Biotransformation of newly synthesized coumarin derivatives by Candida albicans as potential antibacterial, antioxidant and cytotoxic agents

Article abstract of DOI:10.1016/j.procbio.2019.08.024

In this study, one bioactive coumarin analog was obtained as a result of biotransformation of three inactive coumarin derivatives by free cells of Candida albicans. The bioactive analog was purified by Column chromatography and HPLC. The presence of coumarin moiety in the biotrasformed product was confirmed by λ_max at 350–400 nm and FT-IR spectrum. The structure of the purified compound established by LC–MS and ¹H NMR suggests the chances of biotransformation of 7-(3-(Cyclopropylamino)-2-hydroxypropoxy)-4-methyl-2H-chromen-2-one (MW 289 Da) into 7-(3-Cyclopropylamino-2-hydroxy-propoxy)-4-methoxymethyl-chromen-2-one or 7-(3-Cyclopropylamino-2-methoxy-propoxy)-4-hydroxymethyl-chromen-2-one as a main product (MW 318 Da). The extra peak of 332 Da in LC–MS further confirms the presence of small proportion of 7-(3-Cyclopropylamino-2-methoxy-propoxy)-4-methoxymethyl-chromen-2-one apart from the main product. Oxidation followed by methylation reaction might be responsible for this conversion. The biotransformed product showed antimicrobial activity against Bacillus pumilus, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae and Salmonella typhi followed by decent antioxidant activity (6.756 μg IC₅₀). The efficacy of coumarin-analog on cellular proliferation was found at 40 μM concentration against human breast cancer MDA-MB-231 cells in MTT assay, which is insignificant against normal breast tissue MCF-10A cells at the same concentration. These findings suggest the potential use of C. albicans for achieving pharmacologically active coumarin analogs showing antibacterial, antioxidant and cytotoxic activity.

Full text of DOI:10.1016/j.procbio.2019.08.024

Process Biochemistry xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Biotransformation of newly synthesized coumarin derivatives by Candida
albicans as potential antibacterial, antioxidant and cytotoxic agents
a,1
b,1
a,⁎
c
d
Ambreen , Shafiul Haque , Vineeta Singh , Diksha Katiyar , Mohd Tariq Ali Khan ,
e
f,g
e
a,⁎
Vikash Tripathi , Hesham El Enshasy , Mukesh Pasupuleti , Bhartendu Nath Mishra
Department of Biotechnology, Institute of Engineering and Technology, Dr. A.P.J. Abdul Kalam Technical University, Lucknow, 226021, Uttar Pradesh, India
Research & Scientific Studies Unit, College of Nursing & Allied Health Sciences, Jazan University, Jazan, 45142, Saudi Arabia
Department of Chemistry MMV, Banaras Hindu University, Varanasi, 221005, Uttar Pradesh, India
Endocrinology Division, CSIR - Central Drug Research Institute, Lucknow, 226031, Uttar Pradesh, India
Microbiology Division, CSIR - Central Drug Research Institute, Lucknow, 226031, Uttar Pradesh, India
a
b
c
d
e
f
Institute of Bioproduct Development (IBD), Universiti Teknologi Malaysia (UTM), Skudai, 81310, Johor Bahru, Johor, Malaysia
City of Scientific Research and Technological Applications, New Burg Al Arab, Alexandria, Egypt
g
A R T I C L E I N F O
A B S T R A C T
Keywords:
Bacteria
Biotransformation
Candida albicans
Mass spectrometry
Coumarin
In this study, one bioactive coumarin analog was obtained as a result of biotransformation of three inactive
coumarin derivatives by free cells of Candida albicans. The bioactive analog was purified by Column chroma-
tography and HPLC. The presence of coumarin moiety in the biotrasformed product was confirmed by λmax at
50–400 nm and FT-IR spectrum. The structure of the purified compound established by LC–MS and 1H NMR
3
suggests the chances of biotransformation of 7-(3-(Cyclopropylamino)-2-hydroxypropoxy)-4-methyl-2H-
chromen-2-one (MW 289 Da) into 7-(3-Cyclopropylamino-2-hydroxy-propoxy)-4-methoxymethyl-chromen-2-
one or 7-(3-Cyclopropylamino-2-methoxy-propoxy)-4-hydroxymethyl-chromen-2-one as a main product (MW
318 Da). The extra peak of 332 Da in LC–MS further confirms the presence of small proportion of 7-(3-
Cyclopropylamino-2-methoxy-propoxy)-4-methoxymethyl-chromen-2-one apart from the main product.
Oxidation followed by methylation reaction might be responsible for this conversion. The biotransformed
product showed antimicrobial activity against Bacillus pumilus, Staphylococcus aureus, Bacillus subtilis, Escherichia
coli, Klebsiella pneumoniae and Salmonella typhi followed by decent antioxidant activity (6.756 μg IC50). The
efficacy of coumarin-analog on cellular proliferation was found at 40 μM concentration against human breast
cancer MDA-MB-231 cells in MTT assay, which is insignificant against normal breast tissue MCF-10A cells at the
same concentration. These findings suggest the potential use of C. albicans for achieving pharmacologically
active coumarin analogs showing antibacterial, antioxidant and cytotoxic activity.
