Phytomedicine Plus 2 (2022) 100276

Contents lists available at ScienceDirect

Phytomedicine Plus
journal homepage: www.sciencedirect.com/journal/phytomedicine-plus

Variations in the Anticancer Activity of Free and Bound Phenolics of Finger

Millet (Eleusine coracana (L) Gaertn; Variety KMR-301) Seeds
Mahadevaswamy G. Kuruburu a, Venugopal R. Bovilla a, b, Rimshia Naaz a, c,
Zonunsiami Leihang a, SubbaRao V. Madhunapantula a, d, *
a
Center of Excellence in Molecular Biology and Regenerative Medicine (A DST-FIST Supported Center), Department of Biochemistry (A DST-FIST Supported
Department), JSS Medical College, JSS Academy of Higher Education & Research (JSS AHER), Mysuru, 570015, Karnataka, India
b
Public Health Research Institute of India (PHRII), Yadavagiri, Mysuru, 570020, Karnataka, India
c
Department of Physiology, JSS Medical College, JSS Academy of Higher Education & Research (JSS AHER), Mysuru, 570015, Karnataka, India
d
Special Interest Group in Cancer Biology and Cancer Stem Cells (SIG-CBCSC), JSS Medical College, JSS Academy of Higher Education & Research (JSS AHER),
Mysuru, 570015, Karnataka, India

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Finger millet (FM, Eleusine coracana (L) Gaertn; also known as “ragi” in Kannada language) is one of
Finger millet the widely used millets especially in south India and parts of Africa. FM is consumed in various forms that include
Phenolic compounds porridge, mudde (ball), dosa (a pan cake), idly (a savory rice cake) and biscuits/cookies. FM is also used in the
Free phenolics
form of malted drink known as “malted ragi or ragi malt”. FM is a rich source of dietary fiber, tannins, phenolic
Bound phenolics
Breast cancer
compounds and calcium. Prior studies have shown health beneficial properties such as anti-diabetic and anti-
Colorectal cancer inflammatory effects of FM, and ascribed those properties to the presence of dietary fiber and phenolic com­
pounds. But, very minimal information is available pertaining to the effect of FM phenolic compounds on cell
lines representing carcinomas of colon and rectum; and breast. Hence, we have studied the effect of FM phenolic
compounds on colorectal carcinoma and breast cancer cell lines.
Methods: Sequentially extracting the seeds by 70% ethanol and 10% alkali generated free (FM-FP) and bound
(FM-BP) phenolic compounds respectively. UPLC-QTOF-MS was used to determine the phytochemical compo­
sition. Antioxidant potential was determined by ferric reducing antioxidant power assay (FRAP) and radical
scavenging activity (DPPH assay). Effect of FM-FP and FM-BP on cellular proliferation was determined by
sulforhodamine-B assay. Staining the untreated and treated cells with acridine orange and ethidium bromide
followed by analyzing the stained cells using fluorescence microscope yielded key information about impact of
extracts on cell death. Effect on cell cycle was determined by staining the cells with DAPI followed by analyzing
the stained cells using NC-3000.
Results: Analysis of the UPLC-QTOF -MS data showed the presence of phenolics and phenolic acid derivatives,
flavonoids and aminoacids in the FM-FP and FM-BP fractions. Both fractions exhibited ferric ion reduction ability
and DPPH radical scavenging potential. But, the effect of FM-FP and FM-BP on cell proliferation varied signif­
icantly from cell line to cell line. FM-FP exhibited better cytotoxic potential compared to FM-BP when tested
against breast cancer cell lines. Cytotoxic FM-FP induced G0/G1 or G2/M arrest in a cell line dependent fashion
and increased the fragmentation of DNA leading to accumulation of cells in Sub-G1 phase.

Abbreviations: BC, Breast cancer; CRC, Colorectal cancer; DAPI, 4’,6-diamidino-2-phenylindole; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, Dime­
thylsulfoxide; DNS, 3, 5-dinitrosalicylic acid; DPBS, Dulbecco’s phosphate-buffered saline; DPPH, 2, 2-Diphenyl-1-picrylhydrazyl; ER, Estrogen receptor; FBS, Fetal
bovine serum; F-C, Folin-Ciocalteu; FRAP, Ferric reducing antioxidant power; FM-FP, Finger millet free phenol; FM-BP, Finger millet bound phenol; HER-2, Human
epidermal growth factor receptor-2; IUPAC, International union of pure and applied chemistry; KVK, Krishi vigyan kendra; LC-MS-MS, Liquid chromatography with
tandem mass spectrometry; NC-3000, NucleoCounter 3000; NCCS, National center for cell science; NPC, Nutritional prevention of cancer; PR, Progesterone receptor;
Q-TOF, Quadrupole time-of-flight; SRB, Sulforhodamine B; TPTZ, 2,4,6-Tris(2-pyridyl)-s-triazine; UPLC, Ultra-performance liquid chromatography; VC Farm,
Vishweshwaraiah canal farm.
* Corresponding author at: Professor of Cellular and Molecular Biology, Center of Excellence in Molecular Biology and Regenerative Medicine (A DST-FIST
Supported Center), Department of Biochemistry (A DST-FIST Supported Department), JSS Medical College, JSS Academy of Higher Education & Research (JSS
AHER), Mysuru, 570015, Karnataka, India.
E-mail addresses: 996464kg@gmail.com (M.G. Kuruburu), venu.1726@gmail.com (V.R. Bovilla), rimshanaaz.12@gmail.com (R. Naaz), zonunlh25@gmail.com
(Z. Leihang), mvsstsubbarao@jssuni.edu.in, madhunapantulas@yahoo.com (S.V. Madhunapantula).

