New Insights on Acanthus ebracteatus Vahl: UPLC-ESI-QTOF-MS Profile, Antioxidant, Antimicrobial and Anticancer Activities

This study investigated the antioxidant, antimicrobial, anticancer, and phytochemical profiling of extracts from the leaves and stem/root of Acanthus ebracteatus (AE). The total phenolic content (TPC), total flavonoid content (TFC), 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical-scavenging activity, 2, 2′-azino-Bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical-scavenging activity, metal chelating activities (MCA), ferric reducing antioxidant power (FRAP) and oxygen radical antioxidant capacity (ORAC) were used for antioxidant assessment. The ethanolic extracts of the leaves (AEL-nor) and stem/root (AEWP-nor) without chlorophyll removal and those with chlorophyll removal, using sedimentation process (AEL-sed and AEWP-sed), were prepared. Generally, AEL-sed showed the highest antioxidant activity (FRAP: 1113.2 µmol TE/g; ORAC: 11.52 µmol TE/g; MCA: 47.83 µmol EDTA/g; ABTS 67.73 µmol TE/g; DPPH 498.8 µmol TE/g; TPC: 140.50 mg/GAE g and TFC: 110.40 mg/CE g) compared with other extracts. Likewise, AEL-sed also showed the highest bacteriostatic (MIC) and bactericidal (MBC) effects, as well as the highest anticancer and antiproliferative activity against oral squamous carcinoma (CLS-354/WT) cells. UPLC-ESI-QTOF/MS analysis of AEL-sed and AEWP-sed tentatively identified several bioactive compounds in the extracts, including flavonoids, phenols, iridoids, and nucleosides. Our results provide a potentially valuable application for A. ebracteatus, especially in further exploration of the plant in oxidative stress-related disorders, as well as the application of the plant as potential nutraceuticals and cosmeceuticals.


Introduction
Medicinal plants have played a pivotal role in primary health care over the past few decades, especially in low-and middle-income countries. Several medicinal plants have been the building blocks for the successful discovery of bioactive medicinal agents currently used in the treatment of a wide range of diseases. Furthermore, medicinal plants are generally perceived as safer substituents for the treatment of devastating diseases, including diabetes, cancer, cardiovascular disorders, and neurodegenerative diseases amongst Molecules 2022, 27,1981 2 of 14 others [1][2][3]. The roles of these natural endowments in oxidative stress-related diseases have been extensively explored. The display of excellent antioxidant activities by plant extracts is directly correlated to the existence of bioactive constituents, notably polyphenolic compounds, which make these medicinal plant extracts display properties indicating they are significant antidiabetic, anti-inflammatory, antiaging, and anticancer agents [3,4].
Acanthus ebracteatus Vahl. (Sea Holly) is a multipurpose mangrove medicinal plant belonging to the Acanthaceae family that grows in several southeast Asian countries including Thailand, Malaysia, Indonesia, the Philippines, and Vietnam [5,6]. A. ebracteatus has several traditional folk medicinal uses, especially in the treatment of rheumatism, cough, snakebite, chronic fever, asthma, hepatitis, intestinal worms, preventing hair loss, herpes zoster, leucorrhea, wound, menstrual disorders, rash, and skin diseases [5]. Although there are few reports regarding the phytochemical richness of A. ebracteatus, previous studies have reported the presence of aliphatic alcohol, aliphatic glycosides, phenolic glycosides, terpenes, megastigmane glycosides, flavonoids, and lignan glycosides [6,7]. A. ebracteatus has been pharmacologically reported to show anti-inflammatory, neuroprotection and wound healing effects [5,6,8,9]. However, none of these reports provided detailed information regarding the phytochemical and pharmacological profiles of the leaves and the stem/root extracts of this species. As such, this work evaluated the chemical composition, antioxidant, antibacterial and cytotoxic activities of A. ebracteatus leaves and stem/root extracts.

