Informatics in Medicine Unlocked 10 (2018) 89–99

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Informatics in Medicine Unlocked

journal homepage: www.elsevier.com/locate/imu

Phytochemical screening and in silico studies of flavonoids from

Chlorella pyrenoidosa
Rajasri Yadavalli a, *, John Reddy Peasari a, Priyadarshini Mamindla b, Praveenkumar c,
Sri Mounika a, Jayasree Ganugapati a
a
Department of Biotechnology, Sreenidhi Institute of Science and Technology (Autonomous), Yamnampet, Ghatkesar, Hyderabad, Telangana state, India
b
Athon LLC, Houston, TX, USA
c
Agasthya International Foundation, Bengaluru, India

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

Keywords: The recent explosion of interest in the bioactivity of the flavonoids of microalgae is due to the potential health
Chlorella benefits of the polyphenolic components that are major dietary constituents. The present study focuses on the
Phototrophic phytochemical screening and in silico studies of flavonoids. Total flavonoids content in Chlorella pyrenoidosa was
Heterotrophic estimated in two modes of cultivation (Autotrophic and Heterotrophic) and its implication in anti-proliferation
Flavonoids and anti-inflammatory activity was assessed through in silico approach. H-Ras p21(PDB-4L9S) and Lip-
Auto dock vina
oxygenase (PDB-3V99) involved in proliferation pathway and inflammatory pathway were selected as the target
Schrodinger
proteins for in silico studies. Seven compounds were selected for molecular docking. Pharmacokinetic properties
of these compounds were calculated using online tools and docking was performed using Auto Dock Vina. By
comparing and analyzing their binding energies in Maestro Schrodinger, suite, it was observed that Epi-
gallocatechin gallate exhibited least binding energy of
9.1 kcal/mol and hence has anti-inflammatory activity.
Catechin has best binding affinity with H-Ras p21 and hence has anti proliferative activity.

1. Introduction Information is scarce on the presence of secondary metabolites, respon-

Microalgae are a key natural resource for a vast array of compounds
viz. biodiesel, nutraceuticals, including proteins, vitamins, minerals,
carotenoid pigments, such as xanthophylls and carotenes, Flavonoids etc.
Subspecies of Chlorella are known to have several bioactive secondary
metabolites which can have bacteriostatic, bactericidal, antioxidant anti-
proliferative, antifungal, antiviral and antitumor activity [1,2]. Recent
exploration of anti-proliferative and anti-angiogenic properties of
Chlorella pyrenoidosa,a unicellular fresh water green alga,paves way for
exploring its use in treating inflammation and proliferation associated
with various diseases.
Flavonoids are the largest groups of phenolic compounds are known
to contain a broad spectrum of chemical and biological activities
including antioxidant and free radical scavenging properties [3]. Flavo-
noids include flavonols, flavones, catechins, proanthocyanidins, antho-
cyanidins and isoflavonoids. In the recent times, flavonoids have gained
increasing interest as they exhibit beneficial health effects due to their
potential antioxidant [4], anti-inflammatory and anti-cancer activities.
sible for anti-proliferative and anti-inflammatory properties of C. pyr-
enoidosa. Hence in this study, autotrophic and heterotrophic cultures of
C. pyrenoidosa were grown and HPLC analysis was carried out for the
algal biomass for analyzing various flavonoids. The aim of the present
study is to assess total flavonoids content and to study the role of flavo-
noids as anti-proliferative and anti-inflammatory agents using in silico
analysis.
In this study above mentioned properties of flavonoids were evalu-
ated by in silico methods with lipoxygenases-an enzyme related to
oxidation of various fatty acids and Ras proteins which is a member of a
super family of small GTPase involved in cell growth. The 5-lipoxygenase
protein enzyme (5LO) and its leukotriene metabolites have long been
known to be important modulators of inflammation in other disease
states [5]. The ras oncogene p21 antigen (p21) has been identified in
several epithelial malignancies, including breast, colon, bladder, and
prostate [6].

