Bioactive Azadirachta indica and Melia azedarach leaves extracts with anti-SARS-CoV-2 and antibacterial activities

The leaves of Azadirachta indica L. and Melia azedarach L., belonging to Meliaceae family, have been shown to have medicinal benefits and are extensively employed in traditional folk medicine. Herein, HPLC analysis of the ethyl acetate fraction of the total methanolic extract emphasized the enrichment of both A. indica L., and M. azedarach L. leaves extracts with phenolic and flavonoids composites, respectively. Besides, 4 limonoids and 2 flavonoids were isolated using column chromatography. By assessing the in vitro antiviral activities of both total leaves extracts against Severe Acute Respiratory Syndrome Corona virus 2 (SARS-CoV-2), it was found that A. indica L. and M. azedarach L. have robust anti-SARS-CoV-2 activities at low half-maximal inhibitory concentrations (IC50) of 8.451 and 6.922 μg/mL, respectively. Due to the high safety of A. indica L. and M. azedarach L. extracts with half-maximal cytotoxic concentrations (CC50) of 446.2 and 351.4 μg/ml, respectively, both displayed extraordinary selectivity indices (SI>50). A. indica L. and M. azedarach L. leaves extracts could induce antibacterial activities against both Gram-negative and positive bacterial strains. The minimal inhibitory concentrations of A. indica L. and M. azedarach L. leaves extracts varied from 25 to 100 mg/mL within 30 min contact time towards the tested bacteria. Our findings confirm the broad-spectrum medicinal value of A. indica L. and M. azedarach L. leaves extracts. Finally, additional in vivo investigations are highly recommended to confirm the anti-COVID-19 and antimicrobial activities of both plant extracts.


Introduction
Drug resistance to infections in humans brought on by microorganisms including bacteria, viruses and fungi is a life-endangering phenomenon with considerable number of mortalities worldwide. Medicinal herbs were applied to produce a plenty of pharmaceuticals that are a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 7-benzoylazadiradione, 7-diacetyl-7-benzoylgedunin, 17-hydroxyazadiradione and nimbiol [23]. Traditionally, Neem leaves extract are utilized in folk medicine to prevent infectious bacterial and parasitic diseases. In addition, researchers reported many other biological and pharmacological activities including antiviral, antifungal, anti-inflammatory, antipyretic, antiseptic, and cytotoxic activities [19].
Herein, we evaluated the anti-SARS-CoV-2 and antibacterial activities of A. indica L. (Neem) and M. azedarach L. leaves ethyl acetate fractions and evaluating their phenolic profile against pandemic SARS-CoV-2 virus and some selected pathogenic bacterial strains.

Chemicals and plant materials
All solvents utilized in this current study were of analytical grade (BDH, UK and Merck, Germany). Fresh leaves of A. indica L., and M. azedarach L. were obtained from Orman botanical garden, Giza, Egypt and identified by Mrs. Trease Labib specialist of plant taxonomy. Leaves were immediately wrapped in plastic packaging after becoming air-dried.

Extraction procedure
Extraction was performed using cold extraction method [24]. Briefly, the air-dried leaves of A. indica L., and M. azedarach L. (500 mg) were separately soaked with 80% methanol solvent for 72 h (statically, 2Lx5 at room temperature). Subsequently, the extract was filtered on gauze and the used solvent was vanished entirely using rotary evaporator instrument. The leftover residue was then suspended in double distilled water (ddH 2 O). Extract was moved into a separating funnel to complete the defatting process using petroleum ether (40-60˚C) and fractionation using ethyl acetate repeatedly [25]. Further, the obtained fractions were concentrated by using rotary evaporator under 40˚C till further phytochemical investigations.

HPLC analysis of the ethyl acetate fraction
To characterize the chemical composition of the ethyl acetate fraction, HPLC was done using Agilent Technologies 1100 series liquid chromatograph that is supplemented with an auto sampler and a diode-array detector (DAD). Eclipse XDB-C18 (150 x 4.6 μm; 5 μm) analytical column with a C18 guard column was used (Phenomenex, Torrance, CA). Acetonitrile (solvent A) and 2% acetic acid in water (v/v) made up the mobile phase (solvent B). The gradient was programmed as follows: 0-5 min, 100% B (isocratic step); 30 min, 100-85% B (linear gradient); 20 min, 85-50% B (linear gradient); 5 min, 50-0% B (linear gradient); 5 min, 0-100% B (linear gradient) at a flow rate of 0.8 ml/min. Peaks for the benzoic acid, cinnamic acid derivatives, and flavonoids were sequentially analyzed at 280, 320, and 360 nm for the injection volume of 20 L, correspondingly. Prior to injection, all specimens were purified with an Acrodisc 0.45 μm syringe filter (Gelman Laboratory, Michigan, USA). Peaks were identified by correlating UV spectra and retention times, and these were then compared with the standards that run under the same conditions.

