Synthesis, antihyperglycemic activity and computational studies of antioxidant chalcones and flavanones derived from 2,5 dihydroxyacetophenone

Article abstract of DOI:10.1016/j.molstruc.2017.07.042

Chronic exposure of supraphysiologic glucose concentration to cells and tissues resulted in glucose toxicity which causes oxidative stress. Antioxidants have promising effect in suppressing the oxidative stress in the pathogenesis of diabetes mellitus (DM

Full text of DOI:10.1016/j.molstruc.2017.07.042

Accepted Manuscript
Synthesis, antihyperglycemic activity and computational studies of antioxidant
chalcones and flavanones derived from 2,5 dihydroxyacetophenone
Affifa Tajammal, Majda Batool, Ayesha Ramzan, Malka M. Samra, Idrees Mahnoor,
Francis Verpoort, Ahmad Irfan, Abdullah G. Al-Sehemi, Munawar Ali Munawar,
Muhammad Asim R Basra
PII:
S0022-2860(17)30975-4
10.1016/j.molstruc.2017.07.042
MOLSTR 24069
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 16 January 2017
Revised Date: 29 June 2017
Accepted Date: 19 July 2017
Please cite this article as: A. Tajammal, M. Batool, A. Ramzan, M.M. Samra, I. Mahnoor, F.
Verpoort, A. Irfan, A.G. Al-Sehemi, M.A. Munawar, M. Asim R Basra, Synthesis, antihyperglycemic
activity and computational studies of antioxidant chalcones and flavanones derived from 2,5
dihydroxyacetophenone, Journal of Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.07.042.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, antihyperglycemic activity and computational studies of antioxidant chalcones
and flavanones derived from 2,5 dihydroxyacetophenone
Affifa Tajammal1, Majda Batool1, Ayesha Ramzan1, Malka M Samra1, Idrees Mahnoor1, Francis
Verpoort2, Ahmad Irfan3,4, Abdullah G. Al-Sehemi3,4, Munawar Ali Munawar1*, Muhammad
Asim R Basra1*
1Institute of Chemistry, University of the Punjab, New Campus, Lahore, Pakistan
2Centre for Chemical and Materials Engineering, Wuhan University of Technology, Wuhan
430070, China
3Department of Chemistry, Faculty of Science, King Khalid University, Abha 61413, P.O. Box
9004, Saudi Arabia
4Research Center for Advanced Materials Science (RCAMS), King Khalid University, Abha
61413, P.O. Box 9004, Saudi Arabia
Running title: Antihyperglycemic and antioxidant screening of synthesized chalcones and
flavanones
* Corresponding author
Corresponding author mailing address: Institute of chemistry, University of The Punjab, New
Campus, Lahore, Pakistan.
Tel +924299230463 Ext. 139,
+92-3320388300
E-mail: asimbasra.chem@pu.edu.pk, asimbasra@gmail.com,
munawaralimumawar@yahoo.com
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Abstract
Chronic exposure of supraphysiologic glucose concentration to cells and tissues resulted in
glucose toxicity which causes oxidative stress. Antioxidants have promising effect in suppressing
the oxidative stress in the pathogenesis of diabetes mellitus (DM). Condensation of 2,5-
dihydroxyacetophenone with different nitrobenzaldehydes was used to synthesize antioxidant
nitro substituted chalcones along with nitro substituted flavanones in one step protocol. The
compounds were characterized by IR, 1H NMR and 13C NMR and then screened for their in vitro
antioxidant and in vivo antihyperglycemic activities. Postulated structures of the synthesized
compounds were in agreement with their spectral data. The results indicated that the novel
compound (2E)-1-(2,5-Dihydroxyphenyl)-3-(2-nitrophenyl) prop-2-en-1-one (2a) was potent
antioxidant because of its lower IC50 value compared with trolox and ascorbic acid. Compound
2a also exhibited excellent antihyperglycemic activity in diabetic rats while the compound (E)-1-
(2,5-Dihydroxyphenyl)-3-(4-nitrophenyl)prop-2-one (2c) suppressed the hyperglycemia more
effectively in normal rats. The radical scavenging activity behavior was elucidated on the basis
of hydrogen atom transfer and one-electron transfer mechanisms by density functional theory
(DFT). The compound 2a showed the smallest ionization potential and bond dissociation
enthalpy. Experimental and computational investigations concluded that compound 2a might be
an effective antihyperglycemic agent because of its antioxidative nature and smallest ionization
potential.
Keywords: Chalcones; Flavanones; Antioxidant; Diabetes; Antihyperglycemia.
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1. Introduction
Production of free radical is enhanced in the body due to oxidation of glucose, non-enzymatic
glycation of proteins and the subsequent oxidative degradation of glycated proteins [1]. In
diabetes mellitus (DM), erratically controlled hyperglycemia promotes free radicals
accumulation and cause oxidative stress by enhancing the cascade of the oxidative reaction [2].
Antioxidants can be helpful in suppressing the oxidative stress and its related disorder including
DM [3].
Chalcones and flavanones are well-known for their wide range of pharmacological activities
including antimicrobial [4], anticancer, antioxidant [5], antiangiogenic [6], anti-inflammatory
[7], antidiabetic, antihyperlipidemic [8], inhibition of tyrosinase activities [9] etc. Excellent
antioxidant activity of chalcones and flavanones is due to the presence of hydroxyl groups on ‘A’
or ‘B’ ring of their structure. This antioxidant potential is involved in decreasing the oxidative
stress in various diseases including cancer, atherosclerosis, DM, hypertension and heart diseases
[10].
