Pawinee Piyachaturawat

Interaction of human and rat organic anion transporter 2 with various cephalosporin antibiotics

Cephalosporin antibiotics are thought to be excreted into the urine via organic anion transporters (OATs) and OAT can mediate nephrotoxicity by cephalosporins, particularly by cephaloridine. The purpose of this study was to elucidate the interaction of human-OAT2 and rat-OAT2 with cephalosporin antibiotics using proximal tubule cells stably expressing human-OAT2 and rat-OAT2. Human-OAT2 is localized to the basolateral side of the proximal tubule, whereas rat-OAT2 is localized to the apical side of the proximal tubule. Cephalosporins tested were cephalothin, cefoperazone, cefazolin, ceftriaxone, cephaloridine, cefotaxime, cefadroxil and cefamandole. These cephalosporins dose-dependently inhibited organic anion uptake mediated by human-OAT2 and rat-OAT2. There was no species difference observed for the effects of OAT2 with cephalosporins between human and rat transporters. Kinetic analysis revealed that the inhibitory effects for human-OAT2 were competitive. Cephaloridine significantly decreased the viability of cells stably expressing human-OAT2, human-OAT1, human-OAT3 and human-OAT4. The decreased viability of cells stably expressing human-OAT1, human-OAT3 and human-OAT4 but not human-OAT2 was reversed by probenecid. In conclusion, human-OAT2 interacts with cephalosporins, and thus, human-OAT2 may mediate the uptake of cephalosporins on the basolateral side of the proximal tubule. The interaction of human-OAT2 with cephalosporins was the weakest among the basolateral human-OATs tested. In addition, it is suggested that human-OATs mediate cephaloridine-induced nephrotoxicity.

Diarylheptanoids, new phytoestrogens from the rhizomes of Curcuma comosa: Isolation, chemical modification and estrogenic activity evaluation.

Three new diarylheptanoids, a 1:2 mixture of (3S)- and (3R)-1-(4-methoxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol (13a and 13b) and 1-(4-hydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-one (15), together with two synthetically known diarylheptanoids 1,7-diphenyl-(1E,3E,5E)-1,3,5-triene (9) and 1-(4-hydroxyphenyl)-7-phenyl-(4E,6E)-4,6-heptadien-3-one (16), and nine known diarylheptanoids, 2, 8, 10-12, 14, a 3:1 mixture of 17a and 17b, and 18, were isolated from the rhizomes of Curcuma comosa Roxb. The absolute stereochemistry of the isolated compounds has also been determined using the modified Moshers method. The isolated compounds and the chemically modified analogues were evaluated for their estrogenic-like transcriptional activity using RT-PCR in HeLa cell line. Some of the isolated diarylheptanoids and their modified analogues exhibited estrogenic activity comparable to or higher than that of the phytoestrogen genistein. Based on the transcriptional activation of both estrogenic targets, Bcl-xL and ERbeta gene expression, the structural features for a diarylheptanoid to exhibit high estrogenic activity are the presence of an olefinic function conjugated with the aromatic ring at the 7-position, a keto group at the 3-position, and a phenolic hydroxyl group at the p-position of the aromatic ring attached to the 1-position of the heptyl chain.

Acute and subacute toxicity of piperine in mice, rats and hamsters

Piperine is acutely toxic to mice, rats and hamsters. The LD50 values for a single i.v., i.p., s.c., i.g. and i.m. administration of piperine to adult male mice were 15.1, 43, 200, 330 and 400 mg/kg body wt, respectively. The i.p. LD50 value was increased to 60 mg/kg body wt in adult female and 132 mg/kg body wt in weanling male mice. In adult female rats, the i.p. LD50 value was 33.5 mg/kg body wt whereas the i.g. LD50 value was increased to 514 mg/kg body wt. Most animals given a lethal dose died of respiratory paralysis within 3-17 min. In subacute toxicity studies, the rats died within 1-3 days after treatment. Histopathologic changes included severe hemorrhagic necrosis and edema in gastrointestinal tract, urinary bladder and adrenal glands. Death of these animals may be attributable to multiple dysfunctions in their organs.

