Bioorganic synthesis, characterization and evaluation of a natural phenolic lipid

Article abstract of DOI:10.1016/j.btre.2019.e00375

The first synthesis of a phenolic natural monoacylglycerol (1- [11-(ferulyloxy) undecanoyl)] glycerol) was carried out by bioorganic synthesis starting from ferulic acid. The synthetic route of the target lipidic compound was designed involving a chemo-enzymatic approach using immobilized Candida antarctica lipase as biocatalyst in two of the steps conducted in organic medium. The prepared lipidic compound was characterized by using spectral data and evaluated for antimicrobial, antioxidant and cytotoxic studies to examine its potential. The synthesized compound showed moderate antimicrobial activity and showed very good antioxidant activity in DPPH radical scavenging assay and also in oxidation inhibition in soybean oil by differential scanning calorimetry. The cytotoxic studies of the synthetic lipid showed promising activity against A549 and HeLa cancer cell lines with IC₅₀ values of 9.102 and 9.886 μM respectively. The prepared compound can be useful in designing novel phenolic lipids with potential applications in cosmetic and biomedical fields.

Full text of DOI:10.1016/j.btre.2019.e00375

Biotechnology Reports 24 (2019) e00375
Contents lists available at ScienceDirect
Biotechnology Reports
journal homepage: www.elsevier.com/locate/btre
Bioorganic synthesis, characterization and evaluation of a natural
phenolic lipid
Juliya Johnya, Venkateshwarlu Konthama, Dileep Veeragonib, Sunil Misrab,
Shiva Shanker Kakia,
*
a
Centre for Lipid Science & Technology, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad, 500007, India
Applied Biology, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad, 500007, India
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 23 April 2019
Received in revised form 6 September 2019
Accepted 6 September 2019
The first synthesis of a phenolic natural monoacylglycerol (1- [11-(ferulyloxy) undecanoyl)] glycerol) was
carried out by bioorganic synthesis starting from ferulic acid. The synthetic route of the target lipidic
compound was designed involving a chemo-enzymatic approach using immobilized Candida antarctica
lipase as biocatalyst in two of the steps conducted in organic medium. The prepared lipidic compound
was characterized by using spectral data and evaluated for antimicrobial, antioxidant and cytotoxic
studies to examine its potential. The synthesized compound showed moderate antimicrobial activity and
showed very good antioxidant activity in DPPH radical scavenging assay and also in oxidation inhibition
in soybean oil by differential scanning calorimetry. The cytotoxic studies of the synthetic lipid showed
Keywords:
Phenolic lipid
Monoacylglycerol
Lipase
Antioxidant
Antimicrobial
Cytotoxicity
promising activity against A549 and HeLa cancer cell lines with IC50 values of 9.102 and 9.886
mM
respectively. The prepared compound can be useful in designing novel phenolic lipids with potential
applications in cosmetic and biomedical fields.
© 2019 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/).
1. Introduction
lipid shows that it is a monoacylglycerol derivative of ferulic acid.
This lipid was also isolated recently from the grains of Echinochloa
Natural products are a source of many complex compounds
which are ingredients in many pharmaceuticals, agrochemicals,
nutraceuticals and bioactives [1]. Among the arsenal of several
complex natural products, lipids which are one of the important
biomolecules are of interest due to their multiple applications
ranging from food, pharma, biomedical and oleochemical appli-
cations [2]. The fatty acids which are the building blocks of lipids
are of great importance as they serve as substrate for the
production of many bioactive lipid derivatives with potential in
biomedical applications [3]. Plants and animals are the main
sources of natural lipids which are composed of a mixture of
several simple and complex lipids. Recently, a lipidic compound
was isolated from a plant source where it was found that the
compound had phenolic substituents along with polar hydroxyl
functional groups with an aliphatic chain. The compound was
identified as 2, 3-dihydroxypropyl 11-{[(2E)-3-(4-hydroxy-3-
methoxyphenyl) prop-2-enoyl] oxy} undecanoate or 1- [11-
(ferulyloxy) undecanoyl)] glycerol, which is a naturally occurring
lipidic derivative of ferulic acid [4]. The molecular structure of this
utilis where the authors reported that the compound exhibited
potent NO inhibitory activity against lipopolysaccharide (LPS)-
induced NO release with beneficial effects against inflammation
[5].
The structure of the compound shows that it has both lipidic
nature and also possess phenolic moiety which can have potential
application as a cosmetic agent. Literature reports suggest that any
compound which is intended to be used as an cosmeceutical
ingredient should have some properties such as anti-oxidant, anti-
inflammatory, skin whitening, anti-aging, anti-wrinkling, photo
protective activity for its potential use in cosmetics [6]. It is
reported that ferulic acid based structured lipids like feruloylated
acylglycerols are potent UV absorbing agents and antioxidants that
can be used in food and cosmetics [7]. Based on the structure of the
compound, we anticipate that it could have promising potential in
cosmetic and biomedical fields and can be explored further. Also
several research groups have been working on the modification of
natural phenolics to improve their biological activity and antioxi-
dant potential [8]. The research interest in antioxidants which are
found in natural sources has been increasing due to their health
beneficial properties. This has prompted us to synthesize and
evaluate the compound for antimicrobial, anti-oxidant and
cytotoxicity properties to examine its potential. For the synthesis
* Corresponding author.
