreatment refers to step (3). Add 10 g (10 mg / ml) of the crude extract of phenolic compounds bound to rice bran (the solvent is water, using DMSO as a co-solvent with a content of 1%). After 24 h, rinse with deionized water, and then rinse with ethanol at 30%, 50%, 70%, and 95% by volume respectively. Rotavaporize the obtained liquid fractions at different levels to obtain BPS-1 (30%), BPS-2 (50%), BPS-3 (70%), and BPS-4 (95%) respectively. Take 1 mg of the sample obtained by rotavaporization for the test of polyphenol content.

[0025] Example 3 Determine using a microplate reader, and calculate the total phenolic content (TPC) with ferulic acid as the standard. Add the appropriately diluted sample solution (20 μl) to a 96-well plate, add Folin-Ciocalteu reagent (40 μL), react for 5 min in the dark at room temperature, add saturated sodium carbonate solution (160 μL), and react for 90 min in the dark at room temperature. Record the absorbance value at 750 nm.

[0026] (I) Screening of different milling components of rice bran TPC gradually decreases from BF1 to BF4. Compared with BF1, the polyphenol content in BF0 and BF4 significantly decreases. The total phenolic content in BF1 is 22.63 mg FAE / g DW. Compared with it, the TPC in BF2–BF4 and BF0 decreases by 28.56%, 67.75%, 81.40%, and 70.17% respectively.

[0027] (II) Screening of organic solvents Extract free and bound polyphenols from rice bran with 80% methanol and 70% ethanol, detect and compare the polyphenol content by the Folin-Ciocalteu method. From Figure 2 It can be seen that: 70% ethanol has a higher extraction rate for free phenols in rice bran, and the content of bound polyphenols in rice bran is higher than that of free polyphenols. Subsequently, relevant experiments were carried out with the crude extract of phenolic compounds bound to rice bran extracted with 70% ethanol.

[0028] (III) Macroporous resin screening experiment Put 1.0 g of pretreated macroporous adsorption resin AB-8 and D101 type in each 100 ml conical flask, then add 50 ml of the extraction solution of phenolic compounds bound to rice bran, and place it in a shaker for static adsorption for 24 h. Take the supernatant for measurement. Make corresponding calculations according to the formula respectively. Select a suitable resin for subsequent experiments.

[0029] Calculation of the adsorption rate of macroporous resin: Equation 1 Where: C0: the initial mass concentration of the crude extract of phenolic compounds bound to rice bran before adsorption, mg / ml; C1: the polyphenol concentration of phenolic compounds bound to rice bran after adsorption, mg / ml.

[0030] Two types of resins with different physical properties (AB-8 and D101) were selected for the adsorption experiment of the crude extract of phenolic compounds bound to rice bran. The adsorption rate of AB-8 was higher than that of D101 among the two resins. The adsorption rate reached the highest at 11 hours of sample loading, and the purity of the 70% fraction was the highest. The purity obtained by AB-8 in this fraction was higher than that of D101. Therefore, macroporous resin AB-8 was selected for the separation and purification of phenolic compounds bound to rice bran.

[0031] (IV) Purification experiment with macroporous resin After purification, the polyphenol content of each fraction increased. The purity of the crude extract of phenolic compounds bound to rice bran obtained from BPS-2 (50% fraction) was the highest, reaching 83%.

[0032] (V) Results of DPPH free radical scavenging experiment Precisely weigh 0.01 g of DPPH into 50 mL of 95% ethanol to obtain a 0.5 mmol / L DPPH working solution. After preparation, wrap it with tin foil and store it in the dark, preparing it freshly each time it is used. Then, add 100 μL of the DPPH working solution to a 96-well plate, and add 100 μL of the purified extracts of each fraction, the crude extract of phenolic compounds bound to rice bran, and the VC solution. React in the dark at room temperature for 30 min, and measure the absorbance of each well at 517 nm. The blank control group uses PBS instead of the sample solution, and the positive control group is the VC solution. The formula applied is as follows: Equation 2 In this formula, A0 is the absorbance value of the blank control group, and A1 is the absorbance value of the sample group.

