2020 年 26 巻 4 号 p. 501-507
In previous studies, the non-sugar component (NSC) fraction, analyzed as 50 % methanol extract from Kokuto, showed a high in vitro antioxidant activity and strong anti-stress effect in acutely stressed mice. In addition, p-hydroxybenzaldehyde (HBA) and p-hydroxyacetophenone (HAP) were determined to be some of the antioxidative compounds in the NSC fraction. Therefore, the aim of the present study was to investigate whether NSC fraction, HBA and HAP could suppress stress response and the changes of hepatic lipids in chronically stressed mice fed a high fat diet. The results of the present study show that the NSC fraction has a high antioxidant activity in vitro, and oral administration of the fraction and HAP suppressed the secretion of stress hormone in the chronically stressed mice. We also found that the two phenolic compounds prevented hepatic lipid accumulation and peroxidation in the stressed mice.
Exposure to stress and fat-rich foods is common in modern lifestyles, and chronic stress is linked to an increased risk of metabolic disorders, including fatty liver disease (Ryan, 2014; Cheng et al., 2017). Moreover, chronic stress leads to an increase of reactive oxygen species (ROS) in hepatic lipid, which have been implicated in serious diseases (Boyd and McGuire, 1991). Prevalence of non-alcoholic fatty liver disease is associated with chronic oxidative stress, in general population is estimated to be nearly 23 % in Europe and 27 % in Asia and it is increasing remarkably over the last three decades (Younossi et al., 2018). Combination of stress and high-fat diet (HFD) can lead to lipid accumulation and peroxidation in liver (Kuo et al., 2007). The Chinese medicine formula Sinisan, called Shigyaku-san in Japan, has been shown to reduce depression-like behavior and is known to contain antioxidative compounds (Cheng et al., 2017; Wen et al., 2012). Several studies have reported that administration of the Sinisan adjusted triglyceride distribution and reduced hepatic peroxide damage in stressed non-alcoholic fatty liver disease model (Cheng et al., 2017). These results indicate that the utilization of dietary antioxidants with anti-stress effects can modify the chronic stress induced changes in hepatic lipids.
Non-centrifuged cane sugar, called Kokuto in Japan, has been shown to have an antioxidant effect (Payet et al., 2005). Kokuto may have great potential for use as an effective inhibitor of mental stress, and the Kokuto 50 % methanol fraction has been shown to have a high antioxidant activity and corticosterone suppression effect (Takahashi et al., 2017; Kinjo et al., 2019). In addition, the antioxidative compounds p-hydroxybenzaldehyde (HBA) and p-hydroxyacetophenone (HAP) were detected from the fraction, which are hypothesized to have an anti-stress effect (Bountagkidou et al., 2010; Hacibekiroğlu and Kolak, 2011). However, so far there have been few reports concerning the anti-stress effect of Kokuto, and therefore, the aim of the present study was to investigate whether 50 % methanol non-sugar component (NSC) fraction, HBA and HAP could suppress chronic stress response in restraint stressed HFD-fed mice. We also evaluated the effect of the two phenolic compounds on hepatic lipid accumulation and peroxidation in mice.
Chemicals HBA was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). HAP, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). All other chemicals and reagents were of analytical grade.
Preparation of non-sugar component fraction Kokuto, processed from sugarcane in 2014, was obtained from Okinawa Brown Sugar Cooperative Association (Okinawa, Japan). Preparation of NSC fraction, obtained from the elusion on Diaion® HP-20 resin (Mitsubishi Chemical Corporation, Tokyo, Japan) with 50 % (v/v) aqueous methanol solution, was performed as previously described (Kinjo et al., 2019). As reference, the average contents of HBA and HAP in 1 g of the NSC fraction were 203 µg and 35 µg as determined by LC-MS analysis.
ABTS radical-scavenging assay and oxygen radical absorbance capacity (ORAC) assay Samples were prepared by dissolving NSC fraction, HBA and HAP in 50 % aqueous methanol. The HBA and HAP used as samples were commercially available. The ABTS radical-scavenging assay was performed as described by Miller et al. (1996). The ABTS radical-scavenging activity was calculated using a calibration curve of the trolox standard and expressed as µmol trolox equivalents (TE) per g of each sample. The ORAC assay was performed as described by Prior et al. (2003). ORAC value was determined using a calibration curve of the trolox standard and expressed as mmol TE per g of each sample.