https://doi.org/10.1016/j.phyplu.2022.100276
Received 3 January 2022; Received in revised form 22 March 2022; Accepted 12 April 2022
Available online 14 April 2022
2667-0313/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
M.G. Kuruburu et al.
Conclusion: In summary, results of our study demonstrated the strength of finger millet free- and bound phenolic
compounds for exhibiting antioxidant property, and the potential to modulate the proliferative potential of
breast and colorectal cancer cells.
INTRODUCTION
Seeds of finger millet (FM, Eleusine coracana (L) Gaertn; also known
as “ragi” in kannada language) are widely used in the preparation of a
variety of diets due to various health-beneficial properties associated
with the consumption of FM-based dietary products (Devi et al., 2014;
Shobana et al., 2013). Finger millet is one of the most widely used
millets in various parts of India and Africa. It is a rich source of calcium
and dietary fiber (Puranik et al., 2017). Several dietary products that
include biscuits, snack items, energy-rich drinks and ready-to-eat foods
have been prepared by finger millet and finger millet-derived malts
(Sood et al., 2017). Prior studies have isolated the carbohydrates, pro­
teins, enzymes, minerals and secondary plant products such as phenolics
from native and germinated finger millet and showed health beneficial
effects such as anti-diabetic and anti-inflammatory properties
(Chaudhary and Mudgal, 2020). FM seeds are rich in carbohydrates
(65-75%), dietary fiber (15-20%), proteins (5-8%), ether extractives
(1-2%), minerals (2.5-3.5%) and phenolic compounds (0.3-3.0%)
(Chandra et al., 2016; Devi et al., 2014). FM seeds are of two types;
white ragi (white FM) and brown ragi (brown FM)(Gupta et al., 2017).
Brown ragi, which is widely used, is known to contain hydroxy benzoic
acids, hydroxy cinnamic acids and flavonoids (Okwudili et al., 2017).
Prior studies have demonstrated the health beneficial properties such as
anti-oxidant, anti-inflammatory and anti-diabetic potential of FM
phenolic compounds (Devi et al., 2014). But, not many studies have
assessed the impact of FM phenolic compounds on cancer cells prolif­
eration, viability and survival.
Phenolic compounds in FM are classified into free- and bound forms
based on their extractability with 70% ethanol or 10% alkali, respec­
tively (Subba Rao and Muralikrishna, 2002). Bound phenolics are linked
to cell wall polysaccharides through ester linkages to arabinoxylan
polysaccharides in cell walls (Chandrasekara and Shahidi, 2010). Prior
studies have characterized the free and bound phenolic acids using TLC,
HPLC and LC-MS, and reported the presence of hydroxybenzoic acid and
hydroxycinnamic acid derivatives that include syringic acid, proto­
catecheuic acid, ferulic acid, caffeic acid, and cinnamic acid (Hassan
et al., 2020). In addition, many flavonoids were reported in the free and
bound phenolic extracts of FM (Chandrasekara and Shahidi, 2011). A
part from these preliminary studies; no systematic identification of
phenolic compounds of FM (KMR 301 variety) was reported; hence,
more accurate identification of free and bound phenolic compounds,
using LC-MS-MS is, warranted.
Earlier studies have reported the ability of phenolic acids and
phenolic acid-rich extracts of finger millet to exhibit potent anti-oxidant
and anti-inflammatory activity (Xiang et al., 2019). However, the ability
of these fractions to inhibit the proliferation of cancer cells and the
molecular processes influenced by the FM-FP and FM-BP were not well
explored (Singh et al., 2015) . Thorough characterization of free and
bound phenolic compounds of KMR 301 variety finger millet is critical
not only to assess the health beneficial properties but also suggest this
finger millet variety for treating various diseases originated due to
inflammation and oxidative stress. Secondary metabolites of millets
such as phenolics, flavonoids and flavones are known for their ability to
suppress inflammatory reactions and prevent the diseases originated
due to oxidative stress(Maleki et al., 2019). Due to their radical scav­
enging activity and ability to suppress key oncogenic signaling cascades
(such as PI3K-Akt pathway, MAPK pathway, Nrf2 and NFκB signaling
cascades) various simple and complex phenolic compounds, in partic­
ular, ferulic acid, caffeic acids, catechins, tannins and their derivatives,
could retard the progression of cancer cells(Huang et al., 2010).
2
Phytomedicine Plus 2 (2022) 100276
Structure activity relationship studies have identified key functional
groups of phenolic compounds required for exhibiting antioxidant and
anti-cancer activity properties; and showed that presence of hydroxylic
(-OH) and unsaturated carbon side chains promote radical scavenging
ability and anti-proliferative potential(Singh et al., 2014). Therefore,
identification of phenolic compounds of KMR 301 variety finger millet is
important, hence, the present study.
MATERIALS AND METHODS
Materials
Colorectal carcinoma cell lines HCT-15, HCT-116 and HT-29; breast
cancer cell lines MCF-7, MDA-MB-231, and MDA-MB-468 were procured
from National Center for Cell Science (NCCS), Pune, Maharashtra, India.
Triple positive breast cancer cell line BT-474 was provided by Prof.
Annapoorni Rangarajan, Department of Molecular, Reproduction,
Development and Genetics, Indian Institute of Science, Bengaluru,
Karnataka, India. Human keratinocyte cell line HaCaT was generously
provided by Dr. Gopinath, M.S. Principal Scientist, Department of Mo­
lecular Nutrition, CSIR-CFTRI, Mysore, Karnataka, India. SRB, DPPH,
Trolox, TPTZ, cell culture grade Hybrimax DMSO were purchased from
Sigma Chemical Company, St Louis, MO, USA. DMEM, FBS, PenStrep,
Glutamax, Trypsin, and DPBS were procured from HiMedia Labora­
tories, Mumbai, Maharashtra, India. Plastic ware (T25, T75, 15.0mL and
50.0mL conical tubes, disposable pipettes, micropipettes tips etc) for cell
culture was procured from Techno Plastic Products India, Pvt Ltd,
Bengaluru, Karnataka, India. All other reagents such as solvents, salts,
acids and bases are of Analytical Reagent (AR) grade and are procured
from Sisco Research Laboratories Pvt Ltd, (SRL), Mumbai, Maharashtra,
India
Methods
Millet collection and processing: The KMR 301 variety finger
millet seeds were collected from Krishi Vigyan Kendra – Vishweshwar­
aiah Channel (KVK-VC) Farm, Mandya-571405, Karnataka. KVK-VC
Farm is a part of University of Agricultural Sciences, Bellary Road,
Bengaluru, Karnataka, India. A specimen of the finger millet KM variety
was stored in KVK-VC farm, Mandya, Karnataka (Fig. 1). The collected
seeds were washed with tap water, and dried under shade. The dried
seeds were powdered using a mixer- grinder (Preethi Steel Supreme
brand) and the flour was stored at -80◦ C in a deep freezer.
Extraction of free and bound phenolic acids: Free and bound
phenolic acids were extracted as detailed in (Subba Rao and Mur­
alikrishna, 2002). In brief, 100 grams finger millet seeds powder was
extracted with 500mL of 70% aqueous ethanol (500mL X 3 times), and
the residue remained after extraction was dried using diethyl ether or
processed immediately for the extraction of bound phenolic acids
(Supplementary Figure-1). The pooled 70% ethanol extract was
concentrated to about 200mL using a rotary evaporator (Heidolph,
Schwabach, Germany) and the pH was adjusted to 2.5 using 1N HCl.
Free phenolic compounds were phase separated by the addition of ethyl
acetate (400mL X 3 times). Addition of anhydrous disodium sulfate
(100g / 1000mL of pooled ethyl acetate fraction) to the pooled ethyl
acetate fraction removed the water molecules and provided an organic
fraction rich in phenolic compounds. The ethyl acetate fraction was
concentrated and dried by lyophilization. The dried sample was desig­
nated as FM-FP (Finger millet Free Phenolic acids) and stored in -80◦ C
for further use.
M.G. Kuruburu et al.
Bound phenolic acids were extracted from the residue obtained after
removing the free phenolic acids. Experimentally, the starch was
digested using heat stable α- amylase from Bacillus amyloliquifaciens (25
to 50 Units/100 gram dried residue devoid of free phenolics) at 80◦ C for
2.0h to 4.0h in 20mM phosphate buffer, pH 6.0 (Supplementary Figure-
1). Once the starch was digested (as evidenced by Iodine test (Patel
et al., 2017), the reaction mixture was centrifuged and the supernatant
discarded. Dehydrating the de-starched material with diethyl ether
(100mL X 4 times) yielded a residue (about 50 grams), which was
extracted by the addition of 6 volumes of 10% NaOH drop-wise under a
continuous flow of Nitrogen gas. Oxidation of phenolic acids was pre­
vented by the addition of sodium borohydride (0.25% weight/weight
final concentration). The extraction was carried out for a period of 4h,
and the extract was centrifuged, and the supernatant containing bound
phenolic acids was collected in to a conical flask. The pH of extract was
adjusted to 1.5 using concentrated HCl under cold condition. Phenolic
acids were phase separated by the addition of ethyl acetate (400mL X 3
times) and processed as described in free phenolic acids protocol. The
bound phenolic acid fraction was designated as FM-BP (Finger millet
Bound Phenolic acids).
Fig. 1. Finger millet (Eleusine coracana, (L) Gaertin) KMR301 seeds.
3
Phytomedicine Plus 2 (2022) 100276
FM-FP and FM-BP were generated from three independent experi­
