Daurichromenic Acid Research Articles

Dual High-Resolution α-Glucosidase and PTP1B Inhibition Profiling Combined with HPLC-PDA-HRMS-SPE-NMR Analysis for the Identification of Potentially Antidiabetic Chromene Meroterpenoids from Rhododendron capitatum.

Thirteen previously undescribed chromene meroterpenoids, capitachromenic acids A-M (3-6, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b, and 11b), were identified from an ethyl acetate extract of Rhododendron capitatum, using dual high-resolution α-glucosidase and PTP1B inhibition profiling in combination with HPLC-PDA-HRMS-SPE-NMR. In addition, one known chromene meroterpenoid, daurichromenic acid (15), and its biosynthetic precursor, grifolic acid (12), two C-methylated flavanones, (2S)-5,7,4'-trihydroxy-8-methylflavanone (1) and farrerol (2), and two triterpenoids, oleanolic acid (14a) and ursolic acid (14b), were identified. New structures were elucidated by extensive 1D and 2D NMR analysis, and absolute configurations of new chromene meroterpenoids were assigned by analysis of their ECD spectra on the basis of the empirical chromane helicity rule and from Rh2(OCOCF3)4-induced ECD spectra by applying the bulkiness rule. Compounds 5, 9a, 9b, 12, and 15 showed α-glucosidase inhibitory activity with IC50 values ranging from 8.0 to 93.5 μM, while compounds 3, 5, 8b, 9a, 9b, 10b, 11b, 12, and 15 showed PTP1B inhibitory activity with IC50 values ranging from 2.5 to 68.1 μM.

Daurichromenic Acid from the Chinese Traditional Medicinal Plant Rhododendron dauricum Inhibits Sphingomyelin Synthase and Aβ Aggregation.

Species of the genus Rhododendron have been used in traditional Chinese medicine, with the medicinal herb “Manshanfong” used as an expectorant and for the treatment of acute bronchitis. Daurichromenic acid (DCA), a constituent of Rhododendron dauricum, is a meroterpenoid with antibacterial, anti-HIV, and anti-inflammatory activities. However, the mechanisms underlying these pharmacologic activities are poorly understood. To develop new drugs based on DCA, more information is required regarding its interactions with biomolecules. The present study showed that DCA inhibits the activity of the enzyme sphingomyelin synthase, with an IC50 of 4 µM. The structure–activity relationships between DCA and sphingomyelin synthase were evaluated using derivatives and cyclized hongoquercin A. In addition, DCA was found to inhibit amyloid β aggregation. These results may help in the design of effective drugs based on DCA.

Isolation of three new meroterpenoids and seven known compounds from Albatrellus yasudae and their Aβ-aggregation inhibitory activity

Alzheimer’s disease is a serious neurologic disorder that cannot be cured completely. In this study, we targeted compounds that inhibit amyloid-beta (Aβ) aggregation, based on the amyloid cascade hypothesis. Ten compounds (1–10) were isolated from CHCl3 extracts of the mushroom Albatrellus yasudae using Aβ-aggregation inhibitory activity–guided separation. The structures of these compounds were elucidated from 1D and 2D NMR and MS spectral data. Compounds 1–3 were novel, whereas 4–10 were identified as the known compounds grifolin, grifolic acid, neogrifolin, confluentin, 2-hydroxyneogrifolin, daurichromenic acid, and a cerebroside derivative. Compounds 1–10 were tested for Aβ-aggregation inhibitory activity. Compounds 1, 3, 5, 6, 8, and 9 have potential as Aβ-aggregation inhibitory activity.

An Aromatic Farnesyltransferase Functions in Biosynthesis of the Anti-HIV Meroterpenoid Daurichromenic Acid.

