Antidiabetic Activity of Chemical Constituents in Elaeocarpus Tectorius Fruits -An In Silico Study

Abstract

Diabetes is a metabolic disorder with an increasing global prevalence and incidence. The drugs that are used to treat diabetes are often associated with side effects and adverse reactions. Plants are considered a high esteemed source of medicine since ancient times and could be used as an alternative source for the treatment of diabetes. The present study aims to explore the phytochemical constituents and the antidiabetic potential of Elaeocarpus tectorius fruits using Gas-chromatography-mass spectrometry (GC-MS) analysis and molecular docking studies. The ethanolic extracts of Elaeocarpus tectorius fruits were analysed to determine the total phenol and flavonoid contents and then subjected to GC-MS analysis to identify the bioactive compounds. The selected bioactive compounds were then docked to some therapeutic targets involved in the pathogenesis of type 2 diabetes using Autodock 4.2.6. The drug-likeness and toxicity of the ligands were obtained from SwissADME, Protox-II and admetSAR online server tools. The GC-MS analysis revealed the presence of 28 compounds in the ethanolic extracts of E. tectorius fruits. Molecular docking studies highlighted three potential antidiabetic compounds, dibutyl phthalate, diethyl phthalate and phytol that exhibited good binding affinity with target proteins, PPAR-γ, PTP-1B, glucokinase and IL-1β. This study thus suggests that the fruits of E.tectorius have a good potential as a source of antidiabetic compounds.

Full text

Antidiabetic Activity of Chemical Constituents in Elaeocarpus
Tectorius Fruits - An In Silico Study
Keerthana M 1* & Chitra P1
1 Department of Biochemistry, Sri Ramakrishna College of Arts and Science for Women,
Affiliated to Bharathiar University, Coimbatore, Tamil Nadu, India.
Abstract:
Diabetes is a metabolic disorder with an increasing global prevalence and
incidence. The drugs that are used to treat diabetes are often associated with side effects and
adverse reactions. Plants are considered a high esteemed source of medicine since ancient
times and could be used as an alternative source for the treatment of diabetes. The present
study aims to explore the phytochemical constituents and the antidiabetic potential of
Elaeocarpus tectorius fruits using Gas-chromatography- mass spectrometry (GC-MS)
analysis and molecular docking studies. The ethanolic extracts of Elaeocarpus tectorius fruits
were analysed to determine the total phenol and flavonoid contents and then subjected to
GC-MS analysis to identify the bioactive compounds. The selected bioactive compounds were
then docked to some therapeutic targets involved in the pathogenesis of type 2 diabetes using
Autodock 4.2.6. The drug-likeness and toxicity of the ligands were obtained from
SwissADME, Protox-II and admetSAR online server tools. The GC-MS analysis revealed the
presence of 28 compounds in the ethanolic extracts of E. tectorius fruits. Molecular docking
studies highlighted three potential antidiabetic compounds, dibutyl phthalate, diethyl
phthalate and phytol that exhibited good binding affinity with target proteins, PPAR- γ, PTP-
1B, glucokinase and IL-1β. This study thus suggests that the fruits of E.tectorius have a good
potential as a source of antidiabetic compounds.
Keywords:
Diabetes, Autodock, Phytochemicals, Medicinal plants, Elaeocarpus tectorius
Introduction
Diabetes Mellitus is a metabolic disorder that occurs due to insulin secretion, action
or both. According to the World Health Organization (WHO) about 422 million people live
with diabetes worldwide [1]. Of the two major types of diabetes, Type 2 diabetes mellitus is
particularly an expanding global health problem and is closely linked to the epidemic of
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-342

obesity. It is characterized by high plasma glucose levels and is primarily caused due to
environmental factors like diet, physical activity, obesity, lifestyle and genetic factors [2] [3].
Individuals with type 2 diabetes are at a greater risk for developing complications like
retinopathy, nephropathy, neuropathy, cardiovascular diseases and skin conditions owing to
hyperglycemia and insulin resistance [4].
Type 2 diabetes mellitus (T2DM) is a complex disease involving different cellular
pathways like carbohydrate absorption, insulin secretion and insulin resistance. Some of the
proteins like peroxisome proliferator-activated receptor gamma (PPAR- γ), glucokinase,
interleukin 1 beta (IL- 1β) and protein tyrosine phosphatases are identified to play an
important role in the development of T2DM. The medical strategies that are developed to
help fight T2DM involves dietary modifications, exercises, antidiabetic and antiobesity
medications. However, these medications produce various side effects like nausea and
hypoglycaemia [5].
Herbs are used since ancient times for alleviating ailments and for the maintenance
of general health and they remain as a major natural resource in the world [6]. The
biologically active metabolites of medicinal plants and their possible therapeutic potential has
become a subject of active investigation [7]. Many plants are known for their hypoglycaemic
properties in folk medicines of different cultures and they have been found to be useful in the
treatment of diabetes. Although various synthetic drugs have been developed for treating
diabetes, their use is still limited because of their side effects and cost. Plant based medicines
are becoming popular because of their lesser toxicity and side effects. The phytoconstituents
like flavonoids, glycosides, steroids and alkaloids exhibit hypoglycaemic effects by various
mechanisms like increasing the secretion of insulin, increasing insulin sensitivity, inhibiting
the production of glucose in liver, enhancing the glucose uptake in muscle and adipose
tissues and by inhibiting the intestinal absorption of glucose [8] [9]. Treatment of T2DM with
plant based medication has been observed to protect β cells and has effects on β cell
proliferation [10]. Molecular docking analysis is a computational approach which is used to
predict the binding affinity between two or more molecules. Docking of therapeutic proteins
with ligands of interest has therapeutic applications in modern structure-based drug designing
[11].
Elaeocarpus tectorius (Lour.) Poir belongs to the Elaeocarpaceae family. It is one of
the tree species that bear edible fruits and is scarcely explored. Pharmacological studies on
other trees of Elaeocarpus species showed that they exhibit anti-inflammatory, antimicrobial,
anti-anxiety, analgesic, antidepressant, anti-asthmatic, antidiabetic, antitumor, and
antihypertensive properties. The fruit extracts of E.tectorius exhibit significant antioxidant
and antimicrobial activities [12]. The present study was aimed to investigate the bioactive
compounds and antidiabetic potential of fruits of Elaeocarpus tectorius by GC-MS analysis
and in silico molecular docking to some of the target proteins involved in the pathogenesis of
T2DM.
Materials and Methods
Collection of plant materials and Preparation of extracts:
The fruits of Elaeocarpus tectorius were collected from Coonoor, The Nilgris
district, Tamil Nadu, India. The fruits were washed to remove dust particles; the fruit pulp
was separated from the seeds and shade dried. The dried material was finely powdered using
a mortar and pestle and stored in an airtight container for further use. Five grams of powdered
plant material was macerated with 50 ml of ethanol and incubated for 48 hours in a shaker
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-343