E-mail address: sskaki@iict.res.in (S.S. Kaki).
https://doi.org/10.1016/j.btre.2019.e00375
2215-017X/© 2019 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/).

2
J. Johny et al. / Biotechnology Reports 24 (2019) e00375
of the target compound we have designed reactions where enzyme
catalyzed transformations can be employed. The unique character-
istics of enzymes like milder reaction conditions, better selectivity
have made them as routine catalysts in organic synthesis. These
characteristics of the biocatalyst provide organic chemists an
opportunity to avoid or at least reduce the usage of toxic reagents
which are used in a multi step organic synthesis [9,10]. Also there
has been an increased interest in enzymatic transformations
conducted in non-conventional organic medium such as organic
solvents as solubility of many organic compounds is facilitated in
solvents rather than water [11]. Hence, the aim of the work was to
synthesize the target compound employing a bioorganic route and
evaluate its preliminary activities.
potassium hydroxide solution (150 mL) were stirred at 100 ꢀC for
16 h. The progress of the reaction was monitored by TLC using
hexane and ethyl acetate (70:30; v/v). After complete conversion,
the temperature was brought to about 25 ꢀC and the pH was
adjusted to 1 by adding concentrated hydrochloric acid. The
resulting precipitate was filtered and dried under reduced pressure
to obtain 11-hydroxyundecanoic acid as a white solid in 1.45 g
(95%).
mp: 65–66 ꢀC.
1H NMR (300 MHz, CDCl3)
d 1.28 (m, (CH2)6, 12 H), 1.62 (m,
(CH2)2, 4 H), 2.34 (t, J = 7.6 Hz, CH2, 2H,), 3.63 (t, J = 6.8 Hz, CH2,
2 H).
IR (cmÀ1): 1216.38 cmÀ1, 3022.17 cmÀ1, 3400.55 cmÀ1
.
MS: M-1 = 201.33
2. Materials and methods
2.2.3. Preparation of (E)-11-(3-(4-hydroxy-3-methoxyphenyl)
acryloyloxy) undecanoic acid (5)
2.1. General information
Vinyl ferulate (900 mg; 4.09 mmol) and 11-hydroxyundecanoic
acid (1.03 g; 5.11 mmol; 1.25 eq) were solubilized in 10 mL of
hexane and 2-butanone (2:1; v/v) and to this mixture was added
immobilized lipase from Candida antarctica (15% to wt of
substrates). The reaction mixture was stirred at 60 ꢀC while
monitoring the progress by TLC using hexane and ethyl acetate
(80:20; v/v). Product formation was observed from the third day
onwards and another 5% of lipase was added to the reaction
mixture. After maximum conversion, the reaction mixture was
diluted with ethyl acetate and the lipase was washed several times
with the solvent and separated by filtration. Solvent was
evaporated and the crude product was purified by column
chromatography (hexane and ethyl acetate (90:10; v/v) to obtain
the ferulic acid ester of hydroxy undecanoic acid in 570 mg (37%).
Solketal (DL – 1,2 – Isopropylidineglycerol), ferulic acid, vinyl
acetate and 11-bromoundecanoic acid were purchased from
Sigma. Potassium hydroxide, mercuric acetate, sulphuric acid,
hydrochloric acid, silica gel for column chromatography were
purchased from SRL chemicals, merck, Avera, Advent and Acme
Synthetic Chemicals, Mumbai, India respectively. All solvents were
procured from Merck and were of highest grade of purity.
Immobilized lipase from Candida antarctica (CAL-B) was procured
from Novozymes Bagsvaerd, Denmark).
The proton NMR spectra were recorded on Varian 500/300 and
125/75 MHz instrument and TMS was used as an internal standard.
Mass spectra were recorded using electron spray ionization on
Waters e2695 Seperators module (Waters, Milford, MA, USA) mass
spectrometer. Infra red (IR) spectra were recorded using a Perkin-
Elmer Fourier transform (FT)-IR Spectrum BX instrument (Model:
Spectrum BX; Connecticut, USA). All the synthesized compounds
were purified by silica gel (60–120 mesh) column chromatography
(Acme Synthetic Chemicals, Mumbai, India) and were identified by
thin-layer chromatography (TLC). TLC was performed on precoated
silica gel 60 F254 from Merck (Darmstadt, Germany).
