Isoflavonoid Glycosides Research Articles

Enzymatic synthesis of novel unnatural phenoxodiol glycosides with a glycosyl donor flexible glycosyltransferase MeUGT1

Isoflavonoids are of great interest due to their human health-promoting properties, which have resulted in studies on exploiting these phytochemicals as hotspots in diverse bio -industries. Biocatalytic glycosylation of isoflavonoid aglycones to glycosides has attracted marked interests because it enable the biosynthesis of isoflavonoid glycosides with high selectivity under mild conditions, and also provide an environmentally friendly option for the chemical synthesis. Thus, these inspired us to exploit new flexible and effective glycosyltransferases from microbes for making glycosides attractive compounds that are in high demand in several industries. Most recently, we have reported the functional characterization of a bacterial-origin recombinant glycosyltransferase (MeUGT1). Herein, more detailed kinetic characteristics of this biocatalyst, using a number of glycosyl donor substrates, were examined for further investigation of its biocatalytic applicability, enabling it feasible to biosynthesize new glycosides; phenoxodiol-4′-O-α-glucuronide, phenoxodiol-4′-O-α-(2''-N-acetyl)glucosaminide, phenoxodiol-4′-O-α-galactoside, phenoxodiol-4′-O-α-(2''-N-acetyl)galactosaminide and phenoxodiol-4′-O-α-(2''-deoxy)glucoside. The thorough kinetic analyses revealed that while the recombinant enzyme can utilize, albeit with different substrate preference and catalytic efficiency, a total five different nucleotide sugars as glycosyl donors, exhibiting its promiscuity towards glycosyl donors. This is the first report that a recombinant glycosyltransferase MeUGT1 that can regio-specifically glycosylate C4′-hydroxyl function of semi-synthetic phenoxodiol isoflavene to biosynthesize a series of unnatural phenoxodiol-4′-O-α-glycosides.

RRLC-QTOF/MS-Based Metabolomics Reveal the Mechanism of Chemical Variations and Transformations of Astragali Radix as a Result of the Roasting Process.

Astragali Radix (AR), which is extensively used as a healthy food supplement and medicinal herb, contains two forms of products corresponding to raw Astragalus Radix (RAR) and processed Astragali Radix (PAR), which was obtained by roasting. In this study, a non-targeted rapid resolution liquid chromatography coupled with quadruple time-of-flight mass spectrometry (RRLC-Q/TOF-MS) based metabolomics approach was developed to investigate the chemical changes of AR due to roasting. A total of 63 compounds were identified or tentatively identified. Among them, 23 isoflavonoids (composed of 12 isoflavones, eight pterocarpans, and three isoflavans) and six cycloastragenols were characterized as differential metabolites. Heatmap visualization and high-performance liquid chromatography coupled with photodiode array and evaporative light scattering detector (HPLC–PDA-ELSD) quantitative analysis revealed that malonyl isoflavonoids or cycloastragenols were at higher levels in RAR. These might be converted to corresponding acetyl isoflavonoids and cycloastragenols and related isoflavonoid glycosides during roasting. To prove this prediction, chemical conversion experiments on malonyl isoflavonoids and cycloastragenols were performed to confirm and clarify the chemical transformation mechanism.

Three Isoflavonoid Glycosides from the Rhizomes of Achyranthes bidentata and their Protective Effects on H2O2 Induced H9c2 Cardiomyocytes Injury

Three Isoflavonoid Glycosides from the Rhizomes of Achyranthes bidentata and their Protective Effects on H2O2 Induced H9c2 Cardiomyocytes Injury

Investigation of red clover (Trifolium pratense) isoflavonoid residual complexity by off-line CCS-qHNMR

