Herbal formula SYJN protect PC12 cells from neurotoxicity induced by corticosterone

Aim of the study

SYJN is a Chinese herbal formula, containing four herbs: Bupleurum chinense DC., Curcuma aromatica Salisb., Perilla frutescens (Linn.) Britt. and Acorus tatarinowii Schott. Previous studies on the formula in our laboratory revealed an antidepressant-like effect on animal models of behavioral despair. However,the mechanisms underlying such antidepressant-like effect are yet to be understood. The aim of this work was to verify the previously established antidepressant-like effects on cell level using corticosterone-induced neurotoxicity in rat pheochromocytoma (PC12) cells to see if SYJN possesses any neuroprotective properties.

Materials and methods

PC12 cells were treated with 200 μM corticosterone in the absence or the presence of various concentrations of SYJN for 48 h. Then, cell viability, apoptosis, intracellular Ca²⁺ ([Ca²⁺]i) concentration and caspase-3 activity were determined.

Results

Following the exposure of PC12 cells to 200 μM corticosterone for 48 h, there were reductions in cell survival rate but increases in lactate dehydrogenase (LDH) release. In parallel, corticosterone caused significant elevations in DNA fragmentation, [Ca²⁺]i concentration and caspase-3 activity. However, when the PC12 cells were incubated with SYJN at different concentrations (10, 50 and 100 mg/L) in the presence of 200 μM corticosterone for 48 h, the above effects were evidently alleviated in a dose-dependent manner.

Conclusion

SYJN could generate a neuroprotective effect on corticosterone-induced neurotoxicity in PC12 cells, suggesting a possible action pathway of SYJN in vivo by decreasing the [Ca²⁺]i concentration and caspase-3 activity.     

The expression change of β-arrestins in fibroblast-like synoviocytes from rats with collagen-induced arthritis and the effect of total glucosides of paeony

Aim of the study

To investigate the expression of β-arrestins in fibroblast-like synoviocytes (FLS) from collagen-induced arthritis (CIA) rats and the effect of total glucosides of paeony (TGP).

Materials and methods

TGP and glucosides of tripterygium wilfordii (GTW) were intragastriclly administrated to collagen-induced arthritis (CIA) rats after immunization. The secondary inflammatory reaction was evaluated by hind paw swelling, polyarthritis index and histopathological changes. Antibodies to type II collagen (CII) were determined by enzyme-linked immunosorbent assay (ELISA). Synoviocyte proliferations were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay. The expression of β-arrestins in synoviocytes from CIA rats was measured by western blot.

Results

The administration of TGP (25, 50, 100 mg/kg) depressed hind paw swelling and decreased the arthritis scores of CIA rats. TGP improved the pathologic manifestations of CIA. Serum anti-CII antibodies level increased significantly in CIA rats, while TGP had no effect on it. Fibroblast-like synoviocytes (FLS) proliferation was inhibited by TGP (50, 100 mg/kg). On d14, d28 after immunization, β-arrestins expression greatly up-regulated in synoviocytes from CIA rats and then returned to baseline levels on d42 after immunization. TGP (50, 100 mg/kg) significantly reduced the expression of β-arrestins.

Conclusion

An inflammatory process in vivo induces an up-regulation of β-arrestins in synoviocytes from CIA rats while TGP can inhibit this change, which might be one of the important mechanisms for TGP to produce a marked therapeutic effect on RA.     

Neuroprotective effects of Total Saikosaponins of Bupleurum yinchowense on corticosterone-induced apoptosis in PC12 cells

Ethnopharmacological relevance

The root of Bupleurum yinchowense Shan et Y. Li, a well-known medicinal plant in China, was originally documented in the “Shennong's Herbal”, which is the oldest Chinese materia medica monographs. It has the action of soothing liver and relieving constraint for improving symptoms of emotional instability such as depression, anxiety and phobia. The in vivo experiment of our previous study has showed an efficacy of Total Saikosaponins (TSS) from Bupleurum yinchowense in acute stress and chronic unpredictable mild stress models. Nevertheless, there are no studies on the cytoprotection and potential mechanisms of TSS on corticosterone-induced apoptosis in PC12 cells. The present study focuses on cytoprotection against corticosterone-induced neurotoxicity in PC12 cells and its underlying molecule mechanisms of the antidepressant-like effect of TSS.

