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The fruit aril of Myristica fragrans, commonly known as “mace”, is a popular spice in worldwide. Chromatographic separation of the combined ethyl acetate (EtOAc) and the methanol (MeOH) extracts of the aril of Myristica fragrans furnished malabaricone C (1), 3-(3-methyl-5-pentyl-2-furanyl)-2(E)-propenoic acid (2), licarin A (3), maceneolignan B (4) and elemicin (5). Compounds 1–5 were screened for acetylcholinesterase (AChE), and α-glucosidase enzyme inhibitory activities and DPPH radical scavenging activity. Compound 1 showed the highest AChE inhibitory activity (IC50 2.06 ± 0.04 μg/mL) and antioxidant activity (IC50 6.56 ± 0.02 μg/mL) while compound 2 displayed the most potent α-glucosidase inhibitory activity (IC50 50.91 ± 0.01 μg/mL). This is the first report of α-glucosidase inhibitory activity of 2. Results indicate that the aril of M. fragrans showed good anticholinesterase and α-glucosidase inhibitory activities and antioxidant effect in-vitro that have a potential to be used as the treatment of Alzheimer's disease.

Abstract An endophytic fungus Pestalotiopsis microspora isolated from the fruits of Manilkara zapota was cultured in potato dextrose broth media. Chromatographic separation of the EtOAc extract of the broth and mycelium led to the isolation of a new azaphilonoid named pitholide E (1), in addition to previously identified pitholide B (2), pitholide D (3), pestalotin (LL-P880α) (4), PC-2 (5), LL-P880β (6), tyrosol (7) and 4-oxo-4H-pyran-3-acetic acid (8). An endophytic fungus P. microspora from M. zapota and the isolation of compounds 1–5, 7 and 8 from P. microspora are reported here for the first time. Graphic Abstract

The absolute stereochemistry of ( +)-solaniol (5,8-dihydroxy-7-(2-hydroxypropyl)-2-methoxy-6-methyl-1,4-naphthoquinone) (1) was deduced to be S using Mosher’s method. Our result did not accord with the R configuration of ( +)-solaniol recently proposed by Maharjan et al., but agreed with the S configuration determined by X-ray crystallographic analysis by Kyekyeku et al. In addition, complete and unambiguous assignments of 13C NMR signals for 1 are reported.

Activity-guided fractionation of the methanol extract of Trigonella foenum-graecum seeds furnished three flavone C-glycosides having pancreatic lipase inhibitory activity. Structures of these compounds were established as vicenin-1 (1), isoschaftoside (2) and schaftoside (3) by detail analysis of 1H, 13C NMR and MS data. These compounds showed percentage inhibition 60.3% (1), 33.8% (2) and 95.5% (3) at the concentration of 250 μg/ml and IC50 values of the inhibition were 207 μg/ml (1), 330 μg/ml (2) and 130 μg/ml (3). This is the first report of the isolation of lipase inhibitors from T. foenum-graecum seeds. Temperature dependency of 1H NMR of 3 was studied in detail.

