Antimicrobial and Molecular Docking Studies of Anthrones from the Leaf Latex of Aloe pirottae A. Berger against Some Plant Pathogenic Microbes

Abstract

Objective/Background: Aloe pirottae A. Berger has traditionally been used in Ethiopia to treat various ailments. While previous studies have examined the antimicrobial activity of different parts of A. pirottae against human pathogens, this study aimed to investigate whether its antimicrobial activity extends to plant pathogenic bacteria and fungi. Methods: Compounds were isolated from the leaf latex of A. pirottae using silica gel column chromatography, and their structures were determined using ¹H-NMR, ¹³C-NMR, and ESI-MS spectral data. Both the leaf latex and the isolated compounds were evaluated for their antimicrobial activity against three bacterial (Pseudomonas syringae pv. gaarcae, Ralstonia solanacearum and Xanthomonas vasicola pv. musacearum) and three fungal (Fusarium graminarum, Fusaium oxysporum, and Fusarium solani) plant pathogens. Results: A mixture of two diastereoisomeric anthrones was isolated from the leaf latex of A. pirottae and characterized as aloin A/B using ¹H-NMR, ¹³C-NMR, and ESI-MS. Aloin A/B showed the most potent activity against X. vasicola among the bacteria and F. oxysporum among the fungi strains tested, with minimum inhibitory concentrations (MIC) of 3.12 mg/mL and 6.25 mg/mL, respectively. Further exploration uncovered strong binding affinity of aloin A/B towards key proteins in these pathogens, specifically with topoisomerase II (−10.060 kcal/mol) and UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2, 6-diaminopimelate ligase (murE) (−8.861 kcal/mol) in F. oxysporum and X. vasicola, respectively, through molecular docking studies. Conclusion: The present findings highlight aloin A/B as promising natural antimicrobial agents and lead compounds for new plant pathogen treatments. Further research is recommended to explore their activity against a wider range of plant pathogens.

Keywords

Asphodelaceae Aloin A/B antibacterial antifungal

Introduction

The Food and Agriculture Organization (FAO) estimates that 20–40% of annual global crop losses are due to plant pathogenic infections, both directly and indirectly.¹ This translates to an annual cost of approximately 40 billion dollars to the global economy.² In Ethiopia, crops like bananas (Musa acuminate), enset (Ensete ventricosum), coffee (Coffea Arabica), and ginger (Zingiber officinale) are seriously threatened by plant microbial diseases such as blight and wilt, which significantly affect their economic viability.³ The widespread use of pesticides worldwide, including fungicides,⁴ nematicides,⁵ and antibiotics for bacterial plant pathogens,⁶ has indeed led to significant increases in crop yields. However, this extensive pesticide use brings several challenges, such as the development of pathogen resistance, changes in plant pathogen growth patterns, and concerns about toxicity to both humans and the environment.^7–9 Hence, there is a pressing need to search for new, safe, and effective drugs, including natural products, capable of efficiently preventing and controlling plant pathogens while also overcoming the limitations of current drugs, such as the development of resistance and toxicity.¹⁰

Aloe pirottae A. Berger, a member of the Asphodelaceae family, is indigenous to Ethiopia growing at altitudes ranging from 1300 to 1820 m.¹¹ In Ethiopian traditional medicine, the roots, leaves, and the leaf latex of A. pirottae have been used to treat various ailments, including tropical ulcer, malaria, snake bites, gallstones, hemorrhoids, wounds, eye infections, gonorrhea, parasites, muscular pain, colon cleaner, constipation, diabetes mellitus, gastritis, and social uses such as weaning children from breastfeeding, mosquito and insect repellent effect.^8,12,13 Previous studies have also investigated the antibacterial,¹⁴ anticancer,¹⁵ and antimalarial¹⁶ activities of different parts of A. pirottae. Hence, this study aimed to isolate, characterize, and perform molecular docking of compound(s) isolated from the leaf latex of A. pirottae. The goal was to gain further insights into the enzyme targets of these compounds and to understand how they bind to the active sites of the enzymes.

Results and Discussion

Characterization of the Isolated Compound (1)

Phytochemical analysis of the leaf latex of A. pirottae was conducted using column chromatography, resulting in the isolation of a yellow amorphous compound (1), with an R_f value of 0.56 in CHCl₃/MeOH (4:1) solvent system.

In the negative-mode HRESI-MS (Fig. S1), compound 1 exhibited a pseudomolecular ion at m/z 417.1190 [M-H]^–, corresponding to a molecular formula of C₂₁H₂₂O₉ (exact calcd. = 417.1186 [M-H]⁻). In the end, compound 1 was unequivocally identified as aloin, a natural mixed diastereomer (A/B), based on its spectral data (¹H; Fig. S2 and ¹³C-NMR; Fig. S3), as described in the Materials and Methods section, and through comparison with literature data. (Figure 1).¹⁷

Figure 1.

Chemical structure of aloin A/B.

Antibacterial Activity of the Leaf Latex and Aloin A/B

Table 1 summarizes the antibacterial effects of both the leaf latex and aloin A/B against three bacterial strains: Pseudomonas syringae pv. gaarcae, Ralstonia solanacearum and Xanthomonas vasicola pv. musacearum. In the disk diffusion method, X. vasicola showed the highest susceptibility to both the leaf latex and aloin A/B at a concentration of 25 mg/mL, with the inhibition zone diameters (IZD) of 9.68 mm and 14.47 mm, respectively. Among the strains tested, R. solanacearum exhibited the lowest sensitivity to the leaf latex (IZD = 9.91 mm; 50 mg/mL), while P. syringae showed the least activity against aloin A/B, with IZD of 11.54 mm at the highest concentration tested (25 mg/mL).

Table 1.

Inhibition Zone Diameter (IZD) and minimum Inhibitory Concentration (MIC) of Leaf Latex and Aloin A/B.