Materials and methods

The PC12 cells were treated with 250 μM corticosterone in the absence or presence of different concentrations of TSS for 24 h, then the cell viability, lactate dehydrogenase (LDH) release, Hoechst 33342 and propidium iodide (PI) double staining and the DNA fragmentation of the apoptotic PC12 cells were determined. The mitochondrial permeability transition pore (mPTP), mitochondrial membrane potential (MMP), intracellular Ca²⁺ ([Ca²⁺]i) concentration and western blot analysis of caspase-3, glucose-regulated protein 78 (GRP78), growth arrest and DNA damage inducible proteins 153 (GADD-153), X-box DNA-binding protein-1 (XBP-1), Bax, Bcl-2 were investigated.

Results

Pretreatment of PC12 cells with TSS (3.125, 6.25, 12.5, 25 μg/ml) partly reversed corticosterone-induced neurotoxicity in a dose dependent manner. TSS (25 =g/ml) reversed the increase of dead cells in the Hoechst 33342 stain, the accumulation in LDH leakage and the number of TUNEL positive cells induced by corticosterone to PC12 cells. Moreover, the cytoprotection of TSS was proved to be associated with the homeostasis of intracellular Ca²⁺, the stabilization of ER stress via the down-regulation of GRP78, GADD-153, XBP-1, and the restoration of mitochondrial function, which included mPTP, MMP and caspase-3 activity. Furthermore, TSS (25 μg/ml) markedly ameliorated up-regulation of Bax and down-regulation of Bcl-2 in corticosterone-induced PC12 cells.

Conclusion

The result depicted that antidepressant-like effect of TSS in vivo may be associated with the cytoprotection of neuron, and the neuroprotective mechanisms were correlated with inhibiting the ER stress and the mitochondrial apoptotic pathways.     

NBM-HD-3, a novel histone deacetylase inhibitor with anticancer activity through modulation of PTEN and AKT in brain cancer cells

Ethnopharmacological relevance

Taiwanese green propolis (TGP) extract contains a variety of chemical components and has proven to have broad-spectrum biological activities, including anticancer, antioxidant, and antimicrobial activities. Propolin G, an active anticancer component of TGP, was isolated and characterized in this study. Histone deacetylase inhibitors (HDACis) have been shown to be effective anticancer agents. The aim of this study was to develop a novel HDACi and investigate its anticancer mechanism.

Materials and methods

NBM-HD-3, a novel HDACi, was derived from propolin G. Two brain cancer cell lines (c6 and DBTRG-05MG) were used in the anti-proliferation assay. NBM-HD-3 treated cells were analyzed by flow cytometry in the cell cycle assay. The gene expression of NBM-HD-3 treated cells was determined by real-time quantitative PCR. HDAC enzyme assay, confocal microscopy and Western blot assay were used to validate NMB-HD-3 as HDACi. Western blot assay was used for analyzing cell cycle modulation by PTEN and AKT.

Results

NBM-HD-3 was found to have potent anti-proliferative activity in brain cancer cells (rat C6 glioma and human DBTRG-05MG glioblastoma). Western blot analysis and HDAC enzyme assay indicated that NBM-HD-3 was an HDAC inhibitor. The Western blot data exhibited increased levels of p21, Ac-histone 3, Ac-histone 4, and Ac-tubulin after brain cancer cells being treated with NBM-HD-3. NBM-HD-3 also affected the cell cycle regulators such as p21 and cyclin B1. In the study for its anticancer mechanism, NBM-HD-3 was found to increase PTEN and AKT protein levels significantly, while decreasing p-PTEN and p-AKT levels markedly.

Conclusion

This study demonstrated that the novel compound, NBM-HD-3, is a potent HDAC inhibitor. It produces anticancer activity through modulation of PTEN and AKT in brain cancer cells.     