* Corresponding author. Department of Biotechnology, Sreenidhi Institute of Science and Technology (Autonomous), Yamnampet, Ghatkesar, 501301, Hyderabad, Telangana State,
India.
E-mail address: rajasriy@sreenidhi.edu.in (R. Yadavalli).

https://doi.org/10.1016/j.imu.2017.12.009
Received 11 November 2017; Received in revised form 21 December 2017; Accepted 21 December 2017
Available online 26 December 2017
2352-9148/© 2017 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
R. Yadavalli et al.
2. Materials and methods
2.1. Inoculum preparation
2.1.1. Phototrophic culture
Chlorella pyrenoidosa (NCIM NO: 2738) was obtained from National
Centre for Industrial Microorganisms (NCIM), Pune, India. C. pyrenoidosa
is used for culturing in phototrophic mode by using BG11 as medium [7].
800 ml of media was taken in four different conical flasks and 10% of
inoculum was added to each flask. The cultures were grown for one week
at room temperature under continuous light illumination of
55 μmol m
2s
1. The prevailing conditions like oxygen supply and car-
bon dioxide supply were monitored by providing air continuously at
2 l min
1 and CO2 for 10 min daily.
2.1.2. Heterotrophic culture
100 ml of the phototropic culture is taken as the inoculum for growing
the heterotrophic culture of Chlorella pyrenoidosa. It is added to one liter
of modified BG11 medium with glucose as carbon source in absence of
light. It was grown in dark for one week by providing aeration in a
controlled manner 2 l min
1. Sub culturing of the same was done again to
obtain pure heterotrophic culture of C. pyrenoidosa.
2.2. Sample preparation for HPLC analysis from phototrophic and
heterotrophic cultures
An accurately weighed 2 g of dried biomass obtained from photo-
trophic and heterotrophic cultures of C. pyrenoidosa were taken each and
the samples were extracted with 2 ml of hexane for 30 min at 20  C
temperature. The tubes were centrifuged at 4500 g for 10 min and the
supernatant was recovered. The extraction was repeated with 2 ml of
hexane and the supernatants were collected [8]. The remaining residue
was subsequently extracted twice with ethyl acetate of 2 ml for 30 min at
20  C temperature and the supernatants were again collected. Subse-
quently, the residues were further extracted twice with water 2 ml each
time for 30 min at 80  C and the supernatants were combined. The
hexane, ethyl acetate and together with aqueous extracts were all stored
at
10  C before using them for biochemical analysis and HPLC analysis.
2.3. Biochemical analysis
Both phototrophic and heterotrophic biomass of C. pyrenoidosa was
used individually for performing biochemical analysis to test for the
presence of flavonoids.
2.3.1. Test for flavonoids
5 ml of dilute ammonia solution was added to a portion of the hexane,
ethyl acetate and aqueous extracts of both photo and heterotrophic
samples followed by addition of concentrated H2SO4. A yellow color in
each extract indicated the presence of flavonoids. The yellow color dis-
appears on standing. Few drops of 1% aluminum solution were added to
portion of each extract filtrate.
2.3.2. Determination of total phenolic compounds
According to Slinkard and Singleton [9], total soluble phenolic
compounds were determined with Folin-Ciocalteu reagent using pyro-
catechol as a standard phenolic compound. Briefly, 1 ml of the extract
(1 mg/ml) in a volumetric flask diluted with 46 ml distilled water. One
milliliter of Folin-Ciocalteu reagent was added and the content was
thoroughly mixed. After 3 min, 3 ml of sodium carbonate (2%) was added
and then was allowed to stand for 2 h with intermittent shaking. The
absorbance was measured at 760 nm in UV–vis spectrophotometer [Elico
SL-210]. The total concentration of phenolic compounds in the extract
determined as microgram of pyrocatechol equivalent (PE) per milligram
of dry extract.