The study aimed to explore the anti-inflammatory effect, underlying mechanism, and chemistry of Halodule pinifolia extract. The ethyl acetate (EHP) and methanol (MHP) extracts of Halodule pinifolia were screened for pro-inflammatory cytokine inhibition effect under various in vitro (LPSand crystal-induced inflammation) and in vivo models (LPS-induced endotoxaemia model, carrageenan-induced paw oedema model, and oxalate-induced renal nephropathy model of inflammation). The effect of EHP on the expression of inflammatory markers using western blot analysis (in vitro) was investigated. Chemical constituents of bioactive EHP were isolated through chromatography and characterised using NMR spectroscopy. Furthermore, EHP was standardised for rosmarinic acid, vanillic acid, and ethyl protocatechuate using HPLC. Also, total phytosterols, phenolic, and flavonoid content of EHP were determined by UV spectroscopy. EHP was comparatively more effective than MHP in inhibiting cytokines secretions under LPS-induced in vitro models. Furthermore, EHP was screened under endotoxaemia in vivo model, EHP (250 mg/kg) reduced plasma IL-6, TNF-α, and IL-1β levels by 88.3%, 78.2%, and 74.5%, respectively. In the carrageenan-induced oedema model, EHP (200 mg/kg) reduced paw volume and release of TNF-α (69.3%) and IL-1β (43.1%). EHP (200 mg/kg) further controlled renal nephropathy by inhibiting plasma IL-1β and BUN levels. Also, a significant reduction of mRNA expressions of TNF-α and IL-1β and KIM-1 in renal tissues was observed. Through western blot, EHP was identified to regulate the expression of pro-form as well as mature-form of IL-1β and caspase-1. EHP constituted rosmarinic acid (RA), vanillic acid (VA), ethyl protocatechuate (EP), sitosterol, stigmasterol, campesterol, and dihydrobrassicasterol. It was determined that 4.6 mg/g of RA, 2.92 mg/g of VA, 0.76 mg/g of EP, 21.7 mg/g of total phenolics, 29.8 mg/g of total flavonoids, and 48.2 mg/g of total phytosterols were present in dry EHP. The presence of anti-inflammatory constituents such as RA, VA, and PE in EHP corroborated the in vitro and in vivo anti-inflammatory activity of EHP. The anti-inflammatory property of EHP and its action through attenuation of pan-cytokines suggest that it can be developed into an oral pharmaceutical drug.

Chemical investigation of the chloroform extract of heartwood of Tectona grandis L. f. led to the isolation of three new naphthoquinone derivatives, tectonaquinones A (1), B (2) and C (3), along with six known compounds: barleriaquinone-I (4), tectoquinone (5), tecomaquinone I (6), lapachol (7), obtusifolin (8) and 2-hydroxy-3-methyl anthraquinone (9). The structures of the new compounds were elucidated by spectroscopic methods including 2 D NMR experiments. Tectonaquinone B is the first natural compound that has a hexa-cyclic dinaphthofuran-dione scaffold. Tectonaquinone C has a bicyclic acetal motif that is unusual in nature.

Phialemonium curvatum, an endophytic fungus isolated from the leaves of Passiflora edulis was cultured in potato dextrose broth (PDB) media and chromatographic separation of the EtOAc extract of the broth and mycelium led to the isolation of 4-hydroxybenzoic acid (1), 3-indole acetic acid (2), solaniol (3), uracil (4), uridine (5) and glycerol (6). Compound 2 showed a weak antifungal activity against Cladosporium cladosporioides. This is the first report of the isolation of the endophytic fungus P. curvatum from P. edulis and complete 13CNMR assignments of 3.

Bipolaris sorokiniana, a fungal endophyte, was isolated from the leaves of Costus speciosus, a popular medicinal plant used to control diabetes. Fermentation of the fungus in potato dextrose broth and chromatographic purification of the ethyl acetate (EtOAc) extracts of the culture broth and mycelium yielded two rare sesquiterpenes helminthosporal acid (1) and helminthosporol (2), together with ergosterol. Compounds 1 and 2 showed a strong phytotoxic effect on lettuce seed germination and toxicity against brine shrimps. Compound 1 also showed antifungal activity. Complete assignments of 1H and 13C NMR data of compound 1 are described for the first time.

Context: Pancreatic α-amylase and α-glucosidase inhibitors serve as important strategies in the management of blood glucose. Even though Syzygium cumini (L.) Skeels (Myrtaceae) (SC) is used extensively to treat diabetes; scientific evidence on antidiabetic effects of SC leaves is scarce. Objective: SC leaf extract was investigated for α-amylase inhibitory effect and continued with isolation and identification of α-amylase inhibitors. Materials and methods: Bioassay-guided fractionation was conducted using in vitro α-amylase inhibitory assay (with 20–1000 μg/mL test material) to isolate the inhibitory compounds from ethyl acetate extract of SC leaves. Structures of the isolated inhibitory compounds were elucidated using 1H NMR and 13C NMR spectroscopic analysis and direct TLC and HPLC comparison with authentic samples. Study period was from October 2013 to October 2015. Results: An active fraction obtained with chromatographic separation of the extract inhibited porcine pancreatic α-amylase with an IC50 of 39.9 μg/mL. Furthermore, it showed a strong inhibition on α-glucosidase with an IC50 of 28.2 μg/mL. The active fraction was determined to be a 3:1 mixture of ursolic acid and oleanolic acid. Pure ursolic acid and oleanolic acid showed IC50 values of 6.7 and 57.4 μg/mL, respectively, against α-amylase and 3.1 and 44.1 μg/mL respectively, against α-glucosidase. Discussion and conclusions: The present study revealed strong α-amylase and α-glucosidase inhibitory effects of ursolic acid and oleanolic acid isolated from SC leaves for the first time validating the use of SC leaves in antidiabetic therapy.

