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Abstract

Objective

The rising crisis of antimicrobial resistance necessitates the discovery of bioactive compounds from natural sources. Plants adapted to extreme environments are promising sources of unique secondary metabolites. This study investigates the chemical composition and antimicrobial potential of Atriplex leucoclada (Amaranthaceae), a halophyte native to the Egyptian desert.

Methods

The dried aerial parts of Atriplex leucoclada were exhaustively extracted with 80% aqueous methanol. The resulting crude extract was suspended in water and successively partitioned with n-hexane and ethyl acetate. The ethyl acetate-soluble fraction was subjected to multi-step chromatographic purification, including flash chromatography and preparative HPLC. The structures of purified metabolites were elucidated through comprehensive spectroscopic analysis, including nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HRESIMS), and by comparison with literature data. The in vitro antibacterial activity of the extract and isolated compounds was evaluated against Bacillus subtilis and Escherichia coli using agar well diffusion and broth microdilution assays.

Results

Phytochemical investigation of A. leucoclada led to the isolation of six compounds (1-6) spanning diverse structural classes: three alkaloids— N-trans-caffeoyltyramine (1), N-trans-feruloyltyramine (2), N-trans-feruloyl methoxytyramine (3); the lignan syringaresinol (4), and the ecdysteroids 20-hydroxyecdysone (5) and 20-hydroxyecdysone-20,22-monoacetonide (6). Antimicrobial screening identified N-trans-feruloyltyramine (2) as the most active compound. The isolated ecdysteroids 5 and 6 are of particular interest as phytoecdysteroids, a class of compounds known for their adaptogenic and proposed anabolic properties.

Conclusion

This study highlights the halophyte Atriplex leucoclada as a promising source of bioactive compounds. The significant antibacterial activity of compound 2 warrants future studies to elucidate its precise mechanism of action, assess its efficacy against several multidrug-resistant pathogens, and evaluate its therapeutic potential.

Keywords

Halophytes lignanamides alkaloids phytocdysteroids

Introduction

Halophytes are plants adapted to survive in high-salinity environments. To endure such harsh conditions, they utilize a range of physiological and molecular adaptations, including the biosynthesis of specific enzymes, osmolytes, and secondary metabolites.¹ The Amaranthaceae family comprises the most abundant halophytes throughout the world. The genus Atriplex, belonging to the Amaranthaceae family, includes approximately 300 species of herbaceous halophytic plants. These species are widely distributed worldwide, particularly in the arid and semi-arid regions of Africa, Asia, and Europe.² Currently, there is growing interest in species of the genus Atriplex due to their rich profile of bioactive compounds. Studies have revealed that Atriplex species exhibit high nutritional value, owing to their high protein content, and thus can be used as alternative cereal grains.³ Pharmacological investigations have demonstrated significant potential across several species; for example, A. semibaccata and A. vestita exhibit antifungal and anti-bronchitis properties, respectively, while A. hortensis is a source of vitamin A and has been used in folk medicine for respiratory, digestive, and urinary tract disorders, as well as for rheumatism due to its analgesic effects. Furthermore, A. halimus has shown efficacy in managing type 2 diabetes (T2DM) and is used in veterinary practices as an anthelmintic. Extracts of A. confertifolia display significant cytotoxicity against human breast cancer cell lines, while A. inflata and A. portulacoides have been reported to contain phytochemicals with fungicidal properties.⁴ These biological activities are attributed to a diverse array of specific metabolites, that have been discovered in Atriplex species, including flavonoids,^2,5 phenolics,^6,7 saponins,⁸ ecdysteroids,⁹ and triterpenes.¹⁰ Consequently, biological screening of Atriplex species has revealed anti-butyrylcholinesterase,² anti-inflammatory and antinociceptive,^2,5 antidiabetic,^8,11 antitumor, antimicrobial,¹² and hepatoprotective properties.¹³

Atriplex leucoclada Bioss., commonly known as orache or raghal, is a perennial halophytic shrub renowned for its high salinity tolerance. Native to the Mediterranean region and the deserts of Egypt and Saudi Arabia, this species holds significant ecological value for soil stabilization and serves as a saline-tolerant livestock feed.¹⁴ Despite the importance of this halophyte species, the detailed chemical composition and biological activities of A. leucoclada remain largely unexplored.

