South African Journal of Botany 149 (2022) 320
338

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South African Journal of Botany

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In vitro pharmacological investigations of Oxystelma esculentum R.Br. and

in silico molecular docking analysis of its leaf constituents on diabetic
related target
Thangaraj Francis Xavier*, Rajendren Sabitha, Senthilkumar Balavivekananthan
Ethnopharmacological Research Unit , PG and Research Department of Botany, St. Joseph's College (Autonomous), Bharathidasan University, Tiruchirappalli, Tamil
Nadu 620 002, India

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

Article History: The current investigation is to determine the preliminary chemical compounds present in Oxystelma esculen-
Received 14 September 2021 tum R. Br. (Apocynaceae) leaf extracts and their pharmacological activities such as antioxidant and antibacte-
Revised 3 June 2022 rial anti-diabetic, assays. Total antioxidant capacity was evaluated by DPPH, SO, FRAP, MC, and PHM assays,
Accepted 13 June 2022
and antibacterial activity was determined by the minimal inhibitory concentration method against 10 patho-
Available online xxx
genic bacteria. The a-amylase and a-glucosidase inhibitory assays were used for the enzymatic properties
Edited by: Dr V. Kumar and its molecular docking approaches were examined through the screening of chemical characterization of
Keywords:
the phytocompounds. The highest amount of total TPC and TFC obtained in ethyl acetate extract was
Diabetes mellitus 77.65 § 0.65 mg/g GAE and 130.75 § 0.01 mg/g QE, respectively. All the three extracts used in this study
Flavonoids exhibited significant antioxidant activity at various concentrations and the results mentioned in the 50% inhi-
Free radicals bition and gram equivalents were respected with the assays. Ethyl acetate, ethanol, and acetone extracts
In silico exhibited significant results at the concentration of 118.75 mg/mL against S. flexneri. The highest inhibition
Minimal inhibitory concentration was achieved by both enzyme activities, a-amylase and a-glucosidase activity on ethanol (IC50
Oxystelma esculentum 17.96 § 2.6 mg/ml) and acetone (25.35 § 1.43 mg/ml) extract respectively. GC-MS screening of ethanol leaf
extract revealed the presence of 13 phytocompounds. Among these, 6 compounds possess anti-diabetic
properties namely p-Chloroamphetamine, 1-Heptadecanol, Pentadecane, Hexadecanoic acid-2-hydroxy-1-
(hydroxymethyl) ethyl ester, Octadecanoic acid-2,3-dihydroxy propyl ester, and Solanesol were chosen for
in silico modeling against human diabetic receptor (1B2Y). From these compounds, solanosol shows less
binding affinity with the highest docking score of
7.3 than standard and other compounds. In conclusion,
Oxystelma esculentum leaf extracts have significant pharmacological properties due to the presence of various
phytocompounds.
© 2022 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction
Since ancient days, the flora is an abundant source of essential
needs and effective drugs, pharmacologically active agents have
been investigated from these medicinally important plants
(Srivastava et al., 1996; Verpoorte Et Al., 1999). They are known to be
a natural repository of substances with various biological properties
that are biologically active. Owing to their decreased side effects,
these bioactive natural remedies are increasingly used to treat a wide
Abbreviations: OE, Oxystelma esculentum; TPC, Total phenolic content; TFC, Total fla-
vonoid content; DPPH, 2,2
Diphenyl-1-picrylhydrazyl radical scavenging activity; SO,
Superoxide radical scavenging activity; FRAP, Ferric reducing antioxidant power assay;
MC, Metal chelating assay; PHM, Total antioxidant activity by phosphor-molybdenum
assay, MIC, Minimal inhibitory concentration, LC-MS, Liquid Chromatography Mass
Spectrum
* Corresponding author.
E-mail address: mycofrancis@yahoo.co.in (T.F. Xavier).
range of clinical diseases (Gupta et al., 2004). Plants have developed
an array of defense strategies (antioxidant systems) to manage oxida-
tive stress. In these systems, there is a wide variety of antioxidants
[e.g., ascorbic acid, flavonoids, glutathione, uric acid, tocopherol, car-
otenoids, and (poly) phenols], which are different in their composi-
tion, mechanism, and site of action (Krishnaiah et al., 2011).
Antioxidants have significant inhibition roles on tissue damage in
various human diseases such as inflammation, cancer, atherosclerosis
(Cupta and Sharma, 2006) and also play a significant role against free
radicals that prevent damage caused by reactive oxygen species
(ROS) (Flora, 2007; Acemi et al., 2020). The most abundant secondary
metabolites in plant tissues can be found in almost every part of the
plant, including cuticles, roots, stems, leaves, fruits, flowers, pollen,
and seeds (Venkatratnam et al., 2006; Balasundram et al., 2006). The
main subgroups of plant phenolics are phenolic acids, flavonoids, tan-
nins, stilbenes, and lignans (Dai and Mumper, 2010). These

https://doi.org/10.1016/j.sajb.2022.06.021
0254-6299/© 2022 SAAB. Published by Elsevier B.V. All rights reserved.
T.F. Xavier, R. Sabitha and S. Balavivekananthan
compounds are known for their antioxidant properties and are essen-
tial for alleviating the adverse effects of oxidative stress in plants
(Venkatratnam et al., 2006; Korkina, 2007). For this reason, during
the last few decades, they have become the subject of the study of
bioactive compounds (Loziene et al., 2007). Diabetes mellitus (DM) is
a chronic illness caused by the pancreatic' hereditary and/or acquired
insulin production deficiency or the inefficiency of the insulin
secreted by the pancreas. This affects all the body's key systems, viz.
skin, kidneys, heart, nerves, blood vessels, etc. (Srivastava et al.,
2016). This insulin deficiency leads to a rise in blood glucose levels
and damages many of the body's systems, particularly the blood ves-
sels and nerves. The incidence of diabetes is growing mainly in the
middle-income countries of the world and India will become the
world's diabetic capital by 2030 (Kaveeswar et al., 2014) and it affects
more than 422 million adults worldwide and rises from 4.7 to 8.5%
compared to 1980. Hence, Insulin is effective in improving the life
expectancy of people with diabetes to some degree, but it is not a
permanent solution, as there are some disadvantages to this proce-
dure. Therefore, it would be a need of looking for novel therapeutic
compounds, their molecular mechanism has free of adverse effects
arising from plants used in Traditional medicine (Kannur et al., 2006).
The herbal preparations are complementary and alternative medicine
(CAM) and the search for the discovery of novel compounds derived
from natural sources like herbs or plants is growing mainly due to
acquired resistance, side effects, and adverse events (AE) of allopathic
medication (Asgar, 2013; Prabakaran and Shanmugavel, 2017;
Sarian et al., 2017; Aba and Asuzu, 2018). Oxystelma esculentum R. Br.
is a threatened medicinal plant belonging to the Apocynaceae family
and is also known as ‘Jaldudhi’ (Shahzad et al., 2016; Panda, 2019). In
India, a single species has existed in this genus (Senthilkumar et al.,
2009). It is one of the plants to contain cardenolides and pregnane
glycosides, which are major classes of therapeutically important phy-
toconstituents. O. esculentum (OE) has been reported to possess
good therapeutic actions like antiseptic, depurative, and galactagogue
properties, and the decoction is used as a gargle for throat and mouth
infections (Poornima et al., 2009). This plant also has been to treat
many ailments of the current world but, has not been sufficiently
explored. While synthetic medications have significantly limited the
use of herbal remedies in just about all countries, it is essential to
reexamine this plant using recent approaches and technologies. In
order to uncover possible medications from medicinal herbs, the
present research was taken out with computer-assisted biological
research, considering the direct costs of time and money. Thus, the
present study aimed to investigate the comprehensive study of pre-
liminary phytochemical analysis, Antioxidant, Antimicrobial activi-
ties of ethyl acetate, ethanol, and acetone leaf extracts. The anti-
diabetic activity of the extracts against pancreatic a-amylase was
examined through phytoconstituents OE and in silico molecular
docking approaches. In conclusion, OE leaf extracts have phenolic
and flavonoid compounds that are responsible for total antioxidant,
anti-diabetic, and antibacterial activities. However, extensive
research is required to characterize and evaluate the importance of
documented bioactive compounds in animal models to promote their
use in pharmaceuticals and herbal medicines.
2. Materials and methods
2.1. Chemical reagents and bacterial strains
The chemical substances utilized in antioxidant and enzymatic
assays, solvents (Ethyl acetate, ethanol, and acetone), and nutrient
agar (NA) media have been procured from the local dealers (HI-
MEDIA, Mumbai, India). The buffers and reagents such as Folin-Cio-
calteau reagent, Phosphate buffer, Griess reagent, Nash reagent, Iron-
EDTA solution, Sodium phosphate buffer, Acetate buffer, p-nitro-
phenyl-a-D-glucopyranoside solution, Dimethyl sulphoxide (DMSO),
321
South African Journal of Botany 149 (2022) 320
338
and Nutrient Agar (NA) media were newly prepared while carrying
out the assays. The standard drugs Ascorbic acid, Acarbose, Quercetin,
Gallic acid, EDTA, a-amylase, and a-glucosidase enzymes and strep-
tomycin were purchased (Sigma-Aldrich and Merck Life Science Pri-
vate Limited, India). A micro-plate reader, ROBOniKreadwell Touch
ELISA Plate Analyzer (Robonik India Pvt. Ltd.), and visible spectropho-
tometer (LMSP-320, Labman Scientific Instruments Pvt. Ltd., India)
had been used for the measurements of absorbance for in vitro anti-
oxidant and anti-diabetic activity. The bacterial strains used for Anti-
bacterial activity were purchased from the Department of
Environmental Sciences and Management, Bharathidasan University,
Tiruchirappalli, Tamil Nadu.
2.2. Collection and extraction of plant samples
The plant sample was collected from a natural habitat in the
Trichy district, from September to November 2018. The experimental
plant was identified by Rapinat Herbarium, Center for molecular sys-
tematics, (Voucher No: R. S. 001) St. Joseph’s College, Tiruchirappalli.
The plant material was thoroughly washed and well dried in shade.
Then the dried plant material was ground to the fine powder, which
is used to make extraction by using soxhlet apparatus. 20 g leaf pow-
der has soaked separately in 250 mL of solvents such as ethyl acetate,
ethanol, and acetone for 24 h. Next to that, the extract of ethyl ace-
tate, ethanol, and acetone was concentrated in a rotary vacuum evap-
orator (Evator) under reduced pressure at 40 °C until solid residues
were obtained. Then the dried extracts were collected and stored at

