Changes in anatomical features, phytochemical screening and antibacterial activity of certain Brassicaceae genera in different area of Iraq
محورهای موضوعی : Anatomy
Mohamed Baqer Hussine Almosawi
1
,
Osama Ghazi Abaas
2
,
Heidar Meftahizade
3
1 - College of Education for Pure Science, Al-Muthanna University, Samawah 88001, Iraq.
2 - College of Education for Pure Science, Al-Muthanna University, Samawah 88001, Iraq
3 - Department of Horticultural Science, Faculty of Agriculture and Natural Resources, Ardakan University. Ardakan, Iran
کلید واژه: epidermal, Flavonoids, MIC assays, morphological attributes,
چکیده مقاله :
This study investigates the phytochemical composition, anatomical features, and antibacterial potential of methanolic leaf extracts of three Brassicaceae species, Diplotaxis harra, Eruca vesicaria, and Lepidium coronopus collected from similar desert habitats in southern Iraq's Salman region. Anatomical analysis revealed distinctive epidermal cell morphologies, anisocytic stomata, and non-glandular trichomes between the species. Phytochemical characterization identified the presence of alkaloids (1.02–1.76%), glycosides (0.42–0.64%), tannins (4.0–10.0%), and coumarins (0.03–0.10%) while saponins were not detected in any species. Flavonoids were observed in D. harra and E. vesicaria but not in L. coronopus. The extracts were assayed for antibacterial activities against five clinically relevant bacterial strains by well diffusion assay: Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, and Staphylococcus aureus. The highest antibacterial activity was demonstrated by the methanol extract of D. harra against S. aureus (17.0 mm), and the minimum inhibitory concentration (MIC) of 1.3 mg/mL was recorded for L. coronopus against the same pathogen. The lowest activity was exhibited with the methanol extract of L. coronopus and D. harra against K. pneumoniae (6.5 mm and 6.6 mm, respectively), where MIC assays were 8.0 mg/mL and 16.0 mg/mL, respectively. These findings emphasize the therapeutic promise of Iraq Brassicaceae species, particularly D. harra, as natural sources of antibacterial compounds against antibiotic-resistant pathogens. Also, this study recommends further isolation and characterization of bioactive compounds to harness their pharmaceutical applications.
This study investigates the phytochemical composition, anatomical features, and antibacterial potential of methanolic leaf extracts of three Brassicaceae species, Diplotaxis harra, Eruca vesicaria, and Lepidium coronopus collected from similar desert habitats in southern Iraq's Salman region. Anatomical analysis revealed distinctive epidermal cell morphologies, anisocytic stomata, and non-glandular trichomes between the species. Phytochemical characterization identified the presence of alkaloids (1.02–1.76%), glycosides (0.42–0.64%), tannins (4.0–10.0%), and coumarins (0.03–0.10%) while saponins were not detected in any species. Flavonoids were observed in D. harra and E. vesicaria but not in L. coronopus. The extracts were assayed for antibacterial activities against five clinically relevant bacterial strains by well diffusion assay: Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, and Staphylococcus aureus. The highest antibacterial activity was demonstrated by the methanol extract of D. harra against S. aureus (17.0 mm), and the minimum inhibitory concentration (MIC) of 1.3 mg/mL was recorded for L. coronopus against the same pathogen. The lowest activity was exhibited with the methanol extract of L. coronopus and D. harra against K. pneumoniae (6.5 mm and 6.6 mm, respectively), where MIC assays were 8.0 mg/mL and 16.0 mg/mL, respectively. These findings emphasize the therapeutic promise of Iraq Brassicaceae species, particularly D. harra, as natural sources of antibacterial compounds against antibiotic-resistant pathogens. Also, this study recommends further isolation and characterization of bioactive compounds to harness their pharmaceutical applications.
