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.     

Polyhydroxylated steroids from an endophytic fungus, Chaetomium globosum ZY-22 isolated from Ginkgo biloba

A novel polyhydroxylated C₂₉-sterol, 25ξ-methyl-22-homo-5α-cholest-7,22-diene-3β,6β,9α-triol, designated globosterol (1), together with one known tetrahydroxylated ergosterol (22E, 24R)-ergosta-7,22-diene-3β,5α,6β,9α-tetraol (2) has been isolated from the cultures of an endophytic fungus, Chaetomium globosum ZY-22 originated from the plant Ginkgo biloba. The structures and relative configurations of 1 and 2 were established on the basis of extensive spectroscopic analyses including 1D and 2D NMR (¹H-¹H COSY, HSQC, HMBC, and NOESY) experiments and comparison with the literature. Globosterol (1) possesses an unprecedented 25-methyl Δ²²-C₁₀-side chain and Δ⁷-3β,6β,9α-hydroxy-steroid nucleus, which represents the first example for C₂₉-steroids of the group.     

Three new triterpenes from the lichen Xanthoria resendei

The three new migrated hopene type triterpenes 12α-acetoxy-3β-hydroxyfern-9(11)-ene, 3β,12α-dihydroxyfern-9(11)-ene and 3,12-diketofern-9(11)-ene have been isolated from the ether extracts of the lichen Xanthoria resendei, together with peroxyergosterol and the anthraquinone pigments physcion, fallacinal and fallacinol.     

Incorporation of methionine-[methyl-2H3] and mevalonic acid-[2-14C,(4R)-4-3H1] into Phycomyces blakesleeanus sterols

Ergosterol, episterol, 4α-methyl-5α-ergosta-8,24(28)-dien-3β-ol and 24-methylene-24,25-dihydrolanosterol, isolated from Phycomyces blakesleeanus grown in the presence of methionine-[methyl-²H₃], each contained two deuterium atoms; lanosterol, however, was unlabelled. The ¹⁴C:³H atomic ratio of the following sterols isolated from P. blakesleeanus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁], was: ergosterol, 5:3; episterol, 5:4; ergosta-5,7,24(28)-trien-3β-ol, 5:3; 4α-methyl-5α-ergosta-8,24(28)-dien-3β-ol, 5:4; 24-methylene-24,25-dihydrolanosterol, 6:5; lanosterol, 6:5. The significance of these results in terms of ergosterol biosynthesis is discussed.     

Rare sesquiterpenes from South African Pteronia species

The genus Pteronia consists of approximately 80 species which are widely distributed in southern Africa. The essential oils isolated from the aerial parts of eleven species, analyzed by GC-MS varied both qualitatively and quantitatively. In Pteronia pallens, Pteronia empetrifolia and Pteronia flexicaulis uncommon sesquiterpenes such as presilphiperfolol-7-ene, 7-α-(H)-silphiperfol-5-ene, 7-β-(H)-silphiperfol-5-ene, α-campholene aldehyde, silphiperfol-5-ene, cameroonan-7-α-ol, silphiperfol-7-β-ol, presilphiperfolan-9-α-ol and presilphiperfolan-8-ol (a major compound in P. pallens) were identified. Cluster analysis based of the chemical composition of the oils revealed that individual plants of Pteronia camphorata collected in the same population had similar oil profiles with a high correlation coefficient (S_corr ≈ 0.98). Similarly, the essential oil composition of P. pallens collected from two distinct localities also showed high levels of congruency (S_corr ≈ 0.99).     

