Analysis and identification of phenolic compounds with antiproliferative activity from Chinese olive (Canarium album L.) fruit extract by HPLC-DAD-SPE-TT-NMR.

Chinese olives (Canarium album L.) are rich in phenolic compounds, exhibiting a broad spectrum of potential clinical applications. This study is the first report on the isolation and elucidation of bioactive compounds with high antiproliferative activity from the ethyl acetate fraction of a Chinese olive fruit methanolic extract (CO-EtOAc). We used the WST-1 assay to determine which subfractions of CO-EtOAc had significant antiproliferative activity using the murine colon cancer cell line CT26. Subsequently, the functional compounds were characterized by the hyphenated technique and high-performance liquid chromatography-diode array detector-solid phase extraction-transfer tube-nuclear magnetic resonance (HPLC-DAD-SPE-TT-NMR). Thirteen phenolic constituents were identified from the antiproliferation-enhancing subfractions of CO-EtOAc, including two new compounds, 2,4-didehydrochebulic acid 1,7-dimethyl ester (5) and 1-hydroxybrevifolin (7), which were further purified and found to exhibit marked antiproliferative activity. Chebulic acid dimethyl ester (2), which was isolated from C. album for the first time, also possessed antiproliferative activity.

Isolation of GLP-1 enhancing indolizidine alkaloids from Boehmeria formosana.

Boehmeria formosana, with its related species, demonstrates anti-glycemic effect, inhibition of HBV production, anti-cancer activities, etc. Some indolizidine alkaloids from the same genus are bioactive but sensitive to light. To overcome this problem and obtain more phenanthroindolizidine alkaloids, isolation was performed in darkness, yielding 10 new indolizidine alkaloids and 17 known compounds. Among them, seven enhanced glucagon-like receptor 1 (GLP-1) activity at 50 mM, especially 14 and 6 (3.5- and 2.3-fold than the negative control). This procedure yielded bioactive indolizidine alkaloids with novel structures.

In silico design and synthesis of N-arylalkanyl 2-naphthamides as a new class of non-purine xanthine oxidase inhibitors.

A series of N-arylalkanyl 2-naphthamides (Xa~e), which were predicted from virtual molecular docking on a built xanthine oxidase template as potential inhibitors, were synthesized. Their inhibitory activity against xanthine oxidase was assayed. Among these prepared, compounds Xb (IC50 13.6 µM), Xc (IC50 13.1 µM), and Xd (IC50 12.5 µM) showed comparable inhibitory activity to allopurinol (IC50 22.1 µM). The in vitro assay result correlated well with molecular docking scores, ΔG = -16.99, -17.66, and -17.13 Kcal/mol, respectively. On the potassium oxonate-induced hyperuricemic mice model, oral administration of Xc-Ac (40 mg/ Kg), the per-O-acetylated Xc, could reduce the blood uric acid level by 60% in comparison to the normal control group and is statistically significant (p < .01) while compared with the hyperuricemic mice group.

Characterization of a fluorescent glucose derivative 1-NBDG and its application in the identification of natural SGLT1/2 inhibitors.

Glucose is an important energy source for cells. Glucose transport is mediated by two types of glucose transporters: the active sodium-coupled glucose cotransporters (SGLTs), and the passive glucose transporters (GLUTs). Development of an easy way to detect glucose uptake by the cell can be valuable for research. 1-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-1-deoxy-d-glucose (1-NBDG) is a newly synthesized fluorescent glucose analogue. Unlike 2-NBDG, which is a good substrate of GLUTs but not SGLTs, 1-NBDG can be transported by both GLUTs and SGLTs. Thus, 1-NBDG is useful for the screening of SGLT1 and SGLT2 inhibitors. Here we further characterized 1-NBDG and compared it with 2-NBDG. The fluorescence of both 1-NBDG and 2-NBDG was quenched under alkaline conditions, but only 1-NBDG fluorescence could be restored upon neutralization. HPLC analysis revealed that 2-NBDG was decomposed leading to loss of fluorescence, whereas 1-NBDG remained intact in a NaOH solution. Thus, after cellular uptake, 1-NBDG fluorescence can be detected on a plate reader simply by cell lysis in a NaOH solution followed by neutralization with an HCl solution. The fluorescence stability of 1-NBDG was stable for up to 5 h once cells were lysed; however, similar to 2-NBDG, intracellular 1-NBDG was not stable and the fluorescence diminished substantially within one hour. 1-NBDG uptake could also be detected at the single cell level and inhibition of 1-NBDG uptake by SGLT inhibitors could be detected by flow cytometry. Furthermore, 1-NBDG was successfully used in a high-throughput cell-based method to screen for potential SGLT1 and SGLT2 inhibitors. The SGLT inhibitory activities of 67 flavonoids and flavonoid glycosides purified from plants were evaluated and several selective SGLT1, selective SGLT2, as well as dual SGLT1/2 inhibitors were identified. Structure-activity relationship analysis revealed that glycosyl residues were crucial since the aglycon showed no SGLT inhibitory activities. In addition, the sugar inter-linkage and their substitution positions to the aglycon affected not only the inhibitory activities but also the selectivity toward SGLT1 and SGLT2.

