A HPTLC densitometric determination of luteolin in Thymus vulgaris and its extracts
A. Bazylko ⁎, H. Strzelecka
Department of Pharmacognosy and Molecular Basis of Phytotherapy, Faculty of Pharmacy, Medical University of Warsaw, 1 Banach St., 02-097 Warsaw, Poland Received 22 March 2006; accepted 31 January 2007 Available online 5May 2007
Abstract Luteolin content in Thymus vulgaris and its extracts have been compared. Luteolin was separated on a thin- layer of silica gel with three-step gradient elution and determined by HPTLC-photodensitometry. The proposed method is simple and sensitive and can be used for the routine assay of luteolin in phytomedicines containing thyme extracts. © 2007 Elsevier B.V. All rightsreserved.
Keywords: High-performance thin-layer chromatography; Densitometry; Luteolin; Thymus vulgaris
1. Introduction Thymus vulgaris (Thyme) is commonly used in medicine as an antiseptic, bronchial spasmolytic and expectorant agent. The herb is used internally in upper respiratory tract disorders, and externally in skin disorders. The main compounds of herb of thyme are: volatile oil withthymol as a chief component [1,2], phenolic acids, including caffeic acid and rosmarinic acid [3–6] and flavonoids. More than twenty flavonoids were described in this herb [6–9]. The investigations on T. vulgaris in our Department led to TLC-photodensitometic methods of quantitative determination of thymol in T. vulgaris oleum  and of rosmarinic acid in extracts. Moreover two methoxylated flavoneshave been isolated from T. vulgaris liquid extract  and used as standards in TLC studies.
⁎ Corresponding author. Fax: +48 225720985. E-mail address: firstname.lastname@example.org (A. Bazylko). 0367-326X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2007.01.007
A. Bazylko, H. Strzelecka / Fitoterapia 78 (2007) 391–395
Preliminary studies (TLC,2D-TLC) of flavonoids in herb of T. vulgaris and its extracts show that luteolin is the main flavonoid constituent. Luteolin has antioxidant [12–15], anti-inflammatory and antiallergic [16,17] as well as spasmolytic and antispasmodic  activity. Moreover luteolin is considered an antimutagenic and anticancer agent [19–21]. For these reasons we have decided to develop a method aimed at thedetermination of the content of luteolin in T. vulgaris, and its liquid and dry extracts. 2. Experimental 2.1. Plant T. vulgaris L. (Lamiaceae), collected in June 2005, at the flowering stage in the South-west Region of Poland, near Piotrków Trybunalski (controlled plantation of PHYTOPHARM Kleka). A voucher sample (A0299) is deposited in the Department of Pharmacognosy, Medical University, Warsaw(Poland). 2.2. Extraction T. vulgaris, dried at 35 °C and powdered gave liquid extract (0.5:1) according to DAB X  and dry extract (from 6 parts of raw material 1 part of extract was obtained) according to FP VI monography . 2.3. Preparation of standard solutions A stock solution of luteolin (0.06 mg/ml) obtained from Carl Roth GmbH & Co. was prepared by dissolving 1.5 mg luteolin in MeOH andmaking up the volume to 25 ml with MeOH. 1, 2, 3 and 4 ml of the stock solution were transferred to 10 ml volumetric flasks and filled to the mark with MeOH to obtain standard solutions containing 6, 12,18, 24 μg/ml, respectively. 2.4. Samples preparation Two g of pulverized raw material in 50 ml of MeOH was heated under reflux for 30 min. After filtration the procedure was repeated four times.The combined extracts were concentrated to 25 ml and mixed with distilled water 1:1. The solution was extracted with 4 × 100 ml of petroleum ether. The aq. layer was concentrated to 1 ml, taken in a 10 ml volumetric flask and filled to the mark with MeOH. Two ml of this solution was taken in a 10 ml volumetric flask and filled to the mark with MeOH. Six samples of raw material were prepared. 0.067...