Natural Products & Metabolomics

Morphine is a plant (opium poppy)-derived alkaloid and one of the strongest known analgesic compounds. Studies from several laboratories have suggested that animal and human tissue or fluids contain trace amounts of morphine. Its origin in mammals has been believed to be of dietary origin. Here, we address the question of whether morphine is of endogenous origin or derived from exogenous sources. Benzylisoquinoline alkaloids present in human neuroblastoma cells (SH-SY5Y) and human pancreas carcinoma cells (DAN-G) were identified by GC/tandem MS (MS/MS) as norlaudanosoline (DAN-G), reticuline (DAN-G and SH-SY5Y), and morphine (10 nM, SH-SY5Y). The stereochemistry of reticuline was determined to be 1-(S). Growth of the SH-SY5Y cell line in the presence of 18O2 led to the [18O]-labeled morphine that had the molecular weight 4 mass units higher than if grown in 16O2, indicating the presence of two atoms of 18O per molecule of morphine. Growth of DAN-G cells in an 18O2 atmosphere yielded norlaudanosoline and (S)-reticuline, both labeled at only two of the four oxygen atoms. This result clearly demonstrates that all three alkaloids are of biosynthetic origin and suggests that norlaudanosoline and (S)-reticuline are endogenous precursors of morphine. Feeding of [ring-13C6]-tyramine, [1-13C, N- 13CH3]-(S)-reticuline and [N-CD3]-thebaine to the neuroblastoma cells led each to the position-specific labeling of morphine, as established by GC/MS/MS. Without doubt, human cells can produce the alkaloid morphine. The studies presented here serve as a platform for the exploration of the function of “endogenous morphine” in the neurosciences and immunosciences.

New, partially acetylated dihydroxy fatty acids could be identified in the floral oil of Malpighia coccigera (Malpighiaceae): 7‐OAc,3‐OH 20 : 0, 7‐OAc,3‐OH 22 : 0, 9‐OAc,3‐OH 22 : 0, 9‐OAc,5‐OH 22 : 0, 3,9‐diOAc 22 : 0, 9‐OAc,3‐OH 24 : 0 , and 11‐OAc,5‐OH 24 : 0 . The substitution patterns of all hitherto undescribed dihydroxylated and additionally identified monohydroxylated fatty acids are in agreement with a polyketide analogous biosynthesis. Intermediates may be 3‐acetoxy fatty acids (C16, C18, and C20), known from flower secretions of other phylogenetically unrelated plant families. A possible relationship between plant epicuticular wax and floral oil biosynthesis is discussed. It may explain why an independent but convergent development of oil flowers and flower oils in unrelated plant families was possible.

Eleven collections of Cladonia furcata and fourteen of Cladonia rangiformis, mainly from Saxony Anhalt (Germany) have been analyzed for their lichen substances by HPLC and HPLC-MS. The main compounds of C. furcata are fumarprotocetraric acid, atranorin, protocetraric acid, rangiformic acid, bourgeanic acid, norrangiformic acid and the new lichen metabolite 1-methyl 3,4-dicarboxydecanoate. C. rangiformis is characterized by atranorin, rangiformic acid, norrangiformic acid and 1-methyl 3,4-dicarboxyhexadecanoate, while bourgeanic acid is absent.

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A new cardenolide, (17α)-H-periplogenin-3-O-β-d-glucopyranosyl-(1–4)-2-O-acetyl-3-O-methyl-β-fucopyranoside (1), was isolated from the roots of Streptocaulon tomentosum.