Doubled production of cordycepin analogs in cultured Cordyceps militaris by addition of Andrea droppings.

Cordyceps sinensis is a traditional Chinese medicine with various biological activities. With its limited natural supply, cultured C. militaris has become the major alternative source, and the culture conditions may affect the chemical compositions. To improve the production of chemical ingredients, C. militaris was cultured with three different media, including rice only, rice plus 3% tea leaves, and rice plus 3% droppings of Andraca theae. The fractions of dried C. militaris cultured with rice were chromatographic separated to afford ten compounds: phenylalanine, dimerumic acid, nicotinic acid, tryptophan, N6-(2-hydroxyethyl)-adenosine, uracil, uridine, cordycepin, ergosterol, and mannitol. Of these, in the cultured medium of rice plus 3% Andraca droppings, the amount of one major compound cordycepin is about two folds than the highest reported data, and dimerumic acid and N6-(2-hydroxyethyl)-adenosine were isolated for the first time from this species.[Figure: see text].

Design, synthesis and the structure-activity relationship of agonists targeting on the ALDH2 catalytic tunnel.

ALDH2, a key enzyme in the alcohol metabolism process, detoxifies several kinds of toxic small molecular aldehydes, which induce severe organ damages. The development of novel Alda-1 type ALDH2 activators was mostly relied on HTS but not rational design so far. To clarify the structure-activity relationship (SAR) of the skeleton of Alda-1 analogs by synthesis of the least number of analogs, we prepared 31 Alda-1 analogs and 3 isoflavone derivatives and evaluated for their ALDH2-activating activity. Among these, the ALDH2-activating activity of mono-halogen-substituted (Cl and Br) N-piperonylbenzamides 3b and 3 k, and non-aromatic amides 8a-8c, were 1.5-2.1 folds higher than that of Alda-1 at 20 µM. The relationship between binding affinity in computer aided molecular docking model and the ALDH2-activating activity assays were clarified as follows: for Alda-1 analogs, with the formation of halogen bonds, the enzyme-activating activity was found to follow a specific regression curve within the range between -5 kcal/mol and -4 kcal/mol. For isoflavone derivatives, the basic moiety on the B ring enhance the activating activity. These results provide a new direction of utilizing computer-aided modeling to design novel ALDH2 agonists in the future.

Microbial Transformations of Two Beyerane-Type Diterpenes by Cunninghamella echinulata.

Microbial transformations of two tetracyclic beyerane-type diterpenes, ent-16ß-oxobeyeran-19-oic acid (1) and its chemical reduction product, ent-16ß-hydroxybeyeran-19-oic acid (2), by the filamentous fungus Cunninghamella echinulata ATCC 8688a yielded eight metabolites (3-10). Incubation of the substrate 2 with C. echinulata afforded three new hydroxylated ones (3-5) along with two known ones (6-7), while incubation of 1 gave three known ones (8-10). The new compounds were characterized by 1D and 2D NMR as well as HRESIMS analysis, and the stereostructures of 3 and 4 were confirmed by X-ray crystallography. The bioreactions were involved not only in stereoselective incorporation of hydroxyl groups at inert positions C-7, -9, -12, and -14 of the two beyerane diterpenes but also in glucosidation at C-19 of 2. This is the first report on the biotransformation of the diterpenes by using C. echinulata. All compounds were assayed for their α-glucosidase inhibitory, neurotrophic, anti-inflammatory, and phytotoxic activity, and only in neurotrophic assay compounds, 2 and 9 were found to display nerve growth factor-mediated neurite-outgrowth promoting effects in PC12 cells; the others were inactive.

Diastereomeric identification of neolignan rhamnosides from Trochodendron aralioides leaves by LC-SPE-NMR and circular dichroism.

