The In Vitro Antioxidant and Anti-Inflammatory Activities of Selected Australian Seagrasses

Recent studies have shown that seagrasses could possess potential applications in the treatment of inflammatory disorders. Five seagrass species (Zostera muelleri, Halodule uninervis, Cymodocea rotundata, Syringodium isoetifolium, and Thalassia hemprichii) from the Great Barrier Reef (QLD, Australia) were thus collected, and their preliminary antioxidant and anti-inflammatory activities were evaluated. From the acetone extracts of five seagrass species subjected to 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging antioxidant assay, the extract of Z. muelleri had the highest activity (half minimal concentration of inhibition (IC50) = 138 µg/mL), with the aerial parts (IC50 = 119 µg/mL) possessing significantly higher antioxidant activity than the roots (IC50 ≥ 500 µg/mL). A human peripheral blood mononuclear cells (PBMCs) assay with bacterial lipopolysaccharide (LPS) activation and LEGENDplex cytokine analysis showed that the aerial extract of Z. muelleri significantly reduced the levels of inflammatory cytokines tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6 by 29%, 74%, and 90%, respectively, relative to the LPS treatment group. The aerial extract was thus fractionated with methanol (MeOH) and hexane fraction, and purification of the MeOH fraction by HPLC led to the isolation of 4-hydroxybenzoic acid (1), luteolin (2), and apigenin (3) as its major constituents. These compounds have been previously shown to reduce levels of TNF-α, IL-1β, and IL-6 and represent some of the major bioactive components of Z. muelleri aerial parts. This investigation represents the first study of the antioxidant and anti-inflammatory properties of Z. muelleri and the first isolation of small molecules from this species. These results highlight the potential for using seagrasses in treating inflammation and the need for further investigation.


Introduction
Inflammation describes the natural healing process after the body sustains damage or is exposed to toxins or infection.This process is initiated by the body's production of key inflammatory cytokines such as tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6, which promote key inflammatory responses including vasodilation, immune cell recruitment, and sensitivity to pain [1].Under normal conditions, inflammation is Life 2024, 14, 710 2 of 15 acute in nature, rapidly clearing the infection or healing damaged tissues, and resolves within a few days.In chronic inflammation, however, the response continues for months or years, causing serious damage to the body [2].In addition to a reduction in quality of life experienced by people with rheumatoid arthritis, inflammatory bowel disease (IBD), and acne, more life-threatening chronic inflammatory disorders such as cancer, stroke, diabetes mellitus, kidney disease, and ischemic heart disease have accounted for over 50% of deaths globally in the past few decades [3].
Given the severity and importance of chronic inflammation, there is a continual search for more effective treatments.The most common treatments for inflammatory conditions in modern medicine are glucocorticoids (steroids) and non-steroidal anti-inflammatory drugs (NSAIDs) [4], which are typically taken as oral or topical treatments depending upon the site of inflammation.In addition, dietary changes are also important interventions due to the link between the modern diet and inflammation [5,6].
Seagrasses, like other plants, produce this wide variety of secondary metabolites as active principles and signalling molecules, especially polyphenols, to cope with various ecological stresses such as poor nutrient availability, ionising UV radiation, temperature, salinity, and to prevent predation and microbial infection [15,16].Such secondary metabolites account for the diverse biological activities observed in seagrass extracts including antimicrobial, anti-inflammatory, antioxidant, antiviral, anticancer, anti-ageing, and hepatoprotective properties [17].These bioactive properties of seagrasses have been utilised in traditional medicine systems in Africa [18,19], Indonesia [20,21], and India [22] for the treatment of many ailments such as itchiness, muscle pains, wounds, gastrointestinal complaints, heart and kidney disease, cancer, and as a mosquito repellent.These bioactivities and traditional medicinal uses highlight the potential for seagrasses in lead compound discovery and for the development of new products for the treatment of common diseases.
Seagrasses typically grow in shallow coastal waters, and it has been reported that seagrass meadows are dominant vegetation types along the Great Barrier Reef (GBR), Australia [23].Given that Australia's coastline in its tropical regions is within the most biodiverse region for seagrasses globally [24] and given the current lack of research on seagrasses and their bioactivities, this study aimed to collect several seagrass species from the GBR in Far-North Queensland (FNQ), perform preliminary antioxidant and anti-inflammatory tests, and isolate major secondary metabolites from the most bioactive species.
The deuterated methanol (CD 3 OD) used for nuclear magnetic resonance (NMR) analysis was obtained from NovaChem (Cambridge Isotope Laboratories, Tewksbury, MA, USA).

