Ecdysteroids and bufadienolides from Helleborus torquatus (Ranunculaceae)

Three bufadienolides, hellebortin A (5-[beta-D-glucopyranosyloxy]-10,14,16-trihydroxy-19-nor-[5beta,10beta,14beta,16beta]-bufa-3,20,22-trienolide [1]), hellebortin B (5-[beta-D-glucopyranosyloxy]-3,4-epoxy-14-hydroxy-19-oxo-bufa-20,22-dienolide [2]) and hellebortin C (5-[beta-D-glucopyranosyloxy]-3,4-epoxy-10,14-dihydroxy-19-nor-bufa-20,22-dienolide [3]), together with 20-hydroxyecdysone 3-O-beta-D-glucoside (4) and 20-hydroxyecdysone (5) have been isolated by bioassay- and RIA-directed HPLC analyses of a methanol extract of the seeds of Helleborus torquatus. The structure and relative stereochemistry of the novel bufadienolide hellebortin A (1) and the structures of hellebortin B (2) and hellebortin C (3) were determined unambiguously by comprehensive analyses of their 1D and 2D NMR data. These five compounds are isolated from Hellborus torquatus for the first time. The biological activities of compound 1, 4 and 5 as ecdysteroid agonists and antagonists have been assessed.     

The recently proposed 20,22-epoxycholesterol as the intermediate in the conversion of cholesterol to pregnenolone by adrenal cortex mitochondria must be 5α,6α-epoxycholestan-3β-ol

Recently proposed 20,22-epoxycholesterol as the obligatory intermediate in the NADPH-dependent conversion of cholesterol to pregnenolone by bovine adrenal cortex mitochondria was isolated and found to be a misassigned metabolite. It was identified as 5α,6α-epoxycholestan-3β-ol by gas-liquid chromatography-mass spectrometry as well as by the reverse isotope dilution method. The thin-layer chromatographic behavior of the proposed 20,22-epoxide which had been reported to have a higher polarity than pregnenolone was in good accordance with that of the identified 5α,6α-epoxycholestan-3β-ol. All four diastereoisomers of 20,22-epoxycholesterol had much lower polarity than pregnenolone in thin-layer chromatograms obtained in various solvent systems. Endogeneous cholesterol, contained originally in the mitochondria, was also converted in the presence of NADPH to pregnenolone and 5α,6α-epoxycholestan-3β-ol as observed with radioactive cholesterol added as the exogeneous substrate.     

苍白秤钩风中脱皮甾酮类化学成分的研究

从中国广西药用植物苍白秤钩风(Diploclisia glaucescens)藤茎的95％乙醇提取物中分离得到5个脱皮甾酮类化合物(1-5)。通过NMR、MS以及化学沟通等方法分别将其结构鉴定为paristerone C-20,22-monoacetonide(1),paristerone(2),eodysterone(3),makisterone C(4)和capisterone(5)。化合物1为首次从自然界分得的天然产物,2,4,5亦为首次从该植物中分得。     

Bufadienolides and phytoecdystones from the rhizomes of Helleborus thibetanus (Ranunculaceae)

Two new bufadienolide glycosides with an A/B trans ring structure, 14β,16β-dihydroxy-3β-(β-d-glucopyranosyloxy)-5α-bufa-20,22-dienolide (1), and 14β,16β-dihydroxy-3β-[β-d-glucopyranosyl-(1→4)-(β-d-glucopyranosyloxy)]-5α-bufa-20,22-dienolide (2), two known ecdysteroids (polypodine B and 20-hydroxyecdysone) (3-4), and six known bufadienolide and its glycosides with 5β-OH (hellebrigenin, 16β-hydroxyhellebrigenin-3-O-α-l-rhamnoside, hellebrigenin 3-O-β-d-glucoside, hellebrin, 16β-hydroxyhellebrigenin-3-O-β-d-glucoside, and deglucohellebrin) (5-10) were isolated from the rhizomes of Helleborus thibetanus. The structures of compounds 1 and 2 were elucidated using various spectroscopic methods. All compounds were reported for the first time from the title plant and their chemotaxonomic significance for the genus Helleborus was discussed.     

