Phytochemical Profiling and Antibacterial Activity of Rumex abyssinicus Jacq. Roots and Verbascum sinaiticum Benth. Leaf Extracts

Abstract

Objective

This study evaluated the phytochemical composition and antibacterial activity of extracts of Rumex abyssinicus roots and Verbascum sinaiticum leaves, two medicinal plants traditionally used in Ethiopia.

Methods

Extracts were prepared by cold maceration, and preliminary toxicity was assessed in Galleria mellonella larvae. Antibacterial activity was determined by the disc diffusion method against Staphylococcus aureus (ATCC 25923), Salmonella typhi (ATCC 19430), Escherichia coli (ATCC 25922), and Shigella sonnei (ATCC 25931). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were established by broth microdilution. Qualitative phytochemical screening was conducted, and principal bioactive constituents were characterized by thin-layer chromatography (TLC) and nuclear magnetic resonance (NMR) spectroscopy.

Results

The extracts demonstrated dose-dependent toxicity in G. mellonella, with up to 40% mortality at 100 mg/mL. Ethanol extracts of R. abyssinicus exhibited the strongest antibacterial effects, notably against S. aureus (21.3 ± 1.21 mm at 200 mg/mL), whereas chloroform extracts of V. sinaiticum showed modest inhibition at higher concentrations. The MIC ranged from 16-32 mg/mL for the ethanol extracts and 32-64 mg/mL for the chloroform extracts of both plant materials. Phytochemical analysis identified anthraquinones, saponins, and tannins in R. abyssinicus, and flavonoids and phenols in V. sinaiticum. NMR profiling revealed nine major compounds, including chrysophanol, emodin, physcion, helminthosporin, and chrysophanol-bianthrone from R. abyssinicus, and luteolin, chrysoeriol-7-O-glucoside, aucubin, and ajugol from V. sinaiticum.

Conclusion

These results provide a chemical basis for the observed antibacterial activity and support further investigation of these extracts and isolated metabolites as potential leads for antibacterial drug development.

Keywords

Antibacterial activity minimum inhibitory concentration phytochemical analysis

Introduction

Antimicrobial resistance (AMR) has emerged as a global health challenge, rendering many conventional antibiotics ineffective and leading to prolonged illnesses, higher medical costs, and increased mortality.^1,2 As pathogens continue to evolve mechanisms to withstand synthetic drugs, the urgency to develop alternative antimicrobial agents has intensified. One promising approach is the exploration of natural products, particularly medicinal plants, which have been used for centuries in traditional medical systems and remain an important source of bioactive compounds.^3,4

Medicinal plants represent a rich reservoir of chemically diverse secondary metabolites with significant therapeutic potential. Their long-standing use in traditional medicine suggests the presence of compounds capable of combating bacterial pathogens, including drug-resistant strains. Scientific validation of ethno-medicinal plants is therefore a critical step toward the discovery of novel antimicrobial agents. Among the numerous medicinal plants used in traditional Ethiopian medicine, R. abyssinicus and V. sinaiticum have attracted attention due to their frequent ethno-botanical use and preliminary reports of antibacterial activity.^5,6

R. abyssinicus, locally known as “Mekmeko” in Amharic, is indigenous to the highlands of East Africa and has traditionally been used to treat wounds, gastrointestinal disorders, and bacterial infections.^7,8 Phytochemical investigations have revealed that this species contains substantial amounts of phenolic compounds and flavonoids.^9,10 Similarly, V. sinaiticum, commonly referred to as “Qotetina” in Amharic, is widely distributed across Africa and the Middle East and has long been used in folk medicine to manage respiratory ailments, skin diseases, and inflammatory conditions.^11-13 Previous studies have reported the presence of diverse phytochemicals in V. sinaiticum, including flavonoids and other classes of secondary metabolites such as saponins, iridoid glycosides, and essential oils.¹⁴

Although previous studies have documented antibacterial activity and identified various phytochemical constituents of R. abyssinicus and V. sinaiticum, most investigations have been limited to preliminary bioactivity screening or isolated phytochemical assessments. Comprehensive studies integrating detailed phytochemical profiling, antibacterial evaluation against clinically relevant reference strains, and in vivo toxicity assessment remain limited. Furthermore, comparative analyses of different solvent extracts derived from specific plant parts and their corresponding bioactivity profiles have not been sufficiently characterized. To address these gaps, the present study provides an integrated evaluation of the in vivo toxicity, antibacterial activity against selected bacterial pathogens, and detailed phytochemical profiles of extracts from R. abyssinicus roots and V. sinaiticum leaves collected from Debre Markos, northwest Ethiopia.

Materials and Methods

Plant Collection, Authentication, and Preparation

The roots of R. abyssinicus and the leaves of V. sinaiticum were collected during the peak flowering season in January 2023 from Debre Markos, located 300 km northwest of Addis Ababa (10°20′N, 37°43′E; elevation 2,446 m). Collection sites were selected based on accessibility, plant abundance, and minimal human disturbance to ensure specimen integrity.

For R. abyssinicus, entire root systems were carefully excavated using gardening tools to avoid damage and gently shaken to remove excess soil. Healthy, mature leaves of V. sinaiticum were harvested by hand, ensuring minimal impact on the plant. Collected plant materials were placed in clean, breathable cloth bags to prevent microbial growth and degradation,¹⁵ and transported to the Microbiology Laboratory of Debre Markos University on the same day to preserve freshness and chemical integrity.

Plant identification and authentication were performed at the Institutional Herbarium of Debre Markos University by Dr. Haimanot Reta, a senior plant taxonomist. Collection and handling followed the Plant Material Collection and Reproducibility Manual of Debre Markos University, in compliance with institutional, national, and international guidelines. Voucher specimens of R. abyssinicus and V. sinaiticum were deposited at the herbarium under reference numbers AL001 and AL002, respectively.

The plant materials were thoroughly washed with distilled water to remove debris, air-dried at room temperature in a shaded, well-ventilated area to preserve sensitive compounds, and subsequently ground into a coarse powder using an electrical cross-beater mill (IEC, 158VDE0660, Germany).

