Abstract
This study investigated how citrus yuzu peel ethanol extract (YPE) affected both fatty liver and liver damage in rats fed with chloretone. Chloretone, a xenobiotic, was used to increase the amount of liver triacylglycerol and simultaneously elevate the concentration of serum alanine aminotransferase by which one can estimate the degree of liver damage. Both the water-soluble and fat-soluble fractions of YPE inhibited fatty liver caused by chloretone. Although the fat-soluble fraction had a lesser effect on fatty liver than the water-soluble fraction, increasing the dosage of the fat-soluble fraction significantly ameliorated fatty liver. Amphiphilic components in the fat-soluble fraction played a key role in improvement. Metabolomic analysis suggested that YPE components would suppress fatty acid synthesis and promote fatty acid degradation. The present study revealed that both the water-soluble and amphiphilic fractions of YPE have a novel inhibitory effect on fatty liver.
Introduction
Excessive triacylglycerol accumulation in liver cells without alcohol should be observed in non-alcoholic fatty liver disease (NAFLD). NAFLD is associated with broad morbid states in the liver ranging from simple fat deposition (steatosis and non-alcoholic fatty liver, NAFL) to cancerous liver via fibrosis and cirrhosis as non-alcoholic steatohepatitis (NASH). Recently, the “multiple parallel hit hypothesis”, which proposed that various factors simultaneously occur to give rise to NAFLD, has been widely adopted in return for the “two-hit theory” of the first step of NAFL, followed by the second step of NASH (Tilg et al., 2020). Therefore, it seems to have become instrumental, we believe, to control not only simple NAFL but also the progressive state of it.
A high-fat and high-sucrose diet (Liang et al., 2016; Chijimatsu et al., 2015) allows the establishment of a simple NAFL model animal. Dosing d-galactosamine (Chijimatsu et al., 2008) or carbon tetrachloride (Park et al., 2000) provides NAFLD with liver damage in model animals. In particular, xenobiotic molecules such as 1,1,1-trichloro-2-methyl-2-propanol (chloretone), 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (one of the polychlorinated biphenyl [PCB] molecules), and 2,6-di-tert-butyl-hydroxytoluenec (BHT) are known to cause more a progressive stage than NAFL (Hitomi et al., 1993). Dosing the above xenobiotics in model animals results in various states such as (1) activating liver drug-metabolizing enzymes (Oda and Yoshida, 1994), (2) accelerating fat accumulation in the liver (Sandberg and Glaumann, 1980; Oda et al., 1994; Sun et al., 2019), (3) emerging hypercholesterolemia (Oda et al., 1994) through increasing serum high density lipoprotein cholesterol and apolipoprotein A-I (Oda and Yoshida, 1994), and (4) enhancing lipid oxidation (Kato et al., 1981). In particular, the chloretone diet in model animals increases serum cholesterol and ascorbic acid in the liver and urine (Chijimatsu et al., 2009), liver lipid accumulation and serum alanine aminotransferase ALT which can be used as an indicator of liver damage (Chijimatsu et al., 2015). These phenomena seem to stem from enhancement of gene expressions both for fatty acid synthesis and for fatty acid degradation.
We have been trying to identify new foods to alleviate the more advanced states of NAFL. Unfortunately, since there no optimal animal model for NASH, herein, we used a method of feeding a chloretone diet. By utilizing the chloretone method, in fact, we have already shown that the freshwater clam (Corbicula fluminea) suppresses fatty liver (Chijimatsu et al., 2015). On the other hand, we demonstrated that citrus “Yuzu”, Citrus junos, peel affects the liver as NAFL in rats fed with a high-sucrose diet (Suzuki et al., 2021, in press).
Citrus yuzu peel is characterized by unique healing properties and has comforting aromatic essential oils; therefore, it is increasingly being used in various industries. It has been reported that yuzu inhibited colorectal cancer cell growth (Kim et al., 2014; Abe et al., 2018), alleviated colitis symptoms in mice (Kim et al., 2014), and had an anti-diabetic effect in rats (Yang et al., 2013) and an anti-obesity effect in zebrafish (Zang et al., 2014). Moreover, we have also revealed that yuzu peel ethanol extracts show inhibition of enhanced NF-κB (Iha et al., 2011), an antiallergic effect to control enhanced IgE (Ishikawa et al., 2013), and inhibition of fat accumulation in the liver of rats fed with a high-sucrose diet. A set of facts showing various novel bioactive molecules in yuzu propelled us to study whether yuzu peel could prevent fatty liver and liver damage.
This study demonstrates the effects of YPE on NAFL in rats with liver damage caused by chloretone diet. Herein, we report that each of the water-soluble and amphiphilic fractions of YPE has a novel inhibitory effect on hepatic steatosis.