1H NMR (300 MHz, CDCl3):
d 1.29 (s, (CH2)6, 12 H), 1.63–1.71 (m,
(CH2)2, 4 H), 2.35 (t, J = 7.5 Hz, CH2, 2H), 3.65 (t, ÀOCH2, J = 6.6 Hz,
2 H), 3.94 (s, ÀOCH3, 3 H), 6.26 (d, J = 15.9 Hz, = CH, 1 H), 6.93 (d,
J = 8.1, 5.5 Hz, ArH, 1 H), 7.13–7.02 (m, ArH, 2 H), 7.58 (d,
J = 15.9 Hz, = CH, 1 H); IR (cmÀ1): 1742.56 cmÀ1, 2938.90 cmÀ1
,
2985.89 cmÀ1, 3460.28 cmÀ1
MS: M-1 = 377.34.
2.2. Synthesis
2.2.4. Synthesis of (E)-(2, 2-dimethyl-1,3-dioxolan-4-yl)methyl 11-(3-
(4-hydroxy-3-methoxyphenyl)acryloyloxy)undecanoate (7)
2.2.1. Preparation of vinyl ferulate (2)
Ferulic acid ester of 11-hydroxyundecanoic acid (570 mg;
1.5 mmol) and solketal (250 mg; 1.96 mmol; 1.3 eq) were
solubilised in a mixture of 10 mL of hexane: 2-butanone (2:1; v/
v). To this mixture, immobilized lipase from Candida antarctica
(10% to wt of substrates) was added and the reaction mixture was
magnetically stirred at 60 ꢀC. After maximum conversion, the
reaction mixture was filtered to separate the lipase, extracted with
ethyl acetate, washed with water and dried over anhydrous sodium
sulphate to obtain the crude product. The crude product was
purified by column chromatography (hexane and ethyl acetate
(92:8; v/v) to obtain pure acetonide in 250 mg (34% yield).
Vinyl ferulate was synthesized from ferulic acid and vinyl
acetate following a reported protocol with slight modifications
[12]. To a mixture of ferulic acid (1.5 g, 7.73 mmol) and vinyl acetate
(11.4 mL, 123.68 mmol) in tetrahydrofuran (20 mL), mercury
acetate (4%, w/w) was added and the mixture was stirred for
30 min under nitrogen atmosphere. After 30 min, 2–4 mL of
sulphuric acid was added and the mixture was stirred overnight
at 40 ꢀC. After 12 h, excess of sodium acetate (20 mg) was added
followed by the removal of vinyl acetate and solvent. The
remaining crude reaction product was purified by column
chromatography (hexane and ethyl acetate (92:8 v/v) to obtain
pure vinyl ferulate in 1.2 g (70.6%) at Rf 0.87 on TLC run on hexane:
ethyl acetate (80:20; v/v).
1H NMR (300 MHz, CDCl3):
d 1.29 (s, (CH2)9, 18 H), 1.67–1.71 (m,
(CH2)2, 4 H), 2.30 (t, J = 7.5 Hz, CH2, 2 H), 3.62–3.66 (m, CH2, ÀOCH2,
4 H), 3.93 (s, ÀOCH3, 3 H), 4.05 (t, J = 6.7 Hz, CH, 1 H), 4.19 (t, J = 6.7
H, CH2, 2 H), 5.85 (s, OH, 1 H), 6.29 (d, J = 15.9 Hz, ( = CH), 1 H), 6.89
(d, ArH, 1 H), 7.01–7.08 (m, ArH, 2 H), 7.61 (d, J = 15.9 Hz, ( = CH),
1H NMR (300 MHz, CDCl3)
d 3.93 (s, CH3, 3 H), 4.62 (dd, 7.78 Hz,
1.52 Hz, = CH, 1 H), 4.94 (dd, 15.56 Hz, 1.67 Hz, = CH, 1 H), 5.94 (s,
OH, 1 H), 6.28(d, 15.86, = CH, 1 H), 6.92 (d, 8.08 Hz, = CH, 1 H), 7.04
(d, ArH, 1 H), 7.08–7.11 (m, ArH, 2 H), 7.43–7.45 (m, = CH, 1 H), 7.70
1 H); IR (cmÀ1): 1216.08 cmÀ1 1708.62 cmÀ1 2858.29 cmÀ1
, , ,
2928.19, 3021.31 cmÀ1, 3389.21 cmÀ1; MS: = M + 23 = 515.28
(d, 15.86, = CH, 1 H); IR (cmÀ1): 1718.58 cmÀ1, 3021.04 cmÀ1
,
3417.78 cmÀ1; MS: M-1 = 219.22.