Chronic stress test on HFD-fed mice Thirty male C57BL/6J healthy mice (7 weeks old, weighing 22.51 ± 1.05 g) were purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan). They were housed at 23 ± 2 °C with 50−60 % humidity and 12/12 h light/dark cycle in plastic cage and allowed free access to water and feed. After the adaptation period of 5 d, mice were weighted and divided into five groups based on average body weight (n=6 / group, weighing 22.82 ± 0.09 g) as follows: Blank (BLK), Control (CON), NSC, HBA, and HAP groups. All mice (except BLK group) were restrained for 60 min (10:30–11:30) a day during a period of 28 d, using a crafted restrainer trap. All mice were fed a HFD (56.7 kcal % fat, 507.6 kcal/100 g, High-Fat Diet 32, CLEA Japan, Inc., Tokyo, Japan) and weighted on days 0 and 28. Once a day, the BLK (no stress) and CON (stressed) groups were orally administrated distilled water (150 µL) and NSC, HBA, and HAP (stressed) groups were administered the respective sample dissolved in distilled water (100 mg/kg body weight) at 9:30–10:30. This dose was selected based on a previous study, which consider administration period for 28 d and non-toxic dose in mice (Kinjo et al., 2019). The HBA and HAP used as administration samples were commercially available. Thirty minutes after the final restraint stress, all mice were sacrificed by decapitation to minimize pain, and blood, adipose tissues (perirenal and periepididymal) and liver were collected in laboratory. The adipose tissues were weighted. This study was conducted in accordance with the guidelines of the University of the Ryukyus Policy on Animal Care and Use after approval by the Animal Care and Use Committee of the University of the Ryukyus (Okinawa, Japan). All animal care and experiments were in accordance with ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments).
Preparation of serum and liver homogenates Blood was harvested by decapitation of mice, collected in micro tubes and then allowed to stand overnight at 6 °C. The blood samples were then centrifuged at 1 000 × g for 15 min. Obtained supernatants were centrifuged again at 3 500 × g for 5 min. The obtained serum samples were collected into tubes and stored at −30 °C until analysis. Excised livers were frozen instantly with liquid nitrogen after weighing and stored at −80 °C before homogenized. About 200 mg of the livers were homogenized by Handy micro homogenizer PHYSCOTRON NS-310E3 (Microtec Co., Ltd., Chiba, Japan) in 1 mL of phosphate buffered saline. The homogenates were centrifuged at 3 000 × g for 15 min and supernatants were collected into tubes and stored at −30 °C until analysis.
Measurement of corticosterone level in serum Serum corticosterone level was determined using an enzyme immunoassay kit (DetectX®, Corticosterone Enzyme Immunoassay Kit, Arbor Assays, Ann Arbor, MI, USA) according to the manufacturer's manual.
Quantitation of liver protein, triglyceride, and total cholesterol The liver protein, triglyceride (TG) and total cholesterol (TC) levels were performed according to the method described by Yamasaki et al. (2019). The protein concentration, as well as TG and TC levels in the supernatant of liver homogenates were determined using the Protein Quantification Kit-Rapid (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), Triglyceride E test Wako and Cholesterol E test Wako (FUJIFILM Wako Pure Chemical Corporation) according to the manufacturer's instructions, respectively.
Measurement of the malondialdehyde (MDA) level in the livers MDA was determined as a thiobarbituric acid reactive substance in the liver homogenates according to the method described by Jamall and Smith (1985). Lipid peroxidation in the livers was expressed in nmol of MDA/g protein.
Statistical analysis Experimental results are expressed as means ± SD. Data were analyzed statistically by performing one-way analysis of variance (ANOVA) and the Tukey-kramer test by using BellCurve for Excel 2012 (Social Survey Research Information Co., Ltd., Tokyo, Japan). Differences were considered statistically significant at p < 0.05.
