Adhatoda zeylanica is a widely distributed plant which is popularly used for the treatment of diabetes. A review of literature reveals that a new moiety , 2',4-dihydroxy chalcone-4-glucoside has been identified in the flowers of the plant. Chalcones and their derivatives possess antidiabetic activity.The present investigation was designed to study the antihyperglycemic effect of the chalcone containing flower extract ; also, a series of chalcone derivatives were synthesised and their antihyperglycemic activity was compared with that of the plant extract.The newly synthesised chalcones were also characterized by conventional methods.
The objectives of the present investigation were:
To prepare a chalcone containing methanolic extract of the flowers of adhatoda zeylanica.
To separate the phytoconstituents of the above extract broadly by TLC and HPTLC methods.
Synthesis of a series of chalcone derivatives using substituted acetophenones and substituted benzaldehydes.
To identify the chalcone 2',4-dihydroxy chalcone-4-glucoside in the extract using the synthesised (2Z)-1-(2,4-dihydroxyphenyl)-3-phenylprop-2-en-1-one as marker.
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Characterization of the newly synthesised chalcones by IR, NMR and Mass spectrometry.
Evaluation of the possible and extend of antihyperglycemic activity of the plant extract by using an in vitro model regulating glucose diffusion.
Comparison of the effect of the newly synthesised chalcones on glucose diffusion with that of the plant extract and its intraprelation.
MATERIALS AND METHODS:
Chloroform, Methanol, Sodiumhydroxide, Potassiumhydroxide, Ethanol,2',4-dihydroxyacetophenone,p hydroxybenzaldehyde,Vanillin,3,4,5- trimethoxy benzaldehyde,3-methoxybenzaldehyde,2-methoxybenzaldehyde, Benzaldehyde, p-dimethyl amino benzaldehyde, p-chloro benzaldehyde,4-fluoro benzaldehyde, 4-isopropylbenzaldehyde, Anisaldehyde, o-chlorobenzaldehyde, p-bromo acetophenone, o-hydroxyacetophenone, 4-methylacetophenone, p-hydroxyacetophenone, Acetophenone, 4'-aminoacetophenone, p-fluoroacetophenone, Sodium hydroxide.
Glucose concentration was estimated by glucose oxidase peroxidase method.
Beakers (250 ml, 100 ml, 500 ml and 50ml), conical flasks, round bottom flask, soxhlet apparatus, mortar and pestle, glass rod, mechanical stirrer, thermometer, dialysis tube and dialysis membrane, Pre-coated silica gel 60F25 on aluminium sheets were procured from Merk, Germany.
Melting points were determined by using melting point apparatus MP-DS TID 2000V in college of pharmacy, SRIPMS, Coimbatore.
UV spectra were recorded on JASCO V-530 UV/VIS spectrophotometer at Department of Pharmaceutical Analysis, College Of Pharmacy, SRIPMS, Coimbatore.
IR Spectra were recorded on JASCO FT/IR-140 at Department of Pharmaceutical Analysis, College of Pharmacy, SRIPMS, Coimbatore.
PMR spectra were recorded using BRUCKER FT-NMR 200MHz at SAIF, IIT Chennai.
Mass spectra were recorded using mass spectrometer at Department of Pharmaceutical Analysis, College Of Pharmacy, SRIPMS, Coimbatore.
Concentration of glucose measured with JASCO V-530 UV/Vis spectrophotometer.
Number of compounds present in the flower extract can be separated by TLC method.
The finger printing of the flower extract was performed by HPTLC method.
CAMAG HPTLC system supported with wincat software was used
a. Twin Trough Chamber
b. Linomat 5 sample applicator (CAMAG Linomat _100632")
c. HPTLC Scanner- CAMAG TLC scanner 3 "Scanner 3_100904"
pH meter ELICO Pvt. Limited, India.
PREPARATION OF THE FLOWER EXTRACT OF ADHATODA ZEYLANICA
The flowers of Adhatoda zeylanica were washed and about 150g was air dried. These flowers were extracted with the help of soxhlet apparatus with chloroform (250ml) for 12hours, the chloroform layer discarded, then extracted with methanol (250ml) for 36hours.This extract is concentrated to about 10-20ml61.
THIN LAYER CHROMATOGRAPHIC ANALYSIS OF ADHATODA ZEYLANICA FLOWER EXTRACT61-62
Chromatography is a technique which is widely used to separate a mixture of substances into its component parts.
Thin-Layer chromatography(TLC) is a chromatographic technique that is useful for separating organic compounds. Because of the simplicity and rapidity of TLC, it is often used to monitor the progress of organic reactions and to check the purity of products.
Preparation of thin layer plates
A thin layer plate was prepared by spreading an aqueous slurry of the finely ground solid (silica gel G) on the clean surface of 20x20cm glass plate62.
The plate was activated in an oven at 110áµ’C-120áµ’C for 30 minutes prior to sample spotting.
