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Antioxidative and Radioprotective Properties of Glycosylated Flavonoid,

Xanthorhamnin from Radio-Resistant Bacterium Bacillus indicus Strain TMC-
6

Article in Current Microbiology · July 2020

DOI: 10.1007/s00284-020-01930-7

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Current Microbiology
https://doi.org/10.1007/s00284-020-01930-7

Antioxidative and Radioprotective Properties of Glycosylated

Flavonoid, Xanthorhamnin from Radio‑Resistant Bacterium Bacillus
indicus Strain TMC‑6
Ayesha Ahmed Chaudhri1 · Mahnoor Nadeem1 · Asim ur Rahman1 · Tayyaba Alam1 · Wasim Sajjad2 · Fariha Hasan1 ·
Malik Badshah1 · Samiullah Khan1 · Fazal Rehman3 · Aamer Ali Shah1

Received: 25 October 2019 / Accepted: 20 February 2020

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
A radio-resistant bacterium labeled as strain TMC-6 was isolated from Thal desert, Pakistan and identified through 16S
rRNA gene sequencing as Bacillus indicus strain TMC-6 (MN721293). The isolate was found to be resistant to UV radiation
dose of 6.780 × 103 J/m2 and showed 50% survivability to mitomycin C (6 μg/ml) and ­H2O2 (30 mM). The bacterium showed
yellowish orange coloration when grown on tryptone yeast glucose (TGY) medium. The cellular metabolite was extracted in
methanol and purified through solid phase extraction with C18 column cartridge. The compound was characterized through
UV/Visible spectrophotometry, Fourier Transform Infra-Red (FT-IR) spectroscopy and Liquid Chromatography Mass Spec-
trometry (LC–MS). The LC–MS analysis of the compound revealed a molar mass of 769 [m/z]− that matched the chemical
formula ­C34H42O20 and identified as a glycosylated flavonoid xanthorhamnin. The compound showed significant antioxidant
(77.05%) and metal chelation (79.80%) activities. Xanthorhamnin showed promising oxidative damage inhibitory actions
in bovine serum albumin (65.32%) and mice liver lipids (71.61%) and prevented DNA strand breaks from oxidative stress.
Cytotoxicity in brine shrimp larvae was observed when compared with mitomycin C indicating its effect toward cancerous
cells. These findings concluded that xanthorhamnin from radio-resistant Bacillus indicus strain TMC-6 has high antioxidant,
radioprotective, and antitumor properties against UV-mediated oxidative damages.

Introduction cause damaging effects to all domains of life, in human body

Microorganisms in their natural habitats encounter numerous
stressful conditions and to ensure their persistent survival,
they react to such challenging environments accordingly.
Microorganisms thriving in extreme arid environments have
special protection mechanisms against desiccation stress and
high levels of radiations emitting from sun. In deserts, low
water availability and prolonged exposure of UV radiations
act as natural mutagens that can harm microbial life by
damaging cellular vital molecules [1]. Ultraviolet radiations
* Aamer Ali Shah
alishah@qau.edu.pk
1
Department of Microbiology, Faculty of Biological Sciences,
Quaid-I-Azam University, Islamabad 45320, Pakistan
2
Department of Biological Sciences, National University
of Medical Sciences, Rawalpindi 46000, Pakistan
3
Department of Biotechnology, University of Balochistan,
Quetta, Pakistan
severe mutational effects occur due to prolonged exposure of
high level radiations resulting in malignancies and cataracts
[2]. It is well known that radio-resistant extremophiles have
adapted different mechanisms to survive under high radia-
tion levels by effectively inactivating the reactive oxygen
species (ROS). The protection against ROS-mediated dam-
ages is accomplished by development of different antioxi-
dant systems within the cell such as antioxidant enzymes:
catalase, peroxidases [3], Mn/Fe-mediated regulatory
response, production of antioxidant compounds as second-
ary metabolites (extremolytes), release of extracellular poly-
meric substances for UV absorbance.
The natural occurring antioxidant compounds such as
flavonoids, carotenoids, mycosporin-like amino acids are
responsible for prevention of the oxidation of cellular com-
ponents by scavenging free radicals and diminishing oxida-
tive stress, thus providing protection to cellular proteomes
and genome. Such extremolytes can be used effectively
against various oxidation induced diseases like premature
aging, cataracts, tumor formation, and modification of