Cholesterol side-chain cleavage by mitochondria from the human placenta. Studies using hydroxycholesterols as substrates

The side-chain cleavage of cholesterol by cytochrome P-450_scc in mitochondria from the human placenta was studied using hydroxycholesterol substrates and intermediates of the reaction. 25-Hydroxycholesterol inhibited 3β-hydroxy-5-pregnen-20-one (pregnenolone) production by placental mitochondria. It was converted to pregnenolone at a maximum velocity of only 19% of that for cholesterol. Addition of 20-hydroxycholesterol or 22R-hydroxycholesterol to placental mitochondria caused a lag in pregnenolone synthesis which was concentration dependent. Measurement of the concentration of 20,22R-dihydroxycholesterol during incubation of placental mitochondria with 22R-hydroxycholesterol revealed that the lag in pregnenolone production was caused by accumulation of 20,22R-dihydroxycholesterol. This intermediate of the reaction dissociated from the active site of cytochrome P-450_scc. Only after its concentration had increased, presumably to a level where it could compete with 22R-hydroxycholesterol for binding to cytochrome P-450_scc, was it converted to pregnenolone. These results indicate a lack of kinetic stabilization of the cytochrome P-450_scc-20,22R-dihydroxycholesterol complex with dissociation occurring more rapidly than the final hydroxylation. Similar measurements of side-chain cleavage of 22R-hydroxycholesterol by mitochondria from the bovine adrenal cortex showed that kinetic stabilization of the cytochrome P-450_scc-20,22R-dihydroxycholesterol complex does not occur in that tissue either. The relative hydroxylation rates of 20-hydroxycholesterol, 22R-hydroxycholesterol and 20,22R-dihydroxycholesterol indicate that all three hydroxylations catalysed by human cytochrome P-450_scc occur at approximately the same rate.     

Cytochrome P-450scc-catalyzed production of progesterone from 22R-hydroxycholest-4-en-3-one by way of 20,22-dihydroxycholest-4-en-3-one

Transient accumulation of a dihydroxylated steroid was found when 22R-hydroxycholest-4-en-3-one was used as the substrate for a reconstituted cholesterol side-chain cleavage system derived from bovine adrenocortical mitochondria. The indications were that the accumulated steroid was an intermediate in the cytochrome P-450scc-catalyzed reaction. The retention time of the accumulated intermediate was identical with that of authentic 20,22-dihydroxycholest-4-en-3-one on HPLC. When 22R-hydroxycholesterol and 22R-hydroxycholest-4-en-3-one were incubated simultaneously, the total amount of reaction products was essentially the same as that observed with 22R-hydroxycholest-4-en-3-one alone. Under the conditions employed, the apparent turnover number of cytochrome P-450scc for 22R-hydroxycholesterol was calculated to be 77 nmol/min/nmol P-450 from the amount of pregnenolone formed, whereas the apparent turnover number for 22R-hydroxycholest-4-en-3-one was 64 nmol/min/nmol P-450 with respect to the intermediate formation and 77 nmol/min/nmol P-450 with respect to the progesterone formation. The apparent turnover number for 20,22-dihydroxycholest-4-en-3-one was about 125 nmol/min/nmol P-450, which was not significantly different from that of 20,22-dihydroxycholesterol. The apparent Km for 22R-hydroxycholesterol was about 20 microM and those for 22R-hydroxycholest-4-en-3-one and 20,22-dihydroxycholest-4-en-3-one were 50 and 40 microM, respectively. Thus, 22R-hydroxycholest-4-en-3-one was efficiently metabolized to progesterone by way of 20,22-dihydroxycholest-4-en-3-one by cytochrome P-450scc.     

An improved synthesis of (20R,22R)-cholest-5-ene-3beta,20,22-triol, an intermediate in steroid hormone formation and an activator of nuclear orphan receptor LXR alpha.

Asymmetric dihydroxylation of (20(22)E)-cholesta-5,20(22)-dien-3beta-ol acetate (2a), prepared from pregnenolone, gave a 1:1 mixture (67% yield) of (20R,22R)-cholest-5-ene-3beta,20,22-triol 3-acetate (3a) and its 20S,22S isomer 3b. Highly purified 3a and 3b were obtained by semipreparative silver ion high performance liquid chromatography. Saponification of 3a and 3b gave (20R,22R)-cholest-5-ene-3beta,20,22-triol (4a) and its 20S,22S isomer 4b. This simple approach provided the natural isomer 4a more efficiently than previously described chemical or enzymatic syntheses. Full 1H and 13C nuclear magnetic resonance data were presented for triols 4a and 4b and their synthetic precursors. Side-chain conformations of 2a, its 20(22)Z isomer, 4a, and 4b were studied by molecular mechanics and nuclear Overhauser effect difference spectroscopy.     

