In vitro phytochemical analysis and antibacterial and antifungal efficacy assessment of ethanolic and aqueous extracts of Rumex nervosus leaves against selected bacteria and fungi

Background and Aim: Scientists are interested in identifying natural antibiotic substitutes that are effective against drug-resistant pathogenic microbes and spoilage fungi to counter pathogens and reduce the major public health problem of antibiotic residues in animal products. This study aimed to evaluate the antimicrobial activity of Rumex nervosus leaves (RNLs) as a medicinal herb against four bacterial and two fungal strains using absolute ethanol, 50% ethanol, and aqueous extracts. Materials and Methods: The antimicrobial activities of various RNL extracts against selected microbes were evaluated using the disk diffusion antibiotic susceptibility test, minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), minimum fungicidal concentrations, and the poisoned food technique. Results: The absolute ethanol RNL extract showed the best bacteriostatic/bactericidal activity against Salmonella Typhimurium, Escherichia coli, and Staphylococcus aureus (MIC/MBC: 0.20/0.40, 0.20/0.40, and 0.32/0.65 mg/mL, respectively). The diameter of the zone of inhibition was larger (p < 0.05) for the 100% ethanol RNL extract (8.17 mm) against Salmonella Typhimurium, the 50% ethanol-RNL extract (11.5 mm) against E. coli, and the aqueous RNL extract (14.0 mm) against S. aureus than for any other bacterial isolate. The aqueous RNL extract strongly (p < 0.0001) inhibited the mycelial growth of Aspergillus fumigatus (100%) and Aspergillus niger (81.4%) compared with the control. Conclusion: The results of this study suggest that RNL is a promising new natural antimicrobial agent for food preservation. To date, most research on the antimicrobial properties of natural herbs has been conducted in vitro, with few exceptions in vivo and intervention-based research.


Introduction
Foodborne pathogens such as Escherichia coli, Salmonella, Listeria monocytogenes, and Staphylococcus aureus are among the hazards of major concern in the food sector worldwide [1]. Due to their potential pathogenicity, they have a significant impact on public health, on the cost of treating these infections, and on the economic consequences for exporters of food or meat. Listeria monocytogenes has a high mortality rate (about 20%) in humans and is considered as one of the most serious foodborne diseases [2]. Between 1999 and 2019, Salmonella was the most common foodborne pathogen in African food exports to the European Union [3]. Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, parasites, and other pathogens have been reported to cause illness [1]. Between 1998 and 2008, poultry products accounted for the most foodborne illness and human deaths in the United States out of 17 commodities [4]. Fungal contamination in food, feed, and other agricultural commodities leads to massive spoilage or decay and a range of food safety problems [5]. In addition, mycotoxins are secondary fungal metabolites that are potentially toxic and pose a high risk of ergot poisoning, aflatoxicosis, leukocyte deficiency, and other human diseases [6,7]. Although therapeutic antibiotics are available against infectious diseases, many researchers throughout human history have been interested in treating a wide range of infectious diseases and pain with herbal remedies [8][9][10]. However, Available at www.veterinaryworld.org/Vol.15/November-2022/25.pdf antibiotic resistance is a major public health problem caused by antibiotic residues in animal products. The widespread, excessive, and indiscriminate use of commercially available antimicrobial drugs to cure infectious diseases has led to the emergence of antibiotic-resistant bacteria [11,12]. Therefore, scientists are searching for new sources, such as natural antimicrobial agents (phytocompounds) that are active against drug-resistant pathogenic microbes and spoilage fungi to counteract pathogens and reduce these problems [13].
Various medicines or active ingredients are extracted from herbal and traditional medicines to treat diseases, improve health, and support body functions [14]. Essential oils (EOs) from garlic, onion, and cinnamon show effective anti-biofilm activity against L. monocytogenes and are promising natural antimicrobial alternatives for food processing plants [15]. As a result, medicinal plants have become increasingly important in maintaining global health [13,16]. Researchers are interested in using natural antioxidants found in plants and other biological materials because they are considered safe and effective in overcoming pathogen and nutrient resistance, as well as therapeutically helpful [17]. Due to their extensive biological and therapeutic properties (bioactive compounds), herbal medicines are an important source of health care in both developed and developing countries [13]. According to recent statistics from the World Health Organization, herbal medicines and dietary supplements are used by more than 80% of people worldwide as part of their primary health care [18]. The antimicrobial activity of medicinal plants needs to be tested to identify potential new compounds for therapeutic use [19]. Rumex nervosus is a medicinal shrub used throughout Asia and North Africa to cure a variety of ailments, such as inflammatory diseases [20,21]. Rumex nervosus, a plant used in folk medicine, was selected by Yemenis [21,22] and Saudis [23] to test its antimicrobial activity. It contains active compounds such as gallic acid, which can be used to treat chronic and infectious diseases and inflammatory conditions [24]. Although R. nervosus possesses antibacterial, antioxidant, and anti-inflammatory properties [25], its potential as an antifungal agent remains to be tested. Although the antimicrobial activity of R. nervosus has been investigated, a comparison of the activity of ethanol extracts of R. nervosus leaves (RNL) with different degrees of polarity (100%, 50%, and 0% ethanol) at high concentrations has not yet been studied in detail.
Therefore, this study aimed to evaluate and compare the different RNL extracts for their antimicrobial activity against four bacteria (S. aureus, L. monocytogenes, E. coli, and Salmonella enterica subspecies Typhimurium) and two fungal isolates (Aspergillus fumigatus and Aspergillus niger).

