INTRODUCTION:

The most of the Traditional formulation are lacking in their defined quality control parameters and method of its evaluation.¹ 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^2,3. Chromatography is a powerful analytical method suitable for the separation and quantitative determination of a considerable number of compounds, even from a complex matrix. These include paper chromatography, thin-layer chromatography (TLC), gas chromatography (GC), high performance liquid chromatography (HPLC), and capillary electrophoresis⁴.

The present paper is an effort to develop the routine fingerprinting method for quality control parameter of Nilaavaarai Chooranam by high performance thin layer chromatography using piperine as an internal standard.

Nilaavaarai Chooranam is an important Siddha formulation, is official in formulary of Siddha Medicine is combination of five reputed herbs, comprised of the Cassia angustifolia (Nilaavaarai), Zingiber officinale (Chukku), Piper nigrum (Milagu), Trichyspermum ammi (Omum) and Embelia ribes (Vaividangam). The formulation is dispensed for the gaseous distension of stomach, hiccup, vomiting, constipation and biliousness. It is also useful as mild laxative⁵.

In this connection an effort has been made to develop the quality control parameter of Nilaavaarai Chooranam by HPTLC method for determining piperine as an internal standard which is as a important and major content in the formulation. Piperine as a major constituent. Piperine, 1-[5-(1, 3-Benzodioxol-5-yl)-1-oxo- 2, 4-pentadienyl] piperidine is the alkaloids a biomarker constituent of piper longum responsible for the pungency of piper longum and black pepper^6,7. Estimation of piperine in formulations can be done n order to develop fingerprint of the formulation.

The TLC densitometric analysis of piperine is a simple, precise, and accurate method which can be considered as one of the quality control method for routine analysis of Nilaavaarai Chooranam.

MATERIALS AND METHODS:

Preparation of Nilaavaarai Chooranam

Nilaavaarai Chooranam, three batches name NC-I, NC-II, NC-III, were prepared in laboratory using method described in formulary of Siddha Medicine

Materials

Standard piperine (98% pure) was purchased from Lancaster (England). 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

Syringe: 100 μL Hamilton (Bonaduz, Switzerland)

TLC Chamber: Glass twin trough chamber (20 x 10 x 4 cm); CAMAG

Densitometer: TLC Scanner 3 linked to Win Cats software V.4.06; CAMAG

HPTLC plates: 10 x 10 cm, 0.2 mm thickness precoated with silica gel 60 F₂₅₄; E. Merck KgaA, Cat. no. 1.05548; (Darmstadt, Germany)

Experimental conditions: Temperature 25± 2 ºC, relative humidity 40 %

Solvent system: toluene: ethyl acetate (70:30v/v)

Detection Wavelength: 342.6 nm

Slit dimension: 6.00 x 0.20 mm

Scanning Speed: 20 mm/s and source of radiation deuterium lamp.

Preparation of standard solutions of piperine

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

Preparation of piperine extract of Nilaavaarai Chooranam

Accurately weighed 1 gm of formulations and separately powdered crude drug of Piper nigrum were refluxed with 60 ml of methanol for 1 hour. The extract was filtered and the marc left was re-refluxed with 40 ml of methanol for another 1 hour. The previous filtrate was filtered and combined. The methanol extract was concentrated under vacuum till a semisolid mass was obtained. It was finally dissolved and the volume made up to 100 ml with methanol and filtered through sintered glass funnel (G-2) by vacuum filtration assembly. The filtrate was centrifuged at 2000 rpm for 30 minutes, the supernatant was collected and volume was made up with methanol.

Calibration curve of piperine

A stock solution of piperine (100 µgmL⁻¹) was prepared in methanol. Different volumes of stock solution were spotted on the TLC plate to obtain concentrations of 100 - 600 ng spot⁻¹ of piperine, 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 piperine

Method validation

1 Precision

Repeatability of the sample application and measurement of peak area were carried out using six replicates of the same spot (600 ng spot⁻¹for piperine) was expressed in terms of percent relative standard deviation (%R.S.D.). The intra- and inter-day variation for the determination of piperine was carried out at three different concentration levels of 100, 300, 600 ng spot−1. (Table-1)

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

Amount of Piperine	Intra -day precision		Inter-day precision
(ng/spot)	Area ± S.D.	R.S.D. %	Area ± S.D.	R.S.D. %
100	527.81± 0.0241	0.0046	527.26± 0.1243	0.0236
300	1192.49± 0.0138	0.0012	1191.89± 0.2038	0.0171
600	2643.63± 0.3214	0.0122	2643.13± 0.2347	0.0089

Table 2: % Recovery for piperine in Nilaavaarai Chooranam (n=6)

S.no.	Amount of piperine (ng /spot)			RSD%	SE	Recovery%
S.no.	Sample	Added	Estimated	RSD%	SE	Recovery%
1	100	50	149.39±0.738	0.494	0.301	99.59
2	100	100	199.05±0.347	0.174	0.142	99.53
3	100	150	249.43±0.379	0.152	0.155	99.77
Mean				0.273	0.199	99.63

