Antibacterial Effect of Silver Nanoparticles Synthesized Using Murraya koenigii (L.) against Multidrug-Resistant Pathogens

Development of multidrug resistance among pathogens has become a global problem for chemotherapy of bacterial infections. Extended-spectrum β-lactamase- (ESβL-) producing enteric bacteria and methicillin-resistant Staphylococcus aureus (MRSA) are the two major groups of problematic MDR bacteria that have evolved rapidly in the recent past. In this study, the aqueous extract of Murraya koenigii leaves was used for synthesis of silver nanoparticles. The synthesized MK-AgNPs were characterized using UV-vis spectroscopy, FTIR, XRD, SEM, and TEM, and their antibacterial potential was evaluated on multiple ESβL-producing enteric bacteria and MRSA. The nanoparticles were predominantly found to be spheroidal with particle size distribution in the range of 5–20 nm. There was 60.86% silver content in MK-AgNPs. Evaluation of antibacterial activity by the disc-diffusion assay revealed that MK-AgNPs effectively inhibited the growth of test pathogens with varying sized zones of inhibition. The MICs of MK-AgNPs against both MRSA and methicillin-sensitive S. aureus (MSSA) strains were 32 μg/ml, while for ESβL-producing E. coli, it ranged from 32 to 64 μg/ml. The control strain of E. coli (ECS) was relatively more sensitive with an MIC of 16 μg/ml. The MBCs were in accordance with the respective MICs. Analysis of growth kinetics revealed that the growth of all tested S. aureus strains was inhibited (∼90%) in presence of 32 μg/ml of MK-AgNPs. The sensitive strain of E. coli (ECS) showed least resistance to MK-AgNPs with >81% inhibition at 16 μg/ml. The present investigation revealed an encouraging result on in vitro efficacy of green synthesized MK-AgNPs and needed further in vivo assessment for its therapeutic efficacy against MDR bacteria.


Introduction
Development of multidrug resistance has become a global issue with serious consequences in the management of infectious diseases caused by pathogenic bacteria [1].
is is mainly due to undiscriminating use of antibiotics in human healthcare, agriculture, and veterinary medicine [2]. e most common problematic multidrugresistant pathogens are Acinetobacter baumannii, ESβLproducing E. coli, penicillin-resistant Streptococcus pneumoniae, Klebsiella pneumoniae, vancomycinresistant Enterococcus, methicillin-resistant S. aureus, and extensively drug-resistant Mycobacterium tuberculosis [3]. ESβL groups of β-lactamases which are evolving at an alarming rate have ability to hydrolyse third-generation cephalosporins in addition to aztreonam [4,5]. Methicillin-resistant S. aureus (MRSA) is the paramount cause of nosocomial infections associated with pneumonia, bloodstream infections, and surgical site infections [6]. Considering these problems, researchers are focussing on the development or discovery of novel agents with broad-spectrum therapeutic potency. e literature in the recent past has demonstrated a potential role of metal nanoparticles as antibacterial agents. However, functional properties of metal nanoparticles can be improved through the green synthesis approach. Biological synthesis of nanoparticles is seeking an extraordinary consideration due to the fact that it is eco-friendly as compared to other routes of nanoparticle synthesis [7]. Despite the ability of physical and chemical methods to synthesize nanoparticles of particular size and shape, use of hazardous material and economically lesser feasibility make their application limited [8]. Commonly used chemical and physical methods are chemical reduction, ion sputtering, sol gel, etc., which have higher energy requirements and include improvident purifications [9,10]. Stability of synthesized nanomaterials and reproducibility make green synthesis a preferred technique over other methods [11]. Nanoparticles have been successfully synthesized from algae [12], actinomycetes [13], bacteria [14], plants [15], sugar [16], biodegradable polymers (chitosan) [17], and from many more substrates. Among aforementioned methods, plant-mediated synthesis is considered faster and requires lesser optimization [18].
Murraya koenigii (L.) is a small aromatic tree, commonly known as curry tree, belonging to Rutaceae family, has many traditional medicinal uses. e green leaves of this plant have been used in the Indian medicinal system. e leaves of this plant have also shown antihyperglycemic effects on rats under diabetic conditions [19]. e hydroalcoholic extract of M. koenigii has shown antioxidant and anti-inflammatory activities comparable with the standard drugs in an in vitro study [20]. Aqueous leaf extracts having high content of phenolics and flavonoids have potent free radical scavenging activity [21]. Ningappa and Srinivas isolated a 35 kDa protein called the curry leaf protein that has been found to exhibit high antioxidant activity [22]. e phytochemical characterization discovered the presence of major alkaloids as 9-formyl-3-methylcarbazole, 9-carbethoxy-3-methylcarbazole, and 3-methylcarbazole [23]. e stem bark extract of M. koenigii containing carbazole alkaloids and benzoisofuranone derivatives has potent antibacterial activity against Aspergillus niger, Bacillus subtilis, S. aureus, E. coli, Proteus vulgaris, and Candida albicans [24]. e antioxidant protein from curry leaves have a potent ameliorative effect of DNA damage on human erythrocytes caused by oxidative stress [25].
We have previously explored the antioxidant and antimutagenic potential of M. koenigii [26]. Considering the wellestablished medicinal value of M. koenigii and lack of work on green synthesis of metal nanoparticles, the leaves of M. koenigii were used for the synthesis of silver nanoparticles. e synthesized nanoparticles were characterized and their antibacterial efficacy against multiple Gram-positive and Gram-negative MDR bacteria was assessed in detail.