The importance of Trifolium pratense L. as a dietary supplement and its use in traditional medicine prompted the preparation of a thorough metabolite profile. This included the identification and quantitation of principal constituents as well as low abundant metabolites that constitute the residual complexity (RC) of T. pratense bioactives. The purity and RC of isoflavonoid fractions from standardized red clover extract (RCE) was determined using an off-line combination of countercurrent separation (CCS) and two orthogonal analytical methodologies: quantitative 1H NMR spectroscopy with external calibration (EC-qHNMR) and LC-MS. A single-step hydrostatic CCS methodology (Centrifugal Partition Chromatography [CPC]) was developed that fractionated the isoflavonoids with a hexanes-ethyl acetate-methanol-water (HEMWat) 5.5/4.5/5/5, v/v solvent system (SS) into 75 fractions containing 3 flavonolignans, 2 isoflavonoid glycosides, as well as 17 isoflavonoids and related compounds. All metabolites were identified and quantified by qHNMR spectroscopy. The data led to the creation of a complete isoflavonoid profile to complement the biological evaluation. For example, fraction 69 afforded 90.5% w/w biochanin A (17), with 0.33% w/w of prunetin (16), and 0.76% w/w of maackiain (15) as residual components. Fraction 27 with 89.4% w/w formononetin (13) as the major component had, in addition, a residual complexity consisting of 3.37%, 0.73%, 0.68% w/w of pseudobaptigenin (11), kaempferol (10) and pratensein (8), respectively. Despite the relatively high resolving power of CPC, and not unexpectedly, the chromatographic fractions retained varying degrees of the original metabolomic diversity. Collectively, the extent of metabolomic diversity should be recognized and used to guide the development of isolation strategies, especially when generating samples for bioactivity evaluation. The simultaneous structural and quantitative characterization enabled by qNMR, supported by LC-MS measurements, enables the evaluation of a relatively large number of individual fractions and, thereby, advances both the chemical and biological evaluation of active principles in complex natural products.

Iris domestica (iso)flavone 7- and 3'-O-Glycosyltransferases Can Be Induced by CuCl2.

In many plants, isoflavones are the main secondary metabolites that have various pharmacological activities, but the low water solubility of aglycones limits their usage. The O-glycosylation of (iso)flavones is a promising way to overcome this barrier. O-glycosyltransferases (UGTs) are key enzymes in the biosynthesis of (iso)flavonoid O-glycosides in plants. However, limited investigations on isoflavonoid O-UGTs have been reported, and they mainly focused on legumes. Iris domestica (L.) Goldblatt et Mabberley is a non-legume plant rich in various isoflavonoid glycosides. However, there are no reports regarding its glycosylation mechanism, despite the I. domestica transcriptome previously being annotated as having non-active isoflavone 7-O-UGTs. Our previous experiments indicated that isoflavonoid glycosides were induced by CuCl2 in I. domestica calli; therefore, we hypothesized that isoflavone O-UGTs may be induced by Cu2+. Thus, a comparative transcriptome analysis was performed using I. domestica seedlings treated with CuCl2, and eight new active BcUGTs were obtained. Biochemical analyses showed that most of the active BcUGTs had broad substrate spectra; however, substrates lacking 5-OH were rarely catalyzed. Real-time quantitative PCR results further indicated that the transcriptional levels of BcUGTs were remarkably induced by Cu2+. Our study increases the understanding of UGTs and isoflavone biosynthesis in non-legume plants.

Transformation of Pueraria candollei var. mirifica phytoestrogens using immobilized and free β-glucosidase, a technique for enhancing estrogenic activity.

Pueraria candollei var. mirifica (PM) has a significant beneficial effect on postmenopausal symptoms associated with estrogen deficiency. However, the estrogenic activity and intestinal absorption of isoflavonoid glycosides derived from PM, such as daidzin and genistin, are significantly lower than those of their aglycones. To enhance the estrogenic activity of the PM extract, we developed β-glucosidase and its immobilized form to increase the PM aglycone content (daidzein and genistein). The enzyme immobilization was done by alginate beads, and the resulting β-glucosidase alginate beads have a diameter of about 0.20 cm. Response surface methodology (RSM) was used to optimize certain parameters, such as the pH, temperature, and ethanol concentration. The optimal conditions of β-glucosidase for daidzein and genistein production were pH of 4.8–4.9, a temperature in the range 46.3–49.1 °C, and ethanol concentration of 10.0–11.0%. The ANOVA results indicated that the design experiment involving free and immobilized β-glucosidase was the best fit by quadratic models, which had adjusted R2 values between 0.8625 and 0.9318. Immobilized β-glucosidase can be reused up to nine times and maintained efficacy of greater than 90%. Treatment of the PM extract with β-glucosidase increased the estrogenic activity of the PM extract by 8.71- to 23.2-fold compared to that of the untreated extract. Thus, β-glucosidase has a high potential for enhancing the estrogenic activity of PM constituents, and it can be applied on an industrial scale to increase the utility of these natural products.