Rosmarinus officinalis polyphenols activate cholinergic activities in PC12 cells through phosphorylation of ERK1/2

Aim of the study

This paper aimed to elucidate the traditional use of Rosmarinus officinalis through the investigation of cholinergic activities and neuronal differentiation in rat pheochromocytoma PC12 cells. These effects were examined in relation to the plant's habitat, the extraction procedure, and the major active compounds of R. officinalis.

Materials and methods

Cell viability, cell differentiation, acetylcholinesterase (AChE) activity, total choline, acetylcholine (ACh) and extracellular signal-regulated kinases (ERK1/2) were determined in PC12 cells treated with extracts and HPLC-identified polyphenols of R. officinalis originated from Tunisian semi-arid and subhumid area in comparison with nerve growth factor (NGF).

Results

R. officinalis extracts potentiated cell differentiation and significantly enhanced AChE activity in PC12 cells. The highest AChE activity was induced by semi-arid hydro-ethanolic extract (137% of control). Among HPLC-identified and screened polyphenols, carnosic acid (CA) and rosmarinic acid (RA) significantly induced cell differentiation, increased ACh level, and enhanced AChE activity in PC12 cells. U0126, inhibitor of ERK1/2, significantly reduced CA and RA effects on cell differentiation and AChE activity.

Conclusions

R. officinalis’ CA and RA exhibited neurotrophic effects in PC12 cells through cell differentiation induction and cholinergic activities enhancement. These effects could be regulated by mitogen-activated protein kinase (MAPK), ERK1/2 signaling pathway.     

Herbal formula SYJN increases neurotrophin-3 and nerve growth factor expression in brain regions of rats exposed to chronic unpredictable stress

Aim of the study

SYJN is a Chinese herbal formula that contains four herbs: Bupleurum chinense DC., Curcuma aromatica Salisb., Perilla frutescens (L.) Britt., and Acorus tatarinowii Schott. Previous studies conducted in our laboratory have revealed an antidepressant-like effect of the formula in chronic unpredictable stress (CUS)-induced depression model in rats. The present study aimed to investigate whether neurotrophin-3 (NT-3) and nerve growth factor (NGF) are involved in the antidepressant-like action of SYJN by using the same depressive model in rats.

Materials and methods

Rats were subjected to an experimental setting of CUS. The mechanism underlying the antidepressant-like action of SYJN was examined by measuring protein and mRNA expression of NT-3 and NGF in brain tissues of CUS-exposed rats.

Results

The results showed that NT-3 protein and mRNA expression in the hippocampus and frontal cortex were significantly decreased in CUS-treated rats. CUS treatment also significantly decreased NGF protein and mRNA expression in the frontal cortex of the animals. Daily intragastric administration of SYJN (1300 or 2600 mg/kg/day) during the 4 weeks of CUS significantly suppressed these changes induced by CUS.

Conclusion

The results suggest that the antidepressant-like activity of SYJN is likely mediated by the increases in NT-3 and NGF expression in brain tissues.     

Antidepressant-like effects of an alkaloid extract of the aerial parts of Annona cherimolia in mice

Ethnopharmacological relevance

Several species of Annona (Annonaceae) are used in traditional Mexican medicine by their anti-anxiety, anticonvulsant and tranquilizing properties. It has been reported that the alkaloids isolated from some species of the Annona have affinity to serotonergic 5-HT_1A receptors and modulate dopaminergic transmission, which is involved in depressive disorders.

Aim of the study

To investigate the antidepressant-like effect of an alkaloid extract from the aerial parts of Annona cherimola (TA) in mice.

Materials and methods

The antidepressant-like effect was evaluated in the forced swimming test. To elucidate a possible mechanism of action, experiments of synergism with antidepressant drugs, such as imipramine (IMI), clomipramine (CLIMI), and fluoxetine (FLX), were carried out. The neurotransmitter content (DA: dopamine, 5HT: serotonin and its metabolites, HVA: homovanillic acid and 5HIAA: 5-hydroxyindoleacetic) in the whole brain of mice were also determined by HPLC method. TA chemical composition was determined using high performance liquid chromatography-electrospray mass spectrometry.