Trochodendron aralioides is an old-existing relic plant with limited availability and only a few identified compounds. Accumulative analysis on the methanolic extract from its leaf part by LC-SPE-NMR resulted in the identification of seven new compounds, including three neolignan α-rhamnosides [(7R,8S)-dihydrodehydrodiconiferyl alcohol- 9-O-α-rhamnopyranoside (2) and 9'-O-α-rhamnopyranoside (3), and (7S,8R)-dehydrodiconiferyl alcohol-9'-O-α-rhamnopyranoside (4)], two isomeric oxyneolignan α-rhamnosides [(7R,8S)- (5) and (7R,8R)-icariside E8 (6)), and (7R,8S)- (10) and (7R,8R)-icariside E9 (11)], and two isomeric acylated fructofuranosyl mevalonolactones (13, 14), along with five known compounds (1, 7-9 and 12). The absolute configuration of the C-7 and C-8 positions for the new compounds 2-6 and 10-11 was assigned by comparison of the reported ECD spectra. Compounds 2, 3, 4, and 6 were further isolated by semi-preparative column chromatography for structure confirmation by 2D NMR spectroscopic analysis.

Chemical investigation on the root bark of Bombax malabarica.

Ten compounds were isolated from the root bark of Bombax malabarica, including two new compounds, bombamalin (1) and 3,4,5-trimethoxyphenol-1-[ß-xylopyranosyl-(1 → 2)]-ß-glucopyranoside (3), and shorealactone (4), a rare dehydroascorbic acid fused l-ε-viniferin. Compound 1 is an unusual bissesquiterpene, containing a 1,4-dioxene ring formed by fusing isohemigossypol with ent-cadinen-2-one. Their structures were elucidated by extensive spectroscopic analysis. This chemical reinvestigation is of value for chemotaxonomy of the Bombax genus, in particular the finding of the unusual 1 and rare 4. Among the isolates, shorealactone (4), l-epicatechin 5-O-ß-D-xyloside (5), and 2-C-[ß-D-apiosyl-(1 → 6)]- ß-D-glucosyl]-1,3,6-trihydroxy-7-methoxyxanthone (6) displayed some inhibition against α-glucosidase with IC50 values of 224, 345, and 285 µM, respectively.

Chemical investigation of Hyptis suaveolens seed, a potential antihyperuricemic nutraceutical, with assistance of HPLC-SPE-NMR.

The seed of Hyptis suaveolens, commonly known as wild flour ball (san fen yuan) in Taiwan, serves as a main refreshing drink substance in several regions. This study investigated firstly its secondary metabolites, leading to the isolation of five major caffeoylquinic acid derivatives (1-5) from the ethanol extract. In addition, ten minors, including three caffeoylquinic acid derivatives (12-14), were characterized via assistance of HPLC-SPE-NMR. Of these isolates, sodium 4,5-dicaffeoylquinate (2) and methyl 3,5-dicaffeoylquinate (4) showed moderate inhibitory activity against xanthine oxidase with the respective IC50 values of 69.4 µM and 92.1 µM (c.f. allopurinol IC50 28.4 µM). Quantitative HPLC analysis of the EtOH extract indicates the content of sodium 3,5-dicaffeoylquinate (1) and sodium 4,5-dicaffeoylquinate (2) to be 0.1% and 0.08% (w/w, dry seed), respectively. This study not only discloses the bioactive constituents, but also demonstrates the potential of H. suaveolens seed as an antihyperuricemic nutraceutical.

Cytotoxic protobassic acid saponins from the kernels of Palaquium formosanum.

Bioassay guided fractionation and separation of the EtOH extract of the kernels of Palaquium formosanum against PC-3 cells via Sephadex LH-20 and reverse phase C-18 columns led to the isolation of 13 protobassic saponins. One of these saponins is new and was characterized as 3‴-O-rhamnopyranosyl-arganin C, a bisdesmoside of 16α-hydroxyprotobassic acid at the C-3 and C-28 positions. The structures of these compounds were determined on the basis of 1D NMR (1H, 13C), 2D NMR (1H-1H COSY, HSQC, HMBC, and NOESY), and selectively excited 1D TOCSY spectroscopic analyses and MS data, and comparison with literature data. Bioassay of these compounds and five additional compounds, isolated from Planchonella obovata leaf, against PC-3 prostate cancer cells indicated arganin C to be the most potent one with the IC50 value of 13.8 µM. Some structure and activity relationships were also drawn.

