Development of bioresources in Okinawa: understanding the multiple targeted actions of antioxidant phytochemicals

Abstract

In research to develop healthy foods or preventive medicines from edible and medicinal herbs in Okinawa, we focused on the antioxidant activities of those bioresources. We first confirmed that the herbal antioxidant activities of such herbs increased upon ultraviolet irradiation treatment. This observation explains the high antioxidant activity of Okinawan vegetables, which grow under exposure to stronger ultraviolet light compared with those in other prefectures in Japan. Antidiabetic, hepatoprotective, cancer preventive, and cardioprotective actions were clarified using herbal extracts, and quercetin, chlorogenic acid, and gallic acid derivatives were isolated as antioxidant components from the herbs. Dimerumic acid was also isolated from the mold Monascus anka. All these antioxidants showed strong radical scavenging activities in vitro and beneficial effects in animal models. However, the concentrations of these compounds used in vivo seemed to be too low to have a physiologically important antioxidant effect based on their radical scavenging activities in vitro. Therefore, I performed a literature survey of antioxidant activities in vivo. Accumulating evidence has emerged that antioxidant phytochemicals show not only radical scavenging activities in vitro but also pleiotropic actions in vivo. The multitargeted, beneficial effects of antioxidant phytochemicals can be rationally explained using the xenohormesis concept, in which phytochemicals are the products of plant evolutionary adaptation to stress in plants, and their ability to induce a stress-adaptive response has been evolutionarily conserved in animals.

Keywords: antioxidant; multitargeted action; phytochemicals; stress adaptive response; xenohormesis.

Fig. 1.

Reactive oxygen species (ROS) and antioxidant enzymes. Oxygen (O₂) generates superoxide anion (O₂⁻) through a one-electron reduction, hydrogen peroxide (H₂O₂) through a two-electron reduction, hydroxyl radical (•OH) through a three-electron reduction, and water through a four-electron reduction. The hydroxyl radical is highly reactive and can react with lipid (LH) to generate a peroxyl radical (ROO•), leading to a chain reaction that damages lipid membranes. The ROS thus produced are converted by antioxidant enzymes. O₂⁻ is converted to H₂O₂ by superoxide dismutase, H₂O₂ is converted to H₂O + O₂ by catalase, and peroxide (LOOH) is converted to LOH by glutathione peroxidase. The shift in the balance between oxidants (such as ROS) and antioxidants in favor of oxidants is termed oxidative stress. Gpx, glutathione peroxidase; SOD, superoxide dismutase; GSH, glutathione.

Fig. 2.

Effect of ultraviolet irradiation on antioxidant activity in edible herbs. Herbs were cultivated inside or outside a greenhouse that was covered with special vinyl films that block UV rays. After drying the herbs, each was extracted with hot water (1 g/10 ml), and then measurements were performed to determine the concentration at which 50% of the radical 2,2-diphenyl-1-picrylhydrazyl was scavenged. The black columns show herbs cultivated under UV irradiation conditions, and the white columns show those cultivated under UV-blocked conditions. The antioxidant activities of the herbs were markedly increased by UV irradiation.

Fig. 3.

Structure of antioxidant phytochemicals isolated from herbs. Antioxidant components were isolated: quercetin glucosides from Psidium guajava (guava), gallic acid from Limonium wrightii (ukonisomatsu), neochlorogenic acid from Peucedanum japonicum (botanboufu), isochlorogenic acids from Crassocephalum crepidioides (benibanaborogiku), and chebulagic acid and corilagin from Terminalia catappa (momotamana).

Fig. 4.

Effect of the extract from guava leaves on streptozotocin (STZ)-induced diabetic rats. The extract at the dose showing 50% 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (5 ml/kg) was given orally to STZ-treated rats (5 ml/kg, 3times/week) or control rats for 6 weeks. Each parameter was measured in both tissue and serum. LPO, lipid peroxide; GSH, glutathione; GST, GSH S- transferase; Gpx, GSH peroxidase; TG, triglyceride; Cho, total cholesterol; LDL, low-density lipoprotein; HDL, high-density lipoprotein.*p<0.05 vs. STZ-treated.

Fig. 5.