20 °C use for in further studies. The yield of extract was calculated
using the below formula,
Amount of dried sampleðg Þ
Yield of extract ð%Þ ¼  100
Amount of extract ðg Þ
2.3. Determination of preliminary phytochemical analysis
The phytocompounds present in the yield of crude extracts were
examined by using various bio-chemicals by standard techniques
(Kokate et al., 2004; Joseph et al., 2013; Sindhu and Arora, 2012)
(Table 1). Three solvent extracts viz. ethyl acetate ethanol and ace-
tone extracts of O. esculentum were used for the anti-diabetic, anti-
oxidant and antimicrobial studies.
2.4. Determination of total phenolic content
The TPC in leaf extracts was carried out by the Folin-Ciocalteu
method (Amalraj et al., 2021). Summarily, 1.0 mL of various concen-
trations (20, 40, 60, 80, and 100 mL) of extracts was added with
0.5 mL of Folin-Ciocalteu reagent and mixed well. Then this mixture
was kept under room temperature for a few min and then the sodium
carbonate (5%, w/v) solution of 2.5 mL was added, and which was
remained at room temperature for 40 min then absorbance of blue
colour was measured by using UV-Vis spectrometer (UV-2700i/
2600i, Shimdzu, Japan) in triplicate at 725 nm. Gallic acid was the ref-
erence standard and the sum of TPC was calculated by the Calibration
curve of Gallic acid equivalent to dry extract (mg GAE/g).
2.5. Determination of total flavonoid content
The quantity of TFC in O. esculentum leaf extract was estimated by
the aluminum chloride (AlC13) colorimetric method. (Amalraj et al.,
2021) The various concentrations of the extracts were prepared (20,
40, 60, 80, and 100 mL) and the standard Quercetin was also prepared
in the various concentrations. 1 mL of extract was mixed with 2 ml of
AlC13 and 6 mL of sodium acetate and then at 430 nm, the absor-
bance was measured. To draw the calibration curve, Quercetin was
used and their results were expressed as equivalents of Quercetin
(mg QE/g).
T.F. Xavier, R. Sabitha and S. Balavivekananthan South African Journal of Botany 149 (2022) 320
338

Table 1
Phytoconstituents present in Oxystelma esculentum R. Br., leaf extracts.

S. No PhytoConstituents Chemical tests Ethyl Acetate Ethanol Acetone

1 Carbohydrates Molisch’s test + + +

2 Proteins
3 Amino acids
4 Steroids
5 Alkaloids
6 Flavonoids
7 Phenolic compounds
8 Saponins
9 Glycosides
10 Tannins
‘+’ represents presence of the constituents ‘-’ represents absence of the constituents
(2-3ml extract + Naphthol+Sulphuric acid ! Violet colour ring)
Million’s test + + +
(2ml extract+Million’s reagent ! Red colour precipitation formed)
Ninhydrin test + + +
(Filterate+lead acetate solution+Spotted+ninhydrin spray ! violet spots appeared)
Salkowski test + - +
(Extract, chloroform and con.
H2SO4 (2 ml of each)! shaking for 5 min ! stand for 5 min ! Red colour layer formed)
Mayer’s test + + +
(Extract+mayer’s reagent ! coloured creamy precipitate formed)
Alkaline reagent test + + +
(2ml extract+few drops of H2SO4 ! adding dilute HCl,
Yellow colour disappeared)
Lead acetate test + + +
(Extract+few drops lead acetate solution ! Formation of white precipitate)
Foam test + - +
(2ml extract+diluted dist. H2O 20ml equivalent+stirred for 20 min ! formation of foam layer)
Keller-kilani test + + +
(2ml extract+1ml glacial acetic acid+1 drop 5% ferric chloride+1ml Conc. HCl ! Reddish brown colour
appeared)
Gelatin test + + +
(extract 2ml+gelatin solution+shake well ! white precipitate formed)