Triterpenoid saponins from Aesculus sylvatica W. Bartram

16 triterpenoid saponins including two new compounds were isolated from the seeds of A esculus sylvatica W. Bartram. The two new saponins were assigned as 3-O-[β-D-glucopyranosyl-(1 → 2)]-α-L-arabinofuranosyl-(1 → 3)-β-D-glucuronopyranosyl-21,22-O-ditigloyl-3β,16α,21β,22α,24,28 hexahydroxyolean-12-ene (aesculioside S1, 1) and 3-O-[β-D-glucopyranosyl-(1 → 2)]-α-L-arabinofuranosyl-(1 → 3)-β-D-glucuronopyranosyl-21-O-tigloyl-22-O-angeloyl 3β,16α,21β,22α,24,28-hexahydroxyolean-12-ene (aesculioside S2, 2). Aesculioside S1 and S2 displayed moderate cytotoxicity against human non-small cell lung cancer cells (A549) and prostate cancer cells (PC3) (GI₅₀ ranged from 8.7 to 18.2 μM). The structural analysis of the saponins isolated from Aesculus supports the taxonomic placement of A. sylvatica under the section Pavia of Aesculus genus.     

Pendulaosides A and B. Two acylated triterpenoid saponins from Harpullia pendula seed extract

Two new acylated triterpenoid saponins named pendulaosides A and B as well as the known phenolic compounds methyl gallate, gallic acid, 1,2,3,6-tera-O-galloyl-β-d-glucose and 1,2,3,4,6-penta-O-galloyl-β-d-glucose, were isolated from the seeds of Harpullia pendula. The structures of pendulaosides A and B were determined using extensive 1D and 2D NMR analysis and mass spectrometry as well as acid hydrolysis, as 3-O-β-d-glucopyranosyl-(1→2)-[α-L-arabinofuranosyl-(1→3)]-β-d-glucuronopyranosyl-22-O-angeloyl-3β,16α,22α,24β,28-pentahydroxylolean-12-ene and 3-O-β-d-glucopyranosyl-(1→2)-[α-L-arabinofuranosyl-(1→3)]-β-d-glucuronopyranosyl-16-O-(2-methylbutyroyl)-3β,16α,22α,24β,28-pentahydroxylolean-12-ene, respectively. To the best of our knowledge the two triterpene parts 22-O-angeloyl-3β,16α,22α,24β,28-pentahydroxylolean-12-ene and16-O-(2-methylbutyroyl)-3β,16α,22α,24β,28-pentahydroxylolean-12-ene have never been characterized before. The two isolated saponins were assayed for their in-vitro cytotoxic activity against the three human tumor cell lines HepG2, MCF7 and PC3. The results showed that pendulaoside A exhibited moderate activity on PC3 cell line with IC50value equal to 13.0 μM and weak activity on HepG2 cell line with IC50 value equal to 41.0 μM. Pendulaoside B proved to be inactive against the three used cell lines.     

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.     

New 19α-hydroxyursane-type triterpenes from the leaves of Meyna spinosa (= Vangueria spinosa), Rubiaceae

Two new 19α-hydroxyursane-type triterpenes, 2α,3α,19α,24,28-pentahydroxyurs-12-ene (1) and meyanthic acid, 3β-acetoxy-2β,19α,23-trihydroxyurs-12-en-28-oic acid (2) along with one new aliphatic ester, myricyl pentadecanoate (3) and five known compounds, 19α-hydroxyasiatic acid (4), oleanolic acid (5), myricyl alcohol (6), β-sitosterol (7) and its glycoside (8) were isolated from the methanolic leaf extract of Meyna spinosa Roxb. ex Link (= Vangueria spinosa Roxb., Rubiaceae). The structures of the new compounds were elucidated on the basis of extensive spectroscopic (including 2D NMR) analysis and comparison with literature. Except oleanolic acid, isolation of known compounds was reported for the first time from this plant.     