1. Introduction
Therefore, microbial transformation can make reactions feasible, which
are not likely to be carried out by traditional synthetic procedures.
Biotransformation is an emerging tool of green chemistry and has
Microbial modifications of chemical compounds can be done by oxi-
dation, reduction, hydrolysis, condensation, isomerization reactions,
formation of the new C]C double bonds etc.[4].
Enzymes produced during microbial metabolic activities are gen-
erally involved in the bioconversion of organic compounds. As these
biocatalysts are highly specific for their activity, hence regio-specific or
enantiomer-specific bioconversion leads to the formation of a specific
product. Bioconversion process facilitated by microbial cells and en-
zymes has proven to be superior compared to chemical synthesis and
other conventional methodologies as it can incorporate stereospecificity
increasing recognition in the chemical and pharmaceutical sector for
the conversion of existing ineffective or less effective compounds into
new or useful products [1,2]. The term biotransformation can be de-
fined as a specific modification of a defined compound to a defined
product with structural similarity by using biological catalysts [3]. A
biological catalyst can be an enzyme, free cells or system. Micro-
organisms are capable of producing various enzymes in a short period
of time because of their natural character to multiply and they can also
survive under extreme environments of temperature and pH conditions.
⁎
Corresponding authors.
E-mail addresses: vscdri@gmail.com (V. Singh), profbnmishra@gmail.com (B.N. Mishra).
Ambreen and Shafiul Haque contributed equally to this work.
1
https://doi.org/10.1016/j.procbio.2019.08.024
Received 19 February 2019; Received in revised form 24 July 2019; Accepted 30 August 2019
1359-5113/ © 2019 Published by Elsevier Ltd.
Please cite this article as: Ambreen, et al., Process Biochemistry, https://doi.org/10.1016/j.procbio.2019.08.024

Ambreen, et al.
Process Biochemistry xxx (xxxx) xxx–xxx
or reduce the complexity of the purification steps [5]. Besides this, the
superiority of microbial transformation over other accounts is basically
due to greater surface-volume ratio, high growth rate, sterility main-
tenance, which is comparatively easier than plant or animal cell culture
and above all they possess a high rate of metabolism for efficient
transformation of substrate added [6].
synthesized coumarin derivatives were: C1: 7-(3-(cyclopropylamino)-2-
hydroxypropoxy)-2H-chromen-2-one; C2: 7-(3-(butylamino)-2-hydro-
xypropoxy)-2H-chromen-2-one; C3: 7-(3-(cyclohexylamino)-2-hydro-
xypropoxy)-2H-chromen-2-one; C4: 7-(2-hydroxy-3-morpholinopro-
poxy)-2H-chromen-2-one;
C5:
7-(3-(cyclopropylamino)-2-
hydroxypropoxy)-4-methyl-2H-chromen-2-one; C6: 4-(2-hydroxy-3-
morpholinopropoxy)-2H-chromen-2-one.
In the present work, coumarins have been selected as a parent
molecule, which are non-toxic and non-inhibitory in nature against the
biotransforming organism. Coumarins are chemically known as ben-
zopyran-2-one or chromen-2-one or simply benzo-α-pyrones ring
system. Coumarins can be synthesized via.chemical route or are widely
available in Umbelliferae, Rutaceae and other plant families [7]. These
coumarins have been reported to possess anti-inflammatory, anti-al-
lergic, antioxidant, antiviral, hepatoprotective, antithrombotic, and
anti-carcinogenic activities. There are reports of profound use of cou-
marins in photochemotherapy, anti-HIV therapy and stimulants of CNS,
and apart from this they are also active as anti-coagulant, estrogenic,
vasodilator, molluscacidal, anti-helminthic, sedative, analgesic, dermal
photosensitizing, antimicrobials and possess hypnotic and hypothermic
activity [1,7]. In the present study, the practicability of bio-
transformation of newly synthesized inactive coumarin derivatives into
biologically active coumarin-analogs via.free cells of C. albicans was
evaluated followed by purification, characterization and prospection of
the transformed product for antibacterial, antioxidant and cytotoxic
activity.
2
.2. Activity profiling of coumarins
The antibacterial activity of coumarins was examined against Gram-
positive Bacillus pumilus MTCC 1604, Bacillus subtilis MTCC 441, and
Staphylococcus aureus MTCC 537 and Gram-negative Escherichia coli
MTCC 1304, Klebsiella pneumonia MTCC 3384, and Salmonella typhi
MTCC 537 bacteria. The bacterial cultures were maintained on nutrient
agar (NA) at 4 °C. The working cultures were prepared by inoculating
the sterile NB medium with the overnight grown test strains until the
solution turbidity reached 0.5 McFarland standards. The stock solutions
of the synthesized coumarin derivatives were prepared in methanol.
The activity profile of 200 μg of coumarin compounds against bacterial
and fungal strains was determined by agar plate method [10]. The zone
of inhibition (zoi; measured in mm) was recorded after 24 h for all the
bacterial strains. Drugs, like novobiocin and vancomycin were used as
antibacterial drug standards, whereas, amphotericin B was used as an
antifungal drug standard.
