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Validated high performance thin layer chromatography method for simultaneous determination of quercetin and gallic acid in Leea indica
G Model
ARTICLE IN PRESS
BJP 306 1–4
Revista Brasileira de Farmacognosia xxx (2016) xxx–xxx
www.elsevier.com/locate/bjp
Original Article
Validated HPTLC method for simultaneous determination of quercetin and gallic acid in Leea indica
1
2
3 4
Q1
Ankita A. Patel, Aeshna A. Amin, Arpit H. Patwari, Mamta B. Shah ∗ Department of Pharmacognosy, L. M. College of Pharmacy, Ahmedabad, Gujarat, India
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a r t i c l e
i n f o
a b s t r a c t
7 8 9 10 11
Article history: Received 14 October 2015 Accepted 24 May 2016 Available online xxx
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Keywords: Gallic acid HPTLC Leea indica Quercetin Simultaneous estimation Validation
A sensitive and reliable high performance thin layer chromatography method has been developed for the simultaneous estimation of quercetin and gallic acid in Leea indica (Burm. f.) Merr., Vitaceae. Ethyl acetate extract prepared from hydrolysed aqueous alcoholic extract (70%) was applied on silica gel G 60 F254 plate. The plate was developed using toluene-ethyl acetate-formic acid, 5:4:1 (v/v/v) as a mobile phase and detection and quantification were performed by densitometric scanning at 254 nm. The system was found to give well resolved bands for quercetin (Rf 0.63) and gallic acid (Rf 0.45) from other constituents present in the extract of L. indica. The correlation coefficient was found to be 0.991 and 0.999 with RSD, 0.97–1.23% and 0.1–1.13% for quercetin and gallic acid respectively in the developed method. The accuracy of the method was confirmed by conducting recovery studies at different levels using the standard addition method. The average recovery of quercetin and gallic acid was found close to 99% suggesting the accurateness of the method. The proposed validated HPTLC method offers a new, sensitive, specific and precise gauge for quantification of quercetin and gallic acid in L. indica.
Q2
© 2016 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
20
Introduction
Leea indica (Burm. f.) Merr., (Syn.: L. sambucina Willd., L. gigantea Griff., L. umbraculifera Cl., Stephylea indica Burm. f.) Merr., Vitaceae, 22 a shrub found to be growing in the forests of tropical and sub23 tropical countries including India, Sri Lanka, Malaysia, Thailand, 24 Andaman and Philippines (Stewart and Brandis, 1874; Gamble, 25 1956). In traditional systems of medicine the leaf is valued in ver26 tigo and diabetes, while the root as antidysentric, anthelmintic, 27 spasmolytic and sudorific and to treat cardiac and skin diseases 28 (Chatterjee and Prakashi, 1997; Khare, 2007; Rahman et al., 2007). 29 The leaf is reported to possess antioxidant (Syahida et al., 2004, Sim 30 et al., 2012), analgesic (Islam et al., 2013), antimicrobial (Vijayan 31 et al., 2010), phosphodiesterase inhibitory (Limpeanchob, 2008), 32 sedative, anxiolytic (Manna et al., 2011) and cytotoxic (Lili et al., 33 2008; Ling et al., 2012; Haque et al., 2012) activities. The plant 34 35Q3 is reported to contain ˛-tocopherol, -amyrin (Israf et al., 2005), lupeol, -sitosterol, ursolic acid, farnesol, phloridzin, gallic acid 36 (Vijayan et al., 2008), and quercitrin (Bhobe et al., 2013). Fla37 vanoids, kaempferol, quercetin, quercitrin, and mearnsitrin have 38 21
∗ Corresponding author. E-mail: [email protected] (M.B. Shah).
been reported from other species of genus Leea (L. guineense) (Mariotte et al., 2003). HPTLC is as an acquiescent and commonly used technique for qualitative and quantitative analysis of chemical markers in herbal raw materials. HPTLC has advantages of simplicity, sensitivity, accuracy, and is one the most approached technique for developing fingerprint and marker-based standardization of herbal drugs and is commonly applied not only for the identification, assay and testing for purity but also for stability, dissolution or content uniformity of raw materials and formulated products (Mukherjee et al., 2010). Taking in to consideration the non-existence of a routine method of analysis, a simple HPTLC method for quantitative estimation of quercetin and gallic acid simultaneously is proposed for L. indica. The proposed method was validated by specificity, range, linearity, accuracy, precision, detection limit, quantitative limit, and robustness according to the ICH guidelines (ICH, 1996, 2005).