华麻花头根中的蜕皮甾酮类成分

从华麻花头(Serratula chinensis S．Moore)根中分得7种蜕皮甾酮类化合物,经光谱分析和化学方法,分别鉴定为：20-羟基蜕皮松(1),podecdysone C(2),3-氧-乙酰基-20-羟基蜕皮松(3),20-羟基蜕皮松-20,22．-缩丁醛(4),shidasterone(5),atrotosterone C(6)和carthamosterone(7),其中20-羟基蜕皮松-20,22．缩丁醛为一新的化合物。     

山牡荆的化学成分研究

研究山牡荆根茎的化学成分。山牡荆根茎经80%丙酮提取,采用硅胶和凝胶进行分离纯化,通过波谱分析进行结构鉴定。从山牡荆中分离得到5个化合物,分别鉴定为:β-谷甾醇(1)、牡荆葡基黄酮(2)、20-羟基蜕皮素-20,22-单丙酮化合物(3)、3,5-O-双咖啡酰基奎宁酸(4)和胡萝卜甾醇(5),这5个化合物均为首次从该植物中分离得到。     

Synthesis of ponasterone A derivatives with various steroid skeleton moieties and evaluation of their binding to the ecdysone receptor of Kc cells

A series of ponasterone A (PNA) derivatives with various steroid moieties were synthesized to measure their binding activity to the ecdysone receptors of Drosophila Kc cells. The activity of compounds was evaluated by determining the concentration required to give the 50% inhibition (IC(50) in M) of the incorporation of [(3)H]PNA to Drosophila Kc cells. Compounds with no functional groups such as OH and CO group in the steroid skeleton moiety were inactive. By the introduction of functional groups such as the OH and the CO group in the steroidal structure, these compounds became active. Some compounds containing the A/B-trans ring fusion, which is different from that (A/B-cis) of ecdysteroids were also active. The oxidation of CH(2) at 6-position to CO, enhanced the activity 19 times, but the activity was erased by the reduction of oxo to OH group at 6-position. The activity was enhanced about 250 times by the conversion of A/B ring configuration from trans [(20R,22R)-2beta,3beta,20,22-tetrahydroxy-5alpha-cholestan-6-one: pIC(50)=4.84] to cis [(20R,22R)-2beta,3beta,20,22-tetrahydroxy-5beta-cholestan-6-one: pIC(50)=7.23]. The latter cis-type compound which is the most potent among compounds synthesized in this study was equipotent to the natural molting hormone, 20-hydroxyecdysone, even though it is 1/50 of PNA.     

Side-chain cleavage of C27-3-oxo-4-ene sterols

In this study various C27 sterols with a 3-oxo-4-ene structure were incubated with adrenal cortex mitochondrial preparations. (22R)-22-Hydroxy-4-cholesten-3-one and (20R,22R)-20,22-dihydroxy-4-cholesten-3-one were found to be converted into progesterone. This suggests the existence of a pathway for adrenal progesterone formation analogous to the normal 3 beta-hydroxy-5-ene pathways. (20S)-20-Hydroxy-4-cholesten-3-one was hydroxylated at C25. 4-Cholesten-3-one, 25-hydroxy-4-cholesten-3-one and (22S)-22-hydroxy-4-cholesten-3-one were not converted to a measurable extent. With 3-oxo-4-ene C27 sterols as substrates, the cholesterol side-chain cleaving enzyme system seems to require the presence of a 22R-hydroxyl group in the substrate. The clinical relevance of these observations is discussed.     