Collection of Plant Samples and Preparation of Aqueous
Extracts. Murraya koenigii (curry leaves) were collected in the vicinity of Aligarh Muslim University (AMU), Aligarh, and were identified by the plant biologist at the Department of Botany, AMU, Aligarh. e leaves were washed with distilled water to remove dust and particles, followed by shade-drying at room temperature. e leaves were then grinded to make fine powder using a blender. Five-gram of the leaf powder was mixed in 100 ml of distilled water and heated for one hour at 100°C. e extract was then centrifuged for 10 min at 10,000 rpm and filtered using the Whatman filter. e extract was stored at −20°C for further use.

Detection of Phytochemicals by Colour Test.
Chemical tests were performed to detect the presence of major groups of phytocompounds in the extract as described earlier [27][28][29].

Test for Tannins.
e dried extract (500 mg) was boiled in 20 ml of water and then filtered. Few drops of FeCl 3 (0.1%) was added to observe blue-black or brownish green colour.

Test for Flavonoids.
A colorimetric method for detection of flavonoids was adopted [27,29]. Dilute ammonia solution (5 ml) was mixed with the aqueous filtrate of the extract, and concentrated H 2 SO 4 was added. e development of yellow colour indicates the presence of flavonoids that disappeared on standing. One-hundred microlitres of NH 3 solution (1%) was added to a portion of filtrate. e yellow colour again appeared, which an indicator for the presence of flavonoids.

Test for Glycosides.
A small amount of the plant extract was dissolved in water. 1 ml of % NaOH solution was added. Appearance of yellow colour confirmed the presence of glycosides [30].

Test for Terpenoids by the Salkowski Test.
e aqueous extract (5 ml) was mixed in 2 ml of chloroform, and 3 ml of conc. H 2 SO 4 was added carefully through the wall of the test tube. Development of reddish brown colour at the interface shows the presence of terpenoids.

Test for Detection of Cardiac Glycosides by the Keller-Kiliani Test.
e aqueous extract (5 ml) was treated with glacial acetic acid (2 ml) containing a drop of FeCl 3 solution, followed by addition of 1 ml of conc. H 2 SO 4 . e development of a brown ring at the interface indicates deoxysugar which is a characteristic of cardenolides.

Synthesis of Silver Nanoparticles (AgNPs) by Aqueous
Extracts. For synthesis of AgNPs, aqueous solution of 1 mM silver nitrate (AgNO 3 ) was prepared. First, 20 ml of AgNO 3 solution (1 mM) was added to 0.5, 1.0, 2.0, and 4.0 ml of aliquots of M. koenigii aqueous extracts. e reaction was allowed to take place in 50 ml volumetric flasks at room temperature, and solutions were kept on a magnetic stirrer for vigorous mixing to optimize the reduction of AgNO 3 by the plant extract. Finally, the combination of 0.5 ml of M. koenigii extract and 20 ml of silver nitrate aqueous solution (1 mM) was found to be optimum; therefore, this concentration was further used in this study. e reduction of AgNO 3 to Ag + ions was confirmed by the appearance of dark brown that finally turned black. e AgNPs formed from M. koenigii (MK-AgNPs) were harvested by centrifugation at 11,000 rpm for 25 minutes at 25°C. e pellet obtained was dried in an oven at 60°C. As the combination of 0.5 ml of the aqueous extract and 20 ml of 1 mM AgNO 3 was found to be optimum, AgNPs synthesized with this combination were used in all further experiments.

