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. 2022 Mar 18;27(6):1981. doi: 10.3390/molecules27061981

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

Opeyemi Joshua Olatunji 1,*, Oladipupo Odunayo Olatunde 2, Titilope John Jayeoye 3, Sudarshan Singh 4, Sirinporn Nalinbenjapun 5,6, Sasikarn Sripetthong 5,6, Warangkana Chunglok 4,7, Chitchamai Ovatlarnporn 5,6,*
Editor: Maria Atanassova
PMCID: PMC8954823  PMID: 35335344

Abstract

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.

Keywords: Acanthus ebracteatus Vahl, antioxidant, antibacterial, anticancer, polyphenols

1. 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 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, snake-bite, 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.

2. Results

2.1. 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).

Table 1.

Antioxidant activity of different extracts from A. ebracteatus.

Sample/Assay AEWP-nor AEWP-sed AEL-nor AEL-sed
TPC (mg GAE/g dry extract) 30.49 ± 0.10 e 36.88 ± 0.10 d 138.20 ± 0.10 b 140.50 ± 0.10 a
TFC (mg CE/g dry extract) 20.24 ± 0.20 e 28.82 ± 0.10 d 107.60 ± 0.02 b 110.40 ± 0.50 a
DPPH-RSA (µmol TE/g dry extract) 91.90 ± 0.40 f 104.90 ± 0.08 e 448.10 ± 1.20 b 498.80 ± 0.40 a
ABTS-RSA (µmol TE/g dry extract) 44.01 ± 0.10 d 48.59 ± 0.08 c 57.05 ±0.10 b 67.73 ± 0.50 a
FRAP (µmol TE/g dry extract) 182.80 ± 0.40 c 223.01 ± 0.40 b 1098.20 ± 7.1 a 1113.20 ± 4.20 a
MCA (µmol EDTA/g dry extract) 36.32 ± 0.10 e 37.29 ± 0.05 d 46.87 ± 0.20 b 47.83 ± 0.01 a
ORAC (µmol TE/g dry extract) 3.22 ± 0.20 b 3.32 ± 0.60 b 11.51 ± 0.50 a 11.52 ± 0.30 a
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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.

2.2. Total Phenolic and Flavonoid Content

Likewise, in the total phenolic (TFC) and total flavonoid content (TFC) quantification, AEL extract also showed higher phenolic and flavonoid contents (AEL-sed; TFC: 110.4 ± 0.5 mg CE/g; TPC: 140.5 ± 0.1 mg GAE/g; AEL-nor; TFC: 107.6 ± 0.02 mg CE/g; TPC: 138.2 ± 0.1 mg GAE/g) compared to AEWP (AEL-sed; TFC: 28.82 ± 0.1 mg CE/g; TPC: 136.88 ± 0.1 mg GAE/g; AEL-nor; TFC: 20.24 ± 0.2 mg CE/g; TPC: 30.49 ± 0.1 mg GAE/g) (Table 1).

2.3. 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).

Table 2.

Antibacterial activity of different extracts from A. ebracteatus.

Samples MIC MBC
EC LM EC LM
AEWP-nor 1.00 a 1.00 a 2.00 a 2.00 a
AEWP-sed 1.00 a 1.00 a 2.00 a 2.00 a
AEL-nor 0.25 c 0.50 b 0.50 c 1.00 b
AEL-sed 0.25 c 0.50 b 0.50 c 1.00 b
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EC, Escherichia coli; LM, Listeria monocytogenes; MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration. Different lowercase superscripts within the same column indicate significant difference (p < 0.05). Mean (n = 3).

2.4. 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).

Figure 1.

Figure 1

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Epithelium-like phenotype oral squamous carcinoma cell (CLS-354/WT) death, in percent, treated with AEL-sed and AEWP-sed extracts (1600–12.5 μg/mL), using MTT assay. Data are expressed as mean ± SEM from at least three independent experiments and analyzed via one-way ANOVA with Dunnett’s test. * p < 0.05, *** p < 0.001 vs untreated control.

2.5. 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.

Figure 2.

