JP2006166834A - Method for extracting ferulic acid, disease prevention food and disease prevention drug - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a ferulic acid extraction method enabling easy extraction of ferulic acid from rice bran. <P>SOLUTION: The ferulic acid extraction method contains an ethanol treatment step to add ethanol to rice bran and shaking the mixture, a primary filtration step to filter the treating liquid obtained by the ethanol treatment step, a hot drying step to heat and dry the residue obtained by the primary filtration step, an enzyme treatment step to suspend the treated product obtained by the hot drying step by using a solution containing an enzyme preparation, a secondary filtration step to filter the suspension obtained by the enzyme treatment step, and a freeze-drying step to dry the extract containing ferulic acid as a main component and obtained by the secondary filtration step by freeze-drying. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  Hypertension is one of the risk factors for cardiovascular disease. Do we have rice bran ingredients (Driselase (Drycelase®) fraction and ethanol fraction) have hypotensive action in stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive rats Wistar Kyoto (WKY)? I studied about how. Four-week-old rats were divided into four groups, and two groups, SHRSP and WKY, were fed a diet according to AIN93M (control diet). The remaining 2 groups of SHRSP were fed a test meal (Driselase fraction diet, ethanol fraction diet) with 60 g of rice bran fraction added per kilogram of diet. Between 7 weeks of age and the end of the experiment, both the rice bran fraction test diet group showed a significant decrease in blood pressure compared to the SHRSP control diet group. Plasma angiotensin converting enzyme (ACE) inhibitory activity was significantly increased by feeding the test meal (Driselase fraction diet group 10.83%, ethanol fraction diet group 9.85%), and plasma nitric oxide (NO) concentration decreased. There was a positive correlation between decreased blood pressure, plasma ACE inhibitory activity, and plasma NO concentration (r = 0.627, p <0.01; r = 0.828, p <0.01, respectively). Both test meals also improved renal function and reduced fasting blood sugar. These data showed that both rice bran fractions reduced SHRSP blood pressure through anti-inflammatory effects.

  Hypertension is a cardiovascular disease and is the leading cause of death in developed countries. Antihypertensive drugs such as enalapril, losartan, ramipri are used to reduce hypertension. These drugs lower blood pressure by inhibiting angiotensin converting enzyme (ACE). At present, attempts are being made to search for food ingredients that reduce the risk of cardiovascular disease. Antioxidants such as vitamins E, C, carotenoids and polyphenols have received much attention because they have protective effects against cardiovascular diseases and their risk factors. Most antioxidants, complex carbohydrates and plant compounds, including lipids, proteins, vitamins, minerals and polyphenols, are present in plant embryos and bran parts.

  The rice bran obtained during the milling process contains plant compounds derived from the embryo and is useful as a source of plant-derived compounds. The use of rice bran is limited due to the generation of rapid malodor caused by lipase or lipoxygenase after rice polishing, but rice bran can be effectively utilized by stabilization.

  In this study, we used two fractions of rice bran, an enzyme-treated (Driselase) -treated fraction and an ethanol-extracted fraction. Of the fractions (extracted fractions and residues) obtained after extracting rice bran with ethanol, the extracted fractions contain unsaponifiable matter (tocotrienol, γ-oryzanol, β-sitosterol) and unsaturated fatty acids in rice bran. It is. The residue was treated with Driselase (enzyme preparation) to hydrolyze the polysaccharide contained in rice bran. This treatment releases ferulic acid in the polysaccharide.

  Rice bran contains many lipids (12-13%). This lipid fraction contains tocotrienols, γ-oryzanols, β-sitosterols, and unsaturated fatty acids that have the effect of reducing total cholesterol, LDL cholesterol, and triglyceride levels. In addition, rice bran is rich in dietary fiber (β-glucan, pectin, gum). Various epidemiological studies have shown that higher soluble dietary fiber intake correlates with a reduced risk of cardiovascular disease.