Preparation of Crude Extracts

Crude extracts of the roots of R. abyssinicus and the leaves of V. sinaiticum were prepared by maceration using 80% ethanol and chloroform. For each extraction, 100 g of powdered plant material was accurately weighed using an electronic balance and placed in a clean, dry container. For ethanol extraction, 1000 mL of 80% ethanol was added to fully submerge the plant material. The mixture was sealed and allowed to macerate at room temperature for 72 hours, with occasional shaking to enhance extraction. The same procedure was applied for chloroform extraction.

After maceration, the mixtures were filtered through Whatman No. 1 filter paper to separate the extracts from plant residues. The filtrates were collected in clean, labeled containers, and solvents were removed using a rotary evaporator under reduced pressure at temperatures not exceeding 40°C to prevent thermal degradation. The resulting concentrated crude extracts were transferred to pre-weighed, labeled amber glass bottles and stored at 4°C until further use.¹⁶

Experimental Pathogens

The bacterial strains used in this study included one Gram-positive and three Gram-negative species. Staphylococcus aureus (ATCC 25923) represented the Gram-positive group, while Salmonella typhi (ATCC 19430), Escherichia coli (ATCC 25922), and Shigella sonnei (ATCC 25931) represented the Gram-negative group. All strains were obtained from the Clinical Bacteriology Laboratory of the Amhara Public Health Institute (APHI).

Upon receipt, the bacterial strains were reactivated by sub-culturing in nutrient broth. Each strain was inoculated into separate sterile tubes containing nutrient broth and incubated at 37°C for 24 hours. Quality control was performed by confirming colony morphology on nutrient agar and by Gram staining. Only cultures within three passages from the original ATCC vial were used to maintain strain integrity.

Following reactivation, bacterial cultures were streaked onto nutrient agar plates to obtain isolated colonies, incubated at 37°C for 24 hours, and examined to confirm purity and identity. Confirmed strains were maintained on nutrient agar slants, incubated at 37°C for 24 hours, and subsequently stored at 4°C to preserve viability for antibacterial activity testing.¹⁷

Study Design

This study employed a controlled, laboratory-based experimental design to evaluate in vivo toxicity and antibacterial activities of R. abyssinicus root and V. sinaiticum leaf extracts, alongside preliminary chromatographic characterization of their bioactive constituents. Two extraction solvents of differing polarity, 80% ethanol and chloroform, were selected to maximize the recovery of phytochemicals with a broad range of physicochemical properties and to enable comparative assessment of solvent-dependent bioactivity.

Each experimental condition included appropriate positive and negative controls to ensure internal validity. Ciprofloxacin served as the positive control in antibacterial assays, while solvent-only treatments (10% DMSO) were used as negative or vehicle controls across all experiments. Biological replication was achieved by using independent bacterial cultures and larval groups for each treatment, with technical triplication applied to minimize procedural variability and enhance reproducibility.

Toxicity Assessment of the Plant Extracts

To evaluate the preliminary safety profile of the extracts, G. mellonella larvae were used as an established in vivo model for toxicity screening of antimicrobial candidates. This model is cost-effective, ethically favorable, easy to handle, and provides reliable indications of potential cytotoxic effects that correlate well with mammalian systems, making it suitable for initial safety evaluation before further studies.¹⁸

Final-stage larvae weighing approximately 250-300 mg were obtained from the Laboratory of Microbiology and Medicine, University of Molise, Italy. Plant extracts were prepared at concentrations of 100, 50, 25, and 12.5 mg/mL using 10% dimethylsulfoxide (DMSO) as the solvent. Ten percent DMSO served as the vehicle control. No positive control was included, as the primary objective was a preliminary safety assessment, and the vehicle control was sufficient to detect extract-induced toxicity while adhering to ethical considerations.

Each larva received a 10 μL injection of the test extract or vehicle control into the last pro-leg using Hamilton syringes (Hamilton Inc., USA). Ten larvae per concentration were placed in individual Petri dishes, totaling 50 larvae per extract per replicate. Dishes were maintained in darkness at 37°C throughout the experimental period. Larvae were monitored at regular intervals for visible signs of toxicity, including altered mobility, feeding behavior, or mortality over 72 hours. All experiments were conducted in three independent assays, each performed in triplicate for every extract concentration and the vehicle control.

Antibacterial Paper Disc Preparation

Paper discs (≈6 mm diameter) for antimicrobial susceptibility testing were prepared by punching individual discs from Whatman No. 1 filter paper using a sterilized office two-hole puncher, avoiding overlap to maintain uniform size. The discs were placed on aluminum foil in a beaker, covered, and autoclaved at 121°C for 15 minutes to ensure sterility.

DMSO was used as the solvent due to its excellent solubilizing properties and compatibility with biological systems at concentrations up to 10%. Four serial dilutions of each plant extract (200, 100, 50, and 25 mg/mL) were prepared using a 10% DMSO stock solution (w/v), consistent with established methodologies for crude extracts. Each dilution was thoroughly mixed to ensure homogeneity.

Sterile, autoclaved discs were immersed in the prepared extract solutions using sterile forceps, allowing full submersion for uniform impregnation. Excess solution was removed by gently touching the disc edge against the side of the beaker. The impregnated discs were transferred to clean, sterile Petri dishes and air-dried at room temperature. Dried discs were stored in sterile containers sealed with parafilm until use. For the positive control, commercially available 5 µg ciprofloxacin discs were used.¹⁹

Antibacterial Sensitivity Test

The antibacterial activity of crude extracts from R. abyssinicus roots and V. sinaiticum leaves was evaluated using the agar disc diffusion method. Stock bacterial cultures were maintained on nutrient agar slants at 4°C. Bacterial suspensions were prepared by inoculating nutrient broth with pure colonies and adjusted to 0.5 McFarland standards (≈1.5 × 10⁸ CFU/mL).