Bacteria strains	Inhibition zone diameter (IZD) in mm
Bacteria strains	Latex (12.5 mg/ml)	Latex (25 mg/ml)	Latex (50 mg/ml)	Aloin A/B (6.25 mg/ml)	Aloin A/B (12.5 mg/ml)	Aloin A/B (25 mg/ml)	Streptomycin 10 µg disc
X. vasicola	3.37 ± 0.83^a3b3c3	9.68 ± 1.10^a3b3d3	16.26 ± 0.25^a3c3d3	2.75 ± 0.74^a3b3	7.18 ± 0.33 ^a3b3	14.47 ± 0.42^a3c3d3	25.73 ± .688
P. syringae	4.22 ± 0.57^a3b3	7.97 ± 0.13^a3b3	12.79 ± 0.53^a3b1d3	2.45 ± 0.17^a3b3	5.25 ± 0.59^a3b3	11.54 ± 0.37^a3c3d3	24.44 ± .664
R. solanacearum	2.64 ± 0.65^a3	6.88 ± 0.21^a3	9.91 ± 0.15^a3b2	2.26 ± 0.42^a3b3c3	5.66 ± 0.25^a3b3c3	12.75 ± 0.22^a3c3d3	23.11 ± .944
Percentage mean mycelial growth inhibition (%)
Fungal strains	Latex (12.5 mg/ml)	Latex (25 mg/ml)	Latex (50 mg/ml)	Aloin A/B (6.25 mg/ml)	Aloin A/B (12.5 mg/ml)	Aloin A/B (25 mg/ml)	Tebuconazole 25 μg/mL
F. oxysporum	44.91 ± .79^a3b2	51.31 ± 0.23^a3	63.95 ± 0.24^a3d3	46.35 ± 1.23^a3b2	59.94 ± 1.79^a3	68.05 ± 2.02^a3d2	100.0 ± .000
F. graminaerum	51.68 ± .89^a3b2	61.27 ± 0.45^a3	73.38 ± 1.23^a3d3	41.58 ± 1.43^a3b3c1	53.98 ± 1.53^a3d1	62.62 ± 1.11^a3d3	100.0 ± .000
F. solani	39.25 ± .60^a3b2	48.80 ± 2.05^a3b1	60.99 ± 1.10^a3c1d3	39.92 ± .55^a3b2	48.47 ± 0.54^a3	55.99 ± 1.16^a3d3	100.0 ± .000
Minimum inhibitory concentration (MIC)
Bacteria strains	Latex (mg/ml)	Aloin A/B (mg/ml)	Streptomycin (μg/ml)
X. vasicola	3.12 ± 00	3.12 ± 00	2.5 ± 00
P. syringae	6.25 ± 00	6.25 ± 00	2.5 ± 00
R. solanacearum	6.25 ± 00	6.25 ± 00	1.25 ± 00
Fungal strains	Latex (mg/ml)	Aloin A/B (mg/ml)	Tebuconazole (μg/mL)
F. oxysporum	6.25 ± 00	6.25 ± 00	6.25 ± 00
F. graminaerum	3.12 ± 00	1.25 ± 00	6.25 ± 00
F. solani	6.25 ± 00	1.25 ± 00	6.25 ± 00

Values are expressed as mean ± SEM (n = 3); the comparison of means of different concentration of latex, aloin A/B, and controls were analyzed using one way ANOVA followed by Post hoc Tukey HSD Multiple comparison test; a compared with positive control (streptomycin sulfate), b to 50 mg/mL for the latex and 25 mg/mL for the aloin A/B, c to 25 mg/mL for the latex and 12.5 mg/mL for the aloin A/B, d to 12.5 mg/mL for the latex and 6.25 mg/mL for the aloin A/B for antibacterial activity; whereas in antifungal activity assay, a compared with positive control (tebuconazole), b to 50 mg/mL for the latex and 25 mg/mL for the aloin A/B, c to 25 mg/mL for the latex and 12.5 mg/mL for the aloin A/B and d to 12.5 mg/mL for the latex and 6.25 mg/mL for the aloin A/B; 1p < 0.05, 2 p < 0.01, 3 p < 0.001.

Consistent with the results from the disk diffusion method, both the leaf latex and aloin A/B (Table 1) exhibited the highest antibacterial activity against X. vasicola, with a minimum inhibition concentration (MIC) value of 3.12 mg/mL for each. Furthermore, the pathogen R. solanacearum was found to be more susceptible to A. pirottae leaf latex (MIC = 6.25 mg/mL) compared to a previous study using Aloe vera extract (MIC = 25 mg/mL).¹⁸ It worth mentioning that a product formulated from Aloe vera has been utilized to combat bacterial, fungal, and viral plant pathogens.¹⁹

Antifungal Activity of the Leaf Latex and Aloin A/B

The antifungal activity of the leaf latex and aloin A/B was evaluated against three fungal strains, namely Fusarium graminarum, Fusaium oxysporum, and Fusarium solani using the poisoned food technique.^20,21 The results presented in Table 1 showed that F. graminarum was most susceptible to the leaf latex, with a maximum inhibition rate of 73.38% at a concentration of 50 mg/mL. Meanwhile, aloin A/B exhibited a maximum inhibition rate of 68.05% against F. oxysporum at a concentration of 25 mg/mL. Additionally, the standard drug (tebuconazole; 25 μg/mL) completely suppressed the mycelial growth of all tested fungal strains.

The mean diameter of inhibition zones produced by both the latex and aloin A/B at all dose levels against the test bacterial and fungal strains (p < 0.05) exhibited statistically significant differences compared to those produced by their respective positive controls. In a previous study conducted by Kankamol et al (2021)¹⁸ on Aloe vera extract, a mycelial growth inhibition rate of 54.70% against F. oxysporum was observed at a concentration of 25 mg/mL. Interestingly, this inhibition rate was comparable to the mycelial growth inhibition rate of A. pirottae leaf latex (51.32%) against F. oxysporum at the same concentration level (25 mg/mL), as revealed in the current study. This similarity may be attributed to common phytochemical constituents among the plants. Moreover, when the broth dilution method was employed, leaf latex of A. pirottae demonstrated the strongest activity against F. graminaerum, with an MIC value of 3.12 mg/mL. On the other hand, F. oxysporum exhibited the highest susceptibility to aloin A/B, with MIC value of 6.25 mg/mL.

