Antioxidant activity and laxative effects of tannin-enriched extract of Ecklonia cava in loperamide-induced constipation of SD rats.

To investigate the role of tannin-enriched extracts of Ecklonia cava (TEE) on the regulation of oxidative balance and laxative activity in chronic constipation, we investigated alterations after exposure to TEE, on constipation phenotypes, muscarinic cholinergic regulation, and oxidative stress responses in the transverse colons of SD rats with loperamide (Lop)-induced constipation. This extract contains high levels of total condensed tannin content (326.5 mg/g), and exhibited high inhibitory activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. TEE treatment induced significant improvements in reactive oxygen species (ROS) production, superoxide dismutase (SOD) expression and nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation in primary smooth muscles of rat intestine cells (pRISMCs) and transverse colon of constipation model. Also, Lop+TEE treated groups showed alleviated outcomes for the following: most stool parameters, gastrointestinal transit, and intestine length were remarkably recovered; a similar recovery pattern was observed in the histopathological structure, mucin secretion, water channel expression and gastrointestinal hormones secretion in the transverse colon; expressions of muscarinic acetylcholine receptors M2/M3 (mAChR M2/M3) and their mediators on muscarinic cholinergic regulation were significantly recovered. Taken together, these results provide the first evidence that TEE stimulates oxidative stress modulation and muscarinic cholinergic regulation when exerting its laxative effects in chronic constipation models.

Anti-ulcer effect of Gallarhois extract with anti-oxidant activity in an ICR model of ethanol/hydrochloride acid-induced gastric injury.

Gallarhois (GR) is a traditional oriental herbal medicine with various pharmacological effects; however, its effect on gastric ulcer has not been previously explored. We firstly investigated the component and antioxidant activity of GR extract (EtGR) by HPLC analysis and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The results showed that EtGR consisted of gallotannin (68.7%), gallic acid (27.2%) and methyl gallate (4.1%) and that it had a high antioxidant value (IC50 value; 1.93 µg/mL). To evaluate the possible anti-gastric ulcer potential of EtGR, we investigated the effects of EtGR in the model of ethanol/hydrochloric acid (EtOH/HCl)-induced gastric ulcer. Gross and histological gastric lesions, biochemical and gene expression parameters were taken into consideration. The results showed that EtOH/HCl treatment produced mucosal injuries with morphological and histological damage, whereas EtGR co-treatment reduced the gastric injuries. EtGR treatment also decreased the contents of malonaldehyde (MDA) activity relative to the vehicle group. Moreover, EtGR decreased the levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and cyclo-oxygenase-2 (COX-2) expression. Finally, EtGR did not induce any specific toxicity in the livers or kidneys of the EtOH/HCl-induced gastric ulcer model. These results suggest that EtGR had stronger antioxidant activity and could be a new useful natural drug for gastroprotection against gastric ulcer. Moreover, these findings provide a scientific basis for the development of drugs from traditional oriental herbal medicines.

Asparagus cochinchinensis stimulates release of nerve growth factor and abrogates oxidative stress in the Tg2576 model for Alzheimer's disease.

BACKGROUD: Use of multifunctional drugs with neurotrophic supporting and oxidative stress suppressing activity may be considered a therapeutic strategy to protect or repair cellular damage caused during the progression of Alzheimer's disease (AD). In this study, we investigated the therapeutic effects of aqueous extract of A. cochinchinesis root (AEAC), particularly its role as a nerve growth factor (NGF) stimulator and anti-oxidant in Tg2576 mice showing AD phenotypes of human. METHODS: Tg2576 mice were received 100 mg/kg/day AEAC via oral administration, while mice in the Vehicle treated group received dH2O for 4 weeks. Non-Tg littermates were used as a control group. Following AEAC treatment for 4 weeks, NGF function, anti-oxidantive status, Aß-42 peptide level, γ-secretase expression and neuronal cell functions were analyzed in the brain of Tg2576 mice. RESULTS: AEAC containing flavonoids, phenols, saponins and protodioscin induced enhancement of NGF secretion and decreased intracellular ROS in the neuronal and microglial cell line. These effects as well as enhanced SOD levels were also detected in AEAC treated Tg2576 mice. The expression of p-Akt among downstream effectors of the high affinity NGF receptor was dramatically recovered in AEAC treated Tg2576 mice, while the expression of p75NTR was slightly recovered in the same group. Significant recovery on the level of Aß-42 peptides and the expression of γ-secretase members including PS-2, APH-1 and NCT were detected in AEAC treated Tg2576 mice. Furthermore, AEAC treated Tg2576 mice showed decreased numbers of dead cells and suppressed acetyl choline esterase (AChE) activity. CONCLUSIONS: These results suggest that AEAC contribute to improving the deposition of Aß-42 peptides and neuronal cell injuries during the pathological progression stage of AD in the brain of Tg2576 mice through increased NGF secretion and suppressed oxidative stress.