Evaluation of the Antioxidant Activity
The antioxidant activities of the leaves and stem/root extracts of A. ebracteatus prepared without chlorophyll removal and with the sedimentation chlorophyll removal method described in Section 4.2 were examined using several in vitro assays (DPPH, ABTS, FRAP, MCA, and ORAC). It was observed from the results that the leaves extract (AEL) showed better antioxidant activity in all the assays compared to the extract from the stem/root (AEWP). The leaves extract from the sedimentation process (AEL-sed) showed the highest antioxidant activity in the FRAP (1113.2 ± 4.2 µmol TE/g), ORAC (11.52 ± 0.3 µmol TE/g), MCA (47.83 ± 0.01 µmol EDTA/g), ABTS (67.73 ± 0.5 µmol TE/g) and DPPH (498.8 ± 0.4 µmol TE/g) assays (Table 1). 3.22 ± 0.20 b 3.32 ± 0.60 b 11.51 ± 0.50 a 11.52 ± 0.30 a Different lowercase superscripts within the same column indicate significant difference (p < 0.05). Mean (n = 3); AEWP-nor: extract from the stem/root without dechlorophyllization; AEWP-sed: extract from the stem/root after the dechlorophyllization process using the sedimentation process; AEL-nor: extract from the leaves without dechlorophyllization; AEL-sed: extract from the leaves after the dechlorophyllization process using the sedimentation process.

Evaluation of Antimicrobial Activity
The antimicrobial activity (MIC and MBC) of the extracts against E. coli and L. monocytogenes is shown in Table 2. The results showed that the AEL extract displayed more potent antibacterial activity compared to AEWP, with MIC values ranging from 0.25-0.5 mg/mL and MBC values of 0.5-1.0 mg/mL (Table 2).

Evaluation of Antimicrobial Activity
The antimicrobial activity (MIC and MBC) of the extracts against E. coli and L. monocytogenes is shown in Table 2. The results showed that the AEL extract displayed more potent antibacterial activity compared to AEWP, with MIC values ranging from 0.25-0.5 mg/mL and MBC values of 0.5-1.0 mg/mL (Table 2).

Evaluation of Anticancer Activity
The effects of the extracts on the cellular viability of CLS-354 / WT cells were tested following a tetrazolium-based MTT assay. The results demonstrated that AEL-sed and AEWP-sed resulted in a significant (p < 0.001) reduction in cell viability of CLS-354/WT in a dose-dependent manner. The ED50 was 200 μg/mL and 400 μg/mL for AEL-sed and AEWP-sed, respectively ( Figure 1

Evaluation of the Anti-Proliferative Effect
The AEL-sed and AEWP-sed extracts were further investigated for their anti-proliferative effects on CLS-354/WT. As shown in Figure 2, The results indicated that both extracts significantly (p < 0.001) inhibited the proliferation of cells. Moreover, the extracts also retarded cell migration. However, cell viability was not significantly affected. These results suggested a pronounced anti-proliferative effect of AEL-sed and AEWP-sed extract on CLS-354/WT at the tested concentration.
The AEL-sed and AEWP-sed extracts were further investigated for their anti-proliferative effects on CLS-354/WT. As shown in Figure 2, The results indicated that both extracts significantly (p < 0.001) inhibited the proliferation of cells. Moreover, the extracts also retarded cell migration. However, cell viability was not significantly affected. These results suggested a pronounced anti-proliferative effect of AEL-sed and AEWP-sed extract on CLS-354/WT at the tested concentration.

Identification of Compounds in AEL-sed and AEPW-sed
The profiling of the phytochemical constituents in AEL-sed and AEWP-sed was performed via UPLC-QTOF-MS analysis, in the negative ionization mode. The chromatogram of the peaks of the eluted compounds in AEL-sed and AEPW-sed showed several peaks (within 20 min), suggesting the presence of several constituents in the extract (Figure 3). The compounds with mass error <5 ppm and high relative abundance are presented in Tables 3 and 4. The data shown in Table 3 indicated that the majority of the compounds tentatively identified in AEL-sed were glycosidic constituents, especially flavonoids and phenolic compounds. According to Table 3