The emergence of antimicrobial resistance, driven by the inappropriate use of antibiotics and its harmful impact on the human microbiota, constitutes a major global health challenge.¹⁵ The World Health Organization (WHO) has recognized antibiotic resistance as one of the most critical threats to humanity in the twenty-first century.¹⁶ Notably, Enterobacteriaceae, such as Escherichia coli, Klebsiella spp., and Enterobacter spp., have emerged as superbugs of the twenty-first century, with associated mortality rates reaching 50%. It is estimated that annual deaths from drug-resistant infections could rise to 10 million by 2050.¹⁷ Escherichia coli, an important member of the family Enterobacteriaceae, is a common gut bacterium and a significant human pathogen, capable of causing a wide range of serious infections such as urinary tract infections (UTIs), septicemia, pneumonia, and meningitis. Critically, over 50% of E. coli isolates in hospitals across five out of six WHO regions are now resistant to essential antibiotics including third-generation cephalosporins, making it a major antimicrobial resistance threat and a priority for global public health.¹⁶ This urgent crisis has accelerated the search for natural products as promising therapeutic alternatives. Owing to their broad spectrum of bioactivity, lower cost, and greater accessibility compared to synthetic pharmaceuticals, plant-derived compounds have become a major focus in developing novel antibacterial strategies.¹⁸ Therefore, as part of our ongoing research on bioactive metabolites from halophytes^,^19–24 this study aimed to characterize the chemical profile of the halophyte Atriplex leucoclada and evaluate the antimicrobial potential of its isolated compounds against two bacterial species including the Gram-negative Escherichia coli and the Gram-positive Bacillus subtilis. As a result, six compounds were isolated from the halophytic herb A. leucoclada and their antimicrobial activity were assessed.

Materials and Methods

Chemicals and Reagents

The extraction and purification processes utilized various solvents, including including n-hexane, dichloromethane (CH₂Cl₂), ethyl acetate (EtOAc), methanol, and acetonitrile, purchased from Wako Pure Chemical Industries (Osaka, Japan). Initial fractionation and purification were performed using a Biotage Selekt system (Uppsala, Sweden) equipped with normal-phase silica gel (Sfär HC, 100 g) and reversed-phase C18 (RP-C18) flash chromatography columns (40 g).

Final purification of the metabolites was achieved using a medium-pressure liquid chromatography system (MPLC Pure C-850 Flash Prep^®, Büchi, Flawil, Switzerland) with ultraviolet (UV) and evaporative light scattering (ELSD) detectors. Preparative separation employed a reversed-phase HPLC column (Inertsil ODS-3, 5 µm, 20 × 250 mm, GL Sciences Inc., Japan). Thin-layer chromatography (TLC) was performed on silica gel 60 F₂₅₄ and RP-C18 plates from Merck (Darmstadt, Germany). Deuterated methanol (CD₃OD) for NMR analysis was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). All other analytical-grade chemicals were purchased from Wako Pure Chemical Industries (Japan).

General Experimental Procedures

Nuclear magnetic resonance (NMR) spectra (¹H: 600 MHz, ¹³C or APT: 150 MHz) were recorded on a Bruker DRX-600 spectrometer (Bruker Daltonics, USA) using deuterated methanol (CD₃OD) as solvent, with tetramethylsilane (TMS, δ 0.0 ppm) as an internal standard. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) data were obtained on an Agilent qTOF-LC-MS instrument (Agilent Technologies, USA). Thin-layer chromatography (TLC) analysis was performed on pre-coated silica gel plates, and compounds were visualized by spraying with 5% H₂SO₄ followed by heating at 110 °C.

Collection of Plant Material

Aerial parts of Atriplex leucoclada Boiss. were collected from desert areas in Maadi, Egypt (Wadi Degla desert land, 29°57′35′′ N, 31°19′44′′ E). The plant was identified by Prof. Ibrahim A. El-Garf, Professor of Botany, Faculty of Science, Cairo University, Egypt. A voucher specimen (ALA-2019-1) was deposited at the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt.

Extraction and Isolation of Secondary Metabolites

The dried aerial parts (powder; 1 kg) was macerated three times (at 25 °C) in 80% aqueous methanol and stirred with mechanical stirrer. The combined extracts were concentrated in vacuo to get 135.6 g of a dark brown residue. The residue was then suspended in distilled H₂O and subjected to extraction with n-hexane, ethyl acetate to yield n-hexane-soluble part (8.0 g), ethyl acetate-soluble part (16.0 g), and aqueous part (111.6 g). The ethyl acetate-soluble part (16.0 g) was partitioned with flash chromatography over a Biotage with Sfär column packed with normal-phase silica HC 100 g and eluted with a gradient mixture of n-hexane-ethyl acetate-methanol (1:0:0-0:0:1) to get 10 subfractions Eth-1 to Eth-10. Subfraction Eth-6 (1.6 g) was subjected to purification over a Flash RP-C18 (40 g) and eluted with H₂O-methanol-0.1% HCO₂H (1:0:0.1-0:1:0.1) with a flow rate 30 mL/min to afford compounds 1 (30.0 mg), 2 (15.6 mg), 3 (10.7 mg), and 4 (8.0 mg). Subfraction Eth-8 (5.3 g) was purified over a RP-C18 (60 g) connected to Biotage system and gradually eluted with H₂O-methanl-0.1% HCO₂H (1:0:0.1-0:1:0.1) with flow rate 35 mL/min to get 11 subfractions. Then, the combined subfractions Eth-8 and 9 (450.0 mg) was purified over a preparative HPLC column and eluted with H₂O-acetonitrile-HCO₂H (1:0:0.1-0:1:0.1) to get compound 5 (12.0 mg) and the remaining fraction was combined and further purified over a preparative HPLC column eluted with H₂O-acetonitrile-HCO₂H (4:6:0.1-6:4:0.1) to get compound 6 (11.5 mg).