Molecular Docking Analysis

Both topoisomerase II and MurE are well-established targets in antimicrobial research. The selection of these enzymes for molecular docking studies in Fusarium oxysporum and Xanthomonas vasicola was based on prior in silico and experimental evidence. Studies have shown that naturally derived anthrones inhibit topoisomerase II^22–26 and MurE (UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase).^27,28

Topoisomerase II is essential for DNA replication, transcription, recombination, and repair, making it a valuable target for antifungal and antibacterial agents.^29,30 MurE, crucial in peptidoglycan biosynthesis, plays a key role in bacterial cell wall formation. Inhibiting MurE can weaken the cell wall, leading to cell lysis and death, which makes it a promising target for antibacterial drug development.^31,32

Homology Modeling and Validations of Modeled Proteins

The crystal structures of the target enzymes were unavailable in the Protein Data Bank (PDB). Therefore, homology models were created using SWISS-MODEL and a template library generated from a BLAST search of UniProt, utilizing the closest crystal structures: murE from Xanthomonas vasicola (PDB: 8F5D) and DNA topoisomerase II from Fusarium oxysporum (PDB: 4GFH).

The homology model of murE of Xanthomonas vasicola was built with a 50.76% sequence identity, a GMQE score of 0.73, and a QMEAN score of −2.35, indicating good quality and reliability. Similarly, the homology model of DNA topoisomerase II of Fusarium oxysporum was built with a 53.70% sequence identity, a GMQE score of 0.60, and QMEAN of −1.72, also indicating good quality and reliability. The QMEAN score of below −4.0 depicts low quality of the predicted structure, while the GMQE score ranges between 0 and 1, and a higher score corresponds to higher reliability.^33,34 The Ramachandran plot (Figure 2), which assesses the precision and dependability of the models, showed favorable results for both proteins. The Ramachandran plot demonstrated that murE of Xanthomonas vasicola protein showed a 95.00% Ramachandran favored, and 1.52% Ramachandran outliers. The DNA topoisomerase II of Fusarium oxysporum protein showed 95.03% Ramachandran favored and 0.66% Ramachandran outliers. Consequently, these results indicate that the proposed homology model is reliable and acceptable.

Figure 2.

3D model structure of protein from murE (A) and DNA topoisomerase II DNA topoisomerase II (B) ramachandran plot analysis of the homology modeled residues murE (C) and DNA topoisomerase II DNA topoisomerase II (D).

Docking Analyses

The molecular docking studies revealed a favorable binding affinity between Aloin A/B and the modeled protein structures from the species that showed the highest antimicrobial activity in the experimental study.

Aloin A/B achieved the highest docking score of −10.060 kcal/mol against DNA topoisomerase II of Fusarium oxysporum. The molecular docking analysis revealed important interactions, including hydrogen bonds, polar interactions, hydrophobic interactions, and charged interactions between aloin A/B and the amino acid residues of the enzyme. In Figure 3, aloin A/B exhibited five hydrogen bond interactions with DNA topoisomerase II of Fusarium oxysporum, specifically involving amino acid residues Ser 308, Asn 279, Asn 250, Asn 254, and Gly 283. Additionally, hydrophobic interactions were observed with Ile 286, Ile 300, Ala 251, and Pro 285. Charged interactions were also noted with Lys 316, Arg 257, Lys 282, and Asp 258.

Figure 3.

Molecular docking interactions between Aloin A/B and the binding sites of FUSOX: (A) 2D model of the interactions; (B) 3D model of the interactions.

In Figure 4, the best conformation of Aloin A/B showed a docking score of −8.861 kcal/mol against murE of Xanthomonas vasicola. Aloin A/B formed five hydrogen bond interactions with polar amino acid residues Thr 153, Ser 180, His 206, and Asn 409. Charged interactions were also observed with Glu 463, and a pi-pi interaction occurred with Arg 384. Additionally, hydrophobic interactions were also noted with Phe 152, Tyr 352, and Tyr 465.

Figure 4.

Molecular docking interactions between Aloin A/B and the binding sites of murE: (A) 2D model of the interactions; (B) 3D model of the interactions.

As shown in Figures 2 and 3, the 2D model representations of molecular docking interactions between aloin A/B and the binding sites of murE and DNA topoisomerase 2 indicate that the highest binding affinities for both enzymes occur with the S-configuration at C10, corresponding to aloin B.

Different parts of A. pirottae have been previously investigated for their effects on various human ailments. However, this is the first time it has been studied against plant pathogens. In the present study, a mixture of the two diastereomeric anthrones, aloin A and aloin B, was isolated, characterized, and evaluated against plant pathogenic microbes. Anthrones are among the predominant secondary metabolites present in Aloe species.³⁵ Aloin A/B is commonly found in the leaf latex of several Ethiopian Aloe species, including Aloe sinana,³⁶ Aloe harlana,³⁷ Aloe trichosantha,³⁸ and Aloe trigonantha.³⁹

In this study, aloin A/B demonstrated antimicrobial activity against plant pathogenic microbes. This antimicrobial activity was further supported by its high binding affinity to DNA topoisomerase II of Fusarium oxysporum, evidenced by a docking score of −10.060 kcal/mol. DNA topoisomerase II, also known as DNA gyrase in bacteria, is essential for maintaining the structural stability of the bacterial chromosome, facilitating critical DNA processes, and ensuring proper cellular functions.⁴⁰ The information from key interactions (hydrogen bonds, hydrophobic interactions, π-π stacking) that contribute to binding affinity can be used to modify the lead compound to enhance these interactions. For example, functional groups can be added or substituted to strengthen interactions with the target enzyme or to improve pharmacokinetic properties.