2.2.5. Synthesis of 1- [11-(ferulyloxy) undecanoyl)] glycerol (8)
A mixture of acetonide (7) of previous step (130 mg, 0.003 mol)
dissolved in methanol (10 mL) was placed in ice bath and to this
cooled solution was added 2 M aqueous hydrochloric acid (0.5 mL,
1.5 eq) in one portion. After few minutes, the ice bath was removed
and the reaction mixture was stirred for about 4 h at room
2.2.2. Preparation of 11-hydroxyundecanoic acid (4)
11-hydroxyundecanoic acid prepared from 11-bromoundeca-
noic acid employing a reported protocol [13]. Briefly, a mixture of
11-bromoundecanoic acid (2 g, 7.54 mmol) and a solution of 2 M aq

J. Johny et al. / Biotechnology Reports 24 (2019) e00375
3
temperature (20 ꢀC) while monitoring by TLC using hexane and
ethyl acetate (60:40; v/v). When the conversion was maximum
(3 h), diethyl ether (20 mL) was added to the reaction mixture and
washed with 10 mL of saturated aqueous sodium bicarbonate and
extracted with diethyl ether (3 x 25 mL). The organic phases
containing the product were dried over sodium sulphate and
concentrated to obtain the crude product which was purified by
column chromatography using chloroform to obtain the final
product in 65% yield (130 mg).
Pseudomonas aeruginosa (MTCC 741), Klebsiella pneumonia (MTCC
618) by Agar Well Diffusion Method [15]. The minimum inhibitory
concentrations (MIC) of various synthetic compounds were tested
against by Microdilution method recommended by CLSI Standard
Protocol [16].
2.5. Cytotoxicity studies (MTT assay)
The cell viability (MTT test) of the synthetic lipid was assessed
following our earlier published literature and compared with
Doxorubicin as reference compound [17].
1H NMR (500 MHz, CDCl3):
d 1.28 (s, (CH2)6, 12 H), 1.63–1.72 (m,
(CH2)2, 4 H), 2.35 (t, J = 7.5 Hz, CH2, 2 H), 3.5–3.72 (m, CH2, ÀOCH2,
CH, 3 H), 3.93 (s, ÀOCH3, 3 H), 4.11–4.22 (m, CH2, CH2, 4 H), 5.91 (s,
OH, 1 H), 6.29 (d, J = 15.9 Hz, ( = CH), 1 H), 6.90 (d, ArH, 1 H), 7.01–
7.08 (m, ArH, 2 H), 7.61 (d, J = 15.9 Hz, = CH, 1 H); 13C NMR
2.6. Statistical analysis
(125 MHz, CDCl3):
d
24.91 (s, CH2), 25.96 (s, CH2), 29.30–29.42
Statistical analysis was performed using unpaired Student’s t-
test followed by one-way ANOVA by Dunnett’s test for antioxidant
activity and Tukey's multiple comparison tests for cytotoxicity
activity using Graph Pad Prism version 6.04 for Windows (Graph
Pad Software, San Diego, CA).
(m, (CH2)6), 55.98 (s, ÀOCH3), 63.37 (s, CH2), 64.62 (s, CH2), 65.21 (s,
CH2), 70.29 (s, CH), 109.33 (s, ArCH), 114.73 (s, = CH), 115.66
(s, ArCH), 123.07 (s, ArCH), 127.06 (s, ArC), 144.71 (s, = CH), 146.85
(s, ArC), 147.93 (s, ArC), 167.46 (s, C = O), 174.33 (s, C = O).
IR (cmÀ1): 1731.21 cmÀ1
3424.75 cmÀ1; MS: M + 23 = 475.34
HRMS: M + 23 = observed mass: 475.2281; calculated mass:
475.2308
,
2256.08 cmÀ1
,
3020.16 cmÀ1
,
3. Results and discussion
3.1. Synthesis
2.3. Antioxidant activity
In this work, we demonstrated a chemo-enzymatic approach
towards the synthesis of a natural phenolic lipid which can have
potential applications in food, cosmetic and pharma industries.
The bioorganic synthetic route followed is shown in Scheme 1. The
synthetic route for the target compound involved preparation of
vinyl ferulate (2) which was carried out by the vinyl exchange
between ferulic acid (1) and vinyl acetate which yielded vinyl
ferulate in 70% yield as one of the coupling partner. The structure of
vinyl ferulate was confirmed by spectral analysis and also by
comparing with the earlier reported literature values.
DPPH radical scavenging activity: The preliminary antioxidant
activity of the synthetic lipid was measured by the DPPH radical
scavenging activity following a reported protocol with small
modification [14]. 200
mL of synthesized lipid (1 mM and 2 mM
concentration) was added to a 0.1 mM methanolic DPPH radical
solution (2 mL) in a screw cap tube and the final volume was
made up to 3 mL with methanol. This solution was kept in dark for
about 40 min and the absorbance was measured at 517 nm.
Methanol was used as blank and standard antioxidants like BHT
and alpha tocopherol at 1 mM concentration were used as
positive control. The DPPH radical scavenging activity was
determined by the formula: [(Ac – As) / Ac] Â 100; where Ac
and As are the absorbance of the control and sample respectively.
