Evidence of the involvement of the monoaminergic systems in the antidepressant-like effect of Aloysia gratissima

Ethnopharmacological relevance

Aloysia gratissima (Verbenaceae) is an aromatic plant distributed in South America and, employed in folk medicine for the treatment of nervous systems illness, including depression. The neuroprotective and antidepressant-like activities of the aqueous extract of Aloysia gratissima (AE) administered orally has already been demonstrated.In this study the involvement of monoaminergic systems in the antidepressant-like effect of the AE was investigated.

Materials and methods

The implication of the monoaminergic systems in the antidepressant-like activity of Aloysia gratissima was evaluated using different pharmacological antagonists that were administered previously to the acute oral administration of AE (10 mg/kg). The antidepressant-like effect was assessed in the TST and locomotor activity was evaluated in the open-field test in mice.

Results

The anti‐immobility effect elicited by AE in the TST was prevented by the pre-treatment of mice with the antagonists, NAN‐190 (5‐HT_1A receptor), ketanserin (5‐HT_2A/2C receptor), prazosin (α₁‐adrenoceptor), yohimbine (α₂‐adrenoceptor), SCH23390 (dopamine D₁ receptor), or sulpiride (dopamine D₂ receptor).

Conclusions

These results indicate that the antidepressant‐like effect of AE in the TST is dependent on its interaction with the serotonergic (5‐HT_1A and 5‐HT_2A/2C), noradrenergic (α₁ and α₂₋adrenoceptors) and dopaminergic (D₁ and D₂ receptors) systems, suggesting that this specie might act as a new potential resource for developing antidepressants to treat depressive disorders.     

Antiplatelet effects of Spatholobus suberectus via inhibition of the glycoprotein IIb/IIIa receptor

Ethnopharmacological relevance

The vine stem of Spatholobus suberectus is a widely used blood-activating and stasis-dispelling medicine for the treatment of diseases related to blood stasis syndrome in traditional medicine in Korea, Japan, and China.

Aim of the study

To demonstrate the clinical effects of Spatholobus suberectus against blood stasis syndromes using in vitro and in vivo platelet aggregation studies and to investigate its exact mechanisms.

Materials and methods

We extracted vine stems of Spatholobus suberectus, using 95% EtOH (SSE) and investigated its antiplatelet activity on platelet aggregation induced by collagen and ADP in human platelet-rich plasma (PRP). For the mechanism study, a glycoprotein IIb/IIIa (GP IIb/IIIa) assay using flow cytometric analysis and a thromboxane A₂ (TXA₂) assay were performed. In addition, we investigated the effects of SSE in a thromboembolic mouse model.

Results

SSE significantly inhibited ADP- and collagen-induced platelet aggregation in human PRP concentration-dependently without affecting plasma clotting time. It also significantly inhibited fibrinogen binding to the GP IIb/IIIa receptor and partly inhibited the formation of TXA₂. In the in vivo study, oral administration of SSE dose-dependently suppressed the death of thromboembolism model mice induced by intravenous injection of collagen plus epinephrine.

Conclusions

SSE showed antiplatelet activity without anticoagulant effects mainly through the inhibition of fibrinogen binding to the GP IIb/IIIa receptor. Our current results support the clinical usage of SSE in the East Asian region treating atherothrombotic diseases and may represent a new natural source to develop antiplatelet agents.     

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.     

Neuroprotective effects of Eleutherococcus senticosus bark on transient global cerebral ischemia in rats

Ethnopharmacological relevance

Eleutherococcus senticosus Maxim., classified into the family of Araliaceae, is used in a variety of diseases in traditional Korean medicine including ischemic heart disease.

Aim of the study

To determine the neuroprotective effects of Eleutherococcus senticosus on global cerebral ischemia.

Materials and methods

A four-vessel occlusion (4-VO) rat model was used to evaluate the potential protective effects against transient global cerebral ischemia ethanol extracts of Eleutherococcus senticosus was orally administered at doses of 3, 30, and 300 mg/kg twice at times of 0 and 90 min after reperfusion. The effects on memory deficit were investigated by using a Y-maze neurobehavioral test after brain ischemia, and the effects on hippocampal neuronal damage were measured 7 days after ischemia. The expressions of glial fibrillary acid protein (GFAP), CD11b antibody (OX-42), and cyclooxygenase-2 (COX-2) were investigated by immunohistochemistry.

