The study was carried out after getting approval from the Institutional ethics committee (IEC - 723/2019) between 1 ^st November 2019 to 25 ^th May 2021.

HCT-116 colon cancer cell lines were purchased from NCCS (National Centre for Cell Science), Pune, India and cultured on Dulbecco’s Modified Eagle Medium (procured from Invitrogen, Thermo Fisher Scientific, India) containing penicillin (100 units/mL), streptomycin (100 μg/mL) and 10% fetal bovine serum (FBS) in a humidified incubator (Rivotech Incubator, India) maintained at 37°C and 5% CO ₂ for 24-48 hours. The plates can be stored for 6 months at -80°C for future use.

The leaves of Annona reticulata were procured from the herbal garden of Dhanya Nursary, Koteshwara, Karnataka, India in May 2020. The authentication of the plant was performed by the Department of Plant Sciences, Bangalore Central University, Karnataka, India.

Preparation of ethanolic leaf extract of Annona reticulata

Fresh leaves of Annona reticulata (1 kg) were cleaned using water and air dried for 20 days. Then the leaves were homogenized and subjected to grinding using a blender (Panasonic MX-AC400 1000-Watt Mixer Grinder) to obtain a fine powder. This leaf powder was macerated in a Soxhlet apparatus (Borosil, India) with ethanol (1L) until the solvent became colorless. After the extraction was complete, using a rotary vacuum evaporator (EPS Biosolutions, India), the solvent was evaporated until semisolid crude extract was obtained. This ethanolic leaf extract was stored at -20°C until use. ¹⁴

Fractionation of ethanolic leaf extract of Annona reticulata

The ethanolic leaf extract (100 g) was sequentially fractionated using a series of organic solvents starting with petroleum ether followed by diethyl ether, chloroform, and ethyl acetate in a fractionating column (Borosil, India). The extract obtained with each solvent was filtered using a rotary vacuum evaporator (EPS Biosolutions, India) at 250-280 rpm. The evaporation of the solvent was carried out at a temperature below 35°C. ¹⁵

Preliminary phytochemical analysis of ethanolic leaf extract and its fractions

Annona reticulata ethanolic leaf extract and its fractions were evaluated for the presence of various classes of phytocomponents using specific biochemical tests mentioned below. ^{16 , 17}

Test for Terpenoids

2 mL of Chloroform was added with the 5 mL of alcohol leaf extract. Then 2 mL of concentrated sulphuric acid was added to the solution. If a reddish-brown color formed this would indicate the presence of terpenoids.

Test for Flavonoids

To test for flavonoids, 2 mL of 20% NaOH mixture was mixed with 2 mL of alcohol leaf extract for the development of the yellow color. If the solution turns colorless after the addition of 2 drops of diluted acid confirms the presence of flavonoid in the given sample.

Test for Phenols/polyphenols

2 mL of test solution in alcohol was added with one drop of neutral ferric chloride 5% solution. The formation of an intense blue color indicates the presence of phenols.

Test for Saponins/fatty acids

5 mL of distilled water was mixed with 1 mL alcohol leaf extract in test tube and it was mixed vigorously using vortex mixer. The upper froth layer was separated and mixed with few drops of olive oil and mixed vigorously and the foam appearance showed the presence of saponins.

Test for Tannins

2 mL of test solution was added with bromine water and acetate. Decoloration of bromine water indicates the presence of tannins.

Test for steroids/polysterols

2 mL of chloroform and concentrated sulphuric acid were added with the 5 mL leaf extract. In the lower chloroform layer red color would appear to indicated the presence of steroids.

Test for Alkaloids - Drangendroff’s test

2 mL of the extract was mixed with 1 mL of Dragendroff’s reagent. The formation of orange or orange red precipitate indicates the presence of alkaloids.

Test for carbohydrates

The Anthrone test was used in this study. 2 mg of ethanolic extract was shaken with 10 mL of water, and the filtrate was concentrated. To this 2 mL of anthrone reagent solution was added. Formation of green or blue color indicates the presence of carbohydrates.

