Arunporn Itharat

Cytotoxic activity of Thai medicinal plants against human cholangiocarcinoma, laryngeal and hepatocarcinoma cells in vitro

BackgroundCholangiocarcinoma is a serious public health in Thailand with increasing incidence and mortality rates. The present study aimed to investigate cytotoxic activities of crude ethanol extracts of a total of 28 plants and 5 recipes used in Thai folklore medicine against human cholangiocarcinoma (CL-6), human laryngeal (Hep-2), and human hepatocarcinoma (HepG2) cell lines in vitro.MethodsCytotoxic activity of the plant extracts against the cancerous cell lines compared with normal cell line (renal epithelial cell: HRE) were assessed using MTT assay. 5-fluorouracil was used as a positive control. The IC50 (concentration that inhibits cell growth by 50%) and the selectivity index (SI) were calculated.ResultsThe extracts from seven plant species (Atractylodes lancea, Kaempferia galangal, Zingiber officinal, Piper chaba, Mesua ferrea, Ligusticum sinense, Mimusops elengi) and one folklore recipe (Pra-Sa-Prao-Yhai) exhibited promising activity against the cholangiocarcinoma CL-6 cell line with survival of less than 50% at the concentration of 50 μg/ml. Among these, the extracts from the five plants and one recipe (Atractylodes lancea, Kaempferia galangal, Zingiber officinal, Piper chaba, Mesua ferrea, and Pra-Sa-Prao-Yhai recipe) showed potent cytotoxic activity with mean IC50 values of 24.09, 37.36, 34.26, 40.74, 48.23 and 44.12 μg/ml, respectively. All possessed high activity against Hep-2 cell with mean IC50 ranging from 18.93 to 32.40 μg/ml. In contrast, activity against the hepatoma cell HepG2 varied markedly; mean IC50 ranged from 9.67 to 115.47 μg/ml. The only promising extract was from Zingiber officinal (IC50 = 9.67 μg/ml). The sensitivity of all the four cells to 5-FU also varied according to cell types, particularly with CL-6 cell (IC50 = 757 micromolar). The extract from Atractylodes lancea appears to be both the most potent and most selective against cholangiocarcinoma (IC50 = 24.09 μg/ml, SI = 8.6).ConclusionsThe ethanolic extracts from five plants and one folklore recipe showed potent cytotoxic activity against CL-6 cell. Sensitivity to other cancerous cell lines varied according to cell types and the hepatocarcinoma cell line. HepG2 appears to be the most resistant to the tested extracts.

Mulberry leaf extract restores arterial pressure in streptozotocin-induced chronic diabetic rats

Free radical-induced vascular dysfunction plays a key role in the pathogenesis of vascular disease found in chronic diabetic patients. Morus alba (MA) leaf extract is promoted for good health especially in diabetic patients. Interestingly, antidiabetic and antioxidant activities of MA have been reported in experimental animals. Thus, the hypothesis of this study was that the long-term treatment with MA could improve vascular reactivity of chronic diabetic rats. To test this hypothesis, we examined the effect of long-term treatment with MA on the vascular responses to vasoactive agents in streptozotocin-induced chronic diabetic rats. The diabetic rats were either orally administered with distilled water, MA (0.25, 0.5 and 1 g/kg per day) or subcutaneously injected with insulin (4 U/kg per day) for 8 weeks. After each treatment, the fasting blood glucose, blood pressure, vascular responses to vasoactive agents and tissue malondialdehyde were examined. Morus alba at the doses of 0.5 and 1 g/kg, which significantly reduced blood glucose level, also significantly decreased the high blood pressure in diabetic rats. Vascular responses of the chronic diabetic rats to vasodilators, acetylcholine (3-30 nmol/kg) and sodium nitroprusside (1-10 nmol/kg) were significantly suppressed by 26% to 44% and 45% to 77% respectively, whereas those to vasoconstrictor, phenylephrine (0.01-0.1 micromol/kg) were significantly increased by 23% to 38% as compared to normal rats. Interestingly, the administration of 0.5 and 1 g/kg MA or 4 U/kg insulin significantly restored the vascular reactivities of diabetic rats. Moreover, 8 weeks of diabetes resulted in the elevation of malondialdehyde content in tissues (liver, kidney, heart, and aorta), and MA treatment significantly lessened this increase. These results provide the first evidence for the efficacy of MA in restoring the vascular reactivity of diabetic rats, the mechanism of which may associate with the alleviation of oxidative stress.