Sample application and detection
The flower extract of Adhatoda zeylanica at the concentration of 10mg/10ml was applied as a spot at a distance of 2cm from the edge of the TLC plate using a thin capillary tube. One of the newly synthesised chalcones (discussed in chapter 2) AC6 [(2Z)-1-(2,4-dihydroxyphenyl)-3-(2-methoxyphenyl)prop-2-en-1-one] was spotted near (2cm) the previous spot, which served as a marker.
Always on Time
Marked to Standard
The plate was placed in a developing chamber and after developing the plate upto 3/4th of the length of the plate, it was removed and dried. Then, the plates were examined in ultra-violet chamber for detecti on and the Rf values were calculated.
Ethyl acetate : Formic acid : Glacial acetic acid : Water
100 : 11 : 11 : 27
RESULT AND DISCUSSION:
Detection and Rf value of flower extract
Detection under UV light
Brown colour flouresence
Brown colour flouresence
Pink colour flouresence
Green colour flouresence
TLC OF FLOWER EXTRACT
From the results, it can be seen that the plant extract exhibits a spot of Rf value 0.14 which corresponds to the Rf value of marker 0.14. Both the spots show brown colour fluorescence too. Therefore, it can safely be assumed that the plant extract contains 2',4-dihydroxy chalcone-4-glucoside. Which is revealed in the literature, in addition to other compounds. Which can be examined for antihyperglycemic effect.
HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHC ANALYSIS OF ADHATODA ZEYLANICA FLOWER EXTRACT63-65
The finger printing of Adhatoda zeylanica was performed by HPTLC method.
HPTLC is a powerful, reliable, sensitive and cost effective separation technique for qualitative and quantitative analysis of herbal components63.
HPTLC -FINGER PRINTING - SCHEMATIC REPRESENTATION65
Selection of chromatographic layer
Sample and standard preparations
Application of sample and standard
Detection of spots
Scanning and Documentation of chromatoplate
HPTLC METHOD DEVELOPMENT FOR FINGER PRINTING
HPTLC precoated,silica gel
Mode of application
8mm separation technique
25Â± 3áµ’Saturation time
254nm Detection TLC
(CAMAG 3 scanner)
FIXED CHROMATOGRAPHIC PARAMETERS
Material :TLC aluminium sheets silica gel 60F 280,[E.MERK KGaA]
Plate size : 20Ã-10 cm
Solvent: Ethyl acetate:Formic acid:Glacial acetic acid:Water
100 : 11 : 11 : 27
Development chamber : Twin Trough Chamber
Chamber saturation time : 20mins.
Development time : 15min.
Drying time of the
development plate : 5min.
Calibration mode : Multi level
Evaluation mode : Peak area
Instrument:CAMAG Linomat 5 "Linomat_100632"
Linomat 5 application parameters.
Spray gas:Inert nitrogen gas
Syringe size : 100Î¼l
Number of tracks : 9
Application position Y : 10.0mm
Band length : 8.0mm
Detection-CAMAG TLC Scanner 3
Instrument:CAMAG Scanner 3 "Scanner 3_100904"
Solvent front position : 80.0 mm
Position of first track: : 15.0 mm
Slit dimensions : 5.00Ã-0.45 mm,Micro
Optimize optical system : Light
Scanning speed : 20 mm/s
Data resolution : 100Î¼m/step
Wavelength : 254nm as single wavelength measurement.
Lamp : D2
Measurement type : Remission
Measurement mode : Absorption and fluorescence
Optical Filter : Second order and K400
PM high voltage : 275 V and 313V
Preparation of stock solution for HPTLC
Flower extract of Adhatoda zeylanica (100Î¼g/ml)
10mg of methanolic extract of Adhatoda zeylanica flowers were weighed and taken in a 100ml standard flask. Then the volume is made upto the mark with methanol, to obtain the final concentration of 100Î¼g/ml. It was then filtered through whatmann filter paper. From the filterate 2Î¼g/spot of the solution was applied on the TLC plate using linomat 5 auto sampler.
Preparation of mobile phase
The mixture of ethyl acetate, formic acid, glacial acetic acid and water in the proportional ratio of 100: 11: 11: 27v/v 61, respectively was prepared and used as the mobile phase.The mixture was thoroughly mixed before transferring into the TLC chamber.
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The mobile phase was kept aside till saturation for about 20minutes by placing a filter paper over the solution.
Application of the sample solutions:
The aliquots of sample solution was applied in volumes of 2,4,6,8,10Î¼l over the silica gel TLC plate of size 20x10cm.Then the plate was developed using the mobile phase and analysed using HPTLC scanner 3 64.
Under fixed chromatographic conditions most of the phyto-constituents present in the Adhatoda zeylanica were measured under the absorption mode.
Hptlc Chromatogram at 254 nm
RESULT AND DISCUSSION:
The developed TLC plate was scanned in absorption mode at 254 nm and the chromatogram of the extract was obtained is shown in the figure.
The HPTLC chromatogram showed a peak corresponding to the Rf value of 0.14 which again confirms the presence of 2',4-dihydroxy chalcone-4-glucoside in the methanolic extract of the flower, which is used for further studies on glucose diffusion. Also, it was seen that the chalcone
2',4-dihydroxy chalcone-4-glucoside constitutes approximately 3.4 % of the total phytoconstituents in the extract.