Ten cis-clerodane-type diterpene lactones from Gutierrezia texana

Ten new 5α,10α-cis-clerodane-type diterpene lactones were isolated from the aerial parts of Gutierrezia texana. Using NMR techniques and some chemical transformations, the structures were established as 6α,18-dihydroxy-cis-cleroda-3,13(14)-diene-15,16-olide; 18,19-dihydroxy-cis-cleroda-3,13(14)-diene-15,16-olide; cis-cleroda-3,13(14)-diene-15,16:18,19-diolide; 18,19-epoxy-19α-hydroxy-cis-cleroda-3,13(14)-diene-15,16-olide; 3α,4:18,19-diepoxy-18β,19α-dihydroxy-cis-cleroda-13(14)-ene-15,16-olide; 3α,4-epoxy-19α-hydroxy-cis-cleroda-13(14)-ene-15,16:18,19-diolide; 3α,4:18,19-diepoxy-19α-hydroxy-cis-cleroda-13(14)-ene-15,16-olide; 3α,4β,19α-trihydroxy-18,19-epoxy-cis-cleroda- 13(14)-ene-15,16-olide; 19-O-α-L-arabinopyranosyl-cis-cleroda-3,3,13(14)-diene-15,16-olide-19-oic ester and 2β,6α-dihydroxy-cis-cleroda-3,13(14)-diene-15,16:18,6α-diolide. One of the structures was also confirmed by X-ray crystallographic analysis.     

The structure of leocardin,two epimers of a diterpenoid from Leonurus cardiaca

From the aerial part of Leonurus cardiaca a new labdane diterpenoid, leocardin, has been isolated and its structure was established by spectroscopic and chemical means. It has been shown to be 8β-acetoxy-9α,13α,15,16-bisepoxy-15-hydroxy-7-oxo-labdan-6β,19-olide present as a C-15 epimeric mixture.     

Plectranthoic acid,acetylplectranthoic acid and plectranthadiol,three triterpenoids from Plectranthus rugosus

Sitosterol and three new pentacyclic triterpenoids, plectranthoic acid, acetylplectranthoic acid and plectranthadiol, have been isolated from leaves of P. rugosus. From spectroscopic evidence and chemical behaviour the structure of plectranthoic acid was established as (19S)-3α-hydroxy-18α-urs-12-en-29β-oic acid and acetylplectranthoic acid is the 3α-acetyl derivative of this compound. Plectranthadiol is (19S)- 3α-hydroxy-18α-urs-12-en-29β-ol.     

Ent-kaurane derivatives from the root cortex of yacon and other three Smallanthus species (Heliantheae,Asteraceae)

The metabolites produced by the secretory canals of the root cortex from four Smallanthus species belonging to the yacon group were identified as ent-kaurane-type diterpenes. The dichloromethane root cortex extracts of the four species were treated with diazomethane and analyzed comparatively by GC–MS using a simple and rapid procedure which is very sensitive and reproducible permitting detection of minor components. In all cases, ent-16-kauren-19-oic acid (kaurenoic acid) methyl ester was the main component, differences being observed only in the minor components. The minor components identified were grandiflorenic acid methyl ester, ent-16-kauren-19-al, 16α,17-epoxy-15α-angeloyloxy-kauran-19-oic acid methyl ester and several O-acyl derivatives at C-15 or C-18 of kaurenoic acid. One of the minor components, 18-isobutyroyloxy-ent-kaur-16-en-19-oic acid is a new kaurenoic acid derivative. Grandiflorenic acid and 15-α-angeloyloxy-16,17-α-epoxy-ent-16-kauren-19-oic acid were present only in Smallanthus sonchifolius and Smallanthus siegesbeckius which showed very similar GC traces. The different GC profile of RC diterpenes from Smallanthus connatus and Smallanthus macroscyphus supports the view that they are different taxa. Some chemotaxonomic aspects of the genus Smallanthus and the subtribe Milleriinae are briefly discussed.     