Anticancer activities against cholangiocarcinoma, toxicity and pharmacological activities of Thai medicinal plants in animal models

BackgroundChemotherapy of cholangiocarcinoma (CCA), a devastating cancer with increasing worldwide incidence and mortality rates, is largely ineffective. The discovery and development of effective chemotherapeutics is urgently needed.Methods/DesignThe study aimed at evaluating anticancer activities, toxicity, and pharmacological activities of the curcumin compound (CUR), the crude ethanolic extracts of rhizomes of Zingiber officinale Roscoe (Ginger: ZO) and Atractylodes lancea thung. DC (Khod-Kha-Mao: AL), fruits of Piper chaba Hunt. (De-Plee: PC), and Pra-Sa-Prao-Yhai formulation (a mixture of parts of 18 Thai medicinal plants: PPF) were investigated in animal models. Anti-cholangiocarcinoma (anti-CCA) was assessed using CCA-xenograft nude mouse model. The antihypertensive, analgesic, anti-inflammatory, antipyretic, and anti-ulcer activities and effects on motor coordination were investigated using Rota-rod test, CODA tail-cuff system, writhing and hot plate tests, carrageenan-induced paw edema test, brewers yeast test, and alcohol-induced gastric ulcer test, respectively. Acute and subacute toxicity tests were performed according to the OECD guideline for testing of chemicals with modification.ResultsPromising anticancer activity against CCA in nude mouse xenograft model was shown for the ethanolic extract of AL at all oral dose levels (1000, 3000, and 5000 mg/kg body weight) as well as the extracts of ZO, PPF, and CUR compound at the highest dose level (5000, 4000, and 5000 mg/kg body weight, respectively). PC produced no significant anti-CCA activity. Results from acute and subacute toxicity tests both in mice and rats indicate safety profiles of all the test materials in a broad range of dose levels. No significant toxicity except stomach irritation and general CNS depressant signs were observed. Investigation of pharmacological activities of the test materials revealed promising anti-inflammatory (ZO, PPF, and AL), analgesic (CUR and PPF), antipyretic (CUR and AL), antihypertensive (ZO and AL), and anti-ulcer (CUR, ZO, and AL) activities.ConclusionPlants used in Thai traditional medicine for the treatment of various ailments may provide reservoirs of promising candidate chemotherapeutics for the treatment of CCA.

Mulberry leaf extract stimulates glucose uptake and GLUT4 translocation in rat adipocytes.

Mulberry (Morus alba L.) leaf tea is promoted for its health benefits and the control of diabetes in Asian nations. The blood glucose lowering activity of mulberry leaf extract (MA) has been proven; however, the molecular basis underlying this effect remains unclear. The aim of the present work is to elucidate its mechanism of the antihyperglycemic action, by examining the effect of MA on glucose uptake and the translocation of glucose transporter 4 protein (GLUT4) to the plasma membrane of adipocytes isolated from diabetic rats. The incubation of adipocytes with 5-45 μg/ml MA resulted in 31-54% increase of glucose uptake in a dose-dependent manner. This glucose uptake enhancing effect was inhibited by the phosphoinositol 3-kinase (PI3-K) inhibitor, wortmannin (100 nM). The GLUT4 protein on the plasma membrane fraction of adipocytes was markedly increased after treatment with 15 μg/ml MA extract. Interestingly, gallic acid, one of the phenolic compounds found in MA extract, increased glucose uptake and enhanced the translocation of GLUT4 at concentrations comparable to the amount of gallic acid in the effective concentration ranges of MA. Thus, it is likely that gallic acid contributes, at least in part, to its antihyperglycemic activity. The present results suggest that the antihyperglycemic action of MA is mediated by increasing glucose uptake via the activation of PI3-K signaling pathway and translocation of GLUT4 to the plasma membrane. These findings are the first molecular evidence supporting the mulberry tea as herbal medicine for diabetic patients.