Figure 1. Structure of chalcone and flavanone
Both in vitro and in vivo studies have also demonstrated the effects of hydroxychalcones and
hydroxyflavanones, originating from both natural and synthetic sources on the carbohydrate
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metabolism and regulation of insulin function [11], [8]. Antioxidant compounds are reported to
have potential protective effects on diabetes by improving
β-cells function. So enhancing
antioxidant defense mechanisms in pancreatic islets may be a potential pharmacological
approach to manage the diabetes [12].
Various drugs are on the horizon as well as there is a need to develop a new category of drug
with lesser side effects and improve the variety of medications. In recent years, the high
therapeutic properties of the chalcones and flavanones related drugs have brought attention of
chemists to synthesize various kinds of their derivatives by improving the existing synthetic
methodologies. Claisen-Schmidt condensation is usually used to synthesize the chalcones in the
presence of basic catalysts such as NaOH/ KOH [13], MgO, BaO, K2O, Na2O and ZaO [14]. It
has been reported previously that the hydroxychalcones are difficult to synthesize in the presence
of base due to formation of phenoxide anion. Acidic catalyst such as HCl, BF3, B2O3 [15], para
toluenesulfonic acid [16] and SOCl2/EtOH [17] have been found to be more effective. It is
therefore, the present work was designed to synthesize new chalcones and flavanones in the
presence of acid without the protection of hydroxyl group. Furthermore, the main objective was
to investigate in vitro and in silico antioxidant activities along with in vivo antihyperglycemic
activities of synthesized compounds.
2. Experimental
2.1. Materials and Methods
Chemicals were purchased from well reputed international suppliers. The chemicals were used
mostly as such however when required purified by normal techniques i.e., distillation and
recrystallization. Synthesis of 2, 5-dihydroxyacetophenone was done by already reported method
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13
[18]. Silica gel 60 F254 TLC plates were used to monitor the reaction. FT-IR, 1H NMR and C
NMR spectra were recorded on Agilent Technologies 41630, AVANCE AV-400 MHz and
AVANCE AV-500 MHz, and Bruker 125 MHz respectively while the EIMS data was taken on
JEOL MS 600H-1.
2.2. General method for the synthesis of chalcones and flavanones
2,5-Dihydroxyacetophenone (50 mg, 0.3 mmol) and nitrobenzaldehyde (45 mg, 0.3 mmol) were
dissolved in hot dry benzene (20 mL). Then p-Toluene sulfonic acid (5 mg, 0.03 mmol) was
added. The resulting reaction mixture was refluxed for 48 hours. The reaction was monitored
with TLC. After completion of the reaction, benzene was removed under vacuum and residue
was purified on silica gel column using hexane-ethyl acetate (4:1) as eluent. In case of 3-
nitrobenzaldehyde and 4-nitrobenzaldehyde, corresponding flavanones were also isolated along
with chalcones. Whereas with 2-nitrobenzaldehyde only a novel chalcone (2a) was obtained.
2.3. (2E)-1-(2,5-Dihydroxyphenyl)-3-(2-nitrophenyl) prop-2-en-1-one (2a)
Orange powder; yield 80 %; IR (ν cm-1): 3367; 1648; 1571; 1517. 1H NMR (500 MHz / DMSO-
d6): δ 11.34 (1H, s, 2ʹ-OH), 9.28 (1H, s, 5′-OH), 8.12 (2H, m, 3-H, 6-H), 8.00 (1H, d, J=15.5 Hz,
β-H), 7.87-7.82 (2H, m, α-H, 5-H), 7.72 (1H, t, J=7.2 Hz, 4-H), 7.41 (1H, d, J=3 Hz, 6ʹ-H), 7.05
(1H, dd, J=8.8 Hz, 3.0 Hz, 4ʹ-H), 6.87 (1H, d, J=8.8 Hz, 3ʹ-H). 13C NMR: δ 192.83, 154.56,
150.09, 149.22, 138.70, 134.33, 131.65, 130.11, 129.93, 127.67, 125.22, 125.00, 121.87, 118.92,
115.56. MS (EI+): m/z 137 (100 %), M+ 285 (5), 27 (86), 238 (83).
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2.4. (2E)-1-(2,5-Dihydroxyphenyl)-3-(3-nitrophenyl)prop-2-en-1-one (2b)
Orange powder; yield 53 %; IR (ν cm-1): 3430; 1643; 1561; 1530. 1H NMR (400 MHz / DMSO-
d6): δ 11.60 (1H, s, 2ʹ-OH), 9.29 (1H, s, 5ʹ-OH), 8.73 (1H, s, 2-H), 8.31 (1H, d, j=7.8 Hz, 4-H),
8.28 (1H, m, 4-H, 6-H), 8.10 (1H, d, J=15.7 Hz, β-H), 7.88 (1H, d, J=15.6 Hz, α-H), 7.75 (1H, t,
J=7.9 Hz, 3-H), 7.54 (1H, d, J=2.8 Hz, 6ʹ-H), 7.06 (1H, dd, J=8.8, 2.8 Hz, 4ʹ-H), 6.87 (1H, d,
J=8.8 Hz, 3ʹ-H). 13C NMR: δ 193.41, 154.98, 150.04, 148.89, 142.00, 136.84, 135.64, 130.93,
125.74, 125.30, 125.15, 123.49, 121.54, 118.84, 115.72.