Cucurbitacins are highly oxygenated triterpenoids characteristic of plants in the family Cucurbitaceae and responsible for the bitter taste of these plants. Fruits of bitter melon (Momordica charantia) contain various cucurbitacins possessing an unusual ether bridge between C5 and C19, not observed in other Cucurbitaceae members. Using a combination of next-generation sequencing and RNA-Seq analysis and gene-to-gene co-expression analysis with the ConfeitoGUIplus software, we identified three P450 genes, CYP81AQ19, CYP88L7, and CYP88L8, expected to be involved in cucurbitacin biosynthesis. CYP81AQ19 co-expression with cucurbitadienol synthase in yeast resulted in the production of cucurbita-5,24-diene-3β,23α-diol. A mild acid treatment of this compound resulted in an isomerization of the C23-OH group to C25-OH with the concomitant migration of a double bond, suggesting that a nonenzymatic transformation may account for the observed C25-OH in the majority of cucurbitacins found in plants. The functional expression of CYP88L7 resulted in the production of hydroxylated C19 as well as C5-C19 ether-bridged products. A plausible mechanism for the formation of the C5-C19 ether bridge involves C7 and C19 hydroxylations, indicating a multifunctional nature of this P450. On the other hand, functional CYP88L8 expression gave a single product, a triterpene diol, indicating a monofunctional P450 catalyzing the C7 hydroxylation. Our findings of the roles of several plant P450s in cucurbitacin biosynthesis reveal that an allylic hydroxylation is a key enzymatic transformation that triggers subsequent processes to produce structurally diverse products.

Two new triterpenoids, maquatic acid (1), a 2,3- seco-triterpene having an acetal in its A-ring, and 3- O-benzoyltormentic acid (2), were isolated from underground parts of Mentha aquatica, in addition to twelve known compounds, tormentic acid (3), 1- O-benzoylhyptadienic acid (4), 3- epi-ursolic acid (5), hyptadienic acid (6), 3- epi-maslinic acid (7), 3- epi-tormentic acid (8), ursolic acid (9), β-sitosterol (10), oleanolic acid (11), pomolic acid (12), micromeric acid (13) and 21α-hydroxyursolic acid (14) from aerial and underground parts of the plant. Compounds 4-6, 8, 13 and 14 have been isolated from the genus Mentha for the first time.

Suppression of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) along with nitric oxide reduction in RAW 264.7 cells by 7,8-dihydroxy-4-methylcoumarin, ethyl p-coumarate, ethyl caffeate and ethyl ferulate drove us to search structural-analogues of the aforementioned compounds through structure-based drug design. Docking studies revealed that substituted cinnamic acids and their ethyl esters (2-7c) showed higher GoldScore-fitness (GSF) and non-bonding interactions with target proteins than 7,8-dihydroxy-4-methylcoumarin (1a) and 7,8-dihydroxy-5-methylcoumarin (1b). With this background, the methylcoumarins (1a and 1b) and the cinnamic acid derivatives (2-7c) were fused in different permutations and combinations to generate sixty novel fused-cyclic coumarinolignans (FCLs) (8-13k). Docking studies on 8-13k indicated that several FCLs possess higher GSF, interesting active site interactions and distinctive π-π interactions compared to the standards (cleomiscosin A, diclofenac Na and prednisolone). Based on these findings, four novel FCLs (9d, 10d, 11d and 11e) were synthesized and tested for inhibition effect on TNF-α, IL-1β and IL-6 expressions in LPS and oxalate crystal-induced in-vitro models. Compound 10d exhibited significant effect (P 0.0001 at 100 μM) with an IC

