Development of fingerprinting method for Amlakyadi Churna: Spectrophotometric approach

Tripti Jain, and Kamlesh Dashora*

Institute of Pharmacy, Vikram University, Ujjain (M.P.)

ABSTRACT:

Selective and efficient analytical methods are required not only for quality assurance but also for authentication of herbal formulations. A simple, rapid and valid fingerprint method has been first carried out for estimation of piperine in Amlakyadi churna an herbal formulation. Amlakyadi churna is an important ayurvedic formulation, is official in Ayurvedic formulary of India is combination of four reputed herbs, comprised of the fruits Embelica officinalis, Piper longum, Terminalia chebula and roots of Plumbago zylenica and Sendha namak. The formulation is dispensed for the treatment of digestive impairment, fever and dyspepsia. The estimation was carried out with three laboratory batches and one marketed formulation by spectrophotometric approach at 342.6 nm.

KEYWORDS: Amlakyadi churna, Piperine, ethanol, Spectrophotometer

INTRODUCTION:

The most of the Ayurvedic formulation are lacking in their defined quality control parameters and method of its evaluation. The World Health Organization (WHO) has emphasized the need to ensure the quality of medicinal plant products by using modern controlled technique and applying suitable standards [1].

Amlakyadi churna is an important ayurvedic formulation, is official in Ayurvedic formulary of India is combination of four reputed herbs, comprised of the fruits Embelica officinalis, Piper longum, Terminalia chebula and roots of Plumbago zylenica and Sendha namak. The formulation is dispensed for the treatment of digestive impairment, fever and dyspepsia [2].The present paper is an effort to develop the quality control parameter of Amlakyadi Churna by spectrophotometric determination using Piperine as a internal standard. 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[3].

The present study is an attempt to develop the fingerprint method for Amlakyadi Churna by spectrophotometric determination using Piperine as a standard, which is as an important content in formulation. The UV spectrophotometric analysis, which is a simple, precise, and accurate method that can be considered as one of the quality control method for routine analysis.

MATERIALS AND METHODS:

Procurement of crude drug:

Crude drugs were procured from local market Raipur (C.G.) India and identified on the basis of morphological and microscopical characters and compared with standard Pharmacopoeial Monograph[4-7].

Preparation of the formulation:

Amlakyadi churna, two laboratory batches (named AK-I, AK-II, AK-III) were prepared in laboratory according to reported method of Ayurvedic formulary of India. The available commercially brand M-1 of Amlakyadi churna was procured from local Pharmacy.

Chemicals:

All the chemicals and solvents were used of A.R. grade; Standard Piperine (98%) was procured from Alfa Aesar (A Johnson Matthey Company); Lancaster (England).

Preparation of standard solution of Piperine:

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

Preparation of Piperine extract of Amlakyadi Churna:

Accurately weighed 1 gm of powdered Amlakyadi churna reflux with 60 ml of ethanol for 1 hour. Filtered the extract and re-reflux the marc left with 40 ml of ethanol for another 1 hours. Filter and combine the previous filtrate. Concentrated the ethanolic extract of Amlakyadi churna under vacuum till semisolid mass was obtained. Finally dissolved and make up the volume up to 100 ml with ethanol 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 with ethanol. The same procedure was performed for each batch of Amlakyadi churna and separately powdered drug of Piper longum (Pippali).

Preparation of calibration curve for Piperine

Standard solutions of Piperine (2, 4, 6, 8 and 10 ml) were pipetted into concentration range 2-10 mg/ml in a series of five 25 ml volumetric flask. The absorbance of the Piperine was measured at 342.6 nm against ethanol. The results are shown in Figure 1.

RESULT AND DISCUSSION:

The developed method was found to be reliable, accurate, precise and sensitive. The method involves absorbance measurement at 342.6 nm for Piperine corresponding to the absorption maxima of the herbal formulation Amlakyadi churna. The optical characteristic shows that Piperine obeys Beer Lambert’s law in concentration range 2-10 mg/ml at λ-max 342.6 nm. The correlation coefficient (r²), Regression equation, Precision and Accuracy were calculated for the spectrophometric method(Table-I).

The r² value 0.985 indicates the good linearity between the concentration and absorbance. The estimation of piperine content of Amlakyadi churna (three laboratory and one marketed samples) and powder of piper longum (Pippali) was carried out separately. The concentration of Piperine present in raw material was found to be 0.932 ± 0.045 w/w in Piper longum. Content of piperine in different batches of Amlakyadi churna (laboratories and marketed batches) was found to be 0.183 ± 0.037%, 0.189 ± 0.412% and 0.188± 0.712% w/w respectively for AK-I, AK-II and AK-III. Whereas marketed formulation M-1 shows piperine content 0.132 ± 0.035% w/w. Results are summarized in Table-II.

In order to obtain precision and accuracy the recovery study were performed by adding known amount of piperine with pre-analyzed sample of piperine in Amlakyadi churna. The result shows 99.38 % recovery of Piperine which shows reproducibility of the result.

The proposed method was found to be accurate, simple and rapid for routine analysis of Piperine in herbal formulation Amlakyadi churna.

Table I: Validation parameters of piperine

S.No.

Parameter

Value

Absorption Maxima

Beer’s Law limit

Regression equation (y= bx+a)

Intercept (a)

Slope (b)

Correlation coefficients (r²)

Precision (n=6, % RSD)

Recovery Studies

a)Accuracy( %RSD)

b)SE

c)Recovery%

342.6 nm

2-20mg/ml

y=0.094x- 0.060

0.094

0.060

r² = 0.985

0.351

0.353

0.400

99.38

Figure 1: Calibration curve of Piperine

Table II: Estimation of Piperine content (% (w/w))

S. No.	Name	Piperine Content % (w/w)		Confidence Level (95 %)
1.	Piper longum (Pippali)	0.932 ± 0.045		± 0.3632
2.	Amlakyadi Churna	AK-I	0.183 ± 0.037	± 0.424
3.		AK –II	0.189 ± 0.412	± 0.5845
4.		AK –III	0.188± 0.712	± 0.3845
5.		M-1	0.132 ± 0.035	± 0.7135

ACKNOWLEDGEMENT:

The authors are highly grateful to Head, Institute of Pharmacy, Vikram University, Ujjain (M.P.)

REFERENCES:

1. World Health Organization, Quality Control Methods For Medicinal Plants Materials, Geneva, 1-15, (1998).

2. Ayurvedic formulary of India, Part-1, 2nd, edition, Goveanment of India, Ministry of Health and family Planning, Department of Indian System of Medicine and Homeopathy, Delhi. p 103.

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

4. Indian Herbal Pharmacopoeia, Volume II, Regional Research Laboratory Jammu, Indian drug Manufacturing Association Mumbai, 93-101, (1999).

5. Quality Standards Of Indian Medicinal Plants, Volume I, Indian Council of Medicinal Research, New Delhi, 168-172,(2003).

6. Mukherjee, P., Pharmacological Screening of Herbal Drug, Quality Control of Herbal Drug: An Approach to Evaluation of Botanicals; Eastern Publishers (Business Horizontal Ltd.), New Delhi, 755-760, (2002).

7. The Ayurvedic Pharmacopoeia of India, Part I; vol. II, edn. I^st, Govt. of India, Ministry of health and Family Welfare, New Delhi, 133, (1999)

Received on 12.12.2011

Modified on 11.01.2012

Accepted on 14.02.2012

Research Journal of Pharmacognosy and Phytochemistry. 4(2): March-April 2012, 61-63