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Informatics in Medicine Unlocked 10 (2018) 89–99
2.3.3. Total flavonoids content
Dowd method [10] was used to estimate the total flavonoids content.
Briefly, two milliliters of 2% aluminum trichloride (AlCl3) in methanol
was mixed with the same volume of the extract solution (1 mg/ml). The
mixture was incubated at room temperature for 10 min, and the absor-
bance was measured at 415 nm in spectrophotometer. The total flavo-
noids content determined as microgram of rutin equivalent (RE) per
milligram of dry extract.
2.4. HPLC analysis
The hexane, ethyl acetate and aqueous extracts of both phototrophic
and heterotrophic C. pyrenoidosa were subjected to HPLC analysis [Shi-
madzu LC-10AT vp] using PDA as detector with wavelength 270 nm
using RP C18 as column in an isocratic manner with HPLC grade meth-
anol: water in the ratio 90:10 as solvents at 30  C. The flow rate was
adjusted to 1 ml/min with sample injection volume 10 μl. The run time
was set for 30min. The obtained results were used to carry out in silico
studies to assess the anti-oxidant and anti-proliferation capacity of
C. pyrenoidosa.
2.5. In silico analysis
2.5.1. Retrieval of protein structure
The X ray crystal structure of proteins 3V99 (5-lipoxygenase) at 2.25
Aº resolution and 4L9S (Signaling protein) at 1.61 Aº resolution used in
this study were retrieved from RCSB Protein Data Bank [11]. They play a
major role in inflammation and proliferation pathways respectively. The
proteins were prepared for molecular docking by removing the water
molecules and other hetero molecules from the original crystal structure.
Active site analysis was performed using Swiss Protein Viewer, SPDBV
[12].
2.5.2. Retrieval of ligands and ADME property prediction
3D structures of flavonoids that were identified from HPLC analysis of
Chlorella pyrenoidosa were retrieved from NCBI Pub Chem Compounds in
SDF format [13]. 2D structures were sketched using Chemspider and
Molinspiration. The names and CID numbers of the compounds are
Caffeine (CID: 2519), Protocatechic acid (CID: 528594), Catechin (CID:
73160), Epicatechin (CID: 72276),Epigallocatechin –gallate (CID:
65064), Caffeoyl-D-glucose (CID: 129661118), Dihydroquercetin-7,
40 -dimethyl ether (3D structure was generated using molinspiration).
ADME [14] properties (i.e., absorption, distribution, metabolism and
excretion) of the selected compounds was predicted by Molinspiration.
2.5.3. Grid preparation and molecular docking
Molecular Docking was performed using Autodock Vina [15] and
MGL tools [16and17]. Docking Input files were created using AutoDock
tools batch file of MGL tools. Docking was performed between selected
macromolecules and ligands. Hydrogen atoms, Kollman charges were
added to protein molecules and prepared as PDBQT files. The ligand was
prepared in PDBQT by setting flexible torsion angles at all rotatable
bonds, while the protein was kept as a rigid structure. The Lamarckian
Genetic Algorithm (LGA), a local search algorithm was utilized for li-
gands conformations searching.
Configuration files were created for both the proteins by setting
suitable Cartesian coordinates to generate Grid box. For protein 4L9S
grid box parameters are X ¼ 36.212, Y ¼
11.524, & Z ¼ 5.085 and grid
box dimensions was set at 60*60*60 Aº which covers all the amino acids
in the active site. For protein 3V99 coordinates for X, Y, and Z axis were
11.446,
73.596,
24.378 and dimensions for grid box are 70*70*70 Aº.
The docked complex forming hydrogen bonds and other parameters
like intermolecular energy (Kcal/mol) and inhibition constant (μM) were
analyzed by Autodock tool. Ten best poses were generated for each
ligand and scored using Autodock Vina scoring functions. Based on the
docked energy all the ligands were ranked. The interacting residues with
R. Yadavalli et al. Informatics in Medicine Unlocked 10 (2018) 89–99