  Rice bran contains 4-hydroxy-3-methoxycinnamic acid (ferulic acid). Many plant cell walls contain ferulic acid esterified with polysaccharides. Purified ferulic acid suppresses the toxicity of β-amyloid peptide, protects from UV rays, has antioxidant activity, reduces plasma glucose levels in streptozotocin-induced diabetic rats, and has hypotensive effects in spontaneously hypertensive rats Etc. have been reported.

  Recently, black rice bran inhibited atherosclerotic plaque formation in rabbits, reduced arterial 8-hydroxy-2-deoxyguanosine levels, and reduced serum and arterial malondialdehyde levels caused by hypercholesterolemia . When tocotrienol extracted from rice bran was orally administered to apolipoprotein E-deficient mice, atherosclerosis was suppressed, serum triglycerides were decreased, and total cholesterol and LDL cholesterol were decreased. Studies with humans have also shown that rice bran reduces LDL cholesterol and HDL cholesterol ratios. Based on these reports, we hypothesized that the beneficial effects of improving cardiovascular disease were due to the nutritional components and plant-derived compounds contained in rice bran.

  In this experiment, it was verified whether rice bran has an action to reduce blood pressure. SHRSP rats were used to elucidate the effects on blood pressure and the mechanism of action.

  An embodiment of the present invention will be described below using an experimental method as an example.

  First, the preparation of rice bran ingredients will be described. The following two extracts (Driselase fraction and ethanol fraction) were obtained from rice bran. First, 1 L of 70% ethanol was added to 500 g of rice bran, extracted by shaking for 2 hours, and filtered (filtrate, residue). The residue was dried at room temperature, suspended in 500 mL of 10 mM acetate buffer containing 0.2 mg / L Driselase, and digested overnight at 37 ° C. Then, it filtered and freeze-dried (Driselase fraction). The ethanol fraction used was the filtrate concentrated and lyophilized.

  Next, animals and test meals will be described. Four-week-old stroke-prone spontaneously hypertensive rats / Izumo (SHRSP) and normotensive Wistar Kyoto / Izumo (WKY) were used. Three types of test meals (control meal, Driselase fraction diet, ethanol fraction diet, see Table 1) were given to SHRSP. WKY was given a control diet. (6 animals per group) Food intake and body weight were measured once a week. Blood pressure was measured once a week with a blood pressure measuring instrument (MK-2000, Muromachi Kikai). Animals were transferred to metabolic cages 5 days prior to dissection and 24-hour urine collection was performed. After 16 hours fast, rats were dissected under diethyl ether anesthesia, blood and organs were removed and stored at -80 ° C until analysis.

  The measurement of protein content, creatine concentration, albumin, and glucose concentration in plasma and urine will be described. The protein content was measured by the Bradford method. Creatinine, blood urea nitrogen, albumin, and glucose concentrations were determined using a measurement kit manufactured by Wako Pure Chemical Industries.

  The measurement of plasma angiotensin converting enzyme (ACE) inhibitory activity will be described. Plasma ACE inhibitory activity was measured by the method of Chusman and Cheung. Incubate 150 μL 5 mM hippuryl-L-histidyl-L-leucine (Sigma) in 100 mM borate, 100 mM NaCl, pH 8.3 for 10 min at 37 ° C, add 60 μL plasma sample and 10 μL ACE (0.5 mU / μL), 30 min Incubated. Hippuric acid liberated by ACE was extracted with ethyl acetate and measured at 228 nm.

  The measurement of plasma nitric oxide (NO) concentration will be described. NO was measured by Griess method. Before the measurement, the plasma sample was subjected to centrifugal filtration to remove hemoglobin and other proteins (Amicom 100 UFC 3, 7500 rpm).

  As a result of the experiment, the change in weight will be described first. Table 2 shows the influence of experimental food on body weight gain and the weight of each organ. The ratio of weight gain to experimental food intake (FER) did not differ between the SHRSP experimental food groups. However, there was a difference in FER between SHRSP and WKY. Liver, heart and kidney weights per 100g body weight were greater with SHRSP than with WKY. However, there were no differences between the experimental groups in lung, spleen, or epididymal fat.