ments to determine the reproducibility in yield and phytochemical
composition. Results (percentage yield, total phenol, total protein, total
carbohydrate and DNS reacting substances) from three independent
batches were shown with ± SE in the Supplemental Data files.
Estimation of total phenolic acid content by F-C method: Total
phenol content in the FM-FP and FM-BP extracts was determined by a
modified F-C method as detailed in (Sánchez-Rangel et al., 2013). In
brief, 70.0µL gallic acid standards (2.5µg/mL to 40.0µg/mL) and suit­
ably diluted FM-FP and FM-BP were aliquoted into a 96-well plate and
incubated with 70.0µL F-C reagent (SRL, Mumbai, India; diluted 1:1
with water) and 60.0µL of 4% sodium carbonate for 30minutes at 37◦ C.
Absorbance of developed color was read at 765nm and the total phenolic
acid content (mg/gram extract) quantified using the gallic acid standard
graph.
Determination of total carbohydrate content by phenol-sulfuric
acid method: Total carbohydrate content in free and bound phenolic
acids was determined by following the method detailed by (Subba Rao
and Muralikrishna, 2004). Experimentally, 0.5mL of standard (from
10.0µg/mL to 100.0µg/mL) and suitably diluted FM-FP and FM-BP
M.G. Kuruburu et al.
fractions were aliquoted in to glass test tubes and 0.5mL of 5% phenol
solution added. To this reaction mixture 2.0 mL of concentrated sulfuric
acid was added and vortexed carefully. After 20minutes of incubation at
room temperature the absorbance of developed color was read in a
UV-Visible spectrophotometer at 490nm. The concentration of total
carbohydrate (mg/gram extract) was determined by reading the values
in the calibration curve generated with standard glucose.
Estimation of reducing sugars by 2,4-Dinitrosalicylic acid
(DNS): Amount of reducing sugars in the extracted samples was deter­
mined by DNS method as detailed by (Miller, 1959; Rao and Mur­
alikrishna, 2001). In brief, a series of glucose standards ranging from
100µg/mL to 800µg/mL was prepared in water. Next, 0.5mL of each
standard and a suitably diluted FM-FP and FM-BP were incubated with
0.5mL DNS reagent (prepared by mixing 75.0mL of 6% sodium potas­
sium tartrate with 30.0mL 5% DNS in 2M NaOH and diluting it to a final
volume of 150.0mL with water) in a boiling water bath for 10minutes.
Next, 2.0mL water was added to each test tube, and the absorbance
measured at 540nm in a UV-Visible spectrophotometer. The results were
expressed as mg/gram reducing sugar.
Quantitative analysis of total protein content by Bradford
method: Total protein content in the FM-FP and FM-BP fractions was
carried out by a dye-binding method as detailed by (Bradford, 1976).
Experimentally, 5.0µL BSA standards (50.0µg/mL to 800.0µg/mL) and
suitably diluted FM-FP and FM-BP were incubated with 250.0µL Brad­
ford reagent. The developed color was read in a UV-Visible spectro­
photometer operating at 595nm within 1h. Concentration of total
protein (mg/gram extract) was determined by comparing the absor­
bance of test samples with that of standards generated using BSA
Identification of phytochemical constituents of FM-FP and FM-
BP fractions by UPLC-QTOF-MS: Phytochemical constituents present
in the FM-FP and FM-BP were analyzed by dissolving in methanol at a
concentration of 1.0mg/mL followed by filtration through 0.45µm
membrane filters, and subjecting the completely soluble material to
UPLC-QTOF-MS analysis as detailed by(Chanda et al., 2019). Experi­
mentally, the identification of compounds was carried out in Agilent
1290 Infinity LC coupled to Agilent 6500 Quadrupole Time-of-Flight
(QTOF) with Agilent Jet Stream Thermal Gradient Technology. The
gradient flow was mentioned in the Supplementary Table 1. Based on
high-resolution accurate mass data the compounds were identified by
mining the Agilent Personal Compound Database and Library (PCDL)
from phenol explore database. Detailed method and workflow were
provided in Fig. 3 (Du et al., 2012; Neveu et al., 2010).
Determination of the antioxidant potential using Ferric
Reducing Antioxidant Power (FRAP): FRAP assay was performed
according to (Benzie and Strain, 1999). Experimentally, the FRAP re­
agent containing 2.5mL of 10mM TPTZ (2,4,6-trypyridyl-s-triazine),
2.5mL of 20mM FeCl3 and 25mL of 300mM acetate buffer, pH 3.6, was
prepared by warming at 37◦ C. 190µL FRAP reagent was incubated with
suitably diluted FM-FP and FM-BP (10,15, 20, 25 and 30 µg/mL by total
phenol content) or FeSO4 (standard – 200-1800µM) in dark for 30 min
and the absorbance measured at 593nm. FRAP units (equivalent amount
of ferrous sulfate) were calculated and the data plotted against the
concentration of extract. Trolox (3.1µg/mL to 200µg/mL) was used to
generate the calibration curve(Daga et al., 2021).
Determination of the antioxidant potential by DPPH (2,2-
diphenylpicrylhydrazyl) radical scavenging activity: DPPH radical
scavenging activity was measured according to (Aksoy et al., 2013).
Briefly, 140µL of DPPH solution (6.2mg in 100mL 100% ethanol) was
incubated with 20.0µL of 10,15, 20, 25 and 30 µg/mL (by total phenol)
extract for 30 min in dark and the absorbance measured at 536nm. The
calibration curve was created using Trolox (6.25µg/mL to 100µg/mL).
The results were expressed as % free radical scavenging activity(Piso­
schi et al., 2009).
Percentage (%) DPPH radical scavenging activity = [A0-A1/A0] ×
100 A0= OD of DPPH incubated with solvent. A1= OD of DPPH incu­
bated with Trolox or Extract.
4
Phytomedicine Plus 2 (2022) 100276
Determination of anti-proliferative potential: Anti-proliferative
potential of FM-FP and FM-BP was determined according to the
method detailed by (Madhunapantula and Robertson, 2008). Breast
cancer (ER, PR and HER2 positive BT-474, ER, PR positive but HER2
negative MCF-7, and triple negative MDA-MB-231 and MDA-MB-468)
colorectal cancer (HCT-15, HT-29, HCT-116) cells and HaCaT cells
were trypsinized and 1 × 104 cells per well in a 100µL volume plated in
96 well plate. The cells were allowed to grow in a CO2 incubator till they
reach 60 - 70% confluence. At this point, cells were treated with
increasing concentration of FM-FP (9.37µg/mL to 150µg/mL) and
FM-BP (6.25µg/mL to 100.0µg/mL) for 48h and the number of viable
cells determined by sulforhodamine-B (SRB) assay according to (Skehan
et al., 1990). Based on the anti-proliferative potential assessment, all the
subsequent studies were carried out only with MCF-7 and MDA-MB-468
cell lines
Analysis of cell cycle using a 2-step 4’,6-diamidino-2-phenyl­
indole (DAPI) staining procedure: In order to determine the variations
in cell cycle stages upon treatment with FM-FP and FM-BP, a 2-step cell
cycle assay was carried out according to (Chikkegowda et al., 2021).
Experimentally, about 1 × 106 MCF-7 and MDA-MB-468 cells were
treated with 37.5µg/mL, 75.0µg/mL and 150µg/mL (microgram total
phenol/volume) FM-FP or 50µM cisplatin (as a positive control) for 48h
and DAPI stained cells were quantified by NucleoCounter® NC-3000
system.
DNA Fragmentation Assay: In order to quantify the fragmented
DNA upon treatment of MCF-7 and MDA-MB-468 cells (1.5 × 106 cells)
with 37.5µg/mL, 75.0µg/mL and 150µg/mL (microgram total phenol/
ml) FM-FP or 50µM cisplatin (a positive control) for 48h, the DNA
fragmentation assay was carried out by NucleoCounter® NC-3000
accordingly to (Rok et al., 2020). The collected data was analyzed,
and the percentage population in Sub-G1 phase was quantified (Rok
et al., 2020).
Cell death assay with acridine orange (AO) and ethidium bro­
mide (EtBr) staining. Cell death was assessed using cell staining with
acridine orange (AO) and ethidium bromide (ETBR). MCF-7 and MDA-
MB-468 cells (0.3 × 106 cell/well in 2 ml medium) were grown in 6-
well plates for ~36h and then treated with 37.5, 75 and 150 μg/mL
(Total phenol concentration) or vehicle (controls) for 48h. Cisplatin
100µM was used as positive control. Apoptosis assay was performed as
detailed method In (Bovilla et al., 2021).
RESULTS
Yield of free and bound phenolic fractions FM-FP and FM-BP: Ex­
tracts containing phenolic compounds were prepared by subjecting the
powdered finger millet seeds to 70% ethanol and 10% sodium hydroxide
as detailed in Supplementary Figure-1, which produced FM-FP and FM-
BP. Extracts were generated from three independent batches and the
average yield determined. The yield of FM-FP and FM-BP is 0.57±0.13
(gram%, wt/wt) and 0.53±0.07 (gram%, wt/wt), respectively.
Composition analysis of free and bound finger millet extracts: The
composition of FM-FP and FM-BP was carried out by measuring the total
carbohydrate, total protein, total phenol and DNS reacting substances as
detailed in methods section. FM-FP exhibited predominance of total
phenol (18.00±1.5%), DNS reacting substances (17.91±1.74%) and
total protein (6.33± 0.6%) . Total carbohydrate (3.44±0.33%) was
relatively low in FM-FP compared to other biomolecules. Unlike FM-FP,
the FM-BP had high DNS reacting substances (31.33±0.21%) total
phenol (12.29±1.08%) and relatively low level of total protein (5.82
±0.56%), and total carbohydrate (2.91±0.3%). (Supplementary Figure-
2)
UPLC-QTOF-MS analysis of FM-FP and FM-BP showed the pres­
ence of hydroxybenzoic-, hydroxycinnamic acids and their derivatives
in the extract: FM-FP and FM-BP were analyzed by UPLC-QTOF-MS to
identify and determine the relative quantification of phenolic com­
pounds. The work-flow is depicted in Fig. 3. The system-generated data
M.G. Kuruburu et al. Phytomedicine Plus 2 (2022) 100276

