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Synthesis and Evaluation of Chalcone Derivatives For Its Alpha Amylase Inhibitory Activity PDF
Synthesis and Evaluation of Chalcone Derivatives For Its Alpha Amylase Inhibitory Activity PDF
Organic CHEMISTRY
An Indian Journal
Full Paper
OCAIJ, 10(5), 2014 [192-204]
ABSTRACT
Chalcones represent a group of compounds with interesting biological activities that are formed from Claisen-
Schmidt reaction between a benzaldehyde and an acetophenone in the presence of NaOH as a catalyst and ethanol
as a solvent. Four different chalcones were synthesized using various substituted aldehydes as 4-nitrobenzaldehyde,
4-hydroxybenzaldehyde, 4-chlorobenzaldehyde, and 2-furfuraldehyde. The designed molecules were successfully
synthesized in the laboratory using literature methods and structures were confirmed by NMR and IR spectroscopy.
For anti-diabetic activity detection alpha amylase inhibitory assay was carried out. According to the results obtained,
it can be concluded that of the synthesized compound 3-(4-hydroxyphenyl)-1-phenylprop-2-en-1-one has an Alpha
amylase inhibitory activity. 2014 Trade Science Inc. - INDIA
Full Paper
Figure 1 : Chalcone
Organic CHEMISTRY
An Indian Journal
194 Synthesis and evaluation of chalcone derivatives for its alpha amylase inhibitory activity OCAIJ, 10(5) 2014
Full Paper
Instruments stirred in ethanol (8 ml) was added with 2-furaldehyde
TABLE 2 : List of instrument’s used in the formulation devel-
(Furfural) (3.0 g, 0.025 mole) in a conical flask (25
opment mL) and then NaOH 30% (4 mL) was added drop
wise into it. The mixture was stirred in ice cold water
Sr.No Name of the Equipment Manufacturer
bath until it solidified. Then solidified mass is kept in
1 FT-NMR Shimadzu
cold condition overnight and after that solidified mass
2 Magnetic Stirrer Remi Laboratory
Fourier Transform Infrared
separated and dried on room temperature to give com-
3 Shimadzu pound.
Spectroscopy
4 U V Spectroscopy Shimadzu Synthesis of designed molecules (Compound 2)
6 Melting point apparatus Veego
Hexatec instruments A solution Acetophenone (3.0 mL, 0.025 mole)
7 Hot air oven stirred in ethanol (8 ml) was added with 4-
Pvt. Ltd.
Chlorobenzaldehyde (3.0 g, 0.025 mole) in a conical
General procedures
flask and then NaOH 30% (4 mL) was added drop
Analytical thin-layer chromatography (TLC) was wise into it. The mixture was stirred in ice cold water
carried out on precoated plates SiO2 (silica gel 60, F bath until it solidified. Then solidified mass is kept in
254, Merck).The spots were viewed under ultraviolet cold condition overnight and after that solidified mass
(UV) light. FTIR spectra were recorded on Perkin Elmer separated and dried on room temperature to give com-
RX I spectrometer using KBr pellets. All melting points pound.
(m.p.) were recorded on VMP-DS (VEEGO Instru-
Synthesis of designed molecules (Compound 3)
ments corporation), having oil-heating system and were
uncorrected. The NMR spectra were recorded on A solution Acetophenone (3.0 mL, 0.025 mole)
JEOL AL-300 FT-NMR spectrometer and Schimatzu stirred in ethanol (8 ml) was added with 4-
60 MHz FT-NMR with CDCl3 / DMSO-D6 as sol- Hydroxybenzaldehyde (3.0 g, 0.025 mole) in a conical
vent using tetramethylsilane (TMS) as internal reference. flask and then NaOH 30% (4 mL) was added drop
wise into it. The mixture was stirred in ice cold water
Synthesis of chalcone
bath until it solidified. Then solidified mass is kept in
The synthesis of chalconewas carried out via cold condition overnight and after that solidified mass
crossed Aldol or Claisen- Schmidt condensation of com- separated and dried on room temperature to give com-
mercially available Acetophenone and Benzaldehyde in pound.
the presence of NaOH (30%) as a base in ethanol. The
Synthesis of designed molecules (Compound 4)
reaction was initiated by removal of a proton from the
á-carbon of Acetophenone to form a resonance-stabi- A solution Acetophenone (3.0 mL, 0.025 mole)
lized enolate ion by the base. This was followed by the stirred in ethanol (8 ml) was added with 4-
nucleophilic enolate attacks on the electrophilic carbo- nitrobenzaldehyde (3.0 g, 0.025 mole) in a conical flask
nyl carbon of benzaldehyde resulting in a new carbon- and then NaOH 30% (4 mL) was added drop wise
carbon bond formation. This reaction joined the á-car- into it. The mixture was stirred in ice cold water bath
bon of acetophenone to the carbonyl carbon of benzal- until it solidified. Then solidified mass is kept in cold
dehyde to form intermediate. The final step of this re- condition overnight and after that solidified mass sepa-
action was protonation and deprotonation by hydrox- rated and dried on room temperature to give compound.
