Collection and processing the plant material

Azadirachta indica A. Juss commonly known as neem, nimtree or Indian lilac, is a tree in the mahogany family Meliaceae. It is native to the Indian subcontinent and most of the countries in Africa. It is typically grown in tropical and semi-tropical regions. Neem trees also grow on islands in southern Iran. Its fruits and seeds are the source of neem oil.

The leaves are collected from Capital Central Park (El-Hadaeq Al-Markazeya), located within the New Administrative Capital to the east of Cairo, Egypt (30.011563° N, 31.654947° E) during the autumn season (October 8th, 2023). This park forms an elongated urban green corridor established on reclaimed desert land and bordered by recently developed residential and governmental districts. The site lies in a hot desert climatic zone (Köppen BWh), with very long, dry and hot summers, short mild winters, and extremely low annual rainfall. The native substrate consists of weakly calcareous sandy to sandy-loam desert soils that originally supported sparse natural vegetation but have been reshaped into an irrigated landscaped area with lawns, ornamental shrubs, and regularly spaced shade trees, including A. indica planted along internal pedestrian routes and service roads.

A voucher specimen was deposited at the Department of botany and microbiology, Faculty of Science, Herbarium, Damietta University (DAM000023).

Firstly, the leaves were dried in shade until complete dryness. The leaves branches were removed and only the leaves were ground into a powder.

Preparation of the neem leaves crude extract (NLE)

The powdered plant material (30 g) was soaked in approximately 1 L of absolute ethanol at room temperature, protected from light, under continuous stirring overnight. After this period, the extract was filtered to separate the solid material from the ethanolic extract. The filtrate of the crude leaf extract was then dried in a hot oven at 50 °C for 12 h to obtain a viscous honey-like crude extract. After this process, the crude extract was then stored at -5 °C.

Determination of total phenolic components (HPLC)

An Agilent (1260) series was used to determine the total phenolic components. The Zorbax Eclipse Plus C8 column (46 cm x 2500 cm) was used to perform the separation. Water and 0.05% trifluoroacetic acid in acetonitrile at a flow rate of 0.9 ml/min made up the mobile phase. In a linear gradient, the mobile phase was programmed as the method of Khalil et al.14.

Determination of the antimicrobial activitiesAntimicrobial activities

Using agar well diffusion method, 0.15 mg/ml of the neem extract were dissolved in ethyl alcohol and evaluated as antimicrobial agents15,16. A solvent control, consisting of ethyl alcohol alone at a maximum concentration of 1% (v/v), was also included in all assays and showed no antimicrobial activity against any of the tested microorganisms. The antimicrobial activities were determined towards bacterial and yeast strains that obtained from the Microbiology Laboratory, Faculty of Science, Damietta University, Egypt. Gram-positive bacteria (B. cereus HES3 OR553494 and Staphylococcus epidermidis ATCC 12228), Gram-negative bacteria (E. coli D8 MF062579 and Enterobacter aerogenes ATCC 13048), and yeast (Candida albicans ATCC 10231) were sub cultured on nutrient agar and yeast extract-peptone-dextrose (YEPD) agar media, respectively. Using the spread plate method, the examined microorganisms were inoculated (0.5 McFarland standard) after the culture media had been prepared, autoclaved, and put into sterile Petri plates. The tested extract (150 µg/ml) was aseptically applied in 100 µl portions to 5 mm perforated wells. After 24 h., the inhibition zones were determined and expressed in millimeters. The prescription drugs fluconazole and tetracycline were included as positive controls; each tested at a fixed concentration of 150 µg/ml to assess their maximum potential inhibitory activity against the test organisms.

Minimum inhibition concentration (MIC)

Using the serial dilution method, MIC of neem extract was evaluated. The extract was dissolved at different concentrations ranging from 5 to 50 µg/ml. Nutrient broth and YEPD broth medium was prepared and inoculated by 0.5 MacFarland of bacterial and yeast strains, respectively. After incubation at 37 °C for 24 h or at 30 °C for 48 h for bacterial and yeast strains, respectively. A spectrophotometric analysis was measured at 600 nm to assess the microbial proliferation17.

The enzymatic antioxidant activity

The Whittenbury18 and Sakamoto and Komagata19 methods were used to measure the peroxidase (POX) and catalase (CAT) activities for microbial strains treated with extract. For the enzymatic reaction, 4-aminoantipyrine, potassium phosphate buffer, 2,4-DCP, and H2O2 were mixed with the extract’s MIC for a minute in order to gauge POX activity. To measure CAT activity, extract MIC was mixed with phosphate buffer and H2O2. Using spectrophotometry at 510 and 415 nm, the oxidation-induced increase in absorbance was assessed in relation to the untreated microbes as a control. The number of moles of H2O2 per liter was one-unit mL− 1 of enzyme20.

