Development of HPLC fingerprinting method via estimation of gallic acid and tannic acid for routine quality control of Ayurvedic formulation Bhuvnesvara vati
Vishal Jain*
University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur (C.G.)
*Corresponding Author E-mail: vishaljain123@gmail.com
ABSTRACT:
Bhuvnesvara vati is an important Ayurvedic formulation, is official in Ayurvedic formulary of India. The present study is an attempt to develop Fingerprint method for Bhuvnesvara vati with High Performance Liquid chromatography (HPLC) using gallic acid and tannic acid as a standard was developed, which are important and major content in formulation. The RP- HPLC Fingerprinting method was developed for three laboratory batch of Bhuvnesvara vati, its two marketed formulations and for its raw materials Emblica officinalis, Terminalia belerica, Terminalia chebula, Trichyspermum ammi and Aegle marmelus.The concentration of gallic acid present in raw material is found to be 3.174±0.049% w/w in Emblica officinalis, 8.920±0.173% w/w in Terminalia belerica, 4.092±0.117% w/w in Terminalia chebula, 1.831±0.973% w/w in Aegle marmelus and 0.264±0.365% w/w in Trichyspermum ammi. Gallic acid content in three identical laboratory batch of Bhuvnesvara vati BV-I, BV-II and BV-III, was found to be 2.623±0.746%, 2.589±0.356% and 2.632±0.239% w/w respectively. Two marketed formulation of Bhuvnesvara vati M-I and M-II showed gallic acid concentration to be 2.019±0.872 % and 2.019±0.872 % w/w respectively. The concentration of tannic acid present in raw material was found to be 6.172%±0.365w/w in Emblica officinalis, 8.667%±0.0319w/w in Terminalia belerica, 13.956%±0.745w/w in Terminalia chebula, 4.789±0.983% w/w in Aegle marmelus and 0.668±1.002% w/w in Trichyspermum ammi respectively and in three identical laboratory batch of Bhuvnesvara vati BV-I, BV-II and BV-III, was found to be 2.623±0.746%, 2.589±0.356%, 2.632±0.239% w/w respectively. The HPLC method developed for the simultaneous estimation of gallic acid and tannic acid is a simple, rapid and precise for the routine estimation of Bhuvnesvara vati.
KEYWORDS: Ayurvedic formulation, Gallic acid, Tannic acid, Standardization, HPLC.
INTRODUCTION:
Bhuvnesvara vati is official in Ayurvedic formulary of India (2000) and is among the most common formulas, used for diarrhoea in ayurvedic medicine.1 It has been described in Bhaisajyaratnawali (1961).2 As per the Ayurvedic formulary of India (2000) it comprised of the fruits of five medicinally important plants, Indian gooseberry (Amalaki, Emblica officinalis), Belleric myrobalan (Vibhitaka, Terminalia belerica), Chebulic myrobalan (Haritaki, Terminalia chebula), Yamani (Ajowan, Trichyspermum ammi), Bilvapesika ( Bael, Aegle marmelos) and two mineral ingredients Saindhava lavan ( rock salt) and Grahadhoom (Soot). 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. In Bhuvnesvara vati, gallic acid and tannic acid are believed to contribute to its therapeutic effects. Therefore, their quantification can provide insights into the quality and potency of the formulation. The developed HPLC fingerprinting method can also be used to compare the chemical profiles of Bhuvnesvara vati samples from different sources or batches, which can help in identifying any variations or adulterations. Overall, this study highlights the importance of using modern analytical techniques such as HPLC fingerprinting for ensuring the safety, efficacy, and consistency of traditional herbalformulations.
MATERIALS AND METHODS:
Preparation of Bhuvnesvara vati
Bhuvnesvara vati, three batches name BV-I, BV-II, BV-III, were prepared in laboratory using method described in Ayurvedic Formulary of India and Two Marketed formulations named M-I and M-II was purchased from local pharmacy store.
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.
Chromatographic conditions
The mobile phase consisted acetonotrile: methanol: phosphate buffer (pH 3.0) (10:5:85). 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.
