Yokukansan, a kampo medicine, protects against glutamate cytotoxicity due to oxidative stress in PC12 cells

Aim of the study

Yokukansan is a traditional Japanese medicine consisted of seven medicinal herbs and has been used for treatment of neurosis, insomnia, and behavioral and psychological symptoms of dementia in Japan. The aim of the present study is to clarify the active compounds responsible for the protective effect of yokukansan against glutamate-induced cytotoxicity in PC12 cells.

Materials and methods

PC12 cells which is a tool for selective evaluation of test substances against oxidative stress was used in the present study. The cell survival rates or glutathione (GSH) levels were evaluated by a MTT reduction assay or GSH assay based on the GSH reductase enzymatic recycling method, respectively.

Results

Glutamate (1-17.5 mM) induced cell death of PC12 cells in a concentration- dependent manner. Yokukansan (125-500 μg/ml) inhibited the glutamate-induced PC12 cell death. When the effects of extracts of the seven constituent herbs in yokukansan on the cell death were examined, Uncaria thorn was found to have the highest potency in the protection. To clarify the active compounds in Uncaria thorn, the effects of seven alkaloids (rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine, hirsutine, hirsuteine, and geissoschizine methyl ether) on the cell death were further examined. The protective effects were found in hirsutine, hirsuteine, and geissoschizine methyl ether, which also ameliorated the glutamate-induced decrease in GSH levels.

Conclusion

These results suggest that yokukansan protects against PC12 cell death induced by glutamate-mediated oxidative stress, i.e., reduction of intracellular GSH level, and the effect may be mainly attributed to a synergistic effect of the hirsutine, hirsuteine, and geissoschizine methyl ether in Uncaria thorn.     

Inhibitory effects of Chelidonium majus extract on atopic dermatitis-like skin lesions in NC/Nga mice

Aim of the study

Chelidonium majus (CM) has traditionally been used for treatment of various inflammatory diseases including atopic dermatitis (AD). However its action on atopic dermatitis (AD) is unclear. Therefore, we investigated the effect of CM on AD using NC/Nga mice as an AD model.

Materials and methods

The effect of CM on 1-chloro-2,4-dinitrobenzene (DNCB) induced NC/Nga mice was evaluated by examining skin symptom severity, itching behavior, ear thickness, levels of serum immunoglobulin E (IgE), tumor necrosis factor-α (TNF-α), and interlukin-4 (IL-4), skin histology.

Results

The CM significantly reduced the total clinical severity score, itching behavior, ear thickness and the level of serum IgE in AD mouse model. CM not only decreased TNF-α but also IL-4.

Conclusion

These results suggest that CM may be a potential therapeutic modality for AD.     

The mechanism of vasorelaxation induced by ethanol extract of Sophora flavescens in rat aorta

Aim of the study

Sophora flavescens (SF) is a known medicinal herb for the treatment of cardiovascular symptoms associated with arrhythmia in China. However, the pharmacological action mechanisms involved have not been well studied. The aim of the present study was to define effects of roots of SF on the vascular tension and responsible mechanisms in rat thoracic aorta.

Materials and methods

Ethanol extract of the roots of SF (ESF) was examined for their vascular relaxant effect in isolated phenylephrine-precontracted rat thoracic aorta.

Results

ESF (0.1-100 μg/ml) induced relaxation of the phenylephrine-precontracted aortic rings in a concentration-dependent manner. Endothelium-denudation abolished the ESF-induced vasorelaxation. Pretreatment of the endothelium-intact aortic rings with l-NAME, an inhibitor of nitric oxide synthase, and ODQ, an inhibitor of soluble guanylyl cyclase (sGC), inhibited ESF-induced vasorelaxation. ESF increased cGMP levels of the aortic rings in a concentration-dependent manner and the effect was blocked by l-NAME and ODQ. Inhibition of K⁺ channels with glibenclamide and tetraethylammonium, cyclooxygenase inhibition with indomethacin, and β-adrenergic and muscarinic receptors blockade had no effect on the ESF-induced vasorelaxation.

Conclusion

These findings suggest that ESF relaxes vascular smooth muscle via endothelium-dependent NO-sGC-cGMP signaling pathway.     