Fig. 2. FM-FP and FM-BP are rich in total phenols and exhibited antioxidant properties in vitro: Total phenol content of FM-FP and FM-BP was measured by F-
C method as detailed in method section (Fig. 2A). The antioxidant potential of FM-FP and FM-BP was studied by determining the ability to reduce Fe3+ ions in to Fe2+
ions (FRAP assay, Fig. 2B) and scavenging the DPPH free radical (Fig. 2C). FM-BP and FM-FP exhibited closely similar antioxidant properties with no statistically
significant differences.

Fig. 3. Analysis of FM-FP using UPLC-QTOF-MS: In order to identify various phytochemicals present in the FM-FP, UPLC-QTOF-MS was carried out as detailed in
methods and depicted in work-flow diagram (Fig. 3).

obtained from one of the three independent batch preparations was
shown in Fig. 3. Analysis of the data showed the presence of (a) Hydroxy
benzoic acids - Vanillic acid (4.33%) and Protocatechuic acid (2.95%);
(b) Hydroxy cinnamic acids - Ferulic acid (5.78%), Dimethylcaffeic acid
(1.24%); (c) Flavanoids, Biflavonoids, Flavones, and Flavans –
Procyanidin-B1 (40.08%), 3-O-methylquercetin (24.13%), Rutin
(2.51%), Epigallocatechin (17.79%) and (d) Aromatic amino acid L-
Phenyl alanine (1.19%) etc (Supplementary Table 2 and Supplementary
Figure-3A and 3B, Supporting information 1A)
The FM-BP found to contain (a) Hydroxy benzoic acids - Proto­
catechuic acid (37.64%), Vanillic acid (11.80%) and Syringic acid
(4.19%); (b) Hydroxy cinnamic acids - Caffeic acid (2.56%) and Ferulic
acid (20.33%); (c) Dimeric forms of phenolic acids - Diferulic acid
(0.71%); (d) Sugar derivatives – Formononetin 7-O-glucuronide
(1.34%); (e) Biflavonoids, Flavones, and Flavans: Procyanidin-B1
(9.57%), Tricin (0.46%), Kaempferol (0.44%), Epigallocatechin gallate
(9.62%) and (f) Cis-aconitic acid (1.35%) (Supplementary Table 3 and
Supplementary Figure-4A and 4B, Supporting information 1B). In both
extracts some of the compounds could not be identified due to variations
in their molecular mass with closely matching reference standards.
Additional studies are required to conclusively identify these
compounds.
FM-FP and FM-BP exhibited antioxidant potential, which is com­
parable to standard gallic acid: Phytochemical extracts rich in phenolic
compounds and their derivatives are known to exhibit antioxidant ac­
tivity, hence, the ability of FM-FP and FM-BP for reducing Fe3+ ions in to
Fe2+ ions as well as for scavenging DPPH radical was tested and the
results compared with Trolox, a well-known antioxidant (Fig.s 2B and

5
M.G. Kuruburu et al.
2C, Supplementary Table ST 4A, 4B, 4C, 4D). The data showed the
ability of FM-FP and FM-BP in reducing ferric ions in to ferrous ions
(Fig. 2B). A dose dependent increase in ferric ion reduction ability of
FM-FP and FM-BP was observed (Fig. 2B). No significant difference in
FRAP was observed between FM-FP and FM-BP (P > 0.05). Ten µg/mL
FM-FP and FM-BP are equivalent to about 40µg/mL Trolox (Fig. 2B).
Similarly, the DPPH assay showed a dose dependent increase in the
radical scavenging potential of FM-FP and FM-BP (Fig. 2C), but no
difference in the radical scavenging potential was observed between FM-
FP and FM-BP (Fig. 2C). In summary, both FM-FP and FM-BP exhibited
antioxidant potential.
FM-FP and FM-BP differentially modulated the proliferation of
breast and colorectal cancer cell lines: Since FM-FP and FM-BP were
found to contain derivatives of hydroxyl benzoic acid and hydroxyl
cinnamic acids as well as many biologically active phytochemicals, it is
predicted that these extracts could modulate the proliferative potential
of cancer cells. Hence, the impact of FM-FP and FM-BP on the prolifer­
ation of colorectal- and breast cancer cell lines was tested using SRB
assay as detailed in methods section. Analysis of the data showed a dose
dependent decrease in cell viability, with the addition of FM-FP, in MCF-
7 and MDA-MB-468 cell lines (Fig. 4A, 4B and Table 1). But, no such
effect was observed when tested against MDA-MB-231 and BT-474
breast cancer cell lines and colorectal cancer cell lines HCT-15, HCT-
116 and HT-29 cell lines (Table 1). An increase in the percentage of
viable cells was noticed in HCT-116, MDA-MB-231 and BT-474 cell
lines, and at the lower doses (<37.5µg/mL) in MDA-MB-468 cell line
(Table 1). Unlike FM-FP, the FM-BP did not exhibit any cytotoxic po­
tential in any of the breast cancer and colorectal cancer cell lines HCT-
116 and HCT-15 (Table 1). Only in case of HT-29, the FM-BP showed a
maximum of about 40% growth inhibition at 25µg/mL concentration
(Table 1). In order to determine whether FM-FP and FM-BP exhibit
similar cytotoxic effects even against normal human keratinocyte cell
line HaCaT, the viability assay was carried out as detailed in methods
and results represented in Fig. 4C. FM-FP exhibited 50% decrease in cell
viability only at 150µg/mL concentration. Concentrations lower than
150µg/mL of FM-FP and all the concentrations of FM-BP had no sig­
nificant impact on HaCaT cell viability (Fig. 4C). In summary, free and
bound phenolic extracts of finger millet exhibited cell line dependent
cytotoxic properties.
Fig. 4. Anti-proliferative potential of FM-FP and FM-BP: FM-FP, but not FM-BP, exhibited a dose dependent cytotoxic effect against MCF-7 (ER+, PR+ and
HER− Fig. 4A), MDA-MB-468 (ER− , PR− and HER− ; Fig. 4B) and HaCaT (Fig. 4C).
6
Phytomedicine Plus 2 (2022) 100276
Cell proliferation inhibition by FM-FP is mediated by the induction
of G0 /G1 and G2/M cell cycle arrest, respectively in MCF-7 and MDA-
MB-468 cell lines: To determine the mechanistic basis of anti-
proliferative effect of FM-FP, a cell cycle analysis was carried out by
staining the cells using double stranded DNA binding DAPI, followed by
analyzing the stained cells using a Nucleocounter-3000 as detailed in
methods section. Analysis of the data showed a visible increase in G0/G1
and G2/M cells, respectively upon treating MCF-7 and MDA-MB-468
cells with 37.5µg/mL FM-FP (Fig. 5). At 75.0µg/mL and 150µg/mL
FM-FP a significant increase in apoptotic sub-G0 cell population was
observed compared to control untreated and vehicle DMSO treated cells
(Fig. 5). The positive control 50µM Cisplatin arrested MCF-7 cells in G2/
M phase, however, in case of MDA-MB-468, the Cisplatin induced S-
phase population and increased sub-G0 cell population (Fig. 5).
Cytotoxic FM-FP induced the fragmentation of DNA in MCF-7 and
MDA-MB-468 cells: Fragmentation of chromosomal DNA in to oligo­
nucleosomal products is one of the hallmark features of apoptosis
(Matassov et al., 2004). Since DAPI binds to double stranded DNA,
examining the DAPI stained cells microscopically as well as by using a
nucleocounter helps in determining the level of apoptosis (Liu et al.,
2015; Rok et al., 2020). A reduction in the fluorescence as evidenced by
elevated sub-G1 phase cell population, is a clear indicator of apoptosis
(Murad et al., 2016). Analysis of the impact of treating breast cancer
cells MCF-7 and MDA-MB-468 with FM-FP showed a significant increase
in MCF-7 sub-G1 cell population from about 10.0% to 15.0% in control
and vehicle treated cells to ~23% at 75µg/mL and 35% at 150µg/mL
FM-FP (Fig. 6A). In case of MDA-MB-468, the treatment with increasing
concentration of FM-FP induced more apoptosis. At 37.5µg/mL of
FM-FP, a significant increase in the percentage cells undergoing
apoptosis was observed in MDA-MB-468 cell line (Fig. 6B).
Induction of cell death by FM-FP was further confirmed by staining
the control and treated cells with acridine orange and ethidium bromide
(Fig. 6C). A dose dependent increase in percentage cells stained “RED”
appeared in both MCF-7 and MDA-MB-468 cell lines (Fig. 6C). The
positive control cisplatin (100µM) produced a two fold more dead cells
compared to vehicle treated cells (Fig. 6C) (Supplementary figure-5).
In summary, breast cancer cell lines MCF-7 and MDA-MB-468, but
not other cell lines, showed susceptibility to treatment with FM-FP. The
decreased viability upon treatment with FM-FP is due to induction of cell
M.G. Kuruburu et al. Phytomedicine Plus 2 (2022) 100276

Table 1
Modulation of the viability of breast and colorectal carcinoma cells by FM-FP and FM-BP.
Finger Millet Free phenols (FM-FP)
Cell viability (% of control)
Breast cancer cell lines Colorectal cancer cell lines
Concentration µg/ml MDA-MB-231 MDA-MB-468 MCF-7 BT474 HCT-15 HCT-116 HT29 HaCat