Results

The results showed that repeated treatment with TA produced antidepressant-like effects in mice. This effect was not related to an increase in locomotor activity. Administration of TA facilitated the antidepressant effect of IMI and CLIMI as well as increased the turnover of DA and 5-HT. The alkaloids: 1,2-dimethoxy-5,6,6a,7-tetrahydro-4H-dibenzoquinoline-3,8,9,10-tetraol, anonaine, liriodenine, and nornuciferine were the main constituents of TA.

Conclusions

Results showed that TA produces an antidepressant-like action from a generalized increase in monominergic turnover, supporting the use in tradicional medicine of Annona cherimolia, and strongly suggest its therapeutic potency as an antidepressant agent.     

Antigenotoxic and antioxidant activities of palmarosa and citronella essential oils

Ethnopharmacological relevance

Essential oils of palmarosa and citronella have been extensively used in ancient Indian and South-east Asian traditional medicines.

Aim of the study

These essential oils have been reported to exhibit antimicrobial, anti parasitic effects against bacteria, yeasts, filamentous fungi, and viruses. In the present study the oils were tested for their potential antigenotoxic and antioxidant properties in human lymphocyte cells.

Materials and methods

The antigenotoxic effect on human lymphocyte cells (measurement of cell viability, DNA damage) was studied using trypan blue dye exclusion test, plasmid pBR322 DNA strand scission, and comet assay. Anti-oxidant activity was evaluated by DPPH⁺ free radical scavenging and lipid peroxidation assay.

Results

The essential oils showed a good antigenotoxic activity against methyl methanesulphonate (MMS) and hydrogen peroxide. In addition, a significant dose dependent antioxidant activity was observed.

Conclusion

Our data provide evidence that support the usage of palmarosa and citronella essential oils in traditional herbal preparations. They can constitute a natural source of a new and safe antioxidant.     

Paeoniflorin, a potent natural compound, protects PC12 cells from MPP and acidic damage via autophagic pathway

Ethnopharmacological relevance

Paeoniflorin (PF) is the principal bioactive component of Radix Paeoniae alba, which is widely used in Traditional Chinese Medicine for the treatment of neurodegenerative disorders such as Parkinson's disease (PD).

Aim of the study

To evaluate the neuroprotective effects of PF on MPP⁺- or acid- (pH 5.0) induced injury in cultured PC12 cells and to investigate the activity of autophagy-lysosome pathway (ALP). Amiloride (Ami), a non-selective blocker of acid-sensing ion channels (ASICs), as a positive control drug, since it is neuroprotective in rodent models of PD.

Materials and methods

The cell viability was analyzed with MTT assay. The cell injury was assessed by lactate dehydrogenase (LDH) assay. Flow cytometry and Western blot analysis were used to study the apoptotic, calcium influx and autophagic mechanisms.

Results

Ami (100 μM) and PF (50 μM) both protected PC12 cells against MPP⁺- or acid-induced injury as assessed by MTT assay, lactate dehydrogenase release, and apoptosis rate. The concentrations of cytosolic free Ca²⁺ were raised after exposure to MPP⁺ or acidosis, while Ami and PF both reduced the influx of Ca²⁺. More importantly, we found that the mechanisms of neuroprotective effects of Ami and PF were closely associated with the upregulation of LC3-II protein, which is specifically associated with autophagic vacuole membranes. Furthermore, application of MPP⁺ or acid induced the overexpression of LAMP2a, which is directly correlated with the activity of the chaperone-mediated autophagy pathway. However, Ami and PF inhibited the overexpression of LAMP2a.

Conclusions

Our data provide the first experimental evidence that PF modulates autophagy in models of neuron injury, as well as providing the first indication of a relationship between ASICs and ALP.     

Antidepressant-like activity of the aqueous extract of Allium macrostemon in mice

Aim of the study

The aim of this study was to identify the effects of water extracts of Allium macrostemon Bunge (AM-W), a traditional herb, in mice.

