1.INTRODUCTION:

Forskolin is a labdane, 7b-acetoxy-1a, 6b, 9a-tri-hydroxy-8, 13-epoxy-labd-14-en-11-one, primarily extracted from Coleus forskohlii Briq. Belongs to the family Lamiaceae. This diterpenoid has been investigated as a promising new drug: it shows positive inotropic, hypotensive activity, inhibits thrombocyte aggregation and has been proposed in the treatment of ocularhypertension and stimulation of skin melanogenesis. It acts by directly stimulating adenylatecyclase, resulting in an increase in the ‘‘second messenger’’ c-AMP ^{[1- 6]}. Forskolin (FSK) derivatives have been proposed as a treatment against hair loss and grey hair and their pharmacological properties have also been widely studied.

Nowadays, HPTLC has become a routine analytical technique due to its advantages of reliability in quantitation of analytes at micro and even in nanogram levels and cost effectiveness.

The major advantage of HPTLC is that several samples can be analyzed simultaneously using a small quantity of mobile phase unlike HPLC. This reduces the time and cost of analysis and possibilities of pollution of the environment. HPTLC also facilitates repeated detection (scanning) of the chromatogram with same or different parameters. The aim of the present work is to develop and validated an accurate, speciﬁc and reproducible HPTLC method for determination of forskolin ^[7-10].

2. MATERIAL AND METHODS:

Drug and Chemicals

Forskolin was obtained from Sigma Chemicals, Bangalore, India, and used without further puriﬁcation, certiﬁed to contain 99% (w/w). Analytical grade methanol, chloroform, anisaldehyde, toluene, formic acid and ethyl acetate, hexane, acetone and glacial acetic acid were al lobtained from Qualigens Fine Chemicals, Mumbai, India.

HPTLC instrumentation and chromatographic condition

The chromatographic estimation was performed by spotting standard and sample on pre-coated silica gel aluminums plate F-254 (250 um thickness, E, Merk) using a Camag Linomate IV sample applicator (Camag, Muttenz, Switzerland) and a 100 ul Hamilton syringe. The standard and samples, in the form band of 6 mm, were spotted 15 mm from the bottom, 12 mm from the left margin of the plate and 8 mm apart, at a constant application rate of 10s/µl using nitrogen aspirator. Plates were developed using mobile phase, development was carried out in 10 cm x 10 cm and 20 cm x 10 cm twin through glass chamber (Camag, Muttenz, Switzerland) equilibrated with suitable mobile phase. The optimized chamber saturation time for mobile phase was 1 hour with filter paper at room temperature. The length of chromatogram run was 7.5 cm, 10 ml of mobile phase (5 ml in trough containing the plate and 5 ml in the other trough) was used for each development, which required 20 min. It results in better apparent resolution with more convenient capability of the detecting device to perform integration of peak area. Subsequent to the development, TLC plates were dried in a current air with the help of a hair-dryer. The slit dimension setting of length 4 mm and width 0.30 mm, and a scanning rate of 20 mm/s and data resolution of 100µm/step. Densitometry scanning was performed at 200 nm on Camag TLC scanner III and operated by winCATS Planar Chromatography version 1.4.3. The source of radiation utilized was deuterium lamp. Concentration of the compound chromatographed was determined from the intensity of diffusedly reflected light. Evaluation was via peak area with linear regression.

Sample preparation

Accurately weighed powdered sample were dissolved in methanol individually and kept for sonication for about 15 min at a temperature of 30^oC and filtered, then made up the volume with methanol.

Standard preparation

Accurately weighed 10 mg of standard Forskolin of purity 98%w/w was dissolved in 10ml of methanol by sonication.

Preparation of Mobile phase

Different compositions of mobile phase for HPTLC analysis were tested in order to obtain high resolution, symmetrical and reproducible peaks for colchicine. The desired resolution of compound was achieved by using Ethyl Acetate: Methanol [10: 1.35] as the mobile phase. On this system separation is good and peaks of colchicines are well defined. The scanning wavelength of 350 nm was found to be optimal for high sensitivity for colchicine spots.

Calibration curves of forskolin

Calibration solutions of forskolin in methanol containing concentration of forskolin 1 mg/ml were prepared by individual weighing. One to Five microlitres from solution was spotted on the TLC plate to obtain ﬁnal concentration range of 1000–5000 ng/spot. Each concentration was spotted two times on the TLC plate. The data of peak area versus drug concentration were treated by linear least-square regression analysis.

