Asian Journal of Plant Sciences

Year: 2023 | Volume: 22 | Issue: 1 | Page No.: 48-55
DOI: 10.3923/ajps.2023.48.55

Synergistic Effect of Some Medicinal Plant Extracts on Escherichia coli and Staphylococcus aureus

Mohamad M. Abou Auda , Tarek A. Elbashiti and Samar Y. Alghuff

Abstract: Background and Objective: The discovery of antibiotics helped in the combat against bacterial infections that cause various diseases, but infectious bacteria formed a resistance against these drugs due to their misuse, there has been a growing interest in the study and use of medicinal plants in the world. Eleven plant extracts were screened for antibacterial efficacy and synergistic effect against Escherichia coli and Staphylococcus aureus in this study. Materials and Methods: Ethanolic and aquatic extraction were carried out in the microwave-assisted method. The antimicrobial activities were assessed by disc and agar well diffusion technique, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The synergistic assay was carried out by mixing Coriandrum sativum extract with Artemisia monosperma extract and Cuminum cyminum extract with Ocimum basilicum extract. Results: The findings demonstrated that S. aureus was less susceptible to the plant extracts employed than E. coli. Except for Syzygium aromaticum, which demonstrated a broad zone of inhibition on the bacteria and a confluence between the ethanol extract findings and those of the aquatic extract in both the disc and well diffusion procedures, aqueous extract inhibits more bacteria than ethanolic extract. When compared to Syzygium aromaticum, Hibiscus sabdariffa displayed the lowest MIC value in aqueous extract when compared to ethanolic extract. MBC values against E. coli and S. aureus for the majority of plant species in each ethanolic and aquatic extract were >200 mg mL^–1. Conclusion: Furthermore, the current study looked at the usefulness of synergistic effects of various extracts against pathogenic bacterial isolates. The findings of employing the disc and well diffusion method and a low MIC value demonstrated a mild synergistic impact of various plant extracts.

Keywords: MIC, MBC, ethnobotany, Medicinal plant, synergism, antimicrobial and ethanolic extract

INTRODUCTION

Because traditional herbal medicine is an important part of all cultures’ histories, determining the original uses and local names of plants has significant potential societal benefits¹. Antibiotics are the primary treatment for microbial (bacterial and fungal) infections. Bacterial resistance to currently available antibiotics has necessitated the search for new antibacterial agents². As a result, several countries of the world have exhibited a high degree of interest in studying medicinal plants and their historic uses.

Modern pharmaceuticals are sometimes unavailable or too priced in impoverished nations. Furthermore, as a result of the country’s poor economic situation, drug prices are constantly rising. As a result, emerging nations rely heavily on derivatives of medicinal plants that are more acceptable to the local populace³.

Peptides, aldehydes, alkaloidal components, water, ethanol, chloroform, methanol essential oils and phenols have all been discovered in plants. These plants have been demonstrated to be useful in treating bacterial, fungal and viral infections in humans⁴.

The WHO chose the official terms traditional, complementary and integrative medicine (TCI) in mid-2017 for its traditional and complementary medicine unit to include integrative approaches T&CM in policy, knowledge and practice⁵.

Numerous studies have demonstrated the potential use of plant extracts in the treatment of some diseases caused by bacterial infection, these studies confirmed the effect of these extracts on bacterial growth, demonstrating that some plants in Palestine have a significant impact on this infectious bacteria⁶.

In the late 19th century, research studies were held to study the qualities of particular plants in terms of their resistance to specific bacteria for the first time. These discoveries were currently regarded as a trustworthy source of contemporary medicine⁷.

Even though the Middle East is the World’s Driest Region, there are over 13,500 plant species found in Middle Eastern countries, these plants are distinguished by their capacity to withstand extreme weather conditions. Other environmental conditions, such as temperature and water availability, affect these plant extracts. Because of the climatic circumstances in which these plants grow, extracts from Middle Eastern plants may include several diverse chemicals with a greater range of biological functions⁸.