Rhododendron dauricum produces daurichromenic acid, an anti-HIV meroterpenoid, via oxidative cyclization of the farnesyl group of grifolic acid. The prenyltransferase (PT) that synthesizes grifolic acid is a farnesyltransferase in plant specialized metabolism. In this study, we demonstrated that the isoprenoid moiety of grifolic acid is derived from the 2-C-methyl-d-erythritol-4-phosphate pathway that takes place in plastids. We explored candidate sequences of plastid-localized PT homologs and identified a cDNA for this PT, RdPT1, which shares moderate sequence similarity with known aromatic PTs. RdPT1 is expressed exclusively in the glandular scales, where daurichromenic acid accumulates. In addition, the gene product was targeted to plastids in plant cells. The recombinant RdPT1 regiospecifically synthesized grifolic acid from orsellinic acid and farnesyl diphosphate, demonstrating that RdPT1 is the farnesyltransferase involved in daurichromenic acid biosynthesis. This enzyme strictly preferred orsellinic acid as a prenyl acceptor, whereas it had a relaxed specificity for prenyl donor structures, also accepting geranyl and geranylgeranyl diphosphates with modest efficiency to synthesize prenyl chain analogs of grifolic acid. Such a broad specificity is a unique catalytic feature of RdPT1 that is not shared among secondary metabolic aromatic PTs in plants. We discuss the unusual substrate preference of RdPT1 using a molecular modeling approach. The biochemical properties as well as the localization of RdPT1 suggest that this enzyme produces meroterpenoids in glandular scales cooperatively with previously identified daurichromenic acid synthase, probably for chemical defense on the surface of R. dauricum plants.

Identification and Characterization of Daurichromenic Acid Synthase from Rhododendron dauricum

Daurichromenic acid (DCA) synthase catalyzes the oxidative cyclization of grifolic acid to produce DCA, an anti‐HIV meroterpenoid isolated from Rhododendron dauricum (Ericaceae). We identified a novel cDNA encoding DCA synthase by transcriptome‐based screening from young leaves of R. dauricum. The gene coded for a 533‐amino acid polypeptide with moderate homologies to flavin adenine dinucleotide oxidases from other plants. The primary structure contained an amino‐terminal signal peptide and conserved amino acid residues to form bicovalent linkage to the flavin adenine dinucleotide isoalloxazine ring at histidine‐112 and cysteine‐175. In addition, the recombinant DCA synthase, purified from the culture supernatant of transgenic Pichia pastoris, exhibited structural and functional properties as a flavoprotein. The reaction mechanism of DCA synthase characterized herein partly shares a similarity with those of cannabinoid synthases from Cannabis sativa, whereas DCA synthase catalyzes a novel cyclization reaction of the farnesyl moiety of a meroterpenoid natural product of plant origin.Moreover, in this study, we present evidence that DCA is biosynthesized and accumulated specifically in the glandular scales, on the surface of R. dauricum plants, based on various analytical studies at the chemical, biochemical, and molecular levels. The extracellular localization of DCA also was confirmed by a confocal microscopic analysis of its autofluorescence. These data highlight the unique feature of DCA: the final step of biosynthesis is completed in apoplastic space, and it is highly accumulated outside the scale cells. With respect to this unique extracellular biosynthesis and accumulation of DCA, we speculated the possibility that DCA would serve as a chemical defense component outside of the scale cells, the outermost layer of plants. In addition, we demonstrated that DCA and grifolic acid are phytotoxic meroterpenoids that induce cell death in the cell cultures of their producer plant R. dauricum, and thus have to be sequestered in the apoplast to avoid self‐poisoning.Support or Funding InformationThis work was supported by JSPS/MEXT KAKENHI (nos. 15K07994 and 17H05436) to FT, and a Grant‐in‐Aid for JSPS Fellows (no. 17J10178) to MI.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Daurichromenic acid and grifolic acid: Phytotoxic meroterpenoids that induce cell death in cell culture of their producer Rhododendron dauricum

ABSTRACTDaurichromenic acid (DCA) is a meroterpenoid with anti-HIV activities that is isolated from Rhododendron dauricum L. We recently reported that DCA is biosynthesized and accumulated in the apoplast of glandular scales attached on the surface of young leaves of R. dauricum. In the present study, we confirmed that a cell suspension culture of R. dauricum could not produce DCA and its precursor grifolic acid even after elicitation with methyl jasmonate and β-cyclodextrin. In addition, exogenous supplementation of DCA and grifolic acid effectively induced cell death in the same culture, with apoptosis-associated phenomena such as cytoplasmic shrinkage, chromatin condensation, and genomic DNA degradation. These findings suggested that DCA and grifolic acid are phytotoxic metabolites that have to be sequestered in the apoplast to avoid self-poisoning.

Identification and Characterization of Daurichromenic Acid Synthase Active in Anti-HIV Biosynthesis.

