INTRODUCTION:

Medicinal plants are one of the best resources from nature and their utilization for the benefits of humankind. According to the world health organization, approximately 80% of the world population still depends on traditional medicine, as an important source of medicine.¹ India is a land where various traditional systems of medicine are existed together and based on natural sources.² The most of the Traditional formulation are lacking in their defined quality control parameters and method of its evaluation.³

The World Health Organization (WHO) in its resolution WHA 31.33 (1978), WHA 40.33 (1987), WHA 42.43 (1989) has emphasized the need to ensure the quality of medicinal plant products by using modern controlled technique and applying suitable standards.^4,5,6 Marker compound means chemical constituents within a medicinal that can be used to verify its potency or identity. For sometimes, the marker compounds may be described as active ingredients or chemicals that confirm the correct botanical identity of the starting material. It is very difficult to identify correct marker compounds for all traditional medicine, because some medicine have unknown active constituents and others have multiple active constituents.^7-12 Fingerprints play an important role in quality control of traditonal medicine. It can be used for authentication of raw material or detection of confounded material/ substitutes. Herbal Fingerprinting is the technique used for the qualitative and quantitative analysis of herbal components in drugs. Chromatographic fingerprinting is used for both establishing the identity and quality of the herbs being added into formulation.¹³ These include Paper chromatography, Thin-layer chromatography (TLC), Gas chromatography (GC), High performance liquid chromatography (HPLC), and Capillary electrophoresis⁶. HPTLC technique can be used for herbal fingerprinting. Each plant has a different fingerprint pattern. When the plant extract is run in a suitable mobile phase the separation pattern observed is the herbal fingerprint of the said plant. HPTLC is a preliminary step to identify and quantify phytochemical compounds and secondary metabolites of a plant.¹³

Eladi gutika is official in Ayurvedic formulary of India and used as a remedy for Kasa (Cough), Svasa (Asthma), Bhrama (Vertigo), Raktapitta (Bleeding disorders), Jvara (Fever), Amvata (Rheumatism) etc. It is the combination of seven reputed herbs Elettaria cardamomum, Cinnamomum tamala, Cinnamomum zeylanicum, Piper longum, Glycyrrhiza glabra, Phoenix dactylifera, and Vitis vinifera and jaggery and sugar.¹⁴

In this connection an effort has been made to develop the quality control parameter Eladi gutika by HPTLC method for determining glycyrrhizinic acid as an internal standard which is as a important content in the formulation. Estimation of glycyrrhizinic acid in formulations can be done n order to develop fingerprint of the formulation. The TLC densitometric analysis of glycyrrhizinic acid is a simple, precise, and accurate method which can be considered as one of the quality control method for routine analysis of Eladi gutika.

MATERIAL AND METHODS:

Preparation of Eladi gutika:

Eladi gutika, three batches name EG-I, EG -II and EG -III were prepared in laboratory using method described in Ayurvedic formulary of India.¹³

Materials:

Standard glycyrrhizinic acid was procured from Hi-media. All chemicals and reagents used were of analytical grade and were purchased from Merck Chemicals, India.

Instrumentation and chromatographic conditions:

Spotting device: Linomat V Automatic Sample Spotter; CAMAG (Muttenz, Switzerland), Syringe: 100μL Hamilton (Bonaduz, Switzerland), TLC Chamber: Glass twin trough chamber (20 x 10 x 4cm); CAMAG, Densitometer: TLC Scanner 3 linked to WinCats software V.4.06; CAMAG, HPTLC plates: 10 x 10 cm, 0.2mm thickness precoated with silica gel 60 F254; E. Merck KgaA, Cat. no. 1.05548; (Darmstadt, Germany), Experimental conditions: Temperature 25±2ºC, relative humidity 40%, Solvent system: chloroform: methanol: water (6.5: 3.5: 1, v/v/v), Detection Wavelength: 251 nm, Slit dimension: 6.00 x 0.20mm, Micro, Scanning Speed: 20mm/s, Quantity of mobile phase: 10ml, Application rate: 0.1µl/sec, Space between two bands: 10mm

Preparation of standard solutions of glycyrrhizinic acid

Accurately weighed glycyrrhizinic acid (10mg) was transferred in 100ml volumetric flask, dissolved in, and diluted to 100ml with methanol. The final solution contained 100mg of the glycyrrhizinic acid per ml of the solution.