Mass Determination by Mass Spectrometry
Electrospray ionization (ESI) low-resolution mass spectrometry (LRMS) was performed on a Shimadzu LCMS-2020 mass spectrometer (Kyoto, Japan) in positive (ESI + ) or negative (ESI − ) ionisation mode at James Cook University (JCU) or the University of Wollongong (UOW).Isolated compounds were prepared for LRMS in HPLC-grade MeOH.The ion mass-to-charge (m/z) values are reported as the relative abundance compared to the base peak.The molecular ion is reported as M. All MS data were processed using Shimadzu LabSolutions software (version 5.96).

Large-Scale Preparation of Z. muelleri Aerial and Root Extracts
The Z. muelleri aerial parts (ZMA, 64.74 g) and root parts (ZMR, 27.86 g) were separated and macerated twice in acetone (aerial: 1.2 L; roots: 0.75 L) for 1 day.Both washes were pooled and evaporated to dryness to yield the ZMA (1.07 g, 1.7%) and ZMR (0.52 g, 1.9%) crude extracts.

Purification of Z. muelleri Aerial Extract
The ZMA extract was purified and compounds elucidated based upon previously reported methods [25].

Biological Screening 2.3.1. Radical Scavenging Antioxidant Assay
Using a modified literature method [29][30][31], the DPPH radical scavenging activity of each seagrass root and aerial extract was evaluated (performed in triplicate) by combining 100 µL of root and aerial extracts (and separated root or aerial extract for Z. muelleri) at five different concentrations (500, 250, 125, 62.5, and 32.5 µg/mL in MeOH) with 200 µL of 0.1 mM DPPH (in MeOH).Absorbances were measured using a microplate reader (SPECTROstar ® Omega, BMG Labtech, Sydney, Australia) at 517 nm after 1 h of incubation in the dark condition at room temperature.Methanol was used as a blank, and gallic acid was used as a standard antioxidant compound and was tested at the same five concentrations as the plant extracts.The percentage of DPPH radical scavenged by the sample was calculated using the formula: where A 1 = absorbance of the sample (at 517 nm), A c = absorbance of the control (at 517 nm).All antioxidant assay data were visualized using GraphPad Prism 10.2.2.The half maximal inhibitory concentrations (IC 50 ) values were measured at the concentration of 50% DPPH radical inhibition.Negative DPPH radical scavenging percentages were normalised to 0%.

Anti-Inflammatory Activity and Quantification
All cell viability and anti-inflammatory screening procedures were performed according to previously reported methods [25,32].

PBMC Collection and Culture Conditions
The ethics approval (H8523) for this assay was granted by the Human Research Ethics Committee of James Cook University in 2023.Blood from two healthy donors was supplied by the Red Cross Lifeblood, Australia.The human peripheral blood mononuclear cells (PBMCs) were separated from the blood samples using a Ficoll-Paque PLUS density gradient method according to the manufacturer's instructions and cryopreserved in filtered FBS containing 10% DMSO.

Sample Treatment
According to a previously reported method [25,32], the PBMCs were stimulated with lipopolysaccharide (LPS, 10 ng/mL) and treated with extracts simultaneously.For all tests, 1 × 10 6 cells in 100 µL of R-10 media (RPMI-1640, containing 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin) were seeded into the wells of 96-well U-bottom culture plates.Stimulated PBMCs were treated in triplicate with the ZMA crude extract (100 µg/mL in cell culture media with 0.5% DMSO).Culture plates were incubated overnight at 37 • C in a 5% CO 2 incubator.Following overnight incubation, plates were centrifuged (277× g, 4 • C, 5 min), and the culture supernatants were collected and kept in a −80 • C freezer until further analysis for cytokine profile.The PBMCs left in the culture plate were immediately stained to determine the effect of the ZM crude extract on cell viability, as described in the subsequent section.