The assay was conducted 3 times and the results are presented as
average of three measurements.
Oxidation stability by differential scanning calorimetry: Differen-
tial scanning calorimeter (DSC) was used to determine the
oxidative onset temperature (OT) according to the method
described by ASTM (Standard Test Method for Oxidation Onset
Temperature of Hydrocarbons by Differential Scanning Calo-
rimetry. E2009 À 08). DSC was studied using Q-100 thermal
analyzer from M/s TA instruments under continuous oxygen
atmosphere at a flow of 50 mL minÀ1. Soybean oil sample with
and without test compounds (4.0–5.0 mg) was placed in an
open aluminium pan under constant flow of oxygen and an
empty pan was used as reference. The temperature was raised
from 25 ꢀC to 300 ꢀC at a heating rate of 10 ꢀC minÀ1. Samples of
soybean oil as control and soybean oil with synthetic lipid, BHT
and alpha tocopherol as reference compounds were analyzed
for oxidative onset temperature. The DSC analysis was
determined for 3 times and the results are presented as
average of three measurements.
The transesterification between vinyl ferulate (2) and 11-
hydroxy undecanoic acid (4) was catalyzed by immobilized lipase
from Candida antarctica which resulted in desired ferulic acid ester
of 11-hydroxyundecanoic acid product (5) in 37% yield during
twelve days of reaction time period. It was found that product
formation was observed from third day of the reaction and reached
maximum at a reaction time of twelve days. It is well known that
the vinyl group will influence the enzyme catalyzed step towards
product formation instead of reverse reaction [18]. The trans-
esterification reaction was carried out in organic media consisting
of hexane and 2-butanone mixture which was reported to be the
suitable solvent for enzymatic esterification reactions in organic
media [19]. In the present study, we found that a mixture of 2:1
ratio of hexane and 2-butanone (v/v) was better as the two
substrates were solubilised in this mixture. In addition, this solvent
mixture was also reported to be non-toxic which further influences
the lipase catalyzed reactions [20]. The product of this trans-
esterification reaction had both ester and acid functionalities and
hence we employed a second lipase catalyzed esterification step
between the acid group of fatty acid (5) and solketal (6), which had
primary hydroxyl functionality to obtain the corresponding
acetonide ester (7) in 34% yield. The acetonide ester compound
was finally deprotected using aqueous hydrochloric acid to yield
the desired target compound, 1- [11-(ferulyloxy) undecanoyl)]
glycerol (8) in 65% yield. The products of each step in the synthesis
were purified by silica gel column chromatography and the
purified compounds were characterized by spectral data. The
spectral analysis was compared with the reported literature data
and the structure of the final product was confirmed with HRMS
analysis also. The HRMS data showed a strong peak which
corresponded to [M + Na] at m/z 475.2281 which further confirmed
the structure of the product.
2.4. Antimicrobial evaluation
In vitro antibacterial activity of the newly synthesized
compounds was studied against the bacterial strains, Gram-
positive organisms: Staphylococcus aureus (MTCC 96), Staphylococ-
cus epidermidis (MTCC 9041), Bacillus subtilis (MTCC 441),
Gram-negative organisms viz Escherichia coli (MTCC 443),

4
J. Johny et al. / Biotechnology Reports 24 (2019) e00375
Scheme 1. Bioorganic synthetic route.
3.2. Biological evaluation of the prepared compound
activity. The antioxidant activity in DPPH radical scavenging assay
was in the following order: ferulic acid > synthetic lipid>11-
feruloyl undecanoic acid > BHT.
3.2.1. Antioxidant activity
The antioxidant activity of the synthetic lipid was measured
using preliminary DPPH radical scavenging activity and also by
measuring oxidative stability using DSC in a lipid medium and
compared to BHT and alpha tocopherol as reference compounds in
respective assays. The DPPH radical scavenging activity of the
prepared lipid showed very good antioxidant activity compared to
commercially available BHT. At 0.5 mM concentration BHT
exhibited 17.61% FRSA (Free radical scavenging activity) and
synthetic lipid showed an FRSA of 33.10% and the activity was
increased as the concentration was increased which is shown in
Table 1 and Fig. 1. However, the activity was lower when compared
to ferulic acid at all the tested concentrations. The antioxidant
activity of phenolic compounds was reported to be dependent on
factors like structure of the compound, position of the substituents
on the phenolic ring, and also the medium in which the activity is
determined [21]. These factors could be one of the reasons for the
difference in antioxidant activity observed even though the
number of phenolic hydroxyls is same in all the three tested
compounds. The compound 5 which is the unbound lipid, i.e. 11-
feruloyl undecanoic acid was also screened for DPPHꢁ scavenging
The antioxidant capacity of the prepared lipid was also
examined by its oxidation stability potential in soybean oil by
determination of oxidative onset temperature (OT) using DSC.