2.6. In vitro antioxidant activity
2.6.1. Determination of radical scavenging activity using DPPH assay
DPPH free radical scavenging activity of O. esculentum ethyl ace-
tate and ethanol leaf extracts was determined by using a standard
procedure with slight changes from Amalraj et al. (2021) protocol.
Various concentrations of leaf extracts such as 50, 100,150, 200, and
250mg/mL were prepared and 100 mL of each extract were added to
100 mL of 0.1mM DPPH solution in methanol then, incubated in dark
condition at room temperature for 30 min. The absorbance was mea-
sured at 517 nm. DPPH concentration in the reaction mixture was
determined by a calibration curve (linear regression). For standard
ascorbic acid was used. The percentage of free radical scavenging
activity of OE was estimated and reported as an IC50 value. The calcu-
lation was made by the following equation.
Control OD
Sample OD
% Inhibition ¼  100
Control OD
The DPPH scavenging activities of different concentrations of the
most powerful extracts were evaluated to find the concentration that
induces 50% (IC50) inhibition.
2.6.2. Superoxide radical scavenging activity
The superoxide radical scavenging activity of OE leaf extracts was
calculated by the blue tetrazolium reduction technique according to
Amalraj et al. (2021) standard procedure. Briefly, 3mL of newly pre-
pared reaction mixture containing 20mg riboflavin, 12 mM EDTA,
and 0.1 mg NBT in 50 mM sodium phosphate buffer (pH 7.6) were
added in Hundred mL of different concentrations (50, 100, 150, 200,
and 250 mg/mL) of both leaf extracts and mixed well. Then this mixture
was incubated for 5 min at 25 °C and absorbance was measured at
590nm. For Positive control ascorbic acid was used. By the following
equation, the superoxide radical scavenging activity was calculated.
Control OD
Sample OD
Scavenging activity ð%Þ ¼  100
Control OD
2.6.3. Ferric reducing antioxidant power assay
This assay is used in the presence of 2,4,6-tri(2-pyridyl)-1,3,5-tri-
azine (TPTZ) at 3.6 pH to test the ability of plant extract to reduce Fe3
322
+into Fe2+ion (under 3.6 pH) by Amalraj et al. (2021) Methodology,
the freshly prepared FRAP solution was used by mixing 10 mM TPTZ
in 40 mM Hydrochloric Acid (HCl), 20 mM (Ferric Chloride) FeCl3 and
25 mL of 0.3 M acetate buffer (pH 3.6 using 0.3 M acetic acid and
0.3 M sodium acetate) in 1:1:10 v/v ratio and kept at 37 °C until the
solution for further use. To begin with, take 300 mL of different con-
centrations of leaf extract mixed with 2700 mL of FRAP solution. The
reaction mixture was incubated at 37 °C for 30 min in dark conditions
and 593nm was used for the measurement of absorbance. The refer-
ence of FeSO4.7H2O in the concentrations of 10, 20, 30, 40, and
50 g/mL was used and for standard, ascorbic acid was used. The value
of FRAP was reported as mmol Fe (II) equivalent/mg extract.
2.6.4. Metal chelating activity
The chelating ability of the ferrous ion of OE leaf extracts was
determined by using a standard protocol by Amalraj et al. (2021).
Both the leaf extracts were prepared at various concentrations such
as 50, 100,150, 200, and 250 mg/mL, and these extracts were added
to 50 mL of 2 mM FeCl2 and then to 200 mL of 5 mM ferrozine. This
solution mixture was made into 3mL. Afterward, the reaction mixture
was mixed well and it was kept for 10 min at room temperature. The
absorbance was measured at 562nm after the incubation period. For
positive control ascorbic acid was used. Estimation of chelating abil-
ity is expressed in Ethylenediaminetetraacetic acid (EDTA) equivalent
(mg EDTA/g extract) and with standard graph, EDTA equivalent was
compared.
2.6.5. Total antioxidant activity by phosphomolybdenum assay
The total antioxidant activity of OE leaf extract was calculated by
using phosphomolybdenum assay (Amalraj et al., 2021). The follow-
ing procedure was used for this assay, the 100 mL different concen-
trations of leaf extracts were mixed with 1 mL of reagent solution
containing 0.6 mM sulphuric acid, 28 mM sodium phosphate, 4 mM
ammonium molybdate and desired volume of 0.6 M H2SO4. After
that, the reaction mixtures were kept in a water bath at 95 °C for
90 min of the incubation period. The prepared mixture was cooled at
room temperature and at 765 nm the absorbance was measured.
Here also ascorbic acid was used as a standard. The findings were
expressed as an equivalent of ascorbic acid per gram extract (AAE)/g)
and higher absorption suggests a high capacity of antioxidants.
T.F. Xavier, R. Sabitha and S. Balavivekananthan
2.7. Evaluation of antibacterial activity
2.7.1. Bacterial strains used
The OE leaf extracts were determined against three gram-positive
and seven gram-negative bacteria respectively Staphylococcus epider-
mis MTCC 435, Bacillus subtilis MTCC 441, Rhodococcus equi MTTC
2558, Escherichia coli MTCC 40, Shigella flexneri MTCC 1457, Salmo-
nella typhi MTCC 3224, Pseudomonas aeruginosa MTCC 1748, Proteus
vulgaris MTCC 426, Enterobacter aerogenes MTCC 8559 and Vibrio
cholera MTTC 3904.
2.7.2. Determination of minimum inhibitory concentrations
The Minimum inhibitory concentrations of OE leaf extracts were
carryout by 96 well microdilution methods (Bouaziz et al., 2016).
Concisely, in each well, 50 mL of plant extracts were poured at vari-
ous concentrations (500-0.488 mg/mL obtained by double serial dilu-
tion) and 50 mL of Mueller Hinton broth were poured. Then, in each
well, 50 mL of bacterial inoculum at 106 CFU/ml was added and incu-
bated for 24 h at 37 °C. After 24 h of the incubation period, 20 mL of
0.5 mg/mL of INT (p-iodonitro-tetrazolium or 2-[4-iodophenyl]-3-[4-
nitrphenyl]-5-phenyltetrazolium chloride) was applied to each well
and which is again incubated for 30 min at 37 °C. As a positive con-
trol, ampicillin (30 0.029 mg/mL) was used. The MIC of OE leaf extract
was observed by the colour change after INT dye was applied. The
red-pink colour represents bacterial growth and No colour change
was observed to inhibit bacterial growth in the broth medium using
plant extracts.
2.8. In vitro anti-diabetic assays
Enzymatic inhibitory assays were performed for a-amylase and
a-glucosidase following the Lordon et al. (2013) protocol defined
using the solution of starch and p-nitrophenyl-a-D-glucopyranoside
(PNPG) as substrates respectively with Acarbose as reference
inhibitor.
2.8.1. Inhibitory assay of a-amylase
In this assay, 100 mL of OELE was incubated at 25 °C at different
concentrations for 10 min with 1% starch solution in a 20 mM phos-
phate buffer with a pH is 6.9 and with 6 mM NaCl. Then 100 ml of the
enzyme a-amylase (0.5 mg/ml) enzymes to each tube were added
and the tubes were incubated for ten minutes at 25 °C. 200 mL of
dinitrosalicylic acid reagent was added to terminate the hydrolytic
reaction and the reaction mixture was incubated for 5 min at 100 °C.
The samples then were cooled to room temperature and then the
50 mL reaction mixture was transferred to a micro-plate (96 well)
and diluted by adding 200 mL of distilled water. The absorbance was
estimated at 540 nm and the proportion of OELE of a-amylase inhibi-
tion was determined as follows,
Absorbance of extract
% Activity ¼  100
Absorbance of Control
2.8.2. Inhibitory assay of a- glucosidase
For this analysis, In a 100 mM sodium phosphate buffer (pH 6.9),
50 mL of various concentrations of OELE were combined with 50 mL
of 5 mM PNPG solution (in a phosphate buffer) to start a hydrolytic
reaction. The mixture was incubated for 5 min at 37 °C and then each
well was added with a phosphate buffer (100 mL) containing
0.1 U/mL of a-glucosidase. The absorbance was measured at 405 nm
after 30 min, and the percentage of a-glucosidase inhibition of OELE
was calculated by using the following formula.
Absorbance of extract
% Activity ¼  100
Absorbance of Control
323
South African Journal of Botany 149 (2022) 320
338
2.9. Identification of phytocomponents by GCMS & LC-MS analysis
The phytocompounds present in ethanol leaf extract of OE were
analyzed by Gas chromatography - Mass spectrometry (GC-MS) tech-
nique. Schimadzu (QP2020) mass spectrometer was used for carrying
out the GCMS analysis and this has SH-Rxi-5Sil-MS non-polar,
0.25 mm inner diameter, 100% polydimethylsiloxane coated film
with 0.25mm thickness and 30 m length capillary column. The initial
oven temperature was maintained at 50oC at a rate of 6oC/min with
a 2 min final hold time. The temperature of the injector was adjusted
to 250 °C. Helium was employed as the carrier gas, with a flow rate of
1.2 mL/min and a linear velocity of 39.7 cm/sec, and a pressure of
68.1 kPa. A split ratio of 1:10 was used to inject one mL of leaf extracts
dissolved in hexane into the GC. The mass spectra were measured in
a scan range of 50 to 500 amu and were obtained in electron ioniza-
tion mode at 70 eV. The temperature of the ion source was kept con-
stant at 200 °C. The mass spectra of each chemical found in the
extract were compared and matched with those of standard spectra
in the NIST 2005 MS collection and literature for compound interpre-
tation. The average peak area total area was used to calculate the rel-
ative percentage of each compound.
The chemical constituents of the ethanol extracts were deter-
mined using LC-MS. LC-MS analysis was performed using Electro-
spray ionization mass spectra (ESI-MS) were obtained on a Agilent
6530 Q-TOF-MS (Agilent Technologies, USA). Malvern Nanosizer ZS
instrument was used to determine the hydrodynamic radius MS
spectra were acquired in the negative ion mode. The mass fragmenta-
tions were identified by using a spectrum database for organic com-
pounds in the SDBS application.
2.10. In silico anti-diabetic activity by molecular docking studies
The determinations of chemical interactions of the OE phytocom-
pounds were performed by molecular docking analysis and human
pancreatic alpha-amylase (1B2Y) was used as the targeted protein.
2.10.1. Preparation of ligand
All the two-dimensional chemical structure of selected phytoli-
gands (Fig. 1) was retrieved using ChemDraw Professional 16.0 soft-
ware and it was saved in mol format. Acarbose (PubChem CID:
444254) was selected as a reference drug. The selected standard drug
was downloaded in SDF format using the PubChem database. All the
ligands were converted into their pdbqt format using the OpenBabel
GUI version 2.4.1 molecular converter tool.
2.10.2. Preparation of target protein
The selected target protein was the structure of human pancreatic
alpha-amylase in complex with the carbohydrate inhibitor acarbose
(PDB ID: 1B2Y) (Nahoum et al., 2000). The selected target protein
was classified under hydrolase and its structure was crystallized by

