Potential Antioxidant and Anti-Inflammatory Effects of Spilanthes acmella and Its Health Beneficial Effects: A Review

Oxidative stress and inflammation are two common risk factors of various life-threatening disease pathogenesis. In recent years, medicinal plants that possess antioxidant and anti-inflammatory activities were extensively studied for their potential role in treating and preventing diseases. Spilanthes acmella (S. acmella), which has been traditionally used to treat toothache in Malaysia, contains various active metabolites responsible for its anti-inflammatory, antiseptic, and anesthetic bioactivities. These bioactivities were attributed to bioactive compounds, such as phenolic, flavonoids, and alkamides. The review focused on the summarization of in vitro and in vivo experimental reports on the antioxidant and anti-inflammatory actions of S. acmella, as well as how they contributed to potential health benefits in lowering the risk of diseases that were related to oxidative stress. The molecular mechanism of S. acmella in reducing oxidative stress and inflammatory targets, such as inducible nitric oxide synthase (iNOS), transcription factors of the nuclear factor-κB family (NF-κB), cyclooxygenase-2 (COX-2), and mitogen-activated protein kinase (MAPK) signaling pathways were discussed. Besides, the antioxidant potential of S. acmella was measured by total phenolic content (TPC), total flavonid content (TFC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and superoxide anion radical scavenging (SOD) and thiobarbituric acid reactive substance (TBARS) assays. This review revealed that S. acmella might have a potential role as a reservoir of bioactive agents contributing to the observed antioxidant, anti-inflammatory, and health beneficial effects.


Introduction
Inflammation is a protective mechanism that aims to restore the integrity of damaged or threatened tissues from injury or infectious pathogens [1]. Meanwhile, chronic inflammation is a prolonged pathological condition characterized by mononuclear immune cell infiltration, tissue destruction, and fibrosis. It adversely affects cell function through excessive production of free radicals and depletion of antioxidants [2]. The overproduction of reactive oxygen species (ROS) from activated neutrophils and macrophages leads to tissue injury by damaging the macromolecules and lipid peroxidation of the membrane [3,4]. Environ  This herb is native to Brazil and popularly known as a toothache plant owing to its traditional use to relieve dental pain. It is cultivated throughout the year as an ornamental or medicinal plant. It can also be found in a damp area, at swamp margin, on rocks near the sea, or as a road-side weed [16]. It is an annual or short-lived prostrate plant with ascending cylindrical hairy stems and has no flower petals. All parts of the plant are unpleasantly bitter, with the flower heads being the most pungent part, causing a tingling sensation and numbness [17], itchiness, and salivation [18]. It has been used as a traditional medicine to treat various illnesses, mostly attributed to its secondary bioactive metabolites.
Abd Jalil et al. [19], reported that nowadays researchers and doctors are interested in traditional medicines due to their potential in treating diseases. Many diseases are associated with oxidative stress within the body system, such as osteoporosis [19], cancer [20], cardiovascular disease [21], and diabetes [22]. This oxidative stress is related to the antioxidant and anti-inflammatory activities of the body system. Anti-inflammatory drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs) have adverse effects on the gastrointestinal lining, coagulation of blood and renal systems, due partly to inhibition of housekeeping enzyme COX-1. Therefore, research on natural plants as alternative medicines should be explored comprehensively as alternative anti-inflammatory agents with minimal and no side effects.  This herb is native to Brazil and popularly known as a toothache plant owing to its traditional use to relieve dental pain. It is cultivated throughout the year as an ornamental or medicinal plant. It can also be found in a damp area, at swamp margin, on rocks near the sea, or as a road-side weed [16]. It is an annual or short-lived prostrate plant with ascending cylindrical hairy stems and has no flower petals. All parts of the plant are unpleasantly bitter, with the flower heads being the most pungent part, causing a tingling sensation and numbness [17], itchiness, and salivation [18]. It has been used as a traditional medicine to treat various illnesses, mostly attributed to its secondary bioactive metabolites.
Abd Jalil et al. [19], reported that nowadays researchers and doctors are interested in traditional medicines due to their potential in treating diseases. Many diseases are associated with oxidative stress within the body system, such as osteoporosis [19], cancer [20], cardiovascular disease [21], and diabetes [22]. This oxidative stress is related to the antioxidant and anti-inflammatory activities of the body system. Anti-inflammatory drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs) have adverse effects on the gastrointestinal lining, coagulation of blood and renal systems, due partly to inhibition of housekeeping enzyme COX-1. Therefore, research on natural plants as alternative medicines should be explored comprehensively as alternative anti-inflammatory agents with minimal and no side effects.
This review is aimed at assessing the antioxidant and anti-inflammatory activities of S. acmella in reducing the risks of their related diseases. The outcome of this review revealed that S. acmella has potential as a bioactive agent with antioxidant and anti-inflammatory activities for health beneficial effects.

