Standardization of ‘Avipathikara Churna’: A HPLC Approach
Dubey Darshan* and Dashora Kamlesh
Institute of Pharmacy, Vikram University, Ujjain (M.P.) 456001, India
*Corresponding Author E-mail: darshandubey@gmail.com
ABSTRACT:
Quantification of active principles through modern analytical tools is necessary for establishing the safety, quality, acceptability and usage of herbal formulations. ‘Avipathikara churna’ is one of the oldest and popular Ayurvedic preparations, is official in Ayurvedic formulary of India used widely for disorder of Digestive impairment, Constipation, Dyspepsia, Haemorrhoids, Retention of urine and Urinary disorders. It comprised of the fruits Piper longum (Pippali), Piper nigrum (Marica), Embelica ribes (Vidanga) rhizomes of Zingiber officianalis (Saunth), Cyperus rotundus (Musta) pericarp of Terminelia chebula (Haritaki), Terminelia bellirica (Babhitaka), Embelica officinalis (Amalaki) seed of Elettaria cardamomum (Ela), leaf of Cinnamomum tamala (Tejpatra), Flower bud of Eugenia caryuphyllus (Lavang) and Operculina turpethum (Trivrt).The present study is an attempt to develop the fingerprint method for Avipathikara Churna by simple high-performance liquid chromatography (HPLC) determination using Gallic Acid as a standard, which is as an important and major content in formulation. RP- HPLC methods for determination of Galiic Acid in Vidanga, Haritaki, Babhitaka, Musta and Amalaki have been developed. A C 18 LUNA (5 micron 25 cm×4.6 mm) column from Phenomenex in binary gradient mode with mobile phase acetonitrile: ethyl acetate at flow rate is 1.2ml/min, and effluent was monitored at 264 nm. Validation of the method was done with a view to demonstrate its selectivity, linearity, precision and accuracy. The concentration of gallic acid present in raw material was found to be 3.096 ±0.729w/w in amalaki, 7.612±0.289 in babhitaka 4.658±0.902 in haritaki 0.211%±0.783 in musta and 0.967%±0.439 w/w in vidanga respectively and in three identical laboratory batch of Avipathikara Churna name AP-I, AP-II, AP-III, was 0.136±0.482, 0.135±0.708, 0.136±0.604w/w respectively. The Gallic acid content of all the three batches is found to be in close proximities with each other. Obtained results were compared with marketed formulation.
KEYWORDS: Gallic acid, Avipathikara Churna, HPLC, Ayurvedic Formulation, Fingerprinting.
1. INTRODUCTION:
Standardization of herbal medicines is the process of prescribing a set of standards or inherent characteristics, constant parameters, definitive qualitative and quantitative values that carry an assurance of quality, efficacy, safety and reproducibility. It is the process of developing and agreeing upon technical standards, standardization is a tool in the quality control process [1]. According to WHO, standardization and quality control of herbals include identification of crude drug based on the use of major chemical constituents as markers. Standardization of herbal product drugs, single chemical entities, “marker compounds,” may be used as potency standards in high performance liquid chromatography (HPLC) analysis. Using well-characterized marker compounds, conventional pharmaceutical manufacturing criteria for assay and content uniformity may be applied.
These marker compounds may be used to help identify herbal materials, set specifications for raw materials, standardize botanical preparations during all aspects of manufacturing processes, and obtain stability profiles [2, 3, 4]. HPLC analysis for marker compounds may provide additional information in the form of “chromatographic fingerprints [5]. The present paper is an effort to develop the quality control parameter of Avipathikara Churna by HPLC determination using gallic acid as an internal standard.
Avipathikara Churna is well known Ayurvedic Formulation, comprised twelve important herbs, Piper longum (Pippali), Piper nigrum (Marica), Embelica ribes (Vidanga) Zingiber officianalis (Saunth), Cyperus rotundus (Musta)Terminelia chebula (Haritaki), Terminelia bellirica (Babhitaka), Embelica officinalis (Amalaki) Elettaria cardamomum (Ela), Cinnamomum tamala (Tejpatra), Eugenia caryuphyllus (Lavang) and Operculina turpethum (Trivrt), vida and sugar.
Avipathikara Churna plays an essential role in the treatment of a wide variety of conditions. It is mainly used for digestive impairment and urinary disorder. [6]. The present study is an attempt to develop the chromatographic fingerprint method for Avipathikara Churna by High-performance liquid chromatographic method using gallic acid as a standard, which is as an important and major content in formulation. The RP- HPLC analysis which is a simple, precise, and accurate method and can be considered as one of the quality control method for routine analysis of Avipathikara Churna.
2. EXPERIMENTAL:
2.1. Materials
Dried herbs for Avipathikara churna were procured from local market Ujjain (M.P), India and identified on the basis of morphological and microscopical characters and compared with standard Pharmacopoeial Monograph [7,8]. All the solvents were used of HPLC Grade. Standard gallic acid (98%) was procured from Lancaster England.
