INTRODUCTION
Natural products have been used as remedies worldwide since ancient times for the treatment of various diseases. Current research focuses on the use of medicinal plants for alternative therapeutic strategies due to their bioactive compounds.[1 2 Semicarpus anacardium has also been used in the treatment of dandruff.[17
V. negundo is an aromatic medicinal shrub distributed in many parts of the world including India. All parts of the plant have been extensively used in traditional system of medicine. The plant has been reported to possess several medicinal properties such as anti-inflammatory, antioxidant, antibacterial, antifungal, antimicrobial, and anticancer properties.[3 4
Antimicrobial resistance among bacterial strains has emerged out to be a serious threat and may be responsible for the loss of effectiveness of the common antibiotics within a span of 5 years due to genetic modifications.[5 6 7
Carbapenem-resistant Klebsiella pneumonia and multiple-drug-resistant K. pneumonia have become significant global public health issue in recent years.[8 K. pneumonia , an encapsulated, Gram negative, facultative anaerobic bacillus present normally in the intestines is responsible for many hospital and community infections inclusive of nosocomial pneumonia, urinary tract infections, diarrhea, and intra-abdominal infections.[9 antibiotic resistance to K. pneumonia include drug efflux, biofilm formation, enzyme-mediated drug inactivation, changes to drug targets, and reduced permeability owing to porin loss or mutation.[10
The mounting concern about the alarming increase in multi drug resistant K. pneumonia has led many researchers to focus on the natural products with antimicrobial properties. With this background, this study was designed to evaluate the efficacy of the leaf extract of V. negundo against multi drug resistant bacteria K. pneumonia using in vitro studies.
MATERIALS AND METHODS
Extraction of V. negundo
To prepare the leaf extract of V. negundo , 30 g of the powdered leaves were extracted in 170 mL ethanol by soxhlet apparatus at 50°C. The filtrate was separated and the solvent was removed using rotary vacuum evaporator. Then it was kept at room temperature until complete evaporation of solvent phase. The weight of dried solvent was taken and redissolved in acetone, benzene, ethyl acetate, chloroform, and methanol at 5 mg/mL concentration for further in vitro studies.
Selection of the species
The active stab culture of Klebsiella pneumoniae subsp. pneumoniae (MCC 2451) was purchased from National Centre for Cell Science (NCCS), Pune and was used for the evaluation of antibacterial activity of V. negundo leaf extract. The bacterial strains were grown and maintained on Muller-Hinton Agar (Merck, Germany) medium. Before using the test organism in antibacterial assay, it was cultured overnight at 37°C.
Estimation of antibacterial activity of V. negundo leaf extracts by disc diffusion assay
Muller-Hinton Agar (MHA) preparation:
Thirty gram of MHA (Hi-media) was dissolved in 1000 mL of distilled water and autoclaved. The agar was allowed to cool to hand bearable temperature. Then around 10 to 15 mL of agar was poured into the petri plates and allowed to solidify. One milliliter of 0.5 McFarland reagent (prepared by 99.5% of sulfuric acid and 0.05 mL of 1.175% barium chloride dihydrate (BaCl2 •2H2 O) with 9.95 mL of 1 % sulfuric acid (H2 SO4 ) was used for comparison with 1 mL of the bacterial suspension which is approximately 1.5 × 108 organisms.
Disc diffusion assay
Bacterial suspension diluted in comparison with 0.5 McFarland reagent was streaked on the prepared sterile MHA plates. Sterile discs loaded with known concentration (10, 20, 40, and 100 μg) of the aqueous and ethanolic leaf extracts of V. negundo were placed on the plates. Then the plates were incubated and observed for the zone of inhibition (ZoI) the next day.
The test was repeated with ethanol and ethyl acetate leaf extracts of V. negundo after performing the TLC bioassay. Sterile disc previously loaded with known and increased concentrations (50, 100, and 200 μg) of the plant extracts were placed on the plates along with positive and negative controls. The plates were incubated for 24 hours at 37°C. By measuring the clear ZoI to the nearest millimetre (mm), the antibacterial activities of the extracts were determined.
TLC bioassay for various extracts of V. negundo leaves
The different leaf extracts (acetone, benzene, ethyl acetate, chloroform, and methanol) of the medicinal plant V. negundo were subjected to preliminary TLC analysis following standard methods. A TLC system with sample applicator was used to apply the samples. The sample of 15 µL was applied on 5 cm × 10 cm TLC aluminum plates precoated with silica gel 60 F254 (E. Merck) of uniform thickness of 0.2 mm (stationary phase). The plates were developed in the mobile phase using ethyl acetate: n-butanol (6:4 v/v) in a twin trough glass chamber. The plates were removed when the solvent front has moved from the original extract position to about 15 cm. Then they were allowed to dry and the spots were visualized and scanned under visible and UV light. Then the plates were derivatized using vanillin–sulfuric acid reagent and heated at 105°C when the compounds appeared as colored spots. The movement of each separating spot of the extract was expressed by its retention factor or R f value.