ide ion to form an á,â-unsaturated ketone, chalcone or Enzymatic assay of alpha-amylase
1, 3-diphenylpropenone as a light yellow solid in
78.33% yield. The reaction mechanism for chalcone Reagent preparation
synthesis is illustrated in Scheme 6.1. (A) Starch solution
Synthesis of designed molecules (Compound 1) 0.5% (w/v) Starch Solution (Starch) was prepared
in 20 mM Sodium Phosphate Buffer (pH-6.9)and
A solution Acetophenone (3.0 mL, 0.025 mole) solubilisation of starch was done by heating the starch
Organic CHEMISTRY
An Indian Journal
OCAIJ, 10(5) 2014 Yusuf Kachwala et al. 195
Full Paper
solution in a glass beaker directly on a water bath using tion was kept on boiling water bath exactly for 15 min-
constant stirring for 15 minutes. utes then cool the solution on ice to room temperature
(B) Colour reagent solution and 9 ml water was added.
Colour reagent was prepared by addition of So- Solutions were mixed and absorbance at 540 nm
dium Potassium Tartrate Solution to 96 mM 3, 5- was recorded using uv spectrophotometer.
Dinitrosalicylic Acid SolutionWith stirring, and was Calculation
stored in amber colored bottle and protected from light. Standard curve
(C) 0.2% (w/v) maltose DA540nm Standard = A540nm Std - A540nm Std Blank
Standard Solution was prepared by dissolving mal- Plot the DA540nm of the Standards vs. milligrams
tose monohydrate in to distil water. of Maltose.
(D) Alpha-amylase solution Sample determination
Immediately before use, alpha amylase solution 2mg/
DA540nm Sample = A540nm Test - A540nm Blank
ml in ice cold distil water was prepared.
Determine the milligrams of Maltose liberated us-
Procedure ing the standard Curve.
TABLE 3 : Reagents and their quantity for preparation of Units/ml enzyme =(mg of Maltose released) (df)
standard curve (1)
Std Std Std Std Std Where, df = dilution factor; 1 = Volume (in milliliter) of
Standard Blank
1 2 3 4 5
0.2%w/vs maltose enzyme used;
0.2 0.4 0.6 0.8 1.0 - Units/mg solid = units/mlenzyme
solution
Distilled water 1.8 1.6 1.4 1.2 1.0 2.0 mg solid/ml enzyme
Colour reagent 1.0 1.0 1.0 1.0 1.0 1.0
Alpha amylase inhibitory activity assay
Standard curve method Procedure
A standard curve was plotted by pipetting (in Crude á –amylase was dissolved in ice-cold dis-
millilitres) the following reagents into a volumetric flaskAnd tilled water to give a concentration of 4 unit/ml solution.
kept on boiling water bath for exactly 15 minutes, and Starch (0.5% w/v) in 20mM phosphate buffer (pH 6.9)
then cool on ice to room temperature and Water was containing 6.7mM sodium chloride, was used as a sub-
added 9.00 ml in each flask and Mix by inversion and strate solution. Experiments were performed with three
record the A540nm for the Standards and Standard Blank replicate determinations for each experiment.