Role for monoaminergic systems in the antidepressant-like effect of ethanol extracts from Hemerocallis citrina

Ethnopharmacological relevance

Hemerocallis citrina, a traditional herbal medicine, has been used for the improvement of emotions in Eastern-Asia countries.

Aim of the study

Herein, we explored the antidepressant-like effect and its monoaminergic mechanism of the ethanol extracts from Hemerocallis citrina (HCE).

Materials and methods

Effect of HCE (90, 180 and 360 mg/kg, p.o.) on the immobility time was assessed in the mouse forced swim test (FST) and tail suspension test (TST), and locomotor activity was evaluated in the open-field test (OFT). Additionally, the monoamine neurotransmitters serotonin (5-HT), noradrenaline (NA) and dopamine (DA) levels involved in the antidepressant-like effect of HCE were also measured in the mice brain regions of frontal cortex and hippocampus.

Results

HCE (90, 180 and 360 mg/kg, p.o.) administration significantly reduced the immobility time in both the FST and TST without accompanying changes in locomotor activity in the OFT. The pretreatment of mice with WAY 100635 (0.1 mg/kg, s.c., a 5-HT_1A receptor antagonist), cyproheptadine (3 mg/kg, i.p., a 5-HT₂ receptor antagonist), prazosin (62.5 μg/kg, i.p., an α₁-adrenoceptor antagonist), yohimbine (1 mg/kg, i.p., an α₂-adrenoceptor antagonist), propranolol (5 mg/kg, i.p., a β-adrenoceptor antagonist) or sulpiride (50 mg/kg, i.p., a dopamine D₂ receptor antagonist), but not SCH23390 (0.05 mg/kg, s.c., a dopamine D₁ receptor antagonist) prevented the antidepressant-like effect of HCE (360 mg/kg, p.o.) in the TST. In addition, HCE enhanced 5-HT and NA levels in the frontal cortex and hippocampus as well as elevated DA levels in the frontal cortex.

Conclusion

The results indicate that the antidepressant-like effect of HCE is dependent on the serotonergic (5-HT_1A and 5-HT₂ receptors), noradrenergic (α₁-, α₂- and β-adrenoceptors) and dopaminergic (D₂ receptor) systems as well as the elevation of 5-HT, NA and DA levels in the mouse brain.     

Differential inductive profiles of hepatic cytochrome P450s by the extracts of Sophora flavescens in male and female C57BL/6JNarl mice

Aim of the study

Sophora flavescens has been used as an antipyretic and analgesic agent. To assess the possible herb–drug interaction, effects of S. flavescens extracts on hepatic cytochrome P450 (P450, CYP) enzymes were studied.

Materials and methods

Effects of the extracts prepared by three different pharmaceutical companies on P450 enzymes were investigated in male and female C57BL/6JNarl mice.

Results

In male mice, extract 1 caused a dose- and time-dependent increase of 7-ethoxyresorufin O-deethylation (EROD) activity. Three-day treatment with 3 g/kg extracts 1–3 elevated EROD, 7-pentoxyresorufin O-dealkylation (PROD), coumarin hydroxylation, and nifedipine oxidation (NFO) activities. In female mice, extracts 1 and 2 increased EROD and PROD activities without affecting coumarin hydroxylation and NFO activities. However, extract 3, which lacked prenylated flavonoids, caused an induction profile in females the same as in males. Treatment with extract 3 fortified with prenylated flavonoids restored the gender difference. An alkaloid, oxymatrine was present in all extracts and increased EROD and PROD activities. At a human equivalent dose (0.18 g/(kg day)), all extracts increased EROD activity.

Conclusions

These results revealed that Cyp1a had a lower induction response threshold. Oxymatrine contributed at least partly to the P450 induction by S. flavescens. At a higher dose, Cyp2a, Cyp2b, and Cyp3a could be induced and the male-specific induction of Cyp2a and Cyp3a was associated with the presence of prenylated flavonoids.     

Involvement of heme oxygenase-1 in the anti-inflammatory activity of Chrysanthemum boreale Makino extracts on the expression of inducible nitric oxide synthase in RAW264.7 macrophages

Aim of the study

This study is to elucidate the involvement of anti-inflammatory heme oxygenase-1 (HO-1) in the inhibitory activity of a Chrysanthemum boreale Makino (CB) extract on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages.