Determination of minimum inhibitory concentration (MIC) by Alamar blue assay
The extracts (100, 50, 25, 12.5, 6.5, 3.2, and 1.5 μg/mL) were prepared by diluting two fold concentrations (200 μg) of extract in 1 mL broth. Serial dilutions were made by transferring 1 mL of the sample containing broth. After dilution, 25 μL of culture was inoculated into all tubes and incubated. Growth inhibition was observed after addition of resazurin indicator. Antibiotic was used as positive control and the solvent was used as negative control. The well that contained the lowest sample concentration that prevented the visible bacterial growth as indicated by the change in color was considered as the MIC value.
Determination of antibacterial activity of ethyl acetate extract by time-kill assay
To evaluate the antibacterial activity of the ethyl acetate leaf extract of V. negundo , time-kill assay was performed according to standard protocol. An overnight culture (106 CFU/mL of cells) of resistant bacteria K. pneumonia was used to inoculate fresh Muller Hinton Agar containing ethyl acetate leaf extract of V. negundo . The extract was prepared with the concentration equal to its MIC value. The culture was incubated at 37°C for 24 hours. Surviving bacterial colonies were visualized at frequent intervals to determine the time taken by the extract to kill the resistant bacteria.
RESULTS
Antibacterial activity of V. negundo leaf extract by disc diffusion assay
In the present study, the tested K. pneumoniae was identified as multi drug resistant (MDR) bacteria. The sensitivity test against the standard antibiotics showed that out of eight antibiotics (VA − Vancomycin, DO − Doxycyline, CIP − Ciprofloxacin, OF − Ofloxacin, SPX − Sparfloxacin, GAT − Gatifloxacin, AT − Aztreonam, AZM − Azithromycin), AT was found to moderately active and produced 14 mm ZoI against the test pathogen.
The sensitivity test performed for the MDR bacteria K. pneumoniae using aqueous and ethanolic leaf extracts of V. negundo at various concentrations (10, 20, 50, and 100 μg) recorded no ZoI [Plate 1a–d].
Then from the results of the TLC bioassay, ethyl acetate was selected as the suitable solvent for extraction of V. negundo leaves. The ethyl acetate leaf extract of V. negundo revealed that it had potent antibacterial activity against the test pathogen K. pneumoniae . In the present study, the ZoI was observed starting from the concentration of 50 μg/mL for the V. negundo ethyl acetate leaf extract for the selected bacteria K. pneumoniae . The highest ZoI was observed as 18 mm at the concentration of 200 mg/mL for the ethyl acetate leaf extract of V. negundo . Hence the ethyl acetate leaf extract of V. negundo was selected for further in vitro studies ([Table 1 ] and Plate 3).
Table 1: Antibacterial activity of different concentrations of ethanolic and ethyl acetate leaf extracts of V. negundo showing zone of inhibition (ZoI) against MDR K. pneumonia
TLC bioassay for various extracts of V. negundo leaves
The different leaf extracts (acetone, benzene, ethyl acetate, chloroform, and methanol) of the medicinal plant V. negundo were subjected to preliminary TLC analysis following standard methods. The movement of each separating spot of the extract is expressed by its R f value. The results of the TLC bioassay clearly depict fluorescent spots in the ethyl acetate leaf extract of V. negundo , which represent the separation of active components in the ethyl acetate extract [Plate 2]. The fourth fraction in the ethyl acetate leaf extract seen as bright fluorescent band at R f value 0.3 exhibited positive activity against the tested pathogen MDR K. pneumoniae [Table 2 ].
Table 2: Bioactivity of TLC fractions
Estimation of MIC of V. negundo ethyl acetate leaf extract against MDR K. pneumoniae by Alamar blue assay
The MIC value of V. negundo leaf extract against MDR K. pneumoniae Alamar blue assay is shown in Plate 4 and [Table 3 ]. Ethyl acetate extract of V. negundo leaves expressed MIC value of 1.5 μg/mL against the test pathogen MDR K. pneumoniae ([Table 3 ]).
Table 3: Minimum inhibitory concentration (MIC) and kill-time of ethyl acetate leaf extract of V. negundo by using Alamar blue assay against MDR K. pneumoniae
Determination of kill-time of V. negundo ethyl acetate leaf extract against MDR K. pneumoniae
The killing of MDR K. pneumoniae by ethyl acetate extract of V. negundo leaves at a concentration equivalent to its MIC value is shown in the Table 3 and Plate 5. In the present study, the inhibitory effect of the ethyl acetate leaf extract of V. negundo was apparent from 40 minutes indicating time dependant killing of the tested pathogen MDR K. pneumoniae.
