IN VITRO ASSESSMENT OF ANTIMICROBIAL AND ANTIOXIDANT ACTIVITIES OF AERIAL PARTS EXTRACTS OF YEMENI FAGONIA SCHWEINFURTHII HADIDI

Salwa M. Raweh1image, Hend Ahmed Alhaj2image, Ali Gamal Al-Kaf3image

1Pharmacognosy department. Faculty of Pharmacy, Sana’a University, Yemen.  

2Modern and Global Pharma research Centre, Sana’a, Yemen.

3Medicinal chemistry department, Faculty of pharmacy, Sana’a University, Yemen.

ABSTRACT 

Background: Antioxidant and antimicrobial properties of plant extracts are attributed to bioactive components derived from medicinal plants. This study inspected into the antimicrobial and antioxidant effects of extracts from Fagonia schweinfurthii Hadidi aerial portions.

Method: Several solvents, including n-hexane, ethyl acetate, and methanol, were utilized sequentially to extract secondary metabolites from F. schweinfurthii aerial parts. Antimicrobial activity was evaluated using the well diffusion method and broth serial dilution, whilst antioxidant activity was evaluated using the 2, 2-diphenyl-1-picrylhydrazyl radical scavenging method.

Results: The findings exposed that the studied fungal strains (Candida albicans and Trichophyton rubrum) were resistant to all plant extracts. F. schweinfurthii methanol and ethyl acetate extracts demonstrated inhibitory effects on Gram-positive and Gram-negative tested bacteria with minimum inhibitory concentration between 2.5 and 20 mg/ml. In addition, the most sensitive bacterium was Proteus vulgaris, with an inhibitory concentration (2.5 and 5 mm). While the most resistant bacterium was Staphylococcus epidermidis. Due to the physical and chemical properties of the solvents, different extracts of F. schweinfurthii aerial parts exhibited diverse antioxidant capabilities in the antioxidant activity experiment. Methanol and ethyl acetate extracts exhibited IC50 values of 236 ± 0.2µg/ml and 359.4± 0.6µg/ml, respectively.

Conclusion: According to the findings of this study, the aerial portions of F. schweinfurthii could be considered a possible source of natural antioxidants as well as a valuable source of antibacterial agents against bacteria that cause ear infections. 

Keywords: Antimicrobial, Antioxidant, DPPH, F. schweinfurthii.

 

INTRODUCTION

 

During their physiological processes, plants produce a variety of secondary metabolites, including alkaloids, polyphenols, flavonoids, terpenoids, and carotenes, among others1. These secondary metabolites are a potent source of anticancer2, antioxidant3, antiviral4, anti-inflammatory5, and antimicrobial agents6. The prevalence of life-threatening infections produced by microbial pathogens has increased globally, and among developed regions, it is now a significant cause of death in immunosuppressed patients7.

In order to minimize the spread of infectious diseases worldwide, antimicrobial drugs are crucial8. Although fewer, or even occasionally, ineffective antimicrobial treatments are available for the infection caused by pathogenic bacteria, the development and spread of multidrug-resistant (MDR) strain have become a significant public health threat9,10. Folk medicine offers a valuable and underdeveloped resource for research-ing and developing potential new treatments for microbial infections to reduce the evolution of drug resistance and adverse medication effects. Additionally, because these may be more readily available, more economical, and more accessible, the use of medicinal plants opened the potential for the developing world11. Free radicals can negatively impact lipids and proteins, leading to peroxidation, which can result in unfavorable alterations to cells and gene mutation12, premature aging, tissue damage, and inflammation13. These free radicals ultimately cause diabetes mellitus, Alzheimer's disease, cancer, and other neurological disorders14,15,16. Because of their capacity to stop or reduce oxidative damage, many synthetic dietary antioxidants have gained recognition. The most widely used, such as tert-butyl hydroquinone (TBHQ), butylated hydroxyl toluene (BHT), butylated hydroxyl anisole (BHA), and tocopherol, have been banned in the food industry because they are thought to cause liver damage and cancer17. It was difficult to build the sustainability idea to screen natural, abundant, low-value raw products. Vegetables, fruits, and biomass from forests all provide significant sources of bioactive compounds, notably natural antioxidants13, 18.

Fagonia schweinfurthii Hadidi (family Zyophyllaceae) and its closely related species are found throughout the deserts and dry regions of South West U.S.A., Chile, and tropical Africa19. People in the desert have traditionally used the plant to cure skin eruptions, sores, skin diseases, antipyretic, analgesic, ear infections, venereal ailments, and other conditions20.