Selenium-loaded cellulose film derived from Styela clava tunic accelerates the healing process of cutaneous wounds in streptozotocin-induced diabetic Sprague-Dawley rats.

PURPOSE: Aims of this study is to evaluate the therapeutic effects and toxicity of Se-loaded cellulose film originated from Styela clava tunic (SeSCTF) on cutaneous wounds during diabetic conditions. MATERIALS AND METHODS: Alterations in skin regeneration, angiogenesis and toxicity were examined using streptozotocine (STZ)-induced diabetic Sprague Dawley® (SD) rats with surgical skin wounds after application of SeSCTF for 12 days. RESULTS: SCTF showed high tensile strength (1.64 MPa), low elongation (28.59%), low water vapor transmission rate (WVTR) and outstanding porous structure. Although SeSCTF application did not induce any significant alterations in glucose concentration or toxicity, wound morphology was rapidly recovered in the SeSCTF treated group relative to the gauze (GZ) and SCTF treated group. Moreover, recovery of re-epithelization, wound contraction and number of blood vessel was observed in SeSCTF treated groups when compared with all other groups. Furthermore, the SeSCTF treated group showed complete recovery of key protein expressions of the downstream signaling pathway of vascular endothelial growth factor (VEGF), angiopoietin-2/1 (Ang-2/1), the signaling pathway of insulin receptors and anti-oxidative status. CONCLUSIONS: Overall, the results of this study suggest that SeSCTF accelerates the healing process of cutaneous wounds in STZ-induced diabetic SD rats through stimulation of angiogenesis and the glucose receptor signaling pathway.

Saponin-enriched extract of Asparagus cochinchinensis alleviates airway inflammation and remodeling in ovalbumin-induced asthma model.

Asthma is a chronic inflammatory disease characterized by T-lymphocyte and eosinophil infiltration, mucus overproduction and airway hyper-responsiveness. The present study examined the therapeutic effects and action mechanism of a saponin-enriched extract of Asparagus cochinchinensis (SEAC) on airway inflammation and remodeling in an ovalbumin (OVA)-induced asthma model. To accomplish this, alterations of the nitric oxide (NO) level, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression levels, as well as variations in immune cell numbers, immunoglobulin E (IgE) concentration, histopathological structure and inflammatory cytokine levels were measured in lipopolysaccharide (LPS)-activated RAW264.7 cells or an OVA-induced mouse model of asthma treated with SEAC. The concentration of NO and mRNA levels of COX-2 and iNOS were significantly decreased in the SEAC + LPS-treated RAW264.7 cells compared with the vehicle + LPS-treated RAW264.7 cells. Additionally, in the OVA-induced asthma model, the number of immune cells in the bronchoalveolar lavage fluid, the concentration of OVA-specific IgE, the infiltration of inflammatory cells, the bronchial thickness and the levels of the inflammatory mediators interleukin-4 (IL-4), IL-13 and COX-2 were significantly lower in the OVA + SEAC­treated group compared with the OVA + vehicle­treated group. In addition, a significant reduction in goblet cell hyperplasia, peribronchiolar collagen layer thickness and VEGF expression for airway remodeling was detected in the OVA + SEAC­treated group compared with the OVA + vehicle­treated group. These findings indicate that SEAC is a suppressor of airway inflammation and remodeling, and may therefore be useful as an anti-inflammatory drug for the treatment of asthma.

