Original Article DENSITOMETRIC VALIDATION OF LAPACHOL IN TECOMELLA UNDULATA SEEM BARK BY HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY RAJ RICHA 1 , SIDDIQUI NAHIDA, AERI VIDHU 2* Department of Pharmacognosy and Phytochemistry School of Pharmaceutical Education and Research, Jamia Hamdard, New Dehli 110062 Email: vidhuaeri@yahoo.com Received: 20 Apr 2018 Revised and Accepted: 06 Jun 2018 ABSTRACT Objective: To develop a validated high-performance thin layer chromatography (HPTLC) method for the quantitative estimation of Lapachol in a Soxhlet extracted bark of Tecomella undulata seem (T. undulata). Methods: The bark of T. undulata was extracted with chloroform by Soxhlet apparatus. The separation was achieved on a silica-gel 60 F254 HPTLC plate using toluene-ethyl acetate–glacial acetic acid (8.5:1.5:0.02 v/v/v) as a mobile phase. Densitometric analysis of Lapachol was carried out in absorbance mode at 254 nm. Results: The proposed method was accurate for the separation and resolution between peaks of the standard and Lapachol (5.04µg) with Rf value 0.77. Calibration curves were found to be linear over the concentration range (10-130µg) for Lapachol and correlation coefficient over (R 2 = 0.9973), indicating an excellent correlation between peak areas and concentrations of the marker compound. The experimentally derived LOD and LOQ for Lapachol were determined to be 0.028 µg and 0.086 µg respectively and the developed HPTLC-UV method showed lower %RSD and SEM, value indicating the method to be precise, accurate and robust. Conclusion: The study concludes that HPTLC-UV validation method can be very efficient and promising technique for the identification and quantitative analysis of Lapachol from T. undulata bark. The statistical analysis of data indicates that the developed method is reproducible and specific. Keywords: Tecomella undulata, Lapachol, High-Performance Thin Layer Chromatography (HPTLC) © 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) DOI: http://dx.doi.org/10.22159/ijpps.2018v10i7.26825 INTRODUCTION Tecomella undulata Seem (Bigoniaceae) is a tree species, locally known as Rohida found in Thar Desert regions of India and Pakistan, which is used as a hepatoprotective agent in various ayurvedic formulations such as Rohitakarishta, Rohitakadyachurna, Rohitakaghrita and Rohitakalauha [1]. T. undulata bark consists of various chemical constituents which are primarily used in the treatment of syphilis, painful swellings and cancer. Lapachol (fig. 1) is a naturally occurring 1, 4-naphthoquinone originally isolated from Tabebuia avellanedae (Bignoniaceae) [2], used against, Trypanosome cruzi, cancer, viruses, bacteria, and Leishmania [3]. This constituent can also be found in other plant families such as, Verbenaceae, Leguminosae, Sapotaceae, Scrophulariaceae, Malvaceae and Proteaceae, however, its occurrence is higher in the Bignoniaceae family, particularly in the gender Tabebuia, along with other heterocyclic quinones [3]. Naphthoquinones are natural aromatic compounds that can be found in several plant families, as well as isolated from fungi, algae and bacteria. Traditionally used for their dyeing properties, however, recently a variety of biological activities of these compounds has been reported [3]. They are also categorized in a class of natural phenolic compounds formed on a C6-C4 skeleton. Due to its versatile pharmacological activity, it is isolated and analyzed from natural sources using a simple, efficient and reliable technique. Conventional Soxhlet extraction (CSE) has been used for a long time, especially because of its low cost and easy applicability as compared to the available novel techniques. Extraction of any kind of secondary metabolite by using a wide range of solvents is the added advantage of Soxhlet extraction [17]. Currently, HPTLC is often used as an alternative to HPLC for the quantification of plant products because of its simplicity, accuracy, cost-effectiveness and rapidity [17]. In this investigation, a validated high-performance thin layer chromatography (HPTLC) method was developed for the quantitative estimation of Lapachol in a Soxhlet extracted bark of (T. undulata). Fig. 1: Chemical structure of Lapachol MATERIALS AND METHODS Chemicals and reagents Standard Lapachol were purchased from Sigma Aldrich fine chemicals, chloroform and methanol (HPLC grade) were used for the preparation of standard and sample and toluene: ethyl acetate-glacial acetic acid (8.5:1.5:0.02v/v/v) were used as a mobile phase for HPTLC analysis. All solutions used for analysis were filtered through a 0.1 µm filter. Stem bark of T. undulata was collected from Lodhi Garden, New Delhi. The samples were authenticated by the National Institute of Science Communication and Information Resources (NISCAIR), New Delhi, India, reference no. NISCAIR/RHMD/Consult/2014/2472-51. For HPTLC analysis of Lapachol, an HPTLC system from Chemie Erzeugnisse and Adsorptions Technik (CAMAG) AG (Muttenz, Switzerland) was used, equipped with a Linomat IV sample applicator. Lapachol extract was applied to aluminum, baked TLC plates (20X20 cm) precoated with silica gel 60 F254 (Merck, Darmstadt, Germany). For developing TLC twin-trough CAMAG (vertical development) chamber was used. Extraction of plant material The plant material was collected from Lodhi Garden, New Delhi. Dried fine plant material was subjected to Soxhlet extraction with chloroform for 8 h at 37 ℃ [11]. International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 10, Issue 7, 2018