The antibacterial, antiviral, analgesic, anti-inflammatory, cooling, antioxidant, anticancer, and thrombolytic effects of Fagonia species have been demonstrated21. Phytochemical investigation suggests that F. schweinfurthii extracts contain flavonoids, tannins, saponins, alkaloids, steroids, amino acids, carbohydrates, and cardiac glycosides22. Pareek et al.,23 investigated the DPPH radical scavenging activity of F. schweinfurthii Hadidi aerial parts ethanol extracts, where results showed antioxidant activity with an IC50 of 200.27µg/ml. Mothana et al.,24 reported that methanol extract of Yemeni Fagonia indica leaves displayed a significant concentration-dependent DPPH scavenging activity.

Al Ghanem25 tested F. mollis petroleum ether, methylene chloride, ethyl acetate, acetone and methanol extracts for antibacterial activity against pathogenic bacterial strains (Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus) and fungal strains (Candida albicans, Mucor spp., Aspergillus fumigatus, and A. Aspergillus niger). All microorganisms were suppressed by acetone, except Escherichia coli and Staphylococcus aureus. The plant's methanol extract displayed broad-spectrum antibacterial action on studied microbes. On the other hand, Aspergillus fumigatus and Aspergillus niger were unaffected by Petroleum ether extract, which inhibited the growth of Candida albicans and Mucor spp. While the methylene chloride, ethyl acetate, and methanol showed no antifungal action.  On the other hand, A. fumigatus experienced growth inhibition from acetone extract, whereas the other fungi were unaffected. 

This study aims to investigate F. schweinfurthii's potential as an antioxidant and antimicrobial agent for the treatment of ear infections.

MATERIALS AND METHODS

 

F. schweinfurthii aerial parts were obtained in September 2019 from resident areas in Sana'a, Yemen. Dr. Hassan Ibrahim recognized the plant in the Biology Department, Sciences College, Sana'a University.

Test Organisms

Two-gram +ve bacteria (S. aureusATCC 25923 and S. epidermidis RCMB 009(2), gram-ve bacteria (E. coli ATCC 25922, P. vulgarisATCC 13315), as well as two fungi, including one filamentous fungus belonging to dermatophytes (Trichophyton rubrum RCMB 025002) and one yeast species (Candida albicans ATCC 10231), obtained from the culture collection of the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt, were used in this study.

Preparation of plant extract:

One kilogram of fresh aerial parts was cleaned thoroughly with tap water, dried at room temperature, and ground into a fine powder with an electric blender. Before subsequent applications, the powder was kept in a cool place and away from light. The powdered aerial parts were soaked in 5 L of n-hexane, ethyl acetate, and methanol respectively. The samples were submerged at room temperature for three to seven days with constant stirring. This procedure was repeated three times to increase the extraction efficiency; following filtration, the extracts were concentrated at a temperature of 40°C in a rotary evaporator and dried in an oven at 37°C. For further examination, the dried extracts were weighed and kept at 4°C26. 

In vitro antimicrobial activity assay:

Agar Well Diffusion Assay:

Antimicrobial activity on Sabouraud Dextrose Agar (SDA) for fungi and Mueller Hinton Agar (MHA) for bacteria was investigated using the agar well diffusion method. Both media were made following the manufacturer's instructions, boiled to dissolve, and autoclaved at 121°C for 15 minutes at 15 psi. After cooling to 45°C, the sterilized media was aseptically placed into an appropriate number of labeled sterile Petri dishes and allowed to harden.

The test organisms were subcultured into Sabouraud Dextrose Broth (SDB) for fungi and Nutrient Broth (NB) for bacteria before being incubated at  25°C for 24-48 hours  and 37°C for 18-24 hours, respectively, before being analyzed. Using the McFarland standard, each organism was standardized to a turbidity of 0.5 x 108 cells/ml in saline solution (0.85 % NaCl) (through visual comparison). Using a sterilized cotton swab, 0.1 ml of the standardized suspensions were used to inoculate the surfaces of the 90mm-diameter MHA and SDA plates respectively. Each agar plate was punctured with sterile cork borer tools, each with 6mm diameter well. Each hole was filled with a 50µl, 20mg/ml methanol, ethyl acetate, and n-hexane extract of F. schweinfurthii.

Commercial antibiotics (gentamicin and ketoconazole) were employed as positive controls for bacteria and fungi, respectively, to test the sensitivity of the isolates, while DMSO was used as negative controls. After allowing the extract to diffuse into the agar for 5 hours at room temperature, the plates were incubated at 37°C for 18-24h for bacteria and 25°C for 24-48h for fungi, except yeast (Candida species), which was incubated at 37°C. The inhibition zones were measured in millimeters after incubation using a meter rule. The entire experiment was done three times, and the zone of inhibition's mean values was calculated27.