Figure 2

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Antiproliferative effects of AEL-sed and AEWP-sed extracts (800 μg/mL) in epithelium-like phenotype oral squamous carcinoma cell (CLS-354/WT) at 0 and 24 h (A) Percentage migration calculated using the length of cell migration obtained from microscopic image (B) Microscopic photographs of CLS-354/WT cells migration by scratch technique. Data are expressed as mean ± SEM from at least 3 independent experiments and analyzed by one-way ANOVA followed by Dunnett’s test. *** p < 0.001 vs. untreated control.

2.6. 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 Table 3 and Table 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, simple phenolic and phenolic glycosides, including caffeic acid (Rt: 6.36  min), dihydroferulic acid 4-O-glucuronide (Rt: 5.52 min), 4-glucogallic acid (Rt: 5.532 min), chlorogenic acid (Rt: 7.038 min), kelampayoside A (Rt: 5.269 min) and hydrojuglone glucoside (Rt: 8.018 min) were tentatively identified in AEL-sed.

Figure 3.

Figure 3

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Total ion chromatograms of A. ebracteatus extract using UHPLC-ESI-QTOF-MS in the negative electrospray ionization mode showing the chromatogram intensity against the acquisition time; (A) AEL-sed, (B) AEWP-sed.

Table 3.

Compounds tentatively identified in AEL-sed using UHPLC-ESI-QTOF-MS analysis.

