Abietane diterpenoids from Salvia sahendica--antiprotozoal activity and determination of their absolute configurations.

In a screening of Iranian plants for antiprotozoal activity, an n-hexane extract of the roots of Salvia sahendica potently inhibited the growth of Plasmodium falciparum K1 strain. Subsequent HPLC-based activity profiling led to the identification of seven known and one new abietane-type diterpenoid. Structure elucidation was achieved by analysis of spectroscopic data including 1D and 2D NMR. The absolute configuration of sahandol (7) and sahandone (8) were assigned by comparison of experimental ECD spectra with calculated ECD data, using time-dependent density functional theory and methanol as the solvent. In vitro biological activity against P. falciparum and Trypanosoma brucei rhodesiense STIB 900 strain and cytotoxicity in rat myoblast (L6) cells were determined. The IC50 values of the compounds ranged from 0.8 µM to over 8.8 µM against P. falciparum, and from 1.8 µM to over 32.3 µM against T. brucei rhodesiense. The cytotoxic IC50 values ranged from 0.5-15.5 µM. Selectivity indices for P. falciparum were 0.1 to 18.2, and 0.1 to 1.2 for T. brucei rhodesiense.

HPLC-based activity profiling--discovery of sanggenons as GABAA receptor modulators in the traditional Chinese drug Sang bai pi (Morus alba root bark).

EtOAc extracts from two batches of Morus alba root bark (Sang bai pi) potentiated γ-aminobutyric acid (GABA)-induced chloride influx in Xenopus oocytes, which transiently expressed GABA (A) receptors of the subunit composition α1ß2γ(2S). With the aid of HPLC-based activity profiling of the extract from the first batch, activity was traced to a peak subsequently identified as sanggenon G (3). The second batch had a different phytochemical profile, and HPLC-based activity profiling led to the identification of sanggenon C (4) and a stereoisomer of sanggenon D (2) as positive GABA (A) receptor modulators. The structurally related compound kuwanon L (1) was inactive. The sanggenons represent a new scaffold of positive GABA (A) receptor modulators.

Discovery of GABA(A) receptor modulator aristolactone in a commercial sample of the Chinese herbal drug "Chaihu" (Bupleurum chinense roots) unravels adulteration by nephrotoxic Aristolochia manshuriensis roots.

In a two-microelectrode voltage clamp assay using Xenopus laevis oocytes, a petroleum ether extract prepared from a commercial sample of the traditional Chinese herbal drug labelled as " Chaihu" (Bupleurum chinense DC. roots) enhanced the I(GABA) by 156 % ± 22 % when tested at 100 µg/mL. By means of HPLC-based activity profiling combined with high-resolution LC-MS and microprobe NMR, the germacranolide aristolactone was identified as one of the main active compounds (EC50 56.02 µM ± 5.09 µM). However, aristolactone has been previously reported only from the genus Aristolochia (Aristolochiaceae), suggesting a possible adulteration. With the aid of a validated HPTLC protocol for detection of aristolochic acids and with reference samples, the commercial sample was confirmed to be a mixture of Aristolochia manshuriensis root and Bupleurum chinense root. This finding was corroborated by macroscopic inspection of the drug. This case of adulteration with a highly nephrotoxic drug raises concerns about adequate quality control of TCM drugs commercialized in Europe.

Identification and characterization of GABA(A) receptor modulatory diterpenes from Biota orientalis that decrease locomotor activity in mice.

An ethyl acetate extract of Biota orientalis leaves potentiated GABA-induced control current by 92.6% ± 22.5% when tested at 100 µg/mL in Xenopus laevis oocytes expressing GABA(A) receptors (α1ß2γ(2S) subtype) in two-microelectrode voltage clamp measurements. HPLC-based activity profiling was used to identify isopimaric acid (4) and sandaracopimaric acid (5) as the compounds largely responsible for the activity. Sandaracopimaradienolal (3) was characterized as a new natural product. Compounds 4 and 5 were investigated for GABA(A) receptor subtype selectivity at the subtypes α1ß1γ(2S), α1ß2γ(2S), α1ß3γ(2S), α2ß2γ(2S), α3ß2γ(2S), and α5ß2γ(2S). Sandaracopimaric acid (5) was significantly more potent than isopimaric acid (4) at the GABA(A) receptor subtypes α1ß1γ(2S), α2ß2γ(2S), and α5ß2γ(2S) (EC504: 289.5 ± 82.0, 364.8 ± 85.0, and 317.0 ± 83.7 µM vs EC505: 48.1 ± 13.4, 31.2 ± 4.8, and 40.7 ± 14.7 µM). The highest efficiency was reached by 4 and 5 on α2- and α3-containing receptor subtypes. In the open field test, ip administration of 5 induced a dose-dependent decrease of locomotor activity in a range of 3 to 30 mg/kg body weight in mice. No significant anxiolytic-like activity was observed in doses between 1 and 30 mg/kg body weight in mice.

Positive GABA(A) receptor modulators from Acorus calamus and structural analysis of (+)-dioxosarcoguaiacol by 1D and 2D NMR and molecular modeling.

