HPTLC Method for Estimation of Ellagic Acid and Gallic Acid from Flowers of Woodfordia fruticosa Kurz.
Bhaskar O. Aher, Yogesh T. Sonawane, Vinod A. Bairagi, Parag A. Pathade
Department of Pharmaceutical Chemistry, K.B.H.S.S. Trust’s Institute of Pharmacy,
Malegaon, 423105, District - Nasik, Maharashtra, India.
Department of Pharmacognosy, K.B.H.S.S. Trust’s Institute of Pharmacy,
Malegaon, 423105, District - Nasik, Maharashtra, India.
*Corresponding Author E-mail: bhaskar.aher4@gmail.com, yogeshsonawane02@gmail.com
ABSTRACT:
Woodfordia fruticosa Kurz, belonging to family Lythraceae, commonly known as dhataki pushpa, is a plant of tropical and subtropical region with a long history of medicinal use. Wide range of chemical compounds including tannins, flavonoides, anthraquinones glycosides and polyphenols have been isolated from the plant. The extract from the flowers are used in folklore medicine for treatments of like wound healing, bowel complaint, rheumatism, hematuria. Litreture survey indicates that the flowers are rich in tannins, both gallo and ellagitannins. Hence, it was thought worthwhile to use Ellagic acid and Gallic acid as the marker compounds for the standardization of the flowers. With this background the present study was undertaken to standardize the flowers using Ellagic acid and Gallic acid as marker compounds. The HPTLC method used for the standardization was validated for the parameters like specificity, limits of detection and quantification, linearity, precision, accuracy and recovery. The total content of Ellagic acid and Gallic acid in the Methanolic extract was found to be 4.21%, 6.70% and the total content of Ellagic acid and Gallic acid in the Total aqueous extract was found to be 2.62%, 4.75%.
KEYWORDS: HPTLC, Anthraquinones Glycosides, Dhataki pushpa, Ellagic acid, Gallic acid.
1. INTRODUCTION:
Herbs are plants which enhance our lives, as they serve and delight us. Medicinal herbs have been in use for thousands of year, in one form or another, under the indigeneous system of medicine like Ayurveda, Sidha and Unani.The herbal products today symbolise safety in contrast to the synthetics that are regarded as unsafe to human and environment. The changes run parallel to our relationship with nature.
Among ancient civilisations, India has been known to be rich a repository of medicinal plants. In India, drugs of herbal origin have been used in traditional systems of medicines such as Unani and Ayurveda since ancient times1. A major lacuna in Ayurveda is the lack of drug standardisation, information and quality control. In the absence of pharmacopoeia data on the various plant extracts, it is not possible to isolate or standardise the active contents having the desired effects2.
The flowers of Woodfordia fruticosa Kurz, called as dhataki pushpa, belonging to family Lythraceae are widely used in Ayurvedic medicine as antiseptic. It is also useful in the preparation of Ayurvedic fermented drugs called ‘Aristha’s and Asava’s’, and is useful in thirst, dysentery, leprosy, blood diseases, leucorrhoea, piles and toothache. The dried flowers of W. fruticosa Kurz, has been used as an the treatment of wound healing, bowel complaint, rheumatism, dysuria and hematuria. The fresh flowers to stop bleeding in emergency cuts. W. fruticosa Kurz has been reported to contain ellagic acid and gallic acid (Figure1A and 1B),
Figure 1A: Chemical structure of Ellagic acid
Figure 1B: Chemical structure of Gallic acid
hecogenin, naringenin 7-glucoside, kaempferol 3-Oglucoside, isoschimawalin A woodfordins A, B, C, D, E, and F3. In a separate study, the methanolic and auqeous extract of the flower of W. fruticosa Kurz., It shows Anti-fertility4, Hepatoprotective5, In vitro Anti-bacterial6, Anti–pepticulcer7, Imunomodulatory8 and An Inhibitor of DNA Topoisomerase II9. But studies have shown that the phytochemical may have other health benefits. Hence the quantification of ellagic acid and gallic acid content in W. fruticosa Kurz flowers is done, which provides a therapeutically active chemical marker to standardize this drug.
This paper reports standardization of methanolic and aqueous extract of flowers of W. fruticosa Kur. containing ellagic acid and gallic acid as biomarker by HPTLC technique.
2. MATERIAL AND METHODS:
2.1 Materials:
Flowers of the plant of W. fruticosa Kur. was procured from the local market, Mumbai. The flowers was authenticated at Agharkar Research Institute, Pune. The voucher specimen no. (1/ F 025) is also deposited in the institute for reference. Standard ellagic acid and gallic acid were procured from Sigma Aldrich Co. (U.S.A). Light petroleum ether (b.p. 60–80°C, GR-grade) and toluene, ethyl acetate, formic acid, methanol, hydrochloric acid, sulphuric acid and ethanol (all AR-grade) were from S.D. Fine Chemicals.
