H ESPERIDIN I SOLATED FROM C ITRUS UNSHIU
3. N ARIRUTIN AND H ESPERIDIN FROM C ITRUS P EELS
3.2. Physiological Properties of Narirutin and Hesperidin
3.2.1. Anti-Inflammatory Activities of Narirutin and Hesperidin
Flavonoids in citrus have long been used for their anti-inflammatory potential as either juices or dried peel extracts. In some cases, inflammation is a harmful biological process that involves the overproduction of various pro-inflammatory mediators or cytokines for protecting the host body from foreign molecules and pathogenic substance. For example, chronic inflammation is usually induced by overexpression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) as proinflammatory mediators, which increase the level of nitric oxide (NO) and prostaglandins (PGs), respectively. Moreover, increased levels of proinflammatory cytokines such as TNF-α, interleukin (IL)-1β, and IL-6 in serum can be risk factors for several chronic inflammatory diseases.
NF-κB is one of the major transcription factors that play a primary role in the expression of inflammation by inducing enzymes (iNOS and COX-2) and cytokines (TNF-α, IL-1β, and IL-6). Another signaling pathway of NF-κB is related to activation of mitogen-activated protein kinases (MAPKs), which are intracellular serine/threonine-specific protein kinases. In addition, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) also assist in inflammatory signal transduction by regulation of leukocyte–
endothelial cell interactions [35, 36].
Results of many studies proved the attenuating effect of citrus flavonoids against inflammatory reactions [37]. These in vitro and in vivo studies used inflammatory agents such as lipopolysaccharide [38], benzo(a)pyrene [39] and hydrogen peroxide [40].
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In our study, the narirutin-rich fraction (70.4% purity) isolated from citrus peels through the UAE method exhibited the regulatory activity of inflammatory mediators managing acute or chronic inflammatory disease in mouse macrophage cell line RAW 264.7 [41]. Citrus narirutin fraction (CNF) inhibited the translocation of the p65 subunit of NF-κB from the cytosol to the nucleus by decreasing the IκB-α degradation. These results demonstrate that CNF suppressed the release of NO and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)- stimulated macrophages by regulating the expression of iNOS and COX-2. In addition, CNF inhibited the phosphorylation of MAPKs (p38, ERK, and JNK) without any change in total protein expression indicating that narirutin is effective in suppressing inflammation through the inhibition of MAPKs phosphorylation (Figure 3). In the pre-clinical animal model, inhibiting MAPKs pathway with diverse agents showed effective anti-inflammatory activities [42].
Figure 3. Effect of citrus narirutin fraction on LPS-induced MAPK activation (From Ha et al., Food and Chemical Toxicology 50, 2012, pp 3503).
Physiological Properties of Narirutin and Hesperidin … 77 Hesperidin also regulates the inflammatory response through inhibition of synthesis and release of proinflammatory mediators, inhibition of activated immune cells, or inhibiting such as COX-2, matrix metalloproteinase (MMP)-2, and MMP-9 [39, 43]. Several animal studies showed that hesperidin has preventive effects on inflammatory-associated neuropathology, nephrotoxicity, and cardiovascular complication. Pretreatment of hesperidin (100 and 200 mg/kg) to Swiss male albino mice prevented cognitive impairment induced by intracerebroventricular-streptozotocin (ICV-STZ) injection (2.57 mg/kg) through modulation of acetylcholine esterase activity (AChE), and modulates neuronal cell death by inhibiting the overexpression of inflammatory markers like NF-κB, iNOS, COX-2 and glial fibrillary acidic protein (GFAP) positive astrocytes [44]. In gentamicin-induced nephrotoxic rats, hesperidin (200 mg/kg) supplementation inhibited NF-κB mediated expression of inflammatory genes (IL-6, iNOS, TNF-α, and COX-2) and upregulated nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and Nrf2 dependent phase II antioxidant gene HO-1 [45]. In addition, treatment of hesperidin (100 mg/kg) for 28 days prevented cardiovascular complication in isoproterenol- induced myocardial infarction in normal and streptozotocin-nicotinamide induced diabetic rats by decreasing HbA1c, glucose, CES, LDL, TC, TG, systolic levels, and diastolic blood pressure [46]. These results indicated that hesperidin attenuates the inflammatory response via direct transcriptional effect or decreased tissue lipid deposition.
3.2.2. Preventive Effect of Narirutin and Hesperidin on Alcoholic Liver Disease
Liver-related diseases are still one of the critical causes of disability and morbidity in the world. Fatty liver, an initial stage of alcoholic liver disease (ALD), is one of the most common liver diseases. Several reports have indicated that chronic alcohol consumption causes fatty liver or steatosis [47, 48], and may progress to hepatitis, inflammation, and fibrosis, which may eventually lead to liver cirrhosis [49].