Biologically active abietane and ent-kaurane diterpenoids and other constituents from Erythroxylum suberosum

Bioassay-guided fractionation of the ethanol extract from the branches of Erythroxylum suberosum, which was toxic to brine shrimp larvae, afforded five diterpenes bearing abietane and ent-kaurane-type skeletons from an active fraction. From these, four were new, 7-oxo-16-hydroxy-abiet-15(17)-en-19-al, 16-hydroxyabiet-15(17)-en-7-one, 7α,16-dihydroxy-abiet-15(17)-en-19-al and ent-12α-hydroxy-kaur-16-en-19-al, while methyl ent-7α,15β-dihydroxy-kaur-16-en-19-oate is reported for the first time as a natural product. This is also the first reported occurrence of abietane-type diterpenes in the genus Erythroxylum. The flavonoid ombuin-3-rutinoside was isolated from an inactive fraction, while rutin (quercetin-3-rutinoside) was obtained from the non-toxic ethanol extract of the leaves. The structures of the new and known compounds were established by analyses of 1D- and 2D-NMR and mass spectrometry data.     

Margotianin,a new diterpenoid from Margotia gummifera

From the roots and aerial parts of Margotia gummifera a new natural diterpenic methyl ester, margotianin, has been isolated. Its structure was established as methyl ent-7α-angeloxy-15α-acetoxy-atis-16-en-19-oate almost exclusively by ¹³C NMR spectroscopy.     

Triterpenes from Douglas fir sapwood

The saponified ether-soluble extractives of Douglas fir sapwood contained (24R)- 4α,14α,24-trimethyl-9β,19-cyclo-5α-cholestan-3β-ol(24R-cyclocucalanol),a new natural product; 4α,14α-dimethyl-9β,19-cyclo-24-methylene-5α-cholestan-3β- ol (cycloeucalenol); and (24R)-4α,24-dimethyl-5α-cholest-7-en-3β-ol (24R- methyllophenol); this is the first time they have been reported from Douglas fir.     

Biotransformation of 7α-hydroxy- and 7-oxo-ent-atis-16-ene derivatives by the fungus Gibberella fujikuroi

The microbiological transformation of 7α,19-dihydroxy-ent-atis-16-ene by the fungus Gibberella fujikuroi gave 19-hydroxy-7-oxo-ent-atis-16-ene, 13(R),19-dihydroxy-7-oxo-ent-atis-16-ene, 7α,11β,19-trihydroxy-ent-atis-16-ene and 7α,16β,19-trihydroxy-ent-atis-16-ene, while the incubation of 19-hydroxy-7-oxo-ent-atis-16-ene afforded 13(R),19-dihydroxy-7-oxo-ent-atis-16-ene and 16β,17-dihydroxy-7-oxo-ent-atisan-19-al. The biotransformation of 7-oxo-ent-atis-16-en-19-oic acid gave 6β-hydroxy-7-oxo-ent-atis-16-en-19-oic acid, 6β,16β,17-trihydroxy-7-oxo-19-nor-ent-atis-4(18)-ene and 3β,7α-dihydroxy-6-oxo-ent-atis-16-en-19-oic acid.     

Biotransformation of the diterpene 15β-hydroxy-18(4→3)-abeo-ent-kaur-4(19),16-diene by the fungus Fusarium fujikuroi

15β-Hydroxy-18(4→3)-abeo-ent-kaur-4(19),16-diene (4) was biotransformed by the fungus Fusarium fujikuroi into 3α,11β,15β-trihydroxy-18(4→3)-abeo-ent-kaur-4(19),16-diene (5). The hydroxylation at C-3(α) in this diterpene reminds a similar reaction that occurs at C-13 in the biosynthesis of gibberellic acid in this fungus. The presence of the 15β-alcohol in the substrate directs the second hydroxylation at C-11(β), which had been observed in the incubation of ent-kaur-16-ene derivatives with this fungus when the C-19 hydroxylation was inhibited by the existence in the molecule of a 3α-OH or 3-oxo group. We also show that the angelate of the substrate is an undescribed natural product now identified as a component of the plant Distichoselinum tenuifolium.     

