Sargassum hystrix as a Source of Functional Food to Improve Blood Biochemistry Profiles of Rats under Stress

Abstract

This study was conducted to determine the influence of Sargassum hystrix powder (SHP) as an alternative source of functional food for treating in vivo stress by measuring levels of glucose, triacylglycerol, total cholesterol, and cortisol, and liver histopathology. Wistar rats aged 3 months and weighing 150~200 g were divided into 7 groups: normal control, fasting control, negative control (stress without adaptogen), and 4 experimental conditions (stress+0.18 mg/kg diazepam, stress+450 mg/kg pellet, stress+mixture of pellet with SHP 450 mg/kg, and stress+450 mg/kg of SHP). Intake of liquids and and body weight were measured daily. Blood samples were collected on day 0 (baseline), day 5, and day 10 to analyze levels of glucose, triacylglycerol, cholesterol, and cortisol. On day 10, rats were euthanized and livers were collected to observe the severity of inflammation. The results indicated that rats receiving SHP 450 mg/kg and the mixture of pellet with SHP 450 mg/kg showed a similar ability as those receiving diazepam 0.18 mg/kg to cope with stress, indicated by an improvement in all blood biochemistry parameters. Supplementation with SHP 450 mg/kg can be used as an alternative source of functional food for overcoming oxidative stress, as indicated by its ability to improve levels of blood glucose, triacylglycerol, total cholesterol, and cortisol, and to improve liver histology by decreasing severity of liver inflammation.

INTRODUCTION

Neuron diseases are relatively common in human, comprising approximately 6.3% of other illnesses, estimated to increase to 12% by 2030 according to the World Health Organization. Among neuron diseases, neurodegenerative diseases constitute the higher proportion, reaching 62% (Kalsi, 2015). One disease related to the nervous system is stress. Stress is a biological response that threatens and disturbs the homeostasis in an individual (Castro e Couto et al., 2015). Stress can originate from outside the body; for example, stress may result from physical activities such as swimming restraint (Lailatussifa et al., 2016). A type of stress that is included among the physical stressors is oxidative stress, which has a destructive characteristic.

Oxidative stress is a state of unbalance between prooxidants and antioxidants that results in oxidative damage (Guo et al., 2012). Oxidative stress is caused by free radicals (Chang et al., 2007). Oxidative damage has been implicated in several pathological conditions such as cell, tissue, and organ damage, including the liver, kidney, and heart (Valko et al., 2007). Oxidative stress is marked by dysfunction of the hypothalamus-pituitary-adrenal axis (HPA), therefore resulting in increased levels of cortisol and catecholamines (Castro e Couto et al., 2015).

Marine algae have anti-oxidant, anti-inflammatory, antidiabetic, and anti-cancer potential (Lailatussifa et al., 2016; Gotama et al., 2018). Sargassum sp. is a type of brown algae found readily in the South coastal district of Gunungkidul, Yogyakarta, Indonesia (Lailatussifa et al., 2016; Gotama et al., 2018; Husni et al., 2016). The safety of Sargassum sp. was reported by Tapia-Martinez et al. (2019), Nasmia et al. (2017), and Xiu et al. (2017). Sargassum sp. has alginate content, which is widely used by the food and pharmaceutical industries, in particular in the formation of ice cream, pills, and tablets (Holdt and Kraan, 2011). The bioactive compounds of Sargassum sp. possess pharmacological properties, such as anti-oxidant, anti-inflammatory, anti-hypertensive, anti-bacterial, and anti-viral activities. These pharmacological properties are a result of the biological activity of metabolites such as alkaloids, steroids, terpenoids, saponins, polyphenols, phlorotannin, and fucoidan (Liu et al., 2012). Firdaus et al. (2009) and Lailatussifa et al. (2016) reported that a 450 mg/kg dose of Sargassum echinocarpum and Sargassum polycystum brown algae for in vivo application (applied to animal tests) is most effective in inhibiting oxidative stress. Various studies on anti-stress oxidatives have been conducted, in which the in vivo adaptogen was still administered in the form of extracts and fractions, requiring expensive and complicated equipment to obtain the adaptogen so it can be applied to humans. Meanwhile, Chan et al. (2015) showed that 10% of red algae Gracilaria changii dry powder added to the pellet diet of rats was effective in decreasing cholesterol levels and improving the liver tissue, as a result of feeding with the high-fat compound. However, there are no recent studies on the effect of supplementation with Sargassum hystrix powder (SHP) on reducing oxidative stress through analyzing liver histopathology and the blood biochemistry levels in rats. Therefore, the present study was conducted to investigate the effect of supplementation with SHP as an alternative to functional food against oxidative stress by measuring the levels of blood glucose, triacylglycerol, cortisol, and total cholesterol, and analyzing liverhistopathology.