the X-ray diffraction method having a resolution of 3.2 A. The PDB
format of the selected target protein was retrieved from the RCSB
Protein Data Bank database (Thirumalaisamy et al., 2018). The opti-
mization of the target protein was performed by eliminating the het-
eroatoms and water molecules.
2.10.3. Molecular docking study
The optimized ligands were docked with the human alpha-amy-
lase target protein (PDB ID: 1B2Y) using AutoDock 4.2.6 software.
The grid box was covered the selected flexible residues and the
dimension size of grid box is X = 15.477, Y = 20.347, Z = 50.465 and
the center of ligand molecule: 0.330, 0.004 and 0.031. To explore the
molecular docking analysis, the Lamarckian genetic algorithm (LGA)
with a maximum of 2.5 million energy evaluations was used. The
binding energy for the topmost conformation of the docked complex
was predicted using AutoDock 4.2.6 software. The protein-ligand
T.F. Xavier, R. Sabitha and S. Balavivekananthan South African Journal of Botany 149 (2022) 320
338

Fig. 1. 2D confirmations of selected phytoligands

(a) p Chloramphetamine
b) 1-Heptadecanol
(c) Pentadecane
(d) Hexadecanoic acid-2-hydroxy-1-(hydroxymethyl)ethyl ester
(e) Octadecanoic acid-2,3-dihyroxypropyl ester
(f) Solanesol.

324
T.F. Xavier, R. Sabitha and S. Balavivekananthan
binding interactions for the topmost conformation of the docked
complex were interpreted using the Discovery studio image Generate
tool.
2.10.4. Pharmacokinetics analysis
To predict the pharmacokinetic characteristics of phenolic sub-
stances, two-line methods were used (Alamri et al., 2020). Molecular
weight (MW), hydrogen bond donors (HBD), hydrogen bond accept-
ors (HBA), rotatable bond (RB), and topological polar surface area
were initially determined using the Swiss ADME tool (TPSA), and the
five Lipinski rules were used to evaluate them. The pkCSM server was
then used to determine the absorption, distribution, metabolism,
excretion, and toxicity of docked phytochemicals (Tables 7 and 8).
2.10.5. Molecular mechanics with generalised born and surface area
(MM/GBSA)
Following docking, the docked ligand-protein complex was used
to explore the drug robustness of the phytochemicals using Amber
tools; specifically, it was used to calculate the MM-GBSA energy lev-
els of docked ligand molecules with the a-amylase. This was done
according to the method of Yunta (2016) as follows:

trations (100 mg/ml) of the extracts. Whereas, already mentioned in
DGbind ¼ Gcomplex

Gprotein þ Gligand
2.11. Statistical analysis
The results of all assays were statistically analyzed, each sample of
each assay was expressed in a minimum of three replicates, and the
data were represented as mean § standard deviation. The variation
and multiple comparisons between the samples and assays were ana-
lyzed by one-way ANNOVA followed by Tukey’s multiple comparison
test (SPSS, version 17). p<0.05 (Anti-oxidant), and p<0.0001 (Anti-
diabetic assay) values are accepted as statistically significant.
3. Results and discussions
3.1. The yield of crude extracts
The total yield of crude extracts of OE leaves using ethyl acetate,
ethanol, and acetone solvents were expressed in percentages viz.,
2.61%, 9.76%, and 5.49%, respectively (Table 2). Further studies on
enzymatic and antioxidant activities of OE ethyl acetate, ethanol, and
acetone extracts were conducted based on their polarity, percentage
of total yield, and the presence of phytochemicals in the plant
extracts.
3.2. Preliminary phytochemical analysis
The phytocompounds in OE leaf extracts (Ethyl acetate, ethanol,
and acetone) were determined by various tests. The results showed
that phytocompounds like proteins, alkaloids, flavonoids, tannins,
Table 2
The yield of extract, Total phenolic and flavonoid content, a-amylase and a-glucosidase activities of O. esculentum leaf extracts.
Solvents Yield of extract % TPC mg/g GAE$
Ethyl acetate 2.61 77.65 § 0.65
Ethanol 9.76 46.22 § 0.821
Acetone 5.49 27.76 § 0.05
Acarbose - -
$
milligram per gram equivalent of gallic acid.
#
milligram per gram equivalent of quercetin.
* The Inhibitory concentration of the sample required to inhibit the enzymes activity by 50%. The values of result mentioned as means
(n=3)§standard deviation. Same letters as superscript in same column (for different extracts/samples) are not significantly different
according to Tukey’s test (p<0.0001).
South African Journal of Botany 149 (2022) 320
338
carbohydrates, amino acids, phenolic compounds, and glycosides
were present in all three extracts (Table 1). Whereas, steroids and
saponins were found in ethyl acetate and acetone extracts.
3.3. Determination of total phenolic and flavonoid content
The results of the total phenolic content of the OE leaf extracts
were estimated as milligram per gram equivalent to Gallic acid and
the linear regression equation of the standard curve (y = 0.0222x-
0.0414, R2 = 0.9646) was used for the calculation of the TPC present
in OELE (Table 2). The highest amount of total TPC obtained in ethyl
acetate extract was 77.65§0.65 mg/g GAE followed by ethanol and
acetone extracts at 46.22§0.821 and 27.76§0.05, respectively. The
results of the total flavonoid content of the OE leaf extracts were esti-
mated as milligram per gram equivalent of Quercetin and the linear
regression equation of the standard curve (y = 0.0319x-0.05254,
R2 = 0.9792) was used for the calculation of the TFC present in OELE.
The highest amount of TFC found in ethyl acetate extract of OE was
130.75§0.01 mg/g QE followed by ethanol and acetone extracts was
112.89§0.45 and 102.12§0.03, respectively. The results of TPC and
TFC of OEE the highest results were observed in the highest concen-
the above statement ethyl acetate is the high polarity solvent fol-
lowed by ethanol and acetone extracts, based on this the results also
varied.
3.4. In vitro antioxidant activity
In vitro antioxidant activity of various concentrations of OE leaf
extracts (ethyl acetate, ethanol, and acetone) was analyzed by five
assays; the results of each sample were compared with the standard
ascorbic acid shown in Table 3.
3.4.1. Determination radical scavenging activity by 2, 2,-diphenyl-
1picrylhyrazyl assay
In the DPPH assay, the highest inhibitory activity was obtained in
ethanol extract of OE with IC50 of 69.4§0.41mg/ml (Table 3) followed
by ethyl acetate and acetone extracts 78.66§0.24 and 103.23§0.69
mg/ml these are comparatively lesser than the standard antioxidant
compound ascorbic acid (13.23§0.35mg/ml). The result of the DPPH
assay is worked based on radical scavenging ability or hydrogen
donating principle.
3.4.2. Superoxide radical scavenging activity
The result of the FRAP assay revealed the best inhibition was
obtained in ethanol extract 1680.90§322.45 mM Fe (II) E/mg fol-
lowed by ethyl acetate and acetone extracts (1525.67§360.26 and
492.33§38.9 mM Fe (II) E/mg). Here, ethanol and ethyl acetate
extracts exhibit a highly significant result than the standard antioxi-
dant compound ascorbic acid (1127.10§11.64 mM Fe (II) E/mg)
shown in Table 3.
TFC mg/g QE# a-amylase IC50 (mg/ml)* a-glucosidase IC50 (mg/ml)*
130.75 § 0.01 18.7 § 2.08ab 60.56 § 6.22ab
112.89 § 0.45 17.96 § 2.6ab 35.33 § 1.76ab
102.12 § 0.03 20.28 § 8.97ab 25.35 § 1.43ab
- 15.28 § 0.43 14.51 § 0.23
325
T.F. Xavier, R. Sabitha and S. Balavivekananthan South African Journal of Botany 149 (2022) 320
338

