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.

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].

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 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.

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.

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.

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.

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.

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.

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.

Neolignans as xanthine oxidase inhibitors from Hyptis rhomboides.

The active fraction from the EtOH extract of Hyptis rhomboides against xanthine oxidase was identified by use of an HPLC microfractionation-centrifugal vacuum evaporation-bioassay hyphenated technique. Scale-up separation of the active subfractions using semi-preparative RP-HPLC provided 13 phenylpropanoid compounds, including O-styrenylneolignans, hyprhombins A-C, epihyprhombin B, and hyprhombin B methyl ester, and O-caffeoylnorneolignans, hyprhombins D and E. All of these compounds shared a common 1,4-benzodioxane skeleton, as established by spectroscopic analyses. Hyprhombin C and epihyprhombin B exhibited better anti-xanthine oxidase activity than allopurinol, with IC50 values of 0.6 and 2.0 µM, respectively.

Chemical constituents from Dendropanax dentiger.

Nineteen compounds and an HPLC inseparable mixture, composed of compounds 20 and 21, were isolated from the leaves and twigs of Dendropanax dentiger (Harms ex Diels) Merr. Of these, syringin (1) is the most abundant, 6'-O-apiofuranosyl dendranthemoside A (16) is a new megastigmane glycoside, and 3-methoxy-D-mannono-1,4-lactone (21) is a new hexono-1,4-lactone. Their structures were elucidated based on NMR spectroscopic and MS analyses.

Metabolomic analysis of complex chinese remedies: examples of induced nephrotoxicity in the mouse from a series of remedies containing aristolochic Acid.

Aristolochic acid nephropathy is caused by aristolochic acid (AA) and AA-containing herbs. In traditional Chinese medicine, a principle called "Jun-Chen-Zou-Shi" may be utilized to construct a remedial herbal formula that attempts to mitigate the toxicity of the main ingredient. This study used Bu-Fei-A-Jiao-Tang (BFAJT) to test if the compound remedy based on a principle of "Jun-Chen-Zou-Shi" can decrease the toxicity of AA-containing herbs. We compared the three toxicities of AA standard, Madouling (an Aristolochia herb), and a herbal formula BFAJT. AA standard was given for BALB/c mice at a dose of 5 mg/kg bw/day or 7.5 mg/kg bw/day for 10 days. Madouling and BFAJT were given at an equivalence of AA 0.5 mg/kg bw/day for 21 days. Nephrotoxicity was evaluated by metabolomics and histopathology. The urinary metabolomics profiles were characterized by (1)H NMR spectroscopy. The spectral data was analyzed with partial least squares discriminant analysis, and the significant differential metabolites between groups were identified. The result showed different degrees of acute renal tubular injuries, and metabolomics analysis found that the kidney injuries were focused in proximal renal tubules. Both metabolomics and pathological studies revealed that AA standard, Madouling, and BFAJT were all nephrotoxicants. The compositions of the compound remedy did not diminish the nephrotoxicity caused by AA.

Hypoglycemic action of borapetoside A from the plant Tinospora crispa in mice.

AIM: This study explores the hypoglycemic effects of borapetoside A, the most active principle among three major diterpenoids (borapetosides A, B, and C) isolated from ethanol extract of Tinospora crispa vines. METHODS: We employed mouse mitogenic C2C12 and hepatocellular carcinoma Hep3B cells in this study. Furthermore, the mice were divided into three groups, including streptozotocin-induced type 1 diabetes mellitus, diet-induced type 2 diabetes mellitus, and normal control. The mice in each group were treated with assigned vehicle control, borapetoside A, or other active agents. RESULTS: Borapetoside A was shown to increase the glycogen content and decrease the plasma glucose concentration in a concentration or dose-dependent manner in vitro and in vivo. The hypoglycemic effects in the normal mice and the mice with type 2 diabetes mellitus were associated with the increases of the plasma insulin levels; whereas, the insulin levels remained unchanged in the mice with type 1 diabetes mellitus. Borapetoside A not only attenuated the elevation of plasma glucose induced by an intraperitoneal glucose tolerance test, but also increased the glycogen synthesis of IL-6 treated C2C12 cells. Moreover, the elevated protein expression levels of phosphoenolpyruvate carboxykinase were reversed after borapetoside A treatment twice a day for 7 days. CONCLUSIONS: The hypoglycemic effects of borapetoside A were mediated through both the insulin-dependent and the insulin-independent pathways. Furthermore, borapetoside A was shown to increase the glucose utilization in peripheral tissues, to reduce the hepatic gluconeogenesis, and to activate the insulin signaling pathway; they thereby contributed to the lowering of the plasma glucose. Comparison of the structures of three borapetosides suggests clearly that the C-8 stereochemistry plays a key role in hypoglycemic effect since the active borapetoside A and C possess 8R-chirality but the inactive borapetoside B possess 8S-chirality. The location of glycoside at C-3 for borapetoside A but C-6 for borapetoside C and the formation of lactone between C-4 and C-6 for borapetoside A, could account for the different potency in hypoglycemic action for these two compounds.

Sinoscrewtine, an alkaloid with novel skeleton from the roots of Sinomenium acutum.

An alkaloid with novel skeleton, sinoscrewtine (1), has been isolated from the roots of Sinomenium acutum. Its structure was established by spectral analysis and X-ray crystallographic study, and its possible biosynthetic pathway was delivered. In vitro experiments, 1 showed weak injurious effects against H(2)O(2)/Aß(25-35) induced oxidative injury in PC-12 cells and DPPH radical scavenging activity with IC(50) of 32.6µM.

Quantitative analysis of fructo-oligosaccharides in Gynura divaricata subsp. formosana by high performance anion exchange chromatography-pulsed amperometric detection.

High performance anion exchange chromatography-pulsed amperometric detection was employed in this study to conduct quantitative analysis of the inulin-related fructo-oligosaccharides present in Gynura divaricata subsp. formosana. Result obtained for the 1-kestose (GF2), nystose (GF3) and 1F-beta-fructofuranosyl-nystose (GF4) contents were 146.60 +/- 0.04, 24.70 +/- 0.75 and 16.60 +/- 0.91 microg/g, fresh weight, and 68.90 +/- 0.02, 7.60 +/- 1.34 and 149.30 +/- 0.06 microg/g (mean +/- RSD), dry weight, respectively. Using this method, the limit of quantitation was 20 microg/mL and the linear detectability between 0-250 microg/mL. The developed method provides a practical analysis for these low caloric value, prebiotic and non-digestible carbohydrates in the genus Gynura.