Evaluation of Antimicrobial Activity Using the Disc Diffusion Assay

The antimicrobial activity of the total methanolic extract and compounds 1–6 (1000 µg/mL) was assessed using the agar well diffusion method under strictly aseptic conditions. Standard bacterial strains—Bacillus subtilis ATCC 6633 and Escherichia coli ATCC 25922—were cultured on sterile Mueller-Hinton agar (MHA) plates to achieve a uniform lawn. The inoculum was standardized to 1.5 × 10⁸ CFU/mL. Six evenly spaced wells were created in each inoculated plate using a sterile cork borer. Each well was loaded with 100 µL of a test sample. A 20% dimethyl sulfoxide (DMSO) solution served as the negative control, while gentamicin (30 µg/mL) and vancomycin (50 µg/mL) were used as positive controls. All test solutions were prepared in DMSO to ensure solubility and consistency. To facilitate radial diffusion into the agar, the plates were pre-incubated at 4 °C for 30 min before being transferred to 37 °C for 24 h. After incubation, the diameters of the inhibition zones (IZDs) were measured in millimeters (mm) to quantify antimicrobial activity.²⁵

Determination of Minimum Inhibitory Concentrations (MICs) Using Microdilution Method

The antibacterial activity of the tested samples against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 was evaluated using a quantitative microdilution method to determine the minimum inhibitory concentration (MIC).²⁶ Briefly, stock solutions of the extract and compounds 1–6 were prepared in DMSO and serially diluted two-fold in Mueller-Hinton broth (MHB) across a microtiter plate to achieve final concentrations ranging from 1 to 512 µg/mL. Each well was inoculated with a bacterial suspension to a final density of 1.5 × 10⁸ CFU/mL in a total volume of 200 µL, followed by aerobic incubation at 37 °C for 24 h. The MIC was determined visually after adding a resazurin indicator (0.015%, 40 µL); a color change from blue to pink indicated bacterial growth, and the lowest concentration preventing this change was recorded as the MIC.²⁵ The optical density (OD) was determined at 570 nm.

Statistical Analysis

All results in the present study were obtained from three independent biological experiments. The results were expressed as the means ± SD (n = 3). The statistical significance of differences between means was established using one-way analysis of variance (ANOVA) with Duncan's post hoc tests. p values < 0.05 indicate statistical significance.

Results

Identification of Isolated Compounds from A. leucoclada

The dried aerial parts of A. leucoclada were extracted with 80% aqueous methanol. The obtained crude extract was suspended in distilled H₂O, and solvents of different polarities, including n-hexane and ethyl acetate, were used for partitioning the crude methanol extract. Metabolites were purified through several chromatographic procedures to get compounds 1–6. Six compounds were isolated and identified through a stepwise chromatography purification and extensive spectroscopic analyses (Supporting Information: Figures S1–S22). The compounds were identified by comparing their spectral NMR data, including ¹H, ¹³C, and APT NMR, with those reported in the literature (Figure 1). The identified compounds were categorized into various chemical classes, including alkaloids—N-trans-caffeoyltyramine (1),²² N-trans-feruloyltyramine (2)^,²² N-trans-feruloyl methoxytyramine (3)²²; the lignan syringaresinol (4),²⁷ ecdysteroids 20-hydroxyecdysone (5),²⁸ and 20-hydroxyecdysone-20,22-monoacetonide (6).

Figure 1.

Structures of the isolated compounds from A. leucoclada.