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Changes in anatomical features, phytochemical screening, and antibacterial activity of certain Brassicaceae genera in Iraq
Mohamed Baqer Hussine Almosawi1*, Osama Ghazi Abaas1, Heidar Meftahizade2
1. College of Education for Pure Science, Al-Muthanna University, Samawah 88001, Iraq
2. Department of Horticultural Science, Faculty of Agriculture and Natural Resources, Ardakan University. Ardakan, Iran
________________________________________________________________________________
Abstract
This study investigates the phytochemical composition, anatomical features, and antibacterial potential of methanolic leaf extracts of three Brassicaceae species, Diplotaxis harra, Eruca vesicaria, and Lepidium coronopus collected from similar desert habitats in southern Iraq's Salman region. Anatomical analysis revealed distinctive epidermal cell morphologies, anisocytic stomata, and non-glandular trichomes between the species. Phytochemical characterization identified the presence of alkaloids (1.02–1.76%), glycosides (0.42–0.64%), tannins (4.0–10.0%), and coumarins (0.03–0.10%) while saponins were not detected in any species. Flavonoids were observed in D. harra and E. vesicaria but not in L. coronopus. The extracts were assayed for antibacterial activities against five clinically relevant bacterial strains by well diffusion assay: Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, and Staphylococcus aureus. The highest antibacterial activity was demonstrated by the methanol extract of D. harra against S. aureus (17.0 mm), and the minimum inhibitory concentration (MIC) of 1.3 mg/mL was recorded for L. coronopus against the same pathogen. The lowest activity was exhibited with the methanol extract of L. coronopus and D. harra against K. pneumoniae (6.5 mm and 6.6 mm, respectively), where MIC assays were 8.0 mg/mL and 16.0 mg/mL, respectively. These findings emphasize the therapeutic promise of Iraq Brassicaceae species, particularly D. harra, as natural sources of antibacterial compounds against antibiotic-resistant pathogens. Also, this study recommends further isolation and characterization of bioactive compounds to harness their pharmaceutical applications.
Key words: epidermal cell, flavonoids, MIC assays, morphological attributes
Almosawi, M.B.H., O. Ghazi Abaas, H. Meftahizade. 2025. 'Changes in anatomical features, phytochemical screening, and antibacterial activity of certain Brassicaceae genera in Iraq'. Iranian Journal of Plant Physiology 15 (3), 5515-5526.
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Historically, herbal medicines had a significant role in the management of a wide range of health problems, and today they can be receiving a renaissance in modern medicine owing to the challenge of synthetic drugs (Li and Weng, 2017). Chemical drugs can be riddled with problems such as high side effects, low utility, and microbial resistance that can make them useless (Ventola, 2015). If this is the case, rediscovery of herbal drugs and the discovery of new bioactive compounds from natural products can be one possible approach that brings to market safer and better medicine (Fabricant and Farnsworth, 2001). This potentially solves issues with current prescribed drugs and allows for opportunity to promote less toxic, more efficacious therapies. The distribution of the Brassicaceae family is mainly throughout the Mediterranean region (Martínez-Sánchez et al., 2006). This family consists of more than 2,500 species in about 350 genera around the world. There are 177 species from 80 genera of this family in Iraq, making this family one of the top ten plant families of economic value in the country (Mathree and Aliwy, 2024). This family includes important vegetables such as cabbage, lettuce, broccoli, and kale (Almasoudi and Schlosser, 2021). The genera Diplotaxis DC. and Eruca Mill. have become more popular in recent years for their value as salad vegetables (Martínez-Sánchez et al., 2006; Pasini et al., 2011). Some species of this family have anticancer properties (Heimler et al., 2006). Of these, the genus Lepidium L. is notable for its high content of bioactive alkaloids with notable pharmacological usefulness for things such as antidiabetic and anticancer uses (Muzammil et al., 2022).
Species within this family of plants show common floral features such as having 4 sepals 4 petals that are arranged in cross shape, 6 stamens, and dehiscent fruits (Ahmed and Aliwy, 2023). Much of the research emphasis of this group of species focuses on their medicinal attributes because of the variation in shape and biological activity associated with their natural compounds (Jurinjak Tušek et al., 2022). Some of these species have been traditionally utilized to treat some extremely serious health conditions. For example, Diplotaxis harra (Forssk.) Boiss. species contains cardiac glycosides and other molecules utilized for cancer, heart disease, and asthma (Badalamenti et al., 2024). Eruca vesicaria (L) Cav. also contains bioactive molecules with potentially therapeutic outcomes, though with potential side effects of nausea or decreased heart rate (Al-Snafi, 2022). Lepidium coronopus (L.) Al-Shehbaz contains over 100 different types of alkaloids, some of which exhibit anticancer, antibacterial, and antidiabetic properties. (Muzammil et al., 2022). These plants are also a good source of important nutrients like iron, phosphorus, and potassium (Yuan et al., 2021). In addition, phytochemicals such as dithiolthiones inhibit cancer by improving enzyme activity (Fahey et al., 2001)while sterols, fatty acids, and glucosinolates aid in the identification and classification of species belonging to this group (Bell and Wagstaff, 2014). In this research, phytochemical content and antibacterial activity of some Brassicaceae species from southern Iraq are examined. By comparison of some species and their applicability, this research elucidates their significance in nature and their potential in the future to be utilized in medicine and pharmaceuticals.