Results

Oral administration of Eleutherococcus seticosus at 30, 100 and 300 mg/kg significantly reduced hippocampal CA1 neuronal death by 3.5%, 25.9% and 53.1%, respectively, compared with a vehicle-treated group. Oral administration of Eleutherococcus senticosus at 300 mg/kg inhibited 81.9% of the decrease in spontaneous alternation induced by 4-VOin the Y-maze test, and also attenuated ischemia-induced activation of COX-2, GFAP and OX-42 in the hippocampal CA1 region.

Conclusion

Eleutherococcus senticosus protects delayed neuronal death in the CA1 region of the hippocampus against global cerebral ischemia in rats with the recovery of spatial memory, which can be considered as the normal functioning of the hippocampus. Regarding the immunohistochemical study, the effect of Eleutherococcus senticosus may be attributable to its anti-inflammatory properties through the inhibition of COX-2 expression, microglia and astrocyte expression.     

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.     

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.     

The GABAergic system contributes to the anxiolytic-like effect of essential oil from Cymbopogon citratus (lemongrass)

Ethnopharmacological relevance

The essential oil (EO) from Cymbopogon citratus (DC) Stapf is reported to have a wide range of biological activities and is widely used in traditional medicine as an infusion or decoction. However, despite this widely use, there are few controlled studies confirming its biological activity in central nervous system.

Materials and methods

The anxiolytic-like activity of the EO was investigated in light/dark box (LDB) and marble-burying test (MBT) and the antidepressant activity was investigated in forced-swimming test (FST) in mice. Flumazenil, a competitive antagonist of benzodiazepine binding and the selective 5-HT_1A receptor antagonist WAY100635 was used in experimental procedures to determine the action mechanism of EO. To exclude any false positive results in experimental procedures, mice were submitted to the rota-rod test. We also quantified some neurotransmitters at specific brain regions after EO oral acute treatment.

Results

The present work found anxiolytic-like activity of the EO at the dose of 10 mg/kg in a LDB. Flumazenil, but not WAY100635, was able to reverse the effect of the EO in the LDB, indicating that the EO activity occurs via the GABA_A receptor-benzodiazepine complex. Only at higher doses did the EO potentiate diethyl-ether-induced sleeping time in mice. In the FST and MBT, EO showed no effect. Finally, the increase in time spent in the light chamber, demonstrated by concomitant treatment with ineffective doses of diazepam (DZP) and the EO, revealed a synergistic effect of the two compounds. The lack of activity after long-term treatment in the LDB test might be related to tolerance induction, even in the DZP-treated group. Furthermore, there were no significant differences between groups after either acute or repeated treatments with the EO in the rota-rod test. Neurochemical evaluation showed no amendments in neurotransmitter levels evaluated in cortex, striatum, pons, and hypothalamus.

Conclusions

The results corroborate the use of Cymbopogon citratus in folk medicine and suggest that the anxiolytic-like effect of its EO is mediated by the GABA_A receptor-benzodiazepine complex.     

Oral Uncaria rhynchophylla (UR) reduces kainic acid-induced epileptic seizures and neuronal death accompanied by attenuating glial cell proliferation and S100B proteins in rats

Aim of the study

Epilepsy is a common clinical syndrome with recurrent neuronal discharges in cerebral cortex and hippocampus. Here we aim to determine the protective role of Uncaria rhynchophylla (UR), an herbal drug belong to Traditional Chinese Medicine (TCM), on epileptic rats.

Materials and methods

To address this issue, we tested the effect of UR on kainic acid (KA)-induced epileptic seizures and further investigate the underlying mechanisms.

Results

Oral UR successfully decreased neuronal death and discharges in hippocampal CA1 pyramidal neurons. The population spikes (PSs) were decreased from 4.1 ± 0.4 mV to 2.1 ± 0.3 mV in KA-induced epileptic seizures and UR-treated groups, respectively. Oral UR protected animals from neuronal death induced by KA treatment (from 34 ± 4.6 to 191.7 ± 48.6 neurons/field) through attenuating glial cell proliferation and S100B protein expression but not GABAA and TRPV1 receptors.

Conclusions

The above results provide detail mechanisms underlying the neuroprotective action of UR on KA-induced epileptic seizure in hippocampal CA1 neurons.     