Diarylheptanoid Phytoestrogens Isolated from the Medicinal Plant Curcuma comosa: Biologic Actions in Vitro and in Vivo Indicate Estrogen Receptor–Dependent Mechanisms

Background Diarylheptanoids isolated from Curcuma comosa Roxb. have been recently identified as phyto estrogens. However, the mechanism underlying their actions has not yet been identified. Objectives We characterized the estrogenic activity of three active naturally occurring diarylheptanoids both in vitro and in vivo. Methods We characterized mechanisms of estrogenic action of the diarylheptanoids (3S)-1,7-diphenyl-(6E)-6-hepten-3-ol (D1), 1,7-diphenyl-(6E)-6-hepten-3-one (D2), and (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (D3) by using a real-time polymerase chain reaction assay, a mammalian transfection model, and a uterotrophic assay in mice. Results All diarylheptanoids up-regulated estrogen-responsive genes in estrogen-responsive breast cancer cells (MCF-7). In HepG2 cells transfected with estrogen receptor (ER) β or different ERα functional receptor mutants and the Vit-ERE-TATA-Luc reporter gene, all diarylheptanoids induced transcription through a ligand-dependent human ERα-ERE–driven pathway, which was abolished with ICI 182,780 (ER antagonist), whereas only D2 was active with ERβ. An ERα mutant lacking the functional AF2 (activation function 2) region was not responsive to 17β-estradiol (E2) or to any of the diarylheptanoids, whereas ERα lacking the AF1 domain exhibited wild-type–like activity. D3 markedly increased uterine weight and proliferation of the uterine epithelium in ovariectomized mice, whereas D1 and D2 were inactive. D3, like E2, up-regulated lactoferrin (Ltf) gene expression. The responses to D3 in the uterus were inhibited by ICI 182,780. In addition, D3 stimulated both classical (Aqp5) and nonclassical (Cdkn1a) ER-mediated gene regulation. Conclusions The results suggest that the D3 diarylheptanoid is an agonist for ER both in vitro and in vivo, and its biological action is ERα selective, specifically requiring AF2 function, and involves direct binding via ER as well as ERE-independent gene regulation.

A phloracetophenone glucoside with choleretic activity from Curcuma comosa

Abstract Three known diarylheptanoids, 1,7-diphenyl-5-hydroxy-(1E)-1-heptene, 5-hydroxy-7-(4-hydroxyphenyl)-1-phenyl-(1E)-1-heptene and 7-(3,4-dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene, were isolated from the ethyl acetate extract of Curcuma comosa rhizomes. A phloracetophenone glucoside, 4,6- dihydroxy -2-O-(β- d -glucopyranosyl)acetophenone , was isolated from the ethyl acetate and n-butanol extracts. This compound exhibited choleretic activity in rats.

A phytoestrogen diarylheptanoid mediates estrogen receptor/Akt/glycogen synthase kinase 3β protein-dependent activation of the Wnt/β-catenin signaling pathway.

Background: Diarylheptanoid (ASPP 049) isolated from C. comosa exhibits high estrogenic activity. Results: ASPP 049 rapidly induced β-catenin accumulation in the nucleus and activated TCF/LEF-mediated activation of Wnt/β-catenin signaling. Conclusion: ASPP 049 from C. comosa induces preosteoblastic cell proliferation and differentiation through activation of Wnt/β-catenin signaling. Significance: Providing a scientific rationale for using C. comosa as a dietary supplement to prevent bone loss in postmenopausal women. Estrogen promotes growth in many tissues by activating Wnt/β-catenin signaling. Recently, ASPP 049, a diarylheptanoid isolated from Curcuma comosa Roxb., has been identified as a phytoestrogen. This investigation determined the involvement of Wnt/β-catenin signaling in the estrogenic activity of this diarylheptanoid in transfected HEK 293T and in mouse preosteoblastic (MC3T3-E1) cells using a TOPflash luciferase assay and immunofluorescence. ASPP 049 rapidly activated T-cell-specific transcription factor/lymphoid enhancer binding factor-mediated transcription activity and induced β-catenin accumulation in the nucleus. Interestingly, the effects of ASPP 049 on the transcriptional activity and induction and accumulation of β-catenin protein in the nucleus of MC3T3-E1 cells were greater compared with estradiol. Activation of β-catenin in MC3T3-E1 cells was inhibited by ICI 182,780, suggesting that an estrogen receptor is required. In addition, ASPP 049 induced phosphorylations at serine 473 of Akt and serine 9 of GSK-3β. Moreover, ASPP 049 also induced proliferation and expressions of Wnt target genes Axin2 and Runx2 in MC3T3-E1 cells. In addition, ASPP 049 increased alkaline phosphatase expression, and activity that was abolished by DKK-1, a blocker of the Wnt/β-catenin receptor. Taken together, these results suggest that ASPP 049 from C. comosa induced osteoblastic cell proliferation and differentiation through ERα-, Akt-, and GSK-3β-dependent activation of β-catenin signaling. Our findings provide a scientific rationale for using C. comosa as a dietary supplement to prevent bone loss in postmenopausal women.