Spilanthes acmella 2.1. Traditional Medicinal Uses
Traditional medicine has remained the most affordable and easily accessible source of treatment since prehistoric times. Medicinal plants are still the most important health care source for the vast majority of the population around the world [23,24]. S. acmella, which can also be found worldwide, is widely used, traditionally in the tropics and subtropics countries, mainly India and South America. They are typically chewed to reduce toothache and relieve throat and gum infections, as well as to paralyze the tongue [25]. According to the dictionary of Malay Peninsula plant products, a decoction of this plant is taken internally as a diuretic and to resolve stones in the bladder, while a decoction of the roots is used as purgative. It is also used to prevent scurvy and stimulate digestion [26].
S. acmella is among the most common Amazonian medicinal plants used by the lay population of the Amazon basin for treating tuberculosis [27]. In India, the juice of S. acmella inflorescence is used to treat mouth ulcers [11]. In Western India, the flower head of S. acmella is a popular remedy for stammering in children. It is also used to treat dysentery and rheumatism [28]. Both leaves and flowers of S. acmella are traditionally used to treat leucorrhea by tribes in Bangladesh [29]. In Cameroon, S. acmella is used as a snakebite remedy and articular rheumatism treatment [30]. Apart from its medicinal uses, the Japanese use the flowers of this species as a spice for appetizers while its extract is used as a flavoring material for dentifrices and gum [31]. Meanwhile, the plant leaves are used as a spice in Brazilian cuisine to produce the characteristic tingling paraesthesia of the traditional regional dishes [25].

Phytochemical
Medicinal plants used in traditional medicine may contain various bioactive compounds, which could exert significant physiological actions on the human body. Phytochemically, S. acmella has been reported to contain isobutyl amide derivatives [32], amino acids, αand β-amyrin esters, myricyl alcohol including sitosterol glucosides [33], and triterpenoid [34]. Asteracea is one of the eight plant families that has been documented to have alkamides as secondary metabolites [35]. Alkamides are considered as the most predominant phytochemicals present in genus Spilanthes and the most significant alkamides found is spilanthol ( Figure 2). This review is aimed at assessing the antioxidant and anti-inflammatory activities of S. acmella in reducing the risks of their related diseases. The outcome of this review revealed that S. acmella has potential as a bioactive agent with antioxidant and anti-inflammatory activities for health beneficial effects.

Traditional Medicinal Uses
Traditional medicine has remained the most affordable and easily accessible source of treatment since prehistoric times. Medicinal plants are still the most important health care source for the vast majority of the population around the world [23,24]. S. acmella, which can also be found worldwide, is widely used, traditionally in the tropics and subtropics countries, mainly India and South America. They are typically chewed to reduce toothache and relieve throat and gum infections, as well as to paralyze the tongue [25]. According to the dictionary of Malay Peninsula plant products, a decoction of this plant is taken internally as a diuretic and to resolve stones in the bladder, while a decoction of the roots is used as purgative. It is also used to prevent scurvy and stimulate digestion [26].
S. acmella is among the most common Amazonian medicinal plants used by the lay population of the Amazon basin for treating tuberculosis [27]. In India, the juice of S. acmella inflorescence is used to treat mouth ulcers [11]. In Western India, the flower head of S. acmella is a popular remedy for stammering in children. It is also used to treat dysentery and rheumatism [28]. Both leaves and flowers of S. acmella are traditionally used to treat leucorrhea by tribes in Bangladesh [29]. In Cameroon, S. acmella is used as a snakebite remedy and articular rheumatism treatment [30]. Apart from its medicinal uses, the Japanese use the flowers of this species as a spice for appetizers while its extract is used as a flavoring material for dentifrices and gum [31]. Meanwhile, the plant leaves are used as a spice in Brazilian cuisine to produce the characteristic tingling paraesthesia of the traditional regional dishes [25].