2. Materials and methods
2.3. Biotransformation by free cells method
2
.1. Synthesis of coumarin derivatives
The microbial culture of C. albicans was prepared in potato dextrose
broth (PDB) after 48 h of growth and used for biotransformation stu-
dies. Biotransformation was carried out by the free cells of C. albicans
with the starting reaction mixture of 1 mg coumarin compounds, 1 ml of
The chemicals used in this work were of analytical grade and purity.
Coumarinyl amino alcohols C1–C6 were synthesized following the
method reported earlier [8,9]. The reaction of 7-hydroxycoumarin with
5
free fungal cells (5 × 10 CFU/ml) and 10 ml of trisodium citrate
epichlorhydrin in the presence of K
2
CO
3
led to the formation of oxirane
buffer. The reaction mixture was incubated for at 32 °C for 48 h at
120 rpm. After 48 h, the reaction mixture was withdrawn and cen-
trifuged at 8000 rpm at 4 °C for 10 min to obtain the supernatant. The
aqueous supernatant was extracted with ethyl acetate and the organic
solvent was concentrated to dryness under vacuum condition in a rotary
evaporator (Buchi labs, Switzerland) to obtained the biotransformed
product. The activity of the biotransformed product against the test
strains was checked by the disc diffusion method. Bioconversion was
further evaluated by thin layer chromatography (TLC) using hexane:
methanol (8:2, v/v) as a solvent system and developed it in iodine
derivative, which on regioselective nucleophilic ring opening with cy-
clopropyl amine, butyl amine, cyclohexyl amine and morpholine in the
presence of ethanol at room temperature afforded coumarinyl amino
alcohols 1–4, respectively, with good yield (Fig. 1). The coumarin
compound 5 was synthesized via.nucleophilic opening of oxirane ring
by cyclopropyl amine. Oxirane was synthesized by the reaction of 7-
hydroxy-4-methyl-coumarin with epichlorohydrin. Whereas, the
synthesis of oxirane intermediate was performed by the reaction be-
tween 4-hydroxy coumarin and epichlorohydrin followed by its nu-
cleophilic ring opening by morpholine and yielded coumarin compound
C6 in good amount. The spectral data of all the synthesized compounds
were in good agreement with the previously reported data. The
f
chamber. R value of the substrate and the product was calculated as the
ratio of distance traveled by the compound to the distance traveled by
the solvent.
Fig. 1. Chemical synthesis scheme used for coumarinyl amino alcohols showing the reaction conditions required for the formation of the reaction intermediates.
2

Ambreen, et al.
Process Biochemistry xxx (xxxx) xxx–xxx
2
.4. Purification and characterization
cells (200 μL) were seeded in a 96-well plate and different concentra-
tions (1, 2, 5, 10, 20, 40 and 80 μM) of coumarin derivatives were
added in triplicate. DMSO (0.1%) and centchroman (10 μM) were used
as negative and positive control, respectively. All the group cells were
treated with 0.2% DMSO in triplicate and incubated for 24 and 48 h.
Following the incubation, the experiment was terminated by adding
20 μL of MTT (stock: 5 mg/ml of MTT in PBS, Life technologies catalog
M6469) in each of the treatment as well as the control well. Further, the
The biotransformed product was purified by column chromato-
graphy using silica gel (mesh size 230–400) column (1.8 × 70 cm) as a
packing material. The column was eluted under an isocratic condition
with ethyl acetate and 5 ml fractions were collected. The collected
fractions were dried and checked for antimicrobial activity against
Bacillus pumilus MTCC1607, Staphylococcus aureus MTCC 902, Bacillus
subtilis MTC441, Escherichia coli MTCC 1304, Salmonella typhi MTCC
2
plate (96-well) was incubated in 5% CO incubator at 37 °C for 2 h.
5
37 and Klebsiella pneumonia MTCC3384 by the disc diffusion method.
Afterwards the plate was observed under the microscope to observe the
formation of formazan crystals. The medium from the wells was dis-
carded and 200 μL of DMSO was added in each well. The 96-well plate
was kept on shaker for 10 min for dissolving the crystals in DMSO. The
absorbance was recorded at 595 nm for each well of the plate using
iMARK microplate reader (Bio-rad, Hercules, CA, USA). The data ob-
tained were expressed as percent proliferation compared to the control
and graphs were plotted.
The purity of the active fractions was tested by using HPLC system
(
(
Waters) equipped with 515 HPLC binary pump, 2998 Photodiode array
PDA) detector, and 2707 autosampler. HPLC separation was achieved
on a reverse phase, reverse phase silica column (RP18) with a mobile
phase consisting of the water-acetonitrile gradient at a flow rate of
1
ml/min. Further, the detailed structure of the pure product was es-
tablished by UV–vis spectroscopy, LC–MS, IR, and NMR.
