Aporn Chuncharunee

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.

Re-evaluating acridine orange for rapid flow cytometric enumeration of parasitemia in malaria-infected rodents.

Methods facilitating research in malaria are of pivotal relevance. Flow cytometry offers the possibility of rapid enumeration of parasitemia. It relies on staining the parasite DNA to distinguish between infected and non‐infected red blood cell (RBC) populations. Unfortunately, in rodents abundant reticulocyte RNA interferes with the application of the method. This results in time‐consuming sample preparation protocols that offer no clear advantage over microscopic counting. We re‐evaluated the use of the DNA/RNA discriminating vital fluorochrome acridine orange (AO) for rapid flow cytometric enumeration of parasitemia in rodents.

A protective effect of Curcuma comosa Roxb. on bone loss in estrogen deficient mice.

ETHNOPHARMACOLOGICAL RELEVANCE Curcuma comosa Roxb. or Wan chak motluk is an indigenous medicinal herb and has traditionally been used among postmenopausal women for relief of unpleasant menopausal symptoms. AIM OF THE STUDY Estrogen deficiency is a causative factor in the development of osteoporosis in menopausal women. Phytoestrogens, non-steroidal plant-derived compounds which have an array of beneficial effects, are considered as an effective alternative compound in preventing bone loss caused by the deficiency of estrogen. The present study determined the potential effect of Curcuma comosa Roxb. (C. comosa) hexane extract containing phytoestrogens in protecting bone loss induced by ovariectomy in mice. MATERIALS AND METHODS Mature Swiss albino female mice were ovariectomized and treated with the C. comosa extract for 5 weeks. Bone calcium content, bone mass density, histology, and bone markers were evaluated. RESULTS The ovariectomized mice showed a marked decrease in total bone calcium content and bone mass density of lumbar vertebrae 5-6, femur and tibia bone in comparison with the intact control mice. Bone histology demonstrated the poor development of endochondral bone formation in ovariectomized mice which correlated with a decrease in plasma bone alkaline phosphatase activity. Treatment with C. comosa protected against the loss of total bone calcium content and bone mass density in both trabecular and cortical bones, similar to results observed with estrogen treatment. In addition, C. comosa treatment resulted in less increase in uterine weight compared to estrogen treatment. CONCLUSION Our results suggest that C. comosa prevents bone loss induced by estrogen deficiency. Therefore, C. comosa would be a potential alternative treatment for prevention of postmenopausal osteoporosis.

Bone Sparing Effect of a Novel Phytoestrogen Diarylheptanoid from Curcuma comosa Roxb. in Ovariectomized Rats

Phytoestrogens have been implicated in the prevention of bone loss in postmenopausal osteoporosis. Recently, an active phytoestrogen from Curcuma comosa Roxb, diarylheptanoid (DPHD), (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol, was found to strongly promote human osteoblast function in vitro. In the present study, we demonstrated the protective effect of DPHD on ovariectomy-induced bone loss (OVX) in adult female Sprague-Dawley rats with 17β-estradiol (E2, 10 µg/kg Bw) as a positive control. Treatment of OVX animals with DPHD at 25, 50, and 100 mg/kg Bw for 12 weeks markedly increased bone mineral density (BMD) of tibial metaphysis as measured by peripheral Quantitative Computed Tomography (pQCT). Histomorphometric analysis of bone structure indicated that DPHD treatment retarded the ovariectomy-induced deterioration of bone microstructure. Ovariectomy resulted in a marked decrease in trabecular bone volume, number and thickness and these changes were inhibited by DPHD treatment, similar to that seen with E2. Moreover, DPHD decreased markers of bone turnover, including osteocalcin and tartrate resistant acid phosphatase (TRAP) activity. These results suggest that DPHD has a bone sparing effect in ovariectomy-induced trabecular bone loss and prevents deterioration of bone microarchitecture by suppressing the rate of bone turnover. Therefore, DPHD appears to be a promising candidate for preserving bone mass and structure in the estrogen deficient women with a potential role in reducing postmenopausal osteoporosis.

Choleretic activity of phloracetophenone in rats: structure-function studies using acetophenone analogues.

