Comparative Evaluation of Phytochemical Composition, Antibacterial and Antioxidant Properties of Ruta graveolens L. and Rosmarinus officinalis L.

MATERIALS AND METHODS

Plant collection: Thirty specimens of the aerial parts of Ruta graveolens and Rosmarinus officinalis were collected from three locations in Irbid Region in Northern Jordan. The plants were identified at the Department of Biological Sciences, Al al-Bayt University, Mafraq, Jordan. This study was conducted from April, 2022 to September, 2022.

Plant extracts preparation: The plants were washed to remove dust. Then air-dried at room temperature for two weeks then were ground into a good powder. The powder (100 g) was mixed with 500 mL of different organic solvents, ethanol and methanol with shaking for 72 hrs. After that, the mixture of plant powder and solvent was filtered via Whitman No. 1 filter paper to get clear crude extracts. The solvents have been removed using a rotary evaporator (Buchi R-215, Flawil, Switzerland) under reduced pressure at temperatures below 45°C. The extracts were kept until needed.

Phytochemical analysis: Ethanolic and methanolic extracts of Ruta graveolens and Rosmarinus officinalis were used to screen phytochemicals using standard tests³².

Detection of flavonoids-alkaline reagent test: One milliliter of 2N NaOH was added to 1 mL (100 mg mL^–1 in distilled water) of crude extract. The appearance of yellow color indicated the presence of flavonoids.

Detection of alkaloids-Wagner’s reagent test: Five drops of Wagner’s reagent (potassium iodide (2 g) and iodine (1.27 g) in a total of 100 mL distilled water) was added to 100 mg of crude extract and mixed well. The reddish-brown precipitate indicated the presence of alkaloids.

Detection of phenols-ferric chloride test: Three milliliters of 5% aqueous ferric chloride was added to 1mL (100 mg mL^–1 in distilled water) of crude extract. The appearance of deep blue color indicated the presence of phenols.

Detection of cardiac glycoside-Keller Kellian’s Test: Two milliliters of glacial acetic acid and 0.5 mL of 5% ferric chloride was added to 5 mL (20 mg mL^–1 in distilled water) of crude extract followed by the addition of 5 mL of concentrated sulphuric acid. The appearance of a brown ring at the interface indicated the presence of cardiac glycoside.

Detection of tannins-Braymer’s Test: Plant extract (100 mg) was stirred with 10 mL of distilled water and then filtered. One mL of 5% alcoholic ferric chloride was then added. The appearance of greenish-black color indicated the presence of tannins.

Detection of saponins-Foam’s Test: Five milliliters of distilled water was added to 500 mg of crude extract. Appearance of stable persistent foam after 15 min of shaking indicated the presence of saponins.

Detection of sterols-Liebermann-Burchard’s Test: As 1 mL of chloroform was added to 1 mL of crude extract followed by the addition of 1 mL of acetic anhydride and 1.5 mL of concentrated sulphuric acid. A reddish brown color of the interphase indicated the presence of sterols.

Bacterial cultures: Four Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Micrococcus luteus ATCC 9341 and Bacillus cereus ATCC 11778) and six Gram-negative bacteria (Proteus vulgaris ATCC 29905, Salmonella typhimurium ATCC 13311, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 10031, Enterobacter aerogenes ATCC 13048 and Escherichia coli ATCC 25922) were used to assess the antibacterial activity of the plant extracts. Bacterial strains used in this study were obtained from the Microbiology Laboratory at the Department of Biological Sciences at Yarmouk University, Jordan.

Antimicrobial activity assay: The antimicrobial activity of plant extracts was assessed using the agar well diffusion method³³. Bacteria were grown on nutrient agar and incubated at 37±2°C for 24 hrs. Then, the colonies were picked and inoculated in 10 mL broth media. The bacterial suspension turbidity was adjusted to 0.5 McFarland standards. One hundred microliters of the tested bacteria were inoculated into nutrient agar. Agar wells were created using a sterile cork borer, then wells were filled with extracts (100, 200, 300, 400 and 500 mg mL^–1) dissolved in Dimethylsulfoxide (DMSO). The plates were incubated for 24 hrs at 37±2°C.

