Ethyl Acetate Fraction Treatment Research Articles

Delayed administration of Trichilia catigua A. Juss. Ethyl-acetate fraction after cerebral ischemia prevents spatial memory deficits, decreases oxidative stress, and impacts neural plasticity in rats.

Trichilia catigua A. Juss (Meliaceae) is used in Brazilian folk medicine to alleviate fatigue and emotional stress and improve memory. Previous studies from our laboratory reported that an ethyl-acetate fraction (EAF) of T. catigua that was given before cerebral ischemia in vivo prevented memory loss and reduced oxidative stress and neuroinflammation. Despite the value of these findings of a neuroprotective effect of T. catigua, treatment that was given immediately before or immediately after ischemia limits its clinical relevance. Thus, unknown is whether T. catigua possesses a specific time window of efficacy (TWE) when administered postischemia. Given continuity to previous studies, we investigated whether an EAF of T. catigua maintains its neuroprotective properties if treatment begins at different time windows of efficacy after ischemia. We also evaluated, for the first time, whether T. catigua possesses neuroplasticity/neurotrophic properties. Rats were subjected to transient global brain ischemia (TGCI) and then given a single dose of the EAF (400mg/kg) or vehicle (1ml/kg) orally 1, 4, or 6h postischemia. The levels of protein PCG, GSH, and GSSG, and activity of SOD and CAT were assayed as markers of oxidative stress on the day after ischemia. In another experiment, naive rats underwent spatial learning training in a radial maze task and then subjected to TGCI. Delayed treatment with the EAF began 4 or 6h later and continued for 7 days. Retrograde memory performance was assessed 10, 17, and 24 days postischemia. Afterward, brains were examined for neurodegeneration and neuronal dendritic morphology in the hippocampus and cerebral cortex. Another group received the EAF at 4h of reperfusion, and 4 days later their brains were examined for GFAP and Iba-1 immunoreactivity. Lastly, ischemic rats received the EAF 4h after ischemia and neural plasticity-related proteins, BDNF, SYN, PSD 95, and NeuN were measured in the hippocampus 7 and 14 days after ischemia. A single EAF administration 1, 4, or 6h postischemia alleviated oxidative stress that was caused by ischemia, expressed as a reduction of the amount of the PCG and GSSG, normalization of the GSH/GSSG ratio, and the restoration of SOD activity. Ischemia caused the persistent loss of memory (i.e., amnesia), an outcome that was consistently ameliorated by treatment with the EAF that was initiated 4 or 6h postischemia. The 4h delay in EAF treatment positively impacted dendritic morphology in neurons that survived ischemia. TGCI reduced BDNF, SYN, PSD-95, and NeuN protein levels in the hippocampus and cerebral cortex. The EAF normalized SYN and PSD-95 protein levels. Ischemia-induced neurodegeneration and glial cell activation were not prevented by EAF treatment. The present study corroborates prior data that demonstrated the neuroprotective potential of T. catigua and extends these data by showing that the delayed administration of EAF postischemia effectively prevented memory impairment and decreased oxidative stress, dendritic deterioration, and synaptic protein loss within a TWE that ranged from 1 to 6h. This specific TWE in preclinical research may have clinical relevance by suggesting the possible utility of this plant for the development of neuroprotective strategies in the setting of ischemic brain diseases. Another innovative finding of the present study was the possible neurotrophic/neuroplastic properties of T. catigua.

Ethyl-acetate fraction from a cinnamon-cortex extract protects pancreatic β-cells from oxidative stress damage.

Background: The pathogenesis of diabetes mellitus is mediated mainly by oxidative stress produced by damaged pancreatic β-cells. We identified that an ethyl-acetate fraction (EA) from a cinnamon-cortex extract (CCE) is rich in flavonoid, and showed no toxicity to β cells. Objective: In this study, we evaluated the pharmacologic activities of EA on pancreatic β cells using a model of oxidative stress induced by H2O2 or alloxan. Results: The results showed that EA could significantly reduce reactive oxygen (ROS) accumulation to improve the survival of cells. Western blot showed that EA treatment upregulated expression of nuclear factor erythroid 2 related factor 2, heme oxygenase-1, and gamma glutamylcysteine synthetase. The same model study found that EA also can protect β cells against the apoptosis induced by oxidative stress. Furthermore, EA can enhance insulin secretion in rat and mouse β cell lines treated or not with alloxan or H2O2. The expression of the insulin transcription factor PDX-1 increased in an EA concentration-dependent manner. At last, the major functional compounds of EA analysis showed that three compounds, cinnamyl alcohol, coumarin, and cinnamic acid, had similar effects as EA. Conclusions: In sum, our data suggested that EA fraction from CCE can protect β cells from oxidative stress, and increase insulin secretion to improve the function of β cells. This function might be due to these three compounds found in EA. Our findings provide a theoretical basis and functional molecules for the use of CCE against diabetes mellitus.