13
Vol.:(0123456789)

cellular lipids and proteins [4, 5]. The increase demand of
antioxidant compounds in pharmaceutics and therapeutics
has impelled the scientific interest in these extremolytes
from UV-resistant microorganisms due to easy and high
productivity of the bioactive compounds from bacteria. So
far, many UV-resistant microbes have been exploited for
the isolation and characterization of radio protective com-
pounds. The present study was focused on the radio protec-
tive and antioxidative properties of bioactive compounds
extracted from a radio-resistant bacterium Bacillus indicus
strain TMC-6 (MN721293) isolated from desert soil. The
resistance of the isolate against UV exposure, ­H2O2 and
mitomycin was analyzed. The colored compound extracted
from Bacillus indicus strain TMC6 (MN721293) was puri-
fied and evaluated for its radioprotective and antioxidant
activities in vitro.
Materials and Methods
Isolation of UV‑Resistant Bacteria
Soil sample was aseptically collected according to standard
microbiological sampling protocol [6] from Thal desert,
Pakistan and transported to the laboratory immediately to
prevent any contamination. The serially diluted soil sample
in normal saline was inoculated on TGY (tryptone, glucose,
yeast extract) agar plates through spread plate method. Inoc-
ulated plates were exposed to 280 nm of UV-B radiations
for 2 min in a UV chamber and incubated for 3–5 days. The
isolates obtained were subcultured and irradiated with dif-
ferent UV-B doses for time durations (2–10 min). The UV
dose effect on test culture plates was determined by time of
exposure to the UV fluence rate. The UV fluence rate is the
radiation energy reaching a surface area in a specific time
(energy/area/time) in J/m2 and is determined by the follow-
ing formula:
He = Ee × t
where He is the radiant exposure that reaches a surface
area with energy Ee for a specific time period t.
Among the survived UV-resistant strains, strain TMC-6
was selected on the basis of its high survival rate upon
UV exposure at 280 nm for a radiation dose of 6780 J/m2
(10 min) and higher pigment production.
Identification of UV‑Resistant Bacterial Strain
The identification of strain TMC-6 was done morpho-
logically as well as biochemically according to previously
describe methods [7]. For molecular characterization 16S
rRNA gene sequencing was used. For this purpose, DNA
13
A. A. Chaudhri et al.
was extracted by phenol-chloroform method [8]. The PCR
amplification of 16S rRNA was done by using two univer-
sal primers F27: AGA​GTT​TGATCMTGG​CTC​AG, R1492:
TAC​GGY​TAC​CTT​GTT​ACG​ACTT [9, 10]. The amplicons
obtained were then sequenced at Macrogen Service Center
(Geunchun-gu, Seoul, South Korea); the sequence was com-
puted for nearest possible genus by using BLAST tool at the
National Center for Biotechnology Information NCBI data-
base and homologs found were assessed for their phylogeny
through Molecular Evolutionary Genetic Analysis (MEGA
X) [11]. Based upon distance matrix, a neighbor-joining tree
was constructed.
Resistance of Strain TMC‑6 to Heavy Metal
Stock solutions of transition metals mercury (­ Hg2+), cobalt
­(Co2+), nickel ­(Ni2+), zinc ­(Zn2+), ferrous ­(Fe2+), and man-
ganese ­(Mn2+) were prepared in sterilized distilled water
from their respective metal salts (chlorides). Effect of metal
ions on the growth of strain TMC-6 was determined by sup-
plementing the TGY agar medium with different metals at
concentrations of 150 ppm, 250 ppm and 500 ppm, respec-
tively, and incubated at 30 °C for 72 hours.
Bacterial Survival Curve under UV Radiation
and Oxidative Stress
The survivability of the UV-resistant strain TMC-6 was
checked against UV-B radiations, H ­ 2O2, and mitomycin C
in comparison with E. coli strain ATCC (10536) and sur-
vival curves were plotted [12]. Overnight grown culture of
TMC-6 was diluted in normal saline until an ­OD600 of 0.08
is obtained. 20 μl of cell suspension was inoculated on TGY
agar plates by spread plate technique. Plates were exposed to
UV-B dosage from 2 to 12 min at 280 nm and incubated at
37 °C in dark. Besides this, the cell suspension was treated
against different molar concentration of ­H2O2 (10–60 mM)
and mitomycin C (2–10 μg/ml) for 60 and 20 min at 37 °C,
respectively. 20 μl from each treated cell suspension was
placed on TGY agar plates and incubated at 37 °C for 72 h
in dark. Survival rate was determined by counting number
of colonies on treated and untreated culture plates. The
experiment was run in triplicate and the survival curves were
obtained from the average of triplicated data.
Extraction and Purification of Bioactive Compound
Strain TMC-6 was grown in 1000 ml of TGY broth at
30 °C in shaking incubator at 150 rpm. Cells were col-