Table 1 Table 2
Phenolic and flavonoids content in various extracts of C.pyrenoidosa. Results are HPLC chromatogram retention time of phytochemical constituents obtained in photo-
mean  S.D. of three parallel measurements of different plant extracts. trophic and heterotrophic modes.

Mode of Phytochemical Aqueous Hexane Ethyl acetate Phytochemical constituents Retention time (minutes)
cultivation Content extract extract extract
Caffeine 4.245
Phototrophic Total Phenols 0.92  0.08 0.73  0.09 0.69  0.07 Protocatechic acid 7.155
(mg PE/mg) Catechin 5.269
Flavonoids (mg 0.6  0.07 0.52  0.09 0.50  0.05 Epicatechin 6.548
RE/mg) Epigallocatechin -gallate 11.328
Heterotrophic Total Phenols 1.21  0.06 0.96  0.08 0.81  0.06 Caffeoyl-D-glucose 12.575
(mg PE/mg) Dihydroquercetin-7,40 -dimethyl ether 10.800
Flavonoids (mg 0.87  0.06 0.78  0.07 0.60  0.06
RE/mg)
aqueous extract of Chlorella pyrenoidosa in both photo and heterotrophic
conditions. The number of peaks represents the different biologically
ligands were analyzed using Schrodinger Suite [18]. active phytochemical constituents and the major peak area compounds
could belong to the polyphenols and flavonoids.
3. Results HPLC analysis carried out from biomass of both phototrophic and
heterotrophic Chlorella pyrenoidosa, showed the presence of flavonoids
3.1. Total phenolic and flavonoid contents and HPLC analysis (Table 2) caffeine (Rt-4.320), catechin (Rt-5.269),epicatechin (Rt-6.548),
pigallocatechingallate (ECGC)(Rt-11.328),dihyroquerecetin-7,40 -
Flavonoids with their multiple activities viz. anti-microbial, anti- dimethyl-ether (DQME)(Rt-10.800), caffeoyl-D-Glucose (Rt-12.575),
cancer, and anti-diabetic can play a vital role in today's dietary supple- protocatechuic acid (Rt-7.155)(Table 2). Ludmila Machu et al. [20]
ments. Qualitative analysis performed in the all three extracts of Chlorella evaluated antioxidant capacity of Chlorella pyrenoidosa, and Spirulina
pyrenoidosa in the present study, demonstrated the presence of phyto- platensis. HPLC analysis of their study showed that the most abundant
chemicals like phenolic compounds and flavonoids (Table 1). .When phenolic compound was epicatechin. In another similar study on
compared to Phototrophic mode, heterotrophic mode yielded high microalgal species, Jayshree et al. investigated phenolic and flavonoid
amount of total phenols and flavonoids content. In both the modes, content in C. vulgaris and Chlamydomonas reinhardtii, followed by anti-
aqueous extract gave more phytochemical yield (1.21 mg PE/mg of total cancer, antioxidant and antimicrobial activities. They observed that both
phenols and 0.87 mg RE/mg dry cell weight of flavonoids) when species exhibited free radical scavenging activity and high antioxidant
compared to hexane and ethyl acetate extracts. Our results are similar potency. Both species also revealed that flavonoids present in their
with the findings of Baviskar and Khandelwal [19] who also extracted biomass exhibited potential anticancer activity with high correlation
flavonoids from microalgae grown in pond water and rice fields. coefficient values. They concluded that flavonoids tend to be significant
Fig. 1 represents the HPLC of various phytochemical constituents of

Fig. 1. HPLC separation of Aqueous extract of C. pyrenoidosa a) in heterotrophic culture b) in phototrophic culture.