2.5. 6-hydroxy-2-(3-nitrophenyl)-2,3-dihydro-4H-chromen-4-one (3a)
Yellow crystals; yield 25 %; IR (ν cm-1): 3472, 1670; 1524; 1342. 1H NMR (500 MHz / DMSO-
d6): δ 9.57 (1H, s, 6-OH), 8.40 (1H, s, 2ʹ-H), 8.24 (1H, dd, J=8.2 Hz, 2.0 Hz, 4ʹ-H), 8.00 (1H, d,
J=7.8 Hz, 6ʹ-H), 7.74 (1H, t, J=8 Hz, 5ʹ-H), 7.13 (1H, d, J=3.0 Hz, 5-H), 7.07 (1H, dd, J=8.8,
3.0 Hz, 7-H), 7.02 (1H, d, J=8.8 Hz, 8-H), 5.75 (1H, dd, J=13.1, 2.7 Hz, 2-H), 3.22 (1H, m, Ha),
2.88 (1H, dd, J=16.9, 2.9 Hz, Hb). 13C NMR: δ 191.72, 154.47, 152.28, 148.35, 141.87, 133.56,
130.72, 125.14, 123.73, 121.63, 121.32, 119.54, 110.46.
2.6. (E)-1-(2,5-Dihydroxyphenyl)-3-(4-nitrophenyl)prop-2-one (2c)
Orange powder; yield 65 %; IR (ν cm-1): 3355; 1648; 1584; 1522. 1H NMR (500 MHz / DMSO-
d6): δ 11.44 (1H, s, 2ʹ-H), 9.18 (1H, s, 5ʹ-H), 8.28 (2H, d, J=8.4 Hz, 3-H, 5-H), 8.13 (2H, d,
J=8.8 Hz, 2-H, 6-H), 8.07 (1H, d, J=16 Hz, β-H), 7.82 (1H, d, J=15.6 Hz, α-H), 7.48 (1H, d,
J=2.4 Hz, 6ʹ-H), 7.04 (1H, dd, J=8.8, 2.8 Hz, 4ʹ-H), 6.85 (1H, d, J=8.8 Hz, 3ʹ-H). MS (EI+): m/z
136 (100 %), M+ 285 (91), 266 (9), 238 (8), 163 (45).
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2.7. 6-hydroxy-2-(4-nitrophenyl)-2,3-dihydro-4H-chromen-4-one (3b)
1
Light orange powder; yield 20 %; IR (ν cm-1): 3355; 1648; 1584; 1522. H NMR (400 MHz /
DMSO-d6): δ 9.57 (1H, s, 6-OH), 8.29 (2H, d, J=8.7 Hz, 3ʹ-H, 5ʹ-H), 7.82 (2H, d, J=8.7 Hz, 2ʹ-
H, 6ʹ-H), 7.13 (1H, d, J=3.0 Hz, 5-H), 7.07 (1H, dd, J=8.8, 3.0 Hz, 7-H), 7.01 (1H, d, J=8.8, 8-
H), 5.75 (1H, dd, J=12.9, 2.9 Hz, 2-H), 3.15 (1H, m, Ha), 2.89 (1H, dd, J=16.9, 3.0 Hz, Hb). 13C
NMR: δ 191.56, 154.42, 152.29, 147.78, 146.99, 128.08, 125.16, 124.18, 121.34, 119.54,
110.46, 78.14, 43.92.
2.8. In vitro Antioxidant Activities
The synthesized compounds were tested individually against five different in vitro antioxidant
activities which include 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, iron
chelating, FeCl3 reducing power, phosphomolybdenum (PM) and 2,2ʹ-Azino-bis(3-
ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) activity. These activities were
carried out for each compound in triplicate while trolox and ascorbic acid were used as standard
compounds.
2.8.1. DPPH free radical scavenging activity
Free radical scavenging effect of synthesized compounds was determined with DPPH assay.
Ethanolic solution of DPPH (0.05 mM, 2.85 mL) and the test compounds (final concentration
2000 µM, 0.15 mL) were mixed and incubated in dark at 37 °C. The absorbance was recorded at
517 nm after 15, 30, 45, 60 and 120 minutes of incubation. The percentage activity of the
synthesized compounds was calculated with following equation [19].
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Percentage of scavenging activity = (Control absorbance - Sample absorbance/Control
absorbance) × 100
2.8.2. Iron chelating activity
Different concentrations of methanolic solution of test compounds (final concentration 2000 µM,
2 mL) were mixed with methanolic o-phenanthroline (0.05 % w/v, 1 mL) solution and incubated
with methanolic FeCl3 (200 µM, 2 mL) at ambient temperature. The absorbance was determined
at 512 nm after 10, 30, 45, 60 and 120 minutes. Finally, the percentage of iron chelating activity
was determined with the following formula [20].