MATERIALS AND METHODS

Materials

The primary material used in this study was the brown seaweed S. hystrix obtained from the intertidal zone, the South coastal district of Gunungkidul, Yogyakarta, Indonesia. The general proximate composition of SHP used in this study was as follows: water content 13.43±0.02 %, fat content 0.05±0.02%, protein content 6.54±0.04%, and dietary fiber content 31.53±0.18%. In addition, a standard pellet diet (Japfa Comfeed, Jakarta, Indonesia) was used as a comparative diet. The general proximate composition of the standard pellet diet was as follows: water content 7.01±0.01%, fat content 4.28±0.04%, protein content 15.35±0.03%, and dietary fiber content 39.55%±0.09%. All other chemicals used in this study were of analytical grade.

Sample preparation

The fresh brown seaweed S. hystrix was collected, packed in a coolbox away from the sun, and brought to the laboratory of the Fisheries Departement of Universitas Gadjah Mada, Yogyakarta, Indonesia, where it was washed under flowing water. The sample was dried at room temperature (±26°C) for 5~7 days. The dried sample was cut into small pieces measuring ±0.5 cm using scissors and blended. Dried seaweed powder was stored in the freezer before in vivo use.

Animals

Female Wistar rats aged 3 months and weighing 150~200 g were obtained from the Integrated Research and Testing Laboratory, Universitas Gadjah Mada, Yogyakarta, Indonesia. Rats were housed under standard conditions of 20±3°C, a relative humidity of 70%, a 12-h light and 12-h dark cycle, and with access to water 100 mL/d. The animals were maintained for a week to adapt to the environment, during which their health status was checked and they were fed a homogenized diet. All experimental protocols involving animal studies were approved after scrutiny by the Institutional Animal Ethics Committee (IAEC) of the Integrated Research and Testing Laboratory, Universitas Gadjah Mada (Approval No. 00006/04/LPPT/IX/2016), Yogyakarta, Indonesia.

Experimental method

A swimming restraint stress model was used for evaluating antistress activities. Animals were divided into seven groups, eachconsisting of five animals. In the normal control group (group 1), rats did not receive any stressor and were fed a standard pellet diet. In the fasting normal control group (group 2), rats were fasted every day for 10 days, were provided with water 100 mL/d, but did not receive the swimming stress treatment. In the negative control group (group 3), rats were fasted and received the swimming stress treatment. In group 4, the rats received standard diazepam treatment at 0.18 mg/kg p.o.. This diazepam dose is the minimum dose limit obtained based on human body weight conversion of 70 kg to a rat body weight of 200 g. In group 5, the rats received the standard pellet diet 450 mg/kg p.o.. The standard pellet diet was the standard diet (ration) for rats, consisting of high water, fat, protein, and dietary fiber content. In group 6, the rats received a mixture of SHP and the standard pellet diet at a 1:1 ratio (450 mg/kg p.o.). In group 7, rats received 450 mg/kg p.o. SHP. SHP is a brown algae powder that consists of fat, protein, and dietary fiber, also alkaloids, steroids, terpenoids, phenols, tannins, and saponins (Lailatussifa et al., 2017). Stress was induced in all rats, except the normal control and the fasting normal control groups, by subjecting the fasting animals to swimming restraint stress with immobilization to water at a depth of 25 cm in a tub measuring 37×37×30 cm³ for 5 min/d until 10 days. Stress was applied once-daily for 10 consecutive days. Body weight and drink intake were measured everyday. On day 10, the neck bones of the rats was dislocated, and the rats were dissected immediately after the application of stress.