N-trans-caffeoyltyramine (1) was purified as a white amorphous powder. The molecular formula of 1 was established as C₁₇H₁₇NO₄ from the HRESIMS [M−H]⁻ ion peak at m/z 298.1094 (calcd for C₁₇H₁₆NO₄⁻, 298.1079) and was elucidated from the ¹H and APT NMR analyses (Table 1). The ¹H NMR data of 1 indicated an α,β-unsaturated ketone moiety with (E)-configuration deduced from doublet signals resonated at δ 7.40, d (J = 15.7 Hz) and 6.35, d (J = 15.7 Hz). Besides, one set of ABX pattern revealed a trisubstituted benzene deduced from the chemical shifts at δ 7.02, d (J = 2.1 Hz), 6.79, d (J = 8.1 Hz) and 6.91, dd (J = 8.1, 2.1 Hz) that were assigned for H-2, H-5, and H-6, respectively. On the other hand, one set of AA′-BB′ pattern indicated a disubstituted aromatic ring resonated at δ 7.06, d (J = 8.6 Hz) and 6.74, d (J = 8.6 Hz) that were assigned as H-2′/6′ and H-3′/5′. Furthermore, two signals resonated in the aliphatic region at δ 2.76, t (J = 7.4 Hz) and 3.47, t (J = 7.4 Hz) indicated two methylenes of the tyramine moiety. The APT NMR spectral data of 1 revealed 17 carbon atoms, comprising one carbonyl, two CH₂, nine CH, and five quaternary carbon atoms. From the APT NMR data, signal resonated at δ 169.3 (C-9) revealed an amide moiety. While signals resonated at δ 142.2 (C-7) and 118.4 (C-8) indicated an olefinic bond in the structure. Moreover, a trisubstituted benzene ring was confirmed by signals resonated at δ 128.3, 115.1, 148.7, 146.7, 116.5 and 122.1 that were assigned for C-1, C-2, C-3, C-4, C-5 and C-6, respectively, whereas signals at δ 131.3 (C-1′), 130.7 (C-2′/C-6′), 116.3 (C-3′/C-5′), 156.4 (C-4′), indicated a disubstituted benzene ring. In addition, two aliphatic carbons of the tyramine moiety were determined from two signals at δ 35.8 and 42.5. These data confirmed that 1 was identified as N-trans-caffeoyl tyramine.

Table 1.

¹H NMR Spectral Data of Compounds 1–4 (CD₃OD).

	1		2		3		4
Position	¹H (J in Hz)	APT	¹H (J in Hz)	APT	¹H (J in Hz)	¹³C	¹H (J in Hz)	¹³C
1	—	128.3	—	128.3	—	127.6		131.7
2	7.02 d (2.1)	115.1	7.13 d (2.1)	111.5	7.09 d (1.9)	111.4	6.69 s	103.1
3	—	148.7	—	149.8	—	149.6	—	148.0
4	—	146.7	—	149.3	—	151.0	—	134.9
5	6.79 d (8.1)	116.5	6.81 d (8.1)	116.5	6.77 d (8.1)	116.7	—	148.0
6	6.91 dd (8.1, 2.1)	122.1	7.03 dd (8.1, 2.1)	123.2	7.00 dd (1.9, 8.3)	123.4	6.69 s	103.1
7	7.40 d (15.7)	142.2	7.45 d (15.7)	142.0	7.43 d (15.7)	142.2	4.75 d (4.9)	86.2
8	6.35 d (15.7)	118.4	6.42 d (15.7)	118.7	6.39 d (15.7)	118.3	3.16 m	54.1
9	—	169.3	—	169.2	—	169.3	4.29 m3.91 m	71.4
1′	—	131.3	—	131.3	—	132.0		131.7
2′	7.06 d (8.6)	130.7	7.07 d (8.7)	130.7	6.81 d (1.9)	113.4	6.69 s	103.1
3′	6.74 d (8.6)	116.3	6.74 d (8.7)	116.3	—	144.0	—	148.0
4′	—	156.4	—	156.9	—	146.1	—	134.9
5′	6.74 d (8.6)	116.3	6.74 d (8.7)	116.3	6.72 d (8.0)	116.2	—	148.0
6′	7.06 d (8.6)	130.7	7.07 d (8.7)	130.7	6.66 dd (8.0, 2.0)	122.3	6.69 s	103.1
7′	2.76 t (7.4)	35.8	2.77 t (7.3)	35.8	2.76 t (7.3)	36.2	4.75 d (4.9)	86.2
8′	3.47 t (7.4)	42.5	3.48 t (7.3)	42.5	3.48 t (7.3)	42.5	3.16 m	54.1
9′	—	—	—	—	—	—	4.29 m3.91 m	71.4
3-OMe	—	—	3.89 s	56.4	3.86 s	56.3	3.87 s	55.4
3′-OMe	—	—	—	—	3.82 s	56.3	3.87 s	55.4
5-OMe	—	—	—	—	—	—	3.87 s	55.4
5′-OMe	—	—	—	—	—	—	3.87 s	55.4