incubator at 40ºC. The extracts were then filtered and the solvent was evaporated to get dry
extract. The amount of crude extract recovered after drying was weighed and the percentage
of extraction yield was calculated [12]. The dried extract was stored at -20°C for further use.
Extraction yield (%) = 𝑊𝑒𝑖𝑔 𝑕𝑡 𝑜𝑓 𝑡𝑕𝑒 𝑑𝑟𝑦 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 (𝑔)
𝑊𝑒𝑖𝑔 𝑕𝑡 𝑜𝑓 𝑝𝑙𝑎𝑛𝑡 𝑠𝑎𝑚𝑝𝑙𝑒 𝑢𝑠𝑒𝑑 𝑓𝑜𝑟 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑖𝑜𝑛 (𝑔) × 100
Qualitative phytochemical analysis:
Preliminary phytochemical screening was performed to identify the presence of
different phytochemical constituents such as alkaloids [13], terpenoids [14], phenolic
compounds [15], saponins [16], flavonoids [14], steroids [17] and glycosides [18] using
standard methods.
Determination of total phenolics:
The total phenolic content of E. tectorius fruit extract was estimated using Folin-
Ciocalteau method [19]. 10mg of extract was diluted with 2mL of methanol. 0.1mL of the
extract was taken in test tubes and made up to 1 ml with distilled water. Then 0.5 mL of
Folin- Ciocalteau phenol reagent (1:1 with water) and 2.5 mL of sodium carbonate solution
(20% Na2CO3) were added sequentially in each tube. Soon after vortexing the reaction
mixture, the test tubes were placed in dark for 40 min and the absorbance was recorded at 725
nm using a UV–VIS spectrophotometer against a reagent blank. The analysis was performed
in triplicates and the results were based on the calibration curve prepared using gallic acid as
a standard. The total phenolic content of the extract was expressed as milligrams gallic acid
equivalent (GAE) per gram of extract.
Determination of total flavonoids:
The total flavonoid content of the extract was investigated using the aluminium
chloride method with slight modifications [20]. 10mg of extract was diluted with 2mL of
methanol. 0.5mL of extract was taken in a test tube and added 0.1 mL of 10% aluminium
chloride solution and 0.1 mL of 0.1 mM potassium acetate solution. The mixture was kept at
room temperature for 30 minutes and the absorbance of the mixture was measured at 415 nm
using a UV–VIS spectrophotometer against a reagent blank. The analysis was performed in
triplicates and the results were based on the calibration curve prepared using quercetin as a
standard. The total flavonoid content of the extract was expressed as milligrams quercetin
equivalent (QE) per gram of extract.
Statistical analysis:
The estimation of total phenolics and flavonoids were performed in triplicates and
expressed as means ± standard deviation (SD).
Gas chromatography-mass spectrometry (GC-MS) analysis:
GC-MS analysis was carried out on a GC clarus 500 Perkin Elmer system and gas
chromatograph interfaced to a mass spectrometer (GC-MS) instrument. The following
conditions were employed. Column Elite-1 fused silica capillary column (30mm x 0.25mm
ID x 1μ Mdf, composed of 100% dimethyl poly siloxane), operating in electron impact mode
at 70eV; Helium (99.999%) was used as carrier gas at a constant flow of 1ml /min and an
injection volume of 1 ml was employed (split ratio of 10:1); Injector temperature 250°C; Ion-
source temperature 280°C. The oven temperature was programmed from 110°C (isothermal
for 2 min) with an increase of 10°C / min, to 200°C then 5°C / min, to 280°C, ending with a 9
min isothermal at 280°C. Mass spectra were taken at 70eV; a scan interval of 0.5 seconds and
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-344