After incubation, the inhibition zone diameter was measured in mm and recorded. Standard antibiotics, ampicillin and tetracycline (250 μg mL^–1), were used as positive controls and DMSO as a negative control. Three replicates were carried out for each extract against each of the tested bacteria.

Minimum inhibitory concentration (MIC): The plant extracts (10, 20, 40, 80, 120, 160, 200, 240, 280 and 320 mg mL^–1) were introduced into different test tubes, each tube was inoculated with an overnight bacterial culture, which then incubated at 37±2°C for 24 hrs. The minimum plant extracts concentration that inhibited visible growth of the bacterial cultures was regarded as MIC³⁴.

Antioxidant activity: The 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) free radical scavenging assay was used to determine the antioxidant activity of methanolic plant extracts. Briefly, 7.88 mg of DPPH was dissolved in 100 mL methanol to acquire the stock solution, antioxidant activity of extracts was determined by mixing 0.5 mL of methanolic plant extract (2, 4, 6 and 10 mg mL^–1) in 2.5 mL of DPPH and allowed to stand in the dark for 30 min at room temperature, then the absorbance was measured at 517 nm. Ascorbic acid (4 mg mL^–1) was used as a reference to compare with the efficiency of plant extracts. Antioxidant activity is calculated as follows³⁵:

Statistical analysis: Excel’s two-sample t-test was used to analyze the data. Data were expressed as Means±SD of three replicates. The p>0.05 were considered statistically significant.

RESULTS

Phytochemical analysis: The qualitative screening of the phytochemicals in Ruta graveolens and Rosmarinus officinalis crude extracts indicated the presence of flavonoids, cardiac glycosides, phenols, saponins, sterols and tannins in ethanolic and methanolic extracts of both plants. In addition, alkaloids were present in Ruta graveolens but not detected in Rosmarinus officinalis extracts (Table 1).

Antimicrobial activity of ethanolic and methanolic extracts of Ruta graveolens: Ethanolic extracts of Ruta graveolens showed high growth inhibitory effect against 2 g positive bacteria, Staphylococcus aureus and Staphylococcus epidermidis at all extract concentrations.

Moreover, High growth inhibitory effect against the other two Gram-positive bacteria, Micrococcus luteus and Bacillus cereus, was shown at 500, 400, 300 and 200 mg mL^–1 plant extracts. On the other hand, ethanolic extracts showed a low growth inhibitory effect against Proteus vulgaris at 400 and 500 mg mL^–1 extract concentrations while Salmonella typhimurium and Pseudomonas aeruginosa growth was inhibited only at 500 mg mL^–1 extract. In addition, ethanolic extracts had no antibacterial activity against Klebsiella pneumonia, Enterobacter aerogenes and Escherichia coli at all concentrations (Table 2).

As shown in Table 2, methanolic extracts of Ruta graveolens showed a high inhibitory effect against 2 g positive bacteria, Staphylococcus aureus and Staphylococcus epidermidis at all concentrations. In addition, they showed a high inhibitory effect against Micrococcus luteus and Bacillus cereus at 500 mg mL^–1 plant extract and a lower effect at 400 mg mL^–1 plant extracts. Methanolic extracts showed a growth inhibitory effect against Salmonella typhimurium and Klebsiella pneumonia only at 500 mg mL^–1 concentration and no antibacterial activity against Pseudomonas aeruginosa, Enterobacter aerogenes and Escherichia coli at all extract concentrations.

Antimicrobial activity of ethanolic and methanolic extracts of Rosmarinus officinalis: As shown in Table 3, ethanolic extracts of Rosmarinus officinalis showed a high inhibitory effect against all Gram-positive bacteria at all extract concentrations and against one Gram-negative bacteria, Proteus vulgaris at extract concentrations higher than 200 mg mL^–1. On the other hand, inhibitory effect was shown against Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumonia, Enterobacter aerogenes and Escherichia coli only at 500 mg mL^–1 plant extracts. Methanolic plant extracts follow the same pattern of bacterial growth inhibition as in the case of the ethanolic plant extracts.