In vitro modulatory effects of Andrographis paniculata, Centella asiatica and Orthosiphon stamineus on cytochrome P450 2C19 (CYP2C19)

Ethno pharmacological relevance

Andrographis paniculata (AP), Centella asiatica (CA) and Orthosiphon stamineus (OS) are three popular herbs traditionally used worldwide. AP is known for the treatment of infections and diabetes and CA is good for wound healing and healthy skin while OS is usually consumed as tea to treat kidney and urinary disorders. Interaction of these herbs with human cytochrome P450 2C19 (CYP2C19), a major hepatic CYP isoform involved in metabolism of many clinical drugs has not been investigated to date.

Aim of the study

In this study, the modulatory effects of various extracts and major active constituents of AP, CA and OS on CYP2C19 activities were evaluated.

Materials and methods

S-Mephenytoin, the CYP2C19 substrate probe, was incubated in the presence or absence of AP, CA and OS components. The changes in the rate of metabolite (hydroxymephenytoin) formation were subsequently determined by a high-performance liquid chromatography (HPLC)-based enzyme assay to characterize the modulatory effects.

Results

Among the herbal extracts studied, AP ethanol extract and CA dichloromethane extract exhibited mixed type inhibition towards CYP2C19 with K_i values of 67.1 and 16.4 μg/ml respectively; CA ethanol extract and OS petroleum ether extract competitively inhibited CYP2C19 activity (K_i = 39.6 and 41.5 μg/ml respectively). Eupatorin (a major active constituent of OS) was found to significantly inhibit CYP2C19 by mixed type inhibition (K_i = 7.1 μg/ml or 20.6 μM).

Conclusions

It was observed that AP, CA and OS inhibited CYP2C19 activity with varying potency. While weak inhibitory effect was observed with AP, moderate to strong inhibition was observed with CA dichloromethane extract and eupatorin, the major OS constituent. Therefore care should be taken when these CA and OS components are co-administered with CYP2C19 substrates (such as omeprazole, proguanil, barbiturates, citalopram, and diazepam).     

Biphasic effects of baicalin, an active constituent of Scutellaria baicalensis Georgi, in the spontaneous sleep-wake regulation

Aim of the study

Baicalin is an active compound originating from the root of Scutellaria baicalensis Georgi, which has been used for anti-inflammation, anti-bacteria, anti-hypertension, anti-allergy and sedation since ancient China, though the neuronal mechanisms involved in the sedative effect is still unclear. Baicalin possesses the ability to decrease the expression of pro-inflammatory cytokines and nuclear factor (NF)-κB activity. Furthermore, baicalin has demonstrated an anxiolytic-like effect via activation of γ-aminobutyric acid-A (GABA_A) receptors. Pro-inflammatory cytokines (e.g. interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α) and the GABAergic system promote sleep. This study was designed to determine whether the GABA_A receptor activation and/or the suppression of pro-inflammatory cytokines mediate(s) baicalin-induced sleep alterations.

Materials and methods

Baicalin was intracerebroventricularly (ICV) administered 20 min either prior to the beginning of the light period or before the onset of the dark period. Electroencephalogram (EEG) and gross body movement were acquired for sleep analysis. Pharmacological blockade of IL-1 and GABA_A receptors were employed to elucidate the involvements of IL-1 and GABA_A receptors in baicalin-induced sleep alterations. IL-1β concentrations obtained after baicalin administration in several distinct brain regions were determined by ELISA.

Results

ICV administration of baicalin decreased slow wave sleep (SWS) during the first 2 h of the light period. Rapid eye movement sleep (REMS) was not altered. The blockade of IL-1β-induced SWS enhancement by baicalin suggests that the antagonism of IL-1 receptors is involved in baicalin-induced SWS decrement during the light period. However, IL-1β concentrations during the light period were not altered after baicalin administration. In contrast, baicalin increased both SWS and REMS during hours 8-10 of the dark (active) period when baicalin was administered at the beginning of the dark period, and its effects were blocked by the GABA_A receptor antagonist bicuculline.

Conclusion

Baicalin exhibits biphasic effects on sleep-wake regulation; the decrease of SWS during the light period and increases of SWS and REMS during the dark period. Inhibition of IL-1 action and enhancement of GABA_A receptor activity may mediate baicalin's effects during the light and dark period, respectively.     

Inhibition of the inflammatory and coagulant action of Bothrops atrox venom by the plant species Marsypianthes chamaedrys

Aim of the study

This study investigated the efficacy of Marsypianthes chamaedrys Vahl (Lamiaceae) inflorescence and leaf extracts in inhibiting the inflammatory and coagulant actions of Bothrops atrox venom.