Percentage of iron chelating activity = (Test absorbance - Control absorbance/Test absorbance) ×
100
2.8.3. FeCl3 reducing power activity
In reducing power assay, aqueous solution of potassium ferricyanide (1 %, 2.5 mL) was mixed
with ethanolic solution of each test compound (final concentration 2000 µM, 2 mL) and
incubated at 50 °C for 20 minutes. After cooling, aqueous trichloroacetic acid (10 %, 2.5 mL)
was added and the mixture was centrifuged at 3000 rpm for 10 minutes. The supernatant liquid
(2.5 mL) was mixed with distilled water (2.5 mL) and freshly prepared aqueous ferric chloride
solution (0.1 %, 1 mL). The absorbance was recorded at 700 nm after 10, 30, 45, 60 and 120
minutes of incubation. Percentage increase in reducing power was determined by the following
formula [21].
Percentage increase of reducing power = (Test absorbance/Control absorbance - 1) × 100
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2.8.4. Phosphomolybdenum activity
PM solution was prepared by mixing 100 mL of each sulfuric acid (0.6 M), ammonium
molybdate (4 mM) and sodium phosphate (28 mM). Series of dilutions of sample solution were
prepared from stock solution of 2000 µM in ethanol. Phosphomolybdenum reagent (3 mL) was
mixed with each test compound (0.3 mL) and incubated at 95 °C for 90 minutes. The absorbance
was measured at 765 nm after 15, 30 and 45 minutes of incubation against the blank. Percentage
increase in reducing power was determined by the following formula [22].
Percentage increase of reducing power = (Test absorbance/Control absorbance - 1) × 100
2.8.5. ABTS activity
In ABTS assay, antioxidant activity was performed by preparing aqueous ABTS (7 mM/L).
ABTS+ was produced by reacting ABTS stock solution with 2.45 mmol/L potassium persulfate.
o
The resulting solution was incubated in dark for 12-16 hours at room temperature (25±2 C).
Ethanol was used for diluting the mixture till the absorbance reached to 0.7±0.02 at 734 nm.
Various dilutions of each sample solution were prepared from stock solution (2000 µM) in
ethanol. ABTS+ solution (2.85 mL) was mixed with each test compound solution (0.15 mL) and
recorded the absorbance at 734 nm after the intervals of 5, 15, 30, 45, 60 and 120 minutes of
incubation against the blank. The activity of each compound was then measured by using the
following formula [23].
Percentage of scavenging activity = (Control absorbance - Sample absorbance/Control
absorbance) × 100
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2.9. Antihyperglycemic activity
2.9.1. Experimental Animals
Sprague Dawley albino rats (200-250 g) were selected and maintained in animal house at
controlled temperature (25±5 °C) and humidity (50±10 %). Animals were provided with free
access to autoclaved tap water and pathogen free feed for 24 hours. Animal experiments were
approved by Institutional ethical committee (Approval No. D/017/Chem.). International ethical
guidelines for the care of laboratory animals were used to maintain rats in animal house.
2.9.2. Oral glucose tolerance test in normal rats
The blood glucose level of each rat was inspected by using code free glucometer after 18 hours
of starvation. Animals showing blood glucose levels between 80-100 mg/dL were divided into
groups of five animals in each. Animals in experimental groups were treated orally with
synthesized compounds at single dose of 100 mg/kg body weight after emulsifying in aqueous
carboxymethyl cellulose (0.5 %). Animals in control group were given aqueous carboxymethyl
cellulose (0.5 %) only. An oral glucose load (2.0 g/kg) was given to each animal exactly after 30
minutes per oral administration of the test sample/vehicle. Blood glucose level of each rat was
checked at 1, 3 and 5 hours after the administration of glucose. After checking the results, three
out of five compounds were selected and screened for their most effective dose at 50, 100 and
200 mg/kg by using the above mentioned protocol. The dose which improved glucose tolerance
significantly was selected for further studies [24].
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2.9.3. Determination of anti-hyperglycemic effects of compounds in streptozotocin induced
diabetic rats
Rats were divided randomly into five groups; Control, Diabetes mellitus (DM), Glibenclamide
(GC), 2a and 2c group. Each group contained seven female rats. Rats in DM, GC, 2a and 2c
groups were injected with streptozotocin (STZ) (50 mg/Kg) interaperitoneally which was freshly
prepared in citrate buffer (0.1 M, pH: 4.5). The elevated blood glucose level (>250 mg/dL)
confirmed the hyperglycemic condition in each rats after 48 hours followed by STZ injection.
Glucose solution (5 %) was also given to avoid the hypoglycemic effect of the drug. After 48
hours, rats in GC, 2a, and 2c were treated orally with single dose of GC (20 mg/kg), compound
2a (100 mg/kg) and compound 2c (100mg/kg) respectively. Rats in Control and DM groups were
treated with vehicle carboxymethyl cellulose (0.5 %). Blood glucose level was determined both
before and after the treatment at different intervals (zero time, 1, 3 and 5 hours).
Statistical analysis
Statistical analysis was done by using One Way ANOVA followed by Tukey’s multiple
comparison tests with the help of GraphPad Prism version 7.01. The values were considered
statistically significant at P < 0.05.
2.10. Computational details
The density functional theory (DFT) is being used to shed light on the elector-optical, radical
scavenging activity and other descriptors efficiently [25]. The ground state geometries of the
neutral and charged species as well as frequency calculations were carried out by DFT at
B3LYP/6-31G** level [26]. There are two key mechanisms (a) H-atom transfer (Eq. (1)) and
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one-electron transfer (b) for the radical scavenging processes of chain-breaking antioxidant
(ArOH) [27], [28], details can be seen in the supporting information.