39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
Materials and methods
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Chemicals and solvents
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Ethyl acetate, methanol, toluene, HCl, NaOH, distilled water used were of analytical grade. Reference standards quercetin and gallic acid (purity 99.9%) were purchased from Sigma–Aldrich.
http://dx.doi.org/10.1016/j.bjp.2016.05.017 0102-695X/© 2016 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Patel, A.A., et al. Validated HPTLC method for simultaneous determination of quercetin and gallic acid in Leea indica. Revista Brasileira de Farmacognosia (2016), http://dx.doi.org/10.1016/j.bjp.2016.05.017
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ARTICLE IN PRESS
Plant material
70
The fresh shrub of L. indica (Burm. f.) Merr., Vitaceae, was procured from Waghai Botanical Garden, Dang, Gujarat in the month of September 2012 and authenticated by the taxonomist of South Gujarat University, Surat, India. Voucher specimen of plant (LM 5412) has been deposited in Department of Pharmacognosy and Phytochemistry, L. M. College of Pharmacy, Ahmedabad. The fresh plant was cut, dried properly, powdered, and passed through 60# sieve. The powdered sample was stored in an airtight container at room temperature.
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HPTLC analysis
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Quercetin and gallic acid standard stock solutions were prepared by dissolving 2 mg of the compound in methanol in 10 ml volumetric flask and volume was adjusted up to the mark with methanol, from this solution 1–5 l solutions was applied on precoated TLC plates for quercetin while 1–6 l was spotted for gallic acid for calibration purpose. Accurately weighed 10 g of plant powder was extracted exhaustively with ethanol (100 ml × 3) under reflux for 2 h/cycle. Ethanolic extract was evaporated to dryness under vacuum and residue was dissolved in 80 ml aqueous methanol (70%). The aqueous methanol solution was hydrolysed by using 40 ml 2 N HCl and 40 ml toluene and refluxing for 2 h. After neutralizing the solution by 5% NaOH, it was again refluxed for 1 h. The aqueous phase was successively extracted with toluene (2× 30 ml) and ethyl acetate (4× 50 ml). Ethyl acetate extract was evaporated to dryness and 10 mg of extract was dissolved in 5 ml methanol and 15 l of methanolic solution was applied to a TLC plate. Co-TLC study was conducted using silica gel G 60F254 plate and toluene-ethyl acetate-formic acid, 5:4:1 (v/v/v) as a mobile phase. The plates were observed under UV after derivatizing using Natural products-polyethylene glycol reagent (NP/PEG) for quercetin and 5% FeCl3 for gallic acid. HPTLC analysis was performed on 10 cm × 10 cm aluminum backed HPTLC plates coated with a 0.2 mm layer of silica gel G 60F254 (Merck, India). The plates were washed with methanol and activated in an oven at 110 ◦ C for 5 min prior to analysis. The standard and sample solutions were applied to the plates in form of bands of 5 mm long, 1 mm from bottom of the plate and 1 mm from the edge by means of automatic TLC sampler (CAMAG semiautomatic IV). The plates were developed using toluene-ethyl acetate-formic acid 5:4:1 (v/v/v) as mobile phase in CAMAG twin trough chamber, presaturated with mobile phase vapor for 30 min before development. After development, the plate was dried in air for 5 min and scanned immediately at 254 nm using TLC scanner (CAMAG scanner-III). A TLC visualizer was used for the photodocumentation of the plates.