Standard antibiotics (Ampicillin and Tetracycline) and DMSO were evaluated for their antibacterial activity (Table 1 and 2). Ampicillin zones of inhibition varied from 8±1.1 mm against Enterobacter aerogenes to 50±2.7 mm against Staphylococcus aureus while had no inhibition against Bacillus cereus, Proteus vulgaris, Pseudomonas aeruginosa and Klebsiella pneumoniae. In addition, the tetracycline zone of inhibition varied from 12±0.7 mm against Salmonella typhimurium to 48±1.8 mm against Staphylococcus epidermidis while had no inhibition against Proteus vulgaris and Klebsiella pneumoniae. The DMSO did not show any growth inhibition for all tested bacteria.

Minimal inhibitory concentrations (MIC) of ethanolic and methanolic crude extracts of Ruta graveolens and Rosmarinus officinalis: As shown in Table 4, ethanolic and methanolic extracts of Ruta graveolens showed the most potent inhibition against Staphylococcus aureus and Staphylococcus epidermidis (MIC = 40 mg mL^–1), followed by Bacillus cereus and Micrococcus luteus (MIC = 160 mg mL^–1). In contrast, the lowest inhibition was obtained by ethanolic extract of Ruta graveolens against Proteus vulgaris, Salmonella typhimurium and Pseudomonas aeruginosa (MIC>320 mg mL^–1). Moreover, methanolic extracts of Ruta graveolens showed low inhibition against Salmonella typhimurium and Klebsiella pneumoniae (MIC >320 mg mL^–1).

Ethanolic and methanolic extracts of Rosmarinus officinalis showed the most potent inhibition against Staphylococcus aureus, Staphylococcus epidermidis and Bacillus cereus (MIC = 40 mg mL^–1), followed by Micrococcus luteus and Proteus vulgaris (MIC = 80 mg mL^–1). On the other hand, the lowest inhibition was obtained by ethanolic and methanolic extracts of Rosmarinus officinalis against Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter aerogenes and Escherichia coli (MIC = 320 mg mL^–1) (Table 4).

Fig. 1:

Free radicle scavenging activity of Ruta graveolens and Rosmarinus officinalis methanolic extracts using DPPH (2,2-Diphenyl-1-Picrylhydrazyl) method after 30 min incubation at different concentrations Ascorbic acid (4 mg mL–1), Data are means of three replicate±standard deviations and ****p<0.0001

Antioxidant activity of methanolic crude extract of Ruta graveolens and Rosmarinus officinalis: The radical scavenging action of the methanolic extracts was measured by DPPH assay. As shown in Fig. 1, the results showed that Rosmarinus officinalis methanolic extract had a high DPPH radical scavenging activity at all concentrations. After 30 min of incubation, the highest antioxidant activity was 93.36±0.861% at 10 mg mL^–1 Rosmarinus officinalis methanolic extracts, which was not significantly different from the positive control, 4 mg mL^–1 ascorbic acid (p>0.5). Ruta graveolens methanolic extract had a DPPH radical scavenging activity at all concentrations. However, they were significantly lower that Rosmarinus officinalis methanolic extracts (p<0.0001). The highest antioxidant activity of Ruta graveolens methanolic extract was 23.37±1.102% at 10 mg mL^–1 (Fig. 1).

DISCUSSION

In this study, the phytochemical composition of ethanolic and methanolic crude extracts of Ruta graveolens and Rosmarinus officinalis was determined and their antibacterial and antioxidant activities were evaluated. The qualitative phytochemical screening of the two plant extracts indicated the presence of various phytochemicals such as flavonoids, cardiac glycosides, phenols, saponins, sterols and tannins in both plant extracts, except the alkaloids, which were not detected in both extracts of Rosmarinus officinalis (Table 1).

Previous studies have shown that the quantity and quality of the extracted bioactive compounds from plants differ according to the plants and parts used in the extraction process, as well as the extraction method, especially the solvent used, temperature and extraction time^36-38.