lected after 72 h of incubation through centrifugation and
placed at − 4 °C to provide cold shock. The cellular sus-
pension in methanol was sonicated for 10–15 mins using
probe sonicator and centrifuged at 11,200×g for 10 min at
Antioxidative and Radioprotective Properties of Glycosylated Flavonoid, Xanthorhamnin…
4 °C to obtain a clear supernatant. The metabolic extract
was collected after freeze drying. The dried sample was
then purified by using HC-C18 SPE (solid phase extrac-
tion) cartridge column with an average flow rate of 1 ml/
min. This technique was performed according to previ-
ously describe method [13] by loading 280 mg of the
extract in C18 cartridge and for sample elution different
solvents with an order of polar to non-polar (methanol,
ethyl acetate and dichloromethane) were passed and col-
lected in separate fractions. The collected purified frac-
tion with pigmentation was then selected for further
processing.
Separation and Characterization of Cellular Extract
The separation of bioactive components from strain TMC-6
was carried out through thin layer chromatography (TLC).
Chloroform: methanol [7:3 v/v] solvent system was used as
mobile phase. The spots of cellular extract were made on the
baseline of the TLC plate and the solvent front was let to run
for about 80% of the plate. The running lane was then dried
thoroughly in air and the developed TLC plate was visual-
ized under UV illuminator. The bioactive components were
identified by spraying DPPH [14].
The absorption peaks of the unknown bioactive com-
pounds in the cellular extract were analyzed through
UV–visible light (UV–Vis) spectrophotometer within a
range of 200–800 nm to obtain absorption spectra.
FT‑IR analysis of Cellular Extract
The cellular extract was subjected to Fourier Transform
Infra-Red (FT-IR) spectroscopy analysis. The sample was
mixed with KBr and the spectrum was obtained in the spec-
tral region between 400 and 4000 cm−1 of the FT-IR spec-
trometer (Perkin Elmer Spectrum 65). The IR transmittance
of the pure compound was plotted as intensity versus wave
number.
LC–MS Analysis of Cellular Extract
Liquid chromatography mass spectrometry (LC/MS) analy-
sis for the bioactive metabolite was performed with an Agi-
lent 6310 Ion Trap LC/MS system as previously described
[15]. 5 μl of the filtered sample was injected onto a C18 col-
umn using solvents acetonitrile and water, as mobile phase
with a flow rate of 0.5 ml/min in isocratic mode. The mass
spectrum was acquired using electrospray ionization mass
spectrometry in negative ion mode to detect m/z transitions
[M+H]−.
In vitro Bioassays of Purified Cellular Extract
Radical Scavenging Activity by DPPH Assay
The DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging
effect of the cellular extract was determined by the method
as previously described [16]. The cellular extract in different
concentration (10–50 µg/µl) was treated with DPPH solution
(0.1 mM) in 96 well microtiter plate with a final volume
200 μl and incubated at 30 °C in dark for 30 min. Absorb-
ance was measured at 517 nm with UV–Vis spectrometry.
Ascorbic acid (10–50 µg/µl) was run as positive control
while using DPPH as blank.
Abs of control − Abs of sample
%Scavenging = × 100
Abs of control
Iron Chelation Assay
The chelation of ferrous ions by the metabolic extract was
determined by Dinis [17] method with slight modifications.
50 μl of 2 mM Ferrous chloride was mixed with different
concentrations of purified extract (10–30 μg/μl) in a 96 well
microtiter plate and the reaction was initiated by adding
200 μl of 5 mM ferrozine prior to incubation for 15 min.
The absorbance was measured at 562 nm by UV–Vis spec-
trometer. EDTA (10–30 µg/µl) was used as positive control.
Chelation activity of bioactive extract was calculated by
formula:
Abs of control − Abs of sample
% Chelation = × 100
Abs of control
Protein Oxidation Prevention Assay
The inhibitory effect of the bioactive extract against protein
oxidation was measured by using DNPH (2,4-dinitrophenyl
hydrazine) [18]. Bovine serum albumin was used as standard
protein. 100 μl of BSA (1 mg/ml) was treated with 100 μl
of ­FeSO4 (1 mM), ­H2O2 (80 mM) and different concentra-
tions of bioactive extract (10–50 μg/μl) and incubated at
37 °C for 1 h. 15U of catalase was added to stop the reaction
and 600 µl of DNPH (10 mM) was added before incubation
for 1 h. 10% TCA was added for precipitation of unbound
proteins. The precipitates were washed with ethyl acetate:
ethanol (50:50) and re-dissolved in 1 ml of guanidine hydro-
chloride (6 M). The mixture was centrifuged at 10,000 rpm
for 5 min and absorbance of supernatant was determined at
370 nm. Ascorbic acid (10–50 μg/μl) was used as positive
control.
13