Estrogenic Activity of Diarylheptanoids from Curcuma comosa Roxb. Requires Metabolic Activation

Curcuma comosa Roxb. has traditionally been used as a dietary supplement for health promotion in peri- and postmenopausal women in Thailand. We investigated the estrogenic activity of 7 naturally occurring diarylheptanoids from the extracts of C. comosa both in vitro and in vivo. A yeast recombinant system containing human estrogen receptor alpha, coactivator TIF2 and a beta-galactosidase reporter gene was used to determine estrogenic activity of diarylheptanoids metabolically activated with rat liver S9-fraction prior to the assay. The most potent compound was (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol, with a relative potency of 4% compared to 17beta-estradiol. The metabolic activation of diarylheptanoids markedly enhanced their efficiency. The chemical structure required for estrogenic activity of diarylheptanoids was the presence of a keto group at C3 and absence of hydroxyl moiety in ring B. Only diarylheptanoids showing full estrogenic efficiency in vitro were able to elicit uterotrophic activity of in immature ovariectomized rat. This is the first evidence for in vivo estrogenic activity of diarylheptanoids from C. comosa. This novel class of natural phytoestrogens has the potential to be developed for use as dietary supplement in the treatment of menopausal symptoms.

New substituted C-19-andrographolide analogues with potent cytotoxic activities.

Andrographolide, the major diterpenoid lactone from Andrographis paniculata, is toxic against cancer cells. In the present study, we investigated the structure-activity relationships (SARs) of 19 andrographolide analogues which were synthesized by modification at the three hydroxyl groups. A number of the andrographolide analogues showed much higher cytotoxic activities than that of the parent compound on cancer cells including P-388, KB, COL-2, MCF-7, LU-1 and ASK cells. SAR studies of the synthetic analogues indicated that the introduction of silyl ether or triphenylmethyl ether group into C-19 of the parent compound led to increase in toxicity against the cancer cells. The 19-O-triphenylmethyl ether analogue 18 showed higher cytotoxic activity than the potent anticancer drug ellipticine, and this analogue may serve as a potential structure lead for the development of new anticancer drugs.

Uterotrophic Effect of Curcuma comosa in Rats

Uterotrophic activities of various extracts of Curcuma comosa were investigated in rats. Among the four extracts tested (hexane, ethyl acetate, butanol and aqueous extract), hexane was the most effective in increasing uterine weight and glycogen content of bilaterally ovariectomized immature rats. The responses were dose-dependent. The hexane extract also induced cornification of vaginal epithelium, promote growth and induced keratinization of vaginal mucosa in ovariectomized mature rats. The duration of action and strength of the extract in increasing uterine weight, uterine levels of glycogen, DNA and protein, and inducing of morphological changes were less than those of estradiol. However, when the extract was given prior to, concurrent with, or after estradiol injection, it enhanced the uterine response to estradiol in all cases. These results suggest that the uterotrophic action C. comosa is mediated through weak estrogenic agonistic activity of the plant.

Reduction of plasma cholesterol by Curcuma comosa extract in hypercholesterolaemic hamsters.

The influence of the extract of Curcuma comosa Roxb. (Zingiberaceae) on lipid metabolism was investigated in hypercholesterolaemic hamsters. Intragastric administration of the ethyl acetate extract of C. comosa rhizome (0-500 mg/kg per day) to hypercholesterolaemic animals for 7 days decreased both plasma triglyceride and cholesterol levels in a dose-dependent manner. The reduction of plasma cholesterol levels was accompanied by a significant increase in the hepatic cholesterol content while the triglyceride content was not significantly changed. The increase of the hepatic cholesterol content was brought about by an expansion of the free cholesterol pool which specifically augments biliary cholesterol excretion. The C. comosa extract also increased plasma high density lipoprotein (HDL)-cholesterol and decreased plasma low density lipoprotein (LDL)-cholesterol. These results suggest that the C. comosa extract exerts a hypolipidaemic action by acceleration of lipid mobilization from extrahepatic tissue to the liver which subsequently increases excretion of cholesterol via the bile for excretion.