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Validation of method
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The method was validated as per ICH guidelines. Calibration curve for quercetin and gallic acid were obtained by plotting the peak area against their amount applied to the plate. The limits of detection (LOD) and quantification (LOQ) were determined by equation as per ICH guidelines. Instrumental precision was determined by repeated analysis of standard quercetin and gallic acid. Repeatability was assessed for each reference standard by applying three concentrations of these standards in triplicate on the plate, which were expressed in terms of percentage relative standard deviation (% RSD) and standard error (SE). The intra-day precision was determined at three different concentration levels of different markers, 200, 400 and 600 ng/spot, six times on the same day, and the inter-day precision was determined at three different concentrations of markers, 100, 300 and 500 ng/spot, six times on five
Table 1 Validation parameters for quercetin and gallic acid estimation. Parameters
Results Quercetin
Gallic acid
Correlation coefficient Linearity range (ng)
0.991 200–1000
0.999 200–1200
Precision (C.V.) Repeatability of measurement Repeatability of application Intra day Interday Limit of detection (ng/spot) Limit of quantification (ng/spot) Accuracy (%) Specificity
0.89 0.53 0.23–0.43 0.97–1.23 21.31 64.57 98.02–99.09 Specific
0.359 0.42 0.2–0.6 0.1–1.13 14.8 55.04 99.28–100.26 Specific
different interval days over a period of one week (Kumar et al., 2008). System suitability was tested by applying 600 ng/spot of quercetin and gallic acid standard solution to the plate seven times and measuring the relative standard deviation (RSD%) for the peak areas and RF. To study the accuracy and precision of the method, recovery studies were performed by the method of standard addition. The recovery of added standard was studied at two different levels, each being analyzed in a manner similar to that described for the assay (Biringanine et al., 2006; Wagner et al., 2008). Results and discussion Quercetin and gallic acid were quantified by HPTLC for the first time in L. indica. The identity of quercetin was confirmed by cochromatography and overlain absorption spectra with reference standard, when scanned at 254 nm. Also the bathochromic shift in UV spectra with AlCl3 /HCl relative to methanol spectra asserted presence of quercetin in the plant. The densitometric chromatogram of HPTLC fingerprint of the ethyl acetate extract is shown in Fig. 1. The peaks resolving at Rf 0.63 and 0.45 in test solution were found to be superimposing with those of respective standards of quercetin and gallic acid. Peak purity was studied by UV overlay spectra obtained from the standards and from the corresponding peaks from test (Fig. 2). The content of quercetin and gallic acid in plant was calculated on the basis of peak area and was found to be 0.13% and 0.041% (w/w) respectively. The calibration plots indicate that the peak-area response was a polynomial function of the amount of standards quercetin and gallic acid in the range 200–1000 ng and 200–1200 ng respectively. The correlation coefficient was found to be 0.991 and 0.999 with RSD, 0.97–1.23% and 0.1–1.13% for quercetin and gallic acid respectively in the developed method. Intraday and interday precision was calculated for the developed method by performing the analysis at three different levels on same day as well as on different days respectively. RSD for repeatability of measurement of peak area based on 7 times measurement of 600 ng/spot of standards quercetin and gallic acid were found to be 0.89 and 0.359% respectively. RSD for repeatability of measurement of peak area based on a 7 time measurement of 1 l/spot of sample extract was found to be 0.53 and 0.42% quercetin and gallic acid respectively. The limits of detection (LOD) and limit of quantification (LOQ) were determined to be 21.31 and 64.57 ng/spot for quercetin and 14.86 and 55.04 ng/spot for gallic acid respectively. Percentage recovery of quercetin and gallic acid were found to be in the range of 98.02–99.09 and 99.28–100.26 respectively, which indicated that the developed method was accurate and satisfactory. Summary of all validation parameters for the developed HPTLC method for simultaneous estimation of quercetin and gallic acid are listed in Table 1.
Please cite this article in press as: Patel, A.A., et al. Validated HPTLC method for simultaneous determination of quercetin and gallic acid in Leea indica. Revista Brasileira de Farmacognosia (2016), http://dx.doi.org/10.1016/j.bjp.2016.05.017
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Fig. 1. Densitometric chromatogram of hydrolysed methanol extract of Leea indica (A), reference standards gallic acid (B) and quercetin (C) scanned at 254 nm.
A 100.0
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Spectra comparison
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Fig. 2. UV Overlain spectra of gallic acid (A) and quercetin (B) standards and in test solution track scanned at 254 nm.
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The method was found to be precise, reliable, and suitable because the recovery in each case was more than 98%. Thus the present HPTLC method enables simple, rapid and accurate quantification of quercetin and gallic acid in plant of L. indica simultaneously.