Calibration curve of glycyrrhizinic acid:

Different volumes of stock solution were spotted on the TLC plate to obtain concentrations of 100-500 ng spot⁻¹ of glycyrrhizinic acid respectively. The data of peak areas plotted against the corresponding concentrations were treated by least-square regression analysis method validation.

Figure 1: HPTLC chromatogram of glycyrrhizinic acid

Method validation:

1 Precision:

Repeatability of the sample application and measurement of peak area were carried out using six replicates of the same spot (500 ng spot⁻¹for glycyrrhizinic acid) was expressed in terms of percent relative standard deviation (%R.S.D.). The intra- and inter-day variation for the determination of glycyrrhizinic acid was carried at three different concentration levels of 100, 300, 500 ng spot−1.

Table 1: Intra- and inter-day precision of HPTLC method (n=6)

Amount of glycyrrhizinic acid	Intra -day precision		Inter-day precision
(ng/spot)	Area ± S.D.	R.S.D. %	Area ± S.D.	R.S.D. %
100	388.09± 0.3694	0.0952	388.96± 0.0469	0.0121
300	952.34± 0.0293	0.0031	953.02± 0.1237	0.0129
500	1493.21± 0.1496	0.0101	1493.96± 0.3296	0.0221

2 Robustness of the method:

By introducing small changes in the mobile phase composition, mobile phase volume, duration of mobile phase saturation and activation of pre washed TLC plates with methanol; the effects on the results were examined. Robustness of the method was done in triplicate at a concentration level of 500 ng spot−1 for glycyrrhizinic acid.

3 Limit of detection and limit of quantification:

In order to estimate the limit of detection (LOD) and limit of quantification (LOQ), blank methanol was spotted six times LOD was considered as 3:1 and LOQ as 10:1. LOD and LOQ were experimentally verified by diluting the known concentrations of plumbagin until the average responses were approximately 3 or 10 times the standard deviation of the responses for six replicate determinations.

4 Ruggedness:

A solution of concentration 500 ng spot⁻¹ was prepared and analyzed on day 0 and after 6, 12, 24, 48 and 72 h. Data were treated for % R.S.D. to assess ruggedness of the method for glycyrrhizinic acid.

5 Specificity:

The specificity of the method was confirmed by analyzing the standard drugs and extract. The spot for glycyrrhizinic acid in the sample was confirmed by comparing the Rf values and spectra of the spot with that of both the standard. The peak purity of the glycyrrhizinic acid was assessed by comparing the spectra at three different levels, viz. peak start (S), peak apex (M) and peak end (E) positions of the spot.

6 Recovery:

The pre-analyzed samples were spiked with extra 50, 100 and 150 % of the standard glycyrrhizinic acid and the mixtures were reanalyzed by the proposed method. The experiment was conducted six times. This was done to check for the recovery of the glycyrrhizinic acid at different levels in the formulations.

Table 3: HPTLC validation parameters of glycyrrhizinic acid

S. No.	Parameters	Observations
01	Retention Factor (Rf)	0.58
02	Beer’s law limit (ng/spot)	100-500
03	Correlation coefficient (r2)	0.998
	Regression equation (y*) Slope (a) Intercept (b)	y=21.3x+0.8213 21.3 0.8213
05	LOD (ng/spot) for SD 0.874	27.08 ng/spot
06	LOQ ng/spot) for SD 0.874	82.88 ng/spot
07	Precision (% R.S.D.) (n = 6) Repeatability Intraday precision Interday precision	0.493 0.0361 0.0157
08	Recovery Studies Accuracy (%RSD) SE Recovery%	0.0916 0.0857 99.75
09	Robustness	Robust
10	Specificity	Specific

Estimation of glycyrrhizinic acid in crude drug and formulations:

The appropriate aliquots from glycyrrhizinic acid extract of each batch of Eladi gutika, marketed formulation and G. glabra withdrawn in 10 ml volumetric flask separately. The filtered solution was applied on the TLC plate followed by development and scanning. A single spot at Rf = 0.58 was observed in the chromatogram of the glycyrrhizinic acid, along with other components. There was no interference in analysis from the other components present in the extracts. The total glycyrrhizinic acid content for EG-I, EG -II, EG -III, M-I was reported in table 3T82.