Table 3
Total antioxidant activity of Oxystelma esculentum R. Br. leaf extract.

Solvents DPPH SO FRAP PHM MC

Ethyl acetate 78.66 § 0.24a 30.55 § 9.80b 1525.67 § 360.26c 129.22 § 6.26ab 751.00 § 18..87e
Ethanol 69.40 § 0.41a 27.06 § 5.47b 1680.90 § 322.45d 145.70 § 13.45ab 691.00 § 22.56cd
Acetone 103.23 § 0.69a 19.83 § 4.83b 492.33 § 38.90d 140 § 11.34ab 612.94 § 28.44eh
Ascorbic acid 13.23 § 0.35 11.34 § 0.75 1127.10 § 11.64 147.19 § 0.64 359.33 § 0.00
Values in IC50 (mg/mL); DPPH - Radical scavenging activity using DPPH method; radical scavenging activity; SO - Super-
oxide radical scavenging activity(mg/ml); PHM - Phosphomolybdenum assay (mg/g); FRAP - Ferric reducing-antioxi-
dant power assay (mM Fe (II) E/mg); MC - Metal chelating activity (EDTA E mg/g). Values are expressed as mean (n=3)
§ standard deviation; same letters as superscript in same column (for different extracts/samples) are not significantly
different according to Tukey’s test (p<0.05).

3.4.3. Ferric reducing antioxidant power assay
The result of the FRAP assay revealed the best inhibition was
obtained in ethanol extract 1680.90§322.45 mM Fe (II) E/mg fol-
lowed by ethyl acetate and acetone extracts (1525.67§360.26 and
492.33§38.9 mM Fe (II) E/mg). Here, ethanol and ethyl acetate
extracts exhibit a highly significant result than the standard antioxi-
dant compound ascorbic acid (1127.10§11.64 mM Fe (II) E/mg)
shown in table 3.
extracts were inhibited P. vulgaris, ethanol, and acetone extracts
were inhibited P. aeroginosa as well as S. epidermis in ethanol extracts
(Fig. 3). Here, lesser results were exhibited at the highest concentra-
tion (950 mg/mL) against E. aerogenes shown in Table 4. Whereas, E.
coli and R. equi organisms are susceptible even at higher concentra-
tions of all the extracts (>950 mg/mL).
3.6. In vitro anti-diabetic activity

3.4.4. Determination of chelating ability by metal chelating assay
The ethyl acetate, ethanol and acetone extracts of OE exhibited
significant results by metal chelating assay expressed as EDTA equiv-
alent. Table 3 showed the significant chelating ability was 751.83§
18.87, 691§22.56, and 612.94§28.44 mg EDTA/g (ethyl acetate, etha-
nol, and acetone, respectively). The results of the EDTA standard
were compared with the reference standard of ascorbic acid
(359.33§0 mg EDTA/g).
3.4.5. Phosphomolybdenum assay (PHM)
The total antioxidant activity of OE leaf extracts and with the stan-
dard was determined with IC50 of 147.19§0.64, 129.22§6.26,
140.33§11.34, and 145.7§13.45 mg AAE/g (Ascorbic acid, ethyl ace-
tate, acetone and ethanol extracts, respectively). The results were
obtained by the reduction of PHM (VI) to PHM (V) principle owing to
the leaf extracts antioxidant capacity and the subsequent formation
of green PHM complex at low pH. When compared with the standard,
OELE exhibits significant results (Table 3).
3.5. In vitro antibacterial activity by minimal inhibitory concentration
method
The present study is covered with the ethyl acetate, ethanol, and
acetone extracts that were tested against various pathogenic bacteria
by the Minimal inhibitory concentration method, and the results
were shown in Table 4. S. flexneri was exhibited significant results in
lower concentrations of Ethyl acetate, ethanol, and acetone extracts
(118.75 mg/mL) in the same concentration of ethanol extract were
inhibited V. cholera followed by 237.5 mg/mL concentration of all the
In vitro anti-diabetic activity of OE leaf extracts were assessed by
the enzymatic method viz. a-amylase and a-glucosidase assays. The
present study reveals the inhibitory effect of a-amylase on OE etha-
nol, ethyl acetate and acetone showed the highest results with IC50
values of 17.96§2.6, 18.7§2.08, and 20.28§8.97 mg/ml. While Fig. 2
showed OE extracts (17.96§2.6 97 mg/ml) exhibit a comparatively
similar level of inhibitory effect to standard Acarbose (15.28§0.43
mg/ml). The significant result of the a-amylase activity of OE extract
was shown in Table 2. The a-glucosidase inhibitory activity on ethyl
acetate, ethanol, and acetone extract was mentioned with IC50 of
60.56§6.22, 35.33§1.76, and 25.35§1.43 mg/ml, respectively. Here,
the a- glucosidase inhibitory effect is lower than the standard Acar-
bose with an IC50 of 14.51§0.23 mg/ml.
3.7. Identification of phytoconstituents by GCMS & LC-MS technique
The phytoconstituents present in ethanol leaf extract of OE were
characterized through GCMS analysis. The results of GCMS analysis
revealed the identification and quantification of phytoconstituents
present in ethanol leaf extract of OE with 100% total extract were
shown in Table 5. The GCMS chromatogram represents the peak
areas of the phytoconstituents (Fig. 4). There are 13 compounds viz.
P-Chloroamphetamine, 1-Heptadecanol, Pentadecane, Cycloheptasi-
loxane, Tetradecamethyl-, Cyclooctasiloxane, Hexadecamethyl-,
Cyclononasiloxane, Octadecamethyl-, 2,2,4,4,6,6,8,8,10,10,12,12,14,
14,16,16,18,18,20,20-Icosamethylcyclodecasiloxane, Hexadecanoic
Acid, 2-Hydroxy-1-(Hydroxymethyl)Ethyl Ester, Octadecanoic Acid,
2,3-Dihydroxypropyl Ester, Tetracosamethyl-Cyclododecasiloxane,
Solanesol, 1,2-Benzenedicarboxylic Acid, Dibutyl Ester, 1,2-

Table 4
Antibacterial activity of Oxystelma esculentum leaf extracts by Minimum inhibitory concentration method.

