Phytochemical and Antimicrobial Activity Evaluation of The Water Immiscible Solvent Extracts of Moringa

Antimicrobial resistance (AMR) is the existing global apprehension for the social health. There is a requisite to take counteractive actions regarding the AMR. Based on the literature, it was aimed to perform the phytochemical and antimicrobial activity evaluation of the water immiscible solvent extracts of Moringa peregrina Forssk. Fiori (Family: Moringaceae). Seven extracts of the powdered leaves of M. peregrina were prepared in solvent systems comprising dichloromethane (DCM), dichloroethane (DCE), and their mixtures with ethyl acetate (EA) and chloroform (CH). The extracts were screened for their phytochemicals and antimicrobial potential. All the extracts showed positive tests for alkaloids, saponins, and flavonoids, wherein negative tests were obtained for tannins, cardenolides, and anthraquinone glycosides. The 1:1 mixture of EA:DCM, and EA:DCE provided positive tests for steroids and terpenoids. The most effective antimicrobial extracts were the 1:1 mixture of EA:DCM and EA:DCE. However, the antimicrobial activity of these extract was mild in comparison to ofloxacin and fluconazole. It is concluded that various mixtures of DCM, DCE, and CH along with the higher concentration of other miscible solvents of DCM, DCE, and CH may provide better antimicrobial extracts.


INTRODUCTION
Antimicrobial resistance (AMR), an existing global worry for social health, is frequently linked to the irrational practice of antibiotics 1,2 . The reports of AMR bacterial infections are accumulating in the Kingdom of Saudi Arabia owing to the irrational practice of antibiotics in addition to the societal, economic, and demographic features of Saudi, non-Saudi and visitor population [3][4][5][6] . Additional factor causative to the expansion of AMR is the failure to find out innovative antimicrobial means [7][8][9] . Consequently, there is a necessity to take counteractive actions regarding the issues associated to AMR.
The indiscriminate use of synthetic antibiotics poses a serious threat to humans as multidrug resistance has developed among the disease-causing microbes 10 towards synthetic antibiotics. Therefore, scientists are more focused on plant-based drugs which are not or least toxic to treat infectious diseases. Moringa peregrina Forssk. Fiori (Family: Moringaceae) is reported to possess a wide variety of biological activities that include antioxidant property, antimicrobial property, antidiabetic property, antispasmodic property, hepatoprotective property, antihypertensive property, lipid-lowering activity, anti-inflammatory activity, treatment of the mental disorders, and anticancer activity [11][12][13][14] . The complete plant description, its cultivation, the economic status, the different phytoconstituents, and the biological activity profile of the different solvent extracts of Moringa peregrina has also been disclosed in the published review articles 14,15 . According to one citation of 2018, most of the antimicrobial activity reports of the leaves of M. peregrina are related to the water-miscible solvents extracts and the compounds isolated from these extracts 14 . However, the literature of Moringa peregrina is silent about the phytochemical and antimicrobial activity evaluation of the water immiscible solvent extracts of the leaves of M. peregrina [11][12][13][14][15] . In view of the above facts, it has been decided to perform the phytochemical and antimicrobial activity evaluation of the water immiscible solvent extracts of the leaves of M. peregrina that can lead to the identification of the possible new class of the phytoconstituents as antimicrobial agents.

MATERIALS AND METHODS Collection of the Plant Material
The semi-dried leaves (2 kg) of M. peregrina were obtained from Al Oula region (Saudi Arabia) in January 2019. These leaves were authenticated by Prof. Abdulhakim Bawadekji and specimens were kept in the herbarium with n. Oul 1. The semi-dried leaves were cleaned and air dried for 8 days at 25-30°C. The dried up leaves were broken up into a coarse powder with the help of a grinder. The powder was sieved and stored in polyethene bags for the extraction purpose.

Preparation of the Extracts
The powder of the leaves (50 g) was transferred to a 1000 ml flask. dichloromethane (DCM, 500 ml) was added to the flask. The flask was plugged and the content was stirred at 25-30°C for 30 minutes. The obtained mixture was kept 25-30°C for three days with occasional shaking. The mixture was filtered with Whatman filter paper. The resulting filtrate was concentrated in a rotary evaporator and a semisolid residue was obtained.