Isolation of major compounds
The ethyl acetate fraction (4 g) of both plant leaves was subjected to silica gel column chromatography (Silica gel 60, Merck, Germany) eluted using 100% n-hexane, and different ratios of mixture of ethyl acetate, and methanol. The collected fractions were evaporated under vacuum and examined by thin layer chromatography (TLC) which was carried out on pre-coated silica gel 60 F254 TLC plate (Merck). The plates were visualized under UV at λmax-254 nm and spraying with 10% sulfuric acid and AlCl 3 . Similar fractions were combined together according to R f values. Detailed structure elucidation of the isolated compounds were performed by different spectral analyses (IR, mass spectrometry, and 1 H-NMR (400 MHz), and 13 C-NMR (125 MHz)).

Cytotoxic concentration 50 (CC 50 ) determination.
Solution of the examined plant extracts were established in 10% DMSO and diluted into serial dilutions with DMEM (1 μg/ ml-10 mg/ml) to estimate the half maximum cytotoxic concentration (CC 50 ). The cytotoxic effectiveness of the extracts was investigated utilizing 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay with minimal alterations in Vero E6 cells, an immortalized cell line generated from renal epithelial cells of the African green monkey [5,6,28]. We employed non-linear regression data to compare the cytotoxicity of differing extract concentrations on treated to untreated cells by graphing log inhibition against standardized responsiveness.

Inhibitory concentration 50 (IC 50 ) determination.
The IC 50 values for the examined extracts were determined as previously described [5,6]. Briefly, Vero-E6 cells were dispensed evenly into the 96-well tissue cultivation plates and were cultured overnight at 37˚C with 5% CO 2 to reach 80-90% confluency. The cell monolayers were then exposed to pre-incubated sample/virus mixtures containing 100 TCID50 (Median Tissue Culture Infectious Dose) of the virus. The treated cell monolayers are kept the mixtures containing different sample concentrations (<CC 50 ) for 1 h at ambient temperature and then washed out with 1x phosphate buffer saline (PBS). Subsequently, 100 μl of infection media DMEM supplemented with 1% pen/strep and 0.2% Bovine Serum Albumin (BSA) were added. The cells were then kept for 72 h at 37˚C in an incubator with 5% CO 2 . Eventually, cell monolayers were fixed with 100 μl of 4% para-formaldehyde for 20 minutes, stained further with 0.1% crystal violet in distilled water at RT for 15 min and destained with absolute methanol. The absorption intensity of the solubilized crystal violet color was then measured at 570 nm using an AnthosZenyth 200rt plate reader. In comparison to the virus and cell controls, the compound's 50% inhibitory concentration (IC 50 ) was calculated using GraphPad prism software by plotting the log concentration against normalized responses.

Antibacterial activities of prepared plant extracts
The bactericidalpotential of 50μg/mL of A. indica L. and M. azedarach L. leaves extracts were evaluated using the disc and well-diffusion assays by the formation of inhibition zone. Then, the broth-microdilution approach was used to estimate the values of minimum inhibitory concentration (MIC). Listeria monocytogenes (ATCC 35152), Staphylococcus aureus (ATCC 43300) and Enterococcus faecalis (ATCC 43845), representing Gram-positive (GP) bacteria. Each bacterial culture stock preserved in -80˚C was injected into a tube containing 20 ml of Trypticase soya broth (TSB). Then, all inoculated tubes were incubated for 24 h at 37˚C. Dilution of the cultures with Mueller-Hinton Broth (MHB) was performed corresponding to ten folds serial dilution upto 10 -8 CFU/ml of the bacterial culture.
2.6.2. Agar dilution methods (well and disc diffusions. 2.6.2.1. Disc-diffusion method. The technique was conducted based upon the Clinical and Laboratory Standards Institute (CLSI) (M02-A11) [29]. The prepared bacterial inoculum suspension was briefly swabbed onto MHA medium with a sterile cotton swab. Next, sterile discs made of filter paper (6 mm) (Maidstone, UK) were saturated with 50μg/mLof the tested samples. Both Ciprofloxacin (5μg/ disc) and Vancomycin (30μg/disc) were used as controls. The saturated discs were lightly pushed on the top surfaces MHA and incubated with bacteria overnight at 37˚C. A produced zone of inhibition (ZOI) was measured and given in millimeters (mm) [15].
2.6.2.2. Well-diffusion method. Using sanitized needle tips, a 6 mm-diameter hole was drilled. Afterward when, a sterilized cotton swab was employed to smear the inoculum standard solution of the examined microbes onto the medium. The specimen was then transferred to each well in intervals of 50 μL. Two reference antibiotic drugs (vancomycin and ciprofloxacin) served as positive controls. Filtered water functioned as the negative control. All inoculation plates were then incubated at 37˚C on the plate. For the tested microorganisms, the inhibition areas were observed within 24 h [16].