Phytochemical
Medicinal plants used in traditional medicine may contain various bioactive compounds, which could exert significant physiological actions on the human body. Phytochemically, S. acmella has been reported to contain isobutyl amide derivatives [32], amino acids, α-and β-amyrin esters, myricyl alcohol including sitosterol glucosides [33], and triterpenoid [34]. Asteracea is one of the eight plant families that has been documented to have alkamides as secondary metabolites [35]. Alkamides are considered as the most predominant phytochemicals present in genus Spilanthes and the most significant alkamides found is spilanthol ( Figure 2). Spilanthol, which is N-isobutylamide, is the major pungent compound in S. acmella responsible for various biological activities. The structure of spilanthol is elucidated as (2E, 6Z, 8E)-N-isobutylamide-2,6,8-decatrienamide [36]. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that spilanthol is present in the mother plant, flower heads, and in vitro plantlets of S. acmella [37]. Spilanthol, which is N-isobutylamide, is the major pungent compound in S. acmella responsible for various biological activities. The structure of spilanthol is elucidated as (2E, 6Z, 8E)-N-isobutylamide-2,6,8-decatrienamide [36]. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that spilanthol is present in the mother plant, flower heads, and in vitro plantlets of S. acmella [37].
Phenolics (vanillic acid, trans-ferulic acid and trans-isoferulic acid) and stigmasteryl glucoside are the active metabolites found in S. acmella that elicit a strong antioxidant activity [38]. Literature reveals that the bioactive compounds from all parts of S. acmella possess remarkable pharmacological activities. Pharmacologically, S. acmella exhibits diverse bioactivities including local anaesthetic and antipyretic [39], diuretic [40], antifungal [41], antiplasmodial [42], antimicrobial [43], and insecticidal activities [44]. Therefore, research studies were carried out to discover plants with antioxidant and anti-inflammatory potentials that could treat various kinds of injuries or protect against diseases. S. acmella also exhibited potent anti-inflammatory and antioxidant activities that may contribute to the plant therapeutic value. Table 2 and Figure S1 show phytochemicals and structures of phytochemicals found in S. acmella. Table 2. Phytochemicals in S. acmella.

Anti-Inflammatory Effects of S. acmella
The inflammatory reaction can be acute (rapid and prompt), chronic (persistent), local (limited to a specific region), or systemic (extended to the whole organism) [51]. The release of cell-derived mediators, such as cytokines, prostaglandins, and reactive oxygen species (ROS) in the acute inflammatory response, helps to protect cells and tissues. ROS such as nitric oxide (NO) and hydroxyl radical, which are physiologically produced from various types of immune cells or respiratory burst in neutrophils, play a role in preventing pathogen invasion, depleting malignant cells, and improving wound healing [52]. However, if inflammatory reaction persists for a longer period than usual, the second stage of inflammation or chronic inflammation may set in, which could predispose individuals to various chronic illnesses [53].
The overproduction of mitochondrial ROS could promote the synthesis of proinflammatory cytokines through the activation of the nucleotide-binding domain, leucinerich repeat-containing family (NLR), and pyrin domain-containing 3 (NLRP3) inflammasomes. NLRP3 induces the elevated release of inflammasomes in response to cytoplasmic ROS [52]. Inflammasomes are a set of intracellular protein complexes that enable autocatalytic activation of inflammatory caspases, leading to host and immune responses by releasing cytokines into circulation [54]. It may trigger the release of IL-1β cytokine from the cytoplasm into the extracellular environment and subsequently activates toll-like receptor (TLR)-1 mediated inflammatory signaling. The activation of TLR-1 by IL-1 triggers NF-κB-activated and mitogen-activated protein kinase (MAPK)-induced pro-inflammatory signaling transductions, producing cytokines, such as IL-1β, IL-6, IL-8, TNF-α, and IFN-γ. A series of these reactions could lead to the amplification of inflammatory events resulting in systemic inflammation.
Medicinal plant species that have been used traditionally to treat pain may have good anti-inflammatory activity [55]. The anti-inflammatory effects of S. acmella have been demonstrated by in vitro and in vivo experiments by using several models as summarized in Table 3.