Materials and methods

Cell viability and NO assay were performed. In addition, iNOS expression was detected by Western blotting and real-time PCR. HO-1 expression was also evaluated by Western blotting, and blocking HO-1 activity on NO production was performed.

Results

The CB extract at the highest concentration (100 μg/ml) significantly inhibited NO production by approximately 90% and suppressed iNOS protein expression by approximately 84.8% compared to LPS-stimulated cells. Furthermore, the CB extract (100 μg/ml) inhibited iNOS mRNA expression in a concentration-dependant manner and suppressed iNOS mRNA expression by 94.8%. The CB extract induced the expression of HO-1 in a dose-dependent manner, and blocking HO-1 activity abolished the inhibitory effects of the CB extract. Moreover, the addition of carbon monoxide such as tricarbonyl dichlororuthenium (II) dimmer (RuCO), a byproduct derived from heme degradation, mimicked the inhibitory action of low concentrations of CB extract.

Conclusion

These results suggest that a CB extract has potent anti-inflammatory activity in RAW264.7 macrophages involving the induction of HO-1.     

Dose-response of berberine on hepatic cytochromes P450 mRNA expression and activities in mice

Ethnopharmacological relevance

: Berberine is an isoquinoline alkaloid isolated from the root and bark of plants such as goldenseal, Berberis, and Chinese goldthread. Berberine-containing crude drugs have been used as an antimicrobial remedy against gastrointestinal infections for thousands of years. It is also widely used in Asian countries for diabetes, hypertension, and hypercholesterolemia therapy.

Aim of the Study

: Potential drug-drug interactions are of concern because of the wide usage of berberine. A few studies have reported interactions between berberine and cytochromes P450 (CYPs) in vitro, but little is known about whether berberine influences CYPs in vivo, especially after repeated administration. In this study, eight-week-old male C57BL/6 mice were given berberine orally (0, 10, 30, 100, 300 mg/kg, i.g., daily for 14 days), and the effect of berberine on over 20 major Cyps and related nuclear receptors in mice livers were examined at both the mRNA and enzyme activity levels.

Results

In general, liver function of mice treated with various doses of berberine had no significant change, and repeated oral administration of the 3 lower doses of berberine for 14 days did not affect the expression of genes examined. However, after the highest dose of berberine (300 mg/kg), Cyp3a11 and Cyp3a25 mRNA decreased 67.6 and 87.4%, respectively, whereas Cyp1a2 mRNA increased 43.2%, and enzyme activities of Cyp3a11 and Cyp2d22 decreased 67.9 and 32.4%, respectively. Cyp2a4, 2b10 and Cyp2c29 were not altered at both mRNA and enzyme activity levels.

Conclusions

If studies in mice extrapolate to humans, lower doses of berberine appear to present a low risk of producing drug-drug interactions as a result of changed Cyp enzyme activity. However, high doses of berberine may suppress Cyp activities and result in drug-drug interactions.     

Anti-obesity potential of Clerodendron glandulosum.Coleb leaf aqueous extract

Ethnopharmacological relevance

Clerodendron glandulosum.Coleb leaf aqueous extract (CG) is traditionally used by people of North-East India to alleviate symptoms of diabetes, obesity and hypertension. Previous study from our laboratory have documented anti-diabetic and anti-hypertensive properties of CG extract but, till date there are no pharmacological studies available on its anti-obesity potential. This inventory investigates the underlining molecular mechanism/s of CG induced regulation of in vivo HFD induced obesity and in vitro adipocyte differentiation.

Aim

To evaluate effects of CG extract on (i) expression of genes regulating visceral adiposity and (ii) in vitro adipocyte differentiation and LEP release.

Materials and methods

Body weight, lee index, plasma lipids and LEP, mRNA expression of PPARγ-2, SREBP1c, FAS, CPT-1 and LEP in epididymal adipose tissue of control and experimental groups were evaluated. Also, potential of CG extract on in vitro adipocyte differentiation and LEP release was assessed.