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cause for exhibiting anticancer activity than phenolic compounds in the
biomass [21]. Hence, the flavonoids identified from HPLC analysis in this
study are used as the inhibitors for carrying out the in-silico studies to
assess for the anti-inflammatory and anti-proliferative activity of
C. pyrenoidosa.
3.2. Retrieval of ligand structures
The structures of the compounds identified from the HPLC chro-
matogram (Table 2) are given in (Fig. 2). These compounds were used for
molecular docking studies to assess their anti-proliferative and anti-
inflammatory activity.
3.3. Active site analysis
Active site analysis of 3V99 and 4L9S were carried out using SPDBV.
Active site residues for protein 3V99 (Fig. 3a)were found to be SER171,
ILE406, HIS372, HIS550, ASN554, GLN557, LEU609, GLN528, PHE117
and residues for protein 4L9S (Fig. 3b) were PHE28, LYS147, VAL29,
ASP30, ALA146, SER145, ASP119, ALA18, GLU31, LEU120, TYR32,
SER17, LYS117, ASN116, GLY15, LYS16, VAL14, GLY13, CYS12, ALA11.
3.4. Molinspiration
For the compound to be absorbed efficiently, logP value must be less
than 5.0. Compund having logP value in reasonable range is said to
possess good permeability character. Out of 7 compounds 7 have TPSA
value less than 140 Aº that implies a good optimum drug absorption
capacity. Lipinski's 'Rule of 50 was applied to identify the best lead
Fig. 2. Ligand structures.
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Informatics in Medicine Unlocked 10 (2018) 89–99
compounds. ADME property values are shown in (Table 3).
All the canonical smiles of ligands were submitted online to molins-
piration to analyze ADME properties. Caffeine, cat-
echin,protocatechicacid,Caffeoyl-D-glucose,epicatechin,dihy-
droquercetin-7,40 -dimethyl ether showed 0 violations and obeyed ‘Rule
of 5’, whereas compounds epigallocatechin gallate violated 2 properties.
Epigallocatechin gallate violated from rule by having 11 nOH and 8
nOHNH.
3.5. Molecular docking
Molecular docking was performed using selected proteins and the
ligands in Autodock vina. Binding energy values are indicated in
(Table 4). All the docking poses showed negative binding energy indi-
cating that they have good binding affinity with the target protein.
Caffeine binding energy was found to be
7.0 kcal/mol (Table 4)
with 3V99 and with 4L9S it was found to be
6.0 kcal/mol. It was found
that 3 H bonds were formed between 3V99 with 0 hydrophobic in-
teractions and caffeine (Asp 442,Arg 438,Lys 441),2 H bonds and formed
one non covalent interactions with Arg 438 residue against 4L9S (Ser
17,Asp 119). (Fig 4.1 (A)&4.2 (A)).
Protocatechic acid binding energy was found to be
6.4 kcal/mol
against 3V99,with 4L9S it was found to be
6.3 kcal/mol (Table 4)
which possessed almost equal energy with 3V99. It was found that no H
bonds were formed between 3V99 and protocatechic acid but non co-
valent interactions were formed (Arg 370 (3), Gln 549),3 H bonds against
4L9S (Lys 18,Asp 119,Lys 147) along with non-covalent interactions. (Fig
4.1 (B) & 4.2 (B)).
Catechin binding energy was observed to be
8.3 kcal/mol (Table 4)
R. Yadavalli et al. Informatics in Medicine Unlocked 10 (2018) 89–99

Fig. 3. Active site analysis of proteins. (3.1) Active site of 4L9S (3.2) Active site of 3V99.

Table 3
Molinspiration property values of Compound.

Compound miLogP1 Natoms3 M.Wt4 nON5 nOHNH6 nVio7 Nrotb8 Volume6

Caffeine 0.60 14 194.19 6 0 0 0 167.63

Protocatechic acid 0.88 11 154.12 4 3 0 1 127.08
Catechin 1.37 21 290.27 6 5 0 1 244.14
Epicatechin 1.37 21 290.27 6 5 0 1 244.14
Epigallocatechin 2.25 33 458.38 11 8 2 4 367.57
gallate
Caffeoyl-D-glucose
0.77 24 342.30 9 6 1 5 286.62
Dihydroquercetin-7,40 -dimethyl ether 1.55 24 332.31 7 3 0 3 281.38

Where (1)Mol Log P(Partition coefficient) for octanol/water (
2.0 to 6.5) (2)Number of atoms in a compound. (3)Molecular weight of the molecule should be in range between 160 and
500.(4)Estimated number of H-bond acceptors should not be more than 10. (5)Estimated number of H-bonds donors should not be more than 5. (6)Number of violations. (7)Molecular
volume.