Discussion
In this study, the leaves and bark/root of A. ebracteatus were extracted with and without using a chlorophyll removal process, and the extracts were analyzed using UHPLC-QTOF-MS for their phytochemical profiles and their antioxidant, antimicrobial and anticancer properties. Free radicals are essential by-products generated during metabolic processes by the body. However, these radicals have the ability to form complexes through ionizing radiation, leading to oxidative stress, and they further attack biological molecules such as lipids, nucleic acid, and protein [10,11]. It has been widely acknowledged that reactive oxygen species and oxidative stress are extensively implicated in the pathophysiology of several diseases that plague humankind, including diabetes, cardiovascular diseases, and cancer [12,13]. The ability of a medicinal plant extract to exert any form of bioactivity is largely dependent on the phyto-constituents present in the plant. In addition, many natural products, including plant extracts or isolated bioactive compounds have displayed several pharmacological activities linked to their potential to modulate oxidative stress and exhibit antioxidant properties [13]. As such, the antioxidant activity of an extract plays a vital role in its pharmacological effects. In view of this, the antioxidant activities of A. ebracteatus extracts were evaluated using various established techniques, namely DPPH, ABTS, FRAP, MCA, and ORAC assays. Generally, the leaves extracts (AEL-sed and AEL-nor) exhibited the highest antioxidant properties. The ability of AEL-sed to scavenge DPPH (498.80 µmol TE/g) and ABTS (67.73 µmol TE/g) radicals, as well as reduce (FRAP: 1113.20 µmol TE/g) or chelate (MCA: 47.83 µmol EDTA/g) metal ions was of a greater extent compared to the extract from the stem/root. In a previous study, the ethanolic extract of A. ebracteatus was shown to exert DPPH (IC 50 : 0.12 ± 0.03 mg/L)-scavenging activity [9].
The results obtained from the total phenolics and flavonoids content indicated that AEL was rich in total phenolics (140.5 and 138.2 mg GAE/g for AEL-sed and AEL-nor, respectively) and flavonoids (110.4 and 107.6 mg CE/g for AEL-sed and AEL-nor, respectively). Earlier studies have indicated the presence of high levels of phenolics and flavonoids in A. ebracteatus [6]. The results obtained from our study confirmed the presence of phenolics and flavonoids. However, the TPC and TFC contents reported in our study were markedly higher [6]. The disparity in the phenolic and flavonoid contents may be attributed to the differences in plant origin, growth conditions, extraction methods, and the solvent employed for extraction.
The antimicrobial properties of the A. ebracteatus extracts could be obviously related to their high polyphenolic constituents. Pratoomsoot et al. [9] previously reported that extracts from A. ebracteatus showed significant antimicrobial activity against the A. baumannii DMST 10437, E. coli 4212, S. aureus DMST 8840, methicillin-resistant S. aureus DMST, S. epidermidis DMST 3547, S. epidermidis DMST 4343, and S. pyogenes DMST 30563 strains. The importance of controlling bacterial infections cannot be over-emphasized due to their prevailing negative effects in primary health care as well as the complications that arise from bacterial infections related to other diseases. An increasing number of reports illustrate the importance of medicinal plants in the treatment of bacterial infections [18]. The results indicated that A. ebracteatus extract showed significant antibacterial properties.
Cancer is a major cause of death globally and, unfortunately, there is no known cure for this dreaded disease [19]. As such, finding a cost-effective, alternate, and safer treatment for cancer is warranted. Medicinal plants have gained attention as alternative chemopreventive and therapeutic agents in recent years. In fact, numerous anticancer agents presently approved for cancer treatment or undergoing clinical trials as possible anticancer drugs have direct links to medicinal plants and are building blocks for the emergence of some synthetic anticancer agents [20]. Oral carcinogenesis is a multistep process that includes genetic events which lead to the disruption of the normal regulatory pathways that control cellular functions [21]. Oropharyngeal cancer and its treatment via chemotherapy causes several complications, including dysphagia, mucositis, pain, related infections, and bleeding [22]. Similar to chemotherapeutic agents, natural products such as phenethyl isothiocyanate [23], resveratrol [24], and curcumin [25] have been reported to have excellent anticancer efficacy with no or minimal side effects. The results from our study suggested that AEL-sed showed reasonable anticancer effects. Several phytochemicals, such as diosmetin, esculetin, isoacteoside, baicalin, isoamericanol A, luteolin, apigenin, and genkwanin, among several others identified in the extract, have been reported as promising anticancer agents in several in vitro and in vivo studies [26][27][28][29][30][31][32][33][34]. Therefore, A. ebracteatus extract contains several constituents with promising bioactivities that could be beneficial for the treatment of several disorders.

Plant Material
The leaves, stem and root of A. ebracteatus were collected from Surat Thani Province, Thailand. The plant was authenticated at the Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand. The samples were powdered with a mechanical grinder (Jing Gongyi, JGY-800B, Yongkang, China) to fine particles and the powdered leaves and stem/roots were divided into two equal portions and extracted separately.