Determination antioxidants activities2,22-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity

Scavenging activity of DPPH was done according to the method of Algfri et al.21 with some modification.

0.1 mM DPPH solution was prepared in methyl alcohol. Various amounts of the plant extract 10 mg/ml (1–10 µl) were mixed with DMSO to reach 20 µl, and then 1.48 ml of DPPH solution was added. The reaction mixture was incubated at room temperature in dark for twenty minutes. The absorbance of the prepared mixture was measured at 517 nm. Two milliliters of DPPH solution were used as a control. The extract’s scavenging radical activity was determined using the equation below.

$$\:\mathbf{\%}\:\mathbf{R}\mathbf{a}\mathbf{d}\mathbf{i}\mathbf{c}\mathbf{a}\mathbf{l}\mathbf{s}\:\mathbf{S}\mathbf{c}\mathbf{a}\mathbf{v}\mathbf{e}\mathbf{n}\mathbf{g}\mathbf{i}\mathbf{n}\mathbf{g}\:\mathbf{A}\mathbf{c}\mathbf{t}\mathbf{i}\mathbf{v}\mathbf{i}\mathbf{t}\mathbf{y}\:\left(\mathbf{R}\mathbf{S}\mathbf{A}\right)=\frac{\varvec{A}\varvec{c}-\varvec{A}\varvec{s}}{\varvec{A}\varvec{c}}\varvec{*}100$$

Where Ac is the control absorbance and As is the Sample mixture absorbance.

Superoxide dismutase (SOD)- like activity

The SOD like activity of the extract was estimated by the method of Dechatelet et al.22.

$$\begin{gathered} {\mathbf{Percent}}{\text{ }}{\mathbf{of}}{\text{ }}{\mathbf{inhibition}}{\text{ }}{\mathbf{reduction}}{\text{ }}{\mathbf{of}}{\text{ }}{\mathbf{nitro}}{\text{ }}{\mathbf{blue}}{\text{ }}{\mathbf{dye}} \hfill \\ \;\; = {\raise0.7ex\hbox{${\left( \begin{gathered} \user2{rate~of~the~change~of~blank~} \hfill \\ – \user2{rate~of~the~change~of~~the~sample~} \hfill \\ \end{gathered} \right)}$} \!\mathord{\left/ {\vphantom {{\left( \begin{gathered} \user2{rate~of~the~change~of~blank~} \hfill \\ – \user2{rate~of~the~change~of~~the~sample~} \hfill \\ \end{gathered} \right)} {\left( {\user2{rate~of~the~change~of~blank}} \right)\user2{~}}}}\right.\kern-\nulldelimiterspace} \!\lower0.7ex\hbox{${\left( {\user2{rate~of~the~change~of~blank}} \right)\user2{~}}$}}*{\mathbf{100}} \hfill \\ \end{gathered}$$

Catalase like activity

The extract’s catalase-like activity was determined using this method of Sinha23.

$${\mathbf{Catalase}}{\text{ }}\left( {{\mathbf{U}}/{\mathbf{L}}} \right)={\mathbf{1}}/{\mathbf{t}}{\text{ }}*{\mathbf{log}}{\text{ }}{{\mathbf{S}}_{\mathbf{0}}}/{\mathbf{S}}$$

Where S0is H2O2 initial concentration, S is H2O2 concentration at (t) mins.

Total antioxidants capacity (TAC) of the ethanolic neem extract

The Total antioxidants capacity of the plant extract was estimated following to the method of Koracevic et al.24.

$${\mathbf{TAC}}{\text{ }}\left( {{\mathbf{mM}}/{\mathbf{L}}} \right) = {\mathbf{C}}_{{{\mathbf{uA}}}} {\mathbf{X}}\left( {{\mathbf{K}} – {\mathbf{A}}} \right)/\left( {{\mathbf{K}} – {\mathbf{UA}}} \right)$$

K: the control absorbance

A: sample absorbance

UA: standard uric acid absorbance

CUA: uric acid concentration

Cytotoxicity of neem extract on different cell lines

The mitochondrial dependent reduction of yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to purple formazan25 was used to evaluate the cell viability. The MTT reagent was purchased from Sigma-Aldrich, St. Louis, MO, USA. A stock solution was prepared at 5 mg/mL in sterile phosphate-buffered saline (PBS) and passed through a 0.22 μm syringe filter to ensure sterility. For the cytotoxicity assay, the working solution was added to the cells at a final concentration of 2.5 µg/mL.

