Novel biflavonoids from the leaves of Leucaena diversifolia and Albizia procera and their protein binding efficiency

The leaves of Leucaena diversifolia and Albizia procera have been found to contain two novel birJavonoids, leucaedirJavone (1) and albipronavone (2) which were characterized as (5,2',3' -trihydroxy-5',6' -furano-7.,8-dimethylallyloxy)-(5,3' -dihydroxy-5' ,6' -fu rano-7,8-dimethylallyloxy)-6C -2' -biflavonyl and bis[5,3' ,4' -trihydroxy-2' -hydroxymethyl-5' ,6' -furano-8,7 -(3-hydroxyphenyi]-6C-6-billavonyl, respectively on the basis of spectral (UV, IR, MS, ID and 20 NMR) evidences. Both showed protein binding efficiency 201.81and 253.05 jlg BSA/mg respectively.

Flavonoidal oligomers are well known for their astringent nature causing feed deterrcncy for the herbivores 1 . In our programme on feed deterrent principles from tree leaves, we herein report for the first time the presence of novel biflavonoids, leucaediflavone and albiproflavone from Leucaena diversifolia and Albizia procera (Fam. : Leguminosae) leaves, respectively, and their protein binding efficiency.

Results and discussion
Leucaediflavone 1, was obtained as light brown amorphous solid m.p. 190°. It. responded positive test for fla-vonoid2 and negative test for sugar 3 . The UV spectrum gave an indication of flavonoid skeleton by showing absorption at 258, 270 and 348 nm 4 . The positive FABMS of 1 showed high molecular ion peak at m/z 767 [M + H]+ corresponding to formula C 44 H 30 o 13 in accordance with NMR data indicated it to be biflavonoid. TheIR spectrum of 1 exhibited absorption bands at 3460 (OH), 1650 (chelated carbonyl), 1363 (gem dimethyl group), 1510 and 1166cm-1 (fused furan ring). The UV spectral observations with diagnostic shift reagents (experimental) indicated that there were hydroxylation on 5,2',3' positions 4 . On the basis ofUV data the location of OH group could be assigned on C-5, C-3' in both the monomers of biflavonoid as well as an OH at C-2' in I unit. The pentahydroxy nature of 1 was also supported by the 1 H NMR, exhibiting a downfield chemical shift as a sharp singlet at 8 12.5 integrated for two protons of OH groups (0 2 0 exchangeable) 5 at C-5 in both monomeric units and broad hump between 8 8.1-9.0 suggested the presence of another three phenolic hydroxyl groups on B 392 rings at C-2', C-3'of unit I and C-3' of unit II (all op exchangeable)5. Diagnostic C-3 protons for biflavonoid structure were confirmed by the presence of singlet at 8 6.64 (2H). Location of three aromatic protons were assigned by the chemical shift appearing as singlet 8 7.37 for C-6 of unit II, and C-4' of both units. Gem dimethyl grouping adjacent to oxygen with olefinic cis protons were attributed dU'.! to proton signals at 8 1.21 (6H, s, 2 x Me), 0.83 (6H, s, 2 x Me), 7.4 (2H, d, 19Hz) and 6.8 (2H, d, 19 Hz) 6 . Presence of two equally shielded furan rings was confirmed by the presence of signals at 8 6.42 (2H, d, 1 2 Hz) and 6.16 (2H, d, J 2 Hz) 6 . In FABMS the molecular ion peak at mlz 349 due to double RDA and release of radical CH=C=O indicated linking of ring A of I unit to the ring B of II, and m/z 307 followed by appearance of mass ion at m/z 217 due to breaking interflavonyllinkage also supported the said structure. The substitution pattern of ring A and B as well as interflavonoid linkage were also established by HMBC experiment ( Fig. 1 ).
All the available features, with the absence of AB system in molecule and biosynthetic consideration 7 ted to conclude that the two chromene rings were attached on A rings of biflavonoids on C-7 and C-8 in both the units, and interflavonoid linkage was between C-6 and c .. of C-14. The fusion of 0 ring to 7 and 8 position of A ring was evident from observed cross peak in HMBC (Fig. 2). FABMS of 2 exhibited the fragment at m/z 14~ due to attachment of furan at 5' and 6' positions of B ring and peak at m/z 287 was due to presence of 0-I, 0-II ring fused with A-I, A-II at 7 and 8 positions. In the aliphatic region the presence of singlet at 8 2.04 (0 2 0 exchangeable), with a multiple at 8 3.7 led for assignment of CH 2 0H. It was further supported by existence of 2 J con-elation ship with C-2' and 3 1 with C-1' (Fig. 2). Thus albiproflavone 2 could be characterized as bis [5-3',4' -trihydroxy-2' -hydroxymcthyl-5' ,6'-furano-8, 7 -(3-hydroxyphcnyl)] -6C-6-biflavony I (Fig.   2). Protein binding efficiency : It was determined by the reported method 9 leucaediflavone 1 and albiprotlavone 2 showed the optimum protein binding efficiency as 201.81 l!g BSA/mg and 253.05 l!g BSA/mg, respectively thus show-ing the involvement of the dimeric flavone in protein binding similar to condensed tannin in woody plants.

Albiprollavone (2)
Experimental M.ps. were determined on Bock Monoscope and are uncorrected. UV spectra were recorded on a Unichem UV-2 spectrophotometer, and IR spectra on a Perkin-Elmer infra cord 157 spectrophotometer, NMR spectra (DMSO-d 6 ) on a Brucker 300 spectrometer (300 MHz) with TMS as internal standard and FAB mass spectra on a Jeol SX 102fDA-6000 spectrophotometer data system using argon/xenon (6 KV, 10 MA) as the FAB gas.
The leaves of L. divers1Jolia and A. procera were collected from the Central Research Farm of the Indian Grassland and Fodder Research Institute, Jhansi. (1) : Fresh leaves ( 4 kg) of L.diversifolia were homogenized with acetone water (7: 3) containing 0.1% ascorbic acid 2-3 times. The whole extract was left overnight and filtered. The total acetone extract was reduced to aqueous phase under vacuum. The resultant after washing with each Et 2 0 and EtOAc was diluted with methanol (I : I) to charge on Sephadex LH-20 column (equilibrated with 50% aq. methanol) and eluted with 50% methanol. The pooled eluants (50% methanol) were rechromatographed over Sephadex LH-20 column and eluted with methanol : water in different ratio. The fractions from methanol : water (3 : 7), were concentrated and lyophilized as leucaediflavone (1)