Root Extracts From Ononis spinosa Inhibit IL-8 Release via Interactions With Toll-Like Receptor 4 and Lipopolysaccharide.

Extracts from the roots of Ononis spinosa L. (restharrow roots) are traditionally used for the treatment of patients with urinary tract infections due to its mild diuretic activity, caused by the inhibition of renal human hyaluronidase-1 by isoflavonoids. Preliminary studies also indicated anti-inflammatory effects. The following study aimed at investigating potential anti-inflammatory effects of restharrow extracts, prepared with solvents of different polarity. A dichloromethane extract (OS1), mainly composed of isoflavonoids and triterpenes as characterized by LC-MS, showed a concentration-dependent (25–100 μg/ml) inhibition of IL-8 and TNF-α release from LPS-stimulated human neutrophils. Significant inhibition was also found for the triterpene α-onocerin and the norneolignan clitorienolactone B, isolated from OS1. Further, OS1 and both compounds significantly decreased the expression of the adhesion molecules CD11b/CD18 and conversely increased the expression of CD62L in LPS-stimulated human neutrophils. This finding corresponds to a reduced inflammatory response by the inhibition of adhesion and migration of immune cells. As all of the observed effects are potentially mediated via Toll-like receptor 4 (TLR4) signaling, TLR4 transfected HEK293 cells were incubated with OS1. LPS-induced IL-8 secretion was significantly inhibited in a concentration-dependent manner, confirming TLR4 antagonism. This inhibition, however, was in part caused by an interaction of OS1 with LPS. In addition, also an aqueous extract containing high amounts of isoflavonoid glycosides and saponins from the roots of O. spinosa showed anti-inflammatory effects by interacting with the TLR4 signaling pathway. This study rationalizes the traditional use of extracts from O. spinosa for therapy of urinary tract infections, due to its potential anti-inflammatory effects that are mediated via TLR4 receptor antagonism.

Review of Medicinal uses, Phytochemistry and Biological Activities of Antidesma laciniatum and A. membranaceum

Antidesma laciniatum and A. membranaceum are small trees used as traditional medicines in tropical Africa. This extensive literature review synthesizes the information currently available on the medicinal uses, phytochemistry and biological activities of A. laciniatum and A. membranaceum. The university library and electronic search engines such as Google Scholar, Scopus, Web of Science, ScienceDirect, and PubMed were searched for pertinent information on the medicinal uses, phytochemistry, and biological activities of A. laciniatum and A. membranaceum. Traditionally, the species have been used as aphrodisiac, and traditional medicine for cough, kwashiorkor, mouth ulcers, pneumonia, prevent miscarriage, snakebites, stomachache and wounds. Various phytochemicals such as essential oils, isoflavonoid glycosides, phytosterols, benzopyranones, lignin glucosides, megastigmane, phenolics, steroids, squalene, terpenoids, triterpenoids, and tetrahydroisoquinoline alkaloids have been isolated from A. laciniatum and A. membranaceum. In vitro studies have confirmed the biological activities of A. laciniatum and A. membranaceum which, include antimicrobial, antioxidant, antiplasmodial, antitrypanosomal, leishmanicidal, molluscicidal and cytotoxicity activities. More pharmacological studies including phytochemical, toxicological, in vitro and in vivo experiments are needed to provide evidence for the clinical effectiveness of remedies prepared from the species.

