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Evaluation of Antimicrobial activity and Genotoxic Potential of Capparis spinosa (L.) Plant Extracts

Objective: The aims of this study were to evaluate the antimicrobial activity and the genotoxic effect of both ethanolic and aqueous extracts of stem and leaf of Capparis spinosa (C. spinosa) plant on Escherichia coli (E. coli) ATCC 25922, Staphylococcus aureus (S. aureus) ATCC 6538P, clinical isolate of Methicillin-resistant S. aureus (MRSA) and Klebsiella pneumoniae (K. pneumoniae) and Candida albicans (C. albicans) ATCC 90028. Materials and Methods: The antimicrobial activity was determined using microbroth dilution method, while the genotoxic effect was investigated using randomly amplified polymorphic DNA (RAPD)-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR. Results: The MIC values of both ethanolic and aqueous leaf and stem extracts of C. spinosa plant had a range 6.25 mg/ml to 100 mg/ml. In addition, it was found that ethanolic extract more effective than aqueous extract. The genotoxic activity of aqueous leaf extract, showed changes in both Random Amplified Polymorphic DNA (RAPD)-PCR and Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR profiles of E. coli strain treated with extract compared to untreated (negative) control. These changes included an alteration in the intensity, absence or appearance of new amplified fragments. Original Research Article Adwan and Omar; MRJI, 31(1): 48-57, 2021; Article no.MRJI.67085 49 Conclusions: Results of this study strongly show the genotoxic effect of aqueous leaf extract from C. spinosa plant on E. coli. The findings draw awareness to the possible toxic effect use of C. spinosa plant in traditional medicine and point out the capability of using C. spinosa to treat bacterial or fungal infections. More studies are needed to detect the exact ingredients of this plant as well as the mechanisms responsible for genotoxicity. Further in vivo genotoxicity studies are recommended to ensure and to evaluate the safety of using plants for therapeutic purposes. In addition, results of this study showed that molecular fingerprinting based on ERIC-PCR can be used to evaluate the genotoxic effect in the model bacterial species E. coli.


INTRODUCTION
Plants are considered a rich source of medicinal and nutraceutical agents for centuries [1][2]. In the modern age, approximately 25% of the new drugs originated from plant sources. Among valuable flora, wild plants have gained much awareness in recent decades because of their functional food and potential health benefits [3][4]. Capparis L. is considered the largest genus of the family Capparaceae (or Capparidaceae). This genus includes 350 species and is distributed in many parts of the world, in arid and semi-arid regions of the tropical and subtropical world, many of them distributed in the Mediterranean regions [5]. The Caper (Capparis spinosa (C. spinosa)) is naturally widely distributed from the Atlantic coast of the Canary Island and Morocco to the Black Sea, in Crimea and Armenia, and to the east side of the Caspian Sea and Iran. It is also spread in Europe, North Africa, Australia, West Asia and Afghanistan. This plant might have emerged in the tropic areas, and then extended to other parts of the world such as the Mediterranean basin and Central Asia [5]. Capparis spinosa plant is a perennial shrub, thorny, 0.3-1 m tall and has deep roots, which can extend up to 6-10 m [6][7].
Since C. spinosa has several beneficial health effects on human diseases, the adverse effects of using or consumption certain parts of this plant is not studied [3]. This study was conducted to evaluate the genotoxic potential of the aqueous extract from C. spinosa growing wild in Palestine on Escherichia coli (E. coli) ATCC 25922 strain using Random Amplified Polymorphic DNA (RAPD)-PCR and Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR as well as to determine the antimicrobial activity of both ethanolic and aqueous extracts of stem and leaf of C. spinosa plant.

Plant Collection and Identification
The stem and leaf parts of C. spinosa plant were collected from a natural habitat in Tulkarm province, West Bank-Palestine, during summer, 2019. Identification of the plant was conducted by the plant taxonomist Dr. Ghadeer Omar, Department of Biology and Biotechnology, An-Najah National University, Palestine.
The collected stem and leaf parts of C. spinosa were washed with water to eliminate soil and dust particles, then they were dried. Light exposure was avoided to minimize or prevent possible loss of active molecules. To obtain a fine powder that was ready for ethanolic and aqueous extract preparation, the air dried stem and leaf parts were powdered using an electric blender.