Table 4 Dihydroquercetin-7,40 -dimethyl ether binding energy was found to be

Docking scores of 3V99, 4L9S with ligands.
7.8 kcal/mol against 3V99, with 4L9S it was observed to be
8.6 kcal/
Compound Binding energy (kcal/ Binding energy (kcal/ mol (Table 4). It was found that no H bond was formed between 3V99
mol) 3V99 mol) 4L9S and dihydroquercetin-7,40 -dimethyl ether but non covalent bonding was
Caffeine
7.0
6.0 observed between residues (Arg 518, Asn 328),1 H bond found against
Protocatechic acid
6.4
6.3 4L9S (Asn 116). (Fig 4.1 (E) & 4.2 (E)).
Catechin
8.3
9.1 Caffeoyl-D-glucose binding energy was found to be
8.0 kcal/mol
Epicatechin
8.0
8.8
Epigallocatechin gallate
9.1
8.5
(Table 4) against 3V99, with 4L9S it was found to be
8.6 kcal/mol. It
Dihydroquercetin-7,40 -
7.8
8.6 was observed that 3 H bonds were and formed between 3V99 and Caf-
dimethyl ether feoyl-D-glucose (Asp 442, Arg 438, Lys 441),2 H bonds (Ser 17, 119) and
Caffeoyl-D-glucose
8.0
8.6 three hydrophobic interactions (Ala 18 (2), Gly 15)against 4L9S. (Fig 4.1
(F) & 4.2 (F)).
Epigallocatechin gallate binding energy was found to be
9.1 kcal/
against 3V99,with 4L9S it was found to be
9.1 kcal/mol. It was found mol (Table 4) against 3V99, with 4L9S it was found to be
8.6 kcal/mol.
that only one H bond was formed between 3V99 and catechin (Leu It was observed that 2 H bonds and 7 non covalent bonds (Arg 246
289),2 H bonds against 4L9S (Lys 147,Asp 119) with no non covalent (2),Val243, Tyr 470 (2), Arg 246,Try470)were formed between 3v99 and
interactions. (Fig 4.1 (C) & 4.2 (C)). epigallocatechin gallate with same active site residue (Arg 370, Arg
Epicatechin binding energy was found to be
8.0 kcal/mol (Table 4) 370),2 H bonds and 7 hydrophobic interactions (Gly 13, Ala 18, Ser 17
with 3V99 and with 4L9S it was found to be
8.8 kcal/mol. It was found (2), Gly 15, Ser 17, Gly 15) against 4L9S (Lys 16, Lys 16) with same
that no H bond was formed between 3V99 and epicatechin but non co- amino acid in the active site region of the protein. (Fig 4.1 (G) & 4.2 (G)).
valent bonding was observed (Try 144 (2), Asp 33 (2)), 2 H bonds against It was found that most of the active site residues interacted with the
4L9S (Asp 119, Ser 145). (Fig 4.1 (D) & 4.2 (D)). ligands through covalent and non-covalent bonding. 2D (Fig 4.1, 4.2) and