Ethical approval
This study does not require animal ethics approval because this study was conducted in vitro without using humans, animals, or tissues derived from them.

Study period and location
The study was conducted from February to November 2020. This study was conducted in Poultry Health Lab., at the Animal Production Department, King Saud University (KSU), located at Riyadh, Saudi Arabia, (24.92°N latitude and longitude of 46.72°E, 600 meters above sea level). In this region, the average annual rainfall is 110.6 mm, and the mean temperature of 26.4°C with a humidity of 65%.

Preparation of RNL powder
Rumex nervosus leaves were collected from the valleys and mountains around Bait Al-Aqra village in Ibb region of Yemen. RN exsiccate registration No. 23033, registered by botanist Dr. Jacob Thomas at King Saud University's Department of Botany, Faculty of Science. The harvested RNL was air-dried under sunlight every 5 h for 15 days. Then, the dried leaves were ground into a fine powder (particle size; 0.25-0.30 mm) using a blender (Nima Electric Super Speed Dry Spices Grinder) in our laboratory.

Preparation of RNL extracts
The ground RNL powder extracts for the antimicrobial tests were prepared according to the method described by Muthuswamy et al. [26]. Rumex nervosus leaves extracts were prepared using three solvents: Absolute ethanol (R100EOH), 50% ethanol (R50EOH), and pure distilled water (DW) alone [Rumex water extract ( RWA)]. Then, 150 g of RNL powder was weighed and distributed to each vials (50 g/vial). Each vial was thoroughly mixed in a tightly stoppered flask and dissolved separately at a ratio of 1:10 (w/v) in 500 mL sterile DW for water extraction, 500 mL ethanol/water (2:2, v/v) for ethanol/water extraction, and 500 mL absolute ethanol for ethanol extraction. The flasks were then boiled under reflux at 37°C in a water bath for 30 min, 45 min, and 5 h, respectively. The supernatants were then collected and filtered through 0.22 µm Millipore membrane filters (Whatman filter paper # No. 1) into new sterile flasks. The filtrate was then dried completely or evaporated at 45°C in an oven or with a rotary evaporator (Eyela, Irvine, California, USA). Before in vitro antimicrobial assays, the dried herbal extracts were weighed and dissolved in dimethyl sulfoxide (DMSO) to obtain stock concentrations of 500 mg/mL of each crude plant extract or stored in sealed bottles for later use.

Percentage yield of RNL extraction (% yield)
The yield (%, w/w) of each dry extract was calculated according to the following formula: Yield (%) = (W 1 *100)/W 2 , where W 1 is the weight of extract after drying with solvent (lyophilization) and W 2 is the weight of RNL powder before extraction [27].

Determination of the total phenolics of RNL
The Folin-Ciocalteu colorimetric method was used to determine the concentration of total phenols in an ethanol extract of RNL powder [28]. For the extract, 25 mL of ethanol was added to 1 g of RNL powder and allowed to stand overnight at room temperature (24°C). A 200 µL of the ethanol solution was pipetted into a flask. Then, 4 mL of DW and 1 mL of Folin-Ciocalteu reagent (1 N) were added and shaken for 10 min. After 3 min, 1.0 mL of 20% sodium carbonate was added to the mixture. After 1 h of incubation at 24°C and shaking, absorbance was measured using a spectrophotometer at 760 nm. Measurements were performed in duplicate, the calibration curve was prepared using gallic acid, and the results were expressed in gallic acid equivalent (GAE)/mg.