N-trans-feruloyl tyramine (2) was determined to be C₁₈H₁₉NO₄ as deduced from a pseudo molecular ion peak at m/z 312.1244 [M−H]⁻ (calcd for C₁₈H₁₈NO₄⁻, 312.1236). The structure analyses of 2 were compared to those of 1, and the HR-MS data indicated an additional methyl group in 2. The ¹H NMR of 2 revealed two sets of aromatic protons, including one ABX system δ 7.13 d (J = 2.1 Hz), 6.81 d (J = 8.1 Hz) and 7.03 dd (J = 8.1, 2.1 Hz) and one AA′-BB′ system δ 7.07 d (J = 8.7 Hz) and 6.74 d (J = 8.7 Hz), suggesting a trisubstituted and disubstituted aromatic rings. Additionally, two signals at δ 7.45 d (J = 15.7 Hz) and 6.42 d (J = 15.7 Hz) indicated olefinic protons H-7 and H-8. The presence of singlet signal (integrating for three protons) resonated at δ 3.89 indicated an aromatic methoxy group in 2. The APT NMR spectral data of 2 revealed 18 carbon atoms, comprising one carbonyl, one -OCH₃, two -CH₂, nine -CH, and five quaternary carbons. Carbon signals at δ 142.2 (C-7), 118.4 (C-8), 169.3 (C-9), 56.4, (3-OCH₃) further confirmed the structure. From the ¹H and APT NMR data, compound 2 was identical to those of 1 in having a phenylpropanoid moiety and a tyramine moiety, except for the existence of a methoxy group in 2. Thus, 2 was identified as N-trans-feruloyl tyramine. The complete data for 2 is seen in Table 1.

The molecular formula of N-trans-feruloyl-3-methoxytyramine (3) was characterized to be C₁₉H₂₁NO₅ based on the HRESIMS at m/z 342.1352 [M−H]⁻ and was further confirmed from the ¹H and ¹³C NMR data as seen in Table 1. The ¹H NMR data of 3 revealed an α,β-unsaturated ketone moiety with trans-configuration δ 7.43 d (J = 15.7 Hz) and 6.39 d (J = 15.7 Hz). Additionally, a trisubstituted benzene ring was determined from the chemical shifts at δ 7.09 d (J = 1.9 Hz, H-2), 6.77 d (J = 8.1 Hz, H-5), and 7.00 dd (J = 1.9, 8.3 Hz, H-6). While signals resonated at δ 6.81 d (J = 1.9 Hz), 6.72 d (J = 8.0 Hz), and 6.66 dd (J = 8.0, 2.0 Hz) that were assigned for H-2′, H-5′, and H-6′, respectively, indicated an additional trisubstituted benzene moiety. The presence of two methoxy groups in 3 evidenced by signals at δ 3.86 (3-OMe) and 3.82 (3′-OMe). Signals resonating at δ 2.76 t (J = 7.3 Hz) and 3.48 t (J = 7.3 Hz) indicated two methylenes in the structure. The ¹³C NMR spectral data of 3 revealed 19 carbon atoms, comprising two OCH₃, two CH₂, eight CH, one carbonyl, and six quaternary carbons. An amide carbon atom was determined from the downfield shift at δ 169.3, which was assigned for C-9. Finally, the structure of 3 was identified as N-trans-feruloyl-3-methoxytyramine.

Compound 4 was obtained as a white amorphous powder. The ¹H and ¹³C NMR spectral data was found similar to those published in literature for syringaresinol²⁷ as seen in Table 1.

Compound 5 was obtained as a white amorphous powder. Its molecular formula was estimated as C₂₇H₄₄O₇ from the positive ion HRESIMS with pseudo molecular ion peaks [M + H]⁺ at m/z 481.3166 and [M + Na]⁺ at m/z 503.2988 (calcd for C₂₇H₄₄O₇Na, 503.2985). The ¹H NMR spectrum of 5 revealed the presence of five tertiary methyl resonances at δ 0.90 (s), 0.98 (s), 1.21 (s), 1.22 (s) and 1.23 (s) for protons at C-18, C-19, C-21, C-26 and C-27, respectively. Additionally, an olefinic proton was characterized by signal at δ 5.84 (d, J = 2.7 Hz, H-7), whereas three oxymethine protons were determined at δ 3.87 (ddd, J = 12.1, 1.7, 4.5 Hz, H-2), 3.98 (br d, H-3) and 3.35 (br d, J = 10.6, H-22). Signals resonated at δ 2.39 (dd, J = 13.1, 4.6 Hz) and 2.41 (m) were assigned for H-5 and H-17, respectively. The APT NMR spectrum revealed 27 carbon atoms, including a carbonyl (δ_C 206.5, C-6), five methyls (δ_C 18.0, 21.0, 24.4, 28.9, and 29.7), eight methylenes, seven methines, and six quaternary carbons. The downfield signals at δ_C 206.5 (C-6), 168.0 (C-8) and 122.1 (C-7), three oxygenated methine signals at δ_C 68.7 (C-2), 68.5 (C-3), 78.4 (C-22) and three oxygen-substituted quaternary carbon at δ_C 71.3 (C-25), 77.9 (C-20) and 85.2 (C-14), suggesting that compound 5 was an ecdysteroid with a double bond between C-7/C-8, as reported in the literature.^29–31 The HMBC spectrum revealed significant correlations between H-4/C-6, H-9/C-7 and H-17/C20 and C-22. The full assignment of 2D-NMR including HSQC and HMBC experiments revealed that 5 is 20-hydroxyecdysone. The complete data of compound 5 is summarized in Table 2.

Table 2.

¹H NMR Spectral Data of Compounds 5–6 (CD₃OD).