Blood samples (1.5 mL) were collected in microtubes. The blood was centrifuged (MPW-55, Eppendorf, Hamburg, Germany) at 10,000 rpm for 15 min at 4°C to obtain serum that was collected and stored at −20°C (GC-124GGFP, LG Electronics Inc., Seoul, Korea) before testing. Blood serum levels were analyzed to measure the biochemical parameters, including the levels of glucose, triacylglycerol, cholesterol, and cortisol. Glucose levels were measured using glucose oxidase-phenol 4-amino antipyrine (PAP), triacylglycerol levels were determined using glycerol-3-phosphate-oxidase, cholesterol levels were determined using cholesterol oxidase-PAP, and cortisol levels were measured using the cortisol enzyme-linked immunosorbent assay Fine Test kit (Wuhan Fine Biotech Co., Ltd., Wuhan, China).

The stomach of each animal was opened along the greater curvature and were qualitatively examined macroscopically for the presence of inflammation in the liver tissues. The liver tissue was stored in a 10% formalin solution before testing for inflammation. Inflammation in the liver was analyzed using the method described by Mossa et al. (2015).

Statistical analysis

The anti-stress effect was reported as mean±standard deviation (SD) and was assessed using analysis of variance (ANOVA). Differences between samples were determined based on Duncan’s multiple range test using the Statistical Analysis System (SAS release 6.12, SAS Institute Inc., Cary, NC, USA). A value of P<0.05 was used to determine statistical significance.

RESULTS AND DISCUSSION

Body weight of rats

Rats in the normal control group showed a trend for increased body weight between day 0 and after the swimming restraint stress treatment, as assessed on the last day of treatment (day 10); the mean difference in body weight was 6.90 g. The optimum amount of SHP 450 mg/kg to prevent lowering of body weight was determined to be −30.17 g. Fig. 1 shows the changes in body weight between day 0 and after the final swimming restraint stress treatment (day 10).

Fig. 1.

Effect of Sargassum hystrix powder (SHP) and diazepam on swimming restraint stress in terms of body weight loss in Wistar rats. Values with different letters (a–g) indicate a significant difference (P < 0.05).

Rats in the fasting control group showed a significant decrease body weight of −57.63 g between day 0 and the last day of swimming restraint stress treatment (day 10). Prolonged fasting promotes gluconeogenesis (glucose synthesis from non-carbohydrate substrates) and ketogenesis (ketone synthesis from acetyl Co-A) as the primary sources of fuel in the liver. Ketogenesis greatly relies on adipose tissue lipolysis to supply oxidized fatty acids to the liver, to generate ketogenic acetyl Co-A. The energy produced by the body during prolonged fasting is derived from conversion of glucose and fat into protein in the liver and muscle tissue, preventing deposition of fat around the body. This subsequently leads to significant weight loss (Goldstein and Hager, 2015). In the negative control group, rats showed a significant decrease in body weight of −66.76 g following treatment (P<0.05). This likely resulted from continuous fasting leading to a reduction of hepatic glucose in the body, and, when undertaking physical activity at the time of fasting, a shift in energy usage from sources of fatty acids to glucose and muscle glycogen in the muscles. Physical activity increases glucose transport into cells via glucose transporter-4, which leads to an increased activity of muscle adenosine monophosphate (AMP). AMP kinase induces changes in metabolism, including glucose metabolism hence why frequent but intense recreational activities are often fuelled by breakdown of carbohydrates (Sigal et al., 2004). Diazepam-treated rats showed no significant decreases in body weight after the stressor treatment, with a difference of −38.37 g. Diazepam has high lipophilicity and crosses the brain-blood barrier rapidly, and has a high affinity to gamma-aminobutyric acid (GABA) receptors, causing a sedative effect that stabilizes body weight (Brigo et al., 2016).