VC-Ethanol 98.91 ±1.69 97.51±3.28 103.78 ±7.13 94.66 ±2.49 96.61 ±0.61 96.56 ±1.23 115.63 ±4.77 97.27±0.79
9.37 134.93 ±5.14 141.31 ±2.30 103.55 ±2.61 108.80 ±2.51 107.59 ±0.53 124.62±1.94 122.40 ±3.73 110.36±0.17
18.75 132.29 ±3.79 136.05 ±8.23 100.71 ±3.42 120.29 ±3.04 104.40 ±0.72 126.26±5.13 118.36 ±5.39 114.31±1.3
37.5 141.76 ±6.15 140.05 ±4.59 94.14 ±2.07 131.69 ±3.50 102.74 ±1.77 138.48±8.63 120.04 ±7.18 119.39± 2.97
75 137.52 ±4.07 82.95 ±0.69 67.49 ±2.27 129.93 ±5.20 103.76 ±3.87 148.50±10.12 120.21 ±2.68 101.35 ±3.84
150 141.26 ±8.87 51.66 ±2.25 43.93 ±2.60 105.61 ±1.26 107.38 ±2.96 153.50±5.00 105.27 ±4.90 54.79 ± 5.16
DADS (1mM) 62.55 ±1.44 54.28 ±0.92 42.74 ±3.58 74.56 ±1.72 45.97 ±2.97 60.23 ±3.82 67.39 ±2.80 35.86 ±1.17
Finger Millet Bound phenols (FM-BP)
Cell viability (% of DMSO vehicle control)
Breast cancer cell lines Colorectal cancer cell lines
Concentration µg/ml MDA-MB-231 MDA-MB-468 MCF-7 BT474 HCT-15 HCT-116 HT29 HaCat
VC-Ethanol 98.91 ±1.69 97.51±3.28 103.78 ±7.13 94.66 ±2.49 96.61 ±0.61 96.56 ±1.23 115.63 ±4.77 97.27±0.79
6.25 115.05 ±7.41 113.84 ±4.72 104.60 ±4.40 114.80 ±1.87 117.57 ±2.84 116.03±6.91 92.57 ±5.15 100.11±0.96
12.5 101.05 ±6.45 114.65 ±7.06 101.19 ±2.84 101.70 ±2.95 115.94 ±2.51 107.95±4.91 67.56 ±8.25 97.81± 1.41
25 99.02 ±10.19 125.10 ±17.14 101.55 ±2.22 107.40 ±7.33 117.24 ±3.76 108.27±5.31 56.45 ±11.81 100.27± 0.95
50 112.17 ±6.72 130.27 ±6.98 102.77 ±3.31 111.72 ±4.09 118.67 ±6.54 116.60±7.46 63.58 ±12.77 101.86± 4.1
100 109.04 ±2.01 101.11 ±6.03 99.23 ±4.56 113.45 ±3.28 110.87 ±11.42 127.66±11.23 81.95 ±11.67 86.90± 14.21
DADS (1mM) 62.55 ±1.44 54.28 ±0.92 42.74 ±3.58 74.56 ±1.72 45.97 ±2.97 60.23 ±3.82 67.39 ±2.80 35.86 ±1.17

Fig. 5. FM-FP modulated cell cycle stages in breast cancer cell lines MCF-7 and MDA-MB-468: Anti-proliferative FM-FP differentially effected cell cycle stages
in breast cancer cells MCF-7 (Fig. 5A) and MDA-MB-468 (Fig. 5B). Whereas the lower dose FM-FP induced G2/M and G0/G1 arrest, respectively, in MDA-MB-468 and
MCF-7 cell lines, the higher doses triggered cell death through the induction of apoptotic sub-G1 population. Fifty micromolar Cisplatin was used as positive control.

cycle arrest, accumulation of sub-G1 cell population, and fragmentation
of DNA.
DISCUSSION
Nutrient composition of different finger millet varieties has been
studied by many investigators and showed the predominance of free
sugars in particular, glucose and maltose, phenolics such as caffeic acid,
vanillic acid and ferulic acid, flavonoids and tannins; and amino acids in
the 70% ethanol / methanol extract (Jayawardana et al., 2021; Phuyal
et al., 2020; Subba Rao and Muralikrishna, 2002). Similar to these re­
ports, we have also identified predominance of DNS reducing sub­
stances, total carbohydrates and phenolic acids in the ethanolic extract
(referred as FM-FP in this study). A recent study by Jayawardana et al.,
2021 reported a total phenol content of ~288mg/100g flour to
528mg/100g flour in the ethanolic extract of Finger millet varieties of
Sri Lanka (Jayawardana et al., 2021). In a separate study, Viswanath,
et al., 2009 extracted the finger millet whole flour and seed coat with
acidified methanol and identified gallic, coumaric, syringic and vanillic
acids(Viswanath et al., 2009). Another report by Banerjee et al., 2012
showed the presence of benzoic acid derivatives gallic acid,
protocatecheuic acid, vanillic acid and syringic acid; trans-cinnamic
acid and the derivatives of cinnamic acid such as ferulic acid and cou­
maric acid and the flavonoid quercetin in the 1%HCl-Methanol extract
(Banerjee et al., 2012). LC-MS-MS analysis of the ethanolic extract ie.,
FM-FP showed predominance procyanidin-B1, epigallocatechin, and
O-methylquercetin. Similar to prior studies we have also identified
protocatecheuic acid, vanilli acid, ferulic acid and diferulic acid in the
FM-FP. Variations in the phenolic acid composition among these studies
could be due to finger millet variety as well as differences in the
extractants used.
The bound phenolic acids of Indaf-15 variety of finger millet were
found to contain ferulic acid, coumaric acid and caffeic acid(Subba Rao
and Muralikrishna, 2002). Similar to this report, we have also detected
ferulic acid, vanillic acid, caffeic acid, syringic acid, protocatecheuic
acid and many other phenolic compounds in the FM-BP of finger millet
KMR-301 variety. Studies from other laboratories have also reported the
predominance of cinnamic acid derivatives in the bound phenolic acids
of finger millet (Chandrasekara and Shahidi, 2011).
Extracts rich in phenolic acids are known to exhibit potent anti-
oxidant, anti-inflammatory and anti-microbial activity(Kumar and
Goel, 2019; Viswanath et al., 2009). Prior studies by Jayawardana, et al.,