All experimental procedures were conducted under sterile conditions within a Class II biosafety laminar flow cabinet (Baker, SG403INT, Sanford, ME, USA). The MCF-7 human breast cancer cell line, the A549 lung carcinoma cell line and BNL mouse normal liver cells were cultured in DMEM/F-12 medium supplemented with 1% L-glutamine and 1% antibiotic-antimycotic solution containing 10,000 U/mL penicillin, 10,000 µg/mL streptomycin sulfate, and 25 µg/mL amphotericin B. Cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂ using a water-jacketed CO₂ incubator.

After 10 days of batch culturing, the cells were seeded at a density of 10 × 10³ cells per well into 96-well microplates containing complete growth medium and incubated for 24 h under standard conditions (37 °C, 5% CO₂). The medium was then replaced with serum-free medium to prevent any interactions between serum proteins and the extract, allowing an accurate evaluation of its cytotoxic effect, and the cells were treated with a range of concentrations of the test extract (1000, 500, 250, 125, 62.5, 31.25, 15.6, and 7.8 µg/mL) which was dissolved in DMSO to make a stock solution, and was further diluted in serum-free medium to achieve the desired test concentrations. The final DMSO concentration in the culture wells did not exceed 0.2% to avoid any solvent-induced cytotoxicity. For each concentration of the neem extract, the assay was performed in three wells (technical replicates) within the same experiment. To ensure reproducibility, the entire experiment was independently repeated three times using freshly cultured cells (biological replicates).

Following a 48-hour incubation, the medium was carefully removed, and each well was supplemented with 40 µL of MTT solution at a final concentration of 2.5 µg/mL. The plates were incubated for an additional 4 h under the same conditions. Then 200 µL of 10% sodium dodecyl sulfate (SDS) in deionized water was added to each well, and the plates were left overnight at 37 °C.

As a positive control, 100 µg/mL of a known cytotoxic natural compound (doxorubicin) was used under identical conditions to ensure total cell death, as described by Thabrew et al.26.

The following formula was used to determine the percentage change in viability.

$$\left( {\left( {{\mathbf{Reading}}{\text{ }}{\mathbf{of}}{\text{ }}{\mathbf{extract}}{\text{ }}/{\text{ }}{\mathbf{Reading}}{\text{ }}{\mathbf{of}}{\text{ }}{\mathbf{negative}}{\text{ }}{\mathbf{control}}} \right){\text{ }} – {\mathbf{1}}} \right){\text{ }}{\mathbf{x}}{\text{ }}{\mathbf{100}}$$

Cell cycle analysis

Cell cycle analysis was performed using flow cytometry to assess potential alterations in the distribution of cell cycle phases between treated and untreated A549 lung cancer cells. Three experimental groups were prepared, each containing 1 × 10⁶ A549 cells. The first group served as the negative control (untreated), the second was treated with doxorubicin as a positive control, and the third received neem extract at its IC₅₀ (128 µg/ml) concentration.

Cells from each group were suspended in 0.5 mL of 1× Dulbecco’s Phosphate-Buffered Saline (DPBS) and gently aspirated several times using a Pasteur pipette. The cell suspensions were then fixed by adding 70% ethanol and incubated on ice for 2 h. Following fixation, cells were centrifuged, and the ethanol supernatant was carefully removed. The resulting pellets were resuspended in 5 mL of 1× DPBS for 30 s, then centrifuged. After washing, each pellet was resuspended in 1 mL of propidium iodide (PI) staining solution and incubated at room temperature in dark for 30 min. The distribution of cells across the G0/G1, S, and G2/M phases of the cell cycle was analyzed using CytExpert software27.

Quantitative real-time PCR (qRT-PCR)

Quantitative real-time PCR (qRT-PCR) was conducted to evaluate gene expression changes in A549 cells treated with neem leaf extract. A549 cells were seeded in 6-well plates at a concentration of 4 × 10⁵ cells/mL and exposed for 24 h to 128 µg/mL of neem extract (IC50 determined by MTT assay, this concentration was selected because it allows around 50% of the cells to remain viable, providing sufficient living cells for reliable measurement of apoptotic and anti-apoptotic gene expression, while avoiding excessive cell death that could compromise RNA integrity). Total RNA was isolated from both treated and untreated (control) cells using TRIzol reagent, following the manufacturer’s protocol. Subsequently, 1 µg of total RNA from each sample was reverse-transcribed into complementary DNA (cDNA) using the Bio-Rad SYBR Green PCR Master Mix kit. The qRT-PCR reactions were carried out on a Rotor-Gene real-time PCR system (Corbett Research, Sydney, Australia) using gene-specific primers for BAX, BCL2, and P5328.

Statistical analysis

Data were analyzed using SPSS software (version 22). All results were expressed as mean ± STD. Differences in means between the various treated cells or media and control were analyzed by one-way ANOVA test and the results were considered statistically significant if p ˂0.05.