Plant Material Collection and Identification:
Fresh leaves and plantlet of Annona muricata were obtained from Mahatma Phule Krishi Vidyapeeth, Rahuri, Ahmednagar (MS) in the month of February to April 2022. The herbarium specimen of plant were deposited, identified and authenticated from Botanical Survey of India, Western Regional Centre, Pune. (Authentication Letter No: BSI/WRC/Iden.Cer./2022/3005220027363)
Callus culture of A. muricata:
Apical tip and third leaf nodal portion of axillary branch of A. muricata plant were used as an explant for the initiation of callus culture. Surface sterilization of excised plant material was done with 4-5 drops of Tween 20 followed by 0.1% HgCl2 solution and washes thrice each times with sterilized distilled water. About 1-1.5cm of explants were cut gently with a sterilized scalpel blade and aseptically inoculated in culture bottles with autoclaved modified Basal Murashige and Skoog medium supplemented with plant growth regulators 1.5 mg/L BAP (6 Benzylaminopurine) and 2.0 mg/L 2,4-D (2,4- Dichlorophenoxy acetic acid). All the culture bottles were maintained at 25± 20C and illuminated for 16 hr. light followed by 8 hr. dark condition. [5]
Sample preparation and extraction:
The callus tissues of A. muricata were dried and powdered. 5 gm of powder were suspended in 100 ml of ethanol. Extraction was done using Soxhlet apparatus for 5 hr at specific temperature not exceeding the boiling point. The extract was concentrated using rotary evaporator and extracted sample were preserved in refrigerator for further experiments. [6]
FTIR Analysis of AMCEP:
The ethanol extract fraction of A. muricata callus were taken for FT-IR analysis. Based on the peak value in the region of infrared radiation the functional groups of active components were detected. All spectra were obtained with the aid of Perkin Elmer Spectrum RX1, Range 4400 to 450 cm-1; Resolution: 1 cm-1 in the region including IR spectra of solids (KBr pellets) on FTIR spectrophotometer and computerized the absorbance data for analyses. [7]
GC-HRMS analysis of A. muricata callus extract:
Ethanolic fraction of A. muricata callus extract was taken for GC-HRMS analysis (Gas-chromatograph coupled with High Resolution Mass Spectrometer). It is a combined analyzer used for qualitative and quantitative analysis of organic compounds. The analysis was carried out on a GC Agilent 7890 system having FID detector with head space injector and combipal autosampler. The gas chromatograph interfaced to a mass spectrometer (Jeol, AccuTOF GCV, EI / CI Source, Time of Flight Analyser, Mass range -35 - 800 amu and Mass resolution – 5000). 99.99% Helium gas was used as carrier gas at constant flow of 1 ml/min and an injection volume of 1 μl was delivered. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas.
Anticancer Activity: Anticancer activity of AMCEP was performed on human colorectal cancer cell line HCT-116 by the following cytotoxicity and annexin V apoptosis assay.
Cytotoxicity by MTT Assay:
Cytotoxicity assay was performed on both HCT-116 Human Colorectal Cancer cell line & WI-38 Human lung fibroblast cell line procured from NCCS Pune. Briefly, to each well of the 96 well microtitre plate, 200μl of the cell suspension was added and the plate was incubated at 37oC and 5% CO2 atmosphere for 24 hr. 200μl of different test concentrations of drugs were added to the respective wells and incubated at 37oC and 5% CO2 atmosphere for 24 hr. Thereafter, the cells were treated with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltratrazolium bromide (MTT) (MP Biomedicals, Germany). The entire medium including MTT solution (5 mg/mL) was aspirated from the wells. The remaining formazan crystals were dissolved in DMSO and the absorbance was measured using a microplate reader at a wavelength of 570 nm and also at 630 nm. The percentage growth inhibition was calculated, after subtracting the background and the blank, and concentration of test drug needed to inhibit cell growth by 50% (IC50) was generated from the dose-response curve for the cell line. [8]
Annexin V Apoptosis Assay by Flow Cytometry:
HCT-116 Human colorectal cancer cell line was used for Annexin V apoptosis assay. Culture cells in a 6-well plate at a density of 3 x 105 cells/2 ml and incubate in a CO2 incubator overnight at 37°C for 24 hr. Aspirate the spent medium and wash with 1ml 1X PBS. Treat the cells with required concentration of test compound in 2 ml of culture medium and incubate the cells for 24 hr. and leave one of the well as untreated to be used as negative control. At the end of the treatment, remove the medium from all the flasks into 15ml centrifuge tubes and wash with 1ml PBS. Remove the PBS and add 0.5mL of trypsin-EDTA solution and incubate at 37°C for 3-4 minutes. Pour the culture medium back into their respective wells and harvest the cells directly into the centrifuge tubes. Centrifuge the tubes for five minutes at 300 x g at 25°C. Carefully decant the supernatant. Wash with PBS twice. Decant the PBS completely and resuspend cells in 1X Binding Buffer at a concentration of 1 x 106 cells/ml. Transfer 100 µl of the solution (1 x 105 cells) to a 5 ml culture tube. Add 5 µl of AbFlour 488 Annexin V. Gently vortex the cells and incubate for 15 min at RT (25°C) in the dark. Add 2 µl of PI and 400 µl of 1X Binding Buffer to each tube and vortex gently. Analyze by flow cytometry immediately after addition of PI. [9, 10]