Results

Supplementation of CG extract to HFD fed mice significantly prevented HFD induced increment in bodyweight, lee index, plasma lipids and LEP, visceral adiposity and adipocyte hypertrophy. Also, CG extract supplementation resulted in down regulation of PPARγ-2, SREBP1c, FAS and LEP expression along with up-regulation of CPT-1 in epididymal adipose tissue compared to HFD fed mice. In vitro study recorded significant anti-adipogenic effect of CG extract that resulted in decreased adipogenesis, TG accumulation, LEP release, G3PDH activity along with higher glycerol release without significantly altering viability of 3T3L1 pre-adipocytes.

Conclusions

Clerodendron glandulosum.Coleb extract prevents adipocyte differentiation and visceral adiposity by down regulation of PPARγ-2 related genes and Lep expression thus validating its traditional therapeutic use in controlling obesity.     

Inonotus obliquus extracts suppress antigen-specific IgE production through the modulation of Th1/Th2 cytokines in ovalbumin-sensitized mice

Ethnopharmacological relevance

Chaga mushroom (Inonotus obliquus, IO) has been used as a folk remedy for cancer, digestive system diseases, and other illnesses in Russia and Eastern Europe.

Aim of the study

In the present study, we investigated the immunomodulating effects of IO through in vivo and ex vivo studies.

Materials and methods

Serum immunoglobulins (IgE, IgG₁, and IgG_2a) and cytokines (interleukin (IL)-4, interferon (IFN)-γ, and IL-2) were measured in concanavalin A (ConA)-stimulated splenocytes and CD4⁺ T cells. The nitric oxide (NO) secretion of lipopolysaccharide (LPS)-stimulated peritoneal macrophages was also measured after oral administration of 50, 100, or 200 mg kg⁻¹ d⁻¹ IO hot water extract (IOE) to ovalbumin (OVA)-sensitized BALB/c mice.

Results

We found that the OVA-induced increase in serum IgE and IgG_2a was significantly suppressed when IOE was orally administered after the second immunization with OVA. ConA stimulation in spleen cells isolated from OVA-sensitized mice treated with 100 mg kg⁻¹ IOE resulted in a 25.2% decrease in IL-4 production and a 102.4% increase in IFN-γ, compared to the controls. Moreover, IL-4, IFN-γ, and IL-2 were significantly reduced after ConA stimulation in isolated CD4⁺T cells. We also determined that IOE inhibits the secretion of NO from LPS-stimulated peritoneal macrophages ex vivo.

Conclusions

We suggest that IO modulates immune responses through secretion of Th1/Th2 cytokines in immune cells and regulates antigen-specific antibody production.     

Salacia reticulata inhibits differentiation of 3T3-L1 adipocytes

Aim of the study

The purpose of this study was to define antidiabetic effects of fruit of Vaccinium arctostaphylos L. (Ericaceae) which is traditionally used in Iran for improving of health status of diabetic patients.

Materials and methods

Firstly, we examined the effect of ethanolic extract of Vaccinium arctostaphylos fruit on postprandial blood glucose (PBG) after 1, 3, 5, 8, and 24 h following a single dose administration of the extract to alloxan-diabetic male Wistar rats. Also oral glucose tolerance test was carried out. Secondly, PBG was measured at the end of 1, 2 and 3 weeks following 3 weeks daily administration of the extract. At the end of treatment period the pancreatic INS and cardiac GLUT-4 mRNA expression and also the changes in the plasma lipid profiles and antioxidant enzymes activities were assessed.Finally, we examined the inhibitory activity of the extract against rat intestinal α-glucosidase.

Results

The obtained results showed mild acute (18%) and also significant chronic (35%) decrease in the PBG, significant reduction in triglyceride (47%) and notable rising of the erythrocyte superoxide dismutase (57%), glutathione peroxidase (35%) and catalase (19%) activities due to treatment with the extract. Also we observed increased expression of GLUT-4 and INS genes in plant extract treated Wistar rats. Furthermore, in vitro studies displayed 47% and 56% inhibitory effects of the extract on activity of intestinal maltase and sucrase enzymes, respectively.

Conclusions

Findings of this study allow us to establish scientifically Vaccinium arctostaphylos fruit as a potent antidiabetic agent with antihyperglycemic, antioxidant and triglyceride lowering effects.     

