Biosci. Biotechnol. Biochem, 5 (7), 1686-1688, 2001 Note 3a Acetyl-CoA Carboxylase Inhibitors from Avocado (Persea americana Mill) Fruits Hironori Hasuimura, Chihoko Urpa, Jun Kawasara,’ and Takanori Kasat Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan Received November 17, 2000; Accepted March 19, 2001 A methanol extract of avocado fruits showed potent tory activity against acetyl-CoA carboxylase, a key enzyme in fatty acid biosynthesis. The active p ples were isolated and identified as (5E,12Z,15Z)- 2 hydroxy - 4 - oxoheneicosa - 5, 12, 15- trienyl (1), (2R,12Z,152)-2-hydroxy-4-oxoheneicosa-12,15-dieny! 2), QR" 4R")-2,4-dihydroxyheptadec-16-enyl (3) and QR" AR")-2,4-dihydroxyheptadec-16-ynyl_ (4) acetates by instrumental analyses. The ICs of the compounds were 4.0% 10%, 4.9% 10%, 9.4% 10-*, and 5.1% 10%, respectively. Key words: acetyl-CoA carboxylase inl do; long-chain fatty alcohol Acetyl-CoA carboxylase (ACC) is one of the key ‘enzymes in fatty acid biosynthesis. Hence, the inhibi- tors of ACC would have possibilities to suppress fat accumulation and avoid obesity. In the course of our search for ACC inhibitors in foodstulfs, epigallocate- chin gallate (EGCg) and 9-oxooctadeca-10,12- dienoic acids have been isolated as active principles from green tea” and red pepper,” respectively. In this article, we deal with isolation and characterization of ACC inhibitors from avocado (Persea americana Mill fruits ‘The edible parts of avocado (1.48 kg) were extract- ed with methanol (4 liter) at room temperature. The crude extract showed 133% relative inhibition against ACC at the concentration of the extract from 1g of fresh fruit in a 10-ml reaction mixture, where inhibitory activity is shown as the activity relative to that of Sms EGCg (ICj9=3.1% 10-*M) as 100%.” ‘The methanolic extract was partitioned between ethyl acetate and water, The resulting ethyl acetate-soluble fraction (16.5 g) was put through silica gel column chromatography (Wakogel C-200, Wako Pure Chem. Ind., 50350 mm) and eluted with a hexane- ethyl acetate gradient, The less polar active fraction (2.8 g) eluted with hexane-ethyl acetate (1:1) was re-chromatographed on a silica gel column (20x 420mm) and eluted with a chloroform- methanol gradient. The active fraction eluted with "To whom correspondence should be addressed. PAX: +81 chloroform-methanol (97:3) was further purified by normal-phase HPLC (Inertsil SIL 150-5, GL Science, 4.6% 250 mm; mobile phase, hexane-2-propanol 20:1); flow ‘rate, 1.0 ml/min; detection, UV at 235 nm). Two active peaks eluted at fx~11 and 19 min were collected. Both collected fractions were separately put through reverse-phase HPLC (Inertsil ODS-2, GL Science, 4.6% 250mm; mobile phase, methanol-water (9:1); flow rate, 1.0 ml/min; detec- tion, UY at 235 nm) to yield a major active principle (1, 41.6 mg, t=10 min) from the former fraction and the minor active principle (2, 3.8mg, t=2.5 min) from the latter. The physicochemical properties of the isolates indicated 1 to be (SE,12Z,15Z)-2-hydroxy-4-oxoheneicosa-5,12,15- trienyl acetate (persenone A), which has recently ‘been identified from the same plant as an inhibitor of nitric oxide and superoxide generation,” and 2 was (2R,12Z,152Z)-12-hydroxy-4-oxoheneicosa-12,15- dienyl acetate (persin), a well-known avocado costituent."” The absolute configuration of 2 