Tea silkworm droppings as an enriched source of tea flavonoids.

Andraca droppings is the waste excreted from the tea biter Andraca theae. Its chemical constituents and potential medical use, unlike those of the traditional Chinese medicine silkworm droppings, have not been reported yet. To explore new nutraceuticals, the chemical constituents of this substance were investigated. Since the bioactive ingredients are generally present in the EtOAc-soluble fraction, this fraction, obtained from the ethanolic extract of the dried Andraca droppings by liquid-liquid partitioning, was separated by chromatographic methods, including Sephadex LH-20, centrifugal partition chromatography, and RP-18 columns, to produce 14 compounds (1-14). They were characterized as 1,7-dimethyl xanthine (1), three benzoic acids (2, 3, and 5), and 10 flavonoids (4, 6-14). The amount of compounds 6, 7, 10, 13, and 14 in the droppings were 1.7-15.5-fold compared to those of tea leaves. In addition, 1,7-dimethyl xanthine (1) was found present only in the Andraca droppings but absent in tea leaves. Therefore, except for compound 1, which might be transformed from caffeine by microflora in the insect, the compounds were believed not to be absorbed by the worm gut and excreted directly. The present study suggests the Andraca droppings are an enriched source of the bioactive flavonoids from tea leaves and are potential as a useful nutraceutical.

A Novel 3,4-Secocycloartane Triterpene, Bearing a Tetrahydropyran Moiety, from Pasaniaformosana Leaf.

Bioassay guided fractionation against a-glucosidase and separation of the ethanolic extract of Pasania formosana leaf by chromatographic methods led to the isolation of a novel secocycloartane triterpene. This compound, named pasasecocycloartenoic acid, was elucidated as 21,24(R)-epoxy-25-hydroty-3;4- secocycloart-4(28)-en-3-oic acid through analysis of 1D and 2D NMR spectra and on the basis of HRESIMS data. The compound showed weak activity against α-glucosidase, but its poor solubility hampered the bioassay.

Peracylated Glucosyl Kaempferols from Pasania dodonfifolia Leaf.

Phytochemical investigation of the ethanolic extract of Pasania dodoniifolia leaf led to the isolation of four kaempferol 3-0-peracylated glucosides (1-4), together with four flavonoid glucosides (5-8), epicatechin (9), and (7S, 7'S, 8R, 8'R)-icariol A2 (10). Of these, kaempferol-3-O-(3",4"-di-O-acetyl-2"-O-(Z)-p- coumaroyl)-6"-O-(E)-p-coumaroyl)-beta-glucopyranoside (3) and 3-O-(3",4"-di-O-acetyl-2",6"-di-O-(Z)-p-coumaroyl)-beta-glucopyranoside (4) are new and their structures were elucidated by 2D NMR spectroscopic analyses and MS data.

Zerumbone, a ginger sesquiterpene, induces apoptosis and autophagy in human hormone-refractory prostate cancers through tubulin binding and crosstalk between endoplasmic reticulum stress and mitochondrial insult.

Zerumbone, a natural monocyclic sesquiterpene, is the main component of the tropical plant Zingiber zerumbet Smith. Zerumbone induced antiproliferative and apoptotic effects against PC-3 and DU-145, two human hormone-refractory prostate cancer (HRPC) cell lines. Zerumbone inhibited microtubule assembly and induced an increase of MPM-2 expression (specific recognition of mitotic proteins). It also caused an increase of phosphorylation of Bcl-2 and Bcl-xL, two key events in tubulin-binding effect, indicating tubulin-binding capability and mitotic arrest to zerumbone action. Furthermore, zerumbone induced several cellular effects distinct from tubulin-binding properties. First, zerumbone significantly increased, while paclitaxel (as a tubulin-binding control) decreased, Mcl-1 protein expression. Second, paclitaxel but not zerumbone induced Cdk1 activity. Third, zerumbone other than paclitaxel induced Cdc25C downregulation. The data suggest that, in addition to targeting tubulin/microtubule, zerumbone may act on other targets for signaling transduction. Zerumbone induced mitochondrial damage and endoplasmic reticulum (ER) stress as evidenced by the loss of mitochondrial membrane potential and upregulation of GRP-78 and CHOP/GADD153 expression. Zerumbone induced an increase of intracellular Ca(2+) levels, a crosstalk marker between ER stress and mitochondrial insult, associated with the formation of active calpain I fragment. It induced apoptosis through a caspase-dependent way and caused autophagy as evidenced by dramatic LC3-II formation. In summary, the data suggest that zerumbone is a multiple targeting compound that inhibits tubulin assembly and induces a crosstalk between ER stress and mitochondrial insult, leading to apoptosis and autophagy in HRPCs.

