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hydroxide (NaOH; 5% w/v) was added into the reaction mixtures and incubated at room

temperature for about 15 minutes. The absorbance of the solutions was measured at 510 nm
using UV-Vis spectrophotometer. Quercetin was used for constructing the standard curve

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(Y=0.006X; R =0.998). The concentration of flavonoid contents in the samples was
expressed as milligrams of Quercetin equivalent per gram (mg QE/g) of dry extract.

Gas Chromatography-Mass Spectrometry (GC-MS) analysis
The fingerprints of compounds present in different parts of S. acmella were analyzed

using GC-MS based metabolite profiling technique. Methanol extracts of the leaves, flowers

and stems of the plant were initially filtered through Polyvinyl Difluoride (PVDF) syringe
filters of pore size 0.2 μm and metabolite profiling of the samples was performed on GCMS-

QP2010 Ultra (Shimadzu) using RTX-5 (30m x 0.25mm x 0.25μm) column. Oven

temperature of GC was programmed to raise from 50° to 250°C at the rate of 10°C/min with
2 min hold time, then to 280°C at the rate of 15°C/min. An injection volume of 1μl was fixed

in split mode (10:1) with helium (He) as carrier gas and the column flow was maintained at
1.21 ml/min. The Injector temperature was maintained at 260°C and the ion source

temperature was 230°C. The samples were run at 40-650 m/z range and the compounds
present in the samples were identified using National Institute of Standard and Technology

(NIST) and WILEY Spectral libraries. The relative quantities of the compounds present in the

samples were expressed as percentage based on peak area in the chromatogram.
Viability Assay

Cytotoxic effect of the plant extracts was determined against three cancer cell lines (a)
MCF-7 (Human breast adenocarcinoma) (b) Saos-2 (Osteosarcoma) and (c) A549 (Human

lung adenocarcinoma). The cell lines A549 and Saos-2 were grown in Dulbecco’s Modified
Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1mM L-

glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin. While MCF-7 was grown in

Minimum Essential Medium (MEM) supplemented with FCS and antibiotics as above.
Viability of the cancer cell lines was determined by a 3-(4,5-dimethylthiazol)-2,5

biphenyltetrazolium bromide (MTT) assay [18].
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The cells (5x10 cells per well) were seeded on 96-well plates and incubated for 24 h
at 37°C in a humidified atmosphere with CO2 (5%) in an incubator. The cells were then

treated with different concentrations of the plant extracts (6.25–100 μg/ml) and incubated
again for 48 h in the humidified incubator. Then, the cells in the wells were treated with 10 μl

of MTT solution (5 mg/ml in PBS). After 2 h of incubation at 37°C in 5% CO2, the culture
media were aspirated and 100 μl of DMSO was added to each well. The well plates were

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