Biflavonoids from Lonicera japonica q Neeraj Kumar, Bikram Singh * , Pamita Bhandari, Ajai P. Gupta, Sanjay K. Uniyal, Vijay K. Kaul Natural Plant Products Division, Institute of Himalayan Bioresource Technology, Palampur, HP 176 061, India Received 4 August 2005; received in revised form 30 September 2005 Available online 15 November 2005 Abstract Two biflavonoids, 3 0 -O-methyl loniflavone [5,5 00 ,7,7 00 -tetrahydroxy 3 0 -methoxy 4 0 ,4 000 -biflavonyl ether (1)] and loniflavone [5,5 00 ,7,7 00 ,3 0 - pentahydroxy 4 0 ,4 000 -biflavonyl ether (2)] along with luteolin (3) and chrysin (4) were isolated from the leaves of Lonicera japonica. The structures were established on the basis of UV/vis, 1D, 2D NMR (HMQC and HMBC) and ESI-QTOF-MS/MS spectroscopic methods and chemical evidences. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Lonicera japonica; Caprifoliaceae; Biflavone; 3 0 -O-methyl loniflavone; Loniflavone 1. Introduction Lonicera (Caprifoliaceae) is a genus of more than 150 spe- cies of erect climbing or scrambling shrub distributed chiefly in the sub-tropical and temperate regions of the Northern Hemisphere. Approximately 45 species are known to grow in India and few species are used in indigenous system of medicine as an antipyretic, stomachic and diuretic and in dysentery (Wealth of India, 1962; Kirtikar and Basu, 1935). Lonicera japonica Thunb., Japanese honeysuckle, has been used to treat urinary disorders, fever and headache. It has been known as an anti-inflammatory agent in Korea from ancient times and is used widely for upper respiratory tract infections, diabetes mellitus and rheumatoid arthritis (Lee et al., 1998). A number of compounds such as iridoid glycosides, saponins, flavonoids and tannins have been reported from this species (Mehrotra et al., 1988; Kawai et al., 1988a,b; Machida et al., 2002; Kwak et al., 2003; Son et al., 1992, 1994a,b). Although the chemical composi- tion of L. japonica from few countries is well documented, there has been no report on the chemical composition from India. We, therefore, report here the isolation and structure elucidation of two new biflavonoids (1 and 2)(Fig. 1) together with luteolin (3) and chrysin (4) from the leaves. 2. Results and discussion Dried powdered leaves of L. japonica were extracted with MeOH–H 2 O (80:20; v/v) and fractionated successively with hexane, CHCl 3 , EtOAc, and n-BuOH. EtOAc extract was subjected to repeated chromatographic purification to yield two new biflavones (1 and 2) along with luteolin (3) and chrysin (4). Compound 1 was isolated as a yellowish amorphous powder. The presence of paired signals in 13 C NMR spec- trum (Table 1) suggested either a dimer or a mixture of two closely related compounds. However, negative HR-ESI- QTOF-MS showed a molecular ion peak at m/z 551.4558 [M À H] À and its positive HR-ESI-MS gave a molecular ion peak at m/z 553.4949 [M + H] + , consistent with a dimer and corresponding to molecular formula C 31 H 20 O 10 . The UV spectrum showed absorption maxima at 271 and 330 nm, characteristic of flavone derivatives (Mabry et al., 1970), addition of aluminium chloride produced a large bathochromic shift of band I (60 nm) 0031-9422/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2005.10.002 q IHBT Communication No. 543. * Corresponding author. Tel.: +91 1894 230426; fax: +91 1894 230433. E-mail address: bikram_npp@rediffmail.com (B. Singh). www.elsevier.com/locate/phytochem Phytochemistry 66 (2005) 2740–2744 PHYTOCHEMISTRY