Chaiyo Chaichantipyuth

Cytotoxic labdane diterpenoids from Croton oblongifolius

Three labdane diterpenoids, 2-acetoxy-3-hydroxy-labda-8(17),12(E)-14-triene, 3-acetoxy-2-hydroxy-labda-8(17),12(E)-14-triene, and 2,3-dihydroxy-labda-8(17),12(E),14-triene were isolated from stem bark of Croton oblongifolius. Their structures were established by spectroscopic data, and each was also tested for cytotoxicity against various human tumor cell lines. The latter compound showed non-specific, moderate, cytotoxicities against all the cell lines; whereas the first two compounds were less active.

Comparative analysis of the chemical constituents of two varieties of Pueraria candollei

A high-performance liquid chromatography (HPLC) method was developed to determine the contents of miroestrol and deoxymiroestrol in the tubers of Pueraria candollei var. mirifica and P. candollei var. candollei. The linear detection ranges were 0.78-25.00 μg/mL for miroestrol and 1.56-25.00 μg/mL for deoxymiroestrol. The limit of detection (LOD) and limit of quantification (LOQ) were 0.2 and 0.78 μg/mL, respectively, for miroestrol and 0.78 and 1.56 μg/mL, respectively, for deoxymiroestrol. Our results suggest that both varieties of P. candollei can produce miroestrol and deoxymiroestrol and that the developed HPLC method can be applied for quality control of plants and their products.

CNS inhibitory effects of barakol, a constituent of Cassia siamia Lamk

The present study determined the pharmacological profile of barakol, a major constituent of Cassia siamea Lamk., in rodent behavioral and neurochemical tests. Barakol reduced spontaneous locomotor activity, increased the number of sleeping animals and prolonged the thiopental-induced sleeping time, indicating a sedative effect. As for interactions between barakol and convulsants (pentylenetetrazole (PTZ), picrotoxin, bicuculline and strychnine), only a high dose (100 mg/kg, i.p.) of barakol slightly prolonged the latency of clonic convulsion induced by picrotoxin. This suggests that the sedative effect may not be induced via the GABA or glycine systems. There was no evidence of an anxiolytic effect of barakol in the plus-maze test. However, barakol (25-100 mg/kg, i.p.) could suppress methamphetamine (1 mg/kg, i.p.)-induced hyper-locomotor activity in a dose-dependent manner, indicating an effect on the dopaminergic system. In a microdialysis study, the dose of barakol (100 mg/kg) that inhibited spontaneous locomotor activity in mice did not affect the basal levels of extracellular dopamine (DA) or its metabolites in the striatum. However, pretreatment with barakol (100 mg/kg, i.p.) decreased the maximal dopamine release and dopamine turnover induced by methamphetamine (1 mg/kg, i.p.). This finding indicates that the inhibitory effect of barakol on dopamine release may account for the blocking effect of barakol on the striatum-related behavior induced by methamphetamine.

Quantitative analysis of miroestrol and kwakhurin for standardisation of Thai miracle herb ‘Kwao Keur’ (Pueraria mirifica) and establishment of simple isolation procedure for highly estrogenic miroestrol and deoxymiroestrol

Quantitative analysis of miroestrol (1) and kwakhurin (3) by HPLC, leading to standardisation of commercially available Thai miracle herb ‘Kwao Keur’ which has been identified with Pueraria mirifica, was established using independent solvent systems. The simple isolation procedure of highly estrogenic miroestrol (1) and deoxymiroestrol (2) from P. mirifica was also proposed.

High performance enzyme-linked immunosorbent assay for determination of miroestrol, a potent phytoestrogen from Pueraria candollei

Pueraria candollei associated preparation is widely applied in folk Thai medicine for rejuvenating purpose in aged people, which correlated with its pharmacological activities reported by pre-clinical and clinical trials. Therefore, standardized products of this plant are needed by consumers and health care personnel. Miroestrol, a potent and stable phytoestrogen in P. candollei, exhibited potential to be biomarker for quality control of P. candollei samples in research or industrial levels. Indirect competitive enzyme-linked immunosorbant assay (ELISA) for miroestrol determination was developed and validated by using polyclonal antibody from rabbit immunization. The polyclonal antibody recognized specifically to miroestrol, which exhibited cross-reactivity to deoxymiroestrol and isomiroestrol with 6.68% and 1.05%, respectively. The linearity range of measurement was 0.73-3000 ng mL(-1), which coefficient of variation (CV) of both intra- and inter-plate determination was less than 5%. With spiked samples of known amount miroestrol, the percentages of recovery were 98.80-104.37% and 98.31-106.69% in P. candollei and its involved product samples, respectively. Validated ELISA was comparable with published HPLC method (R(2)=0.9996) (Yusakul et al.) in samples with various miroestrol contents. For application, the P. candollei involved preparations contained miroestrol 0.695±0.037-12.108±0.285 μg g(-1) dry wt. The developed ELISA was high performance for miroestrol determination, which could be applied for P. candollei quality control in research fields and industrial productions.

Cassane diterpenoid from Caesalpinia major.

A new cassane diterpenoid, 14-deoxy-epsilon-caesalpin was isolated from the seed kernels of Caesalpinia major and its structure was determined by spectroscopic data.