In Vitro Study
Spilanthol, the major pungent compound in S. acmella was studied for its antiinflammatory activities. Wu et al. [56] demonstrated that the anti-inflammatory effects of spilanthol on lipopolysaccharide-activated murine macrophage model, RAW 264.7. The results suggested that spilanthol can inhibit pro-inflammatory mediator production at the transcriptional and translational levels. Spilanthol (90 µM and 180 µM) was found to inhibit NO production, through inhibition of inducible nitric oxide synthase (iNOS) protein expression and iNOS gene transcription alteration. Natural compounds or herbal extracts could exhibit anti-inflammatory activities via the inactivation of NF-κB or activator protein 1 (AP-1) [57]. Spilanthol markedly reduced the inflammatory cytokines such as IL-1β, IL-6, and TNF-α, inhibited the expression of COX-2. This could be partly due to the inactivation of NF-κB. The authors stated that one of the underlying anti-inflammatory mechanisms of S. acmella is via the downregulation of NF-κB [56].
The possible mechanism by which S. acmella executed the anti-inflammatory action was elucidated in cultured RAW 264.7 cells [58]. S. acmella was found to suppress the nuclear localization of NF-κB and expression of NF-κB-dependent cytokines genes, which supported the findings of a previous study by Wu et al. [56]. In inflammatory disease models, activation of Nrf2 has also suppressed inflammation [59]. Recent literature revealed that bioactive compounds including sesquiterpenes [60], flavonoids [61], vanillic acid [62], trans-isoferulic acid [63] and scopoletin [64] could activate Nrf2. Since these compounds can be found in S. acmella extract, this may indicate its underlying anti-inflammation mechanisms. In addition to the suppression of NF-κB, S. acmella was also associated with an increased level of Nrf2 in the nucleus and expression of Nrf2-dependent genes [58].