Materials and methods

Marsypianthes chamaedrys, which is used in Brazil as a folk medicine to treat snakebites and local inflammatory reactions, was tested in vitro to determine its ability to block indirect phospholipase A₂ and direct coagulant activities and in vivo to determine its ability to inhibit leukocyte migration and cytokine release.

Results

In vitro, Marsypianthes chamaedrys showed antiphospholipase A₂ and anticoagulant activities; the latter activity was also confirmed by prothrombin time (PT) and activated partial thromboplastin time (aPTT) in the absence of venom. Of the extracts used, those obtained from the crushed plant had the greater inhibitory activity in in vitro tests, showing that biological activity is affected by the way extracts are obtained. In vivo, Marsypianthes chamaedrys inhibited leukocyte migration and the release of the proinflammatory cytokines IL-6 and TNF-α without altering the concentration of the anti-inflammatory cytokine IL-10.

Conclusions

As specific antivenoms are not effective in neutralizing the local action of Bothrops venoms, characterization of the anti-inflammatory mechanisms induced by Marsypianthes chamaedrys is of vital importance if the extracts of this plant species are to be used in future as adjuvants in the treatment of snakebites.     

Evaluation of the effects of Astragalus mongholicus Bunge saponin extract on central nervous system functions

Ethnopharmacological relevance

In traditional medicine, Astragalus mongholicus (AM) has been used for the treatment of general weakness, chronic illness, and to increase overall vitality.

Aim of the study

The present study investigated possible effects of the saponin fraction of AM on the central nervous system. Moreover, its effects on locomotor activity, anxiety, and hippocampal morphology were studied.

Material and methods

AM extract was tested for its effects on locomotor activity using the Moti-Test, for situational anxiety in the elevated plus maze, and for anticonvulsant activity against acute pentylenetetrazole (PTZ)-induced seizures and in the PTZ kindling model.

Results

It was shown that AM (50, 100, 200 mg/kg) did not interfere with locomotor activity and situational anxiety as measured in the elevated plus maze. In these doses, AM significantly suppressed pentylenetetrazole (PTZ)-induced seizures (p < 0.05). Its anticonvulsant efficacy was also evident against repeated PTZ seizures (p < 0.05). This suggests potential therapeutic usefulness. After subchronic application, the number of cells in hippocampal CA1 was reduced, whilst the cell number in CA3 and hilus remained unaffected.

Conclusions

Doses of AM extract which did not interfere with locomotor acticity and situational anxiety appear to be useful in the treatment of convulsive disorders. The mechanisms underlying this effect on hippocampal morphology are not yet understood.     

Hypnotic effects and binding studies for GABAA and 5-HT2C receptors of traditional medicinal plants used in Asia for insomnia

Aim of the study

Many medicinal plants have been used for treatment of insomnia in Asia. However, scientific evidence and precise mechanism for their sedative-hypnotic activity have not been fully investigated. Thus, we investigated the binding activity of the oriental plant extracts (mainly from Korea and Japan) to the well-known molecular targets for sleep regulation, GABA_A and 5-HT_2C receptors. Following the binding assay, sedative-hypnotic effects of the extracts with high affinity were examined in an animal model of sleep.

Materials and methods

Aqueous and ethanol extracts of 15 medicinal plants were tested for binding at the benzodiazepine site of GABA_A receptor and 5-HT site of 5-HT_2C receptor. The sedative-hypnotic effects of selected extracts were evaluated by measuring the sleep latency and sleep duration during pentobarbital-induced sleep in mice after oral administration of extracts.

Results

In the GABA_A assay, the ethanol extracts of licorice and danshen displayed concentration-dependent, high affinity binding, whereas in the 5-HT_2C assay, the ethanol extracts of ginseng and silk tree showed high affinity. Among these extracts we tested previously uncharacterized licorice and silk tree for hypnotic effects. We found the ethanol extracts of licorice and silk tree significantly decreased sleep latency and increased sleep duration in pentobarbital-induced sleep.

Conclusions

We demonstrate for the first time that licorice and silk tree have the sedative-hypnotic activity possibly by modulating GABA_A and 5-HT_2C receptors. We propose that licorice and silk tree might be effective candidates for treatment of insomnia.     

Effect of Tong Luo Jiu Nao on Aβ-degrading enzymes in AD rat brains

Ethnopharmacological relevance

Tong Luo Jiu Nao (TLJN) is a modern Chinese formula based on Traditional Chinese Medicine theory that has been used to treat ischemic cerebral stroke and vascular dementia. TLJN belongs to the ethnopharmacological family of medicines. In this study, we investigated the mechanism of the TLJN effect on Alzheimer's disease (AD).