fragments from 45 to 450 Da. Interpretation of mass spectrum GC-MS was conducted using
the database of the National Institute Standard and Technique (NIST) having more than
62,000 patterns. The spectrum of the unknown compound was compared with the spectrum
of the known compounds stored in the NIST library.
Molecular docking studies:
Molecular docking studies were employed to predict the binding energies between
the target proteins and selected ligands. Docking studies were carried out using Autodock
4.2.6 and the interactions between proteins and the ligands were analysed using Discovery
Studio Visualizer 2020.
Protein preparation:
Docking calculations were performed for four target proteins involved in the
pathogenesis of T2DM. The crystalline structure of proteins was downloaded from the RCSB
Protein Data Bank (https://www.rcsb.org/). All non-protein molecules and the water
molecules were removed and hydrogen atoms were added to the protein molecules and
converted to PDBQT file format (.pdbqt) using Autodock tools.
Ligand preparation:
The selected ligands to be docked with the target proteins were downloaded from the
PubChem database of the National Centre for Biotechnological Information (NCBI)
(https://pubchem.ncbi.nlm.nih.gov/) and converted to PDB file format using CORINA classic
(https://www.mn-am.com/online_demos/corina_demo). Metformin and Pioglitazone were
used as controls. Gasteiger charges were added to the ligands and converted to PDBQT file
format (.pdbqt) using Autodock tools.
Molecular docking:
Autogrid program available from Autodock tools was used for the preparation of
grid maps. The Lamarckian genetic algorithm was chosen to search for the best conformers.
The process was carried out with the default parameters of Autodock. The population size
was set to 150, maximum number of evaluations to 2,500,000, maximum number of
generations 27,000, maximum number of top individual that automatically survived 1, gene
mutation rate 0.02 and crossover rate 0.8. The results were visualized using Discovery Studio
Visualizer 2020.
Drug-likeness of the ligands:
SwissADME, a free web tool was used to generate the physicochemical and drug-
likeness properties of the ligands.
Toxicity of the ligands:
The toxicity profile of the ligands was predicted using free in silico toxicity
predictor softwares, Protox-II server and admetSAR.
Results and Discussion
Extraction yield:
Extraction is the first and main step to recover and isolate the desired
phytochemicals from plant materials [21]. The extraction yield of E.tectorius fruits using
ethanol as solvent was found to be 18.3 %. The extraction efficiency is influenced by the
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-345

chemical nature of phytochemicals, the method used, sample particle size and nature of the
solvent [22].
Qualitative phytochemical analysis:
The preliminary phytochemical screening of ethanolic fruit extract of E. tectorius
revealed the presence of phytochemicals like alkaloids, terpenoids, phenolic compounds,
flavonoids, steroids and glycosides. The screening also revealed that saponins were absent in
the extract (Table 1).
Table 1: Phytochemical screening of E. Tectorius fruit extracts
Phytochemical constituents
E.tectorius fruits
Alkaloids
+
Terpenoids
+
Phenolic compounds
+++
Saponins
-
Flavonoids
+++
Steroids
++
Glycosides
+
((+): Presence of chemical compound, (−): Absence of chemical compound (+: Low intensity
of characteristic colour, ++: Medium intensity of characteristic colour, +++: Higher intensity
of characteristic colour))
The phytochemicals produced by plants provide protection against various medical
conditions like oxidative stress, diabetes and hypertension [23]. Alkaloids have a wide range
of pharmacological effects and are medically known as analgesics, anticancer drugs,
antimalarial and antihypertensive agents [24]. Terpenoids which constitute a largest class of
natural products have numerous biological activities including analgesic, anti-inflammatory,
antioxidant, anticancer and antimicrobial activities [25]. Phenolic compounds and flavonoids
that hold an aromatic ring with at least one hydroxyl group has been reported in several
medicinal plants are widely known for their antidiabetic, antioxidant, anticancer, cardio
protective and skin protective roles [26]. Steroids are biologically important secondary
metabolites produced by plants known for their antioxidant, antibacterial and
immunostimulant activities [27]. Glycosides are organic molecules that have great
therapeutic potential including analgesic, antimicrobial and anticancer activities [28]. These
phytochemicals might contribute to the therapeutic potential of the plant.
Determination of total phenolics and flavonoid content:
The total phenolic content of the ethanolic extract of E.tectorius fruits, calculated
from gallic acid calibration curve (y = 0.009x+ 0.074, R2= 0.995) was found to be 239.63 ±
12.85 milligram gallic acid equivalents (GAE) /gram extract and the total flavonoid content
calculated from quercetin standard curve (y = 0.032x+ 0.220, R2= 0.997) was 124.53 ± 0.94
milligram quercetin (QE)/ gram extract. Phenolic compounds are strong antioxidants that
have the ability to prevent tissue damage caused by free radicals. They exhibit antidiabetic
activity by improving impaired insulin sensitivity and by up regulation of glucose transport
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-346