A cDNA encoding a stilbene synthase, RtSTS, was isolated from the rhizomes of Tatar rhubarb, Rheum tataricum L. (Polygonaceae), a medicinal plant containing stilbenes and other polyketides. Recombinant RtSTS was expressed in E. coli and assayed with acetyl-coenzyme A (CoA), n-butyryl-CoA, isovaleryl-CoA, n-hexanoyl-CoA, cinnamoyl-CoA and p-coumaroyl-CoA as primers of polyketide synthesis. RtSTS synthesized resveratrol and a trace amount of naringenin chalcone from p-coumaroyl-CoA, supporting the enzyme's identification as a resveratrol-type stilbene synthase (EC 2.3.1.95). Bis-noryangonin and p-coumaroyl triacetic acid lactone (CTAL)-type pyrones were observed in minor amounts in the reaction with p-coumaroyl-CoA and as major products with cinnamoyl CoA. As well, such pyrones, and not aromatic polyketides, were identified as the only products in assays with aliphatic and benzoyl CoA esters. Acetonyl-4-hydroxy-2-pyrone, a pyrone synthesized from acetyl-CoA, was identified as a new product of a stilbene synthase. Using Northern blot analysis, RtSTS transcript was found to be highly expressed in R. tataricum rhizomes, with low transcript levels also present in young leaves. This expression pattern correlated with the occurrence of resveratrol, which was detected in higher amounts in R. tataricum rhizomes compared with leaves and petioles using HPLC. Few stilbene synthases have been found in plants, and the identification of RtSTS provides additional sequence and catalytic information with which to study the evolution of plant polyketide synthases.A cDNA encoding a plant polyketide synthase was isolated from Rheum tataricum and functionally charactarized as a resveratrol-forming stilbene synthase.

From the ethyl acetate extract of the tuberous roots of Ophiopogon japonicus (Liliaceae) eight known and five new homoisoflavonoidal compounds were isolated. The new compounds are 5,7-dihydroxy-8-methoxy-6-methyl-3-(2′-hydroxy-4′-methoxybenzyl)chroman-4-one (1), 7-hydroxy-5,8-dimethoxy-6-methyl-3-(2′-hydroxy-4′-methoxybenzyl)chroman-4-one (2), 5,7-dihydroxy-6,8-dimethyl-3-(4′-hydroxy-3′-methoxybenzyl)chroman-4-one (3), 2,5,7-trihydroxy-6,8-dimethyl-3-(3′,4′-methylenedioxybenzyl)chroman-4-one (4) and 2,5,7-trihydroxy-6,8-dimethyl-3-(4′-methoxybenzyl)chroman-4-one (5). Their structures have been elucidated by mass and NMR spectroscopy. Compounds 4 and 5 are the first isolated homoisoflavonoids with a hemiacetal function at position 2.Five new and eight known homoisoflavonoids were isolated from the tuberous roots of the medicinal plant Ophiopogon japonicus (Liliaceae) and identified by spectroscopic data.

The ectomycorrhizal fungi Laccaria amethystina and Lactarius deterrimus grown in liquid culture were used to study the fate of added ferulic acid. Laccaria amethystina degraded ferulic acid to the major metabolite vanillic acid. The intermediate vanillin was not detected. Lactarius deterrimus showed a completely different detoxification pattern. Two dimers and one trimer of ferulic acid could be identified as polymerization products of this fungus. A bioassay of the possible biological activities of ferulic acid and vanillic acid on these fungi revealed that vanillic acid was less toxic than ferulic acid for Laccaria amethystina but that both phenolic acids were toxic for Lactarius deterrimus. The results are discussed with respect to ectomycorrhizal fungal growth in the organic layer of forest soils and between living root cells of ectomycorrhizas.

Recent cell culture experiments indicated that extracts of Vitex agnus-castus (VAC) may contain yet unidentified phytoestrogens. Estrogenic actions are mediated via estrogen receptors (ER). To investigate whether VAC compounds bind to the currently known isoforms ERα or ERß, ligand binding assays (LBA) were performed. Subtype specific ER-LBA revealed a binding of VAC to ERß only. To isolate the ERß-selective compounds, the extract was fractionated by bio-guidance. The flavonoid apigenin was isolated and identified as the most active ERß-selective phytoestrogen in VAC. Other isolated compounds were vitexin and penduletin. These data demonstrate that the phytoestrogens in VAC are ERß-selective.

2,4-Dimethoxy-2-methyl-6H-pyran-3-one (1), a hitherto unknown natural product, and the calcium salt of rehmapicroside (2) have been isolated from rhizomes of the Vietnamese variety of Rehmannia glutinosa Libosch together with a series of known compounds: norcarotenoids (3–5), 2-formyl-5-hydroxymethylfurane (6), the iridoid rehmaglutin D (7), iridoid glycosides (8–12) and phenylethyl alcohol glycosides (13–17). Their structures were determined by mass and NMR spectroscopy.