Immuno-enhancement effects of Shenqi Fuzheng Injection on cyclophosphamide-induced immunosuppression in Balb/c mice

Ethnopharmacological relevance

Radix Codonopsis and Radix Astragali, of which Shenqi Fuzheng Injection (SFI) is composed, are commonly used in traditional Chinese medicine to improve immune function against chronic diseases.

Aim of the study

The present study was thus designed to systematically elucidate the in vivo immuno-enhancement effects of SFI in immunosuppressed mice induced by cyclophosphamide (Cy) treatment.

Materials and methods

Balb/c mice were injected intraperitoneally (i.p.) once daily with low-dose (2.5 g raw materials/kg), intermediate-dose (5 g raw materials/kg), high-dose (10 g raw materials/kg) of SFI for 10 consecutive days, respectively, accompanied by i.p. injection of Cy (80 mg/kg) on Days 4-6.

Results

Compared with vehicle group, low-, intermediate- and high-dose SFI treatment accelerated recovery dose-dependently of spleen index, peripheral white blood cell and bone marrow cell counts, enhanced T cell and B cell proliferation responses, as well as splenic nature killer cell activity and peritoneal macrophage phagocytosis, and restored the level of interleukin-2 in the serum. Furthermore, SFI treatment promoted recovery of the amount of peripheral white blood cells on Day 6, rather than recombinant human Granulocyte Colony-Stimulating Factor (rhG-CSF) did.

Conclusions

These results demonstrate, for the first time, that chronic treatment with SFI results in accelerating recovery of immunosuppression in Cy-treated mice, which is competent in taking into consideration for both precautions and remedy. Our findings provide experimental evidences for further researches and clinical application in cancer patients undergoing chemotherapy.     

The neuroprotective effects of an extract of Gastrodia elata

Ethnopharmacological relevance

Gastrodia elata (GE) Blume (family Orchidaceae) is a traditional Chinese herbal medicine for treating headaches, dizziness, tetanus, and epilepsy, indicating neuronal protective functions.

Aim of the study

To evaluate the neuroprotection of GE and its molecular mechanism in preventing serum deprivation-induced PC12 cell apoptosis.

Materials and methods

An MTT assay and Hoechst staining were used to respectively validate serum deprivation-induced cell death and apoptosis. Cyclic (c)AMP formation and protein kinase (PK)A activity were also measured after GE treatment. Western blotting was used to detect the phosphorylation of the cAMP response element-binding (CREB) protein. Transient transfection of a dominant negative CREB was used to validate the importance of CREB.

Results

GE targeted the adenosine A_2A receptor (A_2A-R). GE increased cAMP formation, PKA activity, and phosphorylation of the CREB protein. GE-induced CREB protein phosphorylation and protection was blocked by a PKA inhibitor and overexpression of the dominant negative CREB, respectively.

Conclusions

These results support the neuroprotective effects of GE. The protective mechanism might be mediated through an A_2A-R/cAMP/PKA/CREB-dependent pathway.     

Anti-inflammatory effect of Lithrea molleoides extracts and isolated active compounds

Aim of the study

In this study the anti-inflammatory activity of aqueous, dichloromethane (CH₂Cl₂) and methanolic (MeOH) extracts and two major compounds isolated from Lithrea molleoides (Vell.) Engl. (Anacardiaceae) were evaluated.

Materials and methods

Two classical experimental models were used, carrageenan-induced rat paw edema and 12-O-tetradecanoylphorbol-13 acetate (TPA) induced mouse ear edema.

Results

MeOH extracts exhibited a significant systemical anti-inflammatory effect in the carrageenan (inhibition of 46% at 3 h) and in the TPA-ear edema test (inhibition of 21%). The presence of methyl gallate (inhibition of 63% in TPA ear edema), as one of the main compounds in the active fraction from MeOH extract may be explained the effect observed. Also, 1,3-dihydroxy-(Z,Z)-5-(tridec-4′,7?dienyl) benzene obtained from CH₂Cl₂ extract showed a significant topical anti-inflammatory activity (inhibition of 68%). Furthermore, no signs of toxicity were observed with doses up to 3 g/kg in an acute toxicity assay.

Conclusions

The results of this study present evidence that Lithrea molleoides given either systemically or topically has anti-inflammatory properties.     