Extracts Minimum inhibitory concentration (mg/ml)

EC PV SE BS RE VE ST SF PA EA

OE A >950 237.5 950 475 >950 950 475 118.75 237.5 950
OE EA >950 237.5 59.375 475 >950 950 475 118.75 950 950
OE E >950 237.5 237.5 237.5 >950 118.75 475 118.75 237.5 950
Ampicillin 7.5 7.5 7.5 7.5 7.5 7.5 1.875 3.75 3.75 7.5
(Standard)
EC - E.coli; PV - Proteus vulgaris; SE - Staphylococus epidermis; BS - Bacillus subtilis; RE - Rhodococcusequi; VC

Vibrio cholera; ST - Salmonella typhi; SF - Shigellaflexneri; PA - Pseudomonas aerogenes; EA
-Enterobacteraeroegenes.

326
T.F. Xavier, R. Sabitha and S. Balavivekananthan South African Journal of Botany 149 (2022) 320
338

Fig. 2. a-amylase and a-glucosidase inhibitory activity of O. esculentum leaf extracts at different concentrations. Values are expressed as means § SD (n = 3).

Fig. 3. Antibacterial activity of O. esculentum leaf using various solvent extracts at different concentrations by Minimal Inhibitory Concentration. Values are expressed as means §
SD (n = 3).
EC- Escherichia coli; PV- Proteus vulgaris; SE- Staphylococus epidermis; BS- Bacillus subtilis; RE- Rhodococcus equi; VC- Vibrio cholerae; ST- Salmonella typhi; SF- Shigella flex-
neri; PA- Pseudomonas aeruginosa; EA-Enterobacter aeroegenes.

Benzenedicarboxylic Acid, Disooctyl Ester were identified. Among
these Solenosol (89.42%) is the major constituent and this is reported
to have antibacterial and antiviral properties (Li and chase, 2010;
Yan et al., 2015). P-Chloramphetamine is the compound (0.38%) pres-
ent in low amounts. But this is the first report of this compound in
the plant species of O. esculentum and this has not been reported in
any other species of plants. Thus, based on the percentage of peak
area 6 compounds were chosen for insilico anti-diabetic molecular
docking studies (Fig. 1). When compare with GC-MS the
327
phytocompound 1-pentadecane only the compound predicted from
LC-MS spectrum in the retention time of 0.5 min with a peak value of
629.13. The peak of the compound is shown in Fig. 11.
3.8. Molecular docking studies
Based on the in vitro study report, a-amylase was chosen for fur-
ther in silico molecular docking analysis. This is the first report of in
silico molecular docking studies of OE phytoconstituents. Totally 6
T.F. Xavier, R. Sabitha and S. Balavivekananthan South African Journal of Botany 149 (2022) 320
338

Table 5
Phytoconstituents of O. esculentum R. Br. screened by CG-MS and their biological activity.

Sl. No Compound Name Rtn. Time % Area Mol. Formula Mol. Wt. Biological Activity Ref.

1 P-Chloroamphetamine 6.107 0.38 C9H12ClN 169 - -

2 1-Heptadecanol (Aliphatic alcohol) 15.921 0.08 C17H36O 256 Antimalarial, antifungal, Antioxi- Farina et al. (2014),
dant, Antidiabetic Ramalakshmi (2019)
3 Pentadecane (Aliphatic hydrocarbon) 16.096 0.13 C15H32 212 Antibacterial Yogeswari et al. (2012).
Rahbar et al. (2012)
4 Cycloheptasiloxane, Tetradecamethyl-(Organo 17.526 0.9 C14H42O7Si7 518 Antimicrobial, anticancer, Markus et al. (2017), Osaro-
Silicone compound) Antioxidant Matthew et al. (2020)
Antiseptic
5 Cyclooctasiloxane, Hexadecamethyl- 20.771 1.03 C16H48O8Si8 592 Antimicrobial Osaro-Matthew et al. (2020)
6 Cyclononasiloxane, Octadecamethyl- 32.324 2.02 C18H54O9Si9 666 Antioxidant Markus et al. (2017)
7 2,2,4,4,6,6,8,8,10,10,12,12,14, 26.07 0.54 C20H60O10Si10 740 Antioxidant Al Bratty et al. (2020)
14,16,16,18,18,20,20- Antimicrobial
Icosamethylcyclodecasiloxane
8 Hexadecanoic Acid, 2-Hydroxy-1-(Hydroxy- 34.66 0.3 C19H38O4 330 Antibacterial Gavamukulya et.al. (2015),
methyl)Ethyl Ester antioxidant, hemolytic, anti- Teh et. al. (2017),
(Palmitoyl glycerol-amino compound) androgenic, nematicide and Tyagi and Agarwal (2017)
pesticidal; antimicrobial
9 Octadecanoic Acid, 2,3-Dihydroxypropyl Ester 37.255 0.29 C21H42O4 358 Antidiabetic Murugesu et. al. (2018)
10 Tetracosamethyl-Cyclododecasiloxane 38.918 4.34 C24H72O12Si12 888 Hepatoprotective, Al Bratty et al. (2020)
antispasmodic, antirheumatic
11 Solanesol 38.59 89.42 C45H74O 630 Antibacterial, antiviral Yan et al. (2015)
Li and chase (2010)
12 1,2-Benzenedicarboxylic Acid, Dibutyl Ester 26.524 0.22 C16H22O4 278 Antibacterial, Antifouling, Ogunlesi et al. (2009),
(Plasticizer compound) Antimicrobial, Pesticide, Mary and Giri (2018)
Repellent.
13 1,2-Benzenedicarboxylic Acid, Disooctyl Ester 34.872 0.35 C24H38O4 Antimicrobial, Solvent, Plasti- Mary and Giri (2018)
(Plastilizer compound) lixer, Pesticide, Repellent.

Fig. 4. GC-MS chromatogram of O. esculentum ethanol leaf extract.

compounds were selected and docked with human pancreatic alpha-
amylase compared with standard reference Acarbose. When com-
pared with Acarbose (reference drug) and also all the other selected
phytoligands, the phytoligand Solanesol has the better binding affin-
ity with a docking score of
7.3 mM towards the human pancreatic
alpha-amylase (1B2Y) (Table 6). The Discovery studio image program
automatically generates schematic 2-D representations of protein-
328
ligand complexes from standard Protein Data Bank file input. The
output is a colour, or black-and-white, PostScript file giving a
simple and informative representation of the intermolecular
interactions and their strengths, including hydrogen bonds,
hydrophobic interactions, and atom accessibilities. Figs. 5
10 pre-
cisely shows the binding contacts among the phytoligands and
the target protein.
T.F. Xavier, R. Sabitha and S. Balavivekananthan South African Journal of Botany 149 (2022) 320
338

Table 6 of binding are -54.532 for solanesol, -37.632 for 2,3-dihydroxypropyl

Docking score of selected phytoligands using Auto dock software. octadecanoate, -32.432 for p-chloroamphetamine, -30.372 for 1-hep-
Ligand Docking Score (kcal/mol) Rank for best drug tadecanol and -29.264 for 2-hydroxyhexadecanoic acid. 1-(hydroxy-
methyl) ethyl ester and pentadecane. On the other hand, the
p Chloramphetamine
5.3 2
corresponding molecule acarbose was found to be -40.263 (Table 9).
1-Heptadecanol
4.9 3
Pentadecane
4.7 4 According to the MM-GBSA calculation, the phytochemicals were
Hexadecanoic acid-2-hydroxy-
4.0 6 estimated at probable binding energy values with a-amylase.
1-(hydroxymethyl)ethyl ester
Octadecanoic acid-2,3-dihyroxy-
4.5 5
propyl ester 4. Discussion
Solanesol
7.3 1
Acarbose
7.7 standard Phytochemicals are non-nutritive plant chemicals with protective
Lesser the binding energy (the more negative of the free binding energy results in the or disease-prevention properties. Such compounds are found in
formation of stronger complexes) is the better binding of the ligand and protein. plants to protect themselves, but current research suggests that they
can also defend humans and animals from many diseases and infec-
tions (Ngbede et al., 2008; Kubmarawa et al., 2008). The phytochemi-
3.8.1. MM-GBSA cal screening of our study revealed similar results to Savitha and
As shown in Table 9, the MM-GBSA exploration showed that the Balamurugan (2014) research findings. Pandya and Anand (2011)
energy values of the phytochemicals were calculated. The free energy reported that alkaloids are absent in petroleum ether extract.