[0033] BPS-2 has the strongest DPPH free radical scavenging ability. Especially at high concentrations (1 mg / mL, 5 mg / mL), its scavenging rate is basically similar to that of VC, and the difference from other treatment groups is relatively significant. BPS-1 and BPS-3 also have good antioxidant effects, but there are still significant differences from BPS-2 at certain concentrations. Crude and BPS-4 have weak antioxidant abilities at each concentration, indicating their low free radical scavenging activities.

[0034] (VI) Results of ABTS free radical scavenging experiment By measuring the change in absorbance, the ability of the substance to be measured to scavenge ABTS can be quantitatively reflected. +The scavenging efficiency of free radicals was thus evaluated to assess their antioxidant properties. The crude extract of rice bran polyphenols and the purified components of each fraction were appropriately diluted to prepare corresponding sample solutions. The blank group used deionized water instead of the sample solution, and the positive control group was the VC solution. The sample solution was A1, and PBS was used instead of the sample solution as the control blank Ao. The formula applied was as follows: Equation 3 In this formula, A0 is the absorbance value of the blank control group, and A1 is the absorbance value of the sample group.

[0035] VC and the purified rice bran polyphenols of different fractions all had a certain scavenging ability for ABTS free radicals, but at the same concentration of 1 mg / ml, the six samples showed different scavenging abilities for hydroxyl free radicals. Among them, the BPS-2 fraction had an effect equivalent to that of the positive control VC.

[0036] (VII) Results of the FRAP reducing power experiment For the samples (2 mL of purified extracts of each fraction and the crude extract of rice bran polyphenols at concentrations of 0.1, 0.2, 0.5, 0.8, 1, 5 μg / mL, and the VC solution), a mixture of 2 mL of 1% potassium ferricyanide [K3Fe(CN)6] and 2 mL of phosphate buffer was heated in a water bath at 50 °C for 20 min, and then quickly cooled at 4 °C. After cooling, 1 mL of 10% trichloroacetic acid was added to terminate the reaction, and centrifuged at 4000 r / min for 10 min. 2 ml of the supernatant was taken and added to a mixture of distilled water and 400 μL of 1% ferric chloride, and the absorbance was measured at 700 nm using an enzyme-linked immunosorbent assay reader. An increase in absorbance indicates an increase in its reducing power.

[0037] The different fractions of rice bran polyphenols had a certain Fe 3+ reducing ability, but the reducing power of each fraction was lower than that of VC. The absorbance of the BPS-2 fraction was higher than that of other fractions, and the absorbance was equivalent to that of VC at a concentration of 1 mg / ml. Therefore, the BPS-2 fraction of rice bran-bound phenols was used for related determinations in subsequent experiments.

[0038] Example 4 (1) Mice: Male C57 mice at 8 weeks of age, weighing 18 - 22 grams. These mice were housed in separate cages in a controlled environment.

[0039] (2) Establishment of a mouse model of liver injury induced by LPS-induced sepsis The anti-inflammatory effect of BPS-2 was well characterized in in vitro experiments and further investigated in vivo using a mouse model of sepsis-related acute liver injury. Mice were divided into a control group without LPS injection (n = 5), a model group injected with LPS (n = 5), and a group injected with LPS and orally administered BPS-2 (n = 5). Experimental animals were orally administered different doses of BPS-2 (100, 150, 200 mg / kg / d) or a single dose of 200 μL PBS for 7 days. Body weight was recorded throughout the study period ( Figure 9 ). On day 7, 2 hours after the last administration, LPS (10 mg / kg) was injected intraperitoneally. Four hours later, the mice were anesthetized and blood was collected. Serum was obtained by centrifugation. We measured the levels of organ injury markers, pro-inflammatory mediators, ALT, and AST. Liver specimens were quickly taken and stored at -80 °C. Body weight was continuously monitored after LPS injection, recorded every 60 minutes for 4 hours.