The phytochemical constituents and protective effect of Fritillaria hupehensis on acute lung injury

Acute lung injury (ALI), a severe respiratory disorder, frequently develops into acute respiratory distress syndrome (ARDS) without timely treatment and scores highly in terms of morbidity and mortality rates. Fritillaria hupehensis is a famous traditional Chinese medicine with antitussive, expectorant and anti-asthmatic effect. Here, the effects of F. hupehensis extracts on lipopolysaccharide (LPS)-induced ALI mice were evaluated for the first time. We showed ethyl acetate fraction (EAF) significantly reduced the leukocytes and neutrophils of bronchoalveolar lavage fluid (BALF) and the lung index as well as pro-inflammatory cytokines (TNF-α and IL-6) of lung homogenates but increasing the anti-inflammatory cytokines (IL-4 and IL-10). Additionally, the alleviation of EAF treatment on lung injury was verified through histopathological observations. Subsequent phytochemical investigation on bioactive fraction led to isolation of 17 compounds including two new, in which compounds 2, 5 and 6 exhibited better anti-inflammatory effect on LPS-induced 16 human airway epithelial (16HBE) cells model by inhibiting the production of CRP and PCT. Furthermore, compound 2 suppressed the LPS-induced upregulation of proteins containing p-p65, COX-2, Caspase-1 and IL-18. In summary, F. hupehensis alleviating LPS-induced ALI in mice may be associated with the anti-inflammatory activity of steroidal alkaloids by suppressing the NF-κB-regulated pro-inflammatory proteins.

Hypoglycemic and antioxidant potency of ethyl acetate fraction of hydro-methanolic extract (60:40) of Tamarindus indica Linn. seed in streptozotocin-induced diabetic experimental animal

Diabetes mellitus is a chronic metabolic syndrome characterized by hyperglycemia due to defective insulin secretion, insulin action or both. Nowadays massive developments in variety of synthetic modern medicines, alternative and traditional medicinal plants are still widely used for the management of diabetes mellitus. In this context when ethyl acetate fraction at the dose of 100 mg body weight/kg body weight was administered for 28 days, significant diminution in the STZ-induced hyperglycaemia was noticed. Plasma insulin and C-peptide levels as well as activities of antioxidant defense enzymes such as catalase (CAT), peroxidase (Px) and superoxide dismutase (SOD) in the hepatic tissue were found to be decreased in diabetic animals. This was corrected after the treatment of ethyl acetate fraction of hydro-methanolic extract of T. indica. Oral glucose tolerance test (OGTT) reveals that the fraction at the above said dose showed a remarkable decrease of blood glucose level in normal and diabetic rat and also increased glucose tolerance. On the other hand, in diabetic animals body weight decreases along with a decrease in total hemoglobin content in blood and increase in HbA1C level.

Ethyl Acetate Fraction from Leandra dasytricha (A. Gray) Cong. Leaves Promotes Vasodilatation and Reduces Blood Pressure in Normotensive and Hypertensive Rats.

Leandra dasytricha (A. Gray) Cong. is widely distributed in the south of Brazil and is commonly used for cardiovascular and kidney ailments. For this study, we used male Wistar normotensive rats (NTRs) and spontaneously hypertensive rats (SHRs) to verify the effects of the ethyl acetate fraction (EAF) obtained from L. dasytricha leaves on isolated aorta relaxation and in the arterial blood pressure. The EAF was analyzed by LC-DAD-MS, and several components were annotated, including hydrolysable tannins, triterpenes, and O- and C-glycosylated dihydrochalcones, such as the most intense ion peak relative to C-hexosyl phloretin (nothofagin; compound number 13). The EAF caused a concentration and endothelium-dependent relaxation of the aorta in both NTRs and SHRs. This effect was abolished in the endothelium-denuded aorta. L-NAME, a nonselective nitric oxide synthase inhibitor, and ODQ, a soluble guanylate cyclase inhibitor, entirely blocked the EAF-induced relaxation. The presence of a muscarinic receptor antagonist or a cyclooxygenase inhibitor did not alter the EAF's effectiveness in relaxing the aorta. The preincubation with tetraethylammonium, a Ca2+-activated K+ channel blocker, and with 4-aminopyridine, a voltage-dependent K+ channel blocker, significantly interfered with the EAF's relaxation. However, the incubation with glibenclamide, an ATP-sensitive K+ channel blocker, and barium chloride, an inward-rectifier K+ channel blocker, did not interfere with the EAF-induced relaxation. The EAF treatment also caused a dose-dependent decrease in the mean arterial pressure, systolic arterial pressure, and diastolic arterial pressure of both NTRs and SHRs, without significantly interfering with heart rate values. In conclusion, this study demonstrated the EAF-induced vasorelaxant and hypotensive actions, primarily dependent on the endothelium function and mainly with the participation of the nitric oxide and Ca2+-activated and voltage-dependent K+ channels.