In a two-microelectrode voltage clamp with Xenopus laevis oocytes, a petroleum ether extract of Acorus calamus rhizomes enhanced the GABA-induced chloride current through GABA(A) receptors of the α1ß2γ(2S) subtype by 277% ± 9.7% (100 µg/mL). ß-Asarone (1), (+)-dioxosarcoguaiacol (2), (+)-shyobunone (3), and (+)-preisocalamenediol (4) were subsequently identified as main active principles through HPLC-based activity profiling and targeted isolation. The compounds induced maximum potentiation of the chloride current ranging from 588% ± 126% (EC50: 65.3 ± 21.6 µM) (2) to 1200% ± 163% (EC(50): 171.5 ± 34.6 µM) (1), whereas (-)-isoshyobunone (5) and (-)-acorenone (6) exhibited weak GABA(A) modulating properties (5: 164% ± 42.9%; EC50: 109.4 ± 46.6 µM and 6: 241% ± 23.1%; EC50: 34.0 ± 6.7 µM). The relative configuration of 2 was established as 4R*8S*10R* by NOESY experiments and conformational analysis.

HPLC-based activity profiling for GABA(A) receptor modulators: a new dihydroisocoumarin from Haloxylon scoparium.

A new dihydroisocoumarin was isolated from a dichloromethane extract of Haloxylon scoparium with the aid of a functional assay with Xenopus oocytes transiently expressing GABA(A) receptors of defined subunit composition (alpha(1)beta(2)gamma(2S)). Compound 1 induced a maximum potentiation of the chloride currents by 144.6 +/- 35.3% with an EC(50) of 140.2 +/- 51.2 muM.

HPLC-based activity profiling: discovery of piperine as a positive GABA(A) receptor modulator targeting a benzodiazepine-independent binding site.

A plant extract library was screened for GABA(A) receptor activity making use of a two-microelectrode voltage clamp assay on Xenopus laevis oocytes. An ethyl acetate extract of black pepper fruits [Piper nigrum L. (Piperaceae) 100 microg/mL] potentiated GABA-induced chloride currents through GABA(A) receptors (composed of alpha(1), beta(2), and gamma(2S) subunits) by 169.1 +/- 2.4%. With the aid of an HPLC-based activity profiling approach, piperine (5) was identified as the main active compound, together with 12 structurally related less active or inactive piperamides (1-4, 6-13). Identification was achieved by on-line high-resolution mass spectrometry and off-line microprobe 1D and 2D NMR spectroscopy, using only milligram amounts of extract. Compound 5 induced a maximum potentiation of the chloride currents by 301.9 +/- 26.5% with an EC(50) of 52.4 +/- 9.4 microM. A comparison of the modulatory activity of 5 and other naturally occurring piperamides enabled insights into structural features critical for GABA(A) receptor modulation. The stimulation of chloride currents through GABA(A) receptors by compound 5 was not antagonized by flumazenil (10 microM). These data show that piperine (5) represents a new scaffold of positive allosteric GABA(A) receptor modulators targeting a benzodiazepine-independent binding site.

Quantitative analysis of anti-inflammatory and radical scavenging triterpenoid esters in evening primrose seeds.

Lipophilic triterpenoidal esters with radical scavenging and cyclooxygenase inhibitory properties were recently found in cold-pressed, nonraffinated evening primrose oil (EPO). A quantitative assay for the analysis of 3-O-trans-caffeoyl derivatives of betulinic, morolic, and oleanolic acid in evening primrose seeds was developed and validated. Extraction efficiency >99% was achieved by means of pressurized liquid extraction with two extraction cycles and 80% (v/v) ethanol at 120 degrees C. Analysis of esters was by normal-phase high-performance liquid chromatography on a Diol column and hexane/ethyl acetate (containing 0.1% formic acid) (65:35) as the eluent. The analytes were determined without further prepurification. Seeds from defined cultures of Oenothera biennis, Oenothera lamarckiana, and Oenothera ammophila, grown under identical conditions, were analyzed. The cultures originated from seeds from eight collections in the wild and from selections from five cultivars. The content of total triterpenoidal esters in seeds varied between 1.34 and 2.78 mg/g. Three types of qualitative patterns were observed for the triterpenoidal esters. The influence of different harvest times and plant treatments was studied with the cultivar Anothera. Variations between 1.5 and 2.3 mg/g were found.

GABAA receptor modulation by piperine and a non-TRPV1 activating derivative.