Medicinal plants play an important role in primary health care in Palestine, as it has application in many developing countries and are widely used as alternative medicine in traditional Palestinian herbal remedies for health care and treatment of a variety of diseases, including chronic diseases⁹.

Plant types indigenous to Palestine have a considerable effect on Palestinian culture and economics. In Palestinian cuisine, several wild plant species are commonly employed. Many people in rural regions continue to rely on medicinal plants to cure burns, sickness and other maladies¹⁰.

It is worth noting that a small number of research have demonstrated that merging antimicrobial medicines with crude plant extracts boosts their efficiency against some pathogenic species¹¹.

As a result, the current study focused on the effect of several extracts on E. coli and S. aureus bacteria as well as the synergistic effect of some plants on these pathogenic bacterial isolates.

MATERIALS AND METHODS

Study area: The study was carried out in the Gaza Strip, an area of 365 km². The Gaza Strip is a narrow strip of land on the mediterranean coast. It borders the occupied lands to the East and North and Egypt to the South. It is approximately 41 km long and between 6 and 12 km wide¹². The study was carried out at the Department of Biology of the Islamic University of Gaza (IUG), Palestine from August, 2017 to March, 2018.

Plant material collection: The following plants were used to make the extracts: The following herbs were purchased from Al-Zawea popular market: Ocimum basilicum (Basil), Hibiscus sabdariffa (Roselle), Trigonella foenum-graecum (Fenugreek seed), Coriandrum sativum (Coriander), Anethum graveolens (Dill), Syzygium aromaticum (Clove), Glycyrrhiza glabra (Licorice), Cuminum cyminum (Cumin), Cymbopogon schoenanthus (Camel grass), Boswellia sacra (Olibanum) and Artemisia herba-alba (white wormwood) (Table 1).

Culture media and chemicals: Mueller Hinton broth and Mueller-Hinton agar were used to culture bacteria. Ethanol and distilled water were also employed in the extraction procedure and triphenyl tetrazolium chloride (TTC) was used as a microbiological growth indicator. As a solvent, Dimethyl Sulfoxide (DMSO) was used.

Microorganisms: Two bacterial isolates, one Gram-positive S. aureus and one Gram-negative E. coli, were studied. The bacterial isolates were taken from the Department of Biology and Biotechnology at the Islamic University of Gaza (IUG) and the Microbiology Department at Al-Shifa Hospital.

Plant extract: Ten grams of the crushing plant were put in a beaker with 150 mL of solvent (ethanol or distilled water) and subjected for 1 min in a microwave extractor. It was taken and chilled at room temperature for 1 min before being placed back into the microwave apparatus 12 times. The same methods were performed eleven times with plant samples using 80% ethanol and distilled water¹³. To eliminate the solvent, the extracts were transferred to a clock glass and baked at 45°C overnight.

In 5 mL of dimethyl sulfoxide, one gram of each extract was dissolved (DMSO). As a result, a standard concentration for extracts of 200 mg mL^–1 stock was achieved¹⁴. The dried extracts were kept in sterile glass vials at -20°C until they were used.

Antimicrobial effect of plant extracts using agar disc diffusion assay: To evaluate the antimicrobial activity of the selected plant extracts against the test bacterial isolates, sterile filter paper discs about 6 mm in diameter were impregnated with stock extracts solution and put on the surface of an MHA infected with the test bacteria. The antibacterial activity of the plant extracts was assessed after 18-24 hrs by detecting zones of growth inhibition around the extracts¹⁵. The assay was repeated twice.

Antimicrobial effect of plant extracts by agar well diffusion assay: The antibacterial activity of chosen plant extracts against test microorganisms was determined using the well diffusion technique. Following solidification, 6 mm diameter wells were cut into the agar and test plant extracts were put into the wells the plant extracts were evaluated at a concentration of 100 mg mL^–1. The antibacterial activity was measured after 24 hrs by measuring the width of the inhibitory zone produced around the plant extracts well¹⁶. The assay was repeated twice.