Identification of chemical constituents and inhibitory effect of Ficus deltoidea fraction against lipopolysaccharide-induced nuclear factor-kappa B inflammatory pathway in murine macrophage 264.7 cells

Background: Ficus deltoidea Jack or locally known as Mas Cotek belongs to the family Moraceae is a conventionally used Malaysian native medicinal plant. Objectives: The aim is to determine the anti-inflammatory mechanism of various solvent fractions of F. deltoidea methanol extract against Lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-κB) inflammatory pathway in murine macrophage 264.7 cells and to identify the chemical constituents present in the active fraction. Materials and Methods: The effect of crude methanolic extract and its fractions (hexane, chloroform, ethyl acetate, and butanol) on murine macrophages against LPS-induced pro-inflammatory cytokines (interleukin [IL]-Iβ, tumor necrosis factor alpha [TNF-α], and IL-6) and biomarkers were tested using enzyme-linked immunosorbent assay and immunoblot analysis. The chemical constituents present in the active fraction were identified using liquid chromatography mass spectrometry and liquid chromatography tandem mass spectrometry analysis. Results: The findings indicated that among all the fractions, the ethyl acetate fraction of F. deltoidea substantially inhibits the LPS-induced inflammatory mediators such as nitric oxide (NO) and pro-inflammatory cytokine production including TNF-α, IL-6, and IL-1 β in a dose-dependent manner. The expression of inducible NO synthase, NO synthase, and cyclooxygenase-2 were also effectively downregulated by the treatment of ethyl acetate fraction. Moreover, it also suppressed the expression of LPS-induced NF-κB translocation correlated with the inhibition of NF-κB (inhibitor of kappa B alpha) degradation. The presence of bioactive phenolics, especially flavonoids such as catechin, vitexin, dodecadienyl coumaric acid, (epi)-afzelechin-(epi)-catechin, genistein, and apigenin derivatives were identified in the ethyl acetate fraction of F. deltoidea. Conclusion: Overall, it has been recommended that the ethyl acetate fraction of F. deltoidea could be utilized as a potential natural anti-inflammatory agent.

Allium cepa fraction attenuates STZ-induced dementia via cholinesterase inhibition and amelioration of oxidative stress in mice.

Background An earlier study demonstrated significant antioxidant and anticholinesterase activities of hydromethanol extract (HME) of Allium cepa. The aim of the study was to investigate the component responsible for these activities followed by an in vivo study. Methods In vitro antioxidant and anticholinesterase activities of standardized ethylacetate fraction (EAF) of HME were assessed. Bioactivity-guided fractionation showed that, as compared with its subfractions, EAF had most significant activity in 2,2-diphenyl-1-picrylhydrazyl and Ellman assays. Thus, EAF was further examined using a streptozotocin (STZ)-induced model of Alzheimer's disease in mice. STZ was injected intracerebroventricularly on days 1 and 3 (3 mg/kg) in mice. EAF was thereafter administered (42, 84, and 168 mg/kg b.w./day p.o.) from days 9 to 22. The Morris water maze test was used to evaluate learning and memory in mice. Acetylcholinesterase (AChE) activity and oxidative stress markers were assessed in the brain homogenates of mice. Additionally, histopathological studies were performed to observe effects in the brain at the cellular level. EAF was standardized based on quercetin and quercetin 4'-O-glucoside content using a validated thin layer chromatography densitometric method. Results STZ produced significant (p < 0.05) memory impairment along with oxidative stress and a cholinergic deficit in mice. EAF treatment ameliorated STZ-induced behavioral deficits and biochemical alterations in mice in a significant and dose-dependent manner. Conclusions Our results show that EAF is efficacious in improving memory and learning via AChE inhibition and antioxidant activity in the mice brain. Thus, AC could be explored further to find out a lead candidate for Alzheimer's disease.

