Cytotoxicity was determined by counting number of dead cells present among the total counted cells on Neubauer chamber and expressed in % dead cells at respective concentrations. The percent dead cells found were 5%, 12%, 18% and 25% at the concentrations of 125, 250, 500 and 1000 µg/mL respectively after incubation with the extract. Extract found mild cytotoxic at highest selected concentration of 1000 µg/mL. The calculated CC50 value was found 4944.772 µg/mL by considering % dead cells at respective concentrations.
In-vitro activity of F. religiosa leaves extract in MDA-MB-231 breast cancer cell lines by using SRB assay
Determination of in-vitro anticancer activity of leaves extract was done by using human breast cancer cell line MDA-MB-231. Growth inhibition of treated cells was investigated by performing three experiments and % control growth of treated cells against untreated cell growth. Percent control growth in Experiments 1 was found to be 97.1, 96.2, 70.2 and 19.6 at 25, 50, 100 and 200 µg/mL concentrations respectively. Percent control growth in Experiment 2 was found to be 103.4, 97.9, 74.9 and 25.5 at 25, 50, 100 and 200 µg/mL concentrations respectively. Percent control growth in Experiment 3 was found to be 97.9, 93.6, 74.4 and 19.5 at 25, 50, 100 and 200 µg/ml concentrations respectively. Average % control growth of 3 experiments was 99.4, 95.9, 73.1 and 21.5 in 25, 50, 100 and 200 µg/mL concentrations respectively (Table 4). It was observed that leaves extract at the concentrations of 100 and 200 µg/mL caused significant retardation of cell growth in 48 hours of incubation as compared to growth of untreated cells. In Adriamycin treated arm, there was no growth observed in all the selected concentrations, the % growth values were found negative due to high rate of cytotoxicity without growth of the cells occurred up to 48 hours of incubation. Hence, results of Adriamycin validate the experimental set-up and its anticancer potential on MDA-MB-31 cell lines. From above parameters, lethal concentration and total inhibition of the cells were calculated. The concentration of 180 µg/mL of leaves extract caused 50% lethality of the cells (LC50), whereas total growth inhibition of the cells occurred at the concentration of 120 µg/mL and the 50% inhibition of the cell growth (GI50) was found to be at the concentration of 70 µg/mL (Table 5). Average % control affected growth was calculated as 100-% control growth values i.e. 0.6, 4.1, 26.9 and 78.5% at the concentrations of 25, 50, 100 and 200 µg/mL, respectively. On the basis of these values, 50% affected growth was found at 130.503 µg/mL (with the confidence interval of (51.489 to 330.774 µg/mL). Breast cancer cells in 48 hours of treatment with leaves extract sample showed reduced density and morphology of cells as compared to control. Overall, the results of the study indicated that the growth inhibition and killing potential of the extract occurred at higher concentrations (Fig. 2, 3.1 and 3.2). Standard drug, Adriamycin showed satisfactory results at selected concentration, which confirms the validity of experiment. Due to absent of growth proteins of the cells from the lowest concentration of 10 µg/mL of Adriamycin, TGI and GI50 parameters were found to be < 10 µg/mL and % control growth values were found negative. Cell growth after 48 hours of Adriamycin treatment showed reduced density (Fig. 2, 3.1 and 3.2).
F. religiosa leaves extraction and identification of phytochemicals
Fine dried powder was sequentially soaked in non-polar to polar solvents. Among all extractions the chloroform extract was further fractionated by column chromatography using n-hexane and ethyl acetate. In this study, total 100 compounds, including Convallatoxin, Umbelliferone, Embelin, Dihydrocoumarin, Fendiline, Hydroxyibuprofen (Table 1) were detected in the chloroform extract of F. religiosa (L.) leaves as active compounds by HR-LCMS. The above work was done at IIT, Mumbai, India.
Convallatoxin is natural glycoside found in many organisms, including plants. This substance is reported as inhibitor of cancer cell growth . It is also reported as effective anti-proliferative agent against some cancer types [25, 26]. In another study, it is demonstrated good activity against colorectal type of cancer . It also exerts good cytotoxic effects against ER+ breast cancer cell lines, MCF-7 cells . (Fig. 4A).
Umbelliferone is a phenylpropanoid and it is coumarin and widely spread in plants28. Umbelliferone has found as antioxidant in the literature . It is used as a sunscreen agent and as optical brightener in textiles . It has anti-inflammatory , anti-diabetic  and anti-tumour activity . Umbelliferone also have glycation inhibitory activity , angiotensin converting enzyme inhibitory activity . Antinociceptive , antimicrobial , neuroprotective , hepatoprotective , anti-inflammatory and pro apoptotic effects in colon carcinogenesis . (Fig. 4B)
Embelin is naturally occurring parabenzoquinone isolated from plant sources. It inhibits cell growth, induces apoptosis, and activates caspase-9 in prostate cancer cells with high levels of inhibitor of apoptosis family of proteins called XIAP, but has a minimal effect on normal prostate epithelial and fibroblast cells with low levels of XIAP . It is used for treating various ailments like chronic inflammatory disorders, heart and urinary conditions, snake and insect bites, and tumour . Embelin would be useful as an adjunct therapy for cerebral stroke and as a potent neuroprotective agent . It possesses all the characteristics of a compound which can cross the blood-brain barrier and elicit an effect on the CNS . Embelin is reported for its CNS effect by diverse mechanisms. (Fig. 4C).
Coumarins are natural and synthetically active components used for various pharmacological actions. It is widely useful in various types of medicinal treatments, such as cancer, inflammation, infection, and enzyme inhibition . Dihydrocoumarin (DHC) is an inhibitor of the Sirtuin family of NAD+-dependent deacetylases  4-arylcoumarins and 3, 4-dihydro-4-arylcoumarins were synthesized and tested for antioxidant activity, antitumor activity . 3, 4-Dihydrocoumarins, considered to be valuable building blocks, have attracted considerable attention due to their various biological activities  such as aldose reductase inhibition , protein kinases  antiherpetic and flavouring agent to a diverse set of foods for example- soft drinks, yogurt, muffins) . In addition, the 3,4dihydrocoumarin scaffold has been discovered in a number of important natural compounds as exemplified by Calomelanol A–C, E–J . (Fig. 4D).
Fendiline inhibits cancer cell proliferation, specifically cells expressing mutant K-Ras, by altering K-Ras cellular distribution and downstream signalling . It inhibits the maintenance and expression of methamphetamine . The effects of Fendiline were produced in the absence of aversive or rewarding properties and without significant impairment on motivated motor behaviour . It was primarily developed as L-type Ca2+ channel blocker for cardiac smooth muscles and approved for human use to treat angina, hypertension and cardiac arrhythmias . (Fig. 4E).
2-Hydroxyibuprofen is a metabolite of ibuprofen. Ibuprofen is an anti-inflammatory drug (NSAID) and act as cyclooxygenase-inhibitor . It is also used for symptoms of arthritis, fever, and as an analgesic. Ibuprofen is known to have an antiplatelet effect as well. (Fig. 4F).