Chemical Fingerprinting, Total Phenolics and Antioxidant Activity of Some Iris Taxa

Some Iris taxa covered in this study were used or are still being used as remedies in European folk medicine. The most common indication for the decoction of Iris rhizomes is sore throat and productive cough, especially in case of the taxon I. domestica (L.) Goldblatt & Mabb., the well-known remedy in traditional Chinese medicine. The aim of this study is to chemically characterize rhizomes of seven wild and medicinal Iris taxa and to assess the antioxidant activity of the rhizome extracts. The HPTLC chromatograms and densitometric spectrum scan of the spots were used for untargeted metabolic profiling and tentative metabolite class identification of Iris taxa. Total number of found spots is 41, three major spots are found in all taxa, and 22 spots are found to be taxon specific. Two xanthones, three isoflavonoid glycosides, 18 isoflavonoid aglycons (isoflavonoids), and five acetophenones/benzophenones are tentatively identified. The taxon I. pallida Lam. contains largest amount of phenolic substances (28.5 ± 2.0 mg GAE / g dry plant material) and was the most effective ABTS scavenger (27.5 ± 2.4 mg TROLOXE / g dry plant material). The taxon I. illyrica Tomm. ex Vis. was the most effective in both DPPH and FRAP assays, 10.1 ± 1.5 and 17.3 ± 0.1 mg TROLOXE / g dry plant material, respectively. The most effective taxa in the metal chelating assay were I. croatica Horvat & M.D.Horvat (6.1 ± 0.1 mg EDTAE / g dry plant material) and I. germanica var. florentina (L.) Dykes (5.8 ± 0.1 mg EDTAE / g dry plant material). European Iris taxa have a relatively similar and partly shared chemical profile with Asian taxon I. domestica, which might be a sign of equivalence in the medicinal value. This work is licensed under a Creative Commons Attribution 4.0 International License .

Structural Identification and Conversion Analysis of Malonyl Isoflavonoid Glycosides in Astragali Radix by HPLC Coupled with ESI-Q TOF/MS.

In this study, four malonyl isoflavonoid glycosides (MIGs), a type of isoflavonoid with poor structural stability, were efficiently isolated and purified from Astragali Radix by a medium pressure ODS C18 column chromatography. The structures of the four compounds were determined on the basis of NMR and literature analysis. Their major diagnostic fragment ions and fragmentation pathways were proposed in ESI/Q-TOF/MS positive mode. Using a target precursor ions scan, a total of 26 isoflavonoid compounds, including eleven malonyl isoflavonoid glycosides coupled with eight related isoflavonoid glycosides and seven aglycones were characterized from the methanolic extract of Astragali Radix. To clarify the relationship of MIGs and the ratio of transformation in Astragali Radix under different extraction conditions, two MIGs (calycosin-7-O-glycoside-6″-O-malonate and formononetin-7-O-glycoside-6″-O-malonate) coupled with related glycosides (calycosin-7-O-glycoside and formononetin-7-O-glycoside) and aglycones (calycosin and formononetin) were detected by a comprehensive HPLC-UV method. Results showed that MIGs could convert into related glycosides under elevated temperature conditions, which was further confirmed by the conversion experiment of MIGs reference compounds. Moreover, the total contents of MIGs and related glycosides displayed no obvious change during the long-duration extraction. These findings indicated that the quality of Astragali Radix could be evaluated efficiently and accurately by using the total content of MIGs and related glycosides as the quality index.