No Rt (min) Accurate Mass (m/z) Calculated Mass (Da) Score (DB) Predicted Formula Compound Identity
1 2.997 337.0775 338.0848 98.14 C12H18O11 L-Ascorbic acid-2-glucoside
2 3.311 225.0015 226.009 63.57 C8H7ClN4S 6-(2-Chloroallylthio)purine
3 4.076 134.0471 135.0544 87.88 C5H5N5 Adenine
4 4.604 330.119 331.1262 98.71 C14H21NO8 5′-O-beta-D-Glucosylpyridoxine
5 4.729 128.0351 129.0426 95.05 C5H7NO3 (R)-(+)-2-Pyrrolidone-5-carboxylic acid
6 4.767 243.0624 244.0697 99.79 C9H12N2O6 Pseudouridine
7 4.805 174.077 175.0843 99.74 C7H13NO4 Calystegine B5
8 4.854 180.0664 181.0736 99.62 C9H11NO3 3-Amino-3-(4-hydroxyphenyl)propanoate
9 5.005 282.0842 283.0915 98.4 C10H13N5O5 Guanosine
10 5.055 150.042 151.0492 98.18 C5H5N5O 8-Hydroxyadenine
11 5.256 405.1395 406.1468 98.28 C17 H26O11 Morroniside
12 5.269 477.1608 478.1681 97.79 C20H30O13 Kelampayoside A
13 5.356 108.0456 109.0529 98.09 C6H7NO 3-Hydroxy-2-Methylpyridine
14 5.52 371.0979 372.1052 98.94 C16H20O10 Dihydroferulic acid 4-O-glucuronide
15 5.532 331.0668 332.0741 99.44 C13H16O10 4-Glucogallic acid
16 5.959 355.1044 356.1116 95.68 C16H20O9 1-O-2′-Hydroxy-4′-methoxycinnamoyl-b-D-glucose
17 6.16 461.1665 462.1738 99.05 C20H30O12 Verbasoside
18 6.26 403.1245 404.1318 96.86 C17H24O11 Oleoside 11-methyl ester
19 6.336 341.0875 342.0947 83.78 C15H18O9 Glucocaffeic acid
20 6.36 179.0349 180.0423 96.04 C9H8O4 Caffeic Acid
21 6.637 339.0721 340.0794 99.12 C15H16O9 Aesculin
22 7.038 353.088 354.0953 99.19 C16H18O9 Chlorogenic acid
23 7.114 593.151 594.1583 98.71 C27H30O15 Saponarin
24 7.164 387.1657 388.1729 97.14 C18H28O9 2-[4-(3-Hydroxypropyl)-2-methoxyphenoxy]-1,3-propanediol-1-xyloside
25 7.39 639.1929 640.2024 57.65 C29H36O16 beta-Hydroxyacteoside
26 7.415 637.1046 638.1119 99.11 C27H26O18 Scutellarein-7-glucuronosyl-(1->2)-glucuronide
27 7.54 563.1406 564.1476 97.71 C26H28O14 Luteolin 3′-methyl ether 7,4′-dixyloside
28 7.565 415.1608 416.1682 95.16 C19H28O10 Phenylethyl primeveroside
29 7.817 177.0195 178.0267 86.68 C9H6O4 Esculetin
30 7.842 399.166 400.1734 95.33 C19H28O9 Corchoionoside B
31 7.917 621.11 622.117 98.15 C27H26O17 Genistein 4′,7-O-diglucuronide
32 7.942 383.0622 384.0693 97.99 C16H16O11 2-O-Feruloylhydroxycitric acid
33 7.967 353.0517 354.0588 98.05 C15H14O10 2-O-p-Coumaroylhydroxycitric acid
34 8.018 337.0932 338.1004 98.79 C16H18O8 Hydrojuglone glucoside
35 8.369 461.0725 462.0797 98.96 C21H18O12 3-Methylellagic acid 8-rhamnoside
36 8.432 623.1996 624.2065 96.46 C29H36O15 Isoacteoside
37 8.62 637.2143 638.2212 98.89 C30H38O15 4′-Hydroxy-5,7,2′-trimethoxyflavanone-4′-rhamnosyl-(1->6)-glucoside
38 8.745 429.1764 430.1837 97.61 C20H30O10 Phenethyl rutinoside
39 8.871 579.1728 580.1799 97.92 C27H32O14 Cascaroside F
40 8.884 445.0774 446.0847 97.07 C21H18O11 Baicalin
41 8.971 475.088 476.0953 98.09 C22H20O12 Diosmetin 7-O-beta-D-glucuronopyranoside
42 9.147 665.2081 666.2159 88.16 C31H38O16 Tetramethylquercetin 3-rutinoside
43 9.273 651.2289 652.2359 97.31 C31H40O15 (-)-Matairesinol-4′-[apiosyl-(1->2)-glucoside]
44 9.373 413.2173 414.2247 95.11 C21H34O8 (4R,5S,7R,11S)-11,12-Dihydroxy-1(10)-spirovetiven-2-one 11-glucoside
45 9.386 433.1498 434.1571 96.09 C22H26O9 Vestitone 7-glucoside
46 9.624 431.1345 432.1419 95.75 C22H24O9 4′-O-Methylglucoliquiritigenin
47 9.8 591.2074 592.2147 96.83 C29H36O13 Osmanthuside B
48 10.151 503.1552 504.1625 72.14 C25H28O11 Sergeolide
49 10.277 473.1438 474.1511 92.54 C24H26O10 Luteolin 7,3′-dimethyl ether 5-glucoside
50 10.327 275.092 276.0993 98.16 C15H16O5 5-De-O-methyltoddanol
51 10.478 285.0405 286.0478 98.52 C15H10O6 Luteolin
52 10.955 329.1037 330.1107 85.17 C18H18O6 Isoamericanol A
53 11.03 207.0666 208.0738 97.72 C11H12O4 5-(3′,5′-Dihydroxyphenyl)-gamma valerolactone
54 11.18 220.0613 221.0685 99.48 C11H11NO4 Methyl dioxindole-3-acetate
55 11.193 329.2332 330.2404 98.67 C18 H34O5 9S,10S,11R-trihydroxy-12Z-octadecenoic acid
56 11.281 269.0455 270.0528 98.69 C15H10O5 Apigenin
57 11.432 299.0563 300.0635 98.53 C16 H12O6 Diosmetin
58 11.482 619.1446 620.1518 96.93 C32H28O13 Apigenin-7-(3″-acetyl-6″-E-p-coumaroylglucoside)
59 11.934 268.0611 269.0682 97.25 C15H11NO4 Evoxanthidine
60 13.088 307.1907 308.198 81.32 C18H28O4 Dihydrocapsiate
61 13.289 675.358 676.3652 94.84 C33H56O14 Gingerglycolipid A
62 13.44 293.1752 294.1825 98.16 C17H26O4 Myrsinone
63 14.067 273.0765 274.0837 98.17 C15 H14O5 2,3,4-Trihydroxy-4′-ethoxybenzophenone
64 14.545 241.0866 242.0938 98.73 C15H14O3 Resveratrol 4′-methyl Ether
65 15.8 291.0425 292.0498 98.05 C15H13ClO4 Chlorosesamone
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Table 4.