[0040] (3) Effect of orally administered BPS-2 on a mouse model of sepsis-related acute liver injury The ability of BPS-2 to reduce inflammation was well characterized in in vitro experiments and further investigated in vivo using a mouse model of sepsis-related acute liver injury. Interestingly, no significant changes in body weight were observed among the groups ( Figure 10 ), indicating that BPS- had no obvious toxicity to mice. On day 7, mice were injected intraperitoneally with LPS (10 mg / kg) 2 hours after BPS-2 administration, and the control group was only given PBS injection. As Figure 10 shown, the body weight of mice injected with LPS decreased sharply, accompanied by diarrhea and dehydration. Four hours later, the decrease in body weight in the 200 mg / kg BPS-2 pretreatment group was much smaller and comparable to that of the control group.

[0041] (4) Effect of BPS-2 on the hepatic tissue morphology of septic mice Figure 11 As shown, microscopic examination showed that the livers of mice in the control group had a central vein and normal hepatocyte structure. In contrast, the livers of LPS-induced mice ( Figure 11 B) showed that the central vein was surrounded by chronic inflammation, accompanied by focal hepatocyte necrosis. There was abundant lymphocyte infiltration and a small amount of neutrophil infiltration around the blood vessels. When the BPS-2 concentration was 150 mg / kg ( Figure 11 D), necrosis returned to normal, blood vessels were congested, and neutrophil infiltration decreased. When the dose was 200 mg / kg ( Figure 11 E), its morphology was basically normal.

[0042] (5) Effect of BPS-2 on inflammatory factors in septic mice Cytokines are crucial in regulating inflammation and affect acute and chronic responses through complex biological networks such as Figure 12 As shown, after BPS-2 treatment, the levels of pro-inflammatory cytokines in the serum of septic mice were significantly reduced. Specifically, compared with the control group, the level of IL-1β in the model group almost tripled. Similar results were observed in the case of IL-6 levels. However, pretreatment with high, medium, and low doses of BPS-2 significantly reduced the levels of inflammatory cytokines in the serum. The levels of IL-6 in the serum of mice decreased by 21.25%, 27.84%, and 42.56%, respectively. In addition, BPS-2 pretreatment alleviated the increase in pro-inflammatory factors in the liver homogenate of LPS-stimulated septic mice ( Figure 13 ). These results were consistent with the in vitro experimental results. Therefore, this indicates that BPS-2 effectively controls the levels of key regulators of inflammation.

[0043] (6) Effects of BPS-2 on AST and ALT in septic mice The LPS-induced liver injury model in septic mice is a commonly used method to study the mechanism of liver inflammation. Serum alanine aminotransferase (ALT) level is the main indicator for evaluating liver function impairment, while the specificity of aspartate aminotransferase (AST) is not as good as that of alanine aminotransferase. An increase in alanine aminotransferase level indicates more severe liver tissue damage and hepatocyte necrosis. Both ALT and AST are important indicators for evaluating liver health. We monitored the levels of ALT and AST after administering LPS (alone or in combination with BPS-2). As Figure 14 shown, intraperitoneal injection of 10 mg / kg LPS significantly increased the levels of ALT and AST. Among them, the ALT concentration in the model group was about 3-4 times that of the control group, and the AST level was about 2 times that of the control group. These increases indicate that the mice had a severe inflammatory response. However, treatment with 200 mg / kg BPS-2 reduced the ALT level by 3.2 times and the AST level by 1.5 times. It is suggested that BPS-2 can reduce hepatocyte damage and liver inflammation by reducing the secretion of ALT and AST.