Influence of follicular maturation on luteinizing hormone-, cyclic 3',5'-adenosine monophosphate-, forskolin- and cholesterol-stimulated progesterone production in hen granulosa cells

The influence of follicular maturation on progesterone production by collagenase-dispersed hen granulosa cells was measured in short-term incubations. Granulosa cells of the largest follicle (F1) produced up to ten times more progesterone than cells from smaller follicles (F3-F5), not only in response to luteinizing hormone (LH), but also when stimulated by exogenous cyclic AMP or forskolin, both of which raise intracellular cyclic AMP levels by nonreceptor-mediated mechanisms. Moreover, when granulosa cell progesterone synthesis was stimulated by incorporating 25-hydroxy-cholesterol into the incubation medium, an identical pattern was obtained. This could be attributed to a corresponding increase in the specific activity of the mitochondrial cholesterol side-chain cleavage enzyme (20,22 desmolase). An increase in the apparent Vmax was observed without a change in the apparent Km values. Pregnenolone substrate at concentrations which raised progesterone production to levels similar to those observed in response to LH stimulation was utilized equally by granulosa cells of mature and developing follicles. However, at high pregnenolone concentrations, granulosa cells of mature follicles converted significantly more of the precursor to progesterone. Assay of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) showed that the enzyme has two Kms: a low Km present in cells of both mature and developing follicles, and a high Km found only in granulosa cells of more mature follicles. It is concluded that LH-promoted progesterone synthesis in granulosa cells of developing chicken follicles is restricted not so much by the availability of receptors and the competence of the adenylate cyclase/cyclic AMP system, but by the activity of key enzymes, principally the cholesterol-20,22 desmolase.     

Steroidal glycosides from the bulbs of Lilium dauricum.

The bulbs of Lilium dauricum yielded 11 compounds, including six new steroidal glycosides. The structures have been determined by spectral analysis and hydrolysis to be (25R,26R)-26-methoxyspirost-5-en-3 beta-ol 3-O-alpha-L-rhamnopyranosyl-(1----2)-O-[alpha-L-arabinopyranosyl-( 1----3)]- beta-D-glucopyranoside, (25R,26R)-26-methoxyspirost-5-en-3 beta-ol 3-O-alpha-L-rhamnopyranosyl-(1----2)-O-[beta-D-glucopyranosyl-(1----4)]- beta-D-glucopyranoside, (25R)-spirost-5-en-3 beta-ol (diosgenin) 3-O-alpha-L-rhamnopyranosyl-(1----2)-O-[alpha-L-arabinopyranosyl- (1----3)]-beta-D-glucopyranoside, (25R)-3 beta,17 alpha-dihydroxy-5 alpha-spirostan-6-one 3-O-alpha-L-rhamnopyranosyl-(1----2)-beta-D-glucopyranoside, (25R)-3 beta, 17 alpha-dihydroxy-5 alpha-spirostan-6-one 3-O-alpha-L-rhamnopyranosyl-(1----2)-O-[alpha-L- arabinopyranosyl-(1----3)]-beta-D-glucopyranoside and (20R,22R)-3 beta,20,22-trihydroxy-5 alpha-cholestan-6-one (tenuifoliol) 3-O-alpha-L-rhamnopyranosyl-(1----2)-beta-D-glucopyranoside. The absolute configurations of C-20 and C-22 of tenuifoliol were further confirmed by detailed analysis of the NOE difference spectrum of the corresponding isopropylidene derivative. Several known compounds were also isolated and identified.     

Ponasterone A: a new ecdysteroid from the embryos and serum of brachyuran crustaceans

The apolar ecdysteroid present in the developing embryo of the blue crab, Callinectes sapidus Rathbun, is tentatively identified as ponasterone A (2 beta, 14 alpha, 20,22-pentahydroxy-5, beta-cholest-7-en-6-one) on the basis of chromatographic, immunological, and mass spectral evidence. The apolar ecdysteroid present in the serum of land crabs, Gecarcinus lateralis, in the late premolt stages of the intermolt cycle is also tentatively identified as ponasterone A on the basis of chromatographic and immunological evidence.     