Ethyl acetate extract from Asparagus cochinchinensis exerts anti­inflammatory effects in LPS­stimulated RAW264.7 macrophage cells by regulating COX­2/iNOS, inflammatory cytokine expression, MAP kinase pathways, the cell cycle and anti-oxidant activity.

Asparagus cochinchinesis (A. cochinchinesis) is a medicine traditionally used to treat fever, cough, kidney disease, breast cancer, inflammatory disease and brain disease in northeast Asian countries. Although numerous studies of the anti­inflammatory effects of A. cochinchinesis have been conducted, the underlying mechanisms of such effects in macrophages remain to be demonstrated. To investigate the mechanism of suppressive effects on the inflammatory response in macrophages, alterations of the nitric oxide (NO) level, the cell viability, inducible nitric oxide synthase (iNOS) and cyclooxygenase­2 (COX­2) expression levels, inflammatory cytokine expression, the mitogen-activated protein kinase (MAPK) signaling pathway, cell cycle arrest and reactive oxygen species (ROS) levels were measured in lipopolysaccharide (LPS)-activated RAW264.7 cells following treatment with ethyl acetate extract from A. cochinchinesis root (EaEAC). RAW264.7 cells pretreated two different concentrations of EaEAC prior to LPS treatment exhibited no significant toxicity. The concentration of NO was significantly decreased in the EaEAC + LPS treated group compared with the vehicle + LPS treated group. A similar decrease in mRNA transcript level of COX­2, iNOS, pro-inflammatory cytokines [tumor necrosis factor­α and interleukin (IL)­1ß] and anti­inflammatory cytokines (IL­6 and IL­10) was detected in the EaEAC + LPS treated group compared with the vehicle + LPS treated group, although the decrease rate varied. Enhancement of the phosphorylation of MAPK family members following LPS treatment was partially rescued in the EaEAC pretreated group, and the cell cycle was arrested at the G2/M phase. Furthermore, the EaEAC pretreated group exhibited a reduced level of ROS generation compared with the vehicle + LPS treated group. Taken together, these results suggest that EaEAC suppresses inflammatory responses through inhibition of NO production, COX­2 expression and ROS production, as well as differential regulation of inflammatory cytokines and cell cycle in RAW264.7 cells. In addition, these results provide strong evidence to suggest that EaEAC may be considered as an important candidate for the treatment of particular inflammatory diseases.

α-Isocubebenol alleviates scopolamine-induced cognitive impairment by repressing acetylcholinesterase activity.

α-Isocubebenol (ICO) isolated from Schisandra chinensis fruit was recently shown to exert neuroprotective properties with significant anti-neuroinflammatory effects. Here, we present evidence of the novel effects of ICO on alleviation of cognitive impairment. To confirm these effects, ICR mice were pretreated with two different doses of ICO for 3 weeks and scopolamine (SP) to induce memory impairment for the last 7days of the period. A passive avoidance test showed that ICO pretreatment recovered memory impairment in SP treated mice, although there was no difference between the two doses. Acetylcholinesterase (AChE) activity was significantly decreased in the SP+ICO treated group compared with the SP+Vehicle treated group. Additionally, significant recovery of the number of apoptotic cells and the ratio of apoptosis proteins (Bcl-2/Bax) were detected in the SP+ICO treated group than the SP+Vehicle treated group. Moreover, ICO treatment attenuated the decrease of ERK phosphorylation by SP treatment. These results indicate that ICO from S. chinensis fruit could be applied as an active pharmaceutical ingredient for cognitive improvement in Alzheimer's disease (AD).

Effects of five candidate laxatives derived from Liriope platyphylla on the 5-HT receptor signaling pathway in three cell types present in the transverse colon.