Materials and Methods
Materials Fully ripe yellow yuzu grown on a tree were chosen for this study. The ripe yuzu were gifted from Tsue AP Co., Inc. (Oita, Japan). Fresh yuzu peel was dried by far-infrared radiation. The dried peel was cut and crushed to less than 5 mm in length, and the crushed peel was continuously extracted with 95% fermented ethanol. Removing the used solvent in vacuo gave a highly viscous dark brown liquid as YPE. About 600 g of YPE was recovered from 1.2 kg of dried pericarp with a yield of about 50% (w/w). Aqueous warm suspension of YPE was treated with chloroform (Kishida Chemical Co., Ltd., Osaka, Japan, 1st Grade) to extract fat-soluble components. The extracted chloroform layer was then washed with ion-exchanged water (ORGANO Co., Tokyo, Japan, PRB-002A). Removing solvents from collected aqueous and chloroform layers in vacuo gave water-soluble fraction I and fat-soluble fraction II, respectively. In experiment 1, both water-soluble fraction I and fat-soluble fraction II were extracted from YPE with a yield of 87.4 and 3.8% (w/w), respectively. In the case of experiments 2 and 3, fraction II was extracted from YPE with 6.4% (w/w) yield. Fraction II was mixed with silica gel powder (Kanto Chemical Co., Inc., Tokyo, Japan, 37562-85) to separate hydrophobic components from amphiphilic ones. Oleophilic fraction IV was eluted with toluene, and then amphiphilic fraction III was eluted with methanol. The yields of III and IV from fraction II were 54.3% (w/w) and 35.3% (w/w), respectively. Each yield is shown in Figure 1. The yield of the extraction ratio was used in calculations to constitute the diet.
Animals and breeding conditions Male Wistar rats (5-weeks-old; Japan SLC, Inc., Hamamatsu, Japan) were housed in individual stainless cages under controlled environmental conditions maintained at 23 °C with a 12-hour light-dark cycle (lights on 20:00–08:00). The number of each diet group was conditioned to be six, except for the basal and control in experiment 3, which comprised five rats a diet group. First, the rats were fed a commercial diet (5L37; Japan SLC, Inc.) to allow them to adapt to the breeding environment for 4–5 days. Then, they were fed a purified diet without chloretone, YPE and its fractions for 3 days followed by the experimental diet for 14 days. Throughout the study, rats had access to food and tap water ad libitum. The experimental procedures used in this study complied with the guidelines set by the Animal Care and Use Committee of Oita University (No. J048002, Oita, Japan).
Sample collection All rats were killed on the last day of the experimental period by decapitation at 10:00 a.m. After 4 h of fasting, livers were removed, weighed, and fixed in aqueous 10% (v/v) formalin for histologic analysis. Three experiments were carried out. Five groups, a single group consisting of five or six rats, were prepared in all experiments. The “Basal” group was fed a diet without any additive such as chloretone or yuzu components. The “Control” group was fed a diet with 0.3% (w/w) chloretone without any yuzu components. Since the amount of each YPE fraction calculated from the yield for 20 g of YPE was lighter than original weight of 20 g, the disparity between 20 g and the calculated weight of a YPE fraction was compensated by mixing with the corresponding weight of α-corn starch. Table 1 summarizes the precise composition of the diet used.
Table 1.
Composition of the experimental diets
|
Chloretone |
| g/kg |
Basal |
Control |
YPE |
I |
II |
| Corn starch1 |
532.0 |
529.0 |
509.0 |
512.4 |
528.3 |
| Casein |
200.0 |
200.0 |
200.0 |
200.0 |
200.0 |
| Sucrose |
100.0 |
100.0 |
100.0 |
100.0 |
100.0 |
| Soybean Oil2 |
70.0 |
70.0 |
70.0 |
70.0 |
70.0 |
| Cellulose |
50.0 |
50.0 |
50.0 |
50.0 |
50.0 |
| AIN-93G mineral mixture3 |
35.0 |
35.0 |
35.0 |
35.0 |
35.0 |
| AIN-93 vitamin mixture3 |
10.0 |
10.0 |
10.0 |
10.0 |
10.0 |
| L-Cystine |
3.0 |
3.0 |
3.0 |
3.0 |
3.0 |
| Chloretone |
- |
3.0 |
3.0 |
3.0 |
3.0 |
| YPE1 |
- |
- |
20.0 |
- |
- |
| Fraction I1 |
- |
- |
- |
16.56 |
- |
| Fraction II1 |
- |
- |
- |
- |
0.75 |
1 Sum of corn starch and yuzu component such as YPE, fraction I or II is condition to be 529 g. Subtract 3.0 g of chloretone from 532 g of corn starch for basal gives the weight to be 529 g.