In Vivo Study
A preliminary study on anti-inflammatory and analgesic activities of S. acmella was carried out by Chakraborty et al. [65] in an experimental model of acute inflammation. The authors have used Carrageenan as one of the standard phlogistic agents for testing anti-inflammatory drugs, to induce hind paw edema in a rat. Carrageenan is antigenic but devoid of any apparent systemic effects. The results demonstrated that aqueous extract of S. acmella (100 mg/kg, 200 mg/kg, and 400 mg/kg) significantly suppressed carrageenaninduced paw edema and increased the pain threshold in the experimental animal. The anti-inflammatory and analgesic activities of aqueous S. acmella extract may be attributed to the presence of flavonoids, which are known to target prostaglandins during the late phase of acute inflammation and pain perception [65]. Inflammatory cells tend to express enzyme COX-2 when activated by the MAPK pathway, which catalyzed the production of prostaglandins (PGs) from arachidonic acid. Overexpression of COX-2 was associated with inflammation and increased PGs production. Flavonoids may interact with the catalytic activity of COX-2 by binding irreversibly to the active site, leaving COX-2 inactive to bind arachidonic acid [66]. Another study by Kim et al. [58], showed the suppression of lung inflammation by methanol extract of S. acmella (1 mg/kg and 10 mg/kg body weight) in an acute lung injury mouse model. The results demonstrated that intratracheal S. acmella administration effectively relieved acute inflammatory lung disease by suppressing neutrophilic inflammation in lipopolysaccharide-induced lung injury. This was indicated by the reduction of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α as well as the suppression of neutrophils infiltration, which is the hallmark of acute lung inflammation.
Huang et al. [70] demonstrated that spilanthol (75 µM to 150 µM) downregulate COX-2 production and decrease TNF-α and MCP-1 production in IL-1β-stimulated lung epithelial cells. The phosphorylation of IκBα and MAPK pathways significantly decreased as compared to IL-1β alone. Spilanthol (>100 µM) promoted HO-1 protein expression by suppressing NF-κB activation and MAPK pathways in IL-1β-activated human lung epithelial cells. Therefore, the author suggested that spilanthol inhibited the expression of the pro-inflammatory cytokines, COX-2, and ICAM-1 by inhibiting the NF-κB and MAPK signaling pathways in human lung epithelial A549 cells. The author also concluded that spilanthol is a natural anti-inflammatory agent. Spilanthol acted as a regulatory factor in MAPK pathways and NF-κB activation of COX-2 and ICAM-1 expression.
A recent study conducted by Bakondi et al. [67] investigated the effects of different parts of S. acmella (flower, leaf, and stem) (12.5 µg/mL to 100 µg/mL) in methanol extracts on RAW264.7 inflammatory macrophages. The results showed that all three parts, especially the flowers, significantly suppressed NO production in RAW macrophages exposed to interferon-γ and lipopolysaccharide. The authors indicated that spilanthol (10 µM to 100 µM) of S. acmella was responsible for the NO-suppressive effects and provided protection from NO-dependent cell death. The expressions of iNOS mRNA and protein were reduced, whereas the activation of several transcription factors, including NF-κB, was inhibited with spilanthol treatment. In the same study, iNOS inhibitory effect was translated into an anti-inflammatory effect in 12-myristate 13-acetate-induced dermatitis and cerulin-induced pancreatitis animal models. The inhibition of iNOS expression and NO production as well as the suppression of inflammatory transcription factors by spilanthol contributed to the anti-inflammatory actions of S. acmella, particularly in the dermatitis model. The histological signs of acute inflammation in the contact dermatitis model were ameliorated by spilanthol treatment.
In another study, Huang et al. [68] examined the effects of spilanthol derived from S. acmella on atopic dermatitis inflammation-related symptoms in 2,4-dinitrochlorobenzene (DNCB)-induced skin lesions in mice at 5 mg/kg and 10 mg/kg of spilanthol. The authors discovered that serum immunoglobulin E and immunoglobulin G2a levels were reduced, while COX-2 and iNOS expression were suppressed. Topical spilanthol treatment has also caused a reduction in epidermal thickness and collagen accumulation, inhibition of mast cells, and eosinophils infiltration into the skin lesions. The results obtained by the study indicated that topical spilanthol treatment protected against atopic dermatitis skin lesions through the inhibition of MAPK signaling pathways and reduction of inflammatory cell infiltration. These actions were enough to block allergic inflammation. Spilanthol (3 µM to 100 µM) was also found to reduce inflammatory response by downregulating MAPK signaling pathways in 3T3-L1 pre-adipocytes [69]. Literature shows that obesity-induced chronic low-grade inflammation could lead to the development of various metabolic diseases.
Huang et al. [69] demonstrated that spilanthol (5 mg/kg and 10 mg/kg) might help in preventing pre-adipocyte inflammatory responses to reduce obesity-related metabolic diseases. Spilanthol significantly suppressed inflammatory mediator COX-2, promoted anti-inflammatory protein heme oxygenase-1 (HO-1) expression, and blocked the phosphorylation of c-Jun N-terminal kinase (JNK), as well as P38 protein in LPS-stimulated murine pre-adipocytes. Therefore the authors concluded that spilanthol could be a natural anti-obesity agent mainly due to its role in the activation of MAPK signaling.
As a whole, these findings supported that inflammation may be alleviated by S. acmella extract and the bioactive compound responsible for this was spilanthol. Systemic inflammation may be prevented by the inhibition of iNOS, inactivation of NF-κB, suppression of inflammatory cytokines, including IL-1β, IL-6, TNF-α, and the inhibition of COX-2. The anti-inflammatory effect of S. acmella was also contributed by its role in the downregulation of MAPK signaling pathways. This evidence proposed that S. acmella can be therapeutically developed for a broad spectrum of inflammatory disorders.

Antioxidant Properties of S. acmella
Overexposure to various stimuli such as pollutants, drugs, xenobiotics, ionizing radiation, and heavy metal ions may induce excessive accumulation of ROS or depletion of antioxidant capacity that could alter the redox balance and cause oxidative stress. The cellular redox system, which comprises catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, and peroxiredoxins, is an integrated cellular defense mechanism that maintains the oxidative equilibrium [71]. In the imbalance between oxidant/antioxidant statuses that result from an excess of ROS, the antioxidant system can be overwhelmed. The highly reactive radicals can irreversibly and permanently damage vital biomolecules, including lipids, proteins, and DNA [51]. This could contribute to many pathological conditions, such as cancer, neurological disorders, cardiovascular disease, pulmonary disease, renal disease, and autoimmune disease [72].