[29, 30]. Flavonoids are a group of hydroxylated phenolic compounds that show antidiabetic
activity by enhancing insulin secretion and promoting proliferation of pancreatic β cells [31].
Since the fruit extract contains significant amount of total phenolic and flavonoid
compounds, it might be responsible for the antidiabetic activity.
Gas-chromatography mass spectrometry (GC-MS) analysis:
The GC-MS analysis of the ethanolic extracts of E. tectorius fruits revealed the
presence of 28 different compounds (Fig.1). The identified phytochemicals with their
retention time, molecular formula and molecular weight are given in Table 2. Among the
identified phytochemicals dibutyl phthalate, 2, 3-dihydro-3, 5- Dihydroxy-6-methyl-4H-
Pyrane-4-one, n-Hexadecanoic acid was found to have anti-inflammatory, antidiabetic,
antimicrobial and antioxidant activities [32-34]. The compound, 2,3-dihydro-3,5- Dihydroxy-
6-methyl-4H-Pyrane-4-one was shown to have anti-proliferative and pro-apoptotic effects
[35]. 2-oxepanone is reported to exhibit antimicrobial, insecticidal, anticancer, antioxidant
and anti- inflammatory activities [36]. The compound, 1, 2 cyclopentanedione has antioxidant
activity [37]. Diethyl phthalate is reported to exhibit antimicrobial, acetylcholinesterase, and
neurotoxic activity [38].
Table 2: Bioactive compounds identified in the ethanolic extract of E.tectorius fruits
Name of the Compound
Retention
time (min)
Molecular
formula
Molecular
weight (g/mol)
2-amino-3-methyl-1-butanol
6.270
C5H13NO
103.16
1,2-Cyclopentanedione
6.496
C5H6O2
98.1
2,4-Dihydroxy-2,5-dimethyl-
3(2H)-furan-3-one
6.756
C6H8O4
144.13
2-ethyl 1- hexanol
6.832
C8H18O
130.23
3,4-dihydro-2H pyran
7.184
C5H8O
84.12
Methyl 3-
hydroxytetradecanoate
7.352
C15H30O3
258.4
3,4Dehydro-dl-proline
7.427
C5H7NO2
113.1
Butyl 23-hydroxy-
3,6,9,12,15,18,21-
heptaoxatricosan-1-oate
7.704
C20H40O10
440.53
Benzyl alcohol
8.140
C7H8O
108.14
2-Oxepanone
8.375
C6H10O2
114.14
4,5-Diamino-2-
8.778
C4H6N4O
126
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-347

hydroxypyrimidine
3,5-Dihydroxy-6-methyl-2,3-
dihydro-4H-pyran-4-one
9.952
C6H8O4
144.12
Azulene
10.506
C10H8
128.17
3,3,6-trimethyl-1,5-
Heptadien-4-ol
11.689
C10H18O
154.25
5-Hydroxymethylfurfural
11.899
C6H6O3
126.11
2-Methoxy-4-vinylphenol
12.377
C9H10O2
150.17
5-Methyl-5-formylamino-6-
imino-hexahydro-2-
thioxopyrimidin-4-one
13.031
C6H8N4O2S
200
1-chloro-2-methoxy- benzene
13.492
C7H7ClO
142.583
Diethyl Phthalate
16.177
C12H14O4
222.24
5-O-hexyl-d- galactitol
17.344
C12H26O6
266.33
1-O-hexyl- lyxitol
17.452
C11H24O5
236.31
1-Nonadecene
18.198
C19H38
266.50
n-Hexadecanoic acid
18.702
C16H32O2
256.42
Tetradecanoic acid
18.702
C14H28O2
228.37
Dibutyl phthalate
19.876
C16H22O4
278.34
Phytol
19.943
C20H40O
296.5310
Oleic Acid
20.614
C18H34O2
282.4614
Cyclotetracosane
24.834
C24H48
336.6379
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-348

Figure 1: GC-MS chromatogram of ethanolic fruit extract of Elaeocarpus tectorius
Phytol, a diterpene alcohol exhibits antidiabetic activity by regulating the enzymes
involved in maintaining insulin levels and thus helps in the management of type 2 diabetes. It
also has antimicrobial, anticancer and antidiuretic properties [39]. 2-Methoxy-4-vinylpehnol
is reported to have antimicrobial, antioxidant and anti-inflammatory activities [40].
Molecular docking studies:
Molecular docking studies are routinely carried out in modern drug designing for
understanding drug-receptor interactions and it is used to study the interactions between a
small molecule and a protein at atomic level [41]. The selected bioactive compounds from E.
tectorius fruit extract were docked with T2DM target proteins using Autodock 4.2.6 and their
binding energies are illustrated in Table 3. Among the selected compounds, diethyl phthalate,
dibutyl phthalate and phytol showed good binding affinity in kcal/mol. Phytol with PPAR- γ
(-7.27 kcal/mol) and dibutyl phthalate with PTP- 1B (-7.27 kcal/mol) showed highest binding
affinity among the ligands. Dibutyl phthalate had a binding affinity of -6.57 kcal/mol with
PPAR- γ and -5.8 kcal/mol with glucokinase. Diethyl phthalate had a binding affinity of -6.48
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-349