R. + ArOH
R. + ArOH
RH + ArO.
(1)
(2)
R- + ArOH+.
All calculations were performed by Gaussian 09 software [29].
3. Results and Discussion
Chalcones (2a, 2b, 2c) and flavanones (3a, 3b) were isolated from the acid catalyzed
condensation reaction of 2,5-dihydroxyacetophenone (1) with nitrobenzaldehydes. Different
solvent systems such as toluene, methanol, ethanol, 2-propanol were endeavored, however
benzene was found to be the best solvent for these condensation reactions. The synthesized
compounds were characterized using, 1H NMR, 13C NMR etc. The most prominent indication for
the synthesis of chalcones was the appearance of two doublets with J = 15.6 corresponding α and
β proton of the ethene linkage. Whereas absence of these signals and appearance of signals at δ
5.75, 3.22, and 2.88 corresponding to 2-H, 3Ha and 3Hb of the hetro ring indicated the formation
of flavanones. The synthetic route of flavanones and chalcones from 2,5-dihydroxyacetophenone
and the physiochemical data of the synthesized compounds was summarized in detail in figure 2
and Table 1 respectively.
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Figure 2. Synthesis of nitro substituted hydroxychalcones and hydroxyflavanones
Table 1. Physicochemical characterization data of the synthesized compounds
Comp.
R
M.F.
M.W.
M.P. (oC)
Yield (%)
no.
2a
2-nitro
3-nitro
4-nitro
3-nitro
4-nitro
C15H11NO5
C15H11NO5
C15H11NO5
C15H11NO5
C15H11NO5
285.25
285.25
285.25
285.25
285.25
200
208
218
153
167
80
53
65
25
20
2b
2c
3a
3b
3.1. In vitro Antioxidant Activities
3.1.1. Interaction with the DPPH stable free radical
The tested compounds interact with the stable free radical DPPH which shows their radical
scavenging ability. This interaction was found to be concentration and time dependent (table 2).
Among the tested chalcones, compound 2a showed excellent radical scavenging activity where
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nitro group is at position 2 of ring ‘B’. It exhibited significantly higher activity as compared to
the reference compounds trolox and ascorbic acid. The compounds 2b and 2c bearing nitro group
at position 3 and 4 of ring ‘B’ respectively displayed significant good activity when compared
with reference compounds but much lower than 2a. It is therefore, DPPH scavenging ability
seems to be dependent on the position of nitro group at ring ‘B’ of the chalcones. The IC50 values
of tested flavanones 3a and 3b were much higher as compared to all other compounds. So the
order of DPPH activity of synthesized compounds was 2a > 2b > 2c >>> 3a, 3b.
Table 2. IC50 (µM) for DPPH radical scavenging activity
Compou
Time (Minutes)
nds
15
30
45
60
120
Trolox
225.12±15.12
216.25±10.41
223.18±15.01
214.83±10.43
252.73±17.22
Ascorbic
acid
215.50±10.43
221.36±14.81
234.44±15.71
244.46±15.91
240.47±15.81
50.88±5.11***
2a
2b
2c
3a
3b
65.52±6.21*** 55.89±5.55*** 57.78±5.57*** 53.57±5.21***
117.19±7.87*** 112.05±7.85*** 113.82±7.83*** 112.59±7.82*** 111.58±7.81***
142.96±8.25*** 145.47±8.44*** 144.58±8.43*** 148.41±8.52*** 167.71±8.95***
> 2000
> 2000
> 2000
> 2000
> 2000
> 2000
> 2000
> 2000
> 2000
> 2000
Data was shown in Mean ± SD (n = 3) for each compound, ***P < 0.001 showed statistically
significant difference when compared with trolox and ascorbic acid.
3.1.2. Iron chelating activity
In Iron chelating activity, the synthesized compounds reduced Fe3+ to Fe2+ which involved in the
formation of chelate with o-phenanthroline. This method is used to determine the extent of
reduction of ferric ions by synthesized compounds. IC50 value for iron chelating activity of each
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compound was determined (Table 3). Among the tested chalcones, IC50 value of compound 2a
was much lower as compared to other chalcones and flavanones showing its greater chelating
ability. No significant difference was found between 2a and reference compounds. The IC50
value of 2a was found to be very close to ascorbic acid value. The order of increasing activity for
synthetic compound was 2a > 2b > 2c > 3b > 3a.