Therapeutic agents have been successfully used for the prevention or treatment of major risk factors for ALD. Particularly, there is a growing interest in natural bioactive compounds that protect against or mitigate liver diseases without the undesirable side effects. Among those biocompounds, flavonoids are one of the best alternatives.
Our previous studies indicated that narirutin and hesperidin have potent anti-ALD activities. Their hepatoprotective effects were exhibited by maintaining antioxidant status, inhibiting lipid accumulation and apoptosis. Levels of hepatic triglyceride (TG) were significantly (p < 0.05) increased by ethanol-mediated oxidative stress, while such excessive formation of hepatic lipids was obviously inhibited by co-administration of citrus flavonoids.
Investigations were carried out to determine whether administration of citrus flavonoids could regulate oxidative stress to inhibit excessive lipid formation in hepatocytes.
In biological systems, both enzymatic and non-enzymatic antioxidants including glutathione (GSH), glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx) and superoxide dismutase (SOD) play critical roles in handling oxidative stress [50]. It is normally observed that levels of these antioxidant components in hepatocytes are decreased by chronic and/or excessive alcohol consumption. Therefore, it is essential to maintain antioxidant systems to overcome alcohol exposure. Administration of narirutin could maintain these antioxidant systems. Co-administration of CNF (300 mg/kg mouse) reinstated the GSH levels (16.7 nmol/mg protein) and SOD activity (96.5% of normal group) to near normal controls [34]. Oral administration of citrus flavonoids (54% of hesperidin and 28% of narirutin) also prevented decrease of antioxidant enzyme activities including GR,
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GST, and GPx in a significant level (p < 0.05) against ethanol consumption (5 g/kg mouse) for 8 weeks [33]. These results demonstrate that oral administration of narirutin and/or citrus flavonoids may suppress alcoholic fatty liver, via the antioxidant defense mechanism against ethanol-induced oxidative stress.
To maintain antioxidant systems is not only important for suppressing steatosis but also suppressing further damage to hepatocytes, which is caused by lipid peroxidation. Lipid peroxidation of hepatic tissues caused by ROS is another main cause of liver damage induced by ethanol and can produce harmful radicals [51, 52]. Setshedi et al. have also demonstrated that mouse liver produced large amounts of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) mediated by CYP2E1, which resulted in increased generation of ROS leading to oxidative damage and cell death after excessive ethanol administration [53]. Likewise, chronic ethanol consumption caused an increase in MDA concentration in hepatocytes, which could be effectively inhibited by administration of citrus flavonoids.
Figure 4. Histopathological changes of hepatic tissues after co-administration of citrus fraction along with 6.5 g ethanol/kg body weight/day for 8 weeks in ICR mice. No histological abnormalities were observed in normal mice (A). Mice induced with ethanol developed typical lesions of liver damage, which were increased in hydropic degeneration (arrows) and lymphocytes (double arrow head) around the portal vein (B-1) and occurrence of necrosis (double arrow) in hepatocytes (B-2). Co-administration of citrus narirutin fraction (150 mg/kg) resulted in suppression of necrosis and alleviation of increasing lymphocytes (C). Especially, 300 mg/kg of citrus narirutin fraction almost blocked the typical lesions of liver damage (D). The tissues were subjected to histological study by staining with hematoxylin and eosin (H&E). All the magnification is 200 × (From Park et al., Food and Chemical Toxicology 55, 2013, pp 642).
Liver inflammation is closely associated with ROS released from lipid peroxidation. As in the cases of other organs, activated NF-κB, a key transcription factor in inflammation, stimulates many inflammatory cytokine genes to trigger mediators in hepatocytes such as TNF-α, ILs, inducible nitric oxide synthase (iNOS), that are very important for ALD [54]. It has been demonstrated that NF-κB is activated by ROS (Schreck et al., 1991) and its activation can be modulated by antioxidants (Omoya et al., 2001). Narirutin can suppress
Physiological Properties of Narirutin and Hesperidin … 79 levels of these inflammatory components through potent antioxidant [55] and anti- inflammatory effects [41].
Moreover, focal hepatocytic necrosis with lymphocytes infiltration observed in ethanol- induced mice might be a basis for cellular damage [56]. Administration of CNF (300 mg/kg) remarkably reduced the histological alterations caused by ethanol, which may be due to attenuation of the ethanol-mediated threat and reduction of the pathological changes (Figure 4). Chronic alcohol exposure showed that hepatocytes near the central vein area were full of hydropic degeneration, and infiltration of lymphocytes and appreciable necrosis were observed in the central vein area. On the other hand, liver plate and hepatocytes structure of CNF administered group were almost intact and the boundary between hepatocytes was clear.