Ethanolic extract
Ethanolic extract was prepared as described previously [21][22] with some modifications. Briefly, approximately 30 g of dried plant powder was mixed thoroughly using a magnetic stirrer in 150 ml of 80% ethanol. The ethanol-plant powder part mixture was incubated on a shaker at room temperature for 48h. The mixture was filtered using muslin cloth to remove large insoluble particles. After that, the plant mixture was centrifuged at 5,000 rpm for 15 min at 4°C, to remove fine particles. Then, the supernatant extract was dried in an incubator at 40°C. The dried plant extract powder was kept in a refrigerator at 4°C. Before starting the assays, the dried plant extract powder was dissolved in 10% dimethyl sulfoxide (DMSO) to obtain a final concentration of 200 mg/ml and stored at 4°C for further experiments.

Aqueous extract
Aqueous extract was prepared as described previously [21,22] with some modifications. Briefly, approximately 30 g of dried plant powder was mixed thoroughly using a magnetic stirrer in 150-ml cold (room temperature) sterile distilled water. The water-plant powder part mixture was incubated on a shaker at room temperature for 48h. The mixture was filtered using muslin cloth to remove large insoluble particles. After that, the mixture was centrifuged at 5,000 rpm for 15 min at 4°C, to remove fine particles. Then, the supernatant extract was dried and concentrated by freeze dryer (lyophilizer). The dried plant extract powder was kept in a refrigerator at 4°C. Before starting the assays, the dried plant extract powder was dissolved in sterile distilled water to obtain a final concentration of 200 mg/ml and stored at 4°C for further experiments.

DNA extraction
The DNA genome of E. coli was prepared for randomly amplified polymorphic DNA (RAPD)-PCR and enterobacterial repetitive intergenic consensus (ERIC) PCR according to the method described previously [24]. Three ml samples were taken from the E. coli growth culture after 2 h, 5 h, and 24 h, centrifuged for five minutes at 14,000 x g where the supernatant of each sample was discarded. Then, each bacterial sample pellet was re-suspended in 0.8 ml of Tris-EDTA (10 mM Tris-HCl, 1 mM EDTA [pH 8]), centrifuged for 5 min at 14,000 x g; after that, the supernatant was discarded. The pellet of each bacterial sample was re-suspended in 300 μl of sterile distilled water and boiled for 15 min. Then, the mixture was incubated in ice for 10 min. The samples were pelleted by centrifugation at 14,000 x g for 5 min, and each sample supernatant was transferred into a new Eppendorf tube. The DNA concentration for each sample was determined using nanodrop spectrophotometer (GenovaNano, Jenway) and the DNA samples were kept at -20°C for RAPD-PCR and ERIC-PCR-based DNA fingerprinting techniques.

Antimicrobial Activity of C. spinosa Extracts
Results of this study showed that both ethanolic and aqueous extracts of stem and leaf of C. spinosa plant had antimicrobial activity.