Table 7
The ADME parameters of compound from O.esculentum via Swissadme.

Molecule No Formula MW Rotatable H-bond H-bond MLOGP Log S Log S (Ali) Log S pharmacokinetis Lipinski
bonds acceptors donors (ESOL) (Silicos-IT) violations
GI absorption CYP enzymes inhibitor

1 C9H12ClN 169 2 1 1 2.76
2.69
2.62
3.49 High No 0

2 C17H36O 256 15 1 1 4.7
5.26
7.91
6.17 High No 1
3 C15H32 212 12 0 0 6.19
5.24
7.58
5.92 Low No 1
4 C19H38O4 330 18 4 2 3.18
4.57
7.32
5.3 High No 0
5 C21H42O4 358 20 4 2 3.63
5.41
8.64
6.1 High No 0
6 C45H74O 888 25 1 1 9.26
12.54
17.16
10.52 Low No 2

Table 8
ADMET profiling parameters of explored compounds.

Ligand p-Chloramphetamine 1-Heptadecanol Pentadecane Hexadecanoic acid-2-hydroxy-1- Solanesol

(hydroxymethyl)ethyl ester

Absorption
Water solubility
1.632
7.221
7.861
5.334
4.644
Caco2 permeability 1.49 1.463 1.376 0.439 1.135
Intestinal absorption (human) 90.949 89.46 91.389 90.277 83.989
Skin Permeability
1.812
2.61
2.117
2.797
2.735
P-glycoprotein substrate No No No No No
P-glycoprotein I inhibitor No No No Yes No
P-glycoprotein II inhibitor No No No No Yes
Distribution
VDss (human) 0.976 0.476 0.664
0.261
0.501
Fraction unbound (human) 0.665 0.08 0.074 0.201 0.165
BBB permeability 0.358 0.817 0.92
0.263 0.988
CNS permeability
1.97
1.683
1.471
3.316
0.578
Permeability
CYP2D6 substrate No No No No No
CYP3A4 substrate No Yes Yes No Yes
CYP1A2 inhibitior Yes Yes Yes No No
CYP2C19 inhibitior No No No No No
CYP2C9 inhibitior No No No No No
CYP2D6 inhibitior No No No Yes No
CYP3A4 inhibitior No No No No No
Excretion
Total Clearance 0.819 1.91 1.811 2.004 1.863
Renal OCT2 substrate No No No No No
Toxicity
AMES toxicity No No No No No
Max. tolerated dose (human) 0.497
0.028 0.179 0.332
0.214
hERG I inhibitor No No No No No
hERG II inhibitor No Yes No No Yes
Oral Rat Acute Toxicity (LD50) 3.065 1.591 1.52 1.537 2.265
Oral Rat Chronic Toxicity (LOAEL) 1.631 1.105 1.342 2.722 0.312
Hepatotoxicity Yes No No No No
Skin Sensitisation Yes Yes Yes Yes No
T.Pyriformis toxicity 0.118 1.808 1.989 0.608 0.285
Minnow toxicity 1.172
1.17
1.168
0.487
5.656

329
T.F. Xavier, R. Sabitha and S. Balavivekananthan
Fig. 5. a. Docking pose of the p-Chlorampetamine with 1B2Y protein
a. Docking pose of the p-Chlorampetamine with 1B2Y protein
b. The 2D template discloses the variety of amino acid residures interactions of p-Chlorampetamine with 1B2Y protein complex.
Similarly, Paul et al. (2017) also mentioned alkaloids are absent in
hexane extract. The phenolic compounds like polyphenols and flavo-
noid contents were affected by the polarity of the solvents. According
to Naima et al. (2015), the solubility of the phytochemicals in plant
extracts is decreased, due to the polarity of the solvents. Several
researchers also found that extremely polar solvents are more
330
South African Journal of Botany 149 (2022) 320
338
effective than less polar solvents in extracting phenolic compounds
from plant materials (Fatin et al., 2012; Hatipoglu et al., 2013). The
researchers discovered that ethyl acetate was the best for extracting
phenolic compounds from the leaves of Tabernaemontana cathari-
nensis, another plant species from the Apocynaceae family
(Boligon et al., 2013). Whereas, the ethanol extract of Amsonia
T.F. Xavier, R. Sabitha and S. Balavivekananthan South African Journal of Botany 149 (2022) 320
338

Fig. 6. a. Docking pose of the heptadecanol with 1B2Y protein

b. The 2D template discloses the variety of amino acid residures interactions of 1-heptadecanol with 1B2Y protein complex.

331
T.F. Xavier, R. Sabitha and S. Balavivekananthan South African Journal of Botany 149 (2022) 320
338

Fig. 7. a. Docking pose of the Pentadecane with 1B2Y protein

b. The 2D template discloses the variety of amino acid residures interactions of Pentadecane with 1B2Y protein complex.

orientalis had the highest phenolic content (60.91 § 0.57 mg GAE g_
1), while its comparatively lesser than our results and also the total
flavonoid contents present in ethanol (30.68 § 0.43 mg QE g_ 1 and
acetone (30.39 § 0.47 mg QE g_ 1) extract of Amsonia orientalis,
which were statistically the very lesser amount of flavonoid content
332
than our study. (Acemi et al., 2020). Rubus ellipticus ethanol extract
contains the highest amount of TFC (215.00 mg RE/g) than the pres-
ent findings (Muniyandi et al., 2019).
The antioxidant activity of medicinal herbs is revealed by their
involvement in reducing free radical
caused tissue injury
T.F. Xavier, R. Sabitha and S. Balavivekananthan South African Journal of Botany 149 (2022) 320
338

Fig. 8. a. Docking pose of theHexadecanoic acid with 1B2Y protein

b. The 2D template discloses the variety of amino acid residures interactions of hexadecanoic acid with 1B2Y protein complex.

333
T.F. Xavier, R. Sabitha and S. Balavivekananthan South African Journal of Botany 149 (2022) 320
338

Fig. 9. a. Docking pose of octadecanoic acid with 1B2Y protein

b. The 2D template discloses the variety of amino acid residures interactions of octadecanoic acid with 1B2Y protein complex.

(Sylvie et al., 2014). Many researchers already investigated the anti-
oxidant activity of the plant extract using methanol solvent and they
reported, that owing to the presence of antioxidant agents’ significant
reductions were achieved in lipid peroxidation (Durairaj et al., 2007;
Durairaj et al., 2009). Based on the above documentation the highest
334
DPPH free radical scavenging activity (77 § 1.5%) was obtained in
ethanol leaf extracts of C. spiralis but comparatively lesser than in
our study (Chavan et al., 2013). Whereas, the methanol extract of OE
has a highly significant scavenging ability with IC50of 11.15 mg/ml
(Ashok kumar et al., 2008). When compared with our standard
T.F. Xavier, R. Sabitha and S. Balavivekananthan South African Journal of Botany 149 (2022) 320
338

Fig. 10. a. Docking pose of solannesol with 1B2Y protein

b. The 2D template discloses the variety of amino acid residures interactions of solanesol with 1B2Y protein complex.

ascorbic acid, which is more efficient. Although, the highest antioxi-
dant potential was found in ethyl acetate extract according to
Shaker and Al-Qahtani (2018) and which is greater than our result.
As a precursor to more reactive species, the superoxide radical is
335
known to be extremely damaging to biological components. Due to
the presence of scavenging ability of superoxide anions, flavonoids
are reported that potent antioxidants (Robak and Gryglewski, 1988).
Contrary to our study MEOE, BHA and BHT extracts showed their
T.F. Xavier, R. Sabitha and S. Balavivekananthan South African Journal of Botany 149 (2022) 320
338