Material and Methods
Sampling sites
Despite being situated within the arid Samawa desert, the three sampling sites in the Salman area exhibit notable ecological variation due to differences in topography, soil composition, moisture availability, and exposure to wind and solar radiation. The northern region (31.5°N, 45.5°E) is slightly elevated, characterized by compact sandy-loam soil with moderate drainage and occasional rainfall runoff, which enhances soil moisture retention and supports more continuous vegetation across partially stabilized sand dunes. In contrast, the southern region (30.5°N, 45.5°E) features looser, sandier soils with low water-holding capacity, heightened solar radiation, and pronounced temperature fluctuations, all of which contribute to increased wind erosion and drought stress, thereby selecting for xerophytic plant adaptations. Meanwhile, the eastern region (31.48°N, 44.9°E), located near sedimentary flatlands, contains fine silt deposits that produce compacted and less permeable soils, often accompanied by saline patches resulting from shallow groundwater. These conditions affect surface temperatures and ion uptake, fostering salt tolerance in local flora. Collectively, these microhabitat differences impose varied environmental pressures that drive morphological, physiological, and genetic adaptations in plant species, contributing to the ecological and evolutionary diversity observed within this desert ecosystem
Plant sample collection
Plants of three Brassicaceae species Diplotaxis harra, Eruca vesicaria, and Lepidium coronopus were collected during their pre-flowering stage (March–April) from three distinct microhabitats within the Salman region of the Samawa desert, southern Iraq. All three species were sampled across the following coordinates: Northern region: 31.5°N, 45.5°E, Southern region: 30.5°N, 45.5°E, and Eastern region: 31.48°N, 44.9°E.
The observed variations in plant traits likely reflect habitat adaptations. Plant samples were washed, and rotten leaves were discarded. Identification was made using the Flora of Iraq and the herbarium of the University of Baghdad. Leaves were air-dried (25-30 °C), powdered, and kept in labeled containers with species name, part used, and date. Samples were kept in the herbarium as well.
Moisture content was measured by weighing 10 grams of fresh leaves, air-drying, and re-weighing, applying the formula: Moisture (%) = [(Initial weight − Dry weight) / Initial weight] × 100
For pH analysis, a mixture of 10 g powder and 50 mL distilled water was prepared, shaken for ten minutes, filtered, and the pH was measured (Agafonova et al., 2019). Crude extracts and alkaloids’ MIC against pathogenic bacteria were tested. ANOVA and LSD at 0.05 levels of significance compared MIC variations (Sokal and Rohlf, 1987).
Plant extract preparation
The hot alcoholic extraction technique in Azwanida (2015) was employed. Through this procedure, 30 g of each powder of a plant species was individually weighed and loaded on top of the Soxhlet equipment loaded with 250 mL of 80% methanol. It was extracted for 7 hours at 60 °C. After filtration with Whatman No. 1 filter paper and evaporation in rotary-evaporator to yield a sticky solution. The solution was left to dry to a solid at room temperature to yield a dry powder, which was used as the crude extract.
Microorganisms
Bacterial reference strains were acquired from Baghdad University of Botany. Five pathogenic strains of clinical isolates (E. coli, P. vulgaris, K. pneumoniae, S. aureus, and P. aeruginosa) were employed. They were preserved at 4 °C and then subcultured on agar plates (Mueller-Hinton agar) at 37 °C for 24 h prior to any experiments were performed.
Test for of alkaloids
Alkaloids were detected following the standard protocol by Das et al.(Das et al., 2020). Ten grams of dried leaves from each plant species were boiled in 50 mL of 4% hydrochloric acid (HCl) prepared in distilled water. The mixture was filtered, cooled, and then assayed using the following reagents: white precipitate formation with Mayer's reagent indicating alkaloid presence (Lifongo et al., 2014).