Detection of MIC and MBC of plant extracts by microdilution assay: The micro dilution technique was used to determine the minimum inhibitory concentration (MIC), which is the lowest concentration of the plant extract at which bacteria does not display detectable growth in the 96-well plates. Each plant sample had a final concentration ranging from (200-0.1953 mg mL^–1). Except for the positive control, 10 μL of overnight growth microorganism inoculum was introduced to each well. Negative control was inoculated with media, while a positive control was extracted with media¹⁷. After 24 hrs of incubation at 37°C, 50 μL of a 0.01% triphenyl tetrazolium chloride (TTC) solution was added to each well as an indicator and the plate was incubated for 1 hr. The colourless tetrazolium salt is converted to a red product by the living bacterial strains, growth inhibition can be identified when the solution in the well remains clear following incubation¹⁶.

To determine MBC, the lowest concentration of the plant extract at which the inoculated bacteria were completely killed, MHA was inoculated with broth from each well and incubated for 24 hrs at 37°C. MBC was detected as the lowest concentration that inhibited bacterial growth after sub-culturing. Negative controls included a 10% DMSO solution¹⁸.

Synergistic assay: The synergistic effect of the extracts was performed according to Elbashiti et al.¹¹ with slight modifications. The antimicrobial activity of the extracts against the tested microorganisms was compared. The antimicrobial activity was evaluated using well diffusion and disc diffusion assays and MIC after mixing 50 g of Coriandrum sativum extract with 50 g of Artemisia monosperma extract and 50 g of Cuminum cyminum extract with 50 g of Ocimum basilicum.

RESULTS

Evaluation of the antibacterial activity of the plant extracts by disc diffusion method: Antibacterial activity of aquatic and ethanolic extracts of all eleven plants was evaluated separately against two isolated bacterial species, each experiment repeated at least 3 times.

Against E. coli: The aqueous extracts of all the plants tested exhibited varying inhibitory effects ranging from (8-17 mm) in diameter, with (Syzygium aromaticum) showing the greatest impact against E. coli (with a 17 mm zone of inhibition), followed by (Hibiscus sabdariffa, Artemisia monosperma and Coriandrum sativum) (with an 8 mm zone of inhibition). Anethum graveolens, Boswellia carterii, Cuminum cyminum, Trigonella foenum-graecum and Ocimum basilicum had the lowest activity (with a 7 mm zone of inhibition). Both Cymbopogon schoenanthus and Glycyrrhiza glabra had no impact (with a zero mm zone of inhibition). The ethanolic extracts of Syzygium aromaticum L., were the most effective against E. coli showing the highest antibacterial activity with an inhibition zone diameter of 17 mm were extracts of Hibiscus sabdariffa with an inhibition zone diameter of 8 mm, no effect was shown for Ocimum basilicum, Boswellia carterii, Cuminum cyminum, Trigonella foenum-graecum, Anethum graveolens, Artemisia monosperma, Coriandrum sativum, Glycyrrhiza glabra and Cymbopogon schoenanthus (Table 2).

Against S. aureus: Hibiscus sabdariffa aqueous extracts had the greatest antibacterial activity against S. aureus bacteria, with an inhibitory zone width of 10 mm, followed by Syzygium aromaticum. With an inhibitory zone width of 9 mm, but no antibacterial activity has been demonstrated for the remaining plant extracts. Only Syzygium aromaticum which was extracted using ethanol showed an inhibition zone diameter of 12 mm and Hibiscus sabdariffa, with a zone of growth inhibition of 7 mm, showed antibacterial action against S. aureus, whereas, the remaining plant extracts showed no antibacterial activity (Table 2).

Antimicrobial effect of plant extracts using agar disc diffusion assay: The antibacterial activity of aqueous and ethanolic extracts of all eleven plants was investigated using the well diffusion technique against two isolated bacterial species. Each experiment was repeated at least 3 times.