Compounds tentatively identified in AEWP-sed using UHPLC-ESI-QTOF-MS analysis.

No Rt (min) Accurate Mass (m/z) Calculated Mass (Da) Score (DB) Predicted Formula Compound Identity
1 2.251 629.1697 630.1768 77.85 C27H34O17 Leucodelphinidin-3-O-(beta-D-glucopyranosyl-(1->4)-alpha-L-rhamnopyranoside)
2 2.302 191.0562 192.0635 99.67 C7H12O6 Quinic acid
3 2.427 827.2658 828.2731 97.02 C30H52O26 Verbascose
4 2.528 503.1612 504.1685 98.71 C18H32 O16 Nephritogenoside
5 2.653 683.225 684.2323 80.52 C37H36N2O11 Citbismine C
6 2.654 341.1091 342.1163 98.89 C12 H22 O11 2-O-a-D-Galactopyranuronosyl-L-rhamnose
7 3.156 290.0878 291.095 99.06 C11H17NO8 Sarmentosin epoxide
8 3.193 665.2136 666.2208 97.88 C24H42O21 Fagopyritol A3
9 3.331 225.0016 226.009 65.47 C8H7ClN4S 6-(2-Chloroallylthio)purine
10 3.381 203.0196 204.0269 99.89 C7H8O7 Daucic acid
11 4.762 243.0623 244.0697 99.5 C9H12N2O6 Pseudouridine
12 4.837 174.077 175.0843 99.62 C7H13NO4 Calystegine B5
13 5.038 282.0839 283.0913 98.71 C10H13N5O5 Guanosine
14 5.264 477.1609 478.1681 98.24 C20H30O13 Kelampayoside A
15 5.904 329.0876 330.0948 84.7 C14H18O9 3′-Glucosyl-2′,4′,6′-trihydroxyacetophenone
16 6.093 359.0981 360.1055 97.46 C15H20O10 6′-Methoxypolygoacetophenoside
17 6.168 461.1666 462.1738 99.21 C20H30O12 Verbasoside
18 6.243 167.0348 168.0421 99.62 C8H8O4 Dihydroxyphenylacetic acid
19 6.268 343.1027 344.1102 97.17 C15H20O9 4′,6′-Dihydroxy-2′-methoxyacetophenone 6′-glucoside
20 6.344 341.0875 342.0947 82.32 C15H18O9 Glucocaffeic acid
21 6.368 403.1241 404.1315 97.32 C17H24O11 Oleoside 11-methyl ester
22 6.469 179.035 180.0416 60.28 C9H8O4 Caffeic Acid
23 6.871 513.2184 514.2256 98.64 C21H38O14 2-O-(beta-D-galactopyranosyl-(1->6)-beta-D-galactopyranosyl) 2S,3R-dihydroxynonanoic acid
24 6.921 431.1556 432.1629 99.13 C19H28O11 Benzyl gentiobioside
25 6.971 387.1293 388.1365 98.02 C17H24O10 Geniposide
26 6.984 326.0886 327.0955 94.43 C14H17NO8 Blepharin
27 7.047 457.1356 458.1427 98.51 C20H26O12 7-Hydroxy-4-methylphthalide O-[arabinosyl-(1->6)-glucoside]
28 7.122 593.1515 594.1587 98.85 C27H30O15 Saponarin
29 7.222 293.124 294.1313 99.71 C12H22O8 Ethyl 3-O-beta-D-glucopyranosyl-butanoate
30 7.272 785.2499 786.257 97.39 C35H46O20 Echinacoside
31 7.461 639.193 640.2006 93.47 C29H36O16 beta-Hydroxyacteoside
32 7.511 563.1404 564.1476 97.59 C26H28O14 Luteolin 3′-methyl ether 7,4′-dixyloside
33 7.524 137.0247 138.0319 99.35 C7H6O3 2,5-Dihydroxybenzaldehyde
34 7.649 327.1085 328.1158 99.26 C15H20O8 Dihydromelilotoside
35 7.787 581.224 582.2314 97.41 C28H38O13 (+)-Lyoniresinol 9-glucoside
36 7.925 383.0619 384.0691 99.1 C16H16O11 2-O-Feruloylhydroxycitric acid
37 7.95 621.1096 622.1166 98.47 C27H26O17 Genistein 4′,7-O-diglucuronide
38 7.975 353.0514 354.0586 99.36 C15H14O10 2-O-p-Coumaroylhydroxycitric acid
39 8.051 337.0925 338.0999 99.16 C16H18O8 Hydrojuglone glucoside
40 8.377 551.2125 552.2198 97.21 C27 H36O12 Prupaside
41 8.427 623.1992 624.2061 98.06 C29H36O15 Isoacteoside
42 8.628 637.2133 638.2204 97.93 C30H38O15 4′-Hydroxy-5,7,2′-trimethoxyflavanone 4′-rhamnosyl-(1->6)-glucoside
43 9.055 563.1037 564.1109 98.35 C25H24O15 Larycitrin 3-(4″-malonylrhamnoside)