Table 4: HPTLC Estimation of glycyrrhizinic acid content (% (w/w)

S. No.	Name	Glycyrrhizinic acid Content % (w/w)	Standard Error
01	G. glabra	8.2201±0.497	0.203
02	EG-I	1.4945 ± 0.293	0.119
03	EG –II	1.4963 ± 0.763	0.311
04	EG –III	1.4944± 0.864	0.353
05	M-I	1.2396 ± 0.118	0.048

RESULTS AND DISCUSSION:

Fingerprint method for Eladi gutika with HPTLC using glycyrrhizinic acid as an internal standard was developed. The standard and the sample were run in different solvent systems. Better results were obtained with mobile phase consisting of chloroform: methanol: water (6.5: 3.5: 1, v/v/v) with R_f values of 0.0.58 for glycyrrhizinic acid (figure 1). The spots were resolved on the chromatogram that showed the good resolution. To a pre-washed activated TLC plate, standard stock solution of glycyrrhizinic acid were spotted with Linomat V semi sample applicator. The plate was developed and scanned. The peak areas of standard were obtained from the software, and a calibration graph of concentration against peak area was plotted. A good linear relationship was obtained over a concentration range of 100-500ng/spot of glycyrrhizinic acid. The correlation coefficient (r²) value was 0.998 for glycyrrhizinic acid indicates the good linearity between the concentration and peak area. The limit of detection and the limit of quantification for glycyrrhizinic acid were found to be 27.08 ng/spot &82.88 ng/spot (Table 3). These values are considered to be good enough for a reasonable accuracy.

Intra-day assay precision was found by analysis of standard drug three times on the same day. Inter-day assay precision was carried out using the standard drug on three different days, and % relative standard deviation (RSD) was calculated. The RSD was found to be less than 1% for both inter-day and intra-day assay precision (Table1). The low values indicate robustness of the method. Repeatability of sample application was assessed by spotting of drug solution for 6 times. From the peak areas, the % RSD was determined. After development, spot was scanned six times without changing position.

The amount of glycyrrhizinic acid present in the raw materials and formulations was calculated using the respective calibration graph. The concentration of glycyrrhizinic acid present in raw material was found to be 8.2201±0.497w/w in G. glabra and in three identical laboratory batch of Eladi gutika EG-I, EG -II and EG -III, was found to be 1.4945 ± 0.293, 1.4963 ± 0.763%, 1.4944± 0.864w/w respectively and marketed formulation (M-I) of Eladi gutika was found to be 1.2396 ± 0.118 (Table 4). The glycyrrhizinic acid content in all the three different batches is found to be in close proximities with each other. The results were comparable to marketed formulations.

The recovery studies were carried out for the accuracy parameter at three levels. To the powdered formulation, the standard drugs of glycyrrhizinic acid were added at 50% 100% and 150% levels; dilutions were made, and analyzed by the method. The mean of % recovery glycyrrhizinic acid was 99.75% (Table 2). This shows significant precision of methods.

The TLC densitometric analysis of glycyrrhizinic acid is a simple, precise, and accurate method which can be considered as one of the quality control method for routine analysis of Eladi gutika.

REFERENCES:

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14. The Ayurvedic Formulary of India. Part I, 2nd edition. Delhi: Govt. of India, Ministry of Health and Family Planning, Dept. of Health; 2003:103.

Received on 08.10.2022 Modified on 21.10.2022

Asian J. Res. Pharm. Sci. 2022; 12(4):277-280.

DOI: 10.52711/2231-5659.2022.00047

S. No.	Amount of glycyrrhizinic acid (ng /spot)			RSD%	SE	Recovery%
S. No.	Sample	Added	Estimated	RSD%	SE	Recovery%
1	100	50	149.71±0.016	0.011	0.0065	99.80
2	100	100	199.01±0.183	0.092	0.0747	99.50
3	100	150	249.91±0.431	0.172	0.1759	99.96
Mean				0.0916	0.0857	99.75