Against E. coli: The aquatic extracts of all the plants showed various inhibitory effects against E. coli ranging in diameter from 11-25 mm. The Syzygium aromaticum exhibited the greatest zone of inhibition and had the largest inhibition zone diameter at 25 mm, followed by Hibiscus sabdariffa, Boswellia carterii, Anethum graveolens, Cuminum cyminum, Ocimum basilicum, Trigonella foenum-graecum and Glycyrrhiza glabra, all of which had inhibition zone diameters of 16, 16, 13, 14, 12 and 11 mm. Coriandrum sativum and Cymbopogon schoenanthus have no antibacterial action. The ethanolic extracts of Syzygium aromaticum were the most effective extract against E. coli, with an inhibition zone of diameter 25 mm, followed tightly by extracts of Hibiscus sabdariffa with an inhibition zone diameter of 13 mm. The remaining plant extracts had no impact (Table 2).

Against S. aureus: The aquatic extracts of the examined plants produce a range of inhibitory zones (11-24 mm). The extract of Syzygium aromaticum had the highest antibacterial effect with an inhibition zone diameter of 24 mm, followed by Ocimum basilicum with an inhibition zone diameter of 23 mm, followed by Hibiscus sabdariffa, Boswellia Carterii, Cuminum cyminum and Trigonella foenum-graecum, all of which have intermediate activity against S. aureus zone of growth inhibition of 16, 14 and 13 mm, respectively. With inhibitory zone diameters of 11 mm, Anethum graveolens, Artemisia monosperma and Coriandrum sativum had the lowest activity.

Cymbopogon schoenanthus and Glycyrrhiza glabra exhibit no antibacterial action against S. aureus. The ethanolic extract of Syzygium aromaticum was the most effective extract against S. aureus, with an inhibition zone diameter of 22 mm, followed by Hibiscus sabdariffa, Anethum graveolens, Cymbopogon schoenanthus, Coriandrum sativum and Artemisia monosperma, which have intermediate antimicrobial activity against S. aureus, with inhibition zones of 17, 16, 14, 12 and 9 mm antibacterial activity against S. aureus was lacking in Boswellia carterii, Glycyrrhiza glabra, Trigonella foenum graecum, Ocimum basilicum and Cuminum cyminum (Table 2).

Determination of MIC and MBC of the plant extracts: Extracts were evaluated for inhibitory activity against bacterial isolates on 96 multiwell microtiter-plates using a standard broth microdilution technique in two fold dilution series. It was made in triplicate as 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.562, 0.781 and 0.390 mg mL^–1 for the aquatic and ethanolic extracts.

Against E. coli: The MIC values of ethanolic extracts of Anethum graveolens and Cuminum cyminum were 12.5 and 6.25 mg mL^–1 for each of Ocimum basilicum, Artemisia monosperma, Glycyrrhiza glabra and Cymbopogon schoenanthus. While, it was 1.56 mg mL^–1 for Boswellia carterii and Coriandrum sativum. The MIC for Hibiscus sabdariffa, Syzygium aromaticum and Trigonella foenum graecum against E. coli was 0.78 mg mL^–1, however, the MIC for Anethum graveolens, Coriandrum sativum and Cymbopogon schoenanthus was 25 mg mL^–1 and the MIC for Artemisia monosperma and Glycyrrhiza glabra, Ocimum basilicum, Trigonella foenum-graecum, Cuminum cyminum, Syzygium aromaticum and Boswellia carterii had MICs of 6.25, 3.125, 1.56, 1.56 and 0.78 mg mL^–1 against E. coli, respectively. Hibiscus sabdariffa had the lowest value of 0.39 mg mL^–1 (Table 3).

The MBC of 100 mg mL^–1 ethanolic extracts of Glycyrrhiza glabra and 200 mg mL^–1 ethanolic extracts of Syzygium aromaticum. Anethum graveolens, Hibiscus sabdariffa, Boswellia carterii, Cuminum cyminum, Trigonella foenum-graecum, Coriandrum sativum, Cymbopogon schoenanthus, Artemisia monosperma and Ocimum basilicum all had MBC values more than 200 mg mL^–1. The MBC values of aquatic extracts were greater than 200 mg mL^–1 in all eleven samples (Table 3).