Classical Ethanol Extraction
The powdered leaves (200 g) and stem/roots (200 g) were extracted with 2 L of 70% ethanol at a solvent/solid ratio of 10:1 (v/w) on a shaker for 24 h. Subsequently, the extraction mixture was filtered, and the resulting filtrate was dried under reduced pressure with a rotary evaporator at 45 • C. The dried extract of the leaves (AEL-nor) and back/root (AEWP-nor) were stored at 4 • C until further use.

Extraction Using the Sedimentation Method
Likewise, 200 g of the powdered leaves and 200 g of the powdered stem/roots were subjected to 70% ethanol extraction at a solvent/solid ratio of 10:1 (v/w) on a shaker for 24 h. Thereafter, the solution obtained after filtration was concentrated to 30% of the initial volume, and the mixture was refrigerated at 4 • C for 24 h to sediment. Thereafter, the solution was decanted, and the top layer (without chlorophyll) was centrifuged (6000 rpm, 30 min at 4 • C). The supernatants obtained from the leaves extract (AEL-sed) and the stem/roots (AEWP-sed) were freeze-dried and stored until further use [35,36].

Total Phenolic and Flavonoid Content
The TPC and TFC of the extracts were determined based on previously reported protocol [37,38]. The TPC of the extracts was spectrophotometrically determined using the Folin-Ciocalteu method. Briefly, 0.1 mL of the extracts were added to 0.75 mL of 10% Folin-Ciocalteu reagent, and the mixture was allowed to stand for 5 min. Subsequently, 0.75 mL of a saturated solution of Na 2 CO 3 was added, and the mixtures were incubated at room temperature for 3 h, while shaking randomly. Thereafter, the absorbance of the blue-colored solution was measured at 760 nm. TPC was expressed as mg gallic acid equivalent (GE)/g dry extract.
For the analysis of the TFC of the extracts, 800 µL of distilled water was mixed with 200 µL of the extract solution, 60 µL of 5% NaNO 2 solution, and 60 µL of 10% AlCl 3 solution. The mixture was allowed to stand for 5 min at room temperature and thereafter 400 µL of 1M NaOH solution was added. The mixture was made up to a volume of 2 mL with distilled water and thoroughly mixed. The absorbance of the solution was measured at 510 nm. TFC was calculated from the standard curve of catechin and expressed as mg catechin equivalent (CE)/g extract.
For ABTS-RSA, the stock solutions included 7.4 mM ABTS solution and 2.6 mM potassium persulfate solution. The working solution was prepared by mixing the two stock solutions in equal quantities. The mixture was allowed to react for 12 h at room temperature in the dark. The solution obtained (1 mL) was then diluted with 50 mL of distilled water to obtain an absorbance of 1.10 ± 0.02 units at 734 nm. The sample (150 µL) was mixed with 2850 µL of ABTS solution, and the mixture was left at room temperature for 1 h in the dark. The absorbance was then measured at 734 nm using a spectrophotometer. The blank was prepared in the same manner, except that distilled water was used instead of the sample. A standard curve of Trolox ranging from 50-600 µM was prepared. The activity was expressed as µmol Trolox equivalent (TE)/g solid.
The extracts sample (0.3 mL) was mixed with 2.7 mL of a methanolic solution containing DPPH (0.15 mM). The mixture was shaken vigorously and left to stand for 60 min in the dark (until stable absorption values were obtained) at room temperature (25 • C). The reduction of the DPPH-RSA was measured by continuously monitoring the decrease in absorbance at 517 nm. The DPPH scavenging activity was expressed as µmol Trolox equivalent (TE)/g solid.
The FRAP reagent was prepared by mixing acetate buffer (30 mM, pH 3.6) and 10 mM TPTZ solution in a 40 mM HCl and 20 mM iron (III) chloride solution in proportions of 10:1:1 (v/v). The sample solution (150 µL) was mixed with 2.85 mL of working FRAP reagent and incubated in dark conditions at room temperature for 30 min. The absorbance of the reaction mixture was read at 593 nm. The standard curve was prepared using Trolox ranging from 0-500 µM. The activity was expressed as µmol Trolox equivalent (TE)/g sample.
For MCA, 1 mL of extract was mixed with 3.7 mL of distilled water and the mixture was reacted with 0.1 mL of 2 mM FeCl 2 and 0.2 mL of 5 mM ferrozine for 20 min. The absorbance was read at 532 nm. One milliliter of distilled water instead of the extract was used as a control. The chelating activity was expressed as µmol EDTA equivalent (EE)/g solid.