Prepared discs were placed onto Mueller-Hinton agar plates previously inoculated with the bacterial suspensions. Each plate contained six discs: a 5 µg ciprofloxacin disc as a positive control, a 10% DMSO disc as a negative control, and four discs impregnated with different concentrations of plant extracts. Discs were gently pressed onto the agar surface using sterile forceps to ensure proper contact and diffusion of the extracts. Plates were inverted and incubated at 37°C for 24 hours to allow bacterial growth and compound diffusion. Following incubation, antibacterial activity was determined by measuring the diameter of clear inhibition zones around each disc using a calibrated ruler.¹⁷

Minimum Inhibitory Concentration (MIC)

The MIC of crude extracts from R. abyssinicus roots and V. sinaiticum leaves against bacterial strains was determined using the broth microdilution method in 96-well microplates. Two-fold serial dilutions of each extract, ranging from 256 to 2 mg/mL, were prepared in nutrient broth, with 10% DMSO used as the solvent to ensure solubility and consistency.

Each well was filled with 80 µL of nutrient broth, followed by 10 µL of the respective extract dilution. Positive controls contained 5 µg ciprofloxacin, and negative controls contained broth with 10% DMSO. Subsequently, 10 µL of standardized bacterial inoculum (0.5 McFarland, ≈1.5 × 10⁸ CFU/mL) was added to each well, resulting in a final volume of 100 µL. Plates were gently mixed to ensure even bacterial distribution, sealed to prevent evaporation, and incubated at 37°C for 24 hours.

After incubation, wells were visually inspected for turbidity as an indicator of bacterial growth. The MIC for each bacterial strain was defined as the lowest extract concentration that inhibited visible growth, providing a quantitative measure of antimicrobial potency.^17,20,21

Minimum Bactericidal Concentration (MBC)

The MBC of crude extracts from R. abyssinicus roots and V. sinaiticum leaves was determined using the broth dilution method in 96-well microplates. Following MIC determination, wells showing no visible bacterial growth were selected. To assess bactericidal activity, 10 µL from each of these wells was inoculated onto fresh nutrient agar plates, which were then incubated at 37°C for 24 hours. The MBC was defined as the lowest extract concentration resulting in no bacterial growth on agar, providing quantitative insight into the bactericidal potential of the extracts against the tested strains.²²

Phytochemical Profiling of the Ethanol Extracts

The ethanol extracts of R. abyssinicus roots and V. sinaiticum leaves were subjected to qualitative phytochemical screening to identify key antibacterial secondary metabolites, including alkaloids, flavonoids, tannins, saponins, phenols, terpenoids, glycosides, anthraquinones, and steroids, following established procedures.^16,23

Spectroscopic Analysis

The ethanol extracts of the plant samples were characterized using UV-Vis and FT-IR spectroscopy. For UV-Vis analysis, extracts were prepared at 1 mg/mL, filtered, and scanned over 200-700 nm using ethanol as a blank to identify characteristic absorption peaks corresponding to phenolic, flavonoids, anthraquinones, and iridoid glycosides. For FT-IR analysis, dried extracts were thoroughly mixed with KBr, pressed into pellets, and scanned over the 4000-400 cm^-1 range to determine major functional groups. All measurements were performed in triplicate, and the reported spectra represent the mean values.

Fractionation of the Crude Extracts and TLC Profile Analysis

Ethanol crude extracts of R. abyssinicus roots and V. sinaiticum leaves were fractionated and profiled by TLC to isolate and characterize potential bioactive compounds. For column chromatography, 5 g of crude extract was loaded onto a silica gel-packed column (60 × 3 cm, 60-120 mesh). The column was wet-packed by slurrying silica gel in petroleum ether and carefully poured to ensure uniform packing.

Elution was performed using 100% petroleum ether, followed by a stepwise petroleum ether-acetone gradient (100:0, 90:10, 80:20, 70:30, 60:40, 50:50 v/v) to separate compounds based on polarity.²⁴ Fractions were collected in 20 mL aliquots at a controlled flow rate of ∼1 mL/min. Fractions with similar TLC profiles (Rf values and spot patterns) were pooled, evaporated under reduced pressure using a rotary evaporator at ≤40°C to prevent degradation of thermo-labile compounds, and stored at 4°C until further analysis.

For TLC analysis, 10 µL of extract solution (10 mg/mL) was applied onto Fluka silica gel F254 plates (20 × 20 cm, 0.25 mm thickness) and developed using three solvent systems: benzene:ethanol:ammonium hydroxide (BEA, 36:4:0.4), chloroform:ethyl acetate:formic acid (CEF, 20:16:4), and ethyl acetate:methanol:water (EMW, 20:10.8:8).^25,26 Plates were visualized under UV light at 254 and 360 nm, followed by spraying with freshly prepared vanillin-sulfuric acid reagent (0.1 g vanillin in 28 mL methanol and 1 mL sulfuric acid) to detect separated compounds.^16,27 TLC was also used to monitor column fractions, ensuring accurate pooling.

Purity of isolated compounds was verified by TLC, and structural identities were confirmed by ¹H-NMR and ¹³C-NMR spectral data. Detailed information on the specific fractions from which each of the nine compounds was isolated is provided in the Supplementary Materials.

Nuclear Magnetic Resonance (NMR) Spectroscopy

Structural elucidation of the isolated compounds was performed using NMR spectroscopy. Analyses were conducted on a Bruker Avance III spectrometer at 400 MHz for ¹H-NMR and 100 MHz for ¹³C-NMR. Approximately 5-10 mg of each purified compound was dissolved in 0.6 mL of deuterated solvent (DMSO-d₆ or CDCl₃), yielding concentrations of 8-15 mg/mL. All measurements were performed at 298 K (25 °C).

Both ¹H and ¹³C-NMR spectra were recorded. Chemical shifts (δ) are reported in parts per million (ppm) relative to the solvent peaks (δH 2.50 and δC 39.52 for DMSO-d₆; δH 7.26 and δC 77.16 for CDCl₃), and coupling constants (J) are given in Hertz (Hz). Spectra were analyzed to determine the molecular structures and functional groups of the isolated compounds.^28-30

Data Analysis

Experimental data were carefully recorded and initially organized in Microsoft Excel to ensure accuracy and consistency. Statistical analyses were conducted using IBM SPSS version 26.0 (IBM Corp., Armonk, NY, USA). For quantitative measurements, including zones of inhibition, MIC, and MBC values, one-way analysis of variance (ANOVA) was performed to evaluate significant differences across extract concentrations and bacterial strains. Before ANOVA, normality of the data was assessed using the Shapiro-Wilk test, and homogeneity of variances was tested using Levene’s test. Where significant differences were detected by ANOVA, Tukey’s post-hoc test with adjustment for multiple comparisons was applied to determine pairwise differences between groups.

