Stereospecific reduction of progesterone by Pisum sativum

[4 -¹⁴C]-Progesterone was applied to the leaves of growing pea plants, Pisum sativum. After 3 weeks, about 50% of the administered steroid was reduced, about 20% being reduced to 5α-pregnane-3α,20β-diol as the major metabolite. The radioactivities of 5α-pregnane-3α,20α-diol and 5α-pregnane-3α,20β-diol after 3 weeks were more than twice those after one week. The following radioactive metabolises were also isolated: 5α-pregnane-3,20-dione; 20α-hydroxy-4- pregnen-3-one; 20β-hydroxy-4-pregnen-3-one; 3α-hydroxy-5α-pregnan-20-one; 3α-hydroxy-5β-pregnan-20-one; 3β-hydroxy- 5α-pregnan-20-one; 20β-hydroxy-5α-pregnan-3-one; 5α-pregnane-3β,20β-diol; and 5β-pregnane-3α,20β-diol. The radioactivities of the 5α-pregnane derivatives were considerably higher than those of the corresponding 5β-pregnane derivatives.     

Guianin: A neolignan from Aniba guianensis

The wood of Aniba guianensis Aubl. (Lauraceae) contains benzyl benzoate, benzyl salicylate, sitosterol, O-methyleugenol, O-methylisoeugenol and the neolignan guianin for which the structure of 1-allyl-8-hydroxy-3-methoxy-7-methyl-4-oxo-6-piperonylbicyclo[3,2,1]oct-2-ene (VI) is proposed.     

Metabolism and conjugation of [4-14C]progesterone by bovine liver and adipose tissues, in vitro

The ability of bovine liver and fat to metabolize progesterone and also to form glucuronide conjugates with these progestins $\text{in vitro}$ was investigated. Tissue supernatants were incubated with [4-¹⁴C] progesterone, UDP-glucuronic acid, and a NADPH generating system for 5 hr, at 37°C. Steroids were identified by thin-layer chromatography, high performance liquid chromatography, and recrystallization to a constant specific activity. The total original radioactivity which could not be removed by exhaustive ether extraction (presumptive conjugates) was 44.7 ± 14.2% in liver, 5.0 ± 3.6% in subcutaneous fat, and 3.7 ± 2.2% in kidney fat samples. Progestins identified in liver samples include 5β-pregnane-3α, 20α-diol (free and conjugate), 5β-pregnane-3α, 20β-diol (free and conjugate), 3α-hydroxy-5sB-pregnan-20-one (free and conjugate), 3β-hydroxy-5β-pregnan-20-one (free), 5β-pregnane-3, 20-dione (free), and progesterone (conjugate). Progestins identified in both the free and conjugate fractions of subcutaneous fat and kidney fat samples include progesterone, 3α-hydroxy-5β-pregnan-20-one, 20β-hydroxy-4-pregnen-3-one, and 20α-hydroxy-4-pregnen-3-one. Differences due to sex of bovine used were noted. These results confirm the ability of bovine liver to readily metabolize progesterone and form glucuronide conjugates of these compounds and suggest that adipose tissues take an active role in these actions in cattle.     

Terpenoids from seeds of Abies firma

Nine monoterpenoids, 16 sesquiterpenoids including two novel compounds, 12 diterpenoids and sitosterol were identified in seeds of Abies firma. Two new selinane-type compounds were isolated and their structures were determined as 4α-methoxy-selina-11-ene and 11-hydroxy-4α-methoxy-selinane on the basis of chemical and spectroscopic evidence.     

Metabolism of 5β-pregnane-3,20-dione and 3β-hydroxy-5β-pregnan-20-one by Digitalis suspension cultures

Digitalis purpurea normal callus suspension culture is capable of metabolizing 5β-pregnane-3,20-dione (1) to 3β-hydroxy-5β-pregnan-20-one (2), 3α-hydroxy-5β-pregnan-20-one (3), 3β-hydroxy-5β-pregnan-20-one glucoside (7) and 3α-hydroxy-5β-pregnan-20-one glucoside (8). Digitalis purpurea habituated callus suspension culture is also capable of metabolizing 1 to 2, 3, 5β-pregnane-3β,20β-diol (5), (7), (8), 5β-pregnane-3β,20α-diol monoglucoside (9) and 5β-pregnane-3α,20α-diol monoglucoside (11). Furthermore, it was observed that 3β-hydroxy-5β-pregnan-20-one (2) is converted to 7, 9 and 11 by both suspension cultures. At the same time, 1, 3, 5 and 8 were detected in normal callus, while 5β-pregnane-3β,20α-diol (4) and 5β-pregnane-3β,20β-diol monoglucoside (10) were present in the habituated callus culture.     