7
M.G. Kuruburu et al.
Fig. 6. FM-FP triggered fragmentation of DNA and caused death of breast cancer cells. Fragmentation of DNA, an indicator of apoptosis, is one of the key
mechanisms induced by phytochemical rich fractions in cancer cells. FM-FP triggered fragmentation of DNA, as evidenced by sub-G1 cells accumulation, in breast
cancer cells MCF-7 (Fig. 6A) and MDA-MB-468 (Fig. 6B) and showed a significant increase in the number of dead cells.
2021, Viswanath et al., 2009 and Banerjee et al., 2012, SubbaRao and
Muralikrishna 2002 have demonstrated the potential of phenolic acid
rich fractions in scavenging free radicals, protecting the oxidation of
lipids and ferric reducing antioxidant power(Banerjee et al., 2012;
Jayawardana et al., 2021; Subba Rao and Muralikrishna, 2002; Viswa­
nath et al., 2009). Similar to these findings, both FM-FP and FM-BP of
KMR-301 variety also exhibited strong antioxidant potential in the in
vitro studies.
The impact of phenolic compounds of finger millet on breast and
colorectal cancers is not much explored. Currently it is unknown
whether finger millet derived phenolic compounds inhibit the cancer
cells proliferation. A preliminary report by Singh, N., et al., 2015
showed cytotoxic potential of finger millet seeds’ ethyl acetate extract
when tested against HepG2 cell line. Even though Singh et al., 2015
identified 1,2-benzenedicarboxylic acid (BDC) in the ethyl acetate
extract by GC-MS, it is unknown whether this compound alone is
responsible for anti-cancer activity (Singh et al., 2015). In our study, we
have observed that in FM-FP of finger millet are rich in flavonoids and
flavones and differentially modulated the viability of breast and colo­
rectal cancer cell lines (Alves et al., 2016). Several prior studies have
shown the anti-cancer potential of flavonoids and flavones that include
EGCG, Quercetin etc (Du et al., 2012; Signorile et al., 2018).
Cancer chemoprevention is one of the widely used approaches for
reducing the incidence and burden of cancers (Steward and Brown,
2013). In chemoprevention, a dietary formulation or supplement is
recommended to reduce the cancer, particularly to individuals who are
at higher risk of developing the disease (Kotecha et al., 2016). Majority
8
Phytomedicine Plus 2 (2022) 100276
of chemopreventive formulations are known to contain potent antioxi­
dant molecules that can reduce or scavenge the reactive oxygen species
(John et al., 2016). For example, ROS neutralizing phytochemicals such
as flavonoids and hydroxyphenolics are usually fortified into many di­
etary formulations that are recommended for patients suffering from
diabetes, obesity, and cardiovascular diseases (Calinoiu and Vodnar,
2018). Mechanistically, these phytochemical fortified diets activate Nrf2
transcription factor thereby upregulate antioxidant enzymes SOD,
Catalase, Peroxidase, GST etc.; and increase the levels of reducing agents
(GSH, NADH and NADPH) and ion transport systems in cells (Su et al.,
2013). As a result, cells are protected by phytochemical rich formula­
tions. In our study, we have observed a visible increase in the prolifer­
ation of cells representing carcinomas of colon and rectum when they
were exposed to FM-BP. In addition to hydroxybenzoic acid and
hydroxycinnamic acids, the FM-BP has compounds such as tricarboxylic
acid derivatives, isoflavonoid-O-glucosides and very high DNS reducing
substances indicating better ROS scavenging potential (Bolour­
i-Moghaddam et al., 2010; Mailloux et al., 2007; Rimbach et al., 2003).
A prior study by Hassan, ZM 2020 showed the presence of catechin,
protocatechuic acid, epicatechin, p-hydroxybenzoic acid etc in the
acidified methanolic extract of finger millet (Hassan et al., 2020).
Another study by Xiang, J et al., 2019 showed the presence of 20
different phenolic compounds (18 flavonoids with catechin and epi­
catechin being predominant) in the free fraction of 4 different varieties
of finger millet from northern Malawi (Xiang et al., 2019). Authors of
this study showed that the bound fraction of these millets contain ferulic
acid as the major phenolic compound. A separate study evaluating the
M.G. Kuruburu et al.
impact of malting on free and bound phenolic acids content and anti­
oxidant potential showed that ferulic acid, caffeic acid and coumaric
acid are the major constituents of finger millet bound phenolics, and the
antioxidant capacity has decreased upon malting (a process of germi­
nation)(Udeh et al., 2018).
Even though many studies have demonstrated the chemopreventive
and chemotherapeutic potential of phenolic compounds controversies
and concerns pertaining to the use of phenolic acid extracts of millets for
treating cancers. For example, some of the prior studies have shown
increased risk of cancer in individuals consuming diets rich in phenolic
compounds (Hodek et al., 2006; Kyselova, 2011). Similarly, a
population-based case-control study by Nandakumar, A et al., 1990
showed an increased risk of oral cancer in individuals consuming ragi as
their staple diet (Nandakumar et al., 1990). These controversies are
partly due to variations in the concentration and the type of phenolic
compounds present in the samples as well as the presence of compounds
other than polyphenols (Cory et al., 2018; Zamora-Ros et al., 2020). In
our current study, we found that colorectal carcinoma cell lines
exhibited increased proliferation when exposed to FM-BP. But, it is
currently unknown whether FM-BP administration triggers colorectal
tumors? Further studies are warranted to test this possibility. Therefore,
caution must be executed while selecting the extracts for treating
cancers.
Breast cancer is the leading causes of deaths due to cancers in women
(Azamjah et al., 2019; Siegel et al., 2021). Several preclinical studies,
observational, meta-analyses, systematic analyses, and epidemiological
studies have shown an inverse correlation between the consumption of
diets rich in polyphenolic compounds, and the incidence and mortality
rate due to breast cancers(Hui et al., 2013; Kapinova et al., 2018).
Mechanistically, phenolic compounds reduce cellular ROS, augment
anti-inflammatory reactions as well as apoptotic cell death to reduce the
tumor growth (Hussain et al., 2016). In our study, we have observed a
visible decrease in the viability of MCF-7 and MDA-MB-468 cell lines
when treated with FM-FP but not with FM-BP. FM-BP failed to inhibit
the growth of breast cancer cells, which requires further investigation.
Moreover, the FM-FP failed to kill triple positive breast cancer cell line
BT-474 and triple negative cell line MDA-MB-231. Reasons for the
failure of FM-FP against these cell lines are not known, hence, require
additional studies.
Induction of apoptosis is one of the key cell death mechanisms
operated when a cell fail to repair the damage (Elmore, 2007). Many
cancer chemotherapeutic agents exhibit anti-cancer effects through the
induction of apoptosis in cancer cells (Pfeffer and Singh, 2018).
Apoptosis is characterized by the shrinkage of cells, appearance of
apoptotic bodies, and DNA breaks (Elmore, 2007). In this study, we have
observed a visible increase in the fragmented DNA as evidenced by
elevated sub-G1 population in DAPI staining and more number of
ethidium bromide (red color) stained cells in MCF-7 and MDA-MB-468
cell lines exposed to FM-FP, indicating the induction of cell death
through apoptosis. Several prior studies have also demonstrated the
induction of apoptosis by the addition of phytochemical rich fractions of
millets and cereals (Ramasamy et al., 2017).
In conclusion, the free and bound phenolic compounds of finger
millet exhibited diverse effects when tested against cancer cell lines.
Whereas the bound phenolic compounds induced proliferation in ma­
jority of cell lines, the free phenolic acids containing FM-FP differen­
tially induced cell death in breast and colorectal cancer cells. Reasons for
these differential properties could be partly due to variations in their
phenolic constituents and the presence of compounds other than
phenolic compounds, which require further investigations.
CONFLICT OF INTEREST
Authors declare no conflict of interest
9
Phytomedicine Plus 2 (2022) 100276
CRediT authorship contribution statement
Mahadevaswamy G. Kuruburu: Conceptualization, Methodology,
Software, Formal analysis, Writing – original draft, Investigation, Visu­
alization, Data curation, Investigation. Venugopal R. Bovilla:
Conceptualization, Methodology, Data curation, Software, Investiga­
tion, Visualization, Writing – review & editing, Supervision. Rimshia
Naaz: Methodology, Visualization, Formal analysis, Software, Writing –
review & editing, Data curation. Zonunsiami Leihang: Methodology,
Visualization, Formal analysis, Validation, Software. SubbaRao V.
Madhunapantula: Conceptualization, Methodology, Writing – original
draft, Software, Validation, Writing – review & editing, Formal analysis,
Investigation, Resources, Data curation, Supervision, Project adminis­