Analysis of total phenolic and flavonoid content of ethanolic leaf extract and its fractions

Total phenolic content (TPC) and total flavonoid content (TFC) of ethanolic leaf extract and its fractions were determined using the Folin-Ciocalteu method and aluminum chloride colorimetric assay respectively. ¹⁸ In brief, to analyze TPC, 12.5 μl of Annona reticulata ethanolic leaf extract and its fractions (1 mg/mL) was added to a 96-well microplate [Thermo Fisher Scientific, India] and mixed with 125 μL of Folin–Ciocalteu reagent and the mixture was incubated for 5 minutes at room temperature. This was followed by the addition of 12.5 μL of 7% Na ₂CO ₃ and the plate was placed in the dark for 90 min. With the help of a microplate reader (Thermo Fisher Scientific, India) the absorbance was measured at 760 nm. Further, utilizing the standard curve of gallic acid, TPC was determined, and the results were expressed as mg of gallic acid equivalent per gram (mg GAE/g) of dry plant material.

For the analysis of TFC, briefly, 100 μL of Annona reticulata ethanolic leaf extract and its fractions (1 mg/mL) was added to a 96-well microplate (Thermo Fisher Scientific, India) and mixed with 100 μL of 2% aluminum chloride, and the final mixture was incubated for 10 min. The absorbance was measured using a microplate reader (Thermo Fisher Scientific, India) at 368 nm. Drawing upon the curve of quercetin, the TFC was determined, and the result was expressed as mg quercetin equivalent per gram (mg QE/g) of dry plant material.

Gas chromatography-mass spectrometry (GC-MS) analysis of petroleum ether fraction of ethanolic leaf extract of Annona reticulata ^{19 , 20}

The GC-MS analysis of the petroleum ether fraction was performed using a gas chromatography-mass spectrometer equipped with an RTX5 capillary column (length: 30 m×0.25 mm; film thickness: 0.25 micron) (RESTEK, PA, USA). An aliquot of 1 μl of the petroleum ether fraction of Annona reticulata ethanolic leaf extract was injected into the capillary column. Helium gas was used as a carrier gas at a flow rate of 1.0 ml/min. Initially, the temperature of the column was set at 60°C for 2 minutes and then was raised to 150°C and maintained at that temperature for 5 minutes. Then the temperature of the oven was increased and maintained at 280°C for 10 min. The ion source temperature was fixed at 200°C and the interface temperature was set at 280°C. Mass spectral scan range of 40-500 m/z was attained. The relative percent quantity of each phytocomponent in the petroleum ether fraction was evaluated by comparing the average peak area of each component to the total area of the chromatogram. The components present in the extract based on the GC-MS result were identified using Wiley-8 library and National Institute of Standard and Technology Mass Spectral Library (NIST). This analysis was carried out at Analytical Research & Metallurgical Laboratories Pvt. Ltd. (ARML), Bengaluru, India.

Growth conditions and Subculture of HCT116 colon carcinoma cells

HCT116 is an adherent human colorectal carcinoma cell line and has an epithelial morphology. The cells were cultured in T-25 (Eppendorf, India) flasks in DMEM, high glucose consisting of 10% Fetal Bovine Serum [FBS]. The cells are grown until 60 to 70% confluency is achieved upon which, the cells are trypsinized by addition of 1ml of 0.2% Trypsin-EDTA solution ((Gibco ^TM, ThermoFischer Scientific, India). The detached cells were collected and counted using conventional neubaur chamber for seeding. The seeding density was dependent on the surface area of the well.