Lipid Peroxidation Inhibition Assay
The activity of purified extract against lipid peroxidation was
assayed by estimating the Thiobarbituric Acid Reactive Sub-
stances (TBARS) as described previously [19], where mice
liver homogenate was used as lipid source. 1 g of mice liver
tissue obtained from the Department of Animal Sciences
was homogenized in tris HCL buffer (pH 7.4). 0.1 ml of the
homogenate was mixed with cellular extract in different con-
centrations (10–50 μg/μl), 0.3 ml of ­FeSO4 (10 M), 0.2 ml of
SDS (8.1%), 0.5 ml of acetic acid (pH 3.4) and TBA aqueous
solution (0.8% v/v). The reaction mixture was incubated at
95 °C for 60 min and absorbance was measured at 532 nm
using ascorbic acid as positive control [20].
DNA Damage Prevention Assay
The DNA damage preventive activity of the extract was
determined by the method as previously described with
minor modification [21]. 2 μl of plasmid DNA pUC18, Fen-
ton’s reagent 7 μl, 1 M sodium nitroprusside 4 µl and cellular
extract (50 µg/µl) with distilled water for final volume 20 µl
were mixed and incubated for 30 min at 37 °C. The untreated
plasmid DNA was used as positive control. Negative control
was run in parallel where the pUC18 was treated only with
damaging or oxidative substances. The reaction mixtures
were loaded on 0.8% agarose gel and electrophoresis was
carried for 1 h prior to staining with ethidium bromide to
examine the pattern of bands.
Cytotoxic Assay
Cytotoxic effect of flavonoid extract was analyzed by com-
paring it with mitomycin C, through brine shrimp lethality
assay [22, 23]. In separated glass tubes 10 hatched brine
shrimp larvae were added to 5 ml artificial seawater. Test
compounds ranges from 5 to 20 μg/μl were added and the
Fig. 1  Phylogenetic analysis
of UV-resistant bacterial strain
TMC-6 by Neighbor-Joining
method with an evolutionary
analysis conducted in MEGAX.
Sequence variation of nucleo-
tides bar 0.010
13
A. A. Chaudhri et al.
mixture was incubated under light at room temperature.
Cytotoxic effect of the samples was analyzed by counting
the number of viable larvae for every 24 h up to 3 days.
Results
Identification of Strain TMC‑6
The strain TMC-6 is a non-motile, rod shaped, Gram-pos-
itive spore forming bacterium with yellowish-orange col-
oration on TGY agar. The strain showed tolerance to NaCl
up to 8% and is positive for catalase, oxidase, and amylase.
16S rRNA gene sequence analysis revealed that the strain
TMC-6 showed maximum alignment with Bacillus sp. The
nucleotide sequence of the strain was submitted to NCBI
GenBank with accession number MN721293. The phylo-
genetic tree was constructed following Neighbor-Joining
approach, which showed 99.9% homology of strain TMC-6
(MN721293) with Bacillus indicus ­CJY07T (MN177175)
(Fig. 1).
Strain TMC-6 when treated with different metal concen-
trations showed sensitivity to Mercury ­(Hg2+) even at least
concentration used (150 ppm). The strain was observed to
be resistant to cobalt ­(Co2+), zinc ­(Zn2+), nickel ­(Ni2+), man-
ganese ­(Mn2+) and ferrous (­ Fe2+) ions at concentration of