  The effect of experimental diet on systolic blood pressure will be described. The main purpose of this study is to clarify whether an experimental diet containing two fractions derived from rice bran can reduce SHRSP blood pressure. The systolic blood pressure of rats fed the experimental diet is shown in FIG. SHRSP increases blood pressure from 5-6 weeks of age compared to WKY. SHRSP fed the Driselase fraction and ethanol fractions had a significant decrease in blood pressure from the 7th week to the end of the experiment compared to the control diet SHRSP. On the other hand, the blood pressure of WKY rats fed the control diet did not increase. The systolic blood pressure of each group at the end of the experiment was 240.17 ± 2.70 (SHRSP control diet), 190.83 ± 2.65 (SHRSP Driselase fraction diet), 190.33 ± 10.50 (SHRSP ethanol fraction diet), 109.17 ± 3.08 mmHg ( WKY control diet). These results indicate that both rice bran fractions reduce blood pressure in SHRSP.

  The plasma ACE inhibitory activity will be described. ACE is involved in the vasoconstriction mechanism by increasing aldosterone secretion from the adrenal glands. Blood pressure has been reported to correlate with plasma ACE inhibitory activity. The plasma ACE inhibitory activity of rats fed the experimental diet is shown in FIG. After 8 weeks of breeding, the ACE inhibitory activity was significantly lower in the SHRSP control diet group than in the Driselase fraction group and the ethanol fraction group. ACE activity decreased by 10.83% (Driselase fraction group) and 9.85% (ethanol fraction group) compared with the SHRSP control diet group. On the other hand, there was no significant difference between the WKY control diet group, the Driselase fraction diet group, and the ethanol fraction diet group. These results indicate that both rice bran fractions increase the plasma ACE inhibitory activity of SHRSP to the same level as WKY, which is normal blood pressure.

  The measurement of a renal function marker is demonstrated. Hypertension is one of the causes of renal failure called end-stage renal disease. Excess water in the blood vessels increases blood pressure. Table 3 shows the effect of the experimental diet on renal function. There was no difference in plasma creatinine concentration between groups. SHRSP blood urea nitrogen (BUN) and BUN / creatinine ratios were significantly lower than those of the SHRSP control and WKY control diet groups. Plasma albumin in both rice bran fraction diet groups was significantly lower than that in the SHRSP control diet group, and no difference was seen from the WKY control diet group. The plasma protein concentration in the Driselase diet group was significantly lower than that in the SHRSP control diet group. The urinary creatinine concentration was significantly lower in the Driselase fraction group and the ethanol fraction group than in the SHRSP and WKY control diet groups. The amount of urine protein was significantly lower in the rice bran fraction group compared to the SHRSP control group. Creatinine clearance was significantly reduced in the driselase and ethanol fractions compared to the SHRSP and WKY control diet groups. From the above results, it is estimated that the rice bran fraction in the diet can protect the filtration function of the kidney from the damage caused by high blood pressure.

  The plasma NO concentration will be described. Nitric oxide (NO) relaxes vascular smooth muscle by activating soluble guanylate cyclase, increasing cGMP levels and decreasing intracellular calcium levels. Thus, changes in NO synthesis are important factors in the pathogenesis of hypertension. The measurement results of plasma NO concentration are shown in FIG. The plasma NO concentration was higher in the SHRSP control group than in the Driselase fraction group and the ethanol fraction group.

  The fasting plasma glucose concentration will be described. We examined whether rice bran fraction reduces fasting plasma glucose concentration in SHRSP rats. The results are shown in FIG. At the end of the experiment, plasma glucose levels were significantly reduced compared to controls (Driselase fraction group 20.46%, ethanol fraction group 26.08%), and both rice bran fractions could reduce SHRSP fasting blood glucose. It was revealed.