Effect of ay-9944 on sterol biosynthesis in Chlorella sorokiniana

When Chlorella sorokiniana was grown in the presence of 4 ppm AY-9944 total sterol production was unaltered in comparison to control cultures. However, inhibition of sterol biosynthesis was shown by the accumulation of a number of sterols which were considered to be intermediates in sterol biosynthesis. The sterols which were found in treated cultures were identified as cyclolaudenol, 4α,14α-dimethyl-9β,19-cyclo-5α-ergost-25-en-3β-ol, 4α,14α-dimethyl -5α-ergosta-8,25-dien-3β-ol, 14α-methyl-9β,19-cyclo-5α-ergost-25-en-3β-ol, 24-methylpollinastanol, 14α-methyl-5α-ergost-8-en-3β-ol, 5α-ergost -8(14)-enol, 5α-ergost-8-enol, 5α-ergosta-8(14),22-dienol, 5α-ergosta-8,22-dienol, 5α-ergosta-8,14-dienol, and 5α-ergosta-7,22-dienol, in addition to the normally occurring sterols which are ergosterol, 5α-ergost-7-enol, and ergosta-5,7-dienol.The occurrence of these sterols in the treated culture indicates that AY-9944 is an effective inhibitor of the Δ⁸ → Δ⁷ isomerase and Δ¹⁴-reductase, and also inhibits introduction of the Δ²²-double bond. The occurrence of 14α-dimethyl-5α-ergosta-8,25-dien-3β-ol and 14α-methyl-9β,19-cyclo-5α-ergost -25-en-3β-ol is reported for the first time in living organisms. The presence of 25-methylene sterols suggests that they, and not 24-methylene derivatives, are intermediates in the biosynthesis of sterols in C. sorokiniana.     

A diterpenoid from Elaeoselinum foetidum

A new diterpene acid has been isolated from the roots of Elaeoselinum foetidum. Its structure was established as ent-7α-senecioxy-15α-hydroxy-atis-16-en-19-oic acid by ¹H NMR and ¹³C NMR spectroscopic studies of its methyl ester derivative and confirmed by correlation with a margotianin derivative.     

The fission yeast rpa17 + gene encodes a functional homolog of AC19, a subunit of RNA polymerases I and III of Saccharomyces cerevisiae

Eukaryotic RNA polymerases I and III consist of multiple subunits. Each of these enzymes includes two distinct and evolutionarily conserved subunits called α-related subunits which are shared only by polymerases I and III. The α-related subunits show limited homology with the α-subunit of prokaryotic RNA polymerase. To gain further insight into the structure and function of α-related subunits, we cloned and characterized a gene from Schizosaccharomyces pombe that encodes a protein of 17?kDa which can functionally replace AC19 – an α-related subunit of RNA polymerases I and III of Saccharomyces cerevisiae– and was thus named rpa17 ⁺. RPA17 has 125 amino acids and shows 63% identity to AC19 over a 108-residue stretch, whereas the N-terminal regions of the two proteins are highly divergent. Disruption of rpa17 ⁺ shows that the gene is essential for cell growth. Sequence comparison with other α-related subunits from different species showed that RPA17 contains an 81-amino acid block that is evolutionarily conserved. Deletion analysis of the N- and C-terminal regions of RPA17 and AC19 confirms that the 81-amino acid block is important for the function of the α-related subunits.     

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.     

Gibberellin A43 and other terpenes in endosperm of Echinocystis macrocarpa

By GC-MS the following acidic constituents of the endosperm of Echinocystis macrocarpa were identified: abscisic acid and its trans,trans-isomer, 4′-dihydrophaseic acid, GA₄, GA₇, iso-GA₇, GA₂₄, GA₂₅, two isomers of GA₁₃, GA₄₃, ent-6α,7α,17-trihydroxy-16αH-kauran-19-oic acid and ent-6α,7α, 16β, 17-tetrahydroxykauran- 19-oic acid. The structures of the last three new natural products were confirmed by partial synthesis. ent-Kaurene was detected in the neutral fraction.     

The microbiological transformation of some trachylobane diterpenoids by Gibberella fujikuroi

The microbiological transformation of ent-trachylobane, ent-7α-hydroxytrachylobane and ent-19-hydroxytrachylobane into trachylobagibberellins A₇, A₉, A₁₃, A₂₅, A₄₀ and A₄₇ by Gibberella fujikuroi is described. Whereas 7β-hydroxy- and 7β,18-dihydroxytrachylobanolides were obtained from ent-trachylobane and ent-trachyloban- 19-ol, the presence of a 7β-hydroxyl group directed metabolism exclusively into the gibberellin pathway. An 18-hydroxyl group as in ent-7α,18-dihydroxytrachylobane inhibited oxidation at C-6 affording ent-7α,18,19-trihydroxytrachylobane as the major metabolite.     

The microbiological transformation of epicandicandiol,ent-7α,18-dihydroxykaur-16-ene,by Gibberella fujikuroi

Incubation of ent-7α,18-dihydroxykaur-16-ene with Gibberella fujikuroi affords ent-7α,18,19-trihydroxykaur-16-ene and ent-7α,18-dihydroxykaur-16-en-19-oic acid. There was no transformation into 7,18-dihydroxykaurenolide.     