Characterization of Nanoparticles Synthesized by Plant
Extracts.
e standard procedure was adopted for characterization of MK-AgNPs as described earlier [31].

UV-Vis Spectrum Analysis.
e MK-AgNPs were preliminary characterized using a UV-visible spectrophotometer. e synthesis of silver nanoparticles by reduction of silver ions was monitored by recording the UV-visible spectra of solutions after one-hour interval until no changes in absorbance were found. e absorbance spectra (300-600 nm) of AgNP solution were monitored using the Cintra 10e spectrophotometer (GBC Scientific Equipment Ltd.), and distilled water was used for baseline correction.

X-Ray Diffraction Analysis.
Silver nanoparticles obtained were characterized in powdered form by an X-ray diffractometer. e diffraction pattern was obtained using CuK α radiation (λ �1.54060Å) with a nickel monochromator from 20°to 80°. e average crystalline size of the nanoparticles was calculated using Scherer's equation: where D is the average crystal size of the nanoparticle, λ is the wavelength of the X-ray source (1.54060Å), β is the width at half of maxima of the diffraction peak, and K is the constant of the Debye-Scherrer equation with the value ranging from 0.9 to 1.0.

FTIR.
FTIR is an important technique for identification and characterization as it gives valuable information regarding the rotational and vibrational modes of a molecule. MK-AgNPs were washed thrice by centrifugation with deionized water to get rid of unbound or loosely attached proteins/enzymes or any other phytocompounds from the surface of silver nanoparticles. Powder of MK-AgNPs was diluted with KBr (1 : 100), and the transmittance spectrum was recorded. FTIR measurements were carried in the diffuse reflectance mode at 4 cm −1 resolution using the PerkinElmer FT-IR spectrometer (Spectrum Two, Perki-nElmer Life and Analytical Sciences, CT, USA).

Transmission Electron Microscopy (TEM).
e grid was prepared by placing aqueous suspension of MK-AgNPs on a TEM grid. It was allowed to air-dry overnight before imaging. Separate images were taken at 200 KV and magnification 20,000x to 100,000x at the University Sophisticated Instrumentation Facility (USIF), AMU, Aligarh, using a transmission electron microscope (JOEL-2100, Tokyo, Japan).

Scanning Electron Microscopy (SEM).
For SEM analysis, fine powder of MK-AgNPs was used. e images were recorded using the JSM-6510LV scanning electron microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 20 kV at the University Sophisticated Instrumentation Facility (USIF), AMU, Aligarh. e analysis of elemental composition of MK-AgNPs was performed using the INCAx-sight EDAX spectrometer (Oxford Instruments) equipped with an SEM.

Bacterial Strains.
e silver nanoparticles synthesized from the extracts (MK-AgNPs) were tested for antimicrobial activity against three Gram-positive bacterial strains and four Gram-negative bacterial strains. Among Gram-positive strains, there were one methicillin-resistant S. aureus (MRSA) and two methicillin-sensitive S. aureus (MSSA1 and MSSA2). In Gram-negative bacterial strains, three extended-spectrum β-lactamase-producing E. coli (ECESβL1, ECESβL2, and ECESβL3) and one sensitive E. coli (ECS) were used in the present study.

Agar Well Diffusion
Assay. Preliminary antibacterial activity of MK-AgNPs was evaluated using the agar well diffusion assay as described earlier [31]. e bacterial test organisms were grown in the nutrient broth overnight to attain the colony-forming unit (CFU) of ∼10 6 per·ml. Onehundred microlitres of each bacteria culture was spread on the Luria-Bertani agar plates. Agar wells (8°mm) were punched with the help of sterilized micropipette tips and loaded with double distilled water, AgNO 3 solution, and MK-AgNPs. e plates were then incubated for 18 h at 37°C, and diameters of zone of inhibition were recorded in millimetre (mm).