Determination of Cell Viability
After collecting the supernatant, cells were stained with viability dye (LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit) following the manufacturer's instructions.Stained cells were rinsed twice with 200 µL 2% FBS/Dulbecco's phosphate-buffered saline after 30 min (kept on ice under the dark condition).Cells were then resuspended in 2% PFA/PBS (100 µL) and analysed by flow cytometry (LSRFortessa™ X20, BD Biosciences, Franklin Lakes, NJ, USA), and the data was examined using the FlowJo software (version 10.8.1).Before determining the %Live/Dead dye-positive cells, doublets were excluded from the analysis by gating forward scatter height (FSC-H) versus forward scatter area (FSC-A).After obtaining the cell viability results, the culture supernatant was further analysed to quantify the amount of pro-inflammatory cytokines.

DPPH Radical Scavenging Activity of Seagrasses
A DPPH radical scavenging antioxidant assay [33] of the homogenised root and aerial parts of Halodule uninervis, Cymodocea rotundata, Syringodium isoetifolium, Thalassia hemprichii, and Zostera muelleri revealed that the Z. muelleri extract had the best antioxidant activity of the five seagrass species tested, with 83% of DPPH radical scavenged at 250 µg/mL and an IC 50 value of 138 µg/mL compared to the other seagrasses tested, which all had less than 40% scavenging effect at 500 µg/mL concentration (Figure 2, Table 1).

DPPH Radical Scavenging Activity of Seagrasses
A DPPH radical scavenging antioxidant assay [33] of the homogenised root and aerial parts of Halodule uninervis, Cymodocea rotundata, Syringodium isoetifolium, Thalassia hemprichii, and Zostera muelleri revealed that the Z. muelleri extract had the best antioxidant activity of the five seagrass species tested, with 83% of DPPH radical scavenged at 250 µg/mL and an IC50 value of 138 µg/mL compared to the other seagrasses tested, which all had less than 40% scavenging effect at 500 µg/mL concentration (Figure 2, Table 1).

In-Depth Investigation of Z. muelleri Aerial and Root Parts
Given the higher antioxidant capacity of Z. muelleri aerial and root extract relative to the other seagrasses and the lack of studies performed in this species previously, additional oven-dried whole plant materials were separated into aerial (ZMA) and root (ZMR) bulk samples, which were macerated in acetone to produce crude extracts.Separate DPPH radical scavenging assays were performed on the root and aerial extracts, which showed that leaves had a better DPPH radical scavenging capacity of 75% (at 250 µg/mL) and an IC 50 value of 119 µg/mL compared to roots, which had a maximum DPPH radical scavenging capacity of 20% at 500 µg/mL (Figure 3).
Given the higher antioxidant capacity of Z. muelleri aerial and root extract relative to the other seagrasses and the lack of studies performed in this species previously, additional oven-dried whole plant materials were separated into aerial (ZMA) and root (ZMR) bulk samples, which were macerated in acetone to produce crude extracts.Separate DPPH radical scavenging assays were performed on the root and aerial extracts, which showed that leaves had a better DPPH radical scavenging capacity of 75% (at 250 µg/mL) and an IC50 value of 119 µg/mL compared to roots, which had a maximum DPPH radical scavenging capacity of 20% at 500 µg/mL (Figure 3).Since the aerial extract of Z. muelleri had a higher antioxidant capacity, its anti-inflammatory properties were subsequently quantified using a human PBMC assay and LEGENDplex cytokine analysis.A cell viability assay demonstrated no statistical significance in the amount of cell death between the unstimulated and ZMA crude extract treatment groups, indicating the extract was not toxic to the PBMCs at 100 µg/mL (Figure 4A).Analysis of the LPS-stimulated PBMCs culture supernatant revealed that treatment with ZMA crude extract significantly reduced the levels of TNF-α, IL-1β, and IL-6 released by the LPS-stimulated PBMCs relative to the LPS control by 29%, 74%, and 90%, respectively (Figure 4B-D, Table 2), while no response was observed for the remaining cytokines tested.Since the aerial extract of Z. muelleri had a higher antioxidant capacity, its antiinflammatory properties were subsequently quantified using a human PBMC assay and LEGENDplex cytokine analysis.A cell viability assay demonstrated no statistical significance in the amount of cell death between the unstimulated and ZMA crude extract treatment groups, indicating the extract was not toxic to the PBMCs at 100 µg/mL (Figure 4A).Analysis of the LPS-stimulated PBMCs culture supernatant revealed that treatment with ZMA crude extract significantly reduced the levels of TNF-α, IL-1β, and IL-6 released by the LPS-stimulated PBMCs relative to the LPS control by 29%, 74%, and 90%, respectively (Figure 4B-D, Table 2), while no response was observed for the remaining cytokines tested.