Position	5		6
Position	¹H (J in Hz)	¹³C	¹H (J in Hz)	¹³C
1	1.80 dd (4.1, 9.4), 1.43 dd (13.8, 6.8)	37.3	1.46 m1.73 m	37.1
2	3.87 ddd (12.1, 1.7, 4.5)	68.7	3.85 ddd (12.1, 1.7, 4.5)	68.7
3	3.98 br d	68.5	3.97 br d (3.1)	68.5
4	1.74 m	32.8	1.74 m	32.8
5	2.39 dd (4.6, 13.1)	51.7	2.40 dd (12.8, 4.5)	51.8
6	−	206.5	−	206.5
7	5.84 d (2.7)	122.1	5.83 d (2.7)	122.1
8	−	168.0	−	167.6
9	3.17 m	35.0	3.16 m	35.1
10	−	39.2	−	39.2
11	1.80 m1.72 m	21.4	1.87 m	22.4
12	2.15 ddd (13.1, 4.9)1.90 br dd (13.0, 3.2)	32.4	2.13 ddd (12.9, 12.8, 4.7)1.86 m	32.3
13	−	48.6	−	48.4
14	−	85.2	−	85.3
15	1.98 m1.61 m	31.7	1.95 m1.62 m	31.7
16	1.75 m1.97 m	21.4	1.81 m1.70 m	21.5
17	2.41 m	50.5	2.33 t (9.0)	50.7
18	0.90 s	18.0	0.84 s	17.7
19	0.98 s	24.4	0.98 s	24.4
20	−	77.9		85.8
21	1.21 s	21.0	1.20 s	22.6
22	3.35 br d (10.6)	78.4	3.70 dd (8.3, 3.9)	83.3
23	1.31 m1.66 m	27.3	1.53 m	24.7
24	1.79 m1.43 m	42.3	1.47 m1.72 m	42.2
25	−	71.3	−	71.1
26	1.22 s	28.9	1.22 s	28.9
27	1.23 s	29.7	1.23 s	29.4
Ketal group
1'	−	−	–	107.9
2'	−	−	1.34 s	27.2
3'	−	−	1.41 s	29.3

Compound 6, 20-hydroxyecdysone-20,22-monoacetonide, was purified from the ethyl acetate-soluble fraction and was identified by comparing its HRESIMS, ¹H NMR and APT NMR data with those reported in the literature.³² The molecular formula of 6 was deduced to be C₃₀H₄₈O₇ from the HRESIMS data based on the [M + H]⁺ ion at m/z 521.3470 (calcd for C₃₀H₄₉O₇⁺, 521.3478). The ¹H and APT NMR data are summarized in Table 2. The NMR spectral data demonstrated that 6 possesses a similar structure with 5 with an additional 20,22-acetonide moiety. The ¹H and APT NMR resonances (Table 2) indicated an ecdysteroid skeleton with a double bond at C-7/C-8 and carbonyl function group at C-6 (δ 206.5). The presence of an acetonide functionality (ketal group) was characterized by signals C-1′ (δ 107.9) and C-2′ (δ 1.34/δ 27.2) and C-3′ (δ 1.41/δ 29.3) at C-20 and C-22 in 6. As seen in Table 2, compound 6 was thus identified as 20-hydroxyecdysone-20,22-monoacetonide.

Importantly, the isolated compounds 5 and 6 belong to phytoecdysteroids which are natural polyhydroxylated compounds with a four-ring skeleton, typically containing 27–29 carbon atoms derived from plant sterols. While their physiological role in plants remains unexplored and their distribution is rare, they accumulate to high concentrations in several species, including some common vegetables. Moreover, ecdysteroids exhibit several pharmacological properties, such as hepatoprotective, hypoglycemic, and non-androgenic anabolic effects. Additionally, they enhance vitality and physical performance, and they are classified as adaptogens that enhance stress resistance.³³

Antimicrobial Activity of Metabolites from A. leucoclada

In the current study, the isolated compounds were evaluated for their antimicrobial activity (Figures 2 and 3). Importantly, the total methanolic extract of A. leucoclada at a concentration of 1 mg/mL showed antimicrobial activity against tested bacterial standard strains, B. subtilis and E. coli displaying clear IZDs of 16 and 14 mm, respectively. Among the tested isolates, compound 2 demonstrated antimicrobial activity against B. subtilis and E. coli with clear IZDs 20 and 18 mm, in comparison to vancomycin (50 µg/mL) and gentamicin (30 µg/mL). Meanwhile, the rest of compounds showed IZDs ranged between 14 to 16 mm against both bacterial strains as seen in Table 3.

Figure 2.

Evaluation of antibacterial effects of total methanolic extract and isolates 1–6. (A) and (B) Different isolated compounds 1–6, total methanolic extract, vancomycin, and gentamicin were assessed for Antibacterial Effect using the Well Diffusion assay. (C) and (D) Bar Graph Showing the Zone of Inhibition (in mm) for the Tested Samples against B. subtilis and E. coli, respectively. The experiment was conducted in triplicate (n = 3).