44 9.18 533.093 534.1001 95.67 C24H22O14 2′-Hydroxygenistein 7-(6″-malonylglucoside)
45 9.256 665.2085 666.2156 98.23 C31H38O16 Tetramethylquercetin 3-rutinoside
46 9.381 503.0817 504.089 92.63 C23H20O13 Gomphrenol 3-methylether 4′-glucuronide
47 9.557 393.1545 394.1624 88.04 C20H26O8 Gibberellin A43
48 9.633 431.134 432.1416 86.12 C22H24O9 4′-O-Methylglucoliquiritigenin
49 9.833 144.0455 145.0528 87.81 C9H7NO 4-formyl Indole
50 9.934 291.0871 292.0943 85.16 C15H16O6 trans-Grandmarin
51 10.197 395.2066 396.214 94.7 C21H32O7 Isopetasoside
52 10.335 275.0919 276.0993 97.91 C15H16O5 5-De-O-methyltoddanol
53 10.36 213.0917 214.099 98.96 C14H14O2 Ethyl 1-naphthylacetic acid
54 10.436 285.0403 286.0476 99.79 C15H10O6 Luteolin
55 10.586 721.2332 722.2405 43.96 C34 H42O17 Amorphigenol O-vicianoside
56 10.687 135.0815 136.0889 94.23 C9H12O 2-(1-Pentenyl)furan
57 11.038 207.0662 208.0734 99.66 C11H12O4 5-(3′,5′-Dihydroxyphenyl)-gamma-valerolactone
58 11.314 269.0454 270.0527 98.57 C15H10O5 Apigenin
59 11.402 299.0558 300.0631 97.59 C16H12O6 Diosmetin
60 11.515 619.1449 620.1521 96.73 C32H28O13 Apigenin 7-(3″-acetyl-6″-E-p-coumaroylglucoside)
61 12.569 375.1447 376.1519 83.09 C20H24O7 alpha-Peroxyachifolide
62 12.946 223.1338 224.1411 99.62 C13H20O3 Methyl jasmonate
63 13.096 381.0973 382.1046 97.02 C21H18O7 Mollicellin B
64 13.247 227.0709 228.0782 98.97 C14H12O3 3,4′,5-Trihydroxystilbene
65 13.373 283.0606 284.0679 84.78 C16 H12O5 Genkwanin
66 13.398 305.1752 306.1825 84.31 C18H26O4 Capsiate
67 13.448 309.2069 310.2141 84.83 C18H30O4 Auxin b
68 13.448 293.1755 294.1828 95.67 C17H26O4 Myrsinone
69 14.076 273.0764 274.0838 96.81 C15H14O5 6′-Hydroxy-O-desmethylangolensin
70 14.565 307.1912 308.1984 84.43 C18H28O4 Dihydrocapsiate
71 15.08 487.3423 488.3494 95.88 C30H48O5 21beta-Hydroxyhederagenin
72 15.959 423.1805 424.1877 97.88 C25H28O6 1,7-Dihydroxy-3,6-dimethoxy-2,8-diprenylxanthone
73 16.737 407.1858 408.193 98.62 C25H28O5 1-Hydroxy-3,5-dimethoxy-2,4-diprenylxanthone
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The several flavonoids and flavonoid glycosides tentatively identified in AEL-sed included luteolin (Rt: 10.478 min), apigenin (Rt: 11.281 min), diosmetin (Rt: 11.432 min), saponarin (Rt: 7.114 min), scutellarein 7-glucuronosyl-(1->2)-glucuronide (Rt: 7.415 min), luteolin 3′-methyl ether 7,4′-dixyloside (Rt: 7.54 min), genistein 4′,7-O-diglucuronide (Rt: 7.917 min), baicalin (Rt: 8.884 min), diosmetin 7-O-beta-D-glucuronopyranoside (Rt: 8.971 min), tetramethylquercetin 3-rutinoside (Rt: 9.147 min) and vestitone 7-glucoside (Rt: 8.386 min). Other flavonoids, including 4′-O-methylglucoliquiritigenin, luteolin 7,3′-dimethyl ether 5-glucoside, and apigenin 7-(3″-acetyl-6″-E-p-coumaroylglucoside were also identified in AEL-sed. Aside from the polyphenolic compounds identified in AEL-sed, the UPLC-QTOF-MS data revealed the presence of several other classes of compounds, such as nucleosides (adenine guanosine and 8-hydroxyadenine), alkaloids (calystegine B5, evoxanthidine and dihydrocapsiate), coumarins (aesculin, esculetin, and osmanthuside B), and quassinoids (sergeolide).