kcal/mol with PPAR- γ and -6.18 kcal/mol with PTP- 1B. Phytol had a binding affinity of
about -6.09 kcal/mol with glucokinase.
Table 3: Molecular docking of selected bioactive compounds from E. tectorius fruits
with T2DM target proteins
Ligands
Estimated free energy of binding
(Kcal/mol)
PPAR- γ
PTP 1B
GK
IL- 1β
1,2-Cyclopentanedione
-4.48
-4.6
-4.68
-4.45
2,4-Dihydroxy-2,5-dimethyl-
3(2H)-furan-3-one
-5.29
-5.07
-5.48
-4.67
2-Oxepanone
-5.08
-5.21
-5.25
-4.33
3,5-Dihydroxy-6-methyl-2,3-
dihydro-4H-pyran-4-one
-5.6
-6.05
-4.91
-4.53
2-Methoxy-4-vinylphenol
-5.28
-5.46
-5.27
-5.15
Diethyl Phthalate
-6.48
-6.18
-5.79
-4.92
1-Nonadecene
-5.7
-3.72
-5.82
-3.69
Tetradecanoic acid
-5.72
-5.52
-4.71
-4.4
Dibutyl phthalate
-6.57
-7.27
-5.8
-4.91
Phytol
-7.27
-5.03
-6.09
-4.92
Metformin
-5.84
-5.98
-7.06
-5.24
Pioglitazone
-8.53
-7.31
-8.27
-7.26
(PPAR- γ- Peroxisome proliferator activated receptor gamma, PTP 1B- Protein tyrosine
phosphatase 1B, GK- Glucokinase, IL- 1β- Interleukin 1 beta)
The docking results were visualized using Discovery Studio Visualizer 2020 for the
analysis of the interactions between the proteins and ligands. Dibutyl phthalate had strong
interaction with the target proteins. Dibutyl phthalate complex interacted with PPAR- γ
forming two hydrogen bonds (TYR 327, LYS 367) and with PTP- 1B forming four hydrogen
bonds (TYR 46, SER 216, ALA 217, ARG 221). Diethyl phthalate interacted with IL- 1β
forming one hydrogen bond (LEU 62) and with PPAR- γ forming three hydrogen bonds
(GLN 286, TYR 327, LYS 367). Diethyl phthalate interacted with PTP-1B forming one
hydrogen bond at ARG 79. Phytol interacted with PPAR- γ by forming one hydrogen bond
(ARG 288) and with PTP-1B by forming two hydrogen bonds (GLU 75, GLN 78).
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-350

Molecular docking results suggest that dibutyl phthalate and diethyl phthalate could
inhibit protein tyrosine phosphatase 1B which plays important roles in the cell growth and
differentiation through phosphorylation and dephosphorylation of tyrosine residues [42]. The
inhibitors of PTP 1B enhance the sensibility of insulin receptor and have favourable curing
effect on insulin resistant conditions associated with T2DM [43]. Phytol, dibutyl phthalate
and diethyl phthalate showed good binding interaction with PPAR- γ, which is a potential
metabolic regulator of peripheral organs and tissues, such as adipose tissue. Upregulation of
the expression of PPAR- γ has been reported to improve insulin sensitivity and glucose
uptake [44].
Figure 2: 3D display of interactions between ligands and protein. 2a: phytol and PPAR-
γ, 2b: dibutyl phthalate with PTP- 1B, 2c: diethyl phthalate and PPAR- γ
Binding of peroxisome proliferator-activated receptor-gamma (PPAR- γ) to agonists
stimulate genes that favours the storage of triglycerides, thereby lowering the circulating free
fatty acid concentrations. This causes a shift from using free fatty acids to glucose as a fuel
substrate and it is one of the mechanisms whereby they improve insulin sensitivity in
peripheral tissues [45]. The ligands also exhibited good binding affinity with glucokinase that
plays an important role in glucose homeostasis and acts as glucose sensor for insulin
secretion by pancreatic β cells [46]. The binding energies between IL- 1β and the ligands
were also appreciable. IL- 1β is a pro-inflammatory cytokine that plays an important role in
the destruction of pancreatic β cells. Interleukin 1-receptor antagonist, a natural competitive
inhibitor of IL- 1β protects pancreatic β cells from the IL-1β induced apoptosis. However, the
expression of interleukin 1-receptor antagonist is found to be decreased in the β cells of
patients with T2DM [47]. Hence natural products that can inhibit IL-1β would be useful in
the treatment of T2DM.
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-351

Figure 3: 2D display of interactions between ligands and protein. 3a: phytol and PPAR-
γ, 3b: dibutyl phthalate with PTP- 1B, 3c: diethyl phthalate and PPAR- γ
Druglikeness of the ligands:
SwissADME is a free web server which predicts whether a particular compound has
the potential to be drug-like by checking various aspects like physicochemical properties such
as molar mass, hydrogen donor, acceptor, log P value and pharmacokinetic properties like
gastro-intestinal absorption and topological polar surface area (TPSA) [48].
Table 4: Druglikeness of the selected ligands predicted by SwissADME
Ligand
Physicochemical Properties and drug-likeness
Molar
mass
(g/mol)
HBD
HBA
Log P
TPSA
(Ų)
GIA
Drug-
likeness
Dibutyl
phthalate
278.34
0
4
3.69
52.60
High
Yes
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-352