Broth-dilution assay and estimation of MICs values.
The minimum inhibitory concentration (MIC) for antibacterial activity of leaves extracts of A. indica L. and M. azedarach L. were assessed against the above mentioned microbial pathogens. Briefly, 100 μl of each fresh grown microbial culture was inoculated into10 mL sterile distilled water (dH 2 O) tubes containing different concentrations (25,50,75, and 100 mg/ml) of the tested leaves extracts. In contrast, the control tubes for each tested strain were free from the tested extracts. The specific conditions of all trials were at ambient temperature (25±0.43˚C) and 250 rpm of agitation in shaker. Samples were collected from each tube at four different time intervals (5, 10, 20, and 30 min). The bacterial cell densities were counted before (initial count) and after exposure to the tested extracts. The viable bacterial counts were calculated as CFU/mL for 24 h at 37˚C using the pour plate method according to APHA [30]. All experimental trials were accomplished in three times intervals and repeated two times on dissimilar periods [17].

Statistical analysis
The inhibitory concentration (IC 50 ) of leaf extracts of A. indica L. and M. azedarach L. was calculated using GraphPad Prism version 9.2.0. Moreover, the Minimum Inhibitory Concentration (MIC) of the two plant extracts against the tested bacterial strains was also computed using a two-way analysis of variance (ANOVA), where ( �� ) denotes a moderate correlation (p �0.01) and ( ��� ) denotes a high correlation (p� 0.001). Additionally, the Person (r) correlation P-Value and R2 value between various plant extract doses and the examined bacterial strains were calculated.

HPLC-DAD analysis
HPLC-DAD analysis was used to recognize and estimate phenolics and flavonoids in ethyl acetate fraction of the methanolic extract of A. indica L. and M. azedarach L. ( Table 1). The analysis discovered the existence of 10 and 11 phenolics in A. indica, L. and M. azedarach L., respectively. Catechin was proved to be the dominant compound in both fractions (315.404 μg/ g in A. indica L. and 198.247 μg/g M. azedarach L.), followed by vanillic acid (151.947 μg/g) in A. indica L. and chlorogenic acid (112.869 μg/g) for M. azedarach L. Additionally, four flavonols were identified in both fractions of which rutin was the major ( Table 1).
It is to be mentioned that phenolics and flavonoids are the dominant bioactive constituents in plant kingdom possessing various medicinal properties such as antioxidant, cytotoxic, antiinflammatory and antimicrobial activities [24,25]. The prime mechanism of action of attained phenolics and flavonoids is attributed to phenolic-protein interactions where their metabolites, which are present in a rather high concentrations, form the complex with functional enzymes and exert a clear radical scavenging action leading to developing their bioactivities [31].

Characterization of M. azedarach L. compounds
Column chromatography technique afforded the isolation of three compounds. Two compounds (4 and 5) from 100% n-hexane fraction and compound 6 eluted from ethyl acetate/ Methanol (80:20 v/v).  10 , 649 for ethoxyl group loss at C-12, then 449 for two tigloyl side chain loss, followed by 389(100%) due to acetyl group loss and subsequently water loss giving 371 which are in accordance to nimbolinin limonoids fragmentation pattern. The 1 H and 13 C NMR data were similar to [36], thus, the structure was elucidated as 1α,7α-ditigloyloxy-3α-acetoxyl-12α-ethoxylnimbolinin.  13 C-NMR data confirmed this compound as 3-α-tigloylmelianol which was previously identified from M. azedarach fruits by [37] but it is the first time to be detected in leaves.

