Inhibitory effects of serratene-type triterpenoids from Lycopodium complanatum on cholinesterases and ß-secretase 1.

Phytochemical investigation of Lycopodium complanatum whole plants led to the isolation of two new serratene-type triterpenoids (1 and 2) along with eight known triterpenoids (3-10). Their structures were established using 1D and 2D NMR spectroscopic techniques and mass spectrometry. These compounds did not inhibit acetylcholinesterases (AChE) and butyrylcholinesterase (BChE), but did inhibit ß-secretase 1 (BACE1). Compounds 1 and 6 showed potent BACE1 inhibition with IC50 values of 2.79 ± 0.28 and 2.49 ± 0.12 µM, respectively. The kinetic study of BACE1 inhibition revealed that compound 1 showed competitive inhibition, whereas 6 showed mixed-type inhibition. Furthermore, molecular docking results showed that the tested inhibitors 1 and 6 exhibited good binding affinities toward BACE1, with binding energies of -8.8 and -10.3 kcal/mol, respectively.

PTP1B inhibitors from Selaginella tamariscina (Beauv.) Spring and their kinetic properties and molecular docking simulation.

Diabetes is one of the most popular worldwide diseases, regulated by the defects in insulin secretion, insulin action, or both. The overexpression of protein tyrosine phosphatase 1B (PTP1B) was found to down-regulate the insulin-receptor activation. PTP1B has been known as a strategy for the treatment of diabetes via the regulation of insulin signal transduction pathway. Herein, we investigated the PTP1B inhibitors isolated from natural sources. The chemical investigation of Selaginella tamariscina (Beauv.) Spring revealed seven unsaturated alkynyl phenols 1-7, four new selaginellins T-W 1-4 together with three known compounds 5-7 isolated from the aerial parts. The structures of the isolates were determined by spectroscopic techniques (1D/2D-NMR, MS, and CD). The inhibitory effects of these isolates on the PTP1B enzyme activity were investigated. Among them, compounds 2-7 significantly exhibited the inhibitory effects with the IC50 values ranging from 4.8 to 15.9µM. Compound 1 moderately displayed the inhibitory activity with an IC50 of 57.9µM. Furthermore, active compounds were discovered from their kinetic and molecular docking analysis. The results revealed that compounds 2 and 4-7 were mixed-competitive inhibitors, whereas compound 3 was a non-competitive inhibitor. This data confirm that these compounds exhibited potential inhibitory effect on the PTP1B enzyme activity.

Ellagitannin and flavonoid constituents from Agrimonia pilosa Ledeb. with their protein tyrosine phosphatase and acetylcholinesterase inhibitory activities.

A new ellagitannin, agritannin (1), a new flavone glycoside, agriflavone (2), and another flavone glycoside with spectroscopic data reported for the first time, kaempferol-3-O-[(S)-3-hydroxy-3-methylglutaryl (1→6)]-ß-d-glucoside (3), along with 16 known compounds were isolated from the aerial parts of Agrimonia pilosa Ledeb. These compounds were evaluated for PTP1B inhibitory activity. Among them, compounds 9 and 18 displayed potential inhibitory activity against PTP1B with IC50 values of 7.14±1.75 and 7.73±0.24µM, respectively. In addition, compound 1 showed significant inhibitory effect with an IC50 value of 17.03±0.09µM. Furthermore, these compounds were tested in AChE inhibitory assays. Most of them were found to have moderate inhibitory effects, with IC50 values ranging from 60.20±1.09 to 92.85±1.12µM. Except compounds 3, 8, and 18 were inactive.

Effects of Constituent Compounds of Smilax china on Nicotine-Induced Endothelial Dysfunction in Human Umbilical Vein Endothelial Cells.