Method validation

The HPTLC method developed was validated for following parameters.

Sensitivity

The sensitivity of the method was determined with respect to LOD, LOQ, linearity range and correlation coefﬁcient. Solutions containing 1000–5000 ng of forskolin were spotted on TLC plate. The LOD was calculated as lowest concentration detected and LOQ was calculated as 3 times the LOD.

Precision

Precision data were sub-divided into intra-day precision and inter-day precision. Intra-day precision was obtained by analyzing the same standard three times within a day. Inter-day precision was obtained by analyzing the same standard three times on different days.

Different amount of standards covering low, medium and higher ranges of calibration curve were spotted on TLC plates. These spots were analyzed by using above described HPTLC method. Precision was expressed as the percentage relative standard deviation (%RSD). For forskolin 1000, 2500 and 5000 ng/spots were taken.

Accuracy and Recovery

The accuracy was determined by the standard addition technique. Known amounts of the reference compound were added to the sample and conducted chromatography under optimized conditions. The accuracy was then calculated from the test results as the percentage of analyte recovered by the assay. Recovery studies were carried out to check accuracy of the method.

Different amount of standards covering low, medium and higher ranges of calibration curve were spotted on TLC plates. These spots were analyzed by using above method. These spots were analyzed. Accuracy was expressed as a percentage (observed concentration x 100/theoretical concentration). The accuracy of a quantitative measurement is commonly assessed in recovery experiment.

Recovery of samples was determined by spiking standard in samples. The recovery was calculated by comparing the resultant peak areas with those obtained from pure standards in methanol at the same concentrations covering low, medium and higher ranges of calibration curve. For forskolin 1000, 2500 and 5000 ng/spots were taken.

Reproducibility

The repeatability was evaluated by analyzing the amount of forskolin spotted on TLC plate covering low, medium and higher ranges of calibration curve in replicates (n =3). The intermediate precision was evaluated by analyzing the same amount of analyte over period of 3 days (n =3) and expressed in terms of % R.S.D.

Linearity

For the determination of linearity curve, different concentrations of stock solution of standards were applied on HPTLC plate and plate was developed and scanned at particular wavelength. The calibration plot of peak area versus concentration should shows linearity. Working range was chosen starting with the lowest quantifiable concentration to avoid or minimize non-linearity regression curve. Linearity and calibration curves were constructed using five analyte concentrations. The linearity range for forskolin 1000-5000 ng/spot was taken.

Analysis of samples

The developed method was employed for the estimation of forskolin in crude samples. To determine the content of forskolin in crude samples, the crude drug powder was extracted with 10 ml of methanol. To ensure complete extraction of the drug, it was sonicated for 15 min at 30^oC. Two microlitres of the samples solution was applied on TLC plate followed by development and scanned as described in Section 2.1.

3. RESULTS AND DISCUSSION:

Sensitivity

Under the experimental conditions employed, the lowest amount of drug which could be detected was found to be 200 ng/spot and the lowest amount of the drug which could be quantified was found to be 600 ng/spot (LOQ = 3 x LOD).

Precision

Five microlitre aliquots of samples containing 1000, 2500 and 5000ng forskolin were analyzed according to the proposed method. In order to control the scanner parameters, one spot was analyzed several times. By spotting and analyzing the same amount several times (n =3) the precision of the automatic spotting device and the derivatization technique, was evaluated. The relative standard deviation (% R.S.D.) for the analysis of three replicates indicated good precision for the proposed TLC method (% R.S.D. consistently less than 2) as shown in Table 1.

Table.1. Precision and Accuracy data of TLC method performed on forskolin

Actual amount of forskolin spotted (ng) (Mean ± S.D.)	Amount detected 3(Mean ± S.D.)	% RSD
1000	922 ± 70	1.61
2500	2603 ± 5.28	1.17
5000	4900 ± 79.3	1.61

Accuracy and Recovery

The accuracy and recovery studies for forskolin were performed by using the above mentioned method by covering the low, medium and higher range of calibration curve. The results obtained are tabulated below in Table no. 2.