2
Tert-butylhydroquinone 0.2 g/L was included
3 Supplied by Oriental Yeast, Tokyo, Japan.
In experiment 1, we evaluated the effects of YPE, fraction I and II on chloretone-fed rats (Figure 3, Table 2). The “YPE” group was fed a diet with both 2% (w/w) YPE and 0.3% (w/w) chloretone. The “I” group was fed a diet with both 1.66% (w/w) water-soluble fraction I and 0.3% (w/w) chloretone. The “II” group was fed a diet with both 0.075% (w/w) fat-soluble fraction II and 0.3% (w/w) chloretone.
Table 2.
Influence on body weight, food intake, and liver weight
Experiment 2 examined the effect of high concentrations of fat-soluble fraction II on a series of chloretone-fed rats (Figure 4, Table 2). The “II (× 1)” group was fed a diet with both 0.075% (w/w) fraction II and 0.3% (w/w) chloretone. The “II (× 5)” group was fed a diet with both 0.375% (w/w) fraction II and 0.3% (w/w) chloretone. The “II (× 10)” group was fed a diet with both 0.750% (w/w) fraction II and 0.3% (w/w) chloretone. That is, the weights of II (× 5) and II (× 10) correspond to five-fold as much and ten-fold as much as fraction II, respectively. The usage of α-corn starch for adjustment was the same as that in Table 1.
Experiment 3 tested the effect of both amphiphilic III and oleophilic IV components of fraction II on a series of chloretone-fed rats (Figure 5, Table 2). The “II” group was fed a diet with both 0.60% (w/w) fraction II and 0.3% (w/w) chloretone. The “III” group was fed a diet with both 0.33% (w/w) amphiphilic fraction III and 0.3% (w/w) chloretone. The “IV” group was fed a diet with both 0.21% (w/w) oleophilic fraction IV and 0.3% (w/w) chloretone.
Measurement for hepatic lipids Hepatic lipids were extracted with the mixture of chloroform and methanol (2/1 v/v) from homogenized livers according to the method described by Folch et al. (1957). The amount of the extracted hepatic triacylglycerol and cholesterol were determined using commercial kits; Triglyceride E-test and T-cholesterol (Wako Pure Chemical Industries, Osaka, Japan).
Histological observation Liver tissues fixed in formalin were embedded in paraffin and stained with hematoxylin and eosin (H & E). Tissue images were obtained through an optical microscope (Keyence, Osaka, Japan).
Analysis of metabolites by gas chromatography-tandem mass spectrometry (GC-MS/MS) Suitable extraction and purification of samples is a prerequisite for GC-MS/MS analysis. Aqueous suspension of the liver (400 µL, 25 mg/mL) was mixed with 500 µL methanol, 500 µL chloroform, and 250 µL pure water, and the mixture was then sonicated. Removal of used solvent from the sonicated suspension gave a solid residue. Pyridine solution of O-methylhydroxylamine hydrochloride (80 µL, 20 mg/mL) was poured into the residue at room temperature. After sonication of the mixed pyridine solution, N-methyl-N-(trimethylsilyl)trifluoroamide (40 µL, GL Sciences) was added into the solution and left standing for 30 min at 37 °C. The reaction mixture was centrifuged at 14 000 rpm for 5 min, being separated a transparent upper layer from lower precipitates. The clear upper layer was applied for GC-MS/MS (Shimadzu) on a DB-5 (30 m × 0.25 mm i.d., film thickness 1 m; Agilent, Santa Clara, USA) column. The results regarding identified molecules were computed with the SHIMADZU Smart Metabolites Database, according to the method described by Sun et al. (2019).
Statistical analysis Values are calculated as mean ± standard error of the mean (SEM) for the five or six rats in each dietary group. The statistical differences among the values were analyzed by analysis of variance (ANOVA) and the Tukey-Kramer test. Correlation among the samples possessing a statistically significant difference at p < 0.05 were discriminated as letters “a, b, c, and d”, with p values increasing in the order of “a, b, c, and d”.
Results
Optical microscopic observation of liver lobules Figure 2 shows optical micrographs of six liver sections stained with H&E by which a fat droplet can be recognized as a white sphere in the pink background. Rat liver fed with the basal diet showed less white spheres in the micrograph (Figure 2 A). On the other hand, the chloretone-dosed liver displayed a lot of fat droplets surrounding the portal vein (pointed out with a black arrow). Fat accumulation did not appear around the central vein (pointed out by a yellow arrow; Figure 2 B). The same situation as Figure 2 B can be seen in Figure 2 F where fraction IV was used. On the contrary, when YPE, II and III were dosed (Figures 2 C, D, and E), the fat accumulation faded away around both the portal and the central veins.