using a suitable spectrophotometer. 40 ìl of plant extract (20 mg/ml in DMSO), 160ìl
For test incubation, test and blank solutions were of distilled water and 400ìl of starch were mixed in a
prepared. screw top plastic tube. The reaction was started by the
For test solution preparation 1 ml starch solution addition of 200ìl of the enzyme solution. The tubes
was equilibrated at 25°C and 1 ml freshly prepared were incubated at 25°C for a total of 3 min. Final con-
enzyme solution was added and solution was mixed centrations in the incubation mixture were plant extract,1
and incubated at 25°C exactly for 0,1,2,3,6,12 and 24 mg/ml, 0.25% (w/v) starch and 1 unit/ml enzyme. At
minutes then 1 ml colour reagent was added and solu- intervals from addition of enzyme (1, 2 and 3 min), 200ìl
tion was kept on boiling water bath exactly for 15 min- mixture was removed and added into a separate tube
utes then cool the solution on ice to room temperature containing 100ìl DNS colour reagent solution (96mM
and 9 ml water was added. 3,5-dinitrosalicylic acid, 5.31M sodium potassium
For blank solution preparation 1 ml starch solution tartarate in 2M NaOH) and placed into a 85°C water
was equilibrated at 25°C and incubated at 25°C ex- bath. After 15 min, this mixture was diluted with 900 ìl
actly for 3 minutes then 1 ml freshly prepared enzyme distilled water and removed from the water bath. á-
solution and 1 ml colour reagent were added and solu- Amylase activity was determined by measuring the ab-
Organic CHEMISTRY
An Indian Journal
196 Synthesis and evaluation of chalcone derivatives for its alpha amylase inhibitory activity OCAIJ, 10(5) 2014
Full Paper
sorbance of the mixture at 540 nm. calculated as:
Control incubations, representing 100% enzyme ac- A540nm control or plant extract = A540nm Test - A540nm
tivity were conducted in an identical fashion replacing Blank
plant extract with DMSO (40 ìl). From the net absorbance obtained, the % (w/v) of
For blank incubations (to allow for absorbance pro- maltose generated was calculated from the equation
duced by the plant extract), the enzyme solution was re- obtained from the maltose standard calibration curve
placed with distilled water and the same procedure was (0.2% w/v maltose).
carried out as above. A separate set of incubations was Percent of inhibition was calculated as 100 – %
prepared for the reaction of t = 0 min, adding samples to reaction at t = 3 min whereby the
the DNS solution immediately after addition of enzyme. % reaction = (mean maltose in sample/mean maltose in con-
trol) ×100
Calculation
The absorbance (A) due to maltose generated was Synthesis of chalcone
Organic CHEMISTRY
An Indian Journal
OCAIJ, 10(5) 2014 Yusuf Kachwala et al. 197
Full Paper
Organic CHEMISTRY
An Indian Journal
198 Synthesis and evaluation of chalcone derivatives for its alpha amylase inhibitory activity OCAIJ, 10(5) 2014
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TABLE 4 : Description for compound synthesized by general The representative IR spectra for the starting ma-
procedure terial & the product were depicted below:
Tests Inference Synthesis of designed molecules (Compound 1)
Colour Yellow
The representative IR spectra for the starting ma-
State Solid
terial & the product were depicted below
Odour Aromatic
Yield 82%
Synthesis of designed molecules (Compound 2)
Melting Point 58-60 °C The representative IR spectra for the starting ma-
UV ëmax 306 nm terial & the product were depicted below
C=O 1664.21 Synthesis of designed molecules (Compound 3)
IR frequencies (cm-1) C=C (Aromatic) 1577.77, 1449.71 The representative IR spectra for the starting ma-
C=C (Olefinic) 1606.26 terial & the product were depicted below
Full Paper
Organic CHEMISTRY
An Indian Journal
200 Synthesis and evaluation of chalcone derivatives for its alpha amylase inhibitory activity OCAIJ, 10(5) 2014
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TABLE 6 : Description of compound-2 synthesized by gen- TABLE 8 : Description of compound-4 synthesized by gen-
eral procedure eral procedure
Tests Inference Tests Inference
Colour Light Yellow Colour Brownish Yellow
State Solid State Solid
Odour Aromatic Odour Aromatic
Yield 77% Yield 81%
Melting Point 110-113 °C Melting Point 154-157 °C
UV ëmax 313.85 nm UV ëmax 315.87 nm
-CL 788.21 -NO2 1513.21
IR frequencies (cm-1): C=C 1190 IR frequencies (cm-1): C=O 1645.56
C=O 1634.26 Ar-CH 1449.23
Organic CHEMISTRY
An Indian Journal
OCAIJ, 10(5) 2014 Yusuf Kachwala et al. 201
Full Paper
Organic CHEMISTRY
An Indian Journal
202 Synthesis and evaluation of chalcone derivatives for its alpha amylase inhibitory activity OCAIJ, 10(5) 2014
Full Paper
Organic CHEMISTRY
An Indian Journal
OCAIJ, 10(5) 2014 Yusuf Kachwala et al. 203
Full Paper
Organic CHEMISTRY
An Indian Journal
204 Synthesis and evaluation of chalcone derivatives for its alpha amylase inhibitory activity OCAIJ, 10(5) 2014
Full Paper
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[4] H.Garcia, S.Iborra; 6-Endo-Dig vs. 5-Exo-Dig ring novel, 266-270 (2012).
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new approach to the synthesis of flavones and chalcone derivatives on á-glucosidase, 410-414
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Organic CHEMISTRY
An Indian Journal