Ethical disclosures Protection of human and animal subjects. The authors declare that the procedures followed were in accordance with the regula- Q4 tions of the relevant clinical research ethics committee and with
Please cite this article in press as: Patel, A.A., et al. Validated HPTLC method for simultaneous determination of quercetin and gallic acid in Leea indica. Revista Brasileira de Farmacognosia (2016), http://dx.doi.org/10.1016/j.bjp.2016.05.017
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those of the Code of Ethics of the World Medical Association (Declaration of Helsinki). Confidentiality of data. The authors declare that they have followed the protocols of their work center on the publication of patient data. Right to privacy and informed consent. The authors have obtained the written informed consent of the patients or subjects mentioned in the article. The corresponding author is in possession of this document. Authors’ contributions
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AAP (M. Pharm. student) contributed in collecting plant sample and identification, confection of herbarium, running the laboratory work, analysis of the data and drafted the paper. AAA and AHP contributed to chromatographic analysis. AAA contributed to critical reading of the manuscript. MBS designed the study, supervised the laboratory work and contributed to critical reading of the manuscript. All the authors have read the final manuscript and approved the submission.
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Conflicts of interest
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The authors declare no conflicts of interest. Uncited references
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Beck et al. (2003), Emran et al. (2012), Joshi et al. (2013), Nurhanan et al. (2008), Raihan et al. (2011), Raihan et al. (2012), Reddy et al. (2012), Saha et al. (2004), Saha et al. (2005), Saifuzzaman et al. (2013), Srinivasan et al. (2008), Srinivasan et al. (2010) and Temkitthawon et al. (2008).
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References
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Beck, O., Cartier, G., David, G.B., Dijoux-Franca, M.G., Mariotte, A.M., 2003. Antioxidant flavonoids and phenolic acids from leaves of Leea guinensis G. Don (Leeaceae). Phytother. Res. 17, 345–347. Biringanine, G., Chiarelli, M.T., Faes, M., Duez, P., 2006. A validation protocol for the HPTLC standardization of herbal products: application to the determination of acteoside in leaves of Plantago palmata Hook f.s. Talanta 69, 418–424.
Chatterjee, A., Prakashi, S.C., 1997. The Treatise on Indian Medicinal Plants, vol. 3. NISCAIR, New Delhi, pp. 174. Emran, T.B., Rahman, M.A., Hosen, S.M., Khanam, U., Saha, D., 2012. Antioxidant, cytotoxic and phytochemical properties of ethanol extract of Leea indica leaf. J. Pharm. Res. 5, 2938–2941. Gamble, J.S., 1956. Flora of Presidency of Madras, vol. 1, Part 2. Sri Gouranga Press, Calcutta, pp. 172. ICH, 1996. Q2B. Validation of Analytical Procedure: Methodology. International Conference on Harmonization, Geneva. ICH, 2005. Q2A. Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonization, Geneva. Joshi, A.B., Tari, P.U., Bhobe, M., 2013. Phytochemical investigation of the roots of Leea indica (Burm. F.) Merr. Int. J. Res. Pharm. Biomed. Sci. 4, 919–925. Khare, C.P. (Ed.), 2007. Indian Medicinal Plants, An illustrated Dictionary. , 1st ed. Springer-Verlag Berlin/Heidelberg, p. 367. Mukherjee, D., Kumar, N.S., Khatua, T., Mukherjee, P.K., 2010. Rapid validated HPTLC method for estimation of betulinic acid in Nelumbo nucifera (Nymphaeaceae) rhizome extract. Phytochem. Anal. 21, 556–560. Nurhanan, M.Y., Asiah, O., MohdIlham, M.A., Siti Syarifah, M.M., Norhayati, I., Lili, S.H., 2008. Antiproliferative activities of 32 Malaysian plant species in breast cancer cell lines. J. Trop. For. Sci. 20, 77–81. Rahman, M.A., Uddin, S.B., Wilcock, C.C., 2007. Medicinal plants used by Chakma tribes in hill tracts districts of Bangladesh. Indian J. Trad. Knowl. 