Adhatoda zeylanica is a widely distributed works which is popularly used for the intervention of diabetes. A reappraisal of literature reveals that a new mediety, 2′,4-dihydroxy chalcone-4-glucoside has been identified in the flowers of the works. Chalcones and their derived functions possess antidiabetic activity.The present probe was designed to analyze the antihyperglycemic consequence of the chalcone incorporating flower infusion ; besides, a series of chalcone derived functions were synthesised and their antihyperglycemic activity was compared with that of the works extract.The freshly synthesised chalcones were besides characterized by conventional methods.
The aims of the present probe were:
To fix a chalcone incorporating methanolic infusion of the flowers of adhatoda zeylanica.
To divide the phytoconstituents of the above infusion loosely by TLC and HPTLC methods.
Synthesis of a series of chalcone derived functions utilizing substituted acetophenones and substituted benzaldehydes.
To place the chalcone 2′,4-dihydroxy chalcone-4-glucoside in the infusion utilizing the synthesised ( 2Z ) -1- ( 2,4-dihydroxyphenyl ) -3-phenylprop-2-en-1-one as marker.
Word picture of the freshly synthesised chalcones by IR, NMR and Mass spectroscopy.
Evaluation of the possible and extend of antihyperglycemic activity of the works infusion by utilizing an in vitro theoretical account modulating glucose diffusion.
Comparison of the consequence of the freshly synthesised chalcones on glucose diffusion with that of the works infusion 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 hydrated oxide.
Glucose concentration was estimated by glucose oxidase peroxidase method.
Beakers ( 250 milliliter, 100 milliliter, 500 milliliter and 50ml ) , conelike flasks, unit of ammunition underside flask, soxhlet setup, howitzer and stamp, glass rod, mechanical scaremonger, thermometer, dialysis tubing and dialysis membrane, Pre-coated silica gel 60F25 on aluminum sheets were procured from Merk, Germany.
Melting points were determined by utilizing runing point setup MP-DS TID 2000V in college of pharmaceutics, 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 utilizing BRUCKER FT-NMR 200MHz at SAIF, IIT Chennai.
Mass spectra were recorded utilizing 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 infusion can be separated by TLC method.
The finger printing of the flower infusion was performed by HPTLC method.
CAMAG HPTLC system supported with wincat package was used
a. Twin Trough Chamber
b. Linomat 5 sample applier ( CAMAG Linomat _100632 ” )
c. HPTLC Scanner- CAMAG TLC scanner 3 “ Scanner 3_100904 ”
pH metre 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 aid of soxhlet setup with trichloromethane ( 250ml ) for 12hours, the trichloromethane bed discarded, so extracted with methyl alcohol ( 250ml ) for 36hours.This infusion 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 divide a mixture of substances into its constituent parts.
Thin-Layer chromatography ( TLC ) is a chromatographic technique that is utile for dividing organic compounds. Because of the simpleness and celerity of TLC, it is frequently used to supervise the advancement of organic reactions and to look into the pureness of merchandises.
Preparation of thin bed home bases
A thin bed home base was prepared by distributing an aqueous slurry of the finely land solid ( silica gel G ) on the clean surface of 20x20cm glass plate62.
The home base was activated in an oven at 110aµ’C-120aµ’C for 30 proceedingss prior to try staining.
Sample application and sensing
The flower infusion of Adhatoda zeylanica at the concentration of 10mg/10ml was applied as a topographic point at a distance of 2cm from the border of the TLC home base utilizing a thin capillary tubing. One of the freshly 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 old topographic point, which served as a marker.
The home base was placed in a underdeveloped chamber and after developing the home base upto 3/4th of the length of the home base, it was removed and dried. Then, the home bases were examined in ultra-violet chamber for detecti on and the Rf values were calculated.
Ethyl ethanoate: Formic acid: Glacial acetic acid: Water
100: 11: 11: 27
RESULT AND DISCUSSION:
Detection and Rf value of flower infusion
Detection under UV visible radiation
[ ( 2Z ) -1- ( 2,4-dihydroxyphenyl ) -3- ( 2methoxyphenyl ) prop-2-en-1-one ] AC6
Brown coloring material flouresence
Brown coloring material flouresence
Tap coloring materials flouresence
Green coloring material flouresence
TLC OF FLOWER EXTRACT
From the consequences, it can be seen that the works infusion exhibits a topographic point of Rf value 0.14 which corresponds to the Rf value of marker 0.14. Both the musca volitanss show brown color fluorescence excessively. Therefore, it can safely be assumed that the works infusion contains 2′,4-dihydroxy chalcone-4-glucoside. Which is revealed in the literature, in add-on to other compounds. Which can be examined for antihyperglycemic consequence.