Non-reversibility of the isoleucine-acetate pathway in Datura

Five-month-old Datura meteloides plants were fed via the roots with 3-hydroxy-2-methylbutanoic acid-[1-¹⁴C] and isoleucine-[U-¹⁴C] as a positive control. After 5 days the plants were collected and in each case the root alkaloids 3α,6β-ditigloyloxytropane, 3α,6β-ditigloyloxytropan-7β-ol, meteloidine, hyoscine and hyoscyamine were isolated. Whereas isoleucine served as a precursor for the tiglic acid moieties 3-hydroxy-2-methylbutanoic acid did not.     

Triterpenoids from Mallotus repandus: Three new δ-lactones

The leaves of Mallotus repandus contain friedelin, 3β-hydroxy-13α-ursan-28,12β-olide (1), its benzoate (2) and ursolic acid. The stems contain friedelin, lupeol, α-amyrin, 2 and 3α-hydroxy-13α-ursan-28, 12β-olide (3), 21α-hop-22(29)-ene-3β,30-diol and ursolic acid. 1–3 are new compounds.     

Hypochaerin: a new sesquiterpene lactone from Hypochaeris setosus

Hypochaeris setosus contains desacetoxymatricarin, achillin, 1-hydroxy-6β,7α,11β-H-eudesm-4-en-6,12 olide, jacquinelin and hypochaerin, a new guaianolide, established as 3-oxo-4β,5α,6β,7α,11β-H-guai-1(2)-en-6,12 olide.     

Two diuretic triterpenoids from Antidesma menasu

Two new pentacyclic triterpenoids characterized as 16α-hydroxy-3-ketoisomultiflorene and 3β-hydroxy-16-ketoisomultiflorene have been isolated from the aerial parts of Antidesma menasu. Both of these compounds displayed diuretic activity in experimental animals.     

团花树皮的化学成分研究

采用硅胶、MCI和Sephadex LH-20层析方法对团花树皮的化学成分进行分离纯化,运用现代波谱技术鉴定了10个化合物:4-carboxy-3-hydroxy-5-methylphenyl 3-methoxy-4-hydroxy-5-methylbenzoate(1),谷甾醇-3-O-(6’-O-棕榈酰基)-β-D-葡萄糖苷(2),喹诺酸-3-O-α-L-鼠李糖苷(3),clethric acid(4),常春藤苷元(5),钩藤苷元C(6),morolic acid(7),咖啡酸甲酯(8),卡丹宾(9)和3α-二氢卡丹宾(10)。其中化合物1为一个新的酚性成分,化合物2～8首次从该属植物中分离得到。     

Myricetin and quercetin methyl ethers from Haplopappus integerrimus var. Punctatus

Nine flavonoids including two new myricetin derivatives, myricetin 3′,4′-dimethyl ether and myricetin 3,3′, 4′-trimethyl ether, were obtained from Haplopappus integerrimus var. punctatus. The known compounds are quercetin 7,3′-dimethyl ether, querectin 3,3′-dimethyl ether, isorhamnetin, quercetin 3,7-dimethyl ether, quercetin 3-methyl ether, quercetin and quercetin 3-β-d-glucoside.     

