INTRODUCTION:

Hab-e-Azarakhi is an important Unani formulation, is official in Unani Pharmacopoeia (formulary of Unani Medicine) is combination of four reputed herbs, comprised of the fruits Piper nigrum (Filfil e Siyah), Piper longum (Filfil e Daraz), Trichyspermum ammi (Arkh e Ajwain) and seeds of Strychnos nuxvomica (Kuchla mudabbar). The formulation is dispensed for the tone up the nervous system and liver. It is also useful for increase the appetite.^1,2

The most of the Traditional formulation are lacking in their defined quality control parameters and method of its evaluation.^3,4 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^3,4,5,6. 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 electrophoresis⁷. The present paper is an effort to develop the routine fingerprinting method for quality control parameter of Hab-e-Azarakhi by high performance liquid chromatography using piperine as an internal standard. Piperine is 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.^3,8,9. HPLC Estimation of piperine in formulations can be done in order to develop fingerprint of the formulation.

MATERIALS AND METHODS:

Preparation of Hab-e-Azarakhi:

Hab-e-Azarakhi, three batches name HA-I, HA-II, HA-III, were prepared in laboratory using method described in Unani Pharmacopoeia (formulary of Unani Medicine) and one marketed formulations of Hab-e- Azarakhi purchased from local pharmacy were coded M-1.

Instrumentation:

Experiments were performed on a HPLC system Shimadzu-10AT_VP, binary gradient equipped with detector Shimadzu UV -VIS SPD-10 A_VP, software Spinchrom, Chennai. The separations were performed on Merck’s column [Lichrospher 100, C-18 (250 x 4.6mm) and ODS RP-18 (250 x 4.6mm, 5μ particle size)] using methanol: ethyl acetate (70:30) mobile phase with flow rate 1.0ml min−1.the wave length of detection was 342.6nm.

Chemicals:

All the chemicals and solvents used were of AR grade; standard piperine (98% pure) was procured from Lancaster (England). Methanol and ethyl acetate was procured from Merck and used as a mobile phase.

Preparation of standard solution of piperine:

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

Linearity:

Serial dilutions containing 2-20µg/ml piperine in methanol were prepared from a stock solution of piperine (10mg/100ml). Each dilution was chromatographed on HPLC and area under the peak of piperine recorded (Figure 2)

Figure 1 RP HPLC chromatogram of piperine

Figure 2: HPLC Calibration curve of piperine

Preparation of piperine extract of Hab-e- Azarakhi:

Accurately weighed 1gm of formulations and separately powdered crude drug of Piper nigrum and Piper longum were refluxed with 60ml of methanol for 1 hour. The extract was filtered and the marc left was re-refluxed with 40ml of methanol for another 1 hours. 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 100ml with methanol and filtered through sintered glass funnel (G-2) by vacuum filtration assembly. The filtrate was centrifuged at 2000rpm for 30 minutes, the supernatant was collected and volume was made up with methanol.

Method validation of quantitative analysis

The method was validated in terms of limits of detection and quantification (LODs and LOQs), precision, repeatability and recovery test. (Table 1)

1. Precision:

The intra-day variation was determined by analyzing the six samples of Hab-e- Azarakhi within 1 day and inter day variation was determined on three consecutive days. To confirm the repeatability, six different working solutions prepared from the same sample of each batch of Hab-e- Azarakhi were analyzed. Variations were expressed as relative standard deviations (R.S.D.).

2. Limits of detection and quantification (LODs and LOQs)

The LODs and LOQs under the present HPLC-UV method were determined at signal-to-noise ratios (S/N) of 3 and 10, respectively. Standard solution containing piperine as a reference compounds was diluted to a series of appropriate concentrations with ethanol and an aliquot of the diluted solution was injected into HPLC for analysis.

3. Recovery:

The recovery test was determined by standard addition method in accordance to ICH guidelines. Piperine was spiked into the each sample, and then, processed and quantified in accordance with the established procedures.

Estimation of piperine in Hab-e- Azarakhi:

The appropriate aliquots from extract of each batch of Hab-e- Azarakhi and its marketed formulations and separately powdered Piper nigrum and Pipe longum were withdrawn in 10 ml volumetric flask separately. The corresponding concentration of piperine against respective peak areas value was determined using the piperine calibration curve (table 2).