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, dependable, sensitive and cost effectual separation technique for qualitative and quantitative analysis of herbal components63.
HPTLC -FINGER PRINTING – Schematic REPRESENTATION65
Choice of chromatographic bed
Sample and standard readyings
Application of sample and criterion
Detection of musca volitanss
Scaning and Documentation of chromatoplate
HPTLC METHOD DEVELOPMENT FOR FINGER Printing
HPTLC precoated, silicon oxide gel
60, F254 ( Merk )
Mode of application
8mm separation technique
25A± 3aµ’Saturation clip
Optical density manner
Coefficient of reflection slit
254nm Detection TLC
( CAMAG 3 scanner )
FIXED CHROMATOGRAPHIC PARAMETERS
Material: TLC aluminum sheets silica gel 60F 280, [ E.MERK KGaA ]
Plate size: 20A-10 centimeter
Solvent: Ethyl ethanoate: Formic acid: Glacial acetic acid: Water
100: 11: 11: 27
Development chamber: Twin Trough Chamber
Chamber impregnation clip: 20mins.
Development clip: 15min.
Drying clip of the
development home base: 5min.
Calibration parametric quantities
Calibration manner: Multi degree
Evaluation manner: Peak country
Instrument: CAMAG Linomat 5 “ Linomat_100632 ”
Linomat 5 application parametric quantities.
Spray gas: Inert N gas
Syringe size: 100I?l
Number of paths: 9
Application place Y: 10.0mm
Band length: 8.0mm
Detection-CAMAG TLC Scanner 3
Instrument: CAMAG Scanner 3 “ Scanner 3_100904 ”
Solvent front place: 80.0 millimeter
Position of first path: : 15.0 millimeter
Slit dimensions: 5.00A-0.45 millimeter, Micro
Optimize optical system: Light
Scaning velocity: 20 mm/s
Data declaration: 100I?m/step
Measurement tabular array
Wavelength: 254nm as individual wavelength measuring.
Measurement type: Remission
Measurement manner: Absorption and fluorescence
Optical Filter: Second order and K400
PM high electromotive force: 275 V and 313V
Preparation of stock solution for HPTLC
Flower infusion of Adhatoda zeylanica ( 100I?g/ml )
10mg of methanolic infusion of Adhatoda zeylanica flowers were weighed and taken in a 100ml standard flask. Then the volume is made upto the grade with methyl alcohol, to obtain the concluding concentration of 100I?g/ml. It was so filtered through whatmann filter paper. From the filterate 2I?g/spot of the solution was applied on the TLC home base utilizing linomat 5 car sampling station.
Preparation of nomadic stage
The mixture of ethyl ethanoate, formic acid, glacial acetic acid and H2O in the relative ratio of 100: 11: 11: 27v/v 61, severally was prepared and used as the nomadic phase.The mixture was exhaustively assorted before reassigning into the TLC chamber.
The nomadic stage was kept aside till impregnation for about 20minutes by puting a filter paper over the solution.
Application of the sample solutions:
The aliquots of sample solution was applied in volumes of 2,4,6,8,10I?l over the silica gel TLC home base of size 20x10cm.Then the home base was developed utilizing the nomadic stage and analysed utilizing HPTLC scanner 3 64.
Under fixed chromatographic conditions most of the phyto-constituents nowadays in the Adhatoda zeylanica were measured under the soaking up manner.
Hptlc Chromatogram at 254 nanometers
Proportional concentration ( % )
RESULT AND DISCUSSION:
The developed TLC home base was scanned in soaking up manner at 254 nanometers and the chromatogram of the infusion was obtained is shown in the figure.
The HPTLC chromatogram showed a peak corresponding to the Rf value of 0.14 which once more confirms the presence of 2′,4-dihydroxy chalcone-4-glucoside in the methanolic infusion of the flower, which is used for farther surveies on glucose diffusion. Besides, it was seen that the chalcone
2′,4-dihydroxy chalcone-4-glucoside constitutes about 3.4 % of the entire phytoconstituents in the infusion.