Additional minor ecdysteroid components of Leuzea carthamoides

Seventeen additional minor ecdysteroid compounds were isolated and identified from the roots of Leuzea carthamoides (Wild.) DC. Eight of them are new phytoecdysteroids: carthamoleusterone (13) is a new side-chain cyclo-ether with five-membered ring; 14-epi-ponasterone A 22-glucoside (12) is a rare and unusual natural 14 beta-OH epimer; 15-hydroxyponasterone A (11) is also new and rare with its C-15 substituted position, as well as 22-deoxy-28-hydroxymakisterone C (18) possessing secondary hydroxyl in position C-28 and 26-hydroxymakisterone C (20) with hydroxy groups in positions 25 and 26. New are also 1 beta-hydroxymakisterone C (21) and 20,22-acetonides of inokosterone (8) and integristerone A (10). Series of already known ecdysteroids: ecdysone (1), 20-hydroxyecdysone 2- and 3-acetates (3 and 4), turkesterone (6), inokosterone (7), 24-epi-makisterone A (14), and amarasterone A (22) are reported here as new constituents of L. carthamoides. Seven earlier reported Leuzea ecdysteroids: 20-hydroxyecdysone (2), ajugasterone C (5), integristerone A (9), 24(28)-dehydromakisterone A (15), 24(28)-dehydroamarasterone B (16), (24Z)-29-hydroxy-24(28)-dehydromakisterone C (17) and makisterone C (19) are also included because they are now better characterized.     

Protein destabilization by electrostatic repulsions in the two-stranded alpha-helical coiled-coil/leucine zipper.

The destabilizing effect of electrostatic repulsions on protein stability has been studied by using synthetic two-stranded alpha-helical coiled-coils as a model system. The native coiled-coil consists of two identical 35-residue polypeptide chains with a heptad repeat QgVaGbAcLdQeKf and a Cys residue at position 2 to allow formation of an interchain disulfide bridge. This peptide, designed to contain no intrahelical or interhelical electrostatic interactions, forms a stable coiled-coil structure at 20 degrees C in benign medium (50 mM KCl, 25 mM PO4, pH 7) with a [urea]1/2 value of 6.1 M. Four mutant coiled-coils were designed to contain one or two Glu substitutions for Gln per polypeptide chain. The resulting coiled-coils contained potential i to i' + 5 Glu-Glu interchain repulsions (denoted as peptide E2(15,20)), i to i' + 2 Glu-Glu interchain repulsions (denoted E2(20,22)), or no interchain ionic interactions (denoted E2(13,22) and E1(20)). The stabilities of the coiled-coils were determined by measuring the ellipticities at 222 nm as a function of urea or guanidine hydrochloride concentration at 20 degrees C in the presence and absence of an interchain disulfide bridge. At pH 7, in the presence of urea, the stabilities of E2(13,22) and E2(20,22) were identical suggesting that the potential i to i' + 2 interchain Glu-Glu repulsion in the E2(20,22) coiled-coil does not occur. In contrast, the mutant E2(15,20) is substantially less stable than E2(13,22) or E2(15,20) by 0.9 kcal/mol due to the presence of two i to i' + 5 interchain Glu-Glu repulsions, which destabilize the coiled-coil by 0.45 kcal/mol each. At pH 3 the coiled-coils were found to increase in stability as the number of Glu substitutions were increased. This, combined with reversed-phase HPLC results at pH 7 and pH 2, supports the conclusion that the protonated Glu side chains present at low pH are significantly more hydrophobic than Gln side chains which are in turn more hydrophobic than the ionized Glu side chains present at neutral pH. The protonated Glu residues increase the hydrophobicity of the coiled-coil interface leading to higher coiled-coil stability. The guanidine hydrochloride results at pH 7 show similar stabilities between the native and mutant coiled-coils indicating that guanidine hydrochloride masks electrostatic repulsions due to its ionic nature and that Glu and Gln in the e and g positions of the heptad repeat have very similar effects on coiled-coil stability in the presence of GdnHCl.     

Effects of restraint stress on plasma LH and testosterone concentrations, Leydig cell LH/hCG receptors, and in vitro testicular steroidogenesis in adult rats