2.2 Sample Preparation:
Sample Preparation:
The crude drug was air dried and pulverized to obtain a coarse powder. The powdered drug was defatted by extracting with petroleum ether (b.p. 60-800C) followed by extraction with methanol using Soxhlet extractor. The extract obtained was concentrated by recovering the solvent by Rotary Flash Evaporator. The concentrated extract was then evaporated to dryness in vacuum oven at temperature not more than 500C. The dried extract was stored at 2-80C in refrigerator. 10mg of the methanolic and 10mg of the aqueous extract was accurately weighed, placed in different 10ml volumetric flask, and was dissolved in methanol and the volume was make up upto the mark. This solution of the methanolic extract (1mg/ml) and aqueous extract (1mg/ml) was used for the quantification. The methanolic and aqoeous extract was hydrolysed and used for standardization of ellagic acid and gallic acid.
Acid hydrolysis:
Weigh accurately about 100mg methanolic and aqueous extract and was dissolved in 5ml methanol and 10ml 1N HCl and heated for 10-15 min. Then transfer it to 25ml volumetric flask and make up the volume with methanol.
Standard Preparation:
Ellagic acid and gallic acid stock solution of 100μg/ml resp. were prepared and used for experimental work.
2.3 HPTLC finger printing:
Chromatography was performed on aluminum-sheets precoated with silica gel GF254 HPTLC plates (Merck # BOL790330). Before use, the plates were prewashed with methanol, dried, and activated at 110şC for 1 hour between two glass plates of larger dimensions to prevent deformation of the plates. Sample (10μl) was applied to the plates, as 6-mm bands, 10mm apart and 10mm from the edge of the plates, by means of a Camag Linomat V sample applicator fitted with a 100μl Hamilton syringe. After drying of the spots, the plates were developed to a distance of 80mm in a Camag twin-trough chamber previously saturated with mobile-phase vapor for 20 min., the mobile phase was toluene: ethyl acetate: formic acid in the ratio 5:4:1 (v/v). Densitometric evaluation of the plates was performed at λmax = 280nm, using a Dueterium lamp, with a Camag Scanner III in conjunction with winCATS Planar Chromatography Manager software, version 1.4.4.6337 for quantification of ellagic acid and gallic acid.
2.4 Validation of the HPTLC Method by Use of Calibration Standard of ellagic acid and gallic acid:
2.4.1 Specificity:
A methanolic solution of reference standard of ellagic acid and gallic acid (100μg/ml, 10μl) and methanolic and aqueous extract solution in methanol (1mg/ml, 10μl) was applied to a silica gel GF254 HPTLC plate and the plate was developed using the mobile phase and conditions described above. Under all the conditions mentioned above, a single spot of ellagic acid (R.S.) at the Rf = 0.38 and gallic acid (R.S.) at the Rf = 0.53 was obtained. Peak purity tests were also performed for the spectra of the spot. Methanol (5μl) was also spotted in parallel as a control (Figure 2,3,4) No additional peak was observed at the Rf of ellagic acid and gallic acid.
Figure 2: HPTLC Plate image for Detection of Eallgic Acid and Gallic Acid in the Methanolic and Total aqueous extract of flowers of W. fruticosa.
A. Wavelength at 254nm:
Track 1- Standard Gallic acid (10µl), Track 2- Methanolic Extract (10µl)
Track 3- Standard Ellagic acid (10µl), Track 4- Total aqueous Extract (10µl)
Figure 3A: Fingerprint of Methanolic extract of W. fruticosa and the Methanolic extract spiked with reference Standards of Ellagic Acid and Gallic Acid
Note:
a: The methanolic extract + ellagic acid (1000ppm)
b: The methanolic extract + gallic acid (1000ppm)
Figure 3B:
Fingerprint of Total aqueous extract of W. fruticosa and the Total aqueous extract spiked with reference Standards of Ellagic Acid and Gallic Acid
Note:
a: The total aqueous extract + ellagic acid (1000ppm)
b: The total aqueous extract + gallic acid (1000ppm)
Figure 4 A: Superimposition of the UV spectra of Ellagic acid (Reference Standard) the Methanolic and the Total aqueous extract of flowers of W. fruticosa Kurz.
1: UV spectrum of the Methanolic extract of flowers of W. fruticosa Kurz. (Rf = 0.38)
2: UV spectrum of the Total aqueous extract of flowers of W. fruticosa Kurz. (Rf = 0.32)
3: UV spectrum of Ellagic acid from reference standard. (Rf = 0.39)
Figure 4B: Superimposition of the UV spectra of Gallic acid (Reference Standard) the Methanolic and the Total aqueous extract of flowers of W. fruticosa Kurz.