Diethyl
phthalate
222.24
0
4
2.29
52.60
High
Yes
Phytol
296.53
1
1
6.22
20.23
low
Yes
(HBD- Hydrogen bond donor, HBA- Hydrogen bond acceptor, TPSA- topological polar
surface area, GIA- Gastro-intestinal absorption)
From the docking studies, dibutyl phthalate, diethyl phthalate and phytol was
selected for druglikeness and toxicity analysis. The SMILES notation of the three
compounds, obtained from PubChem database was submitted to the SwissADME server to
analyse their drug-likeness properties and the results are summarized in Table 4. Toxicity
issues or undesirable pharmacokinetic properties are the main reasons for the failure of drug
candidates at the clinical trials [49]. The concept of drug-likeness has become an important
consideration in the selection of compounds with desirable bioavailability during the early
stages of drug discovery [50]. The ligands were checked for Lipinski’s rule of five,
Gastrointestinal (GI) absorption and topological polar surface area (TPSA). The Lipinski’s
filter of five states that for a molecule to be considered drug-like, it should fulfil four
different physicochemical parameters (H-bond donors ≤ 5, molecular weight ≤ 500, log P ≤
5, H-bond acceptors ≤ 10) [51]. TPSA is an important property for a molecule to be drug-like
since it influences the absorption, bioavailability and blood-brain barrier penetration and
should be less than 140 Ų [52]. The ligands exhibited favourable drug likeness properties.
Toxicity of the ligands:
Toxicity assessment is an important step done during the process of drug
discovery. The ligands were analysed for their toxic properties using Protox-II and
admetSAR and the results are presented in Table 5. Protox-II is a free in silico toxicity
predictor which predicts the lethal dose 50 (LD50) value in mg/kg body weight, based on
which the server has divided the results into six classes. LD50 is the dose at which 50% or
half of the test population will die upon exposure to a compound [53]. In Protox-II, the
compounds belonging to Classes 5 and 6 are generally safe for consumption and non-toxic
and specifically compounds with LD50 value of above than 5000 mg/kg (Class 6) is labelled
as the safest non-toxic class of compounds for oral consumption. admetSAR predicts the
absorption, distribution, metabolism, excretion and toxicity profile of a compound and plays
an important role in the early steps of drug discovery [54].
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-353

Table 5: Toxicity profile of the selected ligands predicted by Protox II and admetSAR
Ligand
Protox-II
admetSAR
LD50
mg/kg
Toxicity
class
BBB
permeable
HIA
Carcinogenicity
Dibutyl
phthalate
3474
5
Yes
HIA+
No
Diethyl
phthalate
6172
6
Yes
HIA+
No
Phytol
5000
5
Yes
HIA+
No
(BBB- Blood Brain Barrier, HIA- Human intestinal absorption)
Diethyl phthalate had LD50 value of 6172 mg/kg and belongs to class 6 of Protox
classification. Dibutyl phthalate had LD50 value of 3474 mg/kg and phytol had LD50 value of
5000 mg/kg and both belongs to class 5 of Protox classification and is considered safe.
admetSAR results revealed that all compounds pass through blood-brain barrier, have good
intestinal absorption and are non- carcinogenic. The ligands passed all the filters of drug-
likeness and toxicity and thus can be used as potential antidiabetic agents.
Conclusion
In this study, molecular docking analysis of some proteins involved in the
pathogenesis of T2DM was performed using Autodock against selected phytochemicals
obtained from the GC-MS analysis of fruit extract of Elaeocarpus tectorius. From the
docking analysis, it can be concluded that dibutyl phthalate, diethyl phthalate and phytol can
be used in the treatment of diabetes. This study suggests that the edible fruits of Elaeocarpus
tectorius could be a rich source of bioactive compounds that could be used to treat metabolic
disorders like diabetes. These compounds were analysed for their drug-likeness and toxic
properties and they passed all the filters. In vitro and in vivo studies are further needed to
understand their molecular mechanisms.
Acknowledgment
The authors thank Dr. R. Ragunathan, Director, Centre for Bioscience and
Nanoscience research, Coimbatore for providing the laboratory facilities to carry out this
research work and for arranging GC-MS facilities.
Conflict of interest
The authors declare that there is no conflict of interest.
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-354