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 600 ng spot−1 for piperine. (Table-3)

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 piperine until the average responses were approximately 3 or 10 times the standard deviation of the responses for six replicate determinations. (Table-3)

4 Ruggedness

A solution of concentration 600 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 piperine. (Table-3)

5 Specificity

The specificity of the method was confirmed by analyzing the standard drugs and extract. The spot for Piperine 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 piperine 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. (Table-3)

6 Recovery

The pre-analyzed samples were spiked with extra 50, 100 and 150 % of the standard piperine 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 piperine at different levels in the formulations. (Table-2)

Estimation of Piperine

The appropriate aliquots from piperine extract of each batch of Nilaavaarai Chooranam, marketed formulation and Piper nigrum 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 = 0.37 was observed in the chromatogram of the piperine, along with other components. There was no interference in analysis from the other components present in the extracts. (Table-4)

Table 3: HPTLC validation parameters of piperine

S. No.	Parameters	Observations
01	Retention Factor (Rf)	0.37
02	Beer’s law limit (ng/spot)	100-600
03	Correlation coefficient (r²)	0.9774
04	Regression equation (y*) Slope (a) Intercept (b)	y=5.29x-661.2 5.29 661.2
05	LOD(ng/spot) for SD 0.989	61.69 ng/spot
06	LOQ ng/spot ) for SD 0.989	188.82 ng/spot
07	Precision (% R.S.D.) (n = 6) Repeatability Intraday precision Interday precision	0.536 0.006 0.0166
08	Recovery Studies Accuracy( %RSD) SE Recovery%	0.273 0.199 99.63
09	Robustness	Robust
10	Specificity	Specific

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

S. No.	Name	Piperine Content % (w/w)	Standard Error
01	Piper nigrum	2.9836±0.497	0.203
02	NC-I	0.1761 ± 0.384	0.157
03	NC –II	0.1759 ± 0.534	0.218
04	NC –III	0.1760 ± 0.019	0.007
05	M-I	0.1642 ± 0.067	0.027

RESULTS AND DISCUSSION:

Fingerprint method for Nilaavaarai Chooranam with TLC Densitometric Methods (HPTLC) using piperine as an internal standard was developed.

The TLC procedure was optimized with a view to develop a stability indicating assay method. The standard and the sample were run in different solvent systems. Better results were obtained with mobile phase consisting of toluene: ethyl acetate (70: 30 v/v) with R_f values of 0.37 for piperine(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 piperine 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 100-600 ng/spot of piperine. The correlation coefficient (r²) value was 0.9774 for piperine indicates the good linearity between the concentration and peak area. The limit of detection and the limit of quantification for piperine were found to be 61.69ng/spot and 188.82ng/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 piperine present in the raw materials and formulations was calculated using the respective calibration graph. The content of piperine present in raw material (in Piper nigrum), and in three identical laboratory batch (NC-I, NC -II, NC –III) and marketed formulation (M-I) of Nilaavaarai Chooranam was discussed in Table 4.

The recovery studies were carried out for the accuracy parameter at three levels. To the powdered formulation, the standard drugs of piperine were added at 50% 100% and 150% levels; dilutions were made, and analyzed by the method. The mean of % recovery piperine was 99.63% (Table2). This shows significant precision of methods.

The developed HPTLC method is simple, rapid, precise and accurate for routine estimation piperine in Nilaavaarai Chooranam. The statistical analysis proved that the method is reproducible and efficient for the analysis of piperine.

REFERENCES:

1. Jain V., Saraf S. and Saraf S. Spectrophotometric determination of Piperine in Trikatu Churna: An Ayurvedic Formulation, Asian Journal of Chemistry. 2007, 19, (7), 5331-5335.

2. World Health Organization, Quality Control Methods for Medicinal Plants Materials, Geneva, 1998, 1-15.

3. WHO general guidelines for methodologies on research and evaluation of traditional medicine. (2000): http://whqlibdoc.who.int/hq/2000/WHO_EDM_TRM_2000.1.pdf.

4. Gupta M., Chaudhary P.H., Mukund G. Shrivastava T.B., Need and scope of standardization of herbal medicines - A review, International Journal of Green Pharmacy. Oct-Dec 2021 ,16 (1), 346-352.

5. Formulary of Siddha Medicine. 4th ed., Published by Indian Medical Practitioners Cooperative Pharmacy and Store Ltd (IMPCOPS), Chennai, 1993: 45.

6. Indian Herbal Pharmacopoeia, Regional Research Laboratory Jammu, Indian drug Manufacturing Association Mumbai. 1999.

7. Gurinderdeep S, Piperine: A Remarkable Marker with Intense Biological Activity, International Journal of Pharmacognosy and Chinese Medicine. 2017, 1(4): 122.

Received on 10.08.2022 Modified on 27.08.2022

Asian J. Research Chem. 2022; 15(5):327-330.

DOI: 10.52711/0974-4150.2022.00058