Antibacterial Activity
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) measurements of the extracts were performed against Listeria monocytogenes and Escherichia coli 0157, using the previously reported protocol [39].

Anticancer Efficacy Compounds
The anticancer efficacy of the extracts was tested against epithelium-like phenotype oral squamous carcinoma cell (CLS-354/WT) by an indirect method [40]. Briefly, carcinoma cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 1% v/v penicillin/streptomycin (U/mL), and 2mM stable L-glutamine. Approximately, 1 × 10 4 (cells/mL) cells were seeded in 96-well plates and incubated in an incubator with 5% CO 2 at 37 • C. The cells were allowed to form a 70% confluent monolayer and treated with the extract (1600-12.5 µg/mL) and supplemented fresh RPMI-1640 as a negative control, in triplicate. The percentage of cell death was analyzed using MTT assay. The insoluble formazan crystals were solubilized with 99.9% DMSO, and the absorbance was measured at 560 nm using a multi-mode plate reader (BioTek, Winooski, VT, USA). The percentage of cell death was calculated.

Anti-Proliferative Effect of Extract
The in vitro scratch assay was evaluated to quantify the anti-migration capabilities of cells treated with the extracts. Briefly, CLS-354/WT cells were seeded at a cell density at 3 × 10 4 cells/well in a 6-well plate. The confluent monolayer (70%) of the cells was scratched using a sterile pipette tip to create a wound of 1 mm width. Subsequently, the cells were washed with phosphate buffer (pH 7.4) to remove cellular debris and replaced with a fresh medium containing the extract above ED 50 (50 % inhibition of cancer cell growth), or with RPMI-1640 medium as a negative control. Images of cell migration were captured at 0 and 24 h using a Carl Zeiss microscope Axio Vert. A1 (Konigsallee, Gottingen, Germany). The residual gap between the migrating cells was measured using Image J software (1.8.0_172).

UHPLC-ESI-QTOF-MS Profiling of the Extracts
The extracts (AEL-sed and AEWP-sed) with significant antioxidant and antimicrobial activities were selected for LCMS profiling. The experimental procedures and instrumental parameters were previously described by Eze and Tola [41]. The analysis was performed using an Agilent 1290 Infinity II LC System (Agilent Technologies, Santa Clara, CA, USA) equipped with an autosampler, a binary pump, a vacuum degasser, and a diode array detector. The extracts were separated on Agilent's ZORBAX Eclipse Plus C18 column (150 × 2.1 mm, 1.8 µmm). The mobile phases consisted of (A) acidified Milli-Q water (0.1% formic acid) and (B) acetonitrile. The following parameters were employed for the elution: 0.50 min: 0% B; 16.50 min: 100% B; 17.50 min: 100% B; 20.00 min: 0.00% B; 22.00 min: 0.00% B; injection volume of 2.0 mL, flow rate of 0.2 µL min −1 , and column temperature of 25 • C. The HPLC system was coupled to an Agilent 6545 LC/Q-TOF MS mass spectrometer equipped with a dual Agilent Jet Stream ESI negative mode, with a mass range of m/z 100 to 1500 at a scan rate of 1.00 spectrum per second. Accurate mass measurements by the instrument were ensured using an automated calibrant delivery system that continuously

Statistical Analysis
The results were expressed as mean ± standard error. Statistical analysis was determined by one-way ANOVA followed by Dunnett's test using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA). Differences of p < 0.05 were considered significant.

Conclusions
In conclusion, the results from this study suggested that A. ebracteatus displayed promising antioxidant, antibacterial and anticancer activities. The leaves of the plant showed better activity in all the tested assays when compared to other extracts. Furthermore, UPLC-ESI-QTOF-MS analysis indicated that the plant is rich in polyphenolic compounds, including phenolic acids, flavonoids, iridoids, and o-glycosyl compounds. These results suggested that A. ebracteatus can be explored as a possible nutraceutical for the treatment of oxidative stress-related disorders. Further studies are needed to validate the in vivo pharmacological and activities, especially in unexplored and valuable aspects of A. ebracteatus.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.