The conversion of 5α-lanost-24-ene-3β,9α-diol and parkeol into poriferasterol by the alga Ochromon as malhamensis

The 4,4-dimethylsterols 4α-lanost-24-ene-3β,9α-diol-[2-³H₂] and parkeol-[2-³H₂] were synthesized from lanosterol and subsequently incubated with cultures of Ochromonas malhamensis. 5α-Lanost-24-ene-3β,9α-diol was converted into poriferasterol with three times the efficiency of parkeol. Clionasterol was also found to be labelled from both parkeol and 5α-lanost-24-ene-3β,9α-diol. No significant incorporation of radioactivity into sterols was obtained after feeding 5α-lanost-24-ene-3β,9α-diol to higher plants, the chlorophyte alga Trebouxia, yeast or a cell free homogenate of rat liver.     

Sesquiterpenes and diterpenes from Ambrosia arborescens

Six compounds, eudesm-11(13)-en-4β,9β-diol, 15R,16-dihydroxy-3-oxoisopimar-9(11)-ene, 15S,16-dihydroxy-3-oxoisopimar-9(11)-ene, 1α-hydroxy-7-oxo-iso-anhydrooplopanone, 10α-hydroxy-11,13-dihydro-5-epi-psilostachyin, and 4β-hydroxypseudoguaian-12,6-olide 4-O-β-d-glucopyranoside, together with 12 known sesquiterpenes, were isolated from the leaves of Ambrosia arborescens. Structures were elucidated by 1D and 2D NMR spectroscopy including 1D-TOCSY, DQF-COSY, 2D-ROESY, HSQC, and HMBC experiments, as well as by ESI mass spectrometry. The absolute configuration of the 15,16-diol moiety in 15R,16-dihydroxy-3-oxoisopimar-9(11)-ene and 15S,16-dihydroxy-3-oxoisopimar-9(11)-ene was determined using Snatzke’s method. All compounds were evaluated for antiproliferative activity.     

Trichothecene mycotoxins from Fusarium sulphureum

Four mycotoxins isolated from moulded maize cultures of Fusarium sulphureum have been characterized as 3α,4β,15-triacetoxy-12,13-epoxytrichothec-9-ene, 4β,15-diacetoxy-3α-hydroxy-12,13-epoxytrichothec-9-ene, 15-acetoxy-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene and 4β-acetoxy-3α,15-dihydroxy-12,13-epoxytrichothec-9-ene.     

Rothmanniamide and other constituents from the leaves of Rothmannia hispida (K.Schum.) fagerl. (Rubiaceae) and their chemophenetic significance

The chemical investigation of the CH₂Cl₂/MeOH (1:1) extract of the leaves of Rothmannia hispida (K. Schum.) Fagerl. (Rubiaceae) led to the isolation of a new ceramide rothmanniamide (1) and a naturally isolated alkyl cinnamate derivative n-heptadecyl-4-hydroxy-trans-cinnamate (2), along with fifteen known compounds including lupeol palmitate (3), lupeol (4), a mixture of uvaol (5) and erythrodiol (6), ursolic acid (7), 30-nor-2α,3β-dihydroxyurs-12-ene (8), hederagenin (9), stigmast-22-en-3-ol (10), a mixture of β-sitosterol (11) and stigmasterol (12), stigmast-4,22-dien-3-ol (13), stigmasterol 3-O-β-D-glucoside (14), triacontan-1-ol (15), kaempferol 3-O-β-D-glucopyranoside (16) and D-mannitol (17). Their structures were elucidated with the help of MS and NMR data. Compounds 8, 10 and 15 were isolated for the first time from the Rubiaceae family. The crude extract and the isolates were assessed in vitro for their antileishmanial activity against Leishmania donovani 1 S (MHOM/SD/62/1 S) promastigotes and cytotoxicity on RAW 264.7 macrophage cells. Compounds 7 and 8 exhibited a highly potent antileishmanial activity with IC₅₀ values of 0.88 and 1.75 μg/mL, respectively, with good selectivity indexes (SI > 57). The chemophenetic significance of these compounds is also discussed.     