500 ppm, which can be related to UV-B radiation resistance.
An increase in pigmentation was observed in M ­ n2+ and C ­ o2+
supplemented plates.
Bacterial Survival Against UV‑B, Oxidative Stress
and Mitomycin C
Survival rate of strain TMC-6 was observed in comparison
to E. coli strain (ATCC 10536) against different doses of
UV-B, ­H2O2 and mitomycin C concentrations. The sur-
vivability of strain TMC-6 was 54% at the energy dose of
Antioxidative and Radioprotective Properties of Glycosylated Flavonoid, Xanthorhamnin…
5.4 × 103 J/m2 of UV-B radiations. Survival rate against
­H 2O 2 decreased with increase in H ­ 2O 2 concentration,
about 58% survivability was observed at 30 mM concen-
tration of H
­ 2O2 for 60 mins and 51% survivability at 6 μg/
ml of mitomycin C for 20 mins was observed. However,
E. coli ATCC strain represented negligible survivability
at these doses and concentrations (Fig. 2).
Fig. 2  % Survivability of Bacillus indicus strain TMC-6 under expo-
sure of (a) UV-B radiations (b) ­H2O2 and (c) mitomycin C in correla-
tion to E. coli ATCC strain (10536)
Characterization of Purified Metabolic Extract
TLC Analysis
A yellow pigmented compound was obtained upon purifica-
tion of cellular extract by solid phase extraction (SPE) C18
cartridge. TLC analysis showed two spots with antioxidant
property against DPPH spray with Rf values 0.25 and 0.56,
respectively.
The UV/Vis spectrum of the extracted compound when
dissolved in methanol showed predominant peaks at 350 nm,
440 nm and 450 nm.
FT‑IR Spectroscopy
FT-IR analysis showed peaks at 3344 cm−1 that corresponds
to stretching of hydroxyl (-OH) groups. While peak at
1564 cm−1 represents C=C– groups of aromatic ring bend-
ing. A peak at 1462 cm−1 exhibits –CH3,–CH2–,CH– bonds
that can be related to alkanes, flavonoids, and aromatic rings
with –CH– bending. Peaks at 1392 cm−1 and 1087 cm−1
predict the vibrations of –C=O– of carbonyl groups and
–C–O–C– of ethers, respectively, that could be present in
flavonoids, polyphenols and others (Fig. 3).
LC–MS Analysis
The extracted bioactive metabolite was subject to LC–MS
for mass identification through electrospray ionization mass
spectrometry (ESI–MS). The purified extract gave signals
in negative mode at m/z 769 [M+H]− (Fig. 4), indicating
our extract as a glycosylated flavonoid particularly flavo-
noid 3-O- glycosides [24]. As per web data bases, Mass-
Bank [25]; ResPect [26]; HMBD [27] and PubChem [28],
the compound was identified as Xanthorhamnin (7-Methyl
quercetin-3-Galactoside-6″-Rhamnoside-3‴-Rhamnoside)
with chemical formula ­C34H40O20.
In vitro Bioassays of Extracted Flavonoid
Radical Scavenging Activity by DPPH Assay
The concentration dependent DPPH radical scavenging
activity of xanthorhamnin was detected by disappearance
of violet color of DPPH. Maximum scavenging activity
(77%) of xanthorhamnin was observed at 50 μg/μl. While
the standard ascorbic acid showed a maximum activity of
81% at 50 μg/μl (Fig. 5).
Iron Chelation Assay
Iron (Ferrous ion) chelation activity of purified xanthor-
hamnin was observed by disappearance of dark color of
13
 A. A. Chaudhri et al.