Bioactive Compounds Isolated Using HPLC
To isolate the potential active principles responsible for the antioxidant and antiinflammatory activities observed in the aerial extract of Z. muelleri, the ZMA extract was dissolved in MeOH and washed several times with hexanes.The MeOH fraction was purified by reverse-phase high-performance liquid chromatography (RP-HPLC), which led to the isolation of 4-hydroxybenzoic acid (1), luteolin (2), and apigenin (3) (Figure 5).The structure of 4-hydroxybenzoic acid (1) was determined by comparison of the NMR spectrum (see Supporting Information Figures S1 and S2) to previously reported spectra [26] and the observation of a peak at m/z 137 (ESI − ) in the LRMS, which was assigned as the [M − H] − ion of (1) (Figure 6).

Bioactive Compounds Isolated Using HPLC
To isolate the potential active principles responsible for the antioxidant and anti-inflammatory activities observed in the aerial extract of Z. muelleri, the ZMA extract was dissolved in MeOH and washed several times with hexanes.The MeOH fraction was purified by reverse-phase high-performance liquid chromatography (RP-HPLC), which led to the isolation of 4-hydroxybenzoic acid (1), luteolin (2), and apigenin (3) (Figure 5).The structure of 4-hydroxybenzoic acid (1) was determined by comparison of the NMR spectrum (see Supporting Information Figures S1 and S2) to previously reported spectra [26] and the observation of a peak at m/z 137 (ESI − ) in the LRMS, which was assigned as the [M−H] − ion of (1) (Figure 6).Life 2024, 14, x FOR PEER REVIEW 10 of 16

Bioactive Compounds Isolated Using HPLC
To isolate the potential active principles responsible for the antioxidant and anti-inflammatory activities observed in the aerial extract of Z. muelleri, the ZMA extract was dissolved in MeOH and washed several times with hexanes.The MeOH fraction was purified by reverse-phase high-performance liquid chromatography (RP-HPLC), which led to the isolation of 4-hydroxybenzoic acid (1), luteolin (2), and apigenin (3) (Figure 5).The structure of 4-hydroxybenzoic acid (1) was determined by comparison of the NMR spectrum (see Supporting Information Figures S1 and S2) to previously reported spectra [26] and the observation of a peak at m/z 137 (ESI − ) in the LRMS, which was assigned as the [M−H] − ion of (1) (Figure 6).