The action of piperine (the pungent component of pepper) and its derivative SCT-66 ((2E,4E)-5-(1,3-benzodioxol-5-yl))-N,N-diisobutyl-2,4-pentadienamide) on different gamma-aminobutyric acid (GABA) type A (GABA(A)) receptors, transient-receptor-potential-vanilloid-1 (TRPV1) receptors and behavioural effects were investigated. GABA(A) receptor subtypes and TRPV1 receptors were expressed in Xenopus laevis oocytes. Modulation of GABA-induced chloride currents (I(GABA)) by piperine and SCT-66 and activation of TRPV1 was studied using the two-microelectrode-voltage-clamp technique and fast perfusion. Their effects on explorative behaviour, thermoregulation and seizure threshold were analysed in mice. Piperine acted with similar potency on all GABA(A) receptor subtypes (EC50 range: 42.8±7.6 µM (α2ß2)-59.6±12.3 µM (α3ß2). I(GABA) modulation by piperine did not require the presence of a γ(2S)-subunit, suggesting a binding site involving only α and ß subunits. I(GABA) activation was slightly more efficacious on receptors formed from ß(2/3) subunits (maximal I(GABA) stimulation through α1ß3 receptors: 332±64% and α1ß2: 271±36% vs. α1ß1: 171±22%, p<0.05) and α3-subunits (α3ß2: 375±51% vs. α5ß2:136±22%, p<0.05). Replacing the piperidine ring by a N,N-diisobutyl residue (SCT-66) prevents interactions with TRPV1 and simultaneously increases the potency and efficiency of GABA(A) receptor modulation. SCT-66 displayed greater efficacy on GABA(A) receptors than piperine, with different subunit-dependence. Both compounds induced anxiolytic, anticonvulsant effects and reduced locomotor activity; however, SCT-66 induced stronger anxiolysis without decreasing body temperature and without the proconvulsive effects of TRPV1 activation and thus may serve as a scaffold for the development of novel GABA(A) receptor modulators.

Phytochemical profiling of Curcuma kwangsiensis rhizome extract, and identification of labdane diterpenoids as positive GABAA receptor modulators.

An ethyl acetate extract of Curcuma kwangsiensis S.G. Lee & C.F. Liang (Zingiberaceae) rhizomes (100 µg/ml) enhanced the GABA-induced chloride current (IGABA) through GABAA receptors of the α1ß2γ2S subtype by 79.0±7.0%. Potentiation of IGABA was measured using the two-microelectrode voltage-clamp technique and Xenopus laevis oocytes. HPLC-based activity profiling of the crude extract led to the identification of 11 structurally related labdane diterpenoids, including four new compounds. Structure elucidation was achieved by comprehensive analysis of on-line (LC-PDA-ESI-TOF-MS) and off-line (microprobe 1D and 2D NMR) spectroscopic data. The absolute configuration of the compounds was established by comparison of experimental and calculated ECD spectra. Labdane diterpenes represent a new class of plant secondary metabolites eliciting positive GABAA receptor modulation. The highest efficiency was observed for zerumin A (maximum potentiation of IGABA by 309.4±35.6%, and EC50 of 24.9±8.8 µM).

HPLC-based activity profiling of Angelica pubescens roots for new positive GABAA receptor modulators in Xenopus oocytes.

A petroleum ether extract of the traditional Chinese herbal drug Duhuo (roots of Angelica pubescens Maxim. f. biserrata Shan et Yuan), showed significant activity in a functional two-microelectrode voltage clamp assay with Xenopus oocytes which expressed recombinant γ-aminobutyric acid type A (GABA(A)) receptors of the subtype α(1)ß(2)γ(2S). HPLC-based activity profiling of the active extract revealed six compounds responsible for the GABA(A) receptor modulating activity. They were identified by microprobe NMR and high resolution mass spectrometry as columbianetin acetate (1), imperatorin (3), cnidilin (4), osthol (5), and columbianedin (6). In concentration-dependent experiments, osthol and cnidilin showed the highest potentiation of the GABA induced chloride current (273.6%±39.4% and 204.5%±33.2%, respectively at 300 µM). Bisabolangelone (2) only showed minor activity at the GABA(A) receptor. The example demonstrates that HPLC-based activity profiling is a simple and efficient method to rapidly identify GABA(A) receptor modulators in a bioactive plant extract.

Identification of GABA A receptor modulators in Kadsura longipedunculata and assignment of absolute configurations by quantum-chemical ECD calculations.

A petroleum ether extract of Kadsura longipedunculata enhanced the GABA-induced chloride current (I(GABA)) by 122.5±0.3% (n=2) when tested at 100 µg/ml in Xenopuslaevis oocytes expressing GABA A receptors (α(1)ß(2)γ(2S) subtype) in two-microelectrode voltage clamp measurements. Thirteen compounds were subsequently identified by HPLC-based activity profiling as responsible for GABA A receptor activity and purified in preparative scale. 6-Cinnamoyl-6,7-dihydro-7-myrceneol and 5,6-dihydrocuparenic acid were thereby isolated for the first time. The determination of the absolute stereochemistry of these compounds was achieved by comparison of experimental and calculated ECD spectra. All but one of the 13 isolated compounds from K. longipedunculata potentiated I(GABA) through GABA A receptors composed of α(1)ß(2)γ(2S) subunits in a concentration-dependent manner. Potencies ranged from 12.8±3.1 to 135.6±85.7 µM, and efficiencies ranged from 129.7±36.8% to 885.8±291.2%. The phytochemical profiles of petroleum ether extracts of Kadsura japonica fruits (114.1±2.6% potentiation of I(GABA) at 100 µg/ml, n=2), and Schisandra chinensis fruits (inactive at 100 µg/ml) were compared by HPLC-PDA-ESIMS with that of K. longipedunculata.