Cytotoxic Activity and Selectivity Index of Solanum Torvum Fruit on T47D Breast Cancer Cells

Solanum torvum has antioksidan. This study aimed to analyse cytotoxic activity and selectivity index of Solanum torvum on T47D breast cancer cells invitro. Identification of Solanum torvum fruit chemical compounds with LCMS containing Clorogenic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 3-O-feruloyquinic acid, 5-O-feruloylquinic acid, 3-O-feruloylquinic acid, 3,4 - O dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 3-O-feruloylquinic acid, 5-O-feruloylquinic acid, putative dicaffeoylquinic lactone. Cytotoxic assays showed that the Solanum torvum Without Fermentation were IC 50 (IGT) 1111.62μg/ mL, IC 50 (MGT)1889.20μg/mL, IC 50 (EGT) 1171.90μg/mL, IC 50 (FEGT) 305.98 μg/mL. Cytotoxic assays showed that the treatment of fermentation was IC 50 (IBT) 1760.36 μg/mL, IC 50 (MBT) 1631.06 μg/mL, IC 50 (EBT) of 1111.58 μg/mL, IC 50 (FEBT) 39.17μg/mL, IC 50 (FHBT) 85.58 μg/mL. Index of selectivity (IS) Solanum torvum (without fermentation) were IS (IGT) 5.23, IS (MGT) 0.61, IS (EGT) 1.06, IS (FEGT) 1.45. Index of selectivity of Solanum torvum (fermentation treatment) were IS (IBT) 2.41, IS (MBT) 0.78, IS (EBT) 0.18, IS (FEBT) 4.64, IS (FHBT) 1.67. The best results are treatment of ethyl acetate fraction of Solanum torvum (FEBT) has IC 50 was 39.17 μg/mL and Index selectivity was 4.64, declared safe/not toxic to normal cells, thus potentially developed as T47D breast anticancer cells.

Cytotoxic Activity and Selectivity Index of &lt;i&gt;Solanum torvum&lt;/i&gt; Fruit on T47D Breast

Solanum torvum has antioksidan. This study aimed to analyse cytotoxic activity and selectivity index of Solanum torvum on T47D breast cancer cells invitro. Identification of Solanum torvum fruit chemical compounds with LCMS containing Clorogenic acid, 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 3-O-feruloyquinic acid, 5-O-feruloylquinic acid, 3-O-feruloylquinic acid, 3,4-O-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 3-O-feruloylquinic acid, 5-O-feruloylquinic acid, putative dicaffeoylquinic lactone. Cytotoxic assays showed that the Solanum torvum Without Fermentation were IC 50 (IGT) 1111.62 μg/ mL, IC 50 (MGT) 1889.20 μg/mL, IC 50 (EGT) 1171.90 μg/mL, IC 50 (FEGT) 305.98 μg/mL. Cytotoxic assays showed thatthe treatment of fermentation was IC 50 (IBT) 1760.36 μg/mL, IC 50 (MBT) 1631.06 μg/mL, IC 50 (EBT) of 1111.58 μg/mL, IC 50 (FEBT) 39.17μg/mL, IC 50 (FHBT) 85.58 μg/mL. Index of selectivity (IS) Solanum torvum (without fermentation) were IS (IGT) 5.23, IS (MGT) 0.61, IS (EGT) 1.06, IS (FEGT) 1.45. Index of selectivity of Solanum torvum (fermentation treatment) were IS (IBT) 2.41, IS (MBT) 0.78, IS (EBT) 0.18, IS (FEBT) 4.64, IS (FHBT) 1.67. The best results are treatment of ethyl acetate fraction of Solanum torvum(FEBT) has IC 50 was 39.17 μg/mL and Index selectivity was 4.64, declared safe/not toxic to normal cells, thus potentially developed as T47D breast anticancer cells.

DURATION DEPENDENT ANTIAPOPTOTIC EFFICACY OF CAMELLIA SINENSIS (GREEN TEA) ON STREPTOZOTOCIN INDUCED DIABETES LINKED TESTICULAR HYPOFUNCTION IN ALBINO RAT: GENOMIC AND FLOW CYTOMETRIC ASSESSMENT

Objective: The study has been conducted to search out the threshold duration of treatment of ethyl acetate fraction of methanolic extract of leaves of Camellia sinensis (L.) Kuntze at the dose of 100 mg/kg body weight for the management of diabetes-induced testicular impairment in streptozotocin-induced diabetic rat in a duration dependent fashion.&#x0D; Methods: In this respect, the glycemic, androgenic, oxidative stress sensors, gene expression of testicular androgenic key enzymes along with apoptotic markers were evaluated in a duration dependent way (14, 28 and 56 d).&#x0D; Results: A significant correction was noted in the levels of glycated haemoglobin (HbA1C), testicular thiobarbituric acid reactive substances (TBARS), conjugated diene (CD), sperm viability, sperm mitochondrial status, serum testosterone, and genomic expression of testicular Δ5, 3β-hydroxysteroid dehydrogenase (HSD), 17β-HSD, Bax, Bcl-2 after treatment for different duration with the said fraction in diabetic groups in compare to respective vehicle-treated diabetic group without any toxicity induction in general. Thin layer chromatography (TLC) study of the fraction showed two spots with retention factors (Rf) of 0.78 and 0.51.&#x0D; Conclusion: The results showed that 28 d treatment was threshold duration of treatment for the correction of diabetes-induced testicular impairment.