Daurichromenic acid (DCA) synthase catalyzes the oxidative cyclization of grifolic acid to produce DCA, an anti-HIV meroterpenoid isolated from Rhododendron dauricum We identified a novel cDNA encoding DCA synthase by transcriptome-based screening from young leaves of R. dauricum The gene coded for a 533-amino acid polypeptide with moderate homologies to flavin adenine dinucleotide oxidases from other plants. The primary structure contained an amino-terminal signal peptide and conserved amino acid residues to form bicovalent linkage to the flavin adenine dinucleotide isoalloxazine ring at histidine-112 and cysteine-175. In addition, the recombinant DCA synthase, purified from the culture supernatant of transgenic Pichia pastoris, exhibited structural and functional properties as a flavoprotein. The reaction mechanism of DCA synthase characterized herein partly shares a similarity with those of cannabinoid synthases from Cannabis sativa, whereas DCA synthase catalyzes a novel cyclization reaction of the farnesyl moiety of a meroterpenoid natural product of plant origin. Moreover, in this study, we present evidence that DCA is biosynthesized and accumulated specifically in the glandular scales, on the surface of R. dauricum plants, based on various analytical studies at the chemical, biochemical, and molecular levels. The extracellular localization of DCA also was confirmed by a confocal microscopic analysis of its autofluorescence. These data highlight the unique feature of DCA: the final step of biosynthesis is completed in apoplastic space, and it is highly accumulated outside the scale cells.

Combinatorial Biosynthesis of (+)-Daurichromenic Acid and Its Halogenated Analogue.

Daurichromenic acid is a meroterpenoid with various pharmacological activities that is biosynthesized from grifolic acid in Rhododendron dauricum. Heterologous expression of grifolic acid synthases from Stachybotrys bisbyi and a daurichromenic acid synthase from R. dauricum in Aspergillus oryzae mediated three-step combinatorial biosynthesis of (+)-daurichromenic acid through enantioselective 6-endo-trig cyclization. Additional introduction of a halogenase from Fusarium sp. into the strain resulted in the biosynthesis of (+)-5-chlorodaurichromenic acid, which exceeds the antibacterial activity of the original compounds.

A Novel Class of Plant Type III Polyketide Synthase Involved in Orsellinic Acid Biosynthesis from Rhododendron dauricum.

Rhododendron dauricum L. produces daurichromenic acid, the anti-HIV meroterpenoid consisting of sesquiterpene and orsellinic acid (OSA) moieties. To characterize the enzyme responsible for OSA biosynthesis, a cDNA encoding a novel polyketide synthase (PKS), orcinol synthase (ORS), was cloned from young leaves of R. dauricum. The primary structure of ORS shared relatively low identities to those of PKSs from other plants, and the active site of ORS had a unique amino acid composition. The bacterially expressed, recombinant ORS accepted acetyl-CoA as the preferable starter substrate, and produced orcinol as the major reaction product, along with four minor products including OSA. The ORS identified in this study is the first plant PKS that generates acetate-derived aromatic tetraketides, such as orcinol and OSA. Interestingly, OSA production was clearly enhanced in the presence of Cannabis sativa olivetolic acid cyclase, suggesting that the ORS is involved in OSA biosynthesis together with an unidentified cyclase in R. dauricum.

Reducing Molecular Flexibility by Cyclization for Elucidation of Absolute Configuration by CD Calculations: Daurichromenic Acid.

Circular dichroism (CD) calculations of flexible natural products have been difficult because of the large number of low-energy conformers and ambiguous Boltzmann distributions. In this article, through electronic (ECD) and vibrational (VCD) studies on a natural product, (+)-daurichromenic acid, we demonstrate that derivatization of a flexible molecule can dramatically reduce its flexibility. This work also shows the usefulness of derivatization for diminishing computational expenses required for optimization and CD calculations, and for increasing the reliability of the assignment of absolute configuration. Chirality 28:453-459, 2016. © 2016 Wiley Periodicals, Inc.

Sequential Ketene Generation from Dioxane-4,6-dione-keto-dioxinones for the Synthesis of Terpenoid Resorcylates.

Trapping of the ketene generated from the thermolysis of 2-methyl-2-phenyl-1,3-dioxane-4,6-dione-keto-dioxinone at 50 °C with primary, secondary, or tertiary alcohols gave the corresponding dioxinone β-keto-esters in good yield under neutral conditions. These intermediates were converted by palladium(0)-catalyzed decarboxylative allyl migration and aromatization into the corresponding β-resorcylates. These transformations were applied to the syntheses of the natural products (±)-cannabiorcichromenic and (±)-daurichromenic acid.

Stereochemistry of a Rhododaurichromanic Acid Derivative

A rhododaurichromanic acid A derivative was synthesized from the naturally occurring daurichromenic acid in enantiomeric pure form. Its absolute configuration was elucidated by applying VCD, ECD and DFT calculations.