Anti-Acetylcholinesterase Alkaloids from Annona glabra Leaf.

Bioassay guided fractionation and separation of the EtOH extract of Annona glabra leaf against acetylcholinesterse led to the characterization of 15 alkaloids. Among them, (-)-actinodaphnine (2) and (-)-(6aS,7R)-7-hydroxyactinodaphnine (9) are new aporphines, although (+)-2 and (±)-2 have been found in several plants. Their structures were established by spectroscopic analysis. (-)-Anolobine (5) and (-)-roemeroline (8) showed moderate inhibitory activity against eel acetylcholinesterase with IC50 values of 22.4 and 26.3 µM, respectively.

Identification of the metabolites of TCM prescription Sinisan, found in miniature pig urine via intragastric administration.

The metabolic profile of the traditional Chinese medicine, Sinisan, in miniature pig urine via intragastric administration was investigated. In total, 50 compounds, including 10 unchanged parent glycosides, which were not found from Sinisan's metabolic profile in rats' urine, were identified. Among these, 36 compounds were characterized by HPLC-SPE-NMR coupled with HPLC-HRESIMS, five of which are new and nine are endogenous metabolites of miniature pig. Most of phase I and phase II metabolites are hydrolytic products of parent glycosides and glucuronide conjugates, respectively, the latter having been reported as sulfate conjugates while the experimental animal is rat. Benzoic acid, obtained from hydrolysis of albiflorin and paeoniflorin, and phenylpropenoic acids, obtained from oxidative cleavage of flavones, formed phase II glycine conjugates.

Acylated glucosylflavones as α-glucosidase inhibitors from Tinospora crispa leaf.

The ethanolic extract of Tinospora crispa leaf had shown inhibitory activity toward α-glucosidase. Bioassay guided fractionation and separation of this extract led to the isolation of 17 flavonoids. Among them, four acylated glycosylflavonoids (6, 8, 9, 15) are new compounds. Their structures were elucidated on the basis of spectroscopic analysis. Among the isolated compounds, isovitexin 2″-(E)-p-coumarate (8) showed the best activity against α-glucosidase with an IC50 value of 4.3±1.4µM. However, isoorientin 2″-(E)-p-coumarate (7), the 3'-hydroxylated 8, is much less active (IC50 35.7µM). Such significant difference was rationalized by CAD study on α-glucosidase.

Flavonoids from Curcuma longa leaves and their NMR assignments.

Chemical investigation of the n-BuOH-soluble fraction of the EtOH extract of the aerial part of Curcuma longa led to the isolation of 11 flavonol glycosides and one dihydroflavonol glucoside (1) via chromatography over Sephadex LH-20 and Lobar RP-18 columns. Although they are known, the 1H and 13C NMR data recorded in CD3OD rather than the common DMSO-d6 are doubly checked via extensive 2D NMR spectroscopic analyses, leading to some revisions of the reported data, especially for the glycon part.

Houttuynia cordata targets the beginning stage of herpes simplex virus infection.

Herpes simplex virus (HSV), a common latent virus in humans, causes certain severe diseases. Extensive use of acyclovir (ACV) results in the development of drug-resistant HSV strains, hence, there is an urgent need to develop new drugs to treat HSV infection. Houttuynia cordata (H. cordata), a natural herbal medicine, has been reported to exhibit anti-HSV effects which is partly NF-κB-dependent. However, the molecular mechanisms by which H. cordata inhibits HSV infection are not elucidated thoroughly. Here, we report that H. cordata water extracts (HCWEs) inhibit the infection of HSV-1, HSV-2, and acyclovir-resistant HSV-1 mainly via blocking viral binding and penetration in the beginning of infection. HCWEs also suppress HSV replication. Furthermore, HCWEs attenuate the first-wave of NF-κB activation, which is essential for viral gene expressions. Further analysis of six compounds in HCWEs revealed that quercetin and isoquercitrin inhibit NF-κB activation and additionally, quercetin also has an inhibitory effect on viral entry. These results indicate that HCWEs can inhibit HSV infection through multiple mechanisms and could be a potential lead for development of new drugs for treating HSV.