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3D overlays of the docked complexes were obtained in XP Visualizer of
Schrodinger Suite (Fig 5.1 and 5.2).
4. Discussion
4.1. Flavonoids from microalgae
Many studies have focused on the biological activities of plant derived
polyphenolic flavonoids but very few were about microalgal species [22].
Hamed Safafar et al. investigated the potential of microalgae species
grown on industrial waste water as a source of antioxidants. They found
from the study that Desmodesmus sp. represented a potentially rich source
of antioxidants, containing Lutein, tocopherols, and phenolic compounds
[23]. Flavonoids are potent water soluble antioxidants which prevent
oxidative cell damage along with strong anticancer activity and
anti-inflammatory activity [3,24]. In general, total phenolic and
Fig. 4. 2D Diagrams of ligand interactions. (4.1) 2D interaction with 4L9S. (A) Caffeine (B) Protocatechic acid (C) Catechin (D)Epicatechin (E)Epigallocatechin gallate(F) Caffeoyl-D-
glucose (G) Dihydroquercetin-7,40 -dimethyl ether. (4.2) 2D interaction with 3V99. (A) Caffeine (B) Protocatechic acid (C) Catechin (D)Epicatechin (E)Epigallocatechin gallate(F) Caf-
feoyl-D-glucose (G) Dihydroquercetin-7,40 -dimethyl ether.
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Informatics in Medicine Unlocked 10 (2018) 89–99
flavonoid contents of different extracts of microalgae are solvent
dependent. In our study, aqueous extracts of C. pyrenoidosa showed
higher amount of phenolics and flavonoids, while their counterparts
showed lower concentration.
Bioactive compounds formation follows a complex pathway in uni-
cellular microorganisms like microalgae. Synthesis of secondary metab-
olites like fatty acids, carotenoids, phenolics and flavonoids, initiates
through the formation of acetyl CoA by the ACCase gene through acetyl
CoA carboxylation. This forms the key step at which carbon is assigned
for the secondary metabolite. In the present study, flavonoids were
observed in Chlorella pyrenoidosa in both autotrophic and heterotrophic
mode of nutrition. However, the production of flavonoids was observed
more in heterotrophic mode than autotrophic mode. This might be
accounted for external carbon source supplemented, which in turn form
more acetyl CoA and lead to the formation of more flavonoids. Formation
of these flavonoids may also be attributed to the dark condition, a stress
R. Yadavalli et al.
Fig. 4. (continued).
factor maintained in heterotrophic mode. In dark condition, pigments
formation is inhibited and leads to enhanced production of flavonoids by
bypassing the pathway.
4.2. Binding mode analysis of ligands with macromolecules
In the present study it was observed that Epigallocatechin gallate had
the best binding energy (BE) indicating the best possible pose with a BE
of
9.1 kcal/mol against the target molecule 3V99 (Table 5). Catechin
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Informatics in Medicine Unlocked 10 (2018) 89–99
had the BE of
9.1 kcal/mol with the protein 4L9 (Table 6). Interactions
of docked complex of each ligand with functional residues were analyzed
and inspected in Schrodinger Suite [18].
4.2.1. Anti-inflammatory activity of flavonoids
Polyphenols are a large class of compounds synthesized by fruits,
vegetables, teas, cocoa and other plants that possess certain health ben-
efits. Polyphenols are divided into several groups, one of which is rep-
resented by flavonoids. Flavonoids are a group of natural compounds that
R. Yadavalli et al. Informatics in Medicine Unlocked 10 (2018) 89–99

Fig. 5. 3D overlays of the docked complex. (5.1) 3D interactions With 3V99. (A) Caffeine (B) Protocatechic acid (C) Catechin (D)Epicatechin (E)Epigallocatechin gallate(F) Caffeoyl-D-
glucose (G) Dihydroquercetin-7,40 -dimethyl ether. (5.2) 3D interactions With 4L9S. (A) Caffeine (B) Protocatechic acid (C) Catechin (D)Epicatechin (E)Epigallocatechin gallate(F)
Caffeoyl-D-glucose (G) Dihydroquercetin-7,40 -dimethyl ether.

are categorized into flavonols, flavones, catechins, flavanones, antho- phospholipase A2, COX, and LOX. Quercetin is reported to be a strong
cyanidins, and isoflavonoids. inhibitor of both COX-2 and 5-LOX [25,26].
They exhibit anti-inflammatory activity by inhibition of Curcumin, a polyphenol of turmeric, has anti-inflammatory activity,

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Fig. 5. (continued).

due to inhibition of cyclooxygenase (COX), lipoxygenase (LOX) and Similar docking studies were conducted by Rina Herowati and Gunawan
tumor necrosis factor (TNF), and nuclear factor kappa B (NF-κB [27]. Pamudji Widodo [28] and they reported that some flavonoids and