Daurichromenic Acid-producing Oxidocyclase in the Young Leaves of Rhododendron dauricum

Rhododendron dauricum L., a flowering tree popular in Hokkaido, produces daurichromenic acid (DCA), a terpenophenol with a potent anti-HIV activity. The DCA-producing enzyme, named DCA synthase, could be detected in the soluble protein fraction prepared from the young leaves of R. dauricum. DCA synthase catalyzed oxidocyclization of the farnesyl group of grifolic acid to form (+)-DCA as the major reaction product. The DCA synthase reaction proceeds without the need for any cofactors and coenzymes except for molecular oxygen. Interestingly, these catalytic properties of DCA synthase are quite similar to those reported for cannabinoid synthases in the marijuana plant Cannabis sativa L.

Molecular characterization of daurichromenic acid synthase from Rhododendron dauricum

Rhododendron dauricum L. (Ericaceae) produces daurichromenic acid (DCA), an anti-HIV component [1]. DCA is a terpenophenol, and would be biosynthesized from grifolic acid via oxidative cyclization of the farnesyl group, the reaction analogous to those reported for cannabinoid biosynthesis [2]. We attempted to amplify cDNA fragments encoding DCA synthase by homology-based RT-PCR with degenerate primers designed from conserved sequences in cannabinoid synthases and related plant oxidases. Then, the 3' and 5'-end regions of cDNA were obtained by rapid amplifications of cDNA ends. Consequently, three cDNA clones, that encode polypeptides named RdOx 1˜3, were cloned. RdOx 1˜3 consisted of 533, 533 and 534 amino acids containing a FAD binding motif. In addition, these polypeptides had >50% identities with cannabinoid synthases. The heterologous expression system for RdOxs was established using Pichia pastoris as a host. The recombinant RdOx1 and 2 could produce DCA from grifolic acid, whereas RdOx-3 showed no DCA-producing activity, suggesting that RdOx 1 and 2 are active DCA synthase in R. dauricum. DCA synthase would be applied for biotechnological production of DCA because the substrate grifolic acid has been isolated from a mushroom Albatrellus dispansus in a large amount [3].