Figure 3.

Antimicrobial activity of tested samples assessed by broth microdilution assay. (A) Activity against Bacillus subtilis ATCC 6633. Wells 1–6: serial dilutions of purified compounds; T: total methanolic extract of A. leucoclada; V: vancomycin (positive control). (B) Activity against Escherichia coli ATCC 25922. Wells 1–6: serial dilutions of purified compounds; T: total methanolic extract of A. leucoclada; G: gentamicin (positive control).

Table 3.

The Inhibition Zone Diameters (IZDs) and Minimum Inhibitory Concentrations (MICs) of A. leucoclada Total Methanolic Extract and Isolates 1–6.

Sample	Inhibition Zone Diameter (IZD, mm)		Minimum Inhibitory Concentration (MIC, µg/mL)
Sample	B. subtilis	E. coli	B. subtilis	E. coli
1	17 ± 0.57	15 ± 0.0	128	256
2	20 ± 0.0	18 ± 0.57	64	128
3	15 ± 0.57	14 ± 0.57	256	256
4	14 ± 0.0	16 ± 0.57	256	256
5	16 ± 0.0	17 ± 0.57	128	128
6	14 ± 0.0	16 ± 0.0.0	256	128
Total extract	15 ± 0.57	14 ± 0.0	256	256
Gentamicin	-	20 ± 0.57	-	32
Vancomycin	25 ± 1.15	-	32	-

Moreover, using the broth microdilution method, the minimum inhibitory concentrations required to inhibit bacterial growth (MIC) against B. subtilis and E. coli were determined. Among the tested samples, the total methanolic extract obtained from A. leucoclada displayed the same MIC value 256 µg/mL against B. subtilis and E. coli. Furthermore, compound 2 demonstrated a promising antimicrobial activity against B. subtilis with lowest MIC 64 µg/mL, compared to vancomycin with MIC 32 µg/mL. Table 3 presents the antimicrobial activity and MICs of all tested compounds. The results of the antimicrobial tests are shown in Figure 2 (disc diffusion) and Figure 3 (MIC determination).

Discussion

Natural products derived from plants are a crucial foundation of human medicine. Bioactive compounds from natural sources—including alkaloids, flavonoids, lignanamides, and ecdysteroids—possess diverse pharmacological activities. Within the Amaranthaceae family, numerous halophytic species are recognized as rich reservoirs of such compounds, holding considerable potential for various applications. Extracts from Amaranthaceae plants frequently demonstrate significant antimicrobial, neuroprotective, anticancer, and anti-inflammatory properties.¹

Previous studies on various Atriplex species (Amaranthaceae) report diverse metabolites, however, the chemical profile of A. leucoclada is poorly investigated. A previous study by Hayam S. Ahmed and colleagues analyzed the composition of defatted methanol and n-hexane extracts obtained from A. leucoclada. The results revealed the isolation of chemical compounds including 20-hydroxy ecdysone, phytol, β-sitosterol, stigmasterol, palmitic acid, luteolin, β-sitosterol-3-O-β-D-glucopyranoside, pallidol and isorhamnetin 3-O-β-galactopyranoside.²⁸ Moreover, the anti-inflammatory activity of the n-hexane-soluble part of A. leucoclada might be associated with the combined effect of 2-hexadecen-1-ol and hexadecanoic acid methyl ester, as well as ursodeoxycholic acid.²⁸ Our study aimed to expand the chemical profile of A. leucoclada and evaluate the antimicrobial activity of its isolated constituents. Hence, our investigation led to the identification of six compounds including lignanamides N-trans-caffeoyltyramine, N-trans-feruloyltyramine, and N-trans-feruloylmethoxytyramine; lignan syringaresinol; and ecdysteroids 20-hydroxyecdysone and 20-hydroxyecdysone-20,22-monoacetonide. Notably, lignanamide derivatives, syringaresinol, and 20-hydroxyecdysone-20,22-monoacetonide are reported from this species for the first time.

The biosynthesis of ecdysteroids is fundamentally controlled by sterol availability. Insects and arthropods cannot synthesize sterols de novo and must acquire C₂₇ or C₂₉ sterols from their diet, subsequently converting them into C₂₇ precursors required for ecdysteroid production. Conversely, plants have sterol biosynthesis pathway, mainly via cytosolic mevalonic acid (MVA) pathway, and can produce phytoecdysteroids (PEs) from C₂₇ sterol precursors such as cholesterol or lathosterol. Importantly, using acetyl-CoA, MVA pathway is considered the primary source of isoprenoid precursors for sterols’ production.³³ The biosynthesis of PEs 5 and 6, as summarized in Figure 4, initiates from sterol precursors generated via the mevalonic acid (MVA) pathway. This pathway produces six activated isoprene units, which undergo condensation to form squalene. Squalene is subsequently epoxidized and cyclized to yield Δ²⁴-cycloartenol, which is then enzymatically processed to form the key C₂₇ sterol precursors: either Δ⁵-cholesterol or Δ⁷-lathosterol. While many plants utilize cholesterol as the primary precursor for PEs, studies in spinach and other chenopods have identified lathosterol as the main precursor. The biosynthesis proceeds via the oxidation of lathosterol or cholesterol to form ecdysone. Ecdysone is then hydroxylated by ecdysone 20-monooxygenase to yield 20-hydroxyecdysone (5). Finally, 5 undergoes further oxidation and cyclization to form compound 6.