Table 3. IC50 (µM) for Iron chelating activity
Time (Minutes)
Compounds
10
30
45
60
120
Trolox
Ascorbic
acid
2a
0.46±0.23
0.49±0.25
0.53±0.32
0.41±0.24
0.28±0.11
3.33±1.19
2.15±1.13
0.33±0.67
0.23±0.52
0.24±0.59
4.21±1.14
15.47±5.78
40.61±3.23
185.09±9.33
121.82±10.12
1.85±1.09
11.37±4.61
56.69±3.98
211.89±5.74
126.59±8.81
0.56±0.76
5.09±3.67
0.22±0.98
1.70±1.37
0.31±1.16
0.41±2.33
2b
2c
46.21±2.31
214.51±4.32
160.70±6.92
16.82±1.12
210.78±6.54
160.34±8.67
11.85±1.92
232.21±3.49
224.59±2.37
3a
3b
3.1.3. FeCl3 reducing power activity
In FeCl3 reducing power assay the chalcones and flavanones reduced potassium ferricyanide
(Fe+3) to potassium ferrocyanide (Fe+2) which then reacted with ferric chloride to form ferric
ferrous complex. The reducing capabilities of all compounds were summarized in the form of
IC50 (Table 4). The IC50 of 2a was found to be lower than the reference compounds while 2b and
2c showed satisfactory results. The IC50 of chalcones was much lower as compared to
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flavanones. The order of FeCl3 reducing power activity of the synthesized compounds was found
to be 2a > 2b > 2c > 3b > 3a.
Table 4. IC50 (µM) for FeCl3 reducing power activity
Time (Minutes)
Compounds
10
30
45
60
120
Trolox
Ascorbic
acid
2a
13.75±2.45
12.04±3.07
11.24±2.74
11.43±1.98
10.06±2.24
14.64±3.34
14.74±3.29
14.02±3.12
13.16±3.54
12.74±2.87
11.28±1.22
15.37±0.24
23.07±5.44
9.98±2.11
14.49±0.23
21.44±5.21
9.51±3.45
14.16±0.23
18.32±4.65
9.21±2.31
13.87±0.26
17.32±4.98
9.11±3.43
13.43±0.21
16.19±4.26
138.45±14.35
28.88±12.51
2b
2c
3a
1305.77±18.81 673.01±16.42 459.85±15.63 180.63±15.24
90.01±15.83 66.56±12.25 53.07±12.66 37.12±13.76
3b
3.1.4. Phosphomolybdenum activity
This activity is based on the reduction of Mo (VI) to Mo (V) by the test compounds, giving a
direct approximation of reducing capacity of the compounds. IC50 values for PM activity of all
compounds were evaluated (Table 5). In PM activity, the IC50 of 2a was found to be lower than
all other compounds. The order of activity was 2a > 2b > 2c > 3b > 3a.
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Table 5. IC50 (µM) for Phosphomolybdenum assay
Time (Minutes)
Compounds
15
30
45
Trolox
Ascorbic acid
16.25±2.35
21.73±1.23
18.04±0.19
19.42±2.78
37.25±1.79
> 2000
15.89±2.73
17.37±3.61
19.13±1.47
20.37±3.77
35.97±2.37
> 2000
15.25±3.43
15.82±1.58
21.65±0.89
24.73±2.89
44.96±1.15
> 2000
2a
2b
2c
3a
3b
533.97±11.33
270.18±8.98
194.30±7.67
3.1.5. ABTS Activity
The synthesized compounds exhibited potent ABTS activity in a concentration-dependent
manner and the IC50 values for ABTS activity of all compounds were evaluated (Table 6).
Compound 2a exhibited statistical prominent ABTS activity than reference compounds after 5,
15, 30, 45, 60 and 120 minutes. Chalcones 2b and 2c showed significant lower IC50 value than
standard ascorbic acid after 5, 15, 30 and 45 minutes. The order of activity of synthetic
compounds was 2a > 2c > 2b > 3b > 3a.
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Table 6. IC50 (µM) for ABTS activity
Time (Minutes)
30
Compounds
5
15
45
60
120
Trolox
32.31±0.21
51.56±0.32
50.38±1.32
97.47±4.36
53.56±1.78
64.19±2.11
31.97±1.43
29.33±1.27
Ascorbic
acid
76.79±0.89
109.46±1.52
45.23±1.92
37.91±1.74
2a
2b
2c
3a
3.19±0.23*** 2.32±0.87***
41.44±0.26### 40.84±1.11###
37.79±0.18### 41.48±0.68###
1665.11±11.32 601.49±7.97
3.43±1.12***
52.24±1.96###
43.96±2.54###
210.69±5.32
1.65±1.65***
53.99±3.28##
46.21±1.56##
145.78±0.16
0.24±0.54*** 3.13±2.23***
54.65±3.42
45.29±2.18
46.66±2.64
37.79±2.43
121.04±4.91 106.03±4.38
145.94±4.22 99.31±6.32
3b
681.63±8.16 307.93±2.23
166.98±1.67
156.29±2.56
Data was shown in Mean ± SD (n = 3) for each compound, ***P < 0.001 showed statistically
##
significant difference when compared with trolox and ascorbic acid. P < 0.01, ###P < 0.001
showed statistically significant difference when compared synthesized compounds with ascorbic
acid.
3.2. Oral glucose tolerance test in normal rats
The synthesized compounds were screened for their antihyperglycemic activity in normal rats.
Compounds 2b, 2c, and 3b were found to be effective at dose of 100 mg/kg in lowering the
hyperglycemia while 2a and 3a appeared to be non-effective in reducing the hyperglycemia in
normal rats (Fig 3A). Because 3a and 3b were synthesized in low yield so 2a, 2b and 2c were
selected for the selection of minimum dose required for antihyperglycemic effect in normal rats.
Three different doses (50 mg/Kg, 100 mg/Kg and 200 mg/Kg) for compounds 2a, 2b and 2c
were selected for dose optimization respectively (Fig 3B-D).