Materials and methods

The antidepressant-like activities of AM-W were evaluated through behavioral despair in forced swimming test and tail suspension test. To elucidate the mode of action of the antidepressant-like effects of AM-W, new born cells in the subgranular zone and the granule cell layer were analyzed by immunostaining for incorporation of 5-bromo-2-deoxyuridine (BrdU). In addition, the effects of AM-W on the expression levels of brain-derived neurotrophic factor (BDNF) were investigated by western blotting and immunohistochemistry.

Results

The administration of AM-W reduced the immobility duration in the forced swimming test and tail suspension test (100 or 200 mg/kg, P < 0.05). Sub-chronic administration of AM-W (100 or 200 mg/kg, p.o., for 14 days) increased the number of BrdU-incorporating cells. The percentage of BrdU-incorporating cells co-localized with NeuN was significantly increased after AM-W administration (100 or 200 mg/kg, P < 0.05). Moreover, the expression levels of BDNF which is reported to be associated with neurogenesis were significantly increased in the hippocampus after administration of AM-W.

Conclusions

These results suggest that AM-W may be a good antidepressant, and that its mechanism of action may be related to its positive effects on neurogenesis and BDNF release.     

Neuroprotective effects of Abacopterin E from Abacopteris penangiana against oxidative stress-induced neurotoxicity

Aim of the study

Abacopterin E (AE) was isolated from Abacopteris penangiana (Hook.) Ching. This study was to elucidate its neuroprotective effects against hydrogen peroxide (H₂O₂) induced oxidative damage in PC12 cells and d-galactose (d-Gal) induced neurotoxicity in mice brain.

Materials and methods

In vitro, the protective effect of AE against H₂O₂-induced oxidative damage in the PC12 was investigated by the method of MTT (3,(4,5-dimethylthiazole-2-yl)2,5-diphenyl-tetrazolium bromide). In vivo, the protective effect of AE against d-Gal-induced neurotoxicity in mice was studied. The mice in the model group and the AE treatment groups were injected with the d-Gal 150 mg/(kg d) for 7 weeks while the mice in the control group were injected with the same volume of saline (0.9%). From the sixth week, the treatment groups were subcutaneously injected 4 or 8 mg/(kg d) of AE. In order to explore the potential mechanism of AE's action, the mice were assessed by behavioral and electrophysiological tests at the end of the administration. Then the mice brain tissues were measured for the levels of superoxide dismutases (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA).

Results

This study showed that AE lowered the H₂O₂-induced cytotoxicity, and AE significantly improved the learning and memory ability in behavioral performance. The biochemical examination revealed that AE restored the activities of SOD and GSH-Px, and attenuated the increase of MDA. Moreover, the electrophysiological analysis evidently showed that AE ameliorated the long-term potentiation (LTP).

Conclusions

These results suggested that AE had neuroprotective effects, and its beneficial effects may be linked with inhibiting the generation of free radical and enhancing the activities of endogenous antioxidant enzymes.     

In vitro antioxidant, antiplatelet and anti-inflammatory activity of Carpobrotus rossii (pigface) extract

Ethnopharmacological relevance

Carpobrotus rossii (CR) has a history of use as a food and therapeutic agent by Australian indigenous peoples and early European settlers and is believed to contain a number of pharmacologically active polyphenolic compounds.

Aims of the study

Oxidation of low density lipoprotein (LDL), platelet aggregation, and inflammation contribute to the development and progression of atherosclerosis. The aim of the present study was to investigate the antioxidant, antiplatelet and anti-inflammatory activity of CR extract using human blood components.

Materials and methods

An assay employing in vitro copper-induced oxidation of serum lipids was used to assess antioxidant activity of CR extract (and tannin, flavonoid and pre- and post-flavonoid fractions). The effects of CR extract on ADP- and collagen-induced platelet aggregation, and on basal (unstimulated) and lipopolysaccharide (LPS)- and phytohaemagglutinin A (PHA)-stimulated cytokine release from peripheral blood mononuclear cells (PBMC) were also investigated.