Phytochemical analysis (gas chromatography-mass spectrometry [GC-MS] and high-performance liquid chromatography [HPLC]) and proximate analysis of RNL
High-performance liquid chromatography was used to identify, separate, and dosing of chemical compounds in the RNL extract mixture. Gas chromatography-mass spectrometry assay was used to determine the chemical composition as described by Adaszyńska-Skwirzyńska and Szczerbińska [29]. To determine the macronutrients and nutritional value of RNL, proximate analysis was performed as described by Melesse et al. [30].

Microorganisms and preparation of bacterial inoculum
The reference strain of two Gram-positive bacteria: S. aureus ATCC 29737 and L. monocytogenes ATCC 13932 and two Gram-negative bacteria: E. coli ATCC 25922 (E. coli) and S. enterica subspecies Typhimurium ATCC 14026 were obtained from standard stocks in our laboratory, College of Food and Agriculture, King Saud University.
Bacterial inoculum was prepared according to the standard protocol of the Clinical and Laboratory Standards Institute (CLSI) [31]. The bacterial stock cultures were activated by suspension in nutrient broth for 24 h before subculturing on sterile nutrient agar plates. To ensure that the bacteria were fully activated, sub cultivation was repeated 3 times. The inoculum suspension for each assay was prepared by taking 4 or 5 pure colonies from a fresh culture. The colony suspensions were emulsified in sterile saline and successively diluted until turbidity was comparable to a 0.5 McFarland turbidity standard. This resulted in inoculum density of nearly 10 8 colony-forming units (CFU)/mL.

Screening for antimicrobial activity (disk diffusion antibiotic susceptibility test)
The RNL extracts (absolute ethanol, 50% ethanol, and water extract) were screened for antibacterial activity against various bacteria (S. aureus, L. monocytogenes, E. coli, and Salmonella Typhimurium) using the agar disk diffusion method (ADDM) based on the standard guidelines of the National Committee for Clinical Laboratory Standards [32] with minimal modifications. After adjusting the turbidity of the inoculum suspension using McFarland turbidity standards, the adjusted microbial suspensions were carefully blotted onto sterile Mueller-Hinton agar plates using a sterile cotton swab. The inoculum was dried in a safety cabinet (Class II biological safety cabinet from NuAire, Plymouth, MN, USA) for 30 min. Circular antibiotic test disks of 6 mm diameter were prepared using Whatman filter paper (No. 3). Following Alhajj et al. [33], the sterile dried disks were aseptically applied to the inoculated agar plates after being soaked in 40 µL of the respective RNL extract. The plates were left at 4°C for 1 h before incubation at 37°C for 24 h. The diameter of the inhibition zones (DIZ) was then measured (in mm). DIZs of <12, 12-16, and >16 mm were classified as having weak, moderate, and strong antibacterial activity, respectively [34]. Microbes were tested for susceptibility to the standard antibiotic piperacillin (100 mg) as a positive control (PC). Paper disks soaked with the appropriate extractant (DMSO) were used as negative control (NC). The tests were performed in triplicate.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) of the tested bacteria
The CLSI method and the microdilution technique (a microplate method) were used to determine the MIC of the RNL extracts [35]. The MIC was determined by selecting the lowest concentration of each RNL extract that did not cause visible bacterial growth [36]. The bacteria in the adjusted inoculum suspensions were further diluted (1:100) to 10 6 CFU/mL. The assay was performed as described by Bekkar et al. [37]. Wells with absorbance values comparable to the PC indicated turbidity or bacterial growth, while wells with the lowest extract concentrations and absorbance comparable to the NC indicated complete growth inhibition and were considered MIC values.
MBC is defined as the lowest antimicrobial concentration required to completely inhibit bacterial growth (without bacterial turbidity). To calculate MBC, 10 µL of inoculum from wells without bacterial growth was inoculated into sterile nutrient agar plates. Plates inoculated with bacteria served as PC, while plates containing only nutrient agar served as NC. Wells that did not show growth were used to determine MBC. Bacterial growth was measured after incubation at 37°C for 48 h. The experiment was performed in triplicate.