Aim of the study

To investigate the effect of TLJN on β-amyloid-degrading enzymes and learning and memory in the AD rat brain.

Materials and methods

AD rats whose disease was induced by Aβ_25-35 injection into the bilateral hippocampus CA1 region were subjected to intragastric administration of various preparations. The experimental animals were healthy male Sprague-Dawley rats which were randomly divided into normal, sham, model, TLJN min, TLJN max and donepezil hydrochloride groups. Spontaneous alternation and passive avoidance behavior, which are regarded as measures of spatial learning and memory, were investigated using Y-maze testing. Western blotting and immunohistochemistry were used to observe the therapeutic effect of TLJN on the deposits of amyloid plaque and on the expression of synaptophysin, insulin-degrading enzyme and neprilysin.

Results

Y-maze results showed that the AD model group presented with spatial learning and memory impairments. Hematoxylin-eosin and Congo red staining indicated neuronal impairment and deposits of amyloid plaque in the model group and these results were consistent with their learning and memory deficits in the Y-maze. The TLJN-treated groups exhibited prolonged a cavity delitescence, decreased arm entries and improvement in learning and memory. Moreover, the structure of the neurons of the treated groups was restored and the expression of synaptophysin increased in both the hippocampus and cortex. In addition, their levels of insulin-degrading enzyme and neprilysin in the cortex and hippocampus were upregulated and the amyloid plaque was decreased.

Conclusion

TLJN can improve learning and memory, up-regulate insulin-degrading enzyme and neprilysin levels, promote the degrading of Aβ and clear amyloid plaque from the AD rat brain. In future, TLJN may have significant therapeutic potential in the treatment of AD patients.     

In vivo anti-inflammatory activities of leaf extracts of Ocimum lamiifolium in mice model

Aim of the study

Ocimum lamiifolium Hochst. ex Benth. (Lamiaceae) has been used in Ethiopian traditional medicine for the treatment of different inflammatory disorders such as oropharyngitis, wound, pain, fever, and others. However, its use has mainly been based on empirical findings. Thus the objective of this study was therefore to evaluate the antiinflammatory effects and acute oral toxicities of the leaf extracts of Ocimum lamiifolium in mice.

Materials and methods

Aqueous and ethanol crude extracts were screened for their antiinflammatory activities in mice using carrageenin induced paw edema. And then the aqueous extract, the most active extract, was further fractionated and the fractions were tested for their anti-inflammatory activities using carrageenin, histamine and serotonin induced mice paw edema. Distilled water and aspirin were employed as negative and positive controls, respectively. Acute oral toxicity of both extracts and fractions were also determined after giving graded doses.

Results

The aqueous and ethanol extracts were able to reduce inflammation significantly, but greater anti-inflammatory activity was observed for the aqueous extract at all dose levels. Of all fractions the water residue showed highly significant anti-inflammatory activity.

Conclusions

Ocimum lamiifolium leaf extracts exhibited significant anti-inflammatory activities with less acute toxicity.     

Sedative, hypnotic and anticonvulsant activities of the ethanol fraction from Rhizoma Pinelliae Praeparatum

Ethnopharmacological relevance

Rhizoma Pinelliae Praeparatum is the product of raw Rhizoma Pinellia processed with alkaline solution and Licorice, which had been widely used for treatment of insomnia in traditional Chinese medicine. The present study aimed to investigate the sedative, hypnotic and anticonvulsant activities of ethanol fraction from Rhizoma Pinelliae Praeparatum (EFRP) and to determine whether these effects were related to GABAergic mechanism.

Materials and methods

The sedative, hypnotic and anticonvulsant activities of EFRP were investigated with locomotion activity, pentobarbital-induced sleeping and nikethamide (NKTM)-induced convulsion tests, respectively. Additionally, the effects of flumazenil (an antagonist of GABA_A receptor) and l-malic acid (blocker of synthetic enzyme for GABA) on the hypnotic activity of EFRP were evaluated.

Results

EFRP at dose of 12 g/kg significantly inhibited the locomotion activity of mice. EFRP showed synergic effect on pentobarbital-induced sleeping by increased numbers of mice falling asleep, reduced the sleep latency and prolonged the sleeping time. l-malic acid and flumazenil inhibited the augment effects of EFRP on pentobarbital-induced sleeping. EFRP promoted a significant protection to NKTM-induced convulsion, by prolonged the death latency and decreased mortality.

