Overproduction of reactive oxygen species (ROS) is the major cause of oxidative stress. This phenomenon arises as a result of an imbalance between the level of ROS and the endogenous enzymatic antioxidants in the human body, and is followed by a series of health disorders, especially cancer, cardiovascular and liver diseases, and inflammation. ROS can oxidize the DNA base guanosine and thus induce point mutations [1
Medicinal plants have a long history in the treatment of health disorders due to their diverse chemical composition of secondary metabolites. Among them, polyphenols are used as naturally occurring antioxidant agents due to their unique aromatic structure with a pronounced hydroxylation pattern [3
(Pers.) B. L. Burtt and R. M. Smit (Zingiberaceae), commonly known as shell ginger, is native to East Asia and is presently being cultivated in tropical and subtropical zones worldwide. A. zerumbet
has been used for a long time in folk and traditional medicine, for the treatment of many diseases and health disorders [4
]. Several bioactive secondary metabolites were reported in A. zerumbet
, including Kava lactones such as dihydro-5,6-dehydrokawain and 5,6-dehydrokawain as well as flavonoids, namely: rutin, kaempferol-3-O
-rutinoside, and kaempferol-3-O
-glucuronide and aliphatic homopolycyclic compounds, among others [6
Furthermore, a broad spectrum of biological activities has been reported from different plant parts, including anti-nociceptive, antioxidant, hepatoprotective, cytotoxic, and antibacterial properties [8
]. In addition, de Moura et al. [15
] reported promising anti-hypertensive activities of leaves from plants grown in Brazil. As for the essential oil, dihydro-5,6-dehydrokawain and methyl cinnamate were the main component in rhizome oil, whereas 1,8-cineol, methyl cinnamate, and camphor dominated in the leaf oil from plants grown in Japan. On the other hand, monoterpenes (4-terpineol, 1,8-cineole and γ-terpinolene) dominated the essential oils from Japanese and Brazilian species [7
The present work was undertaken to evaluate the in vitro antioxidant activities of leaves from A. zerumbet plants cultivated in Egypt, and to investigate its anti-inflammatory, anti-nociceptive, and antipyretic activities in animals. Furthermore, the chemical constituents of a methanol extract were characterized using HPLC-ESI-MS/MS.
In this work, the phytochemical profile of a methanol extract from A. zerumbet
leaves was comprehensively characterized using HPL-ESI-MS/MS. A total of 37 secondary metabolites were characterized. In addition to common phenolic acids such as cinnamic acid, isoferulic acid, eucomic acid, protocatechuic acid, vanillic acid, and sinapic acid, four new ferulic acid derivatives were tentatively annotated. Additionally, several flavonoids, among them apigenin, pinocembrin, kaempferol-3-O
-glucuronide (previously reported from the plant), and genistein were characterized. However, rutin, catechin, and epicatechin were not detected in the current study [6
], and this might be attributed to the geographical origin.
In addition, this study showed that the extract possessed potent antioxidant properties, free radical scavenging activity, and ferric reducing antioxidant power. Furthermore, the total antioxidant capacity (TAC) of the extract was similar to that of ascorbic acid, a well-known direct antioxidant. Previous studies showed that the essential oils from the leaves of A. zerumbet
have potent antioxidant effects, in accordance with our study [17
]. The antioxidant effects of the extract may be attributed to the phenolic acids and flavonoids content of the extract, which possess a potent antioxidant potential.
Free radical generation is implicated in the development of inflammation. Many inflammatory disorders can be alleviated by using antioxidants. The studied extract exhibited anti-inflammatory effects against carrageenan induced rat hind paw edema in the two dose levels (200 and 400 mg/kg). The effect of the highest dose level seems more potent, however, not significantly different from that of diclofenac, a well-known anti-inflammatory drug.
We also tried to confirm the anti-inflammatory properties of the leaf extract, and to investigate its mechanism of action. Therefore, we used another model of inflammation, the carrageenan-induced leukocyte migration into peritoneal cavity in mice. The extract exerted a dose-dependent inhibitory effect against carrageenan induced leukocyte migration into the peritoneal cavity in mice. The highest used dose (400 mg/kg, p.o.) exerted a similar effect to that of diclofenac.