Evaluation of the Genotoxic Potential of C. spinosa Aqueous Leaf Extract
DNA genome was extracted from each E. coli strain, which was treated with different concentrations of aqueous leaf extract of C. spinosa at various time intervals. Changes in the extracted DNA genome from treated E. coli strain were evaluated and compared with negative (untreated) controls at the same time intervals. The effect of aqueous leaf extract on E. coli genome was evaluated using molecular fingerprinting based on PARD-PCR and ERIC-PCR techniques. RAPD-PCR profile showed that a band with an amplicon length of about 700-bp was less intense in E. coli strain treated with 3 doses (250 μg/ml, 125 μg/ml and 62.5 μg/ml) of aqueous leaf extract for 2h (Fig. 1, lanes 1, 2 and 3), compared with the same band that appeared in the negative control. However, the profile showed that a band with an amplicon length of about 1500-bp was more intense in E. coli strain treated with 3 doses for 2h of the same extract ( Fig. 1, lanes 1, 2 and 3), compared with the same band that appeared in the negative control.
The bands with an amplicon length of about 300bp and 400-bp were less intense in E. coli strain treated with 250 μg/mL and 125 μg/ml of the aqueous extract for 2h (Fig. 1, lanes 1 and 2), compared with the same bands that appeared in the negative control. In addition, a band with an amplicon length of about 900-bp was less intense in E. coli strain treated with 250 μg/ml for 2h (Fig. 1, lane 1), compared with the same band that appeared in the negative control. Besides, a band with an amplicon length of about 200-bp appeared in E. coli strain treated with 250 μg/ml of aqueous leaf extract for 2h (Fig. 1, lane 1), compared with negative control. In addition, RAPD-PCR profile showed that a band with an amplicon size of more than 1500-bp appeared in E. coli strain treated with 3 doses of the same extract for 2h (Fig. 1, lanes 1, 2 and 3), compared with negative control. Results of RAPD-PCR showed that bands with an amplicon length of about 1500-bp, 700-bp, 400-bp and 300-bp were less intense in E. coli strain treated with 250 μg/ml of aqueous leaf extract for 5h (Fig. 1, lane 4), compared with the same bands that appeared in the negative control. Additionally, a band with an amplicon length of about 1200-bp was less intense in E. coli strain treated with 3 doses of aqueous leaf extract for 5h (Fig. 1, lane 4, 5 and 6), compared with the same band that appeared in the negative control. Besides, a new band with an amplicon size of about 200-bp appeared in E. coli strain treated with 250 μg/ml and 125 μg/ml of the same extract for 5h (Fig. 1, lanes 4 and 5), compared with negative control. Results of RAPD-PCR also showed that bands with an amplicon length of about 700-bp and 300-bp were less intense in E. coli strain treated with 3 doses of the aqueous leaf extract for 24h (Fig. 1, lanes 7, 8 and 9), compared with the same bands that appeared in the negative control. RAPD-PCR profiles of E. coli strain treated with different concentrations of aqueous leaf extract of C. spinosa and negative control at the different time intervals are shown in Fig. 1.
ERIC-PCR profile showed that two bands with an amplicon fragment size of than 1500-bp appeared in E. coli strain treated with 250 μg/ml and 125 μg/ml of aqueous leaf extract of C. spinosa for 2h (Fig. 2, lanes 1 and 2), compared with negative control. Additionally, the band with an amplicon fragment size of about 700-bp was more intense, while the band with an amplicon fragment size of about 300-bp was less intense in E. coli strain treated with both doses 125 μg/ml and 62.5 μg/ml of the same extract for 2h (Fig. 2,  lanes 2 and 3), compared with the same bands that appeared in the negative control. Besides, the bands with an amplicon fragment length of about 300-bp and 700-bp disappeared in E. coli strain treated with 125 μg/ml of aqueous leaf extract of C. spinosa for 2 h (Fig. 2, lane 1), compared with the same bands that appeared in the negative control. Results of ERIC-PCR showedthat band with an amplicon fragment size of more than 1500-bp was less intense in E. coli strain treated with 3 doses of aqueous leaf extract for 5 h (Fig. 1, lane 4, 5 and 6), compared with the same band that appeared in the negative control. Besides, a new band with an amplicon fragment size of about 1500-bp appeared in E. coli strain treated with 250 μg/ml of the same extract for 5 h (Fig. 1, lane 4), compared with negative control. Additionally, the band with an amplicon fragment size of about 700-bp was more intense in E. coli strain treated with both doses 125 μg/ml and 62.5 μg/ml of aqueous leaf extract, while the same band that disappeared in E. coli strain treated with both doses 250 μg/ml of the same extract for 5 h (Fig.  2, lanes 4, 5 and 6), compared with the same bands that appeared in the negative control. The band with an amplicon fragment length of about 450-bp was less intense in E. coli strain treated with 250 μg/ml and 125 μg/ml for 5 h (Fig. 2,  lanes 5 and 6), compared with the same band that appeared in the negative control. In addition, a new band with an amplicon fragment size of about 100-bp appeared in E. coli strain treated with 3 doses for 5h of the same extract (Fig. 2, lanes 4, 5 and 6), compared with negative control. ERIC-PCR profile also showed that the band with an amplicon fragment length more than 1500-bp was more intense in E. coli strain treated with 3 doses of aqueous leaf extract for 24 h (Fig. 2, lane 7, 8 and 9), compared with the same band that appeared in the negative control. Besides, the band with an amplicon fragment length of about 1300-bp appeared in E. coli strain treated with 250 μg/ml and 125 μg/ml for 24h (Fig. 2, lanes 7 and 8), compared with negative control. Additionally, the band with an amplicon fragment length of about 450-bp was less intense, while the same band was more intense in E. coli strain treated with 250 μg/ml and 62.5 μg/ml of the same extract for 24 h, respectively (Fig. 2, lanes 7 and 9), compared with the same bands that appeared in the negative control. Also, the band with an amplicon fragment size of about 100-bp was less intense in E. coli strain treated with 250 μg/ml and 125 μg/ml for 24 h (Fig. 2, lanes 7 and 8), compared with the same band that appeared in the negative control. ERIC-PCR profiles of E. coli strain treated with different concentrations of aqueous leaf extract of C. spinosa and negative control at the different time intervals are shown in Fig. 2.