Isoflavones and Isoflavone Glycosides: Structural-Electronic Properties and Antioxidant Relations—A Case of DFT Study

Isoflavonoids and isoflavonoid glycosides have drawn much attention because of their antioxidant radical-scavenging capacity. Based on computational methods, we now present the antioxidant potential results of genistein (1), biochanin A (2), ambocin (3), and tectorigenin 7-O-[β-D-apiofuranosyl-(1-6)-β-D-glucopyranoside] (4). The optimized structures of the neutral and radical forms have been determined by the DFT-B3LYP method with the 6-311G(d) basis set. From the findings and thermodynamic point of view, the ring B system of isoflavones is considered as an active center in facilitating antioxidant reactions. Antioxidant activities are mostly driven by O-H bond dissociation enthalpy (BDE) following hydrogen atom transfer (HAT) mechanism. Antioxidant ability can be arranged in the following order: compounds (4) > (3) > (2) > (1). Of comprehensive structural analysis, flavonoids with 4′-methylation and 6-methoxylation, especially 7-glycosylation would claim responsibility for antioxidant enhancement.

Renoprotective effect of tectorigenin glycosides isolated from Iris spuria L. (Zeal) against hyperoxaluria and hyperglycemia in NRK-49Fcells

Oxidative stress has been identified as an underlying factor in the development of insulin resistance, β-cell dysfunction, impaired glucose tolerance and type 2 diabetes mellitus and it also play major role in kidney stone formation. The present study is aimed to elucidate the in vitro nephroprotective activity of two isoflavonoid glycosides, tectorigenin 7-O-β-D-glucosyl-(1→6)-β-D-glucoside (1) and tectorigenin 7-O-β-D-glucosyl-4′-O-β-D-glucoside (2) isolated from the n-BuOH fraction of Iris spuria L. (Zeal) rhizome MeOH extract against oxalate and high glucose-induced oxidative stress in NRK-49F cells. The results revealed that compounds 1 and 2 significantly increased the antioxidant enzyme activities and decreased MDA levels in both oxalate and high glucose stress. Treatment with these phytochemicals effectively down-regulated expression of crystal modulator genes and pro-fibrotic genes in oxalate and high glucose-mediated stress respectively. This study indicates cytoprotective, antioxidant, anti-urolithic and anti-diabetic effects of compounds 1 and 2 against oxalate and high glucose stress.

Three New Isoflavonoid Glycosides from the Mangrove-Derived Actinomycete Micromonospora aurantiaca 110B.

The mangrove ecosystem is a rich resource for the discovery of actinomycetes with potential applications in pharmaceutical science. Besides the genus Streptomyces, Micromonospora is also a source of new bioactive agents. We screened Micromonospora from the rhizosphere soil of mangrove plants in Fujian province, China, and 51 strains were obtained. Among them, the extracts of 12 isolates inhibited the growth of human lung carcinoma A549 cells. Strain 110B exhibited better cytotoxic activity, and its bioactive constituents were investigated. Consequently, three new isoflavonoid glycosides, daidzein-4′-(2-deoxy-α-l-fucopyranoside) (1), daidzein-7-(2-deoxy-α-l-fucopyranoside) (2), and daidzein-4′,7-di-(2-deoxy-α-l-fucopyranoside) (3) were isolated from the fermentation broth of strain 110B. The structures of the new compounds were determined by spectroscopic methods, including 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESIMS). The result of medium-changing experiments implicated that these new compounds were microbial biotransformation products of strain M. aurantiaca 110B. The three compounds displayed moderate cytotoxic activity to the human lung carcinoma cell line A549, hepatocellular liver carcinoma cell line HepG2, and the human colon tumor cell line HCT116, whereas none of them showed antifungal or antibacterial activities.

A new phthalazinone derivative and a new isoflavonoid glycoside from lichen-associated Amycolatopsis sp.

A new phthalazinone derivative, named amycophthalazinone A (1), and a new isoflavonoid glycoside, 7-O-methyl-5-O-α-L-rhamnopyranosylgenestein (2), along with an isoflavonoid glycoside, 7-O-α-D-arabinofuranosyl daidzein (3) firstly found from natural sources, and eight known compounds (4–11), were isolated from the culture broth of the lichen-associated Amycolatopsis sp. YIM 130642. The structures of new compounds were elucidated on the basis of spectroscopic analysis. Compound 1 was the first example of naturally occurring phthalazinone derivative. The antimicrobial activities of all compounds towards five pathogenic strains were evaluated by a broth microdilution assay. Compound 1 exhibited the most potent inhibitory activity against Staphylococcus aureus, Salmonella typhi, and Candida albicans with MIC values of 32, 32, and 64 μg/mL, respectively.