Suppression of dust mite extract and 2,4-dinitrochlorobenzene-induced atopic dermatitis by the water extract of Lindera obtusiloba

Ethnopharmacological relevance

The Lindera obtusiloba has been used in traditional medicine for the treatment of inflammation and dermatitis. In this study, we investigated the effect of topical application of Lindera obtusiloba water extract (LOWE) on the house dust mite extract (Dermatophagoides farinae extract, DFE) and 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD).

Materials and methods

We established AD model in BALB/c mice by repeated local exposure of DFE/DNCB to the ears. After a topical application of LOWE on the skin lesions, the epidermal thickness, mast cell infiltration, and serum immunoglobulin E (IgE) and histamine were measured. In addition, the gene expression of interleukin (IL)-4, IL-13, IL-31, and tumor necrosis factor (TNF)-α in the ears was assayed.

Results

LOWE reduced AD symptoms based on ear thickness, histopathological analysis, and serum IgE levels. LOWE inhibited mast cell infiltration into the ear and elevation of serum histamine in AD model. Moreover, LOWE suppressed DFE/DNCB-induced expression of IL-4, IL-13, IL-31, and TNF-α in the ears.

Conclusions

Our results showed that topical application of LOWE exerts beneficial effects in AD symptoms, suggesting that LOWE might be a candidate for the treatment of AD.     

Analgesic compounds from Scorzonera latifolia (Fisch. and Mey.) DC.

Ethnopharmacological relevance

A traditional mastic named yak? sak?z? prepared from the roots of Scorzonera latifolia (Fisch. and Mey.) DC. is used as a folk remedy for treatment of pain in Turkish folk medicine.

Aim of the study

To isolate and identify the compounds responsible for the antinociceptive activity of S. latifolia using bioassay-guided fractionation.

Materials and methods

The methanolic extract of the S. latifolia roots was prepared and subjected to isolation procedures such as solvent-solvent partitioning and column chromatography. Writhing and tail-flick tests were used to determine the antinociceptive activity.

Results

The n-hexane fraction of the S. latifolia root methanolic extract showed potent antinociceptive activity in both writhing and tail-flick tests. Three compounds were isolated from n-hexane fraction using bioassay-guided chromatographic purification. Isolated compounds were the triterpene taraxasteryl myristate, taraxasteryl acetate, and fern-7-en-3-β-one, structures were elucidated by means of MS and NMR techniques. Both taraxasterol derivatives showed promising antinociceptive activity when compared to reference compounds.

Conclusion

Results of the present study support the usage of S. latifolia in Turkish folk medicine. Both plant root extract and the isolated compounds showed promising antinociceptive activities. Our results suggested that antinociceptive activity of the plant extract is probably caused by the synergistic interaction of the isolated compounds.     

Neuroprotective effect of Alpinia galanga (L.) fractions on Aβ(25-35) induced amnesia in mice

Ethnopharmacological relevance

The rhizomes of Alpinia galanga (L.) Willd (Zingiberaceae), a ginger substitute for flavouring food was traditionally used as nervine tonic and stimulant.

Aim of the study

This investigation is designed to screen cognitive improvement of Alpinia galanga (AG) fractions in Alzheimer's type of amnesia in mice induced by Aβ_(25-35).

Materials and methods

Alzheimer's disease induced mice treated with fractions (n-hexane, chloroform and ethyl acetate) of AG in 200 and 400 mg/kg. Neurotoxicity was induced by intracerebroventricular injection of Aβ_(25-35) on the 14th day of 21 days drug treatment. Open field and water maze were carried to determine habituation memory and hippocampal memory. Na⁺/K⁺-ATPase, acetylcholinesterase (AChE) and antioxidant enzymes (SOD, GPx, catalase and vitamin C) were determined in brain tissue homogenate to estimate the brain biochemical changes and its anti-amnesic potential with intensity of oxidative stress signaling. Further bioactive (chloroform) fraction was eluted through column chromatography to identify the lead molecules.

Results

Increased habituation memory and decreased escape latency in behavioral parameter are the indicative of the cognitive enhancement after treatment with Alpinia galanga fractions. Increment in Na⁺/K⁺-ATPase and antioxidant activity depicts brain membrane integrity improvement and free radical scavenging property. AChE level was decreased to improve the cognition by enhancing cholinergic transmission.