Anthrones, such as aloin A/B, have been reported to disrupt the integrity of microbial cell walls, contributing to their antimicrobial activity.³⁶ Similarly, anthraquinones, which are structurally similar to anthrones and isolated from Aloe barbadensis, have been shown to inhibit bacterial protein synthesis by binding to the ribosomal A site.⁴¹ Additionally, both anthraquinone and 1,8-dihydroxyanthraquinone inhibit the growth of human and plant pathogenic microbes by targeting the cell membrane and crucial enzymes involved in their metabolic processes.^42–44 Several studies also support the anthraquinones inhibitory activity on various cell lines through binding on topoisomerase II enzyme.⁴⁵ Anthraquinones prevent metabolic transformation of fungal aflatoxins to toxic derivatives or promoting their conversion to nontoxic products.⁴⁶ Anthraquinone inhibits DNA topoisomerase II and promotes DNA double-strand breaks through the stabilization of the topoisomerase II–DNA cleavage complex ultimately inhibiting ATP hydrolysis.^29,47

Aloin A/B shows promise in combating plant pathogens, but several limitations still need to be addressed. The effectiveness of aloin A/B in real-world agricultural settings has yet to be thoroughly investigated. Its spectrum of activity against various pathogens is unclear, and the potential for resistance development over time is not addressed. Additionally, the potential toxicity to non-target organisms and the environmental impact of large-scale use are not considered. Addressing these limitations through further research is essential for validating aloin A/B as a viable agricultural antimicrobial agent.

In the end, given the high concentration of aloin A/B in the leaf latex of A. pirottae and its presence in several other Ethiopian Aloe species, aloin A/B holds promising potential for combatting plant pathogens in Ethiopia.

Conclusion

This study demonstrated that the leaf latex of A. pirottae has antibacterial and antifungal properties against plant pathogenic bacteria and fungi in an in vitro model. The results from the assays on plant pathogenic bacteria and fungi indicate that the antimicrobial activity of A. pirottae is, at least in part, due to the presence of aloin A/B. The antimicrobial activity of aloin A/B was evident from molecular docking studies, which showed a strong binding affinity of aloin A/B towards topoisomerase II in Fusarium oxysporum and UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase (murE) in Xanthomonas vasicola. However, the greater antimicrobial activity of the leaf latex compared to aloin A/B against both plant pathogenic bacteria and fungi could be due to the higher availability of phytochemicals in the leaf latex, along with their diverse mechanisms or synergistic activities. Finally, this study provides insights into potential candidates for the proposed molecular targets, based on the binding affinities of aloin A/B. However, experimental characterization is crucial for further validating these protein targets. In the end, the present findings highlight aloin A/B as promising natural antimicrobial agents and lead compounds for new plant pathogen treatments. Further research is recommended to explore their activity against a wider range of plant pathogens.

Materials and Methods

General

The ¹H-NMR (400 MHz), ¹³C-NMR (100 MHz), DEPT-90, and DEPT-135 were run on a Bruker Advance DMX400 FT-NMR spectrometer instrument using tetramethylsilane (TMS) as internal standard. Electrospray Ionization mass spectrum (ESI-MS) was generated using Ultimate 3000 LC-MS instrument, Ultraviolet Spectrophotometer (UV) (Cammag, Switherland), Normal phase column chromatography, pre-coated plates with silica gel 60 F₂₅₄ (Merck KGaA, Darmstandt, Germany) for analytical thin layer chromatography (TLC).

Plant Material

The leaf latex of A. pirottae A. Berger was collected in November 2018 from Erer valley of Babile Wereda, East Hararge zone, Oromia region, Ethiopia. The plant was authenticated by Dr Anteneh Desta at the National Herbarium and given a voucher herbarium specimen of AP01.

Extraction and Isolation

The latex from the leaves of A. pirottae was collected using the methods described in our earlier study.⁴⁸ The yellow sap was collected by cutting the fresh leaves of A. pirottae at the base. The collected sap was then left to stand for a week to allow water to evaporate and the sap to dry, resulting in the formation of the leaf latex. Silica gel column chromatography was employed to isolate compounds from the leaf latex of A. pirottae. The column was initially packed with slurry consisting a mixture of silica gel and chloroform. The methanol soluble portion of the leaf latex (2 g) was then adsorbed on silica gel and loaded onto the column. A total of 100 fractions, each 10 mL, were collected using a gradient elution of chloroform and methanol. The first fraction was eluted with 100% chloroform, and continued with 5% increments of methanol in chloroform. The purity of the fractions was monitored using TLC with CHCl₃/MeOH (4:1 v/v) as a solvent system. Fractions 89–95, eluted with a CHCl₃ and MeOH (9:1) mixture, yielded a yellowish amorphous solid substance (185 mg) and coded as compound 1.