DISCUSSION
Antibacterial activity of V. negundo leaf extract by disc diffusion assay
In the present study, the tested K. pneumoniae was identified as multi drug resistant (MDR) bacteria from the sensitivity test against the standard antibiotics. The tested K. pneumoniae was found to be non-susceptible to all the standard antibiotics except Aztreonam (AT), which was found to moderately active and produced 14 mm ZoI against the test pathogen.
K. pneumoniae is gaining resistance even against the last line antimicrobials and has emerged as an urgent threat to human health by World Health Organisation and Centre for Disease Control and Prevention.[11 12 K. pneumoniae and development of alternative therapeutic strategies to curb the alarming increase in MDR K. pneumonia .
Plants are a potential source of new anti-infective drugs as shown by the studies pertaining to screening plant extracts and products for their antimicrobial properties.[13 26 V. negundo , is a medicinal plant with several reported therapeutic properties.
In the present study, the leaf extract of V. negundo was evaluated for its antimicrobial properties against the tested pathogen MDR K. pneumoniae. The aqueous and ethanolic leaf extracts of V. negundo at various concentrations (10, 20, 50, and 100 μg) recorded no ZoI [Plate 1a–d] by the disc diffusion assay.
But from the results of the TLC bioassay, ethyl acetate was selected as the suitable solvent for extraction of V. negundo leaves. The ethyl acetate leaf extract of V. negundo revealed that it had potent antibacterial activity against the tested pathogen MDR K. pneumoniae .
The differences in the antimicrobial properties of the different leaf extracts of V. negundo could be attributed to the different solvents used for extraction procedures and the various methods of extraction. In agreement, it was recently reported that the different bio-molecules obtained from plant sources depends on the different solvents used for the extraction methods and the different methods of extraction.[14
In the present study, the ZoI was observed starting from the concentration of 50 μg/mL for the V. negundo ethyl acetate leaf extract against the tested MDR bacteria K. pneumoniae . The highest ZoI was observed as 18 mm at the concentration of 200 mg/mL for the ethyl acetate leaf extract of V. negundo. The differences in the ZoI exhibited by the different leaf extracts of V. negundo may be due to the differences in the elution of various phytochemicals with different pharmacological activities while using different solvents and different extraction methods. It was also earlier reported that different ZoI and the differences in the antibacterial activity of the plant extracts depends on the geographical distribution of the plants, the part of the plant used for the extraction procedures and also on the bacterial strain used.[15
In the present study, ethyl acetate leaf extract at a concentration of 200 mg/mL was shown to possess maximum antibacterial activity against the tested MDR bacteria K. pneumoniae , which might be attributed to the elution of maximum concentration of phytochemicals with potent antibacterial properties.
In another study, the ethanolic leaf extract of V. negundo showed antibacterial activity against the tested pathogens Escherichia coli, Staphylococcus aureus , and K. pneumoniae . The maximum ZoI was observed for S. aureus (15 mm) at the concentration of 80 mg/mL, while for E. coli and K. pneumoniae , the maximum ZoI was recorded as 12 and 11 mm respectively, at the concentration of 100 mg/mL.[16
The methanolic extract of V. negundo leaves were also reported to exhibit potent antibacterial activity (significant ZoI ranging from 9.9 to 22.6 mm) against the tested pathogenic enteric bacteria Vibrio cholera , V. parahaemolyticus , V. mimicus , E.coli , Shigella sps. , and Aeromonas sps under in vitro conditions. The extract was also reported to significantly decrease the mortality rate in mice model of V. cholera infection.[18
In an earlier study, it was documented that the different extracts of V. negundo exhibited marked antibacterial activity against MRSA isolates. The non polar solvent, n-hexane extract of V. negundo showed highest ZoI (20 mm) against the ATCC MRSA culture. Petroleum-ether extract of V. negundo showed 17 mm ZoI against the MRSA strains. Similarly, both ethyl acetate and acetone extracts of V. negundo showed 16 mm ZoI individually, while the methanol and chloroform extracts of V. negundo exhibited 12 and 11 mm ZoI respectively.[19
Hence the differences in the zone of inhibition against the tested pathogen MDR K. pneumoniae exhibited by the different extracts of V. negundo in this study may be due to the differences in elution of maximum concentration of phytochemicals with potent antibacterial properties using different solvents and possible synergistic interactions between the various phytochemicals present in the extracts. The phytochemical analysis of the leaf extract of V. negundo revealed the presence of several secondary metabolites such as flavonoids, saponins, polyphenols, and tannins which either alone in combination might be responsible for its anti-microbial activity.