Figure 4.

Proposed biosynthesis of compounds 5 and 6 isolated from A. leucoclada.

Halophytes are a rich source of antimicrobial metabolites, demonstrating efficacy against both Gram-positive and Gram-negative pathogens. Bioactive constituents such as phenolics and flavonoids underlie their ability to inhibit the growth of bacteria including Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa, as evidenced by studies on some species.³⁴ Given the significance of bioactive compounds in plants that combat microbial infections, the evaluation of antimicrobial activities has become a key focus in phytochemical research. In the present study, the total methanolic extract of A. leucoclada (1 mg/mL) and compound 2 (N-trans-feruloyltyramine) demonstrated antimicrobial activity against B. subtilis and E. coli. Additionally, the total methanolic extract exhibited a MIC of 256 µg/mL, while 2 demonstrated promising antimicrobial activity against B. subtilis with the lowest MIC of 64 µg/mL.

The novelty of this study lies in the detailed chemical investigation of Atriplex leucoclada, which led to the isolation of six metabolites and the evaluation of their in vitro antimicrobial activity. The chemical findings underscore the potential impact of environmental conditions on the plant's biosynthetic pathways. However, the exact mechanisms of action for the observed bioactivity remain unknown. Future work should therefore focus on elucidating these mechanisms, expanding the scope of pharmacological testing, and evaluating the clinical potential of this halophyte species and its metabolites. Specifically, biological evaluation against multidrug-resistant (MDR) strains and assays against diverse therapeutic targets are essential to fully reveal its potential as a source of novel therapeutic agents.

Conclusion

This study provides a phytochemical and biological investigation of the halophyte A. leucoclada, contributing to the understanding of its chemical diversity and therapeutic potential. Six compounds were successfully isolated and characterized from the ethyl acetate-soluble fraction of the methanolic extract. The identified metabolites, encompassing the alkaloids 1–3, lignan 4, and ecdysteroids 5 and 6, confirm the presence of diverse and biologically relevant secondary metabolite classes in this species. Antimicrobial evaluation revealed the antibacterial activity of A. leucoclada against Bacillus subtilis and Escherichia coli. Among the isolated compounds, N-trans-feruloyltyramine (2) emerged as the most promising antimicrobial agent, demonstrating the most significant inhibition zone and the lowest minimum inhibitory concentration (MIC) against B. subtilis.

In summary, this research validates A. leucoclada as a source of bioactive agents for further investigation. The significant antibacterial activity of compound 2 warrants future studies to elucidate its precise mechanism of action, assess its efficacy against a broader panel of multidrug-resistant pathogens, and evaluate its therapeutic potential. Furthermore, the rich chemical profile revealed in this study suggests that A. leucoclada may hold additional, yet unexplored, pharmacological properties.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X261427064 - Supplemental material for Chemical Constituents and Antimicrobial Activity of the Halophyte Atriplex leucoclada

Supplemental material, sj-docx-1-npx-10.1177_1934578X261427064 for Chemical Constituents and Antimicrobial Activity of the Halophyte Atriplex leucoclada by Ahmed Othman, Fahd M. Abdelkarem, Yhiya Amen, Mohammed S. Abdulrahman and Kuniyoshi Shimizu in Natural Product Communications

Footnotes

Acknowledgements

The authors are indebted to Prof. Ibrahim A. El-Garf, Department of Botany, Faculty of Science, Cairo University, Egypt, for the identification of plant.

ORCID iDs

Ahmed Othman

Fahd M. Abdelkarem

Yhiya Amen

Kuniyoshi Shimizu

Ethical Approval

Ethical Approval is not applicable to this article.

Author Contributions

Ahmed Othman: Conceptualization, Methodology, Analysis of spectral data, Writing – Original Draft; Fahd M. Abdelkarem: Analysis of spectral data, Writing – Original Draft; Yhiya Amen: Methodology, Writing – review and editing; Mohammed S. Abdulrahman: Biological investigation, Writing – Original Draft; Kuniyoshi Shimizu: Supervision, Writing – review and editing.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability

All data generated or analyzed during this study are included in this published article.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Supplemental Material

Supplemental material for this article is available online ().

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