2.5. Biological activity of transformed product
2.7. Statistical analysis
The antimicrobial activity of the biotransformed coumarins ex-
Student t-test was used to calculate the significance of the data
obtained from MTT assay. In t-test, the control was compared with the
treated values and also among the pairs. All the significant analysis was
done for triplicate values. Where * shows significant (p < 0.05), **
very significant (p < 0.001) and *** highly significant (p < 0.0001).
pressed in terms of minimum inhibitory concentration (MIC) values was
determined by micro-broth dilution method as described by Priyanka
et al. [9]. Stock solutions were prepared as 1 mg/ml concentration of
the biotransformed coumarin derivatives and standard antibiotics, i.e.,
Novobiocin and Vancomycin. The antioxidant activity of the bio-
transformed compound was analyzed using DPPH (2,2-diphenyl-1-pi-
cryl-hydrazyl-hydrate) free radical scavenging assay with minor mod-
ifications [11]. Different concentrations of the coumarin compounds in
ethanol (222.5–2225 μg in 100 μl) were taken in test tubes and 2.9 ml of
DPPH solution (2.5 mg/100 ml of ethanol) was added. The reaction
mixture was vortexed and incubated for 30 min in dark and absorbance
was measured at 520 nm by using spectrophotometer (UV–vis Shi-
madzu). The reaction mix without compound was considered as a
control standard. Ascorbic acid was used as a reference compound and
the experiment was performed in triplicate. The percentage scavenging
effect of DPPH of the tested sample was calculated using the following
formula:
3. Results
3.1. Antimicrobial activity of the substrate/product
Six coumarin derivatives were evaluated for their antimicrobial
activity against the bacterial test strains by measuring the zone of in-
hibition with a concentration of 200 μg. The results of antibacterial
testing indicate that the synthesized coumarin compounds possessed
little antimicrobial potential, with the highest zoi of 12 mm only in the
case of compound C1 against S. typhi (data not shown). From anti-
microbial activity profile of the synthesized coumarin series, the most
inactive derivatives C4–C6 were selected for the biotransformation
purpose. Biotransformation studies were performed for the above
mentioned three compounds using free cells of C. albicans, however,
only compound number C5 was found active. The biotransformed
product was tested against the same group of bacteria and the results of
MIC values expressed in μg/ml of the biotransformed compound C5-P
Percentage scavenging effect of DPPH or % inhibition = (A
) × 100
0
1
̶ A /
A
0
Where, A
0 1
is the absorbance of control reaction and A is the absor-
bance of the test of the standard sample.
(
7-(3-(cyclopropylamino)-2-hydroxypropoxy)-4-methyl-2H-chromen-2-
2
.6. Cytotoxicity assay
one) and the standard drugs are summarized in Table 1. The results
indicated that the biotransformed coumarin derivative is capable of
inhibiting both Gram-positive and Gram-negative bacteria.
MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bro-
mide) assay was performed against human breast carcinoma cell lines
MDA-MB-231) following the protocol as reported by Srisawat et al.,
(
3.2. Antioxidant activity
with minor modifications [12]. MDA-MB-231 cells were maintained in
DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FBS
The results of DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free
radical scavenging experiment suggest that the biotransformed product
possess good antioxidant properties (Table 2). The experimental re-
gression value 0.98 further emphasized the modest antioxidant activity
(
Fetal Bovine Serum) and incubated in a CO
2
incubator for 24 h at
3
7 °C. When the cells reached a confluency of 40–50%, the complete
3
media was replaced with fresh medium. Around 5 × 10 MDA-MB-231
Table 1
Antimicrobial activity profile in terms of MIC values (μg/ml) of coumarin derivatives.
Test strains (MTCC)
C1
C2
C3
C4
C5
C6
C5-P
N
V
Bacillus subtilis MTCC 441
200
> 200
200
100
> 200
100
> 200
> 200
200
100
> 200
200
> 200
> 200
> 200
200
200
200
> 200
> 200
> 200
200
> 200
> 200
> 200
> 200
> 200
> 200
200
> 200
100
> 200
> 200
> 200
> 200
100
25
50
100
100
50
6.25
6.25
6.25
25
25
25
1.56
1.56
1.56
0.35
0.35
R
Bacillus pumilus MTCC 1604
Staphylococcus aureus MTCC 537
Salmonella typhi MTCC 537
Klebsiella pneumonia MTCC 3384
Escherichia coli MTCC 1304
> 200
C1-C6: Coumarin derivatives; C5-P = biotransformed product; N = Novobiocin; V = Vancomycin; R = Resistant.
3

Ambreen, et al.
Process Biochemistry xxx (xxxx) xxx–xxx
Table 2
Percentage DPPH scavenging effect against varying concentrations of the bio-
transformed coumarin derivative.
S. No.
Concentration of
DPPH scavenging
Ascorbic acid DPPH
biotransformed coumarin
effect (% inhibition)
scavenging effect (%)
(μg)
1
2
3
4
5
250
500
750
1000
1250
27.5
43.5
63.5
70..5
89.5
30
51
72
78
91
of the biotransformed coumarin derivative. Effective concentration
EC50 value demonstrated the concentration of coumarin-derivative re-
quired to scavenge 50% of DPPH free radical. In the performed ex-
periment, the EC50 value was 6756.76 μg, means 6.7 mg of the bio-
transformed product of coumarin derivative is effective in showing
antioxidant activity.