References
1) Mamatha SV, Bhat M, Kumara HK, Channe Gowda D, Tirukoti M, Meenakshi SK.
Design, synthesis and SAR evaluation of mercaptooxadiazole analogs as anti-
tubercular, anti-diabetic and anti-bacterial agents. Chem Data Coll 2020;
26:100343.
2) Olokoba AB, Obateru OA, Olokoba LB. Type 2 diabetes mellitus: A review of current
trends. Oman Med J 2012; 27(4):269-273.
3) Farzaei F, Morovati MR, Farjadmand F, Farzaei MH. A Mechanistic Review on
Medicinal Plants Used for Diabetes Mellitus in Traditional Persian Medicine. J Evid-
Based Integr Med 2017;22(4):944-955.
4) DeFronzo RA, Ferrannini E, Groop L, Henry RR, Herman WH, Holst JJ et al. Type 2
diabetes mellitus. Nat Rev Dis Primers 2015; 1:15019.
5) DeFronzo RA, 2009. From the triumvirate to the ominous octet: A new paradigm for
the treatment of type 2 diabetes mellitus. Diabetes 2009;58 (4):773-795.
6) Surya S, Salam AD, Tomy DV, Carla B, Kumar RA, Sunil C. Diabetes Mellitus and
medicinal plants-A review. Asian Pac J Trop Dis 2014;4(5):337-347.
7) Khoushika Rajshree R, Chitra P. A comparative study on the in vitro anti-
inflammatory and antibacterial activities in the leaf extracts of Costus igneus and
Mangifera indica. Int J Curr Res 2016;8(8):36714-36722.
8) Dewanjee S, Das AK, Sahu R, Gangopadhyay M. Antidiabetic activity of Diospyros
peregrina fruit: Effect on hyperglycemia, hyperlipidemia and augumented oxidative
stress in experimental type 2 diabetes. Food Chem Toxicol 2009;47(10):2679-2685.
9) Saad B, Zaid H, Shanak S, Kadan S. Antidiabetic Medicinal Plants and Their
Mechanisms of Action. Anti-Diabetes and Anti-Obesity Medicinal Plants and
Phytochemicals- Safety, Efficacy and Action Mechanisms 2017; 175–213.
10) Ríos JL, Francini F, Schinella GR. Natural Products for the Treatment of Type 2
Diabetes Mellitus. Planta Med 2015;81(12/13):975-994.
11) Middha SK, Goyal AK, Bhardwaj A, Kamal R, Lokesh P, Prashanth HP, Wadhwa G,
Usha T. In silico exploration of cyclooxygenase inhibitory activity of natural
compounds found in Myrica nagi using LC-MS. Symbiosis 2016;70:169-178.
12) Manoharan AL, Thamburaj S, Muniyandi K, Jagadeesan G, Sathyanarayanan S,
Nataraj G, Thangaraj P. Antioxidant and antimicrobial investigations of Elaeocarpus
tectorius (Lour.) Poir. fruits against urinary tract infection pathogens. Biocatal Agric
Biotechnol 2019;20:101260.
13) Evans WC. Pharmacology. Singapore: Harcourt Brace and Company 1997;226.
14) Gul R, Jan SU, Faridullah S, Sherani S, Jahan N. Preliminary Phytochemical
Screening, Quantitative Analysis of Alkaloids, and Antioxidant Activity of Crude Plant
Extracts from Ephedra intermedia Indigenous to Balochistan. Sci World J
2017;5873648.
15) Mace ME. Histochemical localization of phenols in healthy and diseased banana
roots. Physiol Plant 1936;16(4):915-925.
16) Banso A, Adeyemo S, 2006. Phytochemical screening and antimicrobial assessment of
Abutilon mauritianum, Bacopa monnifera and Datura stramonium. Biokemistri
2006:18(1).
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-355

17) Kokate CK. Practical Pharmacognosy, fourth ed. Vallabh Prakashan Publication,
New Delhi, India 1999.
18) Marka R, Talari S, Penchala S, Rudroju S, Swamy Nanna R. Preliminary
phytochemical analysis of leaf, stem, root and seed extracts of Arachis hypogaea L.
Int J Pharm Sci Rev Res 2013;20(1):134-139.
19) Siddhuraju P, Becker K. Antioxidant properties of various solvent extracts of total
phenolic constituents from three different agroclimatic origins of drumstick tree
(Moringa oleifera Lam) leaves. J Agric Food Chem 2003;51: 2144 –2155.
20) Chang CC, Yang MH, Wen HM, Chern JC. Estimation of total flavonoid content in
propolis by two complementary colorimetric methods. J Food Drug Anal
2002;10:178-182.
21) Zhang QW, Lin LG, Ye WC. Techniques for extraction and isolation of natural
products: A comprehensive review. Chin Med 2018;13(20).
22) Stalikas CD. Extraction, separation and detection methods for phenolic acids and
flavonoids. J Sep Sci 2007;30(18):3268-3295.
23) Craig WJ. Phytochemicals: Guardians of health. J Am Diet Assoc 1997;97(10):S199-
S204.
24) Kuete V. Health Effects of Alkaloids from African Medicinal Plants. Toxicological
Survey of African Medicinal Plants 2014;611-633.
25) Sülsen VP, Lizarraga E, Mamadalieva NZ, Lago JHG. Potential of Terpenoids and
Flavonoids from Asteraceae as Anti-Inflammatory, Antitumor, and Antiparasitic
Agents. Evid Based Complementary and Altern Med 2017;6196198.
26) Tungmunnithum D, Thongboonyou A, Pholboon A, Yangsabai A. Flavonoids and
Other Phenolic Compounds from Medicinal Plants for Pharmaceutical and Medical
Aspects: An Overview. Medicines 2018;5(3):93.
27) B. Gunaherath GMK, Gunatilaka AAL. Plant Steroids: Occurrence, Biological
Significance and their Analysis. Encyclopedia of Analytical Chemistry; R.A. Meyers
(Ed.) 2014.
28) Khan H, Pervaiz A, Intagliata S, Das N, Nagulapalli Venkata KC, Atanasov AG et al.
The analgesic potential of glycosides derived from medicinal plants. DARU
2020;28(1):387-401.
29) Striegel L, Kang B, Pilkenton SJ, Rychlik M, Apostolidis R. Effect of Black Tea and
Black Tea Pomace Polyphenols on α- glucosidase and α- amylase Inhibition, Relevant
to Type 2 Diabetes Prevention. Front Nutr 2015;2.
30) Rupasinghe HPV, Balasuriya N, Wang Y. Prevention of Type 2 Diabetes by
Polyphenols of Fruits. Nutritional Antioxidant Therapies: Treatments and
Perspectives 2017;447–466.
31) Vinayagam R, Xu B. Antidiabetic properties of dietary flavonoids: A cellular
mechanism review. Nutr Metab 2015;12:60.
32) Roy RN, Laskar S, Sen SK. Dibutyl phthalate, the bioactive compound produced by
Streptomyces albidoflavus 321.2. Microbiol Res 2006;161(2):121-126.
33) Yu X, Zhao M, Liu F, Zeng S, Hu J. Identification of 2, 3-dihydro-3,5-dihydroxy-6-
methyl-4H-pyran-4-one as a strong antioxidant in glucose–histidine Maillard
reaction products. Food Res 2013;51(1):397-403.
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-356

