Determination Of Stigmasterol From Polyherbal Formulation Biology Essay

The infusions were obtained from consecutive dissolver of clerodendrum serratum. 10mg of each infusion was weighed and made up to 100ml with dissolvers such as crude oil quintessence, trichloromethane, ethyl ethanoate and methyl alcohol severally. The concentrations of 1000 I?g/ml solutions were prepared and filtered over Whatmann filter paper. 20 I?g/spot of each solution was applied on the TLC home base.10 milligram of marker compound was transferred into 10ml standard flask. The volume was made with trichloromethane to acquire the concentration of 1000 I?g/ml. fix 50 I?g/ml from the above stock solution 300-900ng /spot ( 3-15 I?l/spot ) was applied on the HPTLC home base.

Preparation of stock solutions of the infusion of Formulation.

The infusions were obtained from the preparation of clerodendrum serratum.10mg of each infusion was weighed and made up to 100ml methyl alcohol. The concentration of 1000 I?g/ml solutions were prepared and filtered over Whatmann filter paper. 20 I?g/spot of each solution was applied on the TLC home base.

Fixed Chromatographic Parameters:

Stationary stage

Material: TLC aluminum sheets silica gel 60 F 254, [ E.

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MERCK KGaA ]Plate size: 10A-10 centimeter

Mobile stage:

Solvent: CHCl3: MeOH ( 95:5 v/v )Development chamber: Twin Trough ChamberChamber impregnation clip: 20 min.Development clip: 20min.Drying clip of the developed home base: 5 min.

Calibration Parameters:

Calibration manner: Multi degreeEvaluation manner: Peak Area

Sample application:

Instrument: CAMAG Linomat 5 “ Linomat_100632 ”Linomat 5 applier. WINCAT SoftwareSpray gas: Inert N gas.

Dosage velocity: 150 ml/s


Syringe size: 100 I?lNumber of paths: 8Application place Y: 10.0 millimeterBand length: 6.0 mm.tection – CAMAG TLC Scanner 3Instrument: CAMAG Scanner 3 “ Scanner 3_100904 ”Solvent front place: 80.0 millimeterPosition of first path: 15.

0 millimeterSlit dimensions: 5.00 A- 0.45 millimeter, MicroOptimize optical system: LightScaning velocity: 20 mm/sData declaration: 100I?m/step.


Wavelength: 366nm.Lamp: D2Measurement Type: RemissionMeasurement Mode: AbsorptionOptical Filter: Second order and K400PM high electromotive force: 275 V and 313V


In Absorption Mode:

The developed HPTLC home bases were scanned in soaking up manner at 366 nanometers and the chromatograms of marker compound, all four consecutive infusion and preparations are taken. The concentration and peak Area of the standardization graph, sum of stigmasterol nowadays in consecutive infusions and preparation are tabulated.

Choice and Prewashing of Plate

A precoated silicon oxide gel 60 F254 on aluminium sheet with 100-250 millimeter thickness was selected for the survey.

Pre-washing of home base was done with methyl alcohol and so it was activated by maintaining in an oven at 1150C for 10 proceedingss.

Choice of dissolver

The drug is dissolved in a dissolver in which it is soluble and in which the drug shows good stableness. Another standard for choice of dissolver is that it has to be volatile, inexpensive and easy available. stigmasterol is readily soluble in trichloromethane and shown good stableness. Hence trichloromethane was selected as dissolver.

Choice of wavelength

An ideal wavelength is one that give maximal optical density and good response to the drug to be detected. UV spectrum of drug shows maximal optical density at 286 nanometer which was selected as sensing wavelength.

( Fig1 ) .

Fig1: UV spectrum of standard Stigmasterol on TLC home base

Choice of nomadic stage

Solvent system tried


Chloroform: H2ODrug did non travelbutyl ethanoate: trichloromethaneDrug did non traveln-butanol: butyl ethanoateChasingChloroform: MethanolCompact topographic point

Optimization of nomadic stage ratio:

Different ratios of Chloroform: Methanol like 5:5, 4:6, 3:7, 2:8 were tried and the ratio of ( 95:5 v/v ) was selected because it gave compact musca volitanss with good separation from solvent forepart and sample application places and good symmetrical extremum.

Fixed Experimental Conditionss:

Stationary stage – TLC precoated silica gel 60F254Mobile stage – Chloroform: Methanol ( 95:5v/v )Development chamber – Twin trough glass chamberSeparation technique – Rise developmentDistance developed – 85 millimeterDetection wavelength – 366 nanometerRf value – 0.80 A± 0.


Preparation of standard stock solution

Stock solution was prepared by weighing 10 milligram of stigmasterol into a 10ml criterion flask and the volume was made upto 10ml with trichloromethane to acquire a concentration of 1000Aµg/ml.

Preparation of Standard Graph

From the stock solution of stigmasterol, 0.5 to 5 Aµl was spotted on the TLC home base followed by development and scanning of musca volitanss. The chromatograms are shown in Fig 3. The peak countries were recorded. Calibration graph was obtained by plotting the peak countries against the corresponding concentration of the standard solutions. The values are tabulated.