Preparation of extract of Bhuvnesvara vati
The powdered Bhuvnesvara vati (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 Bhuvnesvara vati, two marketed formulation M-I and M-II and separately powdered Emblica officinalis, Terminalia belerica, Terminalia chebula, Aegle marmelus and Trichyspermum ammi, and solution (100 ml) of their extract were prepared.
Preparation of standard solution of gallic acid and tannic acid
The stock solution of gallic acid and tannic acid was prepared by dissolving 10.0 mg of each 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 (2, 4, 6, 8, 10,--------16, 20μg/mL).
Linearity
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 and 2).
Figure 1 : Standard curve of tannic acid Figure2 :Standard curve of gallic acid
Table1: Range of linearity
|
Concentration (μg/mL) |
Gallic acid |
Tannic acid Peak area (mean and S.D.) |
|
2 |
65.01±0.231 |
85.89±0.654 |
|
4 |
132.16±0.569 |
171.12±0.236 |
|
6 |
192.14±0.634 |
251.03±0.453 |
|
8 |
263.43±0.432 |
342.01±0.821 |
|
10 |
312.12±0.526 |
420.02±0.763 |
|
12 |
416.23±0.236 |
505.2±0.367 |
|
14 |
450.27±0.864 |
574.2±0.964 |
|
16 |
521.43±0.651 |
660.25±0.239 |
|
18 |
570.12±0.196 |
761.02±0.546 |
|
20 |
642.12±0.298 |
852.03±0.547 |
Mean ± S.D. (n = 3).
Precision
The precision of the method was tested by injecting a standard solution of gallic acid and tannic acid (20μg/mL and 2μg/mL) three times. Peak areas were determined and compared. Precision was expressed as percentage relative standard deviation (R.S.D.) (Table 2).
Table 2: Validation Parameter of gallic acid and tannic acid
|
S.No. |
Parameter |
Gallic acid |
Tannic acid |
|
1 2 3 4 5 6 7 8 9 10 |
Retention time Beer’s Law limit Regression equation (y= bx+a) Intercept (a) Slope (b) Correlation coefficients (r2) Precision (n=3 % RSD) Accuracy (%) Limit of quantification(LOQ) Limit of detection(LOD) |
3.410min 2-20mg/ml y= 32.033x + 4.14 4.14 32.03 r2 = 0.9967 0.357 99.69 1.456mg/ml 0.478mg/ml |
25.976 min 2-20mg/ml y=41.98x+0.53 0.53 41.68 r2 = 0.9991 0.353 99.38 0.754mg/ml 0.249mg/ml |
Repeatability
Inter and intra-day variation was performed by injecting the standard solutions (2, 4, 6, 8, 10,--------18, 20μg/mL), each in three replicates, twice on the same day, and once on the next day and Peak areas were determined and compared in (Table 3 and table 4).
Table 3: System repeatability of gallic acid
|
Concentration (μg/mL) |
Day 1 peak area |
Day 1 peak area |
Day 2 peak area |
|
2 |
65.01±0.231 |
65.21±0.981 |
65.89±0.456 |
|
4 |
132.16±0.569 |
132.19±0.369 |
132.72±0.652 |
|
6 |
192.14±0.634 |
192.16±0.258 |
193.12±0.832 |
|
8 |
263.43±0.432 |
263.33±0.147 |
264.22±0.624 |
|
10 |
312.12±0.526 |
312.08±0.546 |
312.96±0.496 |
|
12 |
416.23±0.236 |
416.21±0.236 |
417.02±0.238 |
|
14 |
450.27±0.864 |
450.65±0.985 |
451.23±0.234 |
|
16 |
521.43±0.651 |
521.22±0.924 |
522.03±0.941 |
|
18 |
570.12±0.196 |
570.36±0.846 |
571.09±0.743 |
|
20 |
642.12±0.298 |
642.13±0.754 |
643.02±0.725 |
Mean ± S.D. (n = 3).
