477 0009-3130/11/4703-0477  2011 Springer Science+Business Media, Inc. 1) Department of Pharmaceutical Technology, Faculty of Pharmacy, Tbilisi State Medical University, 22 G. Akhvlediani, Tbilisi, Georgia 0108, e-mail: annabozhadze@yahoo.com; 2) Laboratory of Alkaloids, Institute of Pharmacochemisty, 36 Sarajishvili street, Tbilisi, Georgia 0159; 3) Institute of Physical and Organic Chemistry, #5 Jikia str., Tbilisi, Georgia 0186. Published in Khimiya Prirodnykh Soedinenii, No. 3, pp. 424–426, May–June, 2011. Original article submitted March 5, 2010. Chemistry of Natural Compounds, Vol. 47, No. 3, July, 2011 [Russian original No. 3, May–June, 2011] ANALYTICAL STUDIES OF (+)-CHELIDONINE, PROTOPINE, AND l-STYLOPINE IN Chelidonium majus GROWING IN GEORGIA USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY Anna Bozhadze, 1* Vakhtang Mshvildadze, 2 Valentina Vachnadze, 2 UDC 547.944/945 Vladimer Tsitsishvili, 3 and Aliosha Bakuridze 1 Chelidonium majus L. (greater celandine, swallowwort) is a medicinal plant and is the only species in the genus Chelidonium of the Papaveraceae family. Ch. majus contains various isoquinoline alkaloids: protoberberine and compounds with tertiary and quaternary benzo (c) phenanthridine structure [1–8]. The commercial drug (herb of Chelidonium) consists of dried aerial parts harvested during flowering time and is described in several European pharmacopoeias [9]. The major components of the drug are the alkaloids chelidonine, chelerithrine, sanguinarine, coptisine, protopine, desstylopine, and similar compounds. The in vitro cytotoxic activity of these compounds on human and animal tumor cell cultures considered with great interest because they could lead to promising cancer treatments [10–12]. Earlier [13] it was shown that the aerial parts of Ch. majus growing in Georgia can serve as a good raw material for obtaining the equivalent crude drug and the purified components. The present investigation reports the isolation of (+)-chelidonine, protopine, and l-stylopine from roots and shoots of Ch. majus and their determination by HPLC in the crude extract of the aerial and underground parts of Ch. majus collected during and after flowering time. Plant Material. The plant material of Ch. majus was collected in April–May during the flowering phase and in June during seed ripening (in the experimental field of the Institute of Pharmacochemistry, Tbilisi, Georgia, 2008). A voucher specimen of the plant was deposited in the herbarium (# 281) of the Department of Pharmacobotany (head of the Department Sci. D. M. Churadze), Institute of Pharmacochemisty, Tbilisi, Georgia. Sample Preparation. The dried powdered aerial parts (each sample 400 g) and roots (200 g) of Ch. majus were extracted in a Soxhlet apparatus with ethanol. The obtained extract was evaporated under reduced pressure. The residues were dissolved in 0.25 M sulfuric acid and filtered. The acidic layer was alkalized to pH 9 with ammonia (25%). The solution was extracted with chloroform, and the organic fraction was dried with anhydrous sodium sulfate and filtered; the chloroform fraction was concentrated under vacuum. Samples for HPLC analyses were taken from the obtained crude extracts. Isolation of (+)-Chelidonine, l-Stylopine, and Protopine from the Crude Extract of Ch. majus. The crude alkaloid extract was isolated from Ch. majus (4.5 kg dried, powdered plant) by extraction in a percolator by the described method. The extract (10 g) was fractioned on a silica gel column (400 g). Elution was performed with chloroform followed by chloroform gradually enriched with methanol (98:2, 96:4, 92:8, 50:50). Elution was monitored by TLC. N O O O O H H CH 3 HO 1 3 4 13 6 7 9 1 N O O O O H 1 3 5 13 9 10 2 N O O O O O CH 3 3 9 10 4 3