Bioconversion of C19- and C21-steroids with parent and mutant strains of Curvularia lunata

Regio- and stereospecificity of microbial hydroxylation was studied at the transformation of 3-keto-4-ene steroids of androstane and pregnane series by the filamentous fungus of Curvularia lunata VKM F-644. The products of the transformations were isolated by column chromatography and identified using HPLC, massspectrometry (MS) and proton nuclear magnetic resonance (¹H NMR) analyses. Androst-4-ene-3,17-dione (AD) and its 1(2)-dehydro- and 9α-hydroxylated (9-OH-AD) derivatives were hydroxylated by the fungus mainly in position 14α, while 6α-, 6β- and 7α-hydroxylated products were revealed in minor amounts. At the transformation of C₂₁-steroids (cortexolone and its acetylated derivatives) the presence of 17-acetyl group was shown to facilitate further selectivity of 11β-hydroxylation. Original procedures for protoplasts obtaining, mutagenesis and mutant strain selection have been developed. A stable mutant (M4) of C. lunata with high 11β-hydroxylase activity towards 21-acetate and 17α,21-diacetate of cortexolone was obtained. Yield of 11β-hydroxylated products reached about 90% at the transformation of 17α, 21-diacetate of cortexolone (1 g/l) using mutant strain M4.     

Non-stereo-selective cytosolic human brain tissue 3-ketosteroid reductase is refractory to inhibition by AKR1C inhibitors

Cerebral 3α-hydroxysteroid dehydrogenase (3α-HSD) activity was suggested to be responsible for the local directed formation of neuroactive 5α,3α-tetrahydrosteroids (5α,3α-THSs) from 5α-dihydrosteroids. We show for the first time that within human brain tissue 5α-dihydroprogesterone and 5α-dihydrotestosterone are converted via non-stereo-selective 3-ketosteroid reductase activity to produce the respective 5α,3α-THSs and 5α,3β-THSs. Apart from this, we prove that within the human temporal lobe and limbic system cytochrome P450c17 and 3β-HSD/Δ^5–4 ketosteroid isomerase are not expressed. Thus, it appears that these brain regions are unable to conduct de novo biosynthesis of Δ⁴-3-ketosteroids from Δ⁵-3β-hydroxysteroids. Consequently, the local formation of THSs will depend on the uptake of circulating Δ⁴-3-ketosteroids such as progesterone and testosterone. 3α- and 3β-HSD activity were (i) equally enriched in the cytosol, (ii) showed equal distribution between cerebral neocortex and subcortical white matter without sex- or age-dependency, (iii) demonstrated a strong and significant positive correlation when comparing 46 different specimens and (iv) exhibited similar sensitivities to different inhibitors of enzyme activity. These findings led to the assumption that cerebral 3-ketosteroid reductase activity might be catalyzed by a single enzyme and is possibly attributed to the expression of a soluble AKR1C aldo-keto reductase. AKR1Cs are known to act as non-stereo-selective 3-ketosteroid reductases; low AKR1C mRNA expression was detected. However, the cerebral 3-ketosteroid reductase was clearly refractory to inhibition by AKR1C inhibitors indicating the expression of a currently unidentified enzyme. Its lack of stereo-selectivity is of physiological significance, since only 5α,3α-THSs enhance the effect of GABA on the GABA_A receptor, whereas 5α,3β-THSs are antagonists.     

Two new cembranic diterpenoids from the flowers of Nicotiana tabacum L.

Two new cembranoids, together with fifteen known ones, were isolated from the flowers of Nicotiana tabacum L. The structures of the new compounds were established as (1βH,2E,4αOH,6αOH,7E,10αH,11αH,12βOH)-10,11-epoxy-2,7-cembradiene-4,6,12-triol (1) and (1βH,2E,4αOH,6αOH,7E,10βH,11βH,12αOH)-10,11-epoxy-2,7-cembradiene-4,6, 12-triol (2) by using spectroscopic analysis, including HRESIMS, IR, one- and two-dimensional NMR. A plausible biogenetic relationship of the isolated cembranoids was proposed. The antitumor activities of selected compounds against a panel of three human cancer HepG2, A549 and HCT-116 cell lines were evaluated by the MTT assay. Compound 5 exhibited moderate activity against Hep-G2 cell lines with an IC₅₀ value of 14.38 μM.     