Antioxidant activity of NSC fraction, HBA, and HAP Table 1 shows the antioxidant activity of NSC fraction, HBA and HAP as sample characteristics. The antioxidant activity of NSC fraction, HBA and HAP were expressed as ABTS radical-scavenging activity and ORAC value. The ABTS radical-scavenging activity of NSC fraction was significantly higher (p < 0.01) than that of HBA and HAP. However, the ORAC level of HBA and HAP was significantly higher (p < 0.01) than that of NSC fraction. Therefore, NSC fraction showed antioxidant activity in ABTS radical-scavenging and ORAC assays, HAP and HBA showed high antioxidant activity in ORAC assay.
| ABTS radical-scavenging activity (µmol TE/g) |
ORAC value (mmol TE/g) |
|
|---|---|---|
| NSC fraction | 1121.48 ± 7.19a | 4.77 ± 0.40b |
| HBA | 0.96 ± 0.24b | 27.58 ± 3.36a |
| HAP | 9.53 ± 3.00b | 31.32 ± 8.73a |
The data of ABTS radical-scavenging activity and oxygen radical absorbance capacity (ORAC) value are expressed as trolox equivalent (TE) antioxidant capacity. Value are means ± SD (n=3). For each column, different letters indicate significant differences between samples (p < 0.05). NSC = non-sugar component; HBA = p-hydroxybenzaldehyde; HAP = p-hydroxyacetophenone
Serum corticosterone level As shown in Fig. 1, the level of serum corticosterone in the control (subjected restraint stress, CON) group significantly increased (p < 0.01) in comparison with the blank (not subjected restraint stress, BLK) group. Despite being exposed to chronic stress, the corticosterone levels in the non-sugar component (subjected restraint stress and treated with non-sugar component from Kokuto, NSC) and p-hydroxyacetophenone (subjected restraint stress and treated with p-hydroxyacetophenone, HAP) groups significantly (p < 0.05) decreased in comparison with the CON group. On the other hand, the corticosterone level in the p-hydroxybenzaldehyde (subjected restraint stress and treated with p-hydroxybenzaldehyde, HBA) group was about the same as that the level in CON group. Therefore, oral administration of NSC fraction and HAP were suppressed increase of corticosterone secretion by the chronic stress in mice.
Serum corticosterone levels in chronic stressed HFD-fed mice. Different letters indicate significant differences between groups (P < 0.05). HFD = high fat diet; BLK = blank group (no stress); CON = control group (stressed); NSC = group treatment with non-sugar component fraction (stressed); HBA = group treatment with p-hydroxybenzaldehyde (stressed); HAP = group treatment with p-hydroxyacetophenone (stressed)
Body weight, energy intake, adipose tissue weight and liver weight, liver protein content, liver TG and TC levels Table 2 shows body weight, energy intake, adipose tissue weight, liver weight, liver protein content, liver TG and TC levels in HFD-fed mice. At day 28, the body weight was significantly lower (p < 0.05) in the CON group than in the BLK group, whereas those in the NSC and HBA groups did not differ significantly from BLK group. In addition, there were no statistically significant differences between the body weights and energy intake of the groups of CON and treated mice. The results of the adipose tissue weight comparison were quite similar to the results of the body weight comparison. Also, the average liver weight in the CON group was significantly higher (p < 0.05) than the weight in the BLK group, whereas the liver weight in the NSC, HBA and HAP groups did not differ significantly from the CON group. Although the liver protein content in the CON group significantly decreased (p < 0.001) in comparison to the BLK group, the contents in the NSC, HBA and HAP groups significantly increased (p < 0.01) in comparison with the CON group. Moreover, the levels of liver TG and TC were significantly higher (p < 0.001) in the CON group than in the BLK group, and the TG and TC levels in the NSC group were significantly lower (p < 0.05) than in the CON group. The TG level in the HBA group tended to be lower than in the CON group, and the TC level in the HAP group was significantly lower (p < 0.05) than in the CON group. Therefore, oral administration of NSC fraction was suppressed accumulation of TG and TC in liver induced by the chronic stress without affecting changes in adipose tissue and liver weights.