Conclusion

Anti-amnesic effect was exerted by various fractions of Alpinia galanga. Among all fractions, preeminent neuroprotection was exerted by chloroform fraction, which has compound, 1′δ-1′-acetoxyeugenol acetate and it may be a potential therapeutic agent for Alzheimer's type of amnesia. These results further motivate us to explore the activity of lead compound's anti-amnesic effect on transgenic mice model of AD.     

Abrogation of nimesulide induced oxidative stress and mitochondria mediated apoptosis by Fumaria parviflora Lam. extract

Ethanopharmacological relevance

Fumaria parviflora Lam. is used for treating aches and pains, diarrhea, fever, influenza and other complications. The herb mixed with honey is taken to prevent vomiting as per Ayurvedic text.

Aim of the study

In vivo studies were conducted to explore the hepatoprotective potential of Fumaria parviflora Lam. Fp extract against nimesulide induced oxidative stress and regulation of critical events in mitochondria mediated apoptosis.

Materials and methods

Group of Wistar rats were fed with nimesulide for 5 days (80 mg/kg/day, po), another group was pre-treated with Fp extract/silymarin (200 mg/kg/day, po) for 5 days followed by nimesulide exposure. Liver serum biomarkers and histopathology were done to assess hepatotoxicity caused by nimesulide. Antioxidant enzymes (SOD, LPO, GPx, GR) were assessed using biochemical assays as well as gene expression by RT-PCR. GSH content and ROS generation was also evaluated using flow cytometry. Key apoptotic markers like phosphatidyl serine externalization, Bax, Bcl-2 translocation, mitochondrial membrane potential, cytochrome c release, caspases (9/3) activation and DNA damage were also observed in all the groups to confirm involvement of mitochondrial pathway.

Results

Pre-treatment with Fp extract for 5 days significantly reduced the impact of nimesulide induced toxicity as evident from the serum biomarkers of liver damage and histopathology. It also modulated antioxidant enzymes mRNA expression as well as activity (SOD, glutathione peroxidase, glutathione reductase) and reduced lipid peroxidation during nimesulide toxicity. Nimesulide exposure decreased GSH content (92.9%) and increased reactive oxygen species (9.29 fold) which was attenuated in Fp treated rats. Fp pre-treatment significantly altered key apoptotic events like Bcl2 and Bax translocation, inhibited mitochondrial depolarization, prevented cytochrome c release, caspase-9/caspase-3 activation and DNA damage.

Conclusion

Our in vivo findings regarding protection accorded by Fp extract against nimesulide toxicity suggest that Fp not only reduced hepatotoxicity but attenuated critical control points of apoptotic cell death.     

In vitro anti-inflammatory and anti-cancer activities of Cuscuta reflexa Roxb

Aim of the study

To determine anti-inflammatory and anti-cancer activities of Cuscuta reflexa in cell lines (in vitro).

Materials and methods

Anti-inflammatory activity of the water extract was analysed in vitro using lipopolysaccharide (LPS) induced inflammatory reactions in murine macrophage cell line RAW264.7. The expression of COX-2 and TNF-α genes involved in inflammation was analysed by SQ RT-PCR. EMSA was conducted to analyse the influence of the extract on NF-κB signalling. Anti-cancer activity was analysed on Hep3B cells by MTT assay, DAPI staining, annexin V staining and SQ-RT PCR analysis of BAX, Bcl-2, p53 and survivin.

Results

The extract down regulated LPS induced over expression of TNF-α and COX-2 in RAW264.7 cells; blocked NF-κB binding to its motifs and induced apoptosis in Hep3B cells as evidenced from MTT, DAPI staining and annexin V staining assays. The extract up regulated pro-apoptotic factors BAX and p53, and down regulated anti-apoptotic factors Bcl-2 and survivin.

Conclusions

The study showed that Cuscuta reflexa inhibits LPS induced inflammatory responses in RAW264.7 cells through interplay of TNF-α, COX-2 and NF-κB signalling. It induced apoptosis in Hep3B cells through the up regulation of p53, BAX and down regulation of Bcl-2 and survivin.