Table 4 summarizes the tentative chemical constituents identified in AEWP-sed. Likewise, the retention times, absorbance spectra, and the data of MS were used to determine the tentative chemical composition of AEWP-sed. The results also suggested that AEWP possesses several classes of compounds, including alkaloids, terpenoids, flavonoids, and phenols, similarly to AEL-sed. The phytochemical profile of AEWP-sed looked similar to that of AEL-sed. However, the compounds such as quinic acid (Rt: 2.302), citbismine C (Rt: 2.653 min), 3′-glucosyl-2′,4′,6′-trihydroxyacetophenone (Rt: 5.904 min), geniposide (Rt: 6.971 min), 7-hydroxy-4-methylphthalide O-[arabinosyl-(1->6)-glucoside] (Rt: 7.047 min), echinacoside (Rt: 7.272 min), dihydromelilotoside (Rt: 7.649 min), (+)-lyoniresinol 9-glucoside (Rt: 7.787 min), prupaside (Rt: 8.377 min), larycitrin 3-(4″-malonylrhamnoside) (Rt: 9.055 min), 2′-hydroxygenistein 7-(6″-malonylglucoside) (Rt: 9.18 min), gomphrenol 3-methylether 4′-glucuronide (Rt: 9.381 min), trans-grandmarin (Rt: 9.934 min), isopetasoside (Rt: 10.197 min), amorphigenol O-vicianoside (Rt: 10.586 min), alpha-peroxyachifolide (Rt: 12.569 min), mollicellin B (Rt: 13.096 min), 3,4′,5-trihydroxystilbene (Rt: 13.247 min), genkwanin (13.373 min), capsiate (Rt: 13.398 min), 6′-hydroxy-O-desmethylangolensin (Rt: 14.076 min) and 21-beta-hydroxyhederagenin (Rt: 15.08 min) identified in the QTOF-MS data of AEWP-sed, were absent in AEL-sed.

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 (IC50: 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.