Determination of Minimum Inhibitory Concentration (MIC).
e MIC of MK-AgNPs against test bacteria was determined in a 96-well microtitre plate using TTC (2,3,5triphenyl tetrazolium chloride) as described earlier [32]. Twofold dilution of MK-AgNPs (256-08 μg/ml) was made in autoclaved media in a 96-well microtitre plate. Ten microlitres of the overnight-grown culture was inoculated in each well, and the control group did not contain MK-AgNPs. e cultures were then allowed to grow overnight in a bacteriological incubator. To indicate the presence of metabolically active bacterial cells, the presence of colour was observed after 30 min of addition of 10 μl/well of TTC (2 mg/ml). e concentration at which no change in colour was noted was taken as MIC.

Determination of Minimum Bactericidal Concentration (MBC).
e MBC of MK-AgNPs against test bacterial pathogens was assessed by the macrobroth dilution assay as described by Ansari et al. with few modifications [33]. All bacterial cultures were grown in media containing MK-AgNPs, and the group with MK-AgNPs was taken as control. Twofold dilution of varying concentrations (256-16 μg/ml) was selected for the treatment to determine the MBC. e overnight-grown cultures were then streaked on agar plates from each treated concentration to determine the MBC.

Growth Curve Analysis.
e effect of MK-AgNPs on the growth kinetics of E. coli and S. aureus isolates was monitored by optical density measurements [34]. Twofold dilution of silver nanoparticles ranging from 256 μg/ml to 8 μg/ml was made in a sterile 96-well microtitre plate in the nutrient broth. Ten microlitres of the overnight-grown culture (∼10 6 CFU/ml) was inoculated in each well containing varying concentrations of MK-AgNPs. Wells containing only media was taken as the control group, and each concentration was performed in three replicates. All untreated and treated sample plates were incubated for 20 h at 37°C. e OD 620nm was recorded at intervals of 2 h using a microplate reader ( ermo Scientific Multiskan EX, REF 51118170, China). e average of three replicates was taken to plot the growth curve of each culture at each concentration of nanoparticles.

Detection of Phytocompounds by Colour Test and Quantitative Determination of Total Phenolic Content.
Various colour tests carried out on different extracts of M. koenigii revealed the presence of medicinally active phytoconstituents. Aqueous extracts of M. koenigii showed the presence of flavonoids, tannins, and cardiac glycosides, while glycosides were not detected.

Green Synthesis of Silver Nanoparticles Using Plant
Extracts. Several medicinal plants have been successfully tested and used in synthesizing silver nanoparticles [18,35]. In this study, the aqueous extract of M. koenigii was tested for synthesis of AgNPs. e M. koenigii extract was added to AgNO 3 solution, and the changes in colour were monitored.

UV-Visible Analysis of AgNP Synthesis.
e primary characterization of MK-AgNPs synthesized using the aqueous extract of M. koenigii was done by UV-visible spectroscopy. Upon addition of the M. koenigii extract, the colour of silver nitrate solution changed to dark brown after 4 h of incubation, indicating the formation of silver nanoparticles. e change in colour by the extract demonstrates the reducing ability of the plant extract for synthesis of AgNPs [36]. e brownish colour appears due to the coherent oscillation of conduction band electrons at the nanoparticle's surface, resulting in surface plasmon resonance (SPR) [37]. UV-visible spectra of the silver nanoparticle's colloidal solution synthesized using M. koenigii (MK-AgNPs) as a function of time caused by the reduction of AgNO 3 are shown in Figure 1. ere was no remarkable absorption peak just after addition of the extract to silver nitrate solution, while a peak at 400-450 nm started to emerge as the colour of the solution changed. e SPR band at 410 nm indicates the synthesis of silver nanoparticles by the M. koenigii extract that saturated after 4 hours.

XRD Analysis.
e green synthesized MK-AgNPs were further characterized by monitoring the XRD pattern. Figure 2 shows the typical XRD patterns of both MK-AgNPs.
e nanocrystalline nature of the MK-AgNPs is visibly evident by the peak broadening. MK-AgNPs showed a maximum peak at the 2θ value of 33.74°. e average particle size of MK-AgNPs was found to be 13.54 nm. e size range and shape of MK-AgNPs were validated by performing transmission electron microscopy (TEM).