Antifungal activity
To test the activity of RNL extracts against the pure fungal strains A. niger ATCC 6275 and A. fumigatus ATCC 28282, the ADDM, microtiter plate method, and poisoned food technique were used. The MIC and minimum fungicidal concentrations (MFCs) were also determined. Aspergillus fumigatus and A. niger were obtained from the Food Microbiology Laboratory of the Department of Food and Nutritional Sciences, College of Food and Agriculture Sciences, King Saud University.

Preparation of fungal spores
The fungal suspension was prepared according to the methodology of the European Committee on Antimicrobial Susceptibility Testing-Antifungal Susceptibility Testing Subcommittee against Aspergillus spp.
(EUCAST-AST-ASPERGILLUS) [38]. Briefly, fresh, mature (2-5 days old) cultures were grown on sterile potato dextrose agar at 35°C to prepare inoculum suspensions. Colonies were covered with 1 mL of sterile water, to which 0.1% Tween 20 was added. The conidia were collected rationally with a sterile cotton swab and transferred to a sterile tube. The inoculum was homogenized with a rotating vortex blender at 376× g for 15 s. Then, the conidia were diluted with sterile water, counted in a hemocytometer and examined for hyphae and clumps. If a significant number of hyphae or clumps were present (>5% of fungal structures), the inoculum was filtered through a sterile nylon mesh filter with a pore size of 11 mm. Then, the suspension was adjusted to 2-5 × 10 6 CFU/mL with sterile DW by counting the conidia in a hemocytometer. This suspension was then further diluted 1:10 with sterile DW to obtain a final working inoculum of 2-5 × 10 5 CFU/ mL. All adjusted suspensions were quantified by plating on SDA plates.
Miconazole nitrate (20 mg/g) was dissolved in DMSO at a concentration of 20 mg/mL and stored at −20°C. The stored solutions were then diluted to the concentrations used in this study. The final concentration of DMSO in the reaction mixture was limited to <1% (v/v) [39].

Antifungal susceptibility testing using the disk diffusion method
This technique was used to determine the suppressive activities of RNL extracts against A. niger and A. fumigatus, according to Naeini et al. [40]. Briefly, 10 µL of an adjusted suspension of A. niger or A. fumigatus was evenly distributed on potato dextrose agar plates. The inoculated plates were dried at 24°C in a safety cabinet. Then, blank paper disks with a thickness of 5 mm were impregnated with 40 µL of selected herbal extracts on the surface of the previously inoculated plates. The plates were sealed with parafilm and incubated at 35°C for 48-72 h. Each plate was examined after 48-72 h of incubation. The DIZ, as well as the diameter of the disk were measured. Miconazole nitrate and DMSO-soaked paper disks were used as positive and NC disks, respectively. The experiment was performed in triplicate.

Determination of MICs and MFCs of the tested fungi
Minimum inhibitory concentrations of RNL extracts on A. niger and A. fumigatus were determined using a microplate method as described by Lass-Flörl et al. [38]. The adjusted inoculum suspensions (containing conidia or sporangia spores) were mixed with a vortexer (2-5 × 10 5 CFU/mL). Rumex nervosus leaves extracts were dissolved in DMSO (10% of the final volume) and diluted to a concentration of 500 mg/mL. Sterile 96-well apartment microtiter plates (Nunclon, Denmark) were used for this assay. Each well contained 100 µL of sterile Roswell Park Memorial Institute Medium 1640 with 2% glucose as a test medium. Then, serial 2-fold dilutions (the concentration range obtained was between 250 and 0.24 mg/ mL) of each stock solutions of the extracts (500 mg/ mL) were performed. Then, except for the NC, 100 µL of the adjusted inocula were added to each well. The diluted inoculum suspensions without extracts served as active controls, and the medium without extracts was used as NC. The plates were incubated for 48 h at 35°C in the incubator. The MIC was defined as the lowest concentration of extracts in the wells that showed no visible growth.
From the wells that showed no growth, 10 µL were taken and inoculated onto potato dextrose agar plates for the determination of MFC. The plates inoculated with fungi served as PCs, and the plates with potato dextrose agar alone served as NC. Plates were incubated at 35°C for 7 days, and fungal growth was assessed. The MFC was defined as the highest dilution (lowest concentration of extracts) at which no growth occurred on the plates. The test was carried out in three replicates.