  The measurement of plasma and liver lipid parameters will be described. The effect of rice bran fraction on lipid metabolism was analyzed. Table 4 shows the measurement results of plasma total cholesterol, HDL cholesterol, LDL cholesterol, triglyceride, phospholipid, and free fatty acid. Compared with the SHRSP control diet group, the SHRSP Driselase fraction diet group showed a significant decrease in total cholesterol, LDL cholesterol, and triglyceride concentrations. In addition, the HDL / LDL ratio, a marker for cardiovascular disease, also increased significantly. On the other hand, in the SHRSP ethanol fraction diet group, only the plasma triglyceride concentration decreased compared to the SHRSP control diet group. The measurement results of liver lipid parameters are shown in Table 5. Compared to the SHRSP control diet group, the SHRSP Driselase fraction diet group showed no change in total lipid and total cholesterol, whereas a decrease in triglyceride and phospholipid content was observed. On the other hand, no change was seen in the SHRSP ethanol fraction group. From the above, it is estimated that rice bran, especially the Driselase fraction, has an activity to suppress the onset of cardiovascular diseases such as arteriosclerosis.

  When considered, hypertension is a risk factor for stroke, coronary heart disease, and kidney disease. Long-term drugs are usually used to control hypertension. However, the drug has many side effects. Ingestion of more fruits, vegetables and cereals correlates with reducing the incidence of cardiovascular disease.

  The biggest finding in this study is that the blood pressure of SHRSP fed the rice bran fraction test meal decreased compared to the control meal. The blood pressure of SHRSP gradually increases with age. However, the rice bran test meal significantly decreased blood pressure from the third week of feeding until the end of the experiment. This phenomenon is thought to be due to biologically active substances (ferulic acid, etc.) contained in rice bran. Rice bran contains about 0.1% ferulic acid. When free ferulic acid was orally administered to SHR (50 mg / Kg), blood pressure was lowest at 1 hour after administration and returned to predose levels after 6 hours. In addition, a significant reduction in blood pressure was observed when fed on a diet containing ferulic acid at 10 mg or 50 mg / Kg / day for 6 weeks. The decrease in blood pressure caused by the rice bran test meal observed this time is estimated to be through inhibition of ACE activity, improvement of renal function, and reduction of NO production.

  Certain ACE inhibitors, such as captopril and enalapril, are used as antihypertensive agents. However, side effects to be noted have been reported. In recent years, ACE inhibitory peptides derived from food proteins have been isolated and reported. (Indonesian dried salted fish, salmon muscle, fermented soybean, porcine skeletal muscle) There are few reports of ACE inhibitors other than peptides. (Mushroom, Ashitaba, Garlic) In this study, it was shown that the blood pressure lowering effect of rice bran is caused by inhibiting ACE activity in plasma. More importantly, there was a positive correlation between lowering blood pressure and plasma ACE inhibitory activity. This indicates that ACE inhibition by rice bran has a blood pressure lowering effect. This result is consistent with the result of garlic having non-peptide ACE inhibitory activity. When garlic is administered, serum ACE inhibitory activity is significantly increased, and a correlation with a decrease in blood pressure is observed.

  The effects of SHRSP and WKY on the renal function of both rice bran fractions were also analyzed. The kidney is an organ that is damaged by high blood pressure. Therefore, it is important to evaluate whether rice bran has a protective effect on the development of kidney disease. The results of this experiment showed that renal function markers such as BUN, albumin, protein concentration, BUN / creatinine, and creatine clearance were improved by ingestion of rice bran. These results indicate that rice bran reduces blood pressure and improves renal function in SHRSP.

  Oxidative stress is deeply related to the onset of cardiovascular diseases such as hypertension, type II diabetes, hypercholesterolemia, and atherosclerosis and the development of disease states. Reactive oxygen (ROS) is ubiquitous in all aerobic species and is elevated by endogenous and exogenous factors. Overproduction of ROS can cause hypertension and coronary heart disease at different stages, such as vascular endothelial cell damage, foam cell formation, vascular smooth muscle cell proliferation, gene expression, vasoconstriction, and plaque destabilization. Associated with hatred. Antioxidants and superoxide dismutase (SOD) analogs alleviate endothelial cell damage and lower blood pressure in the rat pathologic model system, against increased production of ROS, a major hypertension disorder. Also, in patients with coronary heart disease, inhibition of ACE activity improved endothelial cell function.