6, 508–517. Raihan, M.O., Habib, M.R., Brishti, A., Rahman, M.M., Saleheen, M.M., Manna, M., 2011. Sedative and anxiolytic effects of the methanolic extract of Leea indica (Burm. f.) Merr. leaf. Drug Discov. Ther. 5, 185–189. Raihan, M.O., Tareq, S.M., Brishti, A., Alam, M.K., Haque, A., 2012. Evaluation of antitumor activity of Leea indica (Burm. f.) Merr. extract against Ehrlich ascita carcinoma (EAC) bearing mice. Am. J. Biomed. Sci. 4, 143–152. Reddy, N.S., Navanesan, S., Sinniah, S.K., Wahab, N.A., Sim, K.S., 2012. Phenolic content, antioxidant effect and cytotoxic activity of Leea indica leaves. BMC Complement. Altern. Med. 12, 128–134. Saha, K., Lajis, N.H., Israf, D.A., Hamzah, A.S., Khozirah, S., Khamis, S., Syahida, A., 2004. Evaluation of antioxidant and nitric oxide inhibitory activities of selected Malaysian medicinal plants. J. Ethnopharmacol. 92, 263–267. Saha, K., Lajis, N.H., Shaari, K., Hamzah, A.S., Israf, D.A., 2005. Chemical constituents of Leea indica (Burm. f.) Merr. (Leeaceae). Malays. J. Sci. 24, 75–78. Saifuzzaman, M., Hossain, M.A., Hossain, M.A., Ali, E.S., Islam, M.A., 2013. Evaluation of analgesic activity of ethanolic extract of Leea indica leaves. Int. J. Med. Pharm. Sci. Res. Rev. 1, 1–13. Srinivasan, G.V., Ranjith, C., Vijayan, K.K., 2008. Identification of chemical compounds from leaves of Leea indica. Acta Pharm. 1, 38–45. Srinivasan, G.V., Sharanappa, P., Vijayan, K.K., 2010. Comparison of antimicrobial activity and total phenols of the leaves and root of Leea indica and isolation of compounds from its root. Int. J. Biomed. Res. Anal. 1, 92–95. Stewart, J.L., Brandis, D., 1874. The Forest Flora of North-West and Central India. W. H. Allen & Co., London, pp. 101–102. Temkitthawon, P., Viyoch, J., Limpeanchob, N., 2008. Screening of phosphodiesterase inhibitory activity of Thai medicinal plants. J. Ethnopharmacol. 19, 214–217. Wagner, S., Urena, A., Reich, E., Merfort, I., 2008. Validated HPTLC methods for the determination of salicin in Salix sp. and of harpagoside in Harpogophytum procumbens. J. Pharm. Biomed. Anal. 48, 587–591.
Please cite this article in press as: Patel, A.A., et al. Validated HPTLC method for simultaneous determination of quercetin and gallic acid in Leea indica. Revista Brasileira de Farmacognosia (2016), http://dx.doi.org/10.1016/j.bjp.2016.05.017
210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263
ARTICLE IN PRESS
BJP 306 1–4
Revista Brasileira de Farmacognosia xxx (2016) xxx–xxx
www.elsevier.com/locate/bjp
Original Article
Validated HPTLC method for simultaneous determination of quercetin and gallic acid in Leea indica
1
2
3 4
Q1
Ankita A. Patel, Aeshna A. Amin, Arpit H. Patwari, Mamta B. Shah ∗ Department of Pharmacognosy, L. M. College of Pharmacy, Ahmedabad, Gujarat, India
5
6
a r t i c l e
i n f o
a b s t r a c t
7 8 9 10 11
Article history: Received 14 October 2015 Accepted 24 May 2016 Available online xxx
12 13 14 15 16 17 18 19
Keywords: Gallic acid HPTLC Leea indica Quercetin Simultaneous estimation Validation
A sensitive and reliable high performance thin layer chromatography method has been developed for the simultaneous estimation of quercetin and gallic acid in Leea indica (Burm. f.) Merr., Vitaceae. Ethyl acetate extract prepared from hydrolysed aqueous alcoholic extract (70%) was applied on silica gel G 60 F254 plate. The plate was developed using toluene-ethyl acetate-formic acid, 5:4:1 (v/v/v) as a mobile phase and detection and quantification were performed by densitometric scanning at 254 nm. The system was found to give well resolved bands for quercetin (Rf 0.63) and gallic acid (Rf 0.45) from other constituents present in the extract of L. indica. The correlation coefficient was found to be 0.991 and 0.999 with RSD, 0.97–1.23% and 0.1–1.13% for quercetin and gallic acid respectively in the developed method. The accuracy of the method was confirmed by conducting recovery studies at different levels using the standard addition method. The average recovery of quercetin and gallic acid was found close to 99% suggesting the accurateness of the method. The proposed validated HPTLC method offers a new, sensitive, specific and precise gauge for quantification of quercetin and gallic acid in L. indica.
