INTRODUCTION:

Glycyrrhiza glabra is a vital herb in nature as it has wide therapeutic applications. Glycyrrhizin is main constituent that has anti-inflammatory, antimicrobial, anti-ulcer activity. Glycyrrhizin is saponin glycoside and molecular formula is C₄₂H₆₂0₁₆. Gel containing glycyrrhizin is effective for mouth ulcer and aphatae. The novel gel formulation contains carbopol, chitosan, PEG, glycerin. [1,2]

Various analytical methods reported such as HPLC, HPTLC, and Capillary electrophoresis. The present paper describes precise, accurate, sensitive HPTLC method for determination of glycyrrhizin from the extract and formulation [3].

MATERIALS AND METHODS:

Materials:

A CAMAG TLC system comprising of a Linomat-5 applicator and CAMAG TLC III scanner. Stationary phase used was silica gel G60F254, 20x10cm TLC plate. The Reference standard of Glycyrrhizin was obtained from Rusan Pharmaceutical Limited, Ankleshawar, India. Methanol, Glacial Acetic acid, Formic acid and Chloroform were used of AR Grade. The plates were developed in a CAMAG twin trough glass chamber (20 x 10cm) by ascending method. Distance of solvent front 80mm, band length 6mm, slit dimension 5.00 x 0.45mm and detection wavelength 252nm were used for the present study.

METHOD:

Preparation of Glycyrrhizin Solutions:

The standard solution was prepared by dissolving 10mg in 10ml methanol solution which gives 1000μg/ml. The working standard of 100μg/ml was prepared from standard solution by diluting with methanol. Different concentrations of 2, 5, 10, 15, 20μg/ml were prepared from standard solutions.

Chromatographic Conditions:

Analysis was performed on 20cm × 10cm HPTLC silica gel G60 F254 plates with fluorescent indicator. The plate cleaned by predevelopment to the top with methanol, and dried in an oven 105⁰C for 5 min. Sample and standard zones were applied to the layer as bands by means of a CAMAG. Linomat 5 automated spray-on applicator equipped with a 100μl syringe and operated with the settings band length 6mm, application rate 4 μl/sec, distance between bands 4mm, distance from the plate side edge 6.5mm, and distance from the bottom of the plate 2cm.

Calibration Curve:

2, 5, 10 and 15μl standard solution of Glycyrrhizin was applied onto TLC plate to generate Calibration curve. The chromatograms were developed using said chromatographic conditions. The plate was dried in air and kept in hot air oven at 105°C for 5 min. The standard zones were quantified by linear scanning at 254nm (Fluorescent) by use of a TLC Scanner III CAMAG with a mercury source [4].

Analysis of glycyrrhizin in herbal extracts:

The Glycyrrhiza powder 500mg was taken in conical flask and extracted twice with 10ml of 70% ethanol, and sonicated for 10 min in ultrasonic bath. The solution was filtered through Whatman filter paper No. 44 and the filtrate was used as further analysis. [5,6]

Analysis of glycyrrhizin in herbal formulations:

The weighted amount of gel equivalent to 10mg of glycyrrhizin was mixed with 70% ethanol with vigorous shaking and sonicated for 10 min in ultrasonic bath. The solution was filtered through Whatman filter paper No.44 and the filtrate was used as further analysis.

METHOD VALIDATION [7,8]:

Precision:

Repeatability:

Repeatability of sample application and measurement of peak area was carried out using the three replicates of same spot 1000ng/spot. Repeatability is also termed intra-assay precision.

Intermediate precision:

The intra-day and inter-day variations for determination of glycyrrhizin were carried out at three different concentration levels 2, 5, 10μl/spot.

Specificity:

The specificity of method was ascertained by standard MAG and samples (extracted from powder and extracted from formulation). The spots of diluent ethanol, Placebo of formulation, standard MAG, extracted samples (extracted from powder and extracted from formulation) were spotted on TLC plate in duplicate and run. The spots for glycyrrhizin that eluted were confirmed with Rf value of MAG. [9,10]

Recovery Studies:

Recovery Study was performed by spiking 20, 40 and 60 % of standard drug externally to the pre analyzed samples. The experiment was conducted in triplicate and applied onto the plate in duplicate. This was conducted to check the recovery of drugs at different levels of formulations. [11]

Robustness:

The robustness of method can be performed by allowing variation in sample application time to scanning.