The body weights of rats in group 5 (standard pellet diet intake of 450 mg/kg) showed minor changes between day 0 and after the stress treatment (day 10), with a modest difference of −39.84 g. In groups 6 and 7, rats were fed with a mixture of standard pellet diet+SHP 450 mg/ kg and SHP 450 mg/kg; after the stress treatment, rats in both groups showed minor decreases in body weight of −35.46 g and −30.17 g, respectively. The best treatment for preventing weight loss after stress treatment was SHP 450 mg/kg. Compounds present in SHP, such as saponins and alkaloids, may have increased levels of the central neurotransmitter, helping reduce stress (Ishola et al., 2012) and to adapt to the rats to cope with the stress (Joshi et al., 2012). In addition, tannins and a phenolic compound of S. hystrix can increase uptake of glucose through regulating cellular metabolism via AMP-activated protein kinase (AMPK) (Lailatussifa et al., 2016).

Drink intake

Drink intake of rats in all treatment groups decreases between day 1 and day 10 that they were subjected to the swimming stressor (Fig. 2). On day 1, when the stressor was given rats experienced shock and required a considerable amount of energy to stabilized their body condition during treatment. On day 6 and 10, rats subjected to stressors were able to better adapt, therefore water intake relatively stable so there was a decrease from day 1.

Fig. 2.

Effect of Sargassum hystrix powder (SHP) and diazepam on the drink intake of swimming restraint stress-induced Wistar rats. Values with different letters (a–j) indicate a significant difference (P <0.05).

Drink intake on day 1 was highest in rats in the normal control group subjected to the stressors. Since rats in this group did not have a limit of food intake, rats could consume as much as possible, limited only by stomach size. Consumption of increased amounts of food is accompanied by increased intake of water to dissolved the food consumed. Throughout the 10 days of treatment, rats in the negative control group had the largest drink intake compared with any other group; the smallest drink intake was recorded for rats receiving SHP 450 mg/kg. The swimming restraint stress treatment caused exhaustion in rats, which resulted in an increased need to consume liquids as a source of energy during fasting. An increase in the amount of water lost from the body makes rats consume more water to maintain fluid levels. Water functions as a bond that can contribute hydrogen atoms, hydrogen ions, oxygen atoms, oxide ions, hydroxyl groups, or hydroxide ions to other reactants in the body, promoting biological reactions (Joshi et al., 2012). Interestingly, rats administered other supplements during fasting and induction of swimming stress treatment showed decreases in water intake. Rats receiving the standard pellet diet (450 mg/kg p.o.) were consuming small amounts of reverse osmosis drinking water since the rations orally given had been dissolved in distilled water; the rations also had a moisture content of 7.01±0.01% and a high dietary fiber content of 39.55±0.09%. Moreover, the brown seaweed S. hystrix has high concentrations of water-soluble minerals, notably Na and K, with 10~20 times the amounts of minerals present in land plants; feeding rats with S. hystrix should therefore decrease water consumption (Garcés-Rimón et al., 2016).