Sulforhodamine B assay

Sulforhodamine B (SRB) assay was carried out to determine the effect of the ethanolic leaf extract and its fractions on the viability of the HCT-116 cell line. ²¹ HCT116 cells were seeded in a 96-well plate at a density of 5000 cells/well. The cells were given a 24h window to adhere to the well surface, after which, the cells were treated with compounds of interest at different concentration ranges. The ethanolic leaf extract and its fractions were added at a concentration range of 500 μg/mL - 7.81 μg/mL for a period of 48h to evaluate cytotoxic effect. 0.1% DMSO was used as control as the stock was prepared in DMSO such that, the highest concentration added to the well contains ≤0.1% DMSO. The standard anticancer drug, doxorubicin was added to the wells at concentration range of 24 μM - 0.046 μMand was used as positive control in the assay. Measurement of absorbance was carried out at 510 nm with the help of a microplate reader (Thermo Fisher Scientific, India), and the dose-response curve was used to calculate the half maximal inhibitory concentration (IC ₅₀) of each fraction. The most promising fraction was then chosen for further studies.

Scratch wound healing assay for migration of HTC-116 cell lines

Scratch wound assay was performed to evaluated the migratory properties of cancer cells in response to treatment with our prepared extracts. A monolayer of cells were prepared by seeding 2×10 ⁵ HCT-116 cells/mL in a 6-well plate and allowed to adhere to the well surface during a 24 hr window. Following this, Dulbecco’s phosphate buffered saline (DPBS) was used to wash the cells and then using a sterile 200 μl micropipette tip, a linear wound was created in the cell monolayer. DPBS was used to remove any detached cells and cellular debris. ²² Annona reticulata ethanolic leaf extract (30.16 μg/mL) and petroleum ether fraction (29.07 μg/mL) prepared by appropriate dilution with serum-free DMEM was added to the wells in duplicates To evaluate their ability to inhibit the migration of the cells. The concentrations of the ethanolic leaf extract and petroleum ether fraction used were based on the IC ₅₀ values of these fractions as obtained from the sulforhodamine B cytotoxicity assay. Wound areas were photographed at 0 and 24 hr in 4× magnification using an Labomed (USA, Model -TCM 400) inverted tissue culture microscope with a digital camera. The cells treated with 0.1% DMSO served as control and cells treated with doxorubicin (0.75 μM) served as a positive control. The images were analyzed and photographed at two time points that is at ‘0’ hr (zero) and after 24 hr of treatment using ImageJ software (NIH, USA) RRID:SCR_003070 version 1.53j. The healing of the wound was considered as closure of the wound and rate of migration of the cells was represented as percentage (%) migration for each group and compared with DMSO control % Migration rate = A i − A 24 hr A i × 100

Where, A _i is the initial wound area at ‘0’ (zero) hr and A _24hr is the wound area after 24 hr of extract/drug administration.

Bioinformatics analysis was carried out to determine the potential molecular targets of the major phytocompounds of the most potent fraction. The structures of the top five phytocompounds from GC-MS data of the petroleum ether fraction were retrieved from the NCBI PubChem database. These structures were then uploaded to the prediction of activity spectra for substances (PASS) online server (Version 2.0) to analyze the potential anticancer activities of active compounds using target fishing screening. This screening was based on the chemical similarity between various molecules and on the utilization of current knowledge of the biological activity of small molecules. “Chemical similarity principle” stating that similar molecules are likely to possess equivalent properties form the basis of this methodology. The potential anticancer activity of a phytocomponent using the PASS online server was determined on the basis of the probability activity (Pa) values of the pharmacological properties in the range of 0.03 to 0.99. The results were then filtered for anticancer activity, analyzed and recorded. ²³

All the assays were carried out in triplicate (n=3). The data was analyzed using graph pad prism (V.8.4.3) software with a perpetual license and the data from all the assays are expressed as mean ± standard deviation. One way-Analysis of Variance (ANOVA) followed by Duncan’s multiple range post hoc test was used to determine the statistical significance within various fractions. Student t-test was used to analyze the statistical significance between the treatment and control groups and Duncan’s multiple range test was used to analyze the statistical significance within various groups. p<0.05 was considered statistically significant.

The final dried ethanolic leaf extract yield was 170.3 g and was stored at -20°C until use. Fractionation of ethanolic leaf extract produced dried petroleum ether fraction (0.5 g), diethyl ether fraction (15.06 g), chloroform fraction (47.07 g) and ethyl acetate fraction (35.1 g).