2.2 Apparatus
A C-18 LUNA (5 micron 25 cm×4.6 mm) column from Phenomenex a binary gradient high- pressure liquid chromatograph (Shimadzu HPLC class VP series) with two LC–10 AT VP pumps, variable wavelength programmable UV/Visible SPD 10 AVP were used.
2.3 Chromatographic conditions
The mobile phase consisted acetonitrile: ethyl acetate (70:30). The flow rate was 1.2 ml/min. The wavelength of detection was 264nm.The column temperature was ambient and the injection volume was 10 μl.
Figure 1 HPLC Chromatogram of gallic acid
2.4 Preparation of standard solution of gallic acid
The stock solution of gallic acid was prepared by dissolving 10.0 mg in 100.0 mL methanol, creating a 100 μg/mL solution. This solution was diluted with the solvent as needed to prepare different standard solutions (10, 20, 30, 40, 50,---90, 100μg/mL).
2.5 Preparation of calibration curve of gallic acid
Standard solutions (2, 4, 6, 8, 10,----18, 20μg/mL), each in three replicates, were injected into the system. The method of linear regression was used for data evaluation. Peak area ratios of standard compounds were plotted against theoretical concentrations of standards. Linearity was expressed as a correlation coefficient. (Table 1, figure 1).
Table 1. Range of linearity
|
Concentration (μg/mL) |
Gallic acid Peak area (mean and S.D.) |
|
10 |
304.25±0.986 |
|
20 |
671.78±0.681 |
|
30 |
896.14±0.634 |
|
40 |
1263.43±0.432 |
|
50 |
1412.12±0.526 |
|
60 |
1796.23±0.236 |
|
70 |
2256.27±0.864 |
|
80 |
2521.43±0.651 |
|
90 |
2770.12±0.196 |
|
100 |
3442.12±0.298 |
Mean ± S.D. (n = 3).
Figure 2. Calibration curve of gallic acid
2.6 Preparation of extract of Avipathikara churna
The Avipathikara churna (1gm) was refluxed with 60 ml methanol for 90 min. and filtered. The marc was re reflux with 40 ml of methanol for another 1hours. Filter and the filtrate were combined. The methanol extract was concentrated under vacuum till the semisolid mass is obtained. The residue was dissolved in 75 ml methanol and filtered through sintered glass funnel (G-2) by vacuum filtration assembly. The filtrate was centrifuged at 2000 rpm for 20 minutes. The supernatant was collected in 100 ml volumetric flask and volume was made with methanol.
The same procedure was performed for each batch of Avipathikara churna, one marketed formulation M-I and M-II and separately powdered Emblica officinalis, Terminalia belerica, Terminalia chebula, Cyperus rotundus and Embelica ribes, and solution (100 ml) of their extract were prepared.
2.7 Method Validation
The method was validated for linearity, precision, accuracy, limit of detection, limit of quantification. (Table.2)
Limit of detection and limit of quantitation
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 gallic acid as a reference compounds was diluted to a series of appropriate concentrations with methanol and an aliquot of the diluted solution was injected into HPLC for analysis.
Table 2: Validation Parameter of gallic acid
|
S. No. |
Parameter |
Gallic acid |
|
|
1 |
Retention time |
3.198min |
|
|
2 |
Beer’s Law limit |
10-100µg/ml |
|
|
3 |
Regression equation (y= bx+a) |
y= 333.4x – 80.55 |
|
|
4 |
Intercept (a) |
80.55 |
|
|
5 |
Slope (b) |
333.4 |
|
|
6 |
Correlation coefficients (r2) |
r2 = 0.992 |
|
|
7 |
Limit of quantification(LOQ) |
1.578µg/ml |
|
|
8 |
Limit of detection(LOD) |
0.519µg/ml |
|
|
9
|
Recovery Studies |
Precision |
0.597 |
|
Standard Error |
0.464 |
||
|
Accuracy (%) |
99.62 |
||
2.8 Estimation of gallic acid
The appropriate aliquots from extract of each batch of Avipathikara churna, its one marketed formulations and separately Emblica officinalis, Terminalia belerica, Terminalia chebula, Cyperus rotundus and Embelica ribes, were withdrawn in 10 ml volumetric flask separately. The corresponding concentration of gallic acid against respective peak areas value was determined using the gallic acid calibration curve (Table 4).
3. RESULTS AND DISCUSSIONS:
The fingerprint method for quality control of Avipathikara churna is developed by simple high-performance liquid chromatography (HPLC) determination using gallic acid as a standard, which is important and major content in formulation. RP- HPLC methods for determination of gallic acid from the Emblica officinalis, Terminalia belerica, Terminalia chebula, Cyperus rotundus and Embelica ribes and Avipathikara churna have been developed. The acetonitrile: ethyl acetate (70:30) was selected to obtain a rapid and simple assay method for gallic acid with a reasonable run time, suitable retention time and the sharpness of the peak. The chromatogram of gallic acid under experimental condition showed a single peak of the drug at 3.198 min (Figure 1).