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Table 5 Polyphenols from industrial apple waste have shown efficacy against
Comparison of interactions of selected ligands with the active site residues of 3V99. the proliferation of several human cancers cells, such as human cervical
Compound Number of Hydrogen H-Bond Non Covalent (HeLa), human hepatoma (HepG2), and human colon cancer cells (HT-
Hydrogen bond Length Interactions 29) [30,31].
bonds Interacting (Aº)
Several Molecular docking studies reported that Flavopiridol, a syn-
residues
thetic flavonoid, was best bound to DNA topoisomerase I, Green tea
Caffeoyl-D-glucose 3 Asp 442 2.43 None catechin was best docked with topoisomerase II and VEGFR-2 and
Arg 438 2.31
Lys 441 2.51
quercetin showed very good binding interaction with telomere: G-
Caffine 1 Gln 434 1.87 Arg 438 quadruplex [32].
Catechin 1 Leu 289 1.78 Ile 292, Gly In our present study Catechin exhibited good interaction with the
291 (2) protein 4L9S indicating anti proliferative activity. A similar study by
Ala 307, Asn
Seeram et al. reported that [33] eleven catechins consisting of
328
Glu 334, Arg (þ)-catechin, (
)-catechin, ()-catechin, (þ)-epicatechin, (
)-epi-
518 catechin, (
)-epigallocatechin, (
)-gallocatechin, (
)-epicatechin
Dihydroquercetin- 0 None – Arg 518 gallate, (
)-catechin gallate, (
)-epigallocatechin gallate, and (
)-gal-
7,40 -dimethyl Asn 328 locatechin gallate were tested for their anti-proliferation activity. Their
ether
Epicatechin 0 None – Trp 144, Trp
study reported that the galloyl derivatives of catechins inhibited the
144 proliferation of the cancer cell lines.
Asp 33, Asp 33
Epigallocatechin 2 Arg 370 2.39 Arg 246 (2),
5. Conclusion
gallate Arg 370 2.10 Val 243, Tyr
470 (2), Arg
246, Try 470 The extraction of flavonoids from Chlorella pyrenoidosa were carried
Protocatechic acid 0 None – Arg 370 (3) out using HPLC analysis and the anti-oxidative and anti-proliferative
Gln 549 properties of C. pyrenoiodsa were assessed by performing computa-
tional studies. The extraction of flavonoids from Chlorella pyrenoidosa
Table 6 were carried out using HPLC analysis and the anti-oxidative and anti-
Comparison of interactions of selected ligands with the active site residues of 4L9S. proliferative properties of C. pyrenoiodsa were assessed by performing
Compound Number of Hydrogen H-Bond Non Covalent
in silico studies. Computational studies were carried out using the
Hydrogen bond Length Interactions selected flavonoids with the proteins, 5-lipooxygenase (3V99) and H-ras
bonds interacting (Aº) p21 (4L9S) using Autodock Vina. Molecular docking resulted in the best
residues binding conformations of the ligands. From the docking it is evident that
Caffeoyl-D-glucose 2 Ser 17 2.23 Ala 18 (2) the best pose was obtained with least energy value. Epigallocatechin
Asp 119 1.87 Gly 15 gallate had good binding affinity with 3V99 and Catechin exhibited good
Caffine 1 Asn 116 2.16 None
interaction with the protein 4L9S. The interaction of ligands with active
Catechin 2 Lys 147 2.28 Try 32
Asp 119 2.14
site indicate that, these potential ligands play an important role as anti-
Dihydroquercetin- 1 Asn 116 2.38 Ala 18 inflammatory and anti-proliferative agents. Hence in silico analysis
7,40 -dimethyl gives scope for using C. pyrenoidosa as a target for preparation of drugs for
ether the treatment of various diseases associated with inflammation and
Epicatechin 2 Asp 119 2.27 Lys 147
proliferation such as cancers. Further studies have to be carried out to
Ser 145 2.46 Leu 120
Ala 18 prove their efficacy.
Epigallocatechin 2 Lys 16 2.22 Gly 13, Ala
gallate Lys 16 2.35 18,Ser17(2),
Acknowledgement
Gly 15, Ser 17,
Gly 15
Protocatechic acid 3 Lys 18 2.51 Ala 146 Authors would like to thank Technical Quality Improvement pro-
Asp 119 2.11 gramme (TEQIP-II),World Bank funded project for procuring Schro-
Lys 147 3.89 dinger Software used in this study. We also would like to thank our
management Sreenidhi Institute of Science and Technology for providing
phenolic compounds, i.e., amentoflavone, apigenin, bilobetin, diosmine, necessary facilities in completing our work.
epicatechin gallate, ginkgetin, hesperidin, luteolin, morelloflavon, and
quercetin, showed lower binding energy than that of tolfenamic acid, the References
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