This study investigated the effects of compounds isolated from 70% ethanol (EtOH) extraction of Smilax china L. (SCE), a plant belonging to the family Smilacaceae on nicotine-induced endothelial dysfunction (ED) in human umbilical vein endothelial cells. We isolated 10 compounds from ethyl acetate (EtOAc) fraction of 70% EtOH extract of SCE and investigated their inhibitory effect on nicotine-induced ED in endothelial cells. Kaempferol, kaempferol 7-O-α-L-rhamnopyranoside, puerarin and ferulic acid showed strong inhibition of nicotine-induced vascular cell adhesion molecule (VCAM-1) expression while kaempferol, kaempferin, and caffeic acid attenuated intercellular adhesion molecule (ICAM-1) expression. Lepidoside, caffeic acid and methylsuccinic acid caused the highest up-regulated expression of endothelial nitric oxide synthase at the protein level with caffeic acid and ferulic acid showing strong inhibitory effects on inducible nitric oxide synthase (iNOS) expression. In addition, ferulic acid and kaempferol showed inhibition against interleukin-8 (IL-8) and interleukin-1ß (IL-1ß) expression while ferulic acid and caffeic acid showed comparatively higher inhibition of ED associated tumor necrosis factor-α (TNF-α) expression. These results show the potential of the aforementioned compounds to reverse the toxic effects of nicotine on the endothelium.

Simultaneous analysis of seven marker compounds from Saposhnikoviae Radix, Glehniae Radix and Peucedani Japonici Radix by HPLC/PDA.

A new combination of high performance liquid chromatography (HPLC) method coupled with photodiode array (PDA) analysis has been developed for the simultaneous quantitative determination of seven major components in Saposhnikoviae Radix (SR), Glehniae Radix (GR) and Peucedani Japonici Radix (PR), namely peucedanol 7-O-ß-D-glucopyranoside (1), prim-O-glucosylcimifugin (2), cimifugin (3), 4'-O-ß-D-glucosyl-5-O-methylvisamminol (4), bergapten (5), sec-O-glucosylhamaudol (6), and imperatorin (7). Clear separation of these seven components were achieved on a Phenomenex Kinetex C18 (250 × 4.6 mm, 5 µm) column by gradient elution of water (A) and methanol (B) as mobile phase. The flow rate was 1.0 mL/min and the UV detector wavelength was set at 254 nm. The method was successfully used in the analysis of SR, GR, and PR with relatively simple conditions and procedures, and the results were satisfactory for linearity, recovery, precision, accuracy, stability and robustness. The results indicate that the established HPLC/PDA method is suitable for the classification of SR, GR, and PR.

Anti-inflammatory terpenylated coumarins from the leaves of Zanthoxylum schinifolium with α-glucosidase inhibitory activity.

Nine terpenylated coumarins, namely 7-[(E)-3',7'-dimethyl-6'-oxo-2',7'-octadienyl]oxy-coumarin (1), schinilenol (2), schinindiol (3), collinin (4), 7-[(E)-7'-hydroxy-3',7'-dimethy-locta-2',5'-dienyloxy]-coumarin (5), 8-methoxyanisocoumarin (6), 7-(6'R-hydroxy-3',7'-dimethyl-2'E,7'-octadienyloxy)coumarin (7), (E)-4-methyl-6-(coumarin-7'-yloxy)hex-4-enal (8), and aurapten (9), along with a 4-quinolone alkaloid (10) and integrifoliodiol (11), were isolated from the leaves of Zanthoxylum schinifolium. Of the isolates, compounds 4 and 7 potentially inhibited NO production in lipopolysaccharide (LPS)-stimulated macrophage RAW264.7 cells, with IC50 values of 5.9 ± 0.8 and 18.2 ± 1.8 µM, respectively. Furthermore, compounds 4 and 7 dose-dependently reduced the LPS-induced iNOS expression. Moreover, pre-incubation of cells with 4 and 7 significantly suppressed LPS-induced COX-2 protein expression. In addition, compounds 4, 7, 8, and 10 showed strong α-glucosidase inhibitory effects, with IC50 values of 92.1 ± 0.7, 90.6 ± 0.9, 78.2 ± 0.2, and 82.4 ± 0.8 µM, respectively. Compounds 1, 5, and 11 displayed moderate effects with IC50 values of 161.6 ± 0.3, 164.4 ± 1.1, and 155.4 ± 0.9 µM, while acarbose, a positive control, possessed an IC50 value of 121.5 ± 1.0 µM. This is the first investigation on the α-glucosidase inhibitory effect of components from Zanthoxylum schinifolium. Further studies should be made on active compounds.