Oxidative stress induced apoptosis mediated anticancer activity of Rhus typhina fruits extract in human colon cancer

The present work was tested the anti-proliferation activity of Rhus typhina fruits extract (RTFE) in HEK293 and HT-29 cells, and studied their underlying mechanisms through the analysis of oxidative stress and apoptosis-related genes expressions by flow cytometer and RT-qPCR. In addition, the bioactive constituents were identified from RTFE by chromatography and NMR analysis. Among the various type of the fractions from the RTFE, the ethyl acetate fraction (EAF) was showed the potent anti proliferation effect in HT-29 cells. Thus EAF was selected for the further extensive analysis. Cytotoxcity and RT-qPCR results revealed that the treatment of EAF significantly inhibit the growth of the HT-29 cells by the up regulating of Bax and down regulation of Bcl-2, Survivn, AIF and SOD-2 gene expressions in time and dose dependent manner. Followed by sub G1 accumulation and the G0/G1 phase arrest, ROS production, and mitochondrial transmembrane potential (ΔΨm). Finally, the active anti-proliferations agents such as luteolin and luteolin-7-O-glucuronide were identified from EAF by NMR. This work suggests that EAF could be a functional food to treat the chronic diseases.

Treatment with Trichilia catigua ethyl-acetate fraction improves healing and reduces oxidative stress in TNBS-induced colitis in rats

Beverages containing Trichilia catigua are commonly employed in folk medicine. T. catigua bark extracts possess antioxidant, anti-inflammatory, and bactericidal properties. These properties suggest T. catigua bark extracts as a potential treatment for inflammatory bowel diseases (IBD). Using the 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced model of colitis in rats we evaluated the effect of an ethyl-acetate fraction (EAF) of T. catigua (200 mg/kg) administered by daily oral gavage or intrarectally at different time points after TNBS challenge. TNBS treatment evoked severe colonic inflammation after 24 h that persisted for 7 days, characterized by weight loss, high levels of myeloperoxidase activity, histological and macroscopic damage, and elevated index of oxidative stress in the blood. T. catigua EAF treatment prevented the oxidative stress within 24 h and enhanced tissue recovery observed at day 7, returning histological and macroscopic damage levels to that of the control group. TNBS treatment led to loss of myenteric neurons after 28 days. T. catigua EAF was unable to prevent the neuronal loss. Oral delivery of T. catigua EAF was more effective than intrarectal administration of the extract. In conclusion, T. catigua EAF treatment normalized oxidative stress parameters in blood and reduced the degree of acute inflammation in TNBS colitis.

참소리쟁이 뿌리 추출물 및 분획의 항균 활성과 항생제 증강 활성

Background: The objective of this study were to determine the antibacterial activity and antibiotic activity-enhancing effect of 70% ethanol extract of the root of Rumex japonicus Houtt. and its fractions when used in combination with gentamicin against aerobic skin flora.Methods and Results: The antibacterial activity and antibiotic (gentamicin) activity enhancing effect against aerobic skin flora were determined using the disc diffusion assay. Chloroform fraction (CF) and ethyl acetate fraction (EF) showed higher activities against Staphylococcus aureus and Staphylococcus epidermidis than those shown by other fractions. Regarding the antibiotic (gentamicin) activity-enhancing effect against aerobic skin flora, the n-hexane fraction (HF) and CF showed strong activity. The combination of HF and CF with gentamicin was evaluated using the broth dilution assay to determine the inhibitory effect on the growth of aerobic skin flora. The combination of CF with gentamicin exhibited the highest inhibitory effect on the growth of S. aureus and S. epdermidis. MTT assay performed to determine the viability of L929 cells revealed that EF treatment resulted in viability of 33.96 - 116.76% at the tested concentration. The combination of 70% ethanol extract and its other fractions with gentamicin showed low cell toxicity.Conclusions: Appropriate use of antimicrobial agents is important prior to the development of new antibiotics. The 70% ethanol extract of the root of R. japonicus Houtt. and its fractions showed significant synergism with gentamicin when used in combination against S. aureus and S. epdermidis. Thus, R. japonicus Houtt. could be used as a functional materials in antimicrobial-related fields.

