Free radical scavenging synergism of fucoxanthin with lipophilic plant products

Abstract Fucoxanthin demonstrates potential bioactivity, gaining greater interest with many prospective applications. The fundamental activity of fucoxanthin is antioxidant. However, some findings also report the pro-oxidant potential of carotenoids in particular concentrations and environments. In many applications, fucoxanthin requires additional materials to improve bioavailability and stability, such as lipophilic plant products (LPP). Despite much-growing evidence, little is known how fucoxanthin interacts with LPP, which is susceptible to an oxidative reaction. We hypothesised that lower concentration of fucoxanthin exerts a synergistic effect in combination with LPP. The low molecular weight of LPP may exhibit greater activity than long-chain LPP, and so it does with the concentration of unsaturated moieties. We performed free radical-scavenging assay of fucoxanthin combined with some essential oils and edible oils. Chou-Talalay theorem was employed to depict the combination effect. The current study demonstrates a staple finding and constitutes theoretical viewpoints before further fucoxanthin’s utilization with LPP. Graphical Abstract


Introduction
Numerous natural compounds have shown tremendous biological activity as modulators and/or inhibitors of many protein signaling and enzymes to ameliorate human diseases pathology, including chronic diseases (Alesci et al. , 2022a(Alesci et al. , 2022bFumia et al. 2021). Fucoxanthin, a pigment found exclusively in brown algae, possesses appreciable biological activities (Takahashi et al. 2015;Miyashita and Hosokawa 2017;Os orio et al. 2020;Yang et al. 2020) due to its unique structure consisting of an epoxide bond and hydroxyl groups, an allenic bond, and a conjugated carbonyl group in the polyene chain. However, the structure is susceptible into rearrangement such as cis-isomers that are biological less stable (Zhang et al. 2015).
Most phytotherapy and nutraceutical products comprise more than one bioactive ingredient that synergistically enhances the therapeutic index while reducing toxicity (Singh et al. 2016;Yuan et al. 2017). A growing number of studies have been initiated to combine fucoxanthin with other agents to gain more significant bioactivity and stability (Hu et al. 2012;Gong et al. 2014;L opez-Rios et al. 2018;Menichini et al. 2018;Rodr ıguez-Luna et al. 2019;Chen et al. 2021). Recent findings reported greater bioaccessibility of fucoxanthin by carrier oils containing long and medium-chain triglycerides than indigestible oil (e.g., mineral oil). Carrier oil affects fucoxanthin's absorption into small intestine tissue, although the concentration in rat serum is independent of carrier oil type (Salvia-Trujillo et al. 2015). Zhang et al (2015) also reported that absorption rate of fucoxanthin in the gastrointestinal tract was significantly affected by lipids (Zhang et al. 2015). Although attempts to improve stability and bioavailability of fucoxanthin have emerged, bioactivity property of the combined materials remains unclear.
Many unsaturated fatty acids inhibit superoxide production in human vascular endothelial cells reduce the incidence of inflammation, atherosclerosis, and other risks of cardiovascular disease (Oppedisano et al. 2020). Besides fatty acids, essential oils exhibit a broad spectrum of bioactivity (Amorati et al. 2013). Elicited from the psycho chemistry, lipophilic plant products (LPP) can potentially improve the solubility and stability of carotenoids, including fucoxanthin (Hu et al. 2012). However, the isomer configuration on the hydrocarbon backbone can affect bioactivity and is often associated with stimulating pro-oxidant activity during lipid peroxidation (Shin et al. 2020). In addition, the bioactivity of carotenoids also depends on the concentration that can affect the presence of oxygen and new free radicals (Nurcahyanti et al. 2021a;Kusmita et al. 2022). In the context of biological activity, understanding fucoxanthin's fate combined with LPP remains limited even though some applications have emerged (Salvia-Trujillo et al. 2015;Nurcahyanti et al. 2021bNurcahyanti et al. , 2021c. We performed a free radical scavenging assay in vitro to represent the first groundwork for diverse biological activities. Chou and Talalay's theorem is considered robust to depict the combination effect (Chou 2006).