Glucose level

Rats in the negative control group (rats treated and stressed without the adaptogen) showed significant increases in blood glucose levels after day 10 of stressor treatment, with glucose levels of 222.20±6.71 mg/dL (Table 1). Rats in both the fasting control groups [administered SHP 450 mg/kg, and a mixture of standard pellet diet+SHP (1:1) 450 mg/kg] showed significant (P <0.05) decreases in blood glucose levels, indicating rats experienced hyperglycemia and diabetes mellitus, shown by blood glucose levels of >200 mg/dL. Increased blood glucose levels are associated with the ability of cortisol to decrease insulin secretion and action, and activation of glucagon-secreting cells in the islets of Langerhans. Glucagon responds to decreases in blood glucose concentrations during fasting and due to physical activity. Furthermore, secreted glucagon binds to target receptors on hepatocyte cells, which activates adenyl cyclase to stimulate production of cyclic AMP (cAMP), resulting in glycogenolysis (breakdown of glycogen polymers into glucose monomers). During prolonged fasting, amounts of glucose stored in the liver decreases as it is released into the bloodstream, allowing tissues to utilize free fatty acids and ketones for respiration. This increases gluconeogenesis (conversion of amino acids and fats into glucose, or the formation of glucose from non-carbohydrate sources such as lactate, alanine, and glycerol), resulting in release of large quantities of glucose into the blood (Güemes et al., 2015).

Table 1.

Effect of Sargassum hystrix powder (SHP) and diazepam on swimming restraint stress-induced biochemical changes in fasting rats (unit: mg/dL)

Treatment groups	Glucose	Triacylglycerols	Cholesterol
Normal control	105.70±5.21b	89.93±3.82c	51.77±7.75c
Fasting normal control	78.90±8.00a	37.70±4.11a	33.77±7.04b
Negative control	222.20±6.71d	147.67±7.70d	60.60±2.92c
Diazepam 0.18 mg/kg p.o.	91.10±6.42a	72.30±6.77b	22.00±4.39a
Standard pellet diet 450 mg/kg p.o.	139.43±5.98c	64.93±6.93b	42.53±5.71b
Standard pellet diet+SHP 450 mg/kg p.o.	86.70±6.01a	89.43±9.15c	34.90±1.21b
SHP 450 mg/kg p.o.	88.77±7.48a	76.53±5.88b	35.40±4.10b

Values with different letters (a–d) in the same column indicate a significant difference (P <0.05).

Both SHP and a mixture of SHP with the standard pellet diet contain complete sources of energy, which is required by rats to maintain stable glucose levels. However, saponins, terpenoids, and steroid compounds in SHP have an ability similar to that of diazepam (data not shown). SHP could therefore bind to certain receptors in the brain to increase synaptic transmission of GABA-ergic; this would increase chloride on postsynaptic membranes and decrease levels of norepinephrine, catecholamines, cortisol, serotonin, and others biogenic amines in the brain, allowing stable glucose levels to be maintained (Brigo et al., 2016). The SHP used in this study contains secondary bioactive forms of alkaloids, steroids, triterpenoids, saponins, phenols, and tannins, as shown by phytochemistry screening test results. Triterpenoid and saponin compounds could reduce levels of cortisol and adrenocorticotropic hormone (ACTH) during stress, increase levels of brain neurotropic factors and heat shock protein-70, eliminate effects of stress, and avoid hippocampal damage (Kim et al., 2014). Alkaloids, phenols, and tannins present in S. hystrix brown algae are able to reduce stress by donating hydrogen atoms from hydroxyl groups in compounds to stabilise free radicals (Li et al., 2011). However, activation of AMPK by a phenolic compound of algae can increase Akt activity, increasing glucose uptake via the glucose transporter into muscles, which further lowers the blood glucose levels (Güemes et al., 2015). Polyunsaturated fatty acids present in the algae impact insulin action through transformation of cellular membranes, such as by increasing the affinity of insulin receptor binding and increasing glucose uptake into cells. Moreover, foods containing high levels of algal fiber reduce the risk of diabetes. Soluble dietary fiber decreases the rate of stomach emptying, reducing post-prandial glucose and blood glucose levels. The dietary fiber present in seaweed also decreases short-term energy requirements, stabilizes blood glucose levels, and improves insulin sensitivity (Sharifuddin et al., 2015).