The laxative effects of aqueous extract of Liriope platyphylla (AEtLP) on loperamide (Lop)­induced constipation have been reported; however, the key compounds and the mechanism underlying these effects remain unclear. Therefore, the laxative effects of five candidates derived from L. platyphylla: Diosgenin (DG), 5-hydroxymethylfurfural (5-HMF), adenosine (AD), hydroxypropyl cellulose (HPC) and uridine (UD) were investigated by examining the alteration of G protein α (Gα) expression, protein kinase C (PKC) phosphorylation and inositol triphosphate (IP3) concentration levels in the 5-hydroxytryptamine (5­HT; serotonin) receptor signaling pathway. Primary rat intestine smooth muscle cells (pRISMCs), intestinal epithelial cells (IEC)­18 and B35 cells were cotreated with Lop and the five compounds in order to screen the candidates. AEtLP, prucalopride (PCP) and bisacodyl (BS) served as positive controls. In pRISMCs, Gα expression levels were recovered in the majority of candidate­treated groups, whereas PKC phosphorylation recovery was observed only in the DG, 5­HMF and AD treatment groups. In IEC­18 cells, the AD treatment group mimicked the effects of PCP on PKC phosphorylation levels, whereas the DG, 5­HMF, HPC and UD treatment groups mimicked the effects of AEtLP and BS. In B35 cells, a greater upregulation of PKC phosphorylation levels were observed in the UD treatment group compared with the PCP and BS treatment groups, whereas DG, 5­HMF and AD treatment reduced the PKC phosphorylation levels to a greater extent than AEtLP treatment. However, effects similar to AEtLP, PCP and BS on Gα expression levels were not detected in any treatment groups in IEC­18 and B35 cells. Furthermore, the level of IP3 was enhanced only in pRISMCs, in which all five candidates were effective, while the greatest concentration was observed in the UD treatment group. In conclusion, the results of the present study suggest that UD may be considered the compound with the greatest laxative activity, which may regulate the 5­HT receptor signaling pathway.

Gallotannin-Enriched Extract Isolated from Galla Rhois May Be a Functional Candidate with Laxative Effects for Treatment of Loperamide-Induced Constipation of SD Rats.

Several natural products containing tannins are used as traditional medicines for treatment of constipation; however, their pharmacological mechanism is not well understood. The laxative effects of gallotannin-enriched extract isolated from Galla Rhois (GEGR) were investigated using a constipation model induced by loperamide (Lop) injection. After analysis for antioxidant activity of GEGR, alterations in the excretion parameters, histological structure, mucin secretion, and related protein levels were measured in the transverse colon of Sprague Dawley (SD) rats with Lop-induced constipation following treatment with 250, 500 and 1,000 mg/ml of GEGR. The number and weight of feces increased significantly by 48-79% and 128-159%, respectively, in the Lop+GEGR treated group relative to the Lop+vehicle treated group, while food intake and water consumption were maintained at a constant level. The thickness of mucosa, muscle and flat luminal surface, as well as the number of goblet cells and crypt of lieberkuhn were enhanced in the Lop+GEGR treated group. Moreover, mucin secretion increased significantly in a dose dependent manner in the Lop+GEGR treated group. Furthermore, the downstream signaling pathway of the muscarinic acetylcholine receptors (mAChR) M2 and M3 was recovered by GEGR treatment, although the expression level varied. The levels of Gα expression and inositol triphosphate (IP3) concentration were also recovered in the Lop+GEGR treated group relative to the Lop+vehicle treated group. The results of the present study provide strong evidence that tannins distributed in various medicinal plants are important candidates for improving chronic constipation induced by Lop treatment in animal models.

Neuroprotective effects of fermented soybean products (Cheonggukjang) manufactured by mixed culture of Bacillus subtilis MC31 and Lactobacillus sakei 383 on trimethyltin-induced cognitive defects mice.