Fundamental makers such as body weight More important than the effectiveness against deteriorated fatty liver is the property of showing harmlessness as a food. There was no appreciable difference in body weight and food intake for experiments 1 and 3 (Table 2). There was a significant difference in food intake for experiment 2. It was observed in all experiments that dosed chloretone routinely enhanced liver weight. The increased liver weight caused by chloretone could be lightened by selecting a suitable yuzu component as described below. The improvement in liver weight by means of using a yuzu component is likely to correlate with the results in Figure 3 through Figure 5. Moreover, all the lessened weight by the YPE components were heavier than, or nearly equal to, that of the basal. These facts indicate the harmlessness of YPE.
Effect of YPE, water-soluble I, and fat-soluble II The concentrations of both triacylglycerol and cholesterol in the hepatic lipids were increased by feeding chloretone without any fractions (Figure 3 A, B). The enhanced concentrations of the control, in return, were decreased by feeding YPE, I, or II. For instance, the concentration of triacylglycerol in liver fed chloretone increased more than double than when fed basal diet. Having fed chloretone with YPE, I, or II reduced triacylglycerol by about 20% to 70% the enhanced. Among the three citrus components, the water-soluble fraction I accounted for the strongest reduction.
In the case of serum lipids, feeding chloretone compelled the concentrations of triacylglycerol and total cholesterol to decrease the former and increase the latter (Figure 3 C, D). Fraction I lowered the total cholesterol concentration in serum with a statistically significant difference. On the other hand, there was no significant difference among the four additives in the triacylglycerol maker.
Serum enzyme activity such as ALT and AST was affected by the above additives similar to the hepatic lipids (Figure 3 E, F). That is, feeding chloretone without any fractions activated both ALT and AST, and then dosing the three yuzu fractions helped the activity return to the original state of basal. In the case of ALT, the activity of the control was more than twice of that of the basal. Dosing both YPE and fraction I lessened the activity to nearly the original value of basal.
Concentration effect of fat-soluble II In addition to YPE and water-soluble fraction I, as mentioned above, fat-soluble fraction II was able to decrease the enhanced values of indicators for the lipid concentration and for the serum enzyme activity in spite of the lowest efficiency. Figure 4 shows how the dosage of the II ranging from the standard amount to ten-fold its amount affected the evaluation items.
As for the hepatic lipids, the enhanced concentrations for both cholesterol and triacylglycerol of the control decreased with increasing amounts of II (Figure 4 A, B). In fact, the maximum triacylglycerol concentration (control) declined nearly to that of basal by feeding with II (×10).
Regarding serum lipids, on the other hand, feeding chloretone resulted in altering the concentrations of both total cholesterol and triacylglycerol of the basal regardless of the presence/absence of fraction II (Figure 4 C, D). There was no statistical difference in the altered concentrations of both total cholesterol and triacylglycerol among the control and three fat-soluble fractions II.
The effect of dosed four additives, chloretone and three IIs, for the activity of ALT were analogous to that for the hepatic lipids (Figure 4 E, F). While feeding chloretone gave the maximum activity of ALT, dosing II recovered the activity to the original state of the basal. On the other hand, the activity of AST was not affected by the four additives at all.
Effect of amphiphilic fraction III and oleophilic fraction IV As shown in Figure 1, the fat-soluble fraction II was separated into amphiphilic III and oleophilic IV fractions. Both III and IV concentrations were conditioned to be five-fold as much as the standard amount, II (×1), in Figure 4.
As for triacylglycerol in hepatic lipids, fat-soluble II and the amphiphilic III reduced the enhanced value of the control as well as YPE. In fact, there was not a perceptible difference in the lipid concentration among YPE, II, and III (Figure 5 A). For the enhanced cholesterol in hepatic lipids of the control, the II and III lowered it slightly without significant difference (Figure 5 B). As opposed to II and III, the oleophilic IV did not contribute to recovering the deteriorated value of the control at all. The same situation as Figure 5 A also appeared in the activity of both ALT and AST in serum (Figure 5 E, F). Regarding serum lipids, none of the four yuzu components affected the altered values caused by dosed chloretone at all (Figure 5 C, D).
The serum AST activity of the control exceeded that of the basal in Figure 5 F and also in Figure 3 F. This characteristic is consistent with a previous report to have proved that chloretone increased serum AST activity (Chijimatsu et al., 2015). In Figure 4 F, however, feeding chloretone did not affect the activity of serum AST at all, having showed equal activity between the control and the basal. Despite having applied similar experimental conditions on Figure 3 through Figure 5, the above characteristic showing the enhancement caused by chloretone was not observed only in Figure 4 F. Unlike the AST, the other makers of ALT, cholesterol and triacylglycerol gave rise to the enhancement/reduction caused by chloretone. A lack of consistency as a maker was found in the serum AST activity in the present study.