Effects of antioxidant phytochemicals on Nrf2 and NF-κB signaling pathways. (A) Nrf2/Keap1 pathway. Nrf2 is normally present in the cytoplasm bound to Keap1 and sequestered by proteasomal degradation through Keap1-associated Cul3-Rbx E3 ubiquitin ligase. Nrf2 is activated through two mechanisms. The first mechanism is by modification of the thiols of Keap1, which leads to conformational changes in this protein and subsequently the release of Nrf2. The second mechanism involves the activation of kinases that phosphorylate Nrf2 and thereby free it from Keap1-mediated sequestration. After nuclear translocation, Nrf2 with sMaf binds to antioxidant responsive elements (AREs) on DNA and activates the transcription of antioxidant enzyme genes. Curcumin, sulforaphane, and quercetin activate Nrf2 by the first mechanism, whereas resveratrol and capsaicin function through the second mechanism. (B) NF-κB/IκB pathway. Oxidative stress and ligands of TNFRs and TLRs activate the upstream IκB kinases (IKKs) of NF-κB, resulting in the phosphorylation of IκB, which is usually bound to the inactive NF-κB dimer in the cytoplasm. IκB is then targeted for proteasomal degradation, and NF-κB moves into the nucleus, where it induces the expression of inflammatory cytokines and proteins involved in the adaptive stress response. Phytochemicals can modulate IKKs and thereby inhibit the inflammatory reaction. TLR, Toll-like receptor; TNFR, tumor necrosis factor receptor. Bold arrows indicate the targets of antioxidant phytochemicals.

Fig. 6.

Oxidative stress-induced apoptosis. In response to low levels of oxidative stress, p53 exhibits antioxidant activities that contribute to the elimination of oxidative stress and ensure cell survival, whereas in response to high levels of oxidative stress, p53 exhibits pro-oxidative activities that further increase the levels of stress, leading to cell death via apoptosis. p53 can promote apoptosis by inducing the transcription of pro-apoptotic members of the Bcl-2 family such as Bax and by its direct effects on mitochondrial membranes. TNFα causes apoptosis through death receptors (i.e., the extrinsic apoptotic pathway).

Fig. 7.

Effects of antioxidant phytochemicals on protein kinase signaling pathways. Stressors or ligands bind to receptors on plasma membranes by which the stress signals are transmitted through a consecutive series of phosphorylation events, which is termed the mitogen-activated protein kinase (MAPK) cascade, and finally activate MAPKs, including ERK, JNK, and p38. MAPKs thus activated can phosphorylate multiple proteins, including transcription factors such as p53 and NF-κB, leading to the modulation of cell proliferation, differentiation, cell cycle arrest, apoptosis, and immunocyte activation. In insulin signaling, the signal is transmitted through the PI3K/Akt and MAPK pathways. Antioxidant phytochemicals target multiple kinases that are indicated with bold arrows. MEK, mitogen-activated kinase kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IGF-1, insulin-like growth factor-1; IRS, insulin receptor substrate; GSK, glycogen synthase kinase; PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B.

Fig. 8.

Summary of the multiple targeted actions of antioxidant phytochemicals. Multiple proteins, including protein kinases, deacetylases, transcription factors and their associated proteins, and enzymes, are targeted with phytochemicals, leading to the modulation of intracellular signaling pathways. Bold arrows indicate the targets of these phytochemicals.

Fig. 9.

Hormetic response curve. Antioxidant phytochemicals cause a hormetic response, with a stimulatory or beneficial effect at low doses and an inhibitory or toxic effect at high doses. Within the hormetic dose range, the maximum response is no more than 30–60% greater than the control group. The hormetic response is a stress-adaptive response that is mediated through the Nrf2/Keap1, NF-κB, sirtuin, and protein kinase signaling pathways.

Fig. 10.

Xenohormesis. Because plants cannot physically move away from environmental stresses, including temperature variation, water or nutrient availability, reactive oxygen species generation by UV light, and attack from predators, plants have evolved stress-adaptive responses involving the synthesis of phytochemicals as secondary metabolites. That is, phytochemicals are synthesized in plants as a response to environmental stimuli, by which plants protect themselves against stress through a stress-adaptive response, known as a hormetic action. Animals cannot synthesize the phytochemicals, but their cells can sense them and subsequently undergo a stress-adaptive response that appears to have been evolutionarily conserved between plants and animals. This hormetic action, phytochemical-induced hormetic action, which is found in animals including humans, is recognized as xenohormesis. Xenohormesis is a biological principle that explains how environmentally stressed plants produce bioactive compounds that can confer stress resistance and survival benefits for animals that consume them.