Against S. aureus: Glycyrrhiza glabra ethanolic extracts had MIC values of 50 mg mL^–1 against S. aureus. Boswellia carterii, Cuminum cyminum and Trigonella foenum-graecum each had a MIC of 25 mg mL^–1, whereas, Anethum graveolens, Hibiscus sabdariffa, Ocimum basilicum, Artemisia monosperma, Coriandrum sativum and Cymbopogon schoenanthus each had a MIC of 12.5 mg mL^–1 Syzygium aromaticum had the lowest value of 0.39 mg mL^–1. Cuminum cyminum had a MIC of 50 mg mL^–1, Anethum graveolens, Boswellia carterii, Trigonella foenum-graecum, Ocimum basilicum, Cymbopogon schoenanthus, Coriandrum sativum and Artemisia monosperma had a MIC of 25 mg mL^–1 and Glycyrrhiza glabra had a Hibiscus sabdariffa and Syzygium aromaticum had the lowest value of 6.25 mg mL^–1 (Table 3).

Evaluation the synergistic effect between plant extract: The in vitro synergism of Cuminum cyminum extracts with Ocimum basilicum and Artemisia monosperma extracts with Coriandrum sativum against S. aureus and E. coli was calculated.

Evaluation of the synergistic effect by disc diffusion method
Against E. coli: The aqueous extracts of Cuminum cyminum and Ocimum basilicum showed an inhibitory zone width of 7 mm, while the ethanolic extracts had no impact on E. coli. The ethanolic extract of Artemisia monosperma with Coriandrum sativum demonstrated an inhibitory zone width of 8 mm, but the aquatic extract had no impact (Table 4).

Against S. aureus: In both aqueous and ethanolic extracts, there was no inhibitory zone for Cuminum cyminum with Ocimum basilicum or Artemisia monosperma with Coriandrum sativum (Table 4).

Evaluation of the synergistic effect by well diffusion method
Against E. coli: The aqueous extracts of Cuminum cyminum and Ocimum basilicum showed an inhibitory zone width of 7 mm, while, the ethanolic extracts had no impact on E. coli. The inhibitory zone diameter of Artemisia monosperma and Coriandrum sativum aquatic and ethanolic extracts was 8 mm (Table 4).

Against S. aureus: In both aqueous and ethanolic extracts, Cuminum cyminum with Ocimum basilicum and Artemisia monosperma with Coriandrum sativum had no impact on S. aureus (Table 4).

Detection of MIC of mixed plant extracts against isolated bacteria
Against E. coli: The aqueous extract of Cuminum cyminum with Ocimum basilicum had a MIC of 6.25 mg mL^–1, whereas, the ethanolic extract had a MIC of 25 mg mL^–1. The MIC for Artemisia monosperma in an aquatic extract with Coriandrum sativum was 12.5 mg mL^–1, whereas, the MIC for ethanolic extract was 25 mg mL^–1 (Table 5).

Against S. aureus: The MIC of Cuminum cyminum aqueous extract with Ocimum basilicum was 25 and 50 mg mL^–1 in ethanolic extract. The MIC for Artemisia monosperma with Coriandrum sativum aquatic and ethanolic extract against S. aureus was 25 mg mL^–1 (Table 5).

DISCUSSION

Many naturally occurring plant chemicals have been used as antibacterial agents against infections. The efficacy of 11 plant species’ aqueous and ethanolic extracts to prevent the development of two types of pathogenic bacterial isolates, S. aureus and E. coli were investigated in this study.

To assess the antimicrobial effect of plant extracts, both the disc and well diffusion methods were applied. It was noticed that the well diffusion approach produced many superior results than the disc diffusion method against the two types of bacterial isolates which were consistent with the prior study of Al Talib et al.¹⁹. These findings might be attributed to the fact that plant extracts in the well-diffused approach diffused in the plate more than those in the disc diffusion method. The low observed values of certain plant extracts, however, can be attributable to the fact that the extracts were in crude form, including extremely few levels of physiologically active chemicals²⁰. Part of the preventive or inhibitor substance for microbe development in plant extracts may lose its inhibitory potency during extraction procedures²¹.