The identification and characterization of the c19o3 steroid metabolites of 5α-androstane-3β, 17β-diol produced by the canine prostate: 5α-androstane-3β, 6α, 17β-triol and 5α-androstane-3β, 7α, 17β-triol

This study has identified the polar metabolites of 5α-androstane-3β, 17β-diol(3β-diol) produced by the canine prostate. The major metabolite is 5α-androstane-3β, 7α, 17β-triol (7α-triol) accounting for approximately 80% of the total polar metabolites of 3β-diol. The remaining 20% is accounted for exclusively by another triol, 5α-androstane-3β, 6α, 17β-triol(6α-triol). This study has also characterized two enzymatic hydroxylases responsible for respective triol formation: 5α-androstane-3β, 17β-diol 6α-hydroxylase (6α-hydroxylase) and 5α-androstane-3β, 17β-diol 7α-hydroxylase (7α-hydroxylase). Both of these irreversible hydroxylases are located in the particulate fraction of the prostate and can utilize either NADH or NADPH as cofactor. Several $\text{in vitro}$ steroid inhibitors of these hydroxylases were identified including cholesterol, estradiol and diethylstilbestrol. Neither of the hydroxylases were found to be decreased by castration (3 months) when expressed as activity/DNA. Using a variety of C₁₉ androstane substrates, 6α- and 7α-triol were found to be major components of the total 3β-hydroxy-5α-androstane metabolites produced by the canine prostate.     

Identification of CYP3A4 as the major enzyme responsible for 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol in human liver microsomes

Human liver microsomes catalyze an efficient 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol. The hydroxylation is involved in a minor, alternative pathway for side-chain degradation in the biosynthesis of cholic acid. The enzyme responsible for the microsomal 25-hydroxylation has been unidentified. In the present study, recombinant expressed human P-450 enzymes have been used to screen for 25-hydroxylase activity towards 5β-cholestane-3α,7α,12α-triol. High activity was found with CYP3A4, but also with CYP3A5 and to a minor extent with CYP2C19 and CYP2B6. Small amounts of 23- and 24-hydroxylated products were also formed by CYP3A4. The V_max for 25-hydroxylation by CYP3A4 and CYP3A5 was 16 and 4.5 nmol/(nmol×min), respectively. The K_m was 6 μM for CYP3A4 and 32 μM for CYP3A5. Cytochrome b₅ increased the hydroxylase activities. Human liver microsomes from ten different donors, in which different P-450 marker activities had been determined, were incubated with 5β-cholestane-3α,7α,12α-triol. A strong correlation was observed between formation of 25-hydroxylated 5β-cholestane-3α,7α,12α-triol and CYP3A levels (r²=0.96). No correlation was observed with the levels of CYP2C19. Troleandomycin, a specific inhibitor of CYP3A4 and 3A5, inhibited the 25-hydroxylase activity of pooled human liver microsomes by more than 90% at 50 μM. Tranylcypromine, an inhibitor of CYP2C19, had very little effect on the conversion. From these results, it can be concluded that CYP3A4 is the predominant enzyme responsible for 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol in human liver microsomes.     

6α-Hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The V_max for 6α-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent K_m was 90 μM. Cytochrome b₅ was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6α-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r²=0.97) and testosterone 6β-hydroxylation (r²=0.9). There was also a strong correlation between 6α-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6β-hydroxylation (r²=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6α-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 μM concentration. Other inhibitors, such as α-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent K_m for 6α-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 μM). This might give an explanation for the limited formation of 6α-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver.     

Two 24-nor-triterpenoid carboxylic acids from acanthopanax trifoliatus

From Acanthopanax trifoliatus the new nor-triterpenes 24-nor-3α, 11α-dihydroxy-lup-20(29)-en-28-oic acid and 24-nor-11α-hydroxy-3-oxo-lup-20(29)-en-28-oic acid were isolated. Their structures were determined on the basis of spectroscopic data, X-ray analysis and chemical transformations.     

2-Carboxy-4-hydroxy-α-tetralone,a precursor for catalponol biosynthesis

2-Carboxy-4-hydroxy-α-tetralone (5) and its methyl ester (10) were incorporated into catalponol (1) in Catalpa ovata with retention of C-4 and C-8 tritium atoms. Incorporation of the former two substances into catalpalactone (2) and 4,9-dihydroxy-α-lapachone (12) was also demonstrated.     