Anti-SARS-CoV-2 virus activity
To appraise the antiviral efficacy of leaves extracts against SARS-CoV-2 virus, the half maximal cytotoxic concentration (CC 50 ), the half maximal inhibitory concentration 50 (IC 50 ) and selectivity index (SI)(CC 50 /IC 50 ) against SARS-CoV-2 were individually evaluated (Fig 2 and Table 2). Both leaves extracts of A. indica and M. azedarach showed high safety in Vero E6 cells with CC 50 values of 446.2 μg/mL for A. indica and 351.4 μg/mL for M. azedarach, respectively. Additionally, both extracts exhibited promising inhibition of SARS-CoV-2 replication in Vero E6 cells at potent IC 50 concentrations of 8.541 μg/mL for A. indica and 6.922 μg/mL for M. azedarach (Fig 2). Interestingly, both extracts exhibited extraordinary selectivity indices (SI) (>50) ( Table 2). The determined SI values are meaningfully greater than those recommended for further assessment of the bioactive compounds (> 10) [38,39]. The SI values of 52.8 and 50.8 for A. indica and M. azedarach indicate the bioactive potential of A. indica and M. azedarach leaves extracts. Compared to the reference drug control remedesivir (CC 50 = 473.1 μM, IC 50 = 6.72 μM, and SI = 70.4), both plant extracts showed comparable safety and antiviral activities with slightly lower selectivity indices. These data demonstrate that both extracts can be considered as promising candidates for further in vitro and in vivo studies against SARS-CoV-2 virus and potentially other viruses.
To our knowledge, there is a few previous studies that assessed the antiviral activity of A. indica extracts against the novel SARS-CoV-2 virus [40,41]. In another virtual study, Baildya et al. [42] concluded that, all neem active compounds which extracted from leaves, flowers, seeds and narks have high degree of antiviral activity against Papain-Like Protease (PLpro) of SARS-CoV-2 virus.

PLOS ONE
Borkotoky and Banerjee [43], isolated five compounds from neem (nimbolin A, nimocin, 7-deacetyl-7-benzoylgedunin, 24-methylenecycloartanol and Cycloeucalenone). These compounds showed high stability and well interacted with crucial regions of E and M proteins of SARS-CoV-2. Moreover, Nimbolin A was the strongest compound can bind free energy with both E and M proteins [43]. Additionally, nimocin and cycloartanols (24-Mmethylenecycloartanol and 24-methylenecycloartan-3-one) also showed strong binding to both E and M proteins [44]. In the same line, Paraiso et al. [45] reported that polyphenols play an important role in alleviating the main SARS-CoV-2 infections in different stages, besides boosting the different organs recovery in post-COVID-19 patients.
In this study, we explored the in vitro viral inhibitory activity of M. azedarach L. leaves extract against SARS-CoV-2 which is so far not studied. Nethmini et al. found that M. azedarach L. could turn off the replication of herpes simplex virus, coxsackie virus, enterovirus, influenza A virus, and bovine rhinovirus in both epithelial and macrophage cell lines [46].

Antibacterial activity
In addition to the in vitro anti-SARS-CoV-2 effect in this study for both plant leaves extracts, the inhibitory zone assay was done to assess the in vitro antibacterial properties of the two leaves extracts against E. coli, P. aeruginosa, S. aureus, L. monocytogenes, and E. faecalis using agar well-diffusion assay. The obtained results indicated that 50 μg/ml of A. indica L. and M. azedarach L. displayed high potency for inhibiting a broad spectrum of examined Gram-negative (GN) species (Table 3).
Simultaneously, two reference antibiotics drugs vancomycin (30 μg/disc) and ciprofloxacin (5 μg/disc) were applied in this study as a positive control ( Table 3). Results revealed that the size of ZOI of vancomycin against the selected pathogens; E. coli, S. enterica, P. aeruginosa, S. aureus, L. monocytogenes, and E. faecalis using disc diffusion assay were ranged between 7 and 11 mm as mentioned in Table 3. In comparison, the size of ZOI of ciprofloxacin was ranged between 6 and 14 mm ( Table 3). Abbreviations: "CC 50 " half maximal cytotoxic concentration; "IC 50 " half maximal inhibitory concentration; "SI" Selectivity index.
https://doi.org/10.1371/journal.pone.0282729.t002  These results show the tendency that the GN bacterial species were more susceptible to all tested plant extracts than the GP species. Conversely, the width of ZOI around discs was lower than those around wells. Our data are in agreement with previous study [21], in which the authors demonstrated that the size of ZOI in well-diffusion assay is larger than disc diffusion assay [21]. The ZOI width of vancomycin as a reference drug was less than the ZOI by the tested plant extracts. This indicates that the extracts' inhibitory efficiency is greater than that of two commonly prescribed antibiotics.