Q2
© 2016 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
20
Introduction
Leea indica (Burm. f.) Merr., (Syn.: L. sambucina Willd., L. gigantea Griff., L. umbraculifera Cl., Stephylea indica Burm. f.) Merr., Vitaceae, 22 a shrub found to be growing in the forests of tropical and sub23 tropical countries including India, Sri Lanka, Malaysia, Thailand, 24 Andaman and Philippines (Stewart and Brandis, 1874; Gamble, 25 1956). In traditional systems of medicine the leaf is valued in ver26 tigo and diabetes, while the root as antidysentric, anthelmintic, 27 spasmolytic and sudorific and to treat cardiac and skin diseases 28 (Chatterjee and Prakashi, 1997; Khare, 2007; Rahman et al., 2007). 29 The leaf is reported to possess antioxidant (Syahida et al., 2004, Sim 30 et al., 2012), analgesic (Islam et al., 2013), antimicrobial (Vijayan 31 et al., 2010), phosphodiesterase inhibitory (Limpeanchob, 2008), 32 sedative, anxiolytic (Manna et al., 2011) and cytotoxic (Lili et al., 33 2008; Ling et al., 2012; Haque et al., 2012) activities. The plant 34 35Q3 is reported to contain ˛-tocopherol, -amyrin (Israf et al., 2005), lupeol, -sitosterol, ursolic acid, farnesol, phloridzin, gallic acid 36 (Vijayan et al., 2008), and quercitrin (Bhobe et al., 2013). Fla37 vanoids, kaempferol, quercetin, quercitrin, and mearnsitrin have 38 21
∗ Corresponding author. E-mail: [email protected] (M.B. Shah).
been reported from other species of genus Leea (L. guineense) (Mariotte et al., 2003). HPTLC is as an acquiescent and commonly used technique for qualitative and quantitative analysis of chemical markers in herbal raw materials. HPTLC has advantages of simplicity, sensitivity, accuracy, and is one the most approached technique for developing fingerprint and marker-based standardization of herbal drugs and is commonly applied not only for the identification, assay and testing for purity but also for stability, dissolution or content uniformity of raw materials and formulated products (Mukherjee et al., 2010). Taking in to consideration the non-existence of a routine method of analysis, a simple HPTLC method for quantitative estimation of quercetin and gallic acid simultaneously is proposed for L. indica. The proposed method was validated by specificity, range, linearity, accuracy, precision, detection limit, quantitative limit, and robustness according to the ICH guidelines (ICH, 1996, 2005).
39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
Materials and methods
56
Chemicals and solvents
57
Ethyl acetate, methanol, toluene, HCl, NaOH, distilled water used were of analytical grade. Reference standards quercetin and gallic acid (purity 99.9%) were purchased from Sigma–Aldrich.
http://dx.doi.org/10.1016/j.bjp.2016.05.017 0102-695X/© 2016 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Patel, A.A., et al. Validated HPTLC method for simultaneous determination of quercetin and gallic acid in Leea indica. Revista Brasileira de Farmacognosia (2016), http://dx.doi.org/10.1016/j.bjp.2016.05.017
58 59 60
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Plant material
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The fresh shrub of L. indica (Burm. f.) Merr., Vitaceae, was procured from Waghai Botanical Garden, Dang, Gujarat in the month of September 2012 and authenticated by the taxonomist of South Gujarat University, Surat, India. Voucher specimen of plant (LM 5412) has been deposited in Department of Pharmacognosy and Phytochemistry, L. M. College of Pharmacy, Ahmedabad. The fresh plant was cut, dried properly, powdered, and passed through 60# sieve. The powdered sample was stored in an airtight container at room temperature.