was identified as R by enantioselective synthesis" although no experimental data concerning the stereochemistry of I has been reported. ‘On the other hand, the ACC inhibitory activity was also found in the more polar fraction. The hexane-ethyl acetate (1:4) eluate (1.2 g) of the first column was re-chromatographed on a silica gel column (chloroform-methanol). From the chlo- roform-methanol (97:3) eluates, further active princi- ples were purified by normal-phase HPLC (Inertsil SIL 150-5, GL Science, 4.6 250 mm; mobile phase, hexane-2-propanol (20:1); flow rate, 1.0 ml/min; detection, evaporating light scattering). Two active peaks eluted at te=22 and 23.5 min were obtained. Both collected fractions were separately put through reverse-phase HPLC (Inertsil ODS-2, GL Science, 4.6% 250 mm; mobile phase, methanol-water (9:1); flow rate, 1.0 ml/min; detection, evaporating light scattering) to yield 3 (4.2 mg, ¢,=7-5 min) from the former fraction and 4 (7.8 mg, fa=5 min) from the latter. The physicochemical properties of these iso- lates are as follows. 3: colorless oil; [aff ~ 14.0° ( 106.2496; E-mail: junk chem. agr hoki. ipAcety-CoA Carhoxslase Inhibitors from Avocado 1657 0.5, chloroform); FD-MS m/z (%) 329 (IMHI”, 100); "H-NMR 6 (chloroform-d): 1.24 (18H, m, 6-14-H:), 1.43-1.48 (2H, m, 5-H2), 1.50-1.58 (2H, m, 3-H:), 2.00-2.04 2H, m, 15-H3), 2.09 (s, Ac), 3.85-3.90 (IH, m, 4-H), 3.98 (IH, dd, J=11.9 and 4,5 Hz, one of 1-H), 4.07-4.11 (2H, m, one of 1-H and 2-H), 4.91 (IH, d, J=10.2 Hz, one of 17-H:), 4.97 (1H, d, J=17.1 Hz, one of 17-H:), 5.79 (1H, ddt, J= 17.1 (d), 10.2 (d) and 6.7 (8) Hz, 16-H) and 4: colorless oil; {ali} -18.0° (e=0.5, chloroform); FD- MS m/z (%6) 327 (IMH]*, 100); 'H-NMR 6 (chlo- roform-d): 1.25 (I8H, m, 6-14-H,), 1.44-1.52 QH, im, 5-H), 1.52-1.58 (2H, m, 3-H2), 1.92 (1H, t, 2.7 Hz, I7-H), 2.09 (s, Ac), 2.16 QH, dt, J=7.2 () and 2.7 (d) Hz, 15-H2), 3.85-3.90 (1H, m, 4-H), 3.98 (IH, dd, J= 12.0 and 4.5 Hz, one of I-H;), 4.07-4.11 (QH, m, one of 1-H; and 2-H). These data in addition to the 2D HH COSY results, clarified the structure of Band 4 as (2R*,4R*)-2,4-dihydroxyheptadec-16-enyl acetate” and (2R°,4R*)-2,4-dihydroxyheptadec-16- ynyl acetate,” respectively, both of which have also been found in avocado. The stereochemistry of 3 and 4 was identified as the relative configurations of 2R*.4R* although the absolute configurations are unknown, Measurements of ACC inhibitory activity were done as previously described." The inhibitory activity of the hydroxyoxoalkyl acetates 1 and 2 against ACC. were as high as ICw of 4.0% 10°" and 4.9% 10-*, respectively. The activity of 1 was 20 times higher than that of palmitic acid (ICjo=8.3 x 10 ‘M2? The dihydroxyalkyl acetates, 3 and 4, inhibited ACC with their ICy of 9.4% 10° and 5.1 x 10°* M, respective ly, slightly weaker than those of the hydroxyoxoalky! acetates. Catalytic hydrogenation of 4 with 5% pal- ladium carbon in methanol yielded a saturated diol acetate, (2R*,4R*)-2,4-dihydroxyheptadecy! acetate (3), FD-MS m/z (%) 331 (IMH]”, 100); 'H-NMR 5 (chloroform-d): 0.86 GH, t, J=6.9Hz, 17-Hs), 1.23-1.28 (22H, m, 6-16-H2), 1.42-1.49 QH, m, S-H;), 1511.61 QH, m, 3-H;), 2.09 (5, Ac), 3.85-3.89 (IH, m, 4-H), 3.98 (IH, dd, J=12.0 and 4.5 Hz, one of I-Hz), 4.07-4.11 (2H, m, one of I-Hy and 2-H). The ICs of 5 against ACC was 7.7 10°* M. Consequently, the