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Figure 3. Oral glucose tolerance test of synthesized compounds in normal rats and compounds
administered to the rats before 30 minutes of glucose load (2 g/kg). Rats administered with
compound 2a, 2b, 2c, 3a and 3b at 100 mg/kg each (A), 2a: 50 mg/kg; 100 mg/kg; 200 mg/kg
(B), 2b: 50 mg/kg; 100 mg/kg; 200 mg/kg (C), and 2c: 50 mg/kg; 100 mg/kg; 200 mg/kg (D).
Data was expressed as percentage reduction in blood glucose level while GC was used as
reference drug. Values were expressed as Mean ± SD (n = 5).
The dose optimization study by oral glucose tolerance test (OGTT) in normal rats was revealed
that the compound 2c with nitro group at position 4 in ring ‘B’ of chalcone was more active in
lowering the hyperglycemia at 100 mg/kg dose.
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3.3. Anti-hyperglycemic effects of compounds in STZ induced diabetes
To further evaluate the antihyperglycemic effect of the synthesized compounds, STZ induced
type 2 diabetic rats were used to assess the efficacy of compound 2a and 2c at dose of 100
mg/kg. The reason for selecting 2a was novelty of the compound and excellent antioxidant
abilities than other compounds. In diabetic rats the compound 2a lowered the hyperglycemia
more potentially than compound 2c although 2a was less active in normal rats than 2c. Also, 2a
being structurally similar to 2c, but with a nitro group at position 2 in ring ‘B’, more actively
produced antihyperglycemic effect in diabetic rats. Thus steric conformation of the compounds
is said to be responsible for the anti-hyperglycemic activity of chalcone [30]. It is reported in
literature that the antihyperglycemic activity of chalcone derivatives is mediated via stimulation
of PPAR-γ [31], [32], [33]. The flavonoids, based on 2-phenylchromone or 2-phenyl
benzopyrone, have also shown beneficial effects in the treatment of hyperglycemic disease
probably through changes in the activity of intracellular enzymes such as glucosidase enzyme
[34]. Presently, compound 2a was found to be excellent antioxidant and have shown effective
antihyperglycemic effect in STZ induced diabetic rats. Antioxidants are involved in decreasing
the hyperglycemia by minimizing the oxidative stress [35]. Compound 2a significantly reduced
the hyperglycemia state in STZ induced rats compared with compound 2c and this effect was in
close proximity to the positive control GC (Fig. 4).
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Figure 4. Antihyperglycemic effect of compounds in STZ induced diabetic rats. Values were
expressed in Mean ± SD (n = 7). *P < 0.05 and ***P < 0.001 statistically significant when
compared with diabetic control while ###P < 0.001 statistically significant when compared with
normal Control.
Antibacterial activities were also evaluated for the synthesized compound and only two
compounds, 2a and 2c showed potential activity against Staphylococcus aureus (data was not
shown).
3.4. Computational investigations
With the developments of computational chemistry, the research on the theoretical modeling of
drug design and functional material design has gained much attention of synthetic chemists to
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predict chemical and physical properties of synthetic drugs in biological systems [36]
.
Previously, it was showed that the density functional theory (DFT) [37], [38] is efficient to
limelight on the radical scavenging activity and other physiochemical descriptors of synthetic
compounds [39-45]. Energies of the frontier molecular orbitals (FMOs), structure-activity
relationship (SAR), and molecular electrostatic potential (MEPs) were systematically explored
with the intention to apprehend the biological activity of the studied derivatives.
3.4.1. Frontier molecular analysis and different electronic properties
Highest occupied molecular orbitals (HOMO) and the lowest unoccupied molecular orbitals
(LUMO) are named as Frontier molecular orbitals (FMOs). Electronic and optical properties of
molecules are due to these FMOs. HOMOs and LUMOs were displayed (Figure 5). In chalcone
derivatives 2a-2c, the HOMOs are delocalized at the ring A while the LUMOs are localized on
the ring ‘B’ along with the charge distribution on the keto and nitro group.
A comprehensive intra-molecular charge transfer (ICT) was noticed from ring ‘A’ to ‘B’. In
flavanone derivatives 3a and 3b, the HOMOs are delocalized on the ring ‘A’ and partially on
ring ‘C’ along with the –OH group while the LUMOs are localized at the ring ‘B’ along with the
charge distribution on the nitro group. Here, noticeable ICT was perceived from the rings ‘A/C’
to ring ‘B’. The comprehensive ICT from the HOMOs to LUMOs was due to the strong electron
withdrawing behavior of the –NO2 group at the ring ‘B’. In the reference compound trolox, the
HOMO charge density is at conjugated ring (some charge at –OH and –CH3 groups) whereas the
LUMO is localized at entire compound illuminating ICT.
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Figure 5. The charge density distribution of the HOMOs (bottom) and LUMOs (top) of the
chalcone and flavanone derivatives at ground states
The HOMO energies (EHOMO), LUMO energies (ELUMO), HOMO–LUMO energy gaps (Egap), IP
and BDE at the B3LYP/6-31G** level of theory were presented. Among the studied chalcone
and flavanone derivatives, the highest HOMO and LUMO energy levels were observed for 2a
which was also higher than that of phenol. The Egap of all the studied compounds was found to be
smaller than the reference compounds, i.e.; phenol and trolox. Hitherto, direct relationship
between the HOMO energy levels and the radical scavenging activity of antioxidant compounds
was perceived showing that higher HOMO energy levels of studied compounds might lead to the
strong electron donating ability as compared to phenol. Additionally, the highest HOMO energy
level of 2a would lead better electron donating ability of this derivative among all the studied
chalcone and flavanone derivatives (Table 7). The electronegativity (χ), hardness (η),
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electrophilicity (ω), softness (S) and electrophilicity index (ωi) at the B3LYP/6-31G** level of
theory were presented (Table S1). The smallest χ and ω was observed for the chalcone derivative
2a.