Induction of Apoptosis and Phase-Cell Cycle Inhibition of G0-G1, S, G2-M of T47D Breast Cancer Cells on Treatment with Ethyl Acetate Fraction of Jackfruit Parasite Leaves (Macrosolen cochinensis)

Macrosolen cochinensis is one of parasite types that grows in Indonesia and is empirically known to have anticancer activity. This study aims to explain the anticancer activity of the extracts and fractions of jackfruit mistletoe leaves and describe the cell cycle inhibition and induction of apoptosis on T47D breast cancer cell line. The jackfruit mistletoe is extracted using the maceration method. The extract is then separated by liquid-liquid partition method with n -hexan, chloroform, ethyl acetate, and water solvent. MTT method is used to determine the anticancer activity of crude extract and fractions. Cell cycle inhibition test is conducted using flow cytometry with PI marker. The induction of apoptosis is determined by flow cytometry method using PI-Annexin V and PI double staining acridine orange-ethidium bromide. The results indicated that the IC 50 value of the ethanol extract, chloroform fractions and ethyl acetate fractions of jackfruit mistletoe leaves showed a higher anticancer activity with IC 50 which respectively are 362.8, 356.8, 314.8μg/ml. The treatment of n -hexan and water fraction show no signs of anticancer activity because it has a great value of IC 50 which are respectively 926.0 and 2243 μg/ml. Cell death caused by treatment of ethyl acetate fraction of jackfruit mistletoe leaf (Macrosolen cochinensis) is due to the induction of cell apoptosis and cell cycle inhibition in G0-G1, S and G2-M phase.

Mechanism(s) of action underlying the gastroprotective effect of ethyl acetate fraction obtained from the crude methanolic leaves extract of Muntingia calabura.

BackgroundMuntingia calabura L. (family Muntingiaceae), commonly known as Jamaican cherry or kerukup siam in Malaysia, is used traditionally to treat various ailments. The aim of this study is to elucidate the possible underlying gastroprotective mechanisms of ethyl acetate fraction (EAF) of Muntingia calabura methanolic leaves extract (MEMC).MethodsMEMC and its fractions were subjected to HPLC analysis to identify and quantify the presence of its phyto-constituents. The mechanism of gastroptotection of EAF was further investigated using pylorus ligation-induced gastric lesion rat model (100, 250, and 500 mg/kg). Macroscopic analysis of the stomach, evaluation of gastric content parameters such as volume, pH, free and total acidity, protein estimation, and quantification of mucus were carried out. The participation of nitric oxide (NO) and sulfhydryl (SH) compounds was evaluated and the superoxide dismutase (SOD), gluthathione (GSH), catalase (CAT), malondialdehyde (MDA), prostaglandin E2 (PGE2) and NO level in the ethanol induced stomach tissue homogenate was determined.ResultsHPLC analysis confirmed the presence of quercetin and gallic acid in EAF. In pylorus-ligation model, EAF significantly (p <0.001) prevent gastric lesion formation. Volume of gastric content and total protein content reduced significantly (p < 0.01 and p < 0.05, respectively), while free and total acidity reduced in the doses of 250 and 500 mg/kg (p <0.001 and p <0.05, respectively). EAF also augmented the mucus content significantly (p < 0.001). Pre-treatment with N-nitro-L-arginine methyl ester (L-NAME) or N-ethylmaleimide (NEM) reversed the gastroprotective activity of EAF. EAF treatment markedly ameliorated the SOD, GSH and CAT activity and PGE2 and NO level while attenuating MDA level, relative to the vehicle group.ConclusionsIn conclusion, the underlying gastroprotective mechanisms of EAF could be associated with the antisecretory, participation of mucus, antiperoxidative, improvement of antioxidant status, modulation of NO and SH compounds, stimulation of PGE2 as well as presence of quercetin and gallic acid.

In vitro and in vivo evaluation of anti-leishmanial and immunomodulatory activity of Neem leaf extract in Leishmania donovani infection

The toxicity and emergence of resistance to available chemical drugs against visceral leishmaniasis is evoking to explore herbal treatment. One such attempt with the Neem is being reported here. The current study is primarily focused to evaluate the anti-leishmanial effects of Neem leaf extracts. Among which, ethyl acetate fraction (EAF) alone was found to exhibit leishmanicidal effect validated through cytotoxicity assay and estimated its IC50 to be 52.4 µg/ml on the promastigote stage. Propidium iodide (PI) staining of dead cells substantiated the aforementioned activity. Carboxy fluorescein–diaceate succinimidyl ester (CFSE) staining of promastigotes has affirmed its anti-proliferation activity. The characteristic features such as DNA fragmentation, reduced mitochondrial membrane potential, increased sub G0/G1 phase parasites and increased reactive oxygen species (ROS) production in EAF treated promastigotes indicate the apoptosis like death. In addition, the reduced parasite burden both in vitro (viz. ~45% in human monocytic leukemia cell line (THP-1) and ~50% in peripheral blood mononuclear cells) and in vivo (spleen and liver) provides the evidence for its anti-leishmanial activity on amastigote stage. The increase of ROS levels in THP-1 and nitric oxide (NO) production from J774.1 cell line (mouse macrophages) upon EAF treatment was evidenced for oxidative killing of intracellular amastigotes. Active immunomodulatory activity at m-RNA level (viz. upregulation of Th1 cytokines, and downregulation of Th2 cytokines) both in vitro and in vivo was also shown to be exhibited by EAF. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis of EAF revealed the presence of 14 compounds.