Discussion
Seagrasses grow in marine conditions and produce secondary metabolites for various ecological functions ranging from feeding deterrence [34] to protection from ultraviolet (UV) radiation [35].In defence against pathogens and predators, numerous species of seagrasses produce a wide array of phenolic compounds, thus gaining attention for applications in nutrition, medicine, and cosmetics [36].While considerable phytochemical research has been conducted, a gap persists in our knowledge of seagrass secondary metabolites [34].
The DPPH radical scavenging antioxidant activity of C. rotundata, T. hemprichii, and H. uninvervis (IC 50 > 500 µg/mL, Table 1) in the DPPH assays were significantly weaker compared to previously reported values (IC 50 = 214.68[37], 123.72 [37], and 4.0 µg/mL [38], respectively) while those of S. isoetifolium (IC 50 > 500 µg/mL) correlated with previous values (520.91 µg/mL [37]).This reduced antioxidant activity may be the result of seasonality, with samples collected in this study close to winter instead of summer, which has been reported to result in reduced antioxidant production in seagrasses previously due to the reduced ecological stress (lower temperatures and light intensities) and therefore do secondary in large quantities [39].To the best of our knowledge, this study represents the first investigation of the antioxidant and anti-inflammatory activity of Z. muelleri, which showed good DPPH radical scavenging activities for its aerial and root (138 µg/mL, Table 1) and aerial (119 µg/mL, Table 2) extracts.This potent antioxidant activity observed in the Z. muelleri extracts suggested it may also exert anti-inflammatory effects due to the high oxidative inherent in inflammatory responses [40].These suspicions were confirmed by the significant reduction of TNF-α, IL-1β, and IL-6 cytokine levels in LPS-stimulated PBMCs culture supernatant (29%, 74%, and 90%, respectively, Figure 4).This significant reduction in IL-1β and IL-6 observed coupled with the established correlation between acne vulgaris and IL-1β [41] and IL-6 [42,43] cytokine levels suggest Z. muelleri extracts could be developed into an effective treatment for acne and could be a potential source for the discovery of new anti-inflammatory lead compounds.
Luteolin (2) and apigenin (3) are closely related simple flavone derivatives, the latter known for their broad bioactivities [54].Both of these flavones are extremely common in many plant species and are known to protect plants from UV radiation [55,56] and have anti-microbial, antioxidant, and anti-inflammatory properties [55][56][57][58][59].Both luteolin (2) and apigenin (3) have been reported to exert their antioxidant and inflammatory properties through the deactivation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, modulation of the MAPK signalling pathways and inhibition of many inflammatory cytokines including TNF-α, IL-1β, IL-6, IL-8, IL-17, and nitric oxide (NO) in a host of in vitro, ex vivo, and in vivo models [59][60][61][62][63].Given that both luteolin and apigenin (3) were the major components isolated and the extensive literature on their potent bioactivities, this suggests these compounds are the major bioactive components contributing to the observed antioxidant and anti-inflammatory activities of the Z. muelleri aerial extract.
These results provide a strong case for further research into using seagrasses to treat inflammatory conditions.For example, the UV protection potential of luteolin (2) and apigenin (3) [55,56], coupled with their anti-inflammatory properties in keratinocytes and fibroblasts, [60, 64,65] would make seagrass extracts ideal candidates for the production of topical treatments for the treatment of inflammatory skin conditions such as acne or eczema.In addition, it has been established that the NF-κB pathway and TNF-α, IL-1β, and IL-6 cytokine levels are significantly upregulated within 24 h of experiencing sunburn and remain elevated four days after UV irradiation [66].Given the significant reduction in these pro-inflammatory cytokines observed in the ZMA extract, its antioxidant activity, and its potential UV protective properties, with further research there is the potential for Z. muelleri extracts to be developed into all-in-one sunscreen and sunburn-soothing ointments.
The key limiting factors of this study were insufficient access to Z. muelleri plant materials, which only allowed for the isolation of abundant, known active principles and that the anti-inflammatory investigation of the ZMA extract was performed with a low sample size (n = 6).Therefore, further research with more plant material is required to isolate minor secondary metabolites, and additional screening of the ZMA extract is also needed to confirm these promising preliminary results and justify further study of in vivo models of inflammation.Other research has suggested seagrasses could be consumed for various gastrointestinal ailments [36], with several species also possessing anti-diabetic properties [17,67,68], highlighting additional avenues for future inquiry and showcasing the potential versatility of seagrasses in their uses and the need for further investigation into their seemingly endless list of applications.