Results and discussion
Aging invades oxygen and its by-product progressively, causing excess production of free radicals and later called oxidative stress. Free radicals are very active, triggering chain reactions and causing multiple cellular compartment damage, such as mitochondria, microsomes, peroxysomes, and cytoplasmic membrane, leading to the onset of degenerative diseases such as Alzheimer's, cancer, and metabolic syndrome. The antioxidant defense system can naturally neutralise free radicals and inhibit the propagation chain. However, by age, the enzymes have shown a reduced ability to inhibit radicals. The extracellular antioxidant that acts as a sequencer of free radicals or as a modulator of antioxidant response element signaling pathway, such as NRF-2, is constantly indispensable to be investigated (Pennisi et al. 2017;Miquel et al. 2018).
In the current study, we selected three types of essential oils, namely rosemary oil (RO), orange oil (OO), and eugenol oil (EO) and three types of edible oil, namely canola oil (CO), mustard oil (MO), and grapeseed oil (GO), to represent common and wellused LPP. Identification of composition was performed using GC-MS, and the results corroborated some previous studies (Table S1). Table S2 presents the Combination Index (CI) of fucoxanthin and three essential oils. Table S3 summarises the results of the edible oils. Combining fucoxanthin and essential oils at both ratios produce synergistic effects (Table S2). Eugenol has an aromatic ring and phenolic group, and the latter can stabilise free radicals formed at ⍺-C by conjugation in the eugenol molecule. In addition, the presence of a conjugated double bond and the possible formation of dimers from oxidative coupling of eugenol were reported to increase its antioxidant activity (G€ ulçin 2011;Wojtunik-Kulesza et al. 2018). Combining fucoxanthin and RO or OO also exhibits a synergistic fashion. The presence of unsaturated double bonds can affect antioxidant activity (Zieli nska-Błajet and Feder-Kubis 2020). OO is rich in D-limonene, which contains a monocyclic structure with unsaturated double bonds, explaining the high antioxidant effect. Monoterpene hydrocarbons have high antioxidant activity, although it was found lower than that of oxygenated monoterpenes. A high level of radical scavenging of 1,8-cineole (eucalyptol) could be due to its monoterpene-containing structure (Zengin and Baysal 2014).
Combination of fucoxanthin with GO, especially in lower concentration of fucoxanthin, exhibited highest scavenging activity when compared to the combination with other edible oils (Table S3). Reduced concentration of fucoxanthin is likely to improve synergistic effects in combination with edible oils (Table S3). Edible oils predominantly contain Mono and Poly-Unsaturated Fatty Acids (MUFA and PUFA, respectively) (Mossoba et al. 2013). Oxidation of fatty acids is theoretically dependent on their degree of unsaturation. Compared to polyunsaturated fatty acids (PUFAs), MUFAs are less susceptible to oxidation and hence have better antioxidant activity. Yet some in vitro and in vivo studies suggested the contrary (Richard et al. 2008). It may explain why GO, which is rich in linoleic acid (PUFA), possesses better activity when compared to CO and MO, which is rich in oleic acid (MUFA).
Combination Index (CI) calculations revealed antagonism interaction, especially in a higher concentration of fucoxanthin. It may occur due to the formation of radicals in fucoxanthin after donating proton(s) to DPPH radical. Antioxidant oils can donate their proton to stabilise reactive fucoxanthin. However, when the concentration of fucoxanthin is too high, oils fail to donate sufficient protons, resulting in increased radical concentration. The antagonistic interaction occurred when the concentration of fucoxanthin used in the combination was equal to or above IC 50 (IC 70 , preliminary experiment). Besides, lipid peroxidation of oils during scarcity of proton donors may also contribute to the unfavorable interaction.
Dose Reduction Index (DRI) are summarised in Figure S1. DRI is defined as the level of reduction of the inhibitory concentration of the compound in combination compared to a single compound (Chou 2006). For a particular application, the interaction must be re-tested in suitable models (Caesar and Cech 2019;Di Meo and Venditti 2020). Additive and antagonism effects can underlie further investigation by reducing the concentration of fucoxanthin and maintaining the activity of LPP (Santana-G alvez et al. 2019). Besides, antagonistic interactions related to the excess of free radicals may be potential for therapeutic applications, such as molecular hydrogen can regulate gene expression and function as an anti-inflammatory and anti-apoptotic agent. Chemotherapy-induced reactive oxygen species is one of the effective mechanisms in the treatment of cancer and tumor (Ga sparovi c 2020).

Conclusion
Combination of fucoxanthin and essential oils results in a synergism effect. Among the edible oils, GO demonstrate synergistic interaction when combined with low concentration of fucoxanthin. Additivity and antagonism can be anticipated when there is a prolonged reaction and higher concentration of the combined compounds, especially fucoxanthin. When combined, the number of phenol groups, conjugated double bonds, and double bonds in oils may also affect antioxidant activity. Further study is necessary, including investigating the effect of the combination on biological systems, first employing cell lines and related animal models.

Acknowledgement
Authors thank our colleague, Apt. Untung Gunawan M.Si, for meaningful discussion on the GC-MS.

Disclosure statement
No potential conflict of interest was reported by the authors.