1: UV spectrum of the Methanolic extract of flowers of W. fruticosa Kurz. (Rf = 0.53)
2: UV spectrum of the Total aqueous extract of flowers of W. fruticosa Kurz. (Rf = 0.54)
3: UV spectrum of Gallic acid from reference standard. (Rf = 0.53)
2.4.2 Linearity:
In accordance with ICH recommendations, in order to establish linearity, reference standard ellagic acid and gallic acid (100 μg/ml) was at six different concentrations viz 10, 20, 30, 40, 50 and 60μl containing 2, 4, 6, 8, 10 and 12 μg were loaded on GF254 HPTLC plates respectively. The plate was developed and the detector response for the different concentrations was measured. The experiment was performed in triplicate and the mean was calculated. A graph was plotted of drug peak area against concentration of ellagic acid and gallic acid (Figure 5).
Figure 5A: Standard curve for Ellagic acid
2.4.3 Limits of Detection and Quantification:
Reference standard solutions of ellagic acid and gallic acid at concentrations in the lower part of the linear range of the calibration plot were used to determine limit of detection (LOD) and limit of quantification (LOQ). These were determined from the slope of the calibration plot and standard deviation (SD) of the blank sample by use of the equations:
LOD = 3.3 × SD/S and LOQ = 10 × SD/S
Where SD (standard deviation) of the blank response and S is the slope of the calibration plot (Table 1)
Figure 5B: Standard curve of Gallic Acid
Table 1: Validation Summary
|
Parameter |
Ellagic acid |
Gallic acid |
|
Limit of Detection |
29ng |
15ng |
|
Limit of Quantification |
59ng |
45ng |
|
Linearity range |
0.05 to 0.5µg |
1.25to 6.25 µg |
|
Recovery |
For ME: 96.00 % For HME:96.00 % |
For TAE: 94.00 % for HTAE: 91.00 % |
2.4.4 Precision:
In accordance with ICH recommendations precision was determined at two levels, i.e. repeatability and intermediate precision. Repeatability of sample application was determined as intraday variation whereas interme-diate precision was determined by measuring inter-day variation for triplicates determination of reference standard ellagic acid (20µl, 30µl, 60µl) and gallic acid (20µl, 60µl and 100µl) (Table 2 and 3).
Table 2: Inter- and intra-day precision for quantification of ellagic acid and gallic acid for Methanolic extract
|
Conc. (µg per spot) |
Repeatability (Intraday precision) |
Intermediate precision (Interday) |
||||
|
Mean area ± SD |
Standard error |
RSD (%) |
Mean area ± SD |
Standard error |
RSD (%) |
|
|
Ellagic acid |
||||||
|
2 |
4608.0 ± 0.034 |
0.019 |
1.71 |
4616.60 ± 0.030 |
0.015 |
1.52 |
|
6 |
7759.3 ± 0.032 |
0.018 |
1.62 |
7780.60 ± 0.023 |
0.013 |
1.19 |
|
10 |
8895.6 ± 0.020 |
0.011 |
1.01 |
8895.80 ± 0.021 |
0.012 |
1.06 |
|
Gallic acid |
||||||
|
2 |
5517.4 ± 0.026 |
0.015 |
1.31 |
5555.4 ± 0.018 |
0.010 |
0.91 |
|
6 |
7722.7 ± 0.024 |
0.014 |
1.22 |
7785.5 ± 0.007 |
0.004 |
0.38 |
|
10 |
9617.0 ± 0.006 |
0.003 |
0.30 |
9632.8 ± 0.005 |
0.003 |
0.28 |
Table 3: Inter- and intra-day precision for quantification of ellagic acid and gallic acid for Total aqueous extract
|
Conc. (µg per spot) |
Repeatability (Intraday precision) |
Intermediate precision (Interday) |
||||
|
Mean area ± SD |
Standard error |
RSD (%) |
Mean area ± SD |
Standard error |
RSD (%) |
|
|
Ellagic acid |
||||||
|
2 |
5908.7 ± 0.011 |
0.006 |
0.56 |
5931.2 ± 0.010 |
0.006 |
0.52 |
|
6 |
9219.1 ± 0.006 |
0.003 |
0.30 |
9242.0 ± 0.004 |
0.004 |
0.43 |
|
10 |
11274.2 ± 0.007 |
0.004 |
0.39 |
11278.8 ± 0.003 |
0.003 |
0.30 |
|
Gallic acid |
||||||
|
2 |
6083.3± 0.010 |
0.006 |
0.54 |
6095.8 ± 0.011 |
0.006 |
0.55 |
|
6 |
9332.2 ± 0.008 |
0.004 |
0.40 |
7785.5 ± 0.007 |
0.005 |
0.50 |
|
10 |
12392.2 ± 0.003 |
0.002 |
0.19 |
9632.8 ± 0.005 |
0.005 |
0.47 |
2.4.5 Recovery:
The recovery of the method at low, medium, and high concentrations was determined by six fold replicate application and chromatography of 20μl (4μg), 40μl (8 μg), and 60μl (12μg) of solution of ellagic acid reference standard (100μg/ml) and 20μl (4μg), 40μl (8μg), and 60 μl (12μg) of solution of gallic acid reference standard (100μg/ml) respectively.