The majority of the findings of the disc and well diffusion technique of aqueous plant extracts demonstrated more antibacterial activity against the isolate bacteria than ethanolic extracts. This might be because aquatic extracts were found to be higher in polar phenol than ethanolic extracts²². This was consistent with conventional medicines, which predominantly employ water as a solvent.

It has been demonstrated that Syzygium aromaticum L. and Hibiscus sabdariffa L., have a great antibacterial effect against E. coli and S. aureus that infect the gastrointestinal tract and these findings were consistent with previous research^23,24. Although therapists have not suggested these two plant species for the treatment of gastrointestinal disorders.

According to the data, the MIC value of Syzygium aromaticum L., of the ethanolic extract against S. aureus was determined to be the lowest at 0.39 mg mL^–1. The aqueous extract of this plant species, on the other hand, showed 6.25 mg mL^–1 against S. aureus. It also had the lowest concentration of ethanolic extracts against E. coli, with a value of 0.78 mg mL^–1. In contrast to Syzygium aromaticum L., Hibiscus sabdariffa L., has the lowest MIC value in the aquatic extract. In comparison to ethanolic extract against the two pathogenic bacterial isolates. Whereas, the MIC value of Hibiscus sabdariffa L., in aquatic extract against S. aureus was 6.25 mg mL^–1, the ethanolic extract value was 12.5 mg mL^–1 and the MIC value of this plant species in aquatic extract against E. coli was 0.39 mg mL^–1 and the ethanolic extract value was 0.78 mg mL^–1.

However, some studies have discovered that activity in the disc diffusion method does not always correspond with low MIC and MBC values in the microtiter plate approach²⁵. This data was consistent with current findings, which showed that the MBC of ethanolic extracts of Glycyrrhiza glabra was 100 mg mL^–1 against E. coli. In contrast, the MBC findings for the other plant species against E. coli in both ethanolic and aqueous extracts were greater than 200 mg mL^–1.

The synergistic effects of some plant extracts when combined yielded weak results against pathogenic bacterial isolates when compared to the use of plant extracts alone. This result contradicted²⁶ which found that the antibacterial effect of aqueous and ethanolic leaf extracts of some plants yielded lower zones of growth inhibition when used alone than that of the extract combinations.

In the disc diffusion method, ethanolic and aqueous extracts of Cuminum cyminum combined with Ocimum basilicum and Coriandrum sativum mixed with Artemisia monosperma had an antagonistic impact on S. aureus. These combined plant extracts, on the other hand, had a mild synergistic effect against E. coli with an inhibitory zone of 7-8 mm.

The outcomes of the well diffusion approach were different. The impact of each plant’s aquatic extract on a bacterium yielded average findings between 13-23 mm inhibitory zone, however, when the extracts were blended, there was no synergistic effect against S. aureus. These extracts had an inhibition zone of 7-8 against E. coli in aquatic extracts, which was lower than the inhibition zone of each extract alone.

As a consequence, the prescription’s outcomes were modest when compared to the usage of each extract alone, as seen by the findings of MIC, where all extracts exhibited a drop in MIC to evaluate synergistic impact. This conclusion was consistent with prior research by Adwan and Mhanna²⁷, which indicated that these crude extracts include a variety of phytochemicals that may inhibit bacteria and fungus via several methods.

CONCLUSION

The results of the disc and well diffusion method of the aqueous plant extracts provided more strong antimicrobial activity compared to ethanolic extracts against the bacterial isolates. The strongest effect against E. coli was recorded in MIC in the aquatic extract of Hibiscus sabdariffa L., with a value of 0.39 mg mL^–1. But the Syzygium aromaticum L., observed a strong effect against S. aureus and E. coli of the ethanolic extract by all types of evaluation methods.

SIGNIFICANCE STATEMENT

This study is the current attempt to be the first of its kind in the Gaza Strip. It will identify and give useful information on the classification and identification of the most important medicinal plants in the Gaza Strip used by locals and therapists in traditional medicine in the treatment of some diseases. Moreover, the results of this study should be a top priority for the people in terms of knowledge, management, use, and research potential in the future.

REFERENCES