Isolation of Lignan and Fatty Acid Derivatives from the Grains of Echinochloa utilis and Their Inhibition of Lipopolysaccharide-Induced Nitric Oxide Production in RAW 264.7 Cells.

Two new fatty acid derivatives, echinochlorins A (8) and B (9) and a racemic lignan, (±)-anti-1-(4-hydroxy-3-methoxyphenyl)-2-{4-[(E)-3-acetoxypropen-1-yl]-2-methoxyphenoxy}propan-1,3-diol 3-acetate (1), were isolated from Echinochloa utilis grains, along with six known lignans (2-7) and two fatty acid derivatives (10, 11). Their structures were established by spectroscopic data analyses (IR, UV, HR-FABMS, GC-MS, and 1D and 2D NMR). The configuration of 1 was determined by Mosher's method. Compound 5 displayed potential inhibitory activity on lipopolysaccharide-induced NO production in macrophage RAW 264.7 cells with an IC50 value of 4.8 ± 0.5 µM. These isolated compounds in crude EtOH extract were also quantitated by HPLC.

Protein tyrosine phosphatase 1B (PTP1B) inhibitory activity and glucosidase inhibitory activity of compounds isolated from Agrimonia pilosa.

CONTEXT: Despite phytochemical studies of Agrimonia pilosa Ledeb. (Rosaceae), the antidiabetic effects of this plant are unknown. OBJECTIVE: This study characterizes the isolated compounds from the aerial parts of A. pilosa and evaluates their PTP1B and α-glucosidase inhibitory properties. MATERIALS AND METHODS: Ethanol extract of A. pilosa was found to inhibit 64% PTP1B activity at 30 µg/mL. The ethanol extract was partitioned with methylene chloride, ethyl acetate, n-butanol, and water fractions. Among these, the ethyl acetate fraction displayed the most potent PTP1B activity. The ethyl acetate extract was separated by chromatographic methods to obtain flavonoids and triterpenoids (1-11); which were evaluated for their inhibitory effects on PTP1B activity with p-nitrophenyl phosphate (p-NPP) as a substrate, and also α-glucosidase enzyme. RESULTS: Compounds 1-11 were identified as apigenin-7-O-ß-d-glucuronide-6″-methyl ester, triliroside, quercetin-7-O-ß-d-glycoside, quercetin-3-O-ß-d-glycoside, kaempferol, kaempferol-3-O-α-l-rhamnoside, ß-sitosterol, ursolic acid, tormentic acid, methyl 2-hydroxyl tricosanoate, and palmitic acid. Compounds 8, 9, and 11 displayed inhibitory effects on PTP1B activity with IC50 values of 3.47 ± 0.02, 0.50 ± 0.06, and 0.10 ± 0.03 µM, respectively. Compounds 3, 4, 6, and 9 exhibited inhibition of the α-glucosidase activity with IC50 values of 11.2 ± 0.2, 29.6 ± 0.9, 28.5 ± 0.1, and 23.8 ± 0.4 µM, respectively. DISCUSSION AND CONCLUSION: As major ingredients of A. pilosa, compounds 1, 6, 8, and 9 showed the greatest inhibitory potency on PTP1B activity. Compounds 3, 6, 8, and 9 also showed potent inhibitory effects on α-glucosidase enzyme. This result suggested the potential of these compounds for developing antidiabetic agents.

Simultaneous quantitation and validation of method for the quality evaluation of Eucommiae cortex by HPLC/UV.

A new HPLC/UV method has been developed for the simultaneous quantitative determination of four major components in Eucommiae cortex, namely geniposidic acid (1), geniposide (2), pinoresinol di-O-ß-D-glucopyranoside (3), and liriodendrin (4). Simultaneous separations of these four components were achieved on a J'sphere ODS C(18) column (250 × 4.6 mm, 4 µm). The elution was done using water with 0.1% phosphoric acid (A) and acetonitrile with 0.1% phosphoric acid (B) in a two-step elution of the mobile phase at a flow rate of 1.0 mL/min and a wavelength of 230 nm. The method was validated for linearity, recovery, precision, accuracy, stability and robustness. All calibration curves showed good linear regression (r(2) > 0.999) within the test ranges. This method showed good recovery and reproducibility for the quantification of these four components in 85 species of Eucommiae cortex. The intra-day and inter-day precisions were lower than 0.53% (as a relative standard deviation, RSD) and accuracies between 93.00 and 106.28% for all standards. The results indicate that the established HPLC/UV method is suitable for quantitation and quality evaluation of Eucommiae cortex.