Sonchus asper extract inhibits LPS-induced oxidative stress and pro-inflammatory cytokine production in RAW264.7 macrophages.

BACKGROUND/OBJECTIVESSonchus asper is used extensively as an herbal anti-inflammatory for treatment of bronchitis, asthma, wounds, burns, and cough; however, further investigation is needed in order to understand the underlying mechanism. To determine its mechanism of action, we examined the effects of an ethyl acetate fraction (EAF) of S. asper on nitric oxide (NO) production and prostaglandin-E2 levels in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages.MATERIALS/METHODSAn in vitro culture of RAW264.7 macrophages was treated with LPS to induce inflammation.RESULTSTreatment with EAF resulted in significant suppression of oxidative stress in RAW264.7 macrophages as demonstrated by increased endogenous superoxide dismutase (SOD) activity and intracellular glutathione levels, decreased generation of reactive oxygen species and lipid peroxidation, and restoration of the mitochondrial membrane potential. To confirm its anti-inflammatory effects, analysis of expression of inducible NO synthase, cyclooxygenase-2, tumor necrosis factor-α, and the anti-inflammatory cytokines IL-1β and IL-6 was performed using semi-quantitative RT-PCR. EAF treatment resulted in significantly reduced dose-dependent expression of all of these factors, and enhanced expression of the antioxidants MnSOD and heme oxygenase-1. In addition, HPLC fingerprint results suggest that rutin, caffeic acid, and quercetin may be the active ingredients in EAF.CONCLUSIONSTaken together, findings of this study imply that the anti-inflammatory effect of EAF on LPS-stimulated RAW264.7 cells is mediated by suppression of oxidative stress.

Anti-hepatitis B virus activity of Boehmeria nivea leaf extracts in human HepG2.2.15 cells

Boehmeria nivea (Linn.) Gaudich of the Urticaceae family is a perennial ratoon herbal plant, the root of which is used in traditional Chinese medicine and possesses a variety of pharmacological properties. The 20% ethanol Boehmeria nivea root extract was shown to exert an anti-hepatitis B virus (HBV) effect in vitro and in vivo; however, whether the Boehmeria nivea leaf (BNL) extract possesses similar properties has not been determined. In this study, we aimed to investigate the anti-HBV effects of the BNL extract in HepG2.2.15 cells transfected with human HBV DNA. Our results demonstrated that the secretion of HBsAg and HBeAg was reduced in HepG2.2.15 cells treated with the BNL extract, without any recorded cytotoxic effects. In addition, the chloroform fraction (CF) and ethyl acetate fraction (EAF) of BNL were shown to be more potent compared to the other fractions: CF (100 mg/l) inhibited the secretion of HBsAg by 94.00±1.78% [inhibitory concentration 50 (IC50) = 20.92 mg/l] and that of HBeAg by 100.19±0.35% (IC50=19.67 mg/l) after 9 days of treatment. Similarly, EAF (200 mg/l) inhibited the secretion of HBsAg by 89.95±2.26% (IC50=39.90 mg/l) and that of HBeAg by 98.90±1.42% (IC50=36.45 mg/l). Furthermore, we observed that the content of HBV DNA in the medium secreted by the HepG2.2.15 cells was significantly decreased under CF (100 mg/l) or EAF (200 mg/l) treatment. Thus, we concluded that the BNL extracts exhibited anti-HBV activity, with CF and EAF being the most potent among the fractions.

Effects of the Ethyl Acetate Fraction of Alchornea triplinervia on Healing Gastric Ulcer in Rats.

Alchornea triplinervia (Spreng.) Muell. Arg (Euphorbiaceae) is a medicinal plant commonly used by people living in the Cerrado region of Brazil to treat gastrointestinal ulcers. We previously described the gastroprotective action of methanolic extract (ME) of Alchornea triplinervia and the ethyl acetate fraction (EAF) in increasing of prostaglandin E2 (PGE2) gastric levels in the mucosa. In this work we evaluated the effect of EAF in promoting the healing process in rats with acetic acid-induced gastric ulcers. In addition, toxicity was investigated during treatment with EAF. After 14 days of treatment with EAF, the potent stimulator of gastric cell proliferation contributed to the acceleration of gastric ulcer healing. Upon immunohistochemical analysis, we observed a pronounced expression of COX-2, mainly in the submucosal layer. The 14-day EAF treatment also significantly increased the number of neutrophils in the gastric mucosa regeneration area. The EAF induced angiogenesis on gastric mucosa, observed as an increase of the number of blood vessels supplying the stomach in rats treated with EAF. Oral administration for 14 days of the ethyl acetate fraction from Alchornea triplinervia accelerated the healing of gastric ulcers in rats by promoting epithelial cell proliferation, increasing the number of neutrophils and stimulation of mucus production. This fraction, which contained mainly phenolic compounds, contributed to gastric mucosa healing.