Evaluation of the antifungal activity of RNL extracts by poisoned food technique
According to Shrestha and Tiwari [41], the antifungal activity of the extracts was evaluated by the poisoned food technique. A 0.5 mL of each extract (250 mg/mL concentration) was poured into the Petri plate, followed by the addition of 9.5 mL of sterile melted potato dextrose agar medium to a final volume of 10 mL and then gently swirled to achieve thorough mixing of the contents. After solidification, a 5 mm diameter A. niger or A. fumigatus inoculum disk was aseptically inoculated upside down into the center of the plates and incubated at 30°C for 7 days. The plates with potato dextrose agar without extracts served as NC, and the plates with miconazole nitrate served as PC. Subsequently, the average diameters of fungal growth were measured on the 7 th day of incubation and then used to calculate the inhibition of mycelial growth [42]. The percentage (%) of inhibition of mycelial growth was determined as [(g c -g t )/g c ] × 100, where g c is the average increase in mycelial colony growth in the NC and g t is the average increase in mycelial colony growth on the plates treated with the selected extracts.

Statistical analysis
Statistical Analysis System software 9.4 (SAS Institute Inc., NC, USA) Statistical Analysis System [43] was used to analyze the in vitro data. Antibacterial and antifungal efficacy was expressed as mean ± standard deviation, and one-way variance analysis of variance (ANOVA) was used to distinguish between groups. Means of data were analyzed for statistical differences using Duncan's new multiple range test. p < 0.05 was considered statistically significant.

Extracts yield
The extraction solvent had a significant effect on the extract yield. For each extraction, 150 g of RNL powder was extracted and concentrated in an oven at 45°C. The final weight of the extracts was 32.00, 33.00, and 34.20 g for R100EOH, R50EOH, and RWA extracts, respectively, corresponding to a percent extract yields of 21.23, 22.00, and 22.80 (w/w).

Total phenolic content
The total phenols in the leaves of R. nervosus were 56.31 µg/mg dry weight. The total phenolic content in the ethanolic extract was 171.15 mg GAE/g for RNL.

Nutrient analysis and phytochemical composition of RNL powder
As shown in Table-1, RNL contained 94.3% dry matter or 5.67% moisture. Rumex nervosus leaves contained 82.0% organic matter and 18.01% inorganic matter (crude ash). In addition, RNL contained 8.24% crude fiber, 13.6% crude protein, and 1.54% ether extract. The acid detergent fiber content was 15.5% and the neutral detergent fiber content was 20.2%. The nutritional value was 3273.31 calories. The results of proximate analysis of the dried leaves of R. nervosus indicate that they have the potential to be used as a dietary supplement.

Screening for antimicrobial activity
The activities (growth inhibition zones) of the absolute ethanolic, 50% ethanolic, and aqueous extracts     different plant species were found to have different antimicrobial activities against the tested bacteria.
Depending on the sensitivity of the bacteria tested, all bacterial strains were sensitive to RNL extracts to varying degrees. However, the RNL extracts had no significant effect on L. monocytogenes isolates. R100EOH showed the least antibacterial activity among the extracts, with a DIZ of 6.7-8.2 mm. However, RWA was superior to R100EOH and R50EOH against S. aureus. Antibacterial inhibitory activity against S. aureus increased with increasing polarity (inhibition zone 6.7, 9.0, and 14.0 mm for R100EOH, R50EOH, and RWA, respectively). The diameter of the zone of inhibition was larger (p < 0.05) for R100EOH against Salmonella Typhimurium (8.17 mm), R50EOH against E. coli (11.50 mm), and RWA against S. aureus (14.00 mm) compared to other bacteria tested.

Microplate dilution test
The MIC and MBC values of the tested medicinal plant extracts against the studied microbial strains are shown in Table- were very resistant to RNL extracts. Rumex nervosus leaves extracts had an antimicrobial activity with bactericidal properties observed at different concentration ranges against all clinical bacteria tested in this study. The results suggest that the phenols, flavonoids, and tannins present in RNL extracts may have increased toxicity against pathogenic microorganisms.