Chemical Constituents of Euonymus alatus (Thunb.) Sieb. and Their PTP1B and α-Glucosidase Inhibitory Activities.

Phytochemical study on the corks of Euonymus alatus resulted in the isolation of a novel 3-hydroxycoumarinflavanol (23), along with ten triterpenoids (1-10), ten phenolic derivatives (11-20), and two flavonoid glycosides (21 and 22). Their structures were determined by extensive 1D and 2D-nuclear magnetic resonance spectroscopic and mass spectrometry data analysis. Furthermore, their inhibitory effects against the protein tyrosine phosphatases 1B (PTP1B) and α-glucosidase enzyme activity were evaluated. Compounds 6, 7, 9, 15, 19, and 23 were non-competitive inhibitors, exhibiting most potency with IC50 values ranging from 5.6 ± 0.9 to 18.4 ± 0.3 µm, against PTP1B. Compound 3 (competitive), compounds 5 and 15 (mixed-competitive) displayed potent inhibition with IC50 values of 15.1 ± 0.7, 23.6 ± 0.6 and 14.8 ± 0.9 µm, respectively. Moreover, compounds 15, 20, and 23 exhibited potent inhibition on α-glucosidase with IC50 values of 10.5 ± 0.8, 9.5 ± 0.6, and 9.1 ± 0.5 µm, respectively. Thus, these active ingredients may have value as new lead compounds for the development of new antidiabetic agents.

Quality evaluation and pattern recognition analyses of bioactive marker compounds from Farfarae Flos using HPLC/PDA.

The flower bud of Tussilago farfara L., called Farfarae Flos, has traditionally been used in Oriental medicine for the treatment of bronchitis and asthma. To establish a standard for quality control as well as the reliable identification of Farfarae Flos, the contents of five standards, rutin (1), isoquercetin (2), 3,5-dicaffeoylquinic acid (3), tussilagone (4), and tussilagonone (5), were determined by quantitative high-performance liquid chromatography (HPLC)/photodiode array (PDA) analysis. The five standards were separated on a YoungJinBioChrom Aegispak C18-L (250-mm×4.6-mm, 5-µm) column by gradient elution using 0.03% trifluoroacetic acid in water (A), with acetonitrile (B) as the mobile phase. The flow rate was 1.0 mL/min, and the UV detector wavelength was set at 220 nm. The method was successfully used in the analysis of Farfarae Flos from different geographic origins with relatively simple conditions and procedures, and the results demonstrated satisfactory linearity, recovery, precision, accuracy, stability, and robustness. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of 62 Farfarae Flos samples. This result indicated that the established HPLC/PDA method is suitable for quantitation and pattern recognition analyses for the quality evaluation of Farfarae Flos.

Quality evaluation and pattern recognition analyses of marker compounds from five medicinal drugs of Rutaceae family by HPLC/PDA.

To establish a standard of quality control and to identify different origins for the Rutaceae family [Citri Unshiu Peel (CU), Citri Unshiu Immature Peel (CI), Ponciri Immature Fructus (PI), Aurantii Immature Fructus (AI), and Aurantii Fructus (AU)], 13 standards including rutin (1), narirutin (2), naringin (3), hesperidin (4), neohesperidin (5), neoponcirin (6), poncirin (7), naringenin (8), isosinensetin (9), sinensetin (10), nobiletin (11), heptamethoxyflavone (12), and tangeretin (13) were determined by high performance liquid chromatography (HPLC)/photo-diode array (PDA) analysis. A YMC ODS C18 (250 × 4.6 mm, 5 µm) column was used and the ratio of mobile phases of water (A) and acetonitrile (B) delivered to the column for gradient elution was applied. This method was fully validated with respect to linearity, accuracy, precision, stability, and robustness. The HPLC/PDA method was applied successfully to quantify 13 major compounds in the extracts of CU, CI, PI, AI, and AU. The pattern recognition analysis combined with LC chromatographic data was performed by repeated analysis of 27 reference samples in the above five Rutaceae oriental medicinal drugs. The established HPLC method was rapid and reliable for quantitative analysis and quality control of multiple components in five Rutaceae species with different origins.