Triacylglycerol level

Triacylglycerol levels of rats in the normal control group did not significantly (P>0.05) differ from those of rats fed with a mixture of standard pellet diet+SHP (1:1) 450 mg/kg. Moreover, triacylglycerol levels of rats in the diazepam 0.18 mg/kg group did not significantly differ from those of rats fed the SHP 450 mg/kg diet and the standard pellet diet 450 mg/kg. Triacylglycerol levels were significantly (P<0.05) different between rats in the negative control (fasting+swimming restraint stress-induced) group and the other treatment groups (Table 1).

Stress triggers an increase in triacylglycerol levels because it slows the synthesis of fatty acid and glycerols (lipogenesis process) in the blood (Neelima et al., 2014). The decrease in triacylglycerol levels after administration of adaptogens (diazepam, standard pellet diet, and SHP) was consistent with the study by Duraismi et al. (2010) who suggested that Aegle marmelos extract could decrease the triacylglycerol levels of bridle stress-induced mice. Kim et al. (2012) also reported that administration of Sargassum yezoense methanol extract at doses of 100 and 200 mg/kg to mice with diabetes mellitus could improve levels of triglycerides and decrease levels of plasma free fatty acids; this could help prevent hypertriglyceridemia by activating the transcriptional activity of the peroxisome proliferator-activated receptors (PPAR)α and PPARγ. PPARα and PPARγ are targets of metabolic syndrome and increase expression of the mitochondrial gene UCP3 (uncoupling protein 3) in adipose tissue, which increases fat metabolism, leading to reduced total cholesterol and triglyceride levels in the blood. Moreover, administration of fucoidan (polysaccharide sulfate extract) isolated from Fucus vesiculosus brown algae has been shown to be effective in reducing and improving blood triglyceride levels by increasing lipoprotein lipase (LPL) secretion and increasing ApoC-II via activating LPL release into the blood, allowing free fatty acids to be stored in adipose tissue, skeletal muscle, heart, and other tissues, and to be converted back into triacylglycerols. Administration of adaptogens with antioxidant activities can increase the activity of LPL, thereby decreasing blood triglyceride levels (Yokota et al., 2009).

Total cholesterol

Total cholesterol levels in the blood serum of rats did not significantly (P>0.05) differ between the fasting normal control group, the standard pellet diet 450 mg/kg group, the mixture of standard pellet diet+SHP 450 mg/kg group, and the SHP 450 mg/kg group. However, total cholesterol levels in these groups were significantly (P<0.05) different from those in the normal control and negative control groups (Table 1). Swimming and fasting stressors for 10 days induced increases in serum total cholesterol levels since stress stimulates HPA activity and for the sympathetic nervous system to produce cortisol and catecholamines (epinephrine and norepinephrine). Catecholamine hormone (epinephrine and norepinephrine) increase blood flow through adipose tissue and induce adenoceptor adipose B-2 stimulation, which induces adipose tissue to release free fatty acids into the blood through the lipolysis (the process of breaking down triacylglycerol into glycerol and free fatty acids). This results in formation and accumulation of free fatty acids in the blood circulation (Adekunle, 2011).

The lowest level of cholesterol was observed for rats treated with diazepam 0.18 mg/kg, followed by those receiving the mixture of standard pellet diet with SHP (1:1) at 450 mg/kg, and SHP 450 mg/kg. Supplementation of S. hystrix helped reduce cholesterol levels in the blood. This is because secondary bioactive compounds of algae (polyphenols, phlorotannins, and alkaloids) donate H⁺ ions from hydroxyl groups to reactive oxygen species (ROS) during stress, which neutralizes ROS into stable forms (Lailatussifa et al., 2016). Moreover, the high fiber content in the standard pellet diet and SHP may increase excretion of bile salts and cholesterol in feces, which would decrease the amounts of bile salts in the enterohepatic cycle. Reduced amounts of bile salts and cholesterol entering the liver decrease liver cholesterol levels. This increases cholesterol levels in the blood that can be used to synthesise new bile salts, subsequently leading to reductions in blood cholesterol levels (Astuti et al., 2009). Secondary bioactive compounds, such as terpenoids, steroids, and saponins, play a role in facilitating inhibitory effects of neurotransmitters on the GABA systems (Joshi et al., 2012). Chan et al. (2015) suggested that 10% dried powder of Gracilaria changii algae added to the standard pellet diet of rats was effective in decreasing cholesterol levels that result from a high fat intake. Similar findings were reported by Makkar et al. (2016), who demonstrated that the metabolic ability and the beneficial effects of brown seaweeds have medicinal properties for intestinal disorders, such as hypocholesterolemic and hypoglycemic agents.