Soybean oil was chosen as it contains higher amounts of
unsaturated fatty acids along with linolenic acid. DSC is reported
to be a reliable method for determining the oxidative stability of
oils as it is convenient, simple and rapid technique [22]. Oil was
extracted from soybeans and used for DSC study both as such and
also in the presence of test compounds. The results of the DSC
analysis are shown in Fig. 2.
Table 1
DPPH radical scavenging assay.
Test compound
0.5M
1M
2M
Ferulic acid
11-feruloyl undecanoic
acid
36.34 Æ 0.32*** 62.0 Æ 0.25***
27.30 Æ 0.52*** 49.09 Æ 0.12
(ns)
81.53 Æ 0.14***
68.61 Æ 0.14***
Synthetic lipid
BHT
33.10 Æ 0.98*** 49.70 Æ 0.63*** 72.67 Æ 0.29***
17.61 Æ 0.77 37.4 Æ 0.88 48.33 Æ 0.55
Solvent: methanol; Æ: standard deviation of mean; ns: none significant; *** results
found significantly different from BHT at P < 0.001 (Student’s t-test, followed by
One-way ANOVA).
Fig. 1. DPPH radical scavenging assay of test compounds. *** results found
significantly higher from BHT at P < 0.001 (Student’s t-test, followed by One-way
ANOVA).

J. Johny et al. / Biotechnology Reports 24 (2019) e00375
5
Fig. 2. DSC profiles of test compounds.
Table 2
Antimicrobial activity of the synthesized lipid.
Bacterial sps
Zone of inhibition (150
mg /mL)
MIC (Serial dilution 150 mg /mL to1.17 mg /mL)
Synthetic lipid
–
Streptomycin
Synthetic lipid
Streptomycin
2.34
4.68
2.34
4.68
Staphylococcus epidermidis
Bacillus subtilis
Staphylococcus aureus
Escherichia coli
Pseudomonas aeruginosa
Klebsiella pneumoniae
25
24
23
23
28
26
<150
37.5
37.5
18.75
75
13
12
13
10
11
1.17
4.68
75
In DSC study, soybean oil was found to have oxidative onset
temperature of 160.51 ꢀC which increased in the presence of
antioxidant compounds at 1% concentration. The oxidative onset
temperatures in the presence of the synthetic lipid and alpha
tocopherol were found to be 186.41 ꢀC and 181.30 ꢀC respectively.
The results indicated that the synthetic lipid has better potential in
delaying the oxidative deterioration of soybean oil compared to
alpha tocopherol.
3.2.3. Cytotoxicity studies
The prepared phenolic lipid derivative along with the ferulic
acid and the unbound lipid were studied for its cytotoxicity against
a panel of 7 cell lines with Doxorubicin as standard reference
compound. The results of the cytotoxicity studies are shown in
Table 3. It was observed that the synthesized compound showed
promising cytotoxicity effect against A549 and HeLa cell lines with
IC50 values of 9.102 and 9.866 mM respectively. It exhibited
3.2.2. Antimicrobial activity
Table 3
In vitro cytotoxicity test of compounds against cancerous and non-cancerous cell
lines.
The synthesized lipid along with the ferulic acid and the
unbound lipid were screened for antimicrobial activity against
three Gram positive and three Gram negative bacterial strains to
know whether it can be a potent antimicrobial agent. Antimicro-
bial activity was measured by both zone of inhibition and also by
determination of minimum inhibitory concentrations (MIC) and
the results are presented in Table 2. MIC was determined only for
the compounds which exhibited activity by zone of inhibition
method. It was found that the synthetic lipid exhibited antimicro-
bial activity against both Gram positive and Gram negative
bacterial strains. Ferulic acid and the unbound lipid did not
exhibit antimicrobial activity. Among the tested strains, the
synthetic lipid exhibited highest activity against Escherichia coli
Cell line
IC50 values (mM)
Synthetic lipid
Doxorubicin
0.55 Æ 0.16
0.363 Æ 0.01
0.8 Æ 0.71
A549
DU145
HeLa
HepG2
MCF7
B16
9.102 Æ 0.075***
10.464 Æ 0.572***
9.866 Æ 0.294***
10.282 Æ 0.272***
11.501 Æ 0.045***
19.035 Æ 0.257***
56.180 Æ 0.738
0.72 Æ 0.012
2.0 Æ 0.81
0.7 Æ 0.56
HEK
0.010 Æ 0.001
Cell lines: A549 Homo sapiens lung carcinoma (ATCC1 CCL-185TM); DU145 Homo
sapiens prostrate carcinoma (ATCC1 HTB-81TM); HeLa Homo sapiens cervix
adenocarcinoma (ATCC1 CCL-2.1TM); Hep G2 liver hepatocellular carcinoma
(ATCC1 HB-8065TM); MCF7 human breast adenocarcinoma ((ATCC1 HTB-22TM);
B16-F10 mouse skin melanoma (ATCC1 CRL 6475TM); HEK-293 Homo sapiens
embryonic kidney cells (ATCC1 CRL-1573TM) ; Æ: standard deviation of mean; ***
P < 0.001 is significantly different of cytotoxicity activity in cancer cell lines from
normal cell lines of synthetic lipid (Student’s t-test, followed by One-way ANOVA).
with an MIC value of 18.75 mg/mL followed by Bacillus subtilis and
Staphylococcus aureus. However, the antimicrobial activity was
lower compared to standard antibiotic Streptomycin for all the
bacterial strains in the present study.