Minimum Inhibitory Concentration Determination:

The MIC was obtained specifically for the extracts and isolates that demonstrated inhibitory action. The MIC of the effective extracts was measured using the broth dilution method27. The extracts were tested against the bacteria at various concentrations ranging from 2.5 to 20 mg/ml. The extract-free broth served as a negative control, while the conventional antibacterial drug (gentamycin) served as a positive control. The minimum inhibitory concentration was determined after a 24-hour incubation at 37ºC as the lowest concentration that demonstrated no observable growth using turbidity as a measure.

Determination of antioxidant activity:

The free radical scavenging activity of F. schweinfurthii methanol, ethyl acetate, and n-hexane extracts was determined in vitro using the 2,20-diphenyl-1-picrylhydrazyl (DPPH) test .Sample stock solutions (1.0 mg/mL) in methanol were diluted to final concentrations of (500, 250, 125, 62.5µg/mL).One milliliter of a 0.3 mM DPPH methanol solution was added to 2.5 ml of various concentration sample solutions and allowed to react at room temperature. The absorbance values at 518 nm were taken after 30 minutes and transformed to percentage antioxidant activity (AA) using the following formula28: DPPH solution (1.0 mL; 0.3 mM) plus methanol (2.5 mL) was used as a control and   the reference compound ascorbic acid was   also measured. All

measurements were made in triplicate and averaged.

 

RESULTS

 

Antimicrobial Activity Assay:

Antimicrobial activity of F. schweinfurthii aerial parts was tested against gram +ve bacteria (S. aureus and S. epidermidis), gram -ve bacteria (E. coli and Proteus vulgaris), and fungal strains (Trichophyton rubrum and Candida albicans) causing an ear infection. Antimicrobial activity of F. schweinfurthii methanol, ethyl acetate, and n-hexane extracts at 20 mg/ml was also compared to conventional antibiotics (Gentamicin) and antifungals (Ketoconazole) and results are provided in Table 1 and Table 2. All three F. schweinfurthii aerial extracts were ineffective against Trichophyton rubrum and Candida albicans. On the other hand, Ketoconazole showed an inhibitory zone (11.8±0.6 and 21.3±1.7) against Trichophyton rubrum and Candida albicansrespectively. The methanol extract was the most effective against S. aureus, S. epidermidis, P.vulgaris, and E. coli, with inhibition zone values of 15.3±1.21, 9.13±0.75, 13.4±0.8, and 7.8±0.6 as compared with other tested extracts. Ethyl acetate extract was less efficient against S. aureus (11.2±0.8), P. vulgaris (10.3±1.1), and E. coli (7.5±0.5), With no activity against S. epidermidis. In contrast, n-hexane was inactive against all tested bacteria except P. vulgaris (11.8±1.27). 

Minimum Inhibitory Concentration (MIC):

The bacterial strains sensitive to F. schweinfurthii extracts were tested for the minimum inhibitory concentration (MIC). The MIC for F. schweinfurthii extracts against sensitive bacterial strains is indicated in Table 3. The minimum inhibitory concentration of methanol extract was 20mg/ml against E. coli and 2.5mg/ml against S. aureus and Proteus vulgaris. In contrast, MIC for S. epidermidis was 10 mg/ml. Ethyl acetate extract's MIC against E. coli was 20 mg/ml, and its MIC against S. aureus and P. vulgaris was 5 mg/ml. On the other hand, n-hexane extract had MIC value of 5mg/ml against P. vulgaris.

Antioxidant activity assay:

The antioxidant activity of the plant extracts was assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH). The antioxidant activity of extracts from F. schweinfurthii aerial parts was concentration depen-dent, with methanol extract showed the highest activity level (IC50=236±0.2 µg/ml), followed by ethyl acetate extract (IC50=359.4± 0.6 µg/ml), while, n-hexane extract had no antioxidant activity (Table 3). In contrast to other concentrations examined, the highest DPPH scavenging potential (86.83%, 66.56%) was observed at 500 µg/ml of methanol and ethyl acetate extracts, respectively. While, the lowest DPPH scavenging potential (18.62±0.54%, 9±0.2%) was achieved with the methanol and ethyl acetate extracts at 62.5 µg/ml correspondingly. All the studied extracts had appreciable antioxidant scavenging abilities at lower levels than ascorbic acid.