Compound 1: Yellow amorphous substance; R_f value of 0.56 CHCl₃/MeOH (4:1); -ve HRESI-MS (Fig. S1): m/z = 417.1190 [M-H]⁻, corresponding to a molecular formula of C₂₁H₂₂O₉ (exact calcd. m/z = 417.1186 [M-H]⁻); ¹H-NMR (ppm; Fig. S2): 3.40/3.37 (H-11, d), 4.25/3.95 (H-10, d), 6.84/6.82 (H-2, s), 6.89/6.86 (H-7, d), 7.02/7.00 (H-4, d), 7.07/7.05 (H-5, d), 7.56/7.51 (H-6, d), 11.79/11.77 (OH-8, s), 11.86/11.83 (OH-8, s), 2.72–2.73/2.65–2.71 (Glc-4 & 5), 2.76/2.75 (Glc-2), 2.82/2.76 (Glc-3), 3.10/2.86 (Glc-1), 3.25 & 3.29/3.12 & 3.25 (Glc-6). ¹³C-NMR (ppm; Fig. S3): 44.31/44.57 (C-10), 62.82/61.79 (C-11), 113.12/112.81 (C-2), 115.86/116.17 (C-7), 116.28/116.20 (C-1a), 116.70/117.48 (C-8a), 117.80/118.26 (C-4), 120.67/119.34 (C-5), 136.55/135.68 (C-6), 142.39/142.19 (C-4a), 145.99/146.20 (C-5a), 151.69/152.51 (C-3), 161.31/161.17 (C-1), 161.53/161.31 (C-8), 193.81/193.78 (C-9), 61.79/61.79 (Glc-6), 70.67/70.56 (Glc-2), 78.52/78.59 (Glc-4), 79.51/78.59 (Glc-3), 81.20/81.08 (Glc-5), 85.49/85.38 (Glc-1). ¹H and ¹³C-NMR spectral data of compound 1 were consistent with aloin A/B.¹⁷

Antimicrobial Activity

Bacterial and Fungal Strains

Bacterial strains (Pseudomonas syringae pv. gaarcae, Xanthomonas vasicola pv. musacearum, and Ralstonia solanacearum) and fungal strains (Fusarium graminarum, Fusarium oxysporum, and Fusarium solani) were sourced from the Ambo Agricultural Research Institute in Ambo, Ethiopia.

Antibacterial Activity Test

The antibacterial activity of the test samples were evaluated using the agar well diffusion method.⁴⁹ Sterile nutrient agar plates were prepared and the bacteria were spread over the plates using a sterile swab. Four wells each of 6 mm diameter were punched into the plates using separate sterile borer, and the test samples (100 μL) with three concentrations (50, 25 and 12.5 mg/mL) in 2% dimethyl sulfoxide (DMSO) were added to their respective wells using sterile micropipette. A standard drug streptomycin sulfate (10 µg) disc carefully placed onto the middle part of the media^50,51 and 2% DMSO (100 μL) was added as a negative control. All the plates were then incubated for 24 h at 30 °C, and the resulting zone of inhibitions were measured by digital caliper in mm. All assays were conducted in triplicate.

Antifungal Activity Test

The antifungal activity of the test samples was assessed by the poisoned food technique. The test samples (500 μL) of three concentrations (50, 25 and 12.5 mg/mL) were spread evenly onto three different previously prepared and solidified sterile potato dextrose agar (PDA) plates using a sterile glass spreader. A mycelial disc of 0.5 cm in diameter cut out from the 7-day old fungal culture aseptically placed on the center of different PDA plates containing test samples, negative (DMSO and PDA) and positive control (tebuconazole; 25 μg/mL), respectively. The inoculated plates were incubated at 26 ± 1 °C for 7 days and the mycelial growth inhibition rate was calculated on the seventh day.⁵² Rate of mycelial growth inhibition was calculated as follows:

MGI = \frac{DC - DT}{DC} X 100

Where, MGI: Mycelial growth inhibition rate (%), DC: Diameter of the control sample, DT: Diameter of the test sample.

Determination of Minimum Inhibitory Concentrations (MICs)

MICs of the test samples were determined using the Broth dilution method.⁵³ To prepare the test samples, the latex and isolated compound were dissolved in 2%DMSO solution. The concentrations tested ranged from 50 mg/mL to 0.098 mg/mL for the latex and 25 mg/mL to 0.049 mg/mL for the isolated compound.^54,55 Streptomycin sulfate (10 μg/mL) and 2%DMSO both in Mueller Hinton Broth (MHB) were used as a positive and negative control respectively for antibacterial assay.⁵⁶ Tebuconazole (25 μg/mL) and 2%DMSO both in potato dextrose broth (PDB) were used as a positive and negative control respectively for antifungal assay.¹⁷ The plates were covered with sterile lead and incubated at 30 °C for 24 h for bacteria and 25 °C for 48 h for fungi.^18,57 After the designated incubation periods, ie 24 h for (bacteria) and 48 h (fungi), the MICs were assessed by adding 40 μL of a 2% solution of iodonitrotetrazolium (INT) to each well^58,59 followed by a 25-min incubation at 30 ^oC for bacteria and 25 ^oC for fungi. The metabolically active bacteria and fungi would reduce the colorless test sample to a pink color, and the MIC was considered to be the lowest sample concentration that prevented this change.¹⁸ The assay was conducted in triplicate.

Molecular Docking Studies

Homology Models Building and Validation

The crystal structures of the target enzymes were not available in the PDB. Consequently, homology models were created using SWISS-MODEL and a template library generated from a BLAST search of UniProt, as previously described. The closest crystal structures used as templates were murE of Xanthomonas vasicola (PDB: 8F5D) and DNA topoisomerase II of Fusarium oxysporum (PDB: 4GFH). The amino acid sequence of the DNA topoisomerase II and DNA gyrase subunit B proteins for Fusarium oxysporum and Xanthomonas vasicola was obtained in FASTA format from the Uniprot database UniProtKB (Universal Protein Resource Knowledgebase; http://www.uniprot.org) using the primary accession codes A0A8B5YVD4 and W9KBZ2, respectively.⁶⁰ The identification of suitable templates for homology modeling was conducted using the Basic Local Alignment Search Tool (BLAST) through SWISS-MODEL online workspace (https://www.swiss model.expasy.org). The reliability of the modeled 3D structure was assessed using the Qualitative Model Energy Analysis (QMEAN) and Global Model Quality Estimation (GMQE) scores. A QMEAN score below −4.0 indicated low quality of the predicted structure, while the GMQE score ranges between 0 and 1, with a higher score indicating higher reliability.^33,34

Docking Study

For protein and ligand preparation, the homology modeled target proteins were downloaded in pdb format from the SWISS-MODEL workspace and prepared using the Protein Preparation Wizard module of Schrödinger suite.⁶¹ The protein was prepared by adding hydrogens, fixing any errors such as missing side chains, assigning correct bond orders, adjusting ionization and tautomeric states (via Epik), and deleting water molecules beyond 5 Å. Optimization and subsequent minimization were performed on all atoms using OPLS3 force fields, with the convergence of heavy atoms set to an RMSD of 0.3 Å (default).