GC-MS/MS for identifying metabolites YPE and its fractions except the oleophilic IV could recover the deteriorated fatty liver caused by chloretone. From the viewpoint of the lipid synthesis, various separated yuzu components through GC-MS/MS analysis were comprehensively computed with the metabolite database. Figure 6 addressed six fatty acids and Table 3 covered bioorganic molecules other than the fatty acids.
Table 3.
Metabolomics for various molecules except fatty acids.
|
|
Chloretone |
|
Basal |
Control |
YPE |
II (x5) |
III (x5) |
|
x105 |
x105 |
x105 |
x105 |
x105 |
| Pyruvic acid |
2.25 ± 0.61b |
1.12 ± 0.32ab |
1.03 ± 0.27ab |
0.97 ± 0.29ab |
0.74 ± 0.13a |
| Malic acid |
5.90 ± 1.10b |
4.04 ± 0.94ab |
3.35 ± 0.61ab |
2.76 ± 0.69ab |
2.67 ± 0.38ab |
| Ascorbic acid |
4.00 ± 0.69a |
8.55 ± 1.39ab |
8.66 ± 1.34ab |
9.56 ± 1.03b |
7.31 ± 1.14ab |
| 3 Aminoglutaric acid |
103.8 ± 15.6b |
76.6 ± 15.0ab |
62.3 ± 8.9ab |
54.1 ± 10.9a |
49.5 ± 5.8a |
| Aspartic acid |
94.2 ± 13.9b |
69.5 ± 13.3ab |
56.6 ± 7.9ab |
49.1 ± 9.6a |
44.9 ± 5.1a |
| Glutamic acid |
30.3 ± 2.6b |
20.2 ± 5.0ab |
17.1 ± 2.4a |
12.7 ± 2.4a |
14.8 ± 1.2a |
| Allose |
32.6 ± 4.2b |
18.4 ± 3.0a |
18.1 ± 1.5a |
16.3 ± 2.9a |
16.7 ± 1.2a |
| Glucono 1,5 lactone |
0.04 ± 0.03a |
4.58 ± 0.58b |
4.22 ± 0.74b |
1.47 ± 0.24a |
1.34 ± 0.27a |
| Mannose |
43.4 ± 6.1b |
24.6 ± 4.2a |
24.7 ± 2.2a |
22.0 ± 4.1a |
22.3 ± 1.5a |
| Glucose |
234 ± 14b |
180 ± 19ab |
182 ± 13ab |
151 ± 23a |
155 ± 12a |
| Galactose |
239 ± 16b |
191 ± 26ab |
182 ± 13ab |
157 ± 25a |
155 ± 12a |
| Glucuronic acid |
2.5 ± 0.5a |
16.3 ± 4.48b |
15.4 ± 2.7b |
11.3 ± 1.9ab |
12.3 ± 1.0ab |
| Galacturonic acid |
2 ± la |
177 ± 21c |
166 ± 18c |
81 ± 12b |
70 ± 12b |
| Glucaric acid |
0.06 ± 0.02a |
3.43 ± 1.44b |
2.56 ± 0.54ab |
2.10 ± 0.48ab |
2.02 ± 0.25ab |
| Uridine |
8.73 ± 1.38c |
5.22 ± 0.75b |
3.91 ± 0.46ab |
2.33 ± 0.56ab |
1.61 ± 0.49a |
Effect of YPE and its fractions on rats fed chloretone for 14 days Values are means ± SEM. The statistical differences among the values were analyzed by ANOVA and Tukey-Kramer test. Values in a row with different letters indicate a statistically significant difference (p < 0.05). Values is the peak area.
Each of YPE, II, and III possessed a tendency to decrease the peak area of the control in terms of myristic, palmitic, stearic, and oleic acids, although those were not statistically significantly different. For eicosapentaenoic acid, there was no statistically significant difference among the groups. For linoleic acid, the peak area value of the control was significantly reduced by dosed YPE, II (×5), and III (×5).
The biomolecules shown in Table 3 by referring to metabolite database possessed a peak area of 100 000 or more. Each peak area value for gluconolactone, glucuronic acid, galacturonic acid, and saccharinate was significantly increased by dosed chloretone, while that for allose and mannose were decreased. Each peak area value for gluconolactone and galacturonic acid was significantly decreased with both II (×5) and III (×5).
Discussion
The present study has demonstrated how YPE and its fractions affect fatty liver possessing liver damage. Xenobiotic chloretone is supposed to induce fatty liver, hepatic steatosis, and hypercholesterolemia (Oda et al., 1994; Chijimatsu et al., 2015). Firstly, we confirmed whether added chloretone brought us the above morbid states in liver under our experimental condition. The chloretone (Control) significantly elevated the amount of liver triacylglycerol, liver cholesterol, serum total cholesterol, and serum ALT (Figures 3–5). Moreover, Figure 2 B shows that accumulation of fat droplets can be recognized around the portal vein. These facts are consistent with the report of Chijimatsu et al. (2008). A similar distribution manner of fat droplets was observed in rats fed a high-sucrose diet (Sun et al., 2019).