Antifungal activity
The antifungal activity of RNL extracts against the tested fungi (A. fumigatus and A. niger) using the ADDM and microtiter plate methods is shown in Table-6. The antifungal activity of three RNL extracts tested for A. fumigatus and A. niger showed no practical activity in the sensitivity test. R100EOH had no effect on the fungal growth of A. fumigatus and A. niger when tested with ADDM, but it was the most fungi static and fungicidal when tested with MIC and MFC.
The inhibitory effects of the three RNL extracts on the mycelial growth of selected fungi using the food poisoning method are presented in Table-7. The inhibitory activities of the three RNL extracts against the mycelial growth of A. niger were not significantly different from each other, but they had a potential activity compared with the control. In this in vitro  Rows with different letters indicate statistically significant differences (p < 0.05). The concentrations of each extract were in the range of 500-0.12 mg, 500 mg (1), 125 mg (2) experiment, miconazole was also used as a standard for antifungal activity and it displayed high activity (15 ± 0.82 mm for A. fumigatus and 20 ± 1.87 mm for A. niger). The inhibition of mycelial growth of the tested fungi was 81.43-100%, 28.20-78.27%, and 40.17-80.59% for the extract of RWA, R50EOH, and R100EOH, respectively.

Discussion
The search for herbal remedies with antimicrobial properties has increased due to their potential benefits in alleviating a variety of chronic and acute infectious diseases. The current investigation focused on a well-known medicinal plant, namely, R. nervosus. Rumex nervosus is used in Yemen, Saudi Arabia, and other neighboring countries.
It is well known that phenolic compounds are the major antioxidant plant constituents, and it has been found that the antioxidant activity of plant materials is highly correlated with their phenolic compound content. Phenols have also been shown to be effective in the treatment of chronic diseases [44]. Our results show that the leaves of R. nervosus have a high content of total phenols. In RNL, the total phenolic content in ethanolic extract was 171.15 mg GAE/g [45]. Based on the above findings, RNL can be considered attractive for food and pharmaceutical applications due to its high content of antioxidants. The presence of physiologically active chemicals, especially phenolic compounds, which vary with genetic diversity and the region from which they were collected, explain the differences in antioxidant activity between samples [46]. The ethanolic extracts from RNL showed antimicrobial activity against selected bacteria and fungi. This could be due to the fact that RNL is rich in bioactive compounds and has higher antioxidant activity.
In this study, the antimicrobial activity of RNL extracts was investigated using three solvents (absolute ethanol, 50% ethanol, and water) against Gram-positive bacteria such as S. aureus and L. monocytogenes, Gram-negative bacteria (Salmonella Typhimurium and E. coli), and fungal isolates (A. fumigatus and A. niger). In addition, the extracts were tested for their antimicrobial activity using ADDM and plate dilution. The different RNL extracts showed different levels of inhibition against the selected bacteria. A positive result was observed when the zone of inhibition had a diameter of >6 mm. Based on the results obtained, the RWA extract had the highest antimicrobial activity against S. aureus, which is consistent with the results of this study based on the polarity of the solvents, and it can be assumed that the lower the polarity of the extracts, the higher the antibacterial activity [47]. Although 100% ethanol is a good organic solvent for the extraction of most compounds, it is not as polar as water. The extraction yield, antioxidant properties, radical scavenging activity, and phytochemical content were affected by the polarity of the extraction solvent because the antioxidant compounds in plant parts have a high affinity for more polar solvents compared to nonpolar ones [47]. They found that extraction with more polar solvents (water extract) resulted in a high yield but low flavonoid and phenolic content compared to non-polar solvents. These results indicate that absolute ethanol or a 50% (v/v) water/ethanol mixture are the best solvents for extracting high content of extractable solids from RNL, in contrast to a water solvent alone, which gave a high yield of extractable solids.
The obtained results showed that RWA exhibited significant broad-spectrum antibacterial activity against S. aureus and narrow-spectrum antibacterial activity against Gram-negative bacteria using ADDM. The findings of this study are in agreement with a previous Saudi study [47] that found the higher antibacterial activity of polar (aqueous) extract of R. nervosus against Grampositive bacteria (S. aureus) and low activity against other Gram-negative bacteria (E. coli) using ADDM. In addition, organic extracts such as ethanol and hexane extracts of R. nervosus were found to inhibit the growth of microorganisms. However, the organic extract, especially the hexane extract, had lower efficacy against Gram-negative bacteria. Similarly, the methanol extract of R. nervosus showed lower efficacy against Gramnegative bacteria [48]. Al-Nowihi et al. [21] showed that the ethanolic and methanolic extracts of R. nervosus have high antimicrobial activity against the bacteria S.  [49,50].