For ligand preparation, the compounds drawn in SD format using ChemDraw were imported and prepared using the LigPrep module, which added hydrogens, performed a 2D to 3D conversion, generated ionization and tautomeric states (via Epik) at physiological pH 7.0 ± 2.0, and determined ring conformations using default settings. The docking of the isolated compounds was conducted using the Glide program with the previously prepared receptor grid and ligand molecules. The favorable interactions between the ligand molecules and the receptor were scored using Glide, and all docking calculations were performed in extra precision (XP) mode and using the OPLS-2005 force field.

Statistical Analysis

Data analysis was carried out using IBM SPSS (Statistical Package for Social Sciences) Statistics for Windows, Version 20.0. Results were reported as mean ± standard error of mean (M ± SEM). The statistical significance was determined by one-way ANOVA followed by Tukey post hoc test to compare inhibition activity of the treatment and control groups. p < 0.05 was considered significant.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X241281497 - Supplemental material for Antimicrobial and Molecular Docking Studies of Anthrones from the Leaf Latex of Aloe pirottae A. Berger against Some Plant Pathogenic Microbes

Supplemental material, sj-docx-1-npx-10.1177_1934578X241281497 for Antimicrobial and Molecular Docking Studies of Anthrones from the Leaf Latex of Aloe pirottae A. Berger against Some Plant Pathogenic Microbes by Shewaneh Ayele, Misgana Aragaw, Denberu Kebede, Bayoush Birke and Daniel Bisrat in Natural Product Communications

Footnotes

Acknowledgements

The authors are thankful to Ambo Agriculture and Research Center for their cooperation and the facilities provided for conducting the biological experiment.

Declaration of Conflicting Interests

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

Ethical Approval

Ethical approval is not applicable for this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The present study was financially supported by the School of Graduate Studies of Addis Ababa University research grant (AAU/SGS/Pharm-0022).

School of Graduate Studies of Addis Ababa University, (grant number AAU/SGS/Pharm-0022).

ORCID iDs

Shewaneh Ayele

Daniel Bisrat

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.

References

Pagán

García-Arenal

. Tolerance to plant pathogens: theory and experimental evidence. Int J Mol Sci. 2018;19(3):810. doi:https://doi.org/10.3390/ijms19030810

Ab Rahman

SFS

Singh

Pieterse

Schenk

. Emerging microbial biocontrol strategies for plant pathogens. Plant Sci. 2018;267:102-111. doi:https://doi.org/10.1016/j.plantsci.2017.11.012

Tesfaye

Seid

Getnet

Mamo

. Agriculture under a changing climate in Ethiopia: challenges and opportunities for research. Ethiopian J Agric Sci. 2016; (EIAR 50th Year Jubilee Anniversary Special Issue):67-86.

McDonald

Linde

. The population genetics of plant pathogens and breeding strategies for durable resistance. Euphytica. 2002;124(2):163-180. doi:https://doi.org/10.1023/A:1015678432355

Van der Putten

Cook

Costa

, et al.

Nematode interactions in nature: models for sustainable control of nematode pests of crop plants?

Adv Agron. 2006;89:227-260. doi:https://doi.org/10.1016/S0065-2113(05)89005-4

Ambrico

Trupo

Magarelli

, et al. Effectiveness of Dunaliella salina extracts against Bacillus subtilis and bacterial plant pathogens. Pathogen. 2020;9(8):613. doi: 10.3390/pathogens9080613

Sales

MDC

Costa

Fernandes

PMB

Ventura

Meira

. Antifungal activity of plant extracts with potential to control plant pathogens in pineapple. Asian Pac J Trop Biomed. 2015;6(1):26-31. doi:https://doi.org/10.1016/j.apjtb.2015.09.026

Belayneh

Demissew

Bussa

Bisrat

. Ethno-medicinal and bio-cultural importance of aloes from south and east of the great rift valley floristic regions of Ethiopia. Heliyon. 2020;6(6):1-12. doi:https://doi.org/10.1016/j.heliyon.2020.e04344

Thind

. Role of fungicides in crop health management: prospects and challenges. In: Satyanarayana

Deshmukh

Johri

, eds. Developments in fungal biology and applied mycology. Springer Singapore; 2017:433-447. doi:https://doi.org/10.1007/978-981-10-4768-8-22.

10.

Mosbah

Morsy

Belal

Ashry

. Evaluation of some vital agents, plant extracts, antioxidant and fungicides against onion white rot pathogen (Sclerotium cepivorum) in vitro. Egy J Plant Pro Res. 2017;5(89):30-54.

11.

Demissew

Nordal

. Aloes and Lilies of Ethiopia and Eritrea. Shama Books; 2010.

12.

Mesfin

Seta

Assefa

. An ethnobotanical study of medicinal plants in Amaro Woreda, Ethiopia. Ethnobot Res Appl. 2014;12:341-354. https://ethnobotanyjournal.org/index.php/era/article/view/951

13.

Ayalew

Kebede

Mesfin

Mulualem

. Ethnobotanical study of medicinal plants used by agro pastoralist Somali people for the management of human ailments in jeldesa cluster, dire dawa administration, Eastern Ethiopia. J Med Plants Res. 2017;11(9):171-187. doi:https://doi.org/10.5897/JMPR2016.6292

14.

Gobezie

Manilal

Seid

Lulekal

Tsegaye

. In vitro antibacterial activity of four plant species used in traditional medicine practices of south Omo zone, southern Ethiopia. Acta Microbiol. 2020;65(2):27-41.

15.