In the chloretone-induced fatty liver, the addition of YPE significantly reduced the amount of enhanced triacylglycerol, cholesterol in liver, and serum ALT (Figure 3). This fact was also confirmed by the disappearance of fat droplets in the optical microscopic view depicted in Figure 2. These facts indicate obviously that YPE has inhibitory effects on enhanced hepatic steatosis induced by chloretone.
YPE was divided into water-soluble fraction I and fat-soluble fraction II in order for us to identify the functional fraction of YPE. The fraction I reduced the concentration of serum ALT as well as fatty liver more efficiently than fraction II (Figure 3). When using the high-sucrose diet method in rats, in general, NAFL model rats without enhanced serum ALT are available. Having used NAFL model rats allowed us to find out that myo-inositol, a type of cyclitols, in fraction I strongly suppressed fatty liver (Suzuki et al., 2021, in press). As for triacylglycerol and cholesterol in liver, myo-inositol inhibits chloretone-induced hepatic steatosis, similar to its action in high-sucrose-induced hepatic steatosis (unpublished data). A study on whether myo-inositol affects the enhanced serum ALT in rats fed chloretone is underway.
Although fraction II had less effect on liver steatosis than fraction I, we were aware of the possibility for fraction II to reduce liver triacylglycerol and serum ALT (Figure 3). Hence, the influence of the amount of added II on the fatty liver was investigated. With increasing the amount of added II, the concentration of liver triacylglycerol, liver cholesterol, and serum ALT decreased by around half with a statistically significant difference (p < 0.05) (Figure 4). This indicates that fraction II also contained active compounds to inhibit hepatic steatosis as well as fraction I. To identify the activator, the fat-soluble fraction II was further fractionated into the amphiphilic III and oleophilic IV fractions. As opposed to the oleophilic IV, the amphiphilic III contributes to reduce the value of serum ALT and liver triacylglycerol (Figure 5). Therefore, it is obvious that in addition to the water-soluble fraction I, the amphiphilic fraction II also contained active compounds to suppress hepatic steatosis. These results indicate that both water-soluble fraction I and amphiphilic fraction III in YPE can play an important role in inhibition of hepatic steatosis induced by chloretone.
Herein, an appeared signal peak in the used GC-MS/MS was analyzed, being identified as a known metabolite molecule. Among the six fatty acids, the peak area values of both myristic (14:0) and oleic (18:1) acids were predisposed to increase by dosed chloretone, suggesting that the fatty acid synthesis was enhanced in chloretone-fed rats (Figure 6). Chijimatsu et al., reported that the amount of mRNA for both fatty acid synthase and fatty acid desaturase 1 were increased by chloretone so that fatty acid synthesis was accelerated (Chijimatsu et al., 2015). Unlike chloretone, YPE and fraction II were showing a tendency to decrease the values for myristic, palmitic, stearic, oleic acids, and linoleic acid, probably because of inhibition for fatty acid synthesis. In particular, the value of linoleic acid was significantly reduced by YPE, fraction II and III. As linoleic acid can be originally from the diet, it is thought that fatty acid degradation systems such as β-oxidation was accelerated. These results suggest that YPE and its fractions may have suppressed fatty liver by inhibiting the fatty acid synthesis system in the liver or promoting the degradation system.
Regarding glucuronic acid, chloretone heightened the value to 16.3 from the original basal of 2.52, then the maximum value decreased by a factor of about two-thirds with the fraction II (Table 3). Xenobiotics possessing -OH, -NH2, and COOH functional groups can generally be glucuronidated in the phase II drug-metabolism (Fujiwara et al., 2018). Herein, ingested chloretone, a type of alcohols, probably enhanced the concentration of glucuronic acid through the phase II metabolism, in addition to having facilitated fat accumulation in the liver. While YPE fractions II and III suppressed fat accumulation in the liver, they gradually reduced the enhanced concentration of glucuronic acid. Based on these facts, it could be speculated that YPE fractions II and III might suppress NAFLD by a different pathway from glucuronidation. On the other hand, this effect was not observed in YPE, which are comprised of fraction I and II. For instance, the values for galacturonic acid of the II (×5) to be 81 × 105 was less than half of that of YPE to be 166 × 105, which was very close to that of the control of 177 × 105. This result indicates the myo-inositol isolated from the water-soluble I possessed a different mechanism of action from that of fat-soluble II.