Table 4: System repeatability of tannic acid
|
Concentration (μg/mL) |
Day 1 peak area |
Day 1 peak area |
Day 2 peak area |
|
2 |
85.89±0.654 |
85.94±0.827 |
86.02±1.020 |
|
4 |
171.12±0.236 |
171.22±0.854 |
171.53±0.981 |
|
6 |
251.03±0.453 |
251.16±0.946 |
251.92±0.479 |
|
8 |
342.01±0.821 |
342.09±1.231 |
342.97±0.673 |
|
10 |
420.02±0.763 |
420.13±0.238 |
421.07±0.612 |
|
12 |
505.2±0.367 |
505.29±0.734 |
505.83±0.825 |
|
14 |
574.2±0.964 |
574.24±0.439 |
574.97±0.946 |
|
16 |
660.25±0.239 |
660.31±0.824 |
661.29±0.256 |
|
18 |
761.02±0.546 |
761.14±0.559 |
761.73±0.652 |
|
20 |
852.03±0.547 |
852.18±0.629 |
853.13±0.987 |
Mean ± S.D. (n = 3).
Determination of limit of quantitation and limit of detection
The limit of detection (LOD) is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. The limit of quantitation (LOQ) is the lowest amount of analyte which can be quantitatively determined with suitable precision. The LOD and LOQ of the developed method were determined by injecting progressively low concentration of the standard solution and the lowest concentrations were assayed (Table 2).
Estimation of gallic acid and tannic acid
The appropriate aliquots from extract of each batch of Bhuvnesvara vati, its two marketed formulations and separately Emblica officinalis, Terminalia belerica, Terminalia chebula, Aegle marmelus and Trichyspermum ammi, were withdrawn in 10 ml volumetric flask separately. The corresponding concentration of piperine against respective peak areas value was determined using the gallic acid and tannic acid calibration curve respectively (Table 5).
Table 5: HPLC Estimation of gallic and tannic acid
|
S.no. |
Name |
Gallic acid content %w/w |
Tannic acid content %w/w |
|
|
1 |
Emblica officinalis |
3.174 ± 0.49 |
6.172±0.365 |
|
|
2 |
Terminalia chebula |
4.092± 0.117 |
13.956±0.745 |
|
|
3 |
Terminalia belerica |
8.920± 0.173 |
8.667±0.032 |
|
|
4 |
Aegle marmelus |
1.831±0.973 |
4.789±0.983 |
|
|
5 |
Trichyspermum ammi |
0.264±0.365 |
0.668±1.002 |
|
|
6
|
Bhuvnesvara vati |
BV-I |
2.623±0.746 |
4.914±0. 782 |
|
BV-II |
2.589±0.356 |
4.789±0.636 |
||
|
BV-III |
2.632±0.239 |
4.854±0.698 |
||
|
M-I |
2.019±0.872 |
4.251±0.993 |
||
|
M-II |
2.019±0.872 |
3.987±0.368 |
||
Mean ± S.D. (n = 3).
Recovery Studies
The recovery studies performed at three levels by adding known amount of gallic acid and tannic acid to extract of Bhuvnesvara vati, of which the gallic acid and tannic acid content have been estimated previously. The data were obtained and recovery was calculated (Table 2).
Figure 2: HPLC chromatogram of gallic acid and tannic acid
RESULTS AND DISCUSSION:
The fingerprint method for quality control of Bhuvnesvara vati is developed by simple high-performance liquid chromatography (HPLC) determination using gallic acid and tannic acid as a standard, which are important and major content in formulation. RP- HPLC methods for determination of gallic acid and tannic acid from the fruits of Emblica officinalis, Terminalia belerica, Terminalia chebula, Aegle marmelus and Trichyspermum ammi and Bhuvnesvara vati have been developed. The acetonotrile: methanol: Phosphate buffer (pH 3.0)(10:5:85) was selected to obtain a rapid and simple assay method for gallic acid and tannic acid with a reasonable run time, suitable retention time and the sharpness of the peak. The chromatogram of gallic acid and tannic acid under experimental condition showed a single peak of the drug at 3.410 min and 25.976 min (Figure 2).
The standard curve for gallic acid and tannic acid was linear over the investigated range (2–20 μg/mL) with a percent relative standard deviation (% R.S.D.) of less than 2% based on three successive readings (Table 1, figure 1 and 2). A correlation coefficient (R2) is suggested that the developed HPLC method had an excellent linearity over the concentration range of 2-20μg/m of gallic acid and tannic acid.