Protein tyrosine phosphatase 1B (PTP1B) inhibitory constituents from the aerial parts of Tradescantia spathacea Sw.

Inhibitors of protein tyrosine phosphatase 1B (PTP1B) are promising agents for the treatment of type 2 diabetes and obesity. The bioactivity-guided isolation led to the separation of two new compounds, (±)-tradescantin (13) and tradescantoside (16), along with fourteen known compounds (1-12, 14, and 15) from the aerial parts of Tradescantia spathacea Sw. (Commelinaceae). Their chemical structures were elucidated by spectroscopic methods as well as by comparing with those reported in the literature. The isolated compounds (1-16) were then examined for their inhibitory activity toward PTP1B. The results indicated that compounds 2, 6, 8, and 12 possessed potent inhibition with IC50 values of 7.82±0.79, 6.80±0.89, 4.55±0.92, and 6.38±0.14 µM, respectively. Kinetic study of compounds 2, 6, 8, 12, 13, and 16 was conducted and the structure-activity relationships of the isolated compounds (1-16) were also discussed herein. To the best of our knowledge, all the isolates were separated for the first time from this plant.

Quality evaluation of Perillae Folium by HPLC/PDA.

To establish a standard of quality control for Perillae Folium (Lamiaceae Family), four standard compounds including rosmarinic acid, elemicin, perillaldehyde, and dillapiole were evaluated by high-performance liquid chromatography (HPLC)/photodiode array (PDA). The four standards were analyzed with a Phenomenex Kinetex C18 (250 × 4.6 mm, 5 µm) column by gradient elution using 0.1 % formic acid in water and methanol as the mobile phase. The standards were quantified by HPLC/PDA from Perillae Folium, which included the leaf and twig of Perilla frutescens L. Britton var. acuta (Thunb.) Kudo or Perilla frutescens Britton var. crispa Decne. The method was successfully used in the analysis of Perillae Folium, and the linearity, recovery, precision, accuracy, stability, and robustness were satisfactory according to the validation results. In Perillae Folium samples, the average contents (wt%) of rosmarinic acid, elemicin, perillaldehyde, and dillapiole were 0.540, 0.059, 0.050, and 0.056 %, respectively. The results indicate that the established HPLC/PDA method is suitable for the quantitation and quality evaluation of Perillae Folium.

Insulin-mimetic selaginellins from Selaginella tamariscina with protein tyrosine phosphatase 1B (PTP1B) inhibitory activity.

As part of an ongoing search for new antidiabetic agents from medicinal plants, three new (2, 4, and 5) and two known selaginellin derivatives (1 and 3) were isolated from a methanol extract of Selaginella tamariscina. The structures of the new compounds were determined by spectroscopic data analysis. All isolates showed strong glucose uptake stimulatory effects in 3T3-L1 adipocyte cells at a concentration of 5 µM. Furthermore, these compounds were found to possess inhibitory effects on PTP1B enzyme activity with IC50 values ranging from 4.6 ± 0.1 to 21.6 ± 1.5 µM. Compound 2 showed the greatest potency, with an IC50 value of 4.6 ± 0.1 µM, when compared with the positive control (ursolic acid, IC50 = 3.5 ± 0.1 µM). Therefore, these selaginellin derivatives may have value as new lead compounds for the development of agents against type 2 diabetes.

Pharmacokinetic alteration of baclofen by multiple oral administration of herbal medicines in rats.