Table 3. Antibacterial activity and zone of inhibition (diameter in mm) of the tested plant leaves extracts of
The different concentrations (10-100 mg/mL) of A. indica L. and of M. azedarach L. extracts were examined to estimate MIC values to assess their antibacterial potential during various exposure times (5, 10, 20 and 30 min) to different bacterial strains. As shown in  [22]. This finding is encouraging as the variety of treatment options against multi-resistant GN bacteria is highly restricted as compared to GP bacteria where several reserve antibiotics are available.
Using the same procedure and bacterial strains, we also assessed the antibacterial activity of M. azedarach extract. The results represented in  (Figs 3 and 4).
From statistical analysis results, the potential correlation with significance between various concentrations of particular extract towards all the targeted pathogenic bacterial strains was briefly reported in Table 4. The results gained revealed that there was an exceptional positive correlation with strong significance between GP and GN species and the concentrations of all plant extracts. On the other hand, results perceived that a moderate correlation had been reported for E. coli.
As a public biohazard with millions of deaths every year, antibiotic resistance emerged recently due to the continuous prescription of improper antibiotics at non-lethal doses. Methicillin-resistant S. aureus, vancomycin-resistant enterococci, and Mycobacterium tuberculosis are widely documented as challenging hospital-associated infections to treat with known antibiotics. To this point, medicinal plants are recently applied to provide a supplementary treatment line against these bacterial infections [24]. M. azadirachta L. and A. indica L. showed powerful antibacterial activities against various GP and GN species. The Meliaceae family has a substantial percentage of polyphenolic chemicals that could possess oxidative activities. A. indica L. phytochemicals have such a broad range of biological characteristics, especially antibacterial and cancer-fighting potential [25]. This botanical group has antimicrobial capabilities, particularly towards bacteria that cause ailments with drug tolerance [26]. Further, Jeba Malar and her colleagues described the bactericidal potential of M. azedarach L. towards numerous types of bacterial pathogens [47]. Limonoids are originated from euphane-or tirucallane-type triterpenoid classes and widely distributed in Meliaceae family. They were

PLOS ONE
reported to have many significant bioactivities such as antibacterial, antiviral, antifungal, and anticancer. Their antimicrobial activity were attributed to the destruction of the cell wall unity and coherence resulting exudation of the substance out of the cell and cell lysis [48].
The ethanolic extract of M. azedarach L. was established to be biocidal agent against P. aeruginosa, and E. coli, the lowest activity was documented in aqueous extract of M. azedarach

PLOS ONE
against E. coli (8.5 mm) and S. aureus (8.2 mm). In previous study, E. coli showed the lowest sensitivity to methanolic extract (6 mm), and no activity was observed in 10, 20, 30 μg/ml of methanolic extract of M. azedarach L. [27]. Previous study proved that M. azedarach L. seed extracts efficiently manage illnesses caused on by both GP and GN species and reported that the that ethyl acetate fraction exhibited greatest suppression, followed by aqueous and methanol-based extracts, each of which reduced the proliferation of all the studied pathogenic strains. The petrol and benzene extracts displayed antimicrobial activity versus 15 pathogens, however, in comparison to the polarity extracts. The findings previously illustrate that seed extracts are powerful antibiotics that can effectively prohibit both disease-caused pathogens to human infections.

Conclusions
A. indica L. and M. azedarach L. leaves were chemically characterized and were found to be rich in phenolic compounds and flavonoids. In vitro assessment of both extracts revealed that they have robust antiviral activity against SARS-CoV-2 as well as antibacterial effects against broader spectra of GN and GP bacteria. These data emphasize their broad-spectrum medicinal value and demand further in vitro and in vivo investigations as anti-SARS-CoV-2 candidate and antibacterial agents.