  In this experiment, the plasma NO concentration in the SHRSP control diet group was higher than that in the two rice bran fraction groups. A significant correlation was observed between blood pressure at the end of the 12-week-old experiment and plasma NO concentration (FIG. 6). Alleviation of oxidative stress by rice bran intake indicates that ROS-mediated NO inactivation is reduced. Consistent with these results, polyphenol (ferulic acid) in rice bran contributes as an antioxidant substance.

  A series of studies on ferulic acid show that ferulic acid has an antioxidant effect in vivo and in vitro. Free ferulic acid has the activity of scavenging hydroxy radicals and superoxide anions and inhibits lipid peroxidation. Ferulic acid inhibits rat liver microsomal membrane peroxidation and the production of reactive oxygen induced by tert-butyl hydroperoxide and 2,2'-azobis (2-amidinoprpane) in NIH3T3 cells. Rice bran contains flavonoids that have anti-inflammatory effects in addition to polyphenols. Flavonoids contained in prenyl compounds such as morusin, kuwanon C, and sanggenon D, and biflavonoids such as bilobetin and ginkgetin suppress NO production by lipopolysaccharide (LPS) treatment. Inhibition of NO production is performed by suppressing inducible NO synthase (iNOS), but is not a direct inhibition on iNOS. Since NO production by iNOS plays an important role in inflammatory diseases, inhibition of NO production by flavonoids is thought to be involved in at least the in vivo anti-inflammatory and immune function control systems.

  Rice bran was involved in lowering fasting blood glucose levels of SHRSP. Many researchers have reported that these physiological activities are a synergistic effect of many minor components with biological activity present in rice bran. All trace components, including antioxidants such as tocopherol, tocotrienol, γ-oryzanol, and polyphenols (ferulic acid), are contained in both fractions of rice bran and affect glucose levels by affecting glucose absorption and utilization. The possibility of maintaining These components have free radical scavenging ability and can improve diabetic complications such as glycation, glycooxidation, atherosclerosis and antilipemia.

  In conclusion, our findings indicate that both rice bran fractions, via inhibiting ACE activity, lowered blood pressure that increased in SHRSP, improved renal function, and decreased NO production.

  Traditionally, ferulic acid was obtained from rice bran by alcohol extraction (free ferulic acid). The inventors of the present invention obtained ferulic acid bound to dietary fiber by enzymatic treatment. This enzyme-extracted ferulic acid was found to have a blood pressure increase-inhibiting effect and cholesterol-inhibiting effect, both of which were stronger than free ferulic acid. In addition, it can be obtained in large amounts from rice bran compared to free ferulic acid.

  Even when it is a physiologically active substance contained in foods and strengthened in foods, the onset of lifestyle-related diseases can be suppressed without impairing product safety.

Effects of experimental diet on blood pressure Effect of experimental food on ACE activity Effect of experimental diet on plasma NO concentration Effect of experimental diet on fasting blood glucose Correlation between ACE inhibitory activity and systolic blood pressure Correlation between plasma NO concentration and systolic blood pressure

Claims (3)

  An ethanol treatment process in which ethanol is added to rice bran and shaken, a primary filtration process for filtering the treatment liquid obtained in the ethanol treatment process, and a residue obtained in the primary filtration process are dried by heating. A thermal drying treatment process, an enzyme treatment process in which the treated product obtained in the thermal drying treatment process is suspended using a solution containing an enzyme preparation, and the suspension obtained in the enzyme treatment process is filtered. A method for extracting ferulic acid, comprising: a secondary filtration process; and a freeze-drying process in which an extract mainly containing ferulic acid obtained in the secondary filtration process is freeze-dried.   Extracting ferulic acid by enzymatic treatment of rice bran with an enzyme preparation, and adding ferulic acid to foods mainly to prevent diseases related to the onset and progression of lifestyle and aging Preventive food for diseases characterized.   Extracting ferulic acid by enzymatic treatment of rice bran with an enzyme preparation, and adding ferulic acid to the drug to prevent diseases related to onset and progression, mainly lifestyle and aging A preventive drug for diseases.

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