| Body weight (g) | Energy intake (kcal/d) | Adipose tissue weight (g) | Liver weight (g) | Protein in liver (mg/g tissue) | TG in liver (mg/g protein) | TC in liver (mg/g protein) | ||
|---|---|---|---|---|---|---|---|---|
| Day 0 | Day 28 | |||||||
| BLK | 22.83 ± 0.93a | 29.99 ± 1.75a | 10.94 ± 3.05 a | 1.59 ± 0.21a | 1.14 ± 0.10b | 119.58 ± 4.68a | 12.23 ± 4.38c | 5.22 ± 0.46c |
| CON | 22.85 ± 1.18a | 27.01 ± 1.50bc | 11.42 ± 4.05 a | 0.99 ± 0.22b | 1.36 ± 0.10a | 93.48 ± 2.40b | 29.48 ± 7.19a | 7.62 ± 0.89a |
| NSC | 22.88 ± 1.10a | 27.54 ± 1.27abc | 11.83 ± 3.94 a | 0.88 ± 0.16b | 1.37 ± 0.07a | 108.62 ± 3.70a | 17.77 ± 4.05bc | 6.44 ± 0.15b |
| HBA | 22.88 ± 1.00a | 28.86 ± 1.48ab | 11.20 ± 3.80 a | 1.22 ± 0.20b | 1.39 ± 0.15a | 109.81 ± 4.61a | 21.71 ± 3.02abc | 7.20 ± 0.45ab |
| HAP | 22.89 ± 0.91a | 26.20 ± 0.79c | 10.94 ± 3.45 a | 0.89 ± 0.17b | 1.25 ± 0.09ab | 111.22 ± 11.21a | 26.35 ± 6.72ab | 6.39 ± 0.27b |
Values are means ± SD. For each column, different letters indicate significant differences between groups (p < 0.05). HFD = high fat diet; BLK = blank group (no stress); CON = control group (stressed); NSC = group treatment with non-sugar component fraction (stressed); HBA = group treatment with p-hydroxybenzaldehyde (stressed); HAP = group treatment with p-hydroxyacetophenone (stressed); TG = triglyceride; TC = total cholesterol
Hepatic MDA level As shown in Fig. 2, hepatic MDA level in the CON group was significantly higher (p < 0.01) than that in the BLK group. On the other hand, the levels in the NSC and HAP groups were significantly lower (p < 0.05) than that in the CON group. Oral administration of NSC fraction and HAP were suppressed increase of hepatic peroxidation in chronically stressed mice fed with a high-fat diet.
Hepatic malondialdehyde (MDA) levels in chronic stressed mice fed HFD. Different letters indicate significant differences between groups (p < 0.05). HFD=high fat diet; BLK=blank group (no stress); CON=control group (stressed); NSC= group treatment with non-sugar component fraction (stressed); HBA= group treatment with p-hydroxybenzaldehyde (stressed); HAP= group treatment with p-hydroxyacetophenone (stressed)
We investigated whether NSC fraction, HBA and HAP could prevent chronic stress response, hepatic lipid accumulation and peroxidation in restraint stressed HFD-fed mice. Stress stimuli are known to lead to the secretion of glucocorticoids (cortisol in human, corticosterone in rodents) (Bale et al., 2000). Serum corticosterone level was significantly higher in the CON group (stressed) than in the BLK group (no stress) (Fig. 1). Therefore, our chronic stress assay might have triggered glucocorticoid secretion by activating the hypothalamic-pituitary-adrenal (HPA) axis. Conversely, oral administration of NSC fraction and HAP suppressed the secretion of corticosterone. Watanabe et al. reported that antioxidative flavonoids from buckwheat sprouts exerted anti-stress property (Watanabe and Ayugase, 2008). In addition, the NSC fraction obtained from the elusion with 50 % MeOH is known to have higher antioxidant activity than that of 25 %, 75 % and 100 % MeOH on HP-20 resin, and to suppress the secretion of serum corticosterone in acute stressed mice fed with a normal diet (Kinjo et al., 2019). We hypothesized that the suppression of corticosterone secretion is linked to the in vitro antioxidant activity of the compounds. In this study, the NSC fraction also exhibited high antioxidant activity in vitro (Table 1). HBA and HAP also exhibited strong antioxidant activity in ORAC assay, whereas the two compounds showed weak activity in ABTS radical-scavenging assay. However, HBA and HAP has little contribution in antioxidant activity of the NSC fraction in ORAC assay because there are very few ratios of HBA and HAP (203 µg and 35 µg / g NSC fraction, respectively) for the weight of the fraction. Although the antioxidant activities of HBA and HAP present in the NSC fraction are not enough to allow us to be better understanding of the suppression of corticosterone secretion, there have been few reports about effect of HAP on stress hormone; thus, further insight into this aspect is left to future work. Administration of HBA had no effect on the secretion of serum corticosterone even though HBA has been reported to have an inhibitory effect on the HPA axis (Jung et al., 2006). We suggest that the results could have been affected by a multitude of factors such as administration method and stress terms.