3.3. Cytotoxicity
Fig. 3. Effect of biotransformed coumarin derivative on cellular proliferation
against normal human breast MCF10A cells at different time points (a) 24 h and
at (b) 48 h. Note: ns: non-significant.
The efficacy of the biotransformed coumarin derivative was studied
in a dose-dependent (1, 2, 5, 10, 20, 40 and 80 μM) and time-dependent
manner (24 and 48 h) against cellular proliferation of human breast
MDA-MB-231 cancer cells using MTT assay (Fig. 2). The inhibition of
cellular proliferation by the purified biotransformed compound was not
very significant at the lower concentration, i.e.,up to 10 μM. However,
from 20 μM onwards it was found significant, when compared with the
control under t-test analysis. With the increase in the compound con-
centration, the inhibition of cellular proliferation increased and at
0 μM concentration showed maximum inhibition (p < 0.0001). IC50
value, which depicts 50% inhibition of the cell proliferation, was found
to be at 54 and 40.08 μM of the biotransformed coumarin derivative at
4 and 48 h time points, respectively. The substrate coumarin at a
concentration of 80 μM was able to inhibit only 35 and 40% cellular
proliferation at 24 and 48 h, respectively. Centchroman (CC) was used
as a positive control and the IC50 value of CC was 15 and 10 μM at 24
and 48 h, respectively in MDA-MB-231 cells. The results of 24 and 48 h
treatment suggest that with the increase in time interaction, the effec-
tiveness of the compound increases.
The above experiment was compared by studying the efficacy of the
biotransformed coumarin derivative on cellular proliferation against
human normal breast MCF10A cells using MTT assay for the cytotoxi-
city under the same experimental conditions of dose and time de-
pendency. The compound had no or insignificant influence (p > 0.05)
over cellular proliferation from 10 to 40 μM as they increased cellular
proliferation by 1–9% in 24 h and 9–14% in 48 h. The maximum
experimental dose, i.e.,80 μM was found to show 18% cellular inhibi-
tion.
At the concentration of 40 μM, the biotransformed product inhibited
cellular proliferation by 9 and 14% at 24 and 48 h incubation, respec-
tively; and 18% inhibition at 80 μM concentration; out of which, only
48 h incubation was found significant. Whereas, the original product
(i.e., substrate) inhibited 6 and 2% cells at 80 μM concentration. It is
clear from MTT cytotoxicity assay that biotransformed product has no
cytotoxic effect on human normal MCF10A cells between 10–40 μM
concentration at 24 h and 48 h, respectively, and can be further ex-
plored as anti-cancer compound (Fig. 3).
8
2
3.4. Chemical characterization of the substrate/biotransformed product
The absorption bands observed at 1716 and 3422 cm1 in IR spec-
trum indicated the presence of δ lactone and hydroxyl functions, re-
spectively. The ESI MS spectrum of the coumarin compound showed a
+
I
peak at m/z 290 corresponding to its [M+H] . In H NMR spectrum
signals corresponding to H-5 of benzopyrone ring appeared as doublet
at δ 7.25 with J value of 8.8 Hz, while H-6 and H-8 were observed as
multiplet at δ 6.81. H-3 was observed as singlet at δ 6.12. However,
2
signals for propan-2-ol linker appeared as multiplets at δ 4.10 (C(1′)H ,
Fig. 2. Effect of biotransformed coumarin derivative on cellular proliferation against human breast cancer MDA-MB-231 cells at different time points (a) 24 h and at
(b) 48 h. Note: ns: non-significant; * less than control; ** equal to control; *** greater than control, i.e., centchroman.
4

Ambreen, et al.
Process Biochemistry xxx (xxxx) xxx–xxx
NH, C(2′)H) and 3.12 (C(3′)H
2
). The methylene protons of cyclopropyl
of benzene moiety in the structure; the signals at 0.30 marks the pre-
sence of cyclopropane ring; at 1.93 (t, 3H) shows CH region, 7.58 (d,
ring appeared as multiplets at δ 1.26 and 0.83. However, methine
proton of cyclopropyl ring was observed as multiplet at δ 2.42 merged
4H) denotes CH
confirming the presence of eNH and eCH; signals at 3.9 confirms the
presence of eOH and eCH bonding; signal at 4.04 confirms CH
bonding; at 3.24 it is CH bonding and OH shares peak at 2.0.J coupling
2
bonding; whereas at 2.7 multiple peaks are observed
with signal for methyl protons of the 4-methyl substitutent. The 13
C
NMR spectrum of 5 exhibited signals at δ 102.8, 127.7, 111.7 and 107.6
corresponding to C-3, C-5, C-6 and C-8 of benzopyrone ring. The signals
for quaternary carbons C-2, C-4, C-7, C-9 and C-10 were observed at δ
2
3
region and chemical shift (δ) indicates that either modifying methyl
group or hydroxyl group in the original structure might results in the
161.8, 153.8, 157.6, 151.2 and 116.8, respectively, while signals cor-
responding to carbons of propan-2-ol linker appeared at δ 72.3, 62.8
and 49.8 for C-1′, C-2′, and C-3′, respectively. The methyl carbon of 4-
methyl substituent was observed at δ 18.7. The signal at δ 35.2 was
attributed to methine and at δ 9.3 and 7.8 were attributed to methylene
carbons of cyclopropyl ring.
final product. Therefore, oxidation at CH
oxidation at CH followed by methylation at eOH lead to the molecular
weight of 318 Da, whereas oxidation of CH followed by methylation of
CH and eOH both results in a molecular weight of 332 Da (Fig. 6).