Cytotoxicity, Toxicity, and Anticancer Activity of Zingiber Officinale Roscoe Against Cholangiocarcinoma

Cholangiocarcinoma (CCA) is an uncommon adenocarcinoma which arises from the epithelial cells of the bile ducts. The aim of the study was to investigate the cytotoxicity, toxicity, and anticancer activity of a crude ethanolic extract of ginger (Zingiber officinale Roscoe) against CCA. Cytotoxic activity against a CCA cell line (CL-6) was assessed by calcein-AM and Hoechst 33342 assays and anti-oxidant activity was evaluated using the DPPH assay. Investigation of apoptotic activity was performed by DNA fragmentation assay and induction of genes that may be involved in the resistance of CCA to anticancer drugs (MDR1, MRP1, MRP2, and MRP3) was examined by real-time PCR. To investigate anti-CCA activity in vivo, a total of 80 OV and nitrosamine (OV/ DMN)-induced CCA hamsters were fed with the ginger extract at doses of 1000, 3000, and 5000 mg/kg body weight daily or every alternate day for 30 days. Control groups consisting of 10 hamsters for each group were fed with 5-fluorouracil (positive control) or distilled water (untreated control). Median IC50 (concentration that inhibits cell growth by 50%) values for cytotoxicity and anti-oxidant activities of the crude ethanolic extract of ginger were 10.95, 53.15, and 27.86 μg/ml, respectively. More than ten DNA fragments were visualized and up to 7-9 fold up-regulation of MDR1 and MRP3 genes was observed following exposure to the ethanolic extract of ginger. Acute and subacute toxicity tests indicated absence of any significant toxicity at the maximum dose of 5,000 mg/kg body weight given by intragastric gavage. The survival time and survival rate of the CCA-bearing hamsters were significantly prolonged compared to the control group (median of 54 vs 17 weeks). Results from these in vitro and in vivo studies thus indicate promising anticancer activity of the crude ethanolic extract of ginger against CCA with the absence of any significant toxicity. Moreover, MDR1 and MRP3 may be involved in conferring resistance of CCA to the ginger extract.

Dioscorealide B suppresses LPS‐induced nitric oxide production and inflammatory cytokine expression in RAW 264.7 macrophages: The inhibition of NF‐κB and ERK1/2 activation

Dioscorealide B (DB), a naphthofuranoxepin has been purified from an ethanolic extract of the rhizome of Dioscorea membranacea Pierre ex Prain & Burkill which has been used to treat inflammation and cancer in Thai Traditional Medicine. Previously, DB has been reported to have anti‐inflammatory activities through reducing nitric oxide (NO) and tumor necrosis factor‐α (TNF‐α) production in lipopolysaccharides (LPS)‐induced RAW 264.7 macrophage cells. In this study, the mechanisms of DB on LPS‐induced NO production and cytokine expression through the activation of nuclear factor‐κB (NF‐κB) and ERK1/2 are demonstrated in RAW 264.7 cells. Through measurement with Griesss reagent, DB reduced NO level with an IC50 value of 2.85 ± 0.62 µM that was due to the significant suppression of LPS‐induced iNOS mRNA expression as well as IL‐1β, IL‐6, and IL‐10 mRNA at a concentration of 6 µM. At the signal transduction level, DB significantly inhibited NF‐κB binding activity, as determined using pNFκB‐Luciferase reporter system, which action resulted from the prevention of IκBα degradation. In addition, DB in the range of 1.5–6 µM significantly suppressed the activation of the ERK1/2 protein. In conclusion, the molecular mechanisms of DB on the inhibition of NO production and mRNA expression of iNOS, IL‐1β, IL‐6, and IL‐10 were due to the inhibition of the upstream kinases activation, which further alleviated the NF‐κB and MAPK/ERK signaling pathway in LPS‐induced RAW264.7 macrophage cells. J. Cell. Biochem. 109: 1057–1063, 2010.

Greener liquid chromatography using a guard column with micellar mobile phase for separation of some pharmaceuticals and determination ofparabens

In this research, a greener chromatography employing a short column, Zorbax SB C18 cartridge (12.5 × 4.6 mm, 5 μm) commonly used as a guard column in a reverse phase high performance liquid chromatography (RP-HPLC), was utilized as the analytical column in conjunction with a more eco-friendly micellar mobile phase of sodium dodecyl sulfate (SDS) for separation tertiary mixtures of local anesthetics and antihistamines; and binary mixture of colds drugs; and quaternary mixture of some parabens with different separation conditions. The chromatographic behavior of these analytes was studied to demonstrate separation efficiency of this guard column in a micellar mobile phase. Moreover, this column and SDS mobile phase was exploited for determination of parabens in 64 samples of cosmetic product, both those that were produced locally in the community and those that were commercially manufactured. Linear calibration graphs of the parabens as detected at 254 nm were obtained in the range of 1-100 μmol L(-1) with R(2)>0.9990. Percentage recoveries were 92.4-109.2 with %RSD<3, and the limit of detection and quantitation were 0.04-0.10 and 0.20-0.80 μmol L(-1), respectively. This analytical system is not only greener but also faster and employing simpler sample preparation than a conventional liquid chromatographic system.