terminal unsaturated bond of 3 and 4 can scarcely have an effect on the inhibitory ac- tivity. From the structural similarity to the isolated fatty alcohols, ACC inhibitory activity of monolinoleoyl glycerol (6) was further examined. ‘The result, ICy=7.2 10"*, showed the long-chain monoacyl glycerol had activity comparable to the avocado acetates. Since the ACC inhibitory activity of the isolates has little dependence on their structural variation, total activity of the crude avocado extracts would. ‘come from the mixture of these long-chain fatty alco- hols. Persin (2) and its related hydroxyoxoalkyl acetates are the constituents characteristic of avoca- do leaves and fruits and have been reported to show many biological activities, an growth inhibition against silkworms,” an antifungal activity,* causing ‘mastitis and agalactia for lactating livestock” and an inhibition of nitric oxide and superoxide generation.” However, the biological activity of dihydroxyalkyl acetates has only been reported concerning the an- tifungal activity of 3° other than the currently found ACC inhibitory activity, Acknowledgment We are grateful to Mr. Kenji Watanabe of GC-MS & NMR Laboratory, Faculty of Agriculture, Hokkaido University, for his skillful measurements of FD mass spectra. References 1) Watanabe, J., Kawabata, J., and Niki, R., Isolatio and identification of acetyl-CoA carboxylase inhibi tors from green tea (Camellia sinensis). Biosci Biotechnol. Biochem., 62, 532-534 (1998). 2) Watanabe, J., Kawabata, J., and Kasai, T., 9-Oxooe- tadeca-10,12-dienoic acids as acetyl-CoA carboxylase inhibitors from red pepper (Capsicum annuum L.)- Biosci, Biotechnol, Biochem., 63, 489-493 (199). 3) Kim, 0. K., Murakami, A., Nakamura, Y., Takeda, N., Yoshizumi, H., and Ohigashi, H., Novel nitric oxide and superoxide generation inhibitors, perse- none A and B, from avocado fruit. J. Agric. Food Chem, 48, 1557-1563 (2000), 4) Chang, C. “F., sogai, A., Kamikado, T.,Murakoshi, S., Sakurai, A., and Tamura, S., Isolation and stru ture elucidation of growth inhibitors for silkworm larvae from avocado leaves. Agric, Biol. Chem., 39, 1167-1168 (1975). 5) Prusky, D., Keen, N.., Sims, J. J., and Midland, S. L., Possible involvement of an antifungal diene in the lateney of Colletorricum gloeosporioides on unripe avocado fruits. Phytopathology, 72, 1578-15821658 ° a) H, Hasina era (1982). Prusky, D., Kobiler, I, Fishman, ¥., Sims, J. J., Midland, S. L. and Keen, N. T., Identification of an antifungal compound in unripe avocado fruits and its possible involvement in the quiescent infections of Colletotrichum gloeosporioides. J. Phytophathol., 132, 319-327 (1991), Oeltichs, P. B., Ng, J. C., Seawright, A. A., Ward, A., Schaller, L,, and MacLeod, J. K., Isolation and identification of a compound from avocado (Persea americana) leaves which causes necrosis of the acinar epithelium of the lactating mammary gland and the % » 10) myocardium. Natural Toxins, 3, 344-349 (1995) Macleod, J. and Schatfeler, L., A short enantioselec- tive synthesis ofa biologically active compound from Persea americana. J. Nat. Prod., 58, 1270-1273 (1995). Kashman, Y., Neeman, I., and Lifshitz, A., New compounds from avocado pear. Tetrahedron, 25, 4617-4631 (1969), Kashman, ¥., Neeman, 1, and Lifshitz, A., New compounds from avocado pear-II. Tetrahedron, 26, 1943-1951 (1970).