In this study, RWA showed superior antimicrobial activity against S. aureus, while R50EOH showed superior activity against E. coli compared to other Rumex extracts. However, R. nervosus extracts showed low activity against other organisms, such as Salmonella Typhimurium and L. monocytogenes. Since ethanol is a colorless volatile liquid, the antibacterial activity of R. nervosus extract seems to be more inhibited in ethanol than in methanol and appears to be more inhibitory for Gram-positive bacteria than for Gram-negative bacteria [21].
In this case, the antibacterial activity of R. nervosus extracts was limited to Gram-negative bacteria, although it showed a zone of inhibition against all Gramnegative bacteria, Gram-positive bacteria, and fungal strains. With the exception of S. aureus, R. nervosus was found to be less effective against Gram-negative and Gram-positive bacteria, and not effective against fungi when used with ADDM. The MIC was used to determine the effectiveness of the extracts on the bacterial and fungal strains tested. Only organisms that had a zone of inhibition in the previous ADDM and were sensitive to the herbal extracts were tested for MIC [22]. All the extracts tested were found to have antimicrobial activity. The MIC of the R100EOH extract against Salmonella Typhimurium and E. coli was 0.20 mg/mL, but against S. aureus was 0.32 mg/mL.
Here, the extracts of R. nervosus inhibited Grampositive and Gram-negative bacteria, with no effect on A. fumigatus and A. niger, supporting reports from Ethiopia and Saudi Arabia against S. aureus, Pseudomonas aeruginosa, and Streptococcus pyogenes, but not C. albicans [22,51].
Rumex nervosus extracts showed the highest DIZ (20 mm) against the genus Salmonella compared to Plantago lanceolata, Lepidium sativum, and Solanum incanum [52]. They found that the MIC and MBC of R. nervosus extracts against S. aureus were 1.56 mg/mL. Rumex nervosus also showed the highest MIC against many organisms tested [22].
Several species of Rumex (family Polygonaceae) have antimicrobial activity. However, ADDM results exhibited that the EOs have a broad spectrum of antimicrobial activity against pathogenic bacteria. The spectrum of antimicrobial activity varies due to the presence of different minor and major constituents in EO of each plant, which is due to the extraction method, plant species, plant part, microbial strains, geographical origin and environmental conditions, test conditions, test procedures and concentrations used, etc. [53][54][55]. In studying the antimicrobial activities of 22 EOs, Yousef and Tawil [56] discovered discrepancies between ADDM and dilution tests. Rios et al. [57] found that ADDM is not suitable for samples that are difficult to dissolve in water or for nonpolar samples. In addition, there was no correlation between diffusivity and antimicrobial activity nor between MIC values and growth inhibition diameter. The ADDM, on the other hand, was ideal for screening because it has a small sample size and can test multiple compounds against a single microorganism. A dilution method was recommended to determine MIC and MBC with the greatest precision and suitability for nonpolar samples such as EO [57,58]. Therefore, the microplate dilution assay was used in this study to evaluate the efficacy of antimicrobial activity. Moreover, the R100EOH minimally inhibited the selected bacteria at a much lower concentration than the 50% ethanol extract and the water extract, except for L. monocytogenes. In addition, the R100EOH had a lethal effect (MBC) on most of the bacteria studied at a much lower concentration than the R50EOH extract and the RWA. However, the RWA showed the highest lethal effect on L. monocytogenes. This extract proved to be both inhibitory and bactericidal. As indicated by the lowest MIB and MBC values, Salmonella Typhimurium and E. coli appeared to be the most sensitive bacteria to RNL. As a result, Salmonella Typhimurium and E. coli were more sensitive to R100EOH than S. aureus and L. monocytogenes.
These data indicate that Gram-negative bacteria were more sensitive to RNL extracts than Grampositive bacteria. Nevertheless, comparisons of bacterial susceptibility may differ. This is due not only to the antimicrobial agents in EO but also to the diversity of microorganisms tested [53,59]. Another factor that may have contributed to the different results was the type of media used to test antimicrobial activity. The growth conditions for the bacteria may differ when different media are used. For example, Khakhanang et al. [58] used a nutrient broth medium in their study, Kasimala et al. [60] used a Mueller-Hinton agar medium in their study, and a nutrient agar medium was used in this study. These aforementioned factors may be responsible for the conflicting results in each case. However, the promising antimicrobial properties of EO, especially the extracts of RNL reported here, could recommend their use as a substitute for antibiotics in the treatment of infectious diseases in animals. However, the toxicity and safety of the use of these extracts must be considered. In addition, the results obtained indicate that RNL has antimicrobial activity against bacteria and fungi, which means that they could be used as antimicrobial agents.