All experiments included three independent biological replicates, with each measurement performed in triplicate as technical replicates to ensure reliability and reproducibility. Statistical significance was set at p < 0.05. Qualitative data on secondary metabolites were analyzed descriptively, with the occurrence of each compound type calculated based on positive detection in the extracts. Chromatographic and spectroscopic data from TLC, UV–Vis, FT-IR, and NMR were used for compound identification, and spectra were compared with published references for confirmation.

Results

Extracts Yield

The percentage yields of crude extracts from the roots of R. abyssinicus and the leaves of V. sinaiticum are summarized in Table 1. Using a 1:10 w/v ratio with the cold maceration technique, ethanol extracts consistently yielded higher amounts than chloroform extracts for both plant species. The 80% ethanol extract of R. abyssinicus roots produced the highest yield (22%), whereas the chloroform extract of V. sinaiticum leaves yielded the lowest (11%).

Table 1.

Percentage Yield of 80% Ethanol and Chloroform Crude Extracts of the Roots of R. abyssinicus and the Leaves of V. sinaiticum

Plant species	Plant part	Solvents	Ratio (w/v)	Yield %
R. abyssinicus	Root	80% ethanol	1:10	22
R. abyssinicus	Root	Chloroform	1:10	14
V. sinaiticum	Leaf	80% ethanol	1:10	18
V. sinaiticum	Leaf	Chloroform	1:10	11

Toxicity Study of Extracts

One-way ANOVA followed by Tukey’s post-hoc test demonstrated a statistically significant (p < 0.05) concentration-dependent larvicidal effect of both R. abyssinicus root and V. sinaiticum leaf extracts against G. mellonella larvae (Table 2). At 100 mg/mL, ethanol extracts of R. abyssinicus and V. sinaiticum caused the highest larval mortality (4.10 ± 0.01), which was significantly higher than that observed at 25 mg/mL (1.20-1.30 ± 1.75) and 12.5 mg/mL (0.10-0.90 ± 0.01-0.33). The chloroform extract of V. sinaiticum showed slightly lower activity at 100 mg/mL (3.80 ± 0.11), yet remained significantly more toxic than all lower concentrations. However, no larval mortality was recorded in the DMSO-treated control group.

Table 2.

In vivo Toxicity Result (Larvicidal Effect) of R. abyssinicus Root and V. sinaiticum Leaf Extracts at Different Concentrations Against G. mellonella Larvae

Concentration (mg/mL)	Larvae per group	R. abyssinicus (80% EtOH)	R. abyssinicus (Chloroform)	V. sinaiticum (80% EtOH)	V. sinaiticum (Chloroform)
100	10	4.10 ± 0.01^a	4.10 ± 0.01^a	4.10 ± 0.01^a	3.80 ± 0.11^a
50	10	3.20 ± 1.75^b	3.30 ± 3.21^b	3.10 ± 4.54^b	2.20 ± 2.65^c
25	10	1.30 ± 1.75^c	1.20 ± 1.75^c	1.30 ± 1.75^c	1.10 ± 2.76^c
12.5	10	0.70 ± 0.33^c	0.90 ± 0.01^c	0.10 ± 0.01^d	0.10± 0.01^d
Vehicle control (DMSO)	10	0.00^d	0.00^d	0.00^d	0.00^d

Note: Values are expressed as mean ± SD. Within each column, values sharing different superscript letters indicate statistically significant differences (p < 0.05), as determined by one-way ANOVA followed by Tukey's post hoc test. DMSO served as the negative control.

Antibacterial Sensitivity

The antibacterial sensitivity assay demonstrated that both ethanol and chloroform extracts of R. abyssinicus roots and V. sinaiticum leaves exhibited significant activity (p < 0.05) against all tested bacterial pathogens, with activity varying by extract type, concentration, and bacterial species.

For R. abyssinicus roots, the ethanol extract displayed the highest activity against S. aureus at 200 mg/mL (21.3 ± 1.21 mm) and the lowest activity against E. coli at 25 mg/mL (9.6 ± 0.88 mm) (Table 3). Chloroform extracts showed slightly lower activity, with the weakest inhibition observed against E. coli at 25 mg/mL (11.5 ± 0.28 mm). In general, inhibition zones increased with higher extract concentrations. Among bacterial strains, S. aureus and S. typhi were more sensitive to both extracts, while E. coli showed the lowest susceptibility. The positive control (5 μg Ciprofloxacin) consistently produced the largest inhibition zones (23-24 mm), whereas the negative control (10% DMSO) produced no inhibition.

Table 3.

Antibacterial Sensitivity Result of Crude Extracts of the Roots of R. abyssinicus Against Experimental Bacterial Pathogens (Inhibition Zone Diameter in mm)

Treatments	Dose (mg/mL)	S. aureus	E. coli	S. typhi	S. sonnei
80% Ethanol extracts	200	21.30 ± 1.21^a	17.10 ± 0.58^a	19.30 ± 0.58^a	17.20 ± 1.15^a
	100	18.70 ± 0.88^a	15.30 ± 1.21^a	11.70 ± 0.33^c	14.30 ± 1.15^b
	50	14.40 ± 1.22^b	12.10 ± 0.58^c	11.30 ± 0.34^c	13.10 ± 1.15^b
	25	12.70 ± 0.88^c	9.60 ± 0.88^c	10.10 ± 1.21^c	12.20 ± 0.88^c
Chloroform extracts	200	18.50 ± 0.28^a	16.50 ± 0.29^b	18.33 ± 0.42^a	17.0 ± 0.57^a
	100	17.20 ± 0.44^a	14.20 ± 0.61^b	17.10 ± 0.57^a	17.20 ± 0.16^a
	50	14.50 ± 0.28^b	13.80 ± 0.16^b	15.50 ± 0.28^b	16.2 ± 0.01^b
	25	11.20 ± 0.44^c	11.50 ± 0.28^c	11.30 ± 0.46^c	12.80 ± 0.43^c
Positive control (PC)	5μg Ciprofloxacin	24^a	23^a	23^a	24^a
Negative control (NC)	10% DMSO	NI	NI	NI	NI

Note: NI: No inhibition.