대두 분획물의 항돌연변이 및 항암활성 효과

대두를 먼저 헥산(Hex)으로 지방을 제거한 후 메탄올로 추출한 추출물에서 항돌연변이성 및 항암활성 물질을 분리하기 위해 극성이 다른 용매인 디클로로메탄(<TEX>$CH_2Cl_2$</TEX>), 에틸아세테이트(EtOAC), 부탄올(BuOH)로 분획하여 각 분획물의 항돌연변이 및 항암활성 효과를 검토하였다. <TEX>$AFB_1$</TEX>에 대한 돌연변이 억제효과는 대두의 메탄을 추출물(MeOH)의 분획물들 중 디클로로메탄 분획물(<TEX>$CH_2Cl_2$</TEX>)과 에틸아세테이트 분획물(EtOAc)은 2.5 mg/plate 첨가농도에서 각각 83%로 상당히 높은 항돌연변이 효과를 나타내었다. 중간 분획물(Inter)의 경우는 56%의 항돌연변이 효과를 나타내었으나 다른 분획물들의 경우 항돌연변이 효과가 낮았다. 한편 MNNG의 경우, <TEX>$AFB_1$</TEX>에 비해 돌연변이 저해효과가 떨어지지만 그 중에서도 에틸아세테이트 분획물(EtOAc)이 67%로 가장 높은 항돌연변이 효과를 나타내었으며 중간 분획물과 디클로로메탄 분획물(<TEX>$CH_2Cl_2$</TEX>)은 각각 63%, 49%의 돌연변이 저해효과를 나타내었다. 대두의 메탄을 추출물은 첨가농도 2 mg/assay에서 44%의 인체 위암세포의 증식 억제효과를 가졌고 대두 메탄을 추출물의 분획물들 중 에틸아세테이트층(EtOAC)의 저해 효과가 가장 높았는데 동일농도에서 66%로 위암세포 성장을 저해시키는 효과를 나타내었고 디클로로메탄(<TEX>$CH_2Cl_2$</TEX>)과 중간 분획물(Inter)의 경우에는 각각 54% 및 51%의 저해효과를 나타내었다. Hep 3B 인체 간암세포의 경우, 대두 메탄을 추출물을 2 mg/assay 투여했을 때 60%의 저해효과를 나타내었으며 여기에서도 분획물들 중에서 에틸아세테이트 분획물(EtOAC)이 73%로 가장 높은 저해효과를 보였다. 대두의 메탄을 추출물은 첨가농도 2 mg/assay에서 44%의 인체 결장암세포 증식억제효과를 가졌고 대두 메탄을 추출물의 분획물들 중 에틸아세테이트층(EtOAC)의 저해 효과가 가장 높았는데 동일농도에서 77%의 결장암세포의 성장을 저해시키는 효과를 나타내었다. 이상의 결과로부터 대두 분획물들 중 특히 에틸아세테이트 분획물에 의한 항돌연변이 및 항암활성효과가 가장 높았으므로 이 분획물에 주로 isofavone류가 함유되어 있으므로 에틸아세테이트 분획물을 더욱 정제하여 연구할 필요가 있다고 여겨진다. Inhibitory effects of several solvent fractions from soybean on mutagenicity using Salmonella typhimurium TA 100 in Ames test and growth of human cancer cells (AGS gastric adenocarcinoma, Hep 3B hepatocellular cancinoma and HT-29 colon cancer cells) were studied. The treatment of dichloromethane and ethylacetate fractions (2.5 mg/assay) extracted from soybean to Ames test system inhibited aflatoxin <TEX>$B_1\;(AFB_1)$</TEX> induced mutagenicity by 83%, respectively, and showed a higher antimutagenic effect than other solvent fractions. In case of N-methyl-N#-nitro-N-nitrosoguamidine (MNNG) induced mutagenicity, the ethylacetate fraction showed the highest inhibitory effect (by 67%) among solvent extracts, although the inhibitory effect was not stronger compared with <TEX>$AFB_1$</TEX> induced mutagenicity. In sulforhodamine B (SRB) assay, the treatment of ethylacetate fraction (2 mg/assay) significantly inhibited the growth of AGS, Hep 3B and HT-29 cancer cells by 66%, 73% and 77%, respectively, followed with the intermediate and dichloromethane fractions. These results indicated that soybean fraction extracted with ethylacetate had higher inhibitory effects on <TEX>$AFB_1$</TEX> and MNNG in Ames test and growth inhibition activity to human cancer cells was appeared, suggesting that soybean fraction extracted with ethylacetate may contain the biologically active compounds.