71
HPTLC analysis
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Quercetin and gallic acid standard stock solutions were prepared by dissolving 2 mg of the compound in methanol in 10 ml volumetric flask and volume was adjusted up to the mark with methanol, from this solution 1–5 l solutions was applied on precoated TLC plates for quercetin while 1–6 l was spotted for gallic acid for calibration purpose. Accurately weighed 10 g of plant powder was extracted exhaustively with ethanol (100 ml × 3) under reflux for 2 h/cycle. Ethanolic extract was evaporated to dryness under vacuum and residue was dissolved in 80 ml aqueous methanol (70%). The aqueous methanol solution was hydrolysed by using 40 ml 2 N HCl and 40 ml toluene and refluxing for 2 h. After neutralizing the solution by 5% NaOH, it was again refluxed for 1 h. The aqueous phase was successively extracted with toluene (2× 30 ml) and ethyl acetate (4× 50 ml). Ethyl acetate extract was evaporated to dryness and 10 mg of extract was dissolved in 5 ml methanol and 15 l of methanolic solution was applied to a TLC plate. Co-TLC study was conducted using silica gel G 60F254 plate and toluene-ethyl acetate-formic acid, 5:4:1 (v/v/v) as a mobile phase. The plates were observed under UV after derivatizing using Natural products-polyethylene glycol reagent (NP/PEG) for quercetin and 5% FeCl3 for gallic acid. HPTLC analysis was performed on 10 cm × 10 cm aluminum backed HPTLC plates coated with a 0.2 mm layer of silica gel G 60F254 (Merck, India). The plates were washed with methanol and activated in an oven at 110 ◦ C for 5 min prior to analysis. The standard and sample solutions were applied to the plates in form of bands of 5 mm long, 1 mm from bottom of the plate and 1 mm from the edge by means of automatic TLC sampler (CAMAG semiautomatic IV). The plates were developed using toluene-ethyl acetate-formic acid 5:4:1 (v/v/v) as mobile phase in CAMAG twin trough chamber, presaturated with mobile phase vapor for 30 min before development. After development, the plate was dried in air for 5 min and scanned immediately at 254 nm using TLC scanner (CAMAG scanner-III). A TLC visualizer was used for the photodocumentation of the plates.
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The method was validated as per ICH guidelines. Calibration curve for quercetin and gallic acid were obtained by plotting the peak area against their amount applied to the plate. The limits of detection (LOD) and quantification (LOQ) were determined by equation as per ICH guidelines. Instrumental precision was determined by repeated analysis of standard quercetin and gallic acid. Repeatability was assessed for each reference standard by applying three concentrations of these standards in triplicate on the plate, which were expressed in terms of percentage relative standard deviation (% RSD) and standard error (SE). The intra-day precision was determined at three different concentration levels of different markers, 200, 400 and 600 ng/spot, six times on the same day, and the inter-day precision was determined at three different concentrations of markers, 100, 300 and 500 ng/spot, six times on five
Table 1 Validation parameters for quercetin and gallic acid estimation. Parameters
Results Quercetin
Gallic acid
Correlation coefficient Linearity range (ng)
0.991 200–1000
0.999 200–1200
Precision (C.V.) Repeatability of measurement Repeatability of application Intra day Interday Limit of detection (ng/spot) Limit of quantification (ng/spot) Accuracy (%) Specificity
0.89 0.53 0.23–0.43 0.97–1.23 21.31 64.57 98.02–99.09 Specific
0.359 0.42 0.2–0.6 0.1–1.13 14.8 55.04 99.28–100.26 Specific
different interval days over a period of one week (Kumar et al., 2008). System suitability was tested by applying 600 ng/spot of quercetin and gallic acid standard solution to the plate seven times and measuring the relative standard deviation (RSD%) for the peak areas and RF. To study the accuracy and precision of the method, recovery studies were performed by the method of standard addition. The recovery of added standard was studied at two different levels, each being analyzed in a manner similar to that described for the assay (Biringanine et al., 2006; Wagner et al., 2008). Results and discussion Quercetin and gallic acid were quantified by HPTLC for the first time in L. indica. The identity of quercetin was confirmed by cochromatography and overlain absorption spectra with reference standard, when scanned at 254 nm. Also the bathochromic shift in UV spectra with AlCl3 /HCl relative to methanol spectra asserted presence of quercetin in the plant. The densitometric chromatogram of HPTLC fingerprint of the ethyl acetate extract is shown in Fig. 1. The peaks resolving at Rf 0.63 and 0.45 in test solution were found to be superimposing with those of respective standards of quercetin