Results

CR extract increased the lag time of serum oxidation (maximum of ∼4-fold at 20 μg/ml) in a concentration-dependent manner. The antioxidant activity resided only in the tannin and post-flavonoid fractions. CR had no effect on ADP-induced platelet aggregation, but significantly decreased collagen-induced platelet aggregation. LPS, but not PHA, significantly increased the release of IL-1β and TNF-α from PBMC. CR extract alone inhibited monocyte chemoattractant protein (MCP)-1 release and in the presence of LPS, inhibited IL-10, TNF-α and MCP-1 release compared to LPS alone.

Conclusion

CR has significant in vitro antioxidant, antiplatelet and, potentially, anti-inflammatory activity.     

Protective effects of aqueous extract from Acanthopanax senticosus against corticosterone-induced neurotoxicity in PC12 cells

Ethnopharmacological relevance

Acanthopanax senticosus, classified into the family of Araliaceae, has been known for thousands of years as a remedy and is used to treat various diseases in traditional Chinese medicine system including hypertension, ischemic heart disease and hepatitis.

Aim of the study

This study aimed to examine the protective effects of aqueous extract from Acanthopanax senticosus (ASE) on corticosterone-induced neurotoxicity and its possible mechanisms, using PC12 cells as a suitable in vitro model of depression.

Materials and methods

In this paper, PC12 cells were treated with 200 μM of corticosterone in the absence or presence of ASE in varying concentrations for 24 h. Then, cell viability was measured by MTT assay. The release amount of lactate dehydrogenase (LDH) was quantified using LDH assay kit. Apoptosis of PC12 cells was measured by Annexin V-FITC and PI labeling. The intracellular Ca²⁺ content was tested by fluorescent labeling. The mRNA level of brain-derived neurotrophic factor (BDNF) was examined by real-time RT-PCR, and the expression of cAMP response element binding protein (CREB) was determined by western blotting.

Results

The results showed that treatment with 200 μM of corticosterone could induce cytotoxicity in PC12 cells. However, different concentrations of ASE (50, 100, 200, and 400 μg/mL) significantly increased the cell viability, decreased the LDH release, suppressed the apoptosis of PC12 cells, attenuated the intracellular Ca²⁺ overloading, up-regulated the BDNF mRNA level and CREB protein expression compared with the corresponding corticosterone-treated group.

Conclusion

The present results suggest that ASE exerts a neuroprotective effect on corticosterone-induced neurotoxicity in PC12 cells, which may be one of the acting mechanisms that accounts for the in vivo antidepressant activity of ASE.     

Gastrodia elata prevents huntingtin aggregations through activation of the adenosine A₂A receptor and ubiquitin proteasome system

Ethnopharmacological relevance

Gastrodia elata Blume (Fam. Orchidaceae) is a traditional Chinese herbal medicine for treating headaches, dizziness, tetanus, epilepsy, and numbness of the limbs, which suggests that it has neuroprotective effect.

Aim of the study

To validate the neuroprotection of Gastrodia elata in preventing neurodegenerations, such as Huntington's disease (HD).

Materials and methods

MTT assay was used to validate the protection of Gastrodia elata. In pheochromocytoma (PC12) cell. Transient transfection of mutant huntingtin (Htt) in PC12 cell was used as an in vitro model of HD. Filter retardation assay was used to measure Htt-induced protein aggregations. Proteasome activity was monitored by transfection of pZsProSensor-1 and imaged by a confocal laser scanning microscope.

Results

This protection of Gastrodia elata could be blocked by an A_2A-R antagonist and a protein kinase A (PKA) inhibitor, indicating an A_2A-R signaling event. Gastrodia elata could reverse mutant Htt-induced protein aggregations and proteasome de-activation through A_2A-R signaling. In addition, activation of PKA tended to activate proteasome activity and reduce mutant Htt protein aggregations. The proteasome inhibitor, MG 132, blocked Gastrodia elata-mediated suppression of mutant Htt aggregations.

Conclusion

Gastrodia elata prevented mutant Htt aggregations and increased proteasomal activity by targeting the A_2A-R through PKA-dependent pathway.     

