Evaluation of Antibacterial and Synergistic/Antagonistic Effect of Some Medicinal Plants Extracted by Microwave and Conventional Methods

The purpose of the study was to evaluate the efficiency of the microwave (MW) extraction method by comparing it with a conventional method through evaluation of antimicrobial and synergism/antagonism activity of each aquatic and ethanolic extracts samples, which were extracted from the dried plants (Ficus sycomorus leaves, Lawsonia inermis leaves and Glycerrhiza glabra Linn.). Nine samples of each plant for both solvents were irradiated with MW at several power outputs (180 w, 360 w, and 540 w) in several interval times (1, 2 and 3 minutes). The antibacterial activities of extracts and the synergistic effect between plants and antibiotics were evaluated using disk diffusion method against clinical isolated Staphylococcus aureus and Escherichia coli. The result revealed that the inhibition zone for more than 50% using aquatic and ethanolic samples results (extracted in two minutes and MW power 180 w) had shown the optimum extract and better antibiotic activity for each plant. Also, the results of ethanolic extracts used against selected microorganisms showed antimicrobial and synergistic effect with most antibiotics better than aquatic extracts. Our results indicate the possibility of using MW apparatus as an extractor to obtain bioactive compounds from plants and thus used in the treatment of bacterial infections, and some results of this study were encouraging. However, the antagonistic reactions of some extracts with some antibiotics and their use in combination should be further studied for in vitro activities. It is clearly a need to be furthermore evaluated, to identify the effective components, the mode of action and the possible toxHow to cite this paper: El Kahlout, K.E.M., El Borsh, W., Aksoy, A., El Kichaoi, A.Y., El Hindi, M.W. and El Ashgar, N.M. (2020) Evaluation of Antibacterial and Synergistic/ Antagonistic Effect of Some Medicinal Plants Extracted by Microwave and Conventional Methods. Journal of Biosciences and Medicines, 8, 69-79. https://doi.org/10.4236/jbm.2020.89006 Received: July 11, 2020 Accepted: September 6, 2020 Published: September 9, 2020 Copyright © 2020 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access K. E. M. El Kahlout et al. DOI: 10.4236/jbm.2020.89006 70 Journal of Biosciences and Medicines ic effect in-vivo of these ingredients.


Introduction
Research in herbal medicine has increased in developing countries as a way to rescue ancient traditions as well as an alternative solution to health problems. Therefore, with the increasing acceptance of traditional medicine as an alternative form of health care, the screening of medicinal plants for active compounds has become very important [1].
The emergence and spread of multidrug resistance as a phenomenon among bacterial pathogens has been a major problem confronting the field of antibacterial chemotherapy in modern years. However, it has been found that, in addition to the production of intrinsic antimicrobial compounds, some medicinal plants also produce multidrug resistance inhibitors which enhance the activities of antibiotics against multidrug resistant bacteria pathogens. It is this finding that prompted efforts in screening of crude extracts for synergistic interaction with standard antibiotics against resistant bacteria as this would have the way for possible isolation of multidrug resistance inhibitors of plant origin [2].
Nowadays, microwaves are used for extraction of bioactive compounds from plant materials because of tremendous research interest and potential [3] [4]. Conventional extraction techniques are time-consuming and require more solvent and most of them are not suitable for thermolabile constituents [5]. In MW, the process acceleration and high extraction yield may be the result of a synergistic combination of two transport phenomena: heat and mass gradients working in the same direction. On the other hand, in conventional extractions, the mass transfer occurs from inside to the outside, although the heat transfer occurs from the outside to the inside of the substrate [6]. fruit. It is one of the old and historic plant species in the Palestine coastal valley and the study area as well. It is known and called in Palestine as Balami or Jummaze. The antibacterial activity of F. sycomorus could be related to the presence of bioactive compounds, such as tannins, saponins, flavonoids, steroids, anthraquinone glycosides and reducing sugars [7]. G. glabra Linn, commonly known as liquorice and sweet wood belongs to Leguminosae family. Reported antibacterial activity because of the presence of secondary metabolites such as; saponins, alkaloids, flavonoids [10] [11]. Thereby, this study tried to throw light on the importance of extraction step of bioactive compounds from F. sycomorus, L. inermis and G. glabra by using microwave irradiation through evaluation of antimicrobial and synergism/antagonism activity of each aquatic and ethanolic activity of the extracts.