Stress stimuli promote lipolysis and inhibit proliferation in white adipose tissue, resulting in the loss of body weight (Bowers et al., 2004). Here, the body weight and adipose tissues weight in chronically stressed mice significantly decreased in comparison with the BLK group (Table 2). Moreover, liver weights of the stressed mice were significantly higher in comparison with the BLK group, and the liver protein content of the CON group significantly decreased in comparison with the BLK group. Loss of liver protein could be due to injured hepatocytes resulting from oxidative stress and inflammation (Ochanda et al., 2016). On the other hand, the groups treated with NSC fraction, HBA and HAP showed an increase of protein content in the livers compared with the CON group. Thus, HBA and HAP may contribute to the protective effect of NSC fraction regarding liver injuries induced by chronic stress. Moreover, liver TG and TC in the CON group were significantly higher than in the BLK group. Persistent exposure to glucocorticoids induces portal circulation of many fats and promotes ectopic fat storage in the liver and vascular tissues (Dallman et al., 2003; He et al., 2017). Our data showed that serum corticosterone (Fig. 1), liver TG and TC levels (Table 2) were increased by chronic stress, indicating that hepatic lipid accumulation may be increased by persistent exposure to corticosterone. Liver TG and TC levels were significantly lower in the NSC group than in the CON group, therefore, oral administration of the NSC fraction could help prevent lipid accumulation by suppressing corticosterone secretion (Fig. 1). Moreover, oral administration of HAP had weak influence on the increase of liver TG level but prevented the increase of liver TC level (Table 2). Although the inhibitory effect towards increase of liver TG and TC is different, HAP may have anti-lipid accumulation effect. Oral administration of HBA tended to prevent the increase of liver TG level. HBA has been reported to regulate the expression of genes involved in lipid metabolism in adipocytes (Park et al., 2011). Therefore, HBA and HAP may contribute to the prevention effect of NSC fraction on lipid accumulation in chronically stressed mice through different mechanisms. On the other hand, consumption of fat-rich diet and exposure of chronic stress directly affect the serum lipid profile. Because the alteration of serum lipid levels affects lipid homeostasis in whole body, it would be interesting for further study to investigate the lipid levels in the serum in order to gain more insight of this phenomena.
Chronic stress is known to trigger oxidative stress, caused by increased intracellular levels of ROS (Flaherty et al., 2017). Oxidative damage to the liver can be confirmed by observing the level of MDA, which is a lipid peroxidation product. Hepatic MDA level in the CON group was significantly higher than in the BLK group (Fig. 2). Chronic stress in combination with fat- and sugar-enriched diet can lead to an increase of hepatic MDA levels (Fu et al., 2010). Oxidative stress in the liver can result in progressive cirrhosis and hepatocellular carcinoma (Kawanaka et al., 2004; Adams et al., 2005). The hepatic MDA level in the groups treated with the analyzed compounds tended to be lower than in the CON group. The in vitro antioxidant activity did not differ between HBA and HAP (Table 1), only the MDA level in the HAP group was significantly lower than in the CON group (Fig. 2). This difference in our results of antioxidant activity in vitro and in vivo was likely due to the bioavailability of HBA and HAP. Their metabolism in relation to the active or inactive form and the co-ingestion of other nutrients affect their bioavailability (Martins et al., 2016). The inhibitory effect of HAP on the increase of hepatic MDA is caused by increasing the activity of antioxidant enzymes (Chang et al., 2017). Hence, administration of NSC fraction prevented hepatic lipid peroxidation induced by chronic stress, and this effect could be contributed to the activation of antioxidant enzymes in vivo by administration of HAP.
In conclusion, we investigated the effect of NSC fraction, HBA and HAP on chronic stress responses, and found that oral administration of NSC fraction suppressed the increase of corticosterone secretion in chronically stressed HFD-fed mice. Oral administration of the NSC fraction prevented not only hepatic lipid accumulation but also hepatic lipid peroxidation. These mechanisms were possibly mediated by the antioxidant activity in vivo of HAP. Evaluation of other compounds besides HAP which could influence the suppression effects of NSC fraction are now in progress because other antioxidant phenol compounds detected from the NSC fraction in previous study (Kinjo et al., 2019). This is the first study showing that HAP may contribute to the anti-stress effects of Kokuto, and the present findings support the utilization of Kokuto as a functional food for the prevention of stress and lipid metabolic disease.
Acknowledgments This work was partially supported by a Grant-in-Aid for Scientific Research (17K00819) from Japan Society for the promotion of Science.