Terfa

Tesso

Jung

. Anticancer activity studies of root extract of Aloe pirottae A. Berger endemic plant species of Ethiopia. Int J Pharm Drug Anal. 2019;7(11):49-53.

16.

Dibessa

Engidawork

Nedi

Teklehaymanot

. Antimalarial activity of the aqueous extract of the latex of Aloe pirottae A. Berger (Aloaceae) against Plasmodium berghei in mice. J Ethnopharmacol. 2020;255:1-29. doi:https://doi.org/10.1016/j.jep.2020.112763

17.

Manitto

Monti

Speranza

. Studies on aloe. Part 6. Conformation and absolute configuration of aloins A and B and related 10-C-glucosyl-9-anthrones. J Chem Soc Perkin Trans 1. 1990;1(5):1297-1300. doi:https://doi.org/10.1039/P19900001297

18.

Kankamol

Srikam

Chumsiriwong

. Antimicrobial activities of Aloe vera rind extracts against plant pathogenic bacteria and fungi. Agric Nat Resour. 2021;55(5):715-723. doi:https://doi.org/10.34044/j.anres.2021.55.5.02

19.

Coats

. Compositions and methods to treat plant pathogens. US patent appl. US 15/453. 2017;639.

20.

Ambuse

Bhale

. Antifungal activity of plant latex extracts against resistant isolates of pathogens associated on (Rumex acetosa L.). Int J Plant Sci. 2012;7(2):395-398.

21.

Chinche

Gade

Shinde

Vairagade

Kendhale

. Fractionation of secondary metabolites from tulsi (Ocimum sanctum) and Aloe vera (Aloe barbadensis Mill.) and their antifungal activity against Aspergillus niger and Sclerotium rolfsii. Int J Curr Microbiol App Sci. 2020;9(5):445-452. doi:https://doi.org/10.20546/ijcmas.2020.905.050

22.

Alhadrami

Abdulaal

Hassan

Alhakamy

Sayed

. In silico-based discovery of natural anthraquinones with potential against multidrug-resistant E. coli. Pharmaceuticals. 2022;15(1):86. doi:https://doi.org/10.3390/ph15010086

23.

Malik

Alsantali

Jassas

, et al. Journey of anthraquinones as anticancer agents–a systematic review of recent literature. RSC Adv. 2021;11(57):35806-35827. doi:https://doi.org/10.1039/d1ra05686g

24.

Kalirajan

Sankar

Jubie

Gowramma

. Molecular docking studies and in-silico ADMET screening of some novel oxazine substituted 9-anilinoacridines as topoisomerase II inhibitors. Indian J Pharm Educ Res. 2017;51(1):110-115.

25.

Spencer

Panda

. DNA Gyrase as a target for quinolones. Biomedicines. 2023;11(2):371. doi:https://doi.org/10.3390/biomedicines11020371

26.

Liu

Zhang

, et al. Comparative antibacterial analysis of the anthraquinone compounds based on the AIM theory, molecular docking, and dynamics simulation analysis. J Mol Model. 2023;29(1):16. doi:https://doi.org/10.1007/s00894-022-05406-2

27.

Qun

Zhou

Hao

, et al. Antibacterial activities of anthraquinones: structure–activity relationships and action mechanisms. RSC Med Chem. 2023;14(8):1446-1471. doi:https://doi.org/10.1039/d3md00116d

28.

Cazzaniga

Mori

Chiarelli

Gelain

Meneghetti

Villa

. Natural products against key Mycobacterium tuberculosis enzymatic targets: emerging opportunities for drug discovery. Eur J Med Chem. 2021;224:113732. doi:https://doi.org/10.1016/j.ejmech.2021.113732

29.

Kondaka

Gabriel

. Targeting DNA topoisomerase II in antifungal chemotherapy. Molecules. 2022;27(22):7768. doi:https://doi.org/10.3390/molecules27227768

30.

Wang

. DNA Topoisomerases. Annu Rev Biochem. 1996;65(1):635-692. doi:https://doi.org/10.1146/annurev.bi.65.070196.003223

31.

Barreteau

Kovac

Boniface

Sova

Gobec

Blanot

. Cytoplasmic steps of peptidoglycan biosynthesis. FEMS Microbiol Rev. 2008;32(2):168-207. doi:https://doi.org/10.1111/j.1574-6976.2008.00104.x

32.

El Zoeiby

Sanschagrin

Levesque

. Structure and function of the Mur enzymes: development of novel inhibitors. Mol Microbiol. 2003;47(1):1-12. doi:https://doi.org/10.1046/j.1365-2958.2003.03289.x

33.

Waterhouse

Bertoni

Bienert

, et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 2018;46(W1):W296-W303. doi:https://doi.org/10.1093/nar/gky427

34.

Abdullahi

Adeniji

Arthur

Haruna

. Homology modeling and molecular docking simulation of some novel imidazo[1,2-a]pyridine-3-carboxamide (IPA) series as inhibitors of Mycobacterium tuberculosis. JGEB. 2021;19(1):12. doi:https://doi.org/10.1186/s43141-020-00102-1

35.

Lucini

Pellizzoni

Pellegrino

Molinari

Colla

. Phytochemical constituents and in vitro radical scavenging activity of different Aloe species. Food Chem. 2015;170:501-507. doi:https://doi.org/10.1016/j.foodchem.2014.08.034

36.

Minale

Bisrat

Asres

Mazumder

. In vitro antimicrobial activities of anthrones from the leaf latex of Aloe sinana Reynolds. Int J Green Pharm. 2014;8(1):7-12. DOI: https://doi.org/10.4103/0973-8258.126812

37.

Asamenew

Bisrat

Mazumder

Asres

. In vitro antimicrobial and antioxidant activities of anthrone and chromone from the latex of Aloe harlana Reynolds. Phytother Res. 2011;25(12):1756-1760. doi:https://doi.org/10.1002/ptr.3482

38.