Table 7. HOMO energies (EHOMO), LUMO energies (ELUMO) and HOMO-LUMO gaps (Egap) in
eV, IP and BDE (Kcal/mol) of chalcone and flavanone derivatives at B3LYP/6-31G** level of
theory.
Compounds
EHOMO
-5.42
-6.39
-5.65
-5.74
-5.79
-6.32
-6.34
ELUMO
0.12
Egap
5.54
5.82
2.89
2.93
2.69
3.77
3.79
IP
690d
192e
165
167
168
174
176
BDE
381.8d
Trolox
phenol
2a
-0.57
-2.76
-2.81
-3.10
-2.55
-2.55
83e
82.15a, 68.17b, 447.42c
82.23a, 68.40b, 447.62c
82.11a, 68.62b, 447.71c
71.50
2b
2c
3a
3b
71.60
b
c
d
a BDE of 2a-x, 2b-x and 2c-x; 2a-y, 2b-y and 2c-y; 2a-z, 2b-z and 2c-z; The BDE and IP of trolox is
e
381.8 and 690 Kcal/mol from reference ; The BDE and IP of phenol is 83 and 192 Kcal/mol from
reference
3.4.2. Hydrogen atom transfer mechanism
The radicals were obtained by hydrogen abstraction, see Figure S1. The vital position for
hydrogen atom transfer is hydroxyl. The BDE values presented in Table 7, describes the
hydrogen atom donating ability. These values were compared to the trolox [46] and phenol [28].
The BDE values computed at B3LYP/6-31G** level of theory showed that the mono-abstraction
of hydrogen usually lowers the BDE values more efficiently. The trend to lower the BDE value
in the chalcone derivatives according to the H-abstraction position was observed as y < x < z
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(Fig. S1 and Table 7). On the basis of BDE, it was observed that the chalcone derivatives showed
better radical scavenging activity as compared to the flavanones. The smallest BDE value was
observed for the 2a-y, i.e., 2a chalcone derivative revealing that this compound would be proficient
antioxidant contender. Finally, Hydrogen atom transfer mechanism would be favorable in 2a.
3.4.3. Single electron transfer mechanism
By single electron donation, the scavenging of free radicals may be attained. Conferring to the one-
electron transfer, an electron is removed from the HOMO giving rise to radical cations. The scavenging
of free radical can be evaluated by single electron donation. It can be seen from Table 7 that all
the derivatives have smaller IP values than the trolox and phenol. Among all the studied
derivatives, 2a has the smallest IP value revealing that in aforesaid material electron transfer
mechanism might be more inspiring for the scavenging of free radicals. The smaller IP value of 2a
as compared to the other counterparts revealed that prior compound would be better antioxidant material
(Table 7).
3.4.4. Molecular electrostatic potential
The molecular interaction and reactivity sites were assessed by 3-D surface maps of the MEP. In
Fig. 6, the MEP surface maps of the chalcone and flavanone derivatives as well as trolox were
illustrated to apprehend the positive and negative electrostatic potential (ESP) regions. Negative,
positive and zero potential regions were represented by red, blue and green colors, respectively
(Fig 6). It is expected that the negative and positive ESP regions would be encouraging for the
electrophilic and nucleophilic attack, respectively. In chalcone derivatives, negative ESP
(endorsing electrophilic reactive sites) have been realized on the C=O, nitro and -OH oxygen
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whereas the nitro of the flavanone derivatives. In trolox, negative ESP at –COOH and hydroxyl
oxygen would be promising electrophilic reactive sites.
Figure 6. The molecular electrostatic potential surfaces of the chalcone and flavanone
derivatives
4. Conclusion
Acid catalyzed condensation of 2,5-dihydroxyacetophenone with nitrobenzaldehydes leads to the
formation of chalcones along with flavanones which showed good antioxidant abilities especially
2a, 2b and 2c. The compound 2a showed excellent antihyperglycemic activity in diabetic rats
while 2c exhibited good activity in normal hyperglycemic rats. The determined positions of nitro
group at ring ‘B’ of chalcones exhibited antihyperglycemic activity. The comprehensive intra-
molecular charge transfer has been perceived from the HOMOs to the LUMOs. The smaller IP
and BDE values for the 2a revealed that this drug would show proficient antioxidant behavior
which is in good agreement with the experimental data. In future, compound 2a can be further
used to explore its beneficial impacts at molecular level.
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Acknowledgments
This work was supported by Higher Education Commission (HEC) [pin # 117-6030-PS7-077],
Institute of Chemistry, University of The Punjab, Lahore, Pakistan for their financial support and
providing the chemicals. We should express gratitude to M. Hussnain Khan and Ummaima for
their help in handling the animals.
Conflicts of Interest
The authors declare no conflict of interest.
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