Antioxidant Activity in a Cell-Free System
The significant antioxidant effect exerted by S. acmella is illustrated in Table 4. Wongsawatkul et al. [17] studied the antioxidant activity of S. acmella and its potential role as a natural vasodilator in the phenylephrine-induced contraction of rat thoracic aorta. S. acmella extracts were reported to possess vasorelaxant and antioxidant activities as shown by a partial release of NO from functional endothelial cells and a strong scavenging activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The study also demonstrated that S. acmella ethyl acetate and chloroform extract had the highest DPPH scavenging activity (IC 50 = 216 µg/mL) and superoxide radical activity (57.92% SOD, 200 µg/mL) respectively. However, ethyl acetate extract showed the most potent radical scavenging activity with immediate vasorelaxant effects. In another study, Tanwer et al. [78] evaluated the antioxidant activity of S. acmella extracts and showed that all parts of the S. acmella plant (0.1 mg/mL), which were the callus, root, stem, and leaf, exhibited antioxidative activities that were comparable to butyl hydroxy anisole (BHA), the standard antioxidant. The stem methanolic extract (39.54%) showed the highest superoxide radical scavenging activity, while the leaves (76.42%) showed the highest DPPH radical scavenging activity. S. acmella leaves were reported to have the highest phenolic content (52.3 ± 1.6 mg/g) than other plant parts (roots, 32 ± 0.75 mg/g; stem, 38 ± 1.67 mg/g; callus, 29 ± 1.09 mg/g). The authors concluded that S. acmella possessed a strong antioxidant activity and it was comparable to a butyl hydroxytoluene (BHT) reference compound.
Nabi & Shrivastava [79] investigated the presence of polyphenol compounds in S. acmella leaves via quantification of total flavonoids and their antioxidant activity. In accordance with earlier studies, the leaves of S. acmella extract exhibited the most potent antioxidant activity with the lowest IC 50 values at 134.11 µg/mL for DPPH radical scavenging and 104.51 µg/mL for superoxide radical scavenging assays. The authors documented that the strong antioxidant activity of S. acmella was due to the high concentration of flavonoids (72.14 QE mg/g) and phenols (84.52 GAE mg/g) in the leaves extract. These studies demonstrated that S. acmella could be an important source of natural antioxidants, which might help in preventing the progress of various oxidative stress-related diseases.
A study on bioassay-guided isolation of S. acmella from hexane, ethyl acetate, chloroform, and methanol extracts resulted in a diverse group of bioactive compounds, including phenolics, coumarin, and triterpenoids [38]. Both fractions and bioactive compounds exhibited antioxidant properties in SOD and DPPH assays in the range of 11.29% to 81.50% and 1.90% to 96.05%, respectively. The most potent antioxidant (96.05%) in the DPPH assay was from methanol extract fraction cause the isolation of phenolic compound (trans-isoferulic acid). Meanwhile, the highest antioxidant activity of ethyl acetate extract fraction in DPPH (82.46%) and SOD (81.50%) assays induced the isolation of the phenolic compound (vanillic acid). It was also observed that fractions from methanol and chloroform elution exhibited antimicrobial activity. This study reports the isolation of 3-acetylaleuritolic acid, vanillic acid, β-sitostenone, scopoletin, trans-ferulic acid, trans-isoferulic acid, and a mixture of stigmasteryl and β-sitosteryl glucosides that possessed antioxidant and antimicrobial activities.
A recent study conducted by Swargiary et al. [80] evaluated the phytochemical properties and larvicidal activities of S. acmella against Aedes aegypti. The results showed that S. acmella methanol extract contained high total phenolic content (approximately 67 µg GAE/mg dry weight) and antioxidant activity in DPPH, TBARS, and superoxide anion scavenging activity with IC 50 values of 730 µg/mL, 57 µg/mL, 965 µg/mL and 175.6 µg/mL, respectively. The authors reported that there was a relation between TPC and antioxidant activity. Higher TPC contributed to higher antioxidant activity. The authors suggested that the higher antioxidant activity was due to phenolics' ability to chelate metal ions during free radical productions. However, there was no positive correlation between antioxidant activity and larvicidal property against Aedes aegypti.
Boontha et al. [81] reported the anticancer effect of S. acmella extract on MCF-7 cells. S. acmella extracts contained TPC (62.8 ± 5.2 mg GAE/g), TFC (375.6 ± 20.1 mg rutin equivalent/g) and DPPH with IC 50 (1.2 ± 0.1 mg/mL). Cytotoxicity activity on MCF-7 cells showed IC 50 values of 37.1 ± 1.1 µg/mL in 48 h. S. acmella inhibited formation of colony cells with IC 50 values of 44.9 ± 1.3 µM and demonstrated an anti-migration effect at the concentration of 50 µg/mL. The authors suggested that the high antioxidant activity of S. acmella extract was due to the phenolics and flavonoids contents in the extract that was responsible for its cytotoxic effect.
In addition to that, the antioxidant potential of different extracts (water, methanol, solvent) of S. acmella on different parts (stems, leaves, flowers) was studied by DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay [82]. Methanol extracts of all the plant parts exhibited higher antioxidant capacity with IC 50 values that ranged from 67.34 µg/mL to 127.19 µg/mL when compared to acetone and water extracts. Flower methanol extracts showed the highest antioxidant capacity with an IC 50 value of 67.34 µg/mL. The authors suggested that the antioxidant properties of the S. acmella might be due to the presence of highly valuable bioactive compounds such as phenolics, coumarins, and triterpenoids.