 

DISCUSSION

 

The systematic and proper screening of numerous extracts derived from various medicinal plants is necessary to search for novel antibiotics. The methanol extract of F. schweinfurthii showed potential antibacterial activity against all tested bacterial strains that cause ear infections (S. aureus, S. epidermidis, P. vulgaris, and E. coli). In contrast, the ethyl acetate extract was only effective against S. aureus, P. vulgaris, and E. coli. However, n-hexane extract, was only effective against P. vulgaris. These results supported those of Shad et al.,29, who found that n-hexane extract of F. oliveri had no effect against S. aureus, E. coli, P. aeruginosa, S. typhus, and B. subtilis. Similarly, Ur Rehman et al.,30 reported that methanol extract of Fagonia cretica exhibited the greatest antibacterial activity against tested bacterial strains.  The methanolic extract demonstrated the highest antibacterial activity, indicating that the polar components of the crude extract predominated over the non-polar ones, confirming the traditional methods of use that rely on the aqueous extract as preferable for public use. However, the findings of this investigation contradicted those of Kouser and Quershi31 that the methanol extract of Fagonia indica has no activity against S. aureus or S. epidermis. Still, the n-hexane and ethyl acetate extracts show activity against S. epidermis with MICs of 2.5 and 1 mg/ml, respectively. Different plant species, growing areas, and bacterial strains could take all account for these antibacterial activity variances. The presence of tannin, alkaloids, saponins, and flavonoids in F. schweinfurthii extracts may explain its antibacterial activity32, Which is similar to the findings of Doughari and Manzara33 who found a correlation between antibacterial activity and phytoconstituents (alkaloid, saponin, phenol). In immu-necompromised individuals, persistent opportunistic fungal infections have become a major cause of morbidity and mortality34,38-40. Extracts of F. schweinfurthii aerial parts were tested for antifungal activity against Candida albicans and Trichophyton rubrum. Ketoconazole was employed as an antifungal standard. F. schweinfurthii aerial parts extracts did not inhibit fungal growth, meaning they are inactive against tested fungal strains. These findings matched those25, which indicate that ethyl acetate and methanol extracts of Fagonia mollis exhibited no antifungal activity against Aspergillus fumigatus, Aspergillus niger, Candida albicans, and Mucor spp.

Similarly, Shad et al.,29 found that methanol extract of Fagonia Oliveri had no activity against Candida albicans, and hexane fraction had no activity against many fungal strains tested (T. longifusus, C. albicans, C. glaberata, F. solani, and A. flavus). The current investigation results demonstrated that n-hexane extract lacked antioxidant activity while methanol and ethyl acetate extracts had concentration-dependent antioxidant activity. A similar outcome was obtained by El-Amier & Abo Aisha35 who discovered that as plant extract concentration increased, F. arabica, F. criticus, and F. mollis methanolic extracts' capacity to scavenge free radicals increased constantly. 

The maximum antioxidant activity was found in the methanol extract (IC50=236±0.2µg /ml), followed by the ethyl acetate extract (IC50=359.4±0.6 µg/m), whereas the n-hexane extract had no antioxidant activity. Current findings on antioxidant activity correspond with those of Pareek et al.,23. However, the 

results of the present study contradicted those of a study36. Diverse plant species, locations used for collecting, and extraction techniques could all contribute to this variation in activity. Flavonoids and phenolic compounds may be responsible for F. schweinfurthii's antioxidant action37. Additionally, methanol extract of Yemeni Fagonia indica leaves has been revealed to include flavonoids and saponins responsible for the antioxidant action24.

Limitations of the study

Difficulty getting standard bacteria and fungi.

 

CONCLUSION

 

This study shows that several extracts from F. schweinfurthii aerial parts have various antioxidant and  antibacterial activities. According to the findings, methanol extract had the highest levels of antibacterial and antioxidant capabilities. Additionally, F. schwein-furthii extracts could inhibit bacteria linked to ear infections and may provide scientific support for the plant's traditional usage in folk medicine. Also, it is proposed that additional research be conducted on the isolation and identification of antioxidant components contained in plant materials for application in both the animal and human nutraceutical industries.

 

ACKNOWLEDGMENTS

 

The authors wish to acknowledge Dr. Safwan Al-Aghbari, the manager of Modern and Global Company's development and research center, for his support with this study. 

 

AUTHOR’S CONTRIBUTIONS

 

All authors claim to be held accountable for all aspects of the study, assisted with data analysis, wrote, updated, and evaluated the paper, and provided final approval before publishing. 

 

CONFLICT OF INTEREST

 

There is no conflict of interest related to this work.

 

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