Conclusions
The investigation of the bioactivities of the five selected seagrasses-Zostera muelleri, Halodule uninervis, Cymodocea rotundata, Syringodium isoetifolium, and Thalassia hemprichiishowed that acetone extracts of Z. muelleri had the most potent antioxidant activity in a DPPH free radical scavenging assay.The aerial extract of Z. muelleri possessed significantly higher antioxidant activity than that of the roots.It was also shown to significantly reduce the levels of key inflammatory cytokines TNF-α, IL-1β, and IL-6 in human PBMCs.Purification of the Z. muelleri aerial extract led to the isolation of 4-hydroxybenzoic acid (1), luteolin (2), and apigenin (3), which were suggested to be the major anti-inflammatory components of the aerial extract.These preliminary results emphasise the potential utility of seagrasses in the treatment of inflammatory conditions and highlight the need for further research with additional Z. muelleri plant material to isolate other potential active principles, confirm the anti-inflammatory properties of the ZMA extract in vivo, and evaluate this extract for utility as a topical treatment for inflammatory skin conditions and as a sunscreen.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/life14060710/s1: Figure S1 Author Contributions: M.J.P. assisted with supervision, analysed NMR spectroscopic data, prepared the original manuscript, and edited the final manuscript; M.C. performed the extraction of seagrasses, isolation of compounds and performed the antioxidant assays, performed data analysis, assisted with the preparation of the original manuscript, and proofread the final manuscript; E.R. assisted in seagrass collection, cleaned and freeze-dried, prepared the samples, and proofread the manuscript; A.L.S. collected and identified the seagrasses used and proofread the final manuscript; D.U.C.R. assisted with performing LRMS and proofread the final manuscript, P.A.K. assisted with organising LRMS testing and proofread the final manuscript.M.O.contributed to project conceptualisation and proofread the final manuscript; K.Y. assisted with supervision of extraction and antioxidant experiments, performed the cell viability and PBMCs assays, performed data analysis and visualisation, and proofread the final manuscript; P.W. led and conceptualised the project, provided supervision, assisted with data analysis, provided financial support and research facilities, and proofread the final manuscript.All authors have read and agreed to the published version of the manuscript.

Funding:
The authors acknowledge the National Health and Medical Research Council (NHMRC) Ideas Grant (APP1183323) for supporting this work.
Institutional Review Board Statement: The human ethics approval for experiments using human blood was granted by the Human Research Ethics Committee of James Cook University (Approval number H8523, 2023).

Data Availability Statement:
The data presented in this study is available in the electronic Supplementary Information of this article.

Figure 1 .
Figure 1.A map of seagrass collection locations around Cairns.The aerial and root parts of Halodule uninervis, Cymodocea rotundata, Syringodium isoetifolium, and Thalassia hemprichii were collected from Green Island (A), and Zostera muelleri was collected from Cairns harbour (B).Inset shows the location of Cairns in far north Queensland (FNQ), Australia.This image was created with the use of a map adapted from Google Maps.

Figure 1 .
Figure 1.A map of seagrass collection locations around Cairns.The aerial and root parts of Halodule uninervis, Cymodocea rotundata, Syringodium isoetifolium, and Thalassia hemprichii were collected from Green Island (A), and Zostera muelleri was collected from Cairns harbour (B).Inset shows the location of Cairns in far north Queensland (FNQ), Australia.This image was created with the use of a map adapted from Google Maps.

Figure 3 .
Figure 3.The DPPH-free radical scavenging activity by aerial or root crude extracts (31.25-500 µg/mL concentrations) of Z. muelleri.The data expressed represent mean ± SD from two independent experiments performed in triplicate (n = 6), visualized using GraphPad Prism 10.2.2.Gallic acid was included as a standard antioxidant compound for comparison.

Figure 3 .
Figure 3.The DPPH-free radical scavenging activity by aerial or root crude extracts (31.25-500 µg/mL concentrations) of Z. muelleri.The data expressed represent mean ± SD from two independent experiments performed in triplicate (n = 6), visualized using GraphPad Prism 10.2.2.Gallic acid was included as a standard antioxidant compound for comparison.

Table 1 .
Summary of the DPPH radical scavenging activities and half maximal inhibitory concentration (IC 50 ) values of the five seagrass aerial and root extracts.* Radical scavenging activity observed at 500 µg/mL.† Reported as the mean ± SD.

Table 2 .
Summary of the antioxidant DPPH radical scavenging activities and IC50 values and the anti-inflammatory PBMC assay normalised cytokine levels of the Z. muelleri aerial (ZMA) and root (ZMR) extracts.* Radical scavenging activity observed at 500 µg/mL.† Reported as the mean ± SD.

Table 2 .
Summary of the antioxidant DPPH radical scavenging activities and IC 50 values and the anti-inflammatory PBMC assay normalised cytokine levels of the Z. muelleri aerial (ZMA) and root (ZMR) extracts.* Radical scavenging activity observed at 500 µg/mL.† Reported as the mean ± SD.