For V. sinaiticum leaves, the ethanol extract exhibited the highest activity against S. aureus at 200 mg/mL (18.8 ± 0.44 mm) and moderate activity against E. coli at 25 mg/mL (8.3 ± 0.16 mm). The chloroform extract showed moderate antibacterial activity across all tested strains, with the strongest inhibition against S. sonnei at 100 mg/mL (17.2 ± 0.16 mm) (Table 4). Similar to R. abyssinicus, the antibacterial activity of V. sinaiticum extracts increased with concentration, and strain-specific differences were observed, with S. aureus and S. sonnei being the most susceptible, and E. coli the least.

Table 4.

Antibacterial Sensitivity Result of Crude Extracts of the Leaves of V. sinaiticum Against Experimental Bacterial Pathogens (Inhibition Zone Diameter in mm)

Treatments	Dose (mg/mL)	S. aureus	E. coli	S. typhi	S. sonnei
80% Ethanol extracts	200	18.80 ± 0.44^a	17.30 ± 0.33^a	15.20 ± 0.29^b	15.50 ± 0.29^b
	100	15.50 ± 0.29^b	13.30 ± 0.31^c	10.50 ± 0.31^c	11.50 ± 0.29^c
	50	13.30 ± 0.61^c	9.70 ± 0.33^c	9.10 ± 0.31^c	10.80 ± 0.23^c
	25	11.20 ± 0.72^c	8.30 ± 0.16^c	8.80 ± 0.44^c	9.50 ± 0.31^c
Chloroform extracts	200	14.20 ± 0.29^b	16.50 ± 0.29^b	18.30 ± 0.44^b	16.10 ± 0.57^b
	100	8.17 ± 0.61^c	14.20 ± 0.62^b	11.20 ± 0.57^c	17.20 ± 0.16^a
	50	8.80 ± 0.16^c	13.60 ± 0.16^c	NI	NI
	25	8.10 ± 0.16^c	11.50 ± 0.28^c	NI	NI
PC	5μg Ciprofloxacin	24^a	23^a	21^a	24^a
NC	10% DMSO	NI	NI	NI	NI

Minimum Inhibitory Concentration (MIC)

The MIC assay demonstrated that the ethanol extracts of R. abyssinicus roots and V. sinaiticum leaves were generally more effective at inhibiting bacterial growth at lower concentrations compared to the chloroform extracts. For R. abyssinicus roots, the ethanol extract exhibited MIC values of 16 mg/mL against S. aureus and S. typhi, and 32 mg/mL against E. coli and S. sonnei (Table 5). The chloroform extract of R. abyssinicus roots showed similar activity, with MICs of 32 mg/mL for most strains, except S. typhi, which was inhibited at 16 mg/mL.

Table 5.

MIC Result of Crude Extracts of the Roots of R. abyssinicus and the Leaves of V. sinaiticum (mg/mL)

Treatments		S. aureus	E. coli	S. typhi	S. sonnei
R. abyssinicus	80% Ethanol extracts	16	32	16	32
R. abyssinicus	Chloroform extracts	32	32	16	32
V. sinaiticum	80% Ethanol extracts	32	32	64	32
V. sinaiticum	Chloroform extracts	64	64	32	64

For V. sinaiticum leaves, the ethanol extract displayed MICs of 32 mg/mL against S. aureus, E. coli, and S. sonnei, and a higher MIC of 64 mg/mL against S. typhi, indicating a moderate antibacterial effect. The chloroform extract of V. sinaiticum showed generally higher MIC values, ranging from 32 to 64 mg/mL, suggesting lower antibacterial potency compared to the ethanol extract.

Minimum Bactericidal Concentration (MBC)

The MBC results verified the MIC findings, demonstrating that the bactericidal activity of the plant extracts was both extract and strain-dependent. For R. abyssinicus roots, the ethanol extract required 32 mg/mL to exert bactericidal effects against S. aureus and S. sonnei, whereas 64 mg/mL was necessary to kill E. coli and S. typhi. The chloroform extract of R. abyssinicus roots generally required 64 mg/mL to achieve bactericidal activity for most bacterial strains, except for S. typhi, which was inhibited at 32 mg/mL (Table 6).

Table 6.

MBC Result of Crude Extracts of the Roots of R. abyssinicus and the Leaves of V. sinaiticum (mg/mL)

Treatments		S. aureus	E. coli	S. typhi	S. sonnei
R. abyssinicus	80% Ethanol extracts	32	64	64	32
R. abyssinicus	Chloroform extracts	64	64	32	64
V. sinaiticum	80% Ethanol extracts	64	128	128	64
V. sinaiticum	Chloroform extracts	128	128	128	128

For V. sinaiticum leaves, ethanol extracts demonstrated bactericidal activity at 64 mg/mL against S. aureus and S. sonnei, while E. coli and S. typhi required higher concentrations of 128 mg/mL. The chloroform extract of V. sinaiticum exhibited the lowest potency, requiring 128 mg/mL to kill all tested bacterial strains.

Phytochemical Analysis

Qualitative phytochemical screening of the ethanol extracts of R. abyssinicus roots and V. sinaiticum leaves revealed the presence of diverse secondary metabolites with potential antibacterial activity (Table 7). The roots of R. abyssinicus were particularly rich in anthraquinones, saponins, tannins, and phenols, while steroids were absent. In contrast, the leaves of V. sinaiticum contained higher levels of flavonoids and phenols but lacked anthraquinones.

Table 7.