TLC bioassay for various extracts of V. negundo leaves
The results of the TLC bioassay clearly depict fluorescent spots in the ethyl acetate leaf extract of V. negundo , which represent the separation of active components in the ethyl acetate extract [Plate 2]. The fourth fraction in the ethyl acetate leaf extract seen as bright fluorescent band at R f value 0.3 exhibited positive activity against the tested pathogen MDR K. pneumoniae [Table 2 ] which may be due to the elution of maximum concentration of the secondary metabolites such as flavonoids, saponins, polyphenols, and tannins with potent antimicrobial properties acting either alone or in synergistic combination. There are several reports documenting the antimicrobial properties of the plant secondary metabolites including alkaloids, glycosides, tannins, flavonoids, and saponins using different strategies to inhibit the resistant bacteria.[20 21 22
Estimation of MIC of V. negundo ethyl acetate leaf extract against MDR K. pneumoniae by Alamar blue assay
The lowest concentration of the extract that inhibited the complete growth of bacteria after 48 hours of incubation at 37°C is known as MIC value. For pre-clinical assessment of new antimicrobial agents, MICs are usually measured. Lower MIC values indicate efficient antimicrobial agents.
The MIC value of V. negundo leaf extract against MDR K. pneumoniae by using Alamar blue assay is shown in Plate 4 and [Table 3 ]. Ethyl acetate extract of V. negundo leaves expressed MIC value of 1.5 μg/mL against the test pathogen MDR K. pneumoniae which is in harmony with the zone of inhibition value of 18 mm at the concentration of 200 mg/mL confirming its anti-bacterial activity. In agreement, the potential antimicrobial properties of the crude extracts and the purified compounds of V. negundo has been reported earlier.[23 V. negundo expressed lowest MIC value of 2.5 mg/mL against both X. axonopodis and P. solanacearum , while ethyl acetate extract of fruits of V. negundo showed MIC value of 5 and 10 mg/mL for X. axonopodis and P. solanacearum respectively (Jeyaseelan et al ., 2011[24
In the present study, the antimicrobial activity of the ethyl acetate extract of V. negundo leaves could be contributed by the presence of important bioactive compounds eluted in the extract. There are several reports that has documented the presence of the important bioactive phytochemicals flavonoids, lignans, terpenoids, and steroids in the V. negundo extract.[23 25 27 V. negundo leaves in this study might have also contributed primarily to its antibacterial properties. The differences in the MIC value of the different plant extracts in this study might be due to the variations in their chemical constituents and the volatile nature of certain components.
Determination of kill-time of V. negundo ethyl acetate leaf extract against MDR bacteria K. pneumoniae
Time kill assay studies the rate at which an antibacterial agent kills the bacteria in different concentrations. It examines the concentration-dependent and time-dependent bactericidal activities of antimicrobial agents.[28
In the present study, the inhibitory effect of the ethyl acetate leaf extract of V. negundo was apparent from 40 minutes indicating time dependant killing of the tested pathogen MDR K. pneumoniae. The mechanism of the antimicrobial action of a compound can be determined by evaluating the time it takes to kill the bacterial cell. If the antimicrobial action is instantaneous, the compound affects the membrane integrity and if it is time dependent, it affects the cellular processes.[29 V. negundo was apparent from 40 minutes indicating that the cellular process and the physiological factors inside the tested bacterial cell could be disrupted.
In agreement, the alcoholic extracts of certain medicinal plants were found to exhibit time dependent killing of E. coli due to their effect on several physiological factors inside the bacterial cell.[29 29 30 Phyllanthus muellerianus has been demonstrated against E. coli and Pseudomonas aeruginosa by time killing assay.[31 29 V. negundo could be due to the detergent like properties of its phytoconstituents.
Further studies are warranted to understand the exact underlying mechanism of action of the bactericidal effect of the ethyl acetate leaf extract of V. negundo including other bactericidal mechanisms such as disrupting the bacterial membrane potential, permeabilization, and leakage of cellular contents.
CONCLUSION
From the results of the present study, the bactericidal activity of the ethyl acetate leaf extract of V. negundo was evident, which may be attributed to the presence of secondary metabolites in the leaf extract. To understand the underlying mechanism of the bactericidal activity of the plant extract, further studies are required to provide insights into the pharmacokinetic and dynamic profile of the plant extract.
Financial support and sponsorship
Nil.
Conflicts of interest
The authors declare no conflict of interest
Acknowledgment
The authors acknowledge the financial support from RUSA 2.0 of Bharathidasan University.
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