3
followed by methylation or
3
3
3
The efficiency of biotransformation reaction was found to be 64%,
i.e., biotransformation of 100 mg of coumarin-derivative resulted 64 mg
of the pure product. The product was oily in nature and showed solu-
bility in DMSO. The process of bioconversion held was confirmed by
thin layer chromatography showing spots with different Rf value than
the substrate. The complete structure identification of the bio-
transformed compound was done on the basis of spectral studies. The
absorbance in the UV region for the substrate and product was 350 nm
and 400 nm, respectively (Fig. 4a) falling in the range (between
4
. Discussion
The ability of biocatalysts to selectively produce useful products
under relatively mild conditions compared to its chemical catalyst
counterpart make biocatalysts an interesting and powerful tool.
Biocatalyst can be a free enzyme or a whole cell catalyzing a chemical
reaction and producing highly enantioselective, low toxic product [4].
Recent technology advancements have increased the focus of industry
to discover new biocatalysts and optimize their performance. In order
to create an eco-friendly system, certain studies on the modification of
benzopyrone ring system employing different fungi and bacteria, as
functional group transferring agents, have been undertaken [1]. Cou-
marins are common example of benzopyrone ring system, and popu-
larly named as benzopyran-2-one or chromen-2-one or simply benzo-α-
pyrones ring system. Coumarins are one of the most common phyto-
chemicals present in various natural products. Due to its diverse
bioactivity [13] it is also synthesized chemically [14]. In this direction,
through chemical synthesis a series of coumarins derivatives (coumar-
inyl amino alcohols) have been synthesized; unfortunately, the com-
pounds of this series are not very effective in bioactivity, therefore,
biotransformation method has been used to make an effort to convert
the inactive derivative into an active one. The modifications at the
chemical level of inactive coumarins generally lead the conversion into
active structure which is mainly useful for pharmaceutical industries
and opens the door for novel drug development. In this direction, mi-
croorganisms play an important role for structural modifications [15].
Biotransformation of coumarin-derivatives have been reported in var-
ious literatures using a variety of microorganisms, like Streptomyces
griseus [16], Candida tropicalis [1], Arthrobacter species, Aspergillus niger
3
00–400 nm) of coumarins [8]. IR spectra: peaks at 1072, 1283 in the
product and 1071, 1286 in the crude corresponds to aryl-alkyl-ether
AryleOeR). The peaks, 3425 and 3013 in the product belongs to eOH,
(
eNH bonding, respectively. These similarities suggest that the side
chain present in the product is intact as same as in the crude; a peak at
2
925 in the product corresponds to eCH stretching region; The ab-
−1
sorption bands observed at 1716 and 3422 cm
in the substrate and
product indicate the presence of δ lactone and hydroxyl functions, re-
spectively, which further confirm the intact lactam ring. As, benzene is
a stable structure in comparison to lactam ring, hence the chances of
group modification at the side chain or at pyrone ring are speculated
(
Fig. 4b).
In ESI-MS the major compounds corresponding to the retention time
.41, 9.64 and 9.99 possess molecular weight 290.3, 318.3 and 332 Da,
8
respectively (Fig. 5). Mass distribution from LC–MS predicts the product
mass to be 318 or 332 at a retention time of 9.64 and 9.99 respectively
(
Fig. 5). ESI-MS also establishes the distinct molecular weight of 318; it
is certain that the main product corresponds to the molecular weight
3
3
18 with the smaller fraction being converted to the molecular weight
32. While, FT-IR suggests that the major functional groups and ring
remained intact and provides maximum structural similarity in the
substrate and product; it is likely that there is a modification of either
[
17], Fusarium solani [18] and Cunninghamella elegans [19].
The structure of any molecule plays an important role in order to
3
eOH, eCH , eNH group (Fig. 4b).
reflect its biological activity. Kayser and Kolodziej [20] reported that
the addition of eOH moiety to coumarin core structure significantly
The presence of signals between J = 7.7–7.0 confirm the presence
Fig. 4. (a) UV absorption pattern of the biotransformed product C5-P: showing absorbance near 400 nm; (b) FTIR of the biotransformed product C5-P: showing the
presence of basic functional groups of coumarin derivatives.
5

Ambreen, et al.
Process Biochemistry xxx (xxxx) xxx–xxx
Fig. 5. Mass spectra of the substrate/product reflecting variations in the mass of the substrate and the product endorsing biotransformation process.