Fourier-Transform Infrared Spectroscopy (FTIR).
FTIR analysis was employed to assess the role of various phytoconstituents of M. koenigii which were responsible for synthesis and stabilization of MK-AgNPs. Figure 3 shows the FTIR spectrum of MK-AgNPs. A comprehensive peak near 3404 cm −1 indicates the stretching vibrations of the hydroxyl group. e short absorption peak at 2928 cm −1 is assigned to vibrations of aliphatic C-H stretching. e absorption band at 1642 cm −1 can be attributed to the N-H bond of amines. Presence of few short peaks between 1370 and 1390 cm −1 corresponds to the CH 3 -CH bond of alkanes and alkyl groups. e prominent peak at 1063 cm −1 is due to C-O stretching from carboxylic acid, alcohols, and ethers. e peak around 632 cm −1 might be due to C-H bonding of aromatic compounds. It has been documented that phytocompounds and proteins/enzymes present in the extracts of plants act as a capping agent [38,39].

Transmission Electron Microscopy.
e absorption spectra give information only on formation of AgNPs. e formation of nanoparticles is investigated in detail and validated by using TEM. Figure 4 shows the transmission electron micrograph of MK-AgNPs. TEM images clearly illustrated the different size range (5-20 nm) of MK-AgNPs. Furthermore, the transmission electron micrograph revealed that most of the particles were spheroidal in shape along with few nanoparticles having anisotropic morphology. Few particles were also found to form small aggregates which may possibly be due to agglomeration or improper capping. e variations in shape and size of the nanoparticles synthesized by green synthesis approaches had also been documented earlier [40,41].

Scanning Electron Microscopy.
Scanning electron microscopy was employed for the surface morphology of synthesized nanoparticles. e scanning electron micrograph at 7,000x of silver nanoparticles synthesized using M. koenigii is presented in Figure 5. e surface morphology of most of the nanoparticles was found to be anisotropic in shape. It is clear from the EDX profile that the elemental composition of silver, carbon, oxygen, and chlorine in MK-AgNPs was found to be 60.86%, 19.84%, 12.53%, and 6.77%, respectively, by weight ( Figure 5(b)). However, distribution of particle size is also reliant on their relative nucleation rates, extent of agglomeration, and growth processes [42].

Agar Well Diffusion Assay.
e antimicrobial activity of MK-AgNPs was investigated against E. coli and S. aureus using the agar well diffusion assay. e zones of inhibition (mm) around each well containing MK-AgNPs and AgNO 3 solution are represented in Figure 6. Among the tested bacteria, MSSA2 was found to be most resistant with a minimum inhibition zone of 16 mm for MK-AgNPs and 15 mm for AgNO 3 solution at 100 µg/well. ECS was found to exhibit maximum zones of inhibition of 21 and 18 mm for MK-AgNPs and AgNO 3 solution, respectively. e enhanced antibacterial activity of AgNPs compared to AgNO 3 solution is attributed to their large surface area that provides more surface contact with microorganisms [43]. Another important reason of enhanced antibacterial activity of AgNPs as documented in the literature is the synergistic effect between particles and natural compounds [44]. Cardozo et al. found that synergy between phenazine-1-carboxamide and AgNPs   Bioinorganic Chemistry and Applications increased the antibacterial effect by 32-fold against MRSA strains, causing morphological alterations to the cell wall of bacteria [45]. e mechanism of action of the antibacterial activity of AgNPs is attacking the respiratory chain and cell division that ultimately leads to cell death. e silver nanoparticles have also been reported to release silver ions inside the bacterial cells, further enhancing their bactericidal activity [46].