Fig. 3  FT-IR spectrum of xanthorhamnin from Bacillus indicus strain TMC-6

Fig. 4  LC–MS chromatogram/negative electrospray ionization mass spectrometry (ESI–MS) spectrum of Xanthorhamnin exhibiting high signal
at m/z 769 [M+H]−

ferrozine. A significant percentage of ferrous ion chela-

tion (79%) by the extracted flavonoid xanthorhamnin
was observed at concentration 50 μg/μl when compared
to standard EDTA (10–30 µg/µl) showing 89% chelation
(Fig. 6).

Protein and Lipid Oxidation Inhibition Assay

The protein and lipid oxidation inhibition assays were per-

Fig. 5  Concentration dependent DPPH scavenging activity of xan-
thorhamnin with ascorbic acid as positive control. The bars show
mean ± SD
formed to evaluate oxidation prevention activity of xan-
thorhamnin. The maximum inhibition of oxidative dam-
age to protein and lipid was observed as 65% and 72%,
respectively, at 50 μg/μl of xanthorhamnin in comparison
to ascorbic acid showing 80% and 70% inhibition, respec-
tively (Fig. 7).

13
Antioxidative and Radioprotective Properties of Glycosylated Flavonoid, Xanthorhamnin…
Fig. 6  Concentration dependent Iron (Ferrous ion) chelation by
xanthorhamnin using EDTA as positive control. The bars show
mean ± SD
Fig. 7  Concentration dependent protein carbonylation and lipid per-
oxidation inhibition activity of xanthorhamnin using bovine serum
albumin and mice lipids as test samples using ascorbic acid (50 µg/µl)
as positive control. The bars show mean ± SD
Cytotoxic Assay
Cytotoxic effect of xanthorhamnin was determined by per-
forming brine shrimp cytotoxic assay and compared it with
an anticancer agent Mitomycin C. The cytotoxicity of xan-
thorhamnin was concentration dependent, at 20 μg/μl maxi-
mum cytotoxicity (70%) was observed as compared to stand-
ard which showed (100%) cytotoxicity at 20 μg/μl (Fig. 8).
DNA Damage Prevention Assay
The prevention of DNA damage through extracted xanthor-
hamnin was detected by treating a plasmid pUC18 with
oxidative agents. The DNA strands were broken down by
the hydroxyl radical (⋅OH) generated through Fenton reac-
tion, as observed in lane NC (negative control). In lane PC,
Fig. 8  Percentage survivability of brine shrimp larvae against xan-
thorhamnin and positive control (mitomycin C) after 72 h of incuba-
tion. The bars show mean ± SD
Fig. 9  DNA damage preven-
tive assay. Lane P.C is positive
control containing pUC18. Lane
N.C is negative control without
antioxidant. Lane FLV contains
pUC18 treated with xanthor-
hamnin
untreated DNA was loaded. While lane A indicates DNA
damage prevention by the flavonoid xanthorhamnin (Fig. 9).
Discussion
The objective of this study was to assess the bioactive
compounds (xanthorhamnin) from UV-resistant Bacillus
indicus TMC-6 isolated from the desert soil of Pakistan
that may facilitates the survivability of the organism.
Bacillus indicus TMC-6 was found to produce xanthor-
hamnin, a yellow pigmented glycosylated flavonoid with
3-O glycosylation of quercetin which is methylated at 7
position of the benzene ring. Through FT-IR analysis, the
obtained peaks confirmed the benzene rings bending along
with the methyl groups and hydroxyl groups. Furthermore,
by elemental analysis through ESI–MS the compound pro-
duced signals of a molecular ion at m/z 769. The isolate
showed high resistance to UV radiations and other oxida-
tive stresses (mitomycin C and ­H 2O 2), probably due to
the high production of ROS quenching enzymes catalase
and peroxidases [29]. The production of flavonoid also
provides antioxidant properties to the isolate against such
stresses [30]. Manganese accumulation and antioxidants
13