Table.2. Accuracy and recovery data of TLC method performed on forskolin:

Parameters
Actual amount of forskolin spotted (ng) (Mean ± S.D.)	Amount detected (Mean ± S.D.)	% RSD	% Accuracy
1000	922 ± 70	1.61	92.2
2500	2603 ± 5.28	1.17	104.12
5000	4900 ± 79.3	1.61	98

Results showed high extraction efﬁciency of forskolin from crude samples. The recovery of forskolin ranged from 92.2 to 104.12%, average of 98%. This conﬁrms that the proposed method can be used for the determination of forskolin in crude drugs.

Linearity

For determination of linearity curve, different amounts of standard solution of forskolin were applied on HTLC plate and plate was developed as above and scanned at a wavelength of 200 nm. The calibration plot of peak area versus concentration was linear. The linearity between the detector response and concentration of forskolin (1-5 µg/ml) in HPTLC analysis was evaluated. The linear regression equation was Y = 281.761 + 871.892* X with correlation coefficient r = 0.99928. Peak area and concentration was subjected to least least-square linear regression analysis to calculate the calibration equation and correlation coefficient. The regression data as shown in Table no. 1 shows a good linear relationship over the concentration range studied.

Table.3. Linear regression data for the calibration curve

Linearity range (ng/spot)	r²±S. D.	Slope	Intercept
1000-5000	0.9962 ± 0.0015	87.49 ± 0.68	352.41 ± 70

Fig.1. Linearity graph of forskolin

Reproducibility

Table 4 shows repeatability and intermediate precision studies of forskolin at different levels. The percentage R.S.D. was found to range from 0.70 to 1.85%, averaging to 1.06%.

Table.4. Precision data of HPTLC assay of forskolin

Amount of forskolin spotted (ng)

Amount detected (ng)

(Mean ± S.D.)

% R.S.D.

Intra-day (n=3)

1000

2500

5000

1002 ± 0.012

2470 ± 0.179

4995 ± 0.036

1.20

0.95

0.73

Inter-day (n=3)

1000

2500

5000

987 ± 0.036

2478 ± 0.245

4837 ± 0.450

1.85

0.97

0.70

Analysis of samples

Estimation of the forskolin was done by comparing the peak area of sample with the standard as well as by using the linear equation. The results obtained are tabulated below in Table no. 5.

Table.5. Quantification of forskolin from different samples

S. No.	Samples ID	Peak area	Amount detected in sample (µg)
1	Udaipur	3053.39	3.178867
2	Tuticorin	2322.66	2.34077
3	Dehradhun	1043.30	0.873433
4	Delhi	4039.68	4.310074
5	Bhopal	3250.20	3.404595
6	Bangalore	2514.67	2.560993
7	Mumbai	2528.67	2.57705
8	Salem	4311.46	4.621787

Forskolin content was quantified in raw material of Coleus forskohlii and the values in µg were found per 2µl of sample are Udaipur (3.17), Tuticorin (2.34), Dehradhun (0.87), Delhi (4.31), Bhopal (3.40), Bangalore (2.56), Mumbai (2.57) and Salem (4.62).

The parameters used for validation of HPTLC method were specificity, system precision, method precision, linearity, recovery, limit of detection, limit of quantification. All these parameters were found to be in the limit.

4. CONCLUSION:

The crude raw material of Coleus forskohlii, Salem, Tamil Nadu (4.621 ng) has showed the highest content of Forskolin followed by Delhi (4.310 ng) and Bhopal, MP (3.404 ng).

In the present study an original simple accurate High performance thin layer chromatography method was developed for quantitative determination of forskolin in crude raw material from the collected plant Coleus forskohlii.

The proposed HPTLC methods can be widely used as a standard technique for rapid and accurate qualitative and quantitative determination of forskolin. Moreover, this method has several advantages over the other analytical procedure such as: low cost, rapid analysis and high throughput of samples. The HPTLC method’s analytical performance was established and the method validated in terms of accuracy, precision, recovery, detection and quantification limits.

From the above studies, it can be concluded that HPTLC technique can be successfully used for the estimation of forskolin. The developed HPTLC method for this estimation is accurate, linear, simple and rapid. Because of the possibility of analyzing 8 samples in parallel the proposed methods are very rapid and cost effective.

5. REFERENCES:

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Received on 13.07.2012 Modified on 19.08.2012

Asian J. Research Chem. 5(8): August, 2012; Page 1029-1032