Occurrence of 3-epi-22-deoxy-20-hydroxyecdysone and its phosphoric ester in diapause eggs of the silkworm,Bombyx mori

Abstract

Abstract. Ecdysteroids in diapause eggs of the silkworm, Bornbyx mori, were analyzed using high-performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA). A relatively large amount of an unidentified free ecdysteroid and its phosphoric ester (conjugated form) were detected. These two compounds were isolated by a combination of column chromatography on silicic acid, thin-layer chromatography (TLC), and HPLC using a reverse-phase (RP) column. The purified compounds were identified as 3-epi-22-deoxy-20-hydroxyecdysone (22d20E') and 3-epi-22-deoxy-20-hydroxyecdysone 2-phosphate (22d20E'2P) by means of mass spectrometry and nuclear magnetic resonance spectroscopy. To our knowledge, this is the first report of 22d20E' and 22d20E'2P.Key words. Molting hormone; 3-epi-ecdysteroids; ecdysteroid conjugate; silkworm; Bombyx mori.The occurrence of large amounts of ecdysteroids in the ovaries and eggs of insects including the silkworm, Bombyx mori, has been welI documented ~-3, Ovaries and eggs thus serve as convenient materials for the isolation and identification of new species of ecdysteroids. In the ovaries of the Bombyx silkworm, the Ohnishi group identified six free ecdysteroids: 20-hydroxyecdysone (20E), ecdysone (E), 2-deoxy-20-hydroxyecdysone (2d20E), 2-deoxyecdysone (2dE), 2,22-dideoxy-20-hydroxyecdysone (2,22d20E) and bombycosterol (B), and their phosphoric ester (conjugated form), 20E 22-phosphate (20E22P), E22P, 2d20E22P, 22d20E3P, 2dE22P, 2,22d20E3P and B3P 4-7. We have recently isolated and identified six additional ecdysteroids in the ovaries and diapause eggs of the Bombyx silkworm, 22-deoxy-20-hydroxyecdysone (22d20E), 3-epi-ecdysone (E') and 3-epi-2-deoxyecdysone (2dE') and their phosphoric esters (22d20E3P, E'22P and 2dE'22P), and furthermore indicated the presence of several other unidentified ecdysteroids s,9. In the present communication we report the isolation and identification of two compounds detected in diapause eggs of the Bombyx silkworm, provisionally termed UKE (unknown ecdysteroids in the free eccysteroid fraction) and C-UKE (conjugated form of UKE). Materials and methodsInsects. Hybrid strains (Tokai-Fuyou, Tokai-Asahi, Kinshuu-Shouwa and Shunrei-Shougetu) of the silk-* To whom all correspondence should be addressed.worm, Bombyx mori, were used as a source of diapause eggs. Diapause eggs which were maintained for 10 days at 25 ~ after oviposition (embryos cease their development at the gastrula stage and enter diapause m) were used as the source of ecdysteroids for extraction. The eggs were stored at -80 ~ until extraction. Preparation of ecflysteroids for analytical high-performance liquid chromatography (HPLC). The procedure for extraction of ecdysteroids from diapause eggs was essentially the same as that for the preparation of ovarian ecdysteroids 8. Briefly, 10 g (wet wt) of diapause eggs were homogenized in 23.5 ml of 70% aqueous methanol and the homogenate was centrifuged. The supernatant was shaken with an equal volume of hexane to remove lipids. The methanol layer ...

Ecdysone 20-monooxygenase in eggs of the silkworm,Bombyx mori: Enzymatic properties and developmental changes

“…In the diapause eggs, the bulk of the ecdysteroids are conjugated forms (phosphoric esters), but in the non-diapause eggs free forms co-exist with the conjugated forms (Ohnishi et al, 1971(Ohnishi et al, 1977Mizuno et al, 1981;Mamiya et al, 1995;Sonobe et al, 1997). In the course of quantitative analysis of egg ecdysteroids in the silkworm, we found that the 20-hydroxyecdysone titer, which is thought to be a physiologically active molecule in the silkworm egg (Gharib et al, 1981a), in-creased sharply in the non-diapause eggs as embryonic development proceeded, but remained at a low level in the diapause eggs .…”

Ecdysone 20‐monooxygenase in eggs of the silkworm, Bombyx mori: Enzymatic properties and developmental changes

“…In the diapause eggs, the bulk of the ecdysteroids are conjugated forms (phosphoric esters), but in the non-diapause eggs free forms co-exist with the conjugated forms (Ohnishi et al, 1971(Ohnishi et al, 1977Mizuno et al, 1981;Mamiya et al, 1995;Sonobe et al, 1997). In the course of quantitative analysis of egg ecdysteroids in the silkworm, we found that the 20-hydroxyecdysone titer, which is thought to be a physiologically active molecule in the silkworm egg (Gharib et al, 1981a), in-creased sharply in the non-diapause eggs as embryonic development proceeded, but remained at a low level in the diapause eggs .…”

Novel Ecdysteroids fromSerratula wolffii

“…This compound was earlier isolated from diapause eggs of the silkworm Bombyx mori , but only partial 1 H NMR data supporting the proposed structure were reported [14]. The complete 1 H and 13 C NMR assignments are thus listed in Table 1.…”