Antioxidant Activity in Cell System (Cellular Method)
Literature has revealed that antioxidant compounds have derived from natural products, including flavonoids (epigallocatechin-3-gallate and quercetin) and phenols (phenolic acids and carnosic acid) exhibited neuroprotective activity [85]. Suwanjang et al. [83] recently studied the neuroprotective effect of S. acmella extracts (hexane, chloroform, ethyl acetate, and methanol) on pesticide-induced neuronal death. Dopaminergic cell lines (SH-SY5Y) were pretreated with S. acmella extracts before being exposed to hydrogen peroxide (H 2 O 2 ) or primicarb (pesticide). It was observed that cell viability reduction induced by pirimicarb was attenuated by pretreatment of the cells with S. acmella extracts. It was also noted that S. acmella hexane extract exerted the strongest protective effect in SH-SY5Y cells exposed to H 2 O 2 . The authors suggested that the neuroprotective effect of S. acmella extracts resulted from their antioxidant properties.
Other studies by Gay et al. [84] reported that phenolic compound (5 µM) of vanillic acid and trans-ferulic acid in S.acmella were able to attenuate cell death on SH-SY5, caused by H 2 O 2 -induced toxicity. The study also demonstrated that the ROS level and apoptotic cells were reduced after 24 h treatment of cells with the phenolic compounds. Phenolic compounds were able to upregulate H 2 O 2 -induced depletion of the expressions of sirtuin-1 (SIRT1), activate FoxO signaling, and mediated oxidative stress resistance [84]. The findings suggested that these phenolics might be promising compounds against neurodegenerative disorders. Collectively, these pieces of evidence showed that the antioxidant effect of S. acmella was mainly contributed to by the presence of phenolic compounds.
Several antioxidant mechanisms of flavonoids have been identified. Firstly, the inhibition of enzymes responsible for superoxide anion production and ROS generation. Secondly, the scavenging of ROS and lastly, the chelation of metal ions was responsible for producing ROS [86].

Conclusions
In vitro and in vivo studies have shown that metabolites of S. acmella contributed to their anti-inflammatory and antioxidant activities. S. acmella could reduce NO release, inhibit inflammatory cytokines (IL-1β, IL-6, and TNF-α), attenuate COX-2 and iNOS expressions, suppress NF-κB and inhibit MAPK signaling pathways. S. acmella and its active metabolites contain high TPC and TFC, which contributed to the high antioxidant activities in a cell-free system through chemical method (DPPH, TBARS, and SOD) assays. Few studies have determined the antioxidant activity of S. acmella in a cell system (cellular method). Phenolic compounds of S.acmella have demonstrated strong protective effects in SH-SY5Y cells that were exposed to H 2 O 2 by upregulating SIRT1 and FoxO3a expressions and inducing superoxide dismutase and catalase. Therefore, S. acmella, as well as its metabolites, may have a potential role as a natural antioxidant that is viable for the prevention of various life-threatening diseases. Further clinical studies need to be verified for future prevention and treatment of chronic diseases.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.