Fig. 6. 1H NMR of the biotransformed product C5-P confirms the presence of benzopyrone and cyclopropyl ring.
6

Ambreen, et al.
Process Biochemistry xxx (xxxx) xxx–xxx
reduced the antibacterial activity against all the tested bacteria. Their
findings suggest that the antibacterial activity of oxygenated coumarins
apparently depends on the position of polar (eOH) and less polar (Me/
OMe) functional group at the aromatic nucleus of the coumarin struc-
ture. This assumption match with the present study proving the last
three compounds (C4–C6) from the series were most inactive against a
group of tested bacteria. The addition of methyl group further depicted
fairly low activity of the compound C5 against all the tested strains. The
active compounds favor its permeation more efficiently through the
lipid layer of the fungi [21].
Acknowledgements
The authors are grateful to IET, Lucknow for providing the labora-
tory facility and partial financial support under World Bank assisted
TEQIP-Government of India program-phase II for this research work.
The author, Shafiul Haque, is grateful to Jazan University, Saudi Arabia
for providing the access to the Saudi Digital library. The author,
Hesham El Enshasy would like to acknowledge the support of MOE and
UTM-RMC through HICOE grant no. R.J130000.7846.4J262.
Nigam et al. [1] reported the application of Saccharomyces griseus
and Candida tropicalis for microbial biotransformation purposes for the
modification of coumarin substrates. They reported the bio-
transformations of coumarins derivatives 7-ethoxy-4-methyl coumarin
into 7-dihydroxy-4-methyl coumarin by Saccharomyces griseus and 7-
hydroxy-4-methyl coumarin into 7-methoxy-4-methyl coumarin by
Candida tropicalis. The report of Nigam et al. [1] provides the prime
reason to examine the ability of Candida albicans for bioconversion
process. The results of bioconversion of inactive coumarin derivative
References
[1] S. Nigam, J.V. Rao, B.S. Jayashree, Microbial biotransformation—a novel approach
for modification on coumarin substrates, Ind. J. Biotechnol. 12 (2013) 379–385.
[
2] M.E. Hegazy, T.A. Mohamed, A.I. ElShamy, H.M. Abou-El-Hamd, U.A. Mahalel,
E.H. Reda, A.M. Shaheen, W.A. Tawfik, A.A. Shahat, K.A. Shams, N.S. Abdel-Azim,
Microbial biotransformation as a tool for drug development based on natural pro-
ducts from mevalonic acid pathway: a review, J. Adv. Res. 6 (2015) 17–33.
3] M.D. Lilly, Eighth P. V. Danckwerts memorial lecture presented at Glaziers’ Hall,
London, U.K. 13 May 1993: advances in biotransformation processes, Chem. Engg.
Sci. 49 (1994) 151–159.
[
(
C5) into active product suggest the efficiency of C. albicans towards
[4] F. Garzón-Posse, L. Becerra-Figueroa, J. Hernández-Arias, D. Gamba-Sánchez,
Whole cells as biocatalysts in organic transformations, Mol. 23 (2018) 1265.
biotransformation. The results regarding the structure elucidation of
the product suggest the most possible reaction of the conversion is the
[
5] E.M. Sales, T.F. Barros, E.D.S. Velozo, Biotransformation of coumarins by
Saccharomyces cerevisiae, World J. Pharm. Pharm. Sci. 3 (2014) 209–216.
[6] M.S. Smitha, S. Singh, R. Singh, Microbial Biotransformation: a process for chemical
oxidation at CH
3
moiety present on chromone ring followed by me-
alterations, J. Bacteriol. Mycol. 4 (2017) 85.
thylation at specific positions, which ultimately offer main and side
products. Methylation and oxidation are the common chemical reac-
tions involve in the mechanism of bioconversion using microbes.
Beside antimicrobial activity, the modified coumarin possessed ROS
activity and toxic activity against the breast cancer cell line MDA-MB-
[
7] M.A. Musa, J.S. Cooperwood, M.O.F. Khan, A review of coumarin derivatives in
pharmacotherapy of breast cancer, Curr. Med. Chem. 15 (2008) 2664–2679.
[8] S. Misra, L.K. Singh, Priyanka, J. Gupta, S. Misra-Bhattacharya, D. Katiyar,
Synthesis and biological evaluation of 4-oxycoumarin derivatives as a new class of
antifilarial agents, Eur. J. Med. Chem. 94 (2015) 211–217.
[
9] Priyanka, V. Singh, Ekta, D. Katiyar, Synthesis, antimicrobial, cytotoxic and E. coli
231. Previously, the toxic activity has been diligently mentioned in the
DNA gyrase inhibitory activities of coumarinyl amino alcohols, Bioorg. Chem. 71
(2017) 120–127.
work of Musa et al. [7] regarding coumarin and its active metabolite 7-
hydroxy coumarins particularly against breast cancer, which is a
leading cause of death. Musa et al. [7], have proved that bioactive
analog of coumarin works as a potent selective estrogen receptor
modulators (SERMs) as well as strong chemotherapeutic agent in breast
cancer. Few naturally occurring coumarin derivatives including um-
belliferone, warfarin, aesculetin (6,7 dihydroxycoumarin), 7-methoxy
coumarin, psoralen, and imperatorin have shown their potential in the
past. In addition, coumarins work as lipid-lowering agents and hydro-
xycoumarins are powerful chain-breaking antioxidants [22], and can
prevent free radical injury by scavenging reactive oxygen [7].