Conclusion

EFRP possessed sedative, hypnotic and anticonvulsant activities and these activities may be related to the GABAergic system.     

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.     

Antinociceptive activity of ethanolic extract and isolated compounds of Urtica circularis

Ethnopharmacological relevance

Urtica circularis (Hicken) Sorarú is a medicinal plant commonly used in traditional medicine to relieve pain in inflammatory processes.

Aim of the study

In the present study, the in vivo antinociceptive effect of Urtica circularis ethanolic extract and its isolated compounds has been investigated.

Materials and methods

Antinociceptive activity was evaluated through writhing, formalin and hot plate tests in mice. The phytochemical analysis was performed.

Results

The extract produced significant inhibition on nociception induced by acetic acid (ED50: 72.2 mg/kg, i.p.) and formalin (ED50: 15.8 mg/kg, i.p.) administered intraperitoneally and also orally. Atropine diminished the activity of the extract in the acetic acid test. In this model, at dose of 10 mg/kg i.p., vitexin was the most active of the isolated compounds (inhibition of 91%), and chlorogenic acid, caffeic acid and vicenin-2 (6,8-di-C-glucosyl apigenin) produced an inhibition of 72%, 41% and 41%, respectively, whereas apigenin did not show any activity.

Conclusions

These results suggest that Urtica circularis extract produced antinociception possibly related to the presence of vitexin, chlorogenic, caffeic acid and vicenin-2. The activation of cholinergic systems seems to be involved in the mechanism of antinociception of the extract.     

In vitro growth stimulatory and in vivo wound healing studies on cycloartane-type saponins of Astragalus genus

Aim of the study

The present study was undertaken to evaluate the wound healing effects of the four chief saponins of Astragalus species [cycloastragenol (CA), astragaloside IV (AG), cyclocephaloside I (CCI) and cyclocanthoside E (CCE)].

Material and methods

Effects of cell viability and proliferation of the isolated compounds were evaluated by the MTT assay on human keratinocyte. The wound healing activity was studied by using in vitro wound healing, proliferation and migration scratch assay. In order to see in vivo effectiveness of the compounds, an animal study with Sprague-Dawley male rats at the age of 12 weeks was carried out, and then the main histological outcomes were investigated to observe reepithelization, neovascularization, and presence of inflammatory cells, granulation tissue amount and maturation.

Results

All the compounds increased both fibroblast proliferation and migration, but the effects were much superior for CA at 1 ng/ml concentration. Among the compounds, based on the histological findings, 5% CA preparation was found to be the most remarkable in vivo wound healing agent showing greater cell density, more regularly organized dermis and more newly formed blood vessels.

Conclusion

Results of this study indicate that the cycloartane-type saponins are the principal constituents responsible for wound healing activities of the roots of Astragalus species substantiating its use in traditional medicine.     

Gypsum fibrosum and its major component CaSO4 increase cutaneous aquaporin-3 expression levels

Ethnopharmacological relevance

We have previously reported that Byakkokaninjinto improves cutaneous pruritus by increasing the expression level of aquaporin-3 (AQP3). In this study, we examined the effect of Gypsum fibrosum (main component: CaSO₄), which is the main component of Byakkokaninjinto, on the cutaneous AQP3 expression level.

Materials and methods

KKAy mice were given a diet containing 0.3% Gypsum fibrosum extract, or a diet containing 0.3% CaSO₄ for 4 weeks. The urine volume, plasma glucose levels, cutaneous AQP3 protein expression, and the Ca²⁺ content were measured.

Results

The 24-h urine volumes and the plasma glucose levels in the Gypsum fibrosum extract group were not significantly different from those in the control group. In the Gypsum fibrosum extract group, the cutaneous AQP3 protein levels increased significantly, by approximately 3.2-fold, compared to the control group. The cutaneous Ca²⁺ content in the control group was approximately 35 μg/g. In the Gypsum fibrosum extract group, the Ca²⁺ content increased to approximately 51 μg/g, which was significant compared to the control group. In the CaSO₄ group, an increase in the AQP3 protein expression levels and Ca²⁺ content were observed; the extent of these increases were similar to those in the Gypsum fibrosum extract group.

Conclusions

The results of this study suggest that Gypsum fibrosum plays an important role in the increased levels of cutaneous AQP3 expression enhanced by Byakkokaninjinto. The results also indicate that the increase in AQP3 caused by Gypsum fibrosum is attributable to an increase in the cutaneous Ca²⁺ content from its main component, CaSO₄.