Cortisol hormone

Rats in the negative control group, who were fasted and subjected to swimming stress treatment (group 3), showed increased cortisol levels at day 5 and day 10. The normal control group also showed decreased cortisol levels after 10 days of treatment. Rats receiving the standard pellet diet with SHP 450 mg/kg and SHP 450 mg/kg showed decreased basal serum cortisol concentrations, suggesting both these treatments induced decreases in adrenal hormone secretion. Changes in cortisol hormone levels for all study groups are depicted in Fig. 3.

Fig. 3.

Effect of Sargassum hystrix powder (SHP) and diazepam on the levels of cortisol (ng/mL) in swimming restraint stress-induced Wistar rats. Values with different letters (a–l) indicate a significant difference (P <0.05).

Cortisol exhibits significant fluctuations over time to regulate pulsatile episodic release of ACTH from the anterior pituitary and corticotrophin-releasing factor from the hypothalamus (Sharifuddin et al., 2015). Lowered adrenal function can result in insufficient release of adrenal hormones, which can lead to chronic fatigue. Triterpenoids, steroids, and saponin compounds of algae can decrease the levels of cortisol and ACTH during stress due to their abilities to inhibit GABA neurotransmitters (Joshi et al., 2012). Supplementation of rats with brown seaweed Aschophyllum nodosum decreases levels of plasma cortisol and aldosterone (Neelima et al., 2014). Kannan et al. (2007) also showed that the brown seaweed A. nodosum may decrease levels of cortisol in goats under transportation stress. In addition, sulphated polysaccharides from the brown seaweed Laminaria japonica may regulate and stabilize plasma cortisol levels in rats subjected to psychological stress for 10 days (Li et al., 2014).

Liver inflammation

In Fig. 4, normal liver tissue (0% severity level) is indicated by the arrangement and shape of normal liver cells. Hepatocyte cells appear to be lined radially in liver lobules, in polyhedral-shaped with six or more surfaces containing one or two round nuclei, and form layers consisting of one to two cells, similar to brick arrangement. The cell plate leads from the edges of the lobules to the center and abrasively freezes to form structures such as labyrinth and foam. The gaps between these plates contain capillaries known as sinusoids of the liver. The sinusoid liver is a capillary blood vessel branching from the portal vein and the hepatic artery. The sinusoid shaped channel is tortuous and widened, coated in endothelial cells, has an irregular diameter, and is not intact (Makkar et al., 2016). Low-severity liver tissue (1%~25% severity level) is indicated by a slight degree in parenchymal degeneration, characterized by slight changes in hepatocyte form, slight infiltration of lymphocytes, presence of vacuole degeneration, and cell nuclei shifting to the edges. Liver tissue with moderate severity (26%~75% severity level) is indicated by red tissue because of the presence of several erythrocytes, a considerable amount of parenchymal degeneration with some form of hepatocyte changes, changes to eosinophils, sliding of cell nuclei to the edges, lymphocyte infiltration, irregularly arranged sinusoids, and occurrence of sinusoid constriction (Junquiera, 2007). Rats treated with diazepam 0.18 mg/kg, SHP 450 mg/kg, and the mixture of the standard pellet diet and SHP 450 mg/kg showed reduced liver inflammation (Table 2). The severity of liver inflammation and the effect of each treatment on the histopathological changes of liver hepatocytes are shown in Fig. 4 and Table 2.

Fig. 4.