Polyphenols such as eriocitrin, nobiletin and hesperidin in various foods are known to affect fatty liver (Zang et al., 2014; Saito et al., 2007; Takayanagi et al., 2011; Wang et al., 2011). In particular, it has been reported that hesperidin in the fat-soluble fraction II of Cannabis species inhibit the accumulation of fat droplets in fatty liver (Takayanagi et al., 2011; Wang et al., 2011). As the hesperidin molecule is amphiphilic enough to be dissolved in both the fraction II and the III, the polyphenol seems to be one of candidates that affect the presence fatty liver. We have already reported that fat-soluble fraction II of YPE regulated enhanced NF-κB (Ishikawa et al., 2013). It was reported that curcumin, a hydrophobic polyphenol to inhibit NF-κB activation, ameliorates galactosamine-induced hepatotoxicity (Xiea et al., 2017). Should curcumin be involved in YPE, it would be conceivable that YPE affects the fatty liver via the cascade of NF-κB regulation system. In addition, we have also reported that fraction II contains a coumarin derivative, auraptene, by which an anti-inflammatory effect was observed in an allergy model mouse characterized by the enhanced IgE (Ishikawa et al., 2013). Coumarin derivatives would be one of the plausible molecules that affect fatty liver possessing liver damage. Identification of bioactive molecules to control hepatic steatosis is in progress.
Conclusion
The present paper has demonstrated for the first time that YPE possesses effective components to improve the more progressive morbid states than simple NAFL. The water-soluble fraction I in YPE contains stronger compounds to affect fatty liver than the fat-soluble fraction II in YPE. The regulatory component in the fat-soluble fraction II is characterized as amphiphilic. Metabolomic analysis forecasts the components of YPE would alter the sugar metabolic process by which the lipid metabolism could be affected.
The fact of proving the improvement of fat liver with liver damage would make the citrus an attractive candidate for development of functional foods. However, we have not isolated and identified the bioactive molecules in YPE yet. Studies are currently underway to identify the bioactive molecules and to further elucidate the more detailed mechanism of the inhibition of hepatic steatosis.
References
- Abe, H., Ishioka, M., Fujita, Y., Umeno, A., Yasunaga, M., Sato, A., Ohnishi, S., Suzuki, S., Ishida, N., Shichiri, M., Yoshida, Y., and Nakajima, Y. (2018). Yuzu (Citrus junos Tanaka) peel attenuates dextran sulfate sodium-induced murine experimental colitis. J. Oleo. Sci., 67, 335-344.
- Chijimatsu, T., Iwao, T., Oda, H., and Mochizuki, S. (2009). A Freshwater Clam (Corbicula fluminea) Extract Reduces cholesterol level and hepatic lipids in normal rats and xenobiotics-induced hypercholestelemic rats. J. Agric. Food Chem., 57, 3108-3112.
- Chijimatsu, T., Umeki, M., Kobayashi, S., Kataoka, Y., Yamada, K., Oda, H., and Mochizuki, S. (2015). Dietary freshwater clam (Corbicula fluminea) extract suppressed accumulation of hepatic lipids and increase in serum cholesterol and aminotransferase activities induced by dietary chloretone in rats. Biosci. Biotech. Biochem., 79, 1155-1163.
- Chijimatsu, T., Yamada, A., Miyaki, H., Yoshinaga, T., Murata, N., Hata, M., Abe, K., Oda, H., and Mochizuki, S. (2008). Effect of freshwater clam (Corbicula fluminea) extract on liver function in rat. Nippon Shokuhinkagaku Kougaku Kaishi, 5, 63-68. (in Japanese)
- Folch, J., Lees, M., and Sloane, S.G.H. (1957). A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem., 226, 497-509.
- Fujiwara, R., Yoda, E., and Tukey, H.R. (2018). Species differences in drug glucuronidation: Humanized UDP-glucuronosyltransferase 1 mice and their application for predicting drug glucuronidation and drug-induced toxicity in humans. Drug Metab. Pharmacokinet., 33, 9-16.
- Hitomi, Y., Wakayama, M., Oda, H., and Yoshida, A. (1993). Liver-specific induction of NADPH-generating enzymes by polychlorinated biphenyls in rats. Biosci. Biotech. Biochem., 57, 1134-1136.
- Iha, H., Ichinose, T., and Ishikawa, Y. (2011) Japan, Patent No. 4803553. Aug. 19.
- Ishikawa, Y., Kaoru, N., Ichinose, T., Sadakane, K., Yoshida, S., and Hasebe, T. (2013). Immune modulating extracts from Citrus-Yuzu peel and commercializing special Yuzu deverage/candy for deducing allergies. The Allergy in Practice, 33, 870-875. (in Japanese)
- Kato, K., Kawai, K., and Yoshida, A. (1981). Effect of dietary level of ascorbic acid on the growth, hepatic lipid peroxidation, and serum lipids in guinea pigs fed polychlorinated biphenyls. J. Nutr., 111, 1727-1733.