Fig. 11. The graphical representation of phytocompound from LC-MS spectrum.

superoxide scavenging activity with IC50 74.43, 60.11, and 7.67mg/ both extract and fraction (Baah et al., 2019). While comparing to our
ml, respectively (Ashok kumar et al., 2008). All oxygen metabolizing results S. japonica extract has the highest antioxidant ability and
cells have superoxide dismutase and catalase in the radical-scaveng- IC50 value of 1656.52 mg/mL (Uddin et al., 2016). According to
ing mechanism, and their job is to protect against the potentially Alam et al. (2013) due to chelates formation with ferric ion (Fe2+),
harmful modes of action of superoxide and hydrogen peroxide the ferrozine can form a red colour with a complex, when the red col-
(Rushmore and Picket, 1993). The result of the FRAP assay is deter- our reduces the ferrozine reduced into ferrous ions by the presence of
mined based on the ferric tripridyltriazine complex at lower pH con- chelating agents.
ditions is reduced into ferrous complex and measures the ability of Plant extracts may have an antibacterial effect by inhibiting
electron-donation of antioxidants and to reduce ferric ions microbe cell wall construction, metabolism, protein synthesis, and
(Alam et al., 2013). Pellegrini et al. (2006) also reported, that in the DNA synthesis (Goodman and Gillman, 2001). The variations in anti-
presence of antioxidants, the FRAP assay quantifies the reduction of bacterial activities of crude extracts could be attributed to the num-
Fe3+ to Fe2+. For evaluating the cumulative antioxidant effects of ber of antibacterial agents present in the extracts (Harborne, 1973).
plant diets, the total antioxidant capacity method was found to be By Ashok kumar et al. (2010) the significant minimal inhibitory con-
appropriate. The crude ethanol and ethyl acetate extracts of S. gratus centration of MEOE was noted. Here, Staphylococcus epidermidis,
were 6.7 gA AE/100g and 8.3 gA AE/100g, respectively. There was no Proteus vulgaris, and Escherichia coli are highly inhibited in concen-
significant difference (P < 0.05) between the antioxidant capacities of trations of 400 - 500 mg/mL. The ethyl acetate extract of Dregea volu-
bilis leaves exhibited a significant inhibitory effect against the
bacteria like P. vulgaris and S. epidermis at 62.5-250 ml/mL concen-
Table 9
The energy values of the phytochemicals by MM-GBSA. tration. While Jayaprasad and Sharavanan (2015) reported their find-
ings that gram-positive bacteria like B. subtillis, B. cereus, and S.
S. No. Phytochemicals DockingMM-GBSA Rank aureus have been highly resistant and the gram-negative bacteria
Scores
like K. pneumonia, P. aureuginosa, E. coli, and P. vulgaris were
1. Solanesol
12.3
54.532 1 completely inactive. Hence, Oxystelma esculentum R. Br. is considered
2. Octadecanoic acid-2,3-dihyroxypropyl
6.7
37.632 2
the best and most effective source for natural antioxidants and other
ester
3. p Chloramphetamine
6.3
32.432 3
pharmacological as well as therapeutic applications.
4. 1-Heptadecanol
5.9
30.372 4 The anti-diabetic activity of OELE revealed the significant results
5. Hexadecanoic acid-2-hydroxy-1-
5.9
29.264 4 determined by enzymatic assays. The hydrolysis of polysaccharides
(hydroxymethyl)ethyl ester into oligosaccharides is carried out by the a-amylase enzyme and the
6. Pentadecane
5.7 28.212 5
7. Acarbose
.7.7
40.263 Standard
a-glucosidase enzyme breaks down complex dietary carbohydrates
and starch into monosaccharides, which raises blood sugar levels
336
T.F. Xavier, R. Sabitha and S. Balavivekananthan
(Khan et al., 2019). This is the first study for the evaluation of the
enzymatic activity of OE leaf extracts. When compared with both
enzyme activities, a-amylase activity on an ethanol extract of OE has
the highest inhibitory activity. Several herbal extracts have tradition-
ally been used to treat diabetes and other illnesses as enzyme inhibi-
tors. It's important to remember that bioactive compounds can
provide good ligand-receptor profiles for in silico modeling
(Zhang et al., 2015). Thus, the selected phytoconstituents based on
the area of percentage peak through GC-MS were docked with 1B2Y
receptor with standard Acarbose to find the comparative binding
interaction (Ponce-Cusi and Calaf, 2016). 1B2Y is the best receptor,
and much research has also been done by using this as a target pro-
tein for in silico antidiabetic studies. (Nahoum et al., 2000) In protein-
ligand interactions such as hydrogen bond side chains, backbone, pi-
pi stacking, and salt bridge contacts, understanding the ligand bind-
ing pocket is crucial. Electrostatic interactions between tiny com-
pounds and the targeted protein also were emphasised. It may also
aid in the reduction of stronger bonding interactions as well as the
clear distances among protien and ligand (Prabhu et. al., 2017).
5. Conclusion
The comprehensive study of high polarity solvents like ethyl ace-
tate, ethanol, and acetone extracts of Oxystelma esculentum R. Br.,
leaves about the preliminary phytochemicals, in vitro anti-diabetic,
antioxidant, and antibacterial activities. The result of the determina-
tion of phytochemicals revealed that the secondary metabolites like
proteins, alkaloids, flavonoids, tannins, carbohydrates, amino acids,
phenolic compounds, and glycosides were present in all three
extracts of OE leaf. The total phenolic and flavonoid contents were
obtained in ethyl acetate extract followed by ethanol and acetone
extracts. The total antioxidant assays revealed that the OELE has the
efficiency to inhibit the oxidative agents. Ethyl acetate, ethanol, and
acetone extracts exhibited significant antibacterial results at the min-
imal concentration level. The enzymatic activity of OELE like a-amy-
lase and a-glucosidase activity, the best inhibition was found on
ethanol and acetone extracts, respectively. The phytoconstituents
present in the OELE were identified by GC-MS chromatograms and 6
compounds including Solanesol were chosen for in silico modeling
against human diabetic receptors (1B2Y). From these compounds,
solanosol shows less binding affinity with the highest docking score
ranging from -12.3 to -10.3 than standard and other compounds. In
conclusion to that of Oxystelma esculentum R.Br., leaf extracts are
highly potential in its pharmacological properties and therapeutic
values due to the presence of various phytocompounds.
Funding
Not Applicable
Availability of data and material
During the current study the datasets used and analysed materials
will be getting from corresponding author on reasonable request.
Consent for publication
Not Applicable
CRediT authorship contribution statement
Thangaraj Francis Xavier: Conceptualization, Visualization, For-
mal analysis, Data curation, Writing
review & editing. Rajendren
Sabitha: Methodology, Data curation, Writing
original draft, Visu-
alization, Writing
review & editing. Senthilkumar
337
South African Journal of Botany 149 (2022) 320
338
Balavivekananthan: Methodology, Data curation, Writing
original
draft, Visualization, Writing
review & editing.
Declaration of Competing Interest
The authors were reported that there is no conflict of interest(s).
The contents and writing of the paper are solely the authors' respon-
sibility.
Acknowledgement
Dr.TFX wish to thank Rev.Dr.S.Peter SJ (former Secretary), Rev.Dr.
K.Amal SJ, Secretary, Rev.Dr. M.Arockiasamy Xavier SJ , Principal , St.
Joseph's College, Tiruchirappalli for providing laboratory facilities to
carry out this research work. Authors also thank Dr. S. Prabu, Assis-
tant Professor of Botany, Annai Velainkanni Arts and Science College,
Tanjore for his assistance to prepare the statistical report of insilico
studies.
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