34) Mujeeb F, Bajpai P, Pathak N. Phytochemical evaluation, antimicrobial activity, and
determination of bioactive components from leaves of Aegle marmelos. Biomed Res
Int 2014;497606.
35) Ban JO, Hwang IG, Kim TM, Hwang BY, Lee US, Jeong HS et al. Anti-proliferate
and pro-apoptopic effects of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4H-pyranone
through inactivation of NF-κB in Human Colon Cancer Cells. Arch Pharm Res
2007;30:1455.
36) Ishmuratov GY, Vydrina VA, Galkina YA, Yakovleva MP, Ishumuratova NM,
Tolstikov AG. Natural Seven- Membered Terpene Lactones: Synthesis and Biological
Activity. Chem Nat Compd 2015;51(6):1011-1034.
37) Alghamdi S, Migdadi H, Khan M, El-Harty EH, Ammar M, Farooq M, Afzal M.
Phytochemical profiling of Soyabean (Glycine max (L) Merr.) Genotypes using GC-
MS analysis. Phytochemicals - Source of Antioxidants and Role in Disease Prevention
2018.
38) Dubey D, Patnaik R, Ghosh G, Padhy RN. In Vitro Antibacterial Activity, Gas
Chromatography-Mass Spectrometry Analysis of Woodfordia fruticosa Kurz. Leaf
Extract and Host Toxicity Testing With In Vitro Cultured Lymphocytes From Human
Umbilical Cord Blood. Osong Public Health Res Perspect 2014;5(5):298-312.
39) Venkata Raman B, Samuel LA, Partha Saradhi M, Narashimha Rao B, Naga Vamsi
Krishna A, Sudhakar M, Radhakrishnan TM. Antibacterial, antioxidant activity and
GC-MS analysis of Eupatorium odoratum. Asian J Pharm Clin Res 2012;5(2):99–
106.
40) Guerrero RV, Vargas RA, Petricevich VL. Chemical compounds and biological
activity of an extract from bougainvillea x buttiana (var. Rose) holttum and standl.
Int J Pharm 2017;9(3):42-46.
41) Meng XY, Zhang HX, Mezei M, Cui M. Molecular docking: a powerful approach for
structure-based drug discovery. Curr Comput- Aid Drug 2011;7(2):146–157.
42) Patel AM, Anand IS, Suva MA, 2014. Role of protein tyrosine phosphatase-1B
Inhibitors in Type 2 Diabetes Mellitus. J PharmaSciTech 2014;4(1)
43) Gurzov EN, Stanley WJ, Brodnicki TC, Thomas HE. Protein tyrosine phosphatases:
Molecular switches in metabolism and diabetes. Trends Endocrinol Metab
2015;26(1):30-39.
44) Buse JB, Polonsky KS, Burant CF. Type 2 diabetes mellitus. Williams Textbook of
Endocrinology. WB Saunders 2011;1371–1435.
45) Janani C, Ranjitha Kumari BD, 2015. PPAR gene- A review. Diabetes Metab Syndr
2015;9(1):46-50.
46) Agius L. Targeting hepatic glucokinase in type 2 diabetes: Weighing the benefits and
risks. Diabetes 2009;58(1):18-20.
47) Larsen CM, Faulenbach M, Vaag A, Volond A, Ehses JA, Seifert B, Mandrup-Poulsen
T, Donath MY. Interleukin-1-receptor antagonist in type 2 diabetes mellitus. N Engl J
Med 2007;356:1517-1526
48) Daina A, Michielin O, Zoete V. SwissADME: A free web tool to evaluate
pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small
molecules. Sci Rep 2017;7:42717.
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-357

49) Walker DK. The use of pharmacokinetic and pharmacodynamic data in the
assessment of drug safety in early drug development. Br J Clin Pharmacol
2004;58(6):601-608.
50) Jia CY, Li JY, Hao GF, Yang GF. A drug-likeness toolbox facilitates ADMET study in
drug discovery. Drug Discov Today 2020;25:248-258.
51) Lipinski CA 2004. Lead and drug like compounds: The rule of five revolution. Drug
Discov Today 2004;1:337-41.
52) Veber DF, Johnson SR, Cheng HY, Smith BR, Ward KW, Kopple KD. Molecular
properties that influence the oral bioavailability of drug candidates. J Med Chem
2002;45:2615-23.
53) Banerjee P, Eckert AO, Schrey AK, Preissner R. Protox
‑
II: A webserver for the
prediction of toxicity of chemicals. Nucleic Acids Res 2018;46:W257
‑
W263.
54) Yang H, Lou C, Sun L, Li J, Cai Y, Wang Z, Li W, Liu G, Tang Y. admetSAR 2.0: web-
service for prediction and optimization of chemical ADMET
properties. Bioinformatics 2009;35(6):1067-1069.
Journal of University of Shanghai for Science and Technology
ISSN: 1007-6735
Volume 22, Issue 12, December - 2020
Page-358