[0044] (7) Effects of BPS-2 on the protein expression levels of iNOS and COX-2 in the liver of septic mice In the LPS-induced mouse model of septic liver injury, common features include the release of inflammatory factors and the increase in the levels of inflammation-related enzymes Figure 15 As shown, treatment with LPS alone significantly increased the protein levels of iNOS and COX-2, while BPS-2 at doses of 100, 150, and 200 mg / kg dose-dependently inhibited these increases induced by LPS. Therefore, the purified millet bran polyphenol fraction alleviates liver inflammation in mice by downregulating the production of COX-2 and iNOS proteins.

[0045] (8)Effect of BPS-2 on the expression levels of TLR4-mediated NF-κB signaling pathway proteins in the livers of septic mice LPS activation upregulates the expression of TLR4 in the livers of mice ( Figure 16 ), which can be alleviated by pretreatment with BPS-2 and is consistent with the in vitro experimental results. In addition, LPS stimulation activates the NF-κB pathway, and the ratios of p-IκBα / IκBα and p-p65 / p65 increase by approximately 3.29-fold and 5.61-fold, respectively. BPS-2 reverses this increase, indicating that it regulates cytokine levels (COX-2, iNOS, IL-6, and IL-1β) in the livers of septic mice through the TLR4-mediated NF-κB pathway, contributing to reducing inflammation and providing protection for the organism.

Claims

1. A method for extracting phenolic compounds combined with bran, characterized in that, It includes the following steps: (1) Add the crushed and sieved rice bran into a solvent for thorough mixing. After shaking at room temperature, centrifuge, and rotary evaporate the supernatant to obtain the crude extract of free phenols from rice bran, and dry the precipitate; (2) After drying the precipitate, add NaOH solution for hydrolysis. After centrifugation, take the supernatant. After adjusting the pH of the supernatant, centrifuge, take the supernatant to remove fat, then extract with ethyl acetate, combine the organic phases, evaporate under reduced pressure, and finally collect the dried crude extract of bound phenols from rice bran; (3) Purify the crude extract of bound phenols from rice bran using the pretreated macroporous resin, then rinse with deionized water and gradient ethanol, and rotary evaporate the liquids with different gradients to obtain bound phenols from rice bran.

2. The extraction method according to claim 1, wherein In step (1), the material-liquid ratio of the rice bran to the solvent is 1:20; the solvent is 70% ethanol; the shaking time at room temperature is 1 h.

3. The extraction method according to claim 1, wherein In step (2), the concentration of the NaOH solution is 2 M; the material-liquid ratio of the precipitate to the NaOH solution is 1:40; the volume ratio of the supernatant after defatting to ethyl acetate is 1:1; the number of extractions is 3 times; the hydrolysis is first shaking at room temperature for 1 h, and then magnetic stirring for 3 h; the pH is adjusted to 1.5 - 2.

0.

4. The extraction method according to any one of claims 1 to 3, characterized in that, In step (3), the concentration of the solution of the crude extract of bound phenols from rice bran is 10 mg / ml.

5. The extraction method according to claim 1 or 4, characterized in that In step (3), the pretreatment method of the macroporous resin is: soak the macroporous resin in 95% ethanol, fully expand for 24 h, wash with absolute ethanol until there is no white turbidity, and then rinse with deionized water until there is no alcohol smell. Then soak the resin in 5% hydrochloric acid solution, after 3 h, rinse with deionized water until neutral, then soak the resin in 5% sodium hydroxide solution, after 3 h, rinse with deionized water until neutral, and dry for standby.

6. The extraction method according to claim 5, characterized in that, The macroporous resin is AB-8 or D101; preferably, the macroporous resin is AB-8.

7. The extraction method according to any one of claims 1-6, characterized in that, The volume fractions of the gradient ethanol are successively: 30%, 50%, 70%, 95%; the bound phenols from rice bran are obtained by rotary evaporation of the eluate with 50% ethanol.

8. A bound phenol from rice bran prepared by the extraction method according to any one of claims 1 - 7.

9. An application of the bound phenol from rice bran according to claim 8 in the preparation of antioxidant and anti-inflammatory products.

10. An application of the bound phenol from rice bran according to claim 8 in the preparation of drugs for treating sepsis.

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