Secondary Metabolites Identified From the Ethanol Crude Extracts of the Roots of R. abyssinicus and the Leaves of V. sinaiticum

Phytochemicals	Test methods	R. abyssinicus extracts	V. sinaiticum extracts
Steroids	Salkowski	-	+
Glycosides	Bortanger	+	+
Anthraquinones	NaOH	++	-
Alkaloids	Wagner	+	+
Flavonoid	NaOH	+	++
Tannins	Ferric chloride	++	+
Phenol	Ferric chloride	++	++
Terpenoid	Salkowski	+	+
Saponins	Foam	++	+

Note: (-): absent; (+): present; (++): moderately/highly present.

Phytochemical Characterization

The ethanol extracts of R. abyssinicus roots and V. sinaiticum leaves were subjected to chromatographic separation using gradient solvent systems of petroleum ether and ethyl-acetate to isolate bioactive constituents.

For R. abyssinicus roots, the petroleum ether:ethyl acetate (98:2) system was most effective, yielding nine distinct fractions as visualized by TLC. Fraction 1 afforded compound RA-1, a yellow-orange crystalline solid with an Rf value of 0.46, while Fraction 2 yielded RA-2, a yellowish crystalline solid with an Rf value of 0.35, using a petroleum ether:ethyl acetate (95:5) solvent system. Further purification of Fraction 8 with petroleum ether:ethyl acetate (65:35) produced three additional compounds: RA-3, yellowish-red crystals with an Rf of 0.57; RA-4, with an Rf of 0.68; and RA-5, with an Rf of 0.56. All isolated compounds were comprehensively characterized using ¹H-NMR, ¹³C-NMR, and complementary spectroscopic techniques.

Similarly, the ethanol extract of V. sinaiticum leaves was fractionated into 14 distinct fractions. Among these, Fraction VS-1 was a light green compound (Rf 0.74), Fraction VS-2 a yellow compound (Rf 0.73), Fraction VS-3 a blue crystalline compound (Rf 0.36), and Fraction VS-4 a white to light yellow compound (Rf 0.54). These fractions were also characterized using ¹H-NMR, ¹³C-NMR, and other spectroscopic methods.

Structural Elucidation of Isolated Compounds From the Roots of R. abyssinicus Using NMR Spectroscopy

Compound 1 (Chrysophanol) was isolated as a yellowish crystalline solid. Its RF value was 0.65 in a solvent system of petroleum-ether:ethyl-acetate (7:3). The proton NMR spectrum revealed the presence of five aromatic protons at δ¹H 7.22, δ¹H 7.39, δ¹H 7.55, δ¹H 7.71, and δ¹H 7.81. Additionally, a hydroxyl group was observed at δ¹H 11.93. The ¹H-NMR spectroscopic data showed a proton signal at δ¹H 2.44 ppm, integrating for three protons, indicating a methyl group attached to C-3 of the aromatic ring. Two broad singlet peaks at δ¹H 7.55 (¹H) and δ¹H 7.22 (¹H) represented protons attached to C-2 and C-4 of the aromatic ring, respectively. The aromatic protons attached to C-5 and C-7 appeared as doublets at δ¹H 7.72 (d, J = 5, ¹H) and δ¹H 7.39 (d, J = 5), respectively. A triplet proton was observed at δ¹H 7.82 (t, J = 5, 10, m, ¹H). Additionally, the presence of two chelated protons at the peri-position of C-1 and C-8 of the aromatic ring was noted. These proton and carbon NMR spectral values confirmed the structure of Chrysophanol (Table SM-01).

Compound 2 (Emodin) was isolated as an orange crystalline solid with an RF value of 0.58 in a petroleum-ether:ethyl-acetate (7:3) solvent system. The ¹H-NMR spectrum of Emodin displayed four aromatic protons at δ¹H 7.42 (s), δ¹H 7.11 (br s), δ¹H 7.06 (d, J = 1.2), and δ¹H 6.55 (d, J = 1.2). It also revealed a methyl proton at δ¹H 2.51 and two hydroxyl protons at δ¹H 12.23 (s) and δ¹H 12.32 (Table SM-02).

Compound 3 (Physcion) was identified as a yellow crystalline compound with distinct spectral characteristics. Its IR spectrum revealed absorption bands at 3424 cm^-1 for hydroxyl (OH) stretching, 2936 cm^-1 for C-H stretching, and carbonyl absorption bands at 1680 cm^-1 (unchelated) and 1650 cm^-1 (chelated). These features indicate the presence of hydroxyl and carbonyl groups in the molecule, crucial for its structural identification and potential biological activity (Table SM-03).

Compound 4 (Helminthosporin) appeared as a bright-yellow solid with specific spectral features. Its UV-Vis spectrum showed absorption peaks at λ_max 259 nm, 289 nm, and 433 nm, characteristic of anthraquinone derivatives with conjugated systems. The IR spectrum indicated hydroxyl groups (absorption at 3468 cm^-1) and carbonyl groups (absorptions at 1672 cm^-1 and 1629 cm^-1). The ¹H-NMR spectrum displayed distinctive singlets at δ¹H 12.06 and δ¹H 12.15 ppm, corresponding to chelated hydroxyl protons attached to the aromatic ring. These spectroscopic data provide comprehensive insights into the molecular structure and functional groups of Helminthosporin, facilitating its characterization and potential pharmacological applications (Table SM-04).

Compound 5 (Chrysophanol-bianthrone) was characterized as an orange-red solid with specific spectral properties. Its TLC showed an RF value of 0.280 in n-hexane:ethyl-acetate (8:2). In UV-Vis spectroscopy (acetone), it exhibited absorption maxima at λ_max 265 nm, 330 nm, and 445 nm, indicating the presence of conjugated systems within the molecule. The IR spectrum (KBr) displayed absorption bands at various wavenumbers, including 3391 cm^-1 (hydroxyl group), 2341 cm^-1 (C=O stretching), and others indicative of aromatic and aliphatic functionalities. The ¹H-NMR spectrum (DMSO-d6) revealed signals at δ¹H 2.40 (³H, methyl group), δ¹H 6.60 (¹H, doublet, H-7), δ¹H 7.20 (¹H, broad singlet, H-2), δ¹H 7.18 (¹H, doublet, H-5), δ¹H 7.50 (¹H, singlet), δ¹H 12.01 (¹H, singlet, -OH), and δ¹H 12.15 (¹H, singlet, -OH), confirming the presence of hydroxyl groups attached to the aromatic rings (Table SM-05).