7-Dehydrocholesterol-derived oxysterols and retinal degeneration in a rat model of Smith–Lemli–Opitz syndrome

Smith–Lemli–Opitz syndrome (SLOS) is a recessive disease characterized by markedly elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in tissues and fluids of affected individuals, due to defective 3β-hydroxysterol-Δ⁷-reductase (Dhcr7). Treatment of Sprague Dawley rats with AY9944 (an inhibitor of Dhcr7) leads to similar biochemical features as observed in SLOS. Eighteen oxysterols previously have been identified as oxidation products of 7-DHC (most of them distinct from cholesterol (Chol)-derived oxysterols) in solution, in cells, and in brains obtained from Dhcr7-KO mice and AY9944-treated rats, formed either via free radical oxidation (peroxidation) or P450-catalyzed enzymatic oxidation. We report here the identification of five 7-DHC-derived oxysterols, including 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), 4α- and 4β-hydroxy-7-DHC, 24-hydroxy-7-DHC and 7-ketocholesterol (7-kChol, an oxysterol that is normally derived from Chol), in the retinas of AY9944-treated rats by comparing the retention times and mass spectrometric characteristics with corresponding synthetic standards in HPLC-MS analysis. Levels of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol were quantified using d₇-DHCEO as an internal standard. Among the five oxysterols identified, only 7-kChol was observed in retinas of control rats, but the levels of 7-kChol in retinas of AY9944-rats were 30-fold higher. Intravitreal injection of 7-kChol (0.25 μmol) into a normal rat eye induced panretinal degeneration within one week; by comparison, contralateral (control) eyes injected with vehicle alone exhibited normal histology. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the retinal degeneration associated with the SLOS rat model and in SLOS patients.     

ω-ethyl, ω-ethenyl and ω-ethynyl-α-alkylidene-γ-lactones from Clinostemon mahuba

The trunk wood of Clinostemon mahuba contains eight (3R)-2-alkylidene-3-hydroxy-4-methylenebutanolides, seven (3R,4S)-2-alkylidene-3-hydroxy-4-methylbutanolides and seven (3S,4S)-2-alkylidene-3-hydroxy-4-methylbutanolides distinguished by the alkylidene side chains with respect to their E- or Z-geometry, ethenyl, ethynyl or ethyl terminals and lengths (C₁₆ or C₁₈).     

Flavans from Zephyranthes flava

A new optically active flavan aglucone, 7-hydroxy-3′,4′-methylenedioxyflavan, and its 7-glucoside have been isolated from the bulbs of Zephyranthes flava, collected at flowering. Additionally, two known flavans, 7,4′-dihydroxy-3′-methoxyflavan and 7-methoxy-2′-hydroxy-4′,5′-methylenedioxyflavan, have been isolated for the first time from this species. The structures of these flavans have been established by comprehensive analyses (UV, IR, ¹H NMR, ¹³C NMR, mass spectrometry, [α]_D) of the compounds and their acetates, and also by chemical correlation.     

Influence of dietary bile acids on formation of bile acids in rat

Various taurine-conjugated bile acids were fed to rats at the 1%-level in the diet for 3 or 7 days and the effect on several hydroxylations involved in the biosynthesis and metabolism of bile acids was studied. The hydroxylations studied were all catalyzed by the microsomal fraction of liver homogenate fortified with NADPH. The 7α-hydroxylation of cholesterol was inhibited by feeding taurocholic acid, taurocheno-deoxycholic acid and taurodeoxycholic acid for 3 as well as 7 days. No marked inhibition was obtained with taurohyodeoxycholic acid or taurolithocholic acid. The 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one was inhibited after 3 as well as 7 days by all bile acids except taurohyodeoxycholic acid. With this acid a marked stimulation of 12α-hydroxylation was observed. The effects of the different bile acids on the 7α-hydroxylation of taurodeoxycholic acid were not very marked. The 6β-hydroxylation of lithocholie acid and taurochenodeoxycholic acid was stimulated by taurocholic acid and taurodeoxycholic acid. The reaction was inhibited by taurochenodeoxycholic acid, at least after 7 days. Taurohyodeoxycholic acid inhibited the 6β-hydroxylation slightly and taurolithocholic acid had no effect. The results were discussed in the light of present knowledge concerning mechanisms of regulation of formation and metabolism of bile acids and it was suggested that the mechanisms may be more complex than previously thought.