6
J. Johny et al. / Biotechnology Reports 24 (2019) e00375
assistance under Early Career Research Award [grant number: ECR/
2017/000639] and Director, CSIR-IICT for providing the facilities.
IICT Communication no: IICT/Pubs/2019/137.
References
[1] M.S. Butler, Natural products to drugs: natural product-derived compounds in
clinical trials, Nat. Prod. Rep. 25 (2008) 475–516, doi:http://dx.doi.org/
10.1039/b514294f.
[2] P. Górnas, A. Siger, D. Seglin, Physicochemical characteristics of the cold-
pressed Japanese quince seed oil: new promising unconventional bio-oil from
by-products for the pharmaceutical and cosmetic industry, Indust. Crops Prod.
48 (2013) 178–182, doi:http://dx.doi.org/10.1016/j.indcrop.2013.04.018.
[3] S.S. Kaki, A. Sammaiah, P.V. Korlipara, R.B.N. Prasad, P. Yedla, C.G. Kumar,
Synthesis and biological evaluation of novel lipoamino acid derivatives,
Bioorg. Med. Chem. Lett. 26 (2016) 209–212.
[4] Y. Cong, J.-G. Guo, J. Liu, Two new chemical constituents of Veratrum dahuricum
(Turcz.) loes, F. Helv. Chim. Acta. 96 (2013) 345–349.
[5] M.H. Woo, D.H. Nguyen, B.T. Zhao, U.M. Seo, T.T. Nguyen, D.Y. Jun, Y.H. Kim,
Echinochlorins A-C from the grains of Echinochloa utilis (Barnyard Millet) and
their anti-inflammatory activity, Planta Med. 81 (PX66) (2015), doi:http://dx.
doi.org/10.1055/s-0035-1556510.
[6] I. Antonopoulou, S. Varriale, E. Topakas, U. Rova, P. Christakopoulos, V. Faraco,
Enzymatic synthesis of bioactive compounds with high potential for
cosmeceutical application, Appl. Microbiol. Biotechnol.100 (2016) 6519–6543,
doi:http://dx.doi.org/10.1007/s00253-016-7647-9.
[7] S. Sun, X. Chen, C. Jiang, Enhanced synthesis of feruloylated acylglycerols by
the lipase-catalyzed transesterification of glyceryl monoferulate with
different acyl donors using ionic liquids as reaction solvents, J. Biotechnol. 280
(2018) 31–37, doi:http://dx.doi.org/10.1016/j.jbiotec.2018.05.016.
[8] O.S. Maldonado, R. Lucas, F. Comelles, M.J. Gonzalez, J.L. Parra, I. Medina, J.C.
Morales, Synthesis and characterization of phenolic antioxidants with
surfactant properties: glucosyl- and glucuronosyl alkyl gallates, Tetrahedron
67 (2011) 7268–7279.
[9] Mark W. Empie, Akiva Gross, Enzyme assisted transformations in organic
media, in: Richard C. Allen (Ed.), Annual Reports in Medicinal Chemistry, vol.
23, Academic Press, Inc. Harcourt Brace Jovanovlch, Publishers,1988, pp.1–390
Pages iii-xiv.
[10] F. Rudroff, M.D. Mihovilovic, H. Gröger, R. Snajdrova, H. Iding, U.T. Bornscheuer,
Opportunities and challenges for combining chemo- and biocatalysis, Nat.
Catal. 1 (2018) 12–22, doi:http://dx.doi.org/10.1038/s41929-017-0010-4.
[11] P. Adlercreutz, Biocatalysis in non-conventional media, in: Adrie J.J. Straathof,
Patrick Adlercreutz (Eds.), Applied Biocatalysis, 2nd edn, Harwood Academic
Publishers, 2000, pp. 295–316.
[12] C. Gao, P. Mayon, D.A. MacManus, E.N. Vulfson, Novel enzymatic approach to
the synthesis of flavonoid glycosides and their esters, Biotechnol. Bioeng. 71
(2001) 235–243, doi:http://dx.doi.org/10.1002/1097-0290(2000)71.