Furthermore, the extract was more potent on acetic acid induced vascular permeability in mice than diclofenac. In this model, acetic acid causes an increase in the level of histamine in peritoneal fluids, serotonin, and prostaglandins, which leads to vasodilation of blood vessels and an increase in vascular permeability in the peritoneal cavity [18
]. The current study indicates that the extract can prevent vasodilation and inhibit the release of inflammatory mediators in the acute vascular phase of inflammation. Different compounds from A. zerumbet
fruit essential oil showed anti-inflammatory effects against dimethylbenzene-induced ear edema in rats when compared to aspirin [19
Carrageenan-induced rat hind paw edema occurs in two phases, an early vascular phase and a late cellular phase, in which different mediators operate sequentially to produce the inflammatory response. Histamine, serotonin, and bradykinin represent the first detectable mediators in the early phase of carrageenan-induced inflammation, while the release of prostaglandins (PGs) is characteristic of the late phase of inflammation [20
]. In the present study, the extract was capable of inhibiting COX-1 and thus the production of PGs. These results would explain the ability of the extract to subside carrageenan-induced rat hind paw edema. It was reported recently that the second accelerating phase of edema was not only attributed to the increased production of PGs, but also correlated to the induction of COX-2 in the hind paw. Therefore, we studied the effect of the extract on COX-2. The extract has a potent inhibitory effect on COX-2, and its effect was comparable to celecoxib, a well-known COX-2 inhibitor.
During the acute cellular phase of inflammation, infiltration of leukocytes, particularly neutrophils, from the blood into the tissue occurs. These neutrophils release oxygen-derived free radicals, among them hydroxyl radicals, superoxide anions (O2−
), and other inflammatory mediators [20
]. Another suggested mechanism by which the extract could inhibit the inflammatory response to carrageenan is the inhibition of the cellular phase by reducing the recruitment of neutrophil to the inflammation site. This idea was supported by the ability of the extract to decrease the number of leucocytes in the peritoneum of mice challenged with carrageenan. The previous effect may be attributed to the free radical scavenging power of the extract and the potent antioxidant activity of polyphenols [21
Fever is a thermoregulatory indication of systemic inflammation [22
]. Brewer’s yeast-induced pyrexia is caused by increasing the synthesis of prostaglandins, and therefore is used as a common model for the screening of active antipyretic drugs from natural and synthetic origins [23
]. The present study showed that the high dose level of the extract significantly attenuated rectal temperature of yeast-induced febrile mice, and its effect was even more potent than acetaminophen (150 mg/kg), the well-known antipyretic drug. The effect of the extract may be attributed to the inhibition of prostaglandin synthesis in the hypothalamus. This was also confirmed by the ability of the extract to inhibit both COX-1 and COX-2 in our in vitro study.
The present study also investigated the anti-nociceptive activity of the extract by two methods. These methods included chemical nociception in the test model of acetic acid-induced writhing, and thermal nociception in the hot plate test. The hot plate test is a typical model to evaluate narcotic analgesia or central anti-nociceptive effects. The anti-nociceptive potential of the extract was shown by hot plate test (significant only 3 h post treatment), while nalbuphine, the narcotic anti-nociceptive standard, had a significant effect at all time points. It is assumed that the extract has a peripheral anti-nociceptive effect, but a less potent central anti-nociceptive effect than nalbuphine.
The writhing test is often used to evaluate the peripheral anti-nociceptive activity of drugs. Acetic acid causes secretion of endogenous pain mediators, thus stimulating the neurons responsible for pain sensation [24
]. In this study, the low dose level of the extract demonstrated solid anti-nociceptive effects in the acetic acid-induced writhing test in mice. This result suggests that the extract has peripheral anti-nociceptive properties and they might be attributed by blocking the release of endogenous inflammatory mediators such as leukotriene, prostaglandin, serotonin, and histamine. This assumption was supported by the ability of the extract to inhibit both COX and LOX in the in vitro study. The biological activity data suggest that the anti-inflammatory and different anti-nociceptive effects of the A. zerumbet
extract are directly linked to its flavonoids content such as quercetin 3-O
-glucoside, isorhamnetin 3-O
-glucuronide, apigenin, chrysoeriol, and diosmetin as well as its phenolic acids composition, among them gallic acid, cinnamic acid, eucomic acid, vanillic acid 4-β-d
-glucoside, and protocatechuic acid [21
A previous study by de Araújo et al. [11
] was in accordance with our findings. They showed that orally administered essential oil of A. zerumbet
, at doses of 100 mg/kg and 300 mg/kg, had anti-nociceptive effects against acetic acid-induced writing, formalin-induced paw licking effect, and increased the latency in the hot plate test. They also showed that the central anti-nociceptive effect in the hot plate test was partially mediated through opiate receptors, as it was partially blocked by naloxone, an opioid antagonist.
In summary, we conclude that the polyphenol-rich A. zerumbet extract can exert anti-nociceptive, antipyretic, and anti-inflammatory effects by scavenging of free radicals from the site of injury and inhibiting the synthesis of prostaglandins and leukotrienes.