Antimicrobial Screening
The antimicrobial activity was performed by the serial plate dilution technique 17,20 . Six microorganisms were used to determine the minimum inhibitory concentrations (MIC) of the extracts and the standard drugs, ofloxacin and fluconazole. Different concentrations of the extracts and the standard, ranging from 10-200 µgml -1 , were prepared using sterile dimethylformamide (DMF) as a solvent, which also served as a control group. Agar media and Sabouraod dextrose media were used for antibacterial and antifungal activity assessment, correspondingly. The tested microorganisms were the followings: Escherichia coli Castellani and Chalmers ex. Migula, Klebsiella pneumoniae Trevisan ex. Shroeter, Pseudomonas aeruginosa Migula ex. SCHRUTER, Staphylococcus aureus Rosenbach, Aspergillus niger Tieghem, Candida albicans (Robin) Berkhout.

Statistical Analysis
The data (N = 3, Mean±Standard Error Mean) was analysed by SPSS-software, in which p < 0.05 specified the significant results.

RESULTS AND DISCUSSION
Many reports about the antimicrobial potential of the leaves of M. peregrina have been documented. However, the literature is silent about the phytochemical and antimicrobial activity evaluation of the water immiscible solvent extracts of the leaves of M. peregrina. Accordingly, seven extracts of the powdered leaves of M. peregrina were prepared using dichloromethane, dichloroethane, and their mixtures with ethyl acetate and chloroform. The physical data of these extracts is provided in Table 1. According to this data, all the semisolid extracts had a greenish appearance, and the percentage yield range from 8% to 14%. The highest yield (14%) was obtained for the 1:1 mixture of ethyl acetate and dichloroethane (EA:DCE), wherein the lowest yield (8%) was obtained for the 1:1 mixture of chloroform and dichloromethane (CH:DCM). This variation in the yield might be because of the polarity of the solvents systems used for the extraction.
The phytochemical analysis of the extracts was performed by the standard procedure 15,16 , which is mentioned in Table 2. According to this data, all the extracts showed positive tests for alkaloids, saponins, and flavonoids. This shows that alkaloids, saponins, and flavonoids are among the main chemical constituents of the   [15][16][17][18][19][20][21] .
The antimicrobial activity of the extracts was performed by serial dilution technique 17,18 against four bacteria and two fungi using ofloxacin and fluconazole as standard drugs, respectively. The MIC values of the extracts, ofloxacin, and fluconazole with respect to the tested microorganisms are mentioned in Table 3. For the comparison purpose, the MIC values of ofloxacin and fluconazole are taken as 100%. All the data had a statistically significant (p < 0.5) results. It is evident from Table 3 data that all the seven extracts possess mild antifungal activity in comparison to fluconazole (MIC: 15µgml -1 ; 100%) against C. albicans and A.niger. The most effective antifungal extracts were the 1:1 mixture of ethyl acetate:dichloromethane (EA:DCM) and ethyl acetate:dichloroethane (EA:DCE), which had MIC of 75µgml -1 . However, it was only 20% in comparison to fluconazole against C. albicans and A.niger. It is also evident that all the seven extracts possess mild antibacterial activity in comparison to ofloxacin (MIC: 20 µgml -1 ; 100%) against S. aureus, E. coli, P. aeruginosa, and K. pneumoniae. The most effective antifungal extracts were the 1:1 mixture of ethyl acetate: dichloromethane (EA:DCM) and ethyl acetate: dichloroethane (EA:DCE), which had MIC of 75-100 µgml -1 . However, it was 20-26.66% in comparison to ofloxacin against S. aureus, E. coli, P. aeruginosa, and K. pneumoniae. It was also observed that solvent system, for example, EA:DCE system, having more polarity had better antifungal and antimicrobial activity in comparison to the less polar solvent system, for example, DCM. The higher antimicrobial activity of the EA:DCM and EA:DCE solvent system might be because of the presence of steroids and terpenoids, which are absent in other solvent systems 14 (Table 2). Another possibility of this result is that the extracts of the more polar solvent system might be having more number chemical constituents, which attribute to the higher antimicrobial activity of the extracts of the more polar solvent systems 11 .

CONCLUSION
It is evident from the results that the extracts of 1:1 mixture of EA:DCM and EA:DCE possess more phytoconstituents in comparison to other extracts of DCM, DCE, DCM:DCE (1:1), CH:DCM (1:1), and CH:DCE (1:1). Accordingly, it is apparent that the better antimicrobial potential of the extracts of 1:1 mixture of EA:DCM and EA:DCE is because of the presence of more number of phytoconstituents. It is concluded that various mixtures of DCM, DCE, and CH along with the higher concentration of other miscible solvents of DCM, DCE, and CH may provide better antimicrobial extracts.