MIC and MBC of MK-AgNPs.
MK-AgNPs were found to be an effective antibacterial agent against the test bacteria. e recorded MIC values at which no visible growth of test bacterial strains is found are presented in Figure 6. e minimum inhibitory concentration for MK-AgNPs against MRSA and MSSA was found to be 32 μg/ml. ECS was found to be most sensitive among the tested strains with an MIC value of 16 μg/ml, while ECESβL2 was found to be highly resistant against silver nanoparticles with an MIC value of 64 μg/ml. However, no definite criteria have been established so far to consider the MIC breakpoints on the resistance level.  e MBC is the lowest concentration of any antibacterial agent that kills 100% of the bacterial population and did not show any viable growth when streaked on agar plates. e value of MBC of the test organisms for MK-AgNPs is presented in Figure 6. e value of MBC for MRSA, MSSA1, MSSA2, ECESβL1, ECESβL2, and ECESβL3 was found to be 64 μg/ml, while for ECS, MBC was found to be 32 μg/ml for MK-AgNPs. ese values are in accordance with MIC results in which ECS was found to be most sensitive.
Similar results have been documented earlier where MIC and MBC values of AgNPs synthesized using Eucalyptus globulus against E. coli were found to be 36 and 42 μg/ml [47]. Moreover, Ansari and Alzohairy have reported MIC and MBC values of AgNPs prepared using seed extracts of Phoenix dactylifera as 10.67 and 17.33 μg/ml, respectively, against methicillin-resistant S. aureus [48]. ese variations might be due to the different intrinsic tolerance levels of test strains used in the assays, size and nature of nanoparticles, and methods adopted for the determination of the MIC and MBC.

Effects of Silver Nanoparticles on Growth Kinetics of Bacteria.
e dose-dependent effect of MK-AgNPs against MRSA, MSSA, EsβL-producing E. coli, and a sensitive E. coli (ECS) strain was studied. e growth was monitored at different time intervals by measuring absorbance at 620 nm using a microplate reader. e treatment with nanoparticles to all the bacterial strains was given in twofold dilution, and the wells without any amendment of MK-AgNPs were taken as the control group. e extent of growth inhibition on MRSA by MK-AgNPs was found to be 31.4%, 53.3%, and 85.6% at 8, 16, and 32 μg/ml, respectively (Figure 7), and the concentration above 32 μg/ml was found to be inhibitory to more than 90% of the bacteria. e treatment with 8, 16, and 32 μg/ml of MK-AgNPs resulted in 37.0%, 48.5%, and 90.8% inhibition on MSSA1, respectively, as shown in Figure 8(a). e concentration exceeding 32 μg/ml checked the MSSA1 growth by more than 95%. A similar pattern of inhibition was obtained for MSSA2 as presented in Figure 8(b). For the ECESβL1 strain, the reduction in viability was observed to be 19.8%,  59.6%, and 87.4%, respectively (Figure 9(a)). On the contrary, ECESβL2 showed 21.3%, 57.4%, and 60.4% inhibition showing more resistance to MK-AgNPs as compared to ECESβL1 (Figure 9(b)). ECESβL3 exhibited growth kinetics similar to ECESβL1 at varying concentrations of MK-AgNPs (Figure 9(c)). ECS showed least resistance to MK-AgNPs with percent inhibition of 31 and 81.8 at 8 and 16 μg/ml ( Figure 10). e concentration above 16 μg/ml was found to inhibit ECS by more than ninety percent. e toxicological impact of silver nanoparticles depends not only on its size but also on the test organisms [49]. It has been documented by Brunner et al. that toxicity of nanoparticles may either be due to release of metal ions from nanoparticles such as Ag + or production of reactive oxygen species or direct interaction and disruption of biological macromolecules such as intercalation to DNA [50]. A study has demonstrated that silver nanoparticles  synthesized from the green approach with an average size of 18 nm exhibited the MIC value against ESβL-producing E. coli and P. aeruginosa as 36 μg/ml and 27 μg/ml, respectively [47].

Conclusion
In this study, an eco-friendly method for synthesis of nanoparticles using the aqueous extract of M. koenigii was developed. Characterization of MK-AgNPs revealed that the particles were spheroidal in shape with a particle size distribution range of 5-20 nm along with 60.86% silver content. MK-AgNPs effectively inhibited the growth of the test pathogens on nutrient agar plates with varying zones of inhibition. e MIC for different strains of S. aureus was 32 μg/ml, while different strains of E. coli showed a varying range of MIC (16 to 64 μg/ml). ere was approximately 90% growth inhibition for all strains of S. aureus at 32 μg/ml of MK-AgNPs. e finding revealed that MK-AgNPs exhibited a wider range of activity against both Gram-positive and Gram-negative MDR bacteria. e green approach for synthesis of silver nanoparticles, especially for antibacterial purposes against human pathogens, opens a new path in antibacterial drug discovery. e study clearly reveals an encouraging in vitro efficacy of MK-AgNPs that can be used for topical application against MDR bacteria after careful in vivo investigation.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
All authors declare that there are no conflicts of interest.