RESULTS AND DISCUSSION:

The simple, reliable, accurate, precise and sensitive high-performance liquid chromatography (HPLC) fingerprint method for determination of piperine was developed for quality control of Hab-e- Azarakhi. For RP-HPLC method development mobile phase was used for piperine was methanol: ethyl acetate (70:30) with flow rate 1.0 ml min⁻¹.The chromatogram of piperine under experimental condition showed a single peak of the drug at 2.826 min (at 342.6nm) (Figure 1). An aliquot of each standard working solution was subjected to HPLC-UV analysis. The linearity was established by plotting the peak area (y) versus concentration (x) of piperine and Calibration curves (Figure 2) showed good linear regression within test range. The intercept and slope of the standard plot of piperine were observed to be 57.32 AND 27.60. The LODs and the LOQs were found to be 0.0436mg/ml and 0.1336mg/ml for the piperine.

Piperine revealed the results of the tests of precision and repeatability. It indicated that the R.S.D. values of the overall intra- and inter-day variations and the repeatability of piperine in the formulations. In order to obtain precision and accuracy the recovery study was performed at three levels by adding known amount of piperine with pre-analyzed sample of piperine in Hab-e- Azarakhi. The average recoveries of the piperine were found to be 99.77 respectively. (Table 1)

Table 1: HPLC Validation parameters of piperine in HA

S. No.	Parameters	Observations
01	Retention time (Rt)	2.826 min
02	Beer’s law limit (mg/ml)	0-20
03	Correlation coefficient (r2)	0.996
04	RHAression equation (y*) Slope (a) Intercept (b)	y=57.32x-27.60 57.32 27.6
05	LOD(mg/ml)	0.0436
06	LOQ (mg/ml)	0.1336
07	Precision (% R.S.D.) (n = 6) Repeatability Intraday precision Interday precision	0.521 0.698 1.118
08	Recovery Studies Accuracy (%RSD) SE Recovery %	0.184 0.145 99.77

The concentration of piperine content of Hab-e- Azarakhi (three laboratory batches) and powdered Piper nigrum and Piper longum was carried out separately. The concentration of piperine present in raw material was found to be 2.9876±0.837%w/w in Piper nigrum and 0.9363±0.167% w/w in Piper longum and in three identical laboratory batches of Hab-e- Azarakhi HA-I, HA-II and HA-III 1.8121±0.739%, 1.8806±0.439%, 1.8901±0.779% w/w respectively, while in M-I it was 1.2108±0.182% (Table 2).

Table 2: HPLC Estimation of Piperine content (% (w/w)

S. No.	Name	Piperine Content % (w/w)	Standard Error
01	Piper nigrum	2.9876±0.837	0.342
02	Piper longum	0.9363 ± 0.167	0.068
03	HA-I	1.8121 ± 0.739	0.302
04	HA –II	1.8806 ± 0.439	0.179
05	HA –III	1.8901± 0.779	0.318
06	M-I	1.2108 ± 0.182	0.074

The results were comparable to marketed formulations. The results indicate that the developed method can be used for quantification of piperine in the Unani formulation Hab-e-Azarakhi.

REFERENCES:

1. Unani Pharmacopoeia (formulary of Unani Medicine), 4th , edition,. Formulary of Unani Medicine. 4th ed., published by Indian Medical Practitioners Cooperative Pharmacy and Store Ltd (IMPCOPS), Chennai, 2004:25.

2. Tripti Jain, and Kamlesh Dashora. Spectrophotometric fingerprinting method for Unani formulation Hab-e-Azarakhi. Asian J. Pharm. Tech. 2012; 2(1): 01-03

3. Tripti Jain, Amber Vyas, Darshan Dubey, Kamlesh Dashora, Vishal Jain. Development of fingerprinting method for Siddha formulation Nilaavaarai chooranam: A HPTLC approach, Asian J. Research Chem., 2022:15, (5), 327-330

4. 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.

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

6. 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.

7. 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.

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

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

Received on 11.10.2022 Modified on 26.10.2022

Asian J. Pharm. Ana. 2022; 12(4):258-260.

DOI: 10.52711/2231-5675.2022.00042