tration, Funding acquisition.
ACKNOWLEDGEMENTS
Authors would like to acknowledge (a) the infrastructure support
provided by Department of Science & Technology to CEMR Laboratory
(CR-FST-LS-1/2018/178) and to Department of Biochemistry (SR/FST/
LS-1-539/2012); (b) the laboratory facilities provided by CEMR labo­
ratory (DST-FIST supported center), Department of Biochemistry (DST-
FIST supported department), and Special Interest Group on Cancer
Biology and Cancer Stem Cells (SIG-CBCSC), JSS Academy of Higher
Education & Research (Mysore, Karnataka, India). Mahadevaswamy G.
Kuruburu and Venugopal R. Bovilla are grateful to Indian Council of
Medical Research (ICMR), and Government of India for Senior Research
Fellowship (SRF) award (Fellowship sanction No. 3/2/3/105/2019/
NCD-III and 3/2/2/5/2018/online Onco Fship/NCD-III, respectively).
Zonunsiami Leihang would like to thank Ministry of Tribal Affairs,
Government of India for the award of National Fellowship and Schol­
arship for Higher Education of ST Students (NFST). Venugopal R. Bovilla
would like to also acknowledge the postdoctoral Global Health Equity
Scholars (GHES) fellowship Program funding from the NIH Fogarty In­
ternational Center (Award number D43 TW010540). Rimshia Naaz
would like to thank Directory of Minority, Karnataka Govt for providing
fellowship (Fellowship/C. R-03/2019-2020). Authors would like to
thank Dr. Ashish Pargonkar from Agilent Technologies India Pvt Ltd,
Bangalore, India for assisting in UPLC-QTOF-MS analysis.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.phyplu.2022.100276.
REFERENCES
Aksoy, L., Kolay, E., Agilonu, Y., Aslan, Z., Kargioglu, M., 2013. Free radical scavenging
activity, total phenolic content, total antioxidant status, and total oxidant status of
endemic Thermopsis turcica. Saudi J Biol Sci 20, 235–239. https://doi.org/10.1016/
j.sjbs.2013.02.003.
Alves, A.C., Ribeiro, D., Nunes, C., Reis, S., 2016. Biophysics in cancer: The relevance of
drug-membrane interaction studies. Biochim Biophys Acta 1858, 2231–2244.
https://doi.org/10.1016/j.bbamem.2016.06.025.
Azamjah, N., Soltan-Zadeh, Y., Zayeri, F., 2019. Global Trend of Breast Cancer Mortality
Rate: A 25-Year Study. Asian Pac J Cancer Prev 20, 2015–2020. https://doi.org/
10.31557/APJCP.2019.20.7.2015.
Banerjee, S., Sanjay, K., Chethan, S., Malleshi, N., 2012. Finger millet (Eleusine
coracana) polyphenols: Investigation of their antioxidant capacity and antimicrobial
activity. Afr. J. Food Sci. 6, 362–374. https://doi.org/10.5897/AJFS12.031.
Benzie, I.F., Strain, J.J., 1999. Ferric reducing/antioxidant power assay: direct measure
of total antioxidant activity of biological fluids and modified version for
simultaneous measurement of total antioxidant power and ascorbic acid
concentration. Methods Enzymol 299, 15–27. https://doi.org/10.1016/s0076-6879
(99)99005-5.
Bolouri-Moghaddam, M.R., Le Roy, K., Xiang, L., Rolland, F., Van den Ende, W., 2010.
Sugar signalling and antioxidant network connections in plant cells. FEBS J 277,
2022–2037, 10.1111/j.1742-4658.2010.07633. x.
Bovilla, V.R., Kuruburu, M.G., Bettada, V.G., Krishnamurthy, J., Sukocheva, O.A.,
Thimmulappa, R.K., Shivananju, N.S., Balakrishna, J.P., Madhunapantula, S.V.,
2021. Targeted Inhibition of Anti-Inflammatory Regulator Nrf2 Results in Breast
M.G. Kuruburu et al.
Cancer Retardation In Vitro and In Vivo. Biomedicines 9, 1119. https://doi.org/
10.3390/biomedicines9091119.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72,
248–254. https://doi.org/10.1006/abio.1976.9999.
Calinoiu, L.F., Vodnar, D.C., 2018. Whole Grains and Phenolic Acids: A Review on
Bioactivity, Functionality, Health Benefits and Bioavailability. Nutrients 10, 1–32.
https://doi.org/10.3390/nu10111615.
Chanda, J., Mukherjee, P.K., Biswas, R., Biswas, S., Tiwari, A.K., Pargaonkar, A., 2019.
UPLC-QTOF-MS analysis of a carbonic anhydrase-inhibiting extract and fractions of
Luffa acutangula (L.) Roxb (ridge gourd). Phytochem Anal 30, 148–155. https://doi.
org/10.1002/pca.2800.
Chandra, D., Chandra, S., Sharma, A., 2016. Review of Finger millet (Eleusine coracana
(L.) Gaertn): a power house of health benefiting nutrients. Food Sci. Hum. Wellness
5, 149–155. https://doi.org/10.1016/j.fshw.2016.05.004.
Chandrasekara, A., Shahidi, F., 2010. Content of insoluble bound phenolics in millets and
their contribution to antioxidant capacity. J Agric Food Chem 58, 6706–6714.
https://doi.org/10.1021/jf100868b.
Chandrasekara, A., Shahidi, F., 2011. Determination of antioxidant activity in free and
hydrolyzed fractions of millet grains and characterization of their phenolic profiles
by HPLC-DAD-ESI-MSn. J. Funct. Foods 3, 144–158. https://doi.org/10.1016/j.
jff.2011.03.007.
Chaudhary, J.K., Mudgal, S., 2020. Antidiabetic and Hypolipidaemic Action of Finger
Millet (Eleusine coracana)-Enriched Probiotic Fermented Milk: An in vivo Rat Study.
Food Technol Biotechnol 58, 192–202. https://doi.org/10.1515/pjvs-2017-0106.
Chikkegowda, P., Pookunoth, B.C., Bovilla, V.R., Veeresh, P.M., Leihang, Z.,
Thippeswamy, T., Padukudru, M.A., Hathur, B., Kanchugarakoppal, R.S., Basappa,
Madhunapantula, S.V., 2021. Design, Synthesis, Characterization, and Crystal
Structure Studies of Nrf2 Modulators for Inhibiting Cancer Cell Growth In Vitro and
In Vivo. ACS Omega 6, 10054–10071. https://doi.org/10.1021/acsomega.0c06345.
Cory, H., Passarelli, S., Szeto, J., Tamez, M., Mattei, J., 2018. The Role of Polyphenols in
Human Health and Food Systems: A Mini-Review. Front Nutr 5. https://doi.org/
10.3389/fnut.2018.00087, 87.1-7.
Daga, P., Vaishnav, S.R., Dalmia, A., Tumaney, A.W., 2021. Extraction, fatty acid profile,
phytochemical composition and antioxidant activities of fixed oils from spices
belonging to Apiaceae and Lamiaceae family. J. Food Sci. Technol 1–14. https://doi.
org/10.1007/s13197-021-05036-1.
Devi, P.B., Vijayabharathi, R., Sathyabama, S., Malleshi, N.G., Priyadarisini, V.B., 2014.
Health benefits of finger millet (Eleusine coracana L.) polyphenols and dietary fiber:
a review. J Food Sci Technol 51, 1021–1040. https://doi.org/10.1007/s13197-011-
0584-9.
Du, G.J., Zhang, Z., Wen, X.D., Yu, C., Calway, T., Yuan, C.S., Wang, C.Z., 2012.
Epigallocatechin Gallate (EGCG) is the most effective cancer chemopreventive
polyphenol in green tea. Nutrients 4, 1679–1691. https://doi.org/10.3390/
nu4111679.
Elmore, S., 2007. Apoptosis: a review of programmed cell death. Toxicol Pathol 35,
495–516. https://doi.org/10.1080/01926230701320337.
Gupta, S.M., Arora, S., Mirza, N., Pande, A., Lata, C., Puranik, S., Kumar, J., Kumar, A.,
2017. Finger Millet: A "Certain" Crop for an "Uncertain" Future and a Solution to
Food Insecurity and Hidden Hunger under Stressful Environments. Front Plant Sci 8,
643. https://doi.org/10.3389/fpls.2017.00643, 1-11.
Hassan, Z.M., Sebola, N.A., Mabelebele, M., 2020. Assessment of the phenolic
compounds of pearl and finger millets obtained from South Africa and Zimbabwe.
Food Sci Nutr 8, 4888–4896. https://doi.org/10.1002/fsn3.1778.
Hodek, P., Hanustiak, P., Krizkova, J., Mikelova, R., Krizkova, S., Stiborova, M.,
Trnkova, L., Horna, A., Beklova, M., Kizek, R., 2006. Toxicological aspects of
flavonoid interaction with biomacromolecules. Neuro Endocrinol Lett 27 (Suppl 2),
14–17.
Huang, W.Y., Cai, Y.Z., Zhang, Y., 2010. Natural phenolic compounds from medicinal
herbs and dietary plants: potential use for cancer prevention. Nutr Cancer 62, 1–20.
https://doi.org/10.1080/01635580903191585.
Hui, C., Qi, X., Qianyong, Z., Xiaoli, P., Jundong, Z., Mantian, M., 2013. Flavonoids,
flavonoid subclasses and breast cancer risk: a meta-analysis of epidemiologic studies.
PLoS One 8, e54318. https://doi.org/10.1371/journal.pone.0054318.
Hussain, T., Tan, B., Yin, Y., Blachier, F., Tossou, M.C., Rahu, N., 2016. Oxidative Stress
and Inflammation: What Polyphenols Can Do for Us? Oxid Med Cell Longev 2016,
7432797 doi:10.1002/fsn3.1778.
Jayawardana, S.A.S., Samarasekera, J., Hettiarachchi, G., Gooneratne, J., Choudhary, M.
I., Jabeen, A., 2021. Anti-inflammatory and Antioxidant Properties of Finger Millet
(Eleusine coracana (L.) Gaertn.) Varieties Cultivated in Sri Lanka. Biomed Res Int
2021, 7744961. https://doi.org/10.1155/2021/7744961.
John, A.S., Ankem, M.K., Damodaran, C., 2016. Oxidative Stress: A Promising Target for
Chemoprevention. Curr Pharmacol Rep 2, 73–81. https://doi.org/10.1007/s40495-
016-0052-3.
Kapinova, A., Kubatka, P., Golubnitschaja, O., Kello, M., Zubor, P., Solar, P., Pec, M.,
2018. Dietary phytochemicals in breast cancer research: anticancer effects and
potential utility for effective chemoprevention. Environ Health Prev Med 23 (36),
1–18. https://doi.org/10.1186/s12199-018-0724-1.
Kotecha, R., Takami, A., Espinoza, J.L., 2016. Dietary phytochemicals and cancer
chemoprevention: a review of the clinical evidence. Oncotarget 7, 52517–52529.
https://doi.org/10.18632/oncotarget.9593.
Kumar, N., Goel, N., 2019. Phenolic acids: Natural versatile molecules with promising