Dioscoreanone suppresses LPS‐induced nitric oxide production and inflammatory cytokine expression in RAW 264.7 macrophages by NF‐κB and ERK1/2 signaling transduction

Dioscoreanone, a 1,4‐phenanthraquinone isolated from an ethanolic extract of the rhizome of Dioscorea membranacea, Pierre ex Prain & Burkill, a plant which has been used to treat inflammation and cancer in Thai Traditional Medicine. In this study, the mechanisms of dioscoreanone on LPS‐induced NO production and cytokine expression through the activation of NF‐κB and ERK1/2 are demonstrated in RAW 264.7 cells. Through measurement with Griess reagent, dioscoreanone was found to reduce NO levels with an IC50 value of 2.50 ± 0.64 µM, due to the significant suppression of LPS‐induced iNOS mRNA expression, as well as IL‐1β and IL‐6 levels at a concentration of 6 µM. At the signal transduction level, using the pNFκB‐Luciferase reporter system, dioscoreanone significantly inhibited NF‐κB transcriptional activity, which resulted from the prevention of IκBα degradation. In addition, dioscoreanone in the range of 1.2–5 µM significantly enhanced LPS‐induced ERK1/2 activation and dioscoreanone alone induced the activation of ERK1/2 proteins in a concentration‐ and time‐dependent response. The activation of ERK1/2 proteins by dioscoreanone was due to both an arylating reaction, which was suppressed by N‐acetyl cysteine, and a redox cycling reaction of NQOR, which was inhibited by dicoumarol. In conclusion, the mechanisms of dioscoreanone on the inhibition of NO production and mRNA expression of iNOS, IL‐1β, and IL‐6 were due to both the inhibition of NF‐κB activation and the activation of ERK1/2 proteins. The activation of dioscoreanone may in turn inhibit the binding of NF‐κB to pro‐inflammatory gene promoters in LPS‐induced RAW264.7 macrophage cells. J. Cell. Biochem. 113: 3427–3435, 2012.

Development and Evaluation of Granule and Emulsifiable Concentrate Formulations Containing Derris elliptica Extract for Crop Pest Control

Derris elliptica Benth. extracts containing rotenone have long been used as natural insecticides, but time-consuming preparation processes and the short shelf life of the extract limit their use in pest control. In this study, stable water-dispersible granules and emulsifiable concentrate liquids containing Derris extract (equivalent to 5% w/w of rotenone) were developed with simple techniques. Accelerated degradation kinetics of rotenone in the Derris extract, and in both formulations, indicated that its degradation followed first-order kinetics. The predicted half-life (t(1/2)) and shelf life (t(90%)) at 30 degrees C of rotenone in Derris extract were 520 and 79 days, respectively. Derris granules and emulsifiable concentrate clearly prolong the stability of rotenone 8-fold (t(90%) = 633 days) and 1.4-fold (t(90%) = 110 days), respectively. The study of rotenone degradation after application onto plants indicated that both formulations would be effective for up to 3 days after spraying. Preliminary efficacy testing indicated that the Derris emulsifiable concentrate was clearly more effective than Derris water-dispersible granules in controlling Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae).

Anti-Inflammatory, Analgesic, and Antipyretic Activities of the Ethanol Extract of Piper interruptum Opiz. and Piper chaba Linn.

Piper interruptum Opiz. and Piper chaba Linn. are herbaceous plants in the Piperaceae family. The ethanol extract of P. interruptum and P. chaba inhibited ethyl phenylpropiolate-induced ear edema and carrageenan-induced hind paw edema in rats. Both extracts reduced transudative and granuloma weights as well as body weight gain and thymus weight of the chronic inflammatory model using cotton pellet-induced granuloma formation in rats. Moreover, both extracts exhibited analgesic activity on both early phase and late phase of formalin test in mice and also showed antipyretic activity on yeast-induced hyperthermia in rats.