RESULT AND DISCUSSION:

Mobile Phase Development:

The mixtures of several mobile phases were tried to separate spot of glycyrrhizin from other spots and get stable peak. The solvent system used was ethyl acetate: Glacial acetic acid: Formic acid: water (15:2:1:1 v/v/v/v) was selected for estimation of glycyrrhizin, which gave good resolution. Good chromatogram in Figure 2 was attained with Rf value 0.34 ± 0.04. The wavelength of 252 nm was used for quantification of sample.

Figure 1. Chemical structure of Glycyrrhizin.

Figure 2. Chromatogram of representative sample

Calibration Curve, Linearity and Range:

Linear regression data showed a good linear relationship over concentration range.

The correlation coefficient r2 was 0.998 shown in Table no.2.

Table No. 1. Comparative data of standard, extract and extract from formulation

Track	Rf	Height of Peak	Area	% Area
1	0.33	31.4	402	6.41
2	0.33	139.4	5532.7	62.97
3	0.33	215.5	8226.3	12
4	0.33	293.8	1179.5	10.39
5	0.33	208.6	7333.7	11.51
6	0.33	230.1	8708.1	29.64

Table No. 2. Linearity regression data for calibration curve

Parameters

Value

Linearity Range

Correlation of Coefficient

(According to area)

Slope

200-1000ng/spot

0.999

141.97

Figure 3. Calibration Curve of MAG

Precision:

The repeatability and intermediate precision were studied separately and shown below in table 3 and 4 respectively.

Table no.3. Repeatability study

Sr. No.	Sample applied ng	Area AU	%RSD
1	1000	256.6	0.056
2	1000	251.6	0.064
3	1000	258.2	0.056

Table no. 4. Intraday and Interday Precision

Sr. No.	Sample applied ng	Intraday % RSD	Interday RSD
1	200	2.028	2.17
2	400	1.98	1.9
3	1000	1.54	1.6

Repeatability:

It showed very low % RSD of peak area of drug.

Intermediate precision:

Specificity:

The mobile phase was optimized and it showed good result. Glycyrrhizin was found to be well separated from other constituents present in extracted sample. There was no interference of diluent, placebo and other constituent’s peaks from extracted sample found at the Rf value of peak, indicates specificity of the method.

Robustness of method:

The time from sample application to scanning varied from 0, 20, 40, 60 mins. The standard deviation of peak areas was calculated for each parameter and % RSD was found to be less than 3 %.

Recovery studies:

The proposed method when used for extraction and subsequent estimation of glycyrrhizin from gel after spiking with 20, 80, 120% standard. The afforded recovery is listed below in Table no.5.

Figure 4. Comparision of Extracts peaks with Standard.

Estimation of glycyrrhizin in herbal extracts and in herbal gel:

The optimized solvent system was used for the estimation of the glycyrrhizin from herbal extract. There was no interference in analysis from other components present in extract. The resolution was good and components were observed at different Rf value. The total glycyrrhizin present in herbal extract was found to be 18.52% w/w, 17.92% w/w and 18.26% w/w. There was no interference from in analysis from other active components from extract and inactive components from formulation. The total glycyrrhizin present in formulations are 17.96%, w/w 17.85% w/w, and 18.1% w/w.

CONCLUSION:

The developed HPTLC method is fast, simple, precise, specific and accurate. Statistical analysis proved that method is repeatable and selective for determination of Glycyrrhizin.

ACKNOWLEDGEMENT:

The authors are thankful to the Principal, Annasaheb Ramesh Ajmera College of Pharmacy Nagao Dhule Dist: Dhule 424002, Maharashtra, India, for providing the laboratory facility.

REFERENCES:

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Received on 25.03.2018 Modified on 20.05.2018

Res. J. Pharma. Dosage Forms and Tech.2020; 12(2): 89-92.

DOI: 10.5958/0975-4377.2020.00016.6