Biosynthesis of ditigloyl esters of DI- and tri-hydroxytropanes in Datura

3α-Tigloyloxytropane-[¹⁴CO] [N-¹⁴Me], ratio 1·6:1 and valtropine-[¹⁴CO] [N-¹⁴Me], ratio 1·75:1 were separately fed via cotton wicks to 4-month-old Datura innoxia plants. After 8 days the root alkaloids 3α-tigloyloxytropane, 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol were isolated and the distribution of radioactivity in the acid and alkamine moieties was determined by hydrolysis. The precursor ratios were not maintained in the isolated ditigloyl esters, a result which does not support our hypothesis that the ditigloyl esters are formed by the progressive hydroxylation of 3α-tigloyloxytropane.     

Terpenoids and other constituents from Euphorbia bupleuroides

The phytochemical investigation of the roots of Euphorbia bupleuroides Desf. (Euphorbiaceae) yielded three new compounds named 4,20-dideoxy(4α)phorbol-12-benzoate-13-isobutyrate (1), 25-hydroperoxycycloart-3β-ol (2), and 3β,7β-dihydroxy-4α,14α-dimethyl-8β,9β-epoxy-5α-ergosta-24(28)-ene (3), together with 17 known compounds 4–20. Their structures were established from analysis of 1D (¹H, ¹³C and DEPT) and 2D NMR (COSY, HSQC, HMBC and NOESY) data, and of mass spectrometry (HRESIMS), and by comparison with literature data.     

Biosynthesis of the tigloyl esters in Datura: The role of 2-methylbutyric acid

Datura innoxia plants were wick fed with (±)-2-methylbutyric acid-[1-¹⁴C] and harvested after 7 days. The root alkaloids 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol were isolated and degraded. In each case the radioactivity was located in the ester carbonyl group indicating that this acid is an intermediate in the biosynthesis of tiglic acid from l-isoleucine. On the other hand, (±)-2-hydroxy-2-methylbutyric acid-[1-¹⁴C], which was fed to hydroponic cultures of Datura innoxia alongside isoleucine[U-¹⁴C] positive control plants, is not an intermediate.     

Bifunctional enzyme activity at the same active site: Competitive inhibition kinetics with 3α/20β-hydroxysteroid dehydrogenase

20β-Hydroxy-5α-pregnan-3-one (HPO) is a competitive inhibitor of reduction by 3a/20β-hydroxysteroid dehydrogenase (3α/20β-HSD; E.C.1.1.1.53) of 17β-hydroxy-5α-androstan-3-one (DHT; 3α-activity; K_i = 4.6 × 10^?5M) and of 6β-acetoxyprogesterone (6β-AP; 20β-activity; K_i = 4.34 × 10^?5M). HPO and DHT inhibit affinity alkylation of 3α/20β-HSD by 6β-bromoacetoxyprogesterone (6β-BAP). The facts that 1) enzyme 3α-activity and 20β-activity are both competitively inhibited by HPO with practically identical K_i-values, 2) 6β-BAP is solely a 20β-activity substrate for 3α/20β-HSD, 3) one mole of 6β-BAP reacts with one mole of 30/20β-HSD to simultaneously inactivate 3α- and 20β-activity and 4) inactivation of 3α/20β-HSD by 6β-BAP is inhibited by DHT (a Cig-steroid) or HPO (a C₂₁-steroid), support the view that the same active site of 3α/20β-HSD possesses both 3α- and 20β-activity. Bifunctional activity at the same active site is considered for other steroid-specific enzymes in female mammalian reproductive systems.