Structural Elucidation of Compounds Isolated From the Leaves of V. sinaiticum Using NMR Spectroscopy

Compound 6 (Luteolin) was characterized using UV spectroscopy, revealing absorption bands at 349 nm (Band I) and 267 nm (Band II), typical of flavones. The addition of NaOMe induced a 57 nm bathochromic shift in Band I, indicating a free hydroxyl group at position 4'. Further addition of AlCl₃ caused a 76 nm bathochromic shift in Band I, suggesting additional free hydroxyl groups at positions 5 and/or orthodihydroxyl groups. A 36 nm hypsochromic shift in Band I after HCl addition confirmed the presence of orthodihydroxyl groups. NaOAc addition led to a 13 nm bathochromic shift in Band II, indicating a free hydroxyl group at position 7. NMR analysis revealed characteristic signals, including doublets at δ 6.40 for H-6 and H-8 (A-ring), and a three-spin system in the B-ring. The ¹³C-NMR spectra closely matched those of standard luteolin, confirming Compound 6 as luteolin (Table SM-06).

Compound 7 (Chrysoeriol-7-O-glucoside) exhibits UV spectra with absorption maxima at 347 nm (Band I), 254 nm, and 267 nm (Band II), indicating a flavone structure. NaOMe addition caused a 55 nm bathochromic shift in Band I, suggesting a free hydroxyl group at position 4'. Addition of AlCl₃ resulted in a 71 nm bathochromic shift in Band I, indicating a free hydroxyl group at position 5 and/or orthodihydroxyl groups. No hypsochromic shift was observed with HCl addition, confirming the absence of orthodihydroxyl groups. NaOAc addition did not induce a bathochromic shift in Band II, suggesting the absence of a free hydroxyl group at position 7. The NMR spectrum revealed singlet signals at δ 6.40 and 6.85 for H-8 and H-3, respectively, along with meta-coupled doublets at δ 7.6 (H-2″) and an ortho-coupled doublet at δ 6.95 (H-5′). A singlet at δ 3.9 was attributed to the methoxy group. The ¹³C-NMR chemical shifts were consistent with reported data, confirming Compound 7 as chrysoeriol-7-O-glucoside (Table SM-07).

Compound 8 (Ajugol) was identified based on its ¹H-NMR spectrum in D₂O, revealing a doublet signal at δ 5.8 corresponding to H-1 with a coupling constant (J) of 1.95 Hz. A doublet of doublets was observed at δ 6.48 (J = 6.15, 1.95 Hz) and δ 5.25 (J = 6.15, 3 Hz) for H-3 and H-4, respectively. Protons on C-7 appeared at δ 2.4 (dd, J = 13.5, 6 Hz) and 2.09 (dd, J = 9.75 Hz), while methyl protons were observed as a singlet at δ 1.52. Sugar protons included H-1′ at δ 5.08 (d, J = 7.5 Hz), and H-6′ at δ 4.18 (dd, J = 12, 2.5 Hz) and δ 4.0 (dd, J = 12, 6 Hz). These spectral data were consistent with those reported for ajugol, confirming its identification (Table SM-08).

Compound 9 (Aucubin) was identified using ¹H and ¹³C-NMR spectra in D2O. The ¹H-NMR spectrum showed a doublet signal at δ 5.28 corresponding to H-1 with a J = 5.23 Hz. A doublet of doublets appeared at δ 6.32 (J = 6, 1.5 Hz) and δ 5.14 (J = 6.3 Hz) for H-3 and H-4, respectively. Multiplets were observed at δ 2.8, 4.58, 5.88, and 3.16, corresponding to H-5, H-6, H-7, and H-9, respectively, in the ¹H-NMR spectrum. The ¹³C-NMR spectrum supported these assignments. The spectral data matched those previously reported for aucubin, confirming its identity (Table SM-09).

Discussion

This study evaluated the antibacterial activity, in vivo toxicity, and phytochemical composition of ethanol and chloroform extracts from the roots of R. abyssinicus and the leaves of V. sinaiticum. Ethanol extraction yielded a relatively high product (22 g/100 g dry material), consistent with previous reports using polar solvents for these and related species.^31-33 This high yield likely reflects the abundance of polar secondary metabolites and the efficiency of ethanol in extracting phenolics, flavonoids, glycosides, and anthraquinones. The findings align well with traditional Ethiopian medicinal practices, where polar solvents such as water and locally prepared alcohols are commonly used for plant extraction.^34,35

Toxicity evaluation using G. mellonella larvae revealed moderate dose-dependent effects, with a maximum mortality of 40% at 100 mg/mL and negligible mortality at lower concentrations. These results suggest that the LD₅₀ of both extracts exceeds 100 mg/mL. Importantly, earlier in vivo studies using vertebrate models reported no acute or sub-acute toxicity for R. abyssinicus and V. sinaiticum extracts at comparable or higher doses.^6,36,37 In addition, selective cytotoxicity against cancer cell lines and hepatoprotective effects, particularly for V. sinaiticum extracts, have been documented.^38,39 Taken together, these findings indicate that while high concentrations may exert moderate toxicity in arthropod models, the extracts appear relatively safe in mammalian systems.

Ethanol extracts exhibited stronger antibacterial activity than chloroform extracts across all tested pathogens. The ethanol extract of R. abyssinicus roots showed pronounced activity against S. aureus and S. typhi, with inhibition zones exceeding 19 mm at 200 mg/mL. The finding is consistent with former studies.^6,40-42 Similarly, ethanol extracts of V. sinaiticum leaves demonstrated notable activity against S. aureus and E. coli, in agreement with previous reports.^7,14,43 In contrast, chloroform extracts displayed weak or no antibacterial activity, particularly at lower concentrations, indicating that the principal antibacterial constituents are predominantly polar in nature.