OBJECTIVES: This study aimed to investigate the beneficial effects of Cheonggukjang (CGK) manufactured by mixed culture of Bacillus subtilis MC31 and Lactobacillus sakei 383 on neurotoxic damages. METHODS: The specific aspects of brain functions were measured in Institute for Cancer Research (ICR) mice that had been pretreated for 4 weeks with three difference doses of CGK before trimethyltin (TMT) treatment. RESULTS: The short- and long-term memory loss induced by TMT treatment was significantly improved in the CGK-pretreated group in a dose-dependent manner. The number of dead cells in the granule cell layer of the dentate gyrus was decreased in the TMT/CGK-cotreated group relative to the TMT/vehicle-treated group, whereas significant suppression of acetylcholinesterase (AChE) activity was observed in the same group. Additionally, a dose-dependent increase in nerve growth factor (NGF) concentration, activation of the NGF receptor signaling pathway including the TrkA high affinity receptor and p75(NTR) low affinity receptor, and decline in Bax/Bcl-2 level was measured in all TMT/CGK-treated groups, although a decrease in the active form of caspase-3 was observed in the TMT/H-CGK-treated group. Furthermore, superoxide dismutase (SOD) activity was enhanced in the TMT/CGK-treated group, whereas the level of malondialdehyde (MDA), a marker of lipid peroxidation, was 43-58% lower in the TMT/CGK-treated group than the TMT/vehicle-treated group. DISCUSSION: These results demonstrate that CGK fermented by mixed culture of B. subtilis and L. sakei could exert a wide range of beneficial activities for neurodegenerative diseases, including Alzheimer, Parkinson, and Huntington disease.

Toxicity of antioxidative extract collected from Styela clava tunics in ICR mice.

Some polymers and bioactive compounds derived from Styela clava tunic (SCT) have been reported as traditional medicine for the treatment of inflammation, oxidative stress and surgical wounds although there is little scientific evidence of their liver and kidney toxicity. To investigate the toxicity of ethanol extracts of SCT (EtSCT) in the liver and kidney of ICR mice, alterations in related markers including body weight, organ weight, urine composition, liver pathology and kidney pathology were analyzed following oral administration of 50 and 100 mg/kg body weight/day of EtSCT for 14 days. EtSCT showed a high level of free radical scavenging activity for DPPH (93.1%) and NO (16.2%) as well as the presence of 14.8 mg/mL of flavonoids and 36.2 mg/mL of phenolics, while EtSCT treated groups did not show any significant alterations in the body and organ weight, clinical phenotypes, urine parameters or mice mortality when compared with the vehicle treated group. In addition, constant levels of serum biochemical markers including alanine phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and serum creatinine (CRE) were maintained. Moreover, no specific histopathological features induced by most toxic compounds were observed in liver and kidney sections stained with hematoxilin and eosin. Therefore, the present results indicate that EtSCT with strong antioxidant activity cannot induce any specific toxicity in liver and kidney organs of ICR at doses of 100 mg/kg body weight/day.

Characterization of Changes in Global Genes Expression in the Distal Colon of Loperamide-Induced Constipation SD Rats in Response to the Laxative Effects of Liriope platyphylla.

To characterize the changes in global gene expression in the distal colon of constipated SD rats in response to the laxative effects of aqueous extracts of Liriope platyphylla (AEtLP), including isoflavone, saponin, oligosaccharide, succinic acid and hydroxyproline, the total RNA extracted from the distal colon of AEtLP-treated constipation rats was hybridized to oligonucleotide microarrays. The AEtLP treated rats showed an increase in the number of stools, mucosa thickness, flat luminal surface thickness, mucin secretion, and crypt number. Overall, compared to the controls, 581 genes were up-regulated and 216 genes were down-regulated by the constipation induced by loperamide in the constipated rats. After the AEtLP treatment, 67 genes were up-regulated and 421 genes were down-regulated. Among the transcripts up-regulated by constipation, 89 were significantly down-regulated and 22 were recovered to the normal levels by the AEtLP treatment. The major genes in the down-regulated categories included Slc9a5, klk10, Fgf15, and Alpi, whereas the major genes in the recovered categories were Cyp2b2, Ace, G6pc, and Setbp1. On the other hand, after the AEtLP treatment, ten of these genes down-regulated by constipation were up-regulated significantly and five were recovered to the normal levels. The major genes in the up-regulated categories included Serpina3n, Lcn2 and Slc5a8, whereas the major genes in the recovered categories were Tmem45a, Rerg and Rgc32. These results indicate that several gene functional groups and individual genes as constipation biomarkers respond to an AEtLP treatment in constipated model rats.