Chemical Identification of Isoflavonoids from a Termite-Associated Streptomyces sp. RB1 and Their Neuroprotective Effects in Murine Hippocampal HT22 Cell Line.

Insect-associated bacteria have been recognized as a very promising natural resource for discovering bioactive secondary metabolites with diverse pharmacological effects. One new isoflavonoid glycoside, termisoflavone D (1), together with seven known isoflavonoids (2–8), were identified from MeOH extracts of the fungus-growing termite-associated Streptomyces sp. RB1. The chemical structure of the new compound 1 was elucidated using comprehensive spectroscopic methods including 1D and 2D NMR, along with LC/MS analysis. The existence of two rhamnose moieties in 1 was determined with comparative NMR analysis, and the absolute configuration was elucidated using chemical reactions. The neuroprotective activities of compounds 1–8 were thoroughly investigated using the murine hippocampal HT22 cell line. Compound 5 prevented glutamate-induced HT22 cell death by blocking intracellular reactive oxygen species (ROS) accumulation. The present study provides the first experimental evidence for the potential use of isoflavonoids from termite-associated bacteria as lead compounds that can prevent neuronal damage induced by glutamate.

Termisoflavones A-C, Isoflavonoid Glycosides from Termite-Associated Streptomyces sp. RB1.

Three new isoflavonoid glycosides, termisoflavones A-C (1-3), and eight isoflavonoids (4-11) were isolated from termite-associated Streptomyces sp. RB1 recovered from the cuticle of the South African termite, Macrotermes natalensis. The structures of new compounds were determined by spectroscopic methods including 1D and 2D NMR and HR-MS analysis, as well as comparison of their NMR data with those of related isoflavonoid glycoside derivatives. The absolute configurations of the sugar moieties were clarified by chemical reactions. None of the isolates (1-11) displayed antifungal or antimicrobial activities (MICs > 100 μg/mL), whereas compounds 6 and 11 ameliorated cisplatin-induced kidney cell damage to 80% of the control value at a cisplatin dose of 25 μM.

Antioxidant α-amylase inhibitors flavonoids from Iris germanica rhizomes

A new isoflavonoid glycoside, iridin A (9), along with eight known isoflavonoids: irilone 4′-methyl ether (1), irilone (2), irisolidone (3), irigenin S (4), irigenin (5), irilone 4′-O-β-d-glucopyranoside (6), iridin S (7), and iridin (8) were separated from Iris×germanica L., Iridaceae, rhizomes. The structural elucidation of these flavonoids was achieved with the aid of extensive spectroscopic techniques and comparing with the published data. They were estimated for their α-amylase and 1,1-diphenyl-2-picrylhydrazyl inhibitory capacities. Compounds 3, 5, and 9 showed α-amylase inhibitory activities with % inhibition 70.8, 67.5, and 70.5, respectively compared to acarbose (a reference α-amylase inhibitor). Moreover, 9 exhibited moderate antioxidant activity with IC50 8.91μM.

Molecular Cloning and Functional Characterization of a Novel (Iso)flavone 4',7-O-diglucoside Glucosyltransferase from Pueraria lobata.