Phenolic compounds from Rhododendron dauricum from the Baikal region

Rhododendron dauricum L. (Ericaceae) is a mountainous half-evergreen shrub indigenous to Central Siberia and the Baikal region. The phenolic complex of R. dauricum contained simple phenols (hydroquinone, arbutin, 4-O-methylfloroacetophenone, orcinol, grifolin) [1], phenolcarboxylic acids (p-hydroxybenzoic, anisic, protocatechoic, vanillic, syringic, 3-methyl ether of gallic acid) [1, 2], coumarins (umbelliferone, scopoletin) [3], chromanes (rhododaurichromanic acids A and B) [4], chromenes (daurichromenic acid, daurichromenes A–D, confluentin) [4, 5], and flavonoids (poriol, farrerol, matteucinol, kaempferol, 5-methylkaempferol, quercetin, azaleatin, hyperoside, isohyperoside, avicularin, quercitrin, myricetin, 5-methylmyricetin, gossipetin) [1, 3, 6, 7]. R. dauricum growing in China, Khabarosk Territory, and in the Altai have been studied previously. Our goal was to study the qualitative and quantitative content of phenolic compounds in R. dauricum from the Baikal region. Leaves of R. dauricum were collected in Irkutsk Oblast (Irkutsk, test plot of SIFIBR, SB RAS, June 28, 2008, raw material I; August 28, 2008, II; Olkha village, September 3, 2008, III; Orlenok station, September 9, 2008, IV), in the Lake Baikal basin (Olkhon Island, September 13, 2008, V; November 4, 2008, VI), and in the Republic of Buryatiya (Mukhorshibir Region, Barsk Ridge, July 25, 2009, VII; Soviet Region, Ulan-Ude, August 25, 2009, VIII; Pribaikal Region, Goryachinsk village, September 17, 2009, IX). The speciation was determined by Cand. Pharm. Sci. N. K. Chirikova (IGEB, SB RAS). Raw material samples are preserved in the herbarium of the Division of Biologically Active Compounds, IGEB, SB RAS. Dried ground raw material (0.2 kg, VII) was extracted with EtOH (80%, 5 , 1:25) at 80°C. The EtOH extract was concentrated to a watery residue that was extracted with hexane, CHCl3, EtOAc, and n-BuOH to produce five fractions: hexane (20.07 g, 10.04% of air-dried mass), CHCl3 (2.59 g, 1.30%), EtOAc (16.29 g, 8.15%), n-BuOH (8.25 g, 4.13%) and an aqueous residue (25.22 g, 12.61%). The total yield of extracted compounds was 36.23% of the air-dried mass. The CHCl3 fraction (2.4 g) was separated by column chromatography over SiO2 (2 30 cm) using hexane:CHCl3 (100:0 70:30) and CHCl3:Me2CO (100:0 50:50) with subsequent rechromatography by preparative TLC (toluene:EtOAc:HCOOH 3:3:1) and HPTLC (petroleum ether:Et2O:HCOOH 9:4:1). This afforded 1 (10 mg, farrerol, 6,8-dimethyl-5,7,4 -trihydroxyflavanone) [8, 9], 2 (31 mg, scopoletin) [10], 3 (6 mg, umbelliferone) [10], 4 (4 mg, kaempferol) [11], 5 (10 mg, quercetin) [12], and 6 (11 mg, hyperoside) [12]. The EtOAc (10 g) and n-BuOH extracts (7 g) were chromatographed over columns of SiO2 (3.5 40 cm) using CHCl3:Me2CO (100:0 70:30), Me2CO:EtOH (100:0 50:50), and EtOH:H2O (95:5 40:60). Subfractions were separated over columns of polyamide (Woelm, 2 20 cm, EtOH:H2O, 0:100 95:5, Me2CO) with subsequent preparative TLC (toluene:EtOAc:HCOOH, 5:4:1; EtOAc:MeOH:H2O, 9.6:1.9:1). Chromatographic separation of the EtOAc fraction isolated 7 (67 mg, gallic acid 3-methyl ether) [13], 8 (18 mg, gallic acid) [13], 9 (31 mg, ellagic acid) [14], 10 [11 mg, (+)-catechin], 11 [4 mg, (–)-epicatechin] [15], 6 (214 mg), 12 [2 mg, (–)-epicatechin-3-gallate] [16], 13 (82 mg, avicularin) [12], 14 (41 mg, quercitrin) [12], and 15 (8 mg, procyanidin B1) [17]. HPLC (conditions 1) of the EtOAc fraction also detected p-hydroxybenzoic (16), gentisic (17), and syringic (18) acids. The n-BuOH fraction afforded 8 (10 mg), 10 (5 mg), 12 (6 mg), 15 (12 mg), and 17 (8 mg). The study found in leaves of R. dauricum 18 compounds. Of these, 1-7, 13, 14, 16, and 18 were isolated earlier from this species whereas 8-12, 15, and 17 were isolated for the first time.

Tetracyclic Chromane Derivatives from Rhododendron anthopogonoides

Four new chromane derivatives, anthopogochromane (1), anthopogochromene A (2), anthopogochromene B (3), and anthopogochromene C (4), and two known compounds, daurichromenic acid (5) and ilicicolinic acid B (6), have been isolated from the Chinese medicinal plant Rhododendron anthopogonoides. The S absolute configuration of the stereogenic carbons in the chromane and chromene rings of 1-4 was determined from their circular dichroism spectra. Compounds 1 and 2 inhibited compound 48/80-induced histamine release from rat peritoneal mast cells.

Enantioselective Synthesis of Daurichromenic Acid and Confluentin

AbstractThe first asymmetric synthesis of daurichromenic acid and confluentin is described. The key step of the sequence leading to both natural products is a highly enantioselectivedomino aldol/oxa Michael reaction (97 % ee) of farnesal and 2‐methoxy‐4‐methylsalicylaldehyde.

Efficient Synthesis of Biologically Active (.+‐.)‐Confluentin and (.+‐.)‐Daurichromenic Acid.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Efficient Synthesis of Biologically Active (±)‐Confluentin and (±)‐Daurichromenic Acid

One‐step synthesis of biologically active (±)‐confluentin is described from commercially available orcinol with trans,trans‐farnesal in the presence of ethylenediamine diacetate as a catalyst. Further conversion of synthetic confluentin to highly potent anti‐HIV agent, (±)‐daurichromenic acid, is accomplished by formylation and oxidation in two steps.

Concise Synthesis of Biologically Interesting (.+‐.)‐Cannabichromene, (.+‐.)‐Cannabichromenic Acid, and (.+‐.)‐Daurichromenic Acid.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.