An endophytic fungus Biscogniauxia capnodes was isolated from a popular edible fruit Averrhoa carambola. The fungus was fermented in potato dextrose broth for 3 weeks, and then the culture broth and mycelium were extracted with ethyl acetate. Chromatographic separation of this extract furnished 2 isocoumarins, reticulol (1) and 6- O-methyl-reticulol (2), and 2 dihydroisocoumarins, 5-methylmellein (3) and 7-hydroxy-5-methylmellein (4). Compound 1 showed moderate antioxidant activity against 2,2′-diphenyl-1-picrylhydrazyl radicals (IC50 value, 58 μg/mL). This is the first report of the isolation of B. capnodes as an endophyte, as well as the compounds 1 to 4 from B. capnodes.

Two new isodrimene sesquiterpene derivatives, 2( S )-hydroxyalbicanol ( 1 , =(2 S ,4a S ,8 S ,8a S )-8-(hydroxymethyl)-4,4,8a-trimethyl-7-methylenedecahydronaphthalen-2-ol) and 2( S )-hydroxyalbicanol 11-acetate ( 2 , =((1 S ,4a S ,7 S ,8a S )-7-hydroxy-5,5,8a-trimethyl-2-methylenedecahydronaphthalen-1-yl)methyl acetate) were isolated from the culture broth of the fungus Polyporus arcularius , together with two phenylpropanediols, (1 S ,2 S )- and (1 R ,2 S )-1-phenyl-1,2-dihydroxypropane ( 3 , 4 ). Compound 3 is reported as a naturally occurring compound for the first time. The structures of the compounds were elucidated on the basis of spectroscopic analysis. Compound 1 exhibited growth inhibition of lettuce seedlings with IC 50 values of 1.3 mM to hypocotyl and 1.7 mM to radicle.

Sarcodonin e (2), isolated from Sarcodon scabrosus, was treated with trimethylsilyldiazomethane to yield a crystalline methylated derivative 6. The structure of 6 was determined by X-ray analysis, which confirmed the presence of an unprecedented N(1b)-OMe group, the configuration at N(1b) and the 1,3,4-substituted phenyl ring structure of 2. More importantly, the structures of 6 and 2 have answered the intriguing problem of solving the core structure of the sarcodonin class of natural products, establishing that sarcodonins have a benzodioxazine core structure, rather than the recently proposed benzodioxane aminal core structure.

Onocerin is known for its unusual structure among triterpenoids, with a symmetrical structure that is formed by cyclizations at the both termini of dioxidosqualene. The nature of the enzyme catalyzing these unusual cyclizations has remained elusive for decades. Here, we report the cloning of genes responsible for these reactions; they exhibited unprecedented substrate specificities among oxidosqualene cyclase family members. Two genes, LCC and LCD, were identified from the fern Lycopodium clavatum. Expression in yeast revealed that both were required to produce α-onocerin. LCC, the first dioxidosqualene cyclase, catalyzed the production of a novel intermediate pre-α-onocerin from only dioxidosqualene as a substrate; LCD catalyzed the second half of the cyclization, exclusively from pre-α-onocerin. These results demonstrated that these two most unusual oxidosqualene cyclases were involved in onocerin biosynthesis.

Synopsis A genetic linkage map of guinea grass (Panicum maximum Jacq.) was generated with nine of the AFLP markers found to be associated with apospory. These apospory-associated markers were assigned to a linkage group having previous association with microsporogenesis of the aposporous guineagrass cultivar ‘Natsukaze’. An aposporous linkage group was constructed utilizing 38 AFLP markers. Embryo sac analysis revealed that sexual and apomictic embryo sacs occurred at a frequency of 1 : 1, indicating simple inheritance of a single major gene controlling apospory in guineagrass. In addition, utilizing 56 AFLP primer combinations and 41 RAPD primers, 39 linkage groups and 360 simplex marker loci were assigned to the genetic map of the ‘Natsukaze’ cultivar. These markers covered 1703.5 cM of the autotetraploid guineagrass genome (2n = 4x = 32), with an average spacing of 4.7 cM. These tightly linked markers to apospory locus in guineagrass could be a powerful tool for marker-assisted selection of apospory and map-based cloning of the apospory gene.