In general, at the different concentrations used, methanolic and ethanolic extracts yielded from the two plants showed good antibacterial activity against all studied pathogenic gram-positive compared with the two controls used, ampicillin and tetracycline (Table 2 and 3). However, the two extracts of Rosmarinus officinalis showed higher activity against Micrococcus luteus (MIC = 80 mg mL^–1) and Bacillus cereus (MIC = 40 mg mL^–1) than Ruta graveolens extracts (MIC = 160 mg mL^–1). On the other hand, both plant extracts showed little to no activity against other tested Gram-negative bacteria, except at higher concentrations of Rosmarinus officinalis that showed good activity against Proteus vulgaris (MIC = 80 mg mL^–1) compared with Ruta graveolens extracts (MIC >320 mg mL^–1) (Table 4). Although there were significant differences between ethanolic and methanolic extracts in terms of the diameters of the inhibition zones in both plants, there were no significant differences between the MICs of the two extracting solvents used. The antimicrobial effects of plant extracts may be due to the presence of phenolic acids, flavonoids and other secondary metabolites, which could interfere with microbial membrane function, nutrient uptake, electron transport, nucleic acid and protein synthesis and enzyme activity^39,40.

The results of this study were somewhat consistent with Alzoubi et al.⁴¹, who found that ethanolic extracts of Rosmarinus officinalis had a growth inhibitory effect against clinical strains of Methicillin-Resistant Staphylococcus aureus (MRSA). Al-Shuneigat et al.⁴², concluded that Ruta graveolens essential oils had more inhibitory activity against Gram-positive than Gram-negative bacteria.

Many studies have confirmed that oxidative stress is one of the main risk factors for many chronic diseases, such as cancer, diabetes, Sharifi-Rad et al.⁴³ and Leyane et al.⁴⁴. The use of safe and cheap antioxidants, as in plant extracts, is one of the important goals of many pharmaceutical and food industries⁴⁵. The antioxidant activities of Ruta graveolens and Rosmarinus officinalis extracts were measured by DPPH radical-scavenging assays (Fig. 1). From the analysis, it was concluded that the scavenging effects of Rosmarinus officinalis extract on DPPH radicals were excellent, compared with known antioxidant Ascorbic acid. However, Ruta graveolens extracts revealed a low value of antioxidant activity. Since ancient times, herbal plants considered good antioxidants, which may be due to the presence of phenolic compounds. Toyokuni et al.⁴⁶ found that polyphenols and Flavonoids significantly raised antioxidant enzyme action. The level of antioxidant activity varies according to the total phenolic contents of the medically used parts, as reported by a study by Al-Mustafa and Al-Thunibat⁴⁷, on twenty-one plant samples collected from different Jordanian locations and used for antioxidant evaluation.

The results of this study confirmed the traditional use of Ruta graveolens and Rosmarinus officinalis against bacterial infections and various diseases. Jordan is distinguished by the abundance of medicinal plants with diverse bioactive compounds^47-51 which make them good candidates as an alternative medicine to treat many diseases such as cancer, diabetes, infections and others.

In addition, these plants might be good substitutes for conventional antimicrobial food additives and synthetic antioxidants. However, isolating the active ingredients of plant extracts is needed to justify their biological activities.

CONCLUSION

The present study demonstrated that the crude extracts of Ruta graveolens and Rosmarinus officinalis are rich in secondary metabolites including, flavonoids, cardiac glycosides, phenols, saponins, sterols and tannins. Plant extracts showed potent antibacterial activities against a wide array of pathogenic bacteria. In addition, all extracts showed high free radicle scavenging activities. The biological activities of both plants make them promising candidates for use as natural products-based antimicrobials and antioxidants for human health. Further research is needed to isolate active ingredients and elucidate the mechanisms of actions behind their biological activities.

SIGNIFICANCE STATEMENT

This study discovered the phytochemical content of Ruta graveolens and Rosmarinus officinalis extracts as well as their biological activities. It was found that these plants are rich in bioactive secondary metabolites, which exhibited antibacterial and antioxidant activities due to their structure and redox properties. The current study findings will help researchers to expand their understanding of plant extract components and their interactions as well as the antibacterial and free radical scavenging activities of medicinal plants. Further research is suggested to identify and isolate active ingredients, which might be useful for the development of novel pharmaceutical drugs.

ACKNOWLEDGMENT

The authors would like to thank Al al-Bayt University and Yarmouk University for providing facilities to conduct research.