Antinociceptive activity of Quillaja saponaria Mol. saponin extract, quillaic acid and derivatives in mice

Ethnopharmacological relevance

Quillaja saponaria bark contains a high percentage of triterpene saponins and has been used for centuries as a cleansing and analgesic agent in Chilean folk medicine.

Aim of the study

The topical and systemic analgesic effects of a commercial partially purified saponin extract, 3β,16α-dihydroxy-23-oxoolean-12-en-28-oic acid (quillaic acid), methyl 3β,16α-dihydroxy-23-oxoolean-12-en-28-oate and methyl 4-nor-3,16-dioxoolean-12-en-28-oate.

Materials and methods

The samples were assessed in mice using the topical tail-flick and i.p. hot-plate tests, respectively.

Results

All the samples showed activity in both analgesic tests in a dose-dependent manner. The most active against tail flick test was commercial partially purified saponin extract (EC50 27.9 mg%, w/v) and more than the ibuprofen sodium. On hot-plate test, methyl 4-nor-3, 16-dioxoolean-12-en-28-oate was the most active (ED50 12.2 mg/kg) and more than the ibuprofen sodium.

Conclusions

The results of the present study demonstrated that Quillaja saponaria saponins, quillaic acid, its methyl ester, and one of the oxidized derivatives of the latter, elicit dose-dependent antinociceptive effects in two murine thermal models.     

Olive (Olea europaea L.) leaf extract attenuates early diabetic neuropathic pain through prevention of high glucose-induced apoptosis: in vitro and in vivo studies

Aim of study

Since the leaves of olive have been recommended in the literature as a remedy for the treatment of diabetes and they also contain antioxidant agents, we decided to investigate the possible effects of olive leaf extract (OLE) on in vitro and in vivo models of diabetic pain neuropathy.

Materials and methods

The high glucose-induced cell damage in naive and NGF-treated Pheochromocytoma (PC12) cells and streptozotocin-induced diabetic rats were used. Tail-flick test was used to access nociceptive threshold. Cell viability was determined by MTT assay. Biochemical markers of neural apoptosis were evaluated using immunoblotting.

Results

We found that elevation of glucose (4 times of normal) sequentially increases functional cell damage and caspase-3 activation in NGF-treated PC12 cells. Incubation of cells with OLE (200, 400 and 600 μg/ml) decreased cell damage. Furthermore, the diabetic rats developed neuropathic pain which was evident from decreased tail-flick latency (thermal hyperalgesia). Activated caspase 3 and Bax/Bcl2 ratio were significantly increased in spinal cord of diabetic animals. OLE treatment (300 and 500 mg/kg per day) ameliorated hyperalgesia, inhibited caspase 3 activation and decreased Bax/Bcl2 ratio. Furthermore, OLE exhibited potent DPPH free radical scavenging capacity.

Conclusion

The results suggest that olive leaf extract inhibits high glucose-induced neural damage and suppresses diabetes-induced thermal hyperalgesia. The mechanisms of these effects may be due, at least in part, to reduce neuronal apoptosis and suggest therapeutic potential of olive leaf extract in attenuation of diabetic neuropathic pain.     

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.     

Methanolic extract of Hibiscus asper leaves improves spatial memory deficits in the 6-hydroxydopamine-lesion rodent model of Parkinson's disease

Ethnopharmacological relevance

While the Hibiscus asper Hook.f. (Malvaceae) is a traditional herb largely used in tropical region of the Africa as vegetable, potent sedative, tonic and restorative, anti-inflammatory and antidepressive drug, there is very little scientific data concerning the efficacy of this.

Aim of the study

We investigated antioxidant activity and the effects of methanolic extract of Hibiscus asper leaves on neurological capacity of male Wistar rats subjected to unilateral 6-hydroxydopamine (6-OHDA)-lesion.

Materials and methods

Two model systems: 2,4-dinitrophenyl-1-picryl hydrazyl (DPPH) radical scavenging activity and β-carotene bleaching inhibition assay were used to measure the antioxidant activities of the plan extract. We also investigated the neuroprotective effect of methanolic extract of Hibiscus asper leaves (50 and 100 mg/kg) in male Wistar rats subjected to unilateral 6-hydroxydopamine (6-OHDA)-lesion rat model.