[13] D.Y. Duveau, P.M. Arce, R.A. Schoenfeld, N. Raghav, G.A. Cortopassi, S.M. Hecht,
Synthesis and characterization of mitoQ and idebenone analogues as
mediators of oxygen consumption in mitochondria, Bioorg. Med. Chem. 18
(2010) 6429–6441, doi:http://dx.doi.org/10.1016/j.bmc.2010.06.104.
[14] M. Betty, S.S. Kaki, M. Bo, E. Mulaa, P. Adlercreutz, Enzymatic synthesis of
Lipophilic Rutin and Vanillyl Esters from fish byproducts, J. Agric. Food Chem.
59 (2011) 7021–7027, doi:http://dx.doi.org/10.1021/jf200867r.
[15] M.E. Linday, Practical Introduction to Microbiology, E and F.N. Spon Ltd., United
Kingdom, 1962, pp. 177.
[16] Clinical and Laboratory Standards Institute, Performance Standards for
Antimicrobial Susceptibility Tests; Eighteen Informational Supplement
M100-S18, (2008) .
[17] J. Johny, A. Jatla, V.K. Eruva, S. Misra, S.S. Kaki, Synthesis, characterization and
evaluation of 1-monoacylglycerols of unsaturated fatty acids as potential
bioactive lipids, Grasas Aceites 70 (2019) e325, doi:http://dx.doi.org/10.3989/
gya.1052182.
Fig. 3. Multiple comparisons of cytotoxicity effects of synthesized lipid among
cancer and normal cell lines. *P < 0.05; *** P < 0.001 is significantly different among
the cell lines used (Tukey's Multiple comparison test).
moderate activity against HepG2, DU145, MCF7 and B16 cell lines
where the IC50 values were in the range of 10.464–19.035
prepared compound was least active against HEK cell lines with an
IC50 value of 56.180 M. The mean differences of IC50 value of
mM. The
m
synthesized lipid among the cell lines used (Tukey's multiple
comparison test), showed that there was a statistically significant
lower IC50 value observed among all the cancer cell lines compared
to normal cell line (HEK) (P < 0.001). Whereas, the mean differ-
ences in cytotoxicity of synthesized lipid was compared among the
cancer cell lines, a significant lower IC50 was found in all the cancer
cell lines when compared to B16 cell line (P < 0.001) as shown in
Fig. 3. It was observed that ferulic acid and the unbound lipid did
not show activity against the tested cell lines. However, it was
found that the prepared compound was less effective compared to
the reference drug Doxorubicin against all cell lines.
4. Conclusions
Bioorganic synthesis of a natural phenolic lipid derivative was
carried out for the first time following a chemo-enzymatic
approach. Immobilized Candida antarctica lipase was employed
as a biocatalyst in organic media for two esterification reactions.
The synthesized lipid was characterized by spectral analysis and
the product was screened for antioxidant, antimicrobial and
cytotoxic activities. The synthesized compound has shown
promising antioxidant and cytotoxic activity and moderate
antimicrobial activity. Further studies on the related derivatives
of the phenolic lipid with applications could be promising as the
compounds could have potential in biomedical fields.
[18] Z. Tan, F. Shahidi, Chemoenzymatic synthesis of phytosteryl ferulates and
evaluation of their antioxidant activity, J. Agric. Food Chem. 59 (2011) 12375–
12383, doi:http://dx.doi.org/10.1021/jf2034237.
[19] K. Sabally, S. Karboune, F.K. Yebaoh, S. Kermasha, Enzymatic esterification of
dihydrocaffeic acid with linoleyl alcohol in organic solvent media, Biocatal.
Biotransform. 23 (2005) 37–44, doi:http://dx.doi.org/10.1080/
10242420500043762.
[20] K. Sabally, S. Karboune, R. St-Louis, S. Kermasha, Lipase-catalyzed
transesterification of dihydrocaffeic acid with flaxseed oil for the synthesis
of phenolic lipids, J. Biotechnol. 127 (2006) 167–176, doi:http://dx.doi.org/
10.1016/j.jbiotec.2006.06.010.
Declaration of Competing Interest
We declare that we have no conflict of interests.
Acknowledgements
[21] R. Amorati, A. Cavalli, M.G. Fumo, M. Masetti, S. Menichetti, C. Pagliuca, C.
Viglianisi, Kinetic and thermochemical study of the antioxidant activity of
sulfur-containing analogues of vitamin E, Chem. Eur. J. 13 (2007) 8223–8230,
doi:http://dx.doi.org/10.1002/chem.200700309.
Ms Juliya Johny and Dr. Shiva Shanker Kaki gratefully
acknowledge the Science and Engineering Research Board,
Department of Science & Technology, New Delhi for the financial
ꢀ
[22] M. Kozłowska, E. Gruczynska, Comparison of the oxidative stability of soybean
and sunflower oils enriched with herbal plant extracts, Chem. Pap. 72 (2018)
2607–2615, doi:http://dx.doi.org/10.1007/s11696-018-0516-5.