therapeutic applications. Biotechnol Rep (Amst) 24, e00370. https://doi.org/
10.1016/j.btre.2019.e00370, 1-10.
10
Phytomedicine Plus 2 (2022) 100276
Kyselova, Z., 2011. Toxicological aspects of the use of phenolic compounds in disease
prevention. Interdiscip Toxicol 4, 173–183. https://doi.org/10.2478/v10102-011-
0027-5.
Liu, K., Liu, P.C., Liu, R., Wu, X., 2015. Dual AO/EB staining to detect apoptosis in
osteosarcoma cells compared with flow cytometry. Med Sci Monit Basic Res 21,
15–20. https://doi.org/10.12659/MSMBR.893327.
Madhunapantula, S.V., Robertson, G.P., 2008. Is B-Raf a good therapeutic target for
melanoma and other malignancies? Cancer Res 68, 5–8. https://doi.org/10.1158/
0008-5472.CAN-07-2038.
Mailloux, R.J., Beriault, R., Lemire, J., Singh, R., Chenier, D.R., Hamel, R.D., Appanna, V.
D., 2007. The tricarboxylic acid cycle, an ancient metabolic network with a novel
twist. PLoS One 2, e690. https://doi.org/10.1371/journal.pone.0000690.
Maleki, S.J., Crespo, J.F., Cabanillas, B., 2019. Anti-inflammatory effects of flavonoids.
Food Chem 299, 125124. https://doi.org/10.1016/j.foodchem.2019.125124.
Matassov, D., Kagan, T., Leblanc, J., Sikorska, M., Zakeri, Z., 2004. Measurement of
apoptosis by DNA fragmentation. Methods Mol Biol 282, 1–17. https://doi.org/
10.1385/1-59259-812-9:001.
Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing
sugar. Anal.l chem 31, 426–428. https://doi.org/10.1021/ac60147a030.
Murad, H., Hawat, M., Ekhtiar, A., AlJapawe, A., Abbas, A., Darwish, H., Sbenati, O.,
Ghannam, A., 2016. Induction of G1-phase cell cycle arrest and apoptosis pathway in
MDA-MB-231 human breast cancer cells by sulfated polysaccharide extracted from
Laurencia papillosa. Cancer Cell Int 16 (39), 1–11. https://doi.org/10.1186/s12935-
016-0315-4.
Nandakumar, A., Thimmasetty, K.T., Sreeramareddy, N.M., Venugopal, T.C.,
Rajanna, Vinutha, A.T., Srinivas, Bhargava, M.K., 1990. A population-based case-
control investigation on cancers of the oral cavity in Bangalore, India. Br J Cancer
62, 847–851. https://doi.org/10.1038/bjc.1990.392.
Neveu, V., Perez-Jimenez, J., Vos, F., Crespy, V., du Chaffaut, L., Mennen, L., Knox, C.,
Eisner, R., Cruz, J., Wishart, D., Scalbert, A., 2010. Phenol-Explorer: an online
comprehensive database on polyphenol contents in foods. Database (Oxford) 2010.
https://doi.org/10.1093/database/bap024 bap024.1-9.
Okwudili, U.H., Gyebi, D.K., Obiefuna, J.A.I., 2017. Finger millet bioactive compounds,
bioaccessibility, and potential health effects–a review. Czech J. Food Sci. 35, 7–17.
https://doi.org/10.17221/206/2016-CJFS.
Patel, H., Royall, P.G., Gaisford, S., Williams, G.R., Edwards, C.H., Warren, F.J.,
Flanagan, B.M., Ellis, P.R., Butterworth, P.J., 2017. Structural and enzyme kinetic
studies of retrograded starch: Inhibition of alpha-amylase and consequences for
intestinal digestion of starch. Carbohydr Polym 164, 154–161. https://doi.org/
10.1016/j.carbpol.2017.01.040.
Pfeffer, C.M., Singh, A.T.K., 2018. Apoptosis: A Target for Anticancer Therapy. Int J Mol
Sci 19, 1–10. https://doi.org/10.3390/ijms19020448.
Phuyal, N., Jha, P.K., Raturi, P.P., Rajbhandary, S., 2020. Total Phenolic, Flavonoid
Contents, and Antioxidant Activities of Fruit, Seed, and Bark Extracts of
Zanthoxylum armatum DC. Sci. World J 2020, 1–7. https://doi.org/10.1155/2020/
8780704, 8780704.
Pisoschi, A.M., Cheregi, M.C., Danet, A.F., 2009. Total antioxidant capacity of some
commercial fruit juices: electrochemical and spectrophotometrical approaches.
Molecules 14, 480–493. https://doi.org/10.3390/molecules14010480.
Puranik, S., Kam, J., Sahu, P.P., Yadav, R., Srivastava, R.K., Ojulong, H., Yadav, R., 2017.
Harnessing Finger Millet to Combat Calcium Deficiency in Humans: Challenges and
Prospects. Front Plant Sci 8, 1311. https://doi.org/10.3389/fpls.2017.01311, 1-16.
Ramasamy, S., Anupam, K., Ratnavathi, C., Karunagaran, D., Chandra, T., 2017.
Paspalum scrobiculatum polyphenol rich extracts inhibit human cervical cancer cell
proliferation and induce apoptosis in vitro. The FASEB Journal 31. https://doi.org/
10.1096/fasebj.31.1_supplement.lb162 lb162-lb162.
Rao, M.S., Muralikrishna, G., 2001. Non-starch polysaccharides and bound phenolic
acids from native and malted finger millet (Ragi, Eleusine coracana, Indaf-15). Food
Chem 72, 187–192. https://doi.org/10.1016/S0308-8146(00)00217-X.
Rimbach, G., De Pascual-Teresa, S., Ewins, B.A., Matsugo, S., Uchida, Y., Minihane, A.M.,
Turner, R., VafeiAdou, K., Weinberg, P.D., 2003. Antioxidant and free radical
scavenging activity of isoflavone metabolites. Xenobiotica 33, 913–925. https://doi.
org/10.1080/0049825031000150444.
Rok, J., Rzepka, Z., Beberok, A., Pawlik, J., Wrzesniok, D., 2020. Cellular and Molecular
Aspects of Anti-Melanoma Effect of Minocycline-A Study of Cytotoxicity and
Apoptosis on Human Melanotic Melanoma Cells. Int J Mol Sci 21. https://doi.org/
10.3390/ijms21186917.
Sánchez-Rangel, J.C., Benavides, J., Heredia, J.B., Cisneros-Zevallos, L., Jacobo-
Velázquez, D.A., 2013. The Folin–Ciocalteu assay revisited: improvement of its
specificity for total phenolic content determination. Analytical Methods 5,
5990–5999. https://doi.org/10.1039/C3AY41125G.
Shobana, S., Krishnaswamy, K., Sudha, V., Malleshi, N.G., Anjana, R.M., Palaniappan, L.,
Mohan, V., 2013. Finger millet (Ragi, Eleusine coracana L.): a review of its
nutritional properties, processing, and plausible health benefits. Adv Food Nutr Res
69, 1–39, 10.1016/B978-0-12-410540-9.00001-6.
Siegel, R.L., Miller, K.D., Fuchs, H.E., Jemal, A., 2021. Cancer Statistics, 2021. CA Cancer
J Clin 71, 7–33. https://doi.org/10.3322/caac.21654.
Signorile, P.G., Viceconte, R., Baldi, A., 2018. Novel dietary supplement association
reduces symptoms in endometriosis patients. J Cell Physiol 233, 5920–5925.
Singh, N., Meenu, G., Sekhar, A., Jayanthi, A., 2015. Evaluation of antimicrobial and
anticancer properties of finger millet (Eleusine coracana) and pearl millet
(Pennisetum glaucum) extracts. The Pharma Innovation 3 (82), 82–86.
Singh, P., Anand, A., Kumar, V., 2014. Recent developments in biological activities of
chalcones: a mini review. Eur J Med Chem 85, 758–777. https://doi.org/10.1016/j.
ejmech.2014.08.033.
M.G. Kuruburu et al.
Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J.T.,
Bokesch, H., Kenney, S., Boyd, M.R., 1990. New colorimetric cytotoxicity assay for
anticancer-drug screening. J Natl Cancer Inst 82, 1107–1112. https://doi.org/
10.1093/jnci/82.13.1107.
Sood, S., Kant, L., Pattanayak, A., 2017. Finger Millet [Eleusine coracana (L.) Gaertn.]: A
Minor Crop for Sustainable Food and Nutritional Security. Asian J of Chem 29,
707–710. https://doi.org/10.14233/ajchem.2017.20284.
Steward, W.P., Brown, K., 2013. Cancer chemoprevention: a rapidly evolving field. Br J
Cancer 109, 1–7. https://doi.org/10.1038/bjc.2013.280.
Su, Z.Y., Shu, L., Khor, T.O., Lee, J.H., Fuentes, F., Kong, A.N., 2013. A perspective on
dietary phytochemicals and cancer chemoprevention: oxidative stress, nrf2, and
epigenomics. Top Curr Chem 329, 133–162. https://doi.org/10.1007/128_2012_
340.
Subba Rao, M.V., Muralikrishna, G., 2002. Evaluation of the antioxidant properties of
free and bound phenolic acids from native and malted finger millet (ragi, Eleusine
coracana Indaf-15). J Agric Food Chem 50, 889–892. https://doi.org/10.1021/
jf011210d.
Subba Rao, M.V., Muralikrishna, G., 2004. Structural analysis of arabinoxylans isolated
from native and malted finger millet (Eleusine coracana, ragi). Carbohydr Res 339,
2457–2463. https://doi.org/10.1016/j.carres.2004.07.005.
11
Phytomedicine Plus 2 (2022) 100276
Udeh, H.O., Duodu, K.G., Jideani, A.I.O., 2018. Malting Period Effect on the Phenolic
Composition and Antioxidant Activity of Finger Millet (Eleusine coracana L. Gaertn)
Flour. Molecules 23, 1–12. https://doi.org/10.3390/molecules23092091.
Viswanath, V., Urooj, A., Malleshi, N., 2009. Evaluation of antioxidant and antimicrobial
properties of finger millet polyphenols (Eleusine coracana). Food Chem 114,
340–346. https://doi.org/10.1016/j.fshw.2016.05.004.
Xiang, J., Apea-Bah, F.B., Ndolo, V.U., Katundu, M.C., Beta, T., 2019. Profile of phenolic
compounds and antioxidant activity of finger millet varieties. Food Chem 275,
361–368. https://doi.org/10.1016/j.foodchem.2018.09.120.
Zamora-Ros, R., Cayssials, V., Franceschi, S., Kyro, C., Weiderpass, E., Hennings, J.,
Sandstrom, M., Tjonneland, A., Olsen, A., Overvad, K., Boutron-Ruault, M.C.,
Truong, T., Mancini, F.R., Katzke, V., Kuhn, T., Boeing, H., Trichopoulou, A.,
Karakatsani, A., Martimianaki, G., Palli, D., Krogh, V., Panico, S., Tumino, R.,
Sacerdote, C., Lasheras, C., Rodriguez-Barranco, M., Amiano, P., Colorado-Yohar, S.
M., Ardanaz, E., Almquist, M., Ericson, U., Bueno-de-Mesquita, H.B., Vermeulen, R.,
Schmidt, J.A., Byrnes, G., Scalbert, A., Agudo, A., Rinaldi, S., 2020. Polyphenol
intake and differentiated thyroid cancer risk in the European Prospective
Investigation into Cancer and Nutrition (EPIC) cohort. Int J Cancer 146, 1841–1850.
https://doi.org/10.1002/ijc.32589.