In order to determine the inter day and intraday precision the mean peak area, the standard deviation and the relative standard deviation were calculated and are reported in Table 3.19 and 3.20. After injecting gallic acid and tannic acid standard concentrations (2, 4, 6, 8, 10,--------18, 20μg/mL) in triplicate, on same day, and on two consecutive days the peak areas were compared. The relative standard deviation for the peak areas on same day, and on two consecutive days, were found to be with in the acceptable limits less than 2%.
Under the developed HPLC conditions, the limit of quantitation was determined to be 1.456 and 0.754 μg/mL respectively for gallic acid and tannic acid after three successive injections of the sample. Also, the limit of detection was found to be 0.478 and 0.249 μg/mL for gallic acid and tannic acid respectively (Table 2).
The concentration of gallic acid present in raw material is found to be 3.174±0.049% w/w in Emblica officinalis, 8.920±0.173% w/w in Terminalia belerica, 4.092±0.117% w/w in Terminalia chebula, 1.831±0.973% w/w in Aegle marmelus and 0.264±0.365% w/w in Trichyspermum ammi. Gallic acid content in three identical laboratory batch of Bhuvnesvara vati BV-I, BV-II and BV-III, was found to be 2.623±0.746%, 2.589±0.356% and 2.632±0.239% w/w respectively. Two marketed formulation of Bhuvnesvara vati M-I and M-II showed gallic acid concentration to be 2.019±0.872 % and 2.019±0.872 % w/w respectively (Table 5).
The concentration of tannic acid present in raw material was found to be 6.172%±0.365w/w in Emblica officinalis, 8.667%±0.0319w/w in Terminalia belerica, 13.956%±0.745w/w in Terminalia chebula, 4.789±0.983% w/w in Aegle marmelus and 0.668±1.002% w/w in Trichyspermum ammi respectively and in three identical laboratory batch of Bhuvnesvara vati BV-I, BV-II and BV-III, was found to be 2.623±0.746%, 2.589±0.356%, 2.632±0.239% w/w respectively. The results were comparable to marketed formulations (Table 5). The results indicate that the developed method can be used to quantification of gallic and tannic acid from Bhuvnesvara vati.
In order to obtain precision and accuracy the recovery study was performed at three levels by adding known amount of of gallic and tannic acid with preanalysed sample of of gallic and tannic acid in Bhuvnesvara vati. The experiment was repeated three times (Table 2) and result shows 99.69% and 99.38% for gallic and tannic acid respectively, which prove reproducibility of the result. This shows significant precision of methods at 95% confidence level. The %relative standard deviation (% RSD) value was found to be 0.357 and 0.353 for gallic and tannic acid respectively. The recovery of gallic and tannic acid from the Bhuvnesvara vati was quantitative and there was no interference from the other compounds present in the formulation when compared to the control.
The HPLC method developed for the simultaneous estimation of gallic acid and tannic acid is a simple, rapid and precise for the routine estimation of Bhuvnesvara vati. The method was validated by statistical analysis and recovery studies. As Bhuvnesvara vati is a good source of gallic acid and tannic acid, these findings can be used as routine chromatographic fingerprinting method for the standardization of the raw materials of the Bhuvnesvara vati as well as finished formulation.
REFERENCES:
1. The Ayurvedic Formulary of India. Part II, 1st edition. Delhi: Govt. of India, Ministry of Health and Family Planning, Dept. of Health; 2000:178.
2. Bhaisajyaratnavali. 2nd edition. Varanasi: Chowkhamba Sanskrit Series Office; 1961: Sloke no.149-150,163.
3. Tripti Jain, and Kamlesh Dashora, Spectrophotometric fingerprinting method for Unani formulation Hab-e-Azarakhi. Asian J. Pharm. Tech. 2012; 2(1): 1-3
4. 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.
5. Vishal Jain, Ambar Vyas, Swarnlata Saraf, S. Saraf. HPLC Determination of Piperine in ‘Trikatu Churna’ a Potent Ayurvedic Formulation for Routine Quality Control. Asian J. Research Chem. 2011; 4(2): 183-186.
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7. 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.
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Received on 28.07.2023 Accepted on 16.09.2023 ©A&V Publications all right reserved Research J. Engineering and Tech. 2023; 14(2):51-56. DOI: 10.52711/2321-581X.2023.00005 |
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