Results

Methanolic extract of Hibiscus asper leaves showed potent antioxidant and free radical scavenging activity. Chronic administration of methanolic extract (50 and 100 mg/kg, i.p., daily, for 7 days) significantly reduce anxiety-like behavior and inhibit depression in elevated plus-maze and forced swimming tests, suggesting anxiolytic and antidepressant activity. Also, spatial memory performance in Y-maze and radial arm-maze tasks was improved, suggesting positive effects on memory formation.

Conclusions

Taken together, our results suggest that the methanolic extract of Hibiscus asper leaves have antioxidant effects and might provide an opportunity to management neurological abnormalities in Parkinson's disease conditions.     

Antioxidant, antinociceptive, and anti-inflammatory activities of Xanthii Fructus extract

Ethnopharmacological relevance

Xanthii seeds commonly called Cang-Erzi were used as a traditional Chinese medicine for treating sinusitis, headache due to rheumatism and skin pruritus.

Aim of the study

In order to evaluate the actions of this plant, studies were performed on antioxidant, antinociceptive, and anti-inflammatory activities.

Materials and methods

The aqueous extract of Xanthii Fructus (AXF) was evaluated in mice for anti-inflammatory activity using carrageenan-induced hind paw edema model. The antinociceptive activity of AXF was evaluated by writhing and formalin tests. Antioxidant properties were assayed in terms of antioxidant activity by scavenging abilities on 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS), reducing activity and liposome protection. In addition, the total phenolic content was determined with spectrophotometric method.

Results

AXF exhibited significant radical scavenging and reducing activity. And oral treatment with AXF elicited inhibitory activity on acetic acid effect and reduced the formalin effect at the late-phase. In the anti-inflammatory test, AXF inhibited the development of paw edema induced by λ-carrageenan (Carr). AXF decreased the paw edema at the fifth hour after Carr administration, and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver tissue and decreased the malondialdehyde (MDA) level in the edema paw. AXF decreased the level of serum nitric oxide (NO) and tumor necrosis factor (TNF)-α after Carr injection and AXF decreased the levels of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions in paw edema at the fifth hour.

Conclusions

AXF shows antioxidant, antinociceptive, and anti-inflammatory activities, supporting the folkloric usage of the plant to treat various inflammatory diseases.     

Effects of total glucosides from paeony (Paeonia lactiflora Pall) roots on experimental atherosclerosis in rats

Ethnopharmacological relevance

Total glucosides of paeony (TGP), compounds extracted from the roots of Paeonia lactiflora Pall, have been used as an anti-inflammatory drug for the treatment of rheumatoid arthritis (RA) in China. Inflammation plays a critical role in the development of atherosclerotic vascular disease. Risk of cardiovascular diseases is significantly higher in patients with RA than in normal population. It has a great significance to study the effects of TGP on atherosclerosis.

Aim of the study

To investigate the effects of TGP on atherosclerosis induced by excessive administration of vitamin D and cholesterol in rats and study the mechanisms involved.

Materials and methods

Atherosclerosis was induced by excessive administration of vitamin D and cholesterol in rats. TGP was intragastrically administered for 15 weeks. The serum concentrations of total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C) and high density lipoprotein-cholesterol (HDL-C) were measured by automatic biochemistry analyzer. Apolipoprotein A1 (ApoA1) and apolipoprotein B (ApoB) were determined by immunoturbidimetry method, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and C-reactive protein (CRP) were measured by enzyme-linked immunosorbent assay (ELISA) method. The morphological changes of aorta were observed with optical microscopy.

Results

Compared to controls, TGP significantly lowered the serum level of TC, TG, LDL-C, ApoB, TNF-alpha, IL-6 and CRP, increased the ratios of HDL-C/LDL-C and ApoA1/ApoB, decreased the intima-media thickness (IMT) of abdominal aortal wall and improved the morphological change of the aorta.

Conclusions

TGP may attenuate the development of atherosclerotic disease. The beneficial effects are associated with its lowering blood lipids and inhibiting the expression of inflammatory cytokines.