and gallic acid. Peak purity was studied by UV overlay spectra obtained from the standards and from the corresponding peaks from test (Fig. 2). The content of quercetin and gallic acid in plant was calculated on the basis of peak area and was found to be 0.13% and 0.041% (w/w) respectively. The calibration plots indicate that the peak-area response was a polynomial function of the amount of standards quercetin and gallic acid in the range 200–1000 ng and 200–1200 ng respectively. The correlation coefficient was found to be 0.991 and 0.999 with RSD, 0.97–1.23% and 0.1–1.13% for quercetin and gallic acid respectively in the developed method. Intraday and interday precision was calculated for the developed method by performing the analysis at three different levels on same day as well as on different days respectively. RSD for repeatability of measurement of peak area based on 7 times measurement of 600 ng/spot of standards quercetin and gallic acid were found to be 0.89 and 0.359% respectively. RSD for repeatability of measurement of peak area based on a 7 time measurement of 1 l/spot of sample extract was found to be 0.53 and 0.42% quercetin and gallic acid respectively. The limits of detection (LOD) and limit of quantification (LOQ) were determined to be 21.31 and 64.57 ng/spot for quercetin and 14.86 and 55.04 ng/spot for gallic acid respectively. Percentage recovery of quercetin and gallic acid were found to be in the range of 98.02–99.09 and 99.28–100.26 respectively, which indicated that the developed method was accurate and satisfactory. Summary of all validation parameters for the developed HPTLC method for simultaneous estimation of quercetin and gallic acid are listed in Table 1.
Please cite this article in press as: Patel, A.A., et al. Validated HPTLC method for simultaneous determination of quercetin and gallic acid in Leea indica. Revista Brasileira de Farmacognosia (2016), http://dx.doi.org/10.1016/j.bjp.2016.05.017
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Fig. 1. Densitometric chromatogram of hydrolysed methanol extract of Leea indica (A), reference standards gallic acid (B) and quercetin (C) scanned at 254 nm.
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The method was found to be precise, reliable, and suitable because the recovery in each case was more than 98%. Thus the present HPTLC method enables simple, rapid and accurate quantification of quercetin and gallic acid in plant of L. indica simultaneously.
Ethical disclosures Protection of human and animal subjects. The authors declare that the procedures followed were in accordance with the regula- Q4 tions of the relevant clinical research ethics committee and with
Please cite this article in press as: Patel, A.A., et al. Validated HPTLC method for simultaneous determination of quercetin and gallic acid in Leea indica. Revista Brasileira de Farmacognosia (2016), http://dx.doi.org/10.1016/j.bjp.2016.05.017
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those of the Code of Ethics of the World Medical Association (Declaration of Helsinki). Confidentiality of data. The authors declare that they have followed the protocols of their work center on the publication of patient data. Right to privacy and informed consent. The authors have obtained the written informed consent of the patients or subjects mentioned in the article. The corresponding author is in possession of this document. Authors’ contributions
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AAP (M. Pharm. student) contributed in collecting plant sample and identification, confection of herbarium, running the laboratory work, analysis of the data and drafted the paper. AAA and AHP contributed to chromatographic analysis. AAA contributed to critical reading of the manuscript. MBS designed the study, supervised the laboratory work and contributed to critical reading of the manuscript. All the authors have read the final manuscript and approved the submission.
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Conflicts of interest
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The authors declare no conflicts of interest. Uncited references
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Beck et al. (2003), Emran et al. (2012), Joshi et al. (2013), Nurhanan et al. (2008), Raihan et al. (2011), Raihan et al. (2012), Reddy et al. (2012), Saha et al. (2004), Saha et al. (2005), Saifuzzaman et al. (2013), Srinivasan et al. (2008), Srinivasan et al. (2010) and Temkitthawon et al. (2008).
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Please cite this article in press as: Patel, A.A., et al. Validated HPTLC method for simultaneous determination of quercetin and gallic acid in Leea indica. Revista Brasileira de Farmacognosia (2016), http://dx.doi.org/10.1016/j.bjp.2016.05.017
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