The stabilization effect of the novel self-emulsifying drug delivery systems (SEDDS) type O/W microemulsion on the gastrointestinal absorption of a poorly water soluble new compound, ER-1258 was examined by bile-fistula model rats. In the components of this formulation, medium chain fatty acid triglyceride (MCT), diglyceryl monooleate (DGMO-C), polyoxyethylene hydrogenated castor oil 40 (HCO-40) and ethanol were used as an oil, a lipophilic surfactant, a hydrophilic surfactant and a solubilizer at the mixture ratio of 25/5/45/25 w/w%, respectively. The ratios of AUC in the non-treated rats to that in the bile-fistula rats were 5.1, 12.1 and 3.0 for the suspension, the oily solution and the SEDDS type O/W microemulsion, respectively. The risk from which the difference between individuals of the compound absorption amounts resulting from the flow of the bile secretion serves as the maximum was high in order of oily solution>suspension>SEDDS type O/W microemulsion. Therefore, it was verified that the SEDDS type O/W microemulsion was able to reduce this risk, compared with the other formulations. When short chain fatty acid triglyceride (Triacetin) was used as an oil, the similar effect was demonstrated in the formulation composed of sorbitan sesquioleate (SO-15) as a lipophilic surfactant and polyoxyethylene hydrogenated castor oil 60 (HCO-60) or polyoxyethylene 20 sorbitan monooleate (TO-10M) as a hydrophilic surfactant.

The biosynthesis of the lactone moiety of acaterin was studied using Pseudomonas sp. A92. Feeding experiment with (2R S)-[1-13C]glycerol revealed that glycerol is efficiently incorporated into the branched three-carbon unit (C-3, C-4 and C-5 positions) of acaterin and its precursor, 4,5-didehydro-acaterin. Further feeding studies of (2R S)-[1,1-2H2]- and sn-[3,3-2H2]-glycerols showed that two hydrogens at the sn-C-3 position of glycerol are incorporated into the C-5 position of the two compounds, whereas those at the sn-C-1 position are completely lost during the transformation. These results suggest that acaterin is biosynthesized via a tetronic acid type intermediate.

An olefinic analog of acaterin, named 4,5-didehydroacaterin, was isolated and characterized from a fermentation broth of Pseudomonas sp. A92 cultured in a medium supplemented with lauric acid. The dehydro-compound was shown to be a biosynthetic precursor of acaterin.

The biosynthetic origin of the C3 branched unit of agglomerin A has been investigated. Feeding of sn-(3R)- and sn-(3S)-[3-2H]glycerols to Enterobacter agglomerans PB-6042 followed by 2H NMR analysis of the resulting agglomerin A revealed that pro-R and pro-S hydrogens at sn-C-3 of glycerol were incorporated stereospecifically into 5E and 5Z hydrogens of agglomerin A, respectively. These results imply that the immediate precursor of the C3 branched unit is not pyruvate, but 1,3-bisphosphoglyceric acid or its biological equivalent.

The oxymethine chiral centers, i. e., C-26, C-28 and C-29 positions, of tetrahydrofuranic acetogenins have been determined by advanced Mosher method focusing on the chemical shift of the terminal methyl group.

Tetrahydrofuranic (annonaceous) acetogenins are a new class of bioactive natural products isolated from several genera of Annonaceae plants. We have isolated nearly thirty these acetogenins from the seeds of Annona squamosa and A. reticulata Their structures have been elucidated by the application of precursor-ion scanning method in mass spectrometry. Thus, these compounds were converted into lactam derivatives on treatment with amines such as N, N-dimethylethylenediamine. Precursor-ion scanning spectra of the derivatives from a specific fragment ion due to C-N bond cleavage were measured. This newly developed method allowed to establish unambiguously the planar structures of tetrahydrofuranic acetogenins. Biological activity and stereochemical aspects of annonaceous acetogenins are also reviewed.