The potential of Cyathus africanus for transformation of terpene substrates

The insecticidal sesquiterpenes cadina-4,10(15)-dien-3-one and aromadendr-1(10)-en-9-one were administered to the fungus Cyathus africanus ATCC 35853. Biotransformation of the former produced (4R)-9α-hydroxycadin-10(15)-en-3-one, while the latter gave 2β-hydroxyaromadendr-1(10)-en-9-one, 2α-hydroxyaromadendr-1(10)-en-9-one and 10α-hydroxy-1β,2β-epoxyaromadendran-9-one. The bioconversion of santonin led to the production of two analogues, 11,13-dihydroxysantonin and the hitherto unreported 8α,13-dihydroxysantonin, while cedrol yielded 3β,8β-dihydroxycedrane and 3α,8β-dihydroxycedrane. Stemod-12-ene, a diterpene, was transformed to 2-oxostemar-13-ene, a hitherto unknown analogue with a rearranged carbon framework. When methyl betulonate, a triterpenoid belonging to the lupane family, was supplied to the fungus 18α-ursane and 18α-oleanane derivatives, namely 19β-hydroxy-3-oxo-18α-oleanan-28-oic acid and 19α-hydroxy-3-oxo-18α-ursan-28-oic acids, were generated. There are no previous reports of fungal transformation of a triterpene in which a skeletal rearrangement occurred. All substrate administration experiments were done in the presence of the terpene cyclase inhibitor chlorocholine chloride (CCC), using the single phase – pulse feed method.     

Kaurane and kaurene diterpenes from the stem bark of Xylopia acutiflora

Four diterpenes were isolated from the stem bark of Xylopia acutiflora and characterized as (?)-kauran-16α-ol, 7,8-acetoxy-(?)-kaur-16-en-19-oic acid, 15-oxo-(?)-kaur-16-en-19-oic acid, and 16α- hydroxy-(?)-kauran-19-oic acid.     

Neo-clerodane diterpenoids from Teucrium massiliense

From the aerial part of Teucrium massiliense three new neo-clerodane diterpenoids, deacetylajugarin-II, teumassilin and 6,19-diacetylteumassilin, have been isolated, besides the previously known diterpenes montanin C and teucjaponin A. The structures of deacetylajugarin-II (4α,18-epoxy-6α,19-dihydroxy-neo-clerodan-13-en-15,16-olide), teumassilin (4α,18:15,16-diepoxy-6α,12S,19-trihydroxy-neo-cleroda-13(16),14-diene) and 6,19-diacetylteumassilin (6α,19-diacetoxy-4α,18:15,16-diepoxy-12S-hydroxy-neo-cleroda-13(16),14-diene) were established by chemical and spectroscopic means. In addition, the previously known flavones salvigenin and cirsimaritin have also been obtained from the same source.     

cDNA cloning and bacterial expression of phospholipase A2 inhibitor PLIα from the serum of the Chinese mamushi, Agkistrodon blomhoffii siniticus1

The cDNA encoding of a phospholipase A₂ inhibitor (PLIα) of the Chinese mamushi, Agkistrodon blomhoffii siniticus, was identified from a liver cDNA library by use of a probe prepared by polymerase chain reaction (PCR) on the basis of the amino acid sequence of PLIα. It encoded a polypeptide of 166 amino acid residues, including 19 residues of the signal sequence and 147 residues of the complete mature sequence of PLIα. The PLIα cDNA was subcloned into the expression vector pET-16b and used to transform Escherichia coli strain BL21(DE3)pLysS. The recombinant PLIα expressed as a fusion protein was solubilized and purified to homogeneity by use of a metal affinity resin. The purified PLIα fusion protein underwent folding to form a trimeric structure like the intact PLIα, and showed inhibitory activity against the group II acidic PLA₂ from A. blomhoffii siniticus venom; although its binding constant (1/K_i) value was 30-fold lower than that of the natural PLIα. The elimination of the N-terminal additional peptide from the fusion protein resulted in a marked increase in the inhibition activity with a binding constant comparable to that of the natural PLIα against the acidic PLA₂. Furthermore, the carbohydrate chains of the natural PLIα were found to play an important role in the inhibitory activity against the basic PLA₂.