[
10] S.I.G.H.M. Montalvão, V. Singh, S. Haque, Bioassays for bioactivity screening.
Analysis of Marine samples in search of bioactive compounds, in: T. Rocha-Santos,
A. Duarte (Eds.), Comprehensive Analytical Chemistry Series, Elsevier B.V.,
2
014ISBN9780444633590.
[
11] Shekhar, C. Tailor, A. Goyal, Antioxidant activity by DPPH radical scavenging
method of Ageratum conyzoides Linn. leaves, Am. J. Ethnomed. 1 (2014) 244–249.
[12] T. Srisawat, Y. Sukpondma, P. Graidist, S. Chimplee, K. Kanokwiroon, The dose
dependent in vitro responses of MCF-7 and MDA-MB-231 cell lines to extracts of
vaticadiospyroides symington type SS fruit include effects on mode of cell death,
Pharmacogn. Mag. 11 (Suppl. 1) (2015) S148–S155.
[13] S. Tavakoli, M.R. Delnavazi, R. Hadjiaghaee, S. Jafari-Nodooshan, F. Khalighi-
Sigaroodi, M. Akhbari, A. Hadjiakhoondi, N. Yassa, Bioactive coumarins from the
roots and fruits of Ferulagotrifida Boiss. an endemic species to Iran, Nat. Prod. Res.
3
2 (2018) 2724–2728.
[
14] Priyanka, R.K. Sharma, R.J. Butcher, D. Katiyar, Facile construction of 4H-chro-
menes via Michael addition of phenols to benzylideneoxobutanoates and their
successful conversion into pyranocoumarins, Tetrahedron Lett. 59 (2018)
5. Conclusion
2
347–2351.
In the present study, biotransformation was done by oxidation fol-
lowed by methylation of 7-(3-(Cyclopropylamino)-2-hydroxypropoxy)-
-methyl-2H-chromen-2-one by Candida albicans to 7-(3-
[
[
15] J.S. doNascimento, J.C. Conceição, E. de Oliveira Silva, Biotransformation of cou-
marins by filamentous fungi: an alternative way for achievement of bioactive
analogs, Mini-Rev. Org. Chem. 16 (2019) 1–10.
4
16] F.S. Sariaslani, J.P. Rosazza, Novel biotransformations of 7-ethoxycoumarin by
Cyclopropylamino-2-hydroxy-propoxy)-4-methoxymethyl-chromen-2-
one or 7-(3-Cyclopropylamino-2-methoxy-propoxy)-4-hydroxymethyl-
chromen-2-one as main product with molecular weight 318 Da. In ad-
dition to the main peak, a small peak of molecular weight 332 Da was
also observed in the LC–MS region that indicates the presence of 7-(3-
Cyclopropylamino-2-methoxy-propoxy)-4-methoxymethyl-chromen-2-
one in addition to the main product; and this was confirmed by the
spectral analysis. Besides this, attempts were made to evaluate the
success of bioconversion of the substrate into the final product. The
biotransformed product showed significant results for its biological
activity assessed in terms of decent antibacterial, antioxidant cyto-
toxicity properties, and reflects its therapeutic use in the near future
after several in vivo trials.
Streptomyces griseus, Appl. Environ. Microbiol. 46 (1983) 468–474.
[17] C.B. Aguirre-Pranzoni, G.I. Furque, C.E. Ardanaz, A. Pacciaroni, V. Sosa, C.E. Tonn,
M. Kurina-Sanz, ARKIVOC: Online J. Org. Chem. (2011) 170–181 (vii).
[
18] H.S. Shieh, A.C. Blackwood, Use of coumarin by soil fungi, Can. J. Microbiol. 15
1969) 647–648.
(
[19] G.A. Attia, K.A. Abou-El-Seoud, A.R. Ibrahim, Biotransformation of coumarins by
cunninghamellaelegans, Afr. J. Pharm. Pharmacol. 10 (2016) 411–418.
20] O. Kayser, H. Kolodziej, Antibacterial activity of simple coumarins: structural re-
[
quirements for biological activity, Z. Naturforsch. C 54 (1999) 169–174.
[21] S.U. Rehman, Z.H. Chohan, F. Gulnaz, C.T. Supuran, In-vitro antibacterial, anti-
fungal and cytotoxic activities of some coumarins and their metal complexes, J.
Enzyme Inhib. Med. Chem. 20 (2005) 333–340.
[
22] A.A. Kadhum, A.A. Al-Amiery, A.Y. Musa, A.B. Mohamad, The antioxidant activity
of new coumarin derivatives, Int. J. Mol. Sci. 12 (2011) 5747–5761.
Declaration of Competing Interest
The authors declare no conflicts of interest.
7