presence might be two interesting mechanisms of the strain
TMC-6 for the survival under high UV radiations and salt
concentrations, as ­Mn2+ are reported to prevent the cells
from oxidative damage by blocking the Fenton reactions
and by quenching super oxides. The antioxidant and pro-
tein, lipid peroxidation prevention assay of the extracted
flavonoid xanthorhamnin reveals a significant antioxidant
property which is a substantial property of flavonoids. Free
radical scavenging through high ­Fe2+ chelation activity
supports the ability of xanthorhamnin to scavenge unstable
free radicals. It indicates that these extremolytes can be
useful as therapeutic agents against the diseases associ-
ated with free radical production. Reduced ability of gly-
cosylated flavonoid to bound with protein diminished the
property of protein oxidation prevention as determined
through the protein carbonylation inhibition assay [31].
On the other hand, glycosylation increases the solubility
of the compound within the cell, the deglycosylation of
flavonoids, as previously mentioned in studies, occurs in
the GIT of human body where the aglycone of this fla-
vonoid can present more enhanced activities [32]. The
hydroxyl moieties on the benzene rings are responsible
for the scavenging of the ROS produced due to any exter-
nal stimulus, thus prevent lipid and protein peroxidation.
The free radical chelation by the flavonoids also enhances
the oxidative damage prevention of the bio-molecules
[33]. Xanthorhamnin showed effective preventive action
against the oxidative agents and prevented the DNA dam-
age, thus confirming its antioxidative and DNA protective
properties. Many of the flavonoids and other active natural
compounds like quercetin can directly interact with telom-
erase sequences of human DNA to stabilizes G-quadruplex
structure [34]. One of the two possible mechanisms to
prevent DNA damage is the formation of DNA duplex as
a result of flavonoid-DNA binding, ultimately protecting
the oxidative damages [35]. The other possible mecha-
nism is the quenching of superoxide radicals generated by
­Fe2+ ­(FeSO4) in Fenton pathway thereby block the forma-
tion of 8-oxo-2-deoxyguanosine during the oxidation of
DNA under stress conditions [5] Cytotoxic effect on brine
shrimp larvae at higher concentration defines the poten-
tial of xanthorhamnin as anticancer agent, which could be
further evaluated with anticancer activities on malignant
human cell lines [30].
Based on these results, we conclude that xanthorhamnin
from newly isolated Bacillus indicus strain TMC-6 contrib-
ute significantly in the resistance of the microorganism from
UV radiation induced oxidative stress and protects the pro-
tein, DNA and cellular lipids from oxidative stress. Further
investigations are required for the identification of biosyn-
thetic pathway of this flavonoid for enhanced production and
intensified bioactivities through metabolic engineering.
13
A. A. Chaudhri et al.
Compliance with ethical standards
Conflicts of interest All the authors declare no conflicts of interest as-
sociated with the contents of this article.
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