- Kim, S.H., Shinb, E.J., Hur, H.J., Parka, J.H., Sung, M.J., Kwon, D.Y., and Hwang, J.T. (2014). Citrus junos Tanaka peel extract attenuates experimental colitis and inhibits tumour growth in a mouse xenograft model. J. Funct. Foods, 8, 301-308.
- Liang, H., Yang, J., Tang, J., Wu, C., Li, H., and Chen, S. (2016) Optimization of dosage ratio of chlorogenic acid and gardenia glycosides in the treatment of rats with fatty liver disease induced by high-fat feed. J Tradit Chinese Medical Sci, 36, 683-688.
- Oda, H. and Yoshida, A. (1994). Effect of feeding xenobiotics on serum high density lipoprotein and apolipoprotein A-I in Rats. Biosci. Biotech. Biochem., 58, 1646-1651.
- Oda, H., Matsushita, N., Hirabayashi, A., and Yoshida, A. (1994). Cholesterolrich very low density lipoproteins and fatty liver in rats fed polychlorinated biphenyls. Biosci. Biotech. Biochem., 58, 2152-2158.
- Park, E.J., Jeon, C.H., Ko, G., Kim, J., and Sohn, D.H. (2000). Protective effect of curcumin in rat liver injury induced by carbon tetrachloride. J. Pharm. Pharmacol., 52, 437-440.
- Saito, T., Abe, D., and Sekiya, K. (2007). Nobiletin enhances differentiation and lipolysis of 3T3-L1 adipocytes. Biochem. Biophys. Res. Commun., 357, 371-376.
- Sandberg, P.O., and Glaumann, H. (1980). Studies on the cellular toxicity of polychlorinated biphenyls (PCBs) partial block and alteration of intracellular migration of lipoprotein particles in rat liver. Exp. Mol. Pathol., 32, 1-22.
- Sun, S., Hanzawa, F., Kim, D., Umeki, M., Nakajima, S., Sakai, K., Ikeda, S., Mochizuki, S., and Oda, H. (2019). Circadian rhythm-dependent induction of hepatic lipogenic gene expression in rats fed a high-sucrose diet. J. Biol. Chem., 294, 15206-15217.
- Suzuki, A., Miyajima, S., Mochizuki, S., Umeki, M., Sakai, K., Koya, M., Oda, H., Nobuoka, K., and Ishikawa, Y. (2021). Suppressive effect arising from Yuzu (Citrus junos) peel extract for fatty liver steatosis induced by high-sucrose diet in rat. Nippon Shokuhinkagaku Kougaku Kaishi, In press. (in Japanese)
- Takayanagi, K., Morimoto, S., Shirakura, Y., Mukai, K., Sugiyama, T., Tokuji, Y., and Ohnishi, M. (2011). Mechanism of visceral fat reduction in Tsumura Suzuki obese, diabetes (TSOD) mice orally administered β-cryptoxanthin from Satsuma Mandarin Oranges (Citrus unshiu Marc). J. Agric. Food Chem., 59, 12342-12351.
- Tilg, H., Adolph, T.E., Moschen, A.R. (2020). Multiple parallel hits hypothesis in NAFLD-revisited after a decade. Hepatology. DOI:10.1002/HEP.31518.
- Wang, X., Hasegawa, J., Kitamura, Y., Wang, Z., Matsuda, A., Shinoda, W., Miura, N., and Kimura, K. (2011). Effects of hesperidin on the progression of hypercholesterolemia and fatty liver induced by high-cholesterol diet in rats. J. Pharmacol. Sci., 117, 129-138.
- Xiea, Y.L., Chua, J.G., Jianc, X.M., Dongd, J.Z., Wanga, L.P., Lia, G.X., Yanga, N.B. (2017). Curcumin attenuates lipopolysaccharide/d-galactosamine-induced acute liver injury by activating Nrf2 nuclear translocation and inhibiting NF-kB activation. Biomed. Pharmacother. 91, 70-77.
- Yang, H.J., Hwang, J.T., Kwon, D.Y., Kim, M.J., Kang, S., Moon, N.R., and Park, S. (2013). Yuzu extract prevents cognitive decline and impaired glucose homeostasis in β-Amyloid-infused rats. J. Nutr., 143, 1093-1099.
- Zang, L., Shimada, Y., Kawajiri, J., Tanaka, T., and Nishimura, N. (2014). Effects of Yuzu (Citrus junos Siebold ex Tanaka) peel on the diet-induced obesity in a zebrafish model. J. Funct. Foods, 10, 499-510.