The standard curve for gallic acid was linear over the investigated range (10–100 μg/mL) with a percent relative standard deviation (% R.S.D.) of less than 2% based on three successive readings (Table 4.T.23, figure 4.4). A correlation coefficient (R2) is suggested that the developed HPLC method had an excellent linearity over the concentration range of 10–100 μg/m of gallic acid.Under the developed HPLC conditions, the limit of quantitation was determined to be 1.578 and limit of detection was found to be 0.478 after three successive injections of the sample. (Table 2).
The concentration of gallic acid present in raw material is found to be 3.096± 0.729% w/w in Emblica officinalis, 7.612±0.289% w/w in Terminalia belerica, 4.658% w/w in Terminalia chebula, 0.211%±0.783% w/w in Cyperus rotundus and 0.967%±0.439% w/w in Embelica ribes. Gallic acid content in three identical laboratory batch of Avipathikara churna AP-I, AP-II and AP-III, was found to be 0.136±0.482%, 0.135±0.708% and 0.136±0.604% w/w respectively. Marketed formulation of Avipathikara churna M-I showed gallic acid concentration to be 0.119±0.798% (Table 4).
Table 3. Recovery study
|
S. No. |
Amount of tannic acid (µg/ml) |
RSD% |
SE |
Recovery % |
||
|
Sample |
Added |
Estimated |
||||
|
1 2 3 |
100 100 100 |
50 100 150 |
148.96±0.953 199.63±1.672 249.42 ±0.793 |
0.639 0.837 0.317 |
0.388 0.682 0.323 |
99.30 99.81 99.76 |
|
Mean |
|
0.597 |
0.464 |
99.62 |
||
Mean ± SD of six determinations, RSD =Relative Standard Deviation, SE = Standard Error
|
S. No. |
Formulations and crude drugs |
Gallic acid content % (w/w) |
Standard error |
|
|
1 |
Emblica officinalis |
3.096 ± 0.729 |
0.297 |
|
|
2 |
Terminalia belerica |
7.612± 0.289 |
0.117 |
|
|
3 |
Terminalia chebula |
4.658± 0.902 |
0.368 |
|
|
4 |
Cyperus rotundus |
0.211%±0.783 |
0.319 |
|
|
5 |
Embelica ribes |
0.967%±0.439 |
0.179 |
|
|
6 |
Avipathikara Churna
|
AP-I |
0.136±0.482 |
0.196 |
|
7 |
AP –II |
0.135±0.708 |
0.288 |
|
|
8 |
AP–III |
0.136±0.604 |
0.246 |
|
|
9 |
M-I |
0.119±0.798 |
0.325 |
|
In order to obtain precision and accuracy the recovery study was performed at three levels by adding known amount of gallic acid with preanalysed sample of of gallic in Avipathikara churna. The experiment was repeated three times and results were cited in Table 3, which prove reproducibility of the result.
The HPLC method developed for the estimation of gallic acid is a simple, rapid and precise for the routine estimation of Avipathikara churna. The method was validated by statistical analysis and recovery studies. As Avipathikara churna is a good source of gallic acid, these findings can be used as routine chromatographic fingerprinting method for the standardization of the raw materials of the Avipathikara churna as well as finished formulation.
4. ACKNOWLEDGEMENT:
The authors are highly grateful to Director, Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur for providing sophisticated instrument facility.
5. REFERENCES:
1. Kunle, Oluyemisi Folashade, Egharevba Henry Omoregie and Ahmadu, Peter Ochogu, Standardization of herbal medicines - A review, International Journal of Biodiversity and Conservation Vol. 4(3), pp. 101-112, March 2012
2. WHO (1992). Quality Control Methods for Medicinal Plant Materials. World Health Organization, Geneva.
3. WHO (1996a). Quality Assurance of Pharmaceuticals: A Compendum of Guidelines and Related Materials, Good Manufacturing Practices and Inspection. World Health Organization, Geneva. 2.
4. Jain V., Saraf S., Saraf S. Asian Journal of Chemistry, 19 (2007), 1406-1410
5. N. J. Lazarowych and P. Pekos, Drug Information Journal, 32 (1998) 497–512.
6. The Ayurvedic Formulary of India, Part I, Sec ed., Govt. of India, Ministry of Health and Family Planning, Dept. of Health, Delhi, (1978) xxvii-xxvii, 110.
7. Indian Herbal Pharmacopoeia, Volume II, Regional Research Laboratory Jammu, Indian drug Manufacturing Association, Mumbai, (1999) 93-101,.
8. The Ayurvedic Pharmacopoeia of India. Part I; vol. II, first ed., Govt. of India, Ministry of health and Family Welfare, New Delhi, (1999) 133,
Received on 09.10.2012 Modified on 09.11.2012
Accepted on 01.12.2012 © AJRC All right reserved
Asian J. Research Chem. 5(12): Dec., 2012; Page 1415-1418