Results of the phytochemical analysis of ethanolic leaf extract of Annona reticulata and its fractions are summarized in Table 1. The presence of terpenoids, flavonoids, polyphenols, tannins, steroids and poly-sterols were reported in the extract, whereas alkaloids and saponins were absent.

TPC was expressed as mg of gallic acid equivalent per gram of dry plant material (mg GAE/g) and TFC was expressed as mg of quercetin equivalent per gram of dry plant material (mg QE/g). Ethanolic leaf extract was found to contain highest TPC (46 ± 0.26 mg GAE/g of plant extract) and TFC (19.9 ± 0.3 mg QE/g of plant extract) compared to its fractions. The TPC and TFC of all the fractions were mentioned in Table 2.

The GC-MS analysis of the petroleum ether fraction revealed the presence of 56 volatile compounds ( Figure 1). The most predominant phytocompounds present in the petroleum ether fraction were phytol (13.03%), 2,6-bis (3,4-methylenedioxyphenyl)-3,7-dioxabicyclo (3.3.0) octane (11.95%), gamma.-sitosterol (10.45%), alpha.-tocopherol-beta.-D-mannoside (7.50%) and 3-amino-4-piperonyl-5-pyrazolone (5.84%) were labeled and indicated in Figure 1.

The dose-dependent curve equations were used to determine the IC ₅₀ for ethanolic leaf extract and its fractions. Annona reticulata ethanolic leaf extract and its fractions significantly ( p<0.05) inhibited the proliferation of HCT-116 cells in a dose-dependent manner ( Table 3). The ethanolic leaf extract demonstrated a potent anti-proliferative activity with an IC ₅₀ of 30.16 ± 0.56 μg/mL. Among the fractions tested, petroleum ether fraction exhibited the highest anti-proliferative activity (IC ₅₀-29.07 ± 0.83 μg/mL) followed by diethyl ether fraction (IC ₅₀-32.03 ± 0.78 μg/mL), ethyl acetate fraction (IC ₅₀-33.66 ± 1.20 μg/mL) and chloroform fraction (IC ₅₀-43.0 ± 1.64 μg/mL).

Based on the results of the anti-proliferative assay, ethanolic leaf extract and petroleum ether fraction were chosen for further studies.

There was significant reduction in the migration of HCT-116 colon cancer cells into the wound area upon the treatment with Annona reticulata ethanolic leaf extract ( p<0.05, Figure 2B) and its petroleum ether fraction ( p<0.01, Figure 2C) when compared to standard drug doxorubicin ( Figure 2D) at the end of 24 h.

The migration rate of HCT-116 cells was 15.6 ± 2.9% and 9.34 ± 1.6% at 24 h post treatment with ethanolic leaf extract (30.16 μg/mL) and petroleum ether fraction (29.07 μg/mL) respectively. The petroleum ether fraction demonstrated highest anti-migratory activity on HCT-116 cells compared to ethanolic leaf extract and standard drug doxorubicin ( Figure 2).

The bioinformatics analysis of the five major phytocompounds of the petroleum ether fraction using the PASS online server tool revealed the potential pharmacological activities and molecular targets, indicating the possible interactions of these compounds in cancer signaling pathways. A higher Pa value indicates greater probability of the pharmacological activity. Based on the chemical similarity principle it was seen that the phytocompounds of the petroleum ether fraction have the ability to inhibit enzymes and proteins overexpressed in cancers such as EIF4E, B-Raf, NOS2, ICAM, caspase 3, caspase 8, and MMP-9, demonstrating their potential to inhibit various processes in cancer such as proliferation, migration, invasion, angiogenesis and immune invasion. The phytochemicals also showed a high potential to affect the levels of key proteins driving colon cancer progression such as c-Myc, MMP-9, p53 and B-Raf indicating their potential to be developed as promising anticancer agents for colon cancer. The predicted anticancer activity and the possible molecular targets of these compounds are given in supplementary file.

The study was carried out after getting approval from the Institutional ethics committee (IEC - 723/2019).