Plant Sample Collection
The plant materials used in this study consisted of F. sycomorus, L. inermis and G. glabra which were collected from different areas in Gaza strip (Table 1).

Chemicals and Culture Media
Three types of media were used for carrying out this study, Nutrient broth, Nutrient agar and Muller Hinton agar. Distilled water and ethanol was used for extraction process. Cefotaxime, Ofloxacin, Ciprofloxacin, Levofloxacin, Nitrofurantoin, ceftriaxone, chloramphenicol, and Amikacin used as reference antibiotics (Table 2). Dimethyl sulfoxide (DMSO) and ethanol 80%.

Bacteria
Clinical isolated species of S. aureus, and E. coli were obtained from Biology & Biotechnology Department at the Islamic University of Gaza (IUG), and were maintained on DMSO at −80˚C for further experiments.

Microwave Apparatus
Commercial microwave oven (Panasonic) with ten power levels (80 to 800 W) was used NN-SE996S.

Preparation of Plant Extract
A total of plants extract were used in this study as shown in Table 3.

Paper Disk Diffusion Assay
A modification procedure was followed to evaluate of antibacterial activity of plant extracts. Standardized inoculums of each bacterium, i.e., 10

Synergism/Antagonism between Plant Extract and Antibiotics
A modification procedure was followed to investigate the synergistic effect. The bacterial cultures were grown in sterile nutrient broth medium at 37˚C. After 4 h of growth, standardized inoculums of each bacterium, i.e., 10 6 CFU/ml to 0.1 at Air dried powder (20 g) was added to 150 ml of distilled water, (100˚C), as a solvent for 8 hours using soxhlet equipment. Then the extract was filtered and allowed to evaporate in oven (45˚C) through 48 hr [12].
Air dried powder (20 g) was added to 150 ml of 80 % ethanol, (78.5˚C), as a solvent for 8 hours, using soxhlet equipment. Then the extract was filtered and allowed to evaporate in oven (45˚C) through 48 hr [12]. 4 g of the powder was mixed with 100 ml distilled water. Then the mixture was irradiated with microwave at several power output (180, 360 and 540) in several interval times (1, 2 and 3) minuets to obtain nine samples [13]. The plates were incubated at 37˚C for 24 h. The diameters of cleared zones were measured and compared with that of the antibiotic alone. For each test solution, three replicates were maintained [7].

Evaluation of Antibacterial Activity of Plant Extracts and the Synergistic/Antagonistic Effect
The results in Tables 4-9 showed that ethanolic extracts used against selected microorganisms offered antimicrobial and synergistic effect with most antibiotics better than aquatic extracts. In case of aquatic extracts; G. glabra had the best antibitic activity against E. coli. In case of ethanolic extracts, the best activity was observed with G. glabra against E. coli. Also, synergistic activity of the plant extracts, in case of aquatic extracts; F. sycomorus had the best synergism against S. aureus and E. coli. In case of ethanolic extracts, the best synergism was observed with L. innermis against E. coli, and with F. sycomorus against S. aureus. High levels of antagonism reaction exhibited by all aquatic plant extracts of both methods when combined with antibiotics which showed sensitivity when tested as alone against S. aureus. Also all ethanolic extracts of L. innermis and G. glabra of both methods exhibited antagonism reactions when combined with antibiotics which showed sensitivity when tested as alone against S. aureu. In addition, antagonism reaction occurred against E. coli for antibiotics which were resistant against E. coli when combined with aquatic and ethanolic extracts for both methods of G. glabra which showed sensitivity when tested as alone against E. coli. In addition, dilution reactions against S. aureus occurred with some antibiotics combined with ethanolic extracts of F. sycomorus & L. innermis.

Effect of Irradiation Time
Through evaluation of antimicrobial activity of plant extracts and in synergisms/Antagonism, our results showed that, the inhibition zone is increased by

Effect of Microwave Power and Temperature
Through evaluation of antimicrobial activity of plant extracts and synergisms/Antagonism effect, our results showed that, the inhibition zone is increased

Conclusion
In conclusion, the results from these studies were encouraging to find new anti-