Several bioactive molecules were detected in the A. ebracteatus extracts, including baicalin, apigenin, luteolin, glucocaffeic acid, caffeic acid, aesculin, diosmetin, genkwanin, saponarin, and hydrojuglone glucoside. These identified compounds could be responsible for the observed antioxidant properties since previous reports illustrated the antioxidant potential of these compounds through several mechanism in in vitro and in vivo models [12,14,15,16]. Prasansuklab and Tencomnao [6] reported the antioxidant potential of A. ebracteatus extract and suggested that its protective effects against oxidative stress injury were attributed to the presence of polyphenolic compounds in the extract (verbasoside, leucosceptoside A, isoverbascoside, and Vicenin-2). Furthermore, Ilori et al. [17] noted that polyphenolic compounds, such as verbascoside, leucosceptoside A, martynoside, β-hydroxyacteoside, pteleifoside G, magnolenin C, vecenin-2, shaftoside, luteolin-7-O-β-d glucuronide, and apigenin, which have several reported therapeutic activities such as antimicrobial, anticancer, wound healing, anti-inflammatory, anti-hair loss, and antioxidant properties, were reported to be present in A. ebracteatus [8,17].

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.

4. Materials and Methods

4.1. Chemicals and Reagents

Dimethyl sulfoxide and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Corp., (St. Louis, MO, USA). Fetal bovine serum was procured from Biochrom GmbH (Berlin, Germany). RPMI-1640, phosphate buffer saline, and penicillin/streptomycin (U/mL) were purchased from PAA Laboratories GmbH (Pasching, Austria). 2′,7′-dichlorodihydrofluorescein diacetate, 0.25 % trypsin-EDTA, and stable L-glutamine were purchased from Gibco Life Technologies (Carlsbad, CA, USA). Phenotype oral squamous carcinoma cells (CLS-354/WT) were obtained from the Research Institute for Health Sciences, School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat, Thailand. All other chemicals used were of analytical grade and used as purchased.

4.2. 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.

4.3. Preparation of A. ebracteatus Extracts

4.3.1. 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.

4.3.2. 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].

4.4. 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 Na2CO3 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% NaNO2 solution, and 60 µL of 10% AlCl3 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.

4.5. Antioxidant Activity

Measurements of the DPPH radical-scavenging activity (DPPH-RSA), ABTS radical-scavenging activity (ABTS-RSA), metal chelating activities (MCA), ferric reducing antioxidant power (FRAP), and oxygen radical antioxidant capacity (ORAC) of the extracts were performed using previously reported methods [37,38].

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 FeCl2 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.

4.6. 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].

4.7. 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 ʟ-glutamine. Approximately, 1 × 104 (cells/mL) cells were seeded in 96-well plates and incubated in an incubator with 5% CO2 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.

4.8. 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 x 104 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 ED50 (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).

4.9. 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 introduced a reference solution with a mass mix of m/z 112.985587 (TFA anion) to m/z 1033.988109 (HP-0921) in the ESI-negative mode, while a mass mix of m/z 121.050873 (purine) and m/z 922.009798 (HP-0921) were introduced in the ESI-positive mode. The parameters set for ESI-MS included: drying gas temperature: 325 °C; drying gas flow rate: 13 L minˉ1; nebulizer gas pressure: 35 psig; capillary voltage: 4000 V; fragmentor voltage: 175 V; radiofrequency voltage in the octupole: 750 V, and fixed collision energies of 10.00 eV, 20.00 eV and 40.00 eV. Data acquisition was performed on Mass Hunter Workstation Software Data Acquisition for Q-TOF, version B.08.00 (B8058.3 SP1) and QTOF Firmware, version 20.712.

4.10. 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.

5. 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.

Author Contributions

Conceptualization, O.J.O. and C.O.; methodology, O.O.O., T.J.J. and S.S. (Sudarshan Singh); formal analysis, O.O.O., S.S. (Sudarshan Singh) and O.J.O.; investigation, O.O.O., S.S. (Sudarshan Singh), S.N. and S.S. (Sasikarn Sripetthong); resources, W.C. and C.O.; data curation, O.O.O., S.S. (Sudarshan Singh) and O.J.O.; writing—original draft preparation, S.S. (Sudarshan Singh) and O.J.O.; writing—review and editing, C.O. and O.J.O.; supervision, C.O. and O.J.O.; project administration, C.O. and O.J.O.; funding acquisition, C.O. All authors have read and agreed to the published version of the manuscript.

Funding

The authors are grateful for the grant support from Sura Thani Provincial Health Office (Contract No.361/2564.)

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are not available from the authors.

Footnotes

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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