Test for glycosides
The detection of glycosides was carried out using the Evans (2009) protocol. During the first test, equal quantities of plant extract were used and mixed with Fehling's solution, and a red precipitate formed after 10 minutes of boiling, proving the presence of glycosidic compounds. For confirmatory test, 5 mL of the extract was mixed with a few drops of Kedde's reagent. Purple coloration shows the presence of glycosides.
Test for essential oil
The essential oils were assayed as per the method described in the Indian Ayurveda Pharmacopoeia (Joshi et al., 2017). Ten (10) ml of each filtered leaf extract was spilled over filter paper and subjected to ultraviolet radiation at 365 nm wavelength. The development of a pale pink color was taken as an indicator of the essential oil presence.
Test for tannins
The tannin was detected according to Das et al. (Das et al., 2020) protocol. Five grams of leaf extract from every plant was pounded and boiled afterwards with 50 mL of distilled water. The extract was cooled and filtered. The resulting filtrate was equally divided into two equal portions:
• To the first half, 1% lead acetate solution was poured.
• To the second half, 1% ferric chloride solution was added.
Flavonoid determination
Flavonoid identification was performed according to Evans (Evans, 2009). During the process:
• Solution A: 10 g of leaf powder was dissolved in 5 mL of 95% ethanol
• Solution B: A mixture of 10 mL of 50% ethanol and 10 mL of 50% KOH solution was prepared.
To detect flavonoids, solutions A and B were mixed in equal proportions. The presence of yellow color showed the presence of flavonoid compounds.
Test for coumarin
Coumarin identification was performed as described by Zhang et al. (2024). A small amount each of the plant extracts was pipetted into test tubes and filter paper was wetted with diluted NaOH solution. The test tubes were heated in a water bath for a few minutes. The filter paper, while being heated, was exposed to ultraviolet light. Yellow-green fluorescence in ultraviolet light indicated the presence of coumarins.
Test for resin
Resins were identified according to the process described by Zhang et al. (2024). A 10 mL aliquot of each of the extracts was blended with 20 mL of acidified distilled water (4% HCl). Solution turbidity is evidence of the presence of resins.
Detection of saponin was done according to Cheok et al. (2014). For this test, the plant extract was shaken thoroughly in a test tube. The presence of stable foam indicates the presence of saponins. The addition of 0.5 mL HgCl₂ to 1.5 mL extract resulted in a white precipitation, confirming saponin presence.
Media preparation
Nutrient agar (23 g, Sigma-Aldrich, Germany) was dissolved in 1 L distilled water by boiling. The solution was autoclaved (121 °C, 15 min), left to cool down to room temperature, then poured into Petri dishes at ∼45 °C. Plates were left to solidify for 30-40 min on a flat surface.
Antibacterial assay
The antimicrobial activity of plant extracts and alkaloid fractions was determined by the disc diffusion method. Bacterial strains' overnight culture was diluted to an equivalent turbidity of 0.1 at 600 nm (A600) or approximately 3.2 × 10⁸ CFU/100 μL. Suspension of 20 μL was uniformly spread over sterile plates of nutrient agar (20 mL/plate) with sterile cotton swabs. Plates were dried by incubation for 3 minutes before use.
Sterile 5 mm diameter discs of filter paper were loaded with 100 μL of plant extracts (40 mg/disc) and placed on inoculated agar plates. Reference antibiotic discs (kanamycin, 30 μg/disc) were used. plates were maintained at 37 °C (standard microbiological temperature) for a 24-hour incubation period. Inhibition zone diameters were determined in millimeters by using a ruler after incubation. The experiments were conducted in triplicate, and the results were presented as means ± standard deviation.
Minimum inhibitory concentration (MIC)
Quantitative antibacterial activity was assessed by standardized broth dilution methods. Methanolic extracts of D. harra, L. coronopus, and E. vesicaria were made in serial dilutions between 1.3 and 32 mg/ml and were added to the nutrient broth with test bacterial strains. The tubes were incubated for 24 hours at 37 °C, after which turbidity check showed bacterial growth.
Anatomical methods
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Results
Fig. I. Yield and percentage of different Brassicaceae species |