Effect of Sargassum hystrix powder (SHP) and diazepam on liver inflammation in swimming restraint stress induced Wistar rats. (A) Severity 0%, (B) severity 1%∼25%, and (C) severity 26%∼75%.

Table 2.

Effect of Sargassum hystrix powder (SHP) and diazepam on liver inflammation in swimming restraint stress-induced Wistar rats

Treatment group	Severity of liver inflammation (Replication)
	1	2	3	4	5
Normal control	−	−	−	−	−
Fasting normal control	+	++	+	++	+
Negative control	++	++	++	++	++
Diazepam 0.18 mg/kg p.o.	−	−	+	+	+
Standard pellet diet 450 mg/kg p.o.	+	+	+	+	+
Standard pellet diet+SHP 450 mg/kg p.o.	-	+	−	+	+
SHP 450 mg/kg p.o.	+	−	−	+	+

−, 0%; +, low severity (1%∼25%); ++, severity (26%∼75%).

Hepatic injury is always accompanied by inflammation (Han et al., 2016). Swimming and fasting stressors increases the energy needs of an organism, thus increasing levels of ROS. Excessive accumulation of ROS due to stress will reduce the body’s immune system. This activates Toll-like receptor 4 (TLR4), which plays an important role in activating the innate immune system through downstream signalling pathways, such as activation of p38 mitogen-activated protein kinase (MAPK). p38 MAPK plays an important role in signalling pathways involved in generating proinflammatory cytokines and downstream signals related to inflammation. Signalling via p38 MAPK pathways activate cAMP downstream element that binds proteins (CREB). CREB further regulates genes associated with inflammation through increasing nuclear factor (NF)-κB expression in the nucleus. NF-κB rapidly induces expression of inflammatory genes, including the inflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and cyclooxygenase-2, resulting in liver inflammation (Liu et al., 2016). Diazepam has a hypnotic-sedative effect to regulate cortisol secretion, which reduces glucose consumption and the supply of glucose and fat to the liver tissues, effectively preventing liver inflammation (Pifarré et al., 2015). Saponins, steroids, terpenoids, and alkaloids present in SHP also have functions like those of diazepam in facilitating the inhibitory effect of the neurotransmitter GABA (data not shown). These compounds also reduce levels of adrenaline and noradrenaline in the brain, and reduce the cerebral metabolic rate of both oxygen and cerebral blood flow, thus reducing fatigue and helping the body adapt to stress (Joshi et al., 2012; Pifarré et al., 2015). In addition, phenolic compounds in SHP possess radical-scavenging activity, which prevent oxidative stress by transferring hydroxyl groups, therefore helping prevent liver inflammation (Firdaus et al., 2009). It has been demonstrated that polyphenol extracts of hawthorn peels exhibit hepatoprotective capacities through inducing release of pro-inflammatory cytokines, such as IL-1β and IL-6, and genetic biomarkers involved in the inflammatory response (Han et al., 2016).

Diet composition may be affected by the physiology of animals. Malta et al. (2014) reported that low-protein diets induces weak hallmarks of metabolic malfunctions in rats later in life. Barros et al. (2018) also suggested that a protein-restricted diet during pregnancy does not alter reproductive, biochemical, and hematological parameters of Wistar rats.

In conclusion, supplementation of SHP 450 mg/kg can be used as an alternative source of functional food for overcoming oxidative stress; SHP improves blood glucose, triacylglycerol, total cholesterol, and cortisol levels and liver histology by decreasing the severity of inflammation. SHP 450 mg/kg was the best experimented diet supplement for preventing oxidative stress, indicated by decreases in liver inflammation severity (ranging from low to none, 0%~25%), decreases in fasting blood glucose levels to 86.70 mg/dL, a stable triacylglycerol level of 89.43 mg/dL, decreases in total cholesterol level to 34.90 mg/dL, and a significant decrease in cortisol levels to 1,308.31 ng/mL verses initial values prior to treatment.