Pueraria lobata roots accumulate a rich source of isoflavonoid glycosides, including 7-O- and 4′-O-mono-glucosides, and 4′,7-O-diglucosides, which have numerous human health benefits. Although, isoflavonoid 7-O-glucosyltranferases (7-O-UGTs) have been well-characterized at molecular levels in legume plants, genes, or enzymes that are required for isoflavonoid 4′-O- and 4′,7-O-glucosylation have not been identified in P. lobata to date. Especially for the 4′,7-O-di-glucosylations, the genetic control for this tailing process has never been elucidated from any plant species. Through transcriptome mining, we describe here the identification and characterization of a novel UGT (designated PlUGT2) governing the isoflavonoid 4′,7-O-di-glucosylations in P. lobata. Biochemical roles of PlUGT2 were assessed by in vitro assays with PlUGT2 protein produced in Escherichia coli and analyzed for its qualitative substrate specificity. PlUGT2 was active with various (iso)flavonoid acceptors, catalyzing consecutive glucosylation activities at their O-4′ and O-7 positions. PlUGT2 was most active with genistein, a general isoflavone in legume plants. Real-time PCR analysis showed that PlUGT2 is preferentially transcribed in roots relative to other organs of P. lobata, which is coincident with the accumulation pattern of 4′-O-glucosides and 4′,7-O-diglucosides in P. lobata. The identification of PlUGT2 would help to decipher the P. lobata isoflavonoid glucosylations in vivo and may provide a useful enzyme catalyst for an efficient biotransformation of isoflavones or other natural products for food or pharmacological purposes.

Characterization and identification of isoflavonoid glycosides in the root of Spiny restharrow (Ononis spinosa L.) by HPLC-QTOF-MS, HPLC–MS/MS and NMR

Restharrow root has been used in traditional medicine for thousands of years; however, the active ingredients responsible for the diuretic effect are still unknown. Previous studies have proved that the root extract contains isoflavonoids, however only few derivatives were identified, mostly relying on retention times or UV data. The aim of our work was to perform a detailed structural characterization of the complete isoflavonoid profile in the aqueous-methanolic extract of Ononis spinosa root by high-performance liquid chromatography coupled with electrospray ionization accurate-mass quadrupole time-of-flight and tandem mass spectrometry in positive ionization mode (HPLC-ESI-QTOF-MS, HPLC-ESI–MS/MS) and nuclear magnetic resonance spectroscopy (NMR). On the basis of the accurate masses and fragmentation patterns isoflavones (formononetin, calycosin and pseudobaptigenin) and pterocarpans (maackiain and medicarpin) were identified. Two further dihydroisoflavone aglycones, namely onogenin and sativanone and a unique glucoside were isolated and their structures were elucidated by NMR experiments. Calycosin, onogenin and sativanone were detected in this plant for the first time. In contrast to previous works, the presence of biochanin A could not be confirmed, however its regioisomer calycosin and its derivatives were identified. Similarly, neither tectorigenin derivatives could be detected, however the isobar compound sativanone and its various glucosides were elucidated. The presence of genistein and daidzein could not be confirmed in the extract. Fragmentation pathways for onogenin and sativanone are presented. In the aqueous-methanolic extract 9 glucosides, 6 minor and 8 major glucoside malonates, 4 glucoside acetates and 7 aglycones were found. In total, 34 compounds were successfully identified.

A new isoflavonoid glycoside and other constituents from Tilia amurensis with anti-neuroinflammatory activity

Tilia amuresnsis Rupr. is a tree traditionally used in Korean medicine. Its leaves have been used as tea for health purposes [1]. As part of our ongoing search for bioactive constituents of natural Korean medicinal resources, we found in a preliminary study that the MeOH extract from the trunks of T. amurensis showed an inhibitory effect on nitric oxide (NO) production in an activated murine microglial cell line. A bioassay-guided fractionation and chemical investigation of the MeOH extract resulted in the isolation and identification of a new isoflavonoid glycoside, orobol 4'-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (1) and 16 known compounds (2-17). The structure of the new compound was determined by spectroscopic methods, namely 1D and 2D NMR techniques, HRMS, and chemical methods. Anti-neuroinflammatory activities of the isolated compounds were determined by measuring NO levels in the medium using murine microglia BV-2 cells. Among them, twelve compounds, including compound 1 (most active with an IC50 value of 23.42 µM), inhibited NO production in lipopolysaccharide-stimulated BV-2. Moreover, compounds 1-4 showed moderate anti-proliferative activities against the SK-MEL-2 cell line with IC50 values ranging from 12.31 to 19.67 µM.