The relationship between the chemical structure and choleretic activity of phloracetophenone (2,4,6-trihydroxyacetophenone) was investigated in adult male rats. Fourteen acetophenone analogues, with different substituents on the benzene nucleus, were intraduodenally administered and bile samples were collected via a bile fistula. All of the compounds tested immediately induced choleresis. For the same number of substituents on the benzene ring, hydroxy analogues induced a greater choleresis. The number and position of hydroxy substituents on the benzene nucleus play an important role in determining choleretic activity and biliary secretion of bile acid, but had no relation to biliary excretion of cholesterol. The choleretic activity of the hydroxylated compounds was inversely related to hydrophobicity, as inferred by thin-layer chromatography (TLC). Among the hydroxylated acetophenone analogues, 2,4,6-trihydroxyacetophenone was identified as the most potent, with a choleretic activity of 231.8+/-6.1 microl/mmol/min. It induced both a high bile flow rate and a high bile salt output and led to lower plasma cholesterol levels. This bile had a low lithogenic potential. The results suggest that a structural requirement for high choleretic activity of 2,4,6-trihydroxyacetophenone is a substituent hydroxy group at 4-position. Additional hydroxy groups at 2- and 6-positions are essential for the induction of higher an output of bile acid, and possibly, other solid materials.

Role of ERK1/2 signaling in dengue virus-induced liver injury.

The liver is considered to be an important organ of dengue virus (DENV) replication and pathogenesis. However, molecular mechanisms of hepatic injury are still poorly understood. Modulation of Mitogen Activated Protein Kinases (MAPKs) was previously shown to affect DENV-induced apoptosis of hepatocytes in vitro. However, the in vivo role of ERK1/2, a member of the MAPK family, and the question whether its activation can facilitate cell survival or cell death, has not been thoroughly investigated. Therefore, the role of ERK1/2 in a mouse model of DENV infection was examined. Our results show that DENV induces phosphorylation of ERK1/2 and increases apoptosis. Inhibition of phosphorylated ERK1/2 by the selective ERK1/2 inhibitor, FR180204, limits hepatocyte apoptosis and reduces DENV-induced liver injury. Clinical parameters, including leucopenia, thrombocytopenia, transaminases and histology, show improvements after FR180204 treatment. The expression of cell death genes was further identified using real-time PCR array and Western blot analysis. Caspase-3 was significantly decreased in FR180204 treated DENV-infected mice compared to the levels of untreated DENV-infected mice suggesting the role of ERK1/2 signaling in immune-mediated liver injury during DENV infection.

SB203580 Modulates p38 MAPK Signaling and Dengue Virus-Induced Liver Injury by Reducing MAPKAPK2, HSP27, and ATF2 Phosphorylation.

Dengue virus (DENV) infection causes organ injuries, and the liver is one of the most important sites of DENV infection, where viral replication generates a high viral load. The molecular mechanism of DENV-induced liver injury is still under investigation. The mitogen activated protein kinases (MAPKs), including p38 MAPK, have roles in the hepatic cell apoptosis induced by DENV. However, the in vivo role of p38 MAPK in DENV-induced liver injury is not fully understood. In this study, we investigated the role of SB203580, a p38 MAPK inhibitor, in a mouse model of DENV infection. Both the hematological parameters, leucopenia and thrombocytopenia, were improved by SB203580 treatment and liver transaminases and histopathology were also improved. We used a real-time PCR microarray to profile the expression of apoptosis-related genes. Tumor necrosis factor α, caspase 9, caspase 8, and caspase 3 proteins were significantly lower in the SB203580-treated DENV-infected mice than that in the infected control mice. Increased expressions of cytokines including TNF-α, IL-6 and IL-10, and chemokines including RANTES and IP-10 in DENV infection were reduced by SB203580 treatment. DENV infection induced the phosphorylation of p38MAPK, and its downstream signals including MAPKAPK2, HSP27 and ATF-2. SB203580 treatment did not decrease the phosphorylation of p38 MAPK, but it significantly reduced the phosphorylation of MAPKAPK2, HSP27, and ATF2. Therefore, SB203580 modulates the downstream signals to p38 MAPK and reduces DENV-induced liver injury.