The biosynthesis of the lactone moiety of acaterin was studied using Pseudomonas sp. A92. Feeding experiment with (2R S)-[1-13C]glycerol revealed that glycerol is efficiently incorporated into the branched three-carbon unit (C-3, C-4 and C-5 positions) of acaterin and its precursor, 4,5-didehydro-acaterin. Further feeding studies of (2R S)-[1,1-2H2]- and sn-[3,3-2H2]-glycerols showed that two hydrogens at the sn-C-3 position of glycerol are incorporated into the C-5 position of the two compounds, whereas those at the sn-C-1 position are completely lost during the transformation. These results suggest that acaterin is biosynthesized via a tetronic acid type intermediate.

Two bis-tetrahydrofuran acetogenins, squamocin-O1 (1) and squamocin-O2 (2), were isolated from a MeOH extract of seeds of Annona squamosa L. Their structures were determined by spectral means including precursor-ion scanning mass spectral analysis for their aminal derivatives. The configurations at the oxymethine chiral centers were assigned as 12R,15R,16R,19R,20R,23R,24S,28S,36S for 1 and 12S,15R,16R,19R,20R,23R,24S, 28S,36S for 2, based on 1H NMR analysis of their Mosher's ester derivatives and CD data.

The seeds of Annona squamosa (Annonaceae) furnished squamostanal-A (1), a novel annonaceous acetogenin. The structure of squamostanal-A was established by spectral methods to be (5S)-3-(12-formyldodecyl)-5-methyl-2,5-dihydrofuran-2-one.

The uneven distribution of the glucuronidation and sulfation activity of acetaminophen (APAP) and p-nitrophenol (PNP) in liver was studied using centrilobular and periportal regions of isolated rat hepatocytes obtained by Percoll density gradient centrifugation, in a manner similar to that for harmol reported previously. The glucuronidation of APAP and PNP occurred predominantly in the periportal region. Glucuronidation activity of APAP in the centrilobular region was not detected. This finding may reflect the existence of different forms of uridine diphosphate-glucuronyltransferase (UDPGT) for APAP and PNP. The regional difference in Km values was observed for PNP, but not for harmol as reported in the previous paper. This suggests the existence of multiple forms of UDPGT for PNP as well as the heterogeneous distribution of this enzyme in liver lobules. The sulfation rates of APAP and PNP in the centrilobular region were smaller than those in the periportal region. The regional difference of APAP sulfation was due to different Vmax values. As shown in the harmol experiment, the predominant distribution of sulfation activity of APAP and PNP in the periportal region agreed with the results in the perfused liver previously reported by other investigators but that of glucuronidation activity was at variance with them.

The regional distribution of the conjugation reaction activities of harmol was examined in isolated rat liver cells fractionated to the centrilobular and periportal regions. The heterogeneous distribution in the conjugation rates per unit cell numbers was found only in glucuronidation. The difference in the metabolic intrinsic clearance per total cell numbers in each region was mainly due to the cell numbers, resulting that the metabolic abilities in both reactions are higher in the periportal region than the centrilobular region. This finding in sulfate conjugation is consistent with the results in the isolated perfused liver previously presented by other investigators but the result that in glucuronide conjugation is contradictory to them.

Acetaminophen caused a reversible change in the fluorescence polarization of eosin maleimide and dansyl chloride labeled to rat liver microsomes, indicating that acetaminophen acts on microsomal membrane proteins. Acetaminophen in the presence of nicotinamide adenine dinucleotide phosphate (NADPH), brought about an irreversible change in the fluorescence polarization of the probes. Acetaminophen, with or without NADPH, was also found to change the fluorescence polarization of lipid-soluble fluorescent probes, 2- and 12-(9-anthroyloxy) stearic acid labeled to microsomes, indicating its action on microsomal membrane lipids.