Fig 3: HPTLC chromatogram of Stigmasterol – ( 300ng/spot )Fig 4: HPTLC chromatogram of Stigmasterol – ( 450ng/spot )Fig 5: HPTLC chromatogram of Stigmasterol – ( 600ng/spot )Fig 6: HPTLC chromatogram of Stigmasterol – ( 750ng/spot )Fig 7: HPTLC chromatogram of Stigmasterol – ( 900ng/spot )



Peak country











Fig 8: Calibration graph of STIGMASTEROL


Fig 9: HPTLC Chromatogram of PEE at 366nm

Fig 10: HPTLC Chromatogram of CE at 366 nanometers

Fig 11: HPTLC Chromatogram of EAE at 366 nanometers

Fig 12: HPTLC Chromatogram of ME at 366 nanometers




Sum OF STIGMASTEROL ( milligram )

Petroleum ETHER0.

948Chloroform0.68ETHYL ACETATE0.23Methanol0.10Formulation0.



Preparation of sample solution

10mg of preparation infusion ( triterpens ) each incorporating 0.761mg of clerodendrum serratum was weighed, Quantity equivalent to 10 milligram of criterion was weighed, transferred to a 100 milliliter volumetric flask, extracted and made up to volume with and filtered through a Whatman filter paper and this was used for the survey.The preparation was assayed by descrying 4 Aµl onto the home base followed by development and scanning. The Chromatograms were recorded which are shown in Fig The sum of stigmasterol was calculated from peak country arrested development analysis table:4 and the equation is as followsY = I± + I?xI± = ( I?y ) ( I?x2 ) – ( I?x ) ( I?xy )N I?x2 – ( I?x ) 2I? = NI?xy – ( I?x ) ( I?y )N I?x2 – ( I?x ) 2where, x = concentrationY = optical densityN = Number of braces of values

Table 4: Analysis of preparation


Sum 10mg

% label claim +





35496.93 + 0.3727

Validation OF THE METHOD

The proof of the developed method was carried out in footings of one-dimensionality, truth, bound of sensing ( LOD ) , bound of quantification ( LOQ ) , inter and intraday preciseness, repeatability of sample application, measuring, stableness surveies and selectivity.

One-dimensionality and Range

Linear arrested development informations showed a good linear relationship over a concentration scope of 300 to 900 ng/spot.

( Fig: 8 ) The incline, intercept and correlativity co-efficient values were found to be 0.9976.


Recovery surveies of the drug was carried out for finding truth parametric quantity. It was done by blending known measure of standard drug with the pre-analysed sample preparation and the contents were reanalyzed by the proposed method.Recovery surveies carried out at 50 and 100 % degrees. The per centum recovery and its % RSD were calculated table 5.

Table 5: Recovery surveies


% Recovery

% rsd*

80 %102.

980.96153100 %96.50.3751* Mean of three findings


Preciseness of the method was demonstrated byIntra-day precisenessInter-day precisenessRepeatabilityRepeatability of measuringRepeatability of sample application

Intra-day preciseness

Intra twenty-four hours preciseness was found out by transporting out the analysis of the standard drug for two different concentrations in the one-dimensionality scope of drug for three times on the same twenty-four hours and % RSD was calculated, table 6.

Table 6: Intraday preciseness

Volume applied ( Aµl )

Peak Area

% RSD*




2* Mean of 3 findings

Inter-day preciseness

Inter twenty-four hours preciseness was found by transporting out the analysis of the standard drug for two different concentrations in the one-dimensionality scope of drug for two yearss and % RSD was calculated which is shown in table 7.

Table 7: Inter-day preciseness

Vol applied ( Aµl )

DayPEAK AREA% RSD*312666.60.968722680.332693.2615634.


20.815126145.636114.S9* Mean of 3 findings


Repeatability of sample application

Repeatability of sample application was assessed by descrying 2 and 4Aµl of drug solution five times on pre-coated TLC home base followed by development of home base and % RSD was calculated, table8.

Table 8: Repeatability of sample application

Volume Applied ( AµL )

Peak Area

% RSD*

96128.00.45146132.16122.76121.76135.76130.9* Mean of 5 findings

Repeatability of measuring

Repeatability of measuring of peak country was determined by descrying 2 and 4 Aµl of preparation on pre-coated TLC home base.

After development of the home base, the detached musca volitanss were scanned five times without altering place of the home base and % RSD was calculated. ( table:9 )

Table 9: Repeatability of measuring for preparation

Volume Applied ( Aµl )

Peak Area

% RSD*


66134.56118.6* Mean of 5 findings


LOD and LOQ were determined by using diminishing sum of the drug in triplicate on the home base.

The lowest concentration at which the extremum is detected is called Limit Of Detection and it was found to be 100ng/spot. ( Fig: ) The lowest concentration at which the extremum is quantified is called Limit Of Quantification and it was found to be 300ng/spot ( Fig: 14 )

Fig 14: LOD of stigmasterol ( 100ng/spot )

Fig15: LOQ of Stigmasterol ( 300 ng/spot )

Stability surveies

When the developed chromatographic home base is exposed to atmosphere, the analytes are likely to break up. Hence it is necessary to carry on stableness surveies.Stability of the analyte on the home base was studied at different clip intervals and peak countries were compared with the peak country of newly scanned home base.The developed home base was found to be stable for about 7 hours as the decrease in peak countries was within the bounds. The values are shown in table10.

Table 10: Stability of the analyte on home base

Volume Applied ( I?l )Time ( hours )Peak Area613066.523115.533139.643118.353143.963146.8


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