Oumer

Bisrat

Mazumder

Asres

. A new antimicrobial anthrone from the leaf latex of Aloe trichosantha. Nat Prod Commun. 2014;9(7):949-952. doi:https://doi.org/10.1177/1934578X1400900717

39.

Megeressa

Bisrat

Mazumder

Asres

. Structural elucidation of some antimicrobial constituents from the leaf latex of Aloe trigonantha LC leach. BMC Complement Altern Med. 2015;15:1-7. doi:https://doi.org/10.1186/s12906-015-0803-4

40.

Mun Huang

. Bacterial diversity based on type II DNA topoisomerase genes. Annu Rev Genet. 1996;30(1):79-107. doi:https://doi.org/10.1146/annurev.genet.30.1.79

41.

Pandey

Mishra

. Antibacterial activities of crude extract of Aloe barbadensis to clinically isolated bacterial pathogens. Appl Biochem Biotechnol. 2010;160(5):1356-1361. doi:https://doi.org/10.1007/s12010-009-8577-0

42.

Abu-Darwish

Ateyyat

Salt

. The pharmacological and pesticidal actions of naturally occurring 1, 8-dihydroxyanthraquinones derivatives. World J Agric Sci. 2008;4(4):495-505.

43.

Ouyang

Xiao

Gao

Liu

. Antimicrobial properties and toxicity of anthraquinones by microcalorimetric bioassay. Chin J Chem. 2006;24(1):45-50. doi:https://doi.org/10.1002/cjoc.200690020

44.

Alves

Pérez-Fons

Estepa

Micol

. Membrane-related effects underlying the biological activity of the anthraquinones emodin and barbaloin. Biochem Pharmacol. 2004;68(3):549-561. doi:https://doi.org/10.1016/j.bcp.2004.04.012

45.

Malik

Alsantali

Jassas

, et al. Journey of anthraquinones as anticancer agents – a systematic review of recent literature. RSC adv. 2021;11(57):35806-35827. Doi: https://doi.org/10.1039/d1ra05686g

46.

Martínez

Benito

. Biological activity of quinones. Stud Nat Prod Chem. 2005;30:303-366. doi:https://doi.org/10.1016/S1572-5995(05)80036-5

47.

Raghuveer

Pai

Murali

Nayak

. Exploring anthraquinones as antibacterial and antifungal agents. Chem Select. 2023;8(6):e202204537. doi:https://doi.org/10.1002/slct.202204537

48.

Teka

Bisrat

Mazumder

Asres

. Antimicrobial constituents from the leaf latex of Aloe pulcherrima Gilbert & Sebsebe. Int J Phytopharmacol. 2014;5(4):261-266.

49.

Bhagwat

Datar

. Antibacterial activity of herbal extracts against five plant pathogenic bacteria. Arch Phytopathol Plant Prot. 2014;47(7):892-899. doi:https://doi.org/10.1080/03235408.2013.825398

50.

Khaleel

Sijam

Rashid

Bin Ahmad

. Phytochemical determination and antibacterial activity of Punica granatum peel extracts against plant pathogenic bacteria. Am J Plant Sci. 2016;07(01):159-166. DOI: https://doi.org/10.4236/ajps.2016.71017

51.

Pretorius

Magama

Zietsman

Van Wyk

. Growth inhibition of plant pathogenic bacteria and fungi by extracts from selected South African plant species. S Afr J Bot. 2003;69(2):186-212. doi:https://doi.org/10.1016/S0254-6299(15)30344-6

52.

Praveen

Thippeswamy

Mohana

Manjunath

. Antimicrobial efficacy and phytochemical analysis of Albizia amara (Roxb.) Boiv. An indigenous medicinal plant against some human and plant pathogenic bacteria and fungi. J Pharm Res. 2011;4(3):832-835.

53.

Newton

Lau

Gurcha

Besra

Wright

. The evaluation of forty-three plant species for in vitro antimycobacterial activities; isolation of active constituents from Psoralea corylifolia and Sanguinaria canadensis. J Ethnopharmacol. 2002;79(1):57-67. doi:https://doi.org/10.1016/S0378-8741(01)00350-6

54.

Al-Abrahim

Mohammed

Elobeid

. Assessment of in vitro antifungal potential of ethanolic extract of Calligonum Comosum against two fungal postharvest pathogens of fruits and vegetables in Saudi Arabia. Int J Appl Biol Pharm. 2014;5:90-91.

55.

Marei

GIK

Rasoul

MAA

Abdelgaleil

. Comparative antifungal activities and biochemical effects of monoterpenes on plant pathogenic fungi. Pestic Biochem Physiol. 2012;103(1):56-61. doi:https://doi.org/10.1016/j.pestbp.2012.03.004

56.

Pham

Dao

, et al. Antimicrobial efficacy of extracts and constituents fractionated from Rheum tanguticum Maxim. ex Balf. rhizomes against phytopathogenic fungi and bacteria. Ind Crops Prod. 2017;108:442-450. doi:https://doi.org/10.1016/j.indcrop.2017.06.067

57.

Mdee

Masoko

Eloff

. The activity of extracts of seven common invasive plant species on fungal phytopathogens. S Afr J Bot. 2009;75(2):375-379. doi:https://doi.org/10.1016/j.sajb.2009.02.003

58.

Eloff

. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta med. 1998;64(08):711-713. doi:https://doi.org/10.1055/s-2006-957563

59.

Bitchagno

Sama Fonkeng

Kopa

, et al. Antibacterial activity of ethanolic extract and compounds from fruits of Tectona grandis (Verbenaceae). BMC Complement Altern Med. 2015;15:1-6. doi:https://doi.org/10.1186/s12906-015-0790-5

60.

UniProt Consortium. Uniprot: a worldwide hub of protein knowledge. Nucleic Acids Res. 2019;47(D1):D506-D515. doi:https://doi.org/10.1093/nar/gky1049

61.

Schrödinger Release 2023-2 (2023) Schrödinger Release 2023-2.

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