MIC values further confirmed the superior efficacy of ethanol extracts, which inhibited the growth of most bacterial strains at 16 and 32 mg/mL. Chloroform extracts consistently showed higher MIC values, indicating lower potency. Since MIC reflects growth inhibition,²⁶ these results demonstrate the enhanced antibacterial effectiveness of ethanol-derived extracts. MBC results followed a similar trend. Ethanol extracts of R. abyssinicus achieved bactericidal effects at 32 and 64 mg/mL, with S. aureus showing greater susceptibility, whereas chloroform extracts required uniformly higher concentrations. For V. sinaiticum, ethanol extracts displayed consistent bactericidal activity at 64 mg/mL, while chloroform extracts required up to 128 mg/mL. Given that MBC indicates bacterial killing,²⁷ these findings confirm that ethanol extracts possess stronger bactericidal properties. Comparable solvent-dependent antibacterial effects have been widely reported for medicinal plants rich in polar secondary metabolites.^6,7,14,40-43

Phytochemical screening of ethanol extracts revealed a diverse array of bioactive constituents. R. abyssinicus root extracts contained anthraquinones, saponins, tannins, and phenolic compounds, consistent with previous reports.^{6,8,9,32,40,44} These metabolites are well-known for their antimicrobial, antioxidant, and immunomodulatory activities. In V. sinaiticum, flavonoids and phenols were predominant, compounds widely documented for their antimicrobial and anti-inflammatory properties.^{7,13,14,31,33,36,43,45} The presence of these metabolite classes provides a mechanistic basis for the observed antibacterial activity.

Chromatographic and spectroscopic analyses (TLC, UV-Vis, IR, and NMR) enabled the identification of several pharmacologically important compounds in the ethanol extracts of both species. In R. abyssinicus, chrysophanol and emodin, prominent anthraquinones, were identified. These compounds are known to exert stimulant laxative effects by enhancing intestinal motility,^46-49 and they also demonstrate broad-spectrum antimicrobial and anti-inflammatory activities.^49-53 Additional constituents, including physcion and chrysophanol-bianthrone, further enhance the therapeutic profile of the extract. Physcion has been reported to exhibit cytotoxic effects in experimental cancer models, whereas chrysophanol-bianthrone is associated with significant antioxidant and antibacterial properties.^49,50,52-54 The detection of helminthosporin, recognized for its antifungal activity, further expands the pharmacological relevance of the roots of R. abyssinicus.^55,56

In the extracts of V. sinaiticum leaves, key identified compounds included luteolin and chrysoeriol-7-O-glucoside, both flavonoids with well-established antioxidant and anti-inflammatory activities. These metabolites contribute to cellular protection against oxidative damage, modulation of inflammatory pathways, and potential cardiovascular benefits.^50,55-60 The identification of iridoid glycosides such as ajugol and aucubin has been associated with antimicrobial and hepatoprotective effects, respectively, indicating their potential value in the management of infectious and inflammatory disorders.^61,62 Collectively, these findings provide a robust chemical basis for the observed biological activities and reinforce the therapeutic potential of both plant species.

The present study offers new outlooks into the antibacterial properties of extracts from the roots of R. abyssinicus and leaves of V. sinaiticum, utilizing detailed phytochemical profiling methods and assessing their MIC and MBC against clinically important pathogens. The results enhance the limited scientific knowledge of these medicinal plants and provide support for their traditional therapeutic applications. However, the scope was limited to in vitro assessments of antibacterial activity and preliminary toxicity evaluation, leaving the in vivo efficacy in animal models unexplored. Additionally, the use of relatively high concentrations of crude extracts (up to 200 mg/mL) in the disc diffusion assay, while common for preliminary screening, may not reflect the potency of individual bioactive compounds and should be considered when interpreting the antibacterial results. Furthermore, the study did not extend to evaluating the antibacterial activity of individual isolated compounds, nor did it address critical factors such as the stability and bioavailability of these compounds within biological systems, which are essential for their potential therapeutic application. Finally, a positive control was not included in the G. mellonella toxicity assay, which represents a limitation, as the inclusion of a known toxic agent would have validated the sensitivity of the larval batch and the experimental setup.

Conclusion

The present study demonstrates that extracts from R. abyssinicus roots and V. sinaiticum leaves exhibit substantial antibacterial activity against the tested human pathogens. The phytochemical analysis identified anthraquinones, saponins, tannins, and phenols in extracts of R. abyssinicus, while V. sinaiticum is rich in flavonoids and phenols. Fractionation and compound isolation further identified significant bioactive constituents such as chrysophanol, emodin, physcion, helminthosporin, and chrysophanol-bianthrone in R. abyssinicus, and luteolin, chrysoeriol-7-O-glucoside, aucubin, and ajugol in V. sinaiticum extracts. These findings provide a chemical basis for the observed antibacterial effects and highlight the pharmacological potential of these medicinal plants for further investigation.

Supplemental Material

Supplemental material - Phytochemical Profiling and Antibacterial Activity of Rumex abyssinicus Jacq Roots and Verbascum sinaiticum Benth Leaf Extracts

Supplemental material for Phytochemical Profiling and Antibacterial Activity of Rumex abyssinicus Jacq Roots and Verbascum sinaiticum Benth Leaf Extracts by Agumas Lemlemu, Solomon Yeshanew, Asmamaw Habtamu, Getnet Atenafu in Natural Product Communications

Footnotes

Acknowledgments

We gratefully acknowledge Debre Markos University, the University of Molise, and the Erasmus+ International Credit Mobility project for their financial, technical, and material support.

ORCID iD

Solomon Yeshanew

Ethical Considerations

This study was conducted in compliance with biosafety regulations and was approved by the Biosafety Committee of the Department of Biology, Debre Markos University, Ethiopia. All procedures involving pathogens were performed under appropriate containment conditions to ensure the safety of researchers and the environment.

Consent for Publication

All authors approved the final version for submission and publication.

Author Contributions

AL, AH, and SY conceived the study. AL conducted the laboratory investigations. AL and GA performed the statistical analysis. All authors contributed to interpreting the results and drafting the manuscript.

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 Statement

All data analyzed during this study are included in this manuscript.*

Supplemental Material

Supplemental material for this article is available online.

Appendix

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