The minimum inhibitory concentration of RNL ranged from 0.20 to 10.41 mg/mL for the absolute ethanol extract, 0.82-15.63 mg/mL for the 50% ethanol extract, and 6.51-7.81 mg/mL for the water extract. The MBC of RNL ranged from 0.40 to 20.81 mg/mL for the absolute ethanol extract, 1.63-31.26 mg/mL for the 50% ethanol extract, and 13.02-15.62 mg/mL for the water extract. This is somehow similar to the report of Liang et al. [61], who observed MBC values ranging from 20 to 160 mg/mL for ethanol extracts. Moreover, MIC and MBC of RNL extracts ranged from 0.20 to 13.02 for Gram-negative bacteria and from 0.32 to 31.26 mg/mL for Gram-positive bacteria. In contrast, Alhajj et al. [33] reported that MIC and MBC values ranged from 7.8 to 31.2 mg/mL for both types of bacteria.
Traditional healers could prepare their medicines with water because they do not have access to more lipophilic solvents [62]. This is of concern because it is possible that the healers do not extract all the active ingredients contained in the plant. Dosage is a critical factor in choosing which solvent to use. Several studies have shown that organic solvents are superior to aqueous extracts in terms of antibacterial activity [61,63,64]. Thus, the dosage would be higher if a water extract is used, while the same dosage could be dangerous if a lipophilic solvent extract is used. For rural communities and informal settlements, it is beneficial to determine the antibacterial and antifungal properties of plants used in traditional medicine.
Several researchers have used this method to evaluate the antifungal activity of herbs [65,66]. According to Gwa and Ekefan [67], some plant extracts (neem, tobacco, ginger, papaya, black pepper, and Mancozeb) have fungicidal activity against yam disease caused by A. niger. The results of food poisoning with RNL extracts suggest that RNL extracts can be considered as effective natural agents to inhibit mycelial growth compared to control.
The chemical composition of the methanolic RNL extracts in this study was investigated by HPLC and GC-MS, which led to the identification of gallic acid and 13 compounds, respectively. In the HPLC results, gallic acid has the highest peak area fraction. The presence of various phenolic acids and flavonoids in mushroom ethyl acetate extracts was quantified by HPLC [68]. Due to the negative effects of free radicals and toxic byproducts of their metabolism on various metabolic processes [69] during the immune response of host cells to microbial invasion, phenolic compounds may play an important role in the defense against microbial infections and in averting tissue damage and cytotoxicity [70].
Our GC-MS results for phytochemical analysis show that saturated octadecanoic acid and unsaturated octadecanoic acid, also known as 9, 2-octadecadienoic acid or omega-6 unsaturated fatty acid, have the highest peak area fraction. In addition, other fatty acids, including pentadecanoic acid (pentadecylic acid), hexadecanoic acid (also known as palmitic acid), and other phytochemicals, including methoxyphenyl-oxime, cyclotrisiloxane, 5-methyl-2-phenylindolizine, and 1,2-bis(trimethylsilyl) benzene, were present in moderate amounts. The tested bacteria and fungi can be killed or inhibited by the powdered leaves of R. nervosus. At low concentrations, the powder extracts of the leaves have been found to have antifungal and antibacterial properties. The presence of a secondary plant compound in RNL powder is responsible for its activity. According to Krishnaveni et al. [71], GC-MS analysis of phytochemicals and fatty acid profiles of natural plants have antimicrobial activity in killing bacteria and fungi. Basil hexane extract and ethanol extract had the best antibacterial activity and lowest MIC concentration. Therefore, they were selected as the most effective antibacterial extracts for GC/MS analysis to determine their chemical constituents [72]. In the GC-MS analysis of the methanolic extract of RNL, 13 compounds were detected, whose activity and mode of action against pathogenic microbes, either alone or in combination with other antimicrobial substances, need to be further investigated.

Conclusion
The three extracts of RNL herb had varying degrees of inhibitory activity against the selected bacteria and were resisted by the tested fungi. R100EOH showed good bacteriostatic and bactericidal activity. This antimicrobial activity can be attributed to the presence of the main bioactive constituents of RNL, especially gallic acid. It is concluded that RNL can be developed as natural alternatives for further use in food preservation to prevent microbial contamination in the food industry and to extend the shelf life of food as novel preservatives that can replace synthetic bactericidal or fungicidal agents. Field studies are recommended to test the bioactive extract against the selected bacteria or fungi. In addition, the results of this study would help to develop new antimicrobial agents in the future.