We examined the effect of restraint stress (3 hr) on plasma LH and testosterone levels, on the Leydig cell LH/hCG receptor, and on the activity of enzymes in the testicular steroidogenic pathway of the adult rat. Restraint stress caused a 47% reduction in plasma testosterone concentrations, but had no effect on plasma LH levels. The binding capacity and affinity of Leydig cell LH/hCG receptors were not affected by restraint. Stress did not affect the testicular activity of 20,22 desmolase or 3 beta-hydroxysteroid dehydrogenase, but testicular interstitial cells of stressed rats incubated in vitro with progesterone as a substrate produced more 17 alpha-hydroxyprogesterone but less testosterone than control cells, and when incubated with 17 alpha-hydroxypregnenolone, produced 39% less androstenedione and 40% less testosterone than control cells. These results suggest that restraint stress inhibited 17,20 desmolase but not 17 alpha-hydroxylase activity. When the delta 4 pathway was blocked with cyanoketone (3 beta-HSD inhibitor), stress did not alter the production of pregnenolone or 17 alpha-hydroxypregnenolone, but the production of dehydroepiandrosterone by cells from stressed rats was subnormal, suggesting again a reduction of 17,20 desmolase activity. The data suggest that a major site of the inhibitory action of restraint stress on testicular steroidogenesis is the 17,20 desmolase step. The disruption of androgen production by restraint appears to be LH independent since stress did not affect plasma LH levels, the binding capacity or affinity of LH/hCG receptors, or the activity of 20,22 desmolase.     

Effect of natural and structurally modified bile acids on cholesterol metabolizing enzymes in rat liver microsomes. II

The effect of ursodeoxycholic acid analogues bearing modifications at the side-chain moiety of the molecule was tested on cholesterol 7 alpha-hydroxylase and HMG-CoA reductase in rat liver microsomes. The compounds included 23 R,S mixture and the single isomers 23R and 23S of 23 methylursodeoxycholic acid (23-methyl UDCA), the isomeric mixture (cis + trans) of 3 alpha,7 beta-dihydroxy-20,22-methylen-5 beta-cholan-23-oic acid (norcypro-UDCA) and the corresponding single isomers. Each steroid was added to liver microsomes as the sodium salt, at concentrations ranging from 25 to 200 microM. Isomers 23R and 23S of 23-methyl-UDCA inhibited cholesterol 7 alpha-hydroxylase in a concentration-dependent manner. The inhibitory capacity was similar for the two isomers. The extent of inhibition of the analogues was greater than that of the parent compound UDCA. Shortening of the side-chain in norcypro-UDCA resulted in a partial loss of the inhibitory effect, as compared to cypro-UDCA (3 alpha,7 beta-dihydroxy-22,23-methylen-5 beta-cholan-24-oic acid). None of these bile acid derivatives affected the activity of the enzyme HMG-CoA reductase.     

Pyridoxal phosphate inhibits the DNA-binding activity of the ecdysteroid receptor

The effect of pyridoxal 5'-phosphate on the binding of the ecdysteroid receptor from a nuclear extract of Drosophila melanogaster to DNA-cellulose was studied. The binding of hormone-receptor complexes to DNA-cellulose was completely blocked after a 30-min incubation with 3 mM pyridoxal 5'-phosphate at 0-4 degree C. The effect was specific for pyridoxal 5'-phosphate since related compounds (pyridoxal, pyridoxamine 5'-phosphate and pyridoxamine) were not effective or gave only 17% inhibition (pyridoxal). Under standard conditions, none of the compounds tested exerted a significant effect on the stability of [3H](20R,22R)-2 beta,3 beta, 14 alpha,20,22-pentahydroxy-5 beta-cholest-7-en-6-one ([3H]ponasterone A)-receptor complexes. The loss of DNA-binding activity caused by pyridoxal 5'-phosphate is accompanied by changes in the molecular properties of [3H]ponasterone-A-receptor complexes. A shift of [3H]ponasterone-A binding was observed from the 8.0-8.5 S to the 4.5-5.0 S region, when [3H]ponasterone-A-receptor complexes were exposed to pyridoxal 5'-phosphate during sucrose-gradient centrifugation. The inhibition of DNA-cellulose binding by pyridoxal 5'-phosphate can be reversed. Probably, pyridoxal 5'-phosphate forms a Schiff base with a critical lysine group of the ecdysteroid receptor, presumably at its DNA-binding site. The hormone-receptor complexes obtained after removal of pyridoxal 5'-phosphate had the same affinity for DNA-cellulose as 'native' complexes. DNA-cellulose-bound [3H]ponasterone-A complexes were efficiently eluted from DNA-cellulose with pyridoxal 5'-phosphate in 0.1 M KCl resulting in a 104-fold purification of the ecdysteroid receptor. The results reflect possible structural similarities between ecdysteroid and vertebrate steroid receptors.