DIETARY SUPPLEMENTS Validated HPTLC Method for Quantitative Determination of Gallic Acid in Stem Bark of Myrica esculenta Buch.-Ham. Ex D. Don, Myricaceae KALPANA G. PATEL, VANDANA G. PATEL, KIRTI V. PATEL, and TEJAL R. GANDHI Anand Pharmacy College, Shri Ram Krishna Seva Mandal Campus, Near Town Hall, Anand, Gujarat, India A simple, rapid, and precise HPTLC method was developed for quantitative estimation of gallic acid in stem bark of Myrica esculenta, family Myricaceae. Separation was performed on silica gel 60F 254 HPTLC plates using toluene–ethyl acetate–formic acid–methanol (3 + 3 + 0.6 + 0.4, v/v/v/v) mobile phase for separation of the extracted components. The determination was carried out in the UV densitometric absorbance-reflection mode at 280 nm. The amount of gallic acid in free and combined form in the stem bark powder was found to be 0.276 and 0.541%, respectively, on a dry weight basis. The method was validated in terms of linearity, accuracy, precision, and specificity according to International Conference on Harmonization guidelines. Gallic acid response was found to be linear over a broad concentration range of 0.4–2.0 mg/band. LOD and LOQ were 0.103 and 0.312 mg/spot, respectively. The developed method is capable of quantifying amounts of gallic acid in stem bark powder of M. esculenta. F or the past few decades, compounds from natural sources have been gaining importance because of their vast chemical diversity. This has led to a phenomenal increase in the demand for herbal medicines in the last two decades, and a need to ensure the quality, safety, and efficacy of herbal drugs. Phytochemical evaluation is one of the tools for quality assessment, which include preliminary phytochemical screening, chemoprofiling, and marker compound analysis using modern analytical techniques (1–3). In the last two decades, HPTLC has emerged as an important tool for the qualitative, semiquantitative, and quantitative phytochemical analysis of herbal drugs and formulations. This includes developing HPTLC fingerprint profiles and determination of chemical markers and biomarkers. The major advantage of HPTLC is that numerous samples can be analyzed simultaneously using a small quantity of mobile phase (4–7). Myrica esculenta, family Myricaceae, is a small-to-moderate sized tree varying from 3 to 15 m from place to place in the subtropical Himalayas, from the Ravi River eastward to the Khasi, Jaintia, Naga, and Lushai hills of India at altitudes of 900–2100 m. The plant is known to possess varied medicinal properties, and its stem bark has various reported traditional uses for the treatment of asthma, fever, dyspnoea, throat, and lung affections, chronic bronchitis, and cough (8–10). The bark of M. esculenta is rich in tannin content belonging to the pyrogallol group (11). The stem bark also contains myricetin, myriconol, proanthocyanidin, sitosterol, friedelin, taraxerol, myricadiol, myricanone, and 13-oxomyricanol (12). Preliminary studies performed at our laboratory have established the efficacy of M. esculenta in decreasing bronchial hyperresponsiveness and bronchoconstriction through systematic studies on bronchoalveolar lavage, acetylcholine, and histamine-induced bronchospasm and lung cytology in guinea pigs (13, 14). A literature survey revealed that gallic acid and pyrogallol possess anti-inflammatory activity (15), and hence, further work was focused on identification and quantification of gallic acid in M. esculenta. Various HPLC and TLC methods for analysis of gallic acid in crude extracts have been reported (16–19). The objective of this work was to develop a simple, rapid, and cost-effective HPTLC method for the analysis of gallic acid in the stem bark of M. esculenta. Gallic acid is present both in free and combined form as gallotannin in bark. By hydrolysis with hydrochloric acid, gallotannin was converted to the free form of gallic acid followed by extraction with ethyl acetate and quantification by HPTLC. Shorter processing time, small sample volumes, a single optimized extraction step using inexpensive chemicals, and small mobile phase volumes are the inherent advantages of this method compared to HPLC. Additionally, this HPTLC method also involves estimation of the combined form of gallic acid by hydrolysis, making it superior to the reported HPLC and HPTLC methods. The published method was validated 1422 PATEL ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 93, NO. 5, 2010 Received November 11, 2009. Accepted by AP February 15, 2010. Corresponding author’s e-mail: kalpana_jpatel@yahoo.com Downloaded from https://academic.oup.com/jaoac/article/93/5/1422/5655783 by guest on 08 August 2022