DISCUSSION
In this study broth microdilution method was used to detect the potential antimicrobial effect of both ethanolic and aqueous leaf and stem extracts of C. spinosa against different species of microorganisms. These species included E. coli, S. aureus, MRSA, K. pneumoniae and C. albicans. The results of the current confirmed that both ethanolic and aqueous leaf and stem extracts of C. spinosa showed antimicrobial effect on these microorganisms. Antimicrobial activity of C. spinosa has been reported previously using different types of extracts and plant parts against different types of microorganisms [11,12,[16][17][18][19]. According to the previously conducted studies, diverse phytochemical compounds are the active ingredients of C. spinosa plant [6][7][8][9][10]13].
Most nutraceutical plants are used without any standard safety and toxicological trials. The common hypothesis that the products of these plants are nontoxic. However, this hypothesis is incorrect, so toxicological tests should be conducted for herbal drugs [29]. Recently advances in machine learning and artificial intelligence immensely decoded and empowered, the herbal drug discovery and modeling, which gave medicine modern tool to predict the biosafety and efficacy [30][31] and insilico methods [32][33] to potentially decipher the quantitative nanostructure activity-relationship (Nano-QSAR).
In this study, the potential genotoxic effect of the aqueous extract of C. spinosa plant against E. coli was tested using molecular fingerprinting based on ERIC-PCR and RAPD-PCR techniques. Reviewing the scientific literature showed that this study is the first of its kind that studied the genotoxicity of C. spinosa extract on prokaryotes using molecular fingerprinting based on ERIC-PCR and RAPD-PCR techniques. Besides, many plants were previously examined to investigate their genotoxic potential using different techniques [21,22,28,[34][35][36][37][38][39]. In this study, RAPD-PCR and ERIC-PCR profiles showed many significant differences between the treated and untreated E. coli strain. The alterations in the treated E. coli strain with aqueous leaf extract at different time intervals included the appearance and disappearance of certain bands and the alteration in the band intensity compared with negative control. These alterations in both the RAPD-PCR and ERIC-PCR profiles of the treated E. coli strain compared with the negative control could be explained due to the effect of the genotoxic ingredients that were present in the aqueous leaf extract. These ingredients can induce different alterations and changes such as point mutations and/or rearrangements in chromosomes, damage and chromosomal aberrations. These alterations in the DNA might have a potential change on the primer binding sites and/or interpriming distances [21,22]. Using other techniques such as DNA sequencing or probing can help understand the correct mechanisms that lead to such differences in RAPD-PCR and ERIC-PCR profiles [7,18,24]. Findings of the current study were in contrast to study published previously [20], which showed that C. spinosa buds aqueous extract is non-genotoxic and their study reveals that C. spinosa aqueous extract had antimutagenic potential against Ethyl Methane sulfonate induced chromosomal aberrations in A. cepa root meristem cells. This may be due to differences in plant parts and the techniques used to evaluate genotoxicity. In a literature survey, it is also showed that plant extracts can be mutagenic and antimutagenic depending on the test system used. This indicates that a group of assays is needed before any conclusion can be reached about the genotoxic effect [34].
Results of the current study showed that molecular fingerprinting based on ERIC-PCR is an effective and sensitive technique that can be used to evaluate the genotoxic effects to estimate the chemical compounds or molecules risk connected with their potential mutagenic effects in the model bacterial species E. coli.

CONCLUSION
The results of the current study showed that aqueous leaf extract of C. spinosa possesses genotoxic and mutagenic potential effects on E. coli. In addition, the results also point out the capability of using C. spinosa to treat and prevent infections caused by several microorganisms.
Further studies are recommended to determine the specific ingredients in this plant as well as the correct mechanisms responsible for that genotoxicity. In addition, findings of this study showed that molecular fingerprinting based on ERIC-PCR is an effective and sensitive technique that can be used to evaluate the genotoxic effect in the model bacterial species E. coli.

COMPETING INTERESTS
Authors have declared that no competing interests exist.