Long-term effect of phytoestrogens from Curcuma comosa Roxb. on vascular relaxation in ovariectomized rats.

Phytoestrogens have been implicated as promising therapeutic agents to treat the vascular impairment seen in menopausal women. The present study investigated the long-term effects of phytoestrogens from Curcuma comosa Roxb. on vascular relaxation of isolated thoracic aorta from ovariectomized (OVX) rats. Treatment of OVX rats for 12 weeks with C. comosa powder, hexane extract, and a novel phytoestrogen, diarylheptanoid-D3, [(3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol] prevented impairment of the endothelium-dependent relaxation response to acetylcholine in OVX, but not the endothelium-denude aortic ring relaxation in response to sodium nitroprusside. These data suggest that the vascular relaxation effect of C. comosa is mediated via endothelial cells. Treatment with D3 also increased endothelial nitric oxide synthase (eNOS) and estrogen receptor-α (ERα) protein expression in the aorta of OVX rats and suppressed elevated tumor necrosis factor-α (TNF-α) expression in OVX aortic rings. These results indicate that C. comosa treatment prevents impairment of vascular relaxation in estrogen-deficient animals via the ER-eNOS pathway as well as through its ability to promote an anti-inflammatory response.

Protection of centrilobular necrosis by Curcuma comosa Roxb. in carbon tetrachloride-induced mice liver injury.

AIM OF THE STUDY To investigate the protective effect and possible mechanism of Curcuma comosa hexane extract on CCl(4)-induced liver injury in adult male mice. MATERIALS AND METHODS Hepatotoxicity was induced by an intraperitoneal injection of CCl(4) and was evaluated after 24 h from the elevations of plasma alanine transaminase (ALT) and aspartate transaminase (AST) activities, and histological analysis of liver injuries. Hexane extract of Curcuma comosa was given at different time points from 1 to 72 h, prior to CCl(4) administration and the protection from liver injury was assessed. RESULTS CCl(4)-induced damage to liver cells was resulted in elevations of plasma ALT and AST activities. Pretreatment with Curcuma comosa hexane extract 24 h at a dose of 100, 250, and 500 mg/kg BW resulted in a dose-dependent prevention of the increases in plasma ALT and AST activities as well as time dependent. The protective effect of the extract at a dose of 500 mg/kg BW was seen at 12-24 h. Pretreatment of the extract completely prevented elevation of plasma ALT and AST activities, and centrilobular necrosis. The protective effect of Curcuma comosa was associated with restoration of hepatic glutathione content, and CYP2E1 catalytic activity, and its mRNA and protein levels as well as increase in activity of glutathione-S-transferase (GST). CONCLUSION Curcuma comosa has a potent protective property against CCl(4)-induced hepatic injuries via the activation of detoxifying mechanisms (GST) as well as reduction of the bioactive toxic metabolites. Therefore, Curcuma comosa may be beneficial for prevention of hepatotoxicity.

Evaluation of the acute and subacute toxicity of a choleretic phloracetophenone in experimental animals.

Toxicity of a choleretic compound, phloracetophenone (2,4,6-trihydroxyacetophenone; THA) was investigated in mice, rats and hamsters. Acute toxicity of THA was observed to be dependent on species and route of administration, but not sex and age. LD(50) values for an acute toxicity of a single i.p. administration to adult male hamsters and mice were 338 and 365 mg/kg BW, respectively. It was significantly increased to 489 mg/kg BW in adult male rats and greatly increased by i.g. route. Subacute toxicity was investigated in adult male mice by giving THA at a doses of 37-300 mg/kg BW/day, i.g. for 30 consecutive days. High doses of THA induced periportal hepatocyte degeneration whereas plasma concentrations of alanine and aspartate aminotransferases, bilirubin, and blood urea nitrogen, and hepatic triglyceride content were only slightly increased. The possible therapeutic effect of the choleretic THA was evaluated in the ethinylestradiol (EE)-induced cholestasis. THA enhanced the hepatic clearance of sulfobromophthalein and decreased the elevated plasma alkaline phosphatase in EE-cholestatic rats to control levels. These results suggested that THA at biologically active choleretic dose had low toxicity, it might be safe for further development as a therapeutic agent for a short period of treatment in cholestasis.