The potential pharmacokinetic (PK) interaction of conventional western drug, baclofen, and oriental medications Oyaksungisan (OY) and Achyranthes bidentata radix (AB) extract for the treatment of spasticity has been evaluated. Rats were pretreated with distilled water (DW), OY, or AB extract by oral administration every day for 7 days. After 10 min of the final dose of DW or each herbal medication, baclofen (1 mg/kg) was given by oral administration and plasma concentrations of baclofen were determined by LC/MS/MS. The plasma baclofen concentration-time profiles were then analyzed by noncompartmental analysis and a population PK model was developed. Baclofen was rapidly absorbed, showed biexponential decline with elimination half-life of 3.42-4.10 hr, and mostly excreted into urine. The PK of baclofen was not affected by AB extract pretreatment. However, significantly lower maximum plasma concentration (C max) and longer time to reach C max (T max) were observed in OY pretreated rats without changes in the area under the curve (AUC) and the fraction excreted into urine (F urine). The absorption rate (K a ) of baclofen was significantly decreased in OY pretreated rats. These data suggested that repeated doses of OY might delay the absorption of baclofen without changes in extent of absorption, which needs further evaluation for clinical significance.

Quantitative and pattern recognition analyses of magnoflorine, spinosin, 6'''-feruloyl spinosin and jujuboside A by HPLC in Zizyphi Semen.

Two rapid and simple HPLC methods with UV detector to determine three main compounds (magnoflorine, spinosin and 6'''-feruloyl spinosin) and evaporative light scattering detector (ELSD) to determine jujuboside A were developed for the chemical analyses of Zizyphi Semen. Magnoflorine, spinosin, and 6'''-feruloyl spinosin were separated with an YMC J'sphere ODS-H80 column (250 mm × 4.6 mm, 4 µm) by the gradient elution followed by the isocratic elution using methanol with 0.1 % formic acid and water with 0.1 % formic acid as the mobile phase. The flow rate was 1.0 mL/min. Jujuboside A was separated by HPLC-ELSD with YoungJinBioChrom Aegispak C18-L column (250 mm × 4.6 mm, 5 µm) column in a gradient elution using methanol with 0.1 % formic acid (A) and water with 0.1 % formic acid as the mobile phase. These two methods were fully validated with respect to linearity, precision, accuracy, stability, and robustness. These HPLC methods were applied successfully to quantify four compounds in a Zizyphi Semen extract. The HPLC analytical methods were validated for pattern recognition analysis by repeated analysis of 91 seed samples corresponding to 48 Zizyphus jujuba var. spinosa (J01-J48) and 43 Zizyphus mauritiana (M01-M43). The results indicate that these methods are suitable for a quality evaluation of Zizyphi Semen.

Quantitative determination and pattern recognition analyses of bioactive marker compounds from Dipsaci Radix by HPLC.

In this study, quantitative and pattern recognition analyses were developed using HPLC/UV for the quality evaluation of Dipsaci Radix. For quantitative analysis, five major bioactive compounds were assessed. The separation conditions employed for HPLC/UV were optimized using ODS C18 column (250 × 4.6 mm, 5 µm) with a gradient of acetonitrile and water as the mobile phase at a flow rate of 1.0 mL/min and a detection wavelength of 212 nm. These methods were fully validated with respect to linearity, accuracy, precision, recovery, and robustness. The HPLC/UV method was applied successfully to the quantification of five major compounds in the extract of Dipsaci Radix. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of 17 Dipsaci Radix and four Phlomidis Radix samples. The results indicate that the established HPLC/UV method is suitable for quantitative analysis.

Constituents with DNA topoisomerases I and II inhibitory activity and cytotoxicity from the roots of Rubia cordifolia.

Activity-directed isolation of the ethyl acetate fraction from the roots of Rubia cordifolia resulted in the identification of a new anthraquinone, 1,3,6-trihydroxy-2-hydroxymethyl-9,10-anthraquinone-3- O- α- L-rhamnopyranosyl-(1 → 2)- ß-D-(6'-O-acetyl)-glucopyranoside (1), two new dihydronaphtoquinones, 1,4-dihydroxy-2-carbomethoxy-3-prenylnaphthalene-1-O- ß-D-glucopyranoside (2) and mollugin-1-O- ß- D-glucopyranoside (3), and a new monoterpenoid, 3 R,3a S,4 R,6a R-3,4,6-tris(hydroxymethyl)-3,3a,4,6a-tetrahydro-2 H-cyclopenta[ B]furan-2-one (4), together with nine known compounds (5-13). The structures of these compounds were elucidated on the basis of spectroscopic evidence. In addition, their DNA topoisomerases I and II inhibitory activity and cytotoxicity were measured.