Highly selective and sensitive determination of deoxymiroestrol using a polyclonal antibody-based enzyme-linked immunosorbent assay

Pueraria candollei-associated products are of interest to worldwide consumers for their rejuvenating and cosmetic purposes. In addition, clinical trials have supported the beneficial effects of P. candollei on the alleviation of menopausal symptoms. Deoxymiroestrol, which was reported as the most potent phytoestrogen in the tuberous root of P. candollei, exhibited potential as a biomarker of P. candollei-derived samples and products. A polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for deoxymiroestrol determination. The raised antibody bound specifically to deoxymiroestrol with very low cross reactivities of 1.26 and 0.42% to structurally related miroestrol and isomiroestrol, respectively. The linear range was 0.73-3000.00 ng mL(-1), and the coefficients of variation for both the intra- and inter-plate determinations were less than 5%. In samples spiked with a known amount of deoxymiroestrol, the recoveries were 99.82-102.58% in P. candollei samples and 98.07-106.33% in its products samples. In comparison with other analytical methods, the validated ELISA was comparable to published HPLC-UV methods for samples with high deoxymiroestrol content (R(2)=0.9993). Furthermore, ELISA can be used for samples with deoxymiroestrol concentrations that are too small to detect by HPLC and for conditions when other chemicals cause interference with chromatographic analysis. For the P. candollei-derived products, the preparations contained 0.154-10.998 µg g(-1) dry wt. Our ELISA successfully measured deoxymiroestrol content with high sensitivity, selectivity, accuracy and rapidity. Therefore, this ELISA showed potential for dosage standardization of P. candollei-associated samples.

Barakol-induced apoptosis in P19 cells through generation of reactive oxygen species and activation of caspase-9.

ETHNOPHARMACOLOGICAL RELEVANCE Barakol, an anxiolytic agent isolated from Senna siamea leaves which has been traditionally used for producing natural sleep, has been described as toxic to patients. AIM OF THE STUDY The aim of current study was to investigate the molecular mechanism of barakol-induced toxicity in mouse embryonal carcinoma P19 cell model. MATERIALS AND METHODS XTT assay was used to determine cell viability in P19 cells treated with barakol. Apoptotic cells were detected by Hoechst 33342 staining. Intracellular reactive oxygen species (ROS) generation was analyzed by flow cytometry using a fluorescent dye, DCFH-DA. Detection of apoptotic protein expression in P19 cells was performed by Western blot analysis. Caspase-9 activity was measured using a fluorescent immunosorbent enzyme assay kit. RESULTS Treatment with barakol decreased cell viability in a concentration- and time-dependent manner with an IC(50) value of 1.5mM in 24-h treated cells. A Hoechst 33342 assay revealed that barakol cytotoxicity was due to a significant increase in the number of apoptotic cells. Different scavengers to characterize ROS were utilized and revealed that hydroxyl radicals played a major role in ROS-induced apoptosis in barakol-treated cells. Western blot analysis demonstrated that barakol-induced apoptosis was mediated by the increase in expression ratio of Bax/Bcl-2. Furthermore, increase in caspase-9 activity after exposure to barakol for 24h was also observed. Pretreatment of cells with N-acetyl-l-cysteine (NAC) attenuated intracellular ROS generation, the Bax/Bcl-2 protein expression, and apoptosis. CONCLUSIONS The mechanism of barakol-mediated toxicity in P19 cells is mainly associated with the ROS generation, followed by the imbalance of the Bax/Bcl-2 ratio, and caspase-9 activation leading to apoptotic cell death. Pretreatment of cells with NAC could antagonize the toxicity produced by barakol.

New labdane-type diterpenoids from Croton oblongifolius and their cytotoxic activity

Eight new labdane-type diterpenoids (1-3) and (5-9) and hardwickiic acid (4), a clerodane, were isolated from the stem bark of Croton oblongifolius. Their structures were established to be 3-oxygenated ent-manoyl oxide derivatives with a 8,13-epoxytricyclic ring system and hydroxylabdandienes on the basis of spectroscopic and X-Ray crystallographic analysis. The absolute stereochemistry of the core labdane skeleton was deduced to be (5S,8S,9S,10R,13S) from the X-Ray crystallographic analysis of the p-bromobenzoate of ent-3α-hydroxymanoyl oxide (3) and mutual chemical correlation. Cytotoxicity of these compounds was tested against a panel of human tumor cell lines.

Stability of Barakol under Hydrolytic Stress Conditions and its Major Degradation Product

The aim of the present study was to investigate the stability of barakol, an anxiolytic constituent extracted from leaves of Senna siamea (Lam.) Irwin & Barneby (syn. Cassia siamea Lam.), under the International Conference on Harmonisation suggested conditions using HPLC with photodiode array detection. Extensive degradation of barakol was found to occur under alkaline conditions through base-catalyzed hydrolysis. Mild degradation of barakol was observed under thermal and oxidative stress while it was stable under acidic conditions. The reaction rate constants (Kobs) of barakol degradation under alkaline conditions at pHs 12 and 13 were 3.0x10(-5) and 9.6x10(-3) min(-1), respectively. The activation energy according to the Arrhenius plot was calculated to be 26.9+/-3.3 kcal/mol at pH 13 and temperatures between 12 and 51 degrees C. The major degradation product of barakol under both alkaline and thermal stress conditions was characterized by LC-MS and NMR as cassiachromone.