Reference standard solution (10μl containing 100μg/ml) of ellagic acid and reference standard solution (10μl containing 100μg/ml) of gallic acid was added to solution (100μl containing 1000μg/ml) of methanolic extract of flowers of W. fruticosa Kurz. This was considered as the spiked methanolic extract. Reference standard solution (10μl containing 100μg/ml) of ellagic acid and reference standard solution (10 μl containing 100 μg/ml) of gallic acid was added to solution (100 μl containing 1000μg/ml) of aqueous extract of flowers of W. fruticosa Kurz. This was considered as the spiked aqueous extract. HPTLC analysis was performed for both, the spiked extract and solution (100μl containing 1000 μg/ml) of methanolic and aqueous extract of bark of flowers of W. fruticosa Kurz. Recovery was calculated by use of the equation:
Recovery = [(A – B)/C] × 100
Note:
A – Concentration of std. in the spiked extract
B - Concentration of std in the methanolic extract without added standard
C - Concentration of std added.
3. RESULTS AND DISCUSSION:
An HPLTC method was established to quantify ellagic acid and gallic acid in the methanolic and aqueous extract of flower of W. fruticosa Kurz. The presence of ellagic acid and gallic acid was verified by comparison of Rf values with R.S. Rf = 0.38 and Rf = 0.53 resp., co-elution, and by also comparison of the UV spectra (λmax 275 for ellagic acid and λmax 284 for gallic acid) of the spots corresponding obtained from the sample and the standard. The amount of ellagic acid and gallic acid present in the methanolic and aqueous extract and hydrolysed methanolic, hydrolysed aqueous extract and was calculated from the calibration curve. The methanolic extract of the flower was found to contain 4.21% ellagic acid and 2.62% of gallic acid respectively and hydrolysed methanolic extract of the flower was found to contain 7.99% ellagic acid and 4.69% of gallic acid respectively. The aqueous extract of the flower was found to contain 6.70% ellagic acid and 4.75% of gallic acid respectively and hydrolysed aqueous extract of the flower was found to contain 8.43% ellagic acid and 8.82% of gallic acid respectively.
Validation of the method:
The specificity of the method was established by analysis of the methanolic and aqueous extract, reference standards of ellagic acid, gallic acid and control, i.e. methanol.
Absence of interfering peak originating from the control track when scan at λmax = 280nm at the Rf values corresponding to ellagic acid and gallic acid, the method can be deemed to have been validated for specificity. Also, peak-purity test performed for the spectra of the spot did not reveal the presence of coeluting peaks at the Rf values of to ellagic acid and gallic acid.
Plot of peak area against concentration of ellagic acid was linear in the range of 0.05 to 0.5μg with regression coefficient 0.981 and gallic acid Plot of peak area against concentration was linear in the range of 1.25 to 6.25μg with regression coefficient 0.996.
The limit of detection and quantification for ellagic acid were found to be 29ng per spot and 15ng per spot respectively, and for gallic acid were found to be 59ng per spot and 45 ng per spot respectively.
For methanolic extract, ellagic acid inter-day precision (%RSD) for peak area ranged between 1.06% to 1.52% and Intra-day precision (%RSD) ranged between 1.01% to 1.71% and for gallic acid peak area ranged between 0.28% to 0.91% and Intra-day precision (RSD) ranged between 0.30% to 1.31%. For aqueous extract, ellagic acid inter-day precision (%RSD) for peak area ranged between 0.30% to 0.52% and Intra-day precision (%RSD) ranged between 0.39% to 0.56% and for gallic acid peak area ranged between 0.47% to 0.55% and Intra-day precision (RSD) ranged between 0.19% to 0.54%. The results of inter and intraday precision are well in the range i.e. %RSD is <2%. The Recovery of the method was established by means of a recovery experiment. The mean recovery was close to 100 %, which indicates the accuracy of the method.
4. ACKNOWLEDGEMENT:
The authors are grateful to the authorities of K.B.H.S.S. Trust’s Institute of Pharmacy, Malegaon, Nasik for the facilities.
5. CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 05.11.2020 Modified on 19.03.2021
Accepted on 31.05